UGT79B31 is responsible for the final modification step of pollen-specific flavonoid biosynthesis in Petunia hybrida

Main conclusion 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. Electronic supplementary material The online version of this article (10.1007/s00425-017-2822-5) contains supplementary material, which is available to authorized users.


Introduction
Flavonoids are major plant secondary metabolites with over 9000 compounds distributed widely throughout the plant kingdom (Markham 1988;Richardson 1989;Williams and Grayer 2004;Anderson and Markham 2006). The biosynthetic pathways leading to the core skeletons have been well studied in terms of natural product chemistry, genetics and molecular biology, whereas pathways for subsequent modification steps such as glycosylation, acylation and methylation are being elucidated in several plant species (Anderson and Markham 2006;Saito et al. 2013). Flavonoids play important roles as pigments, UV protectants, attractants for pollinators, phytoalexins, signaling molecules and regulators of fertility and auxin transport (Falcone Ferreyra et al. 2012;Xu et al. 2015). Nevertheless, the enormous chemical diversity of flavonoid structures and the intricate distribution patterns in plant tissues and species make it difficult to Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00425-017-2822-5) contains supplementary material, which is available to authorized users. correlate specific flavonoid structures, including modification patterns, with their physiological functions.
As one of a few exceptions, a relationship between pollen-specific flavonol glycosides and pollen fertility is well established (Mo et al. 1992;van der Meer et al. 1992;Ylstra et al. 1992). Pollen of flavonoid-deficient mutants of petunia (P. hybrida) are unable to germinate, resulting in male sterility (Mo et al. 1992;Napoli et al. 1999). Likewise, maize (Z. mays) mutants deficient in flavonoids are also male sterile (Pollak et al. 1995). This phenotype was rescued by the exogenous addition of flavonol aglycones such as kaempferol and quercetin, indicating that flavonoids are essential for functional pollen in petunia and maize (Mo et al. 1992). In the pollen of petunia mutants, exogenously added flavonol aglycones are rapidly converted into the flavonol diglucosides, kaempferol/quercetin 3-O-glucosyl(1 → 2)galactosides that are identical to those accumulating in the wild type (Zerback et al. 1989;Vogt and Taylor 1995). Diglycosides of quercetin and isorhamnetin including quercetin 3-O-rhamnosyl(1 → 2)glucoside are prominent flavonoids in maize pollen (Ceska and Styles 1984).
To elucidate the role of flavonoids in pollen fertility, we have identified a gene encoding a glycosyltransferase, F3GT, that is responsible for the final modification step in the biosynthesis of petunia pollen-specific flavonoids. Based on an in silico search of the petunia transcriptome database, flavonol analyses in various organs and expression profiles of the candidate genes, we focused on UGT79B31. In vitro characterization of UGT79B31 and functional complementation of Arabidopsis mutants that lacked flavonoid diglycosides indicated that UGT79B31 encodes flavonoid 3-O-glycoside: 2″-O-glucosyltransferase.

Plant materials
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 m 2 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.
Arabidopsis thaliana accession Columbia-0 (Col-0; Lehle Seeds, Texas, USA) was used as the wild type. The Arabidopsis TILLING line CS95581 (ugt79b6-3) was previously described (Yonekura-Sakakibara et al. 2014 Sequences showing over 40% identity with the query were used for the analyses.

Degenerate PCR
Complementary DNA from poly(A) + RNA isolated from P. hybrida V26 anthers at developmental stages 2 and 3 ( Fig. 1a) was synthesized with SuperScript III Reverse Transcriptase (Invitrogen) using an oligo(dT) primer. Degenerate primers UGT79B6G73f and UGT79B6F289r (Table S1) were based on the amino acid sequences, GAETT(A/S) D and ELT(D/G)LPF, respectively. PCR was performed using a Taq polymerase (Takara Bio Inc., Kusatsu, Japan) with thermal cycling conditions as follows: PCR mixture was incubated at 98 °C for 10 s, followed by 30 cycles of PCR (one cycle consists of 98 °C for 10 s, 42 °C for 30 s, and 72 °C for 1 min), and finally incubated at 72 °C for 3 min. The resultant product (ca. 700 bp) was cloned and sequenced.

