Peribacteroid Space Research Articles

Genome-wide identification of nodule-specific transcripts in the model legume Medicago truncatula.

The Medicago truncatula expressed sequence tag (EST) database (Gene Index) contains over 140,000 sequences from 30 cDNA libraries. This resource offers the possibility of identifying previously uncharacterized genes and assessing the frequency and tissue specificity of their expression in silico. Because M. truncatula forms symbiotic root nodules, unlike Arabidopsis, this is a particularly important approach in investigating genes specific to nodule development and function in legumes. Our analyses have revealed 340 putative gene products, or tentative consensus sequences (TCs), expressed solely in root nodules. These TCs were represented by two to 379 ESTs. Of these TCs, 3% appear to encode novel proteins, 57% encode proteins with a weak similarity to the GenBank accessions, and 40% encode proteins with strong similarity to the known proteins. Nodule-specific TCs were grouped into nine categories based on the predicted function of their protein products. Besides previously characterized nodulins, other examples of highly abundant nodule-specific transcripts include plantacyanin, agglutinin, embryo-specific protein, and purine permease. Six nodule-specific TCs encode calmodulin-like proteins that possess a unique cleavable transit sequence potentially targeting the protein into the peribacteroid space. Surprisingly, 114 nodule-specific TCs encode small Cys cluster proteins with a cleavable transit peptide. To determine the validity of the in silico analysis, expression of 91 putative nodule-specific TCs was analyzed by macroarray and RNA-blot hybridizations. Nodule-enhanced expression was confirmed experimentally for the TCs composed of five or more ESTs, whereas the results for those TCs containing fewer ESTs were variable.

Rhizobium colonization induced changes in membrane-bound and soluble hydroxyproline-rich glycoprotein composition in pea.

Abundance and distribution of plant cell surface proteins of the hydroxyproline-rich glycoprotein (HRGP) class were studied in the pea-Rhizobium symbiosis using immunoblot analysis. The MAC 265-epitope was especially abundant in pea root nodules containing nitrogen-fixing Rhizobium bacteria. A 180-kDa MAC 265-HRGP dominated in pea shoot plasma membranes, while almost no MAC 265-HRGP was detected in root plasma membranes. We show here that a major difference between the plant-derived peribacteroid membrane of the symbiosomes and the root plasma membrane was the presence of a 100-kDa MAC 265-HRGP in the former. Arabinogalactan proteins (AGPs), as recognized by the monoclonal antibodies MAC 207 and JIM 8, were not detected in the peribacteroid membrane, while two isoforms (100 and 220 kDa) were detected in shoot and root plasma membranes. Specific MAC 265-HRGP isoforms were found in the peribacteroid space fraction of the symbiosomes and thus as soluble proteins in the interface between the symbionts. The abundance of the MAC 265-epitope was much reduced in non-nitrogen-fixing nodules when this phenotype resulted from a dicarboxylate transport mutation in Rhizobium. There was no reduction in the abundance of the MAC 265-epitope in non-fixing phenotypes resulting from a mutation in the plant. The results suggest that bacterial signals related to the bacterial ability to fix nitrogen, might be responsible for the regulation of HRGP expression in root nodules.

Characterisation by proteomics of peribacteroid space and peribacteroid membrane preparations from pea (Pisum sativum) symbiosomes.

The legume Rhizobium symbiosis leads to the formation of a new compartment in the plant cell, the symbiosome. This compartment harbours the bacteroids surrounded by a peribacteroid membrane (PBM) originating from the plant plasma membrane. The PBM and the space between the PBM and the bacteroid membrane, called peribacteroid space (PS), mediate the exchange of metabolites between the symbionts. Proteome analysis was used as an approach to characterise the proteins in the PBM and the PS. A standard differential centrifugation procedure including a Percoll gradient was used for symbiosome isolation from pea root nodules. Proteins in the PBM and PS fractions obtained from the symbiosomes were separated by two-dimensional gel electrophoresis, and 89 spots were analysed by tandem mass spectrometry. The proteins of 46 spots could be identified by database search. The results showed that PS and even PBM preparations from pea symbiosomes always contain abundant amounts of bacteroid proteins as a contaminate. Interestingly, in addition to a few PS/PBM proteins a number of endomembrane proteins (less likely representing a contaminate), including V-ATPase, BIP, and an integral membrane protein known from COPI-coated vesicles, were found in the PBM fraction, supporting the role of the endomembrane system in PBM biogenesis.

