Tracheary Element Differentiation

气管元件分化

• 批准号: 9807801
• 负责人: Alan Jones
• 金额: $ 19.89万
• 依托单位: University of North Carolina at Chapel Hill
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-10-01 至 2001-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9807801&HistoricalAwards=false
• 关键词: Tracheary; Element; Differentiation

This project focuses on two important developmental processes occurring during the normal differentiation of tracheary elements (TE), namely, the coordination of secondary cell wall synthesis with the cell's programmed cell death (PCD). Tracheary elements are the cellular corpses that form the water conducting vessels of plant xylem. As TEs develop, they lay down a rigid secondary wall. At the conclusion of this process, death of the developing TE is triggered by its own extracytoplasmic signal. Autolysis of the cytoplasm ensues after release of nascent hydrolases, stored in the vacuole, but the synthesis of these hydrolases begins well before the new wall is completed. Strict coordination of the PCD and autolysis with wall formation is critical because inappropriate timing of cell death would have detrimental effects, not only on the function of the developing corpse, but on the entire developing cell flank (vessel). Recent data from the PI's lab support the following model: prior to cell wall formation, hydrolases axe synthesized and sequestered in the vacuole. During the final stage of secondary wall synthesis, a 40-kD serine protease is secreted and induces the influx of calcium that directly or indirectly activates rupture of the tonoplast and subsequent release of the hydrolases. The proposed project will utilize the well-characterized zinnia cell culture system to address 3 questions about the terminal events of tracheary element differentiation: What is the protease, and how does the signal trigger cell death? The secreted protease will be partially purified in order to obtain internal sequence for cloning the corresponding cDNA. The protease will be expressed in E. coli to analyze its proteolytic activity and in both zinnia roots and arabidopsis plants to test its function. The cleavage specificity will be determined using both the partially purified protease and the recombinant protease and artificial and natural substrates. Arabidopsis homologs of the protease will be identified and the effect of regulated overexpression will be examined. For the latter, an estrogen-inducible system will be used to control expression. The extracellular matrix is recognized as a store of information directing the development of plant and animal cells but how it does this is not understood, especially within a single transduction pathway such as PCD. The proposed model system has several advantages over other systems to now determine the mechanism of signal transduction involved in cell differentiation. Furthermore, the mechanism of regulation of PCD in any context is not well understood and the significance of these results could have impact on many developmental processes from coordination of TE development and wood formation to control of neoplasia. Moreover, this work will address how the extracellular matrix provides signals for cellular development and more specifically, how a novel secreted protease regulates cell death.

本项目主要研究发生在气管分子(TE)正常分化过程中的两个重要发育过程，即次生细胞壁合成与细胞程序性死亡(PCD)的协调。导管分子是构成植物木质部导水导管的细胞体。随着TES的发展，它们铺设了一道坚硬的次级墙。在这个过程结束时，发育中的TE的死亡是由它自己的胞外信号触发的。在储存在液泡中的新生水解酶释放后，细胞质自溶，但这些水解酶的合成在新壁完成之前就开始了。PCD和自溶与壁形成的严格协调是至关重要的，因为不适当的细胞死亡时间将对发育中的身体的功能以及整个发育中的细胞侧翼(血管)产生有害的影响。PI实验室的最新数据支持以下模型：在细胞壁形成之前，水解酶被合成并隔离在液泡中。在次生壁合成的最后阶段，40kD的丝氨酸蛋白酶被分泌，并诱导钙的内流，直接或间接地激活液泡膜的破裂和随后的水解酶的释放。拟议的项目将利用具有良好特性的百日草细胞培养系统来解决关于管状分子分化的最终事件的三个问题：什么是蛋白酶，以及信号如何触发细胞死亡？将所分泌的蛋白水解酶进行部分纯化，以获得克隆相应的cdna的内部序列。该酶将在大肠杆菌中表达，以分析其蛋白分解活性，并在百日草和拟南芥中进行表达，以测试其功能。将使用部分纯化的酶和重组酶以及人工和天然底物来确定切割特异性。将鉴定该酶在拟南芥中的同源物，并检测其调控过表达的效果。对于后者，将使用雌激素诱导系统来控制表达。细胞外基质被认为是指导植物和动物细胞发育的信息存储，但它如何做到这一点还不清楚，特别是在单个转导途径，如PCD。与其他系统相比，所提出的模型系统在确定细胞分化所涉及的信号转导机制方面具有几个优势。此外，PCD在任何情况下的调控机制都不是很清楚，这些结果的意义可能会对许多发育过程产生影响，从TE发育和木材形成的协调到肿瘤的控制。此外，这项工作将研究细胞外基质如何为细胞发育提供信号，更具体地说，一种新的分泌型蛋白酶如何调节细胞死亡。