Ethylene Perception Is Associated with Methyl-Jasmonate-Mediated

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Agricultural and Environmental Chemistry

Ethylene Perception Is Associated with Methyl JasmonateMediated Immune Response against Botrytis cinerea in Tomato Fruit Wenqing Yu, Mengmeng Yu, Ruirui Zhao, Jiping Sheng, Yujing Li, and Lin Shen J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 22 May 2019 Downloaded from http://pubs.acs.org on May 27, 2019

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Journal of Agricultural and Food Chemistry

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Ethylene Perception Is Associated with Methyl Jasmonate-Mediated Immune

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Response against Botrytis cinerea in Tomato Fruit

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Wenqing Yu, † Mengmeng Yu, † Ruirui Zhao, † Jiping Sheng, ‡ Yujing Li, † and Lin

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Shen*,†

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† College

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Beijing 100083, China

8

‡

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China, Beijing 100872, China

of Food Science and Nutritional Engineering, China Agricultural University,

School of Agricultural Economics and Rural Development, Renmin University of

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* Corresponding Author

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Lin Shen: Tel: +86-10-62737620; E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Jasmonic acid (JA)- and ethylene- mediated signaling pathways, are reported to have

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synergistic effects on inhibiting gray mold. The present study aimed to explain the

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role of ethylene perception in methyl jasmonate (MeJA)-mediated immune responses.

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Results showed that exogenous MeJA enhanced disease resistance, accompanied by

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the induction of endogenous JA biosynthesis and ethylene production, which led to

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the activation of phenolic metabolism pathway. Blocking ethylene perception by

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using 1-methylcyclopropene (1-MCP), either before or after MeJA treatment could

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differently weaken the disease responses induced by MeJA, including suppressed the

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induction of ethylene production and JA contents, and reduced activities of

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lipoxygenase and allene oxide synthase, compared with MeJA treatment alone.

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Consequently, MeJA-induced elevations in the total phenolic content and the

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activities of phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, 4-coumarate:

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coenzyme A ligase, peroxidase were impaired by 1-MCP. These results suggested that

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ethylene perception participated in MeJA-mediated immune responses in tomato fruit.

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KEY WORDS: methyl jasmonate, ethylene perception, 1-methylcyclopropene,

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immune response, Botrytis cinerea, tomato fruit

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INTRODUCTION

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Tomato (Solanum lycopersicum) is a horticultural commodity with superior

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economic value, which was also widely used as a model in the field of fruit

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postharvest diseases, since it displays greatly vulnerability to postharvest rot caused

34

by various fungal pathogens.1-2 Botrytis cinerea (B. cinerea) is one of the most

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destructive fungi of tomato, which can infect tomato fruit through epicarpic wounds

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caused during harvesting. With time, the disease progresses and a fuzzy gray mold

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develops during storage, which lessens shelf-life and reduces consumer acceptability,

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resulting in significant economic loss.3-4

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Salicylic acid (SA)-, jasmonic acid (JA)-, and ethylene (ET) -mediated signaling

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pathways are important in response to pathogen attack.5 In general, SA-mediated

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signaling is mainly required to defend against biotrophic pathogens, whereas defend

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against necrotrophic pathogens, such as B. cinerea, is mainly dependent on JA- and/or

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ET-mediated signaling.6 The plant hormone, JA and its volatile methyl ester, methyl

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jasmonate (MeJA), are well-studied activators of plant defense.7-8 Evidence has

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shown that MeJA could potentially induce immune response against pathogen

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infection and inhibit various postharvest rots of tomato, such as Alternaria diseases

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caused by Alternaria alternate and Alternaria porri f. sp. solani ,9-10 Fusarium wilt

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caused by Fusarium oxysporum f. sp. lycopersici,11 and grey mold rot caused by B.

