Functional analysis of members of the isoflavone and isoflavanone O-methyltransferase enzyme families from the model legume Medicago truncatula

Abstract

Previous studies have identified two distinct O-methyltransferases (OMTs) implicated in isoflavonoid biosynthesis in Medicago species, a 7-OMT methylating the A-ring 7-hydroxyl of the isoflavone daidzein and a 4'-OMT methylating the B-ring 4'-hydroxyl of 2,7,4'-trihydroxyisoflavanone. Genes related to these OMTs from the model legume Medicago truncatula cluster as separate branches of the type I plant small molecule OMT family. To better understand the possible functions of these related OMTs in secondary metabolism in M. truncatula, seven of the OMTs were expressed in E. coli, purified, and their in vitro substrate preferences determined. Many of the enzymes display promiscuous activities, and some exhibit dual regio-specificity for the 4' and 7-hydroxyl moieties of the isoflavonoid nucleus. Protein structure homology modeling was used to help rationalize these catalytic activities. Transcripts encoding the different OMT genes exhibited differential tissue-specific and infection- or elicitor-induced expression, but not always in parallel with changes in expression of confirmed genes of the isoflavonoid pathway. The results are discussed in relation to the potential in vivo functions of these OMTs based on our current understanding of the phytochemistry of M. truncatula, and the difficulties associated with gene annotation in plant secondary metabolism.

Fig. 1

Isoflavonoid biosynthetic pathways and IOMT activities. (A) Pathway leading to biosynthesis of formononetin (4), medicarpin (5), and sativan (7). The flavanone liquiritigenin (1) is converted to 2,7,4′-trihydroxyisoflavanone (2) in a reaction catalyzed by isoflavone synthase (IFS). Methylation of (2) by 4′IOMT forms 2,7-dihydroxy, 4′-methoxyisoflavanone (3), which is converted to (4) by 2-hydroxyisoflavanone dehydratase (2HID). Formononetin is a precursor of (5) and (7), and therefore the methyl groups at the 9- (5) and 4′- (7) positions of these compounds originate from 4′IOMT. Sativan (7) is most likely synthesized by 2′-methylation of vestitol (6). (B) Isoflavone 7-OMT activity characterized from legumes. I7OMT methylates daidzein (8) on the 7-position to form isoformononetin (9). (C) Proposed pathway for biosynthesis of alfalone (11) and afromosin (12). Flavanone 6-hydroxylase (F6H) has been shown to introduce a 6-hydroxyl group at the level of flavanone. Subsequent steps may be catalyzed according to the pathway in A and would lead to 6,7-dihydroxy, 4′-methoxyisoflavone (10). Methylation by a 7IOMT would form (11), while methylation by a 6IOMT would form (12). (D) Proposed pathway for biosynthesis of irilone (16). Irilone may be formed by methylation of 4′,5,6,7-tetrahydroxyisoflavone (13) at either the 6- (15) or 7- (14) position followed by ring closure

Fig. 2

Relationship tree of (iso)flavonoid OMTs. CrOMT6, Catharanthus roseus flavonoid 4′-OMT (AY343490); CrOMT2, C. roseus 3′/5′-flavonol OMT (AY127569); HvF7OMT, Hordeum vulgare flavonoid 7-OMT (X77467); GeD7OMT, G. echinata daidzein 7-OMT (AB091685); MsI7OMT, M. sativa isoflavone 7-OMT (U97125); LjHI4′OMT, Lotus japonicus 2-hydroxyisoflavanone 4′-OMT (AB091686); GeHI4′OMT, G. echinata 2-hydroxyisoflavanone 4′-OMT (AB091684); PsHMM1, P. sativum 6a–hydroxymaackiain 3-OMT (U69554). The M. truncatula IOMT sequences are available in the GenBank database under the accession numbers: AY942159, MtIOMT1; DQ419910, MtIOMT2; DQ419911, MtIOMT3; DQ419912, MtIOMT4; AY942158, MtIOMT5; DQ419913, MtIOMT6; DQ419914, MtIOMT7; DQ419915, MtIOMT8

Fig. 3

(A) Genomic organization of MtIOMTs 1–3. Black boxes represent exons and arrows indicate relative orientation. Numbering is in reference to BAC clone AC146549. (B) Location of the single intron in the protein sequence of MtIOMT1, MtIOMT2, and MtIOMT3. Numbers in parentheses represents length of intron in bps. Residues involved in SAM-binding (underlined) and catalysis (bold) are indicated (Zubieta et al. 2001). Figure 3B is modeled after Schroder et al. (2004)

Fig. 4

HPLC chromatograms of reaction products of MtIOMT1 (A, D, G, J) and MtIOMT3 (B, E, H, K) with 6,7,4′-trihydroxyisoflavone (A, B), 7,3′,4′-trihydroxyisoflavone (D, E), daidzein (G, H), and genistein (J, K) as substrates. Standards are shown in panels C, F, I, L and methylated standards are labeled according to the position of the methyl group. (C) THI, 6,7,4′-trihydroxyisoflavone; 6, glycitein; 4′, 6,7-dihydroxy, 4′-methoxyisoflavone. (F) THI2, 7,3′,4′-trihydroxyisoflavone; 3′, 3′-methoxydaidzein; 4′, calycosin. (I) D, daidzein; 7, isoformononetin; 4′, formononetin. (L) G, genistein; 7, prunetin; 4′, biochanin A

Fig. 5

Chiral HPLC chromatography of unreacted liquiritigenin present in reactions with MtIOMT5 (B) and MtIOMT7 (C). Racemic liquiritigenin standard is shown in (A)

Fig. 6

Homology models of selected members of the MtI7OMT clade. (A, B) Close-up view of the MtIOMT2 active site with dihydrodaidzein docked for 7-O-methylation (A) or 4-O-methylation (B). (C, D) View of 7,3′,4′-trihydroxyisoflavone (C) and glycitein (D) docked into the MtIOMT3 active site. (E, F) Molecular surface views of the experimentally determined MsI7OMT active site (E) and the modeled MtIOMT4 active site (F)

Fig. 7

Expression of MtIOMTs in healthy tissues of M. truncatula and in leaves infected with the leaf pathogen P. medicaginis. MtIOMT expression was determined by RT-PCR using primers specific for each MtIOMT. IFS, isoflavone synthase

Fig. 8

Expression analysis of MtIOMT and IFS genes in elicitortreated cell suspension cultures of M. truncatula. (A) Microarray analysis of seven MtIOMT genes in response to YE and MeJA treatment. TC numbers from TIGR MtGI v. 8/v.5 are given under the description. (B) RT-PCR analysis confirming induction of MtIOMT2 and MtIOMT6 by MeJA. (C) Microarray analysis of IFS expression in response to YE and MeJA treatment. The TC number is from TIGR MtGI v. 8. Note: for (A) and (C), numbers represent ratios of treatment versus control samples. All ratios are coded according to the color scheme at the bottom of the figure. All color coded values had a “presence call:” by the GCOS program in the higher expressed or both sample groups. The non-coded values indicate absence of a “presence call:” in both control and treatment samples