Changes In Cytosolic pH Research Articles

K+ and pH homeostasis in plant cells is controlled by a synchronized K+ /H+ antiport at the plasma and vacuolar membrane.

Stomatal movement involves ion transport across the plasma membrane (PM) and vacuolar membrane (VM) of guard cells. However, the coupling mechanisms of ion transporters in both membranes and their interplay with Ca2+ and pH changes are largely unclear. Here, we investigated transporter networks in tobacco guard cells and mesophyll cells using multiparametric live-cell ion imaging and computational simulations. K+ and anion fluxes at both, PM and VM, affected H+ and Ca2+ , as changes in extracellular KCl or KNO3 concentrations were accompanied by cytosolic and vacuolar pH shifts and changes in [Ca2+ ]cyt and the membrane potential. At both membranes, the K+ transporter networks mediated an antiport of K+ and H+ . By contrast, net transport of anions was accompanied by parallel H+ transport, with differences in transport capacity for chloride and nitrate. Guard and mesophyll cells exhibited similarities in K+ /H+ transport but cell type-specific differences in [H+ ]cyt and pH-dependent [Ca2+ ]cyt signals. Computational cell biology models explained mechanistically the properties of transporter networks and the coupling of transport across the PM and VM. Our integrated approach indicates fundamental principles of coupled ion transport at membrane sandwiches to control H+ /K+ homeostasis and points to transceptor-like Ca2+ /H+ -based ion signaling in plant cells.

Salinity-Induced Cytosolic Alkaline Shifts in Arabidopsis Roots Require the SOS Pathway.

Plants have evolved elaborate mechanisms to sense, respond to and overcome the detrimental effects of high soil salinity. The role of calcium transients in salinity stress signaling is well established, but the physiological significance of concurrent salinity-induced changes in cytosolic pH remains largely undefined. Here, we analyzed the response of Arabidopsis roots expressing the genetically encoded ratiometric pH-sensor pHGFP fused to marker proteins for the recruitment of the sensor to the cytosolic side of the tonoplast (pHGFP-VTI11) and the plasma membrane (pHGFP-LTI6b). Salinity elicited a rapid alkalinization of cytosolic pH (pHcyt) in the meristematic and elongation zone of wild-type roots. The pH-shift near the plasma membrane preceded that at the tonoplast. In pH-maps transversal to the root axis, the epidermis and cortex had cells with a more alkaline pHcyt relative to cells in the stele in control conditions. Conversely, seedlings treated with 100 mM NaCl exhibited an increased pHcyt in cells of the vasculature relative to the external layers of the root, and this response occurred in both reporter lines. These pHcyt changes were substantially reduced in mutant roots lacking a functional SOS3/CBL4 protein, suggesting that the operation of the SOS pathway mediated the dynamics of pHcyt in response to salinity.

Salt-Induced Changes in Cytosolic pH and Photosynthesis in Tobacco and Potato Leaves.

Salinity is one of the most common factors limiting the productivity of crops. The damaging effect of salt stress on many vital plant processes is mediated, on the one hand, by the osmotic stress caused by large concentrations of Na+ and Cl- outside the root and, on the other hand, by the toxic effect of these ions loaded in the cell. In our work, the influence of salinity on the changes in photosynthesis, transpiration, water content and cytosolic pH in the leaves of two important crops of the Solanaceae family-tobacco and potato-was investigated. Salinity caused a decrease in photosynthesis activity, which manifested as a decrease in the quantum yield of photosystem II and an increase in non-photochemical quenching. Along with photosynthesis limitation, there was a slight reduction in the relative water content in the leaves and a decrease in transpiration, determined by the crop water stress index. Furthermore, a decrease in cytosolic pH was detected in tobacco and potato plants transformed by the gene of pH-sensitive protein Pt-GFP. The potential mechanisms of the salinity influence on the activity of photosynthesis were analyzed with the comparison of the parameters' dynamics, as well as the salt content in the leaves.

