Salicylic-Acid-Induced Chilling - American Chemical Society

Oct 18, 2016 - School of Agricultural Economics and Rural Development, Renmin University of China, Beijing 100872, People's Republic of China. §. Col...
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Salicylic acid-induced chilling- and oxidative-stress tolerance in relation to gibberellin homeostasis, CBF pathway, and antioxidant enzyme systems in cold-stored tomato fruit Yang Ding, Jinhong Zhao, Ying Nie, Bei Fan, Shujuan Wu, Yu Zhang, Jiping Sheng, Lin Shen, Ruirui Zhao, and Xuanming Tang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02902 • Publication Date (Web): 18 Oct 2016 Downloaded from http://pubs.acs.org on October 19, 2016

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Journal of Agricultural and Food Chemistry

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Salicylic acid-induced chilling- and oxidative-stress tolerance in

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relation to gibberellin homeostasis, CBF pathway, and antioxidant

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enzyme systems in cold-stored tomato fruit

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Yang Ding,† Jinhong Zhao,† Ying Nie,† Bei Fan,† Shujuan Wu,† Yu Zhang,† Jiping

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Sheng,‡ Lin Shen,§ Ruirui Zhao,§ and Xuanming Tang*,†

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† Institute of Food Science and Technology, Chinese Academy of Agricultural

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Sciences, Key Laboratory of Agro-Products Processing, Ministry of Agriculture,

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Beijing, 100193, China

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‡ School of Agricultural Economics and Rural Development, Renmin University of

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China, Beijing, 100872, China

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§ College of Food Science and Nutritional Engineering, China Agricultural University,

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Beijing, 100083, China

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* Corresponding Author

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Phone: +86-10-62811868;

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E-mail: [email protected]

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Abstract

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Effects of salicylic acid (SA) on gibberellin (GA) homeostasis, cold-responsive

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transcription factor (CBF) pathway, and antioxidant enzyme systems linked to

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chilling- and oxidative-stress tolerance in tomato fruit was investigated. Mature green

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tomatoes (Solanum lycopersicum L. cv. Moneymaker) were treated with 0, 0.5, and 1

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mM SA solution for 15 min before storage at 4°C for 28 days. Compared with 0 or 0.5

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mM SA, 1 mM SA significantly decreased the chilling injury (CI) index in tomato

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fruit. In the SA-treated fruit, the upregulation of GA biosynthetic gene (GA3ox1)

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expression was followed by gibberellic acid (GA3) surge and DELLA protein

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degradation. CBF1 participated in the SA-modulated tolerance and stimulated the

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expression of GA catabolic gene (GA2ox1). Furthermore, 1 mM SA enhanced

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activities of antioxidant enzymes, thus reduced reactive oxygen species accumulation.

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Our findings suggest that SA might protect tomato fruit from CI and oxidative damage

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through regulating GA metabolism, CBF1 gene expression, and antioxidant enzyme

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activities.

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Keywords: tomato fruit; chilling tolerance; salicylic acid; gibberellin homeostasis;

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CBF pathway; antioxidant enzyme systems

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INTRODUCTION

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Chilling injury (CI) is a physiological disorder that greatly reduces the

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postharvest quality of tropical and subtropical fruit and vegetables at low but not

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freezing temperatures.1 Tomato (Solanum lycopersicum L.) is a typical cold-sensitive

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fruit that has been used as a model to investigate CI-related metabolism.2 CI

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macroscopic symptoms of tomato fruit including pitting, browning, and decay usually

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appear when the fruit are transferred to 20°C to 22°C after storage at 2°C to 6°C for

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more than 2 weeks.1,3 The development of CI restricts refrigerated transportation and

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long-term cold storage of harvested tomato fruit, and reduces consumer acceptability

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leading to substantial economic loss.

