Quercetin-induced H2O2 mediates the pathogen resistance against Pseudomonas syringae pv. Tomato DC3000 in Arabidopsis thaliana

Quercetin is a potent antioxidant and has been extensively used as a therapy intervention to prevent age-associated diseases. However, emerging studies showed it can also act as a prooxidant and induce H₂O₂ under certain conditions. In the current study, our results showed that quercetin contributed to the pathogen resistance in Arabidopsis thaliana (Arabidopsis) in response to the infection of virulent strain Pseudomonas syringae pv. Tomato DC3000 (Pst). Various defense responses, such as H₂O₂ burst, callose deposition, cell death, PR1 (pathogenesis-related 1) and PAL1 (Phe ammonia-lyase 1) gene expression, have been investigated in quercetin-pretreated Pst-inoculated Arabidopsis Col-0 and there was a strong defensive response in quercetin-pretreated Arabidopsis against virulent Pst. However, with the presence of catalase, the protective effects of quercetin on pathogen resistance to virulent Pst disappeared in Arabidopsis, suggesting that H₂O₂ may play a key role in plant defense responses. In addition, we confirmed that quercetin did not show any beneficial effect on pathogen-free leaves in Arabidopsis, indicating that pathogen challenge is also required to induce the defense responses in quercetin-pretreated Arabidopsis. Furthermore, strong defense responses have been observed in quercetin-pretreated Arabidopsis mutant jar1, ein2, and abi1-2 under Pst challenge, whereas no protective effect has been observed in quercetin-pretreated Arabidopsis mutant NahG and npr1. These findings indicate that quercetin induces the resistance to Pst in Arabidopsis via H₂O₂ burst and involvement of SA and NPR1.     

Temperature-related effects of treatments with jasmonic and salicylic acids on Arabidopsis infected with cucumber mosaic virus

Plant-virus interactions are affected by environmental factors, including temperature. Plant defenses are often inhibited by high or low temperature. In this study, oxidative damage and gene expression were detected in Arabidopsis thaliana infected with cucumber mosaic virus (CMV) at different temperatures. Before virus inoculation, plants were treated with jasmonic acid (JA) and salicylic acid (SA), both of which are important signaling molecules in plant defense responses. The levels of MDA and hydrogen peroxide (H₂O₂), and electrolyte leakage were significantly higher in CMV-infected leaves at 15 and 37°C. The accumulation of H₂O₂ and superoxide radical (O ₂ ^·? ) was obviously suppressed by spraying with JA followed by SA (JA → SA) at different temperatures. The CMV-CP expression analysis showed that virus replication was inhibited efficiently in the (JA → SA) treatment. Therefore, many JA- and SA-responsible resistance genes were quantified; MPK4 was expressed highly and steadily in the (JA → SA) treatment. To further confirm the role of MPK4, the CMV-CP gene expression was evaluated in wild-type Arabidopsis and its mpk4 mutant infected with CMV. The results suggested that MPK4 might play an important role in the antagonism between JA and SA at temperature fluctuation.     

Salicylic and Jasmonic acids in regulation of the proantioxidant state in wheat leaves infected by <Emphasis Type="Italic">Septoria nodorum</Emphasis> Berk

Influence of mediators of the signal systems of salicylic (SA) and jasmonic (JA) acids and their mixture on reactive oxygen species’ (ROS) (superoxide radical and O₂^·− H₂O₂) generation and activity of oxidoreductases (oxalate oxidase, peroxidase and catalase) in leaves of wheat Triticum aestivum L. infected by Septoria leaf blotch pathogen Septoria nodorum Berk has been studied. Presowing treatment of seeds by SA and JA decreased the development rate of fungus on wheat leaves. SA provided earlier inductive effect on production of O₂^·− and H₂O₂ compared with JA. The protective effect of the salicylic and jasmonic acids against Septoria leaf blotch pathogen was caused by activation of oxalate oxidase, induction of anion and cation peroxidases, and decrease of catalase activity. Ability of compounds to stimulate ROS in the plant tissues can be used as criteria for evaluation of immune-modulating activity of new substances for protection of the plants.     

