Melatonin Induces Disease Resistance to Botrytis cinerea in Tomato

Subscriber access provided by Bibliothèque de l'Université Paris-Sud

Agricultural and Environmental Chemistry

Melatonin Induces Disease Resistance to Botrytis cinerea in Tomato Fruit by Activating Jasmonic Acid Signaling Pathway Chunxue Liu, Lingling Chen, Ruirui Zhao, Rui Li, Shujuan Zhang, Wenqing Yu, Jiping Sheng, and Lin Shen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00058 • Publication Date (Web): 14 May 2019 Downloaded from http://pubs.acs.org on May 14, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39

Journal of Agricultural and Food Chemistry

1

Melatonin Induces Disease Resistance to Botrytis cinerea

2

in Tomato Fruit by Activating Jasmonic Acid Signaling

3

Pathway

4

Chunxue Liu,† Lingling Chen,† Ruirui Zhao,† Rui Li,† Shujuan Zhang,† Wenqing Yu,†

5

Jiping Sheng,‡ and Lin Shen*,†

6

† College of Food Science and Nutritional Engineering, China Agricultural

7

University, Beijing 100083, China

8

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

9

China, Beijing 100872, China

10

Corresponding Author

11

*E-mail: [email protected]; Phone: +86-10-62737620; Fax: +86-10-62737620.

1

ACS Paragon Plus Environment


12

ABSTRACT

13

Melatonin acts as a crucial signaling molecule with multiple physiological

14

functions in plant response to abiotic and biotic stresses. However, the impact and

15

regulatory mechanism of melatonin on attenuating tomato fruit fungal decay are

16

unclear. In this study, we investigated the potential roles of melatonin in modulating

17

fruit resistance to Botrytis cinerea and explored related physiological and molecular

18

mechanisms. The results revealed that disease resistance was strongly enhanced by

19

melatonin treatment and 50 μM was confirmed as the best concentration. Melatonin

20

treatment increased the activities of defense-related enzymes and decreased hydrogen

21

peroxide (H2O2) content with enhanced antioxidant enzyme activities. Moreover, we

22

found that melatonin treatment increased methyl jasmonate (MeJA) content,

23

up-regulated the expressions of SlLoxD, SlAOC and SlPI II, and reduced the

24

expressions of SlMYC2 and SlJAZ1. We postulated that melatonin played a positive

25

role in tomato fruit resistance to Botrytis cinerea through regulating H2O2 level and

26

JA signaling pathway.

27

Keywords: melatonin, tomato fruit, Botrytis cinerea, jasmonate signaling, hydrogen

28

peroxide

2


Page 2 of 39

Page 3 of 39


29

INTRODUCTION

30

Tomato (Solanum lycopersicum), a vegetable crop cultivated worldwide,

31

constitutes a crucial part of agricultural industry.1 However, biotic stresses such as

32

phytopathogens attack contribute to substantial yield loss of tomato and other crops.2

33

According to the lifestyles, pathogens are generally classified as biotrophs and

34

necrotrophs. Biotrophs grow on living plant tissues, whereas necrotrophs destroy host

35

cells and fend on dead or dying materials.3 Botrytis cinerea (B. cinerea), one kind of

36

necrotrophic pathogen whose infection may cause enormous and constant damage in

37

plants, leads to gray mold disease in more than 200 crops.4

38

Melatonin (N-acetyl-5-methoxytryptamine), an indolic compound derived from

39

serotonin (5-hydroxytryptamine), was first discovered in the pineal gland of cows and

40

makes contribution to the regulation of many physiological events in animals.5 Since

41

the presence of melatonin in plants was identified by Dubbels et al.6 and Hattori et al.7

42

in 1995, continuous studies have led to an accumulation of information about its wide

43

distribution and multiple physiological functions in plants. Melatonin exists in nearly

44

all organs and tissues of plants and acts as a signaling molecule involved in numerous

45

physiological processes such as differentiation, growth, ripening and leaf senescence.5

46

In recent years, the number of studies on melatonin in plants increased markedly and

47

the protective effect of melatonin against an array of abiotic and biotic stresses has

48

been concerned.8 Many studies focused on the ability of melatonin in alleviating the

49

effect of abiotic stresses such as UV rdiation9, temperature fluctuations10,11,

50

drought12,13, and high salinity14. Additionally, the roles of melatonin in phytopathogen 3



Page 4 of 39

51

defense have also been discussed recently. For example, exogenous melatonin

52

contributed to greater resistance to Diplocarpon mali (D. mali) infection in apple

