Proton pumping by tomato roots. Effect of Fe deficiency and hormones on the activity and distribution of plasma membrane H+-ATPase in rhizodermal cells

The immunocytochemical localization of the plasma membrane H⁺‐ATPase in epidermal cells of tomato roots was studied using a monoclonal antibody raised against purified maize P‐type H⁺‐ATPase. Plants subjected to iron starvation exhibited increased proton extrusion that was confined to the root elongation zones. Immunogold labelling of the H⁺‐ATPase on the plasma membrane was considerably higher in rhizodermal cells within zones with intense proton extrusion than in non‐acidifying areas of the roots. Transfer cells were formed in rhizodermal cells of Fe‐deficient plants. Quantitative determination of immunolabelling revealed that the density of PM H⁺‐ATPase in transfer cells was about twice that of ordinary epidermal cells. In transfer cells, H⁺‐ATPase was most abundant on the plasma membrane lining the labyrinthine invaginations of the peripheral cell wall. While the number of immunologically detectable ATPase molecules in transfer cells was not spatially correlated with proton extrusion activity, the frequency of transfer cells was considerably higher in acidifying root areas relative to non‐active segments. Split‐root experiments indicated that both the steady‐state level of plasma membrane H⁺‐ATPase and proton extrusion activity are systemically regulated, indicating inter‐organ regulation of rhizosphere acidification. Exogenous application of the auxin analog 2,4‐dichlorophenoxyacetic acid and the ethylene precursor 1‐aminocyclopropane‐1‐carboxlic acid caused the formation of transfer cells at a frequency similar to that observed in Fe‐deficient roots. However, the number of proton pumps was not affected by the hormone treatment, suggesting that both responses are regulated independently. It is concluded that transfer cells in the rhizodermis may be important but not crucial for rhizosphere acidification.     

Plasma membrane H+-ATPase in sorghum roots as affected by potassium deficiency and nitrogen sources

We studied the influence of inorganic nitrogen sources (NO₃ ^? or NH₄ ⁺) and potassium deficiency on expression and activity of plasma membrane (PM) H⁺-ATPase in sorghum roots. After 15 d of cultivation at 0.2 mM K⁺, the plants were transferred to solutions lacking K⁺ for 2 d. Then, K⁺ depletion assays were performed in the presence or absence of vanadate. Further, PMs from K⁺-starved roots were extracted and used for the kinetic characterization of ATP hydrolytic activity and the immunodetection of PM H⁺-ATPase. Two major genes coding PM H⁺-ATPase (SBA1 and SBA2) were analyzed by real-time PCR. PM H⁺-ATPase exhibited a higher V_max and K_m in NH₄ ⁺-fed roots compared with NO₃ ^? -fed roots. The optimum pH of the enzyme was slightly lower in NO₃ ^? -fed roots than in NH₄ ⁺-fed roots. The vanadate sensitivity was similar. The expressions of SBA1 and SBA2 increased in roots grown under NH₄ ⁺. Concomitantly, an increased content of the enzyme in PM was observed. The initial rate of K⁺ uptake did not differ between plants grown with NO₃ ^? or NH₄ ⁺, but it was significantly reduced by vanadate in NH₄ ⁺-grown plants.     

Plasma membrane ATPase and H+ transport activities in microsomal membranes from mycorrhizal tomato roots

ATPase activity, ATP-dependent H⁺ transport and the amount of antigenic tomato plasma membrane H⁺-APTase have been analysed in membrane vesicles isolated from Glomus mosseae- or Glomus intraradices-colonized roots and from non-mycorrhizal tomato roots. Microsomal protein content was higher in mycorrhizal than in control roots. The specific activity of the plasma membrane H⁺-ATPase was not affected by mycorrhizal colonization, although this activity increased in membranes isolated from mycorrhizal roots when expressed on a fresh weight basis. Western blot analysis of microsomal proteins using antibodies raised against the Arabidopsis thaliana plasma membrane H⁺ - ATPase showed that mycorrhizal colonization did not change the relative amount of tomato plasma membrane ATPase in the microsomes. However, on a fresh weight basis, there was a greater amount of this protein in roots of mycorrhizal plants. In addition, mycorrhizal membranes showed a higher specific activity of the vanadate-sensitive ATP-dependant H⁺ transport than membranes isolated from control roots. These results suggest that mycorrhiza might regulate the plasma membrane ATPase by increasing the coupling efficiency between H⁺ transport and ATP hydrolysis. The observed effects of mycorrhizal colonization on plasma membrane H⁺-ATPase were independent of the AM fungal species colonizing the root system.     

