Ethylene and nitric oxide are involved in maintaining ion homeostasis in <Emphasis Type="Italic">Arabidopsis</Emphasis> callus under salt stress

In the present study, the role of ethylene in nitric oxide (NO)-mediated protection by modulating ion homeostasis in Arabidopsis callus under salt stress was investigated. Results showed that the ethylene-insensitive mutant etr1-3 was more sensitive to salt stress than the wild type (WT). Under 100 mM NaCl, etr1-3 callus displayed a greater electrolyte leakage and Na⁺/K⁺ ratio but a lower plasma membrane (PM) H⁺-ATPase activity compared to WT callus. Application of exogenous 1-aminocyclopropane-1-carboxylic acid (ACC, an ethylene precursor) or sodium nitroprusside (SNP, a NO donor) alleviated NaCl-induced injury by maintaining a lower Na⁺/K⁺ ratio and an increased PM H⁺-ATPase activity in WT callus but not in etr1-3 callus. The SNP actions in NaCl stress were attenuated by a specific NO scavenger or an ethylene biosynthesis inhibitor in WT callus. Under 100 mM NaCl, the NO accumulation and ethylene emission appeared at early time, and NO production greatly stimulated ethylene emission in WT callus. In addition, ethylene induced the expression of PM H⁺-ATPase genes under salt stress. The recovery experiment showed that NaCl-induced injury was reversible, as signaled by the similar recovery of Na⁺/K⁺ ratio and PM H⁺-ATPase activity in WT callus. Taken together, the results indicate that ethylene and NO cooperate in stimulating PM H⁺-ATPase activity to modulate ion homeostasis for salt tolerance, and ethylene may be a part of the downstream signal molecular in NO action.     

Heterotrimeric G protein mediates ethylene‐induced stomatal closure via hydrogen peroxide synthesis in Arabidopsis

Heterotrimeric G proteins function as key players in hydrogen peroxide (H₂O₂) production in plant cells, but whether G proteins mediate ethylene‐induced H₂O₂ production and stomatal closure are not clear. Here, evidences are provided to show the Gα subunit GPA1 as a missing link between ethylene and H₂O₂ in guard cell ethylene signalling. In wild‐type leaves, ethylene‐triggered H₂O₂ synthesis and stomatal closure were dependent on activation of Gα. GPA1 mutants showed the defect of ethylene‐induced H₂O₂ production and stomatal closure, whereas wGα and cGα overexpression lines showed faster stomatal closure and H₂O₂ production in response to ethylene. Ethylene‐triggered H₂O₂ generation and stomatal closure were impaired in RAN1, ETR1, ERS1 and EIN4 mutants but not impaired in ETR2 and ERS2 mutants. Gα activator and H₂O₂ rescued the defect of RAN1 and EIN4 mutants or etr1‐3 in ethylene‐induced H₂O₂ production and stomatal closure, but only rescued the defect of ERS1 mutants or etr1‐1 and etr1‐9 in ethylene‐induced H₂O₂ production. Stomata of CTR1 mutants showed constitutive H₂O₂ production and stomatal closure, but which could be abolished by Gα inhibitor. Stomata of EIN2, EIN3 and ARR2 mutants did not close in responses to ethylene, Gα activator or H₂O₂, but do generate H₂O₂ following challenge of ethylene or Gα activator. The data indicate that Gα mediates ethylene‐induced stomatal closure via H₂O₂ production, and acts downstream of RAN1, ETR1, ERS1, EIN4 and CTR1 and upstream of EIN2, EIN3 and ARR2. The data also show that ETR1 and ERS1 mediate both ethylene and H₂O₂ signalling in guard cells.     