53

leaves.15 Melatonin decreased the infection rate of Penicillium spp. in non-sterilized

54

Lupinus albus seeds.16 Furthermore, the Arabidopsis SNAT knockout mutant with a

55

reduction in endogenous melatonin level suffered from the avirulent pathogen

56

Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) due to the reduced

57

expressions of defense genes (PR1, ICS1, and PDF1.2), while exogenous melatonin

58

treatment restored the pathogen resistance.17 However, the function and action

59

mechanism

60

melatonin-induced fungal resistance in tomato fruit are still unclear.

especially

the

definite

signaling

pathway

responsible

for

61

Jasmonates (JAs), like methyl jasmonate (MeJA) and its free acid, jasmonic acid

62

(JA), are essential phytohormones which regulate many aspects of growth,

63

development, and environmental responses in plants.18 It is generally believed that

64

activating JA signaling pathway can induce resistance against necrotrophs, which

65

always cause great damage to the host through cell-wall-degrading enzymes and

66

phytotoxin.3 The possible mechanism for JA-signaling dependent defenses was that

67

JAs promote the expressions of defense-related genes and induct most of the

68

defense-related secondary metabolites as well as proteins.19,20 Previous study has

69

pointed out that melatonin treatment induced JA accumulation in banana and

70

suggested that melatonin may have an effect on JA signaling pathway.21 Additionally,

71

the studies obtained from npr1, ein2, and mpk6 Arabidopsis mutants suggested that

72

melatonin involved in plant defense responses and suppressed bacterial multiplication 4


Page 5 of 39


73

based on the induction of action through salicylic acid (SA) and ethylene (ET)

74

signaling pathways, however, how melatonin acted in the regulation of JA signaling

75

pathway has not been described in detail yet.22

76

Reactive oxygen species (ROS) are signaling components and byproducts of

77

metabolism in plants, and have several possible functions in defense system.23

78

Previous studies have suggested that oxidative burst in plant was one of the early

79

defense responses to counteract pathogen invasion, and ROS were considered as

80

crosslinkers in plant cell wall to defense pathogens.24 However, other studies have

81

found that fungal produced ROS were crucial for pathogenic development and

82

necrotrophs could also regulate ROS accumulation to achieve full pathogenicity.25 In

83

recent years, many studies have emphasized the importance of melatonin in directly

84

or indirectly scavenging ROS in plants. For example, melatonin increased plant

85

resistance to a variety of abiotic stresses through inducing antioxidant enzyme

86

activities and scavenging H2O2.26,27,28 Additionally, although there was a study

87

indicating that the application of exogenous melatonin on apple trees alleviated the

88

disease damage caused by D. mali partly through keeping the intracellular H2O2

89

concentrations at steady levels and increasing the activities of plant defense-related

90

enzymes15, little has been known about the role that melatonin plays in regulation of

91

ROS to defense necrotrophic pathogens in fruits.

92

Melatonin is environmental and safe to animals and humans, and exogenous

93

melatonin treatment may be a promising strategy to protect postharvest fruits from

94

pathogen infection. To study the possible mechanism of melatonin-induced pathogen 5



95

resistance in tomato fruits, activities of defense enzymes, H2O2 content, antioxidant

96

enzyme activities, MeJA content as well as the relative expressions of JA signaling

97

pathway related genes were detected. The main focus of this study was to explore the

98

underlying regulatory mechanism of melatonin-induced pathogen resistance in tomato

99

fruit.

100

MATERIALS AND METHODS

101

Fruit Materials and Treatments. Tomato (Solanum lycopersicum cv. La-bi) fruits

102

were harvested at a mature green stage from a greenhouse which is special for

103

experiment at Shangzhuang Geothermal Special Vegetable Base, Beijing, China, and

104

immediately transported to the laboratory. Fruits were tagged at 2 days postanthesis

105

(dpa) and harvested at 45 dpa for mature green fruit. Fruits with uniformity in shape,

106

texture, color, size and without pathogens infection or physical injury were selected

107

for the following experiments. Twelve hours after picking, all fruits were

108

surface-disinfected with 2% (v/v) sodium hypochlorite for 2 min, washed with

109

distilled water for 2 times, and air-dried at room temperature. Melatonin

110

(N-acetyl-5-methoxytryptamine) was purchased from Sigma-Aldrich (St. Louis, MO,

111

USA). Three replicates were carried out in this experiment to obtain the results.

