Science 178 (2010) 130–139

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Plant Science

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Jasmonic acid regulates ascorbate and metabolism in Agropyron cristatum leaves under water stress

Changjuan Shan a,b, Zongsuo Liang a,* a College of Life Science, Northwest A & F University, Yangling 712100, China b Henan Institute of Science and Technology, Xinxiang 453003, China

ARTICLE INFO ABSTRACT

Article history: This study investigated the regulation of ascorbate and glutathione metabolism by jasmonic acid (JA) in Received 11 August 2009 Agropyron cristatum leaves under water stress induced by 10% PEG 6000. The results showed that JA level, Received in revised form 1 November 2009 the transcript levels and activities of APX, GR, MDHAR, DHAR, GalLDH and g-ECS, and the contents of AsA, Accepted 7 November 2009 GSH, total ascorbate and total glutathione were increased by water stress. Above increases except for Available online 14 November 2009 total glutathione content and the transcript level and activity of g-ECS were suppressed by application of JA biosynthesis inhibitor ibuprofen (IBU), a lipoxygenase inhibitor, for 12 h before water stress Keywords: treatment. Application of JA to IBU-inhibited prevented the reduction in the transcript levels and Water stress activities of GalLDH, APX, GR, DHAR and MDHAR, and the contents of AsA, GSH, total ascorbate induced Jasmonic acid Ascorbate by IBU under water stress. Meanwhile, pretreatment with IBU increased the malondialdehyde content Glutathione and electrolyte leakage of plants and these increases were suppressed by application of JA under water Agropyron cristatum stress. Our results suggested that water stress-induced JA is a signal that leads to the regulation of ascorbate and glutathione metabolism and has important role for acquisition of water stress tolerance in A. cristatum. ß 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction (GalLDH) is the key in this way [5]. Gamma-glutamylcys- teine synthetase (g-ECS) is the key enzyme for glutathione Water stress is one of the main environmental factors that biosynthesis [6]. Ascorbate–glutathione cycle is the recycling adversely affect plant growth, productivity and survival. Water pathway of ascorbate and glutathione regeneration. Thus, the stress usually induces the accumulation of reactive species ascorbate–glutathione cycle plays an important role in maintain- (ROS), which cause oxidative damage to plants [1,2]. If not ing the contents of ascorbate and glutathione in plants. In this effectively and rapidly removed from plants, ROS can damage a cycle, ascorbate peroxidase (APX) utilizes AsA as electron donor for wide range of cellular macromolecules such as lipids, and reduction of H2O2, monodehydroascorbate is reduced to AsA by DNA [3]. Plants can protect themselves against oxidative damage monodehydroascorbate reductase (MDHAR) and dehydroascor- by system including antioxidative enzymes and bate is reduced to AsA by dehydroascorbate reductase (DHAR). nonenzymatic compounds [4]. Oxidized glutathione (GSSG) produced in this cycle is reduced to Ascorbate and glutathione are two crucial nonenzymatic GSH by glutathione reductase (GR) [7]. By this way, AsA and GSH compounds involved in defence against . It is well are regenerated and H2O2 is scavenged. known that plants can adjust ascorbate and glutathione contents Jasmonic acid and methyl jasmonate (MeJA), collectively by modulating the regeneration and biosynthesis of ascorbate and termed jasmonates, are regarded as endogenous regulators that glutathione. L-Galactose pathway is the main biosynthetic path- play important roles in regulating stress responses, plant growth way of ascorbate in plants, L-galactono-1,4-lactone dehydrogenase and development [8]. Levels of endogenous jasmonates increase during stress conditions such as treatment with elicitors, wounding, ozone stress and water stress [9–18].Evidencehas Abbreviations: APX, ascorbate peroxidase; GR, glutathione reductase; MDHAR, showed that the expression of jasmonate-responsive monodehydroascorbate reductase; DHAR, dehydroascorbate reductase; GalLDH, L- (JRGs) is altered under stress conditions such as pathogen galactono-1,4-lactone dehydrogenase; g-ECS, gamma-glutamylcysteine synthe- infection, wounding and ozone stress, and thus contributes to tase; GSH, reduced glutathione; AsA, reduced ascorbic acid; IBU, ibuprofen. * Corresponding author. Tel.: +86 29 87092373; fax: +86 29 87092262. stress tolerance in plants [9–11,19–23]. It has been reported that E-mail address: [email protected] (Z. Liang). JRGs can also be induced by drought [24,25]. Increasing evidence

0168-9452/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2009.11.002 C. Shan, Z. Liang / Plant Science 178 (2010) 130–139 131 showed that exogenously supplied JA or MeJA increased collected and frozen in liquid nitrogen, and then kept at À80 8C antioxidative ability of plants under water stress [26–28]. until used for analyses. Ascorbate and glutathione metabolism are important parts of the antioxidant metabolism in plants. It has been documented 2.2. Analysis of APX, GR, DHAR, MDHAR that jasmonic acid is involved in the regulation of ascorbate and glutathione metabolism under ozone stress [23].Previous Enzymes were extracted according to Grace and Logan [32] studies have also showed that jasmonates play an important with some modifications. Each frozen sample (0.5 g) was ground role in signaling drought-induced antioxidant responses, includ- into a fine powder in liquid N2 with a mortar and pestle. Fine ing ascorbate [29–31]. expression in response to environ- powder was homogenized in 6 ml 50 mM KH2PO4 (pH 7.5) mental stress is usually studied at the transcript level because containing 0.1 mM ethylenediaminetetraacetic acid, 0.3% (v/v) this gives a more precise estimate of antioxidant gene activation Triton X-100, and 1% (w/v) insoluble polyvinylpolypyrrolidone. than enzyme activity. Previous studies with respect to the The extract was immediately centrifuged at 13,000 Â g for 15 min regulation of ascorbate and glutathione metabolism by JA under at 2 8C. The supernatant was then used immediately for measuring water stress have been focused on the activities of enzymes, the following enzymes. involved in ascorbate and glutathione metabolism. However, the Ascorbate peroxidase (APX, EC 1.11.1.11) activity was measured transcriptional responses of these enzyme genes under water by monitoring the decrease in absorbance at 290 nm [33]. The assay stress are still not clear. mixture (2.5 ml) contained 50 mM phosphate buffer (pH 7.3),

