Art_15702.indd Journal of Applied Botany and Food Quality 94, 132 - 139 (2021), DOI:10.5073/JABFQ.2021.094.016 1Institute of Genetics and Physiology, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang, China 2Hebei Plant Genetic Engineering Center, Shijiazhuang, China The influence of 1-MCP on the fruit quality and flesh browning of ‘Red Fuji’ apple after long-term cold storage Jingang He1,2, Yunxiao Feng1,2, Yudou Cheng1,2, Shen Wang1, Yaxiong Fu1, Junfeng Guan1,2* (Submitted: January 25, 2021; Accepted: June 28, 2021) * Corresponding author Summary This study assessed the influence of 1-MCP treatment on the fruit quality and flesh browning of ‘Red Fuji’ apple at shelf life after long-term cold storage. The ‘Red Fuji’ fruit were stored at 0±0.5 °C for 270 days after treating with 1.0 μL L-1 1-methylcyclopropylene (1-MCP). Fruit quality, browning rate of stem-end flesh, chloro- genic acid content, polyphenol oxidase (PPO) activity were analyzed at shelf-life under 20±0.5 °C, the expression profile of ethylene re- ceptors (MdERS1), phenylalnine ammonia lyase genes (MdPAL1, MdPAL2), quinate hydroxycinnamoyl/hydrxycinnamoyl CoA shi- kimate gene (MdHCT3), polyphenol oxidase genes (MdPPO1, MdPPO5) and lipoxygenase gene (MdLOX) were measured by real- time quantitative PCR. 1-MCP treatment improved the fruit storage quality, decreased stem-end flesh tissue browning, and fruit decay. In addition, the fruit respiration rate and ethylene production rate increased at shelf- life, but this increase could be inhibited by 1-MCP. The same rule was observed in the changes of chlorogenic acid content and PPO activity, the expression of MdERS1, MdPAL1, MdPPO1 and MdLOX were inhibited by 1-MCP as well in the stem-end flesh. Thus, 1-MCP treatment improves the fruit quality of ‘Red Fuji’ apple at shelf-life after long-term cold storage, and inhibits the browning of stem-end flesh by decreasing the chlorogenic acid content and PPO activity. MdPAL1, MdHCT3, MdPPO1 and MdLOX participate in the flesh browning progress. Keywords: Malus × domestica; 1-methylcyclopropylene; storage quality; chlorogenic acid; polyphenol oxidase Introduction 1-methylcyclopropene (1-MCP), an inhibitor of ethylene perception, delays fruit ripening and extends the storage and shelf life by impeding binding of ethylene to its receptors (SISLER et al., 2003). 1-MCP based technology is widely used by apple industries around the world to help maintain fruit quality, especially texture (WATKINS, 2008; MATTHEIS et al., 2008). Apples are characterized by valuable nutritional and taste qualities, and among the most popular fruit species in China. ‘Red Fuji’ apple (Malus × domestica cv. Red Fuji) accounts for about 72% of the total apple yield in China. Low-temperature storage is generally used as commercial application to prolong the storage period. However, a number of physiological disorders limit its quality in cold storage and shelf life, including flesh browning and superficial scald (HE et al., 2016). Flesh browning is a result of membrane damage and it is usually associated with enzymatic oxidation of phenolic compounds by polyphenol oxidase (PPO) to o-quinones, which polymerize non- enzymatically to produce heterogeneous black, brown or red pig- ments commonly called melanins (TOMÁS-BARBERÁN et al., 2010). The occurrence of browning is mainly due to disruption of cellular compartmentalization, allowing the enzymatic oxidation of pheno- lic substrates in the vacuole by PPO located in the cytoplasm. This oxidation generates o-quinones that react with other compounds and result in polymerization to produce the browning appearance (FRANCK et al., 2007; SUN et al., 2011; LIN et al., 2014). Chlorogenic acid is an important substrate for PPO enzymatic browning reaction (TOMÁS-BARBERÁN et al., 2010). The biosynthetic pathway of chlorogenic acid is initiated from the deamination of phenylalanine to cinnamic acid by phenylalanine ammonia lyase (PAL), followed by hydroxylation and methylation of cinnamic acid by cinnamate 4-hydroxylase (C4H) and 4-hydroxycinnamoyl- CoA ligase (4CL), respectively. Thus ρ-coumaric acid is produced, then hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT/HQT) pathway begins, and chlorogenic acid is synthesized finally (NIGGEWEG et al., 2004; MAHESH et al., 2007; ZHAO et al., 2013). Furthermore, PAL, 4CL and HCT/HQT are considered as key regulators related to chlorogenic acid biosynthesis (LALLEMAND et al., 2012; ESCAMILLA-TREVIÑO et al., 2014; YUAN et al., 2014). Damage of membrane integrity is considered the primary event that ultimately leads to the development of browning (KOU et al., 2015). Previous studies have demonstrated that the membrane lipid metabolism is correlated to lipoxygenase (LOX) (MAO et al., 2007). LOX is potentially involved in senescence, membrane alter-actions and lipid degradation in plants under stress (GAO et al., 2015). LOX catalyzes peroxidation of plasma membrane lipids, increases lipid unsaturation, and thus changes membrane fluidity (WANG, 2001), resulting in the loss of membrane integrity and increased membrane permeability (AGHDAM et al., 2013). It has been proven that normal tissue does not develop browning. In fruit suffering from environment stress, membrane lipid composition, the structure and function of cell membranes are all altered, thereby cellular compartmentalization is broken down, which results in the release of enzymes to contact with their substrates, and then promoting browning (PONGPRASERT et al., 2011; LIN et al., 2016). The effects of 1-MCP on the incidence of physiological disorders of apples have been variable (WATKINS, 2007). Our previous research found that 1-MCP can effectively inhibit the flesh softening and reduce the flesh browning in ‘Red Fuji’ apple (HE et al., 2016). The objective of the current study was to investigate the effects of 1-MCP treatment on fruit quality, flesh browning and its regulation mechanism of flesh browning in ‘Red Fuji’ apple during cold storage and shelf life. With regard to ‘Red Fuji’ apples, it is important to discuss the potential relationship of browning, the metabolism of phenols and membrane lipid during cold storage and shelf life. Our specific aim was to elucidate the role of ethylene, chlorogenic acid and membrane lipid metabolism in stem-end browning. To achieve the objective, we assessed the activity of key enzyme PPO and its gene expression, and investigated the gene expression pattens in chlorogenic acid biosynthesis pathway and membrane lipid peroxidation gene. Postharvest 1-MCP treatment reduced apple flesh browning 133 Materials and methods Materials and 1-MCP treatment ‘Red Fuji’ apples were harvested from a commercial orchard during the optimum commercial harvest period in Hebei province of China (N38°23 4́1.92 E114°27´21.66). The apple fruit (average single fruit weight of 237.73 ± 20.42 g) were randomly divided into 2 groups of 48 kg fruit each. Fruit of each group were divided into 3 equal parts and carefully put into 3 sealed plastic boxes (60 L), then were exposed to 0, and 1.0 μL L-1 1-MCP (Dow Chemical Co., Midland, MI, USA) at 25 ± 2 °C for 24 h. The group without 1-MCP (0 μL L-1) was set as control (HE et al., 2016). After treatment, the fruit were packed with microporous plastic film (15 μm in thickness) and packed into paper box. Each box that contained 30 fruits was stored at 0 ± 0.5 °C with 85% - 95% relative humidity. The fruit were stored at 20 ± 0.5 °C as shelf life after 270 days of cold storage. The respiration rate and ethylene production rate were measured at 1, 3, 5 and 7 days of shelf life, and the fruit quality was measured on the harvest day (0 d), the end of cold storage (270 d), 1 d (270 + 1 d) and 7 d (270 + 7 d) at shelf life. Fruit quality Fruit firmness was measured at two equidistant points on the equa- torial region with the skin removed, using a digital fruit hardness meter (Model: GY-4, Top Instruments Co., Ltd., Hangzhou, China) with a pressure head 8 mm in diameter. The firmness was auto- matically calculated and expressed in kg cm-2. The flesh juice was squeezed from two equidistant points and measured for soluble solids content (SSC) by a PAL-1 pocket digital refractometer (Atago Co., Ltd.,Tokyo, Japan). Titratable content (TA) was determined by acid base titration with 0.01 mol L-1 NaOH up to pH 8.1, and the result was calculated as malic acid (%). Browning rate of stem-end flesh was calculated as the percentage of browning fruit number in the total fruit number. Fruit decay rate was calculated as the percentage of rotten fruit in the total fruit number. Three replicates of 10 fruit each were measured for each treatment at every sampling time. Sampling For each treatment, three replicates were set and there were 10 fruits in each replicate at every sampling time (0 d, 270 + 1 d, 270 + 7 d). After firmness and SSC measurement, we cut flesh of 0.5 cm wide and 1.0 cm deep, about 1 cm from the stem of each fruit being tested, and the stem-end flesh was frozen and ground into powder with liquid nitrogen immediately, then stored at -80 °C for chlorogenic acid content, PPO activity analysis and RNA isolation. Respiration rate and ethylene production rate Respiration rate was measured by HFY-1a CO2 infrared analyzer (Kexi Instrument Co., Ltd., Jiangsu, China) after the fruits were sealed in the container for 1 h, 10 mL of gas was withdrawn from the container and injected to the analyzer, respiration rate was expressed as CO2 release rate (μL kg-1 h-1). For ethylene measurements, 1 mL of gas was withdrawn from the container after the fruits were sealed for 3 h, and then injected into a gas chromatograph (Model: GC9790II, Fuli Instruments Technology Co., Ltd., Wenling, China) equipped with a GDX-502 column and a flame ionization detector (FID). The temperatures of column, vaporization oven and FID were 78 °C, 120 °C and 200 °C, respectively. N2 was used as the carrier gas with a rate of 40 mL min-1. Ethylene production rate was expressed as μL kg-1 h-1. 10 fruits were sealed in each container, and three replicates were measured for each treatment. Chlorogenic acid content and PPO activity Chlorogenic acid was extracted from 2 g frozen powder of stem- end flesh and its content was measured via high-performance liquid chromatography (HPLC) according to the method of He et al. (2017). With a HITACHI L2000 HPLC System (Hitachi, Tokyo, Japan), which consisted of a Hitachi pump L-2130, a Hitachi automatic sample injector L-2200, and a Hitachi UV detector L-2400 and equipped with a Lachrom C18 column (250 mm × 4.6 mm, 5 μm), HPLC was performed in a mobile phase of 5% acetic acid (solvent A), H2O (solvent B) and 100% acetonitrile (solvent C) at a flow rate of 1 mL min-1 and a temperature of 30 °C, and monitored at a wave length of 280 nm with injection of 5 μL samples. Samples were tested using external standard method, which used retention time to determine the quality and peak area for quantification. The analysis was performed in triplicates for each sample to obtain the mean value and standard deviation. For PPO (EC 1.14.18.1) extraction and activity measurement, 1 g of frozen stem-end flesh tissue was powdered in liquid nitrogen with a mortar and blended in 5 mL of 100 mmol L-1 phosphate buffer (pH 7.0) containing 0.3 g PVP. Next, the solution was centrifuged at 12 000 × g for 15 min at 4 °C, after which the supernatant was collected as the crude enzyme extract. The reaction mixture con- tained 100 mmol L-1 catechol in 50 mmol L-1 phosphate buffer (pH 6.0) (CHENG et al., 2015). Changes in the absorbance at 420 nm were measured by using a spectrophotometer (Model: UV-2100, UNICO Instrument, Dayton, USA). The PPO active unit (U) was determined as the change value of absorbance increased by 0.01 per minute per gram of flesh sample and the result was expressed as U g-1 FW. RNA isolation and real-time quantitative PCR analysis Total RNAs were isolated from fruit stem-end flesh, using improved hexadecyltrimethylammonium bromide (CTAB) method (GASIC et al., 2004). Genomic DNAs were eliminated with PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa Bio Inc., Japan) and first strand of cDNA was generated by reverse transcription. Primers for real-time quantitative