This proposal presents an experimental plan to investigate biological mechanisms to defend against activated forms of oxygen in soybean root nodules. This defense is centered on the 4 enzymes of the ascorbate-glutathione pathway in wich H2O2 is scavenged. Biochemical studies will concentrate on glutathione reductase. This enzyme will be purified and characterized with regard to amino acid sequence, kinetic properties, pH optimum, inhibitor effects, cofactor requirements, substrate specificity, pI values, and possible isozymes. Polyclonal antibodies will be used to immunologically characterize the enzyme and to determine the subcellular location with electron microscopy. A major component of the proposed work consists of molecular studies to 1) identify the biochemical mechanisms that regulate the genes encoding glutathione reductase and ascorbate peroxidase and 2) describe environomental and developmental factors that affect regulation. The genes for glutathione reductase and ascorbate peroxidase will be cloned in order to identify regulatory sites and study cis- and trans-acting factors involved in express. The regulatory role of various environmental factors (water stress, heat and chilling shock, pO2) and of potential biochemical effectors (GSH, H2O2, paraquat, rhizobiol signals) will be examined. These studies will involved analysis of transcript and protein levels in whole nodules and in soybean protoplasts bearing electroporated reporter genes fused to promoter regions. A long term goal is to assess the criticality of oxygen defense through use of transgenic plants bearing antisense genes.
