Isolation of four heat shock protein cDNAs from grapefruit peel tissue

and characterization of their expression in response to heat and chilling

temperature stresses

Dafna Rozenzviega,1, Cengiz Elmacib,1, Alon Samachc, Susan Luriea and Ron Porata,*

aDepartment of Postharvest Science of Fresh Produce, Agricultural Research Organization, the Volcani Center, PO Box 6, Bet Dagan50250, IsraelbUludag University, Faculty of Agriculture, Department of Animal Science, Gorukle Campus 16059, Bursa, TurkeycThe Kennedy-Leigh Centre for Horticultural Research, Institute for Plant Sciences and Genetics, Faculty of Agricultural, Food andEnvironmental Quality Sciences, the Hebrew University of Jerusalem, Rehovot 76100, Israel1These authors contributed equally to this work.*Corresponding author, e-mail: rporat@volcani.agri.gov.il

Received 24 December 2003; revised 1 February 2004

In order to continuously supply horticultural products for

long periods, it is essential to store them after harvest inlow temperatures. However, many tropical and subtropical

fruits and vegetables, such as citrus, are sensitive to chilling.

In previous studies, the authors have shown that a short hot

water rinsing treatment (at 62�C for 20 s) increased chillingtolerance in grapefruit. In order to gain more insight into

the molecular mechanisms involved in heat-induced chilling

tolerance, PCR cDNA subtraction analysis was performed

which isolated four different PCR fragments whose expres-sion was enhanced 24 h after the heat treatment, and that

showed high sequence homology with various plant HSP18-I,HSP18-II, HSP22 and HSP70 genes. It was found that theshort hot water treatment given at 62�C for 20 s, but not at

lower temperatures of 20 or 53�C, increased the expressionof the various HSP cDNAs in grapefruit peel tissue. How-

ever, when the fruits were kept at ambient temperatures, theincreases in HSP mRNA levels following the hot water

treatment were temporary and lasted only between 6 and

48 h. Similar temporary increases in the HSP mRNA levels

were detected following exposure of the fruit to a hot air

treatment at 40�C for 2 h. Nevertheless, when the fruits weretreated with hot water but afterwards stored at chilling

temperatures of 2�C, the mRNA levels of the various

HSP18-I, HSP18-II, HSP22 and HSP70 cDNAs increasedand remained high and stable during the entire 8-week cold-storage period, suggesting their possible involvement in heat-

induced chilling-tolerance responses. The chilling treatment

by itself increased the expression of the HSP18-I cDNA, buthad no effect on the mRNA levels of any of the other HSPcDNAs. Exposure of fruit to other stresses, such as wound-

ing, UV irradiation, anaerobic conditions and exposure to

ethylene, had no effect on the expression of the variousHSPs. Overall, the study explored the correlation between

the expression and persistence of various HSP cDNAs in

grapefruit peel tissue during cold storage, on the one hand,

and the acquisition of chilling tolerance, on the other hand,and the results suggest that HSPs may play a general role in

protecting plant cells under both high- and low-temperature

stresses.

Introduction

Post-harvest heat treatments are used commercially inorder to reduce decay development, to enhance chillingtolerance, to inhibit ripening and to eradicate insects(Lurie 1998). Recently, we showed that a short hotwater rinsing and brushing treatment (HWB) at 62�Cfor 20 s, increased chilling tolerance and pathogen

resistance in grapefruits (Porat et al. 2000a, b). Theobserved heat-induced increase in pathogen resistancewas temporary and lasted for only 1–3 days after theheat treatment, whereas the increase in chilling tolerancepersisted for up to 6 weeks of cold storage at 2�C (Poratet al. 2000b).

PHYSIOLOGIA PLANTARUM 121: 421–428. 2004 doi: 10.1111/j.1399-3054.2004.00334.x

Printed inDenmark – all rights reserved Copyright#PhysiologiaPlantarum2004

Abbreviations – HSPs, heat shock proteins; HWB, hot water rinsing and brushing; smHSPs, small HSPs.