PCR (qRT-PCR) were designed according to apple (Malus × domestica) nucleotide sequences re- gistered in GenBank of NCBI by DNAMAN 6.0 software. The sequence of primers was shown in Tab. 1. qRT-PCR analysis was performed on ABI 7500 Real-Time System using TB Green™ Pre- mix ex Taq II quantitative PCR kit (TaKaRa Bio Inc., Japan). The relative gene expression amount was calculated with the formula 2-ΔΔCT by Excel 2007 (LIVAK and SCHMITTGEN, 2001). The MdActin gene (GenBank ID: XM_029089583.1) was used as internal refer- ence and the amount of gene expression in the stem-end flesh at 0 d was defined as 1.0. Statistical analysis All statistical data analyses were done by SPSS (18.0). The data were analyzed by one-way analysis of variance (ANOVA). Means were separated using Duncan method at p<0.05. Data are shown as means ± standard errors. Fig. 1-8 were generated by GraphPad Prism 8.0, and different letters in Fig. 1-8 represented significant difference at p<0.05. Results Fruit quality, browning rate and decay rate The fruit firmness, SSC and TA content decreased after 270 days of cold storage, and 1-MCP treatment could maintain higher firmness, SSC and TA, but there was no significant difference in SSC at 270 + 7 d (Fig. 1). This indicated that 1-MCP effectively delayed fruit 134 J. He, Y. Feng, Y. Cheng, S. Wang, Y. F1, J. Guan After 270 d cold storage, the MdPAL1 expression level was nearly 200 times higher than the initial storage fruit, and then down- regulated at shelf life. 1-MCP treatment significantly decreased MdPAL1 expression except that there was no significant difference with the control at 270 + 7 d (Fig. 6A). Different with MdPAL1, the MdPAL2 expression was down-regulated during cold storage, and was gradually up-regulated at shelf life. However, both the down- regulation and up-regulation were alleviated by 1-MCP treatment (Fig. 6B). The expression level of Md4CL2 increased during cold storage, and then decreased during the shelf-life, but the expression of Md4CL2 was inhibited by 1-MCP treatment especially at the end of shelf- life (Fig. 6C). The expression level of MdHCT3 was significantly up-regulated during storage and was nearly 100 times higher at 270 + 7 d than that at the initial storage, and 1-MCP exhibited an obviously inhibition at the HCT3 expression (Fig. 6D). PPO activity and the expression level of MdPPOs PPO activity increased during cold storage and shelf-life, which was significantly inhibited by 1-MCP (Fig. 7). The expression of MdPPO1 reached the peak value at the end of cold storage, decreased at 270 + 1 d and increased again at 270 + 7 d in shelf life (Fig. 8A). There was no obvious change of MdPPO5 expression during cold storage and earlier shelf-life, but a dramatical up-regulation was observed at 270 + 7 d in shelf-life (Fig. 8B). It is worth noting that the Tab. 1: Sequence of primers for qRT-PCR Gene Genbank ID Primer sequences (5’-3’) MdActin XM_029089583.1 5’-TGACCGAATGAGCAAGGAAATTACT-3’ 5’-TACTCAGCTTTGGCAATCCACATC-3’ MdERS1 NM_001328757.1 5’-GGAGATCTCGTTGGACGCAA-3’ 5’-GGATGACTCTGACCACTGGC-3’ MdETR2 XM_008358087.3 5’-ATGTTATGTGGCAGCGCAGG-3’ 5’-GGCACACAATTCTGCCAACA-3’ MdPAL1 XM_008357397.2 5’-GTGTTTAATTAGGCGGGGGA-3’ 5’-AAATTGATTGCCTCATATCACAGAT-3’ MdPAL2 XM_029105821.1 5’-AGCACCACCTCTTTCCATTCC-3’ 5’-GCCGGGAAAAACTAAGTGGG-3’ Md4CL2 XM_008364603.2 5’-CTCGATCGATGCGTGTTGAC-3’ 5’-TGCCTTTGATCCCCGACTTC-3’ MdHCT3 XM_008344601.3 5’-TCACCATTTCCTTGCGCCTC-3’ 5’-ACGTTGACTCCCTCACGGTA-3’ MdPPO1 NM_001319261.1 5’-GAGCTCATGAAGGCCCTACC-3’ 5’-TTTGGAGCTCGAGTTCTGGG-3’ MdPPO5 JQ388482.1 5’-TCAAATCGACGCCAACCTCA-3’ 5’-GTTTCGATTGAACCGGCCC-3’ MdLOX NM_001294093.1 5’-AGCGACAAGAAAGAAGAACC-3’ 5’-TACTGACCGTAGTTGATTGC-3’ Fig. 1: Fruit firmness (A), SSC (B) and TA content (C) of control and 1-MCP treated ‘Red Fuji’ apple at shelf life after cold storage. Data are shown as means ± standard errors, different letters represent significant difference at p<0.05. softening and maintained fruit quality at shelf life in ‘Red Fuji’ apple. In addition, the browning rate of stem-end flesh was as high as 46.7% in the control