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Introduction Most agronomic and horticultural crop species are angiosperms. Angiosperms are vascular plants that produce their seeds enclosed in a matured ovary, a fruit; the fruit arises from a flower. In contrast, some tree crops—such as pine or spruce—are gymnosperms, which are vascular plants possessing “naked seeds” that are not enclosed within fruit structures. There are two groups of angiosperms: monocots and dicots. Although all angiosperms have some reproductive features in common, species vary in their mode of reproduction. A species’ reproductive mode is fundamental to the methods applied to develop improved cultivars. Learning Objectives • Review hereditary mechanisms and flower anatomy. • Understand the sexual reproduction processes of pollination, fertilization, and seed development. • Become familiar with asexual reproduction. • Comprehend the implications of reproductive mode for crop breeding strategies. Hereditary Mechanisms Heredity, Genotypes, and Phenotypes Plant breeders take advantage of the mechanisms of heredity to develop and maintain cultivars. The observable characteristics and performance of cultivar (of a plant), its phenotype, is the result of the cultivar’s genotype, as influenced by the environment. In other words, phenotype is a function of both genotype and environment, plus the interaction between genotype and environment. $P=G+E+(G \times E)$ where • $P= \text{Phenotype}$ • $G= \text{Genotype}$ • $E= \text{Environment}$ Fundamental to effective and efficient plant breeding is an understanding of the hereditary mechanisms that affect genotype: • Nuclear division and chromosomes • Modes of reproduction Deoxyribonucleic Acid (DNA) And Chromosomes Every cell nucleus contains the genetic material of the cell, deoxyribonucleic acid (DNA), located in chromosomes. Each chromosome is a single DNA molecule. Associated with the DNA are special proteins called histones (see the round yellow shapes in Figure 2 around which the DNA is wound like “beads on a string” along the chromosome) that are related to the organization of the DNA, as well as enzymes involved in replication of the DNA strand. In plant cells, DNA and associated genetic information are mainly located in nuclear chromosomes. However, some additional DNA and genetic information is located in specialized cell structures that are “extra-nuclear,” meaning found outside of the cell nucleus. Nuclear vs. Organellar DNA Although by far the main portion of DNA and genetic information is located on nuclear chromosomes, additional DNA (and genetic information) is located in two types of organelles in plant cells: plastids and mitochondria. Each plant cell usually contains multiple plastids and mitochondria, which contain multiple copies of circular DNA each. Different from the inheritance of nuclear DNA, organellar DNA is maternally inherited in most plant species and not undergoing meiosis. Mitochondrial DNA is the small circular chromosome found inside mitochondria, a type of organelle found in cells, and that are the sites of energy production.Illustration by NIH-NHGRI. Chromosomes And Genomes A genome is the basic set of chromosomes inherited as a unit from one parent. Somatic cells (non-germ cells) of diploid species contain two sets (2n) of the basic genome (haploid) (1n) number of chromosomes. Among species, the number of chromosomes varies. Within a species, the chromosome number (2n in somatic cells and 1n in germ cells) is ordinarily constant. However, crop species include both diploids and polyploids, which are plants with more than two sets of chromosomes in their cells. Crop Chromosome Number Crop species include a wide range of chromosome numbers. The genomic formula of crop species (with 2n representing the somatic chromosome number, n, the haploid number, and x, the basic chromosome number) can reveal whether or not the crop is a polyploid. For example, vanilla, coconut, pecans, alfalfa, leek, and sour cherry all have the same number of chromosomes, but the first three are diploids (2n=2x=32), and the last three are polyploids (2n=4x=32). Polyploids can be classified as either autopolyploids (see crop examples labeled as “auto” in the table where their status is known) or allopolyploids (“allo” in the table). Autopolyploidsare polyploids with multiple chromosome sets derived from a single species, whereas allopolyploids are polyploids with genomes derived from different species. Interactive Crop Chromosome Number Click each of the crop groups below to see additional information. Mitosis and Meiosis Mitosis is one phase of the cell cycle. Mitosis is divided into four arbitrary stages. Progress between stages, however, is gradual and continuous. During cell division, DNA is duplicated and distributed to daughter nuclei. The number of chromosomes in the daughter nuclei depends on the process of nuclear division. There are two nuclear division processes by which new cells are formed. Mitosis Let’s follow mitosis, the process by which somatic cells, non-germ cells, are reproduced. We’ll begin with interphase of the cell cycle. This illustration represents a diploid, or 2n, somatic nucleus. It has two sets of chromosomes, shown here as a blue set and a red set. Each chromosome is duplicated at the end of interphase during the synthesis period of the cell cycle that precedes mitosis. Mitosis is a process of nuclear division. It has four stages: • Prophase • Metaphase • Anaphase • Telophase Let’s see what happens during each of these stages. Query $2$ FYI: Cell Cycle • G2-Gap This period occurs after DNA replication is complete, but before mitosis begins. • M-Mitosis The nucleus divides, distributing a complete set of chromosomes to each daughter nucleus. The cell subsequently undergoes cytokinesis, cytoplasmic division, completing the formation of the two daughter cells; each repeats a new cell cycle. Of the cell cycle phases, mitosis is the shortest, typically lasting only 1 to 3 hours. • G1-Interphase Occurs after the completion of mitosis and precedes DNA replication. • S-Synthesis DNA replication occurs. This phase can be identified because it is the only phase during which the cell can incorporate radioactive thymidine into nuclear DNA. Thymidine is related to one of the purine bases of DNA, thymine. Meiosis In contrast to mitosis, meiosis is the process through which germ cells, microspores and megaspores, are derived. Meiosis is similar to mitosis except in two important aspects: • Meiosis involves two successive divisions. • Homologous chromosomes replicate only once during the two divisions. Thus, the diploid microspore and mega spore mother cells are meiotically reduced to the haploid, or 1n, chromosome number of the gametes. In meiosis, there are two successive divisions, called meiosis I and meiosis II. Each of these is divided into four phases analogous to those of mitosis. Like mitosis, these stages progress in a gradual and continuous manner. Similarities to Mitosis Meiosis I resembles mitosis in that: • The division results in the production of two daughter cells. • Cells are derived from a microspore or megaspore mother cell. • Replication of homologous chromosomes precedes it. Review Now review these two processes: mitosis and meiosis. Again, pay attention to commonalities and differences. You should be able to identify key features of each process and stage. Process Divisions Equational division One equational division, one reductional division Results in Two 2n daughter cells Four 1n daughter cells Stages Four: • Prophase • Metaphase • Anaphase • Telophase Eight or nine: • Prophase I • Metaphase I • Anaphase I • Telophase I • Interkinesis (sometimes) • Prophase II • Metaphase II • Anaphase II • Telophase II Study Question 1 Identify the cell division process that best fits each statement by dragging the label to the box next to it. Query $4$ Study Question 2 Barley has a diploid chromosome number of 14. Identify the number of chromosomes in various cell or tissue types. Sexual Reproduction Reproduction enables the propagation of new individuals. Reproduction in crop species may occur sexually, asexually, or both. • Sexual reproduction: Requires the fusion of egg and sperm cells (known as gametes) to obtain the next generation. The life cycle of a typical angiosperm involves sexual reproduction based on the process of meiosis, in which the chromosome number of cells in the female and male reproductive organs is reduced by half to form female and male gametes. Meiosis is the process responsible for the genetic segregation observed in progeny of heterozygous individuals. • Asexual reproduction: Propagation occurs without the fusion of male and female gametes. Asexual reproduction is based on the multiplication of cells by mitosis and results in two new cells that are genetically identical to each other and to the cell from which they originated. Synopsis of the Life Cycle of the Angiosperm Plant In the animal kingdom, the production of gametes follows immediately after the meiotic divisions. Normally, therefore, the gametes are the only haploid (1n) representatives of the animal life cycle. In plants, however, almost invariably (and without exception in higher plants) the immediate products of the meiotic divisions are not gametes but spores. The higher plants (angiosperms) we recognize, e.g., the oak tree, turfgrass, clover, wheat, are the diploid (2n) or sporophytic stage of the plant’s life cycle. In these plants the haploid vegetative or gametophytic stage is short-lived and quite inconspicuous. The sporophyte produces spores as a result of sporogenesis or meiosis. Spores undergo a few nuclear divisions in a process known as gametogenesis to form mature gametophytes. Gametes develop within the gametophytes. All higher plants produce two types of spores, microspores and megaspores. Corresponding to these two types of spores are the two different modes of their development, microgametogenesis and megagametogenesis, which culminate respectively in two dissimilar and relatively simple plants, the mature microgametophytes and megagametophytes. Male Spore Formation Each of many 2n microsporocytes (pollen mother cells) within the anther undergoes meiosis with the result that four haploid (1n) microspores are produced within the anther for each original microsporocyte. FYI: Microsporogenesis Microsporogenesis is the process by which male gametes (pollen grains) are formed. This process can be divided into three parts: 1. Meiosis I 2. Meiosis II 3. Endomitosis Each of these is subdivided into several stages. The stages of microsporogenesis are transitory. The sequence of stages is shown in the following photos (from Chang and Neuffer, 1989). Each photo represents a momentary expression, which may not be a good representation of the complete event. The event in each photo is indicated by an arrow. Microgametogenesis The single nucleus of each microspore divides once by mitosis and one of the two daughter nuclei draws about itself a mass of deeply staining cytoplasm. This nucleus is known as the generative nucleus while the other is the tube nucleus. The generative nucleus undergoes a single mitotic division to form two male gametes, or sperm cells. (In some plants this division does not occur until pollination has taken place and the generative nucleus is moving through the pollen tube.) This constitutes a pollen grain or a mature microgametophyte. Female Spore and Gamete Formation Megasporogenesis A 2n megasporocyte (megaspore mother cell) in each ovule undergoes meiosis. Four megaspores result, each with a haploid chromosome number. Three of these disintegrate; the fourth develops into the mature female gametophyte. Megagametogenesis (development of the female gametophytes and gametes) The surviving megaspore enlarges greatly to form the embryo sac. Three successive mitotic divisions, starting with the original nucleus of this megaspore, produce eight haploid daughter nuclei within the embryo sac. These orient themselves as follows: • The two polar nuclei lie together in the middle of the sac. • Three nuclei are located at the end of the sac where the sperm will enter, the center one becoming the female gamete or egg, and the two flanking ones the synergids. • The remaining three, the antipodal cells, come to lie at the opposite end of the sac. The number of antipodal cells, however varies greatly from zero in Oenothera species to more than 100 in some grass species. The embryo sac with the eight haploid nuclei thus arranged is the mature megagametophyte the female gamete or egg, and the two flanking ones the synergids; the remaining three, the antipodal cells, come to lie at the opposite end of the sac. The number of antipodal cells, however varies greatly from zero in Oenothera species to more than 100 in some grass species. The embryo sac with the eight haploid nuclei thus arranged is the mature megagametophyte. Double Fertilization Pollen grains are freed by opening of the anther wall and are carried to the stigma of the same or other plants. Each pollen grain soon sends out a small thin pollen tube (generated by the tube nucleus) which penetrates the tissues of the stigma and digests its way through these and the stylar tissues down to one of the ovules. The sperm cells pass down the pollen tube behind the tube nucleus. Once the pollen tube reaches the ovule it penetrates the embryo sac, the tube nucleus disintegrates, and the two sperm cells enter. One of the sperms fuses with the haploid egg to produce the 2n zygote, while the other fuses with the two polar nuclei to give a 3n (triploid) product, the endosperm nucleus. Further Development The triploid endosperm tissue grows more rapidly than the embryo at first. Later on, the embryo, which develops from the zygote, grows at the expense of the endosperm. Depending on the species, endosperm tissue may or may not persist at the time the seed has completed growth. With the resumption of growth (seed germination) the embryo continues development until it reaches the mature sporophyte stage, at which time microspores and megaspores are again produced. Study Question 3 For Your Information Endomitosis Interphase occurs between meiosis II and endomitosis. Chromosomes replicate during interphase. Endomitosis is a process of cell division resulting in the production of the pollen grains. Endomitosis is also divided into phases. Figures 23 through 36 show these phases, as well as the gradually increasing accumulation of starch granules in the cell. The starch grains progressively obscure the visibility or resolvability of the cellular structures of the male gametophyte. Interphase The cell nucleus is round, condensed and nondifferentiated . Chromosomes replicate during this period. First Prophase The chromosomes condense into short thick threads surrounding the nucleolus. Middle First Prophase The chromosomes continue to condense into short thick threads which allow identification of individual chromosomes. The nucleolus and the nucleolus-organizing region of chromosome six are visible. Late First Prophase The chromosomes further condense to become short thick rods. The nucleolus and the nucleolus-organizing regions with chromosome six are clearly seen. First Metaphase The nucleolus disappears and the ten chromosomes are arranged in one plane close to one another. First Anaphase The sister chromatids are now separated and moving towards the opposite poles. Late First Anaphase The separated chromosomes have reached the opposite poles and formed two chromosome clusters. First Telophase The chromosomes at each pole are now extended and surround the nucleolus. Binucleate Two nuclei are formed at the opposite poles. The generative nucleus (bottom) is usually located near the germ pole. It will further divide to form two sperm. Second Prophase The generative nucleus underneath the surface of the intine wall becomes cup-shaped and will proceed to the second nuclear division. The vegetative nucleus is not at resting stage and appears to continue its metabolic activities. Second Metaphase The nucleolus disappears, the ten chromosomes are arranged in one single plane. The vegetative nucleus is dark-stained. Second Anaphase The sister chromatids are now separated and moving towards the opposite poles . The vegetative nucleus remains large and clear. Second Telophase The chromosomes at each pole are now extended and surround the nucleolus. The vegetative nucleus remains large and clear. Mature Pollen The mature pollen grain now has three nuclei. The top two condensed, crescent-shaped nuclei, surrounding the germ pore are the sperms. The large one at the bottom is the vegetative nucleus. Meiosis I Meiosis I is a reductional division-the number of chromosomes in the nucleus is reduced to the haploid number. Meiosis I has four phases: prophase I, metaphase I, anaphase I, and telophase I. Premeiotic Interphase The irregularly shaped pollen mother cell has dense protoplasm, no vacuoles, no clear cell wall structure and an undifferentiated nucleus. Prophase I Prophase I is the longest phase of Meiosis I. During Prophase I, the nuclear membrane breaks down, the chromosomes contract, and the spindle forms. Prophase I has several substages: lepotene, zyotene, pachytene, diplotene, and diakinesis. Leptotene Cell becomes round with dense protoplasm. The chromatin threads are greatly extended and coiled around the nucleolus. Synapsis is initiated. Single and double strand configuration is evident. The chromomeres are visible. Late Zygotene-Early Pachytene The pairing of the homologous chromosomes is complete. The condensed chromosomes show details of hetero-chromatin and knobs. The nucleolus and nucleolar-organizing region of chromosome six are visible. Pachytene The paired chromosomes are further condensed to become a very thick thread. Individual chromosomes can be identified by their relative lengths, distinctive chromomere patterns, position of knobs, and other recognizable characteristics. The nucleolar-organizing region of chromosome six is clearly attached to the nucleolus. The chromosomes continue to condense into short, thick threads. The paired chromosomes appear to be repulsing one another, except regions where an actual crossover took place. The chiasmata are frequently seen as X-shaped and looped chromosome configurations. Late Diplotene The chiasmata are terminalized and the very short condensed chromosome pairs are separated from each other. The X-shaped and looped chromosome configurations are still shown. The nucleolar- organizing region of chromosome six is firmly attached to the nucleolus. Diakinesis The condensed chromosome pairs are separated from each other. The chiasmata , the X-shaped and looped configurations are still seen. Late Diakinesis The chromosome pairs are dark, round bodies and the nucleolus starts to disappear. Metaphase I During metaphase I, chromosomes migrate to the spindle equator. Metaphase I (side view) The nucleolus has disappeared. The paired chromosomes lie at the equatorial plate of the spindle structure. The chiasmata have moved to the ends of the paired chromosome. Metaphase I (polar view) The paired chromosomes appear as dense bodies scattered on a single plane of the protoplast. Anaphase I The paired chromosomes separate and move toward the opposite poles. The V-shaped configuration of the chromosome is due to movement of the centromere ahead of the arms. The number of chromosomes at each pole is now reduced to half the number possessed by the microspore mother cell. Telophase I The chromosomes at each pole are now extended. The nucleolus reappears and the cytoplasm divides (cytokinesis) to form two half-mooned cells. Meiosis II Meiosis II is an equational division during which sister chromatids separate and are distributed to daughter nuclei. Thus, each nucleus receives the haploid number of chromosomes. Meiosis II is divided into four phases: prophase II, metaphase II, anaphase II, and telophase II. These phases are analogous to the four phases of Meiosis I. Prophase II The chromosomes condense into short thick threads surrounding the small nucleolus. Metaphase II The chromosomes (each chromosome has two sister chromatids) lie at the equatorial plate of the spindle structure. Nucleoli have again disappeared. Anaphase II The two sister chromatids seen collectively as a dark staining mass, are now separated and have moved towards the opposite poles. Telophase II The chromosomes at each pole are extended, the nucleoli reappear and the cytoplasm divides to form four cone-shaped cells. Four cone-shaped microspores are formed and are enclosed inside the maternal wall, which is being digested and will thus release the four microspores. The newly released free microspores are undifferentiated, cone-shaped, and appear to have no distinct cell walls. Early Uninucleate Cell The shape of the microspores are round with dense cytoplasm. The nucleus is located near the center and the cells are undifferentiated with no vacuoles and no clear wall structure. Late Early-Uninucleate Cell The microspores start to differentiate. The exine and intine structures are being formed. The cytoplasm remains dense, but many small vacuoles are being formed. The nucleus is still near the center of the protoplast. Middle Uninucleate Cell A large vacuole is forming in the protoplast, pushing the nucleus to one side. Late Uninucleate Cell The differentiation of exine and intine, germ pore and annulus are complete. Creases seen are due to pressure of coverslip on rigid spherical pollen wall. Cell volume increases four to six times.

textbooks/bio/Agriculture_and_Horticulture/Crop_Genetics_(Suza_and_Lamkey)/1.01%3A_Reproduction_in_Crop_Plants.txt

Sexual Reproduction Kinds of Flowers Inflorescence type influences the techniques that are used to control pollination in developing cultivars and in maintaining the genetic purity of cultivars. Inflorescence types can also be used to identify plants. Flowers are classified into a couple of categories. Flowers are either complete or incomplete and either perfect or imperfect. A flower having all of the main floral parts (sepals, petals, pistils, and stamens) is said to be complete, whereas a flower lacking one or more of these structures is said to be incomplete. The stamen (male part) and pistil (female part) are not always present together in a single flower. When both are present, the flower is said to be perfect (or bisexual). Imperfect flowers are those that are unisexual, either male or female. Table 1 Examples of plants with complete and incomplete flowers. Complete flowers Incomplete flowers Soybean Alfalfa Clovers Common bean Vetches Cotton Tomato Rapeseed Sunflower Tomato Cabbage Tobacco Maize Sorghum Oat Barley Wheat Sugar beet Fig Date palm Forage grasses Turf grasses Rice Spinach Notice that plants in the legume family (Leguminosae or Fabaceae) have complete flowers, whereas plants belonging to the grass family (Gramineae or Poaceae) have incomplete flowers. Flower Dissection Dissect a complete and incomplete flower. Think about how the presence or absence of a floral structure might influence the pollination process, and thus, the methods that can be used to develop improved cultivars or to maintain the genetic purity of the cultivar. Grass Floret Complete Soybean Flower Dissection 1. Standard petals: Collectively, petals are called the corolla. Petals are typically large and conpicuous and are not required for reproduction. Soybean has five petals: one standard petal, two wing petals and two keel petals 2. Wing petals: The dissected view of the two wing petals. 3. Keel: The keel is composed of two united petals. The keel encloses the stamina column. Stamens are the pollen-bearing organs of the flower. Stamens are composed of slender stalks (filaments) that support anthers. Pollen grains are produced in the anthers. The pistil is the seed-bearing organ of the flower. It consists of stigma, style, and ovaries. The stigma is the part that is receptive to pollen. Following pollination and fertilization, seed form in ovaries. 1. Sepals: Like the petals, sepals are not neccessary for reproduction. Sepals are small and inconspicuous. They enclose and protect the flower while still a bud. Collectively they form the calyx. 2. Pedicel: The pedicel is the stalk of the flower, attaching to the plant Wheat Spike Dissection Grass Floret Study Question 4 Query \(3\) Study Question 5 Select the floral part or parts necessary for reproduction: Query \(4\) Table 2 Examples of crops and different floral systems. Adapted from Lersten (1980). Flower Characteristics Terms Examples Male and female expression in INDIVIDUAL FLOWERS Male and female in ONE flower bisexual, hermaphroditic, monoclinous, perfect Wheat, peach 1. Pollen shed before stigma is receptive protandry (prevent self-pollination) Carrot, walnut 1. Stigma matures and ceases to be receptive before pollen is shed protogyny (prevent self-pollination) Pearl millet, pecan 1. Stigma receptive, and pollen shed, after flower opens chasmogamy (promote self-pollination) Violet, rye 1. Stigma receptive, and pollen shed, in closed flower cleistogamy (ensure self-pollination) Oat, peanut Perfect flowers of TWO types on SAME plant heterostyly Buckwheat, flax 1. Long styles and short stamens pin flower 1. Short styles and long stamens thrum flower Male and female in SEPARATE flowers unisexual, diclinous, imperfect Cucumber, hemp 1. Male flower male, staminate 1. Female flower female, pistillate, carpellate Flower DISTRIBUTION on PLANTS Male and female flowers on one plant monoecious Maize, oak Male and female flowers on separate plants dioecious Yams, asparagus • Male, female, and perfect flowers mixed, polygamous Red maple, papaya 1. On same plants polygamomonoecious Coconut, mango 1. On separate plants polygamodioecious Strawberry, holly Perfect and Imperfect Flowers Perfect flowers have both staminate and pistillate structures in the same flower. Imperfect flowers are either staminate or pistillate. An imperfect flower is staminate if it possesses stamen. Conversely, an imperfect flower is pistillate if it bears a pistil. Staminate flowers are considered “male” because they produce pollen, whereas pistillate flowers are “female” because they possess ovules. Staminate and pistillate flowers may occur on the same or different plants of the same species. Species having such specializations are either: • monoecious — staminate and pistillate flowers are separate but occur on the same plant; or • dioecious — staminate and pistillate flowers are on separate plants. Analogous to the separate sexes in animals, a dioecious plant must have a partner of the opposite type to complete its life cycle. Usually, about half of all individuals of a dioecious species are of each type, staminate or pistillate. Thus, the dioecious condition is reproductively expensive in that only about half of the species’ plants can produce seed. Table 3 Examples of monoecious and dioecious plants. Monoecious Dioecious Maize Walnut Oil palm Squash Cassava Wile rice Castor bean White pine Hemp Hops Spinach Yam Date palm Cottonwood Asparagus Nutmeg The “mono-” prefix indicates one and the “di-” prefix indicates two. The “-oecious” part of the word translates to “house.” Thus, an easy way to remember the distinction between these terms is to remember that in monoecious species, the staminate and pistillate flowers reside in the same house or plant, whereas in dioecious species, these flowers reside in two different houses or plants. Study Question 6 Pollination and Fertilization Pollination occurs when a pollen grain (from the staminate flower) is placed on a receptive stigma (of the pistillate flower), either naturally or artificially. Fertilization requires that a male gamete and a female gamete fuse to form a zygote. These gametes may be from the same or different plants. There are two kinds of pollination processes in sexual reproduction. • Self-pollination — seeds develop from the union of male and female gametes produced on the same plant or clone. The development of seed by self-pollination is also referred to as autogamy. • Cross-pollination — seeds develop from the fusion of gametes produced on different plants. The development of seed by cross-pollination is known as allogamy. Pollination and Fertilization Self-Pollination Several floral mechanisms enforce self-pollination. • Flowers do not open, preventing external pollen from reaching the stigma. • Anthesis occurs before the flower opens. • Stigma elongates through the staminal column (filaments and anthers) immediately after anthesis. • Floral organs may obscure the stigma after the flower opens. Although these mechanisms usually enforce self-pollination, a low frequency of cross-pollination may occur. The frequency of cross-pollination in normally self-pollinating species generally depends on the species and environmental conditions. Soybean is an example of a species that is normally self-pollinated. Before the flower opens, the anthers burst and pollen grains fall out of the anthers on to the receptive stigma contained in the same flower: self-pollination occurs. Cross-Pollination Floral Mechanisms of Promotion Several floral mechanisms promote cross-pollination. • Emergence or maturity of the staminate and pistillate flowers is asynchronous. • Protandry — anthesis occurs before stigma are receptive. • Protogyny — pistillate flower matures before the staminate flower. • Flowers are monoecious or dioecious. Mechanical obstruction between the staminate and pistillate flowers in the same individual prevents self-pollination. Gametes produced on the same plant or clone are unable to effect fertilization. • Mechanical obstruction between the staminate and pistillate flowers in the same individual prevents self-pollination. Alfalfa flowers, for example, have a membrane over the stigma that precludes self-pollination. When a bee lands on the flower, the keel is tripped, rupturing the membrane and exposing the stigma to pollen carried by the bee from other plants it has visited, effecting cross-pollination. • Gametes produced on the same plant or clone are unable to effect fertilization. • Self-sterility — gametes from same individual cannot successfully fuse to form a zygote. Sterility can be caused by lack of function of pollen (male gametes) or ovules (female gametes). • Male sterility — either genetic or cytoplasmic, occurs because the pollen is not viable. Female sterility occurs when the ovule is defective or seed development is inhibited. • Self-incompatibility — self-pollination may occur, but fertilization and seed set fail. Pollen Transportation Pollen is transported from the staminate flower to the pistillate flower by wind, insects, or animals. Occasionally pollen is transported to receptive stigma of the same individual and self-pollination may occur. For example, pollen from the tassel of a maize plant may land on and pollinate silks on the same plant, effecting self-pollination. Sunflower is ordinarily cross-pollinated. Bees often carry pollen from one plant and deposit it on other plants. Classification Plants are classified as either self- or cross-pollinated based on which of these processes most frequently produces its seed. Click each category for more information. Query \(7\) Study Question 7 You encounter an unfamiliar flowering plant. What key floral feature(s) would you check to determine the plant’s likely mode of pollination, self or cross-pollinating? Query \(8\) Study Question 8 For each of the following types, indicate the probable mode of pollination by clicking on the appropriate button. Assume no male sterility or self-incompatibility. Asexual Reproduction Some species can be propagated without a gametophytic stage. The fusion of gametes (fertilization) is omitted from the life cycle. Reduction in chromosome number (meiosis) and seed production may or may not occur. Asexual reproduction produces individuals genetically identical to the maternal parent. There are several mechanisms of asexual reproduction. • Vegetative Propagation • Tissue Culture • Apomixis Vegetative Propagation In some species, new individuals can arise from a group of differentiated or undifferentiated cells of the parent plant; no embryo or seed is produced. Because such new individuals develop asexually from a single parent, they are genetically identical to that parent. These progeny are clones. Numerous tissues and organs may asexually produce progeny. • Rhizomes – Rhizomes are specialized underground stems that can branch at nodes to produce new plants. Banana, bromegrass, hops, and johnsongrass can be reproduced from rhizomes. • Stolons – These “runners” or horizontal-growing, above-ground stems develop adventitious roots whose axillary buds can become independent plants. Strawberry is an example of a crop that can be reproduced from stolons. • Bulbs and bulbils – These short underground stems have thickened or fleshy scales (modified leaves) that can form buds. These buds detach and form “offsets” or new individuals. Onions and garlic are commonly propagated from bulbs. • Tubers – Tubers are also short, enlarged stem tissue, containing food reserves. Nodes or “eyes” in such tissue can give rise to adventitious roots and separate plants. Potatoes are commonly propagated from eyes cut from tubers. • Suckers – Suckers arising as lateral shoots from the base of stems can separate and form new plants. Pineapple, sweet potato, and date palm are examples. Suckers may also derive from adventitious buds on the roots. Roses, poplars, and some other woody species can be propagated from such root cuttings or rootstocks. • Corms – A corm is an underground, tuber-like base of a vertical stem that can also produce a separate plant. Taro, an important starch crop in Southeast Asia and the Pacific Islands, is propagated from corms. Banana also can be propagated from corms. • Stem cuttings – When placed in moist soil, cuttings from aerial stems of some species, such as sugarcane, pineapple, and cassava, can give rise to new plants from the nodes and lateral buds. The usual mode of reproduction of some species is vegetative. However, other species that reproduce sexually are more commonly propagated vegetatively to maintain genetic purity, including some forage cultivars and many horticultural species. Vegetative reproduction does not usually provide opportunity for selection of genetic variants. Tissue Culture Tissue culture is a specialized type of asexual propagation. Tissue culture usually involves excision of undifferentiated cells or meristematic pieces of a plant and growing these in vitro on sterile nutrient agar medium; cell division is by mitosis. By manipulating the components of the medium, the tissue can be prompted to develop roots or shoots. Eventually, new individuals may be separated and transplanted to soil. Tissue culturing takes advantage of the totipotency of somatic cells. That is, these cells contain the plant’s entire genome and have the potential to develop into whole plants. Some species that cannot normally be reproduced vegetatively may be reproduced by tissue culture. Tissue culture is of interest to plant breeders as a technique to • maintain and propagate genetically identical plants that otherwise can only be reproduced sexually; • provide disease-free plants of species that often transmit pathogens to progeny when propagated by conventional vegetative means; and • create novel genetic variation within which selections can be made. Under some conditions, tissue culturing can promote genetic changes. Apomixis Process Apomixis differs from other forms of asexual reproduction in that seed is produced. Unlike sexual reproduction, however, apomictic seed is developed from sexual organs or related structures without fertilization. Pollination is also usually omitted. Agamospermy Apomixis generally involves forms of agamospermy, which is a process through which seeds develop without fertilization. There are two different degrees of agamospermy. • Obligate — Seed produced arises from asexual reproduction. • Advantages: Preserves genotype, including heterozygotic genotypes • Disadvantages: Precludes genetic recombination and variation for selection of improved cultivars • Facultative — Although most of the seed generated is asexually produced, sexual reproduction occurs regularly. • Advantages: Permits development of genetic variation for selection of improved cultivars • Disadvantages: Cultivars may be genetically unstable, making it difficult to maintain the desired genotype Each of these degrees of agamospermy provides advantages and disadvantages from the plant breeding perspective. There are also two general types of agamospermy. • Autonomous — Endosperm forms without pollination or fertilization. • Pseudogamous — Although fertilization (the fusion of gametes) does not occur, pollination is apparently required to stimulate apomictic embryo or embryo sac development to produce seed. Pollination adds no genetic material. Mechanisms of Cause The mechanisms that cause apomixis differ by the cell that undergoes mitosis to produce the embryo of the seed. • Adventitious embryony — The embryo develops directly from diploid sporophytic tissue, skipping the gametophytic stage. This is the simplest form of agamospermy. • Apospory — Nucellus or integument cells, which are somatic cells, undergo mitosis to produce a diploid embryo sac. • Apospory is the most common form of apomixis in angiosperms. • Diplospory — The embryo and endosperm derive from the diploid megaspore mother cell. The megaspore mother cell’s nucleus divides by mitosis, rather than meiosis, resulting in a diploid embryo sac. • Parthenogenesis — The egg cell divides mitotically to form the embryo without fertilization. • Androgenesis — A haploid embryo develops from a male sperm nucleus after it enters the embryo sac. The individual that develops from the seed is haploid and has the genotype of the sperm from which it is derived. Study Question 9 Apomictic embryos may form from reduced (haploid) or unreduced (diploid) cells. For each situation, select the button according to whether the resulting embryo could be homozygous, heterozygous, neither, or both. Answer both parts of this question and then check your answer. Discussion Crops can be self-pollinated, cross-pollinated, or vegetatively propagated. Discuss the breeding consequences of these three different methods of propagation. In addition: previously, a student suggested that with today’s technologies, plants can simply be converted into self- or cross-pollinated or into vegetatively propagated species. Do you agree? Provide arguments in favor or against this statement, and examples, in case you are aware of any. Finally, if it was possible, which type of crops would be your favorite, and why? Study Question 10 For each of the following terms, identify whether the term is associated with sexual, asexual, or both manners of reproduction by clicking on the appropriate button.

textbooks/bio/Agriculture_and_Horticulture/Crop_Genetics_(Suza_and_Lamkey)/1.02%3A_Flower_Morphology_and_Distribution.txt

Introduction Most crop species require seed production for their propagation. Some species, however, possess mechanisms that regulate fertility. Such mechanisms can reduce or prevent seed set, and affect self- or cross-pollination. These fertility-regulating mechanisms may be an obstacle or a benefit to the plant breeder. In this module, we’ll explore these mechanisms and their utility. Learning Objectives • Mechanisms and utility of incompatibility systems. • Modes of sex inheritance in plants and their application in plant breeding. • Male sterility systems and their applications in plant breeding. Self-incompatibility Genetic-based Self-incompatibility Self-incompatibility is the inability of a plant to set seed when self-pollinated, even though it produces viable pollen. In contrast, cross-pollination generally results in seed set in self-incompatible species. Many plant families include species with self-incompatibility systems, such as Fabaceae (Leguminosae), Poaceae (Gramineae), Solanaceae, Brassicaceae (Cruciferae), and Asteraceae (Compositae). Self-incompatibility may be caused by genetic interactions between pistil and pollen, or by physical obstacles that hinder self-fertilization. Self-incompatibility may be caused by genetic interactions between the pistil and pollen-producing physiological factors that interfere with fertilization of female gametes by male gametes produced on the same plant or on a closely-related plant. Typically one or few self-incompatibility (SI) genes are involved in this self/non-self discrimination process, depending on the plant species. Pollen can be rendered ineffectual at several points in the pollination process: 1. Sperm enters the embryo sac but does not fuse with the egg. 2. Pollen tube penetrates the stigma but it grows too slowly in the style to reach the ovary while the ovule is still receptive. 3. Pollen germinates but the tube is unable to penetrate the stigma. 4. Pollen germination on the stigma is inhibited. Pistil receptivity to a particular pollen grain depends on the SI alleles carried by the pistil. The phenotype of the pollen (its capacity to fertilize the female gamete) is determined by either the pollen’s alleles (gametophytic incompatibility) or by the alleles of the plant that produced the pollen (sporophytic incompatibility). There are two self-incompatibility systems that result from genetic interactions: • Gametophytic Self-incompatibility • Sporophytic Self-incompatibility Both incompatibility types influence the rate of pollen tube growth, but their genetic controls and location of effect differ. Both types involve multiple alleles. Gametophytic Self-incompatibility • Gametophytic self-incompatibility involves the allele possessed by the pollen grain. The incompatibility effect occurs in the style. However, in some species with gametophytic SI, incompatibility is expressed on the surface of stigma (in grasses). • Gametophytic self-incompatibility is controlled by a series of alleles. The rate of pollen tube growth responds to the allelic interaction of both the style and the pollen. • If both the stylar tissue and pollen possess identical alleles, pollen tube growth is inhibited. • If stylar and pollen alleles differ, tube growth occurs at normal rates. FYI: Homozygous Flowers What happens if the pistillate flower is homozygous for the S allele? The same rules apply: if the pollen carries an allele that matches the one possessed by the pistil, fertilization will not occur; if they differ, seed can form. However, it should be noted that plants homozygous for a self-incompatibility allele in the gametophytic system are rare. Why are plants homozygous for gametophytic self-incompatibility rare? Homozygotes are unusual because the probability of a pollen grain carrying the same allele as the pistil successfully overcoming the incompatibility of their matching alleles is small—but such events do occur occasionally. Thus, a population may have a few individuals homozygous for an S allele. In Fig 2A, all pollen grains are incompatible with the pistil as they fail to germinate beyond the stigmatic surface. In Fig 2B, nearly all pollen grains are compatible with the pistil as they germinated and pollen tubes grew through the stylar tissues and eventually entered the ovule for fertilization. Sporophytic Self-incompatibility Sporophytic self-incompatibility involves dominance and depends on the allelic composition of the plant that produced the pollen. Incompatibility is expressed at the surface of stigma. In sporophytic self-incompatibility systems, the rate of pollen tube growth depends on the presence or absence of a dominant allele at the SI locus (will be called S locus in the following) carried by the pollen-producing plant. In the pollen, any S allele can exhibit dominance-that dominance is determined by the sporophyte, the plant that produces the pollen (thus, this system of self-incompatibility is termed “sporophytic”). Two important points need to be emphasized about sporophytic self-incompatibility. The genotype of the pollen-producing plant determines self-incompatibility, not the allelic composition of the pollen itself. In other words, every male gamete has the same ability to fertilize a female as every other male gamete, irrespective of the pollen’s individual genotype. There is no dominance in the female stigmatic tissue. How does the genotype of the pollen parent transmit the influence of the dominant allele to the pollen? Although we don’t yet understand this, we describe it as ‘imprinting.’ That is, the pollen “remembers” the genetic environment in which it developed and is conditioned to behave in accordance with that environment. Cell walls of pollen grains are consisted of at least two layers, the intine or inner layer and the exine or the outer layer. The exine is made up of a highly durable organic polymer, sporopollenin. The exine is believed to be derived from the somatic tissues of the pollen-producing parent and likely play a role in pollen-pistil interaction. This could explain why the compatibility response is determined by pollen-producing parent, instead of the pollen itself. Here’s an example of sporophytic self-incompatibility. Assume the species is diploid and that there are four possible self-incompatibility alleles: S1, S2, S3, S4. Let S1 be a dominant allele. Let’s see what happens. (Color indicates the source of the allele, from the female or the male parent.) Why are no offspring produced? The S1 allele in the plant producing the pollen is dominant. Hence, both the gamete types produced by that plant, S1 and S3, will behave as if they were both dominant alleles (S1). As a result, neither pollen type will be able to effect fertilization of this female—neither type of pollen tube will be able to penetrate the style because their growth will be impeded on the S1S2 stigma. 1. In sporophytic systems, hindrance of pollen tube growth is localized on the surface of the stigma. However, an exception is gametophytic self-incompatibility in several grass species such as rye and ryegrass, where pollen tube growth is inhibited on the surface of the stigma. 2. In gametophytic systems, growth is impeded in the style. Pollen tube growth inhibition A feature that distinguishes sporophytic from gametophytic self-incompatibility is the location of pollen tube growth inhibition. When there are multiple S alleles in sporophytic systems, genetic segregation ratios become complex. The presence of a dominant allele in the pollen-producing plant conditions incompatibility if the female carries that same dominant allele. Assume a diploid species and dominance: S1 > S2 > S3 > S4. Female Genotype Male Genotype Pollen Genotypes Offspring Explanation No Matching alleles so incompatible No S2 is imprinted on S3 pollen grains. Yes 1/4 S1S2: 1/4 S1S3: 1/4 S2S3: 1/4 S3S3 S2 is imprinted on S3 pollen grains. Self-incompatibility Systems Many crops have self-incompatibility systems. Why might these systems have evolved? Self-incompatibility is common among naturally cross-pollinated species. Self-incompatibility prevents or limits self-fertilization and promotes out-crossing. Out-crossing maintains heterozygosity and heterogeneity in a population, which often improves plant vigor and productivity. In some species, homozygosity can severely reduce vigor, a phenomenon referred to as ‘inbreeding depression.’ Table 1 Examples of crops with self-incompatibility. Gametophytic Self-incompatibility Sporophytic Self-incompatibility Alsike clover Sunflower (wild populations) Red clover Buckwheat Tall fescue Cacao Potato Brassica species Rye (a two-loci system) Cabbage Sugar beet (a four-loci system) Broccoli Alfafa Kale Tobacco Brussel sprouts Not found in monocots! Techniques to Overcome Self-incompatibility In order to self-pollinate or mate closely-related plants that are normally self-incompatible, plant breeders can employ various techniques that bypass self-incompatibility mechanisms. The technique used depends on the type of self-incompatibility. Uses of Self-incompatibility Genes Some systems have been developed or proposed for utilizing self-incompatibility genes to control pollination and produce F1 hybrid varieties. Gametophytic System Cross-pollination of vegetatively propagated, self-incompatible clones. Using this approach, seed can be obtained from species that are normally self-incompatible, and thus propagated vegetatively. An example is the production of a hybrid variety of bahiagrass, Tifhi. Sporophytic System Bud-pollination can be used in Brassicaceae for self-pollination and thus inbreeding, resulting in inbred lines or families homozygous for an allele at the self-incompatibility (SI) locus (only one SI locus is present in, e.g., cabbage). Hybrids can subsequently be produced by planting two such lines with different fixed SI alleles side by side. Any seed produced is expected to be hybrid seed, since self-pollination within parental lines is prevented by SI. The resulting hybrid will be heterozygous for the SI locus and thus self-incompatible. However, this is not critical since the economic product does not require pollination and is vegetative, e.g., cabbage and kale. Pseudo-Compatibility Produce inbreds in environments that promote pseudo-compatibility and their hybrids in environments that prevent self-fertilization. Sugar beet is normally self-incompatible. However, when grown at high elevations, plants are self-compatible. Low Elevation: Sugar beet inbreds will behave as normal self-incompatible lines at low elevations — self-pollination is genetically eliminated. All seed produced at the lower elevation will be the result of out-crossing among inbred lines, and thus will be hybrid. High Elevation: Self-fertilized seed can be obtained via elevation-induced pseudo-self compatibility. Sufficient quantities of selfed seed can be produced for subsequent hybrid generation. Gametophytic System • Pseudo-compatibility: Expose the plant(s) to lower or higher temperatures, elevated CO2 concentration, or electric shock to induce a pseudo-compatibility response. • Sf alleles: Self-fertility (Sf) alleles, reported to be present in some species can be transferred into a population using conventional breeding methods. The presence of an Sf allele allows self-fertilization. These alleles can be present at self-incompatibility loci, but can also be present at distinct self-fertility loci. Sporophytic System • Removal of stigmatic surfaces: Mechanical (e.g., rupturing) or chemical removal of the stigma surface eliminates the inhibiting factors [believed to be specialized proteins or enzymes, (Barrett, 1998)] that inhibit pollen tube penetration of the style. With those factors absent, pollen tube growth can proceed normally and fertilization can be achieved. • Bud pollination: Pollen is placed on an immature stigma before the inhibiting factor is formed. An example of a gametophytic system is the production of a hybrid variety of bahiagrass, Tifhi. Male Sterility Male sterility is due to the failure of a plant to produce functional anthers or pollen; usually, its female gametes are normal. Thus, male sterility prevents self-pollination and can be used to ensure cross-pollination without emasculation. Male sterility can have genetic causes or be induced chemically. Female sterility, the failure of a plant to produce functional ovaries or eggs, can also occur, but is of little use to plant breeders and will not be discussed. Genetic-based Male sterility Male-sterile gene expression may be complete or partial, and may vary with environment. Breeders desire complete expression that is stable regardless of environment. The extent of sterility is measured by the percentage of viable pollen produced or percentage of seed set. Male sterility is used by breeders to eliminate the necessity of emasculation to control pollination — male-sterile plants cannot self-pollinate. Viable Pollen Measurement The percentage of viable pollen can be estimated using a microscope. Fresh pollen from a plant or group of plants is placed on a microscope slide and viewed at low magnification, 10X. In the field, a hand-held magnifier is often adequate for scoring the percentage of viable pollen. Pollen grains can also be stained and viewed under a microscope in the lab. Pollen viability can also be tested in bioassays. Male-sterility is controlled by nuclear genes, the cytoplasm, or by genetic interaction between the cytoplasm and nucleus. Controlled by the action of specific gene(s) in the nucleus. Usually, the recessive allele(s), designated ms, conditions inhibition of normal anthers or pollen development. Thus, the male sterility phenotype is expressed in plants homozygous for the ms allele. • ms ms = male sterile (therefore, is functionally a female plant) • Ms _ = male fertile (normal) Maintenance of sterility genes in a population is challenging. Uses of Genetic Male Sterility Numerous crops have genes causing male sterility in their gene pools. There are two major uses of genetic male sterility: • Eliminate hand emasculation in making crosses • Increase natural cross-pollination in populations of self-pollinated crops These uses necessitate the transfer of the ms allele into the population being worked with; then the ms allele is maintained in the population through selection for sterility in each subsequent generation. Because genetic male sterility is controlled by a recessive gene(s), it is not possible to get a true-breeding or homozygous ms population. However, the recessive ms allele can be maintained at a high frequency in the population. Genetics of Male Sterility Interactive A male sterile line can be created and maintained by pollination of a line with an identical genotype except for the dominant allele for male fertility. To make the hybrid, male and female rows are planted in a specific pattern and selected plants are allowed to randomly mate with homozygous fertile plants. Query \(2\) Study Question 1: Sterile and Fertile Phenotypes Cytoplasmic Male-sterility The genetic composition of the cytoplasm determines male sterility. The genetic makeup of the cytoplasm results from genes located in mitochondria. Cytoplasm is inherited entirely through the female line. The cytoplasm can be • Normal (N) — normal development of anthers and pollen = male fertile; or • Sterile (S or CMS) — anthers or pollen are non-functional = male sterile Female Line In all organisms, the cytoplasm contains genetic material in the mitochondria. Unlike the genetic material in the nucleus, however, cytoplasmic genetic material is not subject to recombination. Cytoplasm is inherited strictly through the female parent because the male parent does not contribute cytoplasm to the zygote. Recall that the zygote is formed when the sperm nucleus and the egg nucleus fuse; since the zygote’s nucleus is contained in the cytoplasm of the egg and the sperm has no cytoplasm, the zygote inherits its cytoplasm only from its female parent. This fact is used by evolutionary biologists to trace family lines, avoiding the complexity resulting from genetic recombination and segregation that occur with nuclear genetic material. In some systems, the expression of the male sterility phenotype depends on the interaction between cytoplasmic and nuclear genes. In these systems, a plant having sterile cytoplasm, but a nuclear dominant fertility-restorer gene (Rf _), will express a fertile phenotype. The particular combination of cytoplasm and nuclear genes determines the phenotype. Table 2 Phenotype of cytoplasm and nucleus genotype combinations. Cytoplasm Type Nucleus Genotype Rf_ rf rf CMS Male Fertile Male Sterile N Male Fertile Male Fertile Plant breeders must pay close attention to both the cytoplasm type and nuclear genes of the parents used in crosses to generate and maintain cytoplasmic male sterility. Sorghum Example Several crops have cytoplasmic male sterility types available, including sunflower, millet, wheat, maize, and sorghum. Let’s examine the maintenance and use of cytoplasmic male sterility in sorghum. This system involves interaction between the cytoplasm and a nuclear gene. There are two types of cytoplasm possible: S = sterile; N = normal. There are two possible alleles at the restorer locus in the nucleus, Rf (dominant) and rf (recessive). In this example, we’ll look at three types of inbreds. Table 3 Genotypes and phenotypes of sorghum inbred line types. Inbred Type Cytoplasm Type Nuclear Genotype † Male Phenotype A line S rf rf Sterile B line N rf rf Fertile (normal) R line N, or Rf Rf Fertile (normal) S Rf Rf Fertile (normal) † Although we use rf and Rf in this example, sorghum breeders indicate the dominant and recessive nuclear fertility restorer alleles with different symbols: msc = rf and Msc = Rf. How are these inbred (homozygous) lines maintained? A line Assume that the cultivar ‘Martin’ has two versions, one with normal cytoplasm and one that differs only in that it has a sterile type cytoplasm (‘Martin S’). The normal version is a B inbred type and Martin S is an A type. Mate the sterile version as the female with the normal, fertile version. Notice that there is no blending or segregation of the cytoplasm. The F1 always has the cytoplasmic type of its female parent. B line B and R lines can be maintained simply by self-pollination. This is usually accomplished by growing each line in an isolated block and allowing the plants to intermate. Since the line is homozygous and fertile, the seed produced will also be homozygous and fertile. R line Like the B line, the R line can be maintained simply by self-pollination. This is usually accomplished by growing each line in an isolated block and allowing the plants to intermate. Since the line is homozygous and fertile, the seed produced will also be homozygous and fertile. How is single-cross hybrid seed produced using cytoplasmic male sterility? Cross a male sterile line (e.g., A line) by a fertile line (e.g., R line). ‘Caprock’ is an R line. Try This: Cytoplasmic Male Sterility You’re planning to create a set of hybrids carrying various combinations of cytoplasms and fertility restorer alleles. A, B and R lines are available for generating the hybrids.For the cross shown below, predict the type of offspring, if any, will be produced. Engineered Genetic Male Sterility Different systems of male sterility and fertility restoration are being worked on by various companies. All of these involve molecular techniques (genetic engineering) and the development of transgenic plants. One of the first to be utilized was the development of male sterile plant by transforming plant cells with a bacterial gene. This male sterility is dominant, since when the gene is present the plants are male sterile. The normal state for the plants is that the male sterile gene is not present and that gives normal, male fertility. As with the naturally occurring genetic male sterility we discussed earlier, it is impossible to maintain a population of completely male sterile plants as we can in cytoplasmic male sterility; so to identify the male sterile plants from the male fertile ones and to be able to eliminate male fertile plants, an herbicide-resistance gene has been linked to the male sterility gene. This resistance is also dominant, since when present it confers resistance and when absent (the normal state of the untransformed plants) plants are susceptible to the herbicide. System: Linking herbicide-resistant gene to the male sterility gene A plant is transformed to contain linked male sterility and herbicide resistance genes Ms R (The transformed plant will have only one homologue containing both the Ms and R genes, thus will neither be homozygous nor heterozygous, but rather what we call hemizygous for the two genes.) We now must incorporate this linked pair into an elite inbred line by backcrossing. (The symbol “-” indicates that there is no allele for this gene present.) The elite inbred will be developed and then maintained by crossing the male sterile plants with the normal inbred to give 50% sterile and 50% fertile plants. Discussion for Further Thought For the diploid, self-pollinated species tomato (Solanum lycopersicum), discuss from a genetic perspective the strengths and weaknesses of different hybridization systems that use either hand emasculation, male sterility, or genetic engineering, and come to a consensus, on which one you prefer. Chemically-induced Genetic Male Sterility In species that lack genetic male sterility or cytoplasmic male sterility, or in species in which these are difficult to work with, breeders can use chemicals to induce male sterility. These chemicals have at one time or another been called by the following names: • Gametocides • Pollen suppressants • Hybridizing agents These inhibit the production of viable pollen or prevent pollen shed, but do not damage the pistillate flower or interfere with seed development. There are both advantages and disadvantages to chemically-induced male sterility. Advantages • No need to develop and maintain cytoplasmic or genetic male sterility systems • Applied to normally self-pollinated species, the chemicals prevent self-pollination and facilitate cross-pollination without the necessity of hand emasculation. This approach has been used in cotton, maize, sorghum, and various vegetable crops. It was also tested for hybrid wheat production. Disadvantages • Incomplete pollen sterility can occur, resulting in some selfing. Several factors can account for incomplete sterility. • differential genotypic reactions to the chemical agents • timing and dosage of application is critical and varies with genotype • environmental effects on chemical and interaction with genotype • long periods of flowering cause difficulties in maintaining optimum dosage in plants • Does not provide a means of pollen transfer to produce hybrid seed, and so additional methods must still be employed. Hybrid Wheat Since wheat has perfect flowers and is normally self-pollinating, the production of hybrid varieties was impractical without the use of male gametocides. Gametocides were applied to the female plants prior to flowering to kill their pollen and prevent their self-pollination; the treatment was not applied to adjacent rows containing the intended male parent. Thus, seed produced by the female plants resulted from cross-pollination with the desired males. Although hybrid seed could be produced in this manner, the approach has been largely abandoned by most commercial seed companies—farmers did not buy the hybrid seed because the hybrids did not yield sufficiently better than conventional wheat to justify the additional seed costs. Sex Inheritance Description The ultimate biological mechanism to prevent self-pollination, and thus inbreeding, is to have distinct male and female flowers on different plants. In such “dioecious” plant species, which are common in the plant kingdom, male and female plants occur usually at a 1:1 ratio. The decision on whether a plant becomes male or female is genetically determined, and can in some species be influenced by environmental factors. As a consequence, development of inbred lines can be accomplished either by crosses of male and female sister plants, or by employing environmental factors to induce plants with both sexes to allow self-pollination. In addition to dioecious and hermaphrodite plant species, there are various intermediate variants realized in plants (Table 4). Table 4 Sex systems in flowering plants. Data from Charlesworth 2002, Heredity 88, 94-101. Plant Term Definition of Plant Term Sexually monomorphic Hermaphrodite Flowers have both male and female organs (Tomato, potato, sugarcane) Monoecious Separate sex flowers on the same plant (Cassava, maize, banana) Gynomonoecious Individuals have both female and hermaphrodite flowers Andromonoecious Individuals have both male and hermaphrodite flowers Sexually polymorphic Dioecious Male and female plants (spinach, asparagus) Gynodioecious Individual either female or hermaphrodite (papaya) Androdioecious Individuals either male or hermaphrodite Genetics of Sex Inheritance Distinct male and female flowers can be of practical importance, if present on the same plant. Best example is maize, where spatial separation of female and male flowers facilitates both elimination of male flowers to produce “female plants” for hybrid seed production, and self-pollination for inbred development. However, the majority of plants carry either flowers containing both male and female organs, or male and female flowers on distinct plants. In the latter case, pollination will be mediated by wind or insects. Female plants will set seed only if a male plant is located in the vicinity. There are two genetic mechanisms of sex inheritance: • Sex chromosomes — Like in humans, the sex of an individual is determined by specific chromosomes. In the case of humans, carriers of two X-chromosomes are females, whereas males have one copy each of the X- and Y-chromosomes. Since X- and Y-chromosomes differ cytologically, their pairing in meiosis might be incomplete. • Autosomal inheritance — Genes affecting sex inheritance are located on “regular” chromosomes (autosomes), forming bivalents in meiosis. Depending on the plant species, sex determination can be due to one or a limited number of genes (mono-, or oligogenic inheritance). Sex Inheritance Systems Application and Challenges Application Like self-incompatibility or male sterility, sex inheritance can be used for controlled crosses between any pair of genotypes in hybrid seed production schemes. One genotype would be used as female and seed parent, the other parent as pollen donor. Challenges in using sex inheritance for hybrid seed production As plants with only one sex cannot be sexually maintained, it is either required to use environmental or chemical factors to induce the other sex to allow self-pollination (if available), or to develop male and female sister lines for each hybrid parent, comparable to male sterile and maintainer lines when using male sterility. This is described in more detail in the following cucumber and asparagus examples. Cucumber Cucumber (Cucumis sativus) has a wide range of floral types. Staminate (male), pistillate (female), and hermaphrodite flowers can occur in different arrangements. Generally, embryonic flower buds possess both staminate and pistillate initials, and thus the potential to develop into any of the above-mentioned flower types. The cucumber phenotype with regard to floral types depends on autosomal genes and their interaction with environmental factors. Sex inheritance is controlled by a minimum of three major loci: • m+, m — controls the tendency to form hermaphrodite versus male or female flowers. mm homozygotes develop hermaphrodite flowers. • F+, F — controls the female tendency, with F allele being dominant and favoring female flowers. This locus is subject to strong environmental influence. Among environmental factors, in particular photoperiodic conditions and temperature affect flower formation. • a+, a — Homozygotes for a allele intensify male tendency. The effects of a are dependent on the allelic composition at the ‘F’ locus. Male tendency is only pronounced in ‘F+F+‘ homozygotes. Although other loci such as ‘de’ and ‘cp’ have been shown to affect sex types in cucumber, the main sex types are determined by the above mentioned loci m, F, and a (Table 5). Table 5. Phenotypes and Genotypes of basic sex types in cucumber.Note: “-/-” means that any allele can be present at this locus Genotype, locus Phenotype m F a Androecious (male) -/- F/F a/a Monoecious m+/m+ F/F -/- Hermaphroditic m/m F/F -/- Gynoecious (female) m+/m+ F/F -/- CHEMICAL REGULATION OF SEX EXPRESSION Phytohormones can be applied to alter flower phenotypes. Whereas auxin and ethylene promote female flowers, gibberellin promotes male flowers. Moreover, silver nitrate and silver thiosulfate induce male flowers even in strongly gynoecious genotypes. These treatments have become invaluable in cucumber breeding, as they allow efficient development of gynoecious inbreds to be used as female hybrid parents, and even the production of hybrids from crosses of two gynoecious lines. Asparagus Sex inheritance in Asparagus officinalis, is similar to humans, based on sex chromosomes. XX chromosome carriers are female, XY carriers are male. Males and females occur at about equal frequencies in natural populations. Males have been shown to be more productive. For this reasons varieties with only male plants are preferred. At a low frequency and for unknown reasons from a genetic perspective, andromonoecious XY plants occur. These plants can be self pollinated, and will result in a 1:2:1 ratio with regard to the sex chromosomes in resulting offspring. One quarter of those will thus carry two copies of the “male” Y chromosome, and are thus called supermales. As Asparagus can be vegetatively propagated, those YY plants are potential variety candidates. Alternatively, they can be used as male parent in hybrid breeding schemes to be crossed with female XX genotypes, and result in 100% male XY offspring. In contrast, if regular XY males would be crossed to XX females, the offspring would segregate 1:1 into XY and XX plants, and XX plants would need to be eliminated by producers. Controlling Hybridization Knowledge of the breeding system of a crop species is essential to take advantage of the types of gene action that give the most useful cultivars. Self-incompatibility systems are important in many natural species for forcing outcrossing and thus maintaining vigor through heterozygosity. As we have seen, this system can be adapted to help produce F1 hybrid cultivars in domesticated species containing self-incompatibility loci. Similarly, sex inheritance can be employed in controlled F1 hybrid seed production schemes. Male sterility is a rather unimportant method of enforcing outcrossing in natural plant species. It has, however, become such an important tool in the production of hybrid cultivars that it is utilized in many species of both cross- and self-pollinated crops.

textbooks/bio/Agriculture_and_Horticulture/Crop_Genetics_(Suza_and_Lamkey)/1.03%3A_Controlled_Hybridization-_Self-incompatibility_Male-sterility_and_Sex-inheritance.txt

Introduction Plant breeders take advantage of the variation that occurs within a population to develop improved cultivars. Ordinarily, the goal of the plant breeder is to combine the favorable characteristics of one plant or cultivar with the desirable traits of another plant or cultivar to obtain a new combination that has the best of both. Understanding the genetics of desired, as well as undesirable, characteristics enhances the efficiency of the plant improvement process. Learning Objectives • Understand the molecular basis of genes and chromosomes. • Understand the basic principles of transcription and translation. • Understand Mendelian mechanisms and patterns of inheritance. • Be able to differentiate among different types of gene action. • Determine genotypic and phenotypic consequences of independently inherited genes through generations of self-pollination. • Know how epistasis occurs through interaction of genes and alteration of expected phenotypic ratios. Overview of Genetics The Science of Genetics Genetics is one of the principal sciences that underlie plant breeding. Genetics is the study of heredity, genes, chromosomes, and variation in biological organisms. The science of genetics is often divided into four major subdisciplines: • Transmission genetics (also called classical or Mendelian genetics) • Quantitative genetics • Population genetics, and • Molecular genetics Transmission genetics deals with how genes and genetic traits are transmitted from generation to generation and how genes recombine. The foundation of modern genetics is recognized to have occurred in the mid-1800s when Gregor Mendel analyzed the results of crosses he made among garden pea plants. Mendel concluded that inherited characteristics (now called traits or phenotypes) are determined by factors (now known as genes) that he observed. He also realized that each organism contained two copies of each “factor” (gene), one inherited from its mother and one from its father. Mendel discovered the principles of heredity when he noticed how inherited traits (e.g., seed shape round vs. wrinkled; pod color yellow vs. green; flower position axial vs. terminal; or plant height tall vs. short) are passed from parents to offspring. Transmission (Mendelian) genetics is the focus of this module. Genetic Subdisciplines Quantitative genetics focuses on the study of inheritance when phenotypes exhibit continuous variation or distribution. In particular, it considers the effects of many genes that could be simultaneously influencing such traits, as well as the relative contributions of the environment and the interaction between genotype and environment. Quantitative genetics is the focus of the module on Inheritance of Quantitative Traits. Population genetics entails a study of heredity in groups of individuals for traits that are usually determined by one or only a few genes. It deals with gene distribution and genetic diversity within and among populations and subpopulations. Population genetics includes assessment and prediction of response to selection. It describes relationships between allele and genotype frequencies due to four main evolutionary forces: natural selection, genetic drift, mutation, and gene flow. Population genetics is the focus of the module on Inbreeding and Heterosis. Molecular genetics is concerned with the molecular structure and function of genes. It includes the study of DNA structure and replication and deals with gene expression and regulation. Gene Structure Genes and Chromosomes To understand inheritance, it is essential to understand gene structure and action. Let’s review key terminologies and principles. For a more in-depth review, please refer to biology or genetic textbooks, for example, From Genes to Genomes (Hartwell et al. 2011), Genetics: A Conceptual Approach (Pierce 2012), or iGenetics: A Molecular Approach (Russell 2010). Genes are encoded with DNA. Most of the DNA in plants is located in the nucleus of cells and arranged in groups of genes along multiple, linearly-shaped, chromosomes. Nuclear DNA is subject to Mendelian inheritance, which will be discussed later in this module. In addition to its occurrence in chromosomes in the nucleus, DNA is also located in organelles present in the cytoplasm of plant cells. FYI: Cytoplasmic DNA In plants, DNA is not just present in the nucleus of cells. It is also located in other membrane-bound, specialized subunits known as organelles that are found within the cytoplasm, or cell fluid. Two plant cell organelles that contain DNA are chloroplasts (which are plastids or organelles that carry pigments-specifically green chlorophyll) and mitochondria (singular, mitochondrion; organelles that break down complex carbohydrates and sugars into usable forms, and thus supply energy for the plant). The non-nuclear, organellar DNA located in plants follows cytoplasmic inheritance and is not subject to Mendelian inheritance. Cytoplasmic inheritance is also known as extrachromosomal or extranuclear inheritance, and is of significance in certain types of male sterility where the genes for those traits are present in the mitochondria, not in nuclear chromosomes. Molecular Basis of Chromosomes Chromosome – Each chromosome contains a single DNA molecule (Figure 2). DNA DNA (deoxyribonucleic acid) is composed of two chains of polynucleotides. Polynucleotides are also called nucleic acids, and consist of linear polymers that are macromolecules formed by the chemical joining of many identical or similar units called nucleotides. Every nucleotide in each chain consists of a nitrogen-containing base, deoxyribose (a sugar), and a phosphate group. Nucleotides within each chain are held together by sugar-phosphate (phospho-diester) bonds (Figure 3). Nitrogen-containing bases are purines (adenine, A, and guanine, G) and pyrimidines (cytosine, C, and thymine, T). Pairing occurs between one purine and one pyrimidine and is specific. Sequences of consecutive nucleotides constitute genes (Figure 4). C always pairs with G T always pairs with A DNA replication is semiconservative. The process of DNA replication is not yet fully understood. Basically, there are three steps. 1. Two strands of DNA unwind and pull apart. 2. Free (unbound) nucleotides bind to complementary bases on an original strand of DNA. 3. One newly formed strand and a template DNA strand re-coil to form a double helix. This process is semiconservative because each resulting double-stranded DNA molecule is composed of a newly synthesized strand and a template strand (Figure 4). Since one strand of each DNA molecule is an original strand, there is less probability of error occurring during replication. Genes Many genes are present in each chromosome. Each specific gene occurs at a defined point on a chromosome, the gene locus, on each of the two homologous chromosomes. More than one form of a particular gene, alleles, may occupy the same locus on homologous chromosomes. Alleles Alleles are variants that differ slightly in their DNA sequence. Diploid plant species have two sets of chromosomes, each of which can possess a different allele for a particular gene. For example, a gene for seed color might have the two alleles, A and a. Allele A causes one phenotype (e.g., brown seed color) and allele a causes a different phenotype (e.g., white seed color). For that gene, the genotype could be either AA, Aa, or aa. If one allele at a locus on a homologous chromosome partially or completely masks the expression of the other in influencing the phenotype, the allele that is expressed is termed dominant and the allele that is masked is termed recessive. By convention, we often write the dominant form with an uppercase letter, and the recessive form in lowercase. In the example above for seed color, allele A is the dominant allele. If the A allele is completely dominant to the a allele, individuals with either the AA or Aa genotypes would have the brown seed color phenotype, while aa individuals would have white seeds. An individual is heterozygous (Aa) when two different alleles are present at a locus and is homozygous—in this example, either homozygous dominant (AA) or homozygous recessive (aa)—when the same alleles are present on both chromosomes. Alleles at a locus can interact in several ways that are revealed by their phenotype, whether heterozygous or homozygous. FYI: Homozygosity and Heterozygosity For a given locus, an individual with a genotype of either AA or aa is homozygous for that gene and is known as a homozygote; the status of the gene is referred to as homozygosity. An individual with the genotype Aa is heterozygous for that gene and is called a heterozygote; the status is known as heterozygosity. In the case of polyploid individuals, those with the genotypes AAAA (tetraploid) or aaa (triploid) would be examples of homozygotes and those with genotypes of AAaa (tetraploid) or AAaaaa (hexaploid) would be examples of heterozygotes. The terms homozygous and heterozygous are used to describe the status of single genes or all gene loci within an individual, not within a population. There may be many different alleles of a gene present in a population of individuals, but for each diploid individual there are only two alleles per gene. For each individual, there is one allele from each parent and each allele per gene is present at corresponding loci on homologous chromosomes. With regard to populations, a homogeneous population would be one in which all individuals in the population would have the same genotype and possess the same alleles for one or more genes. In contrast, a heterogeneous population would be characterized by differing alleles at one or more loci. Note that a cross between two homozygous parents produces progeny that are homogeneous because all of the individual offspring are genetically identical. However, the offspring would be heterozygous for all loci for which different alleles occurred in the two parents. Terms and Definitions Gene Expression, Translation, and Transcription DNA, Protein, and Other Gene Products In order to have a better understanding of the concept of gene that will be the focus of this and the following lesson on linkage, it is critical to understand the chemical nature of DNA . Let’s review the pathways by which the genetic information in DNA is transferred from one DNA molecule to another (the process termed DNA replication) and from DNA to ribonucleic acid (RNA) molecules (called transcription), and then transferred from RNA to a protein (termed translation) by a code that specifies the amino acid sequence of the protein (see Figure 6). A gene is a stretch of DNA along a chromosome consisting of sequences of consecutive nucleotides. Recall that genetic information in DNA is coded in the sequence of four nucleotides that are abbreviated by the type of nitrogen-containing base that each contains—the purines A and G and the pyrimidines C and T. Through DNA replication, genetic information of an individual is transmitted from cell to cell during development and from generation to generation during reproduction. DNA Structure Examine the following for a better understanding of the chemical structure of the nucleotides that comprise the basic building blocks of DNA and the process of DNA replication: Review the chemical structure of DNA and what occurs during the process of DNA replication. DNA replication occurs within the synthesis phase of the cell cycle. Four types of chemical bases—A, G, C, T—in gene sequences carry the instructions for assembling a protein (Figure8). The base pairs are bonded together by H-bonds to form the “rungs of a DNA ladder” (Figure 8). Nucleotides Nucleotides are the basic building blocks of nucleic acids such as DNA and RNA, which are polymers made of long chains of nucleotides. DNA is double-stranded and RNA is single-stranded (Figure 9). Note that in RNA, the chemical base uracil (U) replaces thymine (T). Genes generally express their effect by coding for polypeptide chains, which are polymers consisting of ten or more aminoacids linked by peptide bonds. One or more polypeptides make up a protein. The DNA sequence of a gene is used as the basis for producing a specific protein sequence. Proteins are the complex molecules responsible for most biological functions in the cell. Gene Expression, RNA, Translation, and Transcription Amino acids are the building blocks of proteins. A protein is composed of one or more long chains of amino acids, the sequence of which corresponds to the DNA sequence of the gene that encodes it. The process of creating proteins from the genetic code in DNA is referred to as gene expression. The general process of gene expression in the cells of eukaryotes such as plants involves numerous steps, which are described below. FYI: Eukaryotes Plants are multicellular organisms known as eukaryotes, which are organisms possessing cells that contain DNA in a nucleus and other membrane-bound, specialized subunits known as organelles that are found within the cytoplasm, or cell fluid. Two plant cell organelles that contain DNA are chloroplasts (which are plastids or organelles that carry pigments—specifically green chlorophyll) and mitochondria (singular, mitochondrion; organelles that break down complex carbohydrates and sugars into usable forms, and thus supply energy for the plant). The non-nuclear, organellar DNA located in plants follows cytoplasmic inheritance and is not subject to Mendelian inheritance. Cytoplasmic inheritance is also known as extrachromosomal or extranuclear inheritance, and is of significance in certain types of male sterility where the genes for those traits are present in the mitochondria, not in nuclear chromosomes. In contrast to eukaryotes, prokaryotes such as bacteria are often unicellular and lack a cell nucleus and usually have their DNA in a single circular molecule. Transcription is a process in which the sequence of nucleotides in one DNA strand of a gene is copied into the nucleotides of an RNA molecule. The order of nucleic acids in RNA complements those on the DNA strand from which it is transcribed. In the RNA strand, however, uracil(U), rather than thymine (T), is the base that complements adenine (A). As the RNA transcript is formed, each base in the DNA is paired with a base in an RNA nucleotide, which is progressively added to the RNA strand as it grows. Transcription occurs in the nucleus of the cell (Figure 13). In a procedure known as RNA processing, intervening sequences or introns are removed from the RNA transcript by splicing. Introns are a special type of so-called non-coding DNA sequences that do not code for amino acids, but are located within genes until such sequences are removed during RNA processing. (Note that aside from intron sequences, most non-coding DNA found in chromosomes is located between (not within) gene loci along the chromosome.) The regions between the introns in the fully processed RNA are called exons, the sequences that code for proteins (Figure 10). The ends of the transcript are also modified. The fully processed RNA is referred to as mRNA (messenger RNA). mRNA is a single-stranded sequence of nucleic acid and it moves from the cell nucleus to the cytoplasm where proteins are made (Figure 10). Translation is the process through which mRNA directs the assembly of amino acids in the proper sequence to synthesize the particular protein. Ribosomes in the cell cytoplasm read the base sequence of the mRNA (Figure 10). In the translated part of the mRNA, each adjacent group of three nucleotides constitutes a coding group or codon. Each codon specifies an amino acid subunit in the polypeptide chain. Adapter molecules, tRNA (transfer RNA) are complexed with the specific amino acid corresponding to the base sequence of the given mRNA. tRNA molecules bring the amino acids specified by the mRNA to the ribosomes where they are added to the growing protein chain. When the polypeptide chain is complete, it is released from the mRNA and forms a protein molecule. The order of amino acids determines the structure of the protein which affects its action. Basic Steps of Transcription These are the basic steps of transcription and translation: 1. During transcription, a region of double-stranded DNA is momentarily pushed open, separating the two strands and allowing an enzyme known as RNA polymerase to build a strand of mRNA corresponding to that region of DNA. 2. The tRNA anticodon attaches to the mRNA codon. The tRNA has a region called the “anticodon” that complements the codon sequence of the mRNA (Figure 14). 3. The specific amino acid complexed with the tRNA is held in place while the tRNA-amino acid complex corresponding to the next codon moves into place. A peptide bond is formed between the adjacent amino acids, building the protein molecule. Inheritance and Gene Action Mechanisms Inheritance is based on the behavior of chromosomes and the genes that they carry. During meiosis and gametogenesis, homologous chromosomes separate. Each gamete receives one (haploid) set of chromosomes. The particular chromosome of a homologous pair that is distributed to a given gamete is random. When two gametes fuse during fertilization, the zygote receives from each parent one set of chromosomes, and the alleles that they each carry. The resulting combination of alleles in the zygote determines its genotype. Because the distribution of homologous chromosomes to gametes is random, the fusion of gametes to form the zygote may produce different genetic combinations. Thus, within a population, variation for specific traits or characters may be observed. If the variation for a given trait is due to contrasting alleles at one or more loci, rather than to responses to the environment, the variation is heritable and can be transmitted from parent to progeny. Plant breeders select plants that exhibit desirable characteristics and those plants carry the desired allele of the gene that encodes the characteristic of interest. Each gene or combination of genes and alleles, as influenced by the environment, determines the phenotype or observed expression of the particular trait. An individual’s allelic composition at corresponding loci on homologous chromosomes confers the expression of that gene. Alleles at corresponding loci interact. One allele may mask the presence of the other allele(s). Alleles at a locus can interact in different ways, including no dominance (also referred to as additive gene action), partial dominance, complete dominance, and over-dominance. Gene Action There are several general types of gene action. The type of gene action and the alleles present for a given gene affect the phenotype. Let’s consider the gene action as indicated by the phenotype of a diploid individual heterozygous at the given single locus compared to the phenotype of its parents. Addictive gene action (no dominance) The progeny’s phenotypic value is at the midpoint between both parents. Complete dominance The phenotype of the heterozygous progeny equals the phenotype of the homozygous dominant parent. Partial (incomplete) dominance The heterozygous progeny has a phenotypic value greater than that of the mid-parent value (MPV), but less than that of the homozygous dominant parent. Over-dominance The phenotype of the heterozygous progeny is greater than either parent. Study Questions 1: Parental and Progeny Value Comparison Two diploid plants having different phenotypes for characters A, B, and C are mated. The progeny are grown out and their phenotypes are evaluated. Assume that both parents are homozygous at each locus. Compare the parental and progeny values for each character. Select the gene action at each locus. A Locus Genotype Phenotype value Parent One AA 75 Parent Two aa 40 Progeny Aa 75 Query $2$ B Locus Genotype Phenotype value Parent One BB 60 Parent Two bb 20 Progeny Bb 55 Deviations from Expected Phenotypes Multiple Alleles With complete dominance of the type that we have been discussing, two different alleles exist for a trait, but only one of the alleles is observed in the phenotype. But it is important to understand that dominance does not affect the way in which genes are inherited. For some characters, there are reasons other than dominance among alleles at the same locus that explain deviations from expected phenotypes. Multiple alleles—rather than just two—can occur at a single locus. Examples of multiple alleles at a single locus include the ABO blood group system in humans or the S alleles that control self-incompatibility in plants. Multiple alleles at a locus are sometimes referred to as an allelic series. However, while there may be more than two alleles per gene present in a population, be aware that the genotype of any given individual diploid plant in the population possesses only two alleles. Penetrance is a measure of the percentage of individuals having a particular genotype that express the expected phenotype. Incomplete penetrance occurs when a genotype does not always produce the expected phenotype. Expressivity is a related concept that describes the degree to which a character is expressed. Incomplete Penetrance Incomplete penetrance and variable expressivity are due to effects of other genes or environmental factors that change the effect of a particular gene. For example, a phenotype produced by an enzyme encoded by a particular gene may be expressed only within a narrow temperature range. In barley, a recessive allele occurs that produces albino plants when they are grown at lower temperatures. The allele inhibits chlorophyll production. But if barley plants that are homozygous recessive for this allele are grown above a critical temperature, the effect is not present so the plants have normal chlorophyll and are green. Lethal alleles can change expected phenotypic ratios as well. Lethal alleles cause death when present, so that one or more genotypes will be missing from the offspring of a cross. Lethal alleles can be recessive (causing death only in homozygotes) or dominant (both homozygotes and heterozygotes with the allele will die). Dominant lethal alleles are rarely maintained in populations. Essential genes are genes that when mutated can result in a lethal phenotype. Study Questions 2: Deviations from Expected Phenotypes An example of a recessive lethal allele is one that controls chlorophyll production in the aurea strain of golden-leaved snapdragons. Aurea plants are heterozygous for the gene. A cross between two aurea plants produces progeny in the ratio of 2:1 golden to green. The expected phenotypic ratios in the progeny would be 1:2:1 white to golden to green. However, the white-leaved offspring die before germination or in the seedling stage due to a lack of ability to make chlorophyll. What are the genotypes for each of these leaf phenotypes in the progeny of a cross between aurea snapdragons? Mendelian Heredity Gregor Mendel analyzed the segregation of hereditary traits. We now know that the genotype is the genetic constitution of an organism and the phenotype is the observable characteristic or set of characteristics of an organism produced by interactions between its genotype and the environment. The phenotype is influenced by not only the genotype but also environmental effects and developmental events and by actions of other genes and their products. Therefore, individuals with the same genotype can have different phenotypes and conversely, individuals with the same phenotype can have different genotypes. Terminology The parental generation of a cross is often called the P generation. Using symbolism based on what is called the F Symbol, the progeny of the mating of two parents is typically called the F1 or first filial generation. The subsequent generation produced by either self-pollination or crossing among the F1 offspring (a type of mating called inbreeding) is referred to as the F2 generation, or the second filial generation. The progeny resulting from self-pollination of each consecutive generation following the F2 is referred to as F3, F4, F5, and so on. Another kind of symbolism is based on the S Symbol. The S symbol is used to describe the offspring of a single cross—specifically the cross between two homozygous parents. F and S symbolism have been developed to describe progeny developed by hybridization and self-pollination. F and S Symbolism It is important to note on pages 28-33 of Fehr’s textbook, plant breeders have developed a variety of systems using either the F or the S symbol to describe progeny developed by hybridization and self-pollination. What is challenging is that depending on the plant breeder, F and S symbols may be used in different, often contradictory, ways. The table below depicts examples of the particular system chosen and the way in which symbols are defined for use (Fehr, 1987, p. 28-33). Table 3 Symbol Description F1 Hybrids produced from the mating of homozygous parents. F2 = S0 First segregating generation produced from the cross of two or more parents F3 = S1 Offspring from self-pollination of F2 (or S0) plants F5 = S3 Offspring from self-pollination of F4 (or S2) plants Syn 1 Synthetic 1 = Offspring from random mating of an F2 population Syn 4 Synthetic 4 = Offspring from random mating of a Syn3 population F2:5 line F2-derived line in F5 = an F5 generation line available for planting that originated from an F2 generation S2:9 line S2 -derived line in S9 = an S9 generation line available fro planting that originated from an S2 generation Crosses A cross involving a single trait (e.g., seed color) is referred to as a monohybrid cross, while one involving two traits (e.g., seed color and plant height) is termed a dihybrid cross. Conventionally, in equations used to symbolize a cross, the female parent is listed first and the male parent second, as in this example involving a single locus in diploid individuals: AA x aa ⇒ Aa Crosses that are done both ways are referred to as reciprocal crosses. For example, the reciprocal cross of the one above would be: aa x AA ⇒ Aa Reciprocal crosses can be used to determine whether a trait is maternally inherited. If a trait is controlled by genes located in cytoplasmic DNA, the segregation ratios between reciprocal crosses would be different because cytoplasmic DNA is inherited only through the female parent. Predicting Segregation Ratios If the genetic basis of a trait is known, principles developed by Mendel can be used to predict the outcome of crosses. There are three common approaches used to analyze segregation results, two of which use the listing of all possible genotypes and phenotypes of zygotes and gametes by systematic enumeration and the other of which uses mathematical rules. • The Punnett Square Method is best for situations involving one or two genes. All possible gametes are written down in a square and then combined systematically to depict an array of genotypes of the offspring. • The Branching or Forked-Line Method [See Appendix C for some examples] also works well for situations involving one or two genes. It uses a tally system in a diagram of branching lines. • The Probability Method is based on two rules in mathematical probability theory—the Multiplicative Rule and the Additive Rule—and deals with the frequency of events. Punnett Square Examples Parental Monohybrid Cross Trait Seed color Alleles Y yellow y green Cross yellow seeds x green seeds YY x yy (homozygous dominant x homozygous recessive) Offspring called F1 generation Genotype all alike Yy (heterozygous) Phenotype all alike Yy (green) Pollen Egg   1/2y 1/2y 1/2Y 1/4Yy 1/4Yy 1/2Y 1/4Yy 1/4Yy F1 Monohybrid Cross Alleles Y yellow y green Cross yellow seeds x green seeds Yy x Yy (heterozygous x heterozygous) Offspring called F2 generation Genotypic ratio 1:2:1 YY (homozygous dominant): Yy (heterozygous): yy (homozygous recessive) Phenotypic ratio 3:1 Y_ (yellow): yy (green) Results: Pollen Egg   1/2Y 1/2y 1/2Y 1/4 YY 1/4 Yy 1/2y 1/4 Yy 1/4 yy Dihybrid Cross Trait Seed shape and seed color Alleles R round, r wrinkled, Y yellow, y green Cross Round, yellow seeds x round, yellow seeds RrYy x RrYy (heterozygous x heterozygous) Offspring called F3 generation Genotypic ratio 1:2:1:2:4:2:1:2:1 RRYY:RRYy:RRyy:RrYY:RrYy:Rryy:rrYY:rrYy:rryy Phenotypic ratio 9:3:3:1 R_Y_ (round, yellow): R_yy (round, green): rrY_ (wrinkled, yellow): rryy (wrinkled, green) Results: Pollen Egg   1/4 RY 1/4 Ry 1/4 rY 1/4 ry 1/4 RY 1/16 RRYY 1/16 RRYy 1/16 RrYY 1/16 RrYy 1/4 Ry 1/16 RRYy 1/16 RRyy 1/16 RrYy 1/16 Rryy 1/4 rY 1/16 RrYY 1/16 RrYy 1/16 rrYy 1/16 rrYy 1/4 ry 1/16 RrYy 1/16 Rryy 1/16 rrYy 1/16 rryy Branching or Forked-Line Method Below is an example of the forked-line or branch diagram method for determining the outcome of an intercross involving three independently assorting genes in peas. Traits Plant height, seed color and seed texture Alleles D tall / d dwarf G yellow / g green W round / w wrinkled Cross Tall plants with yellow, round seeds x dwarf plants with green, wrinkled seeds DDGGWW x ddggww (homozygous dominant x homozygous recessive) F1 DdGgWw Expected F2 phenotypes for each trait Segregation of gene for plant height Segregation of gene for seed color Segregation of gene for seed texture Combined phenotype of all three genes 3/4 D_(tall) 3/4 G_(yellow) 3/4 W_(round) 27/64 D_G_W(tall, yellow, round) 1/4 ww(winkled) 9/64 D_G_ww(tall, yellow, wrinkled) 1/4 gg(green) 3/4 W_(round) 9/64 D_ggW_(tall, green, round) 1/4 ww(wrinkled) 3/64 D_ggww(tall, green, wrinkled) 1/4 dd(dwarf) 3/4 G_(yellow) 3/4 W_(round) 9/64 ddG_W_(dwarf, yellow, round) 1/4 ww(wrinkled) 3/64 ddG_ww(dwarf, yellow, wrinkled) 1/4gg(green) 3/4 W_(round) 3/64 ddggW_(dwarf, green, round) 1/4 ww(wrinkled) 1/64 ddggww(dwarf, green, wrinkled) Rules of Probability Using probability theory can allow for accounting of the frequency of events, such as the chance of obtaining a head on a coin toss or obtaining a dominant homozygote (AA) from the mating between two heterozygotes (Aa). To figure out the probability of an event, all possible outcomes must be determined. For a coin toss, there are two possible events—heads or tails—each with a probability of ½ that it would occur. For the progeny produced by a heterozygote, the probability associated with each type of offspring is ¼ (AA), ½ (Aa) and ¼ (aa). The Multiplicative Rule states that if events X and Y are independent, the probability that they occur together (that is A and B), is the probability of A times the probability of B. It is denoted as: $P(A)\times P(B)$ The Additive Rule states that if events X and Y are independent, the probability that at least one of them occurs (that is A or B), is the probability of A plus the probability of B minus the probability that both A and B occur together. It is denoted as: $P(A) + P(B) - [P(A) \times P(B)]$ Mendel’s Principles Mendel’s analysis of monohybrid crosses identified three key principles: The Principle of Uniformity If both parents are homozygous, their F1 is genetically uniform. To the right is a Punnett Square showing an example of this phenomenon, depicting the genotypic and phenotypic ratios and chromosomes of the diploid parents, haploid gametes, and the F1 generation. The Principle of Segregation In a heterozygote, two different alleles of a gene locus segregate from each other in the formation of gametes. Below are two figures (one using a Punnett Square and the other the fork or branch diagram method) showing an example of Mendel’s law of segregation. The figures depict the genotypic and phenotypic ratios and chromosomes of the F1 heterozygote, haploid gametes, and the F2 generation. The Principle of Independent Assortment Alleles at different gene loci are transmitted independently of one another during the production of gametes. Below are two figures (one using a Punnett Square and the other the fork or branch diagram method) showing an example of Mendel’s law of independent assortment. The figures depict the genotypic and phenotypic ratios and chromosomes of the parents, the F1 heterozygote, haploid gametes, and the F2 generation. Inheritance A trait or characteristic may be under the control of one or more genes. The range of variation for a particular characteristic indicates the mode of inheritance of that characteristic. • Qualitative inheritance — simple inheritance of a characteristic under the control of single gene or a few major genes. The expression of simply inherited characteristics is discrete. That is, the phenotypic variation of the characteristic can be separated into distinct classes. Generally, the environment has little influence on the characteristic’s expression. • Quantitative inheritance — inheritance of characteristics influenced by numerous genes (multiple genes or polygenes). The involved genes have small, cumulative effects on the phenotype of the characteristic. The expression of such characteristics can be measured in quantitative units that are continuous, rather than discrete, and is often considerably influenced by the environment. Quantitative inheritance is the subject of the module on Inheritance of Quantitative Traits. The inheritance of some characteristics cannot easily be categorized as either qualitative or quantitative. These characteristics are usually under the control of one or few major genes as modified by multiple genes with small effects. Together with environmental effects, the phenotype of such characteristics may show continuous variation. Try This! – Trait Graphs Drag the correct inheritance to the appropriate trait graph Progeny Ratios To determine the mode of inheritance of a particular character, plant breeders mate plants and evaluate the performance of their offspring. The proportion of progeny exhibiting different phenotypes provides information about the proportion of progeny possessing different genotypes. • Phenotypic ratio — the proportion of progeny exhibiting different phenotypes • Genotypic ratio — the proportion of progeny possessing different genotypes These ratios are commonly determined by crossing two plants having contrasting phenotypes for a given character. The parents may or may not be homozygous. The progeny are heterozygous for the trait. Self-pollinating the F1 progeny produces the F2 generation, and so forth (Fn). In each generation, the ratio of plants displaying contrasting phenotypes for the particular trait reveals information about the genotypes of the parents, as well as gene action (e.g., dominant or recessive alleles). In the exercise concerning phenotypic and genotypic ratios, with each consecutive generation, the proportion of heterozygotes (Gg) is reduced. With continued self-pollination, the heterozygotes will segregate, decreasing the proportion of heterozygotes in the population by half each generation. Notice that the homozygotes can only produce homozygotes. Try This! – Crossing A cross is made between a plant homozygous for green seeds (GG) and a plant homozygous for white seed (gg) — a monohybrid cross. Assume: the species is diploid and normally self-pollinating, and the G allele is completely dominant. By convention, “X” means cross-pollinating, and the “” symbol indicates self-pollinating. At each generation, you will determine and fill in the missing phenotypic and genotypic ratios. You will drag a fraction from the options provided below to its respective empty box. Successive Generations Table 1 Generation Heterozygosity (%) F1 100.0 F2 50. F3 25.0 F4 12.5 F5 6.25 F6 3.12 For each successive generation of offspring resulting from one F1 individual, by the F8 generation, the population is essentially homozygous. When no further segregation for the trait occurs, all progeny derived from that F1 will “breed true” because they are homozygous for the trait. The proportion of plants that are expected to be heterozygous at any gene when starting with a heterozygous F1 and selfing can be determined by using the formula (½)n, where n = the number of segregating generations, e.g., in F2, n = 1 and in F5, n = 4. Using this we get the following proportions of heterozygous plants in F4: (½)n = (½)3 = ⅛ = 12.5%. The proportion of homozygous plants in any generation is then given by 1 − (½)n which, when algebraically converted, is equal to: $\frac{2^n - 1}{2^n}$ Applying this to F4 we get $\frac{2^3-1}{2^3} = \frac{8 - 1}{8} = {7 \over 8} = 87.5\%$ When working with actual genotypes we must remember that in any segregating generation there are two homozygous genotypes and we expect equal quantities of each. Using the example of an F1 that is Aa, in F2, we expect ¼ AA + ½ Aa + ¼ aa. In F4 we expect to be homozygous with half of those AA and half aa. Thus overall we expect the following F4 genotypic frequencies: ${7 \over 16} \textrm{AA} + {1 \over 8} \textrm{aa} + {7 \over 16} \textrm{aa}$ Scenarios under cross-pollination — with and without selection — will be discussed in more detail in the module on Population Genetics. Progeny Test There are two principal procedures that allow the plant breeder to determine the basis of phenotypes (genetic or environmental), gene action, and the genotypes of individual plants. Which procedure is used depends on the specific objectives of the breeder. Progeny Test The progeny test evaluates the genotype of an individual based on the performance of its offspring. The progeny test can be used to: 1. Distinguish heritable phenotypes from phenotypes attributable to environmental effects. 2. Determine the genotype or the allelic composition of an individual. Steps in Progeny Test 1. Hybridize (mate) two plants, A and B. 2. Grow out and self-pollinate the F1 plants. 3. Grow out and self-pollinate F2 plants. 1. Determine the phenotypic ratio of trait(s) of interest. 2. Harvest seed separately from each plant. 4. Plant a portion of the F3 seed from each phenotype separately. 1. Determine the phenotypic ratio in each group—the phenotypic ratio reveals which of the F2 plants were homozygous and which were heterozygous for the trait(s) of interest. 2. Based on the phenotype information, calculate the genotypic ratio. In this example, the phenotypic ratios of the F3 plants reveal the following genotypic information about each of the F2 parents: F2 Parent Genotype a Homozygous red b Heterozygous red c Homozygous green d Heterozygous red Both the red and green phenotypes occur in ratios consistent with those of heritable traits. Thus, there is a genetic basis for these phenotypes (i.e., these phenotypes are not just the result of environmental conditions). Testcross The testcross procedure is used to determine the genotype of an individual or linkage groups. Linkage is a condition in which genes located on the same chromosome are inherited together due to their close proximity. Linkage will be discussed in greater detail in “Linkage” module. Steps in Testcross 1. Hybridize (mate) two plants. The genotype of Parent 1 is unknown, A (?). Parent 2 is homozygous recessive for the trait of interest, aa. 1. Grow out F1 plants and evaluate the phenotypic ratio: 1. If segregating 1:1, then you know that the genotype of Parent 1 was heterozygous, Aa. 1. If all plants have the phenotype of Parent 1, than you know that Parent 1 was homozygous dominant, AA. The backcross is a special type of progeny test. It is a cross of an F1 to either of the original parents. This procedure is used extensively in basic genetic studies but not often used by plant breeders to determine genotypes of plants. Study Questions 3 For each of the following situations, identify which procedure(s) would be most appropriate. Determine Linkage To determine linkage groups, hybridize two plants: • Parent 1 is heterozygous at two (or more) loci. • Parent 2 is homozygous recessive at these loci. The interpretation of the results of this cross will be discussed in the module on Linkage. Genetic Recombination and Its Effects Develop Improved Cultivars To develop improved cultivars, plant breeders usually combine the favorable characteristics of one plant or cultivar with the desirable traits of another plant or cultivar, accumulating desirable alleles for key characters. To obtain an improved genetic combination, breeders make a series of matings, selecting the best offspring to produce the next generation. Plant breeders rely on several genetic mechanisms to obtain new genetic combinations. 1. Segregation — Homologous chromosomes derived from different parents separate and distribute randomly to cells during meiosis. 2. Recombination — Formation of new gene combinations by mating individuals having differing genotypes. Segregation Segregation is the result of the independent assortment or chance distribution of homologous chromosomes and the genes that they carry to gametes. Through meiosis, allelic pairs are separated and distributed to different cells, which subsequently undergo gametogenesis. Genes located on different chromosome pairs assort independently. That is, the chance distribution of a particular chromosome, say one of these green chromosomes, to one cell, has no effect on the distribution of a yellow chromosome. Independent assortment facilitates recombination and leads to segregation in subsequent generations. Recombination Mating two plants possessing different genotypes results in progeny with genotypes that may differ from the parental types. The progeny having genotypes that differ from the parents are referred to as “recombinants.” Try this! Recombination Exercise Mate two plants, one heterozygous and the other homozygous at the G and H loci. Determine all possible gamete types and then all possible genotypes that would result from this mating progeny. Let Parent 1 be the female and Parent 2 be the male parent in this cross. Check each step and make corrections if needed before proceeding to the next step. Table 2 Parent 1   Parent 2 Genotype GgHh X gghh Step 1: Among the following types, select the possible gamete types for the eggs, and drag the 4 appropriate types into the boxes below. Query $10$ Step 2: Among the following types, select the possible gamete types for the sperm, and drag the 4 appropriate type into the box below. Query $11$ Step 3: Fertilization: When gametes fuse, the zygote receives half of its genes from each parent. Given below are all possible combinations of the genotypes. Select the correct combinations and drag them to their respective places on the table. Query $12$ Combination of genes in sperm Combination of genes in eggs gh gh gh gh GH GgHh GgHh GgHh GgHh Gh Gghh Gghh Gghh Gghh gH ggHh ggHh ggHh ggHh gh gghh gghh gghh gghh Step 4: What is the genotypic ratio of these progeny? Query $13$ Combination of genes in sperm Combination of genes in eggs gh gh gh gh GH GgHh GgHh GgHh GgHh Gh Gghh Gghh Gghh Gghh gH ggHh ggHh ggHh ggHh gh gghh gghh gghh gghh Step 5: What is the phenotypic ratio of these progeny? Query $14$ Ratio Genotype 4/16 GgHh 4/16 Gghh 4/16 ggHh 4/16 gghh Step 6: Identify the parental types and recombinants by clicking on the correct button under each example. Query $18$ Study Questions 4 A homozygous plant that was • high yielding (Y_ = high, yy = low), • low in protein (P_ = high, pp = low), • early maturing (E_ = late, ee = early), and • with white flowers (W_ = purple, ww = white) was crossed with a homozygous plant that was low yielding, high in protein, early maturing, and with purple flowers. Option Genotype Phenotype Yield Protein Maturity Flowers A YyPpEeWw High High Late Purple B YyPPeeWw High High Early Purple C YyPpeeWw High High Early Purple D yyPpeeWW Low High Early Purple E YyppEeWw High Low Late Purple F YYPpeeww High High Early White Query $23$ Helpful Hint • 3/4 will be high yielding (Y_) • 3/4 will be high protein (P_) • all will be early maturing (ee) • 1/4 will have white flowers (ww) Let’s verify this by looking at the combinations of genes possible in the gametes. There are eight combinations. YPeW YPew YpeW Ypew yPeW yPew ypeW ypew To ascertain all the genotypes in the F2, we can create a Punnett Square with these eight combinations for the eggs and for the sperm, producing an 8 x 8 table showing 64 combinations in the F2 zygotes. Only those F2 with a Y_P_eeww genotype (indicated with an X in the table below) will have the phenotype: high yielding, high protein, early maturity, and with white flowers. Pollen Eggs YPeW YPew YpeW Ypew yPeW yPew ypeW ypew yPeW YPew   X   X   X   X YpeW Ypew   X       X yPeW yPew   X   X ypeW ypew   X Restrictions with Independent Assortment Hybrid Characteristics A breeder cannot improve a characteristic unless there is some variability for that characteristic within which to make selections. Hybridizing plants differing in their phenotypes (and genotypes) and selecting from among the recombinants provide the breeder with the opportunity to make progress towards crop improvement. However, recombination and segregation may fail to provide the expected variation for two general reasons. • Population size — A minimum of progeny from a cross must be grown out and evaluated. If the number is too small, the likelihood of the desired recombinant occurring in the population is reduced. As the number of independently assorting genes increases, the number of plants that must be evaluated increases exponentially. Thus, an adequate population is essential to make efficient progress towards the breeding goals. The minimum population size required for all genotypes to be represented in the population can be calculated as follows: 1. Determine the number of segregating gene pairs. Let that number equal “n”. 2. Calculate the minimum population size: minimum population size = 4n • Gene Interaction — Although the genes involved in epistatic and pleiotropic interactions may assort independently, their interactions often affect phenotypic and genotypic ratios. • Linkage — As stated earlier, loci in close proximity on the same chromosome tend to be transmitted together and do not assort independently. Genetic Cross-Data When analyzing data from genetic crosses, it is frequently appropriate to use some kind of statistical analysis because such data is often quantitative. One statistical procedure commonly used for testing results of segregation data is called a chi-square (χ2) test. The chi-square test is also known as a “goodness-of-fit” test. Breeders wonder if data support or fit a particular hypothesis and therefore help to explain the results. For example, does the range of phenotypes observed within the progeny of a cross-fit a particular segregation ratio, e.g., 3:1 or 9:3:3:1? The chi-square procedure helps breeders understand the significance of deviation of observed results from results predicted by the hypothesis being tested. A null hypothesis is formed that states there is no real difference between the observed and expected data. If differences are due to chance, then the hypothesis can be accepted, otherwise, the null hypothesis is rejected and the breeder can modify the hypothesis in favor of a better one. The equation used to calculate the (χ2) statistics is as follows $x^2 = \sum \frac{(\textrm{observed} - \textrm{expected})^2}{expected}$ The chi-square procedure will be covered in more detail in the Quantitative Methods course. Gene Interactions Traits When multiple genes control a particular trait or set of traits, gene interactions can occur. Generally, such interactions are detected when genetic ratios deviate from common phenotypic or genotypic proportions. • Pleiotropy — Genes that affect the expression of more than one character • Epistasisepistasis — Genes at different loci interact, affecting the same phenotypic trait. Epistasis occurs whenever two or more loci interact to create new phenotypes. Epistasis also occurs whenever an allele at one locus either masks the effects of alleles at one or more loci or if an allele at one locus modifies the effects of alleles at one or more loci. There are numerous types of epistatic interactions. Epistasis is expressed at the phenotypic level. It is important to note that genes that are involved in an epistatic interaction may still exhibit independent assortment at the genotypic level. The following slides show some examples of epistasis drawn from various types of plants. Duplicate Recessive Epistasis Duplicate recessive epistasis (also known as complementary action): 9:7 ratio observed in flower color of progeny of crosses between a pure line pea plant with purple flowers (genotype CCPP) with a pure line, homozygous recessive plant with white flowers (ccpp). The F1 plants are all purple and have a genotype of CcPp, but the F2 progeny will have a modified ratio of 9:7 because color is only produced if both genes have at least one dominant allele. These genes control flower color by controlling the expression of biochemical compounds known as anthocyanins that impart pigment to the flower. Pigmentation in this case is controlled by a two-step chemical reaction. One of these genes controls the first step and the other controls the second step. Male Gametes CP Cp cP cp Female Gametes CP CCPP Purple CCPp Purple CcPP Purple CcPp Purple Cp CCPp Purple CCpp White CcPp Purple Ccpp White cP CcPP Purple CcPp Purple ccPP White ccPp White cp CcPp Purple Ccpp White ccPp White ccpp White Dominant Epistasis Dominant epistasis (also known as masking action): 12:3:1 ratio observed in fruit color of progeny of crosses of squash. In the F2, fruits are white if the genotypes are either W_G_or W_gg because the dominant allele for the first gene (W) masks the effect of either allele for the other gene (G or g). Color is present only if the first gene is homozygous recessive (ww). Yellow squash have the genotype wwG_ and green ones have the genotype wwgg. Male Gametes Female Gametes   WG Wg wG wg WG WWGG White WWGg White WwGG White WwGg White Wg WWGg White WWgg White WwGg White WwGg Yellow wG WwGG White WwGg White wwGG Yellow wwGg Yellow wg WwGg White Wwgg White wwGg Yellow wwgg Green Duplicate Dominant Epistasis Duplicate dominant epistasis (also known as duplicate action): 15:1 ratio observed in fruit shape of progeny of crosses of the common shepherds purse. If either of the two genes involved in fruit shape (T or V) are present alone or both together (TV), then the plants will all produce triangular-shaped fruit. Only the homozygous recessive genotype (ttvv) produces a seed capsule with an ovate shape. Male Gametes Female Gametes   TV Tv tV tv TV TTVV Triangular TTVv Triangular TtVV Triangular TtVv Triangular Tv TTVv Triangular TTvv Triangular TtVv Triangular Ttvv Triangular tV TtVV Triangular TtVv Triangular ttVV Triangular ttVv Triangular tv TtVv Triangular Ttvv Triangular ttVv Triangular ttvv Ovate Epistasis Identification Identify the type of epistasis that best explains the observed effect.

textbooks/bio/Agriculture_and_Horticulture/Crop_Genetics_(Suza_and_Lamkey)/1.04%3A_Gene_Segregation_and_Genetic_Recombination.txt

Introduction Genes located on the same chromosome are genetically linked. Genetic linkage analysis can be used to determine the order of genes on chromosomes. Closely linked genes are not segregating independently, like genes located on different chromosomes. This has different implications, e.g., in relation to trait correlations. Moreover, linked genes can be used as genetic markers, which have become an important tool in plant breeding. Learning Objectives • Develop an understanding of the genetic basis of linkage. • Gain awareness on how to detect the occurrence of linkage. • Review the principles of genetic map construction. • Become familiar with the concept of linkage disequilibrium. Crossover and Recombination Genetic Organization Genes are physically organized on chromosomes. Each gene is located at a particular “address” (particular position on a specific chromosome, which can be identified by genetic mapping). Inheritance of genes located on different chromosomes follows the rules of independent assortment. Since plant species have multiple chromosomes, independent assortment is true for the majority of genes. In contrast, linked genes located on the same chromosome are more likely to cosegregate, i.e., being jointly transmitted to offspring more often than expected by independent assortment. The biological process that separates linked genes is the crossing-over (C.O., or crossover), which occurs during meiosis, and leads to genetic recombination. Crossing-Over Query $1$ During meiosis of diploid organisms, the chromatids of homologous chromosomes pair and form bivalents. During Meiosis I, homologous chromatids pair to physically exchange chromosome segments. The chromosomal site, where this reciprocal exchange of homologous chromosome segments takes place, is called a chiasma. Thus, crossing-over involves not completely understood mechanisms for identification of homologous sites of chromatids, breakage and rejoining of chromosomes. Genetic Distance Crossing-over events occur more or less random during meiosis. In most plant species, one to few crossing-over events occur per meiosis and chromosome. Thus, the closer the genes are physically linked on the same chromosome, the less likely they will get separated, and consequently, the less likely genetically recombinant gametes will be produced. This is the underlying principle of genetic maps: the genetic distance between genes reflects the probability of a crossing-over between linked genes. Recombination Observation of crossing-over events requires cytological methods, which can be cumbersome for large populations. In contrast, genetic recombinants can be observed at the phenotype level, or by use of DNA markers. If two linked genes with two alleles each have clear phenotypic effects, e.g., on flower color (A: red, a: white; A is dominant over a) and seed color (B: green, b: yellow; B is dominant over b), then genetic recombinants can easily be identified by determining the fraction of non-parental gametes in the offspring. Note that crossing-over also takes place in meiosis of completely homozygous individuals. However, in this case, genetic recombination cannot be observed as described above. The reason is that observation of recombinant gametes requires two (or more) different alleles at the loci, for which linkage is going to be determined. This explains why offspring saved from pure line cultivars will not segregate whereas seed harvested from F1 hybrid will segregate. The observable fraction of recombination events is also called effective recombination. Linkage Detection Linkage Phase For linkage detection, it is crucial to know the linkage phase of alleles. The linkage phase is the physical arrangement of linked genes in a chromosome. A double heterozygote with a genotype of AaBb could be in one of the two linkage phases. Conventionally, when linked dominant alleles are located on the same homologous chromosome and the linked recessive alleles are on the other homologous chromosome, for example, AB/ab, it is said the genes are linked in the coupling phase. When a dominant allele at one locus is on the same homologous chromosome as a recessive allele of the other linked gene, for example, Ab/aB, it is said that the genes are linked in repulsion phase (Figure 4). This knowledge is crucial, as linkage detection and distance estimation is based on the observed parental and non-parental gametes. Coupling and Repulsion In the case of close linkage, non-parental gametes and respective offspring are underrepresented. An example is the Australian sheep blowfly, Lucilia cuprina. Normal blowflies have a green thorax and surround themselves in a brown cocoon during their pupal stage. However, recessive genes (here marked a and b) can cause the fly to develop a purple thorax and spin a black puparium. Using Testcrosses For detection of linkage, appropriate testcrosses need to be conducted. The linkage phase is known, if two homozygous parental genotypes (AABB and aabb) are crossed to produce the respective F1 (AaBb). In this case, A and B as well as a and b are linked in coupling phase. The non-parental recombinant gametes have the genotype Ab and aB, whereas the parental gametes have the genotype AB and ab. Usually the phenotype cannot be observed in (haploid) gametes, but only in diploid plants. Thus, to determine whether two loci are linked, offspring need to be produced. This can be achieved by self pollination of the AaBb – F1, by production of doubled haploid offspring, or by a testcross. In this particular example, a backcross (BC) of the F1 to the aabb parent would be the best option. Testcross Gametes All offspring from this BC would receive an ab gamete from the aabb parent, and any of the two parental (AB, ab) or non-parental (Ab, aB) gametes from the F1. Because of dominance of A over a and B over b, all four resulting diploid genotypes in the BC1 (backcross generation 1) generation (AaBb, Aabb, aaBb, aabb) can be phenotypically discriminated, and used to count genotypes that received parental or non-parental gametes from the F1. Thus, when using this BC approach, only the crossing-over events that occurred in the F1 are monitored for linkage estimation. Chi-Square Test For detection of linkage, a Chi-Square test can be employed. The Chi-Square test compares observed with expected frequencies. In this case, the null hypothesis to determine expected frequencies is the assumption of independent assortment. Under this assumption, equal frequencies of all four gametes are expected. In the case of linkage, BC1 individuals carrying non-parental gametes are underrepresented, leading to a statistically significant Chi-Square value. This means that the null hypothesis of independent assortment would be rejected and linkage assumed. Table 1 An example of the detection of linkage in Drosophila melanogaster using a Chi-Square test. d: difference between the observed number and expected number. The significantly higher Chi-Square values reject the null hypothesis and strongly indicate the presence of linkage. Phenotypes Observed Number (o) Expected Number (e) d (o – e) d2 d2 / e Parentals: (black-bodied and normal wing plus grey-bodied, vestigial wing) 2,712 1,618 1,094 1,196,836 739.7 Recombinants: (black-bodied and vestigial wing plus grey-bodied and normal wing) 524 1,618 1,094 1,196,836 739.7 Chi-Square Results To better understand the use of Chi-Square in determining linkage, two numerical examples based on the cross schemes described in Figs. 8 and 9 are provided here. In both examples, a sample size of 2,000 BC1 individuals has been used. The Chi-Square test sums up over all squared differences between observed and expected values, divided by expected values. In example A, observed and expected values are equal, thus the Chi-Square value = 0. In example B, the squared differences between observed and expected values is in all cases 90,000, to be divided by the expected 500 = 180. As there are four genotypic classes, the Chi-Square value is 720, which is significantly larger than the tabulated value of 3.81 (p = 5%). In conclusion, example A is in agreement with independent assortment, whereas in example B, linkage has been detected. In conclusion, example A is in agreement with independent assortment, whereas in example B, linkage has been detected. Genetic Distance The same data used to determine linkage can also be used to estimate the recombination frequency between two genes (more precisely, the recombinant frequency). The recombinant frequency = (number of BC1 progeny with recombinant (non-parental) alleles / total number of BC1 progeny) x 100%. In example B of Section 2: Linkage Detection, the recombinant frequency is (200 + 200 / 2000) * 100% = 20%. The recombination frequencies between any pairs of genes provide an estimate of how close they are linked on a chromosome. The recombinant frequency in % is sometimes also called “map units” (M.U.). In this example, the genetic distance in map units between the two genes under consideration is 20 M.U. In the case of complete linkage of two genes, no recombinants would be expected. The recombinant frequency would be 0%, which represents the lower limit of recombinant frequencies. In the case of random segregation, the expected numbers of recombinant and nonrecombinant alleles are equal. Thus, the upper limit of recombinant frequencies in the case of unlinked or loosely linked genes is 50%. Even for gene pairs located at the different ends of the same chromosome, recombination frequency can reach 50%. The procedure to determine recombination frequencies between any pair of genes is called two-point analysis. Study Question 1 You have a F1 plant heterozygous at two loci that are 12 map units apart on the same chromosome. The F1 received linked recessive alleles from one parent and linked dominant alleles from the other parent. Query $4$ Study Question 2 You have a F1 plant heterozygous at two loci that are 12 map units apart on the same chromosome. The F1 received linked recessive alleles from one parent and linked dominant alleles from the other parent. Since linkage cannot be detected in the F1, you self-pollinate the F1 and evaluate the F2. What would be the F2 and testcross percentages if the F1 percentages in the case of repulsion phase of the recessive alleles? Three-Point Analysis Purpose Whereas two-point testcrosses establish linkage between pairs of genes, three-point testcrosses facilitate establishment of the order of genes on chromosomes, as a prerequisite to establishing genetic maps. If a third locus with alleles C and c (C is dominant over c) is added to the case mentioned in Genetic Distance, where A and B are linked in coupling phase and the dominant allele C is in coupling with A and B, then eight different testcross progeny would result from a backcross with the recessive parent. Class Genotype of gamete from heterozygous parent Number Origins 1 A C B 179 } Parentals, no crossover 2 a c b 173 3 A c b 52 } Recombinants, single crossover AC 4 a C B 46 5 A C b 22 } Recombinants, single crossover CB 6 a c B 22 7 A c B 4 } Recombinants, double crossover AC, CB 8 a C b 2 Frequency Chart Pairwise recombination frequencies can be determined as described in the chart. • 20.8% for AC (AC recombinants are in classes 3, 4, 7, and 8; thus, the recombination rate between A and C is (52+46+4+2/500) * 100% = 20.8%) • 10% for CB (CB recombinants are in classes 5-8) • 28.4% for AB (AB recombinants are in classes 3-6). Once linkage between pairs of three (or more) genes has been established, the next question is how they are arranged in linear order on chromosomes, which could be ABC, ACB, or CAB. Class Genotype of gamete from heterozygous parent Number Origins 1 A C B 179 } Parentals, no crossover 2 a c b 173 3 A c b 52 } Recombinants, single crossover AC 4 a C B 46 5 A C b 22 } Recombinants, single crossover CB 6 a c B 22 7 A c B 4 } Recombinants, double crossover AC, CB 8 a C b 2 Gene Order The most likely gene order minimizes the sum of pairwise recombination frequencies within a three-gene interval, which would be: • 38.4 for ABC (28.4% for AB + 10% for CB) • 30.8 for ACB (20.8% for AC + 10% for CB) • 49.2 for CAB (20.8% for AC + 28.4% for AB) Thus, the most likely gene order is ACB. In other words, the interval between A and B can be subdivided into the intervals between AC and CB. Class Genotype of gamete from heterozygous parent Number Origins 1 A C B 179 } Parentals, no crossover 2 a c b 173 3 A c b 52 } Recombinants, single crossover AC 4 a C B 46 5 A C b 22 } Recombinants, single crossover CB 6 a c B 22 7 A c B 4 } Recombinants, double crossover AC, CB 8 a C b 2 Expressed yet another way: incorrectly ordered genes would increase the total map length because part of the recombination events would be counted twice. If ACB is the true order, then the genetic length of, e.g., ABC would be inflated, because recombinants for the segment BC would be counted two times: for the interval BC, in addition to the same interval within the segment A(C)B. Algorithms of mapping programs use this principle (minimizing the genetic distance) for three-point analyses. Double Crossovers The two-point recombination frequency between A and B (28.4%) differs from the sum of recombination frequencies for AC and CB (30.8%). The reason for this discrepancy is the occurrence of double crossover events. These are two crossovers in a single meiosis within an interval of interest and the second crossover reverses the effect of the first crossover, i.e., the second crossover returns the B allele to the original position before the first crossover. For this reason, by only taking recombinants between A and B into consideration, double crossovers cannot be observed. Because a double crossover exchanges chromosome segments within an interval of two genes, the linkage phase (coupling) of those two genes remains unchanged. Observing Double Crossovers Only by adding a gene like C in between A and B, it is possible to observe double crossovers. In the case of the interval between genes A and B, six double crossovers were observed. In consequence, recombination and crossover frequencies are not identical. The larger the genetic interval, the larger the discrepancy between recombination and crossover frequencies, because even-numbered crossover events within a pair of genes go undetected. By adding an additional gene in this interval, at least some double crossovers can be detected. This leads to detection of additional recombination events. For this reason, the recombination frequency between A and B in the example is increased, after adding C in between those two genes, because 6 double crossovers (= 12 additional recombination events) could be detected. Those 12 additional detectable recombination events explain for the 2.4% difference between recombination frequencies detected for the gene pair A and B with or without inclusion of C. Phase Analysis Recombinants resulting from double crossovers are always in the lowest frequency (class 7 and 8, respectively in this table). To determine which allele is in the middle, a convenient method is to find out which allele in the double crossover recombinants has changed its linkage phase with the other parental alleles (in classes 7 and 8, allele C/c has changed its linkage phase with the other alleles). Class Genotype of gamete from heterozygous parent Number Origins 1 A C B 179 } Parentals, no crossover 2 a c b 173 3 A c b 52 } Recombinants, single crossover AC 4 a C B 46 5 A C b 22 } Recombinants, single crossover CB 6 a c B 22 7 A c B 4 } Recombinants, double crossover AC, CB 8 a C b 2 Coefficient of Coincidence and Interference Crossover events in adjacent chromosome regions might affect each other, a phenomenon called interference. Most typically, a crossover event in one region tends to suppress a crossover in the adjacent regions. The extent of interference is expressed by the coefficient of coincidence, which is equal to the observed frequency of double crossovers / expected frequency of double crossovers. Class Genotype of gamete from heterozygous parent Number Origins 1 A C B 179 } Parentals, no crossover 2 a c b 173 3 A c b 52 } Recombinants, single crossover AC 4 a C B 46 5 A C b 22 } Recombinants, single crossover CB 6 a c B 22 7 A c B 4 } Recombinants, double crossover AC, CB 8 a C b 2 The expected frequency of double crossovers is the product of two single crossovers in adjacent regions assuming there is no interference. In this example, this expected frequency is 0.21 (recombination frequency for AC) * 0.10 (recombination frequency for CB) = 0.021. The observed frequency of double crossover events is 6/500 in the example, resulting in 0.012. Thus, the coefficient of coincidence in this example is 0.012/0.021 = 0.58. Interference is defined as 1 − coefficient of coincidence, which would be 0.42 in this example. A value of zero for interference would mean that a crossover in one region does not affect crossovers in the adjacent region. Interference of 1 means, that crossovers in one region suppress crossovers in the adjacent region. Negative values are possible and have been reported in some instances, which means that crossovers in one region stimulate crossovers in the adjacent region. Map Functions Measurement Units The purpose of genetic maps is to report the length of chromosome intervals, chromosomes, and whole genomes. Since recombination frequencies converge to a value of 50% as reported above, indicating the absence of linkage, recombination frequencies are not additive and, thus, not useful to describe the distance between genes that are located far apart. When recombination frequency reaches 50%, it would be impossible to tell whether the genes are located far apart on the same chromosome or on different chromosomes. Instead, estimates of the number of crossover events are used as an additive measure of genetic map distances. The unit for measuring genetic distances is Morgan (M), or usually centiMorgan (cM). In contrast to recombination frequencies, map units expressed in cM are additive. One Morgan reflects the observation of one crossover event per single meiosis. One cM is a distance between genes that produces 1% recombinants in the offspring. Typical lengths of genetic maps in maize, for example, vary between 1,600 to 2,000 cM, which means that on average, 1.6 – 2 crossovers occur per chromosome and single meiosis in maize (maize has 10 homologous chromosome pairs). Frequency Conversion As mentioned above, direct observation of crossover events is cumbersome. For that reason, most genetic maps published to date are based on the conversion of recombination frequencies into crossover frequencies. The main obstacle to translating recombination frequencies into crossover frequencies is the variable and unknown degree of interference in different genome regions. While it has been possible in the earlier example to determine the degree of interference, and thus frequency, of double crossover events, in the genetic interval between A and B by adding C, the degree of interference between AC and CB is unknown. This could be addressed by observing segregation of further genes within these two regions (if available), but this issue could ultimately only be addressed by complete genome sequencing of all offspring in a mapping population, which at this point is still too costly. Visual Relationship Instead, map functions have been developed, that translate recombination frequencies into crossover frequencies, and thus cM (see Figure 14 below). Figure 14 clearly shows, that there is an approximately linear relationship between recombination rates (y-axis) and crossover rates (x-axis). However, with increasing map distances, recombination rates converge to 50%. In other words, gene pairs with crossover rates of 80 cM or 200 cM, respectively, would be nearly indistinguishable based on recombination rates, which would result in recombination rates between 40 and 50%. The various available map functions make different assumptions on the extent of interference. For example, the Haldane mapping function assumes absence of interference. In contrast, the Kosambi function assumes the presence of interference. Other Types of Maps Genetic maps can be generated in other ways than using testcrosses. Examples include somatic cell hybridization and tetrad analysis. In plants, interspecies addition lines such as oat-maize addition lines created by distant hybridization have been developed as tool for mapping of genes. If two genes appear on the same addition segment, they are genetically linked. Besides genetic maps, cytological and physical maps can be established. Cytological maps show gene orders along each chromosome as determined by cytological methods whereas physical maps are measured in base pairs as determined by DNA sequencing. With rapid progress in sequencing technology and an increasing number of sequenced plant genomes, physical maps gain in importance. Complex plant genomes like the maize genome are billions of base pairs long. When comparing genetic and physical maps, the order of genes is conserved. However, the relative distances between genetic and physical maps might vary substantially. The reason is that crossover events are not evenly distributed in genomes. Usually, crossover events tend to be suppressed in centromere and repetitive DNA regions, whereas they are enhanced in gene-rich regions. Factors Influencing Linkage Mapping Linkage mapping based on testcrosses can be affected by selection or incomplete penetrance, among others. Selection in the most extreme case would be due to lethality of gametes (gametic selection) or zygotes (zygotic selection). If a backcross is used for linkage detection, as described above, lethality of male gametes carrying for example the a allele would lead to only two classes of BC progeny, if AaBb is crossed as pollinator to aabb. In that case, only AaBb (parental) and Aabb (recombinant) genotypes would be obtained. Zygotic selection affects the viability of particular genotypes. If the aa genotype in the example above is lethal, then the aa offspring derived from self-pollination of an AaBb genotype would be missing. Incomplete penetrance means that a genotype that is supposed to express, for example, red flowers, has to a certain extent white flowers. In other words, there is no 100% match between genotype and phenotype, but due to environmental factors, the phenotype might differ. As for selection, incomplete penetrance alters the frequency of expected genotypes in testcrosses, which is the basis for detection of linkage. Consequences and Applications of Linkage The main application of linkage is in genetic mapping of genes using molecular markers. Once genes have been mapped and closely-linked markers identified, those markers can be used for marker-aided selection procedures. Technological progress in DNA methods has been and still is rapid, so that thousands of markers can be produced at low cost in any species of interest. Moreover, novel genomic selection strategies addressing complex inherited traits are being developed. Linkage can in some cases be confused with pleiotropy. If a favorable character (e.g. resistance) is always inherited together with an unfavorable trait (e.g., lodging), a negative pleiotropic effect might be assumed, which might alternatively be caused by two closely linked genes. Whereas close linkage can be resolved to find favorable genotypes for both traits, this is not true for pleiotropy. Linkage reduces the possible genetic variation in small populations. With increasing numbers of generations, or population sizes, genetic variation can be increased. Similarly, inbreeding reduces the opportunity for effective recombination. Linkage Disequilibrium Genotype Distribution Although allele frequencies at individual loci are expected to be stable in the case of random mating, genotype frequencies at two or more loci jointly do not achieve this equilibrium after one generation of random mating. To illustrate this point, consider two populations, one consisting of entirely AABB genotypes and the other consisting entirely of aabb genotypes. Assumed they are mixed equally and allowed to randomly mate. The first generation would consist of the three genotypes AABB, AaBb, and aabb in the proportions 1/4 : 1/2 : 1/4. However, for two loci, each with two alleles, nine genotypes are possible. (For n alleles at each locus and k loci, there are: $(\frac{n(n+1)}{2})^k$ possible genotypes). Continued random mating would produce the missing genotypes, but they would not appear at the equilibrium frequencies immediately. Equilibrium Consider the following examples based on two alleles at each of two loci: • Alleles: A a B b • Allele frequencies: PA Pa PB Pb • Gametic Types: AB Ab aB ab • Gametic Frequencies: PAB PAb PaB Pab In linkage equilibrium, the expected gamete frequencies can be calculated from the marginal allele frequencies. For example, in equilibrium, the frequency of gamete AB (PAB) would be expected to be equal to the product of the frequencies of the A allele (PA) and the B allele (PB). This is valid under the following conditions: PA + Pa = 1; PB + Pb = 1; and PAB + PAb + PaB + Pab = 1. If, for example, the allele frequencies of PA = Pa and PB = Pb are 0.5, then the frequencies of all gametes are 0.25. A measure for Disequilibrium, D = PAB – PA*PB. D = 0 in the case of equilibrium. If D differs from 0, it reflects presence of Disequilibrium. In other words, the frequency of a gamete differs from its expected frequency based on marginal probabilities of the respective individual alleles. LD and Mapping Linkage disequilibrium is the non-random association of alleles at different loci. LD is extensively used in mapping human disease genes using natural populations (Association mapping). In plants, gene mapping has been conducted mainly by using mapping families because of the ease with which mapping families are created, but LD mapping using natural populations is increasing rapidly because such populations are large in size and have much greater allelic diversity. LD Statistic D’ |D’| = $\frac{D^2_{AB}}{min (p_Ap_b,p_Ap_b)}$ for DAB < 0 |D’| = $\frac{D^2_{AB}}{min (p_Ap_b,p_Ap_B)}$ for DAB > 0 Dissipation It can be shown that after t generations of random mating, the remaining disequilibrium is given by: $D_t = D_0 (1 - c)^t$ where, D0 is the disequilibrium in generation 0 and c is the recombination fraction, with c = 0.5 for independently segregating loci, which is identical to a recombination frequency of 50% (the range of c is from 0 to 0.5, whereas the range of r is from 0% to 50%). The dissipation of disequilibrium relative to generation 0 is given in Figure 18. Recombination and LD Generally, deviations from independence at multiple loci are referred to as linkage disequilibrium, even if genetic linkage is not the cause (in other words, alleles are not physically linked). Unless two loci are known to reside on the same chromosome, the term Gametic Disequilibrium should be used to describe disequilibrium among loci. Whereas recombination and crossover frequencies, as mentioned initially, are used to describe the distance between genes from a chromosomal perspective, linkage disequilibrium is mostly used to describe a property of populations. However, both terms are closely related. Genetic Markers Overview Genetic variation results from differences in DNA sequences and, within a population, occurs when there is more than one allele present at a given locus. Such populations are referred to as populations that are polymorphic or segregating at that locus. The opposite situation is when all members of the population are homozygous for the same allele, in which case the population is said to be fixed or monomorphic for that allele. A genetic marker is a DNA sequence that exhibits polymorphism among individuals and can thus be used to identify a particular locus (although not necessarily a gene) on a particular chromosome; the marker itself may be part of a gene or may have no known function. Markers are inherited in a Mendelian fashion and facilitate the study of inheritance of a trait or sometimes a linked gene. Markers are used to identify, map, and isolate genes, select desired genotypes, and detect genetic variation or determine genetic relationships among individuals. Markers are regions of genomes that are heritable, often easy to document, and useful for detecting genetic variation. Three Types Genetic markers generally do not represent target genes of interest to a breeding program, but instead are useful as ‘signs’ or ‘tags’, particularly when they are closely linked to genes that control a trait of interest. A genetic map constructed with genetic markers is similar to a road map. Linkage groups in a genetic map represent roads whereas individual markers on each linkage group represent signs or landmarks that help plant breeders to navigate through the plant genome and find the genes of interest. There are three major categories of markers. • Morphological markers • Biochemical markers • Molecular markers Morphological Markers These types of markers (also called visible or classical markers) are phenotypic traits with only a few distinct morphs or variants (e.g., flower color or seed shape), usually due to one or perhaps two gene loci so they are not strongly affected by the environment. Inheritance patterns of visible and morphological characters have been used to map genes to particular chromosome segments and to identify linkage groups. Such markers are limited in number compared to the abundance of DNA markers, however, and may be influenced by developmental stage of the plant. Biological Markers Isozymes (sometimes called allozymes) are allelic variants of a single enzyme that share the same function, but may differ in level of activity due to differences in amino acid sequence. Isozymes are proteins for which variation can be detected by differential separation using electrophoresis, a technique for separating macromolecules (DNA, RNA, protein) on a gel by means of an electric field and specific chemical staining. Isozymes have codominant expression, meaning that both homozygotes can be distinguished from the heterozygote and neither allele is recessive. In contrast to codominant markers, dominant markers are either present or absent. In comparison to visible polymorphisms, they reveal more of the underlying genetic variation. However isozymes are gene products, so they reveal only a small subset of the actual variation in DNA sequences between individuals and do not reveal variation in the non-coding regions of the genome. In general, such markers are limited in number and have limited use in genetic mapping studies. Molecular Markers Molecular or DNA markers reveal sites of variation in DNA. Variability in DNA facilitates finer scale mapping and detection. Mapping is the process of making a representative diagram cataloging genes and other features of a chromosome and showing their relative positions. Many of these molecular markers avoid the limitations associated with visible and biochemical markers. They facilitate the evaluation of genome-wide coverage and are not affected by environmental factors or developmental stages. They allow high resolution of genetic diversity to be detected. Molecular markers have added substantial amounts of information to our genetic maps. FYI: Molecular Markers Any DNA sequence can be genetically mapped, like genes leading to plant phenotypes as long as there is a polymorphism available for the sequence to be mapped, i.e., two or more different alleles. This can basically be a single nucleotide polymorphism (SNP), a single nucleotide variant at a particular position within the target sequence, or an insertion/deletion (INDEL) polymorphism. Any target sequence can be amplified by the Polymerase chain reaction (PCR), and subsequently be visualized to generate “molecular phenotypes” comparable to visual phenotypes, that can be observed by using appropriate equipment. Various molecular methods have been developed to visualize SNPs or INDEL polymorphisms at low cost and high throughput, which will be presented in detail in the Molecular Genetics and Biotechnology course. The main use of those SNPs and INDEL polymorphisms is as molecular markers. By genetic mapping as described above, linkage between genes affecting agronomic traits or morphological characters, and DNA-based SNP or INDEL markers can be established. It can be more effective in the context of plant breeding, to select indirectly for such DNA markers, than directly for target genes. This is due to lower costs for DNA analyses, the ability to run multiple such assays (for multiple target genes) in parallel, the ability to select early and to discard undesirable genotypes or to perform selection before flowering, codominant inheritance of markers, among others.For example, both ginkgo trees (Ginkgo biloba) and asparagus (Asparagus officinalis) are dioecious species. Male plants are preferred for ginkgo tree because fruits produced from female trees have an unpleasant smell whereas male asparagus plants are preferred because of their higher yield potential. Unfortunately, sex expression will take years to occur for both species. If a DNA marker that either directly affects sex expression or is linked to genes that affect sex expression can be identified, selection of male plants can be conducted in early seedling stage rather than waiting for many years. Occurrence of environmental conditions favoring selection for disease, insect-resistant plants or drought-tolerant plants such as the prevalence of the particular disease or insect or drought is not always reliable. Selection using DNA markers can overcome these limitations as they are not affected by the environment. Polymorphism Polymorphism involves one of two or more variants of a particular DNA sequence. The most common type of polymorphism involves variation at a single base pair, also called single nucleotide polymorphism (SNP) (Figure 22). Polymorphisms can also be much larger in size and involve long stretches of DNA. Tandem repeat is a sequence of two or more DNA base pairs that is repeated in such a way that the repeats are generally associated with non-coding DNA. In contrast, SNPs can sometimes be identified that occur within coding sequences (that is within genes), as well as in non-coding DNA. Types of Biochemical/Molecular Markers There are a variety of biochemical and molecular markers available. Table 2 on the next page summarizes features of a number of the common ones: • RFLP — Restriction Fragment Length Polymorphisms • RAPD — Random Amplified Polymorphic DNA • AFLP — Amplified Fragment Length Polymorphisms • SSR — Simple Sequence Repeats (also known as microsatellites) • SNP — Single Nucleotide Polymorphisms • VNTR — Variable Number of Tandem Repeats Widely-Used Markers Table 2. Comparison among widely used molecular markers. Adapted from Nageswara-Rao and Soneji, 2008. Isozymes RFLP RAPD AFLP SSR SNP Protein- or DNA-based Protein-based DNA-based DNA-based DNA-based DNA-based DNA-based No. of loci 30-50 100s ~Unlimited ~Unlimited 10s 10s Degree of polymorphism Low-medium Meduim-high Medium-high Medium-high High High Nature of gene action Codominant Codominant Dominant Dominant Codominant Codominant Reproducibility High High Low-medium Medium-high High High Amount of DNA per sample Not applicable mg ng ng ng ng Method* Biochemical DNA-DNA hybridization PCR PCR PCR PCR Ease of array? Easy Difficult Easy Moderate Easy-moderate Easy Can be automated? Difficult Difficult Yes Yes Yes Yes Equipment cost Inexpensive Expensive Moderate Expensive Expensive Expensive Development cost Inexpensive Expensive Moderate Expensive Very Expensive Assay cost Inexpensive Expensive Moderate Expensive expensive Expensive * ‘PCR’ means Polymerase Chain Reaction amplification of genomic DNA fragments, a method that uses short, single-stranded DNA sequences, known as primers, to hybridize with the sample DNA Table 2. Comparison among widely used molecular markers. Adapted from Nageswara-Rao and Soneji, 2008. SSR and SNP Markers SSR markers remain useful to plant breeders due to their abundance and convenience with which they are assessed, but they serve most likely as linked markers. SNP markers, however can either be linked to or directly reside in a gene of interest and are hugely abundant. For these reasons, they are increasingly becoming the marker of choice. Uses of Molecular Markers Molecular markers are useful for both applied and basic genetic research. Here are some examples: Indirect selection criteria in breeding programs (marker-assisted selection) This is one of the most important and widely used molecular techniques in applied plant breeding programs today. RFLPs, SSRs, and SNPs enable breeders to indirectly select for a desired trait. Ordinarily, the DNA sequence of a molecular marker does not itself code the gene for the trait, but rather, its presence is correlated or linked with the gene for the particular trait. Thus, the breeder can indirectly select for the trait by directly selecting for the molecular marker—the DNA fragment and the gene encoding the trait are linked. The closer their physical proximity on a chromosome, the greater the probability that they will remain linked and not be separated through a recombination event in subsequent generations. As long as the gene for the trait and the marker remain linked, the marker is a useful selection criterion. Ultimately, however, potential lines still must be field-tested to verify the expression of the desired phenotype. Identify quantitative trait loci (QTLs) Molecular markers linked to genes contributing to the expression of polygenic or quantitative traits can be used to more efficiently identify and select individuals possessing the genes. It is more difficult to use conventional breeding approaches to identify plants that have accumulated the genes necessary to obtain the desired quantitative trait. Genetic mapping Molecular markers provide a means to map genes to more specific chromosome segments than is possible using visible markers. Determine genetic relationships The more molecular markers individuals have in common, the more closely related they are. Genetic Diversity and Conservation Genetic relationships within families, genera, species, or cultivars can be determined from molecular markers. Much information about the evolution of crops has been learned using molecular markers. The markers also enable breeders to monitor the genetic diversity among breeding lines to broaden the genetic base and reduce the risk of widespread genetic vulnerability to detrimental conditions. Molecular Marker ‘Fingerprints’ Individuals possessing more markers in common than could occur by random chance are closely related. Such molecular fingerprints have been used successfully in court to prove the misappropriation of proprietary breeding lines. Isolate genes Molecular markers are used to map candidate genes on a much finer scale and can eventually isolate candidate genes by positional cloning. Isolated genes can be used to study gene regulation or to directly improve agronomic performance by genetic transformation. Although molecular markers have many applications and provide useful tools to plant breeders, lines must still be evaluated under normal production conditions before their release.

textbooks/bio/Agriculture_and_Horticulture/Crop_Genetics_(Suza_and_Lamkey)/1.05%3A_Linkage.txt

Introduction Population genetics is a sub-discipline of genetics that characterizes the structure of breeding populations. The forces of mutation, migration, selection and genetic drift will alter the structure of populations. In this introductory module we will focus on characterizing population structure at a single locus. In more advanced modules you will learn how to characterize populations based on the multi-dimensional space determined by multiple loci throughout the genome. Learning Objectives • Understand the importance of a reference population. • Become familiar with modeling and estimation of genetic variation. • Understand the principles of allele frequency, genotype frequency, and genetic equilibrium in populations. • Be aware of the conditions required for Hardy-Weinberg Equilibrium (HWE). • Examine the forces that cause deviations from HWE. Two possible challenges are described in the following scenarios: Scenario 1—Fate of a Transgene Imagine a community of small farms in a valley located in the highlands of Central America. The farmers of this community produce grain from an open-pollinated maize variety that is adapted to their preferred cultural practices. They also select partial ears from about 5% of their better performing plants to be used for seed in their next growing season. One day, a truck filled with seed of a transgenic insect-resistant hybrid overturns on the highway while passing through the valley. 99.999% of the seed is recovered, but about 500 kernels remain in a farmer’s 10-acre field adjacent to the highway. The transgenic seeds germinate and grow to maturity alongside the planted open-pollinated variety. You are asked to determine the fate of an insect-resistant transgene in this valley. Scenario 2—Fixation of an Allele Imagine a naturally occurring allele at a locus that regulates the structure of carbohydrates in the wheat kernel; with the allele the carbohydrates in the kernel have low glycemic indices. For the last 100 years hard-red winter wheat varieties have not been selected for low glycemic indices, but with the emergence of a Type II diabetes epidemic, there is a demand for low glycemic carbohydrates in hard-red winter wheat varieties. How will you develop a breeding population in which this allele is fixed, that is the frequency of this allele = 1.0? Fields of Genetics These challenges are fundamentally about population genetics. In this section, you have the opportunity to successfully address these types of challenges by learning how to model and estimate allelic frequencies and the forces that affect population structures. In the study of population genetics, the focus shifts away from the individual (which is the focus for transmission genetics) and the cell (which is the focus for molecular genetics) to emphasis on a large group of individuals—a Mendelian population—that is defined as a group of interbreeding individuals who share a common set of genes. This module will include a discussion of inbreeding, which is one type of mating of individuals that is often of particular significance to plant breeders. Inbreeding is the mating of individuals that are more closely related than individuals mated at random in a population. Self-pollination (mating of an individual to itself) represents the most extreme form of inbreeding. Reference Population Goals Population genetics has three major goals, all of which are interrelated (Conner and Hartl, 2004): • Explain the origin and maintenance of genetic variation. • Describe the genetic structure of populations, i.e., the patterns and organization of genetic variation. • Recognize the mechanisms that cause changes in allele and genotypic frequencies. Similar to quantitative genetics, population genetics is concerned with application of Mendelian principles and is amenable to mathematical treatment. Understanding population genetics will require you to apply concepts from high school algebra. Description In order to understand the genetic structure of a population, it is necessary to establish a standard reference population so that the breeding population can be characterized relative to the standard. Consider an ‘ideal’ population that is infinitely large. Further consider development of sub-populations as in Figure 6, described in Falconer and Mackay (1996). Note that the sub-populations depicted in the figure above are based on a genetic sampling process that is affected by reproductive biology of the species. The reproductive mode of most plant species can be classified as sexual or asexual Species that reproduce sexually are generally categorized into three types of mating systems — primarily cross-pollinated, primarily self-pollinated, or a mixture of self- and cross-pollinated. Asexual modes of reproduction include three main categories: vegetative or clonal propagation, and apomixis. Under different mating systems (e.g., random vs. inbreeding) different genotypic frequencies will be generated from the same allele frequencies. With sexually reproducing individuals, mating combines alleles in the pool of haploid gametes produced by meiosis into genotypes in the diploid individuals. Query $1$ In the ideal model population depicted in Figure 8, we make the following assumptions: • The base population is extremely large (too large to count) • No migration between sub-populations • Non-overlapping generations • Number of breeding individuals is the same in each sub-population • Random mating within a sub-population • No selection • No mutation Models such as that shown above are theoretical abstractions. Models provide methods to simulate real-life situations and they are used for two principal reasons: 1) to reduce complexity, allowing underlying patterns to become more visible and 2) to make specific predictions to test with experiments or observations (Connor and Hartl 2004). Discussion Discuss the two challenges described earlier with respect to each reference population: For Scenario 1—Fate of a Transgene, characterize the breeding population. Assume that there are 100 10-acre farms in the Central American valley, where farmers plant about 10,000 maize kernels per acre. For Scenario 2—Fixation of an Allele, determine how many hard red winter wheat varieties exist for the Southern Great Plains region. The number can include all historical varieties grown in the region. Assume that you have identified one additional ancient accession of hard red winter wheat that has the desirable allele for low glycemic carbohydrates. Assume that these varieties represent the lines you will use for your basic breeding population. Characterize this breeding population. Allele and Genotypic Frequencies Model We first model a single locus with only two alleles (e.g., presence or absence of a transgene) in an ideal breeding population of diploid individuals. Define the following: • N = Number of breeding individuals in a sub-population (population size) • t = Time in generations with base population at t0 • q = Frequency of a particular allele at a locus within a sub-population • p = 1 – q = Frequency of other allele at a locus within a sub-population • $\bar{p}$ = Frequency in the whole population (the mean of p) • p0 = Frequency of p in the base population • q0 = Frequency of q in the base population Because of the assumptions associated with an ideal reference population, = q0 at any stage or generation of the sampling process, so q0 can be used interchangeably with . Equations The alleles, allele frequencies, genotypes and genotypic frequencies can be represented as follows: Alleles Genotypes A a AA Aa aa Frequency p q PAA PAa Paa Where, $p + q = 1$ and $P_{AA} + P_{Aa} + P_{aa} = 1$ The relationship between allele frequencies and genotype frequencies can be expressed as follows: $p = P_{AA} + \frac{1}{2}P_{Aa}$ and $q = P_{aa} + \frac{1}{2} P_{Aa}$ Hardy-Weinberg Equilibrium Concept of Genetic Equilibrium Plant breeders recombine and select the alleles present in the gene pool. The gene pool of a population is the total of all alleles within a population, and consists of all of the genes shared by individuals in the population. Gene pools are described in terms of allele and genotype frequencies. Knowing the frequency with which desired (or undesirable) alleles occur in the gene pool of the population influences the choice of breeding population(s), breeding method, and likelihood of progress. The breeding population must contain not only sufficient genetic variability to allow selection, but also have favorable alleles present in high enough frequencies to facilitate their selection and allow efficient breeding progress to occur. • Allele frequency (often also called gene frequency) — the proportion of contrasting alleles present in the gene pool of a population. • Genotype frequency — the proportion of various genotypes present in a population. Assumptions The frequencies of specific alleles and genotypes in a large, random mating population will reach equilibrium and will remain in equilibrium with continued random mating. This tendency toward equilibrium is the foundation of a model called the Hardy-Weinberg Law or Hardy-Weinberg Equilibrium (HWE). This law states that The probability of two alleles uniting in a zygote is the product of the frequency of the alleles in the population The law makes several assumptions. • There are two alleles at a gene locus. • The population is large (that is, the number of breeding individuals is in the hundreds, rather than in the tens). • The population is random-mating. Frequencies Hardy-Weinberg Equilibrium mathematically describes the relationship between allele frequency and genotype frequency. According to the Hardy-Weinberg law, if the frequencies of two contrasting alleles at a locus in the parent population are p and q, respectively, then $p + q = 1$, always; and genotype frequency in the progeny is $p^2 + 2qp + q^2 = 1$ or $p^2 + 2qp + q^2 = 1$. Study Question 1 For each of the following populations, indicate whether the Hardy-Weinberg Law would apply. Population Does the Hardy-Weinberg Law apply? Naturally self-pollinating species YES NO Naturally cross-pollinating species YES NO Limited population size YES NO Query $2$ Study Question 2 Locus Alpha has two contrasting gene forms or alleles (A and a) in a large, random-mating population. The population is at equilibrium. Study Question 2 Explanation The correct frequency of aa genotype following selection and random mating is 0.17. Selection for the A_ phenotype (or against the aa phenotype), shifts the allele and genotype frequencies. Here’s how the answer is determined: • Initial population is 0.09 AA + 0.42 Aa + 0.49 aa • Selection removes aa genotypes, so the unselected portion of the population is 0.09 AA + 0.42 Aa and the remaining individuals are all A_. • Thus, setting p equal to the frequency of the A allele, and q equal to frequency of the a allele, the resulting allelic frequencies are now $\textrm{p} = \frac{\textrm{frequency of A in the AA genotype + frequency of A in the Aa genotype}}{\textrm{total allele frequencies of A and a}}$ $p = \frac{0.09 \times 2 + 0.42 \times 1}{0.09 \times 2 + 0.42 \times 2}$ $q=1-p=0.41$ • So, the frequency of the A allele is 0.59 and the frequency of the a allele is 0.41. • Now, we can calculate the frequency of the aa genotype in the population after one generation of selection and subsequent random mating. p2(AA) + 2pq(Aa) + q2(aa) = 1 (0.59)2 + 2 · 0.59 · 0.41 + (0.41)2 = 1 0.35 AA + 0.48 Aa + 0.17 aa = 1 Thus, the correct frequency of the aa genotype is 0.17. Factors Affecting Equilibrium Several factors may disturb the genetic equilibrium of a population. • Mutation of an allele at the locus of interest. • Natural or human selection may favor one allele over the other. • Migration of alleles into or out of the population (for example, via an introduction of a different allele from another population, or loss of an allele through selection). Generally, a population not in genetic equilibrium, but retaining two contrasting alleles at a single, independently-segregating (non-linked) locus, will be restored to equilibrium at that locus after just one generation of random mating. Random-Mating Interference What is the significance of the Hardy-Weinberg Law to plant breeders? The random-mating assumption is often violated in breeding populations because breeding populations are smaller than natural plant populations. Thus, a mating design that minimizes gamete (allele) sampling errors is an important consideration. The breeder must be aware of several factors: • Self-pollinated population — allele frequency will remain in equilibrium (assuming a sufficiently large population, no selection, or other factors that disturb equilibrium). However, with each successive generation of self-pollination, the genotype frequency of homozygous loci will increase and the frequency of heterozygous loci will decrease. Ultimately, the heterozygous genotype will be eliminated from the population with continued selfing. • Cross-pollinated population — sampling errors occur if plants in the population differ in their vigor, time of flowering, or mate more frequently with plants in close proximity. • Selection for or against a particular allele will alter the allele and genotype frequencies of the population. Selection against a dominant allele (i.e., selection for homozygous recessive) will remove the dominant allele from the population in a single generation. Selection against a recessive allele will require more than a few generations to remove the recessive allele from the population because the homozygous dominant and heterozygous genotypes have indistinguishable phenotypes. Scenarios In addition to being able to estimate allele and genotype frequencies, the breeder also needs to understand the gene action affecting the character of interest. The breeding of cross-pollinated crops differs from self-pollinated species because of differences in the structures of their gene pools and opportunity for genetic recombination. Table 1 Natural genetic structure of self- vs. cross-pollinated species. Reproductive mode Individuals Population Self-pollinated Homozygous Homogeneous or heterogeneous Cross-pollinated Heterozygous Heterogeneous Homozygosity and Heterozygosity For a given locus, an individual with a genotype of either AA or aa is homozygous for that gene and is known as a homozygote; the status of the gene is referred to as homozygosity. An individual with the genotype Aa is heterozygous for that gene and is called a heterozygote; the status is known as heterozygosity. In the case of polyploid individuals, those with the genotypes AAAA (tetraploid) or aaa (triploid) would be examples of homozygotes and those with genotypes of AAaa (tetraploid) or AAaaaa (hexaploid) would be examples of heterozygotes. The terms homozygous and heterozygous are used to describe the status of single genes or all gene loci within an individual, not within a population. There may be many different alleles of a gene present in a population of individuals, but for each diploid individual, there are only two alleles per gene. For each individual, there is one allele from each parent and each allele per gene is present at corresponding loci on homologous chromosomes. With regard to populations, a homogeneous population would be one in which all individuals in the population would have the same genotype and possess the same alleles for one or more genes. In contrast, a heterogeneous population would be characterized by differing alleles at one or more loci. Note that a cross between two homozygous parents produces progeny that are homogeneous because all of the individual offspring are genetically identical. However, the offspring would be heterozygous for all loci for which different alleles occurred in the two parents. Maize, the crop found in the first challenge, Scenario 1—Fate of a Transgene, is monoecious and is cross-pollinated. Wheat, the crop found in the second challenge, Scenario 2—Fixation of an Allele, has bisexual flowers and is normally a self-pollinated crop. Mating Systems for Crop Species Table 6 Flower Type Self-Compatability or Dioecy/Monoecy Crop Examples Normally Cross-Pollinated Bisexual Flowers Self-Compatible Sugarcane, Olive, Amaranth, Avocado, Onion, Carrot, Agave, Sunflower, Kiwi, Pearl millet, Reed canarygrass, Sweet potato Bisexual Flowers Self-Incompatible Radish, Kale, Cabbage, Black mustard, Pineapple, Red Clover, White Clover, Apple, Pear, Cacao, Rye, Alfalfa, Birdsfoot trefoil, Sweet Potato, Buckwheat Unisexual Flowers Dioecious Papaya, Fig, Hops, Hemp, Grape Unisexual Flowers Monecious Mango, Cucumber, Squash, Watermelon, Yam, Rubber, Cassava, Castor bean, Maize, Banana, Coconut, Oil palm Normally Self-Pollinated Bisexual Flowers Self-Compatible Barley, Oats, Rice, Triticale, Wheat, Lettuce, Cowpea, Dry bean, Lentil, Chickpea, Peanut, Pea, Soybean, Sesame, Tomato, Tobacco, Coffee, Eggplant, Safflower, Flax, Peach Predominantly Self-Pollinated, but also Cross-Pollinated to fairly high extent Bisexual Flowers Self-Compatible Cotton, Sorghum, Rapeseed, Brown mustard Let’s examine the genetic structure of populations of self- and cross-pollinated species. Scenario 1—Fate of a Transgene Imagine a community of small farms in a valley located in the highlands of Central America. The farmers of this community produce grain from an open-pollinated maize variety that is adapted to their preferred cultural practices. They also select partial ears from about 5% of their better performing plants to be used for seed in their next growing season. One day a truck filled with seed of a transgenic hybrid overturns on the highway while passing through the valley. 99.999% of the seed is recovered, but about 500 kernels remain in a farmer’s 10-acre field adjacent to the highway. The transgenic seeds germinate and grow to maturity alongside the planted open-pollinated variety. You are asked to determine the fate of an insect-resistant transgene in this valley. Scenario 2—Fixation of an Allele Imagine a naturally occurring allele at a locus that regulates the structure of carbohydrates in the wheat kernel; with the allele, the carbohydrates in the kernel have low glycemic indices. For the last 100 years, hard-red winter wheat varieties have not been selected for low glycemic indices, but with the emergence of a Type II diabetes epidemic, there is a demand for low glycemic carbohydrates in hard-red winter wheat varieties. How will you develop a breeding population in which this allele is fixed, that is the frequency of this allele = 1.0? Genetics of Cross-Pollinated Species Because cross-pollinated species have evolved to outcross, individuals tend to be heterozygous at many loci and they usually perform best when that heterozygosity is maintained. This is a characteristic referred to as heterosis or hybrid vigor. When repeated self-pollination occurs in cross-pollinated species, homozygosity increases and plant vigor is reduced, a phenomenon called inbreeding depression. Heterosis and inbreeding depression will be further discussed in Lesson 6. Several morphological and physiological features of cross-pollinated species promote cross-pollination. Let’s briefly review these. • Monoecy — pistillate and staminate flowers occur on different sections of the same plant. • Dioecy — pistillate and staminate flowers occur on different plants. • Protandryorprotogyny — pistillate and staminate flowers mature at different times. • Self-incompatibility — pollen from the same plant cannot effect fertilization or seed set. • Male or female sterility — pollen or ovule does not function normally. Genetics of Self-Pollinated Species Self-pollinated species rarely hybridize naturally. Although cross-pollinating may occasionally occur, ovules of a self-pollinated plant are normally fertilized by pollen produced on that same plant. The result of repeated generations of selfing is that homozygosity is increased or maintained. • Homozygous loci will remain homozygous. • Heterozygous loci will segregate such that the frequency of homozygotes will increase at the expense of the frequency of heterozygotes with each generation of selfing. Frequency of Homozygotes With continued self-pollination, the heterozygotes will segregate, decreasing the proportion of heterozygotes in the population by half each generation. Notice that the homozygotes can only produce homozygotes. Table 5 Change in percent heterozygosity in each successive generation. Generation Heterozygosity (%) F1 100.0 F2 50.0 F3 25.0 F4 12.5 F5 6.25 F6 3.12 For each successive generation of offspring resulting from one F1 individual, by the F8 generation, the population is essentially homozygous. When no further segregation for the trait occurs, all progeny derived from that F1 will “breed true” because they are homozygous for the trait. The proportion of plants that are expected to be heterozygous at any gene when starting with a heterozygous F1 and selfing can be determined by using the formula (½)n, where n = the number of segregating generations, e.g., in F2 n = 1 and in F5 n = 4. Proportion of homozygous plants in any generation is then given by 1 − (½)n which when algebraically converted is equal to: $\frac{2^n - 1} {2^n}$ How does a locus become heterozygous? A contrasting allele can be acquired when a plant out-crosses or when a mutation occurs. Each successive self-pollination thereafter will reduce heterozygosity by half. Breeders rely on the natural tendency of self-pollinated crops to become homozygous to obtain lines that exhibit uniformity in characters that affect appearance and performance. Notice how rapidly populations lose heterozygosity with selfing. For self-pollinated crops, one of the breeder’s objectives is usually to develop pure lines. Since pure lines are homozygous, their rapid loss of heterozygosity speeds cultivar development. Some background heterozygosity may remain in a pure line, but the line is sufficiently homozygous to provide the uniformity in characters required for reliable and predictable appearance and performance. Allelic Effects The tendency of a species to self-pollinate or outcross influences allelic and genotypic frequencies in the population. In a self-pollinated homozygous population, the effect of a gene (allele) is determined by the gene’s effect in combination with itself and with alleles at other loci. What determines the effect of a gene in a cross-pollinated population? Effect or fate of an allele in a cross-pollinated population is determined by its effect • in combination with other alleles at the same locus • additive effects • dominance effects • overdominance effects • in combination with alleles at other independent loci (epistatic effects) • in combination with alleles at closely linked loci One difference between a self-pollinated and a cross-pollinated population is that in the cross-pollinated population there is constant inter-crossing. Thus, recombination and rearrangement of alleles and expression of dominance and epistatic effects occur. Review gene action or gene interactions, such as epistasis in the next screens. Gene Action There are several general types of gene action. The type of gene action and the alleles present for a given gene affect the phenotype. Let’s consider the gene action as indicated by the phenotype of a diploid individual heterozygous at the given single locus compared to the phenotype of its parents. Gene Interactions When multiple genes control a particular trait or set of traits, gene interactions can occur. Generally, such interactions are detected when genetic ratios deviate from common phenotypic or genotypic proportions. • Pleiotropy — Genes that affect the expression of more than one character. • Epistasis — Genes at different loci interact, affecting the same phenotypic trait. Epistasis occurs whenever two or more loci interact to create new phenotypes. Epistasis also occurs whenever an allele at one locus either masks the effects of alleles at one or more other loci or if an allele at one locus modifies the effects of alleles at one or more other loci. There are numerous types of epistatic interactions. Epistasis is expressed at the phenotypic level. It is important to note that genes that are involved in an epistatic interaction may still exhibit independent assortment at the genotypic level. In the case of two completely dominant, non-interacting (i.e., no linkage) genes, all of the deviations observed in results involving epistatic interactions are modifications of the expected 9:3:3:1 ratio. Study Question 3 Describe a natural cross-pollinated population as to its heterozygosity, heterogeneity, and effect of inbreeding. For each of the following, select the best terms to complete the statement. Proof The proof of Hardy-Weinberg Equilibrium (HWE) requires the following assumptions (Falconer and Mackay, 1996): 1. Allele frequency in the parents is equal to the allele frequency in the gametes 1. Assumes normal gene segregation 2. Assumes equal fertility of parents 2. Allele frequency in gametes is equal to the allele frequency in gametes forming zygotes 1. Assumes equal fertilizing capacity of gametes 2. Assumes large population 3. Allele frequency in gametes forming zygotes is equal to allele frequencies in zygotes 4. Genotype frequency in zygotes is equal to genotype frequency in progeny 1. Assumes random mating 2. Assumes equal gene frequencies in male and female parents 5. Genotype frequencies in progeny do not alter gene (allele) frequencies in progeny. 1. Assumes equal viability For a two allele locus in a population in HWE: $P_{AA}= p^2$; $P_{Aa} = 2_{pq}$; $P_{aa} = q^2$ Proof HWE at a given genetic locus is achieved in one generation of random mating. Genotype frequencies in the progeny depend only on the gene (allele) frequencies in the parents and not on the genotype frequencies of the parents. If a population is in HWE, relationships between frequencies of alleles and genotypes may be derived as depicted in figure 13. As shown in figure 13, in HWE: • frequency of heterozygotes does not exceed 0.5 • heterozygotes are most frequent genotype when p or q are between 0.33 and 0.66 • very low allele frequency should result in very low frequency of homozygotes for that allele • if there are only two alleles at a locus in the population, p+q=1. A chi-square test is typically used to determine whether or not a population varies significantly from Hardy-Weinberg expectations. The Hardy-Weinberg formula is useful in describing situations where mating is completely randomized. But more commonly, mating is not at random and populations are subjected to other forces, such as mutation, migration, genetic drift, and selection. Linkage can also have a significant effect on gene frequencies. Forces Affecting Population Structures Descriptions Non-Random Mating Two methods of non-random mating that are important in plant breeding are assortative mating and disassortative mating. Assortative mating occurs when similar phenotypes mate more frequently than they would by chance. One example would be the tendency to mate early x early-maturing plants and late x late maturing plants. The effect of assortative mating is to increase the frequency of homozygotes and decrease the frequency of heterozygotes in a population relative to what would be expected in a randomly mating population. Assortative mating effectively divides the population into two or more groups where matings are more frequent within groups than between groups. Disassortative mating occurs when unlike or dissimilar phenotypes mate more frequently than would be expected under random mating. Its consequences are in general opposite those of assortative mating in that disassortative mating leads to an excess of heterozygotes and a deficiency of homozygotes relative to random mating. Disassortative mating can also lead to the maintenance of rare alleles in a population. For example, in self-incompatible species, an individual will only mate with another individual that differs in the self-incompatibility loci. This is a type of disassortative mating, resulting in a great alleleic diversity in the self-incompatibility loci. It is an effective mechanism to maintain heterozygosity and prevent inbreeding. Study Question 4 Scenario 2—Fixation of an Allele Forces Affecting Allele Frequency Factor Categories The factors affecting changes in allele frequency can be divided into two categories: systematic processes, which are predictable in both magnitude and direction, and dispersive processes, which are predictable in magnitude but not direction. The three systematic processes are migration, mutation, and selection. Dispersive processes are a result of sampling in small populations. Table 2 Systematic Processes Dispersive Processes Migration Small Population Size Mutation Selection Migration Clearly, the first challenge described in the introduction represents a case of migration. A new set of genes in a developed transgenic hybrid have been introduced into an open pollinated variety of maize. When discussing population genetics, migration is also sometimes referred to as gene flow, a concept that is often used interchangeably with migration by population geneticists. However, the term migration means the movement of individuals between populations, whereas gene flow is the movement of genes between populations. New genes would be established in the population if the immigrant successfully reproduces in its new environment, but if it doesn’t reproduce migration would still have occurred while gene flow would not. Assume a population has a frequency of m new immigrants each generation, with 1− m being the frequency of natives. Let qm be the frequency of a gene in the immigrant population and q0 the frequency of that gene in the native population. Then the frequency in the mixed population will be: $q_1 = mq_m +(1-m)q_0$ $q_1 = m(q_m - q_0) + q_0$ The change in gene frequency brought about by migration is the difference between the allele frequency before and after migration $\Delta q = q_1 - q_0$ $\Delta q = m (q_m - q_0)$ Thus the change in gene frequency from migration is dependent on the rate of migration and the difference in allele frequency between the native and immigrant population. Migration or gene flow can introduce new alleles into a population at a rate and at more loci than expected from mutation. It can also alter allele frequencies if the populations involved have the same alleles but not in the same proportions. Thus the effect of migration on changes in allele frequency depends on differences in allele frequencies (migrants vs. residents) and the proportion of migrants in the population. Study Question 5 Scenario 1—Fate of a Transgene Mutation Mutations are the source of all genetic variation. Loci with only one allelic variant in a breeding population have no effect on phenotypic variability. While all allelic variants originated from a mutational event, we tend to group mutational events in two classes: rare mutations and recurrent mutations where the mutation occurs repeatedly. Rare Mutations By definition, a rare mutation only occurs very infrequently in a population. Therefore, the mutant allele is carried only in a heterozygous condition and since mutations are usually recessive, will not have an observable phenotype. Rare mutations will usually be lost, although theory indicates rare mutations can increase in frequency if they have a selective advantage. Fate of a Single Mutation Consider a population of only AA individuals. Suppose that one A allele in the population mutates to a. Then there would only be one Aa individual in a population of AA individuals. So the Aa individual must mate with a AA individual. AA x Aa → 1AA:1Aa From Li (1976; pp 388), this mating has the following outcomes: 1. No offspring are produced in which case the mutation is lost. 2. One offspring is produced: the probability of that offspring being AA is 1/2 so the probability of losing the mutation is 1/2. 3. Two offspring are produced: the probability of them both being AA is 1/4 so the probability of losing the mutation is 1/4. If k is the number of offspring from the above mating then the probability of losing the mutation among the first generation of progeny is (1/2)k. The probability of losing the gene in the second generation can be calculated by making the following assumptions: • Number of offspring per mating is distributed as a Poisson process (which means that they follow a stochastic distribution in which events occur continuously and independently of one another). • With the average number of offspring per mating = 2. • New mutations are selectively neutral. With these assumptions, the probabilities of extinction are: Table 3 Probability of extinction in different generations. Generation Probability of Loss 1 0.37 7 0.79 15 0.89 31 0.94 63 0.97 127 0.98 Recurrent Mutations Let the mutation frequencies be: Mutation rate: $A\xrightarrow{u}a$ Frequency: $p_0\overleftarrow{v}q_0$ Then the change in gene frequency in one generation is: $\Delta q_0 = up_0 - vq_0$ at equilibrium $p_0u = q_0v$ $q_0 = \frac{u}{v + u}$ Conclusions: • Mutations alone produce very slow changes in allele frequency • Since reverse mutations are generally rare, the general absence of mutations in a population is due to selection Selection Selection is one of the primary forces that will alter allele frequencies in populations. Selection is essentially the differential reproduction of genotypes. In population genetics, this concept is referred to as fitness and is measured by the reproductive contribution of an individual (or genotype) to the next generation. Individuals that have more progeny are more fit than those who have less progeny because they contribute more of their genes to the population. The change in allele frequency following selection is more complicated than for mutation and migration, because selection is based on phenotype. Thus, calculating the change in allele frequency from selection requires knowledge of genotypes and the degree of dominance with respect to fitness. Selection affects only the gene loci that affect the phenotype under selection—rather than all loci in the entire genome—but it also would affect any genes that are linked to the genes under selection. Effects of Selection Change in allele frequency The strength of selection is expressed as a coefficient of selection, s, which is the proportionate reduction in gametic output of a genotype compared to a standard genotype, usually the most favored. Fitness (relative fitness) is the proportionate contribution of offspring to the next generation. Partial selection against a completely recessive allele To see how the change in allele frequency following selection is calculated consider the case of selection against a recessive allele: Table 4 Genotypes AA Aa aa Total Initial Frequencies p2 2pq q2 1 Coefficient of Selection 0 0 s Fitness 1 1 1-s Gametic Contribution p2 2pq q2(1-s) 1 – sq2 Frequency Equations The frequency of allele a after selection is: $q_1 = \frac{q-sq^2}{1-sq^2}$ $q_1 = \frac{q-sq^2}{1-sq^2}$ The change in allele frequency is then: $\Delta q = q_1 - q$ $\Delta q = \frac{q-sq^2}{1-sq^2} -q$ In general, you can show that the number of generations, t, required to reduce a recessive from a frequency of q0 to a frequency of qt, assuming complete elimination of the recessive (s = 1) is: $t = \frac{1}{q_t} - \frac{1}{q_0}$ Discussion Review the two challenges at the beginning of the lesson and then answer these questions: • Allele Frequency—For Scenario 1, calculate the frequency of the insect-resistant transgene in the Central American maize farmer’s 10-acre field assuming that it is a) hemizygous and b) homozygous in the spilled hybrid seed. Remember that hemizygous means that the individual has only one single homologous chromosome, and therefore is neither homozygous nor heterozygous; in contrast homozygous means that there are two homologues. • Allele Frequency—For Scenario 2, calculate the frequency of the allele responsible for low glycemic carbohydrates in the wheat breeding population, assuming the allele is not present in any wheat variety except one. • Mutation—For Scenario 2, assume the mutation that produced the low glycemic allele was selectively neutral in the hard red-winter wheat breeding population. Why was that allele lost from all varieties that were developed over the last 100 years? • Selection—In Scenario 1 a transgene (and likely other genes) is introduced into an open-pollinated variety in one farmer’s field. Determine Δq for the transgenic allele assuming that the allele is homozygous in the hybrid seed, the insect-resistant allele is completely dominant and the selective advantage of the allele is a) two to one (2:1) when the insect is present and b) one to one (1:1) when it is absent. Scenario 1: Fate of a Transgene Imagine a community of small farms in a valley located in the highlands of Central America. The farmers of this community produce grain from an open-pollinated maize variety that is adapted to their preferred cultural practices. They also select partial ears from about 5% of their better performing plants to be used for seed in their next growing season. One day a truck filled with seed of a transgenic hybrid overturns on the highway while passing through the valley. 99.999% of the seed is recovered, but about 500 kernels remain in a farmer’s 10-acre field adjacent to the highway. The transgenic seeds germinate and grow to maturity alongside the planted open pollinated variety. You are asked to determine the fate of an insect-resistant transgene in this valley. Scenario 2: Fixation of an Allele Imagine a naturally occurring allele at a locus that regulates the structure of carbohydrates in the wheat kernel; with the allele the carbohydrates in the kernel have low glycemic indices. For the last 100 years hard-red winter wheat varieties have not been selected for low glycemic indices, but with the emergence of a Type II diabetes epidemic, there is a demand for low glycemic carbohydrates in hard-red winter wheat varieties. How will you develop a breeding population in which this allele is fixed, that is the frequency of this allele = 1.0? Small Population Size Unlike the three systematic forces that are predictable in both amount and direction, changes due to small population size are predictable only in amount and are random in direction. The effects of small population size can be understood from two different perspectives. It can be considered a sampling process and it can be considered from the point of view of inbreeding. The inbreeding perspective is more interesting, but looking at it from a sampling perspective lets us understand how the process works. A particular sub-population is a random sample of N individuals or 2N gametes (for a diploid) from the base population. Therefore, the expected gene frequency of a particular allele in the sub-populations is q0 and the variance of q is $\sigma^2_q = \frac{p_0q_0}{2N}$ Since q0 is a constant, the variance of the change in allele frequency (q1 − q0) is also: $\sigma^2_{\Delta q} = \frac{p_0q_0}{2N}$ Examples Example 1: Let q = 0.5 and N = 50, then $\sigma_{q}^{2}= \frac{(0.5)(0.5)}{100}=0.0025$ Example 2: Let q = 0.5 and N = 4, then $\sigma_{q}^{2}= \frac{(0.5)(0.5)}{8}=0.03125$ Consequences of small population size 1. Random genetic drift: random changes in allele frequency within a subpopulation 2. Differentiation between subpopulations 3. Uniformity within subpopulations 4. Increased homozygosity Random Genetic Drift Small Population Size Random genetic drift refers to allelic frequencies that change through time (generations) due to errors and other random factors (i.e., not selection or mutation). When sample sizes are small, all genotypes may not be produced and then mate at expected frequency. The effective population size (Ne) of a population is a term used to describe the number of parents that actually contribute gametes to the next generation; not all individuals may contribute equally, thus resulting in genetic drift. Small populations are susceptible to genetic bottlenecks, which are sudden decreases in breeding population due to deaths, migration, or other factors. Small populations can be subject to so-called founder effects, which occur when a breeding population is small when initially founded, then increases in size but the gene pool is largely determined by the genes present in the original founders. Rate of Change The rate of change due to random genetic drift depends on population size and allele frequency. As illustrated in the figure below, the more frequent the allele, the higher chances of being fixed and the smaller the population, the faster it will either move towards fixation or loss. In the absence of other forces: • genetic drift leads to loss or fixation of alleles • frequency of rare alleles would be expected to go to zero • lower frequency of heterozygotes in later generations • less genetic variation within subpopulations • more genetic variation among subpopulations Inbreeding and Small Populations Inbreeding and Small Populations Inbreeding is the mating together of individuals that are related by ancestry. The degree of relationship among individuals in a population is determined by the size of the population. This can be seen by examining the number of ancestors that a single individual has: Just 50 generations ago note that a single individual would have more ancestors than the number of people that have existed or could exist on earth. Therefore, in small populations individuals are necessarily related to one another. Pairs mating at random in a small population are more closely related than pairs mating together in a large population. Small population size has the effect of forcing relatives to mate even under random mating, thus with small population sizes inbreeding is inevitable. Generation Ancestors 0 1 1 2 2 4 3 8 4 16 5 32 6 64 10 1,024 50 1,125,899,906,842,620 100 1,267,650,600,228,230,000,000,000,000,000 t 2t Identical Types In finite populations there are two sorts of homozygotes: Those that arose as a consequence of the replication of a single ancestral gene — these genes are said to be identical by descent (Bernardo, 1996). If the two genes have the same function, but did not arise from replication of a single ancestral gene, they are said to be alike in state. It is the production of homozygotes that are identical by descent that gives rise to inbreeding in a small population. Study Question 6 Scenario 2—Fixation of an Allele Summary of Factors Hardy and Weinberg discovered mathematically that genotype frequencies will reach an equilibrium in one generation of random mating in the absence of any other evolutionary force. If the conditions of equilibrium are met, the frequencies of different genotypes in the progeny will depend only upon the allele frequencies of the previous generation. If allele frequencies do not accurately predict genotype frequencies, then plants are mating in a non-random way or another evolutionary force is operating. Effect of level of variation Within subpopulations Among subpopulations Affect all loci equally? Mutation Increase Increase No Migration (Gene flow) Increase Decrease Yes Random Genetic Drift Decrease Increase Yes Selection Increase or decrease Increase or decrease Yes Within subpopulations the degree of genetic variation can be assessed by heterozygosity, while variation among subpopulations is measured by population differentiation. Mutation is the ultimate source of all genetic variation and it tends to increase variation both within and among subpopulations. But because most mutations are rare, the effect of mutation is slow relative to the change the other forces can effect. Migration or gene flow and random genetic drift are opposite in their effects: migration tends to increase variation within subpopulations but decrease it among subpopulations, and random drift does the opposite. In contrast, the effects of selection vary both within and between populations. For example, variation can decrease if one homozygote is favored, or may increase or be maintained if heterozygosity is advantageous. Selection acts on the phenotype so it will affect only those genes that control the trait under selection, as well as genes linked to those loci.

textbooks/bio/Agriculture_and_Horticulture/Crop_Genetics_(Suza_and_Lamkey)/1.06%3A_Population_Genetics.txt

Introduction This module focuses on inbreeding, a type of mating of individuals that is often of particular significance to plant breeders. Inbreeding is defined as the mating of individuals that are related by ancestry. Self-pollination (mating of an individual to itself) represents the most extreme form of inbreeding. Inbreeding leads to an increase in homozygosity at the expense of heterozygosity. A key feature of inbreeding is that as homozygosity increases in a population undergoing inbreeding in the absence of selection, the genotype frequency changes while the allele frequency stays unchanged. Inbreeding may occur unintentionally as a result of selection or maintenance of small populations. Inbreeding is also deliberately practiced as a method to create genetic uniformity in populations of interest for genetic or breeding research, for retaining genotypes of inbred cultivars of self-pollinated species through many years of production, or for reliable production of inbred lines to be used in the development of commercial hybrid cultivars. Inbreeding Depression A phenomenon known as inbreeding depression—the reduced survival and fertility of offspring of related individuals—occurs in both plants and animals, showing that variation for heritable fitness traits occurs within populations. The occurrence of inbreeding depression varies across species. Charles Darwin, British naturalist famous for his theories of evolution and natural selection was the first person to make a distinction between plants that are outbreeders (species with reproductive mechanisms promoting cross-pollination, who typically exhibit inbreeding depression and tend to be intolerant of inbreeding, e.g., maize and alfalfa) and inbreeders (species with reproductive mechanisms promoting self-pollination in which inbreeding depression is minimal, who tolerate many generations of inbreeding, e.g., wheat and oat). Darwin noticed that: • outcrossing is more common in nature than self-fertilization • there are complex reproductive systems that promote outcrossing in plants • many plant species have evolved systems that prevent self-fertilization. This module also describes hybrid vigor or heterosis, which is a phenomenon that is functionally the opposite of inbreeding depression. Heterosis is defined as the increased vigor of F1 progeny resulting from the mating of inbred parents. Generally, the performance of the F1 hybrid exceeds the performance of its inbred parents for various traits. The expression of hybrid vigor might affect phenotypes under single gene or polygenic control, e.g., size, growth rate, fertility, and yield. The first-generation offspring of crosses from mating between different pure-line inbreds generally show in a greater amount of desired traits of both parents, but hybrid vigor decreases in F2 and subsequent selfing generations due to inbreeding. The exploitation of hybrid vigor or heterosis is a key feature in the success of hybrid cultivars. Cross-Pollination Several mechanisms promote cross-pollination. • Emergence or maturity of the staminate and pistillate flowers is asynchronous. • Protandry – anthesis occurs before stigma are receptive. • Protogyny – pistillate flower matures before the staminate flower. • Flowers are monoecious or dioecious. • Mechanical obstruction between the staminate and pistillate flowers in the same individual prevents self-pollination. Alfalfa flowers, for example, have a membrane over the stigma that precludes self-pollination. When a bee lands on the flower, the keel is tripped, rupturing the membrane and exposing the stigma to pollen carried by the bee from other plants it has visited, effecting cross-pollination. • Gametes produced on the same plant or clone are unable to effect fertilization. • Self-sterility – gametes from same individual cannot successfully fuse to form a zygote. Sterility can be caused by lack of function of pollen (male gametes) or ovules (female gametes). Male sterility, either genetic or cytoplasmic, occurs because the pollen is not viable. Female sterility occurs when the ovule is defective or seed development is inhibited. • Self-incompatibility – self-pollination may occur, but fertilization and seed set fail. Pollen is transported from the staminate flower to the pistillate flower by wind, insects, or animals. Occasionally pollen is transported to receptive stigma of the same individual and self-pollination may occur. For example, pollen from the tassel of a maize plant may land on and pollinate silks on the same plant, effecting self-pollination. Sunflower is ordinarily cross-pollinated. Bees often carry pollen from one plant and deposit it on other plants. Self-Pollination Several floral mechanisms enforce self-pollination. • Flowers do not open, preventing external pollen from reaching the stigma. • Anthesis occurs before the flower opens. • Stigma elongates through the staminal column (filaments and anthers) immediately after anthesis. • Floral organs may obscure the stigma after the flower opens. Although these mechanisms usually enforce self-pollination, a low frequency of cross-pollination may occur. The frequency of cross-pollination in normally self-pollinating species generally depends on the species and environmental conditions. Soybean is an example of a species that is normally self-pollinated. Before the flower opens, the anthers burst and pollen grains fall out of the anthers on to the receptive stigma contained in the same flower-self-pollination occurs. Learning Objectives • Understand the effects of inbreeding. • Be able to assess the amount of inbreeding by consideration of the inbreeding coefficient. • Compare and contrast systems of mating that promote inbreeding—self-pollination, half-sib mating, full-sib mating, and backcrossing. • Learn why inbreeding depression occurs and know why it happens more commonly in species that are predominately cross-pollinated vs. those that are predominately self-pollinated. • Understand the effects of heterosis and know the difference between the dominance vs. the overdominance hypotheses for explaining its occurrence. Genetics of Inbreeding Inbreeding as a Probability Inbreeding is characterized by a departure from random mating and involves preferential mating between relatives. The most significant effect of inbreeding is that replicates of a single allele in a common ancestor can come together through the mating of relatives to produce homozygous progeny whose alleles are identical by descent. In contrast in a second type of homozygote, two alleles that are said to be alike in state are those that have the same function, but did not derive from replication of a single ancestral gene. These two types of homozygotes can be understood by analyzing pedigrees such as the ones shown in Figure3. The two copies of the A1 allele in the A1A1 offspring individual at the bottom of Figure 3a descend from the same copy in a common ancestor; this type of genotype is termed autozygous. In contrast the two copies of the A1 allele in the A1A1 offspring individual at the bottom in Figure 3b are descended from two different copies in ancestors; this type of genotype is allozygous. Inbreeding can be estimated directly by studying pedigrees or lineages of ancestry showing genetic relationships. Measurement of Inbreeding The probability that two genes are identical by descent is called the coefficient of inbreeding (denoted as F) and will be the measure of relationship between mating pairs. The coefficient of inbreeding (F) is defined as the probability that two alleles at the same locus are identical by descent. At the population level, F describes the average level of homozygosity. The coefficient of inbreeding is always expressed relative to a specified base population. The base population is defined to be non-inbred (F=0). The range of F is 0 to 1, with 0 indicating random mating and no inbreeding, while 1 means prolonged selfing. Consider a base population consisting of N individuals each shedding equal numbers of gametes uniting at random (although see Fehr, 1987, p.113, with respect to the concept of effective population size, or Ne). Because the base is defined to have F=0, each individual in this population carries genes that are non-identical. The only way a homozygote that carries genes that are identical by descent can arise is by the mating of a male and female gamete from the same individual that carries a replication of the same gene. Because there are 2N gametes the probability that two mating gametes are identical by descent is 1 / 2N. Effective Population Size The concept of effective population size, denoted by the symbol Ne, was introduced by Sewall Wright as number of breeding individuals in an idealized (as small as possible, i.e., “redundant” individuals would be eliminated) population showing the same distribution of allele frequencies under random genetic drift or inbreeding as the population under consideration. The effective population size is usually smaller than the absolute population size (N). As noted in pages 112-114 in the Fehr textbook, Ne is a relative measure of the number of parents mating in a population >>> Ne (see Fehr, 1987, p. 112-114). The general equation for Ne is $N_{e} = \frac{2N}{1 = F_{p}}$ where: • N = number of individuals that are mating • Fp = coefficient of inbreeding for parents • Ne is a basic parameter in many models in population genetics, and will be explained in more detail in the Population and Quantitative Genetics for Breeding course. Second Generation In the second generation, there are two ways genes that are identical by descent can be joined: 1) by a new replication of the same ancestral gene; and 2) by the previous replication that occurred in generation 1. The probability of a new replication event is $\frac{1}{2N}$. The remaining proportion of zygotes, $1 − {1\over 2N}$, carry genes that are independent in origin from Generation 1, but may have been identical in their origin in Generation 0. The probability the genes are identical by descent from Generation 1 is the inbreeding coefficient of Generation 1 $(F_{1} = {1\over 2N})$. Note that F1, F2, Ft, etc in this section (written in italics) denote inbreeding coefficients, and not generations as on pages 1 and 2 of this section and in earlier sections. Therefore the probability of identical homozygotes in Generation 2 is: $F_{2} = {1 \over 2N} + (1 - {1 \over 2N})F_{1}$ Where F1 and F2 are the inbreeding coefficients of Generations 1 and 2. The same arguments apply to future generations, so we can write the recurrence equation: $F_{t} = {1 \over 2N} + (1 - {1 \over 2N})F_{t-1}$ Generation Inbreeding The inbreeding of any generation is composed of two components: New inbreeding, which arises from self-fertilization and the “old” that was already there. Note that inbreeding is cumulative and the absence of inbreeding in Generation t does not change the fact that a population had old inbreeding from prior generations. Through a series of algebraic steps, we can write the inbreeding coefficient as a function of the number of generations removed from the base populations: $\textrm{F}_t = 1 - (1 - \Delta F) ^t$ where, $\Delta \textrm{F} = {1 \over 2N}$ Genotype Frequencies The genotype frequencies in a population can then be expressed as: Table 1 Genotype frequencies for a locus with two alleles. Data from Falconer ad Mackay, 1996. Original Frequencies Change due to inbreeding Origin Independent Identical AA $p_{0}^{2}$ $+p_0q_0F$ $=p_{0}^{2}(1 - F)$ $+p_0F$ Aa $2p_0q_0$ $-2p_0q_0F$ $=2p_0q_0(1-F)$ aa $q_{0}^{2}$ $+p_0q_0F$ $=q_{0}^{2}(1-F)$ $+q_0F$ Allozygous genes Autozygous genes We can examine inbreeding or the probability of identity of alleles by descent, by looking at the genotype frequencies in Table 1 under two extremes of the F statistic: • if F = 0 (random mating; no inbreeding) • the equation for genotypic frequencies reduces to the familiar equation for genotypes in Hardy-Weinberg proportions: p2 + 2pq + q2 • if F = 1 (alleles identical by descent) • the genotype equation reduces to a ratio of homozygotes to heterozygotes as p:0:q Inbreeding Coefficient Thus inbreeding leads to homozygosity (all or nearly all loci homozygous), and almost a complete absence of heterozygosity (all or nearly all loci heterozygosity). As noted in Table 1, with inbreeding there is a deficit of heterozygotes equal to 2pqF and an excess of each homozygous class equal to half the deficiency of the heterozygotes. To illustrate one type of mating that promotes inbreeding, consider the case of a population that reproduces by self-fertilization so that F = 1. Another way to express the inbreeding coefficient, F, is to compare the frequency of heterozygotes in the population to the frequency expected under random mating: $F = \frac{H_{0} - H}{H_{0}}$ where, • H = frequency of heterozygotes in the population • H0 = expected frequency under HWE, meaning 2pq Therefore the inbreeding coefficient is the proportional reduction in heterozygosity relative to a random mating population with the same allele frequencies. Inbreeding via Self-Pollination In a population that reproduces by self-fertilization the inbreeding coefficient, F = 1. Let’s assume the population begins with genotypic frequencies in Hardy-Weinberg proportions (p2 + 2pq + q2). With selfing, each homozygote produces only progeny of the same genotype: AA × AA ⇒ all AA aa × aa ⇒ all aa However only half of the progeny of a heterozygote will be like the parent Aa × Aa ⇒ 1/4 AA, 1/2 Aa, 1/4 aa Self-pollination therefore reduces the proportion of heterozygotes in the population by half with each generation until all genotypes in the population are homozygous (Table 2). Table 2 Increased over generations in frequency of homozygous in a self-pollinated population starting with p = q = 0.05>>>frequency of homozygotes. Genotype Frequencies Generation AA Aa aa 1 1/4 1/2 1/4 2 1/4 + 1/8 = 3/8 1/4 1/4 + 1/8 = 3/8 3 3/8 + 1/16 = 7/16 1/8 3/8 + 1/16 = 7/16 4 7/16 + 1/32 = 15/32 1/16 7/16 + 1/32 = 15/32 n $d7f9b3e956676a1d251be02ed8201485.png$ $479add8bfbfdff3bbbbff024bee5812b.png$ $d7f9b3e956676a1d251be02ed8201485.png$ 1/2 0 1/2 Proportion of Homozygotes With multiple segregating loci, the proportion of homozygotes in various selfing generations can be estimated using the following formula: [(2m – 1)/2m]n: where m is the number of selfing generations(m = 1 for F2; m = 2 for F3 and so on) and n is the number of segregating loci. For example, an F1 plant with four independent segregating loci will result in the following frequency of homozygous plants in F2, [(21 – 1)/21]4 = 1/16 = 6.25%. The expected proportion of completely homozygous plants in F2 and later generations of selfing for different numbers of segregating loci are indicated in Table 3. The proportion of homozygotes decreases sharply with increasing heterozygosity (more segregating loci) in the F1. Table 3 Frequency of completely homozygous individuals in various selfing generations in relation to the number of segregating loci in F1. Number of segregating loci in the F1 Frequency of completely homozygous individuals (%) F2 F4 F6 F8 1 50.00 87.5 96.87 99.22 2 25.00 76.56 93.85 98.44 3 12.50 66.99 90.91 97.67 4 6.25 58.62 88.07 86.91 5 3.13 51.29 85.32 96.15 10 0.01 26.31 72.79 92.45 100 7.89 x 10-31 6.1 x 10-4 4.18 45.64 1000 9.33 x 10-302 1.02 x 10-58 1.63 x 10-58 0.04 Study Questions 1 Consequences of Inbreeding Increasing Inbreeding Through increasing homozygosity, inbreeding brings together identical alleles at a locus. Homozygosity permits the expression of recessive alleles that may have been previously masked by heterozygosity in the parent generation. If recessive alleles are less favorable than dominant ones, the overall fitness of the individual decreases. Inbreeding is often detrimental because it increases the appearance of lethal and deleterious recessive traits. The term inbreeding depression describes the decrease in fitness or performance that often accompanies inbreeding or random genetic drift. Recall that fitness is the relative ability of an individual to survive and reproduce to contribute its genes to the next generation. Inbreeding depression is further described in the next section. Plant stature, vigor, yield and other traits decline with increasing inbreeding, although significant differences exist among species for the amount of inbreeding depression expressed—ranging from minimal among self-pollinated crops such as oat and wheat to severe in cross-pollinated polyploid species such as alfalfa, whereby homozygous genotypes do not survive (Figure 4). Table 4 on the next screen summarizes some general differences between outbreeders and inbreeders. Outbreeders vs. Inbreeders Table 4 General features of reproduction and population genetics pertaining to outbreeders vs. inbreeders. Data from Simmonds and Smartt, 1999. Outbreeder Inbreeder Has crossing mechanism, approaches random mating Closed flowering, approaches regular selfing Individuals heterozygous at many loci Individuals approach homozygosity Variability distributed over the population Variability mostly between component lines Carries deleterious recessives Deleterious recessives tend to be eliminated Intolerant of inbreeding Tolerant of inbreeding Much heterozygote advantage (epistasis, overdominance) Less heterozygote advantage Inbreeding Depression Inbreeding Depression and Homozygosity Inbreeding results in increased homozygosity. In cross-pollinated species, increased homozygosity results in inbreeding depression or reduced performance. Symptoms of inbreeding depression may include: • Reduced plant vigor • Smaller plant size • Decline in fertility • Suppressed seed production • Decreased pollen production • Inferior seed quality • Greater susceptibility to insect or pathogen damage or • Poorer standability. Why? With increased homozygosity, expression of deleterious recessive alleles may be revealed by fixation that had been masked by more favorable dominant alleles, or it can be caused by overdominance in which homozygotes are less fit, resulting in poorer performance. The severity of inbreeding depression varies with the species and genotype. If the inbreeding depression is too severe, it may be difficult or impractical to maintain or propagate inbred lines by seed. In such cases, some heterogeneity must be maintained or other propagation means used. Overdominance Hypothesis Over-dominance: The phenotype of the heterozygous progeny is greater than either parent. Mating Systems The frequency of homozygotes in a population can be increased using any of several mating systems: • Self-pollination—Repeated self-pollination is routinely used to develop pure lines of self-pollinating crops. It can also be applied to cross-pollinating species to obtain inbred lines. Like pure-lines in self-pollinated species, inbred lines are homozygous at nearly all loci. • Half-sib mating—Plants having one parent in common are mated. The pollen source is random from the breeding population, but the female plants are identifiable. • Full-sib mating—Plants having both parents in common are crossed. • Backcrossing—A method in which a hybrid is mated to one of its parents, the recurrent parent, resulting in a backcross 1 (BC1) population. The resulting backcross offspring are repeatedly crossed to the recurrent parent. The inbreeding coefficient increases more rapidly with matings among more closely related individuals. The first three mating systems listed above are compared in Figure 5. In each case the initial value of the inbreeding coefficient is assumed to be F0 = 0. Backcrossing—like self-fertilization—can be one of the most extreme forms of inbreeding. Study Questions 2A & 2B Interpret the trends shown on the graph in Figure 5. Query $2$ After the 6th generation, what is the percent homozygosity in the half-sib and in the full-sib mating systems? Query $3$ Study Questions 2C List the circumstance(s) under which the breeder would develop inbred lines using the half-sib mating or the full-sib mating system rather than self-pollination. Query $5$ Study Questions 3 The challenge for the plant breeder is to develop inbred lines that can be maintained and are sufficiently homozygous to generate reliably superior and uniform progeny when mated to produce the hybrid cultivar. Inbred lines can be developed from any heterogeneous population. Heterogeneity is essential to obtain variability for important traits. Without variability for the characters of interest, the breeder cannot make selections or breeding progress. Degree of Relatedness of Individuals The coefficient of relatedness (rIJ) is a measure of the degree of relatedness between individuals (Table 5). It is the proportion of genes shared between two individuals I and J due to common descent (e.g., identical by descent). It ranges from -1.0 (no genes in common, at least among the genetic markers assessed) to +1.0 (vegetative clones). For example, the value of rIJ for a parent and each of their progeny is 0.5 for a sexually reproducing species because half of each offspring’s genes come from each parent. In the case of full-siblings the value of rIJ is also 0.5, but on average because siblings receive half of their genes from each of the same pair of parents, but the haploid set of genes in each parental gamete is a random sample of half of the parental genome due to recombination (Conner and Hartl 2004). Table 5 Values of the coefficient of relatedness (rIJ) among relatives. Data from Conner and Hartl, 2004. Mating Systems rIJ Self-pollination*, vegetative clones, doubled haploids 1.0 Parents and offspring 0.5 Full siblings 0.5 Half siblings 0.25 *After selfing for many generations Heterosis Heterosis or Hybrid Vigor When inbred lines of cross-pollinating species are mated, their progeny are often superior to either or both of the parents for one or more characters, a phenomenon referred to as hybrid vigor or heterosis. Recall that genetic drift is random, so that different inbred lines or subpopulations tend to be fixed for different alleles at various loci; when they are intermated, the F1 population will be highly heterozygous. Heterosis is the opposite of inbreeding depression—heterosis is commonly expressed as • Improved plant vigor, • Greater plant size, and/or • Increased productivity. The performance of a hybrid relative to its parents can be described in two ways: mid-parent heterosis is the performance of a hybrid compared with the average of the performance of its parents; high-parent heterosis is a comparison of the performance of the hybrid with that of the best parent: $\text{mid-point heterosis (%)} = \frac{F_{1} - MP}{MP} \times 100$ Where: • F1 = performance of the hybrid • MP = average performance of the parents (Parent 1 + Parent 2)/2 • HP = performance of the best parent Dominance vs. Overdominance Hypothesis Heterosis tends to be greatest in the progeny of diverse genotypes. There are two common explanations for this phenomenon: 1. Dominance hypothesis—Heterosis results from the complete or partial dominance of favorable alleles at various loci. 2. Overdominance hypothesis—Heterosis reflects the superior performance of the heterozygote over either homozygote. The next few pages provide a summary of the dominance and overdominance hypotheses from Charlesworth and Willis (2009). Note the differences between models for a single locus and those describing multiple loci. They include a third model that involves deleterious alleles at closely linked loci and focuses on so-called “pseudo-overdominance”, which will not be further discussed here. Complete dominance The phenotype of the heterozygous progeny equals the phenotype of the homozygous dominant parent. Partial (incomplete) dominance The heterozygous progeny has a phenotypic value greater than that of the midparent value, but less than that of the homozygous dominant parent. Overdominance The phenotype of the heterozygous progeny is greater than either parent. Heterozygote Advantage Heterozygote advantage is a term that is frequently confused with heterosis. However, heterozygote advantage is a synonym of overdominance, and refers to a condition in which the heterozygous genotype has a higher phenotypic value (especially for fitness) than for either homozygous genotype. Heterosis is usually due to a number of loci that control quantitatively inherited traits, although depending on the situation it could be due to phenotypes controlled by either single genes or polygenic traits. In contrast, heterozygote advantage describes effects at a single locus (Conner and Hartl 2004). Compare the circumstances for these two concepts in Table 6. Table 6 Fitness effects of high and low frequency of heterozygotes. Data from Donner and Hartl, 2004. Subpopulations Small and isolated Crossed with each other Genotypic frequencies Highly homozygous Highly heterozygous Fitness Low High Fitness effects Inbreeding depression Heterosis or hybrid vigor Cause for fitness effects Deleterious recessives expressed or loss of heterozygote advantage Deleterious recessives masked or occurrence of heterozygote advantage Genetics of Heterosis and Inbreeding Depression Whatever genetic mechanisms can explain heterosis must also be able to explain inbreeding depression. Let’s take a look at hypothetical numeric examples of these two hypotheses to show how each can explain both. Heterosis and Inbreeding Depression In the following examples we will use a scenario where six genes control a certain quantitative characteristic. Favorable Dominant Alleles Theory (with complete dominance at each locus) 1. Heterosis The homozygous recessive genotype, aa bb cc dd ee ff has a value of 40. One dominant allele at any of the six genes increases the value by 10, that is, N_=+10.The following two homozygous parents are crossed. Each has a value of 70 because of having three loci with dominant alleles [40+(3)(10)=70]. Note that the F1 has a value of 100 because of having six loci with dominant alleles (40 + (6)(10) = 100). 1. Inbreeding depressionInbreeding depression can be explained with the accumulation of favorable dominant alleles mechanism. If we self the Aa Bb Cc Dd Ee Ff individuals that have a value of 100, we will get a population of individuals as follows: Table 7 Percentage of individuals Number of loci with at least one dominant allele 17.80% 6 35.60% 5 29.66% 4 13.18% 3 3.30% 2 0.44% 1 0.02% 0 This gives an overall population mean value of 85. This is lower than the value of the Aa Bb Cc Dd Ee Ff parental individuals, thus showing depression upon inbreeding. Although conclusive evidence to support either of these hypotheses remains elusive, the dominance hypothesis has been more widely accepted. However, it is recognized that a gene’s effect is determined by its effect in combination with: • Other alleles at the same locus, • Itself (when homozygous), • Genes at other loci (epistasis), and • Closely linked genes. Thus, dominance, overdominance, and epistasis, most likely all contribute to heterosis. Overdominance Theory 1. Heterosis: The genotype aa bb cc dd ee ff has a value of 40.The homozygous dominant allele at any locus increases the value by 7 over the homozygous recessive allele, i.e., NN = +7. Heterozygous at any locus increases the value by 10 over the homozygous recessive, i.e. Nn = +10. The following homozygous parents are crossed; each has a value of 61 because of having three loci with homozygous alleles [40 + (3)(7) = 61]. The F1 has a value of 100 because of having six heterozygous loci. 2. Inbreeding depression: Inbreeding depression can be explained with overdominance. If we self the Aa Bb Cc Dd Ee Ff individuals that have a value of 100, we will get a population of individuals with a mean value of 85 (see table above). As was the case for the “favorable dominant alleles theory”, this value is lower than the value of the Aa Bb Cc Dd Ee Ff parental individuals, thus showing depression upon inbreeding. Genetic Analysis Molecular techniques are now being used to try to shed more light on the mechanisms involved in heterosis. In a study published in 2006, Swanson-Wagner et al. assayed the gene expression of 13,999 maize genes from seedling plants of two inbred parents and their F1 hybrid. When comparing the three genotypes they found 1,367 of the genes produced significantly different amounts of messenger ribonucleic acid (mRNA), which performs an essential role in protein synthesis. Analysis of these genes showed that 78 percent of them had additive gene action, 15 percent showed either high-parent or low-parent dominance, and 3 percent showed either overdominance or underdominance. (The other 45 genes showed non-additive gene action but with the statistical analysis used these genes could not be classified as dominant, overdominant, or underdominant.) The experiment analyzed individual genes and was not designed to evaluate interactions among the genes, so epistasis could not be measured. This study involved only two maize inbred lines and their hybrid offspring and the evaluation was based on tissue from seedling plants. Different genes will be active during different parts of the life cycle of the maize plant and further studies will most likely show differential gene action among different genes. Likewise, different parents and hybrids and different species may well show very different gene action patterns among the same genes that were tested in this study. With just this one experiment, where nearly 14,000 genes were evaluated, we can see that heterosis is complex and that multiple types of gene action are involved. Molecular methods in the context of hybrid breeding will be discussed in detail in the molecular plant breeding course. When two genetically different parents are mated, heterosis is observed in their F1 progeny. • All seed and forage species express some level of heterosis. • Most cross-pollinated species express greater heterosis than do self-pollinated species. Because of the expense in producing hybrid cultivars, hybrid cultivars are generally developed only in those crops in which the level of heterosis results in significantly better performance.

textbooks/bio/Agriculture_and_Horticulture/Crop_Genetics_(Suza_and_Lamkey)/1.07%3A_Inbreeding_and_Heterosis.txt

Introduction Many of the traits that plant breeders strive to improve are quantitatively inherited. For example, breeding efforts targeting quantitative traits have allowed major increases in crop yield during the past 80 years or so. A quantitatively inherited trait is controlled by many genes at different loci, with each gene — known as a polygene — contributing a small effect to the expression of the character. Polygenes are also known quantitative trait loci (QTL). QTLs involved in expression of a quantitative character act cumulatively to determine the phenotype of the trait. Their mode of inheritance is called quantitative genetics. Quantitative genetics describes the connection between phenotype and genotype and provides tools to show how phenotypic selection of complex characters changes allele frequencies. Quantitative genetics focuses on the nature of genetic differences, seeks to determine the relative importance of genetic vs. environmental factors, and examines how phenotypic variation relates to evolutionary change. Typically, quantitative genetic analysis is executed on traits showing a continuous range of values. Analysis of quantitative traits is based on statistical predictions of population response. Examples of quantitatively inherited traits include yield, vigor, rate of photosynthesis, protein content, and drought tolerance. Key Concepts Central to the mathematical modeling of quantitative genetics is the concept of recognition of family resemblance. If genes influence variation in a trait (and sources of environmental variation are minimized or controlled), related individuals would be expected to resemble one another more than unrelated ones. Siblings should resemble each other more than distantly related relatives. A comparison of plant individuals with different degrees of relatedness provides information about how much genes influence the character. Several factors influence the likelihood of progress when breeding for quantitatively inherited traits, including: • Interaction of the multiple genes contributing to the phenotype; • Gene actions of the respective genes involved; and • Frequencies of those genes. Various models are used to distinguish genetic, environmental, and genetic x environmental interaction effects on phenotype and to improve breeding efficiency of quantitative traits. Learning Objectives • Distinguish environmental and hereditary variation and be aware of why detecting the interaction of genetic and environmental factors is important. • Understand the attributes of quantitative inheritance. • Be familiar with statistical methods applied to quantitative inheritance. • Study the types of gene actions and interactions affecting quantitative traits. • Examine the genetic advance from selection formula and be able to explain how each of its components influences the improvement of the selected characteristic. • Learn the types of heritability estimates and their importance in plant breeding. Heritable vs. Environmental Variation The phenotype of a plant or group of plants is modeled as a function of its genotype as modified by the environment. Phenotype = Genotype + Environment + (Genotype x Environment) $P = G + E + (G \times E)$ Some characters are more responsive or sensitive to growing conditions than others. Qualitative traits such as flower color are not strongly influenced by the environment. On the other hand, quantitative traits such as grain yield or abiotic stress tolerance are influenced markedly by the environment. The degree of sensitivity or the range of potential responses to the environment is determined by the genetic composition of the individual plant or population of plants. Genetic variation is essential in order to make progress in cultivar improvement. However, sources of variation include: • Environmental variation • Genetic variation and • Interaction of genetic and environmental variation Plant breeders must distinguish among these sources of variation for the character of interest in order to effectively select and transmit the desired character or assemblage of characters to subsequent generations. GxE Interaction Example A classic example of genotype × environment interaction (GxE) involves studies conducted in the 1930s and 1940s by the ecologists Clausen, Keck, and Hiesey (1940, 1948). They collected plants from natural wild populations — principally yarrow (Achillea millefolia) and sticky cinquefoil (Potentilla glandulosa) — that grew along an east-west elevational transect in California, running from near sea level at the Pacific Ocean to more than 3000 m elevation in the Sierra Nevada mountains. Both species exhibited a vast amount of variation in native populations with respect to growth form and other traits, including such attributes as plant height, winter survival, and number of stems produced. Through a series of reciprocal transplant experiments using cuttings or clonal material from wild populations, they tested the contribution of genetic and environmental variation to observed phenotypic variation among plants established in three main “common gardens” or transplant plots: Stanford, Mather, and Timberline (Figure 1). Conclusions They concluded each species had differentiated into genetically distinct subspecies — which they called ecotypes — that are best suited to their specific environments. In the transplant gardens, no single ecotype performed best at all altitudes. For example, genotypes that produced the tallest plants at the mid-altitude garden site grew poorly at the low and high sites. Conversely, genotypes that grew the best at the low or high sites sometimes performed poorly at the mid-altitude site. Although within a species, all populations were found to be completely interfertile, ecotypes adapted to low or mid-altitude died when transplanted to the high altitude garden, while ecotypes from high elevations along the transect survived through the winter when locally grown in the test plots. GxE interaction was observed for height, among other characters. Significance Illustration Figure 3 shows variation in phenotype between two cultivars of watermelon with regard to a quantitatively inherited trait (yield in this case) in response to variation in an environmental factor (soil salinity in this case) and illustrates the significance of genotype x environmental interaction. Characteristics of Quantitative Traits Inheritance of quantitative traits involves two or more nonallelic genes (multiple genes or polygenes); the combined action of these genes, as influenced by the environment, produces the phenotype. The effect of individual genes on the trait is not apparent. However, early in the 1900s it was discovered that the inheritance of the individual genes contributing to the phenotype of quantitative traits do indeed follow the same Mendelian inheritance principles as simply-inherited genes. Inheritance of Quantitative Traits In 1909, Herman Nilsson-Ehle, a Swedish geneticist and wheat breeder, conducted some of the classic studies on quantitatively inherited traits in wheat. He developed what is known as the “Multiple Factor” or multi-factorial theory of genetic transmission. A key observation made by Nilsson-Ehle was that although a spectrum of continuous variation in kernel color (a quantitatively inherited trait influenced by environmental factors) could be observed in segregating generations, he was able to determine that segregation for these genes fit a model that each separate contributing gene followed a pattern of Mendelian inheritance. Bread wheat is a hexaploid — allopolyploid that contains three slightly different, but similar ancestral genomes (referred to as A, B, and D) in its genome (AABBDD). Depending on the cultivars that Nilsson-Ehle studied, each genome had a single gene that affected kernel color, and each of these loci has a red allele (R) and a white allele (r). Alleles at each locus varied slightly in their effect on kernel color, and will be designated in this example by different superscripts, e.g., R1 or r3. He crossed two cultivars of wheat that varied in kernel color, one with dark red seeds (homozygous dominant genotype R1R1R2R2R3R3, based on the symbols designating the ancestral genomes) and another with white kernels (homozygous recessive r1r1r2r2r3r3). He noted that the F1 of a cross between these parents (heterozygote R1r1R2r2R3r3) was intermediate in color (light red), but the F2 generation could be grouped into seven classes, ranging in color from dark red to white. He explained the distribution on the basis of three pairs of genes segregating independently, with each dominant allele contributing to the intensity of the red color. Table 1 Kernel color in F2 progenies from a wheat cross. F2 genotypes Color Number of dominant alleles Number of plants out of 64 R1R1R2R2R3R3   dark red 6 1 R1R1R2R2R3r3 R1r1R2R2R3R3 R1R1R2r2R3R3 moderately dark red 5 6 R1R1R2R2r3r3 r1r1R2R2R3R3 R1R1r2r2R3R3 R1r1R2r2R3R3 R1r1R2R2R3r3 R1R1R2r2R3r3 red 4 15 R1R1R2r2r3r3 R1R1r2r2R3r3 R1r1R2r2R3r3 R1r1R2R2r3r3 R1r1r2r2R3R3 r1r1R2R2R3r3 r1r1R2r2R3R3 light red 3 20 R1R1r2r2r3r3 r1r1R2R2r3r3 r1r1r2r2R3R3 R1r1R2r2r3r3 R1r1r2r2R3r3 r1r1R2r2R3r3 pink 2 15 R1r1r2r2r3r3 r1r1R2r2r3r3 r1r1r2r2R3r3 light pink 1 6 r1r1r2r2r3r3   white 0 1 With three independent pairs of genes segregating, each with two alleles, as well as environmental effects acting on kernel color, the F2 progeny would contain 63 plants with varying shades of red kernels and one with white kernels. Linkage among the genes restricts independent assortment, so that the required size of the F2 population becomes larger. Characteristics Indicative of Quantitative Inheritance Phenotypic values for the specific trait, resulting from simultaneous segregation of multiple genes, exhibit continuous variation, rather than distinct classes. In general, the distribution of values of quantitatively inherited traits in a population follows a normal distribution (also called a Gaussian distribution or bell curve). These curves are generally characterized by two parameters, the mean and the variance or standard deviation. In the figure below, the mean of the random sample is depicted by the symbol and the standard deviation by the symbol s. If the reference is made to a population instead of a sample from a population, the population mean is usually symbolized by μ and the population standard deviation by σ. There are three general types of traits that are quantitatively inherited: continuous, meristic, and threshold. An example of the first type, a continuous trait, is fruit width of pineapple. The second type, a meristic character, is a countable trait that can take on integer values only, e.g., number of tillers of maize or branches of a rose bush. The third type of quantitative trait is known as a threshold character or “all-or-none” trait. Such traits are typically ranked simply as presence or absence, e.g., Downy Mildew disease in soybean. Threshold Traits Although they have only two phenotypes, threshold traits are considered to be quantitatively inherited because their expression depends on a liability (such as disease susceptibility or tolerance of nicotine levels) that varies continuously. Heritability of these traits is a function of the incidence of the trait in the population, so it is difficult to determine the importance of genetic factors in different environments or in different populations that differ in incidence. Threshold traits are assumed to be represented by an underlying normally distributed “liability trait” that is the sum of the independent genetic and environmental components of the distribution. A disease would have to be present before you could determine if certain genotypes were susceptible or not. For example, plants might be able to tolerate low to moderate levels of nicotine in their tissues until a threshold was crossed, above which the high level of nicotine present would be lethal. Threshold characters exhibit only two phenotypes — the trait is either present or absent — but the susceptibility to the trait varies continuously and environmental components of the distribution. A disease would have to be present before you could determine if certain genotypes were susceptible or not. For example, plants might be able to tolerate low to moderate levels of nicotine in their tissues until a threshold was crossed, above which the high level of nicotine present would be lethal. Threshold characters exhibit only two phenotypes — the trait is either present or absent — but the susceptibility to the trait varies continuously. Environment has a large influence on the trait’s phenotype. That is, for the particular trait, the relative responses of plants change when grown under different environmental conditions. Distinct segregation ratios of individual nonallelic genes are not observed. Recombination and segregation patterns are based on the combined effect of the polygenes on the trait. The more loci controlling the character, the greater the complexity. Genes may differ in their individual gene action, but their effect on the trait is cumulative. Types of gene action include additive, dominance, overdominance and epistasis. Effects of gene action using the concepts of genotypic and breeding values are discussed in Appendix A. Genotypic Value The genotypic value is equal to $G = A + D + I$ G = genotypic value of all loci considered together A = sum of all additive effects (i.e. breeding values) for separate loci D = sum of all dominance deviations (i.e., interaction between alleles at a locus or so-called intralocus interactions) I = interaction of alleles among loci (also referred to as the deviation or epistatic deviation) For an individual, the breeding value is calculated by the summation of the average effects of its genes (also referred to as the additive effect of genes). The average effect of an allele is approximately the average deviation of the mean phenotypic value from the population mean if the allele at a particular locus is substituted by another allele (Falconer and Mackay 1996). Table 2 Summary of interactions among alleles (within or between loci) defining different types of gene action. No interaction among alleles Interaction among alleles Within a locus Additive Dominance Between loci Additive Epistasis Transgressive segregation may occur. These individuals exhibit phenotypes outside the range of those expressed by the parents. Transgressive segregation occurs when progeny contain new combinations of multiple genes with more positive effects or more negative effects for the quantitative trait than found in either parent. One challenge is that strong environmental effects would make it difficult to assess the mean performance in parental plants vs. progeny in order to detect for the presence of any transgressive segregants. Measurement of Continuous Variation Analysis of inheritance of qualitative traits is generally concerned with individual matings and their progeny and is made by counts and ratios. In contrast, analysis of quantitative traits is concerned with populations of organisms that consist of many possible kinds of matings; analysis of such traits is made by use of statistics. Statistical methods provide a tool for describing and evaluating quantitatively inherited characters. Since it is impractical to examine an entire population, plant breeders sample the population(s) of interest. The sample must be representative of the population — the sample must be: • large enough to include the entire range of variability of the trait that occurs in the population, and • random to avoid introducing any bias. Thus, the greater the variability within the population, the larger the sample size that is required to accurately describe that population. (Throughout this and subsequent modules, you can generally assume that we’re referring to a representative sample, rather than a population.) Statistics may be descriptive or analytical. [See Appendix B to briefly review some statistical terms and concepts]. Statistical Terms The study of quantitative traits is sometimes referred to as “statistical genetics” because of its reliance on statistical methods. In order to understand the inheritance of quantitative characters and the methods applied to these characters, it is essential that you become familiar with fundamental statistics. A basic review is provided here. When using symbols to represent these population parameters, it is important to distinguish between information about the population and that concerning a sample representing the population. Similar Statistical Symbols As a shorthand, statistics commonly use symbols to convey concepts. Often there are several symbols that relate to very similar, but slightly differing concepts. Here’s a list of symbols related to means, variance, and standard deviation that you will encounter in this lesson. The latter two parameters describe the variability or dispersion about the mean of the population or sample derived from a population. Although the differences between these are important from a statistical perspective, they are commonly used synonymously. Parameter For a population For a fixed or selected sample of a population Mean $\mu$ or M $\bar{X}$ Variance V or $\delta^{2}$ $s^{2}$ Standard deviation $\delta$ s Descriptive Statistics Range — the lowest and highest phenotypic values in the population or sample for the character. Mean (µ or M for population; $\bar{X}$ for sample) — describes the average performance of a random sample from a population for a trait. The mean is a measure of central tendency — it does not tell anything about the distribution of individual observations. Mean equals the sum of the trait values of each individual divided by the number of samples (n): $\bar{x} = \frac{\Sigma x}{n}$ Variance (V or σ2 for population; s2 for sample) — a measure of the scatter or dispersion of phenotypic values. The greater the variability among individuals, the greater the variance. Two populations with the same mean for the same character could differ greatly in their respective variance for that character. Average the squared deviations from the mean, (X – X)2: $V = \sigma^{2} = \frac{\Sigma [(x - \bar{x})^{2}]}{n - 1}$ Standard deviation (σ for population; s for sample) — also a measure of dispersion around the mean. Standard deviation expresses the dispersion in the same unit as the mean. Standard deviation is the square root of the variance. $\sigma = \sqrt{V} \text{ or } \sqrt{\sigma^{2}}$ A small variance and a small standard deviation tell us that the phenotypic values are near the mean value. In contrast, a large variance and standard deviation indicate that the trait values have a wide range. Samples in which the observations are clustered closely around the mean (red) have a smaller variance and standard deviation than observations dispersed widely (blue). Coefficient of variation (CV) — the standard deviation as a percentage of the mean. Because the units cancel out, CV is a unitless measure. Divide the standard deviation by the mean and multiply by 100: $CV = \frac{\sigma}{\bar{X}} \times 100$ A CV of about 10% or less is desirable in assessing biological systems. When the CV is greater than 10%, the variability in the sample or the population may be too great to sort out the factors contributing to that variability. The mean and the variance are used to describe an individual characteristic, but plant breeders are frequently interested in more than one trait simultaneously. Two or more characteristics can vary together, and are thus not independent of one another. Covariance provides measure of the strength of the correlation or dependent relationship between two or more sets of variables. When two traits are correlated, a change in one trait is likely to be associated with a change in the other trait. Variance is a special case of covariance that is the covariance of a variable with itself. Correlations between characteristics are measured by a correlation coefficient (r). Covariance — the mean value of the product of the deviations of two random variables from their respective means. For example, the covariance of two random variables, x and y is expressed as: $Cov_{x,y} = \frac{\Sigma (x_{i} - \bar{x})(y_{i} - \bar{y})}{n - 1}$ Correlation coefficient (r) — measures the interdependence of two or more variables and is obtained by dividing the covariance of x and y by the product of the standard deviations of x and y. The correlation coefficient can range from -1 to +1. $r = \frac{Cov_{x,y}}{S_{x}S_{y}}$ Correlation is a “scaled” version of covariance and the two parameters always have the same sign—positive, negative, or zero (0). When the sign is positive, the variables are positively correlated; when it’s negative, they are said to be negatively correlated, and when it’s zero, the variable are described as being uncorrelated. But it is important to note that a correlation between variables indicates that they are associated, but it does not imply a cause-and-effect relationship. QTLs and Mapping The relative importance of genetic and environmental factors for a given trait can be estimated by the phenotypic resemblance between relatives. Until recently, quantitative genetics focused on phenotypic information, but increasingly molecular biology tools are being applied in an effort to locate where quantitative trait loci (QTLs) occur in plant and animal genomes. Various genetic markers have been identified and mapped, allowing identification of QTLs by linkage analysis. A common method for mapping QTLs is to cross two homozygous lines that have different alleles at many loci. The F1 progeny are then backcrossed and intercrossed to allow genes to recombine through independent assortment and crossing over. Offspring in segregating generations are examined for correlations between inheritance of marker alleles and phenotypes that are quantitatively inherited. QTL mapping will be covered in more detail in later courses on Molecular Genetics and Biotechnology and Molecular Plant Breeding. Multiple Genes and Gene Action The general types of gene action for quantitative characters (additive, full and partial dominance, and over-dominance) do not differ from those for qualitative traits. However, the genes contributing to the phenotype of a quantitative character may or may not differ in their individual gene action, and their relative effects on the trait’s expression may differ. Some may have major influence and others may have only minor effects on the phenotype. The genes controlling a quantitative trait may also interact. The table below gives examples of types of gene action at two loci. For quantitative traits this would be expanded to multiple loci. Table 3 Gene Action or Interaction Explanation Example Additive Effects Genes affecting a genetic trait in a manner that each enhances the expression of the trait. aabb = 0 Aabb = 1 aaBb = 1 AAbb = 2 AaBb = 2 aaBB = 2 AABb = 3 AaBB = 3 AABB = 4 Dominance Effects Deviations from additivity so the heterozygote is more like one parent than the other. With complete dominance, the heterozygote and the homozygote have equal effects aabb = 0 Aabb = 2 aaBb = 2 AAbb = 2 AaBb = 4 aaBB = 2 AABb = 4 AaBB = 4 AABB = 4 Interaction of Epistasis Effects Two nonallelic genes (e.g., genes at different loci) may have no effect individually, yet have an effect when combined aabb = 0 Aabb = 0 aaBb = 0 AAbb = 0 AaBb = 4 aaBB = 0 AABb = 4 AaBB = 4 AABB = 4 Overdominance Effects Each allele contributes a separate effect and the combined alleles contribute an effect greater than that of either allele separately aabb = 0 Aabb = 2 aaBb = 2 AAbb = 1 AaBb = 4 aaBB = 1 AABb = 3 AaBB = 3 AABB = 2 In the preceding examples, A and B are assumed to have equal effects. However, this often may not be true because genes at different loci may affect the expression of the trait in different ways. Some QTLs may be genes with major effect, while others may contribute only a minor effect. Penetrance or expressivity (refer to Deviations from Expected Phenotypes in the “Gene Segregation and Genetic Recombination” module) may influence trait expression. Likewise, pleiotropic effects (refer to Gene Interactions in the “Gene Segregation and Genetic Recombination” module) may be present, affecting different traits in different ways. Heritability Conceptual Basis for Understanding Heritability Heritability estimates the relative contribution of genetic factors to the phenotypic variability observed in a population. What causes variance among plants and among lines or varieties? Phenotypic variation observed among plants or varieties is due to differences in • their genetic makeup, • environmental influences on each plant or genotype, and • interaction of the genotype and environment. The effectiveness with which selection can be expected to take advantage of variability depends on how much of that variability results from genetic differences. Why? Only genetic effects can be transmitted to progeny. Heritability estimates • the degree of similarity between parent and progeny for a particular trait, and • the effectiveness with which selection can be expected to take advantage of genetic variability. Family Resemblance As mentioned in the introduction of this lesson, central to the understanding of quantitatively inherited traits is the recognition of family resemblance. Two relatives, such as a parent and its offspring, two full or half-siblings, or identical twins, would be expected to be phenotypically more similar to each other than either is to a random individual from a population. Although close relatives may share not only genes (they may also share similar environments for traits that have a large genetic component), resemblance between relatives is expected to increase as closer pairs of relatives are examined because they share more and more genes in common. In this conceptual framework, heritability can be understood as a measure of the extent to which genetic differences in individuals contribute to differences in observed traits. Statistical Basis for Understanding Heritability For plant breeders, heritability can also be understood in a statistical framework by defining it as the proportion of the phenotypic variance that is explained by genetic variance. Heritability indicates the proportion of the total phenotypic variance attributable to genetic effects, the portion of the variance that is transmittable to offspring. A general formula for calculating heritability is $\text{Heritability} = \frac{V_{G}}{V_{P}}= \frac{V_{G}}{V_{G} + V_{E} + V_{GE}}$ $=\frac{\sigma^{2}_g}{\sigma^{2}_{ph}} = \frac{\sigma^{2}_g}{\sigma^{2}_g + \sigma^{2}_c + \sigma^{2}_{ge}}$ where: $V_{P} = V_{G} + V_{E} + V_{GE}$ or $\sigma^{2}_{ph} = \sigma^{2}_{g} + \sigma^{2}_{e} + \sigma^{2}_{ge}$ and $V_{P} = \sigma^{2}_{ph}$ $V_{G} = \sigma^{2}_{g}$ $V_{E} = \sigma^{2}_{e}$ $V_{GE} = \sigma^{2}_{ge}$ Uses of Heritability Estimates Heritability serves as a guide for making breeding decisions. It is generally used to • determine the relative importance of genetic effects which could be transferred from parent to offspring • determine which selection method would be most likely to improve the character • predict genetic advance from selection A key point to understand is that heritability is a population concept — application of heritability estimates is restricted to the population on which the estimate was based and to the environment in which the population was grown. However, some characters exhibit fairly consistent estimates (either high or low) among populations (within species) and environments. When considering characters that have high heritability, what we expect to observe for each genotype is that its phenotype will be quite predictable over a range of environments (growing conditions). In other words, for characters with high heritability, genotype fairly accurately predicts phenotype. This is not so for characters with low heritability. Characters Heritability depends on the range of typical environments experienced by the population under study (if the environment is fairly uniform, then heritability can be high, but if the range of environmental differences is high, then heritability may be low. Even when heritability is high, environmental factors may influence a characteristic. Heritability does not indicate anything about the degree to which genes determine a trait; instead it indicates the degree to which genes determine variation in a trait. Characters having low heritabilities are usually highly sensitive to the environment, presenting greater breeding challenges — low heritability traits often require larger populations and more test environments than do characters having high heritabilities for selection and improvement. Table 4 Average heritability estimates (h2) of maize characters. Average estimates are derived from estimates reported in the literature. The magnitude of these estimates reflects both the complexity of the trait and the number of estimates reported in the literature. Data from Hallauer and Miranda, 1988, p. 118. Heritability Estimate Maize Characters h2 < .70 • Percent Oil • Number of tillers .50 < h2 < .70 • Plant height • Ear height • Kernel-row number • Dates to flowering • Grain moisture .30 < h2 < .50 • Number of ears • Ear length • Ear diameter • Kernel weight • Cob diameter h2 < .30 • Yield • Kernel depth Broad-Sense Heritability Types of Heritability There are two types of heritability: broad-sense and narrow-sense heritability. Broad-Sense Heritability Broad sense heritability, H2, estimates heritability on the basis of all genetic effects. $H^{2} = \frac{V_{G}}{V_{P}} \times 100$ $= \frac{\sigma^{2}g}{\sigma^{2}_{ph}} \times 100$ It expresses total genetic variance as a percentage, and does not separate the components of genetic variance such as additive, dominance, and epistatic effects. Table 5 Genetic variance = Additive variance + Dominance variance + Epistatic variance VG = VA + VD + VI $\sigma^{2}_{e}$ = $\sigma^{2}_{A}$ + $\sigma^{2}_{D}$ + $\sigma^{2}_{I}$ Generally, broad-sense heritability is a relatively poor predictor of potential genetic gain or breeding progress. Its usefulness depends on the particular population. Broad-sense heritability is • more commonly used with asexually propagated crops than with sexually propagated agronomic crops • applied to early generations of self-pollinated crops Narrow-Sense Heritability Narrow-sense heritability, h2, in contrast, expresses the percentage of genetic variance that is caused by additive gene action, VA. $h^{2} = \frac{V_{A}}{V_{P}} \times 100$ $= \frac{\sigma^{2}_{A}}{\sigma^{2}_{ph}} \times 100$ Narrow-sense heritability is always less than or equal to broad-sense heritability because narrow-sense heritability includes only additive effects, whereas broad-sense heritability is based on all genetic effects. The usefulness of broad- vs. narrow-sense heritability depends on the generation and reproductive system of the particular population. In general, narrow-sense heritability is more useful than broad-sense heritability since only additive gene action can normally be transmitted to progeny. This is, because in systems with sexual reproduction, only gametes (alleles) but not genotypes are transmitted to offspring. In contrast, in case of asexual reproduction, genotypes are transmitted to offspring. Table 6 Comparison of broad- and narrow-sense heritability. Broad-sense heritability Narrow-sense heritability Symbols used H2, H, hb2, or hB2 h2, hn2, or hN2 Predictor of Gain Poor Better Genetic Variance Additive, dominance, and epistatic Additive only Generation Early Later Reproductive System Self-pollinated or cloned population Cross-pollinated Estimating Heritability As the formulas presented above indicate, heritability is calculated from estimates of the components of phenotypic variation: genetic, environmental, and genetic x environmental interactions. Two main approaches are described here to help estimate the contribution of different G, E, and GxE components and for calculating heritability. One approach focuses on eliminating one or more variance component, while the other focuses on comparing the resemblance of parents and offspring. These estimates can be determined from an analysis of variance or regression analysis of the character performance of a population grown in several environments (multiple locations and/or years). Several Environments Testing a character’s performance in multiple environments (e.g., more than one location and/or years) is essential to get an accurate estimate of the environmental effects on the character. Test environments should be either random or representative of the target environment the type of environment for which the cultivar under development is intended. Heritability is based on variance, the average of the squared deviations from the mean—a statistical measure of how values vary from the mean. Testing in a single environment provides no measure of variance. The greater the number of environments used in the character’s evaluation, the better is the reliability of the variance estimate, as well as the heritability estimate. Without adequate testing in multiple environments, heritability estimates may be misleading. When evaluating different genotypes for a specific character, if the genotypes vary widely in response to differing environments, environmental variance will be relatively high and heritability for the character low. Conversely, if the different genotypes perform in a similar manner across environments, e.g., certain genotypes are always among the best and others always the poorest regardless of environment, environmental variance will be low and heritability high. Estimation Using Analysis of Variance (ANOVA) Estimating heritability from an analysis of variance provides a way to measure the relative contributions of two or more sources of variability. Analytical Tools Several analytical procedures are commonly used to sort out the sources of variation in the sample, to determine the relationship among factors contributing to the variability, and to estimate the heritability of the character. Analysis of Variance — this procedure identifies the relative contribution of the sources of observed variation in the sample. Sources of variation may include environment, replication, or genotypic effects. The portion of variation that cannot be attributed to known causes is called “error.” Analysis of Variance table. Source of Variation Expected Mean Squares Genotypes $\sigma^{2}_{e} + r \sigma^{2}_{g}$ Error $\sigma^{2}_{e}$ In this example, phenotypic variance is explained by differences in genetic composition, as well as to unknown factors. r stands for the number of replications. $\sigma^{2}_{ph} = \sigma^{2} + r \sigma^{2}_{g}$ The particular steps involved in this procedure and the analysis of variance table that results depend on the design of the experiment. Analysis of variance procedures and interpretation are discussed in the Quantitative Methods course. Estimation using Parent-Offspring Regression Heritability can also be estimated by evaluating the similarities between progeny and parent performance using regression analysis. This analysis is based on several assumptions. • The particular character has diploid, Mendelian inheritance. • There is no linkage among loci controlling the character of interest, or the population is in linkage equilibrium. • The population is random mated. • Parents are not inbred. • There is no environmental correlation between the performance of parents and progeny (to avoid violating this last assumption, randomize parents and progeny within replications; i.e., do not test them in the same plot). The linear regression model is: $Y_{i} = a + bX_{i} + e_{i}$ where: • $Y_{i}$= phenotypic value of progeny of the ith parent • a = mean phenotypic value of all parents tested • b = regression coefficient (slope of the line) • $x_{i}$= phenotypic value of the ith parent • $e_{i}$= experimental error in the measurement of Xi Analytical Tools Several analytical procedures are commonly used to sort out the sources of variation in the sample, to determine the relationship among factors contributing to the variability, and to estimate the heritability of the character. Regression — this procedure examines the strength of the relationship between factors or the influence one factor has on another. The linear regression procedure fits a straight line to a scatterplot of data points. The general equation for the regression line is: $y = a + bx$ where: • y = response or dependent variable • x = predictor or independent variable • a = y-intercept of the line • b = regression coefficient, the slope of the line The regression line always passes through the point ($\bar{x}, \bar{y}$). Relative to the total spread of the data, if most of the datapoints lie on or very near the line, there is a strong relationship between the predictor and response variables—x has a strong influence on y. In contrast, the fewer the points that fall on or near the line, the less influence x has on y. A cautionary note: although the x variable may have strong influence on the y variable, x may not be the cause of the y response, nor the sole factor influencing y. Regression can be used to assess the relative effect of environment on phenotypic value or to obtain information about gene action. The relative scatter about the regression line of a plot of genotype (the predictor variable, x) against phenotypic value (the response variable, y), provides information about gene action. For example, regression analysis of the following two examples suggests that the gene action in example 1 (left panel) is additive (no dominance), whereas there is a complete dominance (by the A2 allele) in the case of example 2 (right panel) (Fehr, 1987). When the heterozygous genotype has a value midway between the two homozygotes and thus all three genotypic values fall on the linear regression line the only gene action contributing to the phenotype is additive. Regression analysis of phenotypic values of progeny (y) against parents (x) provides useful information about the degree of similarity of progeny to the parents. Alternative Formula An alternative formula for calculating the regression coefficient, b, is $b = \frac{\Sigma(X - \bar{X})(Y - \bar{Y})}{\Sigma (X - \bar{X})^{2}}$ where: • b = regression coefficient • X = parent values • Y = progeny values The performance of the progeny is a function of the genetic factors inherited from the parents. (Assume that “parent” means either a random plant or line from a population.) Thus, X, the parent value, is the independent variable, and Y, the progeny value, is the response or dependent variable. Regression Coefficient What does the regression coefficient, b, tell us? If b = 1, then • gene action is completely additive, • negligible environmental effect, • and negligible experimental error. The smaller the value of b, the less closely the progeny resemble their parent(s), indicating • greater environmental influence on the character, • greater dominance and/or epistatic effects, and/or • greater experimental error. An analysis of variance will provide estimates of the relative influence of genetics, environment, and experimental error. The type of heritability and the specific formula used to estimate it depends on the type of progeny evaluated. Types of Progeny The population’s reproductive mode and mating design determine the type of heritability and the formula used to calculate the estimate. Selfed progeny 1. F2 plants are self-pollinated to obtain the F3. All the alleles in the F3 come from the F2 parent. Evaluate the character performance of the F3 2. Regress the performance of the F3 on the performance of their F2 parents. 3. The regression coefficient, b, is equal to H2, broad-sense heritability because its genetic variance includes dominance, additive, and epistatic effects. $H^{2} = b \times 100$ 4. Since it is difficult to obtain information about gene action (dominance, epistasis, additive) in self-pollinated populations, narrow-sense heritability is a poor predictor of genetic gain and rarely used in these populations. Inbreeding causes an upward bias in the heritability estimate. Full-sib progeny 1. Two random F2 plants are mated. Half of the alleles in the F3 come from one parent and half from the other. Evaluate the character performance of the F3. 2. Determine the mid-parent value of the two parents. Mid-parent value, X = (x1 + x2) /2 3. Regress the progeny on the mid-parent value. The regression of progeny on the mid-parent value is $\frac{ \frac{1}{2} V_A}{\frac{1}{2} V_P} = \frac{ \frac{1}{2} \sigma^2 _A}{\frac{1}{2} \sigma_{ph}} = \frac{\sigma^2_A}{\sigma^2_{ph}}$ Since the progeny have both parents in common, only additive variance is included, so the regression coefficient, b, is equal to narrow-sense heritability. $h^{2} = b \times 100$ Half-sib progeny 1. Open-pollinate F2 plants. Seed will be harvested from each F2 individual separately. Progenies from the same F2 plant have the maternal parent (the respective F2 plant) in common, while the paternal parent is pollen from the whole F2 population. Thus all offspring from an F2 plant after open pollination are “half-sibs”. 2. Regress the performance of the half-sib progenies on their parents. $b = \frac{\sigma^{xy}}{\sigma^{2}_{x}}$ where: $\sigma^{xy}$ = covariance between parents, x, and their progeny, y $\sigma^{2}_{x}$ = phenotypic variation among parents 3. The covariance between parents and progeny includes additive variance and some forms of additive epistasis (usually negligible), but no dominance variance. Thus, narrow-sense heritability can be estimated. The regression coefficient, b, is equal to half the heritability value. 4. Multiply the regression coefficient by 2 to obtain narrow-sense heritability. $h^{2} = 2b \times 100$ Heritability Influences Heritability is not an intrinsic property of a trait or a population. As we’ve seen, it is influenced by: • population — generation, reproductive system, and mating design • environment — locations and/or years • experimental design — experimental unit (plant, plot, etc.), replication, cultural practices, techniques for data gathering. Heritability can be manipulated by increasing the number of replicates and number of environments sampled (in space and/or time). Genetic variance can be increased by using diverse parents and by increasing the selection intensity. Heritability is only an indicator to guide the breeder in making selections and is not a substitute for other considerations, such as breeding objectives and resource availability. Genetic Advance from Selection Evolution Estimating Response to Selection Evolution can be defined as genetic change in one or more inherited trait that takes place over time within a population or group of organisms. Plant breeders can use quantitative genetics to predict the rate and magnitude of genetic change. The amount and type of genetic variation affects how fast evolution can occur if selection is imposed on a phenotype. The amount that a phenotype changes in one generation is called the selection response, R. The selection response is dependent on two factors—the narrow-sense heritability and the selection differential, S. The selection differential is a measure of the average superiority of individuals selected to be parents of the next generation. $R = h^{2}S$ The above equation is often called the “breeder’s equation”. It shows the key point that response to selection increases when either the heritability of the trait or the strength of the selection increases. Realized Heritability In an experiment, the observed response to selection allows the calculation of an estimate of the narrow-sense heritability, often called the realized heritability. A low h2 (<0.01) occurs when offspring of the selected parents differ very little from the original population, even though there may be a large difference between the population as a whole and the selected parents. Conversely, a high h2 (> 0.6) occurs when progeny of the selected parents differ from the original population almost as much as the selected parents. In the figure in the next screen, it can be seen that the selection differential (S) in each generation is the difference between the mean of the entire original population and the mean of group of individuals selected to form the next generation. In contrast, the response to selection (R) indicates the differences in population means across generations. The value of R is the difference between the mean of the offspring from the selected parents and the mean of the entire original population: $S = \bar{T}_{S} - \bar{T}$ $R = \bar{T}_{O} - \bar{T}$ where: • T = mean of the entire original population • TS = mean of selected parents • TO = mean of the offspring of the selected parents Adaptive Value The proportionate contribution of offspring of an individual to the next generation is referred to as fitness of the individual. Fitness is also sometimes called the adaptive value or selective value. Note in the figure that the non-selected members of the population do not contribute to the next generation and that selection over time reduced the variance of the population. Types of Selection Artificial selection refers to selective breeding of plants and animals by humans to produce populations with more desirable traits. Artificial selection is typically directional selection because it is applied to individuals at one extreme of the range of variation for the phenotype selected. This type of selection process is also called truncation selection because there is a threshold phenotypic value above which the individuals contribute and below which they do not. In contrast, under natural selection in non-managed populations, other types of selection may occur. Three main types of selection are generally recognized. All three operate under natural selection in natural populations, whereas under artificial selection via selective breeding by humans only directional selection is common. Directional Selection Directional selection acts on one extreme of the range of variation for a particular characteristic. Stabilizing selection Stabilizing selection works against the extremes in the distribution of the phenotype in the population. An example of this type of selection is human birth weight. Infants of intermediate weight have a much higher survival rate than infants who are either too large or too small. Disruptive selection Disruptive selection favors the extremes and disfavors the middle of the range of the phenotype in the population. Genetic Advance from Selection One of the most famous longest-term selection experiments is a study conducted by University of Illinois geneticists who have been selecting maize continuously for over 100 generations since 1896. They have been changing oil and protein content in separate experiments, selecting for either high or low content. In some cases after multiple generations, they have shifted selection from high to low or vice versa. Expected Gain From Selection Because resources are limited, the breeder’s objective is to carry forward as few plants or lines as possible without omitting desirable ones. How does the breeder decide how many and which plants or lines within a population to carry forward to the next generation? The breeder can use heritability estimates to predict the probability that selecting a given percentage of the population or selection intensity, i, will result in progress. The expected progress or gain can be calculated using this formula: $G_C = (k) (\sqrt{V_P})(h^2) = k \sigma_{ph} h^2$ where: • Gc = expected gain or predicted genetic advance from selection per cycle • k = selection intensity — a constant based on the percent selected and obtained from statistical tables (note that some people use hte i symbol instead of k for selection intensity • $\sqrt{V_{p}}$ or $\sigma_{ph}$ = square root of phenotypic variance (equivalent to standard deviation) • h2 = narrow – sense heritability in decimal form (narrow – sense is used for sexually reproduced populations whenever possible, and broad sense heritability, H2, is used for self – pollinating and asexually reproduce populations) Caution: The phenotypic values must exhibit a normal, or bell-curve, distribution for Gc to be valid Selection Intensity Table Representative selection intensity (k) values. % k 1 2.67 2 2.42 5 2.06 10 1.76 20 1.40 50 0.80 90 0.20 100 0 As long as the distribution of phenotypic values is normally distributed, selection intensity values (symbolized by k or sometimes i) can be found in statistical tables. The intensity of selection practiced by plant or animal breeders depends just on the proportion of the population in the selected group. The selection intensity is a standardized selection differential and is a measure of the superiority of the individuals selected as parents for breeding relative to the population from which they were selected. Representative values of k are shown in the table. For Your Information Performance As will be explained in the next section, a key statistic used to describe populations is the mean performance of the population of genotypes. The mean performance of a population can be described by a combination of values for performance of both homozygous and heterozygous genotypes, as well as the relative frequency of alleles. For illustration using a locus with two alleles, A1 and A2, the genotypic value of the homozygotes is designated as A1A1 =+a and A2A2 = -a, while the heterozygous genotype is A1A2 = d. The value of a is the performance of a homozygous genotype minus the average performance of the two homozygous genotypes. $+a = A_1A_1 - \frac{(A_1A_1 + A_2A_2)} {2}$ $-a = A_2A_2 - \frac{(A_1A_1 = A_2A_2)}{2}$ The value of d measures the degree of dominance between alleles, and is the difference between the value of the heterozygote and the mean of the homozygotes. $d = A_1A_2 - \frac{(A_1A_1 + A_2 A_2)}{2}$ Degrees of Dominance Examples of relative genotypic values are given here in depictions showing a and d under different types of gene action. Gene action depicted using a and d in relation to genotypic values. Adapted from Conner and Harti, 2004. Gene Action Degree of Dominance Relative Genotypic Values Additive 0 Complete dominance 1 Partial dominance 3/4 Partial dominance 1/2 Overdominance 2 The difference between the depictions of partial dominance show an example of how the effect of an allele can vary depending on whether the locus is a major gene or minor. Remember, additivity of genetic effects for quantitative traits does not mean that there are equal effects of all alleles at a locus or all loci affecting the trait. The study of quantitative traits is sometimes referred to as “statistical genetics” because of its reliance on statistical methods. In order to understand the inheritance of quantitative characters and the methods applied to these characters, it is essential that you become familiar with fundamental statistics. A basic review is provided here. When using symbols to represent these population parameters, it is important to distinguish between information about the population and that concerning a sample representing the population. Genotypic and Breeding Values Populations can be characterized by the amount and type of genetic variability contained within them. Genetic improvement of a quantitative character is based on effective selection among individuals that differ in what is known as the genotypic value. Variation among the genotypic values represents the genotypic variance of a population. The genotypic value is the phenotype exhibited by a given genotype averaged across environments. A related concept is the breeding value, which is the portion of the genotypic value that determines the performance of the offspring. Genotypic value is property of the genotype and therefore is a concept that describes the value of genes to the individual, whereas breeding value describes the value of genes to progeny and therefore helps us understand how a trait is inherited and transmitted from parents to offspring . Remember that only additive genetic effects can be passed on to progeny. Non-additive genetic effects and environmental effects cannot be inherited by offspring.

textbooks/bio/Agriculture_and_Horticulture/Crop_Genetics_(Suza_and_Lamkey)/1.08%3A_Inheritance_of_Quantitative_Traits.txt

