Plant Physiology Preview. Published on April 22, 2009, as DOI:10.1104/pp.109.138230
Running title: ROS in BR-induced stress tolerance
Corresponding author: Jing Quan Yu
Address: Department of Horticulture, Huajiachi Campus, Zhejiang University, Kaixuan
Road 268, Hangzhou 310029, China.
Telephone: 0086-57186971120
E-mail address: [email protected]
Fax: 0086-571-86971120
Research area: Signal Transduction and Hormone Action
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Copyright 2009 by the American Society of Plant Biologists
Reactive oxygen species are involved in brassinosteroids-induced stress tolerance in
Cucumis sativus
Xiao-Jian Xia,a,1 Yan-Jie Wang,a,1 Yan-Hong Zhou,a, b,1 Yuan Tao, a Wei-Hua Mao,a Kai
Shi,a Tadao Asami, c Zhixiang Chen,d and Jing-Quan Yu a ,b,2
a
Department of Horticulture, Huajiachi Campus, Zhejiang University, Kaixuan Road 268,
Hangzhou, P.R. China 310029
b
Key Laboratory of Horticultural Plants Growth, Development and Biotechnology,
Agricultural Ministry of China, Kaixuan Road 268, Hangzhou, 310029 P.R. China
c
Department of Applied Biological Chemistry, University of Tokyo, Bunkyo Ku, Tokyo
1138657, Japan
d
Department of Botany & Plant Pathology, Purdue University, West Lafayette, IN
47907-2054, USA
1
These authors contributed equally to this work.
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
This work was supported by the National Basic Research Program of China
(2009CB119000), National Natural Science Foundation of China (3050344; 30671428)
and the Program for Promotion of Basic Research Activities for Innovative Bioscience
(PROBRAIN).
Address correspondence to [email protected].
The author(s) responsible for distribution of materials integral to the findings presented in
this article in accordance with the policy described in the Instructions for Authors
(www.plantphysiol.org) is: Jing-Quan Yu ([email protected])
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ABSTRACT
Brassinosteroids (BRs) induce plant tolerance to a wide spectrum of stresses. To study
how BR induces stress tolerance, we manipulated the BR levels in cucumber (Cucumis
sativus) through a chemical genetics approach and found that BR levels were positively
correlated with the tolerance to photo-oxidative and cold stresses and resistance to
cucumber mosaic virus (CMV). We also showed that BR treatment enhanced NADPH
oxidase activity and elevated H2O2 level in apoplast. H2O2 levels were elevated as early
as 3 h and returned to basal levels 3 d after BR treatment. BR-induced H2O2
accumulation was accompanied by increased tolerance to oxidative stress. Inhibition of
NADPH oxidase and chemical scavenging of H2O2 reduced BR-induced oxidative and
cold tolerance and defense gene expression. BR treatment induced expression of both
regulatory genes such as RBOH, MAPK1 and MAPK3, and genes involved in defense and
antioxidant responses. These results strongly suggest that elevated H2O2 levels resulting
from enhanced NADPH oxidase activity are involved in the BR-induced stress tolerance.
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INTRODUCTION
Plants are constantly exposed to a variety of biotic (i.e. pathogen infection and insect
herbivory) and abiotic stresses (i.e. extreme temperature, drought and salinity). To
survive such stresses, plants have evolved intricate mechanisms to perceive external
signals and activate optimal responses to environmental conditions. At the molecular
level, the perception of extracellular stimuli and subsequent activation of appropriate
responses require a complex interplay of signaling cascades. It has been shown that
phytohormones such as salicylic acid (SA), jasmonic acid (JA), and abscisic acid (ABA)
regulate the protective responses of plants to both biotic and abiotic stresses
independently and through synergistic and antagonistic crosstalk (Bostock, 2005;
Lorenzo and Solano, 2005; Mauch-Mani and Mauch, 2005). Moreover, plant responses to
different types of stresses are associated with generation of reactive oxygen species
(ROS), suggesting that ROS may function as a common signal in signaling pathways of
plant stress responses (Apel and Hirt, 2004; Torres and Dangl, 2005). More recent studies
indicate extensive crosstalk of plant signaling pathways for defense against pathogens
with those for responses to abiotic stresses (Fujita et al., 2006).
For a long time, ROS was believed as a harmful byproduct in aerobic organisms.
Extensive studies have shown that while high levels of ROS cause cell death, low levels
of ROS have regulatory roles in plant stress responses. Application of ABA and SA as
well as exposure to low temperature all resulted in a transient elevation of H2O2, leading
to an increased tolerance to salt, high light, heat and oxidative stress (Prasad et al., 1994;
Dat et al., 1998; Zhang et al., 2001). It has been proposed that ROS plays a critical role in
induced tolerance by activating or inducing stress response-related factors such as MAP
kinases, transcription factors, antioxidant enzymes, dehydrins, low-temperature-induced,
heat-shock and pathogenesis-related proteins (Gechev et al., 2006).
Brassinosteroids (BRs) are a group of naturally occurring plant steroids and are important
for a broad spectrum of cellular and physiological processes including stem elongation,
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pollen tube growth, leaf bending and epinasty, root inhibition, fruit development,
ethylene biosynthesis, proton-pump activity, xylem differentiation, photosynthesis and
gene expression (Li et al., 1996; Sasse, 1997; Clouse and Sasse, 1998; Dhaubhadel et al.,
1999; Hu et al., 2000; Arteca and Arteca, 2001; Müssig et al., 2002; Yu et al., 2004; Fu et
al., 2008). Important progress has been made in elucidating the BR signal transduction
pathway. The discovery of BR-insensitive mutants in Arabidopsis, pea, tomato and rice
led to the isolation of the BRI1 gene and its homologues (Li and Chory, 1997; Yamamuro
et al., 2000; Montoya et al., 2002; Nomura et al., 2003). BRI1 is a BR-binding
leucine-rich repeat (LRR) receptor located in the plasma membrane and functions in vitro
as a Ser/Thr kinase (Wang et al., 2001). Recent studies have identified several other
components in the BR signaling pathway (Li and Nam, 2002; Nam and Li, 2002;
Mora-Garcia et al., 2004), including BAK1 (BRI1 associated receptor kinase 1), BIN2 (a
GSK3/SHAGGY-like kinase) and BSU1 (BRI1 suppressor 1, a phosphatase). BR binds to
the extracellular domain of BRI1 and activates its intracellular kinase activity (Kinoshita
et al., 2004). Activated BRI1 interacts with and activates its coreceptor BAK1 (Li et al.,
2002; Nam and Li, 2002). The BIN2 kinase and BSU1 phosphatase function downstream
of the receptor kinases and regulate the phosphorylation status of BZR1 and BZR2/BES1
transcription
factors
(Belkhadir and
Chory,
2006;
Vert
and
Chory,
2006).
Dephosphorylated BZR1 and BZR2/BES1 recognize the promoters of BR target genes
and regulate their expression (He et al., 2002; Yin et al., 2002).
