Yeast genes YOL002C and SUL1 are involved in neomycin resistance

In previous studies we suggested the importance of the control of plasma membrane H⁺-ATPase by a phosphatidylinositol-like pathway for cellular proton extrusion in Saccharomyces cerevisiae (Brandão et al. 1994; Coccetti et al. 1998). The observations that provided the model above include the inhibition of the glucose-induced activation of the plasma membrane H⁺-ATPase as well as the inhibition of the glucose-induced external acidification by neomycin, a known inhibitor of the phosphatidylinositol turnover in eukaryotic cells. In this work, using two libraries, we isolated two yeast clones that were able to prevent the inhibition of glucose-induced activation of the H⁺-ATPase by neomycin. We show that the YOL002C gene, which encodes a protein of unknown function, and the SUL1 gene, which is a sulphate transporter belonging to the major facilitator superfamily, suppress growth inhibition by neomycin. However, they are not required for glucose-induced activation of the plasma membrane H⁺-ATPase. The resistance of the clones to neomycin is probably related to the level and/or activity of proteins functioning as drug extrusion pumps.     

A mutant of Arabidopsis thaliana partially resistant to fusicoccin has reduced plasma membrane H+-ATPase

5-2 is a mutant of Arabidopsis thaliana which is partially resistant to fusicoccin in vivo. We have analysed fusicoccin binding and the activity and amount of H⁺-ATPase in plasma membrane isolated from mature leaves of the wild type and of mutant 5-2. Fusicoccin binding was similar in plasma membrane from the two genotypes, while H⁺-ATPase activity was markedly (c. 50%) lower in plasma membrane from mutant 5-2 than in that from the wild type. The H⁺-ATPase of mutant 5-2 was activated by fusicoccin as much as that of the wild type. In plasma membrane from mutant 5-2, the amount of immunodetectable H⁺-ATPase, quantified by densitometry of Western blots, was about half that in the wild type. These results indicate that the major defect of mutant 5-2 detectable at the plasma membrane level is a reduction in the amount of H⁺-ATPase.     

Early changes of Cl− efflux and H+ extrusion induced by osmotic stress in Arabidopsis thaliana cells

In various plant materials changes in turgor pressure, following hyper- or hypo-osmotic stress, were associated with the activation or inactivation of the plasma membrane H⁺-ATPase, respectively. To see if the turgor changes might indirectly influence H⁺-ATPase activity by regulating ion fluxes through plasma membrane, we investigated, in cultured cells of Arabidopsis thaliana (L.) Heynh., the early effects of hyper- and hypo-osmotic stress on Cl^? fluxes in comparison, in the case of hyper-osmotic treatment, with its effect on net H⁺ extrusion. The results obtained showed that hyper-osmotic stress (200 mM mannitol) quickly reduced Cl^? efflux (?70%) from cells preloaded with ³⁶C1^? for 18 h. This inhibiting effect was independent of the simultaneous mannitol-induced stimulation of Cl^? influx and rapidly reversible after removal of the hyper-osmotic treatment. The inhibition of Cl^? efflux was associated with a stimulation of net H⁺ extrusion, and these two effects showed the same dependence on the external mannitol concentration. Fusicoccin (FC, 20 µM), which stimulated H⁺ extrusion to about the same extent as 200 mM mannitol, did not affect Cl^? efflux. When cells preloaded with ³⁶C1^? for 18 h in the presence of mannitol (from 25 up to 200 mM) were eluted in a mannitol-free medium an early and strong increase in Cl^? efflux was found. The increase of Cl- efflux was already detectable for a small hypo-osmotic jump (25 mM), and was reduced (?50%) by the anion channel inhibitor A9C (300 µM). These results lead to exclude a direct causal relationship mediated by E_m changes between the effects of osmoticum on Cl^? efflux and net H⁺ extrusion, and favour the view that the changes in turgor pressure induced by hyper/hypo-osmotic stress may respectively induce an early inactivation/activation of stretch-sensitive anion channels.     

