I. M. Andreev

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.

Histidine-mediated xylem loading of zinc is a species-wide character in Noccaea caerulescens

Histidine plays a crucial role in nickel (Ni) translocation in Ni-hyperaccumulating plants. Here, we investigated its role in zinc (Zn) translocation in four accessions of the Zn hyperaccumulator, Noccaea caerulescens, using the related non-hyperaccumulator, Thlaspi arvense, as a reference. We compared the effects of exogenous histidine supply on Zn xylem loading, and of Zn-histidine complex formation on Zn uptake in energized tonoplast vesicles. The Zn distribution patterns over root tissues were also compared. Exogenous histidine supply enhanced Zn xylem loading in all the N. caerulescens accessions, but decreased it in T. arvense. Zn distribution patterns over root tissues were similar, apart from the accumulation in cortical and endodermal cells, which was much lower in N. caerulescens than in T. arvense. Zn uptake in energized tonoplast vesicles was inhibited significantly in N. caerulescens, but not affected significantly in T. arvense, when Zn was supplied in combination with histidine in a 1:2 molar ratio. Histidine-mediated Zn xylem loading seems to be a species-wide character in N. caerulescens. It may well have evolved as a component trait of the hyperaccumulation machinery for Zn, rather than for Ni.

Functional identification of electrogenic Na+-translocating ATPase in the plasma membrane of the halotolerant microalga Dunaliella maritima.

The hypothesis that the primary Na+‐pump, Na+‐ATPase, functions in the plasma membrane (PM) of halotolerant microalga Dunaliella maritima was tested using membrane preparations from this organism enriched with the PM vesicles. The pH profile of ATP hydrolysis catalyzed by the PM fractions exhibited a broad optimum between pH 6 and 9. Hydrolysis in the alkaline range was specifically stimulated by Na+ ions. Maximal sodium dependent ATP hydrolysis was observed at pH 7.5–8.0. On the assumption that the ATP‐hydrolysis at alkaline pH values is related to a Na+‐ATPase activity, we investigated two ATP‐dependent processes, sodium uptake by the PM vesicles and generation of electric potential difference (Δψ) across the vesicle membrane. PM vesicles from D. maritima were found to be able to accumulate 22Na+ upon ATP addition, with an optimum at pH 7.5–8.0. The ATP‐dependent Na+ accumulation was stimulated by the permeant NO 3 ‐ anion and the protonophore CCCP, and inhibited by orthovanadate. The sodium accumulation was accompanied by pronounced Δψ generation across the vesicle membrane. The data obtained indicate that a primary Na+ pump, an electrogenic Na+‐ATPase of the P‐type, functions in the PM of marine microalga D. maritima.

Functions of the Vacuole in Higher Plant Cells

Current views of the activities inherent in the vacuole as a multifunctional compartment in higher plant cells are outlined. The available data indicate that the vacuole is involved in ion homeostasis of the cytosol, storing products of the primary and secondary metabolism, osmoregulation, generation of defense responses of plant cells under biotic and abiotic stress, and programmed cell death. Transport of diverse molecules and ions across the vacuolar membrane, i.e., the tonoplast, plays the major role in all these functional activities of the vacuole. Much progress toward the identification of the transport systems located in this membrane and the elucidation of the mechanisms underlying their functioning has been achieved in the last 10–15 years and has given new insights into the role of the vacuole in the integration and regulation of plant cell metabolism.

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.

Penetration of Fullerene C60 derivatives Through Biological Membranes

Abstract Penetration of fullerene C60 in hydrated molecular‐colloidal form (FMC) and various C60 water‐soluble derivatives (FDs) through membranes of human erythrocytes, platelets and symbiosomes (subcellular organelles of plant origin) were tested. The FDs bearing amino acids induced pronounced depolarization of symbiosome membranes energized with Mg‐ATP. In erythrocytes and platelets incubated in K+‐free medium in the presence of FCCP, FDs with malonic acid pendants promoted acidification of the intracellular medium thereby simulating an effect of the K+ ionophore valinomycin. Dissipation of ΔpH artificially induced on the plasma membrane of these cells was observed in the presence of C60‐γ‐aminobutiric acid which, in addition, strongly stimulated Mg‐ATP‐dependent generation of membrane potential on symbiosome membranes. C60‐Arg was shown to dissipate K+‐diffusion potential on erythrocyte membranes induced by valinomycin. Fullerene C60 used in hydrated molecular‐colloidal form (FMC) also entered symbiosomes and platelets as evidenced by the quenching of the fluorescence of the Ca2+ indicator chlorotetracycline localized in the interior of these cells. These findings provide evidence for ease of permeation of these fullerene‐based compounds through biological membranes from different type cells.

