Xian-Zhi He

Structures of two natural product methyltransferases reveal the basis for substrate specificity in plant O-methyltransferases.

Chalcone O-methyltransferase (ChOMT) and isoflavone O-methyltransferase (IOMT) are S-adenosyl-l-methionine (SAM) dependent plant natural product methyltransferases involved in secondary metabolism in Medicago sativa (alfalfa). Here we report the crystal structure of ChOMT in complex with the product S-adenosyl-l-homocysteine and the substrate isoliquiritigenin (4,2′,4′-trihydroxychalcone) refined to 1.8 Å as well as the crystal structure of IOMT in complex with the products S-adenosyl-l-homocysteine and isoformononetin (4′-hydroxy-7-methoxyisoflavone) refined to 1.4 Å. These two OMTs constitute the first plant methyltransferases to be structurally characterized and reveal a novel oligomerization domain and the molecular determinants for substrate selection. As such, this work provides a structural basis for understanding the substrate specificity of the diverse family of plant OMTs and facilitates the engineering of novel activities in this extensive class of natural product biosynthetic enzymes.

Crystal Structures of a Multifunctional Triterpene/Flavonoid Glycosyltransferase from Medicago truncatula

Glycosylation is a ubiquitous reaction controlling the bioactivity and storage of plant natural products. Glycosylation of small molecules is catalyzed by a superfamily of glycosyltransferases (GTs) in most plant species studied to date. We present crystal structures of the UDP flavonoid/triterpene GT UGT71G1 from Medicago truncatula bound to UDP or UDP-glucose. The structures reveal the key residues involved in the recognition of donor substrate and, by comparison with other GT structures, suggest His-22 as the catalytic base and Asp-121 as a key residue that may assist deprotonation of the acceptor by forming an electron transfer chain with the catalytic base. Mutagenesis confirmed the roles of these key residues in donor substrate binding and enzyme activity. Our results provide an initial structural basis for understanding the complex substrate specificity and regiospecificity underlying the glycosylation of plant natural products and other small molecules. This information will direct future attempts to engineer bioactive compounds in crop plants to improve plant, animal, and human health and to facilitate the rational design of GTs to improve the storage and stability of novel engineered bioactive compounds.

Genetic Manipulation of Isoflavone 7-O-Methyltransferase Enhances Biosynthesis of 4′-O-Methylated Isoflavonoid Phytoalexins and Disease Resistance in Alfalfa

4′-O-Methylation of an isoflavonoid intermediate is a key reaction in the biosynthesis of the phytoalexin medicarpin in legumes. However, isoflavone O-methyltransferase (IOMT) from alfalfa converts the isoflavone daidzein to 7-O-methyl daidzein (isoformononetin) in vitro as well as in vivo in unchallenged leaves of transgenic alfalfa ectopically expressing IOMT. In contrast, elicitation of IOMT-overexpressing plants with CuCl2 or infecting these plants with Phoma medicaginis leads to greater accumulation of formononetin (4′-O-methyl daidzein) and medicarpin in the leaves than does elicitation or infection of control plants, and no isoformononetin is detected. Overexpression of IOMT results in increased induction of phenylpropanoid/isoflavonoid pathway gene transcripts after infection but has little effect on basal expression of these genes. IOMT-overexpressing plants display resistance to P. medicaginis. The apparently different regiospecificities of IOMT in vivo and in vitro are discussed in relation to potential metabolic channeling at the entry point into the isoflavonoid pathway.

Functional characterization of the switchgrass (Panicum virgatum) R2R3‐MYB transcription factor PvMYB4 for improvement of lignocellulosic feedstocks

• The major obstacle for bioenergy production from switchgrass biomass is the low saccharification efficiency caused by cell wall recalcitrance. Saccharification efficiency is negatively correlated with both lignin content and cell wall ester-linked p-coumarate: ferulate (p-CA : FA) ratio. In this study, we cloned and functionally characterized an R2R3-MYB transcription factor from switchgrass and evaluated its potential for developing lignocellulosic feedstocks. • The switchgrass PvMYB4 cDNAs were cloned and expressed in Escherichia coli, yeast, tobacco and switchgrass for functional characterization. Analyses included determination of phylogenetic relations, in situ hybridization, electrophoretic mobility shift assays to determine binding sites in target promoters, and protoplast transactivation assays to demonstrate domains active on target promoters. • PvMYB4 binds to the AC-I, AC-II and AC-III elements of monolignol pathway genes and down-regulates these genes in vivo. Ectopic overexpression of PvMYB4 in transgenic switchgrass resulted in reduced lignin content and ester-linked p-CA : FA ratio, reduced plant stature, increased tillering and an approx. threefold increase in sugar release efficiency from cell wall residues. • We describe an alternative strategy for reducing recalcitrance in switchgrass by manipulating the expression of a key transcription factor instead of a lignin biosynthetic gene. PvMYB4-OX transgenic switchgrass lines can be used as potential germplasm for improvement of lignocellulosic feedstocks and provide a platform for further understanding gene regulatory networks underlying switchgrass cell wall recalcitrance.

