Peribacteroid Membrane Research Articles

Endophytic colonisation of Bacillus subtilis in the roots of Robinia pseudoacacia L.

The endophytic colonisation of Bacillus subtilis strain GXJM08, isolated from roots of Podocarpus imbricatus B1. Enum. P1. Jav., in roots of the leguminous plant Robinia pseudoacacia L. was investigated. Ultrastructure observations showed that B.subtilis caused morphological changes in the root hair and colonised the plant through infected root hairs. The structure of the infection thread was similar to that of rhizobia, but the structure of infected cells was different. B.subtilis is also different from rhizobia and plant pathogens in terms of the formation of a peribacteroid membrane and the mode of penetration through the host cell wall. Our results provide a basis for studying development of the mutualistic symbiotic relationship between B.subtilis and plants, and a basis for studying the mechanism of the B.subtilis-plant interaction.

Hypoxic stress and the transport systems of the peribacteroid membrane of bean root nodules

When the roots of Vicia faba L. beans were subjected to hypoxic stress, the activity of H(+)-ATPase on the peribacteroid membrane, as well as the transport of dicarboxylates (malate and succinate) mediated by this enzyme, decreased. Since malate and succinate are the main carbon-containing metabolites involved in the energy supply to bacteroids, this caused a change of the relation type from mutualism to commensalism, and the domination of the eukaryote over the prokaryote consequently increased.

Lack of Galactose or Galacturonic Acid in Bradyrhizobium japonicum USDA 110 Exopolysaccharide Leads to Different Symbiotic Responses in Soybean

Exopolysaccharide (EPS) and lipopolysaccharide (LPS) from Bradyrhizobium japonicum are important for infection and nodulation of soybean (Glycine max), although their roles are not completely understood. To better understand this, we constructed mutants in B. japonicum USDA 110 impaired in galactose or galacturonic acid incorporation into the EPS without affecting the LPS. The derivative LP 3010 had a deletion of lspL-ugdH and produced EPS without galacturonic acid whereas LP 3013, with an insertion in exoB, produced EPS without galactose. In addition, the strain LP 3017, with both mutations, had EPS devoid of both galactosides. The missing galactosides were not replaced by other sugars. The defects in EPS had different consequences. LP 3010 formed biofilms and nodulated but was defective in competitiveness for nodulation; and, inside nodules, the peribacteroid membranes tended to fuse, leading to the merging of symbiosomes. Meanwhile, LP 3013 and LP 3017 were unable to form biofilms and produced empty pseudonodules but exoB suppressor mutants were obtained when LP 3013 plant inoculation was supplemented with wild-type EPS. Similar phenotypes were observed with all these mutants in G. soja. Therefore, the lack of each galactoside in the EPS has a different functional effect on the B. japonicum-soybean symbiosis.

The Signal Peptide of the Medicago truncatula Modular Nodulin MtNOD25 Operates as an Address Label for the Specific Targeting of Proteins to Nitrogen-Fixing Symbiosomes

The nodule-specific MtNOD25 gene of the model legume Medicago truncatula encodes a modular nodulin composed of different repetitive modules flanked by distinct N- and C-termini. Although similarities are low with respect to all repetitive modules, both the N-terminal signal peptide (SP) and the C-terminus are highly conserved in modular nodulins from different legumes. On the cellular level, MtNOD25 is only transcribed in the infected cells of root nodules, and this activation is mediated by a 299-bp minimal promoter containing an organ-specific element. By expressing mGFP6 translational fusions in transgenic nodules, we show that MtNOD25 proteins are exclusively translocated to the symbiosomes of infected cells. This specific targeting only requires an N-terminal MtNOD25 SP that is highly conserved across a family of legume-specific symbiosome proteins. Our finding sheds light on one possible mechanism for the delivery of host proteins to the symbiosomes of infected root nodule cells and, in addition, defines a short molecular address label of only 24 amino acids whose N-terminal presence is sufficient to translocate proteins across the peribacteroid membrane.