Cell-Type-Specific Length and Cytosolic pH Response of Superficial Cells of Arabidopsis Root to Chronic Salinity.

Soil salinity negatively affects the growth, development and yield of plants. Acidification of the cytosol in cells of glycophytes was reported under salinity, while various types of plant cells can have a specific reaction under the same conditions. Transgenic Arabidopsis plants expressing the pH sensor Pt-GFP in the cytosol were used in this work for determination of morphometric changes and cytosolic pH changes in the superficial cells of Arabidopsis roots under chronic salinity in vitro. We did not find changes in the length of the root cap cells, while there was a decrease in the length of the differentiation zone under 50, 75 mM NaCl and the size of the epidermal cells of the differentiation zone under 75 mM NaCl. The most significant changes of cytosolic pH to chronic salinity was noted in columella (decrease by 1 pH unit at 75 mM NaCl) and epidermal cells of the differentiation zone (decrease by 0.6 and 0.4 pH units at 50 and 75 mM NaCl, respectively). In developed lateral root cap cells, acidification of cytosol by 0.4 units occurred only under 75 mM NaCl in the medium. In poorly differentiated lateral cells of the root cap, there were no changes in pH under chronic salinity.

A water-soluble 1,8-naphthalimide-based fluorescent pH probe for distinguishing tumorous tissues and inflammation in mice.

A water-soluble fluorescent probe BPN, by introducing a piperazine as the pH-sensitive fluorescence signaling motif to the hydrophilic propionic acid-substituted 1,8-naphthalimide fluorophore, is highly sensitive to pH changes within cytoplasm matrix in living cells, as well as pH-related diseases models. Owing to the protonation-induced inhibition of the photoinduced electron transfer (PET) from piperazine to naphthalimide fluorophore, BPN displayed a significant fluorescence enhancement (more than 131-fold) upon the pH decreasing from 11.0 to 3.0. The linear range was between pH 6.4 to 8.0 with a pKa value of 6.69 near the physiological pH, which was suitable for cytosolic pH research. Furthermore, BPN exhibited a large Stokes shift (142 nm), good water solubility, excellent photostability, high selectivity and low cytotoxicity. All these advantages were particularly beneficial for intracellular pH imaging. Using BPN, we demonstrated the real-time monitoring of cytosolic pH changes in living cells. Most importantly, BPN has not only been successfully applied for distinguishing inflammation in mice, but also the surgical specimens of cancer tissue, making it of great potential application in cancer diagnosis.

A luminescent Nanoluc-GFP fusion protein enables readout of cellular pH in photosynthetic organisms

pH is one of the most critical physiological parameters determining vital cellular activities, such as photosynthetic performance. Fluorescent sensor proteins capable of measuring in situ pH in animal cells have been reported. However, these proteins require an excitation laser for pH measurement that may affect photosynthetic performance and induce autofluorescence from chlorophyll. As a result, it is not possible to measure the intracellular or intraorganelle pH changes in plants. To overcome this problem, we developed a luminescent pH sensor by fusing the luminescent protein Nanoluc to a uniquely designed pH-sensitive GFP variant protein. In this system, an excitation laser is unnecessary because the fused GFP variant reports on the luminescent signal by bioluminescence resonance energy transfer from Nanoluc. The ratio of two luminescent peaks from the sensor protein was approximately linear with respect to pH in the range of 7.0 to 8.5. We designated this sensor protein as “luminescent pH indicator protein” (Luphin). We applied Luphin to the in situ pH measurement of a photosynthetic organism under fluctuating light conditions, allowing us to successfully observe the cytosolic pH changes associated with photosynthetic electron transfer in the cyanobacterium Synechocystis sp. PCC 6803. Detailed analyses of the mechanisms of the observed estimated pH changes in the cytosol in this alga suggested that the photosynthetic electron transfer is suppressed by the reduced plastoquinone pool under light conditions. These results indicate that Luphin may serve as a helpful tool to further illuminate pH-dependent processes throughout the photosynthetic organisms.

