Peribacteroid Membrane Research Articles

A d-pinitol transporter, LjPLT11, regulates plant growth and nodule development in Lotus japonicus.

Polyol transporters have been functionally characterized in yeast and Xenopus laevis oocytes as H+-symporters with broad substrate specificity, but little is known about their physiological roles in planta. To extend this knowledge, we investigated the role of LjPLT11 in Lotus japonicus-Mesorhizobium symbiosis. Functional analyses of LjPLT11 in yeast characterized it as an energy-independent transporter of xylitol, two O-methyl inositols, xylose, and galactose. We showed that LjPLT11 is located on peribacteroid membranes and functions as a facilitative transporter of d-pinitol within infected cells of L. japonicus nodules. Knock-down of LjPLT11 (LjPLT11i) in L. japonicus accelerated plant growth under nitrogen sufficiency, but resulted in abnormal bacteroids with corresponding reductions in nitrogenase activity in nodules and plant growth in the nitrogen-fixing symbiosis. LjPLT11i nodules had higher osmotic pressure in cytosol, and lower osmotic pressure in bacteroids, than wild-type nodules both 3 and 4 weeks after inoculation of Mesorhizobium loti. Levels and distributions of reactive oxygen species were also perturbed in infected cells of 4-week-old nodules in LjPLT11i plants. The results indicate that LjPLT11 plays a key role in adjustment of the levels of its substrate pinitol, and thus maintenance of osmotic balance in infected cells and peribacteroid membrane stability during nodule development.

Requirements of Qa-SNARE LjSYP132s for Nodulation and Seed Development in Lotus japonicus

SNAREs (soluble N-ethyl maleimide-sensitive factor attachment protein receptors) mediate membrane fusion of vesicle transport in eukaryotic cells. LjSYP132s are the members of Qa-SNAREs in Lotus japonicus. Two isoforms, LjSYP132a and LjSYP132b, are generated by alternative splicing. Immunoblot analysis detected strong expression of LjSYP132s in infected root nodules and seeds by posttranscriptional modification. In either LjSYP132a or LjSYP132b silenced roots (RNAi-LjSYP132a, RNAi-LjSYP132b), the infection thread (IT) was not elongated, suggesting that both LjSYP132a and LjSYP132b have a role in IT progression. The results were consistent with the data of qRT-PCR showing that both genes were expressed at the early stage of infection. However, during the nodulation, only LjSYP132a was induced. LjSYP132s protein was observed in the Mesorhizobium loti-inoculated roots of mutants, nfr1, castor and pollux, suggesting that LjSYP132s can be induced without Nod factor signaling. Accumulation of LjSYP132s in the peribacteroid membrane suggests the function of not only IT formation but also nutrient transport. In contrast, qRT-PCR showed that LjSYP132b was expressed in the seeds. A stable transgenic plant of LjSYP132b, R132b, was produced by RNAi silencing. In the R132b plants, small pods with a few seeds and abnormal tip growth of the pollen tubes were observed, suggesting that LjSYP132b has a role in pollen tube growth and nutrient transport in the plasma membrane of seeds.

Hydrogen sulfide is a crucial element of the antioxidant defense system in Glycine max–Sinorhizobium fredii symbiotic root nodules

H2S is emerging as a signaling molecule involved in the regulation of many physiological processes in plants. Here, we investigated the potential antioxidant role of H2S in soybean (Glycine max)-rhizobia (Sinorhizobium fredii) symbiotic root nodules. An endogenous H2S production deficit rhizobia mutant ∆CSE was constructed to study the effect of decreased content of H2S in soybean nodules. Fluorescent probes and confocal microscope were used to observe the production and accumulation of H2S and reactive oxygen species. Transmission electronic microscopy was conducted to study the structural changes in ∆CSE soybean nodules. Finally, qRT-PCR, enzymatic activity, and oxidative damage parameters were measured. The results demonstrated that abundant H2S was generated in the nitrogen-fixing zone of soybean nodules. The deletion of the cystathionine γ-lyase (CSE) gene in S. fredii (∆CSE) caused a sharp decrease in H2S production in both free-living rhizobia and soybean nodules. We found that decrease in the H2S level in nodule cells inhibited nitrogenase activity. In addition, to elevated H2O2 and malondialdehyde accumulation, increased protein carbonyl content and decreased O2− scavenging ability was observed in ∆CSE root nodules. Transmission electron microscopy revealed that an H2S deficit caused the deformation of bacteroids and damage of peribacteroid membranes in nodule cells. Moreover, the expression of some rhizobial and soybean genes related to antioxidant defense was up-regulated in ∆CSE nodules. H2S is crucial for the nitrogen-fixation ability of soybean nodules by acting as an antioxidant element that protects nodule cells and bacteroids from oxidative damage.

