Nitric oxide induced by hydrogen peroxide mediates abscisic acid-induced activation of the mitogen-activated protein kinase cascade involved in antioxidant defense in maize leaves

Authors


Author for correspondence: Mingyi Jiang Tel: +86 25 84396372 Fax: +86 25 84396542 Email: myjiang@njau.edu.cn

Summary

  • • The role of nitric oxide (NO) and the relationship between NO, hydrogen peroxide (H2O2) and mitogen-activated protein kinase (MAPK) in abscisic acid (ABA)-induced antioxidant defense in leaves of maize (Zea mays) plants were investigated.
  • • Both ABA and H2O2 induced increases in the generation of NO in mesophyll cells of maize leaves, and H2O2 was required for the ABA-induced generation of NO. Pretreatment with NO scavenger and nitric oxide synthase (NOS) inhibitor substantially reduced the ABA-induced production of NO, and partly blocked the activation of a 46 kDa MAPK and the expression and the activities of several antioxidant enzymes induced by ABA. Treatment with the NO donor sodium nitroprusside (SNP) also induced the activation of the MAPK, and enhanced the antioxidant defense systems.
  • • Conversely, SNP treatment did not induce the production of H2O2, and pretreatments with NO scavenger and NOS inhibitor did not affect ABA-induced H2O2 production.
  • • Our results suggest that ABA-induced H2O2 production mediates NO generation, which, in turn, activates MAPK and results in the upregulation in the expression and the activities of antioxidant enzymes in ABA signaling.

Introduction

Increasing evidence indicates that nitric oxide (NO), which was first identified as a unique diffusible molecular messenger in animals, plays important roles in plant various physiological processes, including defense responses and programmed cell death (PCD), hormone responses, abiotic stress, root and xylem development, germination, iron homeostasis and flowering (reviewed in Lamattina et al., 2003; Neill et al., 2003; Wendehenne et al., 2004; Delledonne, 2005). It has been shown that NO is involved in the phytohormone abscisic acid (ABA) signaling. Treatment with ABA induces the generation of NO in guard cells, which is required for ABA-induced stomatal closure (Desikan et al., 2002; Garcia-Mata & Lamattina, 2002; Neill et al., 2002; Desikan et al., 2004). Using pharmacological and genetic approaches, it has been demonstrated that ABA-induced H2O2 production mediates NO generation, which in turn induces stomatal closure (Bright et al., 2006). However, guard cells are highly specialized cells, and it is not clear whether the model is also applicable to other cells.

Abscisic acid, as a stress signal, plays critical roles in the regulation of plant water balance and osmotic stress tolerance under drought, cold and salt stress conditions (Finkelstein et al., 2002; Zhu, 2002). It induces a myriad of cellular responses in plants through complex signal transduction cascades, leading to tolerance towards these stress conditions (Zhu, 2002). Accumulating evidence indicates that ABA-enhanced water stress tolerance results, at least in part, from the induction of antioxidant defense systems (Jiang & Zhang, 2002a,b,c, 2004). Previous study has shown that that Ca2+-calmodulin (CaM), NADPH oxidase and H2O2 are required for ABA-induced upregulation in the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) in leaves of maize plants (Jiang & Zhang, 2003; Hu et al., 2005, 2007). A recent study suggested that NO is also involved in the upregulation in the activities of antioxidant enzymes such as SOD, CAT, and APX induced by ABA in leaves of Stylosanthes guianensis (Zhou et al., 2005). However, studies have also shown that NO inhibits the expression and the activity of thylakoidal APX (tAPX; Murgia et al., 2004b), and inhibits the total activities of APX and CAT (Clark et al., 2000; Murgia et al., 2004a,b). More evidence is required for the involvement of NO in ABA-induced antioxidant defense in plants.

Mitogen-activated protein kinase (MAPK) cascades are believed to be one of the major pathways by which extracellular stimuli are transduced into intracellular responses in plant cells (Tena et al., 2001; Zhang & Klessig, 2001; Jonak et al., 2002; Nakagami et al., 2005). A recent study showed that ABA-induced H2O2 production activates a 46 kDa MAPK, which in turn induces the expression and the activities of antioxidant enzymes, and the activation of MAPK also enhances the production of H2O2, forming a positive feedback loop (Zhang et al., 2006). Although it has been shown that NO is involved in the activation of MAPK activity during the plant defense responses against pathogen infections in tobacco (Nicotiana tabacum; Kumar & Klessig, 2000) and Arabidopsis thaliana (Clarke et al., 2000), and the adventitious root formation induced by indole acetic acid in cucumber (Cucumis sativus; Pagnussat et al., 2004), it is not clear whether NO is involved in the ABA-induced activation of MAPK and if so, what the relationship between NO, H2O2 and MAPK in the ABA signaling is.

In the present study, using pharmacological and biochemical approaches, the role of NO and the relationship between NO, H2O2 and MAPK in ABA-induced upregulation in the expression of several antioxidant genes such as CAT1, encoding CAT isozyme 1, cAPX, encoding a cytosolic isoform of APX, and GR1, encoding a plastidial isoform of GR, and the total activities of the antioxidant enzymes CAT, APX, GR and SOD were investigated in leaves of maize (Zea mays) plants. Our results show that NO is involved in the ABA-induced upregulation in the expression and the activities of antioxidant enzymes. ABA-induced NO generation, which acts downstream of H2O2 production, activates a MAPK, resulting in the induction of antioxidant defense systems in the ABA signaling in leaves of maize plants.

