SE160:/S1/M1

Sample Set Information

ID	TSE1319
Title	Two glycosyltransferases involved in anthocyanin modification delineated by transcriptome independent component analysis in Arabidopsis thaliana.
Description	To identify candidate genes involved in Arabidopsis flavonoid biosynthesis, we applied transcriptome coexpression analysis and independent component analyses with 1388 microarray data from publicly available databases. Two glycosyltransferases, UGT79B1 and UGT84A2 were found to cluster with anthocyanin biosynthetic genes. Anthocyanin was drastically reduced in ugt79b1 knockout mutants. Recombinant UGT79B1 protein converted cyanidin 3-O-glucoside to cyanidin 3-O-xylosyl(1→2)glucoside. UGT79B1 recognized 3-O-glucosylated anthocyanidins/flavonols and uridine diphosphate (UDP)-xylose, but not 3,5-O-diglucosylated anthocyanidins, indicating that UGT79B1 encodes anthocyanin 3-O-glucoside: 2''-O-xylosyltransferase. UGT84A2 is known to encode sinapic acid: UDP-glucosyltransferase. In ugt84a2 knockout mutants, a major sinapoylated anthocyanin was drastically reduced. A comparison of anthocyanin profiles in ugt84a knockout mutants indicated that UGT84A2 plays a major role in sinapoylation of anthocyanin, and that other UGT84As contribute the production of 1-O-sinapoylglucose to a lesser extent. These data suggest major routes from cyanidin 3-O-glucoside to the most highly modified cyanidin in the potential intricate anthocyanin modification pathways in Arabidopsis.
Authors	Yonekura-Sakakibara K, Fukushima A, Nakabayashi R, Hanada K, Matsuda F, Sugawara S, Inoue E, Kuromori T, Ito T, Shinozaki K, Wangwattana B, Yamazaki M, Saito K.
Reference	Plant J. 2012 Jan;69(1):154-67. doi: 10.1111/j.1365-313X.2011.04779.x. Epub 2011 Oct 14.
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Sample Information

ID	S1
Title	Arabidopsis thaliana
Organism - Scientific Name	Arabidopsis thaliana
Organism - ID	NCBI taxonomy:3702
Compound - ID
Compound - Source
Preparation	A. thaliana accession Columbia-0 (Lehle Seeds), or accession Nossen (Fedoroff and Smith, 1993)] were used as wild-types in this study. The mutant brt1-1 (ugt84a2-2) was described previously (Sinlapadech et al., 2007). The Arabidopsis transposon-tagged lines Ds53-4592-1 and Ds54-1263-1 for UGT79B1 (ugt79b1-1 and ugt79b1-2, respectively), and Ds11-5836-1 for UGT84A2 (ugt84a2-1) were obtained from RIKEN Bioresource Center. The T-DNA-insertion mutant GABI_765F10 for UGT84A1 (ugt84a1-1) was obtained from the Arabidopsis Biological Resource Center. Homozygous knockout lines were screened by PCR using specific primers for UGT79B1, UGT84A1, UGT84A2, Ds transposon and T-DNA: UGT79B1f, UGT79B1r, UGT84A1f, UGT84A1r, UGT84A2f, UGT84A2r, Ds5-2a, Ds3-2a, o8409, o3144. PCR products were sequenced to determine the exact insertion points. For analyses of anthocyanin accumulation, plants were cultured on one-half-strength MS-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 14 days with a light intensity of 40 μmol of photons m−2 s−1, then transferred on one-half-strength MS-agar medium containing 12% sucrose for 3 days with a light intensity of 80 μmol of photons m−2 s−1. Plants were harvested, immediately frozen with liquid nitrogen, and stored at −80°C until use. At least three biological replicates were used for anthocyanin analysis.
Sample Preparation Details ID
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Analytical Method Information

ID	M1
Title	HPLC/PDA/ESI–MS
Method Details ID	MS1
Sample Amount
Comment

Analytical Method Details Information

ID	MS1
Title	HPLC/PDA/ESI–MS
Instrument	LC: Agilent HPLC 1100 series MS: Agilent single quadrupole LC-MS 6120 series
Instrument Type
Ionization	ESI
Ion Mode	Positive
Description	Anthocyanin extraction was carried out in triplicate as described previously (Tohge et al., 2005). For anthocyanin profiling, Agilent HPLC 1100 series and Agilent single quadrupole LC-MS 6120 series (Agilent Technologies Inc., http://www.home.agilent.com/) were used with an Atlantis® T3 column (Φ4.6 mm × 150 mm, 5 μm, Waters) at a flow rate of 0.5 ml min−1 at 30°C. Anthocyanins were separated with solvent A (10% acetonitrile, 0.1% trifluoroacetic acid in water) and solvent B (90% acetonitrile, 0.1% trifluoroacetic acid in water) using an elution gradient (0 min, 0% B; 40 min, 40% B, 40.1 min, 100% B; 45 min 100% B; 45.1 min, 0% B; 50 min, 0% B). PDA was used for the detection of UV-visible absorption in the range of 200–600 nm. A mass analyzer was used for the detection of anthocyanin glycosides [M]+, and the peak of fragment ions in a positive ion scanning mode with the following setting: drying gas temperature, 350°C with drying gas flow of 12 L/min; capillary voltage, 4.0 kV; nebulizer pressure, 35 psig; fragmentor, 80 V; detection mode, scan (m/z 100–1400).
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