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cinerea.12-15 MeJA treatment effectively mitigated the disease development of tomato

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caused by pathogens, by (1) activation of the phenylpropanoid pathway and the

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accumulation of total phenolics,10, 15 (2) induction of defense-related enzymes, such as 3



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pathogenesis-related proteins (PRs), and peroxidase (POD)11, 15, and (3) inhibition of

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pathogen-induced oxidative stress by promoting both activities and transcript levels of

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antioxidant enzymes.12 Moreover, previous studies have documented that ethylene

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biosynthesis

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1-aminocyclopropane-1-carboxylic acid oxidase (ACO), which is the key enzyme in

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ethylene biosynthesis, was associated with MeJA-induced disease resistance in

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tomato.13-14 The above results revealed that ethylene biosynthesis is important for

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MeJA-mediated immune responses in tomato, however whether ethylene perception is

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required for MeJA-induced disease resistance needs to be investigated.

could

be

induced

by

exogenous

MeJA,

and

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Ethylene, a simple gaseous phytohormone, has long been proposed to play

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critical roles in plant immune responses.16-17 When infected by B. cinerea, tomato

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plants activated ethylene biosynthesis and signaling transduction in response to

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pathogen attack.18 Indeed, not only ethylene biosynthesis and signaling transduction,

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ethylene perception is also important for plant resistance against necrotrophic

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pathogens,19-20 as attested by the fact that the impaired perception of ethylene via

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silencing ethylene receptor (ETR) gene AtETR1 resulted in increased disease

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resistance.21 Accordingly, tomato Never ripe (Nr) mutant, in which ethylene

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perception is impaired, showed decreased disease symptoms.20, 22 In addition, in the

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SlETR4-silenced plants, disease incidence and severity were reduced, suggesting that

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impaired perception of ethylene via the ETR4 receptor resulted in increased disease

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resistance.22 Although the involvement of ethylene perception in disease resistance

73

has been documented, little is known about the relationship between ethylene 4


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perception and MeJA-mediated disease resistance against gray mold in postharvest

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tomato fruit.

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1-Methylcyclopropene (1-MCP) is thought to block ethylene perception, retard

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ethylene responses, and inhibit ethylene-dependent processes such as ripening and

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senescence due to its effect on competing with ethylene for binding site on ethylene

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receptors.23-24 Extensive studies have proven its inhibition effect on ethylene

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perception, and some studies also showed that 1-MCP application could effectively

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improve disease resistance against pathogens in various fruits, including loquat,24

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jujube,25 sweet potato,26 strawberry,27 and tomato.28-29 Exogenous 1-MCP treatment

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significantly improved fruit disease resistance, by (1) maintaining fruit natural

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resistance, such as delaying senescence development and retaining firmness,24-25, 29 (2)

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inhibiting the increase in electrical conductivity and malondialdehyde content,29 (3)

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decreasing ROS accumulation and increasing the activities of some key antioxidant

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enzymes,24-25 (4) inducing higher activities of defense-related enzymes, such as

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chitinase and β-1,3-glucanase.24,

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pretreatment weakened the disease resistance induced by Cryptococcus laurentii in

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tomato fruit, and downregulated the expression of genes that were related to ethylene

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perception.28 Moreover, a previous study showed that exogenous MeJA could

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enhance ester regeneration in 1-MCP-pretreated apple fruit, accompanied by the

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upregulation of genes related to ethylene perception.30 However, the interaction

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between MeJA and 1-MCP on disease resistance against gray mold was still not clear.

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Previous studies have reported that both ethylene biosynthesis and ethylene

29

In addition, it has been reported that 1-MCP

5



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signaling component Solanum lycopersicum ethylene response factor 2 (SlERF2)

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played crucial roles in MeJA-mediated disease resistance.14-15 The present study

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explored the relationship between ethylene perception and MeJA-mediated disease

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resistance in tomato fruit. This study aims to do the following: (1) determine the

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effect of exogenous MeJA on endogenous JA biosynthesis; (2) investigate whether

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there is a positive relationship between endogenous ethylene and JA after MeJA

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treatment; (3) evaluate the change in MeJA-mediated disease resistance against gray

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mold in tomato fruit, either before or after blocking ethylene perception by 1-MCP.

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MATERIALS AND METHODS

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Fruit Materials and Treatments. Green mature tomato fruit (Solanum

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lycopersicum cv. Lichun) were harvested from a local commercial greenhouse in

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Beijing, China, and then transported to our laboratory within 2 h. The selected fruit of

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uniform shape, ripeness, size, and without calyxes and visible blemishes were used

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for the following experiments. Eighteen hours after harvesting, fruit were

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surface-sterilized with 2% (v/v) sodium hypochlorite aqueous solution for 2 min, and

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then rinsed thoroughly with tap water and air-dried.