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Salicylic acid (SA) is a natural phenolic compound and endogenous signal

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involved in the regulation of plant growth, development, and responses to biotic and

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abiotic stresses.4 Recently, SA exhibits high potential in controlling postharvest losses

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of horticultural crops such as tomatoes,5 cucumbers,6 and table grapes.4 A few studies

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have reported that the exogenous application of SA was able to alleviate postharvest

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CI in tomato fruit.7-10 Mitigation of CI in tomato by SA could be attributed to: (1)

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inhibition of activities of a variety of membranous lipolytic enzymes, such as

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phospholipase D and lipoxygenase;7 (2) enhancement of arginine pathway, which

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leads to the accumulation of signaling molecules, such as polyamines, nitric oxide,

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and proline;8 (3) stimulation of the synthesis of some stress proteins, such as heat

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shock proteins,9 pathogenesis-related proteins, and alternative oxidase associated with

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the avoidance of reactive oxygen species (ROS).10 As chilling stress increases ROS

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accumulation, the ability to scavenge ROS during and after chilling determines the

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resistance and adaptation of plants to low temperatures.2 The coordinated action of

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antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), which

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are important for scavenging ROS, helps reduce oxidative damage and CI.11 It has

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been shown that postharvest SA treatment increased the activities of SOD and CAT in

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cold-sensitive fruit such as mangoes,12 peaches,13 and cucumbers6 at low temperatures.

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However, the functional relationships among SA, ROS, and related signaling

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pathways in the responses of postharvest tomato fruit to chilling stress are still poorly

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understood.

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Gibberellins (GAs) are important, naturally occurring phytohormones regulating

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diverse plant growth and development processes throughout the life cycle of higher

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plants.14 It has been reported that GAs play essential roles in plant responses to

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various abiotic stress conditions such as salt, oxidative, and heat stresses.15 Recent

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studies have revealed that exogenous GA3 treatment could effectively alleviate CI

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symptoms

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C-repeat/dehydration-responsive element binding factors (CBF) pathway has a vital

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role in plant responses to cold stress.17 Exogenous GA3 remarkably elevated the

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expression of CBF1 gene transcripts. Interestingly, GA3 treatment was able to

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stimulate SA biosynthesis via isochorismate synthase (ICS) pathway in chilled tomato

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fruit.18 On the other hand, Lee and Park19 pointed out that the transcript levels of GA

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biosynthetic gene GA3ox1 were elevated by exogenous SA treatment in Arabidopsis

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seed germination under high-salinity conditions. Emerging evidence suggests the

in

postharvest

tomato

fruit

during

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storage.16

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existence of a cross talk between SA and GA in plants under abiotic stresses. However,

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tomato is different from Arabidopsis, in that it is susceptible to CI. Little information

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is available concerning the complex mechanism of hormonal interactions in

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postharvest fruit during long-term cold storage. Knowing how SA is linked with GA

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signaling and how antioxidant capacity is regulated by SA would enhance the

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understanding of the molecular mechanisms underlying SA-GA signaling cross talk in

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postharvest fruit under chilling and oxidative stresses.

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The present study aimed to provide a molecular basis to explain the roles of GA

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homeostasis, CBF1 gene and antioxidant enzyme systems in SA-induced chilling- and

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oxidative-stress tolerance in tomato fruit. It would provide a more comprehensive

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insight into the mechanisms underlying SA-GA signaling cross talk in the

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CBF-mediated stress-response pathway of tomato fruit during long-term cold storage.

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MATERIALS AND METHODS

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Fruit Materials and Treatments. Tomato (Solanum lycopersicum L. cv.

