V H-ATPase along the yeast secretory pathway: Energization of the ER and Golgi membranes

H⁺ transport driven by V H⁺-ATPase was found in membrane fractions enriched with ER/PM and Golgi/Golgi-like membranes of Saccharomyces cerevisiae efficiently purified in sucrose density gradient from the vacuolar membranes according to the determination of the respective markers including vacuolar Ca²⁺-ATPase, Pmc1::HA. Purification of ER from PM by a removal of PM modified with concanavalin A reduced H⁺ transport activity of P H⁺-ATPase by more than 75% while that of V H⁺-ATPase remained unchanged. ER H⁺ ATPase exhibits higher resistance to bafilomycin (I₅₀ = 38.4 nM) than Golgi and vacuole pumps (I₅₀ = 0.18 nM). The ratio between a coupling efficiency of the pumps in ER, membranes heavier than ER, vacuoles and Golgi is 1.0, 2.1, 8.5 and 14 with the highest coupling in the Golgi. The comparative analysis of the initial velocities of H⁺ transport mediated by V H⁺-ATPases in the ER, Golgi and vacuole membrane vesicles, and immunoreactivity of the catalytic subunit A and regulatory subunit B further supported the conclusion that V H⁺-ATPase is the intrinsic enzyme of the yeast ER and Golgi and likely presented by distinct forms and/or selectively regulated.     

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.     

NH(4)(+)-stimulated low-K(+) uptake is associated with the induction of H(+) extrusion by the plasma membrane H(+)-ATPase in sorghum roots under K(+) deficiency

The effect of external inorganic nitrogen and K⁺ content on K⁺ uptake from low-K⁺ solutions and plasma membrane (PM) H⁺-ATPase activity of sorghum roots was studied. Plants were grown for 15 days in full-nutrient solutions containing 0.2 or 1.4 mM K⁺ and inorganic nitrogen as NO₃^-, NO₃^-/NH₄⁺ or NH₄⁺ and then starved of K⁺ for 24, 48 and 72 h. NH₄⁺ in full nutrient solution significantly affected the uptake efficiency and accumulation of K⁺, and this effect was less pronounced at the high K⁺ concentration. In contrast, the translocation rate of K⁺ to the shoot was not altered. Depletion assays showed that plants grown with NH₄⁺ more efficiently depleted the external K⁺ and reached higher initial rates of low-K⁺ uptake than plants grown with NO₃^-. One possible influence of K⁺ content of shoot, but not of roots, on K⁺ uptake was evidenced. Enhanced K⁺-uptake capacity was correlated with the induction of H⁺ extrusion by PM H⁺-ATPase. In plants grown in high K⁺ solutions, the increase in the active H⁺ gradient was associated with an increase of the PM H⁺-ATPase protein concentration. In contrast, in plants grown in solutions containing 0.2 mM K⁺, only the initial rate of H⁺-pumping and ATP hydrolysis were affected. Under these conditions, two specific isoforms of PM H⁺-ATPase were detected, independent of the nitrogen source and deficiency period. No change in enzyme activity was observed in NO₃^--grown plants. The results suggest that K⁺ homeostasis in NH₄⁺-grown sorghum plants may be regulated by a high capacity for K⁺ uptake, which is dependent upon the H⁺-pumping activity of PM H⁺-ATPase.     

Ethylene and nitric oxide are involved in maintaining ion homeostasis in <Emphasis Type="Italic">Arabidopsis</Emphasis> callus under salt stress

In the present study, the role of ethylene in nitric oxide (NO)-mediated protection by modulating ion homeostasis in Arabidopsis callus under salt stress was investigated. Results showed that the ethylene-insensitive mutant etr1-3 was more sensitive to salt stress than the wild type (WT). Under 100 mM NaCl, etr1-3 callus displayed a greater electrolyte leakage and Na⁺/K⁺ ratio but a lower plasma membrane (PM) H⁺-ATPase activity compared to WT callus. Application of exogenous 1-aminocyclopropane-1-carboxylic acid (ACC, an ethylene precursor) or sodium nitroprusside (SNP, a NO donor) alleviated NaCl-induced injury by maintaining a lower Na⁺/K⁺ ratio and an increased PM H⁺-ATPase activity in WT callus but not in etr1-3 callus. The SNP actions in NaCl stress were attenuated by a specific NO scavenger or an ethylene biosynthesis inhibitor in WT callus. Under 100 mM NaCl, the NO accumulation and ethylene emission appeared at early time, and NO production greatly stimulated ethylene emission in WT callus. In addition, ethylene induced the expression of PM H⁺-ATPase genes under salt stress. The recovery experiment showed that NaCl-induced injury was reversible, as signaled by the similar recovery of Na⁺/K⁺ ratio and PM H⁺-ATPase activity in WT callus. Taken together, the results indicate that ethylene and NO cooperate in stimulating PM H⁺-ATPase activity to modulate ion homeostasis for salt tolerance, and ethylene may be a part of the downstream signal molecular in NO action.     