The Ca2+-ATPase in Legume Root Nodule Peribacteroid Membrane as a Potential Key Determinant of Ca-Dependent Regulation of Symbiosome Functioning

Certain aspects of Ca-dependent regulation of symbiosomes functioning mediated by the operation of Ca2+-translocating ATPase on the symbiosome membrane (SM) were considered. As follows from the results recently obtained by us, the mechanism underlying the action of this calcium pump exhibits a certain functional feature caused by that transfer by it of calcium ion through the SM is closely coupled with transmembrane translocation through the same membrane but in opposite direction of H+ ions. The data here outlined show that such the cation-exchanged mechanism of the Ca2+-ATPase operation can putatively lead to corresponding modulation or maintenance of both ionic and redox homeostasis of the symbiosome space (SS) around bacteroids at a proper level and thereby to the regulation of symbiosomes functioning. It is suggested that based on the same mechanism inherent function of the enzyme in question is associated with its involvement in symbiotic partner cells signaling as well. All these potential activities of the Ca2+-ATPase lead to the important question whether it is the factor determining substantial functions of symbiosomes. Although it is evident that research an answer to this question is a complex enough experimental problem, its solution may give significant contribution to elucidating mechanisms of Ca-dependent regulation of the processes occurring in mature symbiosomes.

Classical Soybean (Glycine max (L.) Merr) Symbionts, Sinorhizobium fredii USDA191 and Bradyrhizobium diazoefficiens USDA110, Reveal Contrasting Symbiotic Phenotype on Pigeon Pea (Cajanus cajan (L.) Millsp).

Pigeon pea (Cajanus cajan (L.) Millspaugh) is cultivated widely in semiarid agricultural regions in over 90 countries around the world. This important legume can enter into symbiotic associations with a wide range of rhizobia including Bradyrhizobium and fast-growing rhizobia. In comparison with other major legumes such as soybean and common bean, only limited information is available on the symbiotic interaction of pigeon pea with rhizobia. In this study, we investigated the ability of two classical soybean symbionts—S. fredii USDA191 and B. diazoefficiens USDA110—and their type 3 secretion system (T3SS) mutants, to nodulate pigeon pea. Both S. fredii USDA191 and a T3SS mutant S. fredii RCB26 formed nitrogen-fixing nodules on pigeon pea. Inoculation of pigeon pea roots with B. diazoefficiens USDA110 and B. diazoefficiens Δ136 (a T3SS mutant) resulted in the formation of Fix− and Fix+ nodules, respectively. Light and transmission electron microscopy of Fix- nodules initiated by B. diazoefficiens USDA110 revealed the complete absence of rhizobia within these nodules. In contrast, Fix+ nodules formed by B. diazoefficiens Δ136 revealed a central region that was completely filled with rhizobia. Ultrastructural investigation revealed the presence of numerous bacteroids surrounded by peribacteroid membranes in the infected cells. Analysis of nodule proteins by one- and two-dimensional gel electrophoresis revealed that leghemoglobin was absent in B. diazoefficiens USDA110 nodules, while it was abundantly present in B. diazoefficiens Δ136 nodules. Results of competitive nodulation assays indicated that B. diazoefficiens Δ136 had greater competitiveness for nodulation on pigeon pea than did the wild type strain. Our results suggest that this T3SS mutant of B. diazoefficiens, due to its greater competitiveness and ability to form Fix+ nodules, could be exploited as a potential inoculant to boost pigeon pea productivity.

The symbiotic role of the actin filament cytoskeleton.

This article is a Commentary on Zhang et al., 221: 1049–1059.

