3 and Antioxidant Enzymes Are Associated with H2O2

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SlMAPK1/2/3 and antioxidant enzymes are associated with H2O2-induced chilling tolerance in tomato plants Liu Wang, Ruirui Zhao, Yanyan Zheng, Lin Chen, Rui Li, Junfei Ma, Xiaofeng Hong, Peihua Ma, Jiping Sheng, and Lin Shen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01685 • Publication Date (Web): 10 Jul 2017 Downloaded from http://pubs.acs.org on July 11, 2017

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Journal of Agricultural and Food Chemistry

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SlMAPK1/2/3 and antioxidant enzymes are associated with H2O2-induced chilling

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tolerance in tomato plants

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Liu Wang,† Ruirui Zhao,† Yanyan Zheng,† Lin Chen,† Rui Li,† Junfei Ma,† Xiaofeng

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Hong,† Peihua Ma,† Jiping Sheng, *,‡ and Lin Shen*,†

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

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China, Beijing 100872, China

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ACS Paragon Plus Environment


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ABSTRACT

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Hydrogen peroxide (H2O2) acts as a signaling molecule in response to cold stress.

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Mitogen-activated protein kinases (MAPKs) and C-repeat/dehydration-responsive

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factor (CBF) play important roles in cold response regulation. To investigate the roles

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of MAPKs and CBF in H2O2-induced chilling tolerance, tomato (Solanum

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lycopersicum cv. Ailsa Craig) plants were treated with 1 mM H2O2 before chilling

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treatment. The results showed that H2O2 treatment protected subcellular structure,

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increased concentrations of abscisic acid (ABA), zeatin riboside (ZR), and methyl

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jasmonate (MeJA), but decreased the concentration of gibberellic acid (GA3).

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Furthermore, 1 mM H2O2 treatment enhanced the activities of antioxidant enzymes.

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Meanwhile, relative expressions of SlMAPK1/2/3 and SlCBF1 in H2O2-treated plants

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were higher than those in the control. Our findings suggest that H2O2 treatment might

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enhance chilling tolerance of tomato plants by activating SlMAPK1/2/3 and SlCBF1

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gene expression, and by regulating phytohormone concentrations and antioxidant

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enzyme activities.

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Keywords: tomato plants, chilling tolerance, hydrogen peroxide, SlMAPK1/2/3,

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SlCBF1, antioxidant enzymes

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INTRODUCTION

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Chilling is one of the most severe abiotic stresses that limit the growth and

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productivity of many plant species worldwide. As an important vegetable crop,

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tomato (Solanum lycopersicum) is sensitive to low temperatures and may suffer from

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chilling injury when exposed to temperatures between 0-12 °C.1,2

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Hydrogen peroxide (H2O2), which is a component of reactive oxygen species

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(ROS), is often induced in plants under various stress conditions. ROS overproduction

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may lead to oxidative damages of cellular membranes, proteins, nucleic acids, and

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other cellular components, resulting in cell function impairing.3 However, it is now

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clear that H2O2 not only functions as a toxic cellular metabolite,4 but also acts as a

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signaling molecule in responses to both biotic and abiotic stresses at moderate

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concentrations.5,6 As for abiotic stresses, H2O2 treatment increases tolerance to salt

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stress in maize seedlings7 and enhances chilling tolerance of manilagrass and

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mascarenegrass8 by activating the antioxidative system. Previous studies indicate that

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ROS interacts with other signaling molecules such as mitogen-activated protein

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kinases (MAPKs) and plant hormones to respond to abiotic stresses.3,9 However,

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signaling pathways that are responsible for H2O2-induced chilling tolerance in tomato

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plants is still not clear.

