Cellular glutathione redox homeostasis plays an important role in the brassinosteroid-induced increase in CO2 assimilation in Cucumis sativus

Authors

  • Yu-Ping Jiang,

    1. Department of Horticulture, Zijingang Campus, Zhejiang University, Yuhangtang Road 866, Hangzhou 310058, China
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    • These authors contributed equally to this work.

  • Fei Cheng,

    1. Department of Horticulture, Zijingang Campus, Zhejiang University, Yuhangtang Road 866, Hangzhou 310058, China
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    • These authors contributed equally to this work.

  • Yan-Hong Zhou,

    1. Department of Horticulture, Zijingang Campus, Zhejiang University, Yuhangtang Road 866, Hangzhou 310058, China
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  • Xiao-Jian Xia,

    1. Department of Horticulture, Zijingang Campus, Zhejiang University, Yuhangtang Road 866, Hangzhou 310058, China
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  • Wei-Hua Mao,

    1. Department of Horticulture, Zijingang Campus, Zhejiang University, Yuhangtang Road 866, Hangzhou 310058, China
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  • Kai Shi,

    1. Department of Horticulture, Zijingang Campus, Zhejiang University, Yuhangtang Road 866, Hangzhou 310058, China
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  • Zhixiang Chen,

    1. Department of Horticulture, Zijingang Campus, Zhejiang University, Yuhangtang Road 866, Hangzhou 310058, China
    2. Department of Botany & Plant Pathology, Purdue University, West Lafayette, IN 47907-2054, USA
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  • Jing-Quan Yu

    1. Department of Horticulture, Zijingang Campus, Zhejiang University, Yuhangtang Road 866, Hangzhou 310058, China
    2. Key Laboratory of Horticultural Plants Growth, Development and Quality Improvement, Ministry of Agriculture of China, Zijingang Road 866, Hangzhou 310058, China
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Author for correspondence
Jing Quan Yu
Tel: +86 571 88982381
Email: jqyu@zju.edu.cn

Summary

  • Brassinosteroids (BRs) play a vital role in plant growth, stress tolerance and productivity. Here, the involvement of BRs in the regulation of CO2 assimilation and cellular redox homeostasis was studied.
  • The effects of BRs on CO2 assimilation were studied in cucumber (Cucumis sativus) through the analysis of the accumulation of H2O2 and glutathione and photosynthesis-related enzyme activities using histochemical and cytochemical detection or a spectrophotometric assay, and Rubisco activase (RCA) using western blot analysis and immunogold labeling.
  • Exogenous BR increased apoplastic H2O2 accumulation, the ratio of reduced to oxidized glutathione (GSH:GSSG) and CO2 assimilation, whereas a BR biosynthetic inhibitor had the opposite effects. BR-induced CO2 assimilation was decreased by a H2O2 scavenger or inhibition of H2O2 generation, GSH biosynthesis and the NADPH-generating pentose phosphate pathway. BR-, H2O2- or GSH-induced CO2 assimilation was associated with increased activity of enzymes in the Benson–Calvin cycle. Immunogold labeling and western blotting showed that BR increased the content of RCA and this effect was blocked by inhibitors of redox homeostasis.
  • These results strongly suggest that BR-induced photosynthesis involves an H2O2-mediated increase in the GSH:GSSG ratio, which may positively regulate the synthesis and activation of redox-sensitive enzymes in carbon fixation.

Introduction

Brassinosteroids (BRs) are plant steroidal hormones essential for plant growth and development (Clouse & Sasse, 1998). As potent plant growth regulators, BRs have been used to enhance plant growth and yield of important agricultural crops (Khripach et al., 2000). For example, application of 28-homobrassinolide and 24-epibrassinolide (EBR), two BRs, to potato (Solanum tuberosum) plants increased both the yield and the quality of the crop (Korableva et al., 2002). Similarly, Choe et al. (2001) overexpressed the DWARF4 gene of Arabidopsis in the BR biosynthetic pathway and found increased vegetative growth and seed yield in transgenic plants. In rice (Oryza sativa) stems, leaves and roots, Wu et al. (2008) overexpressed the sterol C-22 hydroxylase, which controls BR levels, and found increased production of tillers and seeds in the transgenic plants. BRs can also enhance plant tolerance to a variety of abiotic and biotic stresses (Nakashita et al., 2003; Xia et al., 2009a). In some studies, BRs proved to be more effective in protecting plants against fungal pathogens than standard fungicides (Khripach et al., 2000). We have recently reported that BRs increase tolerance to chilling and oxidative stress and increase resistance to biotic stress in cucumber (Cucumis sativus; Xia et al., 2009a, 2011). We have also recently discovered that BR treatment increases plant tolerance to pesticides, and reduces pesticide residues, in cucumber (Xia et al., 2009b). Thus, BRs have a high activity in improving a range of important agronomic traits of crop plants.

We have previously shown that application of exogenous 24-epibrassinolide (EBR) increases photosynthetic CO2 assimilation in cucumber and tomato (Solanum lycopersium), which may contribute to increased growth and yield in EBR-treated plants (Yu et al., 2004; Ogweno et al., 2008). EBR increased the maximum Rubisco carboxylation rate (Vc, max), total and, to a greater extent, initial Rubisco activity and the quantum yield of photosystem II (ΦPSII) (Yu et al., 2004). Elevated CO2 assimilation may also contribute to increased grain filling in the transgenic rice plants expressing the sterol C-22 hydroxylase for BR biosynthesis (Wu et al., 2008). Thus, EBR-induced CO2 assimilation is associated with increases in both photosynthetic electron transport and the synthesis and activation of photosynthetic enzymes.