Multiple Nodulation Genes Are Up-Regulated During Establishment of Reniform Nematode Feeding Sites in Soybean

The semi-endoparastic reniform nematode (Rotylenchulus reniformis) infects over 300 plant species. Females penetrate host roots and induce formation of complex, multinucleate feeding sites called syncytia. While anatomical changes associated with reniform nematode infection are well documented, little is known about their molecular basis. We grew soybean (Glycine max) in a split-root growth system, inoculated half of each root system with R. reniformis, and quantified gene expression in infected and control root tissue at four dates after inoculation. Over 6,000 genes were differentially expressed between inoculated and control roots on at least one date (false discovery rate [FDR] = 0.01, |log2FC| ≥ 1), and 507 gene sets were significantly enriched or depleted in inoculated roots (FDR = 0.05). Numerous genes up-regulated during syncytium formation had previously been associated with rhizobia nodulation. These included the nodule-initiating transcription factors CYCLOPS, NSP1, NSP2, and NIN, as well as multiple nodulins associated with the plant-derived peribacteroid membrane. Nodulation-related NIP aquaporins and SWEET sugar transporters were induced, as were plant CLAVATA3/ESR-related (CLE) signaling proteins and cell cycle regulators such as CCS52A and E2F. Nodulins and nodule-associated genes may have ancestral functions in normal root development and mycorrhization that have been co-opted by both parasitic nematodes and rhizobial bacteria to promote feeding site and nodule formation.

Structure of dinitrogen fixing nodules in Erythrina poeppigiana

The development and anatomy of root nodules induced in seedlings of Erythrina poeppigiana by a strain of Bradyrhizobium spp (CR 751) is described. Infection apparently occurred via injuries where lateral roots (devoid of root hairs) emerged. The first sign of infection was a group of epidermal cells (fluffy) on the secondary (emerging) root that covered the nodule primordium. Here, thin-walled cells with plastids and in active division were observed but not infection threads. Infection appears to have occurred via protoxylem which was preceded by a dissolution of the vessels' cell wall. A peribacteroid, membrane-generating organelle in infected cells is described. The adjacent noninfected cells showed electron dense depositions (phenolics 2). Developed nodules were spherical 0.5 to 2 cm in diameter. The surface was corky, with lenticels. The peridermis showed several layers of suberized dead cells. The cortex consisted of external, medium and internal layers. Vascular bundles ran in the medium cortex in a centripetal way to join the root vascular system at the base of the nodule. In these vascular bundles, baciliar bacteria were observed which were confirmed to be Bradyrhizobium sp (results of a separate study using immunocitochemistry). Bacteroids densely filled infected cells of the nodule medulla within peribacteroid membranes. Among infected cells non-infected auxiliary cells were seen loaded with starch granules. During later stages of root development few root hairs appeared on tertiary and secondary roots. Infection also occurred via root hairs, resulting in oblong nodules caused by a tumefaction of the root cortex .

Functional characterization of a soybean growth stimulator Bradyrhizobium sp. strain SR-6 showing acylhomoserine lactone production.

A soybean nodule endophytic bacterium Bradyrhizobium sp. strain SR-6 was characterized for production of acyl homoserine lactones (AHLs) as quorum sensing molecules. Mass spectrometry analysis of AHLs revealed the presence of C6-HSL, 3OH-C6-HSL, C8-HSL, C10-HSL, 3oxoC10-HSL, 3oxo-C12-HSL and 3OH-C12-HSL which are significantly different from those reported earlier in soybean symbionts. Purified AHL extracts significantly improved wheat and soybean seedling growth and root hair development along with increased soybean nodulation under axenic conditions. A positive correlation was observed among in vivo nitrogenase and catalase enzyme activities of the strain SR-6. Transmission electron microscopic analysis showed the cytochemical localization of catalase activity within the bacteroids, specifically attached to the peribacteroidal membrane. Root and nodule colonization proved rhizosphere competence of SR-6. The inoculation of SR-6 resulted in increased shoot length (13%), plant dry matter (50%), grain weight (16%), seed yield (20%) and N-uptake (14%) as compared to non-inoculated soybean plants. The symbiotic bacterium SR-6 has potential to improve soybean growth and yield in sub-humid climate of Azad Jammu and Kashmir region of Pakistan. The production and mass spectrometric profiling of AHLs as well as in vivo cytochemical localization of catalase enzyme activity in soybean Bradyrhizobium sp. have never been reported earlier elsewhere before our these investigations.