Physiol. Plant. 121, 2004 421

Preliminary observations showed that the hot watertreatment-induced heat-shock protein (HSP) accumula-tion in the flavedo (the outer pigmented layer of the fruitpeel tissue). Thus, although the heat treatment was veryshort, it was sufficient to induce a heat shock, and toinitiate biochemical changes in the flavedo layer (Pavoncelloet al. 2001). Moreover, we recently found that applicationof the hot water treatment followed by cold storage alsoinduced the expression of two different grapefruit dehydrincDNAs and of a sodium proton exchanger gene, whichsuggested that the hot water treatment activated variousstress-responsive genes in grapefruit peel tissue (Porat et al.2002a, b, 2003).

To gain more insight into the molecular mechanismsinvolved in the regulation of heat-induced chilling-tolerance responses in grapefruit, we used PCR cDNAsubtraction analysis to isolate four different PCR fragmentswhose expressionwas enhanced 24h after the heat treatment,and which showed high sequence homology with variousplant HSP18-I, HSP18-II, HSP22 and HSP70 genes.

HSPs comprise a diverse group of proteins, ranging inmolecular weight from 15 to 115 kDa, that are expressedin all organisms in response to elevated temperatures,and are believed to play a major role in thermotolerance(Howarth and Ougham 1993, Parsell and Lindquist1993). In plants, HSPs function as molecular chaperonesand assist in protein folding, assembly and transport,and in directing damaged proteins towards proteolysis.Overall, it has been suggested that HSPs protect otherproteins from denaturation and aggregation followingexposure to high temperatures (Vierling 1991, Bostonet al. 1996, Waters et al. 1996, Schoffl et al. 1998).

The expression of HSPs is normally regulated by heatstress, although some HSPs are also constitutivelyexpressed. In addition, the expression of HSPs may bedevelopmentally regulated, regardless of exposure to heat,as during the late stages of seed development (Wehmeyerand Vierling 2000). Apart from the effects of heat, it hasbeen reported that the mRNA levels of various HSPsmay also increase following exposure to low chillingtemperatures; thus, HSPs may have chaperone activityunder low-temperature stress (Li et al. 1999, Sung et al.2001).

In the present study, we isolated four different HSPcDNAs from grapefruit peel tissue, and showed thattheir expression was induced temporarily by heat alone,but continuously when the heat treatment was followedby low-temperature storage, which suggested the possibleinvolvement of these HSPs in heat-induced chilling-tolerance responses. The possible roles of HSPs in theacquisition of high- and low-temperature stress tolerancein grapefruit are discussed below.

Materials and methods

Plant material and post-harvest storage

Grapefruits (Citrus paradisi, cv.0star Ruby0) wereobtained from local orchards and used for the experi-

ments shortly after harvest. For cold-storage experi-ments, the fruits were held at 2�C for up to 8 weeksunder a relative humidity of approximately 90%. Forall other experiments, the fruits were held at 20�C.

Hot water treatment

Hot water treatments at 62�C for 20 s were applied byrinsing fruits as they moved along a set of brush rollers,as described previously (Porat et al. 2000b).

UV irradiation, wounding, low oxygen and ethylene

treatments

Fruits were exposed to UV irradiation by placing themunder a germicidal low-pressure vapour lamp with anemission wavelength peak at 254 nm, for 10min. Thewounding was performed by gently scratching the fruitsexternal surface with a metal brush to a depth of 0.5mm.Exposure to anaerobic stress was carried out by placingthe fruits in airtight 30-l barrels and afterwards flushingout the air with pure N2 for 10min until the O2 concen-tration was below 0.5%. Finally, the fruits were exposedto ethylene by placing them in sealed 30-l plastic tanksand adding pure ethylene to obtain a final concentrationin air of 10 ml l�1. The ethylene concentrations within thetanks were determined daily with a Varian 3300 gaschromatograph (Varian, Palo Alto, CA, USA).

