Role of Nitric Oxide and Hydrogen Peroxide During the Salt Resistance Response

Ion homeostasis is essential for plant cell resistance to salt stress. Under salt stress, to avoid cellular damage and nutrient deficiency, plant cells need to maintain adequate K nutrition and a favorable K to Na ratio in the cytosol. Recent observations revealed that both nitric oxide (NO) and hydrogen peroxide (H₂O₂) act as signaling molecules to regulate K to Na ratio in calluses from Populus euphratica under salt stress. Evidence indicated that NO mediating H₂O₂ causes salt resistance via the action of plasma membrane H⁺-ATPase but that activity of plasma membrane NADPH oxidase is dependent on NO. Our study demonstrated the signaling transduction pathway. In this addendum, we proposed a testable hypothesis for NO function in regulation of H₂O₂ mediating salt resistance.Key Words: hydrogen peroxide, nitric oxide, signaling molecule, salt resistanceUnder salinity conditions, tolerant plant cells achieve ion homeostasis by extruding Na to the external medium and/or compartmentalizing into vacuoles, maintaining K uptake and high K and low Na in the cytosol.^1,2 Control of Na movement across the plasma membrane (PM) and tonoplast in order to maintain a low Na concentration in the cytoplasm is a key factor of cellular adaptation to salt stress.^3,4 Na transport across the PM is dependent on the electrochemical gradient created by the PM H⁺-ATPase.^5,6 It has been proven that the activity of the PM H⁺-ATPase is a key index of plant adaptation to salt stress.⁷ Therefore, the regulation of expression of the PM H⁺-ATPase may represent an important cellular mechanism for salt resistance. In contrast to our understanding of the regulation of PM H⁺-ATPase by other factors, the roles of NO and H₂O₂ act as signals under salt stress have been less known.Previous studies have shown that both NO and H₂O₂ function as stress signals in plants, mediating a range of resistance mechanisms in plants under stress conditions.^8–10 We have previously shown that NO serves as a signal in inducing salt resistance by increasing the K to Na ratio, which is dependent on the increased PM H⁺-ATPase activity in calluses from reed.¹¹ Although NO acts as a signal molecule under salt stress and induces salt resistance by increasing PM H⁺-ATPase activity, our research results also indicated NO can not activate purified PM H⁺-ATPase activity, at least in vitro. Subsequently, we set out to find the other signal molecules and factors between NO and PM H⁺-ATPase activity. Since our studies have indicated that NO can not induce salt resistance directly, what roles dose it play in salt resistance in tolerant cells under salt stress? We initially hypothesized ABA or H₂O₂ might be downstream signal molecules to regulate the activity of PM H⁺-ATPase. Further results indicated H₂O₂ content increased greatly under salt stress. Since H₂O₂ might be the candidate downstream signal molecule, we tested PM H⁺-ATPase activity and K to Na ratio in calluses by adding H₂O₂. The results suggested that H₂O₂ inducing an increased PM H⁺-ATPase activity resulted in an increased K to Na ratio. Summing up this new assay that allows us to speculate NO maybe regulate the H₂O₂ generation.Since H₂O₂ is involved in downstream signal molecule of NO, PM NADPH oxidase, the main source of H₂O₂ production, might be the regulated target of NO. We took a pharmacological approach to examine the speculation. The results indicated that PM NADPH oxidase is required for H₂O₂ accumulation and PM NADPH oxidase activity could attribute to NO in calluses under salt stress. These results also raised another question regarding what concentrations of NO can induce such effects. In our experiments, NO content was induced 1.6 times higher than the control values under salt treatment. We speculated there exists an effective balance point in NO signal system similar to previous reports by Delledonne et al.¹² in disease resistance.Further research work is required to decipher the mechanism through which NO and H₂O₂ acts and how K and Na elements uptake might be connected with salt resistance. We would like to propose a simple testable model that accounts for the results reported in this paper (Fig. 1). According to our model, H₂O₂ rather than NO is the major signaling molecular that mediated directly PM H⁺-ATPase under salt stress. Normally, NO generated from nitric oxide synthase (NOS) acts as a signal molecule to regulate other mechanisms. Under salt stress, accumulated NO activates PM NADPH oxidase activity. Then, a number of H₂O₂ is produced from PM NADPH oxidase. The PM H⁺-ATPase is activated greatly by the accumulated H₂O₂. Eventually, the transmembrane electrochemical gradient is created and K to Na ratio increases. The model we have proposed here is testable and should provide further insights into salt resistance mechanism regulated by NO and H₂O₂ signal molecules.Open in a separate windowFigure 1Hypothetical model for the potential function of NO and H₂O₂ as signaling molecules in inducing salt resistance. Salt stress activates a signal transduction cascade that leads to the increased activity of PM H⁺-ATPase, whose expression produces salt resistance. NO is generated by NOS, and H₂O₂ is produced by NADPH oxidase attributed to NO. The activity of PM H⁺-ATPase is regulated by H₂O₂ directly under salt stress. The model is based on the recent results in calluses from P. euphratica¹² and those previously reported on the NO function in reed.¹¹Research on roles of NO and H₂O₂ under stress conditions in plant is advancing rapidly. Further analysis of salt resistance mechanism with novel technology will certainly increase our knowledge in this field.     

