Ammonia permeability of the soybean nodulin 26 channel

Soybean nodulin 26 (nod26), a member of the aquaporin superfamily, is the major protein component of the symbiosome membrane that encloses nitrogen-fixing bacteroids in root nodules. Previous work has demonstrated that nod26 facilitates the transport of water and glycerol, although a potential additional role as a channel for fixed ammonia efflux has been hypothesized. In the present study it is shown that recombinant nod26 reconstituted into proteoliposomes facilitates NH₃ transport in an Hg²⁺-sensitive manner with a reduced activation energy, hallmarks of protein-facilitated transport characteristic of aquaporins. Comparison of the predicted single-channel transport rates of nod26 suggests a 4.9-fold preference for ammonia compared to water.     

Determination of the site of phosphorylation of nodulin 26 by the calcium-dependent protein kinase from soybean nodules.

Nodulin 26 is a nodule-specific protein that is associated with the symbiosome membrane of soybean root nodules. Nodulin 26 is an endogenous substrate for a novel calcium-dependent protein kinase (CDPK) of soybean root nodules. By phosphopeptide mapping of endoproteinase Lys-C-digested nodulin 26 and automated and manual peptide sequence analyses, we have identified the site on nodulin 26 phosphorylated by CDPK. We have also established that the phosphorylation site of nodulin 26 is identical to the phosphorylation site of CK-15, a synthetic peptide with the carboxyl-terminal sequence of nodulin 26. The phosphorylation of nodulin 26 occurs at position Ser262, and the phosphorylation of CK-15 occurs at the analogous position, Ser,6 in vitro. Thus, the CK-15 sequence apparently contains sufficient structural features of the phosphorylation site of nodulin 26 to be recognized by CDPK. On the basis of peptide mapping analysis of nodulin 26 from nodules that are metabolically labeled with [32P]phosphate, it appears that the site of nodulin 26 that is phosphorylated in vitro is also labeled in vivo. The data indicate that the carboxyl terminus of nodulin 26 is phosphorylated by CDPK and provide initial sequence data for the phosphorylation site of an endogenous substrate for a plant CDPK.     

The structure, function and regulation of the nodulin 26-like intrinsic protein family of plant aquaglyceroporins

The nodulin 26-like intrinsic protein family is a group of highly conserved multifunctional major intrinsic proteins that are unique to plants, and which transport a variety of uncharged solutes ranging from water to ammonia to glycerol. Based on structure-function studies, the NIP family can be subdivided into two subgroups (I and II) based on the identity of the amino acids in the selectivity-determining filter (ar/R region) of the transport pore. Both subgroups appear to contain multifunctional transporters with low to no water permeability and the ability to flux multiple uncharged solutes of varying sizes depending upon the composition of the residues of the ar/R filter. NIPs are subject to posttranslational phosphorylation by calcium-dependent protein kinases. In the case of the family archetype, soybean nodulin 26, phosphorylation has been shown to stimulate its transport activity and to be regulated in response to developmental as well as environmental cues, including osmotic stresses. NIPs tend to be expressed at low levels in the plant compared to other MIPs, and several exhibit cell or tissue specific expression that is subject to spatial and temporal regulation during development.     

The structure, function and regulation of the nodulin 26-like intrinsic protein family of plant aquaglyceroporins

The nodulin 26-like intrinsic protein family is a group of highly conserved multifunctional major intrinsic proteins that are unique to plants, and which transport a variety of uncharged solutes ranging from water to ammonia to glycerol. Based on structure-function studies, the NIP family can be subdivided into two subgroups (I and II) based on the identity of the amino acids in the selectivity-determining filter (ar/R region) of the transport pore. Both subgroups appear to contain multifunctional transporters with low to no water permeability and the ability to flux multiple uncharged solutes of varying sizes depending upon the composition of the residues of the ar/R filter. NIPs are subject to posttranslational phosphorylation by calcium-dependent protein kinases. In the case of the family archetype, soybean nodulin 26, phosphorylation has been shown to stimulate its transport activity and to be regulated in response to developmental as well as environmental cues, including osmotic stresses. NIPs tend to be expressed at low levels in the plant compared to other MIPs, and several exhibit cell or tissue specific expression that is subject to spatial and temporal regulation during development.     

Expression of soybean nodulin 26 in transgenic tobacco. Targeting to the vacuolar membrane and effects on floral and seed development.

