Melatonin inhibits ethylene synthesis via nitric oxide regulation to

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Food and Beverage Chemistry/Biochemistry

Melatonin inhibits ethylene synthesis via nitric oxide regulation to delay postharvest senescence in pears Jianlong Liu, Jie Yang, Haiqi Zhang, Liu Cong, Rui Zhai, Chengquan Yang, Zhigang Wang, Fengwang Ma, and Lingfei Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06580 • Publication Date (Web): 08 Feb 2019 Downloaded from http://pubs.acs.org on February 9, 2019

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Journal of Agricultural and Food Chemistry

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Melatonin inhibits ethylene synthesis via nitric oxide regulation to delay postharvest

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senescence in pears

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Jianlong Liu, Jie Yang, Haiqi Zhang, Liu Cong, Rui Zhai, Chengquan Yang, Zhigang Wang,

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Fengwang Ma, Lingfei Xu*

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College of Horticulture, Northwest A&F University, Taicheng Road NO.3, Yangling, Shaanxi

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Province, China

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*Corresponding author

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Tel.: +86 029 87081023,

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Fax: +86 29 87082613.

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E-mail: [email protected]

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ACS Paragon Plus Environment


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Abstract

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Understanding ripening and senescence processes in postharvest stored fruit is key to the

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identification and implementation of effective treatment methods. Here, we explored the effects

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of exogenous applications of melatonin (MT) and nitric oxide (NO) on ripening and softening

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processes in three cultivars of European pear (Pyrus communis L.). The results showed that MT

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and NO played important roles in the two processes: they decreased the rate of upregulation of

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PcCel and PcPG, inhibited expression of ethylene synthetase genes (PcACS and PcACO), and

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reduced rates of respiration and ethylene production. MT increased activity of NO synthase

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through upregulation of expression of PcNOS that subsequently led to an increase in NO

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content. However, when NO synthesis was inhibited, the delaying effect of MT on fruit

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senescence was almost eliminated. These findings indicate that MT acted on the upstream

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process of NO synthesis that then delayed senescence in pear fruit.

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Keywords

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Nitric Oxide, Melatonin, Pear, Ethylene, Senescence

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INTRODUCTION

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Pear (Pyrus communis L.) is cultivated across Asia, Europe, and America and is consumed after

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the process of postharvest ripening that occurs in climacteric fruits. This physiological ripening

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during postharvest respiration transforms the taste of fruit and represents the completion of the

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fruit development stage; after this stage, fruit enter the irreversible senescence stage that is

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characterized by tissue disintegration and cell death.1

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Cell wall modification and degradation influence fruit flesh firmness, juiciness, and

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crispness during ripening and senescence.2 In plant primary cell walls, hemicellulose covers and

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links cellulose microfibrils, while the gaps between these networks are filled with pectin and

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form a network.3 In pear fruit, polygalacturonase (PG) and cellulase (Cel) are responsible for

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the modification of pectin and cellulose, respectively, and genes that encode these two enzymes

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have been isolated.4 Softening of fruit tissue during ripening varies among pear cultivars, and is

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possibly driven by different levels of endo-PG activity and expression of PG and Cel genes.4,5

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Accelerated respiration rate and enhanced ethylene production are typical of climacteric

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fruit, such as apple, peach, and banana, during the ripening process, in which ethylene

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production and respiration rates influence ripening and senescence.5 Commercial fruit supply

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chains require timely storage and transportation of produce; many types of climacteric fruit are

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harvested in advance of ripening, and measures, such as low temperature and atmospheric

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pressure, and controlled atmosphere, are employed to inhibit post-ripening, thereby extending

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storage time and minimizing economic losses. Shelf life of this type of fruit is often controlled 3



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through the restriction of ethylene production. For example, the application of ethylene

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inhibitor 1-methylcyclopropene (1-MCP) prolongs the shelf life of pear fruit, without affecting

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quality, while treatment with vacuum ultraviolet (VUV) rapidly photolyzes ethylene produced

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by fruit to delay senescence.6,7 However, promotion of ethylene biosynthesis also accelerates

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senescence in fruit, due to production of excess reactive oxygen species (ROS) that shortens

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shelf life.8 In higher plants, ethylene is biosynthesized via catalysis of S-adenosylmethionine

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(SAM) by ACC synthase (ACS) to 1-aminocyclopropane-1-carboxylate (ACC), and the

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subsequent transition from ACC to ethylene by ACC oxidase (ACO). ACS and ACO are the

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critical enzymes that catalyze the rate-limiting step in this biosynthetic pathway and regulation

