Control of anthocyanin and non-flavonoid compounds by anthocyanin-regulating MYB and bHLH transcription factors in Nicotiana benthamiana leaves

Anthocyanins represent the major red, purple, violet and blue pigments in many flowers and fruits. They attract pollinators and seed dispersers and defend plants against abiotic and biotic stresses. Anthocyanins are produced by a specific branch of the flavonoid pathway, which is differently regulated in monocot and dicot species. In the monocot maize, the anthocyanin biosynthesis genes are activated as a single unit by a ternary complex of MYB-bHLH-WD40 transcription factors (MBW complex). In the dicot Arabidopsis, anthocyanin biosynthesis genes can be divided in two subgroups: early biosynthesis genes (EBGs) are activated by co-activator independent R2R3-MYB transcription factors, whereas late biosynthesis genes (LBGs) require an MBW complex. In addition to this, a complex regulatory network of positive and negative feedback mechanisms controlling anthocyanin synthesis in Arabidopsis has been described. Recent studies have broadened our understanding of the regulation of anthocyanin synthesis in flowers and fruits, indicating that a regulatory system based on the cooperation of MYB, bHLH and WD40 proteins that control floral and fruit pigmentation is common to many dicot species. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

The integration of metabolomics and transcriptomics can provide precise information on gene-to-metabolite networks for identifying the function of unknown genes unless there has been a post-transcriptional modification. Here, we report a comprehensive analysis of the metabolome and transcriptome of Arabidopsis thaliana over-expressing the PAP1 gene encoding an MYB transcription factor, for the identification of novel gene functions involved in flavonoid biosynthesis. For metabolome analysis, we performed flavonoid-targeted analysis by high-performance liquid chromatography-mass spectrometry and non-targeted analysis by Fourier-transform ion-cyclotron mass spectrometry with an ultrahigh-resolution capacity. This combined analysis revealed the specific accumulation of cyanidin and quercetin derivatives, and identified eight novel anthocyanins from an array of putative 1800 metabolites in PAP1 over-expressing plants. The transcriptome analysis of 22,810 genes on a DNA microarray revealed the induction of 38 genes by ectopic PAP1 over-expression. In addition to well-known genes involved in anthocyanin production, several genes with unidentified functions or annotated with putative functions, encoding putative glycosyltransferase, acyltransferase, glutathione S-transferase, sugar transporters and transcription factors, were induced by PAP1. Two putative glycosyltransferase genes (At5g17050 and At4g14090) induced by PAP1 expression were confirmed to encode flavonoid 3-O-glucosyltransferase and anthocyanin 5-O-glucosyltransferase, respectively, from the enzymatic activity of their recombinant proteins in vitro and results of the analysis of anthocyanins in the respective T-DNA-inserted mutants. The functional genomics approach through the integration of metabolomics and transcriptomics presented here provides an innovative means of identifying novel gene functions involved in plant metabolism.

Hyphenated full-scan MS technology creates large amounts of data. A versatile easy to handle automation tool aiding in the data analysis is very important in handling such a data stream. MetAlign softwareas described in this manuscripthandles a broad range of accurate mass and nominal mass GC/MS and LC/MS data. It is capable of automatic format conversions, accurate mass calculations, baseline corrections, peak-picking, saturation and mass-peak artifact filtering, as well as alignment of up to 1000 data sets. A 100 to 1000-fold data reduction is achieved. MetAlign software output is compatible with most multivariate statistics programs.