Biological nitrogen fixation represents an economical and environmentally-friendly alternative to chemical fertilization. Anaspect of the rhizobia-legume symbiosis with important implications in other fields of plant biology is the molecular basis thatdiff erentiate symbiosis and pathogenesis. In plants under pathogen attack, there is a rapid and transient increase in theproduction of reactive oxygen species ("oxidative burst"). In contrast, during the symbiotic interaction, rhizobia may escape orinhibit the plant's defense response possibly due to bacterial Nod factors and, possibly, polysaccharides. However, hydrogenperoxide has been detected in the infection threads and nodule apoplast, suggesting that it is involved in several steps ofnodulation, such as the growth of cell walls and infection threads. Our previous studies have also shown that nodules containhigh levels of all three superoxide dismutases (CuZnSOD, FeSOD, MnSOD) and, based on co-localization and inhibitorstudies, we have proposed that CuZnSO D may be a source of hydrogen peroxide in nodules. The general objective of thisthree-year project is to study in detail the functions of SODs in nodule development. Using Medicago sativa and Lotuscorniculatus to compare indeterminate and determinate nodul ation, we will determine: (1) the regulatory mechanisms ofexpression for the three sod genes in nodules; (2) the spatio-temporal patterns of activity for the sod promoters; and (3) theeffect of suppression of SOD expression in nodules. To accomplish these specific objectives, a multidisciplinary and innovativeapproach and state-of-the-art techniques will be used. Methodologies include gene-specific quantification of transcripts forSODs and other antioxidant enzymes by real time RT-PCR, cytochemical localiza tion of superoxide and hydrogen peroxide,fusion constructs between promoters and reporter genes, transgenic roots and nodules ("hairy root" system), and genesil'