SE166:/S1/M1

Sample Set Information

ID	TSE1328
Title	UGT79B31 is responsible for the final modification step of pollen-specific flavonoid biosynthesis in Petunia hybrida.
Description	UGT79B31 encodes flavonol 3- O -glycoside: 2″- O -glucosyltransferase, an enzyme responsible for the terminal modification of pollen-specific flavonols in Petunia hybrida. Flavonoids are known to be involved in pollen fertility in petunia (P. hybrida) and maize (Zea mays). As a first step toward elucidating the role of flavonoids in pollen, we have identified a glycosyltransferase that is responsible for the terminal modification of petunia pollen-specific flavonoids. An in silico search of the petunia transcriptome database revealed four candidate UDP-glycosyltransferase (UGT) genes. UGT79B31 was selected for further analyses based on a correlation between the accumulation pattern of flavonol glycosides in various tissues and organs and the expression profiles of the candidate genes. Arabidopsis ugt79b6 mutants that lacked kaempferol/quercetin 3-O-glucosyl(1 → 2)glucosides, were complemented by transformation with UGT79B31 cDNA under the control of Arabidopsis UGT79B6 promoter, showing that UGT79B31 functions as a flavonol 3-O-glucoside: 2″-O-glucosyltransferase in planta. Recombinant UGT79B31 protein can convert kaempferol 3-O-galactoside/glucoside to kaempferol 3-O-glucosyl(1 → 2)galactoside/glucoside. UGT79B31 prefers flavonol 3-O-galactosides to the 3-O-glucosides and rarely accepted the 3-O-diglycosides as sugar acceptors. UDP-glucose was the preferred sugar donor for UGT79B31. These results indicated that UGT79B31 encodes a flavonoid 3-O-glycoside: 2″-O-glucosyltransferase. Transient expression of UGT79B31 fused to green fluorescent protein (GFP) in Nicotiana benthamiana showed that UGT79B31 protein was localized in the cytosol.
Authors	Knoch, E., Sugawara, S., Mori, T., Nakabayashi, R., Saito, K. and Yonekura-Sakakibara, K. (2017)
Reference	Planta. 2018 Apr;247(4):779-790. doi: 10.1007/s00425-017-2822-5.
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PubMed

Sample Information

ID	S1
Title	Petunia hybrida
Organism - Scientific Name	Petunia x hybrida
Organism - ID	NCBI taxonomy:4102
Compound - ID
Compound - Source
Preparation	Seeds of P. hybrida inbred line, V26 (kindly provided by Dr. M. Nakayama, NARO Institute of Floricultural Science, Tsukuba, Ibaraki, Japan) were used. Petunia seeds were sown on one-half-strength MS-agar medium containing 2% (w/v) sucrose and placed in a 25 °C growth chamber with a light intensity of 70 µmol of photons m2 s−1 and a 16 h light/8 h dark photoperiod. After 8 weeks, the seedlings were transferred to sterile vermiculite and acclimated. After acclimation, plants were transferred to soil and grown for 3 months in a greenhouse.
Sample Preparation Details ID
Comment

Analytical Method Information

ID	M1
Title	UPLC/PDA/QTOF/MS
Method Details ID	MS01
Sample Amount
Comment

Analytical Method Details Information

ID	MS01
Title	Flavonoid profiling by UPLC/PDA/QTOF/MS
Instrument	LC, Waters Acquity UPLC system; MS, Waters Xevo G2 Q-Tof
Instrument Type
Ionization	ESI
Ion Mode	positive
Description	Fresh samples were extracted with 5 μl of 80% MeOH containing 2.5 µM lidocaine and 10-camphor sulfonic acid per mg fresh weight using a mixer mill with zirconium beads for 7 min at 18 Hz and 4 °C. After centrifugation for 10 min, the supernatant was filtered using an HLB μElution plate (Waters). The extracts (1 μl) were analyzed using LC-QTOF-MS (LC, Waters Acquity UPLC system; MS, Waters Xevo G2 Q-Tof). Analytical conditions were as follows: LC column, Acquity bridged ethyl hybrid (BEH) C18 (1.7 μm, 2.1 mm × 100 mm, waters); solvent system, solvent A [water including 0.1% (v/v) formic acid] and solvent B [acetonitrile including 0.1% (v/v) formic acid]; gradient program, 90% A/10% B at 0 min, 90% A/10% B at 0.1 min, 80% A/20% B at 25 min, 0% A/100% B at 25.1 min, 0% A/100% B at 27.5 min, 90% A/10% B at 27.6 min and 90% A/10% B at 30.0 min; flow rate, 0.3 ml/min; column temperature, 40 °C; photodiode array, 200–500 nm; flavonoid detection, 320 nm; MS detection: capillary voltage, + 3.0 keV, cone voltage, 25.0 V, source temperature, 120 °C, desolvation temperature, 450 °C, cone gas flow, 50 l/h; desolvation gas flow, 800 l/h; collision energy, 6 V; mass range, m/z 50‒2000; scan duration, 1.0 s; inter-scan delay, 0.014 s; data acquisition, centroid mode; polarity, positive; Lockspray (leucine enkephalin): scan duration, 1.0 s; inter-scan delay, 0.1 s. MS/MS data were acquired in the ramp mode in the following analytical conditions: (1) MS: mass range, m/z 50–1500; scan duration, 1.0 s; inter-scan delay, 0.014 s; data acquisition, centroid mode; and (2) MS/MS: mass range, m/z 50–1500; scan duration, 0.1 s; inter-scan delay, 0.014 s; data acquisition, centroid mode; collision energy, ramped from 10 to 50 V. In this mode, MS/MS spectra of the top 10 ions (> 10,000 counts) in an MS scan were automatically obtained. If the ion intensity was less than 10,000, MS/MS data acquisition was not performed and moved to the next top 10 ions.
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