Materials and Methods

Plant material and treatments

Seeds of maize (Zea mays L. cv. Nongda 108; from Nanjing Agricultural University, China) were sown in trays of sand in a light chamber at a temperature of 22–28°C, photosynthetic active radiation (PAR) of 200 µmol m−2 s−1 and a photoperiod of 14 h light/10 h dark), and watered daily. Seeds of the ABA-deficient vp5 mutant and wild-type maize were obtained by selfing plants grown from heterozygous seed (Maize Genetics Stock Center, Urbana, IL, USA). Selfed ears with kernels segregating for the mutation were chosen; mutant kernels were identified by the lack of carotenoid pigmentation. Mutant and wild-type seedlings were grown as described above. When the second leaves were fully expanded, the plants were collected and used for all investigations.

The plants were excised at the base of the stem, and placed in the distilled water for 1 h to eliminate wound stress. After treatment, the cut ends of the stems were placed in the beakers wrapped with aluminum foil containing 100 µm ABA or 10 mm H2O2 or 100 µm sodium nitroprusside (SNP) solution for various times up to 12 h at 25°C, with a continuous light intensity of 200 µmol m−2 s−1. To investigate the role of endogenous ABA, the detached mutant and wild-type plants were treated with 10% polyethylene glycol (PEG 6000) for 2 h under the same conditions as described earlier. In order to study the effects of various inhibitors or scavengers, the detached plants were pretreated with 200 µm 2–4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), 200 µm NG-nitro-l-Arg methyl ester (l-NAME), 100 µm 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) 10 µm 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene (U0126), 100 µm diphenylene iodonium (DPI) and 5 mm dimethylthiourea (DMTU) for 2 h, and then exposed to 100 µm ABA or 10 mm H2O2 or 100 µm SNP treatment for various times up to 12 h under the same conditions as described earlier. Detached plants were treated with distilled water under the same conditions for the whole period and served as controls for the above. After treatments of detached maize plants, the second leaves were sampled and immediately frozen under liquid N2 for further analysis.

Nitric oxide detection by confocal laser scanning microscopy (CLSM)

Measurement of NO was performed with the specific NO dye DAF-2DA, using the method as described by Corpas et al. (2004) with slight modifications. Leaf segments of approx. 0.5 cm2 were incubated in loading buffer (0.1 mm CaCl2, 10 mm KCl, 10 mm 2-(N-morpholino) ethanesulfonic acid (MES)-Tris, pH 5.6) and 4,5-diaminofluorescein diacetate (DAF-2DA) at a final concentration of 10 µm for 1 h in the dark at 25°C, followed washing with loading buffer for 1 h. All images were visualized using CLSM (excitation 495 nm, emission 515 nm). Images acquired were analysed using Leica image software (Leica Microsystems, Heerbrugge, Switzerland). Data are presented as average fluorescence intensity.

Protein extraction and in-gel kinase activity assay

Protein was extracted from leaves with an extraction buffer (100 mm N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.5, 5 mm ethylenediaminetetraacetic acid (EDTA), 5 mm ethylene glycol-bis(beta-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 10 mm dithiothreitol (DTT), 10 mm Na3VO4, 10 mm NaF, 1 mm phenylmethylsulfonyl fluoride (PMSF), 5 µg ml−1 leupeptin, 5 µg ml−1 aprotinin, 5% glycerol, 50 mmβ-glycerophosphate) using the method of Zhang & Klessig (1997) with minor modifications. After centrifugation at 15 000 g for 30 min at 4°C, the supernatants were transferred into clean tubes and immediately frozen with liquid N2, and stored at –80°C. Protein content was determined according to the method of Bradford (1976) with bovine serum albumin (BSA) as standard.

In-gel kinase activity assays were performed using the method described by Zhang & Klessig (1997). Extracts containing 20 µg of protein were electrophoresed on 10% SDS-polyacrylamide gels embedded with 0.25 mg ml−1 of myelin basic protein (MBP) in the separating gel as a kinase substrate. After electrophoresis, SDS was removed by washing the gel with washing buffer (25 mm Tris, pH 7.5, 0.5 mm DTT, 0.1 mm Na3VO4, 5 mm NaF, 0.5 mg ml−1 BSA, and 0.1% Triton X-100) three times for 30 min each at room temperature. The kinases were allowed to renature in 25 mm Tris, pH 7.5, 1 mm DTT, 0.1 mm Na3VO4, and 5 mm NaF at 4°C overnight with three changes of buffer. The gel was then incubated at room temperature in 30 ml of reaction buffer (25 mm Tris, pH 7.5, 2 mm EGTA, 12 mm MgCl2, 1 mm DTT, and 0.1 mm Na3VO4) with 200 nm ATP plus 50 µCi γ-32P-ATP (3000 Ci mm1) for 60 min. The reaction was stopped by transferring the gel into 5% trichloroacetic acid (w : v)/1% sodium pyrophosphate (w : v). The unincorporated γ-32P-ATP was removed by washing with the same solution for at least 6 h with five changes. The gel was dried onto Whatman 3 mm paper and exposed to Kodak XAR-5 film (Rochester, NY, USA). Prestained size markers (Bio-Rad, Hercules, CA, USA) were used to calculate the size of the kinases. Quantification of the relative kinase activities was done using a Quantity One (Bio-Rad).