In addition to its critical roles in growth regulation and photo-morphogenesis, BRs can
induce plant tolerance to a variety of abiotic stresses such as high and low temperature
stress, drought and salinity injury (Krishna, 2003; Kagale et al., 2007). However, the
underlying mechanisms for BR-mediated stress responses are not understood. It has been
found that BR-induced increase in the basic thermotolerance is associated with increased
heat shock protein synthesis and accumulation, as well as increased expression of some
components of translational machinery (Dhaubhadel et al., 1999, 2002). The mechanism
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by which BR induces protein synthesis during heat stress is unclear. BR may also play a
role in plant responses to pathogens. BR induces resistance of tobacco and rice to
bacterial and fungal pathogens (Nakashita et al., 2003). BR-induced disease resistance
was not correlated with enhanced SA accumulation or increased expression of genes
associated with SA-regulated systemic acquired resistance (SAR). Additionally,
simultaneous treatment of plants with BR and SAR inducers resulted in additive
protection against pathogen attack (Nakashita et al. 2003). Thus, BR-induced disease
resistance is mediated by a novel signaling pathway distinct 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 unexpectedly that BR also triggers a
periodic increase in H2O2 level in cucumber (Cucumis sativus) leaves. H2O2 can function
as a signaling molecule in responses to various stimuli both in plant and animal cells
(Neill et al., 2002). To study the role of elevated H2O2 level in BR-induced stress
tolerance, we analyzed the effects of exogenous BR, inhibitors of BR biosynthesis and
ROS production and ROS scavengers on stress tolerance and associated gene expression
in cucumber. These studies demonstrated that BR induced tolerance to both biotic and
abiotic stresses in cucumber plants. In addition, we provide strong evidence that H2O2
plays a role in the BR-induced plant stress tolerance.
RESULTS
BR induces plant stress tolerance
To determine whether BR induces stress tolerance in cucumber plants, we obtained four
types of plants with different BR levels by applications of 24-epibrassinolide (EBR), one
of the bioactive BRs and brassinazole (Brz), a specific inhibitor of BR biosynthesis
(Supplemental Fig.S1). We first compared the effects of EBR and Brz on plant sensitivity
to paraquat (PQ), which causes photo-oxidative stress. When grown under continuous
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light, necrotic lesions appeared 1 d after PQ 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 photosynthetic efficiency, we compared the maximum quantum yield of PSII
(Fv/Fm) (Maxwell and Johnson, 2000). Fluorescence images of Fv/Fm showed that PQ
treatment resulted in a significant 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 in Brz-treated plants close
to that of water-treated plants.
Electron transport rates (ETR) was determined at 25℃ after the chilling stress (8℃/200
μmol m-2 s-1) to assess the effects of EBR and Brz on chilling tolerance of cucumber
seedlings. Chilling stress caused significant reduction in ETR. EBR treatment alleviated
chilling stress and enhanced the ETR, whereas Brz treatment reduced ETR as compared
to water-treated plants. In addition, EBR treatment significantly restored ETR of
Brz-treated plants (Fig. 1C).
We also examined the role of BR in plant responses to cucumber mosaic virus (CMV) by
comparing disease symptom development and CMV-induced lipid peroxidation based on
the malondialdehyde (MDA) content after EBR or Brz treatment. Water-treated plants
developed typical CMV symptoms by 10 d post-inoculation (dpi). When CMV disease
severity was rated at 14 dpi, Brz-treated plants had higher disease index and MDA
content than water-treated plants (Fig. 1D), suggesting that BR biosynthesis was
important for plant response to CMV. By contrast, CMV disease severity 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 resistance to CMV (Fig. 1D). These
results indicate that BR enhances plant tolerance or resistance to both abiotic and biotic
stresses.
Changes in gene expressions in response to BR levels
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To analyze the underlying molecular mechanisms for BR-induced stress tolerance, we
examined the effects of BR levels on expression of 18 stress responsive genes. As shown
in Fig. 2, three regulatory genes, RBOH, MAPK1 and MAPK3, were up-regulated upon
treatment with EBR but down-regulated after Brz treatment. Among the three genes,
MAPK1 was most affected by Brz, whose expression was reduced by approximately 70%.
Reductions of RBOH and MAPK3 expressions by Brz were more moderate but also
substantial. Again, application of EBR to Brz-treated plants rescued the repressed
expression of the regulatory genes.
Interestingly, expressions of WRKY6 and MYB were induced more than 30-fold by EBR
application (Fig. 2). EBR also induced expression of two other genes encoding
transcription factors, WRKY30 and MYC. Unexpectedly, expressions of the four
transcription factor genes were also up-regulated after Brz treatment. EBR also induced
expressions of genes encoding proteins involved in heat shock response (HSP, DnaJ),
defense (PR-1, PAL, HPL), detoxification (GST, GPX, POD) and antioxidant (CAT, cAPX,
MDAR) (Fig. 2). Brz treatment also induced expression of HSP and DnaJ but has little
effect on expression of GPX, POD, CAT, PAL, cAPX and MDAR. On the other hand,
expression of PR-1, HPL and GST were substantially reduced in Brz-treated plants. EBR
and Brz had additive effect on the induction of HSP and DnaJ.
Changes in H2O2 by BR levels
Reactive oxygen species (ROS) act as second messengers in stress and hormone
responses (Apel and Hirt, 2004; Kwak et al., 2006). To determine a possible role of ROS
in BR-induced stress tolerance, we attempted to detect in situ accumulation of O2.- and
H2O2 using nitroblue tetrazolium (NBT) and 3,3’-diaminobenzidine (DAB) staining
procedures, respectively. Both procedures detected increased staining in EBR-treated
leaves but decreased staining in Brz-treated leaves relative to that in water-treated leaves
(Fig. 3A). However, EBR application to Brz-treated leaves partially restored O2.- and
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H2O2 levels. Interestingly, we observed enhanced staining on the edges of the leaf disks,
probably due to wounding because it was not affected by EBR or Brz treatment (Fig. 3A).
Similar effects of EBR and Brz on leaf H2O2 accumulation were observed when an
independent spectrophotometric 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 intercellular
spaces but was undetectable in the cytosol or intracellular organelles such as chloroplasts,
mitochondria, nuclei or vacuoles (Fig. 3B). In addition, EBR-induced H2O2 accumulation
was sensitive to diphenyleneiodonium (DPI), a potent inhibitor of NADPH oxidase (Fig.
3C). Dimethylthiourea (DMTU), a H2O2 scavenger, also abolished EBR-induced H2O2
accumulation. These results suggest that BR-induced H2O2 accumulation is caused by
increased activity of NADPH oxidase. To confirm this, we measured the activity of
plasma membrane NADPH oxidase in extracts of leaf tissues. NADPH oxidase activities
increased significantly in EBR-treated plants but decreased significantly in Brz-treated
plants as compared with that of water-treated plants (Fig. 3D). Again, EBR was effective
in rescuing the repressed NADPH oxidase activity in Brz-treated plants.
Involvement of H2O2 in BR-induced stress tolerance
To determine whether H2O2 accumulation contributes to BR-induced stress tolerance, we
analyzed the effects of DPI and DMTU on EBR-induced tolerances to oxidative stress
inflicted upon either PQ treatment or chilling stress (8℃) under 1000 μmol m-2 s-1.
