Jasmonic Acid Effect on Cucumis sativus L. Growth Is Related to Inhibition of Plasma Membrane Proton Pump and the Uptake and Assimilation of Nitrates.

When plants are exposed to environmental stress, their growth is inhibited. Under such conditions, controlled inhibition of growth is beneficial for plant survival. Jasmonic acid (JA) is a well-known phytohormone that limits plant growth, which has been confirmed in several species. However, its role in cucumber seedlings has not yet been comprehensively investigated. For this reason, we aimed to determine the involvement of JA in the regulation of proteins crucial for growth including plasma membrane proton pump (PM H+-ATPase), PM nitrate transporters, and nitrate reductase (NR). Treatment of cucumber seedlings with JA not only limited their growth but also increased the H2O2 content in their roots. The main sources of ROS generated for signalling purposes are PM NADPH oxidase (RBOH) and superoxide dismutase (SOD). Exposure of seedlings to JA induced the expression of some CsRBOH and SOD encoding genes, suggesting that ROS signalling can be activated by JA. As a consequence of JA exposure, the activity of all analysed proteins was inhibited and the expression of their genes was modified. The results indicate that reduction of PM H+-ATPase activity and the related decrease in nitrate uptake and assimilation are responsible for the root growth retardation of JA-treated plants.

Regulation of V-ATPase by Jasmonic Acid: Possible Role of Persulfidation.

Vacuolar H+-translocating ATPase (V-ATPase) is a proton pump crucial for plant growth and survival. For this reason, its activity is tightly regulated, and various factors, such as signaling molecules and phytohormones, may be involved in this process. The aim of this study was to explain the role of jasmonic acid (JA) in the signaling pathways responsible for the regulation of V-ATPase in cucumber roots and its relationship with other regulators of this pump, i.e., H2S and H2O2. We analyzed several aspects of the JA action on the enzyme, including transcriptional regulation, modulation of protein levels, and persulfidation of selected V-ATPase subunits as an oxidative posttranslational modification induced by H2S. Our results indicated that JA functions as a repressor of V-ATPase, and its action is related to a decrease in the protein amount of the A and B subunits, the induction of oxidative stress, and the downregulation of the E subunit persulfidation. We suggest that both H2S and H2O2 may be downstream components of JA-dependent negative proton pump regulation. The comparison of signaling pathways induced by two negative regulators of the pump, JA and cadmium, revealed that multiple pathways are involved in the V-ATPase downregulation in cucumber roots.

Involvement of NO in V-ATPase Regulation in Cucumber Roots under Control and Cadmium Stress Conditions.

Nitric oxide (NO) is a signaling molecule that participates in plant adaptation to adverse environmental factors. This study aimed to clarify the role of NO in the regulation of vacuolar H+-ATPase (V-ATPase) in the roots of cucumber seedlings grown under control and Cd stress conditions. In addition, the relationship between NO and salicylic acid (SA), as well as their interrelations with hydrogen sulfide (H2S) and hydrogen peroxide (H2O2), have been verified. The effect of NO on V-ATPase was studied by analyzing two enzyme activities, the expression level of selected VHA genes and the protein level of selected VHA subunits in plants treated with a NO donor (sodium nitroprusside, SNP) and NO biosynthesis inhibitors (tungstate, WO42- and N-nitro-L-arginine methyl ester, L-NAME). Our results indicate that NO functions as a positive regulator of V-ATPase and that this regulation depends on NO generated by nitrate reductase and NOS-like activity. It was found that the mechanism of NO action is not related to changes in the gene expression or protein level of the V-ATPase subunits. The results suggest that in cucumber roots, NO signaling interacts with the SA pathway and, to a lesser extent, with two other known V-ATPase regulators, H2O2 and H2S.

Structural and Functional Diversity of Two ATP-Driven Plant Proton Pumps.

