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International Journal of Fruit Science

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The Effects of Heat Treatment on the GeneExpression of Several Heat Shock Protein Genes inTwo Cultivars of Strawberry

Reginald Brown, Hehe Wang, Melanie Dennis, Janet Slovin & William W.Turechek

To cite this article: Reginald Brown, Hehe Wang, Melanie Dennis, Janet Slovin & William W.Turechek (2016): The Effects of Heat Treatment on the Gene Expression of Several Heat ShockProtein Genes in Two Cultivars of Strawberry, International Journal of Fruit Science, DOI:10.1080/15538362.2016.1199996

To link to this article: http://dx.doi.org/10.1080/15538362.2016.1199996

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The Effects of Heat Treatment on the Gene Expression ofSeveral Heat Shock Protein Genes in Two Cultivars ofStrawberryReginald Browna, Hehe Wanga, Melanie Dennisb, Janet Slovinb,and William W. Turecheka

aU.S. Department of Agriculture, Agricultural Research Service, U.S. Horticultural Research Unit, FortPierce, Florida, USA; bU.S. Department of Agriculture, Agricultural Research Service, GeneticImprovement of Fruits and Vegetables, Beltsville, Maryland, USA

ABSTRACTHeat treatment has been shown to be an effective method forreducing systemic pathogens in strawberry but the processoften has adverse effects on plant health. Research hasshown that a brief heat treatment of plants at a lower tem-perature prior to the main heat treatment can induce heatshock proteins, which serve to protect the plant from damagewhen treated at higher temperatures. The objective of thisstudy was to determine the relative gene expression of twoheat shock factors (HSFs) and eight heat shock proteins (HSPs)in two strawberry cultivars (Festival and Ventana) known tohave differential tolerance to heat. Strawberry plants weretreated at 37 °C for 1 hour to induce the heat shock response.Total RNA was extracted and reverse transcription polymerasechain reaction (RT-PCR) was used to determine the amount oftarget produced. Relative gene expression was determinedusing the 2−ΔΔct method. Results showed that transcripts ofone HSF and five HSPs were significantly more abundant incv. Festival (p < 0.05) but transcripts from only one gene,sHsp15.96, were significantly more abundant in cv. Ventana.Results of this study have identified gene candidates that mayconfer heat tolerance in strawberry, which may be useful forselecting heat tolerant plants in breeding programs.

KEYWORDSFragaria × ananassa;Xanthomonas fragariae;angular leaf spot; diseasemanagement

Introduction

Heat treatment is a common practice for eliminating endophytic and sys-temic infections in plants (Turechek and Peres, 2009; Uchansji et al., 2004). Ithas been shown to significantly reduce or eliminate bacterial infections inseveral crops, including apple, cherry, grape (Burr et al., 1989; Hall et al.,2002; Keck et al., 1995). The key to employing heat treatment successfully isidentification of temperatures capable of killing the pathogen that will notadversely affect the health of the plant. Unfortunately, it is often difficult to

CONTACT William W. Turechek William.Turechek@ars.usda.gov U.S. Department of Agriculture,Agricultural Research Service, U.S. Horticultural Research Unit, 2001 S. Rock Road, Fort Pierce, FL 34945.

INTERNATIONAL JOURNAL OF FRUIT SCIENCEhttp://dx.doi.org/10.1080/15538362.2016.1199996

This article not subject to US copyright law.

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find a temperature that does both (Lurie and Mitcham, 2007). It is possible,however, to increase a plant’s basal level of tolerance to heat by inducing theheat shock response.

In strawberry (Fragaria × ananassa), angular leaf spot (ALS) caused byXanthomonas fragariae is a particularly costly disease in nursery production(Maas et al., 1995). As the name implies, symptoms of the disease are mostprominent on the foliage, but infection of the calyces is common and canaffect marketability of the berries. Earlier studies have shown that ALS can bereduced by heat treating plants at 44 °C for 4 h or 48 °C for 2 h, but somecultivars show damage in the form of slower growth or stunting (Turechekand Peres, 2009). In order to apply the desired heat treatment for ALScontrol, we investigated applying a more moderate elevated temperature(e.g., 37 °C) to plants before subjecting them to the higher temperaturesknown to be lethal to the target pathogen. The lower-temperature treatmentstep contributed to better plant growth following the higher-temperaturetreatment. It was hypothesized that the heat treatment at a lower temperatureinduced a heat-shock response, which protected plants from the secondtreatment at higher temperature. In the heat-shock response, normal geneexpression is partially repressed in favor of the expression of genes that resultin the synthesis of special proteins known as heat-shock proteins (HSPs)(Larkindale et al., 2005).

