Gene knockout of glutathione reductase 3 results in increased sensitivity to salt stress in rice.

Glutathione reductase (GR) is one of important antioxidant enzymes in plants. This enzyme catalyzes the reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) with the accompanying oxidation of NADPH. Previously, we showed that salt-stress-responsive GR3 is a functional protein localized in chloroplasts and mitochondria in rice. To learn more about the role of GR3 in salt-stress tolerance, we investigated the response to 100 mM NaCl treatment in wild-type rice (WT); GR3 knockout mutant of rice (gr3); and the functional gr3-complementation line (C1). Rice GR3 was primarily expressed in roots at the seedling stage and ubiquitously expressed in all tissues except the sheath at heading stage. GR3 promoter-GUS was expressed in the vascular cylinder and cortex of root tissues in rice seedlings, vascular tissue of nodes, embryo and aleurone layer of seeds, and young flowers. Under both normal and salt-stress conditions, total GR activity was decreased by 20 % in gr3. Oxidative stress, indicated by malondialdehyde content, was greater in gr3 than the WT under salt stress. As compared with the WT, gr3 was sensitive to salt and methyl viologen; it showed inhibited growth, decreased maximal efficiency of photosystem II, decreased GSH and GSSG contents, and the ratio of GSH to GSSG. Conversely, the gr3-complementation line C1 rescued the tolerance to methyl viologen and salinity and recovered the growth and physiological damage caused by salinity. These results reveal that GR3 plays an important role in salt stress tolerance by regulating the GSH redox state in rice.

Identification and characterization of a novel chloroplast/mitochondria co-localized glutathione reductase 3 involved in salt stress response in rice.

Glutathione reductases (GRs) are important components of the antioxidant machinery that plants use to respond against abiotic stresses. In rice, one cytosolic and two chloroplastic GR isoforms have been identified. In this work, we describe the cloning and characterization of the full-length cDNA encoding OsGR3, a chloroplast-localized GR that up to now was considered as a non-functional enzyme because of assumed lack of N-terminal conserved domains. The expression of OsGR3 in E. coli validated that it can be translated as a protein with GR activity. OsGR3 shows 76 and 53 % identity with OsGR1 (chloroplastic) and OsGR2 (cytosolic), respectively. Phylogenetic analysis revealed 2 chloroplastic GRs in Poaceae species, including rice, sorghum and brachypodium, but only one chloroplastic GR in dicots. A plastid transit peptide is located at the N terminus of OsGR3, and genetic transformation of rice with a GR3-GFP fusion construct further confirmed its localization in chloroplasts. Furthermore, OsGR1 and OsGR3 are also targeted to mitochondria, which suggest a combined antioxidant mechanism in both chloroplasts and mitochondria. However, both isoforms showed a distinct response to salinity: the expression of OsGR3 but not OsGR1 was induced by salt stress. In addition, the transcript level of OsGR3 was greatly increased with salicylic acid treatment but was not significantly affected by methyl jasmonate, dehydration or heat shock stress. Our results provide new clues about the possible roles of functional OsGR3 in salt stress and biotic stress tolerance.

Cobalt chloride-induced lateral root formation in rice: the role of heme oxygenase.

Lateral roots (LRs) perform the essential tasks of providing water, nutrients, and physical support to plants. Therefore, understanding the regulation of LR development is of agronomic importance. Recent findings suggest that heme oxygenase (HO) plays an important role in LR development. In this study, we examined the effect of cobalt chloride (CoCl2) on LR formation and HO expression in rice. Treatment with CoCl2 induced LR formation and HO activity. We further observed that CoCl2 could induce the expression of OsHO1 but not OsHO2. CoCl2-increased HO activity occurred before LR formation. Zinc protoporphyrin IX (ZnPPIX, the specific inhibitor of HO) and hemoglobin (the carbon monoxide/nitric oxide scavenger) reduced LR formation, HO activity, and OsHO1 expression. Application of biliverdin, a product of HO-catalyzed reaction, to CoCl2-treated rice seedlings reversed the ZnPPIX-inhibited LR formation and ZnPPIX-decreased HO activity. CoCl2 had no effect on H2O2 content and nitric oxide production. Moreover, application of ascorbate, a H2O2 scavenger, failed to affect CoCl2-promoted LR formation and HO activity. It is concluded that HO is required for CoCl2-promoted LR formation in rice.

