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...

Summary• Here, we analyse the temporal signatures of ozone (O 3 )-induced hydrogen peroxide (H 2 O 2 ) and nitric oxide (NO) and the role of the second messenger guanosine 3′,5′-cyclic monophosphate (cGMP) in transcriptional changes of genes diagnostic for biotic and abiotic stress responses.• Within 90 min O 3 induced H 2 O 2 and NO peaks and we demonstrate that NO donors cause rapid H 2 O 2 accumulation in tobacco (Nicotiana tabacum) leaf. Ozone also causes highly significant, late (> 2 h) and sustained cGMP increases, suggesting that the second messenger may not be required in all early (< 2 h) responses to O 3 , but is essential and sufficient for the induction of some O 3 -dependent pathways.• This hypothesis was tested resolving the time course of O 3 -induced transcript accumulation of alternative oxidase (AOX1a), glutathione peroxidase (GPX), aminocyclopropancarboxylic acid synthase (ACS2) that is critical for the synthesis of ethylene, phenylalanine ammonia lyase (PALa) and the pathogenesis-related protein PR1a.• The data show that early O 3 and NO caused transcriptional activation of the scavenger encoding proteins AOX1a, GPX and the induction of ethylene production through ACS2 are cGMP independent. By contrast, the early response of PALa and the late response of PR1a show critical dependence on cGMP.

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