S. F. Izmailov

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.

Saturation and Utilization of Nitrate Pools in Pea and Sugar Beet Leaves

The critical periods in the saturation of pea and sugar beet leaves with nitrate absorbed by roots were discriminated. In peas, during the first 14 h, all nitrate penetrating leaf cells was concentrated in the cytosol (metabolic pool). During the second period (14–62 h), nitrate began to flow into the vacuole (storage pool), and the filling of the metabolic pool continued. Metabolic pool was saturated by the end of this period (62 h). During the third period (62–110 h), further nitrate accumulation in the cell occurred because of expanding of the storage pool. Its saturation (similarly as total cell saturation) commenced 86 h after the start of nitrate uptake. In sugar beet leaves, both metabolic and storage nitrate pools were saturated by the end of the first period (14 h), and the sizes of these pools did not change during the second period (14–86 h). When pea plants were transferred to the nitrate-free medium, nitrate efflux began from the storage pool until its complete exhausting after 3 days. In sugar beet leaves, nitrate was still present in the storage pool 4 days after plant transfer to the nitrate-free medium. In both crops, nitrate export from the storage pool was aimed at the maintenance of the optimum nitrate concentration in the metabolic pool and, thus, at the maintenance of nitrate reductase activity. A functional diversity of nitrate compartmentation in the cells of various plant species is discussed.

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.

Effect of nitrate on pea sucrose synthase

The in vivo and in vitro nitrate effects on pea (Pisum sativum L.) sucrose synthase (SS) were studied. At the period of plant transition from heterotrophic to autotrophic nutrition, exogenous nitrate (14.2 mM) absorbed in the form of KNO3 and Ca(NO3)2 during 10–20 days activated SS in the roots by 22–100% as compared with plants grown on nitrogen-free medium. Such effect was observed only at plant growing under high light (natural illumination up to 25 klx) and thus their sufficient supplement with sucrose. Under low light (climate-controlled chamber, 2.5 klx), nitrate could not activate SS. In the in vitro experiments, nitrate activated SS exponentially by a dose-dependent mode with the plateau at 3–5 mM, where its activity was increased by 50%. It is supposed that there is a second constituent in SS activation by nitrate, and it carries information about plant carbohydrate status. Possible mechanisms of nitrate-induced SS activation are discussed.

Activities of Sucrose Synthase and Acid Invertase in Pea Seedling Organs

The organ topography of sucrose synthase and soluble acid invertase in pea seedlings at heterotrophic stage (3–14 days) was studied. Sucrose synthase was most active in the roots, with the highest activity on the 6–8th days. In the leaves, its activity decreased from day 3 to day 14. In the stems, sucrose synthase activity was at an invariantly low level. The patterns of sucrose synthase activity in etiolated and green plants were similar. As distinct from sucrose synthase, invertase activity was the highest in the stem, especially in etiolated plants. The peak of its activity was observed on the 6-8th days. In the leaves, invertase activity was lower but its pattern was the same. In the roots, acid invertase activity decreased from the 3rd day and did not depend on illumination. The conclusion is that differences in sucrose synthase and acid invertase activities in roots, leaves, and stem are determined by differences in the import of hydrolytic products of stored compound from the cotyledons as well as by different demands of these organs for these products for the processes of organ expansion and for the maintenance of organ metabolism.

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.

Ca2+/H+ antiport as a possible mechanism of the Ca2+-translocating ATPase functioning in vesicles of bean root nodule’s symbiosome membrane

Using vesicles of symbiosome membrane (SM), it was shown that the Ca2+-ATPase can function as an ATP-energized Ca2+/H+ antiporter. The initial rate of the acidic shift inside the vesicles, as well as the rate of the ITP-dependent alkalization of the medium inside them markedly increased in the presence of valinomycin. This process was rapidly stopped by eosin Y, a known inhibitor of the type IIB Ca2+-ATPase. ITP-dependent uptake of Ca2+ was blocked after the addition to the reaction mixture of nigericin in the presence of K+. Under these conditions, the alkaline shift of pH inside the vesicles occurred, leading to the inhibition of operation of the calcium pump in SM. Evaluation of the pH shifts inside the vesicles by using pH-indicator pyranine confirmed the ion-exchange mechanism of the Ca2+-ATPase functioning in the SM.