(E)-2-hexenal, as a potential natural antifungal compound, inhibits

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Bioactive Constituents, Metabolites, and Functions

(E)-2-hexenal, as a potential natural antifungal compound, inhibits Aspergillus flavus spore germination by disrupting mitochondrial energy metabolism Weibin Ma, Luling Zhao, Wenhong Zhao, and Yanli Xie J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06367 • Publication Date (Web): 07 Jan 2019 Downloaded from http://pubs.acs.org on January 14, 2019

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Journal of Agricultural and Food Chemistry

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(E)-2-Hexenal, as a Potential Natural Antifungal Compound, Inhibits

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Aspergillus flavus Spore Germination by Disrupting Mitochondrial

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Energy Metabolism

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Weibin Maa, Luling Zhaoa, Wenhong Zhaoa, and Yanli Xiea,*

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a

Henan Key Laboratory of Cereal and Oil Food Safety Inspection and Control,

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College of Food Science and Technology, Henan University of Technology,

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Zhengzhou 450001, PR China

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* Corresponding author.

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Correspondence: Telephone/fax: +86 371 67758022. Email address:

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[email protected] (Y. Xie)

1

ACS Paragon Plus Environment


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ABSTRACT

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Fungal contamination imposes threats to agriculture and food production, and human

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health. A method to safely and effectively restrict fungal contamination is still needed.

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Here, we report the effect and mode of action of (E)-2-hexenal, one of green leaf

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volatiles (GLVs), on the spore germination of Aspergillus flavus, which can

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contaminate a variety of crops. The EC50 value, minimum inhibitory concentration

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(MIC) and minimum fungicidal concentration (MFC) of (E)-2-hexenal were 0.26, 1.0

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and 4.0 μL/mL, respectively. As observed by scanning electron microscopy (SEM),

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the surface morphology of A. flavus spores did not change after treatment with the

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MIC of (E)-2-hexenal, but the spores were shrunken and depressed upon treatment

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with the MFC of (E)-2-hexenal. The MIC and MFC of (E)-2-hexenal induced evident

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phosphatidylserine (PS) externalization of A. flavus spores as detected by double

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staining with Annexin V-FITC and propidium iodide, indicating that early apoptosis

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was potentially induced. Furthermore, sublethal doses of (E)-2-hexenal disturbed

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pyruvate metabolism and reduced the intracellular soluble protein content of A. flavus

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spores during the early stage of germination, and MIC treatment decreased

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acetyl-CoA and ATP contents by 65.7 ± 3.7% and 53.9 ± 4.0% (P < 0.05),

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respectively. Additionally, the activity of mitochondrial dehydrogenases was 2


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dramatically inhibited by 23.8 ± 2.2% (P < 0.05) at the MIC of (E)-2-hexenal.

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Therefore, the disruption of mitochondrial energy metabolism and the induction of

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early apoptosis, are involved in the mechanism of action of (E)-2-hexenal against A.

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flavus spore germination.

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KEYWORDS

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Aspergillus flavus, (E)-2-hexenal, antifungal, mitochondrial metabolism, spore

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germination

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INTRODUCTION

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The fungal contamination of agricultural and food products threatens human and

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animal health and results in economic losses.1 Particularly, Aspergillus flavus, a

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saprophytic soil fungus that infects oil-rich crops such as maize, peanut and soybean

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at both preharvest and postharvest stages, has attracted great attention due to its broad

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host range and toxic secondary metabolites.2,3 Aflatoxins, mainly including AFB1, B2,

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G1, G2 and M1, have been classified as Group 1 human carcinogens by the

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International Agency for Research on Cancer.4 Because of the frequent occurrence of

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aflatoxin contamination, many countries restrict the aflatoxin levels in crops and food

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products.5 Thus, there is an urgent demand for developing safe and effective methods

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to control A. flavus contamination in agriculture and food products. Although

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synthetic fungicides are commonly used to control fungal contamination in

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agricultural commodities and foods, the overzealous and indiscriminate use of

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synthetic fungicides has led to potential undesirable biological effects in animals and

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humans, including carcinogenic, teratogenic and toxic threats.6,7 Moreover,

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indiscriminate use of synthetic antifungals could lead to the development of resistant

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strains and high levels of toxic residues in food products.8 In light of these problems,

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efforts are continuing to seek natural safe and effective antifungal agents as promising 5



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alternatives to traditional synthetic antifungals to improve food safety and shelf life.

