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，以及对粮食安全和植物生物量生产至关重要的过程。NO在所有高等生物体中都扮演着信号分子的角色，发现NO在细胞中的作用的人被授予了诺贝尔奖。该项目将在拟南芥中使用生化和遗传方法来研究一种被称为GSNOR的特定蛋白质酶是如何控制植物中NO的数量和影响的。这些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受众提供科学数据书面和口头交流方面的指导。此外，外展计划将侧重于让阿默斯特初中和高中的教师和学生参与进来，目标是为中学生开发涉及植物材料的动手实验，并让高中生接触到马萨诸塞大学STEM本科专业及其校园经历。与Amherst学校的合作也将服务于加强社区与大学联系的重要目标。尽管一氧化氮(NO)明确参与了包括种子萌发、根生长和肥力在内的多种植物过程，但对NO对植物生长和发育至关重要的系统产生影响的机制缺乏基本了解，并将在本项目中得到解决。NO的细胞信号和调节作用是通过可逆的翻译后蛋白质氧化还原修饰来介导的，包括半胱氨酸(Cys)亚硝化(Cys-SNO)和谷胱甘肽基化(Cys-SG)。这些可逆的共价修饰主要是与S-亚硝基谷胱甘肽(GSNO)反应的结果，GSNO是一种丰富的细胞氧化还原缓冲液谷胱甘肽的可溶性NO加合物。它们可以对蛋白质活性产生重大影响，这表明在细胞内的时间和空间上都必须有控制NO和GSNO浓度的机制。本项目研究了S-亚硝基谷胱甘肽还原酶(GSNOR)在调节GSNO和蛋白质亚硝化水平中的主要作用。通过对拟南芥、酵母和人GSNOR的生化研究，建立了一个通过亚硝化特定半胱氨酸残基来调节GSNOR活性的模型。本项目将利用生物化学和遗传学在体外(Aim 1)和体内(Aim 2)对这一模型进行测试，并将研究其他翻译后修饰对GSNOR活性的可能调节。实验还将确定硫氧还蛋白和硫氧还蛋白还原酶家族中特定的胞质氧化还原蛋白如何逆转GSNOR亚硝化(目标1和2)。这些结果将为控制GSNOR活性从而控制细胞NO水平的可能的氧化还原修饰提供一个完整的图景，并提供关于胞浆硫氧还蛋白的活性和特异性的新数据。最后，将通过新的质谱学方法来确定与拟南芥GSNOR缺失突变体育性降低相关的蛋白质亚硝化的变化(目标3)。总而言之，实验解决了如何传播控制单个酶(GSNOR)活性的机制以导致整个有机体水平的表型变化。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.