Abstract

   Tricetin (5,7,3′,4′,5′-pentahydroxyflavone) is a dietary flavone from
   flowers of Myrtales plants with demonstrated functions in promoting
   human health. By contrast, the bioactivity of its glucosylated
   derivative tricetin 4′-O-glucoside has not been extensively explored.
   We conducted metabolite profiling analysis of pomegranate (a Myrtales
   plant) floral tissues and revealed that tricetin and tricetin
   4′-O-glucoside accumulate in anthers, but not petals. In addition, the
   comparative analysis of anther and petal transcriptomes identified 10
   UGTs that are more highly expressed in anthers than petals. Of the 10
   UGTs, PgUGT76Z1 and PgUGT73AL1 glucosylated specifically at the 4′-O
   position of tricetin to form tricetin 4′-O-glucoside. The phylogenetic
   analysis indicated that PgUGT76Z1 and PgUGT73AL1 belong to different
   plant UGT groups, suggesting a convergent evolution of these tricetin
   UGTs. Overall, identification and characterization of PgUGT76Z1 and
   PgUGT73AL1 not only provides evolutionary insights into tricetin
   glucosylation, but also offers an opportunity to produce tricetin
   4′-O-glucoside in large quantities through microbial biotransformation
   or plant metabolic engineering, thus facilitating the investigation of
   tricetin 4′-O-glucoside bioactivities.

   Keywords: anther, petal, tricetin, tricetin 4′-O-glucoside, 4′-O-UGT,
   pomegranate

1. Introduction

   Pomegranate (Punica granatum L.) is an edible medicinal plant in the
   order of Myrtales. Many parts of pomegranate plants, such as flowers,
   fruits, and leaves, have been used for medicinal purposes, largely due
   to the abundant hydrolyzable tannins (HTs; including ellagitannins and
   gallotannins) and flavonoids present in these tissues [[28]1]. However,
   most studies have mainly focused on analyzing the metabolite profiles
   in pomegranate fruits, with those in other tissues relatively
   underexplored. Recently, two flavonoid compounds, tricetin
   (5,7,3′,4′,5′-pentahydroxyflavone) and tricetin 4′-O-glucoside, were
   identified from pomegranate flowers using mass spectrometry (MS) and
   nuclear magnetic resonance (NMR) analyses [[29]2]. Tricetin was shown
   to possess anticancer (breast, liver, and lung cancers) and
   anti-inflammatory activities in studies using human cell lines
   [[30]3,[31]4,[32]5,[33]6]. In addition, anti-diabetic and anti-obesity
   activities in in vitro assays were also reported for tricetin [[34]2].
   Attachment of a glucose moiety to tricetin presumably changes the
   solubility and reactivity of tricetin 4′-O-glucoside, although its
   bioactivity has not been extensively studied.

   To further examine metabolite accumulation in different pomegranate
   floral tissues, high-performance liquid chromatography (HPLC) analysis
   was carried out, indicating that tricetin is present in both anthers
   and filaments, whereas tricetin 4′-O-glucoside accumulates specifically
   in anthers of pomegranate flowers [[35]7]. Glucosylation of tricetin to
   form tricetin 4′-O-glucoside is catalyzed by UDP-dependent
   glycosyltransferases (UGTs). A recently identified UGT in pomegranate,
   PgUGT95B2, displayed a strong specific activity towards tricetin, but
   produced seven glucosylated products, suggesting that PgUGT95B2 can
   glucosylate multiple hydroxyl groups of tricetin and likely generate
   tricetin derivatives with multiple glucosidic linkages [[36]7].
   However, it is unclear as to whether there is a UGT activity in
   pomegranate that can glucosylate specifically at the 4′-O position of
   tricetin.