Exposure to either stress caused necrotic lesions in water-treated plants (Supplemental
Fig.S2). In EBR- or H2O2-treated plants, on the other hand, necrotic lesions were greatly
reduced after PQ and chill treatment. Importantly, pretreatment with DPI or DMTU
completely abolished the protective effects of EBR and H2O2 on plant tolerance to PQ
and the chill (Fig. 4A). EBR and H2O2 treatment also alleviated significantly the decline
of Fv/Fm after PQ treatment and the chill, and these protective effects were again almost
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completely blocked by DPI and DMTU (Figs. 4B & C). These results strongly suggest
that H2O2 is involved in the BR-induced stress tolerance.
The expressions of genes implicated in signal transduction (MAPK1), transcription
(WRKY6) and stress tolerance (PR-1, PAL, CAT, cAPX) were induced by EBR and H2O2
(Fig. 5). Again DPI and DMTU pretreatment abolished or substantially reduced
EBR-induced expression of these genes (Fig. 5). Likewise, both EBR and H2O2 induced
expression of antioxidant genes CAT and cAPX and EBR treatment increased activities of
antioxidant enzymes (SOD, CAT, APX, MDAR, DHAR, GR) (Figs. 5 & 6). The increase
in antioxidant gene expression and activities of antioxidant enzymes upon EBR treatment
were also blocked by DPI and DMTU pretreatment (Figs. 5 & 6). These results support
the involvement of H2O2 in BR-induced gene expressions.
Time course of BR-induced H2O2 accumulation, tolerance, transcript levels and
activities of antioxidant enzymes
H2O2 levels were increased as early as at 3 h and remained elevated up to 24 h after EBR
treatment (Fig. 7A). H2O2 levels were not significantly altered during the first 24 h after
Brz treatment but were substantially reduced at 72 h after Brz treatment and continued to
decline during the remaining period of the experiments (Fig. 7B). To further investigate
the involvement of H2O2 in EBR-induced stress tolerance, we examined the effect of
PQ-mediated oxidative stress applied at different intervals after EBR, H2O2 or Brz
treatment. Plants were first sprayed with EBR, H2O2 or Brz and at various intervals after
the treatment plants were subjected to oxidative stress treatment by applying PQ. The
stress tolerance was then determined by measurement of Fv/Fm 1 d after PQ treatment.
Enhanced tolerance of cucumber seedlings to PQ-induced oxidative stress was observed
at 3 h after treatment with H2O2 and at 6 h after EBR treatment (Fig. 8 and Supplemental
Fig.S3A). Thus, H2O2 induced stress tolerance more rapidly than EBR (Fig. 8A). The
maximum level of stress tolerance was observed at 12 h after treatment with H2O2 and at
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24 h after EBR treatment (Fig. 8A). No significant level of stress tolerance was observed
at 72 h after treatment with H2O2 and at 120 h after EBR treatment (Fig. 8A). The time
course for Brz-induced decline in stress tolerance was highly correlated to that of
Brz-induced decrease in H2O2 levels (r=0.96, P<0.01). Thus, like changes in H2O2 levels,
stress tolerance was not significantly altered during the first 24 h but substantially
reduced at 72 h after Brz treatment and continued to decline during the remaining period
of the experiments (Supplemental Fig.S3B; Fig. 8B).
To characterize further the relationship of BR-induced H2O2 and enhanced stress
tolerance, we analyzed the temporal changes in transcript levels of several stress
responsive genes and activities of antioxidant enzymes. The expressions of RBOH and
CAT started to increase at 3 h after EBR treatment, peaked at 12 h and then declined. The
cAPX expression increased at 3 h after EBR treatment and remained elevated for the
following 9 h before declining to control level. PR-1 and PAL transcript levels also
increased at 3 h, peaked at 12 h and became undetectable at 72 h after EBR treatment
(Fig. 9). Significant increases in activities of SOD, CAT and MDAR were also detected at
3 h after EBR treatment (Fig.10). For other antioxidant enzymes (APX, DHAR and GR),
significant increases occurred at 6 h. The activities of all these antioxidant enzymes
declined to basal levels at 96 h after EBR treatment (Fig. 10). The decline of the
antioxidant enzyme activities at 96 h after EBR treatment 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 plant tolerance to a variety of
environmental stresses (Khripach et 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 action mechanisms of BR in plant stress tolerance because of the strong and
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pleiotropic phenotypes of BR biosynthesis and signaling mutants, including extreme
dwarfism, dark green and epinastic leaves and delayed development (Khripach et al.,
2000; Bishop and Koncz 2002; Müssig et al., 2002; Cao et al., 2005). Moreover, current
techniques allow for measurement of stress-induced changes in the levels of only
intermediates in BR biosynthesis pathway, but not of the bioactive brassinolide
(Nakashita et al., 2003; Jager et al., 2008). As a result, application of Brz, a specific and
potent inhibitor of BR biosynthesis (Asami et al., 2000, 2001), has been used to study the
role of BR in plant stress responses. Consistent with the feedback control of BR
biosynthesis (Bancos et al., 2002), Brz up-regulated BR biosynthetic genes and caused
growth aberrations of cucumber seedlings (Supplemental Figs. S1 & S4). These
observations support that endogenous BR contents in cucumber seedlings were altered by
Brz application.
Our results demonstrated that EBR enhanced and Brz reduced tolerance to oxidative, cold
and CMV stresses in cucumber (Fig.1). However, in comparison to the relatively fast
induction (~3h) of tolerance by EBR application, the negative effect of Brz on stress
tolerance was relatively slow (Fig. 8). Brz is a specific inhibitor for DWF4, a cytochrome
P450 monooxygenase of the BR biosynthetic pathway (Asami et al., 2001) and it could
not inhibit the downstream enzymes in the BR biosynthesis pathway. Accordingly, it
needs a period of time to reduce the bioactive BR level. These results strongly suggest
that BR-induced stress tolerance is quantitative in nature and is correlated with the BR
levels. In other words, normal synthesis of BRs under non-stress conditions is expected to
confer a certain level of stress tolerance but an increase in BR accumulation under certain
types of stress conditions will lead to a corresponding increase in stress tolerance.
Conversely, reducing BR accumulation below its normal levels (e.g. after Brz application)
leads to a corresponding decrease in stress tolerance. These studies support the
involvement of BR in plant responses to various environmental stresses (Krishna, 2003;
Nakashita et al., 2003; Kagale et al., 2007). There were significant increases in several
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intermetabolite levels in BR biosynthesis pathway after TMV inoculation in tobacco
leaves (Nakashita et al., 2003) whereas there were no consistent changes in the
intermetabolite levels in leaves of pea in response to water stress (Jager et al., 2008).
Accordingly, BR biosynthesis may be induced in some but not all types of stress
conditions. Furthermore, we recently found that as in Brz-treated cucumber plants, the
tomato BR biosynthesis dim mutant exhibited enhanced sensitivity to PQ-induced
oxidative stress and this phenotype can be rescued by exogenously applied BR. Most
recently, we also found that EBR application to wild-type and BR deficient mutant all
increased their resistance to Cladosporium fulvum in tomato (data not shown). There was,
however, no significant difference in the tolerance of wild-type and BR-deficient mutant
to water stress in pea (Jager et al., 2008). Thus, BR may be involved in plant responses to
some but not all types of plant stress conditions. Alternatively, the severe morphological
or physiological phenotypes, such as the dwarf shoots with thick leaves and decreased
stomatal conductance in BR-deficient pea plants might complicate the water stress
analysis because these phenotypes could lead to reduced transpiration and increased
drought resistance.
