From a clinical viewpoint, graft rejection is one of the greatest threats faced by an organ transplant recipient (OTR). We propose a middle-range theory (MRT) of Perceived Threat of the Risk of Graft Rejection (PTRGR) as a contribution to the practice of transplant nursing. It could also apply to the detection of risky protective behavior, that is, isolation, avoidance, or non-adherence. The proposed MRT covers the following concepts and the relationship between them: transplant care needs, threat reducing interventions, intervening variables, level of PTRGR, protective strategies, and evidence-based practice. Parts of this theory have been empirically tested and support the suggested relationship between some of the concepts. Further tests are needed to strengthen the theoretical links. The conceptual framework might serve as a guide for transplant nurses in their efforts to promote post-transplant health and reduce threat-induced emotions. Solid organ transplantation is an established and successful treatment for critically ill patients. For many patients with severe organ failure, it is the only option for survival. After transplantation, the treatment consists of medications intended to suppress the immune system of organ transplant recipients (OTRs) to prevent graft rejection. According to clinical practice, the transplanted patient is required to submit to an extensive follow-up program for the purpose of early identification of graft rejection, infection, or other complications, especially during the first post-operative year. The patient is also expected to develop an adequate self-care capacity regarding medication, observing signs of graft rejection as well as dealing with new demands and changes in daily life.A common intervention in different transplant settings worldwide is to educate the patients in “patient awareness,” before as well as after discharge from the transplant unit. The aim of this is to provide OTRs with tools and strategies on how to avoid exposure to risks that could lead to infections or graft rejection. The key message in most awareness training is that OTRs should live as normally as possible, in spite of having received a transplant. The life of the OTR is thus transformed from one of suffering from a life-threatening illness to something more healthy when compared with the situation before the transplantation, but with the underlying necessity of undergoing lifelong immunosuppressive treatment and follow-up because of the still chronic condition. When OTRs are asked about what they fear most, the most common response is graft rejection. The Perceived Threat of the Risk of Graft Rejection (PTRGR) is prominent in the lives of OTRs . The thrWithin the field of transplant nursing, there is a distinct lack of specific transplant-related nursing theories that identify and express the key ideas about the essence of transplant nursing practice. The already existing nursing theories such as Neuman’s system theory on stressors  or the tThe rationale behind our efforts to develop this middle-range theory (MRT) is based on vast clinical experience that clearly shows that the threat of the risk for graft rejection is prominent in the lives of OTRs. Together with infection, graft rejection is viewed as the most common threat for these patients. Previous studies  indicateThe rationale behind this MRT is to support nursing care for OTRs experiencing the threat of the risk for graft rejection and who also show negative psychological reactions. Altogether, this knowledge is important for the health care professionals to improve existing strategies of relieving the condition. There is also a need to develop methods that support those patients where the HRQoL as well as the pattern of daily life is affected. The concept of threat is critical to nurses who are faced with the challenge of caring for those experiencing threat-induced emotions. Regardless of whether the threat is perceived or real, as in the case of a biopsy-proven graft rejection, this threat induces various negative emotional responses and has deep psychophysiological and social consequences . It is tThe foremost rational for this MRT is to create a firm base of knowledge from the OTRs’ perspective that will be useful in both the care and training of patients that suffer from the PTGR[Perceived Threat of the Risk of Graft Rejection]. It will also serve as a basis for future longitudinal and interventional studies. To summarize, the aim of this article is to propose a MRT dealing with the PTRGR among solid OTRs.For many years, our research has focused on the specific event of graft rejection within the context of solid organ transplantation. The results of these studies  have encAlso in qualitative studies, the risk of rejection has been found to be the most stressful event among OTRs. The elements and strategies for theory building proposed by Derivation, where we transposed and redefined the concept threat from a concept analysis performed by Synthesis, where we constructed new statements and a premature theory based on our literature review and the findings of observations made in empirical research .The definitions given below are based on a subjective perspective of the OTR where the individual’s experience is the most important. The purpose of this MRT of PTRGR is to assist transplant nursing in (a) caring for OTRs suffering from threat-induced emotions because of the constant risk of graft rejection that negatively affect and limit their everyday life and HRQoL and (b) detecting risky, protective behavior adopted by the OTR to prevent graft rejection or manage the sense of fear, that is, isolation, avoidance, or non-adherence.The concept PTRGR was derived primarily from the three references below. According to Threat might be defined as “an expression of an intention to inflict pain, injury, evil, or punishment; an indication of impending danger or harm; one that is regarded as a possible danger; a menace” , p. 1801Furthermore, According to In the event of a perceived threat, the individuals’ perception of a threatening event is based on a cognitive appraisal to that event. What is appraised as threatening to one individual might be appraised as challenging to another. And finally, perceived threat exhibits itself as an emotional response that is part of an individual’s stress response. , p. 194The anticipation of graft rejection is based on symptoms, signs or the cognitive appraisal of graft rejection. This anticipation is capable of eliciting the stress response of organ transplant recipients, expressed as intrusive anxiety and fear of negative health implications for the future. (Authors’ definition)To test the concept, we performed a phenomenographic study aimed at exploring perceptions of experiences of graft rejection among OTRs receiving a kidney, liver, heart, or lung. This study provided an in-depth understanding of the patient perspective and confirmed the proposed concept definition in all its parts. These findings are reported in detail elsewhere  and are When testing the theory, we found that kidney transplant recipients reported significantly more graft-related threat than patients receiving a liver, heart, or lung. PTRGR was independent of time since transplantation, experienced graft rejections, and clinical differences among OTRs .In this phase, we used information from the literature review, as well as from our empirical observations to construct a new theory. The next step was to develop an instrument to measure PTRGR (PTGR-instrument) where we named the three factors in the factor structure: (a) graft-related threat, (b) intrusive anxiety, and (c) lack of control. The PTGR-instrument measures the concept by 12 items on a 5-point Likert-type scale . The catThe second factor, intrusive anxiety, meant being constantly aware of the risk of graft rejection, thinking about it constantly. It also meant experiencing great anxiety, which was elevated when taking immune-suppressive medication or going through a biopsy. Thus, this factor showed the extent of the OTRs’ stress response and level of anxiety. The majority, 74%, of the OTRs scored low levels of intrusive anxiety.Finally, the third factor, lack of control, involved perceptions that the threat by means of risk of graft rejection was out of one’s control, revealing the degree of belief that one can control and protect oneself from the threat. A high level of lack of control was experienced by 48%. The deductive stage of theory development began as a form of logical reasoning in which specific conclusions were inferred from more general premises, that is, the basic nursing concepts: person, health, environment, and nursing. The actual theory of PTRGR (PTRGR-theory) is proposed as follows.We started to define the meta-concepts of nursing, as they appear in our theory. The person is an OTR, subjected to lifelong immunosuppressive medication because of a constant risk of graft rejection, to prevent the possible harm of graft rejection. The environment is any context where the OTR is trying to master his or her everyday life. Health is viewed as experienced comfort and cognitive understanding to master the PTRGR, resulting in an experienced well-being and HRQoL. Transplant nursing also involves the following:Assessment: Context-specific deliberative actions to approach and assess threat-induced emotions and actions to relieve intrusive anxiety in the OTR. Assessment can be either subjective or objective.Development: Person-centered nursing care plans, which is a standard intervention.Implementation: Context-specific threat reducing interventions that promote the OTRs’ mastering of graft-related threat and support adoption of useful and reasonable strategies to protect oneself from harm, that is, a graft rejection.Evaluation: The level of the PTRGR as a nursing outcome.Subjective assessment starts with a one-on-one approach, listening to the OTRs’ perceptions of experiences of graft rejection. A simple introductory question might be the following: “When I say graft rejection, what comes to your mind”? The 12-item PTGR-instrument described above is useful for carrying out objective assessment. Repeated instructional conversations can be a useful intervention to promote the mastering of a graft-related threat. An important strategy to support the adoption of useful and reasonable strategies to protect oneself from harm is to assess barriers for adherence . It is tAdditional major concepts of the PTRGR-theory are as follows:Transplant care needs—Occurs after discharge from the transplant unit when the OTR finds it difficult to adopt health-promoting behavior, accepting the new health situation, and remain adherent to immunosuppressive medication and the obtained self-care instructions. This concept was developed inspired by Threat reducing interventions—Context-specific deliberative nursing interventions, that is, assessment, development, implementation, and evaluation. The concept was inspired by the deliberative nursing process originally developed by Orlando When using analysis, we chose to dissect the whole, that is, the tentative framework, into its parts again, so that they could be better understood. We tested the theory by exploring relationships between the factors in the PTGR-instrument, psychological reactions to PTRGR and HRQoL as well as various clinically relevant variables as reported in detail in a previous study . Mainly Transplant care needs, threat reducing interventions, and intervening variables will result in a certain level of PTRGR and HRQoL in everyday life. This level of PTRGR is affected by and affects the OTRs’ use of protective strategies and evidence-based practice as well as transplant nursing interventions performed by the transplant nurse. The level of PTRGR also affects everyday life and HRQoL. The use of protective strategies and the nurse’s transplant nursing interventions and evidence-based practice will both affect the level of PTGR and the transplant care needs, the threat reducing interventions, and the intervening variables. The evidence-based practice strategy that is selected depends on the OTRs’ use of protective strategies. The content of the framework is illustrated in This framework will serve as the proposed theory of PTRGR and will also serve as a foundation for further theory testing.This is the first effort ever to propose a middle-range nursing theory useful in relation to OTRs facing the PTRGR. As such, this proposed theory has of course several limitations. First, not all relationships are tested in robust studies. Second, the effects of threat reducing interventions are not established. Instead, it is based on clinical assumptions and extensive clinical experience. The proposed intervening variables are validated by the literature review and clinical experience. Some of these variables are also established by research of symptoms and non-adherence . Third, We have proposed that this MRT of PTRGR is more specific than a conceptual model  because The theory originally emerged while performing several research studies, reported in the “Introduction” section, and has taken shape stepwise by the use of The proposed theory is a conceptualization of the core aspects of OTRs’ reality that relates to transplant nursing and deals with several specific substantial concepts and propositions. We also argue that the framework might be referred to as a MRT because it has a narrow scope, is substantial and practical, and includes few concepts and propositions . It reprAccording to Finally, evidence-based standardized nursing care plans and protocols can affect the OTRs’ protective strategies. This demands new strategies so that the situation can be approached in a health-promoting manner. The impact of evidence-based practice has echoed across nursing practice, and nurses have responded to launch initiatives that maximize the valuable contributions that nurses can make to fully deliver on the promise of providing evidence-based practice to all patients in need of health promotion nursing interventions. Such initiatives include theory development. Nevertheless, many transplant nursing interventions are useful even if they lack the standard of evidence-based practice.Regarding generality, this MRT is highly contextual. However, person-centered care, deliberative nursing interventions, and evidence-based practice are all essential and universal in nursing practice. The empirical precision has proven to be promising. We have already found that it is possible to test several links in the theory as well as validate the content of the concept of PTRGR. Finally, the importance of the theory is left for reviewers to judge; however, because of a total lack of MRTs offering guidance in the area of transplant nursing and the fact that PTRGR is highly relevant for OTRs, the theory might be of importance.Other important aspects when discussing a theory are the factors of social utility, social congruence, and social significance. In respect of social utility, there might be a need for the transplant nurse to undergo brief training before clinical use. Social congruence considers whether the model will lead to nursing activities that will meet the expectations of the public. We assume that the MRT will inspire nursing practitioners to utilize several deliberative nursing activities that meet the expectations of the OTRs. The MRT will scarcely meet the criterion of social significance or make any difference in the health conditions of the public. Finally, we strongly believe that this MRT, despite its limitations, still offers much to the discipline of transplant nursing by increasing the understanding of the PTRGR.