   To better understand the metabolite profiles of different floral
   tissues of pomegranate, anthers and petals were collected and analyzed
   using liquid chromatography high-resolution electrospray ionization
   mass spectrometry (LC-HR-ESI-MS) in this study. In addition to the
   unique presence of tricetin and tricetin 4′-O-glucoside in anthers,
   more flavonoid and HT compounds were also identified in anthers
   relative to petals. To identify UGTs that can regiospecifically modify
   tricetin and form tricetin 4′-O-glucoside, a comparative transcriptome
   analysis was carried out using anthers and petals of pomegranate
   flowers. Ten UGTs that were more highly expressed in anthers than
   petals were cloned and biochemically characterized. Two of the UGTs,
   PgUGT73AL1 and PgUGT76Z1, demonstrated glucosylation activities
   specific to the 4′-O position of tricetin.

2. Results

2.1. Metabolite Analysis Identified the Unique Accumulation of Tricetin and
Tricetin 4′-O-glucoside, and Abundant Accumulation of Flavonoids and HTs in
Anthers of Pomegranate Flowers

   To examine the compounds that accumulate in anthers and petals of
   pomegranate flowers, metabolites were extracted from both tissues and
   subjected to LC-HR-ESI-MS analysis ([37]Figure 1; [38]Tables S1 and
   S2). In contrast to the previous HPLC analysis of metabolites in
   flowers using several available standards for compound identification
   [[39]7], the LC-HR-ESI-MS analysis obtains high-resolution MS data of
   the metabolites and allows tentative compound identification by
   comparing the retention times, molecular ions, and MS/MS fragment ions
   to those accessible in public databases. This approach enables
   identification of a larger collection of metabolites, as authentic
   standards are not available for many of them. A total of 80 and 52
   metabolites were tentatively identified from anthers and petals,
   respectively ([40]Tables S1 and S2). For HTs, multiple
   hexahydroxydiphenoyl (HHDP) glucoside derivatives, bis-HHDP glucoside
   derivatives, and galloyl-hexoside derivatives, as well as gallic acid,
   galloyl glucoside, tri-galloyl-glucose, tetra-galloyl-glucose, granatin
   A, granatin B, pedunculagin II, strictinin (an ellagitannin), and
   punigluconin were identified in both anthers and petals ([41]Tables S1
   and S2). The following HTs were identified in anthers but not petals:
   casuarinin, digalloyl glucoside, castalagin, punicafolin, ellagic acid,
   galloyl-bis-HHDP-hexoside, and pentagalloyl glucose ([42]Tables S1 and
   S2).

Figure 1.

   [43]Figure 1
   [44]Open in a new tab

   Metabolite analysis of pomegranate anthers and petals. (a)
   Chromatograms of liquid chromatography high-resolution electrospray
   ionization mass spectrometry (LC-HR-ESI-MS) for metabolites extracted
   from pomegranate anthers. (b) Chromatograms of LC-HR-ESI-MS for
   metabolites extracted from pomegranate petals.

   For flavonoids, rutin, flavonoid glycoside, eriodictyol, hovetrichoside
   C (maesopsin 4-O-glucoside; an aurone glucoside), cyanidin
   3-O-rutinoside, and cyanidin 3,5-di-O-glucoside were identified in both
   anthers and petals ([45]Tables S1 and S2). The flavonoids that were
   specifically identified in petals include kaempferol 3-O-glucoside,
   apigenin 4-O-glucoside, and phlorizin. Those were only identified in
   anthers but not petals include luteolin 7-O-glucoside/kaempferol
   4′-O-glucoside, kaempferol 7-O-glucoside, luteolin 3′-O-xylopyanoside,
   naringenin, isorhamnetin, luteolin, apigenin, phloretin, tricetin, and
   tricetin 4′-O-glucoside ([46]Tables S1 and S2).

