Reactive Oxygen Species Are Involved in Brassinosteroid-Induced … · Reactive Oxygen Species Are Involved in Brassinosteroid-Induced Stress Tolerance in Cucumber1[W] ... Vert and

Reactive Oxygen Species Are Involved inBrassinosteroid-Induced Stress Tolerancein Cucumber1[W]

Xiao-Jian Xia2, Yan-Jie Wang2, Yan-Hong Zhou2, Yuan Tao, Wei-Hua Mao, Kai Shi, Tadao Asami,Zhixiang Chen, and Jing-Quan Yu*

Department of Horticulture, Zhejiang University, Hangzhou 310029, China (X.-J.X., Y.-J.W., Y.-H.Z., Y.T.,W.-H.M., K.S., J.-Q.Y.); Key Laboratory of Horticultural Plant Growth, Development, and Biotechnology,Agricultural Ministry of China, Hangzhou 310029, China (Y.-H.Z., J.-Q.Y.); Department of Applied BiologicalChemistry, University of Tokyo, Bunkyo Ku, Tokyo 1138657, Japan (T.A.); and Department of Botany andPlant Pathology, Purdue University, West Lafayette, Indiana 47907–2054 (Z.C.)

Brassinosteroids (BRs) induce plant tolerance to a wide spectrum of stresses. To study how BR induces stress tolerance, wemanipulated the BR levels in cucumber (Cucumis sativus) through a chemical genetics approach and found that BR levels werepositively correlated with the tolerance to photo-oxidative and cold stresses and resistance to Cucumber mosaic virus. We alsoshowed that BR treatment enhanced NADPH oxidase activity and elevated H2O2 levels in apoplast. H2O2 levels were elevatedas early as 3 h and returned to basal levels 3 d after BR treatment. BR-induced H2O2 accumulation was accompanied byincreased tolerance to oxidative stress. Inhibition of NADPH oxidase and chemical scavenging of H2O2 reduced BR-inducedoxidative and cold tolerance and defense gene expression. BR treatment induced expression of both regulatory genes, such asRBOH, MAPK1, and MAPK3, and genes involved in defense and antioxidant responses. These results strongly suggest thatelevated H2O2 levels resulting from enhanced NADPH oxidase activity are involved in the BR-induced stress tolerance.

Plants are constantly exposed to a variety of biotic(i.e. pathogen infection and insect herbivory) andabiotic stresses (i.e. extreme temperature, drought,and salinity). To survive such stresses, plants haveevolved intricate mechanisms to perceive externalsignals and activate optimal responses to environmen-tal conditions. At the molecular level, the perception ofextracellular stimuli and subsequent activation of ap-propriate responses require a complex interplay ofsignaling cascades. It has been shown that phytohor-mones, such as salicylic acid (SA), jasmonic acid, andabscisic acid (ABA), regulate the protective responsesof plants to both biotic and abiotic stresses indepen-dently and through synergistic and antagonistic crosstalk (Bostock, 2005; Lorenzo and Solano, 2005; Mauch-Mani and Mauch, 2005). Moreover, plant responses todifferent types of stresses are associated with genera-

tion of reactive oxygen species (ROS), suggesting thatROS may function as a common signal in signalingpathways of plant stress responses (Apel andHirt, 2004;Torres and Dangl, 2005). More recent studies indicateextensive cross talk of plant signaling pathways fordefense against pathogens with those for responses toabiotic stresses (Fujita et al., 2006).

For a long time, ROS was believed as a harmfulbyproduct in aerobic organisms. Extensive studieshave shown that while high levels of ROS cause celldeath, low levels of ROS have regulatory roles in plantstress responses. Application of ABA and SA as well asexposure to low temperature all resulted in a transientelevation of H2O2, leading to an increased tolerance tosalt, high light, heat, and oxidative stress (Prasad et al.,1994; Dat et al., 1998; Zhang et al., 2001). It has beenproposed that ROS plays a critical role in inducedtolerance by activating or inducing stress response-related factors, such as mitogen-activated proteinkinases (MAPKs), transcription factors, antioxidantenzymes, dehydrins, and low-temperature-induced,heat shock, and pathogenesis-related proteins (Gechevet al., 2006).

Brassinosteroids (BRs) are a group of naturallyoccurring plant steroids and are important for a broadspectrum of cellular and physiological processes, in-cluding stem elongation, pollen tube growth, leafbending and epinasty, root inhibition, fruit develop-ment, ethylene biosynthesis, proton pump activity,xylem differentiation, photosynthesis, and gene ex-

1 This work was supported by the National Basic ResearchProgram of China (grant no. 2009CB119000), the National NaturalScience Foundation of China (grant nos. 3050344 and 30671428), andthe Program for Promotion of Basic Research Activities for Innova-tive Bioscience (PROBRAIN).

2 These authors contributed equally to the article.* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Jing-Quan Yu ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.109.138230

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pression (Li et al., 1996; Sasse, 1997; Clouse and Sasse,1998; Dhaubhadel et al., 1999; Hu et al., 2000; Artecaand Arteca, 2001; Mussig et al., 2002; Yu et al., 2004; Fuet al., 2008). Important progress has been made inelucidating the BR signal transduction pathway. Thediscovery of BR-insensitive mutants in Arabidopsis(Arabidopsis thaliana), pea (Pisum sativum), tomato (So-lanum lycopersicum), and rice (Oryza sativa) led to theisolation of the BRI1 gene and its homologs (Li andChory, 1997; Yamamuro et al., 2000; Montoya et al.,2002; Nomura et al., 2003). BRI1 is a BR-binding Leu-rich repeat receptor located in the plasma membraneand functions in vitro as a Ser/Thr kinase (Wang et al.,2001). Recent studies have identified several othercomponents in the BR signaling pathway (Li andNam, 2002; Nam and Li, 2002; Mora-Garcıa et al.,2004), including BAK1 (for BRI1-associated receptorkinase 1), BIN2 (a GSK3/SHAGGY-like kinase), andBSU1 (for BRI1 suppressor 1, a phosphatase). BR bindsto the extracellular domain of BRI1 and activates itsintracellular kinase activity (Kinoshita et al., 2004).Activated BRI1 interacts with and activates its core-ceptor BAK1 (Li et al., 2002; Nam and Li, 2002). TheBIN2 kinase and BSU1 phosphatase function down-

stream of the receptor kinases and regulate the phos-phorylation status of BZR1 and BZR2/BES1 transcriptionfactors (Belkhadir and Chory, 2006; Vert and Chory,2006). Dephosphorylated BZR1 and BZR2/BES1 rec-ognize the promoters of BR target genes and regulatetheir expression (He et al., 2002; Yin et al., 2002).

