Ogawa Taro, Nishimura Kenji, Aoki Takehiko, Takase Hisabumi, Tomizawa Ken-Ichi, Ashida Hiroki, Yokota Akiho
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan.
Plant Physiol. 2009 Sep;151(1):114-28. doi: 10.1104/pp.109.139683. Epub 2009 Jul 8.
To date, there have been no reports on screening for mutants defective in the massive accumulation of Rubisco in higher plants. Here, we describe a screening method based on the toxic accumulation of ammonia in the presence of methionine sulfoximine, a specific inhibitor of glutamine synthetase, during photorespiration initiated by the oxygenase reaction of Rubisco in Arabidopsis (Arabidopsis thaliana). Five recessive mutants with decreased amounts of Rubisco were identified and designated as nara mutants, as they contained a mutation in genes necessary for the achievement of Rubisco accumulation. The nara5-1 mutant showed markedly lower levels of plastid-encoded photosynthetic proteins, including Rubisco. Map-based cloning revealed that NARA5 encoded a chloroplast phosphofructokinase B-type carbohydrate kinase family protein of unknown function. The NARA5 protein fused to green fluorescent protein localized in chloroplasts. We conducted expression analyses of photosynthetic genes during light-induced greening of etiolated seedlings of nara5-1 and the T-DNA insertion mutant, nara5-2. Our results strongly suggest that NARA5 is indispensable for hyperexpression of photosynthetic genes encoded in the plastid genome, particularly rbcL.
迄今为止，尚未有关于筛选高等植物中在大量积累核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco）方面存在缺陷的突变体的报道。在此，我们描述了一种筛选方法，该方法基于在拟南芥（Arabidopsis thaliana）中由Rubisco的加氧酶反应引发的光呼吸过程中，在蛋氨酸亚砜亚胺（谷氨酰胺合成酶的一种特异性抑制剂）存在的情况下氨的毒性积累。鉴定出了五个Rubisco含量降低的隐性突变体，并将其命名为nara突变体，因为它们在实现Rubisco积累所必需的基因中存在突变。nara5-1突变体显示出包括Rubisco在内的质体编码光合蛋白的水平明显较低。图位克隆表明，NARA5编码一种功能未知的叶绿体磷酸果糖激酶B型碳水化合物激酶家族蛋白。与绿色荧光蛋白融合的NARA5蛋白定位于叶绿体中。我们对nara5-1和T-DNA插入突变体nara5-2的黄化苗在光诱导变绿过程中的光合基因进行了表达分析。我们的结果强烈表明，NARA5对于质体基因组中编码的光合基因的超表达是不可或缺的，尤其是rbcL。
