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Potential key to enhanced disease resistance

Identification of the key enzyme for sakuranetin biosynthesis in rice

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Biotechnology Research Center
2012/06/12

The final step of biosynthetic pathway for sakuranetin, a flavonoid phytoalexin in rice, catalyzed by OsNOMT ©Kazunori Okada
Identification of the OsNOMT gene enables the production of large amounts of sakuranetin through transgenic rice and microorganisms.

Sakuranetin is a major rice phytoalexin and a potential pharmaceutical agent. The research group of Assistant Professor Kazunori Okada and former postdoc Takafumi Shimizu at the Biotechnology Research Center, the University of Tokyo successfully identified the OsNOMT gene encoding naringenin 7-O-methyltransferase (NOMT), which is involved in the final step of sakuranetin biosynthesis in rice. In previous research, OsNOMT was purified to apparent homogeneity from UV treated wild-type rice leaves, but the purified protein, named OsCOMT1, exhibited caffeic acid -O-methyltransferase (COMT) activity and not NOMT activity. In this study, they found that OsCOMT1 does not contribute to sakuranetin production in rice in vivo, and they purified OsNOMT using the oscomt1 mutant. 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. Using SDSPAGE, the purest enzyme preparation showed a minor band at an apparent molecular mass of 40 kDa. Two -O-methyltransferase-like proteins, encoded by Os04g0175900 and Os12g0240900, were identified from the 40-kDa band by MALDI-TOF/TOF analysis. Recombinant Os12g0240900 protein showed NOMT activity, but the recombinant Os04g0175900 protein did not. Os12g0240900 expression was induced by jasmonic acid treatment in rice leaves prior to sakuranetin accumulation, and the Os12g0240900 protein showed reasonable kinetic properties to OsNOMT. On the basis of these results, it was concluded that Os12g0240900 encodes an OsNOMT. Identification of the OsNOMT gene enables the production of large amounts of sakuranetin through transgenic rice and microorganisms. The finding also opens the way to generating disease resistant and sakuranetin bio-fortified rice in the near future.

Press release (Japanese)

Paper

Takafumi Shimizu, Fengqiu Lin, Morifumi Hasegawa, Kazunori Okada, Hideaki Nojiri, Hisakazu Yamane,
“Purification and identification of naringenin 7-O-methyltransferase, a key enzyme in the biosynthesis of the flavonoid phytoalexin sakuranetin in rice”,
Journal of Biological Chemistry 287 (2012): 19315-25, doi: 10.1074/jbc.M112.351270.
Article link

リンク

Graduate School of Agricultural and Life Sciences

Biotechnology Research Center (Japanese)

Laboratory of Environmental Biochemistry

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