Polysaccharides derived from plant foods are major components of the human diet, with limited contributions of related components from fungal and algal sources. In particular, starch and other storage carbohydrates are the major sources of energy in all diets, while cell wall polysaccharides are the major components of dietary fiber. We review the role of these components in the human diet, including their structure and distribution, their modification during food processing and effects on functional properties, their behavior in the gastrointestinal tract, and their contribution to healthy diets. Understanding the relationships between the compositions of raw food materials, the effects of processing on their structures and interactions, and their behavior in the gastrointestinal (GI) tract are crucial for elucidating the relationships between diet and health. Although plant foods provide a range of essential dietary components, they are particularly important as a source of dietary carbohydrates, providing almost all of the carbohydrate, and therefore much of the energy, in the adult diet. For example, Subar et al. Starch is the major storage carbohydrate in plants, and the major source of calories in many plant organs and foods. Its biophysical properties also have major impacts on food texture and other properties. Although available starch is readily digested in the small intestine, resistant starch (RS) and cell wall polysaccharides  are not digested, but are the major components of dietary fiber and are fermented by the colon microbiota to produce short chain fatty acids (SCFAs). NSPs provide a rigid structure surrounding plant cells and therefore affect the release and digestion of the cell contents. Starch is a mixture of two glucose polymers: amylose, which comprises (1→4) α-linked chains of up to several thousand glucose units and amylopectin which is highly branched (with (1→6) α-linkages as well as (1→4) α-linkages) and may comprise over 100,000 glucose residues. Amylose is largely unbranched but may contain a few long branches  in highly organized granules which vary in their abundances, sizes, and shapes between different species  separated by amorphous growth rings .Figure NSPs are made up of the hexose sugars glucose, galactose and mannose, the deoxy-hexoses rhamnose and fucose, glucuronic and galacturonic acids, and the pentose sugars arabinose and xylose -β-D-linked glucose units. The (1→4)-β-D-linked glucose chains associate by hydrogen bonding (both between and within strands) conferring strength to the cell wall. The linear polymers aggregate into either amorphous or crystalline regions. Cellulose is present in fungi, algae and higher plants, ranging from a few percent to 90% of the NSPs; the higher value being found in cotton fiber -β-linked D-xylose unit backbone which is substituted with L-arabinose at either the 3 or the 2 and 3 positions. Further modifications of these chains may occur, including the feruloylation of monosubstituted arabinose units, which may lead to oxidative cross-linking of cell wall components. Arabinoxylans from wheat grain are of particular interest as they constitute the principle source of fiber in flour  -β-linked D-glucose units substituted with (1→6)-α-linked D-xylose, which may be further substituted Fry, . The besGlucomannans comprise glucose and mannose units with β–(1→4) linkages and may also have (1→6)-α-linked glucose substitutions.Galactomannans are (1→4)-β-linked mannans substituted with (1→6)-α-linked galactose. Galactomannans are widely used in the food industry as thickeners and stabilizers; they are derived mainly from guar, locust, and carob beans (seeds). The glucuronomannans are (1→2)-β- linked mannose and glucuronic acid chains with substitutions of galactose and arabinose and are present in some edible fungi and alga. Galactan polysaccharides, (which can be sulfated) consisting of (1→3)-β-D galactose and (1→4)-3, 6-anhydro-α-D-galactose units, are found in numerous red seaweeds, and are used widely in the food industry as gels and thickeners. Carageenans and agars, which differ in whether the (1→4) anhydro galactose is the D- or L- form -β (as in cellulose) but interspersed with (1→3)-β-linkages. The (1→3)-β-linkages generally occur after three or four β-(1→4) linkages, but more extensive cellulose-like stretches of up to 20 (1→4)-β-linked residues have been reported in wheat bran , rhamnogalacturonan-I (RG I), and rhamnogalacturonan-II (RG II) -β-linked oligomers of fructose such as inulin), but also some glucose, which are present in many grass species and 15% of flowering plants Hendry, . Some ofThe solubility of polymers depends on several different factors and molecular properties, with contributions from monomer-solvent interactions as well as entropic contributions. The latter, in the case of polymers, is dominated by the conformational entropy  rather than the entropy of mixing.NSP (dietary fiber) is traditionally divided into two major groups as being either insoluble or soluble in aqueous solution AOAC, . This diA first general requirement for polymer solubility is that the monomers interact favorably with the solvent molecules (in this case water). Hence, the monomers need to have polar properties which enable them to form hydrogen bonds with the surrounding water molecules. The monomers in polysaccharides are monosaccharides which typically display high water solubility. Several NSPs also contain charged monosaccharides  which can have a strong positive influence on the solubility of NSPs. For instance, demethoxylation of pectin (resulting in carboxylic groups) increases the solubility considerably  resulting in segregative phase separation  is beneficial for the thickening ability. The thickening properties may be enhanced further if associative interactions are present in the polysaccharide, as for example, in some modified starches and celluloses.The ability to form gels is dependent on a relatively high solubility of the polysaccharide, in order for the gel structure to be able to hold water, and the formation of a continuous network in the solution. Thus, some interaction between the polysaccharides leading to association is necessary. This association can be mediated either through the hydrophobic effect, through partial local crystallization, through calcium bridges or through the formation of double and triple helices between molecules.It should also be noted that dispersed insoluble polysaccharides may also give rise to increases in viscosity and the formation of gels  and/or cooling under variable moisture conditions, which cause structural changes at the food, granular, and molecular levels. Different processing conditions therefore have different effects on starch structure and accessibility, with implications for digestibility.Heating native starch (50–100°C) in excess water results in gelatinization. During this process, the semicrystalline starch granule becomes completely disrupted. Hydrogen bonds that hold the double helical structure of α-glucan chains (the amylopectin fraction) together are broken, resulting in a greater proportion of amorphous starch material  tend to swell and thicken at lower temperatures than cereal grain starches. Cereal starches  break down more slowly with prolonged cooking. High amylose cereal starches are more resistant to swelling and behave like tuber starches during thermal treatments, with amylose leaching to a greater extent than amylopectin. Also, the concentration of starch when cooked affects its swelling properties. Above certain concentrations, the swollen starch granules entrap all available water and the aqueous phase will not separate from them. Cooked granules, in contrast to unswollen ones, can be disrupted by shearing.The chemical structure of starches can be dramatically altered by heat treatments such as baking, toasting, roasting, and frying. These may result in starch degradation (decomposition) by breaking the glycosidic bonds within the polysaccharide chains. However, the thermal decomposition of starches from different botanical origins showed no significant relationship between microstructure  and the thermal degradation process  and times  of the glucan polymer chains by hydrogen bonds forming starch crystallites which are less favorable to amylolysis  involve the treatment of starch under specific time/temperature and moisture conditions and modify the polysaccharide chain conformation and helicity without destroying the granular morphology or changing the birefringence of the granules  treatment up to 150–250 MPa decreases the gelatinization temperature as a result of alteration in granule structure  and a range of polyphenolic compounds have been shown to limit starch digestion Rehman, . HoweverStarches can be modified to alter their properties. For example, cross-linking of starch gives more stable gels, which are not disrupted by (over)cooking, changes in pH, and shear during processing. Depending on the type and degree of cross-linking, the derived modified starch becomes less affected by acid and sugar, and more resistant to acid, heat, and shear than native starch , bleaching, or oxidation to generate carboxyl groups and formation of esters and ethers. Chemical modifications introduce functional groups into the starch molecule, resulting in altered chemical and physical properties , which are fermented in the colon by bacterial enzymes  during ingestion and digestion can have marked effects on the textural and nutritional properties of foods. Depending on the composition of the walls and the processes that have been applied to them, chewing may induce cell rupture or cell separation. Cell separation occurs when the individual cell walls are stronger than the adhesive force between them, whereas if the adhesive force is stronger, cell rupture occurs. In general, when the cell walls are rich in pectin, cell wall separation will occur after hydrothermal processing, most likely due to calcium ions in the middle lamella being solubilized by the water and/or degradation of the pectin chains. Separation of the cells allows the intracellular nutrients to remain encapsulated in cell walls. If the cell walls are not permeable to digestive enzymes, then minimal digestion of the cell contents will occur until either the cell wall is breached by bacterial degradation in the colon or the digestive conditions change the permeability of the cell wall. In unprocessed foods, cell wall rupture is much more common than separation during chewing. The ruptured cells release their contents, making