Winter wheat hull (husk) is a valuable source for tricin, a potential selective cytotoxic agent.

The flavone, tricin (5,7,4'-trihydroxy-3',5'-dimethoxyflavone) has great potential as an anticancer agent, due to its specific chemopreventive activity. In spite of these characteristics, its use in preclinical studies is still limited, mainly because of its limited availability and high production cost. Tricin is found mainly in cereal grains, such as wheat, rice, barley, oat and maize. However, its concentration in these plants is not sufficient for commercial use. To find a reliable, rich source of tricin, we investigated its distribution in different parts of wheat (Triticum aestivum) and designed an efficient method for its isolation and purification. The highest amount (770 ± 157 mg/kg dry weight) was found in the husks of winter wheat. This concentration is one of the highest in any plant species and is considered as a cheap source of natural tricin. The purified wheat husks tricin was found to be a selective potent inhibitor of two cancer cell lines of liver and pancreas, while having no side effects on normal cells. This selective action suggests that tricin could be considered as a potential candidate for pre-clinical trials as a chemopreventive agent. In addition, fibre-rich crude wheat husk could be used as a natural chemopreventive agent in food supplement.

Tricin biosynthesis during growth of wheat under different abiotic stresses.

In plants, O-methylation is mediated by an enzyme family of O-methyltransferases (OMTs) that transfer the methyl groups from the methyl donor, S-adenosyl-L-methionine (AdoMet) to suitable phenolic acceptor molecules. In a previous study [1], a flavonoid OMT (TaOMT2) was isolated and characterized from wheat (Triticum aestivum L.) leaves. Its novel gene product catalyzes three sequential O-methylations of the flavone tricetin (5,7,3',4',5'-pentahydroxyflavone) to its 3'-monomethyl-(selgin)→3',5'-dimethyl-(tricin)→3',4',5'-trimethyl (TMT) ether derivatives, with tricin being the major product of the reaction. In this report, the biological significance of tricetin methylation was investigated by measuring the OMT activity, its expression level, and the accumulation of its major product (tricin) at different stages of development of wheat plants exposed to different abiotic stresses such as cold, salt and drought. The results showed that tricin accumulates mostly in wheat inflorescences under normal conditions compared to leaves and other developmental stages. Tricin accumulation was associated with increased TaOMT2 expression level and its enzyme activity, suggesting a possible de novo synthesis of the enzyme at this important developmental stage. This phenomenon may be attributed to the putative role of tricin in protecting seeds against biotic and abiotic stresses. The functions of tricin during growth and development of wheat and the importance of tricetin methylation during abiotic stresses are discussed.

Daphnetin methylation stabilizes the activity of phosphoribulokinase in wheat during cold acclimation.

The methylation of daphnetin (7,8-dihydroxycoumarin) to its 8-methyl derivative is catalyzed by a wheat (Triticum aestivum L.) O-methyltransferase (TaOMT1). This enzyme is regulated by cold and photosystem II excitation pressure (plastid redox state). Here, we investigated the biological significance of this methylation and its potential role in modulating the activity of kinases in wheat. To identify the potential kinases that may interact with daphnetin in wheat, the soluble protein extract from aerial parts of cold-acclimated wheat was purified by DEAE-cellulose separation and affinity chromatography on a daphnetin derivative (7,8-dihydroxy-4-coumarin acetic acid)-EAH sepharose column. Mass spectrometric analysis indicated that wheat phosphoribulokinase (TaPRK) is the major kinase that binds to daphnetin. This TaPRK plays an important role in regulating the flow of carbon through the Calvin cycle, by catalyzing the final step in the regeneration of ribulose 1,5-bisphosphate from ribulose-5-phosphate (Ru5P) and ATP. The activities of TaPRK, endogenous or recombinant, are inhibited by daphnetin in a specific and dose-dependent manner, but not by its monomethyl derivative (7-methyl, 8-hydroxycoumarin). Furthermore, HPLC-MS analysis of wheat extracts reveals that 7,8-dimethoxycoumarin is more abundant than its monomethyl derivative. The results also show that cold acclimation does not alter the level of TaPRK mRNA or its enzyme activity, and thus ensures the stable generation of ribulose 1,5-biphosphate.

Changes in wheat leaf phenolome in response to cold acclimation.