Potassium Efflux and Cytosol Acidification as Primary Anoxia-Induced Events in Wheat and Rice Seedlings.

Both ion fluxes and changes of cytosolic pH take an active part in the signal transduction of different environmental stimuli. Here we studied the anoxia-induced alteration of cytosolic K+ concentration, [K+]cyt, and cytosolic pH, pHcyt, in rice and wheat, plants with different tolerances to hypoxia. The [K+]cyt and pHcyt were measured by fluorescence microscopy in single leaf mesophyll protoplasts loaded with the fluorescent potassium-binding dye PBFI-AM and the pH-sensitive probe BCECF-AM, respectively. Anoxic treatment caused an efflux of K+ from protoplasts of both plants after a lag-period of 300–450 s. The [K+]cyt decrease was blocked by tetraethylammonium (1 mM, 30 min pre-treatment) suggesting the involvement of plasma membrane voltage-gated K+ channels. The protoplasts of rice (a hypoxia-tolerant plant) reacted upon anoxia with a higher amplitude of the [K+]cyt drop. There was a simultaneous anoxia-dependent cytosolic acidification of protoplasts of both plants. The decrease of pHcyt was slower in wheat (a hypoxia-sensitive plant) while in rice protoplasts it was rapid and partially reversible. Ion fluxes between the roots of intact seedlings and nutrient solutions were monitored by ion-selective electrodes and revealed significant anoxia-induced acidification and potassium leakage that were inhibited by tetraethylammonium. The K+ efflux from rice was more distinct and reversible upon reoxygenation when compared with wheat seedlings.

Abstract 5166: In silico identification of specific inhibitor(s) of tumor associated Vacuolar ATPase ‘a2' isoform: A critical target for anti-cancer drug development

Abstract The discovery of effective anti-cancer therapies has immense consequences on global health. Vacuolar (H+)-ATPases (V-ATPases) are multi-subunit nano-motors that acidify the lumen of intracellular organelles in normal physiology. In cancer cells, the plasma membrane V-ATPases acidify the extracellular space. This is critical in tumor invasion, metastasis, and chemo-resistance. The `a2` isoform of V-ATPase (a2V) is the major pH sensing unit of this complex and is distinctly overexpressed on the surface of cancer cells making it an excellent drug target. The chemical inhibitors such as bafilomycin/concanamycin target V-ATPase subunits also critical in normal cell function. Currently, there are no selective a2V inhibitors commercially available as anti-cancer agents. Here, we employed in silico molecular modeling to analyze specific a2V binding sites in order to design targeted small molecules a2V inhibitors. We have identified several natural compounds as a2V ligands which inhibit specifically a2V in cancer. Employing human ovarian and breast epithelial cells (normal or tumor), in vitro studies measured each a2V ligand(s) effects on cancer cell proliferation. We selected five a2V ligands showing the most potent anti-proliferative activity in cancer cells and were least toxic to normal cells. We further measured each ligand(s) effects on specific ATPase activity in vitro by determining the decrease in inorganic phosphate levels. Next, the two most potent a2v ligands (KDB001, KDB002) which belonged to alkaloid and mono-terpenoid phenol groups, were selected to measure the changes in cytosolic and extracellular pH- SNARF assay and pH of cell supernatants. KDB001 induced cytosolic pH acidification in a dose-dependent manner indicating inhibition of V-ATPase activity. Based on the above experiments, the best ligand with most prominent effect on a2V activity (KDB-001) was further validated in vivo for anti-ovarian cancer efficacy in an athymic nude mouse model. KDB001 administration resulted in a significant delay in in vivo ovarian tumor growth. Studies are underway to design and synthesize more efficient analogs for KDB001 molecular modelling, toxicity assessment and chemical synthesis approach. In conclusion, the study has discovered new inhibitors of V-ATPase-a2V, positioning these promising compounds to enter the anti-cancer drug development pipeline. Citation Format: Arpita Kulshrestha, Gajendra K. Katara, Awanish Kumar, Kenneth D. Beaman. In silico identification of specific inhibitor(s) of tumor associated Vacuolar ATPase ‘a2' isoform: A critical target for anti-cancer drug development [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5166.

