The Effects of Heat Treatment on the Gene Expression of Several Heat Shock Protein Genes in Two Cultivars of Strawberry

ABSTRACT

Heat treatment has been shown to be an effective method for reducing systemic pathogens in strawberry but the process often has adverse effects on plant health. Research has shown that a brief heat treatment of plants at a lower temperature prior to the main heat treatment can induce heat shock proteins, which serve to protect the plant from damage when treated at higher temperatures. The objective of this study was to determine the relative gene expression of two heat shock factors (HSFs) and eight heat shock proteins (HSPs) in two strawberry cultivars (Festival and Ventana) known to have differential tolerance to heat. Strawberry plants were treated at 37 °C for 1 hour to induce the heat shock response. Total RNA was extracted and reverse transcription polymerase chain reaction (RT-PCR) was used to determine the amount of target produced. Relative gene expression was determined using the 2^−∆∆c_t method. Results showed that transcripts of one HSF and five HSPs were significantly more abundant in cv. 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 may confer heat tolerance in strawberry, which may be useful for selecting heat tolerant plants in breeding programs.

KEYWORDS:

• Fragaria × ananassa
• Xanthomonas fragariae
• angular leaf spot
• disease management

Introduction

Heat treatment is a common practice for eliminating endophytic and systemic infections in plants (Turechek and Peres, 2009; Uchansji et al., 2004). It has been shown to significantly reduce or eliminate bacterial infections in several crops, including apple, cherry, grape (Burr et al., 1989; Hall et al., 2002; Keck et al., 1995). The key to employing heat treatment successfully is identification of temperatures capable of killing the pathogen that will not adversely affect the health of the plant. Unfortunately, it is often difficult to 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 the heat shock response.

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

HSPs constitute a class of proteins that are evolutionarily conserved among organisms indicating the critical function of these proteins. In plants, these proteins are localized in the cytosol and organelles, including the chloroplast, 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 evolutionarily conserved groups based on their functions as molecular chaperones and their molecular masses: HSP100s, HSP90s, HSP70s, HSP60s, and the small heat shock proteins (sHSPs) (Krishna, 2003). Plants are unique in that they produce many different types of sHSPs, ranging from 17–30 kD, with subclasses 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 stress and assists in the refolding of such proteins upon denaturation, precluding irreversible loss of protein function (Garavaglia et al., 2009). Likewise, HSP90, which is constitutively expressed in many organisms, also functions in protein stabilization during heat stress. However, unlike other classes of heat shock proteins, HSP90 shows binding specificity, binding only to protein substrates in their native conformations (Xu et al., 2011). Moreover, many of these protein substrates are implicated in several high priority intracellular events, including gene expression regulation, signaling pathways, and cell cycle control (Wang et al., 2004). A much less characterized group of HSPs are the sHSPs. Unlike the other groups of HSPs, the small heat 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 transcription 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 class A is the best studied and these proteins are well known for their role in positive regulation of HSP gene expression. In contrast to class A HSFs, class B HSFs mostly serve as repressors of gene expression. The function of class C HSFs remains largely unknown (Scharf et al., 2012).

The purpose of this study was to determine whether there is a difference in expression 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 treatment at 37 °C for 1 h followed by a cool-down period for 1 h at room temperature resulted in heat tolerance to subsequent treatments at higher temperatures, 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, we hypothesized that although the expression of heat shock proteins will be up-regulated in both cultivars at 37 °C, the expression of these genes will be greater in the more tolerant cv. Festival.