Isolation of RNA

For each RNA sample, flavedo tissue was collected fromfive different fruits. Total RNA was isolated by phenol/chloroform extraction and precipitation with LiCl,according to standard procedures (Sambrook et al.1992). Poly(A)1 RNA was isolated from total RNA byusing the PolyATract mRNA isolation systems Kit(Promega, Madison, WI, USA).

PCR cDNA subtraction analysis

PCR cDNA subtraction was performed with the ClontechPCR-SelectTM. cDNA Subtraction Kit (Clontech, PaloAlto, CA, USA) according to the manufacturer’s instruc-tions. In general, the subtraction analysis was performedwith cDNA synthesized from 2mg of Poly(A)1 RNAisolated from either control (driver) or HWB-treatedfruit (tester). The final subtracted cDNA fragments werecloned into the pGEM-T Easy vector (Promega).

Sequence analysis

Sequencing was performed by the dideoxynucleotidechain termination method, with an ABI automaticsequencer (Perkin Elmer, Foster City, CA, USA). Thevarious cDNA nucleotide sequences were verified byrepeated sequencing in both sense and antisense direc-tions. Sequence analysis and multiple sequence alignment

422 Physiol. Plant. 121, 2004

(PILEUP) were performed by means of the GeneticsComputer Group’s (GCG) (University of Wisconsin,Madison, WI) and BLAST computer programs (Altschulet al. 1997).

RNA gel blot analysis

Total RNA (10 mg per lane) was separated on a 1%formaldehyde gel and blotted onto a Hybond-N1 mem-brane (Amersham Pharmacia Biotech, Little ChalfontBuckinghamshire, UK). Afterwards, blots were hybrid-ized with the various HSP cDNA probes, labelled tohigh specific activity by random priming (BiologicalIndustries Ltd, Bet Haemek, Israel) with 50 mCi of[32P]dCTP at 42�C in a mixture comprising 50% form-amide, 5�SSC (1�SSC is 0.15M NaCl and 0.015Msodium citrate), 0.05M phosphate buffer, pH6.5,5�Denhardt’s solution (1�Denhardt’s solution is0.02% Ficoll, 0.02% PVP and 0.02% BSA),0.1mgml�1 sheared denatured salmon testes DNA(Sigma, St Louis, MO, USA), and 0.2% SDS. After a16-h hybridization period, the blots were washed threetimes for 20min at 63�C with 0.5�SSC and 0.1% SDS,and exposed to an autoradiography film at �80�C for1–2 days. In all cases, the Northern blots were repeatedtwice, using different RNA extractions.

Results

Isolation of four grapefruit HSP cDNA fragments

In order to isolate ‘Star Ruby’ grapefruit genes respon-sive to the hot water treatment, we performed PCRcDNA subtraction analysis with RNA constructed after24 h from control (driver) and hot water-treated fruits(tester). Sequencing and subsequent nucleotide BLASTsearches of approximately 60 randomly chosen PCRcDNA fragments from the subtracted library revealedthat four different cDNAs shared high sequence hom-ology with various plant HSP genes: three cDNA frag-ments encoded predicted polypeptides homologous tovarious small HSPs (smHSPs) (cytosolic HSP18-I andHSP18-II and chloroplast HSP22), and the fourthcDNA fragment encoded a predicted polypeptide hom-ologous to a high-molecular-weight HSP70 (Fig. 1). Over-all, the various isolated grapefruit HSP18-I, HSP18-II,HSP22 and HSP70 cDNA fragments were 168, 303, 201and 276 bp in length, and encoded predicted polypep-tides of 56, 101, 67 and 92 amino acids, respectively(Fig. 1). The grapefruit HSP18-I polypeptide shared 96,96, 95 and 76% homology with soy, rice, pea and fra-garia HSP18-I proteins, respectively; the grapefruitHSP18-II polypeptide shared 96, 97, 90 and 90% hom-ology with almond, tomato, soy and sunflower HSP18-IIproteins, respectively; the grapefruit HSP22 polypeptideshared 90, 90, 87 and 87% homology with soy, pea,Arabidopsis and tomato HSP22 proteins, respectively;and the grapefruit HSP70 polypeptide showed 96, 96,

95 and 92% homology with apple, cucumber, Arabidop-sis and rice HSP70 proteins, respectively (Fig. 1).