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All tomato fruit were randomly assigned to five groups containing 70 fruit each,

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and all treatments were applied in 50 L airtight containers. 1-MCP can block ethylene

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perception by interacting with ethylene receptors. The fumigation treatments were

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applied as follows:

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(1) Control: tomato fruit were fumigated with distilled water at 25±1°C for 12 h, and 6


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then rinsed with distilled water for 1 min. After drying, fruit were fumigated with

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distilled water for 12 h at 25±1°C.

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(2) 1-MCP: tomato fruit were pre-fumigated with distilled water at 25±1°C for 12 h,

121

and then rinsed with distilled water for 1 min. After drying, fruit were fumigated with

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0.5 μL·L−1 1-MCP at 25±1°C for 12 h.29

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(3) 1-MCP+MeJA: tomato fruit were pre-fumigated with 0.5 μL·L−1 1-MCP at

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25±1°C for 12 h, and then rinsed with distilled water for 1 min. After drying, fruit

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were fumigated with 0.1 mM MeJA for 12 h at 25±1°C.

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(4) MeJA: tomato fruit were pre-fumigated with distilled water at 25±1°C for 12 h,

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and then rinsed with distilled water for 1 min. After drying, fruit were fumigated with

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0.1 mM MeJA for 12 h at 25±1°C.15

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(5) MeJA+1-MCP: tomato fruit were pre-fumigated with 0.1 mM MeJA at 25±1°C

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for 12 h, and then rinsed with distilled water for 1 min. After drying, fruit were

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fumigated with 0.5 μL·L−1 1-MCP for 12 h at 25±1°C.

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After treatment, all fruit were rinsed with distilled water for 1 min and air-dried

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(time 0), and then stored at 25±1°C with 85−90% relative humidity (RH) for 9 d. Five

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fruit from each group were randomly sampled on 0, 0.125, 0.5, 0.75, 1, 3, 6, and 9 d

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during storage for assay of JA content, lipoxygenase (LOX) activity, allene oxide

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synthase (AOS) activity, SlCOI1 relative expression, total phenolic content, and

137

activities of phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H),

138

4-coumarate: coenzyme A ligase (4CL), and POD. Mesocarp tissue from fruit equator

139

area was cut into small pieces, frozen immediately in liquid nitrogen, and then stored 7



140

at −80°C until used. Ten fruit from each group were chosen separately for

141

measurement of disease symptoms. For ethylene production detecting, ten fruit from

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each group were taken on different days. Three biological replicates were carried out

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in this experiment.

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Pathogen Inoculation. The pathogen B. cinerea (ACCC 36028) was purchased

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from the Agricultural Culture Collection of China (Haidian, Beijing), and cultured

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according to Zheng et al.31 The spore suspension of B. cinerea was obtained by

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washing one-week-old fungal cultures with 0.01% Tween-80 solution, and then

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filtered through the sterile gauze. The concentration of spore suspension (2×106 spore

149

mL−1) was modified by the aid of a hemocytometer.31

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Twenty-four hours after treatment, at each fruit equatorial region, three uniform

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holes (2 mm wide × 4 mm deep) were gently made with a sterile nail, and then 10 μL

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of B. cinerea spore suspension were inoculated to each wound.15 Each inoculated fruit

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was placed in a single plastic bag, and then incubated at 25±1°C with 90–95% RH for

154

disease development. Three biological replicates were carried out in this experiment.

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Determination of Disease Symptoms. On the fourth day after inoculation,

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disease incidence and lesion diameter were recorded.15 Disease incidence was

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calculated as the percentage of the inoculated spot showing visible gray mold lesion,

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and lesion diameter was measured with the cross method.15, 32

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Determination of Ethylene Content. Ethylene production was assayed as

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described by Yu et al.15 Ten tomato fruit from each group were sealed in a 9 L airtight

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chamber at 25°C and incubated for 1 h. Triplicate 1 mL of the headspace gas sample 8


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was sucked out and injected into the gas chromatograph (GC-14C, Shimadzu, Kyoto,

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Japan) for ethylene determination. The flow rates used for nitrogen carrier gas, air,

164

and hydrogen were 50 mL·min−1, 400 mL·min−1, and 45 mL·min−1. The temperature

165

of column and injector were 50°C and 120°C. Ethylene content was expressed as

166

nmol·g−1 FW (fresh weight) h−1. All results were replicated three times.

