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The Combined Effect of Drought Stress and Heat Shock on Gene Expression in Tobacco 1 Ludmila Rizhsky, Hongjian Liang, and Ron Mittler* Department of Biology, Technion-Israel Institute of Technology, Technion City, Haifa 32000, Israel (L.R.); and Department of Botany, Plant Sciences Institute, Iowa State University, Room 353 Bessey Hall, Ames, Iowa 50011 (H.L., R.M.) In nature, plants encounter a combination of environmental conditions that may include stresses such as drought or heat shock. Although drought and heat shock have been extensively studied, little is known about how their combination affect plants. We used cDNA arrays, coupled with physiological measurements, to study the effect of drought and heat shock on tobacco (Nicotiana tabacum) plants. A combination of drought and heat shock resulted in the closure of stomata, suppression of photosynthesis, enhancement of respiration, and increased leaf temperature. Some transcripts induced during drought, e.g. those encoding dehydrin, catalase, and glycolate oxidase, and some transcripts induced during heat shock, e.g. thioredoxin peroxidase, and ascorbate peroxidase, were suppressed during a combination of drought and heat shock. In contrast, the expression of other transcripts, including alternative oxidase, glutathione peroxidase, phenylalanine ammonia lyase, pathogenesis-related proteins, a WRKY transcription factor, and an ethylene response transcriptional co-activator, was specifically induced during a combination of drought and heat shock. Photosynthetic genes were suppressed, whereas transcripts encoding some glycolysis and pentose phosphate pathway enzymes were induced, suggesting the utilization of sugars through these pathways during stress. Our results demonstrate that the response of plants to a combination of drought and heat shock, similar to the conditions in many natural environments, is different from the response of plants to each of these stresses applied individually, as typically tested in the laboratory. This response was also different from the response of plants to other stresses such as cold, salt, or pathogen attack. Therefore, improving stress tolerance of plants and crops may require a reevaluation, taking into account the effect of multiple stresses on plant metabolism and defense. Under optimal conditions, cellular homeostasis is achieved by the coordinated action of many biochem- ical pathways. However, different pathways may have different molecular and biophysical properties, making them different in their dependence upon ex- ternal conditions. Thus, during events of suboptimal conditions (stress), different pathways can be af- fected differently, and their coupling, which makes cellular homeostasis possible, is disrupted. This pro- cess is usually accompanied by the formation of re- active oxygen intermediates (ROIs) because of an increased flow of electrons from the disrupted path- ways to the reduction of oxygen (Halliwell and Gut- teridge, 1989; Noctor and Foyer, 1998; Asada, 1999; Dat et al., 2000; Mittler, 2002). One example for this process is the effect of heat shock on mitochondrial electron transfer. It was shown that during heat shock, membrane-bound complexes at the inner mi- tochondrial membrane are uncoupled or disrupted. Electrons from NADH produced by the soluble, and less temperature-sensitive, Krebs cycle enzymes are then channeled to the reduction of O 2 to ROI by different components of the uncoupled electron transport chain (Davidson and Schiestl, 2001). To counter the effects of stress, plants undergo a process of stress acclimation. This process may re- quire changes in the flow of metabolites through different pathways, the suppression of pathways that may be involved in the production of ROI during stress, and the induction of various defense genes such as heat shock proteins (HSPs) and ROI- scavenging enzymes (Vierling, 1991; Dat et al., 2000; Mittler, 2002). The complexity of signaling events associated with the sensing of stress and the activation of defense and acclimation pathways is believed to involve ROI, cal- cium, calcium-regulated proteins, mitogen-activated protein kinase cascades, and cross talk between differ- ent transcription factors (Liu et al., 1998; Xiong et al., 1999; Bowler and Fluhr, 2000; Knight and Knight, 2001; Kovtun et al., 2000; Chen et al., 2002). Interest- ingly, different stress conditions such as drought and cold can result in the activation of similar stress re- sponse pathways (Seki et al., 2001; Chen et al., 2002). Thus, a high degree of overlap may exist between gene clusters activated by different stresses. This over- lap may explain the well-documented phenomena of “cross tolerance,” in which a particular stress can in- duce in plants resistance to a subsequent stress that is different from the initial one (Bowler and Fluhr, 2000). 1 This work was supported by the Israeli Academy of Science, by the Hebrew University Minerva Arid Ecosystem Research Cen- ter, by The Biotechnology Council (Iowa State University), and by the fund for the promotion of research at Technion. * Corresponding author; e-mail [email protected]; fax 515– 294 –1337. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.006858. Plant Physiology, November 2002, Vol. 130, pp. 1143–1151, www.plantphysiol.org © 2002 American Society of Plant Biologists 1143 www.plantphysiol.org on July 28, 2018 - Published by Downloaded from Copyright © 2002 American Society of Plant Biologists. All rights reserved.