Nodulin 26 is an integral membrane protein of the symbiosome membrane of nitrogen-fixing soybean nodules. We expressed a nodulin 26 cDNA in transgenic tobacco (TN26 tobacco) under the control of the cauliflower mosaic virus 35S promoter to study subcellular targeting and the physiological effect(s) of its expression. Based on Northern and Western blots, the expression of nodulin 26 mRNA and protein in transgenic plants is high in apical shoot sections, flowers, and stems, low in mature leaves, and absent in roots. Western blot analysis revealed high levels of transgenic nodulin 26 protein in tonoplast membranes. In contrast, nodulin 26 protein was not found in isolated plasma membranes, the soluble fraction, nor in chloroplast and mitochondria-enriched membrane fractions. About 50-60% of the flowers and pods from TN26 tobacco plants abscised prematurely. Seed capsule size and seed fill per capsule from the remainder of surviving flowers were about 50% of that of control plants. Pollen viability was found to be normal, but flowers from TN26 tobacco plants showed shorter anther filaments compared with control plants. Normal seed production and capsule size was restored by manually crossing the stigmas from TN26 plants with isolated pollen from either transgenic or control plants. Thus, the aberrant filament growth could have resulted in the reproductive defects associated with the plants.     

Transcription of ENOD8 in Medicago truncatula nodules directs ENOD8 esterase to developing and mature symbiosomes

In Medicago truncatula nodules, the soil bacterium Sinorhizobium meliloti reduces atmospheric dinitrogen into nitrogenous compounds that the legume uses for its own growth. In nitrogen-fixing nodules, each infected cell contains symbiosomes, which include the rhizobial cell, the symbiosome membrane surrounding it, and the matrix between the bacterium and the symbiosome membrane, termed the symbiosome space. Here, we describe the localization of ENOD8, a nodule-specific esterase. The onset of ENOD8 expression occurs at 4 to 5 days postinoculation, before the genes that support the nitrogen fixation capabilities of the nodule. Expression of an ENOD8 promoter-gusA fusion in nodulated hairy roots of composite transformed M. truncatula plants indicated that ENOD8 is expressed from the proximal end of interzone II to III to the proximal end of the nodules. Confocal immunomicroscopy using an ENOD8-specific antibody showed that the ENOD8 protein was detected in the same zones. ENOD8 protein was localized in the symbiosome membrane or symbiosome space around the bacteroids in the infected nodule cells. Immunoblot analysis of fractionated symbiosomes strongly suggested that ENOD8 protein was found in the symbiosome membrane and symbiosome space, but not in the bacteroid. Determining the localization of ENOD8 protein in the symbiosome is a first step in understanding its role in symbiosome membrane and space during nodule formation and function.     

Calcium-dependent phosphorylation of symbiosome membrane proteins from nitrogen-fixing soybean nodules : evidence for phosphorylation of nodulin-26

By using a peptide (CK-15) based on the COOH-terminal sequence of nodulin-26, we have demonstrated the presence of a Ca²⁺-dependent protein kinase in soluble as well as particulate fractions of nitrogen-fixing soybean (Glycine max) root nodules. Substantial enzyme activity was found in symbiosome membranes. The soluble enzyme was purified 1570-fold. The enzyme was fractionated from endogenous calmodulin and yet was fully activated by Ca²⁺ (K_0.5 = 0.4 micromolar) in the absence of exogenous calmodulin, phosphatidylserine and 1,2-dioleylglycerol, oleic acid, and platelet activating factor. CK-15 was used to generate a site-specific antibody to nodulin-26. The antibody reacted with a protein in the symbiosome membrane with an apparent molecular mass of 27,000 daltons, consistent with the molecular mass predicted for nodulin-26 from the deduced amino acid sequence. A symbiosome membrane protein with an identical electrophoretic mobility was phosphorylated in vitro in a Ca²⁺-dependent manner. Additionally, this symbiosome membrane protein was phosphorylated when nodules were incubated with ³²P-phosphate. Overall, the results show the existence of a Ca²⁺-dependent and calmodulin/lipid-independent enzyme in nitrogen-fixing soybean root nodules and suggest that nodulin-26 is a substrate for Ca²⁺-dependent phosphorylation.     

Distinct transport selectivity of two structural subclasses of the nodulin-like intrinsic protein family of plant aquaglyceroporin channels

