Regulating Nitric Oxide Homeostasis and its Impact on Plant Growth and Reproduction

该项目将确定一氧化氮（NO）如何调节植物生长，发育和种子产量的重要方面，这些过程对粮食安全和植物生物质生产至关重要。NO在所有高等生物中作为信号分子，NO在细胞中作用的发现者被授予诺贝尔奖。该项目将在拟南芥中使用生物化学和遗传学方法来研究一种称为GSNOR的特定蛋白酶如何控制植物中NO的数量和作用。结果将有助于我们全面了解NO在所有生物体中的作用机制。一名博士后研究员、一名研究生、两名本科生和一名中学教师将接受实验室研究方面的培训，以及为专家和非专业人员提供书面和口头科学数据交流方面的指导。此外，外展计划将侧重于吸引教师和学生在阿默斯特初中和高中的发展动手实验涉及植物材料的中学生和高中学生接触马萨诸塞州的STEM本科专业和他们的校园经验的目标。与阿默斯特学校的参与也将服务于加强社区与大学的联系的重要目标。尽管一氧化氮（NO）明确参与多个植物过程，包括发芽，根生长和生育，一个基本的了解机制，NO对植物生长和发育的关键系统发挥其作用是缺乏的，将在这个项目中得到解决。NO的细胞信号传导和调节作用由可逆的翻译后蛋白质氧化还原修饰介导，包括半胱氨酸（Cys）亚硝化（Cys-SNO）和谷胱甘肽化（Cys-SG）。这些可逆的共价修饰主要来自与S-亚硝基谷胱甘肽（GSNO）的反应，S-亚硝基谷胱甘肽是丰富的细胞氧化还原缓冲剂谷胱甘肽的可溶性NO加合物。它们可以对蛋白质活性产生重大影响，表明必须有机制来控制细胞内时间和空间的NO和GSNO浓度。该项目研究了S-亚硝基谷胱甘肽还原酶（GSNOR）如何在调节GSNO和蛋白质亚硝化水平中发挥重要作用。通过对拟南芥、酵母和人GSNOR的生化研究，已经开发出通过特定Cys残基的亚硝化来调节GSNOR活性的模型。本项目将测试这一模型在体外（目标1）和在体内拟南芥（目标2）使用生物化学和遗传学，和可能的调节GSNOR活性的其他翻译后修饰将进行调查。实验还将确定GSNOR亚硝化如何被硫氧还蛋白和硫氧还蛋白还原酶家族中的特定胞质氧化还原蛋白逆转（目的1和2）。结果将开发一个完整的图片可能的氧化还原修饰控制GSNOR活性，从而细胞NO水平，并提供新的数据胞质硫氧还蛋白的活性和特异性。最后，与拟南芥GSNOR无效突变体的生育力降低相关的蛋白质亚硝化的变化将通过新的质谱方法来确定（目的3）。总之，实验解决了控制单一酶（GSNOR）活性的机制如何传播，从而导致整个生物体水平的表型变化。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This project will determine how nitric oxide (NO) modulates important aspects of plant growth, development and seed yield, processes critical to food security and plant biomass production. NO acts as a signaling molecule in all higher organisms, and the discoverers of the role of NO in cells were awarded the Nobel Prize. This project will use biochemical and genetic approaches in Arabidopsis thaliana to investigate how a specific protein enzyme, known as GSNOR, controls the amount and effects of NO in plants. Results will contribute to our overall understanding of the mechanism of NO action in all organisms. One postdoctoral researcher, a graduate student, two undergraduates and a middle school teacher will receive training in laboratory research, as well as mentoring in written and oral communication of scientific data for specialist and lay audiences. In addition, outreach programs will focus on engaging teachers and students in Amherst middle and high schools with the goals of developing hands on experiments for middle school students involving plant materials and exposing high school students to University of Massachusetts STEM undergraduate majors and their campus experiences. The engagement with Amherst schools will also serve the important goal of strengthening community ties with the university.Despite the clear involvement of nitric oxide (NO) in multiple plant processes, including germination, root growth and fertility, a basic understanding of the mechanisms by which NO exerts its effects on systems critical for plant growth and development is lacking and will be addressed in this project. The cellular signaling and regulatory effects of NO are mediated by reversible, posttranslational protein redox modifications, including cysteine (Cys) nitrosation (Cys-SNO) and glutathionylation (Cys-SG). These reversible, covalent modifications result primarily from reaction with S-nitrosoglutathione (GSNO), a soluble NO-adduct of the abundant cellular redox buffer glutathione. They can have major effects on protein activity, indicating there must be mechanisms to control NO and GSNO concentrations in both time and space within cells. This project investigates how the enzyme S-nitrosoglutathione reductase (GSNOR) plays a major role in modulating GSNO and protein nitrosation levels. A model for regulation of GSNOR activity by nitrosation of specific Cys residues has been developed from biochemical studies of Arabidopsis, yeast and human GSNOR. This project will test this model both in vitro (Aim 1) and in vivo in Arabidopsis thaliana (Aim 2) using biochemistry and genetics, and possible regulation of GSNOR activity by other posttranslational modifications will be investigated. Experiments will also determine how GSNOR nitrosation is reversed by specific cytosolic redox proteins in the thioredoxin and thioredoxin reductase families (Aims 1 and 2). Results will develop a complete picture of the possible redox modifications controlling GSNOR activity and thereby cellular NO levels, and provide novel data on the activity and specificity of cytosolic thioredoxins. Finally, changes in protein nitrosation associated with the reduced fertility of Arabidopsis GSNOR null mutants will be determined by novel mass spectrometry methods (Aim 3). In total, experiments address how mechanisms controlling activity of a single enzyme (GSNOR) are propagated to result in phenotypic changes at the whole organism level.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.