Quantitative reverse transcription PCR
RNA extraction and cDNA synthesis were performed as described previously (Yonekura-Sakakibara et al. 2007) using SuperScript™ III First-Strand Synthesis System (Invitrogen). Real-time PCR was performed as described previously (Yonekura-Sakakibara et al. 2014). The developmental stages of organs used for the analyses are shown in Fig. 1a. The primers, F3GalT9F_523F and F3GalT9R _587R for F3GalT, UGT79B31_196F and UGT79B31_257R for UGT79B31, Ph61074_1F and Ph61074_1R for Phcomp61074_c0_seq1, Ph27832_5F and Ph27832_5R for Phcomp27832_c0_seq1, and phSGN210759_1F and phSGN210759_1R for SGN210759 (Table S1) were designed using Primer Express software (Applied Biosystems). A dissociation program was used to confirm specific product formation. Plasmid DNAs containing the corresponding genes were used to create a calibration curve. The corresponding genes were amplified by PCR using primers, Phcomp61074_(−1)f and Phcomp61074_1390r for Phcomp61074_c0_seq1, Phcomp27832_(−1)f and Phcomp27832_1432r for Phcomp27832_c0_seq1, and PhSGN-U210759_131f and PhSGN-U210759_612r for SGN210759 (Table S1). Cloning of UGT79B31 is described in the next section. Real-time PCR was performed on three biological samples.

Cloning of UGT79B31 and in vitro assays
Full-length UGT79B31 was obtained by PCR using KOD-Plus-Neo DNA polymerase (Toyobo Co. Ltd., Osaka, Japan), petunia pollen cDNA and primers Phcomp17948_(−1)f and Phcomp17948_1368r (Table S1); the amplification product was cloned into the pCR2.1-TOPO vector. The sequence of the resultant plasmid, pKYS479, was confirmed to exclude PCR errors. To construct the protein expression vector, fulllength UGT79B31 was further PCR amplified using primers, UGT79B31/pColdProS2f and UGT79B31/pColdProS2r (Table S1) and pKYS479 as a template; the amplification product was cloned into pColdProS2 using an In-Fusion Advantage PCR Cloning Kit (Clontech). After verifying the sequence of the resultant plasmid, pKYS491, the plasmid was transformed into E. coli strain BL21star™ (DE3). Production and purification of the recombinant protein were performed as described previously (Yonekura-Sakakibara et al. 2014). Transformed cells were grown at 37 °C until A600 reached 0.5. After addition of isopropyl-β-dthiogalactopyranoside to a final concentration of 1 mM, cells were cultivated at 15 °C for 24 h. The cells were corrected and the protein was purified as a His fusion using TALON metal affinity resin (Clontech) according to the manufacturer's instructions. The ProS2 tag was removed using HRV3C protease (Novagen) according to the manufacturer's instructions. After exchanging the buffer for 50 mM Hepes-KOH, pH 7.5, proteins were concentrated using an Amicon Ultra filter (10,000 molecular weight cut-off; Millipore).
The standard enzyme assay reaction mixture was described previously (Yonekura-Sakakibara et al. 2012). The glycosyltransferase assay was performed at 30 °C according to Yonekura-Sakakibara et al. (2014). Flavonoid analyses were performed by UPLC/PDA/QTOF/MS as described above.

Complementation of Arabidopsis ugt79b6 mutants
The UGT79B6 (At5g54010) promoter region was amplified with the primers At5g54010promoter-683 and At5g-54010promoter-R (Table S1) and cloned into the pENTR/ D-TOPO vector to construct the plasmid pKYS449 (Yonekura-Sakakibara et al. 2014). The coding region of UGT79B31 was amplified with the primers 79B6Pro-UGT79B31CDSf and UGT79B31CDS-r (Table S1) and fused to pKYS449 using an In-Fusion HD Cloning Kit (Clontech) to yield pKYS492 (pENTR/D-TOPO/683 bp fragments of the UGT79B6 promoter fused to UGT79B-31CDS). Plasmids pGWB1 and pKYS492 were used for LR reactions to construct the binary vector pKYS498 using Gateway LR Clonase™ II Enzyme Mix (Invitrogen). Plasmid pKYS498 (pGWB1/683 bp fragments of the UGT79B6 promoter fused to UGT79B31 CDS) was used to transform Agrobacterium and Arabidopsis ugt79b6 mutants as described previously (Yonekura-Sakakibara et al. 2014).
For selection of positive transformants, seeds were germinated on one-half-strength MS-agar medium containing 50 μg/ml kanamycin and grown for 10 days at 22 °C with a 16 h light/8 h dark photoperiod before positive transformants were moved to soil. Flowers from three individual F1 plants were harvested and analyzed by UPLC/PDA/ QTOF/MS as described above. in this study correspond to stages 1-2, 3-4, 5-6, 7-8, 9-10, respectively, as described previously (Vogt and Taylor 1995).
and UGT79D1 (f) in petunia organs and tissues