Cellular stress is a prerequisite for glucose-induced mitochondrial matrix alkalinization in pancreatic β-cells

Changes in mitochondrial and cytosolic pH alter the chemical gradient across the inner mitochondrial membrane. The proton chemical gradient contributes to mitochondrial ATP synthesis as well as the uptake and release of metabolites and ions from the organelle. Here mitochondrial pH and ΔpH were studied for the first time in human pancreatic β-cells. Adenoviruses were used for rat insulin promoter dependent expression of the pH sensor SypHer targeted to either the mitochondrial matrix or the cytosol. The matrix pH in resting human β-cells is low (pH = 7.50 ± SD 0.17) compared to published values in other cell types. Consequently, the ΔpH of β-cells mitochondria is small. Glucose stimulation consistently resulted in acidification of the matrix pH in INS-1E insulinoma cells and β-cells in intact human islets or islet monolayer cultures. We registered acidification with similar kinetics but of slightly smaller amplitude in the cytosol of β-cells, thus glucose stimulation further reduced the ΔpH. Infection of human islets with high levels of adenoviruses caused the mitochondrial pH to increase. The apoptosis inducer and broad-spectrum kinase inhibitor staurosporine had similar effects on pH homeostasis. Although staurosporine alone does not affect the mitochondrial pH, glucose slightly increases the matrix pH of staurosporine treated cells. These two cellular stressors alter the normal mitochondrial pH response to glucose in pancreatic β-cells.

Acid-responsive activity of the Helicobacter pylori metalloregulator NikR

Helicobacter pylori is a human pathogen that infects the stomach, where it experiences variable pH. To survive the acidic gastric conditions, H. pylori produces large quantities of urease, a nickel enzyme that hydrolyzes urea to ammonia, which neutralizes the local environment. One of the regulators of urease expression in H. pylori is HpNikR, a nickel-responsive transcription factor. Here we show that HpNikR also regulates urease expression in response to changes in pH, linking acid adaptation and nickel homeostasis. Upon measuring the cytosolic pH of H. pylori exposed to an external pH of 2, similar to the acidic shock conditions that occur in the human stomach, a significant drop in internal pH was observed. This decrease in internal pH resulted in HpNikR-dependent activation of ureA transcription. Furthermore, analysis of a slate of H. pylori genes encoding other acid adaptation or nickel homeostasis components revealed HpNikR-dependent regulation in response to acid shock. This regulation was consistent with pH-dependent DNA binding to the corresponding promoter sequences observed in vitro with purified HpNikR. These results demonstrate that HpNikR can directly respond to changes in cytosolic pH during acid acclimation and illustrate the exquisitely coordinated regulatory networks that support H. pylori infections in the harsh environment of the human stomach.

Real-Time Recording of the Cellular Effects of the Anion Transporter Prodigiosin

Summary The unraveling of the cellular effects of anion transporters is key to their potential development as apoptosis-inducing or autophagy-disrupting therapeutics. Here, we conducted a systematic study of the cellular responses to the anion transporter prodigiosin by using a pH on/off responsive near-infrared (NIR)-fluorescent probe in HeLa and LAMP1-GFP-transfected HeLa cell lines. The sequence of localized and global cellular acidity changes and the resulting outcomes induced by the anion transporter were visualized with high temporal and spatial resolution. The results show that prodigiosin causes the pH within the lysosomal lumen to rise, after which a non-organelle-specific increase in acidity of the cytosol takes place, which prompts cells to undergo apoptosis. This was confirmed by the quantification of NIR-emissive lysosomes, the intra-cellular fluorescence intensity, and fluorescence volume over time. This NIR probe overcame the limitations of acridine orange, which to date have severely restricted researchers in this field.

