A role for Ethylene-Insensitive3 in the regulation of hydrogen peroxide production during seed germination under high salinity in Arabidopsis

The Arabidopsis Ethylene-Insensitive3 (EIN3) has received attention recently and has been shown to be involved in the regulation of multitude of responses ranging from biotic stress defense and development to hormone interaction. To better understand the roles of EIN3 in plants response to salinity stress during germination and post-germination development, seeds of two EIN3 deficient mutant and a EIN3 overexpression mutant of Arabidopsis were analyzed under salinity and compared with Col-0 as control. The results showed that the ein3-1eil1-1 double mutant (lacking EIN3 and EIN3-Like1) and ein3-1 (lacking EIN3) were hypersensitive to high salinity (>150 mM NaCl), while EIN3 overexpression mutant (EIN3ox) displayed enhanced tolerance, indicating that EIN3 plays important roles during seed germination under salinity. In addition, we also found that the two EIN3 deficient mutant seedlings accumulate high levels of hydrogen peroxide (H₂O₂), which was thought to be an inhibitor of germination under salinity before, suggesting that EIN3 may function as a negative regulator of reactive oxygen species metabolism in germinating seeds under salinity. Taken together, our studies provide insights that EIN3 promotes seed germination under salinity, at least in part, through modulating concentration of H₂O₂ in germinating seeds.     

Enhanced oxidative stress in the ethylene-insensitive (ein3-1) mutant of Arabidopsis thaliana exposed to salt stress

To better understand the role of ethylene signaling in plant stress tolerance, salt-induced changes in gene expression levels of ethylene biosynthesis, perception and signaling genes were measured in Arabidopsis thaliana plants exposed to 15 days of salinity. Among the genes analyzed, EIN3 showed the highest expression level increase under salt stress, suggesting a key role for this ethylene-signaling component in response to salt stress. Therefore, we analyzed the salt stress response over 15 days (by adding 100 mM NaCl to the nutrient solution) in the ein3-1 mutant compared to the wild-type (Col-0) in terms of growth, oxidative stress markers (lipid peroxidation, foliar pigments and low-molecular-weight antioxidants) and levels of growth- and stress-related phytohormones (including cytokinins, auxins, gibberellins, abscisic acid, jasmonic acid and salicylic acid). The ein3-1 mutant grew similarly to wild-type plants both under control and salt stress conditions, which was associated with a differential time course evolution in the levels of the cytokinins zeatin and zeatin riboside, and the auxin indole-3-acetic acid between the ein3-1 mutant and the wild-type. Despite showing no signs of physiological deterioration under salt stress (in terms of rosette biomass, leaf water and pigment contents, and PSII efficiency) the ein3-1 mutant showed enhanced lipid peroxidation under salt stress, as indicated by 2.4-fold increase in both malondialdehyde and jasmonic acid contents compared to the wild-type. We conclude that, at moderate doses of salinity, partial insensitivity to ethylene might be compensated by changes in endogenous levels of other phytohormones and lipid peroxidation-derived signals in the ein3-1 mutant exposed to salt stress, but at the same time, this mutant shows higher oxidative stress under salinity than the wild-type.     

Ethylene antagonizes the inhibition of germination in Arabidopsis induced by salinity by modulating the concentration of hydrogen peroxide

The capacity of plants to achieve successful germination and early seedling establishment under high salinity is crucial for tolerance of plants to salt. The gaseous hormone ethylene has been implicated in modulating salt tolerance, but the detailed role of how ethylene modulates the response of early seedling establishment to salt is unclear. To better understand the role of the ethylene signal transduction pathway during germination and seedling establishment, an ethylene insensitive mutation (ein2-5) and an ethylene sensitive mutation (ctr1-1) of Arabidopsis were analyzed under saline conditions and compared with the wild type plant (Col-0) as control. High salinity (>100?mM NaCl) inhibited and delayed germination. These effects were more severe in the ethylene insensitive mutants (ein2-5) and less severe in the constitutive ethylene sensitive plants (ctr1-1) compared with Col-0 plants. Addition of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) or inhibitors of ethylene action implied that ethylene was essential for early seedling establishment under normal and saline conditions. Salt stress increased the endogenous concentration of hydrogen peroxide (H₂O₂) in germinating seeds and ACC reduced its concentration. Our results suggest that ethylene promotes germination under salinity by modulating the endogenous concentration of H₂O₂ in germinating seeds. These findings demonstrate that ethylene is involved in regulating germination as an initiator of the process rather than consequence, and that ethylene promotes germination by modulating the endogenous concentration of H₂O₂ in germinating seeds under salinity.     