Aquaporins and Chloroplast Membrane Permeability

Aquaporins are intrinsic membrane proteins known to facilitate membrane water transport and are found in most living organisms (Agre, 2004). They comprise a large family in plants: Arabidopsis, rice, and maize each have ;35 different aquaporins. These initially were categorized into four subfamilies, largely based on apparent patterns of localization: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), Nodulin26-like intrinsic membrane proteins, present in the peribacteroid membrane ofsoybean rootnodules, and small basic intrinsic proteins, found in the endoplasmic reticulum. However, this classification is somewhat misleading, as PIP aquaporins have been identified in organellar membranes and some TIP aquaporins in the plasma membrane (reviewed in Kaldenhoff et al., 2007; Maurel, 2007). Recent work has suggested that, in addition to water movement, aquaporins might transport other physiologically important molecules across membranes, including CO2 ,H 2O2 ,N H 3/NH4 1 , boron, and silicon, and therefore may be involved in a number of fundamental processes in plants, such as nutrient acquistion, photosynthesis, and stress responses. However, measuring transport via aquaporin channels directly and teasing apart a direct role in transport of these molecules and possible indirect affects associated with water transport has proven to be exceptionally difficult. Uehlein et al. (pages 648–657) show that the aquaporin Nt AQP1 is localized to the inner chloroplast membrane and the plasma membrane in tobacco and present evidence that it facilitates transport of CO2 into the chloroplast and impacts CO2 fixation and photosynthesis. Subcellular localization of Nt AQP1 to the inner chloroplast membrane and plasmamembrane was determined by immunblot analysis, electron microscopy using immungold labeling, and fluorescence microscopy using a green fluorescent protein fusion. The authors investigated AQP1 function in membrane permeability to water and CO2using RNA interference

Developmentally regulated membrane glycoproteins sharing antigenicity with rhamnogalacturonan II are not detected in nodulated boron deficient Pisum sativum.

The peribacteroid membrane (PBM) of symbiosomes from pea root nodules developed in the presence of boron (+B) was labelled by anti-rhamnogalacturonan II (RGII) (anti-rhamnogalacturonan II pectin polysaccharide) antiserum. However, in nodules from plants grown at low boron (-B), anti-RGII pectin polysaccharide did not stain PBMs. Given that RGII pectin binds to borate, and that symbiosomes differentiate aberrantly in -B nodules because of abnormal vesicle traffic, anti-RGII pectin polysaccharide antigens were further analysed. Following electrophoresis and electroblotting, anti-RGII pectin polysaccharide immunostained three bands in +B but not in -B nodule-derived PBMs. A similar banding pattern was observed after the immunostaining of membrane fractions from uninfected roots, indicating that anti-RGII pectin polysaccharide antigens are common to both peribacteroid and plasma membranes. Protease treatment of samples led to disappearance of anti-RGII pectin polysaccharide labelling, indicating that the three immunostained bands correspond to proteins or glycoproteins. The immunochemical study of RGII antigen distribution during nodule development showed that it is strongly present on the PBM of dividing (undifferentiated) symbiosomes but progressively disappeared during symbiosome maturation. In B-deficient nodules, PBMs were never decorated with RGII antigens, and there was an abnormal targeting of vesicles containing pectic polysaccharide (homogalacturanan) to cell membranes. Overall, these results indicate that RGII, boron and certain membrane (glyco)-proteins may interact closely and function cooperatively in membrane processes associated with symbiosome division and general cell growth.

The effect of metabolites on the pH gradient and membrane potential of the bean peribacteroid membrane

The effects of malate, succinate, and glutamate on the kinetics of changes in the pH gradient (delta pH) and membrane potential (delta psi) on the peribacteroid membrane (PBM) of the symbiosomes of bean root nodules varying in age were recorded spectrophotometrically. Addition of all the tested metabolites to potassium-free incubation medium stimulated a passive acidification of the peribacteroid space (PBS) and dissipation of delta psi in PBM of young developing nodules in the presence of the K+/H+ antiporter nigericin in the medium. However, in mature nodules with a high nitrogen-fixing activity, only malate and succinate (but not glutamate) increased delta pH during both passive and ATP-dependent PBS acidification. Dicarboxylates also caused dissipation of both delta pH in the presence of nigericin in the medium and delta psi generated on PBM by H+-ATPase. A decrease in the effects of metabolites on delta pH and the absent activity of the PBM H+ pump were observed in the aging nodules. The obtained data on the changes in deltapH and dlta psi caused by the metabolites in question suggest that PBM is permeable for all these metabolites only in young nodules. Only malate and succinate (but not glutamate) are transported through PBM in mature nodules; and the rate of metabolite translocation through PBM in aging nodules is decreased.

