Isoflavone Malonyltransferases GmIMaT1 and GmIMaT3 Differently Modify Isoflavone Glucosides in Soybean (Glycine max) under Various Stresses

Abstract

Malonylated isoflavones are the major forms of isoflavonoids in soybean plants, the genes responsible for their biosyntheses are not well understood, nor their physiological functions. Here we report a new benzylalcohol O-acetyltransferase, anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase, deacetylvindoline 4-O-acetyltransferase (BAHD) family isoflavone glucoside malonyltransferase GmIMaT1, and GmIMaT3, which is allelic to the previously characterized GmMT7 and GmIF7MaT. Biochemical studies showed that recombinant GmIMaT1 and GmIMaT3 enzymes used malonyl-CoA and several isoflavone 7-O-glucosides as substrates. The K_m values of GmIMaT1 for glycitin, genistin, and daidzin were 13.11, 23.04, and 36.28 μM, respectively, while these of GmIMaT3 were 12.94, 26.67, and 30.12 μM, respectively. Transgenic hairy roots overexpressing both GmIMaTs had increased levels of malonyldaidzin and malonylgenistin, and contents of daidzin and glycitin increased only in GmIMaT1-overexpression lines. The increased daidzein and genistein contents were detected only in GmIMaT3-overexpression lines. Knockdown of GmIMaT1 and GmIMaT3 reduced malonyldaidzin and malonylgenistin contents, and affected other isoflavonoids differently. GmIMaT1 is primarily localized to the endoplasmic reticulum while GmIMaT3 is primarily in the cytosol. By examining their transcript changes corresponding to the altered isoflavone metabolic profiles under various environmental and hormonal stresses, we probed the possible functions of GmIMaTs. Two GmIMaTs displayed distinct tissue expression patterns and respond differently to various factors in modifying isoflavone 7-O-glucosides under various stresses.

Keywords: BAHD family; enzyme kinetics; isoflavone malonate; malonyltransferase; stress response.

FIGURE 1

Scheme of (iso)flavonoid biosynthesis, modification, and transport in soybean. Isoflavonoid biosynthesis starts from legume-specific branches at IFS/CHI-catalyzed steps from phenylpropanoid pathway. Flavonoids and isoflavonoids share naringenin chalcones as common precursors. Enzymes involved in the biosynthesis pathway as following; PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; CHR, chalcone reductase; IFS, isoflavone synthase; I2′H, isoflavone 2′-hydroxylase; IFR, isoflavone reductase; IOMT, isoflavonoid O-methyltransferase; HID, 2-hydroxyisoflavanone dehydratase; F3H, flavanone 3-hydroxylase; FLS, flavonol synthase; FNS, flavone synthase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; UGT, UDP-glucose:flavonoid glycosyltransferase; MaT, Malonyl-CoA: flavonoid glucoside acyltransferase; MATE1/TT12, multidrug and toxic extrusion protein 1/transparent testa 12; ABC, ATP-binding cassette; PTS, pterocarpan synthases.

FIGURE 2

Identification of GmIMaT1 and GmIMaT3 with expression and isoflavone profiles in soybean tissues. (A) Phylogenetic analysis of GmIMaT1 and GmIMaT3 with other benzylalcohol O-acetyltransferase, anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase, deacetylvindoline 4-O-acetyltransferase (BAHD) family acyltransferases. Rooted phylogenetic tree for BAHD malonyltransferases was constructed by using MEGA6 program through neighbor joining method. Bar shows 0.1 amino acid substitution site. (B) Expression patterns of GmIMaT1 and GmIMaT3 in different soybean tissues. Seeds, nodules, and flower from 8 to 11 weeks old plants, root, stem, and leaf from 12 to 15 days old seedling were harvested for quantitative reverse transcription (qRT-PCR) polymerase chain reaction. Transcript levels are expressed relative to that of GmACTIN (Glyma19G147900.1). (C) Isoflavone contents in different tissues of soybean plant. Flavonoids were extracted with 80% methanol and analyzed with high pressure liquid chromatography (HPLC); the isoflavone contents were calculated by standard curves. Data are expressed in means ± SD from at least three independent experiments with duplicates.

