h2o2 paper pmb 8 06
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APA SI HARTIETRANSCRIPT
Heat stress-induced H2O2 is required for effective expressionof heat shock genes in Arabidopsis
Roman A. Volkov Æ Irina I. Panchuk ÆPhillip M. Mullineaux Æ Friedrich Schoffl
Received: 19 May 2005 / Accepted: 15 March 2006
� Springer Science+Business Media B.V. 2006
Abstract The mechanisms of sensing and signalling of
heat and oxidative stresses are not well understood. The
central question of this paper is whether in plant cells
oxidative stress, in particular H2O2, is required for heat
stress- and heat shock factor (HSF)-dependent expression
of genes. Heat stress increases intracellular accumulation
of H2O2 in Arabidopsis cell culture. The accumulation was
greatly diminished using ascorbate as a scavenger or
respectively diphenyleneiodonium chloride (DPI) as an
inhibitor of reactive oxygen species production. The
mRNA of heat shock protein (HSP) genes, exemplified by
Hsp17.6, Hsp18.2, and the two cytosolic ascorbate perox-
idase genes Apx1, Apx2, reached similar levels by moderate
heat stress (37�C) or by treatment with H2O2, butylperox-
ide and diamide at room temperature. The heat-induced
expression levels were significantly reduced in the pres-
ence of ascorbate or DPI indicating that H2O2 is an
essential component in the heat stress signalling pathway.
Rapid (15 min) formation of heat shock promoter element
(HSE) protein-binding complex of high molecular weight
in extracts of heat-stressed or H2O2-treated cells and the
inability to form this complex after ascorbate treatment
suggests that oxidative stress affects gene expression via
HSF activation and conversely, that H2O2 is involved in
HSF activation during the early phase of heat stress. The
heat stress induction of a high mobility HSE-binding
complex, characteristic for later phase of heat shock re-
sponse, was blocked by ascorbate and DPI. H2O2 was
unable to induce this complex suggesting that H2O2 is in-
volved only in the early stages of HSF activation. Signifi-
cant induction of the genes tested after diamid treatment
and moderate expression of the sHSP genes in the presence
of 50 mM ascorbate at 37�C occurred without activation of
HSF, indicating that other mechanisms may be involved in
stress signalling.
Keywords Ascorbate peroxidase Æ DPI Æ Heat shock
factor Æ Heat shock protein Æ Hydrogen peroxide ÆOxidative stress
Introduction
In nature, plants are frequently subject to heat stress and
like other organisms, they have evolved strategies for
preventing and repairing cellular damage caused by heat
stress. In all species studied, heat stress results in the
production of heat shock proteins (HSP), which have been
classified into a number of families based on their molec-
ular mass (HSP100, HSP90, HSP70, HSP60 and small (s)
HSP), most of which have chaperone function (for review
see Jaenicke and Creighton 1993; Boston et al. 1996). The
expression of sHSP is a signature of the heat shock
response in plants. Plants are unique in the number and
Electronic Supplementary Material Supplementary material is
available for this article at http//dx.doi.org/10.1007/s11103-006-0045-4
Roman A. Volkov and Irina I. Panchuk contributed equally
R. A. Volkov Æ I. I. Panchuk Æ F. Schoffl (&)
Zentrum fur Molekularbiologie der Pflanzen – Allgemeine
Genetik, Universitat Tubingen, Auf der Morgenstelle 28, 72076
Tubingen, Germany
e-mail: [email protected]
P. M. Mullineaux
Department of Biological Sciences, University of Essex,
Wivenhoe Park, Colchester, Essex CO4 3SQ, UK
R. A. Volkov Æ I. I. Panchuk
Department of Molecular Genetics and Biotechnology,
University of Chernivtsy, Kotsubinsky str. 2, 58012, Ukraine
Plant Mol Biol (2006) 61:733–746
DOI 10.1007/s11103-006-0045-4
123
complexity of sHSP that they produce upon heat stress, no
isoforms are expressed in vegetative tissue under non-stress
conditions (for review see Jakob and Buchner 1994;
Schoffl et al. 1998). Despite the ubiquitous nature of this
conserved response, little is known how plants sense heat
stress or about the signalling pathways resulting in heat
shock gene expression. In all organisms the heat shock
response is primarily regulated at the transcriptional level
by heat stress transcription factors (HSF), which are acti-
vated by stress for a specific binding to heat shock pro-
moter elements (HSE). In Arabidopsis 21 different HSF
genes have been identified (Nover et al. 2001), but only
few have been functionally characterized. AtHsf1 (HSF-
A1a) and AtHsf3 (HSF-A1b) are the regulators, which
become activated very early in the heat shock response and
are necessary for efficient expression of heat shock genes
because double knock out mutants, hsf1/3 are unable to
form high molecular weight HSE-binding complexes and
mRNA accumulation of HSF target genes is significantly
impaired upon heat stress (Lohmann et al. 2004). Recent
investigations have shown that not only conventional HSP
genes are controlled by HSF. Other genes encoding key
enzymes in biochemical pathways related to environmental
responses have been identified as targets of HSF regulation
in HSF3-transgenic plants (Panchuk et al. 2002; Paniku-
langara et al. 2004; Busch et al. 2005).
Permanent production of reactive oxygen species (ROS)
such as hydrogen peroxide (H2O2), superoxide, hydroxyl
radicals, and singlet oxygen is an unavoidable consequence
of aerobic metabolism. In plant tissues, about 1% of the
total O2 consumption goes to ROS production (Puntarulo
et al. 1988). An excessive generation of ROS leads to the
damage of proteins, lipids, and DNA and causes an oxi-
dative stress, which is a central factor in abiotic and biotic
stress phenomena (Bowler et al. 1992; Chen et al. 1993;
Asada 1999; Finkel and Holbrook 2000; Moller 2001).
There is considerable evidence that oxidative stress induces
expression of HSP and chaperones in plants, which can
provide a protective function against oxidative stress. In
tomato and rice, mitochondrial HSP22 and chloroplastic
HSP26, respectively, are induced by H2O2 (Banzet et al.
1998; Lee et al. 2000). In cyanobacteria and Arabidopsis,
high light and H2O2, respectively, induced the mRNAs of
some chaperones, HSP, and heat shock transcription factors
(Desikan et al. 2001; Hihara et al. 2001). It has also been
shown that thermotolerance can be induced by compounds
that induce oxidative bursts (Dat et al. 1998).
There is ample evidence that different environmental
stresses, including also high temperature, induce oxidative
stress in plants (Foyer et al. 1997; Dat et al. 1998). Very
short heat pulses can result in oxidative bursts of super-
oxide and/or hydrogen peroxide (Vallelian-Bindschedler
et al. 1998). This suggests that there is considerable inter-
linking between heat and oxidative stress signalling and
responses.
There are several possible sources of H2O2 in plants,
which can be activated during abiotic and biotic stress to
induce H2O2 generation and thereby oxidative stress, e.g.
electron transport chains (ETC) in chloroplasts and mito-
chondria, photorespiration in peroxisomes (Noctor and
Foyer 1998; Dat et al. 2000), or enzymatic sources
including plasma membrane-located NAD(P)H oxidases
(Desikan et al. 1998; Keller et al. 1998; Torres et al.
1998), and cell wall bound peroxidases/amine oxidase
(Bolwell and Wojtaszek 1997). Upon severe heat stress the
decrease in enzymatic activities of for example catalase
(Dat et al. 1998) and ascorbate peroxidase (Panchuk et al.
