Post-translational regulation of cell physiology by the circadian clock

植物细胞包含一个生物钟，这是植物生长和生存的基础。我发现，生物钟停止的幼苗只有正常生物钟的一半大小，如果生物钟与环境不同步，幼苗会因光合作用减少而发育不良（Dodd et al. 2005）。最大的种子产量也取决于生物钟。由于这些发现具有巨大的农业重要性，因此了解生物钟的功能是确保未来可持续和安全粮食生产的及时和重要组成部分。重要的是，我们对生物钟增强植物性能的机制的了解非常有限。我们提出的实验将提供新的知识来解决这一缺陷。他们将研究植物生物学中两个尚未解决的问题：（i）生物钟控制细胞功能的哪些方面？(ii)是什么过程将生物钟的定时信息传递到细胞功能的昼夜节律调节方面？(i)细胞功能的哪些方面是由生物钟控制的？我们的初步实验表明，目前的理解植物细胞功能的昼夜节律组织需要广泛的修改与有关蛋白质丰度的信息。我们将鉴定具有丰富的昼夜节律的膜和可溶性蛋白质，以发现通过昼夜节律调节优化的生化机制。(ii)可逆磷酸化如何将生物钟的时间信息传递给细胞内的昼夜节律调节蛋白？蛋白质的可逆磷酸化形成细胞信号传导的重要部分，其直接调节酶的活性，控制基因表达，并控制蛋白质降解。编码大量蛋白激酶的基因是昼夜节律调节的，并且昼夜节律调节的蛋白激酶的实例存在于植物和动物中。这表明可逆磷酸化具有在植物细胞内发出昼夜节律定时信息的潜力。我们将使用两种策略来研究可逆磷酸化在昼夜信号传导中的参与。首先，我们将使用一种称为磷酸化蛋白质组学的技术来鉴定经历磷酸化昼夜节律的蛋白质。其次，我们将确定蛋白激酶和磷酸酶，这些蛋白激酶和磷酸酶是我们所确定的已知对细胞重要的蛋白质的磷酸化节律的基础。这些信息将用于了解生物钟和由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传导的昼夜节律调节方面的专业知识（PI，Dodd博士）与（a）生物学技术设施系蛋白质组学实验室内的专业技术，专业知识和方法以及（B）在拟南芥中发现新蛋白质-蛋白质相互作用的专业知识（Hybridgenics S.A.）和蛋白激酶信号传导（Jörg Kudla教授，明斯特大学），这项研究将大大推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.