Proteomics reveals the overlapping roles of hydrogen peroxide and nitric oxide in the acclimation of citrus plants to salinity

Hydrogen peroxide (H₂O₂) and nitric oxide (˙NO) are key reactive species in signal transduction pathways leading to activation of plant defense against biotic or abiotic stress. Here, we investigated the effect of pre‐treating citrus plants (Citrus aurantium L.) with either of these two molecules on plant acclimation to salinity and show that both pre‐treatments strongly reduced the detrimental phenotypical and physiological effects accompanying this stress. A proteomic analysis disclosed 85 leaf proteins that underwent significant quantitative variations in plants directly exposed to salt stress. A large part of these changes was not observed with salt‐stressed plants pre‐treated with either H₂O₂ or sodium nitroprusside (SNP; a ˙NO‐releasing chemical). We also identified several proteins undergoing changes either in their oxidation (carbonylation; 40 proteins) and/or S‐nitrosylation (49 proteins) status in response to salinity stress. Both H₂O₂ and SNP pre‐treatments before salinity stress alleviated salinity‐induced protein carbonylation and shifted the accumulation levels of leaf S‐nitrosylated proteins to those of unstressed control plants. Altogether, the results indicate an overlap between H₂O₂‐ and ˙NO‐signaling pathways in acclimation to salinity and suggest that the oxidation and S‐nitrosylation patterns of leaf proteins are specific molecular signatures of citrus plant vigour under stressful conditions.     

Involvement of extracellular oxidative burst in salicylic acid-induced stomatal closure in Arabidopsis

Salicylic acid (SA), a ubiquitous phenolic phytohormone, is involved in many plant physiological processes including stomatal movement. We analysed SA‐induced stomatal closure, production of reactive oxygen species (ROS) and nitric oxide (NO), cytosolic calcium ion ([Ca²⁺]_cyt) oscillations and inward‐rectifying potassium (K⁺_in) channel activity in Arabidopsis. SA‐induced stomatal closure was inhibited by pre‐treatment with catalase (CAT) and superoxide dismutase (SOD), suggesting the involvement of extracellular ROS. A peroxidase inhibitor, SHAM (salicylhydroxamic acid) completely abolished SA‐induced stomatal closure whereas neither an inhibitor of NADPH oxidase (DPI) nor atrbohD atrbohF mutation impairs SA‐induced stomatal closures. 3,3′‐Diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) stainings demonstrated that SA induced H₂O₂ and O₂^– production. Guard cell ROS accumulation was significantly increased by SA, but that ROS was suppressed by exogenous CAT, SOD and SHAM. NO scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO) suppressed the SA‐induced stomatal closure but did not suppress guard cell ROS accumulation whereas SHAM suppressed SA‐induced NO production. SA failed to induce [Ca²⁺]_cyt oscillations in guard cells whereas K⁺_in channel activity was suppressed by SA. These results indicate that SA induces stomatal closure accompanied with extracellular ROS production mediated by SHAM‐sensitive peroxidase, intracellular ROS accumulation and K⁺_in channel inactivation.     

Salicylic acid antagonism of EDS1‐driven cell death is important for immune and oxidative stress responses in Arabidopsis

Reactive oxygen species (ROS) have emerged as signals in the responses of plants to stress. Arabidopsis Enhanced Disease Susceptibility1 (EDS1) regulates defense and cell death against biotrophic pathogens and controls cell death propagation in response to chloroplast‐derived ROS. Arabidopsis Nudix hydrolase7 (nudt7) mutants are sensitized to photo‐oxidative stress and display EDS1‐dependent enhanced resistance, salicylic acid (SA) accumulation and initiation of cell death. Here we explored the relationship between EDS1, EDS1‐regulated SA and ROS by examining gene expression profiles, photo‐oxidative stress and resistance phenotypes of nudt7 mutants in combination with eds1 and the SA‐biosynthetic mutant, sid2. We establish that EDS1 controls steps downstream of chloroplast‐derived O₂^?? that lead to SA‐assisted H₂O₂ accumulation as part of a mechanism limiting cell death. A combination of EDS1‐regulated SA‐antagonized and SA‐promoted processes is necessary for resistance to host‐adapted pathogens and for a balanced response to photo‐oxidative stress. In contrast to SA, the apoplastic ROS‐producing enzyme NADPH oxidase RbohD promotes initiation of cell death during photo‐oxidative stress. Thus, chloroplastic O₂^?? signals are processed by EDS1 to produce counter‐balancing activities of SA and RbohD in the control of cell death. Our data strengthen the idea that EDS1 responds to the status of O₂^?? or O₂^??‐generated molecules to coordinate cell death and defense outputs. This activity may enable the plant to respond flexibly to different biotic and abiotic stresses in the environment.     

