Plant Mol Biol (2006) 61:733746 DOI 10.

1007/s11103-006-0045-4

Heat stress-induced H2O2 is required for effective expression of heat shock genes in Arabidopsis
Roman A. Volkov Irina I. Panchuk f Phillip M. Mullineaux Friedrich Scho

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, exemplied by Hsp17.6, Hsp18.2, and the two cytosolic ascorbate peroxidase genes Apx1, Apx2, reached similar levels by moderate heat stress (37C) or by treatment with H2O2, butylperoxide and diamide at room temperature. The heat-induced expression levels were signicantly reduced in the presence 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 response, was blocked by ascorbate and DPI. H2O2 was unable to induce this complex suggesting that H2O2 is involved only in the early stages of HSF activation. Signicant induction of the genes tested after diamid treatment and moderate expression of the sHSP genes in the presence of 50 mM ascorbate at 37C occurred without activation of HSF, indicating that other mechanisms may be involved in stress signalling. Keywords Ascorbate peroxidase DPI Heat shock factor Heat shock protein Hydrogen peroxide Oxidative stress

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 f (&) R. A. Volkov I. I. Panchuk F. Scho r Molekularbiologie der Panzen Allgemeine Zentrum fu t Tu bingen, Auf der Morgenstelle 28, 72076 Genetik, Universita bingen, Germany Tu e-mail: friedrich.schoef@zmbp.uni-tuebingen.de P. M. Mullineaux Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK R. A. Volkov I. I. Panchuk Department of Molecular Genetics and Biotechnology, University of Chernivtsy, Kotsubinsky str. 2, 58012, Ukraine

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 classied into a number of families based on their molecular 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

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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; f et al. 1998). Despite the ubiquitous nature of this Scho 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 activated by stress for a specic binding to heat shock promoter elements (HSE). In Arabidopsis 21 different HSF genes have been identied (Nover et al. 2001), but only few have been functionally characterized. AtHsf1 (HSFA1a) and AtHsf3 (HSF-A1b) are the regulators, which become activated very early in the heat shock response and are necessary for efcient 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 signicantly 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 identied as targets of HSF regulation in HSF3-transgenic plants (Panchuk et al. 2002; Panikulangara 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 oxidative 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 superoxide 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 mitochondria, 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 ow of metabolites, suppression of pathways 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. Arabidopsis plants that have acquired enhanced levels of thermotolerance experienced lower levels of oxidative damage during recovery from heat stress as compared with nonconditioned plants and this protection from oxidative damage 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 ndl et al. 1998). This suggests that one aspect et al. 1995; Pra 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 identication 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). Especially, 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

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protein kinases (MAPKs) that modulate gene expression and transduce cellular responses to extracellular stimuli (Desikan 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 efcient inducer of sHSP mRNA expression. On the other hand we show that heat stress-induced expression of HSP can be counteracted by diphenyleneiodonium chloride (DPI), an inhibitor of avin-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-activation and signalling.

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 ascorbate, DPI, or oxidative compounds were added to the nal concentrations as indicated and cells were incubated in a shaking water bath (60 strokes min-1) in the dark at 20, 37 or 44C, respectively. The following concentrations were tested: ascorbate5, 20, 50 mM; DPI0.5, 10, 25 and 150 lM; H2O20.05, 0.5, 5 and 50 mM, tert-butylperoxide (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

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 lumen m)2) at 20C with shaking (60 strikes min)1). Exponentially growing cells (34 days old) were used for experiments. Before treatments, cell density was adjusted with fresh MS medium to OD 660 = 0.12 and pre-incubated 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 nal concentration of 0.04%, incubated 10 15 min at room temperature and monitored under microscope. 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 37C or 44C for 2 h in a shaking water bath in the dark. Controls were incubated at 20C under otherwise identical condition. Following the treatments samples were immediately assayed for quantications of H2O2 levels, or Arabidopsis (ecotype Columbia 24) plants were cultivated as described by Panchuk et al. (2002). Leaves of sevenweek-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 20C or 37C, respectively. Ascorbate, DPI, or oxidative compounds were added to the nal concentrations as indicated. Measurement of intracellular H2O2 accumulation Intracellular accumulation of ROS (e.g. H2O2) was monitored using in vivo oxidation of carboxy-HDCFDA (56-carboxy-2,7-dichlorodihydrouorescein diacetate: Molecular Probes) uorescence probe (Royall and Ischiropoulos 1993). Five millilitre aliquots of Arabidopsis cell culture were supplemented with carboxy-HDCFDA (end concentration 10 lM) and incubated for 5 min at 20C for the preloading of cells with the uorescence 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 nal concentration of 0.3 mM. Cells were destroyed by sonication (1 min, 30 W) using Sonier B-12 (Branson Sonic Power Company, Dunbury, Connecticut) and uorescence was measured using uorescence 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 accumulation of H2O2 were analyzed for statistical signicance according to t-test (Engel 1997). Intracellular character of oxidation of uorescence probe (Royall and Ischiropoulos