2002) may diminish removal of H2O2 and consequently
contribute to enhanced levels of ROS.
To survive under environmental stress conditions, plants
undergo a process of stress acclimation, which may require
changes in the flow of metabolites, suppression of path-
ways involved in the excessive production of ROS, and the
induction of various defense genes such as HSP and ROS
scavenging enzymes (Vierling 1991; Dat et al. 2000;
Mittler 2002; Panchuk et al. 2002).
Heat stress-induced oxidative damage becomes visible
by bleaching of green tissue, a feature that documents the
interlinkage between the two responses in plants. Arabid-
opsis plants that have acquired enhanced levels of thermo-
tolerance experienced lower levels of oxidative damage
during recovery from heat stress as compared with non-
conditioned plants and this protection from oxidative dam-
age correlates with greater survival rates of such plants
(Larkindale and Knight 2002). Transgenic overexpression
of HSF constructs in Arabidopsis resulted in a moderate
increase in basal thermotolerance that was also associated
with protection from oxidative bleaching of seedlings (Lee
et al. 1995; Prandl et al. 1998). This suggests that one aspect
of thermotolerance in Arabidopsis is an increased ability to
either prevent or repair heat-induced oxidative damage. This
conclusion is in accordance with the heat stress-induced
expression of ascorbate peroxidase (Apx) genes
(Storozhenko et al. 1998; Panchuk et al. 2002) and the
identification of novel heat-tolerant, HSF-dependently
expressed APX isoform in Arabidopsis (Panchuk et al.
2002). Although ROS were originally considered to be
detrimental to cells, it is now widely recognized that redox
regulation involving ROS is a key factor modulating cellular
activities (Allen and Tresini 2000; Dat et al. 2000). Espe-
cially, accumulation of relatively low-toxic H2O2 induces
the expression of various defense-related genes, including
glutathione S transferase, phenylalanine ammonia lyase,
and HSP (Vandenabeele et al. 2003; Levine et al. 1994;
Desikan et al. 1998; Neill et al. 1999; Grant et al. 2000).
H2O2 is also involved in the activation of mitogen-activated
734 Plant Mol Biol (2006) 61:733–746
123
protein kinases (MAPKs) that modulate gene expression and
transduce cellular responses to extracellular stimuli (Desi-
kan et al. 1999; Grant et al. 2000; Kovtun et al. 2000;
Samuel et al. 2000).
In our present analysis we investigate the generation of
H2O2 upon heat stress and the effects of heat stress and
oxidative/antioxidative compounds on the primary
expression level (mRNA) of selected sHSP and APX
genes. We present evidence that at normal temperature,
H2O2 is an efficient inducer of sHSP mRNA expression.
On the other hand we show that heat stress-induced
expression of HSP can be counteracted by diphenylenei-
odonium chloride (DPI), an inhibitor of flavin-dependent
oxidases involved in superoxide radical generation, or by
ascorbate, a peroxide scavenger. Our data demonstrate that
H2O2 is an important component in heat stress-activated
gene expression that appears to be involved in HSF-acti-
vation and signalling.
Material and methods
Cell culture and growth conditions
Arabidopsis thaliana (ecotype Landsberg) cell suspension
culture was grown in MS medium (Murashige and Skoog
1962) containing basal salt mixture, 3% (w/v) sucrose,
0.5 lg/ml NAA, 0.05 lg/ml kinetin, pH 5.7. Three ml of
seven days old suspension culture was added to 97 ml of
fresh medium and cultivated at constant light (3000 lu-
men m)2) at 20�C with shaking (60 strikes min)1). Expo-
nentially growing cells (3–4 days old) were used for
experiments. Before treatments, cell density was adjusted
with fresh MS medium to OD 660 = 0.12 and pre-incu-
bated for 90 min at normal growth condition. Aliquots of
5 ml were used for all further treatments.
Cell viability staining test
To evaluate viability of cells, an aliquot of the suspension
culture was supplemented with Evans Blue Dye (Fluka,
Swiss) to a final concentration of 0.04%, incubated 10–
15 min at room temperature and monitored under micro-
scope. Dead cells are stained with the dye whereas viable
cells remain unstained.
Heat stress treatment of the cell culture
Heat stress was administered by subjecting cell culture
samples to 37�C or 44�C for 2 h in a shaking water bath in
the dark. Controls were incubated at 20�C under otherwise
identical condition. Following the treatments samples were
immediately assayed for quantifications of H2O2 levels, or
frozen in liquid nitrogen and used for isolation of mRNA or
preparation of protein extracts.
Supplementation of oxidative and antioxidative
compounds
All compounds, with the exception of DPI, used for cell
culture treatments were dissolved in MS medium and pH
was subsequently adjusted to 5.7. For DPI treatment a
20 mM stock (in DMSO) was prepared. Reduced ascor-
bate, DPI, or oxidative compounds were added to the final
concentrations as indicated and cells were incubated in a
shaking water bath (60 strokes min-1) in the dark at 20, 37
or 44�C, respectively. The following concentrations were
tested: ascorbate––5, 20, 50 mM; DPI––0.5, 10, 25 and
150 lM; H2O2––0.05, 0.5, 5 and 50 mM, tert-butylperox-
ide (BP)––0.05, 0.5 and 5 mM; diamid (DA)––0.05, 0.5
and 5 mM. All supplements were purchased from Sigma.
Cultivation of Arabidopsis plants, and stress treatments
Arabidopsis (ecotype Columbia 24) plants were cultivated
as described by Panchuk et al. (2002). Leaves of seven-
week-old plants were collected and incubated in section
incubation buffer (SIB: 1 mM potassium phosphate, pH
6.0, and 1% (w/v) sucrose) in a shaking water bath
(60 strokes min)1) in the dark at 20�C or 37�C, respec-
tively. Ascorbate, DPI, or oxidative compounds were ad-
ded to the final concentrations as indicated.
Measurement of intracellular H2O2 accumulation
Intracellular accumulation of ROS (e.g. H2O2) was moni-
tored using in vivo oxidation of carboxy-HDCFDA
(5–6-carboxy-2¢,7¢-dichlorodihydrofluorescein diacetate:
Molecular Probes) fluorescence probe (Royall and Ischir-
opoulos 1993). Five millilitre aliquots of Arabidopsis cell
culture were supplemented with carboxy-HDCFDA (end
concentration 10 lM) and incubated for 5 min at 20�C for
the preloading of cells with the fluorescence probe. Cells
were incubated in the dark in a shaking water bath
(60 strokes min)1) at 20, 37 or 44 �C. After 1 h, samples
were placed on ice and EDTA was added to final con-
centration of 0.3 mM. Cells were destroyed by sonication
(1 min, 30 W) using Sonifier B-12 (Branson Sonic Power
Company, Dunbury, Connecticut) and fluorescence was
measured using fluorescence spectrophotometer F 2000
(Hitachi) at excitation and emission wavelengths of 503
and 525 nm respectively. Three replicates of each sample
were routinely measured in parallel. Changes in the accu-
mulation of H2O2 were analyzed for statistical significance
according to t-test (Engel 1997). Intracellular character of
oxidation of fluorescence probe (Royall and Ischiropoulos
Plant Mol Biol (2006) 61:733–746 735
123
1993) was confirmed in control experiments applying cat-
alase to destroy potential extracellular H2O2. No difference
was found between samples incubated with or without
catalase. Spontaneous oxidation of carboxy-HDCFDA in
the absence of cell culture was below 1% of the levels
observed in the presence of the cells.
mRNA isolation and cDNA preparation
Poly(A)+-mRNA and cDNA were prepared as described by
Panchuk et al. (2002). The amount of poly(A)+-mRNA/
cDNA double-stranded products obtained after reverse
transcription was measured using PicoGreen dsDNA
Quantitation reagent (Molecular Probes). This method of
template quantification improved the reproducibility of data
of subsequent real-time PCR. For monitoring the degree of
potential template degradation during the preparation of
poly(A)+-mRNA/cDNA, two primer pairs spanning proxi-
mal and respectively distal parts of the AtAct2 mRNA were
used. Identical threshold cycles with both pairs of primers
indicated the integrity of mRNA/cDNA.