Signaling Effects of Nitric Oxide, Salicylic Acid, and Reactive Oxygen Species on Isoeuphpekinensin Accumulation in Euphorbia pekinensis Suspension Cells Induced by an Endophytic Fungal Elicitor

Nitric oxide (NO), salicylic acid (SA), and reactive oxygen species (ROS) are important signal molecules that mediate plant resistance reactions and play important roles in secondary metabolism. To research the signal transduction pathway of the endophytic fungal elicitor from Fusarium sp. E5 promoting secondary metabolism in Euphorbia pekinensis suspension cells, the changes in NO, SA, ROS, and isoeuphpekinensin contents in the cells were investigated after elicitor addition to the cell suspension culture. The elicitor did not change H₂O₂ or O₂ ^? contents notably, whereas NO and SA contents were enhanced. Both the NO donator sodium nitroprusside (SNP) and SA enhanced isoeuphpekinensin content in the absence of the fungal elicitor, whereas the NO scavenger cPTIO and SA biosynthesis inhibitor cinnamic acid (CA) inhibited isoeuphpekinensin accumulation in the presence of the elicitor. In addition, cPTIO inhibited SA production induced by the fungal elicitor. CA did not inhibit NO production, but it significantly inhibited isoeuphpekinensin accumulation. The results demonstrated that in Euphorbia pekinensis suspension cells the endophytic fungal elicitor induced increased NO content and SA production, which promoted isoeuphpekinensin accumulation. ROS are clearly not involved in the endophytic fungus–host interaction signaling pathway.     

Nitric oxide interferes with plant photo-oxidative stress by detoxifying reactive oxygen species

Oxidative stress within chloroplasts is originated due to light‐dependent O₂ reduction. This may be exacerbated by bipyridinium herbicides, which act at photosystem I as artificial electron acceptors. Their oxidation produces a superoxide anion that further dismutates to H₂O₂ and then, by the Fenton reaction, H₂O₂ may be reduced to the hydroxyl radical (OH^?). Reactive oxygen species (ROS), when produced in high amounts, provoke severe damage to the plant cell. Herein it is reported that two nitric oxide (NO) donors, sodium nitroprusside (100 µm ) and S‐nitroso‐N‐acetylpenicillamine (200 µm ), greatly reduced lipid peroxidation and the protein loss caused by the application of a high dose of the bipyridinium herbicide diquat to potato leaf pieces or isolated chloroplasts. Nitric oxide donors also protected the RNA against oxidative damage. Photo‐oxidative toxicity was correlated with an increase in photosynthetic electron transport and ROS production, but the rate of electron transport was restored and the ROS free amount was markedly reduced in the presence of NO. The specific activity of superoxide dismutase was not affected by diquat or NO donors, whereas just a small increase in catalase activity was observed after 24 h of treatment. These results provide strong evidence that NO is a potent antioxidant in plants and that its action may, at least in part, be explained by its ability to directly scavenge ROS.     

NMDA Receptor Activation Produces Concurrent Generation of Nitric Oxide and Reactive Oxygen Species: Implications for Cell Death

