SE192:/MS1

Sample Set Information

ID	TSE1351
Title	Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor.
Description	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.
Authors	Tohge, T., Nishiyama, Y., Hirai, M.Y., Yano, M., Nakajima, J., Awazuhara, M., Inoue, E., Takahashi, H., Goodenowe, D.B., Kitayama, M., Noji, M., Yamazaki, M., and Saito, K.
Reference	Plant J. 2005 Apr;42(2):218-35
Comment

PubMed

Analytical Method Details Information

ID	MS1
Title	LC-MS (Targeted flavonoid profiling)
Instrument	LC: Agilent HPLC 1100 series MS: LCQ-DECA, ThermoQuest
Instrument Type
Ionization	ESI
Ion Mode	Positive
Description	Frozen leaves and roots were homogenized in 5 μl extraction solvent (methanol:acetate:H2O = 9:1:10) per 1 mg fresh weight of tissues by mixer mill (MM300; Retsch Gmbl & Co. KG, Haan, Germany) at 30 Hz. After centrifugation at 12000 g, cell debris was discarded and extracts were centrifuged again. Fifty microliters of supernatant was applied to HPLC/PDA/ESI‐MS system comprising a Finnigan LCQ‐DECA mass spectrometer (ThermoQuest, San Jose, CA, USA) and an Agilent HPLC 1100 series (Agilent Technologies, Palo Alto, CA, USA) as described previously (Jones et al., 2003; Yamazaki et al., 2003). HPLC was carried out on a TSK‐GEL RP‐18 (φ4.6 mm × 150 mm; TOSOH, Tokyo, Japan) at a flow rate of 0.5 ml min−1. Elution gradient with solvent A [CH3CN‐H2O‐TFA (10:90:0.1)] and solvent B [CH3CN‐H2O‐TFA (90:10:0.1)] and the following elution profile (0 min 100% A, 40 min 60% A, 40.1 min 100% B, 45 min 100% B, 45.1 min 100% A, 52 min 100% A) using linear gradients in between the time points. PDA was used for detection of UV‐visible absorption in the range of 250–650 nm. Nitrogen was used as sheath gas for the positive‐ion ESI‐MS performed at capillary temperature and voltage of 350°C and 5.0 kV, respectively. The tube lens offset was set at 10.0 V. Full scan mass spectra were acquired from 200–1500 m/z at 2 scans sec−1. Tandem MS analysis was carried out with helium gas as the collision gas. The normalized collision energy was set to 30%.
Comment_of_details

PubMed