Introduction Mutations are the ultimate source of all genetic variation. Mutations can occur at all levels of genetic organization, classified mainly as either chromosome mutations or genome mutations. Chromosome mutations are discussed in this module. Chromosome alterations involve either single nucleotides or fragments of chromosomes and are either small-scale (one or a few nucleotides substituted, inserted, or deleted) or large-scale (deletions, insertions, inversions, or translocations involving large segments of chromosomes or duplications of entire genes). Genome mutations—involving changes in number of whole chromosomes or sets of chromosomes—will be covered separately in the module on Ploidy—Polyploidy, Aneuploidy, Haploidy. Genetic variation—dissimilarity between individuals attributable to differences in genotype—that is generated by mutations is acted upon by various evolutionary forces. Evolutionary processes that alter species and populations include selection, gene flow (migration), and genetic drift—whether or not plants are cultivated or wild. Evolution can be defined as a change in gene frequency over time. The way that plants evolve is dependent on both genetic characteristics and the environment they face. Genetic variation results from differences in DNA sequences and, within a population, occurs when there is more than one allele present at a given locus. Major processes that affect heritable variation in crop plants are topics emphasized throughout the lessons of this course. Changes in gene frequencies within populations caused by natural selection can lead to enhanced adaptation, while changes caused by human-directed selection can facilitate the development of useful genetic variability and selection of superior genotypes. Selection is the differential reproduction of the products of recombination—both within and between chromosomes. Genetic Resources Historically plant breeders seeking sources of variability were constrained in choice of parental materials or plant genetic resources that were interfertile within closely related gene pools. But a range of new techniques such as mutagenesis, genetic engineering (transgenic or transformed plants), and in vitro methods (tissue culture, doubled haploids, induced polyploids) expand the source and scope of variability that can be used in crop improvement. Our expanding understanding of the molecular basis of genetics has provided insights and technologies that further not only our basic understanding of genes and their regulation, but also provide additional tools for crop improvement. Molecular techniques enable breeders to generate genetic variability, transfer genes between unrelated species, move synthetic genes into crops, and make selections at the molecular, cellular, or tissue levels. Combining these laboratory techniques with conventional field approaches can shorten the time required to develop new or improved cultivars. The importance and application of molecular technologies are rapidly increasing. These topics mentioned above—mutations, gene expression, genetic markers, sources of genetic variation, genetic engineering, and molecular breeding methods—will be briefly mentioned in this module, but covered in greater detail in the later courses including Plant Breeding Methods, Molecular Genetics, and Biotechnology and Molecular Plant Breeding. Learning Objectives • Recognize how mutations are classified and inherited, as well as how mutations affect structure, processes, and products of genes and chromosomes. • Understand the basic principles of transcription and translation. • Become familiar with sources of genetic variation for cultivated plants, including crop gene pools and genetic engineering methods. Mutations as Heritable Change Without heritable variation, any trait favored by selection will not be passed on to offspring. Mutation is defined as heritable change in genetic information. Mutations entail modification of the nucleotide sequence of DNA and consist of any permanent alteration of a DNA molecule that can be passed on to offspring. DNA is a highly stable molecule and it replicates with a high degree of accuracy. However changes in DNA structure and replication errors can occur. Mutation involves modifications in the sequence of bases in DNA transmitted through mitosis and meiosis. Nucleotides A nucleotide consists of a sugar molecule (ribose in RNA or deoxyribose in DNA) attached to phosphate group and a nitrogen-containing base. In DNA or RNA molecules, each strand has a backbone of sugar and phosphate groups (Figure 17). CHEMICAL BASES IN DNA AND RNA Two of the four nitrogenous bases in DNA—adenine (Figure 13) and guanine (Figure 14) are known as purines and the other two—cytosine (Figure 15) and thymine (Figure 16) are pyrimidines. Adenine, guanine, and cytosine are also found in RNA. Another pyrimidine known as uracil (Figure 17) is the base used in RNA in place of thymine. Some mutations occur in loci that encode for gene products such as proteins, and thus they may affect the processes of transcription, translation, or gene expression—processes that happen during the creation of proteins from the genetic code in DNA. But mutations also can occur in parts of the genome that do not code for any gene products (called noncoding DNA) or sequences that serve to control regulatory functions in the cell or chromosomes. For most loci, mutation changes allelic frequencies at a very slow rate and therefore consequences are negligible. Mutations may or may not change the phenotype of an organism. The majority of mutations that do occur are neutral in their effect and therefore do not have an influence on fitness. Some mutations are beneficial. But mutations can have deleterious effects, causing disorders or death. Amino Acids and Proteins Amino acids are a set of 20 different molecules used to build proteins. A peptide is one or more amino acids linked by chemical bonds (termed peptide bonds). Linked amino acids form chains of polypeptides (Figure 18). The amino acid sequences of proteins are encoded in genes. One or more polypeptides form the building blocks of proteins (Figure 19). Proteins perform a variety of roles in cells. Primary protein structure is a sequence of a chain of amino acids. Secondary protein structure occurs when the sequence of amino acids is linked by hydrogen bonds. Tertiary protein structure occurs when certain attractions are present between alpha helices and pleated sheets. Quaternary protein structure is a protein consisting of more than one amino acid chain Types of Mutations Classification of Mutations Mutations can occur at all levels of genetic organization, ranging from simple base nucleotide pair alterations to shifts and rearrangements in sequences of nucleotides along fragments of chromosomes to changes in the number and structure of whole chromosomes. A mutation is a change from one hereditary state to another, e.g., allele A mutates to allele a. For a given locus, the normal allele is referred to as the ‘wildtype’. Mutations are usually recessive and therefore their effects are hidden in heterozygotes. There are a number of common ways to classify mutations, including the following: • causal agent • rate or frequency of occurrence • kind of tissue involved and its type of inheritance • impact on fitness or function, or • molecular structure and scale of the mutation. Spontaneous vs. Induced Mutations Depending on the cause, mutations can be either spontaneous or induced: • Spontaneous mutations occur naturally with no intentional exposure to a mutagen. Spontaneous mutations can result from copying errors made during cell division. • Induced mutations are caused by mutagens, either chemicals or radiation. RARE VS. RECURRENT MUTATIONS Recall from the module on Population Genetics, mutational events in a population can be classified into two categories based on frequency of occurrence: Rare mutations (also called non-recurrent mutations) are defined as those that occur infrequently in populations. Rare mutations are usually recessive and occur in a heterozygous condition so that their effect on the phenotype is not apparent. Rare mutations will usually be lost from populations due to random genetic drift. Recurrent mutations are defined as those that occur repeatedly and thus can possibly cause a change in gene frequency in populations. For a given locus, the rate of allele A mutating to allele a can be given as the frequency u per generation; a mutates to A at a rate v. With the frequency of A symbolized as p and that of a symbolized as q, then at equilibrium, pu = qv, or q = p/(u + v) (see the Equation below). $pu = qv$ $q= \frac{u}{v + u}$ Mutation Rates Falconer and Mackay (1996) summarize the following key points about mutation rates and their frequency in populations: • normal spontaneous mutations alone can produce only very slow changes of allele frequency; • mutation rates are generally quite low for most loci in most organisms, occurring about 10-5 to 10-6 per generation or, stated another way, about 1 in 100,000 to 1 in 1,000,000 gametes carry a newly mutated allele at any locus; • with respect to equilibrium in both directions (u and v) in natural populations, forward mutation (from wildtype to mutant; u) is much more frequent than reverse mutation (from mutant to wildtype; v); and • an equilibrium state known as the mutation-selection balance can maintain deleterious alleles at low frequency; selection acts to eliminate deleterious recessive alleles, but very slowly when the allele frequency is low; even if the elimination process of selection is slow, an equilibrium occurs if mutation creates new copies of the deleterious allele. Somatic vs. Germinal Mutations Plants are multicellular organisms, but mutation typically starts from a single cell. There are two broad categories of mutations that are classified according to the type of cell tissue involved. Somatic mutations occur in somatic tissue, which does not produce gametes. Somatic cells divide by mitosis and therefore through that process, mutations can be passed on to daughter cells. Somatic mutations may have no effect on the phenotype if their function is covered by that of normal cells. However somatic cells that stimulate rapid cell division are the basis for tumors in plants and animals. Somatic mutations usually occur as single events (typically in a single cell) in multicellular organisms or organs that lead to chimera, which is a part of a plant with a genetically different constitution as compared to other parts of the same plant. Somatic mutations are not transmitted to progeny (Figure 1). Germinal or germ-line mutations occur in reproductive cells that produce gametes, and therefore can be passed on to future generations. Germ cells or gametes are formed by meiosis. If a germinal mutation is inherited, then it can be carried in all of the somatic and germ-line cells of the offspring (Figure 1). Effects of Mutation on Fitness or Function Mutations can affect fitness in various ways and can therefore be classified based on their effect on individual fitness: • Deleterious mutations are those that are harmful and have a negative effect on phenotype, decreasing the fitness of the individual. • Advantageous mutations are those that are beneficial and have a positive or desirable effect on phenotype, increasing the fitness of the individual. • Neutral mutations have neither beneficial nor harmful effects. • Lethal mutationsare detrimental and lead to the death of the organism when present. Mutations can also be classified by their effect on gene function: • Loss-of-function mutations either result in a gene product that has less function or one that has no function. Phenotypes associated with loss-of-function mutations are usually recessive. Many of the mutations that are associated with crop domestication from wild progenitors involve loss-of-function alleles (Gepts 2002). • Gain-of-function mutations result in a gene product that has novel function. Altered phenotypes associated with gain-of-function mutations are usually dominant. Many of the changes in crop plants brought about by genetic engineering involve gain-of-function mutations (Gepts 2002). However, it is important to underscore that not all mutations occur in genes or protein-coding regions of the chromosome, nor do all mutations that do occur in genes lead to altered proteins. Point vs. Chromosomal Mutations Mutations are often divided into those that affect a single gene, termed a gene mutation—also sometimes called a point mutation—and those that affect the structure of chromosomes, called a chromosomal mutation. These latter two classes of mutations will be covered in more detail after the concept of gene expression is introduced in the following section. Point Mutation Point or Gene Mutation A point mutation is when a single base pair (or just a few) is altered (Figure 2), an alteration at a “micro” level. There are two general types of point mutations: substitutions or insertions and deletions (the latter two are collectively called INDELs). Base pair substitutions involve an alteration of a single nucleotide in the DNA. A substitution mutation can entail either a transition or a transversion: Missense Mutations Some substitution mutations have no effect on the protein coded for. One reason is because of the redundancy of the genetic code (recall that about one fourth of all base pair substitutions code for the same amino acid; such mutations are termed silent mutations since there is no change in amino acid that results from the substitution). Another reason for lack of effect is that even if a change in amino acid occurs (termed missense mutation), it may have no actual influence on the function of a protein (Figure 3). Also any mutation located within a non-coding region of the chromosome will not be translated into a protein. Lastly, an altered gene may be masked by other normal copies of the gene present in the genome. In certain cases, point mutations can have a significant effect—particularly when a substitution produces a stop codon so that the alteration causes the protein synthesis to halt before the protein is entirely translated, altering the entire structure. These are called nonsense mutations (Figure 3). Frameshift Mutation Base pair insertions and deletions are additions (INDELs) or losses of one to several nucleotide pairs in a gene (Figure 2). Mutations that are insertions and deletions tend to have a much greater effect than do mutations that are base pair substitutions because they disrupt the normal reading frame of trinucleotides. Recall that each group of three bases corresponds to one of 20 different amino acids used to build a protein. Mutations involving base pair insertions and deletions are often therefore referred to as frameshift mutations. Under these circumstances the DNA sequence following the mutation is read incorrectly (Figure 4). Chromosomal Mutation MUTATIONS INVOLVING CHROMOSOME SEGMENTS Different cells of the same organism and different individuals of the same species generally have the same number of chromosomes, and homologous chromosomes are typically uniform in number and in the arrangement of genes along them. However, mutations can occur that alter the number or structure of chromosomes. Changes involving chromosomal rearrangements entail the following basic types: deletions, duplications, insertions, inversions, substitutions, and translocations—alterations that occur at a “macro” level (Figure 5). • Chromosomal deletions are when loss of a chromosome segment occurs. • Chromosomal duplications occur when a chromosome segment is present more than once in a genome or along an individual chromosome. Mutations of this type can involve duplication of chromosome fragments of either noncoding regions or genes that do code for a protein or other gene product. Gene duplications have been important events in the evolution of many crop plants, for example in cotton.Both chromosome deletions and duplications generally result from unequal crossing over during meiosis, whereby one gamete receives a chromosome with a duplicated segment or gene and the other gamete receives a chromosome with a missing or “deleted” segment. • Chromosomal inversions happen when two breaks occur in a chromosome and the broken segment turns 180°—reversing the orientation of the sequence—and then reattaches. Such inverted segments may or may not involve the centromere (termed pericentric inversion vs. paracentric inversion). A consequence of chromosomal inversions is that they either prevent crossing over or if crossing over occurs, the recombinants may be eliminated during meiosis. During meiosis, inverted chromosome segments may form loops in order to pair with the same (non-inverted) sequence on homologous chromosomes. • Chromosomal insertions (not pictured) and chromosomal substitutions are when gain of an extra fragment of chromosome occurs. • Chromosomal translocations entail a change in the location of a chromosome segment. Commonly translocations are reciprocal and thus result from exchange of segments between two non-homologous chromosomes. Transpositions Chromosome segments can also be translocated to a new location on the same chromosome or to a different chromosome but without reciprocal exchange; both of the latter types of mutations are termed transpositions. A transposon (also called a transposable element) is a DNA element that can move from one location to another. These mobile DNA sequences commonly occur in some genomes and can themselves cause other mutations to occur, depending on where they “transpose”. Discussion Mutants and mutations are best known in the context of horror films. In the context of plant breeding and more generally crop production, discuss the consequences of mutations—are they good or bad? Which kinds of mutations are desirable and which ones are undesirable? Sources of Variation Sources of Genetic Variability Plant breeding is dependent on differential phenotypic expression. Loci with only one allelic variant (homozygosity) in a breeding population have no effect on the phenotypic variability. Variation can be introduced to breeding populations by various methods: • hybridization and recombination by sexual reproduction within or between species or populations • genetic transformation or genetic engineering using recombinant DNA methods • induced or spontaneous mutations and transposable elements (transposons) • chromosome manipulation via change in chromosome number and structure (ploidization) [to be discussed in the module on Ploidy—Polyploidy, Aneuploidy, Haploidy] • tissue or cell culture techniques [to be discussed in the module on Ploidy—Polyploidy, Aneuploidy, Haploidy] CONCEPT OF CROP GENE POOLS Plant germplasm is a term used to refer to an individual, group of individuals or a clone that represents a genotype, population, or species. With reference to a given crop and its wild and cultivated relatives, the concept of gene pool (all of the genes shared by individuals in a group of interbreeding individuals) has been applied to categorize a broad range of plant genetic resources according to the ease of gene transfer or gene flow to the particular crop species (Harlan and de Wet 1971). Gene Pools Figure 6 depicts three main categories in the original scheme outlined by J. R. Harlan and J.M.J. de Wet (1971) defined as: • primary gene pools (GP-1, consisting of biological species that can be intercrossed easily without problems of fertility in the progeny; including both cultivated varieties and wild progenitors of the crop), • secondary gene pools (GP-2, consisting of more distant relatives that can be intercrossed with difficulty and result in diminished fertility in the hybrids and later generations; including both cultivated and wild relatives of the crop), and • tertiary gene pools (GP-3, consisting of very distant relatives that can be hybridized with the crop only with special techniques, e.g., embryo rescue, due to problems such as sterility, lethality and other abnormalities), With the addition of a fourth gene pool that contains synthetic variants and lines with nucleic acid sequences that do not normally occur in nature. Methods of genetic engineering relevant to this fourth gene pool category will be covered briefly in the next section of this module. Genetic Engineering and Plant Transformation Genetic engineering, also referred to as recombinant DNA or rDNA technology or gene splicing, involves moving a DNA segment from one organism into another to ‘transform’ the recipient or host. Through a broad range of techniques encompassing biotechnology (for example, gene manipulation, gene transfer, cloning of organisms), novel genetic diversity can be generated that extends beyond species boundaries or can be designed and synthesized de novo in molecular laboratories. Potentially, any gene from any species, as well as synthesized segments, could be transferred into a plant using genetic engineering. Gene transfer can be applied for a variety of objectives: • Add different or new functions • Alter existing traits—amplify, suppress, or prevent the expression of a gene already present in the recipient’s genome • ‘Tag’ and isolate genes in the recipient plant • Tool for basic gene regulation and developmental studies Requirements for Gene Transfer In order to efficiently generate transformants (plants that possess DNA introduced via recombinant DNA technologies), the transformation system used must satisfy several requirements. • Ability to get DNA into host cells in high concentration to increase the probability of incorporation into the host genome • Incorporation into the host nucleus (or chloroplast if the objective is to minimize gene flow by pollen or to produce a large quantity of therapeutic proteins) • Integration into host genome (or stabilization as an autonomous replicon—a plasmid or minichromosome) • The introduced gene is expressed and translated properly GENERAL STEPS 1. Identification and isolation of a gene that confers a desired trait. 2. Introduce the gene into a suitable construct and carrier, such as plasmids or bacterial vectors, for delivery into the host. 3. Introduce the DNA into the host. 4. Identify and select transformants. 5. Regenerate plants. 6. Assay for expression of the trait. 7. Test for normal sexual transmission, or asexual propagation, of the transferred gene. Construct and Carrier The carrier that will deliver the DNA into the host should have certain features. • Sites in which to insert passenger DNA sequences (gene of interest, plus a selectable marker gene if the gene of interest does not allow for easy selection of transgenic plants). • Sequences to mediate integration into the host genome • Selectable marker gene for identification and selection of transformants Usually, the DNA sequence to be transferred into the host is joined with other sequences to facilitate transfer, incorporation, and expression of the gene. Here’s a generalized construct for a T-DNA vector, a carrier derived from an Agrobacterium tumefaciens plasmid. Introduce the DNA Several methods are available to introduce the DNA into the host. • Vector-mediated transfer • Direct DNA uptake—DNA cannot be taken directly into cells having a cell wall so protoplast must be used. • Microinjection—DNA is injected directly into the host nucleus • Acceleration of DNA—coated particles – particles are “shot” into the cell (particle bombardment or gene gun) Genetic transformation plays an important role in modern-day crop improvement. The first transgenic plant was created in 1983. By 1996, there were already 1.7 million hectares of genetically modified (GM) crops and this number increased 100-fold to 170 million by 2012 and is still increasing. The majority of the GM crops (soybean, corn, cotton, papaya, canola and sugarbeet) were created by the use of Agrobacterium tumefaciens (vector-mediated transfer) to resist either herbicides or insects. Herbicide-resistant crops greatly simplified weed management where mechanization in agriculture is high. Insect-resistant crop plants produce stable yields. The tremendous expansion of GM crop production, however, is not realized without controversies. There is currently an intense public debate over the impact of GM crops on human and animal health. Besides health issues, other concerns surrounding GM crops are whether they can create superweeds by crossing to related weeds, become invasive or cause unintended harm to wildlife. Bt Gene: Vector Constructs Let’s follow the transfer of a Bt gene into a plant. The Bt gene was identified and isolated from Bacillus thuringiensis, a bacterium. The gene produces a protein that has toxic effects on Diptera (flies), Lepidoptera (butterflies and moths), and Coleoptera (beetles) species. Soybean was transformed using Agrobacterium tumefaciens. Two different T-DNA vector constructs carrying the Bt gene and one control construct were tested for effectiveness in transforming soybean cells and expressing the Bt toxin (Figure 7). Bt Gene: Transformation Leaf disks from soybean plants were infected and cultured on selective (+) and control (-) media—the selective medium was gradually enriched with an antibiotic (Figure 8). At time 1, the leaf disks were infected with the respective constructs. Conditions were used to promote callus production and growth. At time 2, the plates were evaluated for calli formation. Bt Gene: Selection Query $2$ Transformed cells are able to develop into calli. These are selected and transferred to a medium containing both antibiotics and growth regulators that promote the formation of shoots and roots (Figure 9). Bt Gene: Insect Resistance Evaluation Plantlets are regenerated and transferred to pots containing sterilized soil. Nearly all of the regenerated plants exhibited normal morphology and vigor. A few had chlorophyll deficiencies— these were eliminated from the study. The remaining regenerated plants were evaluated for insect resistance. An equal number of insect larvae are placed on each regenerated plant; plants are isolated to prevent insects from moving among plants. Bt Gene: Insect Resistance Data Analysis After several days, the dead larvae on each plant are counted. Here are the data. Table 1 Insecticidal activity as indicated by the number of plants exhibiting different levels of larvae mortality. The total number of plants regenerated from each construct treatment was unequal. Gene Construct No. of Plants Showing % Larvae Mortality < 25 % 25% – 50% 51% – 75% 76% – 100% Intact 14 7 0 0 Fusion 2 6 2 20 Control 29 0 0 0 Try This: Insect Resistance Data Analysis Study the information in the above table. Look for any patterns in the data. Interpret the data by selecting all of the true statements Advantages and Disadvantages Although recombinant DNA technologies have some problems, the technologies offer several advantages. Advantages Disadvantages Characters can be transferred from divergent species without the limitation of sexual compatibility Difficult to identify and isolate gene Single gene or gene sets can be transferred into important breeding lines without the deleterious effects of linked genes. Insertion is random Transferred character ordinarily exhibits dominant, single-gene inheritance Difficult to obtain proper expression Still must screen at whole plant level and under normal production conditions Expensive Despite its limitations, plant transformation has additional advantages over conventional breeding. Millions of cells, with regeneration capacity, can be screened for the desired trait in a few weeks. A desired gene can be transferred without the necessity of generations of breeding to move the trait from one line into another. Recombinant DNA technology can also be used to place synthetic genes into plant genomes. Insertion is Random The insertion point of the transferred gene cannot be controlled. Plants contain an estimated 500,000 to 5,000,000 kilobases (kb) of DNA. The maize genome, for example, has more than 4,000,000 kb. Generally, transferred DNA involves relatively small amounts of DNA, on the order of 10 kb, so the insertion ordinarily has little effect on chromosome pairing, recombination, or mitosis. The incorporated gene may or may not affect other genes in the recipient genome, depending on where it inserts. • In a non-coding region—no effect on the recipient genome • In a gene that occurs in multiple copies in the genome—the effect, if any, is usually not detectable. • In a single-copy gene—inactivates or alters the expression of the single-copy gene. Insertion into a single copy gene is rare. If it does insert into a single-copy gene, inactivation or alteration of the expression of the disrupted gene may be undetected, or may cause favorable or adverse results—albino and other chlorophyll deficiencies are common problems. Dominant, Single-gene Inheritance Why do these characters generally exhibit dominant, single-gene inheritance? Traits acquired via gene transfer often add a function to the transformed plant. Because the transferred trait is unique in the transformant’s genome, the transformant does not possess any contrasting alleles for the character. Thus, its inheritance is expected to be dominant and as a single gene. Query $4$ Gene transfer is also used to suppress or eliminate, or to amplify the expression of genes already possessed by the host plant. These are also usually designed to behave as dominants. Proper Expression Expression involves many steps, all of which must occur properly to obtain the desired phenotype. Expression must be appropriately regulated. • Generation of the gene’s product-requires proper transcription, mRNA processing, and translation. • Location of expression must be in the appropriate plant part. • Timing of expression needs to occur at the right stage of the plant’s development. • Amount needs to be at an effective level or extent of expression to generate the desired phenotype. For Your Information Table of Codons Amino acids are the building blocks of polypeptides, proteins, and enzymes. The order of the nucleotides on a strand of RNA, as transcribed from DNA, determines the order of amino acids in a polypeptide. Each group of three consecutive nucleotides of the RNA codes for a particular amino acid, or the beginning or end of the message. These triplets of nucleotides are called codons (Figure 20). The genetic code, or instructions from a gene that direct the cell to make a specific protein, is usually based on the messenger RNA (mRNA) sequence (Figure 21). In mRNA, uracil (U), rather than thymine (T), is the nucleotide base that complements adenine (A) on the DNA strand; guanine (G) complements cytosine (C) in both DNA and RNA. Here are some examples of codons: • A-A-A and A-A-G signal the amino acid lysine (Lys) • G-A-A and G-A-G code for glutamine (Gln) • A-U-G signals the start of a coding sequencing and codes for methionine (Met) • U-A-A, U-A-G, and U-G-A are stop codons. Of the 64 possible combinations of three bases, 61 specify an amino acid, while the remaining three combinations are stop codons, or trinucleotide sequences that indicate the end of the message, terminate translation of that mRNA section, and signal a “stop” to protein synthesis (Figure 22). The portion of a DNA molecule that, when translated into amino acids, contains no stop codons is referred to as an open reading frame (Figure 23).

textbooks/bio/Agriculture_and_Horticulture/Crop_Genetics_(Suza_and_Lamkey)/1.09%3A_Mutations_and_Variation.txt

Introduction Not all plant species are diploids. In fact, 75% of all angiosperms are polyploids, which are characterized by having more than two sets of chromosomes in their somatic cells. About 70% of undomesticated grasses and 25% of legumes are polyploids. Among domesticated crop species in general, 75% have been found to be polyploid, with roughly similar percentages found for both annual and perennial species. Major crops, such as wheat, alfalfa, potato, cotton, and sugarcane, are polyploids. There are also plants that do not possess complete sets of chromosomes. Aneuploids have abnormal numbers of chromosomes and vary by the addition or deletion of specific individual chromosomes that otherwise would be present in the normal crop genome. Ploidy reduction produces haploids, which have only a single set of homologous chromosomes instead of the pair found in their diploid counterparts. Haploid plants are very valuable in certain breeding applications. The number of chromosome sets possessed by a crop influences its genetics and thus, the strategies applied for its improvement. Plant breeders can alter chromosome numbers to modify and exploit genetic variability. Learning Objectives • Identify types and characteristics of polyploidy, aneuploidy, and haploidy. • Understand how plants that vary in ploidy level occur naturally or are created artificially. • Become familiar with the genetic behavior of polyploids, Aneuploids, haploids, and implications of changes in chromosome number for reproduction and productivity. • Learn about the strengths and weaknesses of variation in ploidy level in plant breeding and genetic applications. • Explore tissue and cell culture methods and their applications. Concepts for Polyploidy Ploidy Polyploidy can be rather complex. The terminology and symbols used in connection with polyploidy communicate much information—understanding these will help clarify and simplify an otherwise complex topic. • Ploidy refers to the number of chromosome sets in a cell. Prefixes are used to specify the number of chromosome sets in a particular organism. The symbol x is used to indicate the number of chromosomes in a set. Monoploids have one set (1x) and diploids have two sets (2x) of chromosomes, and so forth. Table 1 Corresponding number of chromosomes sets to ploidy level. Ploidy Level Number of Chromosome Sets Monoploid 1x Diploid 2x Triploid 3x Tetraploid 4x Pentaploid 5x Hexaploid 6x Heptaploid 7x Octoploid 8x Nonaploid 9x Decaploid 10x Polyploid • Polyploid is a general term indicating multiple (more than two) sets of chromosomes. • Genome is a set of chromosomes that is inherited together, assuming normal meiosis and mitosis. Each genome is composed of the basic chromosome number, x, and each kind of chromosome is represented only once in each set. Several symbols are used as shorthand to convey additional information about the chromosome constitution of the species or individual. • n — indicates the haploid or gametic chromosome number of the species. (Note: n does not tell anything about the number of chromosome sets). • 2n — denotes the diploid or somatic number of the species. • x — indicates the basic number of chromosomes in each set or genome. For example, a tetraploid, 4x, has 4 genomes or sets of chromosomes. The number of chromosomes in its somatic cells is 2n = 4x, and the number in its gametes is n = 2x. In this illustration, let three be the basic chromosome number, so x = 3. This means that each genome or chromosome set consists of 3 chromosomes. Try This: Polyploidy Types of Polyploidy Euploidy There are two general types of ploidy, which include plants that have either one or more complete sets of chromosomes present in their genome (euploids) or those that have partial sets due to the absence of at least one of their individual chromosomes or presence of at least one extra one (aneuploids): Euploidy refers to the number of chromosome sets in a cell. Prefixes are used to specify the number of chromosome sets in a particular organism. The symbol x is used to indicate the number of chromosomes in a set. Monoploids have one set (1x) and diploids have two sets (2x) of chromosomes, and so forth. • Haploidy—individual with half (n=x) of the somatic cell chromosome number. • Diploidy—individual with two sets of the basic, complete genome (2n=2x). • Polyploidy—individual with more than two basic, complete sets of chromosomes in its somatic cells. Among polyploids, there are two main types: • Autoploidy—individual has more than two complete chromosome sets from a single genome. Also known as autopolyploidy. • Alloploidy—individual has two or more genomes contributed from different parental species in their ancestral lineage. Also known as allopolyploidy. Most naturally occurring polyploids are alloploids. Amphidiploids (also called amphiploids) are allotetraploids that contain two sets of genomes from each of two different parents. Amphidiploids are so-called because they behave like diploids during meiosis. Autoploidy Autoploids commonly occur as a result of duplication of the genome(s) of a single species, thus, the genomes possessed by an autoploid are identical. An autotetraploid (4x) has four sets of the same genome, e.g., four sets of the A genome (AAAA). The following are characteristics associated with autoploids: • Greater ability to colonize new habitats than diploid ancestors—Due to gene buffering, autoploids often show a slower response to selection, but more adaptive potential. • Dosage effect of gene expression—Additive effect of the alleles increases the number of phenotypes. There is a linear relationship between gene expression and number of gene copies. • Larger cells and nuclei compared to their respective diploid counterparts—Because of the greater cell size, autoploids tend to have greater vegetative growth and produce larger structures. This feature has been utilized by breeders to increase yield of crops harvested for their vegetative structures. • Reduced fertility—Abnormalities in meiosis may interfere with chromosome pairing, resulting in unbalanced chromosomal distribution between daughter cells, and thus, nonviable gametes. Gametes possessing an extra chromosome or missing a chromosome (i.e., aneuploids) are usually nonviable. This feature is utilized by breeders to develop seedless crops such as seedless watermelon. Alloploidy Alloploids arise when the genomes of two or more unrelated species are combined in a single individual. Several characteristics are associated with alloploidy: • Broadened genetic base. Alloploids (also known as amphiploids) behave like diploids and result in new species. • Increased allele diversity and heterozygousity—Increase in possible allele combinations can provide expanded opportunities for breeding. • Novel phenotypic variation—Genome interactions and changes in gene expression can occur in newly synthesized alloploids. Such gene changes can include transfer of sequences between genomes and gene conversion, loss, or silencing. • Sterile unless genomes are doubled and chromosomes pair correctly. Chromosome doubling also occurs spontaneously in nature, mainly through the fusion of unreduced gametes. Alloploidy has impacted the evolution of some major crops, such as wheat, cotton, tobacco, and various Brassicas (mustards). Information about the ploidy relationships between certain species has improved our understanding of the genetic origin of polyploid crops and facilitated their improvement. In addition to identifying the genetic origin of a crop, genes, as well as whole genomes, can be transferred among related species to obtain novel genotypes and to combine the favorable qualities of different species as is demonstrated in the creation of Triticale. Colchicine Colchicine is a chemical that is commonly used to artificially double chromosomes. Colchicine is a toxic natural product that is found in the bulbs of autumn crocus (Colchicum autumnale). It is used as a medicine to treat the human disease gout, but it is also used to induce polyploidy in plants. Originally it was extracted from crocus bulbs, but is now also manufactured synthetically. When plant seeds or meristem tissues (apical tip, shoots, or suckers) are soaked in colchicine, it makes the cell walls permeable and prevents normal chromosome division. Colchicine functions as a disruptor of mitosis by inhibiting microtubule formation, thus preventing normal chromosome segregation to occur. After seed treatment with the chemical, half the gametes resulting from meiosis contain no chromosomes, while the other half contains diploid instead of the normal haploid number. This disruption leads to embryos with double the usual number of chromosomes. Colchicine is used to make infertile hybrids or haploid plants fertile by restoring doubled chromosomes. The latter plants are known as doubled haploids. An example of the former is development of hexaploid and octaploid triticale. Colchicine is also used in the study of karyotypes, which refer to the chromosome constitution of the cell (specifically, the number and morphological appearance of chromosomes in the nucleus). In order to observe chromosomes under a light microscope, cells are treated with the chemical near the middle of mitosis during metaphase—a point in the cell cycle when chromosomes are most dense and therefore most visible. Colchicine treatment arrests these chromosomes by preventing the formation of the spindle microtubules. Wheat Wheat is an example of a crop whose origin has been extensively studied and is a classic example of a polyploid crop that evolved through the combination of genomes from related species. Wheat has three sets of chromosomes, designated as A, B, and D. Each of these sets contributes 7 chromosomes. • wheat = AABBDD • 1A, 2A, …, 7A; • 1B, 2B, …, 7B; • 1D, 2D, …, 7D. Brassica Evolution—Triangle of U The “Triangle of U” is a theory that the genomes of three ancestral species of Brassica combined through interspecific hybridization. Brassica species include both vegetable and oilseed crops, such as cabbage, broccoli, rapeseed, and various mustards. The original theory was developed by Woo Jang-choon, a Korean botanist who published under the Japanese name Nagaharu U. He made artificial hybrids between closely related, but distinct diploid and polyploid species and examined how chromosomes paired in the progeny. Woo’s theory was later confirmed by examination of DNA from the group of related Brassica species. The different colors in the chromosomes depicted here represent the three ancestral genomes: Genomes of three diploid species AA: 2n = 2x = 20—Brassica rapa (syn. Brassica campestris) – turnip, Chinese cabbage BB: 2n = 2x = 16—Brassica nigra – Black mustard CC: 2n = 2x = 18—Brassica oleracea – cabbage, kale, broccoli, Brussel sprouts, cauliflower AABB: 2n = 4x = 36—Brassica juncea—Indian mustard AACC: 2n = 4x = 38—Brassica napus—Rapeseed, rutabaga BBCC: 2n = 4x = 34—Brassica carinata—Ethiopian mustard Autoploids and Alloploids Many important crops are naturally occurring polyploids—either autoploids or alloploids. Letters are often used to indicate to the different source species that have been identified as contributors to crop genomes. However the same letter does not denote the same genome unless the species are closely related evolutionarily. For example, tall fescue (an alloploid) and perennial ryegrass (a diploid) both share the L genome in common. Pima and Upland cotton (both alloploids) share A and D genomes in common, but only share the A genome with two other cultivated cottons, Levant and Tree cotton (both diploids). Other polyploid crops include sugarcane, yam, white potato, orchardgrass, highbush blueberry, persimmon, basil, nutmeg, and some types of asparagus and celery. Many horticultural species of flowers and ornamentals are polyploids. Some of these polyploids occur naturally and others were manipulated into polyploids through plant breeding efforts. Two examples of artificially created alloploids that are produced commercially include the cereal grain triticale (Tritcosecale) and the vegetable crop known as radicole (Raphanobrassica). Both were developed by intergeneric hybridization among closely related crop genera: wheat-rye and radish-cabbage (or radish plus other Brassicas). Recent studies involving molecular techniques have revealed that many diploid crops with high chromosome numbers (2n>18-20) are actually paleopolyploids—plants whose genomes result from ancient genome duplications that occurred at least several million years ago. Known paleopolyploids of either alloploid or autoploid origins include such crop species as soybean, squash, maize, and lettuce. Diploidization In a process known as diploidization over time polyploid genomes may return to diploid status, although certain genes may be retained in multiple copies. Conversely, the term polyploidization refers to cycles of hybridization and chromosome doubling that result in either allopolyploids or autopolyploids. Table 2 Examples of naturally-occurring euploid crops. Crop Type Diploid or Polyploid Genome Ploidy (x) African rice Diploid A’A’ 2x Asian rice Diploid AA 2x Bread wheat Alloploid AABBDD 6x Durum wheat Alloploid AABB 4x Rye Diploid RR 2x Oats Alloploid AACCDD 6x Broccoli, Cabbage, Kale, Cauliflower, Brussel sprouts Diploid CC 2x Turnip, Chinese cabbage Diploid AA 2x Rapeseed, Rutabaga Alloploid AACC 4x Black Mustard Diploid BB 2x Brown Mustard Alloploid AABB 4x Ethopian Mustard Alloploid BBCC 4x Radish Diploid RR 2x Radicole Alloploid RRCC, RRAA 4x Sweet potato Autoploid BBBB, BBBBBB 4x, 6x Pima cotton Alloploid AADD 4x Tree cotton Diploid AADD 4x Upland cotton Alloploid AADD 4x Levant cotton Diploid AA 2x Safflower Diploid BB 2x Alfalfa Autoploid SSSS 4x Bermuda grass Diploid, Autoploid AA, AAAA 2x, 4x Tall fescue Alloploid PPLLXX 6x Perenial rye grass Diploid LL 2x Timothy Alloploid AAAABB 6x Banana Diploid, Alloploid, Autoploid AAA, AAA, ABB, AB, AAAB, AABB, ABBB 2x-4x Plum, European Alloploid CCSSSS 6x Coffee Alloploid AABB 4x Tobacco Alloploid SSTT 4x Table 3 Examples of artificially developed alloploid crops. Crop Type Hybrid or Parent Genome Triticale Intergeneric hybrid AABBRR, AABBDDRR Bread wheat Parental species AABBDD Durum wheat Parental species AABB Rye Parental species RR Radicole Intergeneric hybrid RRCC, RRAA Broccoli, Cabbage, Kale, Cauliflower, Brussel sprouts Parental species CC Turnip, Chinese cabbage Parental species AA Turnip, Chinese cabbage Parental species RR Aneuploidy Aneuploidy — organism has a partial set of chromosomes due to addition or deletion of specific chromosome(s). or one or more chromosome sets. It is a state in which the number of chromosomes is not an exact multiple of the haploid number. There are numerous possible kinds of aneuploidy. Types of Aneuploids Aneuploids are described relative to the euploid condition. The general genomic formula for aneuploids is somatic chromosome number plus or minus the number of extra or missing chromosomes relative to the euploid somatic number: 2n ± one or more chromosomes. The following table describes the classification system used for aneuploids which includes not only the addition or absence of individual chromosomes (as described in the table below), but also includes the presence or absence of chromosomes characterized by partial rearrangements. Kind of Aneuploid Genomic Formula Interpretation Relative to Euploid Condition Nullisomic 2n – 2 Euploid minus one pair of chromosomes Monosomic 2n – 1 Euploid minus one chromosome Trisomic 2n + 1 Euploid plus one chromosome Tetrasomic 2n + 2 Euploid plus one pair of chromosomes How has aneuploidy been useful in plant breeding? Breeders and geneticists use aneuploids as a tool to • identify the chromosomal location of specific genes, or • substitute a particular chromosome into a genotype. Chromosome transfer or substitution provides an efficient method to transfer specific characters without accompanying adverse traits. It also facilitates chromosome transfer from alien species, enabling the recipient population to acquire genes that are unavailable within its own gene pool. Aneuploids can be used to increase genetic diversity. Try This: Polyploids Induction of Polyploids, Aneuploids, and Haploids Natural Induction of Polyploids Polyploidy occurs spontaneously in nature. There are two mechanisms for natural induction of polyploidy. 1. Unreduced gametes—When the chromosome number fails to be reduced during meiosis, unreduced gametes result and can produce polyploids if the unreduced gamete is successfully fused with reduced or unreduced gametes of the opposite sex. Table 4 Female Gamete X Male Gamete ⇒ Resulting Zygote n (reduced) n (reduced) 2n (normal) 2n (unreduced) n (reduced) 2n + n (1 additional set of chromosomes from female) n (reduced) 2n (unreduced) n + 2n (1 additional set of chromosomes from male) 2n (unreduced) 2n (unreduced) 2n + 2n (2 additional sets of chromosomes, with 1 additional set from each parent) 1. Somatic chromosome doubling—Normal gamete fusion occurs but the chromosomes spontaneously double in the zygote or other somatic tissue. Such chromosome doubling is rare. Ordinarily, conventional crosses between parents having different genomes (referred to as a “wide cross”) are unsuccessful. Their offspring usually exhibit: • Reduced fertility as a result of problems in chromosome pairing during cell division. • Reduced seed set. • Reduced viability associated with genomic instability, especially in those with an odd number of chromosome sets (e.g., triploids, pentaploids). Chromosome doubling generally restores or improves genetic stability, viability and fertility. Artificial Induction of Polyploids Polyploidy can also be induced artificially. Chemicals such as colchicine or environmental shocks can be used to disrupt normal chromosome division and induce polyploidy. Meristematic tissues are especially susceptible to such disruptions. Induction of Aneuploids Aneuploidy occurs during cell division when chromosomes do not segregate properly into daughter cells. When meiosis occurs, germ cells divide to create male and female gametes, typically with each sperm and egg ending up with the same number of chromosomes. However, when nondisjunction occurs, an extra copy may end up in one gamete and the other may not have any copies. In animals, aneuploidy is usually fatal. Plants can tolerate higher levels of aneuploidy, but aneuploidy female gametes are typically more viable than male gametes. Triticale Triticale is a combination of wheat and rye. Efforts to mate these two crops began in 1888. Why was triticale developed? Triticale (Triticosecale) merges the desirable qualities of wheat and rye. • Wheat possesses favorable flour and baking qualities and flavor. • Rye contributes to improved vigor. Rye is winter hardy, adaptable to poorer soils and growing conditions, and resistant to some diseases. Hexaploid triticale was created by combining the genomes of a tetraploid wheat (Triticum turgidum) with rye (Secale cereale). Rye is diploid. In this schematic, the wheat genomes are represented as A and B and the rye genome by R. Octaploid triticale was formed through combination of hexaploid wheat (Triticum aestivum) and rye. The additional wheat genomes are represented by D in this illustration. In the development of hexaploid and octaploid triticale, colchicine was used to double the chromosome number in the newly synthesized interspecific hybrids. Duplication of the genome resulted in restored fertility due to normal pairing of chromosomes in meiosis. With subsequent breeding, triticale was further improved. Today it resembles wheat, except that it has more vigorous growth and produces larger spikes and kernels. Induction of Haploids Spontaneous haploid plants occur at low frequency in many species. In maize, for example, it occurs in the order of less than 1:1000. Haploid plants are of great interest in plant breeding, because genome doubling of haploids by chemical treatment (e.g., by use of colchicine) is possible, which leads to doubled haploid (DH) plants and lines, in which all loci are homozygous. Thus the development of inbred lines can be achieved in a much shorter time than by continued self-pollination starting from a heterozygous plant. To efficiently use DH lines, the spontaneous rates of haploid induction are not sufficient. Thus, procedures have been developed to induce haploidy. Generally, haploids can be: • maternal (based on the egg cell) • paternal (based on microspores / pollen) Moreover, haploids can be induced by using: • Tissue culture techniques, such as anther or pollen culture. • In vivo pollination techniques. In maize, inducer genotypes are available for both paternal and maternal haploids. Current maternal inducer genotypes, when used as pollinator, result in 10% haploid offspring on the female parent. Respective inducer genotypes are available in other crops such as barley and potato. For some crops, other species are used as inducer. In case of wheat, for example, maize is used for induction of haploids. Genetics of Polyploidy Genetic Concepts Polyploid genetics—especially autoploid genetics—is often more complicated than diploid genetics because genetic ratios are more complex. The inheritance of quantitative characters in polyploids is even more complicated. Before examining the complexity of polyploid genetics, let’s become familiar with terminology and associated principles. • homoeologous chromosomes = corresponding chromosomes originating from different but similar genomes. Do not confuse homoeologous chromosomes with homologous chromosomes. Feature Homoeologous Homologous Synapse of pair during the first division of meiosis? Sometimes, but only at homologous or partially homologous regions Yes Have corresponding loci? Sometimes Yes Derived from same genome? No Yes Valency • Valency = the number of homologous or partially homologous chromosomes that associate or pair during meiosis. A prefix is added to indicate the number of chromosomes that associate. Here are some examples. Table 5 Valency levels and corresponding number of meiotically-paired chromosomes. Valency Number of Meiotically Paired Chromosomes Univalent Single Bivalent 2 Trivalent 3 Quadrivalent 4 Pentavalent 5 Hexavalent 6 Polyploid genotypes can be defined by the • number of dominant alleles at a locus, or • number of different alleles at a locus. Chromosomes In each of the following examples, note the valency during meiosis and the chromosome constitution of the gametes. Think about what happens when these gametes fuse with normal or other abnormal gametes. Example 1: Diploid If meiosis proceeds normally, each resulting gamete will receive a full complement of chromosomes and be fertile. In this diploid, assume x = 3, n = 3, and 2n = 2x = 6. Chromosomes form bivalents during meiosis I. One chromosome of each homologous pair migrates to a pole during anaphase I, resulting in a complete chromosome complement in each of two nuclei at the beginning of meiosis II. At anaphase II, sister chromatids separate resulting in a complete chromosome complement in each gamete—gametes are fertile. Example 2: Autotetraploid Normal synapsis in autotetraploids is as quadrivalents, e.g., homologues from all four genomes synapse. Normal first division separation is that bivalents go to each pole, and thus each gamete contains 2x. Random associations of bivalents can also occur. If homologous or partially homologous chromosomes fail to pair properly or fail to disjoin properly during meiosis, there is more likely to be unbalanced distribution of chromosomes to the daughter cells—some gametes will receive an extra chromosome and others will lack a chromosome, usually resulting in non-viable gametes. Thus, fertility is reduced. Reduced fertility is more common in autoploids than alloploids. Inheritance in autoploids is known as polysomic inheritance (or tetrasomic inheritance specifically in autotetraploids) and does not follow typical Mendelian patterns. In this autotetraploid, let x = 4, n = 8, and 2n = 4x = 16. Example 3: Allotetraploid. Alloploids generally have greater fertility than do autoploids. The formation of bivalents during meiosis contributes to greater fertility. Assume this allotetraploid has x = 4, n = 8, and 2n = 4x = 16. Alloploid genetics is complex, too, but usually less so than in autoploids. The formation of bivalents in alloploids contributes to their less complex genetics. In addition, bivalency tends to result in disomic inheritance patterns, where two alleles segregate at a locus. Such inheritance of each individual duplicated loci follows typical Mendelian patterns. Thus, the genetic ratios of alloploids more closely resemble those commonly observed in diploids and it’s the reason why alloploids are often called amphiploids. Alloploids that are derived from divergent progenitors exhibit disomic inheritance and are sometimes called genomic alloploids. Segmental alloploids are derived from partially divergent progenitors and typically exhibit mixed inheritance, consisting of both disomic and polysomic inheritance. Study Questions 1 Select the most likely outcome when these gametes are fused with gametes having the chromosomal constitution indicated. In this question, Gamete A refers to the two upper types of gamete in the previous schematic, and Gamete B refers to the two lower ones. Number of Dominant Alleles The terms used to describe polyploid genotypes encode much information. Let’s define genotypes by the number of times a dominant (A) or recessive (a) allele is present at a particular locus. The most basic genotype is a nulliplex. A nulliplex is a polyploid in which all chromosomes of one homologous type carry the same recessive allele for the particular gene. Table 6 Nulliplex genotypes for a single locus at different ploidy levels. Ploidy Level Genotype Diploid aa Triploid aaa Tetraploid aaaa Octaploid aaaaaaaa Other genotypes have at least one dominant allele. The number of dominant alleles is indicated by the term’s prefix. Let’s look at one locus of an autotetraploid as an example. There are five possible genotypes. Table 7 Genotypes for a single locus of an autotetraploid. Term Genotype Nulliplex aaaa Simplex Aaaa Duplex AAaa Triplex AAAa Quadruplex AAAA If there is complete dominance, then all of the genotypes except the nulliplex will have the dominant phenotype. Only the nulliplex will exhibit the recessive phenotype. The presence of a dominant allele(s) can mask the presence of recessive alleles. The low frequency of recessive phenotypes necessitates the screening of larger populations in order to detect the presence of recessive alleles. If there is partial dominance, the phenotypic ratios are very complex. Genetic linkages are extremely difficult to determine. It may even be difficult to identify autotetraploids as such because of intra- and interallelic interactions. Number of Different Alleles Let’s again use an autotetraploid and consider all the possible genotypes when there are four different alleles at a particular locus. We’ll designate these alleles as a1, a2, a3, and a4 (in this case, however, the use of small letters does not indicate recessive). An individual with all four different alleles present is called a “tetragenic.” Again, the terminology tells us much about the genetic composition. Table 8 Autotetraploid genotypes with four possible alleles at one locus. Term Examples of Genotypes Description Nulliplex a1a1a1a1, a2a2a2a2, a3a3a3a3, a4a4a4a4 Only one allele present Simplex a1a1a1a2, a1a2a2a2, a3a1a1a1, a1a4a4a4, etc. One allele present in three copies with another allele present in one copy Duplex a1a1a2a2, a1a1a3a3, a2a2a4a4, etc. Two alleles, each present in two copies Trigenic a1a1a2a3, a2a2a3a4, a1a2a2a4, etc. One allele present in two copies and two different alleles present in one each copy Tetragenic a1a2a3a4 Four different alleles present Genetics of Polyploidy In an autotetraploid, only four nulliplex genotypes are possible (a1a1a1a1, a2a2a2a2, a3a3a3a3, and a4a4a4a4), and only one tetragenic is possible (a1a2a3a4). Query \(4\) Genotypes Exercise A tetraploid has multiple alleles (a1, a2, a3, and a4) at a particular locus. There is no dominance. A sample of the possible genotypes will be shown. You are required to match each of these genotypes to the term that best describes it. Some terms may have more than one genotype. Autoploid Genetics Now let’s take a closer look at why autoploid genetics is more complex than diploid genetics. Multiple alleles, partial dominance, additive effects, and epistasis make the inheritance in autoploids more complex and linkage relationships “impossible” to decipher. Multi-allelism also affects the response to breeding procedure. FURTHER THOUGHT Many species of crop plants, as well as non-crop plants, are polyploids. Discuss the effects of polyploidy on plants and why this phenomenon is so prevalent. Autotetraploid Genetics Chromosomes assort independently in polyploids, just as they do in diploids. In addition, crossing-over can occur between homologous or partially homologous chromosomes. The higher the ploidy level, the more complex the genetics—the greater the number of chromosomes, the greater the number of possible combinations and genotypes that can be produced. In an autoploid, the number of possible alleles is equal to the ploidy level. Thus, the higher the ploidy level, the greater the possible number of alleles for a given locus. Homologous chromosomes can each carry a different allele at a given locus. Recall that chromatids are the two strands comprising a duplicated chromosome that is still joined at the centromere. When the centromere divides—during anaphase of mitosis or anaphase II of meiosis—then chromatids separate and then are called chromosomes. Since chromosomes and chromatids assort randomly, the inheritance patterns are complicated. Let’s examine the case of an autotetraploid. In this case, assume • two alleles at the locus of interest: the “A” allele confers complete dominance and the “a” allele is recessive; and • a duplex (AAaa) genotype. Plants are self-pollinated and the segregation ratios of the progeny can be determined. Follow the assortment of chromosomes through meiosis to determine the allelic composition of the resulting gametes. Random Chromosome Assortment Chromosome inheritance; two alleles, dominance, duplex (AAaa) What happens when recombination occurs between the gene and the centromere? Let’s follow the random assortment of the chromatids through meiosis and find out the type of gametes produced. Conclusions 1. Genetic segregation is more complex in autopolyploids compared to diploids. Homozygous recessive genotypes are more rare in autopolyploids than in diploids. Thus, if breeders intend to fix a particular allele, larger populations are required. 2. There are different models describing genetic segregation in autopolyploids. Two extreme models have been mentioned above: chromosome and chromatid segregation. As shown for the duplex case AAaa, both models lead to different segregation rates, with homozygous recessives occurring at frequencies of 1/36 (chromosome segregation) and 1/22 (chromatid segregation). Which of these models is more accurate for a given gene, depends on the distance between this gene and the centromere. The larger the genetic distance, the more likely chromatid segregate independently. Plant Cell and Tissue Culture Plant cell and tissue culture methods enable plant breeders to obtain whole plants from somatic or haploid cells or tissues without sexual reproduction. Such methods are beneficial for handling plant material with variation in ploidy that may be otherwise difficult to reproduce. These methods take advantage of the totipotency of cells to generate plants. Under special conditions, cells and tissues extracted from a donor plant can be induced to produce undifferentiated, unorganized cells that are totipotent. Totipotent cells possess all of the genetic information and capabilities needed to produce a whole organism. Because undifferentiated cells have not yet embarked on a developmental track to become a specific tissue or organ, their fate remains undetermined. These totipotent cells can be propagated in vitro and coaxed into developing roots and shoots, ultimately forming a whole plant. Tissue culturing has several attractive features: 1. Large numbers of totipotent cells can be cultured in a small area. 2. The culture medium is usually well-defined—each component in the medium is known. Additions or exclusions of specific substances can provide a means of selection for some phenotypes. 3. Ploidy level can be controlled by selection of explants. 4. Heritable variation can be induced to generate new genotypes. 5. Tissure culture is often required for genetic transformation. General Tissue Culture Procedures Cell and tissue culture procedures are similar. The particulars of this general procedure vary with the cell or tissue source, its age and health, species, genotype, culture type, and culturing objectives. Types of Tissue Culture Cell and tissue culture can be used to maintain genotypes or to obtain novel genotypes. Different tissues or types of culture are used to achieve a variety of objectives. • Clonal propagation or Micropropagation—The general purpose of clonal propagation is to reproduce genetically identical plants without going through a gametophytic or sexual stage. A clone is a population of plants derived from a single plant, or a population of cells descendant from a single cell. This is very important in the production of high-value horticultural crops. • Callus culture—Callus culture is similar to clonal propagation, except that the cultured tissue forms disorganized, undifferentiated masses of cells. Spontaneous variants and mutants can occur during callus culturing. Callus culture is often required for successful genetic transformation. • Suspension culture—Similar to callus culture, except that cells or cell aggregates are in a liquid medium, rather than plated on an agar-solidified medium. These cultures have greater chromosome instability and reduced regeneration potential due to their rapid growth rates. The liquid medium provides relatively uniform exposure of cells to any selecting agents. • Anther Culture—Anthers containing immature pollen are cultured to develop haploid plants or, with chromosome doubling, to obtain homozygous diploids. Anther culturing involves placing anthers containing immature pollen on medium. Generally, anthers are obtained from F1 or F2 plants to maximize the genetic diversity among the haploids generated through culturing. The greater the genetic diversity, the greater the likelihood of obtaining a desirable genotype to use as breeding lines. • Embryo culture—Embryo culture allows the rescue of embryos that would normally be unobtainable or aborted because of some type of incompatibility. From the cultured embryos, whole plants may be regenerated. Embryo Culture Embryo culture is commonly used to bypass genomic incompatibility. Unfertilized embryos can be used to generate haploid plants or they can be pollinated in vitro to bypass self-incompatibility. Genomic Incompatibility Often, embryos resulting from wide crosses (crosses between distantly related parents) are incompatible with the endosperm—the endosperm cannot adequately supply the needs of the growing embryo. As a result of this incompatibility, the embryo will normally abort before the seed is mature. Embryo culture provides a method to circumvent abortion and obtain such hybrids. After fertilization, embryos are excised, with or without the ovule, and grown on a nutrient medium. The nutrient medium substitutes for the endosperm in nourishing the growing embryo. Via culturing, hybrid plants can be generated and grown to maturity. These plants will be sterile and must be treated with colchicine to get chromosome doubling. Flowers resulting from the doubled chromosome number will produce viable gametes much like allopolyploids. Ovule Culture Unfertilized ovules can be excised and cultured to obtain haploid plants. Alternatively, homozygous diploids can be generated by applying colchicine or other treatments to these cultured ovules to induce chromosomal doubling. This process is similar to anther culturing and will be discussed more fully in the next section. Ovule culturing has been used with barley, wheat, rice, and maize. In vitro pollination and fertilization In self-incompatibility systems where pollen tubes fail to grow, in vitro pollination offers an alternative to overcome such incompatibility. An unfertilized ovule is extracted from the pistillate flower and placed on medium. In vitro pollination and fertilization are accomplished by applying pollen directly to the ovule. Thus, the incompatibility factors localized on the stigma or in the style are avoided. The resulting embryo is nourished by the medium to obtain plantlets. Study Questions 2 Uses of Polyploidy, Aneuploidy, and Haploidy Because of the genetic complexities, breeding polyploids, especially autoploids, is challenging and progress is often slower than breeding diploids. Generally larger populations must be evaluated to obtain favorable types and to stabilize the genotype for the desired phenotype. Breeders also must be concerned about and test for the optimum ploidy level for the particular species and desired characters. Although polyploids present breeding challenges, they also offer opportunities for crop improvement. ALLOPLOID USES Alloploids occur more frequently in nature than other types of polyploids. Alloploids can combine the best characters of different species. Table 9 Examples of alloploid uses Alloploid Uses Example 1. Identify genetic origin of crops Wheat evolved through a combination of three related species. Cultivated Brassica species evolved through a series of interspecific hybridizations. 1. Generate new plant genotypes and species Triticale resulted from a cross of wheat and rye. 1. Enable introgression of genes from related species Cotton lint strength was improved through transfer of genes from undomesticated cotton into cultivated cotton. (see Fehr, 1987, p. 82 “Introgression of genes”) Generally, alloploids are more vigorous and fertile than autoploids or aneuploids. AUTOPLOID USES Autoploids are not as common in nature as alloploids, but in some cases, they are developed as a result of artificially doubling the chromosome number of a diploid species. Depending on the crop, the reduced fertility that tends to occur as a result of induced polyploidy can be an advantage or a disadvantage. Reduced fertility found in autoploids is not a problem for species where the target organ is not seed—for example in forage crops. Fertility tends to be low and vegetative growth greater in autoploids than in diploids. This is an advantage in crops produced for vegetative parts; examples include several tuber and forage crops. Autoploidy is also used to generate sterile plants in which seeds are undesirable, such as in banana and watermelon. Autoploids are also used to facilitate interspecific or wide crosses. Such crosses allow traits not otherwise available in the crop’s gene pool to be acquired, enhancing the genetic diversity of the crop. ANEUPLOID USES Aneuploids carry an extra chromosome(s) or are lacking one or more chromosomes — their genomes are unbalanced. Although this usually results in their reduced vigor, their unbalanced genomes provide benefits for genetic studies and increasing the genetic diversity of a crop. Diploids have very low viability in the aneuploid condition and, therefore, have limited utility. Aneuploidy is more useful in alloploids. Aneuploids have been used to: • map genes to specific chromosomes • identify linkage groups • substitute or transfer chromosomes between related or unrelated species With molecular techniques available today aneuploids are seldom used to map genes and identify linkage groups. Chromosome transfer or substitution is a more efficient method than conventional crossing and subsequent segregation and selection because only the particular chromosome carrying the desired gene(s) is transferred. In conventional breeding approaches, both desired and undesired genes are transferred from the donor to the recipient population; subsequently, the desired and adverse traits must be sorted out, often requiring several generations of breeding and selection. Similar to the effects of wide crosses in alloploids, aneuploidy facilitates chromosome transfer from alien species, enabling the recipient population to acquire genes that are otherwise unavailable within its gene pool. Thus, aneuploids can be used to increase genetic diversity. HAPLOID USES Haploid plants are of interest in plant breeding and plant genetics because their genomes can be doubled and homozygous lines can be obtained much faster than by conventional techniques of inbreeding. Genetically homogeneous DH lines that can be quickly obtained make it practicable to establish respective experimental populations for gene mapping. Discussion Wenzel proposed the analytic–synthetic breeding scheme in potato (shown below). Discuss the strengths and weaknesses of this scheme.

textbooks/bio/Agriculture_and_Horticulture/Crop_Genetics_(Suza_and_Lamkey)/1.10%3A_Ploidy-_Polyploidy_Aneuploidy_and_Haploidy.txt

Arti Singh; Jessica Barb; Asheesh Singh; and Anthony A. Mahama Africa produces a diversity of crops including cereal, pulse, oilseed, root, and tuber species (Table 1), but contributes less than a quarter of the world production of root and tuber crops (Ngopya, 2003). In East Africa, bananas (especially cooking bananas) are an extremely important crop. In Uganda, for example, bananas serve as the largest only source of calories. Even though crop production is mostly for subsistence needs, there is tremendous business potential in local and international markets. Table 1 Some major food crops of Africa Cereal Pulse Oilseed Root and Tuber Maize Dry beans Seed cotton Cassava Sorghum Groundnut Sesame Yams Millet Cowpeas Palm Sweet Potatoes Rice Soybean n/a Potatoes Wheat Cocoa beans n/a n/a Barley n/a n/a n/a Learning Objectives • Provide an overview of major crops and production challenges in Africa • Review or introduce basic breeding principles • Understand the concepts of setting plant breeding objectives in a program • Review plant reproductive systems Production Challenges for African Crops Production of African crops is dependent on yield potential and on protection of yield (that is, realization of yield potential). Numerous factors which reduce production include: 1. Biotic constraints to production (i.e., living biological organisms such as disease causing organisms and insects) 2. Abiotic constraints to production (i.e., non-living factors such as heat, drought, poor soil fertility) 3. Lack of incentive to increase production due to poor marketing opportunities, the lack of infrastructure (such as roads, storage facilities, etc.), and policies that fail to encourage increased production. 4. Lack of capital to invest in increasing production Breeding efforts only address the first two constraints of biotic factors and abiotic factors. The ability to store a crop after harvest is also affected by biotic and abiotic factors. In order to improve food security and provide income to farmers, continuous efforts by plant breeders are needed to increase production per unit area of land while maintaining crop quality. Systems of Reproduction in Crops Asexual Reproduction Plant reproductive systems (or mating systems) fall into three main categories: asexual, autogamous (self-fertilizing), and allogamous (cross-fertilizing). These topics are covered in greater detail in Crop Genetics on Reproduction in Crop Plants. Asexual reproduction generates individuals that are genetically identical to the mother parent plant and are referred to as clones. The two main forms of asexual reproduction/propagation are vegetative and apomictic. Vegetative propagation is the creation of clones from stem cuttings, suckers (similar to tillers), tubers, runners (stolons), rhizomes, bulbs, scions, and other plant parts. Cassava, sweet potato, and sugarcane are propagated via stem cuttings. Bananas are typically propagated by suckers, while potatoes are propagated by tubers. Elephant grass (Napier grass, Pennisetum purpureum) is propagated by rhizomes, sets (suckers), and stem cuttings. Apomictic reproduction is the asexual propagation of a plant via clonal seeds formed by one of several means that either bypass meiosis or result in a failure of meiosis. Examples of apomictic crops include Citrus and many perennial forage species. Sexual Reproduction (Self- and Cross-Fertilization) Sexual reproduction involves the union of a male sperm with a female egg cell or ovary. This process is called fertilization. There are two types of pollination: self-pollination and cross-pollination. When the pollen of a plant pollinates a flower on the same plant the process is called self-pollination (Fig. 2A). Pollination is the transfer of male sperm carried in pollen to the female part of a flower called the stigma (Fig. 2B). When the pollen of a plant pollinates a flower on another plant of the same species the process is called cross-pollination. In nature, cross-pollination requires wind, water, insects, birds, or other animals to transfer the pollen. Most cultivars (i.e., cultivated varieties) are created by a process that involves at least one generation of cross-pollination by plant breeders; including both self- and cross-pollinated species. (See Reproduction in Crop Genetics). Both the sperm and the egg are haploid, meaning they contain a single set of chromosomes from the male or female parent, respectively. Fertilization unites the single set of chromosomes in the sperm nucleus with the single set of chromosomes in the egg nucleus to produce a complete pair of chromosomes (diploid) in the zygote. Several crop species, for example, banana, sweet potato, potato, and many grasses have three or more sets of chromosomes and therefore they are, in general, called polyploids (e.g., triploids, tetraploids, pentaploids, hexaploids, octaploids). Flowering plants have a unique process called double-fertilization in which the embryo and the endosperm are fertilized separately. Each pollen grain contains two pollen nuclei; one pollen nucleus fuses with the egg cell to form a diploid zygote and the second pollen nucleus fuses with the two polar nuclei in the ovule, eventually developing into a triploid endosperm. The zygote begins to divide by mitosis forming a multicellular embryo within the ovule. The endosperm provides the energy source that is used by the embryo prior to formation of true leaves that begin photosynthesis. Following fertilization the ovule (with embryo and endosperm) develops into a seed. Genetic Information Transfer Genetic information (i.e., DNA) from both the male and female parents is present in a seed produced by fertilization. It is this union of sperm and egg cell that results in the creation of genetic variation (if the male and female gametes possess different genetic information). Offspring that result from the union of gametes from male and female plants with dissimilar genotypes are known as hybrids. Plant breeders select genotypes (i.e., male and female parents) that complement each other to combine the positive (or desirable) traits from each parent in the hybrid offspring. Genetic information is transferred from generation to generation through seed, which typically consists of an embryo, an endosperm, and a seed coat. The endosperm includes the beginnings of a radicle, a hypocotyl, and one or two cotyledons (seed leaves), which support the formation of roots, stems, and true leaves, respectively. Monocot plant species have a single cotyledon (i.e., mono = one) and dicot plant species have two cotyledons (di = two). The seed coat serves as a protective coat around the seed. Plant Reproductive Systems Autogamous Mating System Crops are capable of both self- and cross-pollination. These crops are classified as either autogamous (self-pollinated) or allogamous (cross-pollinated) depending on the relative frequency of self- or cross-pollination that is observed in the species. Self-fertilization occurs if male and female gametes derived from the same plant unite. Self-fertilization also refers to the union of gametes from the same genotype. One trait, i.e., plant characteristic, that virtually ensures self-pollination is cleistogamy, where pollen shed occurs before the flower opens (i.e., anthesis). Cleistogamy promotes self-pollination and severely limits cross-pollination. Cleistogamy is observed in some legumes (e.g., groundnut, peas, some beans, soybean). In some cereals (e.g., rice, wheat, and barley) the majority of self-pollination occurs before flowers open, but some cross-pollination can occur after the flowers open, even if only partially. This allows for some cross-pollination compared to relatively little or no cross-pollination in cleistogamous species. Most self-fertilizing species undergo a small amount of outcrossing: for example in soybean natural outcrossing of 0.03% to 2% or higher has been observed in some conditions (Caviness, 1966; Ray et al., 2003). Thus, it is critical to understand the mating system of the crops you are working with and how it is affected by different environmental conditions. Examples of important crops with an autogamous mating system include: sorghum, millet, rice, wheat, barley, groundnuts, cocoa, and major pulse crops like cowpeas, and dry beans. Allogamous Mating System Fertilization in cross-pollinated plants occurs via the union of male and female gametes from different plants, which are often different genotypes. A crop is classified as allogamous when it has a higher percentage of pollination and fertilization with different individuals than with itself. There are several traits/plant characteristics that promote cross-pollination: 1. Male sterility: Male sterility is caused by the formation of non-functional pollen grains, which prevents self-pollination and promotes cross-pollination. Two types of male sterility are: cytoplasmic male sterility (CMS), which is caused by mitochondrial genes interacting with nuclear genes; and genic male sterility (GMS), which is caused by nuclear genes alone. These phenomena can be exploited for hybrid seed production. 2. Self-incompatibility: Self-incompatibility refers to the inability of viable pollen to fertilize flowers of the same or similar genotype. Self pollen is rejected on the surface of the stigma or in the style while foreign pollen is unaffected and can germinate, grow, and fertilize the egg cell. Self-incompatibility prevents self-pollination and enforces cross-pollination. More details on self-incompatibility is found in the Crop Genetics – Controlled Hybridization Module. 3. Imperfect flowers: Imperfect flowers are missing either stamens or pistils (i.e., unisex flowers). Unisex flowers either occur on the same plant (i.e., monoecious) or different plants (i.e., dioecious). Monoecious plants have separate male (i.e., staminate) and female (i.e., pistillate) flowers, although they occur on the same plant. In some crops, the male and female flowers are present in the same inflorescence such as in banana (Fig. 4). In some cases, they are on separate inflorescences, as in maize (Fig. 5). Dioecious plants have separate staminate and pistillate flowers present on different plants. Dioecious plants are diclinous (i.e., having flowers of only one sex). Examples of dioecious crops include papaya, date palm, and spinach. Protandry/Protogyny 1. Protandry/Protogyny: Cross-pollination is often observed in crop species that show protogyny (i.e., the pistils/stigmas of a plant mature and become receptive before the anthers of that plant) and protandry (i.e., stamens/anthers of a plant develop and the pollen grains mature and are shed before the pistils/stigma of that plant mature and become receptive). Protogyny is typical in cassava (although this crop is not typically seed propagated) (Fig. 6), and it has also been used to make hybrids in pearl millet (Andrews et al., 1993). Sunflower and coconut are examples of crops that display protandry. Mechanical obstructions such as a membrane around the anthers in alfalfa (also called Lucerne) flowers may also limit normal dehiscence of pollen and limit self-pollination. Understanding Reproductive Systems Breeders must understand the reproductive system of the crop they are working on to make knowledgeable decisions about which breeding methods (i.e., crossing techniques, population maintenance, isolation distances, line and population development) are suitable and which type of cultivar (i.e., hybrid, pure-line, synthetic, clone) is appropriate. The modes of pollination and reproduction of some major crops are shown in Table 2. More information on the reproductive systems of crops is found in Allard (1960; pp. 40-41). Table 2 Methods of pollination and reproduction of crop examples Mode of pollination and reproduction Some examples of crop plants A. Autogamous Species 1. Seed Propagated Rice, wheat, finger, millet, barley, oats, common bean, cowpea, tomato, groundnut, soybean 2. Vegetatively Propagated Potato B. Allogamous Species 1. Seed Propagated Maize, rye, pearl millet, sunflower, oil palm 2. Vegetatively Propagated Sugarcane, coffee, cocoa, banana, cassava, yam, rubber C. Autogamous species with cross pollination Sorghum, cotton, pigeon pea Breeding Populations and Cultivar Types Populations Genetically speaking, a population is a group of individuals that share a common gene pool. If all individuals within the population have the same genotype the population is homogeneous; if the individuals have different genotypes the population is heterogeneous. For example, gene A has alleles A1 and A2 (assuming a diploid). If a population is homogenous then all individuals are the same; all are A1A1 (homozygous), or all are A1A2 (heterozygous), or all are A2A2 (homozygous). If a population is heterogeneous then some individuals have different genotypes; a combination of A1A1, A1A2, and/or A2A2. The genotype of a population and individuals within a population varies depending on the reproductive system of a species. A natural population of a cross-pollinated species consists of a heterogeneous mixture of individuals some or most of which will be heterozygous (A1A2) for individual loci. A natural population of a self-pollinated species will usually also consist of a heterogeneous mixture of individuals, but each individual will be mostly homozygous (A1A1and/or A2A2) at individual loci. Populations of an asexually reproducing species may be homogeneous or heterogeneous and individuals will likely be heterozygous (A1A2) at many loci. Cultivars Some examples of populations are (1) a commercial maize hybrid cultivar (allogamous) which is homogeneous (single cross hybrid) and heterozygous, (2) a commercial soybean pure line cultivar (autogamous) which is homogeneous and homozygous, (3) a commercial maize synthetic cultivar which is heterogeneous and heterozygous, and (4) a commercial potato cultivar (clonal), which is homogeneous and heterozygous. Clonal, synthetic, and hybrid cultivars are heterozygous. Pure-line cultivars are homozygous. Self-pollination is used to achieve homozygosity in an autogamous species. In allogamous species self-pollination is used to develop inbred lines that are used as parents to create hybrids Clonal Cultivars Clonal Cultivars: Most commercial crops are propagated through seed. However, a significant number of agriculturally important species are propagated by using plant parts other than seed which include: stem cuttings, suckers, tubers, and stolons (Fig. 7). As the term ‘clone’ implies, offspring are identical to the mother parent clone plant and are therefore homogenous in the absence of pollination and mutation. Clonal cultivars, although homogenous, are typically heterozygous, therefore ‘hybrid vigor’ is fixed and maintained, unlike a maize hybrid, which is propagated through seed, and loses hybrid vigor with each generation of selfing or sib-mating. The steps to create/develop a clonal cultivar are: 1. develop a genetically variable base/source population; 2. evaluate and select superior clones from the population; and 3. multiply the new cultivar for commercial use. Examples of clonal cultivars include: cassava, sweet potato, potato, cacao, and yam. The cultivars of these crops are homogeneous and heterozygous Synthetic Cultivars Synthetic cultivars: Synthetic cultivars are produced by intermating a population of purposefully selected inbred lines, clones, hybrids, strains, or other populations of cross-pollinated plants. Synthetic cultivars are highly heterozygous and heterogeneous. Inbreeding depression is severe and plants that develop from self-pollinated seed lack the vigor of those obtained by cross-pollination. In a heterogeneous population, each plant is genetically different from another. The components (clones, inbred lines, etc.) of a synthetic cultivar are maintained in their original form so that the cultivar can be reconstituted as needed. A synthetic cultivar is different from an open-pollinated variety because the components are maintained in their original form while with an open-pollinated variety the components are not maintained. Clonally propagated plants or inbred lines with desirable characteristics (traits) are selected and then isolated and allowed to cross-pollinate, randomly or in a structured format in a polycross nursery (Fig. 8). Seed is harvested from the clones or inbred lines and planted in progeny rows for evaluation. The best clones or inbred lines are then selected both for superior plant traits and on the performance of their progeny rows, which measures their general combining ability with the rest of the population. These selected parents are then replanted and permitted to cross pollinate in isolation. Open-pollinated seed harvested from these parental clones or inbred lines (after one or more cycles of intermating) is then sold as a synthetic cultivar. Cultivar Development Example An example of the development of maize synthetics is the development of the maize population HIS1 (Brewbaker, 2009). The development of synthetic cultivars of maize and other crops differs from that described for turfgrass and forage species because of several important biological differences. 1. It is possible to inbreed maize because inbreeding depression is much less than in turfgrass and forage species. 2. There is no self-incompatibility in maize and some self-pollination can occur, even though it is a monoecious species that is wind pollinated. 3. It is an annual crop. Pure-Line Cultivars Pure-line cultivars: Pure-line cultivars are developed for self-pollinated species. Self-pollination leads to homozygosity and homogeneity. Improved self-pollinated cultivars are obtained through three basic approaches. • Introductions: a breeder assembles cultivated varieties currently grown in other regions (domestically or internationally) and identifies lines that exhibit desirable characteristics and are adapted to the new target area. A breeder must be aware of licensing and material transfer agreement (MTA) issues (see next module) before acquiring lines from different sources. Introductions are typically received as pure-lines, but they may contain mixtures of different genotypes or off-types and may require that the breeder rogues these plants out to obtain clean and uniform seed that can be released commercially. [Note: problem of mixture or off-types is common for self-pollinated, cross-pollinated or clonal crops; MTA are required for most if not all crops (self- and cross-pollinating and clonal) when the seed or explant is sent or acquired by breeders]. • Selection: a breeder assembles landraces, identifies the best genotypes, and releases one or more for commercial production. This approach has applicability for orphan crops but has not been widely used for major world crops. • Hybridization: a breeder makes hybridizations between genotypes with desirable characteristics, evaluates, and selects superior genotypes for commercial production. This is the most common approach for developing new cultivars. Cultivar Examples Examples: beans, cowpea, rice, finger millet, tobacco, and wheat. Cultivars are homogeneous and homozygous. Hybrid Cultivars Hybrid cultivars: Hybrid cultivars are produced by crossing inbred lines, typically two inbred lines in the case of a two-way/single-cross hybrid. Inbred lines are chosen for their combining ability to achieve maximum expression of hybrid vigor (i.e., the F1 trait mean is higher than the mid-parent mean and higher than the trait values for the individual inbred parents). Hybrid vigor (i.e., the visible measure of heterosis) is more important in allogamous crop species as the expression is typically lower in autogamous species. Pollen control via the use of mechanical tools, chemicals, or genetic male sterility is necessary to create hybrid seed. Heterosis (or hybrid Vigor) is defined as the difference between the hybrid and the mean of the two parents (Falconer and Mackay, 1996). This definition is also described as midparent heterosis. High-parent heterosis is the superiority of a hybrid over the better parent (Bernardo, 2014). Examples of hybrid cultivars include: commercial single-cross maize hybrids, commercial three-way cross maize hybrids, and sunflower hybrids. Hybrid cultivars are usually utilized for allogamous species but some hybrids are produced for some autogamous species (e.g., sorghum, tomato). Single-cross hybrid cultivars are homogeneous and heterozygous (Fig 10). Three-way hybrids are both heterogeneous and heterozygous. Seed Law Definition of a Cultivar According to UPOV According to seed law a cultivar or cultivated variety is distinct, uniform, and stable. • Distinct: a cultivar is distinct if it can be differentiated by one or more identifiable characteristics from all other cultivars currently available or previously developed. Distinctiveness can involve morphological, physiological, molecular, or other characteristics. • Uniform: a cultivar is uniform if no variation among individuals exists for the distinguishing characteristics that make it distinct from other cultivars. • Stable: a cultivar is stable if plants remain the same from generation to generation. If there are multiple generations in a cycle of propagation the plant characteristics should be the same at the end of each such cycle. These concepts come out of ‘The International Union for the Protection of New Varieties of Plants’ (UPOV), which is an intergovernmental organization based in Geneva, Switzerland. UPOV was established in 1961 by the International Convention for the Protection of New Varieties of Plants (the “UPOV Convention”). Below is an excerpt from The International Union for the Protection of New Varieties of Plants (UPOV): “The mission of UPOV is to provide and promote an effective system of plant variety protection, with the aim of encouraging the development of new varieties of plants, for the benefit of society.” International Union for the Protection of New Varieties of Plants (UPOV) Criterion Distinctness (Article 7) Criterion: A variety is deemed to be distinct if it is clearly distinguishable from any other variety whose existence is a matter of common knowledge at the time of filing of the application. Uniformity (Article 8) Criterion: A variety is deemed to be uniform if, subject to the variation that may be expected from the particular features of its propagation, it is sufficiently uniform in its relevant characteristics. The uniformity requirement within the Convention has been established to ensure that the variety can be defined as far as is necessary for the purpose of protection. Thus, the criterion for uniformity does not seek absolute uniformity and takes into account the nature of the variety itself. Furthermore, it relates only to the relevant characteristics for the protection of the variety. Stability (Article 9) Criterion: A variety is deemed to be stable if its relevant characteristics remain unchanged after repeated propagation or, in the case of a particular cycle of propagation, at the end of each such cycle. As with the uniformity requirement, the criterion for stability has been established to ensure that the identity of the variety, as the subject matter of protection, is kept throughout the period of protection. Thus, the criterion for stability relates only to the relevant characteristics of a variety.” Exercise Read the UPOV Overview online and go through the table of contents (under FAQs) to familiarize yourself with plant variety protection and plant breeder’s rights: • What is UPOV? • What is a plant variety? • Why do farmers and growers need new plant varieties? • How are new plant varieties of benefit to society? • What is Plant Variety Protection? • Who can protect a plant variety? • What are the Exceptions to the Breeder’s Right? • What are the conditions for obtaining protection? • What information is there on the impact of PVP? Visit the main UPOV website for more details on UPOV. Setting Breeding Objectives Plant breeding objectives will depend on geographical adaptation, prevalent biotic and abiotic factors that influence production, uses of a cultivar, crop reproductive system (for example, pureline or hybrid), and factors that are important to farmers, and end-users. It is fundamental that genetic variation exists for the trait that you are attempting to improve and that it is transmissible. Plant breeding programs need to be adequately set up for screening breeding material for the traits that are being improved based on the objectives. Applied learning activity #1 will be used to better understand the considerations involved in setting plant breeding objectives.

textbooks/bio/Agriculture_and_Horticulture/Crop_Improvement_(Suza_and_Lamkey)/1.01%3A_Basic_Principles_of_Plant_Breeding.txt

Asheesh Singh and Anthony A. Mahama Understanding the symbols used to describe the progeny generated following hybridization and self-pollination is critical for clear communication between/among researchers in the breeding community. The two symbols used by plant breeders to enable such communication are F and S, where F is derived from the word filial, defined as the sequence of generations after the mating of two parents. S is the symbol used to denote generations of self-pollination. Different systems exist in the use of the F and S symbols to describe breeding groups – populations, individuals, and inbred lines – making it the more crucial to avoid misleading outcomes by clearly stating the meaning of symbols, and thus underscores that advantages and disadvantages exist in the use of each system. Learning Objectives • Demonstrate the relationship between the “F” and “S” symbols used to designate generations of selfing or sib-mating. • Describe how breeding lines are designated by “F#:#” or “S#:#” according to the generation that they were derived. • Demonstrate pedigree writing. • Demonstrate how selection history is recorded using a Breeder’s Cross Identification (BCID) designation. Using F# and S# Symbology A. Using “F#” and “S#” to designate the number of generations of selfing or sib-mating: In plant breeding, the ‘F’ symbol is used to denote the filial (i.e., family) generation of offspring following a cross between two or more parents. The subscript (#) represents the specific generation (F#). F1 is the first generation following a cross and subsequent generations are designated F2, F3, F4, etc., based on the number of generations the offspring are self-pollinated or sib-pollinated (i.e., pollinated by a sibling plant in the same progeny row). Pollination can occur naturally or artificially if it is imposed by the breeder. If both of the parents of a cross are homozygous then the F1 offspring will be homogeneous (i.e., all plants will be uniform and genetically the same) and heterozygous at individual loci. If either or both of the parents are heterozygous then the F1 offspring will be heterogeneous (non-uniform). In a complex cross involving more than two parents, even if the parents are homozygous), the F1 generation will be heterogeneous and heterozygous. Single Gene Example With Two Homozygous Parents In the example above (Fig. 1) , both parents were developed by the breeder after undergoing several generations of selfing so that they are homozygous at all loci. The cross between these two unrelated parents produces F1 progeny that are all uniformly heterozygous (Aa), and F1 progeny population (all F1 from this cross) will be homogeneous since each F1 will be ‘Aa’ type at this locus. Self-Pollination When an F1 plant is self-pollinated or when two F1 plants are crossed with each other, F2 seed is produced. If the parents were homozygous then the F2 generation is the first generation when the offspring are heterogeneous (i.e., segregating for different parental alleles). The F2 generation is typically the generation when selection for simple traits begins. Self-pollination of F2 plants produces F3 plants, self-pollination of F3 plants produces F4 plants, and so on as shown in Fig. 2. Cross-Pollinated Species In cross-pollinated species, ‘S#’ is used instead of ‘F#’. The symbol S0 can be used to describe the progeny from a single cross between two homozygous parents as either: 1. Similar to F1 (in self-pollinated) which indicates that the plant was not derived from self-pollination or 2. Similar to F2 (in self-pollinated) which indicates that the population is formed by random mating and is therefore heterogeneous and heterozygous Therefore, it is important that the breeder clearly describes what she/he is referring to in a particular situation and then be consistent in usage. Using Fx:y and Sx:y Breeding Lines B. Using “Fx:y” or “Sx:y” to describe breeding lines according to the generation they were derived: Breeding lines (or genotypes) are derived from individual plants at various generations. An F2:4 line refers to an F4 line that was derived from a single F2 plant. The F2 plant was selfed to produce F3 seeds, which were then grown in a single F3 progeny row, self-pollinated, and then harvested as a F4 bulk of many or all of the F3 plants in this row. Based on this scheme each individual F2 plant gives rise to a genetically distinct F2:4 line. These lines are also described as “F2-derived lines in the F4 generation”, or simply “F2-derived F4 lines”. Summary An F3:4 line refers to an F4 line (or progeny row) created from a single F3 plant growing in an F3 progeny row that was produced from the seed of a self pollinated single F2 plant. An individual plant was selected from an F3 row to produce F4 seed and this seed when grown represents an F3:4 line. These are also described as “F3-derived lines in the F4 generation”, or simply “F3-derived F4 lines”. The difference from F2:4 lines is because the first subscript designates the generation of the last individual-plant selection (Fig. 3). To summarize: Fx:y or Sx:y , describes ‘x’ as the generation where single plant was harvested separately to give rise to the derived line, and y represents the current generation of inbreeding of the plants within this derived line. Writing a Standard Pedigree Symbology C. Example of writing a standard pedigree: Each organization follows a different standardized system for recording pedigrees. In this section, we will describe the system adapted from Purdy et al (1968) modified and used by wheat breeders at CIMMYT (CGIAR institute). Depending on the crop you work on and where you are employed you may use a modified system. The female parent is designated by listing it first (starting from the left) followed by the male parent (on the right). For example, A is the female parent and B is the male parent in an (A x B) cross. An (A x B) cross can also be written as A/B. If an F1 (A/B) plant is pollinated with parent C, and the F1 is used as the female and C as the male, the resulting three-way cross would be designated as A/B//C. Subsequent crosses with parental materials D, E, F, and G used sequentially (all as males) are indicated using a number to record the cross order in the following way: A/B//C/3/D/4/E/5/F/6/G. Example If the example above is changed to use D & F as female parents, with E and G remaining as males, the cross would be recorded as follows: • Step 1: A/B is the first cross, • Step 2: A/B//C is the second cross, where A/B is the female. • Step 3: D/3/A/B//C is the third cross, with D as female, and A/B//C as male. • Step 4: D/3/A/B//C/4/E, with E as male, and the 4-parent cross as the female. NOTE: bold and underline text is for information and instructional purpose only. In writing a pedigree, you will not have to bold text. One will simply write the pedigree as D/3/A/B//C/4/E The inclusion of “5/F” as the female and 6/G as a male completes the pattern. Backcross Pedigree In multiple backcrosses, the sequence of these letters from left to right corresponds to the sequence in which the backcrosses are made. Backcross pedigrees include an asterisk (*) and a number indicating the dosage of the recurrent parent. The asterisk and the number are placed next to the crossing symbol (/) that divides the recurrent and donor parents. The following are examples of pedigree formats involving backcrosses: • A is the recurrent parent: A*2/B of the initial cross and has been used as a parent two times. Therefore, A*2/B indicates one backcross or a BC1 cross. • B is the recurrent parent: A/3*B, and has been used as a parent three times. Therefore, A/3*B indicates a BC2 cross. A*2/B is therefore A//A/B and indicates that A was used as a female in both F1 and BC1. A/3*B could be B/3/A/B//B and indicates that A was used as a female, F1 was then used as female, and BC1 was used as male. A/3*B could be A/B//B/3/B and indicates that A was used as a female, F1 was then used as female, and BC1 was used as a female. The F#: derived symbols as previously described for regular crosses will follow the BC# designation. For example, BC1F2:4 or BC2F2:4. Identity Number Assigning an Identity Number to Each Cross or Backcross Every cross should receive a unique ID number that will allow everyone in the breeding group to recognize the year the cross was made (e.g., 2014), a cross number (e.g., 1001), and perhaps the target purpose of the cross (e.g., HO for high-oil, or abbreviation for another specific trait or market segment). Using BCID Recording Selection History D. Recording selection history using a Breeder’s Cross IDentification (BCID) designation: Every F1 plant, segregating line, or advanced line in a program is assigned a Breeders’ Cross IDentification (BCID) and a selection history. This selection history records the process of selection, which describes where and how the initial cross was made and where and how subsequent selection steps occurred for each generation of selection. Example An example of this system is provided below (using CIMMYT’s wheat breeding program). Each BCID begins with a letter designation for the origin of the cross (e.g., CM = crusa Mexicana; Spanish for ‘Mexican cross’). This is followed by an indication of the kind of cross (e.g., BW = bread wheat x winter wheat, SS = spring x spring wheat; SW = spring x winter wheat), an abbreviation of the year when the cross was made (e.g., 00 = 2000), an abbreviation of the location where the cross was made (e.g., Y = Yaqui Valley), and finally a sequential number representing the order in which that cross was made within the crossing cycle (e.g., 0124). The table below shows the letter codes used to indicate the locations in Mexico where crosses were made and the different environmental conditions where CIMMYT breeders carry out selection in wheat. Note that more than abbreviations than shown in table are used to describe locations or nurseries. Table 1 Location and selection environment code in CIMMYT wheat breeding Code Description of Location and/or Environmental Condition B El Batan M Toluca (“M” stands for the State of Mexico) Y Cd. Obregon full irrigation (Cd. = Cuidad = city; “Y” stands for Yaqui valley region in Mexico) KBY Cd. Obregon Karnal bunt HY Cd. Obregon Heat (heat, late planting) SY Cd. Obregon Semi-arid (reduced irrigation) PR Poza Rica PZ Patzcuaro SJ Sierra de Jalisco (El Tigre) AL Selection for tolerance to low pH and aluminum toxicity in laboratory test (El Batan) YDB Selection for BYDV tolerance in El Batan (BYDV = barley yellow dwarf virus) Location Codes Location codes for other countries were determined by the cooperators/breeders in those countries to ensure that everyone was aware and compliant. Hypothetical examples of BCIDs and selection histories are presented below. Table 2 Examples of BCIDs and selection histories for a simple cross using the pedigree method, the modified pedigree/bulk method, and the selected bulk method. Example # Type of cross Breeder’s Cross ID (BCID) Selection history (by generation) n/a n/a n/a F1 F2 F3 F4 F5 F6 F7 1 A/B CMBW08Y0199 n/a 35Y 15M 7Y 5M 12Y 0M 2 A/B CMBW08Y0124 n/a 81Y 010M 010Y 010M 15Y 0M 3 A/B CMSS07Y051 n/a 030Y 030M 030Y 53Y 0M n/a • Example 1: BCID = CMBW08Y0199, a single cross was made in Mexico (“CM”) in 2008 between a bread and winter wheat at location ‘Y’ and this was cross# 0199 that year. The pedigree method of selection was followed for the development of the genotype. The selection history for this example indicates that in the F2, the 35th plant was selected at location “Y”, and in the F3 generation this was the 15th plant selected at location “M”, etc. Finally, in the F7 the “0M” indicates that a single plot was grown at location “M” and harvested in bulk (i.e., all plants in the plot were harvested into one bag or packet). This created the genotype CMBW08Y0199-35Y-15M-7Y-5M-12Y-0M. • Example 2: BCID = CMBW08Y0124, a single cross was made in Mexico (“CM”) in 2008 at location “Y”, with 0124 designating that this cross was number 0124 in the series of crosses made at that location and year. The selection history reflects that a modified pedigree/bulk selection method was used. In the F2 the “81Y” indicates that this genotype was the 81st individual plant among those selected at location “Y.” The F3 designation of “010M” indicates that 10 plants were selected and harvested in bulk from the F3 progeny row grown at location “M.” Seed from the bulked F3 progeny row was planted at location “Y” in the F4 and 10 plants were selected and harvested in bulk. Similar scheme was used in F5. In the F6 a single plant was selected from this genotype (15th plant) at location “Y” and constituted the seed for the next generation. In the F7 (or more appropriately, F6:7) all plants in the progeny row at location “M” were harvested in bulk, as shown by the designation “0M”. This created the genotype CMBW08Y0124-81Y-010M-010Y-010M-15Y-0M. • Example 3: BCID = CMSS07Y051, which describes that a single cross was made in Mexico (“CM”) in 2007 at location “Y”, with 051 designating that this cross was number 051 in the series of crosses made at that location and year. The cross was of type “SS”, spring wheat x spring wheat. The selection history indicates four generations (F2-F4) of selection in which 30 plants were bulked from the progeny row (or plot) for each season at either the “M” or “Y” locations. In the F5 generation the genotype selected was the 53rd plant from the bulk plot at the “Y” location. In the F6 a complete plot bulk was harvested at location “M”. This lead to the creation of the genotype CMSS07Y051-030Y-030M-030Y-53Y-0M. Additional Notes After the BCID, the selection history is presented in which the numbers identify the number of individual plant(s) selected and the letter indicates the location where selection took place and/or under what specific conditions selection was conducted. The zero-letter combinations (e.g., 0Y, 0M, etc.) are reserved for populations harvested in bulk during that generation (i.e., the entire plot was cut and threshed as one unit). A zero followed by a number (e.g., 05…, 010…) and then by a letter indicates that the modified pedigree/bulk selection method was used in which a certain number (e.g., 5 or 10) of selected heads are bulk (0) harvested. The location where the selection was made and, in some cases, the special type of selection performed, is indicated by a letter code.

textbooks/bio/Agriculture_and_Horticulture/Crop_Improvement_(Suza_and_Lamkey)/1.02%3A_Pedigree_Naming_Systems_and_Symbols.txt

Asheesh Singh; Arti Singh; Jessica Barb; and Anthony A. Mahama The presence of genetic variation is a key prerequisite for genetic improvement in plant breeding and plays a pivotal role in germplasm usage in breeding programs. Therefore plant breeders and students in plant breeding can benefit immensely from an understanding of sources of genetic variation present, and ways of creating genetic variability where it is limited. The source of genetic material in a breeding program may come from one’s own breeding program, a colleague’s breeding program with the same or different organizations, or gene banks, among others. Good stewardship needs to be followed by plant breeders to utilize the genetic material. Learning Objectives • Know processes that create genetic variation • Gain an understanding of the concepts of types and origin of genetic variation • Become familiar with plant genetic resources and working with variability in hybridizations • Know the legal issues with germplasm usage and exchange Relationship of Plant Breeding to Natural Selection Creating Genetic Variability Natural selection requires three main processes to function: 1. Processes that create genetic variability: gene mutation, recombination, chromosomal segregation, gene flow are some of the ways to create genetic variability. This provides the potential to change the composition of individuals in the population. Mutations are considered random as they are not created to address a “need” of the organism. Therefore mutations can be neutral, harmful, or beneficial. Somatic mutations (occurring in the non-reproductive cell) are not useful to genetic variability. Gene flow can be an important source of genetic variation if genes are carried to a population where those genes did not previously exist (Fig 1). 1. Processes that rearrange genetic variability: natural selection or random genetic drift. These processes will lead to a change in population, due to the favoring of reproduction of certain individuals over others, thus causing a change in the gene pool (genes possessed by the mating population). Natural selection operates through reproductive fitness (the ability to produce offspring that contribute to the gene pool of the next generation). As the term implies, random genetic drift is random and uncontrollable. For example, in a population, some individuals may leave more offspring by chance than other individuals. Let us consider a hypothetical situation in a forest where there are 50% each of two tree species. Species A is predominant in the western part of the forest and species B is predominant in the eastern part. If fire destroyed 80% of trees in the western part of the forest, species A will be significantly reduced in number, and so species B will leave more offspring, leading to a genetic drift. It is important to note that preponderance of offspring of species B is due to the chance destruction of species A, and not necessarily because species B is healthier or more productive. Unlike natural selection, genetic drift is neutral to adaptation. In the forest fire example above, if species B had wood properties that made them fire-resistant (remember this is a hypothetical example) then a fire will destroy species A and reduce the number of species A offspring in the next generation compared to species B. Because this trait of fire protection is genetic, after repeated fires, species B will have more off-springs and will evolve due to natural selection. This example can be extended to a crop plant and disease. 1. Processes that maintain the product (minimize disturbance). These processes or mechanisms serve to protect the integrity of a population’s gene pool. This functions to maintain the genetic identity of the product, for example, due to reproductive isolating mechanisms. Examples of reproductive isolating mechanisms include sterility or failure of mating due to asynchrony (where males flower and shed pollen before the stigma of the females are receptive or vice versa). Deliberate Choice Artificial selection describes the deliberate choice of individuals for breeding in each generation and the advancement of select individuals. Directional selection is a form of artificial selection in which phenotypically superior plants are chosen for breeding. Artificial selection has been practiced for thousands of years by humans to make improvements in plant species. For example, artificial selection led to the rise of modern maize from its progenitor, teosinte (Fig. 2). Numerous studies show that teosinte (Zea mays ssp. parviglumis, a grass species) is a progenitor of maize (Zea mays L. ssp. mays). Very small differences in morphology (under genetic control) differentiate maize and teosinte. For example, teosinte has a cupulate fruit case protecting each kernel and the rachis segment (internode) and glume (modified bract) cover the kernel (Fig. 3). The cupule and glume are present in maize but they are significantly reduced in size and therefore do not surround the kernel. In maize, these organs form the cob. Ears of teosinte disarticulate at maturity such that the individual fruit cases become the units of seed dispersal. Ears of maize remain intact at maturity, which allows for easy harvest by humans. In teosinte, each cupulate fruit case holds a single-spikelet (kernel-bearing structure). In teosinte, the cupulate fruit cases are borne in two ranks on opposite sides of the longitudinal axis of the ear. In maize, the cupules are borne in four (or more) ranks. Recreating Primitive Maize Previous work by George Beadle has shown that primitive maize can be recreated by crossing teosinte and modern maize. While some of the changes between teosinte and maize may have happened naturally, the rest resulted from domestication and artificial selection as these differences made maize suitable for production for humans. Today we continue to improve the yield of maize using directional selection. Making Progress Artificial selection in genetically heterogeneous populations always leads to a successful outcome (i.e., mean change in population phenotype over generations in the direction of selection). This is true unless a biologically constraining limit is reached. The mean of a trait can be altered in both directions (i.e., an increase or a decrease in a trait’s arithmetic mean value) if genetic variability exists in a population. Genetic variation is ESSENTIAL for making progress using artificial selection. Can we change the mean phenotype of a genetically uniform population (or completely inbred genotype) over generations? What will happen if mutations occur in the genetically uniform population? Will the mean phenotype change over generation in the same genetically uniform population (now with mutations)? Successful Maize Experiment In 1896, C.G. Hopkins started long-term artificial selection experiments looking at oil (Fig. 4) and protein (Fig. 5) content in maize. The open-pollinated corn cultivar Burr’s White was used as the founder population. Four strains were established: Illinois High Oil (IHO), Illinois Low Oil (ILO), Illinois High Protein (IHP), and Illinois Low Protein (ILP) with high and low referring to the direction of the selection. After 48 generations, reverse selection was started in each strain to establish the Reverse High Oil (RHO), Reverse Low Oil (RLO), Reverse High Protein (RHP), and Reverse Low Protein (RLP) strains. After seven generations of selection in RHO, selection was again reversed to create the Switchback High Oil strain (SHO) to study the effect of selection. Protein Content The effects of selection on oil content ceased (i.e., Generation 85) in the ILO strain when the oil content reached a level that was no longer measurable with the analytical tools used in this experiment. Protein content reached a lower limit after approximately 65 generations, likely due to biological (i.e., physiological) limit in this crop species. An upper limit was not reached for oil content in IHO and SHO indicating that significant genetic variance still existed in these strains even after 100 generations of selection. Overview of the Plant Breeding Process Flow Chart Figure 6 presents a broad outline of plant breeding process. For a detailed flow chart on breeding process see Simmonds, 1979. The three main phases of the plant breeding process are: 1. Germplasm development: Generally one trait is improved at a time. Crossed are made between wild accessions or related species and/or between elite breeding line or cultivar. Considerations on commercial suitability is low or non-existent. The intention of the work is to develop improved parental germplasm, not a cultivar. Genetic conservation and genetic variability is improved. Genebanks are more heavily relied on for parental stock material. 2. Cultivar development: Generally several traits are improved simultaneously. The finished product is a genotype or population that has desirable characteristics for release as a cultivar. Crosses are made between elite lines as parents and may include a germplasm line (see above) as one of the parents. In general, both parents are elite lines (See Steps in Cultivar Development). Considerations on commercial suitability are primary. The intention of the work is to develop an improved cultivar to be grown by farmer(s). Breeder will make phenotypic and genotypic selection decisions on multiple traits and in several generations (pure-bred and inbred lines). Wide adaptation and performance testing is done prior to commercialization. 3. Technology Transfer: For germplasm development, there are smaller components of technology transfer for scientists and breeders. For cultivar development, there is a larger component of technology transfer for scientists, breeders, agronomists, pathologists, entomologists, seed merchants, and extension scientists. Cultivar Development Strategies In the public and private sectors, the same individual may be responsible for germplasm development and cultivar development phases. In other situations, two or more individuals may be engaged in these two phases independently but collaboratively within the same or different teams. Germplasm developers will be more interested in working on one or few traits (to transfer them from unadapted or wild relatives) and would not be as concerned about its overall suitability for a fit into a commercial release market. On the other hand, a cultivar development breeder has to consider the commercial requirements of his/her crop and its overall suitability. In the cultivar development strategies, elements that are common in all programs are: 1. Setting objectives 2. Identifying available parents? 3. Creating breeding populations 4. Evaluating and selecting in these population in appropriate environments to meet the objectives 5. Identifying the most suitable genotype for commercial release Setting Breeding Objectives Definition Breeding objectives are based on a mandate (market segment needs), organizational focus, farmer requirement, industry needs, profitability, and sustainability. Objectives need to be clearly defined and based on importance, feasibility and cost-effectiveness. It is not sufficient to set an objective as ‘increase yield’. The breeder should put some quantifiable description, such as “increase yield by x% over check ABC”, where the comparison has to be made head-to-head. Plant breeding is an expensive activity and careful consideration needs to be made prior to setting objectives. The breeding team needs to engage growers, industry, and consumers to decide on objectives. In a large company, this may be done by a different team and the results communicated to the breeder to help her/him define the objectives. A breeder may develop the highest yielding inbred or hybrid parents or population, but without growers’ ability to grow it, this product (inbred, hybrid, population) will not be a commercial success. For example, if a very high yielding genotype has poor storability, growers and industry will not accept this genotype. In plant breeding, multiple objectives are generally set and a prioritization made to decide on ‘must to have’ versus ‘nice to have’ trait. ‘Must to have’ are traits that absolutely need to be included in the product (pure line, or hybrid, or OPV) for it to be suitable for commercial release, whereas ‘nice to have’ are traits, which are not essential but may add value to the product. Exercises Pick three crops common to your agro-ecological zone, and list 2-3 traits that are ‘must to have’ and ‘nice to have’ for each crop. Identifying Parents An important consideration for setting breeding objectives is to identify parents for hybridization that have the necessary traits that the breeder will want in the cultivar to be developed. Sources of parental material will be genotypes or populations from your own program, your colleagues’ programs (within or outside of your organization), international breeding centers, and gene banks. We will learn a little more about sources of parental material in the next few sections. After setting of objectives, a breeder will create breeding populations (i.e., create genetic variability) by crossing two or more parents. In crop species with sexual reproduction, generation advancement is generally occurring in parallel with selection for traits as per defined objectives. Once a finished product (genotype) is ready, broader adaptation testing is performed prior to picking the most suitable cultivars for commercialization. In the next section, we will learn about gene banks, which contain accessions that may be useful to a breeder as sources of genetic variability for use in breeding. GeneBanks: Role, Procedures, Acquisition, and Stewardship Roles of Gene Banks For decades, local, regional, and international efforts have been attempting to preserve valuable agrobiodiversity for future generations by setting up collections of genetic resources, called genebanks. Genebanks contain ‘landraces’ or local varieties of cultivated and non-cultivated wild relatives. This serves to protect and preserve seed diversity as well as provide an accessible source to plant breeders to obtain seed of interest. There are currently about 1,750 institutional crop collections around the world, as well as a number of community-based seed bank initiatives. CGIAR Research Program for Managing and Sustaining Crop Collections is dedicated to maintaining the 706,000 samples of crop, forage, and agroforestry resources held in “genebanks” at 15 CGIAR research centers around the world. Species which include cereals, legumes, roots and tubers, trees, and other essential staple crops are stored in CGIAR international collections. All accessions within these collections are for the international public good, available under the terms and conditions negotiated by the International Treaty on Plant Genetic Resources for Food and Agriculture. In the USA, the National Plant Germplasm System aids scientists and addresses the need for genetic diversity by: • acquiring crop germplasm • preserving crop germplasm • evaluating crop germplasm • documenting crop germplasm • distributing crop germplasm GRIN For example, for the USDA’s Germplasm Resources Information Network (GRIN), the steps are to search for genotypes that you are interested in and then place an order to receive seed: The breeder should determine which genebank has the collection of material in their crops, proceed to search the genebank and order seed. This process involves numerous paperwork (agreements, seed importing or exporting permits and customs documents) and planning ahead is critical to ensure that you receive seed on time. [Note: Many times it is useful to contact the curator or other scientists at a genebank as they can sometimes help to make suggestions on a specific trait or accession you may be looking to obtain. However, one needs to do their groundwork first.] Type of Variability and Sources of Genetic Material Natural Variability – The Gene Pool Concept For plant breeders, it is very important to be aware of available germplasm resources that will be useful to improve traits. The gene pool concept was proposed by Harlan and de Wet (1971) as an attempt to provide a practical guide to place existing classifications into genetic perspective. This information on the relatedness among crop plants and their relatives could be useful to breeders and geneticists wishing to make crosses among them. It is important to note that the gene pool concept did not attempt to change the taxonomy. Its purpose is to serve as a guide to plan breeding activities. Various genetic resources are assigned to different gene pools of a crop species based on ease of hybridization, i.e., ability to move genes between them. The three major gene pools are: primary, secondary and tertiary. Gene pools are not static but change as more information becomes available or as new technologies become available to manipulate genomes. For example, in their paper soybean was reported not to have a secondary or tertiary gene pool. However, we now consider that 26 perennial Glycine species are in tertiary gene pool and G. tomentella has now been used to transfer genes to G. max (Singh et al. 2014; R.J. Singh, USDA-ARS, IL, personal communication). Therefore breeders need to be aware of what is going on around them with the use of unique genetic material. Primary Gene Pool Species in primary gene pool can be cultivated, landraces, farmer developed or maintained population, ecotypes, and spontaneous races (wild or weedy). Among forms of this gene pool, crossing/gene transfer is easy; hybrids are generally fertile (i.e., no sterility issues) with normal chromosome pairing and gene segregation. Most breeders work exclusively within this gene pool which is also the major source of genetic variation for improvement programs. Remember that most breeding programs that are engaged in developing cultivars for commercial production work on elite material exclusively and would spend very little direct efforts on unadapted or wild relatives (because of undesirable linkage blocks, breaking of desirable linkage block and epistatic interactions with undesirable genes from wild relatives). Secondary gene pool Crop’s secondary gene pool will include species between which gene transfer is possible, but difficult. Hybrids tend to be sterile; chromosomes pair poorly during meiosis; F1 plants are weak and develop to maturity with difficult; because of some sterility in F1’s , recovery of desired types in advanced generations is generally difficult. The secondary gene pool includes related species within the same genus, although all species within a genus won’t be in the secondary gene pool and it is also possible that species outside the genus can be in this gene pool. Tertiary Gene Pool Gene transfer between a crop and a species in its tertiary gene pool is very difficult (will require embryo rescue, chromosome doubling, bridging species to obtain hybrids). This gene pool includes distant relatives in other genera or distantly related species within the same species. Hybrid sterility is common, although chromosome doubling may restore fertility by providing homologues for each chromosome. The boundaries of this group are poorly defined and shift as new hybridization techniques are developed. Hybridization A bridging species is a third species that facilitates exchange of germplasm between the other crop species and tertiary gene pool species by developing complex hybrids. In their paper, Harlan and de Wet (1971) described a classic example of the use of bridging species where there was an interest to cross Elymus x Triticum. As expected, hybrid seed could not be obtained. When embryo rescue was used, very few hybrids were obtained and even then these were sterile. However, these researchers found out that if they used Agropyron x Triticum derivative as female parent and then crossed the hybrid to Elymus, introgression of Elymus alleles was possible without need for special technique (See Harlan and de Wet, 1971). Wide Hybridization or Interspecific/Intergenetic Hybridization ‘Wide cross’ refers to crossing that involves individuals outside of cultivated species. This typically involves the secondary and/or tertiary gene pools. Even though it is difficult, it may be useful to transfer vitally important traits, including disease resistance, or other traits simply not found in cultivated genotypes. Many examples exist in wheat and rice. Examples Example 1: In wheat, the T1BL.1RS wheat (Triticum aestivum L.) – rye (Secale cereal L.) has been of particular interest and was widely used in bread wheat breeding programs worldwide. At one point, it was estimated that several million hectares of wheat were planted to cultivars possessing this translocation (tertiary gene pool: rye to wheat crop). This segment had disease resistance cluster for leaf rust, stem rust, stripe rust and powdery mildew, all of which are important diseases of wheat. Additionally, this segment was reported to possess genetic factors that improved grain yield and kernel weight. Resistance to specific genes in the translocation segment have been overcome in some parts of the world, which shows the continual nature of plant breeding where better genetic packages (cultivars) need to be developed. Example 2: In rice, the first example of transfer of a useful gene from wild species was the introgression of a gene for grassy stunt virus resistance from Oryza nivara to cultivated rice. Other examples are transfer of Xa-21 for bacterial blight resistance from O. longistaminata to cultivated rice; CMS sources from O. perennis and O. glumaepatula into rice for hybrid rice production. In 1970’s, grassy stunt virus epidemics were reported in several countries and this was transmitted by brown plant hopper (diseased rice plants produced no panicles or small panicles with deformed grains) leading to severe yield losses. Several thousand accessions of cultivated rice and wild species of Oryza were screened for resistance, which identified one O. nivara accession as resistant. Plant breeders then successfully transferred grassy stunt virus resistance to improved varieties through a backcross breeding method and resistant varieties were released for cultivation. Other examples include, transfer from O. officinalis into elite rice genes for resistance to brown plant hopper, white backed plant hopper (WBPH) and bacterial blight. Some other examples are presented Table 1. Table 1 Introgression of genes from wild Oryza species into cultivated rice. AA = cultivated rice diploid genome. Data from D.S. Brar and G.S. Kush, 1997. Trait transferred to O. sativa (AA Genome) Donor Oryza Species Wild species Genome Grassy stunt resistance O. nivara AA Bacterial blight Resistance O. longistaminata AA O. officinalis CC O. minuta BBCC O. latifolia CCDD O. australiensis EE O. brachyantha FF Blast resistance O. minuta BBCC Brown planthopper resistance O. officinalis CC O. minuta BBCC O. latifolia CCDD O. australiensis EE O. granulataa GG Whitebacked planthopper resistance O. officinalis CC Cytoplasmic male sterility O. sativa f. spontanea AA O. perennis AA O. glumaepatula AA Yellow stemborer resistance O. brachyanthaa FF O. ridleib HHJJ Sheath blight resistance O. minutia BBCC Tungro tolerance O. rufipogona AA O. rufipogona AA Increased elongation ability O. officinalisb CC Tolerance to acid sulfate soils O. rufipogona AA O. rufipogona AA O. rufipogona AA Artificially Created Variability: Mutation and Transgenes Induced Mutation to Augment Genetic Diversity Novel genes are produced by several methods, commonly through the duplication and mutation (Fig. 8) of an ancestral gene, or by recombining parts of different genes to form new combinations with new functions. Lethal mutations do not carry their germline forward, however, nonlethal mutations accumulate within the gene pool and increase the amount of genetic variation. The abundance of some genetic changes within the gene pool can be reduced by natural selection, while other “more favorable” mutations may accumulate and result in adaptive changes. A germline mutation gives rise to a constitutional mutation in the offspring, that is, a mutation that is present in every cell. Mutation Breeding Deletions lead to loss of gene(s) and duplication can lead to an additive effect due to added gene(s). In inversion, linkage block changes occur and other genes in close proximity will co-segregate. In insertion and translocation, a gene moves to a new chromosome, and can have similar effect as duplication. [Note: Mutations can be subdivided into germ line mutations, which are passed on to descendants through their reproductive cells, and somatic mutations, which involve non-reproductive system cells and are therefore not usually transmitted to descendants]. Examples of Success with Mutation Breeding Quality Protein Maize (QPM). Maize endosperm protein is deficient in two essential amino acids, lysine and tryptophan. The opaque 2 mutant gene, together with endosperm and amino acid modifier genes, was used for the development of QPM varieties. Compared to regular maize, QPM has about twice as much lysine and tryptophan, and 30% less leucine, which makes it suitable and useful for human and animal nutrition. QPM varieties are now estimated to be grown on millions of hectares. The high protein content and better amino acid profile is achieved by “opaque-2” single gene mutation. In the early 1960s, a mutant maize with similar total protein content but double the amount of lysine and tryptophan was developed. Subsequent conventional breeding efforts generated numerous cultivars with improved agronomic characteristics, and these were referred to as QPM. Dr. Evangelina Villegas and Dr. Surinder Vasal were awarded the ‘World Food Prize’ in 2000 for their work on development and advancement of QPM cultivars in the world. Approaches If the goal of mutation breeding is to alter only a single trait, the plant breeder needs to be aware that other regions of the genome (i.e., other genes) may have been mutated and also that, that one change may alter other aspects of the plant. Hence extensive agronomic testing of that single mutant is required prior to commercialization or extensive use as a parent in the breeding program. Traditionally, chemical or physical agents were used to induce mutations in crop genomes, and included radiation (X-rays, gamma rays, fast neutrons, etc.), chemicals such as ethyl methane sulfonate (EMS) and others. These mutagens can disrupt chromosomes, causing deletions, insertions, breakage, etc., and will create genetic variation. Major disadvantage of this approach is the non-targeted mutation events. After receiving your M1 seed (one has to send several thousand seed of the same cultivar) plant breeder has to increase the generation to achieve homozygosity (mutant allele will segregated initially) and constantly phenotype for the traits of interest. This can be very resource intensive depending on the cost to phenotype the trait of interest (field for morphological trait or lab for quality trait or chemical component). At low doses, chromosomal changes are not as dramatic (it is desirable not to use high doses as major chromosomal aberrations and lethality can occur) and the mutation frequency is low, therefore warranting large population sizes to be screened. This leads to high expenses to phenotype and sometimes very difficult to identify a target mutant event. Some of the newer approaches include, space light ion irradiation, use of restriction endonucleases, Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas-based RNA-guided DNA endonucleases. These techniques lead to genetic modifications by inducing DNA double-strand breaks that stimulate error-prone nonhomologous end joining or homology-directed repair at specific genomic locations leading to site targeted mutation. Procedure The procedure to create a mutant population is briefly as following: Seed (M0) of an inbred (homozygous, homogenous) genotype is subjected to treatment (chemical, physical, etc). The treated seed are then grown and plants grown from these seeds (treated with a mutagen) form the M1 population (where M stands for Mutant and ‘1’ refers to first the generation in development similar to the concept of filial generation we learned in previous chapter). It is important to realize that although we started from an inbred line, after seed treatment the resultant M1 generation will consist of plants that are heterogeneous, as each M1 plant may have different mutation (or several mutations) in its genome. Each plant will also be heterozygous (or hemizygous) at numerous loci. This would mean that M1 is similar to an F1 developed from noninbred parents. The step includes, self-fertilization of the M1 plants to develop the M2 generation. This will allow the recessive mutants to be made homozygous in order to produce observable phenotypes. Identifying mutants generally requires large populations of M1 plants. Other considerations are size of M1 and M2 family to recover the mutant type, ploidy level, genetically effective cell (GEC) present in germline (these are those cells of the germline that contribute towards formation of gametes and so to offspring), frequency of chimeras. After several M generations, mutant phenotype is confirmed and established as stable (non-segregating). Mutant line then can be used in a forward or backcrossing program to transfer the favorable mutant to elite cultivars using multiple but separate crosses to ensure recovery of the elite phenotype together with the mutant trait. Transgenic Approaches to Increase Genetic Diversity Transgenic technology involves the transfer of cloned genes via transformation or particle bombardment so that the transformed plant expresses the foreign gene. Theoretically, the genes can come from virtually any living organism – from within the same primary gene pool to beyond the tertiary gene pool. Over the past few years, mainly only single genes have been transferred, such as herbicide tolerance in various crops, or corn borer resistance in corn. Creating Breeding Populations – Types of Crosses Types of Crosses Once objectives are set and the breeder has done background investigation to pick the parents that possess the traits that will help meet the objectives, she/he will move to the next step in plant breeding process, which is to develop breeding populations. While some breeding population may start from landraces, most populations will be made by making planned hybridizations (crosses). The primary purpose of crossing is to expand genetic variability by bringing together genes from the parents in the cross to produce offspring that contain genes that will help meet the objectives. Sometimes, multiple crosses are made to generate the variability in the base population to begin the selection process in the program. In most self and cross pollinated species where the product is an inbred line or a hybrid, single crosses are made, while complex crosses are made in population improvement schemes. Parents are selected to have the maximum number of desirable traits and minimize undesirable traits (what is generally called an ‘elite by elite’ cross). This way, recombinants that possess both sets of desirable traits will occur in significant numbers in the F2, the generation of maximum variability (in self-pollinating and inbred line development programs). Several factors will impact the population size include number of genes differing among parents in a cross, number of alleles per locus, and linkage of the gene loci. Major types of crosses made 1. Single cross. This is attempted when a breeder is making a cross between two elite lines. (Line A x Line B). 2. Three-way cross. If two lines are not sufficient to bring together all the necessary traits to meet the objectives, multiple cross with three parents can be used to provide an opportunity to obtain recombinants with all the desirable traits. Three way cross is (Line A x Line B) x Line C. If cultivar development is targeted in a three way cross, the third parent ‘Line C’ in our example, should be an elite and adapted genotype to get at least 50% of favorable genetics. 3. Double cross. A double cross is a cross of two single crosses [(Line A x Line B) x (Line C x Line D)]. The breeder should attempt to make the two single crosses in the same season and then cross the resultant F1 in the next season crossing cycle to minimize the time to obtain F1 from the double cross. More parents start to introduce more opportunities to break linkages (including favorable ones) and there is a low frequency of obtaining desirable recombinants in the early generation selection. The double-cross hybrid is more genetically broad-based than the single-cross hybrid. 4. Diallel or Partial Diallel cross. A diallel cross is one in which each parent is crossed with every other parent in the set (complete diallel), yielding n * (n – 1)/2 different combinations (where n is the number of entries) excluding reciprocal crosses where the female/male order is reversed. For example, where n equals 9, 36 crosses will be made.This method requires making a large number of crosses and it is more suitable for cross-pollinated species. This method is generally used for genetic studies and not for population development. Sometimes, a partial diallel is used in which only certain parent combinations are made. For n equals 9 example, not all 9 parents will be used in crosses. 5. Back cross. The primary goal of this crossing method is to incorporate a specific trait (from a donor parent) into an existing elite cultivar (referred to as recurrent parent in back crossing). The donor parent has one trait that a breeder desires to include into an elite cultivar, which is considered to possess all necessary traits except this one trait. The resultant product (after successive crossings to recurrent parent) is a cultivar that is similar to the recurrent parent with the additional trait from the donor parent. This methods is more efficient if the interest is to improve a current popular cultivar which has an obvious deficiency. Molecular markers have improved the efficiency and reduced the time to develop cultivars through backcrossing. Material Transfer Agreement (MTA) Legal Considerations Seed or plant part material transfer agreement (MTA) is an agreement that allows for transfer of seed or plant part without transfer of title. The agreement is between the provider (one who provides seed) and the recipient (one who receives seed). Provider maintains ownership of the seed transferred at all times during the agreement dates and beyond. Transferred seed is received and used by the recipient according to the terms listed in the legally binding contract. In the agreement, the provider may impose conditions of audit or term bound reports on the usage of material and the recipient has to bind to these conditions. When a plant breeder makes request for seed, she/he will work with their organizational designate in the office of intellectual Property and Commercialization or the office that manages Intellectual Property (IP) and Technology Transfer. Similarly, the plant breeder should discuss with the same office when she/he receives a request for seed/plant part and should NEVER give out material without going through proper steps. MTA is signed by the sending breeder and his/her organizational representative, as well as recipient breeder and his/her organizational representative. Plant breeder or researcher utilizing the material (i.e., seed/plant part) is ultimately responsible for fulfilling the obligations of the MTA and therefore has to follow the regulations. Remember: MTA is a legal document. MTA Sections • Introduction: short text on the type of material or purpose • Parties: describes the sender and recipient and their organizational affiliations • Definitions: describes scientific terms such as material (seed and genotype etc.) • Description of use of the materials: conditions on what can and cannot be done using the material • Confidential information: lists any specific confidentiality clauses • Intellectual Property rights: this is where licensing, royalties, inventions conditions are listed • Warranties: This is to protect sender or provider that stipulates that material does not come with any warranties. • Liability and/or indemnification: Recipient assumes all liability for damages that may arise from how/what recipient does to the material after transfer and sender is not liable. • Publication: provides description on publication rights for receiver. • Governing law: describes which jurisdiction laws will apply (state, country). In most cases where provider and recipient are from separate countries, the two parties may not define this section. • Termination: date of termination of the agreement. It may also describe what the recipient has to do with leftover material from the agreement. Most likely original material sent by provider is expected to be destroyed. • Signatures: Agreement is not considered executed until all necessary signatures are obtained and material should not be sent until MTA is signed and official. Signatories are the official organization representatives, provider breeder and recipient breeder. [note: provider and recipient may or may not be a breeder. For example, recipient or provider may be a geneticist] • Exhibits or appendices: list of material, or data accompanying the material. International Treaty on Plant Genetic Resources for Food and Agriculture SMTA The Standard Material Transfer Agreement (SMTA) is a mandatory model for parties wishing to provide and receive material under the Multilateral System. It is the result of lengthy negotiations among the Contracting Parties to the Treaty and may not be varied or abbreviated in any way. However, as a template, it contains some paragraphs and sections that need to be completed for each use. The material transfer agreements that use the standard template are private agreements between the particular providers and recipients, but the Governing Body, through FAO as the Third Party Beneficiary, is recognized as having an interest in the agreements. The standard template has been developed to ensure that the provisions of the Treaty regarding the transfer of PGRFA under the Multilateral System are enforceable on users.” Farmers’ Rights Farmers’ Rights: In its Article 9, the International Treaty recognizes the enormous contribution that the local and indigenous communities and farmers of all regions of the world, particularly those in the centers of origin and crop diversity, have made and will continue to make for the conservation and development of plant genetic resources which constitute the basis of food and agriculture production throughout the world. It gives governments the responsibility for implementing Farmers’ Rights, and lists measures that could be taken to protect and promote these rights: • The protection of traditional knowledge relevant to plant genetic resources for food and agriculture; • The right to equitably participate in sharing benefits arising from the utilization of plant genetic resources for food and agriculture; and • The right to participate in making decisions, at the national level, on matters related to the conservation and sustainable use of plant genetic resources for food and agriculture. Importance The International Treaty also recognizes the importance of supporting the efforts of farmers and local and indigenous communities in the conservation and sustainable use of plant genetic resources for food and agriculture, including through a funding strategy. In this strategy, priority will be given to the implementation of agreed plans and programs for farmers in developing countries, especially in the least developed countries, and in countries with economies in transition, who conserve and sustainably utilize plant genetic resources for food and agriculture.” It is important that breeders realize that if an MTA was signed to send or receive seed, it is a legal document and they are legally bound to follow the conditions. Uses of germplasm: Methods to use germplasm in breeding programs include direct release as cultivars [less likely in species with breeding efforts, more likely in orphan crops where some selection may be done on a plant introduction or landrace prior to release as a cultivar]. The second and more appropriate use of germplasm is for introgression of single-gene traits from the wild species or unadapted germplasm into the elite cultivars.

textbooks/bio/Agriculture_and_Horticulture/Crop_Improvement_(Suza_and_Lamkey)/1.03%3A_Genetic_Variation_and_Germplasm_Usage.txt

Asheesh Singh and Anthony A. Mahama The presence of genetic variation is a key prerequisite for genetic improvement in plant breeding. Creation of breeding populations with sufficient variability among individuals is key to success of breeding programs. Behind the visual variability in populations are underlying genetic variations and interactions, the understanding of which the plant breeders and students in plant breeding can benefit from to ensure successful use of the sources of genetic variation and their manipulation to maximize improvement of programs. Learning Objectives • Demonstrate understanding of the different populations in plant breeding • Distinguish between qualitative and quantitative traits important in plant breeding • Demonstrate understanding of the concept of variability, phenotype, genotype, and genotype x environment interactions • Describe the concept of heritability and its importance in plant breeding • Discuss selection theory and response to selection (breeder’s equation) • Distinguish between specific versus general combining ability and their calculations • Describe the concept of heterosis and write the equation for estimating heterosis Populations Basic Principles Population genetics deals with the prediction and description of the genetic structure of populations as it relates to Mendel’s laws and other genetic principles. Fundamentals of population genetics were developed for natural outcrossing species, but it is important to note that plant breeders utilize population genetic theories because the breeding methods they use are designed to increase the frequency (proportion) of desirable alleles in the population. The difference between population and Mendelian genetics is that population genetics principles are applied to the total products of all matings that will occur in the population, not just to one specific mating (as is the case with Mendelian genetics). Population refers to any group of individuals sharing a common gene pool. Gene pool refers to the sum total of all genes present in a population. Types of Populations in Breeding Program Simple populations Simple populations consist of two to four parents. The simplest type of population is created by developing a segregating population from a cross of two elite lines, i.e. a two-way cross population. An F2 generation can be produced by self-fertilizing the F1. F2 and higher filial generations are developed (and selections made in these populations) in commercial breeding programs of self-fertilizing crops (such as wheat, rice, and soybean). In many cross-pollinated crops like maize, the commercially marketed products are single cross hybrids (F1) from inbred parents developed following generations of selection and selfing. Another type of segregating population is a backcross population, which can be made from the F1 by crossing it to one of the parents, producing a BC1F1, which can be selfed to form a segregating population. The backcross population is particularly useful if one parent is superior to the other in most traits and the objective is to combine one or two genes from unadapted (or overall undesirable) parent into an elite line. Each backcross F1 seed will be heterogeneous and therefore phenotypic testing or marker-assisted selection will be required to select the desirable plant to use in subsequent backcrossing to fix or enrich for favorable genes. Single Cross A cross involves two parents (Fig. 1), for example the crossing of two elite lines for F1‘s from which an F2 population can be produced by self-fertilizing the F1. Parent ‘A’ and ‘B’ contribute 50% each (genetically) Three-Way Cross Crossing the single cross F1 to a third parent (inbred line) and self-pollinating the resulting three-way hybrid creates a three way cross population (Fig. 2). The generation resulting from the self-fertilization is generally called the F2 population. Note that the third parent contributes 50% of the alleles to the final population therefore should generally always be one of the best parents. A three way cross is useful if one of the parents is less desirable and one or two are more desirable. Double Cross A double cross or four-parent cross population can be produced by crossing two single cross F1 hybrids, each formed from two inbred lines (Fig. 3). Each of the resulting individuals in the double cross generation will be genetically distinct. Complex Populations Some of the reasons to use complex populations include: developing good information for parents and full-sib families, identification of heterotic groups, estimation of general and/or specific combining ability, develop estimates of additive, dominant, and epistatic genetic effects and genetic correlations. Complex populations deriving from more than four parents can be constructed in several ways: • Nested designs • Factorial designs • Diallel cross • Half-diallel • Partial diallel • Polycrosses Nested Designs A North Carolina Design I (nested design) involves mating of each of the male parents to a different subset of female parents as shown in Table 1. Table 1 Mating of four male parents and eight female parents, each male mated to two different female. n/a Male Parent (♂) A B C D Female Parent (♀) 1 X n/a n/a n/a 2 X n/a n/a n/a 3 n/a X n/a n/a 4 n/a X n/a n/a 5 n/a n/a X n/a 6 n/a n/a X n/a 7 n/a n/a n/a X 8 n/a n/a n/a X Note: X means “cross”. n/a means cell is blank. A North Carolina Design II (factorial design), Table 2, involves mating each member of a group of males (A, B, C, D) to each member of the group of females (1, 2, 3, 4, 5, 6, 7, 8). Table 2 Mating of four male parents each to eight female parents in a factorial design. n/a Male Parent (♂) A B C D Female Parent (♀) 1 X X X X 2 X X X X 3 X X X X 4 X X X X 5 X X X X 6 X X X X 7 X X X X 8 X X X X Note: X means “cross” Table 2 Mating of four male parents each to eight female parents in a factorial design. n/a Male Parent (♂) A B C D Female Parent (♀) 1 X X X X 2 X X X X 3 X X X X 4 X X X X 5 X X X X 6 X X X X 7 X X X X 8 X X X X Note: X means “cross”. n/a means cell is blank. Diallel Cross A diallel refers to a crossing scheme in which all pairwise crosses among the parents are made as a series of single crosses (Table 3). Diallels can be “complete,” in which crosses are made in both directions i.e., including reciprocal crosses, as well as self-pollinations of parents. In other words, each parent is mated with every parent in the population (including selfs and reciprocals). Table 3 Mating of a group of eight individuals each crossed to every other individual both as male parent and female parent, and also self-pollinated. n/a Male Parent (♂) 1 2 3 4 5 6 7 8 Female Parent (♀) 1 X X X X X X X X 2 X X X X X X X X 3 X X X X X X X X 4 X X X X X X X X 5 X X X X X X X X 6 X X X X X X X X 7 X X X X X X X X 8 X X X X X X X X Note: X means “cross”. n/a means cell is blank. From a breeding standpoint, selfs do not contribute any interesting recombination if the parents are inbred, and because crosses in different directions are functionally the same in terms of recombination in later generations (unless there are maternal and paternal effects), the complete diallel is usually used only as a research tool. In the example given above, Parent 1 will be selfed, as well as used as male and as female in crosses with the other parents, e.g. parent 2, thus doubling the number of crosses with parent 2, i.e. 1 x 2 and 2 x 1. Half-Diallel Cross In a half-diallel cross, each parent is mated with every other parent in the population excluding selfs and reciprocals (Table 4). Table 4 Mating of a group of eight individuals each crossed to every other individual either as male parent or female parent only, and no self-pollination. n/a Male Parent (♂) 1 2 3 4 5 6 7 8 Female Parent (♀) 1 n/a n/a n/a n/a n/a n/a n/a n/a 2 X n/a n/a n/a n/a n/a n/a n/a 3 X X n/a n/a n/a n/a n/a n/a 4 X X X n/a n/a n/a n/a n/a 5 X X X X n/a n/a n/a n/a 6 X X X X X n/a n/a n/a 7 X X X X X X n/a n/a 8 X X X X X X X n/a Note: X means “cross”. n/a means cell is blank. Partial Diallel Cross Partial diallel refers to a crossing scheme in which only selected subsets of full diallel crosses are made (Table 5). A partial diallel could be made among a large number of parents, followed by a diallel among the single crosses, allowing for sampling more recombination events among favorable parents. This would allow incorporation of a diversity of germplasm without having to make a massive number of crosses. Table 5 Mating of a group of eight individuals using selected individuals as male parent particular female parent only, and no self-pollination. n/a Male Parent (♂) 1 2 3 4 5 6 7 8 Female Parent (♀) 1 n/a X X X n/a n/a n/a n/a 2 n/a n/a X X X n/a n/a n/a 3 n/a n/a n/a X X X n/a n/a 4 n/a n/a n/a n/a X X X n/a 5 X n/a n/a n/a n/a X X X 6 X X n/a n/a n/a n/a X X 7 X X X n/a n/a n/a n/a X 8 X X X X n/a n/a n/a n/a Note: X means “cross”. n/a means cell is blank. Practically making crosses for a diallel (or other methods of population formation) in the field requires careful planting arrangements. There are several things to consider: such as flowering time, length of time pollen and stigma will be viable, and susceptibility to drought or pest. From a planting perspective in the field, the considerations are: (1) physical distance between parents, i.e., distance between rows or plants (how close do you want to have each of the two parents you are crossing), such as side-by-side or not; and (2) how much land area is available for the population development. Planting arrangements vary from unpaired parents, in which parents are not very close to each other but less land is required for example circular crossing (aka, chain crossing), in which parents are crossed in pairs sequentially, A x B, B x C, etc., with the final parent crossed back to A.; or paired parents, in which all parents to be crossed are grown in adjacent rows, however, a large area is required for crossing nursery. Polycrosses Polycrosses are used to intercross a number of selected plants. Polycrosses are primarily used for cross-pollinating crop species allowing natural conditions (e.g., wind or insects) to make the crosses. Thus, the pollen for a polycross comes from the population of selected (or unselected ) individuals as pollen parent source, and no control on the success of any particular parental pairing is known. Some plants will undoubtedly produce more pollen than others, thereby resulting in a higher percentage of pollinations than others. In a clonal crop, parent genotypes may be replicated in a manner to ensure each genotype is adjacent or surrounded by all other genotypes to provide equal frequencies of crossing among all genotypes. If sufficient land is available and not too many entries are included in the crossing, a Latin Square arrangement (each parent is present in each row and each column of the design) is a good way to enhance the equal chance of pollination among the genotypes. Another option will be a randomized complete block design if the number of parents is large. A higher number of replications are planted (two or more); however, each parental genotype will not be surrounded by all other genotypes in equal frequency therefore non-random and unequal mating occurs. A number of other aspects need to be considered for successful intercrossing which include flowering time, wind effects, and insect pollinator activity. Flowering time needs to be similar among the parents to prevent certain parents intercross more frequently (due to overlapping flowering time) than they should by random chance. For polycrosses done in the greenhouse by hand (e.g., in alfalfa), flowering can be controlled more easily than in a field planting and this is something breeders can consider using, resource permitting. If a breeder is relying on wind pollination, the dominant direction of the prevailing wind will affect pollination and lead to a non-random pollination. For insect-pollinated crops, placing bee hives near the field is often done to ensure successful pollination. Polycross Nursery Harvest Procedures To ensure harvested seed of the polycross is representative of seed from random and equal pollinations (which is what is intended) three major procedures exist for harvest: 1. Bulk harvest the entire plot. This is the easiest method but will result in unequal contributions by both paternal and maternal parents to the population because maternal or paternal parent that produces more seed will represent a higher proportion of seed in the lot. 2. If replications were used in the crossing design, bulk each parental genotype’s seed across all the replications. Composite equal amounts of seed from each parent to make the population. This is the most commonly used method but there is an element of unequal contribution. Different clones (or inbred or doubled haploid) of the same parental genotype may not produce the same amount of seed, so this method will skew the population toward the pollen parents surrounding the highest yielding maternal clone. 3. One method that will overcome the problem listed in #2 above is to composite an equal amount of seed from each clone in the polycross – that is, equal amounts from each parental genotype in each replication. This method provides the most balanced contribution to the population possible. If there is a replication that didn’t perform to provide the minimum seed, some adjustments (in either among sample per replication or pulling more from other reps for that genotype) will be needed, leading to some un-equal contribution. Qualitative and Quantitative Traits Qualitative traits are traits that are generally controlled by a single or few genes, the expression of which have phenotype that can be classified into distinct categories. These traits are generally not influenced by environment and are recorded/scored as presence versus absence, or yes versus no, different color, seed shape type, etc. Examples include the presence of awns in wheat (awned versus awnless), flower color (purple versus white), round versus wrinkled seed (Mendel’s garden pea experiment). These traits will be in the ‘yes versus no’ classification and their expression will be the same irrespective of the environment the plants are grown, that is, genotypes with round seed will produce round seed in all environments. Quantitative traits are controlled by several genes, whose expression produce a phenotype that cannot be classified into distinct categories, i.e., there will be a continuum of phenotypes. These traits are influenced by the environment, such that the same genotype will produce different phenotypes in different environments. Examples of such traits are yield, protein %, oil %, and seed weight. Traits such as plant height are described as qualitative because they can be classified as short versus tall. However, it is important to note that plant height can occur across a range of values (cm) meaning that these are not innate categories and most appropriate measurement is on a numerical scale, which makes plant height a quantitative trait from a trait measurement perspective. In most crops, several plant height genes have been identified, again validating that plant height is not a truly qualitative trait. Disease resistance can be qualitative or quantitative, and this distinction between them will be driven by genetic control, influence of environment, and phenotypic expression. Most traits that a plant breeder works to improve are quantitative. Types of Gene Action Expression of genes can be described as additive or non-additive (dominance or epistatic). Additive gene action A gene acts in an additive manner when the substitution of one allele for another allele at a particular gene locus always causes the same effect. For example, A1A1 – A1A2 = A1A2 – A2A2 That is, for this case, the effect of substituting A1 for A2 is the same whether the substitution occurs in genotype A2A2 or in genotype A1A2 (Fig. 4, Table 1). Table 6 Genotypic values of three genotypes at ‘A’ locus. A1A1 8 A1A2 4 A2A2 0 Thus the effect of substituting an A1 allele with an A2 allele is +4. Genotypic Values at ‘B’ Locus As shown in Table 7, if we assume genotypic values at ‘B’ locus as: Table 7 Genotypic value of three genotypes at ‘B’ locus. B1B1 4 B1B2 2 B2B2 0 then, as shown in Table 8, the effect of substituting allele B1 for B2 is +1, indicating an additive effect. Table 8 Hypothetical example to demonstrate additive effects. Genotype Genotypic value A1A1B1B1 12 A1A1B1B2 10 A1A1B2B2 8 A1A2B1B1 8 A1A2B1B2 6 A1A2B2B2 4 A2A2B1B1 4 A2A2B1B2 3 A2A2B2B2 0 When a gene acts additively, the maximum trait expression will occur in the genotype which possesses all the “favorable” alleles. Non-additive Gene Action Non-additive gene action results from the effects of dominance (intra-locus interactions, i.e. A1/A2) and/or the effects of epistasis (inter-locus interactions, i.e. A1/B1 or A2/B2). Dominance effects are deviations from additivity, therefore A1A1 – A1A2 ≠ A1A2 – A2A2. This deviation results in the heterozygote being similar to one of the parents rather than the mean of the homozygotes (Table 9). Table 9 Genotypic value of three genotypes at ‘A’ locus. A1A1 6 A1A2 6 A2A2 1 Note that A1A2 can take different values, and that genotypic values are hypothetical for the purpose of explaining the concept here. Non-Linear Regression Figure 5 describes dominance effects by showing the non-linear regression (with non-common slope) between genotypes to describe non-additive effects (in this case dominance effect). Will selection for maximum expression always lead to selection of true breeding individuals? [Hint: consider individuals in early generations compared to purelines in a self-pollinating crop] Hypothetical Example If we consider a hypothetical example of two gene loci (no linkage), the proportions of F2 genotypes are as shown in Table 10. Table 10 Hypothetical example to demonstrate two gene loci (no linkage). Genotype Ratio A1A1B1B1 1/16 A1A1B1B2 2/16 A1A1B2B2 1/16 A1A2B1B1 2/16 A1A2B1B2 4/16 A1A2B2B2 2/16 A2A2B1B1 1/16 A2A2B1B2 2/16 A2A2B2B2 1/16 Let’s assume genotypic value as: A1A1 = A1A2 = 4, A2A2 = 0 B1B1 = B1B2 = 3, B2B2 = 0. Also assume complete dominance at A and B loci. Then, the genotypic values are as shown in Table 11. Table 11 Hypothetical example to demonstrate complete dominance at A and B loci. Genotype Genotypic value A1A1B1B1 7 A1A1B1B2 7 A1A1B2B2 4 A1A2B1B1 7 A1A2B1B2 7 A1A2B2B2 4 A2A2B1B1 3 A2A2B1B2 3 A2A2B2B2 0 Proportions If a breeder makes selections based only on phenotype, she/he will select plants that have the following genotypes (A1A1B1B1, A1A1B1B2, A1A2B1B1, and A1A2B1B2) in the proportions listed below (with the assumption of independent assortment at the A and B loci). A1A1B1B1 = 1/16 of the total population, A1A1B1B2 = 2/16 of the total population, A1A2B1B1 = 2/16 of the total population, A1A2B1B2 = 4/16 of the total population, In this case, the breeder is not able to distinguish between homozygous and heterozygous individuals of the four genotypes above as they have similar phenotypes. Therefore, if a breeder only wanted homozygous dominant (A1A1B1B1) plants and they only used phenotype to make their selection, they will end up selecting 9 out of 16 plants; however, only 1 out of 16 plants should have been selected. In a self-pollinated species where a cultivar is an inbred line, non-additive gene effects can rarely be fixed, and therefore, selection response is unpredictable when a trait is controlled by genes acting in a non-additive manner. In a cross-pollinated species where hybrid cultivars are used, non-additive gene effects, especially dominance effects, are important. Types of Interaction In the example below (Table 7; Fig. 6), we will look at a hypothetical case of two genes controlling plant height to demonstrate epistatic effects (i.e., the interaction of genes at different loci). Assume that ‘A’ and ‘B’ loci both affect plant height (shown in cm in Table 12). Table 12 Genotypic values for two loci ‘A’ and ‘B’ controlling plant height. B1B1 B1B2 B2B2 A1A1 90 105 110 A1A2 90 95 102 A2A2 90 94 95 Epistatic interactions can be additive*additive, dominance*dominance or additive*dominance, or a higher order for three loci or more. These interactions are important for most traits as these interactions are common. Concept of Variability Johannsen Experiment If you received a seed lot that had variable seed size, you can select for small and large seed types and grow these seed as individual plants. Each individual plant can be harvested separately and a unique ID given to each row of plants of these individual plants. If you grew each row and measured seed size, what will you expect to find among and within lines in the progeny generation if the original seed lot consisted of a homogenous mixture of purelines (true breeding = homozygous)? Johanssen, in 1903, conducted an experiment in beans (Phaseolus vulgaris), a highly self-pollinated species, to study the effect of selection for seed weight using a seed lot from the cultivar ‘Princess’. His experiments showed that: a) selection for seed weight was effective in the original unselected population (i.e., lines selected for differences in seed weight showed consistent differences in seed weight in subsequent generations; large seeded parents produced large seeded progeny and small seeded parents produced small seeded progeny), and b) selection within a line was not effective (i.e., irrespective of whether the parent was small or large seeded, all of the progeny of the selected seed always showed the average seed weight typical of the parent line). Johanssen concluded from this experiment that the original seed lot was composed of a mixture of different genotypes/purelines (that were each homozygous for genes controlling seed weight), and even when the progeny of a seed lot differed phenotypically, each seed of that line possessed the same genotype for seed weight. Mathematical Representations Mathematically, the phenotypic value for an individual (i.e., a single seed in Johanssen’s experiment) in a population is equal to its genotypic value plus an environmental (non-genetic) deviation: $\Large P=G+E$ where: P = phenotype (observed seed weight) G = genotype (genetic potential for seed weight) E = environment (environmental effects, i.e., factors determining the extent to which genetic potential is reached) For a population of seed, the phenotypic variability is represented mathematically by the equation below: $\Large V_P=V_G+V_E$ where: VP = phenotypic variability (total variability observed) VG = genotypic variability (variability due to genetic cause) VE = environmental variability (variability due to environmental causes) Genetic variability is heritable, i.e., variability that can be manipulated by plant breeders and transmitted to progeny. The presence of genetic variability (as we saw earlier) is ESSENTIAL for selection to be effective. Environmental variability is not heritable and it can mask the true expression of a trait. Phenotype Interactions If we assume that the mean value of “E” for all individuals across the population is zero, then the mean phenotypic value equals the mean genotypic value. Thus, the population mean is both the phenotypic and genotypic value. To prove this, consider a theoretical experiment using replicated genotypes – either as clones or as inbred lines – and measure them under “normal” environmental conditions. The mean “E” will be zero across the population, so that the mean phenotypic value would equal the mean genotypic value. However, in reality, plant breeders deal with segregating populations that are not genetically uniform when they are selecting. So let’s explore the types of gene actions and their importance to breeding. Phenotype, Genotype, Environment, and Genotype X Environment Interactions Phenotype is governed by Genotype ($σ_G^2$), and Genotype × Environment interactions ($σ_{GE}^2$). Not all variation for a phenotype is accounted for by Genotype and GxE interaction with the remaining variation attributed to error. Any trait that you observe for a plant, a plant family, or population is a phenotype. Genotype is the genetic basis of a trait (e.g., gene or gene × gene interactions). GxE interaction is the interaction of genotype with environment, where each genotype may perform or look different in different environments. The environment of a single plant consists of all things other than the genotype of the individual. The environment includes differences in soil, temperature, humidity, rainfall, day length, solar radiation, wind, salinity, pathogens, pests, etc. Environment Environment can be micro-environment or macro-environment. A micro-environment refers to a unique set of factors that alter the development of a single plant. Groups of plants growing at the same time in the same space each encountering similar micro-environment are classed under experiencing a macro-environment (i.e. a class of micro-environments). For example, if a field of beans is exposed to excessive moisture stress (i.e., water logging), individual plants may suffer slightly different levels of water logging (micro-envirionment), but all plants would suffer some degree of water logging (macro-environment). This beans field’s macro-environment will be described as water logged. In breeding, we are more interested in the macro-environments and these are classified as location or year, or a combination of location x year, or simply environment. To describe the phenotypic value of a genotype in terms of microenvironment and macro-environment, let’s consider the equation below: $\large P_{ijk} = G_i + E_k + (G*E)_{ik} + e_{ijk}$ where: $G_i$= effect of the $i$th genotype $E_k$= effect of the $k$th macro-environment $(G * E)_{ik}$= effect of interaction between $i$th genotype and $k$th macro-environment $e_{ijk}$= residual composed of deviation of the $j$th micro-environment from the mean of such effects in the macro-environment $k$, and deviation of the interaction from the mean of interactions. Graphical Representations of GxE Interaction Assume two genotypes are tested at two locations. On the y-axis, we present yield/ha, and the x-axis is environment (i.e., locations). Figs. 7, 8, and 9 show different types of GxE interaction: Crossover Interaction Crossover interaction is the most important type of genotype x environment interaction because different genotypes will be selected in different environments. Crossover interactions are often due to differences in how genotypes respond to different environments. For example in Figure 6, let’s consider that environment 1 is disease free, while pathogen ‘A’ is present in environment 2. Genotype 2 is a high yielding, disease susceptible genotype and genotype 1 is a lower yielding, disease resistant genotype. Genotype 2 will yield higher in environment 1, while genotype 1 will yield higher in disease prevalent environment 2. The goal of a breeder should be to combine the better response to both environments into a single genotype. However, a breeder needs to first determine if yield and disease resistance are mutually exclusive. For example, it’s possible that the gene for disease resistance could be linked to gene(s) that reduce yield. In this case, a breeder would need to grow a large segregating population to identify progeny with useful recombination that combines high yield and disease resistance. If however, a single pleiotropic gene controls both disease resistance and yield, a breeder can only improve both traits by complementation (i.e., the building or bringing together of other useful genes to improve the responses). Practical Considerations of GxE Interaction for Plant Breeders 1. Breeders working at international institutions (CGIAR institutes such as CIMMYT, ICARDA, IRRI, and CIAT) have a mandate of a wider adaptation, while a provincial or state breeding institute’s mandate will be more localized (specific area, perhaps one macro-environment). CGIAR breeders often utilize many (more than 20) diverse locations to identify cultivars with wider adaptation, while breeders at a state breeding institute use fewer environments that are representative of one or two macro-environments. 2. If the mandate of the program is to develop cultivars for specific purposes (i.e., disease resistance, stress tolerance, or quality traits), then the testing sites need to be selected by breeders for this objective. For example, malt barley has a very specific crop quality requirement. Stable performance on quality (malt quality for consistent and high quality and better taste for beer-making) is a must and breeders will discard cultivars if they show specific adaptation for malt quality, i.e., only very specific sites produce good malt. 3. Resource allocation: A breeder should be aware of the relative importance (i.e., magnitude) of G × Location, G × Year, and G × Location × Year interactions in order to appropriately allocate resources for cultivar testing. This information will help a breeder decide on how many locations and years should be used for testing materials. 4. While we have not discussed different stability analysis methods, these methods (such as AMMI type analysis) will help to determine which environments are more similar to each other. Each mega-environment will consist of several individual locations or sites. Within a mega-environment, the genotypes perform more similarly compared to genotypes in different mega-environments. In other words there is little or no G×E interaction among environments within a mega-environment. In such a scenario, breeders will gain little by testing in more similar environments, and should aim to test across dissimilar environments to test for stable performance of genotypes in a range of environments. Breeders should therefore aim to sample one or more locations from each mega-environment (or testing zone). Environmental parameters such as rainfall, soil type, pH, etc., may also be a good way to cluster environments. If there are larger agro-ecological regions that grow predominantly a single cultivar and you as a breeder are targeting for that region, the cultivar acreage map may also serve as a good source to identify mega-environments to develop a better yielding alternative to that large acreage cultivar. Another useful exercise is to perform genetic correlations (genotype means analysis) to see if the correlations are high or low. High correlation will mean that predictive ability of those environments is similar and a breeder does not gain as much information on stability as he/she would gain by testing in environments with lower correlation among genotype means. 5. If G×E cannot be measured (due to lack of resources to have more than one site), a breeder should still consider putting that test in two dissimilar conditions, for example dryland versus irrigated nurseries. Selecting a Testing Site The site where you grow your trials may have seasonal patterns, such as cycles of drought at seedling stage, heat stress at flowering stage, and years with no apparent stress. It is advisable to keep track of this information (through the use of long term checks that have known and consistent response to these stresses and weather parameters) and use this site for selection to improve tolerance for these factors. If you are breeding for another trait, say salinity tolerance, a new site more appropriate for screening of this trait will be required. Factors to consider in the selection of testing site include the following: 1. Good correlation with the performance in farmer growing conditions. 2. Ability to handle different tests (infrastructure) and ability to respond to mitigate threats (such as an ability to irrigate if needed to avoid impact of water deficit on response to the selected treatment). 3. Low environmental error, i.e., higher heritability to differentiate ‘keeps’ (i.e., desirables) from discards. 4. Infrastructure to implement breeding decisions of timely planting, maintenance, harvest, processing etc. Adaptation Factors As we previously covered, the debate on wide versus specific adaptation is still ongoing among breeders and really boils down to: • the mandate of the program • target region, and • farmers. Ultimately, breeders need to remember that their job is to ensure that the product (cultivar) that reaches farmers can help them to make a profit, and that it meets the requirement of end-user (e.g. the processing industry) who buys from farmers. If you, a breeder, produce the highest yielding cultivar but it lacks the necessary quality or protection against biotic or abiotic stress, farmers will not grow this variety. Therefore, always think of the cultivar you develop as a “package”. A package needs to have all the ingredients that will make it ready to be adopted by farmers as well as the processing industry. Heritability Concept Definition: Heritability can be defined as the degree to which the characteristics of a plant are repeated in its progeny. Mathematically, we have seen already that it is the proportion of total variability for a character due to genetic causes. Further theoretical information can be obtained here. Broad sense heritability: $dda8ee4e7eda613dda187e589319928f.png$ Narrow sense heritability: $859425a81b3d8f3486d2ae0b362dc0e2.png$ where: $σ^2_G$ is total genotypic variance and $σ^2_a$ is the additive component of the genotypic variance. Narrow sense heritability is more valuable since it indicates how much of the total observed variability is due to additive gene action (which can be selected for effectively and fixed in homozygous condition). Broad sense heritability is less valuable since it also includes dominance and epistasis (these gene actions cannot be fixed and occur only in specific gene combinations). Importance It is important for breeders to have a sense of the heritability of traits that they are selecting for in their programs. This can be obtained by using data from their own experiments, or for a new program, using information available in literature or from previous experiments. The reason heritability is important is that selection response is related to heritability. The higher the heritability, the more the phenotype reflects the genotype and the more effective selection will be. More extensive testing (more environments, more replications) reduces the phenotypic variance and increases heritability. Heritability can be increased more by using higher number of locations rather than by increasing the number of years [this is due to smaller variance component of Genotype × Year, relative to other component such as Genotype × Location]. This suggests that in most cases, a breeder does not need to do selection based on more than one year of data [except in cases where selections are made in each generation as plants are achieving ‘true breeding’ status]. Methods for Estimating Heritability 1. Variance Component method: Comparison of segregating and homogenous populations is applicable to only self-pollinated or clonally propagated species. This method estimates broad sense heritability. This method involves estimating the magnitude of various types of genetic and environmental variability. In such experiments, $σ^2_{P1}=σ^2_{P2}=σ^2_{F1}=σ^2_{E}$ In a self-pollinated species, parent 1 and parent 2 being inbred, their estimates of genetic variability will be similar to each other ($σ^2_{P1}=σ^2_{P2}$) as well as to F1 ( $=σ^2_{F1}$), and these will be equal to environmental variability (as these three are genetically uniform and therefore any variability observed will be due to environment). Variance of F2 or any other segregating generation can then be used to obtain $σ^2_{P}-σ^2_{E}$), where $σ^2_{P}$ is variance estimated from the segregating generations. 2. Covariance between relatives (or resemblance among relatives as measured by regression analysis): Examples are, parent-offspring regression, covariance between half-sibs and full-sibs, covariance between inbred or partially inbred families. Variability estimates can be obtained in other ways such as special mating designs (half-sib, full-sib, North Carolina designs, etc.), or analysis of trials conducted in a range of environments. 3. Realized heritability [see Response to Selection section, below] Example 1 Table 13 is an example of analysis of variance (ANOVA) where a random set of genotypes were evaluated over ‘$l$’ locations for ‘$y$’ years, and ‘$r$’ replications used in each test. Multiple locations, years and reps. Table 13 ANOVA formulas for determining degrees of freedom, mean squares, and expected mean squares for a multi-location, multi-year trial. Source of variation Degrees of freedom Mean square Expected mean square Location ($L$) $(l-1)$ n/a $\sigma ^2_{e} + r\sigma ^2_{gly} + ry\sigma ^2_{gl} + g\sigma ^2_r + gr\sigma ^2_{ly} + gry\sigma ^2_l$ Year ($Y$) $(y-1)$ n/a $\sigma ^2_{e} +r\sigma ^2_{gly} +ry\sigma ^2_{gl} +g\sigma ^2_{r} +gr\sigma ^2_{ly} +grl\sigma ^2_{y}$ $L*Y$ $(l-1)(y-1)$ n/a $\sigma ^2_{e}+r\sigma ^2_{gly}+ry\sigma ^2_{gl}+g\sigma ^2_{r}+gr\sigma ^2_{ly}$ Rep ($L*Y$) $ly(r-1)$ n/a $\sigma ^2_{e}+g\sigma ^2_{r}$ Genotype ($G$) $(g-1)$ $MS_1$ $\sigma ^2_{e}+r\sigma ^2_{gly}+ry\sigma ^2_{gl}+rl\sigma ^2_{gy}+rly\sigma ^2_{g}$ $G*L$ $(g-1)(l-1)$ $MS_2$ $\sigma ^2_{e}+r\sigma ^2_{gly}+ry\sigma ^2_{gl}$ $G*Y$ $(g -1)(y -1)$ $MS_3$ $\sigma ^2_{e}+r\sigma ^2_{gly}+rl\sigma ^2_{gy}$ $G*L*Y$ $(g-1)(l-1)(y-1)$ $MS_4$ $\sigma ^2_{e}+r\sigma ^2_{gly}$ Pooler Error $ly(g-1)(r-1)$ $MS_5$ $\sigma ^2_{e}$ where: $σ^2_{e}=MS_5$ $ea22df1998178f8ccef41bd82879491c.png$ $230fc140e15ea824b22dfcd6b05f9d68.png$ $cf41d9cb00deedf6b6abcc070f609524.png$ $300c32ed76b56b4b27e040f0379ffb17.png$ $σ^2_{P}$ (phenotypic variance of genotypic means) = $σ^2_{g}+\frac{σ^2_{gl}}{l}+\frac{σ^2_{gy}}{y}+\frac{σ^2_{gly}}{ly}+\frac{σ^2_{e}}{rly}$ and if we substitute for Mean squares we will obtain, $σ^2_p =\dfrac {MS_1}{rly}$ Broad Sense heritability can be calculated using the equations above. Example 2 Table 14 is an example of analysis of variance (ANOVA) where a random set of genotypes were evaluated over ‘$e$ environments (can be locations, years or combination of years and locations), and ‘$r$’ replications used in each test. Multiple environments and reps within a year. Table 14 ANOVA formulas for determining degrees of freedom, mean squares, and expected mean squares for a multi-environment trial. Source of variation Degrees of freedom Mean square Expected mean square Environment ($E$) $(e-1)$ n/a $σ^2_{e}+gσ^2_{r(e)}+rσ^2_{ge}+rgσ^2_{e}$ Rep ($E$) $e(r-1)$ n/a $σ^2_{e}+gσ^2_{r(e)}$ Genotype ($G$) $(g -1)$ MS1 $σ^2_{e}+rσ^2_{ge}+reσ^2_{g}$ $G*E$ $(g-1)(e-1)$ MS2 $σ^2_{e}+rσ^2_{ge}$ Pooler Error $e(g-1)(r-1)$ MS3 $σ^2_{e}$ Note: all factors considered random in ANOVA. $σ^2_{e}=MS_3$ $69be6715fb7ff81ad4e7552548f31258.png$ $7f47116a343da7d262536733c9ed27c3.png$ Variables $σ^2_p$ If we substitute for Mean squares we will obtain, $dfdccd039decfb8ac237565e1ab14958.png$ Total phenotypic variance $σ^2_p=σ^2_{g}+σ^2_{ge}+σ^2_{e}$ Heritability on individual experimental unit basis is: $\dfrac{σ^2_{g}}{(σ^2_{g}+σ^2_{ge}+σ^2_{e})}$ Heritability on genotypic mean basis is: $60f35c9b477a485b8f0943d28ceaea44.png$ This estimate of heritability is obtained if the genotypes represent the population and are chosen randomly. If the genotypes are not chosen randomly (e.g. selected genotypes), the ratio between genetic and phenotypic variation is called repeatability and this estimate is a measure of the precision of data and a measure of the proportion of genetic variation, which helps breeders to detect significant difference among genotypes. Summary Each heritability estimate is unique and reflective of the method of calculation, testing environment, generation used in estimation, and genotypes studied. While the heritability estimates are going to somewhat differ based on different conditions described above, a plant breeder can get a good handle on heritability based on published literature, and their or their predecessors’ experiences working on the crop and for various traits. Heritability is used to estimate the expected response to selection and to choose the best breeding approach to improve the target trait(s). Traits with high heritability can be selected on a single-plant basis in an early generation and in fewer (even single) environments. A breeder should consider a range of heritability (rather than absolute value) as well as have some precision around their estimate (confidence interval). Higher heritability, say, 0.7, or higher narrow sense heritability means a breeder can expect that selection in early generation can be effective for that trait. High broad-sense heritability only indicates that effect of environment is smaller but does not provide insight into the relative importance of additive (which can be fixed) or non-additive (which cannot be fixed) gene effects. Selection Theory Truncation When individuals are selected based on their individual phenotypic value, we call this artificial selection or individual selection. Truncation is a type of individual selection and very common in plant breeding programs. The curve in Figure 10a represents the normal distribution of a quantitative trait in a population, and the shaded part ‘T’ (i.e., truncation point) represents the individuals selected for the next generation of breeding – could be cross- or self-pollinated. µ is the mean of the unselected population (or mean of the population in generation 1) and µs is the mean of selected parents. If these selected parents are mated at random, their offspring will have the phenotypic distribution in Figure 10b and a mean equal to µ‘. Generally, µs > µ’ > µ. • µ’ is greater than µ because some of the selected parents have favorable genotypes and therefore pass favorable genes on to their offspring. • µs is greater than µ’ because some of the selected parents did not have favorable genotypes, but instead had superior phenotype due to the favorable environment where they were tested (chance exposure to favorable environment, e.g., low spot in the field that received more water, a spot in the field that received more fertilizer, or a spot in the field that was not exposed to high winds). Secondly, alleles, not genotypes, are transmitted to the offspring and favorable genotypes may segregate or recombination may cause breakage of favorable linkages. Equations The difference in mean phenotype between the selected parents ($µ_s$) and generation 0 ($µ$) is called selection differential ($S$). $\Large {S}=\mu _{\small S}-\mu$ Equation 1 The difference in mean phenotype between the progeny generation (generation 1) ($µ’$) and generation 0 ($µ$) is called the response to selection ($R$). $\Large {R}=\mu' - \mu$ Equation 2 The prediction equation defines the relationship between $S$ and $R$. For truncation selection, the prediction equation is: $\Large {R}={h}^{2}* {S}$ Equation 3 where, $h^2$ is heritability of the trait. Graphical Representation z/A = frequency at the truncation point (in a normal distribution)/Area under the selected portion of the curve. This is equal to selection differential/phenotypic variance, i.e., Equation 4 assuming that the effects of each allele are small relative to phenotypic variation, and phenotypic values are normally distributed. Response to Selection Calculations We cannot get into detailed calculation, but it can be shown that: $\mu' = \mu + 2 \Big[a+(q-p)d \Big] \Delta p$ Equation 5 where: $a$ = genotypic effect $d$ = measure of dominance $p$ = allele frequency of A1 $q$ = allele frequency of A2 $\mu'$ = mean phenotype of progeny generation $\mu$ = progenitor generation Substituting z/A = (µs – µ)/ σ2p and Δp, equation can be written as:\ Equation 6 Since, S = µs – µ and R = µ’ – µ, Equation 7 Additive Genetic Variation We already have seen that R = h2S (Equation 3), therefore, we can now define heritability in the genetic terms of: a, p, q, d, σ2p as: Equation 8 This heritability definition is valid when the trait is under single gene control. This is hardly the case for most of the traits, therefore heritability (narrow sense) can be defined as: Equation 9 Σ2pq[a + (q – p)d]2 = additive genetic variation of the trait = σ2a Selection Intensity Selection Intensity and Response to Selection Equation R = h2S can also be written as: $\LARGE {R}={i}\sigma {h}^{2}$ Equation 10 $cd28276191c7cb056ac56f2983654f92.png$ Equation 11 $i$ Intensity of selection depends on the proportion of the total population selected. For example, Table 15 shows some selection intensities based on % selected (assuming a completely normal trait distribution). The full table can be seen in most plant breeding books. Table 15 Percent of individuals selected and the corresponding standardized selection differential. % selected Standardized selection differential (i) 0.01 3.959 0.1 3.367 0.5 2.892 1 2.665 5 2.063 10 1.755 15 1.554 20 1.400 25 1.271 30 1.159 35 1.058 40 0.966 45 0.880 50 0.798 h2 is narrow sense heritability σp = phenotypic standard deviation (=square root of phenotypic variance) Breeder’s Equation This equation R = iσh2 (Equation 10) is fundamental in plant breeding. Plant breeders generally do not use it to calculate an actual numerical value for selection response. However, this equation is important as it shows that selection response depends on: 1. Selection intensity 2. Heritability 3. Phenotypic variability present in the population, say from a cross. The equation R = µ’ – µ can be written as $\Large \mu '=\mu +{i}\sigma {h}^{2}$ Equation 12 which clearly indicates that to maximize the expression of a trait in the offspring generation, a breeder needs to start with high expression of the trait, maximum heritability, and high selection intensity (although diminishing returns apply beyond a certain level). Square root of heritability (h) is a measure of the correlation between the observed phenotypic value and the underlying genotypic value. In a breeding program, a breeder will try to maximize these factors (higher standardized selection differential, genetic variation, heritability). One has to keep in mind that optimum balance needs to be obtained between increased expected response (which is a good thing and what a breeder is after) and increased variability of that response (undesirable characteristics of selection). We will look at a few examples below to understand the concept of variability of response. $(C.V.\ of\ R) = \sqrt{\frac{\large1+(p\,\times\,h^2)-(h-h^2)}{\large p\,\times\,n\,\times\,i\,\times\,i\,\times\,h^2\,\times\,h^2}}$ Equation 13 (From Baker, 1971.) where: n = total number of lines evaluated p = proportion of lines selected h2 = heritability i = standardized selection differential C.V. = coefficient of variation For example, assume a breeder started with n=1000, % selected (p) = 0.01, heritability (h2) = 0.2, and i = 3.959, then CV (Response to selection) = 39% by substituting values in Equation 10. n = total number of lines evaluated = 1000 p = proportion of lines selected = 0.01 h2 = heritability = 0.2 i = standardized selection differential = 3.959 C.V. (R) = 30% Compared to starting with a smaller number of lines, if a breeder started with n=100, % selected = 0.01, heritability = 0.2, and i = 3.959; CV (Response to selection) = 124%. n = total number of lines evaluated = 100 p = proportion of lines selected = 0.01 h2 = heritability = 0.2 i = standardized selection differential = 3.959 C.V. (R) = 124% Importance of h-squared However, h2 importance can be seen with the same calculations: if a breeder started with n=1000, % selected = 0.01, heritability = 0.7, and i = 3.959; CV (Response to selection) = 8%. n = total number of lines evaluated = 1000 p = proportion of lines selected = 0.01 h2 = heritability = 0.7 i = standardized selection differential = 3.959 C.V. (R) = 8% If breeder had started with n=1000, % selected = 5, heritability = 0.7, and i = 2.063; CV (Response to selection) = 2%. n = total number of lines evaluated = 100 p = proportion of lines selected = 5 h2 = heritability = 7 i = standardized selection differential = 2.063 C.V. (R) = 2% These calculations show that while the response to selection equation is an essential equation for any breeder to consider for trait improvement, it is also worthwhile to consider the extent of variability in relation to the mean of the population (CV). Selection Explanation One of the ways to maximize genetic standard deviation is to cross diverse parents. However, if crosses between diverse parents have lower unselected means than crosses between adapted (elite) parents (which will have lower genetic variability between them, generally), then a breeder may be reducing the mean genotypic value of the subsequent population by crossing diverse parents. Therefore, best x best (or elite x elite) crosses is one way to maximize genotypic mean of the starting population although it may reduce the genotypic variance and even response to selection, R. Most cultivar development programs will work with best x best configuration, or have at least 75% elite, for example, (best x exotic) x best. Standardized selection differential can be increased by selecting fewer lines (but we saw earlier that this can cause increased variability of response, which is undesirable) or testing more units but selecting fewer units (this will require more resources). If a breeder makes compromises between testing more lines to advance a few, it will likely be done at a compromise of not doing a thorough evaluation of units. Less thorough evaluation will result in lower correlation between phenotypic and genotypic values (lower h), therefore a breeder should not compromise on proper trait measurement protocols. Optimum balance needs to be achieved for each trait for more thorough testing to increase the correlation between phenotypic and genotypic values (higher h) as well as increase the standardized selection differential. Expected Genetic Gain Expected genetic gain formula is shown below. $\LARGE \Delta G = \frac{ic}{y} \frac{\sigma^2_G}{\sqrt{\sigma^2_p}}= \frac{ic}{y} \frac{\sigma^2_G}{\sqrt{\frac{\sigma^2_e}{re}+\frac{\sigma^2 _{GE}}e{+ \sigma^2_G}}}$ Equation 14 This formula is an extension of response to selection: $\LARGE R = h^2S = ih^2 \sigma _P = \frac{i \sigma^2_g}{\sqrt{\sigma^2_P}}=\frac{c}{y}=\frac{i\sigma^2_g}{\sqrt{\sigma^2_P}}$ Equation 15 and includes two additional variables: number of years (y) and parental control (c) (Eberhart, 1970) compared to what we have seen so far. Heritability equation is $13aefe00908bb153ab5555b16c301dcc.png$ Equation 16 where: $r$ =number of replications $e$ =number of environments We have already looked at i, which is standardized selection differential, c = parental control, and y = seasons per cycle. Practical Considerations 1. Increase the numerator of this equation by increasing genetic variance (larger population sizes, diverse parents (but keep the proportion of elite parents high), increasing selection intensity (without getting genetic drift problem). 2. Parental control will allow for increased response to selection. Parental control, c, can be increased by recombining genotypes where both sources of gametes originated in selected genotypes (c = 1), which will be generally true in self-pollinated crops. In cross-pollinated species, c = 0.5 if the male gametes are coming from unselected genotypes. Therefore, it is recommended that if possible, conduct selection before pollination so that only selected genotypes contribute to the next generation. C = 2.0 if the selected seed of selected genotypes is used for establishing the next generation. 3. Another way to increase genetic gain is to decrease the value of the denominator. This can be achieved by decreasing the number of seasons per cycle or the phenotypic variance (which can be decreased by reducing g x e and e variances. The phenotypic variance can be decreased by increasing the number of locations (or environments) and by increasing replication. Increasing locations is generally considered to play a more important role in reducing phenotypic variance rather than replications. 4. Usage of proper experimental methods and field design and analysis will reduce error variance and improve confidence in estimate of progeny performance. These methods may include augmented designs, or moving means in earlier generations where no replication is used per environment and using incomplete lattice (for example, alpha-lattice) or RCBD in replicated tests. 5. Different generations and type of progenies have different genetic variance components and therefore affect the equation. The theoretical proportion of additive variance to total genotypic variance of half sib is 0.25, full-sib is 0.5 and 1 for S1 progenies. 6. The number of seasons required to complete a cycle can be reduced by using off-season nurseries, or by using an off-season nursery with high correlation to the home environment to facilitate selection for high to moderate heritability traits and to reduce the ‘y’ in equation above. If resources permit, greenhouse or growth cabinet can be used instead of off-season nursery and complemented with marker assisted selection to increase ‘i’ as well as reduce ‘y’. Reducing Effect of Environment One way to obtain higher heritability is to reduce environmental effects (remember, higher heritability implies that selection will be more effective as ‘what you see is what you will get’). Here are some recommendations to reduce the effect of environment: 1. Use best quality land (uniform area – less gradients in field, highly productive) if selection will be performed (on single plant, rows, or yield trials; in early or later generations). 2. Use best management practices (reflective of the recommended fertilizer, irrigation, crop rotation, time of planting, weeding, harvesting). An advice is to avoid pest or pathogen control as this will provide another trait to select for if naturally present]. 3. Use check cultivars frequently (this will allow breeders to have a better handle on variability). 4. Use appropriate statistical designs (lattice, RCBD, augmented designs as needed). 5. Use replication (improves precision, and provides better handle to measure variation) and randomization (improves accuracy). Multiple Trait Selection 1. Tandem selection: A breeder selects sequentially for each trait in successive generations. In this scenario, population is improved first for one trait, then for the next trait and so on. This will lead to improvement over generations. One disadvantage of this strategy is long selection cycle, and is generally not followed in commercial plant breeding. Another disadvantage of this strategy is the potential reduction in the level of performance of the first trait selected. 2. Independent culling (or truncation selection): Selection is practiced successively in the same generation. This is probably the most common selection strategy deployed by breeders worldwide. In this scheme, a breeder will discard all individuals that fail to meet the desired level for one trait, irrespective of the value for any of the other traits. This will be followed by selecting among the surviving lines for the second trait and the process is repeated until all selections are made. Experienced breeders will know the culling point keeping in mind the trait value of the most important trait, and may allow some relaxation for major traits when culling for traits that have less significance or importance. One issue with independent culling is that with each successive trait cull, the population size and genetic variability is reduced. 3. Index selection: An index is developed based on the combination of the heritability and economic value of each of several traits under selection, simultaneous selection is happening for every trait in the same generation. Each line is given an index score based on the trait expression and weight given to the trait. Most breeders use a “mental” index selection. For example, visual selection may be done for a number of traits, an overall mental assessment done, and selection is made. For example, in a space planted nursery where single plants are growing, a breeder may make a mental assessment on the criteria for different traits such as height, seed fill, plant health, lodging, inflorescence and either keep or discard. Since there are likely several thousand plants in a nursery, “mental” index approach needs to be used, as the most feasible. There are more sophisticated methods described such as Pesek baker index (Pesek and Baker, 1969) but these require estimation of variance and co-variances. Using economic weights is a good compromise to remove the need to know variance and co-variances. However, it is still not an easy task to develop an index. Combining Ability GCA and SCA Combining ability of inbred lines is of paramount importance in determining future usefulness and commercial potential of the inbred lines for hybrid production. Combining ability can be divided into general combining ability (GCA) and specific combining ability (SCA) (Sprague and Tatum, 1942). This concept has been very important in the commercial success of maize breeding and hybrid development. GCA is defined in terms of the average performance of a line in hybrid combinations. The GCA is calculated as the average of all F1s having this particular line as one parent, the value being expressed as a deviation from the overall mean of crosses. SCA is defined in terms of instances in which performance of certain hybrid combinations (between two inbred lines in a single cross) is either better or poorer than would be expected based on the average performance of the parent inbred lines. That is, each cross has an expected value that is the sum of GCAs of its two parental lines. However, each cross may deviate from the expected value to a greater or lesser extent, and the deviation is referred to as the specific combining ability (SCA) of the two lines in combination. Estimates of GCA and SCA are applicable to the particular set that a breeder has used in the crossing. These crossings are generally in a diallel design (full or partial, or other designs such as NC designs). Sprague and Tatum (1942) reported that for unselected inbred lines, GCA was relatively more important than SCA, whereas for previously selected lines SCA was more important than GCA. GCA is an indication of genes having largely additive effects (differences of GCA are due to the additive and additive × additive interactions in the base population) and therefore more important in a population such as synthetics, while SCA is indicative of genes having dominance and epistatic effects (differences in SCA are attributable to non-additive genetic variance) therefore more important in a hybrid combination. Calculations NOTE: GCA is the average performance of a plant in a cross with different tester lines, while the SCA measures the performance of a plant in a specific combination in comparison with other cross combinations. Let us look at some calculations: As we previously described, deviation of the parent mean (X) from the mean of all crosses or population mean (μ) is the general combining ability, therefore we can calculate GCA as: $\Large GCA = X - \mu$ GCA of a parent A can be defined as $\Large Y_i = \mu + GCA_A + e_i$ Example Below is an example to show GCA calculation using an experiment where eight inbreds were mated to produce 16 crosses. Response variable was grain yield. Table 16 Grain yield data from a 4 x 4 cross. Cross mean E F G H Half-sib mean A 91 92 77 84 86 B 83 86 75 120 91 C 82 98 85 103 92 D 104 112 87 101 101 Half-sib mean 90 97 81 102 92.5 GCAA -6.5 (=86 – 92.5) GCAB -1.5 GCAC -0.5 GCAD 8.5 GCAE -2.5 GCAF 4.5 GCAG -11.5 GCAH 9.5 SCAAE = 3 (i.e., full-sib mean – (mid-parent value)) or [91 – (86+90)/2] SCAAF = 0.5 Etc. Diallel Example In the case of a diallel, the calculations of GCA are shown in Table 17 below. Table 17 Example calculation of GCA for a diallel. Cross mean B C D E Total GCA A 91 92 77 84 344 (=91+92+77+84) -7.2 B n/a 86 75 120 372 (=91 + 86 +75 + 120) 2.1 C n/a n/a 85 103 366 (=92 + 86 + 85 +103) 0.1 D n/a n/a n/a 101 338 (=77 + 75 + 85 + 101) -9.2 E n/a n/a n/a n/a 408 (=84 + 120 + 103 + 101) 14.1 n/a n/a n/a n/a n/a 1828 (=344+372+366+408) n/a Note: “n/a” means cell is blank. Expected value of a cross between inbred lines ‘A’ and ‘B’ is $\large \textrm{X}_{AB}=\mu+ \textrm{GCA}_{\textrm{A}}+\textrm{GCA}_{\textrm{B}}+\textrm{SCA}_{\textrm{AB}}$ $μ$ and where: A represents a specific inbred T = Mean of hybrid performance across each parent for a trait n = number of parents used in crosses, which is 5 in this case (From Acquaah, 2007.) GCAA = [344/(5-2)] – [1828/5(5-2)] = -7.2 GCA for other inbreds can be calculated similarly. Expected value of the cross between A and B = -7.2 + 2.1 + 91.4 = 86.3 [91.4 = average of all SCA’s] The SCA is calculated as follows: SCAAB = 91 – 86.3 = 4.7 Heterosis Heterosis is the superior performance of crosses relative to their parents (Shull 1910; Falconer and Mackay, 1996). Mid-parent heterosis is the difference between the hybrid and the mean of the two parents used in developing the hybrid and can be calculated as where: μ = trait mean of the hybrid μ MP = trait average of the two parents High-parent heterosis is the superiority of a hybrid over the better parent. Heterosis is dependent on the presence of dominance and summation of allele frequency differences across loci. In maize and other cross-pollinated crops, heterotic groups have been created such that they maximize the difference in allele frequencies in genes affecting target trait(s) thereby maximizing heterosis. Examples of hybrid cultivars include: commercial single-cross maize hybrids, commercial three-way cross maize hybrids, and sunflower hybrids. Hybrid cultivars are usually utilized for allogamous species but some hybrids are produced for some autogamous species (i.e., sorghum, tomato, rice). Single-cross hybrid cultivars are homogeneous and heterozygous. Three-way hybrids are both heterogeneous and heterozygous.

textbooks/bio/Agriculture_and_Horticulture/Crop_Improvement_(Suza_and_Lamkey)/1.04%3A_Refresher_on_Population_and_Quantitative_Genetics.txt

Asheesh Singh; Arti Singh; and Anthony A. Mahama Cultivar development is one of the four plant breeding projects (other three are genetic improvement, trait integrations and product placement) of many breeding programs. The end product depends on the specific objectives, the mode of propagation and commercial production, and like most other processes, specific steps need to be followed in succession (in parallel in certain cases) to ensure the set objectives are met. Learning Objectives • Describe the basic steps in the development of clonal, inbred, synthetic, hybrid, multilines, and blended cultivars • Distinguish among the different clonal types and source of variation • Know the application of male sterility in hybrid crop development Asexually Propagated or Clonal Cultivars In Chapter 1, we looked at different types of cultivars that are grown by farmers. These cultivars may be sexually or asexually propagated. Clones are types of cultivars that are asexually propagated. We also learned in this module the concept of heterogeneous versus homogenous and heterozygous versus homozygous. Clonal cultivars are heterozygous and homogenous and can be maintained through vegetative plant parts. Figure 1 depicts the gradation of heterogeneity and heterozygosity in different types of varieties. Clones Clones, as the name implies, are identical copies of a genotype. A population of clones of the same genotype is homogeneous (since they are identical). However, individually, they are highly heterozygous. Asexually or clonally propagated plants produce genetically identical progeny. Since the cultivar can be asexually propagated, heterosis can be fixed as long as the propagation continues. Clones can be the product of inter-generic or inter-specific crosses because even sterile hybrids can be maintained in clonally propagated crops. One of the biggest challenges in clonal crops production is to keep parental lines and breeding stocks free of virus and other disease that can be transmitted through vegetative propagation. In-vitro methods (such as, tissue culture) are often used to rapidly increase clonal stocks which can be kept disease free. The tissue culture methods of plant propagation, known as micropropagation, utilizes the culturing of apical shoots, axillary buds and meristems on sterile suitable nutrient medium to grow new clones. Micropropagation offers an ability to continually produce clones (all year round), produce and propagate hybrids, and produce disease free plants. It is a cost effective technique and requires small space. Somaclonal Variation Clones are products of mitosis. Any variation occurring among them is environmental in origin. Micropropagation or tissue culturing can lead to somaclonal variation (SV). SV can be defined as genetically stable variation generated through plant tissue culture (Larkin and Scowcroft, 1981). It has been used by breeders as an approach to create and exploit greater genetic diversity and provides a mechanism to expand germplasm pool for plant improvement and cultivar development. While the resultant success of SV has not been as great as initially promised, it has led to identification of valuable genotypes, for example, Aluminum tolerance in rice (Jan et al 1997). SV can also cause negative effects, therefore mechanism of genotype purity is desirable in clonal crops. Whether natural (spontaneous) or artificial (induced), somatic mutations are characterized by tissue mosaicism, called chimerism. In a chimera, an individual consists of two or more genetically different types of cells, i.e. mosaics, which can only be maintained by vegetative propagation (not transferable to progenies by sexual means as clones are products of mitosis and change did not happen in sex cells). Examples of Clonal Cultivars Many clonal cultivars are in use. Fig. 2 and Fig.3 show some examples. Steps in Development of Clonal Cultivars Clonal cultivars are developed using the micropropagation method, which involves the following steps. 1. Selection and maintenance of stock plants for culture initiation, 2. Initiation and establishment of culture – from an explant like shoot tip, on a suitable nutrient medium, 3. Multiple shoots formation from the cultured explant, 4. Rooting of in vitro developed shoots, and 5. Transplanting and hardening, i.e., acclimatization before transplanting to the field. Breeding Approaches Used In Asexual Crops Clean (disease-free) clonal material is essential starting material for multiplication for propagation. It is very important in clonal crops to maintain disease-free and/or purify an infected cultivar (Fig. 4). Infection can occur due to bacteria or viruses. Viruses are more damaging due their systemic nature. In order to screen for disease-free material plant material is visually inspected for the presence of pathogens, however this is not the most effective method for viruses as there may be no obvious symptoms. Detection of Specific Pathogens Two main methods are used to detect the presence of specific pathogens: • Serological, such as enzyme-linked immunosorbent assay (ELISA) • Nucleic acid based, such as Real Time-PCR. These techniques can detect latent viruses as well as other pathogens. Note that a negative test may not always be proof of the absence of pathogens, and could just be due to ineffective assay. If diseases or viruses are detected, it is important to eliminate them. Methods used include: • Tissue culture: Even when the pathogen is systemic, tissue from the terminal growing points can be used for further propagation as it is often pathogen-free. • Heat treatment: works well for fungal, bacterial, and nematode infections. For viruses, a longer treatment is required relative to other pathogens. • Chemical treatment: surface sterilization with chemicals can be used to eliminate pathogens. • Use of apomictic seed Apomixis Apomixis is the formation of seeds without meiosis, and two forms are present. 1. Gametophytic apomixis in which the asexual embryo is formed from an unfertilized egg; and 2. Adventitious embryony, in which the asexual embryo is formed from nucellus tissue. Steps in Development of Apomictic Cultivars Apomictically produced seeds are genetically identical to the parent plant. Breeding of apomictic species requires developing population improvement by sexual reproduction and subsequent variety development by apomixis. Example of plant species in which apomictic cultivar has been produced is Kentucky bluegrass. Summary of Steps Step 1: Defining objectives Step 2: Develop a segregating population Step 3: Select superior plants (clones) Step 4: Preparation of seedstock for commercial planting After objectives are clearly defined and are considered biologically feasible, genetic improvement in clonal crops starts with the assembly and evaluation of a broad germplasm base, followed by production of new recombinant genotypes derived from selected elite clones and careful evaluation in a set of representative environments. In a crop with few years of breeding efforts, the divergence between landraces and improved germplasm is not as wide as in crops with a more extensive breeding history. As a result, landrace accessions play a more relevant role in clonal crops such as cassava (with fewer years of directed breeding efforts compared to other crops such as potato). Longer-Term Breeding Experience With longer term breeding experience, a breeder will have a better understanding and handle on combining ability of different clones as well as more information on other traits such as quality and pest resistance. Thus it is essential to pick parents that will have a higher chance to produce offspring that will be superior and a commercial success. It is important to recognize the difference between botanical (or biological) seed and vegetative (clonal) parts (used for propagation). The F1 obtained from crossing is biological seed, while the plant parts used from that F1 for further testing in subsequent generations is are clones (identical to the F1 plant). Several cuttings or plant parts taken for testing from a single F1 will be identical to each other and to the F1, while plant parts taken from different F1 plants will be dissimilar to each other. In the case of Cassava, the botanical seed obtained after crossing is either planted directly in the field or first germinated in greenhouse conditions and then transplanted to the field when growth is suitable to transplant. Root systems in plants derived from botanical seed and vegetative cuttings may differ considerably in their starch content and therefore selections should not be made for traits that will differ between biological seed and cuttings due to low correlation between trait data from plants developed from biological seed and vegetative cuttings. Root Data Since the commercial product is derived from vegetative cutting, root data from biological seed is not relevant for use in selection (unless the correlation is high). One way to overcome this issue is to germinate the biological seed and then transplant, which then develops a root system similar to what will be observed with vegetative cuttings. In clonal crops, the area of plant used to get cutting also influences the performance. Therefore plant breeders should be aware if these are issues in the crop they are working on. In Cassava, vegetative cuttings from the mid-section of the stems usually produce better performing plants than those at the top or the bottom. This variation in the performance of the plant, depending on the physiological status of the vegetative cutting, results in larger experimental errors and undesirable variation in the evaluation process. Consistency in vegetative cutting is important to remove this unwanted source of error. Breeders should also be aware of the number of cuttings that can be obtained per plant as one of the biggest constraints in getting to multi-location trial is the inability to produce enough cuttings per plant to put them into replicated evaluations across several locations. Crossing Block Crossing block can be in the field for over a year. In the case of Cassava, synchronization of flowering may require that crossing blocks are maintained for as many as 18 months and on average only one or two seeds per cross can be obtained (in directed crosses). The first selection can be conducted in the third year in the nurseries with plants derived from botanical seed. Due to low correlations between the performance of individual plants and yield plots, selections should be done only on traits with high heritability. Traits with higher heritability can be plant type, branching habits, reaction to diseases. Breeders will be able to reduce their population by up to 60-80%, making the numbers suitable for clonal evaluation trials (generally based on 6-10 vegetative cuttings in Cassava). At this stage, breeders may just use spray paint or tags or another method to identify the discards as some of the traits are visually assessed, and data collection may not be feasible in all cases. From about 100,000 F1 plants a breeder will be able to use single plant selection to reduce the entries going to clonal evaluation trials to about 2,000 to 3,000 clones, where each clone is going to consist of 6-10 plants (coming from vegetative cuttings). In each trial, care is taken to ensure that the same number of plants are grown (for each entry) to avoid bias in selection. Plot Considerations As in row crops, plant breeders will use ways to ensure that plots are of the same size (length and width) so that no plot is given unnecessary advantage just because it had a larger area. Either a GPS planter is used so that plots are of the same size, or a trimmer is used to cut the plots to be equal size, or proper measurements are made prior to planting (i.e., if hand planting). These clones are planted in an un-replicated single row trial generally. Since there is going to be a lot of variability in canopy coverage, height, branching (plant architecture traits), breeders will either try to group more similar entries into same test, or select for high heritability traits (as mentioned above, including harvest index, plant type, branching habit, leaf type, or some other higher heritability quality trait). To reduce this problem of inter-plot competition, the distance between rows can be increased and plant-plant distance reduced, or leave an empty row between plots. This planting strategy increases the competition among plants from the same genotype and reduces the competition between plants from different genotypes. It is also advisable to consider dividing fields into smaller blocks and conducting selection within a block (with commercial or elite checks present regularly across the field within each block). It is advisable to record all trait data as it helps a breeder to assess within (all clones from a cross) and among (between different crosses) family performance, and gives an indication of the parents used in crossing. Trait Assessment Traits with intermediate to lower heritability can be selected for in the evaluation trials including root or tuber dry matter (which are the economic part and most important breeding objective). The clones keep getting included in advanced trials (from clonal evaluation tests > Preliminary yield tests > advanced yield test > regional tests; number of entries reducing at each stage, vigor of testing increasing in terms of locations, replication, traits) to assess their yield and stability. Also processing and consumer preference (i.e. end-use quality) traits are assessed, and since such traits are most expensive or cumbersome to test, only the most advanced material is tested for these traits. As the most promising material is getting evaluated for processing and end use quality, the breeder needs to start the steps to multiple the stock for commercial planting. It is generally accepted that at the PYT and AYT stages, testing at more locations will be more beneficial than using more replications, and 2-3 replications per entry should suffice in most cases. Steps in Development of Self-Pollinated Cultivars Pure Line Cultivars Pure line cultivars are developed in self pollinating species. Pure line cultivars are homogeneous and homozygous and, once created, can be maintained indefinitely by selfing. Inbred lines are different from pure lines, although sometimes people use the terms interchangeably. Inbred lines are developed in cross-pollinated species through inbreeding and these lines are used as parents in the production of hybrid cultivars and synthetic cultivars. Inbred lines are not meant for commercial release to farmers for commercial production because inbred lines suffer from inbreeding depression (yield will be lower than in hybrid and even an open-pollinated variety (OPV). Inbred lines are homogenous and homozygous, similar to pure lines; however, artificial selfing needs to be done at each generation to maintain or increase seed. Sib-mating can be used to avoid severe inbreeding depression. Summary of Steps in Development of Self-Pollinated Cultivars Step 1: Define objectives Step 2: Form the genetic base by creating segregating population(s) Step 3: Perform selection to make pure lines Step 4: Conduct Trials (testing of experimental lines) and Seed Multiplication Clearly Defined Objectives Step 1: It is critical that a plant breeder has clearly defined objectives before any other activity happens. Objectives need to be clearly defined and biologically possible. Also consider the following when defining objectives: 1. Will it meet the needs of the producer, processor and consumer? The best way to accomplish this is by having direct interactions with these three groups. Reading news print and other sources will also give a breeder an indication of the requirements by these groups. If possible, a breeder should attend farm shows, farm group meetings, meet and visit processing companies, colleagues in other disciplines, and marketing companies. 2. Available resources. Do you have the necessary resources to achieve the objectives? For example, if you would like to select for resistance to a disease using molecular markers very closely linked to the gene as you don’t have disease nursery available. However, if you don’t have access to either the disease nursery or marker screening lab, it will be near to impossible to meet the objective of developing cultivars with resistance to that disease. Without clear and logical objectives, a breeder is working aimlessly. It is analogous to driving a car without knowing the destination. Making Strategic Decisions Setting objectives allows a breeder to make strategic decisions, such as: 1. Picking parents that have the necessary complementation of traits to develop progeny that possesses desirable traits from both parents. 2. Which breeding method to use 3. Determine selection strategy and plan for any specialized nursery or tools. 4. Breeder can also make decisions on which traits to select for and in which generations. Step 2: Based on the objectives, a breeder can pick the parental material which can be: 1. Advanced lines from the breeding program 2. Advanced lines from another breeding program 3. Released cultivars 4. Germplasm line from gene bank or a pre-breeding program 5. Introductions (from other countries) from colleagues or genebank 6. Mutant lines, populations (unselected or selected) 7. Wild relative (need to be crossable or resources available to do embryo rescue if needed) Crops that have a long history of breeding efforts will rely on cultivars and advanced breeding lines and in specialized cases, introductions as the choice of parent material. Crops with less breeding effort will rely on populations and introductions. Majority of parents in a breeding program will be best lines (advanced lines or newest cultivars) derived from the continuous breeding cycle of the program. Parents Selection All the traits desired in the cultivar you want to develop need to be present in the parents. Parents selection has to be based on reliable and complete data (yield testing, adaptation testing, specialized nurseries for stress assessment, end use quality, etc.). If a breeder does not have the specialized nurseries, she/he would collaborate with other breeders in the same or different organizations to send material for testing and characterization (material transfer across organizations may need a material transfer agreement (MTA). The number of crosses made by a breeding program depends on various factors, such as objectives, resources available, breeding method (determines number of breeding lines that will be generated). In self pollinated crops with pure-line cultivars, it is assumed that the parent seed being used is a pure-line (homogenous and homozygous), but in case a breeder decided to accelerate incorporation of a trait and uses a line that is still segregating visually for a trait, more number of plants will need to be used to increase the probability of recovering desired recombinants. Cross configuration will also dictate how many F1’s to create. If a cross is made between two pure-lines, all F1’s will be heterozygous but homogenous. If a three way cross is made using three different pure-lines, F1’s of the three way cross will be heterozygous and heterogeneous, necessitating large population size of F1’s to be created. There is considerable debate about the relative importance of number of crosses versus population size per cross. In most scenarios there will be an inverse relationship between number of crosses and population size, primarily due to resources available in a breeding program. In their review on this topic, Witcombe and Virk (2001) suggest that the strategy is to make fewer crosses (but very careful decisions need to be made based on prior information and scientific principles to pick parents) that are considered favorable and produce large sized populations from them to increase the probability of recovering superior genotypes. Appropriate Breeding Method Step 3: Once the crossing scheme is decided and crosses made, the next stage is to choose an appropriate breeding method to develop inbreeding populations which will be composed of an array of different inbred homozygous lines (pure-lines) where genetic variability exists among but not within lines. All breeding methods in pure-line breeding lead to an increase in homozygosity, a reduction in the genetic variance within families, and an increase in the genetic variance between families. Cultivar development is aimed at identifying the best homozygous lines. Selection should commence in an early generation and preferably as early as F2 because it is the generation of maximum variability, and the minimum population size required to observe desirable type is lowest in F2 and progressively increases. This means a breeder will not have to evaluate larger populations as the generations advance in order to recover the desirable types. It is also important that a breeder makes selections in each generation (if possible) so as to continue with the development of pure-lines and to eliminate undesirable types. This may be accomplished through phenotypic or genotypic selection with molecular markers. If a high value marker is, or set of markers are, available and linked to the trait of interest, it will be very beneficial for a breeder to use the marker(s) to enrich and advance the desirable types while eliminating the undesirable types. Selection Considerations Consider for example, a trait under recessive gene control, which, in fixed homozygous recessive state, will never segregate to give the desirable dominant allele. Evaluating lines with the undesirable homozygous recessive genotype in this case will be a drain on resources. Therefore, the use of molecular markers (which are not influenced by environment) lends a breeder confidence in the selections made, provided the molecular marker is robust, tightly linked to or is on the gene, and not background dependent. For selections in early generations, a breeder will likely handle several thousand plants, hills, or rows. It is therefore very important to handle this material in a selection environment that is very similar to the target region or is representative of the target environments of different agro-ecological regions if breeding for a sub-region. In other words ensure that the environment is ideal for selecting for targeted traits. For example, choose dryland environment if breeding for drought tolerance, or irrigated field nurseries if breeding for high-input environments. It is best to grow these early generation trials at a location where a breeder has easy and quick access to observe the material to facilitate making breeding decisions, and also from a logistical viewpoint, to better manage the trial location. It is extremely important that a breeder eliminates controllable sources of variation such as weeds, non-uniform land, animal damage, non-uniform application of chemicals. Generation Trials In early generations (F2 to F4), selection is restricted to traits of high to moderate heritability, whereas in later generations (F4 to F6 or F8 depending on the complexity of the crop genome), evaluation is more detailed and involves multi-location testing and replication. In situations of relatively large number of entries but limited resources, in early generation trials, single replication yield plots may be used to identify material to advance. Statistical approaches such as running mean, partial rep or augmented designs can be used to identify promising lines. As generations advance, more seed is available and population sizes are sufficiently reduced to allow for increased replications and locations. Selection is then done for traits of lower heritability, such as yield (in larger or paired rows) and for end use quality. However, techniques that require small sample sizes such near-infrared spectroscopy (NIRS) can be effectively utilized in earlier generation testing of end use quality traits to select and remove the undesirables based on cut-offs developed in comparison with checks or industry requirement. Note that cut-offs for traits are variable in every test as they are generally developed based on checks or industry requirement. Final Stages Step 4: Final stages in the breeding cycle will involve lines that are considered pure-lines (non-segregating). At this stage, more extensive testing of few best recombinants from a cross is done for agronomic performance and end-use quality. Multi-environment testing is done for adaptation and stability, and environments may be locations or a combination of locations and years. At this stage, trials will be grown using lattice design (incomplete block if the number of entries is large) or RCBD, and detailed observations made and data taken. Since fewer number of lines is tested, more detailed assessment is feasible for an increased number of traits. For optimal use of resources and to ensure timely adoption of pure-lines, a breeder needs to pro-actively initiate seed multiplication alongside advanced yield testing for production of sufficient quantities of certified seed for the launch of pure-lines. In the case of doubled haploid (DH), steps 3 and 4 are very closely aligned and can even be considered as one because once the doubled haploid is generated, lines can go into multiple location replicated testing where seed quantity permits. Steps in Development of Synthetic Cultivars Hybrids and Synthetics Cross-pollinated species have two main types of cultivars: Hybrids and Synthetics. Hybrids are generally a product of a single cross (or two way cross; A x B), and to a much smaller extent three-way crosses (A x B) x (C) or double-crosses (A x B) x (C x D). Synthetic cultivars consist of a mixture of heterogenous and heterozygous individuals (parental lines are generally clones or inbred lines). Synthetics are more common in some developing countries. These parental lines are maintained so that synthetic cultivars can be re-constituted when needed. These parental lines (clones of inbreds) are assessed for their general combining ability and lines exhibiting superior combining ability are crossed in a polycross configuration to produce S0. The S0 plants are allowed to intermate to produce S1, which, in the case of asexual propagated crops such as alfalfa, can be sold as a synthetic cultivar. Sometimes S2 may be sold as commercial cultivar but maximum heterosis is observed in S1 and is therefore a more favorable generation for cultivar development. In annual crops such as maize, asexual propagation is not feasible therefore the progression from Breeder Seed to Foundation Seed to Certified Seed production is done from S2 to S3 to S4, respectively. A synthetic cultivar differs from open-pollinated variety (developed by mass selection). A cultivar developed by mass selection is made up of genotypes bulked together without having undergone preliminary testing to determine their combining ability. This makes an open-pollinated cultivar the same as a landrace cultivar. Synthetic Cultivars Hybrids are preferred over synthetic cultivars in crops where hybrids can be created economically and commercialized. However, in crops that show heterosis but hybrid production is difficult, synthetics are important and preferred. Synthetic varieties are known for their hybrid vigour and for their ability to produce usable seed for succeeding seasons. Because of these advantages, synthetic varieties have become increasingly favored in the cultivation of many species, for examples forage crops such as alfalfa. Summary of Steps in Development of Synthetic Cultivars • Step 1: Define objectives • Step 2: Form the genetic base by creating segregating population(s) • Step 3: Perform selection to make pure lines • Step 4: Conduct Trials (testing of experimental lines) and Seed Multiplication We have already covered in details how to set up objectives. Similar principle can be applied to synthetic cultivars to set reasonable synthetics. Assembly of Parental Lines Assembly of parental lines can be from a previous synthetic cultivar or from other experimental populations. Parental lines can consist of different clones (forages) or inbred lines (maize). Clones will be highly heretogenous and heterozygous and each clone will be unique. These clonal lines are used to establish a source nursery with several thousand individual plants generally grown in a space planted grid system to reduce environmental variance and enable meaningful comparisons of experimental clones amongst each other or to a check within smaller grids. A breeder will select for higher heritability traits such as disease reaction, and morphological traits. Once the superior clones are identified, they are grown in a polycross nursery to either facilitate random pollination among clones or carefully set up clones to facilitate equal chance per clone to contribute pollen to other clones. Clones may be replicated to ensure uniform pollination. Further evaluation may be done, and seed is harvested (in equal amount per clone per replication). In a perennial species, clones may be grown for more than one year and seed harvest each year. This polycross nursery is used to produce seed for progeny testing in performance tests (in corn, one year but several locations and can be replicated; in forages and perennials, one or more years in several locations and can be replicated). Based on the progeny testing, superior clones from the polycross nursery are identified and crossed to each other to produce the synthetic. Syn1 or Syn2 may be released as a synthetic cultivar in a forage species (with clonal propagation). While in maize, two or three more rounds of pollination may be needed to have sufficient seed for commercial launch of a synthetic cultivar. Half-sib Selection Half-sib selection is widely used for breeding perennial forage grasses and legumes. A polycross mating system is used to generate the half-sib families from selected clones maintained vegetatively. The families are evaluated in replicated rows for 2-3 years. Selecting of traits with high heritability, e.g. oil and protein content in maize is effective. Full-sib mating involves the crossing of pairs of plants from a population in which case control is exerted on both male and female parents. A half sib is a plant (or family of plants) with a common but unknown pollen parent (i.e. pollen source). Therefore, in half-sib mating, the pollen source is random from the population, but the female plants are identifiable. Half-sib selection is based on maternal plant selection without pollen control, therefore half-sib selection is less effective for changing traits with low heritability. The methods used by plant breeders in population improvement may be categorized into two groups: one group is based on phenotypic selection alone (no progeny testing), and the second group is based on genotypic selection (with progeny testing). The specific methods include: 1. Simple recurrent selection or mass selection. The procedure does not involve the use of a tester so there is no estimation of general or specific combining ability. Selection is based on phenotypic observations and therefore this method is also known as phenotypic recurrent selection. 2. Recurrent selection for general combining ability. In this method a wide genetic base cultivar (i.e., a population) is used as a tester to cross with identified females, therefore deploying a half-sib progeny test procedure. Based on the test cross progeny performance in replicated or multi-environment trials, selections are made. Selected lines are advanced into the next round of testing. Generations may be advanced by sib-mating while the progeny test is on-going. 3. Recurrent selection for specific combining ability. In this method a narrow genetic base line (i.e., inbred line) is used as a tester to cross with females, also deploying a half-sib progeny test procedure. Similarly, based on the test cross progeny performance in replicated or multi-environment trials, selections are made. Selected lines are advanced into the next round of testing and here also, generation may be advanced by sib-mating while the progeny test is on-going. 4. Reciprocal recurrent selection. This scheme is capable of exploiting both general and specific combining ability. This is achieved by using two heterozygous populations, where each population serves as a tester for the other. [Note: The difference between a synthetic and an open pollinated cultivar is the ability to re-constitute the seed in a synthetic because the parents (inbred, clones, hybrids) are used in a pre-determined manner and configuration, while in an OPV the original population cannot be created due to no control on parent configuration. After a limited number of generations, seed needs to be reconstituted for a synthetic using the breeder selected parental stock, while in OPV the random mating happens in each generation and population can be propogated indefinitely] Steps in Development of Hybrid Cultivars Hybrid Cultivars Hybrid maize (Zea mays L.) in USA is an example of a success story in cross-pollinated crops. Up until the 1930s, open pollinated varieties were the more common type of cultivars in cross-pollinated crops. However since then in USA almost all commercial maize cultivars are hybrids. A hybrid cultivar is the F1 offspring of a planned cross between inbred lines, cultivars, clones, or populations. The hybrids may be the product of a single cross, a three-way cross, or a double cross hybrid. One absolute requirement for a hybrid cultivar is its superior performance over the parents (heterosis) and an ability to economically generate the seed for commercial seed sale. In the case of maize, farmers moved from OPV to hybrids due to several advantages offered by hybrids which include higher yield, improved tolerance to stalk lodging (i.e. better standability), and improved response to drought. Development of hybrids in maize was easier due to the ability to follow a configuration of males and females for seed production – female rows were detasseled and seed was only collected from female rows (in later years, male rows destroyed post-pollination to ensure no contamination). Therefore, the role of male rows is to serve as pollen source, with requirements that the male needs to be a good combiner to the female (higher SCA) and must possess good pollen shed, nicking well with the female, i.e, must have close flowering time to that of the female. Cytoplasmic Male Sterility Discovery of cytoplasmic male sterility (Fig. 5) has also helped in the development of hybrid crops in those species. Plants with sterile cytoplasm plus nuclear non-restorer genes are male sterile; plants with sterile cytoplasm plus nuclear restorer genes produce fertile pollen (fertility restoration). In contrast to plants with sterile cytoplasm, plants with normal cytoplasm are male fertile when they carry either of the nuclear genes: restorer or non-restorer. Fertility restoration may not be needed for crop species in which the vegetative part is of economic value. In addition to straight crossing (male on female) and male sterility, chemical agents have been used to create hybrid cultivars. An important consideration of hybrid cultivars is the expression of heterosis and efficiency (i.e., cost, labor, time) of seed production. Practical Considerations Some practical considerations for hybrid systems: 1. Farm production of field crops such as wheat, barley, rye, sunflower, grain sorghum requires large amounts of seed for planting the crop and return per unit area is relatively low compared to most horticultural crops or oilseed crops (such as canola), so expensive labor intensive methods of producing hybrid seed (such as hand emasculation) are not preferred. Cytoplasmic male sterility is one approach that is useful in field crops to develop hybridization techniques in which seed parents and pollinators could be grown on a field scale. 2. Pollination also needs to be on a field scale, such as with wind or with bees. Successful hybridization via wind pollination requires that males shed abundant amounts of pollen and female lines be receptive (e.g. florets should open at appropriate times). There is a need to synchronous flowering times between females and males. 3. Female inbred line should generally be more productive and with a higher capacity to produce more seed. Male and female need to be chosen for their superior specific combining ability, and are generally distinct i.e. belonging to different heterotic groups as for example stiff stalk and non-stiff stalk in maize in North America, or origins as for example indica and japonica in rice. 4. Expression of heterosis needs to be sufficient to overcome the cost of development and hybrid seed production. For example, in wheat heterosis is not sufficient to warrant development of a hybrid. Floral morphology also prevents easy pollinations. Hybrid Rice Breeding A good example in hybrid rice breeding in China was produced by the International Food Policy Research Institute (Li, Xin, & Yuan, 2009). History of hybrid rice technological development in China 1964 – Research on three-line hybrid rice initiated 1970 – Wild abortive (WA) rice identified on Hainan Island in China 1973 – Photo-thermosensitive genic male sterile (PTGMS) material identified 1974 – First sets of three lines (A, B and R lines) developed for three-line system hybrid rice 1976 – Hybrid rice commercialization started 1977 – Systematic hybrid rice seed production technique developed 1983 – Hybrid rice seed yield more than 1.2 ton/ha 1987 – Hybrid rice seed yield more than 2 ton/ha Hybrid rice area more than 10 million ha National Two-line System Hybrid Rice Program established 1990 – Hybrid rice area more than 15 million ha 1995 – Two-line hybrid rice system developed 1996 – “Super Rice Breeding” national program initiated 1998 – Hybrid rice seed yield more than 2.5 ton/ha 2000 – Super hybrid rice Phase I objective (10.5 ton/ha) achieved 2004 – Super hybrid rice Phase II objective (12.0 ton/ha) achieved 2006 – Super hybrid rice Phase III objective (13.5 ton/ha) initiated Three-line System The three-line system (Fig. 6) includes the following lines: • Male sterile line (A line): The cytoplasmic male sterility trait is controlled by both cytoplasm and nucleus; this line is used as female in hybrid seed production. • Maintainer line (B line): This line is used as a pollinator to maintain the male sterility. The maintainer line has viable pollen grains and sets normal seed. • Restorer line (R line): Any rice cultivar that restores fertility in the F1 when it is crossed to a CMS line. Rice hybrids made with cytoplasmic nuclear male sterility have been grown for several decades in both developed and developing nations. Traditionally, three-line system was used to produce hybrid rice, but more recently an innovative kind of genetic male sterility has been used as well, to make ‘two-line’ hybrid rice. Seed is produced on female inbred lines that are homozygous for environmentally sensitive (photoperiod or temperature or both) recessive male sterility genes. Seed production fields are planted in an environment (e.g. long day and/or high temperature) that enables expression of the male sterility gene in the female and enabling successful hybrid seed production. Seed increase fields of the female lines are grown in an environment (e.g. short day and/or cooler temperatures) that represses expression of the male sterility genes, allowing the female lines to reproduce via self-pollination. Two-line System Two-line system hybrid rice included the following two lines (Fig. 7): • Male sterile line: nuclear gene(s) and environmental conditions such as photoperiod and/or temperature control male sterility. Male sterile lines can be environmental-conditioned genic male sterile (EGMS), photoperiod-sensitive genic male sterile (PGMS), thermo-sensitive genic male sterile (TGMs) or photoperiod- and thermo-sensitive genic male sterile (PTGMS) lines • Restorer line (R line): any rice cultivar that restores fertility in the F1 when it is crossed to the male sterile line. Two-line system has several advantages over three-line system: 1. It is simpler as the need of maintainer is removed, 2. It is more applicable in diverse genetic background and easier to implement, 3. It has reduced cost of breeding program and seed production, 4. There is no detrimental effect of CMS system. However, due to the dependency of trait expression to environmental conditions, problems may arise in hybrid seed production. An important requirement is that environments be chosen that have more consistent temperature and day length at critical times of crop growth. Hybrid Maize Cultivars Summary of Steps in Development of Hybrid Maize Cultivars • Step 1: Development of inbred lines • Step 2: Making the crosses to produce a hybrid • Step 3: Testing the hybrids • Step 4: Development of seed for commercial production Inbred Lines Inbred lines can be developed by inbreeding selected heterozygous plants until sufficient homozygosity is reached without severe inbreeding depression. Sib-mating may be used to maintain inbred lines. With the advent of doubled haploid (DH) technology, this problem is minimized. DH lines provide genetic homozygosity in one generation. Because haploids carry only a single copy of every gene, any gene or genes that have deleterious effects for seed or plant development will have immediate genetic effects to depress or inhibit normal seed or plant development so these plants will be quickly eliminated at the haploid stage. Haploids also provide an advantage of better response to marker assisted selection as markers can be used to identify and select for desirable genes, and upon chromosomal doubling these genes are fixed. This provides an efficient and rapid tool to eliminate unfavorable genes and to enrich favorable genes to improve the genetic pool. DH lines have 100% genetic homozygosity, and the technique significantly reduces the time taken to develop inbred parents for crossing. These DH lines do not show inbreeding depression in the following generations, and in the absence of spontaneous gene mutations or transpositions that may cause certain deleterious influences and segregation, DH lines provide a powerful tool in maize breeding. In the DH process only one round of recombination happens thereby minimizes breakage of desirable linkages, as well as also reduces the chances to break undesirable linkages. Therefore a breeder needs to recognize the need for population size optimum, and so larger population sizes may be useful. Traditionally, open pollinated varieties of maize were the source of inbred lines. However, as the cultivars moved away from OPV to double crosses, three-way crosses or single crosses, which boosted maize yields (Fig. 8), the source of inbred lines also changed. Recurrent selection programs were also a popular source of inbred lines in public breeding programs, but private programs have moved away from recurrent selection programs as sources of inbred. Heterotic Groups In North America, for example, heterotic groups have been developed to classify inbred lines, and modern hybrids are the result of crossing a line from one heterotic group with a line from a different heterotic group (Fig. 9). Classification of heterotic patterns is generally based on several criteria such as pedigree, molecular marker based associations, and performance in hybrid combinations. Most conventional inbred line development involves making crosses within a heterotic group and as the population (within a cross) is advancing, testers belonging to different heterotic groups are used in crosses. Inbred Line Development The majority of inbred development activities in North America involve the use of the pedigree method of breeding (Fig. 10) Breeding crosses tend to be made by crossing inbred lines within a heterotic pattern. Inbred lines from the other heterotic patterns are used to improve the heterotic pattern represented by the breeding cross. Two-parent Cross Two-parent cross (parents belonging to the same heterotic group) are most common in maize inbred line development. An F2 population is formed from the breeding cross, which is then followed by several rounds of inbreeding using ear-to-row with each family tracing back to different F2 plants. During the inbreeding process, genotypes with obvious defects are eliminated. Early generation testing occurs around the F3 or F4 generation, which involves forming topcross hybrids between the F4 lines and an inbred line from a contrasting heterotic group. This cross can be made in the off-season nursery, and then in the summer (in North America), the resulting topcross hybrids can be tested in two or more environments (Fig. 11). Selection will be based on yield, lodging or stalk strength, maturity, test weight, and height, or other trait of interest. Inbred lines can be advanced in the same season through another round of inbreeding. Lines that produce hybrids considered to have merit (similar or better than commercial checks) are advanced through another round of breeding in the off season nursery and perhaps a round of crossing with inbreds of complementary heterotic group. In the summer, selection is made as described above. At this stage, the superior inbreds are forwarded to hybrid development teams for commercial testing with specific testers and in more environments. [Adapted from: Lee and Tollenaar 2007]. Hybrid Commercialization In the hybrid development process, more hybrid combinations are tested in fewer environments during the early testing phase, while in the later testing phases fewer hybrid combinations are tested in more environments. Generally, testing involves growing the hybrids in more locations while reducing the number of replications, thus allowing for more vigorous evaluation for adaptation. Requirements Several requirements need to be met for inbred lines classified as good parents in hybrid production. For example a female parent must be vigorous and produce high quality, healthy seed, and male parents should produce abundant and good quality pollen. An important consideration for choice of testers in the pedigree type breeding approaches is that testers be from a complementary heterotic group, maximize variance (among test crosses), as well as possess high mean (Bernardo 2010; see chapter 9). Steps in Development of Multi-line Cultivars Multi-lines are generally a set of isolines (traditionally created using backcrossing; or can be through transformation) that differ for one trait or more. These are grown in self-pollinating crops, where cultivars are pure-lines, so a mixture of pure-lines (if they are isolines) can form a multi-line cultivar. This approach has been used to provide control over a prevailing pathogen, such as a multi-line cultivar with different rust resistance genes in wheat. This should theoretically provide better protection against pathogen races and prevent a total crop loss. The pure lines are phenotypically uniform for morphological and other traits of agronomic importance (e.g., height, maturity, photoperiod), in addition to genetic resistance to a specific disease (or any other trait, for example, abiotic stress). See Breth (1976). Backcrossing is used to develop isogenic lines which are then combined in a predetermined ratio. Steps in Development of Blends A blend or composite cultivar, like a multiline, is a mixture of different genotypes. The difference between the two lies primarily in the genetic distance between the components of the mixture. Whereas a multiline consists of closely related lines (isolines), a composite may consist of different types of cultivars. It is intended to pick genotypes in a blend to minimize differences in maturity, growth habit, lodging, and disease resistance in the package. This consideration is critical to having uniformity in the cultivar. However, a different approach would be to pick blends that can be phenotypically different if the intention is to maintain some level of percentages. WB Seed Company provides additional information to familiarize yourself with examples of a blend cultivar system in commercial production.

textbooks/bio/Agriculture_and_Horticulture/Crop_Improvement_(Suza_and_Lamkey)/1.05%3A_Steps_in_Cultivar_Development.txt

Asheesh Singh; Arti Singh; and Anthony A. Mahama The results of breeding and selection may be new varieties or clones that are superior to currently used standard commercially grown genotypes (checks) according to some criterion or criteria, or populations that are superior to previous ones. Several breeding strategies exist and though some methods are generally commonly accepted, different methods are applied in different crops as they are more efficient and effective based on the type of mating of different crops, resources and objectives. In other words different breeding strategies are deployed and used to maximize superiority per unit cost and time. Also depending upon the goals of the breeding program, different strategies may be used simultaneously or at different stages of the program. Learning Objectives • Identify and describe different plant breeding methods relevant to crops grown in Africa • Mention and describe innovation used to enhance backcross breeding method • Explain innovation used to enhance recurrent selection method Methods Used in Self-Pollinated Crops In self-pollinated crops, the following breeding methods are commonly used to develop pure-line cultivars: • Bulk method • Pedigree methods • Single Seed Descent • Doubled Haploid Example of self-pollinated crops in which these methods are used include: common bean, soybean, cowpea, groundnut, rice, wheat, barley, millet, and sorghum. In specific situations, for example, when a breeding program is converting pure-lines to contain a specific gene or 2-3 genes (of qualitative inheritance), the backcross breeding method is used. The doubled haploid method is not used in legume crops as these species have so far been recalcitrant to tissue culture and haploid induction and rescue. Methods Used in Cross-Pollinated Crops In cross-pollinated crops, the following breeding methods are used to develop cultivars: • Recurrent selection (for example, maize) • Development of hybrids: a 2-step process where first inbred lines are developed and assessed for their specific combining ability, followed by crossing of the inbred lines (generally, 2 inbred lines, but can be 3 or 4) to produce hybrid, as for example, maize, rice, sorghum, cotton. Few self-pollinated species (such as rice, sorghum, and cotton) have some level of outcrossing and expression of heterosis, which is exploited to develop hybrid cultivars. Recurrent selection methods are used to develop open-pollinated varieties or synthetics. Methods Used in Clonal Crops The crop species that can be clonally propagated present unique advantages: 1. Heterosis can be fixed in F1 and in subsequent crop production cycles, and its clones can be propagated to preserve the high yield advantage. 2. Farmers can harvest the crop and use the vegetative plant part to grow the next crop. For example, potatoes, sugarcane, cassava. In breeding clonal cultivars, hybridization is made between two clones and a large F1 population (remember that parental clones are heterogeneous and heterozygous) is screened as each F1 is unique and different from other F1s. This process is repeated over different crop cycles to identify the superior clone for release as a new cultivar. Breeding Methods Used in Major Crops Pedigree Method The pedigree method of breeding is used in development of both self-pollinated (to develop pure-lines) and cross-pollinated crops (to develop inbreds). It is one of the most commonly used breeding methods. Selection of highly heritable traits is practiced in early generations on individual plants. Yield testing is generally done once homozygous lines are developed (Fig. 1). However, in an early generation testing procedure or a modified pedigree method, yield testing is done in early generations while within-family selection is still ongoing. Explanation of steps in Fig. 1 • Select in F2 and later generations. • Selected F3 plants (or seed from inflorescence of selected plants) grown in next season (in winter nursery if available). • Selected F3 rows (or selected plants within rows) grown as F4 in rows (or yield plot). • Selected F4 plants (or seed from inflorescence of selected plants) grown in next season (in winter nursery if available) as F5. • Repeat this process until selection is effective (remember, additive genetic variance among lines increases but decreases within lines as selfing is used). • Bulk harvest the last generation when a row is grown (and appears homogenous), F6 or Fn and plant in the next season as a yield plot. • Grow through successive seasons of yield testing to select the genotypes that are superior to checks. • Pedigree information is kept to maintain family information, which allows selecting more plants from families that are superior performing or to advance families for yield testing if those families are superior. Additional notes • Number of plants/row and population sizes vary between programs and some estimates can be obtained from text books or plant registration documents. These numbers will depend on the objective of the cross, number of crosses made per year, available resources (technical, infrastructure). • Selection for other specific traits is simultaneously happening (on harvested seed, or specific nurseries). • Single plants or inflorescence per plant are selected at each generation, but in some visibly inferior rows, breeder may not make any within rows selection (i.e., practice among row selection). • Selection can be practiced in winter nursery if genetic correlation is high among home location and off-season location (i.e. winter/dry season nursery locations). • A breeder may combine two or more methods of breeding and these methods will then be called modified pedigree (or modified bulk, or modified single seed descent etc.). Bulk Method Bulk method allows natural selection to act and remove undesirable genotypes from the population (i.e., per cross) (Fig. 2). The choice of growing environment will dictate what kinds of traits will be selected for or against, therefore care needs to be exercised to use environments that are suitable for realizing the objectives of the program. Explanation of steps in Fig. 2 • Generations are advanced to homozygosity through bulks. • It is a low cost, less technical method of breeding. • Natural selection is used to remove undesirable plants. • Artificial selection environment can be used to select for a trait of interest. Bulks can be grown in a disease or another stress nursery to select for that trait. Markers can also be utilized to select for desirable traits to constitute the bulks. These variations will make the scheme as a modified bulk method. • Early generation testing of bulk may be done for yield testing and to make a decision on retention of populations based on ranking among populations. Additional notes • In modified bulk method, single plants or inflorescence per plant are selected at each generation; while in bulk method, plants from the entire population are harvested and seeded (all or sub-sample of seed) in next generation. • Lighter shade yield plot = grown, tested, not selected; darker shade yield plot = grown, tested, selected and advanced to next generation testing. Single-Seed Descent Method Single Seed Descent (SSD) was developed as a breeding method to rapidly advance lines to homozygosity so that selection can be practiced on homozygous lines (Fig. 3). The original intent of this method was to maintain a large population size to mimic the genetic variation in F2 generation for effective selection. However, this method is now used to reduce the time to develop cultivars. (Sleper and Poehlman, 2006). Explanation of steps in Fig. 3 • Generations are advanced to homozygosity rapidly. In case of small grain crops (such as wheat, barley, oats), three seasons can be completed in artificial growing conditions (greenhouse etc.), and limited space is needed to keep a population size of 250-300 seed per cross. • If true single seed descent is practiced (where one seed per plant is grown in successive generations, population size is reduced in each cycle due to losses due to no germination and emergence. As an alternative modified, single seed descent can be used where 2-3 seed per plant are planted in hill plots in each cycle, and 2-3 seed from each hill are collected from an inflorescence. • SSD plots can be grown in a disease or another stress nursery to select for that trait. • It is a cheaper, less technical method of breeding. Rapid inbreeding and homozygosity is achieved. • No need for record keeping of individual plants while advancing through SSD. • Open circle = single plants (or hills in modified SSD) per population. Doubled Haploid Method Doubled haploids (DH) are created by generating haploid plants from microspores (androgenesis) or unfertilized eggs or ovules (gynogenesis). Haploid plants are then subjected to a chemical treatment (with colchicine) to double their chromosome number to produce homozygous diploid plants. Doubled haploids are generated from heterozygous plants, typically F1 plants derived from crossing of two pure-lines or inbred lines. DH can also be developed from selected F2 individuals from a cross. This method is used in development of both self-pollinated (to develop pure-lines) and cross pollinated crops (to develop inbreds). Process is shown in Fig. 4. • Generations are advanced to homozygosity in single generation. DH genotypes are true homozygous. • Specialized lab is needed to create doubled haploids. Can be generated through a service provider. • Population size is an important consideration because only one generation of meiosis occurs (at F1). • This method is suitable for marker assisted breeding to select for traits that are fixed. • Can develop cultivars most quickly. If sufficient seed is available, can go to advanced yield trial in season 3. • It is becoming a preferred method of inbred line development in maize. Backcross Breeding Method The backcross breeding method is used if the objective is to introgress a gene into an elite cultivar or breeding line. Examples are disease resistance gene(s) and herbicide tolerance gene(s) (Fig. 5). By crossing to the recurrent (adapted) parent, the newly developed cultivar will contain the majority of the recurrent parent genome and only the gene of interest from the donor parent. If the gene to transfer is recessive (rr), progeny of crossing with RR recurrent parent will segregate as RR and Rr, and therefore progenies are selfed for one generation to determine the Rr type versus RR types (RR are discarded) before making the next backcross. With the application of molecular markers, this extra step has become redundant and F1 plants can be grown, DNA extracted from young plant tissue to determine Rr and RR types. RR types can be removed and crosses can be made with Rr types. For a backcross breeding program, if the gene to be moved comes from an unadapted or related species, the breeder has to be aware of inadvertently bringing in undesirable genes linked to the desired target gene (termed linkage drag). Larger population sizes will need to be grown to identify recombinants. Innovations, e.g. marker assisted backcrossing, marker assisted recurrent selection, and genomic selection, exist that reduce the need for large population sizes. Innovations in Backcross Breeding Marker-Assisted Recurrent Selection Steps of Marker-Assisted Recurrent Selection 1. One generation of phenotypic selection in the target environment is conducted, 2. Markers with significant effects are used to predict the performance of individual plants, and 3. Several generations of marker-only selection are performed in a year-round nursery or greenhouse Early Generation Testing Early Generation Testing (EGT) describes the procedure for selecting superior lines or families before they are homozygous. It also refers to a specific use where a genetic worth of a population is determined by analyzing yield data from a segregating (early generation) plot and removing entire populations. EGT is used in self- and cross-pollinated species. In the pedigree breeding method we looked at individual plant selection for highly heritable traits in early generations. With high heritability, individual plant selection is still effective, for example traits such as plant height, disease resistance, and morphological traits. Several breeding programs, however, follow a modified method (such as modified pedigree method), in which yield testing is started in an early generation (for example, F3 or F4) to make selections. The early generation lines are grown on yield plots (2 or 4 row plots), therefore, more resources are required to handle EGT. Nonetheless, EGT allows elimination of materials (lines) that are inferior due to use of replication and multi-environment testing. Also, selection for lower heritability can be practiced to discard inferior lines. Other breeders may choose to perform a yield test on populations derived from early generation bulks to identify superior bulks (inferior bulk populations are removed completely from further generation advancement). Thus, EGT testing in this scenario can be done for one or 2 generations followed by selection of superior plants, and then starting yield testing of these lines. Cytoplasmic Male Sterility Systems Plant breeders working with cytoplasmic male sterility (CMS) systems will aim to develop new ‘B-lines’ and ‘R-lines’. In crops where CMS system is used to produce hybrids, different ‘R’ restorer genes are identified and breeders will improve ‘R-lines’ that will be used as males in creation of hybrids. ‘B-lines’ and ‘R-lines’ are developed using the self-pollinated breeding methods we learned about earlier in this module (pedigree, bulk, SSD, DH etc., or a modified method that combines more than one method in the development of breeding line of cultivar). An outline of a CMS system is shown in Fig. 8. Note that the ‘R’ and ‘r’ genes are in the nucleus and the ‘S’ and ‘F’ genes are in the cytoplasm. A breeder who develops ‘B-lines’ will use the backcross method to develop ‘A-lines’ using available CM sterility genes. A hybrid cultivar is produced by crossing of ‘A-lines’ with ‘R-lines’. The A/B and R gene pools are considered separate gene pools (reproductive gene pools) similar to heterotic gene pools we learned about in maize systems. Hybrid Cultivars In the chapter on Steps in Cultivar Development, we looked at the development of maize hybrids using two-way crosses. Crosses are made within a heterotic group to develop superior inbred lines in the heterotic group. These inbred lines are crossed to testers from other heterotic groups to decide on the best specific combing ability. This process is repeated for all heterotic groups that the breeding institution or company works with internally. For evaluation, superior inbred lines from dissimilar heterotic groups are crossed to produce hybrids. Several 100 or 1000’s of hybrids are evaluated each year to finally pick the most superior hybrid(s) for commercial release based on their performance and target area of adaptation (maturity, stress, environment etc.). Hybrid seed is produced by growing inbred female rows (say 6 to 8) from one heterotic group and inbred male rows (1 or 2) from a dissimilar heterotic group interspersed among the sets of female rows, and de-tasseling the female rows (that is, removing male inflorescences from female plant rows) before pollen shed. Manual or mechanical tools are used to de-tassel (prior to pollen being ready or shed to avoid any selfing of plants of the inbred female line). Cobs from female rows are harvested and these constitute the hybrid seed. In some programs, but routinely done in private seed industries, the male rows are usually destroyed when pollination is completed to avoid contamination from cobs from inbred male plants if allowed to grow and produce cobs. Recurrent Selection In recurrent breeding and selection, parents of a crop species are crossed to develop populations using various mating designs described in the chapter on “Refresher on Population and Quantitative Genetics.” Based on one or more selection criteria, and using within family and among family selection strategies, individuals are selected and inter-mated to produce the next generation. This procedure of selection can continue for an indefinite amount of time, hence the term “recurrent”. Recurrent selection method is employed in order to achieve the following: • The goal of recurrent selection is to improve the mean performance of a population of plants and to maintain the genetic variability present in the population. • The underlying principle of recurrent selection is to increase the frequency of desirable genes that the breeder is attempting to improve. • Recurrent selection is used to improve populations in cross pollinated species. Open pollinated varieties are one type of cultivar developed using recurrent selection. Comparison: Mass Selection versus Phenotypic Recurrent Selection • Mass selection: Female plants are selected after pollination with unselected and selected pollen source. • Phenotypic recurrent selection: Male and female are both controlled. ONLY selected plants are intercrossed to obtain seed for the next cycle of selection. Expected genetic gain from selection of only the female parent is one-half compared to expected genetic gain when both parents are selected. Note that the terms mass selection and phenotypic recurrent selection are sometimes used interchangeably and one would have to look at the breeding scheme for details in order to determine which method is being referred to. Comparison: Genotypic versus Phenotypic Recurrent Selection The difference between genotypic and phenotypic recurrent selection is that Genotypic Recurrent Selection is selection based on progeny performance (combining ability), while Phenotypic Recurrent Selection is selection based on the phenotype of the individual. Phenotypic Recurrent Selection Issues There are several problems with selecting individual plants in the field: • Micro-environment variability does not permit assessing breeding value. • Competition effect due to uneven planting. Solutions to these problems include: • Gridding designs (selecting plants within a grid) • Not selecting plants that have missing neighbors The generalized recurrent selection method consists of the following steps: • development of a base population (for selection). • evaluation of individuals from the population • selection of superior individuals from the population • intercrossing the selected individuals to form a new population. Development of Base Population A base population can be an existing population (for example a maize synthetic) which may not have been previously selected for your trait of interest. More commonly, a base population will come from outstanding families from a recurrent selection program. It may also be created with elite inbred lines. Smaller number of inbred lines will ensure use of elite material that are similar morphologically, but inbreeding depression will be greater. Superior inbred lines are identified based on their performance in multi-location tests and superior general combining ability (specific combining ability is not as important in the performance of OPV; it is most important if one is developing a hybrid cultivar). These superior inbred lines are crossed using an appropriate mating design from among available designs (for example, diallel design). Evaluation of Individuals Individuals are evaluated for selection when advancing generations following crossing, and the type of cross made play a key role in the phenotypic and genotypic schemes employed in selecting individuals (Table 1). Table 1 Comparison of phenotypic and genotypic schemes in individual selection Phenotypic scheme Genotypic scheme Evaluation is based on individual plants per se Evaluation is based on the performance of the progeny of the individual Assessment is very variable unless species can be clonally propagated Progeny performance strategy allows for replicated, multi-location testing. Not easy to control environmental variability This provides a more accurate assessment of individual’s breeding value n/a Three types of progenies can be evaluated: self, full sib, or half sib Progeny are produced by self-fertilizing the individuals that are evaluated for selection. Full-sib families are created by crossing the individuals to be evaluated in pairwise combinations. Since in each pairwise cross both parents are common for that family, individuals of that family are full-sibs. Half-sibs are formed by crossing the individuals to be evaluated to a common parent (which can be a population or an inbred line as a tester. Since all progeny have the tester as a common parent, they are half-sibs. Population Improvement As the name implies, the breeding populations creating from crossing parents, need to be improved in performance of the desired traits, in order to continue to make progress in breeding programs. Different methods are used for population improvement, and depending on the breeding program’s specific project goals, can described as intra population or interpopulation improvement. Table 2 shows some methods that are used. Table 2 Methods in recurrent selection. Intrapopulation improvement Interpopulation improvement Mass selection (with or without pollen control) Reciprocal half sibs recurrent Half-sib family Reciprocal full sib Full-sib family Testcross Selfed family n/a Recurrent Phenotypic Selection Steps include: 1. Plant a population (space planting individuals to facilitate note taking on individual plants). 2. Evaluate for trait of interest and identify the best individuals (higher heritability such as flowering time or morphological traits are suitable for this method). 3. Harvest seed of the best individuals and reconstitute seed to form the next cycle of recurrent selection. 4. In this example, pollen control can be exerted if the trait can be evaluated prior to flowering. Undesirables can be removed before they contribute pollen to the rest of population; and this ability to control parental pollen helps improve the response to selection. Recurrent Half-Sib Selection • An intra-population improvement method: cross the individuals in a population to a common tester (population per se, or inbred tester), evaluate the half-sib progeny of each plant, select the best individuals, and intercross the selected individuals. • The main step is evaluation of individuals through their half-sib progeny. There are numerous variations within and among crops based on what is used as a tester (population vs inbred), parental control, intercrossing. • Where possible, it is desirable to control both parents. This can be achieved by evaluating in one season and recombining in another generation (in winter nursery or second season). This necessitates an extra season but genetic gain per year will be higher. While the half-sib are being evaluated, the remnant seed of the individual needs to be kept as reserve so that this seed can be used if the individual is selected based on the half-sib performance to intermate and create material for the next cycle of selection. • In maize, obtaining selfed and half-sib seed from the same plant can be accomplished by self pollinating the single ear on the individual to be tested and using pollen from that individual to pollinate several individuals of the tester (bulk of population per se, or inbred line). The ears on the tester, bulked together from that individual as pollen source, represent the half-sib family to be tested for that individual. • Recombining selfed progeny will require three seasons: (1) selfing and crossing to the tester, (2) evaluation, and (3) intercrossing selfed progeny. Recurrent Half-Sib Examples Female parent selected; population used as tester. • Start with a random mating population • Harvest ears of each plant (say, 200). Grow 200 half-sib progeny plots (with checks) at multiple locations (can be unreplicated or replicated). Traits of interest is yield (for example). At one location, grow in isolation as seed source for the next cycle. At this location, select plants within a half-sib row. At other locations, use for testing. • At the location with isolation, grow the male rows (bulk seed of all half-sib families) adjacent to female half-sib rows. De-tassel the female rows. • At the location where grown in isolation harvest ears from each selected plant by hand. Make selections to pick the best half-sib families. These ears will form the next cycle seed. • Season 2, conduct random mating of selected plants. • Repeat steps • One can use an inbred line as tester instead of bulk seed of population used as male. Female and male parent selected; population used as tester. Cycle 0: (intermate population) • Harvest ears from each plant (selection may be performed) • Divide the seed of half sib plants into two: part 1 for next season field testing, part 2 for remnant to reconstitute selected half-sibs. Season 1: Each half sib (using part 1 seed) is a separate entry in replicated or unreplicated trials with 2 or more locations, with checks. • Select superior half-sib families based on performance. These selections will be used in crossing. Season 2: remnant seed (part 2 of seed bag) of selected individuals is used for intercrossing to form next cycle. Cycle 1: Seasons 3 and 4 – repeat as above. • One can use an inbred line as tester instead of bulk seed of population used as male. Recurrent Half-Sib (Testcross Progeny) • Start with an intermated population • Season 1: plants in an intermated population are selfed and pollen used for selfing and pollinating a tester. • Season 2: testcross progeny are evaluated in replicated tests. Selections made to identify superior performing progenies. • Season 3: selfed seed of selected families are used to form the next intermating cycle. Cycle is repeated as above. Recurrent Full-Sib Process The main steps are listed below. End of first year: • Season 1: Make paired crosses between individuals in the population. • Season 2: Evaluate the full-sib families in the field and identify the best families. • Season 3: Recombine (intercross) the best families using remnant seed from the first season. Start of second year: • Season 4: Begin the second cycle with paired crosses between individuals in the population. An advantage is the completion of one cycle per year. A disadvantage is less recombination between cycles of selection. Recurrent Full-Sib Example Start with an intermated population. Make selections. • Season 1: paired crosses are made between pairs of selected plants in a population. Seed is divided into two parts: Part 1 is for field testing, and Part 2 is to reconstitute next cycle. • Season 2: Part 1 seed used to plant field tests. Evaluate full-sib in field tests (single or multiple locations, unreplicated or replicated, with checks). Select superior families based on performance. • Season 3: Part 2 seed used to intercross selected families. Intermated seed is used to form the next cycle. • Cycle 2: Seasons 4, 5, 6. Recurrent Selection Among Selfed Families • Season 1: S0 plants from the population are selfed to produce S0:1 lines. • Season 2: Evaluate the selfed progenies in field (for trait of interest). • Season 3: Use the remnant S1 seed from season 1 to intercross selected lines. This completes cycle 1 and S0 plants are obtained. The cycle is repeated as described above in season 4-6 for cycle 2, and so on. Variation can include more than one generation of selfing if more seed is required for evaluation. Reciprocal Recurrent Selection Reciprocal recurrent selection (RRS), as a breeding method for open-pollinated crops was first proposed by Comstock et al. 1949 to take advantage of both additive and dominance genetic effects. In brief, plants from one population are mated to plants of another population, and selection of individuals for the next cycle of selection is based on the performance of progeny in hybrid combination. For this breeding method, each cycle requires one generation for selection of individuals and a second generation for intermating of selected individuals to produce materials for the next generation. RRS is a procedure to improve both the general and specific combining ability of two populations simultaneously, and steps involved are as below: • Plants are selected in each of two populations • Plants of population#1 are selfed and outcrossed as the tester to the selected plants in population#2 to generate test cross progeny. • Plants of population#2 are selfed and outcrossed as the tester to the selected plants in population#1. • The resulting test cross progenies are evaluate in each season. Superior plants are identified based on their test cross performance. Selfed seed from these selected plants are used to intercross within each population to generate materials for the next generation. • Cycle is repeated. Maize Open Pollinated Varieties (OPV) OPV Advantages and Disadvantages Table 3 OPV advantages and disadvantages. ADVANTAGES DISADVANTAGES Seed can be re-cycled (if grown in isolation or middle field harvested without a significant yield reduction due to inbreeding depression) Yields lower than hybrids Can have much more broader adaptability compared to hybrids (that are developed for targeted areas) Is not comparable to hybrids in areas where land is fertile and inputs are available to maximize yield May be less costly than hybrid Plants are less uniform May require less inputs than hybrids Seed needs to be harvested properly to use for next year, and even then there will be a yield reduction. OPV may be more accessible in areas where no hybrids are available or seed availability channels are poor n/a Clonal Cultivar Methods Since each clone breeds true (i.e., no gene segregation because no sexual recombination), breeding programs can evaluate a clone in several different tests simultaneously (field testing, disease nursery etc.). In clonal crop breeding, each cross produces unique and distinct F1 seed (true seed). True seed plants are transplanted into field testing and selection commences to identify which F1 of F1’s are suitable for cultivar release. Step-wise reduction process is used to discard undesirable F1 clones each testing season (remember, clones can be propagated for more extensive testing once smaller number of desirable clones are identified. Shown in Table 4 below is an example of sugarcane cultivar CP 03-1912 developed in Florida. Table 4 Summary of process followed in the release of sugarcane cultivar CP 03-1912 in Florida. Data from Gilbert et al., 2011. Year Month Stage and activity completed Number of genotypes in stage Locations 2000 Dec Cross made at USDA–ARS sugarcane field station No data Canal Point, FL 2002 May Germinated true seed transplanted into field (seedlings) 100,000 Canal Point, FL 2003 Jan Advanced from plant–cane seedlings to stage 1 15,000 Canal Point, FL 2003 Sep Assigned name CP 03-1912 in stage 1 15,000 Canal Point, FL 2003 Nov Advanced from plant cane stage 1 to stage 2 1,496 Canal Point, FL 2004 Nov–Dec Advanced from plant cane stage 2 to stage 3 135 Four farms in Florida 2006 Nov–Dec Advanced from plant cane stage 3 to stage 4 sand soils 13 Four farms in Florida 2011 Feb Cultivar release 1 No data Approximately 10% culling rate was practiced in each season after 2003. As seasons advance, clones are grown in replicated yield trials at several locations and comparisons with standard checks is made to identify which clones to advance to the next stage of testing. Synthetic Cultivar Synthetic cultivars are formed by using clones of inbred lines in pre-determined proportions for released to farmers. Farmers can use a synthetic for several generations (as open-pollinated population) but once inbreeding depression causes yield reduction, farmers need to use seed from the breeding institution or company. Therefore synthetics are reconstituted regularly by the breeder. Maize is an example where synthetics have been developed. In crops with self-incompatibility, synthetics are the preferred types of cultivars as the method exploits heterosis for a few generations. Clones or inbred lines used in the formation of synthetic are chosen on the basis of their general combining ability. Crossing is made to ensure random pollination allowing gametes of each component (clone of inbred line) to be equally represented.

textbooks/bio/Agriculture_and_Horticulture/Crop_Improvement_(Suza_and_Lamkey)/1.06%3A_Breeding_Methods.txt

Teshale Mamo; Asheesh Singh; and Anthony A. Mahama Formal or conventional plant breeding programs (centralized breeding programs) are often designed to meet specific requirements of different groups of farmers in different growing environments (regions, countries, soil, or climatic conditions). Formal or conventional plant breeding programs have generally been more beneficial to those farmers who either have good crop growing environments or have the capacity to modify growing environments through the application of additional inputs such as fertilizer, pesticides, and irrigation to create more favorable growing conditions for new varieties. However, the results of formal plant breeding may sometimes not meet the requirements of those farmers who grow their crops under marginal soils and high-stress environmental conditions (Sperling et al., 2001) thus necessitating different breeding approaches to be created to meet the needs of poor farmers. Participatory plant breeding (PPB) and participatory variety selection (PVS) have been developed and implemented over the past 10 years as an alternative and integral part of the breeding approach in traditional plant breeding. It has been mainly implemented in developing countries where farmers with limited resources grow their crops in marginal lands of remote regions. It is practically implemented in areas where the technology transfer or adoption of modern cultivars is low (as farmers are not comfortable with taking the risk to replace their well-known and reliable traditional varieties with new varieties) or where modern cultivars are not available. Therefore PPB has emerged to address the agricultural problems of poor farmers in developing countries where resources and modern technologies are limited. PPB has been widely considered to be more advantageous to use in areas where low yield potential, high stress (drought), and heterogeneous environments exist. The various aspects of PPB described above are depicted in Fig. 1 below. Learning Objectives • Know the goals of participatory plant breeding • State the different types, stages, and requirements of participatory plant breeding • Describe the roles farmers play in participatory plant breeding • Articulate the outcomes and impact of participatory plant breeding and participatory variety selection Participatory Plant Breeding (PPB) PPB Categories It is an approach involving different participants including scientists, farmers, along with consumers, extension agents, farmers’ cooperatives, vendors, traders, processors, government and non-government organizations in plant breeding research (Sperling et al., 2001). It is considered as “participatory” because of the mixture of different people from different organizations involved, especially end-users, having significant research roles in all major stages of the breeding, evaluation and selection process. These different actors participate in setting PPB goals, setting breeding priorities, selecting genotypes from a heterogeneous population, helping in evaluation and selection of cultivars in the farmers’ fields and on research stations, releasing and popularizing high yielding cultivars, and helping in seed multiplication and distributing (McGuire et al., 2003). Participatory plant breeding is grouped into the following categories: 1. Formal-led Participatory Plant Breeding describes a situation when farmers are asked to join in PPB activities which have been initiated, managed and executed by formal breeding programs such as National Agricultural Research System (NARS) or International Agricultural Research Center (IARC). 2. Farmer-led Participatory Plant Breeding describes a situation when scientists and/or development workers seek to contribute or support famers own controlled, managed and executed breeding systems. Scientists can support their own varietal selection and seed system. Goals of PPB In any PPB approach, the first activity involves carrying out a diagnostic survey. The diagnostic survey allows an effective discussion between breeders and farmers and also enables breeders to better understand: 1. agricultural problems of the local farming conditions, 2. farmers’ crop management practices, 3. farmers’ specific needs and preferences. The goals of PPB are to: 1. Increase production and productivity in non-commercial crops in environments that are unpredictable and under abiotic and/or biotic stress. 2. Enhance biodiversity and increase germplasm access to local farmers. This provides benefit to local farmers, especially to disadvantaged user groups (women and poor farmers), for developing adapted genotypes. It also makes the breeding program cost-effective and output-oriented through decentralization that can address more niches. 3. Increase farmer skills to speed up farmer selection and seed production efforts. Types of Participation The types of participation in PPB are: • Conventional: In this approach, there is no farmer participation. • Consultative: Farmers are consulted at every PPB stage but the breeder makes the decisions. The consultation of farmers starts from identifying breeding objectives and selection of appropriate parental materials. In this approach, farmers participate in making joint selections with a breeder among genotypes in breeders’ plots on station. • Collaborative: In this approach, decisions are made jointly by farmer and breeder. Farmers and breeders know each other regarding selection criteria and their priorities for their research through two-way communication. To revoke or override the joint decision made earlier, both farmer and breeder need to agree on the change(s). Usually this type of participation is effective for self-pollinated crops. • Collegial participation: Farmers grow genotypes in their farm fields and make their own plant or genotype selections. In this approach farmers can make decisions in a group or individually but in an organized communication with the breeder. In this approach farmers voluntarily supply some of the seeds of selected genotypes to the breeder for further evaluation and seed multiplication. • Farmer experimentation: In this approach breeders do not participate in selection of genotypes or in any farmers’ research activities. Farmers make their own decision either in a group or as individuals on how to implement their research activities with new genotypes without organized communication with breeders. Stages of Participation In general, participation approaches to choose and implement depends on the resources availability and type of the crop which could be used in PPB. Stages of Participation In PPB Process 1. Set the breeding objectives/targets: Farmers’ participation in setting breeding objectives begins from the participatory rural appraisal. 2. Generate (access) genetic variability from local landraces or using collections for testing with complementary characteristics. 3. Determine the approach (consultative/collaborative). This depends on the availability of resources and on the type of the crop (it is more easily done for collaborative participation if the crop is self-pollinated species) and selecting among segregating populations. 4. Evaluate cultivar and discard inferior genotypes (culling) (this is participatory variety selection if the farmer is involved in selection of genotype). 5. Collaborating with seed system (cultivar release, popularization, diffusion and seed multiplication and finally distribution). Essential Requirements for Success For PPB to be successful, the following requirements must be met: 1. The local farmers should be interested in active participation during plant breeding/ selection process 2. Breeders and farmers have to collaborate with each other during each stage of PPB 3. Importantly, PPB has a better chance of success if: • locally adapted parents are used in the development of crosses made for PPB • selection of desirable or superior genotypes is made in the local environments • cultivars that are selected by farmers should have traits important to the farmers Roles And Contributions of Farmers in PPB Work Farmers: 1. provide technical leadership role in testing cultivars to specific environmental niches. They also contribute their knowledge and experiences. 2. play a role in organizing farmer research groups. 3. provide information on cultivar preferences and important traits that could be maintained or introduced to the existing land races. 4. are involved in skill building through farmer-farmer interactions. 5. provide their landraces or their genetic materials that could be used for further breeding work. 6. provide land for testing the PPB genotypes. Major Possible Outcomes 1. Production gain: significant production gains would be expected through increased yield, increased stability of crop yield, faster uptake of released cultivars, wider diffusion of the varieties and better identification of farmer-preferred quality traits (e.g. taste, ease of processing, etc.). 2. Biodiversity enhancement: Farmers communities get more access to different germplasm, more information related to germplasm as well as getting related knowledge, increases access to inter and intra cultivar diversity. 3. Cost-efficiency and cost effectiveness: The time of selection is short so cultivars are identified within shorter timeframe (3-4 years), i.e. cultivars identified faster. This reduces research cost. The released cultivars do not take a long time to disseminate to the farmers so less expensive for diffusing cultivars. Figure 2 is a timeline comparing conventional and PPB systems in bean breeding and clearly shows the fewer number of years involved in selection for the next cycle or variety release with PPB system. 4. Farmer knowledge increase and capacity is enhancement: this facilitates the development of more PPB lines, gain in extensive experience and increase in agricultural knowledge dissemination, including agronomic practices. 5. Farmers’ needs are met. Farmer satisfaction increases due to fulfillment of their demand. A broader range of users, such as women, men, elders and young, is reached. Impacts of PPB 1. Higher adoption rate of PPB products such as new cultivars, agronomic and crop protection practices. 2. Improved cultivars acceptable by farmers for highly stressed marginal areas. 3. In most remote areas of developing countries where soil is degraded and drought is a major production problem, new varieties developed and immediate adoption of the new technologies and yield increase is achieved. 4. Significant changes in cultivar release procedure and seed multiplication system. In PPB, time for testing to release of cultivar is shorter than conventional breeding. Participatory Variety Selection (PVS) Introduction Generally, participatory variety selection (PVS) is a continuation of PPB. Once potential cultivars are identified through PPB process, farmers can test those cultivars using PVS approach. Usually farmers participate at the end of the cyclical process. More specifically, PVS is an approach where selection of finished or near finished cultivars is made by the farmer on her/his own fields. The finished products/genotypes include released cultivars, advanced stage cultivars, advanced non-segregating lines in self-pollinated crops or advanced populations in cross pollinated crops. PVS includes research and extension methods that help to deploy genotypes (promising advanced lines/released cultivars) on farmers’ fields. Therefore, cultivars that are developed through PVS, would meet the demand of different farmers (men and women, old and young). Participatory variety selection comprises three phases to select farmer preferred cultivars. 1. Clear identification of farmers’ needs. 2. Search for suitable advanced lines or cultivars to test in farmers’ conditions. 3. Implementing the experiment on farmers own fields and dissemination of preferred cultivars. Importance of PVS 1. Provide access to local farmers’ choice of a large number of cultivars and increase in crop diversity. 2. Increase production and productivity which helps to ensure food security. 3. It helps to speed up dissemination and enhances adoption of pre-released and released cultivars in diversified environments. 4. It enables cultivar selection in targeted environmental niches in a short period of time with less cost. Conventional and Participatory Timeline In Fig. 2, conventional and participatory breeding methods are shown, comparing the steps and duration for cultivar release, using beans as an example. Impact Pathway The impacts of participatory breeding are shown in Fig. 3 below.