Effect of octanoic acid on ethylene-mediated processes in Arabidopsis

We have examined whether octanoic acid (OA) one of the short chain saturated fatty acids (SCSFA), increases ethylene response in the following three ethylene-mediated processes: a) hypocotyl growth in darkness; b) formation of new flowers; c) flower abscission. These processes were examined in the presence or absence of exogenous ethylene in Arabidopsis wild type (WT) and in the ethylene-insensitive mutants, etr1-3 and ein2-1 and in the ethylene over-producer mutant eto1-1. Our results show that OA decreased hypocotyl length of WT in the absence or presence of exogenous ethylene, apparently showing that OA acts via augmentation of ethylene action. However, the hypocotyl growth inhibition could not be ascribed to increased ethylene sensitivity since application of inhibitors of ethylene synthesis (aminoethoxyvinylglycine; AVG) or action (1-methylcyclopropene;1-MCP) to WT seedlings did not prevent specifically the OA-induced growth inhibition. Also, OA inhibited hypocotyl growth in the mutants etr1-3 and ein2-1 in a similar pattern to that obtained in WT. On the other hand, OA had no effect on flower formation neither in WT, etr1-3 and eto1-1, in which ethylene reduced flower formation, nor in the ein2-1 mutant, in which ethylene had no effect. OA also did not increase flower abscission in WT or in the mutants etr1-3 and ein2-1 neither in the absence nor in the presence of ethylene. However, OA has augmented flower abscission in the mutant eto1-1 only in the absence of exogenous ethylene. This result might indicate that the effect of OA on eto1-1 is specific to this mutant and is not due to general deleterious effects inflicted by OA. Taken together, our results show that in general OA does not augment ethylene response in Arabidopsis, but it might affect ethylene action in flower abscission of the ethylene-overproducer mutant.     

Ethylene mediates brassinosteroid‐induced stomatal closure via Gα protein‐activated hydrogen peroxide and nitric oxide production in Arabidopsis

Brassinosteroids (BRs) are essential for plant growth and development; however, whether and how they promote stomatal closure is not fully clear. In this study, we report that 24‐epibrassinolide (EBR), a bioactive BR, induces stomatal closure in Arabidopsis (Arabidopsis thaliana) by triggering a signal transduction pathway including ethylene synthesis, the activation of Gα protein, and hydrogen peroxide (H₂O₂) and nitric oxide (NO) production. EBR initiated a marked rise in ethylene, H₂O₂ and NO levels, necessary for stomatal closure in the wild type. These effects were abolished in mutant bri1‐301, and EBR failed to close the stomata of gpa1 mutants. Next, we found that both ethylene and Gα mediate the inductive effects of EBR on H₂O₂ and NO production. EBR‐triggered H₂O₂ and NO accumulation were canceled in the etr1 and gpa1 mutants, but were strengthened in the eto1‐1 mutant and the cGα line (constitutively overexpressing the G protein α‐subunit AtGPA1). Exogenously applied H₂O₂ or sodium nitroprusside (SNP) rescued the defects of etr1‐3 and gpa1 or etr1 and gpa1 mutants in EBR‐induced stomatal closure, whereas the stomata of eto1‐1/AtrbohF and cGα/AtrbohF or eto1‐1/nia1‐2 and cGα/nia1‐2 constructs had an analogous response to H₂O₂ or SNP as those of AtrbohF or Nia1‐2 mutants. Moreover, we provided evidence that Gα plays an important role in the responses of guard cells to ethylene. Gα activator CTX largely restored the lesion of the etr1‐3 mutant, but ethylene precursor ACC failed to rescue the defects of gpa1 mutants in EBR‐induced stomatal closure. Lastly, we demonstrated that Gα‐activated H₂O₂ production is required for NO synthesis. EBR failed to induce NO synthesis in mutant AtrbohF, but it led to H₂O₂ production in mutant Nia1‐2. Exogenously applied SNP rescued the defect of AtrbohF in EBR‐induced stomatal closure, but H₂O₂ did not reverse the lesion of EBR‐induced stomatal closure in Nia1‐2. Together, our results strongly suggest a signaling pathway in which EBR induces ethylene synthesis, thereby activating Gα, and then promotes AtrbohF‐dependent H₂O₂ production and subsequent Nia1‐catalyzed NO accumulation, and finally closes stomata.     

Root Growth of Arabidopsis thaliana Is Regulated by Ethylene and Abscisic Acid Signaling Interaction in Response to HrpNEa, a Bacterial Protein of Harpin Group

HrpN_Ea is a harpin protein from Erwinia amylovora, a bacterial pathogen that causes fire blight in rosaceous plants. Treating plants with HrpN_Ea stimulates ethylene and abscisic acid (ABA) to induce plant growth and drought tolerance, respectively. Herein, we report that both growth hormones cooperate to mediate the role of HrpN_Ea in promoting root growth of Arabidopsis thaliana seedlings. Root growth is promoted coordinately with elevation in levels of ABA and ethylene subsequent to soaking of germinating seeds of wild-type (WT) Arabidopsis in a solution of HrpN_Ea. However, these responses are arrested by inhibiting WT roots from synthesizing ethylene as well as sensing of ABA and ethylene. The effects of HrpN_Ea on roots are also nullified in ethylene-insensitive etr1-1 and ein5-1 mutants and in the ABA-insensitive mutant abi2-1 of Arabidopsis. These results provide evidence for presence of a relationship between root growth enhancement and signaling by ABA and ethylene in response to HrpN_Ea. Nevertheless, when HrpN_Ea is applied to leaves, ethylene signaling is active in the absence of ABA signaling to promote plant growth. This suggests the presence of a different signaling mechanism in leaves from that in roots. X. Ren and F. Liu contributed equally to this study and are regarded as joint first authors     