NaCl increases the activity of the plasma membrane H+-ATPase in C3 halophyte Suaeda salsa callus

Suaeda salsa calli treated with different concentrations of NaCl were used to examine the response of the plasma membrane (PM) H⁺-ATPase to NaCl and its role in salt tolerance. The optimum concentration of NaCl for growth of the calli was 50 mM, while growth was significantly inhibited at 250 mM NaCl. The ion and organic solute contents of calli increased with increasing NaCl. Activity of the PM H⁺-ATPase increased when the calli were treated with NaCl over a certain concentration range (0–150 mM NaCl). However, the activity reached its maximum with 150 mM NaCl. Immunoblotting analysis of the PM H⁺-ATPase protein from calli cultures with anti-Zea mays H⁺-ATPase serum (monoclonal 46E5B11D5) identified a single polypeptide of ~90 kDa. The peptide levels increased in the calli treated with NaCl at 150 mM NaCl compared to control, but the increase at 50 mM NaCl was less pronounced. Northern blot analysis showed that the expression of the PM H⁺-ATPase also increased after the calli were treated with NaCl. These results suggest that the increase in PM H⁺-ATPase activity is due to both an increase in the amount of PM H⁺-ATPase protein and an up-regulation of the PM H⁺-ATPase gene, which is involved in the salt tolerance of S. salsa calli.     

SOS1 gene overexpression increased salt tolerance in transgenic tobacco by maintaining a higher K(+)/Na(+) ratio

Crop productivity is greatly affected by soil salinity, so improvement in salinity tolerance of crops is a major objective of many studies. We overexpressed the Arabidopsis thaliana SOS1 gene, which encodes a plasma membrane Na⁺/H⁺ antiporter, in tobacco (Nicotiana tabacum cv. Xanthi-nc). Compared with nontransgenic plants, seeds from transgenic tobacco had better germination under 120 mM (mmol L⁻¹) NaCl stress; chlorophyll loss in the transgenic seedlings treated with 360 mM NaCl was less; transgenic tobacco showed superior growth after irrigation with NaCl solutions; and transgenic seedlings with 150 mM NaCl stress accumulated less Na⁺ and more K⁺. In addition, roots of SOS1-overexpressing seedlings lost less K⁺ instantaneously in response to 50 mM NaCl than control plants. These results showed that the A. thaliana SOS1 gene potentially can improve the salt tolerance of other plant species.     

Distribution and change patterns of free IAA, ABP 1 and PM H⁺-ATPase during ovary and ovule development of Nicotiana tabacum L

Auxin plays key roles in flower induction, embryogenesis, seed formation and seedling development, but little is known about whether auxin regulates the development of ovaries and ovules before pollination. In the present report, we measured the content of free indole-3-acetic (IAA) in ovaries of Nicotiana tabacum L., and localized free IAA, auxin binding protein 1 (ABP1) and plasma membrane (PM) H⁺-ATPase in the ovaries and ovules. The level of free IAA in the developmental ovaries increased gradually from the stages of ovular primordium to the functional megaspore, but slightly decreased when the embryo sacs formed. Immunoenzyme labeling clearly showed that both IAA and ABP1 were distributed in the ovules, the edge of the placenta, vascular tissues and the ovary wall, while PM H⁺-ATPase was mainly localized in the ovules. By using immunogold labeling, the subcellular distributions of IAA, ABP1 and PM H⁺-ATPase in the ovules were also shown. The results suggest that IAA, ABP1 and PM H⁺-ATPase may play roles in the ovary and ovule initiation, formation and differentiation.     

Type 2C protein phosphatase clade D family members dephosphorylate guard cell plasma membrane H+-ATPase

