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 ...

Summary• Most Azospirillum plant growth-promoting rhizobacteria (PGPR) benefit plant growth through source effects related to free nitrogen fixation and ⁄ or phytohormone production, but little is known about their potential effects on plant physiology. These effects were assessed by comparing the early impacts of three Azospirillum inoculant strains on secondary metabolite profiles of two different maize (Zea mays) cultivars.• After 10 d of growth in nonsterile soil, maize methanolic extracts were analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC) and secondary metabolites identified by liquid chromatography ⁄ mass spectrometry (LC ⁄ MS) and nuclear magnetic resonance (NMR).• Seed inoculation resulted in increased shoot biomass (and also root biomass with one strain) of hybrid PR37Y15 but had no stimulatory effect on hybrid DK315. In parallel, Azospirillum inoculation led to major qualitative and quantitative modifications of the contents of secondary metabolites, especially benzoxazinoids, in the maize plants. These modifications depended on the PGPR strain · plant cultivar combination.• Thus, Azospirillum inoculation resulted in early, strain-dependent modifications in the biosynthetic pathways of benzoxazine derivatives in maize in compatible interactions. This is the first study documenting a PGPR effect on plant secondary metabolite profiles, and suggests the establishment of complex interactions between Azospirillum PGPR and maize.

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