The peribacteroid membrane

The objective of this review is to summarise current knowledge about the structure and function of the peribacteroid membrane from the root nodules of leguminous plants. The information is presented in terms of development of this symbiotic membrane from its origin, through proliferation and in the mature state. There are clear indications that the peribacteroid membrane has a distinct structure and function at each developmental stage. The mature peribacteroid membrane has been the most intensively studied. The lipid and protein content of the mature peribacteroid membrane is discussed with particular emphasis on genetic and functional studies of the proteins. The mechanism and control of peribacteroid membrane biogenesis is also discussed. There is evidence for a specific biogenetic pathway for this membrane which requires both symbiotic partners for its correct functioning.     

Verapamil-Sensitive Calcium Transporter in the Peribacteroid Membrane of Symbiosomes from Vicia faba Root Nodules

Based on electron microscopic studies and visualization of calcium with the Ca indicator pyroantimonate, it was established that a prolonged incubation of the bean (Vicia faba L.) root nodules and isolated symbiosomes in EGTA-containing buffer depletes calcium in these nitrogen-fixing units. Other experiments demonstrated that the induction of calcium deficit in symbiosomes both in vivo and in vitro substantially decreases their nitrogenase activity. The addition of verapamil and ruthenium red, well-known inhibitors of Ca²⁺ channels, to the suspension of root nodules largely prevented both the EGTA-induced calcium efflux from the symbiosomes and the decrease in their nitrogenase activity. Similar effects of verapamil were also observed on isolated symbiosomes. The treatment of isolated symbiosomes with valinomycin in the presence of K⁺ induced a rapid efflux of Ca²⁺ from symbiosomes; this efflux was strongly inhibited by verapamil. The results present evidence for the existence in the peribacteroid membrane of a Ca²⁺-transporting system that exports Ca²⁺ from the symbiosomes.     

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.     

Changes in the Light Scattering by Symbiosomes from Vicia faba Root Nodules as Indicator of Ion and Metabolite Transport across the Peribacteroid Membrane

Passive transport of ions and metabolites across the peribacteroid membrane (PBM) was investigated on symbiosome preparations isolated from the broad bean (Vicia faba L.) root nodules and suspended in a potassium-free medium. Optical density of the symbiosome suspension at 546 nm was monitored as an indicator of light-scattering changes. Depolarization of the PBM with tetraphenylphosphonium cation (TPP⁺) caused an increase in light scattering of symbiosome suspension. This effect was enhanced after adding a K⁺ ionophore valinomycin to the incubation medium. A similar effect was observed after supplementing the symbiosome suspension with nigericin, a K⁺/H⁺ antiporter. Similar experiments on bacteroid suspensions prepared from isolated symbiosomes did not reveal any appreciable changes in light scattering in the presence of the same membrane-active substances. The light scattering by symbiosome suspensions decreased after adding malate or succinate, while the subsequent addition of centimolar concentrations of K⁺ substantially accelerated this process. Light scattering by the symbiosome suspension was insensitive to the addition of glutamate, a substance normally impermeant through the PBM of legume root nodules. These results suggest that the changes in light scattering by symbiosomes reflect the osmotically induced changes of symbiosome volume. These volume changes were assigned to alteration of the peribacteroid space (PBS). The incubation of symbiosomes in a potassium-free medium acidified their the PBS; this acidification was accelerated by valinomycin, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), and nigericin, and it was abolished in the presence of comparatively high concentrations of K⁺ in the incubation medium. The results indicate a relatively high permeability of the PBM to K⁺ ions.     

Calcium-Based Interactions of Symbiotic Partners in Legumes: Role of Peribacteroid Membrane

Based on experimental evidence, a concept is formulated that mutualistic relationships between pro- and eukaryotic cells during nitrogen-fixing legume–rhizobia symbiosis rely both on selective transfer of metabolites and ion transport, Ca²⁺ in particular, across the peribacteroid membrane (PBM). PBM in the nitrogen-fixing cells of yellow lupine (Lupinus luteus L.) and broad bean (Vicia faba L.) is endowed with a calcium-translocating ATPase that pumps Ca²⁺ into the symbiosome. This pumping ensures, on the one hand, calcium homeostasis in the cytosol of infected plant cells and, on the other hand, it optimizes Ca²⁺ level in symbiosomes, first of all in the bacteroids, because Ca²⁺ is one of the main factors controlling their nitrogenase activity. The balance between the symbiotic partners and the maintenance of optimal Ca²⁺ level in the bacteroids also depends on passive Ca²⁺ efflux from symbiosomes to the plant cell cytosol via calcium channels. The Ca²⁺-transporting mechanisms residing at PBM are characterized.     

