Early Defense Responses Involved in Mitochondrial Energy

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Functional Structure/Activity Relationships

Early defense responses involved in mitochondria energy metabolism and reactive oxygen species accumulation in harvested muskmelons infected by Trichothecium roseum Liang Lyu, Yang Bi, Shenge Li, Huali Xue, Zhong Zhang, and Dov Prusky J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06333 • Publication Date (Web): 13 Mar 2019 Downloaded from http://pubs.acs.org on March 18, 2019

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Journal of Agricultural and Food Chemistry

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Early defense responses involved in mitochondrial energy metabolism

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and reactive oxygen species accumulation in harvested muskmelons

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infected by Trichothecium roseum

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Liang Lyu,† Yang Bi,‡* Shenge Li,‡ Huali Xue,‡ Zhong Zhang,‡ Dov B. Prusky‡

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† College of Plant Protection, Gansu Agricultural University, Lanzhou 730070, PR China

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‡ College of Food Science and Engineering, Gansu Agricultural University, Lanzhou 730070, PR

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China

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§ Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, The

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Volcani Center, Beit Dagan, Israel

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*Corresponding author.

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Dr. Yang Bi, E-mail address: [email protected] Tel.:+86-931-7631113;

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Corresponding author's institution: Gansu Agricultural University, Lanzhou , PR China

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ACS Paragon Plus Environment


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ABSTRACT

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Mitochondria play an essential part in fighting against pathogen infection in the defense responses of

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fruits. In this study, we investigated the ROS production, energy metabolism and changes of

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mitochondrial proteins in harvested muskmelon fruits (Cucumis melo cv. Yujinxiang) inoculated

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with Trichothecium roseum. The results indicated that the fungal infection obviously induced the

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H2O2 accumulation in mitochondria. Enzyme activities were inhibited in the first 6 hours post

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inoculation (hpi), including succinic dehydrogenase, cytochrome c oxidase, H+-ATPase and

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Ca2+-ATPase. However, the activities of Ca2+-ATPase and H+-ATPase and the contents of

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intracellular ATP were improved to a higher level at 12 hpi. A total of 42 differentially expressed

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proteins were identified through tandem mass tags-based proteomic analyses, which mainly involved

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in energy metabolism, stress responses and redox homeostasis, glycolysis and tricarboxylic acid

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cycle, transporter and mitochondria dysfunction. Taken together, our results suggest that

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mitochondria play crucial roles in the early defense responses of muskmelons against T. roseum

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infection through regulation of ROS production and energy metabolism.

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KEYWORDS:

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Mitochondrion · Energy metabolism · Reactive oxygen species · Muskmelon · Mitochondrion

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proteomics

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INTRODUCTION

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Fruits have developed complicated immune systems in respond to downstream defense responses

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activation as well as recognition for fighting against pathogen attacks, in which the energy saved by

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regulation of primary metabolism is diverted and adopted for the defense responses.1 During the

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defense responses, substantial energy will need to be expended in multiple defense pathways to

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support hundreds of genes expressions.2 Mitochondria seem not only to produce adenosine

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triphosphate (ATP) through respiratory oxidation of organic acids and transfer of electrons to O2 via

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the electron transport chain (ETC), but also to primarily generate endogenous reactive oxygen

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species (ROS); therefore establishing a favorable energy balance between the stress responses and

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signaling as well as programmed cell death (PCD).3 Mitochondria integrate and amplify diverse

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signals after pathogen perception, such as nitric oxide, salicylic acid, ROS or pathogen elicitors, and

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integrate them into the plant cell defense strategy.4 Destabilized organelle often causes changes in

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ROS production, membrane potential and respiration when signals perceived by mitochondria altered

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normal performance.4 This renders mitochondrial components acutely susceptible to oxidative

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damage.5 Functional integrity of mitochondria is required to form complete functionality for cellular

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energy demands. Some reports suggest that plant mitochondria are involved in the activation of PCD

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and ROS production during both the biotic and abiotic stresses.6 However, few reports were

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available till now on revealing the relationship between mitochondria and the defense responses in

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the fruits host.

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Muskmelon (Cucumis melo L.) is a world-wide horticultural crop and has wide of distribution and

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large amount of output.7 Trichothecium roseum (T. roseum) is a major kind of postharvest fungus

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which causes pink rot on various fruits and vegetables, such as grapes8 apples9, tomatoes and



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oranges10, and especially results in pink rot formation of harvested muskmelons in Northwest China,

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the largest muskmelon production area in the country.11,

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purify mitochondria from the artificially T. roseum fungi infected muskmelon fruits to clarify the

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changes and sources of ROS production, to determinate the activity of enzymes involved in energy

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metabolism, and to analyze the alterations of mitochondrial proteome in the fungi inoculated

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muskmelons. The results showed that mitochondria, through regulating the ROS production and

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energy metabolism, play crucial roles in the early defense responses of T. roseum infected

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muskmelons against the pathogen infection.