A comparative analysis of cytosolic Na+ changes under salinity between halophyte quinoa (Chenopodium quinoa) and glycophyte pea (Pisum sativum)

Sodium (Na+) uptake into the halophyte quinoa (Chenopodium quinoa Willd.) plants was compared with the uptake into pea (Pisum sativum L.), and related to changes in cytosolic pH and potassium (K+) concentration in plant tissues. The total uptake of Na+ and K+ in roots and shoots was analyzed and compared with net ion fluxes at the root xylem parenchyma, determined by ion-specific microelectrodes in a non-invasive way. The cytosolic changes of Na+ concentration, [Na+cyt], and pH, pHcyt, were measured by fluorescent probes, specific to Na+ and H+, using a dual-wavelength fluorescence microscopy. These changes were monitored in protoplasts after cultivation with or without 100mM NaCl, and after addition of NaCl to the protoplasts. Roots and shoots of quinoa controls contained much higher K+ levels than pea roots and shoots, and the K+ levels increased even more after salinity treatments in quinoa. The cytosolic uptake of Na+ in quinoa protoplasts was transient if less than 200mM NaCl was added, while in pea the Na+ concentration increased even upon addition of 50mM Na+ and gradually increased with time. Saline conditions during cultivation increased pHcyt of both species. However, with a direct addition of NaCl to control protoplasts only a small increase was seen in pea pHcyt while in quinoa this increase was much larger. The different reactions of pHcyt to salinity when NaCl was added to salinity-treated seedlings may reflect an increased proton pump activity in quinoa, while this activation is lacking in pea. ABA addition to the root xylem parenchyma cells induced a net efflux of K+ and acidification of the xylem. On the other hand, 20mM NaCl addition induced a net flux of protons in both species, and a net K+ flux in pea, but not in quinoa, probably since such a low concentration is not a stress for quinoa. It is suggested that salinity tolerance in quinoa is achieved by a faster removal of Na+ from the cytosol and a high K+ concentration in roots and shoots under salinity, resulting in a high K+/Na+ ratio, and that this mechanism is driven by a higher proton pump activity, compared with glycophytic pea species.

Ammonium as a signal for physiological and morphological responses in plants.

Ammonium is a major inorganic nitrogen source for plants. At low external supplies, ammonium promotes plant growth, while at high external supplies it causes toxicity. Ammonium triggers rapid changes in cytosolic pH, in gene expression, and in post-translational modifications of proteins, leading to apoplastic acidification, co-ordinated ammonium uptake, enhanced ammonium assimilation, altered oxidative and phytohormonal status, and reshaped root system architecture. Some of these responses are dependent on AMT-type ammonium transporters and are not linked to a nutritional effect, indicating that ammonium is perceived as a signaling molecule by plant cells. This review summarizes current knowledge of ammonium-triggered physiological and morphological responses and highlights existing and putative mechanisms mediating ammonium signaling and sensing events in plants. We put forward the hypothesis that sensing of ammonium takes place at multiple steps along its transport, storage, and assimilation pathways.

Chloroplast Membrane Remodeling during Freezing Stress Is Accompanied by Cytoplasmic Acidification Activating SENSITIVE TO FREEZING2.

Low temperature is a seasonal abiotic stress that restricts native plant ranges and crop distributions. Two types of low-temperature stress can be distinguished: chilling and freezing. Much work has been done on the mechanisms by which chilling is sensed, but relatively little is known about how plants sense freezing. Recently, Arabidopsis (Arabidopsis thaliana) SENSITIVE TO FREEZING2 (SFR2) was identified as a protein that responds in a nontranscriptional manner to freezing. Here, we investigate the cellular conditions that allow SFR2 activation. Using a combination of isolated organelle, whole-tissue, and whole-plant assays, we provide evidence that SFR2 is activated by changes in cytosolic pH and Mg(2+) Manipulation of pH and Mg(2+) in cold-acclimated plants is shown to cause changes similar to those of freezing. We conclude that pH and Mg(2+) are perceived as intracellular cues as part of the sensing mechanism for freezing conditions. This evidence provides a specific molecular mechanism to combat freezing.

