Localization of H+ -ATPase in soybean root nodules

The localization of Hf-ATPases in soybean (Glycine max L. cv. Stevens) nodu les was investigated using antibodies against both P-type and V-type enzyme s. Immunoblots of peribacteroid membrane (PBM) proteins using antibodies ag ainst tobacco and Arabidopsis H+-ATPases detected a single immunoreactive b and at approximately 100 kDa. These antibodies recognized a protein of simi lar relative molecular mass in the crude microsomal fraction from soybean n odules and uninoculated roots. The amount of this protein was greater in PB M from mature nodules than in younger nodules. Immunolocalization of P-type ATPases using silver enhancement of colloidal-gold labelling at the light- microscopy level showed signal distributed around the periphery of non-infe cted cells in both the nodule cortex and nodule parenchyma. In the central nitrogen-fixing zone of the nodule, staining was present in both the infect ed and uninfected cells. Examination of nodule sections using confocal micr oscopy and fluorescence staining showed an immunofluorescent signal clearly visible around the periphery of individual symbiosomes which appeared as v esicles distributed throughout the infected cells of the central zone. Elec tron-microscopic examination of immunogold-labelled sections shows that P-t ype ATPase antigens were present on the PBM of both newly formed, single-ba cteroid symbiosomes just released from infection threads, and on the PBM of mature symbiosomes containing two to four bacteroids. Immunogold labelling using antibody against the B-subunit of V-type ATPase from oat failed to d etect this protein on symbiosome membranes. Only a very faint signal with t his antibody was detected on Western blots of purified PBM. During nodule d evelopment, fusion of small symbiosomes to form larger ones containing mult iple bacteroids was observed. Fusion was preceded by the formation of cone- like extensions of the PBM, allowing the membrane to make contact with the adjoining membrane of another symbiosome. We conclude that the major H+-ATP ase on the PBM of soybean is a P-type enzyme with homology to other such en zymes in plants. In vivo, this enzyme is likely to play a critical role in the regulation of nutrient exchange between legume and bacteroids.