Regulation of HSP gene expression

To confirm the possible regulation of HSP gene expres-sion by the hot water treatments, as observed followingthe PCR cDNA subtraction analysis, we performedRNA gel blot hybridization studies at various timepoints following the heat treatment. Preliminary resultsshowed that when the fruits were kept at room tempera-ture, the mRNA levels of the various HSPs temporarilyincreased 24 h after the heat treatment but subsequentlydeclined (Fig. 2). On the basis of these findings, we exam-ined the expression patterns of the various HSPs atshorter times after application of the heat treatment, inmore detail, and found that the mRNA levels of allHSPs markedly increased 6 h after the hot water treat-ment, and remained high for up to 48 h (Fig. 3). After72 h, the mRNA levels of all HSPs had decreased moreor less to their initial, pre-HWB-treatment levels (Fig. 3).It should be noted that whereas the transcript levels ofthe smHSPs (HSP18-I, HSP18-II and HSP22) could notbe detected at all without the application of the heattreatment, the HSP70 gene was constitutively expressedat low levels, regardless of the heat treatment (Fig. 3).Nevertheless, although the HSP70 gene has constitutiveexpression, application of the hot water treatmentmarkedlyincreased the accumulation of the HSP70 mRNA aboveits basic, pre-treatment levels (Fig. 3).

As the various HSPs were strongly expressed 24h afterthe application of the hot water treatment, we took obser-vations at this time point in order to assess the importanceof the temperature of the hot water rinse for the inductionof HSP gene expression. It was found that the mRNAlevels of the smHSPs (HSP18-I, HSP18-II and HSP22)specifically increased only following the 20-s HWB treat-ment at 62�C but not at lower temperatures of 20 or 53�C(Fig. 4). On the other hand, the high-molecular-weightHSP70 gene was consistently expressed, but its mRNAlevels were also enhanced following the hot water treat-ment at the somewhat lower temperature of 53�C (Fig. 4).The hot water treatment at the highest temperature of62�C was the only one that enhanced the chilling toleranceof the fruits (Porat et al. 2000b).

To compare the inductive effects of the hot watertreatment (20 s at 62�C) with that of a ‘regular’ heatstress that could occur under normal field conditions,we exposed the fruits to hot air at 40�C for 2 h andevaluated the various HSP gene expression patterns.The results show that the mRNA levels of all HSPsmarkedly increased 2 h after the hot air treatment, andthat the mRNA levels of the smHSPs remained high forup to 24 h after the hot air treatment (Fig. 5). Again, theHSP70 mRNA levels were constitutively expressed evenwithout the hot air treatment (Fig. 5).

In order to assess the possible involvement of thevarious HSPs in the acquisition of heat-induced chillingtolerance by grapefruit, we stored the fruits at 2�C for up

Physiol. Plant. 121, 2004 423

to 8 weeks, with or without a pre-storage hot watertreatment, which enhanced chilling tolerance. The resultsshow that exposure of the fruit to chilling alone some-what increased HSP18-I mRNA levels in grapefruitflavedo, but did not affect the transcript levels of any ofthe other HSPs: HSP18-II, HSP22 and HSP70 cDNAs(Fig. 6). On the other hand, the pre-storage hot watertreatment markedly increased the mRNA levels of all ofthe HSPs in the fruits during the entire 8-week cold-storage period, in correlation with the induction of chil-ling tolerance (Fig. 6).