Agropyron cristatum is a native grass with high nutritional value 0.1 mM ethylenediaminetetraacetic acid, 1 mM H2O2,10mMAsA and a strong resistance to the drought stress, which naturally and enzyme extract. The reaction was initiated by adding H2O2.One grows in the semiarid area of loess hilly-gully region on the Loess unit of enzyme was defined as the amount of APX catalyzing the Plateau of northwestern China. In our previous observation, A. oxidation of 1 mmol ascorbate per minute. A molar coefficient of cristatum showed a strong antioxidant ability under water stress 2.8 mMÀ1 cmÀ1 was used for the calculation of enzyme activity. (data not shown). However, the regulation mechanism of Glutathione reductase (GR, EC 1.6.4.2) activity was monitored antioxidant metabolism of A. cristatum is still unknown. Therefore, at 340 nm in 3 ml reaction mixture containing 100 mM Tris–HCl investigating the regulation mechanism of the ascorbate and (pH 8.0), 0.5 mM ethylenediaminetetraacetic acid, 0.5 mM MgCl2, glutathione metabolism is important for elucidating antioxidant 10 mM oxidized glutathione (GSSG), 1 mM NADPH and enzyme mechanism of A. cristatum under water stress. extract. The reaction was initiated by adding NADPH [32]. One unit In this study, we investigated JA levels, the transcript levels and of GR activity was defined as the reduction of 1 mmol NADPH per activities of enzymes involved in ascorbate and glutathione minute. A molar coefficient of 6.2 mMÀ1 cmÀ1 was used for the metabolism, and the contents of AsA, GSH, total ascorbate and calculation of enzyme activity. total glutathione in the leaves of A. cristatum exposed to water Monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) activity stress induced by PEG-6000. The aim of the study was to elucidate was assayed at 340 nm in 3 ml reaction mixture containing 50 mM how jasmonic acid regulates ascorbate and glutathione metabo- Hepes–KOH (pH 7.6), 1 mM NADH, 2.5 mM AsA, 2.5 units AsA lism at both molecular and physiological levels under water stress, oxidase (EC 1.10.3.3) and enzyme extract. The reaction was and provide new knowledge to antioxidant metabolism in plants initiated by adding AsA oxidase [34]. One unit of MDHAR activity under water stress. was defined as the amount of enzyme that oxidizes 1 mmol NADH per minute. A molar coefficient of 6.2 mMÀ1 cmÀ1 was used for the 2. Materials and methods calculation of enzyme activity. Dehydroascorbate reductase (DHAR, EC 1.8.5.1) activity was 2.1. Plant material, growth conditions and treatments measured at 265 nm in 3 ml assay solution containing 100 mM Hepes–KOH (pH 7.0), 20 mM GSH, and 2 mM DHA. The reaction The seeds of native grass A. cristatum were collected from was initiated by adding DHA [35]. One unit of DHAR activity was Yan’an city in the semiarid area of loess hilly-gully region on the defined as the amount of enzyme that produces 1 mmol AsA per Loess Plateau of northwestern China. minute. A molar coefficient of 14.6 mMÀ1 cmÀ1 was used for the Seeds of A. cristatum were sown in plastic trays filled with a calculation of enzyme activity. sand/vermiculite matter mix (2:1, v/v) and grown in a greenhouse The specific enzyme activity for all the above enzymes was under a temperature of 25–30 8C, 500 mmol mÀ2 sÀ1 photosyn- expressed as units mgÀ1 protein. thetic active radiation and a 12-h photoperiod. The seedlings were watered with half-strength Hoagland’s solution every day. When 2.3. Analysis of GalLDH and g-ECS the fifth leaf was fully expanded, the seedlings with uniform height and growth status were selected for all experiments. L-Galactono-1,4-lactone dehydrogenase (GalLDH, EC 1.3.2.3) The plants taken out of trays were studied. The roots of plants was extracted and measured by the method of Tabata et al. [36] were washed softly and thoroughly. After rinsing in distilled with slight modifications. Each frozen sample (0.1 g) was ground water for 12 h, the roots were placed in beakers containing into a fine powder in liquid N2 with a mortar and pestle. Fine 100 ml 10% (w/v) PEG solution and wrapped with aluminium foil powder was homogenized in 0.1 M potassium phosphate buffer for 24 h at 25 8C with a continuous light intensity of (pH 7.4) containing 0.4 M sucrose. The extract was passed through 200 mmol mÀ2 sÀ1. In order to study the effects of JA inhibitor, 2 layers of miracloth and then immediately centrifuged at 300 Â g a group of plants were pretreated with 5 mM IBU for 12 h and for 10 min at 2 8C. The supernatant was centrifuged at 10,000 Â g then exposed to water stress treatment or distilled water for 24 h for 20 min at 2 8C. The sediment was suspended in 0.5 ml of the under the same conditions as described above. To test whether above buffer containing 0.4 M sucrose for the measure of GalLDH the effects of IBU could be reversed by exogenously supplied JA, activity. The assay mixture, in a final volume of 2.4 ml, was another group of plants were pretreated with 5 mM IBU + 1 mM composed of enzyme solution (200 ml), 1.05 mg mlÀ1 Cyt c (2 ml) JA for 12 h, and then exposed to water stress treatment for 24 h. and 56 mM L-Gal (200 ml). Before assay, the mixture was incubated The control plants were treated with distilled water alone under at 25 8C for 1 min. The increase in absorbance at 550 nm was the same conditions as above groups. After treatment of 0 h, 4 h, followed immediately after the addition of L-Gal. One unit of 8h,12h,and24h,thefifthleafof A. cristatum plants were activity is defined as the amount of extract required to oxidize 132 C. Shan, Z. Liang / Plant Science 178 (2010) 130–139

1 nmol of L-Gal (equivalent to the formation of 2 nmol of reduced Thermal Cycler by using primers designed according to the gene Cyt c) per minute. A molar coefficient of 17.3 mMÀ1 cmÀ1 was used fragment of following enzymes in A. cristatum. The gene specific for the calculation of enzyme activity. The specific enzyme activity primers, accession number, melting temperatures, PCR protocols was expressed as units gÀ1 FW. and size of amplified fragments are shown in Table 1.To Gamma-glutamylcysteine synthetase (g-ECS, EC 6.3.2.2) was standardize the results, the relative abundance of b-actin was extracted and measured by the method of Ogawa et al. [37] with also determined and used as the internal standard. some modifications. Each frozen sample (0.1 g) was ground into a

fine powder in liquid N2 with a mortar and pestle. Fine powder was 2.7. Determination of JA content homogenized in 0.1 M HCl. The extract was immediately centrifuged at 20,000 Â g for 10 min at 2 8C. The supernatant was then used for The contents of jasmonic acid in leaves were measured by gas the assay of enzyme activity. The mixture of 200 ml supernatant and chromatograph according to the method described by Lan et al. 400 ml 50 mM Tris–HCl (pH 7.6) containing 0.25 mM glutamate, [14] and Deng et al. [41] with some modifications. Briefly, fresh