Immunoprecipitation kinase activity assay

Protein extract (50 µg) was incubated with 2 µg antiphosphotyrosine monoclonal antibody 4G10 (Upstate Biotechnology, Lake Placid, NY, USA) in immunoprecipitation buffer (20 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 2 mm EGTA, 1 mm Na3VO4, 1 mm NaF, 10 mmβ-glycerophosphate, 2 µg ml−1 antipain, 2 µg ml−1 aprotinin, 2 µg ml−1 leupeptin, 0.5% (v : v) Triton X-100, and 0.5% (v : v) Nonidet P-40) at 4°C for 4 h on a rocker. About 20 µl packed volume of protein G agarose was added, and the incubation was continued for another 2 h. Agarose bead-protein complexes were pelleted by brief centrifugation. After washing with immunoprecipitation buffer three times, 1× SDS sample buffer was added and boiled for 3 min. After centrifugation, the supernatant fraction was electrophoresed on 10% SDS-polyacrylamide gels, and the in-gel kinase assay was performed.

Isolation of total RNA and semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR)

Total RNA was isolated from leaves by using RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the instructions supplied by the manufacturer. Approximately 3 µg of total RNA were reverse transcribed using oligo(dT) primer and SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). cDNA was amplified by PCR using the following primers: CAT1, forward 5′-CCAAGGGTTTCTTTGAGGT-3′ and reverse 5′-AGGGTCGAAGGAACGATAT-3′; cAPX, forward 5′-TCGGCACCATGAAGAACCC-3′ and reverse 5′-TCCTCGTCCGCTGCGTATT-3′; GR1, forward 5′-GAAGGTCGTGGAAAGATA-3′ and reverse 5′-TTGGCAACGAAGACATCA-3′; β-actin, forward 5′-AAATGACGC AGA TTA TGT TTG A-3′ and reverse 5′-GCTCGTAGTGAGGGAGTACC-3′. To standardize the results, the relative abundance of β-actin was also determined and used as the internal standard.

The cycle number of the PCR reactions was adjusted for each gene to obtain barely visible bands in agarose gels. Aliquots of the PCR reactions were loaded on agarose gels and stained with ethidium bromide.

Real-time quantitative RT-PCR

Expression pattern of several antioxidant enzymes was also analyzed by real-time RT-PCR using DNA Engine Opticon 2 (Bio-Rad, Hercules, California, USA) with SYBR ExScript RT-PCR Kit (Takara Bio Inc., Otsu, Shiga, Japan). The cDNA was amplified by real time PCR using the following primers: CAT1, forward 5′-CTAACAGGCTGTCGTGAGAAGTG-3′ and reverse 5′-TGTCAGTGCGTCAACCCATC-3′; cAPX, forward 5′-TCCTATCCTACGCTGACTTCTACC-3′ and reverse 5′-TGGTCGCTCAAACCCATCTG-3′; GR1, forward 5′-TTGCAACAGGTCGCAGACC-3′ and reverse 5′-CTCCAACAGCCCAAATGGAA-3′; β-actin, forward 5′-GATTCCTGGGATTGCCGAT-3′ and reverse 5′-TCTGCTGCTGAAAAGTGCTGAG-3′. Gene-specific primers were designed with the software tool Primer Express (Applied Biosystems, Foster city, CA, USA). The PCR amplification consisted of initial denaturation at 94°C for 1 min, then 44 cycles of 94°C for 5 s, 62°C for 10 s and 68°C 15 s. Standard curves were constructed using PCR products by 10-fold serial dilutions in 1 : 102, 1 : 103, 1 : 104, 1 : 105, 1 : 106, 1 : 107, 1 : 108 along with a nontemplate control. The antioxidant enzymes gene expression profiles obtained from the RT-PCR were subsequently normalized by the amount of β-actin transcript per reaction to correct the differences in RNA amounts and reverse transcription reactions. Three replicates were performed for each sample, and the average values were calculated.

Enzyme assays

Frozen leaf segments (0.5 g) were homogenized in 10 ml of 50 mm potassium phosphate buffer (pH 7.0) containing 1 mm EDTA and 1% polyvinylpyrrolidone, with the addition of 1 mm ascorbate in the case of APX assay. The homogenate was centrifuged at 15 000 g for 20 min at 4°C and the supernatant was immediately used for the following antioxidant enzyme assays.

The total activities of antioxidant enzymes were determined as previously described (Jiang & Zhang, 2001). Total SOD activity was assayed by monitoring the inhibition of photochemical reduction of nitro blue tetrazolium. One unit of SOD activity was defined as the amount of enzyme that was required to cause 50% inhibition of the reduction of nitro blue tetrazolium, as monitored at 560 nm. Total CAT activity was assayed by measuring the rate of decomposition of H2O2 at 240 nm. Total APX activity was measured by monitoring the decrease in absorbance at 290 nm as ascorbate was oxidized. Total GR activity was measured by following the change in absorbance at 340 nm as oxidized glutathione (GSSG)-dependent oxidation of NADPH.

Cytochemical detection of H2O2

H2O2 was visualized at the subcellular level using CeCl3 for localization (Bestwick et al., 1997). Electron-dense CeCl3 deposits are formed in the presence of H2O2 and are visible by transmission electron microscopy. Pieces of issue (c. 1–2 mm2) were excised from the treated and untreated leaves and incubated in freshly prepared 5 mm CeCl3 in 50 mm 3-(N-morpholino)propanesulfonic acid (MOPS) at pH 7.2 for 1 h. The leaf sections were then fixed in 1.25% (v : v) glutaraldehyde and 1.25% (v : v) paraformaldehyde in 50 mm sodium cacodylate buffer, pH 7.2, for 1 h. After fixation, tissues were washed twice for 10 min in the same buffer, postfixed for 45 min in 1% (v : v) osmium tetroxide, and then dehydrated in a graded ethanol series (30–100%; v : v) and embedded in Eponaraldite (Agar Aids, Bishop's Storford, UK). After 12 h in pure resin, followed by a change of fresh resin for 4 h, the samples were polymerized at 60°C for 48 h. Blocks were sectioned (70–90 nm) on a Reichert-Ultracut E microtome (Reichert-Jung, Leica, Bensheim, Germany), and mounted on uncoated copper grids (300 mesh). Sections were examined using a transmission electron microscope at an accelerating voltage of 75 kV.