2.2. Comparative Transcriptome Analysis Coupled with Enzyme Activity Assays
Identified Two Regiospecific UGTs for Tricetin 4′-O-glucoside Biosynthesis

   Because tricetin and tricetin 4′-O-glucoside accumulate in anthers but
   not petals of pomegranate flowers, we hypothesized that gene(s)
   encoding UGT(s) responsible for tricetin 4′-O-glucoside biosynthesis
   could be more highly expressed in anthers than petals. To identify the
   UGT(s) that catalyze 4′-O-glucosylation of tricetin, comparative
   transcriptome analysis was carried out using anther and petal tissues
   of pomegranate (each with three biological replicates). Between 42 and
   46 million raw 2 × 150 bp paired-end sequence reads were obtained for
   each transcriptome with Q30 values ranging from 88.8% to 92.7%
   ([47]Table S3). For all transcriptomes, more than 87% of the cleaned
   sequence reads were mapped to the reference pomegranate genome [[48]8]
   ([49]Table S4). There are 236 genes that are more abundantly expressed
   in petals and 1359 genes more abundantly expressed in anthers according
   to the criteria of Log[2] foldchange > 1 and an adjusted p < 0.05
   ([50]Tables S5 and S6). Consistent with the results from the metabolite
   analysis, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway
   enrichment analysis showed that genes involved in flavonoid
   biosynthesis were enriched in the differentially expressed genes (DEGs)
   ([51]Figure S1).

   Among the more abundantly expressed genes in anthers relative to
   petals, 10 were annotated as UGTs ([52]Table 1). These candidate UGTs
   were cloned and expressed as His-tagged recombinant proteins in E. coli
   ([53]Figure 2a). Enzyme activity assays using purified recombinant UGTs
   showed that only PgUGT73AL1 and PgUGT76Z1 were able to use tricetin and
   UDP-glucose as substrates, and both reactions led to a single product
   tricetin 4′-O-glucoside ([54]Figure 2b,c). No product was formed in the
   control reaction with boiled UGT proteins ([55]Figure 2b). The analysis
   of PgUGT73AL1 and PgUGT76Z1 proteins using the TargetP 2.0 server did
   not identify any sorting signals for subcellular localization (data not
   shown), suggesting that both UGTs are located in the cytosol.

Table 1.

   UGTs that showed higher expression in anthers than petals of
   pomegranate flowers in the transcriptome analysis.
   Gene ID Anther
   (RPKM) Petal
   (RPKM) Fold
   Difference Annotation
   Pgr025859 46.62 0 - UDP-glycosyltransferase 76C4 (Vitis vinifera)
   Pgr010547 1529.03 1.66 917.81 Flavonoid 3-O-glucosyltransferase
   (Medicago truncatula)
   Pgr007463 873.01 3.11 280.04 UDP-glycosyltransferase 79B6 (Arabidopsis
   thaliana)
   Pgr004136 78.25 1.41 55.43 Scopoletin glucosyltransferase (Nicotiana
   tabacum)
   Pgr010383 562.65 12.11 46.44 UDP-glucose iridoid
   glucosyltransferase-like (Eucalyptus grandis)
   Pgr025860
   (PgUGT76Z1) 104.82 2.66 39.32 UDP-glycosyltransferase 76F1 (Arabidopsis
   thaliana)
   Pgr020500 158.14 4.33 36.5 UDP-glycosyltransferase 83A1 (Arabidopsis
   thaliana)
   Pgr003390
   (PgUGT73AL1) 33,879.19 949.73 35.67 Scopoletin glucosyltransferase-like
   (Eucalyptus grandis)
   Pgr010803 72.87 2.82 25.82 UDP-glycosyltransferase 74E2-like
   (Eucalyptus grandis)
   Pgr000716 85.27 10.84 7.86 Zeatin O-glucosyltransferase-like (Jatropha
   curcas)
   [56]Open in a new tab

   RPKM, reads per kilo bases per million reads.

Figure 2.