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1993) was conrmed in control experiments applying catalase 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 quantication 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 proximal 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-specic primers for real-time RT-PCR quantication 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 specicity. The identity of PCR products was conrmed by direct DNA-sequencing. Quantitative real-time RT-PCR Quantication of gene-specic cDNA was performed by real-time PCR monitoring the intercalation of SYBRGreen (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 prepared for two samples of Arabidopsis cells. Using standardized 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 quantication of mRNA levels is based on the comparison to the level of an Act2 mRNA standard, dened 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 signicance 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 centrifuged at 7000 g, 10 min, 4C. The supernatant was cleared again at 21,000 g, 20 min, 4C, 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 puried with QIAquick Nucleotide Removal Kit (Qiagen) and used for EMSA. Samples containing 20 lg protein (1012 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 unspecic 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 0.5TBE (44.5 mM trisboric 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 lm.

Results Heat stress increases H2O2 level in Arabidopsis We have monitored the effect of moderate (37C) and severe (44C) 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 uorescence probe (Royall and Ischiropoulos 1993). The viability of cells in suspension culture, grown under optimal 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

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medium and pre-cultivated prior to application of stress. To avoid light-dependent effects the heat stress was administered in the dark. In addition to heat stress, we investigated also the inuences 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 increase in H2O2 levels: approximately 2.3-fold at 37C and 2.5-fold at 44C within 1-hour treatment. The accumulation of H2O2 is a very fast process, occurring within the rst 15 min of heat stress. Longer exposure, as measured during the rst, the second, and fourth hour of continuous heat stress, resulted in no further increase, rather in a decrease of H2O2 levels. DPI, supplemented at a concentration of 25 lM had a negative effect on the accumulation of H2O2 at 20C and especially at 37C, but not at 44C (Fig. 1B). Higher DPI concentrations of up to 150 lM resulted in only a minor further decrease of the H2O2 level at 20C and 37C. Supplementation of ascorbate caused much stronger negative effects on H2O2 levels at all temperatures tested (Fig. 1B). At a concentration of 50 mM the reduction was approximately 4-, 7- and 5-fold at 20, 37 and 44C, respectively. The effect was dosage-dependent: 25 mM caused a larger reduction than 5 mM ascorbate. In contrast to DPI, ascorbate was also very efcient in reducing the H2O2 levels at normal temperature and at 44C. These data show that heat stress enhances intracellular 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 application 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 exemplify 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 identied as novel HSFdependent heat shock genes, which are signicantly induced 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 20C and compared them with the levels in untreated (20C) or heat-treated (37C or 44C) 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 afnity to cellular membranes. DA is a synthetic SH-groups-oxidizing compound, which changes intracellular redox status and promotes protein disulde 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: approximately 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, 9598% of cells survived after the treatments. In cell culture, several genes tested were induced by moderate heat stress (37C) whereas no induction was

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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 uorescence probe was measured during 15 min (015 min), or 1 h (01 h) immediately after beginning of treatment, or from 1 to 2 h (12 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 44C. All treatments were performed in the dark. Bars show means SD (n = 45)

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738 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 quantied by realtime RT-PCR. Expression levels are represented in comparison to the expression of actin2 mRNA standard, which was dened as 100 relative expression units (REU). Bars show means SD (n = 46). Note that different scales are used in graphs. BP, tertbutylperoxide; DA, diamide

