Tracheary Element Differentiation

该项目的重点是两个重要的发育过程中发生的正常分化的气管元件（TE），即，协调次生细胞壁合成与细胞的程序性细胞死亡（PCD）。导管分子是构成植物木质部导水导管的细胞尸体。 随着TE的发展，它们形成了一个刚性的次级壁。 在该过程结束时，发育中的TE的死亡由其自身的胞质外信号触发。 细胞质包被物的自溶是在储存在液泡中的新生水解酶释放后发生的，但这些水解酶的合成在新壁形成之前就已经开始了。 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机不仅对发育中的尸体的功能，而且对整个发育中的细胞侧翼（血管）都有不利影响。 PI实验室的最新数据支持以下模型：在细胞壁形成之前，水解酶被合成并被隔离在液泡中。在次生壁合成的最后阶段，分泌一种40 kD的丝氨酸蛋白酶，并诱导钙的流入，直接或间接激活液泡膜的破裂和随后的水解酶的释放。 拟议的项目将利用良好的特性百日菊细胞培养系统，以解决3个问题的终端事件的气管元件分化：什么是蛋白酶，以及如何触发细胞死亡的信号？ 将分泌的蛋白酶部分纯化，以获得用于克隆相应cDNA的内部序列。 该蛋白酶将在E. coli中进行蛋白水解活性分析，并在百日菊根和拟南芥中进行功能试验。 切割特异性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。 将鉴定蛋白酶的拟南芥同源物，并检查受调节的过表达的效果。 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储存，但其如何做到这一点尚不清楚，特别是在单个细胞内。 转导途径，如PCD。 所提出的模型系统有几个优势，比其他系统，现在确定参与细胞分化的信号转导机制。 此外，在任何情况下，PCD的调节机制还没有得到很好的理解，这些结果的意义可能会影响到许多发展过程，从TE发展和木材形成的协调控制肿瘤。 此外，这项工作将解决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。

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.