A Functional Genomics Approach to (Iso)Flavonoid Glycosylation in the Model Legume Medicago

A functional genomics approach to (iso)flavonoid glycosylation in the model legume Medicago truncatula

Luzia V. Modolo • Jack W. Blount • Lahoucine Achnine • Marina A. Naoumkina • Xiaoqiang Wang • Richard A. Dixon

SUPPLEMENTARY MATERIAL

Figure S1. HPLC analysis of the products of GT22E09 with biochanin A and UDP-glucose as substrates.

Figure S2. Michaelis–Menten hyperbola and Lineweaver-Burk plot for GT83F with kaempferol as substrate.

Figure S3. Phylogenetic analysis based on amino acid sequence alignment of full-length M. truncatula and Arabidopsis UGTs. M. truncatula genes are designated by Noble Foundation identifiers (GT….), whereas Arabidopsis genes are designated by UGT numbers. Letters on the right indicate the various groups of UGTs described by Ross et al (2001).

Table S1. Phenolic compounds tested as substrates for GT activity in a high throughput screen utilizing mixtures (groups I-IV) of compounds.

Table S2.Clone identifiers, TIGR TC numbers and GenBank Accession numbers for Medicago UGTs

Table S3. Position specificity of Medicago UGTs for glycosylation of flavonoid and isoflavonoid acceptors. Results are based on co-chromatography of products from enzymatic reactions with authentic standards.

Table S4. Substrates for GT83F and their respective products.

Table S5. Amino acid sequence identity between (iso)flavonoid glycosyltransferases from Medicago truncatula and functionally characterized glycosyltransferases from other plant species.

Table S6. Primers used for the quantification of the transcript levels of M. truncatula (iso)flavonoid glycosyltransferase genes by qRT-PCR.

Figure S1. HPLC analysis of the products (P) of GT22E09 with biochanin A (S) as acceptor and UDP-glucose as sugar donor. Solid black line- incubated in the presence of purified GT22E. Long dashed line- incubated without enzyme. Dashed and dotted line- authentic biochanin A and biochanin A 7-O-glucoside standards.

Figure S2 Michaelis-Menten hyperbola (left) and Lineweaver-Burk plot (right) for GT83F and the substrate kaempferol. Substrate concentration [S] and velocity (v) are given in mM and μM/min, respectively. Donor was UDPG.

Fig S3

Phylogenetic analysis based on amino acid sequence alignment of full-length M. truncatula and Arabidopsis UGTs. The group of Medicago enzymes at the bottom of the figure has no obvious homologs in Arabidopsis.

Table S1 Phenolic compounds tested as substrates for GT activity in a high throughput screen utilizing mixtures (groups I-IV) of compounds. Compounds within each group are readily separated by the HPLC method, and each compound has a distinct UV spectrum unlike that of any other compound in that particular group. RT = Retention time (min). (Iso)flavonoid compounds are underlined

Group I / RT / Group II / RT / Group III / RT / Group IV / RT
catechin / 20.44 / caffeic acid / 23.79 / p-hydroxy-benzoic acid / 18.95 / esculetin / 22.37
coumaric acid / 31.17 / coniferyl alcohol / 28.53 / vanillin / 28.15 / epicatechin / 25.69
scopoletin / 33.66 / benzoic acid / 34.46 / ferulic acid / 34.22 / delphinidin / 33.45
dihydro-quercetin / 34.36 / dihydro-kaempferol / 39.09 / cyanidin / 36.82 / salycilic acid / 39.03
pelargonidin / 40.98 / quercetin / 46.93 / cinnamic acid / 45.60 / liquiritigenin / 44.49
daidzein / 44.34 / coumestrol / 54.06 / naringenin / 50.30 / apigenin / 52.87
luteolin / 47.27 / medicarpin / 61.63 / kaempferol / 53.62 / maackiain / 59.41
genistein / 52.13 / biochanin A / 66.20 / formononetin / 57.35 / prunetin / 65.96