In silico search of UGT(s) for pollen-specific flavonols
To identify the UGT(s) catalyzing the terminal glucosylation of pollen-specific flavonols, we conducted an in silico search of the Sol Genomics Network database (https://solgenomics.net/) using UGT79B6 from A. thaliana as a query. To date, the identified flavonoid glycosyltransferases that catalyze glycosylation of the sugar moiety attached to flavonoid aglycones (GGTs) belong to the UGT79 and UGT94 families. The UGT79, UGT91, UGT92 and UGT94 families belong to the same orthologous group that contains genes derived from a common ancestor (Yonekura-Sakakibara and Hanada 2011;Yonekura-Sakakibara et al. 2014). Therefore, we focused on genes in the above UGT families as potential candidate genes. Four UGTs that belong to the UGT79, UGT91 or UGT94 subfamilies (Phcomp61074_c0_seq1, Phcomp17948_c0_seq2, Phcomp27832_c0_seq1, SGN-U210759) were identified as candidates. We also searched for UGT genes expressed in pollen by degenerate PCR using primers based on conserved regions [GAETT(A/S)D and ELT(D/G)LPF] among UGT79B6 and the homologs from P. hybrida (Phcomp61074_c0_seq1 and Phcomp17948_ c0_seq2) ( Table S1). The amplified product (668 bp) corresponded to Phcomp17948_c0_seq2. Phcomp61074_c0_ seq1, Phcomp17948_c0_seq2 and Phcomp27832_c0_seq1 were designated as UGT79D1, UGT79B31 and UGT91S1, respectively, by the UGT nomenclature committee (Mackenzie et al. 1997; https://www.flinders.edu.au/medicine/ sites/clinical-pharmacology/ugt-homepage.cfm). Thus, we obtained four candidate genes (UGT79D1, UGT79B31, UGT91S1 and SGN-U210759) in total.
Expression profiles of the four UGT genes in various organs, tissues and developmental stages of petunia were analyzed by quantitative reverse transcription PCR (Fig. 1). The UGT79B31 transcripts accumulated predominantly in petals and anthers. The expression profile of UGT79B31 transcripts in anthers correlated well with the expression of pollen-specific F3GalT; however, expression of the other three UGT candidates did not correlate with F3GalT expression. SGN-U210759 transcripts were accumulated exclusively in anthers at developmental stage 1. The transcripts of UGT79D1 and UGT91S1 were accumulated in anthers at a negligible level. The UGT79B31 transcripts accumulated in petals and leaves in addition to anthers, consistent with the distribution patterns of flavonol 3-O-glucosyl(1 → 2)  Extracted ion chromatogram at m/z 620 ± 10 was used to detect of flavonols. c Mass spectra of the major peaks are shown glycosides. Therefore, we selected UGT79B31 for further analysis.
The specificity of UGT79B31 as a sugar acceptor was examined. UGT79B31 preferred flavonol 3-O-galactosides to the 3-O-glucosides. The activity with flavonol 3-O-rhamnosyl(1 → 6)glucosides were significantly lower than those for the 3-O-glucosides, suggesting that UGT79B31 had a low affinity for the 3-O-diglycosides as sugar acceptors (Table 1). The sugar donor specificity of UGT79B31 was also examined using kaempferol 3-O-galactoside or kaempferol 3-O-glucoside as potential sugar acceptors; in both cases, UGT79B31 highly preferred UDP-glucose to UDP-galactose. No UGT activity was detected for UDP-rhamnose, UDP-xylose, UDP-arabinose or UDPglucuronic acid ( Table 1). The effect of the end product, UDP, on enzyme activity was also investigated (
The commercial P. hybrida is derived from a whiteflowered P. axillaris and a purple-flowered species of the P. integrifolia clade (Segatto et al. 2014). Recently, the genome sequences of two inbred laboratory accessions regarded as the parents of P. hybrida, P. axillaris N and P. inflata S6, were released (Bombarely et al. 2016). We conducted a BLAST search for petunia OG8 UGTs in the petunia genome databases (P. axillaris and P. inflata, Sol Genomics Network, https://solgenomics.net/) using UGT79B31, UGT92A1 and The activity towards kaempferol 3-O-galactoside or UDP-glucose is taken to be 100%