How BR treatment promotes the synthesis and activation of photosynthetic enzymes is currently unclear. We have recently reported that EBR treatment induces a transient increase in Respiratory burst oxidase homologues 1 (Rboh1) transcript, NADPH oxidase activity and H2O2 in the apoplast and that nitric oxide (NO) and EBR-induced reactive oxygen species (ROS) are important for EBR-induced stress tolerance in cucumber (Xia et al., 2009a, 2011; Cui et al., 2011). Ozaki et al. (2009) have reported that H2O2 application increases CO2 assimilation, the activities of fructose-1,6-bisphosphatase (FBPase), sucrose phosphate synthase and invertases, and sugar content in melon (Cucumis melo) fruits. In light of these findings and known redox regulation of photosynthesis (Ishida et al., 1999; Irihimovitch & Shapira, 2000; Fey et al., 2005; Dietz, 2008; Pfannschmidt et al., 2009), it is possible that ROS, such as H2O2 generated by NADPH oxidase at the apoplast, may function as signal molecules in EBR-induced CO2 assimilation. ROS cause oxidative stress at high concentrations but can also trigger antioxidant responses and affect the redox state, such as glutathione redox homeostasis of the cell. To test this possibility, we analyzed EBR-induced changes in the cellular glutathione pool and the associated ratio of reduced to oxidized glutathione (GSH:GSSG), and determined their roles in EBR-induced photosynthetic CO2 assimilation in cucumber.

Materials and Methods

Plant material and treatments

Cucumber (Cucumis sativus L. cv Jinchun No. 3) seeds were germinated in a growth medium of peat, vermiculite and perlite (6 : 3 : 1, v/v) in a glasshouse. When the first true leaf was fully expanded, seedlings were transplanted into plastic pots (15 cm diameter and 15 cm deep). The seedlings were watered daily with half-strength Enshi nutrient solution (Yu & Matsui, 1997) and kept in growth chambers. The growth conditions were as follows: a 12-h photoperiod, a temperature of 25°C : 17°C (day : night), and a light intensity of 600 μmol m−2 s−1.

Treatment with EBR, H2O2 and GSH at different doses was performed by foliar spraying of cucumber seedlings at the four-leaf stage using distilled water as a control. For comparative analysis of EBR and brassinazole (Brz; an inhibitor of BR biosynthesis; Asami et al., 2000), we first pretreated cucumber seedlings with water or Brz (4 μM) before spraying with water or EBR (0.1 μM). For analysis of the role of H2O2, we pretreated cucumber seedlings with 5 mM dimethylthiourea (DMTU; a H2O2 and OH· scavenger; Fox, 1984) or 100 μM diphenyleneodonium (DPI; an inhibitor of NADPH oxidases and oxidative burst, which produce H2O2; Hancock & Jones, 1987) and then treated the plants with 0.1 μM EBR 8 h later. For analysis of the role of the glutathione redox state in signaling, we pretreated leaves with 1 mM buthionine sulfoximine (BSO; an inhibitor of GSH biosynthesis; Harian et al., 1984) or 5 mM 6-aminonicotinamide (6-AN; an inhibitor of the pentose phosphate pathway, which produces NADPH; Gupte et al., 2002), and then treated the plants with 0.1 μM EBR, 5 mM H2O2 or 5 mM GSH 8 h later. For all the treatments, the chemicals were applied to all leaves. For each treatment, 48 seedlings at the fourth-leaf stage were used. The third leaf from the bottom was used for the gas exchange measurements and other analyses.

Gas exchange measurements and estimation of lipid peroxidation

Gas exchange analysis was performed using LiCor-6400 (Li-Cor, Lincoln, NE, USA). In each experiment, the light-saturated rate of CO2 assimilation (Asat) was measured by maintaining the air temperature, air relative humidity, CO2 concentration and photosynthetic photon flux density (PPFD) at 25°C, 80–90%, 400 μmol mol−1, and 1000 μmol m−2 s−1, respectively. Before the CO2 gas exchange measurement, plants were placed under a photosynthetic photon flux density (PPFD) of 1000 μmol m−2 s−1 for 1 h to activate Rubisco.

The extent of lipid peroxidation was estimated by measuring the amount of malondialdehyde (MDA) equivalents according to Hodges et al. (1999). Leaf samples of 0.3 g were ground with 3 ml of ice-cold 25 mM HEPES buffer (pH 7.8) containing 0.2 mM ethylenediaminetetraacetic acid (EDTA) and 2% polyvinylpyrrolidone (PVP). The homogenates were centrifuged at 4°C for 20 min at 12 000 g, and the resulting supernatants were used for MDA equivalent analysis. Samples were mixed with 10% trichloroacetic acid (TCA) containing 0.65% 2-thiobarbituric acid (TBA) and heated at 95°C for 25 min. The MDA equivalent content was calculated by correcting for compounds, other than MDA equivalent, that absorb at 532 nm, by subtracting the absorbance at 532 nm of a solution containing plant extract incubated without TBA from an identical solution containing TBA.

Quantitative assay and histochemical and cytochemical detection of H2O2

H2O2 was extracted from leaf tissue according to Doulis et al. (1997). Leaf material (0.5 g) was ground in liquid nitrogen and 2 ml of 0.2 M HClO4. After thawing, the mixture was transferred to a 10-ml plastic tube and another 2 ml of 0.2 M HClO4 was added. The homogenate was centrifuged at 2700 g for 30 min at 4°C and the supernatant was collected, adjusted to pH 6.0 with 4 M KOH and centrifuged at 110 g for 1 min at 4°C. The supernatant was placed onto a AG1x8 prepacked column (Bio-Rad, Hercules, CA, USA) and H2O2 was eluted with 4 ml of double-distilled H2O. Recovery efficiencies of H2O2 from different samples were determined by analyzing duplicate samples to which H2O2 was added during grinding at a final concentration of 50 μM. The sample (800 μl) was mixed with 400 μl of reaction buffer containing 4 mM 2,2’-azino-di (3-ethylbenzthiazoline-6-sulfonic acid) and 100 mM potassium acetate at pH 4.4, 400 μl of deionized water, and 0.25 U of horseradish peroxidase. The H2O2 content was measured at OD412 (Willekens et al., 1997).

The histochemical staining of H2O2 was performed as previously described (Thordal-Christensen et al., 1997) with minor modifications. Leaf discs were vacuum-infiltrated with 1 mg ml−1 2,3’-diaminobenzidine (DAB) in 50 mM Tris-acetate (pH 3.8) and incubated at 25°C in the dark for 24 h. Leaf discs were then rinsed in 80% (v/v) ethanol for 10 min at 70°C, mounted in lactic acid/phenol/water (1 : 1 : 1; v/v), and photographed using a microscope (Leica DM4000; Leica, Wetzlar, Germany) at ×5 magnification.