Table S2.Clone identifiers, TIGR TC numbers and GenBank Accession numbers for Medicago UGTs

EST ID by Noble Foundation / Noble Lab Code / TIGR TC Release 8.0 (January 19, 2005) (Blank = singleton) / GenBank ID
Clone NF001E08IN / GT001E / TC109713 / BG449057
Clone NF001F07EC / GT001F / TC97518 / BF643221
Clone NF002E08ST / GT002E / TC100519 / AW688120
Clone NF003G06PL / GT003G / TC108206 / BE323213
Clone NF005F08RT / GT005F / TC106853 / AW687069
Clone NF007C06DT / GT007C / TC100986 / BF631865
Clone NF010A11IN / GT010A / TC107730 / BE322107
Clone NF010D09EC / GT010D / TC101954 / BF643678
Clone NF011D09IN / GT011D / TC95224 / BF639343
Clone NF011H07IN / GT011H / TC106204 / BF639472
Clone NF012E03ST / GT012E / TC107945 / AW688857
Clone NF013H04IN / GT013H / TC95738 / BF639590
Clone NF016F09EC / GT016F / TC94864 / BF644840
Clone NF020D12LF / GT020D / BE249479
Clone NF021F10FL / GT021F / BI271396
Clone NF022D09IN / GT022D / TC107930 / BE321611
Clone NF022E08NR / GT022E_08 / TC107590 / AW684875
Clone NF022E09NR / GT022E_09 / TC101755 / AW684876
Clone NF029C05EC / GT029C / TC101637 / BF647364
Clone NF029H05ST / GT029H / TC95785 / AW690200
Clone NF031E04RT / GT031E_04 / TC108379 / BE320502
Clone NF031E06EC / GT031E_06 / TC112215 / BF645015
Clone NF034D03DT / GT034D / BG450501
Clone NF034F06LF / GT034F / TC101964 / BE319057
Clone NF037D06PL / GT037D / TC96536 / BG457982
Clone NF037F08NR / GT037F / TC102824 / AW686042
Clone NF038E01DT / GT038E / TC102580 / BE247903
Clone NF039B09IN / GT039B / TC102439 / BF640193
Clone NF041H08LF / GT041H / BE317583
Clone NF043G04NR / GT043G / TC95225 / AW686656
Clone NF044A05LF / GT044A / TC108940 / BE318995
Clone NF045H02IN / GT045H / TC95212 / BE321921
Clone NF049F12LF / GT049F / TC100176 / BF636930
Clone NF054H11ST / GT054H / TC101511 / AW692753
Clone NF055D08IN / GT055D / TC99635 / BG449653
Clone NF055F07ST / GT055F / TC101546 / AW692442
Clone NF056E07IN / GT056E / TC104846 / BF640780
Clone NF057E05IN / GT057E / TC98559 / BF642380
Clone NF063F05IN / GT063F / TC101298 / BF642626
Clone NF063G03EC / GT063G / BF644451
Clone NF065D10IN / GT065D / TC108848 / BF641456
Clone NF065F06IN / GT065F / TC111725 / BF641685
Clone NF067A12IN / GT067A / TC108134 / BF642382
Clone NF067E03EC / GT067E / BF646175
Clone NF068E05ST / GT068E / AW693735
Clone NF073B11DT / GT073B / BF633795
Clone NF073H03PL / GT073H / TC100520 / BG455995
Clone NF074C12EC / GT074C / BF646288
Clone NF075G04EC / GT075G / TC96410 / BF646470
Clone NF083F04ST / GT083F / TC105632 / BE325998
Clone NF086C08IN / GT086C / TC107173 / BI266522
Clone NF088C04EC / GT088C / TC94946 / BF650120
Clone NF088G02ST / GT088G / TC101149 / BE325419
Clone NF090F03EC / GT090F / BF650554
Clone NF091H08PL / GT091H / TC104550 / BI263581
Clone NF095B04IN / GT095B / TC105801 / BI265578
Clone NF096D07EC / GT096D / TC97237 / BF650731
Clone NF097G06DT / GT097G / TC96393 / BG451009
Clone NF099D03DT / GT099D / BG451134
Clone NF102C11IN / GT102C / TC100359 / BI266253
Clone NF103F04ST / GT103F / AW696207
Clone NF106F03DT / GT106F / TC97754 / BF635675
Clone NF111E12PL / GT111E / BI263761