HSPs constitute a class of proteins that are evolutionarily conservedamong organisms indicating the critical function of these proteins. In plants,these proteins are localized in the cytosol and organelles, including thechloroplast, mitochondria, peroxisomes, and the endoplasmic reticulum,and they serve to protect the cell during periods of stress (Wang et al.,2004). To date, HSPs in plants have been categorized into five evolutionarilyconserved groups based on their functions as molecular chaperones and theirmolecular masses: HSP100s, HSP90s, HSP70s, HSP60s, and the small heatshock proteins (sHSPs) (Krishna, 2003). Plants are unique in that theyproduce many different types of sHSPs, ranging from 17–30 kD, with sub-classes specific to the cytoplasm or each of the organelles (Waters, 2013).

The functions of the most prominent HSPs are known. For example,HSP70 protects the tertiary conformation of proteins caused by heat stressand assists in the refolding of such proteins upon denaturation, precludingirreversible loss of protein function (Garavaglia et al., 2009). Likewise,HSP90, which is constitutively expressed in many organisms, also functionsin protein stabilization during heat stress. However, unlike other classes ofheat shock proteins, HSP90 shows binding specificity, binding only to pro-tein substrates in their native conformations (Xu et al., 2011). Moreover,many of these protein substrates are implicated in several high priorityintracellular events, including gene expression regulation, signaling path-ways, and cell cycle control (Wang et al., 2004). A much less characterized

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group of HSPs are the sHSPs. Unlike the other groups of HSPs, the smallheat shock proteins have no known enzymatic functions (Wang et al., 2004),but they have been shown to confer heat tolerance in different phytosystems(Chauhan et al., 2012; Malik et al., 1999).

Expression of all heat shock proteins is regulated by a group of transcrip-tion factors, the heat shock factors (HSFs), that bind to a conserved element[the heat shock element (HSE)] found in the promoter of many genes,including the heat shock proteins that are up-regulated in response to heat(Scharf et al., 2012). There are three classes of HSFs in plants, of which classA is the best studied and these proteins are well known for their role inpositive regulation of HSP gene expression. In contrast to class A HSFs, classB HSFs mostly serve as repressors of gene expression. The function of class CHSFs remains largely unknown (Scharf et al., 2012).

The purpose of this study was to determine whether there is a difference inexpression of several HSP and two HSF genes in two cultivars of strawberry(Festival and Ventana) known to have differential tolerance to heat treatment(Turechek and Peres, 2009). Preliminary studies showed that a heat treat-ment at 37 °C for 1 h followed by a cool-down period for 1 h at roomtemperature resulted in heat tolerance to subsequent treatments at highertemperatures, 44 °C for 4 h or 48 °C for 2 h in the strawberry cultivars,Festival and Ventana; however, cv. Festival was generally more tolerant (i.e.,suffering less damage) than cv. Ventana. Based on the earlier observation, wehypothesized that although the expression of heat shock proteins will be up-regulated in both cultivars at 37 °C, the expression of these genes will begreater in the more tolerant cv. Festival.

Materials and methods

Induction of heat shock proteins

Strawberry cultivars, Ventana and Festival, were obtained from LassenCanyon Nursery (Redding, CA, USA) and stored until use at 4 °C at theUnited States Department of Agriculture’s Horticulture Research Laboratoryin Fort Pierce, FL. To induce the heat-shock response in the treatment group,we heat treated bare-root plants using the following protocol. Six plants percultivar in Ziploc bags were removed from 4 °C refrigeration and allowed toacclimate to room temperature for 1 h in the bags. Plants were then indivi-dually placed in stomacher bags and the bags of plants were rolled tightly toremove as much excess air as possible before being sealed with stomacher bagclips. Three of these bags were submerged in a 37 °C water bath for 1 h. Toprevent the bags from floating to the surface, donut-shaped flask weightswere placed on top of them. After removal from the water bath, the plantswere set at room temperature for 1 h. For controls, the remaining three

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plants per cultivar stayed in stomacher bags at room temperature for 2 h. Theexperiment was conducted seven times.