Methyl jasmonate-induced lateral root formation in rice: the role of heme oxygenase and calcium.

Lateral roots (LRs) play important roles in increasing the absorptive capacity of roots as well as to anchor the plant in the soil. Therefore, understanding the regulation of LR development is of agronomic importance. In this study, we examined the effect of methyl jasmonate (MJ) on LR formation in rice. Treatment with MJ induced LR formation and heme oxygenase (HO) activity. As well, MJ could induce OsHO1 mRNA expression. Zinc protoporphyrin IX (the specific inhibitor of HO) and hemoglobin [the carbon monoxide/nitric oxide (NO) scavenger] reduced LR formation, HO activity and OsHO1 expression. LR formation and HO activity induced by MJ was reduced by the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-oxide. The effects of Ca(2+) chelators, Ca(2+)-channel inhibitors, and calmodulin (CaM) antagonists on LR formation induced by MJ were also examined. All these inhibitors were effective in reducing the action of MJ. However, Ca(2+) chelators and Ca(2+) channel inhibitors induced HO activity when combining with MJ further. It is concluded that Ca(2+) may regulate MJ action mainly through CaM-dependent mechanism.

Heme oxygenase is involved in H(2)O (2)-induced lateral root formation in apocynin-treated rice.

KEY MESSAGE : Apocynin is a natural organic compound structurally related to vanillin. We demonstrated that hydrogen peroxide and heme oxygenase participated in apocynin-induced lateral root formation in rice. Apocynin, also known as acetovanillone, is a natural organic compound structurally related to vanillin. Information concerning the effect of apocynin on plants is limited. In this study, we examined the effect of apocynin on lateral root (LR) formation in rice. Treatment with apocynin induced LR formation and increased H(2)O(2) production, but had no effect on nitric oxide production. Diphenyleneiodonium chloride, an inhibitor of H(2)O(2) generating NADPH oxidase, was effective in reducing apocynin-induced H(2)O(2) production and LR formation. Apocynin treatment also increased superoxide dismutase activity and decreased catalase activity. H(2)O(2) application was able to increase the number of LRs. Moreover, H(2)O(2) production caused by H(2)O(2) and apocynin was localized in the root area corresponding to the LR emergence. Treatment with H(2)O(2) and apocynin also increased heme oxygenase (HO) activity and induced OsHO1 mRNA expression. Lateral root formation and HO activity induced by H(2)O(2) and apocynin were reduced by Zn protoporphyrin IX (the specific inhibitor of HO). Our data suggest that both H(2)O(2) and HO are required for apocynin-induced LR formation in rice.

Effect of potassium deficiency on antioxidant status and cadmium toxicity in rice seedlings.

BACKGROUND: Cadmium (Cd) is one of the most toxic heavy metals and inhibits physiological processes of plants. Potassium (K) is an essential macronutrient in plants. K deficiency and Cd stress represent two different abiotic stress conditions that occur in the field simultaneously. In this study, effects of K deficiency on antioxidant status and Cd toxicity in rice seedlings were investigated. RESULTS: K deficiency significantly decreased K concentration in shoots and roots. However, fresh weight and dry weight of rice seedlings were not affected by K deficiency. The activities of antioxidant enzymes (superoxide dismutase, ascorbate peroxidase, glutathione reductase, and catalase) in K-deficient leaves were higher than respective control leaves. However, K deficiency had no effect on the content of antioxidants (ascorbate and glutathione). Cd toxicity was judged by the decrease in biomass production, chlorosis, and induction of oxidative stress. Based on these criteria, we demonstrated that K deficiency protected rice seedling from Cd stress. Moreover, chlorophyll concentration was higher in K-deficient shoots and roots than their respective control shoots and roots. CONCLUSIONS: Our results indicated that K deficiency protects rice seedlings from Cd toxicity. This protective effect of K deficiency is mainly due to enhanced antioxidant enzyme activities but not inhibition of Cd uptake.