   [57]Figure 2
   [58]Open in a new tab

   UGT protein expression and enzyme assays. (a) Expression and
   purification of PgUGT73AL1 and PgUGT76Z1 proteins. U, total lysate from
   uninduced E. coli cells transformed with the PgUGT73AL1 or PgUGT76Z1
   plasmid construct; I, total lysate from E. coli cells transformed with
   the PgUGT73AL1 or PgUGT76Z1 plasmid construct and induced with 0.1 mM
   isopropyl β-D-1-thiogalactopyranoside; M, protein molecular mass
   marker; P, purified recombinant protein; kDa, kilodalton. (b) UGT
   enzyme activity assays with boiled UGT proteins (control) or purified
   recombinant PgUGT73AL1 and PgUGT76Z1 proteins. (c) Glucosylation of
   tricetin by PgUGT73AL1 and PgUGT76Z1 to form tricetin 4′-O-glucoside.

2.3. PgUGT73AL1 and PgUGT76Z1 Belong to Different Plant UGT Phylogenetic
Groups

   To understand the evolutionary relationship of PgUGT73AL1 and PgUGT76Z1
   with UGTs in pomegranate and other plants, a phylogenetic tree of
   selected functionally characterized plant UGTs was constructed using
   the neighbor-joining method ([59]Figure 3). Of the 17 UGT phylogenetic
   groups (A–Q) delineated in plants, PgUGT73AL1 and PgUGT76Z1 belong to
   groups D and H, respectively, suggesting that they have evolved
   independently ([60]Figure 3). Within the group D UGTs, PgUGT73AL1
   clustered closely with NtTOGT1, which glucosylates scopoletin (a
   hydroxycoumarin) to produce scopolin [[61]9]; BvUGT73A4, which
   glucosylates a wide range of flavonoids to form flavonoid glucosides
   [[62]10]; and GuUGAT, which adds glucuronosyl groups to glycyrrhetinic
   acid (a triterpene derivative) to yield glycyrrhizin [[63]11]
   ([64]Figure 3). Within the group H UGTs, PgUGT76Z1 was most closely
   clustered with CsUGT76F1, which has shown flavonoid
   7-O-glucosyltransferase and 7-O-rhamnosyltransferase activities
   [[65]12] ([66]Figure 3).

Figure 3.

   [67]Figure 3
   [68]Open in a new tab

   Phylogenetic analysis of PgUGT73AL1, PgUGT76Z1, and selected plant UGTs
   in phylogenetic groups A-Q. The phylogenetic tree was built using the
   neighbor-joining method and tested with 1000 replicates. PgUGT73AL1 and
   PgUGT76Z1 are highlighted in bold. Bootstrap values greater than 60 are
   shown.

3. Discussion

   In this study, we determined that both tricetin and tricetin
   4′-O-glucoside accumulate in anthers but are absent in petals of
   pomegranate flowers ([69]Tables S1 and S2). In addition, we identified
   10 UGTs from the comparative transcriptome analysis that showed higher
   expression levels in anthers than petals ([70]Table 1). Two of the
   candidate UGTs, PgUGT73AL1 and PgUGT76Z1, carried out regiospecific
   glucosylation of tricetin at the 4′-O position ([71]Figure 2).
   PgUGT73AL1 and PgUGT76Z1 are predicted to be cytosolic proteins,
   suggesting that their substrate tricetin is also accessible in the
   cytosol. Tricetin could be produced in the cytosol or transported to
   the cytosol for the glucosylation reaction from another subcellular
   organelle where it is generated.

   Glucosylation of tricetin is expected to change its hydrophobicity, and
   likely also its reactivity. In contrast to the multiple reports on the
   beneficial functions of tricetin to human health, the bioactivity of
   tricetin 4′-O-glucoside is underexplored. The regiospecific UGTs
   PgUGT73AL1 and PgUGT76Z1 can be expressed as recombinant proteins in
   microbes and used as biocatalysts for the synthesis of tricetin
   4′-O-glucoside for drug discovery. Both UGTs, together with genes
   encoding enzymes for tricetin biosynthesis, can also be overexpressed
   in plants that do not naturally make tricetin and tricetin
   4′-O-glucoside to produce these useful flavone compounds.