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Plant natural compounds with antifungal properties has been receiving increased

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attention because they are biodegradable and not toxic to humans and animals.9,10

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Plant volatiles, widely used as food flavoring agents, are generally recognized as

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safe,11 and their antimicrobial properties have been explored in the prevention of food

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spoilage and against pathogenic microbial species both in model and real systems.12,13

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(E)-2-hexenal, a green leaf volatile (GLV) produced naturally by green plants as a

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defense response,14 is a promising natural antimicrobial agent for food

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preservation.15-19 Previously, we examined the efficacy of 8 GLVs against A. flavus

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and (E)-2-hexenal exhibited a potent inhibitory effect on A. flavus spores via

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fumigation both in vitro and in vivo.20 In general, the development and application of

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antimicrobials for use in foods are closely related to a full understanding of the

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mechanisms of action. Although some evidence showed possible microbicidal actions

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of (E)-2-hexenal on different bacteria and fungi strains,21,22 the precise mechanism of

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action has not yet been clarified. To our best knowledge, the mechanism of action of

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(E)-2-hexenal on A. flavus spore germination is largely elusive. Here, we explore the

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antifungal mechanisms of (E)-2-hexenal on A. flavus spores, including the following

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treatment effects: efficiency of spore germination; changes in the surface 6


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ultrastructure and membrane integrity; intracellular levels of soluble protein and of

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metabolites, such as pyruvate, acetyl-CoA, and ATP; and the level of inhibition of

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mitochondrial dehydrogenase activities.

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MATERIALS AND METHODS

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Chemicals. (E)-2-hexenal (purity 98%; CAS: 67728-26-3), with the chemical

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structure shown in Figure 1, was purchased from Shanghai Aladdin Reagent Co., Ltd.

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(Shanghai, China). (E)-2-hexenal was prepared as a 20% stock solution in ethanol and

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stored at 4 °C. The final concentrations of (E)-2-hexenal added to the experimental

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solution were 0.065, 0.125, 0.25, 0.5, 0.75, 1, 2 and 4 μL/mL, respectively.

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Coomassie brilliant blue G-250, 2,4-dinitrophenylhydrazine (97%), ATP disodium

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salt hydrate (≥ 99%), acetyl coenzyme A sodium salt (≥ 93%), menadione and XTT

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sodium salt were purchased from Sigma Chemical Co., Ltd. Annexin V-FITC

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apoptosis detection kits and PI were purchased from Beijing Solarbio Science &

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Technology Co., Ltd.

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Fungal Strain and Preparation of Spore Suspension. A. flavus (CGMCC

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3.6304) was purchased from the China General Microbiological Culture Collection

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Center (Beijing, China). The fungal strain was cultured in Czapek Dox Agar (CDA)

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medium (sodium nitrate 3 g/L, dipotassium hydrogen phosphate 1 g/L, magnesium 7



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sulfate 0.5 g/L, potassium chloride 0.5 g/L, ferrous sulfate 0.01 g/L, sucrose 30 g/L,

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agar 20 g/L) at 28 ± 2 °C for 7 days. The spore suspension was collected by flooding

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the surface of the 5-day-old culture plates with normal saline containing 0.1% (v/v)

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Tween 80 and gently scraping with a sterile L-shaped spreader. Thereafter, the A.

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flavus spore suspension was diluted to a concentration of approximately 1.0 x 107

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spores per mL in sterile water with 0.1 % Tween 80, using a hemocytometer.

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Effect of (E)-2-hexenal on Spore Germination of A. flavus. According to a

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previously published method with minor modifications,23,24 (E)-2-hexenal was added

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to the A. flavus spore suspensions in sterilized penicillin bottles to make the final

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concentrations of 0.065, 0.125, 0.25, 0.5, 0.75, 1, 2 and 4 μL/mL, and the control was

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a parallel treatment prepared with ethanol replacing (E)-2-hexenal. The cultures were

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incubated at 28 ± 2 °C for 24 h, and the spores were examined under a microscope

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every 6 h. The germination ratio was scored microscopically by measuring the

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percentage of germinated spores in approximately 200 spores. Spores were considered

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germinated when the length of the germ tube was equal to or longer than the spore

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diameter. The lowest concentration of (E)-2-hexenal that did not permit any

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germination of A. flavus spores was taken as the minimal inhibitory concentration

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(MIC). Afterward, the sample without any germination of A. flavus spores was 8


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reincubated in normal saline containing 0.1% (v/v) Tween 80 without (E)-2-hexenal

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for 5 days at 28 ± 2 °C to determine if the inhibition was reversible or permanent. The

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minimal fungicidal concentration (MFC) was the lowest concentration at which A.

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flavus did not germinate during cultivation. The inhibition rate of germination was

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calculated according to the following formula: Inhibition of spore germination (%) =

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[(Gc – Gt) / Gc] × 100, where Gc is the percentage of spore germination with control

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treatment and Gt is the percentage of spore germination with (E)-2-hexenal treatment.