Effects of hyper-osmotic stress on K+ fluxes, H+ extrusion, transmembrane electric potential difference and comparison with the effects of fusicoccin

The stimulation of H⁺ extrusion by hyper-osmotic stress (0.2–0.3 M mannitol) in cultured cells of Arabidopsis thaliana (L.) Heynh. was shown to be associated with an inhibition of Cl^? efflux, whereas hypo-osmotic stress, inhibiting H⁺ extrusion, early and strongly stimulated Cl^? efflux. In this paper, we investigate the contribution of other factors [K⁺ transport and transmembrane electric potential difference (E_m)] to the hyper-osmotic-induced activation of the plasma membrane (PM) H⁺-ATPase. The effects of mannitol (MA) on K⁺ transport and on E_m were compared with those of fusicoccin (FC) since the modes of action of osmotica and of the toxin in stimulating H⁺-ATPase activity seem to differ at least in some steps. The changes in H⁺ extrusion induced by hyper- or hypo-osmotic stress were opposite and could be reversed by the application of the respective opposite stress. The effect of MA on H⁺ extrusion was dependent on the presence of K⁺ (or Rb⁺) similarly to that of FC, while Na⁺ and Li⁺, which also stimulated the FC effect, were ineffective on that of MA. The MA effect was independent of the anions (Cl^?, SO₄^2?, NO₃^?) accompanying K⁺. K⁺ net uptake and K⁺ influx were stimulated by both MA and FC. Tetraethylammonium (TEA⁺) and Cs⁺ inhibited both MA- and FC-induced H⁺ extrusion, suggesting the involvement of K⁺ channels. MA (0.2 M) induced a strong hyperpolarization of E_m both in the absence and in the presence of K⁺. The hyperpolarizing effect of MA was also found when the cells were already hyperpolarized by FC, and was rapidly reversed by removing the osmoticum from the medium. In the presence of the lipophilic cation tributylbenzylammonium (TBBA⁺), MA was no longer able to stimulate H⁺ extrusion, while FC still stimulated it. In cells pretreated with TBBA⁺, which strongly depolarized E_m, the subsequent addition of FC repolarized it, while the hyperpolarizing effect of MA was lacking. On the contrary, in cells pretreated with Erythrosine B (EB), E_m was strongly depolarized and the following addition of FC did not hyperpolarize it, while the hyperpolarizing effect of MA was still observed. These results suggest that the mechanism of MA in activating H⁺ extrusion and K⁺ uptake is different from that of FC. The rise in net K⁺ uptake seems to be driven by the activation of some hyperpolarizing system that does not seem to depend on a direct activation of PM H⁺-ATPase, but rather on the inhibition of Cl^? efflux induced by hyper-osmotic stress.     

Calcium-dependent phosphorylation regulates the plasma-membrane H+-ATPase activity of maize (Zea mays L.) roots

Phosphorylation/dephosphorylation of the plasma-membrane H⁺-ATPase (EC 3.6.1.35) could act as a regulatory mechanism to control its activity. In this work, a plasmalemma-enriched fraction from maize roots and a partially purified H⁺-ATPase were used to investigate the effects of Ca²⁺ and calmodulin on the H⁺-ATPase activity and on its phosphorylation status. Both the hydrolytic and the proton-pumping activities were reduced approximately 50% by micromolar Ca²⁺ concentrations while calmodulin did not show any effect either alone or in the presence of Ca²⁺. The lack of effect of calmodulin antagonists indicated that calmodulin was not involved in this response. The addition of staurosporine, a kinase inhibitor, abolished the inhibitory effect of Ca²⁺. Phosphorylation of plasma membrane and partially purified H⁺-ATPase showed the same behavior. In the presence of Ca²⁺ a polypeptide of 100 kDa was phosphorylated. This polypeptide cross-reacted with antibodies raised against the H⁺-ATPase of maize roots. The autoradiogram of the immunodetected protein clearly showed that this polypeptide, which corresponds to the H⁺-ATPase, was phosphorylated. Additional clear evidence comes from the immunoprecipitation experiments: the data obtained show that the H⁺-ATPase activity is indeed influenced by its state of phosphorylation. Received: 19 October 1998 / Accepted: 23 February 1999     

Ethanol-Induced Activation of ATP-Dependent Proton Extrusion in Elodea densa Leaves

In Elodea densa leaves, ethanol up to 0.17 m stimulates H⁺ extrusion activity. This effect is strictly dependent on the presence of K⁺ in the medium and is suppressed by the presence of the plasmalemma H⁺-ATPase inhibitor vanadate. Stimulation of H⁺ extrusion is associated with (a) a decrease in cellular ATP level, (b) a marked hyperpolarization of transmembrane electrical potential, and (c) an increase in net K⁺ influx. These results suggest that ethanol-induced H⁺ extrusion is mediated by an activation of the plasma membrane ATP-dependent, electrogenic proton pump. This stimulating effect is associated with an increase of cell sap pH and of the capacity to take up the weak acid 5,5-dimethyloxazolidine-2,4-dione, which is interpretable as due to an increase of cytosolic pH. This indicates that the stimulation of H⁺ extrusion by ethanol does not depend on a cytosolic acidification by products of ethanol metabolism. The similarity of the effects of ethanol and those of photosynthesis on proton pump activity in E. densa leaves suggests that a common metabolic situation is responsible for the activation of the ATP-dependent H⁺-extruding mechanism.     