A study of wheat (Triticum aestivum L.) leaves phenolome was carried out during cold acclimation of the winter (Claire) and spring (Bounty) varieties using a combination of HPLC-ESI-MS techniques. A total of 40 phenolic and flavonoid compounds were identified, and consisted mainly of two coumarin derivatives, eight simple phenolic derivatives, 10 hydroxycinnamoyl amides and 20 flavonoid derivatives. Identification and quantification of individual compounds were performed using an HPLC system coupled with a photodiode array detector and two different ESI-MS systems, in combination with a multiple reaction monitoring (MRM) technique. The analyses indicated that, although there were no qualitative differences in their profiles, the winter variety exhibited a higher phenolic content compared to the spring variety when both were grown under non-acclimated (control) conditions. Cold acclimation, on the other hand, resulted in a significant differential accumulation of phenolic compounds in both varieties: mostly as luteolin C-glycosides and their O-methyl derivatives in the winter variety (Claire) and a derivative of hydroxycinnamoyl amide in the spring variety (Bounty). These compounds accumulated in relatively large amounts in the apoplastic compartment. The accumulation of the O-methylated derivatives was associated with a marked increase in O-methyltransferase (OMT) activity. In addition, the trimethylated flavone, 3',4',5'-trimethyltricetin was identified for the first time in the native extracts of both control and cold-acclimated wheat leaves. The accumulation of a mixture of beneficial flavonoids, such as iso-orientin, vitexin and tricin in cold acclimated wheat leaves, attests for its potential as an inexpensive source of a health-promoting supplement to the human diet.

Structure-function relationships of wheat flavone O-methyltransferase: Homology modeling and site-directed mutagenesis.

BACKGROUND: Wheat (Triticum aestivum L.) O-methyltransferase (TaOMT2) catalyzes the sequential methylation of the flavone, tricetin, to its 3'-methyl- (selgin), 3',5'-dimethyl- (tricin) and 3',4',5'-trimethyl ether derivatives. Tricin, a potential multifunctional nutraceutical, is the major enzyme reaction product. These successive methylations raised the question as to whether they take place in one, or different active sites. We constructed a 3-D model of this protein using the crystal structure of the highly homologous Medicago sativa caffeic acid/5-hydroxyferulic acid O-methyltransferase (MsCOMT) as a template with the aim of proposing a mechanism for multiple methyl transfer reactions in wheat. RESULTS: This model revealed unique structural features of TaOMT2 which permit the stepwise methylation of tricetin. Substrate binding is mediated by an extensive network of H-bonds and van der Waals interactions. Mutational analysis of structurally guided active site residues identified those involved in binding and catalysis. The partly buried tricetin active site, as well as proximity and orientation effects ensured sequential methylation of the substrate within the same pocket. Stepwise methylation of tricetin involves deprotonation of its hydroxyl groups by a His262-Asp263 pair followed by nucleophilic attack of SAM-methyl groups. We also demonstrate that Val309, which is conserved in a number of graminaceous flavone OMTs, defines the preference of TaOMT2 for tricetin as the substrate. CONCLUSIONS: We propose a mechanism for the sequential methylation of tricetin, and discuss the potential application of TaOMT2 to increase the production of tricin as a nutraceutical. The single amino acid residue in TaOMT2, Val309, determines its preference for tricetin as the substrate, and may define the evolutionary differences between the two closely related proteins, COMT and flavone OMT.

Biochemical characterization of a putative wheat caffeic acid O-methyltransferase.

A wheat (Triticum aestivum L., near isogenic line of Hamlet) O-methyltransferase (OMT) was previously reported as a putative caffeic acid OMT (TaCOMT1), involved in lignin biosynthesis, based on its high sequence similarity with a number of graminaceous COMTs. The fact that the putative TaCOMT1 exhibits a significantly high sequence homology to another recently characterized wheat flavone-specific OMT (TaOMT2), and that molecular modeling studies indicated several conserved amino acid residues involved in substrate binding and catalysis of both proteins, prompted an investigation of its appropriate substrate specificity. We report here that TaCOMT1 exhibits highest preference for the flavone tricetin, and lowest activity with the lignin precursors, caffeic acid/5-hydroxyferulic acid as the methyl acceptor molecules, indicating that it is not involved in lignin biosynthesis. We recommend its reannotation to a flavone-specific TaOMT1 that is distinct from TaOMT2.

Structure-activity relationships of wheat flavone O-methyltransferase: a homodimer of convenience.