Relationship between chloroplastic H2O2 and the salicylic acid response

Reactive oxygen species (ROS) act as signaling molecules for regulating plant responses to abiotic and biotic stress and there exist source- and kind-specific pathways for ROS signaling. Recently, we created a novel system for producing H₂O₂ in Arabidopsis chloroplasts by chemical-dependent thylakoid membrane-bound ascorbate peroxidase (tAPX) silencing using an estrogen-inducible RNAi method. Microarray analysis revealed that the expression of a large set of genes was altered in response to tAPX silencing, some of which are known to be involved in pathogen response/resistance. Furthermore, we found that tAPX silencing enhances the levels of salicylic acid (SA) and the response to SA, a central regulator for biotic stress response. In this addendum, we describe the relationship between chloroplastic H₂O₂ and SA in stress response, and discuss the function of the kind- and source-specific ROS signaling in SA-mediated stress response.     

A Benzothiadiazole Primes Parsley Cells for Augmented Elicitation of Defense Responses

Systemic acquired resistance is an important component of the disease-resistance arsenal of plants, and is associated with an enhanced potency for activating local defense responses upon pathogen attack. Here we demonstrate that pretreatment with benzothiadiazole (BTH), a synthetic activator of acquired resistance in plants, augmented the sensitivity for low-dose elicitation of coumarin phytoalexin secretion by cultured parsley (Petroselinum crispum L.) cells. Enhanced coumarin secretion was associated with potentiated activation of genes encoding Phe ammonia-lyase (PAL). The augmentation of PAL gene induction was proportional to the length of pretreatment with BTH, indicating time-dependent priming of the cells. In contrast to the PAL genes, those for anionic peroxidase were directly induced by BTH in the absence of elicitor, thus confirming a dual role for BTH in the activation of plant defenses. Strikingly, the ability of various chemicals to enhance plant disease resistance correlated with their capability to potentiate parsley PAL gene elicitation, emphasizing an important role for defense response potentiation in acquired plant disease resistance.     

H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced acclimation of photosystem II to excess light is mediated by alternative respiratory pathway and salicylic acid

Acclimation to excess light is required for optimizing plant performance under natural environment. The present work showed that the treatment of Arabidopsis leaves with exogenous H₂O₂ can increase the acclimation of PSII to excess light. Treatments with H₂O₂ also enhanced the capacity of the mitochondrial alternative respiratory pathway and salicylic acid (SA) content. Our work also showed that the lack in alternative oxidase (AOX1a) in AtAOX1a antisense line and the SA deficiency in NahG (salicylate hydroxylase gene) transgenic mutant attenuated the H₂O₂-induced acclimation of PSII to excess light. It indicates that the H₂O₂-induced acclimation of PSII to excess light could be mediated by the alternative respiratory pathway and SA.     

Identification of dimedone-trapped sulfenylated proteins in plants under stress

In stressed plants, the reactive oxygen species (ROS) levels rise. Key to ROS signaling research are detection and identification of the protein cysteine sulfenylation (-SOH), the ROS-mediated oxidative product of a thiol (-SH). Arabidopsis thaliana seedlings were stressed with hydrogen peroxide (H₂O₂) and the sulfenylated proteins were tagged with dimedone. Dimedone-tagged sulfenic acid proteins were visualized on a two-dimensional electrophoresis (2DE) immunoblot with an anticysteine sulfenic acid antibody and were subsequently detected by mass spectrometry. We optimized the detection method for protein sulfenylation in Arabidopsis. We conclude that dimedone can penetrate the cell wall, does not stress plants, and can “read” the changes in the protein sulfenylation pattern under oxidative stress. We observed that the number of sulfenylated proteins in plants treated with 10 mM H₂O₂ was higher than that in untreated plants. A total of 39 sulfenylated protein spots were found on 2DE immunoblots. By means of mass spectrometry, 11 sulfenylated proteins were discovered involved in primary metabolism, redox regulation, translation and signaling pathways. Hence, by combining an immunochemical 2DE strategy with mass spectrometry, we were able to identify sulfenylated proteins in H₂O₂-stressed Arabidopsis seedlings. The sulfenylated proteins can be considered for further validation as redox regulators in plants.     