Besides heat stresses, exposure of grapefruits to otherstress conditions, such as to wounding, UV irradiationand low oxygen, as well as to the plant hormone ethyl-

ene, had no inductive effect at all on the various HSPsexpression patterns (Fig. 7). Exposure to these stressesindeed stimulated stress responses in the fruit peel tissue,since they activated the expression of other known stressgenes, such as b-1,3-endoglucanase (Fig. 7).

Discussion

Several recent genomic studies have revealed the exist-ence of similarities in gene expression patterns, follow-ing exposure of plant tissues to various temperatures,salinity and drought stresses, indicating extensive cross-talk between their signalling pathways (Iba 2002, Sekiet al. 2002, Sung et al. 2003). One such group of genes,

Fig. 1. Alignment of the amino acid sequences of grapefruit HSP18-I, HSP18-II, HSP22 and HSP70 polypeptides with other plant HSPs.Amino acids that are identical in at least three of the four different proteins are shaded. The GenBank accession numbers of the grapefruitHSP18-I, HSP18-II, HSP22 and HSP70 cDNAs are AY261672, AY261671, AY261673 and AY261674, respectively.

424 Physiol. Plant. 121, 2004

that was hypothesized to play a common role in plantstress tolerance, comprises the HSPs (Sabehat et al.1998a, Hoekstra et al. 2001, Seki et al. 2002, Sung et al.2003). Indeed, several studies performed with transgenic

plants found that overexpression of HSPs increased ther-motolerance (Lee et al. 1995, Malik et al. 1999), as wellas salt and drought stress tolerance (Sugino et al. 1999,Sun et al. 2001). Furthermore, in some cases it wasreported that the mRNA levels of various HSPs increasefollowing exposure to other stresses, such as low tem-peratures or dehydration. Therefore, HSPs may share acommon role in protecting plant cells under variousstress conditions (Li et al. 1999, Hoekstra et al. 2001,Sung et al. 2001).

Fig. 5. Effects of a hot air treatment (40�C for 2h) followed bystorage at 20�C on HSP mRNA levels in grapefruit peel tissue. Eachlane contained 10mg of total RNA isolated 0, 2, 6, 24 and 72h afterthe fruit had been held at 20�C. Blots were probed with the HSP18-I,HSP18-II,HSP22 and HSP70 cDNAs. Hybridization to a 26S rRNAprobe served as a control for equal loading of RNA among lanes.

Fig. 4. Effects of different hot water rinsing temperatures on HSPmRNA levels in grapefruit peel tissue. Fruits were untreated, orwere rinsed and brushed at 20, 53 or 62�C for 20 s and afterwardskept at 20�C for 24 h. Each lane contained 10 mg of total RNA.Blots were probed with theHSP18-I,HSP18-II,HSP22 andHSP70cDNAs. Hybridization to a 26S rRNA probe served as a control forequal loading of RNA among lanes.

Fig. 3. Effects of a short hot water treatment (62�C for 20 s)followed by storage at 20�C, on HSP mRNA levels in grapefruitpeel tissue. Each lane contained 10mg of total RNA isolated 0, 6, 12,24, 48 or 72 h after the fruit had been held at 20�C. Blots wereprobed with the HSP18-I, HSP18-II, HSP22 and HSP70 cDNAs.Hybridization to a 26S rRNA probe served as a control for equalloading of RNA among lanes.

Fig. 2. Effects of a short hot water treatment (62�C for 20 s)followed by storage at 20�C onHSPmRNA levels in grapefruit peeltissue. Each lane contained 10 mg of total RNA isolated 0, 1, 3 or7 days after the fruit had been held at 20�C. Blots were probed withthe HSP18-I, HSP18-II, HSP22 and HSP70 cDNAs. Ethidiumbromide staining of the RNA gels was used to ensure equal loadingamong lanes.