Primer design

Primer pairs designed for quantitative (q)polymerase chain reaction (PCR)amplification of gene-coding regions of several heat shock protein and heatshock factors in strawberry, and primer pair sequences for the reference geneswere designed and are shown in Table 1. Primers were designed from Fragariavesca gene sequences obtained by using BLAST (Altshul et al., 1990) to search theF. vesca reference genome (Shulaev et al., 2011) with Arabidopsis HSP and HSFgenes. The Arabidopsis sequences were obtained from TAIR (Lamesch et al.2011). Of the strawberry homologs identified, eight HSP genes from differentfamilies (HSP90, HSP70, and sHSPs) and two HSF genes were chosen for thestudy based on their expression in response to heat as determined from expressedsequence tag (EST) data (Rivarola et al., 2011). F. vesca sequences for the genes tobe assayed were retrieved from the F. vescaWhole Genome v1.1 Assembly at the

Table 1. Primer pairs designed for qPCR amplification of gene-coding regions of several heatshock protein and heat shock factors in strawberry.z

Gene annotationy Gene IDy Primer sequence

Hsp90 01495 F5′-CTGGATGAGGAAGCCAGAAG-3′R5′-AAAGGTGCTCTTCCCAGTCA-3′

Hsp70 29595 F5′-CTTCCGTCGCTTACACT-3′R5′-AGAAGGTGTTCTCCGGGTTC-3′

Hsp70 26629 F5′-ATTGCTGCCCTTAATGTGCT-3′R5′-CCAACACTGGTTGCCTTCTT-3′

HsfA-4a 23802 F5′-ATGGGAATTTGCGAATGATG-3′R5′-CAGGTTCGGCAAAGAATGAC-3′

HsfA-6b 20347 F5′-CAAGCTGTAGGCCCTTGTGT-3′R5′-CCATCATCAGAACCTGCTTG-3′

sHsp25.1 12739 F5′-TGTCACCAATGCGGACTATG-3′R5′-CACCTCCTGATCGGCTTCTT-3′

sHsp24.5 08959 F5′-CAGGGCACTCTGACTGTGAA-3′R5′-GAGAACCTCCTCCCTCCATC-3′

sHsp17.4 15418 F5′-TCTGGATGCTCCAATCTTGAC-3′R5′-TTGAAGGTCTTCTCGGCATC-3′

sHsp16 11408 F5′-GACTGCCGGGAAATCAGTAG-3′R5′-CCATTCTCCACCTGAGCTTT-3′

sHsp15.96 07764 F5′-CTCCCTGAGAATGCCAAGAC-3′R5′-GGTGTCCTAGCCTCCACCTT-3′

UBQ 09659 F5′-GACCATTACCCTGGAGGTTGAGAG-3′F5′-CCCACCACGAAGCCTGAGC-3′

DBPx 08438 F5′-TTGGCAGCGGGACTTTACC-3′R5′-CGGTTGTGTGACGCTGTCAT-3′

zPrimer pairs for the two reference genes are listed. F and R represent the forward and reverse primers,respectively.

yGene annotations and IDs were obtained from Fragaria vesca Whole Genome v1.1 Assembly andAnnotation at the GDR (https://www.rosaceae.org/species/fragaria/fragaria_vesca/genome_v1.1).

xThe primer sequences of DBP were retrieved from the study of Mehli et al. (2004).

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Genome Database for Rosaceae (https://www.rosaceae.org/species/fragaria/fragaria_vesca/genome_v1.1) (Jung et al., 2014) and gene specific primer pairswere designed using Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/primer3/)(Table 1). Specific amplification was supported by melting curve analysis asdescribed below. The PCR efficiency of each primer pair was calculated fromstandard curves constructed with serial 10-fold dilutions of cDNA samples.

RNA extraction

Total RNA was extracted from crown tissue using the RNeasy Plant Mini Kit(Qiagen, Valencia, CA, USA) according to the manufacturer’s instructionswith several modifications. For each extraction (single plant), 200 mg ofcrown tissue was ground to a powder in liquid nitrogen and transferred toa pre-chilled 2-mL micro-centrifuge tube. RLT lysis buffer master mix (550µL) containing 4% (w/v) polyvinylpyrrolidone (PVP-40, Sigma-Aldrich, St.Louis, MO, USA) was immediately added and the tube was vortexed vigor-ously. PVP-40 is used to remove polysaccharides and polyphenolic com-pounds that irreversibly bind to RNA upon oxidation, adversely affectingdownstream applications (John, 1992). The lysate was transferred to a QIAshredder spin column in a collection tube and centrifuged for 2 min at 22,000g. The supernatant was mixed with a half volume of ethanol (100%) bypipetting in a new micro-centrifuge tube and transferred to an RNeasyMini spin column. Three successive wash steps were carried out with theaddition of 700 µL of buffer RW1 (wash 1) and 500 µL of buffer RPE (washes2 and 3) to the RNeasy spin column, discarding the flow-through after eachwash. The samples were incubated at room temperature for 5 min betweeneach successive wash. Following incubation the samples were centrifuged for15 s at 8000 g. The RNeasy spin columns were transferred to a new 2-mLmicro-centrifuge tube and centrifuged at 22,000 g for 1 min. RNA was elutedwith 50 µL of nuclease-free water and the samples were incubated at roomtemperature for 30 min before centrifugation at 8000 g for 1 min. Afterelution, the quantity and purity of the RNA were determined using aNanodrop 2000 (Thermo Scientific, Wilmington, DE, USA). RNA was storedon ice if moving to the protocol below or stored in a –80 °C freezer untilfurther use.

