Cellular acclimation and signal transduction of the green alga Chlamydomonas reinhardtii under anaerobic conditions

在缺乏分子氧（O2）的情况下，好氧生物必须通过低效的底物水平磷酸化产生足够的能量，同时调整生长以适应受损的生物合成途径。对厌氧的适应是通过改变底物和电子通量以及基因的差异表达来促进的。本项目旨在分析莱茵衣藻（Chlamydomonas reinhardtii）单细胞绿藻对厌氧胁迫条件下代谢和转录水平的响应。我们通过rna测序获得厌氧莱茵梭菌培养物的全基因组转录谱，结果鉴定出两个候选基因。其中一种编码蛋白THB8属于截断血红蛋白家族。我们已经观察到，thb8基因的转录后沉默导致莱茵梭菌在光照条件下难以在厌氧条件下生长，并且几个厌氧关键基因不能正常表达。鉴于转化子的表型和重组THB8的一氧化氮（NO-）结合能力，我们假设THB8参与了一个基于NO的信号转导途径。THB8敲低转化子的表达模式表明，适当的THB8表达与CYG12基因的厌氧诱导之间存在联系，CYG12基因编码血红素/ no结合鸟苷酸环化酶。CYG12在上述全基因组转录谱中也表现突出。在本项目中，将通过确定THB8和CYG12的生化和生物物理特征，以及分析THB8和CYG12敲低菌株的生理和遗传特征，来检验假设的基于no的信号级联。该项目的生化部分将侧重于表征血红素基团，配体（如NO）的结合以及识别THB8和CYG12的相互作用伙伴。这个项目部分还将包括一种发酵酶的分析，丙酮酸：铁氧还蛋白氧化还原酶PFR1。根据我们的RNA-Seq数据，PFR1基因的调控与其他参与发酵的基因有很大不同。PFR1的生物化学和生物物理特性以及可能催化的反应有望阐明其生理作用，从而为PFR1基因表达不同的原因提供线索。本研究的生理-遗传部分将侧重于THB8-和尚待创建的cyg12敲低转化子厌氧作用下的生理和（no依赖性）基因表达的表征。该项目的一个主要部分将是产生敲除菌株的全基因组转录谱，以确定受影响的基因群。

In the absence of molecular oxygen (O2), aerobic organisms have to generate sufficient energy via the inefficient substrate level phosphorylation while adjusting growth to impaired biosynthetic pathways. The acclimation to anaerobiosis is facilitated by altered substrate- and electron fluxes as well as the differential expression of genes. This project aims at analyzing the response of the unicellular green alga Chlamydomonas reinhardtii to the stress condition of anaerobiosis on metabolic and transcriptional levels.Genome-wide transcript profiles from anaerobic C. reinhardtii cultures which we obtained by RNA-sequencing resulted in the identification of two candidate genes. One of the encoded proteins, THB8, belongs to the family of truncated hemoglobins. We could already observe that post-transcriptional silencing of the THB8-gene results in C. reinhardtii transformants which hardly grow under anaerobic conditions in the light and in which several anaerobic key genes are not properly expressed. In view of the phenotype of the transformants and a nitric oxide- (NO-) binding capacity of recombinant THB8 we postulate that THB8 is involved in an NO-based signaling transduction pathway. The expression patterns of the THB8-knockdown transformants indicate a connection between a proper THB8 expression and anaerobic induction of the CYG12 gene which encodes a heme/NO-binding guanylate cyclase. CYG12 was also outstanding in the above mentioned whole-genome transcript profiles.During this project, the postulated NO-based signaling cascade will be examined by determining the biochemical and biophysical features of THB8 and CYG12 as well as by analyzing the physiological and genetic characteristics of THB8- and CYG12-knockdown strains. The biochemical part of this project will focus on characterizing the heme groups, the binding of ligands such as NO and on identifying interaction partners of THB8 and CYG12. This project part will also include the analysis of a fermentative enzyme, the pyruvate:ferredoxin oxidoreductase PFR1. According to our RNA-Seq data, the regulation of the PFR1 gene differs significantly from other genes involved in fermentation. The biochemical and biophysical characteristics as well as the possible reactions catalyzed by PFR1 will hopefully elucidate its physiological role and thereby give clues to the reason for the different PFR1 gene expression.The physiological-genetic part of this study will focus on the characterization of physiology and (NO-dependent) gene expression upon anaerobiosis of the THB8- and yet to be created CYG12-knockdown transformants. A major part of this project will the generation of whole genome transcript profiles of the knockdown strains in order to identify affected gene groups.