In addition to its critical roles in growth regulationand photomorphogenesis, BRs can induce plant tol-erance to a variety of abiotic stresses, such as highand low temperature stress, drought, and salinityinjury (Krishna, 2003; Kagale et al., 2007). However,the underlying mechanisms for BR-mediated stressresponses are not understood. It has been found thatBR-induced increase in the basic thermotolerance isassociated with increased heat shock protein syn-thesis and accumulation as well as increased expres-sion of some components of translational machinery(Dhaubhadel et al., 1999, 2002). The mechanism bywhich BR induces protein synthesis during heatstress is unclear. BR may also play a role in plantresponses to pathogens. BR induces resistance oftobacco (Nicotiana tabacum) and rice to bacterialand fungal pathogens (Nakashita et al., 2003). BR-induced disease resistance was not correlated with

Figure 1. Effects of BR levels on resistance to PQ, chill, or CMV. A, Symptoms (up) and images of the maximum PSII quantumyield (Fv/Fm, down). The false color code depicted at the bottom of the image ranged from 0 (black) to 1.0 (purple). Plants treatedwith water, 0.1mM EBR, 4 mM Brz, and 4 mM Brz + 0.1mM EBRwere challengedwith 10mM PQ at 600mmolm22 s21 light intensityand 25�C for 1 d. Five plants were used for each treatment and the picture of one representative leaf is shown. Bars = 2.5 cm. B,Average Fv/Fm values. Fv/Fm was determined with the whole leaf as area of interest. Fv/Fm for control plants was 0.83. Data are themeans of five replicates (6SD). Means denoted by the same letter did not significantly differ at P, 0.05 according to Tukey’s test.C, ETRs determined after 1-d exposure to chill stress (8�C/200 mmol m22 s21) for plants treated with water, 0.1 mM EBR, 4 mM Brz,and 4 mM Brz + 0.1 mM EBR. Measurements were conducted at 25�C. Data are the means of five replicates (6SD). D, CMVincidence and MDA content determined at 14 dpi for plants treated with water, 0.1 mM EBR, 4 mM Brz, and 4 mM Brz + 0.1 mM

EBR. Disease index is the mean (n = 9 leaves) of disease severities (0, light, to 1, severe). Data for MDA are the means of fivereplicates (6SD). Means denoted by the same letter did not significantly differ at P , 0.05 according to Tukey’s test. FW, Freshweight.

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enhanced SA accumulation or increased expressionof genes associated with SA-regulated systemic ac-quired resistance (SAR). Additionally, simultaneoustreatment of plants with BR and SAR inducers re-sulted in additive protection against pathogen attack(Nakashita et al., 2003). Thus, BR-induced diseaseresistance is mediated by a novel signaling pathwaydistinct from the SA-regulated SAR pathway.We have previously reported that BR enhances

photosynthesis and chill tolerance (Yu et al., 2002,2004). Subsequently, we have observed unexpectedlythat BR also triggers a periodic increase in H2O2level in cucumber (Cucumis sativus) leaves. H2O2can function as a signaling molecule in response tovarious stimuli both in plant and animal cells (Neillet al., 2002). To study the role of elevated H2O2 levelin BR-induced stress tolerance, we analyzed the effectsof exogenous BR, inhibitors of BR biosynthesis and ROSproduction, and ROS scavengers on stress toleranceand associated gene expression in cucumber. Thesestudies demonstrated that BR induced tolerance to bothbiotic and abiotic stresses in cucumber plants. In addi-

tion, we provide strong evidence that H2O2 plays a rolein the BR-induced plant stress tolerance.

RESULTS

BR Induces Plant Stress Tolerance

To determine whether BR induces stress tolerance incucumber plants, we obtained four types of plantswith different BR levels by applications of 24-epibrassi-nolide (EBR), one of the bioactive BRs, and brassinazole(Brz), a specific inhibitor of BR biosynthesis (Supple-mental Fig.S1). We first compared the effects of EBRand Brz on plant sensitivity to paraquat (PQ), whichcauses photo-oxidative stress. When grown undercontinuous light, necrotic lesions appeared 1 d afterPQ treatment on leaves of water-, Brz-, and Brz+EBR-treated plants but not on those of EBR-treated plants(Fig. 1A). To analyze the effects on photosyntheticefficiency, we compared the maximum photochemicalefficiency of PSII in the dark-adapted state (Fv/Fm;Maxwell and Johnson, 2000). Fluorescence images of

Figure 2. Expression of stress-responsive genesin response to BR levels in cucumber seedlings.qRT-PCR analysis was performed to examinesteady-state levels of mRNAs for 18 genes inplants treated with water, 0.1 mM EBR, 4 mM Brz,and 4 mM Brz + 0.1 mM EBR. Data are the meansof three replicates (6SD).

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Fv/Fm showed that PQ treatment resulted in a signif-icant decrease in Fv/Fm in water-treated plants (Fig. 1).PQ-induced reduction in Fv/Fm was less in EBR-treated plants but greater in Brz-treated plants (Fig.1B). Furthermore, EBR treatment restored Fv/Fm inBrz-treated plants close to that of water-treated plants.

Electron transport rates (ETRs) were determinedat 25�C after the chilling stress (8�C/200 mmol m22 s21)to assess the effects of EBR and Brz on chilling toler-ance of cucumber seedlings. Chilling stress caused sig-nificant reduction in ETR. EBR treatment alleviatedchilling stress and enhanced the ETR, whereas Brztreatment reduced ETR compared to water-treatedplants. In addition, EBR treatment significantly re-stored ETR of Brz-treated plants (Fig. 1C).

We also examined the role of BR in plant responsesto Cucumber mosaic virus (CMV) by comparing diseasesymptom development and CMV-induced lipid per-oxidation based on the malondialdehyde (MDA) con-tent after EBR or Brz treatment. Water-treated plantsdeveloped typical CMV symptoms by 10 d postinoc-ulation (dpi). When CMV disease severity was rated at14 dpi, Brz-treated plants had higher disease indexand MDA content than water-treated plants (Fig. 1D),suggesting that BR biosynthesis was important forplant response to CMV. By contrast, CMV diseaseseverity and MDA content in EBR-treated plants were

lower than those in water-treated plants. In addition,application of EBR to Brz-treated plants restored re-sistance to CMV (Fig. 1D). These results indicate thatBR enhances plant tolerance or resistance to bothabiotic and biotic stresses.

