Cloning and characterization of Lotus japonicus formate dehydrogenase: a possible correlation with hypoxia.

Formate dehydrogenases (FDHs, EC 1.2.1.2) comprise a group of enzymes found in both prokaryotes and eukaryotes that catalyse the oxidation of formate to CO(2). FDH1 from the model legume Lotus japonicus (LjFDH1) was cloned and expressed in E. coli BL21(DE3) as soluble active protein. The enzyme was purified using affinity chromatography on Cibacron blue 3GA-Sepharose. The enzymatic properties of the recombinant enzyme were investigated and the kinetic parameters (K(m), k(cat)) for a number of substrates were determined. Molecular modelling studies were also employed to create a model of LjFDH1, based on the known structure of the Pseudomonas sp. 101 enzyme. The molecular model was used to help interpret biochemical data concerning substrate specificity and catalytic mechanism of the enzyme. The temporal expression pattern of LjFDH1 gene was studied by real-time RT-PCR in various plant organs and during the development of nitrogen-fixing nodules. Furthermore, the spatial transcript accumulation during nodule development and in young seedpods was determined by in situ RNA-RNA hybridization. These results considered together indicate a possible role of formate oxidation by LjFDH1 in plant tissues characterized by relative hypoxia.

Characterization of spermidine and spermine synthases in Lotus japonicus: induction and spatial organization of polyamine biosynthesis in nitrogen fixing nodules.

The biosynthesis of the polyamines spermidine (Spd) and spermine (Spm) from putrescine (Put) is catalysed by the consequent action of two aminopropyltransferases, spermidine synthase (SPDS EC: 2.5.1.16) and spermine synthase (SPMS EC: 2.5.1.22). Two cDNA clones coding for SPDS and SPMS homologues in the nitrogen-fixing nodules of the model legume Lotus japonicus were identified. Functionality of the encoded polypeptides was confirmed by their ability to complement spermidine and spermine deficiencies in yeast. The temporal and spatial expression pattern of the respective genes was correlated with the accumulation of total polyamines in symbiotic and non-symbiotic organs. Expression of both genes was maximal at early stages of nodule development, while at later stages the levels of both transcripts declined. Both genes were expressed in nodule inner cortical cells, vascular bundles, and central tissue. In contrast to gene expression, increasing amounts of Put, Spd, and Spm were found to accumulate during nodule development and after maturity. Interestingly, nodulated plants exhibited systemic changes in both LjSPDS and LjSPMS transcript levels and polyamine content in roots, stem and leaves, in comparison to uninoculated plants. These results give new insights into the neglected role of polyamines during nodule development and symbiotic nitrogen fixation (SNF).

Structure, function and regulation of ammonium transporters in plants.

Ammonium is an important source of nitrogen for plants. It is taken up by plant cells via ammonium transporters in the plasma membrane and distributed to intracellular compartments such as chloroplasts, mitochondria and vacuoles probably via different transporters in each case. Ammonium is generally not used for long-distance transport of nitrogen within the plant. Instead, most of the ammonium transported into plant cells is assimilated locally via glutamine synthetases in the cytoplasm and plastids. Ammonium is also produced by plant cells during normal metabolism, and ammonium transporters enable it to be moved from intracellular sites of production to sites of consumption. Ammonium can be generated de novo from molecular nitrogen (N(2)) by nitrogen-fixing bacteria in some plant cells, such as rhizobia in legume root nodule cells, and at least one ammonium transporter is implicated in the transfer of ammonium from the bacteria to the plant cytoplasm. Plant physiologists have described many of these ammonium transport processes over the last few decades. However, the genes and proteins that underlie these processes have been isolated and studied only recently. In this review, we consider in detail the molecular structure, function and regulation of plant ammonium transporters. We also attempt to reconcile recent discoveries at the molecular level with our knowledge of ammonium transport at the whole plant level.

Identification with proteomics of novel proteins associated with the peribacteroid membrane of soybean root nodules.

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.

Characterization of Arabidopsis AtAMT2, a novel ammonium transporter in plants.

