Sakuranetin, the major
flavonoid phytoalexin in rice, can be induced by ultraviolet (UV) irradiation, treatment with CuCl 2 or
jasmonic acid (JA), or phytopathogenic
infection. In addition to
sakuranetin's
biological significance on
disease resistance in rice, its broad bioactivities have recently been described. Results from these studies have shown that
sakuranetin is a useful compound as a plant
antibiotic and a potential
pharmaceutical agent.
Sakuranetin is biosynthesized from
naringenin, a precursor of
sakuranetin, by
naringenin 7-O-methyltransferase (NOMT), but the relevant gene has not yet been identified in rice. Recently, we identified the OsNOMT gene, which is involved in the final step of
sakuranetin biosynthesis in rice. In previous studies, OsNOMT was purified to apparent homogeneity from UV-treated wild-type rice leaves; however, the purified
protein, termed OsCOMT1, exhibited
caffeic acid 3-O-methyltransferase (COMT) activity, but not NOMT activity. Based on the analysis of an oscomt1
T-DNA tagged mutant, we determined that OsCOMT1 did not contribute to
sakuranetin production in rice in vivo. Therefore, we took advantage of the oscomt1 mutant to purify OsNOMT. A crude
protein preparation from UV-treated oscomt1 leaves was subjected to three sequential purification steps resulting in a 400-fold purification from the crude
enzyme preparation with a minor band at an apparent molecular mass of 40 kDa in the purest
enzyme preparation. Matrix-assisted
laser desorption/ionization time of flight/time of flight analysis showed that the 40 kDa
protein band included two O-
methyltransferase-like
proteins, but one of the
proteins encoded by Os12g0240900 exhibited clear NOMT activity; thus, this gene was designated OsNOMT. Gene expression was induced by treatment with
jasmonic acid in rice leaves prior to
sakuranetin accumulation, and the
recombinant protein showed reasonable kinetic properties to NOMT. Identification of the OsNOMT gene enables the production of large amounts of
sakuranetin through transgenic rice and microorganisms. This finding also allows for the generation of disease-resistant and
sakuranetin biofortified rice in the future.