Abstract: The ability of glutamate to stimulate generation of intracellular oxidant species was determined by microfluorescence in cerebellar granule cells loaded with the oxidant-sensitive fluorescent dye 2,7-dichlorofluorescin (DCF). Exposure of cells to glutamate (10 µM) produced a rapid generation of oxidants that was blocked ～70% by MK-801 (a noncompetitive NMDA-receptor antagonist). To determine if nitric oxide (NO) or reactive oxygen species (ROS) contributed to the oxidation of DCF, cells were treated with compounds that altered their generation. NO production was inhibited with N^G-nitro-l -arginine methyl ester (l -NAME) (nitric oxide synthase inhibitor) and reduced hemoglobin (NO scavenger). Alternatively, cells were incubated with superoxide dismutase (SOD) and catalase, which selectively metabolize O₂^?· andH₂O₂. Concurrent inhibition of O₂^?· and NO production nearly abolished intracellular oxidant generation. Pretreatment of cells with either chelerythrine (1 µM, protein kinase C inhibitor) or quinacrine (5 µM, phospholipase A₂ inhibitor) before addition of glutamate also blocked oxidation of DCF. Generation of oxidants by glutamate was significantly reduced by incubating the cells in Ca²⁺-free buffer. In cytotoxicity studies, a positive correlation was observed between glutamate-induced death and oxidant generation. Glutamate-induced cytotoxicity was blocked by MK-801 and attenuated by treatment with l -NAME, chelerythrine, SOD, or quinacrine. It is concluded that glutamate induces concurrent generation of NO and ROS by activation of both NMDA receptors and non-NMDA receptors through a Ca²⁺-mediated process. Activation of NO synthase and phospholipaseA₂ contribute significantly to this response. It is proposed that simultaneous generation of NO and ROS results in formation of peroxynitrite, which initiates the cellular damage.     

Ethylene signaling in salt stress- and salicylic acid-induced programmed cell death in tomato suspension cells

Salt stress- and salicylic acid (SA)-induced cell death can be activated by various signaling pathways including ethylene (ET) signaling in intact tomato plants. In tomato suspension cultures, a treatment with 250 mM NaCl increased the production of reactive oxygen species (ROS), nitric oxide (NO), and ET. The 10^?3 M SA-induced cell death was also accompanied by ROS and NO production, but ET emanation, the most characteristic difference between the two cell death programs, did not change. ET synthesis was enhanced by addition of ET precursor 1-aminocyclopropane-1-carboxylic acid, which, after 2 h, increased the ROS production in the case of both stressors and accelerated cell death under salt stress. However, it did not change the viability and NO levels in SA-treated samples. The effect of ET induced by salt stress could be blocked with silver thiosulfate (STS), an inhibitor of ET action. STS reduced the death of cells which is in accordance with the decrease in ROS production of cells exposed to high salinity. Unexpectedly, application of STS together with SA resulted in increasing ROS and reduced NO accumulation which led to a faster cell death. NaCl- and SA-induced cell death was blocked by Ca²⁺ chelator EGTA and calmodulin inhibitor W-7, or with the inhibitors of ROS. The inhibitor of MAPKs, PD98059, and the cysteine protease inhibitor E-64 reduced cell death in both cases. These results show that NaCl induces cell death mainly by ET-induced ROS production, but ROS generated by SA was not controlled by ET in tomato cell suspension.     

Cadmium-induced subcellular accumulation of O2·− and H2O2 in pea leaves

Cadmium is a toxic metal that produces disturbances in plant antioxidant defences giving rise to oxidative stress. The effect of this metal on H₂O₂ and O₂^·? production was studied in leaves from pea plants growth for 2 weeks with 50 µm Cd, by histochemistry with diaminobenzidine (DAB) and nitroblue tetrazolium (NBT), respectively. The subcellular localization of these reactive oxygen species (ROS) was studied by cytochemistry with CeCl₃ and Mn/DAB staining for H₂O₂ and O₂^·?, respectively, followed by electron microscopy observation. In leaves from pea plants grown with 50 µm CdCl₂ a rise of six times in the H₂O₂ content took place in comparison with control plants, and the accumulation of H₂O₂ was observed mainly in the plasma membrane of transfer, mesophyll and epidermal cells, as well as in the tonoplast of bundle sheath cells. In mesophyll cells a small accumulation of H₂O₂ was observed in mitochondria and peroxisomes. Experiments with inhibitors suggested that the main source of H₂O₂ could be a NADPH oxidase. The subcellular localization of O₂^·? production was demonstrated in the tonoplast of bundle sheath cells, and plasma membrane from mesophyll cells. The Cd‐induced production of the ROS, H₂O₂ and O₂^·?, could be attributed to the phytotoxic effect of Cd, but lower levels of ROS could function as signal molecules in the induction of defence genes against Cd toxicity. Treatment of leaves from Cd‐grown plants with different effectors and inhibitors showed that ROS production was regulated by different processes involving protein phosphatases, Ca²⁺ channels, and cGMP.     

