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Regulation of Respiratory Pathway and Electron Transport Chain in Relation to Senescence of Postharvest White Mushroom (Agaricus bisporus) under high O2/CO2 controlled atmospheres Ling Li, Hiroaki Kitazawa, Xiangyou Wang, and Han Sun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05738 • Publication Date (Web): 14 Mar 2017 Downloaded from http://pubs.acs.org on March 18, 2017
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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Journal of Agricultural and Food Chemistry
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Regulation of Respiratory Pathway and Electron Transport Chain in
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Relation to Senescence of Postharvest White Mushroom (Agaricus
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bisporus) under high O2/CO2 controlled atmospheres
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Ling Li†, ‡, Hiroaki Kitazawa‡, Xiangyou Wang∗†, Han Sun†
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†
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Zibo 255049, People’s Republic of China
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‡
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Ibaraki 305-8642, Japan
School of Agricultural and Food Engineering, Shandong University of Technology,
Food Research Institute, National Agriculture and Food Research Organization,
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*
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E-mail address:
[email protected] Corresponding author (Tel.: +86 13806481993)
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ABSTRACT
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In order to study the respiration metabolism mechanism based on the generation
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of adenosine triphosphate (ATP) and reactive oxygen species (ROS) and nitric oxide
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(NO) by the electron transport chain (ETC) of the white mushroom under high
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O2/CO2 controlled atmospheres, the treatments of 100% O2, 80% O2 + 20% CO2, 60%
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O2 + 40% CO2 and 40% O2 + 60% CO2 at 2±1 °C were employed and natural air was
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used as the control.
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ATP and energy charge can maintain the membrane integrity and function, life
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activities and physic-chemical reactions of higher plants. The results showed that the
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80% O2 + 20% CO2 treatment inhibited the respiration rate, embden-meyerhof-parnas
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or glycolysis pathway, ROS and NO contents. It significantly delayed the reduction of
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the ATP content and energy charge level; tricarboxyfic-acid-cycle and cytochrome
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pathway proportion, their key enzymes activity and gene expression. It also
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maintained a high phosphopentose pathway and moderate alternative pathway.
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Results indicated that the 80% O2 + 20% CO2 prolonged the storage time of
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mushrooms to 24 days and retarded the senescence through retaining the higher
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energy, suppressing the ROS contents, enhancing the endurance capability in
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adversity and regulating the respiration pathways and ETC metabolism.
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KEYWORDS: Agaricus bisporus, high O2, energy, respiration pathway, electron
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transport chain
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Journal of Agricultural and Food Chemistry
INTRODUCTION
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In recent years, fresh white mushroom is increasingly popular owing to its rich
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nutrients and claimed biological properties such as antitumour, antimicrobial,
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anti-inflammatory, thus it gradually becomes desirable daily food for human beings.1,2
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However, white mushroom easily loses water and softens due to its high respiration
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rate.3 Moreover, organoleptic quality attributes such as cap opening and browning
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also occur easily, which consume more nutrients and metabolic substances4 and
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strongly influence the decisions of consumers.2 Only within half of fresh mushrooms
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produced are consumed owing to their short shelf life.5 Consequently, an effective and
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safe storage method is critical to control the respiration rate and preserve white
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mushroom well.
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Although there has been a range of research analyzing deficient energy
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triggering the senescence of fruits and vegetables,6,7 oxidative damage of reactive
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oxygen species (ROS) and nitric oxide (NO) in mitochondria also has a more direct
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correlation with aging.8 The majority of adenosine triphosphate (ATP), ROS and NO
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in mitochondria of products are generated through the electron transport chain (ETC)
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of respiration.6,9 Respiration generates energy that maintains life activities and
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physico-chemical reactions, while it consumes metabolic substances which results in
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the ageing of postharvest products.10,11 Respiration pathways of plants compose
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embden-meyerhof-parnas (EMP) or glycolysis, pentose phosphate pathway (PPP) or
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hexose monophosphate pathway (HMP), tricarboxyfic-acid-cycle (TCA) or Krebs
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cycle, cytochrome pathway (CCP) and alternative pathway (AP). The mitochondrial 3
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respiratory pathway, especially ETC, plays a central and critical part in providing
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ATP for various cellular events, but it is also a major generator of ROS in the
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mitochondria.12 ETC contains complex I (nicotinamide adenine dinucleotide
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dehydrogenase; NADH-DH), complex II (succinate dehydrogenase of the TCA cycle;
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SDH), complex III (cytochrome b/c1oxidoreductases; COD), complex IV (cyt c
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oxidase; COX) and complex V (ATP synthase; ATPase). Importantly, electron
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transport at complexes I, III and IV is coupled with proton translocation，and the
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proton motive force is employed by complex V to form ATP, but complexes I and III
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are the main sites of ROS production.13