Functional Identification of H<Superscript>+</Superscript>-ATPase and Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter in the Plasma Membrane Isolated from the Root Cells of Salt-Accumulating Halophyte <Emphasis Type="Italic">Suaeda altissima</Emphasis>

A membrane fraction enriched in plasma membrane (PM) vesicles was isolated from the root cells of a salt-accumulating halophyte Suaeda altissima (L.) Pall. by means of centrifugation in discontinuous sucrose density gradient. The PM vesicles were capable of generating ΔpH at their membrane and the transmembrane electric potential difference (Δψ). These quantities were measured with optical probes, acridine orange and oxonol VI, sensitive to ΔpH and Δψ, respectively. The ATP-dependent generation of ΔpH was sensitive to vanadate, an inhibitor of P-type ATPases. The results contain evidence for the functioning of H⁺-ATPase in the PM of the root cells of S. altissima. The addition of Na⁺ and Li⁺ ions to the outer medium resulted in dissipation of ΔpH preformed by the H⁺-ATPase, which indicates the presence in PM of the functionally active Na⁺/H⁺ antiporter. The results are discussed with regard to involvement of the Na⁺/H⁺ antiporter and the PM H⁺-ATPase in loading Na⁺ ions into the xylem of S. altissima roots.     

Exogenous IAA and ABA stimulate germination of petunia male gametophyte by activating Ca<Superscript>2+</Superscript>-dependent K<Superscript>+</Superscript>-channels and by modulating the activity of plasmalemma H<Superscript>+</Superscript>-ATPase and actin cytoskeleton

To date, the molecular mechanisms underlying the osmoregulation of pollen grains (PGs) related to the maintenance of their water status and allowing pollen tubes (PTs) to regulate concentrations in them of osmolytes and transmembrane water transport remain to be not so far characterized. In the present work, the data on the participation of IAA and ABA in the osmoregulation of germinating in vitro petunia male gametophyte were obtained. It has been established that the growth-stimulating effect of these phytohormones is due to their action on intracellular pH (pH_c), the membrane potential of plasmalemma (PM), the activity of PM H⁺-ATPase, K⁺-channels in the same membrane and organization of actin cytoskeleton (AC). Two possible targets of the action of these compounds are revealed. These are represented by (1) PM H⁺-ATPase, electrogenic proton pump responsible for polarization of this membrane, and (2) Ca²⁺-dependent K⁺-channels. The findings of the present work suggest that the hormone-induced pH_c shift is involved in cascade of the events including the functioning of pH-dependent K⁺-channels. It was shown that the hormoneinduced hyperpolarization of the PM is a result of stimulation of electrogenic activity of PM H⁺-ATPase and the hormonal effects are mediated by transient elevation in the level of free Ca²⁺ in the cytosol and generation of reactive oxygen species (ROS). The results on the role of K⁺ ions in the control of water-driving forces for transmembrane water transport allowed us to formulate the hypothesis that IAA and ABA stimulate germination of PGs and growth of PTs by activating K⁺-channels. In addition, the studies performed showed that the AC of male gametophyte is sensitive to the action of exogenous phytohormones, with to more extent to the action of IAA. As judged by the action of latrunculin B (LB) the AC may serve as the determinant of the level of endogenous phytohormones that most likely participate in the regulation of the polar growth of PTs impacting on the pool of F-actin in their apical and subapical zones.     

