Ectopic expression of a basic helix-loop-helix gene transactivates parallel pathways of proanthocyanidin biosynthesis. structure, expression analysis, and genetic control of leucoanthocyanidin 4-reductase and anthocyanidin reductase genes in Lotus 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.

Figure 1.

Pathways to PA starter units (catechin type and epicatechin type) from flavan 3,4-diols in two model species. Superscript letters give references for genes encoding these enzymatic activities in given species and/or reference for structure of PA polymers. a, Abrahams et al. (2003); b, Devic et al. (1999); c, Tanner (2004); d, this paper; e, Paolocci et al. (2005); f, Foo et al. (1996); and g, Meagher et al. (2004).

Figure 2.

Phylogenetic tree of LAR proteins from legume species. The sequences from grapevine LAR1 (VvLAR1, AAZ82410) and LAR2 (VvLAR2, AAZ82411) and from apple LAR1 (MdLAR1, AAZ79364) and LAR2 (MdLAR2, AAZ79365 and AAX12186) were used as outgroups. Lu, L. uliginosus (AAU45392); Du, D. uncinatum (CAD79341); Pc, P. coccineus (CAI56322); Mt, M. truncatula (CAI56327). Numbers indicate bootstrap values.

Figure 3.

Structure of ANR, LAR1, and LAR2 genes. Boxes represent exons; lines introns, thicker lines 5′ and 3′ UTRs. The figure is drawn to scale. A, Genomic structure of ANR genes. Dotted box represents the 108-bp-long deletion that characterizes LcANR2 from LcANR1 cDNAs; barred boxes indicate part of exons 1 and 5 common to LcANR gene 1 and LcANR gene 2. B, Genomic structure of LAR1 genes. C, Genomic structure of LAR2 gene.

Figure 4.

Expression of LAR1, LAR2, and ANR genes in different organs of S41 L. corniculatus. Expression was determined by real-time RT-PCR analysis using EF-1α as the housekeeping gene. Values with the same letter do not differ significantly (P < 0.05).

Figure 5.

Expression of LAR1, LAR2, and ANR genes in L. corniculatus lines, polymorphic for leaf PA accumulation. Expression was determined by real-time RT-PCR analysis using EF-1α as the housekeeping gene. Values are average of two S transgenic lines (S50/6 and S50/9), two E lines (S50/10 and S50/11), two S50C lines (recipient genotype and 121.1 transgenic line), and two S41C lines (recipient genotype and 121.1 transgenic lines), replicated four times in each experiment. Values with the same letter do not differ significantly (P < 0.05).

Figure 6.

ANR and LAR enzyme assays with recombinant Lotus LcANR1 and LcLAR1-1, 1-2, 2-1, and 2-2. Thin-layer chomatography separation of assay extractions was done in n-buthanol:HOAc:water. A, Activity assay for LcANR1. Product identification by cochromatography with epicatechin and derivatization by subsequent spraying with DMACA. Lanes 1 and 2, ANR1, 50 and 100 μL protein, respectively; lane 3, denaturated ANR1, 100 μL protein; Ref, authentic epicatechin probe after DMACA spraying. B, Coupled assay of GH_DFR and Lotus LARs with ¹⁴C-DHQ as starter substrate after autoradiography. Lane 1, Denaturated LAR1-1, 50 μL; lane 2, LAR1-1, 50 μL protein; lane 3, LAR1-2, 50 μL protein; lane 4, LAR2-1, 50 μL protein; lane 5, LAR2-2, 50 μL protein; Ref, authentic catechin probe after DMACA spraying.

Figure 7.

Number and distribution of PA-containing cells in leaves of PA polymorphic genotypes. Total leaf PA (mg/g dry weight) for the lines S41 (23.3 ± 0.3), S50 (1.92 ± 0.04), S50/10 (6.64 ± 0.11), and S50/9 (0.80 ± 0.07) determined using the butanol-HCl hydrolysis method as described by Robbins et al. (2003). PM, Palisade mesophyll; VM, vascular mesophyll; SM, spongy mesophyll.

Figure 8.

Comparison of the effects of TT8 in Arabidopsis and Sn expression in Lotus on genes of PA biosynthesis. Gray line indicates the putative activity of the protein coded by LcLAR2.