Changes in Gene Expression in Response to BR Levels

To analyze the underlying molecular mechanismsfor BR-induced stress tolerance, we examined the ef-fects of BR levels on expression of 18 stress-responsivegenes. As shown in Figure 2, three regulatory genes,RBOH, MAPK1, and MAPK3, were up-regulated upontreatment with EBR but down-regulated after Brztreatment. Among the three genes, MAPK1 was mostaffected by Brz, whose expression was reduced byapproximately 70%. Reductions of RBOH and MAPK3expression by Brz were more moderate but also sub-stantial. Again, application of EBR to Brz-treated plantsrescued the repressed expression of the regulatorygenes.

Interestingly, expression of WRKY6 and MYB wasinduced .30-fold by EBR application (Fig. 2). EBRalso induced expression of two other genes encodingtranscription factors, WRKY30 and MYC. Unexpect-edly, expression of the four transcription factor geneswas also up-regulated after Brz treatment. EBR also

Figure 3. The roles of BR in regulation of ROSaccumulation. A, In situ detection of leaf O2

.2 andH2O2. NBT and DAB stains were used to detectthe presence of O2

.2 and H2O2 in leaves treatedwith water, 0.1 mM EBR, 4 mM Brz, and 4 mM Brz +0.1 mM EBR. Leaf discs (1.5 cm in diameter) wereharvested at 6 h after treatment and stainedimmediately. B, Cytochemical localization ofH2O2 accumulation in mesophyll cells of cucum-ber leaves with CeCl3 staining and transmissionelectron microscopy. The plants were treated andharvested as described in (A). Arrows, CeCl3precipitates; C, chloroplast; CW, cell wall; V,vacuole; IS, intercellular space. C, Blockage ofEBR-induced H2O2 accumulation by DPI andDMTU. Plants were pretreated with 100 mM DPIor 5 mM DMTU for 8 h and then treated with 0.1mM EBR. After 6 h, the DAB staining of leaf discswas performed. D, Quantitative measurements ofH2O2 level and NADPH oxidase activity in cu-cumber leaves with different BR levels. Values aremeans 6 SD (n = 6). Means denoted by the sameletter did not significantly differ at P , 0.05according to Tukey’s test. FW, Fresh weight.

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induced expressions of genes encoding proteins in-volved in heat shock response (HSP and DnaJ), de-fense (PR-1, PAL, and HPL), detoxification (GST, GPX,and POD), and antioxidant (CAT, cAPX, and MDAR;Fig. 2). Brz treatment also induced expression of HSPand DnaJ but has little effect on expression of GPX,POD, CAT, PAL, cAPX, andMDAR. On the other hand,expression of PR-1, HPL, and GST were substantiallyreduced in Brz-treated plants. EBR and Brz had addi-tive effect on the induction of HSP and DnaJ.

Changes in H2O2 by BR Levels

ROS act as secondmessengers in stress and hormoneresponses (Apel and Hirt, 2004; Kwak et al., 2006). Todetermine a possible role of ROS in BR-induced stresstolerance, we attempted to detect in situ accumulationof O2

.2 and H2O2 using nitroblue tetrazolium (NBT)and 3,3#-diaminobenzidine (DAB) staining proce-dures, respectively. Both procedures detected in-creased staining in EBR-treated leaves but decreasedstaining in Brz-treated leaves relative to that in water-treated leaves (Fig. 3A). However, EBR application toBrz-treated leaves partially restored O2

.2 and H2O2levels. Interestingly, we observed enhanced stainingon the edges of the leaf discs, probably due to wound-ing because it was not affected by EBR or Brz treatment(Fig. 3A). Similar effects of EBR and Brz on leaf H2O2accumulation were observed when an independentspectrophotometric method was used (Fig. 3D).Using the CeCl3-based procedures, we showed that

EBR-induced H2O2 was predominantly accumulated

on the cell walls of mesophyll cells facing intercellularspaces but was undetectable in the cytosol or intracel-lular organelles, such as chloroplasts, mitochondria,nuclei, or vacuoles (Fig. 3B). In addition, EBR-inducedH2O2 accumulation was sensitive to diphenyleneiodo-nium (DPI), a potent inhibitor of NADPH oxidase (Fig.3C). Dimethylthiourea (DMTU), an H2O2 scavenger,also abolished EBR-induced H2O2 accumulation.These results suggest that BR-induced H2O2 accumu-lation is caused by increased activity of NADPHoxidase. To confirm this, we measured the activity ofplasma membrane NADPH oxidase in extracts of leaftissues. NADPH oxidase activities increased signifi-cantly in EBR-treated plants but decreased signifi-cantly in Brz-treated plants compared with that ofwater-treated plants (Fig. 3D). Again, EBR was effec-tive in rescuing the repressed NADPH oxidase activityin Brz-treated plants.

Involvement of H2O2 in BR-Induced Stress Tolerance

To determine whether H2O2 accumulation contrib-utes to BR-induced stress tolerance, we analyzed theeffects of DPI and DMTU on EBR-induced tolerancesto oxidative stress inflicted upon either PQ treatmentor chilling stress (8�C) under 1,000 mmol m22 s21.Exposure to either stress caused necrotic lesions inwater-treated plants (Supplemental Fig. S2). In EBR- orH2O2-treated plants, on the other hand, necrotic le-sions were greatly reduced after PQ and chill treat-ment. Importantly, pretreatment with DPI or DMTUcompletely abolished the protective effects of EBR and

Figure 4. Requirement of H2O2 for EBR-induced resistance to PQ and chill. A,Images of the maximum PSII quantumyield (Fv/Fm) of PQ-challenged and chilledleaves (bar = 2.5 cm). The false color codedepicted at the bottom of the image rangedfrom 0 (black) to 1.0 (purple). Plants werepretreated with 100 mM DPI or 5 mM

DMTU for 8 h, and then plants weretreated with 0.1 mM EBR or 10 mM H2O2.After 1 d, plants were challenged with 10mM PQ or exposed to chill at high lightintensity (8�C/1,000 mmol m22 s21). Singletreatment of DPI or DMTU was includedas negative control. B and C, Average Fv/Fmvalues of PQ-challenged (B) or chilled (C)leaves. Fv/Fm was determined with thewhole leaf as area of interest. Fv/Fm for con-trol plants was 0.83. Values are means 6SD (n = 5). Means denoted by the sameletter did not significantly differ at P, 0.05according to Tukey’s test.