Table S3. Position specificity of Medicago UGTs for glycosylation of flavonoid (A) and isoflavonoid (B) acceptors. Results are based on co-chromatography of products from enzymatic reactions with authentic standards.

(A)

GT / Flavonoids
22D / Primarily 7-OH, some 4'-OH
22E / 4'-OH
29C / Very low 4'-OH
29H / Multi-site, but primarily 3'-OH
63G / Primarily 3'-OH, some 3-OH
67A / Primarily 3-OH, some 3'-OH
83F / 3-OH, then 7-OH if no 3-OH
99D / 3-OH

(B)

GT / Isoflavonoids
22D / Primarily 7-OH, 4'-OH if no 7-OH
22E / Primarily 7-OH, also 4'-OH
29C / 7-OH, 4'-OH if no 7-OH
29H / Primarily 7-OH, little or no 4'-OH
63G / No activity
67A / No activity
83F / 7-OH
99D / No activity

Table S4Substrates for GT83F and their respective products. The positions of glycosylation were determined by comparison of the retention times of reaction products to those of authentic standards

Substrate / Product
Afromosin / Afromosin 7-O-glucoside
Apigenin / Apigenin-7-O-glucoside
Biochanin A / Biochanin A-7-O-glucoside
Cyanidin / Cyanidin-3-O-glucoside
Daidzein / Daidzein-7-O-glucoside
Formononetin / Formononetin-7-O-glucoside
Genistein / Genistein -7-O-glucoside
Kaempferol / Kaempferol-3-O-glucoside
Pelargonidin / Pelargonidin-3-O-glucoside
Quercetin / Quercetin-3-O-glucoside

Table S5 Amino acid sequence identity between (iso)flavonoid glycosyltransferases from Medicago truncatula and functionally characterized glycosyltransferases from other plant species.

Glycosyltransferases / GT22D / GT22E09 / GT29C / GT29H / GT63G / GT67A / GT83F / GT99D
UGT73C1 (AY573820)
Zeatin O-glucosyltransferase 1 / 54% / 25% / 23% / 24% / 26% / 27% / 50% / 38%
UGT73C5 (NM_129235)
Zeatin O-glucosyltransferase 3 / 55% / 25% / 24% / 26% / 26% / 27% / 26% / 38%
UGT73C6 (NM_129234)
Zeatin O-glucosyltransferase / 55% / 24% / 26% / 24% / 26% / 28% / 27% / 38%
UGT73B3 (NM_119574)
2,5- or 2,4-dihydroxybenzoic acid glucosyltransferase / 41% / 28 / 27% / 26% / 27% / 28% / 28% / 39%
UGT71C1 (NM_128529)
3,4-dihydroxybenzoic acid or 3-hydroxy 3-O-benzoic acidglucosyltransferase / 26% / 29% / 26% / 44% / 23% / 25% / 22% / 25%
UGT71C4 (AY040019)
3,4- or 2,5-dihydroxybenzoic acid glucosyltransferase / 25% / 29% / 27% / 44% / 24% / 25% / 20% / 25%
UGT73B4 (NM_127109)
3,4- or 2,5-dihydroxybenzoic acid glucosyltransferase / 40% / 26% / 27% / 27% / 27% / 28% / 26% / 40%
UGT78D2 (NM_121711)
3,4-dihydroxybenzoic acid or 4-hydroxy benzoic acidglucosyltransferase / 23% / 22% / 23% / 24% / 25% / 27% / 50% / 18%
TOGT1 (AF346431)
Scopoletin glucosyltransferase / 42% / 27% / 36% / 32% / 28% / 26% / 30% / 43%