textbooks/bio/Agriculture_and_Horticulture/Crop_Improvement_(Suza_and_Lamkey)/1.07%3A_Participatory_Plant_Breeding_and_Participatory_Variety_Selection.txt

Teshale Mamo; Asheesh Singh; Arti Singh; and Anthony A. Mahama Common bean, Phaseolus vulgaris, (belongs to the Fabaceae family) is an annual plant that is grown in many parts of the world for the seed (bean) that is eaten immature or mature (after shelling and drying). Common bean is an important plant protein source in human diet. The genus Phaseolus originated across a wide geographical area in the tropics and subtropics of Latin America, from north-central Mexico to northwest Argentina (Fig. 1), and comprises more than 30 species. Among these species, the most widely cultivated species are: • P. vulgaris L. (common bean), • P. coccinus L. (runner bean), • P. accutifolius L. (tepary bean), • P. polyanthus Greenman (year-long bean) and • P. lunatus L. (Lima bean). Among these five species, P. vulgaris (common bean) is the most widely grown (~ 85% of total world production planted). Learning Objectives • Become familiar with the Common bean crop • Know crop biology and classification system • Describe adaptation and usage • Outline production constraints • List breeding institutions working on the crop • Discuss the breeding methods used to develop common bean cultivars Domestication and Diversity Ancestral Origins Common beans were domesticated in two major centers, the Andean and Middle America (Fig. 1). Plants of the wild bean ancestor of Phaseolus vulgaris L. grow as viny herbaceous annual plants found from northern Mexico to northern Argentina (Fig. 1). Bean Gene Pool Genetic analysis using DNA marker diversity and Amplified Fragmented Length Polymorphism (AFLP) suggest that there are four wild bean gene pools, centered in: (1) Middle America (Mexico and Central America); (2) Colombia; (3) Western Ecuador and Northern Peru; and (4) the southern Andes (Tohme et al., 1996). The cultivated bean gene pool is derived mainly from the southern Andean wild bean gene pool and the Middle American gene pool. Wild stands are shown in Fig. 2. Different Races The major gene pools in turn have been divided into different races based on plant morphology, adaptation range and agronomic traits (Fig. 3). Biology of the Crop General Characteristics of the Development of the Bean Plant The biological cycle of the bean plant is divided into a vegetative phase and a reproductive phase following that. The vegetative phase starts from germination of the seed and ends when the first floral buds appear in cultivars of determinate growth habit, or the first racemes in cultivars of indeterminate habit. The reproductive phase ranges from the moment the first floral buds or racemes appear, to maturity. In plants of indeterminate growth habit, vegetative structures continue to appear when the vegetative phase has ended, which makes it possible for a plant to produce leaves, branches, stem, flowers and pods simultaneously. In determinate growth habit, the vegetative phase ends when the floral buds appear. Growth stages of common bean are categorized into four groups (Schwartz et al., 2010). Group 1: Emergence & Early Vegetative Growth • VE: Emergence of hypocotyl from soil (crook stage) • VC: two cotyledons & primary leaves at nodes 1 & 2 • V1: 1st trifoliolate leaf unfolded at node 3 • V2: 2nd trifoliolate leaf unfolded at node 4 • V3: 3rd trifoliolate leaf unfolded at node 5 Group 2:Branching & Rapid Vegetative Growth • V4: 4th trifoliolate unfolded at node 6 + branching • Vn: nth trifoliolate leaf unfolded at node (n+2) Group 3: Flowering & Pod Formation • R1: one open flower (early flower) on the plant • R2: 50% open flowers (mid flower) • R3: one pod at maximum length (early pod set) • R4: 50% of pods at maximum length (mid pod set) Group 4: Pod Fill & Maturation • R5: one pod with fully developed seeds • R6: 50% of pods with fully developed seeds (mid seed fill) • R7: one pod at mature color (physiological maturity) • RH: 80% of pods at mature color (harvest maturity) Photosynthesis Beans are classified in a C3 photosynthetic pathway. The maximum leaf photosynthetic rates at ambient carbon dioxide (CO2) concentrations is estimated from 12 mg CO2 dm2 h-1 to 35 mg CO2 dm2 h-1. Recent report showed relatively high photosynthetic rates in common beans, and this might be due to improved measurement techniques, but still lower photosynthetic rates than soybean (White and Juan, 1991). Photoperiod and Temperature Common bean, like most plants, flowers only in response to a certain amount of exposure to sunlight or photoperiod (termed the critical photoperiod), and are described as being photoperiod-sensitive, while others flower regardless of exposure time, and are described as photoperiod-insensitive or day neutral. Whereas day neutral genotypes occur, most common bean cultivars show a short day response for flowering (i.e. plants of such cultivars flower when the length of night exceeds their critical photoperiod). Genotypes of a high proportion of large seeded and highland germplasm are photoperiod-sensitive. The International Center for Tropical Agriculture (CIAT) reported that the photoperiod effects on common bean phenology increases with temperature. Higher temperatures cause a greater overall rate of growth and development. In general, both temperature and photoperiod have strong effects on growth and development of the bean plant. The inheritance of photoperiod-temperature response of flowering is controlled by few major genes. General Classification System of Beans Classification by Utilization or Mode of Consumption Common beans may be grouped based on the stage of plant maturity when they are consumed: • green or snap beans are horticultural beans grown for, and consumed as, fresh or processed pods • green shell or fresh beans are grown for, and consumed as, fresh, full-sized seeds • dry beans are grown for dried ripe seeds. Classification by Seed Characteristics Dry common beans are primarily characterized by the great diversity of seed types within the species: a rainbow array of colors and color patterns, varying degree of brilliance, and several seed shapes and sizes exist as shown in Fig. 4. • Seed type (color, size, shape, and surface texture) is the character most commonly used to classify beans. Seed size of commercial cultivars may vary from 17 grams (navy beans) to 100 grams per 100 seeds (Faba beans). • Seed shape varies from round to oblong to kidney-shaped with many combinations of color patterns. Surface texture may be shiny (brilliant), opaque, or intermediate. Classification By Growth Habit Growth habit in beans varies from determinate dwarf beans to very vigorous indeterminate climbing beans. Common classification often divides beans into two or three groups: bush and climbing beans, or bush, semi-climbing, and climbing beans (Fig. 5). Classification by Duration of Growth Period Bean varieties are usually grouped as early or late, however, the range of duration of growth-period varies from one region to another, or among varieties of different growth habits. According to growth habit and region, days to maturity among bean cultivars range from 60 to 300. The difference is not only varietal but also environmental, especially for the factors of day-length and temperature. Adaption, Economic Importance, and Uses Adaption Common bean is a widely cultivated grain legume crop in tropical and subtropical areas of the world. Bean is adapted to a wide range of environments, and grows in latitudes between 52oN to 32oS in humid tropics, in the semi-arid tropics, and even in the cold climatic regions (Fig. 6). It is a short-day tropical crop that requires between 300-600 mm precipitation to complete its life cycle, depending on soil, climate, and cultivar (Beebe et al., 2013). Optimum crop production requires temperatures of between 21-24oC during the growing season and soil pH of between 6.3-6.7. According to figures from FAO, world production is around 27.7 million tons (FAO, 2021). Latin America is the largest common bean-producing region, followed by the continent of Africa. Brazil, Mexico, and the USA are the three largest common bean-producing countries in the western hemisphere. In Africa, the majority of bean production is concentrated in the eastern and southern highlands extending from Ethiopia to South Africa. In this region, Kenya is the largest common bean-producing country. Common Bean production mainly occurs on dryland (i.e., depending on rainfall), with smaller land area under irrigated systems. Common Bean in the Human Diet and Nutrition Common bean is mainly grown for human consumption. In some countries it is one of the food security crops providing protein and fiber to more than 100 million people in Africa (Kimani et al. 2001). Common bean is mainly consumed as a mature grain in most parts of the world. Immature seeds, young pods and leaves are also consumed as a vegetable by some communities in sub-Saharan Africa and Latin America. Plant protein is the largest source of protein in human diet of poor people in the developing countries. Common bean therefore plays an important role in human diet due to its high protein content. Common beans are also a key source of minerals in human diet, especially iron. Common Bean in Cropping Systems In Africa, common beans are traditionally grown by farmers with small land holdings. This crop is often grown in complex farming systems as intercropped or in rotation with maize, sorghum, bananas, or other crops (Fig. 7). The range of growth habits (from determinate bush types to vigorous climbers), and the range of growth cycles (from 3 to 10 months in length) allow common beans to fit many production niches. In East and Central Africa, 23% of the production area is monocropped and 77% is grown in association, that is intercropping, with other crops (Katungi et al. 2010). Monocropping is dominant in southern Africa with only 47% of the production area assigned to intercropping with other crops. In this cropping system, common bean has the capacity to break disease and pest cycles usually associated with cereals. Atmospheric Nitrogen The ability to fix atmospheric nitrogen (N) for subsequent crops has made common bean a valuable crop in many smallholder cropping systems. Lunze and Ngongo (2011) reported that climbing beans have the capacity to fix 16-42 kg ha-1 of atmospheric N per season and this can be further increased with good agronomic and cultural practices, thus boosting yields of non-legume crops. For example, in East Africa, sorghum yield improvements of 40-57% were reported when sorghum was grown in rotation with climbing beans. In the eastern region of Central Africa, yield of cereal crops grown after climbing beans increased by 25-40%. In this region farmers have no capacity to purchase inorganic fertilizers, neither do they have enough animals to supply organic fertilizer in the form of manure. As a result, common bean acts as a source of N supply to primary cereal crops. Common bean is therefore important in improving the soil health and helps maintain soil fertility. Production Constraints Biotic Constraints Biotic stresses such as diseases and pests are universal constraints to common bean production, especially fungal pathogens. Under favorable disease conditions, fungal pathogens cause significant yield losses. Yield losses also occur due to insect damage (Table 1). Anthracnose, rust, and angular leaf spot are widely distributed, while rhizoctonia web blight and ascochyta blight can be locally intense in warm-moist environments, respectively. In recent years, root rots have emerged as a significant problem for common bean production, especially those caused by Pythium spp. and Fusarium spp. Insects are occasional problems. In Central America the bean pod weevil, Apion godmani and A. aurichalceum, is the most important pest, while in East Africa the bean stem maggot, aphids, and pod borers cause the most serious problems. Abiotic Constraints Abiotic stress is the major constraint to bean productivity in most tropical countries. Abiotic factor such as extreme limited water stress (drought) cause yield loss in Mexico, Brazil, Central America, and Eastern and Southern Africa. Heat stress adversely affects the cultivation of beans in Central and Southern America and Africa (Beebe et al. 2011). Nutrient deficiencies of phosphorous (P) and nitrogen (N) also reduces yield, while Aluminum and Manganese toxicity associated with acid soil, as well as low Calcium availability, cause significant common bean yield loss (Table 1). Table 1 A schematic comparison of different bean production limitations, classified for their frequency, likely intensity, and risk to farmers. Limitation Frequency Intensity Risk Pests and diseases +++ +++ ++++ Drought ++ ++++ Low soil fertility +++++ +++ + High temperatures +++++ +++ + + : very low +++++ : very high Source: Adapted from Beebe et al. 2006b n/a International Breeding Centers The International Center for Tropical Agriculture (CIAT) was established in Cali, Colombia, under the Consultative Group on International Agricultural Research (CGIAR) system (in 1971) with the mandate to work on common bean (Phaseolus vulgaris L.). CIAT coordinates all common bean research programs at the national level. Strong collaborative and active breeding programs are found in many countries throughout the tropics of the Americas and Africa, with interchanging of improved germplasm among countries. The primary mission of CIAT’s bean program is to contribute to global food security. Their goal includes making bean production more profitable for small scale farmers in Africa, Latin America and the Caribbean countries. CIAT has successfully developed bean varieties with genetic resistance to major diseases and pests, which have helped to minimize yield losses for farmers. More recently breeding programs have focused on breeding for improved bean tolerance to abiotic stresses such as drought and soil problems. These efforts have gained more significance due to more erratic climatic conditions that change the patterns and intensity of both abiotic and biotic stresses. CIAT’s breeding strategy for beans focusses on priority bean grain (market class) types. Supporting Broad Goals The CIAT’s bean program uses tools that allow them to support the broad goals including exploiting the biodiversity of more than 35,000 accessions in CIAT collection, biotechnology, particularly marker assisted selection, and gene discovery. CIAT outlines the technical contributions and responsibilities for various regional bean breeding centers such as ECABREN (East and Central Africa Bean Research Network) and SABREN (South African Bean Research Network), and national bean breeding programs, universities and advanced research institutions. Breeding Methods Improving Seed Yield Common bean is a self-pollinating crop, and thus breeding methods to improve seed yield and other important traits have followed methods similar to those applied to autogamous crops. These include pedigree selection (most commonly used breeding system in common bean improvement), back cross (for highly heritable traits, usually under single gene control), inbred back cross (1 or 2 back cross, followed by selfing), congruity back crossing (alternate crossing to each parent in alternate generations – maintains heterozygosity), recurrent selection (Fig. 8), single seed descent (among closely related elite lines), gamete selection (individual F1 plants of multiple parent crosses give rise to families) have been used. The breeding strategies of common bean have also followed approaches similar to those applied to other crops (Gepts, 2002). These approaches are described in Fig. 9. In addition, a three-tiered breeding strategy has been proposed to accommodate gene exchange between distantly related parents and to have more success for integrated genetic improvement of common bean (Fig. 10).

textbooks/bio/Agriculture_and_Horticulture/Crop_Improvement_(Suza_and_Lamkey)/1.08%3A_Common_Bean_Breeding.txt

Arti Singh; Teshale Mamo; Asheesh Singh; and Anthony A. Mahama Cowpea (Vigna unguiculata L. Walp.) (2n=2x=22) belongs to the Leguminosae family. Cowpea is an important legume crop ranked second after groundnut. It is grown for food and feed in multiple continents (Africa, Asia, Europe, the United States, and Central and South America). The center of origin and domestication is Southern Africa from where is was later carried to East and West Africa and Asia. Wild relatives of cowpea are found all over Africa. With grain comprised of 25% protein and several minerals and vitamins, it is another important crop that is vital for tackling current global food security challenges facing the world. Learning Objectives • Become familiar with the Cowpea crop • List breeding institutions working on it • Know classification system • Describe adaptation and usage • Outline production constraints • Discuss breeding method used to develop pureline cowpea cultivars • Outline a step by step breeding procedure using CB-27 cowpea cultivar as an example Domestication and Diversification Cowpea was domesticated in southern Africa and later spread to East and West Africa and Asia. Baudoin and Marechal (1985) classified domesticated cowpea into five cultivar groups (cultigroups). 1. Unguiculata (seed testa thick and shiny) is the major group. 2. Textilis (long inflorescence peduncle) is mostly found in West Africa. 3. Sesquipedalis (fleshy pod, wrinkled when ripe) is mainly found in East Africa. 4. Melanophthalmus (seed testa thin & often wrinkled, flower & seed partly white) originated in West Africa. 5. Biflora (seed testa thick and shiny, flower and seed most often colored) is grown in South East Asia. Biology of the Crop General Characteristics and Development of the Crop Cowpea is a warm-season, annual, herbaceous and similar in appearance to common bean (Phaseolus vulgaris L.) except that the leaves are generally darker green, shinier, and rarely pubescent. It has twining stems varying in erectness and bushiness. The trifoliate leaves develop alternatively, and petioles are 2 to 12 cm long. A wider range exists for leaf shape and size in cowpea than in common bean. Plant growth habit is categorized as erect to semi-erect, prostrate (trailing type) or climbing, and indeterminate to determinate, depending on the genotype. However, most cowpea accessions have the indeterminate type of growth habit. Cowpea has a strong taproot system and the depth of the root has been measured up to 95 inches after 8 weeks of seeding. Flowers are born in axillary racemes on stalks with 15 to 30 cm peduncles. Usually, a single peduncle has two to three pods, however, under favorable growing conditions, a single peduncle often carries four or more pods. The presence of long peduncles is a unique feature of cowpea among legumes, and this characteristic facilitates hand harvesting. The cowpea flowers vary in color from white, cream and yellow to purple, and the seeds, which are smooth or wrinkled, range in color from white, cream or yellow to red, and are characterized by a marked hilum surrounded by a dark arc (Fig. 1). Photosynthesis, Photoperiod, and Temperature Cowpea is a short-day plant and like other grain legumes, cowpea processes its food using a C3 photosynthetic pathway. Different cowpea genotypes show photoperiod sensitivity in connection with floral bud initiation and development. Some genotypes are day-neutral, while other genotypes display a wider range of photoperiods (Craufurd et al. 1997). In addition, few cowpea genotypes exhibit various degrees of sensitivity to photoperiod (extent of delay in flowering) and temperature (Ehlers and Hall 1996). Warmer temperatures speed up flowering time in both photoperiod sensitive and insensitive cowpea genotypes. The development of improved cowpea genotypes for warm environments requires an understanding of the developmental responses to heat and photoperiod. Cowpea cultivars show a wide range of reproductive characteristics. The flower initiation ranges from 30 to 90 days after planting, and attaining physiological maturity (dry seed maturity) ranges from 55 to 240 days after planting (Wien and Summerfield, 1984). Wien and Summerfield (1984) reported that cowpea cultivars that flower early have a shorter or more concentrated flowering period than cultivars that flower late. In Sub-Saharan Africa, selection for different degrees of photosensitivity has occurred in different climatic zones and this resulted in pod ripening coinciding with the rainy season in some given locations. This condition helps the plant during pod set and ripening to escape damage from excessive rainfall and diseases attack. Therefore, photoperiod and temperature responses of particular cowpea genotypes allow cowpea breeders to make parental choices to best utilize exotic and adapted germplasm to serve particular environments. General Classification Classification by Utilization or Mode of Consumption Cowpea is used as food as well as feed, including forage, hay and silage for livestock in Sub-Saharan Africa, Asia, Europe, USA and Central and South America. In Africa, people consume young leaves, immature pods, immature seeds and dried seeds. The stems, leaves, and vines of the cowpea serve as animal feed. Cowpea is also used as green manure and cover crop for maintaining the productivity of the soil. The grain contains 25% protein and several vitamins, minerals and fibers. Breeding efforts at the International Institute of Tropical Agriculture (IITA) and national programs have resulted in dual-purpose varieties (with good grain and fodder yields). The dual-purpose varieties have provided both grain and fodder while fitting the different cropping systems, economic, and climatic conditions encountered in Africa. In addition, cowpea has great flexibility in terms of its use as farmers can choose to harvest the cowpea for grains or for forage to feed their livestock, depending on economic or climatic conditions. Classification by Seed Characteristics Cowpea seed size ranges from small wild types to 0.5-1 cm long. The 1000 seed weight of cowpea is 150-300 grams. Most of the time, seeds develop a kidney shape if not restricted within the pod. If the development of seed is restricted by the pod, the seed becomes more globular. The seed coat in cowpea can either be smooth or wrinkled and an assortment of colors has been observed (including white, cream, green, buff, red, brown and black). Sometimes, the seed is either speckled or mottled. Many of the cowpea seeds are also referred to as eye bean (black eye, pinkeye purple hull) (Fig 2) where they are covered with a white tissue, with a blackish rim-like aril. In cowpea, the seed size is important because it directly influences productivity, and together with different color standards, can determine grain quality for the market. Therefore, seed size and color should also be considered as major traits of interest for breeding programs. In the United States, different cowpea cultivar classes with a broad range in characteristics are grown for horticultural use. All cultivars that are grown in USA are day neutral members of the subspecies Unguiculata cultivar group Unguiculata. The cultivars grown for seed are classified as Blackeye beans, are known for good yield production), the Crowders type are known for their largest peas, and are often used for canning. Cream peas are the most popular and have become increasingly important for home gardening, while field types have few popular cultivars and most cultivars are old agronomic types. Classification by Growth Habit Cowpea has substantial genetic diversity for growth habit. The major growth habits are categorized as erect to semi-erect, prostrate (trailing) or climbing types. Growth habit in cowpea ranges from indeterminate to fairly determinate with the non-vining types tending to be more determinate. Meanwhile, some of the early maturing groups have a determinate growth types. Classification by duration of Growth Period Cowpea is grouped into early, medium and late maturity group. However, the range for growth-period duration varies from one region to another or among varieties of different growth habits. According to growth habit and region, cowpea cultivars range from 55 to 240 days to physiologically mature. The difference is not only varietal but also environmental, especially for the factors of day-length and temperature. Adaptation and Economic Importance and Uses Adaption Cowpea is widely cultivated throughout the tropics and subtropics between 35°N and 30°S, across Africa, Asia and Oceania, the Middle East, Southern Europe, Southern USA and Central and South America. Cowpea is a crop adapted to hot and dry tropical conditions. It is also considered drought tolerant compared to other legumes. They grow best at low altitude with a precipitation of 400 to 700 mm per annum. Optimum crop production requires temperatures between 20-35°C during the growing season, and soil pH between 5.5 and 8.3. Cowpea is grown on a wide range of soil textures but the crop shows preference to sandy soil. It has low tolerance to salt but somewhat tolerant to aluminium. Like other legumes, the crop does not withstand waterlogged or flooded conditions. Cowpea is sensitive to chilling conditions. The crop is grown in 45 countries across the globe. An estimated 14 million ha is planted to cowpea each year across the globe with total annual production of about 6 million MT, the current average is estimated at about 0.45 tonnes/ha (FAOSTAT, 2010). The production trend of cowpea across the world is shown in a Fig. 3. Cowpea is primarily an African crop. The largest producers are Nigeria, Niger, Brazil, Haiti, India, Myanmar, Sri Lanka, Australia and the United States. Among these high cowpea producing countries, Nigeria and Niger each grow over 4 million ha and account for 45% and 15%, respectively, of the total world production (FAOSTAT, 2010). Cowpea in the Human Diet and Nutrition Cowpea is one of the most widely used legumes in the tropical parts of the world. It can be used at all growth stages as a vegetable crop. The grain is mainly used for human nutrition, making cowpea one of the most important dual purpose legumes. The nutritional content of cowpea grain is comparable to common beans, with relative low fat content. The protein in cowpea grains is rich in tryptophan compared to cereal grains. In Africa, immature green pods are used similar to snap bean in common bean. Cropping System Cowpea grows well in association with cereal crops through intercropping. Cowpea is a major component of the traditional cropping system in Africa, Asia, and Central and South America, where it is mainly grown with other crops in various combinations. It is grown as a millet-cowpea mixture (exhibit 22% of the field sampled), a predominant crop mixture system in the Sudan savanna of Nigeria (Henriet et al., 1997). In the dry savanna cropping system, millets have been grown with different crop mixtures including millet-sorghum-cowpea (represent 19%), sorghum-cowpea (10%) and millet-cowpea-groundnut (8 %) (Olufajo and Singh, 2002). Cowpea grain yield in the mixture is lower than under sole crop condition. The factors contributing to low yields under intercropping systems include low plant population, shading effects, and competition for nutrients. Cowpea is also used as green manure, where it is incorporated into soil and can provide nitrogen to subsequent crops, minimize soil erosion and suppresses weeds. Production Constraints Biotic Constraints Several biotic factors that cause yield reduction in cowpea include insect pests, fungal, bacterial, viral diseases, plant parasites, other organisms. • Insect Pests – Aphids are the main insect pests of cowpea, and are important vectors of cowpea mosaic virus. Other insect pests attacking cowpea are flower thrips and pod borers. • Diseases – Cowpea diseases are due to fungi, bacteria and viruses. Examples of diseases include, Cercospora leaf spot, ashy stem blight, bacterial blight, blackeye cowpea mosaic polyvirus (BICMV), and cowpea mosaic comovirus. • Plant Parasites – Certain weeds are important in cowpea production and most notable examples are the parasitic weedy plants Striga and Alectra. • Nematodes – Nematode also causes root damage to the crop and result in significant yield loss. Abiotic Constraints Extreme drought and heat, soil acidity, low phosphorous are some of the abiotic factors that limit the yield of cowpea. International Breeding Centers The International Institute of Tropical Agriculture (IITA) has a global mandate for the development and improvement of cowpea. Its main duty and responsibility is to develop and distribute improved cowpea varieties to over 65 national cowpea research programs in Africa. Variety requirements for cowpea differ from region to region in respect of the seed color preference, use patterns, maturity and growth habit. Therefore, IITA located additional scientists and breeding centers in Philippines, Nigeria, Burkina Faso, Cameroon, Congo and Brazil in order to address the regional constraints in cowpea production at the global level. A general strategy for IITA is to develop different cowpea breeding lines with diverse maturity (to feed specific adaptation across wide agro-ecological zones where cowpea is grown), plant type, and seed types combined with resistance to major biotic (diseases, insect-pests, and weeds) and abiotic (drought, heat and low phosphorous) stresses. IITA’s genetic resources account for the world’s largest and most diverse pool of cowpea germplasm. The collection consists of over 15,000 cultivated varieties from over 100 countries, and 560 accessions of wild cowpeas (Singh et al., 1997). The IITA collection constitutes a valuable resource for the cowpea improvement worldwide. Scientists from IITA center and regional centers have identified various cowpea genotypes with numerous desirable genes, which govern plant architecture and physiological traits (like plant type, root architecture, growth habit, pod traits, seed traits, photosensitivity, maturity and nitrogen fixation), quality traits (fodder quality and grain quality), abiotic stress (heat and drought tolerances), biotic stress (resistance to major bacterial, fungal and viral diseases, resistance to rootknot nematodes, resistance to aphids, bruchid, thrips, and resistance to parasitic weeds such as Striga gesneriodes, and Alectra vogelii). Breeding Methods and Strategies Introduction Cowpea is a true diploid species with a chromosome number of 2n = 2x = 22. It is primarily a self-pollinating crop in most production environments, although up to 5% outcrossing can occur in some environments, possibly associated with pollen transfer by insects. Different cowpea breeding programs have their own priority of target production zones including the cropping systems, consumption preferences and major constraints to cowpea production in their agro-ecological zones. Most Cowpea breeders at IITA and National programs use bulk, backcross, and pedigree breeding methods to deal with large numbers of segregating populations because cowpea is an autogamous crop and most cultivars grown by farmers are pure lines. The primary objective in all cowpea breeding programs is higher grain yield and improved grain quality. In addition, to yield and quality traits, most breeders seek to breed in a wide range of abiotic and biotic stress resistance traits. The breeding strategy of IITA and regional breeding program is to develop broad range of breeding lines with high yield and adapted to different agro-ecological zones that possess regionally preferred characters for plant type, growth habit, days to maturity, seed type, combined with resistance to biotic and abiotic stress, along with quality. In general, the main focus of breeding programs is to develop extra early maturity (60-70 days) and medium maturity (75-90 days), non-photosensitive lines with good grain quality and possibility for dual purpose use, either for use as sole crop or as intercrop in multiple cropping systems. Example of Cultivar Development Development of Blackeye Cowpea Cultivar “CB27” at University of California Riverside California Blackeye 27 (CB27) was developed by the University of California, Riverside (UCR) following the protocol shown in Fig. 4, and released in 1999 for its better performance in the following characteristics: 1. High yielding 2. Reproductive-stage heat tolerance 3. Broad-based resistance to Fusarium wilt 4. Broad-based resistance to root-knot nematodes 5. Semi-dwarf and less vegetative shoot biomass 6. Bright white seed coat 7. Good seed weight 8. Non-leaky pigments during boiling and excellent canning quality. Actual data from Preliminary Yield Trials (PYT), Advanced Yield Trials (AYT) and Uniform Yield Trials (UYT) along with different test conducted on agronomic, disease and quality traits (from 1989 – 1998) led to the development of CB-27. Tables 1 – 20 show the results of various trials and years in which they were conducted to eventually release CB-27. Note: all cells in Tables 1-20 with “n/a” are blank cells. Year – 1989 Table 1 Preliminary Blackeye Trials at Kearney Agricultural Center (KAC), 1989; H = Heat Tolerance. Entry Origin Score Yield (lbs/acre) Seed weight g/100 H8-14 336 x 1393 H 3281 24.5 H8-9 336 x 1393 H 3236 24.7 H8-8 336 x 1393 n/a 3152 23.5 H8-7 336 x 1393 H 3022 25.9 H8-4 336 x 1393 H 2861 23.6 CB5 n/a n/a 2995 26.3 CB46 n/a n/a 3017 21.5 LSD n/a n/a 715 13 CV (%) n/a n/a 16.2 3.4 Year – 1990 Table 2 Advanced Blackeye Trials at University of California Riverside (UCR), 1990. Entry Origin Yield (lbs/acre) Seed weight (mg) Seed density g/cm3 Lodging Earliness Vigor H8-14 336 x 1393 1805 235 1.10 erect early compact H8-9 336 x 1393 1805 248 1.09 erect early compact H8-8 336 x 1393 1497 233 1.11 erect early compact CB5 CB x Iron 1889 254 1.06 erect med moderate CB46 CB5 x 166146 2274 224 1.09 erect med moderate LSD.05 n/a 266 10 0.03 n/a n/a n/a CV (%) n/a> 10 3 2 n/a n/a n/a Year – 1992 Table 10 Screening for Heat Tolerance data, 1992. Line Total # of Sub-lines # Heat Tolerance – Flowering (CVARS & GH) Heat Tolerance – % Podding (Hot Glasshouse at UCR) # Selected sublines Average Podding H8-14 45 26 57 9 4.7 H8-9 54 54 92 14 9.4 H8-8 46 24 70 12 5.0 Table 11 Summer glasshouse evaluation results for associations among heat Tolerance and Resistance to Root Knot Nematode (non-aggressive Meloidogyne incogonita); day/night temperatures of 34/30 degree centigrade, 1992. Line 1Nematode Resistance #2Heat Tolerance – Floral buds #2Heat Tolerance – Pod set H8-8-1 R N 3 H8-8-2 S S H8-8-3 R N 6 H8-8-4 S S H8-8-5 R N 2 H8-8-6 S S H8-8-8 R N 0 H8-8-9 R N 7 H8-8-10 R N 0 -to- n/a n/a n/a —- n/a n/a n/a H-8-8-27 R N n/a Year – 1993 The blackeye cowpea cultivators follow three management schemes: 1. Single-flush main crop cut after ~ 100 days 2. Single-flush double crop, sown later and cut after ~ 100 days 3. Double-flush main crop, sown early and cut after ~ 140 days Short-term goal – to develop blackeye varieties with resistance to the: 1. common race of Fusarium wilt in California (race #3) 2. wide range of root knot nematodes 3. heat tolerance 4. increased yield potential Medium-term goal – to develop blackeye varieties that have resistance to early cut-out and greater ability to produce pods over an extended season (140 days from planting to cutting) Long-term goal – resistance to lygus, resistance to cowpea aphid Table 12 Mean non-aggressive Meloidogyne incognita egg mass count from four to five replicates on breeding lines using “pouch” tests, 1993. R indicates resistant, S indicates susceptible, and – indicates not tested. Line Date of Test 13-May 27-Feb 25-Aug R/S CB5 2 n/a n/a R CB46 2 n/a n/a R H-8-8-2 2 0 n/a R H-8-8-4 4 <1 n/a R H-8-8-6 <1 <1 0 R H-8-8-8 2 0 n/a R H-8-8-13 0 0 0 R H-8-8-15 0 0 0 R H-8-8-16 10 0 0 R H-8-8-27 <1 <1 <1 R H-8-8-28 5 n/a n/a R H-8-8-30 0 n/a n/a R H-8-8-32 43 n/a n/a S H-8-8-35 0 <1 <1 R Table 13 Mean non-aggressive Meloidogyne incognita egg mass count from four to five replicates on breeding lines using “pouch” tests, in Advanced Yield Trial, 1993. R indicates resistant, S indicates susceptible, and – indicates not tested. Line Date of Test Classification May 13 April 9 Aggressive Non-aggressive CB3 32 190 S S CB46 15 24 S R H-8-8-2 6 n/a R R H-8-8-4 n/a 4 R R H-8-8-6 7 12 R R H-8-8-8 n/a 17 R R H-8-8-13 2 29 R R H-8-8-15 6 7 R R H-8-8-16 11 20 R R H-8-8-27 2 31 R R H-8-8-31 n/a 85 S S H-8-8-35 8 22 S R Table 14 Heat–tolerance results of advanced blackeye breeding lines evaluated in a hot glasshouse (day/night temperature of 35/30 degree Celsius) and Coachella Valley Research Station, 1993. – indicates not tested. Entry Grain Yield g/plant Plots/Plant Seeds/Pod Seed Weight Mg/seed Flower Production Pods/Peduncle # CB5 0 0 n/a n/a NO n/a CB46 2 4 2.7 166 NO n/a H8-8-6 22 28 4.2 192 YES 2.5 H-8-8-13 21 27 4.1 190 YES 2.75 H-8-8-15 22 27 4.1 196 YES 3.00 H-8-8-16 30 34 4.6 195 YES 2.75 H-8-8-27 26 30 4.2 207 YES 2.75 H-8-8-35 28 30 4.6 201 YES 2.75 Table 15 Advanced Blackeye Trial at Riverside, 1993. (Sown June 14, cut September 17 (95-day season)) Entry Grain Yield lbs/ac Seed weight mg/seed Heat tolerance Root Knot Resistance Non-aggressive Aggressive CB5 1975 260 SUS RES SUS CB46 1996 225 SUS RES SUS H8-8-6 1631 240 TOL RES RES H-8-8-13 1951 228 TOL RES RES H-8-8-15 1938 227 TOL RES RES H-8-8-16 2156 231 TOL RES RES H-8-8-27 1767 246 TOL RES RES H-8-8-35 2049 229 TOL RES RES LSD.05 405 22 n/a CV% 15 6 Year – 1994 Table 16 Mean Grain Yields (lbs/ac) under single and double management in multilocation Advanced Yield Trials at UCR and KAC. a – indicates the top yielding group based on statistical analysis. Entry Riverside Single Flush Riverside Double Flush Kearney Single Flush Kearney Double Flush Mean CB-46 1860 2996 3046a 3916 2955 CB-5 2063 2869 2222 3479 2658 H8-8-1N 1985 3199 2629 3786 2900 H8-8-6 2039 2677 2051 3366 2531 H8-8-13 1800 2318 2637 2717 2668 H8-8-15 1703 3040 2589 3175 2627 H8-8-27 1742 2979 2398 3728 2712 H8-8-35 1771 2658 2414 3399 2561 LSD(.05) NS 581 245 NS 288 CV(%) 13 14 15 16 16 Table 17 Seed Weights (mg/seed) under single and double management in multilocation Advanced blackeye Trial at UCR and KAC, 1994. Entry Riverside Single Flush Riverside Double Flush Kearney Single Flush Kearney Double Flush Mean CB-46 234 211 221 211 219 CB-5 273 255 265 246 260 H8-8-1N 249 233 242 225 237 H8-8-6 243 238 230 220 233 H8-8-13 231 206 209 213 215 H8-8-15 239 227 240 218 231 H8-8-27 240 229 242 221 233 H8-8-35 237 220 234 222 228 LSD(.05) 11 12 6 13 6 CV(%) 3.1 3.6 3.9 3.9 3.6 Table 18 Plant growth habit, plant size and Pythium incidence (no. of infected plants/plot) in of entries in advanced trials at KAC and UCR and double flush advanced trial at UCR, 1994. M = medium; M-L = medium-large; L = large plants; L = low; M = medium; H = high; M-L = moderately low in vinyness. Entry Growth habit Plant Size Growth habit vinyness Pythium CB-46 M M-L 5.3 CB-5 L H 7.5 H8-8-1N M-L H 2.3 H8-8-6 L M 2.5 H8-8-13 M M 4.3 H8-8-15 M L 1.3 H8-8-27 M L 1.5 H8-8-35 M L 1.3 LSD(.05) n/a n/a 4.3 Table 19 Average grain yields (lbs/ac) and seed size under single and double management systems for the resistance to three nematode strains in Advanced blackeye trials at KAC, 1994. a – indicates the top-yielding group based on statistical analysis. Entry Grain yield lbs/ac Seed weight mg/seed RKN resistance non-aggr RKN resistance aggr RKN resistance javanica CB-46 3481a 216 R S S CB-5 2851 255 R S S H8-8-1N 3208a 234 R S S H8-8-6 2709 225 R R R H8-8-13 2677 211 R S S H8-8-15 2882 229 R R R H8-8-27 3063a 231 R R R H8-8-35 2907 228 R R R H8-8-35 2907 228 n/a n/a n/a LSD(.05) 472 9 n/a CV(%) 16 3.9 Year – 1995 Table 20 Grain Yields (cwt/ac) in Uniform Trial at Stanislaus and Shafter, 1995 Entry Stanislaus Shafter Mean H8-8-27 24.5 55.5 40.2 H8-8-15 23.0 55.2 39.1 CB46 20.8 55.9 38.4 CB88 11.5 54.3 32.9 LSD(.05) 3.4 3.8 2.5 CV(%) 14.8 6.1 8.5 Table 21 Grain Yields (cwt/ac) of UCR Advanced Blackeye Trials at UCR, 1995. Entry Origin Single Flush Double Flush Mean H8-8-27 CB5/CB3//1393 22.3 29.6 26.0 H8-8-15 CB5/CB3//1393 23.9 29.2 26.6 CB46 CB5/CB3//PI1166146 23.3 25.9 24.6 CB88 CB5/CB3//PI1166146 24.5 29.6 27.1 LSD(.05) 2.1 NS 3.4 n/a CV(%) 8 14 16 Table 22 Grain Yields (cwt/ac) of UCR Advanced Blackeye Trials at KAC, 1995. Entry Origin Single Flush Double Flush Mean H8-8-27 CB5/CB3//1393 38.3 42.8 41.3 H8-8-15 CB5/CB3//1393 38.3 41.9 40.1 CB46 CB5/CB3/PI1166146 31.7 47.2 39.4 CB88 CB5/CB3/PI1166146 34.0 44.4 39.2 LSD(.05) 4.6 8.0 4.8 n/a CV(%) 12 16 15 Table 23 Rootknot nematode infestation from UCR Advanced Blackeye Trials at KAC, 1995. Line Nematodes Non-aggres Nematodes aggres Nematodes M.jay. Fusarium Race 3 Fusarium Race 4 Heat H8-8-27 Yes Yes Yes Yes Yes Yes H8-8-15 Yes Yes Yes Yes Yes Yes CB46 Yes No No Yes Yes No CB5 Yes No No No Yes No Notes: Types of RKN; Non-aggres = Non-aggressive M. incognita – not able to overcome standard ‘Rk’ gene resistance. aggres = strain of M. incognita – able to overcome ‘Rk’ resistance Table 24 Seed Size (mg/seed) from UCR Advanced Blackeye Trials at KAC and UCR, 1995. Line Kearney Riverside Mean H8-8-27 208 215 212 H8-8-15 207 209 208 CB46 203 201 202 CB88 216 210 213 LSD(.05) 11 19 11 CV(%) 4.4 7.7 6.3 Table 25 Comparison of seed size (mg/seed) of high-yielding line, at locations KAC and UCR, 1994 and 1995. Line 1994 1995 Mean H8-8-27 231 212 222 H8-8-15 229 208 219 CB46 216 202 209 CB88 231 213 222 LSD(.05) 6 11 n/a Table 26 Mean Yields of high-yielding lines at locations KAC, UCR, SHAFT and STANI, 1994 and 1995. Line Mean H8-8-27 33 H8-8-15 33 CB46 33 CB88 32 Year – 1996 Table 27 Grain Yields (cwt/ac) of high yielding breeding lines and check varieties CB46 and CB88 in 5 uniform trials and Individual Seed Weight from single flush trial at KAC–1996. Entry Shafter Tulare Kearney double Kearney single Westside Mean Seed weight mg/seed H8-8-27 53.1 42.6 38.0 26.1 24.5 36.9 213 H8-8-15 45.8 40.3 33.3 23.9 23.8 33.4 207 CB46 46.6 49.1 40.5 25.5 24.6 37.3 215 CB88 46.2 41.1 37.0 23.4 20.9 33.7 215 LSD.05 8.3 8.1 NS 2.7 1.4 2.7 7 CV(%) 11.7 12.5 13.8 7.7 4.0 12.1 2.1 Table 28 Grain Yields (cwt/ac), root-knot galling scores and number of nematodes (juveniles per liter of soil) of high yielding UCR blackeye breeding lines in a field at KAC and at the Muller Farm-Chance Field, Stanislaus Co. that are infested with Rk gene virulent stains of M.javanica and M.incognita. Fusarium wild races 3 and 4 from the Chance Field alone, 1996. Entry Grain Yield (KAC) Grain Yield (Muller) Galling (KAC) Galling (Muller) No. juveniles (KAC) No. juveniles (Muller) Resistance Nematode Fusarium Wilt H8-8-27 21.1 21.0 2.3 2.1 1672 328 Rk+ 3 & 4 H8-8-15 19.9 23.1 1.6 2.0 1061 239 Rk+ 3 & 4 CB46 21.2 16.5 4.9 4.7 1833 572 Rk 3 CB88 22.9 10.2 4.5 5.1 2478 572 Rk 3 LSD.05 2.9 3.1 0.6 0.9 750 NS n/a n/a Table 29 Grain Yields (cwt/ac) and seed size (mg/seed) of high-yielding UCR blackeye breeding lines and checks (C46 and CB88) under single and double flush management at UCR, 1996. Entry Grain Yield (Single) Grain Yield (Double) Seed Size (Single) Seed Size (Double) Means Yield Means Seed Size H8-8-27 20.8 32.7 223 249 26.8 236 H8-8-15 22.4 36.1 226 245 29.3 236 CB46 24.7 34.9 223 234 29.8 228 CB88 22.7 34.7 226 232 28.7 229 LSD.05 2.5 NS 9 9 2.5 6 CV(%) 7.5 9.0 2.7 2.5 8.7 2.6 Table 30 Grain Yields of promising blackeye breeding lines and checks (CB46 and CB88) over years and locations in the Central Valley. Overall mean includes data from Tulare and Westside Field Station trials, 1996. Entry Shafter mean Yield 1995 and 1996 Stanislaus mean Yield 1995 and 1996 Kearney Mean Yield 1995 and 1996 Overall Mean H8-8-27 55 23 34 36 H8-8-15 50 23 33 34 CB46 51 19 36 36 CB88 50 11 33 31 Table 31 Summary of grain yields (cwt/ac) of selected blackeye bean breeding lines and check cultivars (CB46 and CB88) from single-flush and double-flush trials at KAC, 1994, 1995, 1996. Entry 1994 Single flush 1994 Double flush 1995 Single flush 1995 Double flush 1996 Single flush 1996 Double flush Mean Single Mean Double H8-8-27 30.5 39.2 31.7 47.2 25.5 40.5 29.2 42.3 H8-8-15 25.1 35.5 34.0 44.4 23.4 37.0 27.5 39.0 CB46 25.9 31.8 38.3 42.8 23.9 33.3 29.4 36.0 CB88 24.0 37.3 38.3 41.9 26.1 38.0 29.5 39.1 LSD.05 2.4 NS 4.6 8.0 2.7 NS 2.2 4.2 CV(%) 15 16 12 16 8 14 10 13 Year – 1997 Table 32 Grain yield, bean weight and bean quality grade of selected H8-8-27 cowpea breeding line in a Strip Trial near Wasco, CA, 1997. Entry Grain Yield (Dirt Wt) Cwt/ac Grain Yield (Clean Wt) Cwt/ac Clean Out % Bean Size Gm/100 seeds Total Damage (%) Splits Grade H8-8-27 49 44 8.1 24.1 2.0 0.3 UN No. 1 CB46 48 44 7.7 21.7 4.2 0.3 US No. 3 Table 33 Grains yield of selected H8-8-27 cowpea breeding line in Uniform Trials, 1997. Entry Westside Riverside Tulare Shafter Mean H8-8-27 1790 3491 2840 4446 3147 CB46 1990 3772 3769 5231 3638 LSD(.05) NS 472 302 NS 241 CV(%) 11.4 7.8 7.0 16.6 11.3 Table 34 Grain Yield and bean size of selected H8-8-27 cowpea breeding line and check (C46) at Shafter and KAC, 1997. Entry Shafter Mean Yield 1995, 1996, and 1997 Kearney mean Yield 1994, 1995, 1996, and 1997 Overall Mean H8-8-27 51 35 42 H8-8-15 51 33 41 Seed size CB46 n/a n/a 21.1 CB88 n/a n/a 22.1 Table 35 Grain Yields and rating for ‘greenness’ after the first pod plush of selected H8-8-27 cowpea breeding line and check (C46) in Uniform Trials at UCR and Tulare, 1997. Entry Riverside Yield Riverside Greenness Tulare Yield Tulare Greenness Mean Yield Means Greenness H8-8-27 3491 0 2840 0.7 3166 0.4 CB46 3772 0 3769 2.2 3771 1.1 LSD.05 472 n/a 699 n/a 302 n/a CV(%) 7.8 n/a 7.0 n/a 7.6 n/a 1998 – Uniform Blackeye Trials Table 36 Grain yields (lb/ac) H8-8-27 and check (CB46) in Uniform Blackeye Trials, 1998. Entry Shafter Tulare Kearney Riverside Mean H8-8-27 5156 4967 4629 3113 4466 CB46 4732 5178 4268 2938 4271 LSD(.05) 521 478 806 529 295 CV(%) 8 6 13 13 10 Table 37 Individual seed weights (grams/100 seeds) and % split seedcoat in Uniform Blackeye Trials, 1998. Entry Shafter Tulare Kearney Riverside Mean % Split H8-8-27 22.8 4967 22.6 24.9 23.0 13 CB46 22.6 5178 21.9 25.8 23.0 18 LSD.05 1.3 478 1.1 1.7 0.7 6 CV(%) 4 6 3 5 4 42 Table 38 Effect of row spacing and play type on yield and yield contributing traits of cowpea lines at UCR, 1998. Genotype Spacing Yield HI % Seed weight g/100 seed Seeds/pod Pods/peduncle Compact type H8-8-27 30″ 3642 47.0 24.7 8.4 1.7 n/a 40″ 3407 50.6 24.6 8.7 1.5 n/a 40″ x 2 4111 47.1 23.8 8.4 1.5 n/a Mean 3721 48.2 24.4 8.5 1.6 CB46 30″ 3583 47.0 23.6 8.5 2.1 n/a 40″ 3081 45.8 24.1 8.3 1.9 n/a 40″ x 2 3692 42.7 24.2 8.6 1.6 n/a Mean 3665 45.2 24.0 8.5 1.9 Table 39 Effect of row spacing and plant type on yield and yield contributing traits of cowpea genotypes at Shafter, 1998. Genotype Spacing Yield HI % Seed weight g/100 seed Seeds/pod Pods/peduncle Compact type H8-8-27 30″ 2717 48 23.8 8.4 2.1 n/a 40″ 2399 48.1 23.3 8.8 2.2 n/a 40″ x 2 2643 48.2 23.7 8.0 2.0 n/a Mean 2587 48.1 23.6 8.4 2.1 CB46 30″ 2472 42.0 23 9.5 1.9 n/a 40″ 2328 45.5 21.9 8.0 2.0 n/a 40″ x 2 2498 44.0 23.3 8.1 1.8 n/a Mean 2432 43.8 22.8 8.5 1.9 Year – 1999 Table 40 Performance of CB27 compared to checks CB5 and CB46 for disease (Fusarium), pest (nematode) and Agronomic performance (heat and chill tolerance), 1999. Entry Fusarium wilt Race 3 Fusarium wilt Race 4 RKN (M. incognita) Avirulent RKN (M. incognita) Virulent RKN (M. javanica) Heat Tolerance Chill Tolerance CB5 No No Yes No No No No CB46 Yes No Yes No No No No CB27 Yes Yes Yes Yes Yes Yes Yes Example Using Participatory Varietal Selection Recent example of Cowpea cultivar released by IITA in parts of Africa using participatory varietal selection. 1. In Burkina Faso, two improved cowpea varieties developed by IITA have been released. 1. IT99K-573-2-1 and 2. IT98K-205-8, 2. Using participatory varietal selection approach, local farmers and researchers choose varieties from various options after two years of trial in the central and northern regions of Burkina Faso. 3. Selected varieties are early maturing (60 days), high yielding (2170 kg/ha), resistant to parasitic weed striga along with big size, preferred color, and cooking qualities pertaining to farmers’ taste. 4. New cowpea varieties also have better adaptability to climate change and can be grown successfully in drier regions with low rainfall. Important Traits Example of Participatory market-led cowpea breeding in Sub-Saharan Africa (Tanzania and Malawi) in assigning importance to traits (Fig. 5) Pathway Based on Preferences Farmers’ and consumers’ preferences of traits in a variety or cultivar play a critical role on the release and adoption of new varieties. It is important to note that the preferences of the two groups differ and therefore, require the close attention needed to address those preferences (Fig. 6). Notes to Consider 1. Alectra vogelii is a parasitic weed that causes considerable damage to cowpea plant by attaching to it and tapping nutrients. 2. In Tanzania and Malawi, Alectra is one of the major weed growing in almost all cowpea growing areas. 3. In Figure 4 is shown important traits of cowpea required by farmers. Out of 11 traits used in selection of best cowpea lines by farmers, only five traits (brown seed color, white seed color, good taste, large seed, many leaves and tender leaves) are specific to the final consumer, while the other six traits (early maturity, high yield, resistance to Alectra, resistance to diseases, tolerant to pest, drought tolerance) are agronomic traits. Large seed size is the most important trait from marketing perspective, whereas high yield, early maturity, and resistance to A. vogelli are the main agronomic traits which are the deciding criteria used by farmers to select varieties for growing on their farm. 4. In Figure 5 is shown an example of value chain approach used to develop cultivars (for example-IT99K-573-2-1) 5. This approach resolves biases and takes care of farmers, consumers and market preference and will not let breeders effort go waste like in past where outstanding varieties with excellent agronomic traits failed due to inability to satisfy needs of farmers, consumers and market at the same time. Marker-Assisted Selection Marker-assisted selection approaches are being developed in cowpea with high-density marker maps and SNP markers becoming available. As cowpea is gaining acreage globally more investment is being made for breeding and marker development. This will assist in further development of MAS in cowpea. Genetic loci controlling important pest and disease resistance genes and agronomic traits have been placed on the genetic map (for example, Kelly et al, 2003). Closely linked markers to some of the biotic traits have been identified (Gowda et al., 2002). Most of these traits are governed by major genes and are potentially good candidates for MAS. Along with MAS for simply inherited traits, the genomic selection approach offers usefulness in future breeding efforts. Currently, joint efforts are being made by IITA, Bean/Cowpea Collaborative Research Support Program (Bean/Cowpea CRSP), advanced laboratories in the USA, Australia, African Agricultural Technology Foundation (AATF), Network for Genetic Improvement of Cowpea for Africa (NGICA) and Monsanto Corporation to exploit biotechnology tools to complement conventional breeding methods for improving resistance to diseases and insects.

textbooks/bio/Agriculture_and_Horticulture/Crop_Improvement_(Suza_and_Lamkey)/1.09%3A_Cowpea_Breeding.txt

Teshale Mamo; Asheesh Singh; and Anthony A. Mahama Millets are tall and vigorous grasses with panicles containing small seeds, and are grouped in the cereal family Gramineae, same category as sorghum and maize. Millets are adapted and used as staple food in the semi-arid tropics of Africa and Asia where other crops generally cannot be grown. Pearl millet (Pennisetum glaucum L.), finger millet (Eleusine coracana L.), foxtail millet (Setaria italic L.) and proso millet (panicum miliaceum) are among the millet species grown widely in Africa, Asia, Europe and North America. Learning Objectives • Become familiar with the Millet crop • List breeding institutions working on this crop • Know crop biology and classification system • Describe adaptation and usage • Outline production constraints • Discuss breeding method used to develop pearl millet cultivars Origin and Domestication History Millet is one of the ancient staple human foods and believed to be the primary domesticated cereal crop. Although the exact origin and domestication of millet remains unclear, it is believed that millet was domesticated and cultivated over 7000 years ago during the Neolithic era in Africa and then distributed throughout the world as human food. To date, a total of 161,708 accessions of millet species have been collected and preserved in gene banks across the globe, and these collections comprise 98.1% of cultivated types (Sangham et al., 2012). Fig. 1 shows a field of millet with a path through it, in the Upper Region of Ghana, which is characterized by generally sandy soils and limited rainfall regimes. Global Production Globally, millets are grown in over 90 countries from 2004 to 2008 and on average contribute 32.3 million tons of food production annually (http://faostat.fao.org/). Major producers of millet include India, China, Nepal, Pakistan, and Myanmar in Asia, and Nigeria, Niger, Senegal, Cameroon, Burkina Faso, Mali, Uganda, Kenya, Namibia, Tanzania, Togo, Senegal, Chad and Zimbabwe in Sub-Saharan Africa (Sangham et al., 2012). Pearl millet is mainly grown in South Asia and Sub-Saharan Africa while Finger millet is grown mainly in South and Southeast Asia and East Africa. Foxtail millet is grown mainly in South and Southeast Asia, while Proso millet is grown mainly in Asia, Europe, and North America. Finger millet, foxtail millet, Pear millet, and Proso millet are the largest collection of cultivated millet germplasm. The International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) is one of the Consultative Group on International Agricultural Research (CGIAR) centers that has a mandate to work on millet, and ICRISAT coordinates all millets research programs in the semi-arid tropics. A large number of millet germplasm is preserved in the ICRISAT gene bank in Patancheru, India. In addition, East African countries (Ethiopia and Kenya), and West African countries (Nigeria and Senegal) preserved significant numbers of millets in their gene banks. In the USA different species of millet are also preserved in gene banks based in Fort Collins, Colorado; Griffin, Georgia; Ames, Iowa; and Pullman, Washington. Morphology Pearl millet (Pennisetum glaucum), commonly known as bulrush millet, is a member of the grass family and originated in the semi dry land tropics of western Africa. The diversity and current distribution of the crop indicate that a large number of cultivated and wild forms of pearl millet is found from western Sudan to Senegal. In western Africa, a higher morphological diversity of pearl millet is found particularly in the south of the Sahara desert (Harlan, 1971; Harlan et al., 1975; Tostain et al. 1987). It is believed that the evolution of the crop under the pressures of high temperature and drought made pearl millet tolerant to moisture stress and high temperatures, in addition to low soil fertility. Such tolerance makes pearl millet a very important crop to farmers in the hot dessert parts of Africa and Asia. Races Pearl millet is grouped into four races based on grain shape. • Race typhoides: This race is typically identified by obovate caryopsis in which the cross sections are obtuse and terete. The shape of the inflorescences is cylindrical and has more diverse morphology among the four races. This group occurs widely in all Africa and it is widely grown in India (Brunken et al. 1977). • Race Nigritarium: the cross-section in caryopsis is angular with three and six facets in each grain. It has candle-like inflorescence and mature grain is longer and protrudes beyond the floral bracts compared to other group of races. Western Sudan and Nigeria are the main places where this race is found. • Race Globosum: It has spherical caryopsis with candle shape inflorescence. It is mainly found in central Nigeria, Niger, Ghana, Togo and Benin • Race Leonis: It has an acute and terete caryopsis. The unique character of this race is its acute apex, which is ended by the remnants of the stylar base. It has candle-like inflorescence shape. It is mainly grown in Sierra Leone but also produced in Senegal and Mauritania. Biology of the Plant Pearl millet is a warm season grass that uses the high efficiency C4 type of photosynthesis to fix carbon and thus has the ability to produce high dry matter. It is a short day plant requiring long nights before flower initiation. It is produced mainly for grain and forage in the semi-arid tropics of Africa and the Indian subcontinent. The crop grows on different types of soil including, light textured soils, sandy, and on acidic less fertile soils. In Pearl millet, a wide variation is observed for vegetative, reproductive and physiological features, and these variations are advantageous in the development of cultivars adapted to different climates, environments and cropping systems. Knowledge of pearl millet biology has enabled breeders to develop different cultivars that are adapted to varied environmental conditions (Khairwal et al. 1990). Growth and Development Vegetative Phase Pearl millet has three well defined growth phases: the vegetative phase; reproductive phase; and the grain filling phase. This phase is from emergence to floral (panicle) initiation of the main stalk. The seed of pearl millet takes 2-3 days to germinate under optimum temperature and moisture. The root has monocotyledonous type of root system consisting of a primary root, and adventurous type roots. It has deep root system penetrating up to 180 cm below the soil surface to absorb water. A research report indicated that in heavy-tillering pearl millet plants, the root system tends to have more horizontal spread than deep penetration. Similarly, those cultivars tolerant to early season moisture stress (3-15 days after sowing) have a 35% more root length than the susceptible cultivars. Pearl millet is an upright annual grass that tillers from the base. The main stems are 1-2 cm in diameter and are solid, attaining a height of 2-4 m with a round and oval shape. Pearl millet stem has slightly swollen nodes with a ring of adventitious roots at the basal end. Usually the internodal length increases upwards from the base of the stem. Pearl millet leaves appear as single leaf on each node in alternate orientation with leaf sheaths open and hairy ligules. Pearl millet has high potential to produce effective tillers enhancing the probability of producing more seeds from the same plant if flowering of tillers is synchronized with that of the main shoot. Different tillers arise from different branches and all can potentially bear productive panicles, a situation that can be important during unfavorable environmental conditions such as extreme drought. Reproductive Phase Reproductive stage is started by the formation of a dome-like structure which leads to the development of spikelets, florets, glumes, stigma and anthers, and finally stigma emergence (flowering) and pollination occurs. This is the time that marks the end of the reproductive stage. The critical time for grain number determination in pearl millet is the period between panicle initiation to anthesis. The inflorescence in pearl millets is a compound terminal spike known as panicle and is often similar in size and shape for a particular genotype. Usually the panicle is compact and cylindrical or conical in shape, 2-3 cm in diameter, and usually 15-45 cm long. Grain Filling Phase This is the stage that fertilization taking place in the panicle of the main shoot and continues until the plant matures. During this phase, plant dry weight increases in the grain (seed). However, in some cultivars, elongation and flowering of tillers takes place during this time and in this case there is some dry matter translocation to the non-grain components, mainly to stems of the tillers. The end of physiological maturity or grain filling stage is clearly marked by the development of a dark layer of tissue on the grain. For most cultivars, this dark layer of tissue occurs in an individual panicle 20-25 days after flowering. Adaptation, Economic Importance and Uses Pearl millet is produced annually on about 29 million ha in the dry land tropical regions of which 16 million hectares are grown in Africa), 11 million hectares in Asia, and 2 million hectares in Latin America (FAO data, 2005). Pearl millet accounts for about half of the world’s millet production. Africa accounts for about 60% of the area under millet cultivation, followed by 35% in Asian countries (primarily India), 4% European countries, and 1% in North America where millet is mainly used as forage and for poultry feed. Pearl millet is the third major cereal crop produced and used as staple food in sub-Saharan Africa which spans Nigeria, Niger, Burkina Faso, Chad, Mali, Mauritania, Senegal, Sudan, and Uganda. Pearl millet is referred to as subsistence staple food of the poor people living in semi-arid and arid environments in Asia and Africa. Ninety-three percent of pearl millet grain is used as food in developing countries of Africa and Asia while the rest (7%) is used for animal and poultry feed in USA, Australia and South Africa (Sangham et al., 2012). The crop is traditionally used to prepare food products such as flat bread, stiff roti and porridge. It is used for bakery products and snacks. Soil Types Pearl millet is grown on wide range of soil types, but light sandy soil is the best suited for the crop with rainfall of 350 to 700 mm per annum. Its high tolerance to drought allows pearl millet to regrow and produce tillers to compensate for losses due to drought stress thereby resulting in faster regeneration of yield of about 4000-5000 kg/ha when conditions are favorable. However, severe drought conditions result in yield reduction in the range between 500 to 600 kg/ha. In marginal areas, pearl millet is more reliable than other cereals such as sorghum and maize. In some parts of the world, pearl millet is produced in warm areas overlapping with other cereals such as sorghum, but it is less tolerant to waterlogging and flooding. Production Constraints Biotic stress: Diseases and insect pests are the major biotic factors significantly reducing grain yield and quality in pearl millet. Among bacterial diseases, bacterial spot (caused by Pseudomonas syringae) and bacterial leaf streak (caused by Xanthomonas campestris pv. pennamericanum) are the major causes of yield loss in pearl millet. Fungal diseases including downy mildew (caused by Sclerospora graminicola and Plasmopara penniseti), blast (caused by Pyricularia grisea), smut (caused by Moesziomyces penicillariae or Tolyposporium penicillariae) and rust (caused by Puccinia substriata var. penicillariae) cause more yield loss than other fungal diseases. Among insect pests, millet head miner and stalk borer cause serious problems to pearl millet plants. Parasitic weeds such as Striga hermonthica and Striga asiatica are major plant pests contributing to yield reduction in pearl millet. These two parasitic weeds are serious problems in sub-Saharan African countries. Parasitic nematodes are also major problems in pearl millet production regions. Abiotic stress: Significant yield losses can result due to abiotic stresses which include drought (in all pearl millet growing regions), high soil salinity and soil acidity, and extreme high temperature at seedling stage and during flowering. Pearl Millet Breeding Basics Pearl millet (chromosome number of 2n=2x=14) is a diploid hermaphrodite with a protogynous type of flower development. Protandry (the stigma is receptive before the anthers are ready to shed pollen) in the hermaphroditic flowers of pearl millet enhances a high rate of cross pollination (> 85% outcrossing). Breeding of pearl millet began in Asia, particularly in India, in the early 1930s with an emphasis on high yield production and productivity, while in the USA the focus was on forage and biomass yield production. In West Africa, early breeding for pearl millet started in the 1950s emphasizing increases in grain yield. The discovery of A1 cytoplasmic-nuclear male sterility (CMS) system in Tifton, Georgia, USA and initiation of breeding of a commercially viable male-sterile line (A-line) is a breakthrough for hybrid cultivar development in pearl millet and this led to the release of the first F1 grain hybrid for production in India. Male Sterility Cytoplasmic-nuclear male sterility also provides control over outcrossing, enabling the application of testcross method where a large number of inbred lines could be crossed with few, better and high general- and specific-combining ability CMS inbred lines. Breeding objectives of pearl millet at ICRISAT include: 1. high grain yield with compact head, more tillers, earliness and reduced plant height; 2. high forage yield with high biomass and good digestibility; 3. resistance to diseases, insect pests and striga; 4. tolerance to drought, heat and acid soils. Pearl millet breeding programs employ both hybrid and population improvement approaches at ICRISAT and West Africa and these methods help breeders to develop open pollinated cultivars (mainly in Africa) and hybrid cultivars (India and China). Breeding Methods Mass selection: This is the most common type of cultivar development method being used in several African and Asian countries. In this method, a group of pearl millet plants are selected from an open-pollinated population and the seeds from selected plants are mixed and planted to begin the next cycle of selection. Mass selection in pearl millet has helped to improve traits with high heritability. The main criteria that have been taken into consideration to improve grain yield in pearl millet are head characteristics such as compactness, length of ear, the weight of grain, and uniform maturity. Synthetic cultivar development: Synthetic varieties are developed in open pollinated crops by mixing several hundred elite genetic stocks/germplasm with one or more important traits in common. The synthetic cultivar developed in the first generation or cycle exhibits considerable heterosis. Hybrid breeding: The hybrid breeding program at ICRISAT and West Africa includes the development of inbred lines and pure line selection and the use of cytoplasmic male sterility. Cytoplasmic male sterility in pearl millet has been used to produce a hybrid for grain production in India and for forage production in the USA. Several sources of male-inducing cytoplasm have been discovered in pearl millet including A1, A2, A3, A4, and A5. A1 is the most commonly used male sterile line for hybrid grain production in India. The CMS system involves the development of three-line systems (A, B, and R) in order to produce hybrid seeds. Line A is male sterile and serves as the seed parent, line B has the recessive form of the fertility restorer gene in the nucleus and does not have the capacity to restore fertility in A system; it maintains sterility. The R line has the dominant form of the fertility restorer genes, and so reverses the effects of the CMS cytoplasm of the A-line, therefore resulting in fertile hybrid seeds when used as a male parent. B and R lines should be multiplied in separate and isolated fields to maintain purity. For details, refer to Crop Improvement modules 5 (Steps in Cultivar Development) and 6 (Breeding Methods).

textbooks/bio/Agriculture_and_Horticulture/Crop_Improvement_(Suza_and_Lamkey)/1.10%3A_Millet_Breeding.txt

Arti Singh; Asheesh Singh; and Anthony A. Mahama Rice is grown on all six continents in the world except Antarctica. The two cultivated rice species are Oryza glaberrima, commonly known as African rice, and Oryza sativa L., commonly known as Asian rice, which has two major subspecies (japonica and indica). Cultivated rice is a diploid species (2n=2x=24) with basic chromosome number of 12. Twenty-one different wild varieties exist. Rice is one of the most important food crops in the world and is the staple food in numerous countries, particularly in Asia. Learning Objectives • Become familiar with the rice crop • Demonstrate knowledge about the crop’s biology and classification system • Know the origin and domestication of the crop • Outline the classification of the different production systems • List breeding institutions working on the crop • Discuss the breeding methods used to develop pureline and hybrid rice cultivars Origin Rice has three distinct cultivated species and 21 different wild varieties. Information on the origin of glaberrima and the two oryza subspecies is as below: • Oryza glabberima – domesticated in West Africa between about 1500 and 800 BC or about 2,000-3,000 years ago (Linares, 2002). • Oryza sativa japonica – domesticated in central China about 7000 BC or about 8,200-13,500 years ago (Molina et al 2011; Huang et. al., 2012; Harris, D. R., 1996; Vaughan et al, 2008) • Oryza sativa indica – domesticated in the Indian subcontinent about 2500 BC (Londo et al, 2006) Major Categories Rice is grouped into four major categories worldwide: • indica is the long-grain type and is non sticky when cooked • japonica is the short-grain type which becomes sticky when cooked • aromatic is the medium to long-grain type which when cooked has nut-like aroma and taste. • glutinous is the type that is especially sticky and glue-like when cooked Domestication and Diversity Wild Ancestor of Rice The wild ancestor of cultivated rice (Oryza rufipogon) existed over a broad range of geographic regions across Asia (Fig. 1). Domestication of O. rufipogon in response to human selection resulted in complete transformation of morphological and physiological traits of the plant. Consequently, cultivated rice (O. sativa) displays reduced dormancy, grain shattering and outcrossing, and reduced loss of pigmentation in the hull and seed coat. Transformation to Cultivated Rice In addition, there is a better synchronization of tiller development and panicle formation in modern rice cultivars along with an increased number of secondary panicle branches (Fig. 2), higher grain yield and weight, and improved photoperiodic response. NERICA NERICA stands for “New Rice for Africa” developed using interspecific hybridization of O. glaberrima (African rice) and O. sativa (Indian rice) at the Africa Rice Center (WARDA) (Fig. 3). NERICA was developed for the purpose of raising the yield of African rice cultivars. Since inter-specific crosses do not result in viable seed, embryo-rescue technique was used for the production of NERICA rice. The resulting hybrid rice cultivar has a higher yield due to increased grain size, better growth and also resistance to biotic (diseases and pest) and abiotic (drought) stresses. Dr. Monty Jones won the 2004 World Food Prize for creating a rice cultivar specifically bred for the ecological and agricultural conditions in Africa. The new rice cultivar was suitable to African drylands including and is grown in Guinea, Nigeria, Côte d’Ivoire, and Uganda. Biology of the Crop General Characteristics of the Development of the Rice Plant The growth of rice plant can be divided into three developmental stages (Fig. 6): 1. vegetative (germination to panicle initiation) 2. reproductive (panicle initiation to heading); 3. grain filling and ripening or maturation (milky stage to maturity) Development Stages The vegetative phase is subdivided into three stages 1. Germination 2. Early seedling growth and 3. Tillering The reproductive phase is subdivided into four stages 1. Stem elongation 2. Panicle initiation 3. Panicle development 4. Flowering The ripening phase is subdivided into four stages 1. Milk grain 2. Dough grain 3. Mature grain In a tropical environment, approximately half of the days of growth (from seeding to harvest) are in vegetative phase, and one-quarter each in the vegetative phase and the ripening phase. Seed Development In 2000, Counce et al., proposed a rice developmental staging system divided into three main phases of development: 1. Seedling 2. Vegetative 3. Reproductive and Ripening The sequence of normally occurring seedling developmental events is presented, noting that there are exceptions to the sequence given. Seedling development can be further divided into four stages (Fig. 7): 1. Unimbibed seed (S0) 2. Coleoptile emergence (S1) 3. Radicle emergence (S2) 4. Prophyll emergence from the coleoptile (S3) Vegetative Development Vegetative development consists of V1, V2…… VN; where N is equal to the final number of leaves with collars on the main stem (Fig. 8). Reproductive Development Reproductive development consists of 10 growth stages based on distinct morphological measures as shown in Table 9. Table 9. Rice reproductive growth stages. R0 Panicle development has initiated R1 Panicle branches have formed R2 Flag leaf collar formation R3 Panicle exertion from boot, tip of panicle above collar of flag leaf R4 R5 R6 R7 One or more florets on the main stem panicle has reached anthesis At least one caryopsis on the main stem panicle is elongating to the end of the hull At least one caryopsis on the main stem panicle has elongated to the end of the hull At least one grain on the main stem panicle has a yellow hull R8 R9 At least one grain on the main stem panicle has a brown hull. The brown hull indicates the grain has begun to dry. All grains which reached R6 have brown hulls Further Characteristics The grain yield of rice is comprised of the following four components: • Number of panicles/m2 • Number of grains / panicle • Percentage of ripened grains • 1000 gm weight. However, plot yield weight remains the best way to determine the yield of lines or genotypes. Photosynthesis Rice is a C3 plant. C3 photosynthetic pathway is not very efficient at transforming inputs to grain, in comparison to the C4 pathway. In order to increase rice yield there are ongoing efforts on development of C4 rice to create a new type of rice with enhanced photosynthetic capacity (Susanne von Caemmerer et al, 2012). The effort to develop C4 rice is worth it as rice is the staple food source in many Asian countries like India, China and Japan, and also it is grown in places where maize cannot be grown. Photoperiod and Temperature Oryza sativa is classified as a short-day plant (i.e., requires long nights to flower). This means that heading date (the number of days it takes for the panicle to begin to exert from the boot, that is, the flag leaf sheath) is accelerated under short-day conditions, while heading date is delays when long-day conditions exist (Garner and Allard, 1920). General Classification of Rice Production Systems According to the International Rice Research Institute (IRRI), rice can be classified into four major production ecosystems (Fig. 10): 1. Irrigated rice – Rice is grown in well watered condition and is flooded throughout the rice growing season. 2. Rainfed lowland rice – Rice grown under this condition is dependent on rainfall only, and land is prepared such that it preserves the rain water. 3. Upland rice – Rice grown without irrigation water and relies completely on rainfall. 4. Flood-prone rice – Rice grown in river areas is deep water rice, with no inbuilt water control system. Table 1 Total production area and total rice production (%) in three Ecosystems, 2009. Rice Ecosystem Total Production area (%) Total rice production (%) Irrigated lowland 55-60 ≈ 75 Rainfed lowland ≈ 30 ≈ 20 Rainfed upland ≈ 10 < 5 Diversity There is huge diversity in Oryza species for shape, color, size as can be seen in Fig. 11. Rice production totals in different regions of the world is shown in Fig 12. Adaptation, Economic Importance and Uses Processing The procedure of milling and consequently polishing rice, results in the highly valued white rice which removes nearly all the outer layers and germ and leaves a product deficient in thiamine. Through fortification and parboiling, adequate quantities of thiamine and other B vitamins can be retained in rice. Parboiling is usually done in the mill where unhusked rice is generally steamed, so that water is absorbed by the whole grain providing an even distribution of vitamins in the whole grain. However, in conventional methods, the paddy is dried and dehusked prior to milling. International Breeding Centers The Consultative Group on International Agricultural Research (CGIAR) has three centers: • The International Rice Research Institute (IRRI) has a global mandate to work on rice and its headquarters is in Los Baños, Laguna in the Philippines. • The West Africa Rice Development Association (WARDA) has mandate to work on rice in West Africa. • The International Centre for Tropical Agriculture (CIAT) has the regional mandate to work on rice in Latin America. Breeding Methods Heterosis Heterosis refers to the superiority of the F1 hybrids resulting from a cross of diverse parents, over their parents in performance of desired traits, for example, vigor, yield, number of productive tillers, panicle size, number of spikelets per panicle. The crosses (×) between rice subspecies showing heterosis in decreasing order are as follows (Fig. 14): Types of Heterosis Intrasubspecific heterosis: It is the most commonly used heterosis which provides around 15% to 20% more yield than the best check grown under similar conditions. For example 1. indica × indica 2. japonica × japonica Intersubspecific heterosis:Oryza sativa ssp. indica and ssp. japonica are two of the most common subspecies of cultivated rice. The cross between these two subspecies shows maximum heterosis in the F1 hybrid. Major limitation in intersubspecific heterosis is the high spikelet sterility and long growth duration. The discovery of wide compatibility (WC) genes has provided a solution to overcome these problems allowing the utilization of these type of crosses. Interspecific heterosis: The crosses in cultivated species refer to only O. sativa and O. glaberrima. However, heterosis of yield is very high and plant stature remains a problem. In rice, the interspecific F1 hybrids cannot be used commercially. Examples of interspecific hybrids as a result of wide hybridization, generates genetic variability and bring together several biotic and abiotic stress resistance genes. 1. O. sativa × O. longistaminata 2. O. sativa × O. rufipogon 3. O. sativa × O. perennis Hybrid Rice For details on hybrid rice research at public and private commercial sectors, refer to the following links: The commercial rice crop grown as a hybrid crop is an F1 hybrid developed from the cross of two genetically diverse pureline parents. Good rice hybrids have the potential to yield 15-20% higher than the best pureline cultivar when these two (hybrid and pureline parents) are grown under similar conditions. Since rice is a self-pollinated crop, the male sterility system has been used to develop commercial rice hybrids. Commercial companies are more interested in developing hybrid cultivars because of the profits accrued from farmers returning each year to buy new seed. The higher cost of hybrid seed is partly due to the increased cost of development of the parents used to make the hybrids. Male Sterility in Rice Male sterility is defined as the inability of a plant to produce functional pollen grains. The use of male sterility in hybrid seed production has a great importance as it eliminates the process of mechanical emasculation. Three forms of male sterility that can be used are: 1. Cytoplasmic genetic male sterility (CGMS) 2. Environment-sensitive genic male sterility (EGMS) EGMS is classified in the following categories 1. TGMS: temperature-sensitive genetic male sterility 2. rTGMS: reverse temperature-sensitive genetic male sterility 3. PGMS: photoperiod-sensitive genetic male sterility 4. rPGMS: reverse photoperiod-sensitive genetic male sterility 5. PTGMS: photothermosensitive genetic male sterility 3. Chemically induced male sterility (CIMS) Cytoplasmic Genetic Male Sterility In CGMS, three lines are involved in hybrid rice development and the process flow is as follows (Fig. 15): 1. Cytoplasmic male sterile line (A line) – The male sterility is controlled by the interaction of sterile cytoplasm (S) and fertility-restoring genes (rf) present in the recessive form in the nucleus. 2. Cytoplasmic male fertile line also known as maintainer (B line) – is iso-cytoplasmic to the CMS A-line since it is similar to it for nuclear genes but differs as it has normal cytoplasmic factor (N). A-lines are developed from B-lines using backcross breeding to transfer the CMS gene. The N gene makes the B-line self-fertile, and is used in crossing with the sterile A-line to maintain A-line seed production. 3. Restorer line (R line) – R line possesses dominant fertility-restoring genes (Rf), and so is different from the A line and B-line; the restorer line is developed separately to maintain genetic dissimilarity from A-line for expression of heterosis. The restorer gene in the dominant homozygous (RfRf) or heterozygous (Rfrf) state has the ability to restore the fertility in the F1 hybrid. Hybrid Seed Production Using CGMS Hybrid seed production involves two steps: 1. CMS line (A line) multiplication (AxB): The seed of the CMS line (A line) is multiplied by crossing with the maintainer line (B line) either by hand (in plant breeding programs where a small quantity of seed is required) or in the field under isolation by space or time (to produce breeder seed for commercial seed production). Remember that the A-line is only a parent in hybrid production and it is not for commercial seed sale for farmer production. Seed companies may be interested to obtain marketing rights of A-line for their hybrid breeding program). Generally, A-line seed production field consists of 6 or 8 rows of A-lines alternating with 2 rows of B-lines. This pattern is repeated throughout the field as depicted below (6 rows of A-lines, alternating with two rows of B-lines and repeating pattern in field). … B B A A A A A A B B A A A A A A B B A A A A A A B B … 2. Hybrid seed production (AxR): Hybrid seed is produced by crossing the A line with the R line in isolation. The hybrid seed is sold to farmers for commercial production so a large increase of parent seed and hence hybrid seed is necessary. More than one location may be planted to make the hybrid seed to minimize the impact of loss of field due to an environmental event or other causes. In the field, 8-10 rows of A-lines are grown interspersed with two rows of R-line to produce hybrid seed. … R R A A A A A A A A R R A A A A A A A A R R A A A A A A A A R R … Additional Information For Hybrid Seed Production • The planting dates may need to be staggered to achieve synchronized flowering of the two parents. • For better pollen dispersal (from male parent) and seed set (on female parent), ropes or sticks are often used. • Hormone treatment, such as Gibberellic acid (GA), can increase the receptivity of female to accept pollen. This happens due to better emergence of female panicles from the sheath, exposing the ovary to male pollen. Hybrid Rice Breeding using EGMS In the two-line method, the male sterile line is (male sterility controlled by a recessive gene) crossed to a pureline that is male fertile (i.e. it possesses the dominant gene for sterility). Male sterility in the female line is genetically controlled by recessive genes and sterility expression is influenced by environment (temperature, photoperiod, or both), and the male parent is selected to be good pureline pollen producer. Classification of the EGMS System Types of EGMS Systems Used in Two-Line Hybrid Rice Breeding 1. Thermo-sensitive genetic male sterility (TGMS) – In TGMS lines, the sterility or fertility expression is controlled by temperature. Regardless of photoperiod, TGMS lines are usually highly sterile under high temperature and highly fertile under low temperature. The first TGMS line was reported by Japanese Scientist in the rice variety Remei where gamma ray induced mutation resulted in sterility (31-24ºC) to partial fertility (28-21ºC) and complete fertility (25-15ºC). 2. Photoperiod-sensitive genetic male sterility (PGMS) – In PGMS lines, the sterility or fertility expression is controlled by daylength. Under long-day conditions, most PGMS lines remain male sterile. Under short-day conditions, they revert back to being fertile. The first spontaneous PGMS mutant, Nongken 58S (NK58S), was reported in 1973 from the japonica (O. sativa ssp. japonica) cultivar Nongken 58 (NK58). NK58S retained male sterility under long day length (longer than 13.75 h) during anther development, while under short day length (less than 13.5 h), partial or complete male fertility was observed. Temperature response was also observed for this line. Under long-day conditions at high temperatures (~29°C) slightly more male sterility was observed. 3. Reverse photoperiod-sensitive genic male sterility (rPGMS) – PGMS lines express sterility under short day length and under long day length revert to being fertile. This system is known as reverse PGMS (rPGMS). 4. Photo-thermosensitive genetic male sterility (PTGMS) – PTGMS lines are sensitive to both photoperiod and temperature. Temperature is the important factor since PTGMS lines become completely male sterile or fertile beyond (over or under) a threshold temperature range, without any influence of photoperiod. In this system the effect of temperature and photoperiod is difficult to separate and under natural conditions both factors interact to determine sterility or fertility. Advantages of EGMS Systems 1. There is no requirement for seed multiplication of a maintainer line, therefore making the seed production system cheaper. 2. No need for backcross breeding to develop a CMS A-line from B-lines. 3. Hybrid breeding efficiency is higher in two-line breeding than three-line breeding since it allows use of any fertile line as a pollen source parent. 4. Undesirable effects of sterility-inducing cytoplasm do not occur. 5. It is ideal for developing indica by japonica hybrids as there is no requirement for restorer lines. Disadvantages of the EGMS Systems 1. The sterility trait is under the control of environmental factors, and any variation such as temperature fluctuation because of a storm, rain etc., will impact the sterility of EGMS lines. 2. Seed production can be done in the latitudes with optimal photoperiod length, therefore limiting options in some cases for which locations can be used. The seed multiplication (lines and hybrids) are constrained by space and season. Conventional Rice Breeding Program In conventional breeding, and for rice programs as well, two parents are crossed and segregating generations are screened for the trait of interest, for example, disease resistance, maturity, height and protein. Uniform lines are tested for yield and along with resistance, desirable varieties are selected and released. The development process from making the initial cross to variety release is shown in Fig. 18.

textbooks/bio/Agriculture_and_Horticulture/Crop_Improvement_(Suza_and_Lamkey)/1.11%3A_Rice_Breeding.txt

Teshale Mamo; Asheesh Singh; and Anthony A. Mahama Sorghum (Sorghum bicolor L. Moench) has historically been a major staple food source globally, and is currently ranked the fifth most important cereal. Recently, it has become a multipurpose crop produced not only for food but for feed, fuel and forage, and being bred for use as a cover crop in pastures, through varieties with compacted internodes. Sorghum is serving as a vital model for tropical grass species for functional genetics and genomic studies, made possible by the availability of genomes of three sorghum lines and numerous genetic stocks and populations. It is therefore a crop of immense importance in tackling current global food security challenges. Learning Objectives • Students become familiar with the Sorghum crop • Know crop biology and classification system • Describe adaptation and usage • Outline production constraints • List breeding institutions working on the crop • Discuss breeding methods used to develop sorghum cultivars Origin, Domestication, and Diversification Sorghum (Sorghum bicolor (L.) Moench) is an ancient crop that originated in North Eastern Africa. These places are also areas where greatest diversity of wild and cultivated species of sorghum are found to this day (Fig. 1). Domestication of sorghum probably took place in Ethiopia and some parts of Congo by selecting wild sorghum, approximately 5,000 years ago. India, Sudan and Nigeria are considered as secondary centers of origin. From these centers of origin, sorghum was probably distributed to other parts of the world (Acquaah, 2007). This early distribution and introduction of the crop helped generate further genetic diversity in other continents, such as Asia. The genus Sorghum has greatest genetic diversity ranging from 20 to 30 species. Cultivated sorghum along with the two perennial species [Sorghum halepense L (2n=40-forage sorghum) and Sorghum propinquum (Kunth)] are included in the genus sorghum. Based on morphological classification, all cultivated sorghums (Sorghum bicolor spp.) are grouped in five races along with ten intermediate races. The five races are: • durra, • kafire, • guinea, • bicolor, and • caudatum. Most of these races differ mainly in their panicle morphology, grain size and yield potential. Durra type of sorghum originated primarily in Ethiopia and the horn of Africa, and then spread to Nigeria and other parts of West Africa where it became popular. Kafir types of sorghum developed in the eastern and southern parts of Africa where they grow well. Guinea types developed in West and Central Africa and grow well in that region, while the bicolor type originated in East Africa but is less important to African production. Biology of the Crop General Characteristics of the Development of the Sorghum Plant Sorghum is an annual grass, and belongs to the graminae family. It reaches up to 5 m in height with one to several tillers, and these tillers emerge first from the base of the plant and sometimes later from the stem nodes. The tillers on stem notes form when growing conditions are favorable. These tillers form on upper or lower nodes and are undesirable because they form later and produce a small amount of grain that is unripe by harvest with higher moisture content. This can cause delayed harvest, as well as problems in storage, delivery and sales. Lower plant density (i.e. sparse planting) causes more tillering and higher plant density in field planting suppresses tillering. Tillering is suppressed when growing conditions are unfavorable. Optimum temperature for germination ranges from 27-35oC, and after germination the plant goes through root and leaves development rapidly. Sorghum has a fibrous root system which is mostly concentrated in the top 90 cm of the soil, but root growth can extend twice that depth under dry environments. Sorghum leaves are alternate with the leaf sheath and ranges from 15-35 cm in length. Total number of leaves on the plant varies between 7 to 24 depending on the variety and environmental conditions. Sorghum leaves have rows of motor cells along the midrib on the upper surface of the leaf which is unique characteristic of sorghum leaves as these cells can help the leaves to roll up rapidly during drought stress to minimize water loss from the leaves. In addition, morphological and physiological characteristics of sorghum such as extensive root system, wax on the leaves (minimize water loss) and the ability to stop growth during moisture stress and resume growth when moisture levels increase (from rain) are inherent characteristics of sorghum to adapt to drought conditions. Growth Stages The inflorescence or head of sorghum is called panicle that may be loose or dense. Under favorable conditions, initiation of panicle takes place after one third of the growth cycle. Each fully developed panicle can contain 800 to 3000 grains, each one usually enclosed by glumes. The color of the seed is variable. Sorghum flowers usually open during the night or early in the morning with those flowers at the top of the panicle opening first, and it takes 6 to 9 days for the whole panicle to flower. Sorghum is a self pollinated crop due to its flower structure but cross pollination (approximately 2-25 %) occurs naturally. In general, once the sorghum seedling emerges, the plant goes through three distinct growth stages represented as growth stage I, II and III. The first growth stage (GS I) is recognized as vegetative growth. During this stage, the plant develops leaves, internodes and tillers. This stage helps the plant to prepare for grain formation and growth. At this stage the plant can tolerate drought stress, heat and freezing temperatures. The second growth stage (GS II) is the reproductive phase in which the panicle is developed and maximum number of seeds per plant are set. This growth stage starts with panicle initiation and it continues to flowering. It is reported that it is the most critical period that determines the level of grain production. This is the stage when the crop’s water requirement is high. Hence if severe moisture stress occurs at this stage, panicle initiation is hindered or delayed, leading to incomplete flowering, seed set and loss in grain yield. The third growth (GS III) is the grain filling period which starts with flowering and continues until the grain is filled with dry matter. Photosynthesis, Photoperiod, and Temperature Sorghum is one of the C4 grasses with high photosynthetic efficiency. It is a short day plant requiring long nights before flower initiation (start of reproductive stage) (Craufurd et al., 1999). The optimum photoperiod for flower initiation ranges from 10 to 11 hours and a photoperiod beyond 12 hours can stimulate vegetative growth. Tropical cultivars are more photoperiod sensitive than short-season sorghum cultivars (quick mature). Sorghum is a dry land crop requiring high temperature ranges from 27 to 300C for its growth and development (Craufurd et al., 1999). Increased day and night temperature beyond plant requirements can delay flower initiation and development of flower primordia, and this reduces yield. The sorghum plant can tolerate a temperature as low as 21oC without significant effect on growth and yield. General Classification Classification by Utilization or Mode of Consumption Grain sorghum is the most widely cultivated type of sorghum in the world, and it is the main staple food in dry land (semi-arid tropical region) areas of Africa and Asia. It is an important part of diet that is prepared in the form of boiled porridge, unleavened bread (pancake), popped (like maize), dumplings, beers and non-alcoholic fermented beverages. Sorghum grain is also used as animal feed, and the stems and leaves are used as green chopped animal feed, hay and pasture feed. It is grown as grain and fodder crop in the USA, Europe and Australia (Berenji and Dahlberg, 2004). Human Food In Africa and Asia, many people consume sorghum grain in unfermented and fermented pancake (breads), porridges, dumplings, snacks, and malted alcoholic and nonalcoholic beverages. White grain sorghum is mostly preferred for cooking while red and brown grain sorghum are preferred for beer making. In some parts of Africa, e.g., around Lake Victoria, where bird pressure is high, farmers may grow red and brown grain sorghum instead of white grain types, because these types of sorghum are rich in tannin and are bitter tasting thus preventing bird feeding and associated losses. In the USA, sorghum is primarily grown as a fuel crop (for ethanol production) and there are few food products available to consumers; however several researchers have developed and introduced products from sorghum into the food market. In addition, several researchers have been working on health benefits associated with sorghum grain that might increase its use in the health food industry. For example, food products made from sorghum grain did not show toxicity to celiac patients (Ciacci et al., 2007), and several gluten-free sorghum products have been developed and are being popularized (Schober et al., 2005). Animal Feed In the United State, Central and Southern America, Europe, Australia and China, sorghum grain is mainly used as cattle, pig and chicken feeds. Similar to the use of silage corn, the sweet sorghum type is also used as cattle feed in Europe. The problem with sorghum as cattle feed is the presence of prussic acid (HCN) which causes death in cattle if the animal consumes fresh sweet sorghum. This problem is eliminated through cultivar choice and proper agronomic practices. Renewable Energy Sorghum is one of the crops that can be used for production of renewable fuels in temperate regions. It is unique among grasses in being used as feed stocks for renewable energy because it can be used in various forms for biofuel production. Starch and sugar are converted to ethanol, and lignocellulose (composed mainly of cellulose, hemicellulose and lignin – inedible parts of the plant) is converted to biogas, making sweet sorghum a unique biofuel crop that is also used as food and fodder. Classification of Grain Sorghum by Intended Purpose Sorghum is classified into four major groups based on the applications. 1. Grain sorghum: this group is used as staple food in the tropical areas of Africa and Asia and is often used as raw materials for making alcoholic beverages, sweets and glucose. 2. Sweet sorghum: this group is mainly produced for sugar production. This sugar is used as material to produce sweet syrup, which is similar to molasses. 3. Broom sorghum: this is recognized by long panicles with fine, elastic branches called fibers with the seed on their tip which is used as material to making brooms. 4. Grass sorghum: this is mainly grown for green feed and forage purpose. Classification of Grain Sorghum by Agronomic Groups Commercial grain sorghum is classified into seven groups. 1. Kafir sorghum: this group of sorghum is originally from South Africa. In this group, the stalk is thick and juicy, have large leaves, and the panicles are cylindrical in shape without awn. The seed are medium in size, color could be white, pink or red. 2. Milo sorghum: came from east Africa, has short, compact and oval panicles, with less juicy stems than kafir, and has light green leaves. The seed size is relatively large with either yellow or white seed color. The plant in this group has more tillers than kafir. The varieties in this group are more heat and drought tolerant than kafir. 3. Hegari sorghum: came from Sudan and is similar to kafir but has more nearly oval panicles. Plants in this group have more leaves than kafir, and the grain produces a sweeter juice which is desirable for forage. Seeds are chalky white in color. 4. Feterita sorghum: is originally from Sudan. Plants have few leaves and dry stems. Panicles are oval compact shape. Seeds are large in size and chalky white in color. 5. Dura sorghum: came from Mediterranean and Middle East regions. Plants have dry stems, flat seeds with pubescent glumes. The panicles in this group are erect but compact or loose. The varieties in this group are mainly grown in North Africa, India and the Near East. 6. Shallu sorghum: This group is from India, and plants have tall, slender and dry stalks. Panicles are loose, and seed color is pearly white. The varieties in this group are late maturing, thus requiring a relatively long growing period. 7. Kaoliang sorghum: This group is mainly grown in China and Japan. The varieties have slender, dry and woody stalks with sparse leaves. They have an open semi compact panicle. Seeds are small in size and white or brown in color and are bitter in taste. Adaption and Economic Importance and Uses Adaption Sorghum is a small cereal crop adapted to wide range of environmental conditions, but is particularly adapted to a warm weather. Sorghum is mainly grown between 40oN and 40oS in arid, semi-arid tropics and subtropics, and it can also be grown at an altitude of up to 2300 m above sea level in the tropics with annual rainfall ranges from 300 -1200 mm. It is also widely grown in temperate regions mainly in South China and USA and some parts of Europe. Cold tolerant sorghums are also grown successfully in Central America. Sorghum is a short day plant requiring 90 to 140 days to mature depending on climate and type of cultivar. Its genetic variation in response to photoperiod and temperature contribute to its adaptation to the wide range of growing environments. Sorghum’s most outstanding characteristics are its heat and drought tolerance and it can also be grown on a wide range of soil types from vertisol (clay soil) in the tropics to light sandy soil. The soil pH requirement ranges from neutral to high pH (5.0 to 8.5) and it is tolerant to salinity compared with corn. Sorghum can be grown in poor soil and can produce grain where other crops fail to produce fruit. Cropping System Intercropping is a cropping system involving the growing of two or more crops in the same space and at the same time. It is a common practice among small scale farmers in the semi-arid areas of Africa and Asia in order to increase productivity per unit area of the land (Kidane et al., 1989). Sorghum is one of the important cereal crops being used for intercropping. It is commonly intercropped with a legume crop such as sorghum-chickpea, sorghum-common bean, sorghum-pigeon pea, sorghum-cowpea and sorghum-mung bean. It is also intercropped with other cereals such as sorghum-millet and maize-sorghum. Several researchers have reported that a significant yield was obtained from intercropping compared to pure stands. Sorghum yield was increased 8-34% in a sorghum-legume intercropping system compared to sole sorghum crop stand (Singh, 1977). In Ethiopia, sorghum-mung bean intercropping gave extra yield of 495 kg/ha of sorghum compared to sole sorghum crop (Kidane et al., 1989). Striga infestation on sorghum was reduced when mung bean was intercropped with sorghum. In general, intercropping of sorghum with legumes has a benefit in that the legume crop allows efficient use of both space and time to optimize effects (increased land productivity). Intercropping promotes diversification of crops so that the farmers can harvest two or more different crops from the same piece of land, and it provides better weed control and reduces diseases and pests incidence. Production Constraints Biotic Constraints Diseases and pests are the main causes of significant yield losses in sorghum. The fungal disease Smut, caused by Sphacelotheca spp., may cause more yield losses than other fungal diseases and is widely important in the eastern, central and southern parts of Africa where sorghum is used as major staple food. The different types of smut are: loose smut, kernel smut, head smut and long smut. They are controlled by seed treatment with fungicides. Through breeding, use of resistant cultivars provide protection against this disease. Rust, which is caused by Puccunia purpurea, is another important fungal disease and is widely distributed in many parts of sorghum producing regions particularly in Africa. Grain mold is caused by several fungi, Curvularia lunata, Fusariumspp, and anthracnose (Colletotrichum graminicola) are the most important sorghum diseases that infect the grain during grain development and can cause severe discoloration of grain and loss of seed quality. Continued rainfall throughout grain maturity period increases the occurrence of grain mold and causes delayed harvest. Grain mold control measures include the use of resistant cultivars and adjustment of planting time to avoid long maturation period during prolonged rainy season. Downy mildew, ergot and bacterial streak are occasional important constraints. Sorghum insect pests important in tropical Africa are stem borer (particularly, Busseola fusca and Chilo partellus) and shoot fly (Antherigona soccata). Yield losses due to these insect pests are significantly high and the problem is widespread in major sorghum producing African countries. Other biotic constraints such as Striga, weeds, and quelea are also considered as major production contains. Striga is rated as causing high yield losses in all regions in Africa. In some countries, the yield losses were estimated to be more than 50%, particularly in Rwanda and Kenya (Wortman et al. 2009). Abiotic Constraints Abiotic stresses such as extreme drought (in all sorghum growing regions), saline soil (some parts of India and Middle East countries) and acidic soil (mostly in Latin America) are major production constraints. International Breeding Center Collections Diversity International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), a member of the Consultative Group on International Agricultural Research (CGIAR), based at Patancheru, India was established in 1972 with sorghum as one of its five mandate crops. A total of 36,774 accessions have been collected from 90 countries (Reddy et al., 2008) and maintained in a gene bank, and these collections exhibit 80% of the diversity present in the crop. ICRISAT coordinates all sorghum research programs in the semi-arid (dry land) tropics of the world. It has strong collaboration and active breeding programs covering over 55 countries in Asia and sub-Saharan Africa with a mission to reduce poverty, hunger, and environmental degradation in the dry land areas of the tropics. ICRISAT has been addressing national, regional and global concerns for sorghum improvement through developing sorghum cultivars with genetic resistance to major diseases and insect pests. ICRISAT is also developing intermediate breeding products such as a wide range of male sterile lines that are widely used by public and private breeding centers for hybrid cultivar development. ICRISAT is more involved in diversification of sorghum breeding populations through incorporation of major abiotic and biotic resistance traits that have not previously been used in sorghum improvement programs. The traits that are currently being given emphasis at the global level include tolerance to drought, heat, Aluminum toxicity, salt, head and stem pests, and grain molds. Earliness with high grain and biomass yield, and tillering capacity are also emphasized. Additional Collections Locations In addition to ICRISAT, large sorghum collections are held in temperate regions including USA (National seed storage lab) and China. Similarly, in tropical Africa, large sorghum germplasm collections are held in Zimbabwe (SADC/ICRISAT sorghum and millet improvement program, Matopos), Ethiopia (Institute of Biodiversity Conservation, Addis Ababa), Kenya (National gene bank, Crop Plant Genetic Center, KARI) and Uganda (Serere Agricultural and Animal Production Research Institute). All these accessions are a valuable genetic resource for further germplasm development efforts. Breeding Methods and Strategies Breeding Opportunities and Objectives Sorghum (2n=2x=20) is predominantly a self-pollinated crop with out-crossing ranging from 2 to 25 %. It has a small genome size (730 Mbp) compared to maize or sugar cane. Sorghum genome is a fully sequenced and provides many useful opportunities to plant breeders and genomics researchers. Breeding objectives of sorghum include: high grain and fodder yield potential, resistance to diseases (smut, rust, grain mold, bacterial blight, anthracnose, and downy mildew etc.), resistance to insect pests (stalk borer, shoot fly, and midge), resistance to drought and extremely acidic soil, wide adaptation and improved quality (for use in bread, porridge, snacks, and beverages). In sorghum breeding programs, breeders are developing two kinds of cultivars: 1) open pollinated (OP) or pure line cultivars (mainly for developing countries), and 2) hybrid cultivars (mainly for industrialized countries where the seed system are well developed). The breeding methods for open pollinated variety (OPV) is different from pure-line or hybrid cultivar development. Recurrent selection schemes are used for OPV, while breeding methods that we learned for self-pollinating crops are used to develop pure-lines. Hybrid development programs will also use pure-lines, however, they use three different kinds of pure-lines: A-line (cms line), B line (maintainer line) and R-line (restorer line) details presented in Breeding Methods module. Open Pollinated and Pure Line Cultivars – Development Methods 1. Population improvement: This is the most common type of breeding method being used in developing countries (Africa), and it includes a group of sorghum plants sharing a common gene pool. Sorghum population improvement program is mainly used for developing broad-based gene pools through recurrent selection methods. In population improvement, the recurrent selection methods are the most useful methods for improving quantitatively inherited traits by increasing frequency of genes that effect trait/traits under selection and to maintain genetic variability by recombining superior genotypes for further and continuous improvement. The method of population improvement is grouped into Intra-population improvement (practiced within specific population for its improvement), and Inter-population improvement methods (selection is based on the intercross performance between two populations). The most convenient population improvement methods in sorghum are mass selection, S1, and S2 progeny testing (ICRISAT annual report, 2010). Details of these methods are covered in the chapter on Breeding Methods. 2. Pedigree method (or another method applicable to self-pollinated crops): In this method, sorghum breeders are hybridizing between desirable complementary parental lines (Fig. 2), followed by selection of desirable plants from segregating populations until homozygosity is achieved. It is applicable for improving specific trait such as disease and insect pest resistance, plant height, early maturity, etc. These methods will lead to the development of pureline cultivars. Note: This method is used to develop B-line and R-lines for Hybrid development and production programs. 1. Backcross breeding: Backcross breeding in sorghum is used to transfer favorable single or few genes including resistance to diseases (grain mold, rust and smut), and resistance to insect pests (stalk borer and shoot fly) from donor genotype, which generally has poor agronomic performance, into elite genotype (recipient). Note: This method is used to develop A-line version of B-lines for Hybrid development and production programs. Hybrid Cultivar Development (Hybrid Breeding) This method of hybrid cultivar development in sorghum closely resembles that of hybrid corn breeding. The two major differences are that (a) heterotic groups are not well defined in sorghum as in maize, so groups are based on fertility restorer genes, however more recently, reproductive groups are emerging with the differentiation and use of nuclear fertility genes. The other difference (b) is that sorghum utilizes cytoplasmic male sterility system (3-line system) to facilitate hybrid seed production unlike maize where manual detasseling, which works very well is employed (detassel female inbred line and allow male inbred line pollen to pollinate) to create hybrid seed on female ears. Briefly, a plant breeder will develop B-line (maintainer) and R-line (restorer) under two separate reproductive groups (to maximize heterosis) using pedigree or Single Seed Descent approach or any other method suitable for a self-pollinating crop. Once new and superior B-lines are developed, backcross breeding is used to convert them to A-lines (CMS lines). As the backcross breeding program continues, general combining ability (GCA) or specific combining ability (SCA) may be assessed to decide which B-line conversion to continue and also to generate information on suitable R line parent in combination. The A-line is cytoplasmic male sterile and serves as the seed bearing parent. The B-line has recessive form of fertility restorer gene and is used as a maintainer for the A-line. The R line has the dominant form of fertility restorer gene in the nucleus and has the capacity to restore fertility in the A system and it is used as the pollen parent. For details, see the Breeding Methods e-module for diagram on fertility and restorer genes in cytoplasm and nucleus, respectively.

textbooks/bio/Agriculture_and_Horticulture/Crop_Improvement_(Suza_and_Lamkey)/1.12%3A_Sorghum_Breeding.txt

Jessica Barb and Anthony A. Mahama Sweetpotato (Ipomoea batatas (L.) Lam., Convovulaceae) is a hexaploid species (2n = 6x = 90) that originated in both Central and South America. Recent evidence (Roullier et al. 2013) suggests that sweetpotato evolved from at least two autopolyploid events involving distinct populations of I. trifida or a now extinct species that was an ancestor to both I. batatas and I. trifida, which is similar to extant populations of wild tetraploid Ipomoea. The primary center of diversity for sweetpotato includes Central and South America (Huang and Sun, 2000; Zhang et al. 2000). Uganda in East Africa (Austin, 1987) (Yada et al 2010) and the region including Papua New Guinea (Fajardo et al. 2002) are both considered secondary centers of diversity. Learning Objectives • Become familiar with the basic biology and importance of sweetpotato • Provide examples of breeding schemes used to develop cultivars of sweetpotato Importance Sweetpotato is a tremendously important low-input crop in Sub-Saharan Africa (SSA) that grows well without fertilizers and limited water and requires little care other than occasional hand weeding. In 2013 sweetpotato was the sixth most important food crop-based on total food crop production after cassava, maize, yams, rice, and sorghum, and 10th based on harvested land area (FAOSTAT). Usage Sweetpotato is grown for its enlarged storage roots and leaves that are harvested for human consumption (i.e., table stock), for animal feed, as a source of starch, and for industrial purposes. It is an important food security crop in African countries, especially in rural areas. Sweetpotato is a hardy crop that can provide reasonable yield in a variety of different environments and requires less external inputs (i.e., fertilizers, water) than most grain crops. Sweetpotato is a useful crop for subsistence purposes because it can be planted and harvested throughout the year in many locations, and a farmer can “store” roots in the ground and harvest the roots as needed. Roots are boiled, baked, fried, and included in a variety of dishes. Roots can also be dried and pulverized to make flour. Sweetpotatoes are extremely variable in size, shape, color, moisture content, and carbohydrate content. Most consumers in SSA prefer the white-fleshed high dry matter types, but efforts are underway to develop orange-fleshed sweetpotato (OFSP) varieties that are both high in bioavailable beta-carotene and dry matter content to alleviate Vitamin A deficiency, especially in children. Sweetpotato leaves and petioles are good sources of protein, fiber, and minerals, especially K, P, Ca, Mg, Fe, Mn, and Cu, and when consumed could prevent malnutrition in developing countries. Purple fleshed types are also available that are usually high in dry matter and have a low level of sweetness. Because sweetpotato can be planted throughout the year and there is a large range in maturity dates, farmers can manage the supply period and ensure continual yield, both for home consumption and for the local market. In most countries in SSA roots are available 4-8 months out of a year and in countries with two rainy seasons (i.e., Rwanda, Burundi, and Uganda) roots are available 11 months of the year. In most SSA countries except South Africa sweetpotato is grown primarily by smallholder producers who often plant a mix of different varieties in the same field, which is typically rainfed. Important Diseases and Pests Infected by Several Different Viruses Sweetpotato is clonally propagated via vines, root slips (i.e., sprouts), or storage roots therefore, it is often infected by several different viruses. Durable resistance to viruses typically requires a combination of different traits that reduce the severity of symptoms, increase the tolerance of the variety in the field (i.e., decent yield despite the presence of the virus and visual symptoms), and allow the plant to recover from symptoms and revert to a virus free condition after being infected (Mwanga et al. 2013). Unfortunately degeneration of clean material often occurs very rapidly (i.e., within weeks to a year) (Gibson and Kreuze 2014) and yield losses are common. Sweetpotato growing in humid, tropical, low- and mid-elevation regions of Eastern and Central Africa are mostly affected by Sweet potato chlorotic stunt virus (SPCSV) and Sweet potato virus disease (SPVD) which is caused by co-infection of Sweet potato chlorotic stunt virus (SPCSV) and Sweet potato feathery mottle virus (SPFMV). SPCSV is more detrimental than SPFMV and typically results in permanent symptoms and yield losses. SPCSV is transmitted by white flies. SPFMV is of less concern because infection of just this single virus only produces transient symptoms and very little loss in yield. This virus is transmitted by aphids. Resistance to SPCSV appears to be conferred by a recessive allele, which occurs in low frequency in the sweetpotato gene pool. However, this resistance still needs to be proven in regions where virus pressure is highest. SPVD (SPCSV + SPFMV) causes devastating yield losses in regions of high humidity though some varieties (e.g., NAPSPOT 11 and Tanzania) are reported to possess some levels of resistance. Other viruses that are less prevalent include: Sweet potato mild mottle virus (SPMMV), Sweet potato latent virus (SPLV), and Sweet potato virus G (SPVG). Diseases and Pests in Africa Sweetpotato growing in the humid, tropical highland regions of Eastern and Central Africa are affected by SPVD but are more affected by Alternaria stem blight which is the dominant disease of sweetpotato in this region. Other diseases that cause problems to a lesser extent throughout Africa include: scurf (caused by Monilochaetes infuscans), foot rot (caused by Plenodomus destruens), chlorotic leaf distortion (caused by Fusarium denticulatum), and Rhizopus soft rot (caused by Rhizopus stolonifer and R. arrhizus) Two of the major pest groups that cause considerable damage to sweetpotato are plant-parasitic nematodes and weevils (Cylas spp.). Weevils are a major problem in drought-prone regions of Southern and Eastern Africa. Currently, there is no resistance to sweetpotato weevils. Breeding Goals Sweetpotato is grown for human consumption (i.e., table stock), processed starch, bioethanol, colorants/dyes, and for foliage for human and animal consumption. Examples of breeding goals include: resistance to sweetpotato virus disease (SPVD) and Alternaria stem blight, weevil resistance, improved yield, improved size, shape, and uniformity of roots, yield stability, high dry matter content, orange-flesh varieties with high nutritional value [i.e., beta-carotene content for combating vitamin A deficiency, improved chemical composition (i.e., starch, cellulose, sugars, protein content, carotenes, anthocyanins), improved micronutrient content (e.g., Zn and Fe)], extended harvest for subsistence cropping, drought tolerance, dense foliage with high protein content, improved palatability, and digestibility, vine survival and vigor after planting (especially during periods of drought), improved storage, resistance to skinning, and lower acrylamide potential. Breeding Centers in Africa and Elsewhere International Potato Center in Peru (CIP), Crops Research Institute (CSIR) in Ghana, Mozambique Institute of Agricultural Research (IIAM), National Crops Resources Research Institute (NaCRRI) in Uganda, North Carolina State University (NCSU) in the USA, Louisiana State University (LSU) in the USA, Agricultural Research Council (ARC) in South Africa, the Kenya Agricultural Research Institute (KARI), the Agricultural Research Institute (ARI) in Tanzania, the Zambia Agricultural Research Institute (ZARI), the Department for Agricultural Research Services (DARS) in Malawi, the Rwanda Agriculture Board (RAB), the National Root Crops Research Institute (NRCRI) in Nigeria, the Ethiopian Institute of Agricultural Research (EIAR) and the Environment and Agricultural Research Institute (INERA) in Burkina Faso. Status of Sweetpotato in Africa (ca. 2015) Constraints on Yield Although acreage in Africa planted with sweetpotatoes has steadily increased from the 1960’s, the average yield per hectare of sweetpotato has remained basically unchanged (Fig. 1) (FAOSTAT, 2014) due to five main constraints (Low et al. 2009): • Farmers’ inability to acquire virus and disease free planting materials • The lack of improved varieties • The damage due to sweetpotato weevils • Poor agronomic practices • The lack of easily accessible markets Average yield of sweetpotato in Africa is 5.6 tons ha-1, which is less than the average yield observed in the USA and China (24.5 and 22.4 tons ha-1, respectively), and far below the maximum achievable yield of 40-50 tons ha-1. Experiments conducted in East Africa suggest that yield could double if farmers had access to clean planting material of improved varieties, and the addition of improved crop and soil fertility management practices could more than triple the yield potential of sweetpotato in Africa (Gruneburg et al. 2004). Yield and Acreage Over Time The lack of improved varieties of sweetpotatoes is mostly the result of limited investment in sweetpotato breeding programs in Africa, though this trend is changing as government and NGO’s are now focusing more efforts towards the training and support of plant breeders working on secondary crops including sweetpotato. Additional effort is also now being focused on linking farmers with breeders to ensure that varieties produced by breeding programs are meeting the needs of farmers and consumers of sweetpotato. Breeding Sweetpotato Breeding Factors The rate of progress that is achievable for a breeding program is dependent on the gene frequencies in the base population, the effectiveness of the breeding methods that are used, and the access the breeder has to field sites, greenhouses, lab equipment, and trained personnel needed to conduct a breeding program. A breeder should consider the following when deciding on which breeding method to use: • The germplasm that is available • The inheritance of the target traits (if known) • Biological constraints of the species (i.e., low seed set per plant, self-incompatibility, etc.) Genetic improvement of sweetpotato is complicated by a number of factors: • Self- and cross-incompatibility • The highly heterozygous nature of individual clones • The large number of chromosomes (2n = 6x = 90) These factors contribute to a low correlation between parent performance and offspring performance. In general the success of a sweetpotato breeding program relies mostly on the ability to grow and evaluate a large number of clones/hybrid progeny in a selective environment that closely resembles the target environment. Thus the development of rapid and reliable screening methods is critical. Hexaploid Nature of Sweetpotato Cultivars of sweetpotato are phenotypically homogenous (because they are clonally reproduced) and genetically heterozygous (because they are self-incompatible outcrossers). Sweetpotato is a hexaploid with 2 non-homologous genomes (B1B1B2B2B2B2) with tetradisomic inheritance (Lebot 2010), so the genetics of simple traits is more complex with up to six alleles per locus. Furthermore, because sweetpotato is a hexaploid, heterozygous genotypes occur in much larger frequencies (Fig. 2) making heterosis more important for quantitatively inherited traits (e.g., yield, yield stability, vigor after planting, etc.). The hexaploid nature of sweetpotato and the fact that this species is self-incompatible makes it extremely difficult to fix recessively inherited traits like resistance to certain viruses and some quality traits e.g., orange flesh) (Fig. 2) even if the frequency of these recessive alleles is greater than 70%. Characteristics of Clonally Propagated Crops Sweetpotato is an open-pollinated clonally propagated crop. Characteristics common to clonally propagated crops include: • A strong positive relationship between productivity/vigor and level of heterozygosity • Selfing reduces vigor due to inbreeding depression • Vigor/heterozygosity can be fixed and maintained for the life of the clone • Polyploids/aneuploids can be maintained via clonal propagation • Often difficult to create a large quantity of clones from one plant, whereas it is relatively easy to produce a large amount of seed from most sexually propagated crops • Clonal propagules are typically bulky and difficult to store A basic breeding procedure for a clonal crop like sweetpotato includes the following steps: 1. Define breeding objectives (i.e., yield stability, adaptation, taste, and pest and disease resistance) 2. Assemble germplasm (i.e., local varieties, wild species, cultivars developed in other parts of the world, etc.) and establish a breeding nursery 3. Develop segregating populations via hybridization (i.e., biparental cross, polycross nursery, or diallel crosses) and/or induced mutagenesis 4. Evaluate and select superior clones • Plot size, the number of replications, and the number of locations where clones are evaluated is increased after each round of selection in an attempt to reduce the variability due to the environment • Select early for traits with high heritability, select later for traits with low heritability 5. Name and release a cultivar and multiply and distribute clones Basic Breeding Scheme for Sweetpotato A basic breeding scheme for sweetpotato (Fig. 3) usually involves one (or more) cycles of hybridization to generate genetically variable progeny (i.e., true seeds) that are first evaluated in a seedling nursery. Superior genotypes are then clonally propagated and selection is conducted in replicated trials in multiple environments over multiple years (i.e., observational, preliminary, and advanced yield trials). Superior genotypes that are selected throughout the process are often recycled back into the system and used as parents for the next cycle which is the basis for recurrent selection. Recurrent Selection Recurrent selection is a common method for improving a breeding population that captures additive variance and is most effective for traits with moderate to high heritability. The basic scheme consists of three phases: 1. Selection of genotypes and their hybridization in an insect-pollinated polycross nursery or using controlled crosses 2. Evaluation of progeny 3. Selection of superior progeny and the creation of a new polycross nursery or controlled crosses with or without the best parents from the initial polycross nursery in Step 1. Recurrent selection is a proven method for increasing the frequency of desired alleles and for creating a genetically broad-based population especially when new germplasm (i.e., new parents/genotypes and/or superior progeny from previous cycles) is added to the polycross nursery/controlled crossing block after each cycle. Recurrent selection allows for the rapid increase in the percentage of minor and recessive alleles, but it requires accurate screening techniques to be successful. Accelerated Breeding Scheme (ABS) Population Improvement and Variety Development Many sweetpotato breeders are now using the Accelerated Breeding Scheme (ABS) for population improvement and for variety development (Table 1, below) (Gruneberg et al. 2009a, Gruneberg et al. 2015). The ABS reduces the total time needed to develop a new variety from 7-8 years to 4 years by compressing the early breeding stages into two years (i.e., 1 year of crosses/seedling multiplication + 3 to 4 years of evaluation in a single location vs. 1 year of crosses/seedling multiplication + only 1 year of evaluation in multiple locations). This method evaluates as many genotypes as possible in the first season of year 2 in 3-4 locations without replication. This allows the breeder to simultaneously select traits in multiple environments in a single year vs. growing clones in one location per year and sequentially selecting clones over several years. This shortens the time needed to develop a variety and conserves resources while still allowing the breeder to estimate the stability of the clones being tested. In sweetpotato, genotype x year variation (i.e., temporal variation) is typically less than genotype x location variation (i.e., spatial variation) (Gruneberg et al. 2004) so the focus is shifted to making selections in more environments in fewer years. For the second season of Year 2 a second replication and more locations are added. G×E Interactions High yield and stability in different environments is not well correlated in sweetpotato, suggesting that GXE interactions are important and yield needs to be evaluated under different conditions. For sweetpotato GXE interactions are often larger than or equal to the genetic variation for some traits (Gruneberg et al. 2009b) (Table 2, below); therefore, reducing replication to increase the number of locations can be beneficial. Because replication is eliminated in Season 1 of Year 2 an accurate understanding of the variability in the field is critical to ensure that the early evaluation stages are adequate for distinguishing superior genotypes from poor-performing genotypes. The inclusion of check varieties throughout the field (i.e., alpha lattice design) is suggested to account for microenvironments in a field that may bias the performance of individual clones. The efficiency of the ABS can be increased by using a low input or high stress (i.e., drought) environment during Season 1 of Year 2 in addition to 2-3 normal input locations. Selection is conducted sequentially during Season 1 such that genotypes that don’t perform well in the low input/high-stress location are eliminated from the normal input locations before selections are made. This procedure is called “independent culling) (Gruneberg et al., 2009a) (Lebot 2010). This allows the breeder to select only genotypes that perform well both under normal and stressful environments in a single season. In general breeding programs allocate >60% of the budget for replicated trials during the later stages when a few clones are evaluated in advanced yield trials in multiple locations. The accelerated breeding scheme shifts the emphasis of the budget to Year 2 to maximize the number of clones evaluated in multiple environments. Accelerated Breeding Scheme Table 1 Accelerated Breeding Scheme for Sweetpotato. Year Season Name of trial # of clones/ genotypes Plot type # of clones/ genotypes # of locations # of reps/location traits Notes 1 1 Crossing block n/a n/a n/a n/a n/a n/a n/a 1 2 Seeding nursery (SN) 2000 single hills (50cm x 50cm) 1 1 1 only highly heritable traits (i.e., disease-resistant, storage root color) n/a 2 1 Observational trial (OT) 2000 single row plot (1m x 1m) 3-4 3-4 1 harvest index, storage root characteristics (i.e., dry matter content, protein, starch, sugars, provitamin A concentrations) superior clones are often selected from OTs and used as parents for controlled crosses where the progeny are added to the next seeding nursery 2 2 Preliminary yield trial (PT) 150-300 2 row plot 30 plants (15 plants/row) minimum of 3 2 all traits RCBD, 1 location can be used to create multiple environments if a treatment (i.e., fertilizer, irrigation) is applied 3 1 Advanced yield trait (AT) 40 5 row plot 75 plants (15 plants/row) minimum of 4 2 all traits n/a 3 2 Advanced yield trial (AT) + On-farm trials (OFT) 5-8 5 row plot 75 plants (15 plants/row) minimum of 4 plus 10 more on-farm locations (OFT) n/a all traits field design/plot size determined by the farmer for OFTs 4 1 Multiplication 1-3 1-3 ha fields n/a n/a n/a n/a n/a Estimation of Variance Component Table 2 Estimation of variance component due to genotypes ($\sigma_G^2$). Genotype by environment interactions ($\sigma_{G \times E}^2$) and the plot error $\sigma_E^2$ from 1146 CIP genebank clones evaluated at three locations: (i) arid irrigated, (ii) humid tropic lowland, and (iii) mineral stress humid tropic lowland with two plot replications per site (95% confidence limits of parameter estimates in brackets). n/a Genotype $\sigma_G^2$ Genotype by environment $\sigma_{G \times E}^2$ Error $\sigma_E^2$ Ratio $\sigma_G^2:\sigma_{G \times E}^2:\sigma_E^2$ Yield (t2/ha) 36.2 (20.6 – 43.6) 39.4 (33.8 – 46.9) 64.2 (60.0 – 68.9) 1 : 1.1 : 1.8 Dry matter (%2) 14.8 (13.3 – 16.6) 5.7 (5.0 – 6.5) 5.7 (5.3 – 6.1) 1 : 0.4 : 0.4 Protein (%2 DM+) 0.21 (0.16 – 0.30) 0.67 (0.59 – 0.78) 0.73 (0.68 – 0.79) 1 : 3.2 : 3.5 Starch (%2 DM+) 21.5 (19.3 – 24.2) 3.2 (2.3 – 4.9) 16.3 (15.2 – 17.5) 1 : 0.2: 0.8 Sucrose (%2 DM+) 5.6 (4.9-6.5) 2.1 (1.6 – 2.8) 7.4 (6.9 – 7.9) 1 : 0.4 : 1.3 Total sugar (%DM+) 17.0 (15.2 – 19.2) 6.0 (5.2 – 7.1) 9.0 (8.4-9.7) 1 : 0.4 : 0.5 Carotene (ppm2 DM+) 6327 (5681-7091) 2462 (2224 – 2740) 1421 (1323 – 1529) 1 : 0.4 : 0.2 Calcium (ppm2 DM+) 74001 (61485-90791) 95.990 (82657 – 112849) 157303 (147021-168708) 1 : 1.3 : 2.1 Magnesium (ppm2 DM+) 143005 (12351 – 16764) 9880 (8360 – 11858) 17638 (16451 – 18960) 1 : 0.05 : 0.1 Iron (ppm2 DM+) 2.33 (1.92 -2.87) 3.46 (3.0 – 3.97) 3.85 (3.59 – 3.97) 1 : 1.7 : 2.2 Zinc (ppm2 DM+) 0.8 (0.62- 0.97) 1.37 (1.20 – 1.59) 1.72 (1.60- 1.85) 1 : 1.7 : 2.2 Establish a Crossing Block YEAR 1 (SEASON 1) – ESTABLISH A CROSSING BLOCK AND/OR MAKE CONTROLLED CROSSES AND HARVEST TRUE (HYBRID) SEED Sweetpotato breeding has traditionally involved making crosses between complementary parents using controlled crosses or a polycross nursery with multiple parents. The major goal of a crossing block or controlled crosses is the improvement of the overall population mean from one cycle to the next. The ability to generate genetic variation is easy in sweetpotato due to the high heterozygosity of individual clones/parents. The creation of genetic variation via hybridization is easy in sweetpotato because this species is generally self-incompatible so seeds do not develop from self-fertilization. If a breeder has limited information about the value/usefulness of individual crosses (i.e., from prior test crosses or data from previous years) then the goal should be to maximize the number of parental combinations. A minimum of 15 parents should be used in a polycross nursery or for controlled crosses. Most breeding programs use 20-30 parents – though larger breeding programs (e.g., CIP) are now using 150 or more different parents that are separated into different gene pools (i.e., heterotic groups). YEAR 1 (SEASON 2) – SEEDLING NURSERY (SN) Hybrid seed from the crossing block or controlled crosses is harvested and planted in 0.5 x 0.5 m plots (depends on quantity of seed available and the size of the breeding program). Selection during this step is usually limited to natural selection for tolerance and resistance to pathogens and pests. Artificial selection by breeders is typically avoided during the “true seed plant stage” because seed plants often grow differently than plants grown from vegetative cuttings and because this stage is often grown in an artificial environment (e.g., shade or glass house in pots) that is not representative of the field environment where the clones will eventually be grown. Stem cuttings are harvested after 10 weeks. YEAR 2 (SEASON 1) – OBSERVATIONAL TRIAL (OT)/HILL TRIAL (HT) Hybrid seed from the crossing block or controlled crosses is harvested and planted in 0.5 x 0.5 m plots (depends on quantity of seed available, size of breeding program). Selection during this step is usually limited to natural selection for tolerance and resistance to pathogens and pests. Artificial selection by breeders is typically avoided during the “true seed plant stage” because seed plants often grow differently than plants grown from vegetative cuttings and because this stage is often grown in an artificial environment (e.g., shade or glass house in pots) that is not representative of the field environment where the clones will eventually be grown. Stem cuttings are harvested after 10 weeks. YEAR 2 (SEASON 2) – PRELIMINARY YIELD TRIAL (PT) Larger 2 row plots (30 plants, 15 plants/row) are planted at a minimum of 3 locations. A second rep is added at each location. Selection for lower heritability traits (e.g., yield, biomass) begins at this stage. A breeder can begin to assess yield stability at this step but this requires 3 or more environments with the best results achieved when more than 6 environments are included (Gruneberg et al. 2009a). YEAR 3 (SEASON 1) – ADVANCED YIELD TRIAL (AT) Larger 5 row plots (75 plants, 15 plants/row) are planted at a minimum of 4 locations. Selection for lower heritability traits (e.g., yield, biomass) continues. YEAR 3 (SEASON 2) – ADVANCED YIELD TRIAL (AT) + ON FARM TRIALS (OFT) On farm trials are added where the field design, plot size, and inputs are determined by the individual farmers. Farmers are asked to rate the clones on a common set 5-6 traits plus any additional traits that are important to them. YEAR 4 (SEASON 1 AND 2) – MULTIPLICATION AND DISTRIBUTION OF VARIETIES Superior genotypes are multiplied and distributed to farmers Trial Conditions A breeder is strongly encouraged to plant check varieties, guard rows, and spreader/susceptible rows and to use an appropriate field design for each location. It is best to harvest cuttings from plants that are ten weeks old, so it is helpful to plant a multiplication plot for each clone about 2.5 months before a trial is planted. These plots should be maintained separately and managed (e.g., weeded, fertilized, irrigated) to promote vegetative growth. A single vine cutting usually produces five vine cuttings. Storage root sprouts can also be used (i.e., an average-sized storage root yields about 20 vine cuttings). A breeder should use tip cuttings of the same length and vigor when possible. If uniform cuttings are not available divide the cuttings up by replication (i.e., best in rep 1, second-best in rep 2, etc.). All field management (e.g., weeding, fertilizer, irrigation, etc.) should be applied uniformly across all reps. If possible each task should be divided so each worker completes an entire rep. For example, if planting an entire field takes 3 people, 1 worker should plant rep 1, a second worker would plant rep 2, and a third worker would plant rep 3, so differences in planting technique can be partitioned out as a rep effect when the data is analyzed. The same procedure should be applied for data/note-taking. If multiple people are making evaluations each individual should be assigned to evaluate specific reps and workers should not collect data for others. It is nearly impossible to simultaneously select the top genotypes for every trait without growing out an impracticably large population because the number of genotypes to be screened increases exponentially as the number of traits increases. For example, if a breeder wanted to select the top 10 clones from among 100 genotypes for 10 traits then 100^10 genotypes need to be grown and evaluated. Thus a breeder usually has to compromise and select 3-5 quality traits at a time while simultaneously maintaining sufficient genetic variation for yield, yield stability, and adaptability. ABS – Mulitple Concurrent Cycles Breeders will usually have 3-5 different cycles of an ABS at various stages (SN, HT/OT, PT, AT) running at the same time (see Fig. 4). This allows a breeder to use advanced clones from later stages of one cycle to create new polycross nurseries/controlled crosses. Sources of Parents/Germplasm Institutions that maintain and distribute Ipomoea germplasm include: CIP (Centro Internacional de la Papa) (> 4,200 accessions), USDA/ARS National Genetic Resources Information Network (>900 accessions), NaCRRI National Crops Resources Research Namulonge/Uganda, and IITA (International Institute of Tropical Agriculture), in Nigeria. Though some interspecific hybrid combinations can be made especially using species within the Batatas section, wild species are typically not used in most breeding programs. Polycross Nursery vs. Controlled Crosses Ensuring Specific Cross Combinations Often a combination of open-pollination (i.e., polycross nursery) and hand-pollination (i.e., controlled crosses) is used to ensure that specific cross combinations are made. Some of the advantages and disadvantages of using a polycross nursery vs. controlled crosses are shown in Table 3. Table 3 Advantages and disadvantages of using an insect-pollinated polycross nursery vs. controlled crosses to generate progeny. Polycross nursery Controlled crosses Advantages Disadvantages Advantages Disadvantages Requires less labor Only the female parent is known so genetic advancement is based on only ½ of the genetic variation that is available Both parents are known so genetic advancement is based on all of the genetic variation that is available Requires more labor to make crosses by hand Makes more seed/more cross combinations Unbalanced contribution from some clones (poor seed set, low pollen production, limited and/or asynchronous flowering) Superior combinations are tracked and can be recreated Less seed/less combination are created Polycross nurseries are theoretically less efficient because genetic advancement is halved because parental control is based only on the female parent while parental control for controlled crosses is based on a control factor of 1 because both parents are known. GS = (c)(i)VA / σP GS is the expected gain or predicted genetic advance from selection c = parental control factor, ½ if only the female parent is known,1 if both parents are known i = the selection intensity, a constant based on the percent selected and obtained from statistical tables VA = additive genetic variance σP= phenotypic standard deviation Note: the phenotypic values must exhibit a normal, or bell-curve, distribution for GS to be valid. Using Different Methods To better understand the impact of using different methods to generate progeny, CIP breeders and collaborators compared the population means (i.e., unselected and after one cycle of selection) of sweetpotato progeny created by 3 different pollination designs (I, II, and III) to determine which method of generating progeny produced the best results. For this experiment the breeders used the same 22 clones but crossed them in different ways: (I) using an open-pollinated polycross nursery, (II) using a partial diallel design where 4 of the best clones were crossed by hand to each other and to the rest of the clones (4 x 22), and (III) using a factorial design where the best 5 clones were crossed by hand to rest of the remaining clones (5 x 17) (“the best by the rest”). The progeny of each of these designs were planted in unreplicated plots in a single location and the population mean was determined for each = average performance of the unselected population. The breeders then selected the best 100 genotypes from each population and averaged their performance as a measure of the achieved increase in root yield after one cycle of selection. The standardized response to selection (R) was then calculated for each method by comparing the unselected mean to the mean of the 100 selected genotypes. The results for this experiment showed that the average root yield of the progeny created by method (II) (18.4 t/ha) and the average root yield of the 100 selected genotypes (23.5 t/ha) after one round of selection were both higher than the unselected and selected progeny generated by the other methods (I and III) (Table 4). These results indicated that for this scenario, controlled crosses made by a breeder guided by a partial diallel design (II) produced better progeny and more genetic advancement per cycle than an open-pollinated polycross nursery (I). The factorial design (III) was the least successful method though it only differed from the partial diallel design in that it did not include progeny from the intermating of the top 5 clones. This suggests that much of the success of the partial diallel design may be attributed to the successful performance of the progeny created by crossing the top 5 clones. Case Study: Comparing the Use of a Polycross Nursery vs. Controlled Crosses Considering Other Factors Table 4 Results from an experiment comparing progeny and genetic advancement made from progeny created using 3 different pollination designs. Design Pollination design Population mean of progeny before selection (t/ha) Population mean of progeny before selection (t/ha) Standardized R I Open pollinated polycross nursery (22) Open-pollinated polycross nursery (22) 21.2 1.35 II Partial diallel (4 X 22) Partial diallel (4 X 22) 23.5 0.904 III Factorial (5 X 17) Factorial (5 X 17) 17.4 0.715 Despite the gains made using controlled crosses vs. a polycross nursery in this particular scenario a breeder must also consider other factors as well when deciding which method to use for their program. For example, not all programs have enough staff to make all of the hand pollinations necessary for a partial diallel design so a polycross nursery may be a better option. If a breeder does choose to use an insect-pollinated polycross nursery it is important that all clones be equally represented so experimental design and replication are crucial. If a polycross nursery contains ten or fewer clones a Latin square design is recommended (Table 5). For a large number of clones a randomized complete block design with replication is recommended (Table 5). Hybridization Using a Polycross Nursery Table 5 Recommended planting designs for polycross nurseries to ensure equal contribution from all parents. Latin square – n × n array, each genotype occurs exactly once in each row and exactly once in each column Randomized complete block design A B F C E D A B C C A E B C A D F E D E F B H F C D B E A F G H I D I G D E C F B A F A G I A B E F D A C B B D H G H F F A E B D C E C I D C E • Planted in isolation • Natural insect cross-pollination & artificial crosses to ensure random pollination • Plant extra reps of less vigorous clones or clones that don’t flower well • Stagger planting times so that all clones flower at the same time Crossing Block/Controlled Crosses • Flowering is best under short day conditions and begins about 1.5 months after the crossing block is planted/established and continues for ~3-5 months • It is advisable to plant extra replications of clones that do not flower well • Clones that don’t flower well can also be grafted onto I. setosa, or I. nil, or on a genotype that flowers prolifically to promote flowering • Clones that flower later than the other clones should be planted earlier to ensure that all clones have an equal chance of contributing to the gene pool of the progeny • Seed harvest begins about 2 months after the crossing block is established and continues for up to 4 months • Plant 2 vines per 1×1 m plot, 5 randomized plots of each clone • Stake/trellis plants and erect a windbreak if needed to protect the crossing block from high winds • Label each stake with the clone number to make identification easier • Avoid using a high N fertilizer to promote flowering • Monitor plants for insect and disease problems, but avoid using pesticides that may injure bees, and other pollinating insects • Most clones of sweetpotato are self-incompatible and do not produce selfed seed • Each flower opens early in the day just after sunrise and lasts for only a few hours before fading around noon • Each pistil contains 1 superior ovary with two carpels and each carpel has two locules that contain 1 or 2 ovules, so a single capsule can produce 1 to 4 seeds • Flowers that are hand-pollinated usually produce 1 or 2 seeds and capsules that are open-pollinated produce 2 to 3 seeds. Seed Harvest Seeds mature 4 to 6 weeks after pollination. The capsule and pedicel will both turn brown and dry and begin to shrivel when the seed is ready to harvest. Capsules that are left too long will dehisce (split open) so care must be taken to harvest seeds before they are lost. Seed Scarification Seeds can be hand-scarified by scratching a small notch in each seed coat with a sharp needle or a small, 3-cornered file or acid scarified with concentrated sulfuric acid for 40 minutes followed by a 5-10 minute rinse under running water. Polycross Nursery vs. Controlled Crosses Cross Incompatibility Self-fertilization is rare because sweetpotato possesses a homomorphic, sporophytic type of self-incompatibility that is not affected by environment, chemical treatment, and cannot be overcome using bud pollination. This system is likely controlled by a single S-locus with multiple alleles with a dominant-recessive relationship. Heterostyly also occurs in sweetpotato; however, it does not appear to affect fertility. Cross incompatibility among different varieties can limit recombination and seed production and hinders targeted breeding especially when the parents with desirable traits of interest, such as disease resistance, drought tolerance, enhanced levels of protein, vitamins, macro- and micro-nutrients and dry matter are closely related and belong to the same incompatibility group. As a result breeders must maintain large populations that contain non-related accessions with complementary traits. Three types of cross compatibility (Table 6) exist depending on the success or failure of reciprocal crosses: reciprocal fertility occurs when fertility is present in both directions, reciprocal incompatibility occurs when incompatibility occurs in both directions, and unilateral fertility/incompatibility occurs when fertility occurs only when a genotype is used as the female but not when used as a male and vice versa. Table 6 Types of cross-compatibilities Type of cross-compatibility Expected outcome; A and B are different genotypes Reciprocal fertility A x B and B x A both produce seed Reciprocal incompatibility A x B and B x A both do not produce seeds Unilateral fertility/incompatibility A x B produces seed but B x A does not OR B X A produces seed but A x B does not Incompatibility and Sterility Incompatibility and sterility are often used interchangeably although this is done incorrectly. Sterility is the failure of reproduction due to the failure of a plant to produce viable gametes and incompatibility is the failure of viable gametes to fertilize one another. Sterility or reduced fertility in sweetpotato is not uncommon as aneuploidy due to multivalent formation among non-homologous chromosome pairs can often lead to an unbalanced number of chromosomes in the gametes. Sterility is also caused by gene action. Participatory Plant Breeding (PPB) Women are responsible for most of the labor when it comes to growing sweetpotato. Although men are usually involved in land preparation, especially when land needs to be cleared or soils are heavy, and in marketing, particularly where sweetpotato is a significant cash crop. Men also significantly contribute to weeding and harvesting, particularly when sweetpotato is intercropped. Traits that are important to farmers (e.g., piecemeal harvest for subsistence farming and resistance to regionally important abiotic and biotic stresses, etc.) are difficult to select for outside of the target environment(s), therefore, it is helpful to cooperate with local farmers/growers to gain access to additional testing locations and to gain insight into farmer/grower preferences. Participatory plant breeding practices are beneficial especially during the earlier steps when genetic diversity is high and most traits (e.g., quality characteristics, disease resistance, plant architecture, etc.) are visually evaluated. PPB practices are less useful at the later stages of selection because those traits that are usually selected based on visual evaluations are typically highly heritable and are already fixed in the population. The involvement of farmers and consumers also helps facilitate the rapid adoption of new varieties and lessens the chance that superior varieties are rejected because they fail to meet the expectations of the farmers. Regional and local preferences for flesh color, dry matter content, and texture, as well as adaptability to the local environment are critical traits in sweetpotato and should be considered during the early stages of selection. CIP’s Convergent-Divergent Scheme CIP breeders are currently using a convergent-divergent approach that is designed to meet the regional needs of growers by developing widely adapted cultivars and promoting collaboration among breeders and consumers (Lebot 2010). Due to the difficulty of breeding for adaptation across multiple agro-ecological zones in Africa a program must be conducted in a decentralized way. Therefore programs in different countries can develop cultivars that work best for their region. This method starts with a diverse base population composed of genotypes from a wide range of sources. This base population is evaluated in a single centralized location and superior genotypes are intercrossed. Seed from this cycle is collected and then sent to collaborators in different regions where it is evaluated and intercrossed with elite germplasm that is adapted for each individual location. This scheme introduces new germplasm while also including elite germplasm carrying alleles for traits that are specific to a region (e.g., disease and pest resistance endemic to that location, a particular taste profile, plant architecture that fits a cultivation technique used in that region, etc.). By allowing breeders to introduce alleles specific to their location and then test the progeny locally GXE interactions are better accounted for. Selection in the centralized location is focused on maintaining high genetic diversity while also introducing desired traits, while selection at the local level focuses more on selecting varieties that will grow well in a specific environment. Exploiting Heterosis Creating Distinct Germplasm Pools Exploiting Heterosis Using 2 Heterotic Gene Pools Breeders are now exploring the idea of exploiting heterosis in sweetpotato by creating two genetically distinct germplasm pools and selecting and recombining within each pool based on the general combining ability (GCA) of clones that are crossed with the other germplasm pool. Work at the international Potato Center (CIP) is focused on creating high yielding orange-fleshed sweetpotato varieties using this approach. For this experiment, two populations were created: the Jewel population and the Zapallo population. The Jewel population consists of orange-fleshed clones released prior to 2004 and includes mostly North American varieties including Jewel and Resisto. The Zapallo population was created using a factorial crossing design using 8 male parents (Jonathan, Zapallo, Huambachero, Tanzania, Yurimaguas, Wagabolige, Xushu18, and Ninshu1) and 200 orange fleshed females. More than 200 families were then produced from cross combinations involving 49 clones from the Jewel population crossed with 31 clones from the Zapallo population (Fig. 5). Mid-parent (MP) heterosis values for dry root yield were generally low to moderate, but some individual cross combinations/families had MP heterosis values. Root yield of the best clone within each family was typically double the average root yield of the parents, with some clones producing 5 times the average yield of the parents, thus indicating success of the approach. Choosing Parents Example of Parent Selection Choosing Parents for a Crossing Block/Controlled Crosses A breeder will often use morphological data, coancestry/pedigree information, and molecular marker data to choose parental combinations. By using a combination of analyses a breeder can select parents that are phenotypically superior, and genetically distant from one another to limit inbreeding depression and possible cross-compatibility issues. Molecular markers are also useful for identifying duplicate clones or instances when genetically different clones are distributed under the same name. Example of Parent Selection Based on Molecular Markers vs. Morphological Markers Clones were characterized using both molecular markers and agronomic characters to identify phenotypically superior yet distantly related clones (Dai-fu et al. 2009). Fifteen clones (Table 7) and 60 open-pollinated offspring per clone were planted in a common garden and phenotyped for 22 traits. The 15 parental clones were genotyped with ISSR and RAPD markers. The dendrograms (Figs. 6 and 7) based on the morphological traits and the molecular markers are shown below along with Table 8, which summarizes the different groupings based on the morphological and molecular marker data. Trial Materials and Parental Dendrograms Choosing Parents for a Crossing Block/Controlled Crosses Table 7 The list of the trial materials. Code Variety Female1) Male Source or distribution location 1 Centennial Unit 1 Porto Rico Pelican Processor Louisiana State University, USA 2 Beijing 553 Okinawa100 OP The Experimental Station of Huabei, China n/a 3 Beauregard L78-21 OP Louisianna State University, USA n/a 4 Chaun 8129-4 Jiangjin Wujianshao Neiyuan The Crop Institute, Sichuan Academy of Agricultural Sciences, China 5 Guangshu 88-70 Zhan75-57 OP The Crop Institute, Guangdong Academy of Agricultural Sciences, China n/a 6 Jianshui Huangxin Landrace Jianshui County, Yunnan Province, China n/a 7 Longshju 1 Yanfen 1 Longyan 7-3 Agricultural Science Institute of Longyan, Fuijian, China 8 Sushu 6 Peng S1-12-48 AIS 0122-2 Xuzhou Sweetpotato Research Centre, Jiangsu 9 Tainung 69 Group crossing Jiayi Agricultural Experimental Station, Taiwan of China n/a 10 Xiangshu 15 Kyushu 5 Xiangshu 6 The Crop Institute, Hunan Academy of Agricultural Sciences, China 11 Xu 22-5 LO323 AIS 0122-2 Xuzhou Sweetpotato Research Centre, Jiangsu, China 12 Xu 34 Huabei 166 Qunli 2 Xuzhou Sweetpotato Research Centre, Jiangsu, China 13 Xushu 23 P616-23 Yanshu 27 Xuzhou Sweetpotato Research Centre, Jiangsu, China 14 Yanshu 27 77183 Centennial Agricultural Science Institute of Yantai, Shandong, China 15 Yanshu 5 Yanchi Red Yan 94-1 Agricultural Science Institute 1) OP, open pollination Summary of Groups of Clones Choosing Parents for a Crossing Block/Controlled Crosses Table 8 A summary of the groups of clones based on their phenotype and genotype. Clone Phenotype Group Genotype Group Centennial 1 1 Choose 1 from this group of 3 Beijing 553 1 1 Chuan 8129-4 1 1 Yanshu 5 1 2 Choose this one Xu 34 1 3 Choose 1 from this group of 3 Xiangshu 15 1 3 Jianshui Huangxin 1 3 Baeuregard 2 1 Choose this 1 Xu 22-5 2 2 Choose this 1 Xushu 23 3 1 Choose 1 from this group of 4 Yanshu 27 3 1 Guangshu 88-70 3 1 Longshu 1 3 1 Tainong 69 3 2 Choose this 1 Sushu 6 3 3 Choose this 1 A breeder can use this information to select clones that are phenotypically similar (i.e., same phenotype group) but genetically different (i.e., different genotype group) thereby reducing the number of parental clones from 15 to 8 without significantly reducing the genetic variation that is available. Combining Ability of Individual Clones Choosing Parents for a Crossing Block/Controlled Crosses When considering which parents to choose for a polycross nursery or controlled crosses it is also important to consider the combining ability of individual clones. The combining ability of a clone can be estimated by comparing the performance of a clone versus the average performance of open-pollinated progeny harvested from that clone when it is grown in a polycross nursery or other population of mixed genotypes (i.e., seedling nursery, yield trial, etc.), for example Figure 10. If the average performance of the progeny is greater than the performance of the maternal clone then a breeder can assume that a particular clone has good combining ability when crossed with other clones. Parent Selection Important to choose parents that possess superior qualities and parents that combine with other clones (superior GCA)

textbooks/bio/Agriculture_and_Horticulture/Crop_Improvement_(Suza_and_Lamkey)/1.13%3A_Sweetpotato_Breeding.txt

Teshale Mamo; Arti Singh; Asheesh Singh; and Anthony A. Mahama Groundnut or peanut (Arachis hypogaea L. Millsp) is a self-pollinated species belonging to the Fabaceae family. It is considered as the most important food legume crop in continental Africa because of its multiple purpose uses in food, feed, paints, lubricants and insecticides as the seeds are comprised of 35–56% oil, 25–30% protein, 9.5–19.0% carbohydrates, several minerals like P, Ca, Mg and K, as well as vitamins E, K and B (Gulluoglu et al. 2016).The crop has various industrial uses including products such as food, feed, paints, lubricants and insecticides. Because of its natural ability to fix atmospheric nitrogen in the soil, it is considered an ideal crop in crop rotation systems and in intercropping under small scale subsistence farming systems. Plagued by many biotic and abiotic growth limiting factors, production has been lower than the potential. Breeding to alleviate or overcome these limiting factors is therefore key new cultivars to ensure increased productivity to contribute toward assured food security. Learning Objectives • Become familiar with the basic biology and importance of groundnut • Provide examples of breeding schemes used to develop cultivars of groundnut Crop Attributes Crop Biology Groundnut or peanut (Arachis hypogaea L. Millsp) is a self-pollinated species belonging to the Fabaceae family. Groundnut is a disomic allotetraploid (2n = 4x = 40). The two sets of chromosomes of A. hypogaea are highly diplodized, meaning there is little recombination between the A and B genomes except when the infrequent quadrivalent is formed. Groundnut is found in the Arachis section along with A. monticola, also a tetraploid, and ~25 diploid species (Dwivedi et al., 2007). Arachis is subdivided into nine taxonomical sections: Arachis,Erectoides, Rhizomatosae, Extranervosae, Heteranthae, Trierectoides, Triseminatae, Caulorrhizae, and Procumbentes with groundnut classified in the Arachis section (Dwivedi et al., 2007). Crop Geography The genus Arachis originated in South America and is comprised 68 species (Dwivedi et al., 2007; Krapovickas and Gregory, 1994). The species of Arachis are easily delineated from other closely related genera because they flower above ground but set seed below ground (Holbrook and Stalker, 2003). There are six centers of diversity for groundnut in South America including geographic regions in Paraguay-Paraná, the upper Amazon, the west coast of Peru, Brazil, and the southwest Amazon region in Bolivia. A secondary center of diversity also exists in Africa (Holbrook and Stalker, 2003; Wynne and Coffelt 1982). Arachis hypogaea is believed to have originated in the South American region encompassing southern Bolivia to northern Argentina (Holbrook and Stalker, 2003). Arachis hypogaea is thought to have arisen ~4000 years ago from a single hybridization event between two diploid Arachis species (i.e., A genome from A. duranensis, B genome from A. ipaensis) followed by a spontaneous chromosome doubling of the sterile hybrid to form a fertile allotetraploid (i.e., AABB) (Kochert et al., 1996; Young et al. 1996). Though the fertility was restored in the resulting allotetraploid it was reproductively isolated from its progenitor species creating a strong genetic bottleneck, which is partially responsible for the low allelic diversity present in modern cultivated peanut (Dwivedi et al., 2007; Kochert et al., 1996; Stalker et al., 2013; Young et al. 1996). Crop Inflorescence Arachis hypogaea or cultivated groundnut has simple or compound (i.e., 1-5 flowers) inflorescences that occur in the leaf axils on both primary and secondary branches. Typically a single flower per inflorescence opens per day (Holbrook and Stalker, 2003). The flower includes a standard, wing, and keel petals. The standard is deep orange to light yellow in color, and in rare cases may be white. The calyx consists of five sepals attached to the elongated hypanthium. Flowers possess an elongated tubular hypanthium or calyx tube so they look like they are borne on stalks but are instead classified as sessile (Holbrook and Stalker, 2003). Arachis hypogaea is divided into two subspecies (e.g., hypogaea and fastigiata) and six botanical varieties (e.g., hypogaea, hirsuta, fastigiata, peruviana , aequatoriana, and vulgaris) (Table 1) (Holbrook and Stalker, 2003). Subspecific and Varietal Classification Table 1 Data from Holbrook and Stalker, 2003, with additional comments from Dwivedi et al., 2003; Okello et al, 2013; Rami et al., 2014. Botanical variety Market type Location Traits hypogeae Virginia/Runner Bolivia, Amazon No flowers on the central stem, alternating pairs of floral and vegetative axes on lateral branches, short branches, spreading growth habit, relatively few trichomes Virginia type: large seeds, less hairy, consumed in the shell or roasted, longer growing cycle Runner type: small seeds, less hairy, used to make groundnut/peanut butter hirsuta Peruvian runner Peru more hairy fastigiata Valencia Brazil, Paraguay, Peru, Uruguay Flowers in the mainstem, sequential/disorganized pairs of floral and vegetative axes on the branches, more erect to procumbent, shorter life cycle, pods with 2+ seeds, smooth pericarp, consumed in the shell, boiled, or canned, primary type in Uganda peruviana n/a Peru, Bolivia Less hairy, deep pod reticulation aequatoriana n/a Ecuador Very hairy, deep pod reticulation, purple stems, more branched, upright vulgaris Spanish Brazil, Paraguay, Uruguay More branched, upright, shorter life cycle, flowers on the central stem, floral and vegetative axes arranged in a disorganized fashion, 2 seeds per pod Importance Groundnut is a legume crop adapted to a hot, semi-arid climate, survives and yields under rainfed conditions, fixes nitrogen, and requires few inputs, making it a crop that is suitable for small shareholder farms. It can be grown in a low input environment and provides a source of fat and protein, and provides many phytonutrients including vitamin E and antioxidants. Usage Groundnut is a multiuse crop that is harvested as a source of edible oil, vegetable protein, and forage (haulms) for horse and cattle (Dwivedi et al., 2003). Groundnut is a rich source of oil, protein, minerals (Ca, Mg, P, and K), and vitamins (E, K, and B1) (Savage and Keenan 1994). Groundnut contains 40-60% oil, 20-30% protein and, and 1020% carbohydrate (Dwivedi et al., 2003; Pandey et al. 2012; Savage and Keenan 1994) In the United States, approximately 70 percent of the groundnuts are runners (small-seeded types of var. hypogaea), 20 percent are virginias (large-seeded types of var. hypogaea), 10 percent are spanish (var. vulgaris), and less than 1 percent are valencia (var. fastigiata) market types (Holbrook and Stalker, 2003; Knauft and Gorbet, 1989. Most of the groundnut produced is for human consumption, but low quality lots (blemished seeds, aflatoxin contamination) are crushed for oil. The quality of groundnut oil is determined by the ratio of oleic (O) to linoleic (L) fatty acids. A higher ratio results in better storage quality of the oil that is less prone to oxidation and the development of undesirable flavors, and has a longer shelf life. For groundnut varieties harvested for oil extraction (i.e., high oil, high O/L ratio) seed size doesn’t matter as much as total yield. For groundnut varieties harvested for human consumption (i.e., low oil, high O/L ratio) larger seed size is desired (Dwivedi et al., 2003). Groundnuts are used in the food and confectionary industries, but is limited by its allergenic properties in both adults and children, and by concerns about aflatoxin contamination caused by Aspergillus niger and A. flavus), which is carcinogenic and act as an immunosuppressant in both animals and humans (Dwivedi et al., 2007). Biotic Constraints Disease Constraints Biotic stresses include early leaf spot (Cercospora arachidicola), late leaf spot (Phaeoisariopsis personata), rust (Puccinia arachidis), groundnut mottle virus (Potyviridae), and groundnut rosette virus (Tombusviridae) (Okello et al., 2013; Pandey et al. 2012). (Table 2) Table 2 Major disease constraints to groundnut production. Data from Dwivedi, 2003. Pathogen Diseases Fungi Rust (Paccinia arachidis Speg.), early leaf spot (ELS) (Cercospora arachidicola Hori), and late leaf spot (LLS) [Phaeoisariopsis personata (Berk. and Curtis) Deighton] Viruses Groundnut rosette disease (GRD), peanut clump virus (PCV), peanut bud necrosis virus (PBNV), and tomato spotted wild virus (TSWV) Bacteria Bacterial wilt [Burkholderia solanacearum (E.F. Smoth) Yabuuchi et al.] Nematodes Meloidogyne, Scutellonema, Pratylenchus, Helicotylenchus, Aphelenchoides, Telotylenchus, and Paralongidorus species Pest Constraints Important insect pests include aphids (Aphis craccivora), jassids (Amrasca devastans), leafminers (Aproarema modicella), termites (Isoptera), army worms (Spodoptera litura), and thrips (Frankliniella spp.) (Okello et al., 2013; Pandey et al., 2012) (Table 3). Thrips and aphids are more detrimental as vectors of viruses versus causing direct damage to the plants. The groundnut leaf miner (A. modicella) causes extensive defoliation in the major groundnut producing regions in Uganda (Mukankusi et al., 2000; Okello et al., 2013). Because groundnut seed is sensitive to heat and high moisture, seed storage must be carefully managed at all stages of the seed production chain (Okello et al., 2013). Table 3 Major insect constraints to groundnut production. Data from Dwivedi, 2003. Environment Insect Field Leaf miner [Aproaerema modicella (Deventer)], army worm (Spedoptera litura Fab.), corn earworm (Helicoverpa armigera Hubner), lesser corn stock borer (Elasmopalpus lingosellus Zeller), southern corn rootworm (Diabrotica undecimpuctata howardi Barber), thrips (Frankinella and Scirtothrips species), jassids (Empoasca kerri Pruthi), aphids (Aphis craccivora Koch.), and termites (Microtermes and Odontotermes species) Storage Bruchid (Caryedon serratus Oliver), red flour beetle (Tribolioum castanem Herbst, rice moth (Corcyra caphalionica Sainton), and pod-sucking bug (Elasmolomus (Aphanus) sordidus Fab.) Resistance Traits Wild Arachis species possess high levels of disease resistance and display a wide range of morphological variation (Dwivedi et al., 2007). e.g., A. diogoi has virus resistance genes that are not present in the cultivated gene pool (Table 4). Table 4 Sources of resistance to rust, leaf spots, sclerotinia blight, groundnut rosette virus, aflatoxin, nematode, defoliator, aphid, and drought reported in cultivated and wild Arachis species. Data from Dwivedi et al., 2007. Peanut accessions with beneficial traits reported Type Trait Cultivated Species Wild Arachis species Rust 169 29 Late leaf spot 69 27 Early leaf spot 37 11 Groundnut rosette virus 116 12 Nematode 21 Seed infection and/or aflatoxin production by Aspergillus flavus 21 4 Sclorotinia blight 51 Defoliator (Leaf miner and Spodoptera) 9 28 Aphid 2 Not evaluated Drought 40 Not Evaluated Resistance in Arachis Species (5 tables) Figures 2 to 7 show Arachis species on the vertical axes and pests and diseases on the horizontal axes. Resistance is indicated by the red shaded boxes. Breeding Properties Breeding Goals Improved yield – less vegetative biomass, shorter main stem, increased flower production (Dwivedi et al., 2003), high yielding potential, high quality, resistance to major pests and diseases (rosette and Cercospora leafspots, peanut bud necrosis, root rot), aflatoxin resistance, short to medium-term maturity periods and tolerance to drought, large seeds for confectionery purposes, high oil content for oil extraction (Okello et al., 2013). Breeding CentersStalker et al., 2013. The largest collection of groundnut germplasm is maintained in India at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT). This collection includes ~15,000 accessions of cultivated groundnut and ~500 accessions of 48 wild Arachis species representing all six botanical varieties: var. hypoagaea ( 45.8%), var. vulgaris (36.6%), var. fastigiata (15.7%), var. aequitoriana (0.10%), var. peruviana ( 1.7%), and var. hirsuta (0.13%). Other major collections include the National Research Center for Groundnut in Junagadh, India (~8,000 accessions), the USDA NPGS, Griffin, GA, USA (~9,000 accessions). Large collections of wild Arachis species are also maintained at Texas A&M and North Carolina State University, Raleigh, NC, USA. The National Center of Genetic Resources (CENARGEN) in Brazil maintains over 1200 accessions of 81 species belonging to 9 sections. Oil Crops Research Institute (OCRI) of the Chinese Academy of Agricultural Sciences (CAAS) (8083 accessions) and the Crops Research Institute of the Guangdong Academy of Agricultural Sciences (4210 accessions) in China Instituto Nacional de Technologia Agropecuaria (INTA) and the Instituto de Botánica del Nordeste (IBONE) in Argentina There are also two well-defined core and minicore collections representing the majority of variation present in the cultivated peanut germplasm. Germplasm Pools The primary gene pool for groundnut consists of cultivated accessions (Arachis hypogaea) and the wild tetraploid species A.monticola. The secondary gene pool includes diploid species of the section Arachis that can be successfully crossed with cultivated groundnut. The tertiary gene pool includes species in sections other than Arachis that cannot be crossed with A. hypogaea using conventional crosses and are limited by both pre- and postzygotic hybridization barriers (Dwivedi et al., 2007) and (Fig. 8). Introgressing Diversity from Wild Arachis Species Wild species of Arachis have been successfully used as sources of resistance to pathogens (i.e., Sclerotinia blight, tomato spotted wilt virus, early and late leaf blights) and pests (root knot nematodes). There are three main methods for the strategic incorporation of this diversity from wild species into cultivated groundnut (Dwivedi et al., 2007; Simpson, 2001). Introgressing Diversity – Method 1 The first method starts with a cross between a diploid wild species and a tetraploid variety of cultivated groundnut (A. hypogaea), which produces a sterile triploid hybrid. The chromosomes of this sterile F1 hybrid are then doubled using colchicine to produce a hexaploid plant, which is then backcrossed with cultivated groundnut producing pentaploid progeny. These progeny are then self-pollinated for several generations usually resulting in aneuploid progeny, which over time lose chromosomes due to problems during meiosis (i.e., mispairing, lagging chromosomes) and eventually stabilize at the normal chromosome number of 40 (i.e., tetraploids) (Dwivedi et al., 2007; Holbrook and Stalker, 2003). Introgressing Diversity – Method 2 For the second method, colchicine is used to double the chromosomes in two wild diploid species (i.e., AA and BB doubled to AAAA and BBBB) (Dwivedi et al., 2007). These autotetraploids are then crossed to produce an allotetraploid hybrid (i.e., AABB), which is then crossed and backcrossed with cultivated groundnut, A.hypogaea for multiple generations during which the breeder selects to recover useful agronomic traits from the cultivated parent while simultaneously selecting for exotic traits from the wild species. For the third method two wild diploid species (i.e., AA and BB) are crossed and the sterile F1 hybrid (i.e., AB) is treated with colchicine to double the chromosomes to produce a fertile allotetraploid (i.e., synthetic amphidiploid, AABB) (Dwivedi et al., 2007). This allotetraploid is then backcrossed with cultivated groundnut A. hypogaea to produce tetraploid hybrids carrying agronomic traits from the cultivated parent and exotic traits from the wild species. Synthetic tetraploid varieties produced via this method are relatively normal and demonstrate normal meiosis, pollen fertility, and genetic recombination. Introgressing Diversity – Method 3 Method 1 is the most useful for developing new varieties (reviewed by Dwivedi et al. 2003a and Holbrook and Stalker 2003). Method 2 is less useful for developing varieties in a breeding program because of problems with sterility in the autotetraploids (i.e., AAAA, BBBB) (Dwivedi et al., 2007). Method 3 is designed to recreate the events that originally gave rise to cultivated groundnut as described previously [i.e., hybridization of two 2x species (A genome from A. duranensis, B genome from A. ipaensis) followed by a spontaneous chromosome doubling of the sterile hybrid to form a fertile allotetraploid (AABB)]. This method has been used to introgress resistance genes from wild species into cultivated groundnut (Simpson and Starr 2001; Simpson et al. 2003). In this example Arachis cardenasii and A. diogoi were used as the A donors crossed with A. batizocoi, which was used as the B donor. The resulting allotetraploid was then crossed with cultivated groundnut (reviewed in Dwivedi et al., 2007). Interspecific crosses are usually more successful when the species with the higher ploidy level (usually A. hypogaea) is used as the female parent when crossed with diploid wild Arachis species (i.e., Method 1). Greater success has also been observed when the annual species is used as the female parent and the perennial species (i.e., smaller stigmas) is used as the pollen parent. However, even when a cross successfully produces hybrid progeny, genetic recombination during meiosis in the hybrids is often restricted, and desired genes are not incorporated due to a lack of crossing over between the different genomes (Holbrook and Stalker, 2003). Because of these problems breeders must make multiple crosses using different parents to increase the probability of success. Summary Biparental crosses or more complex crosses are used to generate variability then pedigree and bulk-pedigree selection methods are used to identify and select superior genotypes. Single seed descent (with or without concurrent selection) is often used to increase the homozygosity of the breeding population during the early generations prior to selection (Dwivedi et al., 2003). Typically breeders select for qualitative traits (such as disease/pest resistance) during the early generations F2-F5 followed by late generation testing for quantitative traits like yield and/or traits that are influenced by the environment (e.g., oil content, O/L ratio, etc.) (Dwivedi et al., 2003). Most groundnut breeding programs begin preliminary yield trials in the F5 or F6 generation, by which time the level of heterozygosity has been minimized through inbreeding and meaningful selection for complex quantitative traits can begin. Recurrent selection is sometimes used to maintain genetic diversity in breeding populations (Dwivedi et al., 2003) though this method is limited by the presence of negative correlations between disease resistance and yield in some populations (Holbrook and Stalker, 2003). Backcross breeding is commonly used for introgressing 1 or 2 genes into a superior genotype. Outcomes Artificial crosses are most successful when made during the early morning hours after sunrise when the pollen is mature and viable and the stigmas are receptive (Holbrook and Stalker, 2003). High humidity levels help ensure adhesion of pollen to the stigmatic surface so it is often helpful to spray down the greenhouse floors with water in the morning to increase the humidity levels especially on dry days (Holbrook and Stalker, 2003). Pod development in cultivated groundnut generally begins 16 to 17 days after pollination. In other species pod development is delayed until 23 to 25 days (Halward and Stalker 1987). Pegs of cultivated groundnut are relatively short and robust and do not break easily, but pegs of wild Arachis species may, since they may be 1 m or more in length and are fragile (Holbrook and Stalker, 2003), so it is preferable to use cultivated groundnut as the female parent whenever possible. Because only a small number of progeny (i.e., usually 1-2 seeds per pod) can be made per artificial cross it is important to make as many crosses as possible especially when making backcrosses to introgress traits from a wild Arachis species. Outcrossing rates in groundnut are typically around 2% though rates near 8% have been identified (Coffelt, 1989; Knauft et al., 1992) Because photoperiod and temperature greatly affect how growth is partitioned between above and below ground structures it is critical to make selections in an environment that is similar to your target environment. This is especially true in genotypes with large seeds. It is also important to select for resistance to pests and pathogens in an environment that has the same photoperiod conditions as your target environment because late maturing types are often more affected by variation in photoperiod (Dwivedi et al., 2003). Limitations Genetic enhancement of groundnut is limited due to the existence of unfavorable linkages between useful disease resistance genes and loci conferring undesirable pod and seed characteristics. A second limitation is due to the fact that most disease resistant accessions are later maturing types that are more sensitive to photoperiod and partition less to below ground pod development. A third limitation is the large genotype by environment interactions for economically important traits. A fourth limitation is the small number of progeny created by artificial crosses (Dwivedi et al., 2003). Because of these limitations a varied approach to breeding groundnut is recommended (Dwivedi et al., 2003). Breeding Strategies Table 5 Traits and breeding strategy suggested for rapid and cost-effective genetic enhancement in groundnut. Data from Dwivedi et al., 2003. X indicates breeding strategy applicable for the trait. Trait Conventional breeding Marker-assisted selection Wide crosses + marker-assisted backcross Genetic transformation Genetic basis Maturity X n/a n/a n/a Polygenic Pod yield X n/a n/a n/a Polygenic Pod size and shape X n/a n/a n/a Polygenic 100-seed weight X n/a n/a n/a Polygenic Shelling outturn X n/a n/a n/a Polygenic Sound Mature seeds X n/a n/a n/a Polygenic Seed dormancy X n/a n/a n/a Monogenic Oleic/Linoleic fatty acid ratio n/a X n/a n/a Oligogenic Aflatoxin n/a n/a n/a X Polygenic Drought n/a X n/a X Polygenic Leaf miner n/a n/a X n/a Unknown Spodoptera n/a n/a X n/a Unknown Rust X n/a n/a n/a Oligogenic Early leaf spot n/a n/a X n/a Polygenic Late leaf spot n/a n/a X n/a Polygenic Bacterial wild X n/a n/a n/a Oligogenic Groundnut rosette disease X n/a n/a X Mono- and diagenic Peanut bud necrosis disease n/a n/a n/a X Unknown Speed Breeding In temperate regions of the world, groundnut breeders are only able to grow 1 generation per year, thus it usually requires 10-15 years from the first cross to the release of a cultivar. When available the use of a winter nursery for increasing homozygosity levels via single seed descent can increase the total number of generations per year to 2, thereby reducing the time needed to develop a new cultivar. Recent efforts have demonstrated that the use of a controlled environment in which continuous light and optimal temperatures and humidity are maintained can be used to advance lines from the F2 to the F4 in a single calendar year, further reducing the time to release a cultivar down to 6-7 years. This concept of “speed breeding” allows the breeder to maximize the efficiency of the breeding program by decreasing the growing period to maturity by ~30% relative to the time needed in the field, thus increasing the number of generations that can be grown per calendar year to 3 (O’Connor et al. 2013). • Strategy 1 (42 months): pedigree breeding with one summer generation per year • Strategy 2 (23 months): pedigree breeding with two generations per year (i.e., 1 in the summer, 1 in a winter nursery) • Strategy 3 (17 months): speed breeding/SSD (i.e., the F2-F4 generations grown in a controlled environment; 24 hour lights, optimal temperatures and humidity) Calendar In all 3 strategies, the F5 is grown in the field to maximize F5:6 seed numbers to facilitate preliminary yield trials in the F6. Strategy 3 of speed breeding/SSD is best suited for programs using a backcrossing breeding strategy focused on incorporating a simply inherited trait controlled by one or two genes into a new variety. This system is also appropriate for the rapid development of RILs which are useful for genetic studies and molecular marker discovery. Breeding Example The following is an example of breeders introgressing alleles from Arachis cardenasii, a wild diploid species, into tetraploid groundnut. Breeding objective: develop small-seeded, tetraploid (2n = 4x = 40) runner-type groundnut (Arachis hypogaea L. subsp. hypogaea var. hypogaea) germplasm lines that possess resistance to multiple diseases [e.g., early leaf spot (ELS), Cylindrocladium black rot (CBR), Sclerotinia blight (SB), and tomato spotted wilt (TSW)]. Registration of ‘Bailey’ Peanut This is an example of breeders using simultaneous selection for resistance to multiple diseases using both early-generation testing for resistance combined with late-generation selection for improved pod and seed characteristics in superior families. Breeding objective: develop a large-seeded virginia-type peanut (Arachis hypogaea L. subsp. hypogaea var. hypogaea) with partial resistance to five diseases that occur commonly in the Virginia-Carolina production area: early leaf spot (caused by Cercospora arachidicola Hori), late leaf spot [caused by Cercosporidium personatum (Berk. & M.A. Curtis) Deighton], Cylindrocladium black rot [caused by Cylindrocladium parasiticum Crous, M.J. Wingf. & Alfenas], Sclerotinia blight ( caused by Sclerotinia minor Jagger), and tomato spotted wilt (caused by Tomato spotted wilt tospovirus). This variety should possess characters that make it suitable for the confectionary market (i.e., sold to consumers as in-shell groundnut or as a shelled kernel). The breeders used a modified version of pedigree selection that included several generations of advancement using single seed descent to develop a pureline cultivar named ‘Bailey’. For the initial cross, NC 12C (a cultivar partially resistant to early leaf spot) was crossed with N96076L (a breeding line resistant to early leaf spot, CBR, and TSWV). Multiple F1 plants were then backcrossed as males to NC 12C to increase the chances of recovering an inbred line with suitable agronomic characteristics as NC 12C possessed more desirable traits relative to N96076L. Extra Questions Why did the breeder backcross the F1s to the NC 12C parent? NC 12C was the superior plant. Groundnut breeding is hampered by the limited genetic diversity in A. hypogeae and difficulty introgressing traits from wild Arachis species into cultivated varieties. The narrow genetic base in groundnut is attributed to multiple genetic bottlenecks that occurred as groundnut evolved into the current day species that it is, as well as, during domestication and as breeders work to develop modern-day cultivars. Consumers want a consistent commercial product so successful cultivars are often recycled back into a breeding program and used as the parents to develop the next round of cultivars.

textbooks/bio/Agriculture_and_Horticulture/Crop_Improvement_(Suza_and_Lamkey)/1.14%3A_Groundnut_Breeding.txt

Shui-Zhang Fei and Anthony A. Mahama Cassava (Manihot esculenta Crantz) is a dicot perennial shrub, belonging to the family of Euphorbiaceae. It is also known as tapioca, manioc, mandioca or yuca in different parts of the world. It can reach a height of 1-4 m (Fig. 1). Its tuberous storage roots are rich in starch (20-40%) and are harvested either for direct human consumption, animal feed, or industrial uses. Learning Objectives Become familiar with cassava, its biology, center of origin and domestication, utilization, breeding objectives and methods, and the application of molecular tools in cassava improvement. Cassava Anatomy Stem The growth habit of cassava has important implications in cassava breeding as it can affect root yield. There are two growth types: 1. Erect growth type, with or without branching at the top. 2. Spreading type, which is not cultivated. Branching occurs as a result of flower induction (Ceballos et al 2010), therefore the branchings are often called “reproductive branchings”. The branches can undergo further branching when flowering occurs, resulting in high order branchings (Fig. 2). Roots Cassava roots are true roots, therefore cannot be used for propagation. Cassava root is the most economically important organ of a cassava plant because of the starch-containing cells in the parenchyma tissue, the edible part of the root. Only 3-10 of the fibrous roots of a cassava plant will eventually bulk and become storage roots through secondary growth while the rest of the fibrous roots remain thin and serve to function in water and nutrient absorption (Fig. 3). Cassava Production Economic importance Cassava is the sixth-most important crop in the world following wheat, rice, maize, potato and barley. It is a staple food for over 500 million people, most of whom live in developing countries where food security is a major concern. Cassava is grown successfully between latitudes of 30°N and 30°S. It is drought tolerant and can grow under annual precipitation of 600mm. Cassava can also be grown on marginally fertile soils with a pH ranging from 3-8. Its roots can be left in the ground without harvest, serving as a good food security crop in cases where other crops fail. Worldwide production The main cassava production areas in the world are Africa, Asia, the Caribbean and South America. Fig. 4 describes the share of each main production area in 2014. Fig. 5 shows the production in tonnes in ten countries in 2014. Main uses Cassava roots are either directly consumed by humans as food, used as animal feed or for industrial use. Food Cassava roots can be eaten raw, cooked after removing the skin and rind or even baked and the charred skin removed before consumption. Cassava roots have numerous culinary uses around the world. Cassava leaves can also be consumed as a vegetable. Animal feed All parts of the cassava plant can be used for animal feeding. In particular, the high energy content of cassava roots makes it an ideal carbohydrate ingredient in animal diet. The majority of the cassava produced in south Asia are used for animal feed in the form of chips and pallets. Industrial use Cassava roots are also used for industrial purposes, primarily for extraction of starch which has a wide variety of uses. Recently bioethanol production from cassava has received great attention due to increased fossil fuel price and concerns over global climate changes caused by burning fossil fuels. Reproductive Biology of Cassava Flower morphology and flowering behavior Cassava is monoecious, producing separate male and female flowers on the same plant. Flowering in cassava is highly genotype and environment dependent. While some early-flowering genotypes can flower one month after planting, others may take 24 months to flower, consequently, synchronization of flowering time can be challenging in cassava breeding programs. Flowering rarely occurs during long dry period, thus irrigation is required for crossing blocks during an extended drought. Cassava inflorescence is developed from the fork of the branchings (Fig. 9). The female flowers (Fig. 10) which are larger in size but smaller in numbers than the male flowers are situated at the base of the inflorescence while the male flowers, often numerous are located on the upper part of the inflorescence. Female flowers open 1-2 weeks earlier than the male flowers on the same inflorescence. However, male and female flowers located on different inflorescences of the same plant can still open at the same time. Consequently, selfing can occur. Pollination behavior Despite the occurrence of self-pollination, cassava is considered a cross-pollinated species and cross-pollination is done by insects. The average size of cassava pollen ranges from 122-148 µm, much larger and heavier than pollen of most other species. The longevity of cassava pollen is relatively short, lasting no more than 2 days. The degree of self-pollination depends on genotype, environmental conditions, and the presence of pollinating insects. Inbreeding depression is severe in cassava, thus seedlings of selfed progeny typically exhibit low vigor and lack competitiveness. Seed formation, dormancy, and storage Cassava fruit is a trilocular capsule (Fig. 11), with each capsule containing a single seed (Fig. 12). It takes 75-90 days after pollination for a cassava fruit to become physiologically mature. Dehiscing of mature cassava fruits is explosive, therefore bagging must be done before fruits become mature to collect seed in controlled crosses. Freshly harvested seeds are generally dormant and a 3-6 month of dry storage at ambient temperature is necessary to break the dormancy. Physiologically active cassava seed can germinate readily in about 15 days. The optimum temperature for germination is between 30-35°C. Cassava seed can be stored at 4-5°C and relative humidity of 60%. A germination percentage of greater than 80% has been reported for seed stored under such conditions for a year. Propagation Methods Cassava can be propagated by either seed or stem cuttings (stakes). Because cassava plants are highly heterozygous, seed propagation will result in highly heterogeneous offspring that no longer possess the desirable traits of the seed parent. Consequently, cassava propagation is done mostly through stem cuttings. Multiplication rate is one of the determining factors that affect whether a new improved cultivar is successfully adopted or not. After one year, a typical mature cassava plant will produce 10-30 stakes sized at about 25cm (Fig. 13). Reducing the stake size to include only two nodes or one per stake will likely produce 100 or 200 stakes from a single plant per year, resulting a multiplication rate of 100 or 200. Such multiplication rates, although high may still not be sufficiently rapid to produce a large quantity of stakes in a short period of time to meet the consumer demand. Even higher multiplication rates have been achieved by growing 2-node stakes in high density in moist chambers and continuously removing sprouting shoots of 15-20cm long for rooting in boiled water. Rooted plantlets are transferred to soil and after a brief period of hardening are transplanted to the field for production. Such a system can produce a multiplication rate of 36,000. Tissue culture methods have also been developed for rapidly multiplying desirable cassava germplasm. Plantlets can be regenerated from either pre-existing meristem or through somatic embryogenesis (Fig. 14). Virus-indexed plants can be obtained by culturing very small meristems and regenerating plants. Origin of Cassava and Genetic Diversity Cassava is widely believed to be originated from the southern rim of Amazonia. It is domesticated about 5,000-7,000 years BC (Allem, 2002) and was introduced to Africa by Portuguese and Spanish explorers, likely in the sixteenth century. Cassava did not become popular in Asia until in the 1960s. The genus Manihot contains more than 100 species, all naturally occurring between 33°N (southwest USA) and 33°S (Argentina). Wild relatives that have been used for interspecific hybridization include M. catingae, M. dichotama, M. glaziovii, M. melanobasis and M. saxicola. Among these, M. glaziovii (ceara rubbertree) is the only species that has made significant contributions in developing cassava germplasm resistant to cassava mosaic disease. Cassava has 36 chromosomes, forming 18 bivalents at meiosis. However, there are cytological and sequence information supporting the paleotetraploidy nature in cassava. Breeding Objectives Yield Developing high-yielding cassava cultivars remains the highest priority of most, if not all cassava breeding programs. Root yield in cassava is, however, a complex trait and is affected by both genetics, environment, and their interactions. Cassava plants with an intermediate branching height have been shown to be highly correlated with high yield. Similarly, plants with good leaf retention (longer leaf life) are also found to be correlated with high root yield. Root quality Root quality is very important as it affects consumer acceptance and successful adoption of a cultivar. Cultivars with highly reduced cyanogenic glucosides and increased dry matter in the roots are desired. Cassava roots are naturally low in protein, therefore cultivars with enhanced protein content are desirable if they are used for animal feed. Cultivars with altered starch content and composition may be developed for specialty use. Biotic stress Cassava production is constrained by many biotic stresses including some of the most devastating viral diseases such as cassava mosaic disease (CMD) (Fig. 15) and cassava brown streak disease (CBD) that can cause significant yield loss. Bacterial diseases such as cassava bacterial blight and root rot can also cause damages. Developing cassava germplasm with resistance to a number of insects including cassava mites, mealybugs, and whiteflies which are responsible for transmitting the devastating CMD is of great importance. Abiotic stress The shelf life of cassava roots is notoriously short, often within 2 days after harvest. This post-harvest deterioration of cassava roots is manifested by internal discoloration which causes the immediate loss of marketability (Fig. 16). Developing cassava cultivars that are resistant to this post-harvest physiological deterioration is therefore highly desirable. Principles of Cassava Cultivar Development: An Overview Cassava is primarily cross-pollinated, therefore individual plants are highly heterozygous. Because cassava can be easily propagated by stem cuttings, improved cassava cultivars are primarily clonal cultivars that are multiplied by stem cuttings for distribution. Therefore, the principle of developing clonally propagated cultivars for other crops also applies to cassava. Briefly, large segregating populations are first created from which initial seedling screening is performed. Plants selected from the initial screening are then evaluated in subsequent replicated, multi-location trials that would eventually produce one or more superior clones possessing desirable traits. The following flowchart describes the general aspects of a cassava breeding procedure: 1. Population development 2. Seedling evaluation and selection 3. Clonal evaluation and selection 4. Preliminary yield trial and selection 5. Advanced yield trials and selection 6. Regional trials While the size of the initial population is large (for example, 50,000 seedlings), the number of entries is drastically reduced following each step of selection, and at the end of the regional trial, only one or a few clones may endure the rigorous selection process and are released as new cultivars. Cassava Hybridization Techniques Preparation of female flowers To determine if a female flower is about to open, Kawano (1980) described a reliable method by which a petal of an unopened female flower is peeled back, if a drop of nectar is observed at the base of the pistil, the flower will open in the afternoon of the same day (Fig. 17). Female flowers ready to be pollinated are then covered with a large (20 x 25 cm) cloth bag to avoid stray pollen. Emasculation is generally not necessary because male flowers on the same inflorescence will not open until 1 to 2 weeks later when the female flowers either developed into fruits or have died. Preparation of male flowers Between noon and 2pm in the afternoon, freshly open male flowers are collected by glass bottle or other suitable devices and pollination can be performed immediately. A single male flower can pollinate up to three female flowers. Pollination performed after 5pm will not be as effective. Pollinated female flowers can be left uncovered to promote seed development with minimal risk of hybridization by stray pollen. However covering of the pollinated flowers with a small cloth bag is needed 1 or 2 weeks after the pollination in order to collect seeds. Breeding Scheme Population development To increase the chance of obtaining a superior genotype, distinct genotypes with diverse genetic backgrounds are selected for creating the base segregating population from which evaluation and selection will be performed. Three methods are used to create the base population: 1. Controlled biparental crosses – In this case, male flowers are collected from the male parent and are used to pollinate female flowers of the chosen female parents. Seed yield from such crosses is limited because of the amount of labor involved. Many such crosses may be needed to produce a large base population. Pollination in cassava, however is viewed as one of easiest among all the crops because of its large flowers, large and sticky pollen and no need of emasculation, therefore, this method can still be productive. 2. Use of crossing blocks – In this case, a set of cultivars are grown in an isolated crossing block. Male flowers are removed from all cultivars that are chosen to be female parents and seed harvested from the female parents are hybrid seeds that can be used to start the selection process. The physical separation of male and female flowers in a cassava plant makes it easy to remove male flowers. This method is commonly used in recurrent selection. 3. Polycross – In this case, each elite parent is replicated several times and they are randomly grown in a polycross nursery. Random pollination among parents ensure a fair representation of each parent in the progeny. This method is more efficient in producing sufficient quantity of hybrid seed than the biparental method. However, it does not prevent self-pollination. Seedling evaluation trial (Year 1) Once a base population is created using one of the methods described earlier, selection can immediately start. Up to 100, 000 hybrid seeds are directly sown in the field and seedlings will be screened for resistance to major disease such as cassava mosaic disease, cassava brown streak disease and for ideal growth habit, i.e., branching at a medium height (100cm for example) and low hydrogen cyanide (HCN) content in leaves. At harvest, additional screening is performed for plants producing compact roots with short necks. In this step, selection is concentrated on eliminating poor genotypes rather than selecting for good genotypes. A total of up to 3,000 individuals may be selected for further testing. Clonal evaluation trial (Year 2) Entries selected from the seedling evaluation trial are multiplied by stem cuttings and each entry is grown in single-row plot without replication. Resistance to major diseases is again screened. In addition, root yield, root dry matter content as well as the HCN content in the root of each entry is evaluated. A locally-adapted leading cultivar is grown as a check to aid selection. A total of up to 100 individuals are selected. Preliminary yield trial (Year 3) Stem cuttings from each of the selected entries are grown into larger plots that are replicated at least twice, along with a locally-adapted check cultivar to evaluate yield potential. This trial is conducted in multiple locations. The clonal entries within each location is randomized. Besides root yield, root quality and resistance to major disease and other pests will be continuously monitored. Evaluation of consumer acceptance is conducted at this stage. A total of 25 genotypes may be selected at the end of the trial. Advanced yield trial (Year 4) Stem cuttings from the selected genotypes are grown into larger plot size with more replications (for example 4) and more locations, along with a locally-adapted check cultivar. Selection is primarily focused on root yield and root quality traits. At the end of the trials, up to 5 genotypes may be selected. Regional yield trial (Year 5) At this stage, the best clones are grown on large-scale farms in multiple locations within the target region. At least four replications are used within each location. Final selection will be made based on data for yield, root quality, and consumer acceptance. Planting materials will be rapidly multiplied and distributed to end-users. Note that “upgraded” base populations can be continuously created by crossing elite entries selected at the end of each step and additional germplasm from other sources. A new cycle of selection can then be performed on the “upgraded” base populations. Farmer Participatory Trial Traditionally, farmers are not involved in the breeding process until a new cultivar is close to being released commercially, therefore farmers have limited influence on how new cultivars are developed. Participatory Plant Breeding (PPB) is a process in which farmers play an active role early in the breeding process and it has become increasingly important in breeding grain crops as well as vegetatively propagated crops such as cassava. For example, in developing early bulking cassava cultivars in Kenya, farmers are invited to participate in the selection process after the 1st clonal evaluation trial. Farmers evaluated root quality traits including appearance, size, taste/texture, and their inputs were used along with those of the breeders in making final selection decisions. A similar effort involving farmers early in the breeding process was also reported in developing cassava mosaic disease-resistant cultivars in Ghana. Subsistence farmers in Africa often grow multiple crops/cultivars to counter the uncertain and adverse climatic conditions. Under such circumstances, PPB has been proven very useful and it also increased the adoption rate of new cultivars released by programs in which farmers have actively participated in selecting. Germplasm Conservation Centers and Practice Genetic diversity is essential to plant breeding. Modern agricultural practices and the destruction of native cassava habitats in the centers of diversity cause significant erosion of cassava genetic resource. Therefore, collection and conservation of cassava germplasm is of paramount importance to sustained successes in cassava breeding. Great efforts have been put forth to collect and conserve cassava germplasm. Table 1 lists the number of accessions maintained at each location. The international institutes, the Centro Internacional de Agricultura Tropical (CIAT), Colombia, and the International Institute for Tropical Agriculture (IITA), Nigeria, both of which hold the UN mandate for cassava maintain a large number of accessions. These accessions are available freely to the public. Cassava germplasm is typically conserved in one of the three methods: Field genebanks Cassava plants representing each accession are grown and maintained in the field. As is practiced at IITA, eleven plants of each accession are grown on a 2.5m row plot with a 25cm space between plants and 50cm space between rows. Field genebanks require a large amount of field space and germplasm may be lost due to various biotic and abiotic stresses. Seed genebank Seed of each accession are maintained in controlled environment with low temperatures and humidity. It has been reported that cassava seeds remain viable after 14 years of hermetic storage at -20°C with 6% moisture content in the seed. As is practiced at IITA, seeds representing each accession are harvested in bulk from all plants of an accession and are therefore heterogeneous. In vitro genebanks Table 1 The second report on the state of the world’s plant genetic resources for food and agriculture. Adapted from FAOSTAT 2010, Rome. Location Number of accessions Type of accession (%) Wild species Landraces/ old cultivars Research materials/ breeding lines Advanced cultivars Others/ mixture CIAT 5436 1 87 11 0 1 Brazil 2889 0 0 0 0 100 IITA 2756 0 28 47 0 25 India 1327 0 0 0 0 100 Nigeria 1174 0 0 0 0 100 Uganda 1136 0 4 89 7 0 Malawi 978 0 22 72 6 0 Indonesia 954 0 0 0 100 0 Thailand 609 0 0 100 0 0 Benin 600 0 100 0 0 0 Togo 435 0 100 0 0 0 Other 14148 6 26 3 14 51 In vitro cultures established from apical buds or nodes containing axillary buds are maintained on culture media or culture conditions that slow down the growth of the culture. Such cultures can be maintained for 8-12 months without the need for subculture. Following disease and virus indexing, these cultures are suitable for exchange among collaborators across countries. Cassava Genetics Marker-assisted breeding in cassava DNA markers are variations in DNA sequences, and such variations among individuals can be easily detected using polymerase chain reaction (PCR) or high throughput sequencing technology. DNA markers, unlike phenotypes are not affected by environment or developmental stages; therefore they can be assessed at any time and from any plant tissue. They are used for cultivar fingerprinting, assessing genetic diversity or marker-assisted selection for traits for which markers are available. In cassava, many simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers have been developed. Recently, a high-resolution composite map covering 2412cM and organizing 22,403 genetic markers on 18 chromosomes has been described for cassava (International Cassava Genetic Map Consortium). This map will greatly facilitate marker-assisted selection in cassava, particularly for selecting plants resistant to the cassava mosaic disease for which linked DNA markers have been developed. Sequencing of the cassava genome The genome size of cassava is estimated to be ~770Mbp. It has recently been sequenced from a partially inbred line AM560-2 developed at CIAT. The complete sequence is made available by the Joint Genome Institute (JGI). A resequencing project in 2012 report a genome size of 760 Mbp. The sequenced genome will undoubtedly enable a whole array of research aimed at improving the crop. Application of Biotechnology in Cassava Traditional plant breeding is a very long and imprecise process and it could take more than a decade to release an improved new cultivar. Genetic transformation, however can introduce a trait very efficiently and rapidly. Furthermore it can break the reproductive barrier and transfer traits from unrelated species to cassava. Because cassava is a vegetatively propagated species, traits introduced into an existing elite cultivar or a farmer-favored landrace can be “fixed” immediately without the need of inbreeding or backcross as is the case in sexually propagated species. Successful genetic transformation has been accomplished in cassava using either Agrobacterium or particle bombardment. The key to the success relies on an efficient plant regeneration protocol. The current standard practice is to use the friable embryogenic callus (FEC) derived from immature leaf explants cultured in vitro for gene transfer. The potential of using a transgenic approach to produce novel cassava germplasm has been demonstrated in transgenic lines with insect and disease resistance, herbicide tolerance, altered starch content and increased protein level as well as reduced cyanogenic content in cassava storage roots. Another noted example is the biofortified cassava generated by the BioCassava Plus (BC+) program supported by the Bill and Melinda Gates Foundation. These biofortified cassava varieties demonstrated the potential of developing cassava as a more nutritionally complete crop with increased zinc, iron, proteins and vitamin A. Major accomplishments in cassava breeding Notable is the consistent increase in production and harvested area despite the low yields in Africa compared to Asia and Latin America/Caribbean (Figs 19, 20, 21). Thus there is potential for even greater increases in Africa with the increased availability of improve cultivars.

textbooks/bio/Agriculture_and_Horticulture/Crop_Improvement_(Suza_and_Lamkey)/1.15%3A_Cassava_Breeding.txt

Teshale Mamo; Asheesh Singh; and Anthony A. Mahama Seed is a basic and fundamental input for agriculture. Accessibility of high-quality seed is one of the basic requirements to increase crop productivity, production and use (Pelmer, 2005). The dissemination and use of high-quality seeds have great benefits to increase and continue crop production, improve household incomes, minimize risks of insect pests and plant diseases, and enhance the crop production patterns, which would increase agriculture sustainability. Therefore, viable seed supply system strategies are important to ensure the availability of good quality varieties of seed to farmers in a timely and affordable fashion (FAO, 1999). Learning Objectives • Differentiate between formal (commercial), informal, and semi-formal (integrated) seed systems and their development • Develop knowledge of seed regulation systems • Know the International Union For The Protection Of New Varieties of Plants (UPOV) • Demonstration knowledge of Breeders’ Right • Know the different classes of seed Current Seed System in Sub-Saharan Africa Formal Seed System The seed system represents involvement and interconnection among different organizations, institutions, and individuals associated with the development of new varieties and producing, testing, processing, storage, certifying and marketing seed to the farmers. Public and private sectors are highly involved in the production of different classes of seed for domestic/local use and export market. In sub-Saharan Africa, the majority of smallholder farmers are involved in various kinds of seed systems, which benefit them to produce and obtain the seed they need. There are three broad categories of seed systems in sub-Saharan Africa: Formal (commercial) seed system, informal seed system (local seed supply system), and integrated seed system (community-based) (Table 1). The detailed description of each seed system is outlined below. The Formal (commercial) Seed System The formal seed supply system is highly regulated and covers seed production and supply mechanisms. This system involves a chain of activities leading to clearly defined products, i.e., certified seed of verified cultivars (Louwaars, 1994). It involves formal plant breeding, multiplication by seed companies, following established procedures including processing, bagging, labeling, and marketing. The formal systems also follow the standard of distinctness, uniformity and stability (DUS) of varieties. The system also assures that the cultivar identity and purity are kept throughout various levels of seed multiplication (Breeder/Prebasic to Foundation/Basic to Registered and/or certified seed). The main participants in a formal seed supply system are private and public sectors, and mainly focus on major economically viable crop species with good recurrent seed demands, such as hybrid maize. This kind of seed system is dominant in developed countries. It is a more complex system compared to the informal seed system. The formal seed system produces about 10-20% of the seed demands in Africa (Wekundah, 2012). Informal Seed System Informal seed supply system is also sometimes called as ‘farmer seed system’ or ‘traditional seed system’. It is a chain of seed multiplication and marketing steps that involve farmers who produce, disseminate or access seed through farmer-to-farmer seed exchange based on barter system and through local grain/seed markets mainly based on indigenous knowledge and local diffusion mechanism. In addition, small private companies and farmers cooperatives are involved in seed production in many countries for example, Tanzania, Uganda and Malawi. The informal seed supply system is mostly characterized by its flexibility and operates under non-law regulated conditions. Cultivars may be landraces/local varieties or mixture of different varieties of the same races or may be heterogeneous. Besides, the seed may be of variable quality in terms of purity, physical and physiological quality (Almekinders and Louwaars, 1999). Though the informal seed supply is not formally framed, it covers the majority of seed related activities in most of sub-Saharan Africa and contributes about 80-100% of seed supply to the farmers (Maredia et al. 1999; Wekundah, 2012). It can also enhance wide diffusion of seed over relatively wide areas and promote the small scale seed businesses in the region (Sperling and Cooper, 2004). However, little is known about the system, production and marketing chain due to lack of regulation. The informal seed system is believed to help the farmers due to the following factors: • Retain seed on farm from previous harvest/farm saved seeds • Farmer-to-farmer exchange networks • The seed do not go under certification process so it is less expensive Seed Sector Development Table 1 Range of seed sector development. Data from FAO, 2010. Formal seed system Informal seed system Medium to large companies Small scale enterprise On-farm management Plant breeding capacity Limited to public sector Farmer selection Variety registration and release process Not applicable Inexistent Early generation seed production capacity Not applicable Inexistent Seed policy and regulatory framework adapted Inexistent or not always adapted Inexistent Seed quality assurance and capacity Inadequate capacity Not applicable Seed production capacity Limited Farmer saved seed Seed conditioning, storage capacity Limited Farmer’s practice Entrepreneurial capacity Limited Not applicable Access to credit Limited Not applicable Market access Limited Seed exchange or local market Integrated Seed Supply Systems (Semi-formal Seed Supply System) This is a mix of informal and formal seed supply systems. Small farmers and community-based organizations such as small famers’ cooperatives multiply and sell a small amount of good affirmed seed of improved cultivars to other farmers within a restricted production area with the least possible quality control (Alemkinders and Louwaars, 1999). Variety Release Regulations Seed Regulation Systems Most sub-Saharan African countries differ highly in seed regulation systems. This seed regulation system is comprised of seed quality control/certification and cultivar regulation. Cultivar regulation system follows steps to control the release of cultivars both by private seed companies and government-owned research institution breeding programs. The cultivar registration requires new cultivars to show distinctiveness, uniformity and stability (DUS), and value for cultivation and use (VCU) before being officially registered (Setimela et al., 2009). Each cultivar registration is performed by national and private breeding programs. Meanwhile, the national cultivar releasing committees have different criteria to register a new variety. Depending on the country’s variety release regulations, the DUS and the VCU tests may take one to three years (three seasons) before enough data are available for cultivar registration. The seed law in terms of evaluation and release of varieties are different and inconsistent among sub-Saharan African countries. These different regulations and inconsistent seed laws (and implementation) among countries make it costly and discouraging for private seed companies to release and market their new cultivars. Most crop breeding programs in sub-Saharan Africa differ in their capacity. Some national and regional crop breeding programs focus on testing lines introduced from other countries, while others have established their own crossing/hybridization programs to develop breeding lines targeting specific and wide crop growing environments. The consultative Group for International Agricultural Research (CGIAR) such as International Maize and Wheat Improvement Center (CIMMYT), International Institute for Tropical Agriculture (IITA), International Rice Research Institute (IRRI), International Crops Research Institute for Semi-Arid Tropics (ICRISAT), International Center for Tropical Agriculture (CIAT) and International Center for Agricultural Research in the Dry Areas (ICARDA) have helped and contributed for crop varieties and other crop technology development (David and Sperlings, 1999). Common Features of Regulations In most African countries the following common features of regulations for cultivar release have been established: • Developed guidelines and standard procedures for testing cultivars proposed for release. • Independent national varietal releasing committee (NVRC) formed with a mandate to recommend for release or reject based on test results. • Officially released cultivars that have been recommended by NVRC should be registered and made available to the public. Sufficient information on morphological description, year of release, variety name, and releasing institutions should be clearly indicated. The International Union For The Protection Of New Varieties of Plants (UPOV) The International Union for the Protection of New Varieties of Plants (UPOV) is an intergovernmental organization with a goal of providing and promoting plant variety protection (UPVO, 2015). The main objective of UPVO is to strengthen the development of new cultivars that benefit the farmers (UPVO, 2015). The UPOV helps to recognize the rights of plant breeders for the varieties they develop. The UPVO convention provides intellectual property rights to the breeder that enables her/him to have full authority on seed multiplication of her/his cultivar. The breeder’s right is implemented if the variety is new, distinct, uniform and stable (UPVO, 2015). Among sub-Saharan African countries, South Africa, Kenya, Morocco, and Tunisia are the only members of UPOV (UPOV 2017). However, Africa has its own regional organization called African regional intellectual property organization (ARIPO) with the main objective of pooling resources of its member countries to solve intellectual property (IP) and related issues through harmonizing IP laws and facilitating IP activities within member countries and distributing of information associated with IP. There are 19 member countries in ARIPO: Botswana, The Gambia, Ghana, Kenya, Lesotho, Malawi, Mozambique, Namibia, Sierra Leone, Liberia, Rwanda, São Tomé and Príncipe, Somalia, Sudan, Swaziland, Tanzania, Uganda, Zambia and Zimbabwe. Plant Breeder’s Right Plant breeder’s right: is an intellectual property right granted to a crop breeder in respect to new plant varieties developed by him/her against exploitation without his/her permission. The breeder has exclusive control over his/her new plant materials such as seed, cuttings, tissue culture and harvested materials including fruit and foliage for a number of years. This provides to a plant breeder a recognition and economic reward for his effort and also energizes the plant breeder to continue developing new and better high yield good quality varieties. According to South Africa’s plant breeders’ Rights Act (Act 15 of 1976), once the plant materials or cultivars are approved then the plant breeder is given a certificate of plant breeder’s right. This plant breeder’s right is valid for 25 years in the case of vines and trees, and for 20 years for annual crops, which is started from the date on which a certificate of registration is given. • Eligibility for protection; the cultivar • must be new, • distinct • uniform • Stable and have acceptable variety name • Distinct: it is distinguishable from any other existing cultivars of common knowledge at the time of application. • The new variety should be uniform: It should be adequately uniform in its unique characteristics. • The new variety should be stable (DUS): The essential characteristics of the new varieties should remain unchanged after repeated propagation or multiplications. Who Can Apply Who can apply for a plant breeder’s right: a breeder who bred the cultivars and the employer of the breeder who bred the varieties. According to the South Africa breeder’s right: the following steps must be authorized by the breeder: • Seed production and reproduction • Permission for sale • Exporting and importing Right of plant breeders: the breeder who developed new varieties has the following rights: • The right to sell his/her new varieties including the right to delegate other persons to sell or multiply his/her new varieties. • Full right to multiply his/her new cultivars including the right to authorize other persons to multiply or propagate his/her varieties for sale Variety Performance Testing This is a variety trial focusing on the selection of new cultivars with desirable traits that could meet the requirements of farmers or consumers. This test ensures that the new cultivar is similar to or better than the existing cultivars in terms of agronomic characteristics such as grain yield and diseases and insect pest resistance. In the majority of sub-Saharan African countries such as in Uganda, Malawi, Ghana, Kenya, Tanzania, Rwanda, Burundi, Nigeria, Cameroon, Angola, and Zambia, multi-environment and multi-year variety trials are conducted across different agro-ecological zones to select better performing cultivars (FAO, 1994). The new cultivars have to show better performance in acceptable number of tests in comparison to existing/commercial cultivar(s). The variety performance testing usually includes testing for two to three years in regional or national varietal trials at least in 3 or 4 locations before being recommended for release (Bishaw and Gastel, 2009). The cultivar which is proposed to be released should be uniform, stable and distinctly better than the existing commercial cultivars in the environments where it is intended to be grown and should have also good agronomic characters and fulfill farmers’ requirements. The decision for variety release is made by National Variety Releasing Committee (NVRC). However, in some countries where not enough number of released varieties are available with the unique quality of a particular crop, the NVRC may consider releasing of varieties despite not being better than the existing commercial varieties (Setimela et al., 2009). All sub-Saharan African countries have their own variety release procedure in place even if it is done by an ad hoc committee or by officially assigned authority (Bishaw and Gastel, 2009). Variety Release After the variety is officially released and registered, the breeder or institution makes appropriate quantities of the breeder seed and basic seed available for commercial seed multiplication and marketing (Bishaw and Gastel, 2009). In a new development, Drought Tolerant Maize for Africa (DTMA) governed under CIMMYT has proposed a regional harmonization of seed laws in eastern, southern and western African countries, and they will get advantages from the free flow or exchange of maize germplasm across the regions if the regional maize variety release process is implemented. The seed laws allow a maize cultivar released in one country to be considered for automatic release in neighboring countries with similar environments. This helps to release varieties in mega-environments that cover large adaptation zones across country boundaries and also helps to link and create a big maize seed market and fast variety release across the regions (Setimela et al., 2009) Condition for Release • Appropriate documents and based on guide, clear morphological description, distinguishing characters, vegetative description and quality test (palatability, taste etc.) • Sufficient season data from multiple sites [check country registration system guidelines], wide adaptation (at national level), specific adaptation (regional level). • The new cultivar has to show better performance compared to existing commercial cultivars in environments where it is intended to be grown. • The cultivars should show distinctiveness, uniformity, Stability (DUS), and value for cultivation and use (VCU). Seed Quality and Certification Seed certification is a tool to produce genetically pure and good quality standard seed of improved varieties for farmers. Also, it is appraised for true to type physical purity, germination, seed health and moisture contents, true labeling, backed with appropriate laws and regulation and DUS. The newly released variety must have excellent seed quality attributes which is critical to crop production whereas if the seed is of poor quality, it lowers the potential yield of the variety. Seed Quality Attributes • Genetic purity: The seeds have to be genetically pure, this means true-to-type of the specific seed lot. For example breeder seeds must be 100%, foundation seed 99.5%, and certified seed varieties 98 % genetically pure (Brijesh Tiwari, 2014). • Physical purity or physical qualities: This is characterized by minimum of damaged seed (broken, cracked or shriveled seed), minimal noxious weed seed or other crop seeds and inert matter, minimal diseased seed (discolored or stained seed) in a sample seed lot. • Physiological attributes/physical qualities: This includes high germination and vigor of the seed. • Seed health: This refers to free from diseases and insect pests. Example, seedborne diseases could have impact on the health and productivity of the crop which may cause contamination of the seed lot. Seed Production Current seed production systems in sub-Saharan African countries include both the formal, which involves both the public institutions and private seed companies, or informal, which includes small scale informal village and community level seed production (Table 3). Hybrid maize seed production is mainly run by both public and private seed companies, whereas legume crops seeds are not extensively produced by public and private seed companies, and they are mostly produced by informal village and community level seed production. Legumes are not widely commercial crops in most Africa countries therefore market demand for good quality and uniform seeds is low (Muigai et al., 2010). In advanced or formal seed production system, five different classes of standard are known, though each country has its own specification based on the affiliate international protocols such as International Seed Testing Association (ISTA) or OECD seed schemes, or Union for the Protection of Varieties (UPOV). For example, the seed laboratory of Zimbabwe and Zambia is mandated for seed quality control and is accredited to the international Seed Testing Association (ISTA) (Muigai et al., 2010). Different Classes of Seed In sub-Saharan Africa, four major classes of seeds are currently being produced by public institutions and private companies. Even some of the countries such as South Africa, Kenya, and Zimbabwe have accredited seed certification by OECD (Organization for Economic Cooperation and Development (Europe) and AOSCA (Association of official seed Certifying Agency (Table 1). Seed Classes 1. Breeder seed: This is the highest purity of the new cultivar and maintained and multiplied by breeder, and provided to the seed companies for multiplication by breeder’s institutions. This class of seed is used to increase foundation seed. 2. Foundation seed: This is a class of seed produced from breeder seed. The breeder and research institutions are the ones who help to keep genetic purity and identity. Depending on the seed regulation of the country, foundation seeds could be produced by public or private seed companies. 3. Registered seed: This class of seed is produced from foundation seed and is produced by selected farmers and seed companies under the seed regulation agency to keep varietal identity and purity. In most countries, production of registered seed undergoes field and seed (lab) inspection by representative seed inspectors to ensure the fulfillment of the standards. 4. Certified seed: This class of seed is produced from foundation or registered seed, or sometimes from certified seed and is available to farmers for general production. It is grown by selected farmers who have experience and capacity to produce the certified seed. This helps to maintain varietal purity. Production of certified seed is subjected to field and seed (lab) inspection priority to approval by certifying agency. Comparing Class Systems Table 2 Seed class system of Organization for Economic Cooperation and Development (OECD) and comparable US seed classes. Data from www.oecd.library.org. US Seed Class Label color Equivalent OECD Seed Classes OECD Label color Breeder n/a Prebasic White with diagonal violet stripe Foundation White Basic White Registered Purple Basic White Certified Blue 1st Generation Certified Seed Blue Certified produced from certified Blue 2nd Generation Certified Seed Red Table 3 Differences between the formal and informal sector. Data from Minot et al. , 2007 Component of seed system Formal seed system Informal seed system Varietal development Plant breeders employed by the public institution or private company select for specific traits and produced varietal pure “breeder seed” Farmers select seed from plants with desirable traits, but the seed is not necessarily varietally pure Seed production State or private seed companies multiply seed under strict conditions to avoid mixture of varieties; sometimes contract farmers. Farmers produce seed along with crops; in some cases the portion of the crop destined for seed is given special management Processing Seed is dried using mechanical dryers. Seed may be cleaned by hand, processing machinery used to remove dirt, dried in the sun, and sometimes rocks, and seeds of other plants. May be treated to extend shelf-life. Seed may be cleaned by hand, dried in the sun, and sometimes treated to extend shelf-life. Certification Seed is usually subjected to some formal quality control procedure based on tests of purity and germination of random samples Seed is generally not tested, certified, or labeled. Distribution Seed is bagged and labeled, and distributed by stockists, extension agents, NGOs, and cooperatives Farmers use seed they save from previous harvest, acquired from other farmers through barter, gift, or sales, or acquired in local grain markets. 02: Applied Learning Activities 2 The following downloadable Applied Learning Activities (ALAs) are associated with the Crop Improvement course:

textbooks/bio/Agriculture_and_Horticulture/Crop_Improvement_(Suza_and_Lamkey)/1.16%3A_Seed_Systems_and_Certification.txt

Hunter-Gatherers Before the agricultural revolution (10,000–12,000 years ago), hunting and gathering was, universally, our species’ way of life. It sustained humanity in a multitude of environments for 200,000 years—95 percent of human history. Why did our ancestors abandon their traditional way of life to pursue agriculture? For a long time, scientists, including Charles Darwin, assumed that primitive humans invented agriculture by chance, and once the secret was discovered, the transition toward agriculture was inevitable. However, this is only possible if we assume that (i) the biggest obstacle to the adoption of agriculture was a lack of knowledge about plants’ life cycles and propagation and (ii) farming was easier than hunting and gathering from its beginnings. We can’t verify or refute these hypotheses directly. However, studies of materials (e.g., tools made of stones and bones, fossilized seeds, rock paintings, and engravings) found in many archaeological excavations, as well as anthropological studies on present-day hunters and gatherers—who still live in various corners of the world—have contributed much to our understanding of the early history of agriculture. Scholars’ interest in the contemporary hunters and gatherers was rekindled after the 1966 Man the Hunter conference, organized by Irven DeVore and Richard Lee in Chicago. Richard Lee, a PhD student studying under DeVore, had lived in Botswana with the !Kung, one of the San (Bushman) clans of Kalahari Desert, for three years (1963–1965). He shared his experience at the conference and reported !Kung men hunt and !Kung women gather. He added, “Although hunting involves a great deal of effort and prestige, plant foods provide from 60–80 per cent of the annual diet by weight. Meat has come to be regarded as a special treat; when available, it is welcomed as a break from the routine of vegetable foods, but it is never depended upon as a staple” (1). He further added that the !Kung had a more than adequate diet achieved by a subsistence work effort of only two or three days per week, a far lower level than that required of wage workers in our own industrial society, and working adults easily take responsibility for children, old people, and the disabled. In these groups, starvation, malnutrition, and crime are nil. He argued that the social lives of the people of the Bushmen clans are more dignified than that of civilized society and concluded, “First, life in the state of nature is not necessarily nasty, brutish, and short” (1). American anthropologist Marshall Sahlins agreed with Richard Lee, stating that Australia’s indigenous people also have substantial resources when compared to the common man of industrial society and work fewer hours per day, with more time for leisure. He explained that the hunter-gatherers consume less energy per capita per year than any other group of human beings, and yet all the people’s material wants were easily satisfied. Sahlins further adds, “Hunters and gatherers work less than we do, and rather than a grind the food quest is intermittent, leisure is abundant, and there is a more sleep in the daytime per capita than in any other condition of society” (2). The prevailing belief till then was that in comparison to civilized societies, the hunters and gatherers were impoverished: their way of life precarious, full of hardship, and the life of people in such a state of nature, short and brutish. When the proceedings of this conference were published in 1968, such prejudices were refuted and put to rest, rousing a new interest in researchers worldwide to study contemporary hunters and gatherers. Over the past fifty years, anthropologists, archeologists, biologists, botanists, demographers, and linguists have studied various tribes of contemporary hunter-gatherers. These studies suggest that hunter-gatherers possess tremendous knowledge about the flora and fauna present in their surroundings. They can identify edible plants from a sea of wild vegetation and know which plants’ parts can be eaten raw and which need cooking or further processing. In their memories, they retain a seasonal calendar: they know when new plants sprout, bloom, and are ready for harvest or when animals and birds breed. They extract medicines, drugs, intoxicants, and poisons from various plants and make fibers for clothing, baskets, and other objects. The marks of seasonal variations and their specific geographical surroundings are visible in their diets. For example, people living in Arctic regions are entirely carnivorous; the Hadza of Tanzania are predominantly vegetarians; and the !Kung San of the Kalahari Desert in South Africa are omnivores. Regardless of their locale, hunters and gatherers consume ~500 varieties of food throughout the year and make the best possible use of the resources available to them. In comparison, today’s rich urban folks hardly sample food items from fifty unique sources. It has also been observed that most hunter-gatherers care about their environment. They do not hunt without need, waste less, and play active roles in managing their resources. For example, natives living in different parts of the world set forest fires at fixed intervals to manage the landscape. Such controlled fires help eliminate weeds and insects and promote the germination of seeds that are enclosed within hard shells (e.g., pinenuts, chestnuts, and walnuts), thus deliberately increasing the number of seed-producing plants for them to eat. Afterward, when the fire is extinguished, grass grows on the ground, and herbivores are attracted to these pastures for several months, thereby making hunting easy. Such multilevel environmental management is just one example of how these people use their knowledge of the natural world to survive outside of agricultural society. Many foragers are also aware of how to produce food and occasionally do so in hours of need. For example, New Guinea tribes weed and prune the Sago palms that grow in the forest to increase their yield. The natives of northern Australia bury the tips of taro and eddoes into the ground to propagate new plants and channel rainwater to plains where many wild grasses grow. Subsequently, they harvest tubers and seeds for their consumption. It is not very difficult to comprehend that compared to foraging, farming is labor-intensive and requires substantial planning to sow, weed, harvest, process, and store crops. Farming must have been a very difficult task in prehistoric times, and crop failures would have been prevalent. Thus as long as needs were fulfilled by hunting and gathering, people likely did not pursue farming despite having the knowledge required for plant propagation. For centuries, humans were sustained instead by a mixed strategy that included hunting, fishing, foraging, and some farming. When resources from the wild were plentiful, farming was abandoned. It was thousands of years before human societies began to completely rely on agriculture. The growth of agriculture was not linear but rather erratic; its adoption was not a coincidence but a slow pursuit full of trials and errors. Archaeological evidence suggests that nomadic human tribes began farming during the Neolithic period, and so it is often referred to as the Neolithic Revolution. About 12,000 years ago, one of the first attempts at farming began in the Fertile Crescent, the Levant region of the Near East that includes the interior areas of present-day Turkey, Israel, Syria, Jordan, Lebanon, Iran, Iraq, Turkmenistan, and Asia Minor. The ancient inhabitants of the Fertile Crescent, known as Natufians, gathered wild wheat, barley, lentils, almonds, and so on and hunted cattle, gazelle, deer, horses, and wild boars (see figure 1.1). Throughout this region, thousands of archeological excavations have been conducted and numerous objects—including grinding stones, flint, bone tools, stone sickles, dentalium, shell ornaments, and many fine tools of polished stone—have been unearthed. These show that prior to the adoption of agriculture, the inhabitants of this region had already acquired knowledge about their surrounding vegetation and used tools for cutting, uprooting, and harvesting wild plants, which could have made their transition toward farming easier. The direct descendants of the Natufians, the prepottery Neolithic people, successfully domesticated more than 150 crops, including barley, wheat, pulses, and so on. They also domesticated animals and built the first villages in human history. Numerous discoveries in recent years indicate that besides the Fertile Crescent, parallel efforts of cultivation began in several other parts of the world. For example, 9,000 years ago, in China’s Yellow River valley, rice cultivation began; 5,000 to 8,000 years ago, in Africa and East Asia, the cultivation of a variety of roots and tubers was underway; and 7,000 to 9,000 years ago, people in South America were growing maize, beans, and squash. Hence the history of agriculture is only 12,000 years old, and it spread around the globe relatively quickly. But how did geographically isolated human groups, unaware of one another’s existence, begin farming within this short period of time, ditching the hunter-gatherer way of life? Agriculture? Australian archaeologist Vere Gordon Childe first linked the beginning of agriculture to climate change. He suggests that at the end of the last ice age (6,000–13,000 years ago), the earth’s average temperature increased and glaciers moved rapidly northward. Additionally, rainfall progressively decreased in Southwest Asia and Africa; thus year after year, this region suffered spells of drought that caused the loss of vegetation and several animal species. Over a prolonged period, the rainforests turned into savannas, where herbivores dwelled only for a few months. Under the changed circumstances, humans were unable to sustain themselves throughout the year by hunting and foraging. The human groups, living on different continents and unaware of one another’s existence, were forced to produce their own food, and farming began within a short span of time all around the globe. The transition toward farming was not easy. It was not a eureka moment; agriculture was adopted under unpleasant circumstances and the obligation to produce food was indeed a farewell to the heaven for mankind. Consider the story of Eve, who, under the influence of a snake, plucks the forbidden fruit and eats it with Adam; as a consequence, they acquire wisdom and develop a sense of good and evil. The Lord God becomes angry, and as a punishment, Adam and Eve are banished from the Garden of Eden to work the ground and grow their own food to survive. To Adam, God says, Because you listened to your wife and ate fruit from the tree about which I commanded you, “You must not eat from it,” Cursed is the ground because of you; through painful toil you will eat food from it all the days of your life. It will produce thorns and thistles for you, and you will eat the plants of the field. By the sweat of your brow you will eat your food until you return to the ground, since from it you were taken; for dust you are and to dust you will return.[1] Farming was not fun. It required tremendous effort and the capacity to endure hardship. Women’s Enterprise It is believed that agriculture was invented by women. The women of the preagrarian societies collected wild fruits, berries, tubers, and roots and had generational experience in identifying edible plants and knowledge about plants’ life cycles and how they grow. It has been suggested that women’s extraordinary vision, more developed motor skills, and ability to process finer details evolved due to the importance of their involvement in foraging activities for millions of years. For example, the average woman’s eyes can distinguish about 250 shades and hues, while an average man’s can only see 40–50. When droughts became regular, the tribes of our ancestors made temporary encampments along the lakes and ponds where men ambushed animals who came to quench their thirst. Their foraging experience helped womenfolk take the initiative in growing food. They sowed the seeds of wild grasses in the surrounding marshes and planted parts of the tubers to propagate new plants. In most traditional societies, even today, this historical association of women in agriculture is revered: often, women sow the first seeds to bestow good luck for a bountiful harvest. Invariably, across all cultures, we find a similar feminine influence in stories related to the origin of farming. For example, in ancient Egypt, Isis was deemed the goddess of agriculture: Once upon a time, a severe drought caused a widespread famine on the earth. There was nothing to eat, and cannibalism began. In such a situation, the goddess Isis offered barley and wheat from the wild to the starving people, and taught them how to produce their own food. Thus farming saved mankind from starvation. In Greek mythology, Demeter was the goddess of fertility and the harvest. After every harvest, the first loaf of bread was offered to her as a sacrifice. She was called Ceres by the Romans, and thus grains were called “cereal.” The legend of Demeter contains an interesting tale about the origin of agriculture. According to this myth, with the blessing of Demeter, the earth was always filled with grains, berries, and fruits. Humans took their share of this bounty and survived happily for a long time. But it came to a sudden end when Hades, the god of the underworld, abducted Demeter’s lovely daughter Persephone. Persephone was Demeter’s only child and the center of all her attention and devotion. Demeter desperately searched everywhere for her daughter, but it was to no avail. She fell into depression, and transformed into an ugly old woman and became unrecognizable. She encountered abuse and ill-treatment from everyone around. Only, Celeus, the king of Eleusis, warmly welcomed her. While she continued her search for Persephone, she ignored her responsibility of making the earth fertile. Her despair had an effect on the crops; famines were prevalent and people starved. Eventually, the gods intervened and plead with Demeter to bless the earth with a good harvest, and in exchange, they forced Hades to free Persephone. However, before releasing Persephone, Hades fed her pomegranate seeds (the food of the underworld) that bound her to the underworld forever. When the gods asked Persephone to choose where she wanted to live, she wished to remain in the underworld. As a result, Demeter was devastated. Finally, Zeus intervened and came up with a compromise that allowed Persephone to spend six months per year on the earth with Demeter and six with Hades in the underworld. When Persephone visits her mother from spring to summer, the earth is full of flowers and fruits, crops grow, and harvests are bountiful. When Persephone heads back underground, Demeter falls into a depressed state, resulting in autumn and winter. Demeter did not get her daughter fully back, so, she did not restore earth’s fertility completely. However, in return for the kindness of King Celeus, she taught his son Triptolemus the art of agriculture for survival, and he later taught it to mankind. In Mexico, Hispaniola, and Latin America (the sites of the great Mayan and Aztec civilizations), we find stories about the origin of man and corn. For example, Man is born from maize; maize is the mother of man. When the gods created man, the Holy Spirits chanted for his well-being and finally maize emerged from the breasts of the mother Earth to sustain humans. Mother Earth gifted her five daughters—white, red, yellow, spiked, and blue maize—to man. Similarly, Hindu mythology has several goddesses, including Bhudevi (the earth goddess), Annapurna (the goddess of grains, who provides nutrition to everyone), and Shakti (the creator of the entire plethora of vegetation). According to the legend of Annapurna, Once, the Lord Shiva (who represents the male power, the Purush) and his wife Parvati (the creator of nature, a.k.a. Prakriti or Shakti) argued about who was superior between the two and the discussion quickly ended on a sour note. Shiva stressed the superiority of Purush (male) over Prakriti (Mother Nature). Enraged, Parvati deserted her husband and disappeared, which resulted in a widespread famine. Shiva’s followers, who were starving, asked for his help. Shiva took a begging bowl, and his band followed him. They went from door to door, but the people themselves had nothing to eat, so they turned the beggars away. Shiva’s band learned about a charity kitchen in the city of Kashi (also known as Varanasi) that was feeding everyone and decided to visit Kashi. To their surprise, Parvati owned that kitchen, and she had become the goddess Annapurna, wearing celestial purple and brown, and was serving food to the starving gods and mankind. Upon Shiva’s turn, she likewise offered food to him and his followers. Shiva realized that the existence of humanity depends on nature; the brute force of male power is not enough to sustain life on Earth. The common thread of all these myths is the appearance of the savior female deity who taught mankind to cultivate grains to survive. Although these myths have no literal value, they serve as metaphors or narratives of human experiences and survived through generational memories. They are also, in fact, consistent with the climate change theory of the origin of agriculture. Slash and Burn: Shifting Cultivation At the dawn of agriculture, the primitive people of the Neolithic era only had some tools made of stones and animal bones at their disposal. These tools were useless for such a monumental task as embarking on the path to farming. Fortunately, mankind’s knowledge and experience of taming fire came in handy. Long before the origins of modern humans, Homo erectus, an ancestor of the hominid branch, had learned to light and control fire. Afterward, the members of genus Homo moved northwards from Africa and survived the cold weather of Europe and Asia with the help of this skill. Humans used their best weapon, fire, to create the first farms. First, they slashed the vegetation, then burned it to clear the small patches in the forests, and finally, sowed seeds in the ashes. This practice of farming, known as slash-and-burn agriculture, still survives in the Amazon rainforests and in many mountainous regions of the world. The plots created by this method are very fertile initially, but with each passing year, they are overtaken by more and more weeds, pests, and parasites, which causes a decline in their fertility. So after three to four years, the people move to another site. Thus this practice is also known as “shifting cultivation” or “swidden agriculture.” In India, it is known as “jhoom” among the native Adivasi tribes (descended from an ancient forest-dwelling people). After people abandon a site, in the fallow field, grass and weeds grow, and it serves as a pasture for herbivores and hunting grounds. Slowly, the fertility of these pastures returns, shrubs and trees grow, and they become part of the forest again. In this way, the field, fallow land, and the surrounding forest are recycled. Also, weeds, insects, and other parasites are kept in balance. Today, we are farming with highly sophisticated machines and have specialized tools for various tasks, from sowing to harvesting, and yet farming is still a tremendous task. We cannot even grasp how difficult it would have been in prehistoric times. For thousands of years, generations of mankind struggled to make farming productive. They also continued to gather and hunt to make up for the shortfall or crop failures. Since farming required much more time and effort, farming would have been abandoned from time to time if nature was bountiful. It has been suggested by various studies that agriculture did not progress smoothly; it took several thousand years before humanity could fully rely on agriculture. So to summarize, the history of agriculture is 12,000 years old, and traces back to a time when changes in the earth’s global climate led to widespread drought and a decline in natural resources, forcing our ancestors to produce their own food. For thousands of years, shifting cultivation supplemented their diets while hunting and gathering remained the main source of sustenance. The discovery of agriculture was not an accident but the product of trial and error as well as improvisation that spanned many centuries, a process that still continues today. Animal husbandry is considered a by-product of agriculture. It has been suggested that during droughts, people survived on stored grains, and they used some grains to feed herbivores to keep them around for easy hunting. The credit to domesticating animals primarily goes to men. Emergence of the First Agricultural Societies Even though agriculture started almost simultaneously in many regions of the world, its progress was not uniform. The biodiversity of various geographical regions (e.g., their flora, animals, birds, insects, and microorganisms) influenced the emergence of stable agricultural societies. In some areas of the tropics, particularly in Africa, people had great success in growing tubers like yams, potatoes, eddoe, sweet potatoes, and cassava. These plants can be propagated by burying a small part of the tuber in the ground and so did not require an understanding of the plant’s life cycle. So these groups had an easy head start thanks to vegetative propagation that produced identical plants (clones), which did not differ significantly in yield. They learned to process many types of tubers and invented a very complex process of separating cyanide and starch from cassava to make starchy tapioca pearls. These undertakings helped folks sustain themselves year-round, but did not accumulate enough surplus to free a section of the population from farming, allowing them to pursue other tasks needed for the further advancement of their societies. In South and Central America, maize was the mainstay of civilization. However, the natural structure of maize plants promotes outcrossing: on this plant, male flowers, known as tassels, hang from the tip, whereas the female flowers (silk) grow on the stem. Maize pollens are very lightweight and reach the female flowers via wind. Thus male flowers can pollinate female flowers of the same plant (self-pollination) or of another plant (cross-pollination). Although in maize, the chances of self-pollination and cross-pollination events are equal, the progeny born of selfing is inferior (gives lower yield) compared to the progeny born of outcrossing. Thus farmers need to plant different varieties of maize in the same fields to ensure maximum yield. In the absence of this knowledge, the productivity of the crop cannot be assured from one year to another. Only after such an understanding developed could people rely fully on maize farming and utilize the surplus to build the great civilizations of the Aztecs and Mayans. In the Fertile Crescent, about thirty-two species of grass—including the wild species of wheat, barley, sorghum, millet, and oats—grow naturally. Incidentally, most grasses have complete flowers with both male and female organs, and self-pollination rules over cross-pollination. As a result, the characteristics of grasses remain stable, and crop yields do not vary from one year to another. If the plants with large grains are picked and carried forward, then crops with large seeds can be harvested for generations. Additionally, the grass seeds can be stored for a very long period of time and year-round dependence on these grains was easily established. So the early farmers of this region benefitted from growing grasses. In the Fertile Crescent, animal husbandry began in parallel with farming. Some groups exclusively pursued this path and so developed a nomadic way of life. They moved with their herds following the availability of grazing grounds and trading the products of one farming community with another. These pastoralists thus further strengthened the stability of farming communities and broadened the region’s resource base. Such advances helped the people of the Fertile Crescent settle in one place permanently and establish the first villages. In China, along the Yellow River, people learned to cultivate rice. Rice is also a self-pollinated grass, and so these early farmers could rely on its harvest for yearlong sustenance and could store surplus seeds. Unlike their contemporaries in the Fertile Crescent, Chinese farmers could harvest two crops of rice per year and so their surplus grew even more rapidly. So they reaped similar advantages and almost simultaneously established permanent settlements. About 5,500 years ago, an independent initiative of paddy cultivation was undertaken in the Indo-Gangetic plains that are spread across South Asia. In addition to rice, the peoples of South Asia and East Asia independently learned to grow a variety of minor grains, pulses, vegetables, fruits, tubers, roots, and oilseed crops. As agriculture progressed, many river-valley civilizations—in the Indus Valley, Egypt, Mesopotamia, and China—came into existence. Meanwhile, the populations of hunter-gatherers remained more or less stable. As agriculture developed and productivity increased, the human population grew proportionally (see figure 1.2). The first minor jump occurred after the discovery of metals that made many more tools like plows, available to farmers. The use of a plow powered by domesticated animals resulted in a significant increase in food production. In this way, 5,000 years ago, farming began to seem like a better alternative to the hunter-gatherer’s way of life. The surplus grain freed a large section of the population from farming and allowed them to invest energy in other tasks that led to the second major change—the division of labor in human society. Subsequently, the collective sharing of resources was replaced with individual ownership—private property—which gave rise to a need to ensure the succession of one’s own bloodline. As a consequence, rules of strict sexual conduct for women were formulated and, like infields and domesticated animals, they became the property of men. As class divisions deepened, the unit of a family, headed by a patriarch, strengthened. Soon, tribes headed by patriarchs began to fight for control over their possessions and to expanding their domains, which gave rise to more complex and organized social and political structures, like states, nations, and religion. In societies dependent on subsistence agriculture—where property has not developed and most of the people remain engaged in agriculture—complex social structures, division of labor, crime, patriarchy, and other sociopolitical structures are also poorly developed. Agriculture transformed human society, but this transformation also, in turn, influenced agricultural practices. While the family, private property, and various institutions were born as by-products of agriculture, these sociopolitical advancements also impacted agriculture. To this day, agriculture continues to be highly entangled with society and human history. In the following chapters, we will review the historical progress of agriculture, advancements in science and technology that influenced farming, and the impact of agriculture on humanity. Further Readings Diamond, J. (1997). Guns, germs, and steel: The fates of human societies. W. W. Norton. Lee, R. B. (1979). The !Kung San: Men, women and work in a foraging society. Cambridge University Press. 1. From Genesis 4:23, from the Holy Bible, New International Version®. NIV®. Copyright © 1973, 1978, 1984 by International Bible Society. Used by permission of Zondervan. All rights reserved.

textbooks/bio/Agriculture_and_Horticulture/History_and_Science_of_Cultivated_Plants_(Naithani)/1.01%3A_The_Origins_of_Agriculture.txt

Domestication of Plants and Animals Tremendous natural variations exist among the individuals of any plant species. The traits that define color, shape, flavor, height, yield, and resistance to pests, pathogens, and environmental stresses are not fixed within a species. Individual plants and animals from the same species can be easily distinguished based on these characteristics. Since the beginning of agriculture, humans have unconsciously been selecting plants and animals with desirable traits, such as large-sized grains, pods, fruits, and vegetables; sweeter and less-seeded fruits; less bitter and nonprickly vegetables; cereals with large panicles and tough rachis; non-seed-shattering plants; and so on. As a consequence of such artificial selections over many generations, unprecedented changes occurred in cultivated plants that set them apart from their ancestors and wild relatives. For example, the relentless efforts of humans led to the development of various crops, such as corn from a wild-grass teosinte; long-spiked, six-row barley from short-spiked, two-row wild barley; large tomatoes from a small berry; and a variety of less-seeded fruits and palatable vegetables from their bitter wild ancestors (see figures 2.1 and 2.2). These plants—enriched in traits that favor higher yields, productive harvest, and increased palatability—would not have come into being without the persistence of humans since the dawn of agriculture. Artificial selection by humans counteracts the process of natural selection. In nature, small fruits are packed with seeds, thistles, thorns, and prickly leaves; have a bitter taste; ripen asynchronously; and have seeds that spontaneously shatter—all traits that favor the survival of the plants. Thus artificially selected and propagated species of cultivated plants, lacking necessary traits for survival, become more vulnerable to diseases, predators, and environmental stresses. These crops cannot survive in nature for a long time without human help. For several millennia, humans have put tremendous effort into providing protection and ensuring the continuous propagation of cultivated plants. We provide fertilizers, pesticides, and water and provide services such as weeding to promote the growth of crop plants. Thus domesticated plants need humans for their survival as much as human survival depends on them. These species cannot survive in nature for a long time by themselves, but with human help, they have spread globally. For some species, this dependency on humans has become total. For example, maize absolutely depends on humans for its survival. If you leave a mature cob in the field, some of its seeds may germinate on the cob, but they will soon die due to the lack of space for emerging seedlings to grow. Furthermore, maize seeds do not fall spontaneously and need human help to be detached from the cob and planted in the soil. This mutual interdependence between crop plants and humans (and, similarly, between humans and artificially selected and bred animals) was achieved over several millennia, and this is the historical process we call “domestication.” Thus to a great extent, all crop plants and domesticated animals are man-made. Landraces of Crop Plants n addition to artificial selection, cultivated plants were continuously subjected to the natural selection imposed by their immediate environment, geographical locality, and agricultural practices. Thus domesticated plants are products of artificial selection operating within environmentally enforced natural selection and the agricultural practices prevalent in a given region. The early domesticated plants flourished in their native environment, but when shifted to new locations, they performed poorly. Over time, the few offspring of the introduced plants acquired characteristics (i.e., via spontaneous mutations or hybridization with related species present in the new locality) that helped them stabilize in their new surroundings. This process has produced diversified varieties of crops known as landraces, each adapted to a specific geography, climate, or environment (seasonal variations in day length, temperature, water availability, soil quality, salinity, etc.) and its associated pests and pathogens. The landraces differ from one another in taste, fragrance, flavor, nutrient composition, and tolerance to pathogens and environmental stresses. Before the industrialization of agriculture, traditional farmers had very small holdings but grew many varieties of the same crops for multiple uses. Typically, in a small village, one could find several cultivars of fruits and vegetables as well as many varieties of grains. Farmers have local names for each of these different varieties based on their special traits or origins. For example, more than 3,000 varieties of rice have been developed around the globe and grow in a range of climatic conditions and geographical locations, from temperate hillsides, to tropical planes, to flooded marshes in coastal regions. Each variety of rice has a name and specific usage in culinary dishes (e.g., sushi, pudding, pilaf, steamed rice, crackers, and baby food). Similarly, more than fifty races of maize are found in Mexico—thirty races in the Oaxaca province alone. Each race includes hundreds to thousands of varieties adapted to the immediate environment and climatic conditions and each has a special use (see figure 2.3). For instance, the popcorn variety’s seeds possess a hard outer cover capable of holding steam for a sufficient amount of time when heated, allowing for the full transformation of starch required for making popcorn. Conversely, the seeds from other corn varieties containing a thin seed cover do not produce good popcorn because, upon heating, they break easily, causing steam leakage. Another example is the sweet corn variety, consumed primarily after boiling or roasting, which lacks an enzyme that efficiently converts sugar to starch and thus has higher sugar content. Dent corn, the most popular corn variety grown today, has soft starch and is lightly sweeter; thus it is used for making chips and tortillas. Before 1920, flint corn was the most widespread variety because it is naturally resistant to many pests and pathogens prevalent in the tropics and is highly productive. For centuries, farmers have been stocking seeds of different varieties separately to ensure their purity and have been careful about not mixing the seeds from multiple varieties. To avoid cross-contamination, farmers usually save seeds for the next year’s sowing from the plants growing in the middle of their fields. This way, the individual varieties are maintained, but their independent evolution and development are also continued. The landraces of the various crops that have survived today are not 100 percent identical to their ancestors that existed 200 or 1,000 years ago. They are constantly changing, keeping in tune with their growing environment and adapting to the changes in it. Centers of Crop Domestication Nikolai Vavilov, a Russian agricultural scientist, was one of the first scientists in the world to infer that the process of domestication—the enrichment of desirable traits by human/artificial selection—also led to the loss of many useful traits (see figure 2.4). He noted that in comparison to their wild relatives, most crops easily succumb to parasites, pests, and pathogens and are less resilient under unfavorable environmental conditions. Vavilov proposed that these lost traits can be traced back to the wild progenitors and related species of crop plants, which are likely to still be present in the regions where crops were first domesticated. He further proposed that useful traits can be reintroduced in crops by employing the kind of systematic plant breeding rooted in the principles of Mendelian genetics (we describe the work of Mendel and the basic principle of genetics in chapter 5). In the 1920s, knowledge about the domestication centers of crop plants was lacking. So Vavilov took this herculean task upon himself and set forth on a mission to collect the germplasm (seeds, tubers, roots) of all domesticated crops and their wild relatives. He led nearly a hundred expeditions to sixty-four countries on five continents over the course of twenty years and built the world’s biggest plant germplasm collection, which included 350,000 accessions of seeds, roots, and tubers representing about 2,500 plants. Based on the study of this comprehensive collection and observations about human cultures and linguistics, Vavilov proposed eight geographic centers as the birthplaces or “centers of origin” of crops—the places where the ancestors, wild relatives, and other related species of crops still live and where mankind first began their cultivation. These eight centers include China, India and the Indo-Malayan region, Central Asia (including Pakistan, Afghanistan, Turkestan, and the northwest Indian provinces of Punjab and Kashmir), the Near East, the Mediterranean, Ethiopia, southern Mexico and Central America, and South America (Ecuador, Peru, Bolivia, Chile, and Brazil-Paraguay; see figure 2.5). Overall, Vavilov associated about 640 crops with their biodiversity centers. Five-sixths of these came from the Old World (Asia, Africa, Europe) and one-sixth from the New World (Australia, North and South America). Contrary to popular belief, Vavilov’s research revealed that crops did not originate in the large river valleys associated with the rise of human civilization. Rather, most crops were born in the kinds of geographically isolated regions (hills, desert-ridden areas, or cold or extremely hot regions) that comprise only 2–3 percent of the world’s area. In these challenging localities, the pressure of natural selection on vegetation is robust and variable compared to other areas, and so we find high biodiversity in these areas. The list below identifies the eight centers of origin of crops and associated plant species that Vavilov identified (along with a ninth, discovered after his death).[1] It is eye-opening to see how the various kinds of produce, grains, fruits, vegetables, and spices available in supermarkets today have originated in such distant places. Since human societies have been continuously in motion, crops have moved with them, away from their centers of origin, and have been subjected to additional artificial and natural selection in their new environments. They continued to evolve and diversify as a result of spontaneous mutations, hybridization, and agricultural practices. Thus most crops also acquired genotypic and phenotypic diversity postdomestication. As a result, for many crops, their primary center of origin and the center of diversification (where many varieties of crops evolved) differ.[2] For example, the center of origin and center of diversification of maize differ significantly. However, compared to the primary centers of origin, the secondary centers occupy a large area and possess less biodiversity of plant species. The secondary centers are relatively rich in domesticated crop varieties (landraces) but lack the immediate wild progenitor and other related wild species. After Vavilov, many archeological digs ascertained the centers of origin of additional crop species, and a new center of crop origin was identified as well (New Guinea). The fundamental research work conducted by Vavilov and his colleagues still stands; however, now scientists recommend the use of center of diversity instead of center of origin because only a crop’s center of biodiversity can be identified based on data—at best, we can only guess at the center of origin. Currently, those identified by Vavilov are known as “Vavilov’s Centers of Biodiversity.” Since, typically, the “centers of origin” of crops are rich in their biodiversity,[3] their exploration helps scientists comprehend the full spectrum of the gene pool available for a given crop, which can then be utilized for breeding experiments. It is important to note that the plant breeders only transfer useful traits from one variety to another by crossing and then selecting the progeny that have desirable combinations of traits. The breeders do not create traits. If a trait is absent in cultivated varieties of a crop species and its ancestors, progenitors, and related species, it cannot be introduced by classical breeding. Biodiversity serves as the resource bank from which scientists can borrow useful traits. It also defines the limits of classical breeding. Therefore, biodiversity centers are the insurance policies for the continuation of today’s crops. Scattered across these centers are the genes/traits that provide resistance to pests and pathogens or the ability to tolerate environmental stresses—the raw materials for breeding advanced varieties of crops. Table 2.1: Centers of origins (biodiversity) of crops Center Remarks 1. China 136 crops were domesticated in this region including rice, sorghum, soybeans, barley, radish, cabbage, mustard, onion, cucumber, pear, apple, apricot, peach, cherry, walnut, litchi, sugarcane, and poppy. Rice was one of the first crop (~ 8,000 years ago) cultivated in the Yangtze River Valley. Pigs, roosters, and dogs were also domesticated here. 2a. Indo-Malay This region includes parts of India, parts of China and the Malay Archipelago. Here, clove, nutmeg, black pepper, coconut, hemp, banana, grapefruit, reed and velvet beans were domesticated. 2b. Indo Burma This center includes North-East region of India and present day Myanmar (Burma). Here, ~117 crops including jute, sandalwood, indigo, bamboo, neem, rice, gram, pigeon pea, mung, cowpea, eggplant, cucumber, radish, carrot, mango, orange, lemon, tamarind, coconut, banana, hemp, pepper, cloves, nutmeg, reed, sesame, and cotton were domesticated. 3. Central Asia This center includes North-Western India (Punjab, Haryana, and Kashmir provinces), Pakistan, Afghanistan, Tajikistan, and Uzbekistan. ~ 43 crops developed in this area, including three varieties of wheat, peas, lentils, horse lentil, gram, mung bean, mustard, linseed, sesame, cotton, hemp, onions, garlic, spinach, carrots, pistachio, almond grapes, pears and apples. 4. Near East (Fertile Crescent) This center includes present day Turkey, Israel, Syria, Jordan, Lebanon, Iran, Iraq, Turkmenistan and the interiors of Asia Minor. ~ 150 crops including rye, barley, oats, Einkorn wheat, Durum wheat, Persian wheat, Pollard wheat, common Bread Wheat, Oriental wheat, lentils, lupine, peas, gram, pomegranate, mulberry, apple, grapes, pears, cherries, walnut, almonds, pistachios, dates, fennel, cumin, carrots, onions, and garlic were domesticated in this region. ~ 10,000 year old fossils of rye have been found at many archeological sites in this area. Evidence of earliest rye cultivation ~13,000 years ago has been found in Syria and the remains of ~ 9000 year old domesticated sheep, goats and pigs have been found in Turkey. 5. Mediterranean This center includes regions around the Mediterranean Sea. Here, 84 crops including durum wheat, Emmer wheat, Polish wheat, oats, peas, lupine, clover, black mustard, olives, beets, cabbage, turnip, lettuce, asparagus, rhubarb, mint, hop, sage, celery, etc. were domesticated. 6. Ethiopian Center This center includes Abyssinia, Eretria, Somaliland, and Ethiopia. 38 important crop plants including Abyssinian and emmer varieties of wheat, millet, sorghum, cowpea, flaxseed, tef, sesame, coffee, okra, indigo, castor, and gum Arabica were domesticated in this region. 7. Southern Mexico and Central America This center includes Southern Mexico, Guatemala, Honduras and Costa Rica. Here, maize, potato, tomato, pumpkin, capsicum, chili, papaya, guava, cashew, chocolate, cotton, passion flower, tobacco, various beans, sisal, sweet potato, arrowroot etc. were domesticated. in this region, ~7000-year-old remains of maize and 10,000-8,000 years old seeds of squash have been found in the archaeological excavation. 8a. Peru, Ecuador, Bolivia Sub center (South America) In this region, 62 species of crop plants including potatoes, maize, lima beans, tomatoes, pumpkin, capsicum, cotton, guava, passion flower, tobacco etc. were domesticated. 8b. Chile (South America) Several varieties of potato and strawberry were domesticated in this center. 8c. Brazilian-Paraguay (South America) Peanuts, pineapples, cashew nuts, Brazil nuts, rubber, etc. developed here. 9. New guinea* (Far East) Recent research proves that ~ 7000 years ago, agriculture started independently in the mountainous region of New Guinea. In this area, bananas, knives, reeds etc. developed. *This center was identified after Vavilov death. Life and Work of Nikolai Vavilov Nikolai Vavilov (1887–1943), a Russian scientist of the early twentieth century, was a pioneer in the field of plant biogeography and germplasm conservation. He was among the first few scientists of the early twentieth century who championed Mendelian genetics for crop improvement. Nikolai Vavilov (see Figure 2.6) was born on November 25, 1887, in Moscow, before the Russian Revolution. He studied agriculture, and after graduating in 1911, he worked with Dmitry Pryanishnikov, a world-renowned soil scientist, for a year while also teaching at Petrovskaya Agricultural Academy. Soon, Vavilov joined a PhD program under the supervision of the Russian professor Robert Eduardovich Regel (of the Bureau of Applied Botany in Leningrad) and famous English biologist William Bateson (the director of the John Innes Horticultural Institution in Norwich, England). In the early years of the twentieth century, Bateson was the champion of Mendelian genetics; he in fact coined the term genetics to describe the study of Mendelian inheritance and the science of variation. Vavilov spent two years in Bateson’s laboratory at John Innes, where he acquired necessary knowledge about Mendelian genetics. In 1914, Vavilov returned to Russia and was appointed as a professor at Moscow University. In 1917, he became the director of the Lenin Academy of Agricultural Sciences in Saratov. He was the first scientist to start work on genetics in the Soviet Union. In this position, he set the primary agenda of the institute: to collect germplasm from all over the world in order to develop advanced crop varieties by employing the principles of Mendelian genetics. For the first decade, the newly formed Soviet government provided Vavilov with plenty of resources and grants for several germplasm collection expeditions. Vavilov, along with his team, traveled to sixty-four countries on five continents to collect seeds, tubers, and germplasm. In Kazakhstan, they found wild apples, and thus the world came to know the birthplace of this fruit. In Peru, they found countless varieties of potatoes, and in Iran, Syria, and Afghanistan, they found relatively quick-ripening varieties of wheat that possessed natural resistance to pests and parasites. Overall, Vavilov led a hundred explorations, which resulted in the collection of over 300,000 samples, accompanied by detailed descriptions. He had remarkable success in managing his extensive field trips in such diverse nations as Ethiopia, Italy, Kazakhstan, Mexico, Brazil, and the United States. In the early 1900s, traveling was rather difficult and required hopping between various modes of transportation, including extensive journeys on foot and horseback. An equally daunting task was the recruitment of local crews of porters, guides, and collaborators. However, many of Vavilov’s personal qualities and his demeanor helped make these missions successful. He was physically fit, mentally sharp, and a polyglot who could converse in eight languages. He was capable of quickly developing friendships and collaborations with strangers, common folk and international scientists alike. On a personal level, Vavilov took care of everyone on his team and the people he interacted with around the world. Among his various field trips, the most historic missions included three visits to North America, Mexico, and South America in 1921, 1930, and 1932; the Mediterranean Sea and Ethiopia expedition in 1926–27; and the trip to Afghanistan in 1924, for which he received a gold medal from the Russian Geographic Society. Later, he also served as the president of that society, from 1931 to 1940. In the first decade of his career, he became the leader of Soviet agricultural policies and headed 111 institutions. In addition to germplasm collection, Vavilov directed projects at Saratov and other research centers aimed at breeding crop varieties that could tolerate the extremely cold climate of the Soviet Union. These projects were deemed of national importance because a large area of the country suffered agricultural losses due to frost, and the damages to the wheat crop were especially devastating. However, the success of these projects required sustained work on classical breeding and the evaluation of the crossings. Unfortunately, year after year, in the Soviet Union, grain remained in short supply and hunger was prevalent. To improve agriculture, in 1929, Stalin announced his Great Break from the Past policy, which led to the establishment of collective farms through the merging of individual family farms. The big government-run farms employed monocropping, which led to more loss of harvest. Before government-run collective farming, individual farmers sowed many varieties of seeds. If the crop was ruined in some areas due to the weather, then it would have been saved in other places, and so a good yield could be obtained elsewhere. With monocropping, the impacts of disease and frost were instead widespread. The failure of collective farms, along with rapid industrialization, caused famine. Millions of people died of hunger in the Soviet Union. Stalin wanted to make the Soviet Union self-sufficient in terms of food production, and the responsibility for improving agricultural yields rested on Vavilov’s team. Stalin gave Vavilov three years to develop advanced varieties of grains that could perform in the extremely cold and unpredictable climate of the USSR. At that time, even the best breeder could succeed only after ten to twelve years. Nikolai Vavilov knew that advanced crops could be developed by following the principles of genetics in a very methodical and systematic manner for a decade, but not in a shorter time frame. Thus he could not promise a miracle to Stalin. But he kept working hard and kept up the morale of his colleagues. Meanwhile, scientist Trofim Lysenko claimed that soaking seeds in cold water for a day or two prior to sowing would result in higher germination rates and could possibly enhance the resilience of a crop in colder climates. Unlike Vavilov, Lysenko was from a poor proletariat background and thus more trustworthy to Soviet policy makers and Stalin, who actively sought to replace bourgeois scientists with party cadres hailing from impoverished families. They deemed Trofim Lysenko a proletarian genius who could provide an alternative to bourgeois science and thought Lysenko’s experiments to be consistent with Darwin’s theory of evolution. In his desperation to bring revolutionary changes to agriculture, in 1938, Stalin appointed Lysenko as the president of Lenin Academy, the highest post for an agricultural scientist in the Soviet Union. Vavilov, as an expert geneticist, knew that Lysenko’s methods would not work. He firmly opposed Lysenko’s claims and, to the best of his ability, provided scientific explanations to Stalin and his cabinet, but they did not trust Vavilov. Vavilov challenged Lysenko to a scientific debate on the merit of his claims, but Lysenko campaigned against Vavilov and the science of genetics with the help of the Communist Party cadres. Stalin and his cabinet took a highly negative stand toward genetics based on their political ideology in response to the inhumane experiments of eugenics in Germany and the US at the time. Since Vavilov was a world-renowned scientist in genetics, he was declared the leader of this unacceptable science. Thus the end of genetics in the Soviet Union meant the end of Vavilov and his colleagues. His funding was ceased, and in 1937, Vavilov’s foreign travels were banned forever. More than a hundred of Vavilov’s fellow scientists were sentenced to death on charges of treason. Stalin also ordered the execution of two of his (Stalin’s) closest associates, Nikolai Bukharin and Nikolai Gorbunov, for supporting the science of genetics. Vavilov’s position was downgraded to the head of the Institute of Applied Botany and New Crops in Leningrad, where he was to work under Lysenko. Even while working in difficult circumstances and without resources, Vavilov stood firmly by his principles. He continued to write letters to the government for the release of his associates, though his correspondence with foreign scientists was blocked. He wrote his last letter to Professor Harry Harlan in 1937. Harry’s son Jack Harlan was interested in doing his PhD under Vavilov’s supervision, and he had sent his application to Vavilov. In reply, Vavilov addressed Harry Harlan (not Jack) and described a variety of wheat found in China. Vavilov normally addressed Harry Harlan as “Dear Doctor Harlan.” This was the first letter in which Vavilov addressed him as “My Dear Doctor Harlan” and derailed from addressing a question. The senior Harlan asked his son to abandon the idea of research with Vavilov. On August 6, 1940, Nikolai Vavilov was arrested while on a field trip in Ukraine, though the information of his arrest was not made public for three years. However, since Vavilov was a scientist of international repute and used to correspond with scientists from all over the world, the scientific fraternity became concerned about Vavilov’s well-being after his correspondence ceased. So in 1942, the Royal Society of London elected him as a member, sending a letter along with the associated certificates to Vladimir Komarov, president of the Soviet Academy. In order to accept this honor, a signature from Nikolai Vavilov was required (which would have confirmed him being alive). In reply, Komarov had the certificate signed merely “Vavilov”—by Sergei Vavilov, the younger brother of Nikolai and the physicist who headed the Soviet nuclear program. The British embassy wrote back to Komarov stating that the signature of Nikolai Vavilov was expected, not that of his brother. However, the Soviet government went silent, and so after this, a rumor was spread in the scientific community that Nikolai Vavilov had been murdered by Stalin. Actually, Nikolai had been accused of misuse of government grants and treason and was sentenced to death in 1942. But from prison, Vavilov appealed to Marshal Leverentia Beria to reconsider his sentence. Beria was one of Stalin’s trusted allies and the head of the Supreme Department of Legal Affairs (NKVD). In addition, Vavilov’s old professor Dmitry Pryanishnikov tried his best to reduce Nikolai’s punishment. He too had friendly relations with the Beria family: Beria’s wife was also a student of Pryanishnikov. Despite the hopelessness, the British Royal Society also continued to press for Vavilov’s safety. Whatever the reason, Beria converted Vavilov’s death sentence to twenty years of rigorous imprisonment. But on January 26, 1943, Vavilov died of starvation in Saratov prison. After Stalin’s death in 1953, Russian scientists pressured Nikita Khrushchev to reinvestigate Vavilov’s case. This time, all the charges against Vavilov were dismissed, and he was awarded a place in the history of the Soviet Union with posthumous honors. Although many of Vavilov’s unpublished research papers and research data were destroyed between 1940 and 1953, some material was saved by his wife, Yelena Berulina. Fatikh Bakhtev, a close associate of Vavilov who was involved in many of his field trips collecting germplasm, also survived the Stalin era. In 1957, Yelena Berulina and Fatikh Bakhtev published Vavilov’s remaining material and data. This fundamental research work conducted by Vavilov’s team of scientists provided insight and the direction to future research on crop domestication. The building of the Bureau of Applied Botany is now known as the Nikolai Vavilov Institute of Plant Genetic Resources, and Vavilov is counted among the greatest scientists of the twentieth century. Vavilov’s collection, his research papers, and information about his life are available on the Nikolai Vavilov Research Institute website (http://www.vir.nw.ru). World’s First Seed Bank In the 1920s, Nikolai Vavilov established the world’s first seed bank in the building of the Bureau of Applied Botany in Leningrad, housing major collections of seeds and germplasm there that included 300,000 varieties of seeds and germplasm of 2,500 plant species. His team of scientists was also involved in conducting the detailed characterization of major crop varieties and systematic crossing experiments in experimental centers across the Soviet Union. Hitler had a keen interest in genetics. During World War II, Germany formed a special commando squad to capture Vavilov’s seed bank. On June 22, 1941, Germany invaded the Soviet Union. As the Germans advanced, they captured small seed banks in the western region, but the main seed bank in Leningrad was still out of their reach. After the incarceration of Vavilov and many of his senior colleagues in 1942, the remaining scientists of the institute voluntarily shared the responsibility of protecting the germplasm collection during World War II. Apart from Hitler, this seed bank was also threatened by the starving people of Leningrad. In those years of war, 700,000 people died of hunger in Leningrad—including many of Vavilov’s colleagues who stood protecting the seed bank. One by one, they starved to death while safeguarding the vast storehouse of seeds and tubers. Stalin was not interested in saving the seed bank, and thus the government did not provide any support. As Vavilov wasted away in prison, his associates successfully protected the germplasm collection for 900 days during the siege of Leningrad. This is a unique story of the sacrifice made by scientists to protect a seed collection for future generations. The Leningrad seed bank also survived by pure chance. Hitler had planned to host a celebration of his victory at the Astoria Hotel on St. Isaac’s Square; thus Germans did not bomb that area. The seed bank was right in front of the hotel. Similarly, when the war ended in 1944, the Germans were defeated before reaching Leningrad. In the years since World War II, more seed banks have been established around the world, many of which also contain large amounts of seeds from Vavilov’s collection. Today, this seed bank exists as part of the N. I. Vavilov Scientific Research Institute of Plant Industry in St. Petersburg (see figure 2.7). For the past century—and still today—the seeds and germplasm of various crops from this collection have been used by plant breeders worldwide to produce improved varieties of cereals, vegetables, and fruits. Much of the raw material for making advanced varieties of various crops, fruits, vegetables, and so on remains in Vavilov’s seed collection for free. Even though the world population has increased more than three times over the last century, the world is now producing more food than people need. Vavilov and his fellows—many of whom themselves died of hunger—made tremendous contributions toward achieving global food security by creating a tremendous genetic resource for future crop improvement. Now that many species of plants are extinct in their centers of origin, Vavilov’s seed and germplasm collection has become invaluable. The work of Vavilov and his colleagues helped further our understanding of the importance of biodiversity and the conservation of germplasm in ensuring food security for mankind. Thanks to them, today, there are more than a hundred large and many thousands of small seed and germplasm banks in the world. Further Readings Darwin, C. (1859). On the origin of species by means of natural selection, or the preservation of favored races in the struggle for life. John Murray, London. Harlan, J. R. (1973). On the quality of evidence for origin and dispersal of cultivated plants. Current Anthropology, 14(1–2), 51–62. Harlan, J. R. (1975). Agriculture origins: Centers and noncenters. Science, 174, 465–74. Nabhan, G. P. (2009). Where our food comes from: Retracing Nickolay Vavilov’s quest to end famine. Island Press. Pringle, P. (2009). The murder of Nikolai Vavilov: The story of Stalin’s persecution of one of the great scientists of the twentieth century. JR Books. Vavilov, N. I. (1931). The problem of the origin of the world’s agriculture in the light of the latest investigation. https://www.marxists.org/subject/science/essays/vavilov.htm. Vavilov, N. I. (1987). Proiskhozhdenie i geografi i a kul’turnykh rastenii [The origin and geography of cultivated plants]. Nauka. 1. A clickable world map showing the center of origin of various crops is available at https://www.biodiversidad.gob.mx/v_ingles/genes/centers_origin/centers_plants1.html 2. This is also sometimes due to reasons other than human migration. One classical example is pine, which has its center of origin in northwestern China but its center of diversification in Central America (Mexico, Guatemala, and Honduras). At present, 49 out of a total of 111 species of pine are found in Mexico. The difference in the center of origin and center of diversification of pine resulted from a geological event (continental drift) long before the beginning of agriculture, which led to the isolation of various flora and fauna and thus their diversification in a new environment. 3. In simple terms, biodiversity refers to all cultivated varieties of crops along with their wild progenitor(s) and evolutionarily related species.