Wheat flavone O-methyltransferase catalyzes three sequential methylations of the flavone tricetin. Like other flavonoid O-methyltransferases, the protein is a homodimer. We demonstrate, using analytical ultracentrifugation, that perchlorate dissociates the dimer into monomers. The resulting monomers retain all their catalytic capacity, including the ability to catalyze the three successive methylations. We show, using isothermal titration calorimetry, that the binding constant for S-adenosyl-L-methionine does not change significantly as the protein dissociates. The second substrate, tricetin, binds to the dimers but could not be tested with the monomers. CD, UV and fluorescence spectroscopy show that there are substantial changes in the structure of the protein as it dissociates. The fact that there are differences between the monomers and dimers even as the monomers maintain activity may be the result of the very low catalytic capacity of this enzyme. Maximal turnover numbers for the dimers and monomers are only about 6-7 per minute. Even though the binding pockets for S-adenosyl-L-methionine, tricetin, selgin and tricin are intact, selection of a catalytically competent structure may be a very slow step during catalysis.

Structure, function, and evolution of plant O-methyltransferases.

Plant O-methyltransferases (OMTs) constitute a large family of enzymes that methylate the oxygen atom of a variety of secondary metabolites including phenylpropanoids, flavonoids, and alkaloids. O-Methylation plays a key role in lignin biosynthesis, stress tolerance, and disease resistance in plants. To gain insights into the evolution of the extraordinary diversity of plant O-methyltransferases, and to develop a framework phylogenetic tree for improved prediction of the putative function of newly identified OMT-like gene sequences, we performed a comparative and phylogenetic analysis of 61 biochemically characterized plant OMT protein sequences. The resulting phylogenetic tree revealed two major groups. One of the groups included two sister clades, one comprising the caffeoyl CoA OMTs (CCoA OMTs) that methylate phenolic hydroxyl groups of hydroxycinnamoyl CoA esters, and the other containing the carboxylic acid OMTs that methylate aliphatic carboxyl groups. The other group comprised the remaining OMTs, which act on a diverse group of metabolites including hydroxycinnamic acids, flavonoids, and alkaloids. The results suggest that some OMTs may have undergone convergent evolution, while others show divergent evolution. The high number of unique conserved regions within the CCoA OMTs and carboxylic acid OMTs provide an opportunity to design oligonucleotide primers to selectively amplify and characterize similar OMT genes from many plant species.

Sequential O-methylation of tricetin by a single gene product in wheat.

Flavonoid compounds are ubiquitous in nature. They constitute an important part of the human diet and act as active principles of many medicinal plants. Their O-methylation increases their lipophilicity and hence, their compartmentation and functional diversity. We have isolated and characterized a full-length flavonoid O-methyltransferase cDNA (TaOMT2) from a wheat leaf cDNA library. The recombinant TaOMT2 protein was purified to near homogeneity and tested for its substrate preference against a number of phenolic compounds. Enzyme assays and kinetic analyses indicate that TaOMT2 exhibits a pronounced preference for the flavone, tricetin and gives rise to three methylated enzyme reaction products that were identified by TLC, HPLC and ESI-MS/MS as its mono-, di- and trimethyl ether derivatives. The sequential order of tricetin methylation by TaOMT2 is envisaged to proceed via its 3'-mono--->3',5'-di--->3',4',5'-trimethyl ether derivatives. To our knowledge, this is the first report of a gene product that catalyzes three sequential O-methylations of a flavonoid substrate.

Molecular characterization and functional expression of flavonol 6-hydroxylase.

BACKGROUND: Flavonoids, one of the major groups of secondary metabolites, play important roles in the physiology, ecology and defence of plants. Their wide range of activities is the result of their structural diversity that encompasses a variety of functional group substitutions including hydroxylations. The aromatic hydroxylation at position 6 of flavonols is of particular interest, since it is catalyzed by a 2-oxoglutarate-dependent dioxygenase (ODD), rather than a cytochrome P450-dependent monooxygenase. ODDs catalyze a variety of enzymatic reactions implicated in secondary metabolite biosynthesis. RESULTS: A cDNA fragment encoding an ODD involved in the 6-hydroxylation of partially methylated flavonols, flavonol 6-hydroxylase (F6H), was isolated and characterized from Chrysosplenium americanum using internal peptide sequence information obtained from the native plant protein. This novel clone was functionally expressed in both prokaryotic and eukaryotic expression systems and exhibited ODD activity. The cofactor and cosubstrate requirements of the recombinant proteins are typical for ODDs, and the recombinant enzymes utilize 3,7,4'-trimethylquercetin as the preferred substrate. The genomic region encoding this enzyme possesses two introns at conserved locations for this class of enzymes and is present as a single copy in the C. americanum genome. CONCLUSIONS: Recombinant F6H has been functionally expressed and characterized at the molecular level. The results demonstrate that its cofactor dependence, physicochemical characteristics and substrate preference compare well with the native enzyme. The N-terminal region of this protein is believed to play a significant role in catalysis and may explain the difference in the position specificity of the 6-hydroxylation reaction.