A symbiotic SNARE protein generated by alternative termination of transcription.

Many microbes interact with their hosts across a membrane interface, which is often distinct from existing membranes. Understanding how this interface acquires its identity has significant implications. In the symbiosis between legumes and rhizobia, the symbiosome encases the intracellular bacteria and receives host secretory proteins important for bacterial development. We show that the Medicago truncatula SYNTAXIN 132 (SYP132) gene undergoes alternative cleavage and polyadenylation during transcription, giving rise to two target-membrane soluble NSF attachment protein receptor (t-SNARE) isoforms. One of these isoforms, SYP132A, is induced during the symbiosis, is able to localize to the peribacteroid membrane, and is required for the maturation of symbiosomes into functional forms. The second isoform, SYP132C, has important functions unrelated to symbiosis. The SYP132A sequence is broadly found in flowering plants that form arbuscular mycorrhizal symbiosis, an ancestral mutualism between soil fungi and most land plants. SYP132A silencing severely inhibited arbuscule colonization, indicating that SYP132A is an ancient factor specifying plant-microbe interfaces.

Peribacteroid solution of soybean root nodules partly induces genomic loci for differentiation into bacteroids of free-living Bradyrhizobium japonicum cells

In leguminous root nodules, rhizobia differentiate into morphology specific to symbiosis, called bacteroids. As bacteroids are surrounded with peribacteroid membranes filled with peribacteroid solution (PBS), it is considered that PBS contains substances inducing differentiation of rhizobia into bacteroids. In this study, genome-wide expression profiles of Bradyrhizobium japonicum cells cultured in PBS purified from root nodule of soybean (Glycine max L.) were compared with those of bacteroids using macroarray. PBS treatment preferentially induced regions in a large symbiosis island including various symbiosis relevant genes such as nod, fix, nol and noe, in which 75% of regions were commonly induced in bacteroids, while general repressions outside of the symbiosis island seen in bacteroids were not observed in PBS treated cells. The present results suggest that PBS contained some, but not all, substances inducing expression of the genes which are involved in differentiation into bacteroids.

Functional analysis of duplicated Symbiosis Receptor Kinase (SymRK) genes during nodulation and mycorrhizal infection in soybean (Glycine max)

Association between legumes and rhizobia results in the formation of root nodules, where symbiotic nitrogen fixation occurs. The early stages of this association involve a complex of signalling events between the host and microsymbiont. Several genes dealing with early signal transduction have been cloned, and one of them encodes the leucine-rich repeat (LRR) receptor kinase (SymRK; also termed NORK). The Symbiosis Receptor Kinase gene is required by legumes to establish a root endosymbiosis with Rhizobium bacteria as well as mycorrhizal fungi. Using degenerate primer and BAC sequencing, we cloned duplicated SymRK homeologues in soybean called GmSymRKα and GmSymRKβ. These duplicated genes have high similarity of nucleotide (96%) and amino acid sequence (95%). Sequence analysis predicted a malectin-like domain within the extracellular domain of both genes. Several putative cis-acting elements were found in promoter regions of GmSymRKα and GmSymRKβ, suggesting a participation in lateral root development, cell division and peribacteroid membrane formation. The mutant of SymRK genes is not available in soybean; therefore, to know the functions of these genes, RNA interference (RNAi) of these duplicated genes was performed. For this purpose, RNAi construct of each gene was generated and introduced into the soybean genome by Agrobacterium rhizogenes-mediated hairy root transformation. RNAi of GmSymRKβ gene resulted in an increased reduction of nodulation and mycorrhizal infection than RNAi of GmSymRKα, suggesting it has the major activity of the duplicated gene pair. The results from the important crop legume soybean confirm the joint phenotypic action of GmSymRK genes in both mycorrhizal and rhizobial infection seen in model legumes.