10 mM ATP, 1 mM dithioerythritol and 2 mM cysteine reacted at leaves were ground to a fine powder in liquid N2 with a mortar and 25 8Cfor1h.Then600ml phosphorus agent containing 3 mM pestle. Extraction was done by adding 10 ml 80% methanol

H2SO4, distilled water, 2.5% ammonium molybdate and 10% vitamin solution with 300 ng internal standard dihydrojasmonic acid c was added and mixed adequately. The mixture was incubated at (dhJA) at 4 8C for 24 h [42]. Samples were centrifuged for 10 min at 45 8C for 25 min. The mixture was cooled at room temperature after 4200  g. The supernatant was evaporated in vacuo to remove reaction finished. The absorbance at 660 nm was measured. One unit methanol and dissolved in phosphate buffer (pH 8.2), and then a of g-ECS activity is defined as 1 mmol cysteine-dependently few polyvinylpyrrolidone (PVP) was added and mixed. After 3À À1 À1 generated PO4 per minute. A molar coefficient of 5.6 mM cm standing for 5 min, above solution was centrifuged at 4200  g for À1 was used for the calculation of enzyme activity. The specific 5 min. The supernatant was then extracted by ether three times. enzyme activity was expressed as units mgÀ1 protein. The water phase was collected and extracted by the same volume of hexane, and concentrated to 1 ml under vacuum. The sample 2.4. Analysis of AsA, GSH, total ascorbate and total glutathione solution was dried by nitrogen and dissolved in the solution containing 1 ml benzene–petroleum ether (1:1, v/v) and 1 ml 1.6% AsA and DHA were measured according to Hodges et al. [38]. For KOH–methanol. After reacting at room temperature for 20 min, the each sample, DHA was estimated from the difference between total solution was extracted by 8 ml distilled water. The upper solution ascorbate and AsA. was dried by nitrogen and redissolved in 200 ml ethanol, and Total glutathione, GSSG and GSH were measured according to immediately injected into gas chromatograph. Griffith [39]. For each sample, GSH was estimated from the A GC-2010A gas chromatograph (Shimadzu Corporation, Japan) difference between total glutathione and GSSG. was used for the analysis. Compounds were separated on an HP- 1MS column (30 m  0.25 mm  0.25 mm). Injection volume was 2.5. Measurement of protein concentration 1 ml. The initial temperature was 50 8C and then temperature programmed at 20 8C/min to 280 8C, with nitrogen as the carrier Protein concentration was measured using bovine serum gas (flow rate 50 ml/min). The identity of the compounds was albumin as standard according to the method of Bradford [40]. determined by the comparison of their retention time with pure commercially available standards. The content of JA was quantified 2.6. Isolation of total RNA and semi-quantitative reverse transcriptase by the method of Deng et al. [41]. polymerase chain reaction (RT-PCR) 2.8. Measurement of malondialdehyde content and electrolyte Total RNA was isolated from leaves by using TRNzol Total RNA leakage Reagent (TIANGEN) according to the instruction supplied by the manufacturer. Approximately 3 mg of total RNA were reverse Malondialdehyde content was measured by thiobarbituric acid transcribed using oligo(dT) primer and TIANScript RT Kit (TIAN- (TBA) reaction as described by Hodges et al. [43]. Electrolyte GEN). cDNA was amplified with the GeneAmp PCR System 9700 leakage was determined according to Zhao et al. [44]. The

Table 1 Gene specific primers, accession number, melting temperatures, PCR protocols and size of amplified fragments.

Gene and accession no. Primer sequences Melting PCR protocols Size of amplified temperatures fragments (bp)

GalLDH (GQ457296) Forward: GGCTGGAGTGATGAAATA 52.7 94 8C/2 min; 94 8C/30 s, 53 8C/30 s, 280 Reverse: GATCCGATGTTGGTAAAT 50.4 72 8C/2 min, 30 cycles; 72 8C/10 min

g-ECS (GQ457297) Forward: TGCCCACTTTGAACGATT 52.7 94 8C/2 min; 94 8C/30 s, 55 8C/30 s, 305 Reverse: CTTGGCTAACTGGAGAACAT 55.7 72 8C/2 min, 30 cycles; 72 8C/10 min

DHAR (GQ457298) Forward: CACTTGTGACCCCTCCTG 53.4 94 8C/2 min; 94 8C/30 s, 53 8C/30 s, 130 Reverse: TTCGCCCCGCCGATGTAG 52.1 72 8C/2 min, 25 cycles; 72 8C/10 min

MDHAR (GQ457299) Forward: GGAAAGTCGCCACATCAC 57.3 94 8C/2 min; 94 8C/30 s, 55 8C/30 s, 276 Reverse: TCTACGAGGGTTACTACGC 57.5 72 8C/2 min, 30 cycles; 72 8C/10 min

APX (GQ457300) Forward: AGATGTCCCTTAGATGTGG 55.4 94 8C/2 min; 94 8C/30 s, 55 8C/30 s, 140 Reverse: GCTTGCTGGAGTAGTTGC 57.3 72 8C/2 min, 25 cycles; 72 8C/10 min

GR (GQ457301) Forward: CAACTGGTAGCCGAGCAA 57.3 94 8C/2 min; 94 8C/30 s, 55 8C/30 s, 166 Reverse: ACTTCAGCACCCAACCCT 57.3 72 8C/2 min, 27 cycles; 72 8C/10 min

b-Actin (GQ457302) Forward: GAGCGTGGTTACTCATTCA 55.4 94 8C/2 min; 94 8C/30 s, 55 8C/30 s, 304 Reverse: TGCCGTACAGATCCTTCC 57.3 72 8C/2 min, 30 cycles; 72 8C/10 min C. Shan, Z. Liang / Plant Science 178 (2010) 130–139 133