We have cloned and characterized the first member of a novel family of ammonium transporters in plants: AtAMT2 from Arabidopsis thaliana. AtAMT2 is more closely related to bacterial ammonium transporters than to plant transporters of the AMT1 family. The protein was expressed and functionally characterized in yeast. AtAMT2 transported ammonium in an energy-dependent manner. In contrast to transporters of the AMT1 family, however, AtAMT2 did not transport the ammonium analogue, methylammonium. AtAMT2 was expressed more highly in shoots than roots and was subject to nitrogen regulation.

Localization of H(+)-ATPases in soybean root nodules.

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.

Characterization of an ammonium transport protein from the peribacteroid membrane of soybean nodules.

Nitrogen-fixing bacteroids in legume root nodules are surrounded by the plant-derived peribacteroid membrane, which controls nutrient transfer between the symbionts. A nodule complementary DNA (GmSAT1) encoding an ammonium transporter has been isolated from soybean. GmSAT1 is preferentially transcribed in nodules and immunoblotting indicates that GmSAT1 is located on the peribacteroid membrane. [14C]methylammonium uptake and patch-clamp analysis of yeast expressing GmSAT1 demonstrated that it shares properties with a soybean peribacteroid membrane NH4^{+ channel described elsewhere. GmSAT1 is likely to be involved in the transfer of fixed nitrogen from the bacteroid to the host.}

Regulation of GmNRT2 expression and nitrate transport activity in roots of soybean (Glycine max).

A full-length cDNA, GmNRT2, encoding a putative high-affinity nitrate transporter was isolated from a Glycine max (L.) root cDNA library and sequenced. The deduced GmNRT2 protein is 530 amino acids in length and contains 12 putative membrane-spanning domains and a long, hydrophilic C-terminal domain. GmNRT2 is related to high-affinity nitrate transporters in the eukaryotes Chlamydomonas reinhardtii and Aspergillus nidulans, and to putative high-affinity nitrate transporters in barley and tobacco. Southern blot analysis indicated that GmNRT2 is part of a small, multigene family in soybean. Expression of GmNRT2 in roots was regulated by the type of nitrogen source provided to plants: GmNRT2 mRNA levels were barely detectable in ammonium-grown plants, higher in nitrogen-deprived plants, and highest in nitrate-grown plants. Induction of GmNRT2 mRNA levels in roots occurred within 1 h after exposure of plants to nitrate. Nitrate induction of GmNRT2 mRNA levels was accompanied by a fourfold increase in net nitrate uptake by soybean roots at 100 microM external nitrate. The molecular and physiological evidence indicates that GmNRT2 is probably a high-affinity nitrate transporter involved in nitrate uptake by soybean roots.

Isolation and characterization of a cDNA encoding NADP(+)-specific isocitrate dehydrogenase from soybean (Glycine max).

A cDNA that encodes an NADP-specific isocitrate dehydrogenase (IDH) was cloned from a soybean nodule cDNA library by complementation of an Escherichia coli mutant that lacked IDH. DNA sequence analysis showed that the 1583 bp soybean cDNA could encode a protein that shares 63.9% amino acid sequence identity with the Saccharomyces cerevisiae NADP-IDH and long sequences of identity to an IDH from pig. Southern blot analysis suggests that this gene corresponds to a gene family made up of no more than two loci. The IDH cDNA hybridized to a 1.7 kb soybean mRNA and the relative amount of this transcript in soybean leaves, nodules and roots was 1:3.4:7.7. In alfalfa, a 1.7 kb mRNA was also found but the ratios for the corresponding tissues were 1:7.4:7.7. IDH activity was detected in the complemented E. coli strain and the electrophoretic mobility of this activity in nondenaturing polyacrylamide gels was identical to that of an IDH in extracts from soybean cotyledons or nodule cytosol. NADP-IDH specific activity in the E. coli host strain varied with growth phase; the highest rates (ca. 180 nmol/min per mg protein) were observed in late-stationary-phase cells. The enzyme had a broad pH optimum of 8.0 to 9.5 and had an absolute metal cofactor requirement, preferring Mn2+ below pH 8.0 and Mg2+ above pH 8.0. The Km for isocitrate and NADP was 21 microM and 11 microM respectively with Mn2+ as cofactor and 13 microM and 12 microM with Mg2+ as cofactor.

