Hydrogen peroxide functions as a secondary messenger for brassinosteroids-induced CO₂ assimilation and carbohydrate metabolism in Cucumis sativus

Hydrogen peroxide functions as a secondary messenger for brassinosteroids-induced CO 2 assimilation and carbohydrate metabolism in Cucumis sativus 1 first-author 1 1 1 1 1 1 3 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:jqyu@zju.edu.cn †E-mail:jqyu@zju.edu.cn jqyu@zju.edu.cn Department of Horticulture, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China Department of Horticulture, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China Key Laboratory of Horticultural Plants Growth, Development and Quality Improvement, Ministry of Agriculture, Hangzhou 310058, China Key Laboratory of Horticultural Plants Growth, Development and Quality Improvement, Ministry of Agriculture, Hangzhou 310058, China Department of Botany and Plant Pathology, Purdue University, West Lafayette 47907-2054, USA Department of Botany and Plant Pathology, Purdue University, West Lafayette 47907-2054, USA Brassinosteroids (BRs) are potent regulators of photosynthesis and crop yield in agricultural crops; however, the mechanism by which BRs increase photosynthesis is not fully understood. Here, we show that foliar application of 24-epibrassinolide (EBR) resulted in increases in CO 2 assimilation, hydrogen peroxide (H 2 O 2 ) accumulation, and leaf area in cucumber. H 2 O 2 treatment induced increases in CO 2 assimilation whilst inhibition of the H 2 O 2 accumulation by its generation inhibitor or scavenger completely abolished EBR-induced CO 2 assimilation. Increases of light harvesting due to larger leaf areas in EBR- and H 2 O 2 -treated plants were accompanied by increases in the photochemical efficiency of photosystem II (Φ PSII ) and photochemical quenching coefficient ( q P ). EBR and H 2 O 2 both activated carboxylation efficiency of ribulose-1,5-bisphosphate oxygenase/carboxylase (Rubisco) from analysis of CO 2 response curve and in vitro measurement of Rubisco activities. Moreover, EBR and H 2 O 2 increased contents of total soluble sugar, sucrose, hexose, and starch, followed by enhanced activities of sugar metabolism such as sucrose phosphate synthase, sucrose synthase, and invertase. Interestingly, expression of transcripts of enzymes involved in starch and sugar utilization were inhibited by EBR and H 2 O 2 . However, the effects of EBR on carbohydrate metabolisms were reversed by the H 2 O 2 generation inhibitor diphenyleneodonium (DPI) or scavenger dimethylthiourea (DMTU) pretreatment. All of these results indicate that H 2 O 2 functions as a secondary messenger for EBR-induced CO 2 assimilation and carbohydrate metabolism in cucumber plants. Our study confirms that H 2 O 2 mediates the regulation of photosynthesis by BRs and suggests that EBR and H 2 O 2 regulate Calvin cycle and sugar metabolism via redox signaling and thus increase the photosynthetic potential and yield of crops. Metabolism Photosynthesis Reactive oxygen species Rubisco Sucrose Introduction 1 In this century, the world faces great challenges such as an exploding human population, global warming, environmental pollution, and shortages of fresh water and arable land. It is predicted by the US Population Division that the global population will increase to 9.1 billion by 2050 (Population Division of the Department of Economic and Social Affairs of the United Nations Secretariat, 2009 2009 ). For the survival of this large population, it is a priority to increase global crop yields, which are mostly determined by the photosynthetic potential of the crops (Long et al., 2006 2006 ). In C3 plants, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes primary carbon fixation, in which CO 2 is converted to 3-phosphoglycerate. Phosphoglycerate is then phosphorylated and reduced by adenosine triphosphate (ATP) and triphosphopyridine nucleotide (NADPH), which are generated in the thylakoid membrane at the expense of the proton motive force generated by photosynthetic electron transport. The product triose phosphate is exported from the chloroplast via the chloroplast envelope phosphate (Pi) transporter to the cytosol for sugar metabolism or is used for starch synthesis or recycling to ribulose-1,5-bisphosphate (RuBP) in chloroplast (Sonnewald et al., 1994 1994 ; Furbank and Taylor, 1995 1995 ). It is clear that there are many constraints limiting the increase of photosynthetic potential from the light reaction through the Calvin cycle and sugar metabolism. During the past thirty years, great efforts have been made to increase photosynthesis efficiency, with special emphasis on the modification of the biochemical characteristics of Rubisco, an enzyme with dual functions of RuBP carboxylation and oxygenation (Parry et al., 2003 2003 ). Researchers have focused on engineering the key enzymes used in the Calvin cycle and sugar metabolism via plant biotechnology (Galtier et al., 1995 1995 ; Miyagawa et al., 2001 2001 ; Lefebvre et al., 2005 2005 ). However, most of these studies failed to significantly increase photosynthetic rate and crop yield (Sinclair et al., 2004 2004 ). It is likely that the regulation of photosynthesis is far more complex than is commonly understood. The regulation of photosynthesis by phytohormones is an approach that figures prominently in research aimed at increasing crop yields. In recent years, brassinosteroids (BRs), a new type of phytohormones, have shown exciting promotion effects on crop yield in both field and greenhouse trials (Khripach et al., 2000 2000 ). Over-expression of genes involved in BR biosynthesis and reduction of plant BR level in BR deficient mutants or by application of inhibitors of BR biosynthesis resulted in enhanced plant growth, reduced rate of photosynthetic CO 2 assimilation, and lower sink strength and carbohydrate metabolism, respectively (Choe et al., 2001 2001 ; Schluter et al., 2002 2002 ; Asami et al., 2003 2003 ; Wu et al., 2008 2008 ). Wu et al. ( 2008 2008 ) generated a BR biosynthetic gene over-expressing rice lines using roots and shoots specific promoters and observed significant increases in biomass accumulation and seed yield. They attributed the larger size of seeds and higher yield per plant to the increased flow of photoassimilates from source leaves. We have previously shown that BR enhances photosynthesis by directly regulating the activation state of Rubisco (Yu et al., 2004 2004 ). However, the mechanism by which BR regulates Rubisco and increases photosynthesis is still unclear. To date, the major framework for BR signaling in plant growth and development has been established, which includes the receptor brassinosteroid-insensitive 1 (BRI1) and associated signaling cascade components BRI1-associated receptor kinase 1 (BAK1), BR-signaling kinase 1 (BSK1), BRI1 suppressor 1 (BSU1), and BZR1/2 (Clouse, 2011 2011 ). A surprisingly wide range of genes involved in cellular processes, environmental responses, development and hormonal responses have been shown to be directly regulated by BZR1 (Sun et al., 2010 2010 ). However, no clues have been provided as to the role they play in the regulation of photosynthetic genes. It is highly likely that enzymes of carbon metabolism are regulated posttranscriptionally by BRs, or there are missing links between BRs and expression of photosynthetic genes. We have previously shown that BR induced a transient oxidative burst, with hydrogen peroxide (H 2 O 2 ) accumulation in the apoplast of mesophyll cells of cucumber (Xia et al., 2009b 2009b ). H 2 O 2 has been implicated as a second messenger in several plant hormone responses (Laloi et al., 2004 2004 ; Kwak et al., 2006 2006 ). Abscisic acid-induced stomata closure was dependent on H 2 O 2 production in the cell membrane and chloroplast (Zhang et al., 2001 2001 ; Kwak et al., 2003 2003 ) and H 2 O 2 crosstalks