H2O2 was visualized at the subcellular level using CeCl3 for localization (Bestwick et al., 1997). Samples were fixed, embedded, sectioned, and stained for conventional electron microscopy (Xia et al., 2009a). Sections were examined using a transmission electron microscope (JEM-1200EX; JEOL, Tokyo, Japan) at an accelerating voltage of 75 kV.

Glutathione content and glutathione reductase (GR) activity assay

For the measurement of GSH and GSSG, plant leaf tissue (0.2 g) was homogenized in 2 ml of 2% metaphosphoric acid containing 2 mM EDTA and centrifuged at 4°C for 10 min at 14 000 g. After neutralization with 0.5 M phosphate buffer (pH 7.5), 0.1 ml of the supernatant was added to a reaction mixture containing 0.2 mM NADPH, 100 mM phosphate buffer (pH 7.5), 5 mM EDTA and 0.6 mM 5,5’-dithio-bis (2-nitrobenzoic acid). The reaction was started by adding 3 U of GR and was monitored by measuring the changes in absorbance at 412 nm for 1 min. For the GSSG assay, GSH was masked by adding 20 μl of 2-vinylpyridine to the neutralized supernatant, whereas 20 μl of water was added for the total glutathione assay. The GSH concentration was obtained by subtracting the GSSG concentration from the total concentration (Rao & Ormrod, 1995).

For the assay of GR activity, leaf tissues (0.3 g) were ground with 3 ml of ice-cold buffer containing 25 mM HEPES (pH 7.8), 0.2 mM EDTA, 2 mM ascorbic acid (AsA) and 2% PVP. The homogenates were centrifuged at 4°C for 20 min at 12 000 g and the resulting supernatants were used for the determination of enzymatic activity. GR activity was measured according to Halliwell & Foyer (1976) based on the rate of decrease in the absorbance of NADPH at 340 nm. Spectrophotometric analyses were conducted on a SHIMADZU UV-2410PC spectrophotometer (Shimadzu Corporation, Kyoto, Japan).

Rubisco, Rubisco activase (RCA), FBPase, 3-phosphoglycerate kinase (PGK) and ribulose-5-phosphate kinase (PRK) activity determination

Rubisco activity was measured spectrophotometrically by coupling 3-phosphoglyceric acid formation with NADH oxidation at 25°C following the method of Lilley & Walker (1974), with some modifications. The total activity was assayed after the crude extract had been activated in a 0.1-ml activation mixture containing 33 mM Tris-HCl (pH 7.5), 0.67 mM EDTA, 33 mM MgCl2 and 10 mM NaHCO3 for 15 min. Initial Rubisco activity measurements were carried out in 0.1 ml of reaction medium containing 5 mM HEPES-NaOH (pH 8.0), 1 mM NaHCO3, 2 mM MgCl2, 0.25 mM dithiothreitol (DTT), 0.1 mM EDTA, 1 U of glyceraldehyde 3-phosphate dehydrogenase, 0.5 mM ATP, 0.015 mM NADH2, 0.5 mM phosphocreatine, 0.06 mM ribulose-1,5-bisphosphate (RuBP), and 10 μl of extract. The change in absorbance at 340 nm was monitored for 90 s. RCA activity was determined using a Rubisco Activase Assay Kit (Genmed Scientifics Inc., Washington, DC, USA). FBPase activity was determined by monitoring the increase in A340 using an extinction coefficient of 6.2 mM−1 cm−1 (Scheibe et al., 1986). Initial activity was assayed immediately after homogenization. The assay mixture consisted of 0.1 M HEPES-NaOH (pH 8.0), 0.5 mM Na2EDTA, 10 mM MgCl2, 0.3 mM NADP+, 0.6 mM Fru-1,6-bisP, 0.6 U Glc-6-P dehydrogenase from bakers’ yeast (Sigma, USA), 1.2 U Glc-P-isomerase from bakers’ yeast (Sigma), and 100 μl of enzyme extract in a final volume of 1 ml. The reaction was initiated by the addition of enzyme extract. PGK activity was determined according to Hatch & Kagawa (1973). The reaction mixture consisted of 100 mM HEPES-KOH (pH 7.8), 10 mM MgCl2, 1 mM NaF, 1 mM KH2PO4, 4 mM phosphoglyceric acid, 4 U ml−1 triosephosphate isomerase and 4 U ml−1 glyceradehyde-3-phosphate dehydrogenase. Reactions were initiated by the addition of 2 mM ATP and 0.1 mM NADH. For the assay of PRK, aliquots of extract were diluted and assayed by coupling the formation of ADP to the oxidation of NADH using pyruvatekinase and lactate dehydrogenase (Kagawa, 1982).

RCA western blot analysis and immunogold labeling

Western blot analysis of RCA was performed according to Feller et al. (1998). The primary antibody used for RCA detection was rabbit monoclonal antibody raised against the rice enzymes.

RCA fixation and immunolocalization were carried out according to Jin et al. (2006). Subcellular protein distribution was analyzed by electron microscopy, on sections from the portions (c. 100 mm2 of area) near the middle rib of the third leaves, similar to those used for gas exchange. The sections were stained with uranyl acetate and lead citrate, observed and photographed with an electron microscope (JEM-1200EX) at 80 kV. The labeled density was determined by counting the gold particles per unit area (μm2) on electron micrographs at ×20 000 magnification from 10 samples.

Statistics

Data were statistically analyzed using analysis of variance (ANOVA), and tested for significant (< 0.05) treatment differences using Tukey’s test.

Results

Dose–response curves for the effects of EBR on Asat and redox metabolites

We have previously reported that BR levels have significant effects on CO2 assimilation and H2O2 accumulation (Yu et al., 2004; Xia et al., 2009a). In the present study, we first analyzed the response of CO2 assimilation at saturated PPFD (Asat), H2O2 concentration and glutathione (GSH and GSSG) accumulation to foliar application of different concentrations of EBR. When applied at very low concentrations (≤ 0.05 μM), EBR treatment increased Asat only marginally (< 20.0%; Table 1). When the EBR concentration was increased to 0.06–0.15 μM, Asat was increased by c. 40–65% as compared with that of control plants. Further increases of applied concentrations (e.g. to 0.2 and 0.5 μM), however, made EBR less beneficial to Asat. At 1.0 μM, the Asat in EBR-treated plants was even slightly lower than that of control plants (Table 1). Thus, EBR enhanced CO2 assimilation only at moderate concentrations.