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Moneymaker) fruit were harvested at the mature green stage in October 2014 in a

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greenhouse under 16 h/8 h (light/dark) photoperiod at 24 °C/19 °C (day/night) in

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Beijing, China, and transported to the laboratory immediately and stored at 4 °C and

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80%–90% relative humidity (RH) for 24 h. The mature green stage was determined

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based on epidermis color (a* = –13 to –10).1 The fruit of uniform size and free from

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visual blemishes and disease were selected and randomly divided into three lots. The

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fruit were disinfected with 1% sodium hypochlorite (v/v) for 2 min, washed, and

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air-dried. Two lots were dipped in aqueous solutions of either 0.5 or 1 mM SA

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solution [prepared in ethanol/distilled water (1:1000, v/v) containing 0.1% (v/v)

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Tween-20] for 15 min at 20 °C, respectively. A third lot was dipped in

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ethanol/distilled water (1:1000, v/v) with 0.1% (v/v) Tween-20 under the same

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conditions and used as the control. The fruit were stored at 4 °C and 80%-90% RH for

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28 days. Three replicates of twenty fruit each for control, 0.5 mM SA or 1 mM SA

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treatment were taken on day 14, 21, and 28 of cold storage for 3 days at 20 °C for CI

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evaluation. Three replicates of five fruit each for control or 1 mM SA treatment were

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picked before treatment (time 0) and on day 3, 7, 14, 21 and 28 of cold storage for

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estimating GA3 content, gene expression, ROS production, and antioxidant enzyme

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activities. The mesocarp from the fruit equator area was cut into small pieces, frozen

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in liquid nitrogen, ground to a fine powder and stored at –80 °C.

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Chilling Injury (CI) Index. The CI index of the chilled fruit was assessed at

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20 °C for 3 days after 14, 21, and 28 days of cold storage for the development of CI

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symptoms. Symptoms included surface pitting according to the method of Zhao et al.

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20

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pitting; 1, pitting covering less than 25% of the fruit surface; 2, pitting covering

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25%-50% of the surface; 3, pitting covering 50%-75% of the surface; 4, pitting

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covering more than 75% of the surface. The average extent of CI was expressed as a

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CI index, which was calculated as following: CI index (%) = ∑ [(CI level) × (Number

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of fruit at the CI level)]/[4 × (Total number of fruit)] × 100.

The severity of the symptoms was assessed visually in a four-stage scale: 0, no

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Gibberellic Acid (GA3) Content. The GA3 content was measured by liquid

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chromatography-tandem mass spectrometry (LC-MS/MS) following the method of

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Shi et al. 21 and Zhu et al.18 Frozen tissue (10 g) was weighed into a 50-mL centrifuge

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tube and extracted using 10 mL of 0.1% (v/v) acetic acid in acetonitrile for 2 min with

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a vortex mixer. Anhydrous magnesium sulfate (4 g) was added into the tube, and the

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mixture was centrifuged at 12,000g for 5 min at 4 °C. The extract (1 mL) was filtered

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with a 0.22-µm membrane, and then analyzed by LC-MS/MS. The chromatographic

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separation was performed on a Waters XBridge C18 reversed-phase column (2.1 mm

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× 150 mm, with a 5.0-µm particle size). The gradient condition was shown in Table 1,

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and the injection volume was 5 µL. The column temperature was maintained at 30 °C

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with a flow rate of 5 µL s–1. The API5500 tandem quadrupole mass spectrometer

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(Applied Biosystems, CA, USA) was used for LC-MS/MS analysis and operated in

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the multiple-reaction monitoring mode. The data was acquired and processed using

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the AB Sciex Analyst 1.4.2 software (Applied Bioscience, CA, USA). The GA3

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content was expressed as mg kg–1 fresh weight (FW).

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RNA Isolation and Reverse Transcription. Total RNA in 2 g of frozen tissue

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was extracted using an RNAprep Pure Plant Kit (Tiangen, Beijing, China). Total RNA

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was dissolved in 40 µL of RNase free water, quantified by using a SMA 4000 UV-Vis

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Spectrophotometer (Merinton, Beijing, China) and stored at -80 °C. Reverse

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transcription was completed to synthesize the firststrand of cDNA with 1 µg of total

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RNA according to the instructions from QuantScript RT Kit (Tiangen).