Cross-talk between salicylic acid and NaCl-generated reactive oxygen species and nitric oxide in tomato during acclimation to high salinity

Hydrogen peroxide (H₂O₂) and nitric oxide (NO) generated by salicylic acid (SA) are considered to be functional links of cross‐tolerance to various stressors. SA‐stimulated pre‐adaptation state was beneficial in the acclimation to subsequent salt stress in tomato (Solanum lycopersicum cv. Rio Fuego). At the whole‐plant level, SA‐induced massive H₂O₂ accumulation only at high concentrations (10^?3–10^?2M), which later caused the death of plants. The excess accumulation of H₂O₂ as compared with plants exposed to 100 mM NaCl was not associated with salt stress response after SA pre‐treatments. In the root tips, 10^?3–10^?2M SA triggered the production of reactive oxygen species (ROS) and NO with a concomitant decline in the cell viability. Sublethal concentrations of SA, however, decreased the effect of salt stress on ROS and NO production in the root apex. The attenuation of oxidative stress because of high salinity occurred not only in pre‐adapted plants but also at cell level. When protoplasts prepared from control leaves were exposed to SA in the presence of 100 mM NaCl, the production of NO and ROS was much lower and the viability of the cells was higher than in salt‐treated samples. This suggests that, the cross‐talk of signalling pathways induced by SA and high salinity may occur at the level of ROS and NO production. Abscisic acid (ABA), polyamines and 1‐aminocyclopropane‐1‐carboxylic acid, the compounds accumulating in pre‐treated plants, enhanced the diphenylene iodonium‐sensitive ROS and NO levels, but, in contrast to others, ABA and putrescine preserved the viability of protoplasts.     

Induction of systemic acquired resistance in Zea mays also enhances the plant’s attractiveness to parasitoids

Plants under attack by caterpillars emit volatile compounds that attract the herbivore’s natural enemies. In maize, the caterpillar-induced production of volatiles involves the phytohormone jasmonic acid (JA). In contrast, pathogen attack usually up-regulates the salicylic acid (SA)-pathway and results in systemic acquired resistance (SAR) against plant diseases. Activation of the SA-pathway has often been found to repress JA-dependent direct defenses, but little is known about the effects of SAR induction on indirect defenses such as volatile emission and parasitoid attraction. We examined if induction of SAR in maize, by chemical elicitation with the SA-mimic benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), attenuates the emission of volatiles induced by Spodoptera littoralis or exogenously applied JA. In addition, we determined how these treatments affected the attractiveness of the plants to the parasitoid Microplitis rufiventris in a six-arm-olfactometer. BTH treatment alone resulted in significant systemic resistance of maize seedlings against the pathogen Setosphaeria turcica, but had no detectable effect on volatile emissions. Induction of SAR significantly reduced the emission rates of two compounds (indole and (E)-β-caryophyllene) in JA-treated plants, whereas no such negative cross-talk was found in caterpillar-damaged plants. Surprisingly, however, BTH treatment prior to caterpillar-feeding made the plants far more attractive to the parasitoid than plants that were only damaged by the herbivore. Control experiments showed that this response was due to plant-mediated effects rather than attractiveness of BTH itself. We conclude that in the studied system, plant protection by SAR activation is compatible with and can even enhance indirect defense against herbivores.     

Salicylic acid,hydrogen peroxide and calcium-induced saline tolerance associated with endogenous hydrogen peroxide homeostasis in naked oat seedlings

This study investigates the role of salicylic acid (SA), hydrogen peroxide (H₂O₂) and calcium chloride (CaCl₂) singly or in combination, in inducing naked oat plant tolerance to sodium chloride (NaCl). Two-week-old naked oat plants were pretreated with both single and double of 0.5 mM SA, 0.5 mM H₂O₂ and 5 mM CaCl₂ by adding them to the culture solution for 24 h. At the end of the pretreatment, the plants were subjected to 200 mM NaCl exposure for 7 days. Data were collected on plant biomass, H₂O₂ level, antioxidant enzyme activity, non-enzymatic antioxidant content and malondialdehyde (MDA) content. Results showed that exposure to salt significantly inhibited plant growth, and the shoot and root dry weights were reduced 47.5% and 63.4%, and the H₂O₂ levels elevated 5.8 and 2.4 times in comparison with those in the control, respectively. Under the saline stress, the activities of superoxide dismutase (SOD) and catalase (CAT) were induced, but the contents of ascorbic acid (AA) and glutathione (GSH) decreased, and MDA largely accumulated. The various pretreatments efficiently counteracted the salt-caused growth inhibition, especially with H₂O₂ + CaCl₂ the shoot and root dry weights reduced only 9.4% and 24.4% of the non salt-stressed plants. The determination of endogenous H₂O₂ level demonstrated that the pretreatments induced H₂O₂ accumulation, with H₂O₂ + CaCl₂ being most efficient, but the effect was transient. After 7 days of saline stress, the H₂O₂ contents in the pretreated shoots and roots accounted for 23.7–41.8% and 31.7–57.3% of the non-pretreated plants, varying according to the different pretreatments. Under saline stress, SOD and CAT further increased, AA and GSH maintained higher levels and MDA decreased in the pretreated plants compared to the untreated plants. With application of diphenylene iodonium (DPI) during the pretreatment, which inhibited the accumulation of H₂O₂, the ameliorative effect of the pretreatment on salt-caused plant growth inhibition was reduced. However, applied DPI at the immediate end of the pretreatment did not alter its favorable role, indicating a H₂O₂ peak formed at the early time of saline stress might play an important role in regulating plant tolerance to saline stress.     