Biliverdin-promoted lateral root formation is mediated through heme oxygenase in rice.

In this study, we examined the effect of biliverdin (BV), a product of heme oxygenase (HO) catalyzed reaction, on lateral root (LR) formation in rice. Treatment with BV induced LR formation and HO activity. As well, BV, could induce OsHO1 mRNA expression. Zn protoporphyrin IX (the specific inhibitor of HO) reduced LR number, HO activity and OsHO1 mRNA level induced by BV. Our data suggest that HO is required for BV-induced LR formation in rice.

Calcium deficiency increases Cd toxicity and Ca is required for heat-shock induced Cd tolerance in rice seedlings.

While growing in the field, plants may encounter several different forms of abiotic stress simultaneously, rather than a single stress. In this study, we investigated the effects of calcium (Ca) deficiency on cadmium (Cd) toxicity in rice seedlings. Calcium deficiency alone decreased the length, fresh and dry weight, and the Ca concentration in shoots and roots. Also, the content of glutathione (GSH), the ratio of GSH/oxidized glutathione, and the activity of catalase were lower in Ca-deficient leaves compared to control leaves. Exogenous Cd caused a decrease in the contents of chlorophyll and protein, and induced oxidative stress. Based on these stress indicators, we found that Ca deficiency enhanced Cd toxicity in rice seedlings. Under exogenous Cd application, internal Cd concentrations were higher in Ca-deficient shoots and roots than in the respective controls. Moreover, we observed that Ca deficiency decreased heat-shock (HS) induced expression of HS protein genes Oshsp17.3, Oshsp17.7, and Oshsp18.0 in leaves thereby weakening the protection system and increasing Cd stress. In conclusion, Ca deficiency enhances Cd toxicity, and Ca may be required for HS response in rice seedlings.

Heme oxygenase is involved in nitric oxide- and auxin-induced lateral root formation in rice.

Lateral root (LR) development performs the essential tasks of providing water, nutrients, and physical support to plants. Therefore, understanding the regulation of LR development is of agronomic importance. In this study, we examined the effect of nitric oxide (NO), auxin, and hemin (Hm) on LR formation in rice. Treatment with Hm [a highly effective heme oxygenase (HO) inducer], sodium nitroprusside (SNP, an NO donor), or indole-3-butyric acid (IBA, a naturally occurring auxin) induced LR formation and HO activity. LR formation and HO activity induced by SNP and IBA but not Hm was reduced by the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. As well, Hm, SNP, and IBA could induce OsHO1 mRNA expression. Zn protoporphyrin IX (the specific inhibitor of HO) and hemoglobin (the carbon monoxide/NO scavenger) reduced LR number and HO activity induced by Hm, SNP, and IBA. Our data suggest that HO is required for Hm-, auxin-, and NO-induced LR formation in rice.

Calcium is involved in nitric oxide- and auxin-induced lateral root formation in rice.

In the present study, the role of nitric oxide (NO) in the regulation of lateral root (LR) formation in rice was examined. Application of sodium nitroprusside (SNP; a NO donor) and indole-3-butyric acid (IBA; a naturally occurring auxin) to rice seedlings induced LR formation. The effect is specific for NO because the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3- oxide (cPTIO) blocked the action of SNP and IBA. Endogenous NO was detected by the specific fluorescence probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. SNP- and IBA-induced NO fluorescence was specifically suppressed by cPTIO. Nitrate reductase (NR) inhibitor sodium tungstate completely inhibited IBA-induced LR formation and NO fluorescence. However, nitric oxide synthase inhibitor N (G)-nitro-L: -arginine methyl ester hydrochloride slightly reduced IBA-induced LR formation and NO generation. It appears that NO generation that occurs in response to IBA might primarily involve NR activity. Moreover, NO production caused by SNP and IBA was localized in root area corresponding to LR emergence. The effects of Ca(2+) chelators, Ca(2+)-channel inhibitors, and calmodulin antagonists on LR formation induced by SNP and IBA were also examined. All these inhibitors were effective in reducing the action of SNP and IBA. However, Ca(2+) chelators and Ca(2+)-channel inhibitors had no effect on SNP- and IBA-induced NO generation. It is concluded that cytosolic levels of Ca(2+) may regulate SNP and IBA action through calmodulin-dependent mechanism.