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The EC50 value was estimated statistically by Probit analysis with Probit package of

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SPSS 20.0 software.

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Scanning Electron Microscopy. Morphological observation of A. flavus spores

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treated with (E)-2-hexenal was performed by scanning electron microscopy (SEM) as

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previously described with minor modifications.25 Briefly, after exposure to the MIC

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and MFC of (E)-2-hexenal for 24 h, the A. flavus spores were fixed with 2.5%

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glutaraldehyde, washed with 1x PBS buffer and dehydrated with a series of ethanol

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gradients (30-100%) and resuspended in tert-butanol. Afterward, the spores were

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freeze-dried and coated with gold in a metallizer. Spores receiving no (E)-2-hexenal

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treatment were included as control. The morphology of the A. flavus spores was

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imaged by a field emission scanning electron microscope (FEI Quanta 250 FEG, FEI, 9



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US).

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Annexin V-FITC/PI Double Staining Assay. The cell membrane integrity of A.

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flavus spores was examined by double staining with Annexin V-FITC and propidium

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iodide (PI), which can also discriminate types of cell death (apoptotic or necrotic cell

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death), using an Annexin V-FITC apoptosis detection kit (Beijing Solarbio Science &

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Technology Co., Ltd., China).26 After exposure to the MIC and MFC of (E)-2-hexenal

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for 6 h, the A. flavus spores were collected and digested with 0.3% lywallzyme, 0.5%

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cellulase and 0.3% snailase for 3.5 h at 30 °C to obtain protoplasts. The gathered

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protoplasts were washed, resuspended in binding buffer, and stained with 5 μL

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Annexin V-FITC and 5 μL PI for 10 and 5 min, respectively, at room temperature in

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the dark, respectively. The samples were imaged by a confocal laser scanning

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microscope (FluoView FV3000, Olympus, Japan).

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Detection of Soluble Protein and Pyruvate Contents. A. flavus spore

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suspensions were treated with 0.25 or 1.0 μL/mL (E)-2-hexenal for 0, 3 and 6 h,

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respectively. The spores at each time point were pelleted, washed and disrupted in

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liquid nitrogen using a mortar and pestle, and then resuspended in Tris-HCl buffer

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(pH 7.5) and centrifuged at 4 °C at 15000 g for 30 min. Finally, the supernatant was

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collected for further determinations of soluble protein and pyruvate contents.27 10


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The soluble protein content was determined using the Bradford protein assay.28

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3.5 mL of Coomassie Brilliant Blue G-250 solution was added to 0.5 mL of

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supernatant and incubated for 5 min. The absorbance at 595 nm was measured by a

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UV-6100 spectrophotometer, using Tris-HCl buffer as control. The pyruvate content

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was measured as previously reported with minor modifications.27 Briefly, the

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supernatant (0.5 mL) was mixed with an equal volume of 2,4-dinitrophenylhydrazine

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and incubated at 37 °C for 10 min, followed by NaOH treatment (5 mL, 0.4 M) at

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room temperature for 30 min. Absorbance at 520 nm was measured by UV-6100

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spectrophotometer, with Tris-HCl buffer as a control.

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Detection of Intracellular Acetyl-CoA and ATP. High-performance liquid

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chromatography (HPLC) was used to determine the intracellular acetyl-CoA29 and

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ATP30 in A. flavus spores at 0, 3 and 6 h after treatment with 0.25 or 1.0 μL/mL

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(E)-2-hexenal, respectively. For intracellular acetyl-CoA, A. flavus spores were

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collected, washed, resuspended in 1.0 M perchloric acid containing 2 mM

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dithiothreitol. The spores were then ground with liquid nitrogen, homogenized in an

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ice bath for 15 min with an ultrasonic homogenizer, and centrifuged at 8,000 g at 4 °C

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for 10 min. The supernatant was collected, adjusted to pH 3.0 with 3 M KCO3, and

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centrifuged at 8,000 g at 4 °C for 20 min to remove potassium perchlorate. The 11



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supernatant was collected for HPLC. The intracellular ATP of A. flavus was extracted

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as described above except that 0.6 M perchloric acid was used for extraction, and the

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supernatant was adjusted to pH 6.5 by 1 M KOH.

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HPLC was performed by an Agilent 1290 liquid chromatography system (1290

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Infinity II, Agilent Technologies Co., Ltd., Palo Alto, USA) with an XB-C18 column

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(4.6 × 250 mm, 5 μm; Welch Ultimate). For acetyl-CoA measurement in this

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experiment, 0.2 M sodium phosphate buffer (pH 5.0) was used as solvent A, and 0.25

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M sodium phosphate buffer (pH 5.0) with 20% acetonitrile was used as solvent B.