with Ca 2+ , nitric oxide, and cyclic guanosine monophosphate (cGMP) (Desikan et al., 2004 2004 ). Interestingly, the kinase domain of BRI1 receptor contains a GC-domain, which potentially generates cGMP (Kwezi et al., 2007 2007 ). This prompts us to consider the link between H 2 O 2 and BR signaling. Recently, we have proposed evidence suggesting that H 2 O 2 is required for BR-induced expressions of photosynthetic genes, which are involved in light harvesting, electron transport, and the Calvin cycle (Jiang et al., 2012 2012 ). It is extremely important to note that the activities of certain enzymes in CO 2 assimilation are regulated by the redox potential via the ferredoxin/thioredoxin system (Schüermann and Buchanan, 2008 2008 ). Although reactive oxygen species (ROS) such as H 2 O 2 could cause posttranslational modifications to proteins and inhibition of enzymes at a high level, evidence suggests that ROS are involved as signal transducing messengers in the regulation of enzyme activities by thioredoxins (Kamata and Hirata, 1999 1999 ). An appropriate level of H 2 O 2 accumulation during plant acclimation to cold, high light, and drought resulted in increased content and redox state of glutathione (Foyer et al., 1997 1997 ). Up-regulation of genes involved in cysteine and glutathione biosynthesis by H 2 O 2 further confirm the role of ROS in modulation of cellular redox state (Queval et al., 2009 2009 ). Glutathione is potentially involved in regulating enzyme activity via electron input into the redox network or protein glutathionylation (Meyer and Hell, 2005 2005 ; Rouhier et al., 2008 2008 ). In this study, we compared the effects of 24-epebrassinolide (EBR) and H 2 O 2 treatments on the growth and photosynthesis of cucumber seedlings. We found that EBR and H 2 O 2 can both cause similar increases in the carboxylation capacity of Rubisco and potential maximum rate of CO 2 assimilation. In addition, accelerated carbohydrate metabolism was also involved in EBR-or H 2 O 2 -induced photosynthesis. Pretreatment with inhibitors of production or scavenger of ROS indicated that ROS act as mediators for the enhancement of photosynthesis. Materials and methods 1 Plant materials and treatments 2 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/v) in a glasshouse. Cucumber seedlings were transplanted into plastic pots (15 cm diameter and 15 cm deep) when the first true leaf was fully expanded. The seedlings were watered daily with half-strength Enshi nutrient solution (Yu and Matsui, 1997 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). Treatment with EBR (Sigma, Santa Clara, USA) at 0.1 μmol/L or H 2 O 2 at 5 mmol/L was performed by foliar spraying of cucumber seedlings at the four-leaf stage using distilled water as a control. For analysis of the role of H 2 O 2 in EBR-induced up-regulation of CO 2 assimilation, we pretreated cucumber seedlings with 5 mmol/L dimethylthiourea (DMTU; a H 2 O 2 and OH· scavenger) (Fox, 1984 1984 ) or 100 μmol/L diphenyleneodonium (DPI; an inhibitor of NADPH oxidases which produce H 2 O 2 (Hancock and Jones, 1987 1987 ) and could be up-regulated by EBR (Xia et al., 2009b 2009b )), and then treated the plants with 0.1 μmol/L EBR 8 h later. For all the treatments, the chemicals were applied to all leaves. For all cases, the solutions were applied at the rate of 80 ml per m 2 leaf area. Gas exchange and chlorophyll fluorescence measurements 2 Leaf gas exchange measurements were coupled with measurements of chlorophyll fluorescence using an open gas exchange with an integrated fluorescence chamber head system (LI-6400, LI-COR, Inc., Lincoln, NE, USA) on the 3rd leaves. Light-saturated rate of CO 2 assimilation ( A sat ) was measured at ambient CO 2 concentration of 360 μmol/mol and saturating photosynthetic photon flux density (1 000 μmol/(m 2 ·s)) with a leaf temperature of (25±1.5) °C and relative air humidity of 80%–90%. An assimilation versus intercellular CO 2 concentration ( A / C i ) curve was measured according to von Caemmerer and Farquhar ( 1981 1981 ). Assimilation was measured as described by Yu et al. ( 2004 2004 ). The maximum Rubisco carboxylation rates ( V c,max ) and maximum RuBP regeneration rates ( J max ) were estimated from the A / C i curves using the method of Ethier and Livingston ( 2004 2004 ). Stomatal limitation ( l ), the proportion of photosynthesis that is limited by stomatal conductance, was calculated according to Farquhar and Sharkey ( 1982 1982 ). Chlorophyll fluorescence parameters were calculated on the basis of the light-adapted fluorescence measurements as described by Zhou et al. ( 2004 2004 ) and Ogweno et al. ( 2008 2008 ). Photochemical efficiency of photosystem II (Ф PSII ), efficiency of excitation capture by open PSII center ( F v ′/ F m ′), and photochemical quenching coefficient ( q P ) were calculated as ( F m ′− F s )/ F m ′, ( F m ′− F o ′)/ F m ′, and ( F m ′− F s )/( F m ′− F o ′), respectively (Genty et al., 1989 1989 ; van Kooten and Snel, 1990 1990 ). F o , F m , F v : minimal, maximal, and variable fluorescence yields; F m ′, F v ′, F s , maximal, variable, and steady-state fluorescence yield in a light-adapted state. Measurements of total chlorophyll, soluble proteins, and carbohydrate content 2 Total chlorophyll content was determined by the method of Arnon ( 1949 1949 ). Total soluble protein content was measured using Bradford ( 1976 1976 ) reagent. Freeze-dried samples were used for the determination of carbohydrate content. Sucrose, starch, and hexose contents were determined using a modified phenol-sulfuric acid method (Buysse and Merckx, 1993 1993 ). Soluble sugars were extracted from 200 mg of dried material with 50 ml of 80% ethanol, using five extraction steps. The supernatant was analyzed for hexose, sucrose, and total soluble sugars. The residue was boiled for 3 h in 10 ml 2% HCl to hydrolyze starch. The supernatant was analyzed for soluble sugars released from starch by acid hydrolysis (Yu et al., 2004 2004 ). Measurement of H 2 O 2 2 Leaf samples (0.5 g) were ground in liquid nitrogen and 2 ml of 0.2 mol/L HClO 4 . After thawing, the mixture was transferred to a 10-ml plastic tube and another 2 ml of 0.2 mol/L HClO 4 was added. The homogenate was centrifuged at 2 700× g for 30 min at 4 °C and the supernatant was collected, adjusted to pH 6.0 with 4 mol/L KOH, and then centrifuged at 110× g for 1 min at 4 °C. The supernatant was placed onto an AG1x8 prepacked column (Bio-Rad, Hercules, CA) and H 2 O 2 was eluted with 4 ml double-distilled H 2 O. Recovery efficiencies of H 2 O 2 from different samples were determined by analyzing duplicate samples to which H 2 O 2 was added during grinding at a final concentration of 50 μmol/L. The sample (800 μl) was mixed with 400 μl reaction buffer containing 4 mmol/L 2,2′-azino-di(3-ethylbenzthiazoline-6-sulfonic acid) and 100 mmol/L potassium acetate at pH 4.4, 400 μl deionized water, and 0.25 U of horseradish peroxidase (HRP). H 2 O 2 content was measured at optical density at 412 nm (OD 412 ) (Willekens et al., 1997 1997 ). Measurement of enzyme activity of carbohydrate metabolism 2 Sucrose phosphate synthase (SPS) and sucrose synthase (SS) were extracted at 0–4 °C according to Lowell et al. ( 1989 1989 ). SPS activity was assayed at 37 °C by the method of Hubbard et al. ( 1989 1989 ) and assayed essentially as described previously (Yu et al., 2004 2004 ). Colour development of the reaction solution was measured at 620 nm and the SPS activity was calculated. SS was assayed in both the synthetic (SS-s) and cleavage (SS-c) directions with the method of Lowell et al. ( 1989 1989 ). The increase in absorbance at 520 nm was measured (Yu et al., 2004 2004 ). Acid invertase (AI) was extracted as described by Miron and Schaffer ( 1991 1991 ). The reaction mixture that consisted of 4% sucrose, 50 mmol/L sodium acetate buffer (pH 