Major intrinsic proteins (MIPs) are a diverse class of integral membrane proteins that facilitate the transport of water and some small solutes across cellular membranes. X-ray structures of MIPs indicate that a tetrad of residues (the ar/R region) form a narrow pore constriction that constitutes the selectivity filter. In comparison with mammalian and microbial species, plants have a greater number and diversity of MIPs with greater than 30 genes encoding four phylogenetic subfamilies with eight different classes of ar/R sequences. The nodulin 26-like intrinsic protein (NIP) subfamily in Arabidopsis can be subdivided into two ar/R subgroups: the NIP subgroup I, which resembles the archetype of the family, soybean nodulin 26, and the NIP subgroup II, which is represented by the Arabidopsis protein AtNIP6;1. These two NIPs differ principally by the substitution of a conserved alanine (NIP subgroup II) for a conserved tryptophan (NIP subgroup I) in the helix 2 position (H2) of the ar/R filter. A comparison of the water and solute tranport properties of the two proteins was performed by expression in Xenopus laevis oocytes. Nodulin 26 is an aquaglyceroporin with a modest osmotic water permeability (P(f)) and the ability to transport uncharged solutes such as glycerol and formamide. In constrast, AtNIP6;1 showed no measurable water permeability but transported glycerol, formamide, as well as larger solutes that were impermeable to nodulin 26. By site-directed mutagenesis, we show that the H2 position is the crucial determinant that confers these transport behaviors. A comparison of the NIPs and tonoplast-intrinsic proteins (TIP) shows that the H2 residue can predict the transport profile for water and glycerol with histidine found in TIP-like aquaporins, tryptophan found in aquaglyceroporins (NIP I), and alanine found in water-impermeable glyceroporins (AtNIP6;1).     

Molecular events leading to enhanced glucose transport in Rous sarcoma virus-transformed cells

Transformation by Rous sarcoma virus results in a dramatic increase in the rate at which the transformed cells transport glucose across the cell membrane. The increased transport rate is a consequence of an increased number of transporters in the transformed cells. Utilizing antibody raised against the purified human erythrocyte glucose transporter, we have identified the glucose transporter as a membrane glycoprotein with a monomer Mr of approximately 41,000. The increased rate of glucose transport is dependent on the activity of pp60src, the transforming protein of Rous sarcoma virus. This protein has been shown to be a protein kinase that phosphorylates on tyrosine residues. We have examined the tyrosine phosphorylation of a major cellular protein of Mr 36,000 in cells infected with a panel of partially transforming mutants of Rous sarcoma virus. One of these mutants (CU2) increases the rate of glucose transport only slightly and does not render the infected cells fully anchorage independent or tumorigenic (although other transformation parameters are fully induced). Cells infected with this mutant display a 36,000-dalton protein that is phosphorylated to a considerably lesser extent than cells infected with wild-type virus. Analyses of this sort may help to identify the cellular targets of pp60src whose phosphorylation is necessary for the increased glucose transport rate.     

A Peanut Nodule Lectin in Infected Cells and in Vacuoles and the Extracellular Matrix of Nodule Parenchyma

Root nodules on peanut (Arachis hypogaea L.) accumulate a galactose/lactose-binding lectin that is similar, but not identical, to the major seed lectin in peanut. The function of the peanut nodule lectin (PNL) is not known. In the current study, we have investigated the location of lectin in the nodule using immunogold labeling and enzyme-linked immunosorbant assays (ELISA). Lectin was most abundant in the nodule parenchyma, where it accumulated in vacuoles, suggesting a possible role as a vegetative storage protein. Lectin was also detected in the extracellular matrix in the nodule parenchyma, a location that corresponds to the tissue layer forming a barrier to oxygen diffusion. The potential for interactions between PNL and other cell wall components, including a previously described high-molecular weight glycoprotein that co-localizes with PNL, is discussed. Within infected cells, lectin was not detectable by immunogold labeling within the cytoplasm, but light labeling was suggestive of lectin localization within the symbiosome lumen. Analysis of fractionated symbiosomes by the more sensitive ELISA technique confirmed that lectin was present within the symbiosome, but was not bound to bacteroids. Our results indicate that PNL probably plays several roles in this nitrogen-fixing symbiosis.     

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.     

GmN70 and LjN70. Anion transporters of the symbiosome membrane of nodules with a transport preference for nitrate

A cDNA was isolated from soybean (Glycine max) nodules that encodes a putative transporter (GmN70) of the major facilitator superfamily. GmN70 is expressed predominantly in mature nitrogen-fixing root nodules. By western-blot and immunocytochemical analyses, GmN70 was localized to the symbiosome membrane of infected root nodule cells, suggesting a transport role in symbiosis. To investigate its transport function, cRNA encoding GmN70 was expressed in Xenopus laevis oocytes, and two-electrode voltage clamp analysis was performed. Ooctyes expressing GmN70 showed outward currents that are carried by anions with a selectivity of nitrate > nitrite > > chloride. These currents showed little sensitivity to pH or the nature of the counter cation in the oocyte bath solution. One-half maximal currents were induced by nitrate concentrations between 1 to 3 mm. No apparent transport of organic anions was observed. Voltage clamp records of an ortholog of GmN70 from Lotus japonicus (LjN70; K. Szczyglowski, P. Kapranov, D. Hamburger, F.J. de Bruijn [1998] Plant Mol Biol 37: 651-661) also showed anion currents with a similar selectivity profile. Overall, these findings suggest that GmN70 and LjN70 are inorganic anion transporters of the symbiosome membrane with enhanced preference for nitrate. These transport activities may aid in regulation of ion and membrane potential homeostasis, possibly in response to external nitrate concentrations that are known to regulate the symbiosis.     