Results

Abscisic acid or H2O2 treatment induces NO generation and H2O2 is required for ABA-induced NO production in maize leaves

Previous studies have shown that ABA treatment can cause increased generation of NO in guard cells (Desikan et al., 2002; Garcia-Mata & Lamattina, 2002; Neill et al., 2002; Bright et al., 2006), and H2O2 treatment can also induce increased production of NO (Lum et al., 2002; He et al., 2005; Bright et al., 2006). In order to investigate the production of NO in leaves of maize plants exposed to ABA or H2O2 treatments, the leaf segments were loaded with the NO-specific fluorescent dye DAF-2DA and CLSM was used to monitor changes in NO-induced fluorescence in mesophyll cells of maize leaves. Treatment with 100 µm ABA led to a rapid increase in DAF-2DA fluorescence (Fig. 1a). The DAF-2DA fluorescence was visible as early as 0.5 h, maximized at 2 h and remained high for 4 h after ABA treatment, and then decreased after 6 h of ABA treatment. Similar changes were observed in H2O2-treated leaves, but a strong increase occurred at 1 h and a substantial reduction began at 4 h after H2O2 treatment (Fig. 1a). The ABA- and H2O2-induced fluorescence was dose-dependent in the concentration range 10–100 µm ABA and 1–10 mm H2O2 (Fig. 1a). By contrast, no increase in fluorescence was observed in ABA- and H2O2-treated leaves when the leaf segments were loaded with the negative probe 4AF-DA, which lacks one of the amino groups that constitutes the NO specificity domain of the DAF-2DA molecule (Fig. 1b). Moreover, pretreatments with 2–4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a specific scavenger of NO, and NG-nitro-l-Arg methyl ester (l-NAME), a well-known inhibitor of nitric oxide synthase (NOS) activity in animals, substantially reduced the DAF-2DA fluorescence intensity induced by ABA or H2O2 treatment (Fig. 1c). These results clearly indicate that both ABA and H2O2 do induce an increased generation of NO in mesophyll cells of maize leaves.

Figure 1.

Nitric oxide (NO) production in response to abscisic acid (ABA) or hydrogen peroxide (H2O2) treatment in maize (Zea mays) leaves. (a) Time-course and dose dependence for ABA- and H2O2-induced NO production in mesophyll cells of maize leaves. The detached plants were treated with 100 µm ABA (closed diamonds) and 10 mm H2O2 (triangles) for various times (left; control, open diamonds), or different concentrations of ABA and H2O2 for 2 h (right), and then the leaf segments were loaded with 4,5-diaminofluorescein diacetate (DAF-2DA) and detected by confocal laser scanning microscopy (CLSM). (b) DAF-2DA fluorescence accumulation in ABA- or H2O2-treated mesophyll cells of maize leaves. The detached plants were treated with distilled water (control), 100 µm ABA or 10 mm H2O2 for 2 h, and the leaf segments were loaded with DAF-2DA (top) or 4AF-DA (bottom) and detected by CLSM. (c) Effects of pretreatments with 2–4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and NG-nitro-l-Arg methyl ester (l-NAME) on ABA- or H2O2-induced NO production. The detached plants were pretreated with 200 µm cPTIO or 200 µm l-NAME for 2 h, and then exposed to 100 µm ABA or 10 mm H2O2 for 2 h. Detached plants treated with distilled water served as controls. (a,c) Data are means ± SE of three different experiments. Means denoted by the same letter did not differ significantly at P < 0.05 according to Duncan's multiple range test. In (b), experiments were repeated at least three times, with similar results.

To investigate whether the generation of NO can be induced by endogenous ABA, the ABA-deficient maize vp5 mutant, which interrupts ABA biosynthesis early in the biosynthetic pathway (Guan & Scandalios, 1998; Sharp, 2002), and wild type were used. Treatment with 10% PEG for 2 h resulted in a significant increase in the production of NO in the wild type, but only a slight increase in the mutant (Fig. 2b). Application of 100 µm ABA restored the level in the production of NO in the leaves of mutant maize plants exposed to water stress, when compared with the wild type. These results indicated that ABA is required for the induction of NO generation in the leaves of maize plants exposed to water stress.

Figure 2.

Hydrogen peroxide (H2O2) is required for abscisic acid (ABA)-induced nitric oxide (NO) production in mesophyll cells of maize (Zea mays) leaves. (a) Effects of pretreatments with diphenylene iodonium (DPI), dimethylthiourea (DMTU) and catalase (CAT) on ABA-induced NO production. The detached plants were pretreated with DPI (100 µm), DMTU (5 mm) and CAT (200 U) for 2 h, respectively, and then exposed to ABA (100 µm) treatment for 2 h. Detached plants treated with distilled water served as controls. (b) Water stress-induced ABA and H2O2 mediate NO generation. The detached vp5 mutant and wild-type maize plants were pretreated with DPI (100 µm), DMTU (5 mm), CAT (200 U), ABA (100 µm), DMTU (5 mm) + ABA (100 µm), and CAT (200 U) + ABA (100 µm) for 2 h, respectively, and then exposed to 10% polyethylene glycol (PEG) for 2 h. Detached plants treated with distilled water served as controls. Data are means ± SE of three different experiments. Means denoted by the same letter did not differ significantly at P < 0.05 according to Duncan's multiple range test.

