Article pubs.acs.org/JAFC
Expression of Defense Genes in Strawberry Fruits Treated with Different Resistance Inducers Lucia Landi, Erica Feliziani, and Gianfranco Romanazzi* Department of Agricultural, Food, and Environmental Sciences via Brecce Bianche, Marche Polytechnic University, Ancona 60131, Italy ABSTRACT: The expression of 18 defense genes in strawberry fruit treated with elicitors: chitosan, BTH, and COA, at 0.5, 6, 24, and 48 h post-treatment was analyzed. The genes were up-regulated differentially, according to the elicitor. Chitosan and COA treatments promoted the expression of key phenylpropanoid pathway genes, for synthesis of lignin and flavonoids; only those associated with flavonoid metabolism were up-regulated by BTH. The calcium-dependent protein kinase, endo-β 1,4glucanase, ascorbate peroxidase, and glutathione-S-transferase genes were up-regulated by BTH. The K+ channel, polygalacturonase, polygalacturonase-inhibiting protein, and β-1,3-glucanase, increased in response to all tested elicitors. The enzyme activities of phenylalanine ammonia lyase, β-1,3-glucanase, Chitinase, and guaiacol peroxidase supported the gene expression results. Similarity of gene expression was >72% between chitosan and COA treatments, while BTH showed lower similarity (38%) with the other elicitors. This study suggests the relationship between the composition of the elicitors and a specific pattern of induced defense genes. KEYWORDS: benzothiadiazole, chitosan, elicitors, Fragaria × ananassa, gene expression
■
INTRODUCTION Strawberry (Fragaria × ananassa) is one of the most widely consumed berries, and it is a good source of natural antioxidants.1 However, strawberry fruits are highly perishable and very susceptible to fungal decay in the field, and even more so during postharvest storage. This can result in severe crop losses. Application of natural compounds known as resistance inducers or elicitors is an innovative approach to prolong the shelf life of fresh fruit, through the reduction of disease incidence and with increased ecological security and safety for consumers. To reduce the postharvest decay of strawberries, the application of these natural compounds has been investigated as an alternative to the use of synthetic fungicides.2−4 These compounds act as elicitors, as they activate the natural phenomenon known as induced resistance, with effects that are localized or, more often, systemic and that promote nonspecific resistance to pathogens.5,6 In the present study, we investigated three different resistance inducers that are based on natural compounds to test their activation of the resistance mechanism. The biopolymer chitosan is an N-deacetylated form of the polysaccharide chitin that is found in the cell wall of many fungi. Chitosan has been shown to have a double action in plant protection: it inhibits the development of decay-causing fungi through the production of a film on treated surfaces,7,8 and it induces resistance responses in plant tissues. Benzothiadiazole (BTH), which is the functional analogue of the plant endogenous hormone-like compound salicylic acid, protects different plant species against diseases caused by viral, bacterial, and fungal pathogens.9 The third product is a commercial formulation that is based on a mixture of calcium and organic acids (COA), according to the well-known effects of calcium in vegetal tissues for the binding of pectins and for strengthening the plant cell wall.10,11 © 2014 American Chemical Society
The signaling pathways that control systemic resistance are multiple component networks with characteristic schemes that lead to plant resistance.12 However, the transcription factors produced as a result of signal transduction can trigger the expression of a large number of genes, with the consequent physiological events usually involving changes in cell-wall composition, ion fluxes, de novo production of pathogenesisrelated (PR) proteins, synthesis of phytoalexins, and reactive oxygen species (ROS) production. 13 Several studies have shown the involvement of phenolic compounds14,15 and cell-wall degradation enzyme activities3,16 in the responses of strawberry fruit exposed to postharvest treatments with elicitors. However, the relationships between resistance inducers and the genes used as potential markers for resistance induction in harvested strawberries have not been investigated. The aim of the present study was to setup a method based on reverse transcription−quantitative real-time polymerase chain reaction (RT-qPCR) to analyze changes in expression of selected defense genes induced in strawberry fruit at 0.5, 6, 24, and 48 h following short (30 s) treatments with the elicitors chitosan, BTH, and COA. The 18 genes analyzed were associated with Ca2+ and K+ ion fluxes; calcium-dependent protein kinase (CDPK); K+ channels; ROS cell responses, including glutathione S-transferase (GST) and ascorbate peroxidase (APX); secondary metabolites of the phenylpropanoid pathway, including phenylalanine ammonia-lyase (PAL); biosynthesis of different flavonoid branches, including chalcone synthase (CHS), chalcone isomerase (CHI), flavanon Received: Revised: Accepted: Published: 3047
