Legumes such as soybean, pea, alfalfa, and clover are valuable crops that are capable of fertilizing themselves through the process of nitrogen fixation. This process provides a source of nitrogen that is environmentally and economically advantageous since it minimizes pollution from the release of excess nitrogen (e.g. nitrates in runoff) and does not require the use of expensive chemical fertilizers. Nitrogen fixation in plants is an energy-intensive, aerobic process that requires substantial amounts of oxygen. This oxygen consumption leads to a fundamental dilemma since the environment within nodules promotes the formation of damaging, activated forms of oxygen such as hydrogen peroxide. Consequently, nodules contain high levels of antioxidants such as vitamin C (ascorbic acid) and numerous enzymes such as ascorbate peroxidase. Recent observations in this laboratory have revealed that soybean nodules also contain glutathione S-transferase (GST). This enzyme is best known for its role in detoxification of herbicides, but it is also capable of serving as an antioxidant. The objectives of this project are to define the role that GST plays in nitrogen-fixing legume nodules. Molecular techniques will be used to determine which forms of the GST gene family are expressed in nodules and what conditions stimulate the production of GST. Microscopic examination of nodule sections treated with fluorescent antibodies will be used to determine precisely where GTS is located. Nodulated plants will be exposed to various regimes involving chemical pro-oxidants to develop a model system to evaluate oxidative stress and to aid in identifying additional novel antioxidant proteins. The function of critical GST genes will be examined in transgenic plants in which the genes are down regulated. The discovery of GST in nodules is significant because it represents a potentially major overlooked aspect of oxygen relations in nodules. Since nitrogen fixation is likely limited by oxygen, any increase in the fundamental knowledge of antioxidants in nodules could have substantial impacts on future attempts to enhance performance of nitrogen-fixing crops. Furthermore, a confirmation of the antioxidant role of GST in nodules will lend credence to a similar role in other systems (including animals and microbes) where GST is present. This project fulfills NSF broader impact criteria by providing valuable preparation of undergraduate students, most of who are destined for graduate studies in plant biology. Students will be encouraged to attend meetings and to remain as a research technician and mentor for a few months following their graduation. Undergraduates engaged in senior thesis or summer projects will conduct the bulk of the research. Over the past 15 years, the P.I. has worked with 66 senior thesis research students and 47 summer research fellows; 35 of the thesis students were women. This grant will also provide summer research opportunities for high school students by establishing collaboration with the Apprenticeships in Science and Engineering (ASE) Program (www.saturdayacademy.pdx.edu/ase/). ASE helps students explore their interests and make informed educational and career decisions.
