Post-translational regulation of cell physiology by the circadian clock

植物细胞含有对植物生长和生存至关重要的生物钟。我发现时钟停止的幼苗只有正常时钟的一半大小，如果时钟与环境不同步，幼苗就会因光合作用降低而发育迟缓(Dodd等人。2005)。最大的种子产量还取决于生物钟。由于这些发现对农业具有巨大的重要性，了解生物钟的功能对于确保未来可持续和安全的粮食生产是及时和必要的部分。关键的是，我们对生物钟提高植物表现的机制的了解非常有限。我们提出的实验将为解决这一缺陷提供新的知识。他们将研究植物生物学中两个悬而未决的问题：(I)细胞功能的哪些方面由生物钟控制？(Ii)什么过程将时钟的计时信息传递到细胞功能的昼夜节律调节的方面？(I)生物钟控制细胞功能的哪些方面？我们的初步实验表明，目前对植物细胞功能的昼夜组织的理解需要广泛修改关于蛋白质丰度的信息。我们将识别具有昼夜节律丰度的膜和可溶性蛋白质，以发现通过昼夜节律调节而优化的生化机制。(Ii)可逆的磷酸化如何将时钟的计时信息传递给细胞内受昼夜节律调节的蛋白质？蛋白质的可逆磷酸化是细胞信号的重要组成部分，直接调节酶的活性，控制基因的表达，控制蛋白质的降解。编码大量蛋白激酶的基因是受昼夜节律调节的，在植物和动物中都存在昼夜调节的蛋白激酶的例子。这表明可逆的磷酸化有可能在植物细胞内发出昼夜节律信息。我们将使用两种策略来研究可逆磷酸化在昼夜节律信号传递中的作用。首先，我们将使用一种名为磷酸蛋白质组学的技术来鉴定经历昼夜节律磷酸化的蛋白质。其次，我们将确定对细胞具有已知重要性的蛋白质的磷酸化节奏所在的蛋白激酶和磷酸酶。这些信息将被用来理解生物钟和受可逆磷酸化调节的蛋白质之间的信号通路。通过将我在细胞生理学和信号转导的昼夜调节方面的专业知识(PI，Dodd博士)与(A)生物系技术设施的蛋白质组学实验室内的专业技术、专业知识和方法以及(B)在拟南芥(Hybrigenics S.A.)中发现新的蛋白质-蛋白质相互作用的专业知识相结合。和蛋白激酶信号传递(Jörg Kudla教授，明斯特大学)，这项研究将极大地促进我们对植物生物钟与细胞功能整合的理解。

Plant cells contain a biological clock that is fundamental to plant growth and survival. I discovered that seedlings in which the clock is stopped are half the size of those with a normal clock, and that if the clock is not synchronized with the environment, seedlings are stunted with reduced photosynthesis (Dodd et al. 2005). Maximum seed production also depends on the biological clock. Since these findings are of enormous agricultural importance, understanding the functions of the circadian clock is a timely and essential part of ensuring sustainable and secure food production in the future. Critically, our knowledge of the mechanisms by which the circadian clock enhances plant performance is very limited. The experiments that we propose will provide new knowledge to address this deficit. They will investigate two unresolved questions in plant biology; (i) what aspects of cell function are controlled by the circadian clock? (ii) what processes communicate timing information from the clock to circadian-regulated aspects of cell function? (i) What aspects of cell function are controlled by the circadian clock? Our preliminary experiments indicate that current understanding of the circadian organization of plant cell function requires extensive revision with information concerning protein abundance. We will identify membrane and soluble proteins that have circadian rhythms of abundance to discover biochemical mechanisms that are optimized by circadian regulation. (ii) How does reversible phosphorylation communicate timing information from the clock to circadian-regulated proteins within the cell? Reversible phosphorylation of proteins forms an important part of cell signalling that regulates the activity of enzymes directly, controls gene expression, and controls protein degradation. Genes encoding a large number of protein kinases are circadian regulated, and examples of circadian-regulated protein kinases are present in both plants and animals. This suggests that reversible phosphorylation has the potential to signal circadian timing information within plant cells. We will use two strategies to investigate the involvement of reversible phosphorylation in circadian signalling. First, we will identify proteins that undergo circadian rhythms of phosphorylation using a technique called phosphoproteomics. Second, we will identify the protein kinases and phosphatases that underlie the rhythms of phosphorylation for the proteins we identify that are of known importance to the cell. This information will be used to understand the signalling pathway between the circadian clock and the protein that is regulated by reversible phosphorylation. By combining my expertise (PI, Dr Dodd) in the circadian regulation of cell physiology and signalling with (a) specialist technology, expertise and methods within the Proteomics Laboratory in the Department of Biology's Technology Facility and (b) expertise in discovering novel protein-protein interactions in Arabidopsis (Hybrigenics S.A.) and protein kinase signalling (Prof. Jörg Kudla, Universität Münster), this research will advance significantly our understanding of the integration of the circadian clock in plants with cell function.