Fusicoccin Binding to its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase. II. Stimulation of the H+-ATPase in a Plasma Membrane Fraction Purified by Phase-partitioning

The effect of fusicoccin (FC) on the activity of the PM H⁺-ATPase was investigated in a plasma membrane (PM) fraction from radish seedlings purified by the phase-partitioning procedure. FC stimulated the PM H⁺-ATPase activity by up to 100 %; the effect was essentially on V_max with only a slight decrease of the apparent K_M of the enzyme for ATP. FC-induced stimulation of the PM H⁺-ATPase was evident within the first minute and maximal within five minutes of membrane treatment with the toxin indicating that transmission of the signal from the activated receptor to the PM H⁺-ATPase is very rapid. Both FC-induced stimulation of the PM H⁺-ATPase and FC binding to its receptor decreased dramatically upon incubation of the membranes in ATPase assay medium at 33 °C in the absence of FC, due to the lability of the free FC receptor. FC-induced stimulation of the PM H⁺-ATPase was strongly pH dependent: absolute increase of activity was maximal at pH 7, while percent stimulation increased with the increase of pH up to pH 7.5; FC binding was scarcely influenced by pH in the pH range investigated. Taken as a whole, these results indicate that FC binding is a condition necessary, but not sufficient, for FC-induced stimulation of the PM H⁺-ATPase.     

Dimorphism-associated changes in plasma membrane H+-ATPase activity of Candida albicans

In situ plasma membrane H⁺-ATPase activity was monitored during pH-regulated dimorphism of Candida albicans using permeabilized cells. ATPase activity was found to increase in both the bud and germ tube forming populations at 135 min which coincides with the time of evagination. Upon reaching the terminal phenotype the mycelial form exhibited higher H⁺-ATPase activity as compared to the yeast form. At the time of evagination H⁺-efflux exhibited an increase. K⁺ depletion resulted in attenuated ATPase activity and glucose induced H⁺-efflux. The results demonstrate that ATPase may play a regulatory role in dimorphism of C. albicans and K⁺ acts as a modulator.Abbreviations PM Plasma membrane - pHi intracellular pH - Pi inorganic phosphorus - TET Toluene: Ethanol: Triton X-100     

Effect of potassium deficiency on the plasma membrane H+-ATPase of the wood ray parenchyma in poplar

The effect of K⁺ deficiency on the plasma membrane (PM) H⁺‐ATPase was studied in young stems of poplar plants (Populus tremula × tremuloides) grown with low or full‐strength K⁺ supply. Immunological assays using different antibodies were applied to test if K⁺ deficiency affects the amount of immunodetectable PM H⁺‐ATPases in the stem tissue. The monoclonal antibody clone 46 E5 B11 revealed an increased abundance of PM H⁺‐ATPases under conditions of low K⁺ supply, and immunolabelling experiments showed that this increase was restricted to vessel‐associated cells (VACs) of the wood ray parenchyma. Replacement of the monoclonal antibody by a polyclonal antibody against PM H⁺‐ATPase gave a specific immunoreactivity on blots as well as tissue sections too, but the labelling intensity showed no difference between plants with low or full‐strength K⁺ supply. Measurements of extracellular H⁺ concentrations using non‐invasive, H⁺‐selective microelectrodes revealed a lowering of the pH at the surface of VACs and an enhancement of net efflux of H⁺ in plants grown with low K⁺ supply. The present results indicate an up‐regulation of specific isoforms of the PM H⁺‐ATPase in VACs under K⁺‐deficient conditions and suggest a key role for these PM H⁺‐ATPases in unloading K⁺ from the xylem stream.     

Jatropha is vulnerable to cold injury due to impaired activity and expression of plasma membrane H+-ATPase

Cold stress is one of the major environmental factors limiting the amount of plant mass for bioenergy production. A chilling-sensitive Jatropha (Jatropha curcas L.) as a bioenergy crop was used to investigate the cold injury process at the physiological and biochemical levels. Various physiological parameters such as leaf length, width, stomatal conductance, chlorophyll fluorescence, and electrolyte leakage were measured to determine the growth rate of leaves cold-treated (7 and 2 °C) for 5 days. These parameters of cold-treated Jatropha were significantly reduced from day 1 compared with control (23 °C). Using the pH indicator bromocresol purple, it was shown that surface pH of Jatropha root in control was strongly acidified by time only from the starting pH 6, while H⁺-efflux of the surface of cold-treated roots did not change. H⁺-ATPase activity of plasma membrane (PM) isolated from leaves and roots of cold-treated Jatropha was decreased in a time-dependent manner. The expression of PM H⁺-ATPase and 14-3-3 protein, which participates in phosphorylation of PM H⁺-ATPase was reduced in the presence of cold stress. Interestingly, fusicoccin, an activator of the PM H⁺-ATPase, alleviated cold-injury by stimulating the enzyme in leaves. These results may suggest that the activity and expression of PM H⁺-ATPase in Jatropha is closely related to the overcoming of cold stress.     