Subcellular organization of N2-fixing nodules of cowpea (Vigna unguiculata) supplied with silicon.

Provision of silicon (0, 0.048, 0.096, 0.24, 0.48, and 0.96 g/l) in the form of silicic acid (H4SiO4) to nodulated cowpea plants (Vignia unguiculata [L.] Walp.) grown in liquid culture resulted in considerable changes in the internal organization of nodule structure. Compared to the control plants which received no added silicate, bacteroid numbers increased significantly (P < or = 0.05) at silicate concentrations of both 0.096 and 0.48 g/l. The number of symbiosomes also increased by 3.2-fold at the silicate concentration of 0.96 g/l compared to the control. In contrast, the size of bacteroids and symbiosomes decreased significantly (P < 0.05) inside nodules of silicate-treated plants. The peribacteroid space was also decreased considerably (P < 0.05) with the application of 0.096 and 0.96 g of silicate per liter to plants. However, the size of intercellular spaces adjacent to infected and uninfected interstitial cells within the nodule medulla increased significantly (P < or = 0.05) at 0.096 g of silicate per liter followed by a sharply marked (P < or = 0.05) decrease with each subsequent increase in silicate application. The result was a large decrease (P < 0.05) in the area of bacteria-infected tissue occupied by intercellular space at the highest silicate concentration, which was caused by a significant (P < or = 0.05) increase in cell wall thickness. Our findings show that the positive effects of silicon on N2 fixation might actually be due to an increased number of bacteroids and symbiosomes.

Novel ultrastructural observations of pea (Pisum sativum) root nodule cells by high-pressure freezing and propane-jet freezing techniques

Pea (Pisum sativum) root nodule cells infected by the diazotrophRhizobium leguminosarum have been well characterized by chemical fixation techniques. Propane-jet freezing and high pressure freezing were used in this study to compare rapidly frozen and chemically fixed pea root nodule cells. Cells that had been incubated in 2-(N-morpholino)ethanesulfonic acid buffer and frozen with the propane-jet freezer were better preserved than cells that had been chemically fixed or frozen with the high-pressure freezer. Rapidly frozen infected nodule cells showed that the rough endoplasmic reticulum had a high frequency of associations with the peribacteroid membrane and the infection thread. The peribacteroid space also varied in size depending on the method of preservation; however, it was most reduced in size and devoid of inclusions in the propane-jet frozen tissue. The biological significance of these observations is discussed.

Origin of de Novo Synthesized Proteins in the Different Compartments of Pea-Rhizobium sp. Symbiosomes

The origin of de novo synthesized proteins in the interface between pea and a Rhizobium sp. in root nodules has been determined. The symbiotic interface is defined as the peribacteroid space including proteins associated with either the symbiosome membrane or the bacteroid outer membrane. Two approaches have been used to study the origin of proteins in the symbiotic interface. First, to determine the localization of de novo synthesized plant-produced proteins in the symbiosomes, an in vitro protein translocation assay was established. To produce plant proteins poly A+ RNA was isolated from root nodules followed by in vitro translation in the presence of [35S]methionine. Subsequently, purified symbiosomes were incubated with the [35S]methionine-labeled plant proteins. The symbiosomes were subfractionated and de novo synthesized plant proteins in the different fractions were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. These import studies demonstrate the presence of plant proteins in the symbiosome membrane, peribacteroid space, and bacteroid membrane pellet. In contrast, no proteins of plant origin were detected in the bacteroid cytosol. Second, the presence of de novo synthesized bacteroid proteins in the interface was examined by incubation of symbiosomes isolated under micro-aerobic or aerobic conditions with [35S]methionine. Analysis of symbiosome compartments by SDS-PAGE and phosphor image revealed that proteins of bacteroid origin are primarily detected in the bacteroid cytosol and bacteroid membrane pellet. However, a few bacteroid-produced proteins are also observed in the symbiosome membrane. Together these data demonstrate that the majority of de novo synthesized proteins in the pea-Rhizobium sp. symbiotic interface are of plant origin.