Metabolite transport across the peribacteroid membrane during broad bean development

A temporal pattern of the peribacteroid membrane (PBM) transport function was studied. Spectrophotometric recording was used for establishing the effect of carbon-and nitrogen-containing substrates (malate, succinate, and glutamate) on the acidification of the peribacteroid space and the intensity of light scattering in the symbiosome suspension from broad bean (Vicia faba L.) root nodules of different age. At the early stages of nodule formation and functioning, PBM is permeable not only for malate and succinate, but also for glutamate, and this permeability fully provides for the active bacteroid division and the nitrogenase complex synthesis in the bacteroids at the expense of the carbon-and nitrogen-containing substrates. Mature nodules are characterized by the greatest nitrogen-fixing activity. In these nodules, PBM is selectively permeable for malate and succinate, but constitutes a barrier for glutamate. Thereby, mutually beneficial relations between the symbiotic partners are achieved. In senescent nodules, a rearrangement of symbiotic interactions is directed toward a minimization of both carbon and nitrogen metabolite consumption by the bacteroids. It is concluded that, in the course of the development of the legume-rhizobia symbiosis, the PBM transport function is changed. This function determines a qualitatively different pattern of symbiotic partner interactions in the following sequence: parasitism-mutualism-commensalism.

Symbiotic nitrogen fixation in legume nodules: process and signaling. A review

The Green Revolution was accompanied by a huge increase in the application of fertilizers, particularly nitrogen. Recent studies indicate that a sizeable proportion of the human population depends on synthetic nitrogen (N) fertilizers to provide the 53 million t N that is harvested globally in food crops each year. Nitrogen fertilizers affect the balance of the global nitrogen cycle, pollute groundwater and increase atmospheric nitrous oxide (N2O), a potent “greenhouse” gas. The production of nitrogen fertilizer by industrial nitrogen fixation not only depletes our finite reserves of fossil fuels, but also generates large quantities of carbon dioxide, contributing to global warming. The process of biological nitrogen fixation offers an economically attractive and ecologically sound means of reducing external nitrogen input and improving the quality and quantity of internal resources. Recent studies show that in irrigated cropping systems, legume N is generally less susceptible to loss processes than fertilizers. Biological nitrogen fixation (BNF) has provided a number of useful paradigms for both basic and applied research. Establishing a fully functional symbiosis requires a successful completion of numerous steps that lead from recognition signals exchanged between the plant and bacteria to the differentiation and operation of root nodules, the plant organ in which nitrogen fixation takes place. The initial sensing of the two organisms by each other starts with the release of root exudates by the plant that include flavonoids and nutrients such as organic acids and amino acids. Flavonoids secreted by the host plant into the rhizosphere function as inducers of the rhizobial nod genes. nod gene induction results in the secretion of lipochitin oligosaccharides that are thought to bind to specific plant receptor kinases that contain LysM motifs, such as NFR1 and NFR5 in Lotus japonicus and LYK3 and LTK4 in Medicago truncatula. This initiates a complex signaling pathway involving calcium spiking in root hairs. The result is that the root hairs curl and trap the rhizobia, which then enter the root hair through tubular structures known as infection threads that are formed by the plant. The infection threads then grow into the developed nodule tissue. Ultimately, the invading bacteria are taken into the plant cell by a type of endocytosis in which they are surrounded by a plant-derived peribacteroid membrane (PBM). The resulting symbiosomes fill the plant cell cytoplasm and as plant and bacterial metabolism develops, the bacteria become mature bacteroids able to convert atmospheric nitrogen to ammonium. To increase knowledge of this system of particular importance in sustainable agriculture, major emphasis should be laid on the basic research. More work is needed on the genes responsible in rhizobia and legumes, the structural chemical bases of rhizobia/legume communication, and signal transduction pathways responsible for the finely orchestrated induction of the symbiosis-specific genes involved in nodule development and nitrogen fixation. This review unfolds the various events involved in the progression of symbiosis.