October 1, 2013 March 14, 2014 March 16, 2014 March 16, 2014 dx.doi.org/10.1021/jf404423x | J. Agric. Food Chem. 2014, 62, 3047−3056
Journal of Agricultural and Food Chemistry
Article
Table 1. Primers Selected for the Gene Expression Analyses of Strawberry Fruits Treated with Chitosan, COA, and BTH Commercial Formulationsa
a
PCR amplification efficiencies and regression coefficients for the standard curves are reported for each primer pair. *, reference genes. 1% (w/v) chitosan (Chito Plant, ChiPro GmbH, Bremen, Germany), 1% (v/v) COA (Fitocalcio, Agrisystem, Lamezia Terme, CZ, Italy), and 1% (w/v) BTH (Bion, Syngenta, Milano, MI, Italy). The commercial products were prepared by dissolving or diluting them in distilled water. Distilled water was used as the control. Strawberry fruits (800 g) were pooled together and randomized, and then they were immersed for 30 s in 1 L of one of the three resistance inducers or water. After the treatments, the strawberries were dried in air for 30 min and then subdivided into 4 groups of 200 g each. After air drying, one group was stored at −80 °C (0.5 h posttreatment), and the other groups were arranged in small plastic boxes that were placed in bigger covered plastic boxes. These groups were stored up to a total of 6, 24, and 48 h post-treatment, at 20 °C and 95%−98% relative humidity. At each time, fruits corresponding to 20 g were randomly selected and stored in two separated tubes at −80 °C, until RNA extraction. The experiments were repeated at least twice. Gene Expression Analysis. Gene expression analysis on the strawberry fruit after the treatments with the different resistance inducers was performed by RT-qPCR, using a SYBR-green dye system, according to Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines.17 The comparative −ΔΔCt method18 was used to evaluate the relative quantities of each of the amplified products in the samples.
3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), flavonol synthase (FLS), anthocyanidin synthase (ANS), and flavonoid 3-O-glucosyltransferase (UFGT); cell-wall degradation enzymes, including endo-β-1,4-glucanase (endoβGlu), polygalacturonase (PG), polygalacturonase-inhibiting protein (PGIP); and encoding of PR proteins, including β-1,3glucanase (βGlu), class III Chitinase (Chi3), and PR-1. In the same way, the enzyme activities related to PAL, βGlu, total Chitinase (Chi), and guaiacol peroxidase (GPX) were tested. The results of this study provide new information on the effect of resistance inducers on strawberry fruit.
■
MATERIALS AND METHODS
Fruit Material. Experiments were carried out on fruits of strawberries cv. Camarosa grown according to organic agriculture practices, from commercial orchards located in the Marche region, central-eastern Italy. The fruits were selected for the absence of defects, uniformity in size, and degree of ripening (2/3 of surface red), and they were used for the experiments on the day of their harvest. Treatments. The physiological changes in the strawberry fruit were analyzed following treatments with commercial elicitor formulations: 3048
dx.doi.org/10.1021/jf404423x | J. Agric. Food Chem. 2014, 62, 3047−3056
Journal of Agricultural and Food Chemistry
Article
according to Derckel et al.22 The Chi and PAL activities were measured according to Trotel-Aziz, et al.23 and the GPX according to Amako et al.24 All of the enzyme activities were analyzed using a UV 1800 spectrophotometer (Shimadzu Corp., Tokyo, Japan). For all of the assays, the absorbance was measured against a blank (crude protein extract in incubation mixture). The specific activities of the enzymes are expressed as Units (U) mg−1 protein. Data Analysis. The gene expression study was performed using the −ΔΔCt method.18 According to this method, the expression of target genes was given as the mean fold-changes in gene expression normalized to an endogenous reference gene and compared to the untreated controls. For each individual sample, three replications were analyzed. The gene expression calculations were performed using the Excel program Gene Expression Analysis for iCycler iQ Real Time PCR Detection System (Bio-Rad). The calculations in this spreadsheet were derived from the algorithms outlined by Vandesompele et al.20 All of the analyses were performed twice. For the analysis of the enzyme activities, for each individual sample three replications were analyzed. The experiments were performed twice. The data from each sampling point are shown as the mean ± SD and were statistically evaluated by ANOVA, followed by individual comparisons using Duncan’s Multiple Range Test, at p ≤ 0.05.