Oxidative stress occurs during soybean nodule senescence

Several markers of oxidative stress were measured in 2- to 10-week-old soybean (Glycine max [L.] Merr.) nodules. There were increases in peroxides, protein carbonyls and modified DNA base concentrations with nodule age. The catalytic iron content also increased significantly during nodule ageing. Iron contained in the peribacteroid space was effective in promoting lipid peroxidation and this might contribute to the degradation of the peribacteroid membrane in senescing nodules. The concentration of the oxidized forms of glutathione and homoglutathione increased significantly during nodule development and the concentration of reduced glutathione and homoglutathione decreased during senescence. Taken together, these results are consistent with the development of oxidative stress in senescing nodules. Significant DNA and protein damage also occurred in the first days of nodule development, suggesting that an earlier period of oxidative stress might occur in the period over which the symbiosis becomes established.

Proline accumulation inside symbiosomes of faba bean nodules under salt stress

A two‐week salt treatment (NaCl, 100 mM) induced a 50% inhibition of acetylene reduction activity (ARA) of faba bean (Vicia faba L. var. minor cv. Soravi) nodules, associated with a large increase in the nodule pool of amino acids. The concentration of proline in the different nodule compartments was determined after calculating their respective volumes from their areas on electron micrographs. The proline concentration exhibited a large increase, especially in the cytosol where its amount was 8‐fold enhanced under salt stress, whereas the low proline content of bacteroids was less affected. Increase of proline concentration in faba bean nodules subjected to salt stress was correlated with an enhancement of the cytosolic Δ1‐pyrroline‐5‐carboxylate synthetase (EC 2.7.2.11 + EC 1.2.1.41; P5CS) activity. Experiments with purified symbiosome preparations showed that the greatest proline content occurred in the peribacteroid space (PBS), where proline was the most abundant amino acid, with a concentration reaching 15.3 mM under salt stress. Proline accumulation in the PBS resulted both from a diffusive transport from the host cell to the symbiosomes through the peribacteroid membrane (PBM) and from the very low rate of uptake by faba bean bacteroids. This accumulation could be partly responsible for the 1.7‐fold enlargement of the symbiosome volume observed in salt‐stressed nodules. In incubations of bacteroids, isolated from salt‐stressed or unstressed plants and supplied with O2 by purified oxyleghemoglobin, addition of proline stimulated neither O2 consumption nor ARA. These results were consistent with proline playing a role as osmoticum, rather than energy source for bacteroid N2 fixation in amide‐exporting legumes such as faba bean.

Effect of elevated calcium concentration in infected cells of soybean (Glycine max (L.) Merr.) nodules on nitrogenase activity and N input to the plant

Recent evidence from other workers indicates that NH4+ transport through the symbiosome membrane of soybean (Glycine max (L.) Merr.) nodules may occur via an ion channel; the transport channel was reported to be inhibited by very low concentrations of Ca. Two approaches were used to assess the Ca-sensitivity of N input from nodules; first, by providing up to 12-fold excess Ca in nutrient solutions. Second, strains of Bradyrhizobium japonicum that synthesize or do not synthesize an anionic polysaccharide in the peribacteroid space were compared; the rationale here was that the polysaccharide may bind Ca in quantities sufficient to avoid Ca inhibition of N transport. After 7-9 weeks of plant growth under the nutrient treatments, plant growth, mineral content, and N variables were measured; results from two similar experiments are reported. The concentration of Ca (and other elements) was estimated in situ in infected cells of nodules by using Energy Dispersive X-ray Analysis. The 12-fold increase in Ca supply led to increases in [Ca] in the infected cells of 2- to 3-fold. Increases in [Ca] were correlated with a decline in [K] in nodules containing polysaccharide. Elevation of [Ca] in infected cells depressed N content of shoots by 17–30% depending on the rhizobial strain and experiment. Effects of Ca on acetylene reduction activity was not always statistically significant, but the estimates of nitrogenase activity were consistent with the results for N content of shoots. The overall results support the idea that N transport from bacteroids is sensitive to Ca concentration in the peribacteroid space. There was no significant difference between bacterial strains, indicating that the polysaccharide deposited in the peribacteroid space does not enhance N transport by binding Ca when Ca concentration is high.

