Katarzyna Kabała - Academia.edu (original) (raw)

Papers by Katarzyna Kabała

Research paper thumbnail of Modification of plasma membrane NADPH oxidase activity in cucumber seedling roots in response to cadmium stress

Plant science : an international journal of experimental plant biology, 2015

The aim of this study was to investigate the effect of cadmium on plasma membrane (PM) NADPH oxid... more 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 und...

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

Physiologia Plantarum, 2014

Research paper thumbnail of Mechanism of Cd and Cu action on the tonoplast proton pumps in cucumber roots

Physiologia Plantarum, 2013

The effect of Cd and Cu on the tonoplast proton pumps, V-ATPase (EC 3.6.3.14) and V-PPase (EC 3.6... more 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.

Research paper thumbnail of Response of plasma membrane H+-ATPase to low temperature in cucumber roots

Journal of Plant Research, 2012

The effect of low temperature (LT, 10°C) on modification of plasma membrane (PM) H ? -ATPase (EC ... more 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.

Research paper thumbnail of Modification of vacuolar proton pumps in cucumber roots under salt stress

Journal of Plant Physiology, 2008

The time-dependent effect of 50 mM NaCl on the activities of two tonoplast proton pumps was inves... more The time-dependent effect of 50 mM NaCl on the activities of two tonoplast proton pumps was investigated in Cucumis sativus L. var. Krak root cells. Distinct activity profiles for vacuolar proton transporting ATPase (V-ATPase) (EC 3.6.3.14) and vacuolar proton transporting pyrophosphatase (V-PPase) (EC 3.6.1.1) under salinity are presented. ATP-dependent proton transport and ATP hydrolysis increased after 24 h of NaCl exposure, and then decreased in roots stressed with NaCl for 4 and 8 d. Both PP i -driven H + transport and PP i hydrolysis were clearly inhibited by NaCl at all times examined. It was demonstrated that changes in enzyme activities were not due to the salt action on the expression of encoding genes. The levels of specific transcripts for subunit A of V-ATPase (CsVHA-A), subunit c of V-ATPase (CsVHA-c) and V-PPase (CsVP) were similar in cucumber roots untreated (control) and treated with salt. Such results suggest that alterations of proton pump activities under salinity are rather due to the post-translational alterations induced by NaCl.

Research paper thumbnail of The role of polyamines in the regulation of the plasma membrane and the tonoplast proton pumps under salt stress

Journal of Plant Physiology, 2010

Polyamine content (PAs) often changes in response to abiotic stresses. It was shown that the accu... more Polyamine content (PAs) often changes in response to abiotic stresses. It was shown that the accumulation of PAs decreased in roots treated for 24 h with 200 mM NaCl. The role of polyamines (putrescine -PUT, spermidine -SPD and spermine -SPM) in the modification of the plasma membrane(PM) H + -ATPase (EC 3.6.3.6) and the vacuolar(V) H + -ATPase (EC 3.6.3.14) activities in cucumber roots treated with NaCl was investigated. 24 h treatment of seedlings with 50 mM PUT, SPD or SPM lowered the activities of proton pumps in both membranes. The decreased H + -ATPase activity in plasma membranes isolated from the PA-treated roots was positively correlated with a lower level of PM-H + -ATPase CsHA3 transcript. However, transcript levels of PM-H + -ATPase CsHA2 and V-ATPase subunit A and c in roots treated with 50 mM PAs were similar to those in the control. Additionally, treatment of plants with salt markedly increased the activity of the PM-and V-H + -ATPases. However, exposure of plants to 20% PEG had no effect on these activities. These data suggest that, under salt stress conditions, the increase in H + -ATPase activities is caused mainly by the ionic component of salt stress. It seems that the main role of the PAs in the 24 h salt-treated cucumber plants could be a result of their cationic character. The PA levels decreased when concentration of Na + increased, so action of PAs contributes to ionic equilibrium. Moreover, the decrease in the concentration of polyamines, which inhibit the PM-H + -ATPase and the V-H + -ATPase, at least under the studied conditions, seems to be beneficial. Thus, plants can increase salinity tolerance by modifying the biosynthesis of polyamines.