them available for digestion. The proportion of nutrients made available will depend on the ratio of ruptured to intact cells, which increases as particle size decreases. Therefore, the smaller the particle the greater the proportion of nutrients available  linked polyglycan chains. These are further digested by disaccharidases, notably maltase-glucoamylase (MGAM), which hydrolyses maltose, β-limit dextrins and cyclodextrins, and sucrase-isomaltase, which hydrolyses sucrose and isomaltose. The resulting monosaccharides  are then absorbed from the intestinal mucosa into the portal blood through GLUT2 transporters and sodium dependant transporters sodium-dependent glucose co-transporter (SGLT)  have shown large effects on the solubility and aggregation state. It has been suggested that β-glucan “solutions” typically consist of both individually dissolved chains and primary aggregates built up of rather few polymer chains  with about 80–90% of the ingested RS being fermented into SCFAs, particularly not only butyrate, but also propionate, acetate, isolvalerate, valerate, and isobutyrate . Conventionally (on the basis of Atwater factors), 1 g of carbohydrate was assumed to provide 17 kJ/g ; however, as carbohydrates vary greatly in their digestibility and metabolic potential, this can no longer be accepted as a reliable figure  was introduced by Jenkins et al. in 1981 as a means of documenting the glycemic response to different carbohydrate foods. GI refers to the glycemic effect of available carbohydrate  in a food relative to the effect of an equal amount of available carbohydrate , which is defined as having a GI of 100. Since then there have been some improvements in the methodology used to determine GI resulting in revised GI tables (Foster-Powell et al., The rate and extent of starch digestion in the intestinal lumen plays a crucial role in regulating the rise in post-prandial blood glucose and insulin concentrations (Warren et al., Foods with a low GI are digested slowly and do not cause large fluctuations in postprandial glycemia. This indicates that circulating glucose levels are effectively reduced, while glucose uptake in the small intestine continues (Jenkins et al., There are many factors that influence the postprandial glycemic and insulinemic response to food. These include the digestibility of the starch; the natural structure of the food matrix (Liljeberg et al., The extent of digestion during transit through the upper GI tract is important as it determines the proportion of starch that is available to the colon for fermentation (Fuentes-Zaragoza et al., The recommended daily intake of dietary fiber in the United Kingdom is 18 g per day (Department of Health, RS has similar attributes to NSPs in that it is fermented by colonic micro-organisms into SCFAs. Both NSPs and RS increase fecal bulk, by increasing the volume of bacteria. However, the effect of RS is minor in comparison with the effects of NSPs in bran, fruit, and vegetables (Cummings et al., As discussed earlier, NSPs and RS are fermented in the colon to produce SCFAs, and butyrate has received the most attention as it has beneficial effects on colonocytes, suggesting a mechanism for beneficial effects on colorectal cancer (Topping et al., Propionate is absorbed and metabolized aerobically in the liver while acetate passes via the liver into the blood from where it is used as an energy source. It is increasingly recognized that the products of colonic fermentation influence the body as a whole, through effects on the immune system mediated by the colonic epithelium, and through neuronal and hormonal feedback from the colon to upstream regions of the digestive tract (Wikoff et al., NSPs have also been suggested to reduce the reabsorption of bile acids (sometimes explained as “binding to bile acids” as demonstrated in vitro (Kritchevsky and Story, The carbohydrate components of the human diet are derived almost exclusively from plant sources and play crucial roles in food processing and in diet and health. Although widely regarded as primarily sources of energy they also have other impacts on diet and health, particularly the cell wall polysaccharides, which are the major components of dietary fiber. It is becoming clear that both dietary fiber and resistant forms of starch play a positive role in reducing risk factors for chronic diseases, including cardiovascular disease and certain types of cancer. These benefits could be exploited by crop and food scientists to develop new foods to combat the epidemic increases in diet-related disease which are occurring in both developed countries and rapidly expanding economies such as China and India.


Litchi chinensis cv. ‘Jingganghongnuo’ homograft (‘J/J’) and ‘Jingganghongnuo’/‘zhuangyuanhong’ heterograft (‘J/Z’) as compatible and incompatible combination, respectively, was used to study transcriptional changes between incompatible and compatible graft during graft union formation. Anatomical observation indicated that three stages  were critical for graft union formation and selected for high-throughput sequencing. Results indicated 6060 DEGs were differentially expressed in the compatible combination and 5267 DEGs exhibiting in the incompatible one. KEGG pathway enrichment analysis revealed that DEGs were involved in metabolism, wound response, phenylpropanoid biosynthesis and plant hormone signal transduction. The expression of 9 DEGs annotated in auxin pathway was up-regulated in compatible combination than that in incompatible combination. The IAA concentration confirmed that the IAA might promote the graft compatibility. In addition, 13 DEGs related to lignin biosynthesis were differentially expressed during graft healing process. Overall, our results provide abundant sequence resources for studying mechanisms underlying graft compatibility and establish a platform for further studies of litchi and other evergreen fruit trees.Plant grafting has been practiced widely in horticulture and proved as a useful tool in science. However, the mechanisms of graft healing or graft incompatibility remain poorly understood. In this study,  An example occurred in the litchi industry. In China, top-grafting generally aims at modifying the litchi variety of a mature tree. Top-grafting litchi trees begin producing fruits in 1–2 years, greatly reducing the juvenile period. In addition, plant grafting has been used as a convenient tool to study the long-distance movement of proteins, hormones and RNAs3. Despite the widespread use in horticulture and science, the mechanism of plant grafting remains poorly understood3.Grafting is an ancient asexual plant propagation technique that has been widely practiced in agriculture and horticulture. Fruit trees are grafted for propagation, avoiding a juvenile state, modifying plant growth or increasing stress resistance4. Several reports have described the key events for graft healing at the graft junction with regard to the histological and physiological aspects7. After grafting, ruptured cells collapse and adhere to the opposing tissue at the graft junction, where a mass of pluripotent cells termed callus is formed. The callus cells differentiate into vascular tissue-phloem and xylem to reconnect the scion and rootstock across the graft junction6. However, successful grafting termed ‘graft compatibility’ only occurs among the scion and rootstock with close taxonomic affinity. Incompatible grafts fail to form a vascular union between the scion and rootstock duo to insufficiently close genetic relationship. The reasons for graft incompatibility are as yet insufficiently understood2.In grafting, the shoot of one plant, termed the scion, is grafted onto the root of a different plant, termed the rootstock. Successful grafting requires the rejoining of a functional vascular system between the scion and rootstock, which is a complex biochemical and structural process1. As a wound healing process for plants, graft union formation presumably requires considerable re-programming of gene expression, protein translation, and metabolism8. Zheng et al.9 used cDNA-AFLP to examine the gene expression in hickory (Caryaca thayensis) during graft healing process, and the results indicated that the genes involved in indole-3-acetic acid (IAA) transport, cell cycle, signal transduction, and metabolism were differentially expressed. Recently, graft union development and vascular reconnection in model plant Arabidopsis has been studied at the histological and transcriptional level10. Microarray data revealed that graft union development was shown to involve wound and hormone signaling10. Melnyk et al.3 identified the genes involved in vascular reconnection at the graft junction and revealed that ALF4 and AXR1 were required locally below the graft junction to promote graft formation. However, grafting in woody fruit trees seem to be different from hypocotyl grafting11. Cookson et al.8 investigated the transcriptome of grapevine rootstock and graft interface tissues sampled 3 d and 28 d after grafting of over-wintering stems in the spring, results indicating that many differentially expressed genes were related to the activation of stem growth and metabolic activity in the spring. More recently, proteomic-based study was performed in watermelon to reveal the proteins involved in vascular connections between the rootstock and scion12.At the molecular level, the mechanism of graft healing process remains unclear, and no genes have been identified to be critical for graft union formationLitchi chinensis Sonn.) is one of the most important tropical and subtropical fruit trees in the Sapindaceae family. Grafting has been practiced in litchi for several hundred years in China. However, grafting success is variable because of rootstock-scion incompatibility. In a previous study, our group has shown that there were significant differences on the survival rate of ‘Jingganghongnuo’ grafted onto different litchi cultivars as rootstocks13. The compatible combinations had higher SOD, POD and PPO activities than incompatible combinations13. However, to the best of our knowledge, the underlying causes of graft incompatibility in litchi are unknown.Litchi  litchi heterograft was incompatible and Jingganghongnuo litchi homograft (‘J/J’) was compatibleTo determine the vital stages of the graft union formation in litchi, the field performance of the graft compatibility and incompatibility was recorded at different stages after grafting Fig. . The sci22. The reads were mapped to the reference sequence. Results indicated that there were at least 58% total mapped reads for each samples  in the six samples based on the method described by Audic & Claverie’ Figure . In the The DEGs were also subjected to KEGG pathway enrichment analysis. 