Poplar MYB115 and MYB134 Transcription Factors Regulate Proanthocyanidin Synthesis and Structure.

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 high-proanthocyanidin 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 3',5'-hydroxylases and cytochrome b5 Overall, this work provides new insight into the complexity of the gene regulatory network for proanthocyanidin synthesis in poplar.

Transgenic upregulation of the condensed tannin pathway in poplar leads to a dramatic shift in leaf palatability for two tree-feeding Lepidoptera.

Transgenic hybrid aspen (Populus tremula x tremuloides) overexpressing the MYB134 tannin regulatory gene show dramatically enhanced condensed tannin (proanthocyanidin) levels, as well as shifts in other phenolic metabolites. A series of insect bioassays with forest tent caterpillars (Malacosoma disstria) and gypsy moth (Lymantria dispar) caterpillars was carried out to determine how this metabolic shift affects food preference and performance of generalist tree-feeding lepidopterans. Both species showed a distinct preference for the high-tannin MYB134 overexpressor plants, and L. dispar performance was enhanced relative to controls. L. dispar reached greater pupal weight and showed reduced time to pupation when reared on the MYB134 overexpressing poplar. These results were unexpected since enhanced condensed tannin levels were predicted to act as feeding deterrents. However, the data may be explained by the observed decrease in the salicinoids (phenolic glycosides) salicortin and tremulacin that accompanied the upregulation of the condensed tannins in the transgenics. We conclude that for these two lepidopteran species, condensed tannin levels are unlikely to be a major determinant of caterpillar food preference or performance. However, our experiments show that overexpression of a single regulatory gene in transgenic aspen can have a significant impact on herbivorous insects.

Interaction analysis of grapevine MIKC(c)-type MADS transcription factors and heterologous expression of putative véraison regulators in tomato.

MIKC(c)-type MADS-domain transcription factors include important regulators of floral development that interact in protein complexes to control the development of floral organs, as described by the ABC model. Members of the SEPALLATA (SEP) and AGAMOUS (AG) MADS clades include proteins involved in stamen and carpel specification and certain members of these families, such as tomato (Solanum lycopersicon) SlRIN and SlTAGL1, have been shown to regulate fruit development and ripening initiation. A number of expression studies have shown that several floral homeotic MADS genes are expressed during grapevine (Vitis vinifera) berry development, including potential homologues of these characterized ripening regulators. To gain insight into the regulation of berry development and ripening in grapevine, we studied the interactions and functions of grapevine floral homeotic MADS genes. Using the yeast 2- and 3-hybrid systems, we determined that the complexes formed during fruit development and ripening may involve several classes of floral homeotic MADS proteins. We found that a heterologously expressed grapevine SEP gene, VviSEP4, is capable of partially complementing the non-ripening phenotype of the tomato rin mutant, indicating that a role for this gene in ripening regulation may be conserved in fleshy fruit ripening. We also found that ectopic expression of a grapevine AG clade gene, VviAG1, in tomato results in the development of fleshy sepals with the chemical characteristics of tomato fruit pericarp. Additionally, we performed 2-hybrid screens on a library prepared from Pinot noir véraison-stage berry and identified proteins that may interact with the MADS factors that are expressed during berry development and that may represent regulatory functions in grape berry development.

Metabolic engineering and potential functions of proanthocyanidins in poplar.

Poplar (Populus spp.) is a widely distributed tree genus of significant economic and ecological importance. Poplar trees accumulate proanthocyanidins (PAs) in leaves, roots, and a variety of other tissues. Damage to leaves by insects causes a rapid accumulation of PAs, both at the site of damage and distally in undamaged leaves. This rapid PA accumulation is mediated by the activation of genes encoding enzymes involved in PA synthesis. PAs have been hypothesized to deter insect feeding and reduce the nutritive value of poplar leaf tissue, but experimental evidence supporting a role for PAs as an effective inducible defense against herbivores is lacking. Our recent paper described the identification of a MYB gene that regulates the PA pathway under multiple stress conditions, and we used this gene to constitutively activate the PA pathway in poplar. Here we describe observations that suggest that poplar PAs may have roles besides insect defense, for example, responses to UV light. The PA-modified trees will be a useful tool for analyzing the biological roles of PAs in this important model tree.

The wound-, pathogen-, and ultraviolet B-responsive MYB134 gene encodes an R2R3 MYB transcription factor that regulates proanthocyanidin synthesis in poplar.

In poplar (Populus spp.), the major defense phenolics produced in leaves are the flavonoid-derived proanthocyanidins (PAs) and the salicin-based phenolic glycosides. Transcriptional activation of PA biosynthetic genes leading to PA accumulation in leaves occurs following herbivore damage and mechanical wounding as well as infection by the fungal biotroph Melampsora medusae. In this study, we have identified a poplar R2R3 MYB transcription factor gene, MYB134, that exhibits close sequence similarity to the Arabidopsis (Arabidopsis thaliana) PA regulator TRANSPARENT TESTA2 and that is coinduced with PA biosynthetic genes following mechanical wounding, M. medusae infection, and exposure to elevated ultraviolet B light. Overexpression of MYB134 in poplar resulted in transcriptional activation of the full PA biosynthetic pathway and a significant plant-wide increase in PA levels, and electrophoretic mobility shift assays showed that recombinant MYB134 protein is able to bind to promoter regions of PA pathway genes. MYB134-overexpressing plants exhibited a concomitant reduction in phenolic glycoside concentrations and other minor alterations to levels of small phenylpropanoid metabolites. Our data provide insight into the regulatory mechanisms controlling stress-induced PA metabolism in poplar, and the identification of a regulator of stress-responsive PA biosynthesis constitutes a valuable tool for manipulating PA metabolism in poplar and investigating the biological functions of PAs in resistance to biotic and abiotic stresses.

The transcriptional response of hybrid poplar (Populus trichocarpa x P. deltoides) to infection by Melampsora medusae leaf rust involves induction of flavonoid pathway genes leading to the accumulation of proanthocyanidins.

The transcriptional response of hybrid poplar (Populus trichocarpa x P. deltoides) to poplar leaf rust (Melampsora medusae) infection was studied using the Populus 15.5K cDNA microarray. Pronounced changes in the transcriptome were observed, with approximately 20% of genes on the array showing either induction or repression of transcription within the 9-day infection timecourse. A small number of pathogen-defense genes encoding PR-1, chitinases, and other pathogenesis-related proteins were consistently upregulated throughout the experimental period, but most genes were affected only at individual timepoints. The largest number of changes in gene expression was observed late in the infection at 6 to 9 days postinoculation (dpi). At these timepoints, genes encoding enzymes required for proanthocyanidin (condensed tannin) synthesis were upregulated dramatically. Phytochemical analysis confirmed that, late in the infection, proanthocyanidin levels increased in infected leaves. Strongly M. medusae-repressed genes at 9 dpi included previously characterized wound- and herbivore-induced defense genes, which suggests antagonism between the tree responses to insect feeding and M. medusae infection. In this highly compatible plant-pathogen interaction, we postulate that the biotrophic pathogen evades detection and suppresses early host responses.