4.5), and an aliquot of enzyme solution in a total volume of 1 ml. Reducing groups formed in the reaction mixture were measured (Endo et al., 1990 1990 ). Measurement of Rubisco activity 2 For measurements of Rubisco activity, frozen leaf samples were ground to a fine powder in liquid nitrogen and then extracted in a solution containing 50 mmol/L Tris-HCl (pH 7.5), 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 mmol/L MgCl 2 , 12.5% glycerin, 10% polyvinylpyrrolidone (PVP), and 10 mmol/L β-mercaptoethanol. After centrifugation at 15 000× g for 15 min at 4 °C, the total Rubisco activity was assayed according to Lilley and Walker ( 1974 1974 ) and Sharkey et al. ( 1991 1991 ), and the analysis was performed essentially as described previously (Yu et al., 2004 2004 ). The oxidation of NADH was followed by changes in absorbance at 340 nm for 90 s. Total RNA extraction and gene expression analysis 2 Total RNA was isolated from cucumber leaves using TRIZOL reagent (Sangon, China) according to the instructions supplied by the manufacturer. After extraction, total RNA was dissolved in diethyl pyrocarbonate-treated water. The complementary DNA (cDNA) template for real time polymerase chain reaction (RT-PCR) was synthesized using a RevertAid™ first strand cDNA synthesis kit (Fermentas, Burlington, Canada) from 2 μg total RNA purified using RNeasy mini kit (Qiagen, Hilden, Germany). For quantitative RT-PCR (qRT-PCR) analysis, PCR products were amplified in triplicate using iQ SYBR Green SuperMix (Bio-Rad, Hercules, CA, USA) in 25 μl qRT-PCR reactions, an iCycler iQ 96-well RT-PCR detection system (Bio-Rad), iCycler software to calculate threshold cycle values and cucumber actin as an internal control. On the basis of expressed sequence tag (EST) sequences, the following gene-specific primers were designed and used for amplification: BAM (β-amylolytic enzyme), 5′-ACCGAATATGGCGAATTCTT-3′ and 5′-TGCCAATGAATTCCTGCTAC-3′; SUS (sucrose synthase), 5′-AAATTGGCCATTCAGGTTGT-3′ and 5′-CTAGTGGTTGTGGCTGGAGA-3′; Invertase , 5′-CTGGAAAGAATATGGTGGCA-3′ and 5′-CCCAAGAGAAGGATCGAGAG-3′; UGDH (UDP-glucose 6-dehydrogenase), 5′-CCTACCCATCTTCAGCCAAT-3′ and 5′-TCCTTGTTGCTTCATACGC-3′; actin , 5′-TGGACTCTGGTGATGGTGTTA-3′ and 5′-CAATGAGGGATGGCTGGAAAA-3′. Relative gene-expression was calculated as described by Livak and Schmittgen ( 2001 2001 ). Statistical analysis 2 Tukey’s test was used for testing the mean difference between treatments by using the SPSS statistical software package. A P -value < 0.05 was considered statistically significant. Results 1 Time course of CO 2 assimilation and H 2 O 2 accumulation in leaves after EBR treatment 2 We first compared the time courses of the effects of EBR on A sat and H 2 O 2 accumulation (Fig. 1 1 ). A sat was significantly increased 3 h after EBR treatment. A sat reached the highest level 6 h after EBR treatment and then gradually declined. At 72 h after treatment, there was no significant difference of A sat between control and EBR-treated leaves. Elevated H 2 O 2 content was observed as early as 1 h after application of 0.1 μmol/L EBR. Thus, elevation of H 2 O 2 preceded the increase in A sat in EBR-treated plants. Induction of H 2 O 2 by EBR was sustained until 72 h after EBR treatment. Four-week-old cucumber plants were treated with distilled water or EBR at 0.1 μmol/L at 9 am. CO 2 assimilation rate at saturated light ( A sat ) was determined at 1 000 μmol/(m 2 ·s) light intensity and 25 °C. Measurements were taken at 1, 2, 3, 6, 9, 24, and 72 h after EBR treatment. Data are expressed as mean±standard deviation (SD) ( n =4). FW: fresh weight Effects of EBR and H 2 O 2 on the growth of cucumber seedlings 2 To investigate whether EBR-induced H 2 O 2 is involved in the enhanced plant growth, we first compared the effects of EBR and H 2 O 2 on leaf mass, leaf area, total chlorophyll, and soluble protein contents. As expected, EBR significantly increased leaf area and leaf mass after 3 d of treatment (Table 1 1 ). Similarly, H 2 O 2 slightly increased leaf area and mass after 3 d of treatment, although the difference was not statistically significant in some cases. Neither EBR nor H 2 O 2 had significant effects on total chlorophyll content. Interestingly, EBR significantly increased the total soluble protein content after 1 d of treatment although there were some exceptions. In contrast, H 2 O 2 had no effects on the total soluble protein content as compared to the control. EBR at 0.1 μmol/L or H 2 O 2 at 5 mmol/L was used, respectively. Values are expressed as mean±SD ( n =4). Significant differences ( P < 0.05) between treatments are indicated by different letters according to Tukey’s test. FW: fresh weight Time after treatment (h) Treatment Leaf area (cm 2 /plant) Leaf mass per area (g FW/m 2 ) Total chlorophyll content (μg/cm 2 ) Soluble protein content (g/m 2 ) 3 Control 314.3±15.6 a 191.8±10.2 a 33.1±1.2 a 7.39±0.48 a EBR 324.6±27.3 a 189.6±7.8 a 31.8±1.2 a 8.01±0.38 a H 2 O 2 321.0±14.7 a 187.8±9.5 a 32.5±1.3 a 7.49±0.32 a 24 Control 417.6±24.7 a 193.6±11.1 a 30.9±2.1 a 7.41±0.40 b EBR 433.6±20.6 a 190.8±8.2 a 31.2±1.8 a 8.29±0.44 a H 2 O 2 434.6±9.1 a 186.5±11.8 a 30.5±2.4 a 7.44±0.13 b 72 Control 472.0±15.5 b 189.6±13.5 a 32.5±0.9 a 7.58±0.39 a EBR 531.0±26.0 a 192.1±9.8 a 30.2±1.9 a 8.32±0.45 a H 2 O 2 519.6±25.2 ab 193.2±9.6 a 31.6±3.2 a 7.56±0.30 a 120 Control 540.8±14.7 b 194.5±8.0 a 33.2±1.6 a 7.39±0.29 b EBR 603.4±11.5 a 188.6±12.2 a 32.8±2.0 a 8.31±0.54 a H 2 O 2 588.6±43.9 ab 188.8±7.2 a 30.6±2.1 a 7.68±0.40 ab 168 Control 630.7±21.2 b 189.9±7.6 a 29.2±2.6 a 7.21±0.39 b EBR 696.6±23.0 a 190.1±6.5 a 27.6±2.5 a 8.18±0.40 a H 2 O 2 660.8±43.2 ab 191.3±9.0 a 28.6±1.8 a 7.41±0.39 ab Effects of EBR and H 2 O 2 on the CO 2 assimilation and chlorophyll fluorescence quenching 2 To examine whether enhanced plant growth by EBR and H 2 O 2 treatment was associated with increased photosynthesis, we analyzed parameters of gas exchange and chlorophyll fluorescence after EBR and H 2 O 2 treatments, respectively. Fig. 2a 2a shows that the maximum carboxylation rate of Rubisco ( V c,max ) increased rapidly at 3 h after treatment with either EBR or H 2 O 2 . V c,max values peaked at 24 h after treatment and then declined. Interestingly, the effects of H 2 O 2 on V c,max declined more rapidly than those of EBR treatment. V c,max for the H 2 O 2 -treated plants had no significant difference with control from 3 d afterward, whereas the effects of EBR sustained until 5 d after treatment. Changes of maximum potential rate of electron transport ( J max ) showed trends similar to those of V c,max except that the induction rate of J max was a little lower than that of V c,max by EBR and H 2 O 2 treatments (Fig. 2b 2b ). EBR and H 2 O 2 treatment, however, had no significant effects on l (Fig. 2c 2c ). Measurements were taken at 1, 2, 3, 6, 9, 24, and 72 h after treatment with 0.1 μmol/L EBR or 5 mmol/L H 2 O 2 , respectively. Data are expressed as mean±SD ( n =4) Actual Φ PSII for control leaves remained almost constant throughout the experiment (Fig. 2d 2d ). Compared with control leaves, EBR-or H 2 O 2 -treated leaves exhibited significantly higher Φ PSII values 3 h after treatment. The value of EBR-treated plants reached maximum at 1 d after treatment. In contrast, the value for the H 2 O 2 -treated plants began to decline at 1 d. Φ PSII of both EBR- and H 2 O 2 -treated plants declined rapidly after 1 d of treatment and showed no significant difference with control afterward. Changes of q P almost paralleled that of Φ PSII (Fig. 2e 2e ), whereas no changes in the efficiency of energy capture by open PSII reaction centers ( F v ′/ F m ′) were observed (Fig. 2f 2f ). Thus, the increase in Φ PSII was mainly due to a significant increase in q P . Effects of EBR and H 2 O 2 on the activity of Rubisco 2 Changes of V c,max reflected the in vivo carboxylation states of