Proline accumulation inside symbiosomes of faba bean nodules under salt stress

A two‐week salt treatment (NaCl, 100 m M ) 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 m M 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.     

ATPase activity and anion transport across the peribacteroid membrane of isolated soybean symbiosomes

Addition of ATP to intact symbiosomes isolated from soybean nodules, resulted in generation of a membrane potential (positive inside) across the peribacteroid membrane (PBM). This energisation was monitored as oxonol fluorescence quenching. The rate of fluorescence quenching was inhibited by the inclusion of permeant anions in the reaction medium. Using this inhibition as a measure of anion uptake across the PBM, the presence of a phthalonate-sensitive dicarboxylate carrier on the PBM was confirmed. Following dissipation of the membrane potential by a permeant anion, a pH gradient, measured using [¹⁴C]methylamine uptake, was slowly established across the PBM. This pH was abolished by addition of an uncoupler but was insensitive to inhibitors of bacteroid respiration. The difference in pH between the external medium and the symbiosome interior was estimated to be in the range of 1–1.6 pH units. The magnitude in planta will depend on the concentrations of ATP and permeant anions in the cytosol of the host cell.Abbreviations PBM peribacteroid membrane - electrical membrane potential - MA methylamine The term symbiosome refers to the peribacteroid unit consisting of bacteroids enclosed in the host-derived peribacteroid membrane     

Metabolite transport across the peroxisomal membrane

In recent years, much progress has been made with respect to the unravelling of the functions of peroxisomes in metabolism, and it is now well established that peroxisomes are indispensable organelles, especially in higher eukaryotes. Peroxisomes catalyse a number of essential metabolic functions including fatty acid beta-oxidation, ether phospholipid biosynthesis, fatty acid alpha-oxidation and glyoxylate detoxification. The involvement of peroxisomes in these metabolic pathways necessitates the transport of metabolites in and out of peroxisomes. Recently, considerable progress has been made in the characterization of metabolite transport across the peroxisomal membrane. Peroxisomes posses several specialized transport systems to transport metabolites. This is exemplified by the identification of a specific transporter for adenine nucleotides and several half-ABC (ATP-binding cassette) transporters which may be present as hetero- and homo-dimers. The nature of the substrates handled by the different ABC transporters is less clear. In this review we will describe the current state of knowledge of the permeability properties of the peroxisomal membrane.     

Calcium Status of Yellow Lupin Symbiosomes as a Potential Regulator of Their Nitrogenase Activity: The Role of the Peribacteroid Membrane

The capacity of symbiosomes from yellow lupin root nodules for active Ca²⁺uptake and the sensitivity of their nitrogenase activity to a disturbance of the symbiotic Ca partition were investigated. The experiments carried out on the isolated symbiosomes and the peribacteroid membrane (PBM) vesicles, using Ca²⁺indicators arsenazo III and chlorotetracycline, and the cytochemical Ca visualization with potassium pyroantimonate (PA) provided evidence that an Mg-ATP-energized pump, most likely Mg²⁺-dependent Ca²⁺-ATPase catalyzing the active transport of Ca²⁺from the cytosol of the plant cell into the symbiosomes across the PBM, functions on this membrane. Depleting the symbiosomes of Ca both in vivoandin vitroby treating the intact nodules of yellow lupin root or the purified symbiosomes isolated from the latter with EGTA and Ca²⁺-ionophore A23187 substantially decreased their nitrogenase activity. The inhibitory effect of calcium deficit in the symbiosomes was not reversed by the addition of calcium to the incubation medium containing the plant tissues under study and was even enhanced under these conditions. The nitrogenase activity of the isolated symbiosomes not experiencing calcium deficit was also inhibited by the addition of relatively high concentrations of exogenous calcium to the incubation medium. These results seem to give evidence that the calcium status of nodule symbiosomes from yellow lupin roots controls their nitrogenase activity. The data obtained suggest that both Ca²⁺transport on PBM and the low passive permeability of this membrane for the given cation play the key role in such a control.     