Plasma membrane (PM) H⁺-ATPase in guard cells is activated by phosphorylation of the penultimate residue, threonine (Thr), in response to blue and red light, promoting stomatal opening. Previous in vitro biochemical investigation suggested that Mg²⁺- and Mn²⁺-dependent membrane-localized type 2C protein phosphatase (PP2C)-like activity mediates the dephosphorylation of PM H⁺-ATPase in guard cells. PP2C clade D (PP2C.D) was later demonstrated to be involved in PM H⁺-ATPase dephosphorylation during auxin-induced cell expansion in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether PP2C.D phosphatases are involved in PM H⁺-ATPase dephosphorylation in guard cells. Transient expression experiments using Arabidopsis mesophyll cell protoplasts revealed that all PP2C.D isoforms dephosphorylate the endogenous PM H⁺-ATPase. We further analyzed PP2C.D6/8/9, which display higher expression levels than other isoforms in guard cells, observing that pp2c.d6, pp2c.d8, and pp2c.d9 single mutants showed similar light-induced stomatal opening and phosphorylation status of PM H⁺-ATPase in guard cells as Col-0. In contrast, the pp2c.d6/9 double mutant displayed wider stomatal apertures and greater PM H⁺-ATPase phosphorylation in response to blue light, but delayed dephosphorylation of PM H⁺-ATPase in guard cells; the pp2c.d6/8/9 triple mutant showed similar phenotypes to those of the pp2c.d6/9 double mutant. Taken together, these results indicate that PP2C.D6 and PP2C.D9 redundantly mediate PM H⁺-ATPase dephosphorylation in guard cells. Curiously, unlike auxin-induced cell expansion in seedlings, auxin had no effect on the phosphorylation status of PM H⁺-ATPase in guard cells.

Type 2C protein phosphatase clade D family members redundantly dephosphorylate the penultimate C-terminal threonine residue of plasma membrane H⁺-ATPase in guard cells to control stomatal movement.     

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.     

A vacuolar H-pyrophosphatase differential activation and energy coupling integrate the responses of weeds and crops to drought stress

Background

Cyperus rotundus L. is a C4 weed of large vegetative and reproductive vigor endowed with competitive advantages over most crop species mainly under adverse environmental conditions. Vacuole functions are critical for the mechanisms of drought resistance, and here the modulation of the primary system of vacuolar ion transport is investigated during a transient water stress imposed to this weed and to C4 crop species (Zea mays L.).

Methods

The vacuolar H⁺ pumps, the H⁺-ATPase and H⁺-PP_iase, expression, activities and the energy coupling were spectrophotometrically investigated as key elements in the differential drought-resistance mechanisms developed by weeds and crops.

Results

In C. rotundus tonoplasts, ATP hydrolysis was more sensitive to drought than its coupled H⁺ transport, which was in turn at least 3-folds faster than that mediated by the H⁺-PP_iase. Its PP_i hydrolysis was only slightly affected by severe water deficit, contrasting with the disruption induced in the PP_i-dependent H⁺-gradient. This effect was antagonized by plant rehydration as the H⁺-PP_iase activity was highly stimulated, reassuming a coupled PP_i-driven H⁺ pumping. Maize tonoplasts exhibited 2–4 times lower hydrolytic activities than that of C. rotundus, but were able to overactivate specifically PP_i-dependent H⁺ pumping in response to stress relief, resulting in an enhanced H⁺-pumps coupling efficiency.

Conclusion

These results together with immunoanalysis revealed profiles consistent with pre- and post-translational changes occurring on the tonoplast H⁺-pumps, which differ between weeds and crops upon water deficit.

General significance

The evidences highlight an unusual modulation of the H⁺-PP_iase energy coupling as a key biochemical change related to environmental stresses adaptive capacity of plants.     

Chilling-Induced Inactivation and Its Recovery of Tonoplast H-ATPase in Mung Bean Cell Suspension Cultures

The processes involved in adaptation to cold temperature were examined by growing suspension cultured cells of mung bean (Vigna radiata [L.] Wilczek) at 2°C for various periods of time and assaying the activities of various membrane-bound enzymes in vitro. The tonoplast H⁺-ATPase activity and the ATP-proton transport extracted from cells incubated at 2°C declined rapidly and reached a minimum level after 10 hours. The inactivation was reversible within 24 hours of chilling. The recovery of the cold-inactivated H⁺-ATPase was found to proceed in two steps, a faster recovery of ATP hydrolysis activity and a slower recovery of the proton transport. The recovery was markedly inhibited by the presence of azide, but not affected by 0.578 millimolar cycloheximide. This suggested the involvement of an energy process that had no requirement for de novo synthesis of protein. The cold-induced inactivation of the H⁺-ATPase may be due to a structural alteration of the enzyme. The slower recovery of proton transport relative to ATP hydrolysis during warming suggests that the protogenic domains in the enzyme may be affected differently by chilling.     