MATE2 Mediates Vacuolar Sequestration of Flavonoid Glycosides and Glycoside Malonates in Medicago truncatula

This work identifies MATE2, which transports glycosylated flavonoids into the vacuole, showing higher transport efficiency with anthocyanins than other flavonoid glycosides, and an increase in transport efficiency for malonylated flavonoid glucosides. Null mutants of MATE2 show decreases of anthocyanins and increases in other flavonoids, such as seed proanthocyanidin. The majority of flavonoids, such as anthocyanins, proanthocyanidins, and isoflavones, are stored in the central vacuole, but the molecular basis of flavonoid transport is still poorly understood. Here, we report the functional characterization of a multidrug and toxin extrusion transporter (MATE2), from Medicago truncatula. MATE 2 is expressed primarily in leaves and flowers. Despite its high similarity to the epicatechin 3′-O-glucoside transporter MATE1, MATE2 cannot efficiently transport proanthocyanidin precursors. In contrast, MATE2 shows higher transport capacity for anthocyanins and lower efficiency for other flavonoid glycosides. Three malonyltransferases that are coexpressed with MATE2 were identified. The malonylated flavonoid glucosides generated by these malonyltransferases are more efficiently taken up into MATE2-containing membrane vesicles than are the parent glycosides. Malonylation increases both the affinity and transport efficiency of flavonoid glucosides for uptake by MATE2. Genetic loss of MATE2 function leads to the disappearance of leaf anthocyanin pigmentation and pale flower color as a result of drastic decreases in the levels of various flavonoids. However, some flavonoid glycoside malonates accumulate to higher levels in MATE2 knockouts than in wild-type controls. Deletion of MATE2 increases seed proanthocyanidin biosynthesis, presumably via redirection of metabolic flux from anthocyanin storage.

Stress responses in alfalfa (Medicago sativa L). XXII. cDNA cloning and characterization of an elicitor-inducible isoflavone 7-O-methyltransferase.

Medicarpin, the major phytoalexin in alfalfa, is synthesized via the isoflavonoid branch of phenylpropanoid metabolism. The methyl group at the 9 position of medicarpin is generally accepted to arise via the methylation of the 4′ position (B-ring) of daidzein. Surprisingly, the isoflavone-O-methyltransferase (IOMT), which is induced along with other enzymes involved in medicarpin biosynthesis, methylates the A-ring 7-hydroxyl group of daidzein in vitro, a reaction that probably does not occur in vivo. Utilizing internal amino acid sequence information from purified alfalfa IOMT, we have isolated three full-length IOMT cDNA clones. A search of the protein databases revealed sequence similarities to O-methyltransferases from various sources. The highest match (50.5% identity) was found between IOMT8 and 6a-hydroxymaackiain 3-O-methyltransferase from Pisum sativum. The molecular weight of alfalfa IOMT expressed in Escherichia coli was similar to that of purified IOMT from alfalfa cell cultures (41 kDa by SDS-PAGE). The recombinant enzyme catalyzed the O-methylation of A-ring hydroxyl group(s) of isoflavones, and could also methylate the pterocarpan (+) 6a-hydroxymaackiain. Alfalfa contains multiple IOMT genes, and closely related sequences are present in the genomes of chickpea and cowpea, species that also produce B-ring methylated isoflavonoids in vivo. Northern blot analysis indicated that IOMT transcripts are rapidly induced following elicitation, prior to the increase in IOMT activity and medicarpin accumulation. The possible role of the isoflavone 7-OMT in the synthesis of formononetin in vivo is discussed.

Mutational Analysis of the Medicago Glycosyltransferase UGT71G1 Reveals Residues That Control Regioselectivity for (Iso)flavonoid Glycosylation

The plant glycosyltransferase UGT71G1 from the model legume barrel medic (Medicago truncatula) glycosylates flavonoids, isoflavonoids, and triterpenes. It can transfer glucose to each of the five hydroxyl groups of the flavonol quercetin, with the 3′-O-glucoside as the major product, and to the A-ring 7-hydroxyl of the isoflavone genistein. The sugar donor and acceptor binding pockets are located in the N and C termini, respectively, of the recently determined crystal structure of UGT71G1. The residues forming the binding pockets of UGT71G1 were systematically altered by site-directed mutagenesis. Mutation of Phe148 to Val, or Tyr202 to Ala, drastically changed the regioselectivity for quercetin glycosylation from predominantly the 3′-O-position of the B-ring to the 3-O-position of the C ring. The Y202A mutant exhibited comparable catalytic efficiency with quercetin to the wild-type enzyme, whereas efficiency was reduced 3-4-fold in the F148V mutant. The Y202A mutant gained the ability to glycosylate the 5-hydroxyl of genistein. Additional mutations affected the relative specificities for the sugar donors UDP-galactose and UDP-glucuronic acid, although UDP-glucose was always preferred. The results are discussed in relation to the design of novel biocatalysts for production of therapeutic flavonoids.

