P. N. Dubrovo

Functional identification of ATP-driven Ca2+ pump in the peribacteroid membrane of broad bean root nodules

A Ca2+ indicator arsenazo III was used to demonstrate calcium uptake activity of symbiosomes and the peribacteroid membrane (PBM) vesicles isolated from broad bean root nodules and placed in the medium containing ATP and Mg2+ ions. This process was shown to be rapidly stopped by vanadate, completely reversed in the presence of the calcium ionophore A23187 but insensitive to agents abolishing electrical potential or pH difference across the PBM. The presence of an endogenous calcium pool within isolated symbiosomes and bacteroids was detected using a Ca2+ indicator chlortetracycline. These results prove a primary active transport of Ca2+ through the PBM of legume root nodules and provide the first functional identification of an ATP‐driven Ca2+‐pump, most likely Mg2+‐dependent Ca2+‐translocating ATPase, in this membrane.

Characterization of ATP-Hydrolyzing and ATP-Driven proton-translocating activities associated with the peribacteroid membrane from root nodules of Lupinus luteus L

Summary In order to detect and characterize PBM H + -ATPase from root nodules of Lupinus luteus L., ATP-hydrolyzing and ATP-dependent proton translocating activities associated with this membrane were studied using isolated symbiosomes, vesicle PBM preparations and intact plant tissues. It has been found that ATP-hydrolyzing activity of isolated PBMs is sufficiently high (about 100–150 μmoles of μmg protein-h) in the selected pH range (4.5–8.5) and is characterized by the absence of any pronounced pH optimum, little substrate specificity, the absence of selectivity in relation to Mg 2+ and Ca 2+ as stimulators of ATP-hydrolysis and even more sensitivity to Ca 2+ . Mg 2+ -dependent ATP-hydrolyzing activity was moderately decreased in the presence of some known inhibitors of H + -ATPases, such as vanadate, DCCD and nitrate. On the other hand, Ca 2+ -dependent ATP-hydrolysis was significandy inhibited by sodium fluoride. ATP-hydrolyzing activity on the PBM of Lupinus luteus L. with many similar properties was also detected cytochemically in electron microscope studies of intact plant tissues. These findings led us to conclude that at least one more Ca 2+ /Mg 2+ -dependent ATP-hydrolase, in addition to H + -ATPase, is associated with the PBM. This enzyme is likely responsible for the observed background that masks ATP-hydrolyzing activity of the PBM H + -ATPase. ATP-dependent electrogenic proton transport across the PBM was detected in isolated symbiosomes and vesicle PBM preparations using oxonol YI and acridine orange as indicators of ΔΨ and ΔpH, respectively. Transport activity of the PBM H + -ATPase was blocked by the above mentioned ATPase inhibitors, except nitrate, which acted only as permeant anion. These results indicate that the PBM of Lupinus luteus L. contains only one H + -translocating ATPase, which belongs to a P-type H + -ATPase.

Calcium uptake by symbiosomes and the peribacteroid membrane vesicles isolated from yellow lupin root nodules

Summary In order to test whether the symbiosomes of infected cells are able to actively take up calcium ions, preparations of these nitrogen-fixing units and the PBM vesicles isolated from yellow lupin ( Lupinus luteus L.) root nodules were investigated to this end. Ca 2+ uptake was recorded with the use of the metallochromic Ca 2+ indicator arsenazo III added to the incubation medium. It was found that the addition of ATP to symbiosomes suspended in the presence of Mg 2+ and Ca 2+ initiated a gradual removal of calcium from the incubation medium. This process was rapidly inhibited with addition of vanadate, but was resistant to protonophores, the Ca 2+ ionophore A23187, valinomycin in the presence of potassium ions, and erythrosin B, and was gready stimulated by nitrate anions. Qualitatively similar results were obtained with preparations of the PBMs, except that in this case the Ca 2+ ionophore A23187 effectively facilitated the calcium release from the PBM vesicles after the uptake. The data obtained demonstrate primary active transport of calcium across the PBM, which is most likely caused by the activity of the Mg 2+ -dependent Ca 2+ -ATPase associated with this membrane.

Calcium Stores in Symbiosomes from Yellow Lupin Root Nodules

Summary The PPA technique was used for the visualization of calcium in the symbiosomes of yellow lupin root nodule cells at the ultrastructural level both in situ and in vitro. Nodulated plants were grown in pots containing 6 kg of sand with nutrients and various calcium concentrations. At relatively low calcium level (without added calcium or 2 mmol of endogenous calcium per pot) the pyroantimonate calcium precipitates were detected on the cytoplasmic side of the PBM, in the bacteroid cytoplasm, to a much lesser extent within the PBS, and, in addition, in some other subcellular compartments of the infected cells. The observed features in symbiosome calcium distribution became much more profound after increasing the calcium concentration (up to 3 and 6 mmol/pot) in the nutrient medium. In addition, under these conditions numerous electron opaque particles associated with the inner surface of the PBM appeared as well. With the same technique it was found that isolated lupin symbiosomes incubated in the presence of ATP, Mg2+ and Ca2+ are able to accumulate Ca2+, as judged by clearly observed deposits of large size in the PBS in this case. In the experiments with calcium- and ΔpH-sensitive dyes (arsenazo III and acridine orange, respectively), the existence of a calcium pool within the symbiosomes was confirmed by their ability to release calcium ions into the suspension medium after permeabilization of the PBM with the Ca 2+ ionophore A23187. These results provide direct evidence for the hypothesis that the symbiosomes behave as calcium stores in infected cells and, in addition, may be involved in calcium homeostasis in the plant cytosol.

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.

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.

Role of the Peribacteroid Membrane in the Interactions of the Symbiotic Partners in the Nitrogen-Fixing Root Nodules of Lupinus Luteus L.

In legume root nodules, symbiotic Rhizobium are separated from the host cell cytosol by the peribacteroid membrane (PBM). The PBM appears to play an essential role in the interaction between two partners of symbiosis. During the early stage of senescence, or when nodulated plants are submitted to some stress, the PBM is one of the first structures to be degraded and its disappearance parallels the loss of nitrogen- fixing activity; indeed, loss of the PBM could well participate in the senescence process and even provoke it. However, PBM biogenesis, the changes of PBM structure, its functioning in the course of development, and the operation of the symbiotic system have never been completely documented. This involves the role of protein-lipid complexes of the PBM in symbiont interrelations.

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.

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 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 together with calcium ionophore A23187 for 3 h under microaerophilic conditions considerably decreased their nitrogenase activity (NA). Under these experimental conditions, cytochemical electron-microscopic analysis revealed considerable calcium depletion of symbiosomes in the infected nodule cells treated with 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 its initial level in symbiosomes. The experiments on isolated symbiosomes with arsenazo III, a Ca2+ indicator, demonstrated that verapamil inhibited Ca2+ release from them induced by valinomycin in the presence of K+ ions. These data suggest the presence on the peribacteroid membrane of a verapamil-sensitive transporter responsible for Ca2+ release from symbiosomes. A possible role of this transporter in the interaction between symbiotic partners in the infected cells of root nodules is discussed.