textbooks/bio/Agriculture_and_Horticulture/History_and_Science_of_Cultivated_Plants_(Naithani)/1.02%3A_The_Origins_of_Crop_Plants.txt

Crop Exchanges before the Industrial Revolution The migration of the human population meant that the movement of crops away from their centers of origin was inevitable. Domesticated plant species first spread to neighboring regions via nomadic pastoralists, who traded with various farming communities. As tribal wars broke out, the grains were also looted and brought to new territories. However, this spread was limited. Credit for the initial global spread of cultivated plants (3,000 BCE to 1,000 CE) goes to the ancient Polynesian seafarers, who sailed between Southeast Asia, Africa, and South America. They helped at least eighty-four cultivated plants travel from South America to Asia and Africa (e.g., maize, amaranth, cashews, pineapples, custard apples, peanuts, pumpkins, gourds, arrowroot, guava, sunflowers, basil, and brahmi). They also brought hemp and another fifteen plants to Africa and South America from Asia. These exchanges occurred long before Europeans were aware of the existence of the Americas (1). The second global wave of exchange ensued via the Silk Road. The first highway that connected Asia to Europe and Africa, it was built by the emperors of the Han dynasty between the second century BCE and the first century CE. It was not a single road but a network of several routes that connected various regions of China. The silk collected from many provinces in China and sent west through its primary northern route gave it its name. Later, this route was extended to Rome via Central Asia, Iran, Iraq, and Syria, while a branch from Tibet also connected India. At its peak, the Silk Road network covered 7,000 miles, much of which ran through large desert areas with sporadic human inhabitation, where water and food sources were scarce and a constant fear of robbers loomed. Thus travel on the Silk Road was not for everyone. Only Arabs, the inhabitants of Central and Middle Eastern Asia who possessed generational experience surviving in the desert and had tribal networks to rely on, were successful in traversing the Silk Road and thus dominated trade. They traveled on camels across the desert in caravans protected by armed squadrons. They stopped at meeting points where traders heading to different destinations exchanged goods, like silk, cotton, sugar, spices, china, ivory, and precious stones. Traveling alone was not an option, so individuals, small groups, missionaries, and pilgrims also joined the traders’ caravans. For centuries, Arab merchants, travelers, and Sufis played a central role in the exchange of seeds and germplasm among the three continents. From India and China, they transported cotton, sugarcane, eggplants, and bananas to Central Asia and Africa; from Central Asia, they carried pomegranates, chickpeas, gram, pears, walnuts, pistachios, dates, fennel, carrots, onions, and garlic to India and China. From Africa, they procured millet, melons, coffee, and many tubers for Asia and Europe. Some sovereigns and their armies also unintentionally assisted in carrying germplasm across the Silk Road.[1] Columbian Exchange The third and the most dominant wave of global plant germplasm exchange occurred between the Age of Exploration and the Industrial Revolution under European imperialists. They promoted and invested in the systematic cataloging and classification of the fauna and flora found across the seven continents, transporting germplasm in bulk and establishing plantations of cash crops worked by enslaved peoples. This massive germplasm exchange between the Old World (Asia, Europe, and Africa) and New World (the Americas and various archipelagoes) is known as the Columbian Exchange (see figure 3.1). You might be surprised to learn that until the eighteenth century, the peoples of Asia, Europe, and Africa had not seen potatoes, tomatoes, corn, sweet potatoes, or peppers, which are now an integral part of their traditional cuisines. Acceptance of the introduced food crops in their new homes was slow, integration in the local cuisines was gradual, and acceptance in some cases was challenging. For instance, potatoes were brought from Peru to Spain in the sixteenth century, reaching Italy from Spain in 1560. However, it took more than a hundred years for Europeans to accept the potato as food. When it first arrived in Europe, there were rumors that eating potatoes cause leprosy. In the seventeenth century, Irish peasants adopted the crop due to difficult circumstances: invasion, famine, and evictions from their fertile land. They embraced the potato because it was a highly productive crop that could be grown even on the smallest amount of cultivated land. During the eighteenth century, potatoes were successfully introduced in Germany due to frequent crop failures, but the French population was still apprehensive despite famine and starvation. The French eventually accepted potatoes in the late eighteenth century after much encouragement from Queen Marie Antoinette. Marie adorned her hair with potato flowers and commissioned a portrait of herself with a potato plant. She also asked gardeners to plant potatoes in the royal gardens, where guards were stationed during the day but cleverly called away at nighttime. Common folks would dig potatoes from the royal garden at night and sow them in their fields. Later, Europeans introduced potatoes to the Ural Mountains and their colonies in Asia and Africa. For example, the British brought them to India, and in the mid-nineteenth century, officials from the Geological Survey of India sowed potatoes in the slopes of the Himalayas. Potatoes proved to be a very productive and nutritional crop in most areas and, by the middle of the nineteenth century, became an integral part of diets across Europe and Asia. Today, the potato is the most popular and affordable vegetable in the world. In Africa and Central Asia, many traditional cuisines use potatoes and chicken as their main ingredients, even though the domesticated animal’s birthplace is China and the vegetable’s is Peru. Similarly, tomatoes, onions, and garlic, used in abundance in Italian cuisine, were introduced to the country only 400 years ago. Red chilies, bell peppers, pumpkins, gourds, squash, and so on spread all over the world from South America over the past three centuries. Likewise, mangoes, jackfruit, eggplants, cotton, sugarcane, and so on spread out of South Asia. This history of the introduction of cultivated plants shows us that many traditional cuisines are not as old as they are thought to be. Despite the strong sense of cultural identity we ascribe to them, they are constantly evolving and incorporating new ingredients. We find that across cultures and religions, some food items are revered, whereas others are prohibited. During Hanukkah, Jewish people traditionally make latkes (a special pancake made of potatoes), and they eat unleavened bread, matzo, during Passover. Hindus offer basil, barley, sugarcane, sesame, and rice to their gods with their prayers. For Africans, cassava and yam are staples, but red rice is sacred and saved for special occasions. It seems that a sense of reverence for particular foods may be linked to historical memories of crop domestication because, in many cases, the revered foods are prepared from crops that are native to the areas those people lived. In contrast, apprehensions are usually associated with crops that were introduced later. For example, some vegetarian Hindu sects abstain from eating garlic and onion, as these plants were introduced from Central Asia. At the root of this prejudice is doubt and fear of the unfamiliar. Such reverence or prejudice may not seem to make logical sense but may trace back to experiences with the domestication and/or introduction of the crop in the past. Scholars estimate one-third of all food crops grown in the world today are of American origin and were unknown to the Old World before the conquest of the Americas. Over time, these crops became integrated into European, Asian, and African cuisines. Likewise, foods previously unknown in the Americas—such as sugarcane, tea, coffee, oranges, rice, wheat, eggplants, bananas, mangoes, and so on—were brought over by European colonists. Overall, this global exchange of plant species and their cultivation at the industrial scale brought uniformity to the consumer market and actually flattened the diversity known to previous generations. In total, to date, 250,000 species of flora are known, out of which humans can identify some 30,000 plant species as edible, but only 120 are cultivated. Of these, eleven crops—wheat, maize, rice, potatoes, barley, sweet potatoes, cassava, soybeans, oats, sorghum, and millet—satisfy 75 percent of human nutritional needs, and wheat, maize, and rice alone make up more than 50 percent of the entire population’s daily caloric intake. Rise of European Imperialism and Plantations From the seventh century until the seventeenth century, Arabs controlled Silk Road trade. The luxurious goods they brought from China and India were first sold to the sultans of Central Asia, and the remaining items were sold in Europe. As a consequence, only a few valuable goods were auctioned in Europe, where the kings and noble classes bid on their favorite items. Arab traders made tremendous profits and controlled the market: they determined what to sell, where to sell, and at what price. In contrast, Europe had nothing of value to barter and thus had to make their payments in gold or silver. European economic thought and policies until the 1700s were based on the mercantile system, wherein economic and political stability rely on restrained imports and excessive exports. Thus unilateral trade with Arabs caused fear among the European sovereigns, who worried about the diminishment of their treasuries of gold and silver. So in the fifteenth century, the rulers of Portugal poured their resources into developing better ships, navigational equipment, watches, maps of the world, and so on and recruited Europe’s best sailors to lead armed naval squads for exploration. The Portuguese managed to reach the Canary and Azores Islands, the Caribbean, Brazil, Africa, China, India, and Southeast Asia by sea. Soon Spain, France, Britain, and Holland also managed to reach the Americas, Asia, and Africa. Thus Christopher Columbus reached the Caribbean islands in 1492, and five years later, in 1497, Vasco da Gama reached the port of Calicut in India. For the first time, Europeans saw many new crops, fruits, vegetables, ornamental plants, herbs, cattle, and other species of animals from Asia, Africa, and the Americas. From the sixteenth to nineteenth century, the various European powers competed with one another for control over the newfound lands and their wealth. Gradually, all the countries of Africa, the Americas, and Asia became European colonies. European rulers employed new management systems in their colonies. They organized agriculture to focus on the establishment of plantations of cash crops, replacing traditional farming to maximize profits. First, sugar plantations were established in the Caribbean by exploiting slave labor, and later, this model was repeated to produce tea, coffee, cocoa, poppy, rubber, indigo, and cotton in Asia, the Americas, and Africa. The products of plantations were for the distant European markets and not for the consumption of local populations. Often, the crops and the laborers were brought to plantations from different continents. Thus colonial agriculture caused the global displacement of many people and plants. From the sixteenth to the nineteenth century, the story of agriculture centered on these plantations’ products and their availability at a cheap price in the global market. Europe’s sovereign class made huge profits, and the continent prospered tremendously, but at the cost of exploiting the natural resources of the colonies and relying on slavery. Increasing trade between Europe and the colonies also affected business and trade practices during the 1500s and 1600s, which had a great impact on all spheres of society and human civilization. With this also came the Enlightenment and the birth of the ideas of democracy and universal equality. We see that massive change in agricultural practices added several new layers of complexity to human civilization, politics, and economics and eventually changed the world order forever. In the rest of this chapter, we will explore colonial agriculture and its impact on human society through the stories of three representative plantation products: sugar, tea, and coffee. Story of Sugar and Slavery Discovery of Sugar Sugarcane, a plant from the grass family, is the major source of sugar. It is a tropical crop, requiring an abundance of water and sunshine for optimum growth, and is easily damaged by frost and low temperatures. It has a very long and prominent stalk or stem (about twelve feet) that is filled with juicy fibers containing large amounts of sucrose. People enjoy chewing small pieces of the stem to consume its juice directly. This juice is also used for making sweets, puddings, and sugar (see figure 3.2). Sugarcane is a plant native to South and East Asia (covering India, Indonesia, China, and New Guinea), where its six species are found. The sweetest and the most cultivated variety of sugarcane, known as Saccharum officinarum (see figure 3.3), is believed to have originated in New Guinea or Indonesia and was introduced to many South Asian countries 3,000–5,000 years ago by Polynesian sailors. Where and how the cultivation of sugarcane began remains unknown. The earliest written reference to the sugarcane reed is a Vedic Hindu text known as the Atharvaveda (which is 3,500 years old), where the use of ikshu (desire), the Sanskrit name for sugarcane, appears several times. It is not surprising that the sweetest plant on earth was called “desire,” as sweetness is the most primitive human craving, the first taste that settles in our consciousness. We use sweet as an adjective to evaluate and describe the most positive, emotionally fulfilling, and abstract aesthetic experiences. The second-oldest written reference to sugarcane is from the Greek philosopher Herodotus (484–425 BCE). Within a hundred years of Herodotus, Kauṭilya (ca. 350–283 BCE) described five types of sugar in his book Arthaśāstra.[2] This is the first ancient description of sugar making in the world. It is likely that in India, people learned to make sugar 2,000 years ago and developed pressing mills to extract large quantities of juice from the cane, as well as facilities for boiling the juice and extracting sugar. However, people outside of the Indian subcontinent remained largely ignorant of it until much later. For the first time, in the seventh century, Arab traders introduced raw sugar (a.k.a. khand) to Central Asia. There, artisans invented tedious protocols for the purification and crystallization of it, thus making white granulated sugar, sugar cubes, and sugar figurines. Initially, white sugar was made in very small quantities and was so very costly that it was known as “white gold.” White granulated sugar was not made available in India for a long time; the raw material continued to come from India, but for centuries, the advanced technology for making granulated sugar was not known in the country. It wasn’t until the thirteenth century that the technique for making it reached India from China, and that is why it is called “Cheeni” in India today. In the eleventh century, both raw khand and white processed sugar reached the royals and lords of Europe. The word candy was coined for khand, and sucre, sacrum, sucrose, and sugar for the white variety. Before this, Europeans had never known such extreme sweetness, and they went crazy for it. It was so addictive that they were willing to plunder their stores of gold and silver in exchange for just a few pounds of it. But while the demand for sugar was high, so little was available that not even 1 percent of that demand could be met. It is said that once, England’s King Henry IV went to considerable lengths to get just four pounds of sugar, but it could not be arranged. The Arabs realized that there is an immense scope for expanding the highly profitable sugar trade. Islamic rulers (of Arab origin) who dominated India and Central Asia from the seventh century onward focused on increasing the production of sugar: they organized large farms for sugarcane cultivation under the leadership of landlords. These landlords set up sugar factories in the reed fields because cut sugarcane deteriorates rapidly and so must be milled within twenty-four hours. In these fields, armies of workers continuously worked, cutting and transporting sugarcane to the factory, where artisans would extract the juice and pass it along for boiling and further processing. This marked the first time in human civilization that excessive human labor was organized for cultivating a cash crop to be sold in distant markets: these were the first plantations. Due to the efforts of the Islamic rulers, sugar production doubled, but demand quadrupled. Arab merchants could not meet the demands of sugar in Central Asia or Europe; fulfilling it was beyond their ability. There were two main obstacles: first, they had a limited amount of fuel for making sugar, and second, only a modest amount could be transported to Europe via camels and horse convoys. Caribbean Sugar Colonies Strange as it may seem, until the sixteenth century, most people in the world did not know about sugar. The sweetest thing they had known was honey, and that too was available only in small quantities. The Portuguese were the first to invest in commercial sugar plantations. They used their newly founded colonies in the Caribbean for sugarcane cultivation, where fuel for running the factories was plentiful and goods could be easily shipped worldwide. However, they were short on the workers needed to establish the plantations and run the factories. So they turned to West Africa, where they were heavily involved in the plunder of ivory. In 1505, they brought the first ship carrying enslaved Africans to the Caribbean to establish a sugar colony. Over the next twenty years, with the constant toiling of slaves, the Portuguese sugar colonies expanded from Hispaniola (present-day Haiti and the Dominican Republic) to Brazil. Soon Spain, France, Britain, and Holland followed their example and established sugar colonies in the Caribbean islands, Guyana, Suriname, Brazil, Cameroon, and elsewhere. As the sugar industry expanded, the slave trade also grew to new peaks. In mainland Europe, many companies were formed around it (see figure 3.4 for a depiction of a regular scene at the slave market). For the next 300 years, the slave trade continued unabated. By the middle of the nineteenth century, a million Africans had been enslaved and brought to the Caribbean islands and Brazil. As a consequence, sugar production increased, and supply reached the common folks of Europe. The owners of the sugar colonies, who profited from the slavery-based sugar industry, were important officials and members of influential families in Europe. While still residing in their native countries, they made tremendous amounts of money from their estates in the colonies. Although they seldom visited, each often maintained a plush bungalow on a high hill within the plantation, known as the “Great House.” The daily management of the sugar estate was left in the hands of a supervisor or slave commander (figure 3.5). The owner did not directly engage in the dirty work of interacting with the people he enslaved on a day-to-day basis. Slavery became an accepted social institution, and surprisingly, the task of its operation was shouldered by the slaves themselves. Usually, they worked for sixteen to eighteen hours a day, did not get enough food, and were not allowed to leave. They were punished and tortured routinely and could be murdered for raising a slight objection. It was common for them to die every day due to torture, hunger, and sickness. New slaves immediately replaced the dead. The eighteen- to twenty-year-old youth who were brought to sugar colonies from Africa usually died within ten years. A sense of the inhumane conditions of slave life can be drawn from the fact that from 1700 to 1810, 252,500 African slaves were brought to Barbados, a small island of 166 square miles, and 662,400 to Jamaica. From 1600 to 1800, the sugar trade was the most profitable business in the world. It was a large part of “triangular trade,” wherein companies involved in the slave trade sold slaves in the Caribbean in exchange for sugars, then sugar was sold in Europe, and from Europe, armed sailors were sent to Africa for more slaves. This cycle continued for 300 years and ensured an uninterrupted supply of sugar was being sent to Europe. By the eighteenth century, sugar production in the Caribbean was so high that it became available to ordinary people at a very affordable price. However, many Europeans remained ignorant of the inhumane conditions of slaves working within the sugar colonies. Slavery in the Caribbean In 1781, a public lawsuit in England that involved a Liverpool-based slave-trading company and an insurance company brought the slave trade under public scrutiny. The slave-trading company’s ship, Jong, lost its way, and officers onboard foresaw a shortage of drinking water in the ship due to the delayed schedule. To avoid inconvenience due to a drinking water shortage on the ship, they threw 142 slaves from the ship into the open sea. The slave-trading company had insurance on the lives of the slaves, and accordingly, they demanded compensation from the insurance company. However, the insurance company did not consider the “property damage” to be justified and refused to pay compensation. In response, the Slave Trading Company sued them. The news of this lawsuit was published in the newspapers of England, and a public conversation on slavery began. Quakers organized and led protests against the killings, and, in 1783, 300 Quakers appealed to the British Parliament to end slavery. The appeal was dismissed. All the influential British citizens of that time, including the members of parliament and their relatives, owned sugar estates in the Caribbean; their economic interests were directly tied to the slave trade. For example, lord mayor of London William Beckford, who was also a member of parliament, had twenty-four sugar plantations in Jamaica; another parliament member, John Gladstone (father of the future British prime minister William Gladstone), owned a sugar colony in British Guiana. Therefore, it was no surprise that the Quakers’ proposal was rejected in parliament. However, the Quakers were not discouraged. They started the abolitionist movement,[3] boycotting sugar on moral grounds and urging the public to do the same. They reached out to various individuals, groups, and organizations to educate people about slavery and recruit supporters. They approached people from both the lower as well as elite classes and tirelessly campaigned against slavery. Among the top activists of the abolitionist movement, Thomas Clarkson (figure 3.6) is particularly notable. In 1785, when he was a student at Cambridge University, he participated in the annual essay competition. The subject of that year’s essay, “The Slavery and Commerce of the Human Species,” was proposed by Peter Peckard, then the chancellor of Cambridge and a well-known abolitionist himself. Thomas Clarkson received first prize in the competition, and in writing the essay, what he learned about the cruel system of slavery made a lasting impact on him. The essay was published, and soon, he was introduced to several prominent leaders of the abolitionist movement, including James Remje and Grenville Sharp. Later, he became a full-time activist and dedicated his life to the cause. Clarkson gathered testimonies from doctors who worked on slave ships and military men who had served in the Caribbean crushing the slave rebellions. He acquired the various instruments of torture (handcuffs, fetters, whips, bars, etc.) used on slaves to educate British citizens about the cruelty inflicted on the slaves within the sugar estates. He traveled 35,000 miles on horseback to collaborate with antislavery organizations and individuals. Clarkson also recruited supporters from influential political circles, such as parliament member William Wilberforce, who appealed twice to the British parliament to abolish the slave trade. His appeal was dismissed the first time, but the second time, in 1806, it passed under the heavy pressure of public opinion. Finally, in 1807, the slave trade was outlawed throughout the British Empire. The famous English film Amazing Grace is based on these efforts. In March 1807, on the final passing of the Bill for the Abolition of the Slave Trade, the poet William Wordsworth wrote the following sonnet in praise of Thomas Clarkson: Clarkson! it was an obstinate Hill to climb: How toilsome, nay how dire it was, by Thee Is known—by none, perhaps, so feelingly; But Thou, who, starting in thy fervent prime, Didst first lead forth this pilgrimage sublime, Hast heard the constant Voice its charge repeat, Which, out of thy young heart’s oracular seat, First roused thee—O true yoke-fellow of Time With unabating effort, see, the palm Is won, and by all Nations shall be worn! The bloody Writing is forever torn, And Thou henceforth wilt have a good Man’s calm, A great Man’s happiness; thy zeal shall find Repose at length, firm Friend of human kind! Although this was a significant triumph, slavery did not end. British companies stopped buying and selling slaves, but over the next thirty years, slaves continued to work as before in the plantations and mines located within the British Empire, and new slaves were supplied by other European companies as needed. Therefore, the struggle against slavery continued. Apart from abolitionists, many others also played a crucial role in the antislavery struggle. In 1823, an idealistic clergyman, John Smith, went on a mission to British Guiana, where John Gladstone had a sugar estate. Pastor Smith narrated the story of Moses to the slaves there, describing how thousands of years ago, the Jewish slaves of Egypt gained their freedom and reached Israel. Inspired by this biblical story, 3,000 African slaves revolted. However, the rebellion was quickly suppressed, most of the rebels were killed, and Pastor Smith was sentenced to death, though Smith died on the ship carrying him back to England for execution. British commoners were enraged by the death of Smith, which provided momentum to the antislavery movement. For the next ten years, they organized frequent antislavery protests and massive rallies while, in the sugar colonies, slave revolts continued. Finally, again under the pressure of public opinion, in 1833, the British Parliament passed the Emancipation Bill, which resulted in the legal abolition of slavery within the British Empire. The owners of the plantations were given seven years and subsidies to free their slaves. Finally, all slaves within the British Empire were legally freed on August 1, 1838. Haiti: The First Free Country of Slaves Around the time the abolitionists were campaigning to end slavery in England, the French Revolution (1789–99) began. At the time, a revolt erupted in the most valued French sugar colony, St. Dominic. Slave commanders ran the plantations in St. Dominic, where there were 25,000 whites, 22,000 freed Africans (including the slave commanders), and 700,000 slaves. The revolt was organized by slave commanders who pledged to liberate Haiti under the leadership of Toussaint, who called himself L’Ouverture (the opening). The slaves’ destiny was tied to the sugar plantations and the factories, so they burned them. In the wake of the ongoing turmoil within France, the temporary government of France, formed after the abolition of the monarchy, saw no way to quell this uprising and liberated St. Dominic in 1793. Thus St. Dominic became Haiti, the first free country of slaves. However, when France withdrew, Britain moved forward to take control of Haiti. British soldiers began capturing the people of Haiti and returning them to the plantations as slaves. The fight continued for another five years. Eventually, in 1798, the British gave up, and Haiti became free again. But this freedom was also short-lived. In 1799, Napoleon came to power, and he overturned the law that had freed the slaves and attacked Haiti. French troops imprisoned the rebel leader Toussaint, who died in prison in 1803. Nonetheless, the people of Haiti continued fighting Napoleon’s army, resulting in the deaths of 50,000 French soldiers, and France suffered substantial economic losses. In 1804, France retreated, and Haiti finally became a free republic. American Sugar Colonies of Hawaii and Louisiana Napoleon sold the French province of Louisiana to President Thomas Jefferson for just \$15 million to make up for the enormous losses in the war with Haiti. Some of the former owners of the Caribbean sugar plantations started fresh in Louisiana. Louisiana is suitable for sugarcane cultivation. However, it has a mild frost in the winter months, and thus sugarcane crop needs to be harvested from October to December. The additional burden of this weather also fell on the slaves, and therefore working conditions for slaves became even worse in Louisiana. Slavery continued in the United States of America until 1862. On January 1, 1863, Abraham Lincoln issued the Emancipation Proclamation and declared “that all persons held as slaves are, and henceforward shall be free.” Abraham Lincoln was also the first US president who established diplomatic relations with Haiti. Some of the former owners of the Caribbean sugar colonies chose to settle in Hawaii. The conditions were very favorable for the planters, as sugarcane farming had been underway for a long time there. It is believed that in the early twelfth century, Polynesians brought sugarcane to Hawaii from South Asia. Hawaiians used sugarcane juice in many ways, although they did not learn to make sugar. The owners of plantations in Hawaii developed a new model for recruiting workers. Instead of African slaves, they employed workers from Asian countries at meager wages. First, only men from China were recruited. When these workers demanded better wages and working conditions, they brought workers from Japan, Korea, the Philippines, Spain, and Portugal. The workers of Hawaii were divided into different linguistic, ethnic, and cultural groups, and hence they could not organize to challenge the owners. The owner successfully managed their plantations and gained tremendous profits from the sugar business for nearly a century and a half. The living and working conditions of the workers were unfair and cruel, but it was an improvement compared to slavery: they received wages for their work, had families, and the owners could not sell them and their children. It was a significant accomplishment that, thanks to abolitionists, in the nineteenth century, legal slavery ended, and plantation owners were obliged to pay wages. In 1959, Hawaii became the fiftieth state of the United States of America. Indentured Labor: Girmitiya In 1836, John Gladstone wrote a letter to the then viceroy of India, asking him to send laborers for his sugar estate. The viceroy gladly accepted Gladstone’s proposal and sent 2,000 workers from India to British Guiana on a five-year work permit, along with the provisions for a paid trip back home. Like Gladstone, others made arrangements to bring indentured laborers from India when the slaves were freed (see figure 3.7). These workers carrying permits were sent to several British colonies—including Guyana, Mauritius, Suriname, the Caribbean islands, and South Africa—to work in plantations and mines. The indentured workers couldn’t pronounce the word permit and instead referred to it as “Girmit,” and themselves as “Girmitiya”. After a long sea voyage of two to three months, when the Girmitiya reached their destination, they were received by the overseers and were assigned the quarters of the former slaves. The Girmitiya were helpless in a foreign land and could survive only at the mercy of the owners. They worked long hours, earned less money than promised, and were treated as slaves in all practical matters. With the arrival of indentured workers, freed slaves lost opportunities for employment, and the owners got the upper hand in setting wages. So despite the freedom they’d earned after tremendous sacrifice and struggle, former slaves remained marginalized. The European owners deliberately created a situation in which indentured workers, former slaves, and other marginalized groups all remained in competition with one another. The masters of the plantations (and also the mines) thought of Indian workers as weak, obedient children and former slaves as foolish and lazy. The owners did not pay fair wages to either: in most places, the indentured workers received even poorer wages than the former slaves. Indentured labor was cheaper than free slaves, and the indentured workers were not in a position to organize and demand fair treatment or familiarized with the historical exploitation of slaves. Thus, the planters were treating them worse than free slaves. They were the replacement of former slaves. the Indentured Workers System From 1860 onward, indentured workers from India were being brought to work in plantations and mines in South Africa. After the end of a five-year contract, indentured workers were offered two options: to return to India or live as a free worker who received a small plot of land in lieu of his passage home. Returning to India was not easy; Hindu workers who had crossed the sea faced social stigma back home. Thus most people chose to stay in South Africa to start their lives afresh. They often still worked in plantations and mines, but as free workers, and on the side, they served as small-time artisans, grew some fruits and vegetables on their plots, and set up small shops. Girmitiya brought many nuts and vegetable seeds with them from India. They grew local fruits, vegetables, and corn as well as Indian crops like mangoes. In the early years, their trade remained limited to their community. While the Indian population was small in number, the chance of their having direct encounters with the whites was negligible. The relationship between whites and Indians was more like that between an owner and a slave. But gradually the number of people of Indian origin increased. Apart from laborers, a large number of Muslim and Parasi traders from Gujarat also started coming to South Africa for business. Indian shopkeepers were polite, fair, and nonintimidating to Africans. As a result, many local Africans became their customers, which infuriated the white business community of South Africa. In 1893, Mohandas Karamchand Gandhi (figure 3.8) reached South Africa for the first time to assist a Gujarati businessman Dada Abdullah in a legal case. At that time, 50,000 Indian workers (freed after contract) and 100,000 indentured workers and their offspring were living in South Africa. He faced many insults and racial discrimination as a person of color despite being highly educated. He soon realized that Indians suffered more racist violence and social discrimination in South Africa than in Europe. Gradually he became an activist and began to lead civil rights movements in South Africa. His encounters with indentured workers were eye-opening, and he realized that the situations Indian indentured workers faced were in some ways worse than those of former slaves. For the next twenty-one years, Gandhi served as an advocate for the Indian traders, small shopkeepers, and plantation and mine workers. He led a struggle against the policies of apartheid, resulting in the abolition of many discriminatory laws. While in South Africa, Gandhi established communication with the leaders of the Indian Freedom Movement and was successful in inviting a senior congressional leader, Gopalakrishna Gokhale, to South Africa. Gokhale witnessed firsthand the plight of indentured workers living outside of India. After returning home, Gokhale appealed to ban the indentured worker system. Finally, in 1917, the practice ended. Unlike the strategies used in previous battles within the colonies, Gandhi relied on satyagraha (emphasizing the human commonality and truth) and the principle of nonviolence to fight the state of South Africa. His satyagraha experiments in Africa were instrumental to his future work. Gandhi returned to India in 1918 and led the freedom struggle there for the next thirty years, guided by the same principles, until India gained freedom in 1947. Sugar from Sugar Beets A method for making sugar from sugar beets was discovered in 1747 by German scientist Andreas Sigismund Marggraf. Marggraf’s student Franz Karl Achard successfully employed selective breeding for improving sugar content in beets and then opened the world’s first factory for extracting it in Silesia in the year 1801. Napoleon Bonaparte became interested in the idea of beet sugar. Sugar beets required less labor and fuel than sugarcane and could be easily grown in the cold climate of Europe. Under Napoleon’s patronage, the cultivation of beet sugar was promoted, and many factories and training schools were established. In 1812, Benjamin Dalesart discovered a method of extracting it on an industrial scale, which made it so that France could rely on beet sugar. In 1813, Napoleon prohibited the import of cane sugar from the Caribbean and further promoted beet sugar production. By 1837, France had 542 sugar mills and became the largest beet sugar producer in the world, producing 35,000 tons annually. Many other European countries, including Germany, followed France’s lead. In the United States, beet sugar production began in 1890 in the states of California and Nebraska. Sugar Production in the Twentieth Century and Beyond In the twentieth century, many former European colonies became independent nations. Today the largest producers of sugar are Brazil, India, China, Thailand, Pakistan, Mexico, the Philippines, and Colombia. Agriculture practices also underwent significant changes; sugar making and various other processes are now automated, with machines replacing manual labor. In most countries, independent farmers grow sugarcane and then sell it to sugar mills. Now sugar is produced everywhere in abundance—200 million tons per year—and is affordable for most people around the world. Nowadays, 70–80 percent of sugar on the market is cane sugar, and about 20–30 percent is beet sugar. Story of Tea Tea is made from the leaves of Camellia sinensis, a small evergreen tree (ten to twelve feet tall) in the Theaceae family that is native to north Burma and southwestern China (see figure 3.9). Two varieties of the tea plant, C. sinensis var. sinensis and C. s. var. assamica, are cultivated around the world for commercial tea production. The best tea is made from a new bud and the two to three leaves adjacent to it, which are newly formed and delicate and contain the most caffeine. Therefore, Camellia trees are pruned into three-to-four-foot-tall bushes to promote branching and the production of new leaves, as well as to facilitate plucking them. Various processing methods are used to attain different levels of oxidation and produce certain kinds of tea, such as black, white, oolong, green, and pu’erh. Basic processing includes plucking, withering (to wilt and soften the leaves), rolling (to shape the leaves and slow drying), oxidizing, and drying. However, depending on the tea type, some steps are repeated or omitted. For example, green tea is made by withering and rolling leaves at a low heat, and oxidation is skipped; for oolong, rolling and oxidizing are performed repeatedly; and for black, extensive oxidation (fermentation) is employed. Discovery of Tea Tea was discovered in 2700 BCE by the ancient Chinese emperor Shen Nung, who had a keen interest in herbal medicine and introduced the practice of drinking boiled water to prevent stomach ailments. According to legend, once, when the emperor camped in a forest during one of his excursions, his servants set up a pot of boiling water under a tree. A fragrance attracted his attention, and he found that a few dry leaves from the tree had fallen accidentally into the boiling pot and changed the color of the water; this was the source of the aroma. He took a few sips of that water and noticed its stimulative effect instantly. The emperor experimented with the leaves of that tree, now called Camellia sinensis, and thus the drink “cha” came into existence. Initially, it was used as a tonic, but it became a popular beverage around 350 BCE. The historian Lu Yu of the Tang dynasty (618–907 CE) has written a poetry book on tea called Cha jing (The Classic of Tea) that contains a detailed description of how to cultivate, process, and brew tea. Tea spread to Japan and Korea in the seventh century thanks to Buddhist monks, and drinking it became an essential cultural ritual. Formal tea ceremonies soon began. However, tea reached other countries only after the sixteenth century. In 1557, the Portuguese established their first trading center in Macau, and the Dutch soon followed suit. In 1610, some Dutch traders in Macau took tea back to the Dutch royal family as a gift. The royal family took an immediate liking to it. When the Dutch princess Catherine of Braganza married King Charles II of England around 1650, she introduced tea to England. Tea passed from the royal family to the nobles, but for an extended period, it remained unknown and unaffordable to common folks in Europe. The supply of tea in Europe was scant and very costly: one pound of tea was equal to nine months’ wages for a British laborer. As European trade with China increased, more tea reached Europe, and consumption of tea increased proportionally. For example, in 1680, Britain imported a hundred pounds of tea; however, in 1700, it brought in a million. The British government allowed the British East India Company to monopolize the trade, and by 1785, the company was buying 15 million pounds of tea from China annually and selling it worldwide. Eventually, in the early eighteenth century, tea reached the homes of British commoners. the “Opium War” China was self-sufficient; its people wanted nothing from Europe in exchange for tea. But in Europe, the demand for tea increased rapidly in the mid-eighteenth century. Large quantities were being purchased, and Europeans had to pay in silver and gold. The East India Company was buying so much of it that it caused a crisis for the mercantilist British economy. The company came up with a plan to buy tea in exchange for opium instead of gold and silver. Although opium was banned within China, it was in demand and sold at very high prices on the black market. After the Battle of Plassey in 1757, several northern provinces in India came under the control of the East India Company, and the company began cultivating poppy in Bengal, Bihar, Orissa, and eastern Uttar Pradesh. Such cultivation was compulsory, and the company also banned farmers from growing grain and built opium factories in Patna and Banaras. The opium was then transported to Calcutta for auction before British ships carried it to the Chinese border. The East India Company also helped set up an extensive network of opium smugglers in China, who then transported opium domestically and sold it on the black market. After the successful establishment of this smuggling network, British ships bought tea on credit at the port of Canton (now Guangzhou), China, and later paid for it with opium in Calcutta (now Kolkata). The company not only acquired the tea that was so in demand but also started making huge profits from selling opium. This mixed business of opium and tea began to strengthen the British economy and made it easier for the British to become front-runners among the European powers. By the 1830s, British traders were selling 1,400 tons of opium to China every year, and as a result, a large number of Chinese became opium addicts. The Chinese government began a crackdown on smugglers and further tightened the laws related to opium, and in 1838, it imposed death sentences on opium smugglers. Furthermore, despite immense pressure from the East India Company to allow the open trading of opium, the Chinese emperor would not capitulate. However, that did not curb his subjects’ addiction and the growing demand for opium. In 1839, by order of the Chinese emperor, a British ship was detained in the port of Canton, and the opium therein was destroyed. The British government asked the Chinese emperor to apologize and demanded compensation; he refused. British retaliated by attacking a number of Chinese ports and coastal cities. China could not compete with Britain’s state-of-the-art weapons, and defeated, China accepted the terms of the Treaty of Nanjing in 1842 and the Treaty of Bog in 1843, which opened the ports of Canton, Fujian, and Shanghai, among others, to British merchants and other Europeans. In 1856, another small war broke out between China and Britain, which ended with a treaty that made the sale of opium legal and allowed Christian missionaries to operate in China. But the tension between China and Europe remained. In 1859, the British and French seized Beijing and burned the royal Summer Palace. The subsequent Beijing Convention of 1860 ended China’s sovereignty, and the British gained a monopoly on the tea trade. Co-option of Tea and the Establishment of Plantations in European Colonies Unlike the British, the Dutch, Portuguese, and French had less success in the tea trade. To overcome British domination, the Portuguese planned to develop tea gardens outside China. Camellia is native to China, and it was not found in any other country. There was a law against taking these plants out of the country, and the method for processing tea was also a trade secret. In the mid-eighteenth century, many Europeans smuggled the seeds and plants from China, but they were unable to grow them. Then, in 1750, the Portuguese smuggled the Camellia plants and some trained specialists out of China and succeeded in establishing tea gardens in the mountainous regions of the Azores Islands, which have a climate favorable for tea cultivation. With the help of Chinese laborers and experts, black and green tea were successfully produced in the Portuguese tea plantations. Soon, Portugal and its colonies no longer needed to import tea at all. As the owners of the first tea plantations outside China, the Portuguese remained vigilant in protecting their monopoly. It was some time before other European powers gained the ability to grow and process tea themselves. In the early nineteenth century, the British began exploring the idea of planting tea saplings in India. In 1824, Robert Bruce, an officer of the British East India Company, came across a variety of tea popular among the Singpho clan of Assam, India. He used this variety to develop the first tea garden in the Chauba area of Assam, and in 1840, the Assam Tea Company began production. This success was instrumental to the establishment of tea estates throughout India and in other British colonies. In 1848, the East India Company hired Robert Fortune, a plant hunter, to smuggle tea saplings and information about tea processing from China. Fortune was the superintendent of the hothouse department of the British Horticultural Society in Cheswick, London. He had visited China three times before this assignment; the first, in 1843, had been sponsored by the horticultural society, which was interested in acquiring important botanical treasures from China by exploiting the opportunity offered by the 1842 Treaty of Nanking after the First Opium War. Fortune managed to visit the interior of China (where foreigners were forbidden) and also gathered valuable information about the cultivation of important plants, successfully smuggling over 120 plant species into Britain. In the autumn of 1848, Fortune entered China and traveled for nearly three years while carefully collecting information related to tea cultivation and processing. He noted that black and green teas were made from the leaves of the same plant, Camellia sinensis, except that the former was “fermented” for a longer period. Eventually, Fortune succeeded in smuggling 20,000 saplings of Camellia sinensis to Calcutta, India, in Wardian cases.[4] He also brought trained artisans from China to India. These plants and artisans were transported from Calcutta to Darjeeling, Assam. At Darjeeling, a nursery was set up for the propagation of tea saplings at a large scale, supplying plantlets to all the tea gardens in India, Sri Lanka, and other British colonies. The British forced the poor tribal population of the Assam, Bengal, Bihar, and Orissa provinces out of their land, and they were sent to work in tea estates. Tamils from the southern province of India were also sent to work in the tea plantation of Sri Lanka. Tea plantations were modeled on the sugar colonies of the Caribbean, and thus the plight of the workers was in some ways similar to that of the slaves from Caribbean plantations. Samuel Davidson’s Sirocco tea dryer, the first tea-processing machine, was introduced in Sri Lanka in 1877, followed by John Walker’s tea-rolling machine in 1880. These machines were soon adopted by tea estates in India and other British colonies as well. As a result, British tea production increased greatly. By 1888, India became the number-one exporter of tea to Britain, sending the country 86 million pounds of tea. After India, Sri Lanka became prime ground for tea plantations. In the last decades of the nineteenth century, an outbreak of the fungal pathogen Hemilia vastatrix, a causal agent of rust, resulted in the destruction of the coffee plantations in Sri Lanka. The British owners of those estates quickly opted to plant tea instead, and a decade later, tea plantations covered nearly 400,000 acres of land in Sri Lanka. By 1927, Sri Lanka alone produced 100,000 tons per year. All this tea was for export. Within the British Empire, fermented black tea was produced, for which Assam, Ceylon, and Darjeeling tea are still famous. Black tea produced in India and Sri Lanka was considered of lesser quality than Chinese tea, but it was very cheap and easily became popular in Asian and African countries. In addition to India and Ceylon, British planters introduced tea plantations to fifty other countries. Story of Coffee Coffee is made from the roasted seeds of the coffee plant, a shrub belonging to the Rubiaceae family of flowering plants. There are over 120 species in the genus Coffea, and all are of tropical African origin. Only Coffea arabica and Coffea canephora are used for making coffee. Coffea arabica (figure 3.10) is preferred for its sweeter taste and is the source of 60–80 percent of the world’s coffee. It is an allotetraploid species that resulted from hybridization between the diploids Coffea canephora and Coffea eugenioides. In the wild, coffee plants grow between thirty and forty feet tall and produce berries throughout the year. A coffee berry usually contains two seeds (a.k.a. beans). Coffee berries are nonclimacteric fruits, which ripen slowly on the plant itself (and unlike apples, bananas, mangoes, etc., their ripening cannot be induced after harvest by ethylene). Thus ripe berries, known as “cherries,” are picked every other week as they naturally ripen. To facilitate the manual picking of cherries, plants are pruned to a height of three to four feet. Pruning coffee plants is also essential to maximizing coffee production to maintain the correct balance of leaf to fruit, prevent overbearing, stimulate root growth, and effectively deter pests. Coffee is also a stimulative, and the secret of this elixir is the caffeine present in high quantities in its fruits and seeds. In its normal state, when our bodies are exhausted, there is an increase in adenosine molecules. The adenosine molecules bind to adenosine receptors in our brains, resulting in the transduction of sleep signals. The structure of caffeine is similar to that of adenosine, so when it reaches a weary brain, caffeine can also bind to the adenosine receptor and block adenosine molecules from accessing it, thus disrupting sleep signals. History of Coffee Coffea arabica is native to Ethiopia. The people of Ethiopia first recognized the stimulative properties of coffee in the ninth century. According to legend, one day, a shepherd named Kaldi, who hailed from a small village in the highlands of Ethiopia, saw his goats dancing energetically after eating berries from a wild bush. Out of curiosity, he ate a few berries and felt refreshed. Kaldi took some berries back to the village to share, and the people there enjoyed them too. Hence the local custom of eating raw coffee berries began. There are records that coffee berries were often found in the pockets of slaves brought to the port of Mokha from the highlands of Ethiopia. Later, the people of Ethiopia started mixing ground berries with butter and herbs to make balls. The coffee we drink today was first brewed in Yemen in the thirteenth century. It became popular among Yemen’s clerics and Sufis, who routinely held religious and philosophical discussions late into the night; coffee rescued them from sleep and exhaustion. Gradually, coffee became popular, and coffeehouses opened up all over Arabia, where travelers, artists, poets, and common folks visited and had a chance to gossip and debate on a variety of topics, including politics. Often, governments shut down coffeehouses for fear of political unrest and revolution. Between the sixteenth and seventeenth centuries, coffeehouses were banned several times in many Arab countries, including Turkey, Mecca, and Egypt. But coffeehouses always opened again, and coffee became ingrained in Arab culture. Arabs developed many methods of processing coffee beans. Usually, these methods included drying coffee cherries to separate the beans. Dried coffee beans can be stored for many years. Larger and heavier beans are considered better. The taste and aroma develop during roasting, which determines the quality and price of the coffee. Dried coffee beans are dark green, but roasting them at a controlled temperature causes a slow transformation. First, they turn yellow, then light brown, while also popping up and doubling in size. After continued roasting, all the water inside them dries up, and the beans turn black like charcoal. The starch inside the beans first turns into sugar, and then sugar turns into caramel, at which point many aromatic compounds come out of the cells of the beans. Roasting coffee beans is an art, and a skilled roaster is a very important part of the coffee trade. Spread of Coffee out of Arabia Coffee was introduced to Europeans in the seventeenth century, when trade between the Ottoman Empire and Europe increased. In 1669, Turkish ambassador Suleiman Agha (Müteferrika Süleyman Ağa) arrived in the court of Louis XIV with many valuable gifts, including coffee. The French subsequently became obsessed with the sophisticated etiquettes of the Ottoman Empire. In the company of Aga, the royal court and other elites of Parisian society indulged in drinking coffee. Aga held extravagant coffee ceremonies at his residence in Paris, where waiters dressed in Ottoman costumes served coffee to Parisian society women. Suleiman’s visit piqued French elites’ interest in Turquerie and Orientalism, which became fashionable. In the history of France, 1669 is thought of as the year of “Turkmenia.” A decade later, coffee reached Vienna, when Turkey was defeated in the Battle of 1683. After the victory, the Viennese seized the goods left behind by the Turkish soldiers, including several thousand sacks of coffee beans. The soldiers of Vienna didn’t know what it was and simply discarded it, but one man, Kolshitsky, snatched it up. Kolshitsky knew how to make coffee, and he opened the first coffeehouse in Vienna with the spoils. By the end of the seventeenth century, coffeehouses had become common in all the main cities of Europe. In London alone, by 1715, there were more than 2,000 coffeehouses. As in Arabia, the coffeehouses of Europe also became the bases of sociopolitical debates and were known as “penny universities.” Coffee Plantations By the fifteenth century, demand for coffee had increased so much that the harvest of berries from the wild was not enough, and thus in Yemen, people began to plant coffee. Following Yemen’s lead, other Arab countries also started coffee plantations. Until the seventeenth century, coffee was cultivated only within North African and Arab countries. Arabs were very protective of their monopoly on the coffee trade. The cultivation of coffee and the processing of seeds was a mystery to the world outside of Arabia. Foreigners were not allowed to visit coffee farms, and only roasted coffee beans (incapable of producing new plants) were exported. Around 1600, Baba Budan, a Sufi who was on the Haj pilgrimage, successfully smuggled seven coffee seeds into India and started a small coffee nursery in Mysore. The early coffee plantations of South India used propagations of plants from Budan’s garden. In 1616, a Dutch spy also succeeded in stealing coffee beans from Arabia, and these were used by the Dutch East India Company as starters for coffee plantations in Java, Sumatra, Bali, Sri Lanka, Timur, and Suriname (Dutch Guiana). In 1706, a coffee plant from Java was brought to the botanic gardens of Amsterdam, and from there, its offspring reached Jardin de plantes in Paris. A clone of the Parisian plant was sent to the French colony Martinique, and then its offspring spread to the French colonies in the Caribbean, South America, and Africa. In 1728, a Portuguese officer from Dutch Guiana brought coffee seeds to Brazil, which served as starters for the coffee plantations there. The Portuguese also introduced coffee to African countries and Indonesia, and the British established plantations in their Caribbean colonies, India, and Sri Lanka from Dutch stock. In summary, all European coffee plants came from the same Arabian mother plant. So the biodiversity within their coffee plantations was almost zero, which had devastating consequences. In the last decades of the nineteenth century, the fungal pathogen Haemilia vestatrix severely infected coffee plantations in Sri Lanka, India, Java, Sumatra, and Malaysia. As a result, rust disease destroyed the coffee plantations one by one. Later, in some of the coffee plantations, Coffea canephora (syn. Coffea robusta), which has a natural resistance to rust, was planted, but others were converted into tea plantations (as in the case of Sri Lanka, discussed earlier). European coffee plantations used the same model as tea or sugar plantations, and so their workers lived under the same conditions. European powers forcefully employed the poor native population in these plantations and used indentured laborers as needed. For example, in Sri Lanka, the Sinhalese population refused to work in the coffee farms, so British planters recruited 100,000 indentured Tamil workers from India to work the farms and tea plantations there. Heritage of Plantations In the twentieth century, most former European colonies became independent countries. In these countries, private, cooperative, or semigovernmental institutions manage plantations of sugarcane, tea, coffee, or other commercial crops. Though these plantations remain a significant source of revenue and contribute significantly to the national GDP of many countries, their workers still often operate under abject conditions. Further Readings Aronson, M., & Budhos, M. (2010). Sugar changed the world: A story of magic, spice, slavery, freedom, and science (1st ed.). Clarion. Bahadur, G. (2014). Coolie woman: The odyssey of indenture. University of Chicago Press. Clarkson, T. (1785). An essay on the slavery and commerce of the human species. abolition.e2bn.org/source_16.html Galloway, J. H. (1989). The sugar cane industry: An historical geography from its origins to 1914. Cambridge University Press. Gandhi, M. K. (1972). Satyagraha in South Africa. Navajivan. (Original work published 1924) Linares, O. F. (2002). African rice (Oryza glaberrima): History and future potential. Proc. Natl. Acad. Sci. USA, 99, 16360–65. doi.org/10.1073/pnas.252604599 Pendergrast, M. (2010). Uncommon grounds: The history of coffee and how it transformed our world. Basic Books. Rose, S. (2011). For all the tea in China: How England stole the world’s favorite drink and changed history. Penguin. Tinker, H. (1974). New system of slavery the export of Indian labor overseas 1830–1920 (Institute of Race Relations). Oxford University Press. YouTube documentary “Black Coffee: The Irresistible Bean.” Part 1—https://youtu.be/XbrF1CmaUDo Part 2—https://youtu.be/bwsyktsWmhE Part 3—https://youtu.be/a_tWx8-7wB4 1. As an example, Ẓahīr al-Dīn Muḥammad Bābur(1483–1530), the founder of the Mughal dynasty, invaded Hindustan (northern India) and established his capital in the city of Agra. His artisans and well-versed gardeners for the first time introduced several crops of Central Asia into India: on the banks of the Yamuna River in the city of Agra, India, they sowed watermelons, melons, musk melon, and so on and planted grapes and roses in the Agra Fort. 2. The Arthaśāstrais is an ancient Indian text on politics, economic policy, and military strategy written in Sanskrit by Kauṭilya, also known as Vishnugupta or Chanakya. He was a scholar at the ancient school Takshashila and the teacher and guardian of Emperor Chandragupta Maurya, who founded the Mauryan Empire in northern India. 3. See the history of the abolitionist movement and related documents at abolition.e2bn.org. 4. The Wardian case, a precursor to the modern terrarium, was a special type of sealed glass box made by British doctor Nathaniel Bagshaw Ward in 1829. The delicate plants within them could thrive for months. Plant hunter Joseph Hooker successfully used Wardian cases to bring some plants from the Antarctic to England. In 1933, Nathaniel Ward also succeeded in sending hundreds of small ornamental plants from England to Australia in these boxes. After two years, another voyage carried many Australian plants back to Dr. Ward as a gift. Despite lengthy and difficult journeys, these plants survived, and so Wardian cases proved extremely useful for plant hunters, such as Robert Fortune.

textbooks/bio/Agriculture_and_Horticulture/History_and_Science_of_Cultivated_Plants_(Naithani)/1.03%3A_Colonial_Agriculture.txt

After the sixteenth century, when Europeans reached Asia, Africa, and the Americas, they saw many numerous new species of domesticated and wild plants and animals. Soon, surveying and systematic cataloging of the natural and biological resources ensued under the supervision of various experts. First, three or four experts went in small ships to survey the coastal areas of South America, Africa, and Asia. Typically, a geologist/naturalist would collect samples of rocks, soil, mineral, fossils, and plants and attach a note with each sample describing its features; an engineer’s job was to draw or update the navigational maps and gather information about ports and structures of strategic importance; an artist painted landscapes and helped experts prepare sketches of valuable items. From time to time, the collections from survey ships were sent to Europe via other ships. As a result of the various surveys and expeditions, the royal gardens of Europe were overwhelmed with the vast collections from around the world, and several experts were recruited for the analysis, classification, and systematic cataloging of fauna, flora, fossils, and other types of samples. Besides experts, both elites and common folks, especially the elites and middle-class Europeans, were taken aback by seeing thousands of varieties of fruits, vegetables, and ornamental plants from around the world. Also, many small institutions got interested in acquiring exotic plants and gardening different plant varieties. Around 1650, when the educated elites got involved in growing ferns, tulips, fruits, and so on, the publication of gardening books and nursery catalogs began, and the first nurseries and seed and landscaping companies were founded. Amateur horticulturists in Europe and around the world started the conscious selection of a variety of fruits, vegetables, flowers, and cereals. Today, many of what we consider “natural” fruits, vegetables, and flowers are the result of this conscious selection in the last 300 years. For example, in the seventeenth century, orange carrots were developed in the Netherlands for the first time by the artificial selection of yellow carrots. In the natural form, wild carrots are white and thin. Around the tenth century, the people of Afghanistan had domesticated light yellow carrots, which the Arabs had taken to Europe. In the same way, larger tomatoes, berries, and corn were developed via conscious selection. The variety selection was upheld by several organizations, and annual exhibitions of fruit and flower products began in many cities. Even today, annual agricultural fairs and exhibitions continue throughout the world, and gardeners and farmers still consciously select varieties. For example, in South Asia, farmers have developed thousands of rice varieties to thrive in different climatic and geographical conditions. Discovery of Sex in Plants: Self- versus Cross-Pollinated Crops The detailed investigation of plant morphology and anatomy and how these features change in relation to the plant’s habitat and/or in response to its environment led to tremendous advances in knowledge about the natural world and unveiled many mysteries. In this series, German botanist Camerarius (1665–1721) discovered sex in plants,[1] identified various parts of the flower (see figure 4.1), and classified plants into seven categories based on sex (flower structure). In the seventeenth century, it became common knowledge in Europe that seeds are the product of sexual union and flowers contain sexual organs.[2] If we look around carefully, we can easily spot the difference in the structure of flowers and identify the following seven types of sexes in flowering plants: 1. Perfect/Hermaphrodite. These flowers contain all four floral organs (sepals, petals, stamens, and carpels). Examples are various flowers of the Brassicaceae family (i.e., mustard, radish, broccoli, etc.). 2. Monoecious. These plants bear both male and female reproductive organs. Monoecious means “one home.” In cucumber, pumpkin, and gourd, separate male and female flowers are distributed randomly, whereas, in maize, the male flowers (tassel) hang on the top, and the female flowers are on the branch, which can be identified by their silk. 3. Dioecious. In some species, separate male and female plants are present (e.g., dates, papaya, and Cannabis). 1. Androecious. These plants contain only male flowers. 2. Gynoecious. These plants contain only female flowers. 4. Andromonoecious. These plants have male and perfect flowers (e.g., saffron). 5. Gynomonoecious. These plants have female and perfect flowers. 6. Trimonoecious. These plants have male, female, and perfect flowers (e.g., Datura). Camerarius stated that pollen is required to reach the stigma to form fruit and seeds; this process is called pollination. Self-pollination (selfing) refers to the process when the pollen from the anther is transferred on the stigma of the same flower or another flower on the same plant. Cross-pollination (outcrossing) occurs when pollen from one plant is deposited on the flower (stigma) of a different plant of the same species. The structure of flowers determines the mode of pollination. Often, one can tell just by looking at a flower whether it self-pollinates or cross-pollinates. The self-pollinating flowers are small and monocolored; are unscented; have anthers close to stigma; and lack nectar guides. In these types of flowers, pollination and fertilization often occur in an unopened flower bud (wheat, barley, peanut), and resulting offspring are uniform. Typically, manual crossing is needed to produce hybrids. In the wild, most flowers promote outcrossing, which supports the continuous mixing of gametes and new combinations of traits. Many species of animals (e.g., bees, butterflies, birds, and mammals) serve as pollinators; however, wind and water can also carry pollen from one plant to another. The outbreeders have many large, scented, bright-colored flowers that have nectaries present and stigmatic areas well defined and away from the anthers. We find extraordinary structural adaptations in flowers to attract a specific pollinator, and the shape and form of pollen are adapted to their mode of pollination (e.g., wind, water, insects, and mammals). For example, insect-pollinated species have sticky, barbed pollen grains, and wind-pollinated species have light, small, and smooth-surfaced pollens. The cross-pollination increases genetic variability and results in strong evolutionary potential for a species, allowing for adaptations to changing environmental and climatic conditions. However, it can be disadvantageous in certain circumstances; for example, cross-pollination can destroy well-adapted genotypes and relies on vectors for effective pollination and seed set. Table 4.1. Examples of self- and cross-pollinated crops. Self-pollinated crops Cross-pollinated crops Cross/self-pollinated crops Rice Alfalfa Cotton Wheat Cassava Oilseed Rape Barley Corn Tomato Millet Pearl Millet Brassicas Oats Rye Potato Flax Safflower Legumes (cowpea, beans, chickpea, lentil, mung bean, etc.) Sugar Beet Tobacco Sugar Cane Potato Sunflower Tomato Carrot Sesame Cucumber family (cucumber, melons, pumpkin, squash, etc.) Jute Classification of Plants From the 1750s to the 1760s, the Swedish government sponsored a number of expeditions under the supervision of Carolus von Linné (Linnaeus), a professor of medicine and botany at Uppsala University (see figure 4.2). His team collected thousands of fauna and flora samples, and after a comprehensive study, Linnaeus proposed the binomial system of naming plants and animals (see figure 4.3). The smallest unit in the Linnaeus system[3] of classification of organisms is species. He proposed that the males and females of one species can give birth to a healthy progeny, but in a different species, they may not. He then organized the species that are closest in their structure under one genus and many genera having similar characteristics into a family. Then based on further similarities, many families were grouped into an order, several orders were grouped into a class, and several classes were grouped into a phylum. Finally, all the plant phyla were grouped into the plant kingdom, and all the animal phyla were grouped into the animal kingdom (see figure 4.3). Linnaeus proposed two Latin names for the scientific nomenclature of organisms, of which the first name indicates genus and the second indicates the species. Thus the scientific name of a human is Homo sapiens, and the rice plant is Oryza sativa. In this system, the genus part of the name comes first and is always capitalized; it is followed by the species name, which is not capitalized. Both names are italicized. Later, Linnaeus’s binomial method was also used for naming and classifying bacteria, fungi, and other microscopic organisms. Linnaeus laid the foundation of taxonomy, which aims to study biological diversity and provides guidelines for classification, description, identification, and naming of living organisms. Classification is the grouping of organisms based on their shared characteristics (anatomy, structure, morphology, reproductive behavior, etc.).[4] In general, organisms that share similar features are closely related and are placed in a group. Thus the binomial system also provides an insight into the evolutionary history (phylogeny) of organisms. Just as Mendeleev’s periodic table of elements helps in understanding the atomic structure of various elements, the basic understanding of the structure and relationship of organisms is derived from the classification schema proposed by Linnaeus. Historically, the binomial method of Linnaeus was the first major theoretical understanding in the field of biology. Linnaeus became Europe’s greatest scientist in his lifetime. Deliberate Hybridizations Understanding the flower structure and the pollination process made artificial pollination possible and paved the path for crop improvement by deliberate hybridization. It became possible to develop new and improved varieties by crossing two varieties with desirable traits. However, in the absence of a theoretical understanding of laws that govern heredity, the selection of desirable hybrids was almost impossible and a work of trial and error. Thomas Fairchild (1667–1729), a nursery owner in England, was likely the first breeder who succeeded in creating a hybrid pink by crossing carnation and sweet William in 1717. His hybrid is also known as Fairchild’s mule. Fairchild gave a lecture on the crossing in the meeting of the Royal Society that was subsequently published in its journal, Philosophical Transactions as “Some New Experiments Relating to the Different and Sometimes Contrary Motion of the Sap in Plants and Trees.” He is also credited for writing the first book on gardening. After forty years of Fairchild’s research, Linnaeus carried out extensive crossing experiments. He described flowers as “marriage beds” of plants and considered the flower structure as one of the important criteria for developing classification schema for plants. In 1757, Linnaeus crossed two species of Tragopogon and identified many plants that were products of spontaneous hybridization. Because of Linnaeus, European scientists, naturalists, teachers, and amateur gardeners got interested in crossing experiments, and the information about flower structure became public knowledge. In the following sections, we describe a brief review of some of the historic crossing experiments and the success of early plant breeders. Story of the Annanasa Strawberry In 1711, Louis XIV sent Captain Amédée-François Frézier (1682–1773) on an espionage mission to Chile and Peru, as France didn’t deem it appropriate to show overt interest in Spain’s colonies. Captain Frézier, an engineer by profession, traveled to South America and gathered information on fortresses, armies, supply routes, governors, and Indians (indigenous people). In Chile, he noticed a very large strawberry (see figure 4.4), almost quadruple the size compared to the alpine or woodland strawberry (Fragaria vesca) found in Europe or the Virginia strawberry (Fragaria virginiana) in North America that was brought to France by Jack Cartier (known for discovering the St. Lawrence River in the US) in the seventeenth century. Both F. vesca and F. virginiana were wild varieties and have not been domesticated. People often used to collect their fruits from the forest during the season. However, from the fourteenth century onward, wild strawberry plants were introduced as ornamental plants in European churches and in royal gardens. Interestingly, the Chilean strawberry (Fragaria chiloensis) spotted by Frézier was the only species of strawberry that humans had domesticated. The indigenous people of Chile were cultivating strawberries for more than 1,000 years. In 1550–51, when Spanish general Pizarro took over Chile, his army included strawberries in the list of plunders. Later, the Spanish expanded the cultivation of strawberries in Chile, and the royalty and elites of Spain enjoyed this delicacy. However, other Europeans remained ignorant of the existence of F. chiloensis (see figure 4.4) for almost 150 years. In 1714, Frézier brought five stolen plants of F. chiloensis (also known as the beach strawberry) into France. On his return voyage, Frézier faced many challenges (the inspection and search by Spanish customs officers, a pirate attack, and a shortage of drinking water) but was successful in keeping the strawberry plants alive. In the history of the modern strawberry, this journey of the Chilean strawberry was the most important event. Surprisingly, for the next fifty years, these strawberry plants never produced a single fruit in the French royal garden despite plenty of flowering. Bernard de Jussieu, head of the king’s garden, preserved and maintained several specimens of F. chiloensis and gifted its plantlets to other gardeners and his acquaintances. Finally, in 1766, Antoine Nicholas Duchesne explained that Frézier had selected fruit-bearing female plants in Chile, and all five plants that reached France were female. These female plants needed pollen to form fruits. Duchesne was appointed the gardener of the royal gardens of France in 1760, and he worked under the supervision of Antoine de Jussieu. In the garden at Versailles, Duchesne noticed that the Chilean strawberry planted in between F. virginiana and the musky strawberry (Fragaria moschata) had produced a large fruit due to accidental cross-pollination. He then pollinated the female flowers of Chilean strawberries with pollens of the musky strawberry, which produced a huge and succulent strawberry fruit with a pineapple aroma. On July 6, 1764, Duchesne presented a bowl full of this large fruit to King Louis XVI. Madeleine Françoise Basseporte, the famous artist, painted strawberries for the royal botanic library, and Duchesne was given a grant to pursue his research on strawberries. Bernard de Jussieu, the mentor of Nicholas Duchesne, was familiar with Linnaeus’s work and had instructed Duchesne about binomial nomenclature and the role of sexes in plants. Duchesne carefully observed the various strawberry plants present in the French royal garden and learned to differentiate between their male and female flowers. After this, he started experimenting with artificial pollination. First, he pollinated Chilean strawberries with the pollen from European woodland strawberry plants, but this did not result in fruit formation, but the pollination of the Chilean strawberry with pollens from F. moschata or F. virginiana did. Today we know that these compatibilities were related to the ploidy level of different strawberry species. F. vesca is diploid (two sets of chromosomes), and both F. virginiana and F. chiloensis are octoploids (eight sets of chromosomes).xiii F. virginiana produces small, highly aromatic fruits in abundance, and F. chiloensis bears big fruits (the size of walnuts). Duchesne crossed F. virginiana with F. chiloensis to make the hybrid Fragaria x ananassa (ananassa strawberry; x indicates that the strawberry is of hybrid origin), which produces large and flavorful fruits with pineapple (ananassa) aroma (see figure 4.5). The widespread cultivation of strawberries began with F. x ananassa. Therefore, the evolution of the strawberry from a wild to a cultivated plant is relatively recent, and it is a by-product of transatlantic exploration that involved three continents. Duchesne also collected strawberry plants from all over Europe, recorded the relevant information, and wrote a report on the natural history of the strawberry. He also interviewed Captain Frézier fifty years later. There are species of strawberry native to regions all around the world, and after Duchesne, many other scientists collected thousands of varieties of strawberries. These varieties of strawberries have different ploidy, fragrance, flavor, and other physical attributes and are adapted to a wide variety of climates. Today, the largest germplasm collection of strawberries is in Corvallis, Oregon. Monks of the Austro-Hungarian Empire Interestingly, the province of Moravia of the Austro-Hungarian Empire became one of the early centers of plant breeding in Europe.[5] The educated elites of Moravia held liberal beliefs in comparison to the remaining Catholic Europe and were invested in improving both education and local agriculture. Many clergymen got inspired by Linnaeus and quickly grasped the utility of deliberate hybridization in improving the local economy. Christian Andre (1763–1831), a social reformer, was one of the pioneers who advocated for plant and animal breeding in Moravia. He founded the Moravian Sheep Breeders Association and the Pomological and Oenological Society of Brno. He collaborated with many of his contemporary horticulturists and breeders, including Jan Sedlacek and G. C. L. Hempel. Abbot F. C. Knapp (1792–1867) of the monastery of Brno was another early influence in Moravia’s breeders and took a keen interest in improving fruit trees and grapevine. He had planted over a hundred varieties of grapes in the monastery’s nursery and encouraged local farmers to plant fruit trees. In 1840, Knapp organized a large gathering of local farmers and forest officials and explained to them how to make hybrids. The breeding efforts in Kuninn were also noteworthy. In 1796, Father J. Schreiber began teaching crossing as a part of the natural science curriculum in Kuninn. This school was founded by the countess Maria Walpurga Truchsess-Zeil and provided education to village children for free. It is said that the greatest concern of the bishop of Brno around 1854 was that his subordinate clergy were paying more attention to natural science, crossing, and nursery than to religious work. It is believed that Brno’s monastery narrowly escaped being closed. Some decades later, one of those priests, Gregor Johann Mendel, discovered the fundamental principles of genetics. However, breeding in those early stages was more of an art than a standardized scientific technique. The common understanding of heredity was similar to the mixing of two colors: the mother’s and father’s traits mix and reach their offspring. It was with this understanding that the breeders chose the desired characteristics from the cross between two varieties. The task was as difficult as finding a needle in a haystack, and using trial and error, the breeders sometimes found the plants and animals they wanted. Only fewer than a dozen successful hybrids were made during that era, and fewer still had any significant traits. The ananassa strawberry has special significance in hybrid plants made in this era, and equally impressive is its story. Professors and Other Professional Breeders Apart from monks and gardeners, many professors, teachers, and other professionals pursued crossings and plant breeding experiments out of their intellectual curiosity. Particularly in Germany, Joseph Gottlieb Kölreuter, Christian Konrad Sprengel, and Karl Friedrich von Gartner have contributed significantly to the current knowledge of pollination, hybridization, and heredity. Kölreuter (1733–1806), a professor of natural history at the University of Karlsruhe, Germany, carried out more than 500 artificial hybridization experiments involving 138 species. He made important observations about the role of insects as pollinators and examined the shape, color, and size of pollen grains from ~1,000 different plant species. He noted that successful hybridization occurs only between closely related species, and in offspring, some parental traits show up more often than others. Although he did not dwell deep enough in quantifying the ratio of various inherited traits, his experimental results foreshadowed Gregor Johann Mendel’s work. Sprengel (1750–1816) followed Kölreuter’s work on hybridization and made the additional observation that normal sexual reproduction in plants leads to the mixing of traits from both male and female parents and thus creates new combinations in the offspring. This was a big conceptual leap toward understanding the source of diversity among the individual of the same species and an acknowledgment that living species are constantly changing rather than being fixed. Gartner (1772–1850) was another contemporary of Sprengel, who pursued hybridization experiments as an amateur breeder. He was the physician son of famous botanist Joseph Gartner, who was familiar with the work of both Kölreuter and Sprengel. Over twenty-five years, he carried out ~10,000 individual crossings on ~700 plants (belonging to over 80 genera) and created 350 different hybrids. He also noticed that many hybrids bore larger flowers and fruits than their parents and appeared to be from a different species from their parents. Over the years, this phenomenon was noticed by many and was later named Hybrid vigor. In England, botanist Thomas Andrew Knights (1759–1838) bred fruit trees and successfully made several hybrids, including those of pears and apples. He successfully crossed apple and Siberian crab apple and produced a hybrid that is known for making good quality cider. Like Sprengel, he observed the differences between plants of the same species and the mutability of various characteristics that constantly emerge and then disappear. In addition, Knights published over a hundred research papers on various aspects of plant physiology and founded the Royal Horticulture Society. He was also a member of the Royal Society and, in 1806, received the prestigious Copley Medal. Thomas Knight also conducted extensive crossing experiments in peas and found many of the same results as Mendel. However, he lacked the mathematical framework to analyze his results and could not decipher laws governing heredity. Overall, by the early decades of the nineteenth century, numerous experiments of crossing drew the attention of experts and amateur gardeners on the variations among members of the same species and the potential of deliberate hybridization in improving crops and animal stock. Darwin’s Insights from Crossing Experiments Charles Darwin (1809–82) conducted extensive crossings in plants to understand the implications of self-pollination and outcrossing in plants and made two important observations: (1) In nature, the floral structure of most plants favor cross-pollination, and several structural and genetic barriers prevent selfing. (2) As a result of cross-pollination, new combinations of traits continue to emerge by mixing parental characteristics, which provides the raw material for natural selection. Since plants lack mobility, the traits promoting outcrossing were enriched as a result of natural selection to ensure the continued survival of the species. Darwin also reviewed the work of his predecessors and contemporary botanists, including Kölreuter, Sprengel, Gartner, and Knights. He highlighted the differences between crops and wild plants and identified the various domestication traits in both crop plants and domesticated animals. He made it clear that the direction of artificial selection moved in the opposite direction of natural selection, which led to the development of man-made crops and domestic animals. Darwin’s most famous treatise, On the Origin of Species (published in 1859), began with a chapter entitled “Variation under Domestication,” which encapsulated his decade-long comparative study of domesticated plants and animals with their wild ancestors, their origins, and artificial selection by humans—all of which provides a basis for his theory of the bioevolution of living organisms using natural selection. In 1868, Darwin independently published The Variation of Animals and Plants under Domestication to cover a detailed description of the improvement in crop plants and livestock through artificial selection, the beneficial effects of outcrossing, and the harmful effects of inbreeding. Star Breeders of the Early Twentieth Century Luther Burbank: A Plant Wizard Overall, the concept of deliberate hybridization and selection for a desired combination of traits in offspring gained popularity during the nineteenth century and continued until the first two decades of the twentieth century. The most successful and well-known breeder of this tradition was American gardener Luther Burbank (1849–1926). Luther Burbank (see figure 4.6) was born in 1849 in the city of Lancaster, Massachusetts. He received only an elementary education before starting work on a market garden. When he was twenty-one, his father died, and with his share of the inheritance, he purchased a seventeen-acre farm in Lunenburg. On this farm, he identified a natural genetic variant of potato that was large with russet-colored skin, famously known as the russet Burbank potato. This potato variety soon became popular and is used for making french fries. Burbank sold the rights to the russet potato for \$150 and bought a four-acre farm in Santa Rosa, California. A few years later, he purchased an eighteen-acre farm in the nearby town. In both these farms, he laid the necessary infrastructure for greenhouses, nursery beds, and so on so that he can carry out crossings and selection work. Although Burbank’s formal education was only up to primary school, he had learned about horticulture and farming from experience and an interest in the field. His breeding experiments were driven more by an artist’s instinct rather than the scientific understanding of the laws of heredity. He read Charles Darwin’s article about the changes in plants and animals as a result of artificial selection during the process of domestication. This was the article that sparked his interest in plant breeding. He understood that crossings could bring together unconnected gene pools and that finding the desired combinations of traits in the progeny is a rare event and thus requires extensive selection. From his crossing experiments, he selected the best, discarded the rest, and did not keep systematic records of crossing results and the parental strains involved. It is said that Luther simultaneously conducted more than 3,000 crossings at a given time, and millions of plants grew on his farms. Burbank perfected grafting, crossings, and hybrid selection. In his fifty-five-year career, he developed more than 800 new varieties of plants derived from 121 genera: 250 new varieties of fruit, including 113 plums and prunes, 10 strawberries, 10 apples, the russet potato; 34 spineless cacti; about 50 varieties of lilies; and the Shasta daisy. He inevitably sold his new varieties to other growers or nurseries for further commercial production. Andrew Carnegie showed a keen interest in Burbank’s work. And the Dale Carnegie Institute provided financial support to Burbank from 1905 to 1911. In 1893, Burbank published a catalog of his best plant varietals, New Creation in Fruits and Flowers, and also wrote other books on breeding experiments. It was the result of Burbank’s efforts that, in 1930, America began to patent advanced varieties of plants. Because Burbank worked from his own experience and did not keep accurate records, it was not possible for other scientists to review and repeat his work. Famous plant breeders and geneticists, such as George Harrison Shull, Hugo de Vries, Liberty Hyde Bailey, and Nikolai Ivanovich Vavilov, visited him to learn his methods. Shull and de Vries invested time to understand and evaluate Burbank’s work and ultimately became frustrated with his random approach and lack of proper record keeping. However, Burbank became a celebrity in his lifetime and was referred to as the “high priest of horticulture” and the “plant wizard.” Burbank died on April 11, 1926, in Santa Rosa from a heart attack. Nikolai Vavilov wrote a lengthy tribute in remembrance of Burbank. In 1940, a US postage stamp was released in his honor, and in 1991, he was elected to the American Society for Horticultural Science Hall of Fame. The Luther Burbank Home and Gardens, in downtown Santa Rosa, was designated a National Historic Landmark in 2003. “Crank Gardener” Ivan Vladimirovich Michurin Ivan Vladimirovich Michurin (1855–1935), a breeder in the Soviet Union, was a contemporary of Luther Burbank, but while Burbank became a celebrity breeder in his youth, Michurin remained hidden from the world until he reached old age. Michurin (see figure 4.7) lived on a rental farm in a small town named Kozlov near Saratov. Michurin was discovered by chance by Nikolai Vavilov. In 1920, Vavilov attended a conference in Saratov. After lunch, a junior scientist from the conference took the delegates for a walk in Michurin’s garden. The first meeting between young Vavilov and the sixty-five-year-old Michurin lasted several hours. Vavilov was deeply impressed by Michurin’s work but was stunned by the financial condition of this breeder, who was reclusive, paid all his attention to plants, and was considered “crazy” by people in the community. Like Burbank, Michurin had little formal education. His mother had died when he was a child, and his father had gone mad as Michurin reached his teens. At the age of twenty, he rented a small piece of land and started planting fruit trees in it. For fifteen years, he earned a living by working as a clerk, a watchmaker, and a telegraph operator, and when he was not working, he would continue to expand his garden. In 1899, he rented thirteen hectares (thirty-two acres) of land in Kozlov with his savings and brought along his plants. Then he spent thirty years grooming the farm. He had the passion of an artist with a keen eye of a clever gardener. He did not socialize with people and seems to be driven constantly by some sense of urgency for his nursery (that was not profitable). His economic condition did not improve, but he became famous as a “crank gardener” among the people. Throughout his life, Michurin’s focus was on developing new varieties of fruit trees. He learned crossing, grafting, and plant training through trial and error. He acquired many varieties of apples, pears, peaches, apricots, and grapes from distant provinces of Russia and used grafting techniques to make them flourish in Kozlov. Typical fruit trees could not quickly grow in this wintry area, but Michurin had worked for forty-five years and developed about 350 new breeds of fruit trees that could thrive in extremely cold climates. Vavilov sent a proposal to the Ministry of Agriculture to have experts review Michurin’s breeding efforts, which was accepted. On top of that, the Soviet government rewarded him with 500 rubles, granted him the lease of the rental farm where he had set up those marvelous collections, and made that farm tax-free for his lifetime. Vavilov’s meetings with Michurin revealed new information and important details such as how to create the best hybrid variety, how to select hybrids, and so on. Vavilov succeeded in extracting and distilling the life experiences of a clever gardener and provided scientific explanations for why a particular technique worked or not. Subsequently, the Soviet government awarded Michurin the Order of Lenin and the Order of the Red Banner of Labor for his contribution to the field of agriculture. In 1928, Michurin’s farm came to be known as the Genetic Station of Fruit and Berries, and in 1932, his town was renamed Michurinsk in his honor. In 1934, his farm was used for the I. V. Michurin Central Genetic Laboratory. In the last years of life, Michurin became a celebrity breeder in the Soviet Union. Summary Overall, in the nineteenth century, it became public knowledge that living organisms change and are not constant. One source of change in living organisms is sexual reproduction, which allows the mixing of parental traits in the offspring. It also became clear that deliberate hybridization and selection can be used for crop improvement and that many species of plants found in nature were the results of the hybridization of related plant species. Linnaeus himself saw this phenomenon and identified many naturally occurring hybrid plants. Later, many success stories of hybrids emerged, and the hit-and-miss approach of hybrid selection reached its prime in the first decades of the twentieth century with Luther Burbank and Ivan Vladimirovich Michurin. However, simultaneously efforts to understand the laws of heredity began, and a new field of genetics came into existence. Further Readings Chisholm, H. (Ed.). (1911). Camerarius, Rudolf Jakob. In Encyclopædia Britannica (11th ed., Vol. 5). Cambridge University Press. Crow, J. F. (2001). Plant breeding giants: Burbank, the artist; Vavilov, the scientist. Genetics, 158, 1391–95. https://journals.ashs.org/hortsci/view/journals/hortsci/50/2/article-p153.xml Darrow, G. M. (1966). The strawberry: History, breeding, and physiology (1st ed.). Holt, Rinehart and Winston. specialcollections.nal.usda.gov/speccoll/collectionsguide/darrow/Darrow_TheStrawberry.pdf Darwin, C. (1868). The variation of animals and plants under domestication. John Murray. Darwin, C. (1876). On the origin of species by means of natural selection, or the preservation of favored races in the struggle for life. John Murray. https://www.darwinproject.ac.uk Janick, J. (2015). Luther Burbank: Plant breeding artist, horticulturist, and legend. HortScience, 50, 153–56. https://doi.org/10.21273/HORTSCI.50.2.153 Mayr, E. (1986). Joseph Gottlieb Kolreuter’s contributions to biology. Osiris, 2, 135–76. https://doi.org/10.1086/368655. JSTOR:301833. Strawberry: A Brief History can be found at https://ipm.missouri.edu/meg/2012/5/Strawberry-A-Brief-History/ 1. Artificial pollination was in use since ancient times: Stone carvings from ancient Assyria (2400–612 BCE) clearly depict the artificial pollination of date palms (Phoenix dactylifera) and suggest that the knowledge of different sexes of date palms and the need for sexual reproduction did exist. However, this knowledge was not extended to other plants for deliberate hybridization and crop improvement. This phenomenon was also unknown to Europeans. Camareus made the first intellectual attempt to understand the structure of flowers and extended this knowledge to all plants 2. Many plants reproduce only asexually and do not produce seeds and flowers (e.g., money plant and duckweed). In addition, some flowering plants can reproduce asexually—including onion, potato, and gladiolus—as well as many trees that are propagated by using cuttings. The plants generated from asexual reproduction are genetically identical to their siblings and parent plant. 3. The original publications of Carolus von Linné (Linnaeus) are available at the Biodiversity Heritage Library website: https://www.biodiversitylibrary.org/browse/collection/37. 4. The schema proposed by Linnaeus served as a primer for the classification of plants and animals as well as provided a framework for comparative studies for the past 300 years. However, with the advancement of gene and genome sequencing, this schema is now being modified/updated. You can find the latest information on the Tree of Life web project at http://tolweb.org/tree. 5. Until World War I, Austria, the Czech Republic, Slovakia, Croatia, and Hungry were part of the Austro-Hungarian Empire ruled by the House of Habsburg. Moravia is now in the Czech Republic.

textbooks/bio/Agriculture_and_Horticulture/History_and_Science_of_Cultivated_Plants_(Naithani)/1.04%3A_Cataloging_Classification_and_Deliberate_Hybridizations.txt

No-one can say why the same peculiarity in different individuals…is sometimes inherited and sometimes not so: why the child often reverts in certain characters to its grandfather, or other much more remote ancestor; why a peculiarity is often transmitted from one sex to both sexes, or to one sex alone, more commonly but not exclusively to the like sex. —Charles Darwin, On the Origin of Species Our ancestors started agriculture with a certain sense that traits are inherited from parents to progeny. Centuries of breeding domestic animals and plants showed that useful traits could be accentuated by controlled mating, and as we have discussed in chapter 2, domestication and artificial selection gave rise to most modern crops. However, there was no rational way to predict the outcome of a cross between two parents or understanding how and why certain traits show up while others remain hidden (see figure 5.1). Until the nineteenth century, the prevailing theory was the blending inheritance, which was similar to the mixing of two different color paints: progeny were expected to have traits that were a blend of those of its two parents. Darwin was puzzled about the mechanism of heredity and how the substance that carries parental traits into offspring could possess two seemingly contradictory qualities: its flexibility to give rise to variations and the stability required to maintain the species. He proposed the pangenesis theory to explain the mechanism of heredity. This theory suggests that an organism continually produces a specific type of small organic particle called a gemmule that accumulates in the gonads and then is transmitted to the gametes. When gametes form embryos after fertilization, their gemmules from the parents mix like the paint of two colors. Interestingly, pangenesis was in direct contradiction with Darwin’s theory of (bio)evolution. If the principles of pangenesis were correct, then the natural variations that spontaneously arise within any species would have been an exception among most individuals. Therefore, as a result of mating between an exceptional variant with “normal” individuals (and mating of its progenies with normal members for generation after generation), the variations would gradually disappear. However, Darwin’s extensive research suggested that variations in different birds of the Galápagos Islands were maintained over generations. Eventually, Darwin realized that pangenesis does not explain evolution, and these two hypotheses—(bio)evolution and pangenesis—were contradictory. But he could not solve the riddle. The greatest challenge to pangenesis came from the German zoologist August Weismann (1834–1914). Weismann experimented with mice to see if cutting off their tails generation after generation would cause any change in their offspring. However, the progeny of those mice had normal tails. He proposed the germplasm theory, which suggests that multicellular organisms consist of two types of cells: (1) germ cells, which are present in the gonads (ovaries and testes) and contain and transmit heritable information from parents to progeny, and (2) somatic cells, which carry out ordinary bodily functions. Thus the gametes (egg cells and sperm cells) produced by the germ cells serve as carriers of heredity information, and other cells of the body do not function as agents of heredity. This hypothesis discredited the ideas of inheritance of acquired characteristics as proposed by Lamarckism and pangenesis. In this way, the distinction between the hardwired inherited traits contained within the germ cells and the soft-wired traits acquired by the somatic cells was established. However, it remained to be known how much biological contribution the mother and father make to their offspring and what rules govern heredity. Gregor Johann Mendel: The Mathematics of Heredity During Darwin’s lifetime, a clergyman in a monastery in Moravia, Gregor Johann Mendel (1822–84), studied the laws of genetics by crossing pea plants. Moravia, where Mendel (figure 5.2) was born and educated, was the center of plant crossing since the eighteenth century. As discussed in the previous chapter, various pastors took on crossing and hybridization experiments for improving crops and domestic animals. They included crossing and breeding experiments in natural science curricula in the classes they taught at schools. Thus Mendel’s curiosity and his experiments aimed at understanding the laws of heredity come as no surprise. From 1856 to 1863, Mendel grew pea plants in the five-acre garden of the monastery and conducted about 29,000 crossing experiments. The pea proved to be the ideal plant for investigating heredity. The pea is a selfing plant, but it is also easy to perform artificial pollination on it. It has a short life cycle of two and a half months and can be grown in large numbers with very few resources. Thus it is possible to study several generations of this plant within a short period. Mendel selected seven contrasting characteristics of the pea plant (see figure 5.3) in his experiment: stem length (tall or dwarf), flower color (purple or white), pod shape (inflated or constricted), pod color (green or yellow), seed shape (round or wrinkled), seed color (yellow or green), and flower position (axil or terminal). For many years, Mendel developed purebreds by self-pollination. All the offspring produced by the selfing of purebreds are the same. For example, tall plants give rise to 100 percent tall progeny, and dwarf plants produce 100 percent dwarf progeny. Subsequently, he used purebreds of peas for generating hybrids and for studying the pattern of inheritance of various characteristics. He observed that the crossing of the purebred purple-flowered plant with the white-flowered plant produced only purple flowers in the hybrid (first hybrid generation, or F1). He repeated these experiments on the seven pairs of pea plants that exhibited contrasting traits and found every time that all the hybrids of the F1 generation showed traits from one parent, while the contrasting trait from the other parent remained hidden. He did not observe the mixing of two contrasting traits. Based on these observations, Mendel proposed the first principle of heredity, known as the law of dominance, postulating that within any (multicellular) organism, at least two factors determine a given trait, of which only the dominant factor appears in the hybrid, and the recessive factor remains hidden or masked by the dominant trait. In the second step, Mendel crossed F1 hybrids and analyzed their progeny (second hybrid generation, or F2) and found that 75 percent of F2 plants contained purple flowers and 25 percent of F2 plants contained white flowers (see figure 5.4). Thus after skipping the F1 generation, the white flower color reappeared in the F2 generation, but the distribution of dominant (purple) versus recessive (white) traits in their flowers was 3:1 (refer back to figure 5.1). Mendel repeated these experiments on all seven traits and always got a ratio of 3:1 between dominant and recessive traits in F2 progeny. For the first time in history, it became known that the contrasting parental traits do not mix like paints of two colors; instead, they are maintained and transmitted as independent entities. During the gamete formation, the two factors separate and are distributed equally in the gametes. The fertilization between egg and sperm cells that both contain the recessive factor gives rise to the progeny containing both recessive factors, and thus the corresponding trait reappears. Mendel suggested the second law of heredity as the following: 1. The individual has two copies of each factor. Each parent contributes one factor of each trait shown in the offspring. 2. Some traits can mask others, but the traits don’t blend. The two members of each pair of factors segregate from each other during gamete formation. Today, these factors are known as the alleles of a gene, which behave like alternatives to each other. Most recessive alleles cause/indicate a functional deficiency. If one allele of the pair works properly, then it hides the other’s deficiency. If both alleles of a gene are effective/functional or if at least one allele of the pair is functional, then in both cases, we see a dominant trait. Subsequently, Mendel studied the inheritance of two different traits simultaneously. For example, he crossed homozygous plants producing yellow, smooth peas with plants producing homozygous green, wrinkled peas. As expected, the F1 generation peas were yellow and smooth; only dominant traits showed up. Mendel made crosses between the F1 individuals and found that of the sixteen plants in the F2 generation, nine produced yellow, smooth peas (resembling the dominant ancestor); one produced green, wrinkled peas (resembling the recessive ancestor); three produced smooth, green peas; and three produced yellow, wrinkled peas (see figure 5.5). In the F2 generation, he found a new combination of smooth, green and yellow, wrinkled peas in equal ratio. The overall result suggested that two different traits (such as the color and shape of a pea) are transmitted independently of each other to future generations. Mendel postulated the third law of heredity (a.k.a. the law of independent assortment), suggesting that different traits—like seed shape and seed color—are inherited independently of each other. Thus Mendel’s experiment revealed the role of sexual reproduction in generating variations within the same species. Today, Mendel’s three laws are known as the fundamental principles of genetics. Mendel was not aware of the physical and chemical properties of the genetic material, but his discoveries led to the first concrete understanding of how the genetic material behaves. Understanding the behavior of heredity required a mathematical approach rooted in deduction, the fragmentation of big questions into small workable hypotheses, and a good model system for studying heredity. Mendel, a trained meteorologist, had the ability to look at the data and apply math to it. In contrast, Darwin and most biologists of his time implemented the cataloging and description of the morphology of plants and animals. Today, Mendel is the father of genetics, but during his lifetime, his contributions were not valued by his peers. In later years, Mendel did the monastery’s administrative work, and his three principles of genetics were forgotten for thirty-five years. Mendel’s work was no less important than Darwin’s. In fact, his work explained Darwin’s theory of evolution in concrete terms. Many people now wonder why Mendel’s discoveries were ignored, while Darwin became one of the most popular scientists during his lifetime. Perhaps, to some extent, Darwin benefited from his family background. Both his father and grandfather were well-known doctors, and he graduated from the University of Cambridge. He developed his academic network at an early age while working with John Henslow and then got an opportunity to join the HMS Beagle as a naturalist (a volunteer who was not paid a salary). In contrast, Mendel was born into a peasant family. After his initial education in the village, he became a monk and pursued further education in the monastery. During his youth, he struggled to become a high school teacher and carried out experiments with pea plants in the garden. He did not get much opportunity to discuss theories with other scientists, as he was not a part of academia; none took cognizance of his work. Also, most of his contemporary scientists were naturalists involved in cataloging and describing plant and animal diversity; they lacked mathematical understanding. Therefore, most academics and scientists ignored Mendel’s published papers. We have no way of knowing if Darwin ever knew about Mendel’s research work. Rediscovery of Mendel’s Laws and the Birth of Genetics In 1900, Hugo de Vries, Carl Correns, and Erich von Tschermak independently rediscovered Mendel’s work. Thus Mendel’s three laws of heredity resurfaced after being buried after thirty-five years, but even then, these principles were not easily accepted. The first few who recognized the importance of Mendel’s laws include Cambridge University biologist William Bateson. Bateson zealously lectured on the laws of heredity in European and American institutions to popularize Mendel’s work and became known as “Mendel’s bulldog.” Bateson established the first genetics laboratory in Cambridge. Bateson proposed the term genetics for a new branch of biology rooted in the Mendelian approach and the study of inheritance and the structure function of genetic material. During this period, Vavilov had joined Bateson’s laboratory and thus became the first geneticist in the USSR. Botanist Wilhelm Johannsen proposed the word gene for the Mendelian units of heredity (factor) and called two different versions of a gene-defining trait alleles, since hybrid plants show only dominant traits and appear similar to purebreds even though their genetic configuration is different. Hence in 1909, Johannsen proposed the terms genotype for the genetic configuration and phenotype for the manifested trait—that is, its outward appearance. Hugo de Vries proposed the term mutant for organisms with rare traits (whose numbers are less than 1 percent in a population or have a defect). In this way, gradually, terminology and vocabulary for genetics increased, and slowly it was established as a subdiscipline of biology. Exceptions and Extension of Mendelian Genetics In the early twentieth century, many scientists repeated Mendel’s experiments and began analyzing their crossing experiments using a framework provided by Mendelian genetics. Often, the laws of heredity successfully explained their results, but less frequently, exceptions were encountered, which added new dimensions to and knowledge of heredity. Here we discuss several of such examples and how they contributed to furthering knowledge in the field. Partial or Incomplete Dominance and Codominance One exception to Mendel’s first law is partial dominance. When scientists crossed the white- and red-flower varieties of the “four o’clock,” they found pink flowers in the F1 hybrids. Thus in this case, the presence of a dominant allele did not completely mask the recessive allele, and the phenotype of the heterozygous F1 hybrid differs from its homozygous dominant parent. Furthermore, the selfed progeny of heterozygous F1 hybrid segregated in a ratio of 1 red: 1 white: 2 pink. However, the pink flower in the hybrid is not a result of the mixing of red and white but is due to the diminished quantity of red pigment. In fact, both allele coding for red and white are transmitted from one generation to another as discrete factors, but the red flowers are produced by plants where both factors are functional. When only one of the two works, only half the pigment is formed, and the pink color is seen. Another exception is codominance, where both alleles (factors) determining a trait are functional. For example, in people with AB blood group, both A and B alleles are equally active and produce A and B antigens. A third allele, i, is also found in some people that do not produce any antigen due to mutations. Therefore, people with AA or Ai have blood group A; those with BB or Bi have blood group B, and others with ii have blood group O (no antigen). Mendel proposed the basic rules of genetics based on the assumption that there are two alleles (dimorphic) of any gene. While it stands true for an individual, multiple alleles for the same trait exist within any population of a plant or animal. Many genes have several common alleles (they are polymorphic), which may show a complicated pattern of dominance and can be placed in a hierarchy. The interactions of these various alleles cannot be understood without concerted efforts of crossing and establishing a dominance series. For example, if we look carefully at the whole lentil grain, there are many patterns of small or big dots on the light-colored surface. If a lentil with a clear surface is crossed with one with a dotted surface, then the F1 hybrid shows a dotted trait. However, a crossing of the dotted lentil with spotted lentil plants produces an F1 hybrid showing a spotted pattern (here the dotted trait behaves recessively). Based on the results from such crossings, the dominance hierarchy of different alleles can be prepared, and then the results can be explained using Mendel’s laws. But without understanding this hierarchy, the results cannot be explained. It is noteworthy to mention that in such cases, the dominance relations affect only the correspondence between genotype and phenotype; however, the alleles still segregate and unite randomly and follow the Mendelian pattern. The difference lies in the complex relationship between genotype and phenotype. Lethal Mutations There are many essential genes found in a living organism, known as the housekeeping genes, that are required for that organism’s survival. Some of these genes are active in all cells of an organism, whereas others act specifically in a particular type of cell, tissue, or organ. Mutations in housekeeping genes can be lethal. Sometimes heterozygotes exhibit a phenotype or a disease, which leads to recessive homozygotes dying (and thus a progeny class is eliminated). In these cases, we do not find the segregation of F2 progeny according to Mendel’s laws. In contrast, the gametes (egg or sperm cells) contain only one allele, and thus the gametes carrying a nonfunctional allele of housekeeping genes are destroyed. As a result, only the gametes carrying the functional alleles are transmitted to the next generation. In such cases, the results of the crossing do not fit into the Mendelian hypothesis, and only the functioning allele is seen in the progeny. However, close observation can reveal decreased pollination and lower seed formation. In other instances, after successful pollination and fertilization, the embryo does not develop because both alleles of a housekeeping gene required for embryonic development are mutants. Similarly, some plants die after seed germination due to a lack of functional alleles (e.g., albino mutants that lack photosynthesis capacity). There are also instances where both alleles are necessary for the normal development of an organism. For instance, in healthy individuals, both alleles of the fibroblast growth receptor gene function, but people with a mutant allele suffer from the most common form of dwarfism (known as achondroplasia, with a normal-length body but shortened limbs). Pleiotropy Sometimes a single gene affects several unrelated phenotypic traits in the same organism, and this phenomenon is known as pleiotropy. For example, specific mutations in the hemoglobin B gene cause changes in the shape of red blood cells (from round to sickle shaped) that causes sickle cell anemia (termed this to differentiate it from dietary deficiency and other causes of anemia). Due to their shape, these cells obstruct the smooth flow of blood and have a short life compared to normal cells. As a result, people carrying the mutant gene become anemic. In addition to anemia, an enlarged spleen, muscle and heart pains, a weak immune system, resistance to malaria, and early death are associated with the mutations in the hemoglobin B gene. Penetrance or Expressivity in Phenotype Researchers also noticed that certain traits are expressed fully only under a certain environment. Thus two new terms were added: 1. Penetrance is whether a trait is expressed or not. 2. Expressivity is the degree to which a trait is expressed (fully or partially). We see many such examples in our day-to-day lives. For instance, genetically identical hydrangeas growing in soils of different acidity (different environments) produce flowers of a different color. Many houseplants change color if their exposure to light changes or the temperature changes. We also find different pigmentation in cats, dogs, and other animals, where the colder body parts are darker while the warm body parts are lighter. Epistasis In many instances, a single phenotype is controlled by the interaction of two or more genes. This phenomenon is known as epistasis. The epistatic genes may code for proteins involved in different steps of a biosynthetic pathway or may have an additive function. In such cases, we see alterations in F2 segregation ratios. Examples of such interactions can explain the various sizes and shapes of squashes, the different skin colors of onions, or the diversity in flower colors of many plants. Unlike animals, plants contain many duplicate genes, and some of these have redundant and/or additive functions. Thus when both alleles of a gene are mutated and are nonfunctional, we see no phenotype due to the presence of a duplicated gene that functions properly. In such cases, a phenotype is only visible if both genes (all four alleles) become nonfunctional. In contrast, the opposite is also true: sometimes, a phenotype is only visible when two or more duplicate genes are simultaneously functional in the organism. For example, for zucchini, two genes (four alleles in total) determine the fruit size. If both genes work or at least one allele of both works, then a very small disk-shaped fruit is produced. If only one gene (or one of its alleles) is functional, then a big, rounded fruit is produced. If both genes (all four alleles) become nonfunctional, then a long fruit is produced. Here crossing between the long and flat breeds of zucchini will give rise to an F1 hybrid bearing round fruits. And the selfing of F1 progeny will result in the segregation of this trait in F2 progeny as 9 disk shaped (A_B_):6 round (A_bb or aaB_):and 1 long (aabb). Genetic Linkage and Chromosome Theory Mendel was extremely fortunate with the plant model he selected for deciphering the basic laws of heredity. He chose seven traits of pea plants that gave a consistent pattern of dominance and recessiveness between two contrasting alleles. Many of Mendel’s contemporaries could not find such a great experimental model or traits, and due to many deviations, their efforts were derailed. Even Bateson, the biggest supporter of Mendelian genetics, could not find an easy path and got entangled with unexpected results not explainable by Mendel’s laws. In 1905, Bateson and Reginald C. Punnett made a dihybrid cross between purebreds of sweet pea plants, where the dominant parent produced purple flowers and long pollen grains (PP LL), and the recessive parent produced red flowers and round pollens (pp ll). Bateson and Punnett observed that the F1 hybrid had purple flowers and long pollen grains (Pp Ll), as expected. However, the F2 population generated by the selfing of F1 hybrids showed a skewed ratio that did not match what was expected based on Mendel’s second law of independent assortment of two independent traits. The data clearly showed that the characteristics of pollen size and flower color did not transmit independent of each other from parents to offspring. The expected ratio of the dihybrid cross was 9:3:3:1, but the results did not fit this pattern. As shown in table 5.1, the gene coding for flower color and pollen shape appears to be linked to some extent: the two phenotypic classes (purple flower + long pollen and red flower + round pollen) are larger than expected, and the number of recombinants is less than expected (1). Bateson was puzzled and could not explain these results in terms of epistasis either. However, Bateson and Punnett proposed that the F1 hybrid produced more P L and p l gametes due to some sort of physical coupling between the dominant alleles P and L and between the recessive alleles p and l (1). Table 5.1: The results of the di-hybrid cross in sweet peas observed by Bateson and Punnett) Phenotypes Observed (12.3:1:1:3.4) Expected (9:3:3:1) Purple flower and long pollen (dominant traits; PP LL+Pp Ll) 4831 3911 Purple flower and, round pollens (recombinant; PP ll+pP ll) 390 (17.8%) 1303 Red flower and long pollen (recombinant; pp LL+pp Ll) 393 (17.7%) 1303 Red flower and round pollen (recessive traits; pp ll) 1338 435 Total plants 6952 6952 American scientist Thomas Hunt Morgan chose the fruit fly (Drosophila melanogaster) to study genetics. The fruit fly has several advantages for this study, including a short life cycle (forty to fifty days), easy propagation within milk bottles, and being amenable to the crossing. Also, with the help of a microscope, changes in the structure of the fruit fly can be easily identified. Thus it was a cheap and accessible model for the study of genetics and since then has served as an important model organism to study eukaryotic genetics. When Morgan crossed a female fly containing a purple eye (pr) and a normal wing (vg; both dominant traits: pr/pr.vg/vg) with a male fly containing a red eye (pr+) and the vestigial wing (vg+; both recessive traits: pr+/pr+.vg+/vg+), he got an F1 hybrid with the purple eye and the normal wing (pr/pr+.vg/vg+), as expected. Then he made a backcross between the F1 hybrid female (pr/pr+.vg/vg+) and the recessive male parent (pr+/pr+.vg+/vg+) (2). In these experiments, the recessive male can only produce one type of gamete, one that carries recessive red eye and vestigial wing traits. Thus the alleles contributed by the F1 female specify the F2 progeny. If both traits are sorted independently of each other, the expected ratio of these traits in the F2 progeny would be 1:1:1:1, representing both parental genotypes and two new recombinant classes. As shown in table 5.2, he found unexpected results: a much larger number of offspring with purple eyes and normal wings or red eyes and vestigial wings was obtained in the F2 progeny. Like Bateson, he also observed a linkage between two independent traits: the linkage was not absolute, and the two types of recombinant class of progeny were in a similar proportion. Table 5.2: The results of fruit fly backcross observed by Morgon Genotypes and Phenotypes Observed pr+/pr. vg/vg+ (pruple eye+ normal wing) 1195 pr+/pr+. vg+/vr+ : (red eye+ vestigial wing) 1339 pr+/pr+. vg/vg+ (reg eye +normal wing) 151 pr+/pr. vg+/vg+ (purple eye + vestigial wing) 154 Total 2839 The progress in the field of cytology[1] helped us understand the phenomenon of linkage between genes that were observed by Bateson and Morgan independently. From the seventeenth century onward, scientists studied the structure of different organisms through microscopes and understood that organisms are made of one or more cells. The simplest forms of life, such as bacteria, are made of only one cell (unicellular), and various animals and plants are made of many cells (multicellular). Therefore, the unit of the structure and the function of life are the cell. In the late nineteenth century, high-resolution microscopes became available, which allowed the identification of various subcellular structures. It was natural that scientists started probing the location of genetic material within the cell. In 1879, Walter Fleming noticed a fine, threadlike structure in the center of salamander cells that shrunk and assumed a clear shape during cell division that was named chromosomes. It was clear from a series of studies that the number of chromosomes is constant for a species, and chromosome numbers vary between species. Typically, multicellular organisms have two copies of each chromosome (two sets of chromosomes) within the somatic cells, and the mother and father each contribute one set of chromosomes to their offspring. For example, human somatic cells have 46 chromosomes (23 pairs; diploid). In contrast, one set of chromosomes (23 chromosomes) is present in the gametes (eggs and sperm cells). At the time of cell division, chromosomes first double in number and then divide equally into two daughter cells. In this way, two cells are made from one mother cell, and both daughter cells have the same number of chromosomes as their mother cell. In contrast, the cell division of a mother germ cell gives rise to gametes that only contain one set of chromosomes (haploid) and thus only one copy of each chromosome. During sexual reproduction, the fusion of male and female gametes produces a diploid embryo, and the two sets of chromosomes are restored in progeny. The behavior of chromosomes during the cell division of the germ cell grossly reminds us of Mendel’s laws. By the beginning of the twentieth century, it was recognized that chromosomes play a role in transmitting genetic traits from parents to offspring. Incidentally, the seven traits chosen by Mendel for his crossing experiments are encoded by seven genes that reside in seven different chromosomes, and thus he did not observe linkage. Therefore, Mendel did not see any contradiction in his experiments. However, besides gene linkage, Bateson and Morgan made another important observation that the linkage between two genes was not absolute and that a few recombinants are present in the F2 population. Morgan hypothesized that during germ cell division (meiosis), homozygous chromosomes interchange small regions before being divided into daughter cells, and thus the linkage between two genes located on the same chromosome occurs, resulting in the formation of recombinants (2). Thus based on the findings of cell biology and results from his laboratory, Morgan provided an explanation of the linkage between genes (2). He proposed the following: • Genes occur in a linear order on chromosomes, and their location on chromosomes is fixed. • Genes on the same chromosome are linked together and do not exhibit an independent assortment. • Genes can be exchanged between chromosomes during meiosis. • The closer genes are located on a chromosome, the less likely they will separate and recombine in meiosis. • Chromosomes undergo segregation and independent assortment. Therefore, genes on different chromosomes follow the law of independent assortment. Chromosome Mapping Several experiments in Morgan’s laboratory revealed that the closer the two genes are, the stronger their association. As the distance between the two genes increases, the probability of crossing over increases, and the number of newly combined progeny (recombinant) increases in the same proportion. Thus the distance between two genes can be estimated by the number of recombinants present among the total progeny. By this method, the distance between genes can be measured in centimorgan (cM). Thus a distance of 1 cM between the two genes meant that in such cases, 1 percent of F2 progeny (of dihybrid cross) would be recombinant. Morgan’s laboratory generated a vast amount of fruit fly crossing data. Eventually, one of his students, Alfred Henry Sturtevant, analyzed this data and constructed the first genetic map of fruit flies’ chromosomes one by one. Subsequently, this method was applied to many other animals and plants to generate their chromosome maps. Morgan did not directly observe the reciprocal exchange of segments between homologous chromosomes, but the assumptions he made about crossing over during meiosis proved to be correct. The crossing over (see figure 5.6) was experimentally confirmed almost two decades later by Barbara McClintock. But by then, scientists had constructed genetic maps of many organisms by following Morgan’s lead. Genetic maps proved to be very useful for breeders. Genes whose phenotypes are visible served as a marker for selecting other nearby/adjacent genes that had no visible phenotype but contributed to important agronomic traits. Similarly, if one desired gene is linked to another undesirable gene, with the help of a genetic map, it became possible to estimate how many crossings will be required to break this linkage. Polyploidy The study of the cells of various animals and plants revealed that the number of chromosomes within a species is fixed. Moreover, most animals are unable to tolerate a slight change in chromosome numbers. Usually, embryos that have lost or gained one or more chromosomes are unable to survive. Unlike animals, plants have a tremendous capacity to harbor multiple sets of chromosomes; this phenomenon is known as polyploidy. Many plant species have two (diploid), three (triploid), four (tetraploid), six (hexaploid), or eight (octoploid) copies of the entire set of chromosomes. Many polyploid crops have come into existence by spontaneous hybridization events between two closely related species naturally. For example, there are 28 chromosomes within emmer wheat (Triticum dicoccoides), 14 of which are from diploid einkorn wheat (Triticum Urartu; AA genome donor) and 14 from a diploid goat grass related to Aegilops speltoides (the BB-genome donor). Therefore, emmer wheat is a tetraploid (AABB) species. Another natural hybridization event between T. dicoccoides and Aegilops tauschii (the DD-genome donor) gave rise to hexaploid modern bread wheat (Triticum aestivum; AABBDD). Thus hexaploid common bread wheat has 42 chromosomes, of which 28 are from the emmer (AABB) and 14 are from A. tauschii. Often the increased ploidy has a direct effect on the structure of the plant in the form of an increase in the size of the leaf, fruit, or grain—thus it adds to the agronomic value of a crop. Multiplication of chromosomes in emmer and common bread wheat had resulted in an increase in grain size and greater tolerance for adverse environmental conditions. Similarly, as discussed in an earlier chapter, the octoploid ananassa strawberry (8 sets of chromosomes) is much larger than diploid wild strawberry varieties. Sometimes, breeders also create sterile hybrids by crossing two closely related species of different ploidy levels that disrupt the formation of seeds in hybrids. The seedless watermelon (a triploid) is one such example that was made by crossing a tetraploid with a diploid variety. Summary In later decades of the nineteenth century, a tremendous amount of geological and biological data suggested that both the earth and the living species inhabiting it have changed over time; in the process of adapting to new environments, new species emerge from the previously existing species, and some existing species also disappear. Mendel extended the knowledge by postulating three fundamental laws of heredity—which provided a correct theoretical explanation for Darwin’s theory of evolution—to describe the origin of species in the natural world. Many new discoveries in cell biology led to the birth of genetics, a subdiscipline of biology that studies heredity and the physical and structural properties of genetic material. By 1900, cells and chromosomes were sufficiently understood to give Mendel’s abstract ideas physical context. It was discovered that chromosomes contain the genes that code the various traits of an organism and that during germ cell division, the reciprocal exchange of chromosomal segments further adds to genetic diversity within a species. The observation of recombination frequencies was exploited to construct chromosomal maps and decipher the location of genes on various chromosomes and their physical relationship with one another. Chromosomal maps provided breeders with the tools for designing a rational experiment for creating improved varieties of plants and animals. Further Readings Landmarks in the History of Genetics. http://www.dorak.info/genetics/notes01.html Mendel, J. G. (1865). Versuche über Pflanzenhybriden Verhandlungen des naturforschenden Vereines in Brünn, Bd. IV für das Jahr. Abhandlungen. 3–47 [for English translation, see http://www.mendelweb.org/Mendel.plain.html]. Mitosis and Meiosis Simulation (video for cell division of two types). https://www.youtube.com/watch?v=zGVBAHAsjJM Winther, R. (2001). August Weismann on germ-plasm variation. Journal of the History of Biology, 34(3), 517–55. https://doi.org/10.1023/A:1012950826540 1. The following are major discoveries in cell biology that played important role in the progress of genetics: 1839: M. J. Schleiden and T Schwann develop the cell theory. 1866: E. H. Haeckel (Häckel) hypothesizes that the nucleus of a cell transmits its hereditary information. 1869: Friedrich Miescher isolates DNA for the first time. 1879: Walter Flemming observes mitosis. 1902: W. S. Sutton and T. Boveri independently propose the chromosome theory of heredity: a full set of chromosomes are needed for normal development; individual chromosomes carry different hereditary determinants; and independent assortments of gene pairs occur during meiosis.

textbooks/bio/Agriculture_and_Horticulture/History_and_Science_of_Cultivated_Plants_(Naithani)/1.05%3A_The_Early_History_of_Genetics.txt

As the field of genetics matured, it became possible for breeders to develop improved crop varieties using Mendelian genetics, genetic maps, and markers. A breeder usually combines the desired genes from two varieties via crossing and then selects progeny containing desired traits. If four different useful genes are found in four different wheat varieties, using a step-by-step approach, a breeder could bring all four genes in the one preferred variety. However, a breeder does not create new genes; he or she only combines preexisting genes present within the genetic pool. Therefore, the biodiversity of any species determines the limits of classical breeding. This chapter summarizes the genetic improvements made in three main cereal crops—maize, wheat, and rice—using the classical breeding approach and their role in making the green revolution successful in the latter part of the twentieth century. In the process, various international agricultural research centers evolved under the Consultative Group for International Agriculture (CGIAR) umbrella, which is playing a critical role in achieving global food security. Story of Maize Maize—more popularly known as corn—is the main staple crop of the Americas. Maize (Zea mays L.) is a member of the grass family Poaceae (Gramineae). Mexico is the center of the domestication and diversification of maize (1). Maize’s ancestor is a wild grass, Teosinte parviglumis(2).[1] Until 1492, people outside of South, Central, and North America were unaware of maize’s existence. Native Americans have been cultivating maize for ~7,000 years. Maize is unique among the cereal crops because it is monoecious (it bears separate male and female inflorescences on the same plant). The main stem terminates in a tassel (male inflorescence), and the silk containing female flowers is on the stem. The natural structure of the maize plant favors cross-pollination. The pollen from the male flowers reaches the female flowers (of the mother plant or other nearby maize plants) via wind. Thus in maize, an equal possibility of self-pollination and cross-pollination exists. The pollen of different plants can reach a female flower, leading to the fertilization of the egg cell independent of one another. After successful pollination, the seeds on a cob are not uniform because each seed results from an independent pollination event. In nature, genes are exchanged continuously between maize plants, which results in new combinations. Discovery of Hybrid Vigor and F1 Hybrids of Maize In 1876, William Beal, a scientist at the Michigan Agricultural College, conducted crossings between maize varieties. To control pollination, Beal cut off the male flowers of the female parent (this process is known as detasseling) to ensure 100 percent cross-pollination from the chosen male parent and 100 percent hybrid progeny. Beal observed that the yields obtained from hybrids were higher than either of their parental varieties. In 1877, Charles Darwin also experimented with crossing maize varieties. He observed that the offspring resulting from outcrossing produced 25 percent larger cobs than from selfing and that the hybrids had a greater ability to tolerate chilling stress. In 1908, George H. Shull and Edward East independently experimented and developed purebreds of maize. They noticed that after successive cycles of selfing, the maize plants’ size and their yields decreased. This phenomenon was named inbreeding depression (see figure 6.1). Like Beal and Darwin, Shull also noticed that F1 hybrids are taller and healthier than their parents and produce higher yields; he named this phenomenon hybrid vigor or heterosis (see figure 6.1). Shull further made a very useful observation that after three generations of inbreeding, maize behaves almost like a purebred that can be used for producing the F1 hybrid. Such an approach could ensure 100 percent human control over pollination and good yields year after year. However, Shull did not have enough resources to continue his work on F1 hybrids of maize. In 1913, Shull migrated to Germany and became the editor of the scientific journal Genetics. East, a Harvard University professor, was also not able to pursue his research on maize, but he encouraged a new generation of scientists. One of his students, Donald F. Jones, crossed the two purebreds of maize to create an F1 hybrid, and afterward, by crossing two different F1 hybrids, he successfully created double-cross hybrids (see figure 6.1). Jones observed that the yield of the double hybrids was higher than single hybrids. Therefore, the most successful corn crop was the double-cross hybrid, and it laid the foundation for the widespread use of double-cross hybrids in the US. The hybrid vigor of maize was only maintained if new seeds were purchased every year, which made the seed industry commercially viable. In the 1930s, the US Department of Agriculture helped farmers adopt F1 double-cross hybrid varieties of maize, leading to progressively higher yields. The first two hybrid varieties were grown side-by-side, and later to ensure 100 percent double hybrids, one row of pollen donor (male) was planted between every four rows of detasseled female plants. Gradually, most American farmers adopted this practice of maize cultivation. In 1933, less than 1 percent of total maize grown in the US was a double hybrid, but by 1944, it increased up to 56 percent. From 1930 to 1990, American scientists released about thirty-six improved hybrid varieties of maize, and farmers gladly adopted better varieties one after the other. US–Mexico Collaboration on Agriculture Research In 1941, the vice president of the United States, Henry A. Wallace (1888–1965), traveled to Mexico to attend the inauguration ceremony of the newly elected president of Mexico, Manuel Ávila Camacho. Roosevelt and Wallace had also won elections in the US that same year. After attending Camacho’s ceremony, Wallace spent the first two days in the Bajio region with outgoing president Lázaro Cárdenas and newly elected agriculture secretary Marte Gómez. Wallace noticed that due to maize’s continuous cultivation, the Mexican soil had lost its fertility, and farming had become very unproductive. While the Iowa farmer spent ten hours to produce ten bushels of corn, it took a Mexican farmer 200 hours for the same. Wallace and his hosts discussed how the United States could help increase the productivity of Mexican farms. Wallace promised to help. Upon his return to the US, Wallace contacted Raymond Fosdick, then the president of the Rockefeller Foundation, about providing US aid to Mexico and developing the appropriate project. Fosdick set up a three-member committee that consisted of maize geneticist Paul Mangelsdorf (Harvard University), agronomist Richard Bradfield (Cornell University), and plant pathologist Elvin Stakman (University of Minnesota) to evaluate a plan on agricultural cooperation between the US and Mexico. In 1942, these three experts recommended a pilot research program that led to the establishment of the Oficina de Estudios Especiales (OEE) program in 1943 with support from the Rockefeller Foundation and the Mexican government. The OEE’s mission was to train Mexican scientists across all disciplines to support the agricultural production of four major crops: maize, beans, wheat, and potatoes. The objective of establishing the Colegio de Postgraduados at Chapingo University in Texcoco, near Mexico City, was added in later years. Mangelsdorf supervised the maize research program, and Stakman was given the responsibility of directing the wheat breeding program. Maize Germplasm Collection and Breeding from the 1950s to the 1960s In 1943, a research project on maize was started under the supervision of Professor Paul Menzaldorf with the support of the Rockefeller Foundation. Menzaldorf’s team promoted F1 and double-cross hybrid varieties of maize developed by American scientists in Mexico. They also developed hybrids of local Mexican maize varieties. By 1947, ten new high-yielding double hybrids were released in Mexico under this program. In 1948, Mexico broke old records in maize production and became self-sufficient. By 1960, one-third of Mexico’s farms were cultivating high-yielding hybrids, and corn production had tripled. Menzaldorf’s team also began the collection of maize germplasm from Mexico and Central America. Since then, 28,000 maize varieties have been collected. This collection, representing almost all local maize varieties from eighty-eight countries, is stored at the Corn Germplasm Bank at the International Maize and Wheat Advancement Center (CIMMYT; https://www.cimmyt.org) located in Mexico City. Story of Wheat Wheat is the second widely grown cereal in the world after maize. However, unlike maize, most of the wheat is consumed as food. Wheat (genus Triticum) has many diploid and polyploid species (see figure 6.2). The einkorn (T. monococcum L.), the oldest among domesticated wheat varieties, is a diploid species that contains the AA genome. A natural hybridization event between einkorn and a diploid weed goat grass with the BB genome gave rise to the emmer/durum wheat (T. turgidum). Therefore, emmer is tetraploid (AABB). Archaeological evidence suggests that the domestication of einkorn and emmer varieties occurred almost simultaneously (10,000–12,000 years ago) in the Fertile Crescent. Wild emmer (T. turgidum ssp. dicoccoides) is the progenitor of today’s tetraploid durum wheat (3). A subsequent spontaneous natural hybridization event between emmer (AABB) and another species of wild grass, Aegilops tauschii (DD), gave rise to the hexaploid (AABBDD) spelt/dinkle or common bread wheat (T. aestivum) (3). Archaeological evidence suggests that spelt’s birthplace is the southeastern mountainous region of present-day Turkey. The polyploid durum and bread wheat produce larger seeds than the diploid species and their wild ancestors and also have a greater capacity to tolerate adverse conditions. Scientists estimate that around 7,000 years ago, the emmer wheat had spread outside its center of domestication and reached India, Central Asia, Egypt, and Europe. Emmer was the main cereal crop in Egypt at the time of the Pharaohs, while spelt was one of the major cereals of the Alemannians in southern Germany, Austria, and Switzerland between the twelfth and nineteenth centuries. Over time, many local varieties of wheat emerged in different regions due to the continued artificial selection of einkorn, emmer, and spelt in a variety of climates. Although these varieties show adaptations to local climates and may differ in morphological traits, most are subspecies of T. turgidum and T. aestivum. The wild einkorn and wild emmer have brittle rachis, while their domesticated descendants have nonbrittle rachis (see figure 6.3). This domestication trait caused the loss of shattering of mature seeds, and it became possible for the farmer to harvest the crop all at the same time. Spelt, emmer, and einkorn are hulled wheat species; their kernels cannot easily be extracted by threshing, and thus at harvest, the chaff remains at the kernels, and a special dehulling of the kernels is required in the mill to separate the chaff from the grain. A mutation in two genes, tenacious glume (Tg1) and q resulted in free-threshing wheat. Thus mutations in the Br1 loci on chromosomes 3A and 3B, Tg1 on 2D, and q on 5A had the most profound impacts on wheat’s domestication. In the last centuries, einkorn, emmer, and spelt were replaced by free-threshing durum and bread wheat. Durum (T. durum) is mainly used in the making of pasta and semolina. The common wheat (T. aestivum) is most suitable for bread, cookies, cakes, crackers, pastries, and noodles (it forms a spongier dough due to its higher gluten content and low gliadins/glutenins ratio). Ninety-five percent of wheat grown worldwide is common bread wheat, and the remaining is mostly durum. Recently, interest in ancient wheat species has been renewed for producing high-value baking goods due to their high nutrition content, and a market for catering to food-conscious elites is slowly growing. Beginning of Wheat Breeding Program in Mexico In 1943, Stakman directed the OEE’s Cooperative Wheat Research and Production Program in Mexico. At that time, agricultural research in Mexico was almost at a standstill. There was no single agriculture scientist with a PhD and no active research program on agriculture in Mexico and South America. He recommended that Norman Ernest Borlaug (1914–2009), his former student, be brought to Mexico for a leading wheat breeding program. Borlaug had finished his PhD under the supervision of Stakman a year earlier, and he was working for DuPont as a scientist. In Mexico, George Harrar, a plant pathologist, oversaw the local unit. The three other scientists in his team (besides Norman Borlaug) were Edward Wellhausen, John Niederhauser, and William Colwell, all from the US. Borlaug arrived in Mexico in 1944 to lead the International Wheat Improvement Program at El Batátan, Texcoco, outside of Mexico City. This was his first time traveling outside the US, and he didn’t know Spanish. He camped at a research center in the Yaqui Valley in Sonora, Mexico. In the 1930s, the governor of Sonora, Rodolfo Elias Calles, had set up that research station to help farmers. The station owned land for experimental research and some animals. The first director of this station, Edmundo Teboada, worked on some varieties of Mexican wheat and built a small wheat germplasm collection. But the research station did not have any modern farm equipment or any trained scientist. There was no maintenance for this research station. So when Borlaug reached there, it was a mess. There was no electricity, and the windows and doors were broken. But Borlaug was not discouraged and managed to set up humble arrangements for his living quarters, and in the early morning, he started working like a farmer. Borlaug established connections with farmers in the Yaqui Valley, who were relatively prosperous and had a good irrigation system compared to other wheat-growing regions of Mexico. Borlaug borrowed tractors and other machinery for research from these farmers as needed. Between 1939 and 1941, the problem farmers faced in this area was primarily rust, a plant disease that would infect their wheat. So Borlaug initiated work on the development of a wheat that can fight rust. First, he selected the wheat varieties from Teboada’s collection, which showed some ability to resist rust. He then began a series of crossing and backcrossing experiments with wheat varieties to develop high-yielding, rust-resistant wheat (see figure 6.4). Shuttle Breeding: Stem Rust-Resistant Wheat Varieties Generally, it takes ten to twelve generations to combine useful desirable traits from two varieties of a crop by implementing classical breeding and selection methods. Thus at least ten years were needed for making a new breed of rust-resistant wheat. Borlaug thought that if two wheat crops could be grown per year, then a new variety of wheat could be made in five years. Fortunately for Borlaug, this opportunity was available in Mexico. In Sonora, wheat is sown in October and harvested in April. In the mountainous region of Toluca Valley in central Mexico, wheat is sown in April and May and harvested in October. Thus taking advantage of this, Borlaug began shuttle breeding. As soon as the wheat crop was harvested in Sonora (in April), Borlaug sent these seeds for sowing in Toluca. Similarly, after the crop harvest in Toluca (in October), new seeds were sent to Sonora. Borlaug’s shuttle breeding program was not well received by his seniors at the Rockefeller Foundation. Until that point, there was a general agreement that the wheat seeds need a resting period after harvest (or before sowing the following year). Stakman advised Borlaug not to waste time and resources. When Borlaug submitted his resignation in response, no one stopped him from using shuttle breeding. Interestingly, the two locations (Yaqui and Toluca) used for shuttle breeding not only were 1,000 kilometers apart but also differed in latitude (by 10°) and elevation (by 2,000 meters; see figure 6.5). The Yaqui Valley is located at latitude 22° north and the Toluca Valley is located at latitude 12° north. Apart from this, the days of wheat cultivation in Yaqui Valley are shorter than in Toluca. As a result, the new wheat varieties developed by shuttle breeding became neutral to day length and regional climates. Those varieties could be grown on long and short days, on highland and valley, and at any elevation. These characteristics proved to be very helpful in spreading the improved varieties of wheat worldwide. The shuttle breeding experiment also reduced the time by half required for producing a variety using traditional breeding. There was no agricultural extension program in Mexico to help Borlaug; he worked directly with the farmers. As he developed new breeds, he gave them directly to the local farmers for preliminary testing. If farmers had positive results, then the seeds of those varieties were sent to experts worldwide for detailed testing. In this way, he built collaborations with local farmers and scientists from all over the world. Between 1948 and 1950, Borlaug released eight new wheat varieties in Mexico, each of which seemed to be more resistant to rust. In addition to developing advanced varieties of wheat, Borlaug advised farmers on the right quantity of fertilizer to use and how to improve irrigation facilities. The best wheat yield in the Bajio region in Mexico without the use of fertilizers and irrigation yielded 1 ton/hectare. But after using sufficient fertilizers, irrigation, and weed management, these same wheat varieties yielded 7.5–8 tons/hectare. In 1951–54, a rust epidemic devastated the wheat crop in North America due to the explosion of a highly pathogenic strain, 15b. By 1955, rust disease became a major challenge for American farmers. Under the pressure of this epidemic, many wheat nurseries were built around the world with the help of scientists from seventeen countries, and research on almost every kind of rust strain began. Also, the collection of wheat germplasm was undertaken afresh. Since Borlaug already had several wheat varieties with rust resistance, he was assigned the responsibility of developing new varieties of American wheat and training other scientists. He built nurseries in Yaqui to test the wheat varieties’ ability to fight rust. He selected those varieties that had larger, more seeded panicles; were rust resistant; and could produce higher yields when a sufficient amount of fertilizer and irrigation was supplied. In this way, rust-resistant, high-yielding varieties were developed. High-Yielding Dwarf Wheat By 1950, the prevalent wheat varieties were six to eight feet long, and their thin stalk was weak, which often collapsed under the weight of their own grain—a trait called lodging. Moreover, the high-yielding varieties were more damaged by thunderstorms, as they grew even taller in response to synthetic fertilizer. Therefore, the breeders wanted to create a rust-resistant dwarf variety of wheat with a strong stem that can bear more weight. Borlaug had developed the rust-resistant, high-yielding wheat varieties, but he did not have any dwarf wheat in his collection. Thus the search for dwarf wheat was ongoing. Finally, S. Sisil Samon of the US Agricultural Research Service spotted a dwarf wheat variety, Norin-10, in Japan. He sent Norin-10 seeds to Professor Orville Fogle, who sent it to Borlaug. In 1959, Borlaug crossed Norin-10 with some of his best North American varieties to create dwarf wheat varieties with a thicker, stronger stalk (e.g., Penjamo 620, Pittic 62, Gaines, Lerma Rojo 64, Siete Cerros, Sonora 64, and Super X). These high-yielding, rust-resistant dwarf varieties can stand the high winds, are amenable to machine harvests, and respond well to the application of fertilizers and irrigation. By 1963, 95 percent of Mexico’s wheat acreage was sown with these dwarf varieties. Thus a complete package was developed to increase wheat production, including improved seeds, synthetic fertilizer, pesticides and weedicides, irrigation, and machines. The implementation of this package in Mexico led to unexpected success in wheat production. In 1958, Mexico became self-sufficient, and in 1960, it started exporting wheat to other countries. The US also shared this success; from 1960 on, it became the leading exporter of wheat. These advancements in US agriculture laid the foundation for the green revolution, and millions of people worldwide were saved from starvation. Green Revolution The Cold War began with the end of World War II. The world was divided into American and Soviet (USSR) camps. Apart from these two poles, there were many Asian, African, and Latin American countries that adopted a policy of remaining neutral to both camps. The nations were part of the Non-aligned Movement (NAM) in 1955, which was under the leadership of Jawaharlal Nehru (prime minister of India), Gamal Abdel Nasser (president of Egypt), and Josip Broz Tito (president of Yugoslavia). Most of the NAM countries had recently emerged from enslavement by former European empires, were economically weak, and were unable to produce enough grain for their populations. Due to continuous starvation and famine, there was a possibility of a “Red revolution” in these countries (like China and regions like Eastern Europe), which could have aligned with the USSR and alienated the US in the global politics; thus the US proposed a “green revolution” to neutralize the threat. The green revolution aimed at making developing countries self-sufficient in grain production. The hope was that if poor people could get enough food, then the need for a Red revolution would automatically disappear, and the US would not be isolated in the world. At the heart of the green revolution were improved maize and wheat varieties developed by American scientists between 1930 and 1960 that yielded eight- to tenfold more grains when sufficient fertilizer, water, pesticides, and weedicides were applied. These modern packages of farming had been successfully implemented in Mexico and the US. The green revolution intended to repeat this experiment in Asia, Africa, and Latin America. The Rockefeller Foundation, Ford Foundation, and several other government and semigovernment organizations were given the responsibility to make the green revolution a success. In 1960, Borlaug represented the Rockefeller Foundation in a Food and Agriculture Organization (FAO) committee. This committee reviewed the wheat-producing capacity of fifteen countries and concluded that except for India, Pakistan, Morocco, Bangladesh, Turkey, and Iran, most of the third world countries did not have agricultural scientists, and even the countries that had agriculture research programs were not capable of achieving food self-sufficiency. Thus the committee recommended training young breeders from developing countries in Mexico under Borlaug’s direction. The OEE project first proposed by Wallace eventually evolved into the International Maize and Wheat Improvement Center, a training center for breeders from all around the globe with the support of the Rockefeller and Ford Foundations and the Mexican government. Borlaug was appointed its director, where he served for the next thirty years. In addition, Menzaldorf was given the responsibility of a maize breeding program for Latin America and Africa. Under the green revolution campaign, advanced varieties of maize developed by American scientists were introduced in Latin American countries. By 1980, high-yielding maize varieties were grown in 50 percent of South America’s arable land. However, the program was not as successful in introducing maize to Africa. Notably, the genetic improvement of rice was included in the green revolution’s agenda, as it was the major cereal crop of Asia. Apart from improved seeds, institutional and infrastructural changes were needed for the green revolution to succeed. Farmers of developing countries used to save the seeds for the next sowing. They did not have the money to buy seeds, synthetic fertilizer, pesticides, and farming equipment. Thus the US entered into policy agreements with developing countries, including India, Pakistan, Bangladesh, Thailand, Indonesia, Egypt, and so on. The US provided seeds, fertilizer, technology, and training. Also, the US promised continued food aid to partner countries until they achieved self-sufficiency in food production. The host governments of developing countries had to build the necessary infrastructure and banking system to provide loans to farmers. Success of the Wheat Breeding Program in Asia In the early 1960s, under Borlaug’s supervision, wheat breeding experiments began in India, Pakistan, and other Asian countries. In 1968, India harvested 16.6 million tons of wheat, for which an extra storage system was needed. Indira Gandhi, the then prime minister of India, used government schools as temporary godowns for grains during the summer months. Despite the drought in 1969 and 1970, 20 million tons of wheat were produced in India. Earlier, the average wheat production from 1963 to 1967 was 9–11 million tons. Pakistan also got about a 60 percent increase in wheat production. Between 1965 and 1970, Pakistan’s wheat yield increased from 4.6 million tons to 8.4 million tons and became self-sufficient in wheat production by 1968. Thus the green revolution had tremendous success in India and Pakistan and was met with similar success in Jordan, Lebanon, Turkey, and Indonesia. In many countries, the average life expectancy and living standards improved, and mortality rates declined significantly. Story of Rice Unlike wheat and maize, very little research and breeding experiments were done on rice when it was included in the green revolution agenda. Thus in 1960, the Ford and Rockefeller Foundations, in collaboration with many international agencies, established the International Rice Research Institute[2] (IRRI; http://irri.org) in Los Baños in the Philippines. Robert Chandler (1907–91) was appointed as the first director of this institute and was responsible for establishing a rice germplasm collection and supervising the rice breeding program. Most of the research about rice’s origin and domestication, its genetic diversity, and its genetic improvement began after the 1960s. However, before the improved rice varieties were made available, the farmers were encouraged to use synthetic fertilizer in rice fields, and an infrastructure for irrigation was developed. From 1950 to 1970, a 25 percent increase in rice production was achieved due to the availability of synthetic fertilization and irrigation. In the 1970s, rice production increased by five to six times due to improved rice germplasm; this helped the green revolution reach a peak because more than one-third of the world’s population is fed with rice. Even though there is more maize and wheat production in the world, maize only fulfills the need of 5 percent of the world’s population, and wheat fulfills 20 percent. In this sense, rice was the most important and representative crop of the green revolution. Here we summarize rice’s origin and genetics and the achievements of rice breeding in Asia and Africa. Origins of Rice In nature, there are twenty-four species of grasses that are closely related to rice and are classified within the genus Oryza. These species are distributed in tropical and temperate regions in Asia, Africa, Australia, and South America. Some of these are diploid (2n = 24 chromosomes), and others are tetraploids (48 chromosomes). However, only two diploid species, Oryza sativa and Oryza glaberrima, were domesticated by humans. Interestingly, rice was independently domesticated in three different continents. O. sativa was domesticated in Asia and Australia. Archaeological and genetic evidence suggests that 9,000 to 11,000 years ago, the O. sativa subspecies japonica was first domesticated in the Yangtze River valley of China from the wild grass O. rufipogon. Another subspecies of rice, O. sativa indica, was independently domesticated 5,000 years ago in the Indo-Gangetic Plain of northern India from the wild grass O. nivara. In Australia, another subspecies of O. sativa was developed 2,000 years ago from O. rufipogon(4). The O. glaberrima was domesticated in Africa 3,500 years ago. O. glaberrima has evolved from the wild grass O. barthii in West Africa (in Mali), and then it was brought to North Africa and the Zanzibar islands (5). This rice arrived in the Americas with African slaves in the seventeenth century and has been grown in North Carolina (5). Among the two cultivated rice species, the O. sativa (Asian rice) is most widely grown globally and is the main staple for one-third of the human population. Diversity of Asian Rice O. sativa is the most widely cultivated rice species in the world. Its japonica subspecies are grown in Southeast Asia, including China, Japan, Korea, Indonesia, Bali, Java, Sumatra, Vietnam, Cambodia, and so on. The japonica subspecies includes several thousand cultivars; some are adapted for the cold climate, while others flourish in the tropics. The varieties of japonica subspecies are broadly known as sticky/sushi rice and have a slightly sweet taste. The indica subspecies of O. sativa is mainly grown in the Indian subcontinent and South Asia. It also has tremendous diversity as well, including varieties adapted to rain-fed highlands and coastal regions. The cooked indica rice grains do not stick to one another and are known as basmati-type. Interestingly, the “sticky” feature is not found in the wild ancestors of rice and related wild species of the grass family. This trait, selected during domestication of the japonica subspecies, is caused by a mutation in the gene Waxi that blocks amylose production within the rice grain. Usually, two types of starch—amylose and amylopectin—are found in the rice grain, but only amylopectin is present in sticky rice. The glucose molecules in amylose form a simple linear structure, while in amylopectin, glucose molecules form branch chains that make amylopectin dissolve faster in hot water than amylose. Thus in sticky rice, the presence of amylopectin makes it sticky. In basmati rice, a high amount of amylose keeps the rice grain separated after cooking. In general, the ratio of amylose and amylopectin varies across cultivars, and accordingly, the different levels of stickiness and the variation in cooking time are observed (for this reason, two varieties of rice are not mixed for cooking). Another interesting fact about rice is that rice grains are naturally red. The white color (or straw color; white is because of polishing) results from a mutation in a gene involved in the anthocyanin pigment’s[3] biosynthesis. For many years, it was a mystery to scientists why humans selected white over red for grain color. This feature was selected independently in both japonica and indica subspecies. It had been speculated that it was for its aesthetics or for some cultural belief. But in both China and India (the two birthplaces of Asian rice), red is considered auspicious, and there is no indication of any cultural preference for white. Genetic studies have recently revealed that the anthocyanin biosynthesis genes are linked to seed-shattering genes, and the loss of shattering also leads to the loss of the red pigment in rice. All the wild ancestors of rice have red seeds that spontaneously shatter upon maturation. This trait is useful for the survival of the wild grasses, but humans can’t harvest the crop at once. Thus a mutation that disrupted both seed shattering and anthocyanin biosynthesis gave rise to the rice plant whose straw-colored seeds did not fall upon maturation. Perhaps early humans picked a straw-colored mutant or collected seeds from more of such plants that favored its selection and propagation. Humans probably carried these plants forward in the process of domestication. Because color and seed-shattering genes are linked, red-colored seeds became less prevalent in the cultivated rice species. The Asian rice O. sativa contains more than one gene for seed shattering and the biosynthesis of anthocyanin pigments. In domestication, two mutant (shattering) genes were selected in japonica that eliminated seed shattering and the red color. In indica, only one (shattering) gene was selected, and thus many cultivars have partial seed-shattering and red-colored seeds. Therefore, farmers in India harvest the paddy before it turns yellow and then dry it in the sun. It is believed that some of the traits of japonica were later introgressed in indica cultivars, and therefore, the modern varieties of indica rice are white. Some varieties of both indica and japonica are found to contain long-grain rice. The comparison of 174 species of O. sativa found in different regions and forty species of its wild ancestor O. rufipogon has shown that the trait of grain length has been fixed in the process of domestication in tropical japonica, indica, and basmati. Basmati rice is the longest among them. The grains of other fragrant rice are also long. In contrast, japonica and Australian rice grown in temperate regions have relatively round and small rice grains (figure 6.6). Like all cereals, humans selected rice for large seeds and panicles and for its adaptation to various climates. This is how thousands of landraces of rice came into existence. Genetic Improvement of Rice at IRRI After World War II, the shortage of grain and an increase in population caused widespread hunger in Asia. In 1949, the FAO established the International Rice Commission with the goal of increasing the yield of rice. The main obstacle in increasing the yield of the indica subspecies was the structure of this plant. Most indica cultivars were tall, had weak stems, and often collapsed on the ground when hit by storms or rains. Thus a strong dwarf stem was required for increasing rice productivity that can bear a heavy grain load. In the 1950s, with FAO’s help, Indian scientists at Rice Research Center, Cuttack, developed rice ADT-27 and Mahsuri rice varieties by crossing Indian rice cultivars with a dwarf Taiwanese Taichung Native 1. In the 1960s, these hybrid rice varieties were grown on a large scale in India but received only a modest increase in yields. After the IRRI opened in the Philippines in the 1960s, Robert Chandler created a team of scientists to develop high-yielding rice varieties. Two members of this team, Peter Jenning and Akiro Tanaka, formulated the strategy for creating improved rice varieties. First, they carefully studied various indica and japonica cultivars. They concluded that if indica plants can be kept standing until maturity (they used bamboo sticks to support these plants), their yields are similar to those of dwarf japonica cultivars. Second, they observed that indica cultivars respond well to the application of synthetic fertilizer. Thus the main problem in the indica subspecies was the tall and weak stem. Jenning crossed dwarf Dee-geo-woo-gen (DGWG) rice variety (Taichung Native 1’s dwarf ancestor) with some indica and tropical japonica cultivars. By 1962, Jenning conducted thirty-nine crossings. He observed that first generation (F1) hybrid progeny was always tall and the F2 population segregated in the ratio of 3 tall:1 dwarf, suggesting that only one gene is responsible for stem’s tallness in Asian rice. Thus dwarfism could be easily introgressed into other rice varieties. Jenning also noticed that some of the dwarf plants matured one month earlier than the traditional rice varieties. The early maturation trait was of great importance because it could save thirty days of land use, fertilizer, and water, and in some regions, three rice crops could be grown a year instead of two. Afterward, Jenning tested seeds of early maturing dwarf plants in the nursery for resistance to blast disease and observed a partial resistance in one plant labeled as IR8-288-3 (IR8). The parents of IR8 were the dwarf DGWG and the Peta rice variety of Indonesia; IR8 was 120 centimeters tall; had a strong, thick stem capable of bearing a huge panicle full of seeds; and was not affected by the change in day length and altitude (thus it could be grown anywhere). Another scientist, S. K. D. Dutta, observed that IR8 yields are 9–10 tons/hectare when synthetic fertilizer, water, and weedicide are applied in adequate amounts. In contrast, the average yield of traditional varieties was 1.2 tons/hectare. In that scenario, the IR8 emerged as a great alternative. It is said that when the news of IR8 reached Ferdinand Marcos (then the president of the Philippines), he immediately went to IRRI and ordered a large-scale multiplication of IR8 seeds. Marcos wanted to showcase this achievement in the next election. The US was also eager to hear some good news in Asia while engaged in the Vietnam War. So amid these pressures, IR8 was released in 1966 without further testing. It is said that 3,000 farmers from the Philippines’ remote islands came to IRRI to procure IR8 seeds. As expected, farmers got an increase of 5-10 times in yields by sowing IR8. With the help of IR8, starvation was temporarily avoided, and the foundation for making high-yielding rice varieties was laid. The yield of IR8 was phenomenal, but it tasted chalky and was hard to chew. IR8 was also susceptible to various diseases, including bacterial blight and viral disease, and had only partial resistance to blast disease. Thus it required a heavy application of chemicals to keep the pests and pathogens away. In 1977, IRRI released IR36, which has a resistance to the blight and tungro viruses, and later IR64, which has an increased resistance to various diseases. Afterward, many rice varieties were developed by IRRI that showed increased resistance against many pathogens. These improved rice varieties were distributed across many rice-growing developing countries using governmental extension centers. Although the arms race between rice and its various pathogens continues, there has been no major famine due to rice shortage. The scientists at IRRI and in several other laboratories around the world are still breeding new rice varieties that can be more tolerant to abiotic stress and can withstand changing climates. Yuan Longping’s Hybrid Rice Varieties In wild ancestors of cultivated rice (O. rufipogon or O. barthii), self- and cross-pollination occur. In contrast, domesticated rice species O. sativa and O. glaberrima favor selfing over cross-pollination. Thus unlike in corn, the phenomenon of hybrid vigor is not easily observed in rice. In 1964, a Chinese scientist, Yuan Longping, decided to change the selfing nature of cultivated rice to make high-yielding hybrids rice varieties. When Longping began his research, the predominant thinking in the field was that hybrid vigor could not be applied to naturally self-fertilizing species such as rice, but he was convinced otherwise. After nine years of hard work, he developed three varieties of rice: the first breed was a male-sterile line incapable of making pollen; the second was a maintainer line that served as a source of pollen, and the third was a restorer line that can rescue a male-sterile line from sterility. Thus a cross between male-sterile and restorer lines produces completely fertile progeny. He demonstrated that hybrid progeny obtained by crossing the male-sterile line with the maintainer line shows hybrid vigor resulting in a significant increase in the grain yield. Generally, under favorable conditions, the yield of the most popular rice varieties is 5–6 metric tons/hectare. However, under similar growing conditions, hybrid rice released by Longping in 1974 gave an average yield of 7.2 metric tons/hectare (a 20 percent increase). Since 1976, this hybrid rice has been grown in China and provides food for an additional 70 million people. Since 1994, hybrid rice varieties have been sown in India, the Philippines, Bangladesh, and Indonesia. However, Longping continued his research on increasing grain yields. In 1997, yields of hybrid rice progeny were 10 metric tons/hectare, and by 2004, it reached 12 metric tons/hectare. Professor Yuan Longping, the father of the hybrid rice, was awarded the UNESCO Science Prize in 1987; the World Food Prize,[4] with Monty Jones (who developed NERICA rice varieties for Africa and is discussed in the following section), in 2004; and the Wolf Prize in 2004 for his contribution to agriculture. In 2007, he was elected a foreign member of the US National Science Academy. NERICA: The New Rice for Africa African rice (O. glaberrima) was developed 3,500 years ago in West Africa from the wild O. barthii in the delta region of the Niger River (present-day Mali). African rice plants are more elongated and have weaker stems than Asian rice. These plants are relatively more susceptible to lodging and cannot bear the load of a heavy panicle. Also, seed shattering in African rice results in a significant loss in yields, as this trait was not selected against during domestication of African rice. However, this rice has the ability to survive in the harsh and challenging environment of Africa; it has a natural resistance against the various pests, parasites, and pathogens prevalent in its environment. Like Asian rice, African rice consists of many cultivars that represent a rich biodiversity, and many useful genes are hidden within these varieties. For example, the O. glaberrima CG-14 variety has a natural ability to tolerate drought, grows faster than weeds, and thrives on marginal land (e.g., deficient in phosphorus or acidic soil). Although the input costs for growing this crop are less, it has less productivity, and thus it is not widely grown. For a long time, the Asian rice O. sativa has been grown in Africa on a large scale, as it is highly productive. Asian rice was introduced about 450 years ago in many countries of Africa. But Asian rice does not possess the ability to cope with Africa’s environment. Its yield decreases in drought conditions, and it cannot protect itself from parasites, pests, pathogens, and insects. The input cost for growing Asian rice is very high, as farmers use large amounts of synthetic fertilizer, pesticides, weedicides, and so on. Thus the sustainable cultivation of Asian rice in Africa poses a challenge. Unfortunately, the useful traits of African and Asian rice cannot be combined using classical breeding methods, as crossings between O. sativa and O. glaberrima yield sterile hybrids. Thus the green revolution failed in improving the productivity of African rice or the resilience of Asian rice for the African environment using classical breeding methods. However, in the 1970s, African scientist Monty Jones, working at the West Africa Rice Advancement Institute (WARDA), collected 1,500 varieties of O. glaberrima and developed genetically improved varieties of African rice that show high resilience in the African environment. In the 1990s, when the technology of tissue culture became easier to apply on rice, Jones’s team created hybrid rice cells by fusing Asian and African rice cells to create hybrid plants from them. These plants were not sterile and can be grown from seeds. This new hybrid variety, called NERICA (New Rice for Africa), has a yield that is five times that of African rice cultivars, is resistant to various biotic and abiotic stress conditions prevalent in Africa, and has fewer input costs. NERICA also matures within three months, while its ancestral African varieties take six months. Thus Jones and his team could combine the useful characteristics of two rice species using advanced in vitro technology of plant tissue culture and surpassed the limits of traditional plant breeding. Farmers who grow NERICA also save three months of land use and labor cost and grow extra short-duration crops such as vegetables. NERICA was Africa’s first successful genetically improved crop. For this work, the United Nations awarded the 2004 World Food Prize to Jones. In the wake of this success, Time magazine included Jones in the list of the most influential people in the world in 2007 (6). Jones’s method was subsequently applied to generate 3,000 new varieties of NERICA, which are being grown in African countries like Benin, Ivory Coast, Gambia, Nigeria, Mali, Guyana, Togo, and so on, and many of these countries have become rice exporters. Green Revolution’s Achievements The green revolution was successful in increasing grain production in Asia, Latin America, and Africa. It saved the lives of millions from hunger and starvation. It can be said that the green revolution played an important role in maintaining peace in the world. It also laid the foundation of mutual cooperation among various international institutes and scientists that lasted beyond the duration and need of the green revolution. Due to the political understanding, various international institutions and government machinery, private foundations, and banks worked together to foster scientific progress and ensure the food security of the world. The green revolution was most successful in Asia because Asian countries already had the infrastructure and roads and had already developed a capitalist market system. During this period, many Asian countries—including India, Pakistan, Turkey, China, Sri Lanka, Indonesia, Malaysia, Vietnam, and Cambodia—became not only self-sufficient in food production but exporters of grains. For example, Pakistan achieved self-sufficiency in grain production in 1969; India followed this success in 1974, which was not expected by the world at that time. Similarly, when the IR8 rice variety was first released in the Philippines, it changed the country from an importer of rice to an exporter within just three years. Overall, due to the increase in the yield of rice and wheat, food grains became cheaper, and the conditions of starvation and famine in developing countries could be avoided. The increase in grain yield per hectare prevented the expansion of cultivated land and helped protect the forests. According to one estimate, in the absence of the green revolution, by the year 2000, the world grain production would have been reduced by 20 percent, an additional 2–2.5 million hectare of agricultural land would have been needed to satisfy the food requirement of the current world population, and the cost of grain would be 30 percent higher. Research in international centers also benefited the US, and the country’s domestic agriculture also increased. Between 1960 and 2000, in developed countries, the yield of wheat increased by 208 percent, rice by 109 percent, maize by 157 percent, potato by 78 percent, and cassava by 36 percent. Overall, the standard of living in developed countries was also improved due to the availability of cheaper food grains. Furthermore, the green revolution brought prosperity to the lives of many farmers and helped agriculture-related businesses flourish. US companies made tremendous profits by selling fertilizers, pesticides, weedicides, and agricultural equipment in the international market, and the green revolution helped recover the US from the post–World War II recession. The green revolution failed in Africa due to various reasons, including the late inclusion of Africa on the agenda and the promotion of maize[5] instead of African crops. Furthermore, African countries lacked the agricultural infrastructure and trained professionals for disseminating adequate training to farmers about the use of fertilizers, pesticides, machines, and so on. Consequently, maize varieties that were successful in the US and Mexico were not successful in Africa. By 1998, while 82 percent of agricultural land in Asian countries, including China, India, and Pakistan, was growing genetically improved varieties of cereal crops, in Africa, only 27 percent of the area had these crops. However, many useful lessons were learned from the first phase of the green revolution (1965–85). The biggest gift of this period has been to lay the foundation for cooperation between agricultural scientists around the world. Later, scientists and policymakers succeeded in solving Africa’s problems in the second phase of the green revolution (1985–2000). During this period, fourteen other CGIAR research centers were opened, each focusing on the prime regional crop (eleven major crops, including potatoes, pulses, cassava, peanuts, beans, millets, and sorghums). The goal of each of these centers, which are spread around the world, is to conserve and improve the germplasm of local crops. From time to time, these international centers release advanced varieties of one or more crops to farmers. Studies show that worldwide production of wheat, rice, and maize has increased by 1 percent, 0.8 percent, and 0.7 percent, respectively, due to germplasm improvement during the second phase of the green revolution. Similarly, yields of minor grains like sorghum and millet have increased about 0.5 percent annually since 2000. International cooperation among scientists and various governmental and private agencies was also beneficial to many African countries, and after 1980, efforts to improve the local African crops, including rice, yam, cassava, and so on, began. Even after the end of the green revolution, these efforts are continuing. Despite a nearly threefold increase in the world’s population in the last fifty years, no major famine occurred. Even today, the world’s population is producing more food than is needed. The green revolution played an important role in achieving global food security. Limits of the Green Revolution The green revolution stood on the maximum exploitation of resources. Therefore, after about fifty to seventy years of intensive grain cultivation, soil fertility has decreased, groundwater levels have fallen significantly, and the overflow of agrochemicals led to the pollution of various water bodies. But by 1980, wheat production showed a slow decline by about 1.5 percent annually, and so far, it has fallen more than 20 percent than the 1970s and ’80s. On average, there has been a one-third decline in wheat production per hectare since the green revolution despite the continuous use of synthetic fertilizer, irrigation, and other mandated agrochemicals required to keep a check on pathogens and weeds. The high-yielding varieties of wheat, rice, and maize absorb large quantities of nutrients from the soil. The synthetic fertilizers replenish three major elements—nitrogen, phosphorus, and potassium (a.k.a. NPK)—but do not replace micronutrients and soil organic matter. Thus in many places, the soil has lost its normal texture, and it has turned almost sand-like. Recuperating the health of the soil is necessary, which cannot be done by continuing the green revolution’s model. The industrial method of farming needs to be reviewed and improved. The goal of the green revolution was to achieve an increase in global food production, and during its implementation, the health of the ecosystem was not taken into consideration. The excessive use of pesticides, fertilizers, and weedicides polluted the groundwater, water bodies, and the air. Today, the entire food chain has become contaminated, and cancer and other diseases have increased in farmers and consumers. In some countries, positive efforts have been made in this direction. Today, breeders around the world are trying to develop varieties of crops that require less synthetic fertilizer, less irrigation, and fewer pesticides to grow. Recently, a super green rice variety has been released in the Philippines, which can maintain its productivity with less fertilizer and water use. Before the green revolution, the staple diet of most Asian countries consisted of a wide variety of coarse cereals such as sorghums, millets, pulses, starchy tubers, and roots, which provided essential nutrients such as iron, calcium, vitamins, and micronutrients. There was no help for farmers growing other crops in the green revolution scheme, so these crops’ production decreased. The majority of the population’s diet has become homogenous, mainly consisting of rice, wheat, and maize. Therefore, malnutrition and devastating lifestyle diseases like diabetes increased despite getting plenty of calories. The varieties of crops that are being grown today are few, and thousands of local varieties of crops have disappeared. Farmers no longer grow them, and thus our food sources are very limited and the diversity of crops has decreased significantly. The outbreak of several diseases has also revealed the importance of biodiversity and the limits of monocropping, which was encouraged by the green revolution. Thus despite achieving a high productivity of cereal crops, the green revolution package was not sustainable. Life of Norman Borlaug Norman Borlaug, the father of the green revolution, was born on a farm near Cresco, Iowa, to Henry and Clara Borlaug.[6] He worked on the 106-acre family farm raising corn, oats, cattle, pigs, and chickens from age seven to nineteen. After completing his primary and secondary education in Cresco, Borlaug enrolled in the University of Minnesota and received a bachelor’s degree in 1937, a master’s degree in 1939, and a PhD in 1942. From 1942 to 1944, he was a microbiologist for DuPont, where he worked on industrial and agricultural bactericides, fungicides, and preservatives. In 1944, he accepted a position as a geneticist and plant pathologist assigned the task of wheat research and production program in Mexico, supported by the Rockefeller Foundation and the Mexican government. As described earlier, in that position, Borlaug successfully developed high-yielding, dwarf, disease-resistant wheat varieties. He built collaborations with local farmers and scientists from all over the world. Borlaug had a deep connection with the farmers, and farming was no stranger to him. Childhood experiences had been with Barlog throughout his life. He was always trying to end hunger in the world. Equally important to his training with Stakman were his life experiences, which helped him sustain hardships for nearly two decades in Mexico’s difficult conditions. When the Rockefeller and Ford Foundations cooperated with the Mexican government to establish CIMMYT, Borlaug was its first director. He is credited with saving more than a billion people around the world from starvation. In 1970, Borlaug received the Nobel Peace Prize. For the first time, a scientist received this award. Shortly after receiving the Nobel Prize, Borlaug established the World Food Prize to honor other scientists and breeders who made outstanding contributions. Borlaug was also awarded the US Congressional Gold Medal, US Presidential Medal of Freedom, Pakistan’s Sitara-e-Imtiaz (1968), and India’s second-highest civilian honor, Padma Vibhushan (2006). Overall, Borlaug was a member of the Agricultural Sciences Academies of eleven countries and received more than sixty honorary doctoral degrees and around fifty other awards. Borlaug also received extensive recognition from universities and organizations in six countries: Canada, India, Mexico, Norway, Pakistan, and the United States. Further Readings Borlaug, N. E. (2007). Sixty-two years of fighting hunger: Personal recollections. Euphytica, 157, 287–97. https://doi.org/10.1007/s10681-007-9480-9 Brown, L. R. (1970). Seeds of change: The green revolution and development in the 1970s. Praeger. Coffman, R. (2010). Mentored by greatness. Indo-US science & technology forum. World Food Prize. https://www.worldfoodprize.org/index.cfm/88533/18129/mentored_by_greatness Myrdal, G. (1970). Agriculture. In The challenge of world poverty: A world anti-poverty program in outline (chap. 4, pp. 78–138). Pantheon. 1. For the story of maize’s domestication and origin, see the video “Popped secret: The mysterious origin of corn—HHMI BioInteractive Video” on YouTube: https://www.youtube.com/watch?v=mBuYUb_mFXA&t=51s. 2. Borlaug and Chandler discuss the origins of the International Rice Research Institute in a discussion filmed in 1994, now available as a multipart series on YouTube: Part 1: https://www.youtube.com/watch?v=17ySNZo3AMs Part 2: https://www.youtube.com/watch?v=TW8hpPi0rqI Part 3: https://www.youtube.com/watch?v=mEjQbo-2nZQ Part 4: https://www.youtube.com/watch?v=rQon8EfQblY Part 5: https://www.youtube.com/watch?v=jhqQwfc0-No Part 6: https://www.youtube.com/watch?v=jtuB2cTEfTU 3. Anthocyanin pigments are also found in many flowers, fruits, and vegetables and are beneficial for health. 4. “A world-brand name: Yuan Longping, the father of hybrid rice,” World Food Prize, 2007, https://www.worldfoodprize.org/index.cfm/87428/40007/a_worldbrand_name_yuan_longping_the_father_of_hybrid_rice. 5. Maize cultivation requires a lot of fertilizer and irrigation, but most areas of Africa lack water, and large dams could not be built for irrigation. 6. For Norman Borlaug’s biography, see https://achievement.org/achiever/norman-e-borlaug and http://nobelprize.org/nobel_prizes/peace/laureates/1970/borlaug-bio.html.

textbooks/bio/Agriculture_and_Horticulture/History_and_Science_of_Cultivated_Plants_(Naithani)/1.06%3A_Genetic_Improvement_in_Cereal_Crops_and_the_Green_Revolution.txt

The knowledge of naturally occurring intra- and interspecies gene transfer events, the advances in recombinant DNA technology, and plant tissue culture helped scientists transcend traditional breeding limits and introduce desired genes from any organism into crops. The crops containing one or more genes from other species (trans/foreign gene) are known as genetically engineered/modified (GE/GM) crops.[1] For example, many insect-resistant GE crops contain the delta-endotoxin gene from a bacterium, Bacillus thurigiensis (Bt). The purpose of making transgenic crops could be to increase the crop yield, prevent damage from pests and pathogens, and increase the nutritional quality. In 1994, the Food and Drug Administration (FDA) approved the first GE crop, Flavr Savr tomato, for human consumption. This tomato variety has a longer shelf life due to delayed fruit ripening. Since then, the FDA has approved more than fifty GE crops.[2] This chapter summarizes the advances made in genetics and other related technology that led to the development of genetically engineered crops and provides examples of a few success stories. Genetic Material In the early 1950s, it was proven that DNA is genetic material. In 1954, Francis Crick and James Watson proposed the double-helix model of DNA based on evidence from Morris Wilkins’s and Rosalind Franklin’s experiments. They suggested that DNA is a double-stranded, helical structure made of four nucleotide bases: adenine (A), thiamine (T), cytosine (C), and guanine (G). The two strands of DNA are antiparallel and are connected by covalent bonds: adenine from one strand binds to thiamine in another strand, whereas cytosine of one strand binds to guanine of the second strand (see figure 7.1). Thus the nucleotide sequence of one strand is complementary to another strand. If the sequence of one strand is known, it is easy to deduce the second strand sequence. Watson and Crick explained that one strand could serve as a template for making the other strand due to their complementary nature. Thus DNA contains information for self-replication—a necessary qualifier for being genetic material. In 1962, Crick, Watson, and Wilkins received the Nobel Prize for the elucidation of DNA structure. In 1958, Arthur Kornberg extracted a DNA polymerase enzyme from E. coli cells that was capable of copying DNA. He demonstrated the replication of a small DNA fragment in vitro using DNA polymerase and proved that either strand of DNA could serve as a template during DNA replication. DNA replication is a semiconservative process: four strands of DNA are made from two parental strands, and eventually, two double helixes form; each helix contains one old and one new strand. Other scientists’ research showed that DNA replication is not completely foolproof, and a proofreading and repair process ensures the removal of mismatched bases and repairs DNA. Thus DNA sequences are preserved for millions of years and ensure the continuity of a biological species. However, a permanent change in the DNA sequence (mutation) can occur due to the extremely low level of proofreading errors during the DNA replication. Most single nucleotide mutations are harmless and don’t affect a phenotype. However, after many generations, the accumulation of mutations could result in phenotypic changes. Thus DNA also acts as raw material for evolution. The complete set of genetic material present in an organism is called its genome.[3] The genome of prokaryotes is made of a single circular chromosome, whereas the genome of eukaryotes consists of more than one chromosome. Individuals of the same species have the same genome size (same number of chromosomes), and chromosomes between different species vary. The structural and functional unit of the genome is a gene. The order of the four nucleotide bases determines the composition of individual genes. Several thousand genes are present within the organisms. Typically, the information from the DNA (gene) is transcribed into RNA, and a subset of RNA (messenger [m] RNA) is then translated into proteins. The proteins carry out most of the structural and functional activities within the cells. This unidirectional flow of genetic information from DNA to protein is called central dogma and serves as the fundamental principle for the basis of life. In addition to protein-coding mRNAs, organisms’ genomes contain genes that encode various other types of structural and regulatory RNA molecules (e.g., rRNA, tRNA, and microRNA). Recombinant DNA Technology After the 1970s, a new branch of biology, molecular biology, came into existence. Its focus is to understand the structure and functions of genes and gene products. It was expected that the sum of the knowledge about all genes could help in understanding the whole organism. The basic principles of molecular biology were discovered by experiments conducted on unicellular bacteria and fungi, and later studies on higher eukaryotes, including plants, began. First, scientists succeeded in extracting DNA from various organisms. The next challenge was to identify the thousands of genes and then determine their function within the cell. The discovery of restriction enzymes and ligase enzymes made the identification and cloning of the individual genes possible. The restriction enzymes recognize a specific sequence of four/six/eight nucleotides within DNA and make a cut in the strand. The sites of restriction enzymes are randomly scattered within the genome, and hence they cut double-stranded DNA into small pieces. So far, more than 3,000 restriction enzymes have been discovered, of which 600 are available in the market. These enzymes were named after the bacteria where they were first identified. For example, the EcoR1 enzyme is found in the Escherichia coli strain RY13, and HindIII was discovered in Hemophilus influenzae. Bacteria make restriction enzymes to protect themselves against virus infection. When viral DNA enters the bacterial cells, these restriction enzymes slice the virus DNA and block the virus’s growth. However, bacterial DNA remains protected from their own restriction enzymes by methylation of the corresponding restriction sites. In 1978, Werner Arbor, Hamilton Smith, and Daniel Nathans were jointly awarded the Nobel Prize for the discovery of restriction enzymes. In 1967, a special enzyme, ligase, capable of joining two DNA fragments, was discovered in Gellert, Lehmann, Richardson, and Hurwitz’s laboratories. For scientists, the restriction enzymes served as molecular scissors, and ligase served as a molecular glue for cutting and joining DNA fragments. The next question was how to amplify small DNA strands for their detailed analysis. Here, the knowledge about a bacteriophage lambda that infects E. coli bacteria explicitly came in handy. Esther Lederberg discovered the bacteriophage lambda in 1951. Esther Lederberg observed that after infecting E. coli, lambda could undergo either an active lytic or a latent lysogenic cycle. When the bacterial cell grows in the rich medium (rapidly dividing), then the lambda enters the lytic cycle, makes 1,000 of its copies by using resources and machinery of bacterial cells, and then destroys the host. These 1,000 lambda offspring then infect 1,000 new cells and produce 1,000,000 new progenies in the next cycle. In this way, the virus grows at the speed of a rocket. However, if bacterial cells are deprived of nutrients and are in the stationary phase (not dividing), lambda enters the lysogenic cycle resulting in the insertion of the lambda DNA into the bacterial genome without causing any trouble. However, if the situation worsens for bacteria (or it experiences heat stress), lambda enters the lytic cycle to makes use of whatever resources are available for making its copies before host cells die. For lambda, going to a lytic or lysogenic state is controlled by the repressor protein encoded in lambda DNA. When lambda DNA enters the bacterial cell, first, the repressor protein is made, which blocks the rest of lambda’s genes. The rapidly dividing bacterial cells contain high levels of proteases, which can break down the repressor and allows the lambda to enter into the lytic cycle. Under nutritional deprivation, bacterial cells come to a stationary stage where the protease levels remain low, and the lambda goes into the dormant lysogenic cycle. When the host cells face stress (e.g., high temperatures), protease protein increases to mitigate the situation and recycle the nutrients. Thus in such a situation, the repressor protein is destroyed, and lambda enters the lytic cycle. Based on this knowledge of the lambda life cycle, scientists created versions of lambda in which researchers could easily turn the lytic and lysogenic cycle on or off. Furthermore, scientists found that only 75 percent of the lambda genome is indispensable; 25 percent of the lambda genome can be replaced with any DNA fragment. Thus if a gene is inserted within the lambda genome, millions of clones can be made quickly in E. coli. Then this DNA could be isolated, stored, and used for further experimentation. The lambda served as the first vector for cloning genes. In the 1950s, Esther and Joshua Lederberg also observed that bacteria have many (10–1,000) small, circular DNA structures called fertility (F) plasmids in addition to a large genome. Before cell division, plasmids undergo autonomous replication and are inherited by the daughter cells. Soon, from a pathogenic bacteria, Shigella, another type of plasmid was discovered that contained antibiotic resistance genes (known as resistance [R] plasmids). It was easy to isolate plasmid DNA from bacterial cells, and also, bacterial cells have a natural capability of uptaking these small plasmids. The introduction of these plasmids into bacterial cells is called transformation. Scientists optimized protocols for increasing the efficiency of transformation and created chimeric plasmids by joining parts of F and R plasmids that contained one or two antibiotic-resistance genes, sites of restriction enzymes, and the autonomous origin of replication for E. coli. Thus these plasmids served as excellent vectors for cloning small DNA fragments. If a mixture of plasmid DNA and bacterial cells are plated in a medium containing a specific antibiotic, then the normal bacterial cells will die, and only transformed cells containing the desired plasmid (clone) will grow. After the 1970s, the cloning of small DNA fragments (less than 20 kilobase pair [Kb]) in plasmid vectors and lambda-based phasmids became routine. Soon methods for DNA sequencing were invented, which made it possible to read the coded information (the order of four nucleotide bases, A, T, G, C) present within the genes. Computer analysis was used for analyzing sequencing data, and the field of bioinformatics was born. By comparing the genes present within living beings, it was found that the genetic code and genetic mechanisms are conserved among bacteria, plants, and animals. Computer algorithms are also used for assessing the similarity between the homologous genes from different organisms. The homologous genes between closely related species are more similar than distantly related species. In the late 1990s, due to advances in sequencing technology, it became possible to sequence whole genomes of bacteria, fungi, plants, and animals. Comparing the entire genome of different organisms allowed quantifying the similarities among the various organisms and deciphering the evolutionary distance between the species (see figure 7.2). In the light of modern genetics, molecular biology, and phylogenomics, we can now understand the process of biological evolution in reverse order.[4] Gene Transfer in Plants We often see swollen, round, tumor-like knots on the roots, twigs, and branches of many plants around us (see figure 7.3). These knots, called crown galls, are caused by a soil bacterium, Agrobacterium tumefaciens. These galls do not cause the death of the host plant but stunt their growth. The virulence of Agrobacterium strains is determined by a Ti plasmid present within the bacterium. Marc Van Montagu and his colleagues studied eleven pathogenic and eight nonpathogenic strains of Agrobacterium and found that all pathogenic strains had Ti plasmids and all nonpathogenic strains lacked Ti plasmids. They also noticed that the introduction of Ti plasmids into nonpathogenic strains transforms those into pathogenic strains. Thus it was concluded that Agrobacterium needs Ti plasmids for infecting the plants. The sequencing of the Ti plasmid revealed that it contains virulence (vir) genes and genes for the biosynthesis of auxin, cytokinins (plant growth hormones), and opines. The vir genes help bacterium infect host plants. The plant hormones promote the rapid growth of the cells at the site of infection (formation of crown galls), and opines serve as sources of energy, carbon, and nitrogen for Agrobacterium. Research in the 1970s and 1980s showed that Agrobacterium’s persistent presence within galls is not required for tumors to grow. In fact, after infecting a plant cell, Agrobacteria leave the Ti plasmid in the host cell, and then a large fragment of Ti plasmid, called T-DNA, gets inserted into the plant genome. The T-DNA in the plant genome continues to provide instructions for promoting the growth of the crown gall tumor and the biosynthesis of opines. If a gene is inserted within the T-DNA of the Ti plasmid using recombinant DNA technology, it can be easily transferred within a plant genome using Agrobacterium. Scientists modified Ti plasmid by replacing genes responsible for tumor growth with restriction enzyme sites, antibiotic resistance genes, and the E. coli plasmid replicons. Thus chimeric Ti plasmids allowed cloning of foreign genes in them and were able to replicate within both E. coli and Agrobacterium. Scientists had the flexibility of efficiently cloning genes in E. coli and then use Agrobacterium for transferring the desired gene into the plant genome (plant transformation). Since Agrobacterium strains have a limited host range, they cannot transform all species of plants. The gene gun was used for transforming plant species, for which agroinfection was not an option. In this alternative method, very fine particles of gold or tungsten coated with desired DNA are bombarded on plant tissue by the gene gun. The advances in plant tissue culture made it possible to regenerate whole plants from small plant parts. Thus the combination of recombinant DNA technology and plant tissue culture allowed the bioengineering / genetic engineering of plants for important quality traits (that were not available in a given plant species’ diversity pool). It became possible to introduce useful genes from bacteria, animals, and other plant species into the crop species. Both methods of plant transformation use small plant parts to start with, and after agroinfection or gene bombardment, the selection of transgenic cells is carried out in the synthetic tissue culture media (see figure 7.4). One or more antibiotic selection makers accompany the transgene; thus only transgenic cells grow in this media while normal cells die. After several rounds of selection and multiplication of transgenic cells in the tissue culture, tests for the desired transgene are carried out. Once the insertion of the transgene is confirmed, the expression analysis and other assays are conducted, and whole plants are regenerated. In general, both plant transformation methods have a low efficiency, and then among the transformed plant cells, only a few show the desired level of transgene expression. Once the transgenic plants are fully developed, the second round of testing begins, and one to five of the best-performing plants are selected. Crops The GE crops made so far can be divided into three classes: (i) pest or pathogen resistant, (ii) herbicide tolerant, and (iii) biofortified with improved nutritional value (see table 7.1). At present, ~15 GE crops are being grown in 12 percent (179.7 million hectares) of the total agricultural land worldwide. In the US alone, ten GE crops are being grown on 70 million hectares. Maize, soybean, and cotton are among the most grown GE crops. Other GE crops include apple, mustard, sugar beet, papaya, potato, pumpkin, eggplant, alfalfa, poplar, rose, golden rice, golden cassava, and so on.[5] Story of Virus-Resistant Rainbow Papaya The delicious papaya fruit is a rich source of vitamins A and C, calcium, and potassium. Papaya (Carica papaya) is the native plant of Central and South America. When Christopher Columbus tasted it for the first time, he was overwhelmed and named it the “fruit of the angels.” After the fifteenth century, papaya spread throughout the world under the umbrella of European colonialism. Today it is grown in India, Brazil, the Philippines, Indonesia, Malaysia, Thailand, Hawaii, and the Caribbean islands. Usually, within six months after sowing the seed, the papaya grows as high as the average tree and starts bearing fruit within a year. Within three years, the papaya tree matures and produces a full yield. The cost of setting up papaya plantations is less than that of other plantations, and the farmers make decent profits from it. The commercial cultivation of papaya first began in the Hawaiian Islands. Around 1940, papaya plantations were first established on the island of Oahu, which provided a livelihood to many local farmers. In the 1950s, an outbreak of the papaya ringspot virus (PRSV) occurred in Oahu’s papaya plantations. This virus causes the stunting of trees, the deformation of leaves, the decline in their yields, and rounded spots in the infected fruit (see figure 7.5). Within a short period, the infected plant dies. Within a few years, the papaya plantations of Oahu were destroyed by the PRSV, so the industry moved to the big island of Hawaii, where the papaya industry flourished and expanded without any problems for the next four decades. In 1984, Hawaii’s papaya production reached its highest point (80.5 million pounds). However, around 1990, the PRSV appeared in Hawaii. As expected, the papaya plantations of Hawaii were devastated. By 1997, the papaya production declined up to 40 percent, Hawaii’s economy staggered, and many farmers’ livelihoods were destroyed. Most of Hawaii’s papaya farmers were first-generation, less-educated immigrant Filipinos. Fortunately, Dennis Gonsalves, a Cornell University professor, grew up in Hawaii and knew the importance of papaya for the local economy. He noticed the PRSV when its outbreak began in Hawaii and started a research project to save the papaya plantations from PRSV. First, he tried to find a PRSV-resistant papaya variety so that trait could be transferred to the high-yielding productive papaya varieties using traditional plant breeding methods. But he didn’t succeed in finding such a variety. After this, Gonsalves identified less harmful strains of PRSV and experimented with them. He observed that if a papaya plant is first infected with a less aggressive strain, then the more harmful PRSV strain does not attack it. Until 1983, Gonsalves kept doing these experiments, but no permanent solution emerged for the disease’s practical management. Around 1983, Roger Beachy made a kind of transgenic tobacco that was resistant to the tobacco mosaic virus (TMV). After entering a living cell, TMV (like most other viruses) makes replicas of its genome using the host cells’ resources, and then genome replication stops, and the synthesis of coat protein begins. The coat protein forms the virus’s outer shell; thus the viral genome gets packed one by one, and the progeny of TMV busts out by destroying the host cells. Beachy cloned the coat protein gene of TMV and then transferred it into tobacco plants. Beachy’s transgenic tobacco expressed high quantities of coat protein, so when TMV infected them, the coat protein present in the transgenic plant prevented replication of the viral genome, and infection was contained. Professor Gonsalves, inspired by Roger Beachy’s success, decided to introduce the PRSV’s coat protein gene in the papaya. In 1986, he successfully cloned this gene and transferred it within papaya plants with the help of a gene gun. As expected, the transgenic papaya showed resistance to the devastating PRSV. Field trials of these GE varieties, known as Rainbow and SunUp, began in 1992, and in late 1996, the US government approved transgenic papaya for commercial planting. In 1998, Hawaiian farmers received Rainbow papaya seeds and the necessary advice free of cost, and Hawaii’s papaya plantations got a new life. Currently, a cooperative society sells seeds of PRSV-resistant papaya varieties in Hawaii to farmers at very low prices. For the past twenty-two years, the papaya of Hawaii has been selling globally and contributes \$50 million annually to Hawaii’s economy. Gonsalves did not receive any grant from any government or private institution to make the GE papaya varieties. The only help he received was a small grant of \$20,000 from Hawaii’s senator Daniel Inouye. He created the world’s first successful GE crop at a very low cost and made it available to farmers free of charge. In 2002, Gonsalves’s research team was awarded the Humboldt Research Award for their contribution to agriculture. Later, others made virus-resistant GE squash, zucchini, potatoes, and plums (see table 7.1). Roundup Ready Soybeans Weed control has always been a challenge for farmers. Weeds are wild plants (sometimes closely related to crops), are well adapted to various environments, and are more resilient than crops. Weeds compete for nutrients, sunshine, and water with crops and negatively affect crop yields (20–40 percent). In traditional societies, farmers used to burn the weeds before sowing and then afterward spent time weeding. However, this is not an option for very large agricultural fields. After 1950, chemicals were discovered that can destroy the foliage. Initially, they were sprayed in the area before sowing to eradicate weeds. But these chemicals are hazardous for human health and have chronic and acute toxicity. Furthermore, many herbicides are carcinogens, and their use poses a risk of polluting water, air, and the food chain. In 1970, John Franz, a scientist at the Monsanto Company, discovered herbicidal activity in glyphosate.[6]Furthermore, it was found that some soil bacteria can convert glyphosate into harmless substances within a few days. Hence it posed relatively less risk of environmental pollution compared to previously used chemicals. In 1974, it was marketed as an herbicide with the trademark Roundup. In the 1970s, Monsanto also invested in plant biotechnology and began developing herbicide-resistant transgenic crops. They already had information that soil microorganisms can metabolize glyphosate into harmless products. Soon scientists discovered a gene within a bacterium that coded for an enzyme capable of degrading glyphosate. Subsequently, this gene was introduced in soybean and corn to create Roundup Ready GE varieties that survive herbicide (glyphosate) sprays while all the weeds die. In 1996, a Roundup Ready soybean was approved by the FDA for its cultivation in the US. In this way, weed management became simpler, cheaper, and more manageable. Also, farmers can plant soybeans in a closer, tighter row and get higher yields. However, the second-generation soybeans are sterile, so farmers must buy new seeds every year from the seed company. This strategy was also used for creating herbicide-tolerant corn, rice, and many other crops. More than 50 percent of GE crops currently grown in the US and worldwide are herbicide resistant (see table 7.1). Today, more than 90 percent of soybeans, corn, cotton, and mustard growing in the US are Roundup Ready varieties. Farmers around the world are increasingly relying on herbicides for weed management. Since 1996, when genetically engineered glyphosate-tolerant Roundup Ready crops were first released, glyphosate use has risen fifteenfold globally; these quantities are beyond the natural capacity of soil bacteria to metabolize glyphosate. Therefore, glyphosate and other herbicides have generated concern among the public and policymakers due to their harmful effects on human health and the environment. Insect-Resistant Bt Crops Most of us do not like to eat any fruit or vegetable that is infested by insects. We see the clean fruit and vegetables in the market because farmers have used plenty of pesticides. If pesticides are not sprayed, one-third of the yields of most crops would be lost due to pest infestation. However, pesticides have harmful effects on human health and pollute the environment. Scientists have been looking for an alternative to pesticides for a long time. In 1901, a Japanese scientist, Shigetane Ishiwata, was investigating the cause of silkworms’ sudden deaths and found the bacterium Bacillus in dead larvae. He concluded that Bacillus is responsible for their deaths. In 1911, Ernst Berliner also noticed that the Mediterranean floor moths’ larvae were dying due to Bacillus’s presence. He named the bacterium Bacillus thuringiensis after the German city of Thuringia. B. thuringiensis (or Bt) is a gram-positive soil bacterium that contains crystals of a delta-endotoxin protein that kills a broad category of insects. Upon ingestion, the crystal protein is cleaved into small fragments by proteases present in the alkaline environment of an insect’s gut. One of the fragments generated from the crystal protein acts as a toxin. This toxin kills insects by forming pores into the cell membranes of the insect midgut. From 1920 onward, European farmers began spraying the Bt bacteria to protect their crops from pests. In 1938, commercial spore-based formulations known as Sporine made it to the market in France. Even today, organic farmers spray a similar formulation of Bt bacteria on their crops. In the 1980s, the Monsanto Company succeeded in cloning the “cry” gene from B. thuringiensis that codes for the crystal protein. Subsequently, this gene was optimized for high expression in plant cells to develop insect-resistant crops (e.g., cotton, maize, eggplant, and rice). When insects feed on Bt plants, the delta endotoxin reaches their intestine, and they die. Therefore, Bt crops can protect themselves from insects. In 1995, the US government approved Bt cotton (Bollgard cotton) and Bt corn for cultivation. Since then, these crops are being grown in the US and many other countries. These seeds of Bt varieties sold in the market are of the heterozygous F1 hybrid. Thus, farmers cannot save seeds of Bt crops for the following year’s sowing, because in the F2 generation, resistance and susceptibility traits segregate, and the insect-resistant traits get diluted in every subsequent generation. The farmers are required to buy these seeds every year, and their input costs increase. The Bt crops do not eradicate insects completely, but their use could reduce the quantities of chemical pesticides manifold. The farm management is crucial for integrating these bioengineered crops and the chemical spray and keeping a sufficient refuge of non-GE crops to reduce the pests’ selection pressure. In the US, Brazil, Argentina, and so on, practicing integrated pest management (IPM) is easy because thousands of acres of the farm are run by a single person or company. But in other developing countries, farmers have smaller agricultural land holdings and do not have enough space for “refuge.” The farms are also surrounded by many neighboring farms. Since living organisms constantly evolve, the protection offered by the Bt gene is not going to last for a long time, and eventually, it will be ineffective against insects. That is why scientists continue to make new versions of the Bt gene and search for other genes with similar properties. Thus it is expected that from time to time, scientists will release new GE varieties as the arms race between hosts and pests continues. Golden Rice The diet of poor people living in Asia, Africa, and Latin America mainly consists of rice and lacks fruits, vegetables, dairy, and meat. Consequently, millions of people have a huge deficiency of vitamin A in their bodies. According to a survey by the World Health Organization, more than 400 million people in twenty-six countries living on rice are deficient in vitamin A, due to which 500,000 children suffer from night blindness/blindness and 1 million children die annually.[7] Vitamin A is essential for the proper development of humans and animals. It supports the healthy development of eyes, bones, and muscles and maintains adequate calcium levels and immunity. Humans cannot biosynthesize vitamin A of their own, but they get it from dairy, meat, and colorful vegetables and fruits. Red, orange, and yellow vegetables and fruits (e.g., sweet potatoes, carrots, oranges, and mangoes) contain provitamin A (β-carotene), which gets converted into vitamin A inside the human body. For a long time, plant breeders have been searching for a rice variety in which β-carotene is found. In 1991, Ingo Potrykus, at the Institute of Plant Sciences of the Swiss Federal Institute of Technology, Zurich, started a project to bioengineer the β-carotene biosynthesis pathway in rice. In plants, the biosynthesis pathway consists of eight reactions, which are catalyzed by four enzymes. Rice contains most of the precursors for making β-carotene, but three out of four enzymes are nonfunctional. In 2000, Potrykus and his colleague Peter Beyer cloned two genes, phytoene synthase and lycopene cyclase, from the daffodil and the phytoene/carotene desaturase (crt1) gene from bacteria and then successfully introduced all three genes in rice plants to create the provitamin-rich variety by reconstructing the β-carotene biosynthesis pathway (see figure 7.6). One kilogram of this rice contains about eight milligrams of β-carotene, which gives them a golden hue, and thus this variety was named golden rice.[8] Once this basic strategy was successful, Potrykus signed a contract with the company Syngenta for enhancing the content of β-carotene and for large-scale experimental testing and handling. Syngenta produced an SGR2 variety of golden rice (~27mg β-carotene/kg) by replacing the phytoene desaturase in a daffodil with maize homolog. The Golden Rice Humanitarian Board has made golden rice free for farmers in developing countries with an annual income below \$10,000. Later, with the help of research institutes of various countries, β-carotene biosynthesis pathways have also been enabled in many rice varieties (IR 64, Boro, PSB, RC82, etc.). After extensive testing, these GE rice varieties were found to be safe for human consumption. Unlike other GE crops, farmers who grow golden rice can save the seeds of the next year’s sowing. It took almost two decades before the golden rice was approved for farmers’ use. In 2018, the FDA, Health Canada, and Food Standards Australia New Zealand recognized the IRRI’s evaluation and approved the release of golden rice’s GR2E variety. Bangladesh and China have begun the cultivation of golden rice. After rice, scientists started to work on biofortifying other crops using a similar strategy. For example, Maria Andrade, Robert Mwanga, and Jan Low created the golden sweet potato[9] with the support of global nonprofit agricultural research program HarvestPlus (https://www.harvestplus.org/), USAID, the Bill and Melinda Gates Foundation. Similarly, the golden cassava variety has also been developed, which has special importance for Africa. Table 7.1 Herbicide-tolerant GE crops Crop Herbicide Year Inventor Canola Glufosinate 1995 Bayer Canola glyphosate 1999 Monsanto Cotton Bromoxynil 1994 Calgene Cotton glyphosate 1996 Monsanto Cotton Sulphonylurea 1996 DuPont Cotton Glufosinate 2003 Bayer Cotton Dicamba 2015 Monsanto Cotton 2,4-Dichlorophenoxyacetic acid (2,4-D) 2015 Dow Maize (corn) Glufosinate 1995 AgrEvo GmbH Maize glyphosate 1996 Monsanto Maize 2,4-D 2014 Dow Sweet corn glyphosate 2011 Monsanto Rice Glufosinate 1999 AgrEvo GmbH Soybean glyphosate 1994 Monsanto Soybean Glufosinate 1996 Bayer Soybean Sulphonylurea  2007 DuPont Soybean Isoxaflutole 2013 Sygenta Soybean Mesotrione 2014 Sygenta Soybean Imidazolinone 2014 BASF Soybean 2,4-D 2015 Dow Soybean Dicamba 2015 Monsanto Sugar beets glyphosate 2005 Monsanto Sugar beets Glufosinate 1998 AgrEvo GmbH Table 7.2 Insect (pest)-resistant crops Crop Transgene Year Inventor Cotton Bt 1995 Monsanto Field corn Bt 1995 Monsanto Sweet corn Bt 1998 Monsanto Potato Bt 1995 Monsanto Soybean Bt 2016 Monsanto Table 7.3 Virus-resistant crops Crop Coat protein gene Year Inventor Papaya (Rainbow and SunUp) Ring Spot Virus 1996 Dannis Gonsalves (Cornell University, and ARS-USDA) Plum Plum pox virus 2007 USDA Potato Potato Virus Y 1999 Monsanto Potato potato leaf roll virus 2000 Monsanto Squash (not widely grown) zucchini yellow mosaic virus 1994 Asgrow Squash (not widely grown) watermelon mottle virus 2 1994 Asgrow Table 7.4 Nutritional value improved crops Crop Trait Year Inventor Canola High Iysine 1994 Calgene Maize High-Iysine 2006 Monsanto Golden Rice Rich in β-carotene (pro-vitamin A) 2005 Sygenta Plenish Soybean High oleic acid content (no trans fat) 2010 DuPont Potato Less acrylamide, when cooked/fried at a high temperature. 2014 J R Simplot Company Arctic Apple The GE Apples resist browning after being cut. 2015 Okanagan Specialty Fruits Inc. Impact of GE Crops Humans have been selecting plants and animals for desired traits that suited their needs since the beginnings of agriculture 10,000 years ago. GE crops are the latest advancements made in this direction, with goals of achieving agricultural productivity and improving the available genetic stocks’ quality. After 1930, these objectives were achieved using traditional breeding. In the early twenty-first century, scientists genetically engineered crops by introducing useful genes from any living forms and/or by rationally designed genes. A GE crop may contain one or more transgenes from bacteria, viruses, animals, or plants. However, more than 99 percent of it is similar to its mother plant (see figure 7.7). In general, crop plant species contain 30,000–50,000 genes, and less than five genes are introduced in any given GE crop. In this sense, GE crops are closer to the domesticated varieties. Interestingly, people have adopted more than 1,000 high-yielding varieties (generated by traditional breeding), but GE crops have aroused significant anxiety around the world and have faced consistent opposition globally. There has been a constant debate about the technology and impacts of GE crops. The first and foremost concern is if GMO food is safe. GE crops undergo extensive safety trials before they are released for cultivation and are approved by the FDA. The data from various GE crops’ safety trials in the last twenty-five years show that GMO food is safe. In 2016, the US National Academy of Sciences published a review of GE crops prepared by an independent committee of eighteen experts. This 600-page report (1) concluded that there is no evidence that anyone has had a problem digesting GMO foods so far. However, people have raised issues related to allergies and the presence of toxins in GMO food. Overall, GMO food is deemed safe by the experts. Today most experts, scientific institutions, and governments agree that GMO food does harm human health. According to an estimate, millions of people in twenty-eight countries have been consuming GMO food products daily for the past twenty-five years, and so far, none died due to eating such foods (1). Contrary to what the experts have revealed, there is increasing anxiety among consumers about GMO products. This issue has not been settled for the people yet. Many nongovernmental organizations, consumer groups, and ordinary people worldwide are worried about the safety of consuming GMO food. No one has currently investigated GMO products’ long-term effects on humans; therefore, both opponents and supporters of GMOs can’t say anything definitively on the matter. For a long time, consumer groups had been advocating to clearly label products that are GMO, since in the US, this had not been required by law. Under consumers’ pressure, the labeling of GMO (and more of non-GMO) products appeared increasingly, and in 2016, US Congress passed a law requiring the labeling of GE food (the bioengineered). Hence, the data on the long-term effects of GMO food on human and animal health may become available in the future. Another issue is how much more productive GE crops are than the high-yielding breeds developed during the green revolution. In the last twenty-five years, the claims made about GE crops’ increased productivity have not been fully met. The 2016 National Science Academy report (1) stated that the productivity of GE crops grown from 1990 to 2015 is not significantly higher than the high-yielding varieties of the green revolution era. Overall, the GE crops made the farmers less prosperous than the green revolution because agriculture’s input cost (for buying seeds, fertilizer, irrigation, pesticides, fungicides, herbicides, etc.) is very high and leaves a small margin for profit. Compared to the green revolution era, today’s farmers have smaller and less fertile farms in developing countries, and government assistance and subsidies have declined. Did GMO technology improve the quality of food? Without a doubt, this technology has created many nutrient-rich crops—like golden rice, cassava, and sweet potato; high oleic acid–containing canola; Plenish soybeans; and so on—and offers an opportunity for further improvements. There is tremendous scope for solving malnutrition by using GMO technology. In addition, the genetic engineering of model and crop plants has provided a conceptual understanding of gene functions and many fundamental biological processes. Besides genetics and genomics, immense progress has been made in vitro tissue culture technology. Together, the new knowledge and advancement in tools served as a foundation for next-generation CRISPR/Cas9 gene-editing technology that has a high potential for developing new biofortified and stress-tolerant varieties of crops. At the beginning of the twenty-first century, GE crops were presented as a promising alternative to a non–environmentally friendly industrial farming system. But looking at the last twenty-five years, it is clear that GE crops have become an integral part of it. The folks who oppose GMOs argue that most GE crops ensure profit to corporations by encouraging increased use of pesticides, weedicides, fungicides, and fertilizers. To date, more than 50 percent of GE crops are herbicide resistant. The use of glyphosate and other herbicides has exceeded the safety threshold and poses threats to human health and the environment. The continuous spray of herbicides has led to the rise of superweeds that are increasingly adapting to various herbicides and require more potent mixtures (including two or more chemicals) or higher doses for effective control. Thus GM crop-based weed management is not sustainable. Additionally, the reliance on both high-yielding and GM crops on heavy inputs of energy and fertilizers also needs careful reevaluation. Toward a Sustainable Future The FAO projects the global population to grow to 9.7 billion by 2050. The biggest challenge of the twenty-first century is to keep the productivity of crops sustainable without causing further destruction to the environment and depletion of natural resources. Despite its overall efficiency, the current industrial agricultural system relies heavily on natural resources and energy. For example, half of the world’s habitable land and more than 70 percent of global freshwater withdrawals are used for agriculture. Also, agriculture is a significant source of global pollution: 26 percent of global greenhouse gas emissions[10] and 78 percent of the global ocean and freshwater eutrophication is caused by agriculture.[11] Agriculture is the prime source of greenhouse gases, including carbon dioxide (CO2) and nitrous oxide (N2O), which cause the depletion of the stratospheric ozone layer and global warming. Therefore, the agricultural production systems have a big impact on global climate change, natural resources, and all living beings’ health. It is important to note that a continuous increase in agriculture productivity alone cannot solve hunger in the world. Natural resources are finite, and their overexploitation will have disastrous consequences. Therefore, we must explore other avenues for bringing efficiency to ensure food security. Today, enough grain is being produced worldwide to meet the need of 10 million people. Theoretically, if food is distributed equally among all the people of the world, then 75 percent of it is enough to feed the entire population of the world, and one-fourth can be saved, although achieving such equity at the global level is difficult to achieve due to various sociopolitical reasons. Nonetheless, it suggests that there are additional avenues where progress can be made beyond agriculture production. It is noteworthy that more than 40 percent of all food produced worldwide is wasted in processing, transportation, supermarkets, and kitchens. Many fruits and vegetables are discarded because of their lack of aesthetic appeal or uniformity. Moreover, readymade, cheap, and readily available food requires extra production to compensate for its regular trashing as it expires on the supermarkets’ shelves. When food is wasted, all the resources to grow, process, package, and transport are wasted along with it. Besides, the waste is a source of significant greenhouse gases. Overall, the current agricultural production systems and consumers’ lifestyles put a burden on natural resources, the environment, and energy. If consumers do not change their habits, then the agricultural production system will remain in its current form. Both farmers and consumers will have to strive together to build a new sustainable agricultural production system. In past decades, the gradual progress in plant genetics, genomics and breeding, precision agriculture, organic agriculture methods, and growing consumer awareness has been paving the way for the agricultural production system’s future. Better management of agriculture, natural resources, the environment, and human resources is required in the agriculture sector. The formula for successful and sustainable agriculture will have elements from industrial farming, GM technology, precision agriculture, and organic farming. Equally important is to foster a culture of conscious consumption and change in consumers’ behavior to create new, sustainable, just, diverse, and healthy agricultural production systems. The issue of agriculture has never been limited to production. It has given rise to human civilization and its polytheistic cultures. Change in agriculture is intricately associated with a change in human society. Only a better society can build a better agricultural system. Further Readings Benbrook, C. M. (2016). Trends in glyphosate herbicide use in the United States and globally. Environ. Sci. Eur., 28, 3. Beyer, et al. (2000). Engineering the provitamin A (-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science, 287(5451), 303–5. doi.org/10.1126/science.287.5451.303 Gillam, C. (2018, May 8). Weedkiller products more toxic than their active ingredient, tests show. The Guardian. https://www.theguardian.com/us-news/2018/may/08/weedkiller-tests-monsanto-health-dangers-active-ingredient Gonsalves, D. (1998). Control of papaya ringspot virus in papaya: A case study. Annu. Rev. Phytopathol, 36, 415–37. Jenkins, M. (2018). Food fight: GMOs and the future of the American diet. Avery. Oerke, E. C. (2006). Crop losses to pests. J. Agric. Sci., 144, 31–43. https://doi.org/10.1017/S0021859605005708 Pingali, P. L. (2012). Green revolution: Impacts, limits, and the path ahead. PNAS, 109, 12302–8. doi.org/10.1073/pnas.0912953109 Pollan, M. (2006). The omnivore’s dilemma: A natural history of four meals. Penguin. Poore, J., & Nemecek, T. (2018). Reducing food’s environmental impacts through producers and consumers. Science, 360(6392), 987–92. Searchinger, T., et al. (2018). Creating a sustainable food future—a menu of solutions to feed nearly 10 billion people by 2050. World Resources Institute. https://www.wri.org Tian, H., et al. (2020). Comprehensive quantification of global nitrous oxide sources and sinks. Nature, 586(7828), 248–56. https://doi.org/10.1038/s41586-020-2780-0 Watson, J. D. (1968). The double helix: A personal account of the discovery of the structure of DNA. Atheneum. 1. An organism containing a transgene is known as a genetically modified organism (GMO). 2. Not all GE crops are in cultivation today. Only a few are grown in the US. To see the updated information on GE crops, visit the FDA’s website: https://www.fda.gov/food/consumers/agricultural-biotechnology. 3. The animals have additional DNA inside mitochondria; plants have DNA inside chloroplast and mitochondria. These are identified separately as organellar genomes. 4. Have a look at the Tree of Life at http://tolweb.org/tree, and see the Tree of Life segment of BBC One’s “Charles Darwin and the Tree of Life,” narrated by David Attenborough, at https://youtu.be/H6IrUUDboZo. 5. See https://www.fda.gov/food/consumers/agricultural-biotechnology. 6. Glyphosate (N-[phosphonomethyl] glycine) was discovered by Henri Martin in 1950. 7. For current data, visit https://ourworldindata.org/micronutrient-deficiency. 8. The story of golden rice, as told by Ingo Potrykus, is available at http://www.agbioworld.org/biotech-info/topics/goldenrice/tale.html. 9. Learn more about the golden sweet potato at www.worldfoodprize.org/en/laureates/20102019_laureates/andrade_mwanga_low_and_bouisold_template/?nodeID=88375&AudienceID=1&preview=1. 10. Agriculture is the prime source of greenhouse gases, including carbon dioxide (CO2), nitrous oxide (N2O), and methane, which cause the depletion of the stratospheric ozone layer and global warming. N2O has 300 times the warming potential of CO2. Usually, N2O is destroyed in the upper atmosphere by solar radiation. But current human activities (e.g., agriculture and livestock) have significantly increased N2O emissions and caused the perturbation in its natural cycle, resulting in its accumulation in the atmosphere. A comprehensive study by the Global Carbon Project and the International Nitrogen Initiative suggests that agriculture has contributed to a 70 percent rise in the global N2O levels. See the Global Carbon Project website for more details: https://www.globalcarbonproject.org/nitrousoxidebudget. 11. See https://ourworldindata.org/environmental-impacts-of-food.

textbooks/bio/Agriculture_and_Horticulture/History_and_Science_of_Cultivated_Plants_(Naithani)/1.07%3A_Genetically_Engineered_Crops.txt

Learning Objectives • Describe the benefits of interior landscaping. The domestication of wild plants for food crops about 10,000 years ago was a major factor in the development of human civilization. However, art and archaeological evidence from early civilizations in Egypt, China, Iran, Greece, and Rome reveal that plants were also selected and cultivated for ornamental purposes.  Foliage and fruit plants in containers adorned the inner courtyards and rooftop gardens of homes, and flowers were cultivated in hothouses for bouquets and garlands. Figure 1.1 shows an example of a fresco tomb painting of the courtyard garden of a wealthy Egyptian homeowner. Figure 1.1 Tomb painting of an ancient Egyptian garden From the 15th to the late 19th century, European world explorers collected many plants for ornamental interest and enjoyment by the wealthy. Exotic plants from the tropics that were cultivated indoors in northern regions became the forerunners  of modern-day foliage, flower, fern, climber, and succulent house plants. By the mid 19th century, indoor gardening was a popular hobby of the wealthy and the emerging middle class. The impact of the Industrial Revolution on building lighting and heating increased the number of plants that could be grown indoors. Developments in building construction methods and heating and ventilation systems in the early to mid 20th century expanded the use of plants to beautify the indoor environments of offices, hospitals, public spaces, and private homes. In response, the horticultural production and hybridization of foliage and flowering plants increased and landscaping companies specialized in interior landscape design, installation, and maintenance services. In addition to providing visual interest and softening the hard edges of structures with foliage texture, studies have shown that plants can improve indoor air quality. For example, during the energy crisis in the late 20th century, the construction of air tight buildings and use of synthetic materials were intended to reduce energy costs. However, toxic air pollutants such as trichloroethylene, benzene, and formaldehyde given off from paint, plywood, insulation, plastic, carpet and fabrics became concentrated in the air tight spaces and made inhabitants feel sick. The NASA Clean Air study demonstrated that indoor plants purified air by removing and trapping air pollutants in leaves, roots, and soil. Learn more about the study at this link to Indoor Air-Wolverton Environmental [New Tab][1][1] Human civilization has developed in conjunction with nature and the psychological and physical health benefits of integrating vegetation into our habitats is well recognized in the 21st century. Research suggests that for people who spend significant time indoors at work, the presence of plants can improve mood, contribute to contentment, and promote motivation and productivity. Read more about the benefits of indoor plants at this link to Houseplants: to support human health [New Tab][2][2]. The benefits of indoor plants have become part of the design of living and working environments in the 21st century. Interior landscapes or plantscapes have diversified the use of tropical foliage and flowering plants in atria and large conservatories, indoor and vertical gardens, potted office plants and hanging baskets, as well as color bowls, dish gardens, and terrariums. Figure 1.2 shows an example of interior landscaping using vertical gardens vegetated with tropical plants. Figure 1.2 Interior vertical gardens vegetated with tropical plants

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_II_(Nakano)/Part_03_Plants_for_Different_Planting_Situations/01.1%3A_Introduction_to_Interior_Landscaping.txt

Learning Objectives • Recognize plants suitable for common tropical and interior landscape situations. Plant species that are native to regions around the equator are described as tropical. They are adapted to climate conditions with an average temperature of 18οC (64.4οF), no chance of frost, and considerable precipitation at least part of the year. Depending on the latitude, plant species may be adapted to tropical humid (rain forest) or tropical dry (savanna) conditions. Rain forest vegetation is lush with tall trees and thick lianas forming a dense canopy that filters sunlight from the smaller trees, vines, palms, orchids, and ferns growing in the understory. Examples of plant adaptations for high humidity and competition for light include large leaves with waxy surfaces and pointed tips that shed water. More information about interesting plant adaptions for this hot, humid climate is available at this link to The Tropical Rainforest [New Tab][1]. Grasses, shrubs and trees of the tropical savanna are well adapted for climate extremes. Long tap roots, thick fire resistant bark, tree trunks that store water, leaf drop during the dry season, and storage organs like bulbs and corms allow plants to survive an extremely hot, long dry season and a very wet season. Learn about some of these unique plants at this link to The Savanna [New Tab] [2]. Many tropical rain forest plants are available year round in temperate climates for use in interior landscaping. Some common indoor plants include Codiaeum variegatum  var. pictum (croton),  Dieffenbachiaseguine (dumb cane), Dypsis lutescens (Areca palm), and Epipremnum aureum (devil’s ivy, golden pothos). The morphology of these plants can be readily recognized as belonging to particular family groups and related genera share identifiable morphology. It is often possible to use vegetative features alone to identify these family groups, relying on reproductive features only when needed. For example, the square stems and opposite leaf arrangement of Solenostemon x hybridus (coleus), an indoor house plant and outdoor bedding plant, can be recognized as a member of the mint family, Lamiaceae. The morphological characteristics used to identify some plant families and genera commonly used in interior landscapes are summarized below. Access to images of the genera is available at this link to the KPU Plant Database [New Tab][3]. Araceae – arum family Members of the arum family are called aroids. There are over 100 genera and 2500 species distributed on every continent, with the majority in North Africa and Mediterranean regions. These moncots are known as much for their magnificent foliage as for their characteristic inflorescence. In natural habitats, they range from shrubs such as Dieffenbachiaseguine and climbers such as Epipremnum aureum to enormous herbs with corms or tubers. The leaves are mostly spirally arranged and often parallel but sometimes net-veined and either simple or compound. The petiole has a membranous, sheathing base. The roots of all species are adventitious (i.e., they can arise anywhere on the stem) and are without root hairs. Climbing and epiphytic aroids have two kinds of roots, ones that are absorbent and grow downwards into the soil, and clasping roots that grow into crevices, away from light. Individual flowers are tiny, and are borne on specialized inflorescence called a spadix. In the majority of species the spadix is surrounded by a leaf-like bract called a spathe. The spathe is frequently colored and serves as a pollinator attractant. In most cases, aroids are pollinated by flies; the resultant fruits are typically berries. Species grown in interior landscapes will prefer bright, indirect light and moist, well drained fertile soils, and evenly warm temperatures. The ARACEAE family has many familiar shrubs and climbers for indoor containers, including: • Aglaonema (Chinese evergreen) • Anthurium (flamingo flower) • Caladium (elephant ear) • Dieffenbachia (dumb cane) • Epipremnum (devil’s ivy) • Monstera (split-leaf philodendron) • Philodendron (philodendron) • Scindapsus (silver pothos) • Spathiphyllum (peace lily) Arecaceae – palm family The palms comprise a large family (more than 200 genera and 2650 species) of evergreen trees and rattans (climbers) with primarily tropical and warm temperate distribution (few in Africa). Palms such as Dypsis lutescens are immediately recognizable to most people, having spirally arranged, often very large leaves in terminal rosettes. The slender, unbranched stem of the coconut palm (of tropical-island-paradise fame) is typical of many palms, but there are other distinctive shapes and sizes of palms. Palms are usually categorized as either feather palms (pinnate leaves) or as fan palms (palmate leaves), and may be stout or slender, solitary or suckering, and from dwarf to full-size. Flowering is rare in indoor cultivation, except with some smaller species (especially Chamaedorea). Flowers are usually small, yellow, 3-parted and partially embedded in the flower stems. After successful pollination, palms generally produce a rounded, fleshy or fibrous drupe (seldom as large as a coconut nor as succulent as a date). Depending on the species, palms grown in interior landscapes will prefer indirect bright to low light and loose well drained soil with regular fertility. Genera commonly cultivated indoors in atriums and containers include: • Caryota (fishtail palm) • Chamaedorea (parlour palms) • Chamaerops (european fan palm) • Chrysalidocarpus (butterfly palm) • Dypsis lutescens (areca palm) • Howea (Kentia, sentry palms) • Phoenix (date palm) • Ravenea (majestic palm) • Rhapis (lady palm) • Trachycarpus (windmill palm) Euphorbiaceae – spurge family The spurge family is large with more than 300 genera and 7500 species of annual and perennial flowering herbs, shrubs, trees, and some climbers growing in tropical and temperate climates.  Some species are succulent and cactus-like and some are characterized by  milky sap that may be poisonous. Genera are commonly used in both indoor and outdoor landscapes for their colorful bracts and unusual forms. Tender species such as Codiaeum variegatum var. pictum, an ornamental shrub with attractive, multicolored foliage, is commonly used in interior landscaping. Family members usually have simple or sometimes palmately compound leaves that may be sessile or petiolate, often with stipules, and alternately arranged on the stem. Species are frequently monoecious with a raceme or cyme inflorescence and often a radially symmetrical cyathium that is composed of 5 colorful bracts surrounding  the reproductive flower parts. The fruit is usually a capsular schizocarp.  Euphorbs grown indoors will prefer bright light and well drained soil with moderate to low moisture and fertility. Some examples of genera used for interior landscaping indoor include: • Acalypha (chenille plant) • Codiaeum (croton) • Euphorbia (spurge)

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_II_(Nakano)/Part_03_Plants_for_Different_Planting_Situations/01.2%3A_Plants_for_Tropical_and_Interior_Landscaping.txt

Learning Objectives • Recognize plants suitable for common floral landscape situations. Worldwide, the floriculture industry grows an enormous range of tropical species in greenhouses and nursery fields for interior and exterior landscape situations. In addition to improving everyday life, potted flowering plants and cut flowers have significance for the celebration of cultural traditions and  events. Examples of potted plants used in traditional seasonal displays include Euphorbia pulcherrima (poinsettia), Chrysanthemum morifolium (garden mum), and Lilium longiflorum (Easter lily). Events such as weddings, graduations, and funerals are usually celebrated with cut flowers and greens in arrangements, garlands, and bouquets. Among the many types of cut flowers available, the tropical species Alstroemeria cvs. (alstroemeria) and Antirrhinum majus (snapdragon) are frequently used in floral design. Key morphological characteristics that distinguish these plants within their family taxon are summarized  below. View detailed images of the plant examples at this link to the KPU Plant Database [New Tab][1] Alstroemeriaceae – alstroemeria family • Erect herbaceous perennial with tuberous roots • Leaves are alternate and simple with parallel veins, an entire margin, and are resupinate (twisted) • Inflorescence is an umbel • Flowers are zygomorphic (bilateral symmetry), funnel-form, the inner whorl of tepals are spotted • Fruit is a capsule • Example: Alstroemeria cvs. (alstroemeria) for growing conditions of part sun to part shade and well drained fertile soils Plantaginaceae – plantain family • Erect herbaceous perennial • Leaves are alternate, simple, with pinnate veins and an entire margin • Inflorescence is a raceme • Flowers are zygomorphic, tubular and bilabiate (two-lipped) • Fruit is a capsule • Example: Antirrhinum majus (snapdragon) for growing conditions of full sun part sun/part shade and well drained soils While tender tropical members of plant families, such as Solenostemon x hybridus (coleus) in the mint family are typically grown in interior landscapes in northern regions, some are used in exterior containers and bed plantings for summer interest. A few examples of the many tender tropical annual species grown for bedding and container displays are Ageratum houstonianum (floss flower), Celosia argentea (cockscomb), and Cleome hassleriana (spider flower). Examples of tropical herbaceous perennials grown for exterior displays include Lantana camara (lantana), Pelargonium spp. (geranium), and Salvia x superba ‘May Night’ (salvia cultivars). As a result of extensive hybridization, tender herbaceous perennials such as Begonia x semperflorens-cultorum (fibrous begonia), Canna x generalis (canna), Pelargonium x hortorum (zonal or bedding geranium), Solenostemon x hybridus (coleus), and Salvia x superba ‘May Night’ (salvia cultivars) may be described as being of garden origin. Key morphological characteristics that distinguish these plants within their family taxon are described below. Amaranthaceae – amaranth family • Erect herbaceous annual • Leaves are alternate, simple, with pinnate veins, an entire margin, and without stipules • Inflorescence is a spike (dense plume or crested) • Flowers are radial and small, with colorful persistent bracts below each flower • Fruit is a capsule • Example: Celosia argentea (cockscomb) for growing conditions of full sun part sun/part shade and well drained soils Asteraceae – aster family • Compact, mounded herbaceous annual • Leaves are simple and opposite, with pinnate venation, a crenate margin, and hirsute (hairy) blade • Inflorescence is a head (capitulum), arranged in corymbs • Flowers are ligulate (ray flowers) • Fruit is a cypsela • Example: Ageratum houstonianum (floss flower) for growing conditions of full sun part sun/part shade and moist, well drained soils Begoniaceae – begonia family • Upright, mounded annual with succulent tissue and fibrous roots • Leaves are alternate, simple, waxy, with pinnate veins and an asymmetrical base • Inflorescence is a cyme • Flowers are monoecious, single or double blooms • Fruit is a capsule • Example: Begonia x semperflorenscultorum (fibrous begonia) for growing conditions of full sun part sun/part shade and well drained soils Cannaceae – canna family • Erect, unbranched herbaceous perennial, with rhizomes • Leaves are alternate, simple, with a sheathed base and pinnate venation • Inflorescence is a raceme • Flowers are asymmetric, with 3 unequal petals basally fused into a tube, 3 sepals are not fused, the modified fertile stamens are petal-like • Fruit is a capsule • Example: Canna x generalis (canna) for growing conditions of full sun and moist, well drained soils Capparidaceae – caper family • Erect, branched herbaceous annual • Leaves are alternate, palmately compound, with glandular hairs and stipules, pinnate venation, and entire, ciliate margins • Inflorescence is a raceme • Flowers are held on upright pedicels, petals are held above long-exerted stamens • Fruit is a cylindrical capsule held spreading or pendulous on the plant • Example: Cleome hassleriana (spider flower) for growing conditions of full sun and well drained soils Geraniaceae – geranium family • Rounded to spreading tender herbaceous perennials and hybrids, with succulent tissue • Leaves are alternate, simple, orbicular with banded markings, palmate venation, and round or acutely lobed margins, leafy stipules are present • Inflorescence is umbel-like • Flowers are zygomorphic, the upper 2 petals differ in shape and/or size from lower 3 petals, with single, semi-double or double blooms • Fruit is an achene or schizocarp, it is often aborted or absent • Examples: Pelargonium spp., Pelargonium x hortorum for growing conditions of full sun and well drained soils Lamiaceae – mint family • Erect to rounded herbaceous perennials and hybrids with squared stems • Leaves are opposite, simple, with pinnate venation, a crenate margin, and are often aromatic • Inflorescence is a spike-like verticillaster • Flowers are zygomorphic and bilabiate • Fruit is a nutlet • Examples: Solenostemon x hybridus(coleus), and Salvia x superba ‘May Night’ for growing conditions of full sun and well drained soils Verbenaceae – verbena family • Upright, arching perennial shrub, stems with prickles, many annual cultivars • Leaves are simple, opposite, with pinnate venation, a serrate margin, and a rough blade surface • Inflorescence is a compact raceme (umbel-like head) • Flowers are small, tubular to salverform opening in four rounded lobes, with variable coloring • Fruit is a berry, it is often aborted in cultivars • Example: Lantana camara (lantana) for growing conditions of full sun part sun/part shade and well drained soils Practice: Recognize plants suitable for common floral landscape situations. An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plantidentification/?p=329

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_II_(Nakano)/Part_03_Plants_for_Different_Planting_Situations/01.3%3A_Plants_for_Floral_Landscape_Situations.txt

Learning Objectives • Describe plant tolerance for difficult planting conditions. Few landscapes and gardens will contain the perfect planting conditions. Environmental stress from variable combinations of light and moisture levels, exposure to wind and cold, soil characteristics, site slopes and drainage can create difficult situations for planting. Some plants will be better suited to tolerate environmental stress because of morphological and physiological adaptations developed in their native habitat. For example, Berberis buxifolia (box leaf barberry), Gleditsia triacanthos f. inermis (thornless honey locust), and Ginkgo biloba (maidenhair tree, ginkgo) are able to tolerate a fairly wide range of planting conditions. When planting in difficult situations such as the examples described below, select plants from similar habitats that are naturally adapted to grow under the existing conditions. Sunny arid conditions Environmental stress associated with arid (xeric) conditions can severely limit plant growth. Climate characteristics include full light exposure, high summer temperatures, low and unpredictable precipitation, and low humidity with drying winds. Soils with poor structure, minimal organic matter or soil biology and low water holding capacity and nutrient availability are common in arid conditions. Where hardiness is a limiting factor for plant selection, local regional native plants adapted to the existing climate, soils, and moisture regimes are often the most suitable choice. Shallow, extensive root systems allow species such as Rudbeckia fulgida (black-eyed Susan) to survive in drought and poor soil conditions. Plant characteristics such as small, compound, and modified leaves and stems, and light or gray colored leaves with hairy or waxy surfaces reflect sunlight, moderate the temperature at the leaf surface, and reduce water loss. Achillea filipendulina  ‘Gold Plate’ (Gold Plate yarrow), Artemisia schmidtiana (silver mound), Festuca ovina glauca (blue fescue), Rosa rugosa (rugosa rose), and Abies concolor (white fir) are some examples of plants with these characteristics. Read more about plant adaptations at this link to Plant Adaptations to Arid Environments [New Tab][1] Shade Shaded areas that may seem problematic are in fact ideal for plants that occur naturally in habitats with low light, such as woodlands and ravines. There are many shrubs, trees, climbers, bulbs, ferns, and ground cover plants that either tolerate or prefer partial to full shade. For example, evergreen species and cultivars of Rhododendron spp. prefer deep to part shade while Rhododendron Northern Lights Group (azalea) prefers full sun to part shade. Characteristics of shade plants such as branched habits, two-ranked leaf arrangement, and broad, thin leaf blades are suited to capture available light. A strategy of some herbaceous plants, such as Crocus cvs. (crocus) is to emerge early, flower, set seed, and die back to resting structures before tree and shrub leaves fill in completely. Some shade tolerant trees, such as Tsuga heterophylla (western hemlock) and Acer saccharum (sugar maple) will germinate and grow as understory species until openings in the canopy allow them to grow to full size. There is a wide array of ornamental plants suitable for planting in partial to full shade. Examples of ferns are Athyrium niponicum var. pictum (Japanese painted fern), and Matteuccia struthiopteris (ostrich fern). Shrubs for shade include Aucuba japonica (Japanese aucuba), Kalmia latifolia (mountain laurel), Kerria japonica (Japanese kerria), and Leucothoe fontanesiana ‘Rainbow’ (Rainbow leucothoe). Shade tolerant ground covers include Pachysandra terminalis (Japanese spurge) and Sarcococca hookeriana  var. humilis  (dwarf sweet box). The woodland understory tree, Cornus florida (Eastern flowering dogwood, pink flowering dogwood) is adapted to growing in partial shade. Learn more about shade gardening at this link to RHS Shade Gardening [New Tab][2] Dry soil Multiple factors can contribute to dry soil conditions on a site. Soils with high sand or aggregate content that drain quickly move available water below the plant root zone, and surface slopes with rapid runoff reduce water infiltration into the soil. Overhead structures that block rainfall, such as building eaves or tree canopies with competing roots below ground can also create dry areas. While few plants will survive in permanently dry areas, drought tolerant native and garden plants can flourish in dry soil once established. Examples include ground covers, Arctostaphylos uva-ursi (bearberry, kinnikinnick) and Thymus serpyllum (mother of thyme), and herbaceous perennials, Arabis caucasica (rock cress) and Echinops bannaticus (globe thistle). A few examples of adapted deciduous shrubs and trees are Chaenomeles japonica (flowering quince), Crataegus laevigata cvs. (English hawthorn), Pyrus calleryana (ornamental pear) and Quercus robur (English oak). A conifer example, Juniperus virginiana (eastern red cedar) is tolerant of dry soil. Read more about suitable species for dry soil conditions at this link to Drought Tolerant Plants For Your Garden [New Tab][3]. Dry shade A combination of shade and dry soil can create a difficult planting situation. Dry shade is typically found under tree canopies where dense fibrous roots close to the surface compete with other plants for water. While plants will not survive extended periods of drought without some watering, there are some such as Berberis spp. that will tolerate dry shade once they are properly established. Alchemilla mollis (lady’s mantle), Epimedium hybrid cvs. (hybrid barrenwort), and Pachysandra terminalis (Japanese spurge) are suitable herbaceous ground covers for planting in dry shade. Learn about some practical approaches to planting in dry shade at this link to RHS The Garden Dry Shade [New Tab][4] Wetlands Natural wetlands with soil that is permanently or seasonally saturated often have anaerobic (low oxygen) conditions. Wetlands are typically vegetated with hydrophytic plants that are adapted to grow wholly or partially in water. Some hydrophytic species float on the surface of water, while others are completely submerged. Emergent species that root in soil underwater and grow shoots up and out of the water are usually found along the shoreline or margin of a wetland. While the roots of many garden plants would rot when deprived of oxygen, hydrophytic plants are suitable choices for sites with water features as well as low areas with seasonal poor drainage or a high water table. Examples of herbaceous perennials suitable for wetland planting include Acorus gramineus ‘Variegatus’ (variegated sweet flag), and Matteuccia struthiopteris (ostrich fern). Aronia melanocarpa (black chokeberry), and Sambucus nigra (elderberry) are adaptable deciduous shrubs for wet conditions as are the deciduous trees  Liquidambar styraciflua (American sweetgum), and Salix x sepulcralis var. chrysocoma (weeping willow). Depending on the available space, the large conifer Metasequoia glyptostroboides (dawn redwood) may be a suitable choice. Learn more at this link to RHS Gardening on Wet soils [New Tab][5]. Compacted soils Compacted soils are common in urban areas that undergo construction damage, or repeated machinery use and foot traffic. Damage to soil structure from tilling or working heavy clay and loam soils when they are too wet or frozen, and crusting of bare soils from the impact of rainfall contribute to compaction. As soil particles become densely packed together pore space is reduced and the movement of air, water, organisms, and plant roots is impeded. Once compacted, poor soil drainage, water logging, low oxygen, and hard surface conditions inhibit plant root growth. Plants symptoms may include poorly formed or rotted roots, stunted growth, discolored leaves, and drought stress. While the addition of compost is a long term solution for compacted garden soils, there are a number of species that are able to tolerate compacted soils reasonably well. For example, Catalpa speciosa (western catalpa) is a tough tree that tolerates poor soils and compaction as well as dry and wet soils. Acer saccharhinum (silver maple), Juglans nigra (black walnut), and Ulmus americana (American elm) tolerate some compaction as do Amelanchier canadensis (serviceberry), Juniperus communis ‘Green Carpet’ (Green Carpet juniper), and Matteuccia struthiopteris (ostrich fern). Read more about adapted species available at this link to Plants for Compacted soils [New Tab][6] Slopes Sloped embankments and hillsides can be difficult planting situations. Successful plant growth will be influenced by soil type, the north to south aspect, the amount of rainfall, and the degree of incline and length of the slope. Steeper slopes increase the risk of erosion and soil loss that exposes roots or buries small plants. In addition, the run off of sediment from eroded slopes can adversely affect drainage systems and waterways that connect to fish habitat. Planting slopes with grasses and shrubs is an effective way to protect soil and prevent erosion. Fast-growing, adaptable species with dense fine roots that hold the soil together and take up water help stabilize slopes and keep soil in place. Complete vegetation coverage will reduce the impact of rainfall and the potential for soil disturbance and erosion. Methods such as planting pockets and terraced steps will slow surface run off and facilitate the infiltration of irrigation for plant establishment. Plants for slopes typically include native and ornamental grasses and low, spreading shrubs and ground covers that leave no areas of bare soil exposed to the elements. On hot, dry southern aspects, drought-tolerant shrubs and grasses such as Juniperus sabina ‘Tamariscifolia’ (tamarix juniper), Rosa rugosa  (rugosa rose), and Festuca ovina glauca (blue fescue) are suitable options. Cooler, moister northern aspects are better suited for shade-tolerant understory shrubs and ground covers such as Gaultheria shallon (salal), and Pachysandra terminalis (Japanese spurge). Read more about slope gardening at this link to Pacific Horticulture Society Dry Slope Gardening in Seattle [New Tab][7].

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_II_(Nakano)/Part_03_Plants_for_Different_Planting_Situations/01.4%3A_Introduction_to_Plants_for_Difficult_Planting_Situat.txt

Learning Objectives • Recognize plants suitable for planting in difficult situations. Practice: Finish the sentences by selecting the matching growing condition for each plant. Click the images for a larger view. Review the detailed information about each plant available at this link to the KPU Plant Database [New Tab][1]. An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plantidentification/?p=707 An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plantidentification/?p=707 An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plantidentification/?p=707 An interactive or media element has been excluded from this version of the text. 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textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_II_(Nakano)/Part_03_Plants_for_Different_Planting_Situations/01.5%3A_Recognize_Plants_Suitable_for_Planting_in_Difficult_.txt

Learning Objectives • Identify plants for favourable planting situations. Horticulturists understand and use the climate and conditions of a given site to select and grow healthy plants. While growing conditions can be optimized by choosing the right plant for the right place, temperature will remain the least controllable of the environmental factors in exterior landscapes. Given suitable plant hardiness,  average conditions of full to part sun or part shade, and well drained, moist soils with appropriate pH will provide favourable growing conditions for many garden plants. Species that prefer average growing conditions are found in a wide range of plant families. Some familiar plant families with members that thrive in average growing conditions include: • Caryophyllaceae – Arenaria verna (Irish moss) • Cupressaceae – Callitropsis nootkatensis ‘Pendula’ (weeping Nootka false cypress) • Ericaceae – Rhododendron Northern Lights Group (azalea) • Fabaceae – Wisteria sinensis (Chinese wisteria) • Lamiaceae – Callicarpa bodinieri var. giraldii ‘Profusion’ (beautyberry) • Ranunculaceae – Clematis cvs. (clematis) • Rosaceae – Prunuslaurocerasus ‘Otto Luyken’ (Otto Luyken laurel), Prunus serrulata ‘Kwanzan’ (flowering cherry) • Sapindaceae – Acer macrophyllum (bigleaf maple) • Pinaceae – Picea abies ‘Nidiformis’ (nest spruce), Picea abies ‘Pendula’ (weeping Norway spruce), Picea glauca (white spruce), Picea glauca ‘Conica’ (dwarf Alberta spruce), Pinus contorta var. contorta (shore pine), and Pinus nigra (Austrian pine) View the images of plant family members available at this link to the KPU Plant Database [New Tab][1]. In addition to shared morphological patterns in flowers and reproductive structures, family members tend to have similar growth characteristics, nutrient needs, and often the same pests. Key characteristics that distinguish members of some additional plant families are summarized below. Betulaceae – birch family • Deciduous trees and some shrub species • Plant stems are mostly smooth, the genus Betula (birch) has bark peeling in layers • Leaves are alternate in arrangement, simple, with double serrate margin and pinnate venation • Inflorescence is a long pendulous catkin (male flower) and short, cone-like pendulous or erect catkin (female flower) • Fruit is a small, single-seeded indehiscent nut in a short-winged samara • Example: Betula papyrifera (paper birch)[New Tab][3] Brassicaceae – mustard family • Herbaceous perennial, and annual and biennial species • Leaves are alternate in arrangement,  simple and pinnately lobed, without stipules • Inflorescence is a raceme • Flower structure is uniform throughout the family with 4 sepals and 4 petals in a cross-like arrangement (note the historical name ‘Cruciferae’) • Fruit is a silique formed by two valves joined by a thin flat membrane that often persists • Example: Arabis caucasica (rock cress)[New Tab][4] Cannabaceae – hop family • Herbaceous perennial, climbing vine, a dioecious plant • Leaves are opposite in arrangement, simple and three-lobed with a serrate margin and prominent venation • Inflorescence is a catkin (male) and cone-like spike (female) • Flowers are small, without petals (wind pollinated), with aromatic glands at the base • Fruit is an achene subtended by a papery floral bract • Example: Humulus lupulus (common hop)[New Tab][5] Caprifoliaceae – honeysuckle family • Deciduous and broadleaf evergreen shrubs, twining lianas, and some herbaceous perennials • Stems have pith inside • Leaves are opposite in arrangement, often simple • Inflorescence a panicle-like corymb in the example. Solitary flowers occur in pairs or in cymes, spikes and racemes in some species • Flowers are tubular, funnel-shaped, or bell-like often with five outward spreading lobes or points • Fruit is a capsule or berry in pairs • Example: Kolkwitzia amabilis (beautybush)[New Tab][6] Cornaceae – dogwood family • Mostly trees and shrubs, rarely rhizomatous perennial herbs • Leaves are opposite in arrangement, simple with undivided, entire margins, and 6-7 pairs of veins • Flower buds are flattened and globose, vegetative buds are narrow and conical • Inflorescence is a dense head of inconspicuous true flowers surrounded by 4-8 showy bracts • Fruit is a drupe, may be multiple in some species • Examples: Hamamelidaceae – witch hazel family • Deciduous shrubs and trees • Leaves are alternate in arrangement, simple with prominent pinnate venation, serrate margins, and a pubescent surface • Inflorescence are clusters of 4-parted cross-shaped florets with small triangular sepals and thin, ribbon-like petals • Fruit is a woody capsule • Example: Hamamelis mollis (Chinese witch hazel)[New Tab][10] Hydrangeaceae – hydrangea family • Deciduous shrubs • Leaves are opposite in arrangement, whorled in some species, simple, with netted venation and serrate to toothed margins • Inflorescence a raceme in the genus Deutzia • Flowers are rotate with 5 separate petals • Fruit is a capsule • Example: Deutzia gracilis (slender deutzia)[New Tab][11] Iridaceae – iris family • Herbaceous perennial monocot from a bulb, other species from bulbs, corms and rhizomes • Leaves are typically basal, sheathing and linear with parallel veins and entire margins • Inflorescence a solitary, 3-parted flower other species, may be a raceme or spike • Fruit is a capsule • Example: Crocus cvs. (Dutch crocus, crocus)[New Tab][12] Magnoliaceae – magnolia family • Deciduous tree, other species are evergreen trees and shrubs • Leaves are alternate in arrangement, simple, with an entire margin • Inflorescence is a solitary flower • Flowers have tepals, with stamens and pistils on a conical receptacle • Fruit is an aggregate of woody follicles • Example: Magnolia stellata (star magnolia)[New Tab][13] Rutaceae – rue or citrus family • Mostly trees and some shrubs • Leaves are opposite in arrangement and trifoliate compound in the genus Choisya. Leaves are alternate and simple in the genus Citrus. Foliage is aromatic, the leaf blade dotted with glands. • Inflorescence a cyme • Flower is rotate with 5 petals, fragrant • Fruit is a capsule • Example: Choisya ternata (Mexican mock orange)[New Tab][14] Theaceae – tea family • Mostly broadleaf evergreen shrubs and trees, few deciduous. • Leaves are alternate and spiral in arrangement, simple, usually glossy, with serrate margin and a gland (hyathode) that excretes water at serration tips • Inflorescence is solitary flower • Flower is radially symmetric, rotate with 11+ petals • Fruit is a capsule • Example: Camellia japonica (common camellia, Japanese camellia)[New Tab][15] Tiliaceae – basswood or linden family • Deciduous tree, some species are shrubs • Leaves are alternate in arrangement, simple with a pubescent surface and hair tufts in vein axils, a serrate margin and a heart-shaped, asymmetrical base • Buds are alternate, oval in shape with 2 scales. • Inflorescence is a cyme with an elongated yellow-green bract • Flowers are small 5-parted, highly scented • Fruit is a nut-like drupe • Example: Tilia cordata (little leaf linden)[New Tab][16] Practice: Identify plants for favourable planting situations. An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plantidentification/?p=752

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_II_(Nakano)/Part_03_Plants_for_Different_Planting_Situations/01.6%3A_Plants_for_Favorable_Planting_Situations.txt

Learning Objectives • Describe native plants common to the horticulture industry. Biodiversity is described as the variety of plants and other living organisms that interact with the non-living environment of a particular habitat or ecosystem. Regardless of the size or type, each organism is dependent on every other, either directly or indirectly through food webs and the natural processes of nutrient cycling and energy flow that sustain ecosystems. Plant biodiversity has an invaluable role in the function of ecosystems and the services that people obtain from them including: • provision of clean air, water, food, materials, and medicines, • regulation of climate, carbon storage, water and waste treatment, and erosion and disease control, • support for pollination, biodiversity and habitat, and • cultural benefits for health, education, recreation, relaxation, and spiritual well being. Read more about the importance of ecosystem services at this link to The Economics of Ecosystems & Biodiversity [New Tab][1] Regardless of whether a plant occurs naturally in a place or has been planted indoors or outdoors for ornament or food value, as a species, it has a native home somewhere in the world. Native plant species originated and co-evolved in communities with other organisms in fourteen identified biomes around the world. Biomes are composed of groups of ecosystems with distinct vegetation types and climate patterns. They are typically named for the dominant vegetation type, such as a forest or grassland. Figure 7.1 shows a map of the distribution of the major biomes around the world. Figure 7.1 Map and legend showing locations and types of global biomes. With an understanding of plant biology, hardiness, and interactions with soils and climate, native species from different biomes can be successfully grown in landscapes and gardens around the world. For example, Gunnera manicata (gunnera) from South America and Impatiens walleriana (impatiens) from South Africa originated in the Tropical, Subtropical Broadleaf Forest biome are often grown as ornamental garden plants. The magnificent specimen tree, Cedrus deodara (Deodar cedar) is native to the Tropical, Subtropical Conifer Forest while hedging plants Thuja occidentalis (white cedar) and Taxus cuspidata ‘Capitata’ (upright yew) originated in the Temperate Conifer Forest biome. Many species of the Temperate Broadleaf Mixed Forest biome are commonly grown in landscapes and gardens. Examples of deciduous specimen trees include Acer tataricum ssp. ginnala (Amur maple, Tatarian maple) and Syringa reticulata ‘Ivory Silk’ (Japanese lilac tree) from Eastern Asia, Cercis canadensis (redbud) and Quercus alba (white oak) from Eastern North America, and the European species Prunus padus var. commutata (European bird cherry), and Sorbus aucuparia (European mountain ash). Viburnum trilobum (highbush cranberry) is an understory shrub from northern North America while the closely related Viburnum opulus (European snowball) is native to Europe and Asia. Some familiar garden plants that originated in the Mediterranean Forest, Woodlands, Scrub biome are Cyclamen persicum (cyclamen), Helictotrichon sempervirens (blue oat grass), Lithodora diffusa ‘Grace Ward’ (blue lithospermum), and Rosmarinus officinalis (rosemary). View images of the plant examples available at this link to the KPU Plant Database [New Tab][2]. Now, complete the following practice exercise. Practice: Recognize plants native to world biomes. An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plantidentification/?p=896 Individual ecosystems within larger biomes are characterized by the naturally occurring communities of native plants and animals and the distinct soil types, land forms, and climate of the area. For example, a plant community of the Temperate Conifer Forest biome located in the Pacific Northwest region of North America would include conifers such as  Pinus contorta var. contorta (shore pine), Pseudostuga menziesii (Douglas Fir), Thuja plicata (western red cedar), and Tsuga heterophylla (western hemlock). Depending on the specific site conditions, deciduous and broadleaf evergreen trees may include Alnus rubra (red alder), Arbutus menziesii (arbutus, madrona), Frangula purshiana (cascara), and Populus trichocarpa (black cottonwood, western balsam-poplar). Associated deciduous shrubs may include Ribes sanguineum (flowering currant, winter currant), Rubus spectabilis (salmonberry), Salix discolor (native pussy willow, pussy willow), and Symphoricarpos albus (snowberry). Some deciduous and evergreen native ferns are Adiantum pedatum (maidenhair fern) and Polystichum munitum (western sword fern). A few examples of the many native herbaceous flowering plants are Asarum caudatum (western wild ginger), Erythronium americanum (trout lily, adder’s tongue), Vancouveria hexandra (inside-out flower), and the semiaquatic Sagittaria latifolia (wapato, arrowhead, duck potato). Read more about plant communities and their distinct ecosystems at this link to Vegetation Regions, The Canadian Encyclopedia [New Tab][3] Historically, horticultural activities have had a significant impact on the geographic distribution of native plant species. Currently, landscape contractors, nurseries, garden centres, and mass-market chain stores are the largest distribution channels of native plants in addition to ornamental and floriculture products. The import and export of species for use in arboriculture, landscape horticulture, floriculture, turf, and food production sectors continues to shape the distribution of species in plant communities and ecosystems. While constructed landscapes and gardens may be considered artificial ecosystems, it is possible to plan and maintain communities of local native species and appropriate ornamental plants that support natural processes and ecosystem services. Practice: Match the images of plants native to the Pacific Northwest. An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plantidentification/?p=896

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_II_(Nakano)/Part_04_Plants_for_Horticultural_Applications/02.1%3A_Native_Plants.txt

Learning Objectives • Describe seasonal plants common to the horticulture industry. Planning combinations of woody and herbaceous plants with different life cycles and high visual impact generates year round interest in exterior and interior plantings. When visual interest is planned for one period such as early summer, borders and containers can have a poor appearance the rest of the year. Optimizing the use of grasses, bulbs, perennials, annuals, biennials, shrubs, climbers, and trees can provide a succession of plant forms, colours, textures, and habits throughout the seasons. In temperate regions, year round interest is maximized by selecting plants with at least two, and even three or four seasons of interest. Conifers and broadleaf evergreens shrubs are often used for year round colour and spatial structure. For example, Taxus cuspidata ‘Capitata’ (upright yew) provides reliable winter colour and a framework that can be enhanced with other shapes, textures, and colours. On the other hand, a planting of broadleaf evergreens such as Skimmia japonica (Japanese skimmia) offers winter colour and structure as well as showy spring flowers and colourful fruit in the autumn. Distinctive plant shapes and the bark of trees such as Cryptomeria japonica (Japanese cedar) and Morus alba ‘Pendula’ or species with persistent fruit like Sorbus aucuparia (European mountain ash) also contribute structure and winter interest. Practice: Recognize woody plants for winter interest. A link to an interactive elements can be found at the bottom of this page. Some deciduous shrubs and trees like Caryopteris x clandonensis (bluebeard), Cercidiphyllum japonicum (katsura), and Rhus typhina (staghorn sumac) have interesting branching patterns throughout all seasons. The bark and buds of Ribes sanguineum (flowering currant, winter currant), Magnolia x soulangeana (saucer magnolia), Liriodendron tulipifera, and Styrax japonicus (Japanese snowbell, Japanese snowcone) provide winter interest and interesting buds forecast the appearance of foliage and flowers. Climbers with variegated or textured foliage and colourful flowers like Actinidia kolomikta (actinitdia) and Campsis radicans (trumpet vine) also contribute vertical structure. View the images seasonal plant characteristics available at this link to the KPU Plant Database [New Tab][1] The appearance of plants before, during, and after flowering is an important consideration for planning seasonal interest. For example, the herbaceous specimen plant Gunnera manicata (gunnera, giant rhubarb) provides a bold shape and texture for at least half to perhaps three quarters of the year. With planning, the eye catching winter stems and seed heads of grasses and perennial species such as Pennisetum alopecuroides (fountain grass), Pennisetum setaceum ‘Rubrum’ (red fountain grass), and Perovskia atriplicifolia (Russian sage) can serve as distractions from seasonal voids. Layering various heights of ground covers, bulbs, annuals and perennials under and around woody shrubs and trees allows a succession of foliage shapes, sizes, textures, and colours to become prominent as the year progresses. In this way, emphasis is placed on year round interest and not only the seasonal show of flowers. A planting calendar is a useful tool for working out the succession of flowers and colour palettes as well as other planting design features. Figure 8.1 shows an example of a basic planting calendar that allows the planner to visualize the times of the year that are most colourful and interesting and those that could use additional development. Figure 8.1 Sample planting calendar As the succession of spring bulbs like Anemone blanda (Greek windflower, blue wood) and Hyacinthus cvs. (hyacinth) finish flowering and foliage fades, deciduous shrubs such as Spiraea x vanhouttei (bridal wreath spirea) and an array of herbaceous annuals, biennials and perennials come into flower in early and mid spring. Examples of spring blooming perennials include Aubrieta x cultorum (common rock cress), Brunnera macrophylla (Siberian bugloss), Papaver orientale (oriental poppy), Pulmonaria saccharata (lungwort), and Dicentra spectabilis (bleeding heart). From late spring and early to mid summer, the flowers and foliage of broadleaf evergreen shrubs such as Daphne cneorum (garland daphne) and herbaceous species like Thymus pseudolanuginosus (woolly thyme), Heuchera cvs. (coralbells, alumroot), and Phlox paniculata (common phlox) take prominence. The progression of seasonal foliage and bloom continues in mid to late summer and through autumn with perennials such as Actaea simplex Atropurpurea Group (cimicifuga), Aster spp. (common aster), Astrantia major (masterwort, astrantia), Coreopsis spp. & cvs. ( coreopsis), Geranium spp. & cvs. (geranium), and Gaillardia cvs. (blanket flower). The texture and seed heads of perennials like Hylotelephium spectabile (autumn joy sedum, stonecrop), and grasses such as Andropogon gerardii (big bluestem), Calamagrostis x acutiflora (feather reed grass), and Molinia arundinacea ‘Skyracer’ (tall moor grass) extend the visual interest from late autumn into winter. Year round interest is fulfilled by evergreens and the flowers of winter blooming shrubs and perennials. View images of the seasonal plant characteristics available at this link to the KPU Plant Database [New Tab][2]. Read more about seasonal plant combinations at this link to Gardenia Seasonal Garden Ideas [New Tab][3]. Practice: Recognize plants for seasonal interest. A link to an interactive elements can be found at the bottom of this page.

