Two-step d-ononitol epimerization pathway in Medicago truncatula.

Methylated inositol, d-pinitol (3-O-methyl-d-chiro-inositol), is a common constituent in legumes. It is synthesized from myo-inositol in two reactions: the first reaction, catalyzed by myo-inositol-O-methyltransferase (IMT), consists of a transfer of a methyl group from S-adenosylmethionine to myo-inositol with the formation of d-ononitol, while the second reaction, catalyzed by d-ononitol epimerase (OEP), involves epimerization of d-ononitol to d-pinitol. To identify the genes involved in d-pinitol biosynthesis in a model legume Medicago truncatula, we conducted a BLAST search on its genome using soybean IMT cDNA as a query and found putative IMT (MtIMT) gene. Subsequent co-expression analysis performed on publicly available microarray data revealed two potential OEP genes: MtOEPA, encoding an aldo-keto reductase and MtOEPB, encoding a short-chain dehydrogenase. cDNAs of all three genes were cloned and expressed as recombinant proteins in E. coli. In vitro assays confirmed that putative MtIMT enzyme catalyzes methylation of myo-inositol to d-ononitol and showed that MtOEPA enzyme has NAD⁺ -dependent d-ononitol dehydrogenase activity, while MtOEPB enzyme has NADP⁺ -dependent d-pinitol dehydrogenase activity. Both enzymes are required for epimerization of d-ononitol to d-pinitol, which occurs in the presence of NAD⁺ and NADPH. Introduction of MtIMT, MtOEPA, and MtOEPB genes into tobacco plants resulted in production of d-ononitol and d-pinitol in transformants. As this two-step pathway of d-ononitol epimerization is coupled with a transfer of reducing equivalents from NADPH to NAD⁺ , we speculate that one of the functions of this pathway might be regeneration of NADP⁺ during drought stress.