Microarray analysis revealed that BR induces the expression of heat shock protein
(HSP83, HSP70, Hsc70-3, Hsc70-G7), heat shock factor (HSF3) and oxidative
stress-related genes (GST, ATPA2, ATP24a) in Arabidopsis (Goda et al., 2002; Müssig et
al., 2002). Our qRT-PCR analysis revealed similar induction by EBR of HSP70, Dnaj,
GST and POD in cucumber (Fig. 2). BR induced defense and antioxidant genes in the
absence of stresses. In contrast, expressions of cold and pathogenesis-related genes are
reduced in Brz-treated cucumber seedlings or BR-deficient Arabidopsis mutants
(Szekeres et al., 1996; Müssig et al., 2002). These results suggest that BR enhances plant
stress tolerance by activating genes involved in plant defense and stress responses.
H2O2 is involved in BR-induced stress tolerance in cucumber
In this study, we have provided several lines of evidences that H2O2 is involved in
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BR-induced stress tolerance. First, EBR-induced stress tolerance was preceded by
increased NADPH oxidase activity and elevated H2O2 levels (Fig. 3). Second, scavenging
of H2O2 by DMTU or inhibiting H2O2 generation by DPI abolished EBR-induced stress
tolerance (Fig. 4). Third, EBR-and Brz-induced changes in H2O2 levels were closely
related to the changes in the tolerance to PQ-mediated oxidative stress (Figs.7 & 8;
Supplemental Fig.S3) and exogenously applied H2O2 also induced the tolerance (Fig. 8A;
Supplemental Fig.S3A). In addition, the temporal changes in H2O2 accumulation,
transcript levels of antioxidant genes and activities of antioxidant enzymes are consistent
with the role of H2O2 in BR-induced stress tolerance (Figs. 7, 9 & 10). Of particular
relevance is the observation that the interval between EBR treatment and stress challenge
is critical for the magnitude of EBR-induced stress tolerance (Figs. 7 & 8). Accordingly,
the variation of the efficiency of BR in enhancing plant stress tolerance in different
studies (Khripach et al., 2000), is likely to be related to time interval between BR
application and stress challenge.
H2O2 is considered as a central signaling molecule in plant responses to biotic and abiotic
stresses (Foyer et al., 1997; Neill et al., 2002). It activates protective mechanisms for
tolerance to chill in maize (Prasad et al., 1994), high temperature in mustard seedlings
(Dat et al., 1998) and light stress in Arabidopsis leaves (Karpinski et al., 1999). Perturbed
H2O2 homeostasis or increased production of H2O2 enhanced the expression of
antioxidant enzymes and acidic PR proteins in the absence of pathogen challenge
(Chamnongpol et al., 1996; Takahashi et al., 1997; Wu et al., 1997). Likewise, we found
that EBR treatment induced increase in both H2O2 and PR-1 mRNA levels (Figs. 2 & 3)
without any significant effect on SA accumulation (Supplemental Fig.S5). However, BR
did not induce changes in expression of PR genes in tobacco (Nakashita et al., 2003).
This discrepancy might be caused by different interval between BR application and
sampling time or the low sensitivity of Northern blot to detect minor changes in
expression of PR genes. In support of this, strong induction of PR-1 was transient and
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observed only within a short period of time after EBR treatment (Fig. 9). Verberne et al.
(2003) have reported that ethylene is also required for the production or transmission of
the mobile SAR signal in TMV-infected leaves. BR induced expression of ACC oxidase
in cucumber (data not shown) and the increase in stress tolerance in BR-treated potato
tubers is associated with increased levels of abscisic acid and ethylene and accumulation
of phenolic and terpenoid compounds (Krishna et al., 2003). Moreover, microarray data
indicate that there is crosstalk between BR and JA signaling pathways (Müssig et al.,
2002; Goda et al., 2002). It is likely that BR-induced PR gene expression and stress
tolerance are mediated by a complex set of signal transcription pathways with H2O2 as a
common signal molecule in the activation of stress responses. It may also not be
completely unexpected that signal transduction pathways mediating plant growth may
cross talk with those mediating plant defense and stress responses.
In higher plants, ROS can be generated by several different pathways, including
plasma-membrane-localized NADPH oxidase, cell wall-localized peroxidases and amine
oxidases (Neill et al., 2002). We have presented evidence that NADPH oxidase is the
potential source of BR-induced H2O2 generation. First, H2O2 accumulated mainly in the
apoplast of mesophyll cells. Second, treatment with EBR significantly increased the
activity of NADPH oxidase. Third, DPI, a potential inhibitor of NADPH oxidase,
blocked EBR-induced production of H2O2. However, because DPI may also inhibit other
oxidases (Bolwell et al., 1998), we cannot completely rule out the possibility that other
oxidases may contribute to BR-induced ROS generation.
It has been reported that BRI1-associated coreceptor BAK1 and BAK1-like 1 (BKK1)
function not only in BR-dependent signaling in plant growth and development but also in
the regulation of plant cell death. BAK1-deficient plants exhibited spreading necrosis
accompanied by enhanced accumulation of ROS after infection by pathogens
(Kemmerling et al., 2007). The bak1 bkk1 double mutants also accumulated enhanced
ROS and exhibited a seedling-lethality (He et al., 2007). However, the role of BAK1 and
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BKK1 in plant cell death control is independent of BR (Kemmerling et al., 2007; He et
al., 2007). It remains to be determined whether 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 transcription factors (Fig. 2). In plants, the
MAPK cascade plays a crucial role in various biotic and abiotic stress responses and in
hormone signaling, which often involves ROS (Nakagami et al., 2005). H2O2 can activate
ANP1, an Arabidopsis MAPKKK to regulate the activities of MPK3 and MPK6 (Kovtun
et al., 2000). A recently identified serine/threonine protein kinase (OXI1) has also been
shown to play a central role in ROS sensing and the activation of MPK3 and MPK6,
which control the activation of different defense mechanisms (Rentel et al., 2004). Thus,
MAPK cascades can mediate H2O2 signaling and may also play an important role in
BR-induced stress tolerance.
In addition to the MAPK genes, BR-induced stress tolerance is associated with
expression of a number of genes encoding WRKY, MYB and MYC transcription factors.
WRKY transcription factors have been implicated in the regulation of transcriptional
reprogramming associated with plant immune responses (Eulgem and Somssich, 2007)
and MYB and MYC transcription factors have been implicated as critical regulators of
ABA-inducible gene expression under drought stress (Abe et al., 2003; Agarwal et al.,
2006). Similarly, genome-wide expression analyses have shown that BR can regulate
expressions of genes encoding MYB and ERF transcription factors (Müssig et al, 2002;
Goda et al, 2002). More recently, Kagale et al (2007) have showed that BR enhances
expression of transcription factors of the CBF/DREB family in both unstressed and stress
plants. The concerted induction of genes encoding these transcription factors suggests
that BR-induced stress tolerance is mediated by transcriptional activation of genes
involved in plant stress responses. Intriguingly, treatment of Brz also resulted in enhanced
expression of transcription factors and heat shock proteins without concomitant induction
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
of other defense genes expression. It is likely that BR deficiency may cause certain stress
response and, as a result, induce expression of some but not all the transcription factors
required for enhanced stress tolerance as observed in BR-treated plants (Szekeres et al.