Alterations induced by glyphosate on lupin photosynthetic apparatus and nodule ultrastructure and some oxygen diffusion related proteins

The effects of glyphosate on protein metabolism, mesophyll cell ultrastructure and nodule ultrastructure and functioning of Lupinus albus cv. Multolupa inoculated with Bradyrhizobium sp. ( Lupinus) were investigated. Young leaves and nodules were especially affected because these organs act as sinks of the herbicide. The alterations on nodular and chloroplast ultrastructure varied depending on herbicide concentration and time of exposure. After 3 days of 2.5 mM glyphosate application some toxic effects were detected. The most important alterations on nodules were the progressive cellular degradation of plant and bacteroidal cytosol and the rupture of bacteroidal membrane, whilst the peribacteroid membrane of the symbiosomes was preserved. This is the first report on the effect of glyphosate on legume-nodule ultrastructure. Glyphosate inhibited B. sp. ( Lupinus) growth at concentrations higher than 62.5 μM. In the mesophyll cells, gradual disorganization of grana and intergrana was observed, loosing the parallel alignment with the chloroplast axis. As in nodules, degradation of membrane systems was observed, with the deformation, and even the rupture, of the tonoplast. These progressive effects were similar to those described in senescence processes. The adverse effects produced on infected zone can be due both to a direct effect of the herbicide on microsymbiont and to an indirect effect of glyphosate action on photosynthetic apparatus. Glyphosate produced changes in nodule cytosol and bacteroid proteins content and polypeptide pattern of leaves and nodules. With respect to proteins related to the oxygen diffusion mechanism, a large decrease in leghemoglobin and glycoproteins (recognized by antibodies MAC236 and MAC265) content was detected, which suggests that the oxygen diffusion mechanisms were also affected by glyphosate.

NH4 + Currents across the Peribacteroid Membrane of Soybean. Macroscopic and Microscopic Properties, Inhibition by Mg2+, and Temperature Dependence Indicate a SubpicoSiemens Channel Finely Regulated by Divalent Cations

The control of ammonium (NH(4)(+)) transport is critical in preventing futile cycles of NH(4)(+)/ammonia transport. An unusual nonselective cation channel with subpicoSiemens single-channel conductance permeable to NH(4)(+) had previously been identified in the peribacteroid membrane (PBM) of symbiosomes from soybean (Glycine max) nodules. Here, we investigate the proposed channel mechanism and its control by luminal magnesium. Currents carried by NH(4)(+) were measured in inside-out PBM patches by patch clamp. NH(4)(+) transport corresponding to the physiological direction of net transfer showed time-dependent activation and associated single-channel-like events. These could not be resolved to discrete conductances but had the same selectivity as the total current. The voltage dependence of the steady-state current was affected by temperature consistent with the rate constant of channel opening being reduced with decreased temperature. This resulted in steady-state currents that were more temperature sensitive at voltages where the current was only partially activated. When fully activated, the current reflected more the ion conduction through open channels and had an activation energy of 28.2 kJ mol(-1) (Q10 = 1.51, 8 degrees C-24 degrees C). Increased Mg(2+) on the symbiosome lumen side blocked the current (ID(50) = 351 microm, with 60 mm NH(4)(+)). Complete inhibition with 2 mm Mg(2+) was relieved with a small increase in NH(4)(+) on the lumen side of the membrane (shift of 60-70 mm). With Mg(2+) the selectivity of the transport for divalent cations increased. From these features, we propose a divalent-dependent feedback regulation of the PBM-nonselective cation channel that could maintain a constant NH(4)(+) gradient across the membrane.

Structural Characterization of a Flavonoid-Inducible Pseudomonas aeruginosa A-Band-Like O Antigen of Rhizobium sp. Strain NGR234, Required for the Formation of Nitrogen-Fixing Nodules

Rhizobium (Sinorhizobium) sp. strain NGR234 contains three replicons, the smallest of which (pNGR234a) carries most symbiotic genes, including those required for nodulation and lipo-chito-oligosaccharide (Nod factor) biosynthesis. Activation of nod gene expression depends on plant-derived flavonoids, NodD transcriptional activators, and nod box promoter elements. Nod boxes NB6 and NB7 delimit six different types of genes, one of which (fixF) is essential for the formation of effective nodules on Vigna unguiculata. In vegetative culture, wild-type NGR234 produces a distinct, flavonoid-inducible lipopolysaccharide (LPS) that is not produced by the mutant (NGRomegafixF); this LPS is also found in nitrogen-fixing bacteroids isolated from V. unguiculata infected with NGR234. Electron microscopy showed that peribacteroid membrane formation is perturbed in nodule cells infected by the fixF mutant. LPSs were purified from free-living NGR234 cultured in the presence of apigenin. Structural analyses showed that the polysaccharide portions of these LPSs are specialized, rhamnose-containing O antigens attached to a modified core-lipid A carrier. The primary sequence of the O antigen is [-3)-alpha-L-Rhap-(1,3)-alpha-L-Rhap-(1,2)-alpha-L-Rhap-(1-]n, and the LPS core region lacks the acidic sugars commonly associated with the antigenic outer core of LPS from noninduced cells. This rhamnan O antigen, which is absent from noninduced cells, has the same primary sequence as the A-band O antigen of Pseudomonas aeruginosa, except that it is composed of L-rhamnose rather than the D-rhamnose characteristic of the latter. It is noteworthy that A-band LPS is selectively maintained on the P. aeruginosa cell surface during chronic cystic fibrosis lung infection, where it is associated with an increased duration of infection.