Role of Serine 286 in cosubstrate binding and catalysis of a flavonol O-methyltransferase.

O-Methyltransferases catalyze the transfer of the methyl groups of S-adenosyl-L-methionine to specific hydroxyl groups of several classes of flavonoid compounds. Of the several cDNA clones isolated from a Chrysosplenium americanum library, FOMT3' encodes the 3'/5'-O-methylation of partially methylated flavonols. The recombinant protein of another clone, FOMTx which differs from FOMT3' by a single amino acid residue (Ser286Arg) exhibits no enzymatic activity towards any of the flavonoid substrates tested. Replacement of Ser 286 in FOMT3' with either Ala, Leu, Lys or Thr, almost abolished O-methyltransferase activity. In contrast with FOMT3', no photoaffinity labeling could be achieved using [(14)CH(3)]AdoMet with the mutant recombinant proteins indicating that Ser 286 is also required for cosubstrate binding. These results are corroborated by isothermal titration microcalorimetry measurements. Circular dichroism spectra ruled out any significant conformational differences in the secondary structures of both FOMT3' and Ser286Arg. Modeling FOMT3' on the structure of chalcone methyltransferase indicates that serine 286 is greater than 10 A from any of the residues of the active site or the AdoMet binding site of FOMT3'. At the same time, residues 282 to 290 are conserved in most of the Chrysosplenium americanum OMTs. These residues form a large part of the subunit interface, and at least five of these residues are within 4 A of the opposing subunit. It would appear, therefore, that mutations in Ser286 exert their influence by altering the contacts between the subunits and that these contacts are necessary for maintaining the integrety of the AdoMet binding site and active site of this group of enzymes.

The three-dimensional structure of Arabidopsis thaliana O-methyltransferase predicted by homology-based modelling.

O-methylation of flavonoid compounds is an important enzymatic reaction since it not only reduces the chemical reactivity of their phenolic hydroxyl groups but also increases their lipophilicity and, hence, their intracellular compartmentation. Several genes encoding flavonoid O-methyltransferases (OMTs) have been isolated and characterized both at the molecular and biochemical levels. In contrast with mammalian enzymes, plant OMTs exhibit narrow substrate specificities as well as position-specific activities, so that the homology comparison, derived using programs such as BLAST can not provide sufficient information on the enzyme function or its substrate preference. In order to study these characteristics, therefore, another approach, homology-based modelling is being carried out. We report here the determination of the 3-D structure of Arabidopsis thaliana O-methyltransferase, AtOMT1 as well as its dynamics when complexed with its substrate. The predicted structure obtained by homology-based modelling is conserved during molecular dynamics simulations. AtOMT1 exhibits a structure similar to that of caffeic acid O-methyltransferase, COMT when the latter was used as a template. Whereas COMT includes 20 alpha-helices and nine beta-sheets, AtOMT1 has 16 and 9, respectively. Although the homology between both proteins is higher than 77% and all amino acids surrounding the active sites, except one residue, are similar in their primary sequences, the two proteins exhibit different substrate preferences. The differences in substrate specificity may be explained on the basis of the predicted structures of the protein and its complex with the substrate. In addition, docking the substrate into the active site of the protein allowed the study of the structural change of the active site on the dihedral angle distribution of the residues surrounding the active site.

Partial purification, kinetic analysis, and amino acid sequence information of a flavonol 3-O-methyltransferase from Serratula tinctoria.

