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Sample Set Information

ID TSE1347
Title Phytochemical genomics in Arabidopsis thaliana: A case study for functional identification of flavonoid biosynthesis genes
Description The completion of the whole genome sequence of Arabidopsis thaliana has made it possible to explore the phytochemical genomics in this species by determining gene-tometabolite correlation through the comprehensive analysis of metabolite accumulation and gene expression. In this study, flavonoid profiling of wild-type plants and T-DNA insertion mutants was analyzed using ultra-performance liquid chromatography (UPLC)/photodiode array detection (PDA)/electrospray ionization (ESI)/multiple-stage mass spectrometry (MSn). Detailed analysis of the metabolite changes in the mutants suggested the functions of genes that have been mutated. In silico coexpression analysis of genes involved in flavonoid metabolism in Arabidopsis was performed using a publicly available transcriptome database of DNA microarrays. We inferred a coexpression framework model of the genes involved in the pathways of flavonol, anthocyanin, and proanthocyanidin synthesis, suggesting specific functions and coregulation of the genes of pathway enzymes and transcription factors. The metabolic profiling of the omt1 mutant lacking a methyltransferase gene narrowed down by the coexpression analysis showed that AtOMT1 (At5g54160) is involved not only in the production of lignins and sinapoyl esters but also in the methylation of flavonols forming isorhamnetin. These results suggest that the functional genomics approach by detailed targetmetabolite profiling with transcriptome coexpression analysis provides an efficient way of identifying novel gene functions involved in plant metabolism.
Authors Tohge, T., Yonekura-Sakakibara, K., Niida, R., Watanabe-Takahashi, A. and Saito, K.
Reference Pure and Applied Chemistry, 2007, 79, 811-823

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Analytical Method Details Information

Instrument LC, Waters Acquity UPLC system; MS, Waters Xevo G2 Q-Tof
Instrument Type UPLC-QTOF-MS
Ionization ESI
Ion Mode Positive
Description Frozen leaves and roots were homogenized in 5 µl extraction solvent (methanol:H2O = 4:1) per 1 mg fresh weight of tissues by using a mixer mill (MM300: Retsch GmbH & Co. KG, Haan, Germany) for 3 min at 30 Hz. After centrifugation at 12 000 x g, the cell debris was discarded, and the extracts were centrifuged again. These supernatants were immediately used for the flavonoid analysis.

For flavonoid profiling, a Waters Acquity UPLC system (Waters Co., Massachusetts) fitted with Q-Tof Premier mass spectrometer (Micromass MS Technologies, Manchester, UK) was used. UPLC was carried out on a UPLC BEH C18 column (Φ2.1 mm x 100 mm, Waters) at a flow rate of 0.5 ml/min at 35 °C. An elution gradient with solvent A (0.1% formic acid in H2O) and solvent B (0.1 % formic acid in acetonitrile) and the elution profile—0 min, 100 % A; 20 min, 20 % B—using linear gradients in between the time points was applied. PDA was used for the detection of UV–vis absorption in the range of 210–600 nm. The time-of-flight (TOF) mass analyzer was used for the detection of flavonoid glycosides [M + H]+ and the peak of fragment ions in a positive ion scanning mode with the following setting: desolvation temperature, 180 °C with a nitrogen gas flow of 500 l/h; capillary spray, 3.2 kV; source temperature, 100 °C; and cone voltage was 35 V.


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