Plant Mol Biol (2006) 61:733746

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found after severe heat treatment (44C) similar to the response observed in Arabidopsis leaves (Panchuk et al. 2002; Volkov et al. 2003). It should be noted that 44C heat stress is lethal to unconditioned Arabidopsis plants (Wunderlich et al. 2003; Lohmann et al. 2004). The current data on tissue culture show that at 20C H2O2, BP, and DA are potent inducers of mRNA expression of the heatinducible genes tested (Fig. 2). The magnitude of mRNA induction by H2O2, BP, or DA, and by moderate heat stress (37C) 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 37C. The optimal concentrations for the induction of the two sHSP genes are 0.5 mM H2O2, 0.55 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 37C; DA-induced levels reach approximately only 50% of the heat-induced levels. The induction factors range between approximately 100-fold for Hsp17.6, 3001000-fold for Hsp18.2 and 1025-fold for Apx2. The expression prole of Apx1 differed from the patterns 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 induction) by heat stress at 37C, (iii) a wider range of effective concentrations of all three different compounds. In common with Apx2 were the higher levels of oxidative stressinduced 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 obtained 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. Interestingly, no signicant changes were detected for mRNA levels of microsomal Apx3 and Apx5. These data show that oxidative compounds, in particular H2O2, cause an efcient increase in transcript levels of sHSP genes. The transcript levels reached, as exemplied by Hsp17.6 and Hsp18.2, approximately the same levels as induced by heat stress. The mRNA expression of Apx1 and Apx2 is more efciently induced by oxidative stress compared to heat stress. H2O2 is required for efcient heat induction of mRNA levels If an increase in H2O2 level is required for heat stressactivated gene expression, it could be expected that inhibition of production or scavenging of ROS should exert profound negative effects on heat-induced mRNA levels. Therefore we tested the inuence 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 20C.

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Plant Mol Biol (2006) 61:733746 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 quantied by realtime RT-PCR. Expression levels are represented in comparison to the expression of actin2 mRNA standard, which was dened as 100 relative expression units (REU). Bars show means SD (n = 46). Note that different scales are used in graphs

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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 20C or DMSO-treated cells (control of the DPI solvent) after heat stress. The heat stress-dependent mRNA expression prole of Apx1 differs from the patterns of the other genes by: (i) Minor but statistically insignicant negative effects after ascorbate treatment, (ii) only a moderate reduction (about 50%) of expression levels after DPI treatment. These data demonstrate that ROS, especially H2O2, are necessary for heat-induced gene expression 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 sevenweak-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 37C 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 maximal 633, 125, 3.2 and 86, respectively. However, in contrast to the cell culture, Hsp17.6, Hsp18.2 and Apx2 mRNA levels induced by heat stress at 37C were respectively 3.0fold, 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 necessary to achieve the maximal induction, and BP was a more effective inducer than H2O2 at 5 mM concentration. Supplementation of the incubation medium with 50 mM

ascorbate during heat stress of leaves at 37C 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 reect the better penetration of compounds in the suspension culture cells and probably tissue-specic differences in leaves. Taken together the data show that the Arabidopsis 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 signalling pathways. The analysis of HSF knock out mutants provided evidence that at least two different HSF, HSFA1a and HSF-A1b (originally designated HSF1 and HSF3 f et al. 1994; Pra ndl et al. 1998), are bel and Scho by Hu involved in the control of Hsp17.6 and Hsp18.2 transcription 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 identied by electrophoretic mobility shift analyses (EMSA). Therefore, we examined the potential of oxidative compounds to induce the formation of heat stressspecic 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

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complexes were formed at 20C as compared to heattreated (37C, 15 min) cells. Ascorbate but not DPI treatment 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 20C, whereas DA treatment was unable to induce this complex; the HSE-binding complex was also not induced at 44C (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 HSFA1a and A1b, which have been identied 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 complex 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 37C, but it was not formed in H2O2-treated cells at 20C, 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 signicantly 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 HSFbinding complex formation correlate well with the intracellular 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 37C in the presence or absence of 0.5 mM hydrogen peroxide, 50 mM ascorbate or 150 lM DPI. (C) Effect of different concentrations of DPI on the induction of DNA-HSF complexes. The stress inducible DNA-HSF complexes specic 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 20C

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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 reect the situation in planta. In our experiments we used exponentially growing Arabidopsis cell culture, which represents a suspension 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 37C, (ii) no induction at 44C, (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 convenient 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 20C indicate tissue specicity of stress response. In order to reduce the complexity of signalling interference 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 rst 15 min with a subsequent decline during longer exposure to heat stress. This response is reminiscent of the oxidative burst occurring after pathogen attack (Desikan et al. 1996; Clarke et al. 2000). Our experiments demonstrate that in Arabidopsis cells the level of H2O2 signicantly increases, 2.32.5 fold, both after moderate (37C) and respectively severe (44C) heat stress. Similar, in mustard seedlings subjected to severe heat stress (55C, 1.5 h) in the dark the level of endogenous H2O2 increased by 65% in comparison with plants grown at 24C (Dat et al. 1998). Severe heat stress (at 60 65C) 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 protonpumping 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-specic nonpumping internal NADPH dehydrogenase, NDin(NADPH) (Moller 2001). Heat treatment at 41C 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 dehydrogenases, NDE1 and NDE2 (Davidson and Schiestl 2001). These two proteins functionally substitute the proton-pumping complex CI present in mitochondria of the majority of eukaryots (Moller 2001). In order to identify possible ROS generating mechanisms in Arabidopsis cells upon heat stress in the dark, we tested the effect of DPI, an inhibitor of avoenzymes (ODonnell 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 2002). affected even by application of 100 lM DPI (Morre 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 effect on H2O2 levels, generated by 25 lM DPI, indicates that CI could be the main site of ROS production at 37C. However, it should be noted, that intracellular concentration 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 concentrations up to 150 lM had only little effect on further decrease of H2O2 levels, which may indicate that NADHoxidases 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 37C is DPI-insensitive and the heat-induced H2O2 levels generated at 44C 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 involved 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 37C, but was severely compromised at 44C (Panchuk et al. 2002). Superoxide dismutase and glutathione reductase activities remained unchanged under these conditions.