Ethylene improves Arabidopsis salt tolerance mainly via retaining K+ in shoots and roots rather than decreasing tissue Na+ content

Ethylene has been reported to play an essential role in the response of Arabidopsis to salinity and K⁺ deficiency. It was proposed that plant's ability to maintain potassium (K⁺) and minimize sodium (Na⁺) in tissues of salinity plants is critical for salt tolerance (ST). It is still unclear how ethylene modulates plant ion homeostasis under saline occasions. We employed Arabidopsis wild type (Col-0), ethylene insensitive mutants (ein2-5 and ein3-1) and constitutive triple response mutant (ctr1-1) plants to compare their phenotypic and physiological responses to salinity. Ethephon applied to plants could convert quickly to ethylene and here was applied exogenously to Col-0 seedlings to validate ethylene role in salt response. We showed that ethylene insensitivity in ein2-5 or ein3-1 plants increased Arabidopsis salt sensitivity than in Col-0. However, the salinity-induced adverse effects on Chlorophyll a/b, photosystem II function (Fv/Fm) and redox state were largely amended in the ctr1-1 than in Col-0 plants with the severe salinity. The compatible solute sucrose and antioxidant system were also up-regulated to improve ST in ctr1-1 plants. The ethephon obviously alleviated the salinity-induced restriction in root length. The subsequent analysis on the Na⁺ and K⁺ homeostasis found that ethylene could help plant retain higher shoot or root K⁺ nutrition in the short- or long-term salt-stressed plants. However, the ethylene did not significantly alter sodium buildup and water relation in the salt-stressed plants. Our observations confirmed the key role of ethylene in improved plant ST and highlighted the ethylene ability to retain K⁺, rather than decreasing Na⁺, in shoots and roots to improve Arabidopsis ST.     

Arbuscular mycorrhizal symbiosis modulates antioxidant response in salt-stressed Trigonella foenum-graecum plants

An experiment was conducted to evaluate the influence of Glomus intraradices colonization on the activity of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), peroxidase (PX), ascorbate peroxidase (APX), and glutathione reductase (GR)] and the accumulation of nonenzymatic antioxidants (ascorbic acid, α-tocopherol, glutathione, and carotenoids) in roots and leaves of fenugreek plants subjected to varying degrees of salinity (0, 50, 100, and 200 mM NaCl) at two time intervals (1 and 14 days after saline treatment, DAT). The antioxidative capacity was correlated with oxidative damage in the same tissue. Under salt stress, lipid peroxidation and H₂O₂ concentration increased with increasing severity and duration of salt stress (DoS). However, the extent of oxidative damage in mycorrhizal plants was less compared to nonmycorrhizal plants. The study reveals that mycorrhiza-mediated attenuation of oxidative stress in fenugreek plants is due to enhanced activity of antioxidant enzymes and higher concentrations of antioxidant molecules. However, the significant effect of G. intraradices colonization on individual antioxidant molecules and enzymes varied with plant tissue, salinity level, and DoS. The significant effect of G. intraradices colonization on antioxidative enzymes was more evident at 1DAT in both leaves and roots, while the concentrations of antioxidant molecules were significantly influenced at 14DAT. It is proposed that AM symbiosis can improve antioxidative defense systems of plants through higher SOD activity in M plants, facilitating rapid dismutation of O₂ ^- to H₂O₂, and subsequent prevention of H₂O₂ build-up by higher activities of CAT, APX, and PX. The potential of G. intraradices to ameliorate oxidative stress generated in fenugreek plants by salinity was more evident at higher intensities of salt stress.     

The Arabidopsis Ethylene-Insensitive 2 gene is required for lead resistance

The Arabidopsis Ethylene-Insensitive 2 (EIN2) gene has been shown to be involved in the regulation of abiotic and biotic stresses, including ozone stress, high salt, oxidative stress and disease resistance. However, little is known about the role of EIN2 gene in lead (Pb) resistance in Arabidopsis. In this study, we showed that EIN2 gene is required for Pb(II) resistance in Arabidopsis. EIN2 gene was induced by Pb(II) treatment, and the ein2-1 mutant showed enhanced sensitivity to Pb(II). A higher Pb content was detected in ein2-1 plants than in wild-type plants when subjected to Pb(II) treatment, which was associated, at least in part, with reduction in expression of AtPDR12 gene, a pump excluding Pb(II) and/or Pb(II)-containing toxic compounds from the cytoplasm. Moreover, the ein2-1 mutation also impaired glutathione (GSH)-dependent Pb(II) resistance, which was related to constitutive reduction of express of GSH1 gene involved in GSH synthesis and consequently reduced GSH content. Taken together, all these results suggest that EIN2 gene mediates Pb(II) resistance, at least in part, through two distinct mechanisms, a GSH-dependent mechanism and a GSH-independent AtPDR12-mediated mechanism.     