Effect of NaCl on leaf H+-ATPase and the relevance to salt tolerance in two contrasting poplar species

During a 30-day period of increasing salinity, we examined the effects of NaCl on leaf H⁺-ATPase and salinity tolerance in 1-year-old plants of Populus euphratica Oliv. (salt resistant) and P. popularis 35–44 (P. popularis) (salt sensitive). Electron probe X-ray microanalysis of leaf mesophyll revealed that P. euphratica had a higher ability to retain lower NaCl concentrations in the cytoplasm, as compared to P. popularis. The sustained activity of H⁺ pumps (by cytochemical staining) in salinised P. euphratica suggests a role in energising salt transport through the plasma membrane (PM) and tonoplast. Salt-induced alterations of leaf respiration, ATP content and expression of PM H⁺-ATPase were compared between the two species. Results show that P. euphratica retained a constant respiratory rate, ATP production and protein abundance of PM H⁺-ATPase (by Western blotting) in salt-stressed plants. P. euphratica was able to maintain a comparatively high capacity of ATP hydrolysis and H⁺ pumping during prolonged salt exposure. By contrast, the activity and expression of PM H⁺-ATPase were markedly decreased in P. popularis leaves in response to salt stress. Furthermore, NaCl-stressed P. popularis plants showed a marked decline of respiration (70%) and ATP production (66%) on day 30. We conclude that the inability of P. popularis to transport salt to the apoplast and vacuole was partly due to the decreased activity of H⁺ pumps. As a consequence, cytosolic ion concentrations were observed to be comparatively high for an extended period of time, so that cell metabolism, in particular respiration, was disrupted in P. popularis leaves.     

Glucose-6-phosphate dehydrogenase-dependent hydrogen peroxide production is involved in the regulation of plasma membrane H+-ATPase and Na+/H+ antiporter protein in salt-stressed callus from Carex moorcroftii

Glucose‐6‐phosphate dehydrogenase (G6PDH) is important for the activation of plant resistance to environmental stresses, and ion homeostasis is the physiological foundation for living cells. In this study, we investigated G6PDH roles in modulating ion homeostasis under salt stress in Carex moorcroftii callus. G6PDH activity increased to its maximum in 100 mM NaCl treatment and decreased with further increased NaCl concentrations. K⁺/Na⁺ ratio in 100 mM NaCl treatment did not exhibit significant difference compared with the control; however, in 300 mM NaCl treatment, it decreased. Low‐concentration NaCl (100 mM) stimulated plasma membrane (PM) H⁺‐ATPase and NADPH oxidase activities as well as Na⁺/H⁺ antiporter protein expression, whereas high‐concentration NaCl (300 mM) decreased their activity and expression. When G6PDH activity and expression were reduced by glycerol treatments, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein level and K⁺/Na⁺ ratio dramatically decreased. Simultaneously, NaCl‐induced hydrogen peroxide (H₂O₂) accumulation was abolished. Exogenous application of H₂O₂ increased G6PDH, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein expression and K⁺/Na⁺ ratio in the control and glycerol treatments. Diphenylene iodonium (DPI), the NADPH oxidase inhibitor, which counteracted NaCl‐induced H₂O₂ accumulation, decreased G6PDH, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein level and K⁺/Na⁺ ratio. Western blot result showed that G6PDH expression was stimulated by NaCl and H₂O₂, and blocked by DPI. Taken together, G6PDH is involved in H₂O₂ accumulation under salt stress. H₂O₂, as a signal, upregulated PM H⁺‐ATPase activity and Na⁺/H⁺ antiporter protein level, which subsequently resulted in the enhanced K⁺/Na⁺ ratio. G6PDH played a central role in the process.     

The Arabidopsis Chaperone J3 Regulates the Plasma Membrane H+-ATPase through Interaction with the PKS5 Kinase

The plasma membrane H⁺-ATPase (PM H⁺-ATPase) plays an important role in the regulation of ion and metabolite transport and is involved in physiological processes that include cell growth, intracellular pH, and stomatal regulation. PM H⁺-ATPase activity is controlled by many factors, including hormones, calcium, light, and environmental stresses like increased soil salinity. We have previously shown that the Arabidopsis thaliana Salt Overly Sensitive2-Like Protein Kinase5 (PKS5) negatively regulates the PM H⁺-ATPase. Here, we report that a chaperone, J3 (DnaJ homolog 3; heat shock protein 40-like), activates PM H⁺-ATPase activity by physically interacting with and repressing PKS5 kinase activity. Plants lacking J3 are hypersensitive to salt at high external pH and exhibit decreased PM H⁺-ATPase activity. J3 functions upstream of PKS5 as double mutants generated using j3-1 and several pks5 mutant alleles with altered kinase activity have levels of PM H⁺-ATPase activity and responses to salt at alkaline pH similar to their corresponding pks5 mutant. Taken together, our results demonstrate that regulation of PM H⁺-ATPase activity by J3 takes place via inactivation of the PKS5 kinase.