Rubisco. To further investigate whether EBR and H 2 O 2 increased photosynthesis by regulating activity of Rubisco, we determined total Rubisco activity and initial Rubisco activity. Table 2 2 shows that EBR and H 2 O 2 had no significant effects on total Rubisco activity, whereas it significantly induced initial activity and activation state of Rubisco. In comparison, the effect of EBR on the initial activity and activation state of Rubisco was more pronounced than that of H 2 O 2 . Samples were taken at 24 h after treatment with EBR at 0.1 μmol/L or H 2 O 2 at 5 mmol/L, respectively. Values are expressed as mean±SD ( n =4). Significant differences ( P < 0.05) between treatments are indicated by different letters according to Tukey’s test Treatment Total Rubisco activity (μmol/(m 2 ·s)) Initial Rubico activity (μmol/(m 2 ·s)) Rubisco activation rate (%) Control 40.37±5.57 a 13.90±1.10 c 34.43 c EBR 47.75±6.37 a 33.69±2.47 a 71.12 a H 2 O 2 43.74±6.97 a 23.24±0.29 b 53.13 b Role of H 2 O 2 in the EBR-induced CO 2 assimilation 2 To determine whether EBR-induced H 2 O 2 accumulation played a role in CO 2 assimilation, we analyzed the changes of potential maximum rate of photosynthetic rate ( A max ) at saturating photosynthetic photon flux density (PPFD) of 1 500 μmol/(m 2 ·s) and CO 2 of 2 000 μmol/mol in plants pretreated with the DPI and DMTU which inhibits NADPH oxidase-dependent H 2 O 2 production and accumulation, respectively, before EBR treatment. The high concentration of CO 2 used in this study excluded the limitation of carboxylation by Rubisco, and therefore, the induction of A max was related to mechanisms other than regulation of activity of Rubisco. Fig. 3 3 shows that A max was significantly increased as compared to control by EBR and H 2 O 2 treatments. Importantly, inhibition of H 2 O 2 production or scavenging of EBR-induced H 2 O 2 by DPI and DMTU, respectively, completely abolished the effects of EBR on A max . A max was determined at saturating PPFD of 1 500 μmol/(m 2 ·s) and CO 2 of 2 000 μmol/mol. Measurements were taken at 24 h after treatment with EBR at 0.1 μmol/L or H 2 O 2 at 5 mmol/L, respectively. DPI at 100 μmol/L and DMTU at 5 mmol/L were used, respectively. Values are expressed as mean±SD ( n =4). Means denoted with different letters showed statistically significant difference ( P < 0.05) according to Tukey’s test Effects of EBR and H 2 O 2 on carbohydrate metabolism 2 Photosynthesis is also limited by inorganic phosphate supply to chloroplasts, which is accompanied by triosephosphate transport and sucrose metabolism. Therefore, we determined the involvement of EBR and H 2 O 2 on carbohydrate metabolism. Treatment of EBR significantly increased total soluble sugar, sucrose, hexose and starch contents (Table 3 3 ). The change of sucrose was most significant, with an increase of about 30%. Treatment with H 2 O 2 had similar effects on contents of the above carbohydrates. Importantly, pretreatment, with DPI and DMTU completely inhibited the induction of carbohydrates by EBR. Increases in carbohydrate metabolite contents were accompanied by increases in acid invertase activity (AI) and activity of SS-s in EBR-or H 2 O 2 -treated leaves, which increased by 82% and 31%, or 116% and 23%, respectively (Table 4 4 ). However, EBR and H 2 O 2 had no influence on the activities of SPS and SS-c direction. Again, pretreatments with DPI and DMTU completely inhibited the induction of activities of AI and SS-s by EBR. DPI at 100 μmol/L and DMTU at 5 mmol/L were applied 8 h before EBR treatment, respectively. Samples were taken at 24 h after treatment with EBR at 0.1 μmol/L or H 2 O 2 at 5 mmol/L, respectively. Values are expressed as mean±SD ( n =4). Significant differences ( P < 0.05) between treatments are indicated by different letters according to Tukey’s test. DW: dry weight 2 Treatment 4 Content (mg/g DW) Total soluble sugar Sucrose Hexose Starch Control 42.12±1.90 b 10.88±0.96 b 26.76±0.47 b 22.41±0.55 b EBR 51.88±3.37 a 14.16±1.40 a 30.79±0.82 a 28.50±1.01 a H 2 O 2 52.26±1.90 a 13.44±0.34 a 31.19±0.62 a 29.32±0.48 a DMTU+EBR 40.34±1.64 b 11.31±1.11 b 23.19±0.97 c 19.00±3.31 c DPI+EBR 42.99±0.58 b 10.33±0.09 b 23.83±0.40 c 19.65±1.13 bc DPI at 100μmol/L and DMTU at 5 mmol/L were applied 8 h before EBR treatment, respectively. Samples were taken at 24 h after treatment with EBR at 0.1 μmol/L or H 2 O 2 at 5 mmol/L, respectively. Values are expressed as mean±SD ( n =4). Significant differences ( P < 0.05) between treatments are indicated by different letters according to Tukey’s test. FW: fresh weight Treatment SPS activity (μmol/(h·g FW)) AI activity (μmol/(h·g FW)) SS-s activity (μmol/(h·g FW)) SS-c activity (μmol/(h·g FW)) Control 34.23±3.21 a 17.66±2.11 c 37.71±1.05 b 18.26±1.20 a EBR 29.59±5.01 a 32.10±2.64 a 49.51±3.02 a 22.80±3.02 a H 2 O 2 38.23±4.55 a 38.06±5.10 a 46.20±4.27 a 24.78±5.23 a DMTU+EBR 30.00±4.20 a 19.00±4.19 bc 29.55±2.19 c 20.58±3.41 a DPI+EBR 37.60±3.90 a 23.44±3.25 b 33.41±5.22 bc 17.28±2.19 a Consistent with changes of AI, the transcription of invertase was induced significantly by H 2 O 2 treatment but was only slightly increased by EBR treatment (Fig. 4 4 ). Meanwhile, EBR and H 2 O 2 significantly inhibited the expressions of BAM and UGDH , encoding β-amylolytic enzyme and UDP-glucose 6-dehydrogenase, which are involved in starch and glucose metabolism, respectively. In contrast, EBR and H 2 O 2 had no significant effects on transcript of SUS . Importantly, pretreatments of DPI and DMTU reversed the effects of EBR on the expressions of BAM and UGDH . These results suggest that sugar metabolism is one of the factors that drive the flux of CO 2 assimilation after EBR or H 2 O 2 treatment, and H 2 O 2 is involved in regulation of carbohydrate metabolism by EBR. Leaf samples were taken at 24 h after treatment with EBR at 0.1 μmol/L or H 2 O 2 at 5 mmol/L, respectively. DPI at 100 μmol/L and DMTU at 5 mmol/L were used respectively. Data are expressed as mean±SD ( n =4). Means denoted with different letters showed statistically significant difference ( P < 0.05) according to Tukey’s test Discussion 1 In this study, we found that both EBR and H 2 O 2 treatments induced an up-regulation of CO 2 assimilation and carbohydrate metabolism. Foliar application of EBR resulted in a H 2 O 2 accumulation while the inhibition of the H 2 O 2 accumulation completely abolished EBR-induced CO 2 assimilation and carbohydrate metabolism. These results suggest that H 2 O 2 functions as a secondary messenger for the EBR-induced changes in CO 2 assimilation and carbohydrate metabolism. The effects of H 2 O 2 and EBR on plant growth and photosynthesis were comparable, which led us to consider whether the effects of EBR were mediated by ROS. Previously, we have shown that EBR induces the transient induction of H 2 O 2 accumulation in the apoplast by activating plasma membrane NADPH oxidase (Xia et al., 2009b 2009b ). Inhibition of ROS production or scavenging of H 2 O 2 significantly inhibited EBR-mediated stress tolerance, which suggests that ROS may act as a second messenger of BR signaling. Recently, we have shown that plant ROS content was highly correlated with endogenous BR content in cucumber and accumulation of ROS increased steadily with increasing concentration of exogenously applied EBR (Jiang et al., 2012 2012 ). Interestingly, the enhancement of CO 2 assimilation after EBR treatment was abolished by pretreatment with DPI or DMTU whilst DPI or DMTU at the applied concentrations had no significant effects on CO 2 assimilation (Jiang et al., 2012 2012 ). In this study, inhibition of H 2 O 2 accumulation inhibited the effects of EBR on A max , accumulation of carbohydrates, and induction of sugar metabolism enzymes. This further confirmed the role of ROS in EBR-mediated plant growth and photosynthesis. Both EBR and H 2 O 2 increased the