SE175:/S01/M01

Sample Set Information

ID	TSE1333
Title	A flavonoid 3-O-glucoside:2"-O-glucosyltransferase responsible for terminal modification of pollen-specific flavonols in Arabidopsis thaliana.
Description	Flavonol 3-O-diglucosides with a 1→2 inter-glycosidic linkage are representative pollen-specific flavonols that are widely distributed in plants, but their biosynthetic genes and physiological roles are not well understood. Flavonoid analysis of four Arabidopsis floral organs (pistils, stamens, petals and calyxes) and flowers of wild-type and male sterility 1 (ms1) mutants, which are defective in normal development of pollen and tapetum, showed that kaempferol/quercetin 3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosides accumulated in Arabidopsis pollen. Microarray data using wild-type and ms1 mutants, gene expression patterns in various organs, and phylogenetic analysis of UDP-glycosyltransferases (UGTs) suggest that UGT79B6 (At5g54010) is a key modification enzyme for determining pollen-specific flavonol structure. Kaempferol and quercetin 3-O-glucosyl-(1→2)-glucosides were absent from two independent ugt79b6 knockout mutants. Transgenic ugt79b6 mutant lines transformed with the genomic UGT79B6 gene had the same flavonoid profile as wild-type plants. Recombinant UGT79B6 protein converted kaempferol 3-O-glucoside to kaempferol 3-O-glucosyl-(1→2)-glucoside. UGT79B6 recognized 3-O-glucosylated/galactosylated anthocyanins/flavonols but not 3,5- or 3,7-diglycosylated flavonoids, and prefers UDP-glucose, indicating that UGT79B6 encodes flavonoid 3-O-glucoside:2″-O-glucosyltransferase. A UGT79B6-GUS fusion showed that UGT79B6 was localized in tapetum cells and microspores of developing anthers.
Authors	Yonekura-Sakakibara, K., Nakabayashi, R., Sugawara, S., Tohge, T., Ito, T., Koyanagi, M., Kitajima, M., Takayama, H. and Saito, K.
Reference	Plant J. 2014 Sep;79(5):769-82. doi: 10.1111/tpj.12580.
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Sample Information

ID	S01
Title	Arabidopsis thaliana accession Columbia-0
Organism - Scientific Name	Arabidopsis thaliana
Organism - ID	NCBI taxonomy:3702
Compound - ID
Compound - Source
Preparation	Arabidopsis thaliana accession Columbia-0 (Col-0; Lehle Seeds, http://www.arabidopsis.com/) was used as the wild-type in this study unless otherwise specified. The Arabidopsis TILLING lines CS88114, CS93994 and CS95581 for UGT79B6 (ugt79b6-1, ugt79b6-2 and ugt79b6-3, respectively), were obtained from the Arabidopsis Biological Resource Center (https://abrc.osu.edu/). The TILLING mutants are in the Col er105 (Big Mama) background (Torii et al., 1996). The services of the Seattle Tilling Project (http://tilling.fhcrc.org/) were used to screen for lines with point mutations in the coding region of UGT79B6. Specific primers for UGT79B6 (UGT79B6 left and UGT79B6 right), yielding a 1274 bp fragment starting 16 bp upstream from the first ATG, were used for screening. Homozygous knockout lines were screened by PCR using primers At5g54010(1f) and At5g54010(1362r). PCR products were sequenced to identify homozygous knockout lines using primer At5g54010(1258r). For phenotypic analyses, we used mutant lines back-crossed with Col–0 for three generations.
Sample Preparation Details ID
Comment

Analytical Method Information

ID	M01
Title	UPLC/PDA/ESI/Q-TOF/MS
Method Details ID	MS01
Sample Amount
Comment	Not written in the paper.

Analytical Method Details Information

ID	MS01
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	Frozen tissues were homogenized in 5 μl extraction solvent (1:1 methanol/H2O) per mg fresh weight of tissue in a mixer mill (MM300; Retsch, http://www.retsch.com/retsch-international/) for 5 min at 30 Hz. Supernatants were immediately used for analysis after centrifugation at 12 000 g. Flavonol analyses were performed essentially as described previously (Yonekura-Sakakibara et al., 2008). Untargeted metabolome analyses of wild-type (Ler) and ms1 mutants were also performed as described previously (Yonekura-Sakakibara et al., 2008).
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PubMed