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Genetically, many cold-induced pathways are activated to protect plants from

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deleterious effects of cold stress. Until now, most studies have been focused on the

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ICE-CBF-COR signaling pathway, which plays an essential role during chilling

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injury.6 C-repeat/dehydration-responsive element binding factor (CBF), which binds

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to cold-induced genes, plays an important role in the regulation of cold response.10

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There are three CBF genes present in tomato, but only the expression of SlCBF1

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increased under cold stress1 and our previous study showed that SlCBF1 gene

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expression was positively correlated with chilling tolerance. Thus, it suggested that

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SlCBF1 expression level could be used to quantify chilling tolerance of tomato

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plants.11

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Mitogen-activated protein kinase (MAPK) cascades consist of MAPK kinase

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kinase (MAPKKK), MAPK kinase (MAPKK), and MAPK, which are common

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signaling pathways to transduce extracellular stimuli into intracellular responses in

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many organisms, including yeast, humans, and plants.12 It has been reported that

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OsMKK6 and OsMPK3 constituted a low-temperature signaling pathway that

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regulated cold tolerance in rice13 and overexpression of SlMAPK3 in tobacco could

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increase low-temperature tolerance.14 MAPKs play important roles in responding to

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cold stress by (1) modulating diverse targets including transcription factors,

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cytoskeletal proteins, and other protein kinases,15 (2) activating antioxidant system,14

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and (3) interacting with signaling molecules such as ROS, NO, plant hormones, and

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cyclic nucleotides.16 Kovtun et al. found that H2O2 activated AtMAPK3 and AtMAPK6,

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which are two stress-related MAPKs, in Arabidopsis thaliana.17 In addition,

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SlMAPK1 and SlMAPK2 were orthologues of AtMAPK6, and SlMAPK3 was highly

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similar to AtMAPK3.12 Thus, we hypothesized that SlMAPK1/2/3 might participate in

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the H2O2-induced chilling resistance, in which plant hormones might also be

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implicated in.

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Plant hormones, such as abscisic acid (ABA), gibberellic acid (GA), methyl

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jasmonate (MeJA), and zeatin riboside (ZR) mediate responses to many stresses,

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which include low temperature stress.18 ABA, MeJA, and ZR positively regulate

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chilling tolerance of plants.19,20 Conversely, GA plays a negative role in plant

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resistance to cold stress.21

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To evaluate the effect of exogenous H2O2 on chilling tolerance of tomato plants, we

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examined the effect of exogenous H2O2 on the ultrastructure, malondialdehyde (MDA)

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content, ion leakage, protein content, soluble sugar content, and the activities of

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antioxidant enzymes that included superoxide dismutase (SOD), catalase (CAT),

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ascorbate peroxidase (APX), and peroxidase (POD) after chilling treatment. Moreover,

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concentrations of plant hormones including ABA, GA3, MeJA, and ZR were detected.

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In addition, relative expressions of SlMAPK1/2/3 and SlCBF1 of H2O2-treated and

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control plants after chilling treatment were explored to study the roles of

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SlMAPK1/2/3 and SlCBF1 in regulation of H2O2-induced chilling tolerance.

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MATERIALS AND METHODS

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Plant Material, H2O2 Application and Chilling Treatment. Tomato (Solanum

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lycopersicum cv. Ailsa Craig) seeds were sown in plastic pots (7cm diameter) that

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contained seedling substrate, soil, and vermiculite (v/v/v = 2:1:1), and then they were

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cultivated in a greenhouse (25 ± 2 °C, 60~65% relative humidity and photoperiod of

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16/8 h light/dark). 6-week-old tomato plants were used in the further experiment. The

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tomato plants were divided into two treatment groups of 30 plants each, plants were

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sprayed with 1 mM H2O2 solution or water (as control), then were placed in a dark

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chamber for 4 h for full absorption of H2O2 solution. Afterwards, these plants were

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transferred to a chamber at 4 ± 0.5 °C with 16/8 h light/dark photoperiod. Three

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biological replicates were carried out in this experiment.

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Functional leaves from the same position were collected after 4 h absorption of

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H2O2 solution (time 0), at 1, 2, 4, 8, 16, and 24 h and on days 1, 3, 5, and 7 under low

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temperature stress, then frozen quickly in liquid nitrogen and stored at -80 °C until

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used for measurements of SlMAPK1/2/3 and SlCBF1 gene expression, MDA content,

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soluble protein content, soluble sugar content, antioxidant enzyme activities, and plant

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hormone concentrations. Meanwhile, ion leakage was measured simultaneously when

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sampling. Additionally, 15 tomato plants each for control and 1 mM H2O2 treatment

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were used to observe the phenotype under 4 °C condition, and three days after

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chilling treatment, photographs of plants with representative effects were taken.

