Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over‐expressing an MYB transcription factor

Abstract

SummaryThe 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 overexpression. 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.

Anthocyaninless1 gene of Arabidopsis thaliana encodes a UDP-glucose:flavonoid-3-O-glucosyltransferase

“…Our result corresponds with the result reported by Tohge et al (2005). They showed that At5g17050 encodes an UDP-glucose:flavonoid-3-O-glucosyltransferase and mutation in the At5g17050 gene decreased the amount of anthocyanin.…”

“…Both SALK_105779 and SALK_049338 accumulated less anthocyanin (f and g in Fig. 1) as reported previously (Kitamura et al 2004;Tohge et al 2005). When these mutants were crossed with the anl1 mutant, SALK_105779 complimented the mutant phenotype but SALK_049338 did not (h and i in Fig.…”

Metabolite profiling of Arabidopsis seedlings in response to exogenous sinalbin and sulfur deficiency

“…In sulfurdeficient plants, tyrosine (29) levels were increased 4.3-fold and phenylalanine (41) was almost unchanged. The accumulation patterns of flavonol diglycosides (93-97) were similar to those described for the ugt78d2 mutant, which lacks the ability to glucosylate flavonols at the 3-position as UDP-glucose: flavonoid 3-O-glucosyltransferase (Tohge et al, 2005). In the sinalbin (1)-supplemented plants, the decreased content of flavonol diglycosides with glucose attached at the 3-position (93, 95, 97) was correlated with increased levels of their downstream products, the flavonol triglycosides (98-100).…”

“…However, many of these secondary metabolites of Arabidopsis are as yet unidentified. Some metabolites were identified from a comprehensive analysis of mass fragmentation patterns and by comparison with published literature (Graham, 1998;Von Roepenack-Lahaye et al, 2004;Tohge et al, 2005;Kerhoas et al, 2006;Routaboul et al, 2006;Stobiecki et al, 2006;Besseau et al, 2007;Stracke et al, 2007). Additionally, compounds were identified by molecular weight determinations from protonated [M+H] + and deprotonated [MÀH] À molecular ions in the first order mass spectra and then MS/MS spectra where sufficient collision energy was applied to obtain maximum structural information.…”

“…UV/VIS absorption spectra were taken in the range of 200-600 nm. Known metabolites were identified by their mass spectrometric fragmentation patterns and relevant data reported in the literature (Graham, 1998;von Roepenack-Lahaye et al, 2004;Tohge et al, 2005;Kerhoas et al, 2006;Routaboul et al, 2006;Besseau et al, 2007;Stracke et al, 2007). The secondary metabolites identified by LC-MS were quantified by integrating the peak areas monitored at 254 nm using the CSASS (Complex Sample Analysis Software System) software and by normalizing them to the fresh wt of the extracted plant material and the peak areas of the internal standard genistein.…”