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The column was equilibrated with 97% solvent A and 3% solvent B for 30 min before

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sample injection. Acetyl-CoA was eluted by a linear gradient from 97% to 82% of

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solvent A for the first 5 min, after which 72% solvent A was applied for 2.5 min. The

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linear gradient continued to drop to 60% solvent A within 5 min and then dropped to

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58% within 5.5 min. This was followed by a linear gradient back up to 97% solvent A

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within 2 min. The flow rate was 1.0 mL/min. A 10 μL sample was automatically

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injected and detected at a 254 nm wavelength. Acetyl coenzyme A sodium salt was

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used to prepare the standard curve. For ATP measurement, the ATP was eluted by 50

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mM KH2PO4–K2HPO4 buffer containing 1 mM EDTA (pH 6.0) and methanol

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(97.5:2.5, v/v). The HPLC was run at a flow rate of 1.0 mL/min with a 20 μL 12


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injection volume. The detection wavelength was 259 nm. ATP-2Na was used to

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prepare the standard curve.

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Detection of Mitochondrial Dehydrogenase Activity. The mitochondrial

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dehydrogenase activity of A. flavus spores was detected according to the XTT

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method.31 Briefly, 200 μL A. flavus spore suspension was aliquoted into a 96-well

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flat-bottom microplate. (E)-2-hexenal was added at a concentration of 0, 0.25 or 1.0

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μL/mL to treat the spores. The mixtures were incubated at 28 ± 2 °C for 0, 3 and 6 h.

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XTT and menadione were added at the final concentrations of 50 μg/mL and 25 μM,

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respectively, and incubated for 2 h. The dehydrogenase activity was then measured at

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450 nm using a microplate reader.

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Statistical Analysis. All assays were performed in triplicate and the results are

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presented as the mean ± SD. The significant differences between mean values were

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determined by one-way ANOVA using Duncan’s multiple range test (P < 0.05). The

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statistical analyses were performed by SPSS 20.0.

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RESULTS

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Effect of (E)-2-hexenal on the Spore Germination of A. flavus. The effect of

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(E)-2-hexenal on the spore germination of A. flavus is presented in Table 1. Spore

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germination of A. flavus was inhibited by (E)-2-hexenal in a dose-dependent manner. 13



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As the (E)-2-hexenal concentration increased, the germination rate of A. flavus spores

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decreased accordingly, and the inhibition effect weakened with the increase of

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incubation time. A. flavus spores without (E)-2-hexenal treatment started to germinate

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after a 6 h incubation, and a 78.2 ± 4.2% germination rate was observed after 24 h

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incubation. After 24 h incubation, treatment with 0.065, 0.125, 0.25, 0.5and 0.75

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μL/mL (E)-2-hexenal caused 21.5 ± 2.0, 27.2 ± 3.2, 49.5 ± 1.4, 81.5 ± 2.0 and 98.5 ±

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0.4% inhibition, respectively, with the EC50 value of 0.26 μL/mL. Furthermore,

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germination was completely inhibited within 24 h of incubation with 1.0, 2.0 and 4.0

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μL/mL treatments of (E)-2-hexenal. However, A. flavus spores could still grow when

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they were reincubated for 5 days without (E)-2-hexenal, except for the group initially

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treated with (E)-2-hexenal at 4.0 μL/mL. Therefore, the MIC and MFC of

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(E)-2-hexenal on A. flavus spores were 1.0 and 4.0 μL/mL, respectively, and these

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two representative concentrations were used in the following experiments to

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determine the morphological impact of (E)-2-hexenal on A. flavus spores.

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Effect of (E)-2-hexenal on the Surface Morphology of A. flavus Spores. The

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morphological impact of (E)-2-hexenal on the surface ultrastructure of A. flavus

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spores was investigated by SEM. As shown in Figure 2, A. flavus spores exposed to

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MIC of (E)-2-hexenal for 24 h exhibited no obvious alterations in surface morphology 14


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compared with the control, showing a normal and typically globular structures.

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However, compared with the control, the morphological structures of spores were

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altered following treatment with the MFC of (E)-2-hexenal. Most of these spores

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exhibited shrunken and depressed spore surfaces.