Primer design and PCR-product identity
Gene-specific primers for real-time RT-PCR quantification
were used as described by Panchuk et al. (2002), Volkov
et al. (2003). The resulting PCR products had the same size
of approximately 300 bp. The quality of PCR products was
visually inspected by electrophoresis; the generation of
only one single band of the expected size was taken as a
criterion for specificity. The identity of PCR products was
confirmed by direct DNA-sequencing.
Quantitative real-time RT-PCR
Quantification of gene-specific cDNA was performed by
real-time PCR monitoring the intercalation of SYBR-
Green (Molecular Probes) essentially as described by
Panchuk et al. (2002). Two concentrations of cDNA (1 ng
and 0.1 ng) were routinely measured in parallel and
duplicate samples were run for each concentration. All
experiments were repeated at least twice for cDNA pre-
pared for two samples of Arabidopsis cells. Using stan-
dardized conditions, deviations of threshold values were
less than 1.0 cycle for independent cDNA preparations and
less than 0.5 cycle for replicates of the same cDNA. The
quantification of mRNA levels is based on the comparison
to the level of an Act2 mRNA standard, defined as 100
relative expression units (REU: Panchuk et al. 2002),
which was determined in separate reactions. Changes in the
relative concentrations of PCR products/steady-state
mRNA levels were checked for statistical significance
according to t-test (Engel 1997).
Preparation of protein extracts
Crude cell extracts were generated by shock freezing cell
culture samples in liquid nitrogen, 1.2 Vol of 1.83 · LEB
(Low salt Extraction Buffer: 1 · LEB = 10 mM KCl,
3.3 mM MgCl2, 0.35 M sucrose, 8% (w/v) glycerol,
15 mM HEPES-KOH, pH 7.9, 2% Ficoll 400, 1 mM
PMSF, 1 · Proteinase Inhibitor Cocktail ‘‘Complett’’
(Roche), 1 · phosphatase inhibibitor Cocktails I and II
(Sigma)) were added, ground on ice for 3 min and centri-
fuged at 7000 g, 10 min, 4�C. The supernatant was cleared
again at 21,000 g, 20 min, 4�C, and protein concentration
was determined (Bradford 1976).
Electrophoretic mobility shift assay (EMSA)
The 5¢-ends of a synthetic double-stranded oligonucleotide
HSE probe were labeled by incubation with a-32P-dATP
and Klenow fragment (MBI Fermentas). The labelled
probe was purified with QIAquick Nucleotide Removal Kit
(Qiagen) and used for EMSA. Samples containing 20 lg
protein (10–12 ll low salt extract) were mixed with 3 Vol
of DB (Dilution Buffer: 15 mM HEPES-KOH, pH 7.9,
1.5 mM EDTA, 0.1 mM EGTA) containing excess of
unspecific DNA (per 1 probe: 5 lg of poly(dI-dC) and
100 ng of PCR product of coding region of NPT gene), and
incubated for 5 min at room temperature. Then 1 ng of
labelled HSE probe was added and samples were incubated
for 25 min at room temperature. To a total volume of 40 ll
binding reaction 2 ll of loading buffer (30% v/v glycerol,
0.2% w/v bromophenol blue) were added. Samples were
loaded onto a pre-run 5% polyacrylamide gel containing
3% v/v glycerol in 0.5ÆTBE (44.5 mM tris–boric acid pH
8.0, 1 mM EDTA) and subjected to electrophoresis for
2.5 hours, 350 V in cold room. Gels were dried on DE81
paper (Whatman Biometra) and exposed to Kodak BioMax
MS film.
Results
Heat stress increases H2O2 level in Arabidopsis
We have monitored the effect of moderate (37�C) and
severe (44�C) heat stress on the intracellular accumulation
of ROS, e.g. H2O2, in Arabidopsis cell culture. The assay
used is based on in vivo oxidation of carboxy-HDCFDA
fluorescence probe (Royall and Ischiropoulos 1993). The
viability of cells in suspension culture, grown under opti-
mal conditions, was tested. Viability staining showed that
approximately 99% of the cells were alive (supplemental
Figure 1). To ensure a good physiological state of the cells,
suspension culture was diluted with an excess of the fresh
736 Plant Mol Biol (2006) 61:733–746
123
medium and pre-cultivated prior to application of stress. To
avoid light-dependent effects the heat stress was adminis-
tered in the dark. In addition to heat stress, we investigated
also the influences of peroxide scavenger (ascorbate) and
inhibitior (DPI) of ROS production by supplementing cell
culture samples with different concentrations of ascorbate
or DPI during temperature treatments. Ascorbate and DPI
treatments had no negative effects on the viability of
Arabidopsis cells.
Figure 1A shows that heat stress causes a strong in-
crease in H2O2 levels: approximately 2.3-fold at 37�C and
2.5-fold at 44�C within 1-hour treatment. The accumula-
tion of H2O2 is a very fast process, occurring within the
first 15 min of heat stress. Longer exposure, as measured
during the first, the second, and fourth hour of continuous
heat stress, resulted in no further increase, rather in a de-
crease of H2O2 levels. DPI, supplemented at a concentra-
tion of 25 lM had a negative effect on the accumulation of
H2O2 at 20�C and especially at 37�C, but not at 44�C
(Fig. 1B). Higher DPI concentrations of up to 150 lM
resulted in only a minor further decrease of the H2O2 level
at 20�C and 37�C. Supplementation of ascorbate caused
much stronger negative effects on H2O2 levels at all tem-
peratures tested (Fig. 1B). At a concentration of 50 mM
the reduction was approximately 4-, 7- and 5-fold at 20, 37
and 44�C, respectively. The effect was dosage-dependent:
25 mM caused a larger reduction than 5 mM ascorbate.
In contrast to DPI, ascorbate was also very efficient in
reducing the H2O2 levels at normal temperature and at
44�C. These data show that heat stress enhances intracel-
lular production of H2O2, which can be blocked completely
by ascorbate supplementation.
Oxidative compounds induce heat shock
gene expression
In order to test whether at normal temperature the appli-
cation of oxidative compounds is capable of inducing heat
shock gene expression, we determined the mRNA levels of
sHSP and APX genes in Arabidopsis cell culture by
quantitative RT-PCR. Small Hsp17.6 and Hsp18.2 exem-
plify typical heat-induced and HSF-dependent expressed
genes in Arabidopsis (Volkov et al. 2003; Lohmann et al.