Elicitor-induced transcription factors for metabolic reprogramming of secondary metabolism in Medicago truncatula

BackgroundExposure of Medicago truncatula cell suspension cultures to pathogen or wound signals leads to accumulation of various classes of flavonoid and/or triterpene defense molecules, orchestrated via a complex signalling network in which transcription factors (TFs) are essential components.ResultsIn this study, we analyzed TFs responding to yeast elicitor (YE) or methyl jasmonate (MJ). From 502 differentially expressed TFs, WRKY and AP2/EREBP gene families were over-represented among YE-induced genes whereas Basic Helix-Loop-Helix (bHLH) family members were more over-represented among the MJ-induced genes. Jasmonate ZIM-domain (JAZ) transcriptional regulators were highly induced by MJ treatment. To investigate potential involvement of WRKY TFs in signalling, we expressed four Medicago WRKY genes in tobacco. Levels of soluble and wall bound phenolic compounds and lignin were increased in all cases. WRKY W109669 also induced tobacco endo-1,3-β-glucanase (NtPR2) and enhanced the systemic defense response to tobacco mosaic virus in transgenic tobacco plants.ConclusionThese results confirm that Medicago WRKY TFs have broad roles in orchestrating metabolic responses to biotic stress, and that they also represent potentially valuable reagents for engineering metabolic changes that impact pathogen resistance.

MtPAR MYB transcription factor acts as an on switch for proanthocyanidin biosynthesis in Medicago truncatula

MtPAR (Medicago truncatula proanthocyanidin regulator) is an MYB family transcription factor that functions as a key regulator of proanthocyanidin (PA) biosynthesis in the model legume Medicago truncatula. MtPAR expression is confined to the seed coat, the site of PA accumulation. Loss-of-function par mutants contained substantially less PA in the seed coat than the wild type, whereas levels of anthocyanin and other specialized metabolites were normal in the mutants. In contrast, massive accumulation of PAs occurred when MtPAR was expressed ectopically in transformed hairy roots of Medicago. Transcriptome analysis of par mutants and MtPAR-expressing hairy roots, coupled with yeast one-hybrid analysis, revealed that MtPAR positively regulates genes encoding enzymes of the flavonoid–PA pathway via a probable activation of WD40-1. Expression of MtPAR in the forage legume alfalfa (Medicago sativa) resulted in detectable levels of PA in shoots, highlighting the potential of this gene for biotechnological strategies to increase PAs in forage legumes for reduction of pasture bloat in ruminant animals.

Functional Characterization of Proanthocyanidin Pathway Enzymes from Tea and Their Application for Metabolic Engineering

Tea contains two anthocyanidin reductases that produce different proportions of two forms of epicatechin. Tea (Camellia sinensis) is rich in specialized metabolites, especially polyphenolic proanthocyanidins (PAs) and their precursors. To better understand the PA pathway in tea, we generated a complementary DNA library from leaf tissue of the blister blight-resistant tea cultivar TRI2043 and functionally characterized key enzymes responsible for the biosynthesis of PA precursors. Structural genes encoding enzymes involved in the general phenylpropanoid/flavonoid pathway and the PA-specific branch pathway were well represented in the library. Recombinant tea leucoanthocyanidin reductase (CsLAR) expressed in Escherichia coli was active with leucocyanidin as substrate to produce the 2R,3S-trans-flavan-ol (+)-catechin in vitro. Two genes encoding anthocyanidin reductase, CsANR1 and CsANR2, were also expressed in E. coli, and the recombinant proteins exhibited similar kinetic properties. Both converted cyanidin to a mixture of (+)-epicatechin and (−)-catechin, although in different proportions, indicating that both enzymes possess epimerase activity. These epimers were unexpected based on the belief that tea PAs are made from (−)-epicatechin and (+)-catechin. Ectopic expression of CsANR2 or CsLAR led to the accumulation of low levels of PA precursors and their conjugates in Medicago truncatula hairy roots and anthocyanin-overproducing tobacco (Nicotiana tabacum), but levels of oligomeric PAs were very low. Surprisingly, the expression of CsLAR in tobacco overproducing anthocyanin led to the accumulation of higher levels of epicatechin and its glucoside than of catechin, again highlighting the potential importance of epimerization in flavan-3-ol biosynthesis. These data provide a resource for understanding tea PA biosynthesis and tools for the bioengineering of flavanols.