Research paper thumbnail of Comparison of heavy metal effect on the proton pumps of plasma membrane and tonoplast in cucumber root cells

Journal of Plant Physiology, 2008

The effects of 10 mM cadmium, copper and nickel on the activities of vacuolar membrane and plasma... more The effects of 10 mM cadmium, copper and nickel on the activities of vacuolar membrane and plasma membrane (PM) ATP-dependent proton pumps was investigated in Cucumis sativus L. root cells. It was demonstrated that vacuolar H + -ATPase (EC 3.6.3.14) and PM H + -ATPase (EC 3.6.3.6) differed in sensitivity to heavy metals. Exposure of cucumber seedlings to Cd, Cu and Ni had no significant effect on the activity of the vacuolar proton pump and, in the case of Ni, also on the activity of the PM proton pump. In contrast, Cd and Cu ions diminished both ATP hydrolysis and proton transport in plasma membranes. Transcript levels of genes encoding PM enzyme as well as the subunit A of tonoplast enzyme in roots stressed with heavy metals were similar to the control. Cd, Cu and Ni were accumulated in cucumber roots with similar efficiency, but their relative distribution between the symplast and apoplast differed. To explain the mechanism of heavy metal action on the plasma membranes of cucumber roots, the MDA content, as a lipid peroxidation product, and fatty acid composition were analyzed. It was shown that exposure of plants to Cd, Cu and Ni did not enhance the lipid peroxidation in the PM fraction. However, all metals caused an increase in the saturation of PM fatty acids and a decrease in the length of the fatty acid chain.

Research paper thumbnail of Nitrate transport across the tonoplast ofCucumis sativus L. root cells

Journal of Plant Physiology, 2003

Nitrate transport across the tonoplast has been studied using vacuole membranes isolated from cuc... more Nitrate transport across the tonoplast has been studied using vacuole membranes isolated from cucumber roots grown in nitrate. The addition of NO 3 ions into the tonoplast with ATP-generated transmembrane proton gradient caused the dissipation of ∆pH, indicating the NO 3 --induced proton efflux from vesicles. NO 3 --dependent H + efflux was almost insensitive to the transmembrane electrical potential difference, suggesting the presence of an electroneutral NO 3 -/H + antiporter in the tonoplast. Apart from saturation kinetics, with respect to nitrate ions, NO 3 --linked H + efflux from the tonoplast of cucumber roots showed other characteristics expected of substrate-specific transporters. Experiments employing protein modifying reagents (NEM, pCMBS, PGO and SITS) indicated that a crucial role in the activity of tonoplast nitrate/proton antiporter is played by lysine residues (strong inhibition of NO 3 -/H + antiport by SITS). None of the ion-channel inhibitors (NIF, ZnSO 4 and TEA-Cl) used in the experiments had a direct effect on the nitrate transport into tonoplast membranes. On the other hand, every protein reagent, as well as NIF and ZnSO 4 , significantly affected the ATP-dependent proton transport in vesicles. Only TEA-Cl, the potassium channel blocker, had no effect on the vacuolar proton pumping activity.

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

Journal of Plant Physiology, 2013

The effect of salt stress (50 mM NaCl) on modification of plasma membrane (PM) H + -ATPase (EC 3.... more The effect of salt stress (50 mM 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 saltstressed 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 H 2 O 2 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.

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

Journal of Experimental Botany, 2012

The effect of heavy metals on plasma membrane (PM) H(+)-ATPase (EC 3.6.3.14) activity in cucumber... more 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.

Research paper thumbnail of Plant Ca2+-ATPases

Acta Physiologiae Plantarum, 2005

Plant Phys i ol ogy De part ment, In sti tute of Plant Bi ol ogy, Wrocław Uni ver sity,

Research paper thumbnail of Characterization of the tonoplast proton pumps in Cucumis sativus L. root cells

