Regulation of CLAVATA1 function in the Arabidopsis shoot apical meristem

描述（由申请人提供）：植物细胞增殖仅限于被称为分生组织的特殊区域。分生组织内的干细胞群产生成体植物的所有组织。在茎分生组织中产生的细胞最终被纳入地上侧枝器官。为了防止茎部分生组织的耗竭，细胞向外侧器官的流失与干细胞的增殖速度相平衡。CLAVATA基因座的突变破坏了这种平衡，导致干细胞的过度积累，额外的器官产生和生育能力和植物生长的降低。CLAVATA1 （CLV1）是一种保守的受体样激酶（RLK），首次在拟南芥中被定义，并被认为是分泌肽CLV3的受体。经过20年的基因分析，我们仍然不知道CLV1在细胞水平上是如何起作用的。遗传证据表明CLV1信号是由clv3介导的内化（LME）调控的。LME是植物和动物中RLK信号衰减的重要机制。在分子水平上了解CLV1 LME将使我们能够解决分生组织中CLV1活跃的位置，CLV1的激活如何受到其他潜在信号伙伴的调节，以及CLV1如何激活调节干细胞生成的下游途径。Meyerowitz实验室的实时成像技术的最新进展现在允许对其进行解剖。本研究旨在了解拟南芥细胞水平的CLV1 LME。具体来说，一种活性的CLV1 YFP融合蛋白将用于解决CLV1在细胞中的定位以及定位如何调节到CLV3感知。这将通过使用clv3突变体、外源性clv3肽和已知的LME抑制剂来检验。LME中对CLV1不同域的需求将使用CLV1 -YFP的突变版本来解决。此外，将详细介绍一种用于创建CLV1的条件抑制激酶版本的系统，这将使我们能够测试CLV3是否直接激活CLV1，并将提供一种在活组织中实时探索CLV1抑制效果的工具。最后，我将详细介绍一种遗传策略，以识别迄今为止难以捉摸的CLV1 LME关键调节因子中的突变体。这项研究将极大地扩展我们对植物中RLK激活的认识，并将作为研究CLV1或CLV3家族其他成员信号传导的模型。这项工作将使我们首次了解CLV1在细胞水平上的功能，这是了解植物干细胞通路如何调节的关键障碍。这项工作将启发我们了解这个王国在干细胞生物学上与人类和其他动物的不同之处。这些知识可能会导致研究干细胞生物学的新方法，并可能导致治疗人类疾病的新方法。

DESCRIPTION (provided by applicant): Plant cell proliferation is restricted to specialized regions called meristems. Stem cell populations within meristems give rise to all tissues of the adult plant. Cells generated in the shoot meristems are eventually incorporated into above ground lateral organs. In order to prevent depletion of shoot meristems, the loss of cells to lateral organs is balanced with the rate of stem cell proliferation. Mutations in the CLAVATA loci disrupt this balance and lead to a hyper-accumulation of stem cells, extra organ production and a reduction in fertility and plant growth. CLAVATA1 (CLV1) is a conserved receptor-like kinase (RLK) defined first in Arabidopsis and proposed to act as a receptor for the secreted peptide CLV3. After 20 years of genetic analysis we still do not know how CLV1 functions at the cellular level. Genetic evidence suggests that CLV1 signaling is regulated by CLV3-mediated internalization (LME). LME is an important mechanism for attenuating RLK signaling in plants and animals. Understanding CLV1 LME at the molecular level would allow us to address where in the meristem CLV1 is active, how CLV1 activation is regulated by other potential signaling partners and how CLV1 might activate downstream pathways that regulate stem cell production. Recent advances in live imaging in the Meyerowitz lab now allow this to be dissected. This proposal aims to understand CLV1 LME at the cellular level in Arabidopsis. In specific, an active CLV1 YFP fusion protein will be used to address where CLV1 is localized in the cell and how localization is regulated to CLV3 perception. This will be examined by using clv3 mutants, exogenous CLV3 peptides and known inhibitors of LME. The requirement for different domains of CLV1 in LME will be addressed using mutant versions of CLV1 -YFP . In addition, a system for creating conditionally inhibitable kinase versions of CLV1 will be detailed which will allow us to test if CLV3 activates CLV1 directly and will provide a tool to explore the effects of CLV1 inhibition in real time in live tissue. Lastly, I will detail a genetic strategy to identify mutants in key regulators of CLV1 LME which have so far proved elusive. This research will greatly expand our knowledge of RLK activation in plants and will serve as a model for studying signaling by other CLV1 or CLV3 family members. The work will provide our first knowledge of how CLV1 functions at the cellular level, a key roadblock for understanding how stem cell pathways are regulated in plants. This work will enlighten our knowledge of how this kingdom differs in stem cell biology from humans and other animals. This knowledge may lead to novel approaches for studying stem cell biology in general and may lead to novel approaches to treating disease in humans.