Physiol. Plant. 121, 2004 425

In the present study, we performed PCR cDNA sub-traction analysis in order to isolate heat-induced genesthat may be involved in the acquisition of chillingtolerance in grapefruit, and cloned four cDNA fragmentsthat showed high sequence homology with various plantHSP18-I, HSP18-II, HSP22 and HSP70 genes. Theactual increase in these HSP mRNA levels 24 h afterthe heat treatment (as observed following the cDNAsubtraction analysis) was further confirmed by RNAgel blot hybridizations (Figs 2–4).

Nevertheless, we found that the heat-induced increasein HSP mRNA levels was temporary when the fruitswere held at 20�C, and could be detected only between6 and 48 h after HWB or hot air treatments (Figs 3 and 5).On the other hand, unlike the temporary effect elicitedby the heat treatment alone, the increases in the variousHSP mRNA levels induced by the combination of heatwith subsequent cold storage was much longer lasting,and was maintained during the entire 8 weeks of coldstorage (Fig. 6). Therefore, the main question that arisesfrom this work is whether the various HSP cDNAsthat are induced by the hot water treatment and thatpersist during cold storage are also involved in the acqui-

sition of heat-induced chilling tolerance, or whether theirpersistence was simply due to a decrease in their mRNAturnover rates during cold storage. The fact thatincreases in the various HSP mRNA levels during coldstorage were observed in chilling-tolerant, heat-treatedfruits but not in chilling-sensitive control fruits (Fig. 6),raises the possibility that HSPs might form a componentof the overall molecular mechanisms associated with theheat-induced, chilling-tolerance responses. Furthermore,the mRNA levels of other stress genes recently isolatedby us, such as the cor15 and cpDHN dehydrin cDNAs,declined during cold storage at 2�C (Porat et al. 2002a,2003), indicating that the increase and persistence of theHSP mRNA levels were probably not just a matter oflow turnover rates under low temperatures, since themRNA levels of other genes did decline.

The increases in the various HSP mRNA levels in heat-treated fruits during cold storage could not be explainedsimply as a general stress response, since the expressionsof most of them were not affected at all by cold storagealone (Fig. 6). Furthermore, the HSP mRNA levels werenot affected either by other stresses, such as wounding,UV irradiation, ethylene or exposure to anaerobic condi-tions (Fig. 7), which indicates that HSP cDNAsresponded specifically to heat and, especially, to the com-bination of heat and subsequent cold storage.

Our findings regarding the increased levels of the vari-ous HSP mRNAs and their persistence in grapefruitpeel tissue, following exposure to heat and subsequentcold storage, in contrast to their rapid decline at ambienttemperatures are in agreement with the findings of otherstudies, in avocado fruits and in apple cell suspensioncultures (Woolf et al. 1995, Wang et al. 2001). In tomatofruits, it was found that the mRNA levels of two HSPs(tom66 and tom111) increased following a hot air treat-ment but decreased and then rose again during subse-quent cold storage, indicating that the combination ofheat with subsequent chilling probably actively inducedHSP gene expression and did not merely slow its mRNAturnover rates (Sabehat et al. 1998b).

The exact roles of HSPs in imparting chilling toleranceto plants are not yet clearly understood. However, asmentioned above, HSPs exhibit general chaperone activ-ities, such as maintaining proteins in their folded orunfolded state, minimizing aggregation of proteins, andtargeting aggregated proteins for degradation andremoval from the cell, and these functions are actuallyessential not only during heat stress but also followingexposure to any other stresses (Hoekstra et al. 2001,Sung et al. 2001, 2003). Regarding the acquisition ofchilling tolerance by plants: a detailed comparison oftranscriptome profiles after transfer of Arabidopsisplants to chilling temperatures revealed that a large num-ber of genes involved in protein synthesis, proper foldingand processing are related to low-temperature acclima-tion (Provart et al. 2003). These last findings match ourpresent results, which suggests that induction of HSPsmight be part of the overall mechanism of thermal induc-tion of chilling tolerance in grapefruit.