Isolation and characterization of a ntrC mutant of Bradyrhizobium (Parasponia) sp. ANU289.

A mutant of Bradyrhizobium (Parasponia) sp. ANU289 affected in the regulation of nitrogen metabolism was isolated. The mutant, designated ANU293, was unable to induce ammonium transport (Amt), nitrate reductase (NR) or glutamine synthetase II (GSII) activities under conditions that induce these activities in the wild-type. However, glutamine synthetase I (GSI), which is expressed constitutively in the wild-type, was present at normal levels in the mutant. The mutant also retained the ability to fix nitrogen in vitro and in planta, although nodule development on siratro (Macroptilium atropurpureum) was retarded. Southern blot analysis showed that ntrC, the product of which is involved in regulation of nitrogen metabolism, is the site of pSUP1021 insertion in ANU293. These results indicate that the transcriptional activator NtrC is required for the expression of Amt, NR and GSII, but not GSI or nitrogenase in Bradyrhizobium (Parasponia) sp. ANU289.

Isolation and analysis of a cDNA clone that encodes an alfalfa (Medicago sativa) aspartate aminotransferase.

We have isolated an alfalfa leaf cDNA clone that encodes aspartate aminotransferase (AAT, EC 2.6.1.1) by direct complementation of an Escherichia coli aspartate auxotroph with a plasmid cDNA library. DNA sequence analysis of the recombinant plasmid, pMU1, revealed that a 1514 bp cDNA was inserted in the correct orientation and in-frame with the start of the lacZ coding sequence in the vector, pUC18. The resulting fusion protein is predicted to be 424 amino acids in length with a molecular weight of 46387 Daltons. The cDNA-encoded protein has a characteristic pyridoxal phosphate attachment site motif and has substantial amino acid sequence homology to both animal and bacterial AATs. Plasmid pMU1 encodes an AAT with a Km for aspartate of 3.3 mM, a Km for 2-oxoglutarate of 0.28 mM, and a pH optimum between 8.0 and 8.5. Several lines of evidence including Western blot analysis, the isoelectric point of the encoded protein, and the effect of pH on the activity of the fusion protein, suggest that the cDNA encodes the isozyme AAT-1 rather than AAT-2. Northern blot analysis showed that the aat-1 clone hybridized to a 1.6 kb transcript present in alfalfa leaves, roots and nodules. The relative concentrations of aat-1 mRNA in these tissues were 1:2:5, respectively. Thus, transcription of aat-1 appears to be induced during nodule development. Southern blot analysis suggested that AAT-1 in alfalfa is encoded by either a single-copy gene or a small, multigene family.

Ammonia (C-Methylamine) Transport across the Bacteroid and Peribacteroid Membranes of Soybean Root Nodules.

[(14)C]Methylamine (MA; an analog of ammonia) was used to investigate ammonia transport across the bacteroid and peribacteroid membranes (PBM) from soybean (Glycine max) root nodules. Free-living Bradyrhizobium japonicum USDA110 grown under nitrogen-limited conditions showed rapid MA uptake with saturation kinetics at neutral pH, indicative of a carrier. Exchange of accumulated MA for added ammonia occurred, showing that the carrier recognized both NH(4) (+) and CH(3)NH(3) (+). MA uptake by isolated bacteroids, on the other hand, was very slow at low concentrations of MA and increased linearly with increasing MA concentration up to 1 millimolar. Ammonia did not inhibit MA by isolated bacteroids and did not cause efflux of accumulated MA. PBM-enclosed bacteroids (peribacteroid units [PBUs]) were qualitatively similar to free bacteroids with respect to MA transport. The rates of uptake and efflux of MA by PBUs were linearly dependent on the imposed concentration gradient and unaffected by NH(4)Cl. MA uptake by PBUs increased exponentially with increasing pH, confirming that the rate increased linearly with increasing CH(3)NH(2) concentration. The results are consistent with other evidence that transfer of ammonia from the nitrogen-fixing bacteroid to the host cytosol in soybean root nodules occurs solely by simple diffusion of NH(3) across both the bacteroid and peribacteroid membranes.