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The Combined Effect of Drought Stress and HeatShock on Gene Expression in Tobacco1

Ludmila Rizhsky, Hongjian Liang, and Ron Mittler*

Department of Biology, Technion-Israel Institute of Technology, Technion City, Haifa 32000, Israel (L.R.);and Department of Botany, Plant Sciences Institute, Iowa State University, Room 353 Bessey Hall, Ames,Iowa 50011 (H.L., R.M.)

In nature, plants encounter a combination of environmental conditions that may include stresses such as drought or heatshock. Although drought and heat shock have been extensively studied, little is known about how their combination affectplants. We used cDNA arrays, coupled with physiological measurements, to study the effect of drought and heat shock ontobacco (Nicotiana tabacum) plants. A combination of drought and heat shock resulted in the closure of stomata, suppressionof photosynthesis, enhancement of respiration, and increased leaf temperature. Some transcripts induced during drought,e.g. those encoding dehydrin, catalase, and glycolate oxidase, and some transcripts induced during heat shock, e.g.thioredoxin peroxidase, and ascorbate peroxidase, were suppressed during a combination of drought and heat shock. Incontrast, the expression of other transcripts, including alternative oxidase, glutathione peroxidase, phenylalanine ammonialyase, pathogenesis-related proteins, a WRKY transcription factor, and an ethylene response transcriptional co-activator, wasspecifically induced during a combination of drought and heat shock. Photosynthetic genes were suppressed, whereastranscripts encoding some glycolysis and pentose phosphate pathway enzymes were induced, suggesting the utilization ofsugars through these pathways during stress. Our results demonstrate that the response of plants to a combination ofdrought and heat shock, similar to the conditions in many natural environments, is different from the response of plants toeach of these stresses applied individually, as typically tested in the laboratory. This response was also different from theresponse of plants to other stresses such as cold, salt, or pathogen attack. Therefore, improving stress tolerance of plants andcrops may require a reevaluation, taking into account the effect of multiple stresses on plant metabolism and defense.

Under optimal conditions, cellular homeostasis isachieved by the coordinated action of many biochem-ical pathways. However, different pathways mayhave different molecular and biophysical properties,making them different in their dependence upon ex-ternal conditions. Thus, during events of suboptimalconditions (stress), different pathways can be af-fected differently, and their coupling, which makescellular homeostasis possible, is disrupted. This pro-cess is usually accompanied by the formation of re-active oxygen intermediates (ROIs) because of anincreased flow of electrons from the disrupted path-ways to the reduction of oxygen (Halliwell and Gut-teridge, 1989; Noctor and Foyer, 1998; Asada, 1999;Dat et al., 2000; Mittler, 2002). One example for thisprocess is the effect of heat shock on mitochondrialelectron transfer. It was shown that during heatshock, membrane-bound complexes at the inner mi-tochondrial membrane are uncoupled or disrupted.Electrons from NADH produced by the soluble, andless temperature-sensitive, Krebs cycle enzymes are

then channeled to the reduction of O2 to ROI bydifferent components of the uncoupled electrontransport chain (Davidson and Schiestl, 2001).

To counter the effects of stress, plants undergo aprocess of stress acclimation. This process may re-quire changes in the flow of metabolites throughdifferent pathways, the suppression of pathways thatmay be involved in the production of ROI duringstress, and the induction of various defense genessuch as heat shock proteins (HSPs) and ROI-scavenging enzymes (Vierling, 1991; Dat et al., 2000;Mittler, 2002).