Early Sr2+-induced effects on membrane potential,proton pumping- and ATP hydrolysis-activities of plasma membrane vesicles from maize root cells

When released in plant environment, strontium (Sr²⁺) can be absorbed predominantly by the plant roots. As the plasma membrane of root cells is amongst the first barriers encountered by Sr²⁺ during its soil/plant transfer and the main entry point of Sr²⁺ into the roots, the main objective of this work aimed to enlighten on some of the Sr²⁺-induced effects at this level in Zea mays L. cv. “Liberal”.Thus this study focused on the Sr²⁺-induced changes on membrane potential of cortical root cells and on proton fluxes in maize roots, in order to determine whether the activity of some of the ion transport systems present in the plasma membrane of maize root cell could be among the first targets of Sr²⁺. We focused in particular on the plasma membrane H⁺-ATPase, known to be one of the major transport systems found in the plasmalemma where it generates a proton motive force (contributing to membrane potential maintaining, and providing energy for ion transport through membrane).The data presented here showed that Sr²⁺ triggered an early and transient membrane depolarisation whose magnitude and duration were dependent on the Sr²⁺-concentration. The time course pattern of a second longer lasting depolarisation could be examined in perspective with the Sr²⁺-induced decrease of the spontaneous proton extrusion observed in root tissues, suggesting a relationship between Sr²⁺-effects on membrane potential and H⁺ excretion. Furthermore, the inhibitory effect exerted by Sr²⁺ on the fusicoccin (FC)-enhanced proton extrusion strongly suggested an inhibition of the plasma membrane H⁺-ATPase. This hypothesis was supported by the inhibition induced by Sr²⁺ on proton pumping- and ATP hydrolysis-activities measured in plasma membrane vesicles (PMV) prepared from maize roots.Taken together the data reported here evidence that, with however a lower efficiency, Sr²⁺ behaved in a quite similar way to Ca²⁺ when inhibiting the H⁺-ATPase activity, and suggest that Sr²⁺ could partially mimic Ca²⁺ onto regulation of the H⁺-ATPase activity.     

Reversible changes of extracellular pH during action potential generation in a higher plant Cucurbita pepo

A pH-sensitive electrode was applied to measure activity of H⁺ ions in the medium surrounding excitable cells of pumpkin (Cucurbita pepo L.) seedlings during cooling-induced generation of action potential (AP). Reversible alkalization shifts were found to occur synchronously with AP, which could be due to the influx of H⁺ ions from external medium into excitable cells. Ethacrynic acid (an anion channel blocker) reduced the AP amplitude but had no effect on the transient alkalization of the medium. An inhibitor of plasma membrane H⁺-ATPase, N,N’-dicyclohexylcarbodiimide suppressed both the AP amplitude and the extent of alkalization. In experiments with plasma membrane vesicles, the hydrolytic H⁺-ATPase activity was subjected to inhibition by Ca²⁺ concentrations in the range characteristic of cytosolic changes during AP generation. The addition of a calcium channel blocker verapamil and a chelating agent EGTA to inhibit Ca²⁺ influx from the medium eliminated the AP spike and diminished reversible alkalization of the external solution. An inhibitor of protein kinase, H-7 alleviated the inhibitory effect of Ca²⁺ on hydrolytic H⁺-ATPase activity in plasma membrane vesicles and suppressed the reversible alkalization of the medium during AP generation. The results provide evidence that the depolarization phase of AP is associated not only with activation of chloride channels and Cl^? efflux but also with temporary suppression of plasma membrane H⁺-ATPase manifested as H⁺ influx. The Ca²⁺-induced inhibition of the plasma membrane H⁺-ATPase is supposedly mediated by protein kinases.     