1996; Kagale et al., 2007).
In conclusion, we have presented strong evidence that H2O2 mediates the transcriptional
induction of defense or antioxidant genes by BR. Following perception of BR signal,
NADPH oxidase may be activated to produce ROS, which initiates a protein
phosphorylation cascade. Transcription factors may be activated via a phosphorylation
cascade by MAPKs. Finally, the products of targets genes participate directly in cellular
protection (Fig. 11). Further studies are needed to provide genetic evidence of the
involvement of NADPH oxidase in BR-induced ROS generation, to identify the critical
signaling components between BR perception and stress responses and to elucidate the
molecular mechanisms of cross-talk between BR and other hormone signaling.
MATERIALS AND METHODS
Plant growth
Cucumber (Cucumis sativus L. cv. Jinyan No. 4) seeds were sown in a growth chamber.
Seven days after sowing, groups of eight seedlings were transplanted into a container (40
cm × 25 cm × 15 cm) filled with Hoagland nutrient solution. The growth conditions were
as follows: a 12-h photoperiod, temperature of 25/17℃ (day/night), and light intensity of
600 μmol m–2 s–1.
Experimental design
To manipulate BR levels, we first treated cucumber seedlings with the BR biosynthesis
inhibitor brassinazole (Brz) by spraying a 4 μM 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
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
24-epibrassinolide (EBR) treatments. Previous tests showed that a relatively moderate
concentration of EBR at 0.1 μM 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) treatments. 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℃
/200 μmol m-2 s-1 for 24 h and then returned to normal growth conditions for a 2-h
recovery. Oxidative stress was induced by spraying with 10 μM paraquat (PQ) at 600
μmol m–2 s–1 and 25℃ for 1 d. CMV was prepared from virus-infected leaf tissues by
grounding in an inoculation buffer containing 0.1 M sodium phosphate (pH 7.5), 2% (w/v)
PVP 25.000, 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 μM PQ at different time points after treatment whereas plants with different
duration of Brz treatment were challenged simultaneously with 10 μM PQ. To investigate
the role of ROS in the resistance, leaves were pretreated with 100 μM DPI
(diphenyleneiodonium, a NADPH oxidase inhibitor) or 5 mM DMTU (dimethylthiourea,
a H2O2 and OH• scavenger) for 8 h and then plants were treated with 0.1 μM BR or 10
mM H2O2. After 1 d plants were sprayed with 10 μM PQ under the same conditions as
described above or exposed to cold at 8℃ and 1000 μmol m–2 s–1 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 imaging PAM (IMAG-MAXI; Heinz Walz,
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Effeltrich, Germany). 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 μmol m–2 s–1. Fv/Fm was determined with the whole leaf as area of
interest. The electron transport rates (ETR) at a given actinic irradiance are determined
according to White and Critchley (1999) and calculated as: (Fm’-Fs)/Fm’× PAR × 0.5 ×α,
where (Fm’-Fs)/Fm’ is the quantum yield of PSII (ΦPSII) in the light, PAR is the actinic
irradiance, 0.5 is the assumed proportion of absorbed quanta used by PSII reaction
centers, and α is the leaf absorptance for cucumber leaves, respectively.
Histochemical staining of O2.- and H2O2
The histochemical staining of O2.- and H2O2 was performed as previously described (Jabs
et al., 1996; Thordal-Christensen et al., 1997) with minor modifications. In the case of
O2.-, leaf disks (1.5cm in diameter) were vacuum infiltrated directly with 0.1 mg mL-1
NBT in 25 mM K-Hepes buffer (pH 7.8) and incubated at 25℃ in the dark for 2 h. In the
case of H2O2, leaf disks were vacuum infiltrated with 1 mg mL-1 3,3’-diaminobenzidine
(DAB) in 50 mM Tris–acetate (pH 3.8) and incubated at 25℃ in dark for 24 h. In both
cases, leaf disks were rinsed in 80% (v/v) ethanol for 10 min at 70℃, 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 3-(N-morpholino)
propanesulfonic acid (Mops) at pH 7.2 for 1 h. The leaf sections were then fixed in 1.25%
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
(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,
Bishop’s UK). After 12 h in pure resin, followed by a change of fresh resin for 4 h, the
samples were polymerized at 60℃ 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 malondialdehyde (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 malondialdehyde (MDA) in leaf homogenates, prepared with 10%
trichloroacetic acid only. Samples were mixed with 10% trichloroacetic acid containing
0.65% 2-thiobarbituric acid (TBA) and heated at 95℃ for 25 min, as in Hodges et al.
(1999). MDA content was calculated by correcting for compounds, other than MDA, 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 sucrose, 1 mM AsA, 1 mM EDTA,
0.6% PVP, and 1 mM PMSF). The homogenate was filtered through four layers of
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
cheesecloth, and the resulting filtrate was centrifuged at 10000 g for 15 min. Microsomal
membranes were pelleted from the supernatant by centrifugation at 50 000 g for 30 min.
The pellet was suspended in 0.33 M sucrose, 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) PEG 3350, 0.33 M sucrose, 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 five-fold in Tris-HCl dilution buffer
(10 mM, pH 7.4) containing 0.25 M sucrose, 1 mM EDTA, 1 mM DTT, 1 mM AsA and 1
mM PMSF. The fractions were centrifuged at 120 000 g for 30 min. The pellets were then
re-suspended in Tris-HCl dilution buffer and used immediately for further analysis. All
procedures were carried out at 4 ℃ . Protein content of plasma membranes was
determined according to the method of Bradford (1976) with BSA as standard.
The NADPH-dependent O2.--generating activity in isolated plasma membrane vesicles
was examined using SOD-inhibitable ferricytochrome c reduction. An aliquot of the
isolated PM vesicles was added to a reaction mixture consisting of 50 mM HEPES-KOH
(pH 7.8), 100 µM EDTA, 50 µM ferricytochrome c and 100 µM NADPH in the presence
or absence of SOD (200 U/mL, SOD from bovine erythrocytes, Sigma) 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 mM-1 cm-1.
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℃ for 20 min at 12,000 g and the resulting supernatants were used
for the determination of enzymatic activity. Superoxide dismutase (SOD) activity was
assayed by measuring the ability to inhibit the photochemical reduction of nitroblue
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
tetrazolium (NBT) following the method of Stewart and Bewley (1980). Catalase (CAT)
activity was measured as a decline in absorbance at 240 nm using the method of Patra et
al. (1978). Ascorbate peroxidase (APX) and DHAR activities were measured by a
decrease in absorbance at 290 nm and an increase in absorbance at 265 nm according to
Nakano and Asada (1981). MDAR activity was measured by using 1 U ascorbate oxidase
and the oxidation rate of NADH was followed at 340 nm (Hossain et al., 1984).
Glutathione reductase (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 RNA was extracted from cucumber leaves using Trizol according to the supplier’s
recommendation. Residual DNA was removed with purifying column. One microgram
total RNA was reverse-transcribed using 0.5 μg 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 1 and
used for amplification.