GFP fused to UGT79B31 localizes to the cytosol
It has been suggested that F3GT, the enzyme corresponding to UGT79B31, may be a membrane-associated protein (Vogt and Taylor 1995). To investigate the subcellular localization of UGT79B31, GFP fused to the C-or N-termini of UGT79B31 (GFP-UGT79B31 and UGT79B31-GFP, respectively) were expressed transiently in N. benthamiana (Fig. 6). GFP fluorescence of GFP-UGT79B31 and UGT79B31-GFP was observed in the cytosol, as was the case with GFP only (Fig. 6). The protein detected by anti-GFP antibody corresponding to GFP-UGT79B31 (lane 2 in Fig. 6e) was slightly smaller than those corresponding to UGT79B31-GFP (lane 3 in Fig. 6e), suggesting that GFP-UGT79B31 may be truncated. GFP signals were also observed in the nuclei (Fig. 6a, b). The proteins smaller than 50-60 kDa were detected by anti-GFP antibody, suggesting that GFP signal in the nuclei may be caused by any cleavage products of GFP fused to UGT79B31. SignalP (Petersen et al. 2011) and TargetP (Emanuelsson et al. 2000) analyses reported that UGT79B31 has no chloroplast transit peptide, mitochondrial targeting peptide or secretory pathway signal peptide. The WoLF PSORT (Horton et al. 2007) analysis indicated that UGT79B31 localizes to the cytosol. These in silico analyses were consistent with our microscopic localization data.

Flavonoid biosynthesis in pollen
Our results indicate that UGT79B31 functions as a flavonol 3-O-glycoside: 2″-O-glucosyltransferase in planta and in vitro. The accumulation patterns of UGT79B31 transcripts and flavonol glycosides in petunia tissues and organs suggest that kaempferol/quercetin 3-O-galactosides and the 3-O-glucosides are the predominant sugar acceptors for UGT79B31 in anthers and petals, respectively. These results also suggest that pollen-specific accumulation of kaempferol/quercetin 3-O-glucosyl(1 → 2)galactoside in petunia is first determined by a glycosylation step catalyzed by F3GalT, not UGT79B31. In contrast, UGT78D2 which catalyzes the first 3-O-glucosylation in Arabidopsis is distributed nearly throughout the plant and UGT79B6 expression is specific to pollens (Yonekura-Sakakibara et al. 2014). Flavonol 3-O-diglycosides with a 1 → 2 inter-glycosidic linkage frequently accumulate as the major flavonoids in pollen of various plant species; however, the key enzymes determining the tissue/organ specificity of flavonol glycosides may be plant-species dependent. Transient expression analyses using leaves of N. benthamiana showed that UGT79B31 fused to GFP localizes in the cytosol. Plant UGTs are thought to be localized in the cytosol (Bowles et al. 2006), and subcellular location prediction programs also support the cytosol localization of UGT79B31; however, petunia flavonol aglycones were proposed to be synthesized in the tapetum, released into the locule, and taken up in the cytosol of developing pollen grains to be glycosylated (Vogt and Taylor 1995;Taylor and Hepler 1997;Xu et al. 1997). F3GT has been postulated to be a membrane-associated protein based on its behavior in salt-or detergent-containing buffers (Vogt and Taylor 1995). In humans and yeast, two membraneassociated UGTs have been reported (Albesa-Jove et al. 2014). In addition, petunia F3GalT catalyzes the reverse reaction at a similar efficiency as the forward reaction (Miller et al. 1999). Further investigations, including tissue localization of F3GalT and UGT79B31 in developing anthers and mutants deficient in F3GalT and UGT79B31, are required to fully describe the pathways for flavonoid metabolism and the role of flavonoids in pollen.