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

TLDR

Integrating metabolomics and transcriptomics yields precise gene‑to‑metabolite networks for uncovering unknown gene functions, provided post‑transcriptional modifications are absent. The study aimed to comprehensively analyze the metabolome and transcriptome of Arabidopsis thaliana over‑expressing the PAP1 MYB transcription factor to identify novel genes involved in flavonoid biosynthesis. Metabolomic profiling combined targeted HPLC‑MS of flavonoids with ultrahigh‑resolution FT‑ICR‑MS, while transcriptomic profiling used a DNA microarray of 22,810 genes to assess PAP1‑induced changes. PAP1 over‑expression caused accumulation of cyanidin and quercetin derivatives, revealed eight new anthocyanins, and induced 38 genes—including two glycosyltransferases confirmed to catalyze flavonoid glucosylation—demonstrating the method’s power to uncover novel metabolic genes.

Abstract

Summary 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.