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of their gene expression may reduce ethylene production and delay fruit senescence.9,10

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Current understanding is that excessive accumulations of ROS, caused by an imbalance of

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their metabolism in fruit, drives senescence in fruit. The isolation and identification of

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melatonin (MT), N-acetyl-5-methoxytryptamine, from the unicellular alga, Gonyaulax

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polyedra,11 confirm its presence in non-animal organisms, including plants and agricultural

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crops.12,13 Studies have identified a range of functions of MT in plants: for example, MT was

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found to scavenge ROS, which led to improved resistance to biotic and abiotic stresses, such as

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pathogen attack, extreme temperatures, chilling, salinity, drought, waterlogging, and

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low-sulfur.13−22 Nitric oxide (NO) is produced endogenously in plants, and may be produced

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non-enzymatically, possibly as a result of chemical reactions between NOs and plant

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metabolites,23 nitrous oxide decomposition, or chemical reduction of nitrite (NO2−) under acid

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conditions.24 NO is also produced enzymatically from NO2− in plants by NAD(P)H-dependent

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nitrate reductase (NR),24,

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and there is increasing evidence for the existence of a 4


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mammalian-type nitric oxide synthase (NOS) in plants. Nitric-oxide synthase catalyzes the

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NADPH-dependent conversion of L-arginine to NO.26, 27 Nomega-Nitro-L-arginine methyl ester

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(L-NAME) is a substrate for arginine of nitric oxide synthase, and when L-NAME is combined

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with NOS, it blocks the binding of arginine to NOS, thereby reducing production of NO. Thus,

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L-NAME is commonly used as an inhibitor of NOS synthase in plants to inhibit NO content. 28,

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is a NO donor, increases NO content and enhances the antioxidant capacity in plants.29, 30

Recent research has shown that exogenous application of sodium nitroprusside (SNP), which

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NO and ROS signals of stress conditions have been extensively studied.31 Not only has NO

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been shown to eliminate damage caused by ROS and reduce effects of stress in plants,32 but its

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relation with ethylene during plant tissue maturation and senescence has been demonstrated,33

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where high levels of NO content result in low levels of ethylene production. This delay effect of

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NO on fruit senescence has led to its use in postharvest fruit to prolong storage time.34, 35Arnao et

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al.36 suggested a model of effects of MT–NO–ROS interactions on plant hormone-mediated

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biotic and abiotic stress responses, which summarizes and compiles much of the information that

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exists about it.

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We have previously reported that MT inhibits ethylene production and removes ROS to

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delay senescence in pear fruit,37 but the mechanisms were unclear. Therefore, in this study, we

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explored the relationship between MT and NO with ethylene production and associated effects

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in three varieties of European pear.

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MATERIALS AND METHODS 5



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Plant material and treatments

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Mature ‘Starkrimson’ (Pyrus communis L.), ‘Abbé Fetel’ (Pyrus communis L.), and ‘Red Anjou’

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(Pyrus communis L.) pear fruit were harvested from a commercial orchard in Wugong County,

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Shaanxi Province, China, at commercial maturity on 10 July (‘Starkrimson’ flesh firmness: c.

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5.5 kg cm–2) and 3 September (‘Abbé Fetel’ flesh firmness: c. 6.0 kg cm–2; ‘Red Anjou’ flesh

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firmness: c. 5.5 kg cm–2) 2018, and stored immediately at 4 °C and > 95% relative humidity.

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After one month of cold storage, 90 fruit of each cultivar were immersed in one of the

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following distilled water solutions at 25 °C for 12 h: distilled water as the negative control (CK);

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100 μM MT (Wako, Wako Pure Chemical Industries, Ltd. Japan); 100 μM NO donor SNP

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(Sigma-Aldrich, Saint Louis, Missouri, USA); 200 μM L-NAME to inhibit NOS activity and

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NO content (Meilunbio, Dalian, China); or, 100 μM MT + 200 μM L-NAME.

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Ethylene production, respiration rate, and flesh firmness

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Following treatment, the fruit from each treatment were randomly placed into five replicate

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bags (N = 18 fruit) and stored at 25 °C; ethylene production and respiration rate were measured

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daily for 4-5 d, until the CK fruit had decayed. Measurement time points were immediately

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after treatment (OS), 1 d prior to the expected climacteric respiration peak of the CK fruit

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(BCR), and 3 d after the climacteric respiration peak (ACR) of the CK fruit. Ethylene

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production and respiration rates, which were measured using the method described by Xie et

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al.,38 were assessed from three replicates of 10 fruit. Mean flesh firmness of five fruit from five 6


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replicates was recorded.