V c,max , Φ PSII and potential maximum photosynthetic rate (Figs. 2 2 and 3 3 ). Photosynthetic CO 2 assimilation is limited by several physiological processes. Stomatal movement controls the entry of CO 2 into the cell, photosynthetic electron transport supplies reducing power of ATP and NADPH for CO 2 reduction, Rubisco is the rate limiting enzyme of the Calvin cycle and sugar metabolisms regulate the balance of triosephosphate transport and Pi translocation (Paul et al., 1992 1992 ; Fryer et al., 1995 1995 ; Allen and Ort, 2001 2001 ). In this study, EBR and H 2 O 2 had no influence on the l value, which excluded the involvement of stomatal limitation (Fig. 2 2 ). Our previous research indicates that EBR enhances photosynthetic CO 2 assimilation by regulating the expressions and activities of Rubisco and Rubisco activase (Xia et al., 2009a 2009a ). Consistent with these results, EBR and H 2 O 2 treatments both caused increased V c,max and initial Rubisco activity, but had no effects on total activity of Rubisco. This indicates that EBR and H 2 O 2 mainly regulate the activation state of Rubisco, possibly through the action of Rubisco activase. EBR and H 2 O 2 treatments also significantly increased the Ф PSII . Ф PSII is determined by q P and F v ′/ F m ′. From our analysis, the inductions of Ф PSII by EBR and H 2 O 2 were mainly attributed to q P but not related to F v ′/ F m ′. This is consistent with the fact that there were no differences in chlorophyll contents between control and EBR or H 2 O 2 -treated leaves (Table 1 1 ). q P has been proposed to be controlled by the demand of ATP and NADPH of Calvin cycle (Nogues and Baker, 2000 2000 ). The increased activity of the Calvin cycle induced by EBR and H 2 O 2 , therefore, contributed to the increased demand and the increased q P . It was interesting to note that A max varied between control and EBR- or H 2 O 2 -treated leaves (Fig. 3 3 ). At saturating PPFD and CO 2 concentration, the capacity for sucrose synthesis ultimately limits the maximal rates of photosynthesis by restricting the rate at which inorganic phosphate can be recycled to support electron transport and carbon fixation in the chloroplast (Stitt, 1986 1986 ). Increased A max for the EBR- or H 2 O 2 -treated leaves suggests that triose-phosphate transport and Pi translocation were in a new balanced state in EBR or H 2 O 2 treated leaves, which could probably be attributed to accelerated carbohydrate metabolisms. An increase in A max was accompanied by increases in sucrose, soluble sugars, and starch contents, together with significant increases in acid invertase and sucrose synthase (Tables 3 3 and 4 4 ) and reductions in the expression of genes of carbohydrate utilization such as BAM and UGDH (Fig. 4 4 ). These results suggest that EBR and H 2 O 2 increased carbohydrate metabolism and kept efficient supply of Pi to the chloroplast. Ozaki et al. ( 2009 2009 ) determined the effects of periodic soil H 2 O 2 drenching on carbohydrate metabolism in leaves and fruits of melon and observed that appropriate dose of H 2 O 2 increased the contents of fructose, glucose, sucrose and starch with concomitant increases of sucrose phosphate synthase and invertase in leaves or fruits. The discrepancy in the effects on SPS between our findings and those of Ozaki et al. ( 2009 2009 ) may be caused by differing application methods and concentration of H 2 O 2 used, because the H 2 O 2 signal could be attenuated rapidly in plant cells due to efficient antioxidant system (Neill et al., 2002 2002 ). Single application of EBR and H 2 O 2 in our experiment system triggered a transient stimulus of metabolism, whereas the periodic application to soil in their application system could ensure sustained translocation of H 2 O 2 signal from roots. The increase of activity of invertase suggests for the enhanced sucrose synthesis and vice versa, as shown by increases in hexose in EBR- or H 2 O 2 -treated leaves. The increase of invertase was related to enhanced sink strength (Roitsch and Gonzalez, 2004 2004 ). Ozaki et al. ( 2009 2009 ) proposed that increases of sugars in melon fruits were related to enhanced carbon flux from source leaves. It is likely that sink strength was increased by EBR and H 2 O 2 treatments, which increased the demand for photo assimilates and CO 2 assimilation. Plants have evolved an efficient oxidation and reduction (redox) system to control their metabolic fluxes (Dietz, 2008 2008 ). Ferredoxin/thioredoxin is well known to be involved in the regulation of Calvin cycle activity and sugar metabolism including fructose-1,6-bisphosphatase (FBPase), SPS, and SUS (Ruelland and Miginiac-Maslow, 1999 1999 ; Buchanan et al., 2002 2002 ; Marino et al., 2008 2008 ). Although higher level of ROS could induce oxidative stress in the cells, ROS could be induced as second messengers during response to EBR at modest concentrations. BRs have been shown to activate the reduction of glutathione pool, resulting in reductive cellular redox state (Jiang et al., 2012 2012 ). Interestingly, EBR had little effects on Rubisco content and transcript levels of SUS and AI, but significantly increased activities of these enzymes, which suggests a role of posttranscriptional regulation. Several studies have shown that sugar metabolism enzymes are subject to direct thiol modification in plants (Balmer et al., 2003 2003 ; Hendriks et al., 2003 2003 ; Ito et al., 2003 2003 ; Lemaire et al., 2004 2004 ). The photosynthesis increase was strictly dependent on the concentration of EBR, which is mostly related to appropriate redox states. It is probable that the reducing redox state induced by EBR application favors the activation of sugar metabolism enzymes. References DJ Allen , DR Ort . Impacts of chilling temperatures on photosynthesis in warm-climate plants . Trends Plant Sci , 2001 . 6 ( 1 ): 36 - 42 . 11164376 DOI: 10.1016/S1360-1385(00)01808-2 http://doi.org/10.1016/S1360-1385(00)01808-2 . Impacts of chilling temperatures on photosynthesis in warm-climate plants DI Arnon . Copper enzymes in isolated chloroplasts: polyphenol oxidase in Beta vulgaris . Plant Physiol , 1949 . 24 ( 1 ): 1 - 15 . 16654194 DOI: 10.1104/pp.24.1.1 http://doi.org/10.1104/pp.24.1.1 . Copper enzymes in isolated chloroplasts: polyphenol oxidase in Beta vulgaris T Asami , T Nakano , H Nakashita , K Sekimata , Y Shimada , S Yoshida . The influence of chemical genetics on plant science: shedding light on functions and mechanism of action of brassinosteroids using biosynthesis inhibitors . J Plant Growth Regul , 2003 . 22 ( 4 ): 336 - 349 . 14676973 DOI: 10.1007/s00344-003-0065-0 http://doi.org/10.1007/s00344-003-0065-0 . The influence of chemical genetics on plant science: shedding light on functions and mechanism of action of brassinosteroids using biosynthesis inhibitors Y Balmer , A Koller , G del Val , W Manieri , P Schurmann , BB Buchanan . Proteomics gives insight into the regulatory function of chloroplast thioredoxins . PNAS , 2003 . 100 ( 1 ): 370 - 375 . 12509500 DOI: 10.1073/pnas.232703799 http://doi.org/10.1073/pnas.232703799 . Proteomics gives insight into the regulatory function of chloroplast thioredoxins MM Bradford . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding . Anal Biochem , 1976 . 72 ( 1-2 ): 248 - 254 . 942051 DOI: 10.1016/0003-2697(76)90527-3 http://doi.org/10.1016/0003-2697(76)90527-3 . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding BB Buchanan , P Schurmann , RA Wolosiuk , JP Jacquot . The ferredoxin/thioredoxin system: from discovery to molecular structures and beyond . Photosynth Res , 2002 . 73 ( 1-3 ): 215 - 222 . 16245124 DOI: 10.1023/A:1020407432008 http://doi.org/10.1023/A:1020407432008 . The ferredoxin/thioredoxin system: from discovery to molecular structures and beyond J Buysse , R Merckx . An improved colorimetric method to quantify sugar content of plant tissue . J Exp Bot , 1993 . 