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，在许多植物生长发育的例子中起着至关重要的作用。尽管它在调节细胞扩张和分化方面很重要，但对壁的合成、组装和拆解的许多方面仍然知之甚少。例如，细胞扩张过程中的壁面重组受到高度调控，虽然已经确定了一些不同的壁面修饰蛋白家族，但它们协同作用以控制方式实现壁面松动的机制尚不清楚。此外，许多新的壁修饰蛋白仍有待鉴定，或目前无法被赋予生化功能。因此，为了了解壁重组的过程，一个重要的目标是确定所有涉及的酶。最近，在发展中的蛋白质组学领域，或研究基因组的蛋白质补体，需要一种系统的方法来鉴定和表征细胞壁蛋白质群体，这已经成为一个现实的考虑。本提案中概述的研究描述了一种蛋白质组学方法来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。这将为了解质外体/细胞壁中发生的生化过程提供有价值的见解，包括对果实快速膨大和成熟过程中壁修饰的调节。需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质量和数量特征是什么？2)在果实个体发育过程中，质外体蛋白的表达模式是如何定性和定量变化的？将通过高分辨率双向凝胶电泳法分离细胞壁蛋白质群体，并通过质谱仪分析单个多肽，从而识别相应的基因。拟议的研究将：(I)提供对质外体/细胞壁蛋白质表达和调控模式的详细和前所未有的见解；(Ii)促进确定细胞扩张或果实软化所需的蛋白质组；(Iii)补充康奈尔大学的现有研究举措，使能够在mRNA和蛋白质水平上并行研究基因表达；(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)确定具有潜在植物生物技术实际应用的新的细胞壁蛋白质组。

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.