SE187:/S1/M1

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