167

Determination of Endogenous JA Content. JA content was measured using an

168

enzyme-linked immunosorbent assay (ELISA) Kit, with the polyclonal anti-JA

169

antibody.13 Frozen tomato tissue (2.0 g, in powder form) was homogenized with 5 mL

170

of extraction buffer (80% methanol, containing 1% (w/v) polyvinylpyrrolidone (PVP)

171

and 0.6% (w/v) benzothiadiazole). After extraction with ultrasonic at 4°C overnight,

172

the homogenate was centrifuged at 10,000×g for 20 min at 4°C. The supernatant was

173

dried with nitrogen, and then dissolved in the extracting solution to a volume of 1.5

174

mL before ELISA. JA content was assayed at 490 nm, which was expressed as ng·g−1

175

FW.

176

Determination of LOX and AOS Activities. To determine the activities of

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LOX (EC 1.13.11.12) and AOS (EC 4.2.1.92), frozen tomato tissue (2.0 g, in powder

178

form) was extracted with 5 mL of 50 mM phosphate buffer (pH 7.0), and centrifuged

179

at 10,000×g for 15 min at 4°C. The supernatant was collected and used for LOX and

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AOS enzymes determination. LOX activity was measured as described by Yu et al.,13

181

by measuring the formation of conjugated diene from hydroxyl fatty acids at 234 nm

182

and using linoleic acid sodium as the substrate. AOS activity was measured as

183

described by Sivasankar et al.,33 with slight modifications, by monitoring the decrease 9



184

in absorbance at 234 nm due to the degradation of the substrate. The activities of LOX

185

and AOS were calculated based on fresh weight, which were expressed as U·g−1 FW.

186

All results were replicated three times.

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Determination of Total Phenolic Content. Frozen tomato tissue (2.0 g, in

188

powder form) was extracted with 10 mL of 1% HCl-methanol (v/v), and then

189

centrifuged at 10,000 × g for 10 min at 4°C. The supernatant was collected and used

190

for total phenolic content assay.15 The total phenolic content was calculated based on

191

a standard curve of gallic acid, and the results was expressed as μg·g−1 FW.

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Determination of Enzymes Activities in the Phenolic Metabolism Pathway.

193

The activities of PAL (EC 4.3.1.5), C4H (EC 1.14.13.11), 4CL (EC 6.2.1.12) and

194

POD (EC 1.11.1.7) were calculated based on fresh weight, which were expressed as

195

U·g−1 FW. For analysis of PAL activity, frozen tomato tissue (2.0 g) was extracted

196

with 5 mL of 0.2 mM boric acid buffer (pH 8.8, containing 10% (w/v) PVP, 1 mM

197

EDTA and 5 μM β-mercaptoethanol).31 For analysis of C4H activity, frozen tomato

198

tissue (2.0 g) was extracted with 5 mL of 50 mM Tris–HCl buffer (pH 8.9, containing

199

15 mM β-mercaptoethanol, 4 mM MgCl2, 5 mM ascorbic acid, 10 μM leupeptin, 1

200

mM PMSF, 0.15% (w/v) PVP, and 10% (v/v) glycerol).34 For analysis of 4CL

201

activity, frozen tomato tissue (2.0 g) was extracted with 5 mL of 0.2 mM Tris–HCl

202

buffer (pH 7.5, containing 8 mM MgCl2, 2% (w/v) PVP, 5 mM DTT, 0.1% (v/v)

203

Triton X-100, and 1 mM PMSF).34 For analysis of POD activity, frozen tomato tissue

204

(2.0 g) was extracted with 5 mL of 0.1 M phosphate-buffered saline (pH 7.0).1 After

205

centrifuging at 10,000 × g for 10 min at 4°C, the supernatants were collected and used 10


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for enzymes activities assay. PAL activity was assayed from the generation of

207

trans-cinnamic acid from L-phenylalanine, and one unit of PAL activity was defined

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as the 0.01 increase in absorbance at 270 nm per hour.31 C4H activity was assayed

209

from the generation of p-coumaric acid from trans-cinnamic acid, and one unit of

210

C4H activity was defined as the 0.01 increase in absorbance at 340 nm per hour.34