Table 1.   The dose–response of the light-saturated rate of CO2 assimilation (Asat), the contents of H2O2 and malondialdehyde (MDA) equivalents, and the glutathione redox status to 24-epibrassinolide (EBR)
EBR (μM)Asat (μmol m−2 s−1)H2O2 content (nmol g−1 FW)GSH content (nmol g−1 FW)GSSG content (nmol g−1 FW)GSH + GSSG content (nmol g−1 FW)GSH:GSSG ratioGR activity (μmol min−1 mg−1 protein)MDA content (nmol g−1 FW)
  1. Four-week-old cucumber (Cucumis sativus) plants were treated with distilled water or EBR at indicated concentrations at 09:00 h. Asat was determined at 1000 μmol m−2 s−1 light intensity and 25°C after activation at 1000 μmol m−2 s−1 light intensity for 1 h. All the measurements were made on leaves at 24 h after EBR treatment. Data are means of four biological replicates (± SD). Means followed by the same letter did not differ significantly at P < 0.05 according to Tukey’s test.

  2. GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione.

09.90 ± 0.36 g96.4 ± 7.3 f574 ± 33b61.6 ± 5.0 c635 ± 28b9.38 ± 1.24 d0.16 ± 0.02 d214 ± 12 bcd
0.0110.0 ± 0.7 fg97.4 ± 4.8 f577 ± 23 b60.2 ± 1.4 c637 ± 24 b9.58 ± 0.17 d0.17 ± 0.01 cd209 ± 9 cd
0.0211.5 ± 0.5 ef95.6 ± 5.3 f581 ± 15 b62.4 ± 1.0 c643 ± 16 b9.31 ± 0.23 d0.19 ± 0.01 cd201 ± 3 cde
0.0512.3 ± 0.5 de107 ± 9 ef585 ± 16 b60.7 ± 3.7 c646 ± 17 b9.67 ± 0.57 d0.19 ± 0.02 cd187 ± 8 de
0.0613.1 ± 0.3 cd133 ± 2 de607 ± 20ab45.9 ± 2.6 d653 ± 19 b13.3 ± 1.0 c0.21 ± 0.01 bc184 ± 134 de
0.0814.5 ± 0.6 bc144 ± 9 d624 ± 6ab31.3 ± 2.0 ef659 ± 8 b18.1 ± 0.7 b0.21 ± 0.01 bc182 ± 15 de
0.1016.3 ± 0.4 0a174 ± 11c644 ± 8 a30.4 ± 1.1 f676 ± 10 b20.1 ± 1.0 a0.25 ± 0.02 ab171 ± 5 e
0.1515.3 ± 0.4 ab189 ± 5 c643 ± 13 a50.0 ± 5.9 de693 ± 15 b14.5 ± 0.4 c0.27 ± 0.03 a200 ± 12de
0.2012.6 ± 0.5 de221 ± 18 b582 ± 33 b167 ± 4 b748 ± 32 a3.49 ± 0.25 e0.27 ± 0.02 a233 ± 11 abc
0.511.8 ± 0.5 de246 ± 17 b574 ± 6 b203 ± 3 a776 ± 4 a2.83 ± 0.07 e0.29 ± 0.02 a243 ± 10 ab
1.009.47 ± 0.67 g281 ± 10 a575 ± 4 b195 ± 7 a770 ± 8 a2.95 ± 0.10 e0.29 ± 0.02 a247 ± 17 a

H2O2 content was not significantly changed by EBR at low concentrations (≤ 0.05 μM). At higher concentrations of 0.08, 0.1, 0.15, 0.2, 0.5 and 1.0 μM, EBR increased H2O2 content by 1.5-, 1.7-, 1.9-, 2.3-, 2.5- and 2.9-fold, respectively, over the control (Table 1). Plants treated with low concentrations of EBR showed no significant changes in the content of MDA equivalents, an indicator of membrane peroxidation, while plants treated with moderate concentrations (i.e. 0.06, 0.08 and 0.1 μM) had decreased MDA equivalent contents. By contrast, plants treated with relatively high EBR concentrations (i.e. 0.5 or 1 μM) had significantly higher MDA equivalent contents than those of the untreated control plants (Table 1). Generally, there was little change in the GSH content after treatment with a range of EBR applications, except from 0.06 to 0.15 μM, at which the GSH content was slightly increased. The GSSG content displayed little change when EBR concentrations were low (≤ 0.05 μM). However, when the EBR concentration was increased to 0.06–0.15 μM, there was a significant drop in GSSG content (Table 1). A further increase in EBR concentration, however, led to a sharp increase in the GSSG content. Accordingly, at low concentrations of EBR (≤ 0.05 μM), there was no significant change in total GSH + GSSH or GSH:GSSG ratio. At 0.06–0.15 μM EBR, the combination of a significant increase in GSH and a significant reduction in GSSG resulted in little change in total GSH + GSSG but a sharp increase in the GSH:GSSG ratio (Table 1). At higher concentrations of EBR (≥ 0.2 μM), the small change in GSH contents and sharp increase in GSSG contents led to an increase in overall GSH + GSSG but a substantial reduction in GSH:GSSG. We also analyzed changes in the activities of GR, a critical enzyme involved in regeneration of GSSG, in EBR-treated plants. Generally, GR activity increased with increased EBR concentration (Table 1). GR activity increased by 78.3% in leaves treated with 0.1 μM EBR. Thus, the promotive effect of EBR on CO2 assimilation at moderate concentrations was associated with increased GSH:GSSG ratios.