The Ca2+ Pump of the Plasma Membrane of Arabidopsis thaliana: Characteristics and Sensitivity to Fluorescein Derivatives

The transport and hydrolytic activities of the plasma membrane (PM) Ca²⁺ pump were characterized in a PM fraction purified from seedlings of Arabidopsis thaliana by the aqueous two-phase partitioning technique. Ca²⁺ uptake could be energized by ATP and by ITP (at about 70% the rate sustained by ATP). This characteristic was used to measure the hydrolytic activity of the enzyme as Ca²⁺-dependent ITPase activity. The PM Ca²⁺ pump displayed a broad pH optimum around pH 7.2, was drastically inhibited by erythrosin B (EB), and was half-saturated by 60 μM ITP. It was stimulated by CaM, specially at low, non-saturating Ca²⁺ concentrations. All of these characteristics closely resemble those of the PM Ca²⁺ pump in other plant materials. Analysis of the effects of EB and other fluorescein derivatives (eosin Y and rose bengal) showed that: i) EB behaved as a competitive inhibitor with respect to ITP; ii) the PM Ca²⁺ pump was drastically inhibited by concentrations of fluorescein derivatives (submicromolar), much lower than those required to inhibit the PM H⁺-ATPase; iii) the different fluorescein derivatives were diversely efficient in inhibiting the activities of the Ca²⁺ pump and of the H⁺-ATPase of the PM (eosin Y was about 10000-fold, EB 1000-fold and rose bengal only 50-fold more active on the Ca²⁺ pump than on the H⁺-ATPase); and iv) the effectiveness of EB in inhibiting the Ca²⁺ pump was strongly affected by the protein concentration in the assay medium.     

Elucidation of antimicrobial activity and mechanism of action by N-substituted carbazole derivatives

Compounds belonging to a carbazole series have been identified as potent fungal plasma membrane proton adenosine triphophatase (H⁺-ATPase) inhibitors with a broad spectrum of antifungal activity. The carbazole compounds inhibit the adenosine triphosphate (ATP) hydrolysis activity of the essential fungal H⁺-ATPase, thereby functionally inhibiting the extrusion of protons and extracellular acidification, processes that are responsible for maintaining high plasma membrane potential. The compound class binds to and inhibits the H⁺-ATPase within minutes, leading to fungal death after 1–3 h of compound exposure in vitro. The tested compounds are not selective for the fungal H⁺-ATPase, exhibiting an overlap of inhibitory activity with the mammalian protein family of P-type ATPases; the sarco(endo)plasmic reticulum calcium ATPase (Ca²⁺-ATPase) and the sodium potassium ATPase (Na⁺,K⁺-ATPase). The ion transport in the P-type ATPases is energized by the conversion of ATP to adenosine diphosphate (ADP) and phosphate and a general inhibitory mechanism mediated by the carbazole derivative could therefore be blocking of the active site. However, biochemical studies show that increased concentrations of ATP do not change the inhibitory activity of the carbazoles suggesting they act as allosteric inhibitors. Furthermore decreased levels of intracellular ATP would suggest that the compounds inhibit the H⁺-ATPase indirectly, but Candida albicans cells exposed to potent H⁺-ATPase-inhibitory carbazoles result in increased levels of intracellular ATP, indicating direct inhibition of H⁺-ATPase.     

Growth cycle stage-dependent NaCl induction of plasma membrane H+-ATPase mRNA accumulation in de-adapted tobacco cells

A cDNA clone encoding an isoform of the plasma membrane H⁺-ATPase was isolated from Nicotiana tabacum. The steady-state plasma membrane H⁺-ATPase message levels were the same in unadapted tobacco cells and tobacco cells adapted to 428 mol m⁻³ NaCl. When cells adapted to 428 mol m⁻³ NaCl maintained in the absence of NaCl (deadapted) for an excess of 100 passages were exposed to 400 mol m⁻³ NaCl for 24 h, there was an increased accumulation of plasma membrane H⁺-ATPase message. The NaCl responsiveness of the deadapted cells was dependent upon the growth cycle stage. Alterations in the levels of plasma membrane FT-ATPase message during the growth cycle support a role for the H⁺-ATPase in cell growth. These results document the induction by NaCl of plasma membrane FT-ATPase message accumulation in tobacco cells, and suggest that enhanced expression of the plasma membrane FT-ATPase has a role in the short term response of cells of NaCl, but is not necessarily involved in long-term adaptation.     