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Effect of (E)-2-hexenal on the Plasma Membrane Integrity of A. flavus

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Spores. To assess the mode of action of (E)-2-hexenal on A. flavus spores, the cell

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membrane integrity of A. flavus spores was investigated using an Annexin V-FITC/PI

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double staining assay. As shown in Figure 3, after 6 h of culture, treatment resulted in

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the positive staining of most cells for Annexin V-FITC with strong green fluorescence

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at the surface of plasma membrane. Furthermore, the green fluorescence gradually

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increased with increasing (E)-2-hexenal concentration. The PI stain, which requires a

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ruptured cell membrane, was negative in almost all the spores treated with MIC.

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However, a number of the spores treated with MFC exhibited red fluorescence,

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indicating that the cell membranes of this A. flavus group was ruptured and unable to

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exclude PI. Phosphatidylserine (PS) externalization from the inner leaflet to the outer

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leaflet of the plasma membrane is a typical marker of early apoptosis, when the cell

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membrane is still intact, and can be detected by the Annexin V-FITC and PI double

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staining assay because Annexin V has a high affinity for PS and PI is unable to pass 15



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through intact cell membranes.32

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Detection of the Intracellular Contents of Soluble Protein, Pyruvate,

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Acetyl-CoA, and ATP During the Germination of A. flavus Spores Treated with

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(E)-2-hexenal. To investigate the effect of (E)-2-hexenal on the energy metabolism of

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A. flavus spores during the early stage of germination, the intracellular contents of

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soluble protein, pyruvate, acetyl-CoA, and ATP were measured. As shown in Figure

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4A, soluble protein content in the control gradually increased within 6 h of incubation,

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whereas it was gradually reduced by (E)-2-hexenal treatment in a dose-dependent

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manner, decreasing by 49.9 ± 9.7% with 0.25 μL/mL (E)-2-hexenal and by 62.1 ± 11%

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with 1.0 μL/mL (E)-2-hexenal (P < 0.05). As shown in Figure 4B, intracellular

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pyruvate levels slightly increased in both the control and treatment groups in the first

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3 h, followed by a downward trend. Notably, the pyruvate levels with (E)-2-hexenal

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treatment were higher than the levels in the control in the first 3 h, and the

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intracellular pyruvate content of spores treated with 0.25 μL/mL (E)-2-hexenal

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maintained an accumulation trend through the 6 h incubation. Intracellular acetyl-CoA

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decreased within 3 h of incubation and started to rise thereafter in the untreated spores.

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In contrast, treatment of (E)-2-hexenal continuously reduced acetyl-CoA up to 6 h in a

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dose-dependent manner, with a sharp decline of 65.7 ± 3.7% in the 1.0 μL/mL 16


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(E)-2-hexenal group (P < 0.05) (Figure 4C). For intracellular ATP, as shown in Figure

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4D, (E)-2-hexenal treatment continuously reduced ATP levels in A. flavus spores

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within 6 h of incubation in a dose-dependent manner, resulting in decreases of 40.5 ±

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1.3% and 53.9 ± 4.0% with treatment of 0.25 μL/mL and 1.0 μL/mL (E)-2-hexenal,

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respectively (P < 0.05).

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Taken together, these results suggest that (E)-2-hexenal interferes with energy

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metabolism in A. flavus spores by disrupting soluble protein synthesis, pyruvate

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metabolism, acetyl-CoA production and ATP synthesis at the early germination stage.

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Effect of (E)-2-hexenal on Mitochondrial Dehydrogenase Activity in A. flavus

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Spores. Finally, the activity of the mitochondrial dehydrogenases in A. flavus spores

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was measured. As shown in Figure 5, the mitochondrial dehydrogenase activity of A.

267

flavus was reduced by increasing (E)-2-hexenal concentration and incubation time.

268

Treatment of (E)-2-hexenal at 0.25 and 1.0 μL/mL for 6 h inhibited mitochondrial

269

dehydrogenase activity by 14.9 ± 2.3% and 23.8 ± 2.2%, respectively (P < 0.05). This

270

is consistent with the results described above that demonstrate that (E)-2-hexenal

271

interferes with the mitochondrial function and energy metabolism of A. flavus spores

272

during early germination.

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DISCUSSION 17



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GLVs, a group of volatile components primarily produced from the

275

hydroperoxide lyase (HPL) and lipoxygenase (LOX) pathways in plants, are more

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promising as antifungal agents compared to synthetic preservatives that have potential

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harmful effects.33,34 (E)-2-hexenal, one of the GLVs, has been widely used as a food

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flavoring agent and is generally recognized as safe (GRAS).31 Moreover, it has been

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reported by the Joint FAO/WHO Expert Committee on Food Additives that there are

280

no safety concerns at current levels of intake when (E)-2-hexenal is used as a

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flavoring agent.35 The antifungal activity of (E)-2-hexenal, against A. flavus was