Two ATP-dependent proton pumps function in plant cells. Plasma membrane H+-ATPase (PM H+-ATPase) transfers protons from the cytoplasm to the apoplast, while vacuolar H+-ATPase (V-ATPase), located in tonoplasts and other endomembranes, is responsible for proton pumping into the organelle lumen. Both enzymes belong to two different families of proteins and, therefore, differ significantly in their structure and mechanism of action. The plasma membrane H+-ATPase is a member of the P-ATPases that undergo conformational changes, associated with two distinct E1 and E2 states, and autophosphorylation during the catalytic cycle. The vacuolar H+-ATPase represents rotary enzymes functioning as a molecular motor. The plant V-ATPase consists of thirteen different subunits organized into two subcomplexes, the peripheral V1 and the membrane-embedded V0, in which the stator and rotor parts have been distinguished. In contrast, the plant plasma membrane proton pump is a functional single polypeptide chain. However, when the enzyme is active, it transforms into a large twelve-protein complex of six H+-ATPase molecules and six 14-3-3 proteins. Despite these differences, both proton pumps can be regulated by the same mechanisms (such as reversible phosphorylation) and, in some processes, such as cytosolic pH regulation, may act in a coordinated way.

Identification and Functional Analysis of Two Mitoferrins, CsMIT1 and CsMIT2, Participating in Iron Homeostasis in Cucumber.

Mitochondria are one of the major iron sinks in plant cells. Mitochondrial iron accumulation involves the action of ferric reductase oxidases (FRO) and carriers located in the inner mitochondrial membrane. It has been suggested that among these transporters, mitoferrins (mitochondrial iron transporters, MITs) belonging to the mitochondrial carrier family (MCF) function as mitochondrial iron importers. In this study, two cucumber proteins, CsMIT1 and CsMIT2, with high homology to Arabidopsis, rice and yeast MITs were identified and characterized. CsMIT1 and CsMIT2 were expressed in all organs of the two-week-old seedlings. Under Fe-limited conditions as well as Fe excess, the mRNA levels of CsMIT1 and CsMIT2 were altered, suggesting their regulation by iron availability. Analyses using Arabidopsis protoplasts confirmed the mitochondrial localization of cucumber mitoferrins. Expression of CsMIT1 and CsMIT2 restored the growth of the Δmrs3Δmrs4 mutant (defective in mitochondrial Fe transport), but not in mutants sensitive to other heavy metals. Moreover, the altered cytosolic and mitochondrial Fe concentrations, observed in the Δmrs3Δmrs4 strain, were recovered almost to the levels of WT yeast by expressing CsMIT1 or CsMIT2. These results indicate that cucumber proteins are involved in the iron transport from the cytoplasm to the mitochondria.

Water and Nutrient Recovery for Cucumber Hydroponic Cultivation in Simultaneous Biological Treatment of Urine and Grey Water.

Water and nutrient deficiencies in soil are becoming a serious threat to crop production. Therefore, usable water and nutrient recovery from wastewater, such as urine and grey water, should be considered. In this work, we showed the possibility of using grey water and urine after processing in an aerobic reactor with activated sludge in which the nitrification process takes place. The resulting liquid (nitrified urine and grey water, NUG) contains three potential factors that can adversely affect plant growth in a hydroponic system: anionic surfactants, nutrient deficits, and salinity. After dilution and supplementation with small amounts of macro- and micro-elements, NUG was suitable for cucumber cultivation. Plant growth on this modified medium (enriched nitrified urine and grey water, NUGE) was similar to that of plants cultivated on Hoagland solution (HS) and reference commercial fertilizer (RCF). The modified medium (NUGE) contained a significant amount of sodium (Na) ions. Therefore, typical effects of salt stress were observed in cucumber plants, including reduced chlorophyll levels, slightly weaker photosynthesis parameters, increased H2O2 levels, lipid peroxidation, ascorbate peroxidase (APX) activity, and proline content in the leaves. In addition, reduced protein levels were observed in plants treated with recycled medium. At the same time, lower nitrate content in tissues was found, which may have resulted from their intensive use by nitrate reductase (NR), the activity of which significantly increased. Although cucumber is a glycophyte, it grew very well in this recycled medium. Interestingly, salt stress and possibly anionic surfactants promoted flower formation, which in turn could positively affect plant yield.