Acta Physiologiae Plantarum, 2001

Large-scale preparation of highly purified tonoplast from cucumber (Cucumis sativus L.) roots was... more Large-scale preparation of highly purified tonoplast from cucumber (Cucumis sativus L.) roots was obtained after centrifngation of microsome pellet (10,000-g0,000 g) on discontinuous sucrose density gradient (20, 28, 32 and 42 %). Lack of PEP carboxylase (cytosol marker) and cytochrome c oxidase (mitochondrial marker) together with a slight activity of VO4-ATPase (plasma membrane marker) and NADH-cytochrome c reductase (ER marker) in tonoplast preparation confirmed its high purity. Using latency of nitrate-inhibited ATPase and H + pumping as criteria it was established that the majority of tonoplast vesicles were sealed and oriented right(cytoplasmic)side-out. Strong acidification of the interior of vesicles observed at the presence of both, ATP and PPi, confirmed that obtained tonoplast contains two classes of proton pumps: V-ATPase and H+PPiase.

Research paper thumbnail of Response of plasma membrane H+-ATPase to heavy metal stress in Cucumis sativus roots

Journal of Experimental Botany, 2008

The effect of heavy metals on the modification of plasma membrane H + -ATPase (EC 3.6.3.14) activ... more The effect of heavy metals on the modification of plasma membrane H + -ATPase (EC 3.6.3.14) activity in cucumber roots was studied. In plants stressed for 2 h with 10 mM or 100 mM Cd, Cu or Ni the hydrolytic as well as the transporting activity of H + -ATPase in the plasma membranes of root cells was decreased. Transcript levels of Cucumis sativus plasma membrane H + -ATPase in roots treated with 10 mM Cd, Cu, or Ni as well as with 100 mM Cu or Ni were similar to the control, indicating that the action of metals did not involve the gene expression level. Only in roots exposed to 100 mM Cd was the level of plasma membrane H + -ATPase mRNA markedly decreased. The inhibition of the plasma membrane proton pump caused by 100 mM Cd, Cu and Ni was partially diminished in the presence of cantharidin, a specific inhibitor of protein phosphatases. Western blot analysis with the antibody against phosphothreonine confirmed that decreased activity of plasma membrane H + -ATPase under heavy metals resulted from dephosphorylation of the enzyme protein. Taken together, these data strongly indicated that alteration of the enzyme under heavy metal stresses was mainly due to the post-translational modification of its proteins in short-term experiments.

Research paper thumbnail of Modification of plasma membrane NADPH oxidase activity in cucumber seedling roots in response to cadmium stress

Plant science : an international journal of experimental plant biology, 2015

The aim of this study was to investigate the effect of cadmium on plasma membrane (PM) NADPH oxid... more 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 und...

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

Physiologia Plantarum, 2014

Research paper thumbnail of Mechanism of Cd and Cu action on the tonoplast proton pumps in cucumber roots

Physiologia Plantarum, 2013

The effect of Cd and Cu on the tonoplast proton pumps, V-ATPase (EC 3.6.3.14) and V-PPase (EC 3.6... more 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.

Research paper thumbnail of Response of plasma membrane H+-ATPase to low temperature in cucumber roots

Journal of Plant Research, 2012

The effect of low temperature (LT, 10°C) on modification of plasma membrane (PM) H ? -ATPase (EC ... more 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.

Research paper thumbnail of Modification of vacuolar proton pumps in cucumber roots under salt stress

Journal of Plant Physiology, 2008

The time-dependent effect of 50 mM NaCl on the activities of two tonoplast proton pumps was inves... more The time-dependent effect of 50 mM NaCl on the activities of two tonoplast proton pumps was investigated in Cucumis sativus L. var. Krak root cells. Distinct activity profiles for vacuolar proton transporting ATPase (V-ATPase) (EC 3.6.3.14) and vacuolar proton transporting pyrophosphatase (V-PPase) (EC 3.6.1.1) under salinity are presented. ATP-dependent proton transport and ATP hydrolysis increased after 24 h of NaCl exposure, and then decreased in roots stressed with NaCl for 4 and 8 d. Both PP i -driven H + transport and PP i hydrolysis were clearly inhibited by NaCl at all times examined. It was demonstrated that changes in enzyme activities were not due to the salt action on the expression of encoding genes. The levels of specific transcripts for subunit A of V-ATPase (CsVHA-A), subunit c of V-ATPase (CsVHA-c) and V-PPase (CsVP) were similar in cucumber roots untreated (control) and treated with salt. Such results suggest that alterations of proton pump activities under salinity are rather due to the post-translational alterations induced by NaCl.