To determine whether H2O2 mediates the ABA-induced NO production, the NADPH oxidase inhibitor DPI and the H2O2 scavenger DMTU and CAT were applied. Pretreatments with DPI, DMTU and CAT substantially blocked the increases in the production of NO induced by exogenously applied ABA and water stress in the leaves of wild-type plants, and in the mutant restored by the addition of ABA (Fig. 2a,b). These results suggested that H2O2 was required for the ABA-induced generation of NO in leaves of maize plants.

Nitric oxide is involved in ABA- and H2O2-induced enhancements in the transcript levels and the activities of antioxidant enzymes

Previous work has been shown that treatments with ABA and H2O2 can enhance the expression of several antioxidant genes such as CAT1, cAPX and GR1, and the total activities of the antioxidant enzymes CAT, APX and GR, and SOD (Zhang et al., 2006). To establish a link between NO production and the antioxidant defense systems in ABA or H2O2 signaling, the detached plants were pretreated with the NO scavenger cPTIO and the NOS inhibitor l-NAME, and then exposed to ABA or H2O2 treatment. Experimental results showed that pretreatments with 200 µm cPTIO and 200 µm l-NAME partly blocked the ABA- and H2O2-induced enhancements in the transcript levels of antioxidant genes analysed by semi-quantitative RT-PCR (Figs 3a and 4a) and real-time quantitative RT-PCR (Figs 3b and 4b) and the total activities (Figs 3c and 4c) of antioxidant enzymes, but these pretreatments alone did not affect the expression and the activities of antioxidant enzymes in the control leaves (data not shown). These results suggested that NO is involved in the ABA- and H2O2-induced upregulation in the expression and the activities of antioxidant enzymes in maize leaves. Moreover, treatment with the NO donor SNP also induced increases in the transcript levels (Fig. 5a) and the total activities (Fig. 5c) of antioxidant enzymes, and the increases were substantially arrested by the pretreatment with the NO scavenger cPTIO (Figs 5b,c). By contrast, treatment with sodium ferricyanide (Fe(III)CN), which shares many structural features with SNP but lacks a nitroso group and thus the ability to generate NO (Bethke et al., 2006), did not induce the increase in the transcript levels of antioxidant enzymes (Fig. 5b). These results clearly indicate that NO itself can induce the upregulation of antioxidant defense systems in plants.

Figure 3.

Effects of pretreatments with 2–4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and NG-nitro-l-Arg methyl ester (l-NAME) on abscisic acid (ABA)-induced increases in the expression and the total activities of antioxidant enzymes in leaves of maize (Zea mays) plants. (a,b) Transcript levels of antioxidant genes CAT1, cAPX and GR1 analysed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) (a) and by real-time RT-PCR (b). (c) The total activities of antioxidant enzymes such as catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and superoxide dismutase (SOD. The detached plants were treated as follows: 1, distilled water (control); 2, 100 µm ABA; 3, 200 µm cPTIO + 100 µm ABA; 4, 200 µml-NAME + 100 µm ABA. The detached plants were pretreated with inhibitors for 2 h, and then exposed to ABA treatment for 8 h (a,b) or 12 h (c). (a) Experiments were repeated at least three times with similar results; (b,c), values are means ± SE of three different experiments. Means denoted by the same letter did not differ significantly at P < 0.05 according to Duncan's multiple range test.

Figure 4.

Effects of pretreatments with 2–4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and NG-nitro-l-Arg methyl ester (l-NAME) on the expression and the total activities of antioxidant enzymes in leaves of maize (Zea mays) plants exposed to hydrogen peroxide (H2O2) treatment. (a,b) Transcript levels of antioxidant genes CAT1, cAPX and GR1 analysed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) (a) and by real-time RT-PCR (b). (c) Total activities of antioxidant enzymes such as catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and superoxide dismutase (SOD. Detached plants were treated as follows: 1, distilled water (control); 2, 10 mm H2O2; 3, 200 µm cPTIO + 10 mm H2O2; 4, 200 µm L-NAME + 10 mm H2O2. The detached plants were pretreated with inhibitors for 2 h, and then exposed to H2O2 treatment for 8 h (a,b) or 12 h (c). (a) Experiments were repeated at least three times with similar results; (b,c), values are means ± SE of three different experiments. Means denoted by the same letter did not differ significantly at P < 0.05 according to Duncan's multiple range test.

Figure 5.

Effects of the nitric oxide (NO) donor sodium nitroprusside (SNP) on the expression and the total activities of antioxidant enzymes in leaves of maize (Zea mays) plants. (a) Time course of changes in the transcript levels of antioxidant genes CAT1, cAPX and GR1 analysed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in maize leaves exposed to 100 µm SNP treatment. (b) Treatment with Fe(III)CN did not induce the enhancement in the transcript levels of antioxidant genes, and pretreatment with 2–4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) blocks SNP-induced increases in the transcript levels of antioxidant genes. The detached plants were pretreated with distilled water or 200 µm cPTIO for 2 h, and then exposed to distilled water, 100 µm Fe(III)CN or 100 µm SNP for 8 h (c) The total activities of antioxidant enzymes such as catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and superoxide dismutase (SOD). (a,b), Experiments were repeated at least three times with similar results; (c) values are means ± SE of three different experiments. Means denoted by the same letter did not differ significantly at P < 0.05 according to Duncan's multiple range test. Closed diamonds, open diamonds, control; SNP; closed squares, cPTIO + SNP.