RNA Extraction. High quality total RNA was obtained from the fruit according to the protocol of Landi and Romanazzi.19 Briefly, 10 g of strawberry tissue samples including both achenes and receptacle was ground in liquid nitrogen, and 400 mg of the resulting fruit powders was randomly collected for RNA extraction. Extraction buffer was added (1 mL; 100 mM Tris-HCl, pH 8.0, 25 mM EDTA, pH 8.0, 2% [w/v] CTAB [Sigma], 2% [v/v] β-mercaptoethanol, 2.5 M NaCl, and 2% [w/v] soluble PVP-40), and the samples were incubated at 65 °C for 30 min. The supernatants were transferred to new tubes with an equal volume of chloroform/isoamyl alcohol (24:1) and centrifuged at 10,000g for 5 min at 4 °C. This last step was repeated two more times. The total RNA were precipitated in 0.25 vol 10 M LiCl, with the reaction left to proceed overnight at 4 °C. The samples were then centrifuged at 10,000g for 30 min at 4 °C, washed in 70% ethanol, dried, and resuspended in 50 μL of double-distilled diethyl pyrocarbonate water. RNA integrity was verified by agarose gels that were stained using SYBRSafe (Invitrogen, Carlsbad, CA, USA). RNA purity was assessed based on an absorbance ratio of 1.80 to 1.90 at 260/280 nm, using BioPhotometer plus (Eppendorf Inc., Westbury, NY, USA) and 1.8 to 2.0 at 230/260 nm. Reverse Transcription. A total of 40 ng to 50 ng RNA was used for cDNA synthesis with reverse-transcription PCR, using iScript TM cDNA synthesis kits (Bio-Rad, Hercules, CA, USA), according to the manufacturer’s instructions. From each RNA extraction, cDNA synthesis was performed twice, and the products were mixed before the gene expression studies. Primers and Reference Gene Selection. Specific primer sets were designed using the Primer3 software (http://biotools. umassmededu/bioapps/primer3_www.cgi) from the specific sequence of Fragaria × ananassa deposited in NCBI GenBank (Table 1). The primer pairs were chosen and validated in silico using primer BLASTspecific analysis (http://www.ncbi.nlm.nih.gov/Blast.cgi) and then according to the melting profiles obtained from the quantitative realtime PCR conditions (qPCR), as described later. The relative expression stabilities of the candidate reference genes of 18S-rRNA, actin, histone H4, and GAPDH2 were validated using the geNorm method, using the 3.5 version.20 Quantitative Real-Time PCR. qPCR reactions were performed in 96-well clear multiplate PCR plates (Bio-Rad, Hercules, CA, USA) using the iQ SYBR Green Supermix (Bio-Rad) on an iCycler iQ RealTime PCR Detection System (Bio-Rad) under the following conditions: an initial denaturating cycle (5 min at 95 °C), followed by 40 cycles of three steps of denaturation, annealing, and polymerization (30 s at 95 °C, 20 s at 55 °C, and 30 s 72 °C). PCR amplification was carried out in a total volume of 22 μL, containing 9 μL of diluted (1:10) cDNA (duplicates), 0.25 μM of each primer, and 11 μL of iQ SYBR Green Supermix. All of the assays included no-RT and no-template controls, to determine the nonspecific amplification. To determine the specificity of the amplicons, melting curve analysis was performed over the range of 55 to 98 °C. The qPCR efficiency (E) of each primer pair was determined using standard curves generated according to the equation E = 10−1/slope of five triplicate cDNA pool dilutions (undiluted, 0.25, 0.0625, 0.015, and 0.003). Enzyme Activities. Spectrophotometric assays were used to determine the βGlu EC 3.2.1.6, Chi EC 3.2.1.14, PAL EC 4.3.1.24, and GPX EC 1.11.1.7 activities of the strawberry fruits at 0.5, 6, 24, and 48 h post-treatment with chitosan, COA, and BTH. Frozen fruits (1 g fresh weight) were ground, and βGlu and Chi were extracted using 1% (w/v) polyvinylpolypyrrolidone in 50 mM sodium acetate buffer (pH 5.0) containing 1 mM dithiothreitol and 0.2% (w/v) phenylmethylsulfonyl fluoride. The PAL was obtained using 100 mM potassium phosphate buffer (pH 8.0) containing 1% (w/v) polyvinylpolypyrrolidone and 1.4 mM β-mercaptoethanol. The GPX was obtained with 100 mM potassium phosphate buffer (pH 7.3) containing 1 mM EDTA. The homogenates were centrifuged at 15000g for 15 min at 4 °C, and the resulting supernatants were used as the crude enzyme extracts. The protein content in the enzyme extracts was determined according to the Bradford assay21 (Sigma-Aldrich), using bovine serum albumin as the standard. βglu activity was assayed
■
RESULTS Selection of Appropriate References Genes for RTqPCR in the Treated Strawberry Fruits. For this study, the RT-qPCR was set up for analysis of the strawberry fruits treated for 30 s with the three elicitors. The specific primer sets identified for all of the genes analyzed showed specific singlepeak melting curves (data not shown), which confirmed the homogeneity and specificity of the amplicons produced in the RT-qPCR for each of the 18 target genes. No amplification was observed in any of the control assays, which confirmed that the samples were free of contamination with genomic DNA or RNA, or the cDNA template (data not shown). The amplification efficiency (E) ranged from 96.2% to 106.2%, according to the standard curve analysis in RT-qPCR of each gene-specific primer pair (Table 1). The validation of four putative candidate reference genes, 18S-RNA, actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH2), and histone H4, according the geNorm method, showed differences within the treatments. However, these reference genes had M values 70%), compared to the similarities between BTH and both chitosan and COA (both