Specificity and regulation of the dicarboxylate carrier on the peribacteroid membrane of soybean nodules.

Malate and succinate were taken up rapidly by isolated, intact peribacteroid units (PBUs) from soybean (Glycine max (L.) Merr.) root nodules and inhibited each other in a competitive manner. Malonate uptake was slower and was severely inhibited by equimolar malate in the reaction medium. The apparent Km for malonate uptake was higher than that for malate and succinate uptake. Malate uptake by PBUs was inhibited by (in diminishing order of severity) oxaloacetate, fumarate, succinate, phthalonate and oxoglutarate. Malonate and butylmalonate inhibited only slightly and pyruvate,isocitrate and glutamate not at all. Of these compounds, only oxaloacetate, fumarate and succinate inhibited malate uptake by free bacteroids. Malate uptake by PBUs was inhibited severely by the uncoupler carbonylcyanidem-chlorophenyl hydrazone and the respiratory poison KCN, and was stimulated by ATP. We conclude that the peribacteroid membrane contains a dicarboxylate transport system which is distinct from that on the bacteroid membrane and other plant membranes. This system can catalyse the rapid uptake of a range of dicarboxylates into PBUs, with malate and succinate preferred substrates, and is likely to play an important role in symbiotic nitrogen fixation. Energization of both the bacteroid and peribacteroid membranes controls the rate of dicarboxylate transport into peribacteroid units.

Electrogenic ATPase Activity on the Peribacteroid Membrane of Soybean (Glycine max L.) Root Nodules.

Electrogenic ATPase activity on the peribacteroid membrane from soybean (Glycine max L. cv Bragg) root nodules is demonstrated. Membrane energization was monitored using suspensions of intact peribacteroid membrane-enclosed bacteroids (peribacteroid units; PBUs) and the fluorescent probe for membrane potential (DeltaPsi), bis-(3-phenyl-5-oxoisoxazol-4yl) pentamethine oxonol. Generation of a positive DeltaPsi across the peribacteroid membrane was dependent upon ATP, inhibited by N,N'-dicyclohexyl-carbodiimide and vanadate, but insensitive to N-ethylmaleimide, azide, cyanide, oligomycin, and ouabain. The results suggest the presence of a single, plasma membrane-like, electrogenic ATPase on the peribacteroid membrane. The protonophore, carbonyl-cyanide m-chlorophenyl hydrazone, completely dissipated the established membrane potential. The extent of reduction in the steady state membrane potential upon addition of ions was used to estimate the relative permeability of the peribacteroid membrane to anions. By this criterion, the relative rates of anion transport across the peribacteroid membrane were: NO(3) (-) > NO(2) (-) > Cl(-) > acetate(-) > malate(-). The observation that 10 millimolar NO(3) (-) completely dissipated the membrane potential was of particular interest in view of the fact that NO(3) (-) inhibits symbiotic nitrogen fixation. The possible function of the ATPase in symbiotic nitrogen fixation is discussed.

Stimulation of respiration and nitrogenase in bacteroids of Siratro (Macroptilium atropurpureum) by plant nodule cytosol.

Nitrogenase activity (acetylene reduction) of isolated Siratro (Macroptilium atropurpureum) bacteroids was stimulated by addition of plant cytosol fractions which also preserved activity at high (up to 3%) O2 tensions. These effects were not due to leghaemoglobin. Boiling removed some, but not all, of the protective capacity of the cytosol. Heat treated cytosol substantially stimulated the respiration of siratro bacteroids. Of a wide variety of compounds tested, only ascorbate could mimic the cytosol. Ascorbate was present in the cytosol fraction, in significant quantities. The effect of ascorbate was evident at low O2 concentrations and in purified bacteroids, and was inhibited by cyanide. Siratro bacteroids appear to possess an oxidase which could serve a protective role in vivo.