4244 DEGs could be annotated in the compatible combination, while 3209 DEGs could be annotated in the incompatible combination. Genes involved in metabolic pathways, biosynthesis of secondary metabolites, plant-pathogen interaction and plant hormone signal transduction were differentially expressed during graft union formation Figure . It is w2Ratio| ≥ 1, false discovery rate (FDR) ≤ 0.001] at 2 h, 14 d and 21 d after grafting in the graft compatibility and incompatibility  validation. The correlation between RNA-seq and qRT-PCR was evaluated. The results showed that there was a significantly positive correlation between the two methods Fig. , thereby24. Using the iTAK pipeline V1.5 (http://bioinfo.bti.cornell.edu/tool/itak) to identify TFs and protein kinases, 30408 DEGs have a high homology index with 485 TFs of that belong to 66 known TF families  play a key role in regulating the secondary metabolism by controlling gene expression1, among which auxin was reported to be involved in vascular formation3. In this study, 9 DEGs annotated as TAA, YUC, Aux/IAA, auxin-responsive protein IAA and auxin-induced protein involved in IAA signal transduction or IAA biosynthesis pathways were differentially expressed, and all were up-regulated in compatible combination initially after grafting. The expression abundance decreased significantly during the late graft stages in compatible combination, however, the expression level in incompatible combination showed low and stable trends , syringyl (S), and p-hydroxyphenyl (H) units. Although researchers have studied lignin for more than a century, many aspects of its biosynthesis remain a matter of debate. 187 DEGs (Supplementary Dataset) involved in phenylpropanoid biosynthesis pathway were found. Overall 27 DEGs were identified, which were related to eight of the enzymes in the general phenylpropanoid pathway: 3 PALs, 2 C4Hs, 1 C3H, 4 4CLs, 4 CCRs, 4 COMTs, 1 F5H and 8 CADs. The DEGs showed increased trend during graft healing process, and the expression level in compatible combination was higher than that in incompatible combination .Lignin is the second most abundant biopolymer in nature contributing to up to 15% to 35% of the dry weight of woodion Fig. . FurtherTo investigate the signal transduction during graft healing process in litchi, the enriched signal transduction pathways and the KEGG pathways related to signal transduction were analyzed Table . 12 DEGsIn addition to wound response, DEGs involved in other signal transduction pathways were also identified Table . CaM and29. However, the molecular mechanisms of graft union formation remain unclear. In the present study, transcriptome changes between compatible and incompatible graft combination of litchi by digital gene expression were investigated, providing the first view of the complex network regulating graft compatibility in litchi.Graft healing procedure is a critical and complicated plant developmental process influenced by numerous endogenous and environmental factors. In graft union development, substantial changes occur, such as cell differentiation and organ regeneration, and even genetic material can be exchanged between the scion and stock at the graft site30. In this study, RNA-Seq technology was used to profile the litchi graft union transcriptome on the Illumina HiSeqTM 2000 platform, and approximately 72 million reads were obtained in total. A total of 6060 DEGs were identified among the three stages in compatible combination and 5267 DEGs were identified in incompatible combination, many of which were functionally annotated. The amount of information obtained from RNA-Seq analysis was, not surprisingly, much larger than that generated using a cDNA -AFLP approach, where only 49 genes were identified from hickory during graft healing process9.Next generation sequencing (NGS) is proved to be of great value for functional genomic study of non-model species11. As a stress situation for plants, there was an oxidative burst at the graft interface. Oxidative stress has been implicated in the graft incompatibility response of pear/quince heterografts31. In our previous study, the activities of antioxidant enzymes  were higher in compatible combination than that in incompatible combination. These antioxidant enzymes might involve in coping with oxidative stress and achieving successful grafting13. Our study here indicated that grafting upregulated wound response and antioxidant activity at the transcriptional level. DEGs involved in oxidative stress in the heterograft and autograft of grapevine was also reported by Cookson et al.11. Moreover, DEGs related to CaM or MAPK which are signal molecules were up-regulated at the early stage during graft healing process. The roles of these signal molecules in graft union formation are deserved to be further studied.Functional categorization of these DEGs revealed that the grafting triggered the expression of a large number of transcripts involved in wound response, stress response, plant hormone signal transduction, metabolic process and phenylpropanoid biosynthesis. Plant grafting is presumably perceived as a considerable stress3. Studies indicated that auxin is important in vascular development33. Yin et al.10 found that cell division commenced after auxin accumulation using a micrografting method in Arabidopsis. Our GC-MS results indicated that the IAA concentration in graft union was enhanced immediately after grafting, the same results were also observed at the early stage of hickory grafting9. Gene expression changes during graft healing process in hickory revealed that the genes related to IAA transport were differentially expressed9. The transport and release of IAA during graft union formation could induce the expression of auxin-response genes, thereby stimulating cell-elongation growth34. Our RNA–seq data showed that IAA related genes were elevated at 2 h after grafting. IAA signal exchange after wounding is indispensable for subsequent events during graft-union healing10. It is conceivable that similar mechanisms activated by IAA after wounding may play a part in the early responses of both graft parts in litchi. The auxin accumulation and auxin-response genes expression remind us of the importance of auxin during the signal-exchange process between scions and rootstocks. Recently, Melnyk et al.3 proposed an inter-tissue communication process that occurred at the graft junction and promoted vascular connection by tissue-specific auxin responses involving ALF4. However, the roles of auxin in the vascular connection during litchi grafting need further analyzed.A common theme to plant wound responses is the involvement of plant hormones, which play important roles in plant growth and developmentArabidopsis35 and poplar36. In the present study, the lignin biosynthesis related genes were upregulated at 21 d after grafting in the compatible combination. In previous study, cell-wall precursor and lignin biosynthesis were also enriched in the grapevine heterograft compared to the autograft control due to differences in plant wounding and defense responses11. The upregulation of PAL gene expression has also been reported during the graft union formation38. The expression of ParPAL1 from Prunus were differentially expressed between compatible and incompatible unions throughout graft development, and ParPAL1 transcripts were 3- fold more abundant in incompatible union than those of compatible one38. The expression of PAL gene in litchi was also differentially expressed between compatible and incompatible unions (data no shown).Cellulose and lignin are two important biopolymers in the vascular bundle, and successful grafting in plants requires the development of a functional vascular system between scion and rootstock. According to the currently accepted lignin metabolic pathway, almost all genes required to encode the related enzymes were found in our transcriptome data set. Many of the genes appear to be from multigene families, which is consistent with related reports of WRKY and 8 NAC were significantly differentially expressed. The NAC family is one of the largest plant-specific TF families with diverse roles in plant development and stress regulation39. The WRKY TFs participate in various plant defense responses and regulate plant growth and development40. As we known, grafting for plant is a kind of stress. We propose that these TFs are involved in graft healing process.In this study, 259 TFs were differentially expressed during graft healing process and, of particular note, 12 2. Yet, despite the numerous studies on grafting dealing with compatible/incompatible union development in vegetables41 and fruits45, the physiological and molecular mechanisms involved in graft incompatibility are still vague2. In the present study, the transcriptome changes between compatible and incompatible graft combinations in litchi were investigated. Based on the functional categorization of DEGs, grafting induced genes involved in wound response, auxin signal transduction, and metabolic process, which may play key roles in vascular differentiation. This hypothesis is supported by the recent study of Melnyk et al.3 who demonstrated the involvement of auxin in graft union formation and vascular reconnection in Arabidopsis.Graft incompatibility is defined as failure of the graft combination to form a strong and lasting functional graft unionThe number of DEGs related to stress response in compatible combination was higher than that in incompatible combination. The largest difference of genes expression profiling between compatible and incompatible combinations occurred at the early stage of graft union formation (2 h after grafting). It is worth noting that the phenylpropanoid biosynthesis pathway may play important roles in graft compatibility/incompatibility. Moreover, the gene involved in metabolic pathways was higher enriched in compatible combination than that in incompatible combination. These DEGs or pathways would provide some clues in understanding the mechanisms involved in the graft incompatibility.Our transcriptome analysis of the graft interfaces of compatible homograft and incompatible heterograft of litchi 2 h, 14 d and 21 d after grafting revealed interesting genes that might be involved in graft healing process. Moreover, a comparative analysis of the DEGs between compatible homograft and incompatible