Research paper thumbnail of The role of polyamines in the regulation of the plasma membrane and the tonoplast proton pumps under salt stress

Journal of Plant Physiology, 2010

Polyamine content (PAs) often changes in response to abiotic stresses. It was shown that the accu... more Polyamine content (PAs) often changes in response to abiotic stresses. It was shown that the accumulation of PAs decreased in roots treated for 24 h with 200 mM NaCl. The role of polyamines (putrescine -PUT, spermidine -SPD and spermine -SPM) in the modification of the plasma membrane(PM) H + -ATPase (EC 3.6.3.6) and the vacuolar(V) H + -ATPase (EC 3.6.3.14) activities in cucumber roots treated with NaCl was investigated. 24 h treatment of seedlings with 50 mM PUT, SPD or SPM lowered the activities of proton pumps in both membranes. The decreased H + -ATPase activity in plasma membranes isolated from the PA-treated roots was positively correlated with a lower level of PM-H + -ATPase CsHA3 transcript. However, transcript levels of PM-H + -ATPase CsHA2 and V-ATPase subunit A and c in roots treated with 50 mM PAs were similar to those in the control. Additionally, treatment of plants with salt markedly increased the activity of the PM-and V-H + -ATPases. However, exposure of plants to 20% PEG had no effect on these activities. These data suggest that, under salt stress conditions, the increase in H + -ATPase activities is caused mainly by the ionic component of salt stress. It seems that the main role of the PAs in the 24 h salt-treated cucumber plants could be a result of their cationic character. The PA levels decreased when concentration of Na + increased, so action of PAs contributes to ionic equilibrium. Moreover, the decrease in the concentration of polyamines, which inhibit the PM-H + -ATPase and the V-H + -ATPase, at least under the studied conditions, seems to be beneficial. Thus, plants can increase salinity tolerance by modifying the biosynthesis of polyamines.

Research paper thumbnail of Comparison of heavy metal effect on the proton pumps of plasma membrane and tonoplast in cucumber root cells

Journal of Plant Physiology, 2008

The effects of 10 mM cadmium, copper and nickel on the activities of vacuolar membrane and plasma... more The effects of 10 mM cadmium, copper and nickel on the activities of vacuolar membrane and plasma membrane (PM) ATP-dependent proton pumps was investigated in Cucumis sativus L. root cells. It was demonstrated that vacuolar H + -ATPase (EC 3.6.3.14) and PM H + -ATPase (EC 3.6.3.6) differed in sensitivity to heavy metals. Exposure of cucumber seedlings to Cd, Cu and Ni had no significant effect on the activity of the vacuolar proton pump and, in the case of Ni, also on the activity of the PM proton pump. In contrast, Cd and Cu ions diminished both ATP hydrolysis and proton transport in plasma membranes. Transcript levels of genes encoding PM enzyme as well as the subunit A of tonoplast enzyme in roots stressed with heavy metals were similar to the control. Cd, Cu and Ni were accumulated in cucumber roots with similar efficiency, but their relative distribution between the symplast and apoplast differed. To explain the mechanism of heavy metal action on the plasma membranes of cucumber roots, the MDA content, as a lipid peroxidation product, and fatty acid composition were analyzed. It was shown that exposure of plants to Cd, Cu and Ni did not enhance the lipid peroxidation in the PM fraction. However, all metals caused an increase in the saturation of PM fatty acids and a decrease in the length of the fatty acid chain.