Temperature Effects on the Activity of the Alternative Respiratory Pathway in Chill-Sensitive Cucumis sativus

After 48 hours at 2°C, hypocotyls from chill-sensitive Cucumis sativus seedlings showed a burst of O₂ uptake. The alternative pathway became engaged to close to 45% full capacity during this postchilling respiratory burst. However, it only accounted for up to 50% of this increased respiratory O₂ uptake. By 24 hours after chilling, when the seedlings were fully recovered from visible symptoms of chilling injury, the flux through the alternative pathway was back to the level (about 10%) found before chilling. Blocking chilling-induced ethylene production with aminoethoxyvinylglycine had no effect on this increased utilization of the alternative pathway.     

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 advances the transition from vegetative growth to flowering in Arabidopsis thaliana

The transition from vegetative growth to flowering is the most drastic change in plant development. In order to examine the involvement of ethylene in growth transition, we compared the development of ethylene-related mutants, eto1, etr1, ein2-1 and ein3-1, with the wild type (WT) in Arabidopsis thaliana. The ethylene sensitivity of two WT and the mutants is decreased in the following order: eto1 = WT < ein3-1 < ein2-1 = etr1-1. Bolting time was also delayed in nearly the same order: eto1 < WT < ein3-1 < ein2-1 < etr1. Leaf numbers increased according to the delay of bolting time, indicating that the delay of bolting time was caused by the delay of transition from vegetative to reproductive growth. Other growth parameters, including leaf area and number of flowers opening at the same time, increased in the same order, indicating that these changes were caused by a single factor, the amount of ethylene signal which was transferred though an ethylene signal transduction pathway. These results suggest that ethylene is involved in the transition from vegetative to reproductive growth in Arabidopsis thaliana.     

Interactions between ethylene,abscisic acid and cytokinin during germination and seedling establishment in <Emphasis Type="Italic">Arabidopsis</Emphasis>

In order to investigate the interaction of the plant hormones ethylene, abscisic acid (ABA) and cytokinin in seed germination and early seedling development, we studied germination in ethylene-related mutants of Arabidopsis. Mutations in the genes etr1 and ein2, which reduce ethylene responses, showed increased dormancy and a delay in germination in comparison with wild type. Mutations in etr1, ein2 and ein6 also resulted in increased sensitivity to ABA with respect to inhibition of germination. Conversely, mutations in ctr1 and eto3, which lead to an increased ethylene response and overproduction of ethylene, respectively, decreased sensitivity to ABA during germination. Increased ABA sensitivity was also effected in wild type seeds by the presence during germination of AgNO₃, an inhibitor of ethylene action. The addition of the cytokinin N-6 benzyl adenine (BA) reversed the increased sensitivity of ethylene-resistant mutants to ABA. The action of cytokinin in reversing increased ABA sensitivity of ethylene-resistant mutants also suggests that at least part of the action of cytokinin in promoting germination is independent of its role in stimulating ethylene production. These observations further extend the evidence in support of interaction between ethylene, ABA and cytokinin signalling in controlling seed germination and early seedling development in Arabidopsis.     

Enhanced chilling tolerance at the booting stage in rice by transgenic overexpression of the ascorbate peroxidase gene, OsAPXa

Low temperatures during the booting stage reduce rice yields by causing cold-induced male sterility. To determine whether antioxidant capacity affects the ability of rice plants to withstand chilling at the booting stage, we produced transgenic rice plants that overexpress OsAPXa and have increased APX activity. The effect of increased APX activity on the levels of H₂O₂ and lipid peroxidation were determined by measuring H₂O₂ levels and malondialdehyde (MDA) contents in spikelets during cold treatments at the booting stage. The levels of H₂O₂ and the MDA content increased by 1.5-fold and twofold, respectively in WT plants subjected to a 12°C treatment for 6 days. In contrast, transgenic lines showed small changes in H₂O₂ levels and MDA content under cold stress, and H₂O₂ levels and MDA content were significantly lower than in WT plants. APX activity showed negative correlations with levels of H₂O₂ and MDA content, which increased during cold treatment. Cold tolerance at the booting stage in transgenic lines and WT plants was evaluated. Spikelet fertility was significantly higher in transgenic lines than in WT plants after a 12°C treatment for 6 days. These results indicate that higher APX activity enhances H₂O₂-scavenging capacity and protects spikelets from lipid peroxidation, thereby increasing spikelet fertility under cold stress.     

