Cold stress causes rapid but differential changes in properties of plasma membrane H-ATPase of camelina and rapeseed

Camelina (Camelina sativa) and rapeseed (Brassica napus) are well-established oil-seed crops with great promise also for biofuels. Both are cold-tolerant, and camelina is regarded to be especially appropriate for production on marginal lands. We examined physiological and biochemical alterations in both species during cold stress treatment for 3 days and subsequent recovery at the temperature of 25 °C for 0, 0.25, 0.5, 1, 2, 6, and 24 h, with particular emphasis on the post-translational regulation of the plasma membrane (PM) H⁺-ATPase (EC3.6.3.14). The activity and translation of the PM H⁺-ATPase, as well as 14-3-3 proteins, increased after 3 days of cold stress in both species but recovery under normal conditions proceeded differently. The increase in H⁺-ATPase activity was the most dramatic in camelina roots after recovery for 2 h at 25 °C, followed by decay to background levels within 24 h. In rapeseed, the change in H⁺-ATPase activity during the recovery period was less pronounced. Furthermore, H⁺-pumping increased in both species after 15 min recovery, but to twice the level in camelina roots compared to rapeseed. Protein gel blot analysis with phospho-threonine anti-bodies showed that an increase in phosphorylation levels paralleled the increase in H⁺-transport rate. Thus our results suggest that cold stress and recovery in camelina and rapeseed are associated with PM H⁺-fluxes that may be regulated by specific translational and post-translational modifications.     

Increased cucumber salt tolerance by grafting on pumpkin rootstock and after application of calcium

Self-grafted and pumpkin rootstock-grafted cucumber plants were subjected to the following four treatments: 1) aerated nutrient solution alone (control), 2) nutrient solution with 10 mM Ca(NO₃)₂ (Ca), 3) nutrient solution with 90 mM NaCl (NaCl), and 4) nutrient solution with 90 mM NaCl + 10 mM Ca(NO₃)₂ (NaCl+Ca). The NaCl treatment decreased the plant dry mass and content of Ca²⁺ and K⁺ but increased the Na⁺ content in roots and shoots. Smaller changes were observed in pumpkin rootstock-grafted plants. Supplementary Ca(NO₃)₂ ameliorated the negative effects of NaCl on plant dry mass, relative growth rate (RGR), as well as Ca²⁺, K⁺, and Na⁺ content especially for pumpkin rootstock-grafted plants. Supplementary Ca(NO₃)₂ distinctly stimulated the plasma membrane (PM) H⁺-ATPase activity which supplies the energy to remove excess Na⁺ from the cells. The expressions of gene encoding PM H⁺-ATPases (PMA) and gene encoding a PM Na⁺/H⁺ antiporter (SOS1) were up-regulated when Ca(NO₃)₂ was applied. The pumpkin rootstock-grafted plants had higher PM H⁺-ATPase activity as well as higher PMA and SOS1 expressions than the self-grafted plants under NaCl + Ca treatment. Therefore, the addition of Ca²⁺ in combination with pumpkin rootstock grafting is a powerful way to increase cucumber salt tolerance.     

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.     

Scanning ion‐selective electrode technique and X‐ray microanalysis provide direct evidence of contrasting Na+ transport ability from root to shoot in salt‐sensitive cucumber and salt‐tolerant pumpkin under NaCl stress

