Tracheary Element Differentiation

本项目重点研究气管元件（TE）正常分化过程中发生的两个重要发育过程，即次级细胞壁合成与细胞程序性死亡（PCD）的协调。管状分子是植物木质部的细胞尸体，构成水的导管。随着TEs的发展，它们形成了坚硬的二次壁。在这一过程结束时，发育中的TE的死亡是由其自身的胞质外信号触发的。储存在液泡中的新生水解酶释放后，细胞质发生自溶，但这些水解酶的合成早在新壁完成之前就开始了。严格协调PCD和自溶与壁形成是至关重要的，因为不适当的细胞死亡时间不仅会对发育中的尸体的功能产生不利影响，而且会对整个发育中的细胞侧面（血管）产生不利影响。来自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。在次级壁合成的最后阶段，分泌一种40kd丝氨酸蛋白酶，诱导钙的流入，直接或间接地激活张力质体的破裂和随后的水解酶的释放。该项目将利用表征良好的百日草细胞培养系统来解决有关气管元件分化终末事件的3个问题：蛋白酶是什么？信号如何触发细胞死亡？分泌的蛋白酶将被部分纯化，以获得内部序列，用于克隆相应的cDNA。该蛋白酶将在大肠杆菌中表达以分析其蛋白水解活性，并在百日草和拟南芥中表达以测试其功能。将使用部分纯化的蛋白酶和重组蛋白酶以及人工和天然底物来确定裂解特异性。将鉴定该蛋白酶的拟南芥同源物，并检查调节过表达的影响。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，但其如何做到这一点尚不清楚，特别是在单一的转导途径中，如PCD。所提出的模型系统在确定细胞分化过程中信号转导的机制方面比其他系统有几个优势。此外，PCD在任何情况下的调节机制尚不清楚，这些结果的意义可能影响从TE发育和木材形成的协调到瘤变控制的许多发育过程。此外，这项工作将探讨细胞外基质如何为细胞发育提供信号，更具体地说，一种新的分泌蛋白酶如何调节细胞死亡。

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.