   Besides applications in biotechnology, cloning and biochemical
   characterization of PgUGT73AL1 and PgUGT76Z1 also provide a glimpse
   into the evolution of UGTs that glucosylate tricetin. PgUGT73AL1 and
   PgUGT76Z1 are members of the plant UGT phylogenetic groups D and H,
   respectively ([72]Figure 3). The previously characterized PgUGT95B2
   that glucosylates tricetin at multiple positions belongs to group Q
   [[73]7]. The observation that UGTs in groups D, H, and Q can all
   glucosylate tricetin suggests the convergent evolution of these UGTs.
   It remains to be determined whether UGTs from other phylogenetic
   groups, besides those that were tested in this study, can also use
   tricetin as a substrate, and whether the glucosylation reaction is
   specific to the 4′-O position of tricetin.

   Abundant HTs and flavonoids were identified in anthers and petals of
   pomegranate flowers, suggesting that they may play a role in these
   reproductive tissues ([74]Tables S1 and S2). On the other hand, the
   unique accumulation of certain HTs and flavonoids in anthers or petals
   suggests that these compounds may function in a tissue-specific manner
   in pomegranate flowers. The transcriptome data could be further
   explored to identify the biosynthetic and/or catabolic genes for HTs
   and flavonoids that may account for the distinctive metabolite
   accumulation in these tissues. For example, an intriguing question
   remains to be answered: what is the mechanistic basis for the
   taxonomically restricted production and accumulation of tricetin in
   pollens of Myrtales plants (including pomegranate) [[75]13]? It is
   possible that a specific hydroxylase that converts luteolin
   (tetrahydroxy flavone) to tricetin (pentahydroxy flavone) is uniquely
   expressed and/or active in pollens of these plants? In the future, the
   transcriptome data from anthers (pollen-bearing structure where
   tricetin is present), in comparison to those from petals (where
   tricetin is absent), of pomegranate flowers can be investigated to
   identify the candidate hydroxylase for tricetin production.

4. Materials and Methods

4.1. Metabolite Analysis

   Anther and petal tissues were collected from blooming flowers of
   pomegranate cv. Wonderful. Three biological replicates (each was pooled
   from many flowers) were harvested for each tissue, ground in liquid
   nitrogen to a fine powder, and kept at −80 °C for metabolite and
   transcriptome analyses. For metabolite analysis, the ground pomegranate
   floral tissues were freeze-dried, and 50 mg of the lyophilized tissue
   was extracted with 1 mL of 70% methanol under sonication. After
   centrifugation at 13,000 rpm for 10 min, the supernatant of the extract
   was passed through a syringe filter (MilliporeSigma, Burlington, MA,
   USA) and subjected to LC-HR-ESI-MS analysis on an ultra-performance
   liquid chromatography (UPLC) (Waters, Milford, MA, USA) coupled to a Q
   Exactive mass spectrometer (Thermo Scientific, Waltham, MA, USA). The
   mass spectra from m/z 120 to 1800 were obtained in the positive (ion
   spray voltage/ISV of 4 kV) and negative (ISV of 3 kV) ion modes. The
   parameters for the LC-HR-ESI-MS analysis and metabolite identification
   using publicly available MS libraries were as previously described
   [[76]7,[77]14].

4.2. Transcriptome Analysis

   For transcriptome analysis, total RNA was extracted from the ground
   anther and petal tissues using the Trizol reagent (Invitrogen,
   Carlsbad, CA, USA). After the quality control of total RNA using
   agarose gel electrophoresis and the Agilent 2100 Bioanalyzer (Agilent,
   Santa Clara, CA, USA), mRNA was enriched using the oligo (dT) magnetic
   beads. RNAseq libraries were constructed from mRNA samples using the
   Illumina TruSeq RNA sample prep kit (Illumina, San Diego, CA, USA) and
   sequenced on an Illumina HiSeq4000 instrument (Illumina).

