Cell and Molecular Biology of High Capacity Calcium Sequestration in Plants

植物高容量钙固存的细胞和分子生物学

• 批准号: 9632027
• 负责人: Vincent Franceschi
• 金额: --
• 依托单位: Washington State University
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-15 至 2000-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9632027&HistoricalAwards=false
• 关键词: Cell; Molecular; Biology; Capacity; Calcium

9632027 Franceschi Calcium (Ca) is involved in the control of many physiological processes in plants, and therefore regulation of Ca activity is critical to normal plant growth, development and productivity. Ca is taken up in the root and transported along with water. The water evaporates from the surfaces of the plant, and over time large amounts of Ca can accumulate in various organs, which necessitates high capacity Ca sequestration systems. Ca oxalate formation in plants has evolved as a high capacity mechanism for removing Ca when it is present at levels that can no longer be controlled by the lower capacity mechanisms common to all cells. This idea is supported by observations that most plant families produce Ca oxalate crystals, that this product can account for up to 85% of the dry weight of some plant species, and that 90% of the Ca in plants can be in this form. The goal of this project is to elucidate features of cells called Ca oxalate crystal idioblasts, which are responsible for large scale Ca accumulation. Crystal idioblasts are structurally and biochemically specialized for Ca accumulation and Ca oxalate crystal formation. Four proteins have been identified that play key roles in crystal formation in crystal idioblasts. These are: "matrix protein," a unique vacuolar Ca binding protein; calreticulin, a high capacity Ca binding protein of the endoplasmic reticulum (ER); an ER Ca ATPase (ERCA) which acts as a Ca pump; and oxalate oxidase, which is involved in release of Ca from crystals when free Ca availability becomes limited. In addition, ultrastructural examination shows that crystal idioblasts have abundant ER and Golgi bodies. The aim of the proposed research is to characterize the structure, function and control of developmental expression of these proteins, and thus to gain a better understanding of how they regulate Ca levels in plant tissues and cells. The specific objectives are to: 1, complete the analysis of cDNAs for matrix protein, calreticulin, ERCA an d oxalate oxidase, and produce antibodies to the proteins isolated from a bacterial expression system; 2, purify matrix protein from an expression system and examine its physical-chemical properties in vitro; 3, characterize the temporal and spatial regulations of the proteins and their mRNA transcripts during changing Ca nutrition; 4, determine the pattern of expression of the proteins and their transcripts at the subcellular level from crystal idioblast initiation through maturation; and 5, characterize the formation and modification of the ER and Golgi systems during idioblast development. Molecular biological techniques will be used to accomplish all or parts of the first 3 objectives. Biochemical techniques, Western and Northern blotting, and high resolution in situ hybridization visualization and immunocytochemistry by the light and electron microscopy will be used for objectives 3 and 4. Objective 5 will be completed using laser scanning confocal and standard transmission electron microscopy. %%% A better understanding of the mechanism of Ca oxalate crystal formation is imperative to our understanding of Ca regulation in plants, as well as for the potential manipulation of this process, or selected features of it, by biochemical or molecular genetic means. This project will significantly enhance our understanding of Ca oxalate crystal formation specifically, and will provide important new information on mechanisms of Ca regulation in plants in general. Furthermore, it should be kept in mind that plants are not the only living organisms that manufacture mineral crystalline structures based on calcium salts; for example, the exquisitely precisely-sculpted spicules and mineralized exoskeletons (e.g., oyster shells) of invertebrates, and the bones and teeth of vertebrates, are also examples of this general process of biomineralization. The Ca oxalate crystals of plant crystal idioblasts represent a relatively simple model system for biomineralization, and it is anticipated that the results of this project will provide important new insights into the general area of biomineralization which will be useful not only to biologists interested in how nature manufactures mineralized materials, but also to materials scientists and engineers who are interested in either harnessing or mimicking natural processes to create novel and useful ceramic structural materials. ***

9632027 Franceschi钙（Ca）参与控制植物中的许多生理过程，因此Ca活性的调节对正常植物生长、发育和生产力至关重要。Ca在根中被吸收并随水沿着运输。水分从植物表面蒸发，随着时间的推移，大量的Ca可以积累在各种器官中，这需要高容量的Ca螯合系统。草酸钙在植物中的形成已经演变为一种高容量的机制，用于去除钙，当它存在的水平，可以不再由所有细胞共同的较低容量的机制控制。这一观点得到了以下观察结果的支持：大多数植物科产生草酸钙晶体，这种产物可以占某些植物物种干重的85%，并且植物中90%的钙可以以这种形式存在。本项目的目标是阐明草酸钙晶体异细胞的特征，这是负责大规模的钙积累。晶体异细胞在结构和生化上专门用于钙积累和草酸钙晶体形成。已经鉴定出四种蛋白质在晶体异基因细胞的晶体形成中起关键作用。这些是：“基质蛋白”，一种独特的空泡Ca结合蛋白;钙网蛋白，一种内质网（ER）的高容量Ca结合蛋白;作为Ca泵的ER Ca ATP酶（ERCA）;和草酸氧化酶，当游离Ca可用性变得有限时，其参与从晶体中释放Ca。此外，超微结构检查显示晶体异细胞具有丰富的ER和高尔基体。拟议研究的目的是表征这些蛋白质的结构，功能和发育表达的控制，从而更好地了解它们如何调节植物组织和细胞中的钙水平。本研究的具体目的是：1.完成基质蛋白、钙网蛋白、ERCA和草酸氧化酶的cDNA分析，并制备从细菌表达系统中分离的蛋白的抗体; 2.从表达系统中纯化基质蛋白，并在体外检测其理化性质; 3.表征蛋白及其mRNA转录在钙营养变化过程中的时空调控; 4、确定从晶体异胚细胞起始到成熟的亚细胞水平上蛋白质及其转录物的表达模式; 5、表征异胚细胞发育过程中ER和高尔基体系统的形成和修饰。分子生物学技术将用于完成前3个目标的全部或部分。生化技术、Western和北方印迹、高分辨率原位杂交可视化和免疫细胞化学（通过光学和电子显微镜）将用于目标3和4。将使用激光扫描共聚焦和标准透射电子显微镜完成目标5。更好地了解草酸钙晶体形成的机制，对于我们理解植物中的钙调节，以及通过生物化学或分子遗传手段对该过程或其选定特征的潜在操纵是至关重要的。该项目将显著提高我们对草酸钙晶体形成的理解，并将为植物钙调节机制提供重要的新信息。此外，应该记住的是，植物并不是唯一制造基于钙盐的矿物晶体结构的生物体;例如，精致精确雕刻的针状体和矿化的外骨骼（例如，无脊椎动物的牡蛎壳和脊椎动物的骨骼和牙齿也是这种生物矿化过程的例子。植物晶体异细胞的草酸钙晶体代表了一个相对简单的生物矿化模型系统，预计该项目的结果将为生物矿化的一般领域提供重要的新见解，这不仅对对自然界如何制造矿化材料感兴趣的生物学家有用，也适用于对利用或模仿自然过程以创造新颖和有用的陶瓷结构材料感兴趣的材料科学家和工程师。***