A Biochemical Genetic Analysis of Inositol Metabolism in Subcellular Organelles

亚细胞器中肌醇代谢的生化遗传分析

• 批准号: 9724117
• 负责人: Margaret Johnson
• 金额: $ 22.5万
• 依托单位: University of Alabama Tuscaloosa
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9724117&HistoricalAwards=false
• 关键词: Biochemical; Genetic; Analysis; Inositol; Metabolism

9724117 Johnson Inositol, a six-carbon cyclitol, is essential for growth, differentiation, and communication of eukaryotic cells. Inositol phosphates convey signals for a wide variety of hormones, growth factors and neurotransmitters. Insitol's metabolism, therefore, is an important concern in many agricultural and clinical disciplines. Although extensive physiological and biochemical data document the importance of inositol to higher plants, less is known of the molecular genetic mechanisms regulating its metabolism. The goal of this research is to understand the genetic mechanisms controlling inositol metabolism during plant growth and development. The investigator's developmental studies of inositol biosynthesis have shown, that isoforms of the pivotal biosynthetic enzyme, myo-inositol-1- phosphate synthase (Ml-1-P synthase), exist and that one form is localized to chloroplast thylakoids in Phaseolus vulgaris and Arabidopsis thaliana. Given this finding, it is imperative that the current two pathway model for phosphatidylinositol (PI) biosynthesis in plans be examined. According to this model, PI(precursor for phosphinositdes) is synthesized in the endoplasmic reticulum (ER) and then transported into other subcellular organelles such as the chloroplasts. The investigator's recent finding (the synthesis of inositol in chloroplasts), however, raises the possibility that PI and the phosphinositides are also being synthesized in these organelles. It is conceivable that inositol and/or the phosphoinositide signal tranduction pathway operates as part of the communication system between chloroplasts and the nucelus ( the hypothesized chloroplast-to-nucleus signal transduction pathway in plants). The investigator will use enzyme assays, western analyses, Immunocytochemical analyses, in situ hybridizations, phospholipid studies, and molecular analyses to (1) correlate sites of inositol biosynthesis with sites of PI biosynthesis and (2) isolate mutants defective in inositol metabolism. This project is expected to yield direct evidence concerning inositol's function in chloroplast and other subcellular organelles shown to synthesize inositol. The findings will be directly applicable to the overall picture (still lacking) of the important plant phosphoinositide signal transduction pathway. This project is concerned with synthesis of inositol in plants. While much is known about inositol phosphate signaling in animals, the corresponding issues in plants have not been well studied. The overall long-term goal of the work is to understand inositol's role in the biology of subcellular organelles. The investigator has shown in previous studies that the key biosynthetic enzyme, myo-inositol-1-phosphate synthase, has temporally and spatially controlled isoforms; this unexpected finding is novel. This project will involve a detailed study of the sites of inositol and phosphionositol synthesis. A combination of immunocytochemical analyses, in situ hybridization studies, and enzyme activity measurements will be performed using Phaseolus vulgaris and Arabidopsis thaliana. This work will help in developing an understanding of cell signaling in plants, as well as of regulation of an important metabolic pathway.

9724117约翰逊肌醇是一种六碳环醇，对真核细胞的生长、分化和通讯至关重要。 肌醇磷酸传递各种激素、生长因子和神经递质的信号。 因此，肌醇的代谢是许多农业和临床学科的重要关注点。 虽然大量的生理和生化数据证明了肌醇对高等植物的重要性，但对其代谢调控的分子遗传机制知之甚少。 本研究旨在了解植物生长发育过程中肌醇代谢的遗传调控机制。研究者对肌醇生物合成的发育研究表明，存在关键生物合成酶肌醇-1-磷酸合酶（MI-1-P合酶）的同种型，并且一种形式定位于菜豆和拟南芥中的叶绿体类囊体。 鉴于这一发现，它是当务之急，目前的两个途径模型磷脂酰肌醇（PI）的生物合成计划进行检查。 根据这一模型，PI（前体磷酯）在内质网（ER）中合成，然后转运到其他亚细胞器，如叶绿体。 然而，研究人员最近的发现（肌醇在叶绿体中的合成）提出了PI和phosphinositides也在这些细胞器中合成的可能性。 可以想象，肌醇和/或磷酸肌醇信号转导途径作为叶绿体和珠心之间的通讯系统的一部分（植物中假设的叶绿体到核的信号转导途径）。 研究者将使用酶测定、蛋白质印迹分析、免疫细胞化学分析、原位杂交、磷脂研究和分子分析来（1）将肌醇生物合成位点与PI生物合成位点相关联，以及（2）分离肌醇代谢缺陷突变体。 该项目有望获得有关肌醇在叶绿体和其他亚细胞细胞器中合成肌醇的功能的直接证据。 这一发现将直接适用于重要的植物磷脂酰肌醇信号转导途径的整体图像（仍然缺乏）。 本课题研究肌醇在植物中的合成。 虽然对动物中的肌醇磷酸信号传导了解很多，但植物中的相应问题尚未得到很好的研究。 这项工作的总体长期目标是了解肌醇在亚细胞器生物学中的作用。 研究者在以前的研究中已经表明，关键的生物合成酶，肌醇-1-磷酸合酶，具有时间和空间控制的异构体;这一意外的发现是新颖的。 本计画将详细研究肌醇与磷酸肌醇合成的位置。 将使用菜豆和拟南芥进行免疫细胞化学分析、原位杂交研究和酶活性测量的组合。 这项工作将有助于发展对植物细胞信号传导的理解，以及对重要代谢途径的调节。