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H2O2 on plant tolerance to PQ and the chill (Fig. 4A).EBR and H2O2 treatment also alleviated significantlythe decline of Fv/Fm after PQ treatment and the chill,and these protective effects were again almost com-pletely blocked by DPI and DMTU (Fig. 4, B and C).These results strongly suggest that H2O2 is involved inthe BR-induced stress tolerance.

The expression of genes implicated in signal trans-duction (MAPK1), transcription (WRKY6), and stresstolerance (PR-1, PAL, CAT, and cAPX) was induced byEBR and H2O2 (Fig. 5). Again, DPI and DMTU pre-treatment abolished or substantially reduced EBR-induced expression of these genes (Fig. 5). Likewise,both EBR and H2O2 induced expression of antioxidantgenes CAT and cAPX, and EBR treatment increasedactivities of antioxidant enzymes (superoxide dismut-ase [SOD], catalase [CAT], ascorbate peroxidase [APX],monodehydroascorbate reductase [MDAR], dehydro-ascorbate reductase [DHAR], and glutathione reductase[GR]; Figs. 5 and 6). The increase in antioxidant gene

expression and activities of antioxidant enzymes uponEBR treatment were also blocked by DPI and DMTUpretreatment (Figs. 5 and 6). These results support theinvolvement of H2O2 in BR-induced gene expressions.

Time Course of BR-Induced H2O2 Accumulation,Tolerance, Transcript Levels, and Activities ofAntioxidant Enzymes

H2O2 levels were increased as early as at 3 h andremained elevated up to 24 h after EBR treatment (Fig.7A). H2O2 levels were not significantly altered duringthe first 24 h after Brz treatment but were substantiallyreduced at 72 h after Brz treatment and continued todecline during the remaining period of the experi-ments (Fig. 7B). To further investigate the involvementof H2O2 in EBR-induced stress tolerance, we examinedthe effect of PQ-mediated oxidative stress applied atdifferent intervals after EBR, H2O2, or Brz treatment.Plants were first sprayedwith EBR, H2O2, or Brz and at

Figure 5. Involvement of H2O2 in EBR-induced up-regulation of stress-respon-sive genes. Plants were pretreated with100 mM DPI or 5 mM DMTU for 8 h andthen treated with 0.1 mM EBR or 10 mM

H2O2. After 6 h, the steady-state tran-script levels were assayed by qRT-PCR.Data are the means of three replicates(6SD).

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various intervals after the treatment plants were sub-jected to oxidative stress treatment by applying PQ.The stress tolerance was then determined by measure-ment of Fv/Fm 1 d after PQ treatment. Enhancedtolerance of cucumber seedlings to PQ-induced oxi-dative stress was observed at 3 h after treatment withH2O2 and at 6 h after EBR treatment (Fig. 8; Supple-

mental Fig. S3A). Thus, H2O2 induced stress tolerancemore rapidly than EBR (Fig. 8A). The maximum levelof stress tolerance was observed at 12 h after treatmentwith H2O2 and at 24 h after EBR treatment (Fig. 8A).No significant level of stress tolerance was observed at72 h after treatment with H2O2 and at 120 h after EBRtreatment (Fig. 8A). The time course for Brz-induced

Figure 6. Involvement of H2O2 in EBR-induced up-regulation of antioxidantenzyme activity. Plants were pretreatedwith 100 mM DPI or 5 mM DMTU for8 h and then treated with 0.1 mM EBR.After 6 h, the activities of antioxidantenzymes were determined. Values aremeans 6 SD (n = 6). Means denoted bythe same letter did not significantlydiffer at P , 0.05 according to Tukey’stest.

Figure 7. Kinetics of changes in H2O2 content in EBR- or Brz-treated plants. A, Plants were treated with distilled water or 0.1 mM

EBR. Leaf samples were harvested at indicated times (h) after EBR treatment. Values are means 6 SD (n = 6). B, H2O2 content inleaves after different duration of Brz treatment. Brz (4 mM) treatment started at indicated times (h) before sampling. Brz treatmentwas repeated on alternative days until sampling. Time zero points were without Brz treatment. Values are means6 SD (n = 6). FW,Fresh weight.



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decline in stress tolerance was highly correlated to thatof Brz-induced decrease in H2O2 levels (r = 0.96, P ,0.01). Thus, like changes in H2O2 levels, stress toler-ance was not significantly altered during the first 24 hbut substantially reduced at 72 h after Brz treatmentand continued to decline during the remaining periodof the experiments (Fig. 8B; Supplemental Fig. S3B).

To characterize further the relationship of BR-induced H2O2 and enhanced stress tolerance, we an-alyzed the temporal changes in transcript levels ofseveral stress-responsive genes and activities of anti-oxidant enzymes. The expression of RBOH and CATstarted to increase at 3 h after EBR treatment, peaked at12 h, and then declined. The cAPX expression in-creased at 3 h after EBR treatment and remainedelevated for the following 9 h before declining tocontrol level. PR-1 and PAL transcript levels alsoincreased at 3 h, peaked at 12 h, and became unde-tectable at 72 h after EBR treatment (Fig. 9). Significantincreases in activities of SOD, CAT, and MDAR werealso detected at 3 h after EBR treatment (Fig. 10). Forother antioxidant enzymes (APX, DHAR, and GR),significant increases occurred at 6 h. The activities ofall these antioxidant enzymes declined to basal levelsat 96 h after EBR treatment (Fig. 10). The decline of theantioxidant enzyme activities at 96 h after EBR treat-ment was accompanied by disappearance of EBR-induced stress tolerance at 120 h after EBR treatment(Fig. 8A).

DISCUSSION

BR Induces Stress Tolerance in Cucumber

Several studies have shown that BR enhances planttolerance to a variety of environmental stresses (Khripachet al., 2000; Dhaubhadel et al., 2002; Nakashita et al.,2003; Krishna, 2003; Kagale et al., 2007). However, it is

difficult to analyze genetically the role and actionmechanisms of BR in plant stress tolerance because ofthe strong and pleiotropic phenotypes of BR biosyn-thesis and signaling mutants, including extremedwarfism, dark green and epinastic leaves, and de-layed development (Khripach et al., 2000; Bishop andKoncz 2002; Mussig et al., 2002; Cao et al., 2005).Moreover, current techniques allow for measurementof stress-induced changes in the levels of only inter-mediates in the BR biosynthesis pathway but not of thebioactive brassinolide (Nakashita et al., 2003; Jageret al., 2008). As a result, application of Brz, a specificand potent inhibitor of BR biosynthesis (Asami et al.,2000, 2001), has been used to study the role of BR inplant stress responses. Consistent with the feedbackcontrol of BR biosynthesis (Bancos et al., 2002), Brz up-regulated BR biosynthetic genes and caused growthaberrations of cucumber seedlings (SupplementalFigs. S1 and S4). These observations support thatendogenous BR contents in cucumber seedlings werealtered by Brz application.

