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，绿色衣藻进化了许多不同的四吡咯生物合成（TBS）的调节机制，这可能是不存在的或难以研究的被子植物，例如由于光的TBS中间体的光敏效应，尽管迄今为止发现的其他功能是相似的。此外，C.莱茵衣藻异养生长提供了探索TBS或光合作用过程的机会，这是不可能在植物中研究的，因为它们的色素生物合成突变体是致命的。因此，C. reinhardtii提出了一个合适的模型来研究TBS，逆行信号和相关过程。我们打算利用C. reinhardtii和应用不同的遗传和生物化学方法（i）分析我们组中可用的一组特定突变体和一组新产生的插入突变体，这些突变体在涉及TBS的调节和催化的基因中具有高的遗传损伤潜力，以及（ii）检查与TBS相关的不同单线态氧介导的逆行信号传导途径的可能串扰。将检查在TBS突变体搜索中预选的突变体的TBS中间体和终产物的水平，这是TBS受损的强烈指示。预计新的基因可以被鉴定，其编码TBS酶的新亚基或调节因子，与植物中的同源对应物相比，其可能在转录和翻译后水平上显示TBS的多样化藻类控制机制。将通过鉴定插入位点和受影响的基因，然后用给定基因的野生型拷贝进行拯救尝试，来确认由诱变引起的给定表型的原因。其编码蛋白在TBS和光合作用中的功能将通过生物化学和遗传学方法阐明。随后，将在转录和代谢水平上探索这些新鉴定的蛋白质的表达失调对整个途径的影响。对于该提案的第二部分，提出的策略，以寻找1 O2-诱导的信号传导组件，最终包括几个步骤的工作流程中，通过选择的启动子-报告构建体，这是诱导的1 O2-积累的插入突变体的屏幕结束。

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.