112

(1) Twenty-four tomato fruits were divided into two groups, the pathogen-treated

113

group and the untreated group. Tomato fruit pericarp tissues from the fruits equatorial

114

region were cut (all seeds and lesion area were removed) into small pieces, and

115

sampled after 2 h absorption of spore suspensions (0 h), at 12 h, 24 h and 48 h to

116

measure melatonin content and the relative expression of SlSNAT1. (2) Fifty fruits 6


Page 6 of 39

Page 7 of 39


117

were divided into five categories, infiltrated with melatonin solutions at different

118

concentrations (0, 1, 25, 50 and 100 μM) for the inoculation experiment to determine

119

the best concentration. (3) The other fifty-four tomato fruits were divided into two

120

groups, infiltrated with 0 μM or 50 μM melatonin solution, respectively, air-dried at

121

room temperature for 2 hours (time 0 h), then stored at 25 ± 1°C with 85–90% relative

122

humidity and sampled at 2 h, 4 h, 8 h, 16 h, 24 h, 72 h, 120 h and 168 h for the

123

measurements of defense enzyme activities, H2O2 content, antioxidant enzyme

124

activities, the level of MeJA as well as the relative expressions of JA signaling

125

pathway related genes. All the fruits infiltrated with melatonin solutions were under

126

−35 kPa for 0.5 min. Sampled tomato fruit pericarp tissues from the fruits equatorial

127

region were cut (all seeds were removed) into small pieces, frozen in liquid nitrogen

128

and stored at −80°C. Before analysis, frozen pericarp tissues were ground to a fine

129

powder in a grinding container (A11 basic, IKA, German), which had been precooled

130

with liquid nitrogen.

131

Pathogen Inoculation and Measurement of Disease Symptoms. B. cinerea

132

(ACCC 36028) was purchased from Agricultural Culture Collection of China

133

(Haidian, Beijing) and was cultured on potato dextrose agar medium at 28°C under

134

darkness for two weeks. The pathogen inoculation was operated following the method

135

described by Zheng et al.29 with some modifications. Spore suspensions (2 × 106

136

conidia mL−1) were collected by brushing the surface of cultures and suspending them

137

in sterile distilled water. After being treated for 24 h, the inoculations were carried

138

out. Fifty fruits with different concentrations of melatonin treatment in (2) were 7



139

inoculated with 10 μL spore suspension of B. cinerea into the wound (2 mm wide × 4

140

mm deep) at two points on the equator of each fruit by using a pipet and the fruits

141

were stored at 25 ± 1°C and 90–95% relative humidity. Necrotic lesions were

142

observed every day after inoculation. On the 3rd and 6th day after inoculation,

143

photographs were taken, and disease incidence as well as lesion diameter was

144

measured. Disease incidence was expressed as the percentage of fruits showing gray

145

mold symptoms. Lesion area was calculated as 3.14 × (lesion diameter/2)2. Three

146

replicates were carried out in this experiment.

147

Assay of Melatonin Content. One gramme of fruit sample was transferred to

148

5-mL extraction mixture (acetone: methanol: water = 89: 10: 1) as Pape et al.30

149

described, and the homogenate was centrifuged at 12 000g for 20 min at 4°C. After

150

centrifugation, the supernatant was used for the measurement of melatonin using the

151

Melatonin Enzyme-Linked Immunosorbent Assay (ELISA) Kit (JL13982; Shanghai

152

Jianglai industrial Limited By Share Ltd., Shanghai, China). All measurements were

153

performed in triplicate with samples collected from three biological replicates.

154

Assay of Defense Enzyme Activities. To determine the activities of CHI

155

(Chitinase, EC 3.2.1.14), GLU (β-1,3-glucanase, EC 3.2.1.39), PPO (polyphenol

156

oxidase, EC 1.10.3.2) and PAL (phenylalanine ammonia-lyase, EC 4.3.1.5), 0.5 g of

157

frozen fruit sample was extracted with 5-mL extraction buffer (100 mM, pH 6.8

158

(PBS) for PPO; 0.2 mM pH 8.8 boric acid buffer, containing 10% (w/v) polyvinyl

159

pyrrolidone and 1 mM EDTA for PAL; 100 mM pH 5.2 acetic acid buffer for CHI

160

and GLU) and centrifuged at 12 000g for 20 min at 4°C. The supernatant was 8


Page 8 of 39

Page 9 of 39


161

collected and used for defense enzyme activities determination. The activities of CHI,

162

GLU, PPO and PAL were measured following the method according to Zheng et al.31

163

and expressed as U·g−1 FW. All measurements were performed in triplicate with

164

samples collected from three biological replicates.