3. Results

3.1. Effect of water stress and pretreatment with JA biosynthesis inhibitor (IBU) on JA content

The effects of water stress and IBU on JA content in A. cristatum leaves are shown in Fig. 1. Water stress led to an increase in JA content within the 24 h of treatment. A significant increase in JA content occurred within 12 h of treatment. After 12 h and 24 h of treatment, the content of JA increased 4.7-fold and 5.0-fold, respectively, compared to the control. Pretreatment with IBU markedly inhibited the accumulation of JA in leaves under water stress. After 12 h and 24 h, pretreatment with IBU inhibited the increase in JA content induced by water stress by 68.9% and 76.8%, respectively. However, the pretreatment with IBU alone did not affect JA content in non-stressed leaves. These results indicated that pretreatment with IBU could significantly inhibited the accumulation of JA in leaves of A. cristatum under water stress. Fig. 1. Effects of water stress and IBU on JA content in leaves of A. cristatum seedlings. The plants were treated as follows: control, distilled water; 5 mM IBU, 3.2. The selection of suitable JA treatment concentration 5 mM ibuprofen; water stress, 10% PEG; 5 mM IBU + water stress, 5 mM ibuprofen + 10% PEG. The plants were pretreated with IBU for 12 h, and then exposed to water stress or distilled water for 0 h, 4 h, 8 h, 12 h, and 24 h. In order to select a suitable JA treatment concentration, we investigated the effects of different JA concentrations on the contents of AsA, total ascorbate, GSH and total glutathione under electrolyte leakage was expressed as the relative ion leakage, a water stress. The JA concentrations are 0.1 mM, 1 mM, 5 mM, and percentage of the total conductivity after boiling. 10 mM, respectively. The results showed that water stress increased the contents of AsA, total ascorbate, GSH and total 2.9. Statistical analysis glutathione (Fig. 2A–D). Pretreatment with IBU significantly decreased the content of AsA, total ascorbate, and GSH in the The results presented were the mean of five replicates. Means water-stressed leaves but not the content of total glutathione, were compared by one-way analysis of variance and Duncan’s compared to water stress treatment. Meanwhile, exogenous JA was multiple range test at the 5% level of significance. added at different concentrations together with IBU to determine

Fig. 2. Effects of different JA concentrations on the contents of AsA (A), total ascorbate (B), GSH (C) and total glutathione (D) in leaves of A. cristatum seedlings exposed to water stress for 12 h. The plants were treated as follows: (1) distilled water (control); (2) 5 mM ibuprofen; (3) water stress (10% PEG); (4) 5 mM ibuprofen + water stress; (5) 5 mM ibuprofen + 0.1 mM JA + water stress; (6) 5 mM ibuprofen + 1 mM JA + water stress; (7) 5 mM ibuprofen + 5 mM JA + water stress; (8) 5 mM ibuprofen + 10 mM JA + water stress. The plants were pretreated with ibuprofen, ibuprofen plus JA for 12 h, and then exposed to water stress or distilled water for 12 h. 134 C. Shan, Z. Liang / Plant Science 178 (2010) 130–139 whether the effects of IBU could be reversed by exogenously supplied JA under water stress. Among different JA concentrations, application of 1 mM JA significantly increased the content of AsA, total ascorbate, and GSH under water stress, but not the content of total glutathione, compared to water stress treatment. These results suggested that 1 mM JA was a suitable concentration to study the regulation of JA in ascorbate and glutathione metabolism in A. cristatum leaves under water stress. Above results also showed that JA could significantly affect the contents of AsA, total ascorbate and GSH but not the content of total glutathione under water stress.

3.3. Effects of water stress and pretreatment with IBU on the transcript levels and activities of key enzymes in ascorbate and glutathione metabolism

Figs. 3–8 show that the transcript levels and activities of key enzymes in ascorbate and glutathione metabolism, including APX, GR, DHAR, MDHAR, g-ECS and GalLDH, increased under water stress. An obvious increase in the transcript levels of APX (Fig. 3A), GR (Fig. 4A), DHAR (Fig. 5A), MDHAR (Fig. 6A), g-ECS (Fig. 7A), and GalLDH (Fig. 8A) occurred from 4 h to 24 h of treatment, compared with the control. The activities of APX (Fig. 3B), GR (Fig. 4B), DHAR (Fig. 5B), MDHAR (Fig. 6B), g-ECS (Fig. 7B) and GalLDH (Fig. 8B) also increased significantly from 4 h to 24 h of treatment, compared with the control. After 12 h of treatment, the activities of APX, GR, DHAR, MDHAR, g-ECS and GalLDH increased by 82.8%, 56.3%,

Fig. 4. Time-course of changes in the transcript level (A) analyzed by RT-PCR and activity (B) of GR in leaves of plants. The plants were treated as follows: control, distilled water; water stress, 10% PEG; 5 mM IBU, 5 mM ibuprofen; 5 mM IBU + water stress, 5 mM ibuprofen + 10% PEG; 5 mM IBU + 1 mM JA + water stress, 5 mM ibuprofen + 1 mM JA + 10% PEG. The plants were pretreated with ibuprofen, ibuprofen plus JA for 12 h, and then exposed to water stress or distilled water for 0 h, 4 h, 8 h, 12 h, and 24 h.

95.4%, 90.5%, 46.9%, and 89.4%, respectively. After 24 h of treatment, the activities of APX, GR, DHAR, MDHAR, g-ECS and GalLDH increased by 68.1%, 54.1%, 77.3%, 90%, 46.3%, and 96.2%, respectively. In order to determine whether the increases in the transcript levels and activities of key enzymes in ascorbate and glutathione metabolism were related to the accumulation of JA in stressed leaves, the effects of IBU on the transcript levels and activities of above enzymes were investigated. At the same time, 1 mM JA was added together with IBU in order to determine whether the effects of IBU could be reversed by exogenously supplied JA. The results showed that the pretreatment with IBU significantly reduced the transcript levels of APX (Fig. 3A), GR (Fig. 4A), DHAR (Fig. 5A), MDHAR (Fig. 6A), and GalLDH (Fig. 8A) induced by water stress. Pretreatment with IBU also markedly reduced the activities of APX (Fig. 3B), GR (Fig. 4B), DHAR (Fig. 5B), MDHAR (Fig. 6B), and GalLDH (Fig. 8B) in stressed leaves of A. cristatum. Pretreatment with IBU alone did not affect the transcript levels and activities of these enzymes in non-stressed leaves (Figs. 3–8). The exogenous application of 1 mM JA prevented the reduction in the transcript Fig. 3. Time-course of changes in the transcript level (A) analyzed by RT-PCR and levels and activities of GalLDH, APX, GR, DHAR and MDHAR activity (B) of APX in leaves of plants. The plants were treated as follows: control, induced by IBU in leaves under water stress. However, pretreat- distilled water; water stress, 10% PEG; 5 mM IBU, 5 mM ibuprofen; 5 mM ment with IBU and exogenous application of JA did not obviously IBU + water stress, 5 mM ibuprofen + 10% PEG; 5 mM IBU + 1 mM JA + water affect the transcript level and activity of -ECS in stressed leaves stress, 5 mM ibuprofen + 1 mM JA + 10% PEG. The plants were pretreated with g ibuprofen, ibuprofen plus JA for 12 h, and then exposed to water stress or distilled (Fig. 7A and B). Pretreatment with IBU alone did not affect the water for 0 h, 4 h, 8 h, 12 h, and 24 h. transcript level and activity of g-ECS in non-stressed leaves, C. Shan, Z. Liang / Plant Science 178 (2010) 130–139 135

