Systemic induction of H2O2 in pea seedlings pretreated by wounding and exogenous jasmonic acid

Mechanical stress was one of stresses with whichplants often met. With the development of fruit andvegetable finish machining in food industry, artificialinjury also appeared. As response to other stresses,plants have evolved with some adaptive mechanismsto cope with wounding[1]. Jasmonic acid (JA) andmethyl jasmonate (MeJA), as important signal mole-cules in plant response to wounding, have attracted agreat deal of attention. The studies on some crops, suchas potato[2], rice[3], and tomato[…     

Relationship between H2O2 and jasmonic acid in pea leaf wounding response

The relationship of H₂O₂ and jasmonic acid (JA) in wound-induced defense response was investigated in the leaves of pea (Pisum sativum L.) plants. The results showed that both wounding and JA treatment led to a significant increase in activities of plasma membrane NADPH oxidase and phenylalanine ammonialyase. However, such an increase was blocked by the pretreatment with plasma membrane NADPH oxidase inhibitors, O ₂ ^? scavengers, or H₂O₂ scavenger, implying that H₂O₂ functions downstream of JA. Furthermore, wounding treatment activated two key enzymes of JA biosynthesis, lipoxygenase and allene oxide synthase, while JA biosynthetic inhibitors impaired the wounding-induced H₂O₂ burst. Thus, it is suggested that H₂O₂ burst depends on JA production in plant wounding response.     

Cytosolic ascorbate peroxidase 1 protects organelles against oxidative stress by wounding- and jasmonate-induced H2O2 in Arabidopsis plants

Background

Reactive oxygen species (ROS) are not only cytotoxic compounds leading to oxidative damage, but also signaling molecules for regulating plant responses to stress and hormones. Arabidopsis cytosolic ascorbate peroxidase 1 (APX1) is thought to be a central regulator for cellular ROS levels. However, it remains unclear whether APX1 is involved in plant tolerance to wounding and methyl jasmonate (MeJA) treatment, which are known to enhance ROS production.

Methods

We studied the effect of wounding and MeJA treatment on the levels of H₂O₂ and oxidative damage in the Arabidopsis wild-type plants and knockout mutants lacking APX1 (KO-APX1).

Results

The KO-APX1 plants showed high sensitivity to wounding and MeJA treatment. In the leaves of wild-type plants, H₂O₂ accumulated only in the vicinity of the wound, while in the leaves of the KO-APX1 plants it accumulated extensively from damaged to undamaged regions. During MeJA treatment, the levels of H₂O₂ were much higher in the leaves of KO-APX1 plants. Oxidative damage in the chloroplasts and nucleus was also enhanced in the leaves of KO-APX1 plants. These findings suggest that APX1 protects organelles against oxidative stress by wounding and MeJA treatment.

General significance

This is the first report demonstrating that H₂O₂-scavenging in the cytosol is essential for plant tolerance to wounding and MeJA treatment.     

Effects of extracellular ATP on local and systemic responses of bean (Phaseolus vulgaris L) leaves to wounding

Wounding increased the extracellular Adenosine 5?-triphosphate (eATP) level of kidney bean leaves. Treatment with wounding or exogenous ATP increased the hydrogen peroxide (H₂O₂) content, activities of catalase and polyphenol oxidase, and malondialdehyde content in both the treated and systemic leaves. Pre-treatment with ATP-degrading enzyme, apyrase, to the wounded leaves reduced the wound-induced local and systemic increases in H₂O₂ content, activities of catalase and polyphenol oxidase, and malondialdehyde content. Application of dimethylthiourea (DMTU) and diphenylene iodonium (DPI) to the wounded and ATP-treated leaves, respectively, reduced the wound- and ATP-induced local and systemic increases in H₂O₂ content, activities of catalase and polyphenol oxidase, and malondialdehyde content. Moreover, the wound- and ATP-induced systemic increases of these physiological parameters were suppressed when DMTU or DPI applied to leaf petiole of the wounded and ATP-treated leaves. These results suggest that eATP at wounded sites could mediate the wound-induced local and systemic responses by H₂O₂-dependent signal transduction.     