165

Assay of H2O2 Content and Activities of Antioxidant Enzymes. For analysis of

166

H2O2, superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC

167

1.11.1.11), and peroxidase (POD, EC 1.11.1.7), 0.5 g of frozen fruit sample was

168

homogenized with 5 mL of cold 100 mM PBS (pH 7.0) using an IKA Disperser (T10

169

basic, IKA, German). All extraction procedures were conducted at 4°C. The

170

homogenate was centrifuged at 12 000g for 15 min at 4°C, and the supernatants were

171

collected. A H2O2 assay kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu,

172

China) was used to measure H2O2 content. APX activity was surveyed by the method

173

of Nakano et al.32 with some modifications. APX activity was assayed by recording

174

the decrease in absorbance of ascorbic acid per minute at 290 nm. POD activity was

175

determined according to Doerge et al.33 and one unit of POD activity was defined as

176

an increase per minute in absorbance at 470 nm. A SOD Detection Kit (A001,

177

Jiancheng, Nanjing, China) was used to measure SOD activity. The absorbance was

178

recorded in a microplate reader (Infinite M200 Pro, Tecan, Switzerland). All

179

measurements were performed in triplicate with samples collected from three

180

biological replicates.

181

Assay of MeJA Content. The content of MeJA was measured following the method

182

according to Zheng et al.29 with some modifications. A 0.5 g portion of frozen 9



183

pericarp tissue in powder form was extracted and homogenized with 4 mL of

184

extraction buffer (80% methanol, containing 1 mM butylated hydroxytoluene) using

185

an IKA Disperser (T10 basic, IKA, German). After the sample was incubated at 4°C

186

overnight, the homogenate was centrifuged at 12 000g for 15 min at 4°C, and the

187

supernatant was collected for further analysis. The supernatant was passed through a

188

C18-SepPak classic cartridge (Waters, Milford, CT, USA) and dried by nitrogen.

189

Then, the residues were dissolved with 2 mL of cold 0.1 mM phosphate buffered

190

saline (PBS, pH 7.5, containing 1% (v/v) Tween-20 and 1% (w/v) gelatin) for further

191

determination of MeJA concentration. The absorbance was recorded in the microplate

192

reader at 490 nm. All measurements were performed in triplicate with samples

193

collected from three biological replicates.

194

Quantitative Real-Time PCR (qRT-PCR). A 200 mg sample of frozen pericarp

195

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

196

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

197

in 20 μL of RNase-free water, quantified by microspectrophotometry, and stored at

198

−80 °C. First-strand cDNA was synthesized according to the instructions from the

199

TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (Beijing

200

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

201

TransStart Top Green qPCR SuperMix (Beijing Transgen Biotech Co.Ltd., Beijing,

202

China) and the Bio-Rad CFX96 real-time PCR system (Bio-Rad, USA) were used to

203

run qRT-PCR. The procedure for qRT-PCR was designed with the following thermal

204

cycling conditions: 94°C for 30 s, followed by 40 cycles at 94°C for 5 s, 60°C for 15 10


Page 10 of 39

Page 11 of 39


205

s, and 72°C for 15 s. The relative gene expression for each sample was normalized

206

and calibrated to the β-actin Ct value and counted using formula 2−

207

experiments were run in triplicate with different cDNAs synthesized from three

208

biological replicates. Primers for qRT-PCR were listed in Table 1.

Δ Δ Ct.

All

209

Statistical Analysis. The data were expressed as the mean ± standard deviation

210

(SD). One-way analysis of variance (ANOVA) and Duncan’s multiple range tests

211

were used for statistical evaluations by SPSS version 18.0 (IBM Corp., Armonk, NY).

212

Significant differences at P < 0.01 and P < 0.05 were respectively marked as double

213

(**) and single (*).

214

RESULTS

215

Induction of Endogenous Melatonin Content and the Relative Expression of

216

SlSNAT1 in Tomato Fruits after B. cinerea Infection

217

To determine the change of endogenous melatonin content in response to B.

218

cinerea infection, expression of SlSNAT1, which is a gene for encoding a key enzyme

219

in melatonin biosynthesis pathway17, and endogenous melatonin content in tomato

220

fruits were examined. After infection with B. cinerea, the endogenous melatonin

221

content increased quickly compared to that in control, and the value of melatonin in

222

infected fruits at 48 h was 82.58% higher than that of control fruits (Figure 1A, p

Melatonin Induces Disease Resistance to Botrytis cinerea in Tomato

Recommend Documents