fruit at 270 d, while the value is 0 in the 1-MCP treatment fruit. The browning became more serious during the shelf life, but 1-MCP significantly delayed and reduced the browning rate (Fig. 2A). Meanwhile, 1-MCP effectively reduced fruit decay (Fig. 2B). Respiration rate, ethylene production rate and the expression of ethylene receptor gene (MdERS1) in the stem-end flesh During the shelf life, the respiration rate of fruit increased at first, reached the peak value at 270 + 5 d and then decreased. The respiration rate was significantly lowered by 1-MCP (Fig. 3A). The ethylene production rate of the control fruit displayed an obvious increase and reached the peak value at 27 + 3 d, but kept at a very low level in 1-MCP treatment fruit during shelf life (Fig. 3B). The ethylene production and the expression of MdERS1 was effectively inhibited by 1-MCP treatment (Fig. 4). Chlorogenic acid content and biosynthesis related genes ex- pression Compared with the initial stage of storage, the chlorogenic acid content of stem-end flesh increased gradually with the extension of storage time, which was markedly inhibited by 1-MCP (Fig. 5). Postharvest 1-MCP treatment reduced apple flesh browning 135 up-regulation of MdPPO1 and MdPPO5 was significantly inhibited by 1-MCP treatment. The expression level of MdLOX gene significantly increased during cold storage, and subsequently decreased. In the control fruit, the expression of MdLOX showed an increasing trend. However, 1-MCP treatment effectively inhibited the expression and kept it at a very low level (Fig. 9). Correlation analysis According to the correlation analysis (Tab. 2), the browning rate of stem-end flesh is significantly correlated with chlorogenic acid content (r=0.962**), PPO activity (r=0.915**), the expression of MdPAL1 (r=0.572**), MdHCT3 (r=0.926**), MdPPO1 (r=0.743**) and MdLOX (r=0.595**). The chlorogenic acid content was sig- nificantly correlated with PPO activity (r=0.865**), the expression amounts of MdPAL1 (r=0.500*), MdHCT3 (r=0.904**), PPO activity and MdPPO1 expression (r=0.841**). Discussions Postharvest treatment with 1-MCP, an inhibitor of ethylene action, has been shown to improve quality characteristics of apples, includ- ing reduced ethylene production and respiration, as well as improved firmness and acidity retention (WATKINS, 2007; DEELL et al., 2007). In our study, 1-MCP could effectively improve the quality of ‘Red Fuji’ apple fruit after long-term cold storage, and significantly reduce the respiration rate and ethylene production rate during cold storage and shelf life (Fig. 3), which was the physiological basis of 1-MCP function in delaying fruit senescence. Therefore, the fruit treated with 1-MCP kept higher fruit firmness and TA content, while maintaining a higher SSC (Fig. 1). 1-MCP treatment also significantly reduced Fig. 5: Chlorogenic acid content of stem-end flesh tissue in ‘Red Fuji’ apple at shelf life after cold storage. Data are shown as means ± standard errors, different letters represent significant difference at p<0.05. Fig. 3: Fruit respiration rate (A) and ethylene production rate (B) of ‘Red Fuji’ apple at shelf life after cold storage. Data are shown as means ± standard errors, different letters represent significant difference at p<0.05. Fig. 4: MdERS1 expression level of stem-end flesh tissue in ‘Red Fuji’ apple at shelf life after cold storage. Data are shown as means ± standard deviations, different letters represent significant difference at p<0.05. Fig. 2: Browning rate (A) of stem-end flesh and decay rate (B) of ‘Red Fuji’ apple at shelf life after cold storage. Data are shown as means ± standard errors, different letters represent significant difference at p<0.05. 