ABSTRACT
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.
Subject(s)
Down-Regulation , Fragaria/genetics , Lotus/metabolism , Plant Leaves/metabolism , Plant Proteins/metabolism , Proanthocyanidins/biosynthesis , Transcription Factors/metabolism , Gene Expression , Gene Expression Regulation, Plant , Lotus/genetics , Plant Leaves/genetics , Plant Proteins/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Transcription Factors/geneticsABSTRACT
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.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Lotus/genetics , Oxidoreductases/genetics , Plant Proteins/genetics , Proanthocyanidins/biosynthesis , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/physiology , Blotting, Southern , Cloning, Molecular , DNA, Complementary/chemistry , Gene Expression Regulation, Plant , Lotus/enzymology , Lotus/metabolism , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Phylogeny , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Proteins/chemistry , Plant Proteins/metabolism , Plants, Genetically Modified/enzymology , Proanthocyanidins/analysis , Proanthocyanidins/chemistry , Signal Transduction , Transcriptional ActivationABSTRACT
A full-length sense Antirrhinum majus dihydroflavonol reductase (DFR) sequence was introduced into birdsfoot trefoil (Lotus corniculatus L.) in experiments aimed at modifying condensed tannin content and polymer hydroxylation in a predictable manner. Analysis of transgenic plants indicated lines that showed enhanced tannin content in leaf and stem tissues. In contrast to previous data from root cultures, levels of propelargonidin units were not markedly elevated in lines with enhanced tannin content. RT-PCR analysis of four selected lines indicated a correlation between enhanced tannin content and expression of the introduced DFR transgene. Using a contrasting approach we introduced a flavonoid 3'5' hydroxylase (F3'5'H) sequence derived from Eustoma grandiflorum into Lotus root cultures. Expression of the transgene was associated with increased levels of condensed tannins and in this case there was also no alteration in polymer hydroxylation. These results suggest that additional mechanisms may exist that control the hydroxylation state of condensed tannins in this model species.