Role of Plasma Membrane NADPH Oxidase in Response to Salt Stress in Cucumber Seedlings.

Plasma membrane NADPH oxidases (RBOHs, EC 1.6.3.1) are known as the main ROS generators involved in plant adaptation to stress conditions. In the present work, regulation of NADPH oxidase was analyzed in cucumber (Cucumis sativus L. var. Krak) seedlings exposed to salinity. RBOH activity and gene expression, as well as H2O2 content, were determined in the roots of plants treated with 50 or 100 mM NaCl for 1 h, and 50 mM NaCl for 1 or 6 days. It was found that enzyme activity increased in parallel with an enhancement in the H2O2 level in roots exposed to 100 mM NaCl for 1 h, and to 50 mM NaCl for 1 day. The expression of some CsRboh genes was induced by salt. Moreover, an increase in the activity of G6PDH, providing the substrate for the NADPH oxidase, was observed. In seedlings subjected to salinity for a longer time, antioxidant enzymes-including superoxide dismutase, catalase, and ascorbate peroxidase-were activated, participating in maintaining a steady-state H2O2 content in the root cells. In conclusion, NADPH oxidase and endogenous H2O2 up-regulation seem to be early events in cucumber response to salinity.

Involvement of Diamine Oxidase in Modification of Plasma Membrane Proton Pump Activity in Cucumis sativus L. Seedlings under Cadmium Stress.

Cucumber (Cucumis sativus L.) is a crop plant being the third most-produced vegetable developed as a new model plant. Heavy metal pollution is a serious global problem that affects crop production. An industrial activity has led to high emissions of Cd into the environment. Plants realize adaptive strategies to diminish the toxic effects of Cd. They can remove excess toxic ions of heavy metals from the cytoplasm to the outside of cells using the metal/proton antiport. The proton gradient needed for the action of the antiporter is generated by the plasma membrane (PM) H+-ATPase (EC 3.6.3.14). We have shown that treatment of cucumber plants with Cd stimulated the diamine oxidase (DAO, EC 1.4.3.6) activity in roots. Under cadmium stress, the PM H+-ATPase activity also increased in cucumber seedlings. The stimulating effect of Cd on the PM H+-ATPase activity and expression of three genes encoding this enzyme (CsHA2, CsHA4, CsHA8) was reduced by aminoguanidine (AG, a DAO inhibitor). Moreover, we have observed that H2O2 produced by DAO promotes the formation of NO in the roots of seedlings. The results presented in this work showed that DAO may be an element of the signal transduction pathway, leading to enhanced PM H+-ATPase activity under cadmium stress.

Interaction between the signaling molecules hydrogen sulfide and hydrogen peroxide and their role in vacuolar H+ -ATPase regulation in cadmium-stressed cucumber roots.

Vacuolar H+ -ATPase (V-ATPase; EC 3.6.3.14) is the main enzyme responsible for generating a proton gradient across the tonoplast. Under cadmium (Cd) stress conditions, V-ATPase activity is inhibited. In the present work, hydrogen sulfide (H2 S) and hydrogen peroxide (H2 O2 ) cross-talk was analyzed in cucumber (Cucumis sativus L.) seedlings exposed to Cd to explain the role of both signaling molecules in the control of V-ATPase. V-ATPase activity and gene expression as well as H2 S and H2 O2 content and endogenous production were determined in roots of plants treated with 100 µM CdCl2 and different inhibitors or scavengers. It was found that H2 S donor improved photosynthetic parameters in Cd-stressed cucumber seedlings. Cd-induced stimulation of H2 S level was correlated with the increased activities of the H2 S-generating desulfhydrases. Increased H2 O2 and lowered H2 S contents in roots were able to reduce V-ATPase activities similar to Cd. H2 O2 and H2 S-induced modulations in V-ATPase activities were not closely related to the transcript level of encoding genes, suggesting posttranslational modifications of enzyme protein. On the other hand, exogenous H2 O2 raised H2 S content in root tissues independently from the desulfhydrase activity. Although treatment of control plants with H2 S significantly stimulated NADPH oxidase activity and gene expression, H2 S did not affect H2 O2 accumulation in roots exposed to Cd. The results suggest the existence of two pathways of H2 S generation in Cd-stressed cucumber roots. One involves desulfhydrase activity, as was previously demonstrated in different plant species. The other, the desulfhydrase-independent pathway induced by H2 O2 /NADPH oxidase, may protect V-ATPase from inhibition by Cd.