Involvement of hydrogen peroxide in heat shock- and cadmium-induced expression of ascorbate peroxidase and glutathione reductase in leaves of rice seedlings.

Hydrogen peroxide (H2O2) is considered a signal molecule inducing cellular stress. Both heat shock (HS) and Cd can increase H2O2 content. We investigated the involvement of H2O2 in HS- and Cd-mediated changes in the expression of ascorbate peroxidase (APX) and glutathione reductase (GR) in leaves of rice seedlings. HS treatment increased the content of H2O2 before it increased activities of APX and GR in rice leaves. Moreover, HS-induced H2O2 production and APX and GR activities could be counteracted by the NADPH oxidase inhibitors dipehenylene iodonium (DPI) and imidazole (IMD). HS-induced OsAPX2 gene expression was associated with HS-induced APX activity but was not regulated by H2O2. Cd-increased H2O2 content and APX and GR activities were lower with than without HS. Cd did not increase the expression of OsAPX and OsGR without HS treatment. Cd increased H2O2 content by Cd before it increased APX and GR activities without HS. Treatment with DPI and IMD effectively inhibited Cd-induced H2O2 production and APX and GR activities. Moreover, the effects of DPI and IMD could be rescued with H2O2 treatment. H2O2 may be involved in the regulation of HS- and Cd-increased APX and GR activities in leaves of rice seedlings.

Effect of magnesium deficiency on antioxidant status and cadmium toxicity in rice seedlings.

Cadmium (Cd) is one of the most toxic heavy metals and inhibits physiological processes of plants. Magnesium (Mg) is known as one of the essential nutrients for plants. Mg deficiency in plants affects metabolic processes. Plants grown in the field may encounter several abiotic stresses, rather than a single stress. Thus, the relationship between Mg nutrition and Cd toxicity is of ecological importance. In this study, effects of Mg deficiency on antioxidant systems and Cd toxicity in rice seedlings were investigated. Mg deficiency significantly decreased Mg concentrations in shoot and roots of rice seedlings. However, fresh weight and dry weight of rice seedlings were not affected by Mg deficiency. The contents of ascorbate and glutathione (GSH), the ratio of GSH/oxidized glutathione, and the activities of superoxide dismutase, ascorbate peroxidase, glutathione reductase, and catalase in Mg-deficient leaves were higher than respective control leaves. Cd toxicity was judged by the decrease in biomass production, decrease in chlorophyll, and induction of oxidative stress. Based on these criteria, we demonstrated that Mg deficiency protected rice seedlings from Cd stress. Moreover, chlorophyll destruction by paraquat was higher in detached leaves from Mg-sufficient than Mg-deficient seedlings. Cd concentration was higher in Mg-deficient shoot and roots than their respective control shoot and roots, suggesting that the protective effect of Mg deficiency against Cd toxicity is not due to reduction of Cd uptake. Moreover, we observed that Cd-decreased Fe and Zn contents in Mg-deficient seedlings were more pronounced than that in Mg-sufficient seedlings. Of particular interest is the finding that the increase in OsIRT1, OsZIP1, and OsZIP3 transcripts caused by Cd in Mg-deficient roots was greater than that in control roots.

Exposure of rice seedlings to heat shock protects against subsequent Cd-induced decrease in glutamine synthetase activity and increase in specific protease activity in leaves.

