CAA: Differential Gene Expression Related to Calcium Oxalate Formation in Grape

在大多数高等植物中，某些细胞专门积累钙并将其沉淀为结晶草酸钙。这些细胞似乎通过隔离多余的钙，从而降低植物内自由离子钙的水平，在钙的大量调节中起作用。植物细胞在隔离草酸钙的过程中表现出明显的结构和生化差异。韦伯博士的研究目标集中在了解是什么刺激植物细胞发育这种特殊功能，以及细胞如何控制结晶过程。她目前研究的主要目标是应用分子方法来鉴定在晶体形成细胞发育的早期阶段差异表达的基因。分子研究将从葡萄的胚乳和根皮层两个组织开始，这两个组织具有截然不同的晶体形态和发育模式。差异表达的基因将通过mRNA差异显示来鉴定，以比较来自这些组织的不同mRNA群体。来自胚乳的信息将在选定的发育阶段进行比较，并在操纵钙营养改变晶体发育后来自根的信息进行比较。在确定所选cdna的差异表达后，将对其进行测序，并与计算机数据库中的已知序列进行比较。全长cDNA克隆将被分离和鉴定。在钙转运，草酸代谢或液泡功能中具有潜在作用的克隆将特别感兴趣。为了鉴定在晶体异母细胞中特异性表达的基因，还将使用原位杂交确定选定的cdna的细胞表达定位。这些研究将为未来的研究奠定基础，以确定特定基因和基因产物的表达在草酸钙结晶和晶体形成细胞的分化中是如何重要的。相关研究将考察葡萄根在分化过程中晶体形成细胞的结构发育。多种方法将探索使用扫描共聚焦显微镜观察这些细胞的独特特征。这项工作的主要目的是研究在液泡内划分草酸钙晶体的膜系统的发展。在职业发展奖下进行的所有研究都强调对Webb博士来说是新的活动和技术，并将显著提高她的研究和咨询能力。

In most higher plants, certain cells become specialized to accumulate calcium and precipitate it as crystalline calcium oxalate. These cells appear to function in bulk regulation of calcium by sequestering excess calcium, thereby reducing levels of free ionic calcium within the plant. Plant cells that sequester calcium oxalate exhibit distinct structural and biochemical differences as they differentiate for this role. Dr. Webb's research goals focus on understanding what stimulates plant cells to develop for this specialized function, and how the cell controls the crystallization process. The primary objective of her current research is the application of molecular approaches to identify genes that are differentially expressed at early stages in the development of crystal-forming cells. Molecular studies will be initiated with two tissues in grape, endosperm and root cortex, which have contrasting patterns of crystal morphology and development. Differentially expressed genes will be identified using mRNA differential display to compare distinct populations of mRNA from each of these tissues. Messages from endosperm will be compared at selected developmental stages, and those from roots after manipulating calcium nutrition to alter crystal development. After differential expression of selected cDNAs is confirmed, they will be sequenced and compared to known sequences in computer databases. Full-length cDNA clones will be isolated and characterized. Clones that have potential roles in calcium transport, oxalate metabolism, or vacuole function will be of particular interest. To identify genes specifically expressed in crystal idioblasts, the cellular localization of expression will also be determined for selected cDNAs using in situ hybridization. These studies will form the basis for future research to determine how expression of particular genes and gene products are important in calcium oxalate crystallization and the differentiation of crystal-forming cells. Correlated studies will examine the structural development of crystal-forming cells in grape roots during their differentiation. A variety of approaches will be explored using scanning confocal microscopy to observe unique features of these cells. A primary objective in this work will be to examine the development of the membrane systems that compartmentalize calcium oxalate crystals within the vacuole. All of the research to be conducted under this Career Advancement Award emphasizes activities and techniques that are new to Dr. Webb and will significantly enhance her research and advisory capabilities.