44 ( 10 ): 1627 - 1629 . DOI: 10.1093/jxb/44.10.1627 http://doi.org/10.1093/jxb/44.10.1627 . An improved colorimetric method to quantify sugar content of plant tissue S Choe , S Fujioka , T Noguchi , S Takatsuto , S Yoshida , KA Feldmann . Over-expression of DWARF4 in the brassinosteroid biosynthetic pathway results in increased vegetative growth and seed yield in Arabidopsis . Plant J , 2001 . 26 ( 6 ): 573 - 582 . 11489171 DOI: 10.1046/j.1365-313x.2001.01055.x http://doi.org/10.1046/j.1365-313x.2001.01055.x . Over-expression of DWARF4 in the brassinosteroid biosynthetic pathway results in increased vegetative growth and seed yield in Arabidopsis SD Clouse . Brassinosteroid signal transduction: from receptor kinase activation to transcriptional networks regulating plant development . Plant Cell , 2011 . 23 ( 4 ): 1219 - 1230 . 21505068 DOI: 10.1105/tpc.111.084475 http://doi.org/10.1105/tpc.111.084475 . Brassinosteroid signal transduction: from receptor kinase activation to transcriptional networks regulating plant development R Desikan , MK Cheung , J Bright , D Henson , JT Hancock , SJ Neill . ABA, hydrogen peroxide and nitric oxide signalling in stomatal guard cells . J Exp Bot , 2004 . 55 ( 395 ): 205 - 212 . 14673026 DOI: 10.1093/jxb/erh033 http://doi.org/10.1093/jxb/erh033 . ABA, hydrogen peroxide and nitric oxide signalling in stomatal guard cells KJ Dietz . Redox signal integration: from stimulus to networks and genes . Physiol Plant , 2008 . 133 ( 3 ): 459 - 468 . 18429942 DOI: 10.1111/j.1399-3054.2008.01120.x http://doi.org/10.1111/j.1399-3054.2008.01120.x . Redox signal integration: from stimulus to networks and genes M Endo , H Nakagawa , N Ogura , T Sato . Size and levels of mRNA for acid invertase in ripe tomato fruits . Plant Cell Physiol , 1990 . 31 ( 5 ): 655 - 659 . . Size and levels of mRNA for acid invertase in ripe tomato fruits GJ Ethier , NJ Livingston . On the need to incorporate sensitivity to CO 2 transfer conductance into the Farquhar-von Caemmerer-Berry leaf photosynthesis model . Plant Cell Environ , 2004 . 27 ( 2 ): 137 - 153 . DOI: 10.1111/j.1365-3040.2004.01140.x http://doi.org/10.1111/j.1365-3040.2004.01140.x . On the need to incorporate sensitivity to CO2 transfer conductance into the Farquhar-von Caemmerer-Berry leaf photosynthesis model GD Farquhar , TD Sharkey . Stomatal conductance and photosynthesis . Annu Rev Plant Physiol , 1982 . 33 317 - 345 . DOI: 10.1146/annurev.pp.33.060182.001533 http://doi.org/10.1146/annurev.pp.33.060182.001533 . Stomatal conductance and photosynthesis RB Fox . Prevention of granulocyte-mediated oxidant lung in rats by a hydroxyl radical scavenger, dimethylthiourea . J Clin Invest , 1984 . 74 ( 4 ): 1456 - 1464 . 6090504 DOI: 10.1172/JCI111558 http://doi.org/10.1172/JCI111558 . Prevention of granulocyte-mediated oxidant lung in rats by a hydroxyl radical scavenger, dimethylthiourea CH Foyer , H Lopez-Delgado , JF Dat , IM Scott . Hydrogen peroxide- and glutathione-associated mechanisms of acclimatory stress tolerance and signaling . Physiol Plant , 1997 . 100 ( 2 ): 241 - 254 . DOI: 10.1111/j.1399-3054.1997.tb04780.x http://doi.org/10.1111/j.1399-3054.1997.tb04780.x . Hydrogen peroxide- and glutathione-associated mechanisms of acclimatory stress tolerance and signaling MJ Fryer , K Oxborough , B Martin , DR Ort , NR Baker . Factors associated with depression of photosynthetic quantum efficiency in maize at low growth temperature . Plant Physiol , 1995 . 108 ( 2 ): 761 - 767 . 12228508 DOI: 10.1104/pp.108.2.761 http://doi.org/10.1104/pp.108.2.761 . Factors associated with depression of photosynthetic quantum efficiency in maize at low growth temperature RT Furbank , WC Taylor . Regulation of photosynthesis in C-3 and C-4 plants: a molecular approach . Plant Cell , 1995 . 7 ( 7 ): 797 - 807 . 12242386 DOI: 10.2307/3870037 http://doi.org/10.2307/3870037 . Regulation of photosynthesis in C-3 and C-4 plants: a molecular approach N Galtier , CH Foyer , E Murchie , R Alred , P Quick , TA Voelker , C Thepenier , G Lasceve , T Betsche . Effects of light and atmospheric carbon-dioxide enrichment on photosynthesis and carbon partitioning in the leaves of tomato ( Lycopersicon esculentum L.) plants over-expressing sucrose-phosphate synthase . J Exp Bot , 1995 . 46 ( SI ): 1335 - 1344 . DOI: 10.1093/jxb/46.special_issue.1335 http://doi.org/10.1093/jxb/46.special_issue.1335 . Effects of light and atmospheric carbon-dioxide enrichment on photosynthesis and carbon partitioning in the leaves of tomato (Lycopersicon esculentum L.) plants over-expressing sucrose-phosphate synthase B Genty , JM Briatais , NR Baker . The relationships between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence . Biochim Biophys Acta , 1989 . 990 ( 1 ): 87 - 92 . DOI: 10.1016/S0304-4165(89)80016-9 http://doi.org/10.1016/S0304-4165(89)80016-9 . The relationships between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence JT Hancock , OTG Jones . The inhibition by diphenyleneiodonium and its analogs of superoxide generation by macrophages . Biochem J , 1987 . 242 ( 1 ): 103 - 107 . 3036079 . The inhibition by diphenyleneiodonium and its analogs of superoxide generation by macrophages JHM Hendriks , A Kolbe , Y Gibon , M Stitt , P Geigenberger . ADP-glucose pyrophosphorylase is activated by posttranslational redox-modification in response to light and to sugars in leaves of Arabidopsis and other plant species . Plant Physiol , 2003 . 133 ( 2 ): 838 - 849 . 12972664 DOI: 10.1104/pp.103.024513 http://doi.org/10.1104/pp.103.024513 . ADP-glucose pyrophosphorylase is activated by posttranslational redox-modification in response to light and to sugars in leaves of Arabidopsis and other plant species NL Hubbard , SC Huber , DM Pharr . Sucrose phosphate synthase and acid invertase as determinants of sucrose accumulation in developing muskmelon ( Cucumis melo L.) fruits . Plant Physiol , 1989 . 91 ( 4 ): 1527 - 1534 . 16667212 DOI: 10.1104/pp.91.4.1527 http://doi.org/10.1104/pp.91.4.1527 . Sucrose phosphate synthase and acid invertase as determinants of sucrose accumulation in developing muskmelon (Cucumis melo L.) fruits H Ito , M Iwabuchi , K Ogawa . The sugar-metabolic enzymes aldolase and triose-phosphate isomerase are targets of glutathionylation in Arabidopsis thaliana : detection using biotinylated glutathione . Plant Cell Physiol , 2003 . 44 ( 7 ): 655 - 660 . 12881492 DOI: 10.1093/pcp/pcg098 http://doi.org/10.1093/pcp/pcg098 . The sugar-metabolic enzymes aldolase and triose-phosphate isomerase are targets of glutathionylation in Arabidopsis thaliana: detection using biotinylated glutathione YP Jiang , F Cheng , YH Zhou , XJ Xia , WH Mao , K Shi , Z Chen , JQ Yu . Cellular glutathione redox homeostasis plays an important role in the brassinosteroid-induced increase in CO 2 assimilation in Cucumis sativus . New Phytol , 2012 . 194 ( 4 ): 932 - 943 . 22432590 DOI: 10.1111/j.1469-8137.2012.04111.x http://doi.org/10.1111/j.1469-8137.2012.04111.x . Cellular glutathione redox homeostasis plays an important role in the brassinosteroid-induced increase in CO2 assimilation in Cucumis sativus H Kamata , H Hirata . Redox regulation of cellular signaling . Cell Signal , 1999 . 11 ( 1 ): 1 - 14 . 10206339 DOI: 10.1016/S0898-6568(98)00037-0 http://doi.org/10.1016/S0898-6568(98)00037-0 . Redox regulation of cellular signaling V Khripach , V Zhabinskii , A de Groot . Twenty years of brassinosteroids: steroidal plant hormones warrant better crops for the XXI century . Ann Bot , 2000 . 86 ( 3 ): 441 - 447 . DOI: 10.1006/anbo.2000.1227 http://doi.org/10.1006/anbo.2000.1227 . Twenty years of brassinosteroids: steroidal plant hormones warrant better crops for the XXI century JM Kwak , IC Mori , ZM Pei , N Leonhardt , MA Torres , JL Dangl , RE Bloom , S Bodde , JD Jones , JI Schroeder . NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis . EMBO J , 2003 . 