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_II_(Nakano)/Part_04_Plants_for_Horticultural_Applications/02.2%3A_Seasonal_Plants.txt

Learning Objectives • Describe plants suitable for green infrastructure projects. The basic infrastructure that supplies a plant with water and food is made up of roots, stems, and leaves. By comparison, an infrastructure that supplies a community with drinking water is made up of a network of parts that include wells, reservoirs, water mains and smaller pipes. Other familiar examples of infrastructure found in communities are transportation, communications, and electrical networks. These constructed networks are often called grey infrastructure. In nature, networks of rivers, streams, lakes, and oceans make up a natural infrastructure that supports the function of ecosystems and the plants and animals that live there. Where human activities lead to the loss of ecosystems and biodiversity, green infrastructure can be planned and managed to conserve ecosystem services and reduce negative environmental impacts. Green infrastructure is made up of vegetation, soils, and bioengineered technologies that provide communities with a wide range of environmental, social, and economic benefits. When connected in a larger framework of natural and urban forests, habitats, streams and rivers, constructed wetlands and floodplains, as well as parks, residential yards, edible landscapes, community gardens, green roofs, green walls, bioswales, and rain gardens, a green infrastructure network is created. Traditional stormwater infrastructure that collects, drains and discharges water from sites as quickly as possible can increase the potential for flash flooding, pollution, and scouring damage downstream. Sealed surfaces like rooftops, parking lots, and roads accelerate surface water runoff and prevent infiltration into soil for cleansing, groundwater recharge, and plant use. In contrast, green infrastructure for stormwater management mimics natural landscapes that intercept, retain, absorb, filter, and slowly release stormwater by evapotranspiration and controlled runoff. Combinations of green infrastructure components such as green roofs, green walls, bioswales, rain gardens, and permeable paving reduce the quantity and improve the quality of stormwater before its release from a site. Read more about the potential benefits of green infrastructure available at this link to Introduction to Green Infrastructure[PDF][New Tab][1] Green infrastructure is designed to optimize the beneficial services provided by plants. Plants and their processes of photosynthesis, water uptake, and respiration contribute to: • oxygen production and carbon sequestration, • pollution removal from air, soil, and water, • flood control, and groundwater and stormwater management, • surface shading and cooling of air temperature by evapotranspiration, and • wildlife and pollinator habitat, and green space for human well being. Plants for green infrastructure projects are often locally available native species, but not always. Both native and non-native plants are used for the ecosystems services a species or plant community provides. Plants suitable for the growing conditions, function, appearance, and maintenance levels associated with green roofs, green walls, bioswales and rain gardens will be selected within the constraints of a particular project. Plants for green roofs Green roofs that are partly or completely covered with vegetation and growing media provide many ecosystem services in urban settings. Services include reducing the volume of rainfall runoff through plant uptake, providing wildlife habitat and green space, and reducing the urban heat island effect through shading and plant evapotranspiration. In addition, the insulating properties of vegetation and growing media dampen noise levels, reduce the heating and cooling costs in buildings, and extend the life of roofing materials. Read more about the benefits of green roofs at this link to Green Roofs for Healthy Cities, About Green Roofs [New Tab][2] Green roofs are categorized as either intensive or extensive depending on the depth of growing media. Intensive green roof systems with growing media depths greater than 150 mm (6”) can support many plant types including ground covers, herbaceous species, shrubs, trees, and climbers. The high structural loading capacity of intensive green roofs also allows for access to amenities like paths, patios, and water features. Like a traditional garden on a roof, intensive plantings have high requirements for maintenance and inputs. In contrast, extensive green roofs with light weight growing media less than 150  mm (6″) in depth support plants with shallow roots and low requirements for maintenance and inputs. Extensive green roofs provide habitat for wildlife but their lower structural loading capacity may restrict human access to maintenance visits. Figure 9.1 shows an example of an extensive green roof that provides visual access to green space in an urban setting. Figure 9.1 Example of an extensive green roof Almost any plant type can be grown on a green roof however, the shallow depth and low organic content of extensive green roof growing media will be the limiting factor for plant selection. In general, suitable species are determined by examining the microclimate of the green roof and comparing it to a species’ native habitat. Extensive green roof features that influence plant selection will include water availability, wind speeds, soil depths and temperatures, as well as solar exposure and climate. Plant growth characteristics for extensive green roofs include fast establishment, long lived with dense coverage, pest and disease resistance, shallow rooting, self-regeneration from seed and vegetative parts, tolerance for extreme weather and very dry to saturated conditions, and low requirements for maintenance and inputs. Examples of native habitats that match the extreme conditions found on extensive green roofs include: • dry grasslands, cliffs and coasts, • arid mountain ranges, • steppe, heath, and alpine communities, • sandy, talus, and cliff communities, and • wastelands, gravel and sand pits, rocky outcrops, other hard surfaces. Plants suitable for extensive green roofs may include succulents, bulbs and corms, annual or biennial self seeders, bunch and stoloniferous grass-like plants, and some wetland and perennial herbs. Succulents, in particular Sedum cvs. (stonecrop) have been extensively used because they are well adapted for growing in the extensive green roof microclimate. Learn more about the interesting characteristics of the genus Sedum at this link to Living Architecture Monitor, Sedum: The Workhorse of Green Roofs Plants [New Tab][3]. In addition to planting sedums, diversified communities may include adapted species such as Aster spp. (common aster), Campanula carpatica (Carpathian harebell, canterbury bells), Heuchera cvs. (coral bells, alumroot), Penstemon cvs. (beardtongue), Phlox subulata (creeping phlox), as well as sedges and grasses like Andropogon gerardii (big bluestem), and Panicum virgatum (switch grass). Practice: Match the images of plants suitable for green roofs. An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plantidentification/?p=1227 Plants for green walls Green walls composed of vertical systems of vegetation, growing media, irrigation, and drainage are increasingly used in exterior and interior landscapes for their aesthetic and environmental benefits. The shading, water management, screening, buffering, and insulating properties of green walls can reduce air temperature, noise levels, and the energy costs for cooling buildings. The three major categories of green walls are green facades, living walls, and retaining living walls. Green facades of cable systems, trellises, arbors, and fences offset from a building face support the growth of vines and lianas or cascading plants that are rooted in ground level or above ground planters. Living wall systems of vegetated modules, panels, or bags containing growing media that are freestanding or attached to structural walls or frames support shallow fibrous rooted and creeping herbaceous and woody plants. Retaining living wall systems that are designed to stabilize slopes incorporate vegetation within interlocking geotextile fabric bags, mats, precast concrete units, or in woven wattles of Salix discolor (native pussy willow, pussy willow). Figure 9.2 shows an example of a living green wall on a building. Read more about the properties and benefits of green walls at this link to Green Roofs for Healthy Cities, About Green Walls [New Tab][4]. Figure 9.2 Example of a living green wall Similar to green roofs, green walls have unique growing conditions that will influence plant selection. Factors for consideration include an indoor or outdoor climate, specialized soil requirements and wall orientation, the green wall design and level of maintenance required. Where green walls are not connected to groundwater, irrigation and intensive maintenance are necessary to ensure appropriate appearance and function. In situations where wall height and desiccation by wind and lack of shade limit plant growth, species adapted to cliff-faces, extreme slopes and thin soil habitats offer suitable choices. Depending on the type of green wall, suitable plants may range from annuals to herbaceous and woody perennials. Some examples of suitable species are  Heuchera cvs. (coral bells, alumroot), Penstemon cvs. (beard tongue), Cotoneaster apiculatus (cranberry cotoneaster), Fragaria x ananassa (garden strawberry), and Gaultheria procumbens (wintergreen), as well as succulents and tropical species. Depending on the need for seasonal shading on buildings, green facades may include evergreen or deciduous climbers such as Actinidia kolomikta (actinidia) and Campsis radicans (trumpet vine). Practice: Recognize plants suitable for green walls. Move cursor over images for plant names. An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plantidentification/?p=1227 Plants for bioswales and rain gardens Green infrastructure uses the process of bioretention to manage stormwater quantity and quality. Bioretention structures such as bioswales and rain gardens are designed to capture, detain, convey, infiltrate, and evaporate water from a planting. Vegetated bioswales are broad shallow straight or meandering channels with porous soil and gently sloped sides and bottom that collect and convey stormwater from one location to another while maximizing soil infiltration and plant uptake. Bioswales are designed to manage short intense periods of rain and flooding followed by dry periods. They reduce the impact of stormwater events and capture the first flush of pollutants from paved and sealed surfaces for remediation by plants and soil microorganisms. Figure 9.3 shows an example of how a vegetated bioswale captures and conveys stormwater runoff from the sealed pavement as well as the turf area. Figure 9.3 Example of a vegetated bioswale Rain gardens are shallow infiltration basins located in depressions and low lying areas that capture and temporarily retain water for infiltration and groundwater recharge. Porous soil filters pollutants and allows for uptake and transpiration by plants to reduce air and water temperatures. Figure 9.4 shows an example of a rain garden. Note that the roof downspout enters the rain garden, and when combined with porous pavement, the site becomes an absorbent landscape. Figure 9.4 Example of a rain garden Learn more about the components and characteristics of green infrastructure for stormwater management available at this link to Capital Regional District, Green Stormwater Infrastructure [New Tab][5]. In addition to specific growing conditions and appearance expectations, plants for green stormwater infrastructure projects must fulfill basic functional requirements that include: • tolerance and resilience to flooding, sediment events, drought, and wilting, • extensive and deep root structure for resistance to heavy water flows, • dense foliage and spreading growth that prevents erosion and increases evapotranspiration, • reliable, vigorous growth without becoming invasive, and • an ability to tolerate and accumulate contaminants from water or saturated soil. Native and ornamental species are usually planted according to their tolerance for the wetter bottom or drier side and upper edges of bioswales and rain gardens. Where space and soil volume permit, planting trees and large shrubs such as Alnus rubra (red alder), Frangula purshiana (cascara), and Salix discolor (native willow, pussy willow) will prevent erosion and transpire great amounts of water. Examples of shrubs for bioswales and raingardens include Clethra alnifolia (summersweet), Ribes sanguineum (flowering currant, winter currant), Rubus spectabilis (salmonberry), and Symphoricarpos albus (snowberry). Where space for trees and shrubs is limited, planting multiple layers of herbaceous vegetation will increase the foliage density and the benefits of transpiration. Some examples of adapted herbaceous species include Aconitum napellus (monkshood), Aster spp. (common aster), Carex oshimensis ‘Evergold’ (Evergold Japanese sedge), Lysimachia clethroides (gooseneck lysimachia), Panicum virgatum (switchgrass), and Pennisetum alopecuroides (fountain grass). Practice: Recognize plants suitable for bioswales and rain gardens. An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plantidentification/?p=1227 In order for green infrastructure to provide ecosystem services as designed, maintenance practices must ensure appropriate vegetation cover for proper function and aesthetic performance. Improper maintenance can defeat the purpose of green infrastructure and lead to costly replacement or restoration. Routine maintenance involves inspection and repair for erosion, appearance, and removal of sediment and potential invasive species. Regular soil testing, weeding, and trash removal will influence the long term efficiency and effectiveness of pollutant removal and stormwater management by vegetated bioretention structures.

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_II_(Nakano)/Part_04_Plants_for_Horticultural_Applications/02.3%3A_Plants_for_Green_Infrastructure_Projects.txt

Learning Objectives • Describe plants suitable for edible landscapes. As part of green infrastructure, gardening for food production offers a wide range of environmental, economic, and social benefits. Growing local food within and around communities supports: • habitat for pollinators and biodiversity, • regulation of local climate and water management, • reduction of energy use and carbon footprints, • food security and local economy, and • physical health and social connections. Urban agriculture is the process of growing, processing, and distributing local food and food products. There are many types of urban agriculture including community gardens, boulevard planting, green roofs, vertical farms, urban chickens, bee keeping, aquaculture, and small scale faming for farmers markets. Some other forms of food production are edible landscapes, food forests, urban orchards, gleaning (public land harvest), grow a row donations and backyard sharing, and guerrilla gardening. Figure 10.1 shows an example of products of urban agriculture available for purchase at a farmers market. Read more about the benefits and different types of urban agriculture at this link to The Urban Farmer [New Tab][1] Figure 10.1 Example of urban agriculture products for sale at a farmers market stall Communities plan and manage urban agriculture through policies, zoning bylaws, and land use regulations that allow certain public green spaces to be used for growing food. For example, community gardens for non-commercial food production that are allowed in some or all land use designations will have guidelines for safety, accessibility, maintenance, and aesthetics. Read about an example of jurisdictional policy and regulations for community gardens available at this link to City of Victoria Community Gardens Policy[PDF][New Tab].[2] Food production in residential landscapes is commonly associated with vegetable plots in backyards. Annual species grown for produce are usually arranged in agricultural patterns of straight lines in designated areas. Soil is often amended with compost, heavily irrigated, and seasonally tilled over for new planting. In contrast, edible landscapes, sometimes called foodscapes, incorporate plants for food as well as ornamental value within existing and new residential and public landscape designs. In general, plants for edible landscapes are herbaceous and woody perennial species that: • are adapted for the climate and naturally resistant to pest and disease, • require less intensive or similar levels of maintenance and inputs as the rest of the planting area, and • provide multiple benefits such as food, aesthetics, shading, and water management. Plants selected for preferred foods and the attributes of form, texture, and colour are integrated with other ornamental plants to achieve a desired garden style and aesthetic appearance. For example, the fruit producing tree, Morus alba ‘Pendula’ (weeping mulberry) serves as a specimen plant with distinctive form. Shrubs with berries and vibrant autumn foliage colour like Vaccinium corymbosum (highbush blueberry) may be planted as hedging. Edible spreaders like Fragaria x ananassa (garden strawberry) and Gaultheria procumbens (wintergreen) provide ground cover while vegetables with fine texture foliage like Daucus carota ssp. sativus (carrot) contrast coarse texture plants like Rheum palmatum (rhubarb). Aromatic herbs such as Origanum laevigatum ‘Herrenhausen’ and Rosmarinus officinalis (rosemary) provide structure, scent, and visual interest alongside edible flowers like Impatiens walleriana (impatiens) and Phlox paniculata (summer phlox, border phlox). Learn more information about the origins, benefits, maintenance, and types of plants for edible landscapes available at this link to Foodscaping-Wikipedia [New Tab][3] Practice: Recognize plants for edible landscapes. An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plantidentification/?p=1443 An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plantidentification/?p=1443 An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plantidentification/?p=1443 An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plantidentification/?p=1443 An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plantidentification/?p=1443 An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plantidentification/?p=1443 Techniques that maximize space use and vegetation cover such as interplanting larger, slow growing food plants with smaller, fast growing plants reduce soil erosion and suppress opportunistic weeds. Combining plants with different heights and structure, nutrient requirements, and rooting depths creates growing microclimates and reduces plant competition for soil nutrients. Certain companion plants such as members of the Fabaceae (pea) family that fix atmospheric nitrogen in available forms in root nodules can benefit nearby nitrogen feeders like leafy vegetables. Aromatic herbs can be used to repel pests attracted to other species by smell, and the deliberate planting of host plants distract pests from other plants and attract beneficial insects and predators that feed on pests. Read more information about the benefits of companion planting available at this link to Companion planting – Wikipedia [New Tab][4]. Edible landscapes that are intended to provide food products for human consumption are distinguished from planted habitats that are intended to attract wildlife. As areas of natural ecosystems are converted to residential, agricultural, industrial, and other uses, the loss of habitat negatively impacts native wildlife. However, where fragments or patches of habitat are not too small and are close together, they can be connected by corridors of vegetation that allow native species to access adequate food, water, shelter and protection. Planting regional native plants that mimic the habitat characteristics of the desired wildlife species in landscapes and gardens can provide the particular needs for food, water, shelter and protection. Creating connections between native and ornamental vegetation and water sources in urban forests, parks, gardens, boulevards, and other plantings allows wildlife to move safely among habitat patches in urban areas. For example, evergreen trees like Cryptomeria japonica (Japanese cedar) with branches close to the ground and deciduous trees with open canopies and multiple branches such as Frangula purshiana (cascara), and Prunus padus var. commutata (European bird cherry) offer shelter and protection, as well as nesting sites and food. Interplanting layers of shrubs like Ribes alpinum (alpine currant), Ribes sanguineum (flowering currant, winter currant), and Rubus spectabilis (salmonberry) with herbaceous species like Andropogon gerardii (big bluestem), Asarum caudatum (western wild ginger), and Polystichum munitum (western sword fern) provides a range of wildlife species with food, shelter, and protection. Review images of plant examples at this link to KPU Plant Database [New Tab][5]w Tab]. Learn more about gardening for wildlife habitat available at this link to Fraser Valley Conservancy Native Plants Guide[PDF][New Tab][6] Habitat loss and invasive species are major threats to wildlife habitats, particularly in wetlands and forests. Selecting ornamental plants for habitat planting includes examining the potential for species to escape, establish, and overtake natural ecosystems. Non-invasive ornamentals and regional native plants are the responsible alternative to invasive plants. For example, an introduced horticultural plant that has become invasive in wetlands is Butomus umbellatus (flowering rush). Alternate choices for this plant include the native species Scirpus microcarpus (small-flowered bulrush), Carex spp. (sedges), and Sagittaria latifolia (wapato, arrowhead).  Alternate choices for another invasive, Euphorbia esula (green spurge, leafy spurge) include species in the genera Delosperma (ice plant) and Helianthemum (rock rose). Species in the genera Salvia (sage), and Penstemon (beardtongue) provide alternate choices for the invasive species, Echium vulgare (blueweed). Another invasive species, Linaria vulgaris (toadflax) can be replaced with selections from the genera Penstemon (beardtongue), Hemerocallis (daylily), Antirrhinum (snapdragon), and Kniphofia (torch lily). Learn more about the threat of invasive horticultural plants and alternative plant choices at this link to Invasive Species Council of BC Grow Me Instead[PDF][New Tab][7] Practice: Name the invasive species. Move the cursor over the image to check your response. An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plantidentification/?p=1443

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_II_(Nakano)/Part_04_Plants_for_Horticultural_Applications/02.4%3A_Plants_for_Edible_Landscapes.txt

Learning Objective • Describe the development of plant identification. All living organisms on earth rely on the process of photosynthesis for food energy and oxygen. Humans depend almost entirely on plants for clean air and a livable climate as well as for food, medicines, materials, and well being. Around the world, groups of people with their own distinct history, culture, and society have learned to identify plants and their properties. For Indigenous peoples, the accumulated traditional knowledge of plants has allowed them to thrive in diverse environments for thousands of years. Traditional knowledge passed among generations through the oral traditions of hunter-gatherers influenced the naming and grouping of plants. With the settlement of agricultural communities and the domestication of plants about 10,000 years ago, written records documented their use. Early systems of plant classification emerged in Eastern and Ancient Egyptian cultures and botany, the scientific study of plants developed in Ancient Greece. Taxonomy, a branch of botany, is defined as the systematic classification, naming, and identification of plants. This orderly system arranges related plants with similar characteristics into groups called taxa and uses a convention called bionomial nomenclature to give a unique name to one group of plants. In addition to classification by morphology (external form) and anatomy (internal structures), botanists now use genetic sequencing and biochemistry to decode the evolutionary history of relationships among plants. As a discipline, taxonomy has continued to develop over centuries of botanical study and this knowledge is available to students of plant identification. The ability to identify plants and their requirements has always been an essential skill for horticulturists who manage plant growth and health. However, with more than 300,000 known species in the world, the plant kingdom – Plantae, is both diverse and complex. No two species of plants will be exactly alike, and while some common characteristics may be easily seen, others are so different that few if any relationships can be observed. Furthermore, the progression of evolution never stops and relationships among plant groups continue to change over time. To address this challenge, this book introduces students of plant identification to a systematic approach to classifying, naming, and identifying unknown species. Review An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plant-identification/?p=158 1.02: Introduction to Taxonomy Learning Objectives • Describe the scientific system of plant classification and naming. A working knowledge of taxonomy is useful for classifying, naming, and identifying unknown plants. Theophrastus (370-285 BC), a Greek philosopher, first used taxonomy to describe and group plants according to their morphology (shape), growth, and reproductive traits. In the 18th century, a scientist named Carl Linnaeus applied binomials (two-term names) and classified known plants into a hierarchical system of classification. Classification and Naming The most effective classification systems are hierarchical and comprised of a nested series of categories or ranks. A good analogy is a computer filing system. Certain kinds of information reside at each level (drive, library, directory or folder, sub-directory, document, etc.), with file names (or labels) that signify the sort of information found there. Every level in the hierarchy is more inclusive than the one below it and the more of the filing system that is investigated, the more related information is uncovered. Similarly, the categories used in plant classification provide an organizational framework into which the names of naturally occurring plants are slotted. In this framework, species of plants that are most similar to each other are grouped together. Groups or taxa (plural) are arranged in a hierarchical sequence of taxons (singular), from least inclusive rank at the bottom to most inclusive rank at the top as shown below in the plant classification hierarchy. In other words, the taxa “family” may include numerous plant genera, and within a genus (singular of genera) there may be any number of species, whereas within a given species, a subspecies may describe only a few populations or individuals. Within taxa – family, genus, species, etc., there are identifiable characteristics common to each group. For example, plants in the cypress family typically have broad, flattened, scale leaves, while plants in the pine family exhibit needle-like leaves. Once organized into a sensible system that recognizes similarities or relatedness, the grouping becomes easier to understand and remember. That is, once characteristics for a given group are known, they can be used to match unknown plants with known taxons. Plant Classification Hierarchy of Taxons • Family • Genus – (plural = genera) • Species • Subspecies or Variety • Forma Other Classification terms • Hybrid • Cultivar • Common Names • Plant Groups Review Identify the hierarchy of plant taxons. An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plant-identification/?p=112 1.03: Introduction to Taxons Learning Objectives • Identify characteristics of taxons. The plant family taxon is a grouping of plants consisting of one or more related genera that are more like each other than to other genera, and that includes the entire surviving lineage of the ancestral population. Family names always end with the suffix -aceae, except in a few notable cases where use of traditional names is also acceptable. Newer family names are based on the “type-genus” concept which means that for every family there is a genus that best represents the characteristics of the family. For example, Brassica (the cabbage genus) is the base for the family Brassicaceae, as is Rosa (the rose genus) for the family Rosaceae. Older family names are still used since many are somewhat descriptive and may be more familiar than their newer counterparts. For example, Cruciferae (from the Latin crucifer, a cross) refers to the four-petal arrangement of flowers characteristic of the mustard family. The revised family names for some familiar plant groups are listed below. Revised Family Names Traditional Name New Name Common Name Compositae Asteraceae Aster Cruciferae Brassicaceae Mustard Graminae Poaceae Grass Labiatae Lamiaceae Mint Leguminosae Fabaceae Pea Umbelliferae Apiaceae Carrot Because of technological advancements for determining plant genetics and other markers, some genera and family names have been reclassified under new names. Reclassified Family Names Family Name Reclassified Name Common Name Aceraceae Sapindaceae Soapberry Asclepiadaceae Apocynaceae Dogbane Taxodiaceae Cupressaceae Cypress Taxonomic Example The list of ten Pacific Northwest native conifers can be grouped into three families. Within each family, there are a different number of genera, as represented by the common names. Within each genus, unless a monospecific (single) genus as with Taxus and Pseudotsuga, there are a number of different species. Pinaceae – pine family Douglas fir ( Pseudotsuga , 1 species) hemlock (Tsuga, 2 species) larch (Larix, 3 species) true fir (Abies, 3 species) spruce (Picea, 4 species) pine (Pinus, 7 species) Cupressaceae – cypress family arborvitae (Thuja, 1 species) yellow cedar (Cupressus, 1 species) juniper (Juniperus, 3 species) Taxaceae – yew family yew (Taxus, 1 species) Review An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=160 An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=160 An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plant-identification/?p=160

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_I_(Nakano)/01%3A_Plant_Identification/1.01%3A_Introduction_to_Plant_Identification.txt

Learning Objectives • Employ correct naming and plant identification terminology. For an orderly system of classification, botanists give each group of plants a name that is recognized by people who know binomial nomenclature, regardless of where they are or the language they speak. This way every plant species will have a unique botanical name based on the binomial system of nomenclature. For example, one of the best-known trees of the Pacific Northwest, the Douglas fir, recognizes botanist Archibald Menzies in its scientific name Pseudotsuga menziesii. While the common name recognizes fellow botanist David Douglas, Archibard Menzies is credited with the first botanical description of the plant. A plant name or binomial is made up of two names: a genus name and a (usually) descriptive specific epithet (species name), both commonly of Latin or Greek origin. For example, of the many species within the group known as pines (genus = Pinus) there is only one named Pinus contorta (contorta = twisted). This species is characterized by often having contorted or twisted young shoots. The “species name” is the binomial; for instance, the species to which we belong is Homo sapiens. Genus A genus (plural = genera) is defined as an assemblage consisting of one or more related species that are more like each other than to other species, and that includes the entire surviving lineage of the ancestral population. Evidence for these relationships is deduced from the fossil record and from comparative techniques in morphological, chemical and molecular (DNA sequencing) analysis. A genus name can be descriptive of the plant, such as Equisetum (common horsetail) which is from two Latin words equus (horse) and saeta (bristle). The genus can be the actual Latin or Greek name such as Erysimum which is derived from the Greek name for the same plant erysimon. It can also be derived from the plant founder’s name such as Davidia, which is from Father David, a famous French plant explorer who lived in China for many years. Species The species is the basic life-unit in biology and can be defined as consisting of one or more related species that are more like each other than to other populations and that presumably come from a single ancestral population. The species name may be an adjective that indicates a distinguishing characteristic of the species, e.g., Quercus alba – white oak, or a noun that honors a person or indicates the species habitat. Species is abbreviated sp. (a single species) or spp. (more than one species). Subspecies Subspecies (ssp. or subsp.) and variety (var.) names are also multinomials. For example, lodgepole pine is known by the botanical name Pinus contorta var. latifolia, or sometimes, P. contorta ssp. latifolia. In other words, a northern variant of Pinus contorta with needles more flattened (lati = broad and folia = leaf) than the typical, coastal variety (P. contorta var. contorta). Note that “variety” is used here at the same rank as “subspecies” while some botanists consider the “variety” as a lower rank. These terms are used to describe naturally occurring plants. Form The rank form or forma (f. or fa.), is used to represent individuals which differ in some specific way from other individuals within the same natural populations. For example, one can find numerous red bract forms throughout populations of the more commonly white bract Cornus florida (Eastern flowering dogwood). These red bract dogwoods are correctly known as Cornus florida f. rubra (rubra = red). Other common, naturally-occurring mutations in other plants include: weeping habit (f. pendula), dissected leaves (f. dissecta), and white flowers (f. alba). Hybrids Hybrids are the offspring of successful mating between plants belonging to different taxa. Known interspecific hybrids (between species in the same genus) are designated by a multiplication sign, as Platanus x acerifolia (P. occidentalis x P. orientalis). Intergeneric hybrids result from crossing plants belonging to separate genera; an intergeneric hybrid name is always preceded by a multiplication sign, as xSolidaster (Solidago x Aster). Cultivars Cultivars are horticultural races or strains of plants which originate under cultivation or may originate in nature as a mutation and subsequently persist under human cultivation. The word cultivar (cv.) comes from “cultivated variety,” a somewhat confusing derivation, since the “variety” represents a naturally occurring entity, while the cultivar does not. As cultivars do not persist in nature, it is not a botanical designation; however, where used, the cultivar is considered part of the botanical name and must be appended to it. Cultivar names are distinguished in text using single quotation marks, as Chamaecyparis pisifera ‘Filifera Aurea’ (filaments or threads of gold). Common names Common names are the local, familiar names given to plants. The same common name may be used for several completely different plants. For example, the common name “cedar” is a name given to a variety of plants with aromatic wood (recalling the “cedar” of antiquity, Cedrus spp.) or to plants that are reminiscent of other plants called “cedars,” for example. In the Pacific Northwest, cedar refers to Thuja (western red cedar) and to Cupressus (yellow cedar). Similarly, a single species may have numerous common names, particularly if known from a variety of locations. For example, yellow cedar is also known as Nootka cypress and Alaska cedar. Clearly, there is potential for much confusion with common names. In text, common names are written out in lower case, except where they include proper names; e.g., Douglas fir, Japanese painted fern, etc. Common names are not botanical names. While botanical names are often, at least initially, difficult to remember and pronounce, they are universally recognized and considerably more accurate than common names.

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_I_(Nakano)/01%3A_Plant_Identification/1.04%3A_Introduction_to_Binomial_Nomenclature.txt

Learning Objectives • Describe conventions for writing botanical names. Botanical nomenclature is the scientific system of naming plants. The naming of plants is governed by two sets of published rules: The International Code of Nomenclature for algae, fungi, and plants and the International Code of Nomenclature for Cultivated Plants. These rules establish a worldwide standard of reference for naming plants. By convention, when written in text a botanical name is always italicized or underlined, and the first letter of the genus name is always capitalized. The following summarizes the basic rules regarding the writing of botanical names for plants: • The generic epithet of a botanical name is always capitalized (e.g., Salvia, Impatiens), and is underlined or italicized except where it is also used as a common name, as in salvia or impatiens. Within text or in a list – but only where unambiguous – the genus name is often abbreviated to the first letter, for example, Rosarugosa, R. moyesii, R. acicularis. • The specific epithet of a botanical name is always lower case, and is underlined or italicized in text, as Gaultheriashallon or Gaultheria shallon. If only the genus of a plant is known, the specific epithet is abbreviated as sp. (designating a single species) or spp. (more than one species). • Hybrids, produced from breeding 2 or more different species (interspecific), are noted by a multiplication sign between the genus and specific epithet, for example, Forsythia x intermedia. • Hybrids produced from crosses between genera (intergeneric), are noted by a multiplication sign before the genus, for example, xSolidaster luteus which has the following parentage, (Solidago canadensis x Aster ptarmicoides). • Subspecies are abbreviated ssp. or subsp. The subspecies epithet is not capitalized but is underlined or italicized, for example Acer glabrum ssp. douglasii • Variety or more officially, varietas, is abbreviated var. The variety epithet is not capitalized but is underlined or italicized, as in this example, Clematismontana var. rubens • Form or more officially, forma, is abbreviated f. (or sometimes fa.). For example, Cornusflorida f. rubra. • Cultivars usually have vernacular names, are not italicized or underlined, and all words are capitalized and usually in single quotes. For example, Astilbechinensis ‘Pumila’ or Ilex aquifolium ‘Ferox Argentea’. • Group: This describes a group of unnamed tree seedlings, for example Picea pungens Glauca Group describes all the un-named seedlings with blue foliage that are available in the nursery trade. Groups names are not italicized or underlined, and all words are capitalized but are not in single quotes. • The ™ designation indicates that the originator of the new plant, for example, Pyrus calleryana Aristocrat™, has applied for a trademarked name. The ® indicates that the plant name is a registered trademark, such as in Pyrus calleryana Chanticleer®. The trademark name is often the “selling name” of the plant, which may differ from the cultivar name. eg. Weigela florida ‘Alexandra’ is sold under the moniker Wine & Roses® weigela. Review An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=1238 1.06: Nomenclature Review Learning Objectives • Apply the conventions for writing botanical names. True or False. Apply binomial nomenclature conventions to each of the plant names. An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plant-identification/?p=127 An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=127 An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=127 An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=127 An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=127 An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=127 An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=127 An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=127 An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=127 An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=127 1.07: The Meaning of Plant Names Learning Objectives • Describe the meaning of botanical names. Botanical names often provide a helpful description of a plant. The origins of Latin and Greek names may be classical, mythological, or commemorative, or they may relate to a place, area, or season. Descriptors for plant surface characteristics or color, habitat, growth habit, and size and shape are other common sources for specific epithets. Familiarity with their meaning is helpful for remembering plant names. References such as the Dictionary of Plant Names by Allen J. Coombes (1994) or The Names of Plants (1996) contain interesting information on the origin and meaning of plant names. Information is also available online at these links to Califlora: Plant Name Meanings and Derivations [New Tab][1] and The Meaning of Latin Plant Names [New Tab][2]. Practice Identify the meaning of each of the specific epithets using the links above to online resources. An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=129

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_I_(Nakano)/01%3A_Plant_Identification/1.05%3A_Conventions_for_Binomial_Nomenclature.txt

Learning Objectives • Identify common classifications of plant growth. Water movement is the most basic classification of plant growth. All plants need water to survive and, based on how they move water, are categorized as either vascular or non-vascular. Vascular plants, such as trees, have a water conducting system, allowing them to supply aerial tissues with water and to grow well above the ground. Non-vascular plants rely on their closeness to water and their own physical absorption to support green tissues above ground. Mosses and liverworts are examples of non-vascular plants. Reproduction is another classification of vascular plants that is based on whether they reproduce themselves asexually or sexually. Vascular plants are subdivided into two major categories, pteridophytes, and spermatophytes. Pteridophytes (Greek for “fern plant”) include ferns and horsetails that reproduce asexually by spores. Spermatophytes (Greek for “seed plant”) include conifers and flowering plants that reproduce sexually by seed. Conifers, from the Latin for “cone-bearing” are woody plants that bear their female and male reproductive structures in separate cones or strobili rather than in flowers. Coniferous trees and shrubs typically bear both female and male cones on the same plant. Pollen produced by male cones is transported on wind currents to the female cones wherein seed development is completed. Conifers belong to the group of seed producing plants called gymnosperms. Gymnosperm literally means ‘naked seed’ as seeds are held on the surface of a cone scale or at the end of a small structure. This is the main differentiation between conifers and the flowering plants (angiosperms) which bear their seeds in an enclosed ovary of a flower that becomes the fruit. Angiosperms are the largest and most diverse group in the plant kingdom. Some angiosperms produce flowers and fruit over many years (polycarpic), while some die after flowering and bearing fruit only once (monocarpic). In addition to the presence of flowers and fruit, angiosperms are classified into two major categories, monocotyledons and eudicotyledons. This classification is based on the number of cotyledons or seed leaves produced at seed germination. Monocotyledons (meaning “single seed-leaf”) include grasses, lilies, orchids, and palms. They develop from a seed with a single seed leaf. Some basic recognizable patterns of monocotyledons include leaf veins arranged in parallel lines; flower parts numbered in 3’s and a herbaceous plant structure. Eudicotyledons (meaning “true dicots”), are an evolutionary line that includes plants such as maples, oaks, roses, buttercups, mints, and sunflowers that develop from seed usually with a pair of seed leaves. Some basic recognizable patterns of dicotyledonous plants include leaves with netted venation; flower parts numbered in 4’s or 5’s and woody or herbaceous plant structure. The classification of plant growth based on methods of water movement and reproduction is summarized in the video How do we classify plants? [New Tab][1]. A video element has been excluded from this version of the text. You can watch it online here: kpu.pressbooks.pub/plant-identification/?p=117 `How do we classify plants? https://bio.libretexts.org/Bookshelves/Introductory_and_General_Biology/Book%3A_Introductory_Biology_(CK-12)/9%3A_Plants/9.6%3A_Plant_Classification.` Type of growth, such as tree versus shrub or woody versus non-woody (herbaceous), is often the first visual recognition of a plant. Plant growth may also be categorized by some aspect of their biology or ecology such as: terrestrial or aquatic habitat (e.g., duckweed), adaptations such as twining stems for climbing (wisteria) or underground storage bulbs (e.g., daffodil), or whether they exhibit seasonal loss of leaves (deciduous) or if they remain evergreen. Plant growth varies from trees with well-defined trunks, to multi-stemmed shrubs and climbers to spreading ground covers and clumping herbaceous plants. The above ground plant structure is typically formed by stems that are either woody or herbaceous. Woody plants such as cedars, oaks, and maples produce more or less permanent structures capable of extension and annual thickening (secondary growth). Non-woody or herbaceous plants such as dandelion, (eudicot) and grasses (monocot), and ferns, (pteridophyte) are limited to only extension growth and do not produce permanent above ground structures. The herbaceous growth habit is common among vascular plants, and many specific plant groups are distinguished on that basis. Herbaceous plants are characterized by a lack of woody tissue, such as bark. Their stems will eventually die back to a live root crown and root structures. Deciduous herbaceous perennials wither and die back to some kind of long-lived, resistant organ (a fleshy crown, bulb, tuber, rhizome, etc.) and enter a state of dormancy when conditions are not suitable for continued growth. In comparison, evergreen herbaceous plants have leaves that persist over one or more seasons of growth. Not all herbaceous plants are seed plants; spore producing plants such as ferns and horsetails are also considered herbaceous. The ability of some perennial plants to propagate themselves non-sexually by means of vegetative reproductive structures such as underground creeping stems (rhizomes) and tubers and bulbs is a competitive advantage over sexually reproduced plants and provides an effective adaptation for spreading. Plants with a climbing growth habit may be woody or herbaceous. Vines (herbaceous) and lianas (woody) have various specialized adaptations for climbing on, through, and over host plants and surrounding objects to gain access to light. Self-clinging climbers attach themselves to supports by aerial (adventitious) roots or by modified leaf structures called tendrils. Tendril climbers twine around or adhere themselves to supports by contact sensitive tendrils with adhesive discs at the tips. Climbers with twining stems or curling leaf stalks coil around supports in a clockwise or counterclockwise spiral habit. Scrambling (scandent) or trailing climbers with long arching stems attach loosely, if at all to supports. Some species, such as roses, are equipped with stem modifications of hooked thorns that allow them to scramble through other plants. Review An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=117 1. bio.libretexts.org/Bookshelves/Introductory_and_General_Biology/Book%3A_Introductory_Biology_(CK-12)/9%3A_Plants/9.6%3A_Plant_Classification ↵

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_I_(Nakano)/01%3A_Plant_Identification/1.08%3A_Plant_Growth.txt

Learning Objectives • Recognize and describe patterns used to classify plants. Classifying unknown plants as identical with or similar to plants within a particular taxonomic group involves observation and comparison. The ability to accurately distinguish and categorize the similarities and subtle differences among plant species relies on at least three interrelated skills: pattern recognition, description, and classification. Pattern recognition includes awareness of visual indicators such as shape, size, habit, etc. as well as other sensory input such as smell, touch, sound, etc. Much pattern recognition depends upon our ability to describe what is perceived. Frequently, people don’t remember those things they can’t describe in words. In contrast, those things, which are related to other more familiar things, are more easily recalled: for example, “it feels like velvet,” “it smells of lemons,” or “it appears to be bigger than a breadbox”. Descriptions allow people to identify and catalog those patterns. “Striped,” “spotted,” “rough” and “smooth” are simple descriptors. It is not difficult to remember such patterns. Other, more complex descriptors are needed for characterizing complex organisms. The “trick” is in recognizing the patterns that indicate important relationships. There is a significant amount of vocabulary involved in describing plants, and the student of plant identification must learn to apply both plant morphology (the study of shape) and the descriptive terminology. Classification is an effective method for organizing data. People naturally classify things according to various categories. Based on their usefulness, some plants may be considered more desirable than others. For example, plants considered to be undesirable for health or economic reasons are often categorized as weeds. Additional categories used for plant classification include their utility (medicinal plants), cultural tolerances (house plants), growth form (trees), leaf shape (needle vs. broad leaves), their assumed evolutionary relationships and genetic sequences (phylogenetics), among others. Review An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=162 1.10: Classify Plants by Life Cycle Learning Objectives • Describe characteristics of plant life cycle classifications. A plant will go through a sequence of stages from seed germination to seed production as a mature plant. For some plants, this sequence, or life cycle may take a few weeks while others continue to grow and flower repeatedly over many years. Plant life cycles are classified as annual, biennial, or perennial. Annuals complete their life cycle of germination from seed, growing, flowering, fruiting and dying within a single season of growth. Biennials require two seasons to complete their life cycle. In the first season, foliage production and storage of food reserves takes place followed by flowering, seed production and death in the next. Perennials typically flower annually once established and may live for several to a great number of years. Types of Annuals While the annual life cycle is completed within a single season of growth, the term annual or bedding plant may also be used to describe any plant that is grown outdoors in the spring and summer for one growing season. Annual flowers differ in their tolerance to cold weather and frost. Hardy annuals are the most cold tolerant; they will take light frost and some freezing weather without being killed. In most cases, hardy annuals can be planted in the fall or in the spring before the last frost date. Examples of hardy annuals include Lathyrus (sweet pea), Viola (pansy), and Tagetes (marigold) cultivars. Most hardy annuals are not heat tolerant and usually decline and die with the onset of hot summer temperatures. Another type of hardy annual is the winter annual that germinates in the fall, overwinters as a rosette of leaves, and flowers in late winter and early spring. Species of Stellaria (chickweed) and Cardamine (snapweed) are examples of winter annuals. Half-hardy annuals will tolerate periods of cold damp weather, but will be damaged by frost. Most half-hardy annuals can be seeded outdoors in early spring since they do not require warm soil temperatures to germinate. Seeds or plants are normally planted after the last spring frost. Examples of plants grown as half-hardy annuals are Cosmos (cosmos) and Tropaeolum (nasturtium). Some half-hardy annuals may decline in the midsummer heat but may re-bloom in late summer or fall. Because most tender annuals are native to warm tropical regions of the world, they are sensitive to cold soil temperatures and are easily damaged by frost. Most seeds will not survive freezing soils temperatures and will not germinate when soil temperatures are below 15°C. It is recommended to wait two to three weeks after the last spring frost to sow seeds or transplant outdoors. Tender annuals include species of Begonia (begonia)and Impatiens (impatiens). While some plants may be perennial in tropical regions, they are categorized as cool- or warm-season annuals when planted in colder regions. Cool-season annuals, such as Pelargonium (geranium), Petunia (petunia), and Antirrhinum (snapdragon), grow best when temperatures are in between 20° and 25° C. during the day. Best flower production is in the spring and fall; flower production tends to decline in the middle of a hot summer. Warm-season annuals, such as Zinnia (zinnia) perform well when day time temperatures are between 26° and 32°C. and night time temperatures are between 15° and 20°C. Biennials The life cycle of biennial plants is completed over two growing seasons. During the first season, they produce only leaves—usually in a rosette. Following a winter cold period, they flower in the second growing season, produce seeds, and then die. Popular biennials include Digitalis (foxglove) and Oenothera (evening primrose). Cultural practices are basically the same as for annuals, except that the plants are alive for two growing seasons. Biennials present the obvious disadvantage of producing only foliage the first year. One solution is to sow biennial seeds in mid-summer so that the plants will develop during the summer and fall. After exposure to the winter cold, they will develop flowers in the spring. Perennials Perennial plants can be either short-lived or long-lived herbaceous or woody plants. Short-lived herbaceous plants such as Gaillardia (blanket flower) may live for only a few years, or they can be long-lived like Paeonia (peony). Woody plants also classify as perennials, though they are rarely referred to as such. Woody species have stems that continue to grow, developing a permanent structure that the plant cannot ‘replace’ once removed. Some woody plants live tremendously long lives, such as the 9500 year old Picea sp. (spruce) in Sweden and British Columbia’s 1000 year old Thujaplicata (western red cedar). Perennials that flower and fruit only once and then die are termed monocarpic. However, most perennials are polycarpic, flowering over many seasons in their lifespan. Common hardy herbaceous plant families include: Asteraceae – sunflowers Brassicaceae – mustards Crassulaceae – sedums Liliaceae – lilies Lamiaceae – mints Poaceae – grasses Ranunculaceae – buttercups Review An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=225

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_I_(Nakano)/01%3A_Plant_Identification/1.09%3A_Introduction_to_Plant_Classification.txt

Learning Objectives • Describe the characteristics of dichotomous keys. A dichotomous key is a useful tool for the identification of things not known to the observer; for example, unfamiliar plant species. The typical dichotomous key, as shown in the example below, is made up of a series of descriptions, features or characteristics, arranged in pairs (couplets) of contrasting alternative choices (e.g., hairy vs. not hairy, bigger than a breadbox vs. not bigger than a breadbox, etc.). Each couplet is worked through sequentially until the correct determination is made. Starting at the first couplet, choose which of the two alternatives best suits an object or thing, and proceed to the couplet number or answer indicated by that choice. The number of things being considered is reduced at each successive step in the key so that by a process of elimination the correct determination is made. A dichotomous key for plant identification is available online at this link to Oregon State University Dichotomous Key [New Tab][1]. Example Dichotomous Key Example objects to identify: apple tree, water-lily, fir tree, dandelion, astroturf, seaweed. 1.a. found in water ………………………………………………………………………………………… go to 2 1.b. found on land ………………………………………………………………………………………….. go to 3 2.a. grows in salt water ………………………………………………………………………………….. seaweed 2.b. does not grow in salt water …………………………………………………………………….. water-lily 3.a. a real plant ………………………………………………………………………………………………. go to 4 3.b. not a real plant ……………………………………………………………………………………….. astroturf 4.a. grows more than 50 m tall ………………………………………………………………………. fir tree 4.b. grows less than 50 m tall ………………………………………………………………………… go to 5 5.a. produces yellow flowers …………………………………………………………………………. dandelion 5.b. does not produce yellow flowers ……………………………………………………………. apple tree Or, the couplets may be grouped like this: 1.a. found in water ……………………………………………………………………………………………………….. 2 2.a. grows in salt water ……………………………………………………………………………………. seaweed 2.b. does not grow in salt water ……………………………………………………………………… water-lily 1.b. found on land ………………………………………………………………………………………………………… 3 3.a. real plant ……………………………………………………………………………………………………………….. 4 4.a. grows more than 50 m tall ……………………………………………………………………………. fir tree 4.b. grows less than 50 m tall ………………………………………………………………………………………. 5 5.a. produces yellow flowers …………………………………………………………………………. dandelion 5.b. does not produce yellow flowers ……………………………………………………………..apple tree 3.b. not a real plant …………………………………………………………………………………………. astroturf Dichotomous keys may be simple or complex depending on what is being identified. For example, distinguishing obvious visible characteristics, such as structures for water movement in woody or herbaceous plant growth is straightforward. However, it should be noted that leaves, flowers, and fruit will not typically be available at the same time and return visits may be needed. Furthermore, differentiating minute plant parts such as reproductive structures requires the use of a hand lens or low magnification microscope for inspection and a thorough understanding of the descriptive terminology used in a dichotomous key. 1.12: Key to Plant Classification Learning Objectives • Use a dichotomous key for plant classification. Dichotomous keys help improve pattern recognition and understanding of the descriptive terminology used to classify important distinctions among plants. The following dichotomous key can be used outdoors to classify a range of plants by type, growth habit, and reproductive method. Practice Use a dichotomous key for plant classification. Key to Plant Classification 1.a. Plants rely on their closeness to water and absorptive green tissues above ground…………………………………………………………………………………. Non-vascular plant (go to 2) 1.b. Plants have a water conducting system that supplies above ground tissues with water and allows growth above ground …………………………………………….Vascular plant (go to 2) 2.a. Plants, (conifers and flowering) that reproduce by seed …….. Spermatophyte (go to 3) 2.b. Plants that reproduce by spores ……………………………………………. Pteridophyte (go to 4) 3.a. Spermatophyte that flowers and develops seeds within ovaries that mature into fruits ………………………………………………………………………………………………..Angiosperm (go to 4) 3.b. Spermatophyte that flowers and develops seeds ‘naked’ in cones (conifer) ……………………………………………………………………………………………………… Gymnosperm (go to 5) 4.a. Plants with primary growth tissue only, lacking woody tissue like bark ……………………………………………………………………………………………………….. Herbaceous (go to 6) 4.b. Plants with secondary growth tissue, like bark …………………………………Woody (go to 5) 5.a. Woody plants with one or few main stems ……………………………………………Tree (go to 7) 5.b. Woody plants with multiple stems emerging from base ……………………. Shrub (go to 7) 6.a. Non-woody tissue does not persist over one or more seasons; withers and dies back to fleshy crown, bulb, tuber, or rhizome ………………………………………………………………………………………. Deciduous herbaceous (go to 8) 6.b. Non-woody tissue and leaves persist over one or more seasons of growth ……………………………………………………………………………………….. Evergreen herbaceous (go to 8) 7.a. Trees or shrubs that lose their leaves every autumn…………………… Deciduous (go to 8) 7.b. Trees or shrubs that are never entirely leafless …………………………………………………………………………….. Evergreen or semi-evergreen (go to 8) 8.a. Herbaceous angiosperm that produces a single seed leaf; leaves have parallel venation; flower parts are in 3’s ………………………………………………………………….… Monocotyledon 8.b. Woody or herbaceous angiosperm that produces a pair of seed leaves; leaves have netted venation; flower parts are in 4’s or 5’s ……………………………………….… Eudicotyledon

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_I_(Nakano)/01%3A_Plant_Identification/1.11%3A_Introduction_to_Dichotomous_Keys.txt

Learning Objectives • Describe the morphological characteristics of herbaceous and woody stems. Plant identification depends on knowledge of taxonomy and understanding of stem, leaf, bud, flower and fruit morphology. Morphology is the Greek word for “the study of shape,” and plant morphology is the study of the external plant structures and shapes. While the original botanical resource, Species plantarum was published by Carolus Linnaeus in 1753, one of the most comprehensive references currently available for plant morphology is Huxley, A. (ed.) The New Royal Horticultural Society Dictionary of Gardening. London, Macmillan Press, 1992. A working knowledge of morphological descriptors for plant identification enables the use of dichotomous keys as well as herbarium samples and digital databases. A herbarium is a collection of pressed and dried plants that is systematically arranged for research and plant identification purposes. Information on the procedure for creating herbarium samples is available at this link to Herbarium: How to Press Plants [New Tab][1] A YouTube element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plant-identification/?p=131 ``` Herbarium: How to Press Plants https://youtu.be/USltmLxNt80 ``` An example of an institutional herbaria is available at this link to the University of British Columbia Beaty Biodiversity Museum [New Tab].[2] Digital databases and apps typically use the morphology of stems, leaves, flowers, and fruit to identify unknown plants . Some regional databases are available at these links to the Kwantlen Polytechnic University Plant Database [New Tab][3], Oregon State University Landscape Plants [New Tab][4], and University of British Columbia E-Flora BC [New Tab][5]. Stem Morphology A morphological description usually starts with the structure of a plant. Plant stems with vascular tissue support leaves and reproductive structures such as flowers. Depending on the type of plant, stems may be woody or herbaceous, and solid or hollow in cross section. Herbaceous (non-woody) stems with solid or hollow stems are typical of forbs (eudicots), grasses, and grass-like plants called rushes and sedges (monocots). The stems are generally filled with a soft spongy tissue called pith, that stores and transports nutrients. The culm (stem) of a grass plant (Poa spp.) is hollow with pith only at the jointed nodes. The base of the leaf circles around the stem forming a series of overlapping sheaths. Sedges (Carex spp.), differ from grasses and rushes in that the stems are triangular (V-shaped) in cross section at the base (“sedges have edges”), have a solid pith, and are not jointed. Rushes differ from grasses in that stems are not jointed (no nodes) and are typically filled with pith. Some rush genera, such as Luzula spp. can look very grass-like with leaf blades while in Juncus spp. the leaves may be reduced to just a rounded sheath. Descriptions of morphological characteristics are illustrated at this link to Grasses, Sedges and Rushes [New Tab][6]. In contrast to herbaceous stems that die at the end of the growing season, woody stems are permanent structures that grow in length and girth (diameter) each year and produce bark as a protective covering. The general features of the woody stem illustrated in Figure 13.1 will be characteristic for a particular plant species. Figure 13.1 External features of a woody stem The shape, size and arrangement of buds and lenticels (small openings in the outer bark that allow for the exchange of gases), are often identifiable in trees and shrubs, as shown in Figure 13.2 and Figure 13.3. The thickness, texture, pattern, and color of the bark of many woody plants is both a distinctive species characteristic for identification and an attractive feature for landscape use. Figure 13.2 Prunus buds Figure 13.3 Prunus bark and lenticels Examples of the morphology of herbaceous stems and woody stems and buds are available at this link to Stems – External KPU.ca/Hort [New Tab][7]. Stem modifications include underground, above ground, and aerial structures that are characteristic to different plant species. Underground structures for spreading and food storage include rhizomes, corms, tubers, and bulbs. Stolons, runners, suckers, and offsets that grow almost parallel to or just above the ground enable plant spread. Aerial modifications include stem tendrils and thorns for climbing and protection. In xeric (dry) conditions, the stem may take over photosynthesis in order to reduce water loss from leaves (Cactus spp.). Examples of different types of stem modifications are shown at this link to Modifications – Stem KPU.ca/Hort [New Tab][8]. True or False Search the plant names available at this link to the KPU Plant Database [New Tab][9] An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=131 An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=131 An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=131 An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=131 An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=131

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_I_(Nakano)/01%3A_Plant_Identification/1.13%3A_Introduction_to_Plant_Morphology.txt

Learning Objectives • Describe the morphological characteristics of plant leaves. Leaves are specialized structures for photosynthesis that provide plants with energy. Leaves arise at nodes just below an axillary bud on woody stems and are usually petiolate, that is composed of a blade and stalk-like petiole. Petioles may have stipules, two small leaf-like flaps that are attached at the base. In some cases, stipules on leaves and stems may become modified into spines, thorns, or prickles. Some leaves are sessile, that is, they lack petioles and have blades directly attached to the stem. When a bud is located in the axil of a single leaf and the stem, as shown in Figure 14.1 the leaf is classified as simple. Figure 14.1 External features of a simple leaf However, when a bud is located in the axil of a structure with more than one leaf (leaflet) on attached to the axis (rachis), the leaf is classified as compound. As shown in Figure 14.2, even or odd numbers of leaflets may be pinnately compound that is, arranged along a central axis (feather-like), or palmately compound from one point on the tip of the petiole, (like fingers on an out-stretched hand). Compound leaves may undergo double (bipinnate) or triple (tripinnate) compounding into finer segments or leaflets. Figure 14.2 Types of compound leaves Phyllotaxy, the arrangement of a leaf or bud in relation to another leaf or bud along a plant stem is a useful basis for classifying plants. Figure 14.3 illustrates common leaf arrangements where leaves and buds on a stem are opposite (directly across from each other on the stem), alternate (spaced alternately along the stem axis), whorled (three or more leaves and buds are positioned at a node), or basal (emerging from the base). Leaf arrangement may also be described as spiral, clustered, decussate (alternating pairs at right angles), and imbricate (overlapping scales). 14.3 Common leaf arrangements Leaf venation refers to the patterns of veins within the leaf blade. In eudicot plants, leaf venation is typically either pinnate or palmate and may have multiple branching that gives an overall netted appearance. In contrast, monocots will have parallel leaf venation. Additional morphological features for description include leaf shape, tip and base features, and margins (edges). Leaf surface characteristics vary and some may be smooth (glabrous) or with hairs (hirsute or pubescent), wrinkles (rugose), pustules (verrucose) or other interruptions of the surface. Additional leaf surface terms are defined at this link to Leaf[New Tab][1]. Figure 14.4 and Figure 14.5 illustrate components of a leaf morphology chart commonly used for plant identification. More detailed information about the external characteristics of leaves is available at this link to Leaf Morphology[New Tab][2]. Figure 14.4 Leaf shapes Figure 14.5 Leaf tips, bases and margins Review. Use the leaf morphology chart to describe leaf parts. Click the image hot spots. An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=295 An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=295 1.15: Plant Morphology - Conifers Learning Objectives • Use a dichotomous key to identify conifers. Both evergreen and deciduous leaves exhibit characteristic broad blades in angiosperms, and narrow needle, scale-like, or awl-shaped leaves in the conifers. Figure 15.1 illustrates the different types of conifer leaves. The awl-shape and scale-like foliage of Juniperus spp. exhibits leaf dimorphism where a juvenile leaf form differs from the mature leaves of the same plant. Leaves may be borne singly on the shoot as in Picea spp. (spruce), in tufts or clusters as in Larix spp. (larch), or in fascicles (bundles) of 2-5 as in Pinus spp. (pines). Figure 15.1 Types of conifer leaves Dichotomous Key for Some Common Conifers. Click the links for plant images. 1.a. leaves long, needle-like ………………………………………………………………………………….. go to 2 1.b. leaves lanceolate, awl or scale-like, overlapping, not needle-like ……………………………………………………………………………………………………………………………… go to 5 2.a. needles in bundles or tufts …………………………………………………………………………….. go to 3 2.b. needles borne singly ………………………………………………………………………………………. go to 7 3.a. needles in bundles of 2 to 5 ……………………………………………………………………………. go to 4 3.b. needles deciduous, many in a tuft …………………………………………………………. Larix decidua [New Tab][1] 4.a. 5 needles per bundle …………………………………………………………………………….. Pinus strobus [New Tab][2] 4.b. 2 needles per bundle …………………………………………………………………………………… go to 10 5.a. scales imbricate (overlapping) cones small, upright………………………………. Thuja plicata [New Tab][3] 5.b. scales imbricate, cones spherical or oval, opening along sutures at maturity ……………………………………………………………………………………………………………………………… go to 6 6.a. cones small, spherical; cone scales with a prominent point …………………………………………………………………………………………………….. Cupressus nootkatensis [New Tab][4] 6.b. cones larger, oval, cone scales thick, deeply pitted ………….. Sequoiadendron giganteum [New Tab][5] 7.a. needles stiff and sharp, 4-sided ……………………………………………………………………. go to 8 7.b. needles flat and pliable ………………………………………………………………………………… go to 9 8.a. needles extremely sharp, new growth coated with bluish wax……………………………………………………………………………………. Picea pungens Glauca Group [New Tab][6] 8.b. needles not extremely sharp, not coated with bluish wax ……………………………………………………………………………………………………………………… Picea abies [New Tab][7] 9.a. needles dull green, 2 cm long, borne on short pegs that persist after the needles fall …………………………………………………………………………………………………………….Tsuga heterophylla [New Tab][8] 9.b. needles shining green, 2 cm long, not borne on pegs ……………………………………………………………………………………………………… Pseudotsuga menziesii [New Tab][9] 10.a. needles < 7 cm long ………………………………………………………………………………….. go to 11 10.b. needles > 7 cm long ……………………………………………………………………………… Pinus nigra [New Tab][10] 11.a. needles dark green, 3-6 cm long, cone scales with a small recurved prickle ………………………………………………………………………………………………………………….. Pinus contorta [New Tab][11] 11.b. needles bluish green 5-7 cm long, slightly twisted, cone scales without a prickle …………………………………………………………………………………………………………………. Pinus sylvestris [New Tab][12]

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_I_(Nakano)/01%3A_Plant_Identification/1.14%3A_Plant_Morphology_-_Leaves.txt

Learning Objectives • Describe the morphological characteristics of flowers and fruit. The most significant patterns, in terms of evolutionary relationships, involve reproductive structures, such as the number and arrangement of flower parts, or the structure of cones. While the size and shape of vegetative structures such as leaves and stems are relatively plastic or changeable, the basic patterns of reproductive structures change little over time. Although access to flowers and fruit may be seasonal, digital resources and herbarium samples allow the identification of patterns and relationships within plant taxa. Flower and Inflorescence Morphology Flower shape, color, and markings are all valuable features for plant identification. Figure 16.1 illustrates some flower shapes that are commonly used for identification purposes. Figure 16.1 Flower corolla shapes A typical angiosperm flower is borne on a peduncle (stalk) and is composed of the receptacle, sepals (calyx), petals (corolla), stamens, and pistil (carpel). Flower parts may be fused or separate and usually exhibit radial (star-shaped) symmetry or bilateral (two-mirror image halves) symmetry as shown in Figure 16.2. Figure 16.2 Flower parts and symmetry In addition to their shape, flowers are often differentiated by further dissections of their structure. For example, complete flowers must have all four main flower parts: sepals, petals, stamens (male) and pistils (female), while incomplete flowers will be missing one or more of these parts. Most flowering plants have perfect flowers that contain both male and female reproductive parts. However, some have imperfect flowers that contain only the male or female part (stamen or pistil) and may or may not contain sepals or petals. A species may have individual plants that are dioecious, producing either male or female flowers or cones on separate plants. Plants that are monoecious produce both female and male flowers and cones on one plant. Flower parts and structures can be examined at this link to Flower Morphology KPU.ca/Hort [New Tab][1]. Angiosperms produce flowers which are arranged on a structure called an inflorescence. An inflorescence may support a solitary flower or display individual flowers (florets) to pollinators or expose flower parts to pollen carried on air currents. Figure 16.3 illustrates types of inflorescence commonly found in both woody and herbaceous plants. Figure 16.3 Inflorescence types Representative characteristics of flowers and inflorescence can be examined at this link to Inflorescence Types KPU.ca/hort [New Tab][2]. Fruit Morphology For the majority of angiosperms, when a flower is pollinated, the pollen joins with an egg to produce a seed. The seed develops within the ovary which is part of the pistil, a female [3]e edible portion of the fruit. The time lapse development of a fruit is captured at this link to Pear flower to young fruit [New Tab][4]. Fruits are classified into one of three main groups: simple, aggregate, or multiple, as shown in Figure 16.4. Simple fruits, which form from a single, ripened ovary, may be either fleshy or dry. Fleshy fruits include the berry (grape), pepo (pumpkin), hesperidium (orange), drupe (plum), and pome (apple). Aggregate fruit develop from a single flower with numerous pistils. Once fertilized, the individual pistils develop into tiny fruitlets clustered on a single receptacle, as in a raspberry or blackberry. Multiple fruits, such as pineapples, form when numerous fertilized flowers in a single inflorescence develop together into a larger fruit. Dry fruits, are either dehiscent (split open at maturity) or indehiscent (remain closed at maturity). Dry fruits that split at maturity include the legume (pea), silique (mustard), follicle (milkweed), and capsule (cotton). Dry fruits that do not split at maturity include the achene (sunflower), nut (pecan, almond), grain (corn), samara (ash), and schizocarp (geranium, carrot). Figure 16.4 Fruit types Figure 16.5 Key to fruit types In addition to an important feature for identification, many fruit types have decorative value and may provide long season interest in the landscape. Images of the morphology of different fruit types are available at this link to Fruit Types KPU.ca/Hort [New Tab][5]. For the majority of gymnosperms, the cone is the reproductive structure. Most familiar is the female cone, which is constructed of many small, rounded, scale-like structures attached to a central stem. The pollen bearing male cone is characteristically smaller than the female cone. Typically, a naked seed will develop on each of the scales of a female cone. As the cone scales ripen and spread the seed is allowed to leave the cone. Conifer cones are described at this link to Gymnosperm pollination [New Tab][6]. Review Match the flower and inflorescence types. An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plant-identification/?p=299 Review Select and place the correct term next to the plant that has that fruit type. Search the type of fruit for each plant at this link to the KPU Plant Database [New Tab][7]. An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=299 1. http://www.horticulturebc.info/labreviews/pdfs/Flower%20Morphology.pdf 2. http://www.horticulturebc.info/labreviews/pdfs/Inflorescence%20Types.pdf 3. reproductive organ of the flower. The expanded and ripened ovary is referred to as the fruit. Commonly, the enlarged ovary becomes th ↵ 4. https://www.youtube.com/watch?v=zVNsCW6eiiw&feature=youtu.be 5. http://www.horticulturebc.info/labreviews/pdfs/Fruit%20Types.pdf 6. https://www.youtube.com/watch?v=KnJMKGnDX0E&feature=youtu.be 7. https://plantdatabase.kpu.ca/

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_I_(Nakano)/01%3A_Plant_Identification/1.16%3A_Plant_Morphology_-_Flowers_and_Fruit.txt

Learning Objectives • Describe key morphological patterns characteristic to plant families. Plant families are separated according to structural differences in flowers, fruit, and seed. Genera that share similar structures are grouped within a particular Family. While some plant families, such as Orchidaceae (orchid) and Asteraceae (sunflower family)have several hundred members, others such as Ginkgoaceae have a single member. As the group with the greatest number of closely related plants, the family taxon provides a starting point for narrowing the search for an unknown plant. In addition to shared morphological characteristics, the family taxon provides information about evolutionary adaptations for growth conditions as well as methods for propagation. One of the most comprehensive references for angiosperms is Flowering Plant Families of the World by V. H. Heywood (2007). The morphological characteristics for some families and genera commonly found in landscapes and gardens are summarized below. Images of the representative genera are available at this link to the KPU Plant Database [New Tab][1] . Asteraceae – aster, sunflower family One of the largest families of flowering plants is the aster or sunflower family, Asteraceae. Most of its members are evergreen shrubs or subshrubs or perennial rhizomatous herbs, but tap-rooted or tuberous-rooted perennials, and biennial and annual herbs are also frequent. Common genera of this family include: • Achillea (yarrow) • Dahlia (dahlia) • Jacobaea (dusty miller) • Leucanthemum (daisy) • Symphyotrichum (aster) • Taraxacum (dandelion) Key identifying characteristics for Asteraceae include an inflorescence that is a composite head with disc florets, (ray florets may or may not be present), and an achene-like cypsela (fruit) with a fringe of hairs or papus. The leaves are alternate or opposite, rarely whorled, and often lobed or toothed and pinnately or palmately veined. Caryophyllaceae – pink, carnation family The pink or carnation family, Caryophyllaceae is a large family of temperate eudicots that are mostly annual, biennial or perennial herbs and a few subshrubs with woody stems. Many members are flowering ornamentals and some, such as Cerastium may be weedy. Common genera include: • Cerastium (snow-in-summer) • Dianthus (pinks, carnations) • Lychnis (campions) • Silene (catchflies) Species in Caryophyllaceae are relatively uniform and recognized by non-succulent stems, swollen stem nodes, and opposite leaves (rarely whorled). Leaf blades are typically simple, lanceolate with entire margins, and without stipules. Flowers are often white or pink, with 4 or 5 petals, and 5 sepals. Petals may be entire, fringed or deeply cleft and sepals may be free or united. There are usually 5-10 stamens or more and the carpels are united in a common superior ovary. Flowers are terminal and bloom singly or branched in cymes. In some species such as Silene spp., the calyx may be cylindrically inflated. The fruit is a capsule with many seeds. Ericaceae – heather family One of the most common groups of plants in the British Columbia and the Pacific Northwest (PNW) is the heather family, Ericaceae. Family members are mostly temperate woody shrubs and trees, and rarely herbs. Species of Arbutus, Arctostaphylos and Gaultheria are indigenous to the PNW. Some common genera in the Ericaceae family include: • Calluna (heather) • Erica (heather or heath) • Pieris (lily-of-the-valley shrub) • Rhododendron (including azaleas and rhododendrons) • Vaccinium (huckleberries and cranberries) For the most part, ericaceous plants have urn-shaped flowers borne in racemes or panicles. Rhododendron is an exception; they have relatively open, bell-shaped flowers in short racemes (trusses). Other shared characteristics include: fine, off-white shallow roots, an affinity for acid soils, leathery leaves arranged alternately or appearing terminally whorled, rough or peeling bark, and dense wood. While many members are deciduous, genera in this family are among the most recognizable of broadleaf evergreens, both in and out of flower. Lamiaceae – mint family The mint family, Lamiaceae is easily recognized because its members exhibit square stems, opposite, often decussate (4-ranked) leaf arrangement, and distinctive two-lipped flowers held in verticillasters (pairs of axillary cymes arising from opposite leaves or bracts and forming a false whorl). The fruit is a nutlet. Family members may be annual or perennial, and are often subshrubs or entirely herbaceous. Many are highly aromatic, vigorous growers and adapted to propagate easily from stem-cuttings. There are a number of broadleaf evergreen members in Lamiaceae, as listed below: • Ajuga (carpet-bugle) • Lamium (dead nettle) • Lavandula (lavender) • Rosmarinus (rosemary) • Salvia (sage) • Thymus (thyme) Liliaceae – lily family Members of the the lily family, Liliaceae are typically perennial herbaceous monocots that grow from bulbs or rhizomes. Leaves are basal, alternate, and sometimes whorled in arrangement with parallel venation. The inflorescence is a raceme or solitary flower. Flowers are radially symmetrical with parts occurring in 3’s, and separate but undifferentiated sepals and petals (tepals) that may be spotted or striped. The fruit is a capsule. Some of the genera in the lily family include: • Erythronium (fawn lily) • Fritillaria (chocolate lily) • Lilium (lily) • Tulipa (tulip) Ranunculaceae – buttercup family The buttercup family, Ranunculaceae is composed of herbaceous annuals or perennials, woody shrubs, and lianas. Leaves are typically alternate, sometimes opposite in arrangement, and simple or compound with lobed or dissected margins. The inflorescence is a cyme or solitary flower. Flower sepals and petals are often similar, separate and radially symmetric. Flowers may have few to many petals, often with many stamens and carpels, and produce follicle fruit. Examples of genera in the buttercup family are: • Aquilegia (columbine) • Clematis (leather flower) • Delphinium (larkspur) • Helleborus (hellebore) • Ranunculus (buttercup) Rosaceae – rose family The rose family, Rosaceae is a large and important family of woody and herbaceous, deciduous and evergreen plants. It is valued for its bush and tree fruits and for many popular horticultural ornamental plants. A few commonly grown rosaceous plants include: • Cotoneaster (cotoneaster) • Fragaria (strawberry) • Malus (crabapple) • Spiraea (spirea) Common features of these genera include simple rotate flowers with 5 separate petals, sepals, and stamens, and simple or multiple fleshy or achene fruits. Leave are alternate or basal, simple or compound, sometimes toothed and often with stipules. Spines, thorns, and prickles are prevalent in the rose family. Sapindaceae – soapberry family The soapberry family, Sapindaceae is a large family of about 140 genera of trees and shrubs, lianas, and vines. Family members such as maples and buckeyes are valued for lumber and ornament. A few examples of sapindaceous plants include: • Acer (maple) • Aesculus (buckeye, horse chestnut) • Koelreuteria (golden rain tree) Some genera in Sapindaceae, including Acer (maple) are lactiferous, i.e. containing a milky sap. Maples and buckeyes include mostly deciduous trees and shrubs with petiolate, opposite leaves that are often simple, lobed or dissected, or pinnate, ternate, or palmately compound. Leaf venation is palmate or pinnate and leaflet margins may be entire, crenate, serrate, or dentate. The flowers are unisexual or bisexual in racemes, panicles or corymbs. The fruit is typically a distinctive samara in the maples, while the buckeyes produce globular dehiscent capsules with poisonous nuts. While there are several hundred plant families, an introduction to some additional families is available at this link to Plant Families [New Tab][2]. Review Identify the family name for each plant genus. An interactive or media element has been excluded from this version of the text. You can view it online here: kpu.pressbooks.pub/plant-identification/?p=133 1. https://plantdatabase.kpu.ca/ 2. http://theseedsite.co.uk/families.html

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_I_(Nakano)/01%3A_Plant_Identification/1.17%3A_Plant_Family_Characteristics.txt

Learning Objectives • Identify plants used in all segments of horticulture. The horticultural use of plants for decoration, food, medicine, and materials spans the history of human development on earth. While early European explorers to North America described the new world as untouched wilderness, generations of Indigenous residents used plants for decoration and ritual and managed growing conditions for food for thousands of years. The relationship between people, plants, and the environment on the Pacific coast of North America is described at this link to the Garry oak ecosystem [New Tab][1]. The early European plant explorer, Archibald Menzies has been credited as the first discoverer, describer, and collector of a number of plants whose provenance is the Pacific Northwest. Provenance refers to the populations of plants that occur naturally in local regions. For example, Pseudotsuga menziesii (Douglas fir) and Arbutus menziesii (Pacific madrone) both occur naturally in the Pacific Northwest. A plant’s nativity or provenance can be determined either geographically or politically. Acer saccharum (sugar maple), is native to central eastern North America, in other words, a Canadian native, but not a Pacific Northwest native. Similarly, Artemisia tridentata (big sagebrush) and Rhusglabra (smooth sumac) are native to interior British Columbia, but only to the dry interior valleys, not to the coast. Native Plants Plants that occur naturally in a place are considered native or indigenous to a place. Native plants have undergone genetic adaptations that have allowed them to evolve within the physical, chemical, and biological conditions of local ecosystems. As such, they function as part of a biodiverse community of organisms that includes plants, animals, and microorganisms adapted to local environmental conditions. In North America, an indigenous designation is usually applied to plants that were present before first contact with Europeans. Thus, Plantago spp. (plantains), although widespread here, are not considered native since they were brought here as a result of immigration by early European settlers. However, the influences of climate change and globalization will likely redefine what it means to be indigenous. Native gardening with indigenous plants that are appropriate to the conditions and geography of a given area can simulate the biodiversity of a natural habitat. Native plant gardens locally frequently include plants that are not native to the Lower Mainland of British Columbia, but also include plants native to other parts of the Pacific Northwest. For instance, Quercusgarryana (Garry oak), also discovered and described by Archibald Menzies, is now grown in gardens in the Lower Mainland, but is only found naturally in rain shadow climates, such as on southern Vancouver Island. While not all native plants may be garden worthy for ornamental impact, those chosen from the regional locality of a garden will often blend appropriately and will be among the best adapted to local moisture, soil, and climatic conditions. Although native plants are not immune to pest and disease problems, the majority of locally native plants seem to attract fewer problems than many exotics do. Efforts to restore natural habitats using provenance-specific plants grown from locally sourced seed perform better than non-natives when established in these areas. However, changing climate patterns and the impacts of urbanization will likely have consequences for plant provenance. Natural Habitats Natural habitats provide the resources that enable indigenous plants to persist and thrive in existing growing conditions. Examples of natural habitats commonly used as horticultural garden themes include alpine, woodland, Mediterranean, and bog. The growth characteristics of plants native to these habitats have been shaped by differences in elevation, temperature range, precipitation, soil types and geology, and biological and chemical factors. Over time, indigenous species successfully adapted to the habitat conditions by developing specialized features for survival. Some features associated with alpine, woodland, Mediterranean, and bog plants are described below. Additional information about how evolution and natural habitats have influenced plant adaptations is available at this link to the Missouri Botanical Garden [New Tab][2]. Alpine plants True alpine plants are well adapted to the harsh environments of high elevations. Above tree line, low temperatures, high sunlight, constant wind, dryness, and a short growing season are typical. Plant adaptations include growth low to the ground, a compact cushion or mat habit, and thick, waxy evergreen or pubescent (hairy), or curly leaves. Alpines, such as Campanula spp. (bell flower) flower in late spring and early summer and may have deep or extensive roots or below ground storage organs to persist in thin, low nutrient mountain soils. Although well adapted for extreme temperatures, alpine plants are typically intolerant of constant wetness around the roots and warm and humid summer conditions. Information about these specialized plants is available at this link to Adaptations to Alpine Plants [New Tab][3]. Woodland understory plants The temperate woodland habitat is characterized by distinct growing seasons, a dormant period, relatively consistent precipitation, and rich soils. Trees dominate this habitat forming an overhead canopy that shades and cools the understory and forest floor to varying degrees. Woodland understory plants include layers of woody shrubs and herbaceous plants that are adapted in size, form, shade tolerance, and slow growth or dormancy when light and water are limited. Understory plants such as Hydrangea quercifolia (oakleaf hydrangea) flower in late winter to early summer, before the leaves of deciduous shade trees fully emerge. Depending on the amount of light available, some understory plants have distinctive leaf color and patterns of ornamental interest in gardens. Examples of understory plants for garden use are shown at this link to Creative Woodland Garden Ideas [New Tab][4]. Mediterranean plants Mediterranean plants, such as Cotinus coggygria (smoke bush) and Lavandula spp. (lavender)are adapted to short, mild, and wet winters and long, warm, and dry summers. Some are short, dense, and shrubby evergreens that are suited to well drained soils, drought, and fire. Leaves may be leathery or reduced in size, and aromatic with thick, waxy or hairy coverings to reduce water loss, and bluish-grey (glaucous) or light in color to reflect excessive light. Some examples of naturally occurring vegetation are listed at this link to the Mediterranean climate Wikipedia [New Tab][5]. Bog plants Bogs and freshwater habitats are typically oxygen and nutrient poor with acidic pH conditions. Quercus palustris (pin oak) is an example of a tree that naturally grows in these conditions. Bog plants are adapted to growing in standing water while marginal plants such as Irissiberica (Siberian iris) and Typha spp. (cattail) thrive in waterlogged soils and shallow waters with short term dryness. Some bog and marginal plants such as Juncus effusus ‘Spiralis’ have striking foliage and make good choices for planting areas with limited or poor drainage. Information about the bog habitat is available at this link to Plants of the Bog [New Tab][6]. 1. http://www.goert.ca/about/why_important.php 2. http://www.mbgnet.net/bioplants/adapt.html 3. https://www.coursera.org/lecture/mountains-101/9-4-adaptations-of-alpine-plants-OFWIc 4. https://www.youtube.com/watch?v=69sN3JOHhDc&feature=youtu.be 5. en.Wikipedia.org/wiki/Mediterranean_climate#Mediterranean_biome ↵ 6. https://youtu.be/kzbhVhXul3E

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_I_(Nakano)/02%3A_Plant_Requirements_and_Use/2.01%3A_Plant_Habitats.txt

Learning Objectives • Describe horticultural plant use categories. Horticulture production provides plant resources for a wide range of functional, cultural, and aesthetic garden purposes. Ornamental plants are used for environmental enhancement, food production, and re-vegetation of damaged ecosystems, as well as for their visual and sensory appeal in landscapes and gardens. Some common categories of plant use are bedding plants and cut flowers, trees and shrubs, and ground covers and climbers. Bedding plants and Cut Flowers Bedding plants, such as Lobelia erinus (lobelia) and Petunia x hybrida (petunia) are grown in greenhouses and nurseries for seasonal interest in gardens and landscapes. They are typically tender and half-hardy annuals, biennials, and some perennials that grow quickly and provide a vibrant display of color in beds, containers, and hanging baskets. Cut flowers produced by the floral industry include both herbaceous and woody flowering plants and cut greens for specialty services. This link provides more information about the Variety of Flowers Grown in Canada [New Tab][1]. Trees and Shrubs Trees such as Acer rubrum (red maple) and Quercus rubra (red oak) grow from single stems while some like Acer circinatum (vine maple) have two or three main stems. This distinguishes trees from shrubs with several or many stems branching from or near soil level. Whether deciduous or evergreen, trees are generally larger than shrubs however, their shape and height can vary from dwarf cultivars 1 meter high, to grafted standards on 2 meter rootstocks and specimens of 90 meters or more. Tree selection must account for mature height and spread to ensure adequate space in the landscape. Trees with year-round interest in form, foliage, flower, fruit, and bark are commonly grown in open sites as specimen plants. They may serve as a focal point for an entrance or as a special accent in the garden. On large sites, trees are often planted in groups to form woodlands or hedging. Strategic planting of trees in urban environments can channel air movement, shade and cool microclimates, and provide barriers for noise and security, as well as frame or screen views. Shrubs such as Cornus alba ‘Elegantissima’ (silverleaf dogwood) and Hibiscus syriacus (hardy hibiscus) are valued for their ornamental features and varied growth forms. Shrub sizes range from 0.15 meter to about 6 meters. Deciduous and broadleaf evergreens, variegated foliage, fragrant and showy flowers and fruits, as well as decorative stems and buds provide year round interest and variety in mixed borders and container planting. Shrubs are commonly massed for effect, planted in small groups in mixed plantings, or used as screens and hedging. The wide selection of shrubs produced by nurseries provides for most garden conditions. Groundcovers Groundcover plants such as Ajuga reptans (bugleweed) are adapted with creeping and carpeting habits and are often used under woodland and shrub plantings, and for covering and stabilizing some slopes. Plant runners (stolons) that root where they touch the ground and spreading underground stems (rhizomes) that send up new shoots and form colonies stabilize and cover bare soil reducing erosion, evaporation, and weed growth. Climbers Climbing plants, whether woody or herbaceous, deciduous or evergreen provide strong vertical elements and year round garden interest. Where space is limited, climbers such as Hydrangea anomala ssp. petiolaris (climbing hydrangea) may be the best option for screening and climate control. It is important to match the vigor, method of attachment, height, and spread of a climber with an appropriately sturdy support and adequate light exposure for flowering and fruiting. 1. https://youtu.be/CE3typCY4rs

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_I_(Nakano)/02%3A_Plant_Requirements_and_Use/2.02%3A_Plant_Use.txt

Learning Objectives • Recognize and describe plant growth characteristics. Plant form and growth habit are among the most noticeable and important features for identification purposes as well as for landscape plant selection. Plant form, the three-dimensional shape or silhouette outline of a plant, is determined by the habit or branching pattern. For example, plants with an excurrent growth habit have single, undivided trunks and lateral branches that typically produce an overall cone or pyramid-shaped form. This plant form and growth habit is characteristic of many gymnosperms such as Thuja plicata (western red cedar) and Pseudotsuga menziesii (Douglas fir). In contrast, decurrent, or sometimes called deliquescent growth habit exhibits several roughly equal branches arising from the trunk or stem that become the main structural system of the plant. This habit results in the typical rounded or spreading form of deciduous trees such as Acer macrophyllum (big leaf maple) or Acer platanoides (Norway maple) as well as many shrubs. Depending on the branching pattern, additional descriptive terms such as upright or horizontal, arching or weeping, open, twiggy or dense may be used for shrubs as well as for trees. Review Describe plant form and habit. Click the image hotspots. An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plant-identification/?p=143 Stem, Bark, and Bud Morphology In addition to plant form and habit, the winter identification of deciduous trees and shrubs depends on the morphology of stems, bark, bark, and buds. Stem color, surface texture and the presence of lenticels, small cork-like spots for gas exchange between plant tissues and air are characteristic for some species of Prunus (cherry). A cross section taken through a stem or shoot reveals soft plant tissue, the pith. The color and texture of the pith may be used for distinguishing between similar plant types, such as species of Cornus (dogwood). Pith may be brown or white, variably shaped, and uniformly solid, chambered or hollow as illustrated in Figure 20.1. Figure 20.1 Pith types Bark, the dead outer protective tissue of woody plants can vary greatly in appearance, thickness, and texture as a tree or shrub matures. In addition to plant identification, plant bark may have highly ornamental value in the landscape. Color change, peeling and exfoliation, and smooth, furrowed (grooved), ridged and plate-like are some common descriptors for bark. The bark of Platanus x acerifolia (London plane tree) is valuable for identification as well ornament, as described in this link to Trees with Don Leopold – London plane tree [NewTab][1]. Buds, condensed shoots containing a new leaf, leaf cluster, or flower are located in leaf axils and at tips of stems. In general, a flower bud appears somewhat larger and rounder than a vegetative bud. While bud shape, size, color, and surface texture vary by species, bud arrangement will be alternate, opposite or whorled on the stem. Bud scales, the protective covering of buds may be single, few, or many, and imbricated (overlapping) or not as shown in the Figure 20.2. The shape of a leaf scar, where a leaf falls off a twig, and the arrangement of vascular bundles within the leaf scar may also provide distinct identification characteristics as in Juglans spp. (walnut). Figure 20.2 Bud types found in woody plants The dichotomous key below differentiates bud characteristics for some common deciduous trees and shrubs. Plant information is available at this link to the KPU Plant Database [New Tab][2]. Dichotomous Key to Buds of Common Deciduous Trees and Shrubs 1.a. buds opposite or whorled on the stem ……………………………………………………….. go to 2 1.b. buds not opposite on the stems ………………………………………………………………….. go to 7 2.a. leaf scars oval or round, vein scars forming a ring ……………………….. Catalpa speciosa [New Tab][3] 2.b. leaf and vein scars not as above …………………………………………………………………. go to 3 3.a. buds valvate, appressed, brownish black …………………….. Cornus kousa var. chinensis [New Tab][4] 3.b. buds acute or swollen, red or green ……………………………………………………………. go to 4 4.a. buds small or narrow, with few obvious scales, the scales more or less valvate (i.e., meeting at the edges) …………………………………………………………………………………………go to 5 4.b. buds large, with several imbricate (overlapping) scales ……………………………… go to 6 5.a. buds conical, the outer scales shiny red, the bud with a short fringe of hairs at its base……………………………………………………………………………………………………….. Acer circinatum [New Tab][5] 5.b. buds conical, the outer bud scales green or red, hairs extending half the height of the bud………………………………………………………………… Acer palmatum Atropurpureum Group [New Tab][6] 6.a. buds brown large, ovoid, and varnished with sticky gum……. Aesculus hippocastanum [New Tab][7] 6.b. buds smooth, leaf scars small, with a single vein scar……………………… Syringa vulgaris [New Tab][8] 7.a. buds narrowly conical and bud scales imbricate ……………………………… Fagus sylvatica [New Tab][9] 7.b. the bud scales imbricate or valvate, or the buds covered by a single scale …………………………………………………………………………………………………………………………… go to 8 8.a. buds stalked ………………………………………………………………………………………….. Alnus rubra [New Tab][10] 8.b. buds not stalked ………………………………………………………………………………………….. go to 9 9.a. twigs yellow, the buds flattened, appressed to stems and covered by a single, silky-downy bud scale………………………………………………………… Salix sepulcralis var. chrysochoma [New Tab][11] 9.b. twigs not yellow, buds not covered by a single scale ………………………………… go to 10 10.a. lateral buds superposed, slightly hairy; leaf scars v-shaped, prominent; pith chambered ………………………………………………………………………. Juglans nigra (black walnut)[New Tab][12] 10.b. buds not superposed; leaf scars not v-shaped; pith not chambered …………………………………………………………………………………………………………………………. go to 11 11.a. buds 2 mm long, rounded to shortly acute, with several reddish-brown scales ………………………………………………………………………………………………………….. Cotinus coggygria [New Tab][13] 11.b. buds 2 mm long, ovoid to acute, with several rows of imbricate scales …………………………………………………………………………………………….………………………….. go to 12 12.a. buds of 2 sizes, bud scales glabrous, leaf scars triangular with 3 vein scars …………………………………………………………………………………………………………… Prunus ‘Kanzan’ [New Tab][14] 12.b. buds of 1 kind; buds scales fringed with hair; leaf scars with 5 or more vein scars ……………………………………………………………………………………………………………….. Quercus robur [New Tab][15]

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_I_(Nakano)/02%3A_Plant_Requirements_and_Use/2.03%3A_Plant_Growth_Characteristics.txt

Learning Objectives • Describe the characteristics of weedy species. Whether a plant is classified as a weed or not depends on its location and relationship to human activities. Plants in gardens, agricultural, and natural settings that are considered undesirable or out of place due to appearance, contamination, or competition with desirable plants are often classed as weeds. Aquatic and terrestrial weedy species transported or migrated beyond their natural range that become established in a new area may pose significant impact or injury to economic, environmental, or human health. These are categorized as invasive, noxious, or nuisance species by governing authorities. Examples of species monitored for management in British Columbia are listed at this link to Invasive Terrestrial Plants [New Tab][1]. Common characteristics of weedy species include aggressive growth, competition with other plants for light, water, nutrients, and space, an ability to grow in a wide range of soils and adverse conditions, and resistance to control measures. Some cultivated plants such as Lythrum salicaria ( purple loosestrife), Vinca minor (periwinkle) and Lamiastrum galeobdolon (yellow archangel) overwhelm and displace other plants and ecosystems. A number of unwanted horticultural plants are identified in this link to the Field Guide to Noxious Weeds and Other Selected Invasive Plants of British Columbia [New Tab][2]. When environmental conditions in a site change there will always be a change in the plant make up. For instance, where the ground is fully covered with vegetation there will be no bare soil available for weeds to inhabit. Disturbances in vegetation cover and changes in environmental conditions due to natural events or human activities and management practices create opportunities for species with adapted life cycles and growth characteristics to become established, reproduce, and colonize a site. Knowledge of family characteristics and life cycles is important for proper landscape and garden plant selection. Species characteristics such as generalist pollination requirements, diverse seed and vegetative dispersal methods, and the ability to adapt quickly to new environmental conditions may be indicators of potential invasive growth. Combinations of these characteristics are commonly found in the Asteraceae (aster), Brassicaceae (mustard), Polygonaceae (knotweed), Fabaceae (pea), and Euphorbiaceae (spurge) families, as well as others. Weeds are typically classified according to their life cycle. Depending on the degree of disturbance to a site, herbaceous plant species with annual and biennial life cycles will be the first to colonize followed by perennial herbaceous and woody plants. Annual weeds such as Galium aparine (cleavers) that produce high numbers of seed occur most frequently in regularly cultivated and disturbed areas such as vegetable gardens or annual borders. Their rapid growth can smother slower-growing plants and compete for moisture and light. An advantage for winter annuals such as Capsella bursa-pastoris (shepherd’s purse) and Cardamine oligosperma (snapweed) is that they germinate in the fall, overwinter as a rosette of leaves, and flower and produce many seeds in late winter and early spring. Biennial weeds such as Echium vulgare (blueweed) usually produce only a rosette of leaves in the first growing season. Energy stored in the roots over a winter cold period enables the plant to bolt (flower), produce seeds, and then die in the next season. Removal of the rosette before flowering stops the biennial life cycle. Herbaceous perennial weeds such as Cirsium vulgare (Canada thistle), Heracleum mantegazzianum (giant hogweed), and Equisetum arvense (horsetail) and woody species such as Buddleja davidii (butterfly bush) and Rubus armeniacus (Himalayan blackberry) survive adverse conditions by storing food reserves in roots, rhizomes, and tubers or bulbs in some species. Important control measures include early identification and removal before establishment. In situations where weed populations remain below established thresholds of impact or injury for a given site and use, they do provide ecosystem benefits. For instance weed cover can provide protection from soil erosion, produce pollen, nectar, and habitat for beneficial organisms and wildlife, serve as indicators of soil conditions, and contribute organic matter for soil enhancement, as well as provide food and medicinal products for human use. Practice For each plant, access the correct common name and family name available at this link to the KPU Plant Database [New Tab][3]. An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plant-identification/?p=145 1. www2.gov.bc.ca/gov/content/environment/plants-animals-ecosystems/invasive-species/plants/terrestrial ↵ 2. https://www.bcinvasives.ca/documents/Field_Guide_to_Noxious_Weeds_Final_WEB_09-25-2014.pdf 3. https://plantdatabase.kpu.ca/plant/search.gsp

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_I_(Nakano)/02%3A_Plant_Requirements_and_Use/2.04%3A_Characteristics_of_weedy_species.txt

Learning Objectives • Explain plant hardiness zones. Over the course of their evolution, plant species adapt to the climate variations of a region. Therefore, the ultimate deciding factor in whether a plant will survive in a given location (with adequate supplies of light, moisture, and nutrients) is quite simply the lowest temperature it will have to endure. Although several factors such as length of frost free period, rainfall, snow cover, wind, and soil type affect the hardiness of a plant, in temperate climates the minimum temperature during the winter is the most important element in plant survival. Plant Hardiness Zones Average annual minimum temperatures are determined for locations throughout North America. Plotting areas with similar average minimum temperatures yields a temperature zone map. Zones numbered 0 to 9 relate to the average annual minimum temperature calculated for that zone. The zones are divided into “a” and “b,” the “b” area representing the mildest part of the zone. Plants designated “a” with the zone number are hardy in the colder part of that zone; those designated “b” in only the milder section. Plant hardiness ratings are determined by testing over several years at agricultural research and testing stations as well as private nurseries and gardens. A plant which is hardy to a particular zone can be expected to survive in all regions on the map which have an average annual minimum temperature equal to or greater than the hardiness zone rating for that plant. Currently, two hardiness zone maps are widely used in North America: • Agriculture Canada • United States Department of Agriculture (U.S.D.A.) In Canada, horticulturists often refer to the Agriculture Canada hardiness zone map. It is similar to the U.S.D.A. system except that the temperature range for each of the 9 zones is given in degrees Celsius instead of degrees Fahrenheit. Examples of hardiness zones for some Canadian communities are listed below. Location Zone Edmonton, Alta. 2 Prince George, B.C. 3 Ottawa, Ont. 3 Fredericton, N.B. 5 Langley, B.C. 7 Vancouver, B.C. 8 A map that outlines all of the different zones is available at this link to Canada’s Plant Hardiness Zones [New Tab][1]. Horticulturists in the United States most commonly use the U.S.D.A. map. It divides the United States into 13 zones based on the average annual extreme minimum temperature with zone 1 being the coldest (-60 F.) and zone 13 being the warmest (above 60 F.). It is included in many books and catalogs, and is available at this link to the USDA Plant Hardiness Zone Map [New Tab][2]. Changing Climate Means Changing Hardiness Zones Natural Resources Canada updated the plant hardiness zones map to include, among other factors, the effects of elevation on plant hardiness. The update provided evidence that there have been marked changes in hardiness zones in Western Canada. While the map expanded the factors affecting plant hardiness, local variability in topography, shelter, and snow cover were not captured. In an effort to increase knowledge about the effect of changing climate climate on the range of species growth in different locales, Natural Resources Canada created an interactive zone map where experts and gardeners contribute information about plant survival at this link to Canada’s Plant Hardiness Site [New Tab][3]. Review Identify the hardiness zone for each plant available at this link to the KPU Plant Database [New Tab][4]. An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plant-identification/?p=147 2.06: Plant Requirements Learning Objectives • Identify plant requirements for woody and non-woody plants. Although plants established in cultivated gardens and landscapes are not the result of long-term evolution, the concept of “right plant, right place” can be effectively applied in these settings. Plant selection and placement that matches a species growth characteristics with existing conditions and available maintenance requirements supports healthy growth and vigor. Cultural requirements are defined as site conditions that influence plant growth and longevity. Conditions typically include diverse combinations of light exposure, moisture conditions, soil types and nutrient availability, and hardiness zones. Depending on the site, conditions may also include plant tolerance for wind, salt, and drought. While garden plants require some routine maintenance for healthy growth, proper plant selection and planting practices decrease unsustainable maintenance inputs and reduce incidences of pest and disease and plant failure or death. Maintenance requirements are related to factors that influence plant growth and development. Light exposure is typically classified as full (six or more hours of direct sunlight per day), part (four to six hours of sunlight) or part shade (two to fours hours of sunlight), and shade (less than two hours of sunlight per day). Water requirements and existing moisture conditions range from dry or xeric, to well-drained, and poorly drained or wet depending on the amount of rainfall or irrigation, the site slope, and the soil type. Soil drainage will be influenced by the amount and arrangement of particles of sand, silt and clay. Gravel or sandy soils tend to drain rapidly and have low nutrient levels because they are made up of relatively large particles with large pores or spaces between them. In contrast, clay and silt soils are composed of tiny particles separated by minute spaces that tend to retain nutrients and drain more slowly. Garden loam that is fertile and well drained is the result of a balanced combination sand, silt, and clay. Information from resources about plant growth requirements for light, water, soils and nutrients combined with knowledge of prevailing conditions and smaller-scale garden variations in temperature, air quality, wind, and humidity, and environmental stresses or pests and disease supports the identification of the right plant for the right place. Review Identify plant requirements for light exposure, soil type, and water use available at this link to the KPU Plant Database [New Tab][1]. An interactive or media element has been excluded from this version of the text. You can view it online here: https://kpu.pressbooks.pub/plant-identification/?p=149

textbooks/bio/Agriculture_and_Horticulture/Red_Seal_Landscape_Horticulturist_Identify_Plants_and_Plant_Requirements_I_(Nakano)/02%3A_Plant_Requirements_and_Use/2.05%3A_Plant_Hardiness.txt

• 1.1: Introduction - Basic Biology The most obvious thing about living organisms is their astounding diversity. Estimates put the number of eukaryotic species at about 8.7 million, while bacteria account for anywhere between 107 and 109 different species. The number of species of archaea is still uncertain, but is expected to be very large. These organisms, representing the three great domains of life, together occupy every environmental niche imaginable. • 1.2: Introduction - Basic Chemistry To understand biochemistry, one must possess at least a basic understanding of organic and general chemistry. In this brief section, we will provide a rapid review of the simple concepts necessary to understand cellular chemistry. Chemistry is chemistry, whether in a cell or outside it, but biological chemistry is a particular subset of organic chemistry that often involves enormous macromolecules, and that happens in the aqueous environment of the cell. • 1.3: Introduction - Water and Buffers When it comes to water, we’re literally drowning in it, as water is by far the most abundant component of every cell. To understand life, we begin the discussion with the basics of water, because everything that happens in cells, even reactions buried deep inside enzymes, away from water, is influenced by water’s chemistry. 01: In The Beginning Figure 1.2 Slices of cork as seen by Hooke YouTube Lectures by Kevin HERE & HERE Graphic images in this book were products of the work of several talented students. Links to their Web pages are below Click HERE for Martha Baker’s Web Page Click HERE for Pehr Jacobson’s Web Page Click HERE for Aleia Kim’s Web Page Click HERE for Penelope Irving’s Web Page Problem set related to this section HERE Point by Point summary of this section HERE To get a certificate for mastering this section of the book, click HERE Kevin Ahern’s free iTunes U Courses - Basic / Med School / Advanced Biochemistry Free & Easy (our other book) HERE / Facebook Page Kevin and Indira’s Guide to Getting into Medical School - iTunes U Course / Book To see Kevin Ahern’s OSU ecampus courses - BB 350 / BB 450 / BB 451 To register for Kevin Ahern’s OSU ecampus courses - BB 350 / BB 450 / BB 451 Biochemistry Free For All Facebook Page (please like us) Kevin Ahern’s Web Page / Facebook Page / Taralyn Tan’s Web Page Kevin Ahern’s free downloads HERE OSU’s Biochemistry/Biophysics program HERE OSU’s College of Science HERE Oregon State University HERE Email Kevin Ahern / Indira Rajagopal / Taralyn Tan 1.02: Introduction - Basic Chemistry Source: BiochemFFA_1_2.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy “Organic chemistry is the chemistry of carbon compounds. Biochemistry is the chemistry of carbon compounds that crawl” -Michael Adams. To understand biochemistry, one must possess at least a basic understanding of organic and general chemistry. In this brief section, we will provide a rapid review of the simple concepts necessary to understand cellular chemistry. Chemistry is chemistry, whether in a cell or outside it, but biological chemistry is a particular subset of organic chemistry that often involves enormous macromolecules, and that happens in the aqueous environment of the cell. Covalent bonds, as you know, are the result of sharing of electrons between two atoms. Ionic bonds, by contrast, are formed when one atom donates an electron to another, such as in the formation of sodium chloride. Single covalent bonds can rotate freely, but double bonds cannot. Single bonds around a carbon atom are arranged in a tetrahedron with bond angles of 109.5° relative to each other, with the carbon at the center (Figure 1.19). Double bonded carbons create a planar structure with bond angles typically of about 120°. Electronegativity Electronegativity is a measure of the affinity a nucleus has for outer shell electrons (Table 1.2). High electronegativity corresponds to high affinity. Electrons in a covalent bond are held closer to the nucleus with a greater electronegativity compared to a nucleus with lower electronegativity. Table 1.2 Image by Aleia Kim For example, in a molecule of water, with hydrogen covalently bonded to oxygen, the electrons are “pulled” toward the oxygen, which is more electronegative. Because of this, there is a slightly greater negative charge near the oxygen atom of water, compared to the hydrogen (which, correspondingly has a slightly higher positive charge). This unequal charge distribution sets up a dipole, with one side being somewhat negative and the other somewhat positive. Because of this, the molecule is described as polar. Hydrogen bonds between water molecules are the result of the attraction of the partial positive and partial negative charges on different water molecules (Figure 1.20). Hydrogen bonds can also form between hydrogens with a partial positive charge and other strongly electronegative atoms, like nitrogen, with a partial negative charge. It is important to remember that hydrogen bonds are interactions between molecules (or parts of molecules) and are not bonds between atoms, like covalent or ionic bonds. Bonds between hydrogen and carbon do not form significant partial charges because the electronegativities of the two atoms are similar. Consequently, molecules containing many carbon-hydrogen bonds will not form hydrogen bonds and therefore, do not mix well with water. Such molecules are called hydrophobic. Other compounds with the ability to make hydrogen bonds are polar and can dissolve in water. They are called hydrophilic. Molecules possessing both characteristics are called amphiphilic. Weak interactions Hydrogen bonds are one kind of electrostatic (i.e., based on charge) interaction between dipoles. Other forms of electrostatic interactions that are important in biochemistry include weak interactions between a polar molecule and a transient dipole, or between two temporary dipoles. These temporary dipoles result from the movement of electrons in a molecule. As electrons move around, the place where they are, at a given time, becomes temporarily more negatively charged and could now attract a temporary positive charge on another molecule. Since electrons don’t stay put, these dipoles are very short-lived. Thus, the attraction that depends on these dipoles fluctuates and is very weak. Weak interactions like these are sometimes called van der Waals forces. Many molecular interactions in cells depend on weak interactions. Although the individual hydrogen bonds or other dipole-dipole interactions are weak, because of their large numbers, they can result in quite strong interactions between molecules. Oxidation/reduction Oxidation involves loss of electrons and reduction results in gain of electrons. For every biological oxidation, there is a corresponding reduction - one molecule loses electrons to another molecule. Oxidation reactions tend to release energy and are a source of bioenergy for chemotrophic cells. Ionization Ionization of biomolecules, by contrast does not involve oxidation/reduction. In ionization, a hydrogen ion (H+) leaves behind its electron as it exits (leaving behind a negative charge) or joins a group (adding a positive charge). Biological ionizations typically involve carboxyl groups or amines, though phosphates or sulfates can also be ionized. A carboxyl group can have two ionization states - a charge of -1 corresponds to the carboxyl without its proton and a charge of zero corresponds to the charge of the carboxyl with its proton on. An amine also has two ionization states. A charge of zero corresponds to a nitrogen with three covalent bonds (usually in the form of C-NH2) and a charge of +1 corresponds to a nitrogen making four covalent bonds (usually X-NH3 +). Stereochemistry A carbon has the ability to make four single bonds (forming a tetrahedral structure) and if it bonds to four different chemical groups, their atoms can be arranged around the carbon in two different ways, giving rise to stereochemical “handedness” (Figure 1.21). Each carbon with such a property is referred to as an asymmetric center. The property of handedness only occurs when a carbon has four different groups bonded to it. Enzymes have very specific 3-D structures, so for biological molecules that can exist in different stereoisomeric forms, an enzyme that synthesizes it would make only one of the possible isomers. By contrast, the same molecules made chemically (not using enzymes) end up with equal amounts of both isomers, called a racemic mix. Gibbs free energy The Gibbs free energy calculation allows us to determine whether a reaction will be spontaneous, by taking into consideration two factors, change in enthalpy (ΔH) and change in entropy (ΔS). The free energy content of a system is given by the Gibbs free energy ($G$) and is equal to the enthalpy ($H$) for a process minus the absolute temperature (T) times the entropy (S) $G = H = TS$ For a process, the change in the Gibbs free energy ΔG is given by $ΔG = ΔH - TΔS$ A negative $ΔG$ corresponds to release of free energy. Reactions that release energy are exergonic, whereas those that absorb energy are called endergonic. The biological standard Gibbs free energy change (ΔG°’) corresponds to the ΔG for a process under standard conditions of temperature, pressure, and at pH = 7. For a reaction $aA + bB \rightleftharpoons cC + dD,$ the equilibrium constant, $K_{eq}$ is equal to $K_{eq} = \dfrac{ [C]^c_{eq} [D]^d_{eq}}{[A]^a_{eq} [B]^b_{eq}}$ where $a$, $b$, $c$, and $d$ are integers in the balanced equation. Large values of $K_{eq}$ correspond to favorable reactions (more C and D produced than A and B) and small values of $K_{eq}$ mean the opposite. At equilibrium, $ΔG^{o\prime} = -RT \ln K_{eq}$ If a process has a $ΔG = Z$ and a second process has a $ΔG = Y$, then if the two processes are linked, $ΔG$ and $ΔG^{o \prime}$ values for the overall reaction will be the sum of the individual ΔG and ΔG°’ values. $ΔG_{total} = ΔG_1+ ΔG_2 = Z + Y$ $ΔG^{o \prime }_{total} = ΔG_1^{o \prime}+ ΔG_2^{o\prime}$ Catalysis Catalysis is an increase in the rate of a reaction induced by a substance that is, itself, unchanged by the reaction. Because catalysts remain unchanged at the end of a reaction, a single catalyst molecule can be reused for many reaction cycles. Proteins that catalyze reactions in cells are called enzymes, while ribozymes are RNA molecules that act as catalysts.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/01%3A_In_The_Beginning/1.01%3A_Introduction_-_Basic_Biology.txt

Source: BiochemFFA_1_3.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy When it comes to water, we’re literally drowning in it, as water is by far the most abundant component of every cell. To understand life, we begin the discussion with the basics of water, because everything that happens in cells, even reactions buried deep inside enzymes, away from water, is influenced by water’s chemistry. The water molecule has wide ‘V’ shape (the HO-H angle is 104°) with uneven sharing of electrons between the oxygen and the hydrogen atoms (Figure 1.23). Oxygen, with its higher electronegativity, holds electrons closer to itself than the hydrogens do. The hydrogens, as a result, are described as having a partial positive charge (typically designated as δ+) and the oxygen has a partial negative charge (written as δ- ). Thus, water is a polar molecule because charges are distributed around it unevenly, not symmetrically. Water as a solvent Water (Figure 1.23) is described as a solvent because of its ability to solvate (dissolve) many, but not all, molecules. Molecules that are ionic or polar dissolve readily in water, but non-polar substances dissolve poorly in water, if at all. Oil, for example, which is non-polar, separates from water when mixed with it. On the other hand, sodium chloride, which ionizes, and ethanol, which is polar, are able to form hydrogen bonds, so both dissolve in water. Ethanol’s solubility in water is crucial for brewers, winemakers, and distillers – but for this property, there would be no wine, beer or spirits. As explained in an earlier section, we use the term hydrophilic to describe substances that interact well with water and dissolve in it and the term hydrophobic to refer to materials that are non-polar and do not dissolve in water. Table 1.3 illustrates some polar and non-polar substances. A third term, amphiphilic, refers to compounds that have both properties. Soaps, for example are amphiphilic, containing a long, non-polar aliphatic tail and a head that ionizes. Table 1.3 Image by Aleia Kim Solubility The solubility of materials in water is based in free energy changes, as measured by ΔG. Remember, from chemistry, that H is the enthalpy (heat at constant pressure) and S is entropy. Given this, \[ΔG = ΔH - TΔS\] where T is the temperature in Kelvin. For a process to be favorable, the ΔG for it must be less than zero. From the equation, lowered ΔG values will be favored with decreases in enthalpy and/or increases in entropy. Let us first consider why non-polar materials do not dissolve in water. We could imagine a situation where the process of dissolving involves the “surrounding” of each molecule of the nonpolar solute in water, just like each sodium and each chloride ion gets surrounded by water molecules as salt dissolves. Water organization There is a significant difference, though between surrounding a non-polar molecule with water molecules and surrounding ions (or polar compounds) with water molecules. The difference is that since non-polar molecules don’t really interact with water, the water behaves very differently than it does with ions or molecules that form hydrogen bonds. In fact, around each non-polar molecule, water gets very organized, aligning itself regularly. As any freshman chemistry student probably remembers, entropy is a measure of disorder, so when something becomes ordered, entropy decreases, meaning the ΔS is negative, so the TΔS term in the equation is positive (negative of a negative). Since mixing a non-polar substance with water doesn’t generally have any significant heat component, the ΔG is positive. This means, then, that dissolving a non-polar compound in water is not favorable and does not occur to any significant extent. Further, when the non-polar material associates with itself and not water, then the water molecules are free to mix, without being ordered, resulting in an increase of entropy. Entropy therefore drives the separation of non-polar substances from aqueous solutions. Amphiphilic substances Next, we consider mixing of an amphiphilic substance, such as a soap, with water (Figure 1.24). The sodium ions attached to the fatty acids in soap readily come off in aqueous solution, leaving behind a negatively charged molecule at one end and a non-polar region at the other end. The ionization of the soap causes in an increase in entropy - two particles instead of one. The non-polar portion of the negatively charged soap ion is problematic - if exposed to water, it will cause water to organize and result in a decrease of entropy and a positive ΔG. Since we know fatty acids dissolve in water, there must be something else at play. There is. Just like the non-polar molecules in the first example associated with each other and not water, so too do the non-polar portions of the soap ions associate with each other and exclude water. The result is that the soap ions arrange themselves as micelles (Figure 1.25) with the non-polar portions on the interior of the structure away from water and the polar portions on the outside interacting with water. The interaction of the polar heads with water returns the water to its more disordered state. This increase in disorder, or entropy, drives the formation of micelles. As will be seen in the discussion of the lipid bilayer, the same forces drive glycerophospholipids and sphingolipids to spontaneously form bilayers where the non-polar portions of the molecules interact with each other to exclude water and the polar portions arrange themselves on the outsides of the bilayer (Figure 1.28). Yet another example is seen in the folding of globular proteins in the cytoplasm. Nonpolar amino acids are found in the interior portion of the protein (water excluded). Interaction of the non-polar amino acids turns out to be a driving force for the folding of proteins as they are being made in an aqueous solution. Hydrogen bonds The importance of hydrogen bonds in biochemistry (Figure 1.30) is hard to overstate. Linus Pauling himself said, “ . . . . I believe that as the methods of structural chemistry are further applied to physiological problems it will be found that the significance of the hydrogen bond for physiology is greater than that of any other single structural feature.” In 2011, an IUPAC task group gave an evidence-based definition of hydrogen bonding that states, “The hydrogen bond is an attractive interaction between a hydrogen atom from a molecule or a molecular fragment X–H in which X is more electronegative than H, and an atom or a group of atoms in the same or a different molecule, in which there is evidence of bond formation.” Partial Charges The difference in electronegativity between hydrogen and the molecule to which it is covalently bound give rise to partial charges as described above. These tiny charges (δ+ and δ- ) result in formation of hydrogen bonds, which occur when the partial positive charge of a hydrogen atom is attracted to the partial negative of another molecule. In water, that means the hydrogen of one water molecule is attracted to the oxygen of another (Figure 1.31). Since water is an asymmetrical molecule, it means also that the charges are asymmetrical. Such an uneven distribution is what makes a dipole. Dipolar molecules are important for interactions with other dipolar molecules and for dissolving ionic substances (Figure 1.32). Hydrogen bonds are not exclusive to water. In fact, they are important forces holding together macromolecules that include proteins and nucleic acids. Hydrogen bonds occur within and between macromolecules. The complementary pairing that occurs between bases in opposite strands of DNA, for example, is based on hydrogen bonds. Each hydrogen bond is relatively weak (compared to a covalent bond, for example - Table 1.4), but collectively they can be quite strong. Table 1.4 Image by Aleia Kim Benefits of weak interactions Their weakness, however, is actually quite beneficial for cells, particularly as regards nucleic acids (Figure 1.33). The strands of DNA, for example, must be separated over short stretches in the processes of replication and the synthesis of RNA. Since only a few base pairs at a time need to be separated, the energy required to do this is small and the enzymes involved in the processes can readily take them apart, as needed. Hydrogen bonds also play roles in binding of substrates to enzymes, catalysis, and protein-protein interaction, as well as other kinds of binding, such as protein-DNA, or antibody-antigen. As noted, hydrogen bonds are weaker than covalent bonds (Table 1.4) and their strength varies form very weak (1-2 kJ/mol) to fairly strong (29 kJ/mol). Hydrogen bonds only occur over relatively short distances (2.2 to 4.0 Å). The farther apart the hydrogen bond distance is, the weaker the bond is. The strength of the bond in kJ/mol represents the amount of heat that must be put into the system to break the bond - the larger the number, the greater the strength of the bond. Hydrogen bonds are readily broken using heat. The boiling of water, for example, requires breaking of H-bonds. When a biological structure, such as a protein or a DNA molecule, is stabilized by hydrogen bonds, breaking those bonds destabilizes the structure and can result in denaturation of the substance - loss of structure. It is partly for this reason that most proteins and all DNAs lose their native, or folded, structures when heated to boiling. Image by Aleia Kim Table 1.5 For DNA molecules, denaturation results in complete separation of the strands from each other. For most proteins, this means loss of their characteristic three-dimensional structure and with it, loss of the function they performed. Though a few proteins can readily reassume their original structure when the solution they are in is cooled, most can’t. This is one of the reasons that we cook our food. Proteins are essential for life, so denaturation of bacterial proteins results in death of any microorganisms contaminating the food. The importance of buffers Water can ionize to a slight extent (10-7 M) to form H+ (proton) and OH- (hydroxide). We measure the proton concentration of a solution with pH, which is the negative log of the proton concentration. pH = -Log[H+] If the proton concentration, [H+]= 10-7 M, then the pH is 7. We could just as easily measure the hydroxide concentration with the pOH by the parallel equation, pOH = -Log[OH- ] In pure water, dissociation of a proton simultaneously creates a hydroxide, so the pOH of pure water is 7, as well. This also means that pH + pOH = 14 Now, because protons and hydroxides can combine to form water, a large amount of one will cause there to be a small amount of the other. Why is this the case? In simple terms, if I dump 0.1 moles of H+ into a pure water solution, the high proton concentration will react with the relatively small amount of hydroxides to create water, thus reducing hydroxide concentration. Similarly, if I dump excess hydroxide (as NaOH, for example) into pure water, the proton concentration falls for the same reason. Acids vs bases Chemists use the term “acid” to refer to a substance which has protons that can dissociate (come off) when dissolved in water. They use the term “base” to refer to a substance that can absorb protons when dissolved in water. Both acids and bases come in strong and weak forms. (Examples of weak acids are shown in Table 1.5.) Strong acids, such as HCl, dissociate completely in water. If we add 0.1 moles (6.02x1022 molecules) of HCl to a solution to make a liter, it will have 0.1 moles of H+ and 0.1 moles of Cl- or 6.02x1022 molecules of each . There will be no remaining HCl when this happens. A strong base like NaOH also dissociates completely into Na+ and OH- . Weak Acids Weak acids and bases differ from their strong counterparts. When you put one mole of acetic acid (HAc) into pure water, only a tiny percentage of the HAc molecules dissociate into H+ and Ac- . Clearly, weak acids are very different from strong acids. Weak bases behave similarly, except that they accept protons, rather than donate them. Since we can view everything as a form of a weak acid, we will not use the term weak base here. Students are often puzzled and expect that [H+] = [A- ] because the dissociation equation shows one of each from HA. This is, in fact, true ONLY when HA is allowed to dissociate in pure water. Usually the HA is placed into solution that has protons and hydroxides to affect things. Those protons and /or hydroxides change the H+ and Aconcentration unequally, since A- can absorb some of the protons and/or HA can release H+ when influenced by the OH- in the solution. Therefore, one must calculate the proton concentration from the pH using the Henderson Hasselbalch equation. \[pH = pKa + log ([Ac- ]/[HAc])\] Image by Aleia Kim Table 1.6 You may wonder why we care about weak acids. You may never have thought much of weak acids when you were in General Chemistry. Your instructor described them as buffers and you probably dutifully memorized the fact that “buffers are substances that resist change in pH” without really learning what Clearing Confusion - this meant. Buffers are much too important to be thought of in this way. UPS Weak acids are critical for life because their affinity for protons causes them to behave like a UPS. We’re not referring to the UPS that is the United Parcel Service, but instead, to the encased battery backup systems for computers called Uninterruptible Power Supplies that kick on to keep a computer running during a power failure. The battery in a laptop computer is a UPS, for example. We can think of weak acids as Uninterruptible Proton Suppliers within certain pH ranges, providing (or absorbing) protons as needed. Weak acids thus help to keep the H+ concentration (and thus the pH) of the solution they are in relatively constant. Consider the bicarbonate/carbonic acid system. Figure 1.35 shows what happens when H2CO3dissociates. Adding hydroxide ions (by adding a strong base like NaOH) to the solution causes the H+ ions to react with OH- ions to make water. Consequently, the concentration of H+ ions would go down and the pH would go up. However, in contrast to the situation with a solution of pure water, there is a backup source of H+ available in the form of H2CO3. Here is where the UPS function kicks in. As protons are taken away by the added hydroxyl ions (making water), they are partly replaced by protons from the H2CO3. This is why a weak acid is a buffer. It resists changes in pH by releasing protons to compensate for those “used up” in reacting with the hydroxyl ions. Henderson-Hasselbalch It is useful to be able to predict the response of the H2CO3 system to changes in H+ concentration. The Henderson-Hasselbalch equation defines the relationship between pH and the ratio of HCO3 - and H2CO3. It is pH = pKa + log ([HCO3- ]/ [H2CO3]) This simple equation defines the relationship between the pH of a solution and the ratio of HCO3- and H2CO3 in it. The new term, called the pKa, is defined as pKa = -Log Ka, just as pH = -Log [H+]. The Ka is the acid dissociation constant and is a measure of the strength of an acid. For a general acid, HA, which dissociates as HA ⇄ H+ + A -, Ka = [H+][A- ]/[HA] Thus, the stronger the acid, the more protons that will dissociate from it when added to water and the larger the value its Ka will have. Large values of Ka translate to lower values of pKa. As a result, the lower the pKa value is for a given acid, the stronger the weak acid is. Constant pKa Please note that pKa is a constant for a given acid. The pKa for carbonic acid is 6.37. By comparison, the pKa for formic acid is 3.75. Formic acid is therefore a stronger acid than acetic acid. A stronger acid will have more protons dissociated at a given pH than a weaker acid. Now, how does this translate into stabilizing pH? Figure 1.35 shows a titration curve. In this curve, the titration begins with the conditions at the lower left (very low pH). At this pH, the H2CO3 form predominates, but as more and more OH- is added (moving to the 45 Why do we care about pH? Because biological molecules can, in some cases, be exquisitely sensitive to changes in it. As the pH of a solution changes, the charges of molecules in the solution can change, as you will see. Changing charges on biological molecules, especially proteins, can drastically affect how they work and even whether they work at all right), the pH goes up, the amount of HCO3- goes up and (correspondingly), the amount of H2CO3 goes down. Notice that the curve “flattens” near the pKa (6.37). Buffering region Flattening of the curve tells us is that the pH is not changing much (not going up as fast) as it did earlier when the same amount of hydroxide was added. The system is resisting a change in pH (not stopping the change, but slowing it) in the region of about one pH unit above and one pH unit below the pKa. Thus, the buffering region of the carbonic acid/ bicarbonate buffer is from about 5.37 to 7.37. It is maximally strong at a pH of 6.37. Now it starts to become apparent how the buffer works. HA can donate protons when extras are needed (such as when OH- is added to the solution by the addition of NaOH). Similarly, A- can accept protons when extra H+ are added to the solution (adding HCl, for example). The maximum ability to donate or accept protons comes when [A- ] = [HA] This is consistent with the Henderson Hasselbalch equation and the titration curve. When [A- ] = [HA], pH = 6.37 + Log(1). Since Log(1) = 0, pH = 6.37 = pKa for carbonic acid. Thus for any buffer, the buffer will have maximum strength and display flattening of its titration curve when [A- ] = [HA] and when pH = pKa. If a buffer has more than one pKa (Figure 1.36), then each pKa region will display the behavior. Buffered vs non-buffered To understand how well a buffer protects against changes in pH, consider the effect of adding .01 moles of HCl to 1.0 liter of pure water (no volume change) at pH 7, compared to adding it to 1.0 liter of a 1M acetate buffer at pH 4.76. Since HCl completely dissociates, in 0.01M (10-2 M) HCl you will have 0.01M H+. For the pure water, the pH drops from 7.0 down to 2.0 (pH = -log(0.01M)). By contrast, the acetate buffer’s pH after adding the same amount of HCl is 4.74. Thus, the pure water solution sees its pH fall from 7 to 2 (5 pH units), whereas the buffered solution saw its pH drop from 4.76 to 4.74 (0.02 pH units). Clearly, the buffer minimizes the impact of the added protons compared to the pure water. Buffer capacity It is important to note that buffers have capacities limited by their concentration. Let’s imagine that in the previous paragraph, we had added the 0.01 moles HCl to an acetate buffer that had a concentration of 0.01M and equal amounts of Ac- and HAc. When we try to do the math in parallel to the previous calculation, we see that there are 0.01M protons, but only 0.005M A- to absorb them. We could imagine that 0.005M of the protons would be absorbed, but that would still leave 0.005M of protons unbuffered. Thus, the pH of this solution would be approximately pH = -log(0.005M) = 2.30 Exceeding buffer capacity dropped the pH significantly compared to adding the same amount of protons to a 1M acetate buffer. Consequently, when considering buffers, it is important to recognize that their concentration sets their limits. Another limit is the pH range in which one hopes to control proton concentration. Multiple ionizable groups Now, what happens if a molecule has two (or more) ionizable groups? It turns out, not surprisingly, that each group will have its own pKa and, as a consequence, will have multiple regions of buffering. Figure 1.36 shows the titration curve for the amino acid aspartic acid. Note that in- stead of a single flattening of the curve, as was seen for acetic acid, aspartic acid’s titration curve displays three such regions. These are individual buffering regions, each centered on the respective pKa values for the carboxyl group and the amine group. Aspartic acid has four possible charges: +1 (α-carboxyl group, α-amino group, and Rgroup carboxyl each has a proton), 0 (α- carboxyl group missing proton, α- amino group has a proton, R-group carboxyl has a proton), -1 (α-carboxyl group and R-group carboxyl each lack a proton, α-amino group retains a proton), -2 (α-carboxyl, R-group carboxyl, and α-amino groups all lack extra proton). Prediction How does one predict the charge for an amino acid at a given pH? A good rule of thumb for estimating charge is that if the pH is more than one unit below the pKa for a group (carboxyl or amino), the proton is on. If the pH is more than one unit above the pKa for the group, the proton is off. If the pH is NOT more than one or less than one pH unit from the pKa, this simple assumption will not work. Further, it is important to recognize that these rules of thumb are estimates only. The pI (pH at which the charge of a molecule is zero) is an exact value calculated as the average of the two pKa values on either side of the zero region. It is calculated at the average of the two pKa values around the point where the charge of the molecule is zero. For aspartic acid, this corresponds to pKa1and pKa2.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/01%3A_In_The_Beginning/1.03%3A_Introduction_-_Water_and_Buffers.txt

Thumbanil: Structure of human hemoglobin. The proteins α and βsubunits are in red and blue, and the iron-containing hemegroups in green. Image used with permission (CC BY-SA 3.0; Richard Wheeler). 02: Structure and Function Source: BiochemFFA_2_1.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy "The man who does not read good books has no advantage over the man who cannot read them." Mark Twain In this chapter, we will examine the structures of the major classes of biomolecules, with an eye to understanding how these structures relate to function. As noted earlier, water is the most abundant molecule in cells, and provides the aqueous environment in which cellular chemistry happens. Dissolved in this water are inorganic ions like sodium, potassium and calcium. But the distinctiveness of biochemistry derives from the vast numbers of complex, large, carbon compounds, that are made by living cells. You have probably learned that the major classes of biological molecules are proteins, nucleic acids, carbohydrates and lipids. The first three of these major groups are macromolecules that are built as long polymers made up of smaller subunits or monomers, like strings of beads. The lipids, while not chains of monomers, also have smaller subunits that are assembled in various ways to make the lipid components of cells, including membranes. The chemical properties and three dimensional conformations of these molecules determine all the molecular interactions upon which life depends. Whether building structures within cells, transferring information, or catalyzing reactions, the activities of biomolecules are governed by their structures. The properties and shapes of macromolecules, in turn, depend on the subunits of which they are built. Interactive 2.1: The enzyme Hexokinase: as for all enzymes, the activity of hexokinase depends on its structure. Protein Database (PDB) We will next examine the major groups of biological macromolecules: proteins, polysaccharides, nucleic acids, and lipids. The building blocks of the first three, respectively, are amino acids, monosaccharides (sugars), and nucleotides. Acetyl-CoA is the most common building block of lipids. 2.04: Structure and Function- Proteins II Source: BiochemFFA_2_3.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy In this section, we hope to bring to life the connection between structure and function of proteins. So far, we have described notable features of the four elements (primary, secondary, tertiary, and quaternary) of protein structure and discussed example proteins/motifs exhibiting them. In this section, we will examine from a functional perspective a few proteins/domains whose function relies on secondary, tertiary, or quaternary structure. It is, of course a bit of a narrow focus to ascribe protein function to any one component of structure, but our hope is by presenting these examples, we can bring to life the way in which a protein’s secondary, tertiary, and quarternary structure lead to the functions it has. Hemoglobin Wikipedia Fibrous proteins - secondary structure Proteins whose cellular or extracellular roles have a strong structural component are composed primarily of primary and second structure, with little folding of the chains. Thus, they have very little tertiary structure and are fibrous in nature. Proteins exhibiting these traits are commonly insoluble in water and are referred to as fibrous proteins (also called scleroproteins). The examples described in this category are found exclusively in animals where they serve roles in flesh, connective tissues and hardened external structures, such as hair. They also contain the three common fibrous protein structures α -helices (keratins), β-strands/sheets (fibroin & elastin) and triple helices (collagen). The fibrous proteins have some commonality of amino acid sequence. Each possesses an abundance of repeating sequences of amino acids with small, non-reactive side groups. Many contain short repeats of sequences, often with glycine. Keratins The keratins are a family of related animal proteins that take numerous forms. α-keratins are structural components of the outer layer of human skin and are integral to hair, nails, claws, feathers, beaks, scales, and hooves. Keratins provide strength to tissues, such as the tongue, and over 50 different keratins are encoded in the human genome. At a cellular level, keratins comprise the intermediate filaments of the cytoskeleton. α- keratins primarily contain α-helices, but can also have β-strand/sheet structures. Individual α-helices are often intertwined to form coils of coiled structures and these strands can also be further joined together by disulfide bonds, increasing structural strength considerably. This is particularly relevant for α-keratin in hair, which contains about 14% cysteine. The odor of burned hair and that of the chemicals used to curl/uncurl hair (breaking/re-making disulfide bonds) arise from their sulfurous components. β-keratins are comprised of β-sheets, as their name implies. Fibroin An insoluble fibrous protein that is a component of the silk of spiders and the larvae of moths and other insects, fibroin is comprised of antiparallel β-strands tightly packed together to form β- sheets. The primary structure of fibroin is a short repeating sequence with glycine at every other residue (Figure 2.57). The small R-groups of the glycine and alanine in the repeating sequence allows for the tight packing characteristic of the fibers of silk. Wikipedia link HERE Elastin As suggested by its name, elastin is a protein with elastic characteristics that functions in many tissues of the body to allow them to resume their shapes after expanding or contracting. The protein is rich in glycine and proline and can comprise over 50% of the weight of dry, defatted arteries. Elastin is made by linking tropoelastin proteins together through lysine residues to make a durable complex crosslinked by desmosine. In arteries, elastin helps with pressure wave propagation for facilitating blood flow. Collagen Collagen is the most abundant protein in mammals, occupying up to a third of the total mass. There are at least 16 types of collagen. Its fibers are a major component of tendons and they are also found abundantly in skin. Collagen is also prominent in cornea, cartilage, bone, blood vessels and the gut. Collagen’s structure is an example of a helix of helices, being composed of three lefthanded helical chains that each are coiled together in a right-handed fashion to make the collagen fiber (Figure 2.60). Each helix is stretched out more than an α-helix, giving it an extended appearance. On the inside of the triple helical structure, only residues of glycine are found, since the side chains of other amino acids are too bulky. Collagen chains have the repeating structure glycinem-n where m is often proline and n is often hydroxyproline (Figure 2.61). Collagen is synthesized in a pre-procollagen form. Processing of the pre-procollagen in the endoplasmic reticulum results in glycosylation, removal of the ‘pre’ sequence, and hydroxylation of lysine and proline residues (see below). The hydroxides can form covalent cross-links with each other, strengthening the collagen fibers. As pro-collagen is exported out of the cell, proteases trim it, resulting in a final form of collagen called tropocollagen. Hydroxylation Hydroxylation of proline and lysine side chains occurs post-translationally in a reaction catalyzed by prolyl-4-hydroxylase and lysyl-hydroxylase (lysyl oxidase), respectively. The reaction requires vitamin C. Since hydroxylation of these residues is essential for formation of stable triple helices at body temperature, vitamin C deficiency results in weak, unstable collagen and, consequently, weakened connective tissues. It is the cause of the disease known as scurvy. Hydrolyzed collagen is used to make gelatin, which is important in the food industry. collagens. Wikipedia link HERE Lamins Lamins are fibrous proteins that provide structure in the cell nucleus and play a role in transcription regulation. They are similar to proteins making up the intermediate filaments, but have extra amino acids in one coil of the protein. Lamins help to form the nuclear lamin in the interior of the nuclear envelope and play important roles in assembling and disassembling the latter in the process of mitosis. They also help to position nuclear pores. In the process of mitosis, disassembly of the nuclear envelope is promoted by phosphorylation of lamins by a protein called mitosis promoting factor and assembly is favored by reversing the reaction (dephosphorylation). Structural domains - tertiary structure Every globular protein relies on its tertiary structure to perform its function, so rather than trying to find representative proteins for tertiary structure (an almost impossible task!), we focus here on a few elements of tertiary structure that are common to many proteins. These are the structural domains and they differ from the structural motifs of supersecondary structure by being larger (25-500 amino acids), having a conserved amino acid sequence, and a history of evolving and functioning independently of the protein chains they are found in. Structural domains are fundamental units of tertiary structure and are found in more than one protein. A structural domain is selfstabilizing and often folds independently of the rest of the protein chain. Leucine zipper A common feature of many eukaryotic DNA binding proteins, leucine zippers are characterized by a repeating set of leucine residues in a protein that interact like a zipper to favor dimerization. Another part of the domain has amino acids (commonly arginine and lysine) that allow it to interact with the DNA double helix (Figure 2.63). Transcription factors that contain leucine zippers include Jun-B, CREB, and AP-1 fos/ jun. Zinc fingers The shortest structural domains are the zinc fingers, which get their name from the fact that one or more coordinated zinc ions stabilize their finger-like structure. Despite their name, some zinc fingers do not bind zinc. There are many structural domains classified as zinc fingers and these are grouped into different families. Zinc fingers were first identified as components of DNA binding transcription factors, but others are now known to bind RNA, protein, and even lipid structures. Cysteine and histidine side chains commonly play roles in coordinating the zinc. Src SH2 domain The Src oncoprotein contains a conserved SH2structural domain that recognizes and binds phosphorylated tyrosine side chains in other proteins (Figure 2.65). Phosphorylation is a fundamental activity in signaling and phosphorylation of tyrosine and interaction between proteins carrying signals is critically needed for cellular communication. The SH2domain is found in over 100 human proteins. Helix-turn-helix domain Helix-turn-helix is a common domain found in DNA binding proteins, consisting of two α-helices separated by a small number of amino acids. As seen in Figure 2.66, the helix parts of the structural domain interact with the bases in the major groove of DNA. Individual α-helices in a protein are part of a helix-turn-helix structure, where the turn separates the individual helices. Pleckstrin homology domain Pleckstrin Homology (PH) domains are protein domains with important functions in the process of signaling. This arises partly from the affinity for binding phosphorylated inositides, such as PIP2 and PIP3, found in Figure 2.66 - Helix-Turn-Helix Domain of a Protein Bound to DNA Wikipedia Figure 2.65 - SH2 Domain Wikipedia biological membranes. PH domains can also bind to G-proteins and protein kinase C. The domain spans about amino acids and is found in numerous signaling proteins. These include Akt/Rac Serine/ Threonine Protein Kinases, Btk/ltk/Tec tyrosine protein kinases, insulin receptor substrate (IRS-1), Phosphatidylinositolspecific phospholipase C, and several yeast proteins involved in cell cycle regulation. Structural globular proteins Enzymes catalyze reactions and proteins such as hemoglobin perform important specialized functions. Evolutionary selection has reduced and eliminated waste so that we can be sure every protein in a cell has a function, even though in some cases we may not know what it is. Sometimes the structure of the proFigure 2.68 - Relationship of basement membrane to epithelium, endothelium, and connective tissue tein is its primary function because the structure provides stability, organization, connections other important properties. It is with this in mind that we present the following proteins. Basement membrane The basement membrane is a layered extracellular matrix of tissue comprised of protein fibers (type IV collagen) and glycosaminoglycans that separates the epithelium from other tissues (Figure 2.68). More importantly, the basement membrane acts like a glue to hold tissues together. The skin, for example, is anchored to the rest of the body by the basement membrane. Basement membranes provide an interface of interaction between cells and the environment around them, thus facilitating signaling processes. They play roles in differentiation during embryogenesis and also in maintenance of function in adult organisms. Actin Actin is the most abundant globular protein found in most types of eukaryotic cells, comprising as much as 20% of the weight of muscle cells. Similar proteins have been identified in bacteria (MreB) and archaeons (Ta0583). Actin is a monomeric subunit able to polymerize readily into two different types of filaments. Microfilaments are major component of the cytoskeleton and are acted on by myosin in the contraction of muscle cells (See HERE). Actin will be discussed in more detail in the next section HERE. Intermediate Filaments Intermediate filaments are a part of the cytoskeleton in many animal cells and are comprised of over 70 different proteins. They are called intermediate because their size (average diameter = 10 nm) is between that of the microfilaments (7 nm) and the microtubules (25 nm). The intermediate filament components include fibrous proteins, such as the keratins and the lamins, which are nuclear, as well as cytoplasmic forms. Intermediate filaments give flexibility to cells because of their own physical properties. They can, for example, be stretched to several times of their original length. Six types There are six different types of intermediate filaments. Type I and II are acidic or basic and attract each other to make larger filaments. They include epithelial keratins and trichocytic keratins (hair components). Type III proteins include four structural proteins - desmin, GFAP (glial fibrillary acidic protein), peripherin, and vimentin. Type IV also is a grouping of three proteins and one multiprotein structure (neurofilaments). The three proteins are α-internexin, synemin, and syncoilin. Type V intermediate filaments encompass the lamins, which give structure to the nucleus. Phosphorylation of lamins leads to their disassembly and this is important in the process of mitosis. The Type VI category includes only a single protein known as nestin. Tubulin A third type of filament found in cells is that of the microbutules. Comprised of a polymer of two units of a globular protein called tubulin, microtubules provide “rails” for motor proteins to move organelles and other “cargo” from one part of a cell to another. Microtubules and tubulin are discussed in more detail HERE. Vimentin Vimentin (Figure 2.70) is the most widely distributed protein of the intermediate filaments. It is expressed in fibroblasts, leukocytes, and blood vessel endothelial cells. The protein has a significant role maintaining the position of organelles in the cytoplasm, with attachments to the nucleus, mitochondria, and endoplasmic reticulum (Figure 2.70). Vimentin provides elasticity to cells and resilience that does not arise from the microtubules or microfilaments. Wounded mice that lack the vimentin gene survive, but take longer to heal wounds than wild type mice. Vimentin also controls the movement of cholesterol from lysosomes to the site of esterification. The result is a reduction in the amount of cholesterol stored inside of cells and has implications for adrenal cells, which must have esters of cholesterol. Mucin Mucins are a group of proteins found in animal epithelial tissue that have many glycosyl residues on them and typically are of high molecular weight (1 to 10 million Da). They are gel-like in their character and are often used for lubrication. Mucus is comprised of mucins. In addition to lubrication, mucins also help to control mineralization, such as bone formation in vertebrate organisms and calcification in echinoderms. They also play roles in the immune system by helping to bind pathogens. Mucins are commonly secreted onto mucosal surfaces (nostrils, eyes, mouth, ears, stomach, genitals, anus) or into fluids, such as saliva. Because of their extensive mucosylation, mucins hold a considerable amount of water (giving them the “slimy” feel) and are resistant to proteolysis. Vinculin Vinculin (Figure 2.72) is a membrane cytoskeletal protein found in the focal adhesion structures of mammalian cells. It is found at cell-cell and cell-matrix junctions and interacts with integrins, talin, paxillins and F-actin. Vinculin is thought to assist (along with other proteins) in anchoring actin microfilaments to the membrane (Figure 2.71). Binding of vinculin to actin and to talin is regulating by polyphosphoinositides and can be inhibited by acidic phospholipids. Syndecans Syndecans are transmembrane proteins that make a single pass with a long amino acid chain (24-25 residues) through plasma membranes and facilitate G proteincoupled receptors’ interaction with Figure 2.71 - Actin filaments (green) attached to vinculin in focal adhesion (red) Wikipedia ligands, such as growth factors, fibronectin, collagens (I, III, and IV) and antithrombin-1. Syndecans typically have 3-5 heparan sulfate and chondroitin sulfate chains attached to them. Heparan sulfate can be cleaved at the site of a wound and stimulate action of fibroblast growth factor in the healing process. The role of syndecans in cell-cell adhesion is shown in mutant cells lacking syndecan I that do not adhere well to each other. Syndecan 4 is also known to adhere to integrin. Syndecans can also inhibit the spread of tumors by the ability of the syndecan 1 ectodomain to suppress growth of tumor cells without affecting normal epithelial cells. Defensin Defensins (Figure 2.73) are a group of small cationic proteins (rich in cysteine residues) that serve as host defense peptides in vertebrate and invertebrate organisms. They protect against infection by various bacteria, fungi, and viruses. Defensins contain between 18 and 45 amino acids with (typically) about 6- 8 cysteine residues. In the immune system, defensins help to kill bacteria engulfed by phagocytosis by epithelial cells and neutrophils. They kill 120 Figure 2.72 - Vinculin Wikipedia bacteria by acting like ionophores - binding the membrane and opening pore-like structures to release ions and nutrients from the cells. Focal adhesions In the cell, focal adhesions are structures containing multiple proteins that mechanically link cytoskeletal structures (actin bundles) with the extracellular matrix. They are dynamic, with proteins bringing and leaving with signals regarding the cell cycle, cell motility, and more almost constantly. Focal adhesions serve as anchors and as a signaling hub at cellular locations where integrins bind molecules and where membrane clustering events occur. Over 100 different proteins are found in focal adhesions. Focal adhesions communicate important messages to cells, acting as sensors to update information about the status of the extracellular matrix, which, in turn, adjusts/ affects their actions. In sedentary cells, they are stabler than in cells in motion because when cells move, focal adhesion contacts are established at the “front” and removed at the rear as motion progresses. This can be very important in white blood cells’ ability to find tissue damage. Ankyrin Ankyrins (Figure 2.74) are a family of membrane adaptor proteins serving as “anchors” to interconnect integral membrane proteins to the spectrin-actin membrane cytoskeleton. Ankyrins are anchored to the plasma membrane by covalently linked palmitoyl-CoA. They bind to the β subunit of spectrin and at least a dozen groups of integral membrane proteins. The ankyrin proteins contain four functional domains: an N-terminal region with 24 tandem ankyrin repeats, a central spectrin-binding domain, a “death domain” interacting with apoptotic proteins, and a C-terminal regulatory domain that is highly varied significantly among different ankyrins. Spectrin Spectrin (Figures 2.75 & 2.76) is a protein of the cellular cytoskeleton that plays an important role in maintaining its structure and the integrity of the plasma membrane. In animals, spectrin gives red blood cells their shape. Spectrin is located inside the inner layer of the eukaryotic plasma membrane where it forms a network of pentagonal or hexagonal arrangements. Spectrin fibers collect together at junctional complexes of actin and is also attached to ankyrin for stability, as well as numerous integral membrane proteins, such as glycophorin. Integrins In multicellular organisms, cells need connections, both to each other and to the extracellular matrix. Facilitating these attachments at the cellular end are transmembrane proteins known as integrins (Figure 2.77). Integrins are found in all metazoan cells. Ligands for the integrins include collagen, fibronectin, laminin, and vitronectin. Integrins function not only in attachment, but also in communication, cell migration, virus linkages (adenovirus, for example), and blood clotting. Integrins are able to sense chemical and mechanical signals about the extracellular matrix and move that information to intracellular domains as part of the process of signal transduction. Inside the cells, responses to the signals affect cell shape, regulation of the cell cycle, movement, or changes in other cell receptors in the membrane. The process is dynamic and allows for rapid responses as may be necessary, for example in the process of blood clotting, where the integrin known as GPIbIIIa (on the surface of blood platelets) attaches to fibrin in a clot as it develops. Integrins work along with other receptors, including immunoglobulins, other cell adhesion molecules, cadherins, selectins, and syndecans. In mammals the proteins have a large number of subunits - 18 α- and 8 β-chains. They are a bridge between its links outside the cell to the extracellular matrix (ECM) and its links inside the cell to the cytoskeleton. Integrins play central role in formation and stability of focal adhesions. These are large molecular complexes arising from clustering of integrin-ECM connections. In the process of cellular movement, integrins at the “front” of the cell (in the direction of the movement), make new attachments to substrate and release connections to substrate in the back of the cell. These latter integrins are then endocytosed and reused. Integrins also help to modulate signal transduction through tyrosine kinase receptors in the cell membrane by regulating movement of adapters to the plasma membrane. β1c integrin, for example, recruits the Shp2 phosphatase to the insulin growth factor receptor to cause it to become dephosphorylated, thus turning off the signal it communicates. Integrins can also help to recruit signaling molecules inside of the cell to activated tyrosine kinases to help them to communicate their signals. Cadherins Cadherins (Figure 2.78) constitute a type-1 class of transmembrane proteins playing important roles in cell adhesion. They require calcium ions to function, forming adherens junctions that hold tissues together (See Figure 2.69). Cells of a specific cadherin type will preferentially cluster with each other in preference to associating with cells containing a different cadherin type. Caderins are both receptors and places for ligands to attach. They assist in the proper positioning of cells in development, separation of different tissue layers, and cell migration. Selectins Selectins (Figure 2.79) are cell adhesion glycoproteins that bind to sugar molecules. As such, they are a type of lectin - proteins that bind sugar polymers (see HERE also). All selectins have an N-terminal calcium-dependent lectin domain, a single transmembrane domain, and an intracellular cytoplasmic tail. There are three different types of selectins, 1) E-selectin (endothelial); 2) L (lymphocytic; and 3) P (platelets and endothelial cells. Selectins function in lymphocyte homing (adhesion of blood lymphocytes to cells in lymphoid organs), in inflammation processes, and in cancer metastasis. Near the site of inflammation, P-selectin on the surface of blood capillary cells interacts with glycoproteins on leukocyte cell surfaces. This has the effect of slowing the movement of the leukocyte. At the target site of inflammation, E- selectin on the endothelial cells of the blood vessel and L-selectin on the surface of the leukocyte bind to their respective carbohydrates, stopping the leukocyte movement. The leukocyte then crosses the wall of the capillary and begins the immune response. Selectins are involved in the inflammatory processes of asthma, psoriasis, multiple scleroris, and rheumatoid arthritis. Laminins Laminins are extracellular matrix glycoproteins that a major components of the basal lamina and affect cell differentiation, migration, and adhesion. They are secreted into the extracellular matrix where they are incorporated and are essential for tissue maintenance and survival. When laminins are defective, muscles may not form properly and give rise to muscular dystrophy. Laminins are associated with fibronectin, entactin, and perlecan proteins in type IV collagen networks and bind to integrin receptors in the plasma membrane. As a consequence, laminins contribute to cellular attachment, differentiation, shape, and movement. The proteins are trimeric in structure, having one α-chain, a β-chain, and a γ-chain. Fifteen combinations of different chains are known. Vitronectin Vitronectin is a glycoprotein (75kDa) found in blood serum (platelets), the extracellular matrix, and in bone. It promotes the process of cell adhesion and spreading and binds to several protease inhibitors (serpins). It is secreted from cells and is believed to play roles in blood clotting and the malignancy of tumors. One domain of vitronectin binds to plasminogen activator inhibitor and acts to stabilize it. Another domain of the protein binds to cellular integrin proteins, such as the vitronectin receptor that anchors cells to the extracellular matrix. Catenins Catenins are a family of proteins interacting with cadherin proteins in cell adhesion (Figure 2.69). Four main types of catenins are known, α-, β-, γ-, and δ-catenin. Catenins play roles in cellular organization before development occurs and help to regulate cellular growth. α-catenin and β-catenin are found at adherens junctions with cadherin and help cells to maintain epithelial layers. Cadherins are connected to actin filaments of the cytoskeleton and catenins play the critical role. Catenins are important for the process whereby cellular division is inhibited when cells come in contact with each other (contact inhibition). When catenin genes are mutated, cadherin cell adhesions can disappear and tumorigenesis may result. Catenins have been found to be associated with colorectal and numerous other forms of cancer. Glycophorins All of the membrane proteins described so far are notable for the connections they make to other proteins and cellular structures. Some membrane proteins, though, are designed to reduce cellular connections to proteins of other cells. This is particularly important for blood cells where “stickiness” is undesirable except where clotting is concerned. Glycophorins (Figure 2.80) are membrane-spanning sialoglycoproteins of red blood cells. They are heavily glycosylated (60%).and rich in sialic acid, giving the cells a very hydrophilic (and negatively charged) coat, which enables them to circulate in the bloodstream without adhering to other cells or the vessel walls. Five glycophorins have been identified - four (A,B,C,and D) from isolated membranes and a fifth form (E) from coding in the human genome. The proteins are abundant, forming about 2% of the total membrane proteins in these cells. Glycophorins have important roles in regulating RBC membrane mechanical properties and shape. Because some glycophorins can be expressed in various nonerythroid tissues (particularly Glycophorin C), the importance of their interactions with the membrane skeleton may have a considerable biological significance. Cooperativity and allosterism - quaternary structure Quaternary structure, of course describes the interactions of individual subunits of a multi-subunit protein (Figure 2.81). The result of these interactions can give rise to important biological phenomena, such as cooperative binding of substrates to a protein and allosteric effects on the action of an enzyme. Allosteric effects can occur by a series of mechanisms, but a common feature is that binding of an effector to an enzyme subunit causes (or locks) the enzyme in either a Tstate (less activity) or an R-state (more activity). Effectors can be enzyme substrates (homotropic effectors) or non-substrates (heterotropic effectors). Allosterism will be covered in more depth in the Catalysis chapter HERE. We begin our consideration of quaternary structure with a discussion of cooperativity, how it arises in the multi-subunit protein hemoglobin and how its properties contrast with those of the related, single subunit protein myoglobin. Cooperativity Cooperativity is defined as the phenomenon where binding of one ligand molecule by a protein favors the binding of additional molecules of the same type. Hemoglobin, for example, exhibits cooperativity when the binding of an oxygen molecule by the iron of the heme group in one of the four subunits causes a slight conformation change in the subunit. This happens because the heme iron is attached to a histidine side chain and binding of oxygen ‘lifts’ the iron along with the histidine ring (also known as the imidazole ring). Movie 2.3 - Hemoglobin’s structural changes on binding oxygen Wikipedia Since each hemoglobin subunit interacts with and influences the other subunits, they too are induced to change shape slightly when the first subunit binds to oxygen (a transition described as going from the T-state to the R-state). These shape changes favor each of the remaining subunits binding oxygen, as well. This is very important in the lungs where oxygen is picked up by hemoglobin, because the binding of the first oxygen molecule facilitates the rapid uptake of more oxygen molecules. In the tissues, where the oxygen concentration is lower, the oxygen leaves hemoglobin and the proteins flips from the R-state back to the Tstate. CO2 transport Cooperativity is only one of many fascinating structural aspects of hemoglobin that help the body to receive oxygen where it is needed and pick it up where it is abundant. Hemoglobin also assists in the transport of the product of cellular respiration (carbon dioxide) from the tissues producing it to the lungs where it is exhaled. Like the binding of oxygen to hemoglobin, binding of other molecules to hemoglobin affects its affinity for oxygen. The effect is particularly pronounced when comparing the oxygen binding characteristics of hemoglobin’s four subunits with the oxygen binding of the related protein myoglobin’s single subunit (Figure 2.83). Different oxygen binding Like hemoglobin, myoglobin contains an iron in a heme group that binds to oxygen. The structure of the globin protein in myoglobin is very similar to the structure of the globins in hemoglobin and hemoglobin is thought to have evolved from myoglobin in evolutionary history. As seen in Figure 2.83, the binding curve of hemoglobin for oxygen is S-shaped (sigmoidal), whereas the binding curve for myoglobin is hyperbolic. What this tells us is that hemoglobin’s affinity for oxygen is low at a low concentration oxygen, but increases as the oxygen concentration increases. Since myoglobin very quickly saturates with oxygen, even under low oxygen concentrations, it says that its affinity for oxygen is high and doesn’t change. Because myoglobin has only a single subunit, binding of oxygen by that subunit can’t affect any other subunits, since there are no other subunits to affect. Consequently, cooperativity requires more than one subunit. Therefore, hemoglobin can exhibit cooperativity, but myoglobin can’t. It is worth noting that simply having multiple subunits does not mean cooperativity will exist. Hemoglobin is one protein that exhibits the characteristic, but many multisubunit proteins do not. Interactive 2.2 - Hemoglobin in the presence (top) and absence (bottom) of oxygen Storage vs. delivery The lack of ability of myoglobin to adjust its affinity for oxygen according to the oxygen concentration (low affinity at low oxygen concentration, such as in tissues and high affinity at high oxygen concentration, such as in the lungs) means it is better suited for storing oxygen than for delivering it according to the varying oxygen needs of and animal body. As we shall see, besides cooperativity, hemoglobin has other structural features that allow it to deliver oxygen precisely where it is needed most in the body. Bohr effect The Bohr Effect was first described over 100 years ago by Christian Bohr, father of the famous physicist, Niels Bohr. Shown graphically (Figures 2.86, 2.87, and 2.88), the observed effect is that hemoglobin’s affinity for oxygen decreases as the pH decreases and as the concentration of carbon dioxide increases. Binding of the protons and carbon dioxide by amino Figure 2.85 - Sequential model of binding. The sequential model is one way to explain hemoglobin’s cooperativity. Squares represent no oxygen bound. Circles represent subunits bound with oxygen and rounded subunits correspond to units whose affinity for oxygen increases by interacting with a subunit that has bound oxygen. Image by Aleia Kim acid side chains in the globin proteins helps to facilitate structural changes in them. Most commonly, the amino acid affected by protons is histidine #146 of the β strands. When this happens, the ionized histidine can form an ionic bond with the side chain of aspartic acid #94, which has the effect of stabilizing the T-state (reduced oxygen binding state) and releasing oxygen. Other histidines and the amine of the amino terminal amino acids in the α-chains are also binding sites for protons. 2,3-BPG Another molecule favoring the release of oxygen by hemoglobin is 2,3- bisphosphoglycerate (also called 2,3-BPG or just BPG - Figure 2.89). Like protons and carbon dioxide, 2,3-BPG is produced by actively respiring tissues, as a byproduct of glucose metabolism. The 2,3-BPG mole cule fits into the ‘hole of the donut’ of adult hemoglobin (Figure 2.89). Such binding of 2,3-BPG favors the T-state (tight - low oxygen binding) of hemoglobin, which has a reduced affinity for oxygen. In the absence of 2,3-BPG, hemoglobin can more easily exist in the R-state (relaxed - higher oxygen binding), which has a high affinity for oxygen. Smokers Notably, the blood of smokers is higher in the concentration of 2,3-BPG than non-smokers, so more of their hemoglobin remains in the T-state and thus the oxygen carrying capacity of smokers is lower than non-smokers.Another reason why smokers’ oxygen carrying capacity is lower than that of non-smokers is that cigarette smoke contains carbon monoxide and this molecule, which has almost identical dimensions to molecular oxygen, effectively outcompetes with oxygen for binding to the iron atom of heme (Figure 2.90). Part of carbon monoxide’s toxicity is due to its ability to bind hemoglobin and prevent oxygen from binding. Carbon dioxide Carbon dioxide binds to form a carbamate when binding the α-amine of each globin chain. The process of forming this structure releases a proton, which helps to further enhance the Bohr effect. Physiologically, the binding of CO2 and H+ has significance because actively respiring tissues (such as contracting muscles) require oxygen and release protons and carbon dioxide. The higher the concentration of protons and carbon dioxide, the more oxygen is released to feed the tissues that need it most. About 40% of the released protons and about 20% of the carbon dioxide are carried back to the lungs by hemoglobin. The remainder travel as part of the bicarbonate buffering system or as dissolved CO2. In the lungs, the process reverses itself. The lungs have a higher pH than respiring tissues, so protons are released from hemoglobin and CO2 too is freed to be exhaled. Fetal hemoglobin Adult hemoglobin releases oxygen when it binds 2,3- BPG. This is in contrast to fetal hemoglobin, which has a slightly different configuration (α2γ2) than adult hemoglobin (α2β2). Fetal hemoglobin has a greater affinity for oxygen than maternal hemoglobin, allowing the fetus to obtain oxygen effectively from the mother’s blood. Part of the reason for fetal hemoglobin’s greater affinity for oxygen is that it doesn’t bind 2,3-BPG. Consequently, fetal hemoglobin remains in the R-state much more than adult hemoglobin and because of this, fetal hemoglobin has greater affinity for oxygen than adult hemoglobin and can take oxygen away from adult hemoglobin. Thus, the fetus can get oxygen from the mother. Sickle cell disease Mutations to the globin genes coding for hemoglobin can sometimes have deleterious consequences. Sickle cell disease (also called sickle cell anemia) is a genetically transmitted disease that arises from such mutations. There are different forms of the disease. It is a recessive trait, meaning that to be afflicted with it, an individual must inherit two copies of the mutated gene. The predominant form of hemoglobin in adults is hemoglobin A, designated HbA (two α chains and two β chains). The mutant form is known as HbS. The most common mutation is an A to T mutation in the middle of the codon for the seventh amino acid (some counting schemes call it the sixth amino acid) of the β-chain. This results in conversion of a GAG codon to GTG and thus changes the amino acid specified at that position from a glutamic acid to a valine. This minor change places a small hydrophobic patch of amino acids on the surface of the β-globin chains. Polymerization Under conditions of low oxygen, these hydrophobic patches will associate with each other to make long polymers of hemoglobin molecules. The result is that the red blood cells containing them will change shape from being rounded to forming the shape of a sickle (Figure 2.94). Rounded red blood cells readily make it through tiny capillaries, but sickleshaped cells do not. Worse, they block the flow of other blood cells. Tissues where these blockages occur are already low in oxygen, so stopping the flow of blood through them causes them to go quickly anaerobic, causing pain and, in some cases, death of tissue. In severe circumstances, sickled red blood cells death may result. The disease is referred to as an anemia because the sickling of the red blood cells targets them for removal by the blood monitoring system of the body, so a person with the disease has chronically reduced numbers of red blood cells. Heterozygote advantage Interestingly, there appears to be a selective advantage to people who are heterozygous for the disease in areas where malaria is prominent. Heterozygotes do not suffer obvious ill effects of the disease, but their red blood cells appear to be more susceptible to rupture when infected. As a consequence, the parasite gets less of a chance to reproduce and the infected person has a greater chance of survival. The protective effect of the mutant gene, though, does not extend to people who suffer the full blown disease (homozygotes for the mutant gene). Treatments for the disease include transfusion, pain management, and avoidance of heavy exertion. The drug hydroxyurea has been linked to reduction in number and severity of attacks, as well as an increase in survival time1,2. It appears to work by reactivating expression of the fetal hemoglobin gene, which typically is not synthesized to any significant extent normally after about 6 weeks of age. Oxygen binding Animals have needs for oxygen that differ from all other organisms. Oxygen, of course, is the terminal electron acceptor in animals and is necessary for electron transport to work. When electron transport is functioning, ATP generation by cells is many times more efficient than when it is absent. Since abundant ATP is essential for muscular contraction and animals move around a lot - to catch prey, to exercise, to escape danger, etc., having an abundant supply of oxygen is important. This is particularly a concern deep inside tissues where diffusion of oxygen alone (as occurs in insects) does not deliver sufficient quantities necessary for long term survival. The issue is not a problem for plants since, for the most part, their motions are largely related to growth and thus don’t have rapidly changing needs/demands for oxygen that animals have. Unicellular organisms have a variety of mechanisms for obtaining oxygen and surviving without it. Two other important oxygen binding proteins besides hemoglobin are myoglobin and hemocyanin. Myoglobin Myoglobin is the primary oxygen-storage protein found in animal muscle tissues. In contrast to hemoglobin, which circulates throughout the body, myoglobin protein is only found in muscle tissue and appears in the blood only after injury. Like hemoglobin, myoglobin binds oxygen at a prosthetic heme group it contains. The red color of meat arises from the heme of myoglobin and the browning of meat by cooking it comes from oxidation of the ferrous (Fe++) ion of myoglobin’s heme to the ferric (Fe+++) ion via oxidation in the cooking process. As meat sits in our atmosphere (an oxygen-rich environment), oxidation of Fe++ to Fe+++ occurs, leaving the brown color noted above. If meat is stored in a carbon monoxide (CO) environment, CO binds to the heme group and reduces the amount of oxidation, keeping meat looking red for a longer period of time. High affinity Myoglobin (Figure 2.97) displays higher affinity for oxygen at low oxygen concentrations than hemoglobin and is therefore able to absorb oxygen delivered by hemoglobin under these conditions. Myoglobin’s high affinity for oxygen makes it better suited for oxygen storage than delivery. The protein exists as a single subunit of globin (in contrast to hemoglobin, which contains four subunits) and is related to the subunits found in hemoglobin. Mammals that dive deeply in the ocean, such as whales and seals, have muscles with particularly high abundance of myoglobin. When oxygen concentration in muscles falls to low levels, myoglobin releases its oxygen, thus functioning as an oxygen “battery” that delivers oxygen fuel when needed and holding onto it under all other conditions. Myoglobin holds the distinction of being the first protein for which the 3D structure was determined by X-ray crystallography by John Kendrew in 1958, an achievement for which he later won the Nobel Prize. Hemocyanin Hemocyanin is the protein transporting oxygen in the bodies of molluscs and arthropods. It is a coppercontaining protein found not within blood cells of these organisms, but rather is suspended in the circulating hemolymph they possess. The oxygen binding site of hemocyanin contains a pair of copper(I) cations directly coordinated to the protein by the imidazole rings of six histidine side chains. Most, but not all hemocyanins bind oxygen non-cooperatively and are less efficient than hemoglobin at transporting oxygen. Notably, the hemocyanins of horseshoe crabs and some other arthropods do, in fact, bind oxygen cooperatively. Hemocyanin contains many subunit proteins, each with two copper atoms that can bind one oxygen molecule (O2). Subunit proteins have atomic masses of about 75 kilodaltons (kDa). These may be arranged in dimers or hexamers depending on species. Superstructures comprised of dimer or hexamer complexes are arranged in chains or clusters and have molecular weights of over 1500 kDa.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/02%3A_Structure_and_Function/2.01%3A_Prelude_to_Structure_and_Function.txt

Source: BiochemFFA_2_4.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy To this point, the proteins we have discussed have not been catalysts (enzymes). The majority of proteins in cells, however, catalyze reactions. In this section we begin our discussion of a subclass of proteins that catalyze reactions releasing energy and convert it into mechanical force. These operate at the cellular and organismal level and are known as motor proteins. Motor proteins rely on globular structural proteins, so it is important that we describe how these cellular “railways” are assembled before discussing the motor proteins themselves. There are two relevant fibrous structures serving as rails for motor proteins. They are: 1. microfilaments (composed of an actin polymer) and 2. microtubules (composed of a polymer of tubulin. Actin The monomeric unit of actin is called G-actin (globular actin) and the polymer is known as F-actin (filamentous actin). Filaments of Factin comprise the smallest filaments of cells known as microfilaments (Figure 2.101). Actin is essential for muscular contraction and also has diverse roles in cellular signaling and maintenance of cell junctions. In conjunction with other proteins, actin has numerous interactions with the cell membrane. The β- and γ-forms of actin are components of the cytoskeleton and facilitate motility inside of cells. α-actin is important in muscle tissues, where it is used by myosin in the mechanical process of contraction (See HERE). Monomeric and polymeric forms of actin play roles in cellular activities relating to motion. Two parallel F-actin strands can pair with each other and create a double helical structure with 2.17 subunits per turn of the helix. Helical F-actin in muscles contains tropomyosin, which covers the actin binding sites for myosin in resting muscles to prevent contraction. Other proteins bound to actin muscle filaments include the troponins (I, T, and C). Actin Cellular Action Examples of actin action at the cellular level include cell motility, cytokinesis, intracellular transport of vesicles and organelles, and cell shape. Each actin monomer is bound to a molecule of ATP or ADP and the presence of one of these is essential for proper G-actin functioning. The role of ATP In the monomer, actin is more commonly bound to ATP, whereas in the filaments, it is typically bound to ADP. Actin is an inefficient ATPase, breaking the molecule down slowly, but the catalysis speeds up as much as 40,000 fold when the monomer begins to polymerize. Actin also has a binding site for divalent cations - either calcium or magnesium. F- Actin binds to structural proteins at the adherens junction (Figure 2.102). These include α-actinin, vinculin (provides a membrane connection and connections to the catenins and cadherin). Polymerization Polymerization of actin begins with a nucleating event (Figure 2.103). One factor known to affect the process is known as the Arp 2/3 complex. It does this by mimicking an actin dimer, starting an autocatalytic process of actin assembly. The Arp 2/3 complex plays roles both in the initiation of polymerization of new actin filaments as well as the formation of branches in the filaments. Two proteins play roles in modulating polymer growth. Thymosin functions on the end of actin filaments to control growth. Profilin works on G-actin monomers exchanging ADP for ATP, promoting addition of monomers to a growing chain. F-actin filaments are held together by relatively weak bonds compared to the covalent bonds of the monomers of nucleic acids, thus allowing for easier disassembly when desired. Actin’s amino acid sequence is optimized, having diverged only a relatively small amount (20%) between algae and humans. Mutations in the actin gene result in muscular diseases and/or deafness. Tubulin Tubulin proteins are the monomeric building blocks of eukaryotic microtubules (Figure 2.104 & 2.105). Bacterial (TubZ) and archaeon (FtsZ) equivalents are known. The α-tubulin and β-tubulin proteins polymerize to make microtubule structures in the cytoplasm of cells. Microtubules are major components of the cytoskeleton of eukaryotic cells, providing structural support, transport within the cell, and functions necessary for segregation of DNAs during cell division. Dimerization of the α-tubulin and β-tubulin proteins is necessary for polymerization and requires that the subunits bind to GTP. Microtubules only grow in one direction. β- tubulin is found on the plus end of the tubule (growth end = plus end) and α-tubulin is exposed on the other end (non-growth end = minus end). Dimers of α-tubulin/β-tubulin are incorporated into growing microtubules in this orientation. If a dimer is bound to GDP instead of GTP, it tends to be unstable and fall apart, whereas those bound to GTP stably assemble into microtubules. Microtubules Microtubules, along with microfilaments and intermediate filaments (see HERE) constitute the cytoskeleton of cells. Found in the cytoplasm, they are found in eukaryotic cells, as well as some bacteria. Microtubules help to give cells structure. They comprise the inner structure of flagella and cilia and provide rail-like surfaces for the transport of materials within cells. Polymerization of α- tubulin and β-tubulin to form microtubules occurs after a nucleating event. Individual units get arranged in microtubule organizing centers (MTOCs), an example of which is the centrosome. Centrosomes are focal points of connection of microtubules. Basal bodies of cilia and flagella also help to organize microtubules. Motor proteins From the transport of materials within a cell to the process of cytokinesis where one cell splits into two in mitosis, a cell has needs for motion at the molecular level. Secretory vesicles and organelles must be transported. Chromosomes must be separated in mitosis and meiosis. The proteins dynein and kinesin (Figure 2.106) are necessary for intracellular movement. These motor proteins facilitate the movement of materials inside of cells along microtubule “rails”. These motor proteins are able to move along a portion of the cytoskeleton by converting chemical energy into motion with the hydrolysis of ATP. An exception is flagellar rotation, which uses energy provided from a gradient created by a proton pump. Kinesins and dyneins As noted, kinesins and dyneins navigate in cells on microtubule tracks (Figure 2.108 & Movie 2.4). Most kinesins move in the direction of the synthesis of the microtubule (+ end movement), which is generally away from the cell center and the opposite direction of movement of dyneins, which are said to do retrograde transport toward the cell center. Both proteins provide movement functions necessary for the processes of mitosis and meiosis. These include spindle formation, chromosome separation, and shuttling of organelles, such as the mitochondria, Golgi apparatuses, and vesicles. Kinesins are comprised of two heavy chains and two light chains. The head motor domains of heavy chains (in the feet) use energy of ATP hydrolysis to do mechanical work for the movement along the microtubules. There are at least fourteen distinct kinesin families and probably many related ones in addition. Dyneins are placed into two groups - cytoplasmic and axonemal (also called ciliary or flagellar dyneins - Figure 2.109). Dyneins are more complex in structure than kinesins with many small polypeptide units. Notably, plants do not have dynein motor proteins, but do contain kinesins. Movie 2.4 The motor protein kinesin walking down a microtubule. Image used with permission (Public Domain; zp706). Myosin An important group of motor proteins in the cell is the myosins. Like kinesins and dyneins, myosins use energy from hydrolysis of ATP for movement. In this case, the movement is mostly not along microtubules, but rather along microfilaments comprised of a polymer of actin (F-actin). Movement of myosin on actin is best known as the driving force for muscular contraction. Myosins are a huge family of proteins, all of which bind to actin and all of which involve motion. Eighteen different classes of myosin proteins are known. Myosin II is the form responsible for generating muscle contraction. It is an elongated protein formed from two heavy chains with motor heads and two light chains. Each myosin motor head binds actin and has an ATP binding site. The myosin heads bind and hydrolyze ATP. This hydrolysis produces the energy necessary for myosin to walk toward the plus end of an actin filament. Non-muscle myosin IIs provide contraction needed to power the action of cytokinesis. Other myosin proteins are involved in movement of non-muscle cells. Myosin I is involved in intracellular organization. Myosin V performs vesicle and organelle transport. Myosin XI provides movement along cellular microfilament networks to facilitate organelle and cytoplasmic streaming in a particular direction. Structure Myosins have six subunits, two heavy chains and four light chains. Myosin proteins have domains frequently described as a head and a tail (Figure 2.111). Some also describe an intermediate hinge region as a neck. The head portion of myosin is the part that binds to actin. It uses energy from ATP hydrolysis to move along the actin filaments. In muscles, myosin proteins form aggregated structures referred to as thick filaments. Movements are directional. Structural considerations of muscular contraction Before we discuss the steps in the process of muscular contraction, it is important to describe anatomical aspects of muscles and nomenclature. There are three types of muscle tissue - skeletal (striated), smooth, and (in vertebrates) cardiac. We shall concern ourselves mostly here with skeletal muscle tissue. Muscles may be activated by the central nervous system or, in the case of smooth and cardiac muscles, may contract involuntarily. Skeletal muscles may be slow twitch or fast twitch. Sarcomeres Sarcomeres are described as the basic units comprising striated muscles and are comprised of thick (myosin) and thin (actin) filaments and a protein called titin. The filaments slide past each other in muscular contraction and then backwards in muscular relaxation. They are not found in smooth muscles. Under the microscope, a sarcomere is the region between two Z-lines of striated muscle tissue (Figure 2.112). The Z-line is the distinct, narrow, dark region in the middle of an I-band. Within the sarcomere is an entire Aband with its central H-zone. Within the Hzone are located tails of myosin fibers, with the head pointed outwards from there projecting all the way to the I-band. The outside of the Aband is the darkest and it gets lighter moving towards the center. Within the Iband are located thin filaments that are not occupied with thick myosin filaments. The Aband contains intact thick filaments overlaying thin filaments except in the central H zone, which contains only thick filaments. In the center of the H-zone is a line, known as the M-line. It contains connecting elements of the cellular cytoskeleton. In muscular contraction, myosin heads walk along pulling their tails over the actin thin filaments, using energy from hydrolysis of ATP and pulling them towards the center of the sarcomere. Sarcolemma The sarcolemma (also known as the myolemma) is to muscle cells what the plasma membrane is to other eukaryotic cells - a barrier between inside and outside. It contains a lipid bilayer and a glycocalyx on the outside of it. The glyocalyx contains polysaccharides and connects with the basement membrane. The basement membrane serves a s scaffolding to connect muscle fibers to. This connection is made by transmembrane proteins bridging the actin cytoskeleton on the inside of the cell with the basement membrane on the outside. On the ends of the muscle fibers, each sarcolemma fuses with a tendon fiber and these, in turn, adhere to bones. Sarcoplasmic reticulum The sarcoplasmic reticulum (Figure 2.114) is a name for the structure found within muscle cells that is similar to the smooth endoplasmic reticulum found in other cells. It contains a specialized set of proteins to meet needs unique to muscle cells. The organelle largely serves as a calcium “battery,” releasing stored calcium to initiate muscular contraction when stimulated and taking up calcium when signaled at the end of the contraction cycle. It accomplishes these tasks using calcium ion channels for release of the ion and specific calcium ion pumps to take it up. Movement direction All myosins but myosin VI move towards the + end (the growing end) of the microfilament. The neck portion serves to link the head and the tail. It also a binding site for myosin light chain proteins that form part of a macromolecular complex with regulatory functions. The tail is the point of attachment of molecules or other “cargo” being moved. It can also connect with other myosin subunits and may have a role to play in controlling movement. Muscular contraction The sliding filament model has been proposed to describe the process of muscular tension/contraction. In this process a repeating set of actions slide a thin actin filament over a thick myosin filament as a means of creating tension/ shortening of the muscle fiber. Steps in the process occur as follows: A. A signal from the central nervous system (action potential) arrives at a motor neuron, which it transmits towards the neuromuscular junction (see more on the neurotransmission part of the process HERE) B. At the end of the axon, the nerve signal stimulates the opening of calcium channels at the axon terminus causing calcium to flow into the terminal. C. Movement of calcium into the axon of the nerve causes acetylcholine (a neurotransmitter) in synaptic vesicles to fuse with the plasma membrane. This causes the acetylcholine to be expelled into the synaptic cleft between the axon and the adjacent skeletal muscle fiber. D. Acetylcholine diffuses across the synapse and then binds to nicotinic acetylcholine receptors on the neuromuscular junction, activating them. E. Activation of the receptor stimulates opening gates of sodium and potassium channels, allowing sodium to move into the cell and potassium to exit. The polarity of the membrane of the muscle cell (called a sarcolemma - Figure 2.111) changes rapidly (called the end plate potential). F. Change in the end plate potential results in opening of voltage sensitive ion channels specific for sodium or potassium only to Figure 2.117 - 3. ATP cleavage by myosin allows actin attachment (J) Wikipediaopen, creating an action potential (voltage change) that spreads throughout the cell in all directions. G. The spreading action potential depolarizes the inner muscle fiber and opens calcium channels on the sarcoplasmic reticulum (Figure 2.115). H. Calcium released from the sarcoplasmic reticulum binds to troponin on the actin filaments (Figure 2.115). I. Troponin alters the structure of the tropomyosin to which is it bound. This causes tropomyosin to move slightly, allowing access to myosin binding sites on the microfilament (also called thin filament) that it was covering (Figure 2.116). J. Myosin (bound to ATP) cleaves the ATP to ADP and Pi, which it holds onto in its head region and then attaches itself to the exposed binding sites on the thin filaments causing inorganic phosphate to be released from the myosin followed by ADP (Figure 2.117). K. Release of ADP and Pi is tightly coupled to a bending of the myosin hinge, resulting in what is called the power stroke. This causes the thin filament to move relative to the thick fibers of myosin (Figures 2.118 & 2.119). L. Such movement of the thin filaments causes the Z lines to be pulled closer to each other. This results in shortening of the sarcomere as a whole (Figure 2.122) and narrowing of the I band and the H zones (Figure 2.123). M. If ATP is available, it binds to myosin, allowing it to let go of the actin (Figures 2.120 & 2.121). If ATP is not available, the muscle will remain locked in this state. This is the cause of rigor mortis in death - contraction without release of muscles . Figure 2.120 - When ATP is present, it binds to myosin (M). Wikipedia N. After myosin has bound the ATP, it hydrolyzes it, producing ADP and Pi, which are held by the head. Hydrolysis of ATP resets the hinge region to its original state, unbending it. This unbent state is also referred to as the cocked position. O.If tropomyosin is still permitting access to binding sites on actin, the process repeats so long as ATP is available and calcium remains at a high enough concentration to permit it to bond to troponin. Relaxation of the muscle tension occurs as the action potential in the muscle cell dissipates. This happens because all of the following things happen 1) the nerve signal stops; 2) the neurotransmitter is degraded by the enzyme acetylcholinesterase; and 3) the calcium concentration declines because it is taken up by the sarcoplasmic reticulum. It should be noted that the sarcoplasmic reticulum is always taking up calcium. Only when its calcium gates are opened by the action potential is it unable to reduce cellular calcium concentration. As the action potential decreases, then the calcium gates close and the sarcoplasmic reticulum “catches up” and cellular calcium concentrations fall. At that point troponin releases calcium, tropomyosin goes back to covering myosin binding sites on actin, myosin loses its attachment to actin and the thin filaments slide back to their original positions relative to the myosin thick filaments. Tropomyosin Tropomyosins are proteins that interact with actin thin filaments to help regulate their roles in movement, both in muscle cells and non-muscle cells (Figure 2.124). Tropomyosins interact to form head-to-toe dimers and perch along the α-helical groove of an actin filament. The isoforms of tropomyosin that are in muscle cells control interactions between myosin and the actin filament within the sarcomere and help to regulate contraction of the muscle. In other cells, nonmuscle tropomyosins help to regulate the cytoskeleton’s functions. The interactions of tropomyosin with the cytoskeleton are considerably more complicated than what occurs in muscle cells. Muscle cells have five tropomyosin isoforms, but in the cytoskeleton of non-muscle cells, there are over 40 tropomyosins. Troponin The troponins involved in muscular contraction are actually a complex of three proteins known as troponin I, troponin C, and troponin T (Figure 2.125). They associate with each other and with tropomyosin on actin filaments to help regulate the process of muscular contraction. Troponin I prevents binding of myosin’s head to actin and thus prevents the most important step in contraction. Troponin C is a unit that binds to calcium ions. Troponin T is responsible for binding all three proteins to tropomyosin. Troponins in the bloodstream are indicative of heart disorders. Elevation of troponins in the blood occurs after a myocardial infarction and can remain high for up to two weeks. Actinin Actinin is a skeletal muscle protein that attaches filaments of actin to Z-lines of skeletal muscle cells. In smooth muscle cells, it also connects actin to dense bodies. Titin Titin (also known as connectin) is the molecular equivalent of a spring that provides striated muscle cells with elasticity. It is the third most abundant protein in muscle cells. The protein is enormous, with 244 folded individual protein domains spread across 363 exons (largest known number), with the largest known exon (17,106 base pairs long), and it is the largest protein known (27,000 to 33,000 amino acids, depending on splicing). Unstructured sequences The folded protein domains are linked together by unstructured sequences. The unstructured regions of the protein allow for unfolding when stretching occurs and refolding upon relaxation. Titin connects the M and Z lines in the sarcomere (Figure 2.123). Tension created in titin serves to limit the range of motion of the sarcomere, giving rise to what is called passive stiffness. Skeletal and cardiac muscles have slight amino acid sequence variations in their ti tin proteins and these appear to relate to differences in the mechanical characteristics of each muscle. Energy backup for muscle energy Myoglobin was described as a molecular batter for oxygen. Muscle cells have a better of their own for ATP. The is important for animals, but not for plants because a plant’s need for energy is different than an animal’s. Plants do not need to access energy sources as rapidly as animals do, nor do they have to maintain a constant internal temperature. Plants can neither flee predators, nor chase prey. These needs of animals are much more immediate and require that energy stores be accessible on demand. Muscles, of course, enable the motion of animals and the energy required for muscle contraction is ATP. To have stores of energy readily available, muscles have, in addition to ATP, creatine phosphate for energy and glycogen for quick release of glucose to make more energy. The synthesis of creatine phosphate is a prime example of the effects of concentration on the synthesis of high energy molecules. For example, creatine phosphate has an energy of hydrolysis of -43.1 kJ/mol whereas ATP has an energy of hydrolysis of -30.5 kJ/mol. Creatine phosphate, however, is made from creatine and ATP in the reaction shown in Figure 2.126. How is this possible? The ∆G°’ of this reaction is +12.6 kJ/mol, reflecting the energies noted above. In a resting muscle cell, ATP is abundant and ADP is low, driving the reaction downward, creating creatine phosphate. When muscular contraction commences, ATP levels fall and ADP levels climb. The above reaction then reverses and proceeds to synthesize ATP immediately. Thus, creatine phosphate acts like a battery, storing energy when ATP levels are high and releasing it almost instantaneously to create ATP when its levels fall.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/02%3A_Structure_and_Function/2.05%3A_Structure_and_Function-_Protein_Function_II.txt

Source: BiochemFFA_2_5.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy The nucleic acids, DNA and RNA, may be thought of as the information molecules of the cell. In this section, we will examine the structures of DNA and RNA, and how these structures are related to the functions these molecules perform. We will begin with DNA, which is the hereditary information in every cell, that is copied and passed on from generation to generation. The race to elucidate the structure of DNA was one of the greatest stories of 20th century science. Discovered in 1869 by Friedrich Miescher, DNA was identified as the genetic material in experiments in the 1940s led by Oswald Avery, Colin MacLeod, and Maclyn McCarty. X-ray diffraction work of Rosalind Franklin and the observations of Erwin Chargaff were combined by James Watson and Francis Crick to form a model of DNA that we are familiar with today. Their famous paper, in the April 25, 1953 issue of Nature, opened the modern era of molecular biology. Arguably, that one-page paper has had more scientific impact per word than any other research article ever published. Today, every high school biology student is familiar with the double helical structure of DNA and knows that G pairs with C and A with T. The double helix, made up of a pair of DNA strands, has at its core, bases joined by hydrogen bonds to form base pairs - adenine always paired with thymine, and guanine invariably paired with cytosine. Two hydrogen bonds are formed between adenine and thymine, but three hydrogen bonds hold together guanine and cytosine (Figure 2.127). The complementary structure immediately suggested to Watson and Crick how DNA might be (and in fact, is) replicated and it further explains how information is DNA is transmitted to RNA for the synthesis of proteins. In addition to the hydrogen bonds between bases of each strand, the double helix is held together by hydrophobic interactions of the stacked, non-polar bases. Crucially, the sequence of the bases in DNA carry the information for making proteins. Read in groups of three, the sequence of the bases directly specifies the sequence of the amino acids in the encoded protein. Structure A DNA strand is a polymer of nucleoside monophosphates held together by phosphodiester bonds. Two such paired strands make up the DNA molecule, which is then twisted into a helix. In the most common Bform, the DNA helix has a repeat of 10.5 base pairs per turn, with sugars and phosphate forming the covalent phosphodiester “backbone” of the molecule and the adenine, guanine, cytosine, and thymine bases oriented in the middle where they form the now familiar base pairs that look like the rungs of a ladder. Building blocks The term nucleotide refers to the building blocks of both DNA (deoxyribonucleoside triphosphates, dNTPs) and RNA (ribonucleoside triphosphates, NTPs). In order to discuss this important group of molecules, it is necessary to define some terms. Nucleotides contain three primary structural components. These are a nitrogenous base, a pentose sugar, and at least one phosphate. Molecules that contain only a sugar and a nitrogenous base (no phosphate) are called nucleosides. The nitrogenous bases found in nucleic acids include adenine and guanine (called purines) and cytosine, uracil, or thymine (called pyrimidines). There are two sugars found in nucleotides - deoxyribose and ribose (Figure 2.128). By convention, the carbons on these sugars are labeled 1’ to 5’. (This is to distinguish the carbons on the sugars from those on the bases, which have their carbons simply labeled as 1, 2, 3, etc.) Deoxyribose differs from ribose at the 2’ position, with ribose having an OH group, where deoxyribose has H. Nucleotides containing deoxyribose are called deoxyribonucleotides and are the forms found in DNA. Nucleotides containing ribose are called ribonucleotides and are found in RNA. Both DNA and RNA contain nucleotides with adenine, guanine, and cytosine, but with very minor exceptions, RNA contains uracil nucleotides, whereas DNA contains thymine nucleotides. When a base is attached to a sugar, the product, a nucleoside, gains a new name. • uracil-containing = uridine (attached to ribose) / deoxyuridine (attached to deoxyribose) • thymine-containing = ribothymidine (attached to ribose) / thymidine (attached to deoxyribose) • cytosine-containing = cytidine (attached to ribose - Figure 2.129) / deoxycytidine (attached to deoxyribose) • guanine-containing = guanosine (attached to ribose) / deoxyguanosine (attached to deoxyribose) • adenine-containing = adenosine (attached to ribose) / deoxyadenosine (attached to deoxyribose) Of these, deoxyuridine and ribothymidine are the least common. The addition of one or more phosphates to a nucleoside makes it a nucleotide. Nucleotides are often referred to as nucleoside phosphates, for this reason. The number of phosphates in the nucleotide is indicated by the appropriate prefixes (mono, di or tri). Thus, cytidine, for example, refers to a nucleoside (no phosphate), but cytidine monophosphate refers to a nucleotide (with one phosphate). Addition of second and third phosphates to a nucleoside monophosphate requires energy, due to the repulsion of negatively charged phosphates and this chemical energy is the basis of the high energy triphosphate nucleotides (such as ATP) that fuel cells. Note: Ribonucleotides as Energy Sources Though ATP is the most common and best known cellular energy source, each of the four ribonucleotides plays important roles in providing energy. GTP, for example, is the energy source for protein synthesis (translation) as well as for a handful of metabolic reactions. A bond between UDP and glucose makes UDP-glucose, the building block for making glycogen. CDP is similarly linked to several different molecular building blocks important for glycerophospholipid synthesis (such as CDP-diacylglycerol). The bulk of ATP made in cells is not from directly coupled biochemical metabolism, but rather by the combined processes of electron transport and oxidative phosphorylation in mitochondria and/or photophosphorylation that occurs in the chloroplasts of photosynthetic organisms. Triphosphate energy in ATP is transferred to the other nucleosides/nucleotides by action of enzymes called kinases. For example, nucleoside diphosphokinase (NDPK) catalyzes the following reaction \[\ce{ATP + NDP <-> ADP + NTP}\] where ‘N’ of “NDP” and “NTP corresponds to any base. Other kinases can put single phosphates onto nucleosides or onto nucleoside monophosphates using energy from ATP. Deoxyribonucleotides Individual deoxyribonucleotides are derived from corresponding ribonucleoside diphosphates via catalysis by the enzyme known as ribonucleotide reductase (RNR). The deoxyribonucleoside diphosphates are then converted to the corresponding triphosphates (dNTPs) by the addition of a phosphate group. Synthesis of nucleotides containing thymine is distinct from synthesis of all of the other nucleotides and will be discussed later. Building DNA strands Each DNA strand is built from dNTPs by the formation of a phosphodiester bond, catalyzed by DNA polymerase, between the 3’OH of one nucleotide and the 5’ phosphate of the next. The result of this directional growth of the strand is that the one end of the strand has a free 5’ phosphate and the other a free 3’ hydroxyl group (Figure 2.130). These are designated as the 5’ and 3’ ends of the strand. Figure 2.131 shows two strands of DNA (left and right). The strand on the left, from 5’ to 3’ reads T-C-G-A, whereas the strand on the right, reading from 5’ to 3’ is T-C-G-A. The strands in a double-stranded DNA are arranged in an anti-parallel fashion with the 5’ end of one strand across from the 3’ end of the other. Hydrogen bonds Hydrogen bonds between the base pairs hold a nucleic acid duplex together, with two hydrogen bonds per A-T pair (or per A-U pair in RNA) and three hydrogen bonds per G-C pair. The B-form of DNA has a prominent major groove and a minor groove tracing the path of the helix (Figure 2.132). Proteins, such as transcription factors bind in these grooves and access the hydrogen bonds of the base pairs to “read” the sequence therein. Other forms of DNA besides the B-form (Movie 2.5) are known (Figure 2.133). One of these, the A-form, was identified by Rosalind Franklin in the same issue of Nature as Watson and Crick’s paper. Though the A-form structure is a relatively minor form of DNA and resembles the B-form, it turns out to be important in the duplex form of RNA and in RNA-DNA hybrids. Both the A form and the B-form of DNA have the helix oriented in what is termed the right-handed form. Movie 2.5 - B-form DNA duplex rotating in space Wikipedia Z-DNA The A-form and the B-form stand in contrast to another form of DNA, known as the Z-form. ZDNA, as it is known, has the same base-pairing rules as the B and A forms, but instead has the helices twisted in the opposite direction, making a left-handed helix (Figure 2.133). The Z-form has a sort of zig-zag shape, giving rise to the name Z-DNA. In addition, the helix is rather stretched out compared to the A- and B-forms. Why are there different topological forms of DNA? The answer relates to both superhelical tension and sequence bias. Sequence bias means that certain sequences tend to favor the “flipping” of Bform DNA into other forms. ZDNA forms are favored by long stretches of alternating Gs and Cs. Superhelical tension is discussed below. Superhelicity Short stretches of linear DNA duplexes exist in the B-form and have 10.5 base pairs per turn. Double helices of DNA in the cell can vary in the number of base pairs per turn they contain. There are several reasons for this. For example, during DNA replication, strands of DNA at the site of replication get unwound at the rate of 6000 rpm by an enzyme called helicase. The effect of such local unwinding at one place in a DNA has the effect increasing the winding ahead of it. Unrelieved, such ‘tension’ in a DNA duplex can result in structural obstacles to replication. Such adjustments can occur in three ways. First, tension can provide the energy for ‘flipping’ DNA structure. Z-DNA can arise as a means of relieving the tension. Second, DNA can ‘supercoil’ to relieve the tension (Figures 2.134 & 2.135). In this method, the duplex crosses over itself repeatedly, much like a rubber band will coil up if one holds one section in place and twists another part of it. Third, enzymes called topoisomerases can act to relieve or, in some cases, increase the tension by adding or removing twists in the DNA. Topological isomers As noted, so-called “relaxed” DNA has 10.5 base pairs per turn. Each turn corresponds to one twist of the DNA. Using enzymes, it is possible to change the number of base pairs per turn. In either the case of increasing or decreasing the twists per turn, tension is introduced into the DNA structure. If the tension cannot be relieved, the DNA duplex will act to relieve the strain, as noted. This is most easily visualized for circular DNA, though long linear DNA (such as found in eukaryotic chromosomes) or DNAs constrained in other ways will exhibit the same behavior. Parameters To understand topologies, we introduce the concepts of ‘writhe’ and ‘linking number’. First, imagine either opening a closed circle of DNA and either removing one twist or adding one twist and then re-forming the circle. Since the strands have no free ends, they cannot relieve the induced tension by re-adding or removing the twists at their ends, respectively. Instead, the tension is relieved by “superhelices” that form with crossing of the double strands over each other (figure 8 structures in Figure 2.136). Though it is not apparent to visualize, each crossing of the double strands in this way allows twists to be increased or decreased correspondingly. Thus, superhelicity allows the double helix to reassume 10.5 base pairs per turn by adding or subtracting twists as necessary and replacing them with writhes. We write the equation L= T + W where T is the number of twists in a DNA, W is the number of writhes, and L is the linking number. The linking number is therefore the sum of the twists and writhes. Interestingly, inside of cells, DNAs typically are in a supercoiled form. Supercoiling affects the size of the DNA (compacts it) and also the expression of genes within the DNA, some having enhanced expression and some having reduced expression when supercoiling is present. Enzymes called topoisomerases alter the superhelical density of DNAs and play roles in DNA replication, transcription, and control of gene expression. They work by making cuts in one strand (Type I topoisomerases) or both strands (Type II topoisomerases) and then add or subtract twists as appropriate to the target DNA. After that process is complete, the topoisomerase re-ligates the nick/cut it had made in the DNA in the first step. Topoisomerases may be the targets of antibiotics. The family of antibiotics known as fluoroquinolones work by interfering with the action of bacterial type II topoisomerases. Ciprofloxacin also preferentially targets bacterial type II topoisomerases. Other topoisomerase inhibitors target eukaryotic topoisomerases and are used in anti-cancer treatments. RNA The structure of RNA (Figure 2.137) is very similar to that of a single strand of DNA. Built of ribonucleotides, joined together by the same sort of phosphodiester bonds as in DNA, RNA uses uracil in place of thymine. In cells, RNA is assembled by RNA polymerases, which copy a DNA template in the much same way that DNA polymerases replicate a parental strand. During the synthesis of RNA, uracil is used across from an adenine in the DNA template. The building of messenger RNAs by copying a DNA template is a crucial step in the transfer of the information in DNA to a form that directs the synthesis of protein. Additionally, ribosomal and transfer RNAs serve important roles in “reading” the information in the mRNA codons and in polypeptide synthesis. RNAs are also known to play important roles in the regulation of gene expression. RNA world The discovery, in 1990, that RNAs could play a role in catalysis, a function once thought to be solely the domain of proteins, was followed by the discovery of many more so-called ribozymes- RNAs that functioned as enzymes. This suggested the answer to a long-standing chicken or egg puzzle - if DNA encodes proteins, but the replication of DNA requires proteins, how did a replicating system come into being? This problem could be solved if the first replicator was RNA, a molecule that can both encode information and carry out catalysis. This idea, called the “RNA world” hypothesis, suggests that DNA as genetic material and proteins as catalysts arose later, and eventually prevailed because of the advantages they offer. The lack of a 2’OH on deoxyribose makes DNA more stable than RNA. The double-stranded structure of DNA also provides an elegant way to easily replicate it. RNA catalysts, however, remain, as remnants of that early world. In fact, the formation of peptide bonds, essential for the synthesis of proteins, is catalyzed by RNA. Secondary structure With respect to structure, RNAs are more varied than their DNA cousin. Created by copying regions of DNA, cellular RNAs are synthesized as single strands, but they often have self-complementary regions leading to “foldbacks” containing duplex regions. These are most easily visualized in the ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) (Figure 2.138), though other RNAs, including messenger RNAs (mRNAs), small nuclear RNAs (snRNAs), microRNAs (Figure 2.139), and small interfering RNAs (siRNAs) may each have double helical regions as well. Base pairing Base pairing in RNA is slightly different than DNA. This is due to the presence of the base uracil in RNA in place of thymine in DNA. Like thymine, uracil forms base pairs with adenine, but unlike thymine, uracil can, to a limited extent, also base pair with guanine, giving rise to many more possibilities for pairing within a single strand of RNA. These additional base pairing possibilities mean that RNA has many ways it can fold upon itself that single-stranded DNA cannot. Folding, of course, is critical for protein function, and we now know that, like proteins, some RNAs in their folded form can catalyze reactions just like enzymes. Such RNAs are referred to as ribozymes. It is for this reason scientists think that RNA was the first genetic material, because it could not only carry information, but also catalyze reactions. Such a scheme might allow certain RNAs to make copies of themselves, which would, in turn, make more copies of themselves, providing a positive selection. Stability RNA is less chemically stable than DNA. The presence of the 2’ hydroxyl on ribose makes RNA much more prone to hydrolysis than DNA, which has a hydrogen instead of a hydroxyl. Further, RNA has uracil instead of thymine. It turns out that cytosine is the least chemically stable base in nucleic acids. It can spontaneously deaminate and in turn is converted to a uracil. This reaction occurs in both DNA and RNA, but since DNA normally has thymine instead of uracil, the presence of uracil in DNA indicates that deamination of cytosine has occurred and that the uracil needs to be replaced with a cytosine. Such an event occurring in RNA would be essentially undetectable, since uracil is a normal component of RNA. Mutations in RNA have much fewer consequences than mutations in DNA because they are not passed between cells in division. Catalysis RNA structure, like protein structure, has importance, in some cases, for catalytic function. Like random coils in proteins that give rise to tertiary structure, single-stranded regions of RNA that link duplex regions give these molecules a tertiary structure, as well. Catalytic RNAs, called ribozymes, catalyze important cellular reactions, including the formation of peptide bonds in ribosomes (Figure 2.114). DNA, which is usually present in cells in strictly duplex forms (no tertiary structure, per se), is not known to be involved in catalysis. RNA structures are important for reasons other than catalysis. The 3D arrangement of tRNAs is necessary for enzymes that attach amino acids to them to do so properly. Further, small RNAs called siRNAs found in the nucleus of cells appear to play roles in both gene regulation and in cellular defenses against viruses. The key to the mechanisms of these actions is the formation of short foldback RNA structures that are recognized by cellular proteins and then chopped into smaller units. One strand is copied and used to base pair with specific mRNAs to prevent the synthesis of proteins from them. Denaturing nucleic acids Like proteins, nucleic acids can be denatured. Forces holding duplexes together include hydrogen bonds between the bases of each strand that, like the hydrogen bonds in proteins, can be broken with heat or urea. (Another important stabilizing force for DNA arises from the stacking interactions between the bases in a strand.) Single strands absorb light at 260 nm more strongly than double strands. This is known as the hyperchromic effect (Figure 2.141)and is a consequence of the disruption of interactions among the stacked bases. The changes in absorbance allow one to easily follow the course of DNA denaturation. Denatured duplexes can readily renature when the temperature is lowered below the “melting temperature” or Tm, the temperature at which half of the DNA strands are in duplex form. Under such conditions, the two strands can re-form hydrogen bonds between the complementary sequences, returning the duplex to its original state. For DNA, strand separation and rehybridization are important for the technique known as the polymerase chain reaction (PCR). Strand separation of DNA duplexes is accomplished in the method by heating them to boiling. Hybridization is an important aspect of the method that requires single stranded primers to “find” matching sequences on the template DNA and form a duplex. Considerations for efficient hybridization (also called annealing) include temperature, salt concentration, strand concentration, and magnesium ion levels (for more on PCR, see HERE). DNA packaging DNA is easily the largest macromolecule in a cell. The single chromosome in small bacterial cells, for example, can have a molecular weight of over 1 billion Daltons. If one were to take all of the DNA of human chromosomes from a single cell and lay them end to end, they would be over 7 feet long. Such an enormous molecule demands careful packaging to fit within the confines of a nucleus (eukaryotes) or a tiny cell (bacteria). The chromatin system of eukaryotes is the best known, but bacteria, too, have a system for compacting DNA. DNA in Bacteria In bacteria, there is no nucleus for the DNA. Instead, DNA is contained in a structure called a nucleoid (Figure 2.142). It contains about 60% DNA with much of the remainder comprised of RNAs and transcription factors. Bacteria do not have histone proteins that DNA wrap around, but they do have proteins that help organize the DNA in the cell - mostly by making looping structures. These proteins are known as Nucleoid Associated Proteins and include ones named HU, H-NS, Fis, CbpA, and Dps. Of these, HU most resembles eukaryotic histone H2B and binds to DNA non-specifically. The proteins associate with the DNA and can also cluster, which may be the origin of the loops. It is likely these proteins play a role in helping to regulate transcription and respond to DNA damage. They may also be involved in recombination. Eukaryotes The method eukaryotes use for compacting DNA in the nucleus is considerably different, and with good reason - eukaryotic DNAs are typically much larger than prokaryotic DNAs, but must fit into a nucleus that is not much bigger than a prokaryotic cell. Human DNA, for example, is about 1000 times longer than c DNA. The strategy employed in eukaryotic cells is that of spooling - DNA is coiled around positively charged proteins called histones. These proteins, whose sequence is very similar in cells as diverse as yeast and humans, come in four types, dubbed H1, H2a, H2b, H3, and H4. A sixth type, referred to as H5 is actually an isoform of H1 and is rare. Two each of H2a, H2b, H3, and H4 are found in the core structure of what is called the fundamental unit of chromatin - the nucleosome (Figure 2.143). Octamer The core of 8 proteins is called an octamer. The stretch of DNA wrapped around the octamer totals about 147 base pairs and makes 1 2/3 turns around it. This complex is referred to as a core particle (Figure 2.144). A linker region of about 50-80 base pairs separate core particles. The term nucleosome then refers to a a core particle plus a linker region (Figure 2.143). Histone H1 sits near the junction of the incoming DNA and the histone core. It is often referred to as the linker histone. In the absence of H1, non-condensed nucleosomes resemble “beads on a string” when viewed in an electron microscope. Histones Histone proteins are similar in structure and are rich in basic amino acids, such as lysine and arginine (Figure 2.145). These amino acids are positively charged at physiological pH, with enables them to form tight ionic bonds with the negatively charged phosphate backbone of DNA. For DNA, compression comes at different levels (Figure 2.146). The first level is at the nucleosomal level. Nucleosomes are stacked and coiled into higher order structures. 10 nm fibers are the simplest higher order structure (beads on a string) and they grow in complexity. 30 nm fibers consist of stacked nucleosomes and they are packed tightly. Higher level packing produces the metaphase chromosome found in meiosis and mitosis. The chromatin complex is a logistical concern for the processes of DNA replication and (particularly) gene expression where specific regions of DNA must be transcribed. Altering chromatin structure is therefore an essential function for transcriptional activation in eukaryotes. One strategy involves adding acetyl groups to the positively charged lysine side chains to “loosen their grip” on the negatively charged DNA, thus allowing greater access of proteins involved in activating transcription to gain access to the DNA. The mechanisms involved in eukaryotic gene expression are Ames test The Ames test (Figure 2.147) is an analytical method that allows one to determine whether a compound causes mutations in DNA (is mutagenic) or not. The test is named for Dr. Bruce Ames, a UC Berkeley emeritus professor who was instrumental in creating it. In the procedure, a single base pair of a selectable marker of an organism is mutated in a plasmid to render it nonfunctional. In the example, a strain of Salmonella is created that lacks the ability to grow in the absence of histidine. Without histidine, the organism will not grow, but if that one base in the plasmid’s histidine gene gets changed back to its original base, a functional gene will be made and the organism will be able to grow without histidine. A culture of the bacterium lacking the functional gene is grown with the supply of histidine it requires. It is split into two vials. To one of the vials, a compound that one wants to test the mutagenicity of is added. To the other vial, nothing is added. The bacteria in each vial are spread onto plates lacking histidine. In the absence of mutation, no bacteria will grow. The more colonies of bacteria that grow, the more mutation happened. Note that even the vial without the possible mutagenic compound will have a few colonies grow, as a result of mutations unlinked to the potential mutagen. Mutation happens in all cells at a low level. If the plate with the cells from the vial with the compound has more colonies than the cells from the control vial (no compound), then that would be evidence that the compound causes more mutations than would normally occur and it is therefore a mutagen. On the other hand, if there was no significant difference in the number of colonies on each plate, then that would suggest it is not mutagenic. The test is not perfect - it identifies about 90% of known mutagens - but its simplicity and inexpensive design make it an excellent choice for an initial screen of a compound. 2.07: Structure and Function- Carbohydrates Endogenous glycation, on the other hand, arises with a frequency that is proportional to the concentration of free sugar in the body. These occur most frequently with fructose, galactose, and glucose in that decreasing order and are detected in the bloodstream. Both proteins and lipids can be glycated and the accumulation of endogenous advanced glycation endproducts (AGEs) is associated with Type 2 diabetes, as well as in increases in cardiovascular disease (damage to endothelium, cartilage, and fibrinogen), peripheral neuropathy (attack of myelin sheath), and deafness (loss of myelin sheath). The formation of AGEs increases oxidative stress, but is also thought to be exacerbated by it. Increased oxidative stress, in turn causes additional harm. Damage to collagen in blood cells causes them to stiffen and weaken and is a factor in hardening of the arteries and formation of aneurysms, respectively. One indicator of diabetes is increased glycation of hemoglobin in red blood cells, since circulating sugar concentration are high in the blood of diabetics. Hemoglobin glycation is measured in testing for blood glucose control in diabetic patients. Homopolymer Monomeric Unit Glycogen Glucose Cellulose Glucose Amylose Glucose Callose Glucose Chitin N-acetylglucosamine Xylan Xylose Mannan Mannose Chrysolaminarin Glucose Function in skin Hyaluronic acid is a major component of skin and has functions in tissue repair. With exposure to excess UVB radiation, cells in the dermis produce less hyaluronan and increase its degradation. For some cancers the plasma level of hyaluronic acid correlates with malignancy. Hyaluronic acid levels have been used as a marker for prostate and breast cancer and to follow disease progression. The compound can to used to induce healing after cataract surgery. Hyaluronic acid is also abundant in the granulation tissue matrix that replaces a fibrin clot during the healing of wounds. In wound healing, it is thought that large polymers of hyaluronic acid appear early and they physically make room for white blood cells to mediate an immune response. Breakdown Breakdown of hyaluronic acid is catalyzed by enzymes known as hyaluronidases. Humans have seven types of such enzymes, some of which act as tumor suppressors. Smaller hyaluronan fragments can induce inflammatory response in macrophages and dendritic cells after tissue damage. They can also perform proangiogenic functions. Proteoglycans Glycosaminoglycans are commonly found attached to proteins and these are referred to as proteoglycans. Linkage between the protein and the glycosaminoglycan is made through a serine side-chain. Proteoglycans are made by glycosylation of target proteins in the Golgi apparatus.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/02%3A_Structure_and_Function/2.06%3A_Structure_and_Function_-_Nucleic_Acids.txt

Source: BiochemFFA_2_7.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Lipids are a diverse group of molecules that all share the characteristic that at least a portion of them is hydrophobic. Lipids play many roles in cells, including serving as energy storage (fats/oils), constituents of membranes (glycerophospholipids, sphingolipids, cholesterol), hormones (steroids), vitamins (fat soluble), oxygen/ electron carriers (heme), among others. For lipids that are very hydrophobic, such as fats/ oils, movement and storage in the aqueous environment of the body requires special structures. Other, amphipathic lipids, such as glycerophospholipids and sphingolipids spontaneously organize themselves into lipid bilayers when placed in water. Interestingly, major parts of many lipids can be derived from acetyl-CoA. Fatty acids The most ubiquitous lipids in cells are the fatty acids. Found in fats, glycerophospholipids, sphingolipids and serving as as membrane anchors for proteins and other biomolecules, fatty acids are important for energy storage, membrane structure, and as precursors of most classes of lipids. Fatty acids, as can be seen from Figure 2.190 are characterized by a polar head group and a long hydrocarbon tail. Fatty acids with hydrocarbon tails that lack any double bonds are described as saturated, while those with one or more double bonds in their tails are known as unsaturated fatty acids. The effect of double bonds on the fatty acid tail is to introduce a kink, or bend, in the tail, as shown for oleic acid. Stearic acid, a saturated fatty acid, by contrast has a straight hydrocarbon tail. Figures 2.190-2.194 show the most common saturated and unsaturated fatty acids. Fatty acids with unsaturated tails have a lower melting temperature than those with saturated tails of the same length. Shorter tails also decrease melting temperature. These properties carry over to the fats/oils containing them. Fatty acids with more than one double bond are called polyunsaturated. Plants are excellent sources of unsaturated and polyunsaturated fatty acids. The position of the double bond(s) in fatty acids has important considerations both for their synthesis and for their actions in the body. Biochemically, the double bonds found in fatty acids are predominantly in the cis configuration. So-called trans fats arise as a chemical by-product of partial hydrogenation of vegetable oil. In humans, consumption of trans fat raises low density lipoprotein (LDL) levels and lowers high density lipoprotein (HDL) levels. Each is thought to contribute to the risk of developing coronary artery disease. The most Figure 2.194 - Fatty acid models. Carboxyl end labeled in red Wikipedia common fatty acids in our body include palmitate, stearate, oleate, linolenate, linoleate, and arachidonate. Two notable shorter fatty acids are nonanoic (9 carbons) and decanoic acid (10 carbons), both of which appear to have anti-seizure effects. Decanoic acid directly inhibits excitatory neurotransmission in the brain and may contribute to the anticonvulsant effect of the ketogenic diet. Numbering Figure 2.195 shows two different systems for locating double bonds in a fatty acid. The ω system counts carbons starting with the methyl end (shown in red) while the Δ system counts from the carboxyl end (shown in blue). For example, an ω-3 (omega 3) fatty acid would have a double bond at the third carbon from the methyl end. In the Δ system, a fatty acid that has a cis double bond at carbon 6, counting from the carboxyl end, would be written as cis-Δ6. Fatty acids are described as essential fatty acids if they must be in the diet (can’t be synthesized by the organism) and nonessential fatty acids if the organism can synthesize them. Humans and other animals lack the desaturase enzymes necessary to make double bonds at positions greater than Δ-9, so fatty acids with double bonds beyond this position must be obtained in the diet. Linoleic acid and linolenic acid, both fall in this category. Related unsaturated fatty acids can be made from these fatty acids, so the presence of linoleic and linolenic acids in the diet eliminates the need to have all unsaturated fatty acids in the diet. Both linoleic and linolenic acid contain 18 carbons, but linoleic acid is an ω-6 fatty acid, whereas linolenic acid is an ω-3 fatty acid. Notably, ω-6 fatty acids tend to be proinflammatory, whereas ω-3 fatty acids are lesser so. Fats/oils Fats and oils are the primary energy storage forms of animals and are also known as triacylglycerols and triglycerides, since they consist of a glycerol molecule linked via ester bonds to three fatty acids (Figure 2.196). Fats and oils have the same basic structure. We give the name fat to those compounds that are solid at room temperature and the name oil to those that are liquid at room temperature. Note that biological oils are not the same as petroleum oils. Increasing the number of unsaturated fatty acids (and the amount of unsaturation in a given fatty acid) in a fat decreases the melting temperature of it. Organisms like fish, which live in cool environments, have fats with more unsaturation and this is why fish oil contains polyunsaturated fatty acids. Adipocytes Fats are stored in the body in specialized cells known as adipocytes. Enzymes known as lipases release fatty acids from fats by hydrolysis reactions (Figure 2.197). Triacylglycercol lipase (pancreatic - Figure 2.198) is able to cleave the first two fatty acids from the fat. A second enzyme, monoacylglycerol lipase, cleaves the last fatty acid. Fats can be synthesized by replacing the phosphate on phosphatidic acid with a fatty acid. Glycerophospholipids Glycerophospholipids (phosphoglycerides) are important components of the lipid bilayer of cellular membranes. Phosphoglycerides are structurally related to fats, as both are derived from phosphatidic acid (Figure 2.199). Phosphatidic acid is a simple glycerophospholipid that is usually converted into phosphatidyl compounds. These are made by esterifying various groups, such as ethanolamine, serine, choline, inositol, and others (Figure 2.200) to the phosphate of phosphatidic acid. All of these compounds form lipid bilayers in aqueous solution , due to their amphiphilic nature. Phosphatidylethanolamines Since all glycerolipids can have a variety of fatty acids at positions 1 and 2 on the glycerol, they all are families of compounds. The phosphatidylethanolamines are found in all living cells and are one of the most common phosphatides, making up about 25% of them. They are common constituents of brain tissue and in the spinal cord, making up as much as 45% of the total phospholipids. Phosphatidylethanolamines are asymmetrically distributed across membranes, being preferentially located on the inner leaflet (closest to the cytoplasm) of the plasma membrane. Metabolically, phosphatidylethanloamines are precursors of phosphatidylcholines. Phosphatidylserines Phosphatidylserines are another group of phosphatidyl compounds that are preferentially distributed across the lipid bilayer of the plasma membrane. Like the phosphatidylethanolamines, phosphatidylserines are preferentially located on the inner leaflet of the plasma membrane. When apoptosis (cell suicide) occurs, the preferential distribution is lost and the phosphatidylserines appear on the outer leaflet where they serve as a signal to macrophages to bind and destroy the cell. Phosphatidylcholines Phosphatidylcholines (Figure 2.201) are another group of important membrane components. They tend to be found more commonly on the outer leaflet of the plasma membrane. Nutritionally, the compounds are readily obtained from eggs and soybeans. Phosphatidylcholines are moved across membranes by Phosphatidylcholine transfer protein (PCTP). This protein, which is sensitive to the levels of phosphatidylcholines, acts to stimulate the activity of a thioesterase (breaks thioester bonds, such as acyl-CoAs) and activates PAX3 transcription factors. Cardiolipins Cardiolipins are an unusual set of glycerophospholipids in containing two diacylglycerol backbones joined in the middle by a diphosphoglycerol (Figure 2.202). It is an important membrane lipid, constituting about 20% of the inner mitochondrial membrane and is found in organisms from bacteria to humans. In both plants and animals, it is found almost totally in the inner mitochondrial membrane. The molecules appear to be required for both Complex IV and Complex III of the electron transport chain to maintain its structure. The ATP synthase enzyme (Complex V) of the oxidative phosphorylation system also binds four molecules of cardiolipin. It has been proposed that cardiolipin functions as a proton trap in the process of proton pumping by Complex IV. Cardiolipin also plays a role in apoptosis. As shown in Figure 2.203, oxidation of cardiolipin by a cardiolipin-specific oxygenase causes cardiolipin to move from the inner mitochondrial membrane to the outer one, helping to form a permeable pore and facilitating the transport of cytochrome c out of the intermembrane space and into the cytoplasm - a step in the process of apoptosis. Diacylglycerol Diacylglycerol (also called diglyceride and DAG - Figure 2.204) is an important intermediate in metabolic pathways. It is produced, for example, in the first step of the hydrolysis of fat and is also produced when membrane lipids, such as PIP2 (phosphatidylinositol-4,5-bisphosphate) are hydrolyzed by phospholipase C in a signaling cascade. DAG is itself a signaling compound, binding to protein kinase C to activate it to phosphorylate substrates. Synthesis of DAG begins with glycerol-3-phosphate, which gains two fatty acids from two acyl-CoAs to form phosphatidic acid. Dephosphorylation of phosphatidic acid produces DAG. DAG can also be rephosphorylated by DAG kinase to re-make phosphatidic acid or another fatty acid can be added to make fat. Inositol Though technically not a lipid itself, inositol is found in many lipids. Inositol is a derivative of cyclohexane containing six hydroxyl groups - one on each carbon (Figure 2.205. It has nine different stereoisomers of which one, cis-1,2,3,5-trans-4,6- cyclohexanehexol (called myo-inositol) is the most common. It has a sweet taste (half that of sucrose). Numerous phosphorylated forms of the compound exist, from a single phosphate to six (one on each carbon). Phytic acid, for example, in plants, has six phosphates (Figure 2.206) that it uses to store phosphate. Inositol is produced from glucose and was once considered vitamin B8, but is made by the body in adequate amounts, so it is not now considered a vitamin. Phosphorylated forms of inositol are found in phosphoinositides, such as PIP2 and PIP3, both of which are important in signaling processes. Some of these include insulin signaling, fat catabolism, calcium regulation, and assembly of the cytoskeleton. Phosphoinositides Compounds based on phosphatidylinositol (PI) are often called phosphoinositides. These compounds have important roles in signaling and membrane trafficking. Hydroxyls on carbons 3,4, and 5 of the inositol ring are targets for phosphorylation by a variety of kinases. Seven different combinations are used. Steric hindrance inhibits phosphorylation of carbons 2 or 6. Naming of these phosphorylated compounds follows generally as PI(#P)P, PI(#P, #P)P, or PI(#P, #P, #P)P where #P refers to the number of the carbon where a phosphate is located. For example, PI(3)P refers to a phosphatidyl compound with a phosphate added to carbons 3 of the inositol ring, whereas PI(3,4,5)P is a phosphatidyl compound with a phosphate added to carbons 3,4,and 5. Phosphatidylinositol-4,5- bisphosphate Phosphatidylinositol-4,5-bisphosphate (PIP2 - Figure 2.207) is a phospholipid of plasma membranes that functions in the phospholipase C signaling cascade. In this signaling pathway, hydrolysis catalyzed by phospholipase C releases inositol-1,4,5- trisphosphate (IP3) and diacylglycerol. Synthesis of PIP2 begins with phosphatidylinositol, which is phosphorylated at position 4 followed by phosphorylation at position 5 by specific kinases. PIP2 can be phosphorylated to form the signaling molecule known as phosphatidylinositol (3,4,5)-trisphosphate (PIP3). Along with PIP3, PIP2 serves as a docking phospholipid for the recruitment of proteins that play roles in signaling cascades. Binding of PIP2 is also required by inwardly directed potassium channels. Phosphatidylinositol (3,4,5)- trisphosphate Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) is an important molecule for the activation of signaling proteins, such as AKT, which activates anabolic signaling pathways related to growth and survival. PIP3 can be dephosphorylated by phosphatase PTEN to yield PIP2 and can be synthesized from PIP2 by kinase action of Class I PI 3- kinases. Kinase activity to synthesize PIP3 results in movement of PIP3-binding proteins to the plasma membrane. They include Akt/ PKB, PDK1, Btk1, and ARNO and each is activated by binding to PIP3. Plasmalogens A special class of the glycerophospholipids are the plasmalogens (Figure 2.209). They differ in containing a vinyl ether linkage at position 1 of glycerol, in contrast to other glycerophopsholipids, which have an ester linkage at this position. Position 2 of each is an ester. The precursor for the ether linkage is typically a 16 or 18 carbon saturated alcohol or an 18 carbon unsaturated alcohol. At the phosphate tail, the most commonly attached groups are ethanolamine or choline. Plasmalogens are found abundantly in humans in heart (30-40% of choline phospholipids). 30% of the glycerophospholipids in brain are plasmalogens and 70% of the ethanolamine lipids of the myelin sheath of nerve cells are plasmalogens. Though their function is not understood, it is believed that plasmalogens may provide some protection against reactive oxygen species and have roles in signaling. Lecithin Lecithin is a generic term for a combination of lipid substances that includes phosphoric acid, glycerol, glycolipids, triglycerides, and phospholipids. Lecithin is a wetting agent helpful with emulsification and encapsulation and is even used as an anti-sludge additive in motor lubricants. Lecithin is used in candy bars to keep cocoa and cocoa butter from separating. Though considered safe as a food ingredient, lecithin can be converted by gut bacteria to trimethylamine-N-oxide which may contribute to cholesterol deposition and atherosclerosis. Sphingolipids Fatty acids are also components of a broad class of molecules called sphingolipids. Sphingolipids are structurally similar to glycerophospholipids, though they are synthesized completely independently of them starting with palmitic acid and the amino acid serine. Sphingolipids are named for the amino alcohol known as sphingosine (Figure 2.210), though they are not directly synthesized from it. Figure 2.211 shows the generalized structure of sphingolipids. If the R-group is a hydrogen, the molecule is called a ceramide. When the R-group is phosphoethanolamine the resulting molecule is sphingomyelin, an important component of the myelin sheath and lipid membranes. If a single, simple sugar is instead added, a cerebroside is created (Figure 2.212). Addition of a complex oligosaccharide creates a ganglioside. Complex sphingolipids may play roles in cellular recognition and signaling. Sphingolipids are found most abundantly in plasma membrane and are almost completely absent from mitochondrial and endoplasmic reticulum membranes. In animals, dietary sphingolipids have been linked to reduced colon cancer, reductions in LDLs, and increases in HDLs. Like the glycerophospholipids, sphingolipids are amphiphilic. Most sphingolipids except sphingomyelin do not contain phosphate. Eicosanoids Fatty acids made from omega-6 and omega-3 fatty acids include three important fatty acids containing 20 carbons. They include arachidonic acid (an ω-6 fatty acid with four double bonds (Δ-5,8,11,14) - Figure 2.213), eicosapentaenoic acid (an ω-3 fatty acid with five double bonds, and dihomo-γ-linolenic acid (an ω-6 fatty acid with three double bonds). The class of compounds known as eicosanoids is made by oxidation of these compounds. Subclasses include include prostaglandins, prostacyclins, thromboxanes, lipoxins, leukotrienes, and endocannabinoids (Figures 2.214-2.219). Eicosanoids play important roles affecting inflammation, immunity, mood, and behavior. Prostaglandins A collection of molecules acting like hormones, prostaglandins are derived from arachidonic acid and have many differing (even conflicting) physiological effects. These include constriction or dilation of vascular smooth muscle cells, induction of labor, regulation of inflammation, and action on the thermoregulatory center of the hypothalamus to induce fever, among others. Prostaglandins are grouped with the thromboxanes (below) and prostacyclins (below), as prostanoids. The prostanoids, which all contain 20 carbons are a subclass of the eicosanoids. Prostaglandins are found in most tissues of higher organisms. They are autocrine or paracrine compounds produced from essential fatty acids. The primary precursor of the prostaglandins is the fatty acid known as arachidonic acid and the prostaglandin made from it is known as PGH2 (Figure 2.214), which, in turn is a precursor of other prostaglandins, as well as the prostacyclins and thromboxanes. Interesting prostaglandins PGD2 - inhibits hair follicle growth, vasodilator, causes bronchial constriction, higher in lungs of asthmatics than others. PGE2 (Figure 2.215) - exerts effects in labor (soften cervix, uterine contraction), stimulates bone resorption by osteoclasts, induces fever, suppresses T-cell receptor signaling, vasodilator, inhibits release of noradrenalin from sympathetic nerve terminals. It is a potent activator of the Wnt signaling pathway. A prostaglandin can have opposite effects, depending on which receptor it binds to. Binding of PGE2 to the EP1 receptor causes bronchoconstriction and smooth muscle contraction, whereas binding of the same molecule to the EP2 receptor causes bronchodilation and smooth muscle relaxation. PGF (Figure 2.216)- uterine contractions, induces labor, bronchoconstriction. PGI2 - vasodilation, bronchodilation, inhibition of platelet aggregation. Thromboxanes Thromboxanes play roles in clot formation and named for their role in thrombosis. They are potent vasoconstrictors and facilitate platelet aggregation. They are synthesized in platelets, as well. The anti-clotting effects of aspirin have their roots in the inhibition of synthesis of PGH2, which is the precursor of the thromboxanes. The most common thromboxanes are A2 (Figure 2.217) and B2. Prostacyclin Prostacyclin (also known as prostaglandin I2 or PGI2 - Figure 2.218) counters the effects of thromboxanes, inhibiting platelet activation and acting as vasodilators. It is produced from PGH2 by action of the enzyme prostacyclin synthase. Leukotrienes Another group of eicosanoid compounds are the leukotrienes (Figure 2.219). Like prostaglandins, leukotrienes are made from arachidonic acid. The enzyme catalyzing their formation is a dioxygenase known as arachidonate 5-lipoxygenase. Leukotrienes are involved in regulating immune responses. They are found in leukocytes and other immunocompetent cells, such as neutrophils, monocytes, mast cells, eosinophils, and basophils. Leukotrienes are associated with production of histamines and prostaglandins, which act as mediators of inflammation. Leukotrienes also trigger contractions in the smooth muscles of the bronchioles. When overproduced, they may pay a role in asthma and allergic reactions. Some treatments for asthma aim at inhibiting production or action of leukotrienes. Cholesterol Arguably, no single biomolecule has generated as much discussion and interest as has cholesterol (Figure 2.220). Certainly, from the perspective of the Nobel Prize committee, no small molecule even comes close, with 13 people having been awarded prizes for work on it. Evidence for cholesterol’s importance comes from the study of brain tissue where it comprises 10-15% of the dry mass. Membrane flexibility In animal cells, cholesterol provides for membrane flexibility that allows for cellular movement that is in contrast to plant and bacterial cells with fixed structures. Cholesterol is made in many cells of the body, with the liver making the greatest amount. The anabolic pathway leading to synthesis of cholesterol is known as the isoprenoid pathway and branches of it lead to other molecules including other fat-soluble vitamins. Cholesterol is only rarely found in prokaryotes (Mycoplasma, which requires it for growth, is an exception) and is found in only trace amounts in plants. Instead, plants synthesize similar compounds called phytosterols (Figure 2.221). On average, the body of a 150 pound adult male makes about 1 gram of cholesterol per day, with a total content of about 35 grams. Packaging Cholesterol’s (and other lipids’) hydrophobicity requires special packaging into lipoprotein complexes (called chylomicrons, VLDLs, IDLs, LDLs, and HDLs) for movement in the lymph system and bloodstream. Though cholesterol can be made by cells, they also take it up from the blood supply by absorbing cholesterol-containing LDLs directly in a process called receptor-mediated endocytosis. Oxidative damage to LDLs can lead to formation of atherosclerotic plaques and this is why cholesterol has gotten such a negative image in the public eye. The liver excretes cholesterol through the bile for elimination into the digestive system, but the compound is recycled there. Reducing cholesterol levels Strategies for reducing cholesterol in the body focus primarily on three areas - reducing consumption, reducing endogenous synthesis, and reducing the recycling. Dietary considerations, such as saturated fat versus unsaturated fat consumption are currently debated. Dietary trans fats, though, correlate with incidence of coronary heart disease. Consumption of vegetables may provide some assistance with reducing levels of cholesterol recycled in the digestive system, because plant phytosterols compete with cholesterol for reabsorption and when this happens, a greater percentage of cholesterol exits the body in the feces. Drugs related to penicillin are also used to inhibit cholesterol recycling. One of these is ezetimibe, shown in Figure 2.224. Genetic defects in the cholesterol movement system are a cause of the rare disease known as familial hypercholesterolemia in which the blood of afflicted individuals contains dangerously high levels of LDLs. Left untreated, the disease is often fatal in the first 10-20 years of life. While LDLs have received (and deserve) a bad rap, another group of lipoprotein complexes known as the HDLs (high density lipoprotein complexes) are known as “good cholesterol” because their levels correlate with removal of debris (including cholesterol) from arteries and reduce inflammation. Membrane function In membranes, cholesterol is important as an insulator for the transmission of signals in nerve tissue and it helps to manage fluidity of membranes over a wide range of temperatures. Stacked in the lipid bilayer, cholesterol decreases a membrane’s fluidity and its permeability to neutral compounds, as well as protons and sodium ions. Cholesterol may play a role in signaling by helping with construction of lipid rafts within the cell membrane. Vitamin A Vitamin A comes in three primary chemical forms, retinol (storage in liver - Figure 2.225), retinal (role in vision - Figure 2.226), and retinoic acid (roles in growth and development). All vitamin A forms are diterpenoids and differ only in the chemical form of the terminal group. Retinol is mostly used as the storage form of the vitamin. Retinol is commonly esterified to a fatty acid and kept in the liver. In high levels, the compound is toxic. Retinol is obtained in the body by hydrolysis of the ester or by reduction of retinal. Importantly, neither retinal nor retinol can be made from retinoic acid. Retinoic acid is important for healthy skin and teeth, as well as bone growth. It acts in differentiation of stem cells through a specific cellular retinoic acid receptor. Sources Good sources of vitamin A are liver and eggs, as well as many plants, including carrots, which have a precursor, β-carotene (Figure 2.227) from which retinol may be made by action of a dioxygenase. Light sensitivity The conjugated double bond system in the side chain of vitamin A is sensitive to light and can flip between cis and trans forms on exposure to it. It is this response to light that makes it possible for retinal to have a role in vision in the rods and cones of the eyes. Here, the aldehyde form (retinal) is bound to the protein rhodopsin in the membranes of rod and cone cells. When exposed to light of a particular wavelength, the “tail” of the retinal molecule will flip back and forth from cis to trans at the double bond at position 11 of the molecule. When this happens, a nerve signal is generated that signals the brain of exposure to light. Slightly different forms of rhodopsin have different maximum absorption maxima allowing the brain to perceive red, green and blue specifically and to assemble those into the images we see (Figure 2.228). Cones are the cells responsible for color vision, whereas rods are mostly involved in light detection in low light circumstances. Deficiency and surplus Deficiency of vitamin A is common in developing countries and was inspiration for the design and synthesis of the geneticallymodified golden rice, which is used as a source of vitamin A to help prevent blindness in children. Overdose of vitamin A, called hypervitaminosis A is dangerous and can be fatal. Excess vitamin A is also suspected to be linked to osteoporosis. In smokers, excess vitamin A is linked to an increased rate of lung cancer, but non-smokers have a reduced rate. Vitamin D The active form of vitamin D plays important roles in the intestinal absorption of calcium and phosphate and thus in healthy bones. Technically, vitamin D isn’t even a vitamin, as it is a compound made by the body. Rather, it behaves more like a hormone. Derived from ultimately from cholesterol, vitamin D can be made in a reaction catalyzed by ultraviolet light. In the reaction, the intermediate 7-dehydrocholesterol is converted to cholecalciferol (vitamin D3) by the uv light (Figure 2.229). The reaction occurs most readily in the bottom two layers of the skin shown in Figure 2.230. Forms of vitamin D Five different compounds are referred to as vitamin D. They are Vitamin D1 - A mixture of ergocalciferol and lumisterol Vitamin D2 - Ergocalciferol Vitamin D3 - Cholecalciferol Vitamin D4 - 22-Dihydroergocalciferol Vitamin D5 - Sitocalciferol Vitamin D3 is the most common form used in vitamin supplements and it and vitamin D2 are commonly obtained in the diet, as well. The active form of vitamin D, calcitriol (Figure 2.231), is made in the body in controlled amounts. This proceeds through two steps from cholecalciferol. First, a hydroxylation in the liver produces calcidiol and a second hydroxylation in the kidney produces calcitriol. Monocyte macrophages can also synthesize vitamin D and they use is as a cytokine to stimulate the innate immune system. Mechanism of action Calcitriol moves in the body bound to a vitamin D binding protein, which delivers it to target organs. Calcitriol inside of cells acts by binding a vitamin D receptor (VDR), which results in most of the vitamin’s physiological effects. After binding calcitriol, the VDR migrates to the nucleus where it acts as a transcription factor to control levels of expression of calcium transport proteins (for example) in the intestine. Most tissues respond to VDR bound to calcitriol and the result is moderation of calcium and phosphate levels in cells. Deficiency/excess Deficiency of vitamin D is a cause of the disease known as rickets, which is characterized by soft, weak bones and most commonly is found in children. It is not common in the developed world, but elsewhere is of increasing concern. Excess of vitamin D is rare, but has toxic effects, including hypercalcemia, which results in painful calcium deposits in major organs. Indications of vitamin D toxicity are increased urination and thirst. Vitamin D toxicity can lead to mental retardation and many other serious health problems. Vitamin E Vitamin E comprises a group of two compounds (tocopherols and tocotrienols - Figure 2.232) and stereoisomers of each. It is commonly found in plant oils. The compounds act in cells as fat-soluble antioxidants. α-tocopherol (Figure 2.233), the most active form of the vitamin, works 1) through the glutathione peroxidase protective system and 2) in membranes to interrupt lipid peroxidation chain reactions. In both actions, vitamin E reduces levels of reactive oxygen species in cells. Action Vitamin E scavenges oxygen radicals (possessing unpaired electrons) by reacting with them to produce a tocopheryl radical. This vitamin E radical can be converted back to its original form by a hydrogen donor. Vitamin C is one such donor. Acting in this way, Vitamin E helps reduce oxidation of easily oxidized compounds in the lipid peroxidation reactions (Figure 2.234). Vitamin E also can affect enzyme activity. The compound can inhibit action of protein kinase C in smooth muscle and simultaneously activate catalysis of protein phosphatase 2A to remove phosphates, stopping smooth muscle growth. Deficiency/excess Deficiency of vitamin E can lead to poor conduction of nerve signals and other issues arising from nerve problems. Low levels of the vitamin may be a factor in low birth weights and premature deliveries. Deficiency, however, is rare, and not usually associated with diet. Excess Vitamin E reduces vitamin K levels, thus reducing the ability to clot blood. Hypervitaminosis of vitamin E in conjunction with aspirin can be life threatening. At lower levels, vitamin E may serve a preventative role with respect to atherosclerosis by reducing oxidation of LDLs, a step in plaque formation. Vitamin K Like the other fat-soluble vitamins, Vitamin K comes in multiple forms (Figure 2.235) and is stored in fat tissue in the body. There are two primary forms of the vitamin - K1 and K2 and the latter has multiple sub-forms . Vitamins K3, K4, and K5 are made synthetically, not biologically. Action Vitamin K is used as a co-factor for enzymes that add carboxyl groups to glutamate side chains of proteins to increase their affinity for calcium. Sixteen such proteins are known in humans. They include proteins involved in blood clotting (prothrombin (called Factor II), Factors VII, IX, and X), bone metabolism (osteocalcin, also called bone Gla protein (BGP), matrix Gla protein (MGP), and periostin) and others. Modification of prothrombin is an important step in the process of blood clotting (see HERE). Reduced levels of vitamin K result in less blood clotting, a phenomenon sometimes referred to as blood thinning. Drugs that block recycling of vitamin K (Figure 2.236) by inhibiting the vitamin K epoxide reductase, produce lower levels of the vitamin and are employed in treatments for people prone to excessive clotting. Warfarin (coumadin) is one such compound that acts in this way and is used therapeutically. Individuals respond to the drug differentially, requiring them to periodically be tested for levels of clotting they possess, lest too much or too little occur. Sources Vitamin K1 is a stereoisomer of the plant photosystem I electron receptor known as phylloquinone and is found abundantly in green, leafy vegetables. Phylloquinone is one source of vitamin K, but the compound binds tightly to thylakoid membranes and tends to have low bioavailability. Vitamin K2 is produced by microbes in the gut and is a primary source of the vitamin. Infants in the first few days before they establish their gut flora and people taking broad spectrum antibiotics may have reduced levels, as a result. Dietary deficiency is rare in the absence of damage to the small bowel. Others at risk of deficiency include people with chronic kidney disease and anyone suffering from a vitamin D deficiency. Deficiencies produce symptoms of easy bruising, heavy menstrual bleeding, anemia, and nosebleeds. Steroids Steroids, such as cholesterol are found in membranes and act as signaling hormones in traveling through the body. Steroid hormones are all made from cholesterol and are grouped into five categories - mineralocorticoids (21 carbons), glucocorticoids (21 carbons), progestagens (21 carbons), androgens (19 carbons), and estrogens (18 carbons). Mineralocorticoids Mineralocorticoids are steroid hormones that influence water and electrolyte balances. Aldosterone (Figure 2.238) is the primary mineralocorticoid hormone, though other steroid hormones (including progesterone) have some functions like it. Aldosterone stimulates kidneys to reabsorb sodium, secrete potassium, and passively reabsorb water. These actions have the effect of increasing blood pressure and blood volume. Mineralocorticoids are produced by the zona glomerulosa of the cortex of the adrenal gland. Glucocorticoids Glucocorticoids (GCs) bind to glucocorticoid receptors found in almost every vertebrate animal cell and act in a feedback mechanism in the immune system to reduce its activity. GCs are used to treat diseases associated with overactive immune systems. These include allergies, asthma, and autoimmune dis- Figure 2.237 - Steroid numbering scheme Image by Pehr Jacobson eases. Cortisol (Figure 2.239) is an important glucocorticoid with cardiovascular, metabolic, and immunologic functions. The synthetic glucocorticoid known as dexamethasone has medical applications for treating rheumatoid arthritis, bronchospasms (in asthma), and inflammation due to its increased potency (25-fold) compared to cortisol. Glucocorticoids are produced primarily in the zona fasciculata of the adrenal cortex. Progestagens Progestagens (also called gestagens) are steroid hormones that work to activate the progesterone receptor upon binding to it. Synthetic progestagens are referred to as progestins. The most common progestagen is progesterone (also called P4 - Figure 2.240) and it has functions in maintaining pregnancy. Progesterone is produced primarily in the diestrus phase of the estrous cycle by the corpus luteum of mammalian ovaries. In pregnancy, the placenta takes over most progesterone production. Androgens Androgens are steroid hormones that act by binding androgen receptors to stimulate development and maintenance of male characteristics in vertebrates. Androgens are precursors of estrogens (see below). The primary androgen is testosterone (Figure 2.241). Other important androgens include dihydrotestosterone (stimulates differentiation of penis, scrotum, and prostate in embryo) and androstenedione (common precursor of male and female hormones). Estrogens The estrogen steroid hormones are a class of compounds with important roles in menstrual and estrous cycles. They are the most important female sex hormones. Estrogens act by activating estrogen receptors inside of cells. These receptors, in turn, affect expression of many genes. The major estrogens in women include estrone (E1), estradiol (E2 - Figure 2.242), and estriol (E3). In the reproductive years, estradiol predominates. During pregnancy, estriol predominates and during menopause, estrone is the major estrogen. Estrogens are made from the androgen hormones testosterone and androstenedione in a reaction catalyzed by the enzyme known as aromatase. Inhibition of this enzyme with aromatase inhibitors, such as exemestane, is a strategy for stopping estrogen production. This may be part of a chemotherapeutic treatment when estrogenresponsive tumors are present. Cannabinoids Cannabinoids are a group of chemicals that bind to and have effects on brain receptors (cannabinoid receptors), repressing neurotransmitter release. Classes of these compounds include endocannabinoids (made in the body), phytocannabinoids (made in plants, such as marijuana), and synthetic cannabinoids (man-made). Endocannabinoids are natural molecules derived from arachidonic acid. Cannabinoid receptors are very abundant, comprising the largest number of G-protein- 247 Figure 2.243 - Tetrahydrocannabinol - Active ingredient in marijuana coupled receptors found in brain. The best known phytocannabinoid is Δ-9- tetrahydrocannabinol (THC), the primary psychoactive ingredient (of the 85 cannabinoids) of marijuana (Figure 2.243). Anandamide Anandamide (N-arachidonoylethanolamine - Figure 2.244) is an endocannabinoid neurotransmitter derived from arachidonic acid. It exerts its actions primarily through the CB1 and CB2 cannabinoid receptors, the same ones bound by the active ingredient in marijuana, Δ9-tetrahydrocannabinol. Anandamide has roles in stimulating eating/appetite and affecting motivation and pleasure. It has been proposed to play a role in “runners high,” an analgesic effect experienced from exertion, especially among runners. Anandamide appears to impair memory function in rats. Anandamide has been found in chocolate and two compounds that mimic its effects (N-oleoylethanolamine and Nlinoleoylethanolamine) are present as well. The enzyme fatty acid amide hydrolase (FAAH) breaks down anandamide into free arachidonic acid and ethanolamine. Lipoxins Lipoxins (Figure 2.245) are eicosanoid compounds involved in modulating immune responses and they have anti-inflammatory effects. When lipoxins appear in inflammation it begins the end of the process. Lipoxins act to attract macrophages to apoptotic cells at the site of inflammation and they are engulfed. Lipoxins further act to start the resolution phase of the inflammation process. At least one lipoxin (aspirin-triggered LX4) has its synthesis stimulated by aspirin. This occurs as a byproduct of aspirin’s acetylation of COX-2. When this occurs, the enzyme’s catalytic activity is re-directed to synthesis of 15R-hydroxyeicosatetraenoic acid (HETE) instead of prostaglandins. 15R-HETE is a procursor of 15-epimer lipoxins, including aspirin-triggered LX4. Heme Heme groups are a collection of protein/ enzyme cofactors containing a large heterocyclic aromatic ring known as a porphyrin ring with a ferrous (Fe++) ion in the middle. An example porphyrin ring with an iron (found in Heme B of hemoglobin), is shown in Figure 2.246. When contained in a protein, these are known collectively as hemoproteins (Figure 2.247). Heme, of course, is a primary component of hemoglobin, but it is also found in other proteins, such as myoglobin, cytochromes, and the enzymes catalase and succinate dehydrogenase. Hemoproteins function in oxygen transport, catalysis, and electron transport. Heme is synthesized in the liver and bone marrow in a pathway that is conserved across a wide range of biology. Porphobilinogen Porphobilinogen (Figure 2.248) is a pyrrole molecule involved in porphyrin metabolism. It is produced from aminolevulinate by action of the enzyme known as ALA dehydratase. Porphobilinogen is acted upon by the enzyme porphobilinogen deaminase. Deficiency of the latter enzyme (and others in porphyrin metabolism) can result in a condition known as porphyria, which results in accumulation of porphobilinogen in the cytoplasm of cells. The disease can manifest itself with acute abdominal pain and numerous psychiatric issues. Both Vincent van Gogh and King ` George III are suspected to have suffered from porphyria, perhaps causing the “madness of King George III.” Porphyria is also considered by some to be the impetus for the legend of vampires seeking blood from victims, since the color of the skin in non-acute forms of the disease can be miscolored, leading some to perceive that as a deficiency of hemoglobin and hence the “thirst” for blood imagined for vampires. Dolichols Dolichol is a name for a group of non-polar molecules made by combining isoprene units together. Phosphorylated forms of dolichols play central roles in the N-glycosylation of proteins. This process, which occurs in the endoplasmic reticulum of eukaryotic cells, begins with a membrane-embedded dolichol pyrophosphate (Figure 2.249) to which an oligosaccharide is attached (also see HERE). This oligosaccharide contains three molecules of glucose, nine molecules of mannose and two molecules of N-acetylglucosamine. Interestingly, the sugars are attached to the dolichol pyrophosphate with the pyrophosphate pointing outwards (away from) the endoplasmic reticulum, but after attachment, the dolichol complex flips so that the sugar portion is situated on the inside of the endoplasmic reticulum. There, the entire sugar complex is transferred to the amide of an asparagine side chain of a target protein. The only asparagine side chains to which the attachment can be made are in proteins where the sequences Asn-X-Ser or Asn-X-Thr occur. Sugars can be removed/added after the transfer to the protein. Levels of dolichol in the human brain increase with age, but in neurodegenerative diseases, they decrease. Terpenes Terpenes are members of a class of nonpolar molecules made from isoprene units. Terpenes are produced primarily by plants and by some insects. Terpenoids are a related group of molecules that contain functional groups lacking in terpenes. Terpenes have a variety of functions. In plants, they often play a defensive role protecting from insects. The name of terpene comes from turpentine, which has an odor like some of the terpenes. Terpenes are common components of plant resins (think pine) and they are widely used in medicines and as fragrances. Hops, for example, gain some of their distinctive aroma and flavor from terpenes. Not all terpenes, however have significant odor. Synthesis Terpenes, like steroids, are synthesized starting with simple building blocks known as isoprenes. There are two of them - dimethylallyl pyrophosphate and the related isopentenyl pyrophosphate and (Figures 2.252 and 2.253) which combine 1-2 units at a time to make higher order structures. Terpene synthesis overlaps and includes steroid synthesis. Terpenes and terpenoids are classified according to how many isoprene units they contain. They include hemiterpenes (one unit), monoterpenes (two units), sesquiterpenes (three units), diterpenes (four units), sesterterpenes (five units), triterpenes (six units), sesquarterpenes (seven units), tetraterpenes (eight units), polyterpenes (many units). Another class of terpene-containing molecules, the norisoterpenoids arise from peroxidase-catalyzed reactions on terpene molecules. Examples Common terpenes include monoterpenes of terpineol (lilacs), limonene (citrus), myrcene (hops), linalool (lavender), and pinene (pine). Higher order terpenes include taxadiene (diterpene precursor of taxol), lycopene (tetraterpenes), carotenes (tetraterpenes), and natural rubber (polyterpenes). Steroid precursors geranyl pyrophosphate (monoterpene derivative), farnesyl pyrophosphate (sesquiterpene derivative), and squalene (triterpene) are all terpenes or derivatives of them. Vitamin A and phytol are derived from diterpenes. Caffeine Caffeine is the world’s most actively consumed psychoactive drug (Figure 2.255). A methylxanthine alkaloid, caffeine is closely related to adenine and guanine and this is responsible for many effects on the body. Caffeine blocks the binding of adenosine on its receptor and consequently prevents the onset of drowsiness induced by adenosine. Caffeine readily crosses the blood-brain barrier and stimulates release of neurotransmitters. Caffeine stimulates portions of the autonomic nervous system and inhibits the activity of phosphodiesterase. The latter has the result of raising cAMP levels in cells, which activates protein kinase A and activates glycogen breakdown, inhibits TNF-α and leukotriene synthesis, which results in reduction of inflammation and innate immunity. Caffeine also has effects on the cholinergic system (acetylcholinesterase inhibitor), is an inositol triphosphate receptor 1 antagonist, and is a voltage independent activator of ryanodin receptors (a group of calcium channels found in skeletal muscle, smooth muscle, and heart muscle cells). The half-life of caffeine in the body varies considerably. In healthy adults, it has a half-life of about 3-7 hours. Nicotine decreases the half-life and contraceptives and pregnancy can double it. The liver metabolizes caffeine, so the health of the liver is a factor in the halflife. CYP1A2 of the cytochrome P450 oxidase enzyme is primarily responsible. Caffeine is a natural pesticide in plants, paralyzing predator bugs. Lipoprotein complexes and lipid movement in the body Lipoprotein complexes are combinations of apolipoproteins and lipids bound to them that solubilize fats and other non-polar molecules, such as cholesterol, so they can travel in the bloodstream between various tissues of the body. The apolipoproteins provide the emulsification necessary for this. Lipoprotein complexes are formed in tiny “balls” with the water soluble apolipoproteins on the outside and non-polar lipids, such as fats, cholesteryl esters, and fat soluble vitamins on the inside. They are categorized by their densities. These include (from highest density to the lowest) high density lipoproteins (HDLs), Low Density Lipoproteins (LDLs), Intermediate Density Lipoproteins (IDLs), Very Low Density Lipoproteins (VLDLs) and the chylomicrons. These particles are synthesized in the liver and small intestines. Apolipoproteins Each lipoprotein complex contain a characteristic set of apolipoproteins, as shown in Figure 2.256. ApoC-II and ApoC-III are notable for their presence in all the lipoprotein complexes and the roles they play in activating (ApoC-II) or inactivating (ApoC-III) lipoprotein lipase. Lipoprotein lipase is a cellular enzyme that catalyzes the breakdown of fat from the complexes. ApoE (see below) is useful for helping the predict the likelihood of the occurrence of Alzheimer's disease in a patient. Gene editing ApoB-48 and ApoB-100 are interesting in being coded by the same gene, but a unique mRNA sequence editing event occurs that converts one into the other. ApoB-100 is made in the liver, but ApoB-48 is made in the small intestine. The small intestine contains an enzyme that deaminates the cytidine at nucleotide #2153 of the common mRNA. This changes it to a uridine and changes the codon it is in from CAA (codes for glutamine) to UAA (stop codon). The liver does not contain this enzyme and does not make the change in the mRNA. Consequently, a shorter protein is synthesized in the intestine (ApoB-48) than the one that is made in the liver (ApoB-100) using the same gene sequence in the DNA. Movement The movement of fats in the body is important because they are not stored in all cells. Only specialized cells called adipocytes store fat. There are three relevant pathways in the body for moving lipids. As described below, they are 1) the exogenous pathway; 2) the endogenous pathway, and 3) the reverse transport pathway. Exogenous pathway Dietary fat entering the body from the intestinal system must be transported, as appropriate, to places needing it or storing it. This is the function of the exogenous pathway of lipid movement in the body. All dietary lipids (fats, cholesterol, fat soluble vitamins, and other lipids) are moved by it. In the case of dietary fat, it begins its journey after ingestion first by being solubilized by bile acids in the intestinal tract. After passing through the stomach, pancreatic lipases clip two fatty acids from the fat, leaving a monoacyl glycerol. The fatty acids and monoacyl glycerol are absorbed by intestinal cells (enterocytes) and reassembled back into a fat, and then this is mixed with phospholipids, cholesterol esters, and apolipoprotein B-48 and processed to form chylomicrons (Figures 2.258 & 2.259) in the Golgi apparatus and smooth endoplasmic reticulum. Exocytosis These are exocytosed from the cell into lymph capillaries called lacteals. The chylomicrons pass through the lacteals and enter the bloodstream via the left subclavian vein. Within the bloodstream, lipoprotein lipase breaks down the fats causing the chylomicron to shrink and become what is known as a chylomicron remnant. It retains its cholesterol and other lipid molecules. The chylomicron remnants travel to the liver where they are absorbed (Figure 2.260). This is accomplished by receptors in the liver that recognize and bind to the ApoE of the chylomicrons. The bound complexes are then internalized by endocytosis, degraded in the lysosomes, and the cholesterol is disbursed in liver cells. Endogenous pathway The liver plays a central role in managing the body’s needs for lipids. When lipids are needed by the body or when the capacity of the liver to contain more lipids than is supplied by the diet, the liver packages up fats and cholesteryl esters into Very Low Density Lipoprotein (VLDL) complexes and exports them via the endogenous pathway. VLDL complexes contain ApoB-100, ApoC-I, ApoC-II, ApoC-III, and ApoE apolipoproteins. VLDLs enter the blood and travel to muscles and adipose tissue where lipoprotein lipase is activated by ApoC-II. In the muscle cells, the released fatty acids are taken up and oxidized. By contrast, in the adipoctyes, the fatty acids are taken up and reassembled back into triacylglycerides (fats) and stored in fat droplets. Removal of fat from the VLDLs causes them to shrink, first to Intermediate Density Lipoprotein (IDL) complexes (also called VLDL remnants) and then to Low Density Lipoprotein (LDL) complexes. Shrinking of VLDLs is accompanied by loss of apolipoproteins so that LDLs are comprised primarily of ApoB-100. This lipoprotein complex is important because cells have receptors for it to bind and internalize it by receptor-mediated endocytosis (Figure 2.261). Up until this point, cholesterol and cholesteryl esters have traveled in chylomicrons, VLDLs, and IDLs as fat has been stripped stripped away. For cholesterol compounds to get into the cell from the lipoprotein complexes, they must be internalized by cells and that is the job of receptormediated endocytosis. Reverse transport pathway Another important consideration of the movement of lipids in the body is the reverse transport pathway (Figure 2.260). It is also called the reverse cholesterol transport pathway, since cholesterol is the primary molecule involved. This pathway involves the last class of lipoprotein complexes known as the High Density Lipoproteins (HDLs). In contrast to the LDLs, which are commonly referred to as “bad cholesterol” (see below also), the HDLs are known as “good cholesterol.” HDLs are synthesized in the liver and small intestine. They contain little or no lipid when made (called depleted HDLs), but serve the role of “scavenger” for cholesterol in the blood and from remnants of other (damaged) lipoprotein complexes in the blood. To perform its task, HDLs carry the enzyme known as lecithincholesterol acyl transferase (LCAT), which they use to form cholesteryl esters using fatty acids from lecithin (phosphatidylcholine) and then they internalize them. The cholesterol used for this purpose comes from the bloodstream, from macrophages, and from foam cells (macrophage-LDL complexes - Figure 2.262). Addition of cholesteryl esters causes the HDL to swell and Figure 2.261 - The process of receptor-mediated endocytosis Image by Aleia Kim when it is mature, it returns its load of cholesterol back to the liver or, alternatively, to LDL molecules for endocytosis. HDLs have the effect of lowering levels of cholesterol and it is for that reason they are described as “good cholesterol.” Regulation of lipid transfer It is important that cells get food when they need it so some control of the movement of nutrients is critical. The liver, which plays the central role in modulating blood glucose levels, is also important for performing the same role for lipids. It accomplishes this task the use of specialized LDL receptors on its surface. Liver LDL receptors bind LDLs that were not taken up by other cells in their path through the bloodstream. High levels of LDLs are a signal to the liver to reduce the creation of VLDLs for release. People with the genetic disease known as familial hypercholesterolemia, which manifests with dangerously high levels of LDLs, lack properly functioning LDL receptors on their liver cells.Figure 2.263 - Progression of atherosclerosis Wikipedia Figure 2.263 - Progression of atherosclerosis Wikipedia Figure 2.263 - Progression of atherosclerosis Wikipedia In sufferers of this disease, the liver never gets the signal that the LDL levels are high. In fact, to the liver, it appears that all VLDLs and LDLs are being taken up by peripheral tissues, so it creates more VLDLs to attempt to boost levels. Untreated, the disease used to be fatal early, but newer drugs like the statins have significantly increased life spans of patients. Cellular needs for the contents of LDLs are directly linked to the levels of synthesis of LDL receptors on their membranes. As cells are needing more cholesterol, their synthesis of components for receptors goes up and it decreases as need diminishes. Good cholesterol / bad cholesterol It is commonly accepted that “high cholesterol” levels are not healthy. This is due, at least indirectly, to the primary carriers of cholesterol, the LDLs. A primary function of the LDLs is to deliver cholesterol and other lipids directly into cells by receptor mediated endocytosis (Figure 2.237). High levels of LDLs, though, are correlated with formation of atherosclerotic plaques (Figure 2.263 & 2.264) and incidence of atherosclerosis, leading to the description of them as “bad cholesterol.” This is because when LDL levels are very high, plaque formation begins. It is thought that reactive oxygen species (higher in the blood of smokers) causes partial oxidation of fatty acid groups in the LDLs. When levels are high, they tend to accumulate in the extracellular matrix of the epithelial cells on the inside of the arteries. Macrophages of the immune system take up the damaged LDLs (including the cholesterol). Since macrophages can’t control the amount of cholesterol they take up, cholesterol begins to accumulate in them and they take on appearance that leads to their being described as “foam cells.” With too much cholesterol, the foam cells, however, are doomed to die by the process of programmed cell death (apoptosis). Accumulation of these, along with scar tissue from inflammation result in formation of a plaque. Plaques can grow and block the flow of blood or pieces of them can break loose and plug smaller openings in the blood supply, ultimately leading to heart attack or stroke. Good cholesterol On the other hand, high levels of HDL are inversely correlated with atherosclerosis and arterial disease. Depleted HDLs are able to remove cholesterol from foam cells. This occurs as a result of contact between the ApoA-I protein of the HDL and a transport protein on the foam cell (ABC-G1). Another transport protein in the foam cell, ABCA-1 transports extra cholesterol from inside the cell to the plasma membrane where it is taken up into the HDL and returned to the liver or to LDLs by the reverse transport cholesterol pathway. Deficiency of the ABCA-1 gene leads to Tangier disease. In this condition, HDLs are almost totally absent because they remain empty as a result of not being able to take up cholesterol from foam cells, so they are destroyed by the body. ApoE and Alzheimer’s disease ApoE is a component of the chylomicrons and is also found in brain, macrophages, kidneys, and the spleen. In humans, it is found in three different alleles, E2, E3, and E4. The E4 allele (present at about 14% of the population) is associated with increased likelihood of contracting Alzheimer's disease. People heterozygous for the allele are 3 times as likely to contract the disease and those homozygous for it are 15 times as likely to do so. It is not known why this gene or allele is linked to the disease. The three alleles differ only slightly in amino acid sequence, but the changes do cause notable structural differences. The E4 allele is associated with increased calcium ion levels and apoptosis after injury. Alzheimer’s disease is associated with accumulation of aggregates of the β- amyloid peptide. ApoE does enhance the proteolytic breakdown of it and the E4 isoform is not as efficient in these reactions as the other isoforms.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/02%3A_Structure_and_Function/2.08%3A_Structure_and_Function_-_Lipids_and_Membranes.txt