The complexity of signaling events associated withthe sensing of stress and the activation of defense andacclimation pathways is believed to involve ROI, cal-cium, calcium-regulated proteins, mitogen-activatedprotein kinase cascades, and cross talk between differ-ent transcription factors (Liu et al., 1998; Xiong et al.,1999; Bowler and Fluhr, 2000; Knight and Knight,2001; Kovtun et al., 2000; Chen et al., 2002). Interest-ingly, different stress conditions such as drought andcold can result in the activation of similar stress re-sponse pathways (Seki et al., 2001; Chen et al., 2002).Thus, a high degree of overlap may exist betweengene clusters activated by different stresses. This over-lap may explain the well-documented phenomena of“cross tolerance,” in which a particular stress can in-duce in plants resistance to a subsequent stress that isdifferent from the initial one (Bowler and Fluhr, 2000).

1 This work was supported by the Israeli Academy of Science,by the Hebrew University Minerva Arid Ecosystem Research Cen-ter, by The Biotechnology Council (Iowa State University), and bythe fund for the promotion of research at Technion.

* Corresponding author; e-mail [email protected]; fax 515–294 –1337.

Article, publication date, and citation information can be foundat www.plantphysiol.org/cgi/doi/10.1104/pp.006858.

Plant Physiology, November 2002, Vol. 130, pp. 1143–1151, www.plantphysiol.org © 2002 American Society of Plant Biologists 1143 www.plantphysiol.orgon July 28, 2018 - Published by Downloaded from Copyright © 2002 American Society of Plant Biologists. All rights reserved.

Although the study of abiotic stress response hasadvanced considerably in recent years, analyzing theeffect of a single stress on plants can be very differentfrom the conditions encountered by plants in thefield in which a number of different stresses mayoccur simultaneously (Merquiol et al., 2001; Mittler etal., 2001). These can alter plant metabolism in a novelmanner that may be different from that caused byeach of the different stresses applied individually,and may require a new type of response that wouldnot have been induced by each of the individualstresses.

To characterize some of the mechanisms involvedin the response of plants to a combination of stresses,applied simultaneously, we studied the effect ofdrought and heat shock on tobacco (Nicotiana taba-cum) plants. A combination of drought and heatshock can represent the conditions encountered bymany plants and crops growing within arid andsemiarid environments (Mittler et al., 2001); there-fore, its understanding may be critical for the devel-opment of new strategies and tools to enhance stresstolerance via genetic manipulations.

RESULTS

Physiological Characterization of Drought Stress, HeatShock, and a Combination of Drought Stress and HeatShock in Tobacco

To mimic the conditions encountered by plantsduring extended periods of drought, accompanied bybrief exposures to heat shock (typically occurringbetween midday to late afternoon; Merquiol et al.,2001), we subjected tobacco plants to drought stressuntil they reached a relative water content (RWC) of65% to 70%. Plants were then exposed to a heat shocktreatment and sampled. As controls, we used well-watered plants (control), drought-stressed plants thatwere not subjected to heat shock (drought), and well-watered plants that were subjected to heat shock(heat shock). All plants were analyzed and sampledat the same time (after the heat shock treatment).Recovery tests indicated that plants subjected to acombination of drought stress and heat shock couldrecover within a few days upon watering and chang-ing of temperature to 23°C (not shown). The condi-tions used in our study, therefore, were not lethal toplants.

As shown in Figure 1, drought stress resulted in thesuppression of respiration and photosynthesis. In con-trast, heat shock resulted in the enhancement of res-piration, but did not significantly alter photosynthesis.Interestingly, the combination of drought stress andheat shock resulted in the suppression of photosyn-thesis, similar to drought stress, but the enhancementof respiration to levels that were comparable withthose measured in plants after heat shock. Measure-ments of stomatal conductance, shown in Figure 2A,indicated that heat shock is accompanied by opening

Figure 1. Measurements of photosynthesis and respiration in plantssubjected to heat shock, drought stress, and a combination of heatshock and drought stress. Plants were subjected to stresses as de-scribed in “Materials and Methods,” and photosynthetic activity anddark respiration were measured with an LI-6400 apparatus (LI-COR,Lincoln, NE). Photosynthetic activity is shown to be suppressed afterdrought stress or a combination of drought and heat shock, whereasrespiration is enhanced after heat shock and a combination ofdrought and heat shock. A combination of drought and heat shock,therefore, is different from drought or heat shock by having a highrate of respiration and a low rate of photosynthetic activity. Resultsare presented as mean and SD of five individual measurements.