Grafting onto salt‐tolerant pumpkin rootstock can increase cucumber salt tolerance. Previous studies have suggested that this can be attributed to pumpkin roots with higher capacity to limit the transport of Na⁺ to the shoot than cucumber roots. However, the mechanism remains unclear. This study investigated the transport of Na⁺ in salt‐tolerant pumpkin and salt‐sensitive cucumber plants under high (200 mM) or moderate (90 mM) NaCl stress. Scanning ion‐selective electrode technique showed that pumpkin roots exhibited a higher capacity to extrude Na⁺, and a correspondingly increased H⁺ influx under 200 or 90 mM NaCl stress. The 200 mM NaCl induced Na⁺/H⁺ exchange in the root was inhibited by amiloride (a Na⁺/H⁺ antiporter inhibitor) or vanadate [a plasma membrane (PM) H⁺‐ATPase inhibitor], indicating that Na⁺ exclusion in salt stressed pumpkin and cucumber roots was the result of an active Na⁺/H⁺ antiporter across the PM, and the Na⁺/H⁺ antiporter system in salt stressed pumpkin roots was sufficient to exclude Na⁺. X‐ray microanalysis showed higher Na⁺ in the cortex, but lower Na⁺ in the stele of pumpkin roots than that in cucumber roots under 90 mM NaCl stress, suggesting that the highly vacuolated root cortical cells of pumpkin roots could sequester more Na⁺, limit the radial transport of Na⁺ to the stele and thus restrict the transport of Na⁺ to the shoot. These results provide direct evidence for pumpkin roots with higher capacity to limit the transport of Na⁺ to the shoot than cucumber roots.     

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.     

Hydrogen sulfide is involved in maintaining ion homeostasis via regulating plasma membrane Na+/H+ antiporter system in the hydrogen peroxide-dependent manner in salt-stress Arabidopsis thaliana root

Hydrogen sulfide (H₂S) and hydrogen peroxide (H₂O₂) function as the signaling molecules in plants responding to salt stresses. The present study presents a signaling network involving H₂S and H₂O₂ in salt resistance pathway of the Arabidopsis root. Arabidopsis roots were sensitive to 100 mM NaCl treatment, which displayed a great increase in electrolyte leakage (EL) and Na⁺/K⁺ ratio under salt stress. The treatment of H₂S donors sodium hydrosulfide (NaHS) enhanced the salt tolerance by maintaining a lower Na⁺/K⁺ ratio. In addition, the inhibition of root growth under salt stress was removed by H₂S. Further studies indicated that H₂O₂ was involved in H₂S-induced salt tolerance pathway. H₂S induced the production of the endogenous H₂O₂ via regulating the activities of glucose-6-phosphate dehydrogenase (G6PDH) and plasma membrane (PM) NADPH oxidase, with the treatment with dimethylthiourea (DMTU, an ROS scavenger), diphenylene iodonium (DPI, a PM NADPH oxidase inhibitor), or glycerol (G6PDH inhibitor) removing the effect of H₂S. Treatment with amiloride (an inhibitor of PM Na⁺/H⁺ antiporter) and vanadate (an inhibitor of PM H⁺-ATPase) also inhibited the activity of H₂S on Na⁺/K⁺ ratio. Through an analysis of quantitative real-time polymerase chain reaction and Western blot, we found that H₂S promoted the genes expression and the phosphorylation level of PM H⁺-ATPase and Na⁺/H⁺ antiporter protein level. However, when the endogenous H₂O₂ level was inhibited by DPI or DMTU, the effect of H₂S on the PM Na⁺/H⁺ antiporter system was removed. Taken together, H₂S maintains ion homeostasis in the H₂O₂-dependent manner in salt-stress Arabidopsis root.     

Adaptation of plasma membrane H+ ATPase and H+ pump to P deficiency in rice roots

The plasma membrane (PM) H⁺ ATPase is involved in the plant response to nutrient deficiency. However, adaptation of this enzyme in monocotyledon plants to phosphorus (P) deficiency lacks direct evidence. In this study, we detected that P deficient roots of rice (Oryza Sativa L.) could acidify the rhizosphere. We further isolated the PM from rice roots and analyzed the activity of PM H⁺ ATPase. In vitro, P deficient rice roots showed about 30% higher activity of PM H⁺ ATPase than the P sufficient roots at assay of pH 6.0. The P deficiency resulted in a decrease of the substrate affinity value (K _m ) of PM H⁺ ATPase. The proton pumping activity of membrane vesicles from the P deficient roots was about 70% higher than that from P sufficient roots. Western blotting analysis indicated that higher activity of PM H⁺ ATPase in P deficient roots was related to a slightly increase of PM H⁺ ATPase protein abundance in comparison with that in P sufficient roots. Taken together, our results demonstrate that the P deficiency enhanced activities of both PM H⁺-ATPase and H⁺ pump, which contributed to the rhizosphere acidification in rice roots.     

