Assay of Plasma Membrane H+-ATPase in Plant Tissues under Abiotic Stresses.

Plasma membrane (PM) H+-ATPase, which generates the proton gradient across the outer membrane of plant cells, plays a fundamental role in the regulation of many physiological processes fundamental for growth and development of plants. It is involved in the uptake of nutrients from external solutions, their loading into phloem and long-distance transport, stomata aperture and gas exchange, pH homeostasis in cytosol, cell wall loosening, and cell expansion. The crucial role of the enzyme in resistance of plants to abiotic and biotic stress factors has also been well documented. Such great diversity of physiological functions linked to the activity of one enzyme requires a suitable and complex regulation of H+-ATPase. This regulation comprises the transcriptional as well as post-transcriptional levels. Herein, we describe the techniques that can be useful for the analysis of the plasma membrane proton pump modifications at genetic and protein levels under environmental factors.

The plasma membrane H(+) -ATPase AHA2 contributes to the root architecture in response to different nitrogen supply.

In this study the role of the plasma membrane (PM) H(+) -ATPase for growth and development of roots as response to nitrogen starvation is studied. It is known that root development differs dependent on the availability of different mineral nutrients. It includes processes such as initiation of lateral root primordia, root elongation and increase of the root biomass. However, the signal transduction mechanisms, which enable roots to sense changes in different mineral environments and match their growth and development patterns to actual conditions in the soil, are still unknown. Most recent comments have focused on one of the essential macroelements, namely nitrogen, and its role in the modification of the root architecture of Arabidopsis thaliana. As yet, not all elements of the signal transduction pathway leading to the perception of the nitrate stimulus, and hence to anatomical changes of the root, which allow for adaptation to variable ion concentrations in the soil, are known. Our data demonstrate that primary and lateral root length were shorter and lower in aha2 mutant lines compared with wild-type plants in response to a variable nitrogen source. This suggests that the PM proton pump AHA2 (Arabidopsis plasma membrane H(+) -ATPase isoform 2) is important for root growth and development during different nitrogen regimes. This is possible by controlling the pH homeostasis in the root during growth and development as shown by pH biosensors.

NO3⁻/H⁺ antiport in the tonoplast of cucumber root cells is stimulated by nitrate supply: evidence for a reversible nitrate-induced phosphorylation of vacuolar NO3⁻/H⁺ antiport.

Studies in the last few years have shed light on the process of nitrate accumulation within plant cells, achieving molecular identification and partial characterization of the genes and proteins involved in this process. However, contrary to the plasma membrane-localized nitrate transport activities, the kinetics of active nitrate influx into the vacuole and its adaptation to external nitrate availability remain poorly understood. In this work, we have investigated the activity and regulation of the tonoplast-localized H(+)/NO3(-) antiport in cucumber roots in response to N starvation and NO3(-) induction. The time course of nitrate availability strongly influenced H(+)/NO3(-) antiport activity at the tonoplast of root cells. However, under N starvation active nitrate accumulation within the vacuole still occurred. Hence, either a constitutive H(+)-coupled transport system specific for nitrate operates at the tonoplast, or nitrate uses another transport protein of broader specificity to different anions to enter the vacuole via a proton-dependent process. H(+)/NO3(-) antiport in cucumber was significantly stimulated in NO3(-)-induced plants that were supplied with nitrate for 24 hours following 6-day-long N starvation. The cytosolic fraction isolated from the roots of NO3(-)-induced plants significantly stimulated H(+)/NO3(-) antiport in tonoplast membranes isolated from cucumbers growing on nitrate. The stimulatory effect of the cytosolic fraction was completely abolished by EGTA and the protein kinase inhibitor staurosporine and slightly enhanced by the phosphatase inhibitors okadaic acid and cantharidin. Hence, we conclude that stimulation of H(+)/NO3(-) antiport at the tonoplast of cucumber roots in response to nitrate provision may occur through the phosphorylation of a membrane antiporter involving Ca-dependent, staurosporine-sensitive protein kinase.

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.

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.

[Plant P-type ATPases]. / Roslinne ATPazy typu P.