Figure 2. Stomatal conductance (A) and leaf temperature (B) ofplants subjected to heat shock, drought stress, and a combination ofheat shock and drought stress. Measurements were performed asdescribed in “Materials and Methods.” The temperature of leavessubjected to a combination of drought and heat shock is shown to behigher than that of plants subjected to heat shock in the absence ofdrought. This difference may result from the inability of plants,subjected to the stress combination, to cool their leaves by transpi-ration because their stomata are closed.

Rizhsky et al.

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of stomata, probably to enable the cooling of leaves viaan enhanced transpiration stream. In contrast, stomataremained closed after drought or a combination ofdrought and heat shock, suggesting that plants sub-jected to a combination of drought and heat shockmay be unable to cool their leaves by enhanced tran-spiration. Measurements of leaf temperature, shownin Figure 2B, revealed that the leaf temperature ofplants subjected to a combination of drought and heatshock was higher by 2°C to 3°C compared with that ofplants subjected to heat shock without drought. Inaddition, measurements of leaf transpiration con-firmed that during heat shock transpiration is en-hanced, whereas during a combination of drought andheat shock, transpiration is almost completely abol-ished (not shown). The results presented in Figures 1and 2 suggest that a combination of drought and heatshock affects plants differently from drought or heatshock applied individually. The differences includedchanges in photosynthesis, respiration, stomatal con-ductance, and leaf temperature.

Molecular Characterization of Gene Expression duringDrought Stress, Heat Shock, and a Combination ofDrought Stress and Heat Shock in Tobacco

To examine the effect of drought and heat shockon gene expression in tobacco, we designed andused cDNA arrays composed of 170 cDNA clones

encoding different defense and metabolic genes.These were spotted in duplicates on nylon filtersand used to assay changes in the steady state levelof their corresponding transcripts during drought,heat shock, and a combination of drought and heatshock. Identical filters were hybridized with radio-labeled cDNAs obtained from total RNA isolatedfrom plants subjected to the different stresses. Theoverall pattern of gene expression detected by thefilter arrays was different among control, droughtstress, heat shock, and a combination of droughtstress and heat shock (not shown). A summary ofthe changes in gene expression calculated as percentof control and averaged over five different experi-ments, each analyzed individually, is shown in Ta-bles I through III. To compare the changes in ex-pression during heat shock, drought stress, and acombination of drought stress and heat shock withother stresses, we subjected plants to salt stress, coldstress, PQ application, TMV infection, treatmentwith MJ, or to the expression of bO (Mittler et al.,1995). A summary of the changes in gene expressionduring these stresses is also shown in Tables Ithrough III. As described previously, TMV infectionand bO expression result in the activation of thehypersensitive response and the enhanced genera-tion of ROI (Mittler et al., 1998). Because each ofthese additional stresses requires a different treat-ment, e.g. spraying with Tween 20 for PQ, mock

Table I. Changes in the steady-state level of transcripts encoding heat shock proteins and ROI removal enzymes

Drought and Heat Shock in Tobacco

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infection for TMV, or growth in liquid culture forsalt stress, an adequate control was designed foreach treatment. These were critical because, asshown in Figures 3 and 4, and as described previ-ously (Mittler and Zilinskas, 1992), different controltreatments alter the expression of key genes such asAPX and catalase. To confirm that these results,obtained with the cDNA arrays, adequately repre-sent changes in steady-state transcript levels, wetested the expression of nine different cDNAs byRNA blots. These, shown in Figures 3 and 4, werefound to be in good agreement with the resultspresented in Tables I through III (the results shownin Figs. 3 and 4 are from one experiment, whereasthe results shown in Tables I through III are theaverage and sd of five different experiments, forthe drought and heat experiments [n � 5], and theaverage of two different experiments each repeatedtwice for the other stresses [n � 4], including theexperiments shown in Figs. 3 and 4). Because theleaf temperature of plants subjected to drought andheat shock was higher than that of plants subjectedto heat shock in the absence of drought (Fig. 2), weperformed additional experiments of heat shock at ahigher temperature (i.e. 46°C); however, we did notfind a significant difference between the induction

of HSPs in tobacco plants subjected to heat shock at46°C or heat shock at 44°C (not shown).