Materials and methods

Induction of heat shock proteins

Strawberry cultivars, Ventana and Festival, were obtained from Lassen Canyon Nursery (Redding, CA, USA) and stored until use at 4 °C at the United States Department of Agriculture’s Horticulture Research Laboratory in 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 per cultivar in Ziploc bags were removed from 4 °C refrigeration and allowed to acclimate to room temperature for 1 h in the bags. Plants were then individually placed in stomacher bags and the bags of plants were rolled tightly to remove as much excess air as possible before being sealed with stomacher bag clips. Three of these bags were submerged in a 37 °C water bath for 1 h. To prevent the bags from floating to the surface, donut-shaped flask weights were placed on top of them. After removal from the water bath, the plants were set at room temperature for 1 h. For controls, the remaining three plants per cultivar stayed in stomacher bags at room temperature for 2 h. The experiment 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 heat shock factors in strawberry, and primer pair sequences for the reference genes were designed and are shown in Table 1. Primers were designed from Fragaria vesca gene sequences obtained by using BLAST (Altshul et al., 1990) to search the F. vesca reference genome (Shulaev et al., 2011) with Arabidopsis HSP and HSF genes. The Arabidopsis sequences were obtained from TAIR (Lamesch et al. 2011). Of the strawberry homologs identified, eight HSP genes from different families (HSP90, HSP70, and sHSPs) and two HSF genes were chosen for the study based on their expression in response to heat as determined from expressed sequence tag (EST) data (Rivarola et al., 2011). F. vesca sequences for the genes to be assayed were retrieved from the F. vesca Whole Genome v1.1 Assembly at the Genome Database for Rosaceae (https://www.rosaceae.org/species/fragaria/fragaria_vesca/genome_v1.1) (Jung et al., 2014) and gene specific primer pairs were designed using Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/primer3/) (Table 1). Specific amplification was supported by melting curve analysis as described below. The PCR efficiency of each primer pair was calculated from standard curves constructed with serial 10-fold dilutions of cDNA samples.

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

Gene annotation^y	Gene ID^y	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′
DBP^x	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 and Annotation 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).

RNA extraction

Total RNA was extracted from crown tissue using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions with several modifications. For each extraction (single plant), 200 mg of crown tissue was ground to a powder in liquid nitrogen and transferred to a pre-chilled 2-mL micro-centrifuge tube. RLT lysis buffer master mix (550 µL) containing 4% (w/v) polyvinylpyrrolidone (PVP-40, Sigma-Aldrich, St. Louis, MO, USA) was immediately added and the tube was vortexed vigorously. PVP-40 is used to remove polysaccharides and polyphenolic compounds that irreversibly bind to RNA upon oxidation, adversely affecting downstream applications (John, 1992). The lysate was transferred to a QIA shredder spin column in a collection tube and centrifuged for 2 min at 22,000 g. The supernatant was mixed with a half volume of ethanol (100%) by pipetting in a new micro-centrifuge tube and transferred to an RNeasy Mini spin column. Three successive wash steps were carried out with the addition of 700 µL of buffer RW1 (wash 1) and 500 µL of buffer RPE (washes 2 and 3) to the RNeasy spin column, discarding the flow-through after each wash. The samples were incubated at room temperature for 5 min between each successive wash. Following incubation the samples were centrifuged for 15 s at 8000 g. The RNeasy spin columns were transferred to a new 2-mL micro-centrifuge tube and centrifuged at 22,000 g for 1 min. RNA was eluted with 50 µL of nuclease-free water and the samples were incubated at room temperature for 30 min before centrifugation at 8000 g for 1 min. After elution, the quantity and purity of the RNA were determined using a Nanodrop 2000 (Thermo Scientific, Wilmington, DE, USA). RNA was stored on ice if moving to the protocol below or stored in a –80 °C freezer until further 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 genomic DNA contamination. A 60-µL reaction contained 5 µg of RNA based on Nanodrop readings, 1x RQ1 RNAse-free DNase Reaction Buffer, and 2.5 U of RQ1 RNase-Free DNase. The reaction was incubated at 37 °C for 30 min. After incubation, 6 µL of RQ1 DNase stop solution was added to terminate the reaction, followed by incubation at 65 °C for 10 min to inactivate the DNase. The samples were immediately placed on ice when proceeding with the cDNA synthesis reaction or stored at –80 °C until further use.

Reverse transcription of RNA samples (cDNA synthesis)