The integral membrane protein SEN1 is required for symbiotic nitrogen fixation in Lotus japonicus nodules

Legume plants establish a symbiotic association with bacteria called rhizobia, resulting in the formation of nitrogen-fixing root nodules. A Lotus japonicus symbiotic mutant, sen1, forms nodules that are infected by rhizobia but that do not fix nitrogen. Here, we report molecular identification of the causal gene, SEN1, by map-based cloning. The SEN1 gene encodes an integral membrane protein homologous to Glycine max nodulin-21, and also to CCC1, a vacuolar iron/manganese transporter of Saccharomyces cerevisiae, and VIT1, a vacuolar iron transporter of Arabidopsis thaliana. Expression of the SEN1 gene was detected exclusively in nodule-infected cells and increased during nodule development. Nif gene expression as well as the presence of nitrogenase proteins was detected in rhizobia from sen1 nodules, although the levels of expression were low compared with those from wild-type nodules. Microscopic observations revealed that symbiosome and/or bacteroid differentiation are impaired in the sen1 nodules even at a very early stage of nodule development. Phylogenetic analysis indicated that SEN1 belongs to a protein clade specific to legumes. These results indicate that SEN1 is essential for nitrogen fixation activity and symbiosome/bacteroid differentiation in legume nodules.     

The phosphorylation state and expression of soybean BiP isoforms are differentially regulated following abiotic stresses

The mammalian BiP is regulated by phosphorylation, and it is generally accepted that its unmodified form constitutes the biologically active species. In fact, the glycosylation inhibitor tunicamycin induces dephosphorylation of mammalian BiP. The stress-induced phosphorylation state of plant BiP has not been examined. Here, we demonstrated that soybean BiP exists in interconvertible phosphorylated and nonphosphorylated forms, and the equilibrium can be shift to either direction in response to different stimuli. In contrast to tunicamycin treatment, water stress condition stimulated phosphorylation of BiP species in soybean cultured cells and stressed leaves. Despite their phosphorylation state, we demonstrated that BiP isoforms from water-stressed leaves exhibit protein binding activity, suggesting that plant BiP functional regulation may differ from other eukaryotic BiPs. We also compared the induction of the soybean BiP gene family, which consists of at least four members designated soyBiPA, soyBiPB, soyBiPC, and soyBiPD, by tunicamycin and osmotic stress. Although all soybean BiP genes were induced by tunicamycin, just the soyBiPA RNA was up-regulated by osmotic stress. In addition, these stresses promoted BiP induction with different kinetics and acted synergistically to increase BiP accumulation. These results suggest that the soybean BiP gene family is differentially regulated by abiotic stresses through distinct signaling pathways.     

Soybean nodulin-26 gene encoding a channel protein is expressed only in the infected cells of nodules and is regulated differently in roots of homologous and heterologous plants.

Nodulin-26 (N-26) is a major peribacteroid membrane protein in soybean root nodules. The gene encoding this protein is a member of an ancient gene family conserved from bacteria to humans. N-26 is specifically expressed in root nodules, while its homolog, soybean putative channel protein, is expressed in vegetative parts of the plant, with its highest level in the root elongation zone. Analysis of the soybean N-26 gene showed that its four introns mark the boundaries between transmembrane domains and the surface peptides, suggesting that individual transmembrane domains encoded by a single exon act as functional units. The number and arrangement of introns between N-26 and its homologs differ, however. Promoter analysis of N-26 was conducted in both homologous and heterologous transgenic plants. The cis-acting elements of the N-26 gene are different from those of the other nodulin genes, and no nodule-specific cis-acting element was found in this gene. In transgenic nodules, the expression of N-26 was detected only in the infected cells; no activity was found in nodule parenchyma and uninfected cells of the symbiotic zone. The N-26 gene is expressed in root meristem of transgenic Lotus corniculatus and tobacco but not in untransformed and transgenic soybean roots, suggesting the possibility that this nodulin gene is controlled by a trans-negative regulatory mechanism in homologous plants. This study demonstrates how a preexisting gene in the root may have been recruited for symbiotic function and brought under nodule-specific developmental control.