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reported previously by our laboratory,20 with an unclear mechanism. In this study, we

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found that (E)-2-hexenal exhibited remarkable antifungal activities by inhibiting the

284

germination of A. flavus spores. The MIC and MFC of (E)-2-hexenal on spore

285

germination were 1.0 and 4.0 μL/mL, respectively. (E)-2-hexenal was more effective

286

on A. flavus than some earlier reported plant natural compounds, such as essential oils

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extracted from Cicuta virosa L.var. latisecta Celak and turmeric.1,23 Therefore, to gain

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an in-depth understanding of its mechanism of action, observation of pathologic

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morphology and investigation of energy metabolic changes induced by (E)-2-hexenal

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in A. flavus spores were conducted.

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The plasma membrane plays important physiological roles in maintaining a 18


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homeostatic environment, exchanging materials, and transferring energy and

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information in the cell,23 and antifungal agents that target the fungal cell membrane

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have been developed and utilized for many years.36 Previous studies have shown that

295

the damage to fungal membranes can be involved in the antifungal activity of

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(E)-2-hexenal.37-39 In our study, the A. flavus spores treated with MFC exhibited a

297

shrunken and wrinkled cell surface. However, A. flavus spores exposed to the MIC of

298

(E)-2-hexenal for 24 h exhibited unaltered surface ultrastructure. Similarly, the cell

299

membranes of A. flavus spores were damaged by the MFC of (E)-2-hexenal for 6 h,

300

but the A. flavus spores treated with the MIC for 6 h maintained cell membrane

301

integrity. Therefore, our results indicated that the (E)-2-hexenal does not directly

302

disrupt the integrity of fungal cell membranes at the MIC and that the cell membrane

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is not sensitive to (E)-2-hexenal.

304

Our finding that (E)-2-hexenal potentially promotes PS externalization, a typical

305

characteristic of early apoptosis (Annexin V+/PI−), as evidenced by double staining

306

with Annexin V-FITC and PI, suggests that (E)-2-hexenal presumably targets fungal

307

mitochondria and induces early apoptosis. Mitochondria play an essential role in the

308

life

309

apoptosis-programmed cell death.40 Thus, similar to the plasma membrane, the

cycle

of

cells,

including

ATP

synthesis,

19


calcium

mobilization

and


310

mitochondria are another potential antifungal target.41-45 Furthermore, several

311

previous studies have demonstrated that mitochondria-mediated apoptosis occurs not

312

only in multicellular organisms but also in single cell organisms such as fungi, 26,32

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and the antifungal mechanisms associated with the form of apoptosis triggered by

314

mitochondrial dysfunction and disruption have been proposed.45-47 In this study, it was

315

found that PS externalization as an early marker of apoptosis occurred in the A. flavus

316

spores inhibited by (E)-2-hexenal. However, systematic studies on the apoptosis of A.

317

flavus spores induced by (E)-2-hexenal need to be further investigated, including

318

mitochondrial membrane potential, cytochrome c release, intracellular Ca2+ levels,

319

activation of metacaspases, and DNA damage.

320

Consequently, we further investigated the effect of (E)-2-hexenal on the

321

mitochondrial function of A. flavus spores during germination by detecting changes in

322

energy metabolism and in the activity of mitochondrial dehydrogenase. Pyruvate and

323

acetyl-CoA, the intermediate mediators of the glycolysis pathway and the

324

tricarboxylic acid cycle, play important roles in energy metabolism under aerobic

325

conditions.48 Pyruvate can be oxidized to acetyl-CoA, and eventually converted to

326

ATP, which occurs in mitochondria.49,50 In our study, (E)-2-hexenal initially induced

327

the accumulation of pyruvate in A. flavus spores during germination (Figure 4B), 20


Page 20 of 47

Page 21 of 47


328

implying that downstream pathways of pyruvate might be inhibited. Interestingly, the

329

content of pyruvate induced by 1.0 μL/mL (E)-2-hexenal was far below that of

330

pyruvate treated with lower concentrations of (E)-2-hexenal (0.25 μL/mL). Based on

331

these results, we hypothesized that 1.0 μL/mL (E)-2-hexenal completely inhibits the

332

downstream pathway of pyruvate and further triggers the feedback regulation of cells.

