SE187:/S1/M1/D1

Sample Set Information

ID	TSE1346
Title	Identification of a flavonol 7-O-rhamnosyltransferase gene determining flavonoid pattern in Arabidopsis by transcriptome coexpression analysis and reverse genetics.
Description	Glycosylation plays a major role in the remarkable chemical diversity of flavonoids in plants including Arabidopsis thaliana. The wide diversity encoded by the large family-1 glycosyltransferase (UGT) gene family makes it difficult to determine the biochemical function of each gene solely from its primary sequence. Here we used transcriptome coexpression analysis combined with a reverse genetics approach to identify a gene that is prominent in determining the flavonoid composition of Arabidopsis. Using transcriptome coexpression analysis accessible on the ATTED-II public data base, the expression pattern of a UGT gene, UGT89C1, was found to be highly correlated with known flavonoid biosynthetic genes. No C-7 rhamnosylated flavonols were detected in either of two T-DNA ugt89c1 mutants. This specific metabolite deficiency in the mutants was complemented by stable transformation with the genomic fragment containing intact UGT89C1. Glutathione S-transferasefused recombinant UGT89C1 protein converted kaempferol 3-O-glucoside to kaempferol 3-O-glucoside-7-O-rhamnoside and recognized 3-O-glycosylated flavonols and UDP-rhamnose as substrates, but not flavonol aglycones, 3-O-glycosylated anthocyanins or other UDP-sugars. These results show that UGT89C1 is a flavonol 7-O-rhamnosyltransferase. The abundance of UGT89C1 transcripts in floral buds was consistent with the flavonoid accumulation of C-7 rhamnosylated flavonols in Arabidopsis organs. Our present study demonstrates that the integration of transcriptome coexpression analysis with a reverse genetic approach is a versatile tool for understanding a multigene family of a metabolic pathway in Arabidopsis.
Authors	Yonekura-Sakakibara, K., Tohge, T., Niida, R. and Saito, K.
Reference	J Biol Chem. 2007 May 18;282(20):14932-41
Comment

PubMed

Sample Information

ID	S1
Title	Arabidopsis thaliana
Organism - Scientific Name	Arabidopsis thaliana
Organism - ID	NCBI taxonomy:3702
Compound - ID
Compound - Source
Preparation	A. thaliana plants (ecotype Columbia-0) were grown on germination medium (24) at 22 °C under 16-h/8-h light and dark cycles. The light intensity was 40 μmol of photons m-2 s-1. T-DNA insertion lines for UGT89C1 were obtained from the SALK Institute and were screened by PCR using specific primers for T-DNA and UGT89C1, UGT89C1f, UGT89C1r, LBa1, and RBa1. PCR products were sequenced to determine the exact insertion points.
Sample Preparation Details ID
Comment

Analytical Method Information

ID	M1
Title	UPLC/PDA/ESI-Q-TOF/MS
Method Details ID	MS1
Sample Amount	2 μL
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
Ionization	ESI
Ion Mode	positive
Description	Frozen Arabidopsis leaves were homogenized in extraction solvent (methanol: H2O = 4:1) with 5 μl of solvent/mg of fresh weight in a mixer mill (MM300; Retsch GmbH & Co. KG) for 3 min at 30 Hz. After centrifugation at 12,000 × g for 10 min, cell debris was discarded, and supernatants were recentrifuged. The resultant supernatants were immediately analyzed with a Waters Acquity UPLC system (Waters Corp.) fitted with a Q-TOF Premier mass spectrometer (Micromass MS Technologies). A 2-μl sample was applied to an ACQUITY UPLC BEH C18 column (Φ2.1 × 100 mm, 1.7 μm, Waters) at a flow rate of 0.5 ml/min with linear gradients of solvent A (0.1% formic acid in H2O) and solvent B (0.1% formic acid in methanol) set according to the following profile: 0 min, 95% solvent A +5% solvent B; 9 min, 60% solvent A + 40% solvent B; 11 min, 100% solvent B; 13 min, 95% solvent A +5% solvent B. The column temperature was 35 °C. Photodiode array (PDA) was used for detection of UV-visible absorption in the range of 210–500 nm. Electrospray ionization (ESI) with positive mode was used. The TOF mass analyzer was used for detection of flavonoid glycosides [M+H]+ and fragment ion peak in a positive ion mode scanning with the following setting; desolvation temperature was 450 °C at a nitrogen gas flow rate of 600 liters/h, capillary spray 3.2 kV, source temperature 150 °C, and cone voltage 35 V.
Comment_of_details

Data Analysis Information

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

Data Analysis Details Information

ID	DS1
Title	Data analysis
Description	Identification of the peaks in the plant extracts was based on comparisons of retention times, UV-visible absorption spectra, and mass fragmentation patterns by tandem MS analysis of the flavonoid standards. Other flavonoids with no standard compounds were annotated by comparison with the reported data in the UV-visible absorption spectra, elution time, m/z values, and MS2 fragmentation patterns.
Comment_of_details