SYMBIOTIC NODULE SENESCENCE IN LEGUMES: MOLECULAR-GENETIC AND CELLULAR ASPECTS (review)

Senescence is the natural stage in development of symbiotic nodule. As a result of senescence, reutilization of different nutrients from nodule to the other plant organs occurs. Generally senescence in legumes is triggered after flowering finishing, although the first traits of senescence can be observed very early during nodule development. A delay of the triggering of senescence program will allow to prolong the active nitrogen-fixating period and therefore to increase the amount of symbiotrophic nitrogen in plants and, finally, to elevate legume productivity. That is why no wonder that in the recent years the senescence of nitrogen-fixing nodules is actively studied. In this review the main developmental stages of nitrogen-fixing symbiotic nodule of legumes, particularities of symbiotic nodule development of determinate and indeterminate types are considered. In legumes with indeterminate nodules, the symbiosomes are not long-leaving as the infected tissues are permanently renewing due to apical meristem. There are two subsequent stages identified in an indeterminate nodule senescent. First a bacteroid degradation and the death of some infected cells occur, and then both symbiosomes and all infected cells are destroyed. In determinate nodules, the senescence initiated in the central part of a nodule, then extends to the peripheral zone. In this review morphological characters of nodule senescence at ulatrstructural level are analyzed. The role of cysteine and threonine proteases is discussed. Reutilization of nitrogen and other products of protein degradation are probably the most important during senescence. There are the evidences that in the root nodules of legumes the cysteine proteases are involved into nodule functions, adaptation of the host plant cells to physiological stresses, and the nodule senescence control. By a large-scale analysis of nodule transcriptome of Medicago truncatula Gaertn. several groups of genes expressed at successive stages of the senescence of indeterminate nodule are revealed. In this review the role of phytohormones, such as ethylene, abscisic acid, jasmonic acid, gibberellins and nitrogen monooxide in senescence of symbiotic nodule is considered. Nevertheless, until recent days our knowledge about hormonal control of nodule senescence is still incomplete. The oxidative stress, accompanying the process of nodule senescence is discussed. On the nodule aging, the concentrations of peroxides, protein carbonyls, modified DNA nucleotides and catalytic Fe increase. Iron activates lipids peroxidation in a peribacteroid space, resulting in degradation of the peribacteroid membrane in senescent nodules. The concentrations of oxidized glutathione and homoglutathione rise significantly during the nodule development, and the reduced forms decrease under senescence, indicating an oxidative stress in the senescing nodules. In this review the role of genes, encoding proteins involved in transport of wide-range of molecules, and genes, whose products are involved in regulatory and signal functions in cell; differences between stress-induced senescence and natural senescence are considered. Using model legumes, Lotus japonicus (Regel) K. Larsen and M. truncatula, several genes were cloned the mutations of which caused early senescence. It is emphasized that these genes encode different proteins involved into functioning of a symbiotic nodule. Until now, two transcription factors in M. truncatula are described, which are involved into nodule senescence. An induced senescence is more rapid, comparing to natural senescence, and manifests the signs of an oxidative stress and programmed cell death.

СТАРЕНИЕ СИМБИОТИЧЕСКОГО КЛУБЕНЬКА У БОБОВЫХ РАСТЕНИЙ: МОЛЕКУЛЯРНО-ГЕНЕТИЧЕСКИЕ И КЛЕТОЧНЫЕ АСПЕКТЫ (обзор)