Table I summarizes results obtained for cDNAsencoding different HSPs, and different ROI removalenzymes. As shown in Table I, a number of HSPswere induced during a combination of drought andheat shock. These included cytosolic HSP90, HSP70,and HSP100, and sHSPs (cytosolic, mitochondrial,and chloroplastic). Overall, the induction of HSPswas higher in drought and heat shock comparedwith heat shock or drought. Analyzing the changesin ROI removal enzymes revealed interesting differ-ences among the different stresses. During heat shock,cytosolic APX and thioredoxin peroxidase appeared tobe dominant. In contrast, during drought stress, CATand GPX appeared to be specifically induced. Duringa combination of drought and heat shock, however,AOX, GPX, glutathione reductase, CuZn-SOD, andglutathione-S-transferase were induced. Thus, thepanel of transcripts encoding ROI-detoxifying en-zymes induced during each of the different stressesappeared to be different, and ROI detoxification mayoccur via different routes during the different stresses.The induction of cytosolic APX during heat shock wasin agreement with previous reports on the presenceof a heat shock factor-binding sequence at the pro-

Table II. Changes in the steady-state level of transcripts encoding metabolic enzymes and proteins

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moter of ApxI (Mittler and Zilinskas, 1992; Storo-zhenko et al., 1998).

Changes in the steady-state transcript level of dif-ferent metabolic genes are shown in Table II. Asshown in Table II, many of the photosynthetic geneswere suppressed during stress. Exceptions weretranscripts encoding a PSI reaction center protein,the large subunit of Rubisco, and a subunit of cyto-chrome B6F. Because cyclic electron flow involvesPSI and cytochrome B6F, it is possible that duringstress some energy dissipation is obtained via thispathway. Glycolate oxidase, a key enzyme of thephotorespiratory pathway induced during drought,was suppressed during a combination of droughtand heat shock. In contrast to the suppression ofphotosynthetic genes, some transcripts encoding en-zymes of the pentose phosphate pathway and gly-colysis were induced during a combination ofdrought and heat shock. These included Glc-6-phosphate dehydrogenase and pyruvate kinase. Theinduction of these transcripts may suggest that dur-ing a combination of drought and heat shock, theflow of sugars through these pathways is enhanced,possibly for the production of reducing energy, suchas NAD(P) H, in the absence of photosynthesis. Incontrast to the suppression of transcripts involvedin photosynthesis during a combination of droughtand heat shock, transcripts encoding different com-ponents of the mitochondrial respiration pathwaywere not suppressed during a combination ofdrought stress and heat shock (Table II).

Table III summarizes changes in the expressionpattern of different stress response genes. In con-trast to drought or heat shock, a combination ofdrought and heat shock resulted in the induction ofa number of different stress response transcripts.These included transcripts encoding PR proteinsand PAL. In contrast to PR proteins that were notinduced to the same extent as during TMV infection,PAL was induced to levels that were similar to oreven higher than those found during pathogen in-fection. DHN, highly induced during droughtstress, was only moderately induced during a com-bination of drought and heat shock (Table III; seealso Fig. 3). In contrast, the induction of a differentdrought-induced protein (DI-19) was augmented bythe combination of drought and heat shock. How-ever, unlike DHN, this transcript was also inducedduring heat shock. The induction of transcripts en-coding different components of the UB protein deg-radation pathway was also elevated during a com-bination of drought and heat shock. The inductionof the different stress and pathogen response tran-scripts during a combination of drought and heatshock suggests that this combination may have ac-tivated a signal transduction pathway that is alsoactivated during wounding or pathogen infection.This activation might have resulted from the com-bined synthesis of different plant hormones such asabscisic acid, ethylene, and MJ. The expression oflipoxygenase, involved in jasmonic acid synthesis,was elevated during drought and heat shock.