Our results demonstrated that EBR enhanced andBrz reduced tolerance to oxidative, cold, and CMVstresses in cucumber (Fig. 1). However, in comparisonto the relatively fast induction (approximately 3 h) oftolerance by EBR application, the negative effect of Brzon stress tolerance was relatively slow (Fig. 8). Brz is aspecific inhibitor for DWF4, a cytochrome P450 mono-oxygenase of the BR biosynthetic pathway (Asamiet al., 2001), and it could not inhibit the downstreamenzymes in the BR biosynthesis pathway. Accordingly,it needs a period of time to reduce the bioactive BRlevel. These results strongly suggest that BR-inducedstress tolerance is quantitative in nature and is corre-lated with the BR levels. In other words, normalsynthesis of BRs under nonstress conditions is ex-pected to confer a certain level of stress tolerance, butan increase in BR accumulation under certain types of

Figure 8. Levels of tolerance to PQ-induced oxidative stress at different times after EBR, H2O2, and Brz treatment. A, Oxidativestress tolerance induction curves of EBR or H2O2. PQ (10 mM) was applied at indicated times (h) after water, 0.1 mM EBR, or 10mM

H2O2 treatment. Time zero points indicate PQ treatment only. Fv/Fm was determined with the whole leaf as area of interest after1 d at 600 mmol m22 s21 and 25�C. Fv/Fm for PQ-untreated leaves was 0.83. Values are means 6 SD (n = 5). B, Oxidative stresstolerance of plants after different duration of Brz treatment. Brz (4 mM) treatment started at indicated times (h) before 10 mM PQchallenge. Brz treatment was repeated on alternative days until PQ challenge. Time zero points indicate PQ treatment only. Fv/Fmwas determined using the whole leaf as area of interest after 1 d at 600 mmol m22 s21 and 25�C. Fv/Fm for PQ-untreated leaveswas 0.83. Values are means 6 SD (n = 5).

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stress conditions will lead to a corresponding increasein stress tolerance. Conversely, reducing BR accu-mulation below its normal levels (e.g. after Brz ap-plication) leads to a corresponding decrease in stresstolerance. These studies support the involvement ofBR in plant responses to various environmental stresses(Krishna, 2003; Nakashita et al., 2003; Kagale et al.,2007). There were significant increases in severalintermetabolite levels in the BR biosynthesis pathwayafter Tobacco mosaic virus inoculation in tobacco leaves(Nakashita et al., 2003), whereas there were no consis-tent changes in the intermetabolite levels in leaves ofpea in response to water stress (Jager et al., 2008).Accordingly, BR biosynthesis may be induced in somebut not all types of stress conditions. Furthermore, werecently found that as in Brz-treated cucumber plants,

the tomato BR biosynthesis dim mutant exhibited en-hanced sensitivity to PQ-induced oxidative stress, andthis phenotype can be rescued by exogenously appliedBR. Most recently, we also found that EBR applicationto wild-type and BR-deficient mutant all increasedtheir resistance to Cladosporium fulvum in tomato (datanot shown). There was, however, no significant differ-ence in the tolerance of wild-type and BR-deficientmutant to water stress in pea (Jager et al., 2008). Thus,BRmay be involved in plant responses to some but notall types of plant stress conditions. Alternatively, thesevere morphological or physiological phenotypes,such as the dwarf shoots with thick leaves and de-creased stomatal conductance in BR-deficient peaplants, might complicate the water stress analysisbecause these phenotypes could lead to reduced tran-spiration and increased drought resistance.

Microarray analysis revealed that BR induces theexpression of heat shock protein (HSP83, HSP70,Hsc70-3, and Hsc70-G7), heat shock factor (HSF3),and oxidative stress-related genes (GST, ATPA2, andATP24a) in Arabidopsis (Goda et al., 2002; Mussiget al., 2002). Our quantitative reverse transcription(qRT)-PCR analysis revealed similar induction by EBRof HSP70, DnaJ, GST, and POD in cucumber (Fig. 2).BR induced defense and antioxidant genes in theabsence of stresses. In contrast, expressions of coldand pathogenesis-related genes are reduced in Brz-treated cucumber seedlings or BR-deficient Arabidop-sis mutants (Szekeres et al., 1996; Mussig et al., 2002).These results suggest that BR enhances plant stresstolerance by activating genes involved in plant defenseand stress responses.

H2O2 Is Involved in BR-Induced Stress Tolerancein Cucumber

In this study, we have provided several lines ofevidences that H2O2 is involved in BR-induced stresstolerance. First, EBR-induced stress tolerance was pre-ceded by increased NADPH oxidase activity and ele-vated H2O2 levels (Fig. 3). Second, scavenging of H2O2by DMTU or inhibiting H2O2 generation by DPI abol-ished EBR-induced stress tolerance (Fig. 4). Third, EBR-and Brz-induced changes in H2O2 levels were closelyrelated to the changes in the tolerance to PQ-mediatedoxidative stress (Figs.7 and 8; Supplemental Fig. S3),and exogenously applied H2O2 also induced the toler-ance (Fig. 8A; Supplemental Fig.S3A). In addition, thetemporal changes in H2O2 accumulation, transcriptlevels of antioxidant genes, and activities of antioxidantenzymes are consistent with the role of H2O2 in BR-induced stress tolerance (Figs. 7, 9, and 10). Of par-ticular relevance is the observation that the intervalbetween EBR treatment and stress challenge is criticalfor the magnitude of EBR-induced stress tolerance (Figs.7 and 8). Accordingly, the variation of the efficiency ofBR in enhancing plant stress tolerance in different stud-ies (Khripach et al., 2000) is likely to be related to timeinterval between BR application and stress challenge.

Figure 9. Time-course analysis of steady-state transcript levels ofRBOH, CAT, cAPX, PR-1, and PAL in response to EBR. Leaf sampleswere harvested at indicated times (h) after 0.1 mM EBR treatment, andthe steady-state transcript levels were assayed by qRT-PCR. Data are themeans of three replicates (6SD).