22 ( 11 ): 2623 - 2633 . 12773379 DOI: 10.1093/emboj/cdg277 http://doi.org/10.1093/emboj/cdg277 . NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis JM Kwak , V Nguyen , JI Schroeder . The role of reactive oxygen species in hormonal responses . Plant Physiol , 2006 . 141 ( 2 ): 323 - 329 . 16760482 DOI: 10.1104/pp.106.079004 http://doi.org/10.1104/pp.106.079004 . The role of reactive oxygen species in hormonal responses L Kwezi , S Meier , L Mungur , O Ruzvidzo , H Irving , C Gehring . The Arabidopsis thaliana brassinosteroid receptor ( AtBRI1 ) contains a domain that functions as a guanylyl cyclase in vitro . PLoS One , 2007 . 2 ( 5 ): e449 17520012 DOI: 10.1371/journal.pone.0000449 http://doi.org/10.1371/journal.pone.0000449 . The Arabidopsis thaliana brassinosteroid receptor (AtBRI1) contains a domain that functions as a guanylyl cyclase in vitro C Laloi , K Apel , A Danon . Reactive oxygen signalling: the latest news . Curr Opin Plant Biol , 2004 . 7 ( 3 ): 323 - 328 . 15134754 DOI: 10.1016/j.pbi.2004.03.005 http://doi.org/10.1016/j.pbi.2004.03.005 . Reactive oxygen signalling: the latest news S Lefebvre , T Lawson , OV Zakhleniuk , JC Lloyd , CA Raines . Increased sedoheptulose-1,7-bisphosphatase activity in transgenic tobacco plants stimulates photosynthesis and growth from an early stage in development . Plant Physiol , 2005 . 138 ( 1 ): 451 - 460 . 15863701 DOI: 10.1104/pp.104.055046 http://doi.org/10.1104/pp.104.055046 . Increased sedoheptulose-1,7-bisphosphatase activity in transgenic tobacco plants stimulates photosynthesis and growth from an early stage in development SD Lemaire , B Guillon , P le Marechal , E Keryer , M Miginia-Maslow , P Decottignies . New thioredoxin targets in the unicellular photosynthetic eukaryote Chlamydomonas reinhardtii . PNAS , 2004 . 101 ( 19 ): 7475 - 7480 . 15123830 DOI: 10.1073/pnas.0402221101 http://doi.org/10.1073/pnas.0402221101 . New thioredoxin targets in the unicellular photosynthetic eukaryote Chlamydomonas reinhardtii RM Lilley , DA Walker . An improved spectrophotometric assay for ribulose-bisphosphate carboxylase . Biochim Biophys Acta , 1974 . 358 ( 1 ): 226 - 229 . 4368401 DOI: 10.1016/0005-2744(74)90274-5 http://doi.org/10.1016/0005-2744(74)90274-5 . An improved spectrophotometric assay for ribulose-bisphosphate carboxylase KJ Livak , TD Schmittgen . Analysis of relative gene expression data using real-time quantitative PCR and the 2 −∆∆ C T method . Methods , 2001 . 25 ( 4 ): 402 - 408 . 11846609 DOI: 10.1006/meth.2001.1262 http://doi.org/10.1006/meth.2001.1262 . Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆CT method SP Long , XG Zhu , SL Naidu , DR Ort . Can improvement in photosynthesis increase crop yields? . Plant Cell Environ , 2006 . 29 ( 3 ): 315 - 330 . 17080588 DOI: 10.1111/j.1365-3040.2005.01493.x http://doi.org/10.1111/j.1365-3040.2005.01493.x . Can improvement in photosynthesis increase crop yields? CA Lowell , PT Tomlinson , KE Koch . Sucrose-metabolising enzymes in transport tissue and adjacent sink structures in developing citrus fruit . Plant Physiol , 1989 . 90 ( 4 ): 1394 - 1402 . 16666942 DOI: 10.1104/pp.90.4.1394 http://doi.org/10.1104/pp.90.4.1394 . Sucrose-metabolising enzymes in transport tissue and adjacent sink structures in developing citrus fruit D Marino , N Hohnjec , H Kuster , JF Moran , EM Gonzalez , C Arrese-Igor . Evidence for transcriptional and post-translational regulation of sucrose synthase in pea nodules by the cellular redox state . Mol Plant-Microbe Interact , 2008 . 21 ( 5 ): 622 - 630 . 18393622 DOI: 10.1094/MPMI-21-5-0622 http://doi.org/10.1094/MPMI-21-5-0622 . Evidence for transcriptional and post-translational regulation of sucrose synthase in pea nodules by the cellular redox state AJ Meyer , R Hell . Glutathione homeostasis and redox-regulation by sulfhydryl groups . Photosynth Res , 2005 . 86 ( 3 ): 435 - 457 . 16315075 DOI: 10.1007/s11120-005-8425-1 http://doi.org/10.1007/s11120-005-8425-1 . Glutathione homeostasis and redox-regulation by sulfhydryl groups D Miron , AA Schaffer . Sucrose phosphate synthase, sucrose synthase, and invertase activities in developing fruit of Lycopersicon esculentum Mill. and the sucrose accumulating Lycopersicon hirsutum Humb. and Bonpl . Plant Physiol , 1991 . 95 ( 2 ): 623 - 627 . 16668028 DOI: 10.1104/pp.95.2.623 http://doi.org/10.1104/pp.95.2.623 . Sucrose phosphate synthase, sucrose synthase, and invertase activities in developing fruit of Lycopersicon esculentum Mill. and the sucrose accumulating Lycopersicon hirsutum Humb. and Bonpl Y Miyagawa , M Tamoi , S Shigeoka . Over-expression of a cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase in tobacco enhances photosynthesis and growth . Nat Biotechnol , 2001 . 19 ( 10 ): 965 - 969 . 11581664 DOI: 10.1038/nbt1001-965 http://doi.org/10.1038/nbt1001-965 . Over-expression of a cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase in tobacco enhances photosynthesis and growth S Neill , R Desikan , J Hancock . Hydrogen peroxide signalling . Curr Opin Plant Biol , 2002 . 5 ( 5 ): 388 - 395 . 12183176 DOI: 10.1016/S1369-5266(02)00282-0 http://doi.org/10.1016/S1369-5266(02)00282-0 . Hydrogen peroxide signalling S Nogues , NR Baker . Effects of drought on photosynthesis in Mediterranean plants grown under enhanced UV-B radiation . J Exp Bot , 2000 . 51 ( 348 ): 1309 - 1317 . 10937707 DOI: 10.1093/jexbot/51.348.1309 http://doi.org/10.1093/jexbot/51.348.1309 . Effects of drought on photosynthesis in Mediterranean plants grown under enhanced UV-B radiation JO Ogweno , XS Song , K Shi , WH Hu , WH Mao , YH Zhou , JQ Yu , S Nogues . Brassinosteroids alleviate heat-induced inhibition of photosynthesis by increasing carboxylation efficiency and enhancing antioxidant systems in Lycopersicon esculentum . J Plant Growth Regul , 2008 . 27 ( 1 ): 49 - 57 . DOI: 10.1007/s00344-007-9030-7 http://doi.org/10.1007/s00344-007-9030-7 . Brassinosteroids alleviate heat-induced inhibition of photosynthesis by increasing carboxylation efficiency and enhancing antioxidant systems in Lycopersicon esculentum K Ozaki , A Uchida , T Takabe , F Shinagawa , Y Tanaka , T Takabe , T Hayashi , T Hattori , AK Rai , T Takabe . Enrichment of sugar content in melon fruits by hydrogen peroxide treatment . J Plant Physiol , 2009 . 166 ( 6 ): 569 - 578 . 18845360 DOI: 10.1016/j.jplph.2008.08.007 http://doi.org/10.1016/j.jplph.2008.08.007 . Enrichment of sugar content in melon fruits by hydrogen peroxide treatment MAJ Parry , PJ Andralojc , RAC Mitchell , PJ Madgwick , AJ Keys . Manipulation of Rubisco: the amount, activity, function and regulation . J Exp Bot , 2003 . 54 ( 386 ): 1321 - 1333 . 12709478 DOI: 10.1093/jxb/erg141 http://doi.org/10.1093/jxb/erg141 . Manipulation of Rubisco: the amount, activity, function and regulation MJ Paul , SP Driscoll , DW Lawlor . Sink-regulation of photosynthesis in relation to temperature in sunflower and rape . J Exp Bot , 1992 . 43 ( 2 ): 147 - 153 . DOI: 10.1093/jxb/43.2.147 http://doi.org/10.1093/jxb/43.2.147 . Sink-regulation of photosynthesis in relation to temperature in sunflower and rape Population Division of the Department of Economic and Social Affairs of the United Nations Secretariat World Population Prospects: The 2008 Revision . 2009 . Available from: http://www.un.org/esa/population/publications/popnews/Newsltr_87.pdf http://www.un.org/esa/population/publications/popnews/Newsltr_87.pdf . World Population Prospects: The 2008 Revision G Queval , D Thominet , H Vanacker , M Miginiac-Maslow , B Gakiere , G Noctor . H 2 O 2 -activated up-regulation of glutathione in Arabidopsis involves induction of genes encoding enzymes involved in cysteine synthesis in the chloroplast . Mol Plant , 2009 . 2 ( 2 ): 344 - 356 . 