MeJA诱导的拟南芥保卫细胞H2O2积累与质膜H+-ATPase的关系

茉莉酸类物质(JAs)作为与昆虫啃噬及损伤相关的植物激素和信号分子在植物防御反应中起重要作用,但是茉莉酸引起的早期防御反应的机理仍不清楚。该研究以拟南芥叶片保卫细胞为材料,结合非损伤微测(NMT)及激光共聚焦技术探讨了茉莉酸诱导的保卫细胞中质膜H+-ATPase与H2O2积累的调控关系。结果表明:茉莉酸甲酯(MeJA)处理导致H+迅速跨膜外排和H2O2积累,H+外排和H2O2积累能够被钒酸钠抑制,而二苯基碘(DPI)处理则对MeJA诱导的H+跨膜外排无显著影响。研究结果证明,在MeJA诱导的早期信号事件中,质膜H+-ATPase的激活先于H2O2的产生。     

UV-B对拟南芥叶片不同来源H2O2的活化和气孔关闭的诱导

在UV-B调控植物许多生理过程中过氧化氢(H2O2)作为第二信使发挥着重要作用,但H2O2来源途径并不清楚。该研究借助气孔开度分析和激光扫描共聚焦显微镜技术,探讨H2O2在介导不同剂量UV-B诱导拟南芥叶片气孔关闭过程中的酶学来源途径。结果发现:0.5W.m-2 UV-B能诱导野生型拟南芥叶片保卫细胞的H2O2产生和气孔关闭,且该效应能被NADPH氧化酶抑制剂二苯基碘(DPI)抑制,而不能被细胞壁过氧化物酶抑制剂水杨基氧肟酸(SHAM)抑制,同时该剂量UV-B也不能诱导NADPH氧化酶功能缺失单突变体AtrbohD和AtrbohF以及双突变体AtrbohD/F保卫细胞的H2O2产生和气孔关闭;相反,0.65 W.m-2 UV-B既能诱导野生型也能诱导NADPH氧化酶突变体保卫细胞的H2O2产生和气孔关闭,且该效应能被SHAM抑制,却不能被DPI抑制。结果表明,不同剂量UV-B通过活化不同生成途径的H2O2来诱导拟南芥叶片气孔关闭,即低剂量UV-B主要诱导NADPH氧化酶AtrbohD和AtrbohF途径来源的H2O2生成,而高剂量UV-B主要活化细胞壁过氧化酶途径来源的H2O2。     

Transportation of de novo synthesized jasmonoyl isoleucine in tomato

In plants, jasmonic acid (JA) and its derivatives are thought to be involved in mobile forms of defense against biotic and abiotic stresses. In this study, the distal transport of JA-isoleucine (JA-Ile) that is synthesized de novo in response to leaf wounding in tomato (Solanum lycopersicum) plants was investigated. JA-[¹³C₆]Ile was recovered in distal untreated leaves after wounded leaves were treated with [¹³C₆]Ile. However, as [¹³C₆]Ile was also recovered in the distal untreated leaves, whether JA-Ile was synthesized in the wounded or in the untreated leaves was unclear. Hence, stem exudates were analyzed to obtain more detailed information. When [¹³C₆]Ile and [²H₆]JA were applied separately into the wounds on two different leaves, JA-[¹³C₆]Ile and [²H₆]JA-Ile were detected in the stem exudates but [²H₆]JA–[¹³C₆]Ile was not, indicating that JA was conjugated with Ile in the wounded leaf and that the resulting JA-Ile was then transported into systemic tissues. The [²H₃]JA-Ile that was applied exogenously to the wounded tissues reached distal untreated leaves within 10 min. Additionally, applying [²H₃]JA-Ile to the wounded leaves at concentrations of 10 and 60 nmol/two leaves induced the accumulation of PIN II, LAP A, and JAZ3 mRNA in the distal untreated leaves of the spr2 mutant S. lycopersicum plants. These results demonstrate the transportation of de novo synthesized JA-Ile and suggest that JA-Ile may be a mobile signal.     