FIGURE 3

GmIMaT1 and GmIMaT3 activity assay. (A) The purified recombinant GmIMaT1 and GmIMaT3. GmIMaT1 and GmIMaT3 were expressed in E. coli in His-tagged fusions and partially purified with nickel resin (A). Proteins were resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), followed by staining with Coomassie Blue R-250. (B–H) GmIMTa1 and GmIMaT3 activity assay. The recombinant proteins were incubated in malonyltransferase reaction with various isoflavone glucosides and malonyl-CoA as substrates. The enzymatic products were analyzed with HPLC as compared with isoflavonoid standards (B), reaction analysis for recombinant GmIMaT1 with daidzin (C), glycitin (D), genistin (E) as substrates. The reaction analysis for recombinant GmIMaT3 with daidzin (F), glycitin (G), genistin (H) as substrates. The symbols are D, daidzin; G, glycitin; Ge, genistin; MD, malonyldaidzin; MG, malonylglycitin; MGe, malonylgenistin. (I–R) Verification of malonylation products from corresponding substrates with mass spectrometry for recombinant GmIMaT1 and GmIMaT3. Mass spectra for daidzin and malonyldaidzin (I), genistin and malonylgenistin (L), glycitin and malonylglycitin (O) in GmIMaT1-catalyzed reactions. Mass spectra for glucoside daidzin (J), genistin (M), and glycitin (P). Mass spectra for malonyldaidzin (K), malonylgenistin (N), and malonylglycitin (Q). D, daidzin; G, glycitin; Ge, genistin; MD, malonyldaidzin; MG, malonylglycitin; MGe, malonylgenistin. (R) The malonylation of isoflavone 7-O-glucosides (daidzin, genistin, and glycitin) at 6′′ position catalyzed by malonyltransferases (GmIMaT1/GmIMaT3) to generate malonylglucosides (malonyldaidzin, malonylgenistin, and malonylglycitin).

FIGURE 4

GmIMaT1 and GmIMaT3 overexpression affects isoflavone contents in hairy roots. (A–C) Representative hairy roots expressing GUS (A), GmIMaT1-OE (B), GmIMaT3-OE (C). (D) Semi-quantitative RT-PCR examination of overexpression of GmIMaT1-OE and GmIMaT3-OE in representative transgenic hairy root lines. Soybean GmACTIN was used as an internal control. L-1, transgenic hairy root line-1; L-2, transgenic hairy root line-2. (E,F) qRT-PCR confirmation of GmIMaT1 and GmIMaT3 expression in transgenic hairy root lines. (G–J) HPLC chromatograms for isoflavone profiles in GmIMaT1 and GmIMaT3 overexpression hairy root lines. The numbered peaks are: 1, daidzin; 2, glycitin; 3, genistin; 4, malonyldaidzin; 5, malonylglycitin 6, malonyl genistin; 7, daidzein; 8, glycitein; 9, genistein. (K) The contents of isoflavone compounds in GmIMaT overexpressed hairy root lines. Data are expressed in means ± SD from at least three independent transgenic lines. Differences between control and GmIMaT transgenic lines were analyzed by using Student’s t-test in two-tailed comparison. ^∗P < 0.05 and ^∗∗P < 0.01.

FIGURE 5

Effects of GmIMaT1 and GmIMaT3 knockdown on isoflavone contents in hairy roots. (A–C) Representative hairy roots expressing GUS (A), GmIMaT1-RNAi (B), GmIMaT3-RNAi (C). (D,E) Expression of GmIMaT1 (D) and GmIMaT3 (E) in transgenic hairy root lines. (F–I) HPLC chromatographs for isoflavone profiles in GmIMaT1 and GmIMaT3 knockdown hairy root lines. (F) The numbered standard peaks are: 1, daidzin; 2, glycitin; 3, genistin; 4, malonyldaidzin; 5, malonylglycitin; 6, malonyl genistin; 7, daidzein; 8, glycitein; 9, genistein. (J) Contents of isoflavones in GmIMaT knockdown hairy root lines. Data are expressed in means ± SD from at least three independent transgenic lines. Differences between control and GmIMaT transgenic lines were analyzed by using Student’s t-test in two-tailed comparison. ^∗P < 0.05 and ^∗∗P < 0.01.

FIGURE 6

Subcellular localization of GmIMaT1 and GmIMaT3. Transient expression of GFP-GmIMaT1, GFP-GmIMaT3 and ER marker CD3-959-mCherry, driven by 35S promoter, was done in epidermal cells of tobacco leaf. Confocal microscopy was used for imaging. Bars = 10 μm. (A–C) ER marker CD3-959-mCherry in blue (A), GFP-GmIMaT1 (B), and the merge of both (C). (D–F) ER marker CD3-959-mCherry in blue (D), GFP-GmIMaT3 (E), and the merge of both (F).

FIGURE 7

Altered GmIMaT expression and isoflavone profiles in response to stresses. Differential expression patterns of GmIMaT1 and GmIMaT3 in soybean tissues under various conditions were examined, simultaneously with measurement of isoflavone contents in the same samples. Eight-week-old soybean plants were treated under cold (4°C) (A,B) or heat (42°C) (C–F) stress and pods (A–D) and leaves (E,F) were sampled in different time intervals. Six-week-old soybean plants were subjected to drought stress and roots were sampled after 10 days of treatment for analyses (G,H). For acidic condition (pH 4.0) treatment and 50 μM Al³⁺ stresses (under pH 4.0), hydroponically cultivated seedlings were transferred to these media for 10 days before harvesting roots for analysis of both gene expression and isoflavone profiling (I,J). Soybean seedlings with nine trifoliate were sprayed with hormones (100 μM ABA) (K,L), or their detached leaves floated on 50 μM MeJA solution and water (control) (M,N). Data are expressed in means ± SD from at least three independent experiments. Differences between control and treated samples were analyzed by using Student’s t-test in two-tailed comparison. ^∗P < 0.05, and ^∗∗P < 0.01.