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Quantitative Real-Time Polymerase Chain Reaction (qPCR). The gene

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expression changes of GA3ox1 (AB010991), GAI (AY269087), CBF1(AY034473),

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and GA2ox1 (EF441351) in tomato fruit were studied by quantitative real-time

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polymerase chain reaction (qPCR) using SuperReal PreMix Plus (SYBR Green)

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(Tiangen) according to the method of Zhu et al.18 β-actin (SGN-U144149) was used

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as the reference gene. Specific primers selected from Nation Center for Biotechnology

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Information (NCBI) were designed from their nucleotide sequences and synthesized

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by Sangon Biotech Co., Ltd., Company (Shanghai, China) (Table 2). The qPCR

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amplifications were carried out using an ABI 7500 real-time PCR system (Applied

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Biosystems). The relative gene expression of the target gene was calculated using the

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2-∆∆Ct method.22

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Superoxide Anion (O2•−) Production Rate. The rate of O2•− production was

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measured by monitoring the formation of nitrite from hydroxylamine as described by

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Jiang et al.23 with some modifications. Frozen tissue (1 g) was homogenized in an ice

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bath with 6 mL of 65 mM potassium phosphate buffer (pH 7.8), 2 mL of 10 mM

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hydroxylamine hydrochloride and 2 mL of 0.1 M ethylenediaminetetraacetic acid

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(EDTA), and then centrifuged at 12000g for 30 min at 4 °C. The obtained supernatant

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(2 mL) was mixed with 2 mL of 17 mM 4-aminobenzenesulfonic acid and 2 mL of 7

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mM α-naphthylamine, and then incubated for 15 min at 40 °C. Ethyl ether (2 mL) was

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added, and the resulting mixture was centrifuged at 3000g for 15 min. The optical

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density was recorded immediately at 530 nm. A standard curve for potassium nitrite

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was used to estimate the production rate of O2•−. The O2•− production rate was

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expressed as µmol min–1 g–1 FW.

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Hydrogen Peroxide (H2O2) Content. The assay of H2O2 content was carried out

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by the procedure previously described by Patterson et al.24 Frozen tissue (1 g) was

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homogenized in an ice bath with 2 mL of cold acetone and centrifuged at 12,000g for

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30 min at 4 °C. The supernatant (1 mL) was mixed with 0.1 mL of 5% (w/v) titanium

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sulfate and 0.2 mL of 17 M ammonia, and then centrifuged at 12,000g for 15 min at

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4 °C. The precipitate was dissolved in 5 mL of 2 M H2SO4, and then centrifuged for 5

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min at 12,000g and at 4 °C. The absorbance of the supernatant was measured at 410

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nm, and H2O2 was quantified using a standard curve generated from known

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concentrations of H2O2. The H2O2 content was expressed as mmol g–1 FW.

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Enzyme Assessment. Enzyme extracts for enzyme activity assays were prepared

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as described previously,2,18 and activities of SOD and CAT expressed as unit U mg–1

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protein were spectrophotometrically measured using an SOD Detection Kit (A001,

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Jiancheng, Nanjing, China) and CAT (A007, Jiancheng, Nanjing, China) according to

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the manufacturer’s instructions.

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Statistical Analysis. Experiments were performed in triplicate according to a

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completely randomized design. One-way analysis of variance (ANOVA) and

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Duncan's multiple range tests were used for statistical evaluations using SPSS

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(version 19.0) statistical analysis software (IBM SPSS, Inc., IL, USA) with

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differences being considered significant at P < 0.05. Pearson correlation analysis was

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used to assess the relationships between GA3 content, gene expressions and enzyme

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activities.