Involvement of NR and PM-NR in NO biosynthesis in cucumber plants subjected to salt stress.

Nitrate reductase (NR) mainly reduces nitrate to nitrite. However, in certain conditions it can reduce nitrite to NO. In plants, a plasma membrane-associated form of NR (PM-NR) is present. It produces NO2- for nitrite NO/reductase (Ni-NOR), which can release NO into the apoplastic space. The effect of 50 mM NaCl on NO formation and the involvement of NR in NO biosynthesis were studied in cucumber seedling roots under salt stress. In salt-stressed roots, the amount of NO was higher than in control. The application of tungstate abolished the increase of NO level in stressed roots, indicating that NR was responsible for NO biosynthesis under the test conditions. The involvement of other molybdoenzymes was excluded using specific inhibitors. Furthermore, higher cNR and PM-NR activities were observed in NaCl-treated roots. The increase in NR activity was due to the stimulation of CsNR genes expression and posttranslational modifications, such as enzyme dephosphorylation. This was confirmed by Western blot analysis. Moreover, the increase of nitrite tissue level in short-term stressed roots and the nitrite/nitrate ratio, with a simultaneous decrease of nitrite reductase (NiR) activity, in both short- and long-term stressed roots, could promote the production of NO by NR in roots under salt stress.

Modification of plasma membrane NADPH oxidase activity in cucumber seedling roots in response to cadmium stress.

The aim of this study was to investigate the effect of cadmium on plasma membrane (PM) NADPH oxidase activity in cucumber roots. Plants were treated with cadmium for 1, 3 or 6 days. Some of the plants after 3-day exposure to cadmium were transferred to a medium without the heavy metal for the next 3 days. Treatment of plants with cadmium for 6 days stimulated the activity of NADPH oxidase. The highest stimulation of O2(•-) production by NADPH oxidase was observed in post-stressed plants, which was correlated with the stimulation of activity of PM H(+)-ATPase in the same conditions. In order to examine the effects of cadmium stresses on the expression level of genes encoding NADPH oxidase, putative cucumber homologs encoding RBOH proteins were selected and a real-time PCR assay was performed. NADPH is a substrate for oxidase; thus alterations in the activity of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP-isocitrate dehydrogenase and NADP-malic enzyme under cadmium stress were studied. The activity of NADPH dehydrogenases was increased under cadmium stress. The results indicate that PM NADPH oxidase could be involved in plants' response to cadmium stress by affecting the activity of PM H(+)-ATPase, and NADPH-generating enzymes could play important roles in this process.

Transcriptional regulation of the V-ATPase subunit c and V-PPase isoforms in Cucumis sativus under heavy metal stress.

Two electrogenic proton pumps, vacuolar H(+) transporting ATPase (V-ATPase, EC 3.6.3.14) and vacuolar H(+) transporting inorganic pyrophosphatase (V-PPase, EC 3.6.1.1), co-exist in the vacuolar membrane of plant cells. In this work, all CsVHA and CsVHP genes encoding V-ATPase and V-PPase, respectively, were identified in the cucumber genome. Among them, three CsVHA-c genes for V-ATPase subunit c and two CsVHP1 genes for type I V-PPase were analyzed in detail. Individual isogenes were differentially regulated in plant tissues and during plant development as well as under changing environmental conditions. CsVHA-c1 and CsVHA-c2 showed similar tissue-specific expression patterns with the highest levels in stamens and old leaves. CsVHP1;1 was predominantly expressed in roots and female flowers. In contrast, both CsVHA-c3 and CsVHP1;2 remained in a rather constant ratio in all examined cucumber organs. Under heavy metal stress, the transcript amount of CsVHA-c1 and CsVHP1;1 showed a pronounced stress-dependent increase after copper and nickel treatment. CsVHA-c3 was upregulated by nickel only whereas CsVHA-c2 was induced by all metals with the most visible effect of copper. Additionally, CsVHP1;2 showed a tendency to be upregulated by copper and zinc. We propose that CsVHA-c1, CsVHA-c2 and CsVHP1;1 are essential elements of mechanisms involved in adaptation of cucumber plants to copper toxicity.