   The raw reads were cleaned by removing the 3′-end adapter and
   low-quality sequences using Cutadapt
   ([78]https://cutadapt.readthedocs.io/en/stable/, accessed on 30 July
   2018). For mapping of sequence reads, the reference pomegranate genome
   (ASM220158v1; GenBank accession number GCA_002201585.1) was downloaded
   from National Center for Biotechnology Information (NCBI). The trimmed
   reads were mapped to the reference pomegranate genome using Bowtie2
   (v2.2.3; [79]http://bowtie-bio.sourceforge.net, accessed on 18 November
   2021) and Tophat2 (v2.1.1;
   [80]https://ccb.jhu.edu/software/tophat/index.shtml, accessed on 18
   November 2021) [[81]15]. The read counts were quantified using HTSeq
   [[82]16], and the reads per kilo bases per million reads (RPKM) values
   were calculated using RSeQC [[83]17]. Differential gene expression
   analysis between anthers and petals was performed using DESeq2, with an
   adjusted p-value < 0.05 and |log[2] FC| > 1 [[84]18]. The KEGG pathway
   enrichment analysis was conducted by mapping DEGs to the KEGG pathways
   ([85]http://www.genome.jp/kegg/, accessed on 18 November 2021)
   [[86]19]. The transcriptome data were deposited in the NCBI sequence
   reads archive (SRA) under the accession number PRJNA806846.

4.3. Protein Sequence Analysis and Enzyme Assays

   The open reading frames (ORFs) of the candidate UGTs were codon
   optimized for expression in E. coli, synthesized by Genewiz (Suzhou,
   China), and then cloned in the pET28a vector. The recombinant plasmids
   were transformed into E. coli BL21 (DE3) cells. Expression of UGT
   proteins was induced by adding isopropyl β-D-1-thiogalactopyranoside
   (IPTG) to a final concentration of 0.1 mM. Procedures for protein
   expression, purification of His-tagged recombinant proteins, UGT enzyme
   assays (0.25 mM tricetin was used in the assays), and HPLC analysis
   were as previously described [[87]20]. The TargetP 2.0 server
   ([88]https://services.healthtech.dtu.dk/service.php?TargetP-2.0,
   accessed on 16 December 2021) was used for prediction of subcellular
   sorting signals in protein sequences.