2004) and cytosolic ascorbate peroxidase genes Apx1 and
Apx2 have been previously identified as novel HSF-
dependent heat shock genes, which are significantly in-
duced at the mRNA level in leaf tissues upon heat stress
(Panchuk et al. 2002).
In our analysis we determined the mRNA levels of these
genes when induced by different concentrations of H2O2,
BP, or DA at 20�C and compared them with the levels in
untreated (20�C) or heat-treated (37�C or 44�C) cells
(Fig. 2). Two of the oxidative compounds used, H2O2 and
BP, are peroxides. While H2O2 is located mainly in
aqueous phase, BP has relatively high affinity to cellular
membranes. DA is a synthetic SH-groups-oxidizing com-
pound, which changes intracellular redox status and pro-
motes protein disulfide cross-linking. These differences
may result in different cellular responses after treatment.
The viability staining test revealed that 50 mM H2O2
has a slightly negative effect on cell viability: approxi-
mately 90% of the cells survived after 2 hours treatment.
Lower concentrations (5 mM or less) of the oxidative
compounds tested appeared to be non-toxic, 95–98% of
cells survived after the treatments.
In cell culture, several genes tested were induced by
moderate heat stress (37�C) whereas no induction was
Ascorbate
28 36
100
238240
948544 51
0
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400
mM
Flu
ore
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ce(a
rbit
ary
un
its)
0 5 50
DPI
141
61112130
240 238
100 95
243 259
70
251275
293280
0
100
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300
400
µM
Flu
ore
scen
ce(a
rbit
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un
its)
0 5 50 0 5 50
20˚C 37˚C 44˚C
20˚C 37˚C 44˚C0
0.5 25 150
DM
SO 0
0.5 25 150
DM
SO 0
0.5 25 150
DM
SO
96139
79108
278
216238240
100
152101
71
0
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Flu
ore
scen
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its)
0-15
min
0-1h
1-2h
3-4h
0-15
min
0-1h
1-2h
3-4h
0-15
min
0-1h
1-2h
3-4h
(A)
(B)
Fig. 1 Intracellular oxidation levels in Arabidopsis suspension cell
culture upon heat-stress. (A) Time-dependent changes after prolonged
heat-treatment: the mean rate of oxidation of fluorescence probe was
measured during 15 min (0–15 min), or 1 h (0–1 h) immediately after
beginning of treatment, or from 1 to 2 h (1–2 h), or from 3 to 4 h (3–
4 h); (B) Effects of application of different concentration of DPI
(dissolved in DMSO), or ascorbate, on the intracellular oxidation levels
in Arabidopsis cell incubated for 1 h at 20, 37 or 44�C. All treatments
were performed in the dark. Bars show means SD (n = 4–5)
Plant Mol Biol (2006) 61:733–746 737
123
found after severe heat treatment (44�C) similar to the
response observed in Arabidopsis leaves (Panchuk et al.
2002; Volkov et al. 2003). It should be noted that 44�C
heat stress is lethal to unconditioned Arabidopsis plants
(Wunderlich et al. 2003; Lohmann et al. 2004). The cur-
rent data on tissue culture show that at 20�C H2O2, BP, and
DA are potent inducers of mRNA expression of the heat-
inducible genes tested (Fig. 2). The magnitude of mRNA
induction by H2O2, BP, or DA, and by moderate heat stress
(37�C) was very similar for Hsp17.6 whereas for Hsp18.2
the oxidative stress-induced levels reach approximately 1/
3rd of the level induced by heat stress at 37�C. The optimal
concentrations for the induction of the two sHSP genes are
0.5 mM H2O2, 0.5–5 mM BP, and 0.5 mM DA. The same
concentrations are also optimal for the induction of Apx2
mRNA, but interestingly, the levels induced by H2O2 or BP
were approximately 3-fold higher compared to the levels
after heat stress at 37�C; DA-induced levels reach
approximately only 50% of the heat-induced levels. The
induction factors range between approximately 100-fold
for Hsp17.6, 300–1000-fold for Hsp18.2 and 10–25-fold
for Apx2.
The expression profile of Apx1 differed from the pat-
terns of the three other genes by: (i) A relatively high basal
level of mRNA present under non-stress condition, (ii) a
moderate stimulating effect (maximum about 3-fold
induction) by application of oxidative stress compounds,
and only a weak stimulating effect (about 1.5-fold induc-
tion) by heat stress at 37�C, (iii) a wider range of effective
concentrations of all three different compounds. In com-
mon with Apx2 were the higher levels of oxidative stress-
induced mRNA of Apx1 compared to heat stress. Among
other members of APX gene family, only Apx4 was
induced by heat stress and 3 genes, Apx4, Apx6 and tApx
were induced by application of oxidative compounds
(supplemental Figure 2). The maximal induction was ob-
tained by application of 5 mM H2O2, which was one order
of magnitude higher than the optimal concentration for the
induction of Apx1, Apx2 and both sHSP genes. Interest-
ingly, no significant changes were detected for mRNA
levels of microsomal Apx3 and Apx5.
These data show that oxidative compounds, in particular
H2O2, cause an efficient increase in transcript levels of
sHSP genes. The transcript levels reached, as exemplified
by Hsp17.6 and Hsp18.2, approximately the same levels as
induced by heat stress. The mRNA expression of Apx1 and
Apx2 is more efficiently induced by oxidative stress com-
pared to heat stress.
H2O2 is required for efficient heat induction
of mRNA levels
If an increase in H2O2 level is required for heat stress-
activated gene expression, it could be expected that inhi-
bition of production or scavenging of ROS should exert
profound negative effects on heat-induced mRNA levels.
Therefore we tested the influence of DPI and ascorbate on
mRNA levels of Hsp17.6, Hsp18.2, Apx1, and Apx2 during
heat stress. Figure 3 shows that ascorbate supplementations
of 5 and 20 mM had a moderate effect on Hsp17.6,
Hsp18.2, 50 mM a profound negative effect on the heat
stress-induced mRNA levels of Hsp17.6, Hsp18.2 and
Apx2. 50 mM ascorbate reduced the mRNA levels of
Hsp17.6, Hsp18.2 and Apx2 by factors of 23 and 28,
respectively. There was no negative effect of 50 mM
ascorbate treatment on the basal mRNA levels of all tested
genes: the levels increased slightly by a factor of less than
2, when cells were treated at 20�C.