Growth,photosynthesis and H+-ATPase activity in two Jerusalem artichoke varieties under NaCl-induced stress

In order to evaluate differential growth, photosynthesis and H⁺-ATPase activity responses to salt-induced stress, two Jerusalem artichoke (Helianthus tuberosus L.) genotypes (Nanyu No. 1 and Qingyu No. 2) were used in sand-culture experiment with different concentrations of NaCl (0, 30, 60, 90, 120 and 150 mM). After 20 days of growth, the NaCl stress resulted in a decrease of biomass accumulation, relative leaf expansion rate and photosynthetic rate, but an increase of proline content in both genotypes. Compared with Qingyu No. 2, Nanyu No. 1 had lower biomass, photosynthetic rate, gas exchange and transpiration rate, but higher proline content, activities of plasma membrane H⁺-ATPase (PM H⁺-ATPase) and vacuolar membrane H⁺-ATPase (VM H⁺-ATPase). Hence, the NaCl adaptation strategy in Nanyu No. 1 was by lowering photosynthetic rate, stomatal conductance and transpiration rate while maintaining high H⁺-ATPase activities, whereas the adjustment of Qingyu No. 2 was by keeping much higher rate of proline accumulation and concentration of chlorophyll. The differences in salt tolerance showed that different adaptation mechanisms existed between cultivars of Jerusalem artichoke. The findings offered the possibility of selecting salt-tolerant genotypes of Jerusalem artichoke.     

Salinity adaptation of plasma membrane H+-ATPase in the salt marsh plant Spartina patens: ATP hydrolysis and enzyme kinetics

Spartina patens, an intertidal C4 grass, grows in the upper salt marsh and tolerates coastal seawater salinity. The regulation of ion movement across the plasma membrane (PM) for plant salt tolerance is thought to be achieved by an electrochemical gradient generated by plasma membrane H⁺-ATPase. In this study, the change of PM H⁺-ATPase in response to NaCl was characterized for S. patens callus. Callus was cultured for 10 weeks under salinity levels of 0 mM, 170 mM, 340 mM, and 510 mM NaCl. Plasma membrane was isolated from a Dextran/PEG aqueous polymer two-phase system and the purity was demonstrated with membrane enzyme markers. There was a significant increase (up to 2-3 fold) of PM H⁺-ATPase activity when callus was grown on media containing NaCl. The incremental activation of PM H⁺-ATPase activity would enable the cell to tolerate higher cytoplasmic NaCl concentrations. PM H⁺-ATPase appeared to have a higher Vmax and a lower substrate concentration (Km to reach Vmax. When growth medium salinity increased from 0 mM to 170 and 340 mM, the Vmax of H⁺-ATPase increased from 0.64 to 1.00 and 1.73, respectively, while the Km decreased from 3.58 to 2.07 and 2.44 mM, respectively. In vitro NaCl inhibition kinetic data revealed a pattern of non-competitive inhibition by NaCl on PM H⁺-ATPase. The response of PM H⁺-ATPase in S. patens callus suggests that this species has evolved mechanisms that can regulate this important enzyme when cells are exposed to NaCl.     

Effects of salt stress and exogenous Ca2+ on Na+ compartmentalization,ion pump activities of tonoplast and plasma membrane in Nitraria tangutorum Bobr. leaves

Nitraria tangutorum Bobr. is a typical halophyte with superior tolerance to salinity. However, little is known about its physiological adaptation mechanisms to the salt environment. In the present study, N. tangutorum seedlings were treated with different concentrations of NaCl (100, 200, 300 and 400 mmol L^?1) combined with five levels of Ca²⁺ (0, 5, 10, 15 and 20 mmol L^?1) to investigate the effects of salt stress and exogenous Ca²⁺ on Na⁺ compartmentalization and ion pump activities of tonoplast and plasma membrane (PM) in leaves. Na⁺ and Ca²⁺ treatments increased the fresh weight and dry weight of N. tangutorum seedlings. The absorption of Na⁺ in roots, stems and leaves was substantially increased with the increases of NaCl concentration, and Na⁺ was mainly accumulated in leaves. Exogenous Ca²⁺ reduced Na⁺ accumulation in roots but promoted Na⁺ accumulation in leaves. The absorption and transportation of Ca²⁺ in N. tangutorum seedlings were inhibited under NaCl treatments. Exogenous Ca²⁺ promoted Ca²⁺ accumulation in the plant. Na⁺ contents in apoplast and symplast of leaves were also significantly increased, and symplast was the main part of Na⁺ intracellular compartmentalization. The tonoplast H⁺-ATPase and H⁺-PPase activities were significantly promoted under salt stress (NaCl concentrations ≤300 mmol L^?1). PM H⁺-ATPase activities gradually increased under salt stress (NaCl concentrations ≤200 mmol L^?1) followed by decreases with NaCl concentration increasing. The tonoplast H⁺-ATPase, H⁺-PPase and PM H⁺-ATPase activities increased first with the increasing exogenous Ca²⁺ concentration, reached the maximums at 15 mmol L^?1 Ca²⁺, and then decreased. The tonoplast and PM Ca²⁺-ATPase activities showed increasing trends with the increases of NaCl and Ca²⁺ concentration. These results suggested that certain concentrations of exogenous Ca²⁺ effectively enhanced ion pump activities of tonoplast and PM as well as promoted the intracellular Na⁺ compartmentalization to improve the salt tolerance of N. tangutorum.     

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.