Serratula tinctoria (Asteraceae) accumulates mainly 3,3'-dimethylquercetin and small amounts of 3-methylquercetin as an intermediate. The fact that 3-methylquercetin rarely accumulates in plants in significant amounts, and given its important role as an antiviral and antiinflammatory agent that accumulates in response to stress conditions, prompted us to purify and characterize the enzyme involved in its methylation. The flavonol 3-O-methyltransferase (3-OMT) was partially purified by ammonium sulfate precipitation and successive chromatography on Superose-12, Mono-Q, and adenosine-agarose affinity columns, resulting in a 194-fold increase of its specific activity. The enzyme protein exhibited an expressed specificity for the methylation of position 3 of the flavonol, quercetin, although it also utilized kaempferol, myricetin, and some monomethyl flavonols as substrates. It exhibited a pH optimum of 7.6, a pI of 6.0, and an apparent molecular mass of 31 kD. Its K(m) values for quercetin as the substrate and S-adenosyl-l-Met (AdoMet) as the cosubstrate were 12 and 45 microm, respectively. The 3-OMT had no requirement for Mg(2+), but was severely inhibited by p-chloromercuribenzoate, suggesting the requirement for SH groups for catalytic activity. Quercetin methylation was competitively inhibited by S-adenosyl-l-homo-Cys with respect to the cosubstrate AdoMet, and followed a sequential bi-bi reaction mechanism, where AdoMet was the first to bind and S-adenosyl-l-homo-Cys was released last. In-gel trypsin digestion of the purified protein yielded several peptides, two of which exhibited strong amino acid sequence homology, upon protein identification, to a number of previously identified Group II plant OMTs. The availability of peptide sequences will allow the design of specific nucleotide probes for future cloning of the gene encoding this novel enzyme for its use in metabolic engineering.

Daphnetin methylation by a novel O-methyltransferase is associated with cold acclimation and photosystem II excitation pressure in rye.

In plants, O-methylation of phenolic compounds plays an important role in such processes as lignin synthesis, flower pigmentation, chemical defense, and signaling. However, apart from phenylpropanoids and flavonoids, very few enzymes involved in coumarin biosynthesis have been identified. We report here the molecular and biochemical characterization of a gene encoding a novel O-methyltransferase that catalyzes the methylation of 7,8-dihydroxycoumarin, daphnetin. The recombinant protein displayed an exclusive methylation of position 8 of daphnetin. The identity of the methylated product was unambiguously identified as 7-hydroxy-8-methoxycoumarin by co-chromatography on cellulose TLC and coelution from high performance liquid chromatography, with authentic synthetic samples, as well as by UV, mass spectroscopy, (1)H NMR spectral analysis, and NOE correlation signals of the relevant protons. Northern blot analysis and enzyme activity assays revealed that the transcript and corresponding enzyme activity are up-regulated by both low temperature and photosystem II excitation pressure. Using various phenylpropanoid and flavonoid substrates, we demonstrate that cold acclimation of rye leaves increases O-methyltransferase activity not only for daphnetin but also for the lignin precursors, caffeic acid, and 5-hydroxyferulic acid. The significance of this novel enzyme and daphnetin O-methylation is discussed in relation to its putative role in modulating cold acclimation and photosystem II excitation pressure.

Molecular and biochemical characterization of a cold-regulated phosphoethanolamine N-methyltransferase from wheat.

A cDNA that encodes a methyltransferase (MT) was cloned from a cold-acclimated wheat (Triticum aestivum) cDNA library. Molecular analysis indicated that the enzyme WPEAMT (wheat phosphoethanolamine [P-EA] MT) is a bipartite protein with two separate sets of S-adenosyl-L-Met-binding domains, one close to the N-terminal end and the second close to the C-terminal end. The recombinant protein was found to catalyze the three sequential methylations of P-EA to form phosphocholine, a key precursor for the synthesis of phosphatidylcholine and glycine betaine in plants. Deletion and mutation analyses of the two S-adenosyl-L-Met-binding domains indicated that the N-terminal domain could perform the three N-methylation steps transforming P-EA to phosphocholine. This is in contrast to the MT from spinach (Spinacia oleracea), suggesting a different functional evolution for the monocot enzyme. The truncated C-terminal and the N-terminal mutated enzyme were only able to methylate phosphomonomethylethanolamine and phosphodimethylethanolamine, but not P-EA. This may suggest that the C-terminal part is involved in regulating the rate and the equilibrium of the three methylation steps. Northern and western analyses demonstrated that both Wpeamt transcript and the corresponding protein are up-regulated during cold acclimation. This accumulation was associated with an increase in enzyme activity, suggesting that the higher activity is due to de novo protein synthesis. The role of this enzyme during cold acclimation and the development of freezing tolerance are discussed.