P-type ATPases are a superfamily of membrane proteins involved in many physiological processes that are fundamental for all living organisms. Using ATP, they can transport a variety of ions and other substances across all types of cell membranes against a concentration electrochemical gradient. P-type ATPases form a phosphorylated intermediate and are sensitive to vanadate. Based on evolutionary relations and sequence homology, P-type ATPases are divided into five major families. All P-type ATPases share a simple structure and mechanism, but also possess domains characteristic for each family, which are crucial for substrate specificity. These proteins usually have a single subunit with eight to twelve transmembrane segments, a large central cytoplasmic domain with the conservative ATP binding site along with N and C termini exposed to the cytoplasm. Because of variety of proteins that belong to P-type ATPase superfamily, in this review the comparison of functional and structure properties of plant cells P-type ATPases is presented, as well as their important role in adaptation to environmental stress.

Comparative study of the active cadmium efflux systems operating at the plasma membrane and tonoplast of cucumber root cells.

The strategies developed by plants to avoid the toxicity of cadmium (Cd) and other heavy metals involve active sequestration of metals into the apoplast and vacuoles. The protein systems excluding heavy metals from the cell cytosol localize to the plasma membrane and tonoplast and are energized either by ATP or by the electrochemical gradient generated by H(+)-ATPase or by V-ATPase and pyrophosphatase (PPase), respectively. In this work, a comparative study on the contribution of both the plasma membrane and tonoplast in the active detoxification of plant cells after treatment with Cd was performed. The studies using plants treated and untreated with Cd reveal that both, H(+)-coupled and MgATP-driven efflux of Cd across plasma membranes and tonoplast is markedly stimulated in the presence of Cd in the environment. Previous studies on plasma-membrane localized H(+)-coupled Cd efflux together with the present data demonstrating tonoplast H(+)/Cd(2+) antiport activity suggest that H(+)-coupled secondary transport of Cd displays a lower affinity for Cd when compared with Cd primary pumps driven by MgATP. In addition, it is shown that MgATP-energized Cd efflux across both membranes is significantly enhanced by cysteine, dithiothreitol, and glutathione. These results suggest that Cd is excluded from the cytosol through an energy-dependent system as a free ion as well as a complexed form. Although both membranes contribute in the active exclusion of ionized and complexed Cd from the cytosol, the overall calculation of Cd accumulation in the everted plasma membranes and vacuolar vesicles suggests that the tonoplast and vacuole have a major function in Cd efflux from the cytosol in the roots of cucumber subjected to Cd stress.

Effect of short-term salinity on the nitrate reductase activity in cucumber roots.

In short-term experiments, the effect of high salinity on cucumber (Cucumis sativus) nitrate reductase activity was studied. The 60-min exposure of cucumber roots to 200 mM NaCl resulted in significant increase of the actual NR activity (measured in the presence of Mg²+), whereas the total enzyme activity (measured with EDTA) was not affected. NaCl-induced stimulation of the actual NR activity was rapidly reversed upon transfer of roots to salt-free solution. The increase in actual activity was completely prevented by microcystin-LR and cantharidin, protein phosphatases inhibitors. In addition, a significant decrease in ATP level was also observed in roots incubated with NaCl. These data suggest that the reversible protein phosphorylation is involved in the induction of NR activity during the first hour of salt stress. The effect of short-term salinity on the expression of genes encoding for nitrate reductase in cucumber roots was also studied. 200 mM NaCl diminished the increase in CsNR1 expression observed in control roots. During the same time period, the expression of CsNR2 was not affected, whereas the expression of CsNR3 decreased significantly after 1h incubation of the excised roots in both, control and salt-containing nutrient solutions. Incubation of roots in the presence of iso-osmotic concentration of PEG had no effect on both, NR activity and expression. This indicates that only the ionic component of salt stress was involved in the salt-induced modifications of nitrate reductase activity.

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.

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

Polyamine content (PAs) often changes in response to abiotic stresses. It was shown that the accumulation of PAs decreased in roots treated for 24h 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. 24h treatment of seedlings with 50 microM 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 microM 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 24h 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.

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

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 microM or 100 microM 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 microM Cd, Cu, or Ni as well as with 100 microM 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 microM Cd was the level of plasma membrane H(+)-ATPase mRNA markedly decreased. The inhibition of the plasma membrane proton pump caused by 100 microM 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.

Modification of vacuolar proton pumps in cucumber roots under salt stress.

The time-dependent effect of 50mM 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 24h of NaCl exposure, and then decreased in roots stressed with NaCl for 4 and 8d. 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.