Time course of EBR-induced changes in photosynthesis, H2O2 accumulation and GSH:GSSG ratio

We next analyzed the time courses of the effects of EBR on Asat, H2O2 accumulation, GSH and GSSG contents, and GSH:GSSG ratio (Fig. 1). Asat was not significantly changed during the first 2 h after EBR application but was substantially increased 3 h after the treatment. Elevated H2O2 content was observed as early as 1 h after application of 0.1 μM EBR. Thus, elevation of H2O2 preceded the increase in Asat in EBR-treated plants. The GSH content was little changed during the whole process after EBR treatment. However, the GSSG content in EBR-treated plants was substantially reduced 3 h after the EBR treatment (Fig. 1). As a result, while the overall GSH + GSSG content was not significantly changed, the GSH:GSSG ratio was substantially increased after 3 h following EBR treatment. Thus, the kinetics of the change of the GSH:GSSG ratio was similar to that of Asat after EBR treatment.

Figure 1.

The time-course response of the light-saturated rate of CO2 assimilation (Asat), the contents of H2O2, malondialdehyde (MDA) equivalents, reduced glutathione (GSH) and oxidized glutathione (GSSG), and the glutathione redox status to 24-epibrassinolide (EBR). Open circles, control; closed circles, EBR. Four-week-old cucumber (Cucumis sativus) plants were treated with distilled water or 0.1 μM EBR at 09:00 h. Asat was determined at 1000 μmol m−2 s−1 light intensity and 25°C. Data are means of four biological replicates (±SD).

Association of BR-regulated CO2 assimilation with differential H2O2 accumulation and GSH:GSSG ratio

To further analyze the involvement of ROS and redox state in EBR-stimulated photosynthesis, we compared the effects of EBR and Brz (an inhibitor of BR biosynthesis) on CO2 assimilation and the contents of H2O2, GSH and GSSG. Consistent with our previous results, treatment with EBR at 0.1 μM resulted in a c. 30.1% increase in Asat, while inhibition of BR biosynthesis with Brz at 4 μM resulted in a 23.0% reduction in Asat. The reduction in Asat in Brz-treated plants could be recovered by EBR application (Fig. 2). Similar to the changes in Asat, EBR induced a 1.9-fold increase in H2O2 accumulation, while Brz treatment resulted in a 32.5% decrease in H2O2 accumulation, and again the reduction in H2O2 by Brz was recovered by EBR application. Neither EBR nor Brz application had significant effects on the GSH + GSSG content. However, the GSH:GSSG ratio was increased by 106.6% by EBR treatment but decreased by 68.8% by Brz treatment. The decreased GSH:GSSG ratio after Brz treatment could again be restored by EBR.

Figure 2.

The effects of brassinosteroid (BR) on CO2 assimilation, H2O2 content and glutathione redox status (grey bars, GSSG; white bars, GSH). Cucumber (Cucumis sativus) plants were treated with distilled water, 4 μM brassinazole (Brz), 4 μM Brz + 0.1 μM 24-epibrassinolide (EBR) or 0.1 μM EBR. Brz was applied to the shoot tip and whole plants every 2 d from the cotyledon stage to the four-leaf stage, while EBR was applied once every 5 d for the Brz + EBR treatment and once for the EBR treatment at 09:00 h. All measurements were made on leaves after exposure to EBR for 24 h. Data are means of four biological replicates (±SD). Means denoted by the same letter did not significantly differ at < 0.05 according to Tukey’s test.

Histochemical DAB staining confirmed increased H2O2 accumulation after foliar application of 0.1 μM EBR, which was blocked by pretreatment with DMTU (a H2O2 and OH· scavenger) or DPI (an inhibitor of NADPH oxidase and oxidative burst) (Fig. 3a). Cytochemical detection showed that EBR-induced H2O2 mostly accumulated on the cell walls of mesophyll cells facing intercellular spaces (Fig. 3b). Again, both DMTU and DPI abolished EBR-induced H2O2 accumulation detected using cytochemical staining.

Figure 3.

The effects of brassinosteroid (BR) on H2O2 accumulation. (a) H2O2 histochemical staining. 2,3′-diaminobenzidine (DAB) stains were used to detect the presence of H2O2 in cucumber (Cucumis sativus) leaves. Leaf discs (2 cm in diameter) were harvested at 24 h after 24-epibrassinolide (EBR) treatment and stained immediately. Photographs were taken at a magnification number of 5. (b) Cytochemical detection of H2O2 accumulation in mesophyll cells of cucumber leaves. Arrows, CeCl3 precipitates; C, chloroplast; CW, cell wall; V, vacuole; IS, intercellular space. Control, plants sprayed with distilled water; EBR, plants treated with 0.1 μM EBR; DMTU + EBR, plants pretreated with 5 mM dimethylthiourea (DMTU) for 8 h and then 0.1 μM EBR for 24 h; DPI + EBR, plants pretreated with 100 μM diphenyleneodonium (DPI) for 8 h and then 0.1 μM EBR for 24 h.

Involvement of H2O2 and GSH:GSSG ratio in EBR-induced photosynthesis

H2O2 concentrations were rapidly elevated after EBR treatment and, therefore, may be critical for the EBR-induced increase of both the GSH:GSSG ratio and Asat. To examine this possibility, we first investigated the dose effects of H2O2 on Asat, GSH + GSSG contents and GSH:GSSG ratios. As shown in Fig. 4, Asat was significantly increased by 5 mM H2O2. A similar response was observed for the effect of H2O2 on the GSH:GSSG ratio. However, the GSH + GSSG content was not affected by 5 mM H2O2. To further determine the role of H2O2, we analyzed the effects of DMTU or DPI on EBR-induced changes in Asat, GSH + GSSG and GSH:GSSG (Table 2). In EBR-treated plants, Asat was increased by 24.2%. Pretreatment with DMTU and DPI, however, abolished the positive effects of EBR on Asat. Similarly, the increased GSH:GSSG ratios in EBR- or H2O2-treated leaves were abolished by pretreatment with DMTU or DPI. However, DMTU and DPI treatment alone or together with EBR had no significant effects on the GSH + GSSG content. Thus, an elevated H2O2 concentration is important for the EBR-induced changes in the GSH:GSSG ratio and photosynthesis.

Figure 4.

The dose–response curves of the effects of H2O2 on the light-saturated rate of CO2 assimilation (Asat), and reduced glutathione (GSH) and oxidized glutathione (GSSG) contents. Four-week-old cucumber (Cucumis sativus) plants were treated with H2O2 at the indicated concentrations at 09:00 h. All measurements were made on leaves after 24 h of treatment with H2O2. Data are means of four biological replicates (±SD).

