Identification and analysis of genes involved in tetrapyrrole metabolism and plastid-derived ROS-mediated signaling in Chlamydomonas reinhardtii

由于在黑暗中合成叶绿素的能力，绿藻衣藻进化出了许多不同的四吡咯生物合成(TBS)的调节机制，这些机制在被子植物中可能不存在或很难研究，例如由于TBS中间体在光中的光敏效应，尽管到目前为止发现的大多数其他特征是相似的。此外，莱茵梭菌异养生长的能力为探索TBS或光合作用过程提供了机会，而这些过程是不可能在植物中研究的，因为它们的色素生物合成突变将是致命的。因此，莱茵哈蒂尔提出了一个合适的模型来研究TBS、逆行信号转导及相关过程。我们打算利用莱茵梭菌的这些特点，并应用不同的遗传和生化方法(I)分析我们小组中可用的一组特定突变和新产生的一组插入突变，这些突变具有参与TBS调控和催化的基因的高遗传损伤潜力，以及(Ii)检测与TBS相关的不同单线态氧介导的逆行信号通路的可能串扰。在寻找TBS突变体时预先选择的突变体将被检测其TBS中间产物和最终产物的水平，这是TBS受损的强烈迹象。与植物中的同源基因相比，新的基因可能编码TBS酶的新亚基或调控因子，从而在转录和翻译后水平上显示TBS不同的藻类控制机制。由突变导致的给定表型的原因将通过鉴定插入位置和受影响的基因来确认，然后用给定基因的野生型拷贝进行抢救尝试。编码的蛋白质在TBS和光合作用中的功能将通过生化和遗传方法来阐明。随后，将在转录和代谢水平上探讨这些新发现的蛋白质的解除调控表达对整个途径的影响。对于提案的第二部分，所提出的寻找IO2诱导的信号成分的策略最终包括工作流程中的几个步骤，该工作流程以通过选择的启动子-报告结构插入突变体而结束，该结构是由IO2-积累诱导的。

Because of the ability to synthesize chlorophyll in the dark, the green alga Chlamydomonas reinhardtii evolved a number of diverse regulatory mechanisms of tetrapyrrole biosynthesis (TBS), which may not be present or are difficult to study in angiosperms, e.g. due to the photosensitizing effect of TBS intermediates in light, although a great majority of other features discovered so far is similar. Additionally, the ability of C. reinhardtii to grow heterotrophically gives an opportunity to explore TBS or photosynthetic processes, which are not possible to study in plants, because their pigment biosynthesis mutants would be lethal. Therefore, C. reinhardtii presents an appropriate model to study TBS, retrograde signaling and related processes. We intend to take advantage of these characteristics of C. reinhardtii and apply different genetic and biochemical approaches (i) to analyze a specific set of mutants available in our group and a newly generated collection of insertional mutants with a high potential of genetic lesions in genes involved in regulation and catalysis of TBS, and (ii) to examine a possible cross-talk of different singlet-oxygen-mediated retrograde signaling pathways related to TBS. The mutants preselected in the search for TBS mutants will be examined for their levels of intermediates and endproducts of TBS, which are strong indication for impaired TBS. It is expected that novel genes can be identified, which encode either new subunits of TBS enzymes or regulatory factors which may show diversified algal control mechanisms of TBS at the transcriptional and posttranslational level in comparison to their homologous counterparts in the plants. The cause of a given phenotype resulting from mutagenesis will be confirmed by identification of the insertion site and the affected genes, followed by the rescue attempt with the wild-type copy of a given gene. The function of the encoded proteins in TBS and photosynthesis will be elucidated by biochemical and genetic methods. Subsequently, the impact of deregulated expression of these newly identified proteins will be explored on the entire pathway at the transcriptional and metabolic level. For the second part of the proposal, the strategy proposed to search for 1O2-inducible signaling components finally includes several steps in a workflow that ends with the screen for insertion mutants by means of a selected promoter-reporter constructs, which is induced by 1O2-accumulation.