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Quantitative Real-Time PCR (qRT -PCR) Analysis. A 150 mg sample of frozen

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leaves in powder form was used to extract the total RNA with EasyPure Plant RNA

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Kit (Beijing Transgen Biotech Co. Ltd., Beijing, China). Total RNA was dissolved in

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30 µL of RNase-free water, quantified by a microspectrophotometry (NanoDrop

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Technologies, Inc.), and stored at -80 °C. Reverse transcription was completed to

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synthesize the first strand of cDNA with 2 µg total RNA, according to the instructions

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from TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit

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(Beijing Transgen Biotech Co. Ltd., Beijing, China).

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The Bio-Rad CFX96 real-time PCR system (Bio-Rad, USA) and TransStart Top

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Green qPCR SuperMix (Beijing Transgen Biotech Co. Ltd., Beijing, China) were used

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according to the supplier’s instructions to perform qRT-PCR. The thermal cycling

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conditions for qRT-PCR were as following: 94 °C for 30 s, followed by 40 cycles at

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94 °C for 5 s, 60 °C for 15 s, and 72 °C for 15 s. Specific primers used for qRT-PCR

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including SlCBF1, SlMAPK1, SlMAPK2, SlMAPK3, and β-Actin were designed (Table1),

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and synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). β-Actin was used as an

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internal control and the relative gene expression was calculated using the 2−∆∆CT

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method.22

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Transmission Electron Microscopy. On the third day after chilling treatment, leaf

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tissues (about 1 mm2) were taken using a scalpel. Samples were processed according to

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Han et al. with some modifications.23 Leaf samples were fixed with 2.5% (v/v)

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glutaraldehyde at room temperature overnight, and then they were washed with 0.1 M

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phosphate buffer (PBS, pH 7.2) three times. After that, samples were post-fixed with 1%

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(w/v) osmic acid for 3 h, dehydrated with a gradient ethanol solution. Samples were

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washed with propylene oxide and later embedded in Spurr resin at 60 °C for 24 h. An

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ultramicrotome (EM UC6, Leica, Germany) was used to cut the embedded samples into

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80 nm ultrathin sections. These sections were mounted on the mesh grids and stained

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with uranyl acetate and lead citrate. Finally, leaf cells were observed with a transmission

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electron microscope (HITACH7500, Japan) at 80 KV and photos were taken

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simultaneously.

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Assay of Ion Leakage. Ion leakage was measured before chilling (time 0) and on

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days 1, 3, 5, and 7 after chilling treatment. The measurement method was that of Zhao

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with some modifications.11 Ten leaf discs were excised with a 1 cm diameter stainless

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steel cork borer, then they were immersed in vials that contained 40 mL deionized

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water, and shaken at 150 rpm for 2 h. The conductivity of the solution (L1) was

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measured with a conductivity meter (DDS-11A, Shanghai Leici Instrument Inc.,

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Shanghai, China). Later, the solution was boiled for 15 min, cooled to room

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temperature, and conductivity of the solution was measured as L2. Ion leakage was

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calculated as (L1/L2) ×100%.

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Assay of Malondialdehyde (MDA) Content. MDA content was measured

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according to the thiobarbituric acid method of Ding et al.24 Absorbance at 532 nm was

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recorded and corrected for nonspecific absorbance at 600 nm. The content of MDA

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was calculated by its extinction coefficient of 155 mM-1cm-1 and expressed as

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mmol·g-1 FW.