Quantitative real time PCR was performed using the iCycler iQTM Real-time PCR
Detection System (Bio-Rad, Hercules, CA, USA). PCRs were performed using the SYBR
Green PCR Master Mix (Applied Biosystems). The PCR conditions consisted of
denaturation at 95℃ for 3 min, followed by 40 cycles of denaturation at 95℃ for 30 s,
annealing at 58℃ for 30 s and extension at 72℃ 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 iQTM 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, USA). To minimize sample
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
variations, mRNA expression of the target gene was normalized relative to the expression
of the housekeeping 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 ΔCt value. The Ct value
of untreated control sample was subtracted from the ΔCt value to obtain a ΔΔCt value.
The fold changes in expression level relative to the control were expressed as 2-ΔΔCt.
Accession Numbers
Sequence data from this article can be found in the EMBL/GenBank data libraries under
the following accession numbers: RBOH (FJ036897); MAPK1 (FJ036898); MAPK3
(FJ0368902); WRKY30 (FJ036895); WRKY6 (FJ036899); 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); 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 level.
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 (A) and different duration of
Brz treatment (B).
Supplemental Figure S4. DWRF gene expression in four types of plants with different
BR level.
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Supplemental Figure S5. SA accumulation after EBR or Brz treatment.
Supplemental Table S1. Primers used for real time RT-PCR assays.
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32
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Figure legend
Figure 1. Effects of BR levels on resistance to PQ, chill or CMV
(A) Symptoms (up) and images of the maximum PSII quantum yield (Fv/Fm, down). The
false color code depicted at the bottom of the image ranged from 0 (black) to 1.0
(purple). Plants treated with water, 0.1 μM EBR, 4 μM Brz and 4 μM Brz+0.1 μM
EBR were challenged with 10 μM PQ at 600 μmol m-2 s-1 light intensity and 25℃
for 1 d. Five plants were used for each treatment and the picture of one representative
leaf was shown. Scale bar=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 the means of five replicates(±SD). Means
denoted by the same letter did not significantly differ at P <0.05 according to Tukey's
test.
(C) Electron transport rates (ETR) determined after 1-d exposure to chill stress (8℃/200
μmol m-2 s-1) for plants treated with water, 0.1 μM EBR, 4 μM Brz and 4 μM Brz+0.1
μM EBR. Measurements were conducted at 25℃. Data are the means of five
replicates(±SD).
(D) CMV incidence and malondialdehyde (MDA) content determined at 14 d
post-inoculation for plants treated with water, 0.1 μM EBR, 4 μM Brz and 4 μM
Brz+0.1 μM EBR. Disease index is the mean (n=9 leaves) of disease severities (0;
light to 1; severe). Data for MDA are the means of five replicates(±SD). Means
denoted by the same letter did not significantly differ at P <0.05 according to Tukey's
test.
33
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Figure 2. Expressions of stress-responsive genes in response to BR levels in cucumber
seedlings. qRT-PCR analysis was performed to examine steady-state levels of mRNAs
for 18 genes in plants treated with water, 0.1 μM EBR, 4 μM Brz and 4 μM Brz+0.1 μM
EBR. Data are the means of three replicates(±SD).
Figure 3. The roles of BR in regulation of ROS accumulation
(A) In situ detection of leaf O2.- and H2O2. NBT and DAB stains were used to detect the
presence of O2.- and H2O2 in leaves treated with water, 0.1 μM EBR, 4 μM Brz and 4
μM Brz+0.1 μM EBR. Leaf discs (1.5cm in diameter) were harvested at 6 h after
treatment and stained immediately.
(B) Cytochemical localization of H2O2 accumulation in mesophyll cells of cucumber
leaves with CeCl3-staining and transmission electron microscopy. The plants were
treated and harvested as described in (A). Arrows, CeCl3 precipitates; C, chloroplast;
CW, cell wall; V, vacuole; IS, intercellular space.
(C) Blockage of EBR-induced H2O2 accumulation by DPI and DMTU. Plants were
pretreated with 100 μM DPI or 5 mM DMTU for 8 h and then treated with 0.1 μM
EBR. After 6 h the DAB staining of leaf discs was performed.
(D) Quantitative measurements of H2O2 level and NADPH oxidase activity in cucumber
leaves with different BR levels. Values are means ± SD (n=6). Means denoted by the
same letter did not significantly differ at P <0.05 according to Tukey's test.
Figure 4. Requirement of H2O2 for EBR-induced resistance to PQ and chill
(A) Images of the maximum PSII quantum yield (Fv/Fm) of PQ-challenged and chilled
leaves (scale bar=2.5 cm). The false color code depicted at the bottom of the image
ranged from 0 (black) to 1.0 (purple). Plants were pretreated with 100 μM DPI or 5
mM DMTU for 8 h and then plants were treated with 0.1 μM EBR or 10 mM H2O2.
34
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
After 1 d plants were challenged with 10 μM PQ or exposed to chill at high light
intensity (8℃/1000 μmol m-2 s-1). Single treatment of DPI or DMTU was included as
negative control.
(B) and (C) Average Fv/Fm values of PQ-challenged (B) or chilled (C) leaves. Fv/Fm
was determined with the whole leaf as area of interest. Fv/Fm for control plants was
0.83. Values are means ± SD (n=5). Means denoted by the same letter did not
significantly differ at P <0.05 according to Tukey's test.
Figure 5. Involvement of H2O2 in EBR-induced upregulation of stress responsive genes.
Plants were pretreated with 100 μM DPI or 5 mM DMTU for 8 h and then treated with
0.1 μM EBR or 10 mM H2O2. After 6 h the steady-state transcript levels were assayed by
qRT-PCR. Data are the means of three replicates(±SD).
Figure 6. Involvement of H2O2 in EBR-induced upregulation of antioxidant enzyme
activity. Plants were pretreated with 100 μM DPI or 5 mM DMTU for 8 h and then
treated with 0.1 μM EBR. After 6 h the activities of antioxidant enzymes were
determined. Values are means ± SD (n=6). Means denoted by the same letter did not
significantly differ at P <0.05 according to Tukey's test.
Figure 7. Kinetics of changes in H2O2 content in EBR- or Brz-treated plants
(A) Plants were treated with distilled water or 0.1 μM EBR. Leaf samples were harvested
at indicated times (h) after EBR treatment. Values are means ± SD (n=6).
(B) H2O2 content in leaves after different duration of Brz treatment. Brz (4 μM) treatment
started at indicated times (h) before sampling. Brz treatment was repeated on
alternative days until sampling. Time zero points were without Brz treatment. Values
are means ± SD (n=6).
35
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Figure 8. Levels of tolerance to PQ-induced oxidative stress at different times after EBR,
H2O2 and Brz treatment.
(A) Oxidative stress tolerance induction curves of EBR or H2O2. PQ (10 μM) was applied
at indicated times (h) after water, 0.1 μM EBR or 10 mM H2O2 treatment. Time zero
points indicate PQ treatment only. Fv/Fm was determined with the whole leaf as area
of interest after 1 d at 600 μmol m-2 s-1 and 25℃ . Fv/Fm for PQ-untreated leaves
was 0.83. Values are means ± SD (n=5).