Table III. Changes in the steady-state level of transcripts encoding general stress, ubiquitin, and “housekeeping” proteins

Drought and Heat Shock in Tobacco

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Expression of Stress Response Transcripts withHomology to Transcripts Isolated from the Desert PlantRetama raetam during a Combination of DroughtStress and Heat Shock in Tobacco

We recently cloned, by a subtraction cDNA cloningmethod, a number of stress response cDNAs inducedin the desert plant R. raetam in response to a combi-nation of different naturally occurring stresses, ofwhich drought and heat shock appear to be the mostprominent (Pnueli et al., 2002). To test whether ho-mologs of these transcripts are also involved in theresponse of laboratory-grown plants to a combina-

tion of stresses, we studied their expression in to-bacco plants subjected to drought, heat shock, and acombination of drought and heat shock.

As shown in Figure 5, the expression of two tran-scripts with a high degree of homology to transcriptsinduced in the desert plant, i.e. those encoding aWRKY transcription factor, and an ethylene responsetranscriptional co-activator (ERTCA), was specifi-cally induced during a combination of drought andheat shock in tobacco. The specific induction of thesetranscription factor homologs during a combinationof drought and heat shock may suggest that thiscombination is accompanied by the activation of aunique genetic program different from the programsactivated in plants during drought or heat shock. Theexpression of another transcript, i.e. a homolog PR-10, induced in the desert plant (Pnueli et al., 2002),was also induced during a combination of droughtand heat shock. However, this transcript was alsoinduced during heat shock in the absence of drought.In contrast, a homolog of a novel transcript corre-sponding to the Arabidopsis gene AC007508.2, in-duced in the desert plant (Pnueli et al., 2002), was notspecifically induced during a combination of droughtand heat shock in tobacco (Fig. 5).

DISCUSSION

We performed an initial characterization of theresponse of tobacco plants to a combination ofdrought stress and heat shock. Our results stronglysuggest that the effect of this combination on plants isvery different from that of drought or heat shockapplied individually. Because in the field or in natureplants are often subjected to a combination of stressessuch as drought and heat shock, studying the re-sponse of plants to a combination of different stressesmay be critical to our understanding of stress toler-

Figure 3. Changes in the steady-state level of transcripts encodingstress response and metabolic proteins and enzymes during a com-bination of drought and heat shock. RNA gel blots were used to assaythe steady-state level of selected transcripts during a combination ofdrought and heat shock. Many of the transcripts shown in this figurehave a distinct expression pattern during a combination of droughtand heat shock. RNA isolation, blots, and analysis are described in“Materials and Methods.”

Figure 4. Changes in the steady-state level of transcripts encodingstress response and metabolic proteins and enzymes after differentenvironmental stresses. RNA gel blots were used to assay the steady-state level of selected transcripts during different stresses. RNA iso-lation, blots, and analysis are described in “Materials and Methods.”

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ance in plants. Thus, stress combinations such asdrought and cold, heat shock and high light, ordrought and heat shock should be studied before asuccessful manipulation of plant metabolism can beachieved, to artificially enhance stress tolerance. Fu-ture studies using full-scale genome arrays con-ducted on Arabidopsis plants subjected to similarstress combinations may reveal key regulators ofgene clusters activated during a combination ofstresses. The identification of two transcripts encod-ing homologs of proteins involved in the transcrip-tional regulation of gene expression, i.e. WRKY andERTCA, specifically induced during a combination ofdrought and heat shock (Fig. 5), supports the pres-ence of key regulators involved in this response. Thefinding that a combination of drought and heat shockresults in the activation of wound and pathogen re-sponse pathways, not activated by each of thesestresses applied individually, can also be viewed asan evidence for the induction of a unique geneticprogram upon stress combination. Our results, there-fore, may provide an entry point and a reference tofuture analysis of gene expression during a combina-tion of stresses. In addition, our results can suggestpossible targets for the enhancement of stress toler-

ance in crops by genetic engineering. Thus, it may bepossible to enhance the tolerance of plants to multi-ple stresses by manipulating the expression of differ-ent enzymes of the pentose phosphate pathway,AOX, GPX, and/or homologs of the transcriptionfactors identified by our study (i.e. WRKY andERTCA).