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Assay of Protein Content and Soluble Sugar Content. Protein content was

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determined by absorbance at 595 nm using bovine serum albumin as standard using the

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method of Bradford.25 The content of soluble sugar was measured by anthrone

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colorimetry according to Cao et al.26

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Assay of Antioxidant Enzymes. To determine the activity of antioxidant enzyme,

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superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate

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peroxidase (APX, EC 1.11.1.11), and peroxidase (POD, EC 1.11.1.7) were analyzed

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as representatives. Frozen leaf samples (0.5 g) in powder form were extracted with 5

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mL cold 100 mM PBS (pH 7.0), and an IKA Disperser (T10 basic, IKA, German) was

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used to homogenize the mixture. The homogenates were centrifuged at 13000g for 15

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min at 4 °C, and the supernatants were collected for enzyme analysis. Activities of the

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four enzymes were expressed as U·g-1 FW. SOD Detection Kit (A001, Jiancheng,

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Nanjing, China) was used to measure SOD activity. CAT activity was determined by

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the method of Larrigaudiere et al.27 One unit of CAT activity was defined as the

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amount of enzyme that caused 0.01 decrease of absorbance at 240 nm per minute.

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APX activity was measured following the method of Nakano et al. with some

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modifications.28 One unit of APX activity was expressed as 0.01 OD decreased at 290

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nm per minute. POD activity was surveyed according to Doerge et al. with some

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modifications,29 and one unit of POD activity was defined as 1 increase of absorbance

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per minute at 470 nm.

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Assay of Plant Hormone Concentrations. Indirect competitive enzyme-linked

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immunosorbent assay (icELISA) was used to determine concentrations of ABA, GA3,

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ZR, and MeJA according to the method described by Deng et al.30 and Wang et al.31

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with some modifications. About 0.2 g sample in powder form was extracted and

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homogenized in cold 80% (v/v) methanol, which contained 1 mM butylated

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hydroxytoluene, and then the sample was incubated at 4 °C overnight. The homogenates

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were centrifuged at 13000g for 15 min at 4 °C and the supernatants were collected for

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further analysis. The supernatants were passed through C18 Sep-Pak cartridges (Waters

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Corp., Millford, MA, USA) and dried in nitrogen. Then, the residues were dissolved

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with 2 mL cold 0.1 mM phosphate buffer sodium (pH 7.5, which contained 1% (v/v)

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Tween-20 and 1% (w/v) gelatin) for further determination of hormone concentrations.

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The absorbance was recorded in a microplate reader at 490 nm.

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Statistical Analysis. One-way analysis of variance (ANOVA) and Duncan’s multiple

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range tests were used for statistical evaluations using the statistical analysis software

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SPSS 18.0 (IBM Corp., Armonk, NY); differences with P < 0.05 were considered

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significant.

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RESULTS

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Effects of H2O2 Treatment on the Phenotype of Tomato Plants. No obvious

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differences in morphology of H2O2-treated and control plants could be observed

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before chilling treatment. However, three days’ exposure to 4 °C caused wilting in

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tomato plants, especially in the control plants. Significant differences could be

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observed in the phenotype between the two groups after chilling treatment.

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Photographs of representative plants were taken, and we could see easily that the

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control plant wilted seriously, while only two leaves of H2O2-treated plant wilted

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(Figure 1).

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Effects of H2O2 Treatment on the Subcellular Structure of Tomato Plants.

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Subcellular structure of tomato plants cells was observed on the third day after

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chilling treatment. Cell membrane was located tightly against the cell wall in most

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leaf cells of the H2O2-treated plants while a large proportion of cells in the control

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plants showed obvious plasmolysis (Figure 2A and C). On the third day after treated

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at 4 °C, the majority of chloroplasts in the control group swelled obviously, and most

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thylakoids became distorted, except for a small proportion remained normal (Figure

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2B). In contrast, most chloroplasts in the H2O2-treated plants remained spindle-shaped,

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except for a few that were swollen to some extent, but their condition was

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significantly better than the control. Moreover, the chloroplast thylakoids in the

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H2O2-treated leaves were well-organized (Figure 2D).

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Effects of H2O2 Treatment on Ion Leakage, MDA Content, Protein Content,

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and Soluble Sugar Content. Ion leakage of H2O2-treated and control plants

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increased during the chilling treatment. Ion leakage of H2O2-treated plants was higher

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than the control from the beginning, but after the first day under 4 °C, ion leakage of

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control plants increased rapidly and became higher than the H2O2-treated plants

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(Figure 3A, P

3 and Antioxidant Enzymes Are Associated with H2O2

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