Proanthocyanidins (PAs) are plant secondary metabolites and are composed primarily of catechin and epicatechin units in higher plant species. Due to the ability of PAs to bind reversibly with plant proteins to improve digestion and reduce bloat, engineering this pathway in leaves is a major goal for forage breeders. Here, we report the cloning and expression analysis of anthocyanidin reductase (ANR) and leucoanthocyanidin 4-reductase (LAR), two genes encoding enzymes committed to epicatechin and catechin biosynthesis, respectively, in Lotus corniculatus. We show the presence of two LAR gene families (LAR1 and LAR2) and that the steady-state levels of ANR and LAR1 genes correlate with the levels of PAs in leaves of wild-type and transgenic plants. Interestingly, ANR and LAR1, but not LAR2, genes produced active proteins following heterologous expression in Escherichia coli and are affected by the same basic helix-loop-helix transcription factor that promotes PA accumulation in cells of palisade and spongy mesophyll. This study provides direct evidence that the same subclass of transcription factors can mediate the expression of the structural genes of both branches of PA biosynthesis.Flavonoids represent one of the oldest, largest, and most diverse families of plant secondary metabolites. These compounds fulfill a multitude of functions during plant development (Winkel-Shirley, 2001) and are also of significant interest as antioxidant and anticancer agents in the human diet Tohge et al., 2005). Proanthocyanidins (PAs), also known as condensed tannins, are oligomeric and polymeric end products of the flavonoid biosynthetic pathway. They are widespread in nature, occurring in numerous plant species, including many important plant-derived food materials (Tanner, 2004).The beneficial effects of PAs on human health and the significant pharmacological activities of PAs have been recently reviewed (Marles et al., 2003;Dixon et al., 2005). In planta, PAs act as protectants against pathogens, pests, and diseases and additionally control seed permeability and dormancy . These compounds strongly affect plant quality traits, and the palatability and nutritive value of forage legumes are highly influenced by their concentration and structure (Barry and McNabb, 1999). High concentrations of PAs can decrease the palatability and digestibility of plants. By contrast, moderate quantities of PAs (2%-4% dry matter) in forage prevent proteolysis during ensiling and rumen fermentation, thereby protecting ruminants against pasture bloat (Tanner et al., 1995). In fact, by binding to proteins in the rumen, PAs reduce the rate of fermentation and increase the levels of proteins passing through the rumen of grazing animals. PAs therefore make the conversion of plant protein into animal protein more efficient, with reduced methane production (Kingston-Smith and Thomas, 2003). However, PAs only accumulate in the seed coats of the most valuable forage species, such as alfalfa (Medicago sativa) and clovers (Trifolium spp.), and are absent fro...

DNAs complementary to poly(A)+ RNA present in elicitor-treated cells of Phaseolus vulgaris L. were inserted into pBR325 and used to transform Escherichia coli strain JA221. A clone was identified that contained sequences complementary to mRNA encoding chalcone synthase, a regulatory enzyme of phenylpropanoid biosynthesis, which catalyzes the first reaction of a branch pathway specific to flavonoid and isoflavonoid biosynthesis. Rapid, marked but transient increases in chalcone synthase mRNA in response to elicitor treatment were observed by RNA blot hybridization with 32P-labeled chalcone synthase cDNA sequences. Induction of chalcone synthase mRNA governs the rate of enzyme synthesis throughout the phase of rapid increase in enzyme activity at the onset of accumulation of isoflavonoid-derived phytoalexins. The data are consistent with the hypothesis that elicitor causes a rapid transient stimulation of transcription of chalcone synthase gene(s) as an early event in the expression of the phytoalexin defense response.

Proanthocyanidins (PAs) are agronomically important biopolymers in higher plants composed primarily of catechin and epicatechin units. The biosynthesis of these natural products is regulated by transcription factors including proteins of the R2R3MYB class. To gain insight into the genetic control of the catechin and epicatechin branches of the PA pathway in forage legumes, here the effects of the expression of FaMYB1, a flavonoid R2R3MYB repressor from strawberry, in Lotus corniculatus (birdsfoot trefoil), were tested. It was found that in leaves of T(0) transgenic lines the degree of PA inhibition correlated with the level of FaMYB1 expression. These effects were heritable in the transgene-positive plant T(1) generation and were tissue specific as the suppression of proanthocyanidin biosynthesis was most pronounced in mesophyll cells within the leaf, whereas other flavonoid and phenolic compounds were substantially unaltered. The data suggest that FaMYB1 may counter-balance the activity of the endogenous transcriptional MYB-bHLH-WD40 (MBW) complex promoting proanthocyanidin biosynthesis via the catechin and epicatechin branches and that FaMYB1 does not interfere with the expression levels of a resident R2R3MYB activator of PAs. It is proposed that in forage legumes leaf cell commitment to synthesize proanthocyanidins relies on the balance between the activity of activator and repressor MYBs operating within the MBW complex.