333

However, 0.25 μL/mL (E)-2-hexenal only inhibits the downstream pathway of

334

pyruvate and does not exhibit an adequate effect on the normal physiological

335

activities of the cells to cause feedback regulation. The pyruvate content of A. flavus

336

spores treated with 1.0 μL/mL (E)-2-hexenal was even lower than that of the control,

337

which might be explained by the fact that (E)-2-hexenal further interferes with the

338

glycolytic pathway. Moreover, acetyl-CoA and ATP levels were reduced by

339

(E)-2-hexenal (Figures 4C and 4D), suggesting that energy metabolism in the

340

mitochondria of A. flavus spores has been disrupted. Several previous studies have

341

demonstrated that the enzymatic activity of respiration, including the condensation

342

reaction of acetyl-CoA and ATP synthesis, in some fungi could be disturbed by plant

343

natural compounds, such as benzoic acid, cuminic acid and citral.48,49 In this study,

344

(E)-2-hexenal might also interfere with the tricarboxylic acid cycle and oxidative

345

phosphorylation pathway in A. flavus spores, resulting in the reduction of ATP level 21



346

and protein biosynthesis, which are essential for spore germination. In addition,

347

mitochondrial dehydrogenases, which contain various key dehydrogenases for ATP

348

production such as malate dehydrogenase (MDH) and succinate dehydrogenase

349

(SDH), were greatly inhibited by (E)-2-hexenal (Figure 5). This also provides a

350

source of mitochondrial dysfunction. Some previous studies have reported similar

351

results that mitochondrial dehydrogenases involved in the antifungal action of

352

plagiochin E and essential oil from dill (Anethum graveolens L.).31,32 Therefore, the

353

disruption of mitochondrial energy metabolism is closely related to the mechanism of

354

action of (E)-2-hexenal against A. flavus spore germination, which to the best of our

355

knowledge has not previously been reported in the literature. Future work needs to

356

focus on the identification of the direct target(s) of (E)-2-hexenal and its role in

357

mitochondrial functions. Moreover, based on our previous study which showed that

358

volatile phase effect of (E)-2-hexenal exhibited more effective antifungal efficacy

359

than contact phase effect,20 the difference in mechanisms of action of (E)-2-hexenal

360

between volatile and contact phase effects also need to be elucidated in further work.

361

In summary, (E)-2-hexenal exerted significant inhibitory effects on A. flavus

362

spore germination, and its mechanism of action may be associated with the activation

363

of early apoptosis and concomitant mitochondrial dysfunction, such as disruption of 22


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Page 23 of 47


364

pyruvate metabolism and decreased acetyl-CoA, ATP synthesis, and mitochondrial

365

dehydrogenase activity. In future studies, further emphasis should be given to

366

clarifying the active site of (E)-2-hexenal and the relationship between disruption of

367

mitochondrial energy metabolism and apoptosis in A. flavus spores.

368

ABBREVIATIONS USED

369

GLVs, green leaf volatiles; MIC, minimal inhibitory concentration; SEM, scanning

370

electron microscopy; PS, phosphatidylserine; ATP, adenosine triphosphate; XTT,

371

(2,3)-bis-(2-methoxy-4-nitro-5-sul-phenyl)-(2H)-tetrazolium-5-carboxanilide;

372

acetyl-CoA, acetyl coenzyme A; PI, propidium iodide; HPLC, high-performance

373

liquid chromatography; CLSM, confocal laser scanning microscope; MDH, malate

374

dehydrogenase; SDH, succinate dehydrogenase.

375

ACKNOWLEDGMENTS

376

We are grateful for the financial support of the Natural Science Research Projects of

377

Education Department of Henan Province (19A550004), the Doctor Research Fund of

378

Henan University of Technology (2016BS012) and Fundamental Research Funds for

379

the Henan Provincial Colleges and Universities in Henan University of Technology

380

(No. 2016QNJH16).

23



381

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547

Figure Legends

548

Figure 1. Chemical structure of (E)-2-hexenal.

549

Figure 2. SEM images of A. flavus spores treated with (E)-2-hexenal at MIC and

550

MFC for 24 h. (Control) nontreated cells, (MIC) cells treated with (E)-2-hexenal at

551

1.0 μL/mL, and (MFC) cells treated with (E)-2-hexenal at 4.0 μL/mL.

552

Figure 3. CLSM images of A. flavus spores treated with (E)-2-hexenal at MIC and

553

MFC for 6 h. (Control) nontreated cells, (MIC) cells treated with (E)-2-hexenal at 1.0

554

μL/mL, and (MFC) cells treated with (E)-2-hexenal at 4.0 μL/mL. Spores were

555

stained with Annexin V-FITC and PI before CLSM imaging. Each set of images

556

represents Annexin V staining, PI staining, and a bright-field view. Scale bars

557

represent 15 μm.

558

Figure 4. Effect of (E)-2-hexenal on the intracellular levels of soluble protein (A),

559

pyruvate (B), acetyl-CoA (C) and ATP (D) of A. flavus spores. The results are

560

presented as the mean ± SD (n =3). The data at the same time point with different

561

superscripts indicate significant differences (P < 0.05).