Continuation

Glycosyltransferases / GT22D / GT22E09 / GT29C / GT29H / GT63G / GT67A / GT83F / GT99D
TOGT2 (AF346432)
Phenylpropanoid glucosyltransferase 2 / 42% / 28% / 32% / 31% / 27% / 27% / 31% / 42%
Betanidin 5-O-glucosyltransferase from Dorotheanthus bellidiformis (Y18871) / 43% / 28% / 32% / 28% / 27% / 26% / 30% / 42%
CaUGT2 (AB159213)
Curcumin glucosyltransferase / 40% / 27% / 37% / 31% / 27% / 28% / 29% / 42%
SaGT4A (AB182385)
Saponin glucosyltransferase / 38% / 29% / 32% / 32% / 29% / 27% / 30% / 41%
UBGAT-I (AB042277)
Baicalein 7-O-glucuronyltransferase / 32% / 38% / 40% / 35% / 27% / 26% / 28% / 33%
LGTase (AB033758)
Limonoid glucosyltransferase / 24% / 29% / 31% / 34% / 51% / 30% / 27% / 28%
BnSGT1 (AF287143)
Sinapate glucosyltransferase / 27% / 30% / 31% / 26% / 49% / 30% / 26% / 23%
Sgt1 (U82367)
Solanidine/sterol alkaloid glucosyltransferase / 37% / 27% / 30% / 30% / 28% / 27% / 26% / 39%
Sgt2 (DQ218276)
Solanidine glucosyltransferase / 38% / 27% / 34% / 30% / 28% / 28% / 29% / 39%
UGT84A3 (NM_117939)
4-coumarate glucosyltransferase / 23% / 24% / 23% / 24% / 45% / 29% / 29% / 22%
UGT84A4 (NM_117640)
Sinapate glucosyltransferase / 22% / 24% / 23% / 21% / 44% / 28% / 25% / 20%
UGT85A1 (NM_102089)
Cytokinins glucosyltransferase / 29% / 24% / 24% / 26% / 27% / 53% / 26% / 24%

Continuation

Glycosyltransferases / GT22D / GT22E09 / GT29C / GT29H / GT63G / GT67A / GT83F / GT99D
UGT85C2 (AY345978)
Steviol glucosyltransferase / 26% / 26% / 25% / 28% / 23% / 46% / 24% / 25%
UGT76G1 (AY345984)
Stevioside glucosyltransferase / 28% / 28% / 27% / 27% / 28% / 43% / 25% / 25%
UGT85B1 (AF199453)
-Hydroxymandelonitrile O-glucosyltransferase / 25% / 28% / 31% / 28% / 29% / 43% / 24% / 25%
AOG (AB065190)
Abscisic acid glucosyltransferase / 37% / 28% / 31% / 27% / 28% / 28% / 27% / 42%
Acgat (AB103471)
Anthocyanin 3-O-galactosyltransferase / 26% / 26% / 29% / 29% / 28% / 30% / 51% / 32%
Anthocyanidin 3-O-glucoside from Vitis vinifera (AF000371) / 26% / 31% / 30% / 29% / 28% / 25% / 49% / 35%
Gs3’GT1 (AB189922)
Anthocyanin 3-O-glucosyltransferase / 39% / 28% / 34% / 30% / 25% / 27% / 30% / 41%
Gs3’GT2 (AB189923)
Anthocyanin 3-O-glucosyltransferase / 39% / 28% / 33% / 30% / 25% / 27% / 30% / 41%
3’GT (AB076697)
Anthocyanin 3-O-glucosyltransferase / 39% / 28% / 34% / 29% / 25% / 27% / 30% / 42%
Ih3GT (AB161175)
Anthocyanidin 3-Oglucosyltransferase / 26% / 31% / 35% / 29% / 29% / 28% / 42% / 37%
PGT8 (AB027454)
Anthocyanidin 3-O-glucosyltransferase / 30% / 30% / 30% / 28% / 28% / 28% / 46% / 27%
RhGT1 (AB201048)
Anthocyanidin 3,5-O-glucosyltransferase / 31% / 47% / 50% / 34% / 26% / 26% / 26% / 28%