Fig. 6. Effects of cold storage, with or without a pre-storage hotwater treatment (62�C for 20 s), on HSP mRNA levels in grapefruitpeel tissue. Each lane contained 10mg of total RNA isolated 0, 1, 2,3, 4, 6 or 8weeks after storage at a low temperature of 2�C. Blotswere probed with the HSP18-I, HSP18-II, HSP22 and HSP70cDNAs. Hybridization to a 26S rRNA probe served as a control forequal loading of RNA among lanes.

426 Physiol. Plant. 121, 2004

Acknowledgements – This research was partially funded by grantsfrom the European Union, FAIR CT-4096, and from the ChiefScientist Ministry of Agriculture of Israel, 406-0813-03. Thepresent paper is a contribution from the Agricultural ResearchOrganization, the Volcani Center, Bet Dagan, Israel, no. 421/03,2003 series.

References

Altschul SF, Madden TL, Schiffer AA, Zhang J, Zhang Z, Miller W,Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs. Nucl AcidsRes 25: 3389–3402

Boston RS, Vitanen VP, Vierling E (1996) Molecular chaperons andprotein folding in plants. Plant Mol Biol 32: 191–122

Hoekstra FA, Golovina EA, Buitink J (2001) Mechanisms of plantdesiccation tolerance. Trends Plant Sci 6: 431–438

Howarth CJ, Ougham HJ (1993) Gene expression under tempera-ture stresses. New Phytol 125: 1–26

Iba K (2002) Acclimative response to temperature stress in higherplants: approaches of gene engineering for temperature toler-ance. Annu Rev Plant Biol 53: 225–245

Lee HJ, Hubel A, Schoffl F (1995) Derepression of the activity ofgenetically engineered heat shock factor causes constitutivesynthesis of heat shock proteins and increased thermotolerancein transgenic Arabidopsis. Plant J 8: 603–612

Li QB, Haskell DW, Guy CL (1999) Coordinate and non-coordinateexpression of the stress 70 family and other molecular chaperonesat high and low temperature in spinach and tomato. Plant MolBiol 39: 24–34

Lurie S (1998) Postharvest heat treatments of horticultural crops.Hortic Rev 22: 91–121

Fig. 7. Effects of wounding, UV irradiation, low oxygen, and exposure to ethylene on HSP mRNA levels in grapefruit peel tissue. Each lanecontained 10mg of total RNA isolated after exposure to the various stresses followed by 0, 1, 2, 3 or 6 days at 20�C. Blots were probed with theHSP18-I,HSP18-II,HSP22 andHSP70 cDNAs. Hybridization to a 26S rRNA probe served as a control for equal loading of RNA among lanes.

Physiol. Plant. 121, 2004 427

Malik K, Slovin JP, Hwang CH, Zimerman JL (1999) Modifiedexpression of a carrot small heat shock protein gene, Hsp17.7,results in increased or decreased thermotolerance. Plant J 20:89–99

Parsell DA, Lindquist S (1993) The function of heat shock proteinsin stress tolerance: degradation and reactivation of damagedproteins. Annu Rev Genet 27: 437–496

Pavoncello D, Lurie S, Droby S, Porat R (2001) A hot watertreatment induces resistance to Penicillium digitatum and pro-motes the accumulation of heat shock and pathogenesis-relatedproteins in grapefruit flavedo. Physiol Plant 111: 17–22

Porat R, Pasentsis K, Rozenzvieg D, Gerasopoulos D, Falara V,Samach A, Lurie S, Kanellis AK (2003) Isolation of a dehydrincDNA from orange and grapefruit citrus fruit that is specificallyinduced by the combination of heat followed by chilling tem-peratures. Physiol Plant in press

Porat R, Pavoncello D, Lurie S, McCollum TG (2002a) Identi-fication of a grapefruit cDNA belonging to a unique classof citrus dehydrins and characterization of its expressionpatterns under temperature stress conditions. Physiol Plant115: 598–603