Dependence of the CO<Subscript>2</Subscript> Uptake in a Plant Cell on the Plasma Membrane H<Superscript>+</Superscript>-ATPase Activity: Theoretical Analysis

A decrease of the plasma membrane H⁺-ATPase activity in plant cells is associated with the formation of response to adverse factors and reception of signals. A theoretical analysis of the influence of the H⁺-ATPase activity on the flow of CO₂ into a plant cell has been conducted. With this purpose the model of transport processes and electrogenesis in the plant cell developed previously was used, which takes into account transport systems, including H⁺-ATPase, in the plasma membrane and tonoplast. The CO₂ fraction (\({P_{c{o_2}}}\)) in the total amount of inorganic carbon (C_i) in the external medium was used as an indicator of the CO₂ amount entering the plant cell; this parameter depends on the extracellular pH, which, in particular, is influenced by the H⁺-ATPase activity. Excitable and non-excitable cells were simulated. It was shown that a decrease of the H+-ATPase activity causes a \({P_{c{o_2}}}\)reduction in both variants of the model, and this reduction has an extremum: after passing through minimal values, \({P_{c{o_2}}}\)reaches a stationary level. The dynamics of \({P_{c{o_2}}}\)decrease may be related to the Ca²⁺ influx into the cytoplasm of the plant cell. The reduction of \({P_{c{o_2}}}\)depended on the extent of the H⁺-ATPase inactivation and on its initial activity. As a whole, it was shown that the inactivation of the H⁺-ATPase can affect the CO₂ uptake in a plant cell and thereby regulate photosynthetic processes.     

Electrogenic activity of plasma membrane H<Superscript>+</Superscript>-ATPase in germinating male gametophyte of petunia and its stimulation by exogenous auxin: Mediatory role of calcium and reactive oxygen species

A lipophilic potential-sensitive cationic dye, safranin O was employed to examine the influence of exogenous IAA on plasma membrane electric potential in germinating pollen grains of petunia (Petunia hybrida L.) with the aim of elucidating whether the electrogenic H⁺-ATPase activity of the plasma membrane is sensitive to this phytohormone. The addition of IAA to pollen grains suspended in a K⁺-free medium was found to induce significant hyperpolarization of the plasmalemma. This effect was fully blocked by orthovanadate, Ca²⁺-active reagents (EGTA and verapamil), and by the inhibitor of NADPH oxidase of plasmalemma, diphenyleneiodonium (DPI). It was also strongly inhibited by the presence of K⁺ at centimolar concentrations in the medium. The hyperpolarizing influence of IAA was mimicked by application of hydrogen peroxide; furthermore, the H₂O₂-induced shift of the membrane potential was inhibited by the same agents that suppressed the IAA-induced hyperpolarization of the pollen plasmalemma. It is concluded that the IAAinduced hyperpolarization of the plasma membrane in male gametophytes of petunia is caused by the enhanced electrogenic activity of ATP-dependent proton pump in the presence of this phytohormone. It is supposed that the effect of IAA is mediated by the transient increase in cytosolic Ca²⁺ level and by generation of reactive oxygen species (ROS). Possible mechanisms underlying the mediatory role of calcium and ROS in the auxin signal transduction and the resulting stimulation of electrogenic activity of the plasma membrane H⁺-ATPase are discussed.     

Nitrogen deprivation induces cross-tolerance of Poa annua callus to salt stress

Alternative respiration pathway (AP) is an important pathway which can be induced by environment stresses in plants. In the present study, we show a new mechanism involving the AP in nitrogen deprivation-induced tolerance of Poa annua callus to salt stress. The AP capacity markedly increased under a 600 mM NaCl treatment or nitrogen deprivation pretreatment and reached a maximum under the nitrogen deprivation pretreatment combined with the NaCl treatment (–N+NaCl). Malondialdehyde (MDA) and H₂O₂ content and Na⁺/K⁺ ratio significantly increased under the 600 mM NaCl treatment but less under the–N+NaCl treatment. Moreover, both the nitrogen deprivation and the NaCl stress stimulated the plasma membrane (PM) H⁺-ATPase activity and increased pyruvate content. The maximal stimulating effect was found under the–N+NaCl treatment. When the AP capacity was reduced by salicylhydroxamic acid (SHAM, an inhibitor of AP), content of MDA and H₂O₂ and Na⁺/K⁺ ratio dramatically increased, whereas PM H⁺-ATPase activity decreased. Moreover, exogenous application of pyruvate produced a similar effect as the nitrogen deprivation pretreatment. The effects of SHAM on the Poa annua callus were counteracted by catalase (a H₂O₂ scavenger) and diphenylene iodonium (a plasma membrane NADPH oxidase inhibitor). Taken together, our results suggest that the nitrogen deprivation enhanced the capacity of AP by increasing pyruvate content, which in turn prevented the Poa annua callus from salt-induced oxidative damages and Na⁺ over-uptake.     