In the present study, we investigated the effect of heat shock (HS) on the subsequent Cd-induced decrease in the activity of glutamine synthetase (GS) and increase in the specific activity of protease in rice leaves. HS exposure of rice seedlings for 3h in the dark was effective in reducing subsequent Cd-induced decrease in the activity of glutamine synthetase and increase in the specific activity of protease. The effect of HS can be mimicked by pretreatment of rice seedlings with exogenous H(2)O(2) or reduced glutathione (GSH) under non-HS conditions. We also found that HS protected against subsequent Cd-induced decrease in the activity of GS and increase in the specific activity of protease can be counteracted by imidazole, a NADPH oxidase inhibitor. Pretreatment with buthione sulfoximine (a GSH synthesis inhibitor) under HS conditions enhanced subsequent Cd effects on the activity of GS and the specific activity of protease. Moreover, the effect of BSO can be reversed by the addition of GSH. The mechanisms of the protective effect of HS effect against subsequent Cd effects are discussed.

The decline in ascorbic acid content is associated with cadmium toxicity of rice seedlings.

Cadmium (Cd) toxicity of rice (Oryza sativa L. cv. Taichung Native 1) seedlings was evaluated by the decrease in chlorophyll content and the increase in malondialdehyde (MDA) in the second leaves of rice seedlings. CdCl2 (5 microM) treatment was accompanied by a decrease in the contents of ascorbic acid (AsA) and AsA + dehydroascorbate (DHA) and in the ratios of AsA/DHA in leaves. However, CdCl2 treatment resulted in an increase in DHA content in leaves. Moreover, the decrease in AsA content was prior to the occurrence of chlorosis and associated with the increase in MDA content in the leaves of seedlings treated with Cd. Pretreatment with 0.5 mM AsA or L-galactono-1,4-lactone (GalL), the biosynthetic precursor of AsA, for 6 h resulted in an increase in the contents of AsA and reduced glutathione (GSH), the ratios of AsA/DHA and GSH/oxidized glutathione, and the activities of ascorbate peroxidase (APX) and glutathione reductase (GR) in the leaves of rice seedlings. Quantitative RT-PCR was applied to quantify the mRNA levels for OsAPX and OsGR genes from rice leaves to examine the effect of AsA or GalL pretreatment on the expression of OsAPX and OsGR genes in rice leaves. The expression of OsAPX2, OsAPX3, OsAPX4, OsAPX5, OsAPX6, OsAPX7, and OsGR1 was increased by AsA or GalL pretreatment. Rice seedlings pretreated with AsA or GalL were observed to reduce the subsequent Cd-induced toxicity. Our results suggest that AsA content may play a role in regulating Cd toxicity of rice seedlings.

Abscisic acid-induced hydrogen peroxide is required for anthocyanin accumulation in leaves of rice seedlings.

The role of hydrogen peroxide (H(2)O(2)) in abscisic acid (ABA)-induced anthocyanin accumulation in detached and intact leaves of rice seedlings was investigated. Treatment with ABA resulted in an accumulation of anthocyanins in detached rice leaves. Dimethylthiourea, a chemical trap for H(2)O(2), was observed to be effective in inhibiting ABA-induced accumulation of anthocyanins. Inhibitors of NADPH oxidase (diphenyleneiodonium chloride and imidazole), phosphatidylinositol 3-kinase (wortmannin and LY 294002), and a donor of nitric oxide (N-tert-butyl-alpha-phenylnitrone), which have previously been shown to prevent ABA-induced H(2)O(2) accumulation in detached rice leaves, inhibited ABA-induced anthocyanin increase. Exogenous application of H(2)O(2), however, was found to increase the anthocyanin content of detached rice leaves. In terms of H(2)O(2) accumulation, intact (attached) leaves of rice seedlings of cultivar Taichung Native 1 (TN1) are ABA sensitive and those of cultivar Tainung 67 (TNG67) are ABA insensitive. Upon treatment with ABA, H(2)O(2) and anthocyanins accumulated in leaves of TN1 seedlings but not in leaves of TNG67. Our results, obtained from detached and intact leaves of rice seedlings, suggest that H(2)O(2) is involved in ABA-induced anthocyanin accumulation in this species.

NaCl-induced expression of ASCORBATE PEROXIDASE 8 in roots of rice (Oryza sativa L.) seedlings is not associated with osmotic component.

