RUI: Antioxidant Defenses of Legume Root Nodules

9507491道尔顿，拟议的工作涉及生物固氮和对过氧化氢和超氧化物等活性形式的氧的防御之间的关系。固氮系统，如豆科植物根瘤和蓝藻，由于需要强烈的还原条件将氮素还原为氨，因此受到活性(部分还原)形式的氧气的高风险。根瘤中自由基的一个主要来源是豆状血红蛋白，这是一种极其丰富的蛋白质，具有自氧化和释放超氧化物和过氧化氢的不幸趋势。在豆类根瘤和一些蓝藻中，对活性氧的防御是通过一系列耦合的还原-氧化反应提供的，这些反应涉及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。这一途径有效地将过氧化氢化物从细胞质中移除。这项工作的一个主要目标是确定抗坏血酸-谷胱甘肽途径在多大程度上是固氮过程中必不可少的。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，以确定受损的过氧化氢清除作用。此外，还将构建一个包含功能类杆菌、豆类血红蛋白和抗坏血酸-谷胱甘肽途径成分的体外系统。这一途径的临界性将通过检查遗漏各种成分的后果来研究，如抗坏血酸、谷胱甘肽或个别酶。我们还将研究在不同氮和光条件下培养的蓝藻中抗坏血酸-谷胱甘肽途径的作用，以期开发一种灵活的原核遗传操作系统。最后，我们将利用根瘤血氧仪来研究在反义和野生型豆科植物根瘤中，抗坏血酸-谷胱甘肽途径在可变的氧扩散屏障中的作用。由于固氮是受氧气限制的，增加对氧气防御过程的了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.