Source: BiochemFFA_2_1.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy All of the proteins on the face of the earth are made up of the same 20 amino acids. Linked together in long chains called polypeptides, amino acids are the building blocks for the vast assortment of proteins found in all living cells. "It is one of the more striking generalizations of biochemistry ...that the twenty amino acids and the four bases, are, with minor reservations, the same throughout Nature." - Francis Crick All amino acids have the same basic structure, which is shown in Figure 2.1. At the “center” of each amino acid is a carbon called the α carbon and attached to it are four groups - a hydrogen, an α- carboxyl group, an α-amine group, and an R-group, sometimes referred to as a side chain. The α carbon, carboxyl, and amino groups are common to all amino acids, so the R-group is the only unique feature in each amino acid. (A minor exception to this structure is that of proline, in which the end of the R-group is attached to the α-amine.) With the exception of glycine, which has an R-group consisting of a hydrogen atom, all of the amino acids in proteins have four different groups attached to them and consequently can exist in two mirror image forms, L and D. With only very minor exceptions, every amino acid found in cells and in proteins is in the L configuration. There are 22 amino acids that are found in proteins and of these, only 20 are specified by the universal genetic code. The others, selenocysteine and pyrrolysine use tRNAs that are able to base pair with stop codons in the mRNA during translation. When this happens, these unusual amino acids can be incorporated into proteins. Enzymes containing selenocysteine, for example, include glutathione peroxidases, tetraiodothyronine 5' deiodinases, thioredoxin reductases, formate dehydrogenases, glycine reductases, and selenophosphate synthetase. Pyrrolysine-containing proteins are much rarer and are mostly confined to archaea. Essential and non-essential Nutritionists divide amino acids into two groups - essential amino acids (must be in the diet because cells can’t synthesize them) and non-essential amino acids (can be made by cells). This classification of amino acids has little to do with the structure of amino acids. Essential amino acids vary considerable from one organism to another and even differ in humans, depending on whether they are adults or children. Table 2.1 shows essential and non-essential amino acids in humans. Some amino acids that are normally nonessential, may need to be obtained from the diet in certain cases. Individuals who do not synthesize sufficient amounts of arginine, cysteine, glutamine, proline, selenocysteine, serine, and tyrosine, due to illness, for example, may need dietary supplements containing these amino acids. Table 2.1 - Essential and non-essential amino acids Non-protein amino acids There are also α-amino acids found in cells that are not incorporated into proteins. Common ones include ornithine and citrulline. Both of these compounds are intermediates in the urea cycle. Ornithine is a metabolic precursor of arginine and citrulline can be produced by the breakdown of arginine. The latter reaction produces nitric oxide, an important signaling molecule. Citrulline is the metabolic byproduct. It is sometimes used as a dietary supplement to reduce muscle fatigue. R-group chemistry Table 2.2 - Amino acid categories (based on R-group properties) We separate the amino acids into categories based on the chemistry of their R-groups. If you compare groupings of amino acids in different textbooks, you will see different names for the categories and (sometimes) the same amino acid being categorized differently by different authors. Indeed, we categorize tyrosine both as an aromatic amino acid and as a hydroxyl amino acid. It is useful to classify amino acids based on their R-groups, because it is these side chains that give each amino acid its characteristic properties. Thus, amino acids with (chemically) similar side groups can be expected to function in similar ways, for example, during protein folding. Non-polar amino acids • Alanine (Ala/A) is one of the most abundant amino acids found in proteins, ranking second only to leucine in occurrence. A D-form of the amino acid is also found in bacterial cell walls. Alanine is non-essential, being readily synthesized from pyruvate. It is coded for by GCU, GCC, GCA, and GCG. • Glycine (Gly/G) is the amino acid with the shortest side chain, having an R-group consistent only of a single hydrogen. As a result, glycine is the only amino acid that is not chiral. Its small side chain allows it to readily fit into both hydrophobic and hydrophilic environments. • Glycine is specified in the genetic code by GGU, GGC, GGA, and GGG. It is nonessential to humans. • Isoleucine (Ile/I) is an essential amino acid encoded by AUU, AUC, and AUA. It has a hydrophobic side chain and is also chiral in its side chain. • Leucine (Leu/L) is a branched-chain amino acid that is hydrophobic and essential. Leucine is the only dietary amino acid reported to directly stimulate protein synthesis in muscle, but caution is in order, as 1) there are conflicting studies and 2) leucine toxicity is dangerous, resulting in "the four D's": diarrhea, dermatitis, dementia and death . Leucine is encoded by six codons: UUA,UUG, CUU, CUC, CUA, CUG. • Methionine (Met/M) is an essential amino acid that is one of two sulfurcontaining amino acids - cysteine is the other. Methionine is non-polar and encoded solely by the AUG codon. It is the “initiator” amino acid in protein synthesis, being the first one incorporated into protein chains. In prokaryotic cells, the first methionine in a protein is formylated. • Proline (Pro/P) is the only amino acid found in proteins with an R-group that joins with its own α-amino group, making a secondary amine and a ring. Proline is a non-essential amino acid and is coded by CCU, CCC, CCA, and CCG. It is the least flexible of the protein amino acids and thus gives conformational rigidity when present in a protein. Proline’s presence in a protein affects its secondary structure. It is a disrupter of α-helices and β-strands. Proline is often hydroxylated in collagen (the reaction requires Vitamin C - ascorbate) and this has the effect of increasing the protein’s conformational stability. Proline hydroxylation of hypoxia-inducible factor (HIF) serves as a sensor of oxygen levels and targets HIF for destruction when oxygen is plentiful. • Valine (Val/V) is an essential, non-polar amino acid synthesized in plants. It is noteworthy in hemoglobin, for when it replaces glutamic acid at position number six, it causes hemoglobin to aggregate abnormally under low oxygen conditions, resulting in sickle cell disease. Valine is coded in the genetic code by GUU, GUC, GUA, and GUG. Carboxyl Amino Acids • Aspartic acid (Asp/D) is a non-essential amino acid with a carboxyl group in its Rgroup. It is readily produced by transamination of oxaloacetate. With a pKa of 3.9, aspartic acid’s side chain is negatively charged at physiological pH. Aspartic acid is specified in the genetic code by the codons GAU and GAC. • Glutamic acid (Glu/E), which is coded by GAA and GAG, is a non-essential amino acid readily made by transamination of α- ketoglutarate. It is a neurotransmitter and has an R-group with a carboxyl group that readily ionizes (pKa = 4.1) at physiological pH. Amine amino acids • Arginine (Arg/R) is an amino acid that is, in some cases, essential, but non-essential in others. Premature infants cannot synthesize arginine. In addition, surgical trauma, sepsis, and burns increase demand for arginine. Most people, however, do not need arginine supplements. Arginine’s side chain contains a complex guanidinium group with a pKa of over 12, making it positively charged at cellular pH. It is coded for by six codons - CGU, CGC, CGA, CGG, AGA, and AGG. • Histidine (His/H) is the only one of the proteinaceous amino acids to contain an imidazole functional group. It is an essential amino acid in humans and other mammals. With a side chain pKa of 6, it can easily have its charge changed by a slight change in pH. Protonation of the ring results in two NH structures which can be drawn as two equally important resonant structures. • Lysine (Lys/K) is an essential amino acid encoded by AAA and AAG. It has an Rgroup that can readily ionize with a charge of +1 at physiological pH and can be posttranslationally modified to form acetyllysine, hydroxylysine, and methyllysine. It can also be ubiquitinated, sumoylated, neddylated, biotinylated, carboxylated, and pupylated, and. O-Glycosylation of hydroxylysine is used to flag proteins for export from the cell. Lysine is often added to animal feed because it is a limiting amino acid and is necessary for optimizing growth of pigs and chickens. Aromatic amino acids • Phenylalanine (Phe/ F) is a non-polar, essential amino acid coded by UUU and UUC. It is a metabolic precursor of tyrosine. Inability to metabolize phenylalanine arises from the genetic disorder known as phenylketonuria. Phenylalanine is a component of the aspartame artificial sweetener. • Tryptophan (Trp/W) is an essential amino acid containing an indole functional group. It is a metabolic precursor of serotonin, niacin, and (in plants) the auxin phytohormone. Though reputed to serve as a sleep aid, there are no clear research results indicating this. • Tyrosine (Tyr/Y) is a non-essential amino acid coded by UAC and UAU. It is a target for phosphorylation in proteins by tyrosine protein kinases and plays a role in signaling processes. In dopaminergic cells of the brain, tyrosine hydroxylase converts tyrosine to l-dopa, an immediate precursor of dopamine. Dopamine, in turn, is a precursor of norepinephrine and epinephrine. Tyrosine is also a precursor of thyroid hormones and melanin. Hydroxyl amino acids • Serine (Ser/S) is one of three amino acids having an R-group with a hydroxyl in it (threonine and tyrosine are the others). It is coded by UCU, UCC, UCA, UGC, AGU, and AGC. Being able to hydrogen bond with water, it is classified as a polar amino acid. It is not essential for humans. Serine is precursor of many important cellular compounds, including purines, pyrimidines, sphingolipids, folate, and of the amino acids glycine, cysteine, and tryptophan. The hydroxyl group of serine in proteins is a target for phosphorylation by certain protein kinases. Serine is also a part of the catalytic triad of serine proteases. • Threonine (Thr/T) is a polar amino acid that is essential. It is one of three amino acids bearing a hydroxyl group (serine and tyrosine are the others) and, as such, is a target for phosphorylation in proteins. It is also a target for Oglycosylation of proteins. Threonine proteases use the hydroxyl group of the amino acid in their catalysis and it is a precursor in one biosynthetic pathway for making glycine. In some applications, it is used as a pro-drug to increase brain glycine levels. Threonine is encoded in the genetic code by ACU, ACC, ACA, and ACG. Tyrosine - see HERE. Other amino acids • Asparagine (Asn/N) is a non-essential amino acid coded by AAU and AAC. Its carboxyamide in the R-group gives it polarity. Asparagine is implicated in formation of acrylamide in foods cooked at high temperatures (deep frying) when it reacts with carbonyl groups. Asparagine can be made in the body from aspartate by an amidation reaction with an amine from glutamine. Breakdown of asparagine produces malate, which can be oxidized in the citric acid cycle. • Cysteine (Cys/C) is the only amino acid with a sulfhydryl group in its side chain. It is nonessential for most humans, but may be essential in infants, the elderly and individuals who suffer from certain metabolic diseases. Cysteine’s sulfhydryl group is readily oxidized to a disulfide when reacted with another one. In addition to being found in proteins, cysteine is also a component of the tripeptide, glutathione. Cysteine is specified by the codons UGU and UGC. • Glutamine (Gln/Q) is an amino acid that is not normally essential in humans, but may be in individuals undergoing intensive athletic training or with gastrointestinal disorders. It has a carboxyamide side chain which does not normally ionize under physiological pHs, but which gives polarity to the side chain. Glutamine is coded for by CAA and CAG and is readily made by amidation of glutamate. Glutamine is the most abundant amino acid in circulating blood and is one of only a few amino acids that can cross the blood-brain barrier. • Selenocysteine (Sec/U) is a component of selenoproteins found in all kingdoms of life. It is a component in several enzymes, including glutathione peroxidases and thioredoxin reductases. Selenocysteine is incorporated into proteins in an unusual scheme involving the stop codon UGA. Cells grown in the absence of selenium terminate protein synthesis at UGAs. However, when selenium is present, certain mRNAs which contain a selenocysteine insertion sequence (SECIS), insert selenocysteine when UGA is encountered. The SECIS element has characteristic nucleotide sequences and secondary structure base-pairing patterns. Twenty five human proteins contain selenocysteine. • Pyrrolysine (Pyl/O) is a twenty second amino acid, but is rarely found in proteins. Like selenocysteine, it is not coded for in the genetic code and must be incorporated by unusual means. This occurs at UAG stop codons. Pyrrolysine is found in methanogenic archaean organisms and at least one methane-producing bacterium. Pyrrolysine is a component of methane-producing enzymes. Ionizing groups pKa values for amino acid side chains are very dependent upon the chemical environment in which they are present. For example, the R-group carboxyl found in aspartic acid has a pKa value of 3.9 when free in solution, but can be as high as 14 when in certain environments inside of proteins, though that is unusual and extreme. Each amino acid has at least one ionizable amine group (α- amine) and one ionizable carboxyl group (α- carboxyl). When these are bound in a peptide bond, they no longer ionize. Some, but not all amino acids have R-groups that can ionize. The charge of a protein then arises from the charges of the α-amine group, the α- carboxyl group. and the sum of the charges of the ionized R-groups. Titration/ionization of aspartic acid is depicted in Figure 2.10. Ionization (or deionization) within a protein’s structure can have significant effect on the overall conformation of the protein and, since structure is related to function, a major impact on the activity of a protein. Most proteins have relatively narrow ranges of optimal activity that typically correspond to the environments in which they are found (Figure 2.11). It is worth noting that formation of peptide bonds between amino acids removes ionizable hydrogens from both the α- amine and α- carboxyl groups of amino acids. Thus, ionization/ deionization in a protein arises only from 1) the amino terminus; 2) carboxyl terminus; 3) R-groups; or 4) other functional groups (such as sulfates or phosphates) added to amino acids post-translationally - see below. Carnitine Not all amino acids in a cell are found in proteins. The most common examples include ornithine (arginine metabolism), citrulline (urea cycle), and carnitine (Figure 2.12). When fatty acids destined for oxidation are moved into the mitochondrion for that purpose, they travel across the inner membrane attached to carnitine. Of the two stereoisomeric forms, the L form is the active one. The molecule is synthesized in the liver from lysine and methionine. From exogenous sources, fatty acids must be activated upon entry into the cytoplasm by being joined to coenzyme A. The CoA portion of the molecule is replaced by carnitine in the intermembrane space of the mitochondrion in a reaction catalyzed by carnitine acyltransferase I. The resulting acylcarnitine molecule is transferred across the inner mitochondrial membrane by the carnitineacylcarnitine translocase and then in the matrix of the mitochondrion, carnitine acyltransferase II replaces the carnitine with coenzyme A (Figure 6.88). Catabolism of amino acids We categorize amino acids as essential or non-essential based on whether or not an organism can synthesize them. All of the amino acids, however, can be broken down by all organisms. They are, in fact, a source of energy for cells, particularly during times of starvation or for people on diets containing very low amounts of carbohydrate. From a perspective of breakdown (catabolism), amino acids are categorized as glucogenic if they produce intermediates that can be made into glucose or ketogenic if their intermediates are made into acetyl-CoA. Figure 2.13 shows the metabolic fates of catabolism of each of the amino acids. Note that some amino acids are both glucogenic and ketogenic. Post-translational modifications After a protein is synthesized, amino acid side chains within it can be chemically modified, giving rise to more diversity of structure and function (Figure 2.14). Common alterations include phosphorylation of hydroxyl groups of serine, threonine, or tyrosine. Lysine, proline, and histidine can have hydroxyls added to amines in their R-groups. Other modifications to amino acids in proteins include addition of fatty acids (myristic acid or palmitic acid), isoprenoid groups, acetyl groups, methyl groups, iodine, carboxyl groups, or sulfates. These can have the effects of ionization (addition of phosphates/sulfates), deionization (addition of acetyl group to the R-group amine of lysine), or have no effect on charge at all. In addition, N-linked- and O-linkedglycoproteins have carbohydrates covalently attached to side chains of asparagine and threonine or serine, respectively. Some amino acids are precursors of important compounds in the body. Examples include epinephrine, thyroid hormones, Ldopa, and dopamine (all from tyrosine), serotonin (from tryptophan), and histamine (from histidine). Building Polypeptides Although amino acids serve other functions in cells, their most important role is as constituents of proteins. Proteins, as we noted earlier, are polymers of amino acids. Amino acids are linked to each other by peptide bonds, in which the carboxyl group of one amino acid is joined to the amino group of the next, with the loss of a molecule of water. Additional amino acids are added in the same way, by formation of peptide bonds between the free carboxyl on the end of the growing chain and the amino group of the next amino acid in the sequence. A chain made up of just a few amino acids linked together is called an oligopeptide (oligo=few) while a typical protein, which is made up of many amino acids is called a polypeptide (poly=many). The end of the peptide that has a free amino group is called the N-terminus (for NH2), while the end with the free carboxyl is termed the C-terminus (for carboxyl). As we’ve noted before, function is dependent on structure, and the string of amino acids must fold into a specific 3-D shape, or conformation, in order to make a functional protein. The folding of polypeptides into their functional forms is the topic of the next section.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/02%3A_Structure_and_Function/202%3A_Structure__Function_-_Amino_Acids.txt

Source: BiochemFFA_2_2.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Proteins are the workhorses of the cell. Virtually everything that goes on inside of cells happens as a result of the actions of proteins. Among other things, protein enzymes catalyze the vast majority of cellular reactions, mediate signaling, give structure both to cells and to multicellular organisms, and exert control over the expression of genes. Life, as we know it, would not exist if there were no proteins. The versatility of proteins arises because of their varied structures. Proteins are made by linking together amino acids, with each protein having a characteristic and unique amino acid sequence. To get a sense for the diversity of proteins that can be made using 20 different amino acids, consider that the number of different combinations possible with 20 amino acids is 20n, where n=the number of amino acids in the chain. It becomes apparent that even a dipeptide made of just two amino acids joined together gives us 202 = 400 different combinations. If we do the calculation for a short peptide of 10 amino acids, we arrive at an enormous 10,240,000,000,000 combinations. Most proteins are much larger than this, making the possible number of proteins with unique amino acid sequences unimaginably huge. Levels of Structure The significance of the unique sequence, or order, of amino acids, known as the protein’s primary structure, is that it dictates the 3-D conformation the folded protein will have. This conformation, in turn, will determine the function of the protein. We shall examine protein structure at four distinct levels (Figure 2.17) - 1) how sequence of the amino acids in a protein (primary structure) gives identity and characteristics to a protein (Figure 2.18); 2) how local interactions between one part of the polypeptide backbone and another affect protein shape (secondary structure); 3) how the polypeptide chain of a protein can fold to allow amino acids to interact with each other that are not close in primary structure (tertiary structure); and 4) how different polypeptide chains interact with each other within a multi-subunit protein (quaternary structure). At this point, we should provide a couple of definitions. We use the term polypeptide to refer to a single polymer of amino acids. It may or may not have folded into its final, functional form. The term protein is sometimes used interchangeably with polypeptide, as in “protein synthesis”. It is generally used, however, to refer to a folded, functional molecule that may have one or more subunits (made up of individual polypeptides). Thus, when we use the term protein, we are usually referring to a functional, folded polypeptide or peptides. Structure is essential for function. If you alter the structure, you alter the function - usually, but not always, this means you lose all function. For many proteins, it is not difficult to alter the structure. Proteins are flexible, not rigidly fixed in structure. As we shall see, it is the flexibility of proteins that allows them to be amazing catalysts and allows them to adapt to, respond to, and pass on signals upon binding of other molecules or proteins. However, proteins are not infinitely flexible. There are constraints on the conformations that proteins can adopt and these constraints govern the conformations that proteins display. Subtle changes Even very tiny, subtle changes in protein structure can give rise to big changes in the behavior of proteins. Hemoglobin, for example, undergoes an incredibly small structural change upon binding of one oxygen molecule, and that simple change causes the remainder of the protein to gain a considerably greater affinity for oxygen that the protein didn’t have before the structural change. Sequence, structure and function As discussed earlier, the number of different amino acid sequences possible, even for short peptides, is very large. No two proteins with different amino acid sequences (primary structure) have identical overall structure. The unique amino acid sequence of a protein is reflected in its unique folded structure. This structure, in turn, determines the protein’s function. This is why mutations that alter amino acid sequence can affect the function of a protein. Protein Synthesis Synthesis of proteins occurs in the ribosomes and proceeds by joining the carboxyl terminus of the first amino acid to the amino terminus of the next one (Figure 2.19). The end of the protein that has the free α-amino group is referred to as the amino terminus or N-terminus. The other end is called the carboxyl terminus or C-terminus , since it contains the only free α-carboxyl group. All of the other α-amino groups and α-carboxyl groups are tied up in forming peptide Figure 2.19 Linking of amino acids through peptide bond formation bonds that join adjacent amino acids together. Proteins are synthesized starting with the amino terminus and ending at the carboxyl terminus. Schematically, in Figure 2.18, we can see how sequential R-groups of a protein are arranged in an alternating orientation on either side of the polypeptide chain. Organization of R-groups in this fashion is not random. Steric hindrance can occur when consecutive R-groups are oriented on the same side of a peptide backbone (Figure 2.20) Primary Structure Primary structure is the ultimate determinant of the overall conformation of a protein. The primary structure of any protein arrived at its current state as a result of mutation and selection over evolutionary time. Primary structure of proteins is mandated by the sequence of DNA coding for it in the genome. Regions of DNA specifying proteins are known as coding regions (or genes). The base sequences of these regions directly specify the sequence of amino acids in proteins, with a one-to-one correspondence between the codons (groups of three consecutive bases) in the DNA and the amino acids in the encoded protein. The sequence of codons in DNA, copied into messenger RNA, specifies a sequence of amino acids in a protein. (Figure 2.21). The order in which the amino acids are joined together in protein synthesis starts defining a set of interactions between amino acids even as the synthesis is occurring. That is, a polypeptide can fold even as it is being made. The order of the R-group structures and resulting interactions are very important because early interactions affect later interactions. This is because interactions start establishing structures - secondary and tertiary. If a helical structure (secondary structure), for example, starts to form, the possibilities for interaction of a particular amino acid Rgroup may be different than if the helix had not formed (Figure 2.22). R-group interactions can also cause bends in a polypeptide sequence (tertiary structure) and these bends can create (in some cases) opportunities for interactions that wouldn’t have been possible without the bend or prevent (in other cases) similar interaction possibilities. Secondary Structure As protein synthesis progresses, interactions between amino acids close to each other begin to occur, giving rise to local patterns called secondary structure. These secondary structures include the well known α- helix and β-strands. Both were predicted by Linus Pauling, Robert Corey, and Herman Branson in 1951. Each structure has unique features. α-helix The α-helix has a coiled structure, with 3.6 amino acids per turn of the helix (5 helical turns = 18 amino acids). Helices are predominantly right handed - only in rare cases, such as in sequences with many glycines can left handed α- helices form. In the α-helix, hydrogen bonds form between C=O groups and N-H groups in the polypeptide backbone that are four amino acids distant. These hydrogen bonds are the primary forces stabilizing the α-helix. We use the terms rise, repeat, and pitch to describe the parameters of any helix. The repeat is the number of residues in a helix before it begins to repeat itself. For an α-helix, the repeat is 3.6 amino acids per turn of the helix. The rise is the distance the helix elevates with addition of each residue. For an α-helix, this is 0.15 nm per amino acid. The pitch is the distance between complete turns of the helix. For an α-helix, this is 0.54 nm. The stability of an α-helix is enhanced by the presence of the amino acid aspartate. β strand/sheet A helix is, of course, a three-dimensional object. A flattened form of helix in two dimensions is a common description for a β- strand. Rather than coils, β-strands have bends and these are sometimes referred to as pleats, like the pleats in a curtain. β-strands can be organized to form elaborately organized structures, such as sheets, barrels, and other arrangements. Higher order β-strand structures are sometimes called supersecondary structures), since they involve interactions between amino acids not close in primary sequence. These structures, too, are stabilized by hydrogen bonds between carbonyl oxygen atoms and hydrogens of amine groups in the polypeptide backbone (Figure 2.28). In a higher order structure, strands can be arranged parallel (amino to carboxyl orientations the same) or anti-parallel (amino to carboxyl orientations opposite of each other (in Figure 2.27, the direction of the strand is shown by the arrowhead in the ribbon diagrams). Turns Turns (sometimes called reverse turns) are a type of secondary structure that, as the name suggests, causes a turn in the structure of a polypeptide chain. Turns give rise to tertiary structure ultimately, causing interruptions in the secondary structures (α- helices and β-strands) and often serve as connecting regions between two regions of secondary structure in a protein. Proline and glycine play common roles in turns, providing less flexibility (starting the turn) and greater flexibility (facilitating the turn), respectively. There are at least five types of turns, with numerous variations of each giving rise to many different turns. The five types of turns are • δ-turns - end amino acids are separated by one peptide bond • γ-turns - separation by two peptide bonds •β-turns - separation by three peptide bonds •α-turns - separation by four peptide bonds •π-turns - separation by five bonds Of these, the β-turns are the most common form and the δ-turns are theoretical, but unlikely, due to steric limitations. Figure 2.29 depicts a β- turn. 310 helices In addition to the α-helix, β-strands, and various turns, other regular, repeating structures are seen in proteins, but occur much less commonly. The 310 helix is the fourth most abundant secondary structure in proteins, constituting about 10-15% of all helices. The helix derives its name from the fact that it contains 10 amino acids in 3 turns. It is right-handed. Hydrogen bonds form between amino acids that are three residues apart. Most commonly, the 310 helix appears at the amine or carboxyl end of an α-helix. Like the α-helix, the 310 helix is stabilized by the presence of aspartate in its sequence. π-helices A π-helix may be thought of as a special type of α- helix. Some sources describe it as an α-helix with an extra amino acid stuck in the middle of it (Figure 2.32). π-helices are not exactly rare, occurring at least once in as many as 15% of all proteins. Like the α- helix, the π-helix is right-handed, but where the α-helix has 18 amino acids in 5 turns, the π-helix has 22 amino acids in 5 turns. π-helices typically do not stretch for very long distances. Most are only about 7 amino acids long and the sequence almost always occurs in the middle of an α-helical region. Ramachandran plots In 1963, G.N. Ramachandran, C. Ramakrishnan, and V. Sasisekharan described a novel way to describe protein structure. If one considers the backbone of a polypeptide chain, it consists of a repeating set of three bonds. Sequentially (in the amino to carboxyl direction) they are 1) a rotatable bond (ψ) between α-carbon and α-carboxyl preceding the peptide bond (see HERE), 2) a non-rotatable peptide bond (ω) between the α-carboxyl and α-amine groups), and 3) a rotatable bond (φ) between the α-amine and α-carbon following the peptide bond (see HERE). Note in Figures 2.33 and 2.34 that the amino to carboxyl direction is right to left. The presence of the carbonyl oxygen on the α-carboxyl group allows the peptide bond to exist as a resonant structure, meaning that it behaves some of the time as a double bond. Double bonds cannot, of course, rotate, but the bonds on either side of it have some freedom of rotation. The φ and ψ angles are restricted to certain values, because some angles will result in steric hindrance. In addition, each type of secondary structure has a characteristic range of values for φ and ψ. Ramachandran and colleagues made theoretical calculations of the energetic stability of all possible angles from 0° to 360° for each of the φ and ψ angles and plotted the results on a Ramachandran Plot (also called a φ-ψ plot), delineating regions of angles that were theoretically the most stable (Figure 2.35). Three primary regions of stability were identified, corresponding to φ-ψ angles of β-strands (top left), right handed α- helices (bottom left), and lefthanded α-helices (upper right). The plots of predicted stability are remarkably accurate when compared to φ-ψ angles of actual proteins. Secondary structure prediction Table 2.3 - Relative tendencies of each amino acid to be in a secondary structure. Higher values indicate greater tendency Image by Penelope Irving By comparing primary structure (amino acid sequences) to known 3D protein structures, one can tally each time an amino acid is found in an α-helix, β-strand/sheet, or a turn. Computer analysis of thousands of these sequences allows one to assign a likelihood of any given amino acid appearing in each of these structures. Using these tendencies, one can, with up to 80% accuracy, predict regions of secondary structure in a protein based solely on amino acid sequence. This is seen in Table 2.3. Occurrence in primary sequence of three consecutive amino acids with relative tendencies higher than one is an indicator that that region of the polypeptide is in the corresponding secondary structure. An online resource for predicting secondary structures called PSIPRED is available HERE. Hydrophobicity Table 2.4 - Hydropathy Scores The chemistry of amino acid Rgroups affects the structures they are most commonly found in. Subsets of their chemical properties can give clues to structure and, sometimes, cellular location. A prime example is the hydrophobicity (wateravoiding tendencies) of some Rgroups. Given the aqueous environment of the cell, such R-groups are not likely to be on the outside surface of a folded protein. However, this rule does not hold for regions of protein that may be embedded within the lipid bilayers of cellular/ organelle membranes. This is because the region of such proteins that form the transmembrane domains are are buried in the hydrophobic environment in the middle of the lipid bilayer. Not surprisingly, scanning primary sequences for specifically sized/spaced stretches of hydrophobic amino acids can help to identify proteins found in membranes. Table 2.4 shows hydrophobicity values for R-groups of the amino acids. In this set, the scale runs from positive values (hydrophobic) to negative values (hydrophilic). A KyteDoolittle Hydropathy plot for the RET protooncogene membrane protein is shown in Figure 2.36. Two regions of the protein are very hydrophobic as can be seen from the peaks near amino acids 5-10 and 630-640. Such regions might be reasonably expected to be situated either within the interior of the folded protein or to be part of transmembrane domains. Random coils Some sections of a protein assume no regular, discernible structure and are sometimes said to lack secondary structure, though they may have hydrogen bonds. Such segments are described as being in random coils and may have fluidity to their structure that results in them having multiple stable forms. Random coils are identifiable with spectroscopic methods, such as circular dichroism Wikipedia and nuclear magnetic resonance (NMR) in which distinctive signals are observed. See also metamorphic proteins (HERE) and intrinsically disordered proteins (HERE). Supersecondary structure Another element of protein structure is harder to categorize because it incorporates elements of secondary and tertiary structure. Dubbed supersecondary structure (or structural motifs), these structures contain multiple nearby secondary structure components arranged in a specific way and that appear in multiple proteins. Since there are many ways of making secondary structures from different primary structures, so too can similar motifs arise from different primary sequences. An example of a structural motif is shown in Figure 2.37. Tertiary structure Proteins are distinguished from each other by the sequence of amino acids comprising them. The sequence of amino acids of a protein determines protein shape, since the chemical properties of each amino acid are forces that give rise to intermolecular interactions to begin to create secondary structures, such as α-helices and β-strands. The sequence also defines turns and random coils that play important roles in the process of protein folding. Since shape is essential for protein function, the sequence of amino acids gives rise to all of the properties a protein has. As protein synthesis proceeds, individual components of secondary structure start to interact with each other, giving rise to folds that bring amino acids close together that are not near each other in primary structure (Figure 2.38). At the tertiary level of structure, interactions among the R-groups of the amino acids in the protein, as well as between the polypeptide backbone and amino acid side groups play a role in folding. Globular proteins Folding gives rise to distinct 3-D shapes in proteins that are non-fibrous. These proteins are called globular. A globular protein is stabilized by the same forces that drive its formation. These include ionic interactions, hydrogen bonding, hydrophobic forces, ionic bonds, disulfide bonds and metallic bonds. Treatments such as heat, pH changes, detergents, urea and mercaptoethanol overpower the stabilizing forces and cause a protein to unfold, losing its structure and (usually) its function (Figure 2.39). The ability of heat and detergents to denature proteins is why we cook our food and wash our hands before eating - such treatments denature the proteins in the microorganisms on our hands. Organisms that live in environments of high temperature (over 50°C) have proteins with changes in stabilizing forces - additional hydrogen bonds, additional salt bridges (ionic interactions), and compactness may all play roles in keeping these proteins from unfolding. Protein stabilizing forces Before considering the folding process, let us consider some of the forces that help to stabilize proteins. Hydrogen bonds Hydrogen bonds arise as a result of partially charged hydrogens found in covalent bonds. This occurs when the atom the hydrogen is bonded to has a greater electronegativity than hydrogen itself does, resulting in hydrogen having a partial positive charge because it is not able to hold electrons close to itself (Figure 2.40). Hydrogen partially charged in this way is attracted to atoms, such as oxygen and nitrogen that have partial negative charges, due to having greater electronegativities and thus holding electrons closer to themselves. The partially positively charged hydrogens are called donors, whereas the partially negative atoms they are attracted to are called acceptors. (See Figure 1.30). Individual hydrogen bonds are much weaker than a covalent bond, but collectively, they can exert strong forces. Consider liquid water, which contains enormous numbers of hydrogen bonds (Figure 2.41). These forces help water to remain liquid at room temperature. Other molecules lacking hydrogen bonds of equal or greater molecular weight than water, such as methane or carbon dioxide, are gases at the same temperature. Thus, the intermolecular interactions between water molecules help to “hold” water together and remain a liquid. Notably, only by raising the temperature of water to boiling are the forces of hydrogen bonding overcome, allowing water to become fully gaseous. Hydrogen bonds are important forces in biopolymers that include DNA, proteins, and cellulose. All of these polymers lose their native structures upon boiling. Hydrogen bonds between amino acids that are close to each other in primary structure can give rise to regular repeating structures, such as helices or pleats, in proteins (secondary structure). Ionic interactions Ionic interactions are important forces stabilizing protein structure that arise from ionization of R-groups in the amino acids comprising a protein. These include the carboxyl amino acids (HERE), the amine amino acids as well as the sulfhydryl of cysteine and sometimes the hydroxyl of tyrosine. Hydrophobic forces Hydrophobic forces stabilize protein structure as a result of interactions that favor the exclusion of water. Non-polar amino acids (commonly found in the interior of proteins) favor associating with each other and this has the effect of excluding water. The excluded water has a higher entropy than water interacting with the hydrophobic side chains. This is because water aligns itself very regularly and in a distinct pattern when interacting with hydrophobic molecules. When water is prevented from having these kinds of interactions, it is much more disordered that it would be if it could associate with the hydrophobic regions. It is partly for this reason that hydrophobic amino acids are found in protein interiors - so they can exclude water and increase entropy. Disulfide bonds Disulfide bonds, which are made when two sulfhydryl side-chains of cysteine are brought into close proximity, covalently join together different protein regions and can give great strength to the overall structure (Figures 2.42 & 2.43). An Ode to Protein Structure by Kevin Ahern The twenty wee amino A's Define a protein many ways Their order in a peptide chain Determines forms that proteins gain And when they coil, it leaves me merry Cuz that makes structures secondary It's tertiary, I am told That happens when a protein folds But folded chains are downright scary When put together quaternary They're nature's wonders, that's for sure Creating problems, making cures A fool can fashion peptide poems But proteins come from ribosoems These joined residues of cysteine are sometimes referred to as cystine. Disulfide bonds are the strongest of the forces stabilizing protein structure. van der Waals forces van der Waals forces is a term used to describe various weak interactions, including those caused by attraction between a polar molecule and a transient dipole, or between two temporary dipoles. van der Waals forces are dynamic because of the fluctuating nature of the attraction, and are generally weak in comparison to covalent bonds, but can, over very short distances, be significant. Post-translational modifications Post-translational modifications can result in formation of covalent bonds stabilizing proteins as well. Hydroxylation of lysine and proline in strands of collagen can result in cross-linking of these groups and the resulting covalent bonds help to strengthen and stabilize the collagen. Folding models Two popular models of protein folding are currently under investigation. In the first (diffusion collision model), a nucleation event begins the process, followed by secondary structure formation. Collisions between the secondary structures (as in the β-hairpin in Figure 2.37) allow for folding to begin. By contrast, in the nucleation-condensation model, the secondary and tertiary structures form together. Folding in proteins occurs fairly rapidly (0.1 to 1000 seconds) and can occur during synthesis - the amino terminus of a protein can start to fold before the carboxyl terminus is even made, though that is not always the case. Folding process Protein folding is hypothesized to occur in a “folding funnel” energy landscape in which a folded protein’s native state corresponds to the minimal free energy possible in conditions of the medium (usually aqueous solvent) in which the protein is dissolved. As seen in the diagram (Figure 2.44), the energy funnel has numerous local minima (dips) in which a folding protein can become trapped as it moves down the energy plot. Other factors, such as temperature, electric/magnetic fields, and spacial considerations likely play roles. If external forces affect local energy minima during folding, the process and end-product can be influenced. As the speed of a car going down a road will affect the safety of the journey, so too do energy considerations influence and guide the folding process, resulting in fully functional, properly folded proteins in some cases and misfolded “mistakes” in others. Getting stuck As the folding process proceeds towards an energy minimum (bottom of the funnel in Figure 2.44), a protein can get “stuck” in any of the local minima and not reach the final folded state. Though the folded state is, in general, more organized and therefore has reduced entropy than the unfolded state, there are two forces that overcome the entropy decrease and drive the process forward. The first is the magnitude of the decrease in energy as shown in the graph. Since ΔG = ΔH -TΔS, a decrease in ΔH can overcome a negative ΔS to make ΔG negative and push the folding process forward. Favorable (decreased) energy conditions arise with formation of ionic bonds, hydrogen bonds, disulfide bonds, and metallic bonds during the folding process. In addition, the hydrophobic effect increases entropy by allowing hydrophobic amino acids in the interior of a folded protein to exclude water, thus countering the impact of the ordering of the protein structure by making the ΔS less negative. Structure prediction Computer programs are very good at predicting secondary structure solely based on amino acid sequence, but struggle with determining tertiary structure using the same information. This is partly due to the fact that secondary structures have repeating points of stabilization based on geometry and any regular secondary structure (e.g., α-helix) varies very little from one to another. Folded structures, though, have an enormous number of possible structures as shown by Levinthal’s Paradox. Spectroscopy Because of our inability to accurately predict tertiary structure based on amino acid sequence, proteins structures are actually determined using techniques of spectroscopy. In these approaches, proteins are subjected to varied forms of electromagnetic radiation and the ways they interact with the radiation allows researchers to determine atomic coordinates at Angstrom resolution from electron densities (see X-ray crystallography) and how nuclei spins interact (see NMR). Levinthal’s paradox In the late 1960s, Cyrus Levinthal outlined the magnitude of the complexity of the protein folding problem. He pointed out that for a protein with 100 amino acids, it would have 99 peptide bonds and 198 considerations for φ and ψ angles. If each of these had only three conformations, that would result in 3198 different possible foldings or 2.95x1094. Even allowing a reasonable amount of time (one nanosecond) for each possible fold to occur, it would take longer than the age of the universe to sample all of them, meaning clearly that the process of folding is not occurring by a sequential random sampling and that attempts to determine protein structure by random sampling were doomed to fail. Levinthal, therefore, proposed that folding occurs by a sequential process that begins with a nucleation event that guides the process rapidly and is not unlike the funnel process depicted in Figure 2.44. Diseases of protein misfolding The proper folding of proteins is essential to their function. It follows then that misfolding of proteins (also called proteopathy) might have consequences. In some cases, this might simply result in an inactive protein. Protein misfolding also plays a role in numerous diseases, such as Mad Cow Disease, Alzheimers, Parkinson’s Disease, and CreutzfeldJakob disease. Many, but not all, misfolding diseases affect brain tissue. Insoluble deposits Misfolded proteins will commonly form aggregates called amyloids that are harmful to tissues containing them because they change from being soluble to insoluble in water and form deposits. The process by which misfolding (Figure 2.45) occurs is not completely clear, but in many cases, it has been demonstrated that a “seed” protein which is misfolded can induce the same misfolding in other copies of the same protein. These seed proteins are known as prions and they act as infectious agents, resulting in the spread of disease. The list of human diseases linked to protein misfolding is long and continues to grow. A Wikipedia link is HERE. Prions Prions are infectious protein particles that cause transmissible spongiform encephalopathies (TSEs), the best known of which is Mad Cow disease. Other manifestations include the disease, scrapie, in sheep, and human diseases, such as CreutzfeldtJakob disease (CJD), Fatal Familial Insomnia, and kuru. The protein involved in these diseases is a membrane protein called PrP. PrP is encoded in the genome of many organisms and is found in most cells of the body. PrPc is the name given to the structure of PrP that is normal and not associated with disease. PrPSc is the name given to a misfolded form of the same protein, that is associated with the development of disease symptoms (Figure 2.45). Misfolded The misfolded PrPSc is associated with the TSE diseases and acts as an infectious particle. A third form of PrP, called PrPres can be found in TSEs, but is not infectious. The ‘res’ of PrPres indicates it is protease resistant. It is worth noting that all three forms of PrP have the same amino acid sequence and differ from each other only in the ways in which the polypeptide chains are folded. The most dangerously misfolded form of PrP is PrPSc, because of its ability to act like an infectious agent - a seed protein that can induce misfolding of PrPc , thus converting it into PrPSc. Function The function of PrPc is unknown. Mice lacking the PrP gene do not have major abnormalities. They do appear to exhibit problems with long term memory, suggesting a function for PrPc . Stanley Prusiner, who discovered prions and coined the term, received the Nobel Prize in Medicine in 1997 for his work. I think that if I chanced to be on A protein making up a prion I’d twist it and for goodness sakes Stop it from making fold mistakes Amyloids Amyloids are a collection of improperly folded protein aggregates that are found in the human body. As a consequence of their misfolding, they are insoluble and contribute to some twenty human diseases including important neurological ones involving prions. Diseases include (affected protein in parentheses) - Alzheimer’s disease (Amyloid β), Parkinson’s disease (α-synuclein), Huntington’s disease (huntingtin), rheumatoid arthritis (serum amyloid A), fatal familial insomnia (PrPSc), and others. Amino acid sequence plays a role in amyloidogenesis. Glutamine-rich polypeptides are common in yeast and human prions. Trinucleotide repeats are important in Huntington’s disease. Where sequence is not a factor, hydrophobic association between β-sheets can play a role. Amyloid β Amyloid β refers to collections of small proteins (36-43 amino acids) that appear to play a role in Alzheimer’s disease. (Tau protein is the other factor.) They are, in fact, the main components of amyloid plaques found in the brains of patients suffering from the disease and arise from proteolytic cleavage of a larger amyloid precursor glycoprotein called Amyloid Precursor Protein, an integral membrane protein of nerve cells whose function is not known. Two proteases, β-secretase and γ- secretase perform this function. Amyloid β proteins are improperly folded and appear to induce other proteins to misfold and thus precipitate and form the amyloid characteristic of the disease. The plaques are toxic to nerve cells and give rise to the dementia characteristic of the disease. It is thought that aggregation of amyloid β proteins during misfolding leads to generation of reactive oxygen species and that this is the means by which neurons are damaged. It is not known what the actual function of amyloid β is. Autosomal dominant mutations in the protein lead to early onset of the disease, but this occurs in no more than 10% of the cases. Strategies for treating the disease include inhibition of the secretases that generate the peptide fragments from the amyloid precursor protein. Huntingtin Huntingtin is the central gene in Huntington’s disease. The protein made from it is glutamine rich, with 6-35 such residues in its wild-type form. In Huntington’s disease, this gene is mutated, increasing the number of glutamines in the mutant protein to between 36 and 250. The size of the protein varies with the number of glutamines in the mutant protein, but the wild-type protein has over 3100 amino acids and a molecular weight of about 350,000 Da. Its precise function is not known, but huntingtin is found in nerve cells, with the highest level in the brain. It is thought to possibly play roles in transport, signaling, and protection against apoptosis. Huntingtin is also required for early embryonic development. Within the cell, huntingtin is found localized primarily with microtubules and vesicles. Trinucleotide repeat The huntingtin gene contains many copies of the sequence CAG (called trinucleotide repeats), which code for the many glutamines in the protein. Huntington’s disease arises when extra copies of the CAG sequence are generated when the DNA of the gene is being copied. Expansion of repeated sequences can occur due to slipping of the polymerase relative to the DNA template during replication. As a result, multiple additional copies of the trinucleotide repeat may be made, resulting in proteins with variable numbers of glutamine residues. Up to 35 repeats can be tolerated without problem. The number of repeats can expand over the course of a person’s lifetime, however, by the same mechanism. Individuals with 36-40 repeats begin to show signs of the disease and if there are over 40, the disease will be present. Molecular chaperones The importance of the proper folding of proteins is highlighted by the diseases associated with misfolded proteins, so it is no surprise, then, that cells expend energy to facilitate the proper folding of proteins. Cells use two classes of proteins known as molecular chaperones, to facilitate such folding in cells. Molecular chaperones are of two kinds, the chaperones, and the chaperonins. An example of the first category is the Hsp70 class of proteins. Hsp stands for “heat shock protein”, based on the fact that these proteins were first observed in large amounts in cells that had been briefly subjected to high temperatures. Hsps function to assist cells in stresses arising from heat shock and exposure to oxidizing conditions or toxic heavy metals, such as cadmium and mercury. However, they also play an important role in normal conditions, where they assist in the proper folding of polypeptides by preventing aberrant interactions that could lead to misfolding or aggregation. The Hsp70 proteins are found in almost all cells and use ATP hydrolysis to stimulate structural changes in the shape of the chaperone to accommodate binding of substrate proteins. The binding domain of Hsp70s contains a β-barrel structure which wraps around the polypeptide chain of the substrate and has affinity for hydrophobic side chains of amino acids. As shown in Figure 2.50, Hsp70 binds to polypeptides as they emerge from ribosomes during protein synthesis. Binding of substrate stimulates ATP hydrolysis and this is facilitated by another heat shock protein known as Hsp40. The hydrolysis of ATP causes the Hsp70 to taken on a closed conformation that helps shield exposed hydrophobic residues and prevent aggregation or local misfolding. After protein synthesis is complete, ADP is released and replaced by ATP and this results in release of the substrate protein, which then allows the full length polypeptide to fold correctly. In heat shock In times of heat shock or oxidative stress, Hsp70 proteins bind to unfolded hydrophobic regions of proteins to similarly prevent them from aggregating and allowing them to properly refold. When proteins are damaged, Hsp70 recruits enzymes that ubiquitinate the damaged protein to target them for destruction in proteasomes. Thus, the Hsp70 proteins play an important role in ensuring not only that proteins are properly folded, but that damaged or nonfunctional proteins are removed by degradation in the proteasome. Chaperonins A second class of proteins involved in assisting other proteins to fold properly are known as chaperonins. There are two primary categories of chaperonins - Class I (found in bacteria, chloroplasts, and mitochondria) and Class II (found in the cytosol of eukaryotes and archaebacteria). The best studied chaperonins are the GroEL/GroES complex proteins found in bacteria (Figure 2.51). GroEL/GroES may not be able to undo aggregated proteins, but by facilitating proper folding, it provides competition for misfolding as a process and can reduce or eliminate problems arising from improper folding. GroEL is a double-ring 14mer with a hydrophobic region that can facilitate folding of substrates 15-60 kDa in size. GroES is a singlering heptamer that binds to GroEL in the presence of ATP and functions as a cover over GroEL. Hydrolysis of ATP by chaperonins induce large conformational changes that affect binding of substrate proteins and their folding. It is not known exactly how chaperonins fold proteins. Passive models postulate the chaperonin complex functioning inertly by preventing unfavorable intermolecular interactions or placing restrictions on spaces available for folding to occur. Active models propose that structural changes in the chaperonin complex induce structural changes in the substrate protein. Protein breakdown Another protein complex that has an important function in the lifetime dynamics of proteins is the proteasome (Figure 2.52). Proteasomes, which are found in all eukaryotes and archaeans, as well as some bacteria, function to break down unneeded or damaged proteins by proteolytic degradation. Proteasomes help to regulate the concentration of some proteins and degrade ones that are misfolded. The proteasomal degradation pathway plays an important role in cellular processes that include progression through the cell cycle, modulation of gene expression, and response to oxidative stresses. Degradation in the proteasome yields short peptides seven to eight amino acids in length. Threonine proteases play important roles. Breakdown of these peptides yields individual amino acids, thus facilitating their recycling in cells. Proteins are targeted for degradation in eukaryotic proteasomes by attachment to multiple copies of a small protein called ubiquitin (8.5 kDa - 76 amino acids). The enzyme catalyzing the reaction is known as ubiquitin ligase. The resulting polyubiquitin chain is bound by the proteasome and degradation begins. Ubiquitin was named due to it ubiquitously being found in eukaryotic cells. Ubiquitin Ubiquitin (Figure 2.53) is a small (8.5 kDa) multi-functional protein found in eukaryotic cells. It is commonly added to target proteins by action of ubiquitin ligase enzymes (E3 in Figure 2.54). One (ubiquitination) or many (polyubiquitination) ubiquitin molecules may be added. Attachment of the ubiquitin is through the side chain of one of seven different lysine residues in ubiquitin. The addition of ubiquitin to proteins has many effects, the best known of which is targeting the protein for degradation in the proteasome. Proteasomal targeting is seen when polyubiquitination occurs at lysines #29 and 48. Polyubiquitination or monoubiquitination at other lysines can result in altered cellular location and changed protein-protein interactions. The latter may alter affect inflammation, endocytic trafficking, translation and DNA repair. Ubiquitin ligase malfunction Parkin is a Parkinson’s disease-related protein that, when mutated, is linked to an inherited form of the disease called autosomal recessive juvenile Parkinson’s disease. The function of the protein is not known, but it is a component of the E3 ubiquitin ligase system responsible for transferring ubiquitin from the E2 protein to a lysine side chain on the target protein. It is thought that mutations in parkin lead to proteasomal dysfunction and a consequent inability to break down proteins harmful to dopaminergic neurons. This results in the death or malfunction of these neurons, resulting in Parkinson’s disease. Intrinsically disordered proteins Movie 2.1 - Dynamic movement of cytochrome C in solution Wikipedia As is evident from the many examples described elsewhere in the book, the 3-D structure of proteins is important for their function. But, increasingly, it is becoming evident that not all proteins fold into a stable structure. Studies on the so-called intrinsically disordered proteins (IDPs) in the past cou- ple of decades has shown that many proteins are biologically active, even thought they fail to fold into stable structures. Yet other proteins exhibit regions that remain unfolded (IDP regions) even as the rest of the polypeptide folds into a structured form. Intrinsically disordered proteins and disordered regions within proteins have, in fact, been known for many years, but were regarded as an anomaly. It is only recently, with the realization that IDPs and IDP regions are widespread among eukaryotic proteins, that it has been recognized that the observed disorder is a "feature, not a bug". Movie 2.2 SUMO-1, a protein with intrinsically disordered sections Wikipedia Comparison of IDPs shows that they share sequence characteristics that appear to favor their disordered state. That is, just as some amino acid sequences may favor the folding of a polypeptide into a particular structure, the amino acid sequences of IDPs favor their remaining unfolded. IDP regions are seen to be low in hydrophobic residues and unusually rich in polar residues and proline. The presence of a large number of charged amino acids in the IDPs can inhibit folding through charge repulsion, while the lack of hydrophobic residues makes it difficult to form a stable hydrophobic core, and proline discourages the formation of helical structures. The observed differences between amino acid sequences in IDPs and structured proteins have been used to design algorithms to predict whether a given amino acid sequence will be disordered. What is the significance of intrinsically disordered proteins or regions? The fact that this property is encoded in their amino acid sequences suggests that their disorder may be linked to their function. The flexible, mobile nature of some IDP regions may play a crucial role in their function, permitting a transition to a folded structure upon binding a protein partner or undergoing post-translational modification. Studies on several wellknown proteins with IDP regions suggest some answers. IDP regions may enhance the ability of proteins like the lac repressor to translocate along the DNA to search for specific binding sites. The flexibility of IDPs can also be an asset in protein-protein interactions, especially for proteins that are known to interact with many different protein partners. For example, p53 has IDP regions that may allow the protein to interact with a variety of functional partners. Comparison of the known functions of proteins with predictions of disorder in these proteins suggests that IDPs and IDP regions may disproportionately function in signaling and regulation, while more structured proteins skew towards roles in catalysis and transport. Interestingly, many of the proteins found in both ribosomes and spliceosomes are predicted to have IDP regions that may play a part in correct assembly of these complexes. Even though IDPs have not been studied intensively for very long, what little is known of them suggests that they play an important and underestimated role in cells. Metamorphic proteins Another group of proteins that have recently changed our thinking about protein structure and function are the so-called metamorphic proteins. These proteins are capable of forming more than one stable, folded state starting with a single amino acid sequence. Although it is true that multiple folded conformations are not ruled out by the laws of physics and chemistry, metamorphic proteins are a relatively new discovery. It was known, of course, that prion proteins were capable of folding into alternative structures, but metamorphic proteins appear to be able to toggle back and forth between two stable structures. While in some cases, the metamorphic protein undergoes this switch in response to binding another molecule, some proteins that can accomplish this transition on their own. An interesting example is the signaling molecule, lymphotactin. Lymphotactin has two biological functions that are carried out by its two conformers- a monomeric form that binds the lymphotactin receptor and a dimeric form that binds heparin. It is possible that this sort of switching is more widespread than has been thought. Refolding denatured proteins All information for protein folding is contained in the amino acid sequence of the protein. It may seem curious then that most proteins do not fold into their proper, fully active form after they have been+++ denatured and the denaturant is removed. A few do, in fact. One good example is bovine ribonuclease (Figure 2.55). Its catalytic activity is very resistant to heat and urea and attempts to denature it don’t work very well. However, if one treats the enzyme with β-mercaptoethanol (which breaks disulfide bonds) prior to urea treatment and/or heating, activity is lost, indicating that the covalent disulfide bonds help stabilize the overall enzyme structure and when they are broken, denaturation can readily occur. When the mixture cools back down to room temperature, over time some enzyme activity reappears, indicating that ribonuclease re-folded under the new conditions. Interestingly, renaturation will occur maximally if a tiny amount of β-mercaptoethanol is left in the solution during the process. The reason for this is because β- mercaptoethanol permits reduction (and breaking) of accidental, incorrect disulfide bonds during the folding process. Without it, these disulfide bonds will prevent proper folds from forming. Irreversible denaturation Most enzymes, however, do not behave like bovine ribonuclease. Once denatured, their activity cannot be recovered to any significant There are not very many ways Inactivating RNase It’s stable when it’s hot or cold Because disulfides tightly hold If you desire to make it stall Use hot mercaptoethanol extent. This may seem to contradict the idea of folding information being inherent to the sequence of amino acids in the protein. It does not. Most enzymes don’t refold properly after denaturation for two reasons. First, normal folding may occur as proteins are being made. Interactions among amino acids early in the synthesis are not “confused” by interactions with amino acids later in the synthesis because those amino acids aren’t present as the process starts. Chaperonins’ role In other cases, the folding process of some proteins in the cell relied upon action of chaperonin proteins (see HERE). In the absence of chaperonins, interactions that might result in misfolding occur, thus preventing proper folding. Thus, early folding and the assistance of chaperonins eliminate some potential “wrong-folding” interactions that can occur if the entire sequence was present when folding started. Quaternary structure A fourth level of protein structure is that of quaternary structure. It refers to structures that arise as a result of interactions between multiple polypeptides. The units can be identical multiple copies or can be different polypeptide chains. Adult hemoglobin is a good example of a protein with quaternary structure, being composed of two identical chains called α and two identical chains called β. Though the α-chains are very similar to the β- chains, they are not identical. Both of the α- and the β-chains are also related to the single polypeptide chain in the related protein called myoglobin. Both myoglobin and hemoglobin have similarity in binding oxygen, but their behavior towards the molecule differ significantly. Notably, hemoglobin’s multiple subunits (with quaternary structure) compared to myoglobin’s single subunit (with no quaternary structure) give rise to these differences. References 1. https://en.wikipedia.org/wiki/Van_der_W aals_force 105

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/02%3A_Structure_and_Function/203%3A_Structure__Function-_Proteins_I.txt

Thumbnail: The cell membrane, also called the plasma membrane or plasmalemma, is a semipermeable lipid bilayer common to all living cells. It contains a variety of biological molecules, primarily proteins and lipids, which are involved in a vast array of cellular processes. It also serves as the attachment point for both the intracellular cytoskeleton and, if present, the cell wall. Image used with permission (CC BU-SA 3.0; Dhatfield and LadyofHats). 03: Membranes Source: BiochemFFA_3_1.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Lipid bilayers The protective membrane around cells contains many components, including cholesterol, proteins, glycolipids, glycerophospholipids, and sphingolipids. The last two of these will, when mixed vigorously with water, spontaneously form what is called a lipid bilayer (Figure 3.1), which serves as a protective boundary for the cell that is largely impermeable to the movement of most materials across it. With the notable exceptions of water, carbon dioxide, carbon monoxide, and oxygen, most polar/ionic require transport proteins to help them to efficiently navigate across the bilayer. The orderly movement of these compounds is critical for the cell to be able to 1) get food for energy; 2) export materials; 3) maintain osmotic balance; 4) create gradients for secondary transport; 5) provide electromotive force for nerve signaling; and 6) store energy in electrochemical gradients for ATP production (oxidative phosphorylation or photosynthesis). In some cases, energy is required to move the substances (active transport). Facilitated Diffusion In other cases, no external energy is required and they move by diffusion through specific cellular channels. This is referred to as facilitated diffusion. Before we discuss movement of materials across membranes, it is appropriate we discuss the composition of cellular membranes. Plasma membranes differ from cell walls both in the materials comprising them and in their flexibility. Cell walls will be covered near the end of this chapter. Though some cells do not have cell walls (animal cells) and others do (bacteria, fungi, and plants), there is commonality among cells in that they all possess plasma membranes. There is also commonality in the components of the membranes, though the relative amount of constituents varies. Figures 3.1 and 3.2 illustrate the structure and environments of plasma membranes. All plasma membranes contain a significant amount of amphiphilic substances linked to fatty acids. These include the glycerophospholipids and the sphingolipids. The fatty acid(s) are labeled as hydrophobic tails in the figures. Hydrophilic heads The composition of the hydrophilic heads varies considerably. In glycerophospholipids, a phosphate is always present, of course, and it is often esterified to another substance to make a phosphatide (Figure 3.3). Common compounds linked to the phosphate (at the X position) include serine, ethanolamine, and choline. These vary in the their charges so in this way, the charge on the external or internal surface can be controlled. Cells tend to have more negative charges on the exterior half of the lipid bilayer (called the outer leaflet) and more positive charges on the interior half (inner leaflet). Sphingolipids In sphingolipids (Figure 3.4), the hydrophilic head can contain a phosphate linked to ethanolamine or choline and this describes the structure of sphingomyelin, an important component of neural membranes. Most sphingolipids lack the phosphate and have instead a hydrophilic head of a single sugar (cerebrosides) or a complex oligosaccharide (gangliosides). Water exclusion In each case, the glycerophospholipid or sphingolipid has one end that is polar and one end that is non-polar. As we saw in the organization of amino acids with hydrophobic side chains occurring preferentially on the inside of a folded protein to exclude water, so too do the non-polar portions of these amphiphilic molecules arrange themselves so as to exclude water. Remember that the cytoplasm of a cell is mostly water and the exterior of the cell is usually bathed in an aqueous layer. It therefore makes perfect sense that the polar portions of the membrane molecules arrange themselves as they do - polar parts outside interacting with water and non-polar parts in the middle of the bilayer avoiding/excluding water. Composition Bias The plasma membrane has distinct biases of composition relative to its inside and the outside (Figure 3.7). First, glycosylation (of lipids and proteins) has the sugar groups located almost exclusively on the outside of the cell, away from the cytoplasm (Figure 3.8). Among the membrane lipids, sphingolipids are much more commonly glycosylated than glycerophospholipids. In addition, some of the glyerophospholipids are found preferentially on one side or the other (Figure 3.7). Phosphatidylserine and phosphatidylethanolamine are found preferentially within the inner leaflet of the plasma membrane, whereas phosphatidylcholine tends to be located on the outer leaflet. In the process of apoptosis, the phosphatidylserines appear on the outer leaflet where they serve as a signal to macrophages to bind and destroy the cell. Sphingolipids are found preferentially in the plasma membrane and are almost completely absent from mitochondrial and endoplasmic reticulum membranes (Figure 3.9). Organelle membranes Bias of lipid composition also exists with respect to organelle membranes. The unusual diphosphodiglycerolipid known as cardiolipin, for example, is almost only found in mitochondrial membranes (see HERE) and like phosphatidylserine, its movement is an important step in apoptosis. In signaling, phosphatidylinositols play important roles providing second messengers upon being cleaved (see HERE). Lateral Diffusion Movement of lipids within each leaflet of the lipid bilayer occurs readily and rapidly due to membrane fluidity. This type of movement is called lateral diffusion and can be measured by the technique called FRAP (Figure 3.10, see HERE also). In this method, a laser strikes and stains a section of the lipid bilayer of a cell, leaving a spot as shown in B. Over time, the stain diffuses out ultimately across the entire lipid bilayer, much like a drop of ink will diffuse throughout when added to a glass of water. A measurement of the rate of diffusion gives an indication of the fluidity of a membrane. Transverse Diffusion While the movement in lateral diffusion occurs rapidly, movement of molecules from one leaflet over to the other leaflet occurs much more slowly. This type of molecular movement is called transverse diffusion and is almost nonexistent in the absence of enzyme action. Remember that there is a bias of distribution of molecules between leaflets of the membrane, which means that something must be moving them. There are three enzymes that catalyze movement of compounds in transverse diffusion. Flippases move membrane glycerophospholipids/ sphingolipids from outer leaflet to inner leaflet (cytoplasmic side) of cell. Floppases move membrane lipids in the opposite direction. Scramblases move in either direction. Other components of lipid bilayer Besides glycerophospholipids and sphingolipids, there are other materials commonly found in lipid bilayers of cellular membranes. Two important prominent ones are cholesterol (Figure 3.13) and proteins. Besides serving as a metabolic precursor of steroid hormones and the bile acids, cholesterol’s main role in cells is in the membranes. The flatness and hydrophobicity of the sterol rings allow cholesterol to interact with the nonpolar portions of the lipid bilayer while the hydroxyl group on the end can interact with the hydrophilic part. Membrane fluidity Cholesterol’s function in the lipid bilayer is complex (Figure 3.13). It influences membrane fluidity. Figure 3.14 shows the phase transition for a membrane as it is heated, moving from a more “frozen” character to that of a more “fluid” one as the temperature rises. The mid-point of this transition, referred to as the Tm, is influenced by the fatty acid composition of the lipid bilayer compounds. Longer and more saturated fatty acids will favor higher Tm values, whereas unsaturation and short fatty acids will favor lower Tm values. It is for this reason that fish, which live in cool environments, have a higher level of unsaturated fatty acids in them - to use to make membrane lipids that will remain fluid at ocean temperatures. Interestingly, cholesterol does not change the Tm value, but instead widens the transition range between frozen and fluid forms of the membrane, allowing it to have a wider range of fluidity. Lipid Rafts Cholesterol is also abundantly found in membrane structures called lipid rafts. Depicted in Figure 3.15, lipid rafts are organized structures within the membrane typically containing signaling molecules and other integral membrane proteins. Lipid rafts affect membrane fluidity, neurotransmission, and trafficking of receptors and membrane proteins. Features Distinguishing features of the rafts is that they are more ordered than the bilayers surrounding them, containing more saturated fatty acids (tighter packing and less disorganization, as a result) and up to 5 times as much cholesterol. They also are relatively rich in sphingolipids, with as much as 50% greater quantities of sphingomyelin than surrounding areas of the bilayer. The higher concentration of cholesterol in the rafts may be due to its greater ability to associate with sphingolipids (Figure 3.16). Some groups, such as prenylated proteins, like RAS, may be excluded from lipid rafts. Lipid rafts may provide concentrating platforms after individual protein receptors bind to ligands in signaling. After receptor activation takes place at a lipid raft, the signaling complex would provide protection from nonraft enzymes that could inactivate the signal. For example, a common feature of signaling systems is phosphorylation, so lipid rafts might provide protection against dephosphorylation by enzymes called phosphatases. Lipid rafts appear to be involved in many signal transduction processes, such as T cell antigen receptor signaling, B cell antigen receptor signaling, EGF receptor signaling, immunoglobulin E signaling, insulin receptor signaling and others. For more on signaling, see HERE. Barrier Transport of materials across membranes is essential for a cell to exist. The lipid bilayer is an effective barrier to the entry of most molecules and without a means of allowing food molecules to enter a cell, it would die. The primary molecules that move freely across the lipid bilayer are small, uncharged ones, such as H2O, CO2, CO, and O2, so larger molecules, like glucose, that the cell needs for energy, would be effectively excluded if there were not proteins to facilitate its movement across the membrane. Figure 3.17 depicts the barrier that the lipid bilayer provides to movement across it and the pressures (ionic attraction, in this case) that can affect movement. Potential energy from charge and concentration differences are harvested by cells for purposes that include synthesis of ATP, and moving materials against a concentration gradient in a process called active transport. Membrane proteins Proteins in a lipid bilayer can vary in quantity enormously, depending on the membrane. Protein content by weight of various membranes typically ranges between 30 and 75% by weight. Some mitochondrial membranes can have up to 90% protein. Proteins linked to and associated with membranes come in several types. Transmembrane proteins Transmembrane proteins are integral membrane proteins that completely span from one side of a biological membrane to the other and are firmly embedded in the membrane (Figure 3.18). Transmembrane proteins can function as docking sites for attachment (to the extracellular matrix, for example), as receptors in the cellular signaling system, or facilitate the specific transport of molecules into or out of the cell. Example of integrated/ transmembrane proteins include those involved in transport (e.g., Na+/K+ ATPase), ion channels (e.g., potassium channel of nerve cells) and signal transduction across the lipid bilayer (e.g., GProtein Coupled Receptors). Peripheral membrane proteins interact with part of the bilayer (usually does not involve hydrophobic interactions), but do not project through it. A good example is phospholipase A2, which cleaves fatty acids from glycerophospholipids in membranes. Associated membrane proteins typically do not have external hydrophobic regions, so they cannot embed in a portion of the lipid bilayer, but are found near them. Such association may arise as a result of interaction with other proteins or molecules in the lipid bilayer. A good example is ribonuclease. Anchored membrane proteins Anchored membrane proteins are not themselves embedded in the lipid bilayer, but instead are attached to a molecule (typically a fatty acid) that is embedded in the membrane (Figure 3.19). The oncogene family of proteins known as ras are good examples. These proteins are anchored to the lipid bilayer by attachment to non-polar farnesyl groups catalyzed by the enzyme farnesyltransferase. Finer classification A more detailed classification scheme further categorizes the integral and anchored proteins into six different types (Figure 3.20). Type I and Type II have only one portion of the protein pass through the membrane. They differ in the orientation of the amine and carboxyl end with respect to inside/outside. Type I transmembrane proteins have the amino terminus on the outside and carboxy terminus on the inside, whereas Type II proteins have this reversed. Type III proteins are a single polypeptide chain that has multiple regions of it cross back and forth across the membrane, often to form a channel. Type IV is a multi-polypeptide protein which has multiple crossings of the membrane. Type V transmembrane proteins do not have a part of them that crosses the membrane, but they are anchored to the membrane by a lipid (such as a fatty acid) embedded in the lipid bilayer. Type VI transmembrane proteins both have a portion of them that crosses the membrane and they are attached to a lipid embedded in the lipid bilayer. Blood Types Cells have hundreds-thousands of membrane proteins and the protein composition of a membrane varies with its function and location. Glycoproteins embedded in membranes play important roles in cellular identification. Blood types, for example, differ from each other in the structure of the carbohydrate chains projecting out from the surface of the glycoprotein in their membranes (Figure 3.21). Osmotic Pressure Membranes provide barriers/boundaries for most molecules, but the permeability of water across a lipid bilayer creates a variable that must be considered. The variable here is osmotic pressure. Osmotic pressure (loosely) refers to the tendency of a solution to take in water by the process of osmosis. In Figure 3.22, one can see a visual representation of the concept of the pressure. A U-shaped tube has at its bottom a semipermeable membrane. Water can pass through the membrane, but sugar molecules (C6H12O6) cannot. On the left side, sugar is added creating a concentration difference between the right and left chambers. Water diffuses across the membrane from right to left in an attempt to equalize the concentrations, causing the level of the right side to decrease and the left side to increase. The pressure resulting from the differences in height is felt at the membrane. Equalizing concentrations The liquid on the right does not completely move to the left, though, as might be expected if the only force involved is equalizing the concentration of sugar across the membrane (no sugar on right = no water). Instead, an equilibrium of sorts of water levels is reached even though the concentrations don’t equal out. The pressure existing at the membrane then from the differences in level corresponds to the osmotic pressure of the mixture. The osmotic pressure of a solution is the pressure difference needed to halt the flow of solvent across a semipermeable membrane. Osmotic pressure can also be thought of as the pressure required to counter osmosis. The osmotic pres- Figure 3.21 - Blood types arise from cell surface glycoproteins Figure 3.22 - Osmotic pressure. Water diffuses leftwards to try to equalize the solute concentration. The pressure realized at the membrane in the right figure is the osmotic pressure sure of a dilute solution mathematically behaves like the ideal gas law \[P_{osmotic} = \dfrac{nRT}{V}\] where n is the number of moles, R is the gas constant, T is the temperature in Kelvin, and V is the volume. It is more convenient in solutions to work with molarity, so \[P_{osmotic}= MR^* T\] where M is the molarity of the dissolved molecules, R* is the gas constant expressed in (L atm)/(K mol), and T is the temperature. The Greek letter Π is used to refer to the Posmoticterm, so \[Π = MR^* T\] Remember when calculating the molarity to include the molarity of each particle. For example, when one dissolves sucrose in solution, it does not split into smaller particles, so \[Molarity_{Particles} = Molarity_{Sucrose}\] However, for salts, like KOH, which forms two ions in solution (K+ and OH- ), MolarityParticles= 2* MolarityKOH. Significant consideration Osmotic pressure is a significant consideration for cells. Consider the fact that water can move freely across cellular membranes, but most of the contents of the cell, such as proteins, DNA, ions, sugars, etc., cannot. Second, the concentration of these compounds inside the cell is different than the concentration of them outside of the cell. Third, since water can move through the lipid bilayer, it will tend to move in the direction that will tend to equalize solute. Hypotonic, hypertonic, isotonic We consider three situations (Figure 3.23). First, if the concentration of solutes is greater inside the cell than outside, water will tend to move into the cell, causing the cell to swell. This circumstance is called hypotonic. Conversely, if the solute concentration is greater outside the cell than inside of it, water will exit the cell and the cell with shrink. This is a hypertonic situation. Last, if the concentrations of solutes into and outside of the cell is equal, this is called an isotonic solution. Here, no movement of water occurs across the cell membrane and the cell retains its size. If the osmotic pressure is greater than the forces holding together a cellular membrane, the cell will rupture. Because of this, some cells have built in defenses to prevent problems. Plant cells, for example have a fairly rigid cell wall that resists expansion in hypotonic solutions (Figure 3.24). Bacteria also have a cell wall that provides protection.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/03%3A_Membranes/3.01%3A_Basic_Concepts_in_Membranes.txt

Source: BiochemFFA_3_2.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Movement of materials across membranes As noted earlier, it is essential for cells to be able to uptake nutrients. This function along with movement of ions and other substances is provided by proteins/protein complexes that are highly specific for the compounds they move. Selective movement of ions by membrane proteins and the ions’ extremely low permeability across the lipid bilayer are important for helping to maintain the osmotic balance of the cell and also for providing for the most important mechanism for it to make ATP - the process of oxidative phosphorylation. Terminology A protein involved in moving only one molecule across a membrane is called a uniport (Figure 3.25). Proteins that move two molecules in the same direction across the membrane are called symports (also called synporters, synports, or symporters). If two molecules are moved in opposite directions across the bilayer, the protein is called an antiport. Proteins involved in moving ions are called ionophores. If the action of a protein in moving ions across a membrane results in a net change in charge, the protein is described as electrogenic and if there is no change in charge the protein is described as electroneutral (Figure 3.26). When the driving force for movement through the membrane protein is simply diffusion, the process is called facilitated diffusion or passive transport and when the process requires other energy input, the process is called active transport. Channels and transporters With respect to movement of materials through membrane proteins, there is a difference between channels (sometimes called pores) and transporters. Channels largely provide openings with some specificity and molecules pass through them at close to the rate of diffusion. They usually involve movement of water or ions. Examples would be the sodium or potassium channels of nerve cells. Transporters have high specificity and transfer rates that are orders of magnitude slower. Transport proteins include the sodium-potassium pump, the sodium-calcium exchanger, and lactose permease, amongst many others). Facilitated diffusion As noted, the driving force for facilitated diffusion is concentration, meaning that in facilitated diffusion, materials will only move from a higher concentration to a lower concentration and that at the end of the process, the concentration of materials on each side of a bilayer will be equal (Figure 3.28). This may work well in many cases. For example, the blood concentration of glucose is sufficiently high that red blood cells can use facilitated diffusion as a means of acquiring glucose. Other cells, further removed from the blood supply where the glucose concentration is lower, must use active transport mechanisms because there is not a sufficient concentration of glucose to provide cells with the glucose they need. Ion channels Ion channels are pore-forming membrane proteins in the membranes of all cells that regulate movement of selected ions across a membrane (Figures 3.29 & 3.30). They help to establish the resting membrane potential and to affect action potentials and other electrical signals. They are very important in the process of nerve transmission. Ion channels control the flow of ions across secretory and epithelial cells, and consequently help to regulate cell volume by affecting osmotic pressure. Ion channels are essential features of almost all cells, functioning as selective “tunnels” that restrict movement through them to ions with specific characteristics (typically size). The size of the opening is very narrow (usually one or two atoms wide) and is able to select even against ions that are too small. Control mechanisms Ion channels are controlled by mechanisms that include voltage, ligands, light, temperature, and mechanical deformation (stretch activated). Ligand-gated ion channels (LGICs) are transmembrane proteins which open to selectively allow ions such as Na+, K+, Ca++, or Cl− to pass through the membrane in response to the binding of a ligand messenger. Sound waves cause mechanical deformation of hair cells in the ear. This results in the opening of ion channels and initiation of a nerve signal to the brain. Sodium ion channels in the tongue for sugar receptors open in response to binding of sucrose, allowing sodium concentration in the nerve cell to increase and initiate a nerve signal to the brain. In this case, the default for the gate is to be closed and it opens in response to binding of a ligand (sucrose). In light sensing cells of the eye, calcium gates are open by default, but stimulation by light causes them to close, triggering a series of events that result in a signal being sent the brain about the perception of light. Thus, in this case, the stimulus (light) causes an open channel to close. Moving the other direction, nerve signals originating in the brain travel to muscle tissue and through a complicated set of exchanges, result in the opening of calcium gates of muscle cells, increasing the concentration of calcium and stimulating muscular contraction (see HERE). Voltage gated channels are essential for transmission of nerve signals, a process discussed in more depth HERE. Ion movement through channels The ability of ion channels to select against ions too large is intuitive - the size of the opening in the ion channel simply isn’t big enough for a larger ion to fit through the opening. Potassium, for example, passes through sodium channels rarely because the opening is too small. Potassium channels that are selective for potassium ions must be big enough to allow potassium to enter, but if size were the only selection means, then sodium ions would also readily pass through potassium channels, since sodium ions (0.95 Å) are smaller than potassium ions (1.33 Å). In order for potassium channels to select against sodium ions and favor potassium ions, other considerations come into play. Hydration shell To understand this unique selectivity, it is important to understand how ions move through channels. Before an ion can pass through a channel, it must first be dissociated from (stripped of) the water molecules in its hydration shell - water molecules surrounding ions in aqueous solutions (Figure 3.32). This process requires an input of energy. The initial energy required to strip the water molecules from the hydration shell has been compared to the activation energy of an enzymatic reaction. Comparable to enzymes Just as enzymes lower the activation energy of enzymatic reactions and thus allow them to more readily occur, so too do channel proteins lower the energy requirements for a molecule to traverse a lipid bilayer. In the absence of the channel protein, the dehydration energy is mostly prohibitive for most polar molecules to occur, so very few make it across the lipid bilayer without the channel protein. This is why ion channel/transport proteins are so important to the cell. After the water has been stripped, the ion can pass through the channel and when it arrives at the other side of the channel, the diffusing ion becomes rehydrated, thus regaining the energy that was required initially to strip away the water molecules from the ion. Selectivity of the potassium channel The potassium channel (Figure 3.33) uses the dimensions of the potassium ion precisely to shepherd it through the channel. The sodium ion, which has different dimensions has a more difficult time making it through the channel despite its smaller size. The reason this is rooted in the energy required for dehydration. For potassium ions, after the water has been stripped off, precisely positioned carbonyl groups along the channel help to stabilize the ion as it moves. The sodium ion, on the other hand is too small and does not make efficient connections with carbonyl groups and thus has a more difficult path. Because of this, the energy difference between dehydration and rehydration of a sodium ion in a potassium channel is energetically unfavorable (requires net input of energy) but the same process for a potassium ion is energetically favorable (results in a net gain of energy). Movie 3.1 - Gramicidin A Wikipedia (animated gif, download to view) Energy factor Thus the selection in favor of potassium and against sodium ions in a potassium channel is based on energy, not physical size, whereas in the selection of sodium ions over potassium ions in a sodium channel, size is the primary consideration. Ion balance The movement of ions across a lipid bilayer is tightly regulated, and with good reason. Maintaining a proper balance of ions inside and outside of cells is important for maintaining osmotic balance. It is also important inside and outside of organelles like the mitochondria and chloroplasts for energy generation. If the ionic balance of a cell is sufficiently disturbed by an uncontrolled ionophore, a cell may die. Gramicidin Gramicidins (Movie 3.1) are antibiotic polypeptides synthesized by the soil bacterium known as Bacillus brevis. These small pentadecapeptides (15 amino acids) are synthesized by the bacterium to kill other bacteria. When released by the Bacillus brevis, the gramicidins insert themselves in the membranes of Gram positive bacteria and allow the movement of sodium ions into the target cells, ultimately killing them. Gramicidins can also cause hemolysis in humans so they cannot be used internally, but instead are used topically. Aquaporins Aquaporins are pore-containing integral membrane proteins that selectively permit passage of water molecules in and out of the cell, while preventing ions and other solutes from moving (Figures 3.34 & 3.35). Some aquaporins called aquaglyceroporins, also transport other small uncharged entities, such as glycerol, ammonia, urea, and CO2, across the membrane,. The water pores are completely impermeable to charged molecules, such as protons, which is important for the preserving the membrane's electrochemical potential difference. Porins Porins are proteins containing a β-barrel structure that crosses the cell membrane/wall and acts as a pore/channel through which specific molecules diffuse. Porins are found in the outer membrane of Gram-negative bacteria and some Gram-positive bacteria, mitochondria, and chloroplasts. Porins typically transport only one group of molecules or, in some cases, one specific molecule. Antibiotics, such as β-lactam and fluoroquinolone pass through porins to reach the cytosol of Gram negative bacteria. Bacteria may develop resistance to these antibiotics when a mutation occurs to the porin involved that results in exclusion of the antibiotics that would otherwise pass through. Transporter proteins Not all facilitated transport occurs through ion channel proteins. Transporter proteins, as noted earlier (HERE and Figure 3.27) facilitate movement of materials across a lipid bilayer, but are slower than ion channels. Figure 3.36 illustrates a transporter protein in action. As can be seen, transporter proteins rely on a specific receptor site for proper recognition of the molecule to be moved. Binding of the proper molecule causes a conformational change in the shape of the protein (an eversion) which results in a flipping of the open side of the protein to the other side of the lipid bilayer. In this way, the molecule is moved. Like ion channels, transporter proteins facilitate movement of materials in either direction, driven only by the concentration difference between one side and the other. Active transport All of the transport mechanisms described so far are driven solely by a concentration gradient - moving from higher concentrations in the direction of lower concentrations. These movements can occur in either direction and, as noted, result in equal concentrations on either side of the bilayer, if allowed to go to completion. Many times, however, cells must move materials against a concentration gradient and when this occurs, another source of energy is required. This process is known as active transport. A good definition of active transport is that in active transport, at least one molecule is being moved against a concentration gradient. A common, but not exclusive, energy source is ATP (see Na+/K+ ATPase), but other energy sources are also employed. For example, the sodium-glucose transporter uses a sodium gradient as a force for actively transporting glucose into a cell. Thus, it is important to know that not all active transport uses ATP energy. Na+/K+ ATPase An important integral membrane transport protein is the Na+/K+ ATPase antiport (Figures 3.37 and 3.38), which moves three sodium ions out of the cell and two potassium ions into the cell with each cycle of action. In each case, the movement of ions is against the concentration gradient. Since three positive charges are moved out for each two positive charges moved in, the system is electrogenic. The protein uses the energy of ATP to create ion gradients that are important both in maintaining cellular osmotic pressure and (in nerve cells) for creating the sodium and potassium gradients necessary for signal transmission. Failure of the system to function results in swelling of the cell due to movement of water into the cell through osmotic pressure. The transporter expends about one fifth of the ATP energy of animal cells. The cycle of action occurs as follows: 1. Pump binds ATP followed by binding of 3 Na+ ions from cytoplasm of cell 2. ATP hydrolysis results in phosphorylation of aspartate residue of pump. ADP is released 3. Phosphorylated pump undergoes conformational change to expose Na+ ions to exterior of cell. Na+ ions are released. 4. Pump binds 2 extracellular K+ ions. 5. Pump dephosphorylates causing it to expose K+ ions to cytoplasm as pump returns to original shape. 6. Pump binds 3 Na+ ions, binds ATP and releases 2 K+ ions to restart process The Na+/K+ ATPase is classified as a P-type ATPase. This category of pump is notable for having a phosphorylated aspartate intermediate and is present across the biological kingdoms - bacteria, archaeans, and eukaryotes. ATPase types ATPases have roles in either the synthesis or hydrolysis of ATP and come in several different forms. • F-ATPases (F1FO-ATPases) are present in mitochondria, chloroplasts and bacterial plasma membranes and are the prime ATP synthesizers for these systems. Each uses a proton gradient as its energy source for ATP production. Complex V of the mitochondrion is an F-type ATPase. • V-ATPases (V1VO-ATPases) are mostly found in vacuoles of eukaryotes . They utilize energy from ATP hydrolysis to transport solutes and protons into vacuoles and lysosomes, thus lowering their pH values. The V-type and F-type ATPases are very similar in structure. The V-type (Figure 3.39) uses ATP to pump protons into vacuoles and lysosomes, whereas F-types use proton gradients of the mitochondria and chloroplasts to make ATP. • A-ATPases (A1AO-ATPases) are found in archaeans and are similar to F-ATPases in function. • P-ATPases (E1E2-ATPases) are in bacteria, fungi and in eukaryotic plasma membranes and organelles. They transport a diversity of ions across membranes. Each has a common mechanism of action which include autophosphorylation of a conserved aspartic acid side chain within it. Examples of P-type ATPases include the Na+/K+ ATPase and the calcium pump. • E-ATPases are enzymes found on the cell surface. They hydrolyze a range of extracellular nucleoside triphosphates, including ATP. Nerve transmission Now that you have seen how the Na+/K+ ATPase functions, it is appropriate to discuss how nerve cells use ion gradients created with it to generate and transmit nerve signals. Neurons are cells of the nervous system that use chemical and electrical signals to rapidly transmit information across the body (Figure 3.40). The sensory nerve system links receptors for vision, hearing, touch, taste, and smell to the brain for perception. Motor neurons run from the spinal cord to muscle cells. These neurons have a cell body and a very long, thin extension called an axon, that stretches from the cell body in the spinal cord all the way to the muscles they control. Nerve impulses travel down the axon to stimulate muscle contraction. Signals travel through neurons, ultimately arriving at junctions with other nerve cells or target cells such as muscle cells. Note that neurons do not make physical contact with each other or with muscle cells. The tiny space between two neurons or between a neuron and a muscle cell is called the synaptic cleft. At the synaptic cleft, the neuron releases neurotransmitters that exit the nerve cell and travel across the junction to a recipient cell where a response is generated. That response may be creating another nerve signal, if the adjacent cell is a nerve cell or it may be a muscular contraction if the recipient is a muscle cell (Figure 3.41). In considering information movement via nerve cells, then, we will discuss two steps - 1) creation and propagation of a signal in a nerve cell and 2) action of neurotransmitters exiting a nerve cell and transiting a synaptic junction. Signal source Creation of a nerve signal begins with a stimulus to the nerve cell. In the case of muscle contraction, the motor cortex of the brain sends signals to the appropriate motor neurons, stimulating them to generate a nerve impulse. How is such an impulse generated? Resting potential In the unstimulated state, all cells, including nerve cells, have a small voltage difference (called the resting potential) across the plasma membrane, arising from unequal pumping of ions across the membrane. The Na+/K+ ATPase, for example, pumps sodium ions out of the cell and potassium ions into cells. Since three sodium ions get pumped out for every two potassium ions pumped in, a charge and chemical gradient is created. It is the charge gradient that gives rise to the resting potential. Altering the gradients of ions across membranes provide the driving force for nerve signals. This happens as a result of opening and closing of gated ion channels. Opening of gates to allow ions to pass through the membrane swiftly changes the ionic balance across the membrane resulting in a new voltage difference called the action potential. It is the action potential that is the impetus of nerve transmission. Initiation of signal The signal generated by a motor neuron begins with opening of sodium channels in the membrane of the nerve cell body causing a rapid influx of sodium ions into the nerve cell. This step, called depolarization (Figure 3.42), triggers an electrochemical signal - the action potential. Remember that the Na+/K+ ATPase has created a large sodium gradient, so sodium ions rush into the cell when sodium channels open. After the initial depolarization, potassium channel gates, responding to the depolarization, open, allowing potassium ions to rapidly diffuse out of the cell (remember K+ ions are more abundant inside of the cell). This phase is called the repolarization phase and during it, the sodium gates close. The rapid exit of potassium ions causes the voltage difference to “overshoot” the resting potential and potassium gates close. This followed by the so-called refractory period, when the Na+/K+ ATPase begins its work to re-establish the original conditions by pumping sodium ions out and potassium ions into the nerve cell. Eventually, the system recovers and the resting potential is re-established. The initiating end of the nerve cell is then ready for another signal. Propagation of action potential What we have described here is only the initiation of the nerve signal in one part of the nerve cell. For the signal to be received, the action potential must travel the entirety of the length of the nerve cell (the axon) and cause a chemical signal to be released into the synaptic cleft to get to its target. Propagating the nerve signal (action potential) in the original nerve cell is the function of all of the rest of the gated ion channels (Figure 3.43) positioned on the sides of the nerve cell. The sodium and potassium gates involved in propagation of the signal all act in response to voltage changes created by the electrochemical gradient moving down the nerve cell (Figure 3.44). Remember that opening of the initial gates at initiation of the signal created an influx of sodium ions and an efflux of potassium ions. Moving signal This chemical and electrical change that creates the action potential leaves the end of the nerve cell where it started and travels down the axon towards the other end of the nerve cell. Along the way, it encounters more sodium and potassium gated channels. In each case, these respond simply to the voltage change of the action potential and open and close, exactly in the same way the gates opened to start the signal. Thus, a rapid wave of increasing sodium ions and decreasing potassium ions moves along the nerve cell, propagated (and amplified) by gates opening and closing as the ions and charges move down the nerve cell. Eventually, the ionic tidal wave reaches the end of the nerve cell (axon terminal) facing the synaptic cleft. Crossing the synaptic cleft For the signal to be received by the intended target (postsynaptic cell) from the originating neuron (presynaptic neuron), it must cross the synaptic cleft and stimulate the neighboring cell (Figure 3.45). Communicating information across a synaptic cleft is the job of neurotransmitters. These are small molecules synthesized in nerve cells that are packaged in membrane vesicles called synaptic vesicles in the nerve cell. Neurotransmitters come in all shapes and chemical forms, from small chemicals like acetylcholine to peptides like neuropeptide Y. The most abundant neurotransmitter is glutamate, which acts at over 90% of the synapses in the human brain. Movie 3.2 - Movement of an action potential down a nerve cell - Wikipedia Into the cleft As the action potential in the presynaptic neuron approaches the axon terminus, synaptic vesicles begin to fuse with the membrane and their neurotransmitter contents spill into the synaptic cleft. Once in the cleft, the neurotransmitters diffuse, some of them reaching receptors on the postsynaptic cell. Binding of the neurotransmitter to the receptors on the membrane of the postsynaptic cell stimulates a response. For motor neurons, the postsynaptic cell will be a muscle cell, and the response will be muscle contraction/relaxation. At this point, the originating nerve cell has done its job and communicated its information to its immediate target. If the postsynaptic cell is a nerve cell, the process repeats in that cell until it gets to its destination. Na+/glucose transporter Absorbing nutrients from the digestive system is necessary for animal life. The sodium/glucose transport protein is an electrogenic symporter that moves glucose into intestinal cells. It is found in the intestinal mucosa and the proximal tubule of the nephron of the kidney. The sodium/glucose transport system functions in the latter to promote reabsorption of glucose. The pump works in conjunction with the Na+/K+ transport system. The gradient of sodium ions built up by the Na+/K+ pump is used as an energy source to drive movement of glucose into cells (see Figure 3.38). Use of an ion gradient established by a separate pump is known as secondary active transport. For intestinal mucosa, the pump transports glucose out of the gut and into gut cells. Later, the glucose is exported out the other side of the gut cells to the interstitial space for use in the body. Calcium pumps Calcium ions are necessary for muscular contraction and play important roles as signaling molecules within cells. In addition, when calcium concentrations rise too high, DNA in chromosomes can precipitate. Calcium concentration in cells is therefore managed carefully. It is kept very low in the cytoplasm as a result of action of pumps, both in the plasma membrane, which pump calcium outwards from the cytoplasm and in organelles, such as the endoplasmic reticulum (sarcoplasmic reticulum of muscle cells), which pump calcium out of the cytoplasm and into these organelles. Opening of calcium channels, then, increases calcium concentration quickly in the cytoplasm resulting in a quick response, whether the intention is signaling or contraction of a muscle. After the response is generated, the calcium is pumped back out of the cytoplasm by the respective calcium pumps. Some calcium pumps use ATP as an energy source to move calcium and others use ion gradients, such as sodium for the same purpose. Na+/Ca++ transporter One calcium pump of interest uses the sodium gradient as an energy source. It is the sodium/calcium pump. This electrogenic antiport system uses sodium’s movement into the cell as a driving force to move calcium out of the cell, although its direction can reverse in some circumstances. The pump is a high capacity system to move a lot of calcium quickly, moving up to 5000 calcium ions per second and is found in many tissues with many functions. Digitalis One important function of the Na+/Ca++ pump occurs in heart cells. Ca++ is important for contraction of heart muscle. Calcium efflux from the cells is the normal operation of the pump, however, during the upstroke of the cycle, there is a large movement of sodium ions into the heart cell. When this occurs, the pump reverses and pumps Na+ out and Ca++ in briefly. Since calcium helps stimulate contraction of cardiac muscle, this can help make the heart beat stronger and is the focus of the use of digitalis to treat congestive heart failure. Digitalis blocks the sodium-potassium ATPase and interferes with the sodium ion gradient. As noted above, when the Na+ gradient is oriented in the wrong direction, calcium is pumped in. Digitalis is therefore used to treat congestive heart failure because it increases the concentration of calcium in the heart cells, favoring more forceful beats. ABC transporters ABC transporters are another class of transmembrane proteins that use ATP energy to transport things against concentration gradients (Figures 3.47 & 3.48). This protein superfamily includes hundreds of proteins (48 in humans alone) and spans all extant phyla from prokaryotes to humans. These proteins function not only in membrane transport, but also in processes that include DNA repair and the process of translation. Transport Substances that ABC transporters move across membranes include metabolic products, lipids, sterols, and drugs. ABC transporters function in multi-drug resistance of many cells, and provide resistance to antibiotics in bacteria as well as resistance to chemotherapy in higher cells by exporting drugs used to treat both of these types of cells. ABC transporters are divided into three main groups - 1) importers (prokaryotes only); 2) exporters (prokaryotes and eukaryotes), and 3) non-transporters with roles in DNA repair and translation. All ABC transport proteins have four protein domains - two that are cytoplasmic and two that are membrane bound. They are alternately open to the cytoplasmic or extracellular (or periplasmic) regions and this is controlled by hydrolysis of ATP. Disease ABC transporters have roles in cystic fibrosis and other inherited human diseases. They are very involved in development of resistance to multiple drugs by a diverse group of cells. ABC transporters provide multi-drug resistance by expelling drug(s) from cells. ABCB1 protein, for example, pumps tumor suppression drugs out of the cell. Another ABC transporter known as Pgp transports organic cationic or neutral compounds. Cystic fibrosis Cystic fibrosis (CF) is a an autosomal recessive genetic disorder arising from mutations in both copies of the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. This ABC transporter system, which moves chloride and thiocyanate ions across epithelial tissue membranes exerts its effect mostly in the lungs but the pancreas, liver, kidneys, and intestine are all also affected by it. Function CFTR has roles in the production of sweat, mucus, and digestive fluids. Manifestations of the disease include breathing difficulty and overproduction of mucus in the lungs. When CFTR is functional, these fluids are normally thin, but when the gene is non-functional, they become much thicker and are points of infection. CFTR contains two ATP-hydrolyzing domains and two cell membrane-crossing domains with 6 α-helices each. It can be activated by phosphorylation by a cAMP-dependent protein kinase. The carboxyl end of CFTR is linked to the cytoskeleton by a PDZ domain. Lactose permease Another integral membrane protein performing active transport is lactose permease. It facilitates the movement of the sugar lactose across the lipid bilayer of the cell membrane (Figures 3.49- 3.51). The transport mechanism is classified as a secondary active transport since it exploits the inwardly directed H+ electrochemical gradient as an energy source. When lactose is transported into cells, it is broken down into its substituent monosaccharide sugars - glucose and galactose - for energy creation. The enzyme catalyzing this reaction is known as lactase and deficiency of it in humans leads to lactose intolerance (see HERE). GLUTs GLUTs (GLUcose Transport proteins) are uniport, type III integral membrane proteins that participate in the transport of glucose across membranes into cells. GLUTs are found in all phyla and are abundant in humans, with 12 GLUT genes. GLUT1, in erythrocytes is well-studied. Through GLUT 1, glucose enters and passes through it via facilitated diffusion at a rate that is 50,000 higher than in its absence. GLUTs of various types are found in different cells of the body. The one in red blood cells is known as GLUT 1 and has 12 membrane-spanning hydrophobic helices. Though the structure of GLUT 1 is not known, it is speculated that the 12 helices form a chamber able to form hydrophilic bonds with glucose to facilitate its passage. GLUT 1 levels in erythrocytes go up as glucose levels decrease and decrease when glucose levels go down. GLUT 1 can also transport ascorbate (vitamin C) in addition to glucose in mammals (such as humans) that do not produce their own vitamin C. Glut 4 GLUT 4 is regulated by insulin and is found primarily in adipose and striated muscle tissue. Insulin alters intracellular trafficking pathways in response to increases in blood sugar to favor movement of various GLUT proteins (including GLUT 4) from intracellular vesicles to the cell membrane, thus stimulating uptake of the glucose. GLUT 4 is also found in the hippocampus where, if trafficking is disrupted, the result can be depressive behavior and cognitive dysfunction. For all of the GLUT proteins, a key to keeping the glucose in the cell is phosphorylation of it by the glycolysis enzyme, hexokinase, in the cytoplasm. Phosphorylated molecules cannot enter GLUTs and don’t have an easy means of exiting the cell. Distance Ed To the tune of “Mister Ed” Metabolic Melodies Website HERE A course is a source, of course, of course Of all of the knowledge that we endorse A major force for better/worse is the campus Distance Ed It’s true to outsource a college course There are a few standards to be enforced The long and short’s we reinforce the campus Distance Ed Bridge A classroom class meets every week the same time every day But Distance Ed is most unique - its flexible schedule’s okay E-course is a source, of course, of course Of online assistance for lab reports You’re not enrolled in an online course? Then sign up for this! “You’ll love Distance Ed” Recording by David Simmons Lyrics by Kevin Ahern Recording by David Simmons Lyrics by Kevin Ahern 313 It's one o'clock and​ Ahern's talkin'​ Henderson and​ Hasselbalch and​ pKa's and​ Buffers I should know​ This song's for BB three five oh I hope that maybe​ He'll think the way we​ Wrote our answers​ Wasn't crazy​ I really need the​ Partial credit - so​ This song's for BB three five oh It's really groovy​ That it improves me​ Watching lectures​ In Quicktime movies​ I really need to​ Go and download those​ Podcasts for BB three five oh This Song's For BB 3-5-0​ To the tune of "This Land is My Land" Metabolic Melodies Website HERE I'm feeling manic​ I'm in a panic​ I'd better study​ My old organic​ It has reactions​ That I need to know​ This song's for BB three five oh I know he said it​ That's why I dread it​ 'cause I skipped Friday's​ Extra credit​ 'twil pro'bly haunt me​ That lowly ze-ro​ Grade in BB three five oh It could be steric​ Or esoteric​ That carbons get so​ Anomeric​ I'm too hysteric​ Better let it go​ This song's for BB three five oh Recording by Tim Karplus Lyrics by Kevin Ahern Recording by Tim Karplus Lyrics by Kevin Ahern

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/03%3A_Membranes/3.02%3A_Transport_in_Membranes.txt

Source: BiochemFFA_3_3.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy There are many functions and factors relating to cell membranes that don’t fit into broad categories. Those items will be the focus of this section. Endocytosis Besides transporter proteins and ion channels, another common way for materials to get into cells is by the process of endocytosis. Endocytosis is an alternate form of active transport for getting materials into cells. Some of these processes, such as phagocytosis, are able to import much larger particles than would be possible via a transporter protein. Like transporter proteins, endocytosis uses energy for the purpose (though it is not as visible as with protein transporters), but unlike protein transporters, the process is not nearly as specific for individual molecules. As a result, the process usually involves the importation of many different molecules each time it occurs. The list of compounds entering cells in this way includes LDLs and their lipid contents, but it also include things like iron (packaged in transferrin), vitamins, hormones, proteins, and even some viruses sneak in by this means. There are three types of endocytosis we will consider (Figure 3.53). Receptor mediated endocytosis The process of receptor mediated endocytosis is a relatively specific means of bringing molecules into cells because it requires the incoming material to be somehow associated with a specific cell surface receptor. In the example of Figure 3.53, the receptor is the cellular LDL receptor. Clathrin-coated invaginations, as shown in the figure are known as “coated pits.” The mechanism proceeds with an inward budding of the plasma membrane receptor (coated vesicles). Binding of the ligand (ApoB-100 of the LDL, for example, in Figure 3.54) to the LDL receptor leads to formation of a membrane invagination. The absorbed LDL particle fuses to form an early endosome (Figure 3.55) and contents are subsequently sorted and processed for use by the cell. The components from the coated vesicle are recycled to the plasma membrane for additional actions. Receptor mediated endocytosis can also play a role in internalization of cellular receptors that function in the process of signaling. Here, a receptor bound to a ligand is brought into the cell and may ultimately generate a response in the nucleus. While receptor mediated endocytosis of receptors can have the effect of communicating a signal inwards to the cell, it can also reduce the total amount of signaling occurring, since the number of receptors on the cell surface is decreased by the process. Non-clathrin endocytosis There are three types of endocytosis occurring in cells that do not involve clathrin. They are 1) caveolae-based endocytosis, 2) macropinocytosis, and 3) phagocytosis. Caveolae-based endocytosis is based on a receptor molecule known as caveolin. Caveolins are a class of integral membrane proteins that compartmentalize and concentrate signaling molecules in the process of endocytosis. They are named for the cave-like caveolae structures of the plasma membrane where they are found. Caveolins Caveolins have affinity for cholesterol and associate with it in the membrane of cells, causing the formation of membrane invaginations of about 50 nm. The caveolin proteins can oligomerize and this is important for the coating and formation of the cave-like structures. There are three caveolin genes found in vertebrate cells, CAV1, CAV2, and CAV3. Down-regulation of caveolin-1 results in less efficient cellular migration in vitro. Caveolins are implicated in both formation and suppression of tumors. High expression of them inhibits cancer-related growth factor signaling pathways, but some caveolin-expressing cancer cells are more aggressive and metastatic, possible due to an enhanced capacity for anchorage-independent growth. Macropinocytosis A phenomenon known as “cell drinking,” macropinocytosis literally involves a cell “taking a gulp” of the extracellular fluid. It does this, as shown in Figure 3.56, by a simple invagination of ruffled surface features of the plasma membrane. A pocket results, which pinches off internally to create a vesicle containing extracellular fluid and dissolved molecules. Within the cytosol, this internalized vesicle will fuse with endosomes and lysosomes. The process is non-specific for materials internalized. Phagocytosis Phagocytosis is a process whereby relatively large particles (0.75 µm in diameter) are intenalized. Cells of the immune system, such as neutrophils, macrophages, and others, use phagocytosis to internalize cell debris, apoptotic cells, and microorganisms. The process operates through specific receptors on the surface of the cell and phagocytosing cell engulfs its target by growing around it. The internalized structure is known as a phagosome, which quickly merges with a lysosome to create a phagolysosome (Figure 3.58), which subjects the engulfed particle to toxic conditions to kill it, if it is a cell, and/or to digest it into smaller pieces. In some cases, as shown in the figure, soluble debris may be released by the phagocytosing cell. Endosomes Internalized material from endocytosis that doesn’t involve phagocytosis passes through an internalized structure called an endosome. Endosomes are membrane bounded structures inside of eukaryotic cells that play a role in endocytosis (Figure 3.59). They have a sorting function for material internalized into the cell, providing for retrieval of materials not destined for destruction in the lysosomes. LDLs, for example, are targeted after endocytosis to the endosomes for processing before part of them is delivered to the lysosome. The endosomes can also receive molecules from the trans-Golgi network. These can be delivered to the lysosomes, as well, or redirected back to the Golgi. Endosomes come in three forms - 1) early, 2) late, and 3) recycling. Figure 3.59 - Internalization of the epidermal growth factor receptor (EGFR) into endosomes. Early (E) and late (M) endosomes and lysosomes (L) are labeled. - Wikipedia Exocytosis The process of exocytosis is used by cells to export molecules out of cells that would not otherwise pass easily through the plasma membrane. In the process, secretory vesicles fuse with the plasma membrane and release their contents extracellularly. Materials, such as proteins and lipids embedded in the membranes of the vesicles become a part of the plasma membrane when fusion between it and the vesicles occurs. Membrane fusion Fusion is a membrane process where two distinct lipid bilayers merge their hydrophobic cores, producing one interconnected structure. Membrane fusion involving vesicles is the mechanism by which the processes of endocytosis and exocytosis occur. When the fusion proceeds through both leaflets of both bilayers, an aqueous bridge results and the contents of the two structures mix. Common processes involving membrane fusion (Figure 3.60) include fertilization of an egg by a sperm, separation of membranes in cell division, transport of waste products, and neurotransmitter release (Figure 3.61). Artificial membranes such as liposomes can also fuse with cellular membranes. Fusion is also important for transporting lipids from the point of synthesis inside the cell to the membrane where they are used. Entry of pathogens can also be governed by fusion, as many bilayer-coated viruses use fusion proteins in entering host cells. SNARE proteins Mediation of fusion of vesicles in exocytosis is carried out by proteins known as SNAREs (Soluble NSF Attachment Protein REceptor). This large superfamily of proteins spans a wide biological range, from yeast to mammals. Common vesicle fusions occur when synaptic vesicles dock with neurons (Figure 3.61) and release neurotransmitters. These are well-studied. The SNAREs involved in this process can be proteolytically cleaved by bacterial neurotoxins that give rise to the conditions of botulism and tetanus. SNAREs are found in two locations. v-SNAREs are found in the membranes of transport vesicles during the budding process, whereas t-SNARES can be found in the membranes of targeted compartments. The act of vesicle fusion coincides with increases of intracellular calcium. Fusion of synaptic vesicles in neurotransmission results in activation of voltage-dependent calcium channels in the targeted cell. Influx of calcium helps to stimulate vesicle fusion. In the endocrine system, binding of hormones to G protein coupled receptors activate the IP3/DAG system to increase levels of calcium. In the process of membrane fusion (Figure 3.62), the v-SNAREs of a secretory vesicle (upper left) interact with the t-SNAREs of a target membrane (bottom). The v- and t-SNAREs “zipper” themselves together to bring the membrane vesicle and the target membrane closer together. Zippering also causes flattening and lateral tension of the curved membrane surfaces, favoring hemifusion of the outer layers of each membrane. Continued tension results in subsequent fusion of the inner membranes as well, yielding opening of the contents of the vesicle chamber to its target (usually outside the cell). Shuttles Another way to transport items across a membrane for which there is no specific transport system available is the use of shuttles. Shuttles are important when there is no transport mechanism for moving material across a membrane for which no transport system exists. A great example is NADH. NADH is an important electron carrier that is produced in the cytoplasm and mitochondria of the cell. NADH produced in the mitochondrion goes directly to the electron transport system and delivers electrons to Complex I. NADH produced in the cytoplasm (such as from glycolysis) does not have this option, since the inner membrane of the mitochondrion is impermeable to the molecule and no transporter exists to move it across. The important part of the NADH is its electron cargo, so cells have evolved two ways to move the electrons into the mitochondrial matrix apart from NADH. Both methods involve shuttles. In each case, an acceptor molecule receives electrons from NADH and the reduced form of the acceptor molecule is transported. It gets transported into the matrix where it is oxidized (electrons are lost) and then donated to the electron transport system. Glycerol phosphate shuttle The first of these methods is the least efficient, but it is rapid. It found commonly in muscles which have needs for rapid energy and brain tissue. This shuttle is referred to as the glycerol phosphate shuttle and is shown in Figure 3.63. It operates in the intermembrane space between the inner and outer mitochondrial membranes. The outer mitochondrial membrane is very porous, allowing many materials to pass freely through it. In the intermembrane space, the cytoplasmic enzyme, glyceraldehyde-3-phosphate dehydrogenase (cGPD) catalyzes transfer of electrons from NADH to dihdydroxyacetone phosphate (#2 in the figure), yielding NAD+ and glyceraldehyde-3-phosphate (#1 in the figure). The glyceraldehyde-3-phosphate then binds to a glyceraldehyde-3-phosphate dehydrogenase (mGPD) embedded in the outer portion of the inner mitochondrial membrane. mGPD catalyzes the transfer of electrons from glyceraldehyde-3-phosphate to FAD, producing dihycroxyacetone phosphate and FADH2. FADH2 then transfers its electrons to the electron transport system through CoQ (Q above), forming CoQH2 (QH2 above). As will be discussed in the section on electron transport, this is not an efficient shuttle system because it does not result in production of as much ATP as occurs when electrons are transferred to NAD+ instead of FAD. Malate-aspartate shuttle A more efficient system of transferring electrons is the malate-aspartate shuttle and it is shown in Figure 3.64. As is apparent in the figure, this shuttle involves more steps than the glycerol phosphate shuttle, but the advantage of the malate-aspartate shuttle is that it is more efficient. NADH outside of the mitochondrion transfers its electrons to the shuttle and then NADH is re-made on the inside of the shuttle. No energy is expended in the process. When NADH accumulates in the cytoplasm, it moves to the intermembrane space where the enzyme malate dehydrogenase catalyzes the transfer of electrons to oxaloacetate to yield NAD+ and malate. A transport system for malate moves malate into the mitochondrial matrix in exchange for α-ketoglutarate. Inside the mitochondrion, malate is reoxidized to oxaloacetate and electrons are given to NAD+ to recreate NADH. NADH then donates electrons to Complex I of the electron transport system. That’s really all there is to the shuttle. The remaining steps are simply to balance the equation of the process. Oxaloacetate accepts an amine group from glutamic acid to yield aspartic acid and α-ketoglutarate. Aspartate then moves out of the mitochondrion through an antiport transport protein that swaps it for glutamate. A series of reactions in the intermembrane space balance the equation. It is easy to get lost in the mess of balancing equations. The most important thing to understand here is that oxaloacetate accepts electrons on the outside to become malate which is the carrier of electrons across the membrane. Once inside the matrix, malate is converted back to oxaloacetate and its electrons are given to NAD+, forming NADH. Everything else is simple equation balancing. Acetyl-CoA shuttle Another kind of shuttle also involves the mitochondrion and in this case, the item being moved is a molecule, not a pair of electrons. The molecule of interest here is acetyl-CoA, for which no transport system operates, but which is needed in the cytoplasm for fatty acid synthesis when the cell has abundant energy. Acetyl-CoA is mostly in the mitochondrion and so long as the citric acid cycle is operating efficiently, its concentration is relatively stable. However, when the citric acid cycle slows, acetyl-CoA and the citrate made from it in the cycle begin to accumulate. A membrane transport system for citrate exists, so it gets moved out to the cytoplasm. In the cytoplasm, an enzyme known as citrate lyase cleaves citrate to acetyl-CoA and oxaloacetate. Oxaloacetate can be reduced to malate and moved back into the mitochondrion. As for acetyl-CoA, the more of that cells have in the cytoplasm, the more likely they will begin making fatty acids and fat, since acetyl-CoA is the starting material for fatty acid synthesis, which occurs in the cytoplasm. When does this process occur? As noted above, it occurs when the citric acid cycle stops and this occurs when levels of NADH and FADH2 increase. These, of course, increase when one is not burning off as many calories as one is consuming as a byproduct of respiratory control. Lack of exercise leads to higher levels of reduced electron carriers. Cell junctions Cells in multicellular organisms are in close contact with each other and links between them are called junctions. In vertebrate organisms, there are three main types of cell junctions and one of them (gap junctions) is important for movement of materials between cells. The three types are 1. Gap junctions 2. Adherens junctions, (Anchoring Junctions, desmosomes and hemidesmosomes) 3. Tight junctions Cell junctions in multicellular plants are structured differently from those in vertebrates and are called plasmodesmata. They too function in exchange of materials between individual cells. Gap junctions Gap junctions are specialized structures made up of two sets of structures called connexons (one from each cell - see Figure 3.65) directly link the cytoplasms of the connected cells. Gap junctions are regulated to control the flow of molecules, ions, and electrical impulses between cells. In plants, similar structures known as plasmodesmata traverse the cell wall (Figure 3.66) and perform similar functions. Adherens junctions Adherens junctions (Figure 3.67) are protein complexes on the cytoplasmic side of the cell membranes of epithelial and endothelial tissues that link cells to each other or to the extracellular matrix. They correspond to the fascia adherens found in non-epithelial/non-endothelial cells. Adherens junctions contain the following proteins - 1) α-catenin (binds cadherin through β-catenin); 2) β-catenin (attachment for α-catenin to cadherin; 3) γ-catenin (binds to cadherin); 4) cadherins (group of transmembrane proteins that dimerize with cadherins on adjacent cells; 5) p120 (also called Δ-catenin - binds to cadherin); 6) plakoglobin (catenin family protein homologous to and acting like β-catenin); 7) actin; 8) actinin; and 9) vinculin. Adherens junctions may help to maintain the actin contractile ring which forms in the process of cytokinesis. Tight junctions Tight junctions (Figure 3.68) are a network of protein strands that seal cells together and restrict the flow of ions in the spaces between them. The effect of their structure is to restrict the movement of materials through tissues by requiring them to pass through cells instead of around them. Tight junctions join together the cytoskeletons of cells and through their structure maintain their apical and basolateral polarity. GPI anchors Membrane proteins attached to glycosylphosphatidylinositol (also known as a GPI anchor) are referred to as being glypiated. The proteins, which play important roles in many biochemical processes, are attached to the GPI anchor at their carboxyl terminus. Phospholipases, such as phospholipase C can cut the bond between the protein and the GPI, freeing the protein from the outer cell membrane. Proteins destined to be glypiated have two signal sequences. They are 1) An N-terminal signal sequence and 2) A C-terminal signal sequence that is recognized by a GPI transamidase (GPIT). The N-terminal signal sequences is responsible for directing co-translational transport into the endoplasmic reticulum. The C-terminal sequence is recognized by GPI transamidase, which links the carboxy terminus of a protein to the GPI anchor. Liposomes The spontaneous ability of phosphoglycerolipid and sphingolipid compounds to form lipid bilayers is exploited in the formation of artificial membranous structures called liposomes (Figure 3.69). Liposomes are useful for delivering their contents into cells via membrane fusion. In the figure, items targeted for delivery to cells would be encased in the middle circular region of the liposome and when the liposome fuses with the cell membrane, it will deliver the contents directly into the cytoplasm. Hydropathy index The interior portion of the lipid bilayer is very hydrophobic, which makes it very restrictive to movement of ions and polar substances across it. This property also places limitations on the types of amino acids that will interact with it as well. Because of this, transmembrane protein domains found in integral membrane proteins are biased in the amino acids that interact with either the lipid bilayer or the aqueous material on either side of it. Hydrophobic amino acids are found within the bilayer, whereas hydrophilic amino acids are found predominantly on the surfaces. An additional clue to identifying membrane crossing regions of a protein is that tryptophan or tyrosine is commonly positioned at non-polar/polar interface(s) of the lipid bilayer for integral proteins. Such an organization of amino acids can be recognized by computer analysis of amino acid sequences using what is called a hydropathy index/score (Figure 3.71). Though the names and the scorings vary, the idea is to assign a number (usually positive) to amino acid side chains with higher hydrophobicity and negative to those that are ionic. With these scores, a computer program can easily find the average scores of short amino acid segments (say 3 amino acids long) and then plot those values on a graph of hydrophobicity index versus position in polypeptide chain. Doing that for a transmembrane protein such as glycophorin results in the plot shown in Figure 3.72. It is apparent in the analysis that this is a transmembrane protein that has seven domains crossing the lipid bilayer, as labeled. Cell walls Cells walls are found in many cells, including plants, fungi, and bacteria, but are not found in animal cells. They are designed to provide strength and integrity and at least some protection against bursting from osmotic pressure (Figures 3.73-3.75). Gram positive bacteria (Figure 3.75) have the simplest cell wall design. Moving from outside the cell towards the cytoplasm there is an outer peptidoglycan layer for the cell wall followed by a periplasmic space, a plasma membrane, and then the cytoplasm. Gram negative bacteria add a second protective layer external to all of this, so for them, starting at the outside and moving inwards, one encounters an outer lipopolysaccharide layer, a periplasmic space, the peptidoglycan cell wall, a second periplasmic space, a plasma membrane and then the cytoplasm. Herbaceous plants have a rigid outer cell wall (primarily composed of cellulose, hemicellulose, and pectin) and an inner plasma membrane. Woody plants add a second level of wall with lignin between the cellulosic wall and the plasma membrane of herbaceous plants. BB Wonderland To the tune of “Winter Wonderland” Metabolic Melodies Website HERE Milam Hall - It’s 12:30 And Ahern’s gettin’ wordy He walks to and fro’ While not talkin’ slow Givin’ it to B-B-4-5-0 I was happy when the term got started Lecture notes and videos galore MP3s got added to my iPod But recitations sometimes were a bore And exams bit me roughly When the curve turned out ugly I don’t think it’s so My scores are too low Slidin’ by in B-B-4-5-0 Final-LY there’s an examination On December 9th at 6:00 pm I’ll have my card packed with information So I don’t have to memorize it then And I’ll feel like a smarty With my jam-packed note-cardy Just one more to go And then ho-ho-ho I’ll be done with B-B-4-5-0 Recording by David Simmons Lyrics by Kevin Ahern Recording by David Simmons Lyrics by Kevin Ahern 334 Thank God There's a Video To the tune of "Thank God I'm a Country Boy" Metabolic Melodies Website HERE There's a bundle of things a student oughta know And Ahern's talk isn't really very slow Learnin' ain't easy / the lectures kinda blow Thank God there's a video Well we’ve gone through the cycles and their enzymes too​ Studying the regulation everything is new​ I gotta admit that I haven’t got a clue​ What am I gonna do? So I got me a note card and bought me a Stryer​ Got the enzymes down and the names he requires​ I hope that I can muster up a little more desire​ Thank God there's a video Just got up to speed about the N-A-D​ Protons moving through Complex Vee​ Electrons dance in the cytochrome C​ Gotta hear the MP3 Fatty acid oxidation makes acetyl-CoA​ Inside the inner matrix of the mitochondri-ay​ It's very complicated, I guess I gotta say​ Thank God there's a video So I got me a note card and bought me a Stryer​ Got the enzymes down and the names he requires​ I hope that I can muster up a little more desire​ Thank God there's a video Replication's kind of easy in a simple kind of way​ Copyin' the bases in the plasmid DNAs​ Gs goes with Cs and Ts go with As​ Thanks to polymerase And the DNA's a template for the RNA​ Helices unwinding at T-A-T-A​ Termination happens, then the enzyme goes away​ Don't forget the poly-A So I got me a note card and bought me a Stryer​ Got the enzymes down and the names he requires​ I think that I can muster up a little more desire​ Thank God there's a video Recording by David Simmons Lyrics by Kevin Ahern Recording by David Simmons Lyrics by Kevin Ahern

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/03%3A_Membranes/3.03%3A_Other_Considerations_in_Membranes.txt

Thumbnail: Enzyme changes shape by induced fit upon substrate binding to form enzyme-substrate complex. Hexokinase has a large induced fit motion that closes over the substrates adenosine triphosphate and xylose. Binding sites in blue, substrates in black and Mg2+ cofactor in yellow. (PDB: 2E2N​, 2E2Q​). Image used with permission (CC BY 4.0l Thomas Shafee) 04: Catalysis A printable version of this section is here: BiochemFFA_4_1.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy If there is a magical component to life, an argument can surely be made for it being catalysis. Thanks to catalysis, reactions that can take hundreds of years to complete in the uncatalyzed “real world,” occur in seconds in the presence of a catalyst. Chemical catalysts, such as platinum, can speed reactions, but enzymes (which are simply super-catalysts with a “twist,” as we shall see) put chemical catalysts to shame (Figure 4.1). To understand enzymatic catalysis, it is necessary first to understand energy. Chemical reactions follow the universal trend of moving towards lower energy, but they often have a barrier in place that must be overcome. The secret to catalytic action is reducing the magnitude of that barrier. Before discussing enzymes, it is appropriate to pause and discuss an important concept relating to chemical/biochemical reactions. That concept is equilibrium and it is very often misunderstood. The “equi" part of the word relates to equal, as one might expect, but it does not relate to absolute concentrations. What happens when a biochemical reaction is at equilibrium is that the concentrations of reactants and products do not change over time. This does not mean that the reactions have stopped. Remember that reactions are reversible, so there is a forward reaction and a reverse reaction: if you had 8 molecules of A, and 4 of B at the beginning, and 2 molecules of A were converted to B, while 2 molecules of B were simultaneously converted back to A, the number of molecules of A and B remain unchanged, i.e., the reaction is at equilibrium. However, you will notice that this does not mean that there are equal numbers of A and B molecules. Concentration Matters So, contrary to the perceptions of many students, the concentrations of products and reactants are not equal at equilibrium, unless the ΔG°’ for a reaction is zero, because when this is the case, $ΔG = \ln \left(\dfrac{[\rm{Products}]}{[\rm{Reactants}]} \right)$ since the ΔG°’ is zero. Because ΔG itself is zero at equilibrium, then $[Products] = [Reactants].$ This is the only circumstance where $[Products] = [Reactants]$ at equilibrium. Reiterating, at equilibrium, the concentrations of reactant and product do not change over time. That is, for a reaction $A \rightleftharpoons B [A]$ at time zero when equilibrium is reached, $[A]_{T_0}$, will be the same 5 minutes later (assuming A and B are chemically stable). Thus, $[A]_{T_0} = [A]_{T+5}$ Similarly, $[B]_{T_0} = [B]_{T+5}$ For that matter, at any amount of time X after equilibrium has been reached, $[A]T0 = [A]T+5 = [A]TX$ and $[B]T0 = [B] T+5 = [B]TX$ However, unless ΔG°’ = 0, it is wrong to say [A]T0 = [B]T0 As we study biochemical reactions and reaction rates, it is important to remember that 1) reactions do not generally start at equilibrium; 2) all reactions move in the direction of equilibrium; and 3) reactions in cells behave just like those in test tubes - they do not begin at equilibrium, but they move towards it. Dynamic reactions The reactions occurring in cells, though, are very dynamic and complex. In a test tube, they can be studied one at a time. In cells, the product of one reaction is often the substrate for another one. Reactions in cells are interconnected in this way, giving rise to what are called metabolic pathways. There are, in fact, thousands of different interconnected reactions going on continuously in cells. Attempts to study a single reaction in the chaos of a cell is daunting to say the least. For this reason, biochemists isolate enzymes from cells and study reactions individually. It is with this in mind that we begin our consideration of the phenomenon of catalysis by describing, first, the way in which enzymes work. Activation energy Figure 4.2 schematically depicts the energy changes that occur during the progression of a simple reaction. In order for the reaction to proceed, an activation energy must be overcome in order for the reaction to occur. In Figure 4.3, the activation energy for a catalyzed reaction is overlaid. As you can see, the reactants start at the same energy level for both catalyzed and uncatalyzed reactions and that the products end at the same energy for both as well. The catalyzed reaction, however, has a lower energy of activation (dotted line) than the uncatalyzed reaction. This is the secret to catalysis - overall ΔG for a reaction does NOT change with catalysis, but the activation energy is lowered. Figure 4.3 - Energy changes during the course of an uncatalyzed reaction (solid green line) and a catalyzed reaction (dotted green line). Image by Aleia Kim Reversibility The extent to which reactions will proceed forward is a function of the size of the energy difference between the product and reactant states. The lower the energy of the products compared to the reactants, the larger the percentage of molecules that will be present as products at equilibrium. It is worth noting that since an enzyme lowers the activation energy for a reaction that it can speed the reversal of a reaction just as it speeds a reaction in the forward direction. At equilibrium, of course, no change in concentration of reactants and products occurs. Thus, enzymes speed the time required to reach equilibrium, but do not affect the balance of products and reactants at equilibrium. Exceptions The reversibility of enzymatic reactions is an important consideration for equilibrium, the measurement of enzyme kinetics, for Gibbs free energy, for metabolic pathways, and for physiology. There are some minor exceptions to the reversibility of reactions, though. They are related to the disappearance of a substrate or product of a reaction. Consider the first reaction below which is catalyzed by the enzyme carbonic anhydrase: $CO_2 + H_2O \rightleftharpoons H_2CO_3 \rightleftharpoons HCO_3^- + H^+$ In the forward direction, carbonic acid is produced from water and carbon dioxide. It can either remain intact in the solution or ionize to produce bicarbonate ion and a proton. In the reverse direction, water and carbon dioxide are produced. Carbon dioxide, of course, is a gas and can leave the solution and escape. When reaction molecules are removed, as they would be if carbon dioxide escaped, the reaction is pulled in the direction of the molecule being lost and reversal cannot occur unless the missing molecule is replaced. In the second reaction occurring on the right, carbonic acid (H2CO3) is “removed” by ionization. This too would limit the reaction going back to carbon dioxide in water. This last type of “removal” is what occurs in metabolic pathways. In this case, the product of one reaction (carbonic acid) is the substrate for the next (formation of bicarbonate and a proton). In the metabolic pathway of glycolysis, ten reactions are connected in this manner and reversing the process is much more complicated than if just one reaction was being considered. General mechanisms of action As noted above, enzymatically catalyzed reactions are orders of magnitude faster than uncatalyzed and chemical-catalyzed reactions. The secret of their success lies in a fundamental difference in their mechanisms of action. Every chemistry student has been taught that a catalyst speeds a reaction without being consumed by it. In other words, the catalyst ends up after a reaction just the way it started so it can catalyze other reactions, as well. Enzymes share this property, but in the middle, during the catalytic action, an enzyme is transiently changed. In fact, it is the ability of an enzyme to change that leads to its incredible efficiency as a catalyst. Changes These changes may be subtle electronic ones, more significant covalent modifications, or structural changes arising from the flexibility inherent in enzymes, but not present in chemical catalysts. Flexibility allows movement and movement facilitates alteration of electronic environments necessary for catalysis. Enzymes are, thus, much more efficient than rigid chemical catalysts as a result of their abilities to facilitate the changes necessary to optimize the catalytic process. Substrate binding Another important difference between the mechanism of action of an enzyme and a chemical catalyst is that an enzyme has binding sites that not only ‘grab’ the substrate (molecule involved in the reaction being catalyzed), but also place it in a position to be electronically induced to react, either within itself or with another substrate. The enzyme itself may play a role in the electronic induction or the induction may occur as a result of substrates being placed in very close proximity to each other. Chemical catalysts have no such ability to bind substrates and are dependent upon them colliding in the right orientation at or near their surfaces. Active site Reactions in an enzyme are catalyzed at a specific location within it known as the ‘active site’. Substrates bind at the active site and are oriented to provide access for the relevant portion of the molecule to the electronic environment of the enzyme where catalysis occurs. Enzyme flexibility As mentioned earlier, a difference between an enzyme and a chemical catalyst is that an enzyme is flexible. Its slight changes in shape (often arising from the binding of the substrate itself) help to optimally position substrates for reaction after they bind. Figure 4.5 - Lysozyme with substrate binding site (blue), active site (red) and bound substrate (black). Wikipedia Induced fit These changes in shape are explained, in part, by Koshland’s Induced Fit Model of Catalysis (Figure 4.6), which illustrates that not only do enzymes change substrates, but that substrates also transiently change enzyme structure. At the end of the catalysis, the enzyme is returned to its original state. Koshland’s model is in contrast to the Fischer Lock and Key model, which says simply that an enzyme has a fixed shape that is perfectly matched for binding its substrate(s). Enzyme flexibility also is important for control of enzyme activity. Enzymes alternate between the T (tight) state, which is a lower activity state and the R (relaxed) state, which has greater activity. Induced Fit The Koshland Induced Fit model of catalysis postulates that enzymes are flexible and change shape on binding substrate. Changes in shape help to 1) aid binding of additional substrates in reactions involving more than one substrate and/or 2) facilitate formation of an electronic environment in the enzyme that favors catalysis. This model is in contrast to the Fischer Lock and Key Model of catalysis which considers enzymes as having pre-formed substrate binding sites. Ordered binding The Koshland model is consistent with multi-substrate binding enzymes that exhibit ordered binding of substrates. For these systems, binding of the first substrate induces structural changes in the enzyme necessary for binding the second substrate. There is considerable experimental evidence supporting the Koshland model. Hexokinase, for example, is one of many enzymes known to undergo significant structural alteration after binding of substrate. In this case, the two substrates are brought into very close proximity by the induced fit and catalysis is made possible as a result. Reaction types Enzymes that catalyze reactions involving more than one substrate, such as $A + B \rightleftharpoons C + D$ can act in two different ways. Enzymatic reactions can be of several types, as shown in Figure 4.7. In one mechanism, called sequential reactions, at some point in the reaction, both substrates will be bound to the enzyme. There are, in turn, two different ways in which this can occur - random and ordered. Figure 4.7 - Categories of enzymatic reactions Types of Reactions Single Substrate - Single Product A ⇄ B Single Substrate - Multiple Products A ⇄ B + C Multiple Substrates - Single Products A + B ⇄ C Multiple Substrates - Multiple Products A + B ⇄ C + D Consider lactate dehydrogenase, which catalyzes the reaction below: $NADH + Pyruvate → Lactate + NAD^+$ This enzyme requires that NADH must bind prior to the binding of pyruvate. As noted earlier, this is consistent with an induced fit model of catalysis. In this case, binding of the NADH changes the enzyme shape/environment so that pyruvate can bind and without binding of NADH, the substrate cannot access the pyruvate binding site. This type of multiple substrate reaction is called sequential ordered binding, because the binding of substrates must occur in the right order for the reaction to proceed. Random binding A second mechanism of binding/catalysis is exhibited by creatine kinase which catalyzes the following reaction: $Creatine + ATP → \text{Creatine phosphate} + ADP$ For this enzyme, substrates can bind to it in any order. Creatine kinase displays sequential random binding. It is worth mentioning that random binding is not inconsistent with Koshland’s induced fit model. Rather, random binding simply means that the enzyme’s induced fit doesn’t affect substrate binding sites and involves other parts of the enzyme. In summary, sequential binding can occur as ordered binding or as random binding. Double displacement reactions Not all enzymes that catalyze multi-substrate reactions, though, bind A and B by the sequential mechanisms above. This other category of enzyme includes those that exhibit what are called “ping-pong” (or double displacement) mechanisms. In these enzymes, the enzyme functions as both a catalyst and a carrier of a group between individually bound substrates. Examples of this type of enzyme include the class of enzymes known as transaminases. A general form of the reactions catalyzed by these enzymes is shown in Figure 4.8. In reversible transaminase reactions, an oxygen and an amine are swapping between the molecules. It can be represented as follows (where N is the amine and O is the oxygen). A=O + C=N ⇄ B-N + D=O This reaction occurs not as one transfer reaction swapping the N and the O, but rather as a set of two half-reactions. In this case, the enzyme is a both donor and a carrier of the group being swapped. The first half-reaction goes as follows A=O + Enz-N ⇄ B-N + Enz=O Next a second half-reaction goes as C-N + Enz=O ⇄ D=O + Enz=N The sum of these half-reactions then is A=O + C=N ⇄ B-N + D=O Note that at no time did the enzyme bind both A and C simultaneously. It is also important to recognize that the enzyme existed in two states - Enz=O and Enz-N. The shuffling of the enzyme between these two states is what gives rise to the ping-pong name of this mechanism - it literally goes back and forth like a ping-pong ball in a table tennis match. Enzyme kinetics To understand how an enzyme enhances the rate of a reaction, we must understand enzyme kinetics. We present a model here proposed by Leonor Michaelis and Maud Menten. In order to understand the model, it is necessary to understand a few parameters. First, we describe a reaction in simple terms proceeding as follows E + S ⇄ ES -> E + P where E is enzyme, S is substrate, and P is product. In this scheme, ES is the Enzyme-Substrate complex, which is simply the enzyme bound to its substrate. We could define the ES state a bit further with E + S ⇄ ES -> ES* -> EP -> E + P where ES* is the activated state and EP is the enzyme-product complex before release of the product. The first consideration we have is velocity. The velocity of a reaction is the rate of creation of product over time, measured as the concentration of product per time. The time is a critical consideration when measuring velocity. In a closed system (in which an enzyme operates), all reactions will advance towards equilibrium. Enzymatically catalyzed reactions are no different in the end result from non-enzymatic reactions, except that they get to equilibrium faster. Equilibrium At equilibrium, the ratio of product to reactant does not change. That is a property of equilibrium. Since the system is closed, the concentration of product over time will not change. The velocity will thus be zero under these conditions and we will have learned nothing about the reaction if we wait too long to study it. Velocity Consequently, in Michaelis-Menten kinetics, velocity is measured as initial velocity (V0). This is accomplished by measuring the rate of formation of product early in the reaction before equilibrium is established and under these conditions, there is very little if any of the reverse reaction occurring. The other two assumptions are related. First, we use conditions where there is much more substrate than enzyme. This makes sense. If the substrate is not in great excess, then the enzyme’s conversion of substrate to product will occur much faster than the enzyme can bind substrate. Waiting for substrate Thus, the enzyme would “wait” for substrate to bind if there were not sufficient amounts of it to bind to the enzyme in a timely fashion (when substrate concentration is low). This would not give an accurate measure of velocity, since the enzyme would be inactive a good deal of the time. Because of this, we assume saturation of the enzyme with substrate will give a maximal velocity of the reaction. Steady state Figure 4.17 - Steady state versus non-steady state conditions Last, the high concentration of substrate combined with measuring initial conditions results in studying reactions that are under so-called steady state conditions (Figure 4.17). When steady state occurs, the concentration of the ES complex over time is not significantly changing during the period of analysis. Reiterating, the three assumptions for Michaelis-Menten kinetics are 1. Measurement of initial velocity of a reaction 2. Substrate in great excess compared to enzyme 3. Reaction conditions occurring under steady state Experimental considerations Now we turn our attention to how studies of the kinetic properties of an enzyme are conducted. To perform an analysis, one would do the following experiment - 20 different tubes would be set up with enzyme buffer (to keep the enzyme stable), the same amount of enzyme, and then a different amount of substrate in each tube, ranging from tiny amounts in the first tubes to very large amounts in the last tubes. The reaction would be allowed to proceed for a fixed, short amount of time and then the reaction would be stopped and the amount of product contained in each tube would be determined. The initial velocity (V0) of the reaction then would be the concentration of product found in each tube divided by the time that the reaction was allowed to run. Data from the experiment would be plotted on a graph using initial velocity (V0) on the Y-axis and the concentration of substrate on the X-axis, each tube, of course having a unique reaction velocity corresponding to a unique substrate concentration. For an enzyme following Michaelis-Menten kinetics, a curve like that shown in Figure 4.18 or 4.19 would result. At low concentration of substrate, it is limiting and the enzyme converts it into product as soon as it can bind it. Consequently, at low concentrations of substrate, the rate of increase of [P] is almost linear with [S] (Figure 4.19). Figure 4.19 - Linear relationship between [P] and [S] at low [S] Non-linear increase As the substrate concentration increases, however, the velocity of the reaction in tubes with higher substrate concentration ceases to increase linearly and instead begins to flatten out, indicating that as the substrate concentration gets higher and higher, the enzyme has a harder time keeping up to convert the substrate to product. Saturation Not surprisingly, when the enzyme becomes completely saturated with substrate, it will not have to wait for substrate to diffuse to it and will therefore be operating at maximum velocity. For an enzyme following Michaelis-Menten kinetics will have its velocity (v) at any given substrate concentration given by the following equation: Vmax Two terms in the equation above require explanation. The first is Vmax. It refers to the maximum velocity of an enzymatic reaction. Maximum velocity for a reaction occurs when an enzyme is saturated with substrate. Saturation is important because it means (per the assumption above) that none of the enzyme molecules are “waiting” for substrate after a product is released. Saturation ensures that another substrate is always instantly available. The unit of Vmax is concentration of product per time = [P]/time. On a plot of initial velocity versus substrate concentration (V0 vs. [S]), Vmax is the value on the Y axis that the curve asymptotically approaches (dotted line in Figure 4.20). It should be noted that the value of Vmax depends on the amount of enzyme used in a reaction. If you double the amount of enzyme used, you will double the Vmax. If one wanted to compare the velocities of two different enzymes, it would be necessary to use the same amounts of enzyme in the reaction each one catalyzes. Km The second term is Km (also known as Ks). Referred to as the Michaelis constant, Km is the substrate concentration that causes the enzyme to work at half of maximum velocity (Vmax/2). What it measures, in simple terms, is the affinity an enzyme has for its substrate. The value of Km is inversely related to the affinity of the enzyme for its substrate. Enzymes with a high Km value will have a lower affinity for their substrate (will take more substrate to get to Vmax/2) whereas those with a low Km will have high affinity and take less substrate to get to Vmax/2. The unit of Km is concentration. Affinities of enzymes for substrates vary considerably, so knowing Km helps us to understand how well an enzyme is suited to the substrate being used. Measurement of Km depends on the measurement of Vmax. Common mistake A common mistake students make in describing Vmax is saying that Km = Vmax /2. This is, of course, not true. Km is a substrate concentration and is the amount of substrate it takes for an enzyme to reach Vmax /2. On the other hand Vmax /2 is a velocity and a velocity certainly cannot equal a concentration. Kcat It is desirable to have a measure of velocity that is independent of enzyme concentration. Remember that Vmax depended on the amount of enzyme used. For this, we use the Kcat, also known as the turnover number. Kcat is a number that requires one to first determine Vmax for an enzyme and then divide the Vmax by the concentration of enzyme used to determine Vmax. Thus, Kcat = Vmax /[Enzyme] Since Vmax has units of concentration per time and [Enzyme] has units of concentration, the units on Kcat are time-1. While that might seem unintuitive, it means that the value of Kcat is the number of molecules of product made by each molecule of enzyme in the time given. So, a Kcat value of 1000/sec means each enzyme molecule in the reaction at Vmax is producing 1000 molecules of product per second. Note that since Kcat is a calculated value, it cannot be read from a V vs [S] graph as Vmax and Km can. Amazing Kcat values A Kcat value of 1000 molecules of product per enzyme per second might seem like a high value, but there are enzymes known (carbonic anhydrase, for example) that have a Kcat value of over 600,000/second (Figure 4.21). This astonishing value illustrates clearly why enzymes seem almost magical in their action. In contrast to $V_{max}$, which varies with the amount of enzyme used, Kcat is a constant for an enzyme under given conditions. As seen earlier, enzymes that follow Michaelis-Menten kinetics produce hyperbolic plots of Velocity (V0) versus Substrate Concentration [S] (Figure 4.18). Not all enzymes, though, follow Michaelis-Menten kinetics. Many enzymes have multiple protein subunits and these sometimes interact differently upon binding of a substrate or an external molecule. See ATCase (HERE) for an example. Perfect enzymes Now, if we think about what an ideal enzyme might be, it would be one that has a very high velocity and a very high affinity for its substrate. That is, it wouldn’t take much substrate to get to Vmax/2 and the Kcat would be very high. Such enzymes would have values of Kcat / Km that are maximum. Interestingly, there are several enzymes that have this property and their maximal Kcat / Km values are all approximately the same. Such enzymes are referred to as being “perfect” because they have reached the maximum possible value. Figure 4.22 - Kcat/Km values for perfect enzymes. Image by Aleia Kim Diffusion limitation Why should there be a maximum possible value of Kcat / Km? The answer is that movement of substrate to the enzyme becomes the limiting factor for perfect enzymes. Movement of substrate by diffusion in water has a fixed rate at any temperature and that limitation ultimately determines the maximum speed an enzyme can catalyze at. In a macroscopic world analogy, factories can’t make products faster than suppliers can deliver materials. It is safe to say for a perfect enzyme that the only speed limit it has is the rate of substrate diffusion in water. Given the “magic” of enzymes alluded to earlier, it might seem that all enzymes should have evolved to be “perfect.” There are very good reasons why most of them have not. Speed Speed is a dangerous thing. The faster a reaction proceeds in catalysis by an enzyme, the harder it is to control. As we all know from learning to drive, speeding causes accidents. Just as drivers need to have speed limits for operating automobiles, so too must cells exert some control on the ‘throttle’ of their enzymes. In view of this, one might wonder then why any cells have evolved any enzymes to perfection. There is no single answer to the question, but a common one is illustrated by triose phosphate isomerase, which catalyzes a reaction in glycolysis shown in Figure 4.24. The enzyme appears to have evolved this ability because at lower velocities, there is breakdown of an unstable enediol intermediate that then readily forms methyl glyoxal, a cytotoxic compound (Figure 4.25). Speeding up the reaction provides less opportunity for the unstable intermediate to accumulate and fewer undesirable byproducts to be made. Dissociation constant In studying proteins and ligands, it is important to understand the “tightness” with which a protein (P) “holds onto” a ligand (L). This is measured with the dissociation constant ($K_d$). The formation of a ligand-protein complex $LP$ occurs as $L + P \rightleftharpoons LP$ The dissociation of the complex, therefore, is the reverse of this reaction, or $LP \rightleftharpoons L + P$ so the corresponding dissociation constant is defined as $K_d = \dfrac{[L][P]}{[LP]}$ where $[L]$, $[P]$, and $[LP]$ are the molar concentrations of the protein, ligand and the complex when they are joined together. Smaller values of $K_d$ indicate tight binding, whereas larger values indicate loose binding. The dissociation constant is the inverse of the association constant. $K_a = \dfrac{1}{K_d}$ Where multiple molecules bond together, such as $J_xK_y \rightleftharpoons xJ + yK$ The complex $J_xK_y$ is breaking down into $x$ subunits of $J$ and $y$ subunits of $K$. The dissociation constant is then defined as $K_d = \dfrac{[J]^x[K]^y}{[J_xK_y]}$ where $[J]$, $[K]$, and $[J_xK_y]$ are the concentrations of J, K, and the complex $J_xK_y$, respectively. Lineweaver-Burk plots The study of enzyme kinetics is typically the most math intensive component of biochemistry and one of the most daunting aspects of the subject for many students. Although attempts are made to simplify the mathematical considerations, sometimes they only serve to confuse or frustrate students. Such is the case with modified enzyme plots, such as Lineweaver-Burk (Figure 4.26). Indeed, when presented by professors as simply another thing to memorize, who can blame students? In reality, both of these plots are aimed at simplifying the determination of parameters, such as $K_m$ and $V_{max}$. In making either of these modified plots, it is important to recognize that the same data is used as in making a V0 vs. [S] plot. The data are simply manipulated to make the plotting easier. Figure 4.26 - A Lineweaver-Burk plot of $1/V_0$ vs $1/[S]$. Image by Aleia Kim Double reciprocal For a LineWeaver-Burk plot, the manipulation is using the reciprocal of the values of both the velocity and the substrate concentration. The inverted values are then plotted on a graph as 1/V0 vs. 1/[S]. Because of these inversions, Lineweaver-Burk plots are commonly referred to as ‘double-reciprocal’ plots. As can be seen in Figure 4.26, the value of Km on a Lineweaver Burk plot is easily determined as the negative reciprocal of the x-intercept , whereas the Vmax is the inverse of the y-intercept. Other related manipulation of kinetic data include Eadie-Hofstee diagrams, which plots V0 vs V0/[S] and gives Vmax as the Y-axis intercept with the slope of the line being -Km. Molecularity of reactions The term molecularity refers to the number of molecules that must come together in order for a reaction to take place. Reactions of the sort of A -> B (where ‘A’ is the reactant and ‘B’ is the product) are unimolecular, since A is directly changed into B. The rate of the reaction is related only to the concentration of reactant A. For a bimolecular reaction where A + B ⇄ C the reaction depends on the concentration of both A and B and its rate will be related to the product of the concentration of A and of B. Coenzymes Organic molecules that assist enzymes and facilitate catalysis are co-factors called coenzymes. The term co-factor is a broad category usually subdivided into inorganic ions and coenzymes. If the coenzyme is very tightly or covalently bound to the enzyme, it is referred to as a prosthetic group. Enzymes without their co-factors are inactive and referred to as apoenzymes. Enzymes containing all of their co-factors are called holoenzymes. Pre-steady state kinetic studies In the study of kinetic rates of enzymatic reactions, time zero is a very critical point. It establishes when the mixing of substrate with enzyme and measurement of formation of product begins. At time zero, there is no product. As shown in Figure 4.29, the appearance of product (on a short time scale) goes through an early burst phase with a steep slope for [product]/time and then changes. Figure 4.29 - Burst phase of product formation This change occurs during a critical period in an enzymatic reaction and gives information about the rate of reaction cycles. The duration of the burst phase tells how long a single reaction turnover occurs, whereas the slow of the line post-burst phase tells the amount of “functional” enzyme performing the reaction. After the burst phase, the slope of the line of the amount of product versus time decreases. This is due to the reaction entering conditions of steady state, used to study Michaelis-Menten kinetics. In steady state conditions, the amount of the enzyme-substrate complex (ES) is relatively constant over time. In simple terms, this occurs when the rate of formation of the ES complex equals the rate of conversion of the substrate to product by the enzyme with release of the product. Earlier events Events occurring prior to the conditions of steady state are referred to as pre-steady state. Depending on the enzyme, in as short as a few milliseconds, steady state conditions can be present meaning that if one hopes to study formation of reaction intermediates in pre-steady state, tools for this analysis must work very rapidly. One instrument commonly used for studying pre-steady state kinetics is called a stopped flow instrument. It loads an enzyme solution and a substrate into separate syringes whose output is pointed into a mixing chamber. The solutions are rapidly mixed and measurements of product concentration begin. With a stopped flow instrument, dead times (time between mixing and detection) can be achieved of as small as 0.3 msec. Ribozymes Proteins do not have a monopoly on acting as biological catalysts. Some RNA molecules are also capable of speeding reactions. The most famous of these molecules was discovered by Tom Cech in the early 1980s Studying excision of an intron in Tetrahymena, Cech was puzzled at his inability to find any proteins catalyzing the process. Ultimately, the catalysis was recognized as coming from the intron itself. It was a self-splicing RNA and since then, many other examples of catalytic RNAs have been found. Catalytic RNA molecules are known as ribozymes. Not unusual Ribozymes, however, are not rarities of nature. The protein-making ribosomes of cells are essentially giant ribozymes. The 23S rRNA of the prokaryotic ribosome and the 28S rRNA of the eukaryotic ribosome catalyze the formation of peptide bonds. Ribozymes are also important in our understanding of the evolution of life on Earth. They have been shown to be capable via selection to evolve self-replication. Indeed, ribozymes actually answer a chicken/egg dilemma - which came first, enzymes that do the work of the cell or nucleic acids that carry the information required to produce the enzymes. As both carriers of genetic information and catalysts, ribozymes are likely both the chicken and the egg in the origin of life.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/04%3A_Catalysis/4.01%3A_Basic_Principles_of_Catalysis.txt

A printable version of this section is here: BiochemFFA_4_2.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Regulation of enzyme activity Apart from their ability to greatly speed the rates of chemical reactions in cells, enzymes have another property that makes them valuable. This property is that their activity can be regulated, allowing them to be activated and inactivated, as necessary. This is tremendously important in maintaining homeostasis, permitting cells to respond in controlled ways to changes in both internal and external conditions. Inhibition of specific enzymes by drugs can also be medically useful. Understanding the mechanisms that control enzyme activity is, therefore, of considerable importance. Inhibition We will first discuss four types of enzyme inhibition – competitive, non-competitive, uncompetitive, and suicide inhibition. Of these, the first three types are reversible. The last one, suicide inhibition, is not. Competitive inhibition Probably the easiest type of enzyme inhibition to understand is competitive inhibition and it is the one most commonly exploited pharmaceutically. Molecules that are competitive inhibitors of enzymes resemble one of the normal substrates of an enzyme. An example is methotrexate, which resembles the folate substrate of the enzyme dihydrofolate reductase (DHFR). This enzyme normally catalyzes the reduction of folate, an important reaction in the metabolism of nucleotides. Figure 4.33 - Competitive inhibitors resemble the normal substrate and compete for binding at the active site. Image by Aleia Kim Inhibitor binding When the drug methotrexate is present, some of the DHFR enzyme binds to it, instead of to folate, and during the time methotrexate is bound, the enzyme is inactive and unable to bind folate. Thus, the enzyme is inhibited. Notably, the binding site on DHFR for methotrexate is the active site, the same place that folate would normally bind. As a result, methotrexate ‘competes’ with folate for binding to the enzyme. The more methotrexate there is, the more effectively it competes with folate for the enzyme’s active site. Conversely, the more folate there is, the less of an effect methotrexate has on the enzyme because folate outcompetes it. No effect on Vmax How do we study competitive inhibition? It is typically done as follows. First, one performs a set of V0 vs. [S] reactions without inhibitor (20 or so tubes, with buffer and constant amounts of enzyme, varying amounts of substrate, equal reaction times). V0 vs. [S] is plotted (Figure 4.35 red line), as well as 1/V0 vs. 1/[S] (Figure 4.36 green line). Next, a second set of reactions is performed in the same manner as before, except that a fixed amount of the methotrexate inhibitor is added to each tube. At low concentrations of substrate, the methotrexate competes for the enzyme effectively, but at high concentrations of substrate, the inhibitor will have a much reduced effect, since the substrate outcompetes it, due to its higher concentration (remember that the inhibitor is at fixed concentration). Graphically, the results of these inhibitor experiments are shown in Figure 4.35 (blue line) and Figure 4.36 (orange line). Notice that at high substrate concentrations, the competitive inhibitor has essentially no effect, causing the \(V_{max}\) for the enzyme to remain unchanged. To reiterate, this is due to the fact that at high substrate concentrations, the inhibitor doesn’t compete well. However, at lower substrate concentrations, it does. Increased \(K_m\) In competitively inhibited reactions, the apparent Km of the enzyme for the substrate increases (\(-1/K_m\) gets closer to zero - red line in Figure 4.36) when the inhibitor is present compared to when the inhibitor is absent, thus illustrating the better competition of the inhibitor at lower substrate concentrations. It may not be obvious why we call the changed Km the apparent Km of the enzyme. The reason is that the inhibitor doesn’t actually change the enzyme’s affinity for the folate substrate. It only appears to do so. This is because of the way that competitive inhibition works. When the competitive inhibitor binds the enzyme, it is effectively ‘taken out of action.’ Inactive enzymes have NO affinity for substrate and no activity either. We can’t measure Km for an inactive enzyme. The enzyme molecules that are not bound by methotrexate can, in fact, bind folate and are active. Methotrexate has no effect on them and their Km values are unchanged. Why then, does Km appear higher in the presence of a competitive inhibitor? The reason is that the competitive inhibitor is having a greater effect of reducing the amount of active enzyme at lower concentrations of substrate than it does at higher concentrations of substrate. When the amount of enzyme is reduced, one must have more substrate to supply the reduced amount of enzyme sufficiently to get to Vmax/2. It is worth noting that in competitive inhibition, the percentage of inactive enzyme changes drastically over the range of [S] values used. To start, at low [S] values, the greatest percentage of the enzyme is inhibited. At high [S], no significant percentage of enzyme is inhibited. This is not always the case, as we shall see in non-competitive inhibition. Non-competitive inhibition A second type of inhibition employs inhibitors that do not resemble the substrate and bind not to the active site, but rather to a separate site on the enzyme (Figure 4.37). The effect of binding a non-competitive inhibitor is significantly different from binding a competitive inhibitor because there is no competition. In the case of competitive inhibition, the effect of the inhibitor could be reduced and eventually overwhelmed with increasing amounts of substrate. This was because increasing substrate made increasing percentages of the enzyme active. With non-competitive inhibition, increasing the amount of substrate has no effect on the percentage of enzyme that is active. Indeed, in non-competitive inhibition, the percentage of enzyme inhibited remains the same through all ranges of [S]. This means, then, that non-competitive inhibition effectively reduces the amount of enzyme by the same fixed amount in a typical experiment at every substrate concentration used The effect of this inhibition is shown in Figure 4.38 & 4.39. As you can see, \(V_{max}\) is reduced in non-competitive inhibition compared to uninhibited reactions. This makes sense if we remember that Vmax is dependent on the amount of enzyme present. Reducing the amount of enzyme present reduces \(V_{max}\). In competitive inhibition, this doesn’t occur detectably, because at high substrate concentrations, there is essentially 100% of the enzyme active and the \(V_{max}\) appears not to change. Additionally, Km for non-competitively inhibited reactions does not change from that of uninhibited reactions. This is because, as noted previously, one can only measure the \(K_m\) of active enzymes and \(K_m\) is a constant for a given enzyme. Uncompetitive inhibition A third type of enzymatic inhibition is that of uncompetitive inhibition, which has the odd property of a reduced Vmax as well as a reduced Km. The explanation for these seemingly odd results is rooted in the fact that the uncompetitive inhibitor binds only to the enzyme-substrate (ES) complex (Figure 4.40). The inhibitor-bound complex forms mostly under concentrations of high substrate and the ES-I complex cannot release product while the inhibitor is bound, thus explaining the reduced \(V_{max}\). The reduced Km is a bit harder to conceptualize. The reason is that the inhibitor-bound complex effectively reduces the concentration of the ES complex. By Le Chatelier’s Principle, a shift occurs to form additional ES complex, resulting in less free enzyme and more enzyme in the forms ES and ESI (ES with inhibitor). Decreases in free enzyme correspond to an enzyme with greater affinity for its substrate. Thus, paradoxically, uncompetitive inhibition both decreases \(V_{max}\) and increases an enzyme’s affinity for its substrate (\(K_m\) - Figures 4.41 & 4.42). Suicide inhibition In contrast to the first three types of inhibition, which involve reversible binding of the inhibitor to the enzyme, suicide inhibition is irreversible, because the inhibitor becomes covalently bound to the enzyme during the inhibition. Suicide inhibition rather closely resembles competitive inhibition because the inhibitor generally resembles the substrate and binds to the active site of the enzyme. The primary difference is that the suicide inhibitor is chemically reactive in the active site and makes a bond with it that precludes its removal. Such a mechanism is that employed by penicillin (Figure 4.43), which covalently links to the bacterial enzyme, DD transpeptidase and stops it from functioning. Since the normal function of the enzyme is to make a bond necessary for the peptidoglycan complex of the bacterial cell wall, the cell wall cannot properly form and bacteria cannot reproduce. Control of enzymes It is appropriate to talk at this point about mechanisms cells use to control enzymes. There are four general methods that are employed: 1. allosterism, 2. covalent modification, 3. access to substrate, and 4. control of enzyme synthesis/breakdown. Some enzymes are controlled by more than one of these methods. Allosterism The term allosterism refers to the fact that the activity of certain enzymes can be affected by the binding of small molecules. Molecules causing allosteric effects come in two classifications. Ones that are substrates for the enzymes they affect are called homotropic effectors and those that are not substrates are called heterotropic effectors. The homotropic effectors usually are activators of the enzymes they bind to and the results of their action can be seen in the conversion of the hyperbolic curve typical of a V0 vs. [S] plot for an enzyme (Figure 4.18), being converted to a sigmoidal plot (Figure 4.44). This is due to the conversion of the enzyme from the T-state to the R-state on binding the substrate/homotropic effector. The V0 vs. [S] plot of allosteric enzyme reactions resembles the oxygen binding curve of hemoglobin (see Figure 2.83). Even though hemoglobin is not an enzyme and is thus not catalyzing a reaction, the similarity of the plots is not coincidental. In both cases, the binding of an external molecule is being measured – directly, in the hemoglobin plot, and indirectly by the V0 vs. [S] plot, since substrate binding is a factor in enzyme reaction velocity. Allosteric inhibition Allosterically, regulation of these enzymes works by inducing different physical states (shapes, as it were) that affect their ability to bind to substrate. When an enzyme is inhibited by binding an effector, it is converted to the T-state (T=tight), it has a reduced affinity for substrate and it is through this means that the reaction is slowed. Allosteric activation On the other hand, when an enzyme is activated by effector binding, it converts to the R-state (R=relaxed) and binds substrate much more readily. When no effector is present, the enzyme may be in a mixture of T- and R-states. Feedback inhibition An interesting kind of allosteric control is exhibited by HMG-CoA reductase, which catalyzes an important reaction in the pathway leading to the synthesis of cholesterol. Binding of cholesterol to the enzyme reduces the enzyme’s activity significantly. Cholesterol is not a substrate for the enzyme, so it is therefore a heterotropic effector. Notably, though, cholesterol is the end-product of the pathway that HMG-CoA reductase catalyzes a reaction in. When enzymes are inhibited by an end-product of the pathway in which they participate, they are said to exhibit feedback inhibition. Feedback inhibition always operates by allosterism and further, provides important and efficient control of an entire pathway. By inhibiting an early enzyme in a pathway, the flow of materials (and ATP hydrolysis required for their processing) for the entire pathway is stopped or reduced, assuming there are not alternate supply methods. Pathway control In the cholesterol biosynthesis pathway, stopping this one enzyme has the effect of shutting off (or at least slowing down) the entire pathway. This is significant because after catalysis by HMG-CoA reductase, there are over 20 further reactions necessary to make cholesterol, many of them requiring ATP energy. Shutting down one reactions stops all of them. Another excellent example of allosteric control and feedback inhibition is the enzyme ATCase, discussed below. ATCase Another interesting example of allosteric control and feedback inhibition is associated with the enzyme Aspartate Transcarbamoylase (ATCase). This enzyme, which catalyzes a step in the synthesis of pyrimidine nucleotides, has 12 subunits. These include six identical catalytic subunits and six identical regulatory subunits. The catalytic subunits bind to substrate and catalyze a reaction. The regulatory subunits bind to either ATP or CTP. If they bind to ATP, the enzyme subunits arrange themselves in the R-state. R-state The R-state of ATCase allows the substrate to have easier access to the six active sites and the reaction occurs more rapidly. For the same amount of substrate, an enzyme in the R-state will have a higher velocity than the same enzyme that is not in the R-state. By contrast, if the enzyme binds to CTP on one of its regulatory subunits, the subunits will arrange in the T-state and in this form, the substrate will not have easy access to the active sites, resulting in a slower velocity for the same concentration of substrate compared to the R-state. ATCase is interesting in that it can also flip into the R-state when one of the substrates (aspartate) binds to an active site within one of the catalytic subunits. Aspartate has the effect of activating the catalytic action of the enzyme by favoring the R-state. Thus, aspartate, which is a substrate of the enzyme is a homotropic effector and ATP and CTP, which are not substrates of the enzyme are heterotropic effectors of ATCase. Allosteric models There are three models commonly used to explain how allosterism regulates multi-subunit enzyme activity. They are known as • the Monod-Wyman-Changeux (MWC) model (also known as the concerted model), • the sequential model (also known as KNF), • and the morpheein model. All models describe a Tense (T) state that is less catalytically active and a Relaxed (R) state that is more catalytically active. The models differ in how the states change. Sequential model In the sequential model, binding of an allosteric effector by one subunit causes it to change from T to R state (or vice versa) and that change makes it easier for adjacent subunits to similarly change state. With this model , there is a cause/effect relationship between binding of an effector by one subunit and change of state by an adjacent subunit. In hemoglobin, for example, binding of one oxygen by one unit of the complex may induce that unit to flip to the R-state and, through interactions with other subunits, cause them to favor adopting the R configuration before they bind to oxygen. In this way, binding of one subunit favors binding of others and cooperativity can be explained by the change in binding affinity as oxygen concentration changes. MWC model The MWC model is less intuitive. In it, the entire complex changes state from T to R (or vice versa) independently of the binding of effectors. Flipping between T-states and R-states is postulated to be in an equilibrium of states in the absence of effector (for example, a 50 to 1 ratio of T/R. This ratio is referred to as L, so L = T/R). Binding of effector by the enzyme complex has the tendency of “locking” the complex in a state. Binding of inhibitors will increase the ratio of T/R whereas binding of activators will increase R and thus decrease the ration of T/R. Morpheein model The morpheein model is similar to the MWC model, but with an added step of dissociation of the subunits. The MWC model proposes that flipping between R and T states occurs by the complex as a whole and occurs on all units simultaneously. The morpheein model instead proposes that the multi-subunit enzyme breaks down to individual units which can then flip in structure and re-form the complex. In the morpheein model, only identically shaped units (all R, for example) can come together in the complex, thus explaining the “all-R-” or “all-T-” state found in the MWC model. A large number of enzymes, including prominent ones like citrate synthase, acetyl-CoA carboxylase, glutamate dehydrogenase, ribonucleotide reductase and lactate dehydrogenase have behavior consistent with the morpheein model. Covalent control of enzymes Some enzymes are synthesized in a completely inactive form and their activation requires covalent bonds in them to be cleaved. Such inactive forms of enzymes are called zymogens. Examples include the proteins involved in blood clotting and proteolytic enzymes of the digestive system, such as trypsin, chymotrypsin, pepsin, and others. Synthesizing some enzymes in an inactive form makes very good sense when an enzyme’s activity might be harmful to the tissue where it is being made. For example, the painful condition known as pancreatitis arises when digestive enzymes made in the pancreas are activated too soon and end up attacking the pancreas. Cascades For both the blood clotting enzymes and the digestive enzymes, the zymogens are activated in a protease cascade. This occurs when activation of one enzyme activates others in a sort of chain reaction. In such a scheme the first enzyme activated proteolytically cleaves the second zymogen, causing it to be activated, which in turn activates a third and this may proceed through several levels of enzymatic action (Figure 4.50). The advantage of cascades is that they allow a large amount of zymogens to become activated fairly quickly, since there is an amplification of the signal at each level of catalysis. Zymogens are also abundant in blood. Blood clotting involves polymerization of a protein known as fibrin. Since random formation of fibrin is extremely hazardous because it can block the flow of blood, potentially causing heart attack/stroke, the body synthesizes fibrin as a zymogen (fibrinogen) and its activation results from a “cascade” of activations of proteases that arise when a signal is received from a wound. Similarly, the enzyme catalyzing removal of fibrin clots (plasmin) is also synthesized as a zymogen (plasminogen), since random clot removal would also be hazardous (see below also). Phosphorylation/dephosphorylation Another common mechanism for control of enzyme activity by covalent modification is phosphorylation. The phosphorylation of enzymes (on the side chains of serine, threonine or tyrosine residues) is carried out by protein kinases. Enzymes activated by phosphorylation can be regulated by the addition of phosphate groups by kinases or their removal by phosphatases. Thus, this type of covalent modification is readily reversible, in contrast to proteolytic cleavage. Reduction/oxidation An interesting covalent control of enzymes using reduction/oxidation is exhibited in photosynthetic plants. In the light phase of photosynthesis, electrons are excited by light and flow through carriers to NADP+, forming NADPH. Thus, in the light, the NADPH concentration is high. When NADPH concentration is high, the concentration of reduced ferredoxin (a molecule donating electrons to NADP+) is also high. Reduced ferredoxin can transfer electrons to thioredoxin, reducing it. Reduced thioredoxin can, in turn, transfer electrons to proteins to reduce their disulfide bonds. Four enzymes related to the Calvin cycle can receive electrons from thioredoxin and become activated, as a result. These include sedoheptulose 1,7-bisphosphatase, ribulose-5-phosphate kinase, fructose 1,6-bisphosphatase, and glyceraldehyde 3-phosphate dehydrogenase. Thus, in the light, electrons flow, causing NADPH to accumulate and ferredoxin to push electrons in the direction of these enzymes above, activating them and favoring the Calvin cycle. In the dark, the concentration of reduced NADPH, reduced ferredoxin, and reduced thioredoxin fall, resulting in loss of electrons by the Calvin cycle enzymes (oxidations that re-form disulfide bonds) and the Calvin cycle inactivates. Other enzyme control mechanisms Other means of controlling enzymes relate to access to substrate (substrate-level control) and control of enzyme synthesis. Hexokinase is an enzyme that is largely regulated by availability of its substrate, glucose. When glucose concentration is low, the product of the enzyme’s catalysis, glucose-6-phosphate, inhibits the enzyme’s function. Regulation of enzymes by controlling their synthesis is covered later in the book in the discussion relating to control of gene expression.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/04%3A_Catalysis/4.02%3A_Control_of_Enzymatic_Activity.txt

A printable version of this section is here: BiochemFFA_4_3.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy The magic of enzymes, as noted, is in their ability to create electronic environments conducive to initiation of a reaction. There are more mechanisms of reaction than we could ever hope to cover in a book like this, and comprehensive discussion of these is not our aim. Instead, we will cite some examples and go into detail on one of them - the mechanism of action of serine proteases. Chymotrypsin We will begin with mechanism of action of one enzyme - chymotrypsin. Found in our digestive system, chymotrypsin’s catalytic activity is cleaving peptide bonds in proteins and it uses the side chain of a serine in its mechanism of catalysis. Many other protein-cutting enzymes employ a very similar mechanism and they are known collectively as serine proteases (Figure 4.52). These enzymes are found in prokaryotic and eukaryotic cells and all use a common set of three amino acids in the active site called a catalytic triad (Figure 4.53). It consists of aspartic acid, histidine, and serine. The serine is activated in the reaction mechanism to form a nucleophile in these enzymes and gives the class their name. With the exception of the recognition that occurs at the substrate binding site, the mechanism shown here for chymotrypsin would be applicable to any of the serine proteases. Specificity As a protease, chymotrypsin acts fairly specifically, cutting not all peptide bonds, but only those that are adjacent to relatively non-polar amino acids in the protein. One of the amino acids it cuts adjacent to is phenylalanine. The enzyme’s action occurs in two phases – a fast phase that occurs first and a slower phase that follows. The enzyme has a substrate binding site that includes a region of the enzyme known as the S1 pocket. Let us step through the mechanism by which chymotrypsin cuts adjacent to phenylalanine. Substrate binding The process starts with the binding of the substrate in the S1 pocket (Figure 4.54). The S1 pocket in chymotrypsin has a hydrophobic hole in which the substrate is bound. Preferred substrates will include amino acid side chains that are bulky and hydrophobic, like phenylalanine. If an ionized side chain, like that of glutamic acid binds in the S1 pocket, it will quickly exit, much like water would avoid an oily interior. Shape change on binding When the proper substrate binds in the S1 pocket, its presence induces an ever so slight change in the shape of the enzyme. This subtle shape change on the binding of the proper substrate starts the steps of the catalysis. Since the catalytic process only starts when the proper substrate binds, this is the reason that the enzyme shows specificity for cutting at specific amino acids in the target protein. Only amino acids with the side chains that interact well with the S1 pocket start the catalytic wheels turning. The slight changes in shape involve changes in the positioning of three amino acids (aspartic acid, histidine, and serine) in the active site known as the catalytic triad. The shift of the negatively charged aspartic acid towards the electron rich histidine ring favors the abstraction of a proton by the histidine from the hydroxyl group on the side chain of serine, resulting in production of a very reactive alkoxide ion in the active site (Figure 4.55). Since the active site at this point also contains the polypeptide chain positioned with the phenylalanine side chain embedded in the S1 pocket, the alkoxide ion performs a nucleophilic attack on the peptide bond on the carboxyl side of phenylalanine sitting in the S1 pocket (Figure 4.56). This reaction breaks the peptide bond (Figure 4.57) and causes two things to happen. First, one end of the original polypeptide is freed and exits the active site (Figure 4.58). The second is that the end containing the phenylalanine is covalently linked to the oxygen of the serine side chain. At this point we have completed the first (fast) phase of the catalysis. Slower second phase The second phase of the catalysis by chymotrypsin is slower. It requires that the covalent bond between phenylalanine and serine’s oxygen be broken so the peptide can be released and the enzyme can return to its original state. The process starts with entry of water into the active site. Water is attacked in a fashion similar to that of the serine side chain in the first phase, creating a reactive hydroxyl group (Figure 4.59) that performs a nucleophilic attack on the phenylalanine-serine bond (Figure 4.60), releasing it and replacing the proton on serine. The second peptide is released in the process and the reaction is complete with the enzyme back in its original state (Figure 4.61). Serine proteases The list of serine proteases is quite long. They are grouped in two broad categories - 1) those that are chymotrypsin-like and 2) those that are subtilisin-like. Though subtilisin-type and chymotrypsin-like enzymes use the same mechanism of action, including the catalytic triad, the enzymes are otherwise not related to each other by sequence and appear to have evolved independently. They are, thus, an example of convergent evolution - a process where evolution of different forms converge on a structure to provide a common function. The serine protease enzymes cut adjacent to specific amino acids and the specificity is determined by the size/shape/charge of amino acid side chain that fits into the enzyme’s S1 binding pocket (Figure 4.62). Examples of serine proteases include trypsin, chymotrypsin, elastase, subtilisin, signal peptidase I, and nucleoporin. Serine proteases participate in many physiological processes, including blood coagulation, digestion, reproduction, and the immune response. Cysteine proteases Cysteine proteases (also known as thiol proteases) catalyze the breakdown of proteins by cleaving peptide bonds using a nucleophilic thiol from a cysteine (Figure 4.63). The cysteine is typically found in a catalytic dyad or triad also involving histidine and (sometimes) aspartic acid (very much like serine proteases). The sulfhydryl group of cysteine proteases is more acidic than the hydroxyl of serine proteases, so the aspartic acid of the triad is not always needed. The mechanism of action is very similar to that of serine proteases. Binding of proper substrate results in activation of the thiol (removal of the proton by the histidine group). The activated thiol acts as a nucleophile, attacking the peptide bond and causing it break. One peptide is released and the other peptide becomes covalently linked to the sulfur. Hydrolysis by water releases the second peptide and completes the cycle. Examples of cysteine proteases include papain, caspases, hedgehog protein, calpain, and cathepsin K. Caspases Caspases (Cysteine-ASPartic ProteASEs) are a family of cysteine proteases that play important roles in the body. At the cellular level they function in apoptosis and necrosis and in the body, they are involved in inflammation and the immune system. Maturation of lymphocytes is one such role. They are best known, however, for their role in apoptosis, which has given rise to descriptions of them as “executioner” proteins or “suicide proteases” that dismantle cells in programmed cell death. There are 12 known human caspases. The enzymes are synthesized as pro-caspase zymogens with a prodomain and two other subunits. The prodomain contains regions that allow it to interact with other molecules that regulate the enzyme’s activity. The caspases come in two forms. The initiator caspases, when activated, activate the effector caspases. The effector caspases cleave other proteins in the cell. Targets for effector caspase cleavage action include the nuclear lamins (fibrous proteins providing structural integrity to the nucleus), ICAD/DFF45 (an inhibitor of DNAse), PARP (flags areas where DNA repair needed), and PAK2 (apoptotic regulation). The caspase activation cascade can itself be activated by granzyme B (a serine protease secreted by natural killer cells and cytotoxic T-cells), cellular death receptors, and the apoptosome (large protein structure in apoptotic cells stimulated by release of cytochrome C from the mitochondria). Each of these activators is responsible for activating different groups of caspases. Metalloproteases Metalloproteases (Figure 4.64) are enzymes whose catalytic mechanism for breaking peptide bonds involves a metal. Most metalloproteases use zinc as their metal, but a few use cobalt, coordinated to the protein by three amino acid residues with a labile water at the fourth position. A variety of side chains are used - histidine, aspartate, glutamate, arginine, and lysine. The water is the target of action of the metal which, upon binding of the proper substrate, abstracts a proton to create a nucleophilic hydroxyl group that attacks the peptide bond, cleaving it (Figure 4.64). Since the nucleophile here is not attached covalently to the enzyme, neither of the cleaved peptides ends up attached to the enzyme during the catalytic process. Examples of metalloproteases include carboxypeptidases, aminopeptidases, insulinases and thermolysin. Aspartyl proteases As the name suggests, aspartyl proteases use aspartic acid in their catalytic mechanism (Figures 4.63 & 4.65). Like the metalloproteases, aspartyl proteases activate a water to create a nucleophile for catalysis (Figure 4.65). The activated water attacks the peptide bond of the bound substrate and releases the two pieces without the need to release a bound intermediate, since water is not covalently attached to the enzyme. Common aspartyl proteases include pepsin, signal peptidase II, and HIV-1 protease. Threonine proteases Though threonine has an R-group with a hydroxyl like serine, the mechanism of action of this class of proteases differs somewhat from the serine proteases. There are some similarities. First, the threonine’s hydroxyl plays a role in catalysis and that is to act as a nucleophile. The nucleophile is created, however, not by a catalytic triad, but rather as a result of threonine’s own α-amine group abstracting a proton. Because of this, the nucleophilic threonine in a threonine protease must be at the n-terminus of the enzyme. Nucleophilic attack of the peptide bond in the target protease results in breakage of the bond to release one peptide and the other is covalently attached to serine, like the serine proteases. Also, as with the serine proteases, water must come in to release the covalently linked second peptide to conclude the catalytic mechanism. Examples Examples of threonine proteases include the catalytic subunits of the proteasome. Some acyl transferases (such as ornithine acyltransferase) have evolved the same catalytic mechanism by convergent evolution. The latter enzymes use ornithine instead of water to break the enzyme-substrate covalent bond, with the result that the acyl-group becomes attached to ornithine, instead of water. Protease inhibitors Molecules which inhibit the catalytic action of proteases are known as protease inhibitors. These come in a variety of forms and have biological and medicinal uses. Many biological inhibitors are proteins themselves. Protease inhibitors can act in several ways, including as a suicide inhibitor, a transition state inhibitor, a denaturant, and as a chelating agent. Some work only on specific classes of enzymes. For example, most known aspartyl proteases are inhibited by pepstatin. Metalloproteases are sensitive to anything that removes the metal they require for catalysis. Zinc-containing metalloproteases, for example, are very sensitive to EDTA, which chelates the zinc ion. One category of proteinaceous protease inhibitors is known as the serpins. Serpins inhibit serine proteases that act like chymotrypsin. 36 of them are known in humans. Serpins are unusual in acting by binding to a target protease irreversibly and undergoing a conformational change to alter the active site of its target. Other protease inhibitors act as competitive inhibitors that block the active site. Serpins can be broad in their specificity. Some, for example, can block the activity of cysteine proteases. One of the best known biological serpins is α-1-anti-trypsin (A1AT - Figure 4.66) because of its role in lungs, where it functions to inhibit the elastase protease. Deficiency of A1AT leads to emphysema. This can arise as a result of genetic deficiency or by cigarette smoking. Reactive oxygen species produced by cigarette smoking can oxidize a critical methionine residue (#358 of the processed form) in A1AT, rendering it unable to inhibit elastase. Uninhibited, elastase can attack lung tissue and cause emphysema. Most serpins work extracellularly. In blood, for example, serpins like antithrombin can help to regulate the clotting process. Figure 4.67 - Incidence of α-1-antitrypsin (PiMZ) deficiency in Europe by percent. Wikipedia Anti-viral Agents Protease inhibitors are used as anti-viral agents to prohibit maturation of viral proteins - commonly viral coat proteins. They are part of drug “cocktails” used to inhibit the spread of HIV in the body and are also used to treat other viral infections, including hepatitis C. They have also been investigated for use in treatment of malaria and may have some application in anti-cancer therapies as well.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/04%3A_Catalysis/4.03%3A_Mechanisms_of_Catalysis.txt

A printable version of this section is here: BiochemFFA_4_4.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Clotting is a process in which liquid blood is converted into a gelatinous substance that eventually hardens. The aim is to stop the flow of blood from a vessel. The formation of a clot is the result of a series of enzymatic reactions that are triggered upon injury. The process involves: 1. a step of activation (wounding) followed by 2. a cellular response (aggregation of blood platelets) and 3. a molecular response (polymerization of the protein called fibrin to create a meshwork that hardens). Factors released in the cellular response help activate the molecular response. The process is highly conserved across species. Cellular Response Injury to the epithelial lining of a blood vessel begins the process of coagulation almost instantly. The cellular response has an initial action followed by an amplification step. In the cellular response (Figure 4.68), the platelets bind directly to collagen using Ia/IIa collagen-binding surface receptors and glycoprotein VI to form a plug. The signal to the platelets to take this action is exposure of the underlying collagen, something that would not happen in the absence of a wound. Upon injury, platelet integrins get activated and bind tightly to the extracellular matrix to anchor them to the site of the wound. The von Willebrand factor (see below also) assists by forming additional links between the platelets’ glycoprotein Ib/IX/V and the fibrils of the collagen. Amplification In the amplification part of the cellular response, the activated platelets release a large number of factors, including platelet factor 4 (a cytokine stimulating inflammation and moderating action of the heparin anticoagulant) and thromboxane A2, The latter has the effect of increasing the “stickiness” of platelets, favoring their aggregation. In addition, a a Gq-protein linked receptor cascade is activated, resulting in release of calcium from intracellular stores. This will play a role in the molecular response. Molecular response The molecular response results in the creation of a web comprised of polymers of fibrin protein. Like the cellular pathway, the molecular pathway begins with an initiation phase and continues with an amplification phase. Polymerization of fibrin results from convergence of two cascading catalytic pathways. They are the intrinsic pathway (also called the contact activation pathway) and the extrinsic pathway (also referred to as the tissue factor pathway). Of the two pathways, the tissue factor pathway has recently been shown to be the more important. Serine Protease Cascade In both pathways, a series of zymogens of serine proteases are sequentially activated in rapid succession. The advantage of such a cascading system is tremendous amplification of a small signal. At each step of the cascade, activation of a zymogen causes the production of a considerable amount of an active serine protease, which is then able to activate the next zymogen which, in turn, activates an even larger amount of the next zymogen in the system. This results in the ultimate activation of a tremendous amount more fibrin than could be achieved if there were only a single step where an enzyme activated fibrinogen to fibrin. Nomenclature The zymogen factors in the molecular response are generally labeled with Roman numerals. A lowercase, subscripted ‘a’ is used to designate an activated form. The tissue factor pathway functions to create a thrombin burst, a process in which thrombin is activated very quickly. This is the initiation phase. It is fairly straightforward because it has one focus - activation of thrombin. Thrombin, which converts fibrinogen into the fibrin of the clot, is central also to the amplification phase, because it activates some of the factors that activate it, creating an enormous increase in signal and making a lot of thrombin active at once. Initiation phase The initiation phase of the molecular response begins when Factor VII (the letter ‘F’ before the Roman numeral is often used as an abbreviation for ‘factor’) gets activated to FVIIa after damage to the blood vessel (Figure 4.69 & 4.70). This happens as a result of its interaction with Tissue Factor (TF, also called coagulation Factor III) to make a TF-FVIIa complex. The combined efforts of TF-FVIIa, FIXa, and calcium (from the cellular response) inefficiently convert FX to FXa. FXa, FV, and calcium inefficiently convert prothrombin (zymogen) to thrombin (active). A tiny amount of thrombin has been activated at the end of the initiation phase. Figure 4.69 - Intrinsic and extrinsic pathways of blood coagulation. The aim is making a fibrin clot (lower right). Wikipedia Figure 4.70 - Another view of the molecular response of the blood clotting pathway. Wikipedia Amplification phase To make sufficient thrombin to convert enough fibrinogen to fibrin to make a clot, thrombin activates other factors (FV, FXI, FVIII) that help to make more thrombin. This is the amplification phase of the molecular process and is shown in the light blue portion in the upper right part of Figure 4.68. The amplification phase includes factors in both the intrinsic and extrinsic pathways. FVIII is normally bound in a complex with the von Willebrand factor and is inactive until it is released by action of thrombin. Activation of FXI to FXIa helps favor production of more FIXa. FIXa plus FVIIIa stimulate production of a considerable amount of FXa. FVa joins FXa and calcium to make a much larger amount of thrombin. Factors FVa and FVIIIa are critical to the amplification process. FVIIIa stimulates FIXa’s production of FXa by 3-4 orders of magnitude. FVa helps to stimulate FXa’s production of thrombin by about the same magnitude. Thus, thrombin stimulates activation of factors that, in turn, stimulate activation of more thrombin. Transglutaminase In addition to helping to amplify product of itself and conversion of fibrinogen to fibrin, thrombin catalyzes the activation of FXIII to FXIIIa. FXIIIa is a transglutaminase that helps to “harden” the clot (Figure 4.71 & 4.73). It accomplishes this by catalyzing formation of a covalent bond between adjacent glutamine and lysine side chains in the fibrin polymers. Not all of the factors involved in the clotting process are activated by the pathway, nor are all factors serine proteases. This includes FVIII and FV which are glycoproteins, and FXIII, which is the transglutaminase described above. The blood clotting process must be tightly regulated. Formation of clots in places where no damage has occurred can lead to internal clots (thrombosis) cutting off the flow of blood to critical regions of the body, such as heart or brain. Conversely, lack of clotting can lead to internal bleeding or, in severe cases, death due to unregulated external bleeding. Such is a danger for people suffering from hemophilia. Figure 4.72 - α-thrombin. Wikipedia Diseases of Blood Clotting: Hemophilia Hemophilia is a hereditary genetic disorder affecting the blood clotting process in afflicted individuals. The disease is X-linked and thus occurs much more commonly in males. Deficiency of FVIII leads to Hemophilia A (about 1 in 5000 to 10,000 male births) and deficiency of FIX produces Hemophilia B (about 1 in 20,000 to 35,000 male births). Hemophilia B spread through the royal families of Europe, beginning with Queen Victoria’s son, Leopold. Three of the queen’s grandsons and six of her great-grandsons suffered from the disease. Hemophilia is treated by exogenous provision of missing clotting factors and has improved life expectancy dramatically. In 1960, the life expectancy of a hemophiliac was about 11 years. Today, it is over 60. Diseases of Blood Clotting: von Willebrand’s disease A related disease to hemophilia that is also genetically linked is von Willebrand’s Disease. The von Willebrand factor plays a role in both the cellular and the molecular responses in blood clotting. First, the factor is a large multimeric glycoprotein present in blood plasma and also is produced in the endothelium lining blood vessels. The von Willebrand factor helps to anchor platelets near the site of the wound in the cellular response. It binds to several things. First, it binds to platelets’ Ib glycoprotein. Second, it binds to heparin and helps moderate its action. Third, it binds to collagen and fourth, the factor binds to FVIII in the molecular response, playing a protective role for it. In the absence of the von Willebrand factor, FVIII is destroyed. Fifth, the von Willebrand factor binds to integrin of platelets, helping them to adhere together and form a plug. Defects of the von Willebrand factor lead to various various bleeding disorders. Blood “thinners” The clotting of blood is essential for surviving wounds that cause blood loss. However, some people have conditions that predispose them to the formation of clots that can lead to stroke, heart attack, or other problems, like pulmonary embolism. For these people, anti-clotting agents (commonly called blood thinners) are used to reduce the likelihood of undesired clotting. The first, and more common of these is aspirin. Aspirin is an inhibitor of the production of prostaglandins. Prostaglandins are molecules with 20 carbons derived from arachidonic acid that have numerous physiological effects. Metabolically, the prostaglandins are precursors of a class of molecules called the thromboxanes. Thromboxanes play roles in helping platelets to stick together in the cellular response to clotting. By inhibiting the production of prostaglandins, aspirin reduces the production of thromboxanes and reduces platelet stickiness and the likelihood of clotting. Vitamin K action Another approach to preventing blood clotting is one that interferes with an important molecular action of Vitamin K. A pro-clotting factor found in the blood, vitamin K is necessary for an important modification to prothrombin and other blood clotting proteins. Vitamin K serves as an enzyme cofactor that helps to catalyze addition of an extra carboxyl group onto the side chain of glutamic acid residues of several clotting enzymes (see HERE). This modification gives them the ability to bind to calcium (Figure 4.77), which is important for activating the serine protease cascade. During the reaction that adds carboxyl groups to glutamate, the reduced form of vitamin K becomes oxidized. In order for vitamin K to stimulate additional carboxylation reactions to occur, the oxidized form of vitamin K must be reduced by the enzyme vitamin K epoxide reductase. Figure 4.77 - γ-carboxylglutamic acid (left) has a calcium binding Site. Unmodified glutamic acid (right) does not. Warfarin blocks reduction The compound known as warfarin (brand name = coumadin - Figure 4.78) interferes with the action of vitamin K epoxide reductase and thus, blocks recycling of vitamin K. As a consequence, fewer prothrombins (and other blood clotting proteins) get carboxylated, and less clotting occurs. Vitamin K-mediated carboxylation of glutamate occurs on the γ carbon of the amino acid’s side chain, for 16 different proteins, 7 of which are involved in blood clotting, including prothrombin. When the carboxyl group is added as described, the side chain is able to efficiently bind to calcium ions. In the absence of the carboxyl group, the side chain will not bind to calcium. Calcium released near the site of the wound in the cellular response to clotting helps to stimulate activation of proteins in the serine protease cascade of the molecular response. Vitamin K comes in several forms. It is best described chemically as a group of 2-methyl-1,4-naphthoquinone derivatives. There are five different forms recognized as vitamin Ks (K1, K2, K3, K4, and K5). Of these, vitamins K1 and K2 come from natural sources and the others are synthetic. Vitamin K2, which is made from vitamin K1 by gut microorganisms, has several forms, with differing lengths of of isoprenoid side-chains. The various forms are commonly named as MK-X, where X is a number, and MK stands for menaquinone, which is the name given to this form of vitamin K. Figure 4.79 shows a common form known as MK-4 (menatetrenone). Figure 4.79 - MK-4 (menatetrenone) Hemorrhaging danger It is very critical that the proper amount of warfarin be given to patients. Too much can result in hemorrhaging. Patients must have their clotting times checked regularly to ensure that they are taking the right dose of anti-coagulant medication. Diet and the metabolism of Vitamin K in the body can affect the amount of warfarin needed. Vitamin K is synthesized in plants and plays a role in photosynthesis. It can be found in the highest quantities in vegetables that are green and leafy. Patients whose diet is high in these vegetables may require a different dose than those who rarely eat greens. Dietary vitamin K is also, as mentioned earlier, metabolized by bacteria in the large intestine, where they convert vitamin K1 into vitamin K2. Plasmin Clots, once made in the body, do not remain there forever. Instead, a tightly regulated enzyme known as plasmin is activated, when appropriate, to break down the fibrin-entangled clot. Like many of the enzymes in the blood clotting cascade, plasmin is a serine protease. It is capable of cleaving a wide range of proteins. They include polymerized fibrin clots, fibronectin, thrombospondin, laminin, and the von Willebrand factor. Plasmin plays a role in activating collagenases and in the process of ovulation by weakening the wall of the Graafian follicle in the ovary. Plasmin is made in the liver as the zymogen known as plasminogen. Several different enzymes can activate it. Tissue plasminogen activator (tPA), using fibrin as a co-factor, is one. Others include urokinase plasminogen activator (using urokinase plasminogen activator receptor as a co-factor), kallikrein (plasma serine protease with many forms and blood functions), and FXIa and FXIIa from the clotting cascade. Plasmin inhibition Plasmin’s activity can also be inhibited. Plasminogen activator inhibitor, for example, can inactivate tPA and urokinase. After plasmin has been activated, it can also be inhibited by α2-antiplasmin and α2-macroglobulin (Figure 4.80). Thrombin also plays a role in plasmin’s inactivation, stimulating activity of thrombin activatable fibrinolysis inhibitor. Angiostatin is a sub-domain of plasmin produced by auto-proteolytic cleavage. It blocks the growth of new blood vessels and is being investigated for its anti-cancer properties. Figure 4.80 - Regulation of fibrin breakdown. Activators in blue. Inhibitors in red. Wikipedia Fibronectin Fibronectin is a large (440 kDa) glycoprotein found in the extracellular matrix that binds to integral cellular proteins called integrins and to extracellular proteins, including collagen, fibrin, and heparan sulfate. It comes in two forms. The soluble form is found in blood plasma and is made by the liver. It is found in high concentration in the blood stream (300 µg/ml). The insoluble form is found abundantly in the extracellular matrix. The protein is assembled in the extracellular matrix and plays roles in cellular growth, adhesion, migration, and differentiation. It is very important in wound healing. Figure 4.82 - Fibronectin 1. Wikipedia Assists in blood clot formation Fibronectin from the blood plasma is localized to the site of the wound, assisting in formation of the blood clot to stop bleeding. In the initial stages of wound healing, plasma fibronectin interacts with fibrin in clot formation. It also protects tissue surrounding the wound. Later in the repair process, remodeling of the damaged area begins with the action of fibroblasts and endothelial cells at the wound site. Their task is to degrade proteins of the blood clot matrix, replacing them with a new matrix like the undamaged, surrounding tissue. Fibroblasts act on the temporary fibronectin-fibrin matrix, remodeling it to replace the plasma fibronectin with cellular fibronectin. This may cause the phenomenon of wound contraction, one of the steps in wound healing. Secretion of cellular fibronectin by fibroblasts is followed by fibronectin assembly and integration with the extracellular matrix. Embryogenesis Fibronectin is essential for embryogenesis. Deleting the gene in mice causes lethality before birth. This is likely due to its role in migration and guiding the attachment of cells as the embryo develops. Fibronectin also has a role in the mouth. It is found in saliva and is thought to inhibit colonization of the mouth by pathogenic bacteria. Platelet activating factor Platelet Activating Factor (PAF) is a compound (Figure 4.83) produced primarily in cells involved in host defense. These include platelets, macrophages, neutrophils, and monocytes, among others. It is produced in greater quantities in inflammatory cells upon proper stimulation. The compound acts like a hormone and mediates platelet aggregation/degranulation, inflammation, and anaphylaxis. It can transmit signals between cells to trigger and amplify inflammatory and clotting cascades. When unregulated, signaling by PAF can cause severe inflammation resulting in sepsis and injury. Inflammation in allergic reactions arises partly as a result of PAF and is an important factor in bronchoconstriction in asthma. In fact, at a concentration of only 10 picomolar, PAF can cause asthmatic inflammation of the airways that is life threatening. Figure 4.83 - Platelet Activating Factor. Wikipedia I’m feeling so sad ‘Cuz I cut . . . . myself bad Now I’m all worried ‘bout . . . . consequences It’s starting to bleed There’s some clo . . . . sure I need So the body kicks . . . . in its defenses It’s happened all so many times before The blood flows out and then it shuts the door Thank goodness my blood is clotting Enmeshing the fibrin chains Thank goodness my blood is clotting The zymogens Are activating and all is well So I’ll stop bleeding again The vitamin K’s Help to . . . . bind to cee-ays Adding C-O-. . . . O-H to amend things Um-m-um-um-um-um It hardens and stays When a glu. . . . taminase Creates co. . . . valent bonds . . . . for cementing In just a moment, things are good to go The clot’s in place and it has stopped the flow But what about clot dissolving? Untangling fibrin chains? This calls for some problem solving There is a way Just activate up some t-PA Get plasmin active in veins Oh, oh, oh. And thanks to the dis-enclotting’ As part of repairin’ veins It’s part of my body’s plotting The wound is gone I’m back where I started and Nothing’s wrong My blood flow is normal again. Thank Goodness My Blood is Clotting To the tune of "Don't Sleep in the Subway Darling" Metabolic Melodies Website HERE Recording by Liz Bacon and David Simmons Lyrics by Kevin Ahern Recording by Liz Bacon and David Simmons Lyrics by Kevin Ahern

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/04%3A_Catalysis/4.04%3A_Blood_Clotting.txt

• 5.1: Basics of Energy Living organisms are made up of cells, and cells contain a horde of biochemical components. Living cells, though, are not random collections of these molecules. They are extraordinarily organized or "ordered". By contrast, in the nonliving world, there is a universal tendency to increasing disorder. Maintaining and creating order in cells takes the input of energy. Without energy, life is not possible. • 5.3: Energy - Photophosphorylation The third type of phosphorylation to make ATP is found only in cells that carry out photosynthesis. This process is similar to oxidative phosphorylation in several ways. A primary difference is the ultimate source of the energy for ATP synthesis. In oxidative phosphorylation, the energy comes from electrons produced by oxidation of biological molecules. In photosynthesis, the energy comes from the light of the sun. • 5.2: Electron Transport and Oxidative Phosphorylation In eukaryotic cells, the vast majority of ATP synthesis occurs in the mitochondria in a process called oxidative phosphorylation. Even plants, which generate ATP by photophosphorylation in chloroplasts, contain mitochondria for the synthesis of ATP through oxidative phosphorylation. 05: Energy Source: BiochemFFA_5_1.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Living organisms are made up of cells, and cells contain a horde of biochemical components. Living cells, though, are not random collections of these molecules. They are extraordinarily organized or "ordered". By contrast, in the nonliving world, there is a universal tendency to increasing disorder. Maintaining and creating order in cells takes the input of energy. Without energy, life is not possible. Oxidative Energy The primary mechanism used by non-photosynthetic organisms to obtain energy is oxidation and carbon is the most commonly oxidized energy source. The energy released during the oxidative steps is “captured” in ATP and can be used later for energy coupling. The more reduced a carbon atom is, the more energy can be realized from its oxidation. Fatty acids are highly reduced, whereas carbohydrates are moderately so. Complete oxidation of both leads to carbon dioxide, which has the lowest energy state. Conversely, the more oxidized a carbon atom is, the more energy it takes to reduce it. In the series shown in Figure $1$, the most reduced form of carbon is on the left. The energy of oxidation of each form is shown above it. The reduction states of fatty acids and carbohydrates can be seen by their formulas. • Palmitic acid: $\ce{C16H34O2}$ • Glucose: $\ce{C6H12O6}$ Palmitic acid only contains two oxygens per sixteen carbons, whereas glucose has six oxygen atoms per six carbons. Consequently, when palmitic acid is fully oxidized, it generates more ATP per carbon (128/16) than glucose (38/6). It is because of this that we use fat (contains fatty acids) as our primary energy storage material. Figure $2$: Photosynthesis: The primary source of biological energy. Image by Aleia Kim Oxidation vs. Reduction in Metabolism Biochemical processes that break things down from larger to smaller are called catabolic processes. Catabolic processes are often oxidative in nature and energy releasing. Some, but not all, of that energy is captured as ATP. If not all of the energy is captured as ATP, what happens to the rest of it? The answer is simple. It is released as heat and it is for this reason we get hot when we exercise. By contrast, synthesizing large molecules from smaller ones (for example, making proteins from amino acids) is referred to as anabolism. Anabolic processes are often reductive in nature (Figures 5.3 & 5.4) and require energy input. By themselves, they would not occur, as they are reversing oxidation and decreasing entropy (making many small things into a larger one). To overcome this energy barrier, cells must expend energy. For example, if one wishes to reduce $\ce{CO2}$ to carbohydrate, energy must be used to do so. Plants do this during the dark reactions of photosynthesis (Figure $3$). The energy source for the reduction is ultimately the sun. The electrons for the reduction come from water, and the $\ce{CO2}$ is removed from the atmosphere and gets incorporated into a sugar. Energy Coupling The synthesis of the many molecules needed by cells needs the input of energy to occur. Cells overcome this energy obstacle by using ATP to “drive” the reaction (Figure $6$). The energy needed to drive reactions is harvested in very controlled conditions in enzymes. This involves a process called ‘coupling’. Coupled reactions rely on linking an energetically favorable reaction (i.e., one with a negative ∆G°’) with the reaction requiring an energy input, which has a positive ∆G°’. As long as the overall ∆G°’ of the two reactions together is negative, the reaction can proceed. Hydrolysis of ATP is a very energetically favorable reaction that is commonly linked to many energy requiring reactions in cells. Without the hydrolysis of ATP (or GTP, in some cases), the reaction would not be feasible. Entropy and energy Most students who have had some chemistry know about the Second Law of Thermodynamics with respect to increasing disorder of a system. Cells are very organized or ordered structures, leading some to mistakenly conclude that life somehow violates the second law. In fact, that notion is incorrect. The second law doesn’t say that entropy always increases, just that, left alone, it tends to do so, in an isolated system. Cells are not isolated systems, though, in that they obtain energy, either from the sun, if they are autotrophic, or food, if they are heterotrophic. To counter the universal tendency towards disorder on a local scale requires energy. As an example, take a fresh deck of cards which is neatly aligned with Ace-King-Queen . . . . 4,3,2 for each suit. Throw the deck into the air, letting the cards scatter. When you pick them up, they will be more disordered than when they started. However, if you spend a few minutes (and expend a bit of energy), you can reorganize the same deck back to its previous, organized state. If entropy always increased everywhere, you could not do this. However, with the input of energy, you overcame the disorder. This illustrates an important concept: the cost of fighting disorder is energy. Biological energy There are, of course, other reasons that organisms need energy. Muscular contraction, synthesis of molecules, neurotransmission, signaling, thermoregulation, and subcellular movements are examples. Where does this energy come from? The currencies of energy are generally high-energy phosphate-containing molecules. ATP is the best known and most abundant, but GTP is also an important energy source (energy source for protein synthesis). CTP is involved in synthesis of glycerophospholipids and UTP is used for synthesis of glycogen and other sugar compounds. In each of these cases, the energy is in the form of potential chemical energy stored in the multi-phosphate bonds. Hydrolyzing those bonds releases the energy in them. Of the triphosphates, ATP is the primary energy source, acting to facilitate the synthesis of the others by action of the enzyme NDPK. ATP is made by three distinct types of phosphorylation – oxidative phosphorylation (in mitochondria), photophosphorylation (in chloroplasts of plants), and substrate level phosphorylation (in enzymatically catalyzed reactions). Gibbs free energy in Biology ATP is generally considered the “storage battery” of cells (See also ‘Molecular Battery Backups for Muscles HERE). In order to understand how energy is captured, we must first understand Gibbs free energy and in doing so, we begin to see the role of energy in determining the directions chemical reactions take. Gibbs free energy may be thought of as the energy available to do work in a thermodynamic system at constant temperature and pressure. Mathematically, the Gibbs free energy is given as: $G = H – TS$ where $H$ is the enthalpy, $T$ is the temperature in Kelvin, and $S$ is the entropy. At standard temperature and pressure, every system seeks to achieve a minimum of free energy. Thus, increasing entropy, $S$, will reduce Gibbs free energy. Similarly, if excess heat is available (reducing the enthalpy, $H$), the free energy can also be reduced. Cells must work within the laws of thermodynamics, as noted, so all of their biochemical reactions, too, are ruled by these laws. Now we shall consider energy in the cell. The change in Gibbs free energy ($∆G$) for a reaction is crucial, for it, and it alone, determines whether or not a reaction goes forward. $∆G = ∆H – T ∆S.$ There are three cases • ∆G < 0: the reaction proceeds as written • ∆G = 0: the reaction is at equilibrium • ∆G > 0: the reaction runs in reverse For a reaction $\ce{aA <=> bB}$ (where ‘a’ and ‘b’ are integers and A and B are molecules) at pH 7, ∆G can be determined by the following equation, $∆G = ∆G°’ + RT \ln(\frac{[B]^b}{[A]^a})$ For multiple substrate reactions, such as $\ce{aA + cC <=> bB + dD}$ $∆G = ∆G°’ + RT \ln(\frac{[B]^b [D]^d}{[A]^a[C]^c})$ The ∆G°’ term is called the change in Standard Gibbs Free energy, which is the change in energy that occurs when all of the products and reactants are at standard conditions and the pH is 7.0. It is a constant for a given reaction. In simple terms, we can collect all of the terms of the numerator together and call them {Products} and all of the terms of the denominator together and call them {Reactants}, $∆G = ∆G°’ + RT \ln(\frac{\rm{\{Products\}}}{\rm{\{Reactants\}}})$ For most biological systems, the temperature, T, is a constant for a given reaction. Since ∆G°’ is also a constant for a given reaction, the ∆G is changed almost exclusively as the ratio of {Products}/{Reactants} changes. Importance of ∆G°’ If one starts out at standard conditions, where everything except protons is at 1M, the RTln({Products}/{Reactants}) term is zero, so the ∆G°’ term equals the ∆G, and the ∆G°’ determines the direction the reaction will take (only under those conditions). This is why people say that a negative ∆G°’ indicates an energetically favorable reaction, whereas a positive ∆G°’ corresponds to an unfavorable one. Increasing the ratio of {Products}/{Reactants} causes the value of the natural log (ln) term to become more positive (less negative), thus making the value of ∆G more positive. Conversely, as the ratio of {Products}/{Reactants} decreases, the value of the natural log term becomes less positive (more negative), thus making the value of ∆G more negative. System response to stress Intuitively, this makes sense and is consistent with Le Chatelier’s Principle – a system responds to stress by acting to alleviate the stress. If we examine the ∆G for a reaction in a closed system, we see that it will always move to a value of zero (equilibrium), no matter whether it starts with a positive or negative value. Another type of free energy available to cells is that generated by electrical potential. For example, mitochondria and chloroplasts partly use Coulombic energy (based on charge) from a proton gradient across their membranes to provide the necessary energy for the synthesis of ATP. Similar energies drive the transmission of nerve signals (sodium and potassium gradients) and the movement of some molecules in secondary active transport processes across membranes (e.g., H+ differential driving the movement of lactose). From the Gibbs free energy change equation, $∆G = ∆H – T∆S$ it should be noted that an increase in entropy will help contribute to a decrease in ∆G. This happens, for example when a large molecule is being broken into smaller pieces or when the rearrangement of a molecule increases the disorder of molecules around it. The latter situation arises in the hydrophobic effect, which helps drive the folding of proteins. Chemical and electrical potential It is said that absence makes the heart grow fonder. We won’t tackle that philosophical issue here, but we will say that separation provides potential energy that cells can and do harvest. The lipid bilayer of cell and (in eukaryotic cells) organelle membranes provide the necessary barrier for separation. Impermeable to most ions and polar compounds, biological membranes are essential for processes that generate cellular energy. Consider Figure 5.8. A lipid bilayer separates two solutions with different concentrations of a solute. There is a greater concentration of negative ions in the bottom and a greater concentration of positive ions on the top. Whenever there is a difference in concentration of molecules across a membrane, there is said to be a concentration gradient across it. A difference in concentration of ions across a membrane also creates a charge (or electrical) gradient. Because there is a difference in both the chemical concentration of the ions and in the charge on the two sides of the membrane, this is described as an electrochemical gradient (Figures 5.8 -5.10). Potential energy Such gradients function like batteries and contain potential energy. When the potential energy is harvested by cells, they can create ATP, transmit nerve signals, pump molecules across membranes, and more. It is important, therefore, to understand how to calculate the potential energy of electrochemical gradients. First, we consider chemical (solute) gradients. In Figure 5.9, two concentrations of glucose are separated by a lipid bilayer. Let’s assume C2 be the concentration of glucose inside the cell (bottom) and C1 be the glucose concentration outside (top). The Gibbs free energy associated with moving glucose in the direction of C2 (into the cell) is given by ∆G = RTln[C2/C1] To move it in the direction of C1 (to the outside of the cell) the expression would be $∆G = RT\ln[C_1/C_2]$ Since C2 is smaller than C1 (i.e., there are fewer glucose molecules inside the cell) then the ∆G is negative and diffusion would be favored into the cell, if the glucose could traverse the bilayer. Conversely, if C2 was greater than C1 (more glucose was in the cell than outside) the ∆G would be positive, so movement in the direction of C2 would not be favored and instead the glucose would tend to move towards C1 , that is, out of the cell. If C2 = C1, with the same concentration of glucose inside and outside, then the ∆G would be zero and there would be no net movement, as the system would be at equilibrium. In the example above, we considered glucose, which is an uncharged molecule. When ions are involved, their charges must also be taken into consideration. Figure $1$0 depicts a similar situation across a lipid bilayer. In this case, a difference of concentration and charge exists. There are more positive charges inside the cell than outside. Using C2 to indicate the concentration of materials inside the cell and C1 for the concentration outside the cell (as before), then the free energy for movement of an ion from top to bottom is given by the following equation $∆G = RT\ln[C_2/C_1] + ZF∆ψ$ Note here that this equation must take into consideration both the concentration differences and the charge differences. Z refers to the charge of the transported species, F is the Faraday constant (96,485 Coulombs/mol), and ∆ψ is the electrical potential difference (voltage difference) across the membrane. If we were to calculate the ∆G for movement of the potassium ion from top to bottom, it would be positive, since [C2/C1] is greater than 1 (making for a positive ln term), and the ZF∆ψ is positive because positively charged ions (Z) are moving against a positive charge gradient given by ∆ψ (greater concentration at the target (bottom) than the starting point (top)). If we were to calculate the concentration of ions moving from bottom to top, then the ln term would be negative (C2<C1) and the ZF∆ψ would be negative as well (Z=positive, but ∆ψ negative). Reduction Potential In discussing chemical potential, we must also consider reduction potential. Reduction potential measures the tendency of a chemical to be reduced by electrons. It is also designated by several other names/variables. These include redox potential, oxidation/reduction potential, ORP, pE, ε, E, and Eh. Reduction potential is measured in volts, or millivolts. A substance with a higher reduction potential will have a greater tendency to accept electrons and be reduced. If two substances are mixed in an aqueous solution, the one with the greater (more positive) reduction potential will tend to take electrons away, thus being reduced, from the one with the lower reduction potential, which becomes oxidized. Relative measures Absolute reduction potentials are difficult to measure, so reduction potentials are typically defined relative to a reference electrode. In aqueous solutions, reduction potentials are measured as the potential difference between an inert sensing electrode (typically platinum) in contact with the test solution and a stable reference electrode (measured as a Standard Hydrogen Electrode: SHE) as shown in Figure $1$1. The standard of reference for measurement is the half-reaction H+ + e→ ½ H2 The electrode where this reaction occurs (referred to as a half-cell) is given the value of E° (Standard Reduction Potential) of 0.00 volts. The hydrogen electrode is connected via an external circuit to another half cell containing a mixture of the reduced and oxidized species of another molecule (for example, Fe++ and Fe+++) at 1M each and standard conditions of temperature (25°C) and pressure (1 atmosphere). Direction and voltage measured The direction and magnitude of electron movement is then measured. If the test mixture takes electrons from the hydrogen electrode, the sign of the voltage is positive and if the direction is reversed, the voltage is negative. Thus, compounds which have greater affinity for electrons than hydrogen will register a positive voltage and negative voltages correspond to compounds with lesser affinity for electrons than hydrogen. Movement of electrons Under standard conditions, electrons will move from compounds generating lower voltages to ones generating higher (more positive) voltages. Just as the standard Gibbs free energy change is the Gibbs free energy change under standard conditions, so, too, is the standard reduction potential E° the reduction potential E under standard conditions. The actual reduction potential of a half cell will vary with the concentration of each chemical species in the cell. The relationship between the reduction potential E and the standard reduction potential E° is given by the following equation (also called the Nernst equation) where F is the Faraday constant (96,480 J/(Volts*moles), R is the gas constant (8.315 J/(moles*K), n is the number of moles of electrons being transferred, and T is the absolute temperature in Kelvin. At 25°C, this equation becomes As for Gibbs free energy, it is useful to measure values at conditions found in cells. This means doing measurements at pH = 7, which differs from having all species at 1M. Adjustment Because of this adjustment, a slightly different standard reduction potential is defined and we designate it by E°’, just as we defined a special standard Gibbs free energy change at pH 7 as ΔG°’. There is a relationship between the change in Gibbs free energy ΔG and the change in reduction potential (ΔE). It is $ΔG = -nFΔE$ Similarly, the relation between the change in standard Gibbs free energy and the change in standard reduction potential is \]ΔG°’ = -nFΔE°’\] Energy Storage in Triphosphates Movie 5.1: ATP: The fuel of the cell Formation of triphosphates, like ATP, is essential to meeting the cell’s energy needs for synthesis, motion, and signaling. In a given day, an average human body makes and breaks down more than its weight in triphosphates. This is especially remarkable considering that there is only about 250 g of the molecule present in the body at any given time. Energy in ATP is released by hydrolysis of a phosphate from the molecule. The three phosphates, starting with the one closest to the sugar are referred to as α, β, and γ (Figure $1$2). It is the γ phosphate that is cleaved in hydrolysis and the product is ADP. In a few reactions, the bond between the α and β is cleaved. When this happens, a pyrophosphate (β linked to γ) is released and AMP is produced. This latter reaction to produce AMP releases more energy (ΔG°’ = -45.6 kJ/mol) than the first reaction which produces ADP (ΔG°’ = -30.5 kJ/mol). Since triphosphates are the “currency” that meet immediate needs of the cell, it is important to understand how triphosphates are made. There are three phosphorylation mechanisms – 1) substrate level; 2) oxidative; and 3) photophosphorylation. We consider them here individually. Substrate level phosphorylation The easiest type of phosphorylation to understand is that which occurs at the substrate level. This type of phosphorylation involves the direct synthesis of ATP from ADP and a high energy intermediate, typically a phosphate-containing molecule. Substrate level phosphorylation is a relatively minor contributor to the total synthesis of triphosphates by cells. An example substrate phosphorylation comes from glycolysis. Phosphoenolpyruvate (PEP) + ADP ⇌ ​ Pyruvate + ATP This reaction has a very negative ∆G°’ (-31.4 kJ/mol), indicating that the PEP contains more energy than ATP, thus tending to energetically favor ATP’s synthesis. Other triphosphates can be made by substrate level phosphorylation, as well. For example, GTP can be synthesized by the following citric acid cycle reaction. Succinyl-CoA + GDP + Pi ​ ⇌ Succinate + GTP + CoA-SH Triphosphates can be interchanged readily in substrate level phosphorylations catalyzed by the enzyme Nucleoside Diphosphate Kinase (NDPK). A generalized form of the reactions catalyzed by this enzyme is as follows: XTP + YDP ⇌ XDP + YTP where X = adenosine, cytidine, uridine, thymidine, or guanosine and Y can be any of these as well. Further, XTP and YDP can be any of the deoxynucleotides as well. Last, an unusual way of synthesizing ATP by substrate level phosphorylation is via the reaction catalyzed by adenylate kinase 2 ADP ⇌ ATP + AMP ATP source This reaction is an important means of generating ATP when the cell doesn’t have other sources of energy. Accumulation of AMP resulting from this reaction activates enzymes, such as phosphofructokinase, of glycolysis, which will catalyze reactions to give the cell additional, needed energy. It is important to note that enzymes cannot make reactions happen that are energetically unfavorable. Enzymes speed reactions, but do not change their direction. Cells are thus bound by the rules of Gibbs free energy. So, how do energetically unfavorable reactions happen in a cell? Reaction coupling Reactions that are energetically unfavorable, can be made favorable by coupling them with the hydrolysis of ATP, a very energetically favorable reaction. There are numerous parallels in the “real world.” Movement of automobiles is energetically unfavorable, but coupling movement of the automobile to oxidation of gasoline makes an unfavorable process favorable. Another approach to making an unfavorable reaction favorable is to manipulate the concentration of reactants and products. Consider the reaction below, which occurs in pyrimidine nucleotide metabolism: orotate + PRPP ⇌ OMP + PPi The ΔG°’ for this reaction is -0.8 kJ/mol, meaning that if one starts with equal concentrations of reactants and products, at equilibrium, there will be a small excess of products. In the cell, however, this reaction moves strongly to the right (ΔG = very negative). Given that the ΔG°’ is very close to zero, a very negative ΔG can only occur if the concentrations of reactants and products are altered, since $ΔG = ΔG°’ + RT \ln(\frac{[\rm{OMP}][\rm{PP_i}]}{[\rm{Orotate}][\rm{PRPP}]})$ Manipulation is exactly what happens here. The key item whose concentration is adjusted in this reaction is the pyrophosphate (PPi). This is possible because cells contain an enzyme called pyrophosphorylase that catalyzes the following reaction PPi + H2O ⇌ 2 Pi Hydrolysis of pyrophosphate is very energetically favored, causing the PPi produced in the reaction to be quickly hydrolyzed. As a result, the concentration of PPi in the cell is kept very low. A low concentration of a product (PPi) causes the natural log (ln) term of the orotate equation to become more negative, driving the ΔG term for the overall reaction to become much more negative. Pushing and pulling Reactions that yield pyrophosphate as a product are produced in the synthesis of DNA and RNA, as well as many other molecules. As shown in the previous example, this pyrophosphate is rapidly hydrolyzed, causing the overall reaction to move in the direction of pyrophosphate production. When reactants are removed/reduced in a metabolic reaction to decrease the concentration of a product, we say that the reaction is “pulled”, to represent the increase in the forward reaction as a result of product depletion. Pushing happens when reactants in a reaction are added/increased. This too has the effect of reducing the ΔG of a reaction and making it more favorable because the ratio of [Products]/[Reactants] is decreased with increasing [Reactants]. Pushing and pulling of reactions are additional tools for cells to overcome energy barriers, just like coupling energetically favorable processes to energetically unfavorable ones.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/05%3A_Energy/5.01%3A_Basics_of_Energy.txt

Source: BiochemFFA_5_3.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Photophosphorylation The third type of phosphorylation to make ATP is found only in cells that carry out photosynthesis. This process is similar to oxidative phosphorylation in several ways. A primary difference is the ultimate source of the energy for ATP synthesis. In oxidative phosphorylation, the energy comes from electrons produced by oxidation of biological molecules. In photosynthesis, the energy comes from the light of the sun. Photons from the sun interact with chlorophyll molecules in reaction centers in the chloroplasts (Figures \(1\) and \(2\)) of plants or membranes of photosynthetic bacteria. The similarities of photophosphorylation to oxidative phosphorylation include: • a membrane associated electron transport chain • creation of a proton gradient • harvesting energy of the proton gradient by making ATP with the help of an ATP synthase. Some of the differences include : • the source of the electrons – H2O for photosynthesis versus NADH/FADH2 for oxidative phosphorylation • direction of proton pumping – into the thylakoid space of the chloroplasts versus outside the matrix of the mitochondrion • movement of protons during ATP synthesis – out of the thylakoid space in photosynthesis versus into the mitochondrial matrix in oxidative phosphorylation • nature of the terminal electron acceptor – NADP+ in photosynthesis versus O2 in oxidative phosphorylation. Electron transport: chloroplasts vs mitochondria In some ways, the movement of electrons in chloroplasts during photosynthesis is opposite that of electron transport in mitochondria. In photosynthesis, water is the source of electrons and their final destination is NADP+ to make NADPH. In mitochondria, NADH/FADH2 are electron sources and H2O is their final destination. How do biological systems get electrons to go both ways? It would seem to be the equivalent of going to and from a particular place while always going downhill, since electrons will move according to potential. Solar power The answer is the captured energy of the photons from the sun (Figure 5.59), which elevates electrons to an energy where they move “downhill” to their NADPH destination in a Z-shaped scheme. The movement of electrons through this scheme in plants requires energy from photons in two places to “lift” the energy of the electrons sufficiently. Last, it should be noted that photosynthesis actually has two phases, referred to as the light cycle (described above) and the dark cycle, which is a set of chemical reactions that captures CO2 from the atmosphere and “fixes” it, ultimately into glucose. The dark cycle is also referred to as the Calvin Cycle and is discussed HERE. Photosynthesis Photosynthesis is an energy capture process found in plants and other organisms to harvest light energy and convert it into chemical energy. This photochemical energy is stored ultimately in carbohydrates which are made using ATP (from the energy harvesting), carbon dioxide and water. In most cases, a byproduct of the process is oxygen, which is released from water in the capture process. Photosynthesis is responsible for most of the oxygen in the atmosphere and it supplies the organic materials and most of the energy used by life on Earth. Steps The steps in the photosynthesis process varies slightly between organisms. In a broad overview, it always starts with energy capture from light by protein complexes, containing chlorophyll pigments, called reaction centers. Plants sequester these proteins in chloroplasts, but bacteria, which don’t have organelles, embed them in their plasma membranes. Energy from the light is used to strip electrons away from electron donors (usually water) and leave a byproduct (oxygen, if water was used). Electrons are donated to a carrier and ultimately are accepted by NADP+, to become NADPH. As electrons travel towards NADP+, they generate a proton gradient across the thylakoid membrane, which is used to drive synthesis of ATP. Thus NADPH, ATP, and oxygen are the products of the first phase of photosynthesis called the light reactions. Energy from ATP and electrons from NADPH are used to reduce CO2 and build sugars, which are the ultimate energy storage directly arising from photosynthesis. Chloroplasts Chloroplasts are found in almost all aboveground plant cells, but are primarily concentrated in leaves. The interior of a leaf, below the epidermis is made up of photosynthesis tissue called mesophyll, which can contain up to 800,000 chloroplasts per square millimeter. The chloroplast’s membrane has a phospholipid inner membrane, a phospholipid outer membrane, and a region between them called the intermembrane space (Figure 5.61). Within the inner chloroplast membrane is the stroma, in which the chloroplast DNA and the enzymes of the Calvin cycle are located. Also within the stroma are stacked, flattened disks known as thylakoids which are defined by their thylakoid membranes. The space within the thylakoid membranes are termed the thylakoid spaces or thylakoid lumen. The protein complexes containing the light-absorbing pigments, known as photosystems, are located on the thylakoid membrane. Besides chlorophylls, carotenes and xanthophylls are also present, allowing for absorption of light energy over a wider range. The same pigments are used by green algae and land plants. Brown algae and diatoms add fucoxanthin (a xanthophyll) and red algae add phycoerythrin to the mix. In plants and algae, the pigments are held in a very organized fashion complexes called antenna proteins that help funnel energy, through resonance energy transfer, to the reaction center chlorophylls. A system so organized is called a light harvesting complex. The electron transport complexes of photosynthesis are also located on the thylakoid membranes. Figure \(6\): Complexes in the thylakoid membrane. Image by Aleia Kim Light reactions of photosynthesis In chloroplasts, the light reactions of photosynthesis involving electron transfer occur in the thylakoid membranes (Figure \(6\)). Separate biochemical reactions involving the assimilation of carbon dioxide to make glucose are referred to as the Calvin cycle, also sometimes referred to as the “dark reactions”. This will be discussed elsewhere in the section on metabolism (HERE). The chloroplasts are where the energy of light is captured, electrons are stripped from water, oxygen is liberated, electron transport occurs, NADPH is formed, and ATP is generated. The thylakoid membrane corresponds to the inner membrane of the mitochondrion for transport of electrons and proton pumping (Figure \(4\)). The thylakoid membrane does its magic using four major protein complexes. These include Photosystem II (PS II), Cytochrome b6f complex (Cb6f), Photosystem I (PS I), and ATP synthase. The roles of these complexes, respectively, are to capture light energy, create a proton gradient from electron movement, capture light energy (again), and use proton gradient energy from the overall process to synthesize ATP. Light harvesting Harvesting the energy of light begins in PS II with the absorption of a photon of light at a reaction center. PS II performs this duty best with light at a wavelength of 680 nm and it readily loses an electron to excitation when this occurs, leaving PS II with a positive charge. This electron must be replaced. The ultimate replacement source of electrons is water, but water must lose four electrons and PS II can only accept one at a time. Manganese centers An intermediate Oxygen Evolving Complex (OEC) contains four manganese centers that provide the immediate replacement electron that PSII requires. After four electrons have been donated by the OEC to PS II, the OEC extracts four electrons from two water molecules, liberating oxygen and dumping four protons into the thylakoid space, thus contributing to the proton gradient. The excited electron from PS II must be passed to another carrier very quickly, lest it decay back to its original state. It does this, giving its electron within picoseconds to pheophytin (Figure \(8\)). Pheophytin passes the electron on to protein-bound plastoquinones . The first is known as PQA. PQA hands the electron off to a second plastoquinone (PQB), which waits for a second electron and collects two protons to become PQH2, also known as plastoquinol (Figure \(9\)). PQH2 passes these to the Cytochrome b6f complex (Cb6f) which uses passage of electrons through it to pump protons into the thylakoid space. ATP synthase makes ATP from the proton gradient created in this way. Cb6f drops the electron off at plastocyanin, which holds it until the next excitation process begins with absorption of another photon of light at 700 nm by PS I. Absorption of light at PS I With absorption of a photon of light by PS I, a process begins, that is similar to the process in PS II. PS I gains a positive charge as a result of the loss of an excited electron and pulls the electron in plastocyanin away from it. Meanwhile, the excited electron from PS I passes through an iron-sulfur protein, which gives the electron to ferredoxin (another iron sulfur protein). Ferredoxin then passes the electron off to the last protein in the system known as Ferredoxin:NADP+ oxidoreductase, which gives the electron and a proton to NADP+, creating NADPH. Note that reduction of NADP+ to NADPH requires two electrons and one proton, so the four electrons and two protons from oxidation of water will result in production of two molecules of NADPH. At this point, the light cycle is complete - water has been oxidized, ATP has been created, and NADPH has been made. The electrons have made their way from water to NADPH via carriers in the thylakoid membrane and their movement has released sufficient energy to make ATP. Energy for the entire process came from four photons of light. The two photosystems performing all of this magic are protein complexes that are similar in structure and means of operation. They absorb photons with high efficiency so that whenever a pigment in the photosynthetic reaction center absorbs a photon, an electron from the pigment is excited and transferred to another molecule almost instantaneously. This reaction is called photo-induced charge separation and it is a unique means of transforming light energy into chemical forms. Cyclic photophosphorylation Besides the path described above for movement of electrons through PS I, plants have an alternative route that electrons can take. Instead of electrons going through ferredoxin to form NADPH, they instead take a backwards path through the the proton-pumping b6f complex. This system, called cyclic photophosphorylation (Figure \(8\)) which generates more ATP and no NADPH, is similar to a system found in green sulfur bacteria. The ability of plants to switch between non-cyclic and cyclic photosystems allows them to make the proper ratio of ATP and NADPH they need for assimilation of carbon in the dark phase of photosynthesis. This ratio turns out to be 3 ATPs to 2 NADPHs. Figure \(9\) - Photosystem II of cyanobacteria. Wikipedia Photosynthetic energy The output of the photophosphorylation part of photosynthesis (O2, NADPH, and ATP), of course, is not the end of the process of photosynthesis. For the growing plant, the NADPH and ATP are used to capture carbon dioxide from the atmosphere and convert it (ultimately) into glucose and other important carbon compounds. This, as noted previously, occurs in the Calvin Cycle (see HERE) in what is called the dark phase of the process. The oxygen liberated in the process is a necessary for respiration of all aerobic life forms on Earth. Indeed, it is believed that essentially all of the oxygen in the atmosphere today is the result the splitting of water in photosynthesis over the many eons that the process has existed.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/05%3A_Energy/5.03%3A_Energy_-_Photophosphorylation.txt