Passive potassium transport by symbiosomes from broad bean root nodules

Passive potassium release from symbiosomes isolated from broad bean root nodules and placed into K +-free assay medium was investigated by following acidification of the symbiosome interior and tetraphenylphosphonium (TPP +)-induced change in turbidity of symbiosome suspension observed under the chosen experimental conditions. The kinetics of acid pH shift inside the symbiosomes was substantially accelerated by the K +-specific ionophore valinomycin, the protonophore carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP), but rapidly reversed by centimolar concentrations of K + as well as by TPP + added to the assay medium. In addition, the acidification process was shown to be markedly inhibited by tetraethylammonium (TEA), the known blocker of K +-channels in cell membranes, and to be strongly suppressed by removal of free calcium ions from the assay medium with the Ca 2+ chelator EGTA. TPP + was found to rapidly trigger the increase in turbidity of symbiosome suspension in the same assay medium. The kinetics of the TPP +-triggered process appeared to be markedly inhibited by TEA as well. Taken together, these results indicate relatively high potassium permeability of the peribacteroid membrane (PBM) of broad bean root nodules and provide evidence that the responses tested are due to K + release from the symbiosomes through TEA-sensitive K +-permeable channel in the PBM down transmembrane electrochemical gradient of K + ions. The data obtained suggest that such a channel is voltage-gated, and Ca 2+ ions on the cytosolic side of the PBM are required for functioning of this transporter.

The Galactolipid Digalactosyldiacylglycerol Accumulates in the Peribacteroid Membrane of Nitrogen-fixing Nodules of Soybean and Lotus

The peribacteroid membrane (PBM) surrounding nitrogen fixing rhizobia in the nodules of legumes is crucial for the exchange of ammonium and nutrients between the bacteria and the host cell. Digalactosyldiacylglycerol (DGDG), a galactolipid abundant in chloroplasts, was detected in the PBM of soybean (Glycine max) and Lotus japonicus. Analyses of membrane marker proteins and of fatty acid composition confirmed that DGDG represents an authentic PBM lipid of plant origin and is not derived from the bacteria or from plastid contamination. In Arabidopsis, DGDG is known to accumulate in extraplastidic membranes during phosphate deprivation. However, the presence of DGDG in soybean PBM was not restricted to phosphate limiting conditions. Complementary DNA sequences corresponding to the two DGDG synthases, DGD1 and DGD2 from Arabidopsis, were isolated from soybean and Lotus. The two genes were expressed during later stages of nodule development in infected cells and in cortical tissue. Because nodule development depends on the presence of high amounts of phosphate in the growth medium, the accumulation of the non-phosphorus galactolipid DGDG in the PBM might be important to save phosphate for other essential processes, i.e. nucleic acid synthesis in bacteroids and host cells.

Symbiotic relationships and root nodule ultrastructure of the pasture legume Biserrula pelecinus L.—a new legume in agriculture

