RUI: Antioxidant Defenses of Legume Root Nodules

拟议的工作涉及生物固氮与抵抗活性形式的氧（如过氧化氢和超氧化物）之间的关系。固氮系统，如豆科根瘤和蓝藻，由于将二氮还原为氨所需的强还原条件，受到活性（部分还原）氧形式的高风险。结节中自由基的主要来源是豆红蛋白，这是结节中极其丰富的蛋白质，不幸的是，它有自氧化和释放超氧化物和过氧化氢的倾向。在豆科结核和一些蓝藻中，对活性氧的防御是由一系列偶联的还原-氧化反应提供的，这些反应包括抗坏血酸过氧化物酶、单脱氢抗坏血酸还原酶、脱氢抗坏血酸还原酶和谷胱甘肽还原酶。这一途径有效地从细胞质溶胶中去除过氧化氢。这项工作的一个主要目的是确定抗坏血酸-谷胱甘肽途径在多大程度上对固氮过程至关重要。这将通过构建抗坏血酸过氧化物酶反义的转基因植物来检验。这些植物的根瘤将在分子和生理水平上进行检查，以确定过氧化氢清除受损的影响。此外，一个体外系统包含功能性类杆菌，豆血红蛋白和抗坏血酸-谷胱甘肽途径的组成部分将被构建。这一途径的重要性将通过检查各种成分如抗坏血酸、谷胱甘肽或个别酶遗漏的后果来研究。抗坏血酸-谷胱甘肽途径在各种氮和光体制下培养的蓝藻中的作用也将被检查，希望开发一个灵活的原核系统的遗传操作。最后，利用根瘤血氧测定法研究反义和野生型豆科根瘤中抗坏血酸-谷胱甘肽途径对可变氧扩散屏障的作用。由于固氮是氧气受限的，增加对氧气防御过程的理解可以促进通过增加氧气供应来改善固氮的长期目标。

9507491 Dalton The proposed work deals with the relation between biological nitrogen fixation and defenses against active forms of oxygen such as hydrogen peroxide and superoxide. Nitrogen- fixing systems, such as legume root nodules and cyanobacteria, are at high risk from active (partially reduced) forms of oxygen due to the strong reducing conditions required to reduce dinitrogen to ammonia. A major source of free radicals in nodules is leghemoglobin, an extremely abundant protein in nodues with the unfortunate tendency to autoxidize and release superoxide and hydrogen peroxide. Defense against active oxygen in legume nodules and in some cyanobacteria is provided by a series of coupled reduction-oxidation reactions involving ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase. This pathway effectively removes hydrogen peroide from the cytosol. A major objective of this work is to determine the extent to which ascorbate-glutathione pathway is essential for the process of nitrogen fixation. This will be examined by constructing transgenic plants that are antisense for ascorbate peroxidase. Nodules of these plants will be examined on molecular and physiological levels to determine the effects of impaired peroxide scavenging. In addition, an in vitro system containing functional bacteroids, leghemoglobin and the components of the ascorbate- glutathione pathway will be constructed. Criticality of this pathway will be investigated by examining the consequences of omission of various components such as ascorbate, glutathione or the individual enzymes. The role of the ascorbate-glutathione pathway in cyanobacteria cultured under various nitrogen and light regimes will also be examined in hopes of developing a flexible prokaryotic system for genetic manipulations. Finally, nodule oximetry will be used to investigate the role of the ascorbate- gluthathione pathway vis-a-vis the variable oxygen diffusi on barrier in antisense and wild type legume root nodules. Since nitrogen fixation is oxygen-limited, an increased understanding of oxygen defense processes could facilitate long range goals of improving nitrogen fixation by increasing the oxygen supply.