Transport of [2-14C]jasmonic acid from leaves to roots mimics wound-induced changes in endogenous jasmonic acid pools in Nicotiana sylvestris

Jasmonic acid (JA) is part of a long-distance signal-transduction pathway that effects increases in de-novo nicotine synthesis in the roots of Nicotiana sylvestris Speg et Comes (Solanaceae) after leaf wounding. Elevated nicotine synthesis increases whole-plant nicotine pools and makes plants more resistant to herbivores. Leaf wounding rapidly increases JA pools in damaged leaves, and after a 90-min delay, root JA pools also increase. The systemic response in the roots could result from either: (i) the direct transport of JA from wounded leaves, or (ii) JA synthesis or its release from conjugates in roots in response to a second, systemic signal. We synthesized [2-¹⁴C]JA, and applied it to a single leaf in a quantity (189 μg) known to elicit both a whole-plant nicotine and root JA response equivalent to that found in plants subjected to leaf wounding. We quantified radioactive material in JA, and in metabolites both more and less polar than JA, from treated and untreated leaves and roots of plants in eight harvests after JA application. [2-¹⁴C]Jasmonic acid was transported from treated leaves to roots at rates and in quantities equivalent to the wound-induced changes in endogenous JA pools. The [2-¹⁴C]JA that had been transported to the roots declined at the same rate as endogenous JA pools in the roots of plants after leaf wounding. Most of the labeled material applied to leaves was metabolized or otherwise immobilized at the application site, and the levels of [2-¹⁴C]JA in untreated leaves did not increase over time. We measured the free JA pools before and after four different hydrolytic extractions of root and shoot tissues to estimate the size of the potential JA conjugate pools, and found them to be 10% or less of the free JA pool. We conclude that the direct transport of wound-induced JA from leaves to roots can account for the systemic increase in root JA pools after leaf wounding, and that metabolism into less polar structures determines the duration of this systemic increase. However, the conclusive falsification of this hypothesis will require the suppression of all other signalling pathways which could have shoot-to-root transport kinetics similar to that of endogenous JA. Received: 14 April 1997 / Accepted: 9 June 1997     

Nitric oxide activates superoxide dismutase and ascorbate peroxidase to repress the cell death induced by wounding

Wounding caused by rain, wind, and pathogen may lead plants to onset defense response. Previous studies indicated that mechanical wounding stimulates plants to generate nitric oxide (NO) and hydrogen peroxide (H₂O₂). In this study, the functions of NO and H₂O₂ after wounding in sweet potato (Ipomoea batatas cv. Tainung 57) was further analyzed. Mechanical wounding damaged cells and resulted in necrosis, but the presence of NO donors or NO scavenger might reduce or enhance the cell death caused by wounding, respectively. The amount of H₂O₂ induced by wounding was also decreased or increased when plants were incubated with NO donors or NO scavenger, individually. These results indicate that NO may regulate H₂O₂ generation to affect cell death. NO-induced proteins isolated from two-dimensional electrophoresis were identified to be Copper/Zinc superoxide dismutases (CuZnSODs). The activities of CuZnSODs and ascorbate peroxidase (APX) could be enhanced by NO. In addition, the expression of CuZnSOD and APX was induced by wounding via NO, and their expression was further stimulated by NO through the generation of cGMP. The influx of calcium ions and the activity of NADPH oxidase were also involved in the NO signal transduction pathway inducing APX expression. Collectively, the generation of H₂O₂ in wounded plants might trigger cell death. Meanwhile, the production of NO induced by wounding stimulated signal transducers including cGMP, calcium ions, and H₂O₂ to activate CuZnSOD and APX, which further decreased H₂O₂ level and reduced the cell death caused by wounding.     