heterograft during graft union formation provided some clues revealing the mechanisms of graft incompatibility. A hypothetical model of graft-union development in compatible and incompatible grafting of litchi was proposed Fig. . Genes rLitchi chinensis cv. ‘Jingganghongnuo’ and ‘zhuangyuanhong’ used in the present study were planted in the experimental orchard of South China Agricultural University, Guangzhou, China. ‘Jingganghongnuo’ grafted onto ‘zhuangyuanhong’ (heterograft) as incompatible combination13, and ‘Jingganghongnuo’ grafted onto itself (homograft) as compatible combination. Tender branches were collected from the mature trees of litchi for splice grafting. The rootstocks were 10 years old which were considered to be in the adult phase. Each combination was grafted 100 times. Trees used in the experiment were not pruned or chemically treated during the experimental period. The grafting survival rate defined as the percent of plants survived the grafting was calculated 6 months after grafting. All samples were collected from the graft interfaces of compatible homograft and incompatible heterograft on 2 h, 14 d and 21 d after grafting. Samples were stored immediately in liquid nitrogen and then stored at −80 °C until use.46. Then the samples were softened in glycerol: alcohol mixture liquid (1:1) about 1 week and dehydrated in an ethanol series , each dehydrated steps for 90 min. After infiltration in Safranin overnight and decoloration by using dimethylbenzene, the specimens were embedded in paraffin. Samples were sectioned on a sliding microtome at a thickness of 10 μm. Finally, the paraffin sections were observed using the Zeiss photomicroscope II .The samples were collected on 2 h, 7 d, 14 d, 21 d, 32 d, 45 d after grafting and fixed in 2.5% glutaraldehyde in 0.03 M phosphate buffer above 24 hTotal RNA was extracted using the Quick RNA Isolation Kit  according to the manufacturer’s instruction and treated with DNase I  to remove genomic DNA contamination. Then the mRNA is enriched by using the oligo (dT) magnetic beads (for eukaryotes). Each library was pooled by mixing equal quantities of RNA from three biological replications for each stage. The steps of mRNA enrichment, mRNA fragmentation, second-strand cDNA synthesis, size selection, PCR amplification and subsequent sequencing using an Illumina HiSeq™ 2000  were performed at the Beijing Genome Institute .47. No more than 2 mismatches were allowed in the alignment. All tags that mapped to reference sequences of multiple genes were filtered out and the remaining tags were designated as unambiguous tags for gene expression analysis. The number of unambiguous tags of each gene was calculated and then normalized to RPKM (Reads per kb per Million reads).Raw reads were filtered to remove low quality tags (tags with unknown sequences ‘N’), empty tags (sequence with only the adaptors but no tags), and tags with only one copy number (probable sequencing error). Clean reads were mapped to the reference sequences (litchi non-redundancy genome) using SOAP aligner/SOAP223. The false discovery rate (FDR) was used to determine the threshold P-value in multiple tests. We used FDR ≤ 0.001 and the absolute value of log2Ratio ≥1 as the threshold to judge the significance of gene expression difference.To compare the differently expressed genes, the frequency of each tag in different digital gene expression libraries was statistically analyzed using the method of Audic & Claveriehttp://www.ncbi.nlm.nih.gov), Nt , COG , KO  and GO (Gene Ontology). GO functional annotation was performed by Blast2GO (v2.5.0) software48.Functional annotation of the differentially expressed genes was predicted based on the highest similarity in the following databases: Nr  according to the manufacturer’s recommendations. Transcript levels were analyzed using quantitative RT-PCR with the DyNAmo Flash SYBR Green qPCR kit  and the CFX96 qPCR System  according to the manufacturer’s instructions. Gene-specific primers were designed using the Primer 5.0 program  (Table Lcactin gene (HQ615689). Unigene expression levels were calculated using the 2ΔΔCt method49.Total RNA was isolated as described above and reverse transcribed with oligo (dT)a) Table . All reaGC-MS was used to determine the concentrations of indole-3-acetic acid (IAA) of the graft interfaces of compatible/incompatible combinations. Abundant cold methanol  was used to extract IAA, and internals standards, 2H-IAA 100 ng were simultaneously introduced into the samples. Crude extract was condensed by vacuum evaporating and hormones were re-extracted by ethyl acctate at pH 2.5–3.0. Then condensed IAA extracts were purified by Sep-Pak C18 columns. Thereafter, purified hormones were methylated using trimethylsilyldizomethane, then analyzed by GC-MS after an additional TMS reaction.P value was lower than 0.05.Statistical analyses were performed with SPSS software . One-way analysis of variance (ANOVA) was used to evaluate the Unigene expression levels on the different graft combinations. Two-way repeated measures anova was used to compare IAA concentrations in compatible and incompatible combinations at different time points. Heatmap diagram were analyzed with pheatmap methods using R software. Significant correlations between qRT-PCR and data obtained from transcriptome analysis were analyzed with SPSS software using Pearson’s correlation as the statistical metric. Significant correlations were considered only when an adjusted The clean data for the digital gene expression analysis were also deposited in the NCBI Sequence Read Archive under accession numbers SRR5313174 (JGHN_ZYH21d), SRR5313175 (JGHN_ZYH14d), SRR5313176 (JGHN_ZYH2h), SRR5313177 (JGHN_JGHN21d), SRR5313178 (JGHN_JGHN14d), and SRR5313179 (JGHN_JGHN2h).Supplementary PDF FileSupplementary XLS File


How organ-shaping mechanical imbalances are generated is a central question of morphogenesis, with existing paradigms focusing on asymmetric force generation within cells. We show here that organs can be sculpted instead by patterning anisotropic resistance within their extracellular matrix (ECM). Using direct biophysical measurements of elongating Drosophila egg chambers, we document robust mechanical anisotropy in the ECM-based basement membrane (BM) but not in the underlying epithelium. Atomic force microscopy (AFM) on wild-type BM in vivo reveals an anterior–posterior (A–P) symmetric stiffness gradient, which fails to develop in elongation-defective mutants. Genetic manipulation shows that the BM is instructive for tissue elongation and the determinant is relative rather than absolute stiffness, creating differential resistance to isotropic tissue expansion. The stiffness gradient requires morphogen-like signaling to regulate BM incorporation, as well as planar-polarized organization to homogenize it circumferentially. Our results demonstrate how fine mechanical patterning in the ECM can guide cells to shape an organ.DOI:http://dx.doi.org/10.7554/eLife.24958.001 All organs have specific shapes and architectures that are necessary for them to work properly. Many different factors are responsible for arranging the right cells into the correct positions to make an organ. These include physical forces that act within and around cells to pull them into the right shape and location.A structure called the extracellular matrix surrounds cells and provides them with support; it can also guide cell movements. It is not clear whether the extracellular matrix plays only a passive role or a more active, instructive role in shaping organs, in part, because it is difficult to measure the physical forces within densely packed cells.Drosophila melanogaster provide a simple system in which to study how organs take their shape. Crest et al. developed a method to measure forces in the fly ovary as it changes from being an initially spherical group of cells to its final elongated tube shape. The results revealed that, during this process, the extracellular matrix becomes gradually stiffer from one end of the ovary to the other. This change is the main factor responsible for the cell rearrangements that shape the developing organ.The ovaries of the fruit fly This work reveals that, along with providing structural support to cells, the mechanical properties of the matrix also actively guide how organs form. In the future, these findings may aid efforts to grow organs in a laboratory and to regenerate organs in human patients.DOI:http://dx.doi.org/10.7554/eLife.24958.002 Drosophila embryo, planar cell polarized (PCP) myosin contractility at the cell cortex generates junctional rearrangements that extend the germband, whereas in vertebrate embryos, PCP actin-based protrusions drive cell movements that extend the neural plate , including the basement membranes (BMs) that line all epithelia. In comparison to the action of cellular forces, the role of non-cellular influences on morphogenesis is poorly understood.Drosophila egg chamber (or ‘follicle’) is such a tissue , and found that germline growth resulted in a nearly spherical follicle at stages when elongation would normally have initiated  axes, an anisotropy associated with polarized Myosin II accumulation  to measure BM stiffness, calculating the Young’s modulus from the deflection of a cantilevered probe indenting into the basal follicle surface . TreatmeAFM measurements at the center of staged wild-type (WT) follicles showed that the BM gradually stiffens as oogenesis proceeds, increasing from ~30 KPa at stage 3 to ~40 KPa at stage 5 and ~70 KPa at stage 7 . Interesfat2, which encodes an atypical cadherin that controls basal PCP organization in the follicle epithelium (misshapen (msn), which encodes a kinase that negatively regulates integrin-mediated adhesion  relocalized swelling and bursting to this region  of Collagen IV along with other BM components. We asked whether any of these processes are controlled by an organizer-like activity that exists at the follicle poles, in which secretion of a cytokine signal activates JAK/STAT to distinguish cell fates along the A–P axis . Interesfat2, the prototypical egg elongation regulator, and their mispolarization of PCP elements such as BM fibrils  and ColIVα2 (Flybase: vkg). For ease, both are referred to in the text and figures as Collagen IV; detailed genotypes for all experiments are listed in UAS-DT-A, UAS-Perlecan (Flybase: Trol), UAS-DomeDN, and UAS-EHBP1  was provided by Dan Kiehart. Strains showing ectopic expression of ColIV–GFP (UAS–GFP–ColIVα1 + UAS–GFP–ColIVα2) were provided by S. Noselli .BM stiffness was