Melatonin Induces Disease Resistance to Botrytis ... - ACS Publications

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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

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Journal of Agricultural and Food Chemistry

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Melatonin Induces Disease Resistance to Botrytis cinerea

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in Tomato Fruit by Activating Jasmonic Acid Signaling

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Pathway

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

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Jiping Sheng,‡ and Lin Shen*,†

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† College of Food Science and Nutritional Engineering, China Agricultural

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University, 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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Corresponding Author

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

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ACS Paragon Plus Environment


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ABSTRACT

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Melatonin acts as a crucial signaling molecule with multiple physiological

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

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regulatory mechanism of melatonin on attenuating tomato fruit fungal decay are

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

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fruit resistance to Botrytis cinerea and explored related physiological and molecular

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

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

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

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

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

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

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

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

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JA signaling pathway.

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

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peroxide

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INTRODUCTION

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

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

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

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

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

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

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necrotrophic pathogen whose infection may cause enormous and constant damage in

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

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

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

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

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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reduction in endogenous melatonin level suffered from the avirulent pathogen

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

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

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

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mechanism

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

especially

the

definite

signaling

pathway

responsible

for

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

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

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

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activating JA signaling pathway can induce resistance against necrotrophs, which

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

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

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

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

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pointed out that melatonin treatment induced JA accumulation in banana and

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

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

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melatonin involved in plant defense responses and suppressed bacterial multiplication 4


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

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

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pathway has not been described in detail yet.22

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

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

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

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

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

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found that fungal produced ROS were crucial for pathogenic development and

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

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

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

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resistance to a variety of abiotic stresses through inducing antioxidant enzyme

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

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

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

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

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

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ROS to defense necrotrophic pathogens in fruits.

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

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

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



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

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

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

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underlying regulatory mechanism of melatonin-induced pathogen resistance in tomato

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fruit.

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MATERIALS AND METHODS

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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measurements of defense enzyme activities, H2O2 content, antioxidant enzyme

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

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

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

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

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

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

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with liquid nitrogen.

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Pathogen Inoculation and Measurement of Disease Symptoms. B. cinerea

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

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

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

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

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

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

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



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

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

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

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

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

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

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

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replicates were carried out in this experiment.

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

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

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

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

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

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

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performed in triplicate with samples collected from three biological replicates.

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

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

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

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

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

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

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


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

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

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

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samples collected from three biological replicates.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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measurements were performed in triplicate with samples collected from three

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biological replicates.

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

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



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

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

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

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

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

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

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

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

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

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

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collected from three biological replicates.

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

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tissue 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). The total RNA was dissolved

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

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

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

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

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

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

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

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


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

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

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experiments were run in triplicate with different cDNAs synthesized from three

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

Δ Δ Ct.

All

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

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

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

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

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(**) and single (*).

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RESULTS

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Induction of Endogenous Melatonin Content and the Relative Expression of

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SlSNAT1 in Tomato Fruits after B. cinerea Infection

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

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

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in melatonin biosynthesis pathway17, and endogenous melatonin content in tomato

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

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

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

Melatonin Induces Disease Resistance to Botrytis ... - ACS Publications

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