The unsaturation of phosphatidylglycerol in thylakoid membrane alleviates PSII photoinhibition under chilling stress

Over-expression of chloroplast glycerol-3-phosphate acyltransferase gene (LeGPAT) in tomato increased cis-unsaturated fatty acid content in phosphatidylglycerol (PG) of the thylakoid membrane. Under chilling stress, the oxygen evolving activity, the maximal photochemical efficiency of PSII (F _v/F _m), and superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities decreased less in sense lines than in antisense lines compared to wild-type (WT) plants. Consistently, the relative electric conductivity, \textO₂ ^{. -} {\text{O}}_{2} ^{{. - }} and H₂O₂ contents in sense lines were lower than those of WT and antisense lines. The antisense lines with low level of unsaturated fatty acids in PG were extremely susceptible to photoinhibition of PSII and had a significant reduction in the D1 protein content of PSII reaction center under chilling stress. However, in the presence of streptomycin (SM), the degradation of D1 protein was faster in sense lines than in WT and antisense plants. These results suggested that, under chilling stress conditions, increasing cis-unsaturated fatty acids in PG through over-expression of LeGPAT can alleviate PSII photoinhibition by accelerating the repair of D1 protein and improving the activities of antioxidant enzymes in chloroplasts.     

The influence of ethylene perception on sex expression in melon (<Emphasis Type="Italic">Cucumis melo </Emphasis>L.) as assessed by expression of the mutant ethylene receptor, <Emphasis Type="Italic">At-etr1-1</Emphasis>, under the control of constitutive and floral targeted promoters

Sexual diversity expressed by Curcurbitaceae species is a primary example of developmental plasticity in plants. Ethylene, which promotes femaleness (carpel development), plays a key role in sex determination. We sought to determine the critical location for ethylene perception in developing floral primodia. The dominant negative Arabidopsis ethylene response mutant gene, etr1-1, was introduced into melon (Cucumis melo L.) plants under control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter, or floral-targeted Apetela3 (AP3) and Crab’s Claw (CRC) promoters, which in Arabidopsis, promote expression in petal and stamen, and carpel and nectary primordia, respectively. Based on effects of exogenous ethylene, it was predicted that inhibition of ethylene perception by carpel primordia would inhibit carpel development. Constitutive expression of etr1-1 caused several phenotypes associated with ethylene insensitivity, verifying that etr1-1 inhibits ethylene perception in the heterologous melon system. Carpel-bearing bud production was essentially abolished in 35S::etr1-1 melons, providing direct demonstration of the requirement for ethylene perception for carpel development. CRC::etr1-1 plants, however, showed enhanced femaleness as manifested by earlier and increased number of carpel-bearing buds, and production of female (rather than bisexual) buds. Despite increased carpel-bearing bud formation, a greater proportion of the CRC::etr1-1 carpel-bearing buds aborted before anthesis. AP3::etr1-1 plants showed increased maleness by nearly exclusive staminate flower production, and poorly developed carpels in the rare bisexual flowers. These results indicate that ethylene perception by the stamen (or petal) primordia plays a critical role in promoting carpel development at the time of sex determination, while ethylene perception by the carpel is important for maturation of carpel-bearing flowers to anthesis.     

Genetic and transformation studies reveal negative regulation of ERS1 ethylene receptor signaling in Arabidopsis

Background  

Ethylene receptor single mutants of Arabidopsis do not display a visibly prominent phenotype, but mutants defective in multiple ethylene receptors exhibit a constitutive ethylene response phenotype. It is inferred that ethylene responses in Arabidopsis are negatively regulated by five functionally redundant ethylene receptors. However, genetic redundancy limits further study of individual receptors and possible receptor interactions. Here, we examined the ethylene response phenotype in two quadruple receptor knockout mutants, (ETR1) ers1 etr2 ein4 ers2 and (ERS1) etr1 etr2 ein4 ers2, to unravel the functions of ETR1 and ERS1. Their functions were also reciprocally inferred from phenotypes of mutants lacking ETR1 or ERS1. Receptor protein levels are correlated with receptor gene expression. Expression levels of the remaining wild-type receptor genes were examined to estimate the receptor amount in each receptor mutant, and to evaluate if effects of ers1 mutations on the ethylene response phenotype were due to receptor functional compensation. As ers1 and ers2 are in the Wassilewskija (Ws) ecotype and etr1, etr2, and ein4 are in the Columbia (Col-0) ecotype, possible effects of ecotype mixture on ethylene responses were also investigated.     