5.9 5.64.3
40
1.96.5
110
3.8 3.3
28
10571
24
0
50
100
150
200
mR
NA
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EU
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24
2640
15 38
1890
29 9.2288
27003100
46
2600
110
1000
2000
3000
4000
mR
NA
leve
l (R
EU
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272250
340290
628800
590
230
630541491
640
0
250
500
750
1000
mR
NA
leve
l (R
EU
)
3.21.1
920
0.8 0.6
370
6.12
10
320150
0.8
340
0
300
600
900
1200
mR
NA
leve
l (R
EU
)Hsp 17.6
Apx 1
Hsp 18.2
Apx 2
T,(˚C) 20˚37˚44˚ 20˚ 20˚ 20˚
0.05 0.5 5 50
0.05 0.5 5
0.05 0.5 5Treatment
+BP,mM +DA,mM
T,(˚C) 20˚37˚44˚ 20˚ 20˚ 20˚
0.05 0.5 5 50
0.05 0.5 5
0.05 0.5 5Treatment
+ H2O2,mM+ H2O2,mM + BP,mM + DA,mM
Fig. 2 Messenger RNA levels
of Hsp17.6, Hsp18.2, Apx1 and
Apx2 in Arabidopsis cells after
different stress treatments. All
treatments were performed in
the dark. Poly(A)+-mRNA
levels were quantified by real-
time RT-PCR. Expression levels
are represented in comparison to
the expression of actin2 mRNA
standard, which was defined as
100 relative expression units
(REU). Bars show means SD
(n = 4–6). Note that different
scales are used in graphs. BP,
tertbutylperoxide; DA, diamide
738 Plant Mol Biol (2006) 61:733–746
123
DPI treatment resulted also in a suppression of heat
stress-induced mRNA levels of Hsp17.6, Hsp18.2 and
Apx2. 25 lM had only little effect, whereas 150 lM DPI
caused 5.5-, 6.6-, and 3-fold reduction of heat-induced
mRNA levels of Hsp17.6, Hsp18.2 and Apx2. There was no
negative effect of DPI on basal gene expression of cells
incubated at 20�C or DMSO-treated cells (control of the
DPI solvent) after heat stress. The heat stress-dependent
mRNA expression profile of Apx1 differs from the patterns
of the other genes by: (i) Minor but statistically insignifi-
cant negative effects after ascorbate treatment, (ii) only a
moderate reduction (about 50%) of expression levels after
DPI treatment. These data demonstrate that ROS, espe-
cially H2O2, are necessary for heat-induced gene expres-
sion of sHSP genes and Apx2.
Regarding to the possibility that the effects observed for
the suspension culture may differ from that in planta, we
have tested changes of mRNA levels in leaves of seven-
weak-old Arabidopsis plants after incubation in dark for
2 h in SIB (i) at room temperature in the presence of
5 mM, 50 mM H2O2 or 5 mM BP and (ii) at 37�C in the
presence of 50 mM ascorbate (supplemental Figure 3) in
comparison to the respective controls. In leaves, similar to
the cell culture, H2O2 and BP markedly induced expression
of Hsp17.6, Hsp18.2, Apx1 and Apx2, by factors of maxi-
mal 633, 125, 3.2 and 86, respectively. However, in con-
trast to the cell culture, Hsp17.6, Hsp18.2 and Apx2 mRNA
levels induced by heat stress at 37�C were respectively 3.0-
fold, 60-fold and 2.7-fold higher than the maximal levels
induced by H2O2 or BP. Comparing with cell culture, a
much higher concentration of H2O2 (50 mM) was neces-
sary to achieve the maximal induction, and BP was a more
effective inducer than H2O2 at 5 mM concentration. Sup-
plementation of the incubation medium with 50 mM
ascorbate during heat stress of leaves at 37�C reduced the
heat-inducible mRNA levels of Hsp17.6, Hsp18.2 and
Apx2 by factors of 1.5, 1.9 and 2.8, respectively. This
shows that the effect of ascorbate is less pronounced in
leaves than in cell culture. These quantitative differences in
responses between Arabidopsis cell culture and leaves may
reflect the better penetration of compounds in the suspen-
sion culture cells and probably tissue-specific differences
in leaves. Taken together the data show that the Arabid-
opsis suspension culture cells, as compared to leaves,
exhibit similar but more pronounced effects (changes at
mRNA of target genes) and are therefore a useful model
system for investigating stress signalling.
Oxidative stress-induced HSF-DNA binding complexes
The common induction of gene expression by heat stress
and oxidative stress raises the possibility of common sig-
nalling pathways. The analysis of HSF knock out mutants
provided evidence that at least two different HSF, HSF-
A1a and HSF-A1b (originally designated HSF1 and HSF3
by Hubel and Schoffl et al. 1994; Prandl et al. 1998), are
involved in the control of Hsp17.6 and Hsp18.2 tran-
scription during the initial phase of the heat shock response
(Wunderlich et al. 2003; Lohmann et al. 2004). This early
phase is characterized by the formation of high molecular
weight DNA-protein binding complexes, which were
identified by electrophoretic mobility shift analyses
(EMSA). Therefore, we examined the potential of oxida-
tive compounds to induce the formation of heat stress-
specific HSF complexes with a double-stranded HSE probe
(Fig. 4A) containing six copies of active pentanucleotide
HSE-modules (Scharf et al. 2001). Figure 4B shows that
in H2O2-treated cells similar high molecular weight
250
340
286 202 246 238339
280173 187
0
250
500
750
1000
mR
NA
leve
l(R
EU
)
4.3
40
7.6
35 311.4 2.9
3213
39
0
50
100
150
200
mR
NA
leve
l(R
EU
)
24
2640
45
14001500
11312
1650
478
2080
0
1000
2000
3000
4000
mR
NA
leve
l (R
EU
)
1.1
920
1.5
500600
400.7
550
140
0
300
600
900
1200
mR
NA
leve
l (R
EU
)Hsp 17.6
Apx 1
Hsp 18.2
Apx 2
T,˚C 20 37˚ 20 37˚ 37 37˚ 20 37˚ 37 37˚
50 5 20 50 25 25 150
+ Ascorbate,mM +DPI,µM
DM
SO
Treatment
T,˚C 20˚ 37˚ 20˚ 37˚ 37˚ 37˚ 20˚ 37˚ 37˚ 37˚
50 5 20 50 25 25 150
+ Ascorbate,mM +DPI, µM
DM
SO
Treatment
990
Fig. 3 Messenger RNA levels
of Hsp17.6, Hsp18.2, Apx1 and
Apx2 in Arabidopsis cells after
heat treatment in the presence of
different concentration of
ascorbate, DPI or DMSO
(solvent control for DPI). All
treatments were performed in
the dark. Poly(A)+-mRNA
levels were quantified by real-
time RT-PCR. Expression levels
are represented in comparison to
the expression of actin2 mRNA
standard, which was defined as
100 relative expression units
(REU). Bars show means SD
(n = 4–6). Note that different
scales are used in graphs
Plant Mol Biol (2006) 61:733–746 739
123
complexes were formed at 20�C as compared to heat-
treated (37�C, 15 min) cells. Ascorbate but not DPI treat-
ment blocked the formation of the heat stress-induced
complex. Interestingly, light grown cells, when assayed
immediately after harvest, show also a high molecular
weight HSE-binding complex that is typical for heat
stressed cells, but which is not detectable anymore after
15 min incubation in the dark. Similar to H2O2, application
of BP also results in formation of high molecular complex
at 20�C, whereas DA treatment was unable to induce this
complex; the HSE-binding complex was also not induced
at 44�C (data not shown).
Since it was known that longer periods of heat stress
cause a change in the pattern of HSE-protein binding
complexes in leaves (Lohmann et al. 2004), we tested also
cells treated for 15 min, 1 and 2 h in the dark. It should be
noted that the components forming the late higher mobility
HSF-DNA binding complex are unknown. Evidently HSF-
A1a and –A1b, which have been identified as early
response regulators, are not part of the late complex. After
2 h (Fig. 4B and C) the high molecular weight complexes
that are typical for the initial phase of the heat shock
response have disappeared or were reduced in all lanes
except for light grown cells. An other HSE-binding com-
plex of higher mobility, which is typical for the later stages
of the heat shock response (Lohmann et al. 2004), was
observed in cells treated for 2 h at 37�C, but it was not
formed in H2O2-treated cells at 20�C, or heat treated cells
incubated in the presence of 50 mM ascorbate or 150 lM
DPI. Application of different concentrations demonstrated
that 10 lM DPI moderately and 25 lM DPI significantly
reduced intensity of the higher mobility complex (Fig. 4C).