Catalytic activity of human carbonic anhydrase isoform IX is displayed both extra- and intracellularly.

Most carbonic anhydrases catalyse the reversible conversion of carbon dioxide to protons and bicarbonate, either as soluble cytosolic enzymes, in or at intracellular organelles, or at the extracellular face of the cell membrane as membrane-anchored proteins. Carbonic anhydrase isoform IX (CA IX), a membrane-bound enzyme with catalytic activity at the extracellular membrane surface, has come to prominence in recent years because of its association with hypoxic tissue, particularly tumours, often indicating poor prognosis. We have evaluated the catalytic activity of CA IX heterologously expressed in Xenopus laevis oocytes by measuring the amplitude and rate of cytosolic pH changes as well as pH changes at the outer membrane surface (pHs ) during addition and removal of 5% CO2 /25 mm HCO3-, and by mass spectrometry. Our results indicate both extracellular and intracellular catalytic activity of CA IX. Reduced rates of CO2 -dependent intracellular pH changes after knockdown of CA IX confirmed these findings in two breast cancer cell lines: MCF-7 and MDA-MB-231. Our results demonstrate a new function of CA IX that may be important in the search for therapeutic cancer drugs targeting CA IX.

Carbonic anhydrase IX inhibition is an effective strategy for osteosarcoma treatment

Objective: Hypoxia-inducible factor 1, a regulator of CA IX activity, is often overexpressed in human osteosarcoma (OS) but not in normal tissues, and its expression levels correlate with prognosis. In this study, we investigated the therapeutic potential of newly synthesized CA IX sulfonamide inhibitors in OS.Methods: CA IX expression was evaluated in OS cell lines and bone marrow stromal cells (BMSC). After treatment with CA IX inhibitors, cell proliferation, apoptosis, cell cycle, extracellular and cytosolic pH changes were evaluated both in vitro and in mouse OS xenografts.Results: CA IX expression levels were significantly higher in OS than in BMSC. Accordingly, CA IX inhibitor 3 induced remarkable cytotoxicity on OS cells without affecting BMSC proliferation. This activity was increased under hypoxia, and was mediated by cell cycle arrest and by the modulation of cytosolic and extracellular pH. In vivo, CA IX inhibitor 3 reduced tumor growth by inducing significant necrosis.Conclusions: Our results provide a strong rationale for the clinical use of the newly synthesized CA IX inhibitor 3 in human OS.

Analysis of cytosolic pH changes in thymocytes during early apoptosis with improved three-channel real-time fluorescence imaging.

Cytosolic pH changes in single thymocytes in the early stages of apoptosis were examined with spectral-crosstalk-improved three-channel real-time fluorescence imaging. Spectral crosstalk was greatly reduced by this improved image processing method. Compared with normal thymocytes, the improved image processing method showed that the cytosolic pH value of thymocytes undergoing apoptosis was lower. A slight diversity in the intracellular pH values of samples subjected to different drug treatments was also observed. This new image processing method is beneficial to the application of the three-channel real-time fluorescence imaging to biological processes.

Inhibition of ABA-induced stomatal closure by fusicoccin is associated with cytosolic acidification-mediated hydrogen peroxide removal.