Nitric oxide mediates ABA- and H2O2-induced activation of MAPK involved in antioxidant defense

It has been shown that MAPK cascade is involved in ABA-induced antioxidant defense and acts downstream of H2O2 production in leaves of maize plants (Zhang et al., 2006). To investigate the role of NO in ABA- and H2O2-activated MAPK, the detached plants were pretreated with cPTIO and l-NAME, and in-gel kinase assays were performed on protein extracts, using MBP as a substrate. The activation of a 46 kDa MBP kinase induced by ABA and H2O2 was partly blocked by the pretreatments with the NO scavenger cPTIO and the NOS inhibitor l-NAME in leaves of maize plants (Fig. 6a), suggesting that NO mediates the ABA- and H2O2-induced activation of MBP kinase. Moreover, SNP treatment led to a rapid activation of the MBP kinase (Fig. 6b). A significant increase in the activity of the kinase occurred within 30 min and maximized at 60 min after SNP treatment, and then decreased after 2 h of SNP treatment. The activation of MBP kinase by SNP treatment was completely blocked by the pretreatment with the NO scavenger cPTIO (Fig. 6d). Meanwhile, treatment with Fe(III)CN did not induce the activation of MBP kinase (Fig. 6d). These results indicate that NO itself can induce the activation of MBP kinase.

Figure 6.

Nitric oxide (NO) is involved in abscisic acid (ABA)- and hydrogen peroxide (H2O2)-induced activation of myelin basic protein (MBP) kinase and reactive oxygen species (ROS) are not required for NO-induced activation of MBP kinase in maize (Zea mays) leaves. (a) Effects of pretreatments with 2–4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and NG-nitro-l-Arg methyl ester (l-NAME) on ABA- or H2O2-induced activation of MBP kinase. The detached plants were pretreated with 200 µm cPTIO and 200 µm L-NAME for 2 h, and then exposed to 100 µm ABA treatment for 4 h or 10 mm H2O2 treatment for 1 h, respectively. Detached plants treated with distilled water served as controls. (b) Time-course of SNP-induced activation of MBP kinase. The detached plants were treated with 100 µm SNP for various times. (c) Tyrosine phosphorylation of SNP-activated MBP kinase. The detached plants were treated with 100 µm SNP for 1 h. Protein extracts from control- or SNP-treated leaves were immunoprecipitated with 4G10, and then subjected to in-gel kinase assay. (d) Treatment with Fe(III)CN does not activate MBP kinase and pretreatments with diphenylene iodonium (DPI) and dimethylthiourea (DMTU) do not affect the SNP-induced activation of MBP kinase. Detached plants were pretreated with distilled water, 200 µm cPTIO, 100 µm DPI, and 5 mm DMTU for 2 h, and then exposed to 100 µm SNP or 100 µm Fe(III)CN for 1 h. Detached plants treated with distilled water served as controls. (a,d) Values are means ± SE of three different experiments. Means denoted by the same letter did not differ significantly at P < 0.05 according to Duncan's multiple range test. (b,c) Experiments were repeated at least three times with similar results.

To demonstrate the 46 kDa MBP kinase is a MAPK-like enzyme, immunoprecipitation was performed on protein extracts using the antiphosphotyrosine monoclonal antibody 4G10, which has been widely used to demonstrate tyrosine phosphorylation of MAPKs, an important characteristic of MAPKs (Zhang & Klessig, 1997; Zhang et al., 1998; Desikan et al., 1999; Burnett et al., 2000; Hoyos & Zhang, 2000; Ichimura et al., 2000). As shown in Fig. 6c, the immunoprecipitated tyrosine phosphorylated-MBP kinase activity correlated with the MBP kinase activity in the protein extracts (Fig. 6b), suggesting that the 46 kDa MBP kinase is a MAPK.

To establish a link between NO-activated MAPK and the antioxidant defense systems, the detached plants were pretreated with PD98059 and U0126, two widely used specific inhibitors of MAPK kinase (MAPKK), and then exposed to SNP treatment. As shown in Fig. 7, the SNP-induced increases in the transcript levels (Fig. 7a) and the total activities (Fig. 7b) of antioxidant enzymes were almost completely blocked by pretreatments with the MAPKK inhibitors, suggesting that NO-activated MAPK is involved in NO-induced upregulation in antioxidant defense systems in the leaves of maize plants.

Figure 7.

Effects of pretreatments with mitogen-activated protein kinase kinase (MAPKK) inhibitors and reactive oxygen species (ROS) inhibitor or scavenger on sodium nitroprusside (SNP)-induced increases in the expression and the total activities of antioxidant enzymes. (a) Transcript levels of antioxidant genes CAT1, cAPX and GR1 analysed by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). (b) Total activities of antioxidant enzymes such as catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and superoxide dismutase (SOD). Detached plants were treated as follows: 1, distilled water (control); 2, 100 vm SNP; 3, 100 µm 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) + 100 µm SNP; 4, 10 µm 1,4-diamino-2,3-dicyano- 1,4-bis)o-aminophenylmercapto)butadiene (U0126) + 100 µm SNP; 5, 100 µm diphenylene iodonium (DPI) + 100 µm SNP; 6, 5 mm dimethylthiourea (DMTU) + 100 µm SNP. The detached plants were pretreated with inhibitors for 2 h, and then exposed to SNP treatment for 8 h (a) or 12 h (b). (a) Experiments were repeated at least three times with similar results; (b) values are means ± SE of three different experiments. Means denoted by the same letter did not differ significantly at P < 0.05 according to Duncan's multiple range test.