562

Figure 5. Effect of (E)-2-hexenal on the mitochondrial dehydrogenase activity of A.

563

flavus spores. The date are presented as the mean ± SD (n =3). The data at the same

564

time point with different superscripts indicate significant differences (P < 0.05). 34


Page 34 of 47

Page 35 of 47


565

Table 1. Effect of (E)-2-hexenal on spore germination of A. flavus

Dose

Spore germination

Inhibition

(%, mean ± SD, n = 3)

rate (%)

(μL/mL) 6h

12 h

24 h

24 h

EC50

Reincubation

(95% CI,

in CDA

μL/mL)

mediuma

0.000 4.8 ± 0.5 d 24.5 ± 2.8 d 78.2 ± 4.2 d

0.0 ± 0.0 h

+b

0.065 0.0 ± 0.0 e 20.7 ± 2.0 d 61.3 ± 2.0 e

21.5 ± 2.0 g

+

0.125 0.0 ± 0.0 e 13.7 ± 2.2 e 56.8 ± 2.4 e

27.2 ± 3.2 g

0.26

+

0.250 0.0 ± 0.0 e

5.6 ± 1.5 f

39.3 ± 1.8 f

49.5 ± 1.4 f

(0.23–0.29),

+

0.500 0.0 ± 0.0 e

0.0 ± 0.0 g 14.5 ± 1.5 g

81.5 ± 2.0 e

R2 = 0.984

+

0.750 0.0 ± 0.0 e

0.0 ± 0.0 g

1.2 ± 0.3 h

98.5 ± 0.4 d

+

1.000 0.0 ± 0.0 e

0.0 ± 0.0 g

0.0 ± 0.0 h

100.0 ± 0.0 d

+

2.000 0.0 ± 0.0 e

0.0 ± 0.0 g

0.0 ± 0.0 h

100.0 ± 0.0 d

+

4.000 0.0 ± 0.0 e

0.0 ± 0.0 g

0.0 ± 0.0 h

100.0 ± 0.0 d

–c

566

a

567

MFC of (E)-2-hexenal on A. flavus spores; b “+” = visible growth; c “–” = no visible

568

growth; d–h Significant differences at P < 0.05 according to Duncan’s multiple range

569

test.

Reincubated without (E)-2-hexenal in CDA medium for 5 days to test the MIC and

35



570

Figure 1.

571 572

36


Page 36 of 47

Page 37 of 47

573


Figure 2.

574

37



576

Figure 3.

577

38


Page 38 of 47

Page 39 of 47

578


Figure 4.

579 580

39



581

Figure 5.

582

40


Page 40 of 47

Page 41 of 47

583


Graphic for table of contents (TOC)

584

41



Figure 1. Chemical structure of (E)-2-hexenal. 59x10mm (600 x 600 DPI)


Page 42 of 47

Page 43 of 47


Figure 2. SEM images of A. flavus spores treated with (E)-2-hexenal at MIC and MFC for 24 h. (Control) nontreated cells, (MIC) cells treated with (E)-2-hexenal at 1.0 μL/mL, and (MFC) cells treated with (E)-2hexenal at 4.0 μL/mL. 439x98mm (300 x 300 DPI)



Figure 3. CLSM images of A. flavus spores treated with (E)-2-hexenal at MIC and MFC for 6 h. (Control) nontreated cells, (MIC) cells treated with (E)-2-hexenal at 1.0 μL/mL, and (MFC) cells treated with (E)-2hexenal at 4.0 μL/mL. Spores were stained with Annexin V-FITC and PI before CLSM imaging. Each set of images represents Annexin V staining, PI staining, and a bright-field view. Scale bars represent 15 μm. 299x299mm (300 x 300 DPI)


Page 44 of 47

Page 45 of 47


Figure 4. Effect of (E)-2-hexenal on the intracellular levels of soluble protein (A), pyruvate (B), acetyl-CoA (C) and ATP (D) of A. flavus spores. The results are presented as the mean ± SD (n =3). The data at the same time point with different superscripts indicate significant differences (P < 0.05) 199x160mm (600 x 600 DPI)



Figure 5. Effect of (E)-2-hexenal on the mitochondrial dehydrogenase activity of A. flavus spores. The date are presented as the mean ± SD (n =3). The data at the same time point with different superscripts indicate significant differences (P < 0.05). 99x73mm (600 x 600 DPI)


Page 46 of 47

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Graphic for table of contents 93x48mm (600 x 600 DPI)


(E)-2-hexenal, as a potential natural antifungal compound, inhibits

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