A number of new findings were uncovered by ouranalysis. For example, a role for mitochondrial AOXand GPX in the protection of cells from ROI-relateddamage during a combination of stresses can be sug-gested. In addition, the finding that the expression ofDHN is suppressed during a combination of droughtand heat shock may suggest that during this combi-nation, HSPs can replace the stabilizing function ofDHN, and it is no longer required for drought-related cellular protection. The source of NAD(P) Hused for the removal of ROI during stress is mostlyunknown. Our results suggest that the reduction ofNAD(P)� to NAD(P) H during stress, in the absenceof photosynthesis, may occur via the pentose phos-phate pathway. This suggestion is supported by anumber of studies in animal cells and yeast (Saccha-romyces cerevisiae), linking the pentose phosphatepathway to the removal of ROI during normal me-tabolism and stress (Pandolfi et al., 1995; Juhnke etal., 1996), and by our recent findings that plants withsuppressed expression of APX and CAT have en-hanced expression of transcripts encoding enzymesof the pentose phosphate pathway (Rizhsky et al.,2002). The expression of transcripts encoding en-zymes of the pentose phosphate pathway was alsoelevated during other stresses such as PQ and salt(Table II).

Drought stress and heat shock may affect plantmetabolism in a different manner when applied in-dividually. However, it is not entirely clear how theyaffect plant metabolism when occurring simulta-neously. Our analysis suggests that the mitochondriamay be critical during a combination of drought andheat shock. During this combination photosynthesisis suppressed, whereas respiration is enhanced (Fig.1). In addition, the expression of photosyntheticgenes is suppressed, whereas the expression of genesinvolved in respiration is unchanged or induced (Ta-ble II). Moreover, the expression of mitochondrialAOX, implicated in the defense of plants frommitochondria-generated ROI during stress (Maxwellet al., 1999), is specifically elevated during a combi-nation of drought and heat shock (Table I; Fig. 3).However, the exact role of the mitochondria, asidefrom energy supply in the absence of photosynthesis,is unknown.

The response of plants to a combination of droughtand heat shock is composed of suppression of pho-tosynthesis, enhancement of respiration, induction ofa large number of defense genes, including genesinduced during pathogen defense, and changes ingenes involved in sugar metabolism. The overall bal-

Figure 5. Expression of transcripts with homology to stress responsecDNAs isolated from the desert plant R. raetam. RNA gel blots wereused to study the expression of different transcripts that hybridized tocDNAs isolated from the desert plant R. raetam subjected to acombination of drought and heat shock in its natural environment.Hybridizations were performed at a high stringency (60°C) usingfull-length R. raetam cDNA clones as described in “Materials andMethods.”

Drought and Heat Shock in Tobacco

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ance between the expression of transcripts encodingdifferent ROI removal enzymes and HSPs is alsoaltered during a combination of drought and heatshock. These changes strongly suggest that the com-bination of drought and heat shock results in theactivation of a unique genetic program that is differ-ent from that activated during drought or heat shock.Comparing the expression pattern of the differenttranscripts shown in Tables I through III between thecombination of drought and heat shock and otherstresses, such as cold, salt, PQ, or pathogen attack,suggests that the response of plants to the stresscombination is also different from the response ofplants to these stresses.

Drought and heat shock combination resulted inthe induction of at least one senescence-associatedtranscript (SAG12; Table III). An overlap in the acti-vation of at least 28 different transcription factorswas recently reported between senescence and envi-ronmental stresses such as cold, salt, and pathogenattack (Chen et al., 2002). Therefore, it is possible thatsome overlap may also exist between senescence anda combination of drought and heat shock. Interest-ingly, the study of Chen et al. (2002), although verycomprehensive, could not assign a function to a spe-cific WRKY protein, identified as the Arabidopsishomolog of NtWRKY4, also a homolog of the R.raetam WRKY used for the hybridizations shown inFigure 5. From our results (Pnueli et al., 2002; Fig. 5),it is possible that this WRKY is involved in the re-sponse of plants to a combination of stresses such asdrought and heat shock, or drought and cold stress.

MATERIALS AND METHODS

Growth Conditions and Physiological Measurements

Growth of tobacco (Nicotiana tabacum cv Xanthi-nc NN) plants and ex-periments were conducted under controlled environmental conditions at23°C or 44°C. Plants were individually potted in equal amounts of Pro-Mix,and watered with 0.5� Hoagland solution. Continuous illumination wasprovided by cool-white fluorescent lamps (150 �mol m�2 s�11). Photosyn-thetic activity, dark respiration, leaf temperature, and stomatal conductancewere measured with a LI-COR LI-6400 apparatus using the following mea-suring cell (6 cm2) parameters: 23°C or 44°C, 150 �mol photons m�2 s�1, andan air flow of 300 �L s�1, as previously described (Mittler et al., 2001). RWCwas determined as described by Mittler and Zilinskas (1994).