Modification of plasma membrane proton pumps in cucumber roots as an adaptation mechanism to salt stress.

The effect of salt stress (50mM NaCl) on modification of plasma membrane (PM) H(+)-ATPase (EC 3.6.3.14) activity in cucumber roots was studied. Plants were grown under salt stress for 1, 3 or 6 days. In salt-stressed plants, weak stimulation of ATP hydrolytic activity of PM H(+)-ATPase and significant stimulation of proton transport through the plasma membrane were observed. The H(+)/ATP coupling ratio in the plasma membrane of plants subjected to salt stress significantly increased. The greatest stimulation of PM H(+)-ATPase was in 6-day stressed plants. Increased H2O2 accumulation under salt stress conditions in cucumber roots was also observed, with the greatest accumulation observed in 6-day stressed plants. Additionally, during the sixth day of salinity, there appeared heat shock proteins (HSPs) 17.7 and 101, suggesting that repair processes and adaptation to stress occurred in plants. Under salt stress conditions, fast post-translational modifications took place. Protein blot analysis with antibody against phosphothreonine and 14-3-3 proteins showed that, under salinity, the level of those elements increased. Additionally, under salt stress, activity changes of PM H(+)-ATPase can partly result from changes in the pattern of expression of PM H(+)-ATPase genes. In cucumber seedlings, there was increased expression of CsHA10 under salt stress and the transcript of a new PM H(+)-ATPase gene isoform, CsHA1, also appeared. Accumulation of the CsHA1 transcript was induced by NaCl exposure, and was not expressed at detectable levels in roots of control plants. The appearance of a new PM H(+)-ATPase transcript, in addition to the increase in enzyme activity, indicates the important role of the enzyme in maintaining ion homeostasis in plants under salt stress.

Mechanism of Cd and Cu action on the tonoplast proton pumps in cucumber roots.

The effect of Cd and Cu on the tonoplast proton pumps, V-ATPase (EC 3.6.3.14) and V-PPase (EC 3.6.1.1) was investigated in cucumber roots subjected to 10 µM metals for 3 and 6 days. Both hydrolytic and transporting activities of V-ATPase as well as V-PPase increased under copper stress. In contrast, all activities examined were inhibited after the exposure of plants to cadmium. Cd and Cu changed the efficiency of coupling between proton transport and ATP hydrolysis whereas H(+) /PP(i) stoichiometry was not modified. Pre-incubation of control tonoplast vesicles with copper caused the stimulation of V-ATPase as well as V-PPase, indicating direct activation by Cu ions. Pre-treatment with cadmium had no significant effect on the activities of both enzymes. The gene expression and western blot analyses showed that observed modifications in enzyme activities were not related to the changes in the transcript levels of genes encoding V-ATPase subunit A and c, and V-PPase or in amounts of enzyme proteins. Moreover, the addition of reduced or oxidized glutathione (GSH and GSSG) to the reaction medium containing tonoplast vesicles isolated from stressed roots did not change the activity level of either enzyme when compared with the controls, suggesting that heavy metal-induced modifications are not simple reversible redox modulations.

Abscisic acid and hydrogen peroxide induce modification of plasma membrane H(+)-ATPase from Cucumis sativus L. roots under heat shock.