Involvement of plasma membrane potential in the tolerance mechanism of plant roots to aluminium toxicity

H⁺-ATPase activity of a plasma membrane-enriched fraction decreased after the treatment of barley (Hordeum vulgare) seedlings with Al for 5 days. A remarkably high level of Al was found in the membrane fraction of Al-treated roots. A long-term effect of Al was identified as the repression of the H⁺-ATPase of plasma membranes isolated from the roots of barley and wheat (Triticum aestivum) cultivars, Atlas 66 (Al-tolerant) and Scout 66 (Al-sensitive). To monitor short-term effects of Al, the electrical membrane potentials across plasma membranes of both wheat cultivars were compared indirectly by measuring the efflux of K⁺ for 40 min under various conditions. The rate of efflux of K⁺ in Scout was twice that in Atlas at low pH values such as 4.2. Vanadate, an inhibitor of the H⁺-ATPase of the plasma membrane, increased the efflux of K⁺. Al repressed this efflux at low pH, probably through an effect on K⁺ channels, and repression was more pronounced in Scout. Al strongly repressed the efflux of K⁺ irrespective of the presence of vanadate. Ca²⁺ also had a repressive effect on the efflux of K⁺ at low pH. The effect of Ca²⁺, greater in Scout, might be related to the regulation of the net influx of H⁺, since the effect was negated by vanadate. The results suggest that extracellular low pH may cause an increase in the influx of H⁺, which in turn is counteracted by the efflux of K⁺ and H⁺. These results suggest that the ability to maintain the integrity of the plasma membrane and the ability to recover the electrical balance at the plasma membrane through a net influx of H⁺ and the efflux of K⁺ seem to participate in the mechanism of tolerance to Al stress under acidic conditions.     

Physiological evidence for a proton pump and sodium exclusion mechanisms at the plasma membrane of the marine angiosperm Zostera marina L

The basic electrical plasma membrane characteristics of leaf cells from the seagrass Zostera marina L. have been investigated with respect to its primary transport system and its Na⁺/K⁺ selectivity. In natural seawater Z. marina exhibits a membrane potential of -15610 mV. The phytotoxin fusicoccin stimulates H⁺ extrusion and hyperpolarizes the plasma membrane. Ouabain, an inhibitor of the mammalian Na⁺K⁺-ATPase did not depolarize the plasma membrane of Z.marina. Both flushing the leaves with CO2 and 'light off' acidified the cytoplasm and hyperpolarized the cells. It is suggested that a H⁺-ATPase rather than a Na⁺-ATPase is the primary pump in Z.marina. In the presence of cyanide plus salicylhydroxamic acid the membrane potential changed to -6411 mV. This so-called diffusion potential was sensitive to external [K⁺] from 0.05 to 0.5 mM in the presence of 0.5 M Na⁺ and revealed a relative permeability PK⁺/PNa⁺ of 303. We suggest that this high ratio is the basic adaptation which permits Z. marina to grow in high [Na⁺] conditions and to exhibit a rather negative resting potential. Since amiloride, an inhibitor of the nH⁺/Na⁺ antiporter, hyperpolarized the plasma membrane, it is suggested that this transporter could be present in the plasma membrane of Z. marina acting as an overflow valve for Na⁺ which leaks into the cell.     

N-Cyclo-N'-(4-Dimethylamino-alpha-Naphthyl)Carbodiimide Inhibits Membrane-Bound and Partially Purified Tonoplast ATPase from Maize Roots

Certain carboxylic acid groups within the primary structure of proton translocating proteins are thought to be involved in the proton pathway. In this report, the effects of a lipophilic carboxylic acid reactive reagent, N-cyclo-N′(4-dimethylamino-α-naphthyl)carbodiimide (NCD-4), on the two types of proton pumps in maize (Zea mays L.) root microsomes were investigated. NCD-4 was found to inhibit the vacuolar-type H⁺-ATPase in microsomal preparations; however, the plasma membrane-type H⁺-ATPase was unaffected. The H⁺-ATPase in highly purified tonoplast vesicles was also inhibited by NCD-4. Inhibition was dependent on the concentration and length of exposure to the reagent. However, there was little, if any, increase in the fluorescence of treated vesicles, indicating few carboxylic acid residues were reacting. Inhibition of the tonoplast H⁺-ATPase by NCD-4 was examined further with a partially purified preparation. The partially purified H⁺-ATPase also showed sensitivity to the NCD-4, supporting the hypothesis that this carboxylic acid reagent is an inhibitor of the tonoplast ATPase from maize roots.     