Considering a dose-dependent inhibition of intracellular
production of H2O2 by DPI (see Fig. 1) the effects on HSF-
binding complex formation correlate well with the intra-
cellular H2O2 levels.
Thus, our experiments indicate that peroxides (H2O2 and
BP) stimulate the formation of high molecular weight
HSE-binding complexes in a similar way as heat stress.
Interestingly, formation of HSE-binding complexes was
not found in DA-treated cells, although this compound
effectively induces expression of heat stress genes. In
contrast to heat stress, the formation of the ‘‘late’’ high
mobility HSE-binding complex (after 2 h) is not initiated
in H2O2-treated cells and prevented in heat-stressed cells
by simultaneous treatment with ascorbate or DPI. This
indicates that H2O2 is involved only in the early stages of
HSF activation. Upon moderate heat-stress H2O2 appears
to be essential for the induction of heat stress response but
by exogenous application it seems to either prolong the
initial phase or an additional signal (e.g. denaturation of
proteins upon heat stress) may be necessary for the late
phase of induction.
Discussion
Arabidopsis cell culture as a model system
Our analysis provides evidence that oxidative stress has a
profound effect on the heat-stress-dependent induction of
HSF target genes in plant cells. The experiments were
conducted with Arabidopsis thaliana suspension culture
cells, which are, with respect to the application of inducers
and scavengers of ROS, particularly hydrogen peroxide,
convenient compared to whole plant or organs. Although
cell culture was used by several authors (Desikan et al.
(A)
(B)
(C)
Fig. 4 Stress-dependent increase in the DNA-binding activity of
HSF. (A) Oligonucleotide probe used for gel mobility shift assay,
HSE modules are underlined. (B) Gel mobility shift assay with
protein extracts prepared from Arabidopsis cells incubated at 20 or
37�C in the presence or absence of 0.5 mM hydrogen peroxide,
50 mM ascorbate or 150 lM DPI. (C) Effect of different concentra-
tions of DPI on the induction of DNA-HSF complexes. The stress
inducible DNA-HSF complexes specific for early and late phases of
stress response are indicated by black and open arrows, respectively;
constitutive DNA-HSF complexes are indicated by asterisks. All
treatments were performed in the dark except the light control
treatment at 20�C
740 Plant Mol Biol (2006) 61:733–746
123
1996; 1998; 1999; Clarke et al. 2000; Vacca et al. 2004)
for investigation of plant cell stress response, the question
arose wether the effects observed really reflect the situation
in planta. In our experiments we used exponentially
growing Arabidopsis cell culture, which represents a sus-
pension of microcolonies of chlorophyll-containing callus
cells originally obtained from leaves. Our data show that
genes investigated in cell culture respond to a heat stress in
a similar way as in leaves by (i) induction of mRNA of
known heat-inducible genes upon moderate heat-treatment
at 37�C, (ii) no induction at 44�C, (iii) rapid formation of a
high molecular weight HSE-binding complex during early
phase of heat shock response and the dynamic changes in
the pattern of binding complexes during the later phase
(after 2 h) of the response. Hence, regulation of the heat
stress response appears to be similar in cell culture and
leaves. Accordingly, the cell culture represents a conve-
nient model system for studies of stress signalling. On the
other hand, quantitative differences between leaves and
cell culture in the expression of stress genes and the
presence of HSE-binding complexes in light growing cells
at 20�C indicate tissue specificity of stress response.
In order to reduce the complexity of signalling inter-
ference between light and heat stress we have routinely
used heat stress treatment in the dark for studying the
expression of heat shock and HSF-dependent target genes
in Arabidopsis (Lohmann et al. 2004, Busch et al. 2005).
With this experimental design we intended to exclude the
effects of photo-oxidative stress generated by heat stress in
chloroplasts.
Possible source of heat-induced H2O2
We have shown that heat stress generates enhanced levels
of H2O2 in tissue culture cells. The data indicate that the
level rises very rapidly within the first 15 min with a
subsequent decline during longer exposure to heat stress.
This response is reminiscent of the oxidative burst occur-
ring after pathogen attack (Desikan et al. 1996; Clarke
et al. 2000).
Our experiments demonstrate that in Arabidopsis cells
the level of H2O2 significantly increases, 2.3–2.5 fold, both
after moderate (37�C) and respectively severe (44�C) heat
stress. Similar, in mustard seedlings subjected to severe
heat stress (55�C, 1.5 h) in the dark the level of endoge-
nous H2O2 increased by 65% in comparison with plants
grown at 24�C (Dat et al. 1998). Severe heat stress (at 60–
65�C) that induces programmed cell death in tobacco tissue
culture cells leads to much higher and sustained levels of
H2O2 (Vacca et al. 2004). There are different possibilities
for the generation of H2O2 within plant cells. In the
absence of light, mitochondria may be the source of
intracellular generation of ROS, in particular the proton-
pumping complexes I and III (CI, NADH dehydrogenase,
and CIII, ubiquinol-cytochrome bc1 reductase) located in
the inner membrane (Moller 2001), and it was proposed
that ROS could be also generated by a plant-specific non-
pumping internal NADPH dehydrogenase, NDin(NADPH)
(Moller 2001). Heat treatment at 41�C increases oxygen
respiration rate and results in excessive production of ROS
in yeast mitochondria (Sugiyama et al. 2000). The main
sites of ROS production appear to be external NADH de-
hydrogenases, NDE1 and NDE2 (Davidson and Schiestl
2001). These two proteins functionally substitute the pro-
ton-pumping complex CI present in mitochondria of the
majority of eukaryots (Moller 2001).
In order to identify possible ROS generating mecha-
nisms in Arabidopsis cells upon heat stress in the dark, we
tested the effect of DPI, an inhibitor of flavoenzymes
(O’Donnell et al. 1994). It has been demonstrated that DPI
inhibits NDin(NADPH), CI and NDin(NADH) activities in
plant mitochondria with a Ki of 0.17, 3.7, and 63 lM,
respectively (Agius et al. 1998). In the plasma membrane
of soybean DPI inhibits NADPH oxidase with a Ki of 0.1
lM, whereas activity of NADH oxidase was only slightly
affected even by application of 100 lM DPI (Morre 2002).
Our data show that the application of 0.5 lM DPI, which
should extensively inhibit NADPH-oxidases, had no effect
on the intracellular H2O2 level. However, the negative ef-
fect on H2O2 levels, generated by 25 lM DPI, indicates
that CI could be the main site of ROS production at 37�C.
However, it should be noted, that intracellular concentra-
tion may be higher than that of exogenous DPI in the
medium (Moller 2001). Therefore, it cannot entirely be
excluded that NADH oxidase may be partially inhibited by
application of 25 lM DPI. Further increase in DPI con-
centrations up to 150 lM had only little effect on further
decrease of H2O2 levels, which may indicate that NADH-
oxidases play a minor role in ROS generation. However,
other enzymes (e.g. mitochondrial CIII) seem to participate
in the generation of H2O2, which is indicated by the fact
that a large fraction (58%) of H2O2 generated at 37�C is
DPI-insensitive and the heat-induced H2O2 levels gener-
ated at 44�C heat stress are completely unaffected by DPI.