DNase treatment of RNA samples

RNA samples were further processed with the RQ1 RNase-Free DNase Kit(Promega, Madison, WI, USA) to enzymatically hydrolyze any genomicDNA contamination. A 60-µL reaction contained 5 µg of RNA based onNanodrop readings, 1x RQ1 RNAse-free DNase Reaction Buffer, and 2.5 Uof RQ1 RNase-Free DNase. The reaction was incubated at 37 °C for 30

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min. After incubation, 6 µL of RQ1 DNase stop solution was added toterminate the reaction, followed by incubation at 65 °C for 10 min toinactivate the DNase. The samples were immediately placed on ice whenproceeding with the cDNA synthesis reaction or stored at –80 °C untilfurther use.

Reverse transcription of RNA samples (cDNA synthesis)

Reverse transcription (RT) was performed using the iScript cDNA SynthesisKit (Bio-Rad, Hercules, CA, USA). A 90-µL RT reaction contained: 5 µg ofDNase-treated RNA, 1x iScript reaction mix, and 5 µL of iScript reversetranscriptase. The reactions were incubated in an MJ Mini thermocycler(Bio-Rad) set to the following program: 5 min at 25 °C, 30 min at 42 °C,and 5 min at 85 °C followed by a 4 °C hold step.

qPCR reaction

qPCR assays were carried out using the KAPA SYBR FAST qPCR Kit (KAPABiosystems, Philadelphia, PA, USA). The qPCR reactions were run in tripli-cate using the iQ5 iCycler qPCR platform from Bio Rad (Hercules, CA,USA). A 20-µL reaction master mix contained: 1x KAPA SYBR FASTUniversal mix, forward and reverse primers at a final concentration of 300nM each, and 2 µL of template cDNA. The qPCR conditions were: 95 °C for3 min, 40 cycles of 95 °C for 10 s, 55 °C for 45 s, followed by a melting curveanalysis at 95 °C for 1 min, 55 °C for 1 min, and a ramping cycle from 55 °Cto 95 °C in 0.5 °C increments of 10 s each (Wang et al., 2010). To normalizethe quantity of RNA among samples, we used ubiquitin (UBQ) and a DNA-binding protein (DBP, Mehli et al., 2004) as reference genes.

Data analysis

Relative gene expression was determined using the 2−ΔΔCt method (Livak andSchmittgen, 2001). The method calculates, as a ratio, the expression of thetarget gene of interest relative to the expression of a reference gene. Theexpression ratio is calculated as: amount of target = [(2ΔCt

target]/[(2ΔCt

ref],where 2 represents 100% PCR efficiency of both target and reference genesand ΔCt is the difference in Ct values between the control and treatedsamples. The 2ΔCt

ref was calculated from the geometric mean of the rawratios of the two reference genes. Differences in cultivar gene expressionratios were analyzed by fitting generalized linear mixed models to the datausing the GLIMMIX procedure of SAS (SAS Institute, Cary, NC, USA).Replication was treated as a random effect, and a gamma error distributionand log link function were specified. Parameters were estimated using

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maximum likelihood estimation based on the Laplace approximation(METHOD = LAPLACE).

Results and discussion

The target genes in this study were chosen based on their up-regulatedexpression upon heat treatment in F. vesca (Rivarola et al., 2011). In thisstudy, expression of all the tested HSP genes were also up-regulated for morethan two-fold upon heat in both strawberry cultivars (Table 2). Among them,five HSPs had ~2–10-fold higher levels of up-regulation in cv. Festivalcompared to cv. Ventana upon heat treatment (p < 0.05, Table 2). Thesefive genes include one HSP90 (gene 1495), one HSP70 (gene 26629), andthree sHSP genes (a peroxisomal sHSP16 gene 11408, a cytosolic Class IIsHSP17.4 gene 15418, and a chloroplast sHSP25.1 gene 12739). They maycontribute to the higher level of heat tolerance observed in cv. Festivalcompared to cv. Ventana (Turechek and Peres, 2009).

