Poplar MYB115 and MYB134 Transcription Factors Regulate Proanthocyanidin Synthesis and Structure

Abstract

The accumulation of proanthocyanidins is regulated by a complex of transcription factors composed of R2R3 MYB, basic helix-loop-helix, and WD40 proteins that activate the promoters of biosynthetic genes. In poplar (genus Populus), MYB134 is known to regulate proanthocyanidin biosynthesis by activating key flavonoid genes. Here, we characterize a second MYB regulator of proanthocyanidins, MYB115. Transgenic poplar overexpressing MYB115 showed a highproanthocyanidin phenotype and reduced salicinoid accumulation, similar to the effects of MYB134 overexpression. Transcriptomic analysis of MYB115-and MYB134-overexpressing poplar plants identified a set of common up-regulated genes encoding proanthocyanidin biosynthetic enzymes and several novel uncharacterized MYB transcriptional repressors. Transient expression experiments demonstrated the capacity of both MYB134 and MYB115 to activate flavonoid promoters, but only in the presence of a basic helix-loop-helix cofactor. Yeast two-hybrid experiments confirmed the direct interaction of these transcription factors. The unexpected identification of dihydromyricetin in leaf extracts of both MYB115-and MYB134-overexpressing poplar led to the discovery of enhanced flavonoid B-ring hydroxylation and an increased proportion of prodelphinidins in proanthocyanidin of the transgenics. The dramatic hydroxylation phenotype of MYB115 overexpressors is likely due to the up-regulation of both flavonoid 39,59-hydroxylases and cytochrome b 5 . Overall, this work provides new insight into the complexity of the gene regulatory network for proanthocyanidin synthesis in poplar.Proanthocyanidins (PAs), also known as condensed tannins, are widespread polyphenols with diverse ecological functions. They are polymers of flavan-3-ols and, thus, end products of the phenylpropanoid and flavonoid pathways (Dixon et al., 2005). The PAs are the most broadly distributed secondary metabolites and are especially prominent in forest trees and woody plants ( Barbehenn and Constabel, 2011). PA accumulation in trees can be substantial; for example, in some species of poplar (genus Populus), PAs can constitute 25% of leaf dry weight. However, the accumulation of PAs also is highly plastic and varies with genotype and growth conditions (Hwang and Lindroth, 1997;Osier and Lindroth, 2006). In trees, PAs are common constituents of vegetative organs, including roots, leaves, bark, and flowers. Seasonal leaf drop in autumn and turnover of roots thus lead to substantial tannin input into forest soils, where it has been shown to slow litter decomposition and nutrient cycling (Schweitzer et al., 2008). In herbaceous plants, PAs are more restricted in distribution, but they can be found in leaves of legumes, 1 This work was supported by the Natural Sciences and Engineering Research Council of Canada, the Max Planck Society, the Academy of Finland, and the Canadian Genomics R&D Initiative.2 These authors contributed equally to the article. * Address correspondence to cpc@uvic.ca. The author responsible for distribution ...

Salicylic acid activates poplar defense against the biotrophic rust fungusMelampsora larici‐populinavia increased biosynthesis of catechin and proanthocyanidins

“…CHS , chalcone synthase; CHI , chalcone isomerase; F3H, flavanone 3‐hydroxylase; DFR , dihydroflavonol reductase; FLS , flavonol synthase; LAR , leucoanthocyanidin reductase; ANS , anthocyanidin synthase; ANR , anthocyanidin reductase. Note that MYB 134 and MYB 115 positively regulate PA synthesis in association with the basic helix–loop–helix enzyme bHLH 131 (James et al ., ). MYB 182 acts as a negative regulator of PA synthesis in poplar (Yoshida et al ., ).…”

“…Recently, MYB115 was shown to be another positive regulator of PA synthesis (James et al ., ; Wang et al ., ). These two transcription factors work in association with a bHLH131 transcription factor and a WD40 protein in the poplar MBW complex (James et al ., ). We showed that transcript levels of these regulatory genes increased in both BTH‐treated and SA‐hyperaccumulating lines.…”

Two R2R3‐MYB proteins are broad repressors of flavonoid and phenylpropanoid metabolism in poplar

“…MYB165 and MYB194 were first identified by being upregulated in MYB134 ‐overexpressing transgenic poplars, with overexpression ratios of 3.55‐ and 7.97‐fold, respectively (James et al ., ). MYB165 and MYB194 are paralogs from a whole genome duplication (Chai et al ., ), with 87% DNA sequence similarity and 79% protein sequence similarity between them.…”

“…MYB165 showed higher expression in young leaves than in older leaves, stems, and roots. Transcript levels were not significantly different between plants grown in the greenhouse and natural sunlight, a treatment previously shown to induce PA and anthocyanin synthesis (James et al ., ).…”

“…MYB182 was included for comparison and as a positive control. In the presence of bHLH131, MYB134 strongly activated DFR2 , ANR1 , and LAR1 promoters as previously shown (Figure a; James et al ., ). When MYB165, MYB194, or MYB182 expression constructs were added, the activation was repressed (Figure a).…”