Taking into account that the intracellular level of H2O2
represents a balance between production and elimination,
the heat inactivation of scavenging enzymes may be in-
volved in changing the steady state levels of H2O2 during
heat stress. It was shown that in wild type Arabidopsis
leaves the activity of APX was not changed following
treatment at 37�C, but was severely compromised at 44�C
(Panchuk et al. 2002). Superoxide dismutase and glutathi-
one reductase activities remained unchanged under these
conditions.
Plant Mol Biol (2006) 61:733–746 741
123
H2O2-dependent expression of heat-inducible genes
Our experiments show that externally added H2O2 and
other oxidative compounds are effective inducers of heat-
inducible genes, Hsp17.6, Hsp18.2, Apx1, and Apx2. The
effective concentration of H2O2 was 0.5 mM. This con-
centration is similar to the one (0.2 mM) being effective in
driving the induction of an AtHsp18.2 promoter–LUC re-
porter construct in Arabidopsis protoplasts (Kovtun et al.
2000). However, the effective concentration of H2O2 at its
cellular site is unknown, but probably much lower than the
externally applied concentration. It has been shown that the
half-life of exogenous 20 mM H2O2 is 2 min, because
Arabidopsis cultures have a high scavenging capacity
(Desikan et al. 1998, 2001). The effect of scavenging is
strongly dependent on cell growth and experimental con-
ditions. Cell cultures used in our experiments had the
capacity to reduce 0.5 mM exogenous H2O2 within 1 hour
to approximately 10%, 5 mM to about 30% (see supple-
mental Figure 4).
The induction of heat stress genes by oxidative stress
has been previously reported, however, the mRNA levels
have not been compared with those after induction by heat
stress. We have used real-time PCR quantification of
mRNA levels of Hsp17.6, Hsp18.2, Apx1 and Apx2. The
sHSP genes and Apx2, which are practically not expressed
in unstressed cells, are strongly induced to comparable
levels by moderate heat stress (37�C) or by application of
H2O2 (0.5 mM). It was previously shown that in Arabid-
opsis leaves the mRNA levels of heat-inducible sHSP and
Apx2 reach the maximum after 1–2 h treatment at 37�C
with a decline thereafter (Panchuk et al. 2002; Volkov
et al. 2003). This fast accumulation and transient expres-
sion is a signature of HSF-regulated heat shock genes
(Panchuk et al. 2002; Lohmann et al. 2004). The Apx1
expression profile is different. It shows a significant
expression at normal temperature, its mRNA level after
heat stress is only about 2-fold increased after heat stress
(Panchuk et al. 2002), and Apx1 shows a strong induction
by H2O2, which is also demonstrated for Apx2 (this paper).
Previously, photo-oxidative stress-induced Apx2 expres-
sion restricted to bundle sheath cells was found in Ara-
bidopsis leaves (Fryer et al. 2003).
H2O2 exerts a pivotal role in plant life, it is widely
recognized as a key signalling compound that can mediate
cross tolerance in plants towards other stresses (Bolwell
1999; Bowler and Fluhr 2000), but it is also a toxic com-
pound that causes detrimental effects and cell death in
plants. It is known that heat stress stimulates the accumu-
lation of H2O2 in plant cells (Foyer et al. 1997; Dat et al.
1998; Vacca et al. 2004). Conversely, transcriptome anal-
ysis of Arabidopsis tissue culture cells subjected to H2O2
revealed the induction of a number of genes with roles in
biotic and abiotic stress responses, including also several
genes encoding HSP and HSF (Desikan et al. 2001). The
expression of sHSP genes following application of H2O2
was also reported for tomato (Banzet et al. 1998) and rice
(Lee et al. 2000). Similar, upon high light stress, which
results in excessive production of H2O2, representatives of
all HSP gene families, e.g. HSP101, HSP90, HSP81,
HSP70 and sHSP, were induced in wild type Arabidopsis
(Rossel et al. 2002) and further upregulated upon high light
in Apx1 (Pnueli et al. 2003) and catalase (Vandenabele
et al. 2003) deficient mutants. It was proposed that upon
oxidative stress chaperone function of HSP may be nec-
essary to limit oxidation-mediated disulfide bridge-induced
protein aggregation (Rossel et al. 2002).
Our data show that heat treatment at 37�C has an effect
on gene expression, similar to exogenous 0.5 mM H2O2. It
was shown that this concentration did not affect viability of
Arabidopsis suspension culture cells. These data are con-
sistent with the observation that the induction of Arabid-
opsis cell death requires H2O2 concentration of more than
5 mM (Neil et al. 1999). Thus the intracellular H2O2 levels
induced by moderate heat treatment seems to be not toxic
but may play a regulatory role in the cellular network that
leads to altered gene expression the adaptation of cells to
different biotic and abiotic stresses.
Heat and oxidative stress signalling
In order to test whether oxidative stress and heat stress
induction share common components in signalling stress
responses, we have studied the influence of inhibitors and
scavengers of H2O2 production on heat-induced levels of
mRNAs. Both DPI and ascorbate exert profound negative
effects on the mRNA induction of Hsp17.6, Hsp18.2, and
Apx2. By contrast the mRNA levels of Apx1 are compro-
mised to a much lower extent. This result is a clear indi-
cation for the involvement of H2O2 in the heat stress and
HSF-dependent expression of typical heat shock genes.
Ascorbate scavenging appears to be much more potent in
blocking heat-induced expression of genes compared to
DPI (Fig. 3). This is in accordance with the higher levels of
H2O2 present after DPI treatment, which is in contrast to
ascorbate, unable to completely block heat-induced gen-
eration of H2O2 (Fig. 1B). It was also demonstrated that
programmed cell death (PCD), triggered by heat stress in
tobacco tissue culture cells, is prevented by antioxidants,
also by ascorbate (Vacca et al. 2004). This phenomenon is
linked to rapid ROS production in PCD cells, which show
an early inhibition of glucose oxidation that was accom-
panied by a strong impairment of mitochondrial function.
In our experiments application of 25–150 lM DPI or
5 mM ascorbate at 37�C reduces intracellular H2O2 to the
level in control untreated cells at 20�C. This correlates with
742 Plant Mol Biol (2006) 61:733–746
123
a decrease of Hsp17.6, Hsp18.2, and Apx2 mRNA level but
it remains considerably higher than in control cells. Only
50 mM ascorbate (which results in H2O2 level of 36%
comparing to untreated control at 20�C––see Fig. 1) is able
to reduce Apx2 expression to the control level, but mRNA
levels of both sHSP genes remain still increased. To ex-
plain the data two possibilities should be considered: (i) An
additional H2O2-independent stress signalling pathway is
active in heat-treated cells, resulting in an induction of
sHSP genes in the presence of 50 mM ascorbate; (ii) heat
shock causes a ‘‘sensitization’’ of H2O2-dependent sig-
nalling pathway allowing expression of heat shock genes in
cells with reduced H2O2 levels.
It is well known that the heat-inducible binding of
transcription factor HSF to the HSE promoter sequences
control heat stress-dependent expression of HSP genes.
