Biotechnology Research Center; The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Bioengineered. 2012 Nov-Dec;3(6):352-7. doi: 10.4161/bioe.21546. Epub 2012 Aug 16.
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.
樱花素是水稻中主要的类黄酮植物抗毒素，可被紫外线（UV）照射、CuCl 2 或茉莉酸（JA）处理或植物病原菌感染诱导。除了在水稻抗病性方面的生物学意义外，樱花素的广泛生物活性最近也有描述。这些研究的结果表明，樱花素作为一种植物抗生素和潜在的药物制剂是一种有用的化合物。樱花素是由柚皮素合成的，柚皮素是樱花素的前体，由柚皮素 7-O-甲基转移酶（NOMT）合成，但在水稻中尚未鉴定出相关基因。最近，我们鉴定了参与水稻樱花素生物合成最后一步的 OsNOMT 基因。在之前的研究中，从 UV 处理的野生型水稻叶片中纯化到了表观均一的 OsNOMT；然而，纯化的蛋白，称为 OsCOMT1，表现出咖啡酸 3-O-甲基转移酶（COMT）活性，但没有 NOMT 活性。基于对 oscomt1 T-DNA 标记突变体的分析，我们确定 OsCOMT1 没有在体内为水稻樱花素的产生做出贡献。因此，我们利用 oscomt1 突变体来纯化 OsNOMT。从 UV 处理的 oscomt1 叶片粗蛋白制备物中进行了三个连续的纯化步骤，从粗酶制剂中获得了 400 倍的纯化，在最纯的酶制剂中出现了一个 40 kDa 的小带。基质辅助激光解吸/电离飞行时间/飞行时间分析表明，40 kDa 蛋白带包括两种 O-甲基转移酶样蛋白，但 Os12g0240900 编码的一种蛋白表现出明显的 NOMT 活性；因此，该基因被命名为 OsNOMT。在樱花素积累之前，茉莉酸处理会诱导基因表达，重组蛋白表现出合理的 NOMT 动力学特性。OsNOMT 基因的鉴定使通过转基因水稻和微生物大量生产樱花素成为可能。这一发现也为未来生产抗病和富含樱花素的水稻提供了可能。