BackgroundFusicoccin (FC), a fungal phytotoxin produced by Fusicoccum amygdale, causes the inhibition of ABA-induced stomatal closure. The mechanism of inhibition is remaining unclear. We analyzed the role of hydrogen peroxide (H2O2) and relationship between H2O2 removal and cytosolic pH changes during inhibition of ABA-induced stomatal closure by FC.ResultsAccording to the results, ABA treatment induced H2O2 production and stomatal closure, but FC inhibited the effects of ABA on these two parameters. Treatment with catalase (CAT) and NADPH oxidase inhibitor diphenylene iodonium (DPI) mimicked the effect of FC. These data suggest that inhibition of ABA effect by FC is related to the decrease of H2O2 levels in guard cells. Furthermore, similar to CAT, FC not only suppressed stomatal closure and H2O2 levels in guard cells treated with exogenous H2O2, but also reopened the stomata which had been closed by ABA and reduced the level of H2O2 that had been produced by ABA, indicating that FC causes H2O2 removal in guard cells. The butyric acid treatment simulated the effects of FC on the stomatal aperture and H2O2 levels in guard cells treated with exogenous H2O2 and had been closed by ABA, and both FC and butyric acid reduced cytosolic pH in guard cells of stomata treated with H2O2 and had been closed by ABA, which demonstrate that cytosolic acidification mediates FC-induced H2O2 removal.ConclusionThese results suggest that FC causes cytosolic acidification in guard cells, then induces H2O2 removal and reduces H2O2 levels in guard cells, finally inhibits stomatal closure induced by ABA.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-55-33) contains supplementary material, which is available to authorized users.

Cadmium uptake in Elodea canadensis leaves and its interference with extra‐ and intra‐cellular pH

This study investigated cadmium (Cd) uptake in Elodea canadensis shoots under different photosynthetic conditions, and its effects on internal (cytosolic) and external pH. The plants were grown under photosynthetic (light) or non-photosynthetic (dark or in the presence of a photosynthetic inhibitor) conditions in the presence or absence of CdCl2 (0.5 μm) in a medium with a starting pH of 5.0. The pH-sensitive dye BCECF-AM was used to monitor cytosolic pH changes in the leaves. Cadmium uptake in protoplasts and leaves was detected with a Cd-specific fluorescent dye, Leadmium Green AM, and with atomic absorption spectrophotometry. During cultivation for 3 days without Cd, shoots of E. canadensis increased the pH of the surrounding water, irrespective of the photosynthetic conditions. This medium alkalisation was higher in the presence of CdCl2 . Moreover, the presence of Cd also increased the cation exchange capacity of the shoots. The total Cd uptake by E. canadensis shoots was independent of photosynthetic conditions. Protoplasts from plants exposed to 0.5 μm CdCl2 for 3 days did not exhibit significant change in cytosolic [Cd(2+)] or pH. However, exposure to CdCl2 for 7 days resulted in increased cytosolic [Cd(2+) ] as well as pH. The results suggest that E. canadensis subjected to a low CdCl2 concentration initially sequesters Cd into the apoplasm, but under prolonged exposure, Cd is transported into the cytosol and subsequently alters cytosolic pH. In contrast, addition of 10-50 μm CdCl2 directly to protoplasts resulted in immediate uptake of Cd into the cytosol.

Consequences of Loss of Vph1 Protein-containing Vacuolar ATPases (V-ATPases) for Overall Cellular pH Homeostasis

In yeast cells, subunit a of the vacuolar proton pump (V-ATPase) is encoded by two organelle-specific isoforms, VPH1 and STV1. V-ATPases containing Vph1 and Stv1 localize predominantly to the vacuole and the Golgi apparatus/endosomes, respectively. Ratiometric measurements of vacuolar pH confirm that loss of STV1 has little effect on vacuolar pH. Loss of VPH1 results in vacuolar alkalinization that is even more rapid and pronounced than in vma mutants, which lack all V-ATPase activity. Cytosolic pH responses to glucose addition in the vph1Δ mutant are similar to those in vma mutants. The extended cytosolic acidification in these mutants arises from reduced activity of the plasma membrane proton pump, Pma1p. Pma1p is mislocalized in vma mutants but remains at the plasma membrane in both vph1Δ and stv1Δ mutants, suggesting multiple mechanisms for limiting Pma1 activity when organelle acidification is compromised. pH measurements in early prevacuolar compartments via a pHluorin fusion to the Golgi protein Gef1 demonstrate that pH responses of these compartments parallel cytosolic pH changes. Surprisingly, these compartments remain acidic even in the absence of V-ATPase function, possibly as a result of cytosolic acidification. These results emphasize that loss of a single subunit isoform may have effects far beyond the organelle where it resides.