211

4CL activity was assayed from the generation of p-coumaroyl-CoA from p-coumaric

212

acid, and one unit of 4CL activity was defined as the 0.01 increase in absorbance at

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334 nm per hour.34 POD activity was assayed from the oxidation of guaiacol, and one

214

unit of POD activity was defined as the 0.1 increase in absorbance at 470 nm per

215

minute.1

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Quantitative Real-Time PCR (qRT-PCR) Analysis. Total RNA was extracted

217

from frozen tomato tissue (0.15 g, in powder form) using an EasyPure Plant RNA Kit

218

(Beijing Transgen Biotech Co. Ltd., Beijing, China). The total RNA was quantified

219

by a NanoDrop 2000 Photometer spectrophotometer (Thermo Fisher Scientific,

220

Waltham, MA, USA), and then stored at -80°C. A total of 2 μg of RNA was used for

221

first-strand cDNA synthesis, using the TransScript One-Step gDNA Removal and

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cDNA Synthesis SuperMix Kit (Beijing Transgen Biotech Co. Ltd., Beijing, China).

223

The cDNA was stored at -20°C until used.15

224

qRT-PCR was performed using the TransStrat Top Green qPCR SuperMix

225

(Beijing TransGen Biotech Co., Ltd, Beijing, China) on a Bio-Rad CFX96 real-time

226

PCR system (Bio-Rad, USA). The qRT-PCR reaction mixture (in a total volume of 10

227

μL per reaction) was as follows: 5 μL of 2× SuperMix, 0.3 μL of specific primers 11



228

(Table 1), 1 μL of cDNA, and 3.4 μL of RNase-free water. The qRT-PCR thermal

229

cycling condition was as follows: initial denaturation for 30 s at 94°C, followed by 40

230

cycles of 5 s at 94°C, 15 s at 60°C, and 15 s at 72°C, and a melt cycle from 65 to

231

95°C.15 SlUbi3 was served as an endogenous reference gene, and the relative

232

expression was normalized to SlUbi3 Ct value using the 2 −ΔΔC t method.

233

Statistical Analysis. All data were obtained from three independent replicates,

234

and the data were expressed as the mean ± standard deviation (SD). Statistical

235

evaluations were conducted by one-way analysis of variance and Duncan’s multiple

236

range tests with the aid of SPSS20.0 (IBM Corp., Armonk, NY, USA). Differences at

237

P < 0.05 were considered as statistical significance. Pearson’s correlation analysis

238

was used to evaluate the relationships among ethylene content, JA biosynthesis, total

239

phenolic content, and enzyme activities related to phenolic metabolism pathway.

240 241

RESULTS

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Effects of 1-MCP, MeJA, (1-MCP+MeJA), and (MeJA+1-MCP) Treatments

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on Disease Incidence and Lesion Diameter. On the fourth day after inoculation with

244

B. cinerea, 1-MCP treatment decreased disease incidence and lesion diameter, which

245

were 40.4% and 45.6% lower than those in control (Figure 1, P < 0.05). Consistent

246

with our previous report,15 in comparison with control, disease incidence and lesion

247

diameter were significantly decreased by 35.1% and 37.7% after single MeJA

248

treatment (Figure 1, P < 0.05). However, either before or after fumigation with

249

1-MCP exerted a negative influence on the MeJA-induced disease symptoms. By 12


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pre-fumigation with 1-MCP, MeJA-induced decrease in disease symptoms was

251

impaired, and the disease incidence and lesion diameter in (1-MCP+MeJA)-treated

252

fruit were 1.16 and 1.41 times higher than those in single MeJA-treated fruit (Figure

253

1, P < 0.05). In addition, MeJA-induced disease resistance could also be abolished

254

after 1-MCP fumigation, and disease incidence in (MeJA+1-MCP)-treated fruit was

255

inhibited by 8.8% compared with control, whose inhibitory effect was 75.0% lower

256

than that in single MeJA-treated fruit (Figure 1A, P < 0.05). Contrastingly, as for

257

lesion diameter, no significant difference was observed between single MeJA-treated

258

and (MeJA+1-MCP)-treated fruit (Figure 1B, P > 0.05). These results suggested that

259

ethylene perception could be associated with MeJA-mediated disease resistance in

260

tomato fruit.

