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，负责水生环境中初级生产力的很大一部分。此外，它们代表着特殊的进化嵌合细胞，因为它们是通过将一种真核藻吸收到另一种真核细胞中而进化出来的，因此具有与绿藻和植物非常不同的遗传背景。与大多数生物体一样，硅藻经历日常的明/暗变化，并表现出基本生物过程的节奏，以适应它的生命周期。尽管最近的研究表明，这些节律在连续光照条件下持续存在，表明硅藻中存在内源昼夜节律振荡器，但这些藻类中调控细胞节律的机制仍然不清楚。有趣的是，在硅藻基因组中还没有发现细菌、植物或动物中已知的生物钟成分的同源基因，这表明这些藻类的生物钟机制存在多样性。因此，本项目的目的是阐明模式硅藻三角褐指藻依赖光的节律机制，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它们的生理相关性。该项目基于两个团队最近的重要发现：i)硅藻整合来自环境和内源生物钟的光信号，以调节Diel细胞的活动；ii)bHLH-PAS转录因子RITMO1已被确定为硅藻昼夜节律的第一调节者；iii)多种参与转录调控的硅藻光感受器，如金色素和动物样隐花色素(PtCPF1)作为光输入到生物钟的关键角色；iv)两个实验室独立产生了RITMO1和光受体KO突变体，并建立了研究硅藻昼夜节律的系统。这些资源的结合提供了一个前所未有的机会来描述硅藻生物钟系统及其受光的影响。因此，通过表征野生型细胞(WT)和一系列突变株系的昼夜节律，该项目将允许对硅藻昼夜节律特征的扩展表征，并将识别输入路径的光感受器。它将通过对WT和RITMO1突变体的转录分析以及通过研究全基因组蛋白质/DNA相互作用分析中的Ritmo结合位点来进一步研究RITMO1伙伴。通过酵母单杂交和免疫沉淀法检测候选的光受体/转录因子与潜在结合位点的相互作用以及蛋白质/蛋白质的相互作用，将有助于鉴定海洋硅藻中产生细胞节律性的第一个调控环(S)。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.