Flavonoids form a large group of polyphenolic compounds, based on a common diphenylpropane skeleton, which occur naturally in plants. Included within this class of compounds are flavonols, flavones, flavanones, catechins, anthocyanins, isoflavonoids, dihydroflavonols and stilbenes. The flavonoids are mostly present as glycosides.
In tomato fruits, the main flavonoid found is narichalcone (naringenin chalcone) (Hunt et al, Phytochemistry, 19, (1980), 1415-1419). It is known to accumulate almost exclusively in the peel and is simultaneously formed with colouring of the fruit. In addition to naringenin chalcone, glycosides of quercetin and, to a lesser extent, kaempferol are also found in tomato peel.
Reports in the literature suggest that there is increasing evidence that flavonoids, especially flavonols are potentially health-protecting components in the human diet. Epidemiological studies suggest a direct relationship between cardioprotection and increased consumption of flavonoids, in particular flavonols of the quercetin and kaempferol type, from dietary sources such as onion, apples and tea (see, for example, Hertog et al, Lancet, 342 (1993), 1007-1011).
Flavonoids have been reported to exhibit a wide range of biological activities in vitro including anti-inflammatory, anti-allergic and vasodilatory activity (Cook et al, Nutritional Biochemistry, 7, (1996), 66-76). Such activity has been attributed in part to their ability to act as antioxidants, capable of scavenging free radicals and preventing free radical production. Within this group of compounds, those having the most potent antioxidant activity are the flavonols (Rice-Evans et al, Free Radical Research, 22, (1995), 375-383). In addition, flavonoids can also inhibit the activity of key processes such as lipid peroxidation, platelet aggregation and capillary permeability (see Rice-Evans et al, Trends in Plant Science, 2, (1997), 152-159).
Based on studies of this type, there is presently considerable interest in the development of food products from plants rich in such protective flavonoids.
It would be desirable to produce plants which intrinsically possess elevated levels of health protecting compounds such as flavonoids in order to develop food products with enhanced protective properties. Traditionally, the approach to improving plant varieties has been based on conventional cross-breeding techniques, but these are slow as they require time for breeding and growing successive plant generations. More recently, recombinant DNA technology has been applied to the general problem of modifying plant, genomes to produce plants with desired phenotypic traits. Whilst reference has been made in the literature to the use of genetic manipulation techniques in modifying the flavonoid biosynthetic pathway, as discussed beneath, it is notable that these attempts have been directed in general towards modifying pigmentary anthocyanin production. For example, several studies have attempted to modify the flavonoid pathway by the introduction of genes encoding for enzymes in the flavonoid pathway. Examples of these are EP522880, WO 90/11682, Goldsbrough et al in Plant Physiology (1994) 105:191-194 and Yoder et al, in Euphytica (1994) 79: 163-167.
Other studies have attempted to modify the anthocyanin biosynthesis pathway by altering the expression of a single transcription factor. Examples of these are WO 91/2059, Goldsbrough et al, (1996), Plant Journal, 9(6), 927-933, Mooney et al (1995), Plant Journal (1995), 7(2), 333-339, W093/14211, W093/18171 and Moyano et al, in Plant Cell, Vol 8, 1519-1532, 1996.
The flavonoid biosynthetic pathway is well established and has been widely studied in a number of different plant species (see, for example, Koes et al, BioEssays, 16, (1994), 123-132). Briefly, three molecules of malonyl-CoA are condensed with one molecule of coumaroyl-CoA, catalysed by the enzyme chalcone synthase, to give naringenin chalcone which rapidly isomerises, catalysed by chalcone isomerase, to naringenin. Subsequent hydroxylation of naringenin catalysed by flavanone 3-hydroxylase leads to dihydrokaempferol. Dihydrokaempferol itself can be hydroxylated to produce either dihydroquercetin or dihydromyricetin. All three dihydroflavonols subsequently can be converted to anthocyanins (by the action of dihydroflavonol reductase and flavonoid glucosyltransferase) or alternatively converted to flavonols such as kaempferol, quercetin and myricetin by the action of flavonol synthase.