(B) Oxidative stress tolerance of plants after different duration of Brz treatment. Brz (4
μM) treatment started at indicated times (h) before 10 μM PQ challenge. Brz
treatment was repeated on alternative days until PQ challenge. Time zero points
indicate PQ treatment only. Fv/Fm was determined using the whole leaf as area of
interest after 1 d at 600 μmol m-2 s-1 and 25℃. Fv/Fm for PQ-untreated leaves was
0.83. Values are means ± SD (n=5).
Figure 9. Time-course analysis of steady-state transcript levels of RBOH, CAT, cAPX,
PR-1 and PAL in response to EBR. Leaf samples were harvested at indicated times (h)
after 0.1 μM EBR treatment and the steady-state transcript levels were assayed by
qRT-PCR. Data are the means of three replicates(±SD).
Figure 10. Time-course analysis of antioxidant enzymes activities in response to EBR
Leaf samples were harvested at indicated times (h) after 0.1 μM EBR treatment and the
activities of antioxidant enzymes were analyzed. Values are means ± SD (n=6).
Figure 11. A model for the induction of stress tolerance by BR in cucumber plants.
Perception of BR by receptors results in the activation of plasma membrane-bound
NADPH oxidase (RBOH). Activated NADPH oxidase results in elevated levels of H2O2,
36
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
which functions as a signal molecule to activate stress response pathways.
37
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
A
B
Water
Brz+EBR
Brz
EBR
0.8
Fv/Fm
a
b
b
0.7
c
0.6
0.5
Water
EBR
Brz
Brz+EBR
D
0.9
a
ab
Disease index
-2
-1
ETR (μmol m s )
40
90
Disease index
MDA
Water
EBR
Brz
Brz+EBR
30
20
0.6
a
bc
b
ab ab
-1
50
60
c
0.3
30
10
0
0
100
200
300
400
-2
500
600
700
0.0
Water
EBR
Brz
Brz+EBR
0
-1
PAR (μmol m s )
Figure 1. Effects of BR levels on resistance to PQ, chill or CMV
(A) Symptoms (up) and images of the maximum PSII quantum yield (Fv/Fm, down).
The false color code depicted at the bottom of the image ranged from 0 (black) to
1.0 (purple). Plants treated with water, 0.1 μM EBR, 4 μM Brz and 4 μM Brz+0.1
μM EBR were challenged with 10 μM PQ at 600 μmol m-2 s-1 light intensity and
25℃ for 1 d. Five plants were used for each treatment and the picture of one
representative leaf was shown. Scale bar=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 the means of five
replicates(±SD). Means denoted by the same letter did not significantly differ at
P <0.05 according to Tukey's test.
(C) Electron transport rates (ETR) determined after 1-d exposure to chill stress (8℃
/200 μmol m-2 s-1) for plants treated with water, 0.1 μM EBR, 4 μM Brz and 4 μM
Brz+0.1 μM EBR. Measurements were conducted at 25℃. Data are the means of
five replicates(±SD).
(D) CMV incidence and malondialdehyde (MDA) content determined at 14 d
post-inoculation for plants treated with water, 0.1 μM EBR, 4 μM Brz and 4 μM
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
MDA (nmol g FW )
C
Brz+0.1 μM EBR. Disease index is the mean (n=9 leaves) of disease severities (0;
light to 1; severe). Data for MDA are the means of five replicates(±SD). Means
denoted by the same letter did not significantly differ at P <0.05 according to
Tukey's test.
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Figure 2
25
Relative transcript abundance
3
Water
EBR
Brz
Brz+EBR
2
1
0
RBOH
MAPK1
MAPK3
40
Relative transcript abundance
Relative transcript abundance
Relative transcript abundance
4
35
30
10
5
0
WRKY30
WRKY6
MYB
20
2
1
0
PR-1
PAL
HPL
GST
GPX
POD
CAT
cAPX
MDAR
8
6
4
2
0
Relative transcript abundance
Relative transcript abundance
16
12
8
4
0
MYC
HSP70-1
DnaJ
4
3
2
1
0
Figure 2. Expressions of stress-responsive genes in response to BR levels in
cucumber seedlings. qRT-PCR analysis was performed to examine steady-state levels
of mRNAs for 18 genes in plants treated with water, 0.1 μM EBR, 4 μM Brz and 4
μM Brz+0.1 μM EBR. Data are the means of three replicates(±SD).
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Figure 3
A
B
Water
EBR
Brz
Brz+EBR
Water
CW
NBT
V
DAB
C
IS
C
Water
DPI
DMTU
EBR
Water
C
V
IS
EBR
D
H2O2 content
a
-1
H2O2 content (nmol g FW )
a
NADPH oxidase
150
100
b
30
b
b
c
b
*
c
10
50
0
20
Water
EBR
Brz
Brz+EBR
NADPH oxidase activity
-1
-1
.(O2 μmol min mg protein )
40
200
Brz
IS
CW
0
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
C
Figure 3. The roles of BR in regulation of ROS accumulation
(A) In situ detection of leaf O2.- and H2O2. NBT and DAB stains were used to detect
the presence of O2.- and H2O2 in leaves treated with water, 0.1 μM EBR, 4 μM Brz
and 4 μM Brz+0.1 μM EBR. Leaf discs (1.5cm in diameter) were harvested at 6 h
after treatment and stained immediately.
(B) Cytochemical localization of H2O2 accumulation in mesophyll cells of cucumber
leaves with CeCl3-staining and transmission electron microscopy. The plants were
treated and harvested as described in (A). Arrows, CeCl3 precipitates; C,
chloroplast; CW, cell wall; V, vacuole; IS, intercellular space.
(C) Blockage of EBR-induced H2O2 accumulation by DPI and DMTU. Plants were
pretreated with 100 μM DPI or 5 mM DMTU for 8 h and then treated with 0.1 μM
EBR. After 6 h the DAB staining of leaf discs was performed.
(D) Quantitative measurements of H2O2 level and NADPH oxidase activity in
cucumber leaves with different BR levels. Values are means ± SD (n=6). Means
denoted by the same letter did not significantly differ at P <0.05 according to
Tukey's test.
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Figure 4
A
PQ
Chill
B
C
0.8
0.9
Chill stress
PQ stress
a
a
Fv/Fm
0.8
a
b
b
b
b
b
Fv/Fm
0.7
0.6
a
b
b
b
b
b
0.7
0.5
TU
PI
D
M
D
R
M
TU
+E
B
2
R
EB
D
R
O
2
PI
+
D
H
EB
at
e
W
TU
PI
M
D
D
R
+E
B
D
M
TU
2
R
O
2
+E
B
H
D
PI
R
EB
er
at
W
r
0.4
0.6
Figure 4. Requirement of H2O2 for EBR-induced resistance to PQ and chill
(A) Images of the maximum PSII quantum yield (Fv/Fm) of PQ-challenged and
chilled leaves (scale bar=2.5 cm). The false color code depicted at the bottom of
the image ranged from 0 (black) to 1.0 (purple). Plants were pretreated with 100
μM DPI or 5 mM DMTU for 8 h and then plants were treated with 0.1 μM EBR
or 10 mM H2O2. After 1 d plants were challenged with 10 μM PQ or exposed to
chill at high light intensity (8℃/1000 μmol m-2 s-1). Single treatment of DPI or
DMTU was included as negative control.
(B) and (C) Average Fv/Fm values of PQ-challenged (B) or chilled (C) leaves. Fv/Fm
was determined with the whole leaf as area of interest. Fv/Fm for control plants
was 0.83. Values are means ± SD (n=5). Means denoted by the same letter did
not significantly differ at P <0.05 according to Tukey's test.