Changes in plasma membrane lipids, aquaporins and proton pump of broccoli roots, as an adaptation mechanism to salinity

Salinity stress is known to modify the plasma membrane lipid and protein composition of plant cells. In this work, we determined the effects of salt stress on the lipid composition of broccoli root plasma membrane vesicles and investigated how these changes could affect water transport via aquaporins. Brassica oleracea L. var. Italica plants treated with different levels of NaCl (0, 40 or 80 mM) showed significant differences in sterol and fatty acid levels. Salinity increased linoleic (18:2) and linolenic (18:3) acids and stigmasterol, but decreased palmitoleic (16:1) and oleic (18:1) acids and sitosterol. Also, the unsaturation index increased with salinity. Salinity increased the expression of aquaporins of the PIP1 and PIP2 subfamilies and the activity of the plasma membrane H⁺-ATPase. However, there was no effect of NaCl on water permeability (P_f) values of root plasma membrane vesicles, as determined by stopped-flow light scattering. The counteracting changes in lipid composition and aquaporin expression observed in NaCl-treated plants could allow to maintain the membrane permeability to water and a higher H⁺-ATPase activity, thereby helping to reduce partially the Na⁺ concentration in the cytoplasm of the cell while maintaining water uptake via cell-to-cell pathways. We propose that the modification of lipid composition could affect membrane stability and the abundance or activity of plasma membrane proteins such as aquaporins or H⁺-ATPase. This would provide a mechanism for controlling water permeability and for acclimation to salinity stress.     

Branchial ammonia excretion in the Asian weatherloach Misgurnus anguillicaudatus

The weatherloach, Misgurnus anguillicaudatus, is a freshwater, facultative air-breathing fish that lives in streams and rice paddy fields, where it may experience drought and/or high environmental ammonia (HEA) conditions. The aim of this study was to determine what roles branchial Na⁺/K⁺-ATPase, H⁺-ATPase, and Rhcg have in ammonia tolerance and how the weatherloach copes with ammonia loading conditions. The loach's high ammonia tolerance was confirmed as was evident from its high 96 h LC₅₀ value and high tissue tolerance to ammonia. The weatherloach does not appear to make use of Na⁺/NH₄⁺-ATPase facilitated transport to excrete ammonia when exposed to HEA or to high environmental pH since no changes in activity were observed. Using immunofluorescence microscopy, distinct populations of vacuolar (V)-type H⁺-ATPase and Na⁺/K⁺-ATPase immunoreactive cells were identified in branchial epithelia, with apical and basolateral staining patterns, respectively. Rhesus C glycoprotein (Rhcg1), an ammonia transport protein, immunoreactivity was also found in a similar pattern as H⁺-ATPase. Rhcg1 (Slc42a3) mRNA expression also increased significantly during aerial exposure, although not significantly under ammonia loading conditions. The colocalization of H⁺-ATPase and Rhcg1 to the similar non-Na⁺/K⁺-ATPase immunoreactive cell type would support a role for H⁺-ATPase in ammonia excretion via Rhcg by NH₄⁺ trapping. The importance of gill boundary layer acidification in net ammonia excretion was confirmed in this fish; however, it was not associated with an increase in H⁺-ATPase expression, since tissue activity and protein levels did not increase with high environmental pH and/or HEA. However the V-ATPase inhibitor, bafilomycin, did decrease net ammonia flux whereas other ion transport inhibitors (amiloride, SITS) had no effect. H⁺-ATPase inhibition also resulted in a consequent elevation in plasma ammonia levels and a decrease in the net acid flux. In gill, aerial exposure was also associated with a significant increase in membrane fluidity (or increase in permeability) which would presumably enhance NH₃ permeation through the plasma membrane. Taken together, these results indicate the gill of the weatherloach is responsive to aerial conditions that would aid ammonia excretion.     