Biserrula pelecinus is a pasture legume species new to Australian agriculture. The potential N benefit from B. pelecinus pastures in agricultural systems may not be realised if its symbiotic interactions with Mesorhizobium spp. are not well understood. This study evaluated the symbiotic interactions of four strains of Biserrula root-nodule bacteria (WSM1271, WSM1283, WSM1284, WSM1497) with four genotypes of B. pelecinus (cv. Casbah, 93GRC4, 93ITA33, IFBI1) and with a range of related legumes, including species known to be nodulated by strains of Mesorhizobium loti and other Mesorhizobium spp. Structures of root nodules were studied using light and electron microscopy enabling the ultrastructure of effective and ineffective nodules to be compared. B. pelecinus always formed typical indeterminate, finger-like nodules. The number of bacteroids inside symbiosomes varied between host×strain combinations, however, nodules formed by ineffective associations had well developed peribacteroid membranes and abundant bacteroids. Considerable variation was found in N 2-fixing effectiveness of strains isolated from B. pelecinus on the four B. pelecinus genotypes. Strains WSM1271, WSM1284 and WSM1497 nodulated Astragalus membranaceus, only strains WSM1284 and WSM1497 nodulated Astragalus adsurgens. Strain WSM1284 also nodulated Dorycnium rectum, Dorycnium hirsutum, Glycyrrhiza uralensis, Leucaena leucocephala, Lotus edulis, Lotus glaber, Lotus maroccanus, Lotus ornithopodioides, Lotus pedunculatus, Lotus peregrinus, Lotus subbiflorus and Ornithopus sativus. The four strains from B. pelecinus did not nodulate Amorpha fruticosa, Astragalus sinicus, Cicer arietinum, Hedysarum spinosissimum, Lotus parviflorus, Macroptilium atropurpureum or Trifolium lupinaster. M. loti strain SU343 nodulated all four genotypes of B. pelecinus. However, M. loti strain CC829 only nodulated B. pelecinus genotypes 93ITA33 and IFBI1 and the nodules were ineffective. The root nodule isolates from H. spinosissimum (E13 and H4) nodulated B. pelecinus cv. Casbah whereas the commercial inoculant strain for Cicer (CC1192) could not nodulate any genotype of B. pelecinus. These results indicate that strains WSM1271, WSM1283 and WSM1497 isolated originally from B. pelecinus have a specific host range while strain WSM1284 is promiscuous in its capacity to nodulate with a broad range of related species. As B. pelecinus can be nodulated by Mesorhizobium spp. from other agricultural legumes, particularly Lotus, there is an opportunity to utilise this trait in cultivar development.

Symbiotic relationships and root nodule ultrastructure of the pasture legume Biserrula pelecinus L.?a new legume in agriculture

Biserrula pelecinus is a pasture legume species new to Australian agriculture. The potential N benefit from B. pelecinus pastures in agricultural systems may not be realised if its symbiotic interactions with Mesorhizobium spp. are not well understood. This study evaluated the symbiotic interactions of four strains of Biserrula root-nodule bacteria (WSM1271, WSM1283, WSM1284, WSM1497) with four genotypes of B. pelecinus (cv. Casbah, 93GRC4, 93ITA33, IFBI1) and with a range of related legumes, including species known to be nodulated by strains of Mesorhizobium loti and other Mesorhizobium spp. Structures of root nodules were studied using light and electron microscopy enabling the ultrastructure of effective and ineffective nodules to be compared. B. pelecinus always formed typical indeterminate, finger-like nodules. The number of bacteroids inside symbiosomes varied between host×strain combinations, however, nodules formed by ineffective associations had well developed peribacteroid membranes and abundant bacteroids. Considerable variation was found in N2-fixing effectiveness of strains isolated from B. pelecinus on the four B. pelecinus genotypes. Strains WSM1271, WSM1284 and WSM1497 nodulated Astragalus membranaceus, only strains WSM1284 and WSM1497 nodulated Astragalus adsurgens. Strain WSM1284 also nodulated Dorycnium rectum, Dorycnium hirsutum, Glycyrrhiza uralensis, Leucaena leucocephala, Lotus edulis, Lotus glaber, Lotus maroccanus, Lotus ornithopodioides, Lotus pedunculatus, Lotus peregrinus, Lotus subbiflorus and Ornithopus sativus. The four strains from B. pelecinus did not nodulate Amorpha fruticosa, Astragalus sinicus, Cicer arietinum, Hedysarum spinosissimum, Lotus parviflorus, Macroptilium atropurpureum or Trifolium lupinaster. M. loti strain SU343 nodulated all four genotypes of B. pelecinus. However, M. loti strain CC829 only nodulated B. pelecinus genotypes 93ITA33 and IFBI1 and the nodules were ineffective. The root nodule isolates from H. spinosissimum (E13 and H4) nodulated B. pelecinus cv. Casbah whereas the commercial inoculant strain for Cicer (CC1192) could not nodulate any genotype of B. pelecinus. These results indicate that strains WSM1271, WSM1283 and WSM1497 isolated originally from B. pelecinus have a specific host range while strain WSM1284 is promiscuous in its capacity to nodulate with a broad range of related species. As B. pelecinus can be nodulated by Mesorhizobium spp. from other agricultural legumes, particularly Lotus, there is an opportunity to utilise this trait in cultivar development.