Involvement of ethylene in reversal of salt‐inhibited photosynthesis by sulfur in mustard

Sulfur (S) assimilation results in the synthesis of cysteine (Cys), a common metabolite for the formation of both reduced glutathione (GSH) and ethylene. Thus, ethylene may have regulatory interaction with GSH in the alleviation of salt stress. The involvement of ethylene in the alleviation of salt stress by S application was studied in mustard (Brassica juncea cv. Pusa Jai Kisan). First, the effects of 0, 0.5, 1.0 and 2.0 mM SO₄^2? were studied on photosynthetic and growth parameters to ascertain the S requirement as sufficient‐S and excess‐S for the plant. In further experiments, the effects of sufficient‐S (1 mM SO₄^2?) and excess‐S (2 mM SO₄^2?) were studied on the alleviation of salt stress‐induced by 100 mM NaCl, and ethylene involvement in the alleviation of salt stress by S. Under non‐saline condition, excess‐S increased ethylene with less content of Cys and GSH and adversely affected photosynthesis and growth. In contrast, excess‐S maximally alleviated salt stress due to high demand for S and optimal ethylene formation, which maximally increased GSH and promoted photosynthesis and growth. The involvement of ethylene in S‐mediated alleviation of salt stress was further substantiated by the reversal of the effects of excess‐S on photosynthesis by aminoethoxyvinylglycine (AVG), ethylene biosynthesis inhibitor. The studies suggest that plants respond differentially to the S availability under non‐saline and salt stress and excess‐S was more potential in the alleviation of salt stress. Further, ethylene regulates plants' response and excess S‐induced alleviation of salt stress and promotion of photosynthesis.     

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.     

Oligochitosan induced Brassica napus L. production of NO and H2O2 and their physiological function

NO (nitric oxide) and H₂O₂ (hydrogen peroxide) are important signaling molecule in plants. Brassica napus L. was used to understand oligochitosan inducing production of NO (nitric oxide) and H₂O₂ (hydrogen peroxide) and their physiological function. The result showed that the production of NO and H₂O₂ in epidermal cells of B. napus L. was induced with oligochitosan by fluorescence microscope. And it was proved that there was an interaction between NO and H₂O₂ with L-NAME (N^G-nitro-l-arg-methyl eater), which is an inhibitor of NOS (NO synthase) in mammalian cells that also inhibits plant NO synthesis, and CAT (catalase), which is an important H₂O₂ scavenger, respectively. It was found that NO and H₂O₂ induced by oligochitosan took part in inducing reduction in stomatal aperture and LEA protein gene expression of leaves of B. napus L. All these results showed that oligochitosan have potential activities of improving resistance to water stress.     

Salicylic acid and nitric oxide increase photosynthesis and antioxidant defense in wheat under UV-B stress

The effects of exogenous salicylic acid (SA), sodium nitropusside (SNP, a nitric oxide donor), or their combination on dwarf polish wheat (Triticum polonicum L.) seedlings under UV-B stress were studied. The UV-B stress significantly decreased plant height, shoot dry mass, pigment content, net photosynthetic rate, intercellular CO₂ concentration, stomatal conductance, transpiration rate, and variable to maximum chlorophyll fluorescence ratio (F_v/F_m) in all plants, but less in the presence of SA, SNP, and their combination. On the other hand, there were considerable increases in malondialdehyde (MDA), proline, O₂ ^?-, and H₂O₂ content under the UV-B stress. When SA, SNP, and their combination were applied, content of MDA, proline, H₂O₂, and O₂ ^?- were less increased. Moreover, there were considerable increases in activities of superoxide dismutase, peroxidase, ascorbate peroxidase, and glutathione reductase under the UV-B stress and more in the presence of SA, SNP, and their combination. Therefore, it is considered that SA, SNP, and especially their combination could alleviate UV-B stress in dwarf polish wheat.