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High O2 controlled atmospheres (CA) has been receiving increasing attention in
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food storage research.14,15 Previous reports showed that high O2 could preserve
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mushrooms relatively well,16,17 but the effect of high O2 combined with CO2 on white
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mushroom is still unknown. In our previous study, proper high O2 /CO2 could inhibit
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the respiration rate and change the respiration pathway of broccoli and preserve it
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well.11,18 Nevertheless, different fruits and vegetables might have different respiration
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metabolic mechanism under external environmental conditions or with some
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particular treatments,19 especially the white mushroom, which has quite a high
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respiration rate and special structure without cuticle to protect it.20 Previously, several
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studies have focused on the physiological mechanism, on deficient energy, and on the
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senescence of fruits,21,22 but its underlying mechanism is still unclear. It is worthwhile
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to show that NADH and nicotinamide adenine dinucleotide phosphate (NADPH)
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contents were utilized for evaluating the EMP and TCA pathways of longan fruit 4
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during energy metabolism.10 In our study, we directly determined the respiration
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pathways such as EMP, TCA and HMP, along with activity of their key enzymes,
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regulation of the pathways and genes expression of ETC complexes during white
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mushrooms storage. Moreover, each respiration pathway has a particular function in
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various life activities, and little is currently known regarding how they alter in
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response to different high O2 /CO2 CA, whether these conditions have a biological
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relevance and the electron transport is more flexible in white mushrooms under high
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O2 /CO2 CA.
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However, the exact mechanism by which high O2/CO2 affects respiration
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pathway and ETC has not been fully elucidated. In this study, we applied high O2
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/CO2 CA to investigate the changes of ATP content, energy charge, ROS, NO,
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respiration rate, respiration pathways and their key enzymes, genes expression of ETC
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complexes of the white mushroom at 2±1 °C. The aim of this paper is to elucidate the
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senescence mechanism of the insufficiency of energy and ROS through the regulation
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of respiration metabolism, and regulation of ETC under high O2/CO2 CA storage.
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MATERIALS AND METHODS
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Mushroom Materials and Treatments. White mushrooms (Agaricus bisporus,
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As2796) were freshly harvested from Zibo City in Shandong Province, China.
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Mushrooms were selected for uniformity of size, and were without any damage or
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blemishes. They were randomly divided into 5 groups with 5 kg of mushrooms per
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group. Thereafter, they were stored at 2±1 °C and 90-95% relative humidity (RH).
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Three replicates were used for each of the following treatments: 100% O2, 80% 5
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O2+20% CO2, 60% O2+40% CO2 and 40% O2+60% CO2. The mushrooms exposed to
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air at 2±1 °C were used as the control. Each treatment was respectively put into a 0.45
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m3 sealable container. The containers were linked with continuous mixed gas flows
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(0.65 L s-1) according to each treatment. Gases were checked regularly with
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FBI-Dansensor CheckPoint O2/CO2 (MR-07825-00, FBI-Dansensor America Inc.).
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Fresh weight (FW) of white mushrooms was used for determining parameters.
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Mushrooms from each treatment were taken to measure parameters such as ATP
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content, energy charge, ROS, NO, respiration rate, respiration pathways and their key
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enzymes, genes expression of ETC complexes at 3 day intervals during storage.
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Determination of ATP, Energy Charge, H+-ATPase and Ca2+-ATPase
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Activities. In brief, 2.5 g of tissue from 18 mushrooms was ground with 9 mL of 0.6
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mol L-1 perchloric acid. The ATP, ADP and AMP contents were measured using a
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high-performance liquid chromatography according to the methods described in our
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previous study.11 Energy charge was calculated by [ATP + 1/2ADP]/ [ATP + ADP +
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AMP]. H+-ATPase and Ca2+-ATPase activities were measured using the method of Jin
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et al.23
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Determination of Respiration Rate and Proportion of Five Types of
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Respiration Pathways. Respiration rate and the proportion of five types of
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respiration pathways of the white mushroom were measured using a liquid-phase
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oxygen measurement system (Chlorolab-2, Hansatech Company, UK) according to
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the methods described in our previous study.11 In addition, 0.1 mL of 0.1 mol L-1
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salicylhydroxamic acid (SHAM) was added to the reaction cup for AP measurement. 6
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The EMP, HMP, TCA, CCP and AP values were marked as Ve, Vh, Vt, Vc and Va,
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respectively. The total respiration rate was defined as Rt, and was expressed as μ mol
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O2 min-1 g-1. The proportions of EMP, HMP, TCA, CCP and AP were expressed as Pe,
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Ph, Pt, Pc and Pa, and their proportions were calculated as follows , Pe = (Rt-Ve)
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/Rt×100%, Ph = (Rt-Vh) /Rt×100%, Pt = (Rt-Vt) /Rt×100%, Pc = (Rt-Vc) /Rt×100%,
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and Pa = (Rt-Va) /Rt×100%, respectively.
