Department of Plant and Microbial Biology, University of California, Berkeley, 94720-3102, USA.
BMC Plant Biol. 2010 Dec 17;10:279. doi: 10.1186/1471-2229-10-279.
When photosynthetic organisms are exposed to harsh environmental conditions such as high light intensities or cold stress, the production of reactive oxygen species like singlet oxygen is stimulated in the chloroplast. In Chlamydomonas reinhardtii singlet oxygen was shown to act as a specific signal inducing the expression of the nuclear glutathione peroxidase gene GPXH/GPX5 during high light stress, but little is known about the cellular mechanisms involved in this response. To investigate components affecting singlet oxygen signaling in C. reinhardtii, a mutant screen was performed.
Mutants with altered GPXH response were isolated from UV-mutagenized cells containing a GPXH-arylsulfatase reporter gene construct. Out of 5500 clones tested, no mutant deficient in GPXH induction was isolated, whereas several clones showed constitutive high GPXH expression under normal light conditions. Many of these GPXH overexpressor (gox) mutants exhibited higher resistance to oxidative stress conditions whereas others were sensitive to high light intensities. Interestingly, most gox mutants produced increased singlet oxygen levels correlating with high GPXH expression. Furthermore, different patterns of altered photoprotective parameters like non-photochemical quenching, carotenoid contents and α-tocopherol levels were detected in the various gox mutants.
Screening for mutants with altered GPXH expression resulted in the isolation of many gox mutants with increased singlet oxygen production, showing the relevance of controlling the production of this ROS in photosynthetic organisms. Phenotypic characterization of these gox mutants indicated that the mutations might lead to either stimulated triplet chlorophyll and singlet oxygen formation or reduced detoxification of singlet oxygen in the chloroplast. Furthermore, changes in multiple protection mechanisms might be responsible for high singlet oxygen formation and GPXH expression, which could either result from mutations in multiple loci or in a single gene encoding for a global regulator of cellular photoprotection mechanisms.
当光合生物暴露在恶劣的环境条件下，如高光强或冷胁迫时，叶绿体中会刺激产生活性氧物种，如单线态氧。在莱茵衣藻中，单线态氧被证明是一种特异性信号，可在高光胁迫下诱导核谷胱甘肽过氧化物酶基因 GPXH/GPX5 的表达，但对此反应涉及的细胞机制知之甚少。为了研究影响莱茵衣藻中单线态氧信号的因素，进行了突变体筛选。
从含有 GPXH-芳基硫酸酯酶报告基因构建体的 UV 诱变细胞中分离出 GPXH 反应改变的突变体。在测试的 5500 个克隆中，没有分离到 GPXH 诱导缺陷的突变体，而几个克隆在正常光照条件下表现出组成型高 GPXH 表达。这些 GPXH 过表达（gox）突变体中有许多对氧化应激条件具有更高的抗性，而其他突变体对高光强敏感。有趣的是，大多数 gox 突变体产生的单线态氧水平增加，与高 GPXH 表达相关。此外，在各种 gox 突变体中检测到不同模式的光保护参数改变，如非光化学猝灭、类胡萝卜素含量和α-生育酚水平。
筛选 GPXH 表达改变的突变体导致了许多 gox 突变体的分离，这些突变体产生的单线态氧增加，表明控制这种 ROS 在光合生物中的产生是很重要的。对这些 gox 突变体的表型特征分析表明，这些突变可能导致叶绿素三重态和单线态氧形成的刺激，或叶绿体中单线态氧的解毒减少。此外，多种保护机制的变化可能是导致高单线态氧形成和 GPXH 表达的原因，这可能是由于多个基因座的突变或单个基因编码细胞光保护机制的全局调节剂。
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