We examined the effect of heat shock (HS), for 2 h at 48°C, on plasma membrane H(+)-ATPase (PM-H(+)-ATPase) measured as the hydrolytic and H(+)-pumping activity. Some of the plants were transferred after 2 h HS to control temperature for another 24 h, as post-stressed (PS) plants. A significant increase of PM-H(+)-ATPase in plants subjected to HS was observed. The stimulation of PM-H(+)-ATPase was higher in PS plants. Estimation of transcript levels of cucumber PM-H(+)-ATPase in roots indicated that the action of HS affected gene expression levels. Transcript levels of two isoforms, CsHA4 and CsHA8, in PS plants were elevated. The expression of PM-H(+)-ATPase genes was not affected in plants treated for 2 h with HS. HS elevated the endogenous level of abscisic acid (ABA) both in plants treated for 2 h with HS and in PS plants. Moreover, in PS plants, a distinctly higher level of H(2)O(2) was observed. It was also demonstrated that transcript levels of PM-H(+)-ATPase were elevated in cucumber roots after 24-h treatment of plants with ABA or H(2)O(2). Both of these compounds seem to play an important role in increasing ATPase activity during heat stress, because the use of the inhibitors tungstate and DPI restrained stimulation of PM-H(+)-ATPase activity by heat. Moreover, protein blot analysis with an antibody against phosphothreonine and 14-3-3 protein indicated that increased activity of PM-H(+)-ATPase under HS resulted from phosphorylation of the enzyme. Taken together, the data presented here suggest that, under post-heat stress conditions, abscisic acid and hydrogen peroxide are involved in PM-ATPase modification, through stimulation of gene expression of that PM proton pump. Moreover, heat treatment of cucumber plants results in increased phosphorylation of PM-ATPase and thus fast post-translational modification, leading to activation of the enzyme protein.

Different effect of cadmium and copper on H+-ATPase activity in plasma membrane vesicles from Cucumis sativus roots.

The effect of heavy metals on plasma membrane (PM) H(+)-ATPase (EC 3.6.3.14) activity in cucumber (Cucumis sativus) roots was studied. The aim of this work was to explain the mechanism of modification of the PM H(+)-ATPase activity in plants subjected to heavy metals. Plants were treated with 10 µM Cd or Cu for 6 d. After 3 d exposure to the heavy metals, some of the plants were transferred to control conditions for a further 3 d (3/3 plants). The activity of PM H(+)-ATPase was found to be increased in plants treated with heavy metals. The highest activity measured as proton transport was observed in 3/3 plants. Estimation of transcript levels of C. sativus PM H(+)-ATPase in roots indicated that the action of Cd, but not Cu, affected the gene expression level. Transcript levels of C. sativus PM H(+)-ATPase (CsHA2, CsHA3, CsHA4, CsHA8, and CsHA9) genes increased in roots treated with Cd. Moreover, Western blot analysis with antibody against phosphothreonine and 14-3-3 protein indicated that increased activity of PM H(+)-ATPase under heavy-metal stress resulted from phosphorylation of the enzyme. It was found that Cu markedly increased the activity of catalase and ascorbate peroxidase and reduced the level of H(2)O(2) in cucumber roots. In contrast, Cd did not affect these parameters. These results indicate that Cd and Cu can, in different ways, lead to modification of PM H(+)-ATPase activity. Additionally, it was observed that treatment of plants with heavy metals led to an increased level of heat-shock proteins in the tissues. This suggests that the plants had started adaptive processes to survive adverse conditions, and increased PM H(+)-ATPase activity could further enhance the repair processes in heavy-metal-stressed plants.

Response of plasma membrane H(+)-ATPase to low temperature in cucumber roots.