The Effects of Exogenous Ascorbic Acid on the Mechanism of Physiological and Biochemical Responses to Nitrate Uptake in Two Rice Cultivars (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) Under Aluminum Stress

As a major antioxidant in plants, ascorbic acid (AsA) plays a very important role in the response to aluminum (Al) stress. However, the effect of AsA on the mitigation of Al toxicity and the mechanism of nitrate nitrogen (NO₃ ^?–N) uptake by plants under Al stress are unclear. In this study, a hydroponic experiment was conducted using peak 1 A rice (sterile line, Indica) with weaker resistance to Al and peak 1 superior 5 rice (F1 hybrid, Indica) with stronger resistance to Al to study the effects of exogenous AsA on the physiological and biochemical responses to NO₃ ^?–N uptake by rice roots exposed to 50 μmol L^?1 Al. Al stress induced increases in the concentrations of H₂O₂ and malondialdehyde (MDA) and in the activities of antioxidant enzymes [such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX)]. Plasma membrane (PM) H⁺-ATPase and H⁺-pump activities, endogenous AsA content and NO₃ ^?–N uptake in rice roots decreased under Al stress. After treatment with 2 mmol L^?1 exogenous AsA combined with Al, concentrations of H₂O₂ and MDA in roots notably decreased, and endogenous AsA content and activities of SOD, POD, CAT, and APX in rice roots increased significantly; furthermore, the interaction of PM H⁺-ATPase and the 14-3-3 protein was also enhanced significantly compared with that in control plants without AsA treatment, which clearly increased NO₃ ^?–N uptake. Based on all of these data, the application of AsA significantly reduced the accumulation of H₂O₂ and MDA and increased the activities of PM H⁺-ATPase and the H⁺-pump by increasing the endogenous AsA content, the antioxidant enzyme activities, and the interaction of PM H⁺-ATPase and the 14-3-3 protein in the roots of the two rice cultivars under Al stress, thereby improving the uptake of NO₃ ^?–N in rice.     

In vivo and in vitro effects of copper and cadmium on the plasma membrane H+-ATPase from cucumber (Cucumis sativus L.) and maize (Zea mays L.) roots

The plasmalemma vesicles isolated from cucumber and maize roots were used to study the effect of Cu²⁺ and Cd²⁺ on the hydrolytic and proton pumping activities of ATPase. In vivo application of metal ions to the plant growth solutions resulted in stimulation of the proton transport in maize. In cucumber roots the action of metals was not the same: cadmium stimulated the H⁺ transport through plasmalemma whereas Cu²⁺ almost completely inhibited it. Copper ions decreased the hydrolytic activity of H⁺-ATPase in cucumber, without any effect on this activity in membranes isolated from maize roots. The effect of cadmium on the hydrolytic activities was opposite: ATP-hydrolysis activity in plasmalemma was not altered in cucumber, whereas in maize its stimulation was observed. The amount of accumulated metals was not the main reason of different influence of metals on H⁺-ATPase activity in tested plants. In in vitro experiments Cu²⁺ inhibited H⁺ transport in the cucumber, to a higher degree than Cd²⁺ and both metals did not change this H⁺-ATPase activity of plasmalemma isolated from corn roots. Cu²⁺ added into the incubation medium reduced the hydrolytic activity of ATPase in the plasma membrane isolated from cucumber as well as from corn roots. Cd²⁺ diminished the hydrolytic activity of ATPase in cucumber, and no effect of Cd²⁺ in the plasmalemma isolated from corn roots was found. Our results indicated different in vitro and in vivo action of both metals on H⁺-ATPase and different response of this enzyme to Cu²⁺ and Cd²⁺ in maize and cucumber.     

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.     

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.