Properties of plasma membrane H<Superscript>+</Superscript>-ATPase in salt-treated <Emphasis Type="Italic">Populus euphratica</Emphasis> callus

The plasma membrane (PM) vesicles from Populus euphratica (P. euphratica) callus were isolated to investigate the properties of the PM H⁺-ATPase. An enrichment of sealed and oriented right-side-out PM vesicles was demonstrated by measurement of the purity and orientation of membrane vesicles in the upper phase fraction. Analysis of pH optimum, temperature effects and kinetic properties showed that the properties of the PM H⁺-ATPase from woody plant P. euphratica callus were consistent with those from herbaceous species. Application of various thiol reagents to the reaction revealed that reduced thiol groups were essential to maintain the PM H⁺-ATPase activity. In addition, there was increased H⁺-ATPase activity in the PM vesicles when callus was exposed to NaCl. Western blotting analysis demonstrated an enhancement of H⁺-ATPase content in NaCl-treated P. euphratica callus compared with the control.     

cGMP regulates hydrogen peroxide accumulation in calcium-dependent salt resistance pathway in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> roots

3′,5′-cyclic guanosine monophosphate (cGMP) is an important second messenger in plants. In the present study, roles of cGMP in salt resistance in Arabidopsis roots were investigated. Arabidopsis roots were sensitive to 100 mM NaCl treatment, displaying a great increase in electrolyte leakage and Na⁺/K⁺ ratio and a decrease in gene expression of the plasma membrane (PM) H⁺-ATPase. However, application of exogenous 8Br-cGMP (an analog of cGMP), H₂O₂ or CaCl₂ alleviated the NaCl-induced injury by maintaining a lower Na⁺/K⁺ ratio and increasing the PM H⁺-ATPase gene expression. In addition, the inhibition of root elongation and seed germination under salt stress was removed by 8Br-cGMP. Further study indicated that 8Br-cGMP-induced higher NADPH levels for PM NADPH oxidase to generate H₂O₂ by regulating glucose-6-phosphate dehydrogenase (G6PDH) activity. The effect of 8Br-cGMP and H₂O₂ on ionic homeostasis was abolished when Ca²⁺ was eliminated by glycol-bis-(2-amino ethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA, a Ca²⁺ chelator) in Arabidopsis roots under salt stress. Taken together, cGMP could regulate H₂O₂ accumulation in salt stress, and Ca²⁺ was necessary in the cGMP-mediated signaling pathway. H₂O₂, as the downstream component of cGMP signaling pathway, stimulated PM H⁺-ATPase gene expression. Thus, ion homeostasis was modulated for salt tolerance.     

The Na<Superscript>+</Superscript>-translocating ATPase in the plasma membrane of the marine microalga<Emphasis Type="Italic"> Tetraselmis viridis</Emphasis> catalyzes Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange

Our previous investigations have established that Na⁺ translocation across the Tetraselmis viridis plasma membrane (PM) mediated by the primary ATP-driven Na⁺-pump, Na⁺-ATPase, is accompanied by H⁺ counter-transport [Y.V. Balnokin et al. (1999) FEBS Lett 462:402–406]. The hypothesis that the Na⁺-ATPase of T. viridis operates as an Na⁺/H⁺ exchanger is tested in the present work. The study of Na⁺ and H⁺ transport in PM vesicles isolated from T. viridis demonstrated that the membrane-permeant anion NO₃^– caused (i) an increase in ATP-driven Na⁺ uptake by the vesicles, (ii) an increase in (Na⁺+ATP)-dependent vesicle lumen alkalization resulting from H⁺ efflux out of the vesicles and (iii) dissipation of electrical potential, , generated across the vesicle membrane by the Na⁺-ATPase. The (Na⁺+ATP)-dependent lumen alkalization was not significantly affected by valinomycin, addition of which in the presence of K⁺ abolished at the vesicle membrane. The fact that the Na⁺-ATPase-mediated alkalization of the vesicle lumen is sustained in the absence of the transmembrane is consistent with a primary role of the Na⁺-ATPase in driving H⁺ outside the vesicles. The findings allowed us to conclude that the Na⁺-ATPase of T. viridis directly performs an exchange of Na⁺ for H⁺. Since the Na⁺-ATPase generates electric potential across the vesicle membrane, the transport stoichiometry is mNa⁺/nH⁺, where m>n.Abbreviations BTP Bis-Tris-Propane, 1,3-bis[tris(hydroxymethyl)methylamino]-propane - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DTT Dithiothreitol - NCDC 2-Nitro-4-carboxyphenyl N,N-diphenylcarbamate - PMSF Phenylmethylsulfonyl fluoride - PM Plasma membrane     