4.4. Phylogenetic Analysis

   Protein sequences of PgUGT76Z1 and PgUGT73AL1, along with those of
   selected plant UGTs, were aligned using Multiple Sequence Comparison by
   Log-Expectation (MUSCLE) [[89]21]. A neighbor-joining tree was
   constructed based on the protein sequence alignment using Molecular
   Evolutionary Genetics Analysis (MEGA) [[90]22]. The Arabidopsis Genome
   Initiative (Arabidopsis sequences) and GenBank (non-Arabidopsis
   sequences) accession numbers for the UGTs are as follows: AcUGT73G1
   ([91]AAP88406), AcUGT73J1 ([92]AAP88407), AsUGT74H5 ([93]ACD03250),
   AtUGT71B1 (AT3G21750), AtUGT71C1 (AT2G29750), AtUGT71C4 (AT1G07250),
   AtUGT71D1 (AT2G29730), AtUGT72B1 (AT4G01070), AtUGT72C1 (AT4G36770),
   AtUGT72D1 (AT2G18570), AtUGT72E1 (AT3G50740), AtUGT73B1 (AT4G34138),
   AtUGT73C1 (AT2G36750), AtUGT74B1 (AT1G24100), AtUGT74C1 (AT2G31790),
   AtUGT74D1 (AT2G31750), AtUGT74E2 (AT1G05680), AtUGT74F1 (AT2G43840),
   AtUGT75B1 (AT1G05560), AtUGT75C1 (AT4G14090), AtUGT75D1 (AT4G15550),
   AtUGT76B1 (AT3G11340), AtUGT76C1 (AT5G05870), AtUGT76D1 (AT2G26480),
   AtUGT76E1 (AT5G59580), AtUGT78D1 (AT1G30530), AtUGT79B6 (AT5G54010),
   AtUGT82A1 (AT3G22250), AtUGT83A1 (AT3G02100), AtUGT84B1 (AT2G23260),
   AtUGT85A1 (AT1G22400), AtUGT86A1 (AT2G36970), AtUGT87A1 (AT2G30150),
   AtUGT88A1 (AT3G16520), AtUGT89A2 (AT5G03490), AtUGT89B1 (AT1G73880),
   AtUGT89C1 (AT1G06000), AtUGT90A1 (AT2G16890), AtUGT92A1 (AT5G12890),
   BvUGT71F1 ([94]AY526081), BvUGT73A4 ([95]AY526080), CaUGT73AH1
   ([96]AUR26623), CoUGT78B3 ([97]AEB61484), CoUGT85N1 ([98]AEB61489),
   CpPGT11 ([99]AIS39477), CsUGT76F1 ([100]KDO69246), CtUGT78K6
   ([101]BAF49297), GbUGT92K1 ([102]ASK39406), GeUGT73F1 ([103]BAC78438),
   GmUGT72 × 4 ([104]KRH46505), GmUGT79A6 ([105]BAN91401), GmUGT91H9
   ([106]NP_001348424), GmUGT92G4 ([107]KRH14708), GtUF6CGT
   ([108]AB985754), GuUGAT ([109]ANJ03631), LgUGT78J1 ([110]AEB61487),
   MeUGT85K4 ([111]AEO45781), MtUGT71G1 ([112]AAW56092), MtUGT72L1
   ([113]ACC38470), MtUGT73K1 ([114]AAW56091), MtUGT73P1 ([115]ABI94026),
   MtUGT78G1 ([116]ABI94025), MtUGT84F1 ([117]ABI94023), MtUGT85H2
   ([118]ABI94024), MtUGT88E1 ([119]ABI94021), MtUGT95B4
   ([120]XP_003612636), NtTOGT1 ([121]AAB36653), OsUGT709A4
   ([122]BAC80066), OsZOGT1 ([123]BAS90436), OsZOGT3 ([124]BAS90518),
   PgUGT72BD1 ([125]MN124519), PgUGT73AL1 ([126]XP_031381080), PgUGT76Z1
   ([127]XP_031382549), PgUGT84A23 ([128]ANN02875), PgUGT84A24
   ([129]ANN02877), PgUGT95B2 ([130]MH507175), PjGAT ([131]AYA60333),
   PoUGT90A7 ([132]EU561019), PoUGT95A1 ([133]ACB56927), ScUGT5
   ([134]BAJ11653), SrUGT73E1 ([135]AAR06917), SrUGT76G1 ([136]AAR06912),
   SrUGT85C2 ([137]AAR06916), VpUGT88D8 ([138]BAH47552), VpUGT94F1
   ([139]BAI44133), VvGT7 ([140]XP_002276546), VvGT15 ([141]XP_002281513),
   VvUGT1 ([142]CBI34463), VvUGT95B6 ([143]XP_010664783), ZmUFGT1
   ([144]P16167), ZmUGT74A1 ([145]NP_001105326), ZmUGT91L1
   ([146]NP_001347041), and ZmcisZOG1 ([147]AAK53551). Ac, Allium cepa;
   As, Avena strigosa; At, Arabidopsis thaliana; Bv, Beta vulgaris; Ca,
   Centella asiatica; Co, Consolida orientalis; Cp, Citrus paradise; Cs,
   Citrus sinensis; Ct, Clitoria ternatea; Gb, Ginkgo biloba; Ge,
   Glycyrrhiza echinata; Gm, Glycine max; Gt, Gentiana triflora; Gu,
   Glycyrrhiza uralensis; Lg, Lamium galeobdolon; Me, Manihot esculenta;
   Mt, Medicago truncatula; Nt, Nicotiana tabacum; Os, Oryza sativa; Pg,
   Punica granatum; Pj, Panax japonicus; Po, Pilosella officinarum; Sc,
   Sinningia cardinalis; Sr, Stevia rebaudiana; Vp, Veronica persica; Vv,
   Vitis vinifera; Zm, Zea mays.

Acknowledgments