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Figure 5
R
B
R
PI
TU
+E
+E
B
2
O
M
D
R
B
R
+E
B
TU
+E
PI
D
M
D
B
R
M
D
D
PI
TU
+E
+E
B
2
O
2
H
EB
er
D
M
R
0
R
1
at
+E
TU
+E
PI
2
O
2
H
R
2
W
B
R
B
2
cAPX
3
R
0
D
EB
at
W
Relative transcript abundance
1
O
er
R
+E
2
2
0
M
D
3
H
1
TU
+E
PI
D
CAT
R
2
4
4
EB
PAL
3
B
R
B
2
O
2
H
EB
at
W
R
0
Relative transcript abundance
1
er
2
D
M
D
2
at
H
er
at
W
+E
TU
+E
PR-1
W
0
4
3
er
Relative transcript abundance
4
Relative transcript abundance
2
B
R
B
2
O
2
D
PI
H
EB
at
W
R
0
R
1
4
R
2
WRKY6
6
EB
MAPK1
3
Relative transcript abundance
8
er
Relative transcript abundance
4
Figure 5. Involvement of H2O2 in EBR-induced upregulation of stress responsive
genes. Plants were pretreated with 100 μM DPI or 5 mM DMTU for 8 h and then
treated with 0.1 μM EBR or 10 mM H2O2. After 6 h the steady-state transcript levels
were assayed by qRT-PCR. Data are the means of three replicates(±SD).
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Figure 6
0.3
(U mg protein
SOD activity
-1
)
a
8
b
b
b
6
4
2
MDAR activity
-1
-1
(μmol min mg protein )
10
EBR
b
b
DHAR activity
-1
-1
(μmol min mg protein )
CAT activity
-1
-1
(μmol min mg protein )
b
0.1
0.0
EBR
a
ab
bc
c
1.5
1.0
0.5
b
EBR
DPI+EBR DMTU+EBR
b
0.2
0.1
Water
EBR
DPI+EBR DMTU+EBR
a
b
b
0.10
c
0.05
0.00
Water
DPI+EBR DMTU+EBR
a
0.15
2.0
EBR
b
DPI+EBR DMTU+EBR
GR activity
-1
-1
(μmol min mg protein )
APX activity
-1
-1
(μmol min mg protein )
Water
0.3
0.0
Water
2.5
0.0
c
0.4
a
b
0.1
DPI+EBR DMTU+EBR
0.3
0.2
b
0.0
Water
a
0.2
Water
EBR
DPI+EBR DMTU+EBR
Figure 6. Involvement of H2O2 in EBR-induced upregulation of antioxidant enzyme
activity. Plants were pretreated with 100 μM DPI or 5 mM DMTU for 8 h and then
treated with 0.1 μM EBR. After 6 h the activities of antioxidant enzymes were
determined. Values are means ± SD (n=6). Means denoted by the same letter did not
significantly differ at P <0.05 according to Tukey's test.
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Figure 7
B
A
200
-1
H2O2 content (nmol g FW )
Water
EBR
-1
H2O2 content (nmol g FW )
160
140
120
100
80
150
100
50
0
0
3
6
9
24
72
120
Time after EBR treatment (h)
0
3
12
24
72
120
168
Intervals between Brz application and sampling (h)
Figure 7. Kinetics of changes in H2O2 content in EBR- or Brz-treated plants
(A) Plants were treated with distilled water or 0.1 μM EBR. Leaf samples were
harvested at indicated times (h) after EBR treatment. Values are means ± SD
(n=6).
(B) H2O2 content in leaves after different duration of Brz treatment. Brz (4 μM)
treatment started at indicated times (h) before sampling. Brz treatment was
repeated on alternative days until sampling. Time zero points were without Brz
treatment. Values are means ± SD (n=6).
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Figure 8
A
B
0.7
Fv/Fm
Fv/Fm
0.8
Water
EBR
H2O2
0.8
0.4
0.6
0.5
0.6
0
3
6
12
24
72
120
Intervals between PQ challenge and EBR or H2O2 treatment (h)
0.2
0
3
12
24
72
120
168
Time of Brz treatment before PQ challenge (h)
Figure 8. Levels of tolerance to PQ-induced oxidative stress at different times after
EBR, H2O2 and Brz treatment.
(A) Oxidative stress tolerance induction curves of EBR or H2O2. PQ (10 μM) was
applied at indicated times (h) after water, 0.1 μM EBR or 10 mM H2O2 treatment.
Time zero points indicate PQ treatment only. Fv/Fm was determined with the
whole leaf as area of interest after 1 d at 600 μmol m-2 s-1 and 25℃ . Fv/Fm for
PQ-untreated leaves was 0.83. Values are means ± SD (n=5).
(B) Oxidative stress tolerance of plants after different duration of Brz treatment. Brz
(4 μM) treatment started at indicated times (h) before 10 μM PQ challenge. Brz
treatment was repeated on alternative days until PQ challenge. Time zero points
indicate PQ treatment only. Fv/Fm was determined using the whole leaf as area of
interest after 1 d at 600 μmol m-2 s-1 and 25℃. Fv/Fm for PQ-untreated leaves was
0.83. Values are means ± SD (n=5).
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Figure 9
4
Rboh
3
2
1
0
4
CAT
3
Relative transcript abundance
2
1
0
cAPX
4
3
2
1
0
18
PR-1
12
2
0
24
PAL
20
6
4
2
0
0
3
6
12
24
72
120
Time after EBR treatment (h)
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Figure 9. Time-course analysis of steady-state transcript levels of RBOH, CAT, cAPX,
PR-1 and PAL in response to EBR. Leaf samples were harvested at indicated times (h)
after 0.1 μM EBR treatment and the steady-state transcript levels were assayed by
qRT-PCR. Data are the means of three replicates(±SD).
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Figure 10
0.3
Water
EBR
12
A
8
4
MDAR activity
-1
-1
(μmol min mg protein )
SOD activity
-1
(Unit mg protein )
16
0.3
0.2
0.1
DHAR activity
-1
-1
(μmol min mg protein )
CAT activity
-1
-1
(μmol min mg protein )
B
0.1
0.4
E
0.3
0.2
0.1
0.0
C
4
3
2
1
0
3
6
12 24 48 72 96 120 144
Time after EBR treatment (h)
GR activity
-1
-1
(μmol min mg protein )
0.0
APX activity
-1
-1
(μmol min mg protein )
0.2
0.0
0
0
D
F
0.3
0.2
0.1
0.0
0
3
6
12 24 48
72 96 120 144
Time after EBR treatment (h)
Figure 10. Time-course analysis of antioxidant enzymes activities in response to EBR
Leaf samples were harvested at indicated times (h) after 0.1 μM EBR treatment and
the activities of antioxidant enzymes were analyzed. Values are means ± SD (n=6).
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Figure 11
Figure 11. A model for the induction of stress tolerance by BR in cucumber plants.
Perception of BR by receptors results in the activation of plasma membrane-bound
NADPH oxidase (RBOH). Activated NADPH oxidase results in elevated levels of
H2O2, which functions as a signal molecule to activate stress response pathways.
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