Department of Biology, University of Saskatchewan, Saskatoon, SK S7N 5E2, Canada.
Planta. 2012 Oct;236(4):1289-303. doi: 10.1007/s00425-012-1683-1. Epub 2012 Jun 21.
In the green alga Chlamydomonas reinhardtii, the cytosolic Glutathione Peroxidase 5 gene (GPX5) is known to be transcriptionally up-regulated in response to singlet oxygen ((1)O(2)). As demonstrated by previous studies, fusion of the promoter region of GPX5 to the Arylsulfatase 2 gene (ARS2) creates an effective reporter system that can be used to monitor (1)O(2)-driven GPX5 expression. This system was also used in this study to generate a stably transformed C. reinhardtii strain which expresses ARS2 in a (1)O(2)-dependent manner, resulting in the synthesis of a functional protein with detectable activity. Using the strain of C. reinhardtii harboring a (1)O(2)-sensitive reporter construct, a secondary mutagenic screen was performed. This allowed identification of mutant cell lines that were unable to up-regulate expression of the GPX5-ARS2 fusion in response to (1)O(2). In one of these lines, the mutation was subsequently localized to the first exon of the PSBP-like gene (PSBP2). The PSBP2 gene is part of a small protein family in C. reinhardtii, also present in all angiosperms studied thus far. While each member of the PSBP protein family contains a similar domain to the PSBP1 protein, which is a member of the oxygen evolving complex of photosystem II (PSII), the PSBP2 protein does not appear to be involved in PSII function, but may function as a sensor and/or signal mediating molecule of the (1)O(2) generated in the chloroplast.
在绿藻莱茵衣藻中,细胞溶质谷胱甘肽过氧化物酶 5 基因(GPX5)已知可响应单线态氧((1)O(2))而上调转录。正如先前的研究表明,将 GPX5 的启动子区域与芳基硫酸酯酶 2 基因(ARS2)融合,创建了一个有效的报告系统,可用于监测(1)O(2)驱动的 GPX5 表达。该系统也用于本研究中,以生成稳定转化的 C. reinhardtii 菌株,该菌株以(1)O(2)依赖性方式表达 ARS2,导致合成具有可检测活性的功能性蛋白质。使用带有(1)O(2)敏感报告构建体的 C. reinhardtii 菌株,进行了二次诱变筛选。这允许鉴定无法上调 GPX5-ARS2 融合表达以响应(1)O(2)的突变细胞系。在这些细胞系之一中,突变随后被定位到 PSBP 样基因(PSBP2)的第一个外显子。PSBP2 基因是 C. reinhardtii 中小蛋白家族的一部分,也存在于迄今为止研究的所有被子植物中。虽然 PSBP 蛋白家族的每个成员都含有类似于 PSBP1 蛋白的类似结构域,PSBP1 蛋白是光合作用系统 II (PSII)氧气进化复合物的成员,但 PSBP2 蛋白似乎不参与 PSII 功能,但可能作为传感器和/或信号转导分子参与叶绿体中产生的(1)O(2)。