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.     

Activation of an oxidative burst is a general feature of sensitive plants exposed to the air pollutant ozone

Ozone exposure stimulates an oxidative burst in leaves of sensitive plants, resulting in the generation and accumulation of hydrogen peroxide (H₂O₂) in tobacco and tomato, and superoxide (O₂^–?) together with H₂O₂ in Arabidopsis accessions. Accumulation of these reactive oxygen species (ROS) preceded the induction of cell death, and both responses co‐occurred spatially in the periveinal regions of the leaves. Re‐current ozone exposure of the sensitive tobacco cv. Bel W3 in closed chambers or in the field led to an enlargement of existing lesions by priming the border cells for H₂O₂ accumulation. Open top chamber experiments with native herbaceous plants in the field showed that Malva sylvestris L. accumulates O₂^–? at those sites that later exhibit plant cell death. Blocking of ROS accumulation markedly reduced ozone‐induced cell death in tomato, Arabidopsis and M. sylvestris. It is concluded that ozone triggers an in planta generation and accumulation of H₂O₂ and/or O₂^–? depending on the species, accession and cultivar, and that both these reactive oxygen species are involved in the induction of cell death in sensitive crop and native plants.     

Defence-related biochemical changes by elicitor of malformation pathogen,Fusarium mangiferae,in mango

Malformation of mango (Mangifera indica L.) induced by Fusarium moniliforme var. subglutinans is a plant disease of international importance. The paper reports the downstream defence responses at the initial stage in a susceptible host (cultivar Amrapali) after treatment with biotic (isolated from the pathogen cell wall) (BEL) and abiotic (salicylic acid, SA) elicitors, and inoculation of vegetative buds with the pathogen (IVB). The SA was further tested to induce resistance in field trials. The inoculation and application of elicitors increased β-1, 3 glucanase that causes lysis of fungal hyphae by many folds. Hydrogen peroxide (H₂O₂) (active oxygen species) that induces hypersensitive cell death was reduced to the minimum level after treatment with BEL. The reduction of H₂O₂ in the inoculated vegetative buds was also substantial; however, comparatively less with SA treatment. Consequently, there was no hypersensitive cell death in the malformed mango. Salicylic acid that enhances H₂O₂ content by suppressing H₂O₂-degradation by catalase, increased marginally with the SA treatment and in the IVB, but reduced with the BEL. The reduction of SA in BEL-treated buds concomitantly reduced its H₂O₂ content. The activity of catalase, suppressor of resistance mechanism, was reduced in all the treatments, but the reduction was not enough to arrest H₂O₂-degradation. Magiferin (1, 3, 6, 7-tetrahdroxyxanthone C₂-β-D glucoside), a defence metabolite of mango, increased substantially in all the treatments; maximum with the BEL. A pathogenesis-related (PR) protein of 20 KDa that resists symptom development appeared in all the treatments except the control. But light colour of the spots for the PR-protein indicated low protein accumulation. The maximum accumulation was with the IVB followed by SA and BEL treatments. The amount of total protein reduced considerably in all the treatments. The SA treatment on healthy plants failed to induce defence against malformation. Contrarily, the treatment on malformed seedlings restored normal growth within two months. Hence, SA acted better over the infected plants in presence of the pathogen. Thus, a signal transduction system involving SA and H₂O₂ remained nonfunctional and enough defence chemicals could not be synthesised. Defence genes that produce phenolic and β-1, 3 glucanase, however, became activated and saved the plants from death although could not prevent symptom manifestations.     