19825619 DOI: 10.1093/mp/ssp002 http://doi.org/10.1093/mp/ssp002 . H2O2-activated up-regulation of glutathione in Arabidopsis involves induction of genes encoding enzymes involved in cysteine synthesis in the chloroplast T Roitsch , MC Gonzalez . Function and regulation of plant invertases: sweet sensations . Trends Plant Sci , 2004 . 9 ( 12 ): 606 - 613 . 15564128 DOI: 10.1016/j.tplants.2004.10.009 http://doi.org/10.1016/j.tplants.2004.10.009 . Function and regulation of plant invertases: sweet sensations N Rouhier , SD Lemaire , JP Jacquot . The role of glutathione in photosynthetic organisms: Emerging functions for glutaredoxins and glutathionylation . Annu Rev Plant Biol , 2008 . 59 143 - 166 . 18444899 DOI: 10.1146/annurev.arplant.9.032607.092811 http://doi.org/10.1146/annurev.arplant.9.032607.092811 . The role of glutathione in photosynthetic organisms: Emerging functions for glutaredoxins and glutathionylation E Ruelland , M Miginiac-Maslow . Regulation of chloroplast enzyme activities by thioredoxins: activation or relief from inhibition? . Trends Plant Sci , 1999 . 4 ( 4 ): 136 - 141 . 10322547 DOI: 10.1016/S1360-1385(99)01391-6 http://doi.org/10.1016/S1360-1385(99)01391-6 . Regulation of chloroplast enzyme activities by thioredoxins: activation or relief from inhibition? U Schluter , D Kopke , T Altmann , C Mussig . Analysis of carbohydrate metabolism of CPD antisense plants and the brassinosteroid-deficient cbb1 mutant . Plant Cell Environ , 2002 . 25 ( 6 ): 783 - 791 . DOI: 10.1046/j.1365-3040.2002.00860.x http://doi.org/10.1046/j.1365-3040.2002.00860.x . Analysis of carbohydrate metabolism of CPD antisense plants and the brassinosteroid-deficient cbb1 mutant P Schüermann , BB Buchanan . The ferredoxin/thioredoxin system of oxygenic photosynthesis . Antioxid Redox Signal , 2008 . 10 ( 7 ): 1235 - 1273 . 18377232 DOI: 10.1089/ars.2007.1931 http://doi.org/10.1089/ars.2007.1931 . The ferredoxin/thioredoxin system of oxygenic photosynthesis TD Sharkey , LV Savitch , ND Butz . Photometric method for routine determination of kcat and carbamylation of Rubisco . Photosynth Res , 1991 . 28 ( 1 ): 41 - 48 . DOI: 10.1007/BF00027175 http://doi.org/10.1007/BF00027175 . Photometric method for routine determination of kcat and carbamylation of Rubisco TR Sinclair , LC Purcell , CH Sneller . Crop transformation and the challenge to increase yield potential . Trends Plant Sci , 2004 . 9 ( 2 ): 70 - 75 . 15102372 DOI: 10.1016/j.tplants.2003.12.008 http://doi.org/10.1016/j.tplants.2003.12.008 . Crop transformation and the challenge to increase yield potential U Sonnewald , J Lerchl , R Zrenner , W Frommer . Manipulation of sink-source relations in transgenic plants . Plant Cell Environ , 1994 . 17 ( 5 ): 649 - 658 . DOI: 10.1111/j.1365-3040.1994.tb00156.x http://doi.org/10.1111/j.1365-3040.1994.tb00156.x . Manipulation of sink-source relations in transgenic plants M Stitt . Limitation of photosynthsis by carbon metabolism. I. Evidence fro excess electron-transport capacity in leaves carrying out photosynthsis in saturating light and CO 2 . Plant Physiol , 1986 . 81 ( 4 ): 1115 - 1122 . 16664953 DOI: 10.1104/pp.81.4.1115 http://doi.org/10.1104/pp.81.4.1115 . Limitation of photosynthsis by carbon metabolism. I. Evidence fro excess electron-transport capacity in leaves carrying out photosynthsis in saturating light and CO2 Y Sun , XY Fan , DM Cao , WQ Tang , K He , JY Zhu , JX He , MY Bai , SW Zhu , E Oh , 等 . In tegration of brassinosteroid signal transduction with the transcription network for plant growth regulation in Arabidopsis . Dev Cell , 2010 . 19 ( 5 ): 765 - 777 . 21074725 DOI: 10.1016/j.devcel.2010.10.010 http://doi.org/10.1016/j.devcel.2010.10.010 . In tegration of brassinosteroid signal transduction with the transcription network for plant growth regulation in Arabidopsis O van Kooten , J Snel . The use of chlorophyll fluorescence nomenclature in plant stress physiology . Photosynth Res , 1990 . 25 ( 3 ): 147 - 150 . DOI: 10.1007/BF00033156 http://doi.org/10.1007/BF00033156 . The use of chlorophyll fluorescence nomenclature in plant stress physiology S von Caemmerer , GD Farquhar . Some relationships between the biochemistry of photosynthesis and the gas-exchange of leaves . Planta , 1981 . 153 ( 4 ): 376 - 387 . DOI: 10.1007/BF00384257 http://doi.org/10.1007/BF00384257 . Some relationships between the biochemistry of photosynthesis and the gas-exchange of leaves H Willekens , S Chamnongpol , M Davey , M Schraudner , C Langebartels , M van Montagu , D Inze , W van Camp . Catalase is a sink for H 2 O 2 and is indispensable for stress defence in C-3 plants . EMBO J , 1997 . 16 ( 16 ): 4806 - 4816 . 9305623 DOI: 10.1093/emboj/16.16.4806 http://doi.org/10.1093/emboj/16.16.4806 . Catalase is a sink for H2O2 and is indispensable for stress defence in C-3 plants CY Wu , A Trieu , P Radhakrishnan , SF Kwok , S Harris , K Zhang , JL Wang , JM Wan , HQ Zhai , S Takatsuto , 等 . Brassinosteroids regulate grain filling in rice . Plant Cell , 2008 . 20 ( 8 ): 2130 - 2145 . 18708477 DOI: 10.1105/tpc.107.055087 http://doi.org/10.1105/tpc.107.055087 . Brassinosteroids regulate grain filling in rice XJ Xia , LF Huang , YH Zhou , WH Mao , K Shi , JX Wu , T Asami , ZX Chen , JQ Yu . Brassinosteroids promote photosynthesis and growth by enhancing activation of Rubisco and expression of photosynthetic genes in Cucumis sativus . Planta , 2009 . 230 ( 6 ): 1185 - 1196 . 19760261 DOI: 10.1007/s00425-009-1016-1 http://doi.org/10.1007/s00425-009-1016-1 . Brassinosteroids promote photosynthesis and growth by enhancing activation of Rubisco and expression of photosynthetic genes in Cucumis sativus XJ Xia , YJ Wang , YH Zhou , Y Tao , WH Mao , K Shi , T Asami , Z Chen , JQ Yu . Reactive oxygen species are involved in brassinosteroid-induced stress tolerance in cucumber . Plant Physiol , 2009 . 150 ( 2 ): 801 - 814 . 19386805 DOI: 10.1104/pp.109.138230 http://doi.org/10.1104/pp.109.138230 . Reactive oxygen species are involved in brassinosteroid-induced stress tolerance in cucumber JQ Yu , Y Matsui . Effects of root exudates of cucumber ( Cucumis sativus ) and allelochemicals on ion uptake by cucumber seedlings . J Chem Ecol , 1997 . 23 ( 3 ): 817 - 827 . DOI: 10.1023/B:JOEC.0000006413.98507.55 http://doi.org/10.1023/B:JOEC.0000006413.98507.55 . Effects of root exudates of cucumber (Cucumis sativus) and allelochemicals on ion uptake by cucumber seedlings JQ Yu , LF Huang , WH Hu , YH Zhou , WH Mao , SF Ye , S Nogues . A role for brassinosteroids in the regulation of photosynthesis in Cucumis sativus . J Exp Bot , 2004 . 55 ( 399 ): 1135 - 1143 . 15107450 DOI: 10.1093/jxb/erh124 http://doi.org/10.1093/jxb/erh124 . A role for brassinosteroids in the regulation of photosynthesis in Cucumis sativus X Zhang , L Zhang , FC Dong , JF Gao , DW Galbraith , CP Song . Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba . Plant Physiol , 2001 . 126 ( 4 ): 1438 - 1448 . 11500543 DOI: 10.1104/pp.126.4.1438 http://doi.org/10.1104/pp.126.4.1438 . Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba YH Zhou , JQ Yu , LF Huang , S Nogues . The relationship between CO 2 assimilation, photosynthetic electron transport and water-water cycle in chill-exposed cucumber leaves under low light and subsequent recovery . Plant Cell Environ , 2004 . 27 ( 12 ): 1503 - 1514 . DOI: 10.1111/j.1365-3040.2004.01255.x http://doi.org/10.1111/j.1365-3040.2004.01255.x . The relationship between CO2 assimilation, photosynthetic electron transport and water-water cycle in chill-exposed cucumber leaves under low light and subsequent recovery 10.1631/jzus.B1200130 Project supported by the National Basic Research Program (973) of China (No. 2009CB119000), the National Natural Science Foundation of China (No. 30972033), and the Program for New Century Excellent Talents in University, China 2012-08-03 2012-09-11 2012-09-10 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2012 2012 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 10 13