Of the HSP genes investigated, only a Class I cytoplasmically localizedsmall HSP gene, sHSP15.96 (gene 07764) showed significantly higher tran-script levels in cv. Ventana compared to cv. Festival (Table 2). Althoughgenerally not as heat tolerant as cv. Festival, cv. Ventana does exhibit the

Table 2. Quantitative data for the eight HSP and two HSF genes investigated showing theLSMEANS of gene expression ratio ± standard error for each cultivar normalized with tworeference genes: ubiquitin (UBQ) and DNA binding protein (DBP).z

Gene annotationy Gene IDy Cultivar Expression ratio ± SE p-Value

Hsp90 01495 Festival 32.16 ± 16.25 0.0215Ventana 7.51 ± 3.78

Hsp70 26629 Festival 95.19 ± 42.35 0.0363Ventana 26.61 ± 11.14

Hsp70 29595 Festival 64.40 ± 21.43 0.2327Ventana 120.71 ± 45.94

HsfA4a 23802 Festival 2.32 ± 0.55 0.5439Ventana 1.87 ± 0.44

HsfA6b 20347 Festival 3.56 ± 0.87 0.0117Ventana 1.13 ± 0.28

sHsp15.96 07764 Festival 9.26 ± 1.89 0.0004Ventana 22.98 ± 4.68

sHsp16 11408 Festival 123.80 ± 61.52 0.0366Ventana 47.61 ± 23.85

sHsp17.4 15418 Festival 153.55 ± 75.05 0.0085Ventana 10.72 ± 5.24

sHsp24.5 8959 Festival 29.48 ± 7.94 0.2089Ventana 20.15 ± 5.45

sHsp25.1 12739 Festival 264.74 ± 140.27 0.0209Ventana 83.38 ± 43.50

zThe observed differences in the gene expression ratios between cultivars was determined to be statisticallysignificant for p-values < 0.05.

yGene annotations and IDs were obtained from Fragaria vesca Whole Genome v1.1 Assembly andAnnotation at the GDR (https://www.rosaceae.org/species/fragaria/fragaria_vesca/genome_v1.1).

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classic induced thermotolerance response, increased (induced) thermotoler-ance to high, lethal temperatures (44 °C or 48 °C) when exposed to 37 °C for1 h prior to the high temperature treatment (data not shown). It is possiblethat heat tolerance in cv. Ventana is conferred through the expression of adifferent set of genes than those expressed in cv. Festival. sHSP15.96 may beexpressed in cv. Ventana at higher levels than that in cv. Festival in anattempt to compensate for the lower abundance of other HSPs.

In contrast to the HSPs, the two HSFs investigated were only up-regulated(more than 2-fold) in cv. Festival and the significantly higher level of up-regulation was only observed with the HSFA6b (p < 0.05, Table 2). HSFAs actas transcriptional activators for HSP genes (Scharf et al., 2012) and thus thegreater abundance of HSFA6b transcript in cv. Festival may be responsiblefor the higher expression of five HSPs in cv. Festival than in cv. Ventana.

HSP and HSF genes responding to heat treatment have been identified inother strawberry cultivars (e.g., Camarosa) and in F. vesca, the ancestor ofcultivated strawberry (Christou et al., 2014; Hu et al., 2015; Lin et al., 2013;Rivarola et al., 2011). However, in these studies, differential expressionbetween strawberry cultivars with differential heat tolerance level was notstudied. Therefore, the focus of this study was to determine which heat shockresponse genes are associated with a higher level of heat tolerance in themore heat-tolerant cultivar. Although differences were found, the exact rolethat each of the genes assayed plays in strawberry heat tolerance is unknown,and functional genomics and proteomics analyses would be needed to iden-tify their roles. Understanding more about how each of the proteins encodedby these genes’ functions could enable us to identify and develop cultivarsadapted for heat treatment to eliminate pathogens. Heat treatment couldbecome a sustainable method for managing not only ALS, but a number ofdiseases in strawberry, including anthracnose, powdery mildew, and a multi-tude of viral diseases. The ability to identify and/or develop strawberryvarieties with heat tolerance would be a first step in realizing such anapproach.

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