Arabidopsis nudix hydrolase 7 plays a role in seed germination

Arabidopsis nudix hydrolase 7 (Atnudt7) mutants exhibit reduced seed germination phenotype following after-ripening. The role of AtNUDT7 in seeds and during early stages of imbibition was examined. Seeds of Atnudt7-1 and Col-0 following 3 days of imbibition were used to profile changes in NADH- and ADP-ribose pyrophosphohydrolase enzyme activities, expression of nudix family genes closely related to AtNudt7, and AtNUDT7 protein levels. Changes in pyridine nucleotides, phytohormones, reactive oxygen species and poly(ADP-ribose) levels in after-ripened seeds and 1 day after imbibition were also analyzed. Changes in AtNUDT7 gene expression, protein levels and enzyme activities in WT seeds and during early stages of imbibition were correlated. Atnudt7-1 seeds lacked NADH pyrophosphohydrolase activity that led to very high catabolic redox charge. Abscisic acid (ABA) levels were higher in Atnudt7-1 mutant while salicylic acid, gibberellic acid, and reactive oxygen species (ROS) levels were higher in WT seeds. In Atnudt7-1, there was excess ROS accumulation 1 day after imbibition. PAR levels were significantly higher in Atnudt7-1 mutant when compared to WT during imbibition. Based on these observations, we conclude NADH pyrophosphohydrolase activity conferred by AtNUDT7 is important for NAD:NADH homeostasis in seeds. Perturbations to this key redox couple alter ABA and ROS levels in the seeds that in turn lowers germination.     

Salinity-induced changes in two cultivars of Vigna radiata: responses of antioxidative and proline metabolism

The influence of increasing salinity stress on plant growth, antioxidant enzymes and proline metabolism in two cultivars of Vigna radiata L. (cv. Pusa Bold and cv. CO 4) was investigated. Salt stress was imposed on 30-days-old cultivars with four different concentrations of NaCl (0, 100, 200 and 300 mM). The roots and shoots of CO 4 showed greater reduction in fresh weight, dry weight and water content when compared to Pusa Bold with increasing salt stress. Under salinity stress, the roots and shoots of CO 4 exhibited higher Na⁺: K⁺ ratio than Pusa Bold. The activities of reactive oxygen species (ROS) scavenging enzymes and reduced glutathione (GSH) concentration were found to be higher in the leaves of Pusa Bold than in CO 4, whereas oxidized glutathione (GSSG) concentration was found to be higher in the leaves of CO 4 compared to those in Pusa Bold. Our studies on oxidative damage in two Vigna cultivars showed lower levels of lipid peroxidation and H₂O₂ concentration in Pusa Bold than in CO 4 under salt stress conditions. High accumulation of proline and glycine betaine under salt stress was also observed in Pusa Bold when compared to CO 4. The activities of proline biosynthetic enzymes were significantly high in Pusa Bold. However, under salinity stress, Pusa Bold showed a greater decline in proline dehydrogenase (ProDH) activity compared to CO 4. Our data in this investigation demonstrate that oxidative stress plays a major role in salt-stressed Vigna cultivars and Pusa Bold has efficient antioxidative characteristics which could provide better protection against oxidative damage in leaves under salt-stressed conditions.     