NADPH oxidase inhibitor diphenyleneiodonium and reduced glutathione mitigate ethephon-mediated leaf senescence,H2O2 elevation and senescence-associated gene expression in sweet potato (Ipomoea batatas)

Ethephon, an ethylene releasing compound, promoted leaf senescence, H₂O₂ elevation, and senescence-associated gene expression in sweet potato. It also affected the glutathione and ascorbate levels, which in turn perturbed H₂O₂ homeostasis. The decrease of reduced glutathione and the accumulation of dehydroascorbate correlated with leaf senescence and H₂O₂ elevation at 72 h in ethephon-treated leaves. Exogenous application of reduced glutathione caused quicker and significant increase of its intracellular level and resulted in the attenuation of leaf senescence and H₂O₂ elevation. A small H₂O₂ peak produced within the first 4 h after ethephon application was also eliminated by reduced glutathione. Diphenyleneiodonium (DPI), an NADPH oxidase inhibitor, delayed leaf senescence and H₂O₂ elevation at 72 h, and its influence was effective only within the first 4 h after ethephon treatment. Ethephon-induced senescence-associated gene expression was repressed by DPI and reduced glutathione at 72 h in pretreated leaves. Leaves treated with l-buthionine sulfoximine, an endogenous glutathione synthetase inhibitor, did enhance senescence-associated gene expression, and the activation was strongly repressed by reduced glutathione. In conclusion, ethephon-mediated leaf senescence, H₂O₂ elevation and senescence-associated gene expression are all alleviated by reduced glutathione and NADPH oxidase inhibitor DPI. The speed and the amount of intracellular reduced glutathione accumulation influence its effectiveness of protection against ethephon-mediated effects. Reactive oxygen species generated from NADPH oxidase likely serves as an oxidative stress signal and participates in ethephon signaling. The possible roles of NADPH oxidase and reduced glutathione in the regulation of oxidative stress signal in ethephon are discussed.     

Induced sink strength as a prerequisite for induced tannin biosynthesis in developing leaves of Populus

Induced defenses occur predominately in young, developing plant tissues that rely upon carbohydrate import to support their growth and development. To test the hypothesis that the induced production of carbon-based defenses is dependent upon photoassimilate import, we examined the response of developing leaves of hybrid poplar (Populus deltoides 2 P. nigra) saplings to wounding by gypsy moth caterpillars (Lymantria dispar L.) and exogenous jasmonic acid (JA). Growth rates, condensed tannin contents and acid invertase activities were measured for individual leaves and the translocation of ¹³C-labeled resources between orthostichous source-sink pairs was quantified. Results showed a substantial increase in the activity of cell wall invertase in sink leaves wounded by gypsy moth caterpillars and treated with JA. JA-induced sink leaves also imported 3-4 times as much ¹³C-labeled carbon from orthostichous source leaves relative to controls and allocated a significant portion of this imported ¹³C to condensed tannin biosynthesis. Reduced carbohydrate flow to these leaves, caused by source leaf removal, resulted in reduced condensed tannin levels and the emergence of a growth-defense tradeoff. These results indicate that (1) induced sink strength is elicited by insect wounding and JA application in hybrid poplar foliage, (2) imported resources are allocated to the production of carbon-based defenses, and (3) the level of induced defense in leaves can be constrained by the ability of leaves to import carbohydrates from source tissues. Together, these results suggest that within-canopy variations in induced resistance may arise in part because of uneven distribution of resources to induced foliage.     

