Ectopic Expression of aBasic Helix-Loop-HelixGene Transactivates Parallel Pathways of Proanthocyanidin Biosynthesis. Structure, Expression Analysis, and Genetic Control ofLeucoanthocyanidin 4-ReductaseandAnthocyanidin ReductaseGenes inLotus corniculatus 

Abstract

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...

Functional Characterization of Proanthocyanidin Pathway Enzymes from Tea and Their Application for Metabolic Engineering

“…3A, top panel). These data are consistent with the functional identification of LAR from other species (Bogs et al, 2005;Pang et al, 2007;Paolocci et al, 2007).…”

“…Although the LAR from D. uncinatum was confirmed to be active in vitro, transgenic tobacco and white clover (Trifolium repens) plants overexpressing this LAR did not produce detectable levels of (+)-catechin in vivo (Tanner et al, 2003), even though LAR activity could be detected in extracts from the leaves of these transgenic plants. Likewise, the expression of other LARs in transgenic plants also failed to result in the accumulation of catechin (Bogs et al, 2005;Pang et al, 2007;Paolocci et al, 2007).…”

“…The LAR enzymes characterized to date from D. uncinatum (Tanner et al, 2003), grape (Bogs et al, 2005;Maugé et al, 2010), M. truncatula (Pang et al, 2007), and L. corniculatus (Paolocci et al, 2007) produce transflavan-3-ols from the corresponding leucoanthocyanidin in vitro. CsLAR, which shares more than 60% amino acid identity with the above LARs, likewise produces (+)-catechin from leucocyanidin in vitro.…”

Transcription Factors of Lotus: Regulation of Isoflavonoid Biosynthesis Requires Coordinated Changes in Transcription Factor Activity

“…A Lotus gene belonging to this subgroup, bHLH114, decreased 60-fold in expression after elicitation. Members of this subgroup from other species encode proteins that interact with R2R3MYB TFs to coregulate the anthocyanin pathway (Goff et al, 1992) or the proanthocyanidin pathway (Nesi et al, 2001;Paolocci et al, 2007). The Lotus bHLHs belonging to subgroup 3f likely interact with MYB proteins regulating anthocyanin/proanthocyanin biosynthesis, some of which were also observed to decrease in expression in microarray and quantitative RT-PCR experiments: LjMYB5 (AtMYB5-like), LjMYB90 (subgroup 6), LjMYB133, and LjMYB134 (subgroup 4 sister).…”

Overexpression of CsDFR and CsANR enhanced flavonoids accumulation and antioxidant potential of roots in tobacco

“…Among them CsDFR and CsANR genes encoding enzymes have been reported as NADPH dependent enzymes and have their expression correlation with flavonoids accumulation (Singh et al 2009a;Kumar and Yadav 2012). The DFR and ANR enzymes have also been characterized from a number of other plant species (Shimada et al 2005;Shen et al 2006;Piero et al 2006;Paolocci et al 2007). Transgenic plants raised with some of these genes were analyzed primarily for change in flavonoids content in leaf or shoot part (Xie et al 2004;Takahashi et al 2006;Xie et al 2006;Kovinich et al 2011Han et al 2012Kumar and Yadav 2013;Kumar et al 2013b).…”