Reverse transcription (RT) was performed using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). A 90-µL RT reaction contained: 5 µg of DNase-treated RNA, 1x iScript reaction mix, and 5 µL of iScript reverse transcriptase. 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 (KAPA Biosystems, Philadelphia, PA, USA). The qPCR reactions were run in triplicate using the iQ5 iCycler qPCR platform from Bio Rad (Hercules, CA, USA). A 20-µL reaction master mix contained: 1x KAPA SYBR FAST Universal mix, forward and reverse primers at a final concentration of 300 nM each, and 2 µL of template cDNA. The qPCR conditions were: 95 °C for 3 min, 40 cycles of 95 °C for 10 s, 55 °C for 45 s, followed by a melting curve analysis at 95 °C for 1 min, 55 °C for 1 min, and a ramping cycle from 55 °C to 95 °C in 0.5 °C increments of 10 s each (Wang et al., 2010). To normalize the 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−∆∆Ct method (Livak and Schmittgen, 2001). The method calculates, as a ratio, the expression of the target gene of interest relative to the expression of a reference gene. The expression ratio is calculated as: amount of target = [(2ΔCttarget]/[(2ΔCtref], where 2 represents 100% PCR efficiency of both target and reference genes and ΔCt is the difference in Ct values between the control and treated samples. The 2ΔCtref was calculated from the geometric mean of the raw ratios of the two reference genes. Differences in cultivar gene expression ratios were analyzed by fitting generalized linear mixed models to the data using the GLIMMIX procedure of SAS (SAS Institute, Cary, NC, USA). Replication was treated as a random effect, and a gamma error distribution and log link function were specified. Parameters were estimated using 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-regulated expression upon heat treatment in F. vesca (Rivarola et al., 2011). In this study, expression of all the tested HSP genes were also up-regulated for more than 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. Festival compared to cv. Ventana upon heat treatment (p < 0.05, Table 2). These five genes include one HSP90 (gene 1495), one HSP70 (gene 26629), and three sHSP genes (a peroxisomal sHSP16 gene 11408, a cytosolic Class II sHSP17.4 gene 15418, and a chloroplast sHSP25.1 gene 12739). They may contribute to the higher level of heat tolerance observed in cv. Festival compared to cv. Ventana (Turechek and Peres, 2009).

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

Gene annotation^y	Gene ID^y	Cultivar	Expression ratio ± SE	p-Value
Hsp90	01495	Festival	32.16 ± 16.25	0.0215
		Ventana	7.51 ± 3.78
Hsp70	26629	Festival	95.19 ± 42.35	0.0363
		Ventana	26.61 ± 11.14
Hsp70	29595	Festival	64.40 ± 21.43	0.2327
		Ventana	120.71 ± 45.94
HsfA4a	23802	Festival	2.32 ± 0.55	0.5439
		Ventana	1.87 ± 0.44
HsfA6b	20347	Festival	3.56 ± 0.87	0.0117
		Ventana	1.13 ± 0.28
sHsp15.96	07764	Festival	9.26 ± 1.89	0.0004
		Ventana	22.98 ± 4.68
sHsp16	11408	Festival	123.80 ± 61.52	0.0366
		Ventana	47.61 ± 23.85
sHsp17.4	15418	Festival	153.55 ± 75.05	0.0085
		Ventana	10.72 ± 5.24
sHsp24.5	8959	Festival	29.48 ± 7.94	0.2089
		Ventana	20.15 ± 5.45
sHsp25.1	12739	Festival	264.74 ± 140.27	0.0209
		Ventana	83.38 ± 43.50

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

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

Of the HSP genes investigated, only a Class I cytoplasmically localized small HSP gene, sHSP15.96 (gene 07764) showed significantly higher transcript levels in cv. Ventana compared to cv. Festival (Table 2). Although generally not as heat tolerant as cv. Festival, cv. Ventana does exhibit the classic induced thermotolerance response, increased (induced) thermotolerance to high, lethal temperatures (44 °C or 48 °C) when exposed to 37 °C for 1 h prior to the high temperature treatment (data not shown). It is possible that heat tolerance in cv. Ventana is conferred through the expression of a different set of genes than those expressed in cv. Festival. sHSP15.96 may be expressed in cv. Ventana at higher levels than that in cv. Festival in an attempt 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 act as transcriptional activators for HSP genes (Scharf et al., 2012) and thus the greater abundance of HSFA6b transcript in cv. Festival may be responsible for the higher expression of five HSPs in cv. Festival than in cv. Ventana.

HSP and HSF genes responding to heat treatment have been identified in other strawberry cultivars (e.g., Camarosa) and in F. vesca, the ancestor of cultivated strawberry (Christou et al., 2014; Hu et al., 2015; Lin et al., 2013; Rivarola et al., 2011). However, in these studies, differential expression between strawberry cultivars with differential heat tolerance level was not studied. Therefore, the focus of this study was to determine which heat shock response genes are associated with a higher level of heat tolerance in the more heat-tolerant cultivar. Although differences were found, the exact role that each of the genes assayed plays in strawberry heat tolerance is unknown, and functional genomics and proteomics analyses would be needed to identify their roles. Understanding more about how each of the proteins encoded by these genes’ functions could enable us to identify and develop cultivars adapted for heat treatment to eliminate pathogens. Heat treatment could become a sustainable method for managing not only ALS, but a number of diseases in strawberry, including anthracnose, powdery mildew, and a multitude of viral diseases. The ability to identify and/or develop strawberry varieties with heat tolerance would be a first step in realizing such an approach.