measured  using ei20. Images were collected at 15 s or 30 s intervals on a Zeiss Axioimager with a Plan-Neofluor 10x/0.38NA objective.Follicles dissected in complete media were adhered to a poly-D lysine glass-bottomed dish (MatTek) before replacing the medium twice with dHData were analyzed and displayed using Microsoft Excel. All error bars represent standard errors and centers represent means. At least three biological replicates were undertaken for each experiment and the results are given in  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Marianne Bronner (Senior Editor) and three reviewers, one of whom, Allan Spradling (Reviewer #1), is a member of our Board of Reviewing Editors. The following individuals involved in review of your submission have agreed to reveal their identity: Eric F Wieschaus (Reviewer #2); Nick Brown (Reviewer #3).Thank you for submitting your article "Organ sculpting by patterned extracellular matrix stiffness" for consideration by Summary:Drosophila oocyte during its growth, thereby molding its final elongated shape. The system is specialized and builds on much previous work, but ultimately addresses a question of great generality and importance for understanding cell shape, namely whether anisotropies in (passive) resistance to an isotropic contractions or expansions are sufficient to result in polarized force distributions and morphogenetic movements. Previous works in Drosophila have long indicated that the follicle cells play the predominant role in shaping the oocyte. The initial expectation was that this would be a structure in the follicle cells themselves. What is exciting about the paper is the demonstration that the constraint is really in the ECM secreted by the follicles and the biophysical measurements that quantify and support that view. The demonstration that reprogrammed expression of ECM components and signaling regulators can alter stiffness is also highly significant. With several relatively minor changes described below the paper would be suitable for acceptance.The manuscript investigates the mechanisms that restrict circumferential expansion of the Essential revisions:1) The initial experiments to implicate a role for the follicle cells are weak and unnecessary given previous research on this system. The remarkably crude approach of completely ablating the follicular epithelium adds nothing of value and is not currently even interpreted correctly. The ablation of follicle cell junctions along the AP and DV axis is not described in detail nor is much data provided. The failure to detect junction-mediated anisotropy does not strongly implicate the ECM because it does not exclude anisotropy in cellular structures other than junctions, such as basal actin fibers. A better approach would be to cite classical papers by Wieschaus, and by Gutzeit where mosaics are used to show that the length vs circumference ratio is governed by an ovarian cell type that is not germ line derived, most likely the follicle cells.2) The authors should clarify whether their experiments apply to egg elongation generally or only to early elongation. The authors study the initial elongation of the follicle that takes place from about stage 3 to stage 7. However, most previous papers have studied elongation throughout oogenesis, including significant additional elongation during stages 8-14.3) The measurement of ECM stiffness using atomic force microscopy is a great strength of the paper. However, relatively few details or controls are shown to validate these measurements. Does the size, shape and exact placement of the tip matter? A large tip might be more relevant to the large scale morphogenetic forces under consideration. The force is applied at right angles to the material, but Young's modulus is conventionally determined by the deformation along the linear dimension of the material. Do the measurements depend on the questionable assumption that the ECM is locally isotropic in all dimensions? More primary data should be shown, rather than just ratios.4) It is not entirely clear that from the experiments presented whether additional mechanisms are needed for egg elongation beyond the reductions in collagen deposition at the poles expected for geometric reasons due to rotation. Artificially changing polar BM stiffness causes problems, but the authors’ experiments and arguments fail to delineate how precise the gradient must be, and whether graded JAK/STAT signaling is essential because of its effects on the stiffness profile or on BM synthesis. The authors should more clearly justify statements such as: "[these data]...reveal that PCP fibrils are insufficient alone to anisotropically resist tissue growth; the organ-shaping stiffness gradient requires patterned A-P BM levels". Essential revisions:1) The initial experiments to implicate a role for the follicle cells are weak and unnecessary given previous research on this system. The remarkably crude approach of completely ablating the follicular epithelium adds nothing of value and is not currently even interpreted correctly. The ablation of follicle cell junctions along the AP and DV axis is not described in detail nor is much data provided. The failure to detect junction-mediated anisotropy does not strongly implicate the ECM because it does not exclude anisotropy in cellular structures other than junctions, such as basal actin fibers. A better approach would be to cite classical papers by Wieschaus, and by Gutzeit where mosaics are used to show that the length vs circumference ratio is governed by an ovarian cell type that is not germ line derived, most likely the follicle cells.short egg, although we have previously examined seg mutants and cannot detect follicle shape defects before st. 8; the gene seems to regulate elongation at later stages (see point 2). Furthermore, osmotic bursting experiments revealed no significant differences between stage 8 WT and seg mutant follicles by  The authors should clarify whether their experiments apply to egg elongation generally or only to early elongation. The authors study the initial elongation of the follicle that takes place from about stage 3 to stage 7. However, most previous papers have studied elongation throughout oogenesis, including significant additional elongation during stages 8-14.The reviewers make an important point. Briefly, our goal here was to use the highly regular architecture of the early follicle to address the general question of whether basement membrane mechanics can directly instruct organ shape. Following stage 8, the non-uniform epithelial morphology, changes in nurse cell/oocyte volume, and coordinated actomyosin contractions in follicle cells create significant complications to analysis, particularly the mechanical assays at the heart of the manuscript. We note that most manipulations that cause follicle elongation defects at stage 8 also result in significant changes in final egg shape . We have3) The measurement of ECM stiffness using atomic force microscopy is a great strength of the paper. However, relatively few details or controls are shown to validate these measurements. Does the size, shape and exact placement of the tip matter? A large tip might be more relevant to the large scale morphogenetic forces under consideration. The force is applied at right angles to the material, but Young's modulus is conventionally determined by the deformation along the linear dimension of the material. Do the measurements depend on the questionable assumption that the ECM is locally isotropic in all dimensions? More primary data should be shown, rather than just ratios.Figure 2—figure supplement 2 now lays out how primary force-extension curves are used to derive stiffness scores, and the Methods have been expanded with additional detail.We have also now repeated the measurements in WT follicles, using a cantilever with a 10 µm round bead. As shown in Because local features of the matrix can change positional stiffness values, we made four indentations at a given position to obtain an accurate average measurement. The AFM measurements, involving calculations of values generated by indentations normal to the plane of the material, are derived using standard approaches within the field . Isotropy is an important assumption. As a reviewer inferred, the Hertzian model used to calculate stiffness assumes that the sample is homogenous and isotropically linearly elastic; this is nearly never the case with biological samples, and a particular concern given the apparent fibril-like structure of the follicle BM. However, to date it is the best approximation that mechanobiology has. Anisotropy of the matrix at the length scale of individual BM fibrils will certainly be interesting to investigate, but we do not currently have a good technique to do so.4) It is not entirely clear that from the experiments presented whether additional mechanisms are needed for egg elongation beyond the reductions in collagen deposition at the poles expected for geometric reasons due to rotation. Artificially changing polar BM stiffness causes problems, but the authors’ experiments and arguments fail to delineate how precise the gradient must be, and whether graded JAK/STAT signaling is essential because of its effects on the stiffness profile or on BM synthesis. The authors should more clearly justify statements such as: "[these data]…reveal that PCP fibrils are insufficient alone to anisotropically resist tissue growth; the organ-shaping stiffness gradient requires patterned A-P BM levels".2 between BM stiffness anisotropy and follicle aspect ration (vkg pattern suggests that it is not regulated by JAK/STAT signaling. However, JAK/STAT is indeed required for relatively lower incorporation of Col IV into the anterior BM, along with the lowered anterior stiffness that creates a mechanical gradient.Although we are struck by the relatively strong RWhile we acknowledge that EHBP1 overexpression at poles could be interpreted as neomorphic stiffening, we also see follicle rounding when the physiological JAK/STAT gradient is disrupted (by Dome-DN, We have now revised the text to clarify our conclusions about the necessity for both the circumferential PCP fibrils and the AP incorporation gradient. The former is based on the fact that follicles overexpressing either Dome-DN or Perlecan rotate appropriately and maintain circumferential collagen organization and stiffness, but nevertheless fail to elongate. The latter of course is based on the fact that Dome-DN overexpressing follicles also flatten the AP Collagen incorporation gradient.


