SE159:/S1/M1/D1

Sample Set Information

ID	TSE1316
Title	Comprehensive flavonol profiling and transcriptome coexpression analysis leading to decoding gene-metabolite correlations in Arabidopsis.
Description	To complete the metabolic map for an entire class of compounds, it is essential to identify gene-metabolite correlations of a metabolic pathway. We used liquid chromatography-mass spectrometry (LC-MS) to identify the flavonoids produced by Arabidopsis thaliana wild-type and flavonoid biosynthetic mutant lines. The structures of 15 newly identified and eight known flavonols were deduced by LC-MS profiling of these mutants. Candidate genes presumably involved in the flavonoid pathway were delimited by transcriptome coexpression network analysis using public databases, leading to the detailed analysis of two flavonoid pathway genes, UGT78D3 (At5g17030) and RHM1 (At1g78570). The levels of flavonol 3-O-arabinosides were reduced in ugt78d3 knockdown mutants, suggesting that UGT78D3 is a flavonol arabinosyltransferase. Recombinant UGT78D3 protein could convert quercetin to quercetin 3-O-arabinoside. The strict substrate specificity of UGT78D3 for flavonol aglycones and UDP-arabinose indicate that UGT78D3 is a flavonol arabinosyltransferase. A comparison of flavonol profile in RHM knockout mutants indicated that RHM1 plays a major role in supplying UDP-rhamnose for flavonol modification. The rate of flavonol 3-O-glycosylation is more affected than those of 7-O-glycosylation by the supply of UDP-rhamnose. The precise identification of flavonoids in conjunction with transcriptomics thus led to the identification of a gene function and a more complete understanding of a plant metabolic network.
Authors	Yonekura-Sakakibara K, Tohge T, Matsuda F, Nakabayashi R, Takayama H, Niida R, Watanabe-Takahashi A, Inoue E, Saito K.
Reference	Plant Cell. 2008 Aug;20(8):2160-76. doi: 10.1105/tpc.108.058040. Epub 2008 Aug 29.
Comment

PubMed

Sample Information

ID	S1
Title	Arabidopsis thaliana (accession Columbia-0; Lehle Seeds)
Organism - Scientific Name	Arabidopsis thaliana
Organism - ID	NCBI taxonomy:3702
Compound - ID
Compound - Source
Preparation	Arabidopsis thaliana (accession Columbia-0; Lehle Seeds) was used as the wild type in this study. The mutants ugt75c1, ugt78d1, ugt78d2, ugt89c1, rol1-1, rol1-2, and rhm2-1 were described previously (Jones et al., 2003; Usadel et al., 2004; Tohge et al., 2005; Diet et al., 2006; Yonekura-Sakakibara et al., 2007). The T-DNA–inserted knockout mutants of omt1 (CS25167) and tt7 (CS6509) were obtained from the ABRC. The tt4 (C1) mutant was obtained from tt mutant lines induced by ion beam irradiation of Arabidopsis (Shikazono et al., 2003). The T-DNA–inserted mutant of Arabidopsis, lines SALK_114099 for UGT78D3 and SALK_085051 (rhm2-3, a homozygous knockout line) for RHM2, were obtained from the Salk Institute. T-DNA insertion lines were screened by PCR using specific primers for UGT78D3, RHM2 ,and T-DNA: UGT78D3f, UGT78D3r, RHM2f, RHM2r, LBa1, and RBa1 (see Supplemental Table 1 online). PCR products were sequenced to determine the exact insertion points. Plants were cultured on Murashige and Skoog–agar medium containing 1% sucrose (Valvekens et al., 1988) in a growth chamber at 22°C with 16 h/ 8 h light and dark cycles for 18 d or in a greenhouse at 22°C with 16 h/ 8 h light/dark for 4 weeks. Plants were harvested, immediately frozen with liquid nitrogen, and stored at −30°C until use. Five biological replicates were used for flavonoid profiling.
Sample Preparation Details ID
Comment

Analytical Method Information

ID	M1
Title	UPLC-PDA-ESI/Q-TOF/MS
Method Details ID	MS1
Sample Amount
Comment

Analytical Method Details Information

ID	MS1
Title	UPLC-PDA-ESI/Q-TOF/MS
Instrument	LC, Waters Acquity UPLC system; MS, Q-ToF Premier mass spectrometer
Instrument Type	UPLC-QTOF-MS
Ionization	ESI
Ion Mode	Positive
Description	Flavonol analyses were performed in quintuplicate. Frozen leaves were homogenized in 5 μL of extraction solvent (methanol: CH3COOH:H2O = 9:1:10, 0.02 mM naringenin-7-O-glucoside) per milligram of fresh weight of tissue in a mixer mill (MM300; Retsch) for 5 min at 30 Hz. After centrifugation at 12,000g, the supernatants were immediately used for flavonoid analysis. For flavonol profiling, a Waters Acquity UPLC system (Waters) fitted with a Q-TOF Premier mass spectrometer (Micromass MS Technologies) was used. UPLC was performed on a UPLC phenyl C18 column (Φ2.1 mm × 100 mm; Waters) at a flow rate of 0.5 mL/min at 35°C. Compounds were separated with a linear elution gradient with solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) from 0 min, 100% A to 10 min, 40% B. PDA was used for the detection of UV-visible absorption in the range of 210 to 500 nm. The TOF mass analyzer was used for the detection of flavonol glycosides [M + H]+ and the peak of fragment ions in a positive ion scanning mode with the following setting: desolvation temperature, 400°C with a nitrogen gas flow of 600 L/h; capillary spray, 3.0 kV; source temperature, 150°C; and cone voltage of 10 V for flavonoid glycosides [M + H]+ or 30 V for fragment ions.
Comment_of_details

Data Analysis Information

ID	D1
Title	Data Processing and Analysis
Data Analysis Details ID	DS1
Recommended decimal places of m/z
Comment

Data Analysis Details Information

ID	DS1
Title	Data processing and analisys
Description	For comprehensive flavonol profiling, nonprocessed MS data were converted to NetCDF format by MassLynx software (Micromass MS Technologies). Data analyses including principal component analysis were performed by the Phenomenome Profiler (Phenomenome Discoveries). Flavonol glycoside standards were used for the identification of the peaks in the plant extracts based on retention times, UV-visible absorption spectra, and mass fragmentation by tandem MS analysis. Other flavonol peaks were annotated by comparing their UV-visible absorption spectra, elution times, m/z values, and MS2 fragmentation patterns with 85 reference flavonoid compounds and the reported data (Tohge et al., 2005, 2007; Routaboul et al., 2006; Yonekura-Sakakibara et al., 2007). The mass spectrum data of standard compounds (see Supplemental Data Set 2 online) were recorded in the MASSBANK Database (http://www.massbank.jp/index-e.html).
Comment_of_details