Symbiotic properties of antibiotic-resistant mutants of Rhizobium galegae

Mutagenesis provoked by exposure to increased concentration of antibiotics of five indigenous Rhizobium galegae strains resulted in the generation of several antibiotic-resistant mutants. The mutants differed from the wild type and one from another in respect to the nodulation capacity, the nitrogenase activity, the nodule ultrastructure, and the plant growth response. Galega plants inoculated with mutants resistant to streptomycin and rifampicin formed nodules with higher nitrogenase activity and accumulated more shoot dry biomass than plants inoculated with the parent strains. Resistance to kanamycin and nalidixic acid was associated with significant decrease of nitrogenase activity. A correlation between nitrogen-fixing efficiency and nodule infected cell ultrastructure was found. When the bacteroids occupied about 10 times higher area in infected cells of nodule than peribacteroid spaces and host cytosol had electron dense and homogenous structure, the nitrogenase activity was the highest.

Comparative study of two cryotechniques to elucidate real functional aspects of legume nodules development

Legume nodules from Lupinus albus L. cv. Multolupa and Phaseolus vulgaris L. cv. Tormesina plants were subjected to two different cryotechniques (High Pressure Freezing, HPF, and Fast Freeze Fixation, FFF) in order to compare the structure obtained with respect to previous features and to deepen the relationships between the microsymbiont and the host cell in the native state. Comparing both cryotechniques, HPF yielded better general nodule ultrastructure than FFF. HPF allowed the observation of the native nodule structure since it permitted an excellent preservation. The result obtained showed that the peribacteroidal membrane was in close contact with the bacteroids, which is in contrast with the observations after chemical fixation. The narrow peribacteroid space observed between bacteroid and peribacteroid membranes appears as a dense matrix which is in agreement with the former knowledge on the existence of some enzymes and proteins in this space. O 2 -released by leghemoglobin will occur in close proximity to the bacteroid membranes. The spatial relationship between Golgi apparatus and ER with the peribacteroidal membrane observed along the development of the nodule indicates that these structures are very much related with the formation of the symbiosome membrane. HPF could be the right technique to continue studying real functional aspects in developing nodules.

Iron Uptake by Symbiosomes from Soybean Root Nodules.

To identify possible iron sources for bacteroids in planta, soybean (Glycine max L. Merr.) symbiosomes (consisting of the bacteroid-containing peribacteroid space enclosed by the peribacteroid membrane [PBM]) and bacteroids were assayed for the ability to transport iron supplied as various ferric [Fe(III)]-chelates. Iron presented as a number of Fe(III)-chelates was transported at much higher rates across the PBM than across the bacteroid membranes, suggesting the presence of an iron storage pool in the peribacteroid space. Pulse-chase experiments confirmed the presence of such an iron storage pool. Because the PBM is derived from the plant plasma membrane, we reasoned that it may possess a ferric-chelate reductase activity similar to that present in plant plasma membrane. We detected ferric-chelate reductase activity associated with the PBM and suggest that reduction of Fe(III) to ferrous [Fe(II)] plays a role in the movement of iron into soybean symbiosomes.