We describe the production and characterization of bread wheat with grains that lack small B-type starch granules, have near-normal weight and starch content, and have novel grain and starch physicochemical properties. Aegilops peregrina, predicted that a single gene per haploid genome, Bgc-1, controls B-type starch granule content in the grain. To test whether bread wheat (Triticum aestivum L.) has orthologous Bgc-1 loci, we screened a population of γ-irradiated bread wheat cv. Paragon for deletions of the group 4 chromosomes spanning Bgc-1. Suitable deletions, each encompassing ~600–700 genes, were discovered for chromosomes 4A and 4D. These two deletions are predicted to have 240 homoeologous genes in common. In contrast to single deletion mutant plants, double deletion mutants were found to lack B-type starch granules. The B-less grains had normal A-type starch granule morphology, normal overall starch content, and normal grain weight. In addition to variation in starch granule size distribution, the B-less wheat grains differed from controls in grain hardness, starch swelling power, and amylose content. We believe that these B-less wheat plants are the only Triticeae cereals available that combine substantial alterations in starch granule size distribution with minimal impact on starch content.Our previous genetic analysis of a tetraploid wild wheat species,  Triticum aestivum L.), barley (Hordeum vulgare L.), and rye  as well as wild wheat or ‘goat grass’ species (Aegilops spp), have endosperm starch with a bimodal granule size distribution  differ in granule size distribution. The synthetic Aegilops has both A- and B-type starch granules like most Triticeae, whilst Ae. peregrina has A-granules but lacks B-granules. A population segregating for B-granule number was generated by crossing these two lines and was used to study the genetic basis of B-granule content. A combination of bulked segregant analysis and quantitative trait locus (QTL) mapping identified a major QTL located on the short arm of chromosome 4S that accounted for 44.4% of the phenotypic variation. The peak of this QTL was between a conserved orthologous marker called 4G and the end of the short arm of chromosome 4S, and was called Bgc-1 for B-granule content  and combined these in a single plant. This resulted in wheat plants with grains containing starch of a unimodal size distribution: the starch has A-granules but lacks B-granules. To our knowledge, these are the first domesticated Triticeae plants that have grains with A-type starch granules only. A preliminary characterization of the growth metrics of the B-granule-less wheat plants, the starch content of the grains, and the physicochemical properties of B-granule-less wheat starch is presented.Here, we have tested the hypothesis that mutation or deletion of all copies of orthologous 4 generation) and DNA (from the M2 generation) were obtained from Simon Griffiths and Simon Orford, John Innes Centre, Norwich, UK and were produced and distributed on behalf of the Wheat Genetic Improvement Network (WGIN). The deletions were induced by γ-irradiation as described by Bread wheat (cv. Paragon) deletion mutant lines (MJXB online). Homoeologue-specific markers were designed to the bread wheat orthologues of marker 4G, the conserved orthologous gene marker previously shown to flank the QTL for B-granule content in Aegilops  were designed to wheat genes between 4G and the telomere, and a D-genome-specific marker (4NS5.5) was designed to a gene between 4G and the centromere . The PCR products of KT71 were separated by capillary electrophoresis  of the lines containing putative homozygous deletions (as identified using M2 DNA) were grown and screened by PCR as above. Those plants containing confirmed homozygous deletions were grown, allowed to self-fertilize, and deletion mutant seed was collected. The A-genome deletion lines were grown and crossed pairwise with the D-genome deletion lines, in all possible combinations. The F2 plants resulting from self-fertilized F1 plants were screened using the KT71 marker only, to identify plants with homozygous deletions in both the A- and the D-genomes.Seeds  to check the quality and by q-PCR/fluorometer (Qubit) to check the quantity. Each pool was run using Illumina v3 chemistry on an Illumina HiSeq 2500 with 100 bp paired-end reads.DNA was extracted using a DNeasy Plant Mini Kit (Qiagen), according to the manufacturer’s instructions. DNA libraries and subsequent exome captures were performed at the Earlham Institute, Norwich, UK. For this, the NimbleGen v5.0 protocol (http://broadinstitute.github.io/picard/) compatibility (option: -M) and a read group header was assigned to each alignment (option: -R). The resulting SAM file was converted to BAM format and sorted using samtools (version 1.3). The MarkDuplicates tool of Picard (version 1.134) was then used to predict PCR and optical duplicates within the sorted BAM file. Suspected duplicate reads were marked (SAM flag 0x400), but not removed from the file. Finally, the output was filtered again to contain only reads mapped in proper pairs (SAM flag 0x2) and then indexed using samtools (version 1.3).The reads were aligned to the IWGSC RefSeq v1.0_parts pseudomolecule reference genome  using bwa-mem (version 0.7.12) . Short sThe reads were counted as follows. The multicov tool from the bedtools package . Likewise, the borders of the predicted deletion consist of at least four adjacent 1 Mb bins, each with >10% of the read count of the Paragon wild type; that is, at least four bins must have a value of >0.1 before we consider the deletion finished. The alignments at the borders of the predicted deletions were examined manually to identify the physical position at which coverage in the mutant line drops off.The normalized data were used to plot whole-genome coverage graphs in R for Windows (version 3.4) using ggplot2 facet_grid . Each chGrain size, weight, and dimensions were measured using a MARVIN seed analyser , according to the manufacturer’s instructions. Each grain sample was from a different plant.Mature grains were fractured in mid-section using a scalpel blade and mounted on the surface of an aluminium pin stub using double-sided adhesive carbon discs . The stubs were then sputter coated with ~15 nm gold particles in a high-resolution sputter-coater (Agar Scientific Ltd) and transferred to a Zeiss Supra 55 VP FEG scanning electron microscope . The samples were viewed at 3 kV and digital TIFF files were stored.Bgc-1 wild-type F2 line, with B-granules), and mutant . Starch content was measured using the Megazyme Total starch assay Kit (AA/AMG)  and the small-scale assay method as described by For these experiments, we used grains from three different genotypes: normal (Paragon), control  and secondly with values of 0.785–78.5 µm2 . The results were used to calculate the percentage of small granules per image and the mean values for each starch sample.Two samples of purified starch were placed on a microscope slide and 20 µl of Lugol’s solution (Sigma Aldrich) was added to each, followed by a coverslip. The slide was observed under a light microscope (Leica DM2500) set to bright field with a ×10 objective. From both starch samples on the slide combined, a minimum of 10 representative images were saved as high-resolution jpeg files with scale bars. The images were analysed using ImageJ software (imagej.nih.gov). Having set the scale, images were made binary, the ‘watershed’ function applied (to separate touching granules), and then the ‘analyse particles’ function was applied twice, first with values of 0.785 µmA FOSS 6500 wavelength scanning near-infrared microscope incorporating ISIScan software was used to measure the protein content, moisture content, and grain hardness for each sample according to the manufacturer’s instructions. Each sample of ~5 g of grain was run in duplicate using a ring cup. The sample spectra were compared with calibration set spectra taken from samples with known protein, moisture, and hardness compositions.–1.DSC was performed by Campden BRI . Three replicate DSC runs were performed per starch sample. The samples were hydrated at a ratio of 2:1 water to starch and left to equilibrate for a minimum of 1 h at ambient temperature. The instrument used was a Mettler Toledo DSC 1. Triplicate aliquots of 30 mg of starch were encapsulated in stainless steel pans and scanned from 25 °C to 115 °C at a rate of 10 °C ming for 5 min. The supernatant was removed and the tube and pellet were weighed. The weight of the starch pellet was calculated by subtracting the weight of the empty tube. Swelling power was calculated by dividing the weight of the starch plus water after swelling by the starch dry weight.Approximately 10 mg of starch was added to a pre-weighed, 2 ml round-bottomed tube and the weight of starch recorded to the nearest 0.1 mg. A 1 ml aliquot of water was added to the tube, which was inverted several times before and during incubation in an oven at 80 °C for 30 min. Tubes were allowed to cool to room temperature and then centrifuged at 1500 2, the vial was capped, vortexed, and incubated at room temperature for 10 min. After addition of 0.5 ml of 6 mM I2 in DMSO, the sample was vortexed and incubated whilst rotating slowly at 70–75 °C for 30 min. After vortexing, a 10 µl aliquot was transferred to a fresh glass vial and 100 µl of 6 mM I2 in DMSO and 800 µl of water were added. The sample was mixed and incubated at room temperature for 10 min before the absorbance at 600 nm was measured using a glass cuvette and a spectrophotometer. Mixtures of potato amylose and maize amylopectin  containing 0–50% amylose were prepared and assayed as above. Amylose content was estimated with reference to the amylose/amylopectin standard curve.Amylose content was measured in purified starch samples using a method based on that of Bgc-1, on the short arm of chromosome 4S in Ae. peregrina accounted for 44.4% of the control of B-granule content. The Bgc-1 locus lies between marker 4G and the telomeric end of the short arm of chromosome 4S. To test whether there is a similar locus responsible for B-granule content in T. aestivum, we screened a bread wheat cv. Paragon deletion mutant population for lines carrying deletions in the Bgc-1-orthologous regions of chromosome arms 4AL, 4BS, and 4DS.Our previous QTL analysis (Bgc-1 region (A1–A3) and five lines carried deletions in the D-genome Bgc-1 region (D1–D5)  lacked all of the genes tested  gave PCR products with marker 4G but not with any of the other markers tested. This suggests that in these lines, the deletion is close to, but does not include, 4G. One line (D5) lacked a PCR product with 4G only, suggesting that the deletion in this line may not include Bgc-1. Line D5 was not used in subsequent experiments.Out of a population of ~1000 Paragon deletion mutant lines, three lines carried deletions in the A-genome  (D1–D5) . No lines tested , suggestBgc-1 regions of both the A- and D-genomes, mutant lines A1–A3 were crossed pairwise to lines D1–D4, in all combinations. All crosses were successful except for A2×D3. The resulting F1 plants were grown and allowed to self-fertilize to generate F2 progeny. For each F2 family, 24 grains were sown. DNA was prepared from seedling leaves and was PCR