Senescence is the natural stage in development of symbiotic nodule. As a result of senescence, reutilization of different nutrients from nodule to the other plant organs occurs. Generally senescence in legumes is triggered after flowering finishing, although the first traits of senescence can be observed very early during nodule development. A delay of the triggering of senescence program will allow to prolong the active nitrogen-fixation period and therefore to increase the amount of symbiotrophic nitrogen in plants and, finally, to elevate legume productivity. That is why no wonder that in the recent years the senescence of nitrogen-fixing nodules is actively studied. In this review the main developmental stages of nitrogen-fixing symbiotic nodule of legumes, particularities of symbiotic nodule development of determinate and indeterminate types are considered. In legumes with indeterminate nodules, the symbiosomes are not long leaving as the infected tissues are permanently renewing due to apical meristem. There are two subsequent stages identified in an indeterminate nodule senescent. First a bacteroid degradation and the death of some infected cells occur, and then both symbiosomes and all infected cells are destroyed. In determinate nodules, the senescence initiated in the central part of a nodule, then extends to the peripheral zone. In this review morphological characters of nodule senescence at ulatrstructural level are analysed. The role of cysteine and threonine proteases is discussed. Reutilization of nitrogen and other products of protein degradation are probably the most important during senescence. There are the evidences that in the root nodules of legumes the cysteine proteases are involved into nodular functions, adaptation of the host plant cells to physiological stresses, and the nodule senescence control. By a large-scale analysis of nodule transcriptome of Medicago truncatula Gaertn. several gene groups expressed at successive stages of the senescence of indeterminate nodule are revealed. In this review the role of phytohormones, such as ethylene, abscisic acid, jasmonic acid, gibberellins and nitrogen monooxide in senescence of symbiotic nodule is considered. Nevertheless, until recent days our knowledge about hormonal control of a nodule senescence is still incomplete. The oxidative stress, accompanying the process of nodule senescence is discussed. On the nodules aging, the concentrations of peroxides, protein carbonils, modified DNA nucleotides and catalytic Fe increase. Iron activates lipids peroxidation in a peribacteroid space, resulting in degradation of the peribacteroid membrane in senescent nodules. The concentrations of oxidized glutathione and homoglutathion rise significantly during the nodule development, and the reduced forms decrease under senescence, indicating an oxidative stress in the senescing nodules. In this review the role of genes, encoding proteins involved in transport of wide-range of molecules, and genes, whose products are involved in regulatory and signal functions in cell; differences between stress-induced senescence and natural senescence are considered. Using model legumes, Lotus japonicus (Regel) K. Larsen and M. truncatula, several genes were cloned the mutations of which caused early senescence. It is emphasized that these genes encode different proteins involved into functions of a symbiotic nodule. Until now, two transcription factors in M. truncatula are described, which are involved into nodule senescence. An induced senescence is more rapid, comparing to natural senescence, and manifests the signs of an oxidative stress and programmed cell death.

Legume nodulation

Legume nodulation

A comparative study of phase States of the peribacteroid membrane from yellow lupin and broad bean nodules.

A comparative study of the lipid bilayer phase status and structure of the outer membrane of free-living Bradyrhizobium strain 359a (Nod+Fix+) and 400 (Nod+FixL) or Rhizobium leguminosarum 97 (Nod+Fix+, effective) and 87 (Nod+FixL, ineffective) has been carried out. Also, the effect of the symbiotic pair combination on the lipid bilayer structure of the bacteroid outer membrane and peribacteroid membrane, isolated from the nodules of Lupinus luteus L. or Vicia faba L., has been studied. As a result, it is shown that the lipid bilayer status of the bacteroid outer membrane is mainly determined by microsymbiont, but not the host plant. In the contrast, the lipid bilayer status of the peribacteroid membrane and, as a consequence, its properties depend on interaction of both symbiotic partners.

A correlative study of hydrogen peroxide accumulation after mercury or copper treatment observed in root nodules of Medicago truncatula under light, confocal and electron microscopy

Heavy metal stress affects both, nodulation and nitrogen fixation of legumes. Mercury triggers disturbances in cellular structure and metabolism but its influence on ROS generation is poorly understood. Copper is redox active metal which in opposition to mercury is an essential micronutrient for plants. Excess of copper is cytotoxic, as it participates in ROS generation via Fenton-type reaction.The present work describes changes in hydrogen peroxide (H2O2) accumulation in response to monthly stress caused by mercury (6mg/L HgCl2) or copper (60mg/L CuCl2) in root nodules.H2O2 accumulation viewed with a light microscopy was detected by the use of diaminobenzidine (DAB). 2′,7′-Dichlorofluorescein diacetate (H2DCF-DA) was used as a probe for the intracellular localization of H2O2 with a confocal laser scanning system. H2O2 detection under transmission electron microscopy was performed by the use of cerium method.Histochemical localization and light and confocal microscopy investigations revealed that under Hg or Cu treatments distinct amount of H2O2 accumulated mainly in the interzone and nitrogen-fixing zone. Under normal conditions H2O2 accumulated predominantly in the interzone. Electron microscopy observations showed H2O2 accumulation under Hg or Cu- treatments around peribacteroid membranes of mature symbiosomes located within nitrogen-fixing zone. It should be underlined that under normal conditions H2O2 was not detected at the peribacteroid membranes.The main result of our observations is increased accumulation of H2O2 in response to mercury and copper treatments at the peribacteroidal membranes, to our knowledge shown for the first time. Therefore, our results revealed that an overproduction of H2O2 in response to copper or mercury-treatment may account for lowering of nitrogen fixation rates in heavy-metal affected root nodules.