Central Role of Salicylic Acid in Resistance of Wheat Against <Emphasis Type="Italic">Fusarium graminearum</Emphasis>

Head blight caused by Fusarium graminearum (F. graminearum) is one of the major threats to wheat and barley around the world. The importance of this disease is due to a reduction in both grain yield and quality in infected plants. Currently, there is limited knowledge about the physiological mechanisms involved in plant resistance against this pathogen. To reveal the physiological mechanisms underlying the resistance to F. graminearum, spikes of resistant (Sumai3) and susceptible (Falat) wheat cultivars were analyzed 4 days after inoculation, as the first symptoms of pathogen infection appeared. F. graminearum inoculation resulted in a greater induction level and activity of salicylic acid (SA), callose, phenolic compounds, peroxidase, phenylalanine ammonia lyase (PAL), and polyphenol oxidase in resistant versus susceptible cultivars. Soil drench application to spikes of SA, 24 h before inoculation with F. graminearum alleviated Fusarium head blight symptoms in both resistant and susceptible cultivars. SA treated plants showed a significant increment in hydrogen peroxide (H₂O₂) production, lipid peroxidation, SA, and callose content. SA-induced H₂O₂ level seems to be related to increased superoxide dismutase and decreased catalase activities. In addition, real-time quantitative PCR analysis showed that SA pretreatment induced expression of PAL genes in both infected and non-infected head tissues of the susceptible and resistant cultivars. Our data showed that soil drench application of SA activates antioxidant defense responses and may subsequently induce systemic acquired resistance, which may contribute to the resistance against F. graminearum. These results provide novel insights about the physiological and molecular role of SA in plant resistance against hemi-biotrophic pathogen infection.     

HYR1-mediated detoxification of reactive oxygen species is required for full virulence in the rice blast fungus

During plant-pathogen interactions, the plant may mount several types of defense responses to either block the pathogen completely or ameliorate the amount of disease. Such responses include release of reactive oxygen species (ROS) to attack the pathogen, as well as formation of cell wall appositions (CWAs) to physically block pathogen penetration. A successful pathogen will likely have its own ROS detoxification mechanisms to cope with this inhospitable environment. Here, we report one such candidate mechanism in the rice blast fungus, Magnaporthe oryzae, governed by a gene we refer to as MoHYR1. This gene (MGG_07460) encodes a glutathione peroxidase (GSHPx) domain, and its homologue in yeast was reported to specifically detoxify phospholipid peroxides. To characterize this gene in M. oryzae, we generated a deletion mutantΔhyr1 which showed growth inhibition with increased amounts of hydrogen peroxide (H₂O₂). Moreover, we observed that the fungal mutants had a decreased ability to tolerate ROS generated by a susceptible plant, including ROS found associated with CWAs. Ultimately, this resulted in significantly smaller lesion sizes on both barley and rice. In order to determine how this gene interacts with other (ROS) scavenging-related genes in M. oryzae, we compared expression levels of ten genes in mutant versus wild type with and without H₂O₂. Our results indicated that the HYR1 gene was important for allowing the fungus to tolerate H₂O₂ in vitro and in planta and that this ability was directly related to fungal virulence.     

Role of salicylic acid in induction of plant defense system in chickpea (Cicer arietinum L.)

Salicylic acid (SA), a plant hormone plays an important role in induction of plant defense against a variety of biotic and abiotic stresses through morphological, physiological and biochemical mechanisms. A series of experiments were carried out to evaluate the biochemical response of the chickpea (Cicer arietinum L.) plants to a range of SA concentrations (1, 1.5, and 2 mM). Water treated plants were maintained as control. Activities of peroxidase (POD) and polyphenol oxidase (PPO) were evaluated and amounts of total phenols, hydrogen peroxide (H₂O₂), and proteins were calculated after 96 h of treatment. Plants responded very quickly to SA at 1.5 mM and showed higher induction of POD and PPO activities, besides the higher accumulation of phenols, H₂O₂ and proteins. Plants treated with SA at 2 mM showed phytotoxic symptoms. These results suggest that SA at 1.5 mM is safe to these plants and could be utilized for the induction of plant defense.