Research paper thumbnail of Nitrate transport across the tonoplast ofCucumis sativus L. root cells

Journal of Plant Physiology, 2003

Nitrate transport across the tonoplast has been studied using vacuole membranes isolated from cuc... more Nitrate transport across the tonoplast has been studied using vacuole membranes isolated from cucumber roots grown in nitrate. The addition of NO 3 ions into the tonoplast with ATP-generated transmembrane proton gradient caused the dissipation of ∆pH, indicating the NO 3 --induced proton efflux from vesicles. NO 3 --dependent H + efflux was almost insensitive to the transmembrane electrical potential difference, suggesting the presence of an electroneutral NO 3 -/H + antiporter in the tonoplast. Apart from saturation kinetics, with respect to nitrate ions, NO 3 --linked H + efflux from the tonoplast of cucumber roots showed other characteristics expected of substrate-specific transporters. Experiments employing protein modifying reagents (NEM, pCMBS, PGO and SITS) indicated that a crucial role in the activity of tonoplast nitrate/proton antiporter is played by lysine residues (strong inhibition of NO 3 -/H + antiport by SITS). None of the ion-channel inhibitors (NIF, ZnSO 4 and TEA-Cl) used in the experiments had a direct effect on the nitrate transport into tonoplast membranes. On the other hand, every protein reagent, as well as NIF and ZnSO 4 , significantly affected the ATP-dependent proton transport in vesicles. Only TEA-Cl, the potassium channel blocker, had no effect on the vacuolar proton pumping activity.

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

Journal of Plant Physiology, 2013

The effect of salt stress (50 mM NaCl) on modification of plasma membrane (PM) H + -ATPase (EC 3.... more The effect of salt stress (50 mM 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 saltstressed 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 H 2 O 2 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.

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

Journal of Experimental Botany, 2012

The effect of heavy metals on plasma membrane (PM) H(+)-ATPase (EC 3.6.3.14) activity in cucumber... more 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.

Research paper thumbnail of Plant Ca2+-ATPases

Acta Physiologiae Plantarum, 2005

Plant Phys i ol ogy De part ment, In sti tute of Plant Bi ol ogy, Wrocław Uni ver sity,

Research paper thumbnail of Characterization of the tonoplast proton pumps in Cucumis sativus L. root cells

Acta Physiologiae Plantarum, 2001

Large-scale preparation of highly purified tonoplast from cucumber (Cucumis sativus L.) roots was... more Large-scale preparation of highly purified tonoplast from cucumber (Cucumis sativus L.) roots was obtained after centrifngation of microsome pellet (10,000-g0,000 g) on discontinuous sucrose density gradient (20, 28, 32 and 42 %). Lack of PEP carboxylase (cytosol marker) and cytochrome c oxidase (mitochondrial marker) together with a slight activity of VO4-ATPase (plasma membrane marker) and NADH-cytochrome c reductase (ER marker) in tonoplast preparation confirmed its high purity. Using latency of nitrate-inhibited ATPase and H + pumping as criteria it was established that the majority of tonoplast vesicles were sealed and oriented right(cytoplasmic)side-out. Strong acidification of the interior of vesicles observed at the presence of both, ATP and PPi, confirmed that obtained tonoplast contains two classes of proton pumps: V-ATPase and H+PPiase.

Research paper thumbnail of Response of plasma membrane H+-ATPase to heavy metal stress in Cucumis sativus roots

Journal of Experimental Botany, 2008

The effect of heavy metals on the modification of plasma membrane H + -ATPase (EC 3.6.3.14) activ... more The effect of heavy metals on the modification of plasma membrane H + -ATPase (EC 3.6.3.14) activity in cucumber roots was studied. In plants stressed for 2 h with 10 mM or 100 mM Cd, Cu or Ni the hydrolytic as well as the transporting activity of H + -ATPase in the plasma membranes of root cells was decreased. Transcript levels of Cucumis sativus plasma membrane H + -ATPase in roots treated with 10 mM Cd, Cu, or Ni as well as with 100 mM Cu or Ni were similar to the control, indicating that the action of metals did not involve the gene expression level. Only in roots exposed to 100 mM Cd was the level of plasma membrane H + -ATPase mRNA markedly decreased. The inhibition of the plasma membrane proton pump caused by 100 mM Cd, Cu and Ni was partially diminished in the presence of cantharidin, a specific inhibitor of protein phosphatases. Western blot analysis with the antibody against phosphothreonine confirmed that decreased activity of plasma membrane H + -ATPase under heavy metals resulted from dephosphorylation of the enzyme protein. Taken together, these data strongly indicated that alteration of the enzyme under heavy metal stresses was mainly due to the post-translational modification of its proteins in short-term experiments.