Table 2.   Role of H2O2 and the glutathione redox status in 24-epibrassinolide (EBR) -induced increase in CO2 assimilation and changes in total glutathione contents
TreatmentAsat (μmol m−2 s−1)GSH + GSSG content (nmol g−1 FW)GSH:GSSG ratio
  1. Diphenyleneodonium (DPI) (100 μM), dimethylthiourea (DMTU) (5 mM), buthionine sulfoximine (BSO) (1 mM) and 6-aminonicotinamide (6-AN) (5 mM) were applied to cucumber (Cucumis sativus) leaves 8 h before EBR treatment. EBR (0.1 μM), H2O2 (5 mM) and reduced glutathione (GSH) (5 mM) were applied at 09:00 h. All measurements were made on leaves after 24 h of H2O2 or EBR treatment. Data are means of four biological replicates (± SD). Means followed by the same letter did not differ significantly at < 0.05 according to Tukey’s test.

Control12.9 ± 0.5 b682 ± 38 b16.1 ± 1.2 bc
EBR16.0 ± 0.9 a732 ± 42 b28.6 ± 1.7 a
H2O215.1 ± 0.1a723 ± 26 b26.7 ± 2.1 a
GSH14.9 ± 10.2 a1131 ± 49 a24.8 ± 2.0 a
DPI12.2 ± 0.6 b668 ± 65 b13.9 ± 1.6 c
DMTU11.2 ± 1.5 b720 ± 75 b15.5 ± 2.0 bc
BSO12.4 ± 0.7 b561 ± 46 c17.0 ± 1.5 bc
6-AN12.1 ± 1.3 b691 ± 21 b16.0 ± 2.0 bc
DPI + EBR12.6 ± 0.4 b699 ± 64b14.8 ± 2.7 bc
DMTU + EBR12.3 ± 0.9b682 ± 71 b14.7 ± 1.3 c
BSO + EBR12.1 ± 0.6 b697 ± 32 b13.9 ± 1.3 c
BSO + H2O211.8 ± 0.8 b735 ± 46 b15.7 ± 1.8 bc
6-AN + EBR11.7 ± 0.7 b745 ± 38 b18.4 ± 0.8 b
6-AN + H2O212.1 ± 0.8 b688 ± 44 b15.1 ± 1.8 c

To determine the role of increased GSH:GSSG ratio in EBR- and H2O2-stimulated photosynthesis, we analyzed the effects of GSH, BSO (an inhibitor of GSH biosynthesis) and 6-AN (an inhibitor of the pentose phosphate pathway). In our preliminary experiments, we found that BSO at 1.0 mM decreased GSH and GSSG contents but had little effect on the GSH:GSSG ratio or Asat (Supporting Information Fig. S1), and thus this concentration was used in the following experiment. Exogenous application of GSH significantly increased the Asat. BSO or 6-AN treatment alone had no significant effects on Asat, but completely abolished the increase of Asat in EBR- and H2O2-treated plants (Table 2). As expected, both GSH and GSH + GSSG were increased by GSH application but decreased by BSO treatment. In contrast, 6-AN alone had little effect on GSH, GSSG, GSH + GSSG or GSH:GSSG. Pretreatment with BSO or 6-AN blocked the EBR- and H2O2-induced increase in the GSH:GSSG ratio by decreasing the content of newly synthesized GSH and suppressing the regeneration of the GSSG content, respectively. Thus, maintaining a reduced cellular environment was important for the enhancement of CO2 assimilation by EBR.

Involvement of H2O2 and GSH:GSSG ratio in EBR-induced activity of Calvin cycle enzymes

To determine the role of high GSH:GSSG ratio in EBR-induced CO2 assimilation, we analyzed the time courses of the changes in the activities of RCA, FBPase, PGK and PRK, four important enzymes involved in CO2 assimilation. As shown in Fig. 5(a), at 3 h after EBR treatment, we observed increased enzymatic activities for the Calvin cycle-related enzymes. The changed activities of these enzymes showed a time course similar to those of Asat and GSH:GSSG. However, the increase in the activity for RCA, FBPase, PGK and PRK in EBR-treated leaves was substantially decreased by pretreatment with DPI, DMTU, BSO or 6-AN. In addition, treatments with GSH and H2O2 at the tested concentrations resulted in increased activities of RCA, FBPase, PGK and PRK and the increases induced by H2O2 were again decreased by pretreatment with BSO or 6-AN (Fig. 5b). We also analyzed the transcript levels of 10 Calvin–Benson cycle genes in cucumber leaves harvested at 3 h after EBR or H2O2 application with or without DPI, DMTU, BSO and 6-AN pretreatment. The transcripts for the Calvin cycle-related genes showed responses to the inhibitors of redox homeostasis similar to those of corresponding enzyme activities (Figs S2, S3). The time courses of expression of these genes were also similar to those of the activities of corresponding enzymes. These results further supported the conclusion that H2O2 and increased GSH:GSSG ratio played positive roles in regulation of the Calvin–Benson cycle enzymes in EBR- or H2O2-treated leaves.

Figure 5.

Involvement of H2O2 and glutathione redox status in the 24-epibrassinolide (EBR)-induced up-regulation of Rubisco activase (RCA), fructose-1,6-bisphosphatase (FBPase), 3-phosphoglycerate kinase (PGK) and ribulose-5-phosphate kinase (PRK) activity. (a) Time course analysis of RCA, FBPase, PGK and PRK activity. Four-week-old cucumber (Cucumis sativus) plants were sprayed with water or 0.1 μM 24-epibrassinolide (EBR). Leaf samples were harvested at the indicated time-points for assays of RCA, FBPase, PGK and PRK activity. Open circles, control; closed circles, EBR. (b) RCA, FBPase, PGK and PRK activity as influenced by 24-epibrassinolide (EBR), H2O2, glutathione redox status. Diphenyleneodonium (DPI; 100 μM), dimethylthiourea (DMTU; 5 mM), buthionine sulfoximine (BSO; 1 mM) and 6-aminonicotinamide (6-AN; 5 mM) were applied to leaves 8 h before EBR treatment. EBR, H2O2 and GSH were applied at 0.1 μM, 5 mM and 5 mM, respectively. All measurements were made on leaves after 3 h of EBR treatment. Data are means of four biological replicates (±SD). Means denoted by the same letter did not significantly differ at < 0.05 according to Tukey’s test.