261


262

on Ethylene Production. In single 1-MCP-treated fruit, ethylene levels remained

263

nearly unchanged throughout the experiment period, which were significantly lower

264

than those in control (Figure 2A, P < 0.05). In single MeJA-treated fruit, ethylene

265

began to increase at the beginning of 0.5 d, and one peak was exhibited on the first

266

day, which was 3.17 times higher than that in control (Figure 2A, P < 0.05). However,

267

blocking ethylene perception, either before or after fumigation with 1-MCP weakened

268

the effect of MeJA on ethylene production. In 1-MCP pre-fumigated and

269

MeJA-treated fruit, ethylene production increased gradually and reached a maximum

270

on the third day (Figure 2B, P < 0.05). In fruit treated with MeJA followed by 1-MCP

271

fumigation, ethylene production shared a trend similar to the single MeJA-treated 13



272

fruit, while the peak value was 15.0% lower than that in single MeJA-treated fruit on

273

the first day (Figure 2B, P < 0.05).

274


275

on JA Pathway. MeJA treatment induced endogenous JA biosynthesis, and then

276

upregulated SlCOI1 (Solanum lycopersicum coronatine-insensitive 1) expression.

277

Moreover, blocking ethylene perception either before or after treatment with MeJA

278

suppressed the activation of JA pathway (Figure 3).

279

JA contents in single MeJA-treated fruit were drastically elevated, which

280

retained significantly higher levels than those in control (Figure 3A, P < 0.05).

281

Blocking ethylene perception with 1-MCP influenced the effect of MeJA-improved

282

content of JA. 1-MCP pre-fumigation inhibited the increase in JA contents induced by

283

MeJA, and JA contents in (1-MCP+MeJA)-treated fruit were 13.4%, 21.4%, 38.4%,

284

31.7%, and 32.4% lower than those in single MeJA-treated fruit (Figure 3A, P
0.05).

288

LOX activity in single MeJA-treated fruit increased rapidly and peaked on day

289

0.5 at 11.28 U·g−1 FW, and then gradually declined during the remaining time (Figure

290

3B, P < 0.05). Treatment with 1-MCP suppressed MeJA-induced elevation in LOX

291

activity. In 1-MCP pre-fumigated and MeJA-treated fruit, LOX activity increased

292

gradually and reached a maximum of 7.40 U·g−1 FW on the first day (Figure 3B).

293

Moreover, LOX activities in fruit treated with MeJA followed by 1-MCP fumigation 14


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were 29.2% and 20.5% lower than those in single MeJA-treated fruit on days 0.5 and

295

9 (Figure 3B, P < 0.05).

296

After the first day, single MeJA-treated fruit maintained remarkably higher AOS

297

activities, which was 54.4%, 49.3% and 14.3% higher than those in control on days 3,

298

6 and 9 (Figure 3C, P < 0.05). However, fumigation with 1-MCP weakened the effect

299

of MeJA. AOS activities in (1-MCP+MeJA)-treated fruit were 19.2%, 21.0% and

300

26.5% lower than those in single MeJA-treated fruit on days 1, 3 and 6 (Figure 3C, P

301

< 0.05). The change pattern in (MeJA+1-MCP)-treated fruit was similar to the single

302

MeJA-treated fruit, but the elevated AOS activity decreased after fumigation with

303

1-MCP, which were 9.2%, 19.3%, and 11.9% lower than MeJA treatment alone on

304

days 1, 3, and 6 (Figure 3C, P < 0.05).

305

SlCOI1 transcript levels were significantly induced by MeJA, which were 1.21-,

306

1.68-, 1.68-, 1.60-, 1.67-, and 1.42-fold higher than those in control (Figure 3D, P
0.05).

312


313

on Phenolic Metabolism Pathway. In comparison with control, MeJA treatment

314

maintained higher total phenolic content, and elevated activities of four key enzymes

315

related to phenolic metabolism pathway. Moreover, either before or after blocking 15



316

ethylene perception by 1-MCP, could weaken the effect of MeJA on phenolic

317

metabolism pathway (Figure 4).

318

Total phenolic contents were 16.2%, 19.5%, 24.1%, and 21.5% higher after

319

MeJA treatment than those in control on days 0.5, 1, 3, and 6, but the elevated effect

320

decreased differently either before or after fumigation with 1-MCP (Figure 4A, P

Ethylene Perception Is Associated with Methyl-Jasmonate-Mediated

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