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Key Enzymes of Respiration Pathways Measurement. Phosphohexose
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isomerase (PGI), succinic dehydrogenase (SDH), cytochrome oxidase (COX) and
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glucose-6-phosphate dehydrogenase (G-6-PDH) +6-phosphogluconate dehydrogenase
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(6-PGDH) activity were determined according to the method of our previous study.11
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AOX activity was measured by the method of Vanlerberghe et al.24 and it was
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expressed by the unit n mol O2 mg-1 protein min-1.
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Determination of Genes Expression of complexes I, II, III, IV and V. The
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total RNA isolation and relative expression of genes were measured according to the
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method of Meng et al.25 with some modifications. The primer mix containing Oligo
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dT and randam 6 mers of the Kit from TaKaRa was employed to synthesize the cDNA.
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The EF1-α gene (Genbank X97204; GI 170088178) was employed to normalize the
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amount of gene-specific for quantitative Real-Time PCR (qPCR). The sequences of
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other genes were obtained in GenBank (http://www.ncbi.nlm.nih.gov) and JGI
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Genome Portal (http://genome.jgi.doe.gov ) (Table 1). The real-time PCR reactions
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were carried out in a total volume of 20 μl containing 10 μl 2×SG Fast qPCR Master
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Mix, 0.4 μl (200 nM) of each primer, 1.5 μl diluted cDNA (20 ng of template DNA), 7
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2μl DNF Buffer and 5.7 μl PCR-grade water. The reactions of PCR were performed as
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follows: 95 °C for 1 min, 40 cycles of 10 s at 95 °C and 30 s at 60 °C and the
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fluorescence signal of the SYBR Green I Master Mix was determined during the
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60 °C annealing step. Reported values are averages of three replicates.
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Assays of ROS and NO Content. The methods of Liu and Wang16 were utilized
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to determine contents of O2. - and H2O2. NO content was measured according to the
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Cvetkovska et al.26 method.
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Statistical Analyses. Experiments were performed randomly and data expressed as
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mean standard deviation. Data were studied by analysis of variance (ANOVA) using
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SPSS 16.0 statistical software (IBM SPSS, Inc., Chicago, IL, USA). Tukey’s test and
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t-test were carried out for multiple and pair-wise comparison. Significant differences
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are shown by different letters among all treatments from 0 to 12th day during storage
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by Tukey’s test (P < 0.05). Means followed by * and ** are significantly different at
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P < 0.05 and P < 0.01 by t-test on the 16th day during storage. NS = not significant.
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RESULTS AND DISCUSSION
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Storage Period
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The 80% O2 + 20% CO2 treatment prolonged the storage period of mushrooms to
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24 days, while 100% O2, 60% O2+40% CO2, 40% O2+60% CO2 and the control were
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only 12, 16, 12 and 8 days, respectively (Table 2). As postharvest white mushroom
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only has a short storage life of 1-3 days at ambient temperature, the mushrooms
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treated with 5% O2 + 10% CO2 at 2 °C had a maximum storage time of 14 days.5 In
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addition, 100% O2 + 2% alginate coating at 4 °C extended the storage time period to 8
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16 days.17 However, in our study, the 80% O2 + 20% CO2 treatment could extend the
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longer storage time of 24 days at least, which promoted mushroom conservation well.
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Effects of HOC-CA on ATP, Energy Charge, H+-ATPase and Ca2+-ATPase Activities
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Insufficient ATP and energy depletion appeared to block electron transfer at the
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terminal of the ETC, to make mitochondria dysfunction, to lose the repairing capacity
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of proteins after injury, to activate the apoptotic signaling pathway, thus leading to the
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senescence of products.6,7,11 On the whole, the ATP content and energy charge of all
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treatments decreased during storage (Fig. 1A, B). The ATP content and energy charge
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of 80% O2 + 20% CO2 treatment exhibited the highest level and the 60% O2 + 40%
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CO2 treatment also showed a higher level, while the 40% O2 + 60% CO2 and the
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control had a lower level.
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As complex V of ETC, ATPase is a bottleneck in ATP synthesis, and subunit β
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(AtpB) is the key catalytic component in the catalytic domain of ATP synthase.27 The
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activity of H+-ATPase, while Ca2+-ATPase activity declined at 0-4 d and then
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increased and showed a downward tendency at the end of storage (Fig. 1C,
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D). Compared with others, 80% O2 + 20% CO2 maintained a higher activity of
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H+-ATPase and Ca2+-ATPase (P