Siderophore-bound iron in the peribacteriod space of soybean root nodules

Water-soluble, non-leghemoglobin iron (125 µmol kg-1 wet weight nodule) is found in extracts of soybean root nodules. This iron is probably confined to the peribacteroid space of the symbiosome, where its estimated concentration is 0.5 – 2.5 mM. This iron is bound by siderophores (compounds binding ferric iron strongly) which are different for each of the three strains of Bradyrhizobium japonicum with which the plants were inoculated. One of these, that from nodules inoculated with strain CC 705, is tentatively identified as a member of the pseudobactin family of siderophores. Leghemoglobin is present in only very small amounts in the peribacteroid space of symbiosomes isolated from soybean root nodules, and may be absent from the peribacteroid space of the intact nodule.

1995 and Beyond

We present the following hypothesis; that lipo­ oligochitin Nod-factors can act in an elicitor-Iike fash­ ion inducing, amongst other effects, a plant chitolytic enzyme, capable of hydrolysing the oligochitin chain of the Nod-factor. Decorative groups on the oligochitin chain, e.g. SUlphate, may confer partial resistance to hydrolysis upon particular Nod-factors. After entry into the plant, Nod-factor synthesis must be down­ regulated in order to avoid further, unwanted, ell· citation and the consequent abortion of the symbiosis. The plant-derived compounds Inhibiting the synthesis of bacterial Nod-factors are limiting in root tissue, leading to residual elicitation and the abortion of infec­ tion thread formation. Nod-gene anti-induction is, fur­ thermore, inactivated by both light and nitrate, thus contributing to the inhibition of nodulation under these conditions. In nitrogen-fixing nodules, the bacteroids are exposed to both nOd-gene inducing and repressing compounds. The slow accumulation of Nod-factors within the peribacteroid space eventually results in the elicitation of phytoalexin synthesis and nodule

Artificially Induced Senescence of Soybean Root Nodules Affects Different Polypeptides and Nodulins in the Symbiosome Membrane Compared to Physiological Ageing

Summary The central unit of all nitrogen-fixing nodules is the symbiosome, which can be considered as a temporary but independent nitrogen-fixing endosymbiotic organelle consisting of the bacteroid, the peribacteroid space and the surrounding symbiosome membrane. Ageing and induced senescence of this membrane were investigated with respect to its protein components. No qualitative changes in the protein and nodulin composition could be detected in mature and ageing root nodules. When root nodule senescence was induced by plant decapitation, the symbiosome membrane persisted as a structure over the examined period. However, in the symbiosome membrane some nodule-specific proteins (nodulins) were extensively degraded, suggesting the presence of selective proteolytic activities against the symbiosome membrane nodulins. Enhanced proteolytic activities were observed during induced nodule senescence but not during physiological ageing. During induced senescence, activities and protein levels of glutamine synthetase (GS) and nitrogenase (N 2 ase) were significantly reduced. Glutamine synthetase activity decreased to 50% of the initial activity, while the immunodetectable amount of the respective polypeptide disappeared completely.

A Soybean Peribacteroid Space Component, Riboflavin, Represses Daidzein-Induced Common Nod-Gene Expression in Bradyrhizobium japonicum

Summary The nodulation of soya (Glycine max) roots could be severely inhibited by treating the symbiotic bacteria (Bradyrhizobium japonicum) with nodule-derived peribacteroid space fractions. Riboflavin, a prominent peribacteroid space component, could mimic this effect. Daidzein-stimulated beta-galactosidase expression, associated with a plasmid (pRJ458) bearing a B. japonicum nodD2D1YABC'-'lacZ construct in a B. japonicum background, was repressed in the presence of both peribacteroid space fractions and riboflavin.

NOD-Active Compounds in Soya Nodules

From the 11 spots resulting after 2-D TLC of ethanolic extracts of nodules, 2 were tentatively identifled as daidzein and daidzin by virtue of their Rf values and nod A-inducing ability. After nodule fractionation, bacteroids were found to contain 3.51 nmol daidzein per gFW nodule (=66.34 µM), but no daidzin. Peribacteroid space contained 24.82 nmol daidzin per gFW nodule (=314 µM), but no daidzein. The rest of the nodule contained 57.30 µM daidzein and 301.09 µM daidzin. The total nodule content of daidzein and daidzin was 18.25 and 102.27 nmol per g FW respectively.