GTR1 is a jasmonic acid and jasmonoyl-l-isoleucine transporter in Arabidopsis thaliana

Jasmonates are major plant hormones involved in wounding responses. Systemic wounding responses are induced by an electrical signal derived from damaged leaves. After the signaling, jasmonic acid (JA) and jasmonoyl-l-isoleucine (JA-Ile) are translocated from wounded to undamaged leaves, but the molecular mechanism of the transport remains unclear. Here, we found that a JA-Ile transporter, GTR1, contributed to these translocations in Arabidopsis thaliana. GTR1 was expressed in and surrounding the leaf veins both of wounded and undamaged leaves. Less accumulations and translocation of JA and JA-Ile were observed in undamaged leaves of gtr1 at 30 min after wounding. Expressions of some genes related to wound responses were induced systemically in undamaged leaves of gtr1. These results suggested that GTR1 would be involved in the translocation of JA and JA-Ile in plant and may be contributed to correct positioning of JA and JA-Ile to attenuate an excessive wound response in undamaged leaves.     

Distal transport of exogenously applied jasmonoyl-isoleucine with wounding stress

Determining the mobile signal used by plants to defend against biotic and abiotic stresses has proved elusive, but jasmonic acid (JA) and its derivatives appear to be involved. Using deuterium-labeled analogs, we investigated the distal transport of JA and jasmonoyl-isoleucine (JA-Ile) in response to leaf wounding in tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum) plants. We recovered [(2)H(2)-2]JA ([(2)H(2)]JA) and [(2)H(3)-12]JA-Ile ([(2)H(3)]JA-Ile) in distal leaves of N. tabacum and S. lycopersicum after treating wounded leaves with [(2)H(2)]JA or [(2)H(3)]JA-Ile. We found that JA-Ile had a greater mobility than JA, despite its lower polarity, and that application of exogenous JA-Ile to wounded leaves of N. tabacum led to a higher accumulation of JA and JA-Ile in distal leaves compared with wounded control plants. We also found that exudates from the stem of S. lycopersicum plants with damaged leaflets contained JA and JA-Ile at higher levels than in an undamaged plant, and a significant difference in the levels of JA-Ile was observed 30 min after wounding. Based on these results, it was found that JA-Ile is a transportable compound, which suggests that JA-Ile is a signaling cue involved in the resistance to biotic and abiotic stresses in plants.     

Kinetics of the Accumulation of Jasmonic Acid and Its Derivatives in Systemic Leaves of Tobacco (Nicotiana tabacum cv. Xanthi nc) and Translocation of Deuterium-Labeled Jasmonic Acid from the Wounding Site to the Systemic Site

In plants, the mobile signal needed for wound-induced systemic acquired resistance (WSR) has been elusive. The signal compound involved in WSR is supposed to be JA or its derivatives. On the basis of kinetic study of the accumulation of JA or its derivatives, it was discovered that JA, JA-Ile, tuberonic acid (TA, 12-OH epi-JA), and tuberonic acid glucoside (TAG) accumulated in systemic tissues in response to mechanical wounding stress in the tobacco plant (Nicotiana tabacum). Attempts to recover deuterium-labeled JA in systemic leaves after feeding the wounded leaves with deuterium-labeled JA were successfully done. It was also found that the translocated deuterium-labeled JA was metabolized to TA in systemic leaves under feeding of deuterium-labeled JA to the wounding leaves.     

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.     

局部灼伤对小麦幼苗外源茉莉酸吸收与运转的影响

采用示踪技术探索了3H-JA的运输和分配规律及其受伤害胁迫的影响.外源3H-JA能够在小麦幼苗体内向上和向下运输,局部灼伤其运输与分配都发生了改变.从小麦根系饲喂的3H-JA,在植株内的分布量依序为根＞茎＞叶,时间较长(4h)时分配于心叶的3H-JA大大增加.当叶片受到局部灼伤时3H-JA向地上部的输出量减少;但局部灼伤可加快由心叶饲喂的3H-JA的向下运输,改变3H-JA在小麦幼苗各部位的分配比率.心叶饲喂短时间(5min)时,3H-JA主要积累在受到伤胁迫的展开叶(第2叶)中.向展开叶(第2叶)饲喂的3H-JA向下运输的速率高于向上运输的速率.推测JA运输及分配的变化可能在植株的防御反应中起重要作用.     