Ascorbate peroxidase (APx; EC 1.11.1.11) plays an important role in scavenging the toxic effects of H(2)O(2) in higher plants. Eight types of APx have been described for Oryza sativa: two cytosolic (OsAPx1 and OsAPx2), two putative peroxisomal (OsAPx3 and OsAPx4), and four chloroplastic isoforms (OsAPx5, OsAPx6, OsAPx7 and OsAPx8). We have recently demonstrated that Na(+) but not Cl(-) is required for the NaCl-induced expression of OsAPx8 in rice roots. Evidence is also provided to show that Na(+)-induced expression of OsAPx8 is mediated through an accumulation of ABA. In addition to its known component of ion toxicity, there is an osmotic effect resulting from salt concentration in the soil. Here we show that ABA level but not OsAPx8 expression was enhanced at a concentration of mannitol iso-osmotic with 150 mM NaCl suggests that NaCl-enhanced OsAPx8 expression is not associated with osmotic component.

Expression of ASCORBATE PEROXIDASE 8 in roots of rice (Oryza sativa L.) seedlings in response to NaCl.

Reactive oxygen species are thought to play an important role in NaCl stress. Therefore, the expression patterns of the gene family encoding the H(2)O(2)-scavenging enzyme ascorbate peroxidase (APx; EC1.11.1.11) were analysed in roots of etiolated rice (Oryza sativa L.) seedlings in response to NaCl stress. Applying semi-quantitative RT-PCR, the mRNA levels were quantified for two cytosolic (OsAPx1 and OsAPx2), two peroxisomal (OsAPx3 and OsAPx4), and four chloroplastic (OsAPx5, OsAPx6, OsAPx7, and OsAPx8) isoforms identified in the rice genome. NaCl at 150 mM and 200 mM increased the expression of OsAPx8 and the activities of APx, but had no effect on the expression of OsAPx1, OsAPx2, OsAPx3, OsAPx4, OsAPx5, OsAPx6, and OsAPx7 in rice roots. However, NaCl at 300 mM up-regulated OsAPx8 expression, increased APx activity, and down-regulated OsAPx7 expression, but had no effect on the expression of OsAPx1, OsAPx2, OsAPx3, OsAPx4, OsAPx5, and OsAPx6. The accumulation of abscisic acid (ABA) in response to NaCl was observed in rice roots. Exogenously applied ABA also specifically enhanced the expression of OsAPx8 in rice roots. The accumulation of ABA in rice roots in response to NaCl was inhibited by fluridone (Flu), an inhibitor of carotenoid biosynthesis. Flu treatment also suppressed NaCl-enhanced OsAPx8 expression and APx activity. The effect of Flu on the expression of OsAPx8 and increase in APx activity was reversed by the application of ABA. It appears that NaCl-enhanced expression of OsAPx8 in rice roots is mediated through an accumulation of ABA. Evidence is provided to show that Na(+) but not Cl(-) is required for enhancing OsAPx8 expression, APx activity, and ABA accumulation in rice roots treated with NaCl. H(2)O(2) treatment resulted in an enhancement of OsAPx8 induction but no accumulation of ABA. Diphenylene iodonium treatment, which is known to inhibit NaCl-induced accumulation of H(2)O(2) in rice roots, did not suppress OsAPx8 induction and ABA accumulation by NaCl. It appears that H(2)O(2) is not involved in the regulation of NaCl-induced OsAPx8 expression in rice roots.

The participation of hydrogen peroxide in methyl jasmonate-induced NH(4)(+) accumulation in rice leaves.