To determine whether H2O2 mediates NO-induced antioxidant defense, the NADPH oxidase inhibitor DPI and the H2O2 scavenger DMTU were applied. Pretreatments with DPI and DMTU did not affect the activation of MBP kinase (Fig. 6d) and the increases in the transcript levels (Fig. 7a) and the total activities (Fig. 7b) of antioxidant enzymes induced by SNP treatment, suggesting that H2O2 is not required for NO-activated MBP kinase and NO-induced antioxidant defense.

Nitric oxide does not induce H2O2 synthesis and the inhibition of MAPK cascade does not affect NO production

To further determine the interrelationship between H2O2, NO and MAPK in the ABA signaling, the effects of NO on H2O2 production, and MAPK and H2O2 on NO production were investigated. Figure 8a shows that during the 6-h SNP treatment, no visible H2O2 accumulation, detected by CeCl3 staining and transmission electron microscopy, was observed in the mesophyll cells, indicating that NO does not induce H2O2 synthesis. Furthermore, pretreatments with cPTIO and l-NAME also did not affect the ABA-induced H2O2 production in the mesophyll cells of maize leaves (Fig. 8b). Pretreatments with the MAPK inhibitors PD98059 and U0126 did not affect the increase in the generation of NO induced by ABA (Fig. 9).

Figure 8.

Sodium nitroprusside (SNP) treatment does not induce hydrogen peroxide (H2O2) production and pretreatment with 2–4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and NG-nitro-l-Arg methyl ester (l-NAME) does not affect abscisic acid (ABA)-induced H2O2 accumulation in maize (Zea mays) leaves with CeCl3-staining and transmission electron microscopy. (a) Effect of 100 µm SNP treatment on H2O2 production. (b) Effects of pretreatments with cPTIO and l-NAME on ABA-induced H2O2 production. Detached plants were pretreated with 200 µm cPTIO and 200 + m l-NAME for 2 h, and then exposed to 100 µm ABA treatment for 2 h. Detached plants treated with distilled water served as controls. All experiments were repeated at least three times with similar results. Arrows, CeCl3 precipitates; C, chloroplast; CW, cell wall; N, nucleus; IS, intercellular space. Bar, 1 µm.

Figure 9.

Effects of pretreatments with DP98059 and U0126 on abscisic acid (ABA)-induced nitric oxide (NO) production in leaves of maize (Zea mays) plants. The detached plants were pretreated with 100 µm 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) and 10 µm 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene (U0126) for 2 h, and then exposed to 100 µm ABA treatment for 2 h. Detached plants treated with distilled water served as controls. Experiments were repeated at least three times with similar results. Data are means ± SE of three different experiments. Means denoted by the same letter did not differ significantly at P < 0.05 according to Duncan's multiple range test.

Discussion

A previous study has suggested that ABA-induced NO can upregulate the activities of antioxidant enzymes such as SOD, CAT and APX in leaves of Stylosanthes guianensis (Zhou et al., 2005). In the present study, our results showed that ABA treatment induced an increase in the generation of NO in mesophyll cells of maize leaves (Fig. 1) and enhanced the expression of the antioxidant genes CAT1, cAPX and GR1 and the total activities of the antioxidant enzymes CAT, APX, GR and SOD (Fig. 3). Such enhancements were blocked by the pretreatments with the NO scavenger cPTIO and the NOS inhibitor l-NAME (Figs 1 and 3). Moreover, treatment with the NO donor SNP (100 µm) also induced increases in the transcript levels and the total activities of antioxidant enzymes, and the increases were substantially arrested by the pretreatment with the NO scavenger cPTIO, but treatment with sodium ferricyanide, which shares many structural features with SNP but lacks a nitroso group and thus the ability to generate NO (Bethke et al., 2006), did not induce an increase in the transcript levels of antioxidant enzymes (Fig. 5). Our results clearly suggested that ABA-induced NO production not only upregulates the activities of antioxidant enzymes, but also induces the expression of antioxidant genes in leaves of maize plants. In previous studies, however, it was also reported that NO produced by SNP treatment inhibited the activities of CAT and APX in vitro (Clark et al., 2000) and in vivo (Murgia et al., 2004a,b). The discrepancy between the studies by Murgia et al. (2004a,b) and our data may be related to the applied concentrations of SNP. Higher concentrations of SNP in Arabidopsis (e.g. 5 mm; Murgia et al., 2004b) and in tobacco (0.5 mm; Murgia et al., 2004a) may produce higher amounts of NO that inhibit the activities of these antioxidant enzymes. It is also possible that different plant materials may have different responses to SNP treatment.

Water stress, including drought, salt or cold stress, can induce ABA accumulation in plant cells (Zhu, 2002), and induce NO generation (Gould et al., 2003; Neill et al., 2003). However, the relationship between ABA accumulation and NO production in plant cells under water stress has not yet been identified. In the present study, using the ABA-deficient vp5 mutant and its wild type, our data show that the water stress-induced increase in the production of NO in the wild-type was substantially reduced in the mutant, and the application of 100 µm ABA, which increased the content of endogenous ABA in maize leaves to a similar extent to water stress (Jiang & Zhang, 2002a), fully restored the production of NO in the mutant (Fig. 2). These results indicate that the water stress-induced increase in the production of NO in leaves of maize plants results mainly from the accumulation of ABA induced by water stress. Our data suggest that the model described above for ABA, NO and antioxidant defense might also exist in maize plants exposed to water stress.