Biochemical characteristics of the Ca2+ pumping ATPase in the peribacteroid membrane from broad bean root nodules

Ca(2+)-ATPase in the peribacteroid membrane (PBM) of symbiosomes isolated from Vicia faba root nodules was characterized in terms of its hydrolytic and transport activities. Both activities were found to be pH-dependent and exhibit pH optimum at pH7.0. Translocation of Ca(2+) through the PBM by the Ca(2+)-ATPase was shown to be fueled by ATP and other nucleotide triphosphates in the following order: ATP > ITP ≅ GTP ≅ UTP ≅ CTP, the K m of the enzyme for MgATP being about 100μM. Ca-dependent ITP-hydrolytic activity of symbiosomes was investigated in the presence of the Ca-EGTA buffer system and showed the affinity of PBM Ca(2+)-ATPase for Ca(2+) of about 0.1μM. The transport activity of Ca(2+)-ATPase was inhibited by erythrosin B as well as orthovanadate, but markedly stimulated by calmodulin from bovine brain. These results allowed us to conclude that this enzyme belongs to IIB-type Ca(2+)-ATPases which are present in other plant membranes.

Copper microlocalisation, ultrastructural alterations and antioxidant responses in the nodules of white lupin and soybean plants grown under conditions of copper excess

Copper (Cu) is a heavy metal which, at high concentrations, is toxic to organisms. Some plants, however, possess systems for dealing with excess Cu, such as its cell localisation, and have antioxidant enzymes that help to reduce the oxidative stress. The present work examines the microlocalisation of Cu and the antioxidant enzyme activity present in the nodules of white lupin and soybean plants grown hydroponically for 35 days in the presence of 1.6μM (control) or 192μM (excess) Cu. The effect of these conditions on nodule (ultra)structure was also examined. Energy-dispersive X-ray microanalysis showed the cell walls to be the main area of Cu binding in the inner and outer cortex and infected zone of white lupin nodules grown under the excess Cu conditions, while in soybean a high Cu signal was detected inside cells (cytoplasm or vacuoles) both in the inner cortex and infected zone. At the tissue level, an increasing Cu gradient was seen from the outer towards the inner nodule cortex in white lupin nodules, while the opposite was seen in soybean. Cu excess also induced oxidative stress and promoted damage to the ultrastructure of nodules. In the white lupin infected cells, a breakdown of the peribacteroidal membrane was seen, along with an increased number of vesicles in the cytosol of these cells. In the infected cells of the soybean nodules, the bacteroidal membrane became degraded and precipitation was seen within the vacuoles of the infected and uninfected cells. Finally, the white lupin nodules seemed to be more sensitive to Cu excess than those of soybean, with the nodulation process, N2 fixation, and the ultrastructure of bacteroids more strongly affected. A less effective antioxidative stress response against Cu was also seen in white lupin than in soybean nodules: the excess copper treatment induced a smaller increase in the total thiol content and ascorbate peroxidase activity in white lupin nodules than in soybean nodules, and promoted a greater reduction in catalase activity.

Bacteroid Development in Legume Nodules: Evolution of Mutual Benefit or of Sacrificial Victims?

Symbiosomes are organelle-like structures in the cytoplasm of legume nodule cells which are composed of the special, nitrogen-fixing forms of rhizobia called bacteroids, the peribacteroid space and the enveloping peribacteroid membrane of plant origin. The formation of these symbiosomes requires a complex and coordinated interaction between the two partners during all stages of nodule development as any failure in the differentiation of either symbiotic partner, the bacterium or the plant cell prevents the subsequent transcriptional and developmental steps resulting in early senescence of the nodules. Certain legume hosts impose irreversible terminal differentiation onto bacteria. In the inverted repeat-lacking clade (IRLC) of legumes, host dominance is achieved by nodule-specific cysteine-rich peptides that resemble defensin-like antimicrobial peptides, the known effector molecules of animal and plant innate immunity. This article provides an overview on the bacteroid and symbiosome development including the terminal differentiation of bacteria in IRLC legumes as well as the bacterial and plant genes and proteins participating in these processes.