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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

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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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1

ACS Paragon Plus Environment


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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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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

Regulation of Respiratory Pathway and Electron Transport Chain in

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