Abstract

Wheat (Triticum aestivum) O-methyltransferase (TaOMT2) catalyzes the sequential methylation of the flavone,tricetin (5,7,3',4',5'-pentahydroxyflavone) to its 3'-methyl-(selgin), 3',5'-dimethyl-(tricin) and 3',4',5'-trimethyl ether derivatives, although tricin is the major product of this reaction. The novelty of TaOMT2 to perform three sequential methylations of tricetin as a substrate, the chemopreventive properties of its major product, tricin, and the compelling interest in the protein’s structure-function relationships, prompted us to further investigate this novel protein at the biochemical, molecular and structural levels. A 3-D model of this protein was constructed 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. Homology modeling experiments in which each of the substrates tricetin, selgin and tricin, was docked into the model revealed a number of amino acid residues putatively involved in substrate binding and catalysis. Results suggest that 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. A possible reaction mechanism is discussed.
The biological significance of this methylation reaction was also investigated by analyzing its expression, enzyme activity patterns at different wheat developmental stages, in response to cold acclimation and to different abiotic stresses such as salt and drought. Results show that TaOMT2 predominantly accumulates in wheat influorescences compared to leaves, coinciding with the increased methyltransferase activity in the influorescence tissues. The effect of abiotic stresses on wheat reveals that TaOMT2 accumulates in cold-acclimated winter wheat leaves. In contrast, TaOMT2 activity with tricetin as a substrate shows a tendency to decrease during cold acclimation. Other abiotic stresses, such as salt and drought have no effects on TaOMT2 accumulation in wheat leaves, but a slight decrease in activity. The importance of tricetin methylation during developmental stages and during abiotic stresses is discussed.