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H2O2-dependent expression of heat-inducible genes Our experiments show that externally added H2O2 and other oxidative compounds are effective inducers of heatinducible genes, Hsp17.6, Hsp18.2, Apx1, and Apx2. The effective concentration of H2O2 was 0.5 mM. This concentration is similar to the one (0.2 mM) being effective in driving the induction of an AtHsp18.2 promoterLUC reporter 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 conditions. 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 supplemental 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 quantication 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 (37C) or by application of H2O2 (0.5 mM). It was previously shown that in Arabidopsis leaves the mRNA levels of heat-inducible sHSP and Apx2 reach the maximum after 12 h treatment at 37C with a decline thereafter (Panchuk et al. 2002; Volkov et al. 2003). This fast accumulation and transient expression is a signature of HSF-regulated heat shock genes (Panchuk et al. 2002; Lohmann et al. 2004). The Apx1 expression prole is different. It shows a signicant 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 expression restricted to bundle sheath cells was found in Arabidopsis 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 compound that causes detrimental effects and cell death in plants. It is known that heat stress stimulates the accumulation of H2O2 in plant cells (Foyer et al. 1997; Dat et al. 1998; Vacca et al. 2004). Conversely, transcriptome analysis 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) decient mutants. It was proposed that upon oxidative stress chaperone function of HSP may be necessary to limit oxidation-mediated disulde bridge-induced protein aggregation (Rossel et al. 2002). Our data show that heat treatment at 37C 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 consistent with the observation that the induction of Arabidopsis 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 inuence 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 compromised to a much lower extent. This result is a clear indication 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 generation 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 accompanied by a strong impairment of mitochondrial function. In our experiments application of 25150 lM DPI or 5 mM ascorbate at 37C reduces intracellular H2O2 to the level in control untreated cells at 20C. This correlates with

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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 20Csee Fig. 1) is able to reduce Apx2 expression to the control level, but mRNA levels of both sHSP genes remain still increased. To explain 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 signalling 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 f et al. 1998) in the majority of eukaryotes, 1998; Scho 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 identication of HSFHSE 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). Furthermore, 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 reects the fact that heat stress-induced H2O2 production is not completely suppressed by DPI. Upon heat treatment at 37C 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 20C), whereas no high molecular weight complex was found at 20C. 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 treatment 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 transcriptional activation. Such a cooperation would explain a sensitization of H2O2-dependent signalling that leads to heat shock gene expression after DPI treatment at 37C. 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 reversed 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 HSEbinding 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 activation would be AtHSFA1a or AtHSFA1b, which are involved in the formation of the high molecular weight HSEbinding complexes and which seem to be able to functionally 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 negative 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 conditions, 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.

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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 HSFHSE binding and transcription 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 f, unpublished results). Neverkov, I.I. Panchuk, F. Scho theless, H2O2 is required for the activation of the later phase of heat shock response because application of ascorbate or DPI at 37C in concentration dependent manner suppressed induction of the late high mobility HSE binding complex. Hence, a combination of H2O2-dependent 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 H2O2independent, 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 intramolecular disulde 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 Arabidopsis, which are unable to form high molecular weight HSEbinding complexes, mRNA accumulation of HSF target genes is signicantly but not completely impaired upon heat stress (Lohmann et al. 2004). Still, little is known about transcription factors modulating oxidative stress response in plants. Besides HSF, WRKY and bZIP proteins were proposed as possible candidates (Vranova et al. 2002). In Arabidopsis, the zinc nger protein Zat12 appears to be involved in Apx1 expression (Rizhsky et al. 2004). The nding 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 relatively 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 environmental 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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