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.     

Structural characteristics and chemical composition of birch (Betula pendula) leaves are modified by increasing CO2 and ozone

Impacts of ozone and CO₂ enrichment, alone and in combination, on leaf anatomical and ultrastructural characteristics, nutrient status and cell wall chemistry in two European silver birch (Betula pendula Roth) clones were studied. The young soil‐growing trees were exposed in open‐top chambers over three growing seasons to 2 × ambient CO₂ and/or ozone concentrations in central Finland. The trees were measured for changes in altogether 35 variables of leaf structure, nutrients and cell wall chemistry of green leaves, and 20 of the measured variables were affected by CO₂ and/or O₃. Elevated CO₂ increased the size of chloroplasts and starch grains, number of mitochondria, P : N ratio, and contents of cell wall hemicellulose. Elevated CO₂ decreased the total leaf thickness, specific leaf area, concentrations of N, K, Cu, S and Fe, and contents of cell wall α‐cellulose, uronic acids, acid‐soluble lignin and acetone‐soluble extractives. Elevated ozone led to thinner leaves, higher palisade to spongy ratio, increased number of peroxisomes and mitochondria, reduced content of Mn, Zn, Cu, hemicellulose and uronic acids, and lower Mn : N and Zn : N ratios. In the combined exposure, interactions were antagonistic. Ultrastructural changes became more evident towards the end of the exposure. Young leaves were tolerant against ozone‐caused oxidative stress, whereas oxidative H₂O₂ accumulation was found in older leaves. CO₂ enrichment improved ozone tolerance not only through increased photosynthesis rates, but also through changes in cell wall chemistry (hemicellulose, in particular). However, nutrient imbalances due to ozone and/or CO₂ may predispose the trees to other biotic and abiotic stresses. Down‐regulation and up‐regulation of photosynthesis under elevated CO₂ through anatomical changes is discussed.     

Hydrogen peroxide is involved in methyl jasmonate-induced senescence of rice leaves

The role of H₂O₂ in the senescence of detached rice leaves induced by methyl jasmonate (MJ) was investigated. MJ treatment resulted in H₂O₂ production in detached rice leaves, which was prior to the occurrence of leaf senescence. Dimethylthiourea, a chemical trap of H₂O₂, was observed to be effective in inhibiting MJ‐induced senescence and MJ‐increased malondialdehyde (MDA) content in detached rice leaves. Diphenyleneiodonium chloride (DPI) and imidazole (IMD), inhibitors of NADPH oxidase, prevented MJ‐induced H₂O₂ production, suggesting that NADPH oxidase is a H₂O₂‐generating enzyme in MJ‐treated detached rice leaves. DPI and IMD also inhibited MJ‐promoted senescence and MJ‐increased MDA content in detached rice leaves. Phosphatidylinositol 3‐kinase inhibitors wortmannin (WM) or LY 294002 (LY) inhibited MJ‐induced H₂O₂ production and senescence of detached rice leaves. Exogenous H₂O₂ reversed the inhibitory effect of WM or LY. In terms of leaf senescence, it was observed that rice seedlings of cultivar Taichung Native 1 (TN1) are jasmonic acid (JA)‐sensitive and those of cultivar Tainung 67 (TNG67) are JA‐insensitive. On treatment with JA, H₂O₂ accumulated in the leaves of TN1 seedlings but not in the leaves of TNG67. Evidence was also provided to show that MJ‐induced H₂O₂ production in detached rice leaves is abscisic acid (ABA)‐independent. Ethylene action inhibitor, silver thiosulfate, was observed to inhibit MJ‐ and ABA‐induced H₂O₂ production and senescence of detached rice leaves, suggesting that the action of MJ and ABA is ethylene‐dependent.