Fig. 5. Time-course of changes in the transcript level (A) analyzed by RT-PCR and activity (B) of DHAR in leaves of plants. The plants were treated as follows: control, Fig. 6. Time-course of changes in the transcript level (A) analyzed by RT-PCR and distilled water; water stress, 10% PEG; 5 mM IBU, 5 mM ibuprofen; 5 mM activity (B) of MDHAR in leaves of plants. The plants were treated as follows: IBU + water stress, 5 mM ibuprofen + 10% PEG; 5 mM IBU + 1 mM JA + water control, distilled water; water stress, 10% PEG; 5 mM IBU, 5 mM ibuprofen; 5 mM stress, 5 mM ibuprofen + 1 mM JA + 10% PEG. The plants were pretreated with IBU + water stress, 5 mM ibuprofen + 10% PEG; 5 mM IBU + 1 mM JA + water stress, ibuprofen, ibuprofen plus JA for 12 h, and then exposed to water stress or distilled 5 mM ibuprofen + 1 mM JA + 10% PEG. The plants were pretreated with ibuprofen, water for 0 h, 4 h, 8 h, 12 h, and 24 h. ibuprofen plus JA for 12 h, and then exposed to water stress or distilled water for 0 h, 4 h, 8 h, 12 h, and 24 h.

compared with the control. These results suggested that the The changes in the redox state of ASA and GSH are shown in accumulation of JA was involved in the regulation of ascorbate and Fig. 9E and F. Water stress caused a decrease in the ratios of AsA/ glutathione metabolism under water stress. DHA and GSH/GSSG, compared with the control. Pretreatment with IBU significantly decreased the ratios of AsA/DHA and GSH/ 3.4. Effects of water stress and pretreatment with IBU on the content GSSG of stressed leaves, compared with the control and water of AsA, total ascorbate, GSH, total glutathione and the redox state of stress treatment. Exogenous application of JA could prevent the ASA and GSH decrease in the ratios of AsA/DHA and GSH/GSSG induced by IBU in stressed leaves. These results suggested that JA had an important To further investigate whether the ascorbate and glutathione role in maintaining the redox state of ASA and GSH under water metabolism were related to the accumulation of JA in the leaves of stress. A. cristatum under water stress, the effects of pretreatment with IBU on the contents of AsA, total ascorbate, GSH and total 3.5. Effects of water stress and pretreatment with IBU on the glutathione were studied. Exogenous JA was added together with malondialdehyde content and electrolyte leakage IBU to determine whether the effects of IBU could be reversed. Pretreatment with IBU significantly reduced the contents of AsA, To further investigate whether JA has an important role for GSH and total ascorbate induced by water stress. Pretreatment water stress tolerance in A. cristatum, the effects of pretreatment with IBU alone did not affect the contents of these in with IBU on the malondialdehyde content and electrolyte leakage non-stressed leaves (Fig. 9A–C). Exogenous application of JA were studied under water stress. Water stress caused an increase prevented the reduction in the contents of AsA, GSH and total in the malondialdehyde content and electrolyte leakage (Fig. 10A ascorbate induced by IBU in stressed leaves (Fig. 9A–C). However, and B), suggesting that oxidative stress was induced in leaves. the content of total glutathione was not obviously affected by Pretreatment with IBU significantly increased the malondialde- supplying JA or pretreating with IBU under water stress (Fig. 9D). hyde content and electrolyte leakage of stressed leaves compared These results suggested that the accumulation of JA increased the with the control and water stress treatment, suggesting that contents of AsA, GSH and total ascorbate but not the content of oxidative stress was enhanced in IBU-inhibited plants. Exogenous total glutathione under water stress. Above results suggested once application of JA could reverse the effects of IBU. These results again that the accumulation of JA was involved in the regulation of suggested that JA had an important role for acquisition of water ascorbate and glutathione metabolism under water stress. stress tolerance in A. cristatum. 136 C. Shan, Z. Liang / Plant Science 178 (2010) 130–139

Fig. 8. Time-course of changes in the transcript level (A) analyzed by RT-PCR and Fig. 7. Time-course of changes in the transcript level (A) analyzed by RT-PCR and activity (B) of GalLDH in leaves of plants. The plants were treated as follows: control, activity (B) of g-ECS in leaves of plants. The plants were treated as follows: control, distilled water; water stress, 10% PEG; 5 mM IBU, 5 mM ibuprofen; 5 mM distilled water; water stress, 10% PEG; 5 mM IBU, 5 mM ibuprofen; 5 mM IBU + water stress, 5 mM ibuprofen + 10% PEG; 5 mM IBU + 1 mM JA + water IBU + water stress, 5 mM ibuprofen + 10% PEG; 5 mM IBU + 1 mM JA + water stress, 5 mM ibuprofen + 1 mM JA + 10% PEG. The plants were pretreated with stress, 5 mM ibuprofen + 1 mM JA + 10% PEG. The plants were pretreated with ibuprofen, ibuprofen plus JA for 12 h, and then exposed to water stress or distilled ibuprofen, ibuprofen plus JA for 12 h, and then exposed to water stress or distilled water for 0 h, 4 h, 8 h, 12 h, and 24 h. water for 0 h, 4 h, 8 h, 12 h, and 24 h.