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H2O2 is considered as a central signaling molecule inplant responses to biotic and abiotic stresses (Foyeret al., 1997; Neill et al., 2002). It activates protectivemechanisms for tolerance to chill in maize (Zea mays;Prasad et al., 1994), high temperature in mustard(Sinapis alba) seedlings (Dat et al., 1998), and lightstress in Arabidopsis leaves (Karpinski et al., 1999).Perturbed H2O2 homeostasis or increased productionof H2O2 enhanced the expression of antioxidant en-zymes and acidic PR proteins in the absence of path-ogen challenge (Chamnongpol et al., 1996; Takahashiet al., 1997; Wu et al., 1997). Likewise, we found thatEBR treatment induced increase in both H2O2 and PR-1mRNA levels (Figs. 2 and 3) without any significanteffect on SA accumulation (Supplemental Fig. S5).However, BR did not induce changes in expression ofPR genes in tobacco (Nakashita et al., 2003). This dis-crepancy might be caused by different interval be-tween BR application and sampling time or the lowsensitivity of northern blots to detect minor changes inexpression of PR genes. In support of this, stronginduction of PR-1 was transient and observed onlywithin a short period of time after EBR treatment (Fig.9). Verberne et al. (2003) have reported that ethylene isalso required for the production or transmission of themobile SAR signal in Tobacco mosaic virus-infectedleaves. BR induced expression of 1-aminocyclopropane-1-carboxylic acid oxidase in cucumber (data not shown),and the increase in stress tolerance in BR-treated potato

(Solanum tuberosum) tubers is associated with in-creased levels of ABA and ethylene and accumulationof phenolic and terpenoid compounds (Krishna, 2003).Moreover, microarray data indicate that there is crosstalk between BR and jasmonic acid signaling pathways(Goda et al., 2002; Mussig et al., 2002). It is likely thatBR-induced PR gene expression and stress toleranceare mediated by a complex set of signal transcriptionpathways with H2O2 as a common signal molecule inthe activation of stress responses. It may also not becompletely unexpected that signal transduction path-ways mediating plant growth may cross talk withthose mediating plant defense and stress responses.

In higher plants, ROS can be generated by severaldifferent pathways, including plasma membrane-localized NADPH oxidase, cell wall-localized peroxi-dases, and amine oxidases (Neill et al., 2002). We have

Figure 10. Time-course analysis ofantioxidant enzymes activities inresponse to EBR. Leaf samples wereharvested at indicated times (h) after0.1 mM EBR treatment, and the activi-ties of antioxidant enzymes were ana-lyzed. Values are means 6 SD (n = 6).

Figure 11. A model for the induction of stress tolerance by BR incucumber plants. Perception of BR by receptors results in the activationof plasma membrane-bound NADPH oxidase (RBOH). ActivatedNADPH oxidase results in elevated levels of H2O2, which functionsas a signal molecule to activate stress response pathways.

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presented evidence that NADPH oxidase is the po-tential source of BR-induced H2O2 generation. First,H2O2 accumulated mainly in the apoplast of meso-phyll cells. Second, treatment with EBR significantlyincreased the activity of NADPH oxidase. Third, DPI,a potential inhibitor of NADPH oxidase, blocked EBR-induced production of H2O2. However, because DPImay also inhibit other oxidases (Bolwell et al., 1998),we cannot completely rule out the possibility thatother oxidases may contribute to BR-induced ROSgeneration.It has been reported that BRI1-associated coreceptor

BAK1 and BKK1 (for BAK1-like 1) function not only inBR-dependent signaling in plant growth and develop-ment but also in the regulation of plant cell death.BAK1-deficient plants exhibited spreading necrosisaccompanied by enhanced accumulation of ROS afterinfection by pathogens (Kemmerling et al., 2007). Thebak1 bkk1 double mutants also accumulated enhancedROS and exhibited seedling lethality (He et al., 2007).However, the role of BAK1 and BKK1 in plant celldeath control is independent of BR (He et al., 2007;Kemmerling et al., 2007). It remains to be determinedwhether BRI1, BAK1, and BKK1 are required for BR-induced ROS production and stress tolerance.

The Signaling Pathways for BR-Induced Stress Tolerance

EBR induced genes encoding MAPK and transcrip-tion factors (Fig. 2). In plants, the MAPK cascade playsa crucial role in various biotic and abiotic stressresponses and in hormone signaling, which ofteninvolves ROS (Nakagami et al., 2005). H2O2 can acti-vate ANP1, an Arabidopsis MAPK kinase kinase toregulate the activities of MPK3 and MPK6 (Kovtunet al., 2000). A recently identified Ser/Thr proteinkinase (OXI1) has also been shown to play a centralrole in ROS sensing and the activation of MPK3 andMPK6, which control the activation of different de-fense mechanisms (Rentel et al., 2004). Thus, MAPKcascades can mediate H2O2 signaling and may alsoplay an important role in BR-induced stress tolerance.In addition to the MAPK genes, BR-induced stress

tolerance is associated with expression of a numberof genes encoding WRKY, MYB, and MYC transcrip-tion factors. WRKY transcription factors have beenimplicated in the regulation of transcriptional repro-gramming associated with plant immune responses(Eulgem and Somssich, 2007), and MYB and MYCtranscription factors have been implicated as criticalregulators of ABA-inducible gene expression underdrought stress (Abe et al., 2003; Agarwal et al., 2006).Similarly, genome-wide expression analyses haveshown that BR can regulate expression of genes en-codingMYB and ERF transcription factors (Goda et al.,2002; Mussig et al., 2002). More recently, Kagale et al.(2007) showed that BR enhances expression of tran-scription factors of the CBF/DREB family in bothunstressed and stress plants. The concerted inductionof genes encoding these transcription factors suggests

that BR-induced stress tolerance is mediated by tran-scriptional activation of genes involved in plant stressresponses. Intriguingly, treatment of Brz also resultedin enhanced expression of transcription factors andheat shock proteins without concomitant induction ofother defense genes expression. It is likely that BRdeficiency may cause certain stress response and, as aresult, induce expression of some but not all thetranscription factors required for enhanced stress tol-erance as observed in BR-treated plants (Szekereset al., 1996; Kagale et al., 2007).

In conclusion, we have presented strong evidencethat H2O2 mediates the transcriptional induction ofdefense or antioxidant genes by BR. Following per-ception of BR signal, NADPH oxidase may be acti-vated to produce ROS, which initiates a proteinphosphorylation cascade. Transcription factors maybe activated via a phosphorylation cascade byMAPKs.Finally, the products of targets genes participate di-rectly in cellular protection (Fig. 11). Further studiesare needed to provide genetic evidence of the involve-ment of NADPH oxidase in BR-induced ROS genera-tion, to identify the critical signaling componentsbetween BR perception and stress responses, and toelucidate the molecular mechanisms of cross talkbetween BR and other hormone signaling.