screened using the KT71 marker only. Out of a total of 457 F2 plants that were successfully screened, only one plant carried homozygous deletions on both 4AL and 4DS (2 for deletions transmitted without bias is 6% (1/16). The observed proportion of homozygous double mutants in the F2 was 0.2%. F2 plants with single homozygous deletions on either the A- or D-genomes were also under-represented in the population.To generate plants with deletions in the  and 4DS . This AD2 plants with homozygous single deletions on either 4AL or 4DS were allowed to self-fertilize and the F3 seeds were each cut in half. Plants recovered from the embryo halves of the F3 seeds were screened for deletions using the KT71 marker. Four additional homozygous double mutant plants were discovered, and the non-embryo half-seeds were phenotyped for starch granule morphology (see below). Unfortunately, only one of these additional double mutants survived and was fertile. This second double mutant plant, like the first double mutant, derived from a cross between mutant lines A1 and D4. The original F2 AD double mutant plant and its progeny only were used in subsequent experiments.To screen for additional AD double deletion mutant plants, selected F2 plants that were PCR positive (i.e. wild type or heterozygous) for both deletions were grown and allowed to self-fertilize. An F2 plant which gave progeny that were all PCR positive (i.e. homozygous wild type) for both Bgc-1 deletions was selected.To screen for a wild-type segregant control line for the AD double deletion mutant plant, the FNext-generation sequencing was used to investigate the size and location of deletions in the genomes of Paragon lines A1 and D4. For this, libraries were created and hybridized to a set of exome capture probes before sequencing using an Illumina platform. We aligned the reads to the latest hexaploid wheat genome assembly (IWGSC RefSeq1.0) and counted the number of reads aligning within 1 Mb sized contiguous segments (bins) along the chromosomes. The read counts were normalized both within each mutant line and to the Paragon wild-type line.To predict the location of large deletions within the mutant lines, we applied several criteria to the normalized data. First, if the normalized read count within a bin was ≤10% of the level of the wild type, we highlighted said bin. Second, if at least four adjacent bins were highlighted, we considered them to belong to a single large deletion. Third, we considered the deletion to be finished if at least four adjacent bins had a normalized read count of >10% of the level of the wild type. Using these criteria, we predicted four large deletions within Paragon mutant A1 and six large deletions within Paragon mutant D4 ; Table 2To better define the extent of the predicted deletions, we examined the alignments at the borders of the deletions to determine the precise position at which the coverage in the mutant line drops off. We then identified the genes closest to the borders of each deletion. For this analysis, we used the TGAC_v1 gene models and only considered transcripts showing coverage in Paragon wild type . Using tThe positions of the markers used to discover the deletions on chromosome arms 4AL and 4DS ; Table 2The analyses described above showed that line A1 had four large (≥4 Mb) deletions  whilst line D4 had six large deletions . Note that the deletion on chromosome 4BL in line D4 is not in the region syntenic to the 4DS/4AL deletions. Thus, exome capture suggests that there are multiple large-scale, γ-induced deletions in each of the lines. The estimated sizes of these deletions ranged from 4 Mb to 110 Mb, the latter constituting a deletion of approximately a sixth of chromosome 4B . None of2 AD double mutant line, however, lacked small B-type starch granules when examined by light microscopy or SEM  plant were grown together with replicate wild-type segregant (control) and wild-type Paragon plants. The height of the primary tillers of both the control and B-less plants was significantly less than that of Paragon  bread wheat, like Aegilops, has Bgc-1 loci on the group 4 chromosomes that control B-granule content; (ii) there are active Bgc-1 loci on the A- and D-genomes; and (iii) there may be no active Bgc-1 locus on the B-genome of wheat. The deletions of the Bgc-1 regions on 4AL and 4DS were each predicted to contain 600–700 genes. Amongst these are 240 genes with homoeologues in both deletions. We hypothesize that one of these 240 genes is Bgc-1, assuming that the gene content in cv. Paragon is similar to that in cv. Chinese Spring.We hypothesized that wheat and other Triticeae may have a locus controlling B-type starch content on the group 4 chromosomes orthologous to the Bgc-1 deletions present in these lines, it is perhaps not surprising that their transmission from one generation to the next was selected against. Two double deletion mutant lines survived. These were derived from crosses between the same A- and D-genome deletions (A1×D4) but from different F1 plants. The mutant lines are therefore independent and this suggests that it is the combination of the two deletions on chromosomes 4A and 4D that is responsible for the lack of B-granules, rather than the combination of any of the other deletions present in lines A1 and D4.We found only a few double deletion mutant plants amongst the progeny of crosses between lines A1 and D4. Given the size of the Aegilops which we had examined previously using a similar method . This discrepancy implies that in the double-deletion mutant that appears to be unable to initiate B-type starch granules, more starch than normal is being deposited in the form of large A-granules.The reduction in small granule content in the mutant was not matched by equivalent reductions in starch content or grain weight. In wheat, the contribution of B-type starch granules to the total starch content is estimated to be ~30% . This result is predicted by the work of In other respects, the physicochemical properties of B-less and control starches did not vary. The gelatinization enthalpy of B-less mutant starch was the same as that of control starch. Our values are within the range expected for wheat starch  grew less well than wild-type Paragon. This could be due to the multiple large deletions (other than the deletions of the Bgc-1 regions) that are scattered throughout the genomes of the parent plants, A1 and D4 , that cause elimination of the amylose component of starch but which have a minimal effect on grain weight and yield , whose existence and location in the genome were inferred from work on wild wheat (Aegilops spp.). Similar approaches include the direct introgression of genes/traits from wild species to bread wheat, as in Ph1 locus in bread wheat (Herein, we have described the deletion of a locus, JXB online.Supplementary data are available at Fig. S1. Genotyping using marker KT71, by separation of PCR products by capillary electrophoresis.Fig. S2. Exome sequencing data for Chr 1B, line A1 with data for four genes close to the deletion borders.Fig. S3. Exome sequencing data for Chr 4A, line A1 with data for four genes close to the deletion borders.Fig. S4. Exome sequencing data for Chr 5D, line A1 with data for four genes close to the deletion borders.Fig. S5. Exome sequencing data for Chr 6A, line A1 with data for four genes close to the deletion borders.Fig. S6. Exome sequencing data for Chr 1A, line D4 with data for four genes close to the deletion borders.Fig. S7. Exome sequencing data for Chr 1B, line D4 with data for four genes close to the deletion borders.Fig. S8. Exome sequencing data for Chr 4B, line D4 with data for four genes close to the deletion borders.Fig. S9. Exome sequencing data for Chr 4D, line D4 with data for four genes close to the deletion borders.Fig. S10. Exome sequencing data for Chr 6D, line D4 with data for four genes close to the deletion borders.Fig. S11. Exome sequencing data for Chr 7A, line D4 with data for four genes close to the deletion borders.Table S1. Oligonucleotide primers for PCR.Table S2. Physical map positions of markers used for genotyping deletions.supplementary_figuresClick here for additional data file.supplementary_tablesClick here for additional data file.


Eurosurveillance has provided the European public health community with a platform to exchange relevant findings on communicable disease surveillance, prevention and control. From the outset, the journal has been open access and has not charged article processing costs.Since 1995, when a first pilot issue was published, Eurosurveillance archives demonstrates how the journal has matured over the years in terms of format and content. It shows, for example, the merging of the formerly weekly and monthly issues, acceptance of the ‘weekly’ for indexing in PubMed/MEDLINE and the evolution from a print and online journal to a full online journal and a gradual geographical expansion of the origin of published articles.In 2016, we celebrate 20 years of regular publication. A glimpse at the Eurosurveillance ran a commentary in its weekly edition, followed, two months later, by one entitled ‘Further evidence that MMR vaccine, inflammatory bowel disease, and autism are not linked’ [However, already from the start, topics covered were remarkably similar to those that are high on the public health agenda today. One of the articles in the pilot issue in 1995 gave an overview of immunisation schedules in Europe . Just on linked’ ,4. The pSince the early days of the journal, surveillance network outputs and outbreak reports have been regular content , with toRapid communications were an early feature for the journal at a time when rapid processing of articles was not a common element of scientific journals. The evolution, growth and opportunities offered by the Internet facilitated timely communication and fast turnaround times tremendously. The initially short news-like items are the element of the journal that has most evolved. Today, rapid communications are well-recognised short scientific dispatches. Several of them are among our most highly cited articles, but more importantly, their value has been in their impact on public health practice.While we have been able to present ‘firsts’ on several occasions ,23 and tEurosurveillance is rewarding. The journal has many supporters and collaborators in Europe and beyond whom we are not able to name individually. We would like to express our gratitude to them and also thank our board members, colleagues and publisher wholeheartedly for their continued support. Our 20th anniversary is a reason to celebrate. We marked the occasion on Wednesday 30 November with a lunchtime seminar ‘20 years of communicating facts and figures in a changing environment’, held on the margins of the European Scientific Conference on Applied Infectious Diseases Epidemiology (ESCAIDE). Two eminent speakers, David Heymann and Lawrence Madoff, highlighted changes in sharing information about communicable diseases from a public health perspective over the past 20 years. In addition, we present this selection of articles as a snapshot of the journal’s publications and evolution. The topics covered match those that have remained relevant over two decades and we hope our readers will enjoy browsing through this compilation.Working with