We also analyzed the Rubisco activity and the protein level of RCA. Total Rubisco activities in EBR- and H2O2-treated plants were not significantly different from those of the control plants (data not shown). Rubisco initial activities in EBR- and H2O2-treated plants, however, were 142.4% and 67.1% higher than those of the control, respectively. As a result, EBR and H2O2 treatments increased the Rubisco activation rate by 106.0% and 54.1%, respectively (Fig. 6a). To study the mechanism by which EBR and H2O2 enhanced Rubisco activation, we determined the influence of EBR and H2O2 on the protein levels of RCA in leaves. Western blot analysis showed that EBR- and H2O2-treated plants had higher levels of RCA than the control plants (Fig. 6b). DMTU and DPI pretreatment, however, substantially reduced EBR-induced increases in the RCA content. Immunogold labeling electron microscopy with antibody against RCA produced similar results. The gold particle densities increased from 87 ± 13 μm−2 for control plants to 146 ± 17 and 165 ± 14 μm−2 after EBR and H2O2 treatments, respectively (Fig. 6c). Again, DMTU pretreatment abolished the EBR-induced increase in gold particle density (99 ± 11 μm−2).

Figure 6.

The role of 24-epibrassinolide (EBR) and H2O2 in the regulation of Rubisco activation and Rubisco activase (RCA) level. (a) Initial Rubisco activity and Rubisco activation state. Data are means of four biological replicates (±SD). (b) Western blot analysis of RCA. Diphenyleneodonium (DPI) and dimethylthiourea (DMTU) were applied to cucumber (Cucumis sativus) leaves 8 h before EBR treatment. Both a representative RCA western blot (upper panel) and the mean abundances of RCA proteins from three biological triplicates (lower panel) are shown. Total soluble proteins (8 μg) were separated by 15% SDS-PAGE in each lane. (c) Immunogold labeling of RCA in the chloroplasts of the leaves from the plants under various treatments. DPI (100 μM) and DMTU (5 mM) were applied to leaves 8 h before EBR treatment. EBR and H2O2 were applied at a concentration of 0.1 μM and 5 mM, respectively. (d) RCA gold particle density in the chloroplasts of the leaves from the plants with indicated treatments. Results are the average for 15 chloroplasts for each treatment. All measurements were made on leaves after 3 h of EBR or H2O2 treatment. Means denoted by the same letter did not significantly differ at < 0.05 according to Tukey’s test.

Discussion

There are increasing numbers of reports on the positive effects of BRs in the regulation of photosynthesis. Here we present evidence that BR-induced apoplast H2O2 and associated glutathione redox homeostasis play critical roles in BR-enhanced CO2 assimilation in cucumber. BR at moderate concentrations induced a modest increase in apoplast H2O2 and a subsequent rise in the GSH:GSSG ratio, which promoted carbon fixation, probably by enhancing the synthesis and activation of redox-sensitive enzymes (Fig. 7).

Figure 7.

A model for the role of brassinosteroid (BR) in the regulation of CO2 assimilation.

H2O2 and GSH:GSSG ratio mediate the BR-induced increase in CO2 assimilation

EBR enhanced photosynthetic CO2 assimilation, which probably contributes to the positive effects of BRs on plant growth and yield (Choe et al., 2001; Yu et al., 2004; Wu et al., 2008). In the present study, we revealed that EBR at moderate concentrations increased while Brz, an inhibitor of BR biosynthesis, reduced both the H2O2 content and the GSH:GSSG ratio in plant leaves (Figs 1, 2). Through exogenous application of H2O2 and GSH and inhibitors of H2O2 or GSH production, we also showed that EBR induction of CO2 assimilation was closely related to accumulation of appropriate H2O2 concentrations and an increased ratio of GSH:GSSG (Figs 2, 4, Table 2).

BR signaling in the regulation of plant growth starts with the binding of BR to the Brassinosteroid insensitive 1 (BRI1) receptor (Kim et al., 2009; Li, 2010). Interestingly, BRI1 from tomato also binds the peptide hormone systemin (Malinowski et al., 2009), which also induces DPI-sensitive H2O2 generation (Orozco-Cardenas et al., 2001). Therefore, H2O2 may be generated after binding of BR by BRI1 and may play important roles in both BR-mediated growth and stress tolerance. Low concentrations of ROS can serve as second messengers in plant growth and defense signaling pathways (Noctor & Foyer, 1998). ABA induces ROS generation by NADPH oxidases in the regulation of the aperture of guard cells (Noctor & Foyer, 1998; Gechev et al., 2006; Wong & Shimamoto, 2009) and plant growth and development (Foreman et al., 2003; Carol et al., 2005). ROS have been proposed as an amplifier or transducer of multiple developmental or external signals. Therefore, ROS may be a mediator of BR-triggered plant growth, while increased CO2 assimilation was found to be one of the molecular, cellular and physiological responses induced by BRs.

Elevated H2O2 in EBR-treated leaves could affect the GSH:GSSG ratio and in turn alter CO2 assimilation through the thiol group regulation of regulatory proteins or enzymes involved in photosynthesis (Moreno et al., 2008). Consistent with this order of events, EBR-induced H2O2 accumulation preceded the increases in the GSH:GSSG ratio and Asat (Fig. 1). Furthermore, like EBR, H2O2 at appropriate concentrations enhanced CO2 assimilation and increased the GSH:GSSG ratio (Fig. 4). Interestingly, the time course of GSH:GSSG paralleled those of activities of Calvin cycle enzymes (Figs1, 5). Importantly, the increase in GSH:GSSG in EBR-treated plants could be effectively abolished by DPI and DMTU (Table 2) and the alteration of GSH:GSSG was closely related to changes in Asat and activities of Calvin cycle-related enzymes (Fig. 5), suggesting that ROS generated by NADPH oxidase was involved in the regulation of CO2 assimilation and the glutathione redox state.