The effect of low temperature (LT, 10°C) on modification of plasma membrane (PM) H(+)-ATPase (EC 3.6.3.14) activity in cucumber roots was studied. Plants were grown under LT for 3 or 6 days. Some of the plants after 3 days exposure to LT were transferred to control conditions for another 3 days (post-cold, PC). The activity of PM-H(+)-ATPase was decreased in plants treated for 3 days with LT. However, the activity of PM-H(+)-ATPase was higher in plants treated with LT for a longer time and in PC plants as well. Estimation of transcript levels of cucumber PM-H(+)-ATPase in roots indicates that the action of LT involves the gene expression level. The level of PM-H(+)-ATPase mRNA was markedly decreased in roots exposed to LT for 3 days. Moreover, the increased H(+)-ATPase activity in PM isolated from plants treated for 6 days with LT and from PC plants was positively correlated with higher levels of CsHA transcripts. Western blot analysis with an anti-phosphothreonine antibody showed that modification of the activity of PM-H(+)-ATPase under LT stress did not result from phosphorylation/dephosphorylation of the enzyme protein. However, the stimulation of PM-H(+)-ATPase activity in the case of PC plants could partially have emanated from increased activity of PM NAD(P)H oxidoreductase. In addition, modification of the transcript level of proton pump genes could have resulted from the action of H(2)O(2). In PC plants, an increase in H(2)O(2) level was observed. Moreover, treatment of plants with H(2)O(2) induced expression of PM H(+)-ATPase genes.

Differential regulation of vacuolar H+-ATPase and H+-PPase in Cucumis sativus roots by zinc and nickel.

Zinc and nickel, as micronutrients, are essential for all organisms. We investigated the effect of 10 and 100 µM Zn and Ni on two tonoplast proton pumps, vacuolar H+-ATPase (V-ATPase) (EC 3.6.3.14) and vacuolar H+-pyrophosphatase (V-PPase) (EC 3.6.1.1), in cucumber roots. ATP-dependent proton transport as well as ATP hydrolysis, catalyzed by V-ATPase, decreased in roots after exposure of plants to both Zn and Ni under all the examined conditions. In contrast, V-PPase activities, measured as PP(i) hydrolysis and PP(i)-driven H+ transport, were stimulated by lower concentration of metals. However, at higher metal concentration, hydrolytic activity of V-PPase remained unchanged, while PP(i)-dependent proton pumping into the tonoplast vesicles was reduced. When heavy metals were introduced into the enzyme reaction medium, both V-ATPase and V-PPase activities were lowered by Zn and Ni in a similar manner. As the gene expression and immunoblot analyses depicted, observed changes in the activity of both tonoplast proton pumps in response to zinc and nickel were not due to the modification in the expression of the CsVHA-A, CsVHA-c and CsVP genes encoding V-ATPase subunit A and c, and V-PPase, respectively, in cucumber roots or in amounts of enzyme proteins. Moreover, Zn as well as Ni ions did not enhance the lipid peroxidation in the root tonoplast fractions. Comparison of ATP and pyrophosphate contents in the control roots and roots treated with heavy metals revealed that Zn and Ni do not affect the ATP amount but reduce the PP(i) level.

Different responses of tonoplast proton pumps in cucumber roots to cadmium and copper.

Cadmium (Cd) and copper (Cu) effects on the two tonoplast proton pumps were compared in cucumber roots. Different alterations of vacuolar H+ transporting ATPase (V-ATPase) (EC 3.6.3.14) and vacuolar H+ transporting pyrophosphatase (V-PPase) (EC 3.6.1.1) activities under heavy metal stress were investigated. ATP-dependent proton transport and ATP hydrolysis increased after exposure of seedlings to Cu, whereas both decreased in plants stressed with Cd. PP(i) hydrolysis was relatively insensitive to both heavy metals. However, cadmium, but not copper, clearly inhibited PP(i)-driven H+ transport. Changes in enzyme activities were not due to the metal action on the expression of CsVHA-A, CsVHA-c and CsVP genes encoding V-ATPase subunit A and c, and V-PPase, respectively, in cucumber roots. Moreover, immunoblot analysis using specific antibodies against V-ATPase holoenzyme, phosphoserine and phosphothreonine suggested that the phosphorylation at Ser residue in regulatory subunit B of cucumber V-ATPase was not regulated by metals. Oxidative alterations of membrane lipids were measured as malondialdehyde (MDA) content. Cu ions, in contrast to Cd, visibly enhanced the lipid peroxidation in the root tonoplast fractions. Because ATP and PP(i) are absolutely required by V-ATPase and V-PPase, respectively, for proton transport, their contents were determined in the control roots and roots treated with cadmium and copper. Both ATP and pyrophosphate amounts decreased under heavy metal stress.