Stress Treatments

Heat shock was applied by raising the temperature in the growth cham-ber to 37°C for 1 h, followed by another increase to 44°C for 6 h. Droughtstress was imposed by withdrawing water from plants until they reached aRWC of 65% to 70% (typically 6–7 d). A combination of drought and heatshock was performed by subjecting drought-stressed plants (RWC of 65%–70%) to the heat hock treatment. All plants, i.e. drought-stressed plants,well-watered plants subjected to heat shock, drought- and heat-shockedplants, and control well-watered plants kept at 23°C were sampled at thesame time for analysis. Cold stress was imposed by changing the tempera-ture in the growth chamber to 4°C for 48 h. Control plants were kept at 23°C.Mock TMV infection plants expressing the bO gene and treatment of plantswith MJ were performed as described previously (Mittler et al., 1998). PQtreatment was performed as described by Mittler and Zilinskas (1992). Saltstress was induced by subjecting 7-d-old tobacco seedlings, grown in culturein a medium containing 0.5� Hoagland, to 250 mm NaCl for 3 d. Control

seedlings were grown in the same culture media without NaCl. For allstresses, control and stressed tissue were sampled at the same time.

RNA Isolation and RNA Gel Blots

Total RNA was isolated as previously described (Mittler et al., 1998) andsubjected to RNA gel-blot analysis (Mittler and Zilinskas, 1992). A probe for18S rRNA was used to ensure equal loading of RNA. Hybridization condi-tions were as follows: 0.25 m Na2HPO4, 1 mm EDTA, 7% (w/v) SDS, and 1%(w/v) casein (pH 7.4) at 60°C to 65°C, overnight, and washes were at 1� SSCand 0.1� SSC in the presence of 0.1% (w/v) SDS.

Filter Array Hybridization

Clones for the production of filter arrays were ordered from the tomato(Lycopersicon esculentum) expressed sequence tag library at Clemson Univer-sity (SC), or obtained from the laboratories of Drs. Dirk Inze (University ofGent, Belgium), Barbara A. Zilinskas (Rutgers University, NJ), Pierre Golou-binoff (Hebrew University, Jerusalem, Israel), and Gadi Schuster (Technion,Haifa, Israel). Filter cDNA arrays were prepared from the clones by spottingPCR products in duplicates on nylon membranes at the Hadassah MedicalSchool DNA Facility of the Hebrew University. Filters were hybridized withradiolabeled cDNAs prepared from total RNA isolated from the differentplants using oligo-dT and Superscript reverse transcriptase (LifeTechnologies/Gibco-BRL, Cleveland) as suggested by the manufacturer.Hybridization conditions were as follows: 57°C, 5� SSC, 5� Denhart, 0.5%(w/v) SDS, and 100 �g mL�1 salmon sperm DNA, overnight. Washingconditions were as follows: 57°C, 2� SSC, and 0.1% (w/v) SDS for 20 min,followed by 0.2� SSC and 0.1% (w/v) SDS, 57°C, for 20 min. After hybrid-ization and washes, the signals were assayed with a phosphor imager(BAS1000, Fuji Photo Film, Tokyo) and analyzed with TINA software (Ray-test, Pittsburgh). A number of control “housekeeping” genes, animal-specific genes (as negative controls), and empty spots (for background) werealso spotted on the membrane. These were used to normalize the intensityof signals between the different filters and calculate the changes in geneexpression presented in Tables I through III. When pertinent, the expressionlevel of specific genes was verified by RNA blots.

ACKNOWLEDGMENTS

We thank Drs. Dirk Inze, Barbara A. Zilinskas, Pierre Goloubinoff, andGadi Schuster for gift of cDNA clones. We also thank Dr. Daniel Goldenbergfor his help with preparing filter arrays.

Received April 7, 2002; returned for revision May 17, 2002; accepted July 5,2002.

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