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The aims of this study are to isolate and

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MATERIALS AND METHODS

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Experimental plant materials preparation

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The muskmelons (Cucumis melo L. cv. Yujinxiang) used in the experiments were harvested at the

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start of physiological maturity, i.e. 35 days after full blossom, in Shichuan Town located in Gaolan

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County of Gansu Province, China. The experimental fruits were picked out with uniform size and

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shape and with no obvious signs of damage or infection and packed in cartons and transported to

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Gansu Agricultural University laboratory within six hours, and stored in normal conditions (i.e. room

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temperature 22±2°C and

relative humidity 55-60%) ready for use.

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Pathogen preparation

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Trichothecium roseum (Pers.: Fr.) Link was separated from infected muskmelons and kept on potato


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dextrose agar (PDA) at 4 ºC. We sourced conidia of the fungi from PDA cultures that were of

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10-days old and incubated at 25ºC. A hemacytometer was used to determine the number of spores,

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and modulate the concentration to 1 × 105 spores/mL.

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Pathogen inoculation and sample collection

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Artificial inoculation (pathogen challenged - PC) was conducted in accordance with the method as

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proposed by Li et al.13 The muskmelons were first washed with tap water and distilled water, and

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then sterilized with 70% alcohol before inoculation. For inoculation, six spots on the surface of the

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fruits around the equator region were placed with 20-ul spore suspensions (1 × 105 spores ml–1). The

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control fruits (control check – CK) were treated with the same wounds but placed only with 20-ul

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sterile water. The inoculated fruits were stored in cellophane covered storage boxes to ensure the

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required assay condition of 22 ±2 ºC and high relative humidity (RH) of 85-90%. Tripartite

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experiments, each with 12 inoculated and control muskmelons, were performed under same

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conditions.

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The sample collection was performed by following Ren’s et al method.14 For sample collection, a

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depth of 1.5 cm peel was removed at 1.0 cm away from the junction of disease lesion and health

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tissue of the muskmelons using a 70% ethanol sterilized stainless steel cork borer. The sampling

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process started in sequence immediately after the inoculation (0 hours post inoculation, hpi) and

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continued every 6 hours during the following 2 days of storage. The peel of sampled discs of the

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fruits was removed by slicing the peel off at a depth of 0.5 to 1.5 cm below the peel, leaving a fruit

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disc of 2 cm diameter and 7-8 mm depth.



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Mitochondria isolation and purity assessment

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Mitochondria were isolated by using the method of Qin et al.15 A stirring blender was used to

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homogenize one hundred grams of frozen samples at 4 ºC in 200 mL of 0-4 ºC extraction solution

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comprised of 10 mM β-mercaptoethanol, 0.1% w/v BSA, 0.5% w/v polyvinylpyrrolidone-40, 1 mM

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EDTA, 250 mM sucrose and 50 mM Tris-HCl, and with pH of 7.5. Four layers of sterile cheesecloth

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was used to filter the homogenate, which was then centrifuged at 4 000×g for 15 minutes at 4 ºC.

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The supernatants were again centrifuged in another centrifuge at 17 000×g for 20 minutes. A small

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and soft brush was used to re-suspend the pellets in the 0.1 percent w/v BSA, 250 mM sucrose and

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50 mM Tris-HCl, pH 7.5 wash medium. The centrifugation steps were repeated and the pellet

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re-suspended in a small quantity of wash medium to lower the contamination from other organelles.

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A step Percoll gradient at 6, 12, and 30% (1:2:1) in wash medium were used to separate the crude

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fraction, followed by 40 000×g centrifugation for 45 minutes at 4 ºC. The 12% and 30% Percoll

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gradient interface showed mitochondria bands (Supporting Figure S1). The mitochondria layer was

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aspirated by centrifugation at 15 000×g for 15 minutes with a washing solution. Mitochondria

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centrifuged at 40 000×g for 30 minutes were later sanitized through top layering on a self-generated

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20% v/v Percoll gradient. The mitochondria bands that formed close to the gradient center were

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removed and thinned ten times with the wash solution, and then pelletized using Percoll at 15 000×g

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for 15 minutes. Through repeated washes the mitochondria was disinfected for final re-suspension in

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a small quantity of wash solution. The mitochondria marker enzymes (cytochrome c oxidase),

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peroxisomes (catalase) and cytosol (alcohol dehydrogenase) were evaluated for purity, respectively.

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(Supporting Figure S2)


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Assay and localization for hydrogen peroxide (H2O2) production

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The method of Ferguson and Patterson16 was used to determine the H2O2 content in the fungal

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inoculated muskmelon fruits and controls. Four grams of fresh flesh tissue, homogenized with

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10-mL of cooled acetone, was centrifuged at 10 000×g for 20 minutes at 4 ºC. The H2O2 in flesh

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tissue was established by monitoring of the titanium peroxide complex absorbance at 410 nm.