Effects of magnesium availability on the activity of plasma membrane ion transporters and light-induced responses from broad bean leaf mesophyll

Considering the physiological significance of Mg homeostasis in plants, surprisingly little is known about the molecular and ionic mechanisms mediating Mg transport across the plasma membrane and the impact of Mg availability on transport processes at the plasmalemma. In this study, a non-invasive ion-selective microelectrode technique (MIFE) was used to characterize the effects of Mg availability on the activity of plasma membrane H⁺, K⁺, Ca²⁺, and Mg²⁺ transporters in the mesophyll cells of broad bean (Vicia faba L.) plants. Based on the stoichiometry of ion-flux changes and results of pharmacological experiments, we suggest that at least two mechanisms are involved in Mg²⁺ uptake across the plasma membrane of bean mesophyll cells. One of them is a non-selective cation channel, also permeable to K⁺ and Ca²⁺. The other mechanism, operating at concentrations below 30 M, was speculated to be an H⁺/Mg⁺ exchanger. Experiments performed on leaves grown at different levels of Mg availability (from deficient to excessive) showed that Mg availability has a significant impact on the activity of plasma-membrane transporters for Ca²⁺, K⁺, and H⁺. We discuss the physiological significance of Mg-induced changes in leaf electrophysiological responses to light and the ionic mechanisms underlying this process.     

Recognition of phlorizin by the carriers of sucrose and hexose in broad bean leaves

Phlorizin (1-[2-(β- d -glucopyranosyloxy)-4, 6-dihydroxyphenyl]-3-(4-hydroxyphenyl)-1-propanone) is a well-known non-transported inhibitor of glucose uptake in animal cells. The effects of this compound were studied on the transmembrane potential difference (PD) of broad bean ( Vicia faba L. cv. Aguadulce) mesophyll cells, and on the uptake of amino acids and sugars by the leaf tissues. Phlorizin (5 m M ) induced a marginal depolarization (7 to 10 mV) of the normal PD (-140 mV), and a slight decrease in the uptake of glycine and serine. By contrast, phlorizin induced a stronger inhibition of the uptake of glucose and 3–O-methylglucose, and more particularly of sucrose uptake and phloem loading. In tissues aged for 12 h, 5 m M phlorizin inhibited the absorption of sucrose from a 1 m M solution by 70%. Kinetic experiments demonstrated that phlorizin acted as a competitive inhibitor for the sucrose carrier and for the hexose carrier. Efflux experiments showed that the counter-exchange of sucrose and of 3–O-methylglucose was also phlorizin-sensitive. Overall, the data show that phlorizin is recognized by the hexose carrier and, more efficiently, by the sucrose carrier of the material investigated, but that it is not transported across the membrane.     

环磷酰胺诱发蚕豆体细胞遗传损伤的研究

利用蚕豆根尖研究环磷酰胺的遗传毒性效应, 结果表明:环磷酰胺(0.1~5.0 mg/mL)能够降低蚕豆根尖细胞有丝分裂指数, 使根尖细胞中具有微核、核出芽及核固缩的细胞明显增多, 并诱发染色体结构和行为异常, 产生染色体断片、滞后和桥。环磷酰胺处理组根尖中具有核固缩和微核的细胞数呈剂量依赖性增加, 且与作用时间呈正相关, 而分裂指数的降低也具有剂量和时间效应关系。研究结果表明, 低浓度长时间接触或高浓度短时间接触环磷酰胺均可产生遗传毒害, 因此, 有关的作业人员应注意防护。     

Carrier-mediated uptake of glyphosate in broad bean (Vicia faba) via a phosphate transporter

The possibility that the herbicide glyphosate (N-phosphonomethylglycine) may be taken up in plant cells via a phosphate transporter of the plasma membrane was investigated using protoplasts of broad bean leaves ( Vicia faba L.). Phosphonoformic acid, a powerful inhibitor of phosphate transport in animal cells, was first demonstrated to be a competitive inhibitor of phosphate uptake inbroad bean protoplasts. Glyphosate was able to inhibit phosphate uptake into the protoplasts, and to protect partially the phosphate transporter from inhibition by phosphonoformic acid. Concentration dependence studies showed that glyphosate uptake exhibited a saturable phase at low glyphosate concentrations (0. 5 to 3 μ M ), superimposed by a linear uptake at higher concentrations (up to 100 μ M ). Inhibition of glyphosate uptake by para -chloromercuribenzene sulphonic acid, sodium azide and carbonyl-cyanide- m -chlorophenylhydrazone was much stronger at 1 than at 100 μ M glyphosate. Kinetics indicated that the saturable component of glyphosate transport was competitively inhibited by either phosphate or phosphonoformic acid. It is concluded that glyphosate can be absorbed via a phosphate transporter of the plasma membrane     