4. Discussion [23]. Xiang and Oliver [46] also reported that jasmonic acid treatment increased the transcript levels of g-ECS and GSH2, as well AsA is an important compound of plant antioxidant system and as GR. Our results also showed that JA increased the transcript level a major redox compound in plants. A novel AsA biosynthetic and activity of GR under water stress. However, our results gave no pathway in plants has been proposed by Wheeler et al. [5]. GalLDH evidence that JA affected the transcript level and activity of g-ECS is the key enzyme in this AsA biosynthetic pathway. Thus, the under water stress obviously. In this study, we did not investigate content of AsA in plants is closely related to the GalLDH activity. whether the transcript level and activity of GSHS was regulated by Besides, the activation of the AsA recycling pathway is also JA under water stress. Thus, further investigation of the transcript important for increasing the cellular content of AsA [45]. In this level and activity of GSHS can help us to elucidate the regulation of study, we found that JA regulated the ascorbate metabolism by glutathione metabolism by JA. increasing the transcript levels and activities of GalLDH, APX, Previous study showed that expression of GSH1 gene in DHAR and MDHAR, and the contents of AsA and total ascorbate Arabidopsis was increased and no increase of APXs and GRs genes under water stress. Under ozone stress, it has been documented was observed by JA treatment [23]. These results are different from that JA induces the accumulation of AsA and increases the our data by using A. cristatum. Since JA was not completely transcript level and activity of DHAR [23], which was consistent inhibited by IBU in our study, perhaps the low levels of JA present with our experimental results under water stress. Besides, our were able to upregulate the expression of GSH1 gene and thus study also indicated that the accumulation of JA increased the trigger total glutathione metabolism responses but not ascorbic transcript levels and activities of MDHAR, APX and GalLDH under acid responses. The discrepancy in the expression of GSH1 gene water stress. between Arabidopsis and A. cristatum may be due to above reason. GSH is another important compound of plant antioxidant While the discrepancy in the expression of APXs and GRs genes system. The cellular content of GSH can be determined by g-ECS between Arabidopsis and A. cristatum may be due to the difference and GR, which are the key enzymes for glutathione biosynthetic of plant species. and recycling pathway, respectively. The results of our study The plant hormone abscisic acid (ABA), as a stress signal, also showed that JA could regulate the GSH metabolism by increasing increases as a result of water stress. Increasing evidence indicated the transcript level and activity of GR, and the content of GSH that one mode of ABA action may be related to its role in the under water stress. It has been reported that JA induces glutathione oxidative stress in plants [47]. It has been documented that ABA biosynthesis by increasing the transcript levels of g-ECS (GSH1) and can induce the expression of antioxidant genes encoding APX and GSH2 encoding glutathione synthetase (GSHS) under ozone stress GR, and increase the activities of APX and GR in maize seedlings C. Shan, Z. Liang / Plant Science 178 (2010) 130–139 137

Fig. 9. Time-course of changes in the contents of AsA (A), total ascorbate (B), GSH (C), total glutathione (D), and the ratios of AsA/DHA (E) and GSH/GSSG (F) in leaves of plants. The plants were treated as follows: control, distilled water; water stress, 10% PEG; 5 mM IBU, 5 mM ibuprofen; 5 mM IBU + water stress, 5 mM ibuprofen + 10% PEG; 5 mM IBU + 1 mM JA + water stress, 5 mM ibuprofen + 1 mM JA + 10% PEG. The plants were pretreated with ibuprofen, ibuprofen plus JA for 12 h, and then exposed to water stress or distilled water for 0 h, 4 h, 8 h, 12 h, and 24 h.

[47,48]. ABA can also increase the contents of AsA and GSH [49,50]. enhanced JA levels which stimulated ABA biosynthesis and ABA These results suggested that ABA was involved in the regulation of then increased drought-resistant ability of apple plants. Bandurska ascorbate and glutathione metabolism under water stress. In the et al. reported that application of exogenous JA increased the present study, our results also showed that JA increased the content of ABA under water deficit in two barley genotypes [26]. transcript levels and activities of APX and GR, and the contents of There is also evidence showing that jasmonate signaling can be AsA and GSH in leaves under water stress. Besides, evidence has uncoupled from abscisic acid signaling in barley [53]. Up to now, showed that JA has many similar characteristics to ABA, such as the relationship between ABA and JA in regulating ascorbate and inducing stomatal closure, inhibiting plant growth, promoting glutathione metabolism in plants under water stress is still senescence and being involved in stress response, and they can unknown. Therefore, it is very interesting to investigate the cooperate dependently or independently with each other [51]. relationship between ABA and JA in regulating ascorbate and Pen˜a-Corte´s et al. discovered that ABA and MeJA operated through glutathione metabolism in plants under water stress. For DHAR, a common signaling pathway for the activation of the wound- MDHAR and GalLDH genes, to our knowledge, there was no other inducible expression of Pin2 in potato and tomato [52]. Wounding signaling pathway that activates their transcription except JA. For led to enhanced ABA levels which stimulate jasmonate biosynth- g-ECS gene, NO and JA signaling pathways have been reported to esis and jasmonate then caused accumulation of Pin2 transcripts. activate its transcription [23,46,54]. The cross-talks between JA However, Lan et al. [27] presumed that water stress led to and NO signaling pathways, which are responsible for the 138 C. Shan, Z. Liang / Plant Science 178 (2010) 130–139

metabolism by JA. These results provide new knowledge to the antioxidant metabolism in plants under water stress conditions.

Acknowledgements

This study was financially supported by the grant of the Knowledge Innovation Program of the Chinese Academy of Science (KZCX2-YW-443) and the Knowledge Innovation Project of Chinese Academy of Science (KZCX2-XB2-05-01).