ROS may directly oxidize and modulate signaling proteins (Kovtun et al., 2000; Neill et al., 2002). Our present study suggests that H2O2 may also act as a regulatory molecule by affecting the cellular glutathione redox state, which is known to regulate the activity of a variety of proteins through thiol group modification (Moreno et al., 2008). ROS-induced cellular redox changes have been previously reported in the regulation of nonexpressor of pathogenesis-related genes 1 (NPR1), an essential regulator of systemic acquired resistance (SAR; Mou et al., 2003). In cucurbit species, the heat-sensitive but cool-tolerant species displayed a higher GSH:GSSG ratio at 14°C, while the heat-tolerant but cool-sensitive species had a higher GSH:GSSG ratio at 34°C (Zhang et al., 2007). Similarly, Kocsy et al. (2004) found that a drought-tolerant wheat (Triticum aestivum) genotype displayed increases in both total GSH content and GSH:GSSG ratio under drought conditions. Thus, a change in the glutathione redox status may be a critical and common mechanism regulating a variety of biological processes in plants.

EBR and H2O2 dose-dependent alteration of GSH:GSSG ratio

An interesting and generally significant finding of this study is that different concentrations of EBR had different effects on the cellular H2O2 content, the glutathione redox state and CO2 assimilation (Fig. 7). At very low EBR concentrations (< 0.05 μM), no significant increase in H2O2, GSH:GSSG ratio or Asat was observed (Table 1). When EBR was increased to 0.06–0.15 μM, there was a significant increase in the H2O2 content, the GSH:GSSG ratio and Asat. At these relatively low concentrations, the EBR-induced increase in H2O2 content was accompanied by a slight decrease in the content of MDA equivalents (Table 1), suggesting that the small increase in H2O2 content was not sufficient to cause significant oxidative stress. Instead, the significantly elevated H2O2 may act as a signal to trigger increased reduction of GSSG to GSH, probably through enhanced activity of GR (Table 1). A transient increase in the GSH:GSSG ratio has also been observed in plants after pathogen infection, SAR inducer and ROS treatments (Vanacker et al., 2000; Mou et al., 2003).When EBR was increased above 0.2 μM, H2O2 concentrations were further increased, which could then cause oxidative stress. Under such conditions, plants would increase synthesis of glutathione, as indicated by the elevated GSH + GSSG content as part of cellular anti-oxidative responses. The ratio of GSH:GSSG under high ROS concentrations also decreased, probably as a result of increased oxidation of GSH for the scavenging of ROS or other detrimental compounds (Mano et al., 2009; Table 1, Fig. 7). Thus, the response of cellular redox state to EBR and H2O2 exhibited distinct phases and, as a result, the positive effects of both EBR and H2O2 on CO2 assimilation were observed only within a moderate range of concentrations. In addition, a high EBR concentration could trigger H2O2 accumulation to a level that impaired the activities of photosynthetic proteins, leading to a decrease in CO2 assimilation (Wan & Liu, 2008; Table 1).

There is often variability in the effects of BRs and ROS on various biological processes of plants (Clouse & Sasse, 1998). ROS have been shown to be pro-cell death by some studies but to be pro-growth by other studies (Prasad et al., 1994; Dat et al., 1998; Karpinski et al., 1999; Bechtold et al., 2005). Some of the variability could be attributable to difference in concentrations used in these studies. In addition, factors including the type of inducers, physiological state of plants and environmental conditions may affect how ROS elicit changes in the pools of GSH and GSSG. Therefore, the sensitive and dynamic changes of the cellular glutathione redox state could be an underlying mechanism for many of the variable or even opposite effects of ROS and ROS-inducing compounds.

Possible mechanisms for enhancement of photosynthesis through change in the cellular glutathione redox state

A number of critical regulatory proteins or enzymes involved in photosynthesis are subjected to redox regulation through thiol group modification (Foyer et al., 1997; Neill et al., 2002; Dietz, 2008). A classic example is the thioredoxin (TRX)-dependent activation and deactivation of enzymes of the Calvin–Benson cycle and the oxidative pentose-phosphate cycle (Schurmann & Jacquot, 2000; Buchanan & Balmer, 2005). Glutathionylation of chloroplast thioredoxin is a redox signaling mechanism in plants (Michelet et al., 2005). The activity of Rubisco or FBPase is also affected by redox through the thioredoxin-dependent RCA large isoform (Zhang et al., 2002; Ozaki et al., 2009). The GSH:GSSG-dependent changes in RCA, FBPase, PGK and PRK activity suggested that they might also be subjected to redox regulation in EBR-treated plants (Figs 5, 6). Furthermore, chloroplast redox state is important for the synthesis of Calvin cycle enzymes. Irihimovitch & Shapira (2000) showed that increased ROS accumulation under high light oxidized the GSH and arrested the translation of Rubisco large subunit (rbcL). Using immunogold labeling and western blotting of RCA (Fig. 6), we showed that the higher GSH:GSSG ratio induced by an appropriate accumulation of H2O2 was important for the BR-induced RCA accumulation. An increase in the content of RCA in turn increased the activation level of Rubsico and consequently CO2 assimilation. Finally, a reducing redox state is important for the stability of Calvin cycle enzymes. Large subunits of Rubisco are susceptible to oxidative damage (Ishida et al., 1999). Therefore, regulation of Calvin cycle enzymes at the translational or post-translational level may be an important mechanism of BR-enhanced CO2 assimilation. There have been many reports on the redox control of gene transcripts and post-transcriptional processes in the chloroplasts (Fey et al., 2005; Pfannschmidt et al., 2009). We also observed that expression of Calvin cycle genes was positively regulated by an increased GSH:GSSG ratio (Fig. S3). Modulation of the gene expression machinery in the chloroplast and the activity of transcription factors in the nucleus may be another mechanism for the control of photosynthesis by BRs.

Acknowledgements

We thank Dr J. Hong and Dr J. H. Zhao for excellent technical help with the immunogold labeling experiment and chlorophyll a fluorescence image analysis. We are also grateful to Prof. C. Foyer of Leeds University for kind advice on H2O2 analysis. This work was supported by the National Basic Research Program of China (2009CB119000), National Natural Science Foundation of China (30972033), and Foundation for the Author of National Excellent Doctoral Dissertation of PR China (200766).

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