Molecular analysis of the light-dependent rhythmic regulation in a marine diatom

硅藻属于世界上最多样化的藻类群体，负责水生环境中大部分的初级生产。此外，它们代表了独特的进化嵌合细胞，因为它们通过将真核生物摄取到另一种真核细胞中而进化，因此具有与绿色藻类和植物非常不同的遗传背景。硅藻和大多数生物一样，每天都经历着光/暗的变化，并表现出基本的生物学过程的节律性，以适应光/暗的变化。尽管最近的研究表明，这些节律性在连续光照条件下持续存在，表明硅藻体内存在内源性昼夜节律振荡器，但调控硅藻细胞节律性的机制仍然不清楚。有趣的是，在硅藻基因组中没有发现细菌、植物或动物的生物钟组件的同源基因，这表明这些藻类的生物钟机制多样化。因此，本项目的目的是阐明模式硅藻三角褐指藻中的光依赖性节律机制，并通过整合德国和法国两个独立研究小组的互补兴趣和专业知识来评估其生理相关性。该项目基于两个团队最近的重要发现：i）硅藻整合来自环境以及内源性昼夜节律钟的光信号来调节昼夜细胞活动; ii）bHLH-PAS转录因子RITMO 1已被确定为硅藻昼夜节律的第一个调节因子; iii）参与转录调节的多种硅藻光感受器，如Aureochromes和动物样隐花色素（PtCPF 1），似乎是光输入到生物钟的关键参与者; iv）这两个实验室已经独立产生了RITMO 1和感光细胞KO突变体，并建立了研究硅藻昼夜节律的系统。结合资源提供了一个前所未有的机会，以表征硅藻昼夜节律钟系统及其夹带的光。因此，通过表征野生型细胞（WT）和一系列突变株系中的昼夜节律，该项目将允许对硅藻昼夜节律钟特征进行扩展表征，并将识别输入途径的光感受器。 它将通过WT和RITMO 1突变体中的转录组学分析以及通过研究全基因组蛋白质/DNA相互作用测定中的RITMO结合位点来进一步研究RITMO 1伙伴。通过酵母单杂交和酵母双杂交及免疫沉淀检测候选光感受器/转录因子与潜在结合位点的相互作用，以及蛋白质/蛋白质相互作用，将有助于确定海洋硅藻中产生细胞节律性的第一个调节环。拟议的分子分析将为生物节律的演变及其对海洋环境中生命的意义提供新的见解。

Diatoms belong to the world’s most diverse group of algae, being responsible for a large part of the primary production in aquatic environments. In addition, they represent peculiar evolutionary chimeric cells, as they have evolved by the uptake of a eukaryotic alga into another eukaryotic cell, thus having a very different genetic background than green algae and plants. As most organisms, diatoms experience daily light/dark changes and show rhythms of basic biological processes to adjust their life cycle to it. Although recent studies revealed that these rhythms persist in continuous light conditions, indicating the existence of an endogenous circadian oscillator in diatoms, the mechanisms regulating cellular rhythmicity in these algae are still obscure. Interestingly, no genes for homologs of the circadian clock components known from bacteria, plants or animals have been found in the diatom genomes, indicating a diversification of the circadian clock machinery in these algae. Therefore, the aim of this project is to elucidate the light-dependent rhythmic mechanisms in the model diatom Phaeodactylum tricornutum and to assess their physiological relevance by integrating complementary interests and expertise of two independent research groups in Germany and France. This project is based on important recent discoveries by the two teams: i) diatoms integrate light signals from the environment as well as from an endogenous circadian clock to regulate diel cellular activities; ii) the bHLH-PAS transcription factor RITMO1 has been identified as the first regulator of the diatom circadian rhythms; iii) diverse diatom photoreceptors involved in transcription regulation, such as the Aureochromes and the animal-like Cryptochrome (PtCPF1) appear as key players for light input to the clock; iv) the two labs have independently generated RITMO1 and photoreceptor KO mutants and established a system for studying diatom circadian rhythms. The combined resources offer an unprecedented opportunity to characterize the diatom circadian clock system and its entrainment by light. Therefore, by characterizing circadian rhythms in wild-type cells (WT) and a collection of mutant lines, the project will allow an extended characterization of the diatom circadian clock features, and will identify the photoreceptors of the input pathways. It will further investigate the RITMO1 partners by transcriptomic analyses in WT and RITMO1 mutants and by studying RITMO-binding sites in genome-wide protein/DNA interaction assays. Testing interactions of candidate photoreceptor/transcription factors to potential binding sites by Yeast one hybrid and protein/protein interactions by Yeast two-hybrid and immunoprecipitation will contribute to identify the first regulatory loop(s) generating cellular rhythmicity in marine diatoms. The proposed molecular analysis will provide new insights into the evolution of biological rhythms and their significance for life in the marine environment.