Calcium in the Control of Nitrogenase Activity in Vicia faba L. Root Nodules: Calcium Release from Symbiosomes and the Accompanying Decrease in Their Nitrogenase Activity Is Blocked by Verapamil

Treatment of root nodules or symbiosomes isolated from them with calcium chelator EGTA alone or with calcium ionophore A23187 for 3 h under microaerophilic conditions considerably decreased their nitrogenase activity (NA). Under these experimental conditions, cytochemical electron microscopy demonstrates a considerable calcium depletion in symbiosomes of the infected nodule cells by EGTA and A23187. Ca2+ channel blockers, verapamil and ruthenium red, inhibited EGTA-induced Ca2+ release from symbiosomes. In this case, NA insignificantly increased in the whole nodules and reached the baseline in symbiosomes. The experiments on isolated symbiosomes and arsenazo III demonstrated that verapamil inhibited Ca2+ transport induced by valinomycin at the background of K+ ions. These data suggest the presence of a verapamil-sensitive transporter on the peribacteroid membrane responsible for Ca2+ release from symbiosomes. A possible role of this transporter in the interaction between symbiotic partners in the infected cells of the root nodules is discussed.

Cell surface interactions of Rhizobium bacteroids and other bacterial strains with symbiosomal and peribacteroid membrane components from pea nodules.

Samples of Rhizobium bacteroids isolated from pea nodule symbiosomes reacted positively with a monoclonal antibody recognizing N-linked glycan epitopes on plant glycoproteins associated with the peribacteroid membrane and peribacteroid fluid. An antiserum recognizing the symbiosomal lectin-like glycoprotein PsNLEC-1 also reacted positively. Samples of isolated bacteroids also reacted with an antibody recognizing a glycolipid component of the peribacteroid membrane and plasma membrane. Bacterial cells derived from free-living cultures then were immobilized on nitrocellulose sheets and tested for their ability to associate with components of plant extracts derived from nodule fractionation. A positive antibody-staining reaction indicated that both PsNLEC-1 and membrane glycolipid had become associated with the bacterial surface. A range of rhizobial strains with mutants affecting cell surface polysaccharides all showed similar interactions with PsNLEC-1 and associated plant membranes, with the exception of strain B659 (a deep-rough lipopolysaccharide mutant of Rhizobium leguminosarum). However, the presence of a capsule of extracellular polysaccharide apparently prevented interactions between rhizobial cells and these plant components. The importance of a close association between peribacteroid membranes, PsNLEC-1, and the bacterial surface is discussed in the context of symbiosome development.

Soybean ( Glycine max. L.) and bacteroid glyoxylate cycle activities during nodular senescence

Soybean (Glycine max. L.) nodular senescence results in the dismantling of the peribacteroid membrane (PBM) and in an increase of soybean isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (MS; EC 4.1.3.2) mRNA and protein levels. This suggests that in senescing soybean nodular cells, the specific glyoxylate cycle enzyme activities might be induced to reallocate carbon obtained from the PBM degradation. In order to evaluate as well the carbon metabolism of the nitrogen-fixing Bradyrhizobium japonicum endosymbiotic bacteroids during nodular senescence, their glyoxylate cycle activities were also investigated. To this end, partial DNA sequences were isolated from their icl and ms genes, but the corresponding mRNAs were not detected in the microorganisms. It was also observed that the bacteroid ICL and MS activities were negligible during nodular senescence. This suggests that glyoxylate cycle activities are not reinitiated in the bacteroids under these physiological conditions. In case the microorganisms nevertheless feed on the PBM degradation products, this might occur via the citric acid cycle exclusively.

The soybean NRAMP homologue, GmDMT1, is a symbiotic divalent metal transporter capable of ferrous iron transport

Iron is an important nutrient in N2-fixing 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 N2-fixing 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 identified 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 fixation in developing nodules. Antibodies raised against a partial fragment of GmDmt1 confirmed its presence on the peribacteroid membrane (PBM) of soybean root nodules. GmDmt1 was able to both rescue growth and enhance 55Fe(II) uptake in the ferrous iron transport deficient 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 fixation is discussed.