Continuation

Glycosyltransferases / GT22D / GT22E09 / GT29C / GT29H / GT63G / GT67A / GT83F / GT99D
RhGT2 (AB201049)
Anthocyanidin 3,5-O-glucosyltransferase / 31% / 46% / 49% / 34% / 26% / 26% / 25% / 27%
RhGT3 (AB201050)
Anthocyanidin 3,5-O-glucosyltransferase / 31% / 47% / 50% / 34% / 26% / 26% / 26% / 28%
DicGT4 (AB191248)
Chalcononaringenin 2’-O-glucosyltransferase / 39% / 27% / 35% / 31% / 28% / 27% / 31% / 39%
L4’CGT (AB198666)
Chalcone 4’-O-glucosyltransferase / 29% / 42% / 41% / 36% / 35% / 32% / 26% / 23%
Am4’CGT (AB198665)
Chalcone 4'-O-glucosyltransferase / 29% / 42% / 43% / 32% / 32% / 25% / 27% / 33%
Kaempferol 3-O-galactosyltransferase from Petunia x hybrida (AF165148) / 29% / 28% / 30% / 29% / 29% / 27% / 49% / 23%
UF3GaT (AY954034)
Flavonoid 3-O-galactosyltransferase / 32% / 32% / 31% / 30% / 30% / 26% / 46% / 26%
Flavonol 3-O-galactosyltransferase from Petunia x hybrida (AF316552) / 29% / 29% / 33% / 31% / 36% / 28% / 52% / 34%
UGT78D1 (AY087785)
Flavonol 3-O-rhamnosylglucosyltransferase / 20% / 24% / 23% / 24% / 26% / 26% / 44% / 20%
DicGT1 (AB191245)
Flavonoid 3-O-glucosyltransferase / 24% / 30% / 31% / 27% / 24% / 24% / 41% / 31%
DicGT2 (AB191246)
Flavonol 3-O-glucosyltransferase / 29% / 33% / 33% / 44% / 31% / 28% / 28% / 30%
DicGT3 (AB191247)
Flavonol 3-O-glucosyltransferase / 26% / 29% / 27% / 29% / 26% / 26% / 43% / 30%

Continuation

Glycosyltransferases / GT22D / GT22E09 / GT29C / GT29H / GT63G / GT67A / GT83F / GT99D
RhGT4 (AB201051)
Flavonol 3-O-glucosyltransferase / 27% / 30% / 29% / 27% / 30% / 28% / 56% / 27%
UGT73A1 (X77459)
Flavonol 3-O-glucosyltransferase / 27% / 31% / 31% / 40% / 25% / 25% / 31% / 26%
UGT73A2 (X77461)
Flavonol 3-O-glucosyltransferase / 36% / 35% / 35% / 47% / 31% / 29% / 29% / 31%
UGT73A6 (X77463)
Flavonol 3-O-glucosyltransferase / 28% / 31% / 35% / 43% / 25% / 29% / 28% / 31%
UGT73B2 (NM_119575)
Flavonoid 7-O-glucosyltransferase / 40% / 27% / 27% / 26% / 27% / 27% / 27% / 40%
UGT73F1 (AB098614)
Isoflavonoid glucosyltransferase / 37% / 26% / 32% / 29% / 27% / 25% / 28% / 39%
UGT73G1 (AY262062)
Flavonoid 7-O-glucosyltransferase / 40% / 28% / 28% / 31% / 29% / 27% / 28% / 41%
Flavonoid 3-O-glucosyltransferase from Citrus sinesis (AY519364) / 25% / 32% / 31% / 30% / 25% / 24% / 44% / 24%
Flavonoid 3-O-glucosyltransferase from Fragaria x ananassa (AY695815) / 27% / 30% / 32% / 31% / 28% / 27% / 49% / 35%
Flavonol 3-O-glucosyltransferase from Forsythia x intermedia (AF127218) / 28% / 32% / 30% / 26% / 28% / 28% / 45% / 27%
Flavonol 3-O-glucosyltransferase from Gentiana triflora (D85186) / 28% / 30% / 32% / 28% / 28% / 26% / 49% / 33%
Flavonol 3-O-glucosyltransferase from Malus x domestica (DQ156906) / 26% / 32% / 31% / 26% / 27% / 24% / 45% / 31%

