SummaryIron is an important nutrient in N 2 -®xing legume root nodules. Iron supplied to the nodule is used by the plant for the synthesis of leghemoglobin, while in the bacteroid fraction, it is used as an essential cofactor for the bacterial N 2 -®xing enzyme, nitrogenase, and iron-containing proteins of the electron transport chain. The supply of iron to the bacteroids requires initial transport across the plant-derived peribacteroid membrane, which physically separates bacteroids from the infected plant cell cytosol. In this study, we have identi®ed Glycine max divalent metal transporter 1 (GmDmt1), a soybean homologue of the NRAMP/Dmt1 family of divalent metal ion transporters. GmDmt1 shows enhanced expression in soybean root nodules and is most highly expressed at the onset of nitrogen ®xation in developing nodules. Antibodies raised against a partial fragment of GmDmt1 con®rmed its presence on the peribacteroid membrane (PBM) of soybean root nodules. GmDmt1 was able to both rescue growth and enhance 55 Fe(II) uptake in the ferrous iron transport de®cient yeast strain (fet3fet4). The results indicate that GmDmt1 is a nodule-enhanced transporter capable of ferrous iron transport across the PBM of soybean root nodules. Its role in nodule iron homeostasis to support bacterial nitrogen ®xation is discussed.

Soybean peribacteroid membrane (PBM) proteins were isolated from nitrogen-fixing root nodules and subjected to N-terminal sequencing. Sequence data from 17 putative PBM proteins were obtained. Six of these proteins are homologous to proteins of known function. These include three chaperones (HSP60, BiP [HSP70], and PDI) and two proteases (a serine and a thiol protease), all of which are involved in some aspect of protein processing in plants. The PBM homologs of these proteins may play roles in protein translocation, folding, maturation, or degradation in symbiosomes. Two proteins are homologous to known, nodule-specific proteins from soybean, nodulin 53b and nodulin 26B. Although the function of these nodulins is unknown, nodulin 53b has independently been shown to be associated with the PBM. All of the eight proteins with identifiable homologs are likely to be peripheral rather than integral membrane proteins. Possible reasons for this apparent bias are discussed. The identification of homologs of HSP70 and HSP60 associated with the PBM is the first evidence that the molecular machinery for co- or post-translational import of cytoplasmic proteins is present in symbiosomes. This has important implications for the biogenesis of this unique, nitrogen-fixing organelle.

The importance of zinc in organisms is clearly established, and mechanisms involved in zinc acquisition by plants have recently received increased interest. In this report, the identification, characterization and location of GmZIP1, the first soybean member of the ZIP family of metal transporters, are described. GmZIP1 was found to possess eight putative transmembrane domains together with a histidine-rich extra-membrane loop. By functional complementation of zrt1zrt2 yeast cells no longer able to take up zinc, GmZIP1 was found to be highly selective for zinc, with an estimated K m value of 13.8 M. Cadmium was the only other metal tested able to inhibit zinc uptake in yeast. An antibody raised against GmZIP1 specifically localized the protein to the peribacteroid membrane, an endosymbiotic membrane in nodules resulting from the interaction of the plant with its microsymbiont. The specific expression of GmZIP1 in nodules was confirmed by Northern blot, with no expression in roots, stems, or leaves of nodulated soybean plants. Antibodies to GmZIP1 inhibited zinc uptake by symbiosomes, indicating that at least some of the zinc uptake observed in isolated symbiosomes could be attributed to GmZIP1. The orientation of the protein in the membrane and its possible role in the symbiosis are discussed.Zinc is an essential micronutrient for all organisms, including plants. More than 3% of the proteins of Saccharomyces cerevisiae and Caenorhabditis elegans are predicted to contain sequence motifs characteristic of zinc binding structural domains (1). Zinc deficiency is a widespread micronutrient deficiency limiting crop production (2). In recent years, genes encoding zinc transporters have been identified in various organisms (3-11). These studies have shed some light on zinc uptake and regulation, particularly at the plasma membrane level. However, with the exception of the recently identified Zrt3p transporter on the vacuole membrane in yeast (9), little is known about intracellular zinc transport systems, nor about the mechanisms of the transporters identified. Here we investigate zinc transport at the symbiotic interface between legumes and rhizobia, which presents an additional level of complexity.Many legumes form a symbiosis with nitrogen-fixing soil bacteria (rhizobia) that enables the plants to utilize atmospheric N 2 for growth. Infection of the legume root by rhizobia results in the formation of specialized organs called nodules that provide the microaerobic conditions required for operation of the nitrogenase enzyme. Within the infected cells of nodules, the N 2 -fixing bacteroids are enclosed in a plant membrane to form an organelle-like structure termed the symbiosome (12). The envelope of the symbiosome is called the peribacteroid membrane (PBM) 1 and effectively controls the exchange of metabolites between the symbiotic partners. The PBM, although originating from the plasma membrane of root cells, evolves over the course of nodule organogenesis to become a new and specialized membrane containing symbiosi...

The Toc and Tic translocon facilitate import of preproteins into chloroplasts. In the past, it was speculated that several translocon subunits act specifically for different types of precursor proteins or in different tissues. To generate a comprehensive picture of the expression and tissue-specific localization of the translocon subunits, their transcript levels were analyzed in roots and leaves. Certain Tocs and Tics were found to be tissuespecific. The protein composition of the transloci in the envelope membranes of chloroplasts was analyzed to describe the function and possible stoichiometry. In contrast to Tic subunits, several Toc subunits seem to have a high turnover.

The localization of H(+)-ATPases in soybean (Glycine max L. cv. Stevens) nodules was investigated using antibodies against both P-type and V-type enzymes. Immunoblots of peribacteroid membrane (PBM) proteins using antibodies against tobacco and Arabidopsis H(+)-ATPases detected a single immunoreactive band at approximately 100 kDa. These antibodies recognized a protein of similar relative molecular mass in the crude microsomal fraction from soybean nodules and uninoculated roots. The amount of this protein was greater in PBM 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-infected cells in both the nodule cortex and nodule parenchyma. In the central nitrogen-fixing zone of the nodule, staining was present in both the infected and uninfected cells. Examination of nodule sections using confocal microscopy and fluorescence staining showed an immunofluorescent signal clearly visible around the periphery of individual symbiosomes which appeared as vesicles distributed throughout the infected cells of the central zone. Electron-microscopic examination of immunogold-labelled sections shows that P-type ATPase antigens were present on the PBM of both newly formed, single-bacteroid 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 detect this protein on symbiosome membranes. Only a very faint signal with this antibody was detected on Western blots of purified PBM. During nodule development, fusion of small symbiosomes to form larger ones containing multiple 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(+)-ATPase on the PBM of soybean is a P-type enzyme with homology to other such enzymes in plants. In vivo, this enzyme is likely to play a critical role in the regulation of nutrient exchange between legume and bacteroids.