References

[1] K. Apel, H. Hirt, : metabolism, oxidative stress, and signal transduction, Annu. Rev. Plant Biol. 55 (2004) 373–399. [2] A.K. Papadakis, K.A. Roubelakis-Angelakis, Polyamines inhibit NADPH oxidase- mediated superoxides generation and putrescine prevents programmed death syndrome induced by the polyamine oxidase generated , Planta 220 (2005) 826–837. [3] C.H. Foyer, G. Noctor, Oxygen processing in photosynthesis: regulation and signaling, New Phytol. 146 (2002) 359–388. [4] R. Mittler, Oxidative stress, antioxidants and stress tolerance, Trends Plant Sci. 7 (2002) 405–410. [5] G.L. Wheeler, M.A. Jones, N. Smirnoff, The biosynthetic pathway of vitamin C in higher plants, Nature 393 (1998) 365–369. [6] R. Dringen, Glutathione metabolism and oxidative stress in neurodegeneration, Eur. J. Biochem. 267 (2000) 4903. [7] G. Noctor, C.H. Foyer, Ascorbate and glutathione: keeping active oxygen under control, Annu. Rev. Plant Physiol. 49 (1998) 249–279. [8] R.A. Creelman, J.E. Mullet, Biosynthesis and action of jasmonates in plants, Annu. Rev. Plant Physiol. 48 (1997) 355–381. [9] M. Kanna, M. Tamaoki, A. Kubo, Isolation of an ozone-sensitive and jasmonate- semi-insensitive Arabidopsis mutant (oji1), Plant Cell Physiol. 44 (2003) 1301– 1310. [10] M.V. Rao, H. Lee, R.A. Creelman, J.E. Mullet, K.R. Davis, Jasmonic acid signaling modulates ozone-induced hypersensitive cell death, Plant Cell 12 (2000) 1633– 1646. [11] C. Wasternack, B. Hause, Jasmonates and octadecanoids: signals in plant stress Fig. 10. Effects of water stress and IBU on the malondialdehyde content and responses and development, Prog. Nucleic Acid Res. Mol. Biol. 72 (2002) 165–221. electrolyte leakage of A. cristatum leaves. The plants were treated as follows: (1) [12] Z.Y. Xin, X. Zhou, P.E. Pilet, Level changes of jasmonic, abscisic, and indole-3yl- distilled water; (2) 5 mM ibuprofen; (3) 10% PEG; (4) 5 mM ibuprofen + 10% PEG; acetic acids in maize under desiccation stress, J. Plant Physiol. 152 (1997) 21–26. (5) 5 mM IBU + 1 mM JA + water stress. The plants were pretreated with IBU for [13] B. Parthier, Jasmonates: hormone regulators or stress factor in leaf senescence, J. 12 h, and then exposed to water stress or distilled water for 24 h. Plant Growth Regul. 9 (1990) 57–63. [14] Y.P. Lan, Z.H. Han, X.F. Xu, Accumulation of jasmonic acid in apple seedlings under water stress, Acta Hortic. Sin. 31 (2004) 16–20. expression of specific genes, have been reported [55]. Therefore, it [15] W.E. Finch Savage, P.S. Black, H.A. Clay, Desiccation stress in recalcitrant Quercus is also very interesting to investigate the relationship between NO robur L. seeds results in lipid peroxidation and increased synthesis of jasmonates and abscisic acid, J. Exp. Bot. 47 (1996) 661–667. and JA in regulating glutathione metabolism in plants under water [16] J. Lehmann, R. Atzorn, C. Bruckner, Accumulation of jasmonate, abscisic acid, stress. Although some signaling pathways that activate transcrip- specific transcripts and proteins in osmotically stressed barley leaf segments, tion of each enzyme gene have been reported, whether there are Planta 197 (1995) 156–162. other signaling pathways are still not clear. Thus, the signaling [17] R.A. Creelman, J.E. Mullet, Jasmonic acid distribution and action in plants: regulation during development and response to biotic and abiotic stress, Proc. pathways that regulate the transcript levels of key enzymes in Natl. Acad. Sci. U.S.A. 92 (1995) 4114–4119. ascorbate and glutathione metabolism are still needed to be [18] H. Pedranzani, R. Sierra-De-Grado, A. Vigliocco, O. Miersch, G. Abdala, Cold and further studied. JA, as a stress signal, increases as a result of water water stresses produce changes in endogenous jasmonates in two populations of Pinus pinaster, Plant Growth Regul. 52 (2007) 111–116. stress and plays important roles in the regulation of plant [19] K. Overmyer, H. Tuominen, R. Kettunen, C. Betz, C. Langebartels, H. Sandermann, J. responses. However, the signal transduction of JA in regulating Kangasjarvi, Ozone-sensitive Arabidopsis rcd1 mutant reveals opposite roles for the ascorbate and glutathione metabolism is still unknown. It has ethylene and jasmonate signaling pathways in regulating superoxide-dependent cell death, Plant Cell 12 (2000) 1849–1862. been reported that NO, H2O2, and MAPK all participated in the [20] B.P.H.J. Thomma, K. Eggermont, I.A.M.A. Penninckx, B. Mauch-Mani, R. Vogelsang, process of JA signal transduction [56–58]. However, whether NO, B.P.A. Cammue, W.F. Broekaert, Separate jasmonate-dependent and salicylate- dependent defence-response pathways in Arabidopsis are essential for resistance H2O2, and MAPK participate in the signal transduction of jasmonic to distinct microbial pathogens, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 15107– acid in regulating the transcript levels and activities of key 15111. enzymes in ascorbate and glutathione metabolism remains [21] E.E. Farmer, E. Alme´ ras, V. Krishnamurthy, Jasmonates and related oxylipins in unknown. Therefore, it is necessary to study the roles of NO, plant responses to pathogenesis and herbivory, Curr. Opin. Plant Biol. 6 (2003) 372–378. H2O2, and MAPK in the ascorbate and glutathione metabolism [22] S. Berger, E. Bell, J.E. Mullet, Two methyl jasmonate insensitive mutants show regulated by JA, in order to provide more new knowledge to the altered expression of AtVsp in response to methyl jasmonate and wounding, Plant antioxidant metabolism in plants. Physiol. 111 (1996) 525–531. In conclusion, our results clearly suggested that water stress- [23] Y. Sasaki-Sekimoto, N. Taki, T. Obayashi, Coordinated activation of metabolic pathways for antioxidants and defence compounds by jasmonates and their roles induced JA accumulation participated in the regulation of in stress tolerance in Arabidopsis, Plant J. 44 (2005) 653–668. ascorbate and glutathione metabolism by increasing the transcript [24] G.K. Agrawal, S. Tamogami, H. Iwahashi, V.P. Agrawal, R. Rakwal, Transient levels and activities of APX, GR, MDHAR, DHAR, GalLDH, and the regulation of jasmonic acid-inducible rice MAP kinase gene (OsBWMK1)by diverse biotic and abiotic stresses, Plant Physiol. Biochem. 41 (2003) 355–361. contents of AsA, GSH, total ascorbate, which, in turn, enhanced the [25] P.M. Schenk, K. Kazan, I. Wilson, J.P. Anderson, T. Richmond, S.C. Somerville, J.M. antioxidant ability and protects A. cristatum against oxidative Manners, Coordinated plant defense responses in Arabidopsis revealed by micro- stress induced by water stress. Our results suggested that the array analysis, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 11655–11660. [26] H. Bandurska, A. Stroinski, K. Jan, The effect of Jasmonate on the accumulation of transcriptional control of antioxidant metabolism enzymes was a ABA, proline and its influence on membrane injury under water deficient in two main way in the regulation of ascorbate and glutathione barley genotypes, Acta Physiol. Plant. 25 (2003) 279–285. C. Shan, Z. Liang / Plant Science 178 (2010) 130–139 139