Alfalfa nodules elicited by a flavodoxin-overexpressing Ensifer meliloti strain display nitrogen-fixing activity with enhanced tolerance to salinity stress

Nitrogen fixation by legumes is very sensitive to salinity stress, which can severely reduce the productivity of legume crops and their soil-enriching capacity. Salinity is known to cause oxidative stress in the nodule by generating reactive oxygen species (ROS). Flavodoxins are involved in the response to oxidative stress in bacteria and cyanobacteria. Prevention of ROS production by flavodoxin overexpression in bacteroids might lead to a protective effect on nodule functioning under salinity stress. Tolerance to salinity stress was evaluated in alfalfa nodules elicited by an Ensifer meliloti strain that overexpressed a cyanobacterial flavodoxin compared with nodules produced by the wild-type bacteria. Nitrogen fixation, antioxidant and carbon metabolism enzyme activities were determined. The decline in nitrogenase activity associated to salinity stress was significantly less in flavodoxin-expressing than in wild-type nodules. We detected small but significant changes in nodule antioxidant metabolism involving the ascorbate–glutathione cycle enzymes and metabolites, as well as differences in activity of the carbon metabolism enzyme sucrose synthase, and an atypical starch accumulation pattern in flavodoxin-containing nodules. Salt-induced structural and ultrastructural alterations were examined in detail in alfalfa wild-type nodules by light and electron microscopy and compared to flavodoxin-containing nodules. Flavodoxin reduced salt-induced structural damage, which primarily affected young infected tissues and not fully differentiated bacteroids. The results indicate that overexpression of flavodoxin in bacteroids has a protective effect on the function and structure of alfalfa nodules subjected to salinity stress conditions. Putative protection mechanisms are discussed.     

Defence Signalling Pathways Involved in Plant Resistance and Phosphite-Mediated Control of Phytophthora Cinnamomi

Phytophthora cinnamomi is one of the most devastating plant pathogens worldwide. Current control of P. cinnamomi in natural ecosystems primarily relies on chemical phosphite (Phi). To investigate host- and Phi-mediated resistance, A. thaliana ecotypes and mutants defective in salicylic acid (SA), jasmonic acid (JA), ethylene (ET) and abscisic acid (ABA) signalling pathways were screened for susceptibility to P. cinnamomi. In contrast to Col-0, the aba2-4 mutant, deficient in the synthesis of ABA, was susceptible suggesting a role for ABA in resistance to P. cinnamomi. Phi treatment increased resistance in aba2-4, but not to the level of Col-0, suggesting that Phi may act through both ABA-dependent and ABA-independent pathways. Phi treatment or P. cinnamomi inoculation of Col-0 down-regulated AtMYC2, a positive regulator of ABA signalling, which negatively regulates JA/ET-related pathogenesis-related genes, such as PDF1.2, whilst positively regulating JA-mediated herbivore responses such as VSP and PI. Consistent with this, P. cinnamomi or Phi treatment caused up-regulation of PDF1.2 and THI2.1 and down-regulation of VSP2 and the ABA-responsive gene RD22. Despite the up-regulation of JA/ET-dependent defence genes, the JA-defective mutant, jar1-1 and ET-defective mutants, ein2-1 and etr1-3, showed wild-type levels of resistance to P. cinnamomi, suggesting that these JA/ET defences are not required for resistance to P. cinnamomi. These results suggest that the resistance response of Col-0 act, at least in part, through a mechanism dependent on ABA synthesis, which appears independent of the interaction between ABA and elements of the JA/ET pathway, whilst Phi-mediated resistance, although inducing a response resembling the resistance response of Col-0, is independent of ABA signalling.     

Arabidopsis thaliana Metallothionein, AtMT2a, Mediates ROS Balance during Oxidative Stress

Cold stress has been shown to induce the production of reactive oxygen species (ROS), which can elicit a potentially damaging oxidative burden on cellular metabolism. Here, the expression of a metallothionein gene (AtMT2a) was upregulated under low temperature and hydrogen peroxide (H₂O₂) stresses. The Arabidopsis T-DNA insertion mutant, mt2a, exhibited more sensitivity to cold stress compared to WT plants during the seed germination, and H₂O₂ levels in mt2a mutant were higher than that in WT plants during the cold stress. Synthetic GFP fused to AtMT2a was observed to be localized in cytosol. These results indicated that AtMT2a functions in tolerance against cold stress by mediating the ROS balance in the cytosol. Interestingly, mRNA level of AtMT2a was increased in seedlings of Arabidopsis cat2 mutant after cold treatment compared to WT seedlings, and overexpression of AtMT2a in cat2 could improve CAT activity under chilling stress. Moreover, the enzymatic activity of CAT in mt2a was higher than that in WT plants after cold treatment, suggesting that AtMT2a and CAT might complement each other in antioxidative process potentially in Arabidopsis. Taken together, our results provided a novel insight into the relationship between MTs and antioxidative enzymes in the ROS-scavenging system in plants.