Subcellular Localization of Oxygen Defense Enzymes in Soybean (Glycine max [L.] Merr.) Root Nodules.

Soybean (Glycine max [L.] Merr.) root nodules contain the enzymes of the ascorbate-glutathione pathway to minimize oxidative damage. In the present study, fractionation and immunocytochemistry were used to determine the subcellular location of the enzymes of this pathway. All four enzymes (ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase) were present in the soluble fraction from nodule plant cells and in isolated mitochondria. No activity was detected in peroxisomes. Bacteroids contained glutathione reductase but not the other enzymes of this pathway. Immunogold localization indicated that ascorbate peroxidase was present in the cytosol of infected and uninfected cells but not in the peribacteroid space. Results of immunogold and immunofluorescence studies indicated that monodehydroascorbate reductase was located primarily in the cell wall, suggesting that ascorbate regeneration in the cytoplasm may proceed primarily through the action of dehydroascorbate reductase. The possible roles of monodehydroascorbate reductase in cell wall metabolism are discussed.

High flavin/protein ratio in the symbiosome space of soybean nodules

Flavins in different compartments of effective nodules fromGlycine max cv Maple Arrow xBradyrhizobium japonicum strains were studied by spectrophotometry and chromatographic techniques. Flavins in the peribacteroid space were riboflavin (80%) and FMN (20%), as identified by TLC and HPLC. Flavin concentrations in the soybean root nodule cytoplasm, in the symbiosome space (PBS) and in the cytosol of bacteroids were monitored between 20 and 40 days post infection (d.p.i.) Between the 20th and 29th d.p.i. an at least four times higher flavin/protein ratio was found in PBS of effective nodules compared with the nodule cytoplasm. Between nitrogenase activity in the free-living state and bacterial flavin accumulation, no correlation could be observed. Flavin accumulation is not restricted to an effective symbiosis, as indicated by the analysis of ineffective nodules with strainB. japonicum RH-31 Marburg. Flavin accumulation is absent in uninfected soybean root tissue and in free-living rhizobia, thus indicating that flavin accumulation is a result of symbiotic interaction. Flavin accumulation is also missing in nodules with a hypersensitive response against the bacteria.

Characterization of symbiosis specific forms of alpha-glucosidase from Glycine max root nodules

Summary Three forms of α-glucosidase (EC 3.2.1.20) were isolated from soybean ( Glycine max L. Merr. cv. Maple Arrow) root nodules and two of them were further purified by a procedure including fractionation with PEG-8,000 followed by Q-Sepharose, S-Sepharose, Chromatofocusing and Superose 6 column chromatography. Chromatofocusing of the plant soluble fraction yielded two different isoforms, which were designated as I and II. Their isoelectric points were 7.3 (I) and 6.5 (II) as determined by native isoelectric focusing. The α-glucosidase I was further purified 502-fold to a specific activity of 1.8 units (mgprotein) -1 . In the peribacteroid space only one form, designated III, was resolved by chromatofocusing. The isoelectric point was 7.2 estimated by native isoelectric focusing. The α-glucosidase III was purified 86-fold to a specific activity of 3.1 units (mg protein) -1 . The α-glucosidases I and II had similar properties. The pH-optima were 5.2 (I) and 4.8 (III). The subunit molecular weights for α-glucosidase I and α-glucosidase III were estimated by SDS-PAGE to be 65 kDa and 66 kDa, respectively. Both enzymes hydrolyzed maltose, maltotriose, isomaltose and dextrin. Starch was only weakly hydrolyzed and neither a-glucosidase acted on sucrose. K m values for maltose were 0.91 mM (I) and 0.67 mM (III), and for PNPG 0.37 mM (I) and 0.76 mM (III). Ag + and Fe 2+ were the most potent inhibitors of both enzymes. The functions of these enzymes in the symbiotic compartments are discussed.