Ammonium is a central intermediate in the nitrogen metabolism of plants. We have previously shown that methyl jasmonate (MJ) not only increases the content of H(2)O(2), but also causes NH(4)(+) accumulation in rice leaves. More recently, H(2)O(2) is thought to constitute a general signal molecule participating in the recognition of and the response to stress factors. In this study, we examined the role of H(2)O(2) as a link between MJ and subsequent NH(4)(+) accumulation in detached rice leaves. MJ treatment resulted in an accumulation of NH(4)(+) in detached rice leaves, which was preceded by a decrease in the activity of glutamine synthetase (GS) and an increase in the specific activities of protease and phenylalanine ammonia-lyase (PAL). GS, PAL, and protease appear to be the enzymes responsible for the accumulation of NH(4)(+) in MJ-treated detached rice leaves. Dimethylthiourea (DMTU), a chemical trap for H(2)O(2), was observed to be effective in inhibiting MJ-induced NH(4)(+) accumulation in detached rice leaves. Scavengers of free radicals (sodium benzoate, SB, and glutathione, GSH), nitric oxide donor (N-tert-butyl-alpha-phenylnitrone, PBN), the inhibitors of NADPH oxidase (diphenyleneiodonium chloride, DPI, and imidazole, IMD), and inhibitors of phosphatidylinositol 3-kinase (wortmannin, WM, and LY 294002, LY), which have previously been shown to prevent MJ-induced H(2)O(2) production in detached rice leaves, inhibited MJ-induced NH(4)(+) accumulation. Similarly, changes in enzymes responsible for NH(4)(+) accumulation induced by MJ were observed to be inhibited by DMTU, SB, GSH, PBN DPI, IMD, WM, or LY. Seedlings of rice cultivar Taichung Native 1 (TN1) are jasmonic acid (JA)-sensitive and those of cultivar Tainung 67 (TNG67) are JA-insensitive. On treatment with JA, H(2)O(2) accumulated in the leaves of TN1 seedlings but not in the leaves of TNG67. Ethylene action inhibitor, silver thiosulfate, was observed to inhibit MJ- and abscisic acid-induced accumulation of NH(4)(+) and changes in enzymes responsible for NH(4)(+) accumulation in detached rice leaves, suggesting that the action of MJ and ABA is ethylene dependent.

Hydrogen peroxide is required for abscisic acid-induced NH4+ accumulation in rice leaves.

The role of H2O2 in abscisic acid (ABA)-induced NH4+ accumulation in rice leaves was investigated. ABA treatment resulted in an accumulation of NH4+ in rice leaves, which was preceded by a decrease in the activity of glutamine synthetase (GS) and an increase in the specific activities of protease and phenylalanine ammonia-lyase (PAL). GS, PAL, and protease seem to be the enzymes responsible for the accumulation of NH4+ in ABA-treated rice leaves. Dimethylthiourea (DMTU), a chemical trap for H2O2, was observed to be effective in inhibiting ABA-induced accumulation of NH4+ in rice Leaves. Inhibitors of NADPH oxidase, diphenyleneiodonium chloride (DPI) and imidazole (IMD), and nitric oxide donor (N-tert-butyl-alpha-phenylnitrone, PBN), which have previously been shown to prevent ABA-induced increase in H2O2 contents in rice leaves, inhibited ABA-induced increase in the content of NH4+. Similarly, the changes of enzymes responsible for NH4+ accumulation induced by ABA were observed to be inhibited by DMTU, DPI, IMD, and PBN. Exogenous application of H2O2 was found to increase NH4+ content, decrease GS activity, and increase protease and PAL-specific activities in rice leaves. Our results suggest that H2O2 is involved in ABA-induced NH4+ accumulation in rice leaves.

Expression of ascorbate peroxidase and glutathione reductase in roots of rice seedlings in response to NaCl and H2O2.

The accumulation of H2O2 by NaCl was observed in the roots of rice seedlings. Treatment with NaCl caused an increase in the activities of ascorbate peroxidase (APX) and glutathione reductase (GR) and the expression of OsAPX and OsGR in rice roots. Exogenously applied H2O2 also enhanced the activities of APX and GR and the expression of OsAPX and OsGR in rice roots. The accumulation of H2O2 in rice roots in response to NaCl was inhibited by the NADPH oxidase inhibitors, diphenyleneiodonium chloride (DPI) and imidazole (IMD). However, DPI, IMD, and dimethylthiourea, a H2O2 trap, did not reduce NaCl-enhanced activities of APX and GR and expression of OsAPX and OsGR. It appears that H2O2 is not involved in the regulation of NaCl-induced APX and GR activities and OsAPX and OsGR expression in rice roots.