Porat R, Pavoncello D, Ben-Hayyim G, Lurie S (2002b) A heattreatment induced the expression of a Na1/H1 antiport gene incitrus fruit. Plant Sci 162: 957–963

Porat R, Pavoncello D, Peretz J, Ben-Yehoshua S, Lurie S (2000a)Effect of various heat treatments on the induction of chillingtolerance and postharvest qualities of ‘Star Ruby’ grapefruit.Postharv Biol Technol 18: 159–165

Porat R, Pavoncello D, Peretz J, Weiss B, Daus A, Cohen L,Ben-Yehoshua S, Droby S, Lurie S (2000b) Induction of resist-ance to Penicillium digitatum and chilling injury in Star Rubygrapefruit by a short hot water rinse and brushing treatment.J Hortic Sci Biotechnol 75: 428–432

Provart NJ, Gil P, Chen W, Han B, Chang H, Wang X, Zhu T(2003) Gene expression phenotypes of Arabidopsis associatedwith sensitivity to low temperatures. Plant Physiol 132: 893–906

Sabehat A, Weiss D, Lurie S (1998a) Heat-shock proteins andcross-tolerance in plants. Physiol Plant 103: 437–441

Sabehat A, Lurie S, Weiss D (1998b) Expression of small heat shockproteins at low temperature: a possible role in protecting againstchilling injuries. Plant Physiol 117: 651–658

Sambrook J, Fritsh EF, Maniatis T (1992) Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Laboratory, ColdSpring Harbor, NY

Schoffl F, Prandl R, Reindl A (1998) Regulation of the heat shockresponse. Plant Physiol 117: 1135–1141

SekiM,NarusakaM, Ishida J, Nanjo T, FujitaM,OonoY,KamiyaA,Nakajima M, Enju A, Sakurai T, Satou M, Akiyama K, Taji T,Yamaguchi-Shinozaki K, Carninci P, Kawai J, Hayashizaki Y,Shinozaki K (2002) Monitoring the expression profiles of 7000Arabidopsis genes under drought, cold and high-salinity stressesusing a full-length cDNA microarray. Plant Cell 31: 279–291

Sugino M, Hibino T, Tanaka Y, Nii N, Takabe T (1999) Over-expression of DnaK from a halotolerant cyanobacterium Apha-nothece halophytice acquires resistance to salt stress in transgenictobacco plants. Plant Sci 146: 81–88

SunW, Bernard C, Van De Cotte B, VanMuntaguM, Verbruggen N(2001) At-HSP17.6A, encoding a small heat shock protein inArabidopsis, can enhance osmotolerance upon overexpression.Plant J 27: 407–415

Sung DY, Kaplan F, Lee KJ, Guy CL (2003) Acquired tolerance totemperature extremes. Trends Plant Sci 8: 179–187

Sung DY, Vierling E, Guy C (2001) Comprehensive expressionprofile analysis of the Arabidopsis HSP70 gene family. PlantPhysiol 123: 789–800

Vierling E (1991) The roles of heat shock proteins in plants. AnnuRev Plant Physiol Plant Mol Biol 42: 579–620

Wang CY, Brown JH, Weir IE, Allan AC, Ferguson IB (2001) Heatinduced protection against death of suspension cultures of applefruit cells exposed to low temperature. Cell Stress Environ 24:1199–1207

Waters ER, Lee GJ, Vierling E (1996) Evolution, structure andfunction of the small heat shock proteins in plants. J Exp Bot47: 325–338

Wehmeyer N, Vierling E (2000) The expression of small heat shockproteins in seeds responds to discrete developmental signals andsuggests a general protective role in desiccation tolerance. PlantPhysiol 122: 1099–1108

Woolf AB, Watkins CB, Bowen JH, Lay-Yee M, Maindonald JH,Ferguson IB (1995) Reducing external chilling injury in stored‘Hass’ avocados with dry heat treatments. J Am Soc Hort Sci120: 1050–1056

Edited by C. Guy

428 Physiol. Plant. 121, 2004