Continuation

Glycosyltransferases / GT22D / GT22E09 / GT29C / GT29H / GT63G / GT67A / GT83F / GT99D
Flavonol 3-O-glucosyltransferase from Perilla frutescens (AB002818) / 30% / 32% / 33% / 29% / 29% / 26% / 44% / 28%
Flavonoid 3-O-glucosyltransferase from Solanum tuberosum (AY954034) / 32% / 32% / 31% / 30% / 30% / 26% / 46% / 26%
Flavonoid 7-O-glucosyltransferase from Scutellaria baicalensis (AB031274) / 40% / 31% / 38% / 33% / 28% / 29% / 28% / 41%
Flavonoid glucosyltransferase from Beta vulgaris (AY526080) / 42% / 28% / 31% / 31% / 29% / 28% / 28% / 43%
Flavonoid glucosyltransferase from Beta vulgaris (AY526081) / 27% / 33% / 35% / 40% / 25% / 25% / 28v / 27%
Flavonoid glucosyltransferase from Glycine max (AF198453) / 33% / 60% / 65% / 33% / 31% / 27% / 29% / 48%
Flavonoid glucosyltransferase from Vigna mungo (AB012115) / 36% / 61% / 67% / 38% / 36% / 31% / 38% / 38%
Flavonoid glucosyltransferase from Vigna mungo (AB012116) / 35% / 40% / 43% / 33% / 30% / 27% / 32% / 35%

Table S6 Primers used for the quantification of the transcript levels of M. truncatula (iso)flavonoid glycosyltransferase genes by qRT-PCR.

Gene / Primer
Actin (upstream) / 5’-TCAATGTGCCTGCCATGTATGT-3’
Actin (downstream) / 5’-ACTCACACCGTCACCAGAATCC-3’
GT22D(upstream) / 5’-GAAGAGTTCACAACACCAGCAA-3’
GT22D (downstream) / 5’-GCATATTGAAGAGATGGAAGCA-3’
GT22E09 (upstream) / 5’-GAACCACTCCGTTGCTTTTT-3’
GT22E09 (downstream) / 5’-ACGCTTTGACCTGGCTGT-3’
GT29C (upstream) / 5’-GGACAAAGGACAAGGGAATG-3’
GT29C (downstream) / 5’-AATCCACCCACGGAGTTATG-3’
GT29H (upstream) / 5’-GGGGTTAGGTTCTTGTGGTCT-3’
GT29H (downstream) / 5’-CCTTCCAATTCCATCCATTCT-3’
GT63G (upstream) / 5’-GTGACCAACTTACAAACGCAAA-3’
GT63G (downstream) / 5’-ACGAGCCAATCTAATCCCAAC-3’
GT67A (upstream) / 5’-ACAGTTATGACACCCGAGCAA-3’
GT67A (downstream) / 5-CTGAGCCACCAATGACAAGA-3’
GT83F (upstream) / 5’-GGCAGAGACAGGGAAGAACA-3’
GT83F (downstream) / 5’-TAAATCCGCACCAAACCAA-3’
GT99D (upstream) / 5’-GGAGTGTGGGACCAGTTTCTT-3’
GT99D (downstream) / 5’-GCACCCCTTCTTCTTCATCTT-3’