Analysis of the Apoplast Sub-Proteome During Tomato Fruit Development

植物初生细胞壁是一种高度动态和复杂的结构，在植物生长和发育的许多实例中起着关键作用。尽管它在调节细胞扩增和分化中的重要性，但对细胞壁合成、组装和拆卸的许多方面仍然知之甚少。例如，细胞扩张期间的细胞壁重构受到高度调控，虽然已经鉴定了许多不同的细胞壁修饰蛋白家族，但它们协同作用以受控方式实现细胞壁松动的机制尚不清楚。此外，许多新类型的壁修饰蛋白仍有待鉴定，或目前不能被分配一个生化功能。因此，为了了解细胞壁重组的过程，一个重要的目标是鉴定所有参与的酶。需要一个系统的方法来识别和表征细胞壁蛋白质群体最近已成为一个现实的考虑，在蛋白质组学的发展领域的形式，或基因组的蛋白质互补的研究。本提案中概述的研究描述了一种蛋白质组学方法，用于表征番茄果实发育过程中细胞壁和周围质外体或细胞壁连续体中的蛋白质群体。这将提供有价值的洞察发生在质外体/细胞壁的生化过程，包括在果实快速膨胀和成熟过程中的壁修饰的调节。要解决的广泛问题包括：1）番茄果实细胞壁中存在的蛋白质群体的定性和定量特征是什么？2)在果实发育过程中，质外体蛋白质的表达模式是如何定性和定量变化的？细胞壁蛋白群体将通过高分辨率2-D凝胶电泳分离，并通过质谱分析单个多肽，从而鉴定相应的基因。拟议的研究将：（i）提供详细的和前所未有的深入了解质外体/细胞壁蛋白的表达和调节模式;（ii）促进细胞扩增或果实软化所需的蛋白质组的鉴定（iii）补充康奈尔大学现有的研究计划，允许在mRNA和蛋白质水平上进行基因表达的平行研究;（iv）展示蛋白质组学作为研究细胞壁和质外体生物学的工具的效用;（v）鉴定对植物生物技术具有潜在实际应用的新细胞壁蛋白质。

The plant primary cell wall is a highly dynamic and complex structure that plays a critical role in numerous examples of plant growth and development. Despite its importance in regulating cell expansion and differentiation, many aspects of wall synthesis, assembly and disassembly remain poorly understood. For example, wall restructuring during cell expansion is highly regulated and while a number of divergent families of wall-modifying proteins have been identified, the mechanism by which they act in concert to achieve wall loosening in a controlled manner is unknown. Furthermore, many new classes of wall-modifying proteins remain to be identified, or cannot currently be assigned a biochemical function. To understand the process of wall reorganization, an important objective is therefore to identify all of the enzymes involved. The need for a systematic approach to the identification and characterization of cell wall protein populations has recently become a realistic consideration in the form of the developing field of proteomics, or the study of the protein complement of the genome. The research outlined in this proposal describes a proteomics approach to the characterization of protein populations in the cell wall and surrounding apoplast, or cell wall continuum, during tomato fruit development. This will provide valuable insight into biochemical processes occurring in the apoplast/cell wall, including the regulation of wall modification during rapid fruit expansion and ripening. Broad questions to be addressed include:1) What are the qualitative and quantitative characteristics of the protein populations present in the tomato fruit cell wall?2) How do the patterns of apoplastic protein expression change qualitatively and quantitatively during fruit ontogeny?Cell wall protein populations will be separated by high-resolution 2-D gel electrophoresis and individual polypeptides analyzed by mass spectrometry, leading to the identification of the corresponding genes. The proposed research will: (i) provide detailed and unprecedented insight into the patterns of expression and regulation of apoplastic/cell wall proteins; (ii) facilitate the identification of groups of proteins that are required for cell expansion or fruit softening (iii) complement existing research initiatives at Cornell, allowing parallel studies of gene expression at the mRNA and protein levels; (iv) demonstrate the utility of proteomics as a tool to study the biology of the cell wall and apoplast; (v) identify new cell wall proteins with potential practical applications for plant biotechnology.