Hitherto, studies in maize have identified two regulatory genes, C1 and R which are required for the production of anthocyanin (see Lloyd et al, Science, (1992), 258, 1773-1775). The C1 gene encodes a protein which has a myb DNA binding domain (Paz-Ares, et al, (1987) EMBO Journal, 6, 3553-3558) whilst the R gene encodes a protein with a basic helix-loop-helix domain characteristic of the myc family of transcriptional regulators (Ludwig et al, Proc. Natl. Acad. Sci., USA, 86 (1989) 7092-7096).
In Lloyd et al, referred to above, the expression of these anthocyanin pathway-specific transcriptional factors from the monocot maize in the dicots Arabidopsis thaliana and Nicotiana tabacum is disclosed. It is reported that anthocyanin production in both plant species is activated by R (Lc allele) in those tissues that normally produce anthocyanins but that C1 alone has no effect. Hybrid transgenic Arabidopsis expressing both transcription factors, placed under transcriptional control of the cauliflower mosaic virus 35S promoter, was reported to produce anthocyanins in tissues which would not normally express anthocyanins such as root, petal and stamen.
Crosses were performed using one R(Lc)-expressing line to pollinate three plants expressing C1. As all four parents were heterozygous, it would be expected that one in four of the progeny would contain both R and C1. In one cross, thirty six progeny were produced, of which four plants displayed anthocyanin accumulation in the roots and gave a small amount of anthocyanin in the petal and stamen tissue, the exact magnitude of the fold increase over wild type being unrecorded. Of the thirty eight progeny resulting from another cross, three plants gave anthocyanin in the petal tissue whereas in a third cross, no progeny with pigmentation in root or petal tissue were produced. Plants producing anthocyanins in the root and petals are assumed to contain both C1 and R, although this is not confirmed experimentally, nor is there any explanation as to why none of the progeny resulting from the third cross displayed anthocyanin accumulation in roots and petals. The authors of the study do not report whether the presence of both R and C1 in Arabidopsis leads to the constitutive production of anthocyanin in the whole plant or if anthocyanin production is restricted to certain areas of the plant.
Reports in the literature suggest that the introduction of a transcription factor from another species into Arabidopsis may cause it to behave atypically with respect to upregulation of anthocyanin production, (see for example, Mooney et al, Plant Journal, (1995), 7, 333-339). Here, overexpression of the gene encoding the Antirrhinum DELILA transcription factor under the control of the cauliflower mosaic virus 35S promoter gave rise to enhanced anthocyanin levels in both tomato and tobacco but in Arabidopsis, no obvious phenotype occurred. In tomato, increased pigmentation was produced in hypocotyl, cotyledon, leaves, stem and roots but no detectable enhancement of normal pigmentation in tomato fruits and testa of the seed was found. Enhanced pigmentation was seen in the flowers in tobacco but no vegetative parts were pigmented.
As reported by Goldsbrough et al, (1996) Plant Journal, 9(6), 927-933, expression of the Lc gene in tomato under the control of the cauliflower mosaic virus 35S promoter led to accumulation of anthocyanin only in those tissues which would normally be expected to produce anthocyanins such as leaves, stems, sepals, and the main vein of petals. In leaves, all the anthocyanin production occurred in the epidermal layer only. It is further reported that overexpression of homozygous Lc is lethal to the plant.
Quattrocchio et al, in Plant Cell, Vol 5, 1497-1512 (1993) describes the introduction of genes into parts of petunia leaves by particle bombardment. In these experiments the combination of the Lc and C1 transcription factors leads to anthocyanin accumulation in the leaves. No teaching is provided as to what the effects of this methodology are on the production of flavonoids other than anthocyanin such as flavonols. Furthermore it is well known in the art that experiments carried out by particle bombardment techniques cannot be used as reliable predictors for the effects which could be obtained by transgene incorporation of genes.
There are no reports in the literature which confirm that levels of anthocyanins are directly correlated to the levels of flavonols or other flavonoids. Also there is no disclosure in the literature of the manipulation of, flavonoids other than anthocyanins in plants by means of expression of transcriptional regulatory factors.
Accordingly, there remains a continuing need for the development of methods for enhancing the levels of flavonoids other than anthocyanins; in particular flavonols, in plants.
Furthermore there is a need to enhance the levels of flavonoids other than anthocyanins in plants or specific parts thereof, while avoiding a substantial increase in anthocyanin production, such that on the one hand the amount of desirable ingredients is increased, but the colour of the plant remains substantially the same.