Regulation of Plasma Membrane H+-Pump Activity by 14-3-3 Proteins in Barley (Hordeum disticum) Roots under Salt Stress

Regulatory changes in the activity of the plasma membrane H⁺-ATPase in salt-stressed roots were investigated using seven-day-old seedlings of two cultivars of barley (Hordeum disticum L.) with different salt tolerances: Moskovskii-121 (salt-tolerant) and Elf (salt-sensitive). During the first hour of salt stress, the rate of proton extrusion from the excised roots increased in parallel with the ATP hydrolase activity and the amount of 14-3-3 proteins bound to H⁺-ATPase in isolated plasma membranes. Subsequently, all these parameters decreased and dropped after 3–6 h below the initial levels. The initial stimulation of proton extrusion from the detached barley roots was caused by osmotic stress, whereas the subsequent retardation of proton extrusion was probably caused by a toxic effect of excessive Na⁺ content in the cytoplasm. The salt-stress responses showed similar trends in both cultivars, with the exception that Moskovskii-121 responded faster than cv. Elf. The results indicate that 14-3-3 proteins regulate the H⁺-ATPase activity in the plasma membranes of barley root cells during salt stress; furthermore, the response time might be a useful indicator to discriminate cultivars with different salt tolerances.     

两相法分离纯化橡胶树树皮质膜

橡胶树树皮质膜H~+-ATPase在橡胶树产排胶过程中扮演着重要角色,制备高纯度及高活性的质膜是研究质膜H~+-ATPase特性和功能的必要条件。该研究以一年生巴西橡胶树(Hevea brasiliensis)树皮为材料,利用差速离心法获得粗膜微粒体,通过两相分配法分离纯化质膜,并研究两相体系中不同浓度聚合物(5.9%、6.1%、6.3%、6.5%、6.7%,W/W)和KCl(2、5、8、11、14 mmol·L~(-1))对质膜蛋白得率和纯化效率的影响。通过Bradford法对质膜蛋白得率进行检测,同时采用酶活性检测法对质膜纯度进行检测,分析结果表明选用6.4%(W/W)聚合物浓度和5mmol·L~(-1)KCl组成的两相体系可获得较高纯度和得率的橡胶树树皮质膜。通过电镜观察法在形态学上对质膜纯度进一步评价,利用铅铀能侵染全部膜组分使其染色,而磷钨酸只能专一性地侵染质膜并使其染色这一特性,分别使用铅铀和磷钨酸对切片进行染色,并通过透射电镜对切片染色程度进行直接观察,结果表明提取的粗膜微粒体中质膜组分较少,存在大量的细胞器膜污染,而纯化后的质膜膜组分较单一,其他膜组分污染较少,而且质膜大小较均一,可以用于进行后续橡胶树树皮质膜H~+-ATPase特性和功能的研究。     

Boric acid stimulates the plasma membrane H+-ATPase of ungerminated lily pollen grains

The stimulation of the plasma membrane (PM) H⁺-ATPase by boric acid was studied on a microsomal fraction (MF) obtained from ungerminated, boron-dependent pollen grains of Lilium longiflorum Thunb. which usually need boron for germination and tube growth. ATP hydrolysis and H+ transport activity increased by 14 and 18%, respectively, after addition of 2-4 mM boric acid. The optimum of boron stimulation was at pH 6.5-8.5 for ATP hydrolysis and at pH 6.5-7.5 for H⁺ transport. No boron stimulation was detected when vanadate was added to the MF, whereas an increase of 10-20% in ATP hydrolysis and H⁺ transport was still measured in the presence of inhibitors specific for V -type ATPase (nitrate and bafilomycin) and F-type ATPase (azide), respectively. A vanadate-sensitive increase in ATP hydrolysis activity was also observed in partially permeabilized vesicles (0.001%[w/v] Triton X-100) suggesting a direct interaction between borate and the PM H⁺-ATPase rather than a weak acid-induced stimulation. Additionally, we measured the effect of boron on membrane voltage (V_m) of ungerminated pollen grains and observed small hyperpolarizations in 48% of all experiments. Exposing pollen grains to a more acidic pH of 4 caused a depolarization, followed in some experiments by a repolarization (21%). In the presence of 2 mM boron such hyperpolarizations, perhaps caused by an enhanced activity of the H⁺-ATPase, were measured in 58% of all tested pollen grains. The effects of boron on V_m may be reduced by additional stimulation of a K⁺ inward current of opposite direction to the H⁺-ATPase. All experiments indicate that boron stimulates an electrogenic transport system in the plasma membrane which is sensitive to vanadate and has a pH optimum around 7, i.e. the plasma membrane H⁺-ATPase. A boron-increased PM H⁺-ATPase activity in turn may stimulate germination and growth of pollen tubes.