Propofol Protects Against H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Oxidative Injury in Differentiated PC12 Cells via Inhibition of Ca<Superscript>2+</Superscript>-Dependent NADPH Oxidase

Propofol (2,6-diisopropylphenol) is a widely used general anesthetic with anti-oxidant activities. This study aims to investigate protective capacity of propofol against hydrogen peroxide (H₂O₂)-induced oxidative injury in neural cells and whether the anti-oxidative effects of propofol occur through a mechanism involving the modulation of NADPH oxidase (NOX) in a manner of calcium-dependent. The rat differentiated PC12 cell was subjected to H₂O₂ exposure for 24 h to mimic a neuronal in vitro model of oxidative injury. Our data demonstrated that pretreatment of PC12 cells with propofol significantly reversed the H₂O₂-induced decrease in cell viability, prevented H₂O₂-induced morphological changes, and reduced the ratio of apoptotic cells. We further found that propofol attenuated the accumulation of malondialdehyde (biomarker of oxidative stress), counteracted the overexpression of NOX core subunit gp91^phox (NOX2) as well as the NOX activity following H₂O₂ exposure in PC12 cells. In addition, blocking of L-type Ca²⁺ channels with nimodipine reduced H₂O₂-induced overexpression of NOX2 and caspase-3 activation in PC12 cells. Moreover, NOX inhibitor apocynin alone or plus propofol neither induces a significant downregulation of NOX activity nor increases cell viability compared with propofol alone in the PC12 cells exposed to H₂O₂. These results demonstrate that the protective effects of propofol against oxidative injury in PC12 cells are mediated, at least in part, through inhibition of Ca²⁺-dependent NADPH oxidase.     

MeJA诱导的拟南芥保卫细胞H2O2积累与质膜H+-ATPase的关系

茉莉酸类物质(JAs)作为与昆虫啃噬及损伤相关的植物激素和信号分子在植物防御反应中起重要作用,但是茉莉酸引起的早期防御反应的机理仍不清楚。该研究以拟南芥叶片保卫细胞为材料,结合非损伤微测(NMT)及激光共聚焦技术探讨了茉莉酸诱导的保卫细胞中质膜H+-ATPase与H2O2积累的调控关系。结果表明:茉莉酸甲酯(MeJA)处理导致H+迅速跨膜外排和H2O2积累,H+外排和H2O2积累能够被钒酸钠抑制,而二苯基碘(DPI)处理则对MeJA诱导的H+跨膜外排无显著影响。研究结果证明,在MeJA诱导的早期信号事件中,质膜H+-ATPase的激活先于H2O2的产生。     

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.     

Changes in the expression pattern of the plasma membrane H<Superscript>+</Superscript>-ATPase in developing<Emphasis Type="Italic"> Ricinus communis</Emphasis> cotyledons undergoing the sink/source transition

The plasma membrane (PM) H⁺-ATPase is thought to play a key role in generating the proton motive force used to drive the uptake and accumulation of solutes in plant cells. Changes in its expression pattern were studied in the Ricinus communis L. cotyledon as it changed from a sink to a source organ. Expression was monitored in 3-, 10- and 14-day-old cotyledons using an antibody to the maize PM H⁺-ATPase. The antibody labelled a 100-kDa protein in membrane fractions prepared from cotyledons and this protein occurred at higher levels in the PM-enriched fractions compared to those enriched in intracellular membranes. Immunostaining of tissue sections of 3-day-old Ricinus cotyledons (sinks) with this antibody demonstrated that the PM H⁺-ATPase was highly expressed in the lower epidermal cells and also in the vascular bundles, particularly the phloem. The high expression in the epidermis suggests that these cells may be important in the initial active uptake of solutes from the endosperm. A similar distribution was observed in the 10-day-old seedlings but, in addition, larger, more spherical cells (idioblasts) had developed in the lower and upper epidermal layers and these were also labelled. In 14-day-old seedlings the cotyledons are no longer reliant on nutrients from the endosperm (which has totally degraded) and they are functioning as source organs. This is reflected in a decrease in PM H⁺-ATPase expression in the lower epidermal cells, apart from idioblasts and stomatal guard cells. The latter were also observed in the upper epidermis. Expression remained high in the vascular bundles of 14-day-old seedlings with strong staining in the phloem.Abbreviation PM Plasma membrane