Source: BiochemFFA_5_2.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy In eukaryotic cells, the vast majority of ATP synthesis occurs in the mitochondria in a process called oxidative phosphorylation. Even plants, which generate ATP by photophosphorylation in chloroplasts, contain mitochondria for the synthesis of ATP through oxidative phosphorylation. Oxidative phosphorylation is linked to a process known as electron transport (Figure 5.14). The electron transport system, located in the inner mitochondrial membrane, transfers electrons donated by the reduced electron carriers NADH and FADH2 (obtained from glycolysis, the citric acid cycle or fatty acid oxidation) through a series of electrons acceptors, to oxygen. As we shall see, movement of electrons through complexes of the electron transport system essentially “charges” a battery that is used to make ATP in oxidative phosphorylation. In this way, the oxidation of sugars and fatty acids is coupled to the synthesis of ATP, effectively extracting energy from food. Chemiosmotic model Dr. Peter Mitchell introduced a radical proposal in 1961 to explain the mechanism by which mitochondria make ATP. It is known as the chemiosmotic hypothesis and has been shown over the years to be correct. Mitchell proposed that synthesis of ATP in mitochondria depends on an electrochemical gradient, across the mitochondrial inner membrane, that arises ultimately from the energy of reduced electron carriers, NADH and FADH2. Electron transport Further, the proposal states that the gradient is created when NADH and FADH2 transfer their electrons to an electron transport system (ETS) located in the inner mitochondrial membrane. Movement of electrons through a series of of electron carriers is coupled to the pumping of protons out of the mitochondrial matrix across the inner mitochondrial membrane into the space between the inner and outer membranes. The result is creation of a gradient of protons whose potential energy can be used to make ATP. Electrons combine with oxygen and protons at the end of the ETS to make water. ATP synthase In oxidative phosphorylation, ATP synthesis is accomplished as a result of protons re-entering the mitochondrial matrix via the transmembrane ATP synthase complex, which combines ADP with inorganic phosphate to make ATP. Central to the proper functioning of mitochondria through this process is the presence of an intact mitochondrial inner membrane impermeable to protons. Tight coupling When this is the case, tight coupling is said to exist between electron transport and the synthesis of ATP (called oxidative phosphorylation). Chemicals which permeabilize the inner mitochondrial membrane to protons cause uncoupling, that is, they allow the protons to leak back into the mitochondrial matrix, rather than through the ATP synthase, so that the movement of electrons through the ETS is no longer linked to the synthesis of ATP. Power plants Mitochondria are called the power plants of the cell because most of a cell’s ATP is produced there in the process of oxidative phosphorylation. The mechanism by which ATP is made in oxidative phosphorylation is one of the most interesting in all of biology. Considerations The process has three primary considerations. The first is electrical – electrons from reduced electron carriers, such as NADH and FADH2, enter the electron transport system via Complex I and II, respectively. As seen in Figure 5.16 and Figure 5.17, electrons move from one complex to the next, not unlike the way they move through an electrical circuit. Such movement occurs a a result of a set of reduction-oxidation (redox) reactions with electrons moving from a more negative reduction potential to a more positive one. One can think of this occurring as a process where carriers “take” electrons away from complexes with lower reduction potential, much the way a bully takes lunch money from a smaller child. In this scheme, the biggest “bully” is oxygen in Complex IV. Electrons gained by a carrier cause it to be reduced, whereas the carrier giving up the electrons is oxidized. Entry of electrons to system Movement of electrons through the chain begins either by 1) transfer from NADH to Complex I (Figure 5.16) or 2) movement of electrons through a covalently bound FADH2 (Figure 5.17) in the membrane-bound succinate dehydrogenase (Complex II). (An alternate entry point for electrons from FADH2 is the Electron Transferring Flavoprotein via the electron-transferring-flavoprotein dehydrogenase, not shown). Traffic cop Both Complex I and II pass electrons to the inner membrane’s coenzyme Q (CoQ - Figures 5.18 & 5.19). In each case, coenzyme Q accepts electrons in pairs and passes them off to Complex III (CoQH2-cytochrome c reductase) singly. Coenzyme Q thus acts as a traffic cop, regulating the flow of electrons through the ETS. Docking station Complex III is a docking station or interchange for the incoming electron carrier (coenzyme Q) and the outgoing carrier (cytochrome c). Movement of electrons from Coenzyme Q to Complex III and then to cytochrome C occurs as a result of what is referred to as the Q-cycle (see below). Complex III acts to ferry electrons from CoQ to cytochrome c. Cytochrome c takes one electron from Complex III and passes it to Complex IV (cytochrome oxidase). Complex IV is the final protein recipient of the electrons. It passes them to molecular oxygen (O2) to make two molecules of water. Making two water molecules requires four electrons, so Complex IV must accept, handle, and pass to molecular oxygen four separate electrons, causing the oxidation state of oxygen to be sequentially changed with addition of each electron. Proton pumping As electrons pass through complexes I, III, and IV, there is a release of a small amount of energy at each step, which is used to pump protons from the mitochondrial matrix (inside of mitochondrion) and deposit them in the intermembrane space (between the inner and outer membranes of the mitochondrion). The effect of this redistribution is to increase the electrical and chemical potential across the membrane. Potential energy As discussed earlier, electrochemical gradients have potential energy. Students may think of the process as “charging the battery.” Just like a charged battery, the potential arising from the proton differential across the membrane can be used to do things. In the mitochondrion, what the proton gradient does is facilitate the production of ATP from ADP and Pi. This process is known as oxidative phosphorylation, because the phosphorylation of ADP to ATP is dependent on the oxidative reactions occurring in the mitochondria. Having understood the overall picture of the synthesis of ATP linked to the movement of electrons through the ETS, we will take a closer look at the individual components of the ETS. Complex I Complex I (also called NADH:ubiquinone oxidoreductase or NADH dehydrogenase (ubiquinone)) is the electron acceptor from NADH in the electron transport chain and the largest complex found in it. Complex I contains 44 individual polypeptide chains, numerous iron-sulfur centers, a molecule of flavin mononucleotide (FMN) and has an L shape with about 60 transmembrane domains. In the process of electron transport through it, four protons are pumped across the inner membrane into the intermembrane space and electrons move from NADH to coenzyme Q, converting it from ubiquinone (no electrons) to ubiquinol (gain of two electrons). An intermediate form, ubisemiquinone (gain of one electron), is found in the Q-cycle. Electrons travel through the complex via seven primary iron sulfur centers. The best known inhibitor of the complex, rotenone, works by binding to the CoQ binding site. Other inhibitors include ADP-ribose (binds to the NADH site) and piericidin A (rotenone analog). The process of electron transfer through complex I is reversible and when this occurs, superoxide (a reactive oxygen species) may be readily generated. Complex II Complex II (also called succinate dehydrogenase or succinate-coenzyme Q reductase ) is a membrane bound enzyme of the citric acid cycle that plays a role in the electron transport process, transferring electrons from its covalently bound FADH2 to coenzyme Q. The process occurs, as shown in Figure 5.20 and Figure 5.21, with transfer of electrons from succinate to FAD to form FADH2 and fumarate. FADH2, in turn, donates electrons to a relay system of iron-sulfur groups and they ultimately reduce ubiquinone (CoQ) along with two protons from the matrix to ubiquinol. The role of the heme group in the process is not clear. Inhibitors of the process include carboxin, malonate, malate, and oxaloacetate. The role of citric acid cycle intermediates as inhibitors is thought to be due to inhibition of the reversal of the transfer process which can produce superoxide. Coenzyme Q Coenzyme Q (Figure 5.23) is a 1,4 benzoquinone whose name is often given as Coenzyme Q10, CoQ, or Q10. The 10 in the name refers to the number of isoprenyl units it contains that anchor it to the mitochondrial inner membrane. CoQ is a vitamin-like lipid substance found in most eukaryotic cells as a component of the electron transport system. The requirement for CoQ increases with increasing energy needs of cells, so the highest concentrations of CoQ in the body are found in tissues that are the most metabolically active - heart, liver, and kidney. Three forms CoQ is useful because of its ability to carry and donate electrons and particularly because it can exist in forms with two extra electrons (fully reduced - ubiquinol), one extra electron (semi-reduced - ubisemiquinone), or no extra electrons (fully oxidized - ubiquinone). This ability allows CoQ to provide transition between the first part of the electron transport system that moves electrons in pairs and the last part of the system that moves electrons one at a time. Complex III Complex III (also known as coenzyme Q : cytochrome c — oxidoreductase or the cytochrome bc1 complex - Figure 5.24) is the third electron accepting complex of the electron transport system. It is a transmembrane protein with multiple subunits present in the mitochondria of all aerobic eukaryotic organisms and and the cell membrane of almost all bacteria. The complex contains 11 subunits, a 2-iron ferredoxin, cytochromes b and c1 and belongs to the family of oxidoreductase enzymes. It accepts electrons from coenzyme Q in electron transport and passes them off to cytochrome c. In this cycle, known as the Q cycle, electrons arrive from CoQ in pairs, but get passed to cytochrome c individually. In the overall process, two protons are consumed from the matrix and four protons are pumped into the intermembrane space. Movement of electrons through the complex can be inhibited by antimycin A, myxothiazol, and stigmatellin. Complex III is also implicated in creation of superoxide (a reactive oxygen species) when electrons from it leak out of the chain of transfer. The phenomenon is more pronounced when antimycin A is present. Q-cycle In the Q-cycle, electrons are passed from ubiquinol (QH2) to cytochrome c using Complex III as an intermediary docking station for the transfer. Two pair of electrons enter from QH2 and one pair is returned to another CoQ to re-make QH2. The other pair is donated singly to two different cytochrome c molecules. Step one The Q-cycle happens in a two step process. First, a ubiquinol (CoQH2) and a ubiquinone (CoQ) dock at Complex III. Ubiquinol transfers two electrons to Complex III. One electron goes to a docked cytochrome c, reducing it and it exits (replaced by an oxidized cytochrome c). The other goes to the docked uniquinone to create the semi-reduced semiubiquinone (CoQ.-) and leaving behind a ubiquinone, which exits. This is the end of step 1. Step two The gap left behind by the ubiquinone (Q) that departed is replaced by another ubiquinol (QH2). It too donates two electrons to Complex III, which splits them. One goes to the newly docked oxidized cytochrome c, which is reduced and exits. The other goes to the ubisemiquinone. Two protons from the matrix combine with it to make another ubiquinol. It and the ubiquinone created by the electron donation exit Complex III and the process starts again. In the overall process, two protons are consumed from the matrix and four protons are pumped into the intermembrane space. Cytochrome c Cytochrome c (Figure 5.26) is a small (12,000 Daltons), highly conserved protein, from unicellular species to animals, that is loosely associated with the inner mitochondrial membrane where it functions in electron transport. It contains a heme group which is used to carry a single electron from Complex III to Complex IV. Cytochrome c also plays an important role in apoptosis in higher organisms. Damage to the mitochondrion that results in release of cytochrome c can stimulate assembly of the apoptosome and activation of the caspase cascade that leads to programmed cell death. Complex IV Complex IV, also known as cytochrome c oxidase is a 14 subunit integral membrane protein at the end of the electron transport chain (Figure 5.27). It is responsible for accepting one electron each from four cytochrome c proteins and adding them to molecular oxygen (O2) along with four protons from the mitochondrial matrix to make two molecules of water. Four protons from the matrix are also pumped into the intermembrane space in the process. The complex has two molecules of heme, two cytochromes (a and a3), and two copper centers (called CuA ad CuB). Cytochrome c docks near the CuA and donates an electron to it. The reduced CuA passes the electron to cytochrome a, which turns it over to the a3-CuB center where the oxygen is reduced. The four electrons are thought to pass through the complex rapidly resulting in complete reduction of the oxygen-oxygen molecule without formation of a peroxide intermediate or superoxide, in contrast to previous predictions. Respirasome There has been speculation for many years that a supercomplex of electron carriers in the inner membrane of the mitochondrion may exist in cells with individual carriers making physical contact with each other. This would make for more efficient transfer reactions, minimize the production of reactive oxygen species and be similar to metabolons of metabolic pathway enzymes, for which there is some evidence. Now, evidence appears to be accumulating that complexes I, III, and IV form a supercomplex, which has been dubbed the respirasome1. Oxidative phosphorylation The process of oxidative phosphorylation uses the energy of the proton gradient established by the electron transport system as a means of phosphorylating ADP to make ATP. The establishment of the proton gradient is dependent upon electron transport. If electron transport stops or if the inner mitochondrial membrane’s impermeability to protons is compromised, oxidative phosphorylation will not occur because without the proton gradient to drive the ATP synthase, there will be no synthesis of ATP. ATP synthase The protein complex harvesting energy from the proton gradient and using it to make ATP from ADP is an enzyme that has several names - Complex V, PTAS (Proton Translocating ATP Synthase), and ATP synthase (Figure 5.29). Central to its function is the movement of protons through it (from the intermembrane space back into the matrix). Protons will only provide energy to make ATP if their concentration is greater in the intermembrane space than in the matrix and if ADP is available. It is possible, in some cases, for the concentration of protons to be greater inside the matrix than outside of it. When this happens, the ATP synthase can run backwards, with protons moving from inside to out, accompanied by conversion of ATP to ADP + Pi. This is usually not a desirable circumstance and there are some controls to reduce its occurrence. Normally, ATP concentration will be higher inside of the mitochondrion and ADP concentration be higher outside the mitochondrion. However, when the rate of ATP synthesis exceeds the rate of ATP usage, then ATP concentrations rise outside the mitochondrion and ADP concentrations fall everywhere. This may happen, for example, during periods of rest. It has the overall effect of reducing transport and thus lowering the concentration of ADP inside the matrix. Reducing ADP concentration in the matrix reduces oxidative phosphorylation and has effects on respiratory control (see HERE). Another important consideration is that when ATP is made in oxidative phosphorylation, it is released into the mitochondrial matrix, but must be transported into the cytosol to meet the energy needs of the rest of the cell. This is accomplished by action of the adenine nucleotide translocase, an antiport that moves ATP out of the matrix in exchange for ADP moving into the matrix. This transport system is driven by the concentrations of ADP and ATP and ensures that levels of ADP are maintained within the mitochondrion, permitting continued ATP synthesis. One last requirement for synthesis of ATP from ADP is that phosphate must also be imported into the matrix. This is accomplished by action of the phosphate translocase, which is a symport that moves phosphate into the mitochondrial matrix along with a proton. There is evidence that the two translocases and ATP synthase may exist in a complex, which has been dubbed the ATP synthasome. In summary, the electron transport system charges the battery for oxidative phosphorylation by pumping protons out of the mitochondrion. The intact inner membrane of the mitochondrion keeps the protons out, except for those that re-enter through ATP Synthase. The ATP Synthase allows protons to re-enter the mitochondrial matrix and harvests their energy to make ATP. ATP synthase mechanism In ATP Synthase, the spinning components, or rotor, are the membrane portion (c ring) of the F0 base and the γ-ε stalk, which is connected to it. The γ-ε stalk projects into the F1 head of the mushroom structure. The F1 head contains the catalytic ability to make ATP. The F1 head is hexameric in structure with paired α and β proteins arranged in a trimer of dimers. ATP synthesis occurs within the β subunits. Rotation of γ unit Turning of the γ shaft (caused by proton flow) inside the α-β trimer of the F1 head causes each set of β proteins to change structure slightly into three different forms called Loose, Tight, and Open (L,T,O - Figure 5.31). Each of these forms has a function. The Loose form binds ADP + Pi. The Tight form “squeezes” them together to form the ATP. The Open form releases the ATP into the mitochondrial matrix. Thus, as a result of the proton flow through the ATP synthase, from the intermembrane space into the matrix, ATP is made from ADP and Pi. Respiratory control When a mitochondrion has an intact inner membrane and protons can only return to the matrix by passing through the ATP synthase, the processes of electron transport and oxidative phosphorylation are said to be tightly coupled. Interdependence In simple terms, tight coupling means that the processes of electron transport and oxidative phosphorylation are interdependent. Without electron transport going on in the cell, oxidative phosphorylation will soon stop. The reverse is also true, because if oxidative phosphorylation stops, the proton gradient will not be dissipated as it is being built by the electron transport system and will grow larger and larger. The greater the gradient, the greater the energy needed to pump protons out of the mitochondrion. Eventually, if nothing relieves the gradient, it becomes too large and the energy of electron transport is insufficient to perform the pumping. When pumping stops, so too does electron transport. ADP dependence Another relevant point is that ATP synthase is totally dependent upon a supply of ADP. In the absence of ADP, the ATP synthase stops functioning and when it stops, so too does movement of protons back into the mitochondrion. With this information, it is possible to understand the link between energy usage and metabolism. The root of this, as noted, is respiratory control. At rest To illustrate these links, let us first consider a person, initially at rest, who then suddenly jumps up and runs away. At first, the person’s ATP levels are high and ADP levels are low (no exercise to burn ATP), so little oxidative phosphorylation is occurring and thus the proton gradient is high. Electron transport is moving slowly, if at all, so it is not using oxygen and the person’s breathing is slow, as a result. Exercise When running starts, muscular contraction, which uses energy, causes ATP to be converted to ADP. Increasing ADP in muscle cells favors oxidative phosphorylation to attempt to make up for the ATP being burned. ATP synthase begins working and protons begin to come back into the mitochondrial matrix. The proton gradient decreases, so electron transport re-starts. Electron transport needs an electron acceptor, so oxygen use increases and when oxygen use increases, the person starts breathing more heavily to supply it. When the person stops running, ATP concentrations get rebuilt by ATP synthase. Eventually, when ATP levels are completely restored, ADP levels fall and ATP synthase stops or slows considerably. With little or no proton movement, electron transport stops because the proton gradient is too large. When electron transport stops, oxygen use decreases and the rate of breathing slows down. Electron transport critical The really interesting links to metabolism occur relative to whether or not electron transport is occurring. From the examples, we can see that electron transport will be relatively slowed when not exercising and more rapid when exercise (or other ATP usage) is occurring. Remember that electron transport is the way in which reduced electron carriers, NADH and FADH2, donate their electrons to the ETS , becoming oxidized to NAD+ and FAD, respectively. Oxidized carriers, such as NAD+ and FAD are needed by catabolic pathways, like glycolysis, the citric acid cycle, and fatty acid oxidation. Anabolic pathways, such as fatty acid/fat synthesis and gluconeogenesis rely on reduced electron carriers, such as FADH2, NADH, and the related carrier, NADPH. Links to exercise High levels of NADH and FADH2 prevent catabolic pathways from operating, since NAD+ and FAD levels will be low and these are needed to accept the electrons released during catabolism by the oxidative processes. Thanks to respiratory control, when one is exercising, NAD+ and FAD levels increase (because electron transport is running), so catabolic pathways that need NAD+ and FAD can function. The electrons lost in the oxidation reactions of catabolism are captured by NAD+ and FAD to yield NADH and FADH2, which then supply electrons to the electron transport system and oxidative phosphorylation to make more needed ATP. Thus, during exercise, cells move to a mode of quickly cycling between reduced electron carriers (NADH/FADH2) and oxidized electron carriers (NAD+/FAD). This allows rapidly metabolizing tissues to transfer electrons to NAD+/FAD and it allows the reduced electron carriers to rapidly become oxidized, allowing the cell to produce ATP. Rest When exercise stops, NADH and FADH2 levels rise (because electron transport is slowing) causing catabolic pathways to slow/stop. If one does not have the proper amount of exercise, reduced carriers remain high in concentration for long periods of time. This means we have an excess of energy and then anabolic pathways, particularly fatty acid synthesis, are favored, so we get fatter. Altering respiratory control One might suspect that altering respiratory control could have some very dire consequences and that would be correct. Alterations can take the form of either inhibiting electron transport/oxidative phosphorylation or uncoupling the two . These alterations can be achieved using compounds with specific effects on particular components of the system. All of the chemicals described here are laboratory tools and should never be used by people. The first group for discussion are the inhibitors. In tightly coupled mitochondria, inhibiting either electron transport or oxidative phosphorylation has the effect of inhibiting the other one as well. Electron transport inhibitors Common inhibitors of electron transport include rotenone and amytal, which stop movement of electrons past Complex I, malonate, malate, and oxaloacetate, which inhibit movement of electrons through Complex II, antimycin A which stops movement of electrons past Complex III, and cyanide, carbon monoxide, azide, and hydrogen sulfide, which inhibit electron movement through Complex IV (Figure 5.33). All of these compounds can stop electron transport directly (no movement of electrons) and oxidative phosphorylation indirectly (proton gradient will dissipate). While some of these compounds are not commonly known, almost everyone is aware of the hazards of carbon monoxide and cyanide, both of which can be lethal. ATP synthase inhibitor It is also possible to use an inhibitor of ATP synthase to stop oxidative phosphorylation directly (no ATP production) and electron transport indirectly (proton gradient not relieved so it becomes increasingly difficult to pump protons out of matrix). Oligomycin A (Figure 5.34) is an inhibitor of ATP synthase. Rotenone Rotenone, which is a plant product, is used as a natural insecticide that is permitted for organic farming. When mitochondria are treated with this, electron transport will stop at Complex I and so, too, will the pumping of protons out of the matrix. When this occurs, the proton gradient rapidly dissipates, stopping oxidative phosphorylation as a consequence. There are other entry points for electrons than Complex I, so this type of inhibition is not as serious as using inhibitors of Complex IV, since no alternative route for electrons is available. It is for this reason that cyanide, for example, is so poisonous. 2,4-DNP Imagine a dam holding back water with a turbine generating electricity through which water must flow. When all water flows through the turbine, the maximum amount of electricity can be generated. If one pokes a hole in the dam, though, water will flow through the hole and less electricity will be created. The generation of electricity will thus be uncoupled from the flow of water. If the hole is big enough, the water will all drain out through the hole and no electricity will be made. Bypassing ATP synthase Imagine, now, that the proton gradient is the equivalent of the water, the inner membrane is the equivalent of the dam and the ATP synthase is the turbine. When protons have an alternate route, little or no ATP will be made because protons will pass through the membrane’s holes instead of spinning the turbine of ATP synthase. It is important to recognize, though, that uncoupling by 2,4 DNP works differently from the electron transport inhibitors or the ATP synthase inhibitor. In those situations, stopping oxidative phosphorylation resulted in indirectly stopping electron transport, since the two processes were coupled and the inhibitors did not uncouple them. Similarly, stopping electron transport indirectly stopped oxidative phosphorylation for the same reason. Such is not the case with 2,4 DNP. Stopping oxidative phosphorylation by destroying the proton gradient allows electron transport to continue unabated (it actually stimulates it), since the proton gradient cannot build no matter how much electron transport runs. Consequently, electron transport runs like crazy but oxidative phosphorylation stops. When that happens, NAD+ and FAD levels rise, and catabolic pathways run unabated with abundant supplies of these electron acceptors. The reason such a scenario is dangerous is because the body is using all of its nutrient resources, but no ATP is being made. Lack of ATP leads to cellular (and organismal) death. In addition, the large amounts of heat generated can raise the temperature of the body to unsafe levels. Thermogenin One of the byproducts of uncoupling electron transport is the production of heat. The faster metabolic pathways run, the more heat is generated as a byproduct. Since 2,4 DNP causes metabolism to speed up, a considerable amount of heat can be produced. Controlled uncoupling is actually used by the body in special tissues called brown fat. In this case, brown fat cells use the heat created to help thermoregulate the temperature of newborn children. Permeabilization of the inner membrane is accomplished in brown fat by the synthesis of a protein called thermogenin (also known as uncoupling protein). Thermogenin binds to the inner membrane and allows protons to pass through it, thus bypassing the ATP synthase. As noted for 2,4 DNP, this results in activation of catabolic pathways and the more catabolism occurs, the more heat is generated. Dangerous drug In uncoupling, whether through the action of an endogenous uncoupling protein or DNP, the energy that would have normally been captured in ATP is lost as heat. In the case of uncoupling by thermogenin, this serves the important purpose of keeping newborn infants warm. But in adults, uncoupling merely wastes the energy that would have been harvested as ATP. In other words, it mimics starvation, even though there is plenty of food, because the energy is dissipated as heat. This fact, and the associated increase in metabolic rate, led to DNP being used as a weight loss drug in the 1930s. Touted as an effortless way to lose weight without having to eat less or exercise more, it was hailed as a magic weight loss pill. It quickly became apparent, however, that this was very dangerous. Many people died from using this drug before laws were passed to ban the use of DNP as a weight loss aid. Alternative oxidase Another approach to generating heat that doesn’t involve breaking respiratory control is taken by some fungi, plants, and protozoa. They use an alternative electron transport. In these organisms, there is an enzyme called alternative oxidase (Figure 5.36). Alternative oxidase is able to accept electrons from CoQ and pass them directly to oxygen. The process occurs in coupled mitochondria. Its mechanism of action is to reduce the yield of ATP, since fewer protons are being pumped per reduced electron carrier. Thus NAD+ concentrations increase, oxygen consumption increases, and the efficiency of ATP production decreases. Organisms using this method must activate catabolic pathways by the increase in NAD+ concentration. This, in turn produces quantities of NADH and FADH2 necessary to make sufficient amounts of ATP. The byproduct of this increased catabolism is more heat. Not surprisingly, the alternative oxidase pathway can be activated by cold temperatures. Energy efficiency Cells are not 100% efficient in energy use. Nothing we know is. Consequently, cells do not get as much energy out of catabolic processes as they put into anabolic processes. A good example is the synthesis and breakdown of glucose, something liver cells are frequently doing. The complete conversion of glucose to pyruvate in glycolysis (catabolism) yields two pyruvates plus 2 NADH plus 2 ATPs. Conversely, the complete conversion of two pyruvates into glucose by gluconeogenesis (anabolism) requires 4 ATPs, 2 NADH, and 2 GTPs. Since the energy of GTP is essentially equal to that of ATP, gluconeogenesis requires a net of 4 ATPs more than glycolysis yields. This difference must be made up in order for the organism to meet its energy needs. It is for this reason that we eat. In addition, the inefficiency of our capture of energy in reactions results in the production of heat and helps to keep us warm, as noted. You can read more about glycolysis (HERE) and gluconeogenesis (HERE). Metabolic controls of energy It is also noteworthy that cells do not usually have both catabolic and anabolic processes for the same molecules occurring simultaneously inside of them (for example, breakdown of glucose and synthesis of glucose) because the cell would see no net production of anything but heat and a loss of ATPs with each turn of the cycle. Such cycles are called futile cycles and cells have controls in place to limit the extent to which they occur. Since futile cycles can, in fact, yield heat, they are used as sources of heat in some types of tissue. Brown adipose tissue of mammals uses this strategy, as described earlier. See also HERE for more on heat generation with a futile cycle. Reactive oxygen species Endogenous production of ROS is directed towards intracellular signaling (H2O2 and nitric oxide, for example) and defense. Many cells, for example, have NADPH oxidase (Figure 5.38) embedded in the exterior portion of the plasma membranes, in peroxisomes, and endoplasmic reticulum. It produces superoxides in the reaction below to kill bacteria . In the immune system, cells called phagocytes engulf foreign cells and then use ROS to kill them. ROS can serve as signals for action. In zebrafish, damaged tissues have increased levels of H2O2 and this is thought to be a signal for white blood cells to converge on the site. In fish lacking the genes to produce hydrogen peroxide, white blood cells do not converge at the damage site. Sources of hydrogen peroxide include peroxisomes, which generate it as a byproduct of oxidation of long chain fatty acids. Aging Reactive oxygen species are at the heart of the free radical theory of aging, which states that organisms age due to the accumulation of damage from free radicals in their cells. In yeast and Drosophila, there is evidence that reducing oxidative damage can increase lifespan. In mice, increasing oxidative damage decreases life span, though in Caenorhabditis, blocking production of superoxide dismutase actually increases lifespan, so the role of ROS in aging is not completely clear. It is clear, though, that accumulation of mitochondrial damage is problematic for individual cells. Bcl-2 proteins on the surface of mitochondria monitor damage and if they detect it, will activate proteins called Bax to stimulate the release of cytochrome c from the mitochondrial membrane, stimulating apoptosis (programmed cell death). Eventually the dead cell will be phagocytosed. A common endogenous source of superoxide is the electron transport chain. Superoxide can be produced when movement of electrons into and out of the chain don’t match well. Under these circumstances, semi-reduced CoQ can donate an electron to O2 to form superoxide (O2-). Superoxide can react with many molecules, including DNA where it can cause damage leading to mutation. If it reacts with the aconitase enzyme, ferrous iron (Fe++) can be released which, in turn, can react in the Fenton reaction to produce another reactive oxygen species, the hydroxyl radical (Figure 5.39) . Countering the effects of ROS are enzymes, such as catalase, superoxide dismutase, and anti-oxidants, such as glutathione and vitamins C and E. Glutathione protects against oxidative damage by being a substrate for the enzyme glutathione peroxidase. Glutathione peroxidase catalyzes the conversion of hydrogen peroxide to water (next page). Catalase 2 H2O2 <=> 2 H2O + O2 The enzyme, which employs four heme groups in its catalysis, works extremely rapidly, converting up to 40,000,000 molecules of hydrogen peroxide to water and oxygen per enzyme per second. It is abundantly found in peroxisomes. In addition to catalase’s ability to break down hydrogen peroxide, the enzyme can also use hydrogen peroxide to oxidize a wide variety organic compounds, including phenols, formic acid, formaldehyde, acetaldehyde, and alcohols, but with much lower efficiency. Health The importance of catalase for health is uncertain. Mice deficient in the enzyme appear healthy and humans with low levels of the enzyme display few problems. On the other hand, mice engineered to produce higher levels of catalase, in at least one study, lived longer. The ability of organisms to live with lower levels or no catalase may arise from another group of enzymes, the peroxiredoxins, which also act on hydrogen peroxide and may make up for lower quantities of catalase. Last, there is evidence that reduced levels of catalase with aging may be responsible for the graying of hair. Higher levels of H2O2 with reduced catalase results in a bleaching of hair follicles. Superoxide dismutase Another important enzyme for protection against reactive oxygen species is superoxide dismutase (SOD), which is found, like catalase, in virtually all organisms living in an oxygen environment. Superoxide dismutase, also like catalase, has a very high Kcat value and, in fact, has the highest Kcat/Km known for any known enzyme. It catalyzes the reactions at the top of the next column (superoxides shown in red): The enzyme thus works by a ping-pong (double displacement) mechanism (see HERE), being converted between two different forms. The hydrogen peroxide produced in the second reaction is easily handled by catalase and is also less harmful than superoxide, which can react with nitric oxide (NO) to form very toxic peroxynitrite ions (Figure 5.43). Peroxynitrite has negative effects on cells, as shown in Figure 5.45. In addition to copper, an ion of Zn++ is also bound by the enzyme and likely plays a role in the catalysis. Forms of superoxide dismutase that use manganese, nickel, or iron are also known and are mostly found in prokaryotes and protists, though a manganese SOD is found in most mitochondria. Copper/zinc enzymes are common in eukaryotes. Three forms of superoxide dismutase are found in humans and localized to the cytoplasm (SOD1 - Figure 5.45), mitochondria (SOD2 - Figure 5.46), and extracellular areas (SOD3 - Figure 5.47). Mice lacking any of the three forms of the enzyme are more sensitive to drugs, such as paraquat. Deficiency of SOD1 in mice leads to hepatocellular carcinoma and early loss of muscle tissue related to aging. Drosophila lacking SOD2 die before birth and those lacking SOD1 prematurely age. In humans, superoxide dismutase mutations are associated with the genetically-linked form of Amyotrophic Lateral Sclerosis (ALS) and over-expression of the gene is linked to neural disorders associated with Down syndrome. Mixed function oxidases Other enzymes catalyzing reactions involving oxygen include the mixed function oxidases. These enzymes use molecular oxygen for two different purposes in one reaction. The mixed function part of the name is used to indicate reactions in which two different substrates are being oxidized simultaneously. Monooxygenases are examples of mixed function oxidases. An example of a mixed function oxidase reaction is shown below. AH + BH2 + O2 <=> AOH + B + H2O In this case, the oxygen molecule has one atom serve as an electron acceptor and the other atom is added to the AH, creating an alcohol. Cytochrome P450 enzymes Cytochrome P450 enzymes (called CYPs) are family of heme-containing mixed function oxidase enzymes found in all domains of life. Over 21,000 CYP enzymes are known. The most characteristic reaction catalyzed by these enzymes follows Monooxygenase reactions such as this are relatively rare in the cell due to their use of molecular oxygen. CYPs require an electron donor for reactions like the one shown here and frequently require a protein to assist in transferring electrons to reduce the heme iron. There are six different classes of P450 enzymes based on how they get electrons 1. Bacterial P450 - electrons from ferredoxin reductase and ferredoxin 2. Mitochondrial P450 - electrons from adrenodoxin reductase and adrenodoxin 3. CYB5R/cyb5 - electrons come from cytochrome b5 4. FMN/Fd - use a fused FMN reductase 5. Microsomal P450 - NADPH electrons come via cytochrome P450 reductase or from cytochrome b5 and cytochrome b5 reductase 6. P450 only systems - do not require external reducing power The CYP genes are abundant in humans and catalyze thousand of reactions on both cellular and extracellular chemicals. There are 57 human genes categorized into 18 different families of enzymes. Some CYPs are specific for one or a few substrates, but others can act on many different substrates. CYP enzymes are found in most body tissues and perform important functions in synthesis of steroids (cholesterol, estrogen, testoterone, Vitamin D, e.g.), breakdown of endogenous compounds (bilirubin), and in detoxification of toxic compounds including drugs. Because they act on many drugs, changes in CYP activity can produce unexpected results and cause problems with drug interactions. Bioactive compounds, for example, in grapefruit juice, can inhibit CYP3A4 activity, leading to increased circulating concentrations of drugs that would normally have been acted upon by CYP3A4. This is the reason that patients prescribed drugs that are known to be CYP3A4 substrates are advised to avoid drinking grapefruit juice while under treatment. St. Johns Wort, an herbal treatment, on the other hand, induces CYP3A4 activity, but inhibits CYP1A1, CYP1B1, and CYP2D6. Tobacco smoke induces CYP1A2 and watercress inhibits CYP2E1. Cytochromes Cytochromes are heme-containing proteins that play major roles in the process of electron transport in the mitochondrion and in photosynthesis in the chloroplast. They exist either as monomers (cytochrome c) or as subunits within large redox complexes (Complex III and Complex IV of electron transport. An atom of iron at the center of the heme group plays a central role in the process, flipping between the ferrous (Fe++) and ferric (Fe+++) states as a result of the movement of electrons through it. There are several different cytochromes. Cytochrome c (Figure 5.47) is a soluble protein loosely associated with the mitochondrion. Cytochromes a and a3 are found in Complex IV. Complex III has cytochromes b and c1 and the plastoquinol-plastocyanin reductase of the chloroplast contains cytochromes b6 and f. Another important class of enzymes containing cytochromes is the cytochrome P450 oxidase group (see above). They get their name from the fact that they absorb light at 450 nm when their heme iron is reduced. Iron-Sulfur Proteins Iron-sulfur proteins contain iron-sulfur clusters in a variety of formats, including sulfide-linked di-, tri-, and tetrairon centers existing in different oxidation states (Figures 5.48 & 5.49). The clusters play a variety of roles, but the best known ones are in electron transport where they function in the redox reactions involved in the movement of electrons. Complexes I and Complex II contain multiple Fe-S centers. Iron-sulfur proteins, though, have many other roles in cells. Aconitase uses an iron-sulfur center in its catalytic action and the ability of the enzyme to bind iron allows it to function as a barometer of iron concentration in cells. Iron-sulfur centers help to generate radicals in enzymes using S-Adenosyl Methionine (SAM) and can serve as a source of sulfur in the synthesis of biotin and lipoic acid. Some iron-sulfur proteins even help to regulate gene expression. Ferredoxin Ferredoxins are iron-sulfur containing proteins performing electron transfer in a wide variety of biological systems and processes. They include roles in photosynthesis in chloroplasts. Ferredoxins are classified structurally by the iron-sulfur clustered centers they contain. Fe2S2 clusters (Figure 5.50) are found in chloroplast membranes and can donate electrons to glutamate synthase, nitrate reductase, and sulfite reductase and serve as electron carriers between reductase flavoproteins and bacterial dioxygenase systems. Adrenodoxin is a soluble human Fe2S2 ferredoxin (also called ferredoxin 1) serving as an electron carrier (to cytochrome P450) in mitochondrial monooxygenase systems. Fe4S4 ferredoxins are subdivided as low and high potential ferredoxins, with the latter ones functioning in anaerobic electron transport chains. Ferritin Ferritin is an intracellular iron-storage protein found in almost all living organisms, from bacteria to higher plants and animals. It is a globular protein complex with 24 subunits and is the primary intracellular iron-storage protein in eukaryotes and prokaryotes. Ferritin functions to keep iron in a soluble and non-toxic form. Its ability to safely store iron and release it in a controlled fashion allow it to act like the prime iron buffer and solubilizer in cells - keeping the concentration of free iron from going to high or falling too low. Ferritin is located in the cytoplasm in most tissues, but it is also found in the serum acting as an iron carrier. Ferritin that doesn’t contain any iron is known as apoferritin. Monoamine oxidases Monoamine oxidases are enzymes that catalyze the oxidative deamination of monoamines, such as serotonin, epinephrine, and dopamine. Removal of the amine with oxygen results in the production of an aldehyde and ammonia. The enzymes are found inside and outside of the central nervous system. There are two types of monoamine oxidase enzymes - MAO-A and MAO-B. MAO-A is particularly important for oxidizing monoamines consumed in the diet. Both MAO-A and MAO-B play important roles in inactivating monoaminergic neurotransmitters. Both enzymes act on dopamine, tyramine (Figure 5.50), and tryptamine. MAO-A is the primary enzyme for metabolizing melatonin, serotonin, norepinephrine, and epinephrine, while MAO-B is the primary enzyme for phenethylamine (Figure 5.51) and benzylamine. MAO-B levels have been reported to be considerably reduced with tobacco usage. Actions of monoamine oxidases thus affects levels of neurotransmitters and consequently are thought to play roles in neurological and/or psychiatric disorders. Aberrant levels of MAOs have been linked to numerous psychological problems, including depression, attention deficit disorder (ADD), migraines, schizophrenia, and substance abuse. Medications targeting MAOs are sometimes used to treat depression as a last resort - due to potential side effects. Excess levels of catecholamines, such as epinephrine, norepinephrine, and dopamine, can result in dangerous hypertension events. DNA damage theory of aging The DNA Damage Theory of Aging is based on the observation that, over time, cells are subject to extensive oxidative events. As already noted, these afford opportunities for the formation of ROS that can damage cellular molecules, and it follows that accumulation of such damage, especially to the DNA would be deleterious to the cell. The build-up of DNA damage could, thus, be responsible for the changes in gene expression that we associate with aging. Numerous damage events The amount of DNA damage that can occur is considerable. In mice, for example, it is estimated that each cell experiences 40,000 to 150,000 damage events per day. The damage, which happens to nuclear as well as to mitochondrial DNA, can result in apoptosis and/or cellular senescence. DNA repair systems, of course, protect against damage to DNA, but over time, unrepairable damage may accumulate. Oxidative damage DNA damage can occur in several ways. Oxidation can damage nucleotides and alter their base-pairing tendencies. Oxidation of guanine by reactive oxygen species, for example, can produce 8-oxo-guanine (Figures 5.52 and 5.53). This oxidized nucleobase commonly produced lesion in DNA arising from action of reactive oxygen species like superoxides. 8-oxoguanine is capable of forming a stable base pairing interaction within a DNA duplex with adenine, potentially giving rise to a mutation when DNA replication proceeds. 8-oxoguanine can be repaired if recognized in time by a DNA glycosylase, which acts to clip out the damaged base and it can then be replaced by the proper one. Polycyclic aromatic hydrocarbons from cigarette smoke, diesel exhaust, or overcooked meat can covalently bind to DNA and, if unrepaired, lead to mutation. Chemical damage to DNA can result in broken or cross-linked DNAs. Diseases of DNA repair The importance of DNA repair in the aging process is made clear by diseases affecting DNA repair that lead to premature aging. These include Werner syndrome, for whom the life expectancy is 47 years. It arises as a result of loss of two enzymes in base excision repair. People suffering from Cockayne syndrome have a life expectancy of 13 years due to mutations that alter transcription-coupled nucleotide excision repair, which is an important system for fixing oxidative damage. Further, the life expectancies of 13 species of mammalian organisms correlates with the level of expression of the PARP DNA repair-inducing protein. Interestingly, people who lived past the age of 100 had a higher level of PARP than younger people in the population. Antioxidants There is a growing interest in the subject of antioxidants because of health concerns raised by our knowledge of problems created as a result of spontaneous oxidation of biomolecules by Reactive Oxygen Species (ROS), such as superoxide. Antioxidants have the chemical property of protecting against oxidative damage by being readily oxidized themselves, preferentially to other biomolecules. Biologically, cells have several lines of antioxidant defense. They include molecules, such as vitamins C, A, and E, glutathione, and enzymes that destroy ROS such as superoxide dismutase, catalase, and peroxidases. Health effects Oxidation by ROS is mutagenic and has been linked to atherosclerosis. Nonetheless, randomized studies of oral supplementation of various vitamin combinations have shown no protective effect against cancer and supplementation of Vitamin E and selenium has revealed no decrease in the risk of cardiovascular disease. Further, no reduction in mortality rates as a result of supplementation with these materials has been found, so the protective effects, if any, of antioxidants on ROS in human health remain poorly understood. Glutathione The thiol group of cysteine is a reducing agent that reduces disulfide bonds to sulfhydryls in cytoplasmic proteins. This, in turn, is the bridge when two glutathiones get oxidized and form a disulfide bond with each other (Figure 5.56). Glutathione’s two oxidative states are abbreviated as follows: GSH (reduced) and GSSG (oxidized). Disulfide-joined glutathiones can be separated by reduction of their bonds with glutathione reductase, using electrons from NADPH for the reduction. Non-ribosomal synthesis Glutathione is not made by ribosomes. Rather, two enzymes catalyze its synthesis. The enzyme γ-glutamylcysteine synthetase catalyzes the joining of the glutamate to the cysteine and then glutathione synthetase catalyzes the peptide bond formation between the cysteine and the glycine. Each step requires energy from ATP. Essential for life Glutathione is important for life. Mice lacking the first enzyme involved in its synthesis in the liver die in the first month after birth. In healthy cells, 90% of glutathione is in the GSH state. Higher levels of GSSG correspond to cells that are oxidatively stressed. Besides reducing disulfide bonds in cells, glutathione is also important for the following: • Neutralization of free radicals and reactive oxygen species. • Maintenance of exogenous antioxidants such as vitamins C and E in their reduced forms. Regulation of the nitric oxide cycle Resveratrol Some data indicates resveratrol may improve the functioning of mitochondria. It also acts as an antioxidant and causes concentration of another anti-oxidant, glutathione, to increase. The compound appears to induce expression of manganese superoxide dismutase (protects against reactive oxygen species) and inhibits several phosphodiesterases. This causes an increase in cAMP which results in increases in oxidation of fatty acids, mitochondria formation, gluconeogenesis, and glycogen breakdown. It has been claimed to be the cause of the French Paradox in which drinking of red wine is supposed to give protection for the cardiovascular system. Research data is lacking in support of the claim, however. Resveratrol is known to activate Sirtuin proteins, which play roles in gene inactivation. Summary In summary, energy is needed for cells to perform the functions that they must carry out in order to stay alive. At its most basic level, this means fighting a continual battle with entropy, but it is not the only need for energy that cells have. References 1. Winge, D.R., Mol Cell Biol. 2012 Jul; 32(14): 2647–2652. doi: 10.1128/MCB.00573-12 Energy: Electron Transport & Oxidative Phosphorylation 429 YouTube Lectures by Kevin HERE & HERE YouTube Lectures by Kevin HERE & HERE 430 Figure 5.14 - Overview of electron transport (bottom left and top right) and oxidative phosphorylation (top left - yellow box) in the mitochondrion 431 Figure 5.15 - Loss of electrons by NADH to form NAD+. Relevant reactions occur in the top ring of the molecule. 432 Figure 5.16 - Flow of electrons from NADH into the electron transport system. Entry is through complex I Image by Aleia Kim Figure 5.17 - Flow of electrons from FADH2 into the electron transport chain. Entry is through complex II. Image by Aleia Kim Interactive Learning Module HERE 433 Figure 5.18 - Complex I embedded in the inner mitochondrial membrane. The mitochondrial matrix at at the top Wikipedia 434 Figure 5.19 - Complex II embedded in inner mitochondrial membrane. Matrix is up. Wikipedia YouTube Lectures by Kevin HERE & HERE 435 Figure 5.20 - Movement of electrons through complex I from NADH to coenzyme Q. The mitochondrial matrix is at the bottom Image by Aleia Kim Figure 5.21 - Movement of electrons from succinate through complex II (A->B->C->D->Q). Mitochondrial matrix on bottom. Image by Aleia Kim 436 Figure 5.22 - Complex II in inner mitochondrial membrane showing electron flow. Matrix is up. Wikipedia Figure 5.23 - Coenzyme Q 437 Movie 5.2 - The Q-cycle Wikipedia Figure 5.24 - The Q-Cycle Image by Aleia Kim Figure 5.24 - Complex III Wikipedia 438 YouTube Lectures by Kevin HERE & HERE Figure 5.25 - The Q-cycle. Matrix is down. Image by Aleia Kim 439 Figure 5.26 - Movement of electrons and protons through complex IV. Matrix is down Image by Aleia Kim Figure 5.25 - Cytochrome c with bound heme Group Wikipedia 440 Figure 5.27 - Mitochondrial anatomy. Electron transport complexes and ATP synthase are embedded in the inner mitochondrial membrane Image by Aleia Kim 441 Figure 5.28 - ATP synthase. Protons pass from intermembrane space (top) through the complex and exit in the matrix (bottom). Image by Aleia Kim Interactive Learning Module HERE 442 Movie 5.3 - ATP Synthase - ADP + Pi (pink) and ATP (red). The view is end-on from the cytoplasmic side viewing the β subunits Movie 5.3 - ATP Synthase - ADP + Pi (pink) and ATP (red). The view is end-on from the cytoplasmic side viewing the β subunits 443 Figure 5.29 - Important structural features of the ATP synthase Image by Aleia Kim 444 Figure 5.30 - Loose (L), Tight (T), and Open (O) structures of the F1 head of ATP synthase. Change of structure occurs by rotation of γ-protein (purple) in center as a result of proton movement. Individual α and β units do not rotate Image by Aleia Kim 445 Figure 5.31 - Respiration overview in eukaryotic cells Wikipedia YouTube Lectures by Kevin HERE & HERE 446 Rest ATP High / ADP Low Oxidative Phosphorylation Low Electron Transport Low Oxygen Use Low NADH High / NAD+ Low Citric Acid Cycle Slow Exercise ATP Low / ADP High Oxidative Phosphorylation High Electron Transport High Oxygen Use High NADH Low / NAD+ High Citric Acid Cycle Fast Interactive Learning Module HERE 447 Figure 5.32 - Three inhibitors of electron transport Image by Aleia Kim 448 Figure 5.33 - Oligomycin A - An inhibitor of ATP synthase Figure 5.34 - 2,4 DNP - an uncoupler of respiratory control 449 In Cells With Tight Coupling O2 use depends on metabolism NAD+ levels vary with exercise Proton gradient high with no exercise Catabolism depends on energy needs ETS runs when OxPhos runs and vice versa In Cells That Are Uncoupled O2 use high NAD+ Levels high Little or no proton gradient Catabolism high OxPhos does not run, but ETS runs rapidly YouTube Lectures by Kevin HERE & HERE 450 451 Figure 5.35 - Alternative oxidase (AOX) of fungi, plants, and protozoa bypasses part of electron transport by taking electrons from CoQ and passing them to oxygen. 452 Figure 5.36 - Structure of an oxygen free radical Wikipedia NADPH + 2O2 NADP+ + 2O2− + H+ Figure 5.37 - Three sources of reactive oxygen species (ROS) in cells Wikipedia 453 454 YouTube Lectures by Kevin HERE & HERE Figure 5.38 A hydroxyl radical Wikipedia 455 Reduced Glutathione (GSH) + H2O2 Oxidized Glutathione (GSSG) + H2O Figure 5.40 - Detoxifying reactive oxygen species Figure 5.39 - Catalase 456 1. O2- + Enzyme-Cu++ O2 + Enzyme-Cu+ 2. O2- + Enzyme-Cu+ + 2H+ H2O2 + Enzyme-Cu++ Figure 5.41 - SOD2 of humans Figure 5.42 3 - Peroxynitrite Ion Figure 5.44 - SOD1 of humans Wikipedia Figure 5.45 - SOD3 of humans 457 Figure 5.43 - Peroxynitrite’s effects on cells lead to necrosis or apoptosis Wikipedia 458 RH + O2 + NADPH + H+ ROH + H2O + NADP+ 459 Figure 5.46 - Cytochrome c with its heme group 460 YouTube Lectures by Kevin HERE & HERE Figure 5.47 - Fe2S2 Cluster Figure 5.48 - Redox reactions for Fe4S4 clusters 461 Figure 5.49 - Tyramine Figure 5.50 - Phenethylamine 462 Figure 5.51 - Guanine and 8-oxo-guanine Figure 5.52 - Adenine-8-oxo-guanine base pair. dR = deoxyribose 463 Figure 5.53 - Good antioxidant sources 464 Figure 5.55 - Oxidized glutathiones (GSSG) joined by a disulfide bond Wikipedia Figure 5.54 - Structure of reduced glutathione (GSH) 465 Figure 5.56 - Resveratrol YouTube Lectures by Kevin HERE & HERE 466 Graphic images in this book were products of the work of several talented students. Links to their Web pages are below Click HERE for Martha Baker’s Web Page Click HERE for Pehr Jacobson’s Web Page Click HERE for Aleia Kim’s Web Page Click HERE for Penelope Irving’s Web Page Problem set related to this section HERE Point by Point summary of this section HERE To get a certificate for mastering this section of the book, click HERE Kevin Ahern’s free iTunes U Courses - Basic / Med School / Advanced Biochemistry Free & Easy (our other book) HERE / Facebook Page Kevin and Indira’s Guide to Getting into Medical School - iTunes U Course / Book To see Kevin Ahern’s OSU ecampus courses - BB 350 / BB 450 / BB 451 To register for Kevin Ahern’s OSU ecampus courses - BB 350 / BB 450 / BB 451 Biochemistry Free For All Facebook Page (please like us) Kevin Ahern’s Web Page / Facebook Page / Taralyn Tan’s Web Page Kevin Ahern’s free downloads HERE OSU’s Biochemistry/Biophysics program HERE OSU’s College of Science HERE Oregon State University HERE Email Kevin Ahern / Indira Rajagopal / Taralyn Tan I'm a little mitochondrion​ Who gives you energy​ I use my proton gradient​ To make the ATPs​ He's a little mitochondrion​ Who gives us energy​ He uses proton gradients​ To make some ATPs​ Electrons flow through Complex II​ To traffic cop Co-Q​ Whenever they arrive there in​ An FADH-two​ Electrons flow through Complex II​ To traffic cop Co-Q​ Whenever they arrive there in​ An FADH-two Tightly coupled is my state​ Unless I get a hole​ Created in my membrane by​ Some di-ni-tro-phe-nol​ Yes tightly coupled is his state​ Unless he gets a hole​ Created in his membrane by​ Some di-ni-tro-phenol​ Both rotenone and cyanide​ Stop my electron flow​ And halt the calculation of​ My "P" to "O" ratio​ Recording by Tim Karplus Lyrics by Kevin Ahern Recording by Tim Karplus Lyrics by Kevin Ahern I’m a Little Mitochondrion To the tune of “I’m a Lumberjack” Metabolic Melodies Website HERE In the catabolic pathways that our cells employ​ Oxidations help create the ATP​ While they lower Gibbs free energy​ Thanks to enthalpy If a substrate is converted from an alcohol​ To an aldehyde or ketone it is clear​ Those electrons do not disappear​ They just rearrange – very strange​ N-A-D is in my ears and in my eyes ​ Help-ing mol-e-cules get oxidized​ Making N-A-D-H then And the latter is a problem anaerobically​ ‘Cuz accumulations of it muscles hate​ They respond by using pyruvate​ To produce lactate​ Catalyzing is essential for the cells to live​ So the enzymes grab their substrates eagerly​ If they bind with high affinity​ Low Km you see, just as me​ N-A-D is in my ears and in my eyes ​ Help-ing mol-e-cules get oxidized​ Making N-A-D-H then N-A-D To the tune of “Penny Lane” Metabolic Melodies Website HERE Recorded by Tim Karplus Lyrics by Kevin Ahern Recorded by Tim Karplus Lyrics by Kevin Ahern When oxygen’s electrons all are in the balanced state There’s twelve of them for oh-two. The molecule is great But problems sometimes happen on the route to complex IV Making reactive species that the cell cannot ignore Oh superoxide dismutase is super catalytic Keeping cells from getting very peroxynitritic Faster than a radical, its actions are terrific Superoxide dismutase is super catalytic Enzyme, enzyme deep inside Blocking all the bad oxides The enzyme’s main advantage is it doesn’t have to wait By binding superoxide in a near-transition state It turns it to an oxygen in mechanism one Producing “h two oh two” when the cycle is all done Oh superoxide dismutase you’re faster than all them You’ve got the highest ratio of kcat over KM This means that superoxide cannot cause too much mayhem Superoxide dismutase is faster than all them Superoxide dismutase Stopping superoxide’s ways The enzyme’s like a ping-pong ball that mechanistic-ly Bounces between two copper states, plus one and two you see So S-O-D behaves just like an anti-oxidant Giving as much protection as a cell could ever want Oh superoxide dismutase, the cell’s in love with you Because you let electron transport do what it must do Without accumulation of a radical oh two Superoxide dismutase - that’s why a cell loves you Superoxide Dismutase To the tune of “Supercalifragilistiexpialidocious” Metabolic Melodies Website HERE Lyrics by Kevin Ahern No Recording Yet For This Song

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/05%3A_Energy/5.2%3A_Electron_Transport_and_Oxidative_Phosphorylation.txt

• 6.1: Metabolism - Sugars • 6.2: Citric Acid Cycle & Related Pathways The primary catabolic pathway in the body is the citric acid cycle because it is here that oxidation to carbon dioxide occurs for breakdown products of the cell’s major building blocks - sugars, fatty acids, and amino acids. The pathway is cyclic and thus, does not really have a starting or ending point. All of the reactions occur in mitochondria, though one enzyme is embedded in the organelle’s inner membrane. Cells may use a subset of the reactions of the cycle to produce a desired molecule. • 6.3: Fats and Fatty Acids There is a tremendous amount of interest in the metabolism of fat and fatty acids. Fat is the most important energy storage form of animals, storing considerably more energy per carbon than carbohydrates, but its insolubility in water requires the body to package it specially for transport. Surprisingly, fat/fatty acid metabolism is not nearly as tightly regulated as that of carbohydrates. Neither are the metabolic pathways of breakdown and synthesis particularly complicated, either. • 6.4: Other Lipids Sugars are the building blocks of carbohydrates, amino acids are the building blocks of proteins and nucleotides are the building blocks of the nucleic acids - DNA and RNA. Another crucial building block is acetyl-CoA, which is used to build many lipid substances, including fatty acids, cholesterol, fat soluble vitamins, steroid hormones, prostaglandins, endocannabinoids, and the bile acids. Indeed, acetyl-CoA goes into more different classes of molecule than any other building block. • 6.5: Amino Acids and the Urea Cycle In contrast to some of the metabolic pathways described to this point, amino acid metabolism is not a single pathway. The 20 amino acids have some parts of their metabolism that overlap with each other, but others are very different from the rest. In discussing amino acid metabolism, we will group metabolic pathways according to common metabolic features they possess (where possible). • 6.6: Nucleotides Nucleotides are most often thought of as the building blocks of the nucleic acids, DNA and RNA. While this, is, of course, a vital function, nucleotides also play other important roles in cells. Ribonucleoside triphosphates like ATP, CTP, GTP and UTP are necessary, not just for the synthesis of RNA, but as part of activated intermediates like UDP-glucose in biosynthetic pathways. ATP is also the universal “energy currency” of cells. Thumbnail: Metabolic Metro Map. Image used with permission (CC BY-SA 4.0; Chakazul).​​​​​ 06: Metabolism Glycolysis Carbohydrates, whether synthesized by photosynthetic organisms, stored in cells as glycogen, or ingested by heterotrophs, must be broken down to obtain energy for the cell’s activities as well as to synthesize other molecules required by the cell. Starch and glycogen, polymers of glucose, are the main energy storage forms of carbohydrates in plants and animals, respectively. To use these sources of energy, cells must first break down the polymers to yield glucose. The glucose is then taken up by cells through transporters in cell membranes. The metabolism of glucose, as well as other six carbon sugars (hexoses) begins with the catabolic pathway called glycolysis. In this pathway, sugars are oxidized and broken down into pyruvate molecules. The corresponding anabolic pathway by which glucose is synthesized is termed gluconeogenesis. Neither glycolysis nor gluconeogenesis is a major oxidative/reductive process, with one step in each one involving loss/gain of electrons, but the product of glycolysis, pyruvate, can be completely oxidized to carbon dioxide (Figure 6.2). Indeed, without production of pyruvate from glucose in glycolysis, a major energy source for the cell would not be available. Glucose is the most abundant hexose in nature and is traditionally used to illustrate the reactions of glycolysis, but fructose (in the form of fructose-6- phosphate) is also readily metabolized, while galactose can easily be converted into glucose for catabolism in the pathway as well. The end metabolic products of glycolysis are two molecules of ATP, two molecules of NADH and two molecules of pyruvate (Figure 6.3), which, in turn, can be oxidized further in the citric acid cycle. Entry points for glycolysis Glucose and fructose are the sugar ‘funnels’ serving as entry points to the glycolytic pathway. Other sugars must be converted to either of these forms to be metabolized in glycolysis. Some pathways, including the Calvin Figure 6.2 - Metabolic fates of glucose Image by Aleia Kim Your cells may have a mounting crisis Should they not go through glyco-lye-sis No glucose energy releases Until it’s fractured into pieces Figure 6.3 - Glycolysis and its Regulators Image by Ben Carson Cycle and the Pentose Phosphate Pathway (PPP) contain intermediates in common with glycolysis, so in that sense, almost any cellular sugar can be metabolized here. Other pathways Intermediates of glycolysis and gluconeogenesis that are common to other pathways include glucose-6-phosphate (PPP, glycogen metabolism), Fructose-6-phosphate (Calvin Cycle, PPP), Glyceraldehyde-3- phosphate (Calvin Cycle, PPP), dihydroxyacetone phosphate (PPP, glycerol metabolism, Calvin Cycle), 3- phosphoglycerate (Calvin Cycle, PPP), phosphoenolpyruvate (C4 plant metabolism, Calvin Cycle), and pyruvate (fermentation, acetyl-CoA genesis, amino acid metabolism). It is worth noting that glycerol from the breakdown of fat can readily be metabolized to dihydroxyacetone phosphate (DHAP) and thus enter the glycolysis pathway. It is the only part of a fat that is used in these pathways. Reaction 1 Glucose gets a phosphate from ATP to make glucose-6-phosphate (G6P) in a reaction catalyzed by the enzyme hexokinase, a transferase enzyme. Glucose + ATP ⇄ G6P + ADP + H+ Hexokinase is one of three regulated enzymes in glycolysis and is inhibited by one of the products of its action - G6P. Hexokinase has flexibility in its substrate binding and is able to phosphorylate a variety of hexoses, including fructose, mannose, and galactose. Why phosphorylate glucose? Phosphorylation of glucose serves two important purposes. First, the addition of a phosphate group to glucose effectively traps it in the cell, as G6P cannot diffuse across the lipid bilayer. Second, the reaction decreases the concentration of free glucose, favoring additional import of the molecule. G6P is a substrate for the pentose phosphate pathway and can also be converted to glucose-1-phosphate (G1P) for use in glycogen synthesis and galactose metabolism (Figure 6.5). It is worth noting that the liver has an enzyme like hexokinase called glucokinase, which Figure 6.4 - Reaction #1 - Phosphorylation of glucose - catalyzed by hexokinase has a much higher Km (lower affinity) for glucose. This is important, because the liver is a site of glucose synthesis (gluconeogenesis) where cellular concentrations of glucose can be relatively high. With a lower affinity glucose phosphorylating enzyme, glucose is not converted to G6P unless glucose concentrations get high, so the liver is able to release the glucose it makes into the bloodstream for the rest of the body to use. Reaction 2 Next, G6P is converted to fructose-6-phosphate (F6P), in a reaction catalyzed by the enzyme \[\ce{G6P ⇄ F6P}\] The reaction has a low ΔG°’ , so it is readily favorable in either direction with Figure 6.6 - Mechanism of conversion of G6P to F6P in reaction #2 Figure 6.5 - The centrality of glucose-6-phosphate in metabolism Image by Aleia Kim only slight changes in concentration of reactants. Reaction 3 \[ce{F6P + ATP ⇄ F1,6BP + ADP + H+}\] The second input of energy occurs when F6P gets another phosphate from ATP in a reaction catalyzed by the enzyme phosphofructokinase-1 (PFK-1 - another transferase) to make fructose-1,6- bisphosphate (F1,6BP). PFK-1 is a very important enzyme regulating glycolysis, with several allosteric activators and inhibitors (see HERE). Like the hexokinase reaction the energy from ATP is needed to make the reaction energetically favorable. PFK-1 is the most important regulatory enzyme in the pathway and this reaction is the ratelimiting step. It is also one of three essentially irreversible reactions in glycolysis. A variant enzyme found in plants and some bacterial uses pyrophosphate rather than ATP as the energy source and due to the lower energy input from hydrolysis of the pyrophosphate, that reaction is reversible. Reaction 4 \[\ce{F1,6BP ⇄ D-GLYAL3P + DHAP}\] With the glycolysis pump thus primed, the pathway proceeds to split the F1,6BP into two 3-carbon intermediates. This reaction catalyzed by the lyase known as aldolase is energetically a “hump” to overcome in the glycolysis direction (∆G°’ = +24 kJ/mol Figure 6.7 - Reaction #3 - Conversion of F6P to F1,6BP by PFK Wikipedia Figure 6.8 - Reaction #4 - Breakdown of F1,6BP into GLYAL3P (left) and DHAP (right) by aldolase °K) so to get over the energy hump, cells must increase the concentration the reactant (F1,6BP) and decrease the concentration of the products, which are D-glyceraldehyde- 3-phosphate (D-GLYAL3P) and dihydroxyacetone phosphate (DHAP). A novel scheme facilitates decreasing concentration of the products (see below). Aldolases cut the ketose ring by two different mechanisms and these enzymes are grouped as Class I (in animals and plants) and Class II (in fungi and bacteria). Reaction 5 \[\ce{DHAP ⇄ D-GLYAL3P}\] In the next step, DHAP is converted to DGLYAL3P in a reaction catalyzed by the enzyme triosephosphate isomerase. At this point, the six carbon glucose molecule has been broken down to two units of three carbons each - D-GLYAL3P. From this point forward each reaction of glycolysis contains two of each molecule. Reaction #5 is fairly readily reversible in cells. The enzyme is of note because it is one example of a “perfect enzyme.” Enzymes in this category have very high ratios of Kcat/Km that approach a theoretical maximum limited only by the diffusion of substrate into the active site of the enzyme. The apparent reason for the enzyme evolving in this way is that the mechanism of the reaction produces an unstable, toxic intermediate (Figure 6.9). With the reaction proceeding as rapidly as it does, there is less chance of the intermediate escaping and causing damage in the cell. Reaction 6 \[\ce{D-GLYAL3P + NAD+ + Pi D-1,3BPG + NADH + H+}\] Figure 6.9 - Reaction #5 - Triose phosphate isomerase with unstable, toxic intermediate (methyl glyoxal) Image by Ben Carson In this reaction, D-GLYAL3P is oxidized in the only oxidation step of glycolysis catalyzed by the enzyme glyceraldehyde-3- phosphate dehydrogenase, an oxidoreductase. The aldehyde in this reaction is oxidized, then linked to a phosphate to make an ester - D-1,3-bisphospho-glycerate (D- 1,3BPG). Electrons from the oxidation are donated to NAD+, creating NADH. NAD+ is a critical constituent in this reaction and is the reason that cells need a fermentation option at the end of the pathway (see below). Note here that ATP energy was not required to put the phosphate onto the oxidized D-GLYAL3P. The reason for this is because the energy provided by the oxidation reaction is sufficient for adding the phosphate. Reaction 7 \[\ce{D-1,3BPG + ADP ⇄ 3PG + ATP}\] The two phosphates in the tiny 1,3BPG molecule repel each other and give the molecule high potential energy. This energy is utilized by the enzyme phosphoglycerate kinase (another transferase) to phosphorylate ADP and make ATP, as well as the product, 3-phosphoglycerate (3-PG). This is an example of a substrate-level phosphorylation. Such mechanisms for making ATP require an intermediate with a high enough energy to phosphorylate ADP to make ATP. Figure 6.10 - Reaction #6 - Oxidation of GLYAL3P, catalyzed by glyceraldehyde-3-phosphate dehydrogenase Figure 6.11 - Reaction #7 - Substrate-level Phosphorylation by 1,3-BPG Though there are a few substrate level phosphorylations in cells (including another one at the end of glycolysis), the vast major of ATP is made by oxidative phosphorylation in the mitochondria (in animals). In addition to oxidative phosphorylation, plants also make ATP by photophosphorylation in their chloroplasts. Since there are two 1,3 BPGs produced for every glucose, the two ATPs produced in this reaction replenish the two ATPs used to start the cycle and the net ATP count at this point of the pathway is zero. Reaction 8 \[\ce{3-PG ⇄ 2-PG }\] Conversion of the 3-PG intermediate to 2-PG (2- phosphoglycerate) occurs by an important mechanism. An intermediate in this readily reversible reaction (catalyzed by phosphoglycerate mutase - a mutase enzyme) is 2,3-BPG. This intermediate, which is stable, is released with low frequency by the enzyme instead of being con- Figure 6.13 - Two routes to formation of 2,3-BPG Figure 6.14 - 2,3- Bisphosphoglycerate (2,3-BPG) Figure 6.12 - Reaction #8 - Conversion of 3-PG to 2-PG verted to 2-PG. 2,3BPG is important because it binds to hemoglobin and stimulates release of oxygen. The molecule can also be made from 1,3-BPG as a product of a reaction catalyzed by bisphophglycerate mutase (Figure 6.13). Cells which are metabolizing glucose rapidly release more 2,3-BPG and, as a result, get more oxygen, supporting their needs. Notably, cells which are metabolizing rapidly are using oxygen more rapidly and are more likely to be deficient in it. Reaction 9 \[\ce{2-PG ⇄ PEP + H2O}\] 2-PG is converted by enolase (a lyase) to phosphoenolpyruvate (PEP) by removal of water, creating a very high energy intermediate. The reaction is readily reversible, but with PEP, the cell has one of its highest energy molecules and that is important for the next reaction. Reaction 10 \[\ce{PEP + ADP + H+ ⇄ PYR + ATP}\] Conversion of PEP to pyruvate by pyruvate kinase is the second substrate level phosphorylation of glycolysis, creating ATP. This reaction is what some refer to as the “Big Bang” of glycolysis because there is almost enough energy in PEP to stimulate production of a second ATP (ΔG°’ = 31.6 kJ/ mol), but it is not used. Consequently, this energy is lost as heat. If you wonder why you get hot when you exercise, the heat produced in the breakdown of glucose is a prime contributor and the pyruvate kinase reaction is a major source. Figure 6.16 - Reaction #10 - The big bang - PEP phosphorylates ADP with a lot of energy to spare Wikipedia Figure 6.15 - Reaction #9 - Enolase-catalyzed removal of water Wikipedia Pyruvate kinase is the third and last enzyme of glycolysis that is regulated (see below). The primary reason this is the case is to be able to prevent this reaction from occurring when cells are making PEP while going through gluconeogenesis (see more HERE). Catabolism of other sugars Though glycolysis is a pathway focused on the metabolism of glucose and fructose, the fact that other sugars can be readily metabolized into glucose means that glycolysis can be used for extracting energy from them as well. Galactose is a good example. It is commonly produced in the produced in the body as a result of hydrolysis of lactose, catalyzed by the enzyme known as lactase (Figure 6.17). Deficiency of lactase is the cause of lactose intolerance. Galactose begins preparation for entry into glycolysis by being converted to galactose-1- phosphate (catalyzed by galactokinase - Figure 6.18). Galactose-1-phosphate swaps with glucose-1-phosphate from UDP-glucose to make UDP-galactose (Figure 6.19). An epimerase converts UDPgalactose back to UDP-glucose and the cycle is complete. Each turn of the cycle thus takes in one galactose-1-phosphate and releases one glucose-1-phosphate. Deficiency of galactose conversion enzymes results in accumulation of galactose (from breakdown of lactose). Excess galactose is converted to galactitol, a sugar alcohol. Galactitol in the human eye lens causes it to absorb water and this may be a factor in formation of cataracts.Figure 6.17 - Breakdown of lactose to glucose and galactose by lactase Image by Pehr Jacobson Figure 6.18 - Galactokinase Reaction Image by Penelope Irving Free fructose can also enter glycolysis by two mechanisms. First, it can be phosphorylated to fructose-6-phosphate by hexokinase. A more interesting alternate entry point is that shown in Figure 6.20. Phosphorylation of fructose by fructokinase produces fructose-1-phosphate and cleavage of that by fructose-1- phosphate aldolase yields DHAP and glyceraldehyde. Phosphorylation of glyceraldehyde by triose kinase yields GLYAL3P. This alternative entry means for fructose may have important implications because DHAP and GLYAL3P are introduced into the glycolysis pathway while bypassing PFK-1 regulation. Some have proposed this may be important when considering metabolism of high fructose corn syrup, since it forces production of pyruvate, a precursor of acetyl-CoA, which is itself a precursor of fatty acids when ATP levels are high. Mannose metabolism Mannose can also be metabolized in glycolysis. In this case, it enters via fructose by the following two-step process - 1) phosphoryla- Figure 6.19 - Conversion of galactose-1-phosphate into glucose-6-phosphate Image by Aleia Kim tion by hexokinase to make mannose-6- phosphate followed by its conversion to fructose-6-phosphate, catalyzed by phosphomannoisomerase (Figure 6.21). Glycerol metabolism Glycerol is an important molecule for the synthesis of fats, glycerophospholipids, and other membrane lipids. Most commonly it is made into glycerol-3- phosphate (Figure 6.22) and the glycolysis/gluconeogenesis pathways are important both for producing the compound and for metabolizing it. The relevant intermediate in these pathways both for producing and for using glycerol-3-phosphate is DHAP. The enzyme glycerol-3-phosphate dehydrogenase reversibly converts glycerol-3- phosphate into DHAP (Figure 6.22). This reaction, which is an oxidation, transfers electrons to NAD+ to produce NADH. In the reverse reaction, production of glycerol-3- phosphate from DHAP, of course, requires electrons from NADH for the reduction. Both glycolysis and gluconeogenesis are sources DHAP, meaning when the cell needs glycerol- 3-phosphate that it can use sugars (glucose, fructose, mannose, or galactose) as sources in glycolysis. For gluconeogenesis, sources include pyruvate, alanine and Figure 6.20 - Entry of fructose into glycolysis, bypassing PFK-1 Image by Penelope Irving Figure 6.21 - Entry of other sugars into glycolysis Image by Penelope Irving lactate (both can easily be made into pyruvate), oxaloacetate, aspartic acid (which can be made into oxaloacetate by transamination), and others. All of the intermediates of the citric acid cycle (and glyoxylate cycle) can be converted ultimately to oxaloacetate, which is a gluconeogenesis intermediate, as well. It is worth noting that animals are unable to use fatty acids as materials for gluconeogenesis in net amounts, but they can, in fact, use glycerol in both glycolysis and gluconeogenesis. It is the only part of the fat molecule that can be so used. Pyruvate metabolism As noted, pyruvate produced in glycolysis can be oxidized to acetyl-CoA, which is itself oxidized in the citric acid cycle to carbon dioxide. That is not the only metabolic fate of pyruvate, though (Figure 6.23). Pyruvate is a “starting” point for gluconeogenesis, being converted to oxaloacetate in the mitochondrion in the first step. Pyruvate in animals can also be reduced to lactate by adding electrons from NADH (Figure 6.24). This reaction produces NAD+ and is critical for generating the latter molecule to keep the glyceraldehyde-3-phosphate dehydrogenase reaction of glycolysis (reaction #6) going under conditions when there is no oxygen. This is because oxygen is necessary for the electron transport system (ETS) to operate and it performs the important function of converting NADH back to NAD+. When the ETS is running, NADH donates electrons to Complex I and is oxidized to NAD+ in the process, generating the intermediate needed for oxidizing GLYAL-3P. In the absence of oxygen, however, NADH cannot be converted to Figure 6.22 - Reactions in glycerol metabolism Image by Penelope Irving NAD+ by the ETS, so an alternative means of making NAD+ is necessary for keeping glycolysis running under low oxygen conditions (fermentation). Bacteria and yeast generate NAD+ under oxygen deprived conditions by doing fermentation in a different way (Figure 6.25). They use NADH-requiring reactions that regenerate NAD+ while producing ethanol from pyruvate instead of making lactate. Thus, fermentation of pyruvate is essential to keep glycolysis operating when oxygen is limiting. It is also for these reasons that brewing of beer (using yeast) involves depletion of oxygen and muscles low in oxygen produce lactic acid (animals). Pyruvate is a precursor of alanine which can be easily synthesized by transfer of a nitrogen from an amine donor, such as glutamic acid. Pyruvate can also be converted into oxaloacetate by carboxylation in the process of gluconeogenesis (see below). The enzymes involved in pyruvate metabolism include pyruvate dehydrogenase (makes acetyl-CoA), lactate dehydrogenase (makes lactate), transaminases (make alanine), pyruvate carboxylase (makes ox- Figure 6.23 - Pyruvate metabolism. When oxygen is absent, pyruvate is converted to lactate (animals) or ethanol (bacteria and yeast). When oxygen is present, pyruvate is converted to acetyl-CoA. Not shown - Pyruvate transamination to alanine or carboxylation to form oxaloacetate. aloacetate), and pyruvate decarboxylase (a part of pyruvate dehydrogenase that makes acetaldehyde in bacteria and yeast). Catalytic action and regulation of the pyruvate dehydrogenase complex is discussed in the section on the citric acid cycle (HERE). Gluconeogenesis The anabolic counterpart to glycolysis is gluconeogenesis (Figure 6.26), which occurs mostly in the cells of the liver and kidney and virtually no other cells in the body. In seven of the eleven reactions of gluconeogenesis (starting from pyruvate), the same enzymes are used as in glycolysis, but the reaction directions are reversed. Notably, the ∆G values of these reactions in the cell are typically near zero, meaning their direction can be readily controlled by changing substrate and product concentrations by small amounts. The three regulated enzymes of glycolysis all catalyze reactions whose cellular ∆G values are not close to zero, making manipulation of reaction direction for their reac- Figure 6.24 - Formation of lactate in animal fermentation produces NAD+ for G3PDH Image by Ben Carson Figure 6.25 - Formation of ethanol in microbial fermentation produces NAD+ for G3PDH Image by Ben Carson tions non-trivial. Consequently, cells employ “work-around” reactions catalyzed by four different enzymes to favor gluconeogenesis, when appropriate. Bypassing pyruvate kinase Two of the enzymes (pyruvate carboxylase and PEP carboxykinase - PEPCK) catalyze reactions that bypass pyruvate kinase. F1,6BPase bypasses PFK-1 and G6Pase bypasses hexokinase. Notably, pyruvate carboxylase and G6Pase are found in the mitochondria and endoplasmic reticulum, respectively, whereas the other two are found in the cytoplasm along with all of the enzymes of glycolysis. Biotin An important coenzyme used by pyruvate carboxylase is biotin (Figure 6.27). Biotin is commonly used by carboxylases to carry CO2 to incorporate into the substrate. Also known as vitamin H, biotin is a water soluble B vitamin (B7) needed for many metabolic processes, including fatty acid synthesis, gluconeogenesis, and amino acid metabolism. Deficiency of the vitamin is rare, since it is readily produced by gut Gluconeogenesis and glycolysis. Only the enzymes differing in gluconeogenesis are shown Image by Aleia Kim teria. There are many claims of advantages of taking biotin supplements, but there is no strong indication of benefits in most cases. Deficiencies are associated with inborn genetic errors, alcoholism, burn patients, and people who have had a gastrectomy. Some pregnant and lactating women may have reduced levels due to increased biotin catabolism. Reciprocal regulation All of the enzymes of glycolysis and nine of the eleven enzymes of gluconeogenesis are all in the cytoplasm, necessitating a coordinated means of controlling them. Cells generally need to minimize the extent to which paired anabolic and catabolic pathways are occurring simultaneously, lest they produce a futile cycle, resulting in wasted energy with no tangible product except heat. The mechanisms of controlling these pathways have opposite effects on catabolic and anabolic processes. This method of control is called reciprocal regulation (see above). Reciprocal regulation is a coordinated means of simultaneously controlling metabolic pathways that do opposite things. In reciprocal regulation, a single molecule (allosteric regulation) or a single covalent modification (phosphorylation/dephosphorylation, Allosteric Regulation of Glycolysis & Gluconeogenesis Reciprocal Regulation AMP - Activates PFK-1, Inhibits F1,6BPase F2,6BP - Activates PFK-1, Inhibits F1,6BPase Citrate - Activates PFK-1, Inhibits F1,6BPase Glycolysis Only ATP - Inhibits PFK-1 and Pyruvate Kinase Alanine - Inhibits Pyruvate Kinase Gluconeogenesis Only ADP - Inhibits Pyruvate Carboxylase and PEPCK Acetyl-CoA - Activates Pyruvate Carboxylase Figure 6.27 - Biotin carrying carbon dioxide (red) Wikipedia for example) has opposite effects on the different pathways. Reciprocal allosteric effects For example, in glycolysis, the enzyme known as phosphofructokinase (PFK-1) is allosterically activated by AMP and a molecule known as F2,6BP (Figure 6.28). The corresponding enzyme from gluconeogenesis catalyzing a reversal of the glycolysis reaction is known as F1,6BPase. F1,6BPase is inhibited by both AMP and F2,6BP. Reciprocal covalent effects In glycogen metabolism, the enzymes phosphorylase kinase and glycogen phosphorylase catalyze reactions important for the breakdown of glycogen. The enzyme glycogen synthase catalyzes the synthesis of glyco- Directional velocity Inverts with reciprocity If glycolysis is flowing Glucose synthesis awaits But when the latter is a-going Sugar breakdown then abates Figure 6.28 - Regulation of glycolysis (orange path) and gluconeogenesis (black path) Image by Aleia Kim gen. Each of these enzymes is, at least partly, regulated by attachment and removal of phosphate. Phosphorylation of phosphorylase kinase and glycogen phosphorylase has the effect of making them more active, whereas phosphorylation of glycogen synthase makes it less active. Conversely, dephosphorylation has the reverse effects on these enzymes - phosphorylase kinase and glycogen phosphorylase become less active and glycogen synthase becomes more active. Simple and efficient The advantage of reciprocal regulation schemes is that they are very efficient. It doesn’t take separate molecules or separate treatments to control two pathways simultaneously. Further, its simplicity ensures that when one pathway is turned on, the other is turned off. This is especially important with catabolic/ anabolic regulation, because having both pathways going on simultaneously in a cell is not very productive, leading only to production of heat in a futile cycle. A simple futile cycle is shown on Figure 6.29. If unregulated, the cyclic pathway in the figure (shown in black) will make ATP in creating pyruvate from PEP and will use ATP to make oxaloacetate from pyruvate. It will also use GTP to make PEP from oxaloacetate. Thus, each turn of the cycle will make one ATP, use one ATP and use one GTP for a net loss of energy. The process will start with pyruvate and end with pyruvate, so there is no net production of molecules. (see HERE for one physiological use of a futile cycle). Specific gluconeogenesis controls Besides reciprocal regulation, other mechanisms help control gluconeogenesis. First, PEPCK is controlled largely at the level of synthesis. Overexpression of PEPCK (stimulated by glucagon, glucocorticoid hormones, and cAMP and inhibited by insulin) produces symptoms of diabetes. Pyruvate carboxylase is sequestered in the mitochondrion (one means of regulation) Figure 6.29 - A simple futile cycle - follow the black lines Image by Aleia Kim Interactive Learning Module HERE and is sensitive to acetyl-CoA, which is an allosteric activator. Acetyl-CoA concentrations increase as the citric acid cycle activity decreases. Glucose-6- phosphatase is present in low concentrations in many tissues, but is found most abundantly and importantly in the major gluconeogenic organs – the liver and kidney cortex. Specific glycolysis controls Control of glycolysis and gluconeogenesis is unusual for metabolic pathways, in that regulation occurs at multiple points. For glycolysis, this involves three enzymes: 1. Hexokinase (Glucose ⇄ G6P) 2. Phosphofructokinase-1 (F6P ⇄ F1,6BP) 3. Pyruvate kinase (PEP ⇄ Pyruvate). Regulation of hexokinase is the simplest of these. The enzyme is unusual in being inhibited by its product, glucose-6-phosphate. This ensures when glycolysis is slowing down hexokinase is also slowing down to reduce feeding the pathway. Pyruvate kinase It might also seem odd that pyruvate kinase, the last enzyme in the pathway, is regulated (Figure 6.30), but the reason is simple. Pyruvate kinase catalyzes the most energetically rich reaction of glycolysis. The reaction is favored so strongly in the forward direction that cells must do a ‘two-step’ around it in the reverse direction when making glucose in the gluconeogenesis pathway. In other words, it takes two enzymes, two reactions, and two triphosphates (ATP and GTP) to go from one pyruvate back to one PEP in gluconeogenesis. When cells are needing to make glu- igure 6.30 - Regulation of pyruvate kinase For cells a glucose cycling’s cost Is energy in reams Four ATPs each time is lost From breaking/making schemes So use for metabolic heat To make it warm inside your feet Else it’s of no utility To practice such futility cose, they can’t be sidetracked by having the PEP they have made in gluconeogenesis be converted directly back to pyruvate by pyruvate kinase. Consequently, pyruvate kinase must be inhibited during gluconeogenesis or a futile cycle will occur and no glucose will be made. Another interesting control mechanism called feedforward activation involves pyruvate kinase. Pyruvate kinase is activated allosterically by the glycolysis intermediate, F1,6BP. This molecule is a product of the PFK-1 reaction and a substrate for the aldolase reaction. Reactions pulled As noted above, the aldolase reaction is energetically unfavorable (high positive ∆G°’), thus allowing F1,6BP to accumulate. When this happens, some of the excess F1,6BP binds to pyruvate kinase, which activates and jump- Figure 6.31 - Regulation of Synthesis and Breakdown of F2,6BP Image by Penelope Irving starts the conversion of PEP to pyruvate. The resulting drop in PEP levels has the effect of “pulling” on the reactions preceding pyruvate kinase. As a consequence, the concentrations of GLYAL3P and DHAP fall, helping to pull the aldolase reaction forward. PFK-1 regulation PFK-1 has a complex regulation scheme. First, it is reciprocally regulated (relative to F1,6BPase) by three molecules. F2,6BP activates PFK-1 and inhibits F1,6BPase. PFK-1 is also allosterically activated by AMP, whereas F1,6BPase is inhibited. On the other hand, citrate inhibits PFK-1, but activates F1,6BPase. PFK-1 is also inhibited by ATP and is exquisitely sensitive to proton concentration, easily losing activity when the pH drops only slightly. PFK- 1’s inhibition by ATP is noteworthy and odd at first glance because ATP is also a substrate whose increasing concentration should favor the reaction instead of inhibit it. The root of this conundrum is that PFK-1 has two ATP binding sites - one at an allosteric site that binds ATP relatively inefficiently and one that the active site that binds ATP with high affinity. Thus, only when ATP concentration is high is binding at the allosteric site favored and only then can ATP turn off the enzyme. F2,6BP regulation Regulation of PFK-1 by F2,6BP is simple at the PFK-1 level, but more complicated at the level of synthesis of F2,6BP. Despite having a name sounding like a glycolysis/ gluconeogenesis intermediate (F1,6BP), F2,6BP is not an intermediate in either pathway. Instead, it is made from fructose-6-phosphate and ATP by the enzyme known as phosphofructokinase-2 (PFK- 2 - Figure 6.31). Cori cycle With respect to energy, the liver and muscles act complementarily. The liver is the major or- Figure 6.32 - The Cori cycle Image by Aleia Kim gan in the body for the synthesis of glucose. Muscles are major users of glucose to make ATP. Actively exercising muscles use oxygen faster than the blood can deliver it. As a consequence, the muscles go anaerobic and produce lactate. This lactate is of no use to muscle cells, so they dump it into the blood. Lactate travels in the blood to the liver, which takes it up and reoxidizes it back to pyruvate, catalyzed by the enzyme lactate dehydrogenase (Figure 6.32). Pyruvate in the liver is then converted to glucose by gluconeogenesis. The glucose thus made by the liver is dumped into the bloodstream where it is taken up by muscles and used for energy, completing the important intercellular pathway known as the Cori cycle. Glucose alanine cycle The glucose alanine cycle (also known as the Cahill Cycle), has been described as the amine equivalent of the Cori cycle (Figure 6.33). The Cori cycle, of course, exports lac- Figure 6.33 - Overlap between the Cori cycle and the glucose alanine cycle tate from muscles (when oxygen is limiting) to the liver via the bloodstream. The liver, in turn, converts lactate to glucose, which it ships back to the muscles via the bloodstream. The Cori Cycle is an essential source of glucose energy for muscles during periods of exercise when oxygen is used faster than it can be delivered. In the glucose-alanine cycle, cells are generating toxic amines and must export them. This is accomplished by transaminating pyruvate (the product of glycolysis) to produce the amino acid alanine. The glucose-alanine process requires the enzyme alanine aminotransferase, which is found in muscles, liver, and intestines. Alanine is exported in the process to the blood and picked up by the liver, which deaminates it to release the amine for synthesis of urea and excretion. The pyruvate left over after the transamination is a substrate for gluconeogenesis. Glucose produced in the liver is then exported to the blood for use by cells, thus completing the cycle. Polysaccharide metabolism Sugars are metabolized rapidly in the body and that is one of the primary reasons they are used. Managing levels of glucose in the body is very important - too much leads to complications related to diabetes and too little gives rise to hypoglycemia (low blood sugar). Sugars in the body are maintained by three processes - 1) diet; 2) synthesis (gluconeogenesis); and 3) storage. The storage forms of sugars are, of course, the polysaccharides and their metabolism is our next topic of discussion. Amylose and amylopectin The energy needs of a plant are much less dynamic than those of animals. Muscular contraction, nervous systems, and information processing in the brain require large amounts of quick energy. Because of this, the polysaccharides stored in plants are somewhat less complicated than those of animals. Plants store glucose for energy in the form of amylose (Figure 6.34 and see HERE) and amylopectin and for structural integrity in the form of cellulose (see HERE). These structures differ in that cellulose contains glucose units solely joined by β-1,4 bonds, whereas amylose has only α-1,4 bonds and amylopectin has α-1,4 and α-1,6 bonds. Figure 6.34 Amylose, a polymer of glucose in plants Glycogen Animals store glucose primarily in liver and muscle in the form of a compound related to amylopectin known as glycogen. The structural differences between glycogen and amylopectin are solely due to the frequency of the α-1,6 branches of glucoses. In glycogen they occur about every 10 residues instead of every 30-50, as in amylopectin (Figure 6.35). Glycogen provides an additional source of glucose besides that produced via gluconeogenesis. Because glycogen contains so many glucoses, it acts like a battery backup for the body, providing a quick source of glucose when needed and providing a place to store excess glucose when glucose concentrations in the blood rise. The branching of glycogen is an important feature of the molecule metabolically as well. Since glycogen is broken down from the "ends" of the molecule, more branches translate to more ends, and more glucose that can be released at once. Just as in gluconeogenesis, the cell has a separate mechanism for glycogen synthesis that is distinct from glycogen breakdown. As noted previously, this allows the cell to separately control the reactions, avoiding futile cycles, and enabling a process to occur efficiently (synthesis of glycogen) that would not occur if Figure 6.35 - Glycogen Structure - α-1,4 links with α-1,6 branches every 7-10 residues it were simply the reversal of glycogen breakdown. Glycogen breakdown Breakdown of glycogen involves 1) release of glucose-1-phosphate (G1P), 2) rearranging the remaining glycogen (as necessary) to permit continued breakdown, and 3) conversion of G1P to G6P for further metabolism. G6P can be 1) used in glycolysis, 2) converted to glucose by gluconeogenesis, or 3) oxidized in the pentose phosphate pathway. Glycogen phosphorylase (sometimes simply called phosphorylase) catalyzes breakdown of glycogen into glucose-1- Phosphate (G1P - Figure 6.36). The reaction that produces G1P from glycogen is a phosphorolysis, not a hydrolysis reaction. The distinction is that hydrolysis reactions use water to cleave bigger molecules into smaller ones, but phosphorolysis reactions use phosphate instead for the same purpose. Note that the phosphate is just that - it does NOT come from ATP. Since ATP is not used to put phosphate on G1P, the reaction saves the cell energy. Glycogen debranching enzyme Glycogen phosphorylase will only act on nonreducing ends of a glycogen chain that are at least 5 glucoses away from a branch point. A second enzyme, Glycogen Debranching Enzyme (GDE) (also called debranching enzyme), is therefore needed to convert α (1-6) branches to α (1-4) branches. GDE acts on glycogen branches that have reached their limit of phosphorylysis with glycogen phosphorylase. Figure 6.36 - Breaking of α-1,4 bonds of glycogen by glycogen phosphorylase Image by Aleia Kim Interactive Learning Module HERE GDE acts to transfer a trisaccharide from an α-1,6 branch onto an adjacent α-1,4 branch, leaving a single glucose at the 1,6 branch. Note that the enzyme also catalyzes the hydrolysis of the remaining glucose at the 1,6 branch point (Figure 6.37). Thus, the breakdown products from glycogen are G1P and glucose (mostly G1P). Glucose can, of course, be converted to Glucose-6-Phosphate (G6P) as the first step in glycolysis by either hexokinase or glucokinase. G1P can be converted to G6P by action of an enzyme called phosphoglucomutase. This reaction is readily reversible, allowing G6P and G1P to be interconverted as the concentration of one or the other increases. This is important, because phosphoglucomutase is needed to form G1P for glycogen synthesis. Regulation of glycogen metabolism Regulation of glycogen metabolism is complex, occurring both allosterically and via hormone-receptor controlled events that result in protein phosphorylation or dephosphorylation. In order to avoid a futile cycle of glycogen synthesis and breakdown simultaneously, cells have evolved an elaborate set of controls that ensure only one pathway is primarily active at a time. Regulation of glycogen metabolism is managed by the enzymes glycogen phosphorylase and glycogen synthase. Glycogen phosphorylase is regulated by both allosteric factors (ATP, G6P, AMP, and glucose) and by covalent modification (phosphorylation / dephosphorylation). Its regulation is consistent with the energy needs of the cell. High energy molecules (ATP, G6P, glucose) al- Figure 6.37 - Catalytic activity of debranching enzyme losterically inhibit glycogen phosphorylase, while the low energy molecule AMP allosterically activates it. GPa/GPb allosteric regulation Glycogen phosphorylase exists in two different covalent forms – one form with phosphate (called GPa here) and one form lacking phosphate (GPb here). GPb is converted to GPa by phosphorylation by an enzyme known as phosphorylase kinase. GPa and GPb can each exist in an 'R' state and a 'T' state (Figure 6.38). For both GPa and GPb, the R state is the more active form of the enzyme. GPa's negative allosteric effector (glucose) is usually not abundant in cells, so GPa does not flip into the T state often. There is no positive allosteric effector of GPa. When glucose is absent, GPa automatically flips into the R (more active) state (Figure 6.39). It is for this reason that people tend to think of GPa as being the more active covalent form of the enzyme. GPb can convert from the GPb T state to the GPb R state by binding AMP. Unless a cell is low in energy, AMP concentration is low. Thus GPb is not converted Figure 6.38 - Glycogen phosphorylase regulation - covalent (horizontal) and allosteric (vertical) Image by Aleia Kim to the R state very often. This is why people think of the GPb form as less active than GPa. On the other hand, ATP and/or G6P are usually present at high enough concentration in cells that GPb is readily flipped into the T state (Figure 6.40). GPa/GPb covalent regulation The relative amounts of GPa and GPb largely govern the overall process of glycogen breakdown, since GPa tends to be active more often than GPb. It is i Phosphorylase kinase itself has two covalent forms – phosphorylated (active) and dephosphorylated (inactive). It is phosphorylated by the enzyme Protein Kinase A (PKA - ). Another way to activate the enzyme is allosterically with calcium (Figure 6.41). Phosphory- Figure 6.39 - Allosteric regulation of GPa Image by Aleia Kim Figure 6.40 - Allosteric regulation of GPb Image by Aleia Kim lase kinase is dephosphorylated by phosphoprotein phosphatase, the same enzyme that removes phosphate from GPa. PKA and cAMPcAMP PKA is activated by cAMP, which is, in turn, produced by adenylate cyclase after activation by a G-protein (See HERE for overview). G-proteins are activated ultimately by binding of ligands to specific membrane receptors called 7-TM receptors, also known as Gprotein coupled receptors. These are discussed in greater detail HERE. Common ligands for 7-TM receptors include epinephrine (binds β- adrenergic receptor) and glucagon (binds glucagon receptor). Epinephrine exerts its greatest effects on muscle and glucagon works preferentially on the liver. Thus, epinephrine and glucagon can activate glycogen breakdown by stimulating synthesis of cAMP followed by the cascade of events described above. Turning off glycogen breakdown Turning off signals is as important, if not more so, than turning them on. Glycogen is a precious resource. If its breakdown is not controlled, a lot of energy used in its synthesis is wasted. The steps in the glycogen breakdown regulatory pathway can be reversed at every level. First, the ligand (epinephrine or glucagon) can leave the receptor, turning off the stimulus. Second, the G-proteins have an inherent GTPase activity. GTP, of course, is what activates Gproteins, so a GTPase activity converts the GTP it is carrying to GDP and the G-protein becomes inactive. Thus, G-proteins turn off Figure 6.41 - Activation of phosphorylase kinase Image by Aleia Kim their own activity. Interfering with their ability to convert GTP to GDP can have dire consequences, including cancer in some cases. Third, cells have phosphodiesterase enzymes (inhibited by caffeine) for breaking down cAMP. cAMP is needed to activate PKA, so breaking it down stops PKA from activating phosphorylase kinase. Fourth, the enzyme known as phosphoprotein phosphatase (also called PP1) plays a major role. It can remove phosphates from phosphorylase kinase (inactivating it) and form GPa, converting it to the less likely to be active GPb. Regulation of phosphoprotein phosphatase activity occurs at several levels. Two of these are shown in Figures 6.42 & 6.43. In Figure 6.42, phosphoprotein phosphatase is shown being inactivated by phosphorylation of an inhibitor (called PI-1 - see below). This happens as a result of cascading actions from binding of epinephrine (or glucagon) to a cell’s β-adrenergic receptor. Reversal of these actions occurs when insulin binds to the cell’s insulin receptor, resulting in activation of phosphoprotein phosphatase. PI-1 The inhibitor PI-1 can block activity of phosphpoprotein phosphatase only if it (PI-1) is phosphorylated. When PI-1 gets dephosphorylated, it no longer functions as an inhibitor, so phosphoprotein phosphatase be- Figure 6.42 - Inactivation of phosphoprotein phosphatase by protein kinase A via phosphorylation of PI-1 (Inhibitor) and the GM binding protein Image by Pehr Jacobson Interactive Learning Module HERE comes active. Now, here is the clincher - PI-1 gets phosphorylated by PKA (thus, when epinephrine or glucagon binds to a cell) and gets dephosphorylated when insulin binds to a cell. Another regulatory mechanism Another way to regulate phosphoprotein phosphatase in the liver involves GPa directly (Figure 6.43). In liver cells, phosphoprotein phosphatase is bound to a protein called GL. GL can also bind to GPa. As shown in the figure, if the three proteins are complexed together (top of figure), then PP1 (phosphoprotein phosphatase) is inactive. When glucose is present (such as when the liver has made too much glucose), then the free glucose binds to the GPa and causes GPa to be released from the GL. This has the effect of activating phosphoprotein phosphatase, which begins dephosphorylating enzymes. As shown in the figure, two such enzymes are GPa (making GPb) and glycogen synthase b, making glycogen synthase a. These dephosphorylations have opposite effects on the two enzymes, making GPb, which is less active and glycogen synthase a, which is much more active. Glycogen synthesis The anabolic pathway opposing glycogen breakdown is that of glycogen synthesis. Just Figure 6.43 - Regulation of phosphoprotein phosphatase (PP-1) activity by GPa Image by Penelope Irving as cells reciprocally regulate glycolysis and gluconeogenesis to prevent a futile cycle between these pathways, so too do cells use reciprocal schemes to regulate glycogen breakdown and synthesis. Synthesis of glycogen starts with G1P, which is converted to an 'activated' intermediate, UDPglucose. This activated intermediate is what 'adds' the glucose to the growing glycogen chain in a reaction catalyzed by the enzyme known as glycogen synthase (Figure 6.44). Once the glucose is added to glycogen, the glycogen molecule may need to have branches inserted in it by the enzyme known as branching enzyme (Figure 6.45). Steps Let us first consider the steps in glycogen synthesis. 1) Glycogen synthesis from glucose involves phosphorylation to form G6P, and isomerization to form G1P (using phospho- igure 6.45 - Branch formation in glycogen by branching enzyme Image by Penelope Irving Figure 6.44 - Catalytic activity of glycogen synthase Image by Penelope Irving glucomutase, common to glycogen breakdown). G1P is reacted with UTP to form UDP-glucose in a reaction catalyzed by UDP-glucose pyrophosphorylase. Glycogen synthase catalyzes synthesis of glycogen by joining carbon #1 of the UDP-derived glucose onto the carbon #4 of the non-reducing end of a glycogen chain, to form the familiar α(1,4) glycogen links. Another product of the reaction is UDP. “Primer” requirements It is also worth noting, in passing, that glycogen synthase will only add glucose units from UDP-Glucose onto a preexisting glycogen chain that has at least four glucose residues. Linkage of the first few glucose units to form the minimal "primer" needed for glycogen synthase recognition is catalyzed by a protein called glycogenin, which attaches to the first glucose and catalyzes linkage of the first eight glucoses by α(1,4) bonds. 3) The characteristic α(1,6) branches of glycogen are the products of the enzyme known as branching enzyme. Branching enzyme breaks α(1,4) chains and carries the broken chain to the carbon #6 and forms an α(1,6) linkage (Figure 6.45). Regulation of glycogen synthesis The regulation of glycogen biosynthesis is reciprocal to that of glycogen breakdown. It also has a cascading covalent modification system similar to the glycogen breakdown system described above. In fact, part of the system is identical to glycogen breakdown. Epinephrine or glucagon signaling stimulates adenylate cyclase to make cAMP, which activates PKA. Figure 6.46 - Reciprocal regulation by the phosphorylation cascade - glycogen breakdown activated / glycogen synthesis inhibited Image by Penelope Irving Effect of phosphorylation In glycogen synthesis, protein kinase A phosphorylates the active form of glycogen synthase (GSa), and converts it into the usually inactive b form (called GSb). Note the conventions for glycogen synthase and glycogen phosphorylase. For both enzymes, the more active forms are called the 'a' forms (GPa and GSa) and the less active forms are called the 'b' forms (GPb and GSb). The major difference, however, is that GPa has a phosphate, but GSa does not and GPb has no phosphate, but GSb does. Thus phosphorylation and dephosphorylation have opposite effects on the enzymes of glycogen metabolism (Figure 6.46). This is the hallmark of reciprocal regulation. It is of note that the less active glycogen synthase form, GSb, can be activated by G6P. Recall that G6P had the exactly opposite effect on GPb. Glycogen synthase, glycogen phosphorylase (and phosphorylase kinase) can all be dephosphorylated by the same enzyme - phosphoprotein phosphatase - and it is activated when insulin binds to its receptor in the cell membrane. Big picture In the big picture, binding of epinephrine or glucagon to appropriate cell receptors stimulates a phosphorylation cascade which simultaneously activates breakdown of glycogen by glycogen phosphorylase and inhibits synthesis of glycogen by glycogen synthase. Epinephrine, is also known as adrenalin, and the properties that adrenalin gives arise from a large temporary increase of blood glucose, which powers muscles. On the other hand, insulin stimulates dephosphorylation by activating phosphoprotein phosphatase. Dephosphorylation reduces action of glycogen phosphorylase (less glycogen breakdown) and activates glycogen synthase (starts glycogen synthesis). Our bodies make glycogen when blood glucose levels rise. Since high blood glucose levels are harmful, insulin stimulates cells to take up glucose. In the liver and in muscle cells, the uptaken glucose is made into glycogen. Figure 6.47 - Cotton - the purest natural form of cellulose Wikipedia Interactive Learning Module HERE Cellulose synthesis Cellulose is synthesized as a result of catalysis by cellulose synthase. Like glycogen synthesis it requires an activated intermediate to add glucose residues and there are two possible ones - GDP-glucose and UDPglucose, depending on which cellulose synthase is involved. In plants, cellulose provides support to cell walls. The reaction catalyzed is shown next where Cellulosen = a polymer of [(1→4)-β-Dglucosyl] n units long. The GDP-glucose reaction is the same except with substitution of GDP-glucose for UDP-Figure 6.48 - The Pentose Phosphate Pathway - Enzymes - 1 = G6P dehydrogenase / 2 = 6-Phosphogluconolactonase / 3 = 6-PG dehydrogenase / 4a = Ribose 5- phosphate isomerase / 4b = Ribulose 5-phosphate 3-epimerase / 5,7 = Transketolase / 6 = Transaldolase UDP-glucose + Cellulosen UDP + Cellulosen+1 glucose. UDP-glucose for the reaction is obtained by catalysis of sucrose synthase. The enzyme is named for the reverse reaction. Pentose phosphate pathway The pentose phosphate pathway (PPP - also called the hexose monophosphate shunt) is an oxidative pathway involving sugars that is sometimes described as a parallel pathway to glycolysis. It is, in fact, a pathway with multiple inputs and outputs (Figure 6.48). PPP is also a major source of NADPH for biosynthetic reactions and can provide ribose-5-phosphate for nucleotide synthesis. Though when drawn out, the pathway’s “starting point” is often shown as glucose-6-phosphate (G6P), in fact there are multiple entry points including other glycolysis intermediates, such as fructose-6-phosphate (F6P) and glyceraldehyde-3-phosphate (GLYAL-3-P), as well as less common sugar compounds with 4,5, and 7 carbons. The multiple entry points and multiple outputs gives the cell tremendous flexibility to meet its needs by allowing it to use a variety of materials to make any of these products. Oxidation #1 Beginning with G6P, PPP proceeds through its oxidative phase as follows: The enzyme catalyzing the reaction is G6P dehydrogenase. It is the rate limiting step of the pathway and the enzyme is inhibited both by NADPH and acetyl-CoA. NADPH is important for anabolic pathways, such as fatty acid synthesis and also for maintaining glutathione in a reduced state. The latter is important in protection against damage from reactive oxygen species. Deficiency of the G6P dehydrogenase enzyme is not rare, leading to acute hemolytic anemia, due to reduced NADPH concentration, and a reduced ability of the cell to disarm reactive oxygen species with glutathione. Reduced activity of the enzyme appears to have a protective effect against malarial infection, likely due to the increased fragility of the red blood cell membrane, which is then unable to sustain an infection by the parasite. Hydrolysis Reaction #2 is a hydrolysis and it is catalyzed by Hydrolysis Reaction #2 is a hydrolysis and it is catalyzed by 6-phosphogluconolactonase. The reac- Sucrose + UDP UDP-glucose + Fructose G6P + NADP+ 6-Phosphoglucono-δ-lactone + NADPH tion converts the circular 6-phosphoglucono- δ-lactone into the linear 6- phosphogluconate (6-PG) in preparation for oxidation in the next step. Decarboxylation Reaction #3 is the only decarboxylation in the PPP and the last oxidative step. It is catalyzed by 6-phosphogluconate dehydrogenase. Mutations disabling the protein made from this gene negatively impact red blood cells. At this point, the oxidative phase of PPP is complete and the remaining reactions involve molecular rearrangements. Ru5P has two possible fates and these are each described below. Isomerization Reaction 4a: The enzyme catalyzing this reversible reaction is Ru5P isomerase (top of next column). It is important because this is the way cells make R-5-P for nucleotide synthesis. The R-5-P can also be used in other PPP reactions shown elsewhere. Epimerization Reaction 4b (catalyzed by Ru-5-P epimerase) is another source of a pentose sugars and provides an important substrate for subsequent reactions. Transketolase reactions The other reactions don’t really have an order to them and whether they occur or not depends on cellular needs. The first enzyme, transketolase, is flexible in terms of its substrate/product combinations and is used not only in PPP, but also in the Calvin cycle of plants. It catalyzes the next two reactions In the first reaction (above), two phosphorylated sugars of 5 carbons each are converted into one phosphorylated sugar of 3 carbons and one of 7 carbons. In the second (next page), a five carbon sugar phosphate and aRu-5-P Xylulose-5-phosphate (Xu-5-P) Xu-5-P + R-5-P GLYAL-3-P + Sedoheptulose-7-phosphate (S-7-P) 6-PG + NADP+ Ribulose-5-phosphate (Ru-5-P) + NADPH + CO2 6-Phosphoglucono-δ-lactone + H2O 6-phosphogluconate (6-PG) + H+ Ru-5-P Ribose-5-phosphate (R-5-P) four carbon sugar phosphate are rearranged into sugar phosphates with 3 and 6 carbons. Glycolysis intermediates In the reversible reactions of the pentose phosphate pathway, one can see how glycolysis intermediates can easily be rearranged and made into other sugars. Thus, GLYAL-3-P and F6P can be readily made into Ribose-5- phosphate for nucleotide synthesis. Involvement of F6P in the pathway permits cells to continue making nucleotides (by making R-5-P) or tryptophan (by making E- 4-P) even if the oxidative reactions of PPP are inhibited. The last reaction is catalyzed by the enzyme known as transaldolase. TPP co-factor Transketolase uses thiamine pyrophosphate (TPP) to catalyze reactions. TPP’s thiaFigure 6.49 - Intermediates of the pentose phosphate pathway Xu-5-P + Erythrose-4-phosphate (E-4-P) GLYAL-3-P + F6P GLYAL-3-P + S-7-P E-4-P + F6P zole ring’s nitrogen and sulfur atoms on either side of a carbon, allow it to donate a proton and act as an acid, thus forming a carbanion, which gets stabilized by the adjacent tetravalent nitrogen (Figures 6.50 & 6.51)). The stabilized carbanion plays important roles in the reaction mechanism of enzymes, such as transketolase that use TPP as a cofactor. Commonly, the carbanion acts as a nucleophile that attacks the carbonyl carbon of the substrate. Such is the case with transketolase. Attack by the carbanion breaks the carbonyl bond on the substrate and covalently links it to the ionized carbon of TPP, thus allowing it to “carry” the carbonyl group to the other substrate for attachment. In this way, two carbons are moved from Xu- 5-P to E-4-P to make F6P (from E-4-P) and GLYAL-3-P (from Xu-5-P). Similarly, S-7-P and GLYAL-3-P are made from R-5-P and Xu-5-P, respectively. Thiamines Thiamines are a class of compounds involved in catalysis of important respiration-related The Pentose Phosphate Pathway by Kevin Ahern I need erythrose phosphate And don’t know what to do My cells are full of G-6-P And NADP too But I just hit upon a plan As simple as can be I’ll run reactions through the path That’s known as PPP In just two oxidations There’s ribulose-5P Which morphs to other pentoses Each one attached to P The next step it is simple Deserving of some praise The pentose carbons mix and match Thanks to transketolase Glyceraldehyde’s a product Sedoheptulose is too Each with a trailing phosphate But we are not quite through Now three plus seven is the same As adding six and four By swapping carbons back and forth There’s erythrose-P and more At last I’ve got the thing I need From carbons trading places I’m happy that my cells are full Of some transaldolases Figure 6.50 - Thiamine pyrophosphate reactions in the citric acid cycle, pyruvate metabolism, the pentose phosphate pathway, and the Calvin cycle. Thiamine was the first water-soluble vitamin (B1) to be discovered via association with the peripheral nervous system disease known as Beriberi. Thiamine pyrophosphate (TPP) is an enzyme cofactor found in all living systems derived from thiamine by action of the enzyme thiamine diphosphokinase. TPP facilitates catalysis of several biochemical reactions essential for tissue respiration. Deficiency of the vitamin is rare today, though people suffering from Crohn’s disease, anorexia, alcoholism or undergoing kidney dialysis may develop deficiencies. TPP is required for the oxidative decarboxylation of pyruvate to form acetyl-CoA and similar reactions. Transketolase, an important enzyme in the pentose phosphate pathway, also uses it as a coenzyme. Besides these reactions, TPP is also required for oxidative decarboxylation of α-keto acids like α-ketoglutarate and branched-chain α-keto acids arising from metabolism of valine, isoleucine, and leucine. Figure 6.51 - Mechanism of action of thiamine pyrophosphate (TPP) - 1) Carbanion formation; 2) Nucleophilic attack; 3) Covalent attachment of carbonyl; 4) Transfer to second group; 5) Release of product and regeneration of TPP TPP acts in the pyruvate dehydrogenase complex to assist in decarboxylation of pyruvate and “carrying” the activated acetaldehyde molecule to its attachment (and subsequent oxidation) to lipoamide. Central to TPP’s function is the thiazolium ring, which stabilizes carbanion intermediates (through resonance) by acting as an electron sink (Figure 6.51). Such action facilitates breaking of carbon-carbon bonds such as occurs during decarboxylation of pyruvate to produce the activated acetaldehyde. Thiamine deficiency Thiamine is integral to respiration and is needed in every cell. Acute deficiency of thiamine leads to numerous problems - the best known condition is beriberi, whose symptoms include weight loss, weakness, swelling, neurological issues, and irregular heart rhythms. Figure 6.52 - The Calvin cycle - The resynthesis phase has multiple steps and is described below. Image by Aleia Kim Causes of deficiency include poor nutrition, significant intake of foods containing the enzyme known as thiaminase, foods with compounds that counter thiamine action (tea, coffee), and chronic diseases, including diabetes, gastrointestinal diseases, persistent vomiting. People with severe alcoholism often are deficient in thiamine. Calvin cycle The Calvin cycle (Figure 6.52) is a metabolic pathway occurring exclusively in photosynthetic organisms. Commonly referred to as the “Dark Cycle” or the Light-Independent Cycle, the Calvin cycle does not actually occur in the dark. The cell/chloroplast simply is not directly using light energy to drive it. Assimilation It is in the Calvin cycle of photosynthesis that carbon dioxide is taken from the atmosphere and ultimately built into glucose (or other sugars). Reactions of the Calvin cycle take place in regions of the chloroplast known as the stroma, the fluid areas outside of the thylakoid membranes. The cycle can be broken into three phases 1) assimilation of CO2 2) reduction reactions 3) regeneration of the starting material, ribulose 1,5 bisphosphate (Ru1,5BP). Though reduction of carbon dioxide to glucose ultimately requires electrons from twelve molecules of NADPH (and 18 ATPs), it is confusing because one reduction occurs 12 times (1,3 BPG to GLYAL-3P) to input the overall reduction necessary to make one glucose. Carbon dioxide Another reason students find the pathway confusing is because the carbon dioxide molecules are absorbed one at a time into six different molecules of Ru1,5BP. At no point are the six carbons ever together in the same molecule to make a single glucose. Instead, six molecules of Ru1,5BP (30 carbons) gain six more carbons via carbon dioxide and then split into 12 molecules of 3- phosphoglycerate (36 carbons). The gain of six carbons allows two three carbon molecules to be produced in excess for each turn of the cycle. These two molecules molecules are then converted into glucose using the enzymes of gluconeogenesis. The other ten molecules of 3-PG are used to regenerate the six molecules of Ru1,5BP. Figure 6.53 - Rubisco, the most abundant enzyme on Earth Cyclic pathway Like the citric acid cycle, the Calvin cycle doesn’t really have a starting or ending point, but can we think of the first reaction as the fixation of carbon dioxide to Ru1,5BP. This reaction is catalyzed by the enzyme known as ribulose-1,5 bisphosphate carboxylase (RUBISCO - Figure 6.53). The resulting six carbon intermediate is unstable and is rapidly converted to two molecules of 3- phosphoglycerate. As noted, if one starts with 6 molecules of Ru1,5BP and makes 12 molecules of 3-PG, the extra 6 carbons that are a part of the cycle can be shunted off as two three-carbon molecules of glyceraldehyde-3-phosphate (GLYAL3P) to gluconeogenesis, leaving behind 10 molecules to be reconverted into 6 moleFigure 6.54 - Resynthesis phase of the Calvin cycle - All paths lead to regenerating Ru1,5BP, which is the aim of the resynthesis phase. Glycolysis/gluconeogenesis intermediates shown in blue. Enzyme numbers explained in text. cules of Ru1,5BP. This occurs in what is called the resynthesis phase. Resynthesis phase The resynthesis phase (Figure 6.54) requires multiple steps, but only utilizes two enzymes unique to plants - sedoheptulose-1,7 bisphosphatase and phosphoribulokinase. RUBISCO is the third (and only other) enzyme of the pathway that is unique to plants. All of the other enzymes of the pathway are common to plants and animals and include some found in the pentose phosphate pathway and gluconeogenesis. Enzymes shown as numbers in Figure 6.54 are as follows (enzymes unique to plants in green): 1 - Phosphoglycerate kinase 2 - G3PDH 3 - Triosephosphate Isomerase 4 - Aldolase 5 - Fructose 1,6 bisphosphatase 6 - Transketolase 7 - Phosphopentose Epimerase 8 - Phosphoribulokinase 9 - Sedoheptulose 1,7 bisphosphatase 10 - Phosphopentose Isomerase Reactions The resynthesis phase begins with conversion of the 3-PG molecules into GLYAL3P (there are actually 10 GLYAL3P molecules involved in resynthesis, as noted above, but we are omitting numbers to try to help students to see the bigger picture. Suffice it to say that there are sufficient quantities of all of the molecules to complete the reactions described). Some GLYAL3P is converted to DHAP by triose phosphate isomerase. Some DHAPs are converted (via gluconeogenesis) to F6P (one phosphate is lost for each F6P). Two carbons from F6P are given to GLYAL3P to create E-4P and Xu-5P (reversal of PPP reaction). E- 4P combines with DHAP to form sedoheptulose-1,7 bisphosphate (S1,7BP). The phosphate at position #1 is Figure 6.55 - Use of CO2 (Calvin cycle) vs. O2 (photorespiration) by RUBISCO. Image by Pehr Jacobson cleaved by sedoheptulose-1,7 bisphosphatase to yield S-7-P. Transketolase (another PPP enzyme) catalyzes transfer to two carbons from S-7-P to GLYAL3P to yield Xu-5P and R5P. Phosphopentose isomerase catalyzes conversion of R5P to Ru5P and phosphopentose epimerase similarly converts Xu-5P to Ru5P. Finally, phosphoribulokinase transfers a phosphate to Ru5P (from ATP) to yield Ru1,5BP. Photorespiration In the Calvin cycle of photosynthesis, the enzyme ribulose-1,5-bisphosphate carboxylase (RUBISCO) catalyzes the addition of carbon dioxide to ribulose-1,5- bisphosphate (Ru1,5BP) to create two molecules of 3-phosphoglycerate. Molecular oxygen (O2), however, competes with CO2 for this enzyme, so about 25% of the time, the molecule that gets added is not CO2, but rather O2 (Figure 6.55). When this happens, the following reaction occurs This is the first step in the process known as photorespiration. The process of photorespiration is inefficient relative to the carboxylation of Ru1,5BP. Phosphoglycolate is converted to glyoxylate in the glyoxysome and then transamination of that yields glycine. Two glycines can combine in a complicated coupled set of reactions in the mitochondrion shown next. Figure 6.56 - Maize - a C4 plant Ru1,5BP + O2 Phosphoglycolate + 3-phosphoglycerate + 2H+ 2 Glycine + NAD+ + H2O Serine + CO2 + NH3 + NADH + H+ Deamination and reduction of serine yields pyruvate, which can be then be converted back to 3-phosphoglycerate. The end point of oxygenation of Ru1,5BP is the same as the carboxylation of Ru1,5BP reactions, but there are significant energy costs associated with it, making the process less efficient. C4 plants The Calvin cycle is the means by which plants assimilate carbon dioxide from the atmosphere, ultimately into glucose. Plants use two general strategies for doing so. The first is employed by plants called C3 plants (most plants) and it simply involves the pathway described above. They are called C3 plants because the first stable intermediate after absorbing carbon dioxide contains three carbons - 3-phosphoglycerate. Another class of plants, called C4 plants (Figure 6.56) employ a novel strategy for concentrating the Figure 6.57 - Assimilation of CO2 by C4 plants Image by Aleia Kim CO2 prior to assimilation. C4 plants are generally found in hot, dry environments where conditions would otherwise favor the wasteful photorespiration reactions of RUBISCO and loss of water. Capture by PEP In C4 plants, carbon dioxide is captured in special mesophyll cells first by phosphoenolpyruvate (PEP) to make oxaloacetate (contains four carbons and gives the C4 plants their name - Figure 6.57). The oxaloacetate is converted to malate and transported into bundle sheath cells where the carbon dioxide is released and captured by Ru1,5BP, as in C3 plants. The Calvin cycle proceeds from there. The advantage of the C4 plant scheme is that it allows concentration of carbon dioxide while minimizing loss of water and photorespiration. Peptidoglycan synthesis Bacterial cell walls contain a layer of protection known as the peptidoglycan layer. Assembly of the layer begins in the cytoplasm. Steps in the process follow 1. Donation of an amine from glutamine to fructose-6- phosphate and isomerization to make glucosamine-6- phosphate. 2. Donation of an acetyl group from acetyl-CoA to make N-acetylglucosamine-6- phosphate 3. Isomerization of N-acetylglucosamine-6- phosphate makes N-acetylglucosamine-1- phosphate Figure 6.58 - Peptidoglycan layer in a bacterial outer cell wall Wikipedia 4. UTP combines with N-acetylglucosamine-1-phosphate to make UDP-N-acetyl-glucosamine-1- phosphate 5. Addition of PEP and electrons from NADPH yields UDP-Nacetylmuramic acid 6. A pentapeptide or tetrapeptide chain is attached to the UDP-Nacetylmuramic acid. The sequence varies a bit between species, but commonly is L-Ala - D-Glu - L-Lys - DAla - D-Ala 7. Dolichol phosphate replaces UMP on the UDP-N-acetylmuramic acid-pentapeptide. 8. UDP-N-acetyl-glucosamine donates a glucose to the Nacetylmuramic acid part of the Dolichol-PP-N-acetylmuramic acidpentapeptide 9. A pentapeptide chain of glycines (pentaglycine) is linked to lysine of the pentapeptide chain to create a Dolichol-PP-Nacetylmuramic acid-N-acetylglucosaminedecapeptide. The pentaglycine serves as cross links in the overall structure. 10. Dolichol-PP is removed to yield Nacetylmuramic acid-N-acetylglucosaminedecapeptide Figure 6.60 - Catalytic activity of DDtranspeptidase Wikipedia Figure 6.59 - Penicillin 11. This last group is added to the growing peptidoglycan network by joining the pentaglycine of one chain to the tetrapeptide/ pentapeptide of another. The enzyme catalyzing the addition of the N-acetylmuramic acid-N-acetylglucosaminedecapeptide to the network in the last step is DD-transpeptidase. This is the cellular enzyme targeted by penicillin and its derivatives. One reason penicillin is so effective is because synthesis of a peptidoglycan cell wall for a single bacterium requires millions of cycles of reactions above. Even slowing down the process can have a major effect on bacterial growth. On the flip side, resistance to penicillin and derivatives arises as a result of mutations in one enzyme - the transpeptidase. Metabolons At this point, it is appropriate to bring up the concept of metabolons. Metabolons are cellular complexes containing multiple enzymes of a metabolic pathway that appear to be arranged so that the product of one enzymatic reaction is passed directly as substrate to the enzyme that catalyzes the next reaction in the metabolic pathway. The structural complexes are temporary and are held together by non-covalent forces. Metabolons appear to offer advantages of reducing the amount of water needed to hydrate enzymes. Activity of enzymes in the complex is increased. Most of the basic metabolic pathways are thought to use metabolons. They include glycolysis, the citric acid cycle, nucleotide metabolism, glycogen synthesis, steroid synthesis, DNA synthesis, RNA synthesis, the urea cycle, and the process of electron transport. Hypoxia Hypoxia occurs when the body or a region of it has an insufficient oxygen supply. Varia- Figure 6.61 - Hypoxia inducible factor tions in arterial oxygen concentrations in normal physiology may lead to hypoxia, for example, during hypoventilation training or strenuous physical exercise. Generalized hypoxia may appear in healthy people when at high altitudes. Cancer cells, which may be undergoing faster respiration than surrounding tissues may also tend to be hypoxic. Hypoxia is an important consideration for sugar metabolism due to the ability of cells to change their sugar metabolism (fermentation) when these conditions exist. The body’s response to hypoxia is to produce Hypoxia-Inducible Factors (HIFs), which are transcription factors that induce expression of genes to help cells adapt to the hypoxic conditions. Many of the genes activated by HIFs are enzymes of glycolysis and GLUTs (glucose transport proteins). The combination of these gene products allows cells to 1) import more glucose and 2) metabolize it more rapidly when it arrives. This is to be expected because anaerobic sugar metabolism is only about 1/15th as efficient as aerobic metabolism. Consequently, it requires much more sugar metabolism to keep the cancer cells alive. A recently discovered protein called cytoglobin is believed to help assist in hypoxia by facilitating transfer of oxygen from arteries to the brain. Covalent modification HIFs are regulated partly by an interesting covalent modification. When oxygen concentration is high, the enzyme prolyl hydroxylase will hydroxylate proline residues in HIFs. This stimulates the protein degradation system (proteasome) to degrade them. When oxygen concentration is low, the hydroxylation occurs to a much lower extent (or does not occur at all), reducing/stopping degradation of HIFs and allowing them to activate genes. In this way, the concentration of HIFs is kept high under low oxygen concentration (to activate HIF genes) and low under high oxygen concentrations (to stop synthesis of HIF genes).

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/06%3A_Metabolism/6.01%3A_Metabolism_-_Sugars.txt

Source: BiochemFFA_6_2.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Citric acid cycle The primary catabolic pathway in the body is the citric acid cycle because it is here that oxidation to carbon dioxide occurs for breakdown products of the cell’s major building blocks - sugars, fatty acids, and amino acids. The pathway is cyclic (Figure 6.63) and thus, doesn’t really have a starting or ending point. All of the reactions occur in mitochondria, though one enzyme is embedded in the organelle’s inner membrane. As needs change, cells may use a subset of the reactions of the cycle to produce a desired molecule rather than to run the entire cycle (see HERE). Acetyl-CoA The molecule “feeding” the citric acid cycle is acetyl-CoA and it can be obtained from pyruvate (from glycolysis), from fatty acid β-oxidation, from ketone bodies, and from amino acid metabolism. Molecules from other pathways feeding into the citric acid cycle for catabolism make the citric acid cycle ‘cataplerotic’. It is worth noting that acetyl-CoA has very different fates, depending on the cell’s energy status/needs (see HERE). The description below describes oxidation (catabolism) in citric acid cycle. Anabolically, acetyl-CoA is also very important for providing building blocks for synthesis of fatty acids, ketone bodies, amino acids and cholesterol. Other citric acid cycle intermediates are also important in amino acid metabolism (Figure 6.63), heme synthesis, electron shuttling, and shuttling of acetyl-CoA across the mitochondrial inner membrane. The ability of the citric acid cycle to supply intermediates to pathways gives rise to the term ‘anaplerotic.’ It means ‘to fill up.’ Before discussing the citric acid cycle, it is important to first describe one important enzyme complex that is a major source of acetyl-CoA for the cycle. Figure 6.64 - E1 Subunit of Pyruvate Dehydrogenase. Wikipedia Pyruvate decarboxylation The pyruvate dehydrogenase enzyme is a complex of multiple copies of three subunits that catalyze the decarboxylation of pyruvate to form acetyl-CoA. The reaction mechanism requires use of five coenzymes. Pyruvate dehydrogenase is an enormous complex in mammals with a size five times greater than ribosomes. Subunits The three subunits are designated by E1, E2, and E3. E2 is also referred to as dihydrolipoamide acetyltransferase and E3 is more precisely called dihydrolipoyl dehydrogenase. Confusion arises with the name for E1. Some call it pyruvate dehydrogenase and others give it the name pyruvate decarboxylase. We will use pyruvate decarboxylase solely to refer to E1 and pyruvate dehydrogenase only to refer to the complex of E1, E2, and E3. The catalytic actions of pyruvate dehydrogenase can be broken down into three steps, each taking place on one of the subunits. The steps, sequentially occurring on E1, E2, and E3, are 1) decarboxylation of pyruvate; 2) oxidation of the decarboxylated product; and 3) transfer of electrons to ultimately form NADH (Figure 6.65). Figure 6.65 - Mechanism of action of pyruvate decarboxylation and oxidation by pyruvate dehydrogenase. Catalysis The catalytic process begins after binding of the pyruvate substrate with activation of the thiamine pyrophosphate coenzyme through formation of an ylide intermediate. The nucleophilic carbanion of the ylide attacks the electrophilic ketone carbon on the pyruvate, releasing carbon dioxide and creating an enol that loses a proton on the carbon to become a 1,3 dipole that includes the positively charged nitrogen of the thiamine. The reaction (step A in Figure 6.65) is a non-oxidative decarboxylation. Oxidation of the two carbon hydroxyethyl unit occurs in the transfer to the lipoamide. Reductive acetylation Reductive acetylation occurs next (Step B) as the 2-carbon hydroxyethyl unit is transferred to lipoamide on E2. (Lipoamide is the name for a molecule of lipoic acid covalently attached to a lysine side chain in the E2 subunit). In prokaryotes in the absence of oxygen, the hydroxyethyl group is not passed to lipoamide, but instead is released as free acetaldehyde , which can accept electrons from NADH (catalyzed by alcohol dehydrogenase) and become ethanol in the process of fermentation. In the presence of oxygen in almost all aerobic organisms, the process continues with transfer of the hydroxyethyl unit to E2 and continuation of the cycle below. Oxidation step Transfer of the hydroxyethyl group from E1 to the lipoamide coenzyme in E2 is an oxidation, with transfer of electrons from the hydroxyethyl group to lipoamide’s disulfide (reducing it) and formation on the lipoamide of an acetyl-thioester (oxidizing it). The acetyl group is then transferred from lipoamide to coenzyme A in E2 (Step C in Figure 6.65), forming acetyl-CoA, which is released and leaving reduced sulfhydryls on the lipoamide. In order for the enzyme to return to its original state, the disulfide bond on lipoamide must be re-formed. This occurs with transfer of electrons from reduced lipoamide to an FAD covalently bound to E3 (Step D). This reduces FAD to FADH2. Formation of NADH In the last step in the process, electrons from FADH2 are transferred to external NAD+, forming NADH (Step E) and completing the overall cycle. Then enzyme can then begin another catalytic round by binding to a pyruvate. Pyruvate dehydrogenase regulation Pyruvate deyhdrogenase is regulated both allosterically and by covalent modification - phosphorylation / dephosphorylation. Regulation of pyruvate dehydrogenase, whether by allosteric or covalent mechanisms has the same strategy. Indicators of high energy shut down the enzyme, whereas indicators of low energy stimulate it. For allosteric regulation, the high energy indicators affecting the enzyme are ATP, acetyl-CoA, NADH, and fatty acids, which inhibit it. AMP, Coenzyme A, NAD+, and calcium, on the other hand, stimulate it (Figure 6.67). Covalent modification Covalent modification regulation of pyruvate dehydrogenase is a bit more complicated. It occurs as a result of phosphorylation by pyruvate dehydrogenase kinase (PDK - Figure 6.67) or dephosphorylation by pyruvate dehydrogenase phosphatase (PDP). PDK puts phosphate on any one of three serine residues on the E1 subunit, which causes pyruvate kinase to not be able to perform its first step of catalysis - the decarboxylation of pyruvate. PDP can remove those phosphates. PDK is allosterically activated in the mitochondrial matrix when NADH and acetyl-CoA concentrations rise. Product inhibition Thus, the products of the pyruvate dehydrogenase reaction inhibit the production of more products by favoring its phosphorylation by PDK. Pyruvate, a substrate of pyruvate dehydrogenase, inhibits PDK, so increasing concentrations of substrate activate pyruvate dehydrogenase by reducing its phosphorylation by PDK. As concentrations of NADH and acetyl-CoA fall, PDP associates with pyruvate kinase and removes the phosphate on the serine on the E1 subunit. Low concentrations of NADH and acetyl-CoA are necessary for PDP to remain on the enzyme. When those concentrations rise, PDP dissociates and PDK gains access to the serine for phosphorylation. Insulin and calcium can also activate the PDP. This is very important in muscle tissue, since calcium is a signal for muscular contraction, which requires energy. Insulin also also activates pyruvate kinase and the glycolysis pathway to use internalized glucose. It should be noted that the cAMP phosphorylation cascade from the β-adrenergic receptor has no effect on pyruvate kinase, though the insulin cascade does, in fact, affect PDP and pyruvate kinase. Citric acid cycle reactions Focusing on the pathway itself (Figure 6.69), the usual point to start discussion is addition of acetyl-CoA to oxaloacetate (OAA) to form citrate. Acetyl-CoA for the pathway can come from a variety of sources. The reaction joining it to OAA is catalyzed by citrate synthase and the ∆G°’ is fairly negative. This, in turn, helps to “pull” the malate dehydrogenase reaction preceding it in the cycle. In the next reaction, citrate is isomerized to isocitrate by action of the enzyme called aconitase. Isocitrate is a branch point in plants and bacteria for the glyoxylate cycle (see HERE). Oxidative decarboxylation of isocitrate by isocitrate dehydrogenase produces the first NADH and yields α-ketoglutarate. This five carbon intermediate is a branch point for synthesis of the amino acid glutamate. In addition, glutamate can also be made easily into this intermediate in the reverse reaction. Decarboxylation of α-ketoglutarate produces succinyl-CoA and is catalyzed by α-ketoglutarate dehydrogenase. The enzyme α-ketoglutarate dehydrogenase is structurally very similar to pyruvate dehydrogenase and employs the same five coenzymes – NAD+, FAD, CoA-SH, thiamine pyrophosphate, and lipoamide. Regeneration of oxaloacetate The remainder of the citric acid cycle involves conversion of the four carbon succinyl-CoA into oxaloacetate. Succinyl-CoA is a branch point for the synthesis of heme (see HERE). Succinyl-CoA is converted to succinate in a reaction catalyzed by succinyl-CoA synthetase (named for the reverse reaction) and a GTP is produced, as well – the only substrate level phosphorylation in the cycle. The energy for the synthesis of the GTP comes from hydrolysis of the high energy thioester bond between succinate and the CoA-SH. Evidence for the high energy of a thioester bond is also evident in the citrate synthase reaction, which is also very energetically favorable. Succinate is also produced by metabolism of odd-chain fatty acids (see HERE). Succinate Oxidation Oxidation of succinate occurs in the next step, catalyzed by succinate dehydrogenase. This interesting enzyme both catalyzes this reaction and participates in the electron transport system, funneling electrons from the FADH2 it gains in the reaction to coenzyme Q. The product of the reaction, fumarate, gains a water across its trans double bond in the next reaction, catalyzed by fumarase to form malate. Fumarate is also a byproduct of nucleotide metabolism and of the urea cycle. Malate is important also for transporting electrons across membranes in the malate-aspartate shuttle (see HERE) and in ferrying carbon dioxide from mesophyll cells to bundle sheath cells in C4 plants (see HERE). Rare oxidation Conversion of malate to oxaloacetate by malate dehydrogenase is a rare biological oxidation that has a ∆G°’ with a positive value (29.7 kJ/mol). The reaction is ‘pulled’ by the energetically favorable conversion of oxaloacetate to citrate in the citrate synthase reaction described above. Oxaloacetate intersects other important pathways, including amino acid metabolism (readily converted to aspartic acid), transamination (nitrogen movement) and gluconeogenesis. It is worth noting that reversal of the citric acid cycle theoretically provides a mechanism for assimilating CO2. In fact, this reversal has been noted in both anaerobic and microaerobic bacteria, where it is called the Arnon-Buchanan cycle (Figure 6.73). Regulation of the citric acid cycle Allosteric regulation of the citric acid cycle is pretty straightforward. The molecules involved are all substrates/products of the pathway or molecules involved in energy transfer. Substrates/products that regulate or affect the pathway include acetyl-CoA and succinyl-CoA . Inhibitors and activators High energy molecular indicators, such as ATP and NADH will tend to inhibit the cycle and low energy indicators (NAD+, AMP, and ADP) will tend to activate the cycle. Pyruvate dehydrogenase, which catalyzes formation of acetyl-CoA for entry into the cycle is allosterically inhibited by its product (acetyl-CoA), as well as by NADH and ATP. Regulated enzymes Regulated enzymes in the cycle include citrate synthase (inhibited by NADH, ATP, and succinyl-CoA), isocitrate dehydrogenase (inhibited by ATP, activated by ADP and NAD+), and α-ketoglutarate dehydrogenase (inhibited by NADH and succinyl-CoA and activated by AMP). Anaplerotic/cataplerotic pathway The citric acid cycle is an important catabolic pathway oxidizing acetyl-CoA into CO2 and generating ATP, but it is also an important source of molecules needed by cells and a mechanism for extracting energy from amino acids in protein breakdown and other breakdown products. This ability of the citric acid cycle to supply molecules as needed and to absorb metabolic byproducts gives great flexibility to cells. When citric acid cycle intermediates are taken from the pathway to make other molecules, the term used to describe this is cataplerotic, whereas when molecules are added to the pathway, the process is described as anaplerotic. Cataplerotic molecules The citric acid cycle’s primary cataplerotic molecules include α-ketoglutarate, succinyl-CoA, and oxaloacetate. Transamination of α-ketoglutarate and oxaloacetate produces the amino acids glutamate and aspartic acid, respectively. Oxaloacetate is important for the production of glucose in gluconeogenesis. Glutamate plays a very important role in the movement of nitrogen through cells via glutamine and other molecules and is also needed for purine synthesis. Aspartate is a precursor of other amino acids and for production of pyrimidine nucleotides. Succinyl-CoA is necessary for the synthesis of porphyrins, such as the heme groups in hemoglobin, myoglobin and cytochromes. Citrate is an important source of acetyl-CoA for making fatty acids. When the citrate concentration is high (as when the citric acid cycle is moving slowly or is stopped), it gets shuttled across the mitochondrial membrane into the cytoplasm and broken down by the enzyme citrate lyase to oxaloacetate and acetyl-CoA. The latter is a precursor for fatty acid synthesis in the cytoplasm. Anaplerotic molecules Anaplerotic molecules replenishing citric acid cycle intermediates include acetyl-CoA (made in many pathways, including fatty acid oxidation, pyruvate decarboxylation, amino acid catabolism, and breakdown of ketone bodies), α-ketoglutarate (from amino acid metabolism), succinyl-CoA (from propionic acid metabolism), fumarate (from the urea cycle and purine metabolism), malate (carboxylation of PEP in plants), and oxaloacetate (many sources, including amino acid catabolism and pyruvate carboxylase action on pyruvate in gluconeogenesis) Glyoxylate cycle A pathway related to the citric acid cycle found only in plants and bacteria is the glyoxylate cycle (Figures 6.74 & 6.75). The glyoxylate cycle, which bypasses the decarboxylation reactions while using most of the non-decarboxylation reactions of the citric acid cycle, does not operate in animals, because they lack two enzymes necessary for it – isocitrate lyase and malate synthase. The cycle occurs in specialized plant peroxisomes called glyoxysomes. Isocitrate lyase catalyzes the conversion of isocitrate into succinate and glyoxylate. Because of this, all six carbons of the citric acid cycle survive each turn of the cycle and do not end up as carbon dioxide. Succinate continues through the remaining reactions to produce oxaloacetate. Glyoxylate combines with another acetyl-CoA (one acetyl-CoA was used to start the cycle) to create malate (catalyzed by malate synthase). Malate can, in turn, be oxidized to oxaloacetate. It is at this point that the glyoxylate pathway’s contrast with the citric acid cycle is apparent. After one turn of the citric acid cycle, a single oxaloacetate is produced and it balances the single one used in the first reaction of the cycle. Thus, in the citric acid cycle, there is no net production of oxaloacetate in each turn of the cycle. Net oxaloacetate production On the other hand, thanks to assimilation of carbons from two acetyl-CoA molecules, each turn of the glyoxylate cycle results in two oxaloacetates being produced, after starting with one. The extra oxaloacetate of the glyoxylate cycle can be used to make other molecules, including glucose in gluconeogenesis. This is particularly important for plant seed germination (Figure 6.76), since the seedling is not exposed to sunlight. With the glyoxylate cycle, seeds can make glucose from stored lipids. Because animals do not run the glyoxylate cycle, they cannot produce glucose from acetyl-CoA in net amounts, but plants and bacteria can. As a result, plants and bacteria can turn acetyl-CoA from fat into glucose, while animals can’t. Bypassing the oxidative decarboxylations (and substrate level phosphorylation) has energy costs, but, there are also benefits. Each turn of the glyoxylate cycle produces one FADH2 and one NADH instead of the three NADHs, one FADH2, and one GTP made in each turn of the citric acid cycle. Carbohydrate needs Organisms that make cell walls, such as plants, fungi, and bacteria, need large quantities of carbohydrates as they grow to support the biosynthesis of the complex structural polysaccharides of the walls. These include cellulose, glucans, and chitin. Notably, each of the organisms can operate the glyoxylate cycle using acetyl-CoA from β-oxidation. Coordination of the glyoxylate cycle and the citric acid cycle The citric acid cycle is a major catabolic pathway producing a considerable amount of energy for cells, whereas the glyoxylate cycle’s main function is anabolic - to allow production of glucose from fatty acids in plants and bacteria. The two pathways are physically separated from each other (glyoxylate cycle in glyoxysomes / citric acid cycle in mitochondria), but nonetheless a coordinated regulation of them is important. The enzyme that appears to provide controls for the cycle is isocitrate dehydrogenase. In plants and bacteria, the enzyme can be inactivated by phosphorylation by a kinase found only in those cells. Inactivation causes isocitrate to accumulate in the mitochondrion and when this happens, it gets shunted to the glyoxysomes, favoring the glyoxylate cycle. Removal of the phosphate from isocitrate dehydrogenase is catalyzed by an isocitrate dehydrognease-specific phosphoprotein phosphatase and restores activity to the enzyme. When this happens, isocitrate oxidation resumes in the mitochondrion along with the rest of the citric acid cycle reactions. In bacteria, where the enzymes for both cycles are present together in the cytoplasm, accumulation of citric acid cycle intermediates and glycolysis intermediates will tend to favor the citric acid cycle by activating the phosphatase, whereas high energy conditions will tend to favor the glyoxylate cycle by inhibiting it. Acetyl-CoA metabolism Acetyl-CoA is one of the most “connected” metabolites in biochemistry, appearing in fatty acid oxidation/synthesis, pyruvate oxidation, the citric acid cycle, amino acid anabolism/catabolism, ketone body metabolism, steroid/bile acid synthesis, and (by extension from fatty acid metabolism) prostaglandin synthesis . Most of these pathways will be dealt with separately. Here we will cover ketone body metabolism. Ketone body metabolism Ketone bodies are molecules made when the blood levels of glucose fall very low. Ketone bodies can be converted to acetyl-CoA by reversing the reaction of the pathway that makes them (Figure 6.78). Acetyl CoA, of course, can be used for ATP synthesis via the citric acid cycle. People who are very hypoglycemic (including some diabetics) will produce ketone bodies (Figure 6.79) and these are often first detected by the smell of acetone on their breath. Overlapping pathways The pathways for ketone body synthesis and cholesterol biosynthesis (Figure 6.80 and see HERE) overlap at the beginning. Each of these starts by combining two acetyl-CoAs together to make acetoacetyl-CoA. Not coincidentally, that is the next to last product of β-oxidation of fatty acids with even numbers of carbons (see HERE for fatty acid oxidation). In fact, the enzyme that catalyzes the joining is the same as the one that catalyzes its breakage in fatty acid oxidation – thiolase. Thus, these pathways start by reversing the last step of the last round of fatty acid oxidation. HMG-CoA formation Both pathways also include addition of two more carbons to acetoacetyl-CoA from a third acetyl-CoA to form hydroxy-methyl-glutaryl-CoA, or HMG-CoA, as it is more commonly known. It is at this point that the two pathways diverge. HMG-CoA is a branch point between the two pathway and can either go on to become cholesterol or ketone bodies. In the latter pathway, HMG-CoA is broken down into acetyl-CoA and acetoacetate. Acetoacetate is itself a ketone body and can be reduced to form another one, D-β-hydroxybutyrate (not actually a ketone, though). Alternatively, acetoacetate can be converted into acetone. This latter reaction can occur either spontaneously or via catalysis by acetoacetate decarboxylase. Acetone can be converted into pyruvate and pyruvate can be made into glucose. D-β-hydroxybutyrate travels readily in the blood and crosses the blood-brain barrier. It can be oxidized back to acetoacetate, converted to acetoacetyl-CoA, and then broken down to two molecules of acetyl-CoA for oxidation in the citric acid cycle. Ketosis When a body is producing ketone bodies for its energy, this state in the body is known as ketosis. Formation of ketone bodies in the liver is critical. Normally glucose is the body’s primary energy source. It comes from the diet, from the breakdown of storage carbohydrates, such as glycogen, or from glucose synthesis (gluconeogenesis). Since the primary stores of glycogen are in muscles and liver and since gluconeogenesis occurs only in liver, kidney, and gametes, when the supply of glucose is interrupted for any reason, the liver must supply an alternate energy source. From fatty acid breakdown In contrast to glucose, ketone bodies can be made in animals from the breakdown of fat/fatty acids. Most cells of the body can use ketone bodies as energy sources. Ketosis may arise from fasting, a very low carbohydrate diet or, in some cases, diabetes. Acidosis The term acidosis refers to conditions in the body where the pH of arterial blood drops below 7.35. It is the opposite of the condition of alkalosis, where the pH of the arterial blood rises above 7.45. Normally, the pH of the blood stays in this narrow pH range. pH values of the blood lower than 6.8 or higher than 7.8 can cause irreversible damage and may be fatal. Acidosis may have roots in metabolism (metabolic acidosis) or in respiration (respiratory acidosis). There are several causes of acidosis. In metabolic acidosis, production of excess lactic acid or failure of the kidneys to excrete acid can cause blood pH to drop. Lactic acid is produced in the body when oxygen is limiting, so anything that interferes with oxygen delivery may create conditions favoring production of excess lactic acid. These may include restrictions in the movement of blood to target tissues, resulting in hypoxia (low oxygen conditions) or decreases in blood volume. Issues with blood movement can result from heart problems, low blood pressure, or hemorrhaging. Strenuous exercise can also result in production of lactic acid due to the inability of the blood supply to deliver oxygen as fast as tissues require it (hypovolemic shock). At the end of the exercise, though, the oxygen supply via the blood system quickly catches up. Respiratory acidosis arises from accumulation of carbon dioxide in the blood. Causes include hypoventilation, pulmonary problems, emphysema, asthma, and severe pneumonia. Figure 6.81 - Symptoms of acidosis

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/06%3A_Metabolism/6.02%3A_Citric_Acid_Cycle__Related_Pathways.txt

Source: BiochemFFA_6_3.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy In the modern Western world, which is fat and getting fatter, there is a tremendous amount of interest in the metabolism of fat and fatty acids. Fat is the most important energy storage form of animals, storing considerably more energy per carbon than carbohydrates, but its insolubility in water requires the body to package it specially for transport. Surprisingly, fat/fatty acid metabolism is not nearly as tightly regulated as that of carbohydrates. Neither are the metabolic pathways of breakdown and synthesis particularly complicated, either. Movement of dietary fat Before we discuss the breakdown and synthesis of fat, let us first discuss the movement of dietary fat and oil (triglycerides - Figure 6.82) in the body. Upon consumption of triglycerides in the diet, they first are solubilized in the digestive system by the churning action of the stomach and the emulsifying properties of the bile acids. Upon passing into the lumen of the intestine, the triglycerides are acted on first by enzymes known as lipases that use water twice on each triglyceride to release two fatty acids, leaving behind a monoacylglyceride. As shown in Figure 6.83, the fatty acids and the monoacylglyceride are moved across the intestinal wall into the lymph system where they are reassembled back into a triglyceride. In the lymph system triglycerides and other insoluble lipids are packaged into lipoprotein complexes called chylomicrons that enter the blood stream and travel to target cells. The journey of lipids in the body after leaving the digestive system is long and is discussed in more depth HERE. In the body, fat is stored in specialized cells known as adipocytes. When these cells receive appropriate signals, they begin the breakdown of fat into glycerol and fatty acids. Breakdown of fat Breakdown of fat in adipocytes requires catalytic action of three enzymes. The first of these is controlled by binding of hormones to the cell membrane (Figure 6.84). It is the only regulated enzyme of fat breakdown and is known as hormone sensitive triacylglycerol lipase. It removes the first fatty acid from the fat. Diacylglyceride lipase removes the second one and monoacylglyceride lipase removes the third. As noted, only the first one is regulated and it appears to be the rate limiting reaction when active. Epinephrine activation As shown in Figure 6.84, activation of hormone sensitive triacylglycerol lipase (HSTL) is accomplished by epinephrine stimulation process and that it overlaps with the same activation that stimulates glycogen breakdown and gluconeogenesis. This coordination is very important. Each of the pathways stimulated by the epinephrine signaling system aims to provide the body with more materials to catabolize for energy - sugars and fatty acids. The HSTL is inhibited by dephospohrylation and this is stimulated by binding of insulin to its cell membrane receptor. Perilipin A protein playing an important roles in regulation of fat breakdown is perilipin. Perilipin associates with fat droplets and helps regulate action of HSTL, the enzyme catalyzing the first reaction in fat catabolism. When perilipin is not phosphorylated, it coats the fat droplet and prevents HSTL from getting access to it. Activation of protein kinase A in the epinephrine cascade, however, results in phosphorylation of both perilipin and HSTL. When this occurs, perilipin loosens its tight binding to the fat droplet, allowing digestion of the fat to begin by HSTL. Perilipin expression is high in obese organisms and some mutational variants have been associated with obesity in women. Another mutation reduces perilipin expression and is associated with greater lipolysis (fat breakdown) in women. Mice lacking perilipin eat more food than wild-type mice, but gain 1/3 less weight when on the same diet. Fat synthesis Synthesis of fat requires action of acyl transferase enzymes, such as glycerol-3 O-phosphate acyl transferase, which catalyzes addition of fatty acids to the glycerol backbone (reaction #1 above). The process requires glycerol-3-phosphate (or DHAP) and three fatty acids. In the first reaction, glycerol-3-phosphate is esterified at position 1 with a fatty acid, followed by a duplicate reaction at position 2 to make phosphatidic acid (diacylglycerol phosphate). This molecule, which is an intermediate in the synthesis of both fats and phosphoglycerides, gets dephosphorylated to form diacylglycerol before the esterification of the third fatty acid to the molecule to make a fat. Fatty acids released from adipocytes travel in the bloodstream bound to serum albumin. Arriving at target cells, fatty acids are taken up by membrane-associated fatty acid binding proteins, which help control cellular fatty acid uptake by transport proteins. Players in this process include CD36, plasma membrane-associated fatty acid-binding protein, and a family of fatty acid transport proteins (called FATP1-6). Fatty acid oxidation Upon arrival inside of target cells, fatty acids are oxidized in a process that chops off two carbons at a time to make acetyl-CoA, which is subsequently oxidized in the citric acid cycle. Depending on the size of the fatty acid, this process (called β-oxidation) will begin in either the mitochondrion (Figure 6.86) or the peroxisomes (see HERE). Transport To be oxidized in the mitochondrion, fatty acids must first be attached to coenzyme A (CoA-SH or CoA) and transported through the cytoplasm and the outer mitochondrial membrane. In the mitochondrion’s intermembrane space, the CoA on the fatty acid is replaced by a carnitine (Figure 6.87) in order to be moved into the matrix. After this is done, the fatty acid linked to carnitine is transported into the mitochondrial matrix and in the matrix the carnitine is replaced again by coenzyme A. It is in the mitochondrial matrix where the oxidation occurs. The fatty acid linked to CoA (called an acyl-CoA) is the substrate for fatty acid oxidation. Steps The process of fatty acid oxidation (Figure 6.88) is fairly simple. The reactions all occur between carbons 2 and 3 (with #1 being the one linked to the CoA) and sequentially include the following steps 1) dehydrogenation to create FADH2 and a fatty acyl group with a double bond between carbons 2 and 3 in the trans configuration; 2) hydration across the double bond to put a hydroxyl group on carbon 3 in the L configuration; 3) oxidation of the hydroxyl group to make a ketone; and 4) thiolytic cleavage to release acetyl-CoA and a fatty acid two carbons shorter than the starting one. Enzymes of β-oxidation Two of the enzymes of β-oxidation are notable. The first is acyl-CoA dehydrogenase, which catalyzes the dehydrogenation in the first reaction and yields FADH2. The enzyme comes in three different forms – ones specific for long, medium, or short chain length fatty acids. The first of these is sequestered in the peroxisomes of animals (see below) whereas the ones that work on medium and shorter chain fatty acids are found in the mitochondria. Αction of all three enzymes is typically needed to oxidize a fatty acid. Plants and yeast perform β-oxidation exclusively in peroxisomes. The most interesting of the acyl-CoA dehydrogenases is the one that works on medium length fatty acids. This one, which is the one most commonly deficient in animals, has been associated with sudden infant death syndrome. Reactions two and three in β-oxidation are catalyzed by enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase, respectively. The latter reaction yields an NADH. Thiolase The second notable enzyme of β-oxidation is thiolase because this enzyme not only catalyzes the formation of acetyl-CoAs in β-oxidation, but also the joining of two acetyl-CoAs (essentially the reversal of the last step of β-oxidation) to form acetoacetyl-CoA – essential for the pathways of ketone body synthesis and cholesterol biosynthesis. Similarity to citric acid cycle oxidation It is worth noting that oxidation of fatty acids is chemically very similar to oxidation of the four carbon compounds of the citric acid cycle (Figure 6.89). In fatty acid oxidation, dehydrogenation between carbons 2 and 3 generates electrons which are donated to FAD to make FADH2 and a trans-bonded intermediate is formed. The same thing happens in the citric acid cycle reaction catalyzed by succinate dehydrogenase - the trans-bonded molecule is fumarate. Addition of water in the second step of fatty acid oxidation occurs also in the next step of the citric acid cycle catalyzed by fumarase to create malate. Oxidation of the hydroxyl on carbon 3 in β-oxidation is repeated in the citric acid cycle reaction catalyzed by malate dehydrogenase yielding oxaloacetate. Oxidation of odd chain fatty acids Though most fatty acids of biological origin have even numbers of carbons, not all of them do. Oxidation of fatty acids with odd numbers of carbons ultimately produces an intermediate with three carbons called propionyl-CoA, which cannot be oxidized further in the β-oxidation pathway. Metabolism of this intermediate is odd. Sequentially, the following steps occur (Figure 6.90) – 1) carboxylation to make D-methylmalonyl-CoA; 2) isomerization to L-methylmalonyl-CoA; 3) rearrangement to form succinyl-CoA. The last step of the process utilizes the enzyme methylmalonyl-CoA mutase, which uses the B12 coenzyme in its catalytic cycle. Succinyl-CoA can be metabolized in the citric acid cycle. Peroxisomal oxidation Long chain fatty acids (typically 22 carbons or more - Figure 6.91) have their oxidation initiated in the peroxisomes, due to the localization of the long acyl-CoA dehydrogenase in that organelle. Peroxisomal fatty acid oxidation is chemically similar to β-oxidation of mitochondria, but there are some differences in the overall process. Differences First, since there is no electron transport system in peroxisomes, the reduced electron carriers produced in oxidation there must have their own recycling process. Peroxisomes accomplish this by transferring electrons and protons from FADH2 to O2 to form hydrogen peroxide (H2O2). As a result of this, the lack of electron transport means no proton pump and, consequently, no ATP produced from FADH2 for peroxisomal fatty acid oxidation, making it less efficient that mitochondrial β-oxidation. Electrons from NADH produced in the third step of the fatty acid oxidation must be shuttled to the cytoplasm and ultimately to the mitochondrion for ATP generation. Peroxisomal oxidation is increased for individuals on a high fat diet. In addition to long chain fatty acids, peroxisomes are also involved in oxidation of branched chain fatty acids, leukotrienes, and some prostaglandins. Unsaturated fatty acid oxidation Unsaturated fatty acids complicate the oxidation process a bit (see below), primarily because they have cis bonds, for the most part, if they are of biological origin, and these must be converted to the relevant trans intermediates for β-oxidation. Sometimes the bond must be moved down the chain, as well, in order to be positioned properly. Two enzymes (described below) handle all the necessary isomerizations and moves necessary to oxidize all of the unsaturated fatty acids (Figure 6.92). Extra enzymes As noted above, oxidation of unsaturated fatty acids requires two additional enzymes to the complement of enzymes for β-oxidation. If the β-oxidation of the fatty acid produces an intermediate with a cis bond between carbons three and four, cis-∆3-enoyl-CoA isomerase will convert the bond to a trans bond between carbons two and three and β-oxidation can proceed as normal. On the other hand, if β-oxidation produces an intermediate with a cis double bond between carbons four and five, the first step of β-oxidation (dehydrogenation between carbons two and three) occurs to produce an intermediate with a trans double bond between carbons two and three and a cis double bond between carbons four and five. 2,4 dienoyl-CoA reductase The enzyme 2,4 dienoyl CoA reductase reduces this intermediate (using NADPH) to one with a single cis bond between carbons three and four. The newly created cis-bonded molecule is then identical to the one acted on by cis-∆3-enoyl-CoA isomerase above, which converts it into a regular β-oxidation intermediate, as noted above. α-oxidation Yet another consideration for oxidation of fatty acids is α-oxidation. This pathway, which occurs in peroxisomes, is necessary for catabolism of fatty acids that have branches in their chains. For example, breakdown of chlorophyll’s phytol group yields phytanic acid (Figure 6.93), which undergoes hydroxylation and oxidation on carbon number two (in contrast to carbon three of β-oxidation), followed by decarboxylation and production of an unbranched intermediate that can be further oxidized by the β-oxidation pathway. Though α-oxidation is a relatively minor metabolic pathway, the inability to perform the reactions of the pathway leads to Refsum’s disease where accumulation of phytanic acid leads to neurological damage. ω-oxidation of fatty acids In addition to β-oxidation and α-oxidation of fatty acids, which occur in the mitochondria and peroxisomes of eukaryotic cells respectively, another fatty acid oxidation pathway known as ω-oxidation also occurs in the smooth endoplasmic reticulum of liver and kidney cells. It is normally a minor oxidation pathway operating on medium chain fatty acids (10-12 carbons), but gains importance 1) when β-oxidation is not functional or 2) for production of long chain intermediates, such as 20-HETE (20-hydroxyeicosatetraenoic acid), that can function in signaling. Steps in the process involve 1) oxidation of the terminal methyl group of the fatty acid to an alcohol; 2) oxidation of the alcohol to an aldehyde, and 3) oxidation of the aldehyde group to a carboxylic acid (Figure 6.94). The first oxidation is catalyzed by a mixed function oxidase, and yields 20-HETE if the starting material is arachidonic acid. The last two reactions are catalyzed by alcohol dehydrogenase and each requires NAD+. After the last oxidation, the fatty acid has carboxyl groups at each end and can be attached to coenzyme A at either end and subsequently oxidized, ultimately yielding succinate. Regulation of fatty acid oxidation Breakdown of fatty acids is controlled at different levels. The first is by control of the availability of fatty acids from the breakdown of fat. As noted above, this process is by regulating the activity of hormone-sensitive triacylglycerol lipase (HSTL) activity by epinephrine (stimulates) and insulin (inhibits). A second level of control of fatty acid availability is by regulation of carnitine acyl transferase (Figure 6.87 - see HERE). This enzyme controls the swapping of CoA on an acyl-CoA molecule for carnitine, a necessary step for the fatty acid to be imported into the mitochondrion for oxidation. The enzyme is inhibited by malonyl-CoA, an intermediate in fatty acid synthesis. Thus, when fatty acids are being synthesized, import of them into the mitochondrion for oxidation is inhibited. Last, the last enzyme in the β-oxidation cycle, thiolase, is inhibited by acetyl-CoA. Fatty acid synthesis Synthesis of fatty acids occurs in the cytoplasm and endoplasmic reticulum of the cell and is chemically similar to the reverse of the β-oxidation process, but with a couple of key differences (Figure 6.95). The first of these occur in preparing substrates for the reactions that grow the fatty acid. Fatty acid synthesis occurs in the cytoplasm of eukaryotic cells. Transport of acetyl-CoA from the mitochondrial matrix occurs when it begins to build up. This happens when the citric acid cycle slows or stops from lack of exercise. Two molecules can play roles in moving acetyl-CoA to the cytoplasm – citrate and acetylcarnitine. Joining of oxaloacetate with acetyl-CoA in the mitochondrion creates citrate which gets transported across the membrane, followed by action of citrate lyase in the cytoplasm of the cell to release acetyl-CoA and oxaloacetate. Additionally, when free acetyl-CoA accumulates in the mitochondrion, it may combine with carnitine and be transported out to the cytoplasm. Fatty acid synthase In animals, six different catalytic activities necessary to fully make palmitoyl-CoA are contained in a single complex called Fatty Acid Synthase. As shown in Figures 6.96 and 6.97, these include 1) transacylases (MAT) for swapping CoA-SH with ACP-SH on acetyl-CoA and malonyl-CoA; 2) a synthase (KS) to catalyze addition of the two carbon unit from the three carbon malonyl-ACP in the first step of the elongation process; 3) a reductase (KR) to reduce the ketone; 4) a dehydrase (DH) to catalyze removal of water; 5) a reductase (ER) to reduce the trans double bond and 6) a thioesterase (TE) to cleave the finished palmitoyl -CoA into palmitic acid and CoA-SH. In the middle of the complex is a site for binding the ACP portion of the growing fatty acid chain to hold it as the other part of the fatty acid is rotated into positions around the enzyme complex for each catalysis. In bacteria, these six activities are found on separate enzymes and are not part of a complex. Cytoplasmic reactions The process of making a fatty acid in the cytoplasm starts with two acetyl-CoA molecules. One is converted to malonyl-CoA by adding a carboxyl group. This reaction is catalyzed by the enzyme acetyl-CoA carboxylase (ACC), the only regulated enzyme of fatty acid synthesis (see below) and the only one separate from the fatty acid synthase. Next, both acetyl-CoA and malonyl-CoA have their CoA portions replaced by a carrier protein known as ACP (acyl-carrier protein) to form acetyl-ACP (catalyzed by acetyl-CoA : ACP transacylase - MAT in Figure 6.97) and malonyl-ACP (catalyzed by malonyl-CoA : ACP transacylase - MAT in Figure 6.97). Joining of a fatty acyl-ACP (in this case, acetyl-ACP) with malonyl-ACP splits out the carboxyl group from malonyl-ACP that was added to it and creates the acetoacyl-ACP intermediate (catalyzed by β-ketoacyl-ACP synthase - KS on Figure 6.97) . From this point forward, the chemical reactions resemble those of β-oxidation reversed. First, the ketone is reduced to a hydroxyl using NADPH (catalyzed by β-ketoacyl-ACP reductase - KR on Figure 6.97). In contrast to the hydroxylated intermediate of β-oxidation, the intermediate here (D-β- hydroxybutyryl-ACP) is in the D-configuration. Dehydration Next, water is removed from carbons 2 and 3 of the hydroxyl intermediate in a reaction catalyzed by 2,3-trans-enoyl-ACP dehydrase - DH on Figure 6.97. This yields a trans doubled-bonded molecule. Last, the double bond is hydrogenated to yield a saturated intermediate by 2,3-trans-enoyl-ACP reductase - ER on Figure 6.97. This completes the first cycle of synthesis. Additional cycles involve addition of more two-carbon units from malonyl-ACP to the growing chain until ultimately an intermediate with 16 carbons is produced (palmitoyl-ACP). At this point, a thioesterase cleaves the ACP from the palmitoyl-ACP to yield palmitic acid and the cytoplasmic synthesis ceases. Regulation of fatty acid synthesis Acetyl-CoA carboxylase, which catalyzes synthesis of malonyl-CoA, is the only regulated enzyme in fatty acid synthesis. Its regulation involves both allosteric control and covalent modification. The enzyme is known to be phosphorylated by both AMP Kinase and Protein Kinase A. Dephosphorylation is stimulated by phosphatases activated by insulin binding. Dephosphorylation activates the enzyme and favors its assembly into a long polymer, while phosphorylation reverses the process. Citrate acts as an allosteric activator and may also favor polymerization. Palmitoyl-CoA allosterically inactivates it. Elongation past 16 carbons Elongation to make fatty acids longer than 16 carbons occurs in the endoplasmic reticulum and is catalyzed by enzymes described as elongases. Mitochondria also can elongate fatty acids, but their starting materials are generally longer than 16 carbons long. The mechanisms in both environments are similar to those in the cytoplasm (a malonyl group is used to add two carbons, for example), but CoA is attached to the intermediates, not ACP. Further, whereas cytoplasmic synthesis employs the fatty acid synthase complex, the enzymes in these organelles are separable and not part of a complex. Desaturation of fatty acids Fatty acids are synthesized in the saturated form and desaturation occurs later - in the endoplasmic reticulum. Reactions to elongate the fatty acid (with elongases) may also occur to make unsaturated fatty acids of varying lengths. Desaturases are named according to the location of the double bonds they introduce in fatty acids. The delta (Δ) system numbers the carbon at the carboxyl end as number 1 and the omega (ω) number system numbers the carbon at the methyl end as number 1 (Figure 6.98). Humans have desaturases named as Δ5, Δ6, and Δ9. A Δ9 desaturase, for example, could convert stearic acid into oleic acid, because stearic acid (see HERE) is a saturated 18 carbon fatty acid and oleic acid is an 18 carbon fatty acid with only one double bond - at position Δ9. Polyunsaturated fatty acids Polyunsaturated fatty acids require the action of multiple enzymes and (in some cases) the action of elongases. Arachidonic acid, for example, is a 20 carbon fatty acid with four double bonds and its synthesis requires both an elongase (to increase the length of the fatty acid from 16 to 20) and multiple desaturases - one for each desaturated double bond. Animals are limited in the fatty acids they can make, due to an inability of their desaturases to catalyze reactions beyond carbons Δ9. Thus, humans can make oleic acid, but cannot synthesize linoleic acid (Δ9,12) or linolenic acid (Δ9,12,15). Consequently, these two must be provided in the diet and are referred to as essential fatty acids. Almost all desaturases make cis, not trans double bonds. There are a few minor exceptions to this, in cattle, for example (Figure 6.99). The trans fatty acids found in trans fat of prepared food are produced not by biological processes, but rather by the process of partial hydrogenation of unsaturated fats. Unusual oxidation reaction Removal of electrons and protons from a fatty acid to create a double bond is an oxidation reaction and these electrons, must have a destination. The path they take is a bit complex. It involves NAD(P)H, O2, two membrane-bound cytochromes, the membrane bound desaturase, and the fatty acid. In the electron transfer, the O2 is reduced to two molecules of H2O. This reduction requires four electrons and four protons. Two electrons and two protons come from the fatty acid to form the double bond on it. Two electrons come from the NAD(P)H via the cytochromes and two protons come from the aqueous solution. Prostaglandin synthesis The pathway for making prostaglandins and related molecules, such as the leukotrienes, prostacyclin, and thromboxanes is an extension of the synthesis of fatty acids (Figure 6.100). Prostaglandins, known as eicosanoids because they contain 20 carbons, are synthesized in cells from arachidonic acid whenever it has been cleaved from membrane lipids. Prostaglandins are important for many physiological phenomena in the body, including swelling and pain and reduction of their levels is a strategy of some painkillers, such as aspirin (see below). Inflammation arising from bee stings, for example, occurs because bee (and snake) venom contains mellitin, an activator of PLA2 activity (Figure 6.100). There are two strategies for reducing prostaglandin production and the pain associated with it. Phospholipase A2 Action of phospholipase enzymes on glycerophospholipids produces fatty acids and either glycerol-3-phosphate or other substances. Figure 6.101 shows cleavage sites on phospholipids that are targeted by different phospholipases. Phospholipase A1 (PLA1), for example, cleaves the fatty acid from position one of the glycerophospholipid and phospholipiase D (PLD) cleaves the R group from the phosphate part of the molecule. Since the fatty acid on position #2 (where PLA2 cuts) is most commonly unsaturated, PLA2 is an important phosopholipase for hydrolyzing the unsaturated fatty acid known as arachidonic acid from glycerophospholipids. Release of arachidonic acid from membranes is necessary for synthesis of prostaglandins. Inhibition of the release of arachidonic acid from membranes is the mechanism of action of steroidal anti-inflammatory drugs. They block action of phospholipase A2 (PLA2 - Figure 6.101) which cleaves arachidonic acid from membrane lipids. Lipocortin Lipocortin (also called annexin) is a protein that inhibits action of PLA2. Synthesis of lipocortin is stimulated by glucocorticoid hormones, such as cortisol, and is used in some treatments to reduce swelling/inflammation when it is severe and untreatable by non-steroidal drugs. Second strategy Synthesis of the prostanoid compounds (prostaglandins, prostacyclin, and thromboxanes) depends on conversion of arachidonic acid to prostaglandins G2 and H2 by COX enzymes. A non-steroidal strategy for decreasing production of prostaglandins then is to inhibit the enzyme that catalyzes their synthesis from arachidonic acid (Figure 6.102). This enzyme is known as prostaglandin synthase, but is more commonly referred to as a cyclooxygenase (or COX) enzyme. COX enzymes come in at least two forms in humans - COX-1, COX-2. A third form known as COX-3 has been reported as a splice variant of COX-1, but information about it is unclear. COX-1 and COX-2 are very similar in structure (70 kD and 72 kD, respectively, and 65% amino acid sequence homology), but coded by different genes. COX-1 is synthesized constitutively whereas COX-2 displays inducible expression behavior and has a more specific pattern of tissue expression. COX-2 enzymes are expressed in increasing amounts in areas of growth and inflammation. Non-steroidal drugs Molecules inhibiting cyclooxygenases are known as non-steroidal anti-inflammatory drugs (NSAIDs). Molecules in this class include aspirin, ibuprofen, vioxx, and celebrex. Targeting inhibitors Some NSAID inhibitors, such as aspirin, bind to all types of COX enzymes. Newer COX inhibitors target the COX-2 enzyme specifically because it was believed to be a better target for relief of joint pain than COX-1 enzymes which are synthesized by most cells. COX-2 enzymes are found more specifically in joints so the thinking was that specific inhibition of them would not affect the COX-1 enzymes which are important for producing prostaglandins that help maintain gastric tissue. Numerous COX-2 - specific inhibitors were developed - celecoxib, etoricoxib, and rofecoxib (Vioxx), for example. Unfortunately, the COX-2 specific inhibitors are associated with some serious side effects, including a 37% increase in incidence of major cardiovascular events in addition to some of the gastrointestinal problems of NSAIDs. Imbalance The increased risk of heart attack, thrombosis, and stroke are apparently due to an imbalance between prostacyclin (reduced by inhibitors) and thromboxanes (not reduced by the inhibitors). Prostacyclin (made from prostaglandin H2 by prostacyclin synthase) is a special prostaglandin that inhibits activation of blood platelets in the blood clotting process and acts as a vasodilator. Thromboxanes counter prostacyclin, causing vasoconstriction and activating blood platelets for clotting. Due to imbalances in these opposite acting molecules resulting from COX-2-specific inhibition, Vioxx, was withdrawn from the market in September, 2004, due to health concerns. Other compounds known to inhibit COX enzymes include some flavonoids, some components of fish oil, hyperforin, and vitamin D. Connections to other pathways There are several connections between fats and fatty acid metabolism and other metabolic pathways. Diacylglycerol (DAG - Figure 6.105), which is produced by removal of a phosphate from phosphatidic acid, is an intermediate in fat synthesis and also a messenger in some signaling systems. Phosphatidic acid, of course, is a branch intermediate in the synthesis of triacylglycerols and other lipids, including phosphoglycerides. Fatty acids twenty carbons long based on arachidonic acid (also called eicosanoids) are precursors of the leukotrienes, prostaglandins, thromboxanes, and endocannabinoids. Acetyl-CoA from β-oxidation can be assembled by the enzyme thiolase to make acetoacetyl-CoA, which is a precursor of both ketone bodies and the isoprenoids, a broad category of compounds that include steroid hormones, cholesterol, bile acids, and the fat soluble vitamins. In plants, acetyl-CoA can be made into carbohydrates in net amounts via the glyoxylate cycle. Fat, obesity, and hunger Obesity is an increasing problem in the western world. It is, in fact, the leading preventable cause of death worldwide. In 2014, over 600 million adults and 42 million children in the world were classified as obese, a condition when their body mass index is over 30 kg/m2 (Figure 6.106). The body mass index of a person is obtained by dividing a person’s weight by the square of their height. At a simple level, obesity arises from consumption of calories in excess of metabolic need, but there are many molecular factors to consider. Adipokines Adipokines are adipose tissue-synthesized cytokines. The class of molecules includes leptin (first discovered adipokine) and hundreds of other such compounds. These include adiponectin (regulates glucose levels and fatty acid oxidation), apelin (control of blood pressure, angiogenesis promotion, vasodilator release, increased water intake), chemerin (stimulation of lipolysis, adipocyte differentiation, link to insulin resistance), and resistin (links to obesity, type II diabetes, LDL production in liver), among others. Resistin Resistin is an adipokine peptide hormone with numerous associated negative health effects. Injection of the hormone into mice results in increased resistance to insulin, a phenomenon of type 2 diabetes. Resistin is linked to increased inflammation and serum levels of it correlate with increased obesity, though direct linkage of it to obesity is controversial. Resistin stimulates production of LDLs in the liver, supporting increased levels in the arteries. Resistin also adversely impacts the effects of statin drugs used to control levels of cholesterol in the body. Leptin Leptin is a peptide hormone (adipokine) made in adipose cells that negatively impacts hunger and regulates energy balance. It is countered by ghrelin, also known as the hunger hormone. Both hormones act in the hypothalamus where hunger is controlled. When leptin levels are higher due to higher levels of body fat, hunger is suppressed, but when levels of leptin are lower (less body fat), then appetite increases. Notably, leptin is also made in places besides adipose tissue and leptin receptors are found in places besides the hypothalamus, so the hormone has other effects in the body. When sensitivity to leptin changes, increased obesity can result. In mice, deletion of leptin function by mutation results in mice with voracious appetites and extreme obesity. Deletion of the leptin receptor gene in mice results in the same phenotype. Eight humans with leptin mutations all suffer from extreme obesity in infancy. Physiology Leptin is produced primarily by cells in white adipose tissue, but is also made in brown adipose tissue, ovaries, skeletal muscle, stomach, mammary epithelial cells and bone marrow. Leptin levels Leptin levels in the body are highest between midnight and early morning, presumably to suppress appetite. Though it is produced by fat cells, levels of leptin in humans do not strictly reflect levels of fat. For example, early in fasting, leptin levels fall before fat levels fall. Sleep deprivation can reduce leptin levels, as can increasing levels of testosterone and physical exercise. Increasing estrogen, however, increases leptin levels. Emotional stress and insulin can increase leptin levels. Obesity increases leptin levels, but doesn’t fully suppress appetite. Leptin resistance in these individuals is an important consideration, lessening the effects of the hormone on appetite. Blocking leptin action In the medial hypothalamus, leptin stimulates satiety and in the lateral hypothalamus, leptin inhibits hunger. Lesions in the lateral hypothalamus that block the ability to sense hunger result in anorexia (there are other causes of anorexia, though) and lesions in the medial hypothalamus cause excess hunger (no satiety). Neuropeptide Y is a potent hunger promoter whose receptors in the arcuate nucleus can be bound and blocked by leptin. Leptin levels are more sensitive to decreasing food intake than increasing food intake meaning that in humans the hormone plays a bigger role with respect to appetite than to levels of fat in the body. At the molecular level, binding of leptin to the Ob-Rb receptor causes down-regulation of synthesis of endocannabinoids, whose normal function is to increase hunger. High fructose diets have been associated with reduced levels of leptin and of leptin receptor. Ghrelin Ghrelin is a peptide hormone made by cells in the gastrointestinal tract when the stomach is empty. Stretching of the stomach reduces the expression of the hormone. Ghrelin exerts its effects on the central nervous system to increase appetite and it is an unusual peptide in being able to cross the blood-brain barrier. The ghrelin receptor in the brain is found on the same cells as the leptin receptor (arcuate nucleus). Leptin can counter the ghrelin effect by decreasing hunger. Behavioral effects Activation of ghrelin occurs after processing the zymogen form of the hormone (pre-proghrelin) followed by linkage of an octanoic acid to a serine at position 3. Circulating levels of ghrelin increase before eating and decrease afterwards. There appears to be a dose dependence for ghrelin on the amount of food consumed. Ghrelin increases food seeking behavior and there is a negative correlation between levels of ghrelin and weight. Neuropeptide Y Neuropeptide Y is a neuropeptide neurotransmitter produced by neurons of the sympathetic nervous system. It acts as a vasoconstrictor and favors growth of fat tissue. It appears to stimulate food intake, fat storage, relieve anxiety/stress, reduce pain perception, and lower blood pressure. Blockage of neuropeptide Y receptors in the brain of rats decreases food intake. Stress effects In mice and monkeys, repeated stress and high fat, high sugar diets stimulate neuropeptide Y levels and cause abdominal fat to increase. High levels of neuropeptide Y may also help individuals to recover from post-traumatic stress disorder and to reduce the fear response. It may also protect against alcoholism. Mice lacking the ability to make neuropeptide Y have a higher voluntary consumption of alcohol and are less sensitive to its effects. The neuropeptide Y receptor is a G-protein-coupled receptor in the 7-transmembrane domain family. Metabolism: Fats and Fatty Acids 564 YouTube Lectures by Kevin HERE & HERE 565 Figure 6.83 - Movement of dietary triglycerides Image by Aleia Kim Figure 6.82 - Trimyristin - A triacylglyceride 566 Figure 6.84 - Breakdown of fat in adipocytes Image by Pehr Jacobson 567 Figure 6.85 - Synthesis of fat from phosphatidic acid (phosphatidate) Image by Penelope Irving Synthesis of Phosphatidic Acid from Glycerol-3-phosphate 1. Glycerol-3-phosphate + Acyl-CoA <=> Monoacylglycerol phosphate + CoA-SH 2. Monoacylglycerol phosphate + Acyl-CoA <=> Phosphatidic acid + CoA-SH 568 Figure 6.86 - Mitochondria - site of β-oxidation Figure 6.87 - Transport of fatty acid (acyl group) across mitochondrial inner membrane Image by Aleia Kim 569 Figure 6.88 - Four reactions in β-oxidation Image by Aleia Kim YouTube Lectures by Kevin HERE & HERE 570 Figure 6.89 - Similar reactions for fatty acid oxidation and oxidation of 4-carbon compounds in the citric acid cycle Image by Aleia Kim 571 Figure 6.90 - Metabolism of propionyl-CoA Image by Pehr Jacobson 572 In beta oxidation, it just occurred to me The process all takes place ‘tween carbons two and three Some hydrogens are first removed to FADH2 Then water adds across the bond, the H to carbon two Hydroxyl oxidation’s next, a ketone carbon three Then thiolase catalysis dissects the last two C’s The products of the path, of course, are acetyl-CoAs Unless there were odd carbons, hence propionyl-CoA Figure 6.91 - Cerotic acid - A long chain fatty acid with 26 carbons 573 Figure 6.92 - Unsaturated fatty acid oxidation YouTube Lectures by Kevin HERE & HERE 574 Figure 6.93 - Phytanic acid 575 Figure 6.94 - ω Oxidation Wikipedia 576 Figure 6.95 - Fatty acid synthesis is the reverse of fatty acid oxidation chemically 577 Figure 6.96 - One round of fatty acid synthesis image by Aleia Kim Figure 6.97 - Fatty acid synthase complex 578 579 For fatty acid synthesis, I must reverse the path Of breaking fatty acids, though you’ll wonder ‘bout the math Each cycle of addition starts with carbons one two three Yet products of reactions number carbons evenly The reason is that CO2 plays peek-a-boo like games By linking to an Ac-CoA then popping off again Reactions are like oxidations ‘cept they’re backwards here Reduction, dehydration, then two hydrogens appear The product of the process is a 16 carbon chain The bonds are saturated. No double ones remain For them desaturases toil to put in links of cis In animals to delta nine, but no more go past this And last there’s making longer ones eicosanoidic fun They’re made by elongases in the e. reticulum Kevin Ahern˙ YouTube Lectures by Kevin HERE & HERE Figure 6.98 - Carbon numbering schemes for fatty acids Image by Pehr Jacobson 580 Stearoyl-CoA + 2 Cytochrome b5 (red) + O2 + 3 H+ + NADPH Oleoyl-CoA + 2 Ferricytochrome b5 (ox) + 2 H2O + NADP+ Desaturase Reaction to Oxidize Stearic Acid Figure 6.99 - Elaidic acid - A rare trans fatty acid in biology 581 Figure 6.100 - Eicosanoid synthesis pathways Image by Pehr Jacobson 582 583 Figure 6.101 - Cleavage sites for four phospholipiases on a glycerophospholipid - phospholipases A1 (PLA1), A2 (PLA2), C (PLC), and D (PLD) YouTube Lectures by Kevin HERE & HERE 584 Figure 6.102 - Catalytic activity of cyclooxygenase and peroxidase in making prostaglandins Image by Pehr Jacobson 585 Figure 6.103 - Synthesis of prostaglandins from prostaglandin H2 (red) Image by Pehr Jacobson Figure 6.104 - Two NSAIDs 586 Figure 6.105 - Diacylglycerol Figure 6.106 - Obesity worldwide - females (top) and males (bottom) Wikipedia 587 Figure 6.107 Leptin Wikipedia 588 YouTube Lectures by Kevin HERE & HERE Figure 6.108 Neuropeptide Y 589 590 Figure 6.109 - Pre-proghrelin 591 Graphic images in this book were products of the work of several talented students. Links to their Web pages are below Click HERE for Martha Baker’s Web Page Click HERE for Pehr Jacobson’s Web Page Click HERE for Aleia Kim’s Web Page Click HERE for Penelope Irving’s Web Page Problem set related to this section HERE Point by Point summary of this section HERE To get a certificate for mastering this section of the book, click HERE Kevin Ahern’s free iTunes U Courses - Basic / Med School / Advanced Biochemistry Free & Easy (our other book) HERE / Facebook Page Kevin and Indira’s Guide to Getting into Medical School - iTunes U Course / Book To see Kevin Ahern’s OSU ecampus courses - BB 350 / BB 450 / BB 451 To register for Kevin Ahern’s OSU ecampus courses - BB 350 / BB 450 / BB 451 Biochemistry Free For All Facebook Page (please like us) Kevin Ahern’s Web Page / Facebook Page / Taralyn Tan’s Web Page Kevin Ahern’s free downloads HERE OSU’s Biochemistry/Biophysics program HERE OSU’s College of Science HERE Oregon State University HERE Email Kevin Ahern / Indira Rajagopal / Taralyn Tan 592 When Acids Get Oxidized To the tune of "When Johnny Comes Marching Home" Metabolic Melodies Website HERE​ The fatty acids carried by​ CoA, CoA​ Are oxidized inside the ​ mi-to-chon-dri-ay They get to there as you have seen​ By hitching rides on carnitine​ Then it goes away​ When acids get oxidized Electrons move through membranes, yes​ It’s true, it’s true​ They jump from complex I onto​ Co-Q, Co-Q The action can be quite intense​ When building proton gradients​ And its good for you​ When acids get oxidized The protons pass through complex V​ You see, you see​ They do this to make lots of ​ A-TP, TP The mechanism you should know​ Goes through the stages L-T-O​ So there's energy​ When acids get oxidized Recording by Tim Karplus Lyrics by Kevin Ahern Recording by Tim Karplus Lyrics by Kevin Ahern 593 When Acids Are Synthesized To the tune of “When Johnny Comes Marching Home” Metabolic Melodies Website HERE The 16 carbon fatty acid, palmitate​ Gets all the carbons that it needs from acetate​ Which citric acid helps release​ From mitochondri - matrices​ Oh a shuttle's great ​ When acids are synthesized Carboxylase takes substrate and it puts within​ Dioxy carbon carried on a biotin​ CoA's all gain a quick release​ Replaced by larger ACPs​ And it all begins​ When acids are synthesized A malonate contributes to the growing chain​ Two carbons seven times around again, again​ For saturated acyl-ates​ There's lots of N-A-DPH ​ That you must obtain​ When acids are synthesized Palmitic acid made this way all gets released​ Desaturases act to make omega-threes​ The finished products big and small​ Form esters with a glycerol​ So you get obese​ When acids are synthesized Recording by Tim Karplus Lyrics by Kevin Ahern Recording by Tim Karplus Lyrics by Kevin Ahern

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/06%3A_Metabolism/6.03%3A_Fats_and_Fatty_Acids.txt

Source: BiochemFFA_6_4.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Sugars are the building blocks of carbohydrates, amino acids are the building blocks of proteins and nucleotides are the building blocks of the nucleic acids - DNA and RNA. Another crucial building block is acetyl-CoA, which is used to build many lipid substances, including fatty acids, cholesterol, fat soluble vitamins, steroid hormones, prostaglandins, endocannabinoids, and the bile acids. Indeed, acetyl-CoA goes into more different classes of molecule than any other building block. Isoprenoids We focus our attention here on a group of molecules made from acetyl-CoA that are known as as the isoprenoids. Isoprenoids are a large, diverse and ancient group of molecules that are found in all three domains of life. As noted earlier, they are components of membrane lipids in the cell membranes of archaebacteria, but beyond this, they serve an astonishing variety of functions. From photosynthetic pigments to plant defense compounds, from flavor compounds in cinnamon, mint, ginger and cloves to plant and animal hormones, from the cannabinoids in marijuana to the lycopene that gives tomatoes their color, and from heme to the quinones in the electron transport chain, isoprenoids are ubiquitous in cells. Isoprenoids derive their name from the fact that they are, in fact, made from five carbon building blocks called isoprenes that are derived from acetyl-CoA. The synthesis of the two isoprene units - isopentenyl pyrophosphate and dimethylallyl pyrophosphate is shown in Figure 6.111 and Figure 6.112. The pathway leading up to isoprene synthesis overlaps with that of ketone body synthesis, for the two reactions (Figure 6.112), as has been discussed earlier in this book (see HERE). Thiolase catalyzes the initial reaction, joining together two acetyl-CoA molecules to make acetoacetyl-CoA. In the second reaction catalyzed by HMG-CoA synthase, a third acetyl-CoA is joined to form the six carbon compound known as hydroxymethyl glutaryl-CoA (HMG-CoA). Reaction three is an important one biologically and medically because of the enzyme catalyzing it - HMG-CoA reductase. Statins Medically, HMG-CoA reductase is the target of a class of drugs known as statins (Figure 6.113 & Movie 6.1), which are used to reduce cholesterol levels in people. These competitive inhibitors, which compete with HMG-CoA for binding have two effects. First, they reduce the production of mevalonate, which restricts the amount of substrate available to make cholesterol. Second, and perhaps more importantly, they increase production of LDL receptors in the liver, which favors uptake and destruction of LDLs, thus lowering serum cholesterol levels. Regulation Biologically, the HMG-CoA reductase enzyme is also of importance because it is the primary regulatory point in cholesterol synthesis. Control of it is complex. First, it is feedback inhibited by cholesterol itself. High levels of glucose in the blood activate the enzyme. Phosphorylation by AMP-activated protein kinase inhibits its activity. Interestingly, the same enzyme phosphorylates and inactivates acetyl-CoA carboxylase - the only regulatory enzyme controlling fatty acid synthesis. Transcription of the gene encoding HMG-CoA reductase is enhanced by binding of the sterol regulatory element binding protein (SREBP) to the sterol recognition element (SRE ) located near the gene coding sequence. As cholesterol levels rise, SREBP is proteolytically cleaved and transcription stops. From HMG-CoA, the enzyme HMG-CoA reductase catalyzes the formation of mevalonate. This reaction requires NADPH and results in release of coenzyme A. Mevalonate gets phosphorylated twice and then decarboxylated to yield the five carbon intermediate known as isopentenyl-pyrophosphate (IPP). IPP is readily converted to the other important isoprenoid unit, dimethylallylpyrophosphate (DMAPP). Isoprenes These two five carbon compounds, IPP and DMAPP, are also called isoprenes (Figure 6.115) and are the building blocks for the synthesis of cholesterol and related compounds. This pathway proceeds in the direction of cholesterol starting with the joining of IPP and DMAPP to form geranyl-pyrophosphate. Geranyl-pyrophosphate combines with another IPP to make farnesyl-pyrophosphate, a 15-carbon compound. Squalene Two farnesyl-pyrophosphates join to create the 30-carbon compound known as squalene. Squalene, in a complicated rearrangement involving reduction and molecular oxygen forms a cyclic intermediate known as lanosterol (Figure 6.116) that resembles cholesterol. Conversion of lanosterol to cholesterol is a lengthy process involving 19 steps that occur in the endoplasmic reticulum. The cholesterol biosynthesis pathway from lanosterol is a long one and requires significant amounts of reductive and ATP energy. As noted earlier (see HERE), cholesterol has an important role in membranes. It is also a precursor of steroid hormones and bile acids and its immediate metabolic precursor, 7-dehydrocholesterol (Figure 6.117), branches to form vitamin D (Figure 6.118). All steroid hormones in animals are made from cholesterol and include the progestagens, androgens, estrogens, mineralocorticoids, and the glucocorticoids. The branch molecule for all of the steroid hormones is the cholesterol metabolite (and progestagen) known as pregnenalone (Figure 6.119). The progestagens are thus precursors of all of the other classes of steroid hormones. The estrogens are derived from the androgens in an interesting reaction that required formation of an aromatic ring (Figure 6.120). The enzyme catalyzing this reaction is known as an aromatase and it is of medical significance. The growth of some tumors is stimulated by estrogens, so aromatase inhibitors are prescribed to prevent the formation of estrogens and slow tumor growth. Two commonly used inhibitors include exemestane (a suicide inhibitor - Figure 6.121) and anastrozole (a competitive inhibitor). Other fat-soluble vitamins Synthesis of other fat soluble vitamins and chlorophyll also branches from the isoprenoid synthesis pathway at geranyl pyrophosphate. Joining of two geranylgeranyl pyrophosphates occurs in plants and bacteria and leads to synthesis of lycopene, which, in turn is a precursor of β-carotene, the final precursor of Vitamin A (see below also). Vitamins E and K, as well as chlorophyll are all also synthesized from geranylgeranyl pyrophosphate. Bile acid metabolism Another metabolic pathway from cholesterol leads to the polar bile acids, which are important for the solubilization of dietary fat during digestion. Converting the very non-polar cholesterol to a bile acid involves oxidation of the terminal carbon on the side chain off the rings. Other alterations to increase the polarity of these compounds include hydroxylation of the rings and linkage to other polar compounds. Common bile acids include cholic acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid, and deoxycholic acid (Figure 6.123). Another important consideration about bile acids is that their synthesis reduces the amount of cholesterol available and promotes uptake of LDLs by the liver. Normally bile acids are recycled efficiently resulting in limited reduction of cholesterol levels. However, inhibitors of the recycling promote reduction of cholesterol levels. Vitamin A Synthesis Vitamin A is important for many cellular functions related to growth, differentiation and organogenesis during embryonic development, tissue maintenance, and vision, to name a few. There are three main active forms of the vitamin, retinal, retinol and retinoic acid, each with its own set of functions. Retinal, complexed with the protein, opsin, is found in the rod cells of the retina and is necessary for vision. Retinol and retinoic acid both function as signaling molecules that can modulate gene expression during development. Synthesis of vitamin A occurs as a branch in synthesis of isoprenoids. Addition of isopentenyl pyrophosphate to farnesyl pyrophosphate creates a 20-carbon intermediate, geranylgeranyl pyrophosphate (GGPP - Figure 6.124). Joining of two GGPPs creates a 40 carbon intermediate that is unstable and decomposes to phytoene. Desaturases oxidize two single bonds in phytoene, creating lycopene. Lycopene is a linear 40 carbon unsaturated molecule found in tomatoes and other red vegetables and it gives them their color. Cyclization of end portions of lycopene give rise to β-carotene, the precursor of vitamin A (retinal/retinol - Figure 6.124). β-carotene is found in carrots and other orange vegetables, and is converted in the body to vitamin A. Catalytic action by β-Carotene 15,15’ monooxygenase cleaves β-carotene to form retinal (the aldehyde form used in vision). The enzyme retinol dehydrogenase catalyzes reduction of retinal to retinol (storage form). Oxidation of retinal creates another important retinoid known as retinoic acid. This form of vitamin A cannot be reduced back to retinal and thus cannot be used for vision or storage. Instead, retinoic acid has roles in embryonic development. Retinoic acid acts through binding to the Retinoic Acid Receptor (RAR). RAR binds to DNA and affects transcription of several important sets of genes important for differentiation. These include the Hox genes, which control anterior/posterior patterning in early embryonic development. Sphingolipid synthesis Synthesis of sphingolipids, which are found primarily in brain and nerve tissue, begins with palmitoyl-CoA and serine that combine to make an 18-carbon amine called 3-keto-sphinganine (Figure 6.125). Reduction of that by NADPH yields dihydrosphingosine and addition of a fatty acid from an acyl-CoA yields N-acylsphinganine, which is a ceramide (Figure 6.126). A ceramide can be converted into a cerebroside by addition of a glucose from UDP-glucose (Figure 6.127). If a few other simple sugars are added to the cerebroside, a globoside is created. If, instead of adding sugar, a phosphocholine is added from phosphatidylcholine, then sphingomyelin is created (Figure 6.127). If a complex set of sugars are added to to a cerebroside, then a ganglioside results (Figure 6.127). Sphingolipid breakdown In the overall metabolism of sphingolipids, the greatest problems arise with their catabolism. Figure 6.128 illustrates the numerous genetic diseases arising from mutations in DNA coding for some of these enzymes. All are lysosomal storage diseases and many of these are quite severe. GM1 glandiosidoses (arising from inability to breakdown GM1 gangliosides) cause severe neurodegeneration and seizures. Individuals suffering from them typically die by age 3. Tay-Sachs disease usually causes death by age 4, though late-onset forms of the disease in adults are known. With Gaucher’s disease, three different types have been described with widely varying effects. In some, the disease is fatal by age four and in others, it does not manifest until teens or even adulthood. Fabry’s disease patients can live into their 50s, on average. Glycerophospholipid metabolism Glycerophospholipids are the major components of membranes. Synthesis of glycerophospholipids begins with glycerol-3-phosphate. In the first reaction, glycerol-3-phosphate gains a fatty acid at position one from an acyl-CoA, followed by a duplicate reaction at position two to make phosphatidic acid (Figure 6.129). This molecule, which can branch to other reactions to form fats, is an important intermediate in the synthesis of many glycerophospholipids. Glycerophospholipid compounds can often be made by more than one pathway. The nucleotide CDP plays an important role in glycerophospholipid synthesis, serving as part of an activated intermediate for synthesis of phosphatidyl compounds. This is necessary, because formation of the phosphodiester bonds of these compounds requires higher energy input. Cells use two strategies to accomplish this. Both involve CDP. In the first, CTP combines with phosphatidic acid to make CDP-diacylglcyerol with release of a pyrophosphate. The reaction is catalyzed by phosphatidate cytidylyltransferase. CDP-diacylglycerol then serves as an activated intermediate to donate the phosphotidate part of itself to another molecule. The reaction below illustrates one example The second strategy is to make a CDP derivative of the group being added to phosphatidic acid. An example is shown next Then the CDP donates the phosphocholine to a diacylglycerol to made phosphatidylcholine and CMP Synthesis of other important glycerophospholipids follows from these basic strategies. Phosphatidylethanolamine can be easily made from phosphatidylserine by decarboxylation. Phosphatidylethanolamine can serve as a precursor in an alternative pathway for making phosphatidylcholine (SAM = S-Adenosyl Methioinine / SAH = S-Adenosyl Homocysteine) Phosphatidylserine and phosphatidylethanolamine can swap groups reversibly in the reaction below Similarly, phosphatidylserine and phosphatidylcholine can be interchanged as follows: Phosphatidylglycerol can be made from glycerol-3-phosphate and CDP-diacylglycerol Cardiolipin, which is essentially a diphosphatidyl compound can be made by joining CDP-diacylglyerol with phosphatidylglycerol Phosphtidylinositol can be made from CDP-diacylglycerol and inositol . Heme synthesis The porphyrin ring found in the hemes of animals, fungi, and protozoa (Figure 6.130) is synthesized starting from very simple compounds (Figure 6.131). The process is a bit complicated, occurring between the cytoplasm and the mitochondrion. The first step is the creation of δ-aminolevulinic acid (also called aminolevulinic acid or dALA) from glycine and Succinyl-CoA. Joining of two δ-aminolevulinic acid molecules together with splitting out of two molecules of water yields porphobilinogen. Joining of four molecules of porphobilinogen together yields hydroxylmethylbilane (Figure 6.132). Next, a series of reactions involving 1) loss of water; 2) loss of four molecules of carbon dioxide; 3) loss of two more carbon dioxides, loss of six protons and electrons and (finally) 4) addition of Fe++ with loss of two protons yields heme. Individual heme molecules may be further processed. Two enzymes in heme synthesis are sensitive to the presence of lead, and this is one of the primary causes of lead toxicity in humans. Inhibition of the enzymes leads to 1) anemia and 2) accumulation of δ-aminolevulinic acid, which can be harmful to neurons in development, resulting in learning deficiencies in children. Porphyria Defects in enzymes of the pathway can also lead to porphyrias, diseases in which one or more of the intermediates in the heme synthesis pathway accumulate due to deficiency of the enzyme necessary to convert the accumulating material into the next molecule in the pathway. The accumulation of purplish intermediates gave the diseases the name porphyria from the Greek word for purple. Severe porphyrias can lead to brain damage, nerve damage, and mental disturbances. The “madness” of King George III may have been due to a form of porphyria. In other manifestations of the disease, cutaneous porphyrias cause skin problems on exposure to light. This need, for patients with certain forms of porphyria, to avoid light, coupled with the fact that porphyrias can be treated by blood tranfusions, may have led to the legend of vampires. Breakdown of heme Catabolism of heme (Figure 6.133) begins in macrophages within the spleen . Targets for degradation are hemes within damaged red blood cells, which get removed from the blood supply due to their appearance. It is because of this system, for example, that sickle cell anemia is classified as an anemia (decrease in red blood cells or hemoglobin in the blood). After cells have sickled, they lose their shape and are more likely to be removed from the blood by this process, leaving the patient weakened from low blood cell counts. The first biochemical step in catabolism is conversion of heme to biliverdin. This reaction is catalyzed by heme oxygenase and requires electrons from NADPH. In the process, Fe++ is released. Interestingly, carbon monoxide is also produced and it acts as a vasodilator. Next, biliverdin is converted to bilirubin by biliverdin reductase and is secreted from the liver into bile. Bacteria in the intestine convert bilirubin to urobilinogens, some of which is absorbed intestinal cells and transported into kidneys and excreted. The yellow color of urine arises from the compound known as urobilin, which is an oxidation product of urobilinogen. The remainder of the urobilinogens are converted in the intestinal tract to stercobilinogen whose oxidation product is stercobilin and it gives the color associated with feces.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/06%3A_Metabolism/6.04%3A_Other_Lipids.txt

Source: BiochemFFA_6_5.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy In contrast to some of the metabolic pathways described to this point, amino acid metabolism is not a single pathway. The 20 amino acids have some parts of their metabolism that overlap with each other, but others are very different from the rest. In discussing amino acid metabolism, we will group metabolic pathways according to common metabolic features they possess (where possible). First, we shall consider the anabolic pathways. Transamination Before beginning discussion of the pathways, it is worthwhile to discuss a reaction common to the metabolism of most of the amino acids and other nitrogen-containing compounds and that is transamination. In cells, nitrogen is a nutrient that moves from one molecule to another in a sort of hand-off process. A common transamination reaction is shown on the next page. A specific reaction of this type is shown in Figure 6.134. Glutamate and glutamine play central roles in transamination, each containing one more amine group than α-ketoglutarate and glutamate, respectively. Transamination reactions, as noted earlier, occur by a ping-pong mechanism and involve swaps of amines and oxygens in Schiff base reactions. Two amino acids, glutamine and asparagine are the products of gaining an amine in their respective R-groups in reactions involving ammonium ion. Synthesis varies It is also important to recognize that organisms differ considerably in the amino acids that they can synthesize. Humans, for example, cannot make 9 of the 20 amino acids needed to make proteins, and the number of these that can be synthesized in needed amounts varies between adults and children. Amino acids that cannot be made by an organism must be in the diet and are called essential amino acids. Non-essential amino acids are those an organism can make in sufficient quantities (Figure 6.135). Though amino acids do not have a common pathway of metabolism, they are often organized in “families” of amino acids with overlapping metabolic reactions common to members of each group. To designate amino acid families in the text we will use a blue font for headings to distinguish them. α-ketoglutarate family This family of amino acids arises from α-ketoglutarate of the citric acid cycle. It includes the amino acids glutamic acid, glutamine, proline, and arginine. It is also called the glutamate family, since all the amino acids in it derive from glutamate. Glutamate α-ketoglutarate is readily converted to glutamate in transamination reactions, as noted above. It can also be produced by the enzyme glutamate dehydrogenase, which catalyzes the reaction below (in reverse) to make glutamate. In the forward direction, the reaction is a source of ammonium ion, which is important both for the urea cycle and for glutamine metabolism. Because it is a byproduct of a citric acid cycle intermediate, glutamate can therefore trace its roots to any of the intermediates of the cycle. Citrate and isocitrate, for example, can be thought of as precursors of glutamate. In addition, glutamate can be made by transamination from α-ketoglutarate in numerous transamination reactions involving other amino acids. Glutamine Synthesis of glutamine proceeds from glutamate via catalysis of the enzyme glutamine synthetase, one of the most important regulatory enzymes in all of amino acid metabolism (Figure 6.136). Regulation of the enzyme is complex, with many allosteric effectors. It can also be controlled by covalent modification by adenylylation of a tyrosine residue in the enzyme (Figure 6.137). In the figure, PA and PD are regulatory proteins facilitating conversion of the enzyme. Ammonia used in the reaction catalyzed by glutamate synthetase commonly arises from nitrite reduction, amino acid breakdown, or photorespiration. Because it builds ammonia into an amino acid, glutamine synthetase helps reduce the concentration of toxic ammonia - an important consideration in brain tissue. Some inhibitors of glutamine synthetase are, in fact, the products of glutamine metabolism. They include histidine, tryptophan, carbamoyl phosphate, glucosamine-6-phosphate, CTP, and AMP. The glutamate substrate site is a target for the inhibitors alanine, glycine, and serine. The ATP substrate site is a target for the inhibitors GDP, AMP, and ADP. Complete inhibition of the enzyme is observed when all of the substrate sites of the multi-subunit enzyme are bound by inhibitors. Lower levels of inhibitors results in partial or full activity, depending on the actual amounts. Proline Synthesis of proline starts with several reactions acting on glutamate. They are shown below in the green text box. The L-glutamate-5-semialdehyde, so produced, is a branch point for synthesis of proline or ornithine. In the path to make proline, spontaneous cyclization results in formation of 1-pyrroline-5-carboxylic acid (Figure 6.138). This, in turn, is reduced to form proline by pyrroline-5-carboxylate reductase. Arginine Arginine is a molecule synthesized in the urea cycle and, thus, all urea cycle molecules can be considered as precursors. Starting with citrulline, synthesis of arginine can proceed as shown on the next page. The urea cycle can be seen HERE. An alternate biosynthetic pathway for making arginine from citrulline involves reversing the reaction catalyzed by nitric oxide synthase. It catalyzes an unusual five electron reduction reaction that proceeds in the following manner Yet another way to synthesize arginine biologically is by reversal of the arginase reaction of the urea cycle Arginine can also be made starting with glutamate. This 5 step pathway leading to ornithing is illustrated at the top of the next page (enzymes in blue). Ornithine, as noted above can readily be converted to arginine. The last means of making arginine is by reversing the methylation of asymmetric dimethylarginine (ADMA - Figure 6.140). ADMA is a metabolic byproduct of protein modification. It interferes with production of nitric oxide and may play a role in cardiovascular disease, diabetes mellitus, erectile dysfunction, and kidney disease. Serine family Serine is a non-essential amino acid synthesized from several sources. One starting point is the glycolysis intermediate, 3-phosphoglycerate, (3-PG) in a reaction catalyzed by 3-PG dehydrogenase. Transamination by phosphoserine aminotransferase produces O-phosphoserine. The phosphate is then removed by phosphoserine phosphatase, to make serine. These reactions are shown below. Phosphoserine phosphatase is missing in the genetic disease known as Williams-Beuren syndrome. Serine can also be derived from glycine and vice versa. Their metabolic paths are intertwined as will be seen below. Serine is important for metabolism of purines and pyrimidines, and is the precursor for glycine, cysteine, and tryptophan in bacteria, as well as for sphingolipids and folate. Serine in the active site of serine proteases is essential for catalysis. A serine in the active site of acetylcholinesterases is the target of nerve gases and insecticides. Covalent modification target Serine in proteins can be the target of glycosylation or phosphorylation. D-serine is the second D-amino acid known to function in humans. It serves as a neuromodulator for NMDA receptors, by serving as a co-agonist, together with glutamate. D-serine is being studied as a schizophrenia treatment in rodents and as a possible biomarker for Alzheimers. Glycine As noted, glycine’s metabolism is intertwined with that of serine. This is apparent in the reaction catalyzed by serine hydroxymethyltransferase. Notably, the previous reaction is also needed for recycling of folate molecules, which are important for single carbon reactions in nucleotide synthesis. Vertebrates can also synthesize glycine in their livers using the enzyme glycine synthase. Glycine is a very abundant component of collagen. It is used in the synthesis of purine nucleotides and porphyrins. It is an inhibitory neurotransmitter and is a co-agonist of NMDA receptors with glutamate. Glycine was detected in material from Comet Wild 2. Cysteine Cysteine can be synthesized from several sources. One source is the metabolism of the other sulfur-containing amino acid, methionine. This begins with formation of S-Adenosyl-Methionine (SAM), catalyzed by methionine adenosyltransferase. SAM is a methyl donor for methyl transfer reactions and that is the next step in the pathway - donation of a methyl group (catalyzed by transmethylase) SAH (S-Adenosylhomocysteine) is cleaved by S-adenosylhomocysteine hydrolase, Homocysteine can be recycled back to methionine by action of methionine synthase On the path to making cysteine, homocysteine reacts as follows (catalyzed by cystathionine β-synthase). Last, cystathionase catalyzes release of cysteine β-ketobutyrate can be metabolized to propionyl-CoA and then to succinyl-CoA to be used ultimately in the citric acid cycle. Another route to making cysteine is a two-step process that begins with serine, catalyzed first by serine-O-acetyltransferase and then by cysteine synthase Cysteine can be also released from cystine by cystine reductase Finally, cysteine can be made from cysteic acid by action of cysteine lyase Aspartate family Metabolism of aspartic acid is similar to that of glutamate. Aspartic acid can arise from transamination of a citric acid cycle intermediate (oxaloacetate). Aspartate can also be generated from asparagine by the enzyme asparaginase. Further, aspartate can be produced by reversal of a reaction in the urea cycle (see HERE) Aspartate is also a precursor to four amino acids that are essential in humans. They are methionine, isoleucine, threonine, and lysine. Because oxaloacetate can be produced from aspartate, aspartate is an important intermediate for gluconeogenesis when proteins are the energy source. Asparagine Asparagine, too, is an amino acid produced in a simple transamination reaction. In this case, the precursor is aspartate and the amine donor is glutamine (catalyzed by asparagine synthetase) Methionine Metabolism of methionine overlaps with metabolism of the other sulfur-containing amino acid, cysteine. Methionine is not made in humans (essential) so the pathway shown in Figure 6.141 is from bacteria. The process begins with phosphorylation of aspartate. Numbers for each catalytic step in the figure are for the enzymes that follow: 1 - Aspartokinase​ 2 - Aspartate-semialdehyde dehydrogenase​ 3 - Homoserine dehydrogenase​ 4 - Homoserine O-transsuccinylase​ 5 - Cystathionine-γ-synthase​ 6 - Cystathionine-β-lyase​ 7 - Methionine synthase Though humans cannot make methionine by the pathway shown in the figure, they can recycle methionine from homocysteine (a product of S-adenosylmethionine metabolism). This reaction requires the enzyme methionine synthase and Vitamin B12 as a co-factor. An alternative pathway of converting homocysteine to methionine involves a prominent liver enzyme, betaine-homocysteine methyltransferase. This enzyme catalyzes the reaction below. In this reaction, a methyl group is transferred to homocysteine from glycine betaine to make the methionine. Glycine betaine is a trimethylated amine of glycine found in plants. It is a byproduct of choline metabolism. Bacteria, mitochondria, and chloroplasts use a modified form of methionine, N-formyl-methionine (Figure 6.142), as the first amino acid incorporated into their proteins. Formylation of methionine occurs only after methionine has been attached to its tRNA for translation. Addition of the formyl group is catalyzed by the enzyme methionyl-tRNA formyltransferase Threonine Though threonine is chemically similar to serine, the metabolic pathway leading to threonine does not overlap with that of serine. As seen in the figure, aspartate is a starting point for synthesis. Two phosphorylations/dephosphorylations and two reductions with electrons from NADPH result in production of threonine. Enzymes in Figure 6.143 are as follows: 1 Aspartokinase​ 2 β-aspartate semialdehyde dehydrogenase​ 3 Homoserine dehydrogenase​ 4 Homoserine kinase​ 5 Threonine synthase Breakdown of threonine produces acetyl-CoA and glycine. It can also produce α-ketobutyrate, which can be converted to succinyl-CoA for oxidation in the citric acid cycle. Lysine To get from aspartate to lysine, nine reactions and two non-enzymatic steps are involved, as seen in Figure 6.144. Enzymes involved in lysine biosynthesis include (numbers correspond to numbered reactions in Figure 6.144): 1 - Aspartokinase​ 2 - Aspartate-semialdehyde dehydrogenase​ 3 - 4-hydroxy-tetrahydrodipicolinate synthase​ 4 - 4-hydroxy-tetrahydrodipicolinate reductase​ 5 - 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase ​ 6 - Succinyl-diaminopimelate transaminase ​ 7 - Succinyl-diaminopimelate desuccinylase ​ 8 - Diaminopimelate epimerase​ 9 - Diaminopimelate decarboxylase Low in cereal grains Lysine is the essential amino acid found in the smallest quantity in cereal grains, but is found abundantly in legumes. Besides its synthesis and breakdown, lysine can be methylated, acetylated, hydroxylated, ubiquitinated, sumoylated, neddylated, biotinylated, pupylated, and carboxylated within proteins containing it. Hydroxylation of lysine is important for strengthening collagen and acetylation/methylation of lysine in histone proteins play roles in control of gene expression and epigenetics. Besides being used to make proteins, lysine is important for calcium absorption, recovery from injuries, and for production of hormones. Oral lysine has been used as a treatment for herpes infections (cold sores) but its efficacy is not established and it is not clear by what mechanism is would reduce the duration of the infection or reduce the number of outbreaks of viral infection.. Aromatic amino acids The aromatic amino acids, tryptophan, phenylalanine, and tyrosine can all be made starting with two simple molecules - PEP and erythrose-4-phosphate (Figure 6.145). All three aromatic amino acids are also important sources of hormones, neurotransmitters, and even the skin pigment melanin. Tryptophan synthesis The proteogenic amino acid with the largest R-group, tryptophan is an essential amino acid distinguished structurally by its indole group. The amino acid is made in bacteria and plants from shikimic acid or anthranilate and serine is used in its synthesis. Erythrose-4-phosphate and phosphenolpyruvate (PEP) also serve as building blocks of tryptophan. The pathway of its synthesis is shown in Figures 6.146 to 6.148. Erythrose-4-phosphate and phosphoenolpyruvate (PEP) are joined and then, after one hydrolysis, one dehydration, one oxidation and one reduction, the product is shikimic acid (Figure 6.147). Shikimic acid is converted to chorismic acid in three steps, as shown in Figure 6.147. Finally, synthesis of tryptophan from chorismic acid is shown in Figure 6.148. Regulation Regulation of tryptophan synthesis in bacteria occurs partly via a process called attenuation that operates through the trp operon. In this mechanism, low levels of tryptophan slow ribosomal movement (and translation) through the operon. This is particularly important because bacteria can have transcription and translation occurring simultaneously. Slowing translation due to low tryptophan levels allows a transcription termination mechanism to be inhibited. Since translation only slows when tryptophan is in short supply, premature termination of transcription occurs when tryptophan is abundant (see also HERE). Besides its importance for making proteins, tryptophan is an important precursor of serotonin (neurotransmitter), melatonin (hormone), niacin (vitamin), and auxin (plant hormone). The two pathways leading from tryptophan to three of these molecules is shown in Figure 6.149. Melatonin Melatonin is a compound made from tryptophan that is found in a wide spectrum of biological systems, including plants, animals, fungi, and bacteria. In animals, it acts as a hormone for circadian rhythm synchronization, signaling the onset of darkness each day. It has effects on the timing of sleep, seasonal effects, and can affect blood pressure, among other physiological phenomena. It can cross cell membranes, as well as the blood-brain barrier. Melatonin is a potent anti-oxidant and provides protective functions for nucleic acids. It is used sometimes to help in treatment of sleep disorders. Some reports have indicated that children with autism have abnormal melatonin pathways with low levels of the hormone. Blue light Melatonin production is affected by blue light and may be linked to sleep abnormalities for people using computer monitors after dark. To protect against this, some computer programs are available that reduce the screen’s blue light output in the evenings. Special eyeglasses that block blue light are also available. Though melatonin is linked to sleep in some animals (including humans), nocturnal animals are activated by increasing melatonin levels. Varying day/night lengths during the year alter melatonin production and provide biological signals of the seasons. These are especially important in the seasonal coloring and breeding habits of some animals. Melatonin is present in cherries, bananas, grapes, rice, cereals, olive oil, wine, and beer. Serotonin Serotonin, or 5-hydroxytryptamine, is a monoamine neurotransmitter derived from tryptophan. Blood platelets store serotonin and release it when they bind to a clot, causing vasoconstriction. Serotonin plays a role in cognitive functions and enhances memory and learning. Serotonin is widely thought to be a contributor to feelings of happiness and well-being. Some common anti-depressant drugs, including Prozac, Paxil and Zoloft, act to modulate action of serotonin at synapses. Niacin Niacin is also known as Vitamin B3 and nicotinic acid. Niacin can be made from tryptophan and people who have the inability to absorb tryptophan in the digestive system exhibit symptoms similar to niacin deficiency. Extreme deficiency of niacin in the diet leads to the disease known as pellagra, while insufficient amounts of niacin in the diet are linked with nausea, anemia, headaches, and tiredness. A diet that is primarily composed of grains like corn can lead to niacin deficiency, because the niacin in these sources is not readily bioavailable. Treatment of the grain with alkali, as in the traditional Mexican practice of soaking corn in lime, can make the niacin more easily absorbed from food. Niacin is related to pyridine and the amide form of it is nicotinamide, an important component of NAD+/NADH and NADP+/NADPH. The last pairs of molecules are essential as electron acceptors/carriers for most cellular oxidation-reduction reactions. Auxins Auxins are plant growth hormones derived from tryptophan. The most important of these is indole-3-acetic acid (Figure 6.151). Auxins are involved in almost every aspect of plant growth and development. They activate proteins, such as expansins and various enzymes that modify the structure of cell wall components, to loosen the cell walls of a plant and stimulate elongation of cells. In the presence of cytokinins, auxins stimulate cell division. Auxins are also involved in the maintenance of meristems and in cell patterning and organogenesis. Auxins are crucial for establishing root primordia as well as for elongation of root hairs. Auxins play important roles in organizing the xylem and phloem of plants, and it has long been known that plant callus tissue can be made to differentiate into shoots or roots, depending on the relative concentrations of auxins and cytokinins supplied in the medium. Agrobacterium tumefaciens, a bacterium which infects a wide variety of plants, inserts its own DNA, including genes necessary for the synthesis of plant hormones, into its host’s cells. The subsequent overproduction of auxins stimulates the growth of tumors (called crown galls) on the plant (Figure 6.153). Phenylalanine Phenylalanine is an essential, hydrophobic amino acid in humans that is a precursor of tyrosine and since tyrosine is a precursor of several important catecholamines, phenylalanine is, thus, a precursor of them as well. PKU Phenylalanine is linked to the genetic disease phenylketonuria (PKU) which arises from an inability to metabolize the amino acid in people lacking (or deficient in) the enzyme phenylalanine hydroxylase. If left untreated, the disease can cause brain damage and even death, but if detected early, it can be easily managed by carefully monitoring dietary intake of the amino acid. Because of this, newborns are routinely tested for PKU. Phenylalanine is a component of the artificial sweetener known as aspartame (Nutrasweet - Figure 6.154) and is consequently dangerous for people suffering from this disorder. Biosynthesis of phenylalanine in bacteria overlaps with synthesis of tryptophan. The branch occurs at chorismic acid where the enzyme chorismate mutase catalyzes a molecular rearrangement to produce prephenate. Proton attack on prephenate results in loss of water and carbon dioxide to yield phenylpyruvate. Transamination of phenylpyruvate yields phenylalanine. Alternatively, phenylalanine can obtain its amine group in a transamination reaction from alanine. Hydroxylation of phenylalanine by aromatic amino acid hydroxylase (phenylalanine hydroxylase) yields tyrosine. Tyrosine Because tyrosine is made from phenylalanine and the latter is an essential amino acid in humans, it is not clear whether to classify tyrosine as essential or non-essential. Some define it as a conditionally essential amino acid. Others simply categorize it as non-essential. As noted above, tyrosine can arise as a result of hydroxylation of phenylalanine. In addition, plants can synthesize tyrosine by oxidation of prephenate followed by transamination of the resulting 4-hydroxyphenylpyruvate (Figure 6.155). The hydroxyl group on tyrosine is a target for phosphorylation by protein kinase enzymes involved in signal transduction pathways (Figure 6.156). When located in membranes, these enzymes are referred to as receptor tyrosine kinases and they play important roles in controlling cellular behavior/response. In photosystem II of chloroplasts, tyrosine, at the heart of the system, acts as an electron donor to reduce oxidized chlorophyll. The hydrogen from the hydroxyl group of tyrosine is lost in the process, requiring re-reduction by four core manganese clusters. Tyrosine is also important in the small subunit of class I ribonucleotide reductases where it forms a stable radical in the catalytic action of the enzyme (see HERE). Tyrosine metabolites Tyrosine is a precursor of catecholamines, such as L-dopa, dopamine, norepinephrine, and epinephrine (Figure 6.157). The thyroid hormones triiodothyronine (T3) and thyroxine (T4) are also synthesized from tyrosine. As shown in Figure 6.158, this involves a series of iodinations of tyrosines side-chains of a protein known as thyroglobulin. Combinations of iodinated tyrosines give rise to thyroxine and triiodothyronine. These are subsequently cleaved from the protein and released into the bloodstream. Oxidation and polymerization of tyrosine is involved in synthesis of the family of melanin pigments. Tyrosine is involved in the synthesis of at least two types - eumelanin and pheomelanin (Figure 6.159). Another molecule derived from tyrosine is the benzoquinone portion of Coenzyme Q (CoQ). This pathway requires the enzyme HMG-CoA Reductase and since this enzyme is inhibited by cholesterol-lowering statin drugs, CoQ can be limited in people being treated for high cholesterol levels. Dopamine Dopamine plays several important roles in the brain and body. A member of the catecholamine and phenethylamine families, its name comes from the fact that it is an amine made by removing a carboxyl group from L-DOPA. Dopamine is synthesized in the brain and kidneys. It is also made in plants, though its function in plants is not clear. Conversion of dopamine to norepinephrine (Figure 6.157) requires vitamin C. Dopamine is a neurotransmitter, being released by one nerve cell and then traveling across a synapse to signal an adjacent nerve cell. Dopamine plays a major role in the brain’s reward-mediated behavior. Rewards, such as food or social interaction, increase dopamine levels in the brain, as do addictive drugs. Other brain dopamine pathways are involved in motor control and in managing the release of various hormones. Chemical messenger Outside the nervous system, dopamine is a local chemical messenger. In blood vessels, it inhibits norepinephrine release and causes vasodilation. In the kidneys, it increases sodium excretion and urine output. It reduces gastrointestinal motility and protects intestinal mucosa in the digestive system and in the immune system, it reduces lymphocyte activity. The effect dopamine has on the pancreas is to reduce insulin production. With the exception of the blood vessels, dopamine is synthesized locally and exerts its effects near the cells that release it. Epinephrine Epinephrine (also called adrenalin) is a catecholamine chemically related to norepinephrine that is a hormone with medical applications. It is used to treat anaphylaxis, cardiac arrest, croup, and, in some cases, asthma, when other treatments are not working, due to its ability to favor bronchodilation. Epinephrine is the drug of choice for treating anaphylaxis. The compound may be given through inhalation, by intravenous injection, or subcutaneous injection and exerts effects through the α- and β-adrenergic receptors. In the body, it is produced and released by adrenal glands and some neurons. Effects Physiological effects of epinephrine may include rapid heart beat, increased blood pressure, heart output, pupil dilation, blood sugar concentration and increased sweating. Other physical effects may include shakiness, increased anxiety, and an abnormal heart rhythm. Norepinephrine Norepinephrine (also called noradrenalin) is a catecholamine molecule that acts as a hormone and neurotransmitter. It is chemically similar to epinephrine, differing only in the absence of a methyl group on its amine. Norepinephrine is made and released by the central nervous system (locus coeruleus of the brain) and the sympathetic nervous system. The compound is released into the blood stream from adrenal glands and affects α- and β-adrenergic receptors. Norepinephrine is at its lowest levels during sleep and at its highest levels during stress (fight or flight response). The primary function of norepinephrine is to prepare the body for action. It increases alertness, enhances memory functions, and helps to focus attention. Norepinephrine increases heart rate and blood pressure, increases blood glucose and blood flow to skeletal muscle and decreases flow of blood to the gastrointestinal system. Medical considerations Norepinephrine may be injected to overcome critically low blood pressure and drugs countering its effects are used to treat heart conditions. α-blockers, for example, are used to battle cardiovascular and psychiatric disorders. β-blockers counter a different set of norepinephrine’s effects than α-blockers and are used to treat glaucoma, migraine headaches and other cardiovascular problems. Pyruvate family The family of amino acids derived from pyruvate has four members, each with a simple aliphatic side chain no longer than four carbons. The simplest of these is alanine. Alanine Alanine is the amino acid that is most easily produced from pyruvate. The simple transamination catalyzed by alanine transaminase produces alanine from pyruvate. Alternative pathways for synthesis of alanine include catabolism of valine, leucine, and isoleucine. Glucose-alanine cycle The glucose-alanine cycle is an important nitrogen cycle related to the Cori cycle that occurs between muscle and liver cells in the body (see HERE). In it, breakdown of glucose in muscles leads to pyruvate. When nitrogen levels are high, pyruvate is transaminated to alanine, which is exported to hepatocytes. In the liver cells, the last transamination of the glucose-alanine cycle occurs. The amine group of alanine is transferred to α-ketoglutarate to produce pyruvate and glutamate. Glucose can then be made by gluconeogenesis from pyruvate. Importantly, breakdown of glutamate yields ammonium ion, which can be made into urea for excretion, thus reducing the body’s load of potentially toxic amines. This pathway may be particularly important in the brain. Another way of removing excess ammonium from a tissue is by attaching it to glutamate to make glutamine. Glutamate is a neurotransmitter, so having an alternative way of removing amines (glucose-alanine cycle) is important, especially in the brain. Leucine Like valine and isoleucine, leucine is an essential amino acid in humans. In adipose tissue and muscle, leucine is used in sterol synthesis. It is the only amino acid to stimulate muscle protein synthesis, and as a dietary supplement in aged rats, it slows muscle degradation. Leucine is an activator of mTOR, a protein which, when inhibited, has been shown to increase life span in Saccharomyces cerevisiae, C. elegans, and Drosophila melanogaster. Metabolism of leucine, valine, and isoleucine (also called Branched Chain Amino Acids - BCAAs) starts with decarboxylation of pyruvate and attachment of the two-carbon hydroxyethyl fragment to thiamine pyrophosphate (Figure 6.161). Metabolism of isoleucine proceeds with attachment of the hydroxylated two carbon piece (hydroxyethyl-TPP) to α-ketobutyrate and is covered in the section describing that amino acid (see HERE). Metabolism of valine and leucine proceeds with attachment of the hydroxyethyl piece from TPP to another pyruvate to create α-acetolactate. Rearrangement of α-acetolactate by acetolactate mutase makes 3-hydroxy-3-methyl-2-oxobutanoate. Reduction with NAD(P)H by acetohydroxy acid isomeroreductase yields α,β-dihydroxyisovalerate. Loss of water, catalyzed by dihydroxyacid dehydratase produces α-ketoisovalerate. This molecule is a branch point for synthesis of leucine and valine. Addition of an acetyl group from acetyl-CoA yields α-isopropylmalate (catalyzed by α-isopropylmalate synthase). Rearrangement, catalyzed by isopropylmalate dehydratase, gives rise to β-isopropylmalate. Oxidation by isopropylmalate dehydrogenase and NAD+, gives α-ketoisocaproate. Transamination of it (catalyzed by leucine aminotransferase and using glutamate) gives the final product of leucine (top of next column). Valine An essential amino acid in humans, valine is derived in plants from pyruvate and shares part of its metabolic synthesis pathway with leucine and a small slice of it with isoleucine. Metabolism of all three amino acids starts with decarboxylation of pyruvate and attachment of the two-carbon hydroxyethyl fragment to thiamine pyrophosphate (Figure 6.161), as noted above. As seen earlier, α-ketoisovalerate is the molecule at the point in the metabolic pathway where synthesis of valine branches from that of leucine. In fact, α-ketoisovalerate is only one step away from valine. Transamination of α-ketoisovalerate catalyzed by valine isoleucine aminotransferase gives valine. Isoleucine Synthesis of isoeleucine (an essential amino acid in humans) begins in plants and microorganisms with pyruvate and α-ketobutyrate (a byproduct of threonine metabolism - threonine deaminase - Figure 6.162). Metabolism of isoleucine proceeds with attachment to α-ketobutyrate of the hydroxyethyl-TPP product of pyruvate decarboxylation to form α-aceto-α-hydroxybutyrate. The reaction is catalyzed by acetolactate synthase. Rearrangement and reduction by acetohydroxy acid isomeroreductase and NAD(P)H yields α,β-dihydroxy-β-methylvalerate. Shown on next page. Loss of water (catalyzed by dihydroxy acid dehydratase) gives α-keto-β-methylvalerate. Τransamination (using glutamate and valine isoleucine transaminase) yields isoleucine. Interestingly, several of the enzymes of valine metabolism catalyze reactions in the isoleucine pathway. Though the substrates are slightly different, they are enough like the valine intermediates that they are recognized as substrates. Isoleucine has a second asymmetric center within it, but only one isomeric form of the four possible ones from the two centers is found biologically. Regulation of synthesis Regulation of synthesis of the branched chain amino acids (BCAAs - valine, leucine, and isoleucine) is complex. The key molecule in the regulation is α-ketobutyrate, which is synthesized in cells as a breakdown product of threonine. The enzyme catalyzing its synthesis is threonine deaminase (Figure 6.162), which is allosterically regulated. The enzyme is inhibited by its own product (isoleucine) and activated by valine, a product of a parallel pathway. Thus, when valine concentration is high, the balances shifts in favor of production of isoleucine and since isoleucine competes with valine and leucine for hydroxyethyl-TPP, synthesis of these two amino acids goes down. When isoleucine concentration increases, threonine deaminase is inhibited, shifting the balance back to production of valine and leucine. Attenuation Another control mechanism for regulation of leucine synthesis occurs in bacteria and is known as attenuation. In this method, accumulation of leucine speeds the process of translation of a portion of the mRNA copy of the leucine operon (coding sequences for enzymes necessary to make leucine). This, in turn, causes transcription of the genes of the leucine operon to terminate prematurely, thus stopping production of the enzymes necessary to make leucine. When leucine levels fall, translation slows, preventing transcription from terminating prematurely and allowing leucine metabolic enzymes to be made. Thus, leucine levels in the cell control the synthesis of enzymes necessary to make it. Histidine family Synthesis of histidine literally occurs in a class by itself - there are no other amino acids in its synthesis family. The amino acid is made in plants (Arabidopsis, in this case) by a pathway that begins with ribose-5-phosphate. The overall pathway is show in the green text boxes on the next two pages. Abbreviations used in the boxes are shown below. Enzyme names 1 = Ribose-phosphate diphosphokinase​ 2= ATP-phosphoribosyltransferase​ 3 = Phosphoribosyl-ATP pyrophospohydrolase​ 4 = Phosphoribosyl-AMP cyclohydrolase​ 5 = ProFAR-I (N’-[(5’phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide isomerase)​ 6 = Imidazole glycerol-phosphate synthase (IGPS)​ 7 = Ιmidazole glycerol-phosphate dehydratase​ 8 = Histidinol-phosphate aminotransferase​ 9 = Histidinol-phosphate phosphatase​ 10 = Histidinol dehydrogenase Abbreviations used 1 - PRPP = Phosphoribosyl Pyrophosphate​ 2. PRATP = Phosphoribosyl ATP​ 3. PRAMP = Phosphoribosyl AMP​ 4. ProFAR = (N′-[(5′-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide) ribonucleotide​ 5. PRFAR = (N′-[(5-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide) ribonucleotide​ 6. IGP = Imidazole glycerol-phosphate​ 7. AICAR = 5′-phosphoribosyl-4-carboximide-5-aminoimidazole​ 8. IAP = Imidazole acetol-phosphate​ 9. α-KG = α-ketoglutarate Histidine is a feedback inhibitor of ATP-phosphoribosyltransferase and thus helps to regulate its own synthesis. Histidine is the only amino acid to contain an imidazole ring. It is ionizable and has a pKa of about 6. As a result, histidine’s R-group can gain/lose a proton at pH values close to cellular conditions. Selenocysteine A cysteine analog commonly referred to as the 21st amino acid, selenocysteine (Figure 6.163) is an unusual amino acid occasionally found in proteins. Although it is rare, selenocysteine has been found in proteins in bacteria, archaea and eukaryotes. In contrast to amino acids such as phosphoserine, hydroxyproline, or acetyl-lysine, which arise as a result of post-translational modifications, selenocysteine is actually built into growing peptide chains in ribosomes during the process of translation. No codon specifies selenocysteine, so to incorporate it into a protein, a tRNA carrying it must bind to a codon that normally specifies STOP (UGA). This alternative reading of the UGA is dependent on formation of a special hairpin loop structure in the mRNA encoding selenoproteins. Selenium is rather toxic, so cellular and dietary concentrations are typically exceedingly low. About 25 human proteins are known to contain the amino acid. These include five glutathione peroxidases, and three thioredoxin reductases. Iodothyronine deiodinase, a key enzyme that converts thyroxine to the active T3 form, also contains selenocysteine in its active site. All of these proteins contain a single selenocysteine. A eukaryotic protein known as selenoprotein P, found in the blood plasma of animals, contains ten selenocysteine residues and is thought to function as an antioxidant and/or in heavy metal detoxification. Besides selenocysteine, at least two other biological forms of a seleno-amino acid are known. These include 1) selenomethionine (Figure 6.164), a naturally occurring amino acid in Brazil nuts, cereal grains, soybeans, and grassland legumes and 2) methylated forms of selenocysteine, such as Se-methylselenocysteine, are found in Astragalus, Allium, and Brassica species. Stop codon The specifics of the process of translation will be described elsewhere in the book, but to get selenocysteine into a protein, the tRNA carrying selenocysteine pairs with a stop codon (UGA) in the mRNA in the ribosome. Thus, instead of stopping translation, selenocysteine can incorporated into a growing protein and translation continues instead of stopping. Four genes are involved in preparation of selenocysteine for incorporation into proteins. They are known as sel A, sel B, sel C, and sel D. Sel C codes for the special tRNA that carries selenocysteine. The amino acid initially put onto the selenocysteine tRNA is not selenocysteine, but rather serine. Action of sel A and sel D are necessary to convert the serine to a selenocysteine. An intermediate in the process is selenophosphate, which is the selenium donor. It is derived from H2Se, the form in which selenium is found in the cell. The tRNA carrying selenocysteine has a slightly different structure than other tRNAs, so it requires assistance in translation. The sel B gene encodes for an EF-Tu-like protein that helps incorporate the selenocysteine into the protein during translation. Recoding the UGA Using UGA codons to incorporate selenocysteine into proteins could wreak havoc if done routinely, as UGA, in fact, almost always functions as a stop codon and is only rarely used to code for selenocysteine. Fortunately, there is a mechanism to ensure that the reading of a UGA codon as selenocysteine occurs only when the mRNA encodes a selenoprotein. Unusual structures in mRNAs The mRNAs for selenocysteine-containing proteins form unusual mRNA structures around the UGA codon that make the ribosome “miss” it as a stop codon and permit the tRNA with selenocysteine to be incorporated instead. Pyrrolysine Like selenocysteine, pyrrolysine is a rare, unusual, genetically encoded amino acid found in some cells. Proteins containing it are enzymes involved in methane metabolism and so far have been found only methanogenic archaeans and one species of bacterium. The amino acid is found in the active site of the enzymes containing it. It is sometimes referred to as the 22nd amino acid. Synthesis of the amino acid biologically begins with two lysines. One is converted to (3R)-3-Methyl-D-ornithine, which is attached to the second lysine. After elimination of an amine group, cyclization, and dehydration, L-pyrrolysine is produced. Pyrrolysine is attached to an unusual tRNA (pylT gene product) by action of the aminoacyl tRNA synthetase encoded by the pylS gene. This unusual tRNA can pair with the UAG stop codon during translation and allow for incorporation of pyrrolysine into the growing polypeptide chain during translation in a manner similar to incorporation of selenocysteine. Urea cycle The urea cycle holds the distinction of being the first metabolic cycle discovered - in 1932, five years before the citric acid cycle. It is an important metabolic pathway for balancing nitrogen in the bodies of animals and it takes place primarily in the liver and kidney. Organisms, like humans, that excrete urea are called ureotelic. Those that excrete uric acid (birds, for example) are called uricotelic and those that excrete ammonia (fish) are ammonotelic. Ammonia, of course, is generated by metabolism of amines and is toxic, so managing levels of it is critical for any organism. Excretion of ammonia by fish is one reason that an aquarium periodically requires cleaning and replacement of water. Liver failure can lead to accumulation of nitrogenous waste and exacerbates the problem. As shown in Figure 1.166, the cycle contains five reactions, with each turn of the cycle producing a molecule of urea. Of the five reactions, three occur in the cytoplasm and two take place in the mitochondrion. (The reaction making carbamoyl phosphate, catalyzed by carbamoyl phosphate synthetase is not shown in the figure.) Ornithine synthesis Though the cycle doesn’t really have a starting point, a common place to begin discussion is with the molecule of ornithine. As discussed elsewhere in this book, ornithine intersects the metabolic pathways of arginine and proline. Ornithine is found in the cytoplasm and is transported into the mitochondrion by the ornithine-citrulline antiport of the inner mitochonrial membrane. In the matrix of the mitochondrion, two reactions occur relevant to the cycle. The first is formation of carbamoyl phosphate from bicarbonate, ammonia, and ATP catalyzed by carbamoyl phosphate synthetase I. Carbamoyl phosphate then combines with ornithine in a reaction catalyzed by ornithine transcarbamoylase to make citrulline. The citrulline is transported out to the cytoplasm by the ornithine-citrulline antiport mentioned above. In the cytoplasm, citrulline combines with L-aspartate using energy of ATP to make citrullyl-AMP (an intermediate) followed by argininosuccinate. The reaction is catalyzed by argininosuccinate synthase. Next, fumarate is split from argininosuccinate by argininosuccinate lyase to form arginine. Water is used by arginase to cleave arginine into urea and ornithine, completing the cycle. Urea is less toxic than ammonia and is released in the urine. Some organisms make uric acid for the same reason. It is worth noting that aspartic acid, ammonia, and bicarbonate enter the cycle and fumarate and urea are produced by it. Points to take away include 1) ammonia is converted to urea using bicarbonate and the amine from aspartate; 2) aspartate is converted to fumarate which releases more energy than if aspartate were converted to oxaloacetate, since conversion of fumarate to malate to oxaloacetate in the citric acid cycle generates an NADH, but direct conversion of aspartate to oxaloacetate does not; and 3) glutamate and aspartate are acting as shuttles to funnel ammonia into the cycle. Glutamate, as will be seen below, is a scavenger of ammonia. Urea cycle regulation The urea cycle is controlled both allosterically and by substrate concentration. The cycle requires N-acetylglutamate (NAG) for allosteric activation of carbamoyl phosphate synthetase I. The enzyme that catalyzes synthesis of NAG, NAG synthetase, is activated by arginine and glutamate. Thus, an indicator of high amine levels, arginine, and an important shuttler of amine groups, glutamate, stimulates the enzyme that activates the cycle. The reaction catalyzed by NAG synthetase is At the substrate level, all of the other enzymes of the urea cycle are controlled by the concentrations of substrates they act upon. Only at high concentrations are the enzymes fully utilized. Complete deficiency of any urea cycle enzyme is fatal at birth, but mutations resulting in reduced expression of enzymes can have mixed effects. Since the enzymes are usually not limiting for these reactions, increasing substrate can often overcome reduced enzyme amounts to a point by simply fully activating enzymes present in reduced quantities. Ammonia accumulation However, if the deficiencies are sufficient, ammonium can accumulate and this can be quite problematic, especially in the brain, where mental deficiencies or lethargy can result. Reduction of ammonium concentration relies on the glutamate dehydrogenase reaction (named for the reverse reaction). Additional ammonia can be taken up by glutamate in the glutamine synthetase reaction. The result of these reactions is that α-ketoglutarate and glutamate concentrations will be reduced and the concentration of glutamine will increase. For the brain, this is a yin/yang situation. Removal of ammonia is good, but reduction of α-ketoglutarate concentration means less energy can be generated by the citric acid cycle. Further, glutamate is, itself, an important neurotransmitter and a precursor of another neurotransmitter - γ-aminobutyric acid (GABA). Energy generation From an energy perspective, the urea cycle can be said to break even or generate a small amount of energy, if one includes the energy produced in releasing ammonia from glutamate (one NADH). There are two NADHs produced (including the one for converting fumarate to oxaloacetate), which give 4-6 ATPs, depending on how efficiently the cell performs electron transport and oxidative phosphorylation. The cycle takes in 3 ATPs and produces 2 ADPs and one AMP. Since AMP is equivalent to 2 ATP, the cycle uses 4 ATP. Thus, the cycle either breaks even in the worst case or generates 2 ATPs in the best case. Amino acid catabolism Amino acids are divided according to the pathways involved in their degradation. There are three general categories. Ones that yield intermediates in the glycolysis pathway are called glucogenic and those that yield intermediates of acetyl-CoA or acetoacetate are called ketogenic. Those that involve both are called glucogenic and ketogenic. These are shown in Figures 6.167 and 6.168. As seen in the two figures, amino acids largely produce breakdown products related to intermediates of the citric acid cycle or glycolysis, but this isn’t the complete picture. Some amino acids, like tryptophan, phenylalanine, and tyrosine yield hormones or neurotransmitters on further metabolism (as noted earlier). Others like cysteine and methionine must dispose of their sulfur and all of the amino acids must rid themselves of nitrogen, which can happen via the urea cycle, transamination, or both. Tyrosine catabolism Breakdown of tyrosine (Figure 6.169) is a five step process that yields acetoacetate and fumarate. Enzymes involved include 1) tyrosine transaminase; 2) p-hydroxylphenylpyruvate dioxygenase; 3) homogentisate dioxygenase; 4) maleylacetoacetate cis-trans-isomerase; and 5) 4-fumaryl acetoacetate hydrolase. Breakdown of leucine is a multi-step process ultimately yielding the ketone body acetoacetate and acetyl-CoA. Branched chain amino acids (BCAAs - valine, leucine, and isoleucine) rely on Branched Chain AminoTransferase (BCAT) followed by Branched Chain α-ketoacid dehydrogenase (BCKD) for catabolism. Breakdown of isoleucine yields intermediates that are both ketogenic and glucogenic. These include acetyl-CoA and propionyl-CoA. Breakdown of valine is a multi-step process ultimately yielding propionyl-CoA.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/06%3A_Metabolism/6.05%3A_Amino_Acids_and_the_Urea_Cycle.txt