According to the chaperone titration model (Morimoto
1998; Schoffl et al. 1998) in the majority of eukaryotes,
HSF are located in the cytoplasm of unstressed cells in an
inactive monomeric form as a complex with HSP70/HSP90
and probably some other proteins. Upon stress, dissociation
of these complexes and activation/trimerisation of HSF
occurs, followed by relocation in the nucleus. However,
little is known about exact molecular mechanisms of HSF
activation in plants. The question arose whether oxidative
stress (e.g. H2O2) or changes in the redox status affect HSF
activation, which is required for initiating the transcription
of target genes. Using EMSA for the identification of HSF-
HSE binding complexes we have shown that both, heat
stress and oxidative stress resulted in the formation of high
molecular weight complexes (Fig. 4), a signature of early
HSFA1a/A1b-dependent gene expression in heat-stressed
leave tissue of Arabidopsis (Lohmann et al. 2004). Fur-
thermore, HSF binding to HSE was prevented if the heat
treatment was performed in the presence of ascorbate but,
interestingly, not when supplemented with DPI (Fig. 4).
The DPI insensitivity of HSE-complex formation probably
reflects the fact that heat stress-induced H2O2 production is
not completely suppressed by DPI. Upon heat treatment at
37�C in the dark the intensity of high molecular weight
complex was practically the same both in the absence of
DPI (increased H2O2 level) and in the presence of 150 lM
DPI (H2O2 level as in control cells at 20�C), whereas no
high molecular weight complex was found at 20�C. This
indicates that upon moderate heat shock the activation of
HSF may occur in the absence of increased of H2O2 levels
(as shown after DPI treatment), although, activation of
HSF-HSE binding appears H2O2-dependent (as shown after
ascorbate treatment). Accordingly, HSF seems to be more
susceptible to H2O2-dependent activation upon heat treat-
ment as at normal temperature. It seems possible that
heat shock per se and H2O2 cooperate in HSF activation
by dissociating cytoplasmic HSF-chaperone complexes,
leading to HSF trimerization, DNA-binding, and tran-
scriptional activation. Such a cooperation would explain a
‘‘sensitization’’ of H2O2-dependent signalling that leads to
heat shock gene expression after DPI treatment at 37�C.
Interestingly, the high molecular weight HSE binding
complexes were also found in untreated light grown tissue
culture cells but rapidly disappeared when cells were
incubated in the dark. This suggests that light-dependent
ROS may activate HSF-binding, that can be rapidly re-
versed in the dark. Whether the light-dependent complex is
functional in transcriptional activation of target genes is
unknown, it may perhaps play a role in the low level basal
expression of genes.
Discrepancies between the HSF-DNA binding complex
formation and only low mRNA levels of target genes, e.g.
observed after heat stress and DPI treatment (Fig. 4), may
indicate that DNA binding and transcriptional activation
are two separate processes. This two step process is a well
known phenomenon in mammalian cells (Hensold et al.
1990; Jurivich et al. 1992) and probably also occur in plant
cells.
What is the mechanism of H2O2 in heat stress signalling
and heat shock gene expression? Our data suggest that
oxidative stress is required for effective transcription of
stress genes, which correlates with the induction of HSE-
binding activity during the early phase of the heat shock
response. It has been shown that in Drosophila and human
cells H2O2 is a potent activator of HSF trimerization and
consequently DNA-binding (Zhong et al. 1998; Ahn and
Thiele 2003). Recombinant human HSF1, but not HSF2,
has the capacity to directly sense heat and oxidative stress
in vitro (Ahn and Thiele 2003). Our data indicate that
Arabidopsis HSF may be also a subject of oxidative stress
activation. The most suitable candidates for H2O2 activa-
tion would be AtHSFA1a or AtHSFA1b, which are in-
volved in the formation of the high molecular weight HSE-
binding complexes and which seem to be able to func-
tionally replace each other in Arabidopsis (Lohmann et al.
2004). The involvement of H2O2 and HSF is further
implicated by the negative effect of a transdominant neg-
ative HSF mutant on the expression of Apx1 in Arabidospis
(Davletova et al. 2005). Alternatively, other components
(e.g., chaperones, composition yet unknown in plants) of
the inactive HSF complex, present under non-stress con-
ditions, may be targets for inactivation by oxidative stress.
Besides, other cellular proteins may be damaged by H2O2,
which will require chaperones for repair. According to the
chaperone titration model a withdrawal of chaperones from
HSF complexes, induced by a higher load of denatured
proteins, would result in trimerization and activation of
HSF (Zou et al. 1998). Hence, H2O2 generated upon heat
stress may both directly and indirectly contribute to the
activation of HSF.
Plant Mol Biol (2006) 61:733–746 743
123
Our data show that H2O2 plays an important role during
the early phase of heat shock response (up to 2 h) being
involved in activation of HSF–HSE binding and tran-
scription of heat shock genes. In contrast, the high mobility
HSE binding complex, characteristic for the later phase of
heat shock response, was not induced by application of
H2O2, BP, or DA at room temperature. Also, mRNA of
heat shock genes induced by application of H2O2 was not
effectively translated as shown by Western analysis using
antibody directed against sHSP of Arabidopsis (R.A. Vol-
kov, I.I. Panchuk, F. Schoffl, unpublished results). Never-
theless, H2O2 is required for the activation of the later
phase of heat shock response because application of
ascorbate or DPI at 37�C in concentration dependent
manner suppressed induction of the late high mobility HSE
binding complex. Hence, a combination of H2O2-depen-
dent and independent mechanisms controls the later phase.
H2O2- and HSF-independent components
of the heat shock response
Heat-inducible expression of sHSP genes, occurring in the
presence of 50 mM ascorbate, seems to be not only H2O2-
independent, but also HSF-independent because high
molecular weight HSE binding complex was not induced by
this treatment. Similar, the complex was not detected after
the treatment with DA, although, the expression of heat
shock genes was induced. It was demonstrated that DA
treatment promotes the formation of the monomer oxidised
form of human HSF1 (Manalo and Liu 2001). This intra-
molecular disulfide cross-linked conformer was resistant to
the in vitro heat-induced trimerisation and activation. The
presumptive involvement of other transcription factors in
the transcription of HSP genes is also implicated by the data
that in the hsfA1a/b double knock out mutants of Arabid-
opsis, which are unable to form high molecular weight HSE-
binding complexes, mRNA accumulation of HSF target
genes is significantly but not completely impaired upon heat
stress (Lohmann et al. 2004).
Still, little is known about transcription factors modu-
lating oxidative stress response in plants. Besides HSF,
WRKY and bZIP proteins were proposed as possible can-
didates (Vranova et al. 2002). In Arabidopsis, the zinc
finger protein Zat12 appears to be involved in Apx1
expression (Rizhsky et al. 2004). The finding that light
stress-induced Apx1 and Zat12 transcript accumulation is
inhibited in plants expressing a dominant-negative HSF21
construct suggest that HSF function is required upstream of
Zat12 and hence, that HSF function is required at a rela-
tively early stage of the oxidative stress signalling and
acclimation response (Davletova et al. 2005). Further
analysis will be required to determine the potential of
AtHSF-A1a and AtHSF-A1b, which at present are the most
likely candidates that may sense and integrate heat and/or
oxidative stress and trigger gene expression in the envi-
ronmental stress responses in Arabidopsis. The roles of
other transcription factors and alternative mechanisms
acting at transcriptional/posttranscriptional levels will be
the target of future investigations
Acknowledgements Part of this work was supported by SFB 446
funded by the Deutsche Forschungsgemeinschaft.
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