Exogenous H<Subscript>2</Subscript>O<Subscript>2</Subscript> improves the chilling tolerance of manilagrass and mascarenegrass by activating the antioxidative system

The effect of foliar pretreatment by hydrogen peroxide (H₂O₂) at low concentrations of 0, 5, 10, and 15 mM on the chilling tolerance of two Zoysia cultivars, manilagrass (Zoysia matrella) and mascarenegrass (Zoysia tenuifolia), was studied. The optimal concentration for H₂O₂ pretreatment was 10 mM, as demonstrated by the lowest malondialdehyde (MDA) content and electrolyte leakage (EL) levels and higher protein content under chilling stress (7°C/2°C, day/night). Prior to initiation of chilling, exogenous 10 mM H₂O₂ significantly increased catalase (CAT), ascorbate peroxidase (APX), glutathione-dependent peroxidases (GPX), and glutathione-S-transferase (GST) activities in manilagrass, and guaiacol peroxidase (POD), APX, and glutathione reductase (GR) activities in mascarenegrass, suggesting that H₂O₂ may act as a signaling molecule, inducing protective metabolic responses against further oxidative damage due to chilling. Under further stress, optimal pretreatments alleviated the increase of H₂O₂ level and the decrease of turfgrass quality, and improved CAT, POD, APX, GR, and GPX activities, with especially significant enhancement of APX and GPX activities from the initiation to end of chilling. These antioxidative enzymes were likely the important factors for acquisition of tolerance to chilling stress in the two Zoysia cultivars. Our results showed that pretreatment with H₂O₂ at appropriate concentration may improve the tolerance of warm-season Zoysia grasses to chilling stress, and that manilagrass had better tolerance to chilling, as evaluated by lower MDA and EL, and better turfgrass quality, regardless of the pretreatment applied.     

Serotonin modulates Arabidopsis root growth via changes in reactive oxygen species and jasmonic acid–ethylene signaling

Serotonin (5‐hydroxytryptamine) is a bioactive indoleamine with neurotransmitter function in vertebrates, which represents an emerging signaling molecule in plants, playing key roles in the development and defense. In this study, the role of reactive oxygen species (ROS) and jasmonic acid (JA)–ethylene (Et) signaling in root developmental alterations induced by serotonin was investigated. An Arabidopsis thaliana mutant defective at the RADICAL‐INDUCED CELL DEATH1 (RCD1) locus was resistant to paraquat‐induced ROS accumulation in primary roots and showed decreased inhibition or root growth in response to serotonin. A suite of JA‐ and Et‐related mutants including coronatine insensitive1, jasmonic acid resistant1 (jar1), etr1, ein2 and ein3 showed tolerance to serotonin in the inhibition of primary root growth and ROS redistribution within the root tip when compared with wild‐type (WT) seedlings. Competence assays between serotonin and AgNO₃, a well‐known blocker of Et action, showed that primary root growth in medium supplemented with serotonin was normalized by AgNO_3, whereas roots of eto3, an Et overproducer mutant, were oversensitive to serotonin. Comparison of ROS levels in WT, etr1, jar1 and rcd1 primary root tips using the ROS‐specific probe 2′,7′‐dichlorofluorescein diacetate and confocal imaging showed that serotonin inhibition of primary root growth likely occurs independently of its conversion into melatonin. Our results provide compelling evidence that serotonin affects ROS distribution in roots, involving RCD1 and components of the JA–Et signaling pathways.     

Evidence for H2O2 as the product of reduction of oxygen by alternative oxidase in mitochondria from potato tubers

Oxygen consumption by alternative oxidase (AOX), present in mitochondria of many angiosperms, is known to be cyanide-resistant in contrast to cytochrome oxidase. Its activity in potato tuber (Solanum tuberosum L.) was induced following chilling treatment at 4 °C. About half of the total O₂ consumption of succinate oxidation in such mitochondria was found to be sensitive to SHAM, a known inhibitor of AOX activity. Addition of catalase to the reaction mixture of AOX during the reaction decreased the rate of SHAM-sensitive oxygen consumption by nearly half, and addition at the end of the reaction released nearly half of the consumed oxygen by AOX, both typical of catalase action on H₂O₂. These findings with catalase suggest that the product of reduction of AOX is H₂O₂ and not H₂O, as previously surmised. In potatoes subjected to chill stress (4 °C) for periods of 3, 5 and ?8 days the activity of AOX in mitochondria increased progressively with a corresponding increase in the AOX protein detected by immunoblot of the protein.