MATERIALS AND METHODS

Plant Growth

Cucumber (Cucumis sativus ‘Jinyan No. 4’) seeds were sown in a growth

chamber. Seven days after sowing, groups of eight seedlings were trans-

planted into a container (40 3 25 3 15 cm) filled with Hoagland nutrient

solution. The growth conditions were as follows: a 12-h photoperiod, tem-

perature of 25�C/17�C (day/night), and light intensity of 600 mmol m22 s21.

Experimental Design

To manipulate BR levels, we first treated cucumber seedlings with the BR

biosynthesis inhibitor Brz by spraying a 4 mM solution (Brz dissolved in DMSO)

to the tip and whole plants every 2 d from the cotyledon stage to the four-leaf

stage. Both water- and Brz-treated plants were then divided equally into two

groups for water or EBR treatments. Previous tests showed that a relatively

moderate concentration of EBR at 0.1 mM is most effective, which was used in

this experiment (Yu et al., 2004). This combination of treatments resulted in four

types of plants: water (BR level unchanged), Brz (BR level reduced), EBR (BR

level increased), and Brz + EBR (BR level reduced and then recovered) treat-

ments. At the four-leaf stage, these four types of seedlings were then exposed to

various forms of biotic or abiotic stresses. The third leaf from the bottom was

used for analysis. For cold stress, seedlings were transferred to 8�C/200 mmol

m22 s21 for 24 h and then returned to normal growth conditions for a 2-h

recovery. Oxidative stress was induced by spraying with 10 mM PQ at 600 mmol

m22 s21 and 25�C for 1 d. CMVwas prepared from virus-infected leaf tissues by

grounding in an inoculation buffer containing 0.1 M sodium phosphate (pH 7.5),

2% (w/v) polyvinylpyrrolidone (PVP), and 0.2% (w/v) Na2SO3 at ratio of 1:100.

The extract was used for inoculation of the cucumber leaves. The injuries or

disease index was evaluated after CMV infection. For time-course analysis of

EBR-, Brz-, and H2O2-induced changes in the tolerance to oxidative stress,

cucumber seedlings were first treated with EBR or H2O2 and challenged with 10

mM PQ at different time points after treatment, whereas plants with different

duration of Brz treatment were challenged simultaneously with 10 mM PQ. To

investigate the role of ROS in the resistance, leaves were pretreated with 100 mM

DPI (an NADPH oxidase inhibitor) or 5 mM DMTU (an H2O2 and OH.

scavenger) for 8 h, and then plants were treated with 0.1 mM BR or 10 mM



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H2O2. After 1 d, plants were sprayed with 10 mM PQ under the same conditions

as described above or exposed to cold at 8�C and 1,000 mmol m22 s21 for 1.5 h.

Stress tolerance was measured based on changes in the maximal quantum yield

of PSII (Fv/Fm).

Analysis of Chlorophyll Fluorescence

Chlorophyll fluorescence was determined with an imaging pulse ampli-

tude modulated fluorometer (IMAG-MAXI; HeinzWalz). For measurement of

maximal quantum yield of PSII (Fv/Fm), plants were dark-adapted for 30 min.

Minimal fluorescence (Fo) was measured during the weak measuring pulses

and maximal fluorescence (Fm) was measured by a 0.8-s pulse light at about

4,000 mmol m22 s21. Fv/Fm was determined with the whole leaf as area of

interest. The ETRs at a given actinic irradiance are determined according to

White and Critchley (1999) and calculated as (Fm#2 Fs)/Fm#3 PAR3 0.53 a,

where (Fm# 2 Fs)/Fm# is the quantum yield of PSII (FPSII) in the light, PAR is

the actinic irradiance, 0.5 is the assumed proportion of absorbed quanta used

by PSII reaction centers, and a is the leaf absorbance for cucumber leaves,

respectively.

Histochemical Staining of O2.2 and H2O2

The histochemical staining of O2.2 and H2O2 was performed as previously

described (Jabs et al., 1996; Thordal-Christensen et al., 1997) with minor

modifications. In the case of O2.2, leaf discs (1.5 cm in diameter) were vacuum

infiltrated directly with 0.1 mg mL21 NBT in 25 mM K-HEPES buffer (pH 7.8)

and incubated at 25�C in the dark for 2 h. In the case of H2O2, leaf discs were

vacuum infiltrated with 1 mg mL21 DAB in 50 mM Tris-acetate (pH 3.8) and

incubated at 25�C in dark for 24 h. In both cases, leaf discs were 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.

Cytochemical Detection of H2O2

H2O2 was visualized at the subcellular level using CeCl3 for localization

(Bestwick et al., 1997). Electron-dense CeCl3 deposits are formed in the

presence of H2O2 and are visible by transmission electron microscopy. Tissue

pieces (1–2 mm2) were excised from the leaves and incubated in freshly

prepared 5 mM CeCl3 in 50 mM MOPS at pH 7.2 for 1 h. The leaf sections were

then fixed in 1.25% (v/v) glutaraldehyde and 1.25% (v/v) paraformaldehyde

in 50 mM sodium cacodylate buffer, pH 7.2, for 1 h. After fixation, tissues were

washed twice for 10 min in the same buffer and postfixed for 45 min in 1%

(v/v) osmium tetroxide and then dehydrated in a graded ethanol series

(30–100%; v/v) and embedded in Eponaraldite (Agar Aids). After 12 h in pure

resin, followed by a change of fresh resin for 4 h, the samples were polymer-

ized at 60�C for 48 h. Blocks were sectioned (70–90 nm) on a Reichert-Ultracut

E microtome and mounted on uncoated copper grids (300 mesh). Sections

were examined using a transmission electron microscope at an accelerating

voltage of 75 kV.

Determination of H2O2 and MDA in Leaf Extracts

The concentration of H2O2 in leaves was measured by monitoring the

absorbance of the titanium-peroxide complex at 415 nm using the method of

Brennan and Frenkel (1977). The absorbance was quantified using a standard

curve generated from known concentrations of H2O2. Oxidative damage to

lipids was estimated by measuring the content of MDA in leaf homogenates,

prepared with 10% TCA only. Samples were mixed with 10% TCA containing

0.65% 2-thiobarbituric acid (TBA) and heated at 95�C for 25 min, as by Hodges

et al. (1999). MDA content was calculated by correcting for compounds, other

thanMDA, 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.