Cloning and expression of amino acid transporters from broad bean

This work describes the isolation of a full-length (VfAAP2) and three partial amino acid transporter genes (VfAAPa, VfAAPb, VfAAPc) from broad bean (Vicia faba L.). The function of VfAAP2 was tested by heterologous expression in a yeast mutant deficient in proline uptake. VfAAP2 mediates proton-dependent proline uptake with an apparent K_m of about 1 mM. Analysis of substrate specificity by competition experiments showed that aromatic amino acids, neutral aliphatic acids and L-citrulline are the best competitors, whereas basic amino acids do not compete with proline. Northern analysis indicates that all VfAAPs exhibit different patterns of expression. VfAAP2 is most strongly expressed in the stem and at a lower level in sink leaves and pods. VfAAPa, VfAAPb and VfAAPc are most strongly expressed in the flowers, but their expression in the other organs varies.     

Effect of Inoculation with Various Rhizobia Strains on the Fatty Acid Composition of Peribacteroid and Bacteroid Membranes of the Nodule Symbiosomes

The fatty acid (FA) composition of bacteroid and peribacteroid membranes was studied in the symbiotic pairs differing in their nitrogen-fixing efficiency; the results are compared with the FA composition of plasmalemma and free-living rhizobia. The experiments involved lupine plants inoculated with strains of Bradyrhizobium lupini359a (Nod⁺Fix⁺) and 400 (Nod⁺Fix L) manifesting high and low nitrogen-fixing efficiency, respectively, and broad bean plants inoculated with strains of Rhizobium leguminosarum97 (Nod⁺Fix⁺) and 87 (Nod⁺Fix L) of high and low nitrogen-fixing efficiency, respectively. We showed that the rhizobia of the strains 359a and 97 were able to form nodules with peribacteroid membranes containing FA mainly or exclusively of plant origin. These strains were able to develop effective symbiotic pairs with legume plants. The use of strains 400 and 87 resulted in the formation of nodules with peribacteroid membranes containing typical bacterial (branched-chain) FAs; these strains were characterized by an ineffective symbiosis.     

Ferrous iron is transported across the peribacteroid membrane of soybean nodules

Symbiosomes and bacteroids isolated from soybean (Glycine max Merr.) nodules are able to take up ferrous iron. This uptake activity was completely abolished in the presence of ferrous-iron chelators. The kinetics of uptake were characterized by initially high rates of iron internalization, but no saturation was observed with increasing iron concentration. This process does not appear to involve the ferric reductase of the peribacteroid membrane. The transport of ferrous iron was inhibited by other transition metals, particularly copper. Ferrous iron was taken up by symbiosomes more efficiently than the ferric form. This indicates that the iron transport from the plant host cell to the microsymbiont in vivo may occur mainly as the ferrous form. Received: 11 February 1998 / Accepted: 29 May 1998     

Ionic and osmotic components of salt stress specifically modulate net ion fluxes from bean leaf mesophyll

Ionic mechanisms of salt stress perception were investigated by non‐invasive measurements of net H⁺, K⁺, Ca²⁺, Na⁺, and Cl^? fluxes from leaf mesophyll of broad bean (Vicia faba L.) plants using vibrating ion‐selective microelectrodes (the MIFE technique). Treatment with 90 m M NaCl led to a significant increase in the net K⁺ efflux and enhanced activity of the plasma membrane H⁺‐pump. Both these events were effectively prevented by high (10 m M ) Ca²⁺ concentrations in the bath. At the same time, no significant difference in the net Na⁺ flux has been found between low‐ and high‐calcium treatments. It is likely that plasma membrane K⁺ and H⁺ transporters, but not the VIC channels, play the key role in the amelioration of negative salt effects by Ca²⁺ in the bean mesophyll. Experiments with isotonic mannitol application showed that cell ionic responses to hyperosmotic treatment are highly stress‐specific. The most striking difference in response was shown by K⁺ fluxes, which varied from an increased net K⁺ efflux (NaCl treatment) to a net K⁺ influx (mannitol treatment). It is concluded that different ionic mechanisms are involved in the perception of the ‘ionic’ and ‘osmotic’ components of salt stress.