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RESULTS AND DISCUSSION

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Effect of SA on CI index in tomato fruit during storage at 4 °C. CI symptoms

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in all the fruit appeared after 14 days of 4 °C storage plus 3 days at 20 °C, and

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continued to progress over time (Figure 1). Compared with the control, 0.5 mM SA

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treatment significantly inhibited the CI development of fruit, with an index 26% less

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than that in the control on day 28 (P < 0.05). One millimolar SA remarkably retarded

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the increase in the CI index, which was 44% less than that in the control at the end of

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cold storage (P < 0.05). As noted in this figure, treatment with 1mM SA led to the

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lower CI index compared with the control or 0.5 mM SA. Based on these results, 1

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mM SA was chosen for further analyses.

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Effect of SA on GA3 content and GA3ox1 gene expression in tomato fruit

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during storage at 4 °C. SA has great agronomic potential to improve the stress

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tolerance of agriculturally important crops.25 However, little information is available

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on the role of bioactive GA and its signaling components in SA-induced cold

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tolerance of postharvest fruit during long-term storage. The present study investigated

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the levels of bioactive GA (GA3) and the expression patterns of a GA biosynthetic

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gene (GA3ox1) in the control and the SA-treated fruit. The levels of endogenous GA3

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in the control fruit reduced gradually from day 3 through day 14, and then increased

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afterward (P < 0.05; Figure 2A). SA treatment notably elevated the GA3 content,

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which was 3.65-, 3.17-, 3.03-, and 1.71-fold higher than that in the control fruit on

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days 3, 7, 14, and 21, respectively (P < 0.05). In the SA-treated fruit, the increased

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GA3 levels might contribute to alleviating chilling symptoms. GA biosynthetic gene

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GA3ox1 and catabolic gene GA2ox1 hold the key to bioactive GA levels in higher

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plants.26 GA homeostasis is maintained through a negative feedback regulation of GA

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anabolism and a positive feedback regulation of GA catabolism.26,27 Low temperature

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disrupts GA homeostasis in postharvest tomato fruit mainly through suppressing the

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gene expression of GA3ox1.16 The present data showed that the GA3ox1 expression

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patterns in both the control and the SA-treated fruit closely paralleled the fluctuations

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of GA3 levels (Figure 2B). A high positive correlation was found between GA3ox1

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gene expression and GA3 content in both the control and the SA-treated fruit with

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Pearson’s coefficients of 0.90 and 0.88 (P < 0.01), respectively (Figure 2C). It

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suggested that the exogenous application of SA stimulated GA biosynthesis through

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upregulating the transcript levels of GA3ox1 in tomato fruit during cold storage.

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Likewise, Lee and Park19 also reported that SA could promote the seed germination of

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Arabidopsis under high salinity by inducing the gene expression of GA3ox1.

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Effect of SA on GAI gene expression in tomato fruit during storage at 4 °C.

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Key components of the GA signaling pathway are the nuclear-localized DELLA

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proteins.28 DELLA proteins are plant growth repressors whose degradation is

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promoted by GA.29 GAI gene expression in the control fruit increased and reached a

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peak on day 14, and then decreased gradually. In the SA-treated fruit, the transcript

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levels of GAI were 50%, 44%, 30%, and 22% lower than those in the control fruit on

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days 3, 7, 14, and 21, respectively (P < 0.05) (Figure 3A). The GAI gene expression

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was negatively correlated with the GA3 levels in both the control and the SA-treated

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fruit with Pearson’s coefficients of 0.85 and 0.61 (P < 0.01), respectively (Figure 3B).

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The present results revealed that SA application stimulated GA biosynthesis, and then

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downregulated GAI expression in postharvest tomato fruit during long-term cold

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storage.