[27] Y.P. Lan, J. Zhou, H. Cao, Effect of jasmonic acid on the drought-resistance in Malus containing anthocyanin and other interfering compounds, Planta 207 (1999) plants, Agric. Res. Arid Areas 19 (2001) 71–74. 604–611. [28] S.Y. Wang, Methyl jasmonate reduces water stress in strawberry, J. Plant Growth [44] L.Q. Zhao, F. Zhang, J.K. Guo, Y.L. Yang, B.B. Li, L.X. Zhang, Nitric oxide functions as a Regul. 18 (1999) 127–134. signal in salt resistance in the calluses from two ecotypes of reed, Plant Physiol. [29] L. Li, J. Van Staden, A.K. Ja¨ger, Effects of plant growth regulators on the antioxidant 134 (2004) 849–857. system in seedlings of two maize cultivars subjected to water stress, Plant Growth [45] Z. Chen, T.E. Young, J. Ling, S.C. Chang, D.R. Gallie, Increasing vitamin C content of Regul. 25 (1998) 81–87. plants through enhanced ascorbate recycling, Proc. Natl. Acad. Sci. U.S.A. 100 [30] L. Li, J. Van Staden, Effects of plant growth regulators on the antioxidant system in (2003) 3525–3530. callus of two maize cultivars subjected to water stress, Plant Growth Regul. 24 [46] C.B. Xiang, D.J. Oliver, Arabidopsis glutathione metabolic genes coordinately (1998) 55–66. respond to heavy metals and jasmonic acid in Arabidopsis, Plant Cell 10 [31] L. Ai, Z.H. Li, Z.X. Xie, X.L. Tian, A.E. Eneji, L.S. Duan, Coronatine alleviates (1998) 1539–1550. polyethylene glycol-induced water stress in two rice (Oryza sativa L.) cultivars, [47] M.Y. Jiang, J.H. Zhang, Water stress-induced abscisic acid accumulation trig- J. Agron. Crop Sci. 194 (2008) 360–368. gers the increased generation of reactive oxygen species and up-regulates the [32] S.C. Grace, B.A. Logan, Acclimation of foliar antioxidant systems to growth activities of antioxidant enzymes in maize leaves, J. Exp. Bot. 53 (2002) 2401– irradiance in three broad-leaved evergreen species, Plant Physiol. 112 (1996) 2410. 1631–1640. [48] X.L. Hu, M.Y. Jiang, J.H. Zhang, A.Y. Zhang, F. Lin, M.P. Tan, Calcium–calmodulin is [33] Y. Nakano, K. Asada, Hydrogen peroxide is scavenged by ascorbate specific required for abscisic acid-induced antioxidant defense and functions both

peroxidase in spinach chloroplasts, Plant Cell Physiol. 22 (1981) 867–880. upstream and downstream of H2O2 production in leaves of maize (Zea mays) [34] C. Miyake, K. Asada, Thylakoid-bound ascorbate peroxidase in spinach chloro- plants, New Phytol. 173 (2007) 27–38. plasts and photoreduction of its primary oxidation product monodehydroascor- [49] M.Y. Jiang, J.H. Zhang, Effect of abscisic acid on active oxygen species, antiox- bate radicals in thylakoids, Plant Cell Physiol. 33 (1992) 541–553. idative defence system and oxidative damage in leaves of maize seedlings, Plant [35] D.A. Dalton, S.A. Russell, F.J. Hanus, G.A. Pascoe, H.J. Evans, Enzymatic reactions of Cell Physiol. 42 (2001) 1265–1273. ascorbate and glutathione that prevent peroxide damage in soybean root nodules, [50] M.Y. Jiang, J.H. Zhang, Role of abscisic acid in water stress-induced antioxidant Proc. Natl. Acad. Sci. U.S.A. 83 (1986) 3811–3815. defense in leaves of maize seedlings, Free Radic. Res. 36 (2002) 1001–1015. [36] K. Tabata, K. Oba, K. Suzuki, M. Esaka, Generation and properties of ascorbic acid- [51] K.Z. Cai, T.X. Dong, T. Xu, The physiological roles and resistance control in stress deficient transgenic tobacco cells expressing antisense RNA of L-galactono-1,4- environment of jasmonates, Ecol. Environ. 15 (2006) 397–404. lactone dehydrogenase, Plant J. 27 (2001) 139–148. [52] H. Pen˜a-Corte´s, J. Fisahn, L. Willmitzer, Signals involved in wound-inducible [37] K. Ogawa, A. Hatano-Iwasaki, M. Yanagida, M. Lwabuchi, Level of glutathione is proteinase inhibitor II in tomato and potato plants, Proc. Natl. regulated by ATP-dependent ligation of glutamate and cysteine through photo- Acad. Sci. U.S.A. 92 (1995) 4106–4113. synthesis in Arabidopsis thaliana: mechanism of strong interaction of light inten- [53] J. Lee, B. Parthier, M. Lijbler, Jasmonate signalling can be uncoupled from abscisic sity with flowering, Plant Cell Physiol. 45 (2004) 1–8. acid signalling in barley: identification of jasmonate-regulated transcripts which [38] D.M. Hodges, C.J. Andrews, D.A. Johnson, R.I. Hamilton, Antioxidant compound are not induced by abscisic acid, Planta 199 (1996) 625–632. responses to chilling stress in differentially sensitive inbred maize lines, Plant [54] I. Gilles, P. Chiara, L.G. Marie, H. Julie, D.S. Matteo, D. Massimo, P. Alain, B. Physiol. 98 (1996) 685–692. Emmanuel, F. Pierre, Glutathione synthesis is regulated by nitric oxide in Med- [39] O.W. Griffith, Determination of glutathione and glutathione disulfide using icago truncatula roots, Planta 225 (2007) 1597–1602. glutathione reductase and 2-vinylpyridine, Anal. Biochem. 106 (1980) 207–212. [55] J.W. Wang, J.Y. Wu, Nitric oxide is involved in methyl jasmonate-induced defense [40] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram responses and secondary metabolism activities of Taxus cells, Plant Cell Physiol. quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 46 (2005) 923–930. 72 (1976) 248–254. [56] X. Liu, W.L. Shi, S.Q. Zhang, C.H. Lou, Nitric oxide participates in the signal [41] W.H. Deng, Y.B. Shen, H.J. Chen, Z.Y. Li, X.N. Jiang, Effects of dendrolimus transduction of stomatal closure induced by Jasmonic acid in Vicia faba L, Chin. punctatus feeding and methyl jasmonate (MeJA)- or terpenes fumigation on Sci. Bull. 50 (2005) 453–458. abscisic acid and jasmonic acid contents in Pinus massoniana seedling needles, [57] D. Suhita, A.S. Raghavendra, J.M. Kwak, A. Vavasseur, Cytoplasmic alkalization Chin. J. Appl. Ecol. 20 (2009) 1166–1170. precedes reactive oxygen species production during methyl Jasmonate- [42] E.A. Schmelz, H.T. Alborn, E. Banchio, J.H. Tumlinson, Quantitative relationships and abscisic acid-induced stomatal closure, Plant Physiol. 134 (2004) 1536– between induced jasmonic acid levels and volatile emission in Zea mays during 1545.

Spodoptera exigua herbivory, Planta 216 (2003) 665–673. [58] H. Bo, P.T. Wang, F.C. Dong, C.P. Song, The methyl jasmonate-induced H2O2 [43] M.D. Hodges, J.M. DeLong, C.F. Forney, R.K. Prange, Improving the thiobarbituric generation and relation to MAPK signal transduction system in Arabidopsis acid-reactive-substances assay for estimating lipid peroxidation in plant tissues thaliana guard cells, Plant Physiol. Commun. 41 (2005) 439–443.