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

The effects of 10 microM 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.

Modification of plasma membrane and vacuolar H+ -ATPases in response to NaCL and ABA.

The role of ABA in the modification of membrane proton pump activity in cucumber roots which had been stressed for 24h with 200 mmol/dm(3) NaCl was investigated. It was shown that treatment of plants with salt distinctly increased the activity of the plasma membrane H(+)-ATPase (EC 3.6.3.6) as well as of the vacuolar H(+)-ATPase (EC 3.6.3.14). In roots treated with NaCl, an increase of ABA level was observed. Moreover, 24-h treatment of seedlings with 50 micro mol/dm(3) ABA increased the activity of proton pumps in both membranes. However, when ABA was added to the reaction medium, no changes in ATPase activities were observed. The increased H(+)-ATPase activity in plasma membranes isolated from the ABA-treated roots was positively correlated with a higher level of PM-H(+)-ATPase transcript. These data have provided evidence that under salt stress conditions, the role of ABA action due to salinity on the induction of the PM-H(+)-ATPase could be at the level of gene expression.

[H(+)-coupled heavy metal transport in plants]. / Wtórne transportery metali ciezkich u roslin.

It has been recently well documented that metal transport systems play a crucial role in the uptake, distribution and detoxification of heavy metals throughout the plant. A range of gene families that are likely to be involved in essential and non-essential metal transport has been now identified and their plasma membrane and/or tonoplast localization in plant cells has been recently confirmed. These include the primary metal transporters, using ATP as the source of energy and H(+)-coupling transporters, utilizing the electrochemical gradient previously generated by plasma membrane and tonoplast proton pumps. As the presence of nucleotide binding domains in the protein sequence may indicate its ATP-hydrolytic activity, it is more difficult to determine the H(+)-coupling activity of protein on the base of its structure. Thus, the H(+)-coupling activity of protein may be only proved by functional analysis of the protein. In this work, we briefly review the structure, regulation and function of the metal transporters operating as H(+)/metal cotransporters.

Modulation by cytosolic components of proton pump activities in plasma membrane and tonoplast from Cucumis sativus roots during salt stress.

The effect of NaCl on the plasma membrane and tonoplast ATPases measured as the hydrolytic and H(+)-pumping activity was studied. Treatment of cucumber seedlings with salt increased the membrane-bound ATPases of the plasma membrane as well as the tonoplast. In both types of membranes the stimulation of ATP-hydrolysis was much higher than the stimulation of H(+)-transport suggesting that the salt- treatment of plants partially uncoupled the membrane proton pumps. It was shown that the soluble fraction obtained from the unstressed or NaCl-stressed roots stimulated the ATPase activities in both membranes isolated from unstressed plants. A stimulatory effect of the soluble fraction on the proton pump activities was considerably enhanced in the salt conditions indicating the presence of a salt-inducible factor (s) in the soluble fraction, which could rapidly modulate the membrane-bound ATPases. Staurosporine, a specific protein kinase inhibitor, totally abolished the stimulatory action of the soluble fractions on the membrane proton pumps, whereas okadaic acid, a phosphatase inhibitor, had no effect. Inclusion of calcium in the mixture of membranes and the soluble fraction from unstressed roots elevated the ATPase activities to the levels determined with the soluble fraction isolated from NaCl-stressed roots. Cation chelators (EGTA), as well as calmodulin antagonist (W7) cancelled the stimulatory effect of calcium ions. The above results strongly suggest the involvement of specific calcium-calmodulin-dependent protein kinases in the activation of the membrane ATPases under salt-stress conditions.

Nitrate transport across the tonoplast of Cucumis sativus L. root cells.

Nitrate transport across the tonoplast has been studied using vacuole membranes isolated from cucumber roots grown in nitrate. The addition of NO3- ions into the tonoplast with ATP-generated transmembrane proton gradient caused the dissipation of delta pH, indicating the NO3(-)-induced proton efflux from vesicles. NO3(-)-dependent H+ efflux was almost insensitive to the transmembrane electrical potential difference, suggesting the presence of an electroneutral NO3-/H+ antiporter in the tonoplast. Apart from saturation kinetics, with respect to nitrate ions, NO3(-)-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 NO3-/H+ antiport by SITS). None of the ion-channel inhibitors (NIF, ZnSO4 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 ZnSO4, 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.