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Absorbance values, denoted as µmol H2O2 g-1FW, were standardized to a curve generated from

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known concentrations of H2O2. And at 24 hpi, the intracellular ROS levels were detected using 10

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mM oxidant-sensitive probe 2′, 7′‐dichlorofluorescein diacetate (10 mM; DCHF-DA, PubChem CID:

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77718; Sigma-Aldrich). Mitochondria were stained with 1 mg/ml rhodamine 123 (Rhod123, Dojindo

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Molecular Technologies, Inc., Kumamoto, Japan), and tissue slices with different dyes for 10

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minutes at 30oC. The latter were rinsed twice with phosphate buffer saline (PBS), and tested under a

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Confocal Laser Scanning Microscope 780 (Carl Zeiss, Oberkochen, Germany). The Axiocam MRc

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(Carl Zeiss) digital camera was used to capture photos.

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Assays for enzyme activity

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The activities of several enzymes involving in energy metabolism were detected through the crude

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mitochondria isolated from the muskmelon fruits. Succinic dehydrogenase (SDH), cytochrome c

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oxidase (CCO), Ca2+-ATPase as well as H+-ATPase were measured according the published method

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by Ge et al.17,18,19 The definition of one unit of SDH activity was the increase of 0.01 in absorbance

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at 600 nm every second in the assay situations. One unit of CCO activity was expressed as U mg−1



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protein, where U = 0.01ΔA550/s. One unit of H+-ATPase activities was expressed as U mg−1 protein,

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where U = 0.01ΔA660/s.

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For ATP assay, 10.0 grams of muskmelon samples were added to 90 ml of boiling double distilled

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(DDS) to obtain 10% homogenate. The homogenates were centrifuged at 10 000×g for 15 minutes.

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Diluting 10 μL supernatant with 115 ml of ATP-free H2O and 125ml of ATP-free Tris-Acetate buffer

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(40 mM, pH 8.0; freshly prepared) helped to establish the ATP contents, with help by an assay kit

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(Nanjing Jiancheng Bioengineering Institute, Jiangsu, China).

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Protein extraction

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The mitochondrial proteins were extracted through the adopted Qin’s et al phenol method.20,21 The

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protein concentrations were determined with the Bradford method.22

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Proteolytic Digestion and Isobaric Labeling

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The filter-aided sample preparation (FASP) was done following a slightly adapted method of

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Wisniewski et al.,23 and the samples were digested with a Trypsin-LysC enzyme mixture (1:100

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w/w, Promega). The resultant biological and technical peptide samples triplicates were refined and

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labeled with reversed-phase C18 and tandem mass tags (TMT) 10-plex, respectively, as per

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manufacturer instruction manuals. Briefly, the tryptic peptides (100 µg each) were labeled with TMT

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10-plex with 126-tag (12hCK-1), 127N-tag (12hCK-2), 127C-tag (12hCK-3), 128N-tag (12hCP-1),

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128C-tag (12hCP-2), 129N-tag (12hCP-3) and 131-tag (MIX).


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Mass Spectrometry Analyses

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Peptides fractionation and mass spectrometry analyses were accomplished as per the method

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proposed by Fang et al. (2017).24 The prepared peptides had been suspended again by nano-RPLC

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buffer A (2 % CAN, 0.1 % FA). The online Nano-RPLC was adopted on the Easy-nLC 1000 System

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(Thermo Fisher Scientific Inc., San Jose, CA, USA). The samples were stowed on a trap C18 column

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(diameter 3 μm, 75 μm × 20 mm, Thermofisher Dionex, NanoViper) and washed with nano-RPLC

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buffer A for 10 minutes at 2 μL/m. Data collection was implemented using a Q Exactive System

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(Thermo Fisher Scientific Inc., San Jose, CA) equipped by a Nanospray.

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Protein Identification and Quantification

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According to Fang et al. (2017),24 Proteome Discoverer 1.3 software analyzed MS/MS data were

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obtained using the SEQUEST search engine, and constrained with a forerunner mass tolerance of 10

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ppm and fragment mass tolerance of 0.02 Da. All quantifiable spectra peptide ratios pertaining to

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corresponding proteins were reported as a median value. Two unique spectra at least are required for

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protein quantitation, whereas differentially expressed proteins were defined by the fold change

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criteria (FC) ≥ 1.2 or ≤ 0.83 (p < 0.05). Both the identifications and TMT quantitation were made

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against

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(https://www.ncbi.nlm.nih.gov/genome/10697).

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implemented on the basis of biological procedures, cellular units as well as molecular functions

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listed in the Gene Ontology (GO) database according to the GO criteria.

the Cucumis

melo.

fasta genome The

recognized


protein proteins’

database annotation

was


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Statistics analysis

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Statistics analysis was accomplished through SPSS 19.0 (SPSS Inc, USA). Duncan’s multiple range

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tests were used to separate the means to determine the treatment effect. And the differences were

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considered as significant at p

Early Defense Responses Involved in Mitochondrial Energy

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