Germination and In Vitro Growth of Petunia Male Gametophyte Are Affected by Exogenous Hormones and Involve the Changes in the Endogenous Hormone Level

The data obtained characterize the changes in the contents of endogenous phytohormones (IAA, cytokinins, GA, and ABA) in germinating pollen grains and growing pollen tubes of a self-compatible clone of petunia (sPetunia hybrida L.) within an 8-h period under in vitro conditions. The hydration and initiation of germination of pollen grains brought the ABA content down to a zero level, while the levels of GA, IAA, and cytokinins increased 1.5–2-fold. Later, in the growing pollen tubes, the GA content increased twofold, while the levels of IAA and cytokinins decreased. The exogenous ABA and GA3 considerably promoted pollen germination and pollen tube growth; however, only the treatment with GA3 produced the maximum length of pollen tubes. The exogenous IAA promoted and the exogenous cytokinins hindered the growth of pollen tubes. The membrane potential, as assessed with a potential-sensitive dye diS-C3-(5), considerably increased in the pollen grains treated with ABA and benzyladenine, whereas IAA and GA3 did not practically affect it. The authors conclude that the mature pollen grains contain the complete set of hormones essential for pollen germination and pollen tube growth. ABA, GA, and IAA together with cytokinins control the processes of pollen grain hydration, germination, and pollen tube growth, respectively.

Functional Identification of H+-ATPase and Na+/H+ Antiporter in the Plasma Membrane Isolated from the Root Cells of Salt-Accumulating Halophyte Suaeda altissima

A membrane fraction enriched in plasma membrane (PM) vesicles was isolated from the root cells of a salt-accumulating halophyte Suaeda altissima (L.) Pall. by means of centrifugation in discontinuous sucrose density gradient. The PM vesicles were capable of generating ΔpH at their membrane and the transmembrane electric potential difference (Δψ). These quantities were measured with optical probes, acridine orange and oxonol VI, sensitive to ΔpH and Δψ, respectively. The ATP-dependent generation of ΔpH was sensitive to vanadate, an inhibitor of P-type ATPases. The results contain evidence for the functioning of H+-ATPase in the PM of the root cells of S. altissima. The addition of Na+ and Li+ ions to the outer medium resulted in dissipation of ΔpH preformed by the H+-ATPase, which indicates the presence in PM of the functionally active Na+/H+ antiporter. The results are discussed with regard to involvement of the Na+/H+ antiporter and the PM H+-ATPase in loading Na+ ions into the xylem of S. altissima roots.

Calmodulin stimulation of Ca2+/nH+ antiport across the vacuolar membrane of sugar beet taproot.

Summary Using preparations of vacuoles and vacuolar membranes isolated from storage tissue of sugar beet ( Beta vulgaris L.), it was shown that Ca 2+ ions caused the dissipation of an ATP-generated or artificially formed transtonoplast pH gradient monitored by the absorption change of acridine orange. Ca 2+ -dependent Δ pH decreasing at the tonoplast is blocked by La 3+ and is due to the action of the Ca 2+ /nH + antiporter associated with this membrane and characterized by an apparent K m of 10 μM for Ca 2+ at pH 8.0. It was found that calmodulin greatly accelerated transtonoplast Δ pH dissipation in the presence of Ca 2+ . This effect is independent of ATP and blocked by La 3+ or removing Ca 2+ from the medium using EGTA. Calmodulin stimulation of Ca 2+ /H + exchange across the tonoplast was shown to be due to a dramatic drop (almost 1000-fold) in apparent K m of Ca 2+ /nH + antiporter for Ca 2+ . This result led us to suggest that calmodulin directly interacts with the carrier in question and such interaction takes place even at Ca 2+ concentrations lower than the K d of the Ca 2+ -calmodulin complex.