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Gene expression

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Levels of expression of cell wall modification, ethylene biosynthetic, and NO biosynthetic

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genes were assessed as three biological replicates from 5 g of fruit flesh sampled from three

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fruit of each replicate at the three time points (OS, BCR, and ACR); samples were frozen in

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liquid nitrogen and stored at –80 °C.

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Total RNA was purified using an RNAprep Pure Plant Kit (Tiangen, Beijing, China), and

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RNA concentration was detected using UV spectrophotometry. Then, 1 mg of total RNA was

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reverse-transcribed to cDNA using a PrimeScript RTreagent kit with gDNA Eraser (TaKaRa,

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Dalian, China). Three replicates of qRT-PCR reactions were performed on an Icycler iQ5

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(Bio-Rad, Berkeley, CA, USA) with the SYBR Premix Ex Taq II (TaKaRa, Dalian, China),

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according to the manufacturer’s instructions. Data were analyzed in iQ5 2.0 software (Bio-Rad,

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Berkeley, CA, USA) using the ddCT algorithm.

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DNA coding sequences of candidate genes for RT-PCR were isolated from the pear

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genome (https://www.ncbi.nlm.nih.gov/genome/12793) using BLAST searches of published

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sequences.

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(XM_018647282.1) were amplified and designated as PcPG and PcCel, respectively; for

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ethylene

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(NM_001302332.1), 1-aminocyclopropane-1-carboxylate oxidase 1 (NM_001302321.1), and

For

cell

biosynthetic

wall

modification

genes,

genes,

PG

(NM_001302285.1)

1-aminocyclopropane-1-carboxylate

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and

synthase

Cel

1


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1-aminocyclopropane-1-carboxylate oxidase 2 (XM_009357643.1) were amplified and

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designated as PcACS1, PcACO1, and PcACO2, respectively; and for NO biosynthetic genes,

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ferredoxin-nitrite reductase (NM_001319809.1), nitric oxide synthase (XM_009374377.2),

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nitrate reductase (XM_009368723.2), and nitrate reductase-like (NM_001319810.1) were

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amplified and designated as PcNi-NOR, PcNOS, PcNR1, and PcNR2, respectively. Primers and

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references for actin, cell wall modification genes, ethylene biosynthetic genes, and NO

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biosynthetic related genes are listed in Table S1.

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NO content and activity of NOS and NR

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As for gene expression analysis, we took 5-g samples of fruit flesh from three fruit of each

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replicate that had been frozen in liquid nitrogen and stored at –80 °C prior to analysis; we ran

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each analysis three times as biological replicates. NO content was measured from a colorimetric

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assay according to the method reported by Lv et al.,39 where 0.3 g of leaf material was

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homogenized in 1.5 mL of glacial acetic acid (pH 3.6) in an ice bath, and then centrifuged at

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10,000 g for 15 min. The reaction mixture containing 1 mL of supernatant and 1mL of Griess

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reagent (Sigma-Aldrich, Saint Louis, Missouri, USA) was allowed to stand at 25 ℃ for 30 min.

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Then, the samples were analyzed spectrophotometrically at 560 nm.

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Activity of NOS and NR was measured using a colorimetric assay kit (Nanjing Jiancheng

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Bioengineering Institute, Jiangsu, China), where 0.5 g of leaf material was homogenized in 1.5

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mL of 0.1M HEPES-KOH buffer (pH 7.4) that comprised 5 mM dithiothreitol, 0.1% Triton

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X-100, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 5 mM Na2MoO4, 10% (v/v) 8


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glycerol, 1 mM leupeptin, 1% polyvinylpyrrolidone, and 20 mM FAD. NR and NOS activities

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were determined from the supernatant after it had been centrifuged at 10,000 g for 30 min at

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4 °C, according to the manufacturer’s instructions.

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Statistical analysis

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Main effect of treatment was analyzed using one-way ANOVA, and between treatment

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differences were evaluated using Duncan’s multiple range tests at P < 0.05. Data are mean ±SD

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of three replicates for gene expression analysis, NO levels, and NOS and NR activities, and five

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replicates for all other evaluations.

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RESULTS

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Pear firmness

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Firmness of all pear cultivars declined with time, regardless of treatment, where levels of

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firmness were greatest in MT and SNP-treated fruit at BCR and ACR; at ACR, firmness was

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consistently lowest in the other treatments and the CK (P < 0.05; Figure 1). By ACR (at 4 d

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after treatment), firmness of ‘Starkrimson’ and ‘Red Anjou’ pears was

Melatonin inhibits ethylene synthesis via nitric oxide regulation to

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