Source: BiochemFFA_6_6.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Diverse functions of nucleotides Nucleotides are most often thought of as the building blocks of the nucleic acids, DNA and RNA. While this, is, of course, a vital function, nucleotides also play other important roles in cells. Ribonucleoside triphosphates like ATP, CTP, GTP and UTP are necessary, not just for the synthesis of RNA, but as part of activated intermediates like UDP-glucose in biosynthetic pathways. ATP is also the universal “energy currency” of cells, and coupling of energetically unfavorable reactions with the hydrolysis of ATP makes possible the many reactions in our cells that require an input of energy. Adenine nucleotides serve as components of NAD(P)+ and FAD. Nucleotides can also serve as allosteric and metabolic regulators. The synthesis and breakdown pathways for nucleotides and the molecules derived from them are thus, of vital importance to cells. Regulation of nucleotide synthesis, especially for deoxyribonucleotides, is important to ensure that the four nucleotides are made in the right proportions, as imbalances in nucleotide concentrations can lead to increases in mutation rates. Pathways of nucleotide metabolism are organized in two major groups and one minor one. These include, respectively, metabolism of 1) purines; 2) pyrimidines; and 3) deoxyribonucleotides. Each group can be further subdivided into pathways that make nucleotides from simple precursors (de novo pathways) and others that use pieces of nucleotides to reassemble full ones (salvage pathways). Notably, de novo synthesis pathways for all of the nucleotides begin with synthesis of ribonucleotides. Deoxyribonucleotides are made from the ribonucleotides. Purine nucleotide metabolism Synthesis of purine nucleotides by the de novo pathway begins with addition of a pyrophosphate to carbon 1 of ribose-5-phosphate, creating phosphoribosylpyrophosphate (PRPP). The reaction is catalyzed by PRPP synthetase. Some number the purine metabolic pathway starting with the next reaction. We have therefore given this reaction the number of zero in Figure 6.172. In the next step (reaction 1 in Figure 6.172), the pyrophosphate is replaced by an amine from glutamine in a reaction catalyzed by PRPP amidotransferase (PPAT). The product is 5-phosphoribosylamine (5-PRA). PPAT is an important regulatory enzyme for purine biosynthesis. The end products of the pathway, AMP and GMP both inhibit the enzyme and PRPP activates it. Interestingly, full inhibition of the enzyme requires binding of both AMP and GMP. Binding of only one of the two nucleotides allows the enzyme to remain partially active so that the missing nucleotide can be synthesized. Through this enzyme, the relative amounts of ATP and GTP are controlled. 5-PRA is very unstable chemically (half-life of 38 seconds at 37°C), so it has been proposed that it is shuttled directly from PRPP amidotransferase to GAR synthetase for the next reaction. In this reaction (#2), glycine is added to the growing structure above the ribose-5-phosphate to create glycineamide ribonucleotide (GAR). This reaction, which requires ATP, is catalyzed, as noted, by the enzyme GAR synthetase. In reaction #3, a formyl group is transferred onto the GAR from N10-formyl-tetrahydrofolate (N10-formyl-THF or fTHF) by phosphoribosylglycinamide formyltransferase (GART). Next, the double bonded oxygen in the ring is replaced with an amine in a reaction catalyzed by phosphoribosylformylglycinamidine synthase (PFAS) that uses glutamine and produces glutamate. The reaction requires energy from ATP (top of next column). In humans the GAR synthetase, phosphoribosylglycinamide formyltransferase, and the enzyme catalyzing the next reaction (#5), AIR synthetase activities are all on the same protein known as trifunctional purine biosynthetic protein adenosine-3. In reaction #6, carboxylation of AIR occurs, catalyzed by phosphoribosylaminoimidazole carboxylase (PAIC) Aspartic acid is then added to donate its amine group and fumarate will be lost in the reaction that follows this one. The enzyme involved here is phosphoribosyl-aminoimidazole-succinocarboxamide synthase (PAICS) In the next reaction, the carbon shell of aspartate is released (as fumarate) and the amine is left behind. The reaction is catalyzed by adenylosuccinate lyase (ADSL). Reaction #9 involves another formylation reaction, catalyzed by phosphoribosylaminoimidazolecarboxamide formyltransferase (ATIC-E1). Next, inosine monophosphate synthase (ATIC-E2) catalyzes release of water to form the first molecule classified as a purine - inosine monophosphate or IMP). Though it doesn’t appear in DNA, IMP does, in fact, occur in the anticodon of many tRNAs where its ability to pair with numerous bases is valuable in reading the genetic code. IMP is a branch point between pathways that lead to GMP or AMP. The pathway to GMP proceeds via catalysis by IMP dehydrogenase as follows: In the last step of GMP synthesis, GMP synthase catalyzes a transamination to form GMP using energy from ATP. The energy source being ATP makes sense, since the cell is presumably making GMP because it needs guanine nucleotides. If the cell is low on guanine nucleotides, GTP would be in short supply. Adenine nucleotide synthesis Synthesis of AMP from IMP follows. First, adenylosuccinate synthetase catalyzes the addition of aspartate to IMP, using energy from GTP. Then, adenylosuccinate lyase splits fumarate off to yield AMP. In humans, the bifunctional purine biosynthesis protein known as PURH contains activities of the last two enzymes above. Abbreviations used above • PRPP = Phosphoribosyl Pyrophosphate​ • 5-PRA = 5-phosphoribosylamine​ • GAR = glycineamide ribonucleotide​ • fGAR = Phosphoribosyl-N-formylglycineamide​ • THF = Tetrahydrofolate​ • fTHF = N10-formyl-Tetrahydrofolate​ • fGAM = 5'-Phosphoribosylformylglycinamidine​ • AIR = 5-Aminoimidazole ribotide​ • CAIR = 5'-Phosphoribosyl-4-carboxy-5-aminoimidazole​ • SAICAR = Phosphoribosylamino-imidazolesuccinocarboxamide​ • AICAR = 5-Aminoimidazole-4-carboxamide ribonucleotide​ • FAICAR = 5-Formamidoimidazole-4-carboxamide ribotide​ • IMP = inosine monophosphate Regulation It is worth repeating that synthesis of GMP from IMP requires energy from ATP and that synthesis of AMP from IMP requires energy from GTP. In addition, the enzymes converting IMP into intermediates in the AMP and GMP pathways are each feedback inhibited by the respective monophosphate nucleotide. Thus, IMP dehydrogenase is inhibited by GMP (end product of pathway branch) and adenylosuccinate synthetase is inhibited by AMP, the end product of that pathway branch. Purine nucleotide levels are balanced by the combined regulation of PRPP amidotransferase , IMP dehydrogenase, adenylosuccinate synthetase and the nucleotides AMP and GMP. The importance of the regulatory scheme of purines is illustrated by two examples. First imagine both AMP and GMP are abundant. When this occurs, PRPP amidotransferase will be completely inhibited and no purine synthesis will occur. Partial activity High levels of GMP and low levels of AMP would result in PRPP amidotransferase being slightly active, due to the fact GMP will fill one allosteric site, but low AMP levels will mean second allosteric site will likely be unfilled. This lowered (but not completely inhibited) activity of PRPP amidotransferase will allow for limited production of 5-PRA and the rest of the pathway intermediates, so it will remain active. At the IMP branch, however, the high levels of GMP will inhibit IMP dehydrogenase, thus shutting off that branch and allowing all of the intermediates to be funneled into making AMP. When the AMP level rises high enough, AMP binds to PRPP amidotransferase and along with GMP, shuts off the enzyme. A reversal will occur if AMP levels are high, but GMP levels are low. Proper balance Regulated in this way, AMP and GMP levels can be maintained in a fairly narrow concentration range. Properly balancing nucleotide levels in cells is critical. It is likely for this reason that cells have numerous controls on the amount of each nucleotide made. Other mechanisms Cells have two other ways of balancing GMP and AMP nucleotides. First, the enzyme GMP reductase will convert GMP back to IMP using electrons from NADPH. The IMP, in turn, can then be made into AMP if its concentration is low. Second, AMP can be converted back to IMP by the enzyme AMP deaminase. In this case, the IMP can then be made into GMP. It is important to maintain appropriate proportions of the different nucleotides. Excess or scarcity of any nucleotide of any nucleotide can result in an increased tendency to mutation. To convert AMP to ATP and GMP to GTP requires action of kinase enzymes. Each monophosphate nucleotide form has its own specific nucleoside monophosphate kinase. For adenine-containing nucleotides (ribose forms and deoxyribose forms), adenylate kinase catalyzes the relevant reaction. The adenylate kinase reaction is reversible and is used to generate ATP when the cell’s ATP concentration is low. When ATP is made from 2 ADPs in this way, AMP levels increase and this is one way the cell senses that it is low on energy. Guanosine monophosphates also have their own kinase and it catalyzes the reaction at the top of the next page. Other monophosphate kinases for UMP and CMP use ATP in a similar fashion. In going from the diphosphate form to the triphosphate form, the picture is simple - one enzyme catalyzes the reaction for all diphosphates (ribose and deoxyribose forms). It is known as nucleoside diphosphate kinase or (more commonly) NDK or NDPK and it catalyzes reactions of the form where X and Y refer to any base. Purine salvage reactions Not all nucleotides in a cell are made from scratch. The alternatives to de novo syntheses are salvage pathways. Salvage reactions to make purine nucleotides start with attachment of ribose to purine bases using phosphoribosylpyrophosphate (PRPP). The enzyme catalyzing this reaction is known as hypoxanthine/guanine phosphoribosyltransferase (HGPRT - Figure 6.175) and is interesting from an enzymological as well as a medical perspective. First, the enzyme is able to catalyze both of the next two important salvage reactions - converting hypoxanthine to IMP or guanine to GMP. HGPRT is able to bind a variety of substrates at its active site and even appears to bind non-natural substrates, such as acyclovir preferentially over its natural ones. Medical perspective From a medical perspective, reduction in levels of HGPRT leads to hyperuricemia, a condition where uric acid concentration increases in the body. Complete lack of HGPRT is linked to Lesch-Nyhan syndrome, a rare, inherited disease in high uric acid concentration throughout the body is associated with severe accompanying neurological disorders. Reduced production of HGPRT occurs frequently in males and has a smaller consequence (gout) than complete absence. Interestingly, gout has been linked to a decreased likelihood of contracting multiple sclerosis, suggesting uric acid may help prevent or ameliorate the disease. Expression of HGPRT is stimulated by HIF-1, a transcription factor made in tissues when oxygen is limiting, suggesting a role for HGPRT under these conditions. Adenine salvage The enzyme known as adenine phosphoribosyltransferase (APRT) catalyzes the reaction corresponding to HGPRT for salvaging adenine bases. Pyrimidine nucleotide metabolism The de novo pathway for synthesizing pyrimidine nucleotides has about the same number of reactions as the purine pathway, but also has a different strategy. Whereas the purines were synthesized attached to the ribose sugar, pyrimidine bases are made apart from the ribose and then attached later. The first reaction is catalyzed by carbamoyl phosphate synthetase (Figure 6.176). Two different forms are found in eukaryotic cells. Form I is found in mitochondria and form II is in the cytoplasm. The reaction catalyzed by carbamoyl phosphate synthetase is the rate limiting step in pyrimidine biosynthesis and corresponds to reaction 1 in Figure 6.178. Balance The enzyme is activated by ATP and PRPP and is inhibited by UMP. This helps to balance pyrimidine vs. purine concentrations. High concentrations of a purine (ATP) activates the synthesis of pyrimidines. PRPP increases in concentration as purine concentration increases, so it too helps to establish that balance. UMP is an end product of pyrimidine metabolism, so the process is self-limiting. The next enzyme in the pathway, aspartate transcarbamoylase (ATCase) also plays a role in the same balance, as we will see. The reaction it catalyzes is shown below and is reaction 2 in Figure 6.178. ATCase is a classic enzyme exhibiting allosteric regulation and feedback inhibition, having both homotropic and heterotropic effectors (Figure 6.179 and see HERE). With 12 subunits (6 regulatory and 6 catalytic units), the enzyme exists in two states - a low activity T-state and a high activity R-state. Binding of the aspartate substrate to the active site shifts the equilibrium in favor of the R-state. Aspartate is a homotropic effector of the enzyme, because it acts allosterically on the enzyme and is a substrate for it as well. Similarly, binding of ATP to the regulatory units favors the R-state, whereas binding of CTP to the regulatory units favors the T-state. ATP and CTP are heterotropic effectors of the enzyme because they are not substrates for it, but act allosterically. Regulation As was seen with the first enzyme of the pathway, high concentration of purine nucleotides stimulates synthesis of pyrimidines and high concentration of pyrimidines turns off the pathway that synthesizes them. Dihydroorotase catalyzes reaction 3 and is found in the cytoplasm, as is ATCase. Reaction 4 occurs in the mitochondrion, so the product of reaction 3, dihydroorotate, must be transported into the mitochondrion from the cytoplasm. In reaction 4, dihydroorotate is oxidized to orotate. The enzyme catalyzing the reaction is dihydroorotate dehydrogenase. Reaction #5, catalyzed by orotate phosphoribosyl transferase, involves connection of orotate to ribose to yield a nucleotide - orotidine-5’-monophosphate (OMP). Last, OMP is converted to uridine-5’-monophosphate (UMP) by action of a fascinating enzyme known as OMP decarboxylase. OMP decarboxylase is frequently cited as an example for the incredible ability of an enzyme to speed a reaction. The decarboxylation of OMP, if allowed to proceed in the absence of an enzyme takes about 78 million years. In the presence of OMP decarboxylase, the reaction takes place in 18 milliseconds, a speed increase of about 1017. Remarkably, the enzyme accomplishes this without any cofactors or coenzymes of any kind. The mechanism of action of the enzyme is shown in Figure 6.180. In mammals, the activities of OMP decarboxylase and orotate phosphoribosyl transferase are contained on the same protein. A monophosphate kinase (UMP/CMP kinase) catalyzes conversion of UMP to UDP. The same enzyme will also phosphorylate CMP to CDP and dCMP to dCDP. Like the reaction of adenylate kinase, the reaction above, when run in the reverse direction, can be a source of ATP when the cell is low on energy. The next step, catalyzed by NDPK, uses energy of any triphosphate nucleotide (XTP) to produce UTP from UDP. CTP Synthase UTP is the substrate for synthesis of CTP via catalysis by CTP synthase. This enzyme is inhibited by its product, ensuring too much CTP is not made and activated by physiological concentrations of ATP, GTP, and glutamine. One human isozyme, CTPS 1, has been shown to be inactivated by phosphorylation by glycogen synthase kinase 3. CTP synthase has two domains and is a heterodimer (Figure 6.183). It exists as an inactive monomer at low enzyme concentrations or in the absence of UTP and ATP. One domain of the enzyme cleaves the amine group from glutamine and transfers it internally to the UTP. The other domain (synthase domain) binds ATP and initiates the mechanism shown in Figure 6.184 for making CTP. CTP is the only nucleotide synthesized de novo directly as a triphosphate, since it arises directly from UTP. Since deoxyribonucleotides are made from ribonucleoside diphosphates, it means deoxycytidine nucleotides must either be made preferentially from salvage nucleotides or CTP must be dephosphorylated first. One enzyme that can do this is a membrane-bound enzyme known as apyrase, which sequentially converts CTP to CDP and then CMP. Pyrimidine salvage reactions Pyrimidine salvage synthesis allows cells to remake pyrimidine triphosphate nucleotides starting from either the C or U pyrimidine bases, nucleosides, or nucleotides. Figures 1.85 & 6.186 depict salvage pathway reactions. As is apparent in Figure 1.86, there are multiple ways of making the same molecules. For example, uracil can be made into uridine by reaction 11 or by reaction 12. The figure depicts not only the synthesis of CTP and UTP from basic components, but also shows how these nucleotides can be broken down into smaller pieces. In many cases, the same enzyme works on cytidine, uridine, and deoxycytidine molecules. Enzymes of note There are several enzymes of note in the salvage pathway. Seven enzymes, for example, work on both uracil and cytosine containing nucleosides/nucleotides. These include NTP phosphatase (reaction 2), NDPK (reaction 3), apyrase (reaction 4), NDP phosphatase (reaction 5), UMP/CMP kinase (reaction 6), pyrimidine-specific 5’ nucleotidase (reaction 7), and uridine/cytidine kinase (reaction 8). The enzymes for reactions 6 and 8 can also use deoxyribonucleosides/deoxyribonucleotides as substrates. Cytidine deaminase (reaction #9) converts cytidine to uridine by removing an amine group from the cytosine base and thus is a counter for the UTP to CTP reaction catalyzed by CTP synthetase. Countered reactions allow cells to balance concentrations of nucleosides/nucleotides in either direction if they should get out of balance. Two other reactions in the figure are worth mentioning. Both UTP and CTP are converted in the breakdown process to UMP and CMP, respectively. Both of these reactions are important for deoxyribonucleotide metabolism. In each case, the monophosphate derivatives are phosphorylated, creating diphosphate derivatives (UDP and CDP) that are substrates for RNR that yield dUDP and dCDP, respectively. dUDP is phosphorylated to dUTP and then pyrophosphate is removed by dUTPase to yield dUMP. dUMP is a substrate for thymidine synthesis (see HERE). dCDP is converted to dCTP by NDPK Deoxyribonucleotide metabolism Deoxyribonucleotides, the building blocks of DNA, are made almost exclusively from ribonucleoside diphosphates. A single enzyme called ribonucleotide reductase (RNR) is responsible for the conversion of each of these to a deoxy form (Figure 6.187). The enzyme’s substrates are ribonucleoside diphosphates (ADP, GDP, CDP, or UDP) and the products are deoxyribonucleoside diphosphates (dADP, dGDP, dCDP, or dUDP). Thymidine nucleotides are synthesized from dUDP. RNR has two pairs of two identical subunits - R1 (large subunit) and R2 (small subunit). R1 has two allosteric binding sites and a catalytic site. R2 forms a tyrosine radical necessary for the reaction mechanism of the enzyme. There are three classes of RNR enzymes and they differ in the nature or means of generating a radical used in the enzyme’s catalytic mechanism. Class I RNRs are found in eukaryotes, eubacteria, bacteriophages, and viruses. They all use a ferrous iron center that loses an electron (converting to ferric iron) to generate a free radical on a tyrosine ring. These enzymes only work in aerobic conditions. Class II RNRs use 5’-deoxyadenosyl cobalamin (vitamin B12) to generate a radical and work under aerobic or anaerobic conditions. They are found in eubacteria, archaebacteria, and bacteriophages. Class III RNRs generate a glycine radical using S-adenosyl methionine (SAM) and an iron-sulfur center. They work under anaerobic conditions and are used by archaebacteria, eubacteria, and bacteriophages. Substrates for class I enzymes are ribonucleoside diphosphates. Class II enzymes work on ribonucleoside diphosphates or ribonucleoside triphosphates. Class III enzymes work on ribonucleoside triphosphates. In class I enzymes, RNR is an iron-dependent dimeric enzyme with each monomeric unit containing a large subunit (known as α or R1) and a small subunit (known as β or R2). The R1 subunit contains regulatory binding sites for allosteric effectors (see below), whereas the R2 subunit houses a tyrosine residue that forms a radical critical to the reaction mechanism of the enzyme. Electrons needed in the reaction are transmitted from NADPH to the enzyme by one of two pathways, reducing a disulfide bond in the enzyme to two sulfhydryls. In the first transfer mechanism, NADPH passes electrons to glutathione, which passes them to glutaredoxin, which then donates them to the RNR enzyme used in the reaction. In the second mechanism, NADPH passes electrons to FAD, which uses them to reduce thioredoxin, which then passes the electrons to RNR with the same end result as in the first pathway - reduction of a suflhydryl in RNR. In the reaction mechanism (Figure 6.188), a tyrosine side chain in the R2 unit must be radicalized to start. This electronic change is transmitted through the small R2 subunit to the active site of the large R1 subunit. Several aromatic amino acid side chains are thought to play a role in that process. Iron atoms in the R2 subunit assist in creation and stabilization of the radical. The tyrosine radical contains an unpaired electron delocalized across its aromatic ring. Transfer of the electronic instability to the R1 unit results in radicalization of a cysteine (to form a thiyl radical) at the active site. The thiyl radical, thus formed, abstracts a hydrogen atom (proton plus electron) from carbon 3 of ribose on the bound ribonucleoside diphosphate, creating a radical carbon atom. Radicalization of carbon #3 favors release of the hydroxyl group on carbon #2 as water. The extra proton comes from the sulfhydryl of the enzyme’s cysteine. In the next step of the process, a proton and two electrons from the same cysteine are transferred to carbon #2 and then carbon #3 takes back the proton originally removed from it to yield a deoxyribonucleoside diphosphate. The enzyme’s thiyl group gains an electron from R2 and the disulfide bond created in the reaction must be reduced by electrons from NADPH again in order to catalyze again. Regulation In addition to RNR’s unusual reaction mechanism, the enzyme also has a complex system of regulation, with two sets of allosteric binding sites, both found in the R1 subunit. Because a single enzyme, RNR, is responsible for the synthesis of all four deoxyribonucleotides, it is necessary to have mechanisms to ensure that the enzyme produces the correct amount of each dNDP. This is a critical consideration, since imbalances in DNA precursors can lead to mutation. Consequently, the enzyme must be responsive to the levels of the each deoxyribonucleotide, selectively making more of those that are in short supply, and preventing additional synthesis of those that are abundant. These demands are met by having two separate control mechanisms on the enzyme - one that determines which substrate will be acted on, and another that controls the enzyme’s activity. Two allosteric sites RNR is allosterically regulated via two molecular binding sites - a specificity binding site (binds dNTPs and induces structural changes in the enzyme that determines which substrates preferentially bind at the catalytic site and an activity control site (controls whether or not enzyme is active). The activity control site functions like a simple on/off switch - ATP activates catalysis, dATP inactivates it. (One subset of class I enzymes, however, is not affected by dATP.) The inactivation of RNR by dATP is an important factor in the disease known as Severe Combined Immunodeficiency Disease (SCID). In SCID, the salvage enzyme adenosine deaminase is deficient, leading to a rise in concentration of dATP in cells of the immune system. dATP shuts down RNR in these cells, thus stopping their proliferation and leaving the affected individual with a very weak or no immune system. Allosteric effectors When dTTP is abundant (Figure 6.189), it binds to RNR’s specificity site and inhibits binding and reduction of CDP and UDP but stimulates binding and reduction of GDP at the active site of the enzyme. Conversely, binding of ATP or dATP at the specificity site stimulates binding and reduction of CDP and UDP at the active site. Last, binding of dGTP to the specificity site (specificity site B) induces binding and reduction of ADP at the active site. Students sometimes confuse the active site of RNR with the activity control site (sometimes called the activity site). The active site is where the reaction is catalyzed, and could better be called the catalytic site, whereas the activity site is an allosteric binding site for ATP or dATP that controls whether the enzyme is active. High levels of dATP are an indicator that sufficient dNTPs are available, so the enzyme gets inhibited to stop production of more. Low levels of dATP allow binding of ATP and activation of the enzyme. In addition to regulation by deoxyribonucleotides and ATP, RNR can be directly inhibited by hydroxyurea. dTTP synthesis Synthesis of dTTP by the de novo pathway involves a multi-step process from UDP to dTTP. It begins with UDP, which is converted to dUDP by RNR. dUDP is phosphorylated by NDPK to yield dUTP, which is quickly broken down by dUTPase to produce dUMP. The remaining reactions are shown in Figure 6.190. Important enzymes in the pathway include dUTPase and thymidylate synthetase. dUTPase is important for keeping the concentration of dUTP low so it does not end up in DNA. DNA polymerase can use dUTP just as it does dTTP, and incorporate it into a DNA strand, across from adenine nucleotides. Thymidylate synthetase is important because it is a target (directly and indirectly) for anticancer therapies. As shown in Figure 6.191, a methyl group from N5,N10-methylene-tetrahydrofolate (often called tetrahydrofolate) is donated to dUMP, making dTMP and dihydrofolate (DHF). Folate molecules are in limited quantities in cells and must be recycled, because if they are not, then the reaction to make dTMP cannot occur. Recycling of dihydrofolate to tetrahydrofolate occurs by the reaction shown in Figure 6.192. The enzyme involved in the conversion of dihydrofolate to tetrahydrofolate, dihydrofolate reductase (DHFR - Figure 6.192), is one target of anticancer drugs because by stopping the regeneration of tetrahydrofolate from dihydrofolate (otherwise a dead end), one can stop production of thymidine nucleotides and, as a result, halt DNA synthesis, thus preventing a cancer cell from dividing. Competitive inhibitors of DHFR include methotrexate (Figure 6.194) or aminopterin. Cells contain numerous folates for performing one carbon metabolism and the pathways by which they are all recycled is shown in Figure 6.193. 5-fluorouracil Yet another important inhibitor of thymidine synthesis is used to treat cancer. This compound, 5-fluorouracil (Figure 6.195 and Movie 6.3) is a suicide inhibitor of thymidylate synthase. Salvage synthesis Besides synthesis from simple precursors, nucleotides can also be made from pieces of existing ones. This is particularly relevant, since consumption of food introduces to the body a large collection of proteins, lipids, and nucleic acids that are all more efficiently recycled than degraded. For proteins, the process is simple. Digestion converts them into constituent building blocks (amino acids) and these are re-assembled into proteins of the consuming organism using the genetic code. Nucleotides The multi-component structure of nucleotides, though (base, sugar, phosphate) means subsections of them may be re-utilized. Phosphate is recycled simply by entering the phosphate pool of the cell. It is typically built back into triphosphate forms (ultimately) by oxidative phosphorylation and kinase actions. Salvage of bases is different for purines and pyrimidines and is discussed separately HERE and HERE. Nucleotide catabolism Besides salvage and being built into nucleic acids, nucleotides can also be broken down into simpler component molecules. Some of these molecules, such as uric acid, can have significant impact on organisms (see HERE). Purine catabolism Breakdown of purine nucleotides starts with nucleoside monophosphates, which can be produced by breakdown of an RNA, for example, by a nuclease (Figure 6.196). Metabolism of AMP and GMP converge at xanthine. First, AMP is dephosphorylated by nucleotidase to create adenosine, which is then deaminated by adenosine deaminase to yield inosine. Alternatively, AMP can be deaminated by AMP deaminase to yield IMP. IMP is also an intermediate in the synthesis pathway for purine anabolism. Dephosphorylation of IMP (also by nucleotidase) yields inosine. Inosine has ribose stripped from it by action of purine nucleotide phosphorylase to release hypoxanthine. Hypoxanthine is oxidized to xanthine in a hydrogen peroxide-generating reaction catalyzed by xanthine oxidase. Catabolism of GMP proceeds independently, though similarly. First, phosphate is removed by nucleotidase to yield guanosine. Guanosine is stripped of ribose to yield free guanine base, which is deaminated by guanine deaminase (also called guanase) to produce xanthine. Xanthine oxidase enters the picture a second time in the next reaction catalyzing a second reaction by a similar mechanism to the hypoxanthine oxidation described previously. It is shown on the next page. Uric acid Uric acid is problematic in some higher organisms (including humans) because it is not very soluble in water. Consequently it precipitates out of solution, forming crystals (Figure 6.198). Those crystals can accumulate in joints and (frequently) in the big toe. Such a condition is known as gout. Interestingly, there may be a negative correlation between gout and contracting multiple sclerosis. This protective effect may be due to the antioxidant protection afforded by uric acid. Uric acid is the primary excretion form of nitrogen for birds. Dalmation dogs also excrete uric acid instead of urea and may suffer from joint pain as a result of gout-like conditions. Gout is treated with a hypoxanthine analog known as allopurinol (Figure 6.199). It inhibits action of xanthine oxidase, which favors increase in the concentration of hypoxanthine. The latter is used in salvage synthesis to make additional purines. Uric acid can be excreted into the urine (in humans) or broken down into allantoin by the uricase enzyme. Since humans lack the enzyme to make allantoin (urea in humans is produced by the urea cycle), its presence in the body means it was produced by non-enzymatic means. This is taken to be an indicator of oxidative stress, since it allantoin is produced non-enzymatically by oxidation of uric acid. Pyrimidine catabolism Catabolism of uridine and thymidine nucleotides is shown above (Figure 6.200). Catabolism of cytidine nucleotides proceeds through uridine by deamination of cytosine. The free bases, thymine and uracil, are released by the enzyme ribosylpyrimidine nucleosidase In the reductive pathway, uracil and thymine reduction by NADPH gives dihydrothymine and dihydrouracil respectively. Addition of water to these creates 3-ureidoisobutyrate and 3-ureidopropionate respectively. Hydrolysis of both these intermediates yields ammonium ion and carbon dioxide (which are made into urea) plus 3-aminoisobutyrate for the thymine pathway and β-alanine for the product of the uracil pathway. 3-aminoisobutyrate is produced during exercise and activates expression of thermogenic genes in white fat cells. β-alanine is a rate-limiting precursor of carnosine, a dipeptide of histidine and β-alanine (Figure 6.201). Carnosine functions as an antioxidant that scavenges reactive oxygen species. It also acts as an anti-glycating agent to prevent against attachment of sugar molecules to proteins. These are factors in degenerative diseases and may play a role in aging. Sugars Last, but not least, the sugars ribose and deoxyribose can be recycled (ribose) or catabolized (ribose and deoxyribose). In the case of ribose, it can be reattached to bases by phosphorylase enzymes, such as uridine phosphorylase, or converted into PRPP for the same purpose, to create nucleosides. Ribose-5-phosphate is an intermediate in the pentose phosphate pathway, allowing it to be converted into other sugars or broken down in glycolysis. Deoxyribose-5-phosphate can be broken into two pieces by deoxyribose-5-phosphate aldolase. The products of this reaction are glyceraldehyde-3-phosphate and acetaldehyde. The former can be oxidized in glycolysis and the latter can be converted into acetyl-CoA for further metabolism.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/06%3A_Metabolism/6.06%3A_Nucleotides.txt

The nature of biological information, how it is copied and passed on, how it is read and interpreted, and how it gives rise to the cellular activities that we can observe, is the subject of this chapter. Another kind of information is also considered, towards the end of the chapter- the molecular information that cells receive from, and send to, each other. Overlaid on the instructions in the genes, this information provides cells with ongoing clues about both their own inner state and the environment around them. The interplay of these two kinds of information is responsible for the form and behavior of all living organisms. • 7.1: Prelude to Information Processing As creatures used to regarding ourselves as exceptional, humans must surely be humbled to realize that the instructions, for making one of our own, reside in a molecule so simple that scientists, for a very long time, did not believe could possibly contain enough information to build even a simple cell. But a large body of evidence, built up over the past century, supports Larison Cudmore’s assertion that the information for making you and me (and all the other kinds of living things in the worl • 7.2: Genes and Genomes For many years, scientists wondered about the nature of the information that directed the activities of cells. What kind of molecules carried the information, and how was the information passed on from one generation to the next? Key experiments, done between the 1920s and the 1950s, established convincingly that this genetic information was carried by DNA. In 1953, with the elucidation of the structure of DNA, it was possible to begin investigating how this information is passed on, and is used • 7.3: DNA Replication The only way to make new cells is by the division of pre-existing cells. Single-celled organisms undergo division to produce more cells like themselves, while multicellular organisms arise through division of a single cell, generally the fertilized egg. Each time a cell divides, all of its DNA must be copied faithfully so that a copy of this information can be passed on to the daughter cell. This process is called DNA replication. • 7.4: DNA Repair It is evident that if DNA is the master copy of instructions for an organism, then it is important not to make mistakes when copying the DNA to pass on to new cells. Although proofreading by DNA polymerases greatly increases the accuracy of replication, there are additional mechanisms in cells to further ensure that newly replicated DNA is a faithful copy of the original, and also to repair damage to DNA during the normal life of a cell. • 7.5: Transcription In the preceding sections, we have discussed the replication of the cell's DNA and the mechanisms by which the integrity of the genetic information is carefully maintained. What do cells do with this information? How does the sequence in DNA control what happens in a cell? If DNA is a giant instruction book containing all of the cell's "knowledge" that is copied and passed down from generation to generation, what are the instructions for? And how do cells use these instructions for? • 7.6: RNA Processing So far, we have looked at the mechanism by which the information in genes (DNA) is transcribed into RNA. The newly made RNA, also known as the primary transcript is further processed before it is functional. Both prokaryotes and eukaryotes process their ribosomal and transfer RNAs. • 7.7: Translation Translation is the process by which information in mRNAs is used to direct the synthesis of proteins. As you have learned in introductory biology, in eukaryotic cells, this process is carried out in the cytoplasm of the cell, by large RNA-protein machines called ribosomes. Ribosomes contain ribosomal RNA (rRNA) and proteins. The proteins and rRNA are organized into two subunits, a large and a small. • 7.8: Gene Expression The processes of transcription and translation described so far tell us what steps are involved in the copying of information from a gene (DNA) into RNA and the synthesis of a protein directed by the sequence of the transcript. These steps are required for gene expression, the process by which information in DNA directs the production of the proteins needed by the cell. • 7.9: Signaling It is intuitively obvious that even unicellular organisms must be able to sense features of their environment, such as the presence of nutrients, if they are to survive. In addition to being able to receive and respond to information from the environment, multicellular organisms must also find ways by which their cells can communicate among themselves. Thumbnail: DNA double helix. Image used with permission (public domain; NIH - Genome Research Institute). 07: Information Processing “The blueprints for the construction of one human being requires only a meter of DNA and one tiny cell. … even Mozart started out this way.” — L.L. Larison Cudmore As creatures used to regarding ourselves as exceptional, humans must surely be humbled to realize that the instructions, for making one of our own, reside in a molecule so simple that scientists, for a very long time, did not believe could possibly contain enough information to build even a simple cell. But a large body of evidence, built up over the past century, supports Larison Cudmore’s assertion that the information for making you and me (and all the other kinds of living things in the world) is encoded in DNA. Tying in with Mendel’s observations about how characteristics are passed on from one generation to the next, the discovery that there was a molecule that carried this information, altered for ever how people thought about heredity. The elucidation of the structure of DNA provided greater insights into how traits might be encoded in a molecule, and the ways in which the information is used by cells. As we learn more about this topic, scientists have remarked on how the information in our DNA resembles the programs that drive computers. While this analogy is a simplification, there is definitely a sense in which, as Richard Dawkins put it, “the machine code of the genes is uncannily computer-like”, with information in our DNA directly determining the properties of the proteins that run our cells. We know, as Ada Yonath described it, that, “DNA is a code of four letters; proteins are made up of amino acids which come in 20 forms. So the ribosome is a very clever machine that reads one language and operates in another. “ If this sounds strange, it is even more intriguing to realize DNA is copied and passed on from cell to cell, from one generation to the next. There is an unbroken line of inheritance from the first cell to every organism alive today. In the words of Lewis Thomas, “All of today’s DNA, strung through all the cells of the earth, is simply an extension and elaboration of [the] first molecule.” The nature of this information, how it is copied and passed on, how it is read and interpreted, and how it gives rise to the cellular activities that we can observe, is the subject of this chapter. Another kind of information is also considered, towards the end of the chapter- the molecular information that cells receive from, and send to, each other. Overlaid on the instructions in the genes, this information provides cells with ongoing clues about both their own inner state and the environment around them. The interplay of these two kinds of information is responsible for the form and behavior of all living organisms.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/07%3A_Information_Processing/7.01%3A_Prelude_to_Information_Processing.txt

Source: BiochemFFA_7_1.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Introduction For many years, scientists wondered about the nature of the information that directed the activities of cells. What kind of molecules carried the information, and how was the information passed on from one generation to the next? Key experiments, done between the 1920s and the 1950s, established convincingly that this genetic information was carried by DNA. In 1953, with the elucidation of the structure of DNA, it was possible to begin investigating how this information is passed on, and how it is used. Genomes We use the word “genome” to describe all of the genetic material of the cell. That is, a genome is the entire sequence of nucleotides in the DNA that is in all of the chromosomes of a cell. When we use the term genome without further qualification, we are generally referring to the chromosomes in the nucleus of a eukaryotic cell. As you know, eukaryotic cells have organelles like mitochondria and chloroplasts that have their own DNA (Figure 7.1 & 7.2). These are referred to as the mitochondrial or chloroplast genomes to distinguish them from the nuclear genome. Starting in the 1980s, scientists began to determine the complete sequence of the genomes of many organisms, in the hope of better understanding how the DNA sequence specifies cellular functions. Today, the complete genome sequences have been determined for thousands of species from all domains of life, and many more are in the process of being worked out by groups of scientists across the world. Global genome initiative The Global Genome Initiative, a collaborative effort to sequence at least one species from each of the 9,500 described invertebrate, vertebrate, and plant families is one of many such ventures. The information from these various efforts is collected in enormous online repositories, so that it is freely available to scientists. As the sequence databases compile ever more information, the fields of computational biology and bioinformatics have arisen, to analyze and organize the data in a way that helps biologists understand what the information in DNA means in the cellular context. Genes It has been known for many years that phenotypic traits are controlled by specific regions of the DNA that were termed “genes”. Thus, DNA was envisioned as a long string of nucleotides, in which certain regions, the genes, were separated by non-coding regions that were simply referred to as intergenic sequences (inter=between; genic=of genes). Early experiments in molecular biology suggested a simple relationship between the DNA sequence of a gene and its product, and led scientists to believe that each gene carried the information for a single protein. Changes, or mutations in the base sequence of a gene would be reflected in changes in the gene product, which in turn, would manifest itself in the phenotype or observable trait. This simple picture, while still useful, has been modified by subsequent discoveries that demonstrated that the use of genetic information by cells is somewhat more complicated. Our definition of a gene is also evolving to take new knowledge into consideration. Figure 7.4 - Human genes sorted by class Matters of size A common-sense assumption about genomes would be that if genes specify proteins, then the more proteins an organism made, the more genes it would need to have, and thus, the larger its genome would be. Comparison of various genomes shows, surprisingly, that there is not necessarily a direct relationship between the complexity of an organism and the size of its genome (Figure 7.5). To understand how this could be true, it is necessary to recognize that while genes are made up of DNA, all DNA does not consist of genes (for purposes of our discussion, we define a gene as a section of DNA that encodes an RNA or protein product). In the human genome, less than 2% of the total DNA seems to be the sort of coding sequence that directs the synthesis of proteins. For many years, non-coding DNA in genomes was believed to be useless, and was described as “junk DNA” although it was perplexing that there seemed to be so much “useless” sequence. Recent discoveries have, however, demonstrated that much of this so-called junk DNA may play important roles in evolution, as well as in regulation of gene expression. Introns So, what is all the non-coding DNA doing there? We know that even coding regions in our DNA are interrupted by non-coding sequences called introns. This is true of most eukaryotic genomes. An examination of genes in eukaryotes shows that non-coding intron sequences can be much longer than the coding sections of the gene, or exons. Most exons are relatively small, and code for fewer than a hundred amino acids, while introns can vary in size from several hundred base-pairs to many kilobase-pairs (thousands of base-pairs) in length. For many genes in humans, there is much more of intron sequence than coding (a.k.a. exon) sequence. Intron sequences account for roughly a quarter of the genome in humans. Other non-coding sequences What other kinds of non-coding sequences are there? One function for some DNA sequences that do not encode RNA or proteins is in specifying when and to what extent a gene is used, or expressed. Such regions of DNA are called regulatory regions and each gene has one or more regulatory sequences that control its expression. However, regulatory sequences do not account for all the rest of the DNA in our genomes, either. Transposable sequences Surprisingly, almost half of the human genome appears to consist of several kinds of repetitive sequences. Many of the repetitive sequences are known to be transposable elements (transposons), sections of DNA that can move around within the genome. Sometimes referred to as “jumping genes” these transposable elements can move from one chromosomal location to another, either through a simple “cut and paste” mechanism that cuts the sequence out of one region of the DNA and inserts it into another location, or through a process called retrotransposition involving an RNA intermediate. LINES & SINES There are millions of copies of each of two major classes of such transposable elements, the LINEs (Long Interspersed Elements) and SINEs (Short Interspersed Elements) in our genomes. LINEs and SINEs are both a kind of transposable element called retrotransposons, sequences that are copied into RNA, then reverse transcribed back into DNA before being inserted into new locations. This movement is typically not sequence specific, meaning that the transposons can be inserted randomly in the genome, in many cases within coding regions. As might be expected, this can disrupt the function of the gene. Transposons may also insert within regulatory regions, and change the expression of the genes they control. As a major cause of mutation in genomes, transposons play an important role in evolution. Finally, recent findings have shown that much of the genome is transcribed into RNAs, even though only about 2% encodes proteins. What are the RNAs that do not encode proteins? Ribosomal RNAs (Figure 7.7) and transfer RNAs, together with the small nuclear RNAs that function in splicing, account for some of these non-translated transcripts, but not all. The remaining RNAs are regulatory RNAs, small molecules that play an important role in regulating gene expression. As we understand more about genomes, it is becoming evident that the so-called “junk” DNA is anything but.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/07%3A_Information_Processing/7.02%3A_Genes_and_Genomes.txt

Source: BiochemFFA_7_2.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Copying instructions The only way to make new cells is by the division of pre-existing cells. Single-celled organisms undergo division to produce more cells like themselves, while multicellular organisms arise through division of a single cell, generally the fertilized egg. Each time a cell divides, all of its DNA must be copied faithfully so that a copy of this information can be passed on to the daughter cell. This process is called DNA replication. It is the means by which genetic information can be transmitted down generations of cells, and it ensures that every new cell has a complete copy of the genome. In the next section, we will examine the process by which the DNA of a cell is completely and accurately copied. The structure of DNA elucidated by Watson and Crick in 1953 immediately suggested a mechanism by which double-stranded DNA could be copied to give two identical copies of the DNA. They proposed that the two strands of the DNA molecule, which are held together by hydrogen bonds between the base-paired nucleotides, would separate and each serve as a template on which a complementary strand could be assembled (Figure 7.8). The base-pairing rules would ensure that this process would result in the production of two identical DNA molecules. The beautiful simplicity of this scheme was shown to be correct in subsequent experiments by Meselson and Stahl, that demonstrated that DNA replication was semi-conservative, i.e., that after replication, each of the two resulting DNA molecules was made up of one old strand and one new strand that had been assembled across from it (Figure 7.9). Building materials What are the ingredients necessary for building a new DNA molecule? As noted above, the original, or parental DNA molecule serves as the template. New DNA molecules are assembled across from each template by joining together free DNA nucleotides as directed by the base pairing rules, with As across from Ts and Gs across from Cs. The nucleotides used in DNA synthesis are deoxyribonucleoside triphosphates or dNTPs. As can be inferred from their name, such nucleotides have a deoxyribose sugar and three phosphates, in addition to one of the four DNA bases, A, T, C or G (Figure 7.10). When dNTPs are added into a growing DNA strand, two of those phosphates will be cleaved off, as described later, leaving the nucleotides in a DNA molecule with only one phosphate per nucleotide. This reaction is catalyzed by enzymes known as DNA polymerases, which create phosphodiester linkages between one nucleotide and the next. Challenges Before examining the actual process of DNA replication, it is useful to think about what it takes to accomplish this task successfully. Consider the challenges facing a cell in this process: • The sheer number of nucleotides to be copied is enormous: e.g., in human cells, on the order of several billions.​ • A double-helical parental DNA molecule must be unwound to expose single strands of DNA that can serve as templates for the synthesis of new DNA strands.​ • Unwinding must be accomplished without introducing topological distortion into the molecule.​ • The unwound single strands of DNA must be kept from coming back together long enough for the new strands to be synthesized.​ • DNA polymerases cannot begin synthesis of a new DNA strand de novo and require a free 3' OH to which they can add deoxynucleotides.​ • DNA polymerases can only extend a strand in the 5' to 3' direction. The 5' to 3' growth of both new strands means that one of the strands is made in pieces.​ • The use of RNA primers requires that the RNA nucleotides must be removed and replaced with DNA nucleotides and the resulting DNA fragments must be joined. • The copying of all the parental DNA must be accurate, so that mutations are not introduced into the newly made DNA. Figure 7.14 - Prokaryotic vs. eukaryotic DNA replication - Wikipedia Addressing challenges With this in mind, we can begin to examine how cells deal with each of these challenges. Our understanding of the process of DNA replication is derived from studies using bacteria, yeast, and other systems. These investigations have revealed that DNA replication is carried out by the action of a large number of proteins that act together as a complex protein machine. Numerous proteins involved in replication have been identified and characterized, including multiple different DNA polymerases in both prokaryotes and eukaryotes. Although the specific proteins involved are different in bacteria and eukaryotes, it is useful to understand the basic considerations that are relevant in all cells. A generalized account of the steps in DNA replication is presented below, focused on the challenges mentioned above. • The sheer number of nucleotides to be copied is enormous: e.g., in human cells, on the order of several billions. Cells, whether bacterial or eukaryotic, have to replicate all of their DNA before they can divide. In cells like our own, the vast amount of DNA is broken up into many chromosomes, each of which is composed of a linear strand of DNA (Figure 7.12). In cells like those of E. coli, there is a single circular chromosome. In either situation, DNA replication is initiated at sites called origins of replication. These are regions of the DNA molecule that are recognized by special proteins called initiator proteins that bind the DNA. In E.coli, origins have small regions of A-T-rich sequences that are “melted” to separate the strands, when the initiator proteins bind to the origin or replication. As you may remember, A-T base-pairs, which have two hydrogen bonds between them are more readily disrupted than G-C base-pairs which have three apiece (Figure 7.15). How many origins of replication are there on a chromosome? In the case of E. coli, there is a single origin of replication on its circular chromosome. In eukaryotic cells there may be many thousands of origins of replication, with each chromosome having hundreds (Figure 7.16). DNA replication is, thus, initiated at multiple points along each chromosome in eukaryotes. Electron micrographs of replicating DNA from eukaryotic cells show many replication bubbles on a single chromosome. This makes sense in light of the large amount of DNA that there is to be copied in cells like our own, where beginning at one end of each chromosome and replicating all the way through to the other end from a single origin would simply take too long. This is despite the fact that the DNA polymerases in human cells are capable of building new DNA strands at the very respectable rate of about 50 nucleotides per second! • A double-helical parental molecule must be unwound to expose single strands of DNA that can serve as templates for the synthesis of new DNA strands. Unwinding Once a small region of the DNA is opened up at each origin of replication, the DNA helix must be unwound to allow replication to proceed. The unwinding of the DNA helix requires the action of an enzyme called helicase. Helicase uses the energy released when ATP is hydrolyzed, to break the hydrogen bonds between the bases in DNA and separate the two strands (Figure 7.17). Note that a replication bubble is made up of two replication forks that "move" or open up, in opposite directions. At each replication fork, the parental DNA strands must be unwound to expose new sections of single-stranded template. • This unwinding must be accomplished without introducing topological distortion into the molecule. What is the effect of unwinding one region of the double helix? Local unwinding of the double helix causes over-winding (increased positive supercoiling) ahead of the unwound region. The DNA ahead of the replication fork has to rotate, or it will get twisted on itself and halt replication. This is a major problem, not only for circular bacterial chromosomes, but also for linear eukaryotic chromosomes, which, in principle, could rotate to relieve the stress caused by the increased supercoiling. Topoisomerases The reason this is problematic is that it is not possible to rotate the entire length of a chromosome, with its millions of base-pairs, as the DNA at the replication fork is unwound. How, then, is this problem solved? Enzymes called topoisomerases can relieve the topological stress caused by local “unwinding” of the extra winds of the double helix. They do this by cutting one or both strands of the DNA and allowing the strands to swivel around each other to release the tension before rejoining the ends. In E. coli, the topoisomerase that performs this function is called gyrase. • The separated single strands of DNA must be kept from coming back together so the new strands to be synthesized. Single-strand DNA binding protein Once the two strands of the parental DNA molecule are separated, they must be prevented from going back together to form double-stranded DNA. To ensure that unwound regions of the parental DNA remain single-stranded and available for copying, the separated strands of the parental DNA are bound by many molecules of a protein called single-strand DNA binding protein (SSB - Figure 7.18). Figure 7.18 - Proteins at a prokaryotic DNA replication fork - Image by Martha Baker • DNA polymerases cannot begin synthesis of a new DNA strand de novo and require a free 3' OH to which they can add DNA nucleotides. Although single-stranded parental DNA is now available for copying, DNA polymerases cannot begin synthesis of a complementary strand de novo. This simply means that DNA polymerases can only add new nucleotides on to the 3' end of a pre-existing chain, and cannot start a chain of nucleotides on their own. Because of this limitation, some enzyme other than a DNA polymerase must first make a small region of nucleic acid, complementary to the parental strand, that can provide a free 3' OH to which DNA polymerase can add a deoxyribonucleotide. This task is accomplished by an enzyme called a primase, which assembles a short stretch of RNA base-paired to the parental DNA template. This provides a short base-paired region, called the RNA primer, with a free 3'OH group to which DNA polymerase can add the first new DNA nucleotide (Figure 7.12). Sliding clamp Once a primer provides a free 3'OH for extension, other proteins get into the act. These proteins are involved in loading the DNA polymerase onto the primed template and keeping it associated with the DNA. The first of these is the clamp loader. As its name suggests, the clamp loader helps to load a protein complex called the sliding clamp onto the DNA at the replication fork (Figure 7.19 and 7.20). The sliding clamp, a multi-subunit ring-shaped protein, is then joined by the DNA Polymerase. The function of the sliding clamp is to keep the polymerase associated with the replication fork - in fact, it has been described as a seat-belt for the DNA polymerase. The sliding clamp ensures that the DNA polymerase is able to synthesize long stretches of new DNA before it dissociates from the template. The property of staying associated with the template for a long time before dissociating is known as the processivity of the enzyme. In the presence of the sliding clamp, DNA polymerases are much more processive, making replication faster and more efficient. Extending the primer The DNA polymerase is now poised to start synthesis of the new DNA strand (in E. coli, the primary replicative polymerase is called DNA polymerase III). As you already know, the synthesis of new DNA is accomplished by the addition of new nucleotides complementary to those on the parental strand. DNA polymerase catalyzes the reaction by which an incoming deoxyribonucleotide, complementary to the template, is added onto the 3' end of the previous nucleotide, starting with the 3'OH on the end of the RNA primer. The importance of the 3’OH group lies in the nature of the reaction that builds a chain of nucleotides. The reaction catalyzed by the DNA polymerase occurs through the nucleophilic attack by the 3’OH group at the end of a nucleic acid strand on the α phosphate of the incoming dNTP (Figure 7.21). The immediate hydrolysis of the pyrophosphate that is cleaved off the incoming dNTP drives the reaction forward. The sequential addition of new nucleotides at the 3’ end of the growing chain of DNA accounts for the fact that the strand grows in a 5’ to 3’ direction. The 5' phosphate on each incoming nucleotide is joined by the DNA polymerase to the 3' OH on the end of the growing nucleic acid chain, to make a phosphodiester bond. Each added nucleotide provides a new 3’OH, allowing the chain to be extended for as long as the DNA polymerase continues to synthesize the new strand. As we already noted, the new DNA strands are synthesized by the addition of DNA nucleotides to the end of an RNA primer. The new DNA molecule thus has a short piece of RNA at the beginning. • DNA polymerases can only extend a strand in the 5' to 3' direction. The 5' to 3' growth of both new strands means that one of the strands is made in pieces. Leading strand We know that DNA polymerases can only build a new DNA strand in the 5' to 3' direction. We also know that the two parental strands of DNA are antiparallel. This means that at each replication fork, one new strand, called the leading strand can be synthesized continuously in the 5' to 3' direction because it is being made in the same direction that the replication fork is opening up. Lagging strand The synthesis of the other new strand, called the lagging strand, also proceeds in the 5’ to 3’ direction. But because the template strands are running in opposite directions, the lagging strand is being extended in the direction opposite to the opening of the replication fork (Figure 7.22). As the replication fork opens up, the region behind the original start point for the lagging strand will need to be copied. This means another RNA primer must be laid down and extended. This process repeats itself as the replication fork opens up, with multiple RNA primers laid down and extended, producing many short pieces that are later joined. These short nucleic acid pieces, each composed of a small stretch of RNA primer and about 1000-2000 DNA nucleotides, are called Okazaki fragments, for Reiji Okazaki, the scientist who first demonstrated their existence. • The use of RNA primers requires that the RNA nucleotides must be removed and replaced with DNA nucleotides. Primer removal We have seen that each newly synthesized piece of DNA starts out with an RNA primer, effectively making a new nucleic acid strand that is part RNA and part DNA. The newly made DNA strand cannot be allowed to have pieces of RNA attached. So, the RNA nucleotides must be removed and the gaps filled in with DNA nucleotides (Figure 7.23). This is done by DNA polymerase I in E. coli. This enzyme begins adding DNA nucleotides at the end of each Okazaki fragment. However, the end of one Okazaki fragment is adjacent to the RNA primer at the beginning of the next Okazaki fragment. DNA polymerase I has an exonuclease activity acting in the 5’ to 3’ direction that removes the RNA nucleotides ahead of it, while the polymerase activity replaces the RNA nucleotides with dNTPs. Once all the RNA nucleotides have been removed, the lagging strand is made up of stretches of DNA. The DNA pieces are then joined together by the enzyme DNA ligase. The steps outlined above essentially complete the process of DNA replication. But one issue still remains. • Ensuring accuracy in the copying of so much information Accuracy How accurate is the copying of information by DNA polymerase? As you are aware, changes in DNA sequence (mutations) can change the amino acid sequence of the encoded proteins and that this is often, though not always, deleterious to the functioning of the organism. When billions of bases in DNA are copied during replication, how do cells ensure that the newly synthesized DNA is a faithful copy of the original information? DNA polymerases, as we have noted earlier work fast (averaging 50 bases a second in human cells and up to 200 times faster in E. coli). Yet, both human and bacterial cells seem to replicate their DNA quite accurately. This is because replicative DNA polymerases have a proofreading function that enables the polymerase to detect when the wrong base has been inserted across from a template strand, back up and remove the mistakenly inserted base, before continuing with synthesis (Figure 7.24). Figure 7.24 - Error corrected by DNA polymerases Multiple activities This is possible because most DNA polymerases are dual-function enzymes. They can extend a DNA chain by virtue of their 5' to 3' polymerase activity. Some polymerases like DNA polymerase I can also remove RNA primers in the 5’ to 3’ direction, though that is not a common activity of polymerases. Many polymerases, however have an ability to backtrack and remove the last inserted base because they possess a 3' to 5' exonuclease activity. The exonuclease activity of a DNA polymerase allows it to excise a wrongly inserted base, after which the polymerase activity inserts the correct base and proceeds with extending the strand. In other words, the DNA polymerase is monitoring its own accuracy (also termed its fidelity) as it makes new DNA, correcting mistakes immediately before moving on to add the next base. This mechanism, which operates during DNA replication, corrects many errors as they occur, reducing by about a 100-fold the mistakes made when DNA is copied. DNA polymerases As noted earlier, both prokaryotic and eukaryotic cells have multiple DNA polymerases. In E.coli, for example, DNA polymerase III is the major replicative polymerase (a.k.a. replicase) while DNA polymerase I is responsible for DNA repair as well as removal of RNA primers and their replacement with DNA nucleotides during replication. DNA polymerase II plays a role in restarting replication after DNA damage stalls replication, while DNA polymerases IV and V are both required in trans-lesion, or bypass, synthesis, which allows replication past sites of DNA damage. Eukaryotic polymerases In eukaryotes, there are over fifteen different DNA polymerases. The primary replicative polymerases in the nucleus are ∂ and ε. DNA polymerase α is also important for replication because it has primase and repair activities. Replication is initiated in eukaryotic cells by DNA polymerase α, which binds to the initiation complex at the origin and lays down an RNA primer, followed by about 25 nucleotides of DNA. It is then replaced by another polymerase, in a step called the pol switch. DNA polymerase ∂ or ε then continues synthesizing DNA, depending on the strand. The role of polymerase ε appears to be synthesis of the leading strand due to its high processivity and accuracy, whereas polymerase ∂ extends Okazaki fragments on the lagging strands. Proteins analogous to the clamp loader and sliding clamp are also present. The protein RFC plays the role of clamp loader, while another protein, PCNA acts like the sliding clamp. Several other DNA polymerases like β, γ and μ function in repairing gaps. Yet others are involved in trans-lesion synthesis following DNA damage and are associated with hypermutation. Despite their diversity, DNA polymerases share some common structural features. X-ray crystallographic studies have shown that these enzymes have a structure that has been compared to a human right hand (Figures 7.25 & 7.26). The «palm» of the hand forms a cleft in which the DNA lies. The cleft is also the where the polymerase catalytic activity resides. This is where the incoming nucleotide is added on to the growing chain. «The fingers» position the DNA in the active site, while the «thumb» holds the DNA as it exits the polymerase. A separate domain contains the exonuclease (proofreading) activity of the enzyme. The enzyme alternates between its polymerizing activity and its proofreading activity. When a mismatched base pair is in the polymerase catalytic site, the 3’end of the growing strand is moved from the polymerase site to the exonuclease active site (Figure 7.26). The mispair at the end is removed by the exonuclease, followed by repositioning of the 3’ end in the polymerase active site to continue synthesis. Termination of replication In circular bacterial chromosomes, there are specific sequences known as terminator or Ter sites. These are multiple short sequences that serve as termination sites, allowing the replication forks traveling clockwise and anticlockwise across the circular chromosome to meet at one of the sites. The binding of a protein, Tus, at a Ter site prevents further movement of the replication fork and ends replication. The parental and newly made circular DNA are, at this point topologically interlinked and must be separated with the help of topoisomerase. The end-replication problem There is no fixed site for termination in linear eukaryotic chromosomes. As the replication forks reach the ends of the chromosome, the leading strand can be synthesized all the way to the end of the template strand. On the lagging strand, the need for an RNA primer to start synthesis creates a challenge. When the RNA primer at the extreme end of the lagging strand is removed, there is a small stretch of the template strand that cannot be copied. As a result, in each round of replication a short sequence at the ends of the chromosome will be lost. Over time, with many cycles of replication, chromosomes would become noticeably shorter. This shortening of chromosomes has been observed in vitro, in cultured mammalian somatic cells. It is also seen in intact organisms, with increasing age. Telomeres What effect does the loss of sequence from the ends of the chromosomes have on cells? We know that the ends of chromosomes are characterized by structures called telomeres (Figure 7.28). Telomeres are made up of many copies of short repeated sequences (in humans, the repeat is TTAGGG) and special proteins that specifically bind to these sequences. This structure of telomeres is useful in distinguishing the ends of chromosomes from double-strand breaks in DNA, thus preventing the DNA repair mechanisms in cells from joining chromosomes end to end. The other advantage of the repeated sequences, which do not encode proteins, is that losing some of the repeats does not lead to loss of important coding information. Thus, the repeats act as a sort of buffer zone, where the loss of sequence does not doom the cell. However, the shortening of chromosomes cannot continue indefinitely. After a certain number of replication cycles, cells are known to stop dividing and enter a state known as replicative senescence. This suggests that the shortening of the telomeres serves as a sort of clock, with the extent of shrinkage of the chromosomes serving as a measure of aging. Eventually cells that enter senescence will die. Figure 7.28 - Chromosomes with telomeres marked in white Problems with sequence loss Even if our cells are able to function with shorter chromosomes during our lifetimes, this leaves us with another problem. If our chromosomes grow shorter with age, then presumably our children, who inherit our chromosomes will be born with shorter chromosomes than we started with. They, in turn, would have their chromosomes shrink as they grew older, and their children would have even shorter chromosomes. Over the course of multiple generations, this would lead to the point where further chromosome shrinkage would result in cells that would enter senescence very early in life and die soon after. This obviously does not happen. Generation after generation of children are born with full-length chromosomes, so there is a mechanism that must ensure that at least in the reproductive cells, chromosomes do not get shorter. To understand this mechanism, it is necessary to first examine the end of a newly made DNA molecule (Figure 7.29). While the leading strand, which grows in the same direction as the movement of the replication fork, can copy its template all the way to the end, the lagging strand encounters a problem. RNA primers are, as we noted, needed to start each of the Okazaki fragments of the lagging strand. The primers must be removed later, and the RNA nucleotides replaced with DNA nucleotides. When the RNA primer across from the end of the parental strand is removed, the RNA nucleotides cannot be replaced by DNA nucleotides because the DNA polymerase has no primer to start from. A short region of the template cannot, therefore be copied. Figure 7.29 - Replication of a linear chromosome results in loss of sequences at the very ends with each round of replication Telomerase How can this problem be solved? It can be seen from Figure 7.29 that the end of the original template strand has a short 3’ overhang resulting from the removal of the RNA primer across from it. In order to fill in this region, another primer would be needed, situated past the end of the template strand. But in order to build such a primer, it would be necessary for the template overhang to be longer. If it were possible to make the template strand longer, then another primer could be placed across from its end and the end of the strand could be copied. Such an extension of the template strand is exactly what happens in our reproductive cells. The parental template strand is extended by the enzyme telomerase, which adds telomere repeats and lengthens the template. We will see shortly how it accomplishes that feat. RNA template Telomerase is an unusual enzyme, in that it is made up of two components, an RNA and a reverse transcriptase. A reverse transcriptase is an RNA-dependent DNA polymerase, an enzyme that copies an RNA template to make DNA. The RNA component of the human telomerase, called hTERC, has a sequence that is complementary to the telomere repeat, TAGGG. As seen in Figure 7.31, this RNA can base-pair with the last telomere repeat on the parental DNA strand, while a portion of the RNA remains unpaired. Template for extension The function of the unpaired region of the RNA is to serve as a template that can be used to extend the overhanging 3’ end of the original DNA molecule. The protein component of telomerase has reverse transcriptase activity and can copy the RNA sequence into DNA. In human telomerase, the protein component is known as hTERT (telomerase reverse transcriptase). As seen in Figure 7.31 and 7.32, the reverse transcriptase extends the original 3’ overhang using the RNA component as its template. The telomerase can then dissociate, and repeat the process multiple times to add many repeats of the telomere sequence. Once the overhang has been extended by the addition of at least several telomere repeats, there is now room for the synthesis of an RNA primer complementary to the newly extended overhang (pointing back towards the rest of the chromosome). This primer can then be extended to complete synthesis of the lagging strand all the way to the end of the original parental DNA strand. Thus, the addition of telomere repeats on the parental DNA strands keeps the newly made DNA strands from becoming shorter with each cycle of replication. The fact that this happens in germ cells (reproductive cells) explains why each generation does not have shorter chromosomes than the parental generation. The proofreading function of DNA polymerases monitors the accuracy of DNA replication while the enzyme telomerase keeps chromosomes that will be passed on to offspring from shortening. Between them, these two activities ensure that the genetic information is copied accurately, and that succeeding generations receive a full complement of the genetic information Disassembly and reassembly of nucleosomes The events of replication have an additional twist in eukaryotes. Recall that DNA is found in eukaryotic cells as chromatin, a complex of the DNA with proteins. At its least condensed, chromatin looks like a string of beads, consisting of the DNA wrapped around histone cores to make nucleosomes. The nucleosome structure must be disrupted to make DNA available for replication and restored after replication is completed (Figure 7.33). Ahead of the replication fork, chromatin structure is disassembled by ATP-dependent chromatin remodeling complexes, allowing access to the DNA template. Once the new strands of DNA have been synthesized, both the original nucleosomes and new nucleosomes must be reassembled behind the replication fork. Since replication gives rise to two DNA molecules where there was one, twice the amount of histones is needed to package the DNA. Preparation for DNA replication, therefore, involves the synthesis of large amounts of histones to supply the need. Interestingly, it appears that newly synthesized DNA is packaged into nucleosomes using the original histones that were displaced to allow the replication fork to pass, as well as newly synthesized histones. We also know that post-translational modifications like acetylation, methylation or phosphorylation of the histones can regulate the degree to which a given region of the genome is accessible for use. One question that remains the subject of intense research is how these modifications are accurately passed on to the new nucleosomes.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/07%3A_Information_Processing/7.03%3A_DNA_Replication.txt

Source: BiochemFFA_7_3.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Safeguarding the genome In the last section we considered the ways in which cells deal with the challenges associated with replicating their DNA, a vital process for all cells. It is evident that if DNA is the master copy of instructions for an organism, then it is important not to make mistakes when copying the DNA to pass on to new cells. Although proofreading by DNA polymerases greatly increases the accuracy of replication, there are additional mechanisms in cells to further ensure that newly replicated DNA is a faithful copy of the original, and also to repair damage to DNA during the normal life of a cell. DNA damage All DNA suffers damage over time, from exposure to ultraviolet and other radiation, as well as from various chemicals in the environment (Figures 7.34 & 7.35). Even chemical reactions naturally occurring within cells can give rise to compounds that can damage DNA. As you already know, even minor changes in DNA sequence, such as point mutations can sometimes have far-reaching consequences. Likewise, unrepaired damage caused by radiation, environmental chemicals or even normal cellular chemistry can interfere with the accurate transmission of information in DNA. Maintaining the integrity of the cell's "blueprint" is of vital importance and this is reflected in the numerous mechanisms that exist to repair mistakes and damage in DNA. Post-replicative mismatch repair We earlier discussed proofreading by DNA polymerases during replication. While proofreading significantly reduces the error rate, not all mistakes are fixed on the fly by DNA polymerases. What mechanisms exist to correct the replication errors that are missed by the proof-reading function of DNA polymerases? Errors that slip by proofreading during replication can be corrected by a mechanism called mismatch repair. While the error rate of DNA replication is about one in 107 nucleotides in the absence of mismatch repair, this is further reduced a hundred-fold to one in 109 nucleotides when mismatch repair is functional. What are the tasks that a mismatch repair system faces? It must:​ • Scan newly made DNA to see if there are any mispaired bases (e.g., a G paired to a T)​ • Identify and remove the region of the mismatch.​ • Correctly fill in the gap created by the excision of the mismatch region. Distinguishing strands Importantly, the mismatch repair system must have a means to distinguish the newly made DNA strand from the template strand, if replication errors are to be fixed correctly. In other words, when the mismatch repair system encounters an A-G mispair, for example, it must know whether the A should be removed and replaced with a C or if the G should be removed and replaced with a T. But how does the mismatch repair system distinguish between the original and the new strands of DNA? In bacteria, the existence of a system that methylates the DNA at GATC sequences is the solution to this problem. E.coli has an enzyme, DNA adenine methylase (Dam) that adds methyl groups on the to adenines in GATC sequences in DNA (Figure 7.36). Newly replicated DNA has not yet undergone methylation and thus, can be distinguished from the template strand, which is methylated. The mismatch repair proteins selectively replace the strand lacking methylation, thus ensuring that it is mistakes in the newly made strand that are removed and replaced. Because methylation is the criterion that enables the mismatch repair system to choose the strand that is repaired, the bacterial mismatch repair system is described as being methyl-directed. Figure 7.36- Dam methylase adds methyl groups at GATC sequences Mismatch repair genes Mismatch repair has been well studied in bacteria, and the proteins involved have been identified. In E.coli, mismatch repair proteins are encoded by a group of genes collectively known as the mut genes. Important components of the mismatch repair machinery are the proteins MutS, MutL and MutH (Figure 7.37). MutS acts to recognize the mismatch, while MutL and MutH are recruited to the mismatch site by the binding of MutS. MutH is an endonuclease that cuts the newly synthesized and, as yet, unmethylated DNA strand at a GATC. This activates a DNA helicase and an exonuclease that help unwind and remove the region containing the mismatch. DNA polymerase III fills in the gap, using the opposite strand as the template, and ligase joins the ends, to restore a continuous strand. Eukaryotes also have a mismatch repair system that repairs not only single base mismatches but also insertions and deletions. Homologs to the E. coli MutS and MutL have been identified in other organisms, including humans: hMSH1 and hMSH2 (human MutS homolog 1 and 2) are homologous to MutS, while hMLH 1 is homologous to MutL. These, together with additional proteins, carry out mismatch repair in eukaryotic cells. DNA methylation is not used by eukaryotic cells as a way to distinguish the new strand from the template, and it is not yet completely understood how the mismatch repair system in eukaryotes "knows" which strand to repair. There is evidence that the newly made DNA may be recognized by the fact that it is nicked, or discontinuous. This suggests that discontinuity resulting from Okazaki fragments that have not yet been joined together may permit the new strand to be distinguished from the old, continuous template strand. Repairing damage to DNA In the preceding section we looked at mistakes made when DNA is copied, where the wrong base is inserted during synthesis of the new strand. But even DNA that is not being replicated can get damaged or mutated. These sorts of damage are not associated with DNA replication, rather they can occur at any time. What causes damage to DNA? ​ Some major causes of DNA damage are:​ a. Radiation (e.g., UV rays in sunlight and in tanning booths, or ionizing radiation)​ b. Exposure to damaging chemicals, such as nitrosamines or polycyclic aromatic hydrocarbons, in the environment (see Figure 7.38)​ c. Chemical reactions within the cell (such as the deamination of cytosine to give uracil, or the methylation of guanine to produce methylguanine). This means the DNA in your cells is vulnerable to damage simply from normal sorts of actions, such as walking outdoors, being in traffic, or from the chemical transformations occurring in every cell as part of its everyday activities. (Naturally, the damage is much worse in situations where exposure to radiation or damaging chemicals is greater, such as when people use tanning beds, or smoke, regularly.) Types of damage What kinds of damage do these agents cause? Radiation can cause different kinds of damage to DNA. Sometimes, as with much of the damage done by UV rays, two adjacent pyrimidine bases in the DNA will be cross-linked to form cyclobutane pyrimidine dimers or CPDs (see Figure 7.39). Note that these are two neighboring pyrimidine bases on the same strand of DNA. UV exposure can also lead to the formation of another type of lesion, known as a (6-4) photoproduct or 6-4PP (Figure 7.39). Ionizing radiation can cause breaks in the DNA backbone, in one or both strands. Figure 7.39 - Possible chemical structures of a pyrimidine dimer - 6-4PP (left) and CPD (right) - Wikipedia Molecules like benzopyrene, found in automobile exhaust, can attach themselves to bases, forming bulky DNA adducts in which large chemical groups are linked to bases in the DNA. Damage like pyrimidine dimers, 6-4PPs or chemical adducts can physically distort the DNA helix, causing DNA and RNA polymerase to stall when they attempt to copy those regions of DNA (Figure 7.40). Chemical reactions occurring within cells can cause cytosines in DNA to be deaminated to uracil. Other sorts of damage in this category include the formation of oxidized bases like 8-oxo-guanine or alkylated bases like O6-methylguanine. These do not actually change the physical structure of the DNA helix, but they can cause problems because uracil and 8-oxo-guanine pair with different bases than the original cytosine or guanine, leading to mutations on the next round of replication. O6-methylguanine similarly can form base pairs with thymine instead of cytosine. Removing damage Cells have several ways to remove the sorts of damage described above. The first of these is described as direct reversal. Many organisms (though, unfortunately for us, not humans) can repair UV damage like CPDs and 6-4PPs because they possess enzymes called photolyases (photo=light; lyase=breakdown enzyme - Figure 7.41). Photolyases work through a process called photoreactivation, and use blue light energy to catalyze a photochemical reaction that breaks the aberrant bonds in the damaged DNA and returns the DNA to its original state. Suicide enzyme O6-methylguanine in DNA can also be removed by direct reversal, with the help of the enzyme O6-methylguanine methyltransferase. This is a very unusual enzyme that removes the methyl group from the guanine and transfers it onto a cysteine residue in the enzyme. The addition of the methyl group to the cysteine renders the enzyme non-functional. As you know, most enzymes are catalysts that remain unchanged over the course of the reaction, permitting a single enzyme molecule to repeatedly catalyze a reaction. Because the O6-methylguanine methyltransferase does not fit this description, it is sometimes not regarded as a true enzyme. It has also been called a suicide enzyme, because the enzyme “dies” as a result of its own activity. Excision repair Excision repair is another common strategy. Excision repair is a general term for the cutting out and re-synthesizing of the damaged region of a DNA. There are several different kinds of excision repair, but they all involve excising the portion of the DNA that is damaged, followed by repair synthesis using the other strand as template, and finally, ligation to restore continuity to the repaired strand. Cells possess several different kinds of excision repair, each geared to specific kinds of DNA damage. Between them, these repair systems deal with the wide variety of insults to the genome. Nucleotide excision repair Nucleotide excision repair (NER) fixes damage such as the formation of chemical adducts, as well as UV damage. Both chemical adducts and the formation of CPDs or 6,4 photoproducts can cause significant distortion of the DNA helix. NER proteins act to cut the damaged strand on either side of the lesion. A short portion of the DNA strand containing the damage is then removed and a DNA polymerase fills in the gap with the appropriate nucleotides. Nucleotide excision repair has been extensively studied in bacteria. In E. coli, recognition and excision of the damage is carried out by a group of proteins encoded by the uvrABC and uvrD genes. The protein products of the uvrA, uvrB and uvrC genes function together as the so-called UvrABC excinuclease. The damage is initially recognized and bound by a complex of the UvrA and UvrB proteins. Once the complex is bound, the UvrA dissociates, leaving the UvrB attached to the DNA, where it is then joined by the UvrC protein. Strand nicking It is the complex consisting of UvrB and C that acts to cut the phosphodiester backbone on either side of the damage, creating nicks in the strand about 12-13 nucleotides apart. A helicase encoded by uvrD then unwinds the region containing the damage, displacing it from the double helix together with UvrBC. The gap in the DNA is filled in by DNA polymerase, which copies the undamaged strand, and the nick is sealed with the help of DNA ligase. Nucleotide excision repair is also an important pathway in eukaryotes. It is particularly important in the removal of UV damage in humans, given that we lack photolyases. A number of proteins have been identified that function in ways similar to the Uvr proteins. The importance of these proteins is evident from the fact that mutations in the genes that encode them can lead to a number of genetic diseases, like Xeroderma pigmentosum, or XP. People with XP are extremely sensitive to UV exposure, because the damage caused by it cannot be repaired, leaving them at a much higher risk of developing skin cancer. Two repair modes Nucleotide excision repair operates in two modes, one known as global genomic repair and the other as transcription-coupled repair. While the function of both is to remove helix-destabilizing damage like cyclobutane pyrimidine dimers or chemical adducts, the way in which the lesions are detected differs. In global genomic repair, damage is identified by surveillance of the entire genome for helix distorting lesions. In the case of transcription-coupled repair, the stalling of the RNA polymerase at a site of DNA damage is the indicator that activates this mode of nucleotide excision repair. Base excision repair Base excision repair (BER) is a repair mechanism that deals with situations like the deamination of cytosine to uracil (Figure 7.43) or the methylation of a purine base. These changes do not typically distort the structure of the DNA helix, unlike chemical adducts or UV damage. In base excision repair a single damaged base is first removed from the DNA, followed by removal of a region of the DNA surrounding the missing base. The gap is then repaired. Uracil-DNA glycosylase The removal of uracil from DNA is accomplished by the enzyme uracil-DNA glycosylase that can recognize uracil in DNA and break the glycosidic bond between the uracil and the sugar in the nucleotide (Figure 7.44). The removal of the base leaves a gap called an apyrimidinic site (AP site) because, in this case, uracil, a pyrimidine was removed. It is important to remember that at this point the backbone of the DNA is still intact, and the removal of a single base simply creates a gap like a tooth that has been knocked out. The formation of the AP site triggers the activity of an enzyme known as an AP endonuclease that cuts the DNA backbone 5’ to the AP site. In the remaining steps, a DNA polymerase binds to the nick, then using its exonuclease and polymerase activities, replaces the sequence in this region. Depending on the situation, a single nucleotide may be replaced (short patch BER) or a stretch of several nucleotides may be removed and replaced (long patch BER). Finally, as always, DNA ligase acts to seal the nick in the DNA. Repair of double-strand breaks While all the repair mechanisms discussed so far fixed damage on one strand of DNA using the other, undamaged strand as a template, these mechanisms cannot repair damage to both strands. What happens if both strands are damaged? Ionizing radiation, exposure to certain chemicals, or reactive oxygen species generated in the cell can lead to double-strand breaks (DSBs) in DNA. DSBs are a potentially lethal form of damage that, in addition to blocking replication and transcription, can also lead to chromosomal translocations, where part of one chromosome gets attached to a piece of another chromosome. Two different cellular mechanisms exist that help repair DSBs (Figure 7.45), homologous recombination (HR) and non-homologous end joining (NHEJ). Figure 7.45 - Non-homologous end joining (left) versus homologous recombination (right) - Wikipedia Homologous recombination repair commonly occurs in the late S and G2 phases of the cell, when each chromosome has been replicated and information from a sister chromatid can be used as a template to achieve error-free repair. Note that in contrast to excision repair, where the damaged strand was removed and the undamaged sister strand served as the template for filling in the damaged region, HR must use the information from another DNA molecule, because both strands of the DNA are damaged in DSBs. Nuclease action Detection of the double-strand break triggers nuclease activity that chews back one strand on each end of the break. This results in the production of single-stranded 3’ overhangs on each end. These single-stranded ends are bound by several proteins, creating a nucleoprotein filament that can then “search” for homologous (matching) sequences on a sister chromatid. When such sequences are found, the nucleoprotein filament invades the undamaged sister chromatid, forming a crossover. This creates heteroduplexes made up of DNA strands from different chromatids. Strand invasion (Figure 7.47) is followed by branch migration, during which the Holliday junction moves along the DNA, extending the heteroduplex away from the original site of the crossover (Figure 7.48). In E. coli, branch migration depends on the activity of two proteins, RuvA and RuvB. The resulting recombination intermediate can be resolved, with the help of RuvC to give complete, error-free strands. Non-homologous end joining In contrast to homologous recombination, Non-homologous end joining (NHEJ) is error-prone. It does not use or require a homologous template to copy, and works by simply chewing back the broken ends of DSBs and ligating them together. Not surprisingly, NHEJ introduces deletions in the DNA as a result. Translesion DNA synthesis As we have seen, cells have a variety of mechanisms to help safeguard the integrity of the information in DNA. One measure of last resort is translesion DNA synthesis, also known as bypass synthesis. Translesion synthesis occurs when a DNA polymerase encounters DNA damage on the template strand, but instead of stalling or skipping past the damage, replication switches to an error-prone mode, ignoring the template and incorporating random nucleotides into the new strand. In E.coli, translesion synthesis is dependent on the activities of proteins encoded by the umuC and umuD genes. Under the appropriate conditions (see SOS response, below) UmuC and UmuD are activated to begin bypass synthesis. Being error-prone, translesion synthesis gives rise to many mutations. The SOS response Named for standard SOS distress signals, the term “SOS repair” refers to a cellular response to UV damage. When bacterial cells suffer extensive damage to their DNA as a result of UV exposure, they turn on the coordinated expression of a large number of genes that are necessary for DNA repair. These include the uvr genes needed for nucleotide excision repair and recA, which is involved in homologous recombination. In addition to these mechanisms, which can carry out error-free repair, the SOS response can also induce the expression of translesion polymerases encoded by the dinA, dinB and umuCD genes. How are all these genes induced in a coordinated way following UV damage? All of the genes induced in the SOS response are regulated by two components. The first is the presence of a short DNA sequence upstream of their coding region, called the SOS box. The second is a protein, the LexA repressor (Figure 7.49), that binds to the SOS box and prevents transcription of the downstream genes. Expression of the genes requires the removal of LexA from its binding site. How is this achieved? When exposure to radiation results in DNA breaks, the presence of single-stranded regions triggers the activation and binding of RecA proteins to the single-stranded region, creating a nucleoprotein filament. The interaction of the RecA with the LexA repressor leads to autocleavage of the repressor, allowing the downstream gene(s) to be expressed (Figure 7.50). The genes controlled by the LexA repressor, as mentioned earlier, encode proteins that are necessary for accurate DNA repair as well as error-prone translesion synthesis. The various genes involved in DNA repair are induced in a specific order. In the initial stages, the repair genes that are derepressed are for nucleotide excision repair, followed by homologous recombination, both error-free mechanisms for repair. If the damage is too extensive to be repaired by these systems, error-prone repair mechanisms may be brought into play as a last resort. SOS response and antibiotic resistance The increased mutation rate in the SOS response may play a role in the acquisition of antibiotic resistance in bacteria (Figure 7.51). An example is the development of resistance to topoisomerase poisons like the fluoroquinolone family of drugs. Fluoroquinolones inhibit the ability of topoisomerases to religate the ends of their substrates after nicking them to allow overwound DNA to relax. This results in accumulation of strand breaks that can trigger the SOS response. As a consequence of error-prone DNA synthesis by low fidelity polymerases during the SOS response, there is a large increase in the number of mutations. While some mutations may be lethal to the bacteria, others can contribute to the rapid development of drug resistance in the population.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/07%3A_Information_Processing/7.04%3A_DNA_Repair.txt

Source: BiochemFFA_7_4.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy In the preceding sections, we have discussed the replication of the cell's DNA and the mechanisms by which the integrity of the genetic information is carefully maintained. What do cells do with this information? How does the sequence in DNA control what happens in a cell? If DNA is a giant instruction book containing all of the cell's "knowledge" that is copied and passed down from generation to generation, what are the instructions for? And how do cells use these instructions to make what they need? Information flow You have learned in introductory biology courses that genes, which are instructions for making proteins, are made of DNA. You also know that information in genes is copied into temporary instructions called messenger RNAs that direct the synthesis of specific proteins. This description of flow of information from DNA to RNA to protein is often called the central dogma of molecular biology and is a good starting point for an examination of how cells use the information in DNA. Consider that all of the cells in a multicellular organism have arisen by division from a single fertilized egg and therefore, all have the same DNA. Division of that original fertilized egg produces, in the case of humans, over a trillion cells, by the time a baby is produced from that egg (that's a lot of DNA replication!). Yet, we also know that a baby is not a giant ball of a trillion identical cells, but has the many different kinds of cells that make up tissues like skin and muscle and bone and nerves. How did cells that have identical DNA turn out so different? The answer lies in gene expression, which is the process by which the information in DNA is used. Although all the cells in a baby have the same DNA, each different cell type uses a different subset of the genes in that DNA to direct the synthesis of a distinctive set of RNAs and proteins. The first step in gene expression is transcription, which we will examine next (Figure 7.52). Transcription Transcription is the process of copying information from DNA sequences into RNA sequences. This process is also known as DNA-dependent RNA synthesis. When a sequence of DNA is transcribed, only one of the two DNA strands is copied into RNA. We will consider what determines which strand of DNA is copied into RNA, later on. But, apart from copying one, rather than both strands of DNA, how is transcription different from replication of DNA? DNA replication serves to copy all the genetic material of the cell and occurs before a cell divides, so that a full copy of the cell's genetic information can be passed on to the daughter cell. Transcription, by contrast, copies short stretches of the coding regions of DNA to make RNA. Different genes may be copied into RNA at different times in the cell's life cycle. RNAs are, essentially, temporary copies of the information in DNA and different sets of instructions are copied for use at different times and in different cell types. Cells make several different kinds of RNA: • mRNAs that code for proteins • rRNAS that form part of ribosomes • tRNAs that serve as adaptors between mRNA and amino acids during translation • Small RNAs that regulate gene expression, including miRNAs and siRNAs • Other small RNAs that have a variety of functions, including the small nuclear RNAs that are part of the splicing machinery. • Long noncoding RNAs (lnc RNAs - Figure 7.53) Building an RNA strand is very similar to building a DNA strand. This is not surprising, knowing that DNA and RNA are very similar molecules. Transcription is catalyzed by the enzyme RNA Polymerase. "RNA polymerase" is a general term for an enzyme that makes RNA. There are several different kinds of RNA polymerases in eukaryotic cells, while in prokaryotes, a single type of RNA polymerase is responsible for all transcription. RNA synthesis Like DNA polymerases, RNA polymerases synthesize new strands only in the 5' to 3' direction, but because they are making RNA, they use ribonucleotides (i.e., RNA nucleotides - Figure 7.54) rather than deoxyribonucleotides. Ribonucleotides are joined in exactly the same way as deoxyribonucleotides, i.e., the 3'OH of the last nucleotide on the growing chain is joined to the 5' phosphate on the incoming nucleotide to make a phosphodiester bond. One important difference between DNA polymerases and RNA polymerases is that the latter do not require a primer to start making RNA. Once RNA polymerases are in the right place to start copying DNA, they just begin making RNA by joining together RNA nucleotides complementary to the DNA template. Starting points This, of course, brings us to an obvious question- how do RNA polymerases "know" where to start copying on the DNA? Unlike the situation in replication, where every nucleotide of the parental DNA must eventually be copied, transcription, as we have already noted, only copies selected portions of the DNA into RNA at any given time. Consider the challenge here: in a human cell, there are approximately 6 billion base-pairs of DNA. Much of this is non-coding DNA, meaning that it will not need to be transcribed. The small percentage of the genome that is made up of coding sequences still amounts to between 20,000 and 30,000 genes in each cell. Of these genes, only a small number will need to be expressed at any given time.What indicates to an RNA polymerase where to start copying DNA to make a transcript? Promoters It turns out that patterns in the DNA sequence indicate where RNA polymerase should start and end transcription. These sequences are recognized by the RNA polymerase or by proteins that help RNA polymerase determine where it should bind the DNA to start transcription. A DNA sequence at which the RNA polymerase binds to start transcription is called a promoter. The DNA sequence that indicates the endpoint of transcription, where the RNA polymerase should stop adding nucleotides and dissociate from the template is known as a terminator sequence. The promoter and terminator, thus, bracket the region of the DNA that is to be transcribed. A promoter is described as being situated upstream of the gene that it controls (Figure 7.57). What this means is that on the DNA strand that the gene is on, the promoter sequence is "before" the gene, or to put it differently, it is on the side of the gene opposite to the direction of transcription. Also notice that the promoter is said to "control" the gene it is associated with. This is because expression of the gene is dependent on the binding of RNA polymerase to the promoter sequence to begin transcription. If the RNA polymerase and its helper proteins do not bind at the promoter, the gene cannot be transcribed and it will therefore, not be expressed. What is special about a promoter sequence? In an effort to answer this question, scientists examined many genes and their surrounding sequences (Figure 7.57). Because the same RNA polymerase has to bind to many different promoters, it would be predicted that promoters would have some similarities in their sequences. As expected, common sequence patterns were seen to be present in many promoters. We will first take a look at prokaryotic promoters. When prokaryotic genes were examined, the following features commonly emerged: • A transcription start site (this the base in the DNA across from which the first RNA nucleotide is paired), which, by convention, is denoted as +1. • A -10 sequence: this is a 6 bp region centered about 10 bp upstream of the start site. The consensus sequence at this position is TATAAT. In other words, if you count back from the transcription start site, the sequence found at roughly -10 in the majority of promoters studied is TATAAT. • A -35 sequence: this is a 6 bp sequence at about 35 basepairs upstream from the start of transcription. The consensus sequence at this position is TTGACA. It is important to understand that each nucleotide in a consensus sequence is simply the one that appeared at that position in the majority of promoters examined, and does not mean that the entire consensus sequence is found in all promoters. In fact, few promoters have -10 and -35 sequences that exactly match the consensus. The box at the left shows the -10 and -35 sequences by percentage of occurrence of each base in the promoter. What is the significance of these sequences? It turns out that the sequences at -10 and -35 are necessary for recognition of the promoter region by RNA polymerase (Figure 7.58). The sequences at -10 and -35 may vary a little in individual promoters, as mentioned above, but the extent to which they are different is limited. It is only when the RNA polymerase has stably bound at the promoter that transcription can begin. The process by which the promoter is recognized and bound stably has been well studied for the RNA polymerase of E. coli. Core polymerase and holoenzyme The E. coli RNA polymerase is made up of a core enzyme of five subunits (α2ββ’and ω) and an additional subunit called the σ (sigma) subunit. Together, the σ subunit and core polymerase make up what is termed the RNA polymerase holoenzyme. The core polymerase is the part of the RNA polymerase that is responsible for the actual synthesis of the RNA, while the σ subunit is necessary for binding of the enzyme at promoters to initiate transcription. Loose association The core polymerase and σ subunit are not always associated with each other. For the most part, the core polymerase is loosely associated with DNA, although it does not discriminate between promoters and other sequences in DNA, and the DNA strands are not opened up to allow transcription in this state. The role of the σ subunit is to reduce the affinity of the core polymerase for non-specific DNA sequences and to help the enzyme specifically bind to promoter sequences. Holoenzyme binding It is when the σ subunit associates with the core polymerase that the holoenzyme is able to bind specifically to promoter sequences. The initial binding of the holoenzyme at the promoter results in what is called a “closed” complex, meaning that the DNA template is still double-stranded and has not opened up to allow transcription. This closed complex is then converted to an “open” complex by the separation of the DNA strands to create a transcription bubble about 12-14 base-pairs long (Figure 7.60). The conversion of the closed complex to the open complex also requires the presence of the σ subunit. Open complex & initiation Once the open complex has formed, the DNA template can begin to be copied, and the core polymerase adds nucleotides complementary to one strand of the DNA. At this stage, known as initiation, the polymerase adds several nucleotides while still bound to the promoter, and without moving along the DNA template. Initially, short pieces of RNA a few nucleotides long may be made and released, without the polymerase leaving the promoter. Eventually, the enzyme makes the transition to the next stage, elongation, when an RNA of 8-9 bases is made and the enzyme moves beyond the promoter region. Elongation Once elongation commences and the RNA polymerase is moving down the DNA template, the σ subunit is no longer necessary and may dissociate from the core enzyme. The core polymerase can move along the template, unwinding the DNA ahead of it to maintain a transcription bubble of 12-15 base-pairs and synthesizing RNA complementary to one of the strands of the DNA. As already mentioned, an RNA chain, complementary to the DNA template, is built by the RNA polymerase by the joining of the 5' phosphate of an incoming ribonucleotide to the 3'OH on the last nucleotide of the growing RNA strand. Behind the RNA polymerase, the DNA template is rewound, displacing the newly made RNA from its template strand. Termination As mentioned earlier, a sequence of nucleotides called the terminator is the signal to the RNA polymerase to stop transcription and dissociate from the template. Some terminator sequences, known as intrinsic terminators, allow termination by RNA polymerase without the help of any additional factors, while others, called rho-dependent terminators, require the assistance of a protein factor called rho (ρ). How does the sequence of the terminator cause the RNA polymerase to stop adding nucleotides and release the transcript? To understand this, it is useful to know that the terminator sequence precedes the last nucleotide of the transcript. In other words, the terminator is part of the end of the sequence that is transcribed (Figure 7.61). Intrinsic terminators In intrinsic terminators, this sequence in the RNA has self-complementary regions that can base-pair with each other to form a hairpin structure that contains a GC-rich run in the “stem” of the hairpin. This hairpin is followed by a single-stranded region that is rich in U’s (Figure 7.62). The secondary structure formed by the folding of the end of the RNA into the hairpin causes the RNA polymerase to pause. Meanwhile, the run of U’s at the end of the hairpin permits the RNA-DNA hybrid in this region to come apart, because the base-pairing between A’s in the DNA template and the U’s in the RNA is relatively weak. This allows the transcript to be released from the DNA template and from the RNA polymerase. Rho-dependent termination In the case of rho-dependent termination, an additional protein factor, rho, is necessary. Rho is a helicase that can separate the transcript from the template it is paired with. As in intrinsic termination, rho-dependent termination requires the formation of a hairpin structure in the RNA that causes pausing of the RNA polymerase. Meanwhile, rho binds to a region of the transcript called the rho utilization site (rut) and moves along the RNA till it reaches the paused RNA polymerase. It then acts on the RNA-DNA hybrid, releasing the transcript from the template. Coupled transcription and translation In prokaryotes, which lack a nucleus, the DNA is not separated from the rest of the cell in a separate compartment, so the mRNA is immediately available to the translation machinery, as the transcript is coming off the template DNA. Indeed, in prokaryotic cells, translation of the mRNA can begin before the entire gene has been transcribed. Ribosomes can assemble at the 5’ end of the transcript, as it is displaced from the template, while the 3’ end of the gene is still being copied. The lag time between transcription and translation is thus, very short in prokaryotes. Transcription in eukaryotes Although the process of RNA synthesis is the same in eukaryotes as in prokaryotes, there are some additional considerations in eukaryotes. One is that in eukaryotes, the DNA template exists as chromatin, where the DNA is tightly associated with histones and other proteins. The "packaging" of the DNA must therefore be opened up to allow the RNA polymerase access to the template in the region to be transcribed (Figure 7.63). The restructuring of chromatin to allow access to regions of DNA is thus an important factor in determining which genes are expressed. Multiple RNA polymerases A second difference is that eukaryotes have multiple RNA polymerases, not just one as in bacterial cells. The different eukaryotic polymerases transcribe different classes of genes. For example, RNA polymerase I transcribes the ribosomal RNA genes, while RNA polymerase III copies tRNA genes. The RNA polymerase we will focus on most is RNA polymerase II, which transcribes protein-coding genes to make mRNAs. All three eukaryotic RNA polymerases need additional proteins to help them get transcription started. In prokaryotes, RNA polymerase by itself can initiate transcription (the σ subunit is a subunit of the RNA polymerase, not an entirely separate protein). The additional proteins needed by eukaryotic RNA polymerases are referred to as transcription factors. We will see below that there are various categories of transcription factors. Transcription and translation are de-coupled Finally, in eukaryotic cells, transcription is separated in space and time from translation. Transcription happens in the nucleus, and the RNAs produced are processed further before they are sent into the cytoplasm.​ Protein synthesis (translation) happens in the cytoplasm. As noted earlier, in prokaryotic cells, mRNAs can be translated as they are coming off the DNA template, and because there is no nuclear envelope, transcription and protein synthesis occur in a single cellular compartment. A representative eukaryotic gene, depicted in Figure 7.64 shows that transcription starts some 25 bp downstream of the TATA box, and creates a transcript that begins with a 5’ untranslated region (5’UTR) followed by the coding region which may include multiple introns and ending in a 3’ untranslated region or 3‘UTR (Figure 7.64). As detailed below, the initial transcript is further processed before it is used. Eukaryotic promoters Like genes in prokaryotes, eukaryotic genes also have promoters that determine where transcription will begin. As with prokaryotes, there are specific sequences in the promoter regions that are recognized and bound by proteins involved in the initiation of transcription. We will focus primarily on the genes encoding proteins that are transcribed by RNA polymerase II. Such promoters commonly have a TATA box, a sequence similar to the -10 sequence in prokaryotic promoters. The TATA box is a sequence about 25-35 basepairs upstream of the start of transcription (+1). (Some eukaryotic promoters lack TATA boxes, and have, instead, other recognition sequences, known as DPE, or downstream promoter elements.) Interestingly, the TATA box is not directly recognized and bound by RNA polymerase II. Instead, this sequence is bound by other proteins that, together with the RNA polymerase, form the transcription initiation complex. Eukaryotic promoters also have, in addition, several other short stretches of sequences, that affect transcription, within about 100 to 200 base-pairs upstream of the transcription start site. These sequences, which are sometimes called upstream elements or promoter-proximal upstream elements, are bound by activator proteins that interact with the transcription complex that forms at the TATA box. Examples of such upstream elements are the CAAT box and the GC box (Figure 7.64). Making transcripts in eukaryotes We noted earlier that all eukaryotic RNA polymerases need additional proteins to bind promoters and start transcription. The proteins that help eukaryotic RNA polymerases find promoter sites and initiate RNA synthesis are termed general transcription factors. We will focus on the transcription factors that assist RNA polymerase II, the enzyme that transcribes protein-coding genes. These transcription factors are named TFIIA, TFIIB and so on (TF= transcription factor, II=RNA polymerase II, and the letters distinguish individual transcription factors). Transcription by RNA polymerase II requires the general transcription factors and the RNA polymerase to form a complex, at the TATA box, called the basal transcription complex or transcription initiation complex (Figure 7.65). This is the minimum requirement for any gene to be transcribed. The first step in the formation of this complex is the binding of the TATA box by a transcription factor, TFIID. TFIID is made up of several proteins, one of which is called the TATA Binding Protein or TBP. Binding of the TBP causes the DNA to bend at this spot and take on a structure that is suitable for the binding of additional transcription factors and RNA polymerase. Interestingly, the binding of the TBP is a necessary step in forming a transcription initiation complex even when the promoter lacks a TATA box. The order of binding of additional proteins after binding of the TBP, as determined through in vitro experiments, appears to be TFIIB, followed by TFIIF and RNA polymerase II, then TFIIE. The final step in the assembly of the basal transcription complex is the binding of a general transcription factor called TFIIH. Some evidence suggests that following the binding of the TBP to DNA, the rest of the proteins in the initiation complex may assemble as a very large complex that then binds directly to the DNA. In any case, the presence of all of these general transcription factors and RNA Polymerase II bound at the promoter is necessary for the initiation of transcription. As in prokaryotic transcription, once the RNA polymerase binds, it can begin to assemble a short stretch of RNA. This must be followed by promoter clearance, in order to move down the template and elongate the transcript. This requires the action of TFIIH. TFIIH is a multifunctional protein that has helicase activity (i.e., it is capable of opening up a DNA double helix) as well as kinase activity. The kinase activity of TFIIH adds phosphates onto the C-terminal domain (CTD) of the RNA polymerase II. This phosphorylation appears to be the signal that releases the RNA polymerase from the basal transcription complex and allows it to move forward on the template, building the new RNA as it goes (Figure 7.66). Termination of transcription is not as well understood as it is in prokaryotes. Termination does not occur at a fixed distance from the 3’ end of mature RNAs. Rather, it seems to occur hand in hand with the processing of the 3’ end of the primary transcript. The polyadenylation signal in the 3’ untranslated region of the transcript appears to play a role in RNA polymerase pausing, and subsequent release of the completed primary transcript. Recognition of the polyadenylation signal triggers the binding of proteins involved in 3’end processing and termination. Information Processing: Transcription 748 YouTube Lectures by Kevin HERE & HERE 749 Figure 7.52 - Overview of eukaryotic transcription Wikipedia 750 Figure 7.53 - Transcripts may code for protein or may be functional as RNAs Wikipedia 751 Figure 7.55 - RNA (green) being synthesized from DNA template (blue strand) by T7 RNA polymerase (purple). The non-template DNA strand is in red. Figure 7.54 - The four ribonucleotides for making RNA 752 Figure 7.56 - Central dogma - DNA to RNA to protein Wikipedia 753 YouTube Lectures by Kevin HERE & HERE Figure 7.57 - Sequences upstream of transcription start site in several prokaryotic genes Image by Martha Baker 754 -10 Sequence T A T A A T 77% 76% 60% 61% 56% 82% -35 Sequence T T G A C A 69% 70% 61% 56% 54% 54% Figure 7.58 - RNA polymerase promoter binding Wikipedia Figure 7.59 - A bacterial RNA polymerase (α2ββ’and ω) 755 Figure 7.60 - Synthesis of RNA in the transcription bubble 756 Figure 7.61 - Promoter and Terminator sequences determine where transcription starts and ends. 757 Figure 7.62 Transcription termination by intrinsic (top) and rho-dependent (bottom) mechanisms Wikipedia 758 Figure 7.63 - Eukaryotic DNA is complexed with proteins in chromatin Wikipedia YouTube Lectures by Kevin HERE & HERE 759 Figure 7.64 - Region surrounding the transcriptional start site in eukaryotic DNA Figure 7.65 - Transcription pre-initiation complex in eukaryotes Wikipedia 760 761

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/07%3A_Information_Processing/7.05%3A_Transcription.txt

Source: BiochemFFA_7_5.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy So far, we have looked at the mechanism by which the information in genes (DNA) is transcribed into RNA. The newly made RNA, also known as the primary transcript is further processed before it is functional. Both prokaryotes and eukaryotes process their ribosomal and transfer RNAs. The major difference in RNA processing, however, between prokaryotes and eukaryotes, is in the processing of messenger RNAs. We will focus on the processing of mRNAs in this section. You will recall that in bacterial cells, the mRNA is translated directly as it comes off the DNA template. In eukaryotic cells, RNA synthesis, which occurs in the nucleus, is separated from the protein synthesis machinery, which is in the cytoplasm. The initial product of transcription of an mRNA is sometimes referred to as the pre-mRNA. After it has been processed and is ready to be exported from the nucleus, it is called the mature mRNA. The three main processing steps for mRNAs are (Figure 7.67): • Capping at the 5' end ​ • Splicing to remove introns​ • Addition of a polyA tail at the 3' end. Although this description suggests that these processing steps occur post-transcriptionally, after the entire gene has been transcribed, there is evidence that processing occurs co-transcriptionally. That is, the steps of processing are occurring as the mRNA is being made. Proteins involved in mRNA processing have been shown to be associated with the phosphorylated C-terminal domain (CTD) of RNA polymerase II. Capping As might be expected, the addition of an mRNA cap at the 5’ end is the first step in mRNA processing, since the 5’end of the RNA is the first to be made. Capping occurs once the first 20-30 nucleotides of the RNA have been synthesized. The addition of the cap involves removal of a phosphate from the first nucleotide in the RNA to generate a diphosphate. This is then joined to a guanosine monophosphate which is subsequently methylated at N7 of the guanine to form the 7mG cap structure (Figure 7.68). This cap is recognized and bound by a complex of proteins that remain associated with the cap till the mRNA has been transported into the cytoplasm. The cap protects the 5' end of the mRNA from degradation by nucleases and also helps to position the mRNA correctly on the ribosomes during protein synthesis. Splicing Eukaryotic genes have introns, noncoding regions that interrupt the gene. The mRNA copied from genes containing introns will also therefore have noncoding regions that interrupt the information in the gene. These noncoding regions must be removed (Figure 7.69) before the mRNA is sent out of the nucleus to be used to direct protein synthesis. Intron removal Introns are removed from the pre-mRNA by the activity of a complex called the spliceosome. The spliceosome is made up of proteins and small RNAs that are associated to form protein-RNA enzymes called small nuclear ribonucleoproteins or snRNPs (pronounced snurps). Splice junctions The splicing machinery must be able to recognize splice junctions (i.e., where each exon ends and its associated intron begins) in order to correctly cut out the introns and join the exons to make the mature, spliced mRNA. What signals indicate exon-intron boundaries? The junctions between exons and introns are indicated by specific base sequences. The consensus sequence at the 5’ exon-intron junction (also called the 5’ splice site) is AGGURAGU. In this sequence, the intron starts with the second G (R stands for any purine). The 3' splice junction has the consensus sequence YAGRNNN, where YAG is within the intron, and RNNN is part of the exon (Y stands for any pyrimidine, and N for any nucleotide). There is also a third important sequence within the intron, about a hundred nucleotides from the 3’ splice site, called a branch point or branch site, that is important for splicing. This site is defined by the presence of an A followed by a string of pyrimidines. The importance of this site will be seen when we consider the steps of splicing. Splicing mechanism There are two main steps in splicing. The first step is the nucleophilic attack by the 2’OH of the branch point A on the 5' splice site (the junction of the 5' exon and the intron). As a result of a trans-esterification reaction, the 5' exon is released, and a lariat-shaped molecule composed of the 3’ exon and the intron sequence is generated (Figure 7.70). In the second step, the 3' OH of the 5’ exon attacks the 3’ splice site, and the two exons are joined together, and the lariat-shaped intron is released . Spliceosome As mentioned earlier, splicing is carried out by a complex consisting of small RNAs and proteins. The five small RNAs crucial to this complex, U1, U2, U4, U5 and U6 are found associated with proteins, as snRNPs. These and many other proteins work together to facilitate splicing. Although many details remain to be worked out, it appears that components of the splicing machinery associate with the CTD of the RNA polymerase and that this association is important for efficient splicing. The assembly of the spliceosome requires the stepwise interaction of the various snRNPs and other splicing factors (Figure 7.71). The initial step in this process is the interaction of the U1 snRNP with the 5’ splice site. Additional proteins such as U2AF (AF = associated factor) are also loaded onto the pre-mRNA near the branch site. This is followed by the binding of the U2 snRNA to the branch site. Next, a complex of the U4/U6 and U5 snRNPs is recruited to the spliceosome to generate a pre-catalytic complex. This complex undergoes rearrangements that alter RNA-RNA and protein-RNA interactions, resulting in displacement of the U4 and U1 snRNPs and the formation of the catalytically active spliceosome. This complex then carries out the two splicing steps described earlier. Alternative splicing On average, human genes have about 9 exons each. However, the mature mRNAs from a gene containing nine exons may not include all of them. This is because the exons in a pre-mRNA can be spliced together in different combinations to generate different mature mRNAs. This is called alternative splicing, and allows the production of many different proteins using relatively few genes, since a single RNA with many exons can, by combining different exons during splicing, create many different protein coding messages. Because of alternative splicing, each gene in our DNA gives rise, on average, to three different proteins. Alternative splicing allows the information in a single gene to be used to specify different proteins in different cell types or at different developmental stages (Figure 7.72). Polyadenylation The 3' end of a processed eukaryotic mRNA typically has a “poly(A) tail” consisting of about 200 adenine-containing nucleotides. These residues are added by a template-independent enzyme, poly(A)polymerase, following cleavage of the RNA at a site near the 3’ end of the new transcript. Components of the polyadenylation machinery have been shown to be associated with the CTD of the RNA polymerase, showing that all three steps of pre-mRNA processing are tightly linked to transcription. There is evidence that the polyA tail plays a role in efficient translation of the mRNA, as well as in the stability of the mRNA. Like alternative splice sites, genes can have alternative polyA sites as well (Figure 7.73). The cap and the polyA tail on an mRNA are also indications that the mRNA is complete (i.e., not defective). Once protein-coding messages have been processed by capping, splicing and addition of a poly A tail, they are transported out of the nucleus to be translated in the cytoplasm. Mature mRNAs are sent into the cytoplasm bound to export proteins that interact with the nuclear pore complexes in the nuclear envelope (Figure 7.74). Once the mature mRNA has been translocated to the cytoplasm, it is ready to be translated. RNA editing In addition to undergoing the three processing steps outlined above, many RNAs undergo further modification called RNA editing. Editing has been observed in not only mRNAs but also in transfer RNAs and ribosomal RNAs. As the name suggests, RNA editing is a process during which the sequence of the transcript is altered post-transcriptionally. A well-studied example of RNA editing is the alteration of the sequence of the mRNA for apolipoprotein B (see also HERE). The editing results in the deamination of a cytosine in the transcript to form a uracil, at a specific location in the mRNA. This change converts the codon at this position, CAA, which encodes a glutamine, into UAA, a stop codon. The consequence of this is that a shorter version of the protein is made, when the edited transcript is translated. It is interesting that the editing of this transcript occurs in intestinal cells but not in liver cells. Thus, the protein product of the apolipoprotein B gene is longer in the liver than it is in the intestine. Insertion/deletion Another kind of RNA editing involves the insertion or deletion of one or more nucleotides. One example of this sort of editing is seen in the mitochondrial RNAs of trypanosomes. Small guide RNAs indicate the sites at which nucleotides are inserted or deleted to produce the mRNA that is eventually translated (Figure 7.75). The effect of either of these kinds of editing on the mRNA is that the encoded protein product is different, providing another point at which the product of expression of a gene can be controlled. tRNA synthesis & processing tRNAs are synthesized by RNA polymerase III, which makes precursor molecules called pre-tRNA that then undergo processing to generate mature tRNAs. The initial transcripts contain additional RNA sequences at both the 5’ and 3’ ends. Some pre-tRNAs also contain introns. These additional sequences are removed from the transcript during processing. The 5’ leader sequence of the pre-tRNA (the additional nucleotides at the 5’-end) is removed by an unusual endonuclease called ribonuclease P (RNase P - Figure 7.76). RNase is a ribonucleoprotein complex composed of a catalytic RNA and numerous proteins. The 3’ trailer sequence (extra nucleotides at the 3’ end of the pre-tRNA) is later removed by different nucleases. All tRNAs must have a 3’ CCA sequence that is necessary for the charging of the tRNAs with amino acids. In bacteria, this CCA sequence is encoded in the tRNA gene, but in eukaryotes, the CCA sequence is added post-transcriptionally by an enzyme called tRNA nucleotidyl transferase (tRNT). Introns As mentioned earlier, some tRNA precursors contain an intron located in the anticodon arm. In eukaryotes, this intron is typically found immediately 3’ to the anticodon. The introns is spliced out with the help of a tRNA splicing endonuclease and a ligase. Base modifications Mature tRNAs contain a high proportion of bases other than the usual adenine (A), guanine (G), cytidine (C) and uracil (U). These unusual bases are produced by modifying the bases in the tRNA to form variants, such as pseudouridine (Figure 7.77) or dihyrouridine. Modifications to the bases are introduced into the tRNA at the final processing step by a variety of specialized enzymes. Different tRNAs have different subsets of modifications at specific locations, often the first base of the anti-codon (the wobble position). rRNA synthesis and processing Cells contain many copies of rRNA genes (between 100 and 2000 copies are seen in mammalian cells). These genes are organized in transcription units separated by non-transcribed spacers. Each transcription unit contains sequences coding for 18S, 5.8S and 28S rRNA, and is transcribed by RNA polymerase I into a single long transcript (47S). The 5S rRNA is separately transcribed. The sizes of ribosomal RNAs are, by convention, indicated by their sedimentation coefficients, which is a measure of their rate of sedimentation during centrifugation. Sedimentation is expressed in Svedberg units (hence the S at the end of the number) with larger numbers indicating greater mass. The initial transcript contains 5’ and 3’ external transcribed spacers (ETS) as well as internal transcribed sequences (ITS). The primary transcript is first trimmed at both ends by nucleases to give a 45S pre-rRNA. Further processing of the pre-rRNA through cleavages guided by RNA-protein complexes containing snoRNAs (small nucleolar RNAs), gives rise to the mature 18S, 5.8S and 28S rRNAs (Figure 7.79). Ribosomal RNAs are also modified both on the ribose sugars and on the bases. Interestingly, methylation of ribose sugars is the major modification in rRNA. The modified base pseudouridine is also common in rRNA. Other modifications include base methylation, and acetylation. These modifications are thought to be important in modulating ribosome function. Information Processing: RNA Processing 767 YouTube Lectures by Kevin HERE & HERE 768 Figure 7.68 - 5’ capping of eukaryotic mRNAs Wikipedia Figure 7.67 - Steps in processing of pre-mRNA 769 Figure 7.69 - Removal of introns from the primary transcript Interactive Learning Module HERE 770 Figure 7.70 - Splicing of introns Wikipedia 771 Figure 7.71 - Assembly of the spliceosome complex Wikipedia YouTube Lectures by Kevin HERE & HERE 772 Figure 7.72 - Alternative splicing leads to different forms of a protein from the same gene sequence Figure 7.73 - Alternative poly-adenylation sites for a gene 773 Figure 7.74 - Structure of a mature eukaryotic mRNA Interactive Learning Module HERE 774 Figure 7.76 - Structure of the RNA component of ribonuclease P Figure 7.75 - Template guided - one mechanism of RNA editing 775 Figure 7.78 - Sequence of a mature tRNA Wikipedia Figure 7.77 - Synthesis of pseudouridine from uridine Wikipedia 776 Figure 7.79 - Processing of ribosomal RNA YouTube Lectures by Kevin HERE & HERE Graphic images in this book were products of the work of several talented students. Links to their Web pages are below Click HERE for Martha Baker’s Web Page Click HERE for Pehr Jacobson’s Web Page Click HERE for Aleia Kim’s Web Page Click HERE for Penelope Irving’s Web Page Problem set related to this section HERE Point by Point summary of this section HERE To get a certificate for mastering this section of the book, click HERE Kevin Ahern’s free iTunes U Courses - Basic / Med School / Advanced Biochemistry Free & Easy (our other book) HERE / Facebook Page Kevin and Indira’s Guide to Getting into Medical School - iTunes U Course / Book To see Kevin Ahern’s OSU ecampus courses - BB 350 / BB 450 / BB 451 To register for Kevin Ahern’s OSU ecampus courses - BB 350 / BB 450 / BB 451 Biochemistry Free For All Facebook Page (please like us) Kevin Ahern’s Web Page / Facebook Page / Taralyn Tan’s Web Page Kevin Ahern’s free downloads HERE OSU’s Biochemistry/Biophysics program HERE OSU’s College of Science HERE Oregon State University HERE Email Kevin Ahern / Indira Rajagopal / Taralyn Tan 778 The Codon Song To the tune of “When I’m Sixty Four” Metabolic Melodies Website HERE Building of proteins, you oughta know​ Needs amino A’s​ Peptide bond catalysis in ribosomes​ Triplet bases, three letter codes Mixing and matching nucleotides​ Who is keeping score?​ Here is the low down​ If you count codons​ You'll get sixty four Got - to - line - up - right​ 16-S R-N-A and​ Shine Dalgarno site You can make peptides, every size​ With the proper code​ Start codons positioned​ In the P site place​ Initiator t-RNAs UGA stops and AUGs go​ Who could ask for more?​ You know the low down​ Count up the codons​ There are sixty four Recording by Tim Karplus Lyrics by Kevin Ahern Recording by Tim Karplus Lyrics by Kevin Ahern

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/07%3A_Information_Processing/7.06%3A_RNA_Processing.txt

Source: BiochemFFA_7_6.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Translation is the process by which information in mRNAs is used to direct the synthesis of proteins. As you have learned in introductory biology, in eukaryotic cells, this process is carried out in the cytoplasm of the cell, by large RNA-protein machines called ribosomes. Ribosomes contain ribosomal RNA (rRNA) and proteins. The proteins and rRNA are organized into two subunits, a large and a small. Ribosomes function by binding to mRNAs and holding them in a way that allows the amino acids encoded by the RNA to be joined sequentially to form a polypeptide. Transfer RNAs are the carriers of the appropriate amino acids to the ribosome. The Genetic Code We speak of genes (i.e., DNA) coding for proteins and the central dogma, which states that DNA makes RNA makes protein. What does this actually mean? A code can be thought of as a system for storing or communicating information. A familiar example is the use of letters to represent the names of airports (e.g., PDX for Portland, Oregon and ORD for Chicago’s O’Hare). When a tag on your luggage shows PDX as the destination, it conveys information that your bag should be sent to Portland, Oregon. To function well, such a set-up must have unique identifiers for each airport and people who can decode the identifiers correctly. That is, PDX must stand only for Portland, Oregon and no other airport. Also, luggage handlers must be able to correctly recognize what PDX stands for, so that your luggage doesn’t land in Phoenix, instead. How does this relate to genes and the proteins they encode? Genes are first transcribed into mRNA, as we have already discussed. The sequence of an mRNA, copied from a gene, directly specifies the sequence of amino acids in the protein it encodes. Each amino acid in the protein is specified by a sequence of 3 bases called a codon in the mRNA (Figure 7.81). For example, the amino acid tryptophan is encoded by the sequence UGG on an mRNA. All of the twenty amino acids used to build proteins have, likewise, 3-base sequences that encode them. Degeneracy Given that there are 4 bases in RNA, the number of different 3-base combinations that are possible is 43, or 64. There are, however, only 20 amino acids that are used in building proteins in cells. This discrepancy in the number of possible codons and the actual number of amino acids they specify is explained by the fact that the same amino acid may be specified by more than one codon. In fact, with the exception of the amino acids methionine and tryptophan, all the other amino acids are encoded by multiple codons. Codons for the same amino acid are often related, with the first two bases the same and the third being variable. An example would be the codons for alanine: GCU, GCA, GCC and GCG all stand for alanine. This sort of redundancy in the genetic code is termed degeneracy. Stop and start codons Three of the 64 codons are what are known as termination or stop codons, and as their name suggests, indicate the end of a protein coding sequence. The codon for methionine, AUG, is used as the initiation or start codon for the majority of proteins. This ingenious system is used to direct the assembly of a protein in the same way that you might string together colored beads in a particular order using instructions that used symbols like UGG for a red bead, followed by UUU for a green bead, CAC for yellow, and so on, till you came to UGA, indicating that you should stop stringing beads. Translating the code While the ribosomes are literally the factories that join amino acids together using the instructions in mRNAs, another class of RNA molecules, the transfer RNAs (tRNAs) are also needed for translation (Figure 7.83 and Interactive 7.1). Transfer RNAs are small RNA molecules, about 75-90 nucleotides long, that function to 'interpret' the instructions in the mRNA during protein synthesis. Transfer RNAs are extensively modified post-transcriptionally and contain a large number of unusual bases. The sequences of tRNAs have several self complementary regions, where the single-stranded tRNA folds on itself and base-pairs to form what is sometimes described as a clover leaf structure. This structure is crucial to the function of the tRNA, providing both the sites for attachment of the appropriate amino acid and for recognition of codons in the mRNA. In terms of the bead analogy above, someone or something has to be able to bring a red bead in when the instructions indicate UGG, and a green bead when the instructions say UUU. This, then, is the function of the tRNAs. They must be able to bring the amino acid corresponding to the instructions to the ribosome. t-RNA specificity A given transfer RNA is specific for a particular amino acid. It is linked covalently at its 3' end to the appropriate amino acid by an enzyme called aminoacyl tRNA synthetase. For example, there is a transfer RNA that is specific to the amino acid tryptophan, and a corresponding aminoacyl tRNA synthetase, called a tryptophanyl tRNA synthetase, that can attach the tryptophan specifically to this tRNA. Likewise, there is an aminoacyl tRNA synthetase specific for each amino acid. A tRNA with an amino acid attached to it is said to be charged (Figure 7.84). A pool of charged tRNAs is necessary to carry out protein synthesis. How do these tRNAs, carrying specific amino acids assist the ribosome in stringing together the correct amino acids, as specified by the sequence of the mRNA? Codon recognition As we already know, the amino acid sequence of the protein is determined by the order of the codons in the mRNA. We also have charged tRNAs carrying the various amino acids present. How are the amino acids attached to each other in the order indicated by the base sequence of the mRNA? This requires recognition of the codons on the mRNA by the appropriate charged tRNAs. The amino acid tryptophan, as we noted, is specified by the codon UGG in the mRNA. This codon must be recognized by a tRNA charged with tryptophan. Every tRNA has a a sequence of 3 bases, the anticodon, that is complementary to the codon for the amino acid it is carrying. When the tRNA encounters the codon for its amino acid on the messenger RNA, the anticodon will base-pair with the codon. For the tryptophan tRNA this is what it would look like: Sequence of tryptophan codon in mRNA: 5’ UGG 3’ Anticodon sequence in tryptophan tRNA: 5’ CCA 3’ Note that the sequences are both written, by convention, in the 5’ to 3’ direction. To base pair, though, they must be oriented in opposite directions (anti-parallel). The codon-anticodon basepair in the antiparallel orientation then would be: 5’ UGG 3’​ 3’ ACC 5’ The base-pairing of the anticodon on a charged tRNA with the codon on the mRNA is what brings the correct amino acids in to the ribosome to be added on to the growing protein chain (Figure 7.85). Making a Polypeptide With an idea of the various components necessary for translation and how they work, we can now take a look at the process of protein synthesis. The main steps in the process are similar in prokaryotes and eukaryotes. As we already noted, ribosomes bind to mRNAs and facilitate the interaction between the codons in the mRNA and the anticodons on charged tRNAs. Ribosomes have two sites (P-site and A-site) for binding and positioning charged tRNAs so each can form base pairs between their anticodon and a codon from the mRNA. The start codon (AUG) is positioned to base pair with the tRNA in the P-site (peptidyl site). Next, the charged tRNA complementary to the codon adjacent to the start codon binds and occupies the A-site (aminoacyl site) in the ribosome (Figure 7.86). At this point, the ribosome joins the amino acids carried on each tRNA by making a peptide bond. The bond between the amino acid and the tRNA in the P-site is broken and the dipeptide is joined to the tRNA on the A-site. The initiator tRNA without its amino acid is then released, moving into a site known as the Exit or E-site, while the second tRNA carrying the dipeptide (and the codon it is base paired to) moves into the P-site. The A-site now is ready with a new codon for the next incoming charged tRNA. This process is repeated, with the ribosome moving on the mRNA one codon at a time, until the stop codon reaches the A-site. At this point, a release factor binds at the A-site, and helps to free the completed polypeptide from the ribosome. The ribosome then dissociates into the small and large subunits, once more. Three steps Having considered the steps of translation in broader terms, we can now look at them in greater detail. We will consider the three steps of translation (bel0w) individually. Initiation (binding of the ribosomal subunits to the transcript and initiator tRNA) Elongation (repeated addition of amino acids to the growing polypeptide, based on the sequence of the mRNA - Figure 7.87) Termination (release of the completed polypeptide and dissociation of the ribosome into its subunits). We already know that processed mRNAs are sent from the nucleus to the cytoplasm in eukaryotic cells, while in prokaryotic cells, transcription and translation occur in a single cellular compartment. The small and large subunits of ribosomes, each composed of characteristic rRNAs and proteins, are found in the cytoplasm and assemble on mRNAs to form complete ribosomes that carry out translation. Both prokaryotic and eukaryotic ribosomal subunits are made up of one or more major rRNAs together with a large number of ribosomal proteins. The small subunits of prokaryotic cells are called the 30S ribosomal subunits, while their counterparts in eukaryotes are the 40S subunits. The large ribosomal subunits in prokaryotes are the 50S subunits, while those in eukaryotic cells are 60S. These differences reflect the larger mass of eukaryotic ribosomes. The rRNA components of ribosomes are important for the recognition of the 5’ end of the mRNA, and also play a catalytic role in the formation of peptide bonds. Initiation Messenger RNAs have non-coding sequences both at their 5' and 3' ends, with the actual protein-coding region sandwiched in between these untranslated regions (called the 5' UTR and 3' UTR, respectively). The ribosome must be able to recognize the 5' end of the mRNA and bind to it, then determine where the start codon is located. It is important to note that both in prokaryotes and eukaryotes, ribosomes assemble at the 5’ end of the transcript by the stepwise binding of the small and large subunits. The small subunit first binds to the mRNA at specific sequences in the 5’ UTR. The large subunit then binds to the complex of the mRNA and small subunit, to form the complete ribosome. Initiator tRNA Initiation also requires the binding of the first tRNA to the ribosome. As we have noted earlier, the initiation, or start codon is usually AUG, which codes for the amino acid methionine. Thus, the initiator tRNA is one that carries methionine and is designated as tRNAmet or methionyl tRNAmet. In bacteria, the methionine on the initiator tRNA is modified by the addition of a formyl group, and is designated tRNAfmet. The initiator tRNA carrying methionine to the AUG is different from the tRNAs that carry methionine intended for other positions in proteins. As such, the initiator tRNA is sometimes referred to as tRNAimet. Prokaryotic initiation In prokaryotes, the 5’ end of the mRNA is the only free end available, as transcription is tightly coupled to translation and the entire mRNA is not transcribed before translation begins. Nevertheless, the ribosome must be correctly positioned at the 5’ end of the messenger RNA in order to initiate translation. How does the ribosome “know” exactly where to bind in the 5’UTR of the mRNA? Shine-Dalgarno sequence Examination of the sequences upstream of the start codon in prokaryotic mRNAs reveals that there is a short purine-rich sequence ahead of the start codon that is crucial to recognition and binding by the small ribosomal subunit (Figure 7.89). This sequence, called the Shine-Dalgarno sequence, is complementary to a stretch of pyrimidines at the 3’ end of the 16S rRNA component of the small ribosomal subunit (Figure 7.90). Base-pairing between these complementary sequences positions the small ribosomal subunit at the right spot on the mRNA, with the AUG start codon at the P-site. Initiation factors The binding of the small ribosomal subunit to the mRNA requires the assistance of three protein factors called Initiation Factors 1, 2 and 3 (IF1, IF2, IF3). These proteins, which are associated with the small ribosomal subunit, are necessary for its binding to mRNA, but dissociate from it when the 50S ribosomal subunit binds. Of these proteins, IF3 is important for the binding of the small subunit to the mRNA, while IF2 is involved in bringing the initiator tRNA to the partial P-site of the small ribosomal subunit. IF1 occupies the A-site, preventing the binding of the initiator tRNA at that site. Once the small ribosomal subunit is bound to the mRNA and the initiator tRNA is positioned at the P-site, the large ribosomal subunit is recruited and the initiation complex is formed. Binding of the 50S ribosomal subunit is accompanied by the dissociation of all three initiation factors. The removal of IF1 from the A-site on the ribosome frees up the site for the binding of the charged tRNA corresponding to the second codon (Figure 7.91). Eukaryotic initiation In eukaryotes, initiation follows a similar pattern, although the order of events and the specific initiation factors are different. Eukaryotes have a large number of IFs that are known as eIFs (eukaryotic initiation factors). These initiation factors are involved in the binding of the initiator tRNA to the small subunit, as well as in association of the small subunit with mRNA and subsequent attachment of the large subunit. Ribosome assembly The assembly of the translation machinery in eukaryotes begins with the binding of the initiator tRNA to the 40S (small) subunit. This step requires the assistance of eIF2 and eIF3. Next the small subunit with the initiator tRNA binds to the 7-methyl G cap on the 5'end of the mRNA. The 40S subunit then moves along the mRNA, scanning for a a start codon. Binding of the ribosomal subunit to the mRNA is dependent not just on finding an AUG, but on the sequences surrounding the codon. Kozak sequences Specific sequences surrounding the AUG, called Kozak sequences for the scientist who defined them, have been shown to be necessary for the binding of the 40S subunit, with the bases at -4 and +1 relative to the AUG being especially important (Figure 7.92). Once the small subunit is properly positioned, the large ribosomal subunit (60S) binds, forming the initiation complex. Elongation After the ribosome is assembled with the initiator tRNA positioned at the AUG in the P-site, the addition of further amino acids can begin. In both prokaryotes and eukaryotes, the elongation of the polypeptide chain requires the assistance of elongation factors. In bacteria, the binding of the second charged tRNA at the A-site requires the elongation factor EF-Tu complexed with GTP (Figure 7.93). When the charged tRNA has been loaded at the A-site, EF-Tu hydrolyzes the GTP to GDP and dissociates from the ribosome. The free EF-Tu can then work with another charged tRNA to help position it at the A-site (Figure 7.94), after exchanging its GDP for a new GTP. The corresponding step in eukaryotic cells is dependent on the elongation factor eEF1α.GTP. Once both P-site and A-site are occupied, the methionine carried by the tRNA in the P-site is joined to the amino acid carried by tRNA in the A-site, forming a peptide bond. The reaction that joins the amino acids occurs in the ribosomal peptidyl transferase center, which is part of the large ribosomal subunit (Figure 7.95). Ribozyme Interestingly, there is strong evidence that this reaction is catalyzed by rRNA components of the large subunit, making the formation of peptide bonds an example of the activity of RNA enzymes, or ribozymes. The result of the peptidyl transferase activity is that the tRNA in the A-site now has two amino acids attached to it, while the tRNA at the P-site has none. This “empty” or deacylated tRNA is moved to the E-site on the ribosome, from which it can exit. The tRNA in the A-site, then moves to occupy the vacated P-site, leaving the A-site open for the next incoming charged tRNA. Yet another elongation factor, EF-G complexed with GTP, is required for the translocation of the ribosome along the mRNA in bacteria, while in eukaryotes, this role is played by eEF2.GTP. Repeated cycles of these steps result in the elongation of the polypeptide by one amino acid per cycle, until a termination, or stop codon is in the A-site. Termination When a stop codon is in the A-site, proteins called release factors (RFs) are needed to recognize the stop codon and cleave and release the newly made polypeptide. In bacteria, RF1 is a release factor that can recognize the stop codon UAG, while RF2 recognizes UGA. Both RF1 and RF2 can recognize UAA. A third release factor, RF3, works with RF1 and RF2 to hydrolyze the linkage between the polypeptide and the final tRNA, to release the newly synthesized protein. This is followed by the dissociation of the ribosomal subunits from the mRNA, ending the process of translation . Polypeptide processing What happens to the newly synthesized polypeptide after it is released from the ribosome? As you know, functional proteins are not simply strings of amino acids. The polypeptide must fold properly in order to perform its function in the cell. It may also undergo a variety of modifications such as the addition of phosphate groups, sugars, lipids, etc. Some proteins are produced as inactive precursors that must be cleaved by proteases to be functional. An additional challenge in eukaryotic cells is the presence of internal, membrane-bounded compartments. Each compartment contains different proteins with different functions. But the vast majority of proteins in eukaryotic cells are made by ribosomes, free or membrane-bound, in the cytoplasm of the cell (the exceptions are a handful of proteins made within mitochondria and chloroplasts). Delivery Each of the thousands of proteins made in the cytoplasm must, therefore, be delivered to the appropriate cellular compartment in which it functions. Some proteins are delivered to their destinations in an unfolded state, and are folded within the compartment in which they function. Others are fully folded and may be post-translationally modified before they are sent to their cellular (or extracellular) destinations. Some proteins are delivered as they are being synthesized (co-translationally - see Movie 7.3) while others are sorted to their compartments post-translationally. But, with the exception of cytosolic proteins, all proteins must cross membrane barriers, through membrane channels or other "gates", or by transport within membrane vesicles that fuse with the membrane of the target organelle to deliver their contents. Folding and post-translational modifications Proper folding of a protein into its 3-dimensional conformation is necessary for it to function effectively. As described in an earlier chapter (HERE), the folding of a protein is largely influenced by hydrophobic interactions that result in folding of the protein in such a way as to position hydrophobic residues in the interior, or core, of the protein, away from the aqueous environment of the cell. Proper folding may also involve the interaction of regions of the polypeptide that are distant from each other, so that portions of the N-terminal region of the polypeptide may be in close proximity to parts of the C-terminus of the final folded molecule. As a polypeptide emerges from the ribosome, however, the N-terminal region of the polypeptide may begin to fold on itself, with adjacent parts of the chain interacting in inappropriate ways, before the entire protein has been made. This can result in misfolding of the protein and consequent malfunction. To prevent misfolding, cells have protein chaperones, whose function is to bind to and shield regions of polypeptides as they emerge from the ribosome, and keep them from improperly interacting with one another or with other proteins in the vicinity, until they can fold into their correct final shape (Figure 7.98). In addition, there are classes of chaperones that are able sequester proteins in such a way as to permit unfolding and refolding of misfolded polypeptides. These proteins ensure that the vast majority of proteins in cells are folded into their correct, functional 3-dimensional shapes. Protein sorting The process by which proteins are identified as belonging to a particular compartment and then correctly delivered to that destination is known as protein sorting. How does a cell know where a particular protein should be sent? Proteins have "address labels" or sorting signals that indicate which cellular compartment they are destined for. Characteristic sorting signals are found on proteins that are sent to the nucleus, the ER (Figure 7.99), the mitochondria, etc. Signal sequences What do these sorting signals look like?​ Most sorting signals (also called signal sequences) are short stretches of amino acid sequence (that is, they are part of the amino acid sequence of the protein). Different cellular compartments have different "address labels". Signal sequences may be found at the N-terminal or C-terminal region of proteins, or they may be within the amino acid sequence of the proteins. The location of the signal sequence for any given protein is fixed, however. Signal sequences for proteins to be delivered to the endoplasmic reticulum (ER) are found at the N-terminus of the protein. Mitochondrial and chloroplast proteins encoded by nuclear genes also have N-terminal signal sequences. Signal sequences for nuclear proteins, by contrast, are internal to the polypeptide, and may consist of one or more stretches of amino acids that will be displayed on the surface of these proteins once they are folded. Free and membrane-bound ribosomes Proteins are synthesized by ribosomes in the cytoplasm or by those that associate with membranes temporarily (membrane-bound ribosomes). The free ribosomes make proteins that are destined for the nucleus, as well as those going to chloroplasts, mitochondria and peroxisomes. Nuclear proteins are delivered in their folded state, while chloroplast and mitochondrial proteins are threaded through translocation channels in the membranes of these organelles, to be folded at their destination. Proteins that are destined for the ER, the Golgi apparatus, lysosomes as well as those that are to be secreted from the cell are first delivered to the ER by ribosomes that associate with the membrane of the rough ER and synthesize the protein directly into the ER. Proteins delivered by this manner into the lumen of the ER undergo folding and modification in the ER. All proteins delivered to the ER, regardless of their final destination, have an N-terminal ER signal sequence of 15-30 amino acids. Protein delivery into the endoplasmic reticulum The N-terminal part of a protein is the first part of a nascent polypeptide that emerges from the ribosome (Figure 7.100). The sequence of amino acids in this region, if it is an ER signal, will be recognized and bound by a ribonucleoprotein complex called the Signal Recognition Particle (SRP). Binding of the SRP to the N-terminal signal sequence causes translation to pause. The SRP, in turn, is bound by an SRP receptor in the ER membrane (Movie 7.3 & Figure 7.101), effectively anchoring the ribosome to the membrane. The location of SRP receptors near membrane channels in the ER positions the ribosome over a translocation channel. Once the ribosome is docked over the channel, the SRP releases the signal sequence, which is threaded through the channel, with its hydrophobic residues interacting with the hydrophobic interior of the membrane. Translation resumes at this point and the rest of the protein is delivered into the lumen of the ER as it is made. The ribosome remains associated with the ER membrane till translation is completed, at which point it dissociates. The signal sequence, which is no longer needed once the protein has been delivered, is cleaved off by a membrane associated signal peptidase, releasing the completed protein into the ER lumen. While soluble proteins are delivered into the ER, integral membrane proteins do not pass all the way through, but, instead, are anchored in the membrane of the ER by hydrophobic stop transfer sequences. Folding and modification Proteins in the lumen of the ER are folded with the help of numerous chaperones resident in the endoplasmic reticulum. The environment within the ER lumen is also more oxidizing than the cytosol, and permits the formation of disulfide bonds to stabilize the folded proteins. Protein disulfide isomerase, an enzyme active in the ER lumen both helps to make disulfide bonds and removes bonds that were incorrectly made during the folding process. In addition, proteins in the ER undergo modifications such as glycosylation and addition of glycolipids. Multimeric proteins are also assembled from their subunits in the ER. Proteins that have been correctly folded and modified are transported from the ER, in membrane vesicles, to their final destinations. Improperly folded proteins are recognized by a surveillance mechanism in the ER and are sent back to the cytoplasm to be degraded in proteasomes. Information Processing: Translation 779 YouTube Lectures by Kevin HERE & HERE 780 Figure 7.81 - The standard genetic code Wikipedia Figure 7.80 - The central dogma in a bacterial cell Wikipedia 781 Figure 7.82 - Coding in DNA, transcribed to RNA, translated to protein 782 Figure 7.83 - tRNA - 3D projection (left) and 2D projection (inset) Wikipedia 783 Interactive 7.1 - Phenylalanyl-tRNA PDB Interactive 7.1 - Phenylalanyl-tRNA PDB Figure 7.84 - Charging of a tRNA by aminoacyl tRNA synthetase Wikipedia 784 Figure 7.85 - Codons in mRNA pair with anticodons on tRNA to bring the appropriate amino acid to the ribosome for polypeptide assembly YouTube Lectures by Kevin HERE & HERE 785 Figure 7.86 - The A, P, and E sites in a ribosome Image by Martha Baker Figure 7.87 - Overview of elongation Wikipedia Interactive Learning Module HERE 786 Movie 7.1 - 30S ribosomal subunit Wikipedia Movie 7.1 - 30S ribosomal subunit Wikipedia Table 7.1 - Location and function of rRNAs. rRNA Name Prokaryotes Eukaryotes Function 5S Large Subunit Large Subunit tRNA binding? 5.8S Large Subunit Translocation? 16S Small Subunit mRNA alignment 18S Large Subunit mRNA alignment 23S Large Subunit Peptide bond formation 28S Large Subunit Peptide bond formation 787 Figure 7.89 - Conserved sequences adjacent to start codons for various bacterial genes Image by Martha Baker Figure 7.88 - Structure of 5S rRNA 788 Figure 7.90 - Base pairing between the Shine-Dalgarno sequence in the mRNA and the 16S rRNA Image by Martha Baker Movie 7.2 - Large ribosomal subunit Wikipedia Movie 7.2 - Large ribosomal subunit Wikipedia 789 YouTube Lectures by Kevin HERE & HERE Figure 7.91 - Initiation - assembly of the ribosomal translation complex Image by Martha Baker 790 Figure 7.92 - Kozak sequence plot showing relative abundance of bases surrounding the AUG (ATG) start codon of human genes Figure 7.93 - EF-Tu (blue) bound to tRNA (red) and GTP (yellow) 791 Figure 7.94 - The process of elongation Image by Martha Baker 792 Figure 7.95 - 50S ribosomal subunit. RNA in brown. Protein in blue. Peptidyl transferase site in red. Wikipedia Interactive Learning Module HERE 793 Figure 7.96 - The process of translation Wikipedia Movie 7.3 Translation of a protein secreted into the endoplasmic reticulum. Small subunit in yellow. Large subunit in green. tRNAs in blue. Wikipedia Movie 7.3 Translation of a protein secreted into the endoplasmic reticulum. Small subunit in yellow. Large subunit in green. tRNAs in blue. Wikipedia 794 Figure 7.97 - Another perspective of translation. The 3’ end of the mRNA is on the left and the ribosome is moving from right to left 795 Figure 7.98 - Action of chaperone to facilitate proper folding of a protein (orange) Image by Aleia Kim YouTube Lectures by Kevin HERE & HERE 796 Figure 7.99 - Rough (ribosome bound) and smooth endoplasmic reticulum Wikipedia 797 Figure 7.100 - N-terminal signal sequence (green) emerging from the ribosome. 798 Figure 7.101 - Translation of a protein into the endoplasmic reticulum Image by Aleia Kim 799 Graphic images in this book were products of the work of several talented students. Links to their Web pages are below Click HERE for Martha Baker’s Web Page Click HERE for Pehr Jacobson’s Web Page Click HERE for Aleia Kim’s Web Page Click HERE for Penelope Irving’s Web Page Problem set related to this section HERE Point by Point summary of this section HERE To get a certificate for mastering this section of the book, click HERE Kevin Ahern’s free iTunes U Courses - Basic / Med School / Advanced Biochemistry Free & Easy (our other book) HERE / Facebook Page Kevin and Indira’s Guide to Getting into Medical School - iTunes U Course / Book To see Kevin Ahern’s OSU ecampus courses - BB 350 / BB 450 / BB 451 To register for Kevin Ahern’s OSU ecampus courses - BB 350 / BB 450 / BB 451 Biochemistry Free For All Facebook Page (please like us) Kevin Ahern’s Web Page / Facebook Page / Taralyn Tan’s Web Page Kevin Ahern’s free downloads HERE OSU’s Biochemistry/Biophysics program HERE OSU’s College of Science HERE Oregon State University HERE Email Kevin Ahern / Indira Rajagopal / Taralyn Tan 801 Translation To the tune of “Maria” (from “West Side Story”) Metabolic Melodies Website HERE Translation​ The most intricate thing I ever saw From five prime to three prime, translation, translation​ The final step that we know about the central dog-ma Amino, carboxyl, translation, translation. . . .​ Translation, translation, translation . . Translation! I just learned the steps of translation​ And all the things they say​ About tRNA​ Are true Translation!​ To form peptide bonds in translation​ The ribosomal cleft​ Must bind to an E-F​ tee-you! Translation!​ A-U-G binds the f-met's cargo​ 16S lines up Shine and Dalgarno​ Translation​ I'll never stop needing translation The most intricate thing I ever saw​ Translationnnnnnnnnnnnnnnnnn Recording by Tim Karplus Lyrics by Kevin Ahern Recording by Tim Karplus Lyrics by Kevin Ahern 802 Good Protein Synthesis To the tune of “Good King Wenceslaus” Metabolic Melodies Website HERE Amino acids cannot join By themselves together They require ribosomes To create the tether All the protein chains get made ‘Cording to instruction Carried by m-R-N-A In peptide bond construction Small subunit starts it all With initiation Pairing up two RNAs At the docking station Shine Dalgarno’s complement In the 16 esses Lines the A-U-G up so Synthesis commences Elongation happens in Ribosomic insides Where rRNA creates Bonds for polypeptides These depart the ribosome Passing right straight through it In the tiny channels there Of the large subunit Finally when the sequence of One of the stop codons Parks itself in the A site Synthesis can’t go on P-site RNA lets go Of what it was holding So the polypeptide can Get on with its folding Recording by David Simmons Lyrics by Kevin Ahern Recording by David Simmons Lyrics by Kevin Ahern

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/07%3A_Information_Processing/7.07%3A_Translation.txt

Source: BiochemFFA_7_7.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy The processes of transcription and translation described so far tell us what steps are involved in the copying of information from a gene (DNA) into RNA and the synthesis of a protein directed by the sequence of the transcript (Figure 7.102). These steps are required for gene expression, the process by which information in DNA directs the production of the proteins needed by the cell. But what determines whether a gene is expressed at a given time? Cells do not, as we know, express all of their genes all of the time. Some genes are expressed in particular cell types but not others, while others may be expressed at specific stages of development. Cells must also be able alter their patterns of gene expression in response to internal and external cues, controlling the production of proteins as needed, to meet their changing needs. Regulating gene expression is, therefore, crucial. Given that there are multiple steps involved in gene expression, there are several different points at which the process could be regulated. Not surprisingly, many regulatory mechanisms are known, each acting at a different stage in the path from DNA to protein. Regulation of Transcription The first step in gene expression is transcription, so regulation of transcription is an obvious way to affect whether a gene is expressed and to what extent. What are the molecular switches that turn transcription on or off? Although there are additional factors that affect transcription, such as the accessibility of a gene to the transcriptional machinery, the basic mechanism by which transcription is regulated depends on highly specific interactions between transcription regulating proteins and regulatory sequences on DNA. What are these regulatory sequences and what proteins bind them? In addition to the promoter sequences required for transcription initiation, genes have additional cis regulatory sequences (sequences of DNA on the same DNA molecule as the gene) that control when a gene is transcribed. Regulatory sequences are bound tightly and specifically by transcriptional regulators, proteins that can recognize DNA sequences and bind to them. The binding of such proteins to the DNA can regulate transcription by preventing or increasing transcription from a particular promoter. Transcriptional regulation in prokaryotes Let us first consider some examples from prokaryotes. In bacteria, genes are often clustered in groups, such that genes that need to be expressed at the same time are next to each other and all of them are controlled as a single unit by the same promoter. Groups of genes that are coordinately regulated by a single promoter are referred to as operons. The entire set of genes in an operon can be controlled through the action of DNA binding proteins that act as either repressors (preventing transcription of the genes) or activators (increasing transcription of the genes). The binding of these proteins to their DNA targets is allosterically controlled by the binding of specific small molecules that signal the state of the cell. Induction of the lac operon The lac operon is one such group of coordinately regulated genes that encode proteins needed for the uptake and breakdown of the sugar lactose. E.coli cells preferentially use glucose for their energy needs, but if glucose is unavailable, and lactose is present, the bacteria will take up lactose and break it down for energy. Since the proteins for taking up and breaking down lactose are only needed when glucose is absent and lactose is available, the bacterial cells need a way to express the genes of the lac operon only under those conditions. The default state of the lac operon is OFF. Removing a repressor Transcription of the lac cluster of genes is primarily controlled by a repressor protein that binds to a region of the DNA just downstream of the -10 sequence of the lac promoter (Figure 7.104). Recall that the promoter is where the RNA polymerase must bind to begin transcription. The location on the DNA where the lac repressor is bound is called the operator (Figure 7.105). When the repressor is bound at this position, it physically blocks the RNA polymerase from transcribing the genes, just as a vehicle blocking your driveway would prevent you from pulling out. Obviously, if you want to leave, the vehicle that is blocking your path must be removed. Likewise, in order for transcription to occur, the repressor must be removed from the operator to clear the path for RNA polymerase (Figure 7.106). How is the repressor removed? When the sugar lactose is present, a small amount of it is taken up by the cells and converted to an isomeric form, allolactose (Figure 7.107). Allolactose binds to the repressor, changing its conformation so that it no longer binds to the operator. When the repressor is no longer bound to the operator, the "road-block" in front of the RNA polymerase is removed, permitting the transcription of the genes of the lac operon What makes this an especially effective control system is that the genes of the lac operon encode proteins that enable the break down of lactose. Turning on these genes requires lactose to be present. Once the lactose has been broken down, the lac repressor binds to the operator once more and the lac genes are no longer expressed. This allows the genes to be expressed only when they are needed. Recruiting RNA polymerase But how do glucose levels affect the expression of the lac genes? We noted earlier that if glucose was present, lactose would not be used. A second level of control is exerted by a protein called Catabolite Activator Protein (CAP - Figure 7.108)). CAP (also sometimes called CBP or cAMP binding protein) binds to a site adjacent to the promoter and is necessary to recruit RNA polymerase to bind the lac promoter. cAMP binding CAP binds to its site only when glucose levels are low. Low glucose levels are linked to the activation of an enzyme, adenylate cyclase, that makes the molecule cyclic AMP (cAMP). The binding of cAMP to the CAP causes a conformational change in CAP that allows it to bind to the CAP-binding site. When CAP is bound at this site, it is able to recruit RNA polymerase to bind at the promoter, and begin transcription. The combination of CAP binding and the lac repressor dissociating from the operator when lactose levels are high ensures transcription of the lac operon just when it is most needed. The binding of CAP may be thought of as a green light for the RNA polymerase, while the removal of lac repressor is like the lifting of a barricade in front of it. When both conditions are met, the RNA polymerase transcribes the downstream genes. Control of the trp operon by repression The lac operon we have just described is a set of genes that are expressed only under the specific conditions of glucose depletion and lactose availability. Other genes may be expressed unless a particular condition is met. For these genes, the default state is ON. An example of this is the trp operon, which encodes enzymes necessary for the synthesis of the amino acid tryptophan. These genes are constitutively expressed (always on), except when tryptophan is available from the cell's surroundings, making its synthesis unnecessary. Under conditions where tryptophan is abundant in the environment, the trp genes can be turned off. This is achieved by a repressor protein that will bind to the operator only in the presence of tryptophan (Figure 7.110). Binding of tryptophan to the repressor causes binding of the repressor to the operator. Because it acts together with the repressor to turn off the trp genes, tryptophan is called a co-repressor. Attenuation Another mechanism that regulates the expression of the trp operon is attenuation. Attenuation is a process by which the expression of an operon is controlled by termination of transcription before the first gene of the operon (Figure 7.111). In the trp operon, this functions as follows: Transcription begins some distance upstream of the first gene in the operon, producing what is termed a 5’ leader sequence. This leader sequence contains an intrinsic terminator that can form a hairpin structure that stops transcription when high levels of tryptophan are available to the cells. It can also form a different structure that permits continued transcription of the genes in the operon when tryptophan levels are low. How does the level of tryptophan influence which of these two structures are formed? Recall that the 5’ end of the RNA is the first part of the transcript to be made and that in bacteria translation is linked to transcription, so the 5’ end of the RNA begins to be translated before the entire transcript is made. It turns out that the 5’ leader sequence of the trp operon mRNA encodes a short peptide that contains two tryptophan codons. If there is plenty of tryptophan available, the leader sequence will be easily translated. Under these conditions, the leader sequence is able to form the termination hairpin, preventing the transcription of the downstream trp genes. If, however, levels of tryptophan are low, then the ribosome stalls as it attempts to translate the leader sequence. Under these conditions, the leader sequence adopts a different conformation that permits continued transcription of the genes of the trp operon. Riboswitches Similar in concept to the attenuation of the trp operon described above, but not dependent on translation, is a control mechanism called a riboswitch (Figure 7.113). Riboswitches are typically found in the 5'UTR of messenger RNAs (i.e., they are part of the sequence of the RNA). These sequences can control transcription of the downstream genes based on the conformation they adopt. One conformation allows continued transcription, while the other terminates it. So, what determines which conformation they adopt? Features Riboswitches have two characteristic features that are important for their function. One is a region of the sequence called an aptamer, which folds into a three-dimensional shape that can bind a small effector molecule. The other is an adjacent region of the RNA, called the expression platform, that can fold into different conformations depending on whether or not the aptamer is bound to the effector. An example of a riboswitch found in bacteria is the guanine riboswitch, which controls the expression of genes required for purine biosynthesis. The aptamer region of this riboswitch binds to the effector, guanine, when levels of the base are high. The binding of the guanine triggers a change in the folding of the downstream expression platform, causing it to adopt a conformation that terminates transcription of the genes needed for the synthesis of guanine. In the absence of guanine, the expression platform assumes a different conformation that allows transcription of the purine biosynthesis genes. Thus, levels of guanine can be sensed and the genes needed for its synthesis can be expressed as needed. Regulation of transcription in eukaryotes Transcription in eukaryotes is also regulated by the binding of proteins to specific DNA sequences, but with some differences from the simple schemes outlined above. For most eukaryotic genes, general transcription factors and RNA polymerase (i.e., the transcription initiation complex) are necessary but not sufficient for high levels of transcription. Promoter-proximal DNA sequences like the CAAT box and GC box bind proteins that interact with the transcription initiation complex, influencing its formation (Figure 7.114). Distant regulatory sequences Additional regulatory sequences called enhancers and the proteins that bind to them are needed to achieve high levels of transcription. Enhancers are short DNA sequences that regulate the transcription of genes, but may be located at a distance from the gene they control (although they are on the same DNA molecule as the gene). Often enhancers are many kilobases away on the DNA, either upstream or downstream of the gene. As the name suggests, enhancers can enhance (increase) transcription of a particular gene. How can a DNA sequence far from the gene being transcribed affect the level of transcription? Transcriptional activators Enhancers work by binding proteins (transcriptional activators) that can, in turn, interact with the proteins bound at the promoter. The enhancer region of the DNA, with its associated transcriptional activator(s) can come in contact with the transcription initiation complex that is bound at a distant site by looping of the DNA (Figure 7.115). This allows the protein bound at the enhancer to make contact with the proteins in the basal transcription complex. The interaction of the activator with the transcription initiation complex may be direct, or it may be through a “middle-man”, a protein complex called mediator. One effect of this interaction is to assist in recruiting proteins necessary for transcription, like the general transcription factors and RNA polymerase to the promoter, increasing the frequency and efficiency of formation of the transcription initiation complex. There is also evidence that at some promoters, following assembly of the transcription initiation complex, the RNA polymerase remains stalled at the promoter. In such cases, the interaction with the transcription initiation complex of an activator bound to an enhancer could play a role in facilitating the transition of the RNA polymerase to the elongation phase of transcription. Chromatin remodeling proteins Another mechanism by which activators bound at the enhancer can affect transcription is by recruiting to the promoter proteins that can modify the structure of that region of the chromosome. In eukaryotes, DNA is packaged with proteins to form chromatin. When the DNA is tightly associated with these proteins, it is difficult to access for transcription. So proteins that can make the DNA more accessible to the transcription machinery can also play a role in the extent to which transcription occurs. Silencers In addition to enhancers, there are also negative regulatory sequences called silencers. Such regulatory sequences bind to transcriptional repressor proteins. Like the transcriptional activators, these repressors work by interacting with the transcription initiation complex. In the case of repressors, the effect they have on the transcription initiation complex is to reduce transcription. DNA binding proteins Transcriptional activators and repressors are modular proteins- they have a part that binds DNA and a part that activates or represses transcription by interacting with the transcription initiation complex (Figure 7.118). The DNA binding domain is the part of the protein that confers specificity for determining which gene(s) will be activated or repressed. The activation domain is the part of the protein that stimulates or represses transcription. The DNA binding domains of transcriptional activators form characteristic structures that recognize their target DNA sequences by making contacts with bases, usually in the major groove of the DNA helix. It is possible to engineer hybrid transcription factors that combine the DNA binding domain of one activator with the activation domain of another. Such proteins retain the specificity dictated by the DNA binding domain. Truncated transcription factors can also be generated that have their DNA binding domain but lack the activation domain. Such transcription factors can be useful tools in studying transcriptional regulation because their DNA binding domains can compete with the endogenous transcription factors for regulatory binding sites without increasing transcription from the target promoters. Multiple factors The description above may suggest that each gene in eukaryotes is controlled by the binding of a single transcriptional activator or repressor to a particular enhancer or silencer site. However, it turns out that the transcription of any given gene may be simultaneously regulated by a combination of proteins, both activators and repressors, bound at multiple regulatory sites on the DNA, all of which interact with the transcription initiation complex. The combinatorial nature of such regulation provides great versatility, with different combinations of regulatory elements and proteins working together in response to a wide variety of conditions and signals. The mechanisms described so far have focused on the sequence elements in DNA that regulate transcription through the activator and repressor proteins bound to them. Following transcription, alternative splicing (see HERE) and editing of the transcripts can also modify the proteins that are produced by the cell. We will now examine some of the other ways in which gene expression is modulated in cells. First, we will consider some so-called epigenetic mechanisms that affect gene expression. The term epigenetics derives from epi (above, or on top of) and genetic (of genes) and refers to the fact that these mechanisms act in addition to, or overlaid on, the information in the gene sequences. Two such epigenetic mechanisms are the covalent modifications of histones in chromatin and the methylation of DNA sequences. Histone modification As noted earlier, transcription in eukaryotes is complicated by the fact that the DNA is packaged with histones to make chromatin. This means that for a gene to be transcribed, the relevant regions of the chromatin must be opened up to allow access to the RNA polymerase and transcription factors. This provides another potential point of control of gene expression. Chromatin remodeling factors, mentioned earlier, assist in reorganizing the nucleosome structure at regions that need to be made accessible. But what determines that a given region of the chromatin will be acted upon by the remodeling complexes? Transcriptional activator proteins bound at enhancers, sometimes work by recruiting histone modifying enzymes to the promoter region. An example of such a modifying enzyme is histone acetyl transferase (HAT) that works to acetylate specific amino acid residues in the tails of the histones forming the nucleosome core (Figures 7.119 & 7.120). Acetylation of histones is thought to be responsible for loosening the interaction between histones and the DNA in nucleosomes and helps to make the DNA more readily accessible for transcription. The opposite effect may be achieved if the enzymes recruited are histone deacetylases (HDAC) which remove acetyl groups from the tails of the histones in the nucleosome, and lead to tighter packing of the chromatin. Writers, readers and erasers In addition to the histone acetyl transferases and the deacetylases, other enzymes may add or remove methyl groups, phosphate groups, and other chemical moieties to specific amino acid side chains on the histone tails. The patterns of these covalent modifications, sometimes called the histone code, are established by the so-called "writers", or enzymes, such as histone methyltransferases, that add the chemical groups on to the histone tails. Yet other enzymes, like the histone demethylases, may act as "erasers," removing the chemical groups added by the "writers." The histone code is interpreted by "readers," proteins that bind to specific combinations of the modifications and assist in either silencing the expression of genes in the vicinity or making the region more transcriptionally active. DNA methylation Gene expression can also be regulated by methylation of the other component of chromatin - DNA. Enzymes called DNA methyltransferases (DNMTs) catalyze the covalent addition of a methyl group to C5 of cytosines in DNA. Patterns of cytosine methylation vary in different organisms, with methylation concentrated in some parts of the genome in some groups and scattered throughout the genome in others. In vertebrates, the cytosines that are methylated are generally next to a guanine (the CG dinucleotide is commonly abbreviated as CpG). Methylation of DNA seems to correlate with gene silencing while demethylation is associated with increased transcription (Figure 7.121). How does methylation of the DNA at CpG sites regulate gene expression? Although the extent of DNA methylation near promoters has been observed to correlate with gene silencing, it is not clear how exactly methylation brings about this effect. It has been suggested that methylation could block the binding of proteins necessary for transcription. Methylation at enhancer sites might also prevent the binding of transcriptional activators to them. Another interesting observation is that certain proteins that bind to methylated CpG sites also seem to interact with histone deacetylases. As noted above, histones deacetylases remove acetyl groups from histones, and promote tighter packing of chromatin and transcriptional silencing. Thus, methylation on DNA likely works in combination with histone modification to affect gene expression. Regulatory RNAs One of the most unexpected discoveries in the past few decades has been the role that RNAs play in regulating gene expression. The classic view that RNA either encoded proteins (mRNA) or assisted in their synthesis (rRNA and tRNA) is now known to be a vast underestimate of the various ways in which RNAs function in gene expression. It is now clear that regulatory RNAs have widespread and significant effects on gene expression, a realization that has revolutionized our understanding of gene regulation. What are some of the ways in which regulatory RNAs function to modulate the expression of genes? Small regulatory RNAs MicroRNAs (miRNAs) and Short Interfering RNAs (siRNAs) are small, non-coding RNAs that act at the post-transcriptional level to regulate gene expression (Figure 7.123 & 7.124). These RNAs appear to silence genes by base-pairing with target mRNAs and marking them for degradation, or by blocking their translation. The functional forms of both miRNAs and siRNAs are from 20-30 nucleotides long and are derived by processing from longer primary transcripts. Mature miRNAs and siRNAs work in association with a class of proteins called Argonaute proteins to form a gene silencing complex. MicroRNAs are transcribed from specific genes by RNA polymerase II. The primary transcript, known as a pri-miRNA folds on itself to form double-stranded hairpin structures that are cleaved by an RNase in the nucleus called Drosha. The products of Drosha cleavage, double-stranded RNAs of roughly 60-70 nucleotides known as pre-miRNAs, are exported to the cytoplasm, where they are further processed into the small 20-30 nucleotide lengths of mature double-stranded miRNAs by an enzyme known as Dicer. The RNA duplexes of miRNAs are not perfectly matched, and have loops and mismatches (Figure 7.124). siRNAs also derive from double-stranded RNAs, but these may arise from either endogenous or exogenous sources (such as viruses). These double-stranded RNAs are processed in the cytoplasm by the same enzyme, Dicer, that generates the mature miRNAs, to produce the small, 20-30 nucleotide double-stranded RNAs. In contrast to miRNAs, the mature siRNAs are perfectly base-paired along their lengths. RISC assembly Both miRNAs and siRNAs then are assembled with Argonaute proteins to form a silencing complex called RISC (RNA-induced silencing complex). Recall that both miRNAs and siRNAs are, at this point double-stranded. One strand of the RNA is referred to as the guide RNA, while the other is called the passenger RNA. During the process of loading the RNA onto the Argonaute protein, the guide strand of the RNA remains associated with the protein, while the passenger strand is removed. The guide RNA associated with the Argonaute protein is the functional gene silencing complex (Figure 7.125). Sequence specific base-pairing of the guide RNA with an mRNA leads to either the degradation of the mRNA by the Argonaute protein (in the case of the siRNAs) or in suppression of translation of the mRNA (for miRNAs). The extent to which these processes play a role in regulating gene expression is impressive. The expression of at least a third of all human genes has already been shown to be modulated by miRNAs, demonstrating clearly that these RNAs play a major role in gene regulation. Long noncoding RNAs Long noncoding RNAs (lncRNAs) are RNAs of greater than 200 nucleotides that do not code for proteins. Some of these RNAs are derived from intron sequences, while others, transcribed from intergenic regions form a subset of lncRNAs called lincRNAs (long intergenic noncoding RNAs). Yet other lncRNAs are produced as antisense transcripts of coding genes. An astounding 30,000 transcripts in humans are thought to be lncRNAs, but little is known of their function. From the few lncRNAs that have been intensively studied, it is evident that they do not all function in the same way. However, they appear to affect gene expression in a variety of ways including modification of chromatin structure, regulation of splicing, or serving as structural scaffolds for the assembly of nucleoprotein complexes. Additional mechanisms will doubtless be uncovered as these fascinating RNAs are investigated in years to come. Regulation of translation The synthesis of proteins is dependent on the availability of the mRNAs encoding them. If an mRNA is blocked at its 5' end, it cannot be translated. The rate of degradation of an mRNA will influence how long it is around to direct the synthesis of the protein it codes for. Gene expression can also, therefore, be regulated by mechanisms that alter the rate of mRNA degradation. Regulation of translation is used to control the production of many proteins. Two examples, ferritin and the transferrin receptor, are important for iron storage and transport in cells. Ferritin is an iron-binding protein that sequesters iron atoms in cells to keep them from reacting. When iron levels are high, there is a need for more ferritin than when iron levels are low. How are ferritin levels regulated? The 5'UTR of the ferritin mRNA contains a 28-nucleotide sequence called the Iron Response Element, or IRE (Figure 7.127). When iron levels are low, the IRE is bound by a protein. The presence of the IRE-binding protein at the 5'UTR blocks translation of the ferritin mRNA. However, if iron levels are high, the iron binds to the IRE-binding protein, which undergoes a conformational change and dissociates from the IRE. This frees up the 5' end of the ferritin mRNA for ribosome assembly and translation, producing more ferritin. The other protein involved in iron transport, the transferrin receptor, is required for uptake of iron into cells, when intracellular iron levels are low. In the case of the transferrin receptor, it is when iron levels are low that more of it is needed. When iron levels are high, there is no need to make more transferrin receptor. The mRNA encoding the transferrin receptor also has IRE sequences, but in this case, the IRE is situated in the 3'UTR of the transcript (Figure 7.128). The IRE is, as in the case of ferritin, bound by the IRE-binding protein. When iron levels in the cell are high, the iron binds the IRE-binding protein, which dissociates from the IRE. This leaves the 3'UTR susceptible to attack by RNases, leading to degradation of the transferrin receptor mRNA. At times when iron levels are low, the IRE-binding protein remains bound to the 3' UTR of the mRNA, stabilizing it and permitting more transferrin receptor to be made by translation. Gene expression is controlled at many steps As can be seen from the examples in this section, regulation of gene expression in eukaryotic cells is a function of multiple mechanisms that act at different stages in the flow of information from DNA to protein, responding to the internal state of the cell as well as external conditions and signals. Information Processing: Gene Expression 803 YouTube Lectures by Kevin HERE & HERE 804 Figure 7.102 - Multiple levels of control of gene expression Wikipedia 805 Figure 7.103 - Prokaryotic genes organized in an operon Wikipedia Figure 7.104 - Protein binding sites in the lac regulatory region Image by Martha Baker Interactive Learning Module HERE 806 Figure 7.105 - Lac operon structure and products Image by Martha Baker Figure 7.106 - Lac operon in the absence (middle) and presence (bottom) of inducer Image by Martha Baker 807 Figure 7.107 - Allolactose (top) and lactose (bottom) Figure 7.108 - CAP (blue) bound to the DNA adjacent to the lac promoter (orange). cAMP shown in pink. Wikipedia Figure 7.109 - Lac operon in the presence (top) and absence (bottom) of glucose Image by Martha Baker 808 Figure 7.110 - Structure and regulation of the trp operon Wikipedia YouTube Lectures by Kevin HERE & HERE 809 Figure 7.111 - Attenuation in regulation of the trp operon Wikipedia Figure 7.112 - Sequence of the leader region of the trp operon XX AUG AAA GCA AUU UUC GUA CUG AAA GGU UGG UGG CGC ACU UCC UGA -XX MET LYS ALA ILE PHE VAL LEU LYS GLY TRP TRP ARG THR SER STOP 810 Figure 7.113 - Riboswitch features 811 Figure 7.114 - Regulatory sequences for a eukaryotic gene Wikipedia 812 Figure 7.115 - DNA looping allows contact between activator bound at a distant enhancer and the basal transcription complex Image by Martha Baker 813 Figure 7.116 - Transcription factors in regulation of eukaryotic transcription Wikipedia YouTube Lectures by Kevin HERE & HERE 814 Figure 7.117 - Binding of c-myc protein to its target DNA sequence Wikipedia Figure 7.118 Activators bound at multiple sites can regulate transcription from a given promoter OpenStax 815 Figure 7.119 - Transcriptional activation (right) and deactivation (left) by histone modification Wikipedia 816 Figure 7.120 - Chromatin configuration affects transcription Wikipedia Interactive Learning Module HERE 817 Figure 7.121 - Inactivation of transcription by CpG methylation Image by Indira Rajagopal YouTube Lectures by Kevin HERE & HERE 818 Figure 7.122 - Epigenetic changes through histone and DNA modification 819 Figure 7.123 - miRNAs function in the regulation of gene expression Wikipedia Figure 7.124 Pre-miRNA hairpin structures with the mature guide miRNAs shown in red Wikipedia 820 Figure 7.125 - Gene silencing by siRNA Image by Pehr Jacobson 821 Figure 7.126 - Processed siRNA duplex with perfect base-pairing, 5’ phosphates and two bases overhanging at each 3’ end 822 Figure 7.127 -Regulation of ferritin mRNA translation Image by Aleia Kim YouTube Lectures by Kevin HERE & HERE 823 Figure 7.128 -Regulation of transferrin receptor mRNA translation Image by Aleia Kim Graphic images in this book were products of the work of several talented students. Links to their Web pages are below Click HERE for Martha Baker’s Web Page Click HERE for Pehr Jacobson’s Web Page Click HERE for Aleia Kim’s Web Page Click HERE for Penelope Irving’s Web Page Problem set related to this section HERE Point by Point summary of this section HERE To get a certificate for mastering this section of the book, click HERE Kevin Ahern’s free iTunes U Courses - Basic / Med School / Advanced Biochemistry Free & Easy (our other book) HERE / Facebook Page Kevin and Indira’s Guide to Getting into Medical School - iTunes U Course / Book To see Kevin Ahern’s OSU ecampus courses - BB 350 / BB 450 / BB 451 To register for Kevin Ahern’s OSU ecampus courses - BB 350 / BB 450 / BB 451 Biochemistry Free For All Facebook Page (please like us) Kevin Ahern’s Web Page / Facebook Page / Taralyn Tan’s Web Page Kevin Ahern’s free downloads HERE OSU’s Biochemistry/Biophysics program HERE OSU’s College of Science HERE Oregon State University HERE Email Kevin Ahern / Indira Rajagopal / Taralyn Tan God Bless These Complexes To the tune of “God Bless America” Metabolic Melodies Website HERE All information in Cells’ DNA Just increases With pieces Mixed and matched in the mRNAs Linking exons All together Using snurps in Complex-ES God bless the spliceosomes And trans-crip-tomes (slow and loud) God bless the spliceosomes And my ge-nome Your blueprint info is In DNA Since you need it Proofread it Or you’ll mutate the mRNA You can translate All the codons With the cells’ gen- et-ic code God bless the ribosomes They translate code (slow and loud) God bless the ribosomes And proteomes Recording by David Simmons Lyrics by Kevin Ahern Recording by David Simmons Lyrics by Kevin Ahern The Book of Life To the tune of “The Look of Love” Metabolic Melodies Website HERE The book of life - the stuff of dreams Is everywhere, it seems The book of life, is biochemistry and Its words fill every day Just what it says is written in the DNA I just want to get to know it How the info’s coded What are all the secrets? Ribosomes can read it Goodness knows it’s needed And so its alphabet’s In codon forms For ribosome bookworms They read it right A protein’s function to its sequence corresponds It’s not just randomly created peptide bonds What a marvel of creation, how they do translation Of m-R-N-A chains, Using bits of glycine Proline and some lysine Translate the code Instrumental I just marvel at the knowledge That I got in college To learn all the secrets Double helix spaces Complementary bases Pyrimidines Paired to purines The book of life Recording by Carol Adriane Smith Lyrics by Kevin Ahern Recording by Carol Adriane Smith Lyrics by Kevin Ahern

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/07%3A_Information_Processing/7.08%3A_Gene_Expression.txt

Source: BiochemFFA_7_8.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy Up to this point we have considered how cells carry out biochemical reactions and how they regulate the expression of the genes in response to their internal and external environments. It is intuitively obvious that even unicellular organisms must be able to sense features of their environment, such as the presence of nutrients, if they are to survive. In addition to being able to receive and respond to information from the environment, multicellular organisms must also find ways by which their cells can communicate among themselves. Coordination Since different cells take on specialized functions in a multicellular organism, they must be able to coordinate activities. Cells grow, divide, or differentiate in response to specific signals. They may change shape or migrate to another location. At the physiological level, cells in a multicellular organism, must respond to everything from a meal just eaten to injury, threat, or the availability of a mate. They must know when to divide, when to undergo apoptosis (programmed cell death), when to store food, and when to break it down. A variety of mechanisms have arisen to ensure that cell-cell communication is not only possible, but astonishingly swift, accurate and reliable. How are signals sent between cells? Like pretty much everything that happens in cells, signaling is dependent on molecular recognition. The basic principle of cell-cell signaling is simple. A particular kind of molecule, sent by a signaling cell, is recognized and bound by a receptor protein in (or on the surface of) the target cell. The signal molecules are chemically varied- they may be proteins, short peptides, lipids, nucleotides or catecholamines, to name a few. Signal properties The chemical properties of the signal determine whether its receptors are on the cell surface or intracellular. If the signal is small and hydrophobic it can cross the cell membrane and bind a receptor inside the cell. If, on the other hand, the signal is charged, or very large, it would not be able to diffuse through the plasma membrane. Such signals need receptors on the cell surface, typically transmembrane proteins that have an extracellular portion that binds the signal and an intracellular part that passes on the message within the cell (Figure 7.130). Receptors are specific for each type of signal, so each cell has many different kinds of receptors that can recognize and bind the many signals it receives. Because different cells have different sets of receptors, they respond to different signals or combinations of signals. The binding of a signal molecule to a receptor sets off a chain of events in the target cell. These events could cause change in various ways, including, but not limited to, alterations in metabolic pathways or gene expression in the target cell. How the binding of a signal to a receptor brings about change in cells is the topic of this section. We will examine a few of the major receptor types and the consequences of signal binding to these receptors. Although the specific molecular components of the various signal transduction pathways differ, they all have some features in common (Figure 7.131): ​ • The binding of a signal to its receptor is usually followed by the generation of a new signal(s) within the cell. The process by which the original signal is converted to a different form and passed on within the cell to bring about change is called signal transduction. ​ • Most signaling pathways have multiple signal transduction steps by which the signal is relayed through a series of molecular messengers that can amplify and distribute the message to various parts of the cell. ​ • The last of these messengers usually interacts with a target protein(s) and changes its activity, often by phosphorylation.​ • When a signal sets a particular pathway in motion, it is acting like an ON switch. This means that once the desired result has been obtained, the cell must have a mechanism that acts as an OFF switch. Understanding this underlying similarity is helpful, because learning the details of the different pathways becomes merely a matter of identifying which molecular component performs a particular function in each individual case. We will consider several different signal transduction pathways, each mediated by a different kind of receptor. Ligand-gated ion channel receptors The simplest and fastest of signal pathways is seen in the case of signals whose receptors are gated ion channels (Figure 7.132). Gated ion channels are made up of multiple transmembrane proteins that create a pore, or channel, in the cell membrane. Depending upon its type, each ion channel is specific to the passage of a particular ionic species. The term "gated" refers to the fact that the ion channel is controlled by a "gate" which must be opened to allow the ions through. The gates are opened by the binding of an incoming signal (ligand) to the receptor, allowing the almost instantaneous passage of millions of ions from one side of the membrane to the other. Changes in the interior environment of the cell are thus brought about in microseconds and in a single step. Swift response This type of swift response is seen, for example, in neuromuscular junctions, where muscle cells respond to a message from the neighboring nerve cell (Figure 7.133). The nerve cell releases a neurotransmitter signal into the synaptic cleft, which is the space between the nerve cell and the muscle cell it is "talking to". An example of such a neurotransmitter signal is acetylcholine. When the acetylcholine molecules are released into the synaptic cleft, they diffuse rapidly till they reach their receptors on the membrane of the muscle cell. The binding of the acetylcholine to its receptor, an ion channel on the membrane of the muscle cell, causes the gate in the ion channel to open. The resulting ion flow through the channel can immediately change the membrane potential of the cell. This, in turn, can trigger other changes in the cell. The speed with which changes are brought about in neurotransmitter signaling is evident when you think about how quickly you remove your hand from a hot surface. Sensory neurons carry information to the brain from your hand on the hot surface and motor neurons signal to your muscles to move the hand, in less time than it took you to read this sentence! Nuclear hormone receptors The receptors for signals like steroid hormones are part of a large group of proteins known as the nuclear hormone receptor superfamily. These receptors recognize and bind not only steroid hormones, but also retinoic acid, thyroid hormone, vitamin D and other signals. The subset of the nuclear hormone receptors that bind steroid hormones are intracellular proteins. Steroid hormones (Figure 7.135), as you are aware, are related to cholesterol, and as hydrophobic molecules, they are able to cross the cell membrane by themselves. This is unusual, as most signals coming to cells are incapable of crossing the plasma membrane, and thus, must have cell surface receptors. Once within the cell, steroid hormones bind to their receptors, which may reside in the cytoplasm or in the nucleus. Steroid hormone receptors are proteins with a double life: they are actually dormant transcription regulators that are inactive till a steroid hormone binds and causes a conformational change in them. When this happens, the receptors, with the hormone bound, bind to regulatory sequences in the DNA and modulate gene expression. Because steroid hormone receptors act by modulating gene expression, the responses to steroid hormones are relatively slow. (There are also some effects of steroid hormones that do not involve transcriptional regulation, but the majority work through changing gene expression.) Like other transcriptional activators, steroid receptors have a DNA-binding domain (DBD) and an activation domain. They also have a ligand-binding domain (LBD) that binds the hormone. Glucocorticoid receptor Examples of such signaling pathways are those mediated by the glucocorticoid receptor (Figures 7.136 & 7.137). Glucocorticoids, sometimes described as stress hormones, are made and secreted by the adrenal cortex. Physiologically, they serve to maintain homeostasis in the face of stress and exhibit strong anti-inflammatory and immunosuppressive properties. Because of these effects, synthetic glucocorticoids are used in the treatment of a number of diseases from asthma and rheumatoid arthritis to multiple sclerosis. All of these effects are mediated through the signaling pathway which starts with the binding of a glucocorticoid hormone to its receptor. Recall that steroids can cross the plasma membrane, so glucocorticoids can diffuse into the cell and bind their receptors which are in the cytoplasm. In the absence of the signal, glucocorticoid receptors are found bound to a protein chaperone, Hsp90 (Figure 7.137). This keeps the receptors from being transported to the nucleus. When a glucocorticoid molecule binds the receptor, the receptor undergoes a conformational change and dissociates from the Hsp90. The receptor, then, with the hormone bound, translocates into the nucleus. In the nucleus, it can increase the transcription of target genes by binding to specific regulatory sequences (labeled HRE for hormone-response elements). The binding of the hormone-receptor complex to the regulatory elements of hormone-responsive genes modulates their expression. Many of these genes encode anti-inflammatory proteins, and their increased production accounts for the physiological effect of corticosteroid therapies. The steroid receptor pathways are relatively simple and have only a couple of steps (Figure 7.138). Most other signaling pathways involve multiple steps in which the original signal is passed on and amplified through a number of intermediate steps, before the cell responds to the signal. Cell surface receptors We will now take a look at two signaling pathways, each mediated by a major class of cell surface receptor- the G-protein coupled receptors (GPCRs) and the receptor tyrosine kinases (RTKs). While the specific details of the signaling pathways that follow the binding of signals to each of these receptor types are different, it is easier to learn them when you can see what the pathways have in common, namely, interaction of the signal with a receptor, followed by relaying and amplification of the signal through a variable number of intermediate molecules, with the last of these molecules interacting with a target or target proteins and modifying their activity in the cell. G-protein coupled receptors G-protein coupled receptors (GPCRs) are involved in responses of cells to many different kinds of signals, from epinephrine, to odors, to light. In fact, a variety of physiological phenomena including vision, taste, smell, and the fight-or-flight response are mediated by GPCRs. What are G-protein coupled receptors? G-protein coupled receptors are cell surface receptors that pass on the signals that they receive with the help of guanine nucleotide binding proteins (a.k.a. G-proteins). Before thinking any further about the signaling pathways downstream of GPCRs, it is necessary to know a few important facts about these receptors and the G-proteins that assist them. Though there are hundreds of different G-protein coupled receptors, they all have the same basic structure (Figure 7.139): They all consist of a single polypeptide chain that threads back and forth seven times through the lipid bilayer of the plasma membrane. For this reason, they are sometimes called seven-pass transmembrane (7TM) receptors. One end of the polypeptide forms the extracellular domain that binds the signal while the other end is in the cytosol of the cell. When a ligand (signal) binds the extracellular domain of a GPCR, the receptor undergoes a conformational change, on its cytoplasmic side, that allows it to interact with a G-protein that will then pass the signal on to other intermediates in the signaling pathway. G-proteins What is a G-protein? As noted above, a G-protein is a guanine nucleotide-binding protein that can interact with a G-protein linked receptor. G-proteins are associated with the cytosolic side of the plasma membrane, where they are ideally situated to interact with the tail of the GPCR, when a signal binds to the GPCR. There are many different G-proteins, all of which share a characteristic structure- they are composed of three subunits called α, β and γ (Figure 7.140). Because of this, they are sometimes called heterotrimeric G proteins (hetero=different, trimeric= having three parts). Ligand binding The guanine nucleotide binding site is on the α subunit of the G-protein. This site can bind GDP or GTP. The α subunit also has a GTPase activity, i.e., it is capable of hydrolyzing a GTP molecule bound to it into GDP. In the unstimulated state of the cell, that is, in the absence of a signal bound to the GPCR, the G-proteins are found in the trimeric form (α-β-γ bound together) and the α subunit has a GDP molecule bound to it. In this form, the α subunit is inactive. With this background on the structure and general properties of the GPCRs and the G-proteins, we can now look at what happens when a signal arrives at the cell surface and binds to a GPCR (Figure 7.141). The signaling pathway The binding of a signal molecule by the extracellular part of the G-protein linked receptor causes the cytosolic tail of the receptor to interact with, and alter the conformation of, a G-protein associated with the inner face of the plasma membrane.​ This has two consequences. First, the α subunit of the G-protein loses its GDP and binds a GTP, instead. Second, the G-protein breaks up into the GTP-bound α part and the β-γ part. The binding of GTP to the α subunit and its dissociation from the β-γ subunits activate the α subunit. The activated α subunit can diffuse freely along the cytosolic face of the plasma membrane and act upon its targets. (The β-γ unit is also capable of activating its own targets.) What happens when G-proteins interact with their target proteins? That depends on what the target is. G-proteins interact with different kinds of target proteins, of which we will examine two major categories: Ion channels We have earlier seen that some gated ion channels can be opened or closed by the direct binding of neurotransmitters to a receptor that is an ion-channel protein. In other cases, ion channels are regulated by the binding of G-proteins. That is, instead of the signal directly binding to the ion channel, it binds to a GPCR, which activates a G-protein that then may cause opening of the ion channel, either directly, by binding to the channel, or indirectly, through activating other proteins that can bind to the channel. The change in the distribution of ions across the plasma membrane causes a change in the membrane potential. Enzyme activation The interaction of G-proteins with their target enzymes can regulate the activity of the enzyme, either increasing or decreasing its activity. The change in activity of the target enzyme, in turn, results in downstream changes in other proteins in the cell, and alters the metabolic state of the cell. This is best understood by examining the well-studied response of cells to epinephrine, mediated through the β-adrenergic receptor, a type of G-protein coupled receptor. Epinephrine (Figure 7.142), also known as adrenaline, is a catecholamine that plays an important role in the body's 'fight or flight' response. In response to stressful stimuli, epinephrine is secreted into the blood, to be carried to target organs whose cells will respond to this signal. If you were walking down a dark alley in an iffy neighborhood, and you heard footsteps behind you, your brain would respond to potential danger by sending signals that ultimately cause the adrenal cortex to secrete epinephrine into the blood stream. The epinephrine circulating in your system has many effects, including increasing your heart rate, but among its prime targets are your muscle cells. The reason for this is that your muscle cells store energy in the form of glycogen, a polymer of glucose. If you need to run or fight off an assailant, your cells will need energy in the form of glucose. But how does epinephrine get your cells to break down the glycogen into glucose? Binding of epinephrine to the β-adrenergic receptor on the surface of the cells causes the receptor to activate a G-protein associated with its cytoplasmic tail. As described above, this leads to the α subunit exchanging its GDP for GTP and dissociating from the β-γ subunits. The activated α subunit then interacts with the enzyme adenylate cyclase (also known as adenylyl cyclase) stimulating it to produce cyclic AMP (cAMP) from ATP. Cyclic AMP is often described as a "second messenger", in that it serves to spread the signal received by the cell. How does cAMP accomplish this? cAMP molecules bind to, and activate an enzyme, protein kinase A (PKA - Figure 7.145). PKA is composed of two catalytic and two regulatory subunits that are bound tightly together. Upon binding of cAMP, the catalytic subunits are released from the regulatory subunits, allowing the enzyme to carry out its function, namely phosphorylating other proteins. Thus, cAMP can regulate the activity of PKA, which in turn, by phosphorylating other proteins can change their activity. In this case, the relevant protein that is activated is an enzyme, phosphorylase kinase. This enzyme can then phosphorylate and activate glycogen phosphorylase, the enzyme ultimately responsible for breaking glycogen down into glucose-1-phosphate - readily converted to glucose. The activation of glycogen phosphorylase supplies the cells with the glucose they need, allowing you to fight or flee, as you might see fit. Simultaneously, PKA also phosphorylates another enzyme, glycogen synthase. In the case of glycogen synthase, phosphorylation inactivates it, and prevents free glucose from being used up for glycogen synthesis, ensuring that your cells are amply supplied with glucose (Figure 7.146). Common pattern Although the steps described above seem complicated, they follow the simple pattern outlined at the beginning of this section:​ • Binding of signal to receptor​ • Several steps where the signal is passed on through intermediate molecules (G-proteins, adenylate cyclase, cAMP, and finally, PKA)​ • Phosphorylation of target proteins by the kinase, leading to changes in the cell. The specific changes depend on the proteins that are phosphorylated by the PKA. Why so many steps? If you need to activate glycogen phosphorylase to break down glucose in a hurry, why not have a system in which binding of a signal to the receptor directly activated the target enzyme? The answer to this puzzle is simple: there is amplification of the signal at every step of the pathway. A single signal molecule binding to a receptor sets in motion a cascade of reactions, with the signal getting larger at each step, so that binding of one epinephrine molecule to its receptor results in the activation of a million glycogen phosphorylase enzyme molecules! Turning signals off If the signal binding to the receptor serves as a switch that sets these events in motion, there must be mechanisms to turn the pathway off. The first is at the level of the receptor itself. A kinase called G-protein receptor kinase (GRK) phosphorylates the cytoplasmic tail of the receptor. The phosphorylated tail is then bound by a protein called arrestin, preventing further interaction with a G-protein. The next point of control is at the G-protein. Recall that the α subunit of the G-protein is in its free and activated state when it has GTP bound, and that it associates with the β-γ subunits and has a GDP bound when it is inactive. We also know that the α subunit has an activity that enables it to hydrolyze GTP to GDP. This GTP-hydrolyzing activity makes it possible for the α subunit, once it has completed its task, to return to its GDP bound state, re-associate with the β-γ part and become inactive again. A third "off switch" is further down the signaling pathway, and controls the level of cAMP. We just noted that cAMP levels increase when adenylate cyclase is activated. When its job is done, cAMP is broken down by an enzyme called phosphodiesterase (Figure 7.147). When cAMP levels drop, PKA returns to its inactive state, putting a halt to the changes brought about by the activation of adenylate cyclase by an activated G-protein. Yet another way that the effects of this pathway can be turned off is at the level of the phosphorylated target proteins. These proteins, which are activated by phosphorylation, can be returned to their inactive state by the removal of the phosphates by phosphatases. Receptor tyrosine kinases Another major class of cell surface receptors are the receptor tyrosine kinases or RTKs. Like the GPCRs, receptor tyrosine kinases bind a signal, then pass the message on through a series of intracellular molecules, the last of which acts on target proteins to change the state of the cell. As the name suggests, a receptor tyrosine kinase is a cell surface receptor that also has a tyrosine kinase activity. The signal binding domain of the receptor tyrosine kinase is on the cell surface, while the tyrosine kinase enzymatic activity resides in the cytoplasmic part of the protein (Figure 7.148). A transmembrane α helix connects these two regions of the receptor. What happens when signal molecules bind to receptor tyrosine kinases? Binding of signal molecules to the extracellular domains of receptor tyrosine kinase proteins causes two receptor molecules to dimerize (come together and associate - Figure 7.149). This brings the cytoplasmic tails of the receptors close to each other and causes the tyrosine kinase activity of these tails to be turned on. The activated tails then phosphorylate each other on several tyrosine residues (Figure 7.150). This is called autophosphorylation. The phosphorylation of tyrosines on the receptor tails triggers the assembly of an intracellular signaling complex on the tails. The newly phosphorylated tyrosines serve as binding sites for a variety of signaling proteins that then pass the message on to yet other proteins to bring about changes in the cell. Receptor tyrosine kinases mediate responses to a large number of signals, including peptide hormones like insulin and growth factors like epidermal growth factor (EGF). We will examine how insulin and EGF act on cells by binding to receptor tyrosine kinases. Insulin receptor Insulin plays a central role in the uptake of glucose from the bloodstream. It increases glucose uptake by stimulating the movement of glucose receptor GLUT4 to the plasma membrane of cells. How does insulin increase GLUT4 concentrations in the cell membrane? The binding of insulin to the insulin receptor (IR - Figure 7.151), results in dimerization of the receptor monomers and subsequent autophosphorylation of the cytosolic kinase domains. The activated tyrosine kinase domains also phosphorylate intracellular proteins called Insulin Receptor Substrates or IRS proteins. These proteins interact with, and activate another kinase called the PI3-kinase. PI3-kinase then catalyzes the formation of the lipid molecule PIP3, which serves to activate yet another kinase, PDK1, which in turn, activates the Akt group of kinases. It is this group of enzymes that appears to increase the translocation of the GLUT4 to the plasma membrane (Figure 7.152), as cells that lack functional Akts exhibit poor glucose uptake and insulin resistance. EGFR pathway Epidermal growth factor, EGF, is an important signaling molecule involved in growth, proliferation and differentiation in mammalian cells. EGF acts through the EGF receptor, EGFR, a receptor tyrosine kinase (Figure 7.153). Because of its role in stimulating cell proliferation and because overexpression of EGFR is associated with some kinds of cancers, EGFR is the target for many anti-cancer therapies. We can trace the signal transduction pathway from the binding of EGF to its receptor to the stimulation of cell division. EGF binding to the EGFR is followed by receptor dimerization and stimulation of the tyrosine kinase activity of the cytosolic domains of the EGFR. Autophosphorylation of the receptor tails is followed by the assembly of a signaling complex nucleated by the binding of proteins that recognize phosphotyrosine residues. An important protein that is subsequently activated by the signaling complexes on the receptor tyrosine kinases is called Ras (Figure 7.154). The Ras protein is a monomeric guanine nucleotide binding protein that is associated with the cytosolic face of the plasma membrane​ (in fact, it is a lot like the α subunit of trimeric G-proteins). Just like the α subunit of a G-protein, Ras is active when GTP is bound to it and inactive when GDP is bound to it. Also, like the α subunit, Ras can hydrolyze the GTP to GDP. Ras activation Activation of Ras accompanies the exchange of the GDP bound to the inactive Ras for a GTP. Activated Ras triggers a phosphorylation cascade of three protein kinases, which relay and distribute the signal. These protein kinases are members of a group called the MAP kinases (Mitogen Activated Protein Kinases). The final kinase in this cascade phosphorylates various target proteins, including enzymes and transcriptional activators that regulate gene expression. The phosphorylation of various enzymes can alter their activities, and set off new chemical reactions in the cell, while the phosphorylation of transcriptional activators can change which genes are expressed. The combined effect of changes in gene expression and protein activity alter the cell's physiological state and promote cell division. Once again, in following the path of signal transduction mediated by RTKs, it is possible to discern the same basic pattern of events: a signal is bound by the extracellular domains of receptor tyrosine kinases, resulting in receptor dimerization and autophosphorylation of the cytosolic tails, thus conveying the message to the interior of the cell. The message is then passed on via a signaling complex to proteins that stimulate a series of kinases. The terminal kinase in the cascade acts on target proteins and brings about in changes in protein activities. What is the OFF switch for RTKs? It turns out that RTKs with the signal bound can be endocytosed into the cell and broken down. That is, the region of the plasma membrane that the RTK is on can be internally pinched off into a vesicle containing the ligand-bound receptor which is then targeted for degradation. Ras, which is activated by GTP binding, can also be deactivated by hydrolysis of the GTP to GDP. The importance of this mechanism for shutting down the pathway is evident in cells that have a mutant ras gene encoding a Ras protein with defective GTPase activity. Unable to shut off Ras, the cells continue to receive a signal to proliferate. The National Cancer Institute estimates that more than 30% of human cancers are driven by mutations in ras genes. The descriptions above provide a very simple sketch of some of the major classes of receptors and deal primarily with the mechanistic details of the steps by which signals received by various types of receptors bring about changes in cells. A major take-home lesson is the essential similarity of the different pathways. Another point to keep in mind is that while we have looked at each individual pathway in isolation, a cell, at any given time receives multiple signals that set off a variety of different responses at once (Figure 7.155). The pathways described above show a considerable degree of "cross-talk" and the response to any given signal is affected by the other signals that the cell receives simultaneously. The multitude of different receptors, signals, and the combinations thereof are the means by which cells are able to respond to an enormous variety of different circumstances. RTKs, cancer and cancer therapies As described above, binding of EGF to its receptor triggers a signaling pathway that results in the activation of a series of Mitogen Activated Protein Kinases (MAP kinases). These kinases are so-called because they are activated by a mitogen, a molecule, like EGF and other growth factors, that stimulates mitosis or cell division. The final kinase in the MAP kinase cascade phosphorylates a number of target proteins, many of them transcription factors, that when activated, increase the expression of genes associated with cell proliferation. Given that the EGF-receptor pathway normally functions to stimulate cell division, it is not surprising that malfunctions in the pathway could lead to uncontrolled cell proliferation, or cancer. Next, we will take a brief look at some examples of such defects. HER2 The human EGF receptor (HER) family has four members, HER1, HER2, HER3 and HER4. These are all receptor tyrosine kinases, cell surface receptors that bind EGF (Figure 7.157) and stimulate cell proliferation. A crucial step in the signal transduction pathway is the dimerization of the receptors following binding of the signal, EGF, to the receptor. While HER1, HER3 and HER4 must bind the signal to dimerize, the structure of the HER2 receptor can, apparently, allow the receptor monomers to dimerize independently of EGF binding. This means that the downstream events of the signaling pathway can be triggered even in the absence of a growth signal. In normal cells, only a few HER2 receptors are expressed at the cell surface, so this property of HER2 plays a relatively minor role in stimulating cell division. However, in about a quarter of breast cancer patients, HER2 receptors are overexpressed, leading to increased dimerization and subsequent uncontrolled cell proliferation. Breast cancers that are HER2-positive can be more aggressive with a greater tendency to metastasize (spread) so therapy that blocks HER2 signaling is key in successful treatment of such cancers. Herceptin, a monoclonal antibody against the HER2 receptor, has been shown to be an effective treatment against Her2-positive breast cancers. Herceptin works by binding specifically to the extracellular domain of the HER2 receptor (Figure 7.158). This prevents dimerization of the receptor and thus blocks downstream signaling. Additionally, the binding of the Herceptin antibody to the receptor signals the immune system to destroy the HER2-positive cells. Bcr-abl Another example of a cancer caused by defects in an RTK signaling pathway is chronic myeloid leukemia (CML). Patients with CML have an abnormal receptor tyrosine kinase that is the product of a hybrid gene called bcr-abl, formed by the breakage and rejoining of chromosomes 9 and 22. This abnormal tyrosine kinase is constitutively dimerized, even when no signal is bound. As a result, it continuously signals cells to divide, leading to the massive proliferation of a type of blood cells called granulocytes. As with HER2, the problem in CML is a receptor tyrosine kinase that dimerizes in the absence of a growth signal. The approach in this case was to target the next step in the signaling pathway. As you know, dimerization of RTKs activates the tyrosine kinase domain of the receptor, which results in the autophosphorylation of the cytoplasmic domains of both monomers. The phosphorylated tyrosines serve to recruit a number of other signaling proteins that pass the signal on within the cell. In the case of the bcr-abl RTK, the drug Gleevec (imatinib) was designed to bind near the ATP-binding site of the tyrosine kinase domain. This "locks" the site in a conformation that inhibits the enzymatic activity of the tyrosine kinase and thus blocks downstream signaling. With no "grow" signal passed on, cells stop proliferating. Information Processing: Signaling 827 YouTube Lectures by Kevin HERE & HERE 828 Figure 7.130 - Schematic representation of a transmembrane receptor protein. E = extracellular; P = plasma membrane; I = intracellular Wikipedia Figure 7.129 - Some examples of signal molecules 829 Figure 7.132 - Ligand-gated ion channel receptor opening in response to a signal (ligand) Wikipedia Figure 7.131 -General features of signal transduction pathways 830 Figure 7.133 - Neuromuscular signaling - A = motor neuron axon; B = axon terminal; C = synaptic cleft; D = muscle cell; E = myofibril . Steps in the process - 1) action potential reaches the axon terminal; 2) voltage-dependent calcium gates open; (3) neurotransmitter vesicles fuse with the presynaptic membrane and acetylcholine (ACh) released into the synaptic cleft; (4) ACh binds to postsynaptic receptors on the sarcolemma; (5) ACh binding causes ion channels to open and allows sodium ions to flow across the membrane into the muscle cell; 6) flow of sodium ions across the membrane into the muscle cell generates action potential which travels to the myofibril and results in muscle contraction. Wikipedia 831 Figure 7.134 - Nerve systems Wikipedia 832 Figure 7.135 - Steroid hormones structures, with the names of their receptors Wikipedia YouTube Lectures by Kevin HERE & HERE 833 Figure 7.136 - Glucocorticoid receptor with its three domains - DNA binding (left), activator domain (top), and ligand binding domain (boxed). Wikipedia Figure 7.137 - Glucocorticoid signaling pathway Wikipedia 834 Figure 7.138 - Steroid hormone signaling Image by Aleia Kim 835 Figure 7.139 - Structure of a G-protein linked receptor Wikipedia 836 Figure 7.140- A heterotrimeric G-protein: α subunit in blue, βγ subunits red and green Interactive Learning Module HERE 837 Figure 7.141 - Cycle of G-protein activation - 1) binding of ligand; 2) change of receptor structure; 3) stimulation of α-subunit; 4) binding of GTP, release of GDP; 5) separation of α-subunit from β-γ; 6) hydrolysis of GTP by α-subunit and return to inactive state. Wikipedia YouTube Lectures by Kevin HERE & HERE 838 Figure 7.142 - β2-adrenergic receptor embedded in membrane (gray) Wikipedia Figure 7.143 - Epinephrine Wikipedia 839 Figure 7.144 - G-protein coupled receptor. Signal starts with ligand binding (orange circle). Gs = G-protein; AC = adenylate cyclase. Wikipedia Figure 7.145 - Activation of Protein Kinase A by cAMP Image by Martha Baker 840 Figure 7.146 - Simultaneous activation of glycogen breakdown and inhibition of glycogen synthesis by epinephrine’s binding of b-adrenergic receptor. Red enzyme names = activated forms; black enzyme names = inactivated forms; GPb = glycogen phosphorylase b; GPa = glycogen phosphorylase a. Image by Penelope Irving 841 β-Adrenergic Signaling Off Switches 1. GRK Phosphorylates Receptor Tail Receptor Tail Bound by Arrestin 2. α Subunit G-protein Cleaves GTP to GDP β-γ subunits Reassociate with α Subunit 3. cAMP Hydrolyzed by Phosphodiesterase PKA Becomes Inactive 4. Dephosphorylation of Phosphorylated Proteins by Phosphoprotein Phosphatase β-Adrenergic Signaling On Switches 1. Binding of Signal Molecule to Receptor 2. Passage of Signal Through Several Molecules (G-proteins, Adenylate Cyclase, cAMP, PKA) 3. Phosphorylation of Target Proteins 842 Figure 7.147 - Cyclic AMP is broken down by phosphodiesterase Figure 7.148 - Structure of a receptor tyrosine kinase YouTube Lectures by Kevin HERE & HERE 843 Figure 7.149 - Signal binding results in receptor dimerization and activation of tyrosine kinase activity Figure 7.150 - Activated tyrosine kinases phosphorylate tyrosines on the receptor tails. Figure 7.151 -The insulin receptor, a receptor tyrosine kinase Wikipedia Figure 7.152 - Effects of insulin binding to its receptor tyrosine kinase: 1) insulin binding; 2) activation of protein activation cascades. These include: 3) translocation of Glut-4 transporter to plasma membrane and influx of glucose; 4) glycogen synthesis; 5) glycolysis; and 6) fatty acid synthesis. Wikipedia 844 Interactive Learning Module HERE 845 Figure 7.153 - EGFR signaling beginning at top with binding of EGF, dimerization of receptor, transmission of signal through proteins, activation of kinases, phosphorylation of transcription factors and effects on transcription Image by Aleia Kim Figure 7.154 - Ras with GTP bound Wikipedia

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/07%3A_Information_Processing/7.09%3A_Signaling.txt

The environment of a cell is very complex, making it difficult to study individual reactions, enzymes, or pathways in situ. The traditional approach used by biochemists for the study of these things is to isolate molecules, enzymes, DNAs, RNAs, and other items of interest so they can be analyzed independently of the millions of other processes occurring simultaneously. Today, these approaches are used side by side with newer methods that allow us to understand events inside cells on a larger scale- for example, determining all the genes that are being expressed at a given time in specific cells. In this section we take a brief look at some commonly used methods used to study biological molecules and their interactions. • 8.1: Cell Lysis To separate compounds from cellular environments, one must first break open (lyse) the cells. Cells are broken open, in buffered solutions, to obtain a lysate. There are several ways of accomplishing this. • 8.2: Fractionation and Chromatography Techniques Fractionation of samples, as the name suggests, is a process of separating out the components or fractions of the lysate. Fractionation typically begins with centrifugation of the lysate. Using low-speed centrifugation, one can remove cell debris, leaving a supernatant containing the contents of the cell. By using successively higher centrifugation speeds (and resulting g forces) it is possible to separate out different cellular components, like nuclei, mitochondria, etc., from the cytoplasm. • 8.3: Electrophoresis Electrophoresis uses an electric field applied across a gel matrix to separate large molecules such as DNA, RNA, and proteins by charge and size. Samples are loaded into the wells of a gel matrix that can separate molecules by size and an electrical field is applied across the gel. This field causes negatively charged molecules to move towards the positive electrode. The gel matrix, itself, acts as a sieve, through which the smallest molecules pass rapidly, while longer molecules are slower-movi • 8.4: Detection, identification and quantitation of specific nucleic acids and proteins One way to detect the presence of a particular nucleic acid or protein is dependent on transferring the separated molecules from the gels onto a membrane made of nitrocellulose or nylon to create a “blot” and probing for the molecule(s) of interest using reagents that specifically bind to those molecules. The next section will discuss how this can be done for nucleic acids as well as for proteins. • 8.5: Transcriptomics Consider a matrix containing all of the known gene sequences in a genome. To make such a matrix for analysis, one would need to make copies of every gene, either by chemical synthesis or by using PCR. The strands of the resulting DNAs would then be separated to obtain single-stranded sequences that could be attached to the chip. Each box of the grid would contain sequence from one gene. One could analyze the transcriptome - all of the mRNAs being made in selected cells at a given time. • 8.6: Isolating Genes Methods to isolate genes were not available till the 1970s, when the discovery of restriction enzymes and the invention of molecular cloning provided, for the first time, ways to obtain large quantities of specific DNA fragments, for study. Although, for purposes of obtaining large amounts of a specific DNA fragment, molecular cloning has been largely replaced by direct amplification using the polymerase chain reaction described later, cloned DNAs are still very useful for a variety of reasons. • 8.7: Polymerase Chain Reaction (PCR) The polymerase chain reaction (PCR) allows one to use the power of DNA replication to amplify DNA enormously in a short period of time. As you know, cells replicate their DNA before they divide, and in doing so, double the amount of the cell’s DNA. PCR essentially mimics cellular DNA replication in the test tube, repeatedly copying the target DNA over and over, to produce large quantities of the desired DNA. • 8.8: Reverse Transcription In the central dogma, DNA codes for mRNA, which codes for protein. One known exception to the central dogma is exhibited by retroviruses. These RNA-encoded viruses have a phase in their life cycle in which their genomic RNA is converted back to DNA by a virally-encoded enzyme known as reverse transcriptase. The ability to convert RNA to DNA is a method that is desirable in the laboratory for numerous reasons. • 8.9: FRET The fluorescence resonance energy transfer (FRET) technique is based on the observation that a molecule excited by the absorption of light can transfer energy to a nearby molecule if the emission spectrum of the first molecule overlaps with the excitation spectrum of the second.  This transfer of energy can only take place if the two molecules are sufficiently close together (no more than a few nanometers apart. • 8.10: Genome Editing (CRISPR) The development of tools that would allow scientists to make specific, targeted changes in the genome has been the Holy Grail of molecular biology. An ingenious new tool that is both simple and effective in making precise changes is poised to revolutionize the field, much as PCR did in the 1980s. Known as the CRISPR/Cas9 system, and often abbreviated simply as CRISPR, it is based on a sort of bacterial immune system that allows bacteria to recognize and inactivate viral invaders. • 8.11: Protein Cleavage Because of their large size, intact proteins can be difficult to study using analytical techniques, such as mass spectrometry. Consequently, it is often desirable to break a large polypeptide down into smaller pieces. Proteases are enzymes that typically break peptide bonds by binding to specific amino acid sequences in a protein and catalyzing their hydrolysis. • 8.12: Membrane Dynamics (FRAP) Understanding the dynamics of movement in the membranes of cells is the province of the Fluorescence Recovery After Photobleaching (FRAP) technique. This optical technique is used to measure the two dimensional lateral diffusion of molecules in thin films, like membranes, using fluorescently labeled probes. It also has applications in protein binding. Thumbnail: A western blot. Image used with permission (CC BY-SA 3.0; Magnus Manske). 08: Basic Techniques Source: BiochemFFA_8_1.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy To separate compounds from cellular environments, one must first break open (lyse) the cells. Cells are broken open, in buffered solutions, to obtain a lysate. There are several ways of accomplishing this. • Osmotic shock and enzymes: One way to lyse cells is by lowering the ionic strength of the medium the cells are in. This can cause cells to swell and burst. Mild surfactants may be used to disrupt membranes. Most bacteria, yeast, and plant tissues are resistant to osmotic shocks, because of the presence of cell walls, and stronger disruption techniques are usually required. Enzymes may be useful in helping to degrade the cell walls. Lysozyme, for example, is very useful for breaking down bacterial walls. Other enzymes commonly employed include cellulase (plants), proteases, mannases, and others. • Mechanical disruption: Mechanical agitation may be employed in the form of beads that are shaken with a mixture of cells. In this method, cells are bombarded with tiny, glass beads that break the cells open. Sonication (20-50 kHz sound waves) provides an alternative type of agitation that can be effective. The method is noisy, however, and generates heat that can be problematic for heat-sensitive compounds. • Pressure disruption: Another means of disrupting cells involves using a “cell bomb”. In this method, cells are placed under very high pressure (up to 25,000 psi) and then the pressure is rapidly released. The rapid pressure change causes dissolved gases in cells to be released as bubbles which, in turn, break open cells. • Cryopulverization: Cryopulverization is often employed for samples having a tough extracellular matrix, such as connective tissue, seed, and cartilage. In this technique, tissues are frozen using liquid nitrogen and then impact pulverization (typically, grinding, using a mortar and pestle or a powerful electric grinder) is performed. The powder so obtained is then suspended in the appropriate buffer. Whatever method is employed to create a lysate, crude fractions obtained from it must be further processed via fractionation.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/08%3A_Basic_Techniques/8.01%3A_Cell_Lysis.txt

Fractionation of samples, as the name suggests, is a process of separating out the components or fractions of the lysate. Fractionation typically begins with centrifugation of the lysate. Using low-speed centrifugation, one can remove cell debris, leaving a supernatant containing the contents of the cell. By using successively higher centrifugation speeds (and resulting g forces) it is possible to separate out different cellular components, like nuclei, mitochondria, etc., from the cytoplasm. These may then be separately lysed to release molecules that are specific to the particular cellular compartment. The soluble fraction of any lysate can, then, be further separated into its constituents using various methods. Column Chromatography One powerful method used for this purpose is chromatography. We will consider several chromatographic approaches. Chromatography is used to separate out the components of a mixture based on differences in their size, charge or other characteristics. During chromatography, the mobile phase (buffer or other solvent) moves through the stationary phase (usually a solid matrix) carrying the components of the mixture. Separation of the components is achieved, because the different components move at different rates, for reasons that vary, depending on the type of chromatography used. We will consider several different kinds of chromatography to illustrate this process. • Ion exchange chromatography • Gel exclusion chromatography • Affinity chromatography • HPLC These variations on chromatography are performed with the stationary phase held within so-called columns (Figure \(1\)). These are tubes containing the stationary phase (also called the “support” or solid phase). Supports are composed of tiny beads suspended in buffer (Figure \(3\)) and are designed to exploit the chemistry or size differences of the components of the samples and thus provide a means of separation. Columns are “packed” or filled with the support, and a buffer or solvent carries the mixture of compounds to be separated through the support. Molecules in the sample interact differentially with the support and, consequently, travel through it at different speeds, thus enabling separation. Ion exchange chromatography In ion exchange chromatography, the support consists of tiny beads to which are attached chemicals possessing a charge. Before use, the beads are equilibrated in a solution containing an appropriate counter-ion to the charged molecule on the bead. Figure \(5\) shows the repeating unit of polystyrolsulfonate, a compound used as a cation exchange resin. As you can see, this molecule is negatively charged, and thus the beads would be equilibrated in a buffer containing a positively charged ion, say sodium. In the suspension, the negatively charged polystyrolsulfonate is unable to leave the beads, due to its covalent attachment, but the counter-ions (sodium) can be “exchanged” for molecules of the same charge. Exchanges Thus, a cation exchange column will have positively charged counter-ions and negatively charged molecules covalently attached to the beads. Positively charged compounds from a cell lysate passed through the column will exchange with the counter-ions and “stick” to the negatively charged compounds covalently attached to the beads. Molecules in the sample that are neutral in charge or negatively charged will pass quickly through the column. At this point, only positively charged molecules from the original sample would be bound to the column. These may then be washed off, or eluted, by using buffers containing high concentrations of salt. Under these conditions, the interaction between the positively charged molecules and the polystyrosulfonate would be disrupted, allowing the molecules that were bound to the column to be recovered. Anion exchange On the other hand, in anion exchange chromatography, the chemicals attached to the beads are positively charged and the counterions are negatively charged (chloride, for example). Negatively charged molecules in the cell lysate will “stick” and other molecules will pass through quickly. To remove the molecules “stuck” to a column, one simply needs to add a high concentration of counter-ions to release them. Uses Ion exchange resins are useful for separating charged from uncharged, or oppositely charged, biomolecules in solution. The resins have a variety of other applications, including water purification and softening. Figure \(5\) shows use of a polystyrolsulfonate polymer in removing calcium for water softening. Figure \(5\): Removal of calcium ions by an ion exchanger. Wikipedia Size Exclusion Chromatography Size exclusion chromatography (also called molecular exclusion chromatography, gel exclusion chromatography, or gel filtration chromatography) is a low resolution separation method that employs beads with tiny “tunnels” in them that each have a precise opening. The size of the opening is referred to as an “exclusion limit,” which means that molecules above a certain molecular weight will not be able to pass through the tunnels. Molecules with physical sizes larger than the exclusion limit do not enter the tunnels and pass through the column relatively quickly, in the spaces outside the beads. Smaller molecules, which can enter the tunnels, do so, and thus, have a longer path that they take in passing through the column and elute last (Figure \(6\)). Figure \(7\) shows a profile of a group of proteins separated by size exclusion chromatography using beads with an exclusion limit of about 30,000 Daltons. Proteins 30,000 in molecular weight or larger elute in the void volume (left) while smaller proteins elute later (middle and right). Affinity chromatography Affinity chromatography is a very powerful and selective technique that exploits the binding affinities of sample molecules (typically proteins) for molecules covalently linked to the support beads. In contrast to ion-exchange chromatography, where all molecules of a given charge would bind to the column, affinity chromatography exploits the specific binding of a protein or proteins to a ligand that is immobilized on the beads in the column. For example, if one wanted to separate all of the proteins in a cell lysate that bind to ATP from proteins that do not bind ATP, one could use a column that has ATP attached to the support beads and pass the sample through the column. All proteins that bind ATP will “stick” to the column, whereas those that do not bind ATP will pass quickly through it. The bound proteins may then be released from the column by adding a solution of ATP that will displace the bound proteins by competing, for the proteins, with the ATP attached to the column matrix. Histidine tagging Histidine tagging (His-tagging) is a special kind of affinity chromatography and is a powerful tool for isolating a recombinant protein from a cell lysate. His-tagging relies on altering the DNA coding region for a protein to add a series of at least six histidine residues to the amino or carboxyl terminal of the encoded protein. This “His-Tag” is useful in purifying the tagged protein because histidine side chains can bind to nickel or cobalt ions. Separation of His-tagged proteins from a cell lysate is relatively easy (Figure \(8\)).Passing the crude cell lysate through a column with nickel or cobalt attached to beads allows the His-tagged proteins to “stick,” while the remaining cell proteins all pass quickly through. The His-tagged proteins are then eluted by addition of imidazole to the column. Imidazole, which resembles the side chain of histidine, competes with the His-tagged proteins and displaces them from the column. Although non-tagged proteins in the lysate may also contain histidine as part of their sequence, they will not bind to the column as strongly as the His-tagged protein and will, thus, be displaced at lower imidazole concentrations than needed to elute the His-tagged protein. Surprisingly, many His-tagged proteins appear to function normally despite the added histidines, but if needed, the histidine tags may be cleaved from the purified protein by treatment with a protease that excises the added histidines, allowing the recovery of the desired protein with its native sequence. Figure \(8\): Affinity chromatographic purification of a protein by histidine tagging.Image by Aleia Kim HPLC High performance liquid chromatography (HPLC) is a powerful tool for separating a variety of molecules based on their differential polarities (Figure \(9\)). A more efficient form of column chromatography, it employs columns with tightly packed supports and very tiny beads such that flow of solvents/buffers through the columns requires high pressures. The supports used may be polar (normal phase separation) or non-polar (reverse phase separation). In normal phase separations, non-polar molecules elute first followed by the more polar compounds. This order is switched in reverse phase chromatography. Of the two, reverse phase is much more commonly employed due to more reproducible chromatographic profiles (separations) that it typically produces. Figure \(9\): HPLC: Pumps on left/Column in center/Detector on the right. Wikipedia

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/08%3A_Basic_Techniques/8.02%3A_Fractionation_and_Chromatography_Techniques.txt

Electrophoresis uses an electric field applied across a gel matrix to separate large molecules such as DNA, RNA, and proteins by charge and size. Samples are loaded into the wells of a gel matrix that can separate molecules by size and an electrical field is applied across the gel. This field causes negatively charged molecules to move towards the positive electrode. The gel matrix, itself, acts as a sieve, through which the smallest molecules pass rapidly, while longer molecules are slower-moving. For DNA and RNA, sorting molecules by size in this way is trivial, because of the uniform negative charge on the phosphate backbone. For proteins, which vary in their charges, a clever trick must be employed to make them mimic nucleic acids - see polyacrylamide gel electrophoresis (PAGE) below. Different kinds of gels have different pore sizes. Like sieves with finer or coarser meshes, some gels do a better job of separating smaller molecules while others work better for larger ones. Gel electrophoresis may be used as a preparative technique (that is, when purifying proteins or nucleic acids), but most often it is used as an analytical tool. Agarose Gel Electrophoresis Agarose gel electrophoresis is a technique used to separate nucleic acids primarily by size. Agarose is a polysaccharide obtained from seaweeds (Figure 8.11). It can be dissolved in boiling buffer and poured into a tray, where it sets up as it cools (Figure 8.12) to form a slab. Agarose gels are poured with a comb in place to make wells into which DNA or RNA samples are placed after the gel has solidified. The gel is immersed in a buffer and a current is applied across the slab. Double-stranded DNA has a uniform negative charge that is independent of the sequence composition of the molecule. Therefore, if DNA fragments are placed in an electric field they will migrate from the cathode (-) towards the anode (+). The rate of migration is directly dependent on the ability of each DNA molecule to worm or wiggle its way through the sieving gel. The agarose matrix provides openings for macromolecules to move through. The largest macromolecules have the most difficult time navigating through the gel, whereas the smallest macromolecules slip through it the fastest. Figure 8.11 - Structure of the agarose polysaccharide. Wikipedia Because electrophoresis uses an electric current as a force to drive the molecules through the matrix, the molecules being separated must be charged. Since the size to charge ratio for DNA and RNA is constant for all sizes of these nucleic acids, the molecules simply sort on the basis of their size - the smallest move fastest and the largest move slowest. All fragments of a given size will migrate the same distance on the gel, forming the so-called “bands” on the gel. Visualization of the DNA fragments in the gel is made possible by addition of a dye, such as ethidium bromide, which intercalates between the bases and fluoresces when viewed under ultraviolet light (Figure 8.13) By running reference DNAs of known sizes alongside the samples, it is possible to determine the sizes of the DNA fragments in the sample. It is useful to note that, by convention, DNA fragments are not described by their molecular weights (unlike proteins), but by their length in base-pairs( bp) or kilobases (kb). Figure 8.13 - DNA bands visualized with ethidium bromide staining. Wikipedia Polyacrylamide gel electrophoresis (PAGE) Like DNA and RNA, proteins are large macromolecules, but unlike nucleic acids, proteins are not necessarily negatively charged. The charge on each protein depends on its unique amino acid sequence. Thus, the proteins in a mixture will not necessarily all move towards the anode. Additionally, whereas double-stranded DNA is rod-shaped, most proteins are globular (folded). Further, proteins are considerably smaller than nucleic acids, so the openings of the matrix of the agarose gel are simply too large to effectively provide separation. Consequently, unaltered (native) proteins are not very good prospects for electrophoresis on agarose gels. To separate proteins by mass using electrophoresis, one must make several modifications. Gel matrix First, a matrix made by polymerizing and cross-linking acrylamide units is employed. A monomeric acrylamide (Figure 8.14) is polymerized and the polymers are cross-linked using N,N’-Methylene-bisacrylamide (Figure 8.15) to create a mesh-like structure. One can adjust the size of the openings of the matrix/mesh readily by changing the percentage of acrylamide in the reaction. Higher percentages of acrylamide give smaller openings and are more effective for separating smaller molecules, whereas lower percentages of acrylamide are used when resolving mixtures of larger molecules. (Note: polyacrylamide gels are also used to separate small nucleic acid fragments, with some acrylamide gels capable of separating pieces of DNA that differ in length by just one nucleotide.) Figure 8.15 - N,N’-Methylenebisacrylamide - acrylamide crosslinking reagent. Wikipedia Charge alteration by SDS A second consideration is that proteins must be physically altered to “present” themselves to the matrix like the negatively charged rods of DNA. This is accomplished by treating the proteins with the anionic detergent, SDS (sodium dodecyl sulfate). SDS denatures the proteins so they assume a rod-like shape and the SDS molecules coat the proteins such that the exterior surface is loaded with negative charges, masking the original charges on the proteins and making the charge on the proteins more proportional to their mass, like the backbone of DNA. Since proteins typically have disulfide bonds that prevent them from completely unfolding in detergent, samples are boiled with mercaptoethanol to break the disulfide bonds and ensure the proteins are as rod-like as possible in the SDS. Reagents like mercaptoethanol (and also dithiothreitol) are sulfhydryl-containing reagents that become oxidized as they reduce disulfide bonds in other molecules (see Figure 8.16) Stacking Gel A third consideration is that a “stacking gel” may be employed at the top of a polyacrylamide gel to provide a way of compressing the samples into a tight band before they enter the main polyacrylamide gel (called the resolving gel). Just like DNA fragments in agarose gel electrophoresis get sorted on the basis of size (largest move slowest and smallest move fastest), the proteins migrate through the gel matrix at velocities inversely related to their size. Upon completion of the electrophoresis, proteins may be visualized by staining with compounds that bind to proteins, like Coomassie Brilliant Blue (Figure 8.17) or silver nitrate. Figure 8.17 - Two SDS-PAGE gels - Proteins are the blue bands (stained with Coomassie Blue). Wikipedia Non-denaturing gel electrophoresis The SDS_PAGE technique described above is the commonest method used for electrophoretic separation of proteins. In some situations, however, proteins may be resolved on so-called “native” gels, in the absence of SDS. Under these conditions, the movement of proteins through the gel will be affected not simply by their mass, but by their charge at the pH of the gel, as well. Proteins complexed with other molecules may move as single entity, allowing the isolation of the binding partners of proteins of interest. Isoelectric focusing Proteins vary considerably in their charges and, consequently, in their pI values (pH at which their charge is zero). This can be exploited to separate proteins in a mixture. Separating proteins by isoelectric focusing requires establishment of a pH gradient in a tube containing an acrylamide gel matrix. The pore size of the gel is adjusted to be large, to reduce the effect of sieving based on size. Molecules to be separated are applied to the gel containing the pH gradient and an electric field is applied. Under these conditions, proteins will move according to their charge. Positively charged molecules, for example, move towards the negative electrode, but since they are traveling through a pH gradient, as they pass through it, they reach a region where their charge is zero and, at that point, they stop moving. They are at that point attracted to neither the positive nor the negative electrode and are thus “focused” at their pI (Figure 8.18). Using isoelectric focusing, it is possible to separate proteins whose pI values differ by as little as 0.01 units. 2D gel electrophoresis Both SDS-PAGE and isoelectric focusing are powerful techniques, but a clever combination of the two is a powerful tool of proteomics - the science of studying all of the proteins of a cell/tissue simultaneously. In 2-D gel electrophoresis, a lysate is first prepared from the cells of interest. The proteins in the lysate are separated first by their pI, through isoelectric focusing and then by size by SDS-PAGE. The mixture of proteins is first applied to a tube or strip (Figure 8.19, Step 1) where isoelectric focusing is performed to separate the proteins by their pI values (Step 2). Next, as shown in the figure, the gel containing the proteins separated by their pIs is turned on its side and applied along the top of a polyacrylamide slab for SDS-PAGE to separate on the basis of size (Step 3). The proteins in the isoelectric focusing matrix are electrophoresed into the polyacrylamide gel and separated on the basis of size. The product of this analysis is a 2-D gel as shown in Figure 8.20.The power of 2-D gel electrophoresis is that virtually every protein in a cell can be separated and appear on the gel as a spot defined by its unique size and pI. In the figure, spots in the upper left correspond to large positively charged proteins, whereas those in the lower right are small negatively charged ones. Every spot on a 2-D gel can be eluted and identified by using high throughput mass spectrometry. This is particularly powerful when one compares protein profiles between different tissues or between control and treated samples of the same tissue. Figure 8.20 - Result of 2-D gel electrophoresis separation. Wikipedia Protein profiles comparison Comparison of 2-D gels of proteins from non-cancerous tissue and proteins from a cancerous tissue of the same type provides a quick identification of proteins whose level of expression differs between the two. Information such as this can be useful in designing treatments or in understanding the mechanism(s) by which the cancer develops.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/08%3A_Basic_Techniques/8.03%3A_Electrophoresis.txt

While gel electrophoresis can be used to resolve molecules in a mixture, by itself, the technique does not permit the detection and identification of specific nucleic acid sequences or proteins. For example, the 2-D gel shown above clearly separates a large number of proteins in a sample into individual spots. However, if we wanted to know whether a specific protein was present, we could not tell by simply looking at the gel. Likewise, in an agarose gel, while bands of DNA could be assigned a size, one could not distinguish between two DNAs of different sequence if they were both the same length in base-pairs. One way to detect the presence of a particular nucleic acid or protein is dependent on transferring the separated molecules from the gels onto a membrane made of nitrocellulose or nylon to create a “blot” and probing for the molecule(s) of interest using reagents that specifically bind to those molecules. The next section will discuss how this can be done for nucleic acids as well as for proteins. Southern and Northern Blots The Southern blot is named for its inventor, Oxford professor, Edwin Southern, who came up with a protocol for transferring DNA fragments from a gel onto a nitrocellulose sheet and detecting a specific DNA sequence among the bands on the blot. As shown in Figure 8.21, the method works as follows. A mixture of DNA molecules (often DNA that has been cut into smaller fragments using restriction endonucleases) is loaded on an agarose gel, as usual. After the gel run is complete, the DNA bands are transferred from the gel onto a membrane. This can be achieved by capillary transfer, where the gel is placed in contact with a piece of membrane and buffer is pulled through the gel by wicking it up into a stack of absorbent paper placed above the membrane. As the buffer moves, it carries with it the DNA fragments. The DNA binds to the membrane leaving a “print” of DNA fragments that exactly mirrors their positions in the gel. The blotting membrane may be treated with UV light, heat, or chemicals to firmly attach the DNA to the membrane. Next, a probe, or visualizing agent specific for the molecule of interest is added to the membrane. In Figure 8.21, this is called a labeled probe. The probes in a Southern blot are pieces of DNA designed to be complementary to the desired target sequence. If the sequence of interest is present on the blot, the probe, which is complementary to it, can base-pair (hybridize) with it. The blot is then washed to remove all unbound probe. Probes are labeled with radioactivity or with other chemical reagents that allow them to be easily detected when bound to the blot, so it is possible to visually determine whether the probe has bound to any of the DNA bands on the blot. Given that the Southern blot relies on specific base-pairing between the probe and the target sequence, it is easy to adapt the technique to detect specific RNA molecules, as well. The modification of this method to detect RNAs was jokingly named a “northern” blot. Figure 8.21 - Northern or Southern blot scheme. Southern blotting adds strand denaturation between steps 4 and 5. Wikipedia Western Blots Proteins cannot, for obvious reasons, be detected through base-pairing with a DNA probe, but protein blots, made by transferring proteins, separated on a gel, onto a membrane, can be probed using specific antibodies against a particular protein of interest. Protein detection usually employs two antibodies, the first of which is not labeled. The label is on the second antibody, which is designed to recognize only the first antibody in a piggyback fashion. The first antibody specifically binds to the protein of interest on the blot and the second antibody recognizes and binds the first antibody. The second antibody commonly carries an enzyme or reagent which can cause a reaction to produce a color upon further treatment. In the end, if the molecule of interest is in the original mixture, it will “light” up and reveal itself on the blot. This variation on the blotting theme was dubbed a western blot (Figure 8.22). Figure 8.22 - Result of a western blot analysis. Wikipedia In each of the blots described above, binding of the probe to the target molecule allows one to determine whether the target sequence or protein was in the sample. Although blots are designed to be used for detection, rather than for precise quantitation, it is possible to obtain estimates of the abundance of the target molecule from densitometry measurements of signal intensity. Microarrays 2-D gels are a way of surveying a broad spectrum of protein molecules simultaneously. One approach to doing something similar with DNA or RNA involves what are called microarrays. Microarrays are especially useful for monitoring the expressions of thousands of genes, simultaneously. Where a northern blot would allow the identification of a single mRNA from a mixture of mRNAs, a microarray experiment can allow the simultaneous identification of thousands of mRNAs that may be made by a cell at a given time. It is also possible to perform quantitation much more reliably than with a blot. Microarrays employ a glass slide, or chip, to which are attached short sequences of single-stranded DNA, arranged in a grid, or matrix (Figure 8.23) Each position in the grid corresponds to a unique gene. That is, the DNA sequence at this spot is part of the sequence of a specific gene. Each spot on the grid has multiple identical copies of the same sequence. The gene sequence immobilized at each position in the grid is recorded. Figure 8.23 - Microarray design. Image by Taralyn Tan To the slide are added a mixture of sample molecules, some of which will recognize and bind specifically to the sequences on the slide. Binding between the sample molecules and the sequences attached to the slide occurs by base pairing, in the case of DNA microarrays. The slide is then washed to remove sample molecules that are not specifically bound to the sequences in the grid. Sample molecules are tagged with a fluorescent dye, allowing the spots where they bind to be identified. The grid is analyzed spot by spot for binding of the sample molecules to the immobilized sequences. The more sample molecules are bound at a spot, the greater the intensity of dye fluorescence that will be observed. Information from this analysis can give information about the presence/absence/abundance of molecules in the sample that bind to the sequences in the grid. Figure 8.24 - Large scale microarray analysis of mouse transcriptome. Wikipedia

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Consider a matrix containing all of the known gene sequences in a genome. To make such a matrix for analysis, one would need to make copies of every gene, either by chemical synthesis or by using the polymerase chain reaction. The strands of the resulting DNAs would then be separated to obtain single-stranded sequences that could be attached to the chip. Each box of the grid would contain sequence from one gene. With this grid, one could analyze the transcriptome - all of the mRNAs being made in selected cells at a given time. For a simple analysis, one could take a tissue (say liver) and extract all the mRNAs from it. This mRNA population represents all the genes that were being expressed in the liver cells at the time the RNA was extracted. These RNAs should be able to hybridize (base-pair) with their corresponding genes on the microarray. Genes that were not being expressed would have no mRNAs to bind to their corresponding genes on the grid. Figure 8.25 - Copying and labeling of transcriptome. Image by Taralyn Tan In practice, the mRNAs are not used directly, but are copied into single-stranded DNA copies called cDNAs. The cDNAs are tagged with a fluorescent dye and added to the microarray under conditions that allow base pairing so that the cDNAs can find and base pair with complementary sequences on the matrix (Figure 8.26). The matrix is then washed to remove unhybridized cDNAs. The presence/absence/abundance of each mRNA is then readily determined by measuring the amount of dye at each box of the grid. Figure 8.26 - Add labeled cDNAs to microarray plate. Image by Taralyn Tan In Figure 8.27, a fluorescent cDNA has bound to the spot on the far right in the third row of the grid. This means that the sequence of the cDNA was complementary to the sequence of the gene sequence immobilized at that spot. Because the identity of the genes at each position on the grid is known, we then know that the sample contained mRNA that corresponded to that particular gene. In other words, that gene was being expressed in the cells from which the mRNAs were obtained. A more powerful analysis could be performed with two sets of mRNAs simultaneously. . One set of cDNAs could come from a cancerous tissue and the other from a non-cancerous tissue, for example. The cDNAs derived from each sample is marked with a different color (say green for normal and red for cancerous) (Figure 8.25). The cDNAs are mixed and then added to the matrix and complementary sequences are once again allowed to form duplexes (Figure 8.27). Figure 8.28 - Microarray analysis comparing gene expression in normal and cancer cells. Wikipedia Unhybridized cDNAs are washed away and then the plate is analyzed. Red grid boxes correspond to an mRNA present in the cancerous tissue, but not in the non-cancerous tissue. Green grid boxes correspond to an mRNA present in the non-cancerous tissue, but not in the cancerous tissue. Yellow would correspond to mRNAs present in equal abundance in the two tissues (Figure 8.28). The intensity of each spot also gives information about the relative amounts of each mRNA in each tissue. Figure 8.29 - Automated high throughput sequencer. Wikipedia The same principle used for nucleic acid microarrays can be adapted for analyzing other molecules. For example, polypeptides could be bonded to the glass slide instead of DNA to create a protein chip. Protein chips are useful for studying the interactions of proteins with other molecules as well as for diagnostics. RNA-Seq Technique Like microarrays, a newer method called RNA-Seq, is a tool for simultaneously detecting and quantitating all of the transcripts in a given sample. This method relies on recently developed sequencing technologies called next-generation sequencing, or deep sequencing. These techniques allow for rapid, parallel sequencing of millions of DNA fragments and can, thus, be used not only for genomic DNA, but also to sequence all of the reverse-transcribed RNAs from a given sample. To determine all the protein-coding genes that were being expressed in a particular set of cells under specific physiological conditions, all of the mRNA would first be extracted and reverse-transcribed into cDNA. This step is similar to the preparation of samples for microarrays. However, at this point, the cDNAs are fragmented into smaller pieces, and have small sequencing adapters attached at either end. The fragments are then subjected to high-throughput sequencing, to obtain short sequences from all of the fragments. These data are aligned against the genome sequence and used to measure the level of expression of different genes. RNA-Seq offers some advantages over microarrays. With microarrays, an RNA can only be detected if the gene sequence corresponding to it is present on the grid. In RNA-Seq every RNA present in the sample is sequenced, so detection of RNAs is not limited by the probes on a chip. RNA-Seq is more sensitive than microarrays and offers a much larger range over which gene expression can be measured accurately.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/08%3A_Basic_Techniques/8.05%3A_Transcriptomics.txt

Earlier in this chapter, we discussed methods such as column chromatography that are used to purify proteins of interest. Using combinations of these methods, it is possible to isolate a protein to a high degree of purity, thus enabling us to study the protein’s activity and properties. This problem is harder to solve for nucleic acids. Genomic DNA can be readily obtained from cells, but is too complex to be analyzed as a whole. Individual genes are the units of DNA that correspond to proteins, and thus, it makes more sense to isolate specific genes for study. Methods to isolate genes were not available till the 1970s, when the discovery of restriction enzymes and the invention of molecular cloning provided, for the first time, ways to obtain large quantities of specific DNA fragments, for study. Although, for purposes of obtaining large amounts of a specific DNA fragment, molecular cloning has been largely replaced by direct amplification using the polymerase chain reaction described later, cloned DNAs are still very useful for a variety of reasons. The development of molecular cloning was dependent on the discovery of restriction endonucleases, described below. Restriction enzymes Restriction enzymes, or restriction endonucleases, are enzymes made by bacteria. These enzymes protect bacteria by degrading foreign DNA molecules that are carried into their cells by, for example, an invading bacteriophage. Each restriction enzyme recognizes a specific sequence, usually of four or six nucleotides in the DNA. These sequences, when they occur in the bacterium's own DNA, are chemically modified by methylation, so that they are not recognized and degraded. Where these sequences occur in foreign DNA, they are cut by the restriction enzyme. The utility and importance of restriction enzymes lies in their ability to recognize specific sequences in DNA and cut near or (usually) at the site they recognize. Over 3000 such enzymes are known. Sequences recognized by these enzymes are typically 4-8 base pairs long and the most commonly used enzymes recognize sequences described as palindromic. Figure \(1\): -A restriction enzyme bound to its recognition sequence on DNA. Wikipedia Palindrome In molecular biology, the term palindrome means that the sequence of the recognition site when read in the 5‘ to 3‘ direction for the top strand is exactly the same as that of the bottom strand. Consider the sequence recognized by the restriction enzyme known as Hind III (pronounced hin-dee-three). It is 5’ -A-A-G-C-T-T-3’​ 3’ -T-T-C-G-A-A-5’ On the top strand, the recognition sequence is 5’ AAGCTT 3’ which is the same as the bottom strand (read in the same 5’ to 3’ direction). While all restriction enzymes must recognize and bind to particular DNA sequences, the exact spot at which they cut the DNA varies. Some enzymes leave a staggered sequence after cutting that has an overhang at the 5’ end of one strand of the duplex; some leave a staggered sequence after cutting that has an overhang at the 3’ end; and some cut both strands in the same place, leaving no overhanging sequence - called blunt end cutters. Consider cutting a DNA sequence that contains the Hind III recognition site, which is 5’ -A-A-G-C-T-T-3’​ 3’ -T-T-C-G-A-A-5’ Embedded within a DNA sequence, the Hind III sequence would look like this (Ns correspond to any base and represent all of the DNA around the recognition site). 5’ -N-N-N-A-A-G-C-T-T-N-N-N-3’​ 3’ -N-N-N-T-T-C-G-A-A-N-N-N-5’ After cutting with Hind III, it would look as follows: 5’ -N-N-N-A 3‘ 5’A-G-C-T-T-N-N-N-N-3’​ 3’ -N-N-N-T-T-C-G-A-5‘ 3’ A-N-N-N-N-5’ where gaps have been inserted to illustrate where cutting has occurred. Hind III cuts between the two ‘A’ containing nucleotides near the 5’ end of the recognition sequence and thus leaves 5’ overhangs (Figure \(2\)). Figure \(2\): Result of cutting DNA with Hind III. Wikipedia The restriction enzyme Pst I, on the other hand, recognizes the following sequence 5’ -N-N-N-C-T-G-C-A-G-N-N-N-N-3’​ 3’ -N-N-N-G-A-C-G-T-C-N-N-N-N-5’ and cuts between the A and the G near the 3’ end of the recognition sequence. 5’ -N-N-N-C-T-G-C-A 3‘ 5’G-N-N-N-N 3’​ 3’ -N-N-N-G 5‘ 3’ A-C-G-T-C-N-N-N-N 5’ As you can see, cutting a DNA with Pst I leaves 3’ overhangs of the recognition sequence. The ends left after cutting by a restriction enzyme that overhang either at the 5’ end or the 3’ end are referred to as being “sticky” because they can form proper base pairs and more readily be joined to a similarly “sticky end”. This means that you can take two unrelated pieces of DNA, cut them with the same restriction enzyme so that they have compatible sticky ends, and then "paste" them together using DNA ligase to form a new hybrid molecule, or recombinant. Making Recombinant DNAs Joining together of DNA fragments from different sources creates recombinant DNA. The ability to cut and paste DNA might seem like purely a technical feat, but one key application that arose out of this is molecular cloning. In molecular cloning a gene of interest can be inserted into a vector, usually a plasmid, by cutting both the vector and the gene (called the insert) with the same enzyme to generate sticky ends and joining the two pieces together to generate a recombinant (Figure \(3\)). A plasmid is a type of autonomously replicating, extrachromosomal DNA. It is quite simple to extract plasmids from the cells, engineer them to contain the gene of interest and re-introduce the recombinant plasmid into the bacteria. The idea was that when the plasmid DNA was replicated, the extra inserted gene would also be copied. Thus, by growing up a lot of the bacteria carrying the plasmid, many copies of the gene of interest could be obtained, to provide sufficient amounts of the gene to use in experiments. While we now have easier methods to accomplish this goal, cloned DNAs remain very useful. For example, it is possible to clone a gene that encodes a protein of interest so that it can be expressed at high levels in the cells into which the recombinant plasmid is introduced. Figure \(3\): Recombinant DNA construction. Wikipedia Whatever the purpose for which the recombinant plasmid is made, it typically carries an antibiotic resistance gene (or genes), called a selectable marker. Cells that take up the plasmid will be able to grow in the presence of the antibiotic. If bacterial cells to which the plasmid has been added are plated on agar containing the antibiotic, the cells which took up the plasmid will be able to grow, while the others will not. Expression cloning As mentioned above, a gene of interest may be inserted into a vector and the recombinant plasmid be placed into a cell where the gene can be expressed. For instance, one might desire to clone the gene coding for human growth hormone or insulin or other medically important proteins and have a bacterium or yeast make large quantities of it very cheaply. Remember that these are human proteins, and thus it is not feasible to extract the proteins in any quantity from human subjects. To clone a gene so that it can be expressed, one needs to set up the proper conditions in order for the human protein to be made in the bacterial cells. This typically involves the use of specially designed plasmids. These plasmids have been engineered to 1) replicate in high numbers; 2) carry markers that allow researchers to identify cells carrying them (antibiotic resistance, for example) and 3) contain sequences (such as a promoter and Shine Dalgarno sequence) necessary for expression of the desired protein, with convenient sites for insertion of the gene of interest in the appropriate place relative to the control sequences. A plasmid which has all of these features is referred to as an expression vector. In addition to plasmids that can be used for expression in bacterial cells, expression vectors are also available that allow protein expression in a variety of eukaryotic cells. Many sophisticated variations on such vectors have been created that have made it easy to produce and purify large amounts of any protein of interest for which the gene has been cloned. A handy feature in some expression vectors is a sequence encoding an affinity tag either up- or downstream of the gene being expressed. This sequence allows a short affinity tag (such as a run of histidine residues) to be fused onto the encoded protein. The tag can be used to readily purify the protein, as described in the section on affinity chromatography.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/08%3A_Basic_Techniques/8.06%3A_Isolating_Genes.txt

Molecular cloning was the first method available to isolate a gene of interest and make many copies of it to obtain sufficient amounts of the DNA to study. Today, there is a faster and easier way to obtain large amounts of a DNA sequence of interest -the polymerase chain reaction (PCR). PCR allows one to use the power of DNA replication to amplify DNA enormously in a short period of time. As you know, cells replicate their DNA before they divide, and in doing so, double the amount of the cell’s DNA. PCR essentially mimics cellular DNA replication in the test tube, repeatedly copying the target DNA over and over, to produce large quantities of the desired DNA. Selective Replication In contrast to cellular DNA replication, which amplifies all of a cell’s DNA during a replication cycle, PCR does targeted amplification to replicate only a segment of DNA bounded by the two primers that determine where DNA polymerase begins replication. Figure 8.34 illustrates the process. Each cycle of PCR involves three steps, denaturing, annealing and extension, each of which occurs at a different temperature. The Starting Materials Since PCR is, basically, replication of DNA in a test-tube, all the usual ingredients needed for DNA replication are required:​ • A template (the DNA containing the target sequence that is being copied)​ • Primers (to initiate the synthesis of the new DNA strands)​ • Thermostable DNA polymerase (to carry out the synthesis). The polymerase needs to be heat stable, because heat is used to separate the template DNA strands in each cycle. ​ • dNTPs (DNA nucleotides to build the new DNA strands). • The template is the DNA that contains the target you want to amplify (the "target" is the specific region of the DNA you want to amplify).​ The primers are short synthetic single-stranded DNA molecules whose sequence matches a region flanking the target sequence. It is possible to chemically synthesize DNA molecules of any given base sequence, to use as primers. To make primers of the correct sequence that will bind to the template DNA, it is necessary to know a little bit of the template sequence on either side of the region of DNA to be amplified. DNA polymerases and dNTPs are commercially available from biotechnology supply companies. First, all of the reagents are mixed together. Primers are present in millions of fold excess over the template. This is important because each newly made DNA strand starts from a primer. The first step of the process involves separating the strands of the target DNA by heating to near boiling. Next, the solution is cooled to a temperature that favors complementary DNA sequences finding each other and making base pairs, a process called annealing. Since the primers are present in great excess, the complementary sequences they target are readily found and base-paired to the primers. These primers direct the synthesis of DNA. Only where a primer anneals to a DNA strand will replication occur, since DNA polymerases require a primer to begin synthesis of a new strand. Figure 8.36 - A PCR thermocycler system. Wikipedia Extension In the third step in the process, the DNA polymerase replicates DNA by extension from the 3’ end of the primer, making a new DNA strand. At the end of the first cycle, there are twice as many DNA molecules, just as in cellular replication. But in PCR, the process is repeated, usually for between 25 and 30 cycles. At the end of the process, there is a theoretical yield of 230 (over 1 billion times) more DNA than there was to start. (This enormous amplification power is the reason that PCR is so useful for forensic investigations, where very tiny amounts of DNA may be available at a crime scene.) The temperature cycles are controlled in a thermocycler, which repeatedly raises and lowers temperatures according to the set program. Since each cycle can be completed in a couple of minutes, the entire amplification can be completed very rapidly. The resulting DNA is analyzed on a gel to ensure that it is of the expected size, and depending on what it is to be used for, may also be sequenced, to be certain that it is the desired fragment. Mutagenesis PCR is frequently used to obtain gene sequences to be cloned into vectors for protein expression, for example. Besides simplicity and speed, PCR also has other advantages. Because primers can be synthesized that differ from the template sequence at any given position, it is possible to use PCR for site-directed mutagenesis. That is, PCR can be used to mutate a gene at a desired position in the sequence. This allows the proteins encoded by the normal and mutant genes to be expressed, purified and compared. Analysis of gene expression PCR can also be used to measure gene expression. Where in PCR the amount of amplified product is not determined till the end of all the cycles, a variation called quantitative real-time PCR is used, in which the accumulation of product is measured at each cycle. This is possible because real-time PCR machines have a detector module that can measure the levels of a fluorescent marker in the reaction, with the amount of fluorescence proportional to the amount of amplified product. By following the accumulation of product over the cycles it is possible to calculate the amount of starting template. To measure gene expression, the template used is mRNA reverse-transcribed into cDNA (see below). This coupling of reverse transcription with quantitative real-time PCR is called qRT-PCR.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/08%3A_Basic_Techniques/8.07%3A_Polymerase_Chain_Reaction_%28PCR%29.txt

In the central dogma, DNA codes for mRNA, which codes for protein. One known exception to the central dogma is exhibited by retroviruses. These RNA-encoded viruses have a phase in their life cycle in which their genomic RNA is converted back to DNA by a virally-encoded enzyme known as reverse transcriptase. The ability to convert RNA to DNA is a method that is desirable in the laboratory for numerous reasons. For example, converting RNAs of interest to cDNA is used in RT-PCR as well as in other applications like microarray analysis. Process First, one creates a DNA oligonucleotide to serve as a primer for reverse transcriptase to use on a target RNA. The primer must, of course, be complementary to a segment (near the 3’ end) of the RNA to be amplified. The RNA, reverse transcriptase, the primer, and four dNTPs are mixed. With one round of replication, the RNA is converted to a single strand of DNA. Denaturation frees the single stranded cDNA, which can be used as is, or converted to double-stranded cDNA, depending on the application. Figure 8.37 - Reverse transcriptase of HIV. The nuclease function is needed for the viral life cycle, but not for lab use. Wikipedia Detecting molecular interactions The study of biochemistry is basically the study of the interactions of cellular molecules. Methods for detecting interactions among biomolecules are, for this reason, very useful to biochemists. We will now discuss a couple of very different methods for detecting these inter-molecular interactions. Yeast two-hybrid system (Y2H) Yeast two-hybrid screening is a sophisticated technique for identifying which protein(s), out of a collection of all of a cell’s proteins, interacts with a specific protein of interest. The method relies on the interaction between two proteins to reconstitute a functional transcriptional activator within yeast cells. You may remember that many transcriptional activators are modular proteins that have a domain that binds to DNA and another domain that activates transcription (Figure 8.38). If the transcription factor is split, so that the binding domain is attached to one protein, and the activation domain to another protein, a functional transcriptional activator can only be re-created if the two “carrier”proteins come into close proximity - that is, they interact. The presence of this functional activator can be detected by the expression of a reporter gene. A simple way to understand this idea is by thinking of a transcriptional activator as a device, like a flashlight, that has two parts, the battery and the lamp, that must be together in order to function. Neither a person who has just a battery nor one who has only the lamp will be able to see in a dark room. But if the two interact by coming close enough to insert the battery in the flashlight, their interaction can be detected by the fact that the flashlight will now be functional as evidenced by the light produced. It takes two to tango Figure 8.39 (A) shows the normal yeast transcriptional activator, GAL4, with both the DNA-binding (DBD) and Activation domains (AD). It is able to stimulate transcription of the downstream reporter gene, lac z. Panels B and C show constructs that produce the GAL4 DBD and AD, respectively, fused to other proteins, one of which is termed the “bait” and the other as “prey”. Neither of these fusion proteins can stimulate transcription of the lac z gene. When constructs encoding both the bait and prey are in the same yeast cell, if the bait protein interacts with the prey, the DBD and AD of the GAL4 will be brought together to reconstitute a functional GAL4. The presence of functional GAL4 is readily detectable because it will stimulate expression of the lac z reporter gene. If the bait and prey proteins do not interact, then there will be no lac z expression. When interaction is detected through expression of the reporter gene, the specific prey protein can then be identified. The yeast two-hybrid system allows for simultaneous screening of many prey proteins, by constructing large collections of fusion constructs, with each potential protein partner of the bait protein fused to the GAL4 activation domain. Figure 8.39 - Four scenarios for the yeast two-hybrid system. UAS = Upstream Activator Sequences - acts like a promoter. Scenario A shows that the two transcription factors start out as one protein. Wikipedia

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/08%3A_Basic_Techniques/8.08%3A_Reverse_Transcription.txt

Another method for detecting molecular interactions is Fluorescence resonance energy transfer (FRET) - also called Förster resonance energy transfer, resonance energy transfer (RET) or electronic energy transfer (EET). The technique is based on the observation that a molecule excited by the absorption of light can transfer energy to a nearby molecule if the emission spectrum of the first molecule overlaps with the excitation spectrum of the second (Figure \(1\)) This transfer of energy can only take place if the two molecules are sufficiently close together (no more than a few nanometers apart). In the technique, a donor fluorophore or an acceptor fluorophore is covalently attached to two molecules of interest. The acceptor fluorophore is designed to accept energy from the donor molecule (orange dotted line in Figure \(2\)) and fluoresce at a unique wavelength (red arrow) when it receives that energy from the donor. Figure \(2\): Fluorescence resonance energy transfer between donor and acceptor chromophores. Image by Pehr Jacobson Further, the wavelength of light that the donor absorbs is uniquely tailored for the donor fluorophore and has no effect on the acceptor fluorophore. The only way the acceptor can fluoresce is if it is close enough to receive energy transferred from the donor (red arrow). This fluorescence will have a unique wavelength, as well. If the donor and acceptor are not close enough together, the donor fluoresces and emits light corresponding to the green or black arrow. These are different wavelengths than that of the red arrow. The experiment begins in the cell with one protein with a donor fluorophore and the other protein with an acceptor fluorophore. Light of a wavelength that excites the donor fluorophore is shined on the cell. If a protein with a donor interacts with the protein carrying an acceptor, then energy transfer occurs from the donor fluorophore to the acceptor and the unique fluorescence (red line) of the acceptor is detected. If the two proteins do not interact, then little or no fluorescence from the acceptor is detected. 8.10: Genome Editing (CRISPR) The development of tools that would allow scientists to make specific, targeted changes in the genome has been the Holy Grail of molecular biology. An ingenious new tool that is both simple and effective in making precise changes is poised to revolutionize the field, much as PCR did in the 1980s. Known as the CRISPR/Cas9 system, and often abbreviated simply as CRISPR, it is based on a sort of bacterial immune system that allows bacteria to recognize and inactivate viral invaders. CRSPR CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, short repeated sequences found in prokaryotic DNA, separated by spacer sequences derived from past encounters with, for example, a bacteriophage. Like the glass slipper left behind by Cinderella that was later used to identify her, the pieces of the invader's sequences are a way for the bacteria to identify the virus if it attacks again. Inserted into the bacterial genome, these sequences can later be transcribed into a guide RNA that matches, and base-pairs with, sections of the viral genome if it was encountered again. A nuclease associated with the guide RNA then cleaves the sequence base-paired with the guide RNA. (The nucleases are named Cas for CRISPR-associated.) The essential elements of this system are a guide RNA that homes in on the target sequence and a nuclease that can make a cut in the sequence that is bound by the guide RNA. By engineering guide RNAs complementary to a target gene, it is possible to target the nuclease to cleave within that gene. In the CRISPR/Cas9 system, the Cas9 endonuclease cuts both strands of the gene sequence targeted by the guide RNA (Figure \(1\)). This generates a double-strand break that the cell attempts to repair. As you may remember, double-strand breaks in DNA can be repaired by simple, nonhomologous end joining (NHEJ) or by homologous recombination. When a break is fixed by NHEJ, there is good chance that there will be deletions or insertions that will inactivate the gene they are in. Thus, targeted cleavage of a site by CRISPR/Cas9 can easily and specifically inactivate a gene, making it easy to characterize the gene's function. But, what if you wished to simply mutate the gene at a specific site to study the effect of the mutation? This, too, can be achieved. If a homologous sequence bearing the specific mutation is provided, homologous recombination can repair the break, and at the same time insert the exact mutation desired. It is obvious that if you can insert a mutation as just described, it should be possible to correct a mutation in the genome by cleaving at the appropriate spot and providing the correct sequence as a template for repair by homologous recombination. The simplicity of the system holds great promise for curing genetic diseases. Scientists have also come up with some creative variations on the CRISPR/Cas9 system. For instance, one variant inactivates the nuclease activity of Cas9. The guide RNA in this system pairs with the target sequence, but the Cas9 does not cleave it. Instead, the Cas9 blocks the transcription of the downstream gene (Figure \(2\)) This method allows specific genes to be turned off without actually altering the DNA sequence. Another variation also uses a disabled Cas9, but this time, the Cas9 is fused to a transcriptional activation domain. In this situation, the guide RNA positions the Cas9-activator domain in a place where it can enhance transcription from a specific promoter (Figure \(3\)). Other variations on this theme attach histone-modifying enzymes or DNA methylases to the inactive Cas9. Again, the guide RNA positions the Cas9 in the desired spot, and the enzyme attached to Cas9 can methylate the DNA or modify the histones in that region. CRISPR has already been used to edit genomes in a wide variety of species (and in human cell cultures). It may not be long before the technique is approved for clinical use. In the meanwhile, CRISPR is transforming molecular biology.

textbooks/bio/Biochemistry/Book%3A_Biochemistry_Free_For_All_(Ahern_Rajagopal_and_Tan)/08%3A_Basic_Techniques/8.09%3A_FRET.txt