The question of the relationship between NO and H2O2 production in plants exposed to various stresses or stimuli appears particularly interesting. In plants, responses to various stresses or stimuli, NO and H2O2 generation occur in parallel, or in short succession one another, and it has been shown that they can act both synergistically and independently (Delledonne et al., 1998, 2001; Clarke et al., 2000; de Pinto et al., 2002; Lum et al., 2002; Murgia et al., 2004a,b; Zeier et al., 2004; He et al., 2005; Bright et al., 2006). In guard cells, it has been shown that H2O2 treatment can induce NO synthesis (Lum et al., 2002; He et al., 2005; Bright et al., 2006). He et al. (2005) also reported that SNP treatment induced H2O2 production, detected by the fluorescent probe 2′,7′-dichlorofluorescein diacetate (H2DCFDA) and confocal laser scanning microscopy (CLSM) However, the H2DCFDA dye has been shown to be not specific for H2O2 and also reacts with NO (Bright et al., 2006). The H2DCF fluorescence monitored in the presence of SNP by He et al. (2005) was attributable to the reaction of the dye with NO, not with H2O2 (Bright et al., 2006). Using pharmacological and genetic approaches, it has been demonstrated that a linear relationship exists between ABA, H2O2 and NO in ABA-induced stomatal closure (Bright et al., 2006). However, guard cells are highly specialized cells, and it is not clear whether the model described above is also applicable to other cells. In the present study, our data showed that both ABA and H2O2 induced NO production in the mesophyll cells of leaves (Fig. 1), and that the ABA-induced NO production was substantially reduced by pretreatments with DPI, DMTU and CAT (Fig. 2), suggesting that ABA-mediated NO generation is dependent on ABA-induced H2O2 production. Furthermore, water stress-induced NO generation that results from the accumulation of ABA also depends on the production of H2O2 (Fig. 2). However, SNP treatment did not induce H2O2 accumulation (detected by CeCl3 staining and transmission electron microscopy, which is a sensitive and specific detection method for H2O2) and ABA-induced H2O2 accumulation was not affected by the pretreatments with cPTIO and l-NAME (Fig. 8). Our results suggest that the linearity between ABA, H2O2 and NO also exists in the nonstomatal tissues. Considering that ABA-induced H2O2 and NO production occur simultaneously in guard cells, mesophyll cells and vascular tissues, the linear relationship between ABA, H2O2 and NO in ABA signaling might have a common significance.

The MAPK cascade has been shown to be one of the major pathways by which extracellular stimuli are transduced into intracellular responses in plant cells (Tena et al., 2001; Zhang & Klessig, 2001; Jonak et al., 2002; Nakagami et al., 2005). Both ABA and H2O2 can activate MAPKs, and the activation of MAPKs play an important role in plant response to multiple stresses, including oxidative stress, drought and salinity (Kovtun et al., 2000; Lu et al., 2002; Samuel & Ellis, 2002; Moon et al., 2003; Xiong & Yang, 2003). Our recent studies has shown that a MAPK is involved in ABA-induced antioxidant defense, and a crosstalk between H2O2 production and MAPK activation play a pivotal role in ABA signaling (Zhang et al., 2006). Although previous studies have revealed that NO is involved in the activation of MAPK activity during plant defense responses to pathogen infections in tobacco (Kumar & Klessig, 2000) and Arabidopsis cells (Clarke et al., 2000), and adventitious root formation induced by indole acetic acid in cucumber (Pagnussat et al., 2004), it is not clear whether NO is involved in the ABA-induced activation of MAPK and if so, what the relationship between NO and MAPK in ABA signaling is. In the present study, our results showed that treatment with ABA and H2O2 induced activation of a 46 kDa MBP kinase, and that activation was suppressed by pretreatment with the NO scavenger cPTIO and the NOS inhibitor l-NAME (Fig. 6). Sodium nitroprusside treatment also induced the activation of the MBP kinase, and the activation was dependent on NO produced by SNP treatment. Tyrosine phosphorylation, with the size (46 kDa), the use of MBP as a substrate, and the inhibition of activation by the MAPKK inhibitors PD98059 and U0126 (Zhang et al., 2006) clearly suggest that the MBP kinase is a MAPK. Our data suggest that NO is involved in the ABA- and H2O2-induced activation of MAPK, and ABA, H2O2 and NO may share the MAPK signaling pathway in ABA signaling, as suggested recently by Desikan et al. (2004) in the regulation of stomatal closure. By contrast, pretreatments with PD98059 and U0126 did not affect the increase in generation of NO induced by ABA (Fig. 9), suggesting that there also exists a linear relationship between NO and MAPK in ABA signaling.

Taken together, our results suggest that ABA-induced H2O2 production mediates NO generation, which in turn activates MAPK and results in upregulation of the expression and activities of antioxidant enzymes in ABA signaling. In a previous study, however, it was suggested that ABA-induced activation of MAPK also enhances H2O2 production, forming a positive feedback loop (Zhang et al., 2006). In the present study, pretreatments with the MAPKK inhibitors PD98059 and U0126 did not affect the generation of NO induced by ABA. These results suggest that a NO-independent signaling is also involved in ABA- and H2O2-induced antioxidant defense. Why the activation of MAPK enhances H2O2 production but does not affect the generation of NO in ABA signaling is currently unknown but is under investigation in our laboratory.

Acknowledgements

This work was supported by the Major State Basic Research Program of China (grant no. 2003CB114302 to MJ), the National Natural Science Foundation of China (grant nos. 30471048 and 30571122 to M.J.), the Key Project of Chinese Ministry of Education (grant no. 104100 to M.J.), the Science Foundation of Doctoral Subject Point of Chinese Ministry of Education (grant no. 20040307011 to M.J.), and Hong Kong Research Grants Council (HKBU 2149/04M to J.Z.).

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