Isolation of Plasma Membrane and the Determination of

NADPH Oxidase Activity

Leaf plasma membranes were isolated using the two-phase aqueous

polymer partition system (Larsson et al., 1987). Samples were homogenized

in four volumes of the extraction buffer (50 mM Tris-HCl, pH 7.5, 0.25 M Suc,

1 mM ascorbic acid (AsA), 1 mM EDTA, 0.6% PVP, and 1 mM PMSF). The

homogenate was filtered through four layers of cheesecloth, and the resulting

filtrate was centrifuged at 10,000g for 15 min. Microsomal membranes were

pelleted from the supernatant by centrifugation at 50,000g for 30 min. The

pellet was suspended in 0.33 M Suc, 3 mM KCl, and 5 mM potassium

phosphate, pH 7.8. The plasma membrane fraction was isolated by adding

the microsomal suspension to an aqueous two-phase polymer system to give a

final composition of 6.2% (w/w) Dextran T500, 6.2% (w/w) polyethylene

glycol 3350, 0.33 M Suc, 3 mM KCl, and 5 mM potassium phosphate, pH 7.8.

Three successive rounds of partitioning yielded the final upper phase. The

upper phase produced was diluted 5-fold in Tris-HCl dilution buffer (10 mM,

pH 7.4) containing 0.25 M Suc, 1 mM EDTA, 1 mM DTT, 1 mM AsA, and 1 mM

PMSF. The fractions were centrifuged at 120,000g for 30 min. The pellets were

then resuspended in Tris-HCl dilution buffer and used immediately for

further analysis. All procedures were carried out at 4�C. Protein content of

plasma membranes was determined according to the method of Bradford

(1976) with BSA as standard.

The NADPH-dependent O2.2-generating activity in isolated plasma mem-

brane vesicles was examined using SOD-inhibitable ferricytochrome c reduc-

tion. An aliquot of the isolated PM vesicles was added to a reaction mixture

consisting of 50 mM HEPES-KOH (pH 7.8), 100 mM EDTA, 50 mM ferricyto-

chrome c, and 100 mM NADPH in the presence or absence of SOD (200 units/

mL, SOD from bovine erythrocytes; Sigma-Aldrich) and incubated at room

temperature for 30 s. The activity was based on difference between A550 with

or without SOD and the absorbance coefficient of 21.0 mM21 cm21.

Antioxidant Enzyme Extraction and Activity Assay

For the enzyme assays, 0.3 g of leaf were ground with 3 mL ice-cold 25 mM

HEPES buffer (pH 7.8) containing 0.2 mM EDTA, 2 mM AsA, and 2% PVP. The

homogenates were centrifuged at 4�C for 20 min at 12,000g, and the resulting

supernatants were used for the determination of enzymatic activity. SOD

activity was assayed by measuring the ability to inhibit the photochemical

reduction of NBT following the method of Stewart and Bewley (1980). CAT

activity was measured as a decline in A240 using the method of Patra et al.

(1978). APX and DHAR activities were measured by a decrease in A290 and an

increase in A265 according to Nakano and Asada (1981). MDAR activity was

measured by using 1 unit ascorbate oxidase, and the oxidation rate of NADH

was followed at 340 nm (Hossain et al., 1984). GR activity was measured

according to Foyer and Halliwell (1976), which depends on the rate of

decrease in the absorbance of NADPH at 340 nm. All spectrophotometric

analyses were conducted on a SHIMADZU UV-2410PC spectrophotometer.

Total RNA Extraction and Gene Expression Analysis

Total RNAwas extracted from cucumber leaves using Trizol according to

the supplier’s recommendation. Residual DNA was removed with purifying

column. One microgram of total RNA was reverse transcribed using 0.5 mg

of oligo(dT)12-18 (Invitrogen) and 200 units of Superscript II (Invitrogen)

following the supplier’s recommendation. On the basis of EST sequences,

the gene-specific primers are shown in Supplemental Table I and used for

amplification.

Quantitative real-time PCR was performed using the iCycler iQ real-time

PCR detection system (Bio-Rad). PCRs were performed using the SYBR Green

PCR Master Mix (Applied Biosystems). The PCR conditions consisted of

denaturation at 95�C for 3 min, followed by 40 cycles of denaturation at 95�Cfor 30 s, annealing at 58�C for 30 s, and extension at 72�C for 30 s. A

dissociation curve was generated at the end of each PCR cycle to verify that a

single product was amplified using software provided with the iCycler iQ

real-time PCR detection system. The identity of the PCR products was verified

by single-strand sequencing using MegaBACE 1000 DNA analysis system

(Amersham Biosciences). To minimize sample variations, mRNA expression

of the target gene was normalized relative to the expression of the house-

keeping gene actin. All experiments were repeated three times for cDNA

prepared for two samples of cucumber leaves. The quantification of mRNA

levels is based on the method of Livak and Schmittgen (2001). The threshold

cycle (Ct) value of actin was subtracted from that of the gene of interest to

obtain a DCt value. The Ct value of untreated control sample was subtracted

from the DCt value to obtain a DDCt value. The fold changes in expression

level relative to the control were expressed as 22DDCt.

Sequence data from this article can be found in the GenBank/EMBL data

libraries under the following accession numbers: RBOH (FJ036897), MAPK1

(FJ036898), MAPK3 (FJ0368902), WRKY30 (FJ036895), WRKY6 (FJ036899),

Xia et al.


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MYB (FJ0368901), MYC (FJ036894), HSP70 (AJ249329), DnaJ (X67695),

PR-1 (DQ641122), PAL (AF475285), HPL (AF229811), GST (FJ0368900), GPX

(FJ036896), POD (M91373), CAT (AY274258), cAPX (D88649),MDAR (D26392),

DWARF (EW968286), and actin (AAZ74666).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Phenotypes of four types of plants with different

BR levels.

Supplemental Figure S2. Oxidative symptoms of the PQ-challenged

leaves after different treatments.

Supplemental Figure S3. Images of the maximum PSII quantum yield

(Fv/Fm) of PQ-challenged leaves after different time of EBR treatment

and different duration of Brz treatment.

Supplemental Figure S4. DWRF gene expression in four types of plants

with different BR levels.

Supplemental Figure S5. SA accumulation after EBR or Brz treatment.

Supplemental Table S1. Primers used for real-time RT-PCR assays.

Received March 8, 2009; accepted April 20, 2009; published April 22, 2009.

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