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Effect of SA on gene expression of CBF1 and GA2ox1 in tomato fruit during

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storage at 4 °C. The transcript levels of cold-responsive gene CBF1 in the control

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fruit increased gradually from day 3 through day 14, and decreased sharply afterward

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(P < 0.05; Figure 4A). Compared with the control, the SA-treated fruit exhibited

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higher levels of CBF1 expression at the corresponding time points. The data were

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supported by the findings of Dong et al.,30 suggesting that exogenous SA application

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elevated expression levels of CBF in chilling responses in cucumber seedlings. As

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shown in Figure 4B, the expression of GA2ox1 in the control fruit reached a

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maximum on day 14, and then decreased gradually under GA deficiency caused by

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low temperatures (P < 0.05). In the SA-treated fruit, the transcript levels of GA2ox1

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were upregulated, which were 1.73-, 1.64-, 1.60-, and 1.56-fold higher than those in

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the control fruit, respectively (P < 0.05). The present study suggested that the positive

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feedback regulation in chilled tomato responded normally to the increased GA3

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content induced by exogenous SA application. In addition, the CBF1 expression

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patterns (Figure 4A) roughly mirrored the observed variation of GA2ox1 expression

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(Figure 4B). A positive correlation was found between CBF1 and GA2ox1 expression

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patterns in both the control and the SA-treated fruit with Pearson’s coefficients of 0.67

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and 0.88 (P < 0.01), respectively (Figure 4C). It is indicated that CBF1 participated in

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SA-modulated chilling tolerance and regulated the expression of GA2ox1. This result

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was consistent with previous studies,28,31-33 which indicated that CBF modulated GA

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catabolism via the induction of GA2ox genes in Arabidopsis, tobacco and soybean

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plants under abiotic stresses.

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Effect of SA on ROS production and antioxidant enzyme activities in tomato

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fruit during storage at 4 °C. Oxidative stress, that is the excess production of ROS

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including O2•− and H2O2, has been reported to be associated with the CI development

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of postharvest fruit.34 In the present study, the O2•− production rate in the control fruit

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increased gradually throughout the storage period (P < 0.05; Figure 5A). SA

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treatment significantly reduced the O2•−production rate in the treated fruit, which were

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12%, 10%, 8%, and 5% lower than that in the control fruit (P < 0.05). The H2O2

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content in the control fruit showed a remarkable increase, while the H2O2 content in

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the SA-treated fruit was significantly lower than that in the control fruit (P < 0.05;

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Figure 5B). These results indicated that the exogenous application of SA inhibited

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ROS overproduction and alleviated ROS-mediated oxidative damages resulting in CI

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in tomato fruit under chilling stress, which is consistent with the studies reporting that

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SA treatment reduced endogenous levels of ROS in lemon flavedo35 and peach fruit36

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during cold storage.

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The antioxidant enzyme system plays a critical role in ROS scavenging to protect

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cell membranes, which is thought to be a major mechanism of resistance to chilling

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stress.37,38 As shown in Figure 5C, the SOD activity in the control fruit increased

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gradually during the early storage period. SA treatment slightly but significantly

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induced the SOD activity, which was 1.21-, 1.13-, 1.12-, and 1.08-fold higher than

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that in the control fruit on days 3, 7, 14, and 21, respectively (P < 0.05). The CAT

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activity in the control fruit reached a maximum on day 14, and then declined sharply

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(Figure 5D). In the SA-treated fruit, the CAT activity showed the same trend as the

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control but was significantly higher from day 3 through day 14 (P < 0.05). It has been

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shown that SOD can catalyze the dismutation of O2•− to H2O2, and H2O2 can be

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scavenged by CAT.34,38 In this study, less ROS accumulation in the SA-treated fruit

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was attributed to increased activities of SOD and CAT during early stages of cold

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storage. A recent study reported that the overexpression of PmhCBFc in Arabidopsis

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improved freezing- and oxidative-stress tolerance, and the SOD activity was elevated

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in the transgenic Arabidopsis during freezing–thawing cycles.39 Hsieh et al.40 pointed

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out that the enhancement of chilling tolerance in transgenic tomato overexpressing

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CBF1 may be due to the induction of the CAT1 gene expression and its enzyme

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activity. In the present study, a high positive correlation was also found between

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CBF1 gene expression and SOD activity, and CAT activity in the control and the

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SA-treated fruit with Pearson’s coefficients of 0.91 and 0.88, and 0.59 and 0.71 (P