136 J. He, Y. Feng, Y. Cheng, S. Wang, Y. F1, J. Guan fruit decay and stem-end flesh browning (Fig. 2). The physiological effect of ethylene begins with the recognition of ethylene signal by ethylene receptors (ETR) located in the endoplasmic reticulum (ALONSO et al., 2003), and 1-MCP can delay and inhibit a series of fruit ripening and senescence processes caused by ethylene through the competition with ethylene binding sites on the receptors (BLANKENSHIP and DOLE, 2003). Ireland et al. (2012) found that 100 μL L-1 ethylene in apples could up-regulate the expression of ERS1 gene, while in our study, 1-MCP significantly inhibited the expression of MdERS1 (Fig. 4), so it was speculated that MdERS1 was involved in the flesh browning caused by ethylene signal. The development of fruit browning depends on the enzymatic action of the PPO. The apple’s susceptibility to flesh browning is thought to be the result of complex interplay between the PPO enzyme and Fig. 6: The expression level of MdPAL1 (A), MdPAL2 (B), Md4CL2 (C) and MdHCT3 (D) of stem-end flesh tissue in ‘Red Fuji’ apple at shelf life after cold storage. Data are shown as means ± standard deviations, different letters represent significant difference at p<0.05. Fig. 7: PPO activity of stem-end flesh tissue in ‘Red Fuji’ apple at shelf life after cold storage. Data are shown as means ± standard errors, dif- ferent letters represent significant difference at p<0.05. Fig. 8: MdPPO1 (A) and MdPPO5 (B) expression level of stem-end flesh tissue in ‘Red Fuji’ apple at shelf life after cold storage. Data are shown as means ± standard deviations, different letters represent significant difference at p<0.05. Fig. 9: MdLOX expression level of stem-end flesh tissue in ‘Red Fuji’ apple at shelf life after cold storage. Data are shown as means ± stan- dard deviations, different letters represent significant difference at p<0.05. Postharvest 1-MCP treatment reduced apple flesh browning 137 polyphenol content (DI GUARDO et al., 2014). However, the effects of 1-MCP on the incidence of physiological disorders of apples have been variable (WATKINS, 2007). Ripening-related disorders such as senescent breakdown are greatly inhibited by 1-MCP (MORAN et al., 2005; DEELL et al., 2007), as are disorders such as superficial scald that is associated with ethylene production (RUPASINGHE et al., 2000; TSANTILI et al., 2007). The present results showed that the content of chlorogenic acid and PPO activity in the stem-end flesh increased after cold storage (Fig. 5, Fig. 7), which was positively correlated with the degree of flesh browning (Fig. 2, Tab. 2). 1-MCP significantly reduced the chlorogenic acid content and PPO activity, which was the biochemical basis of 1-MCP delaying the occurrence of flesh browning. However, it has been previously shown that 1-MCP treatment can make ‘Empire’ apples more sensitive to CO2 concentration, and exacerbate flesh browning because of CO2 injury (JUNG and WATKINS, 2011; LEE et al., 2012). Therefor, we deduced that the stem-end flesh browning in ‘Red Fuji’ apple is the consequence of senescence caused by ethylene, and 1-MCP could effectively inhibit the browning. PAL, 4CL and HCT are involved in chlorogenic acid biosynthesis pathway in apple and pear fruit, and PAL is the enzyme that catalyzes the first step reaction of phenylpropane metabolic pathway (CHENG et al., 2015; DI GUARDO et al., 2014; BOTH et al., 2018). This study demonstrated that 1-MCP treatment significantly decreased the expression of MdPAL1 (Fig. 6A), MdHCT3 (Fig. 6D) and MdPPO1 (Fig. 8A), and displayed a lower chlorogenic acid content (Fig. 5) and PPO activity (Fig. 7) in the stem-end flesh. However, the expres- sion trends of MdPPO5 (Fig. 8B), MdPAL2 (Fig. 6B) and Md4CL2 (Fig. 6C) were not consistent with the changes of chlorogenic acid content and flesh browning. LOX are the crucial enzymes of membrane lipid metabolism, and might induce degradation of phospholipids and unsaturated fatty acids (WANG, 2011; AGHDAM and BODBODAK, 2014). High levels of unsaturated fatty acids could maintain normal cellular function and prevent the interaction of PPO with phenol substrates resulting in enzymatic browning (LIU et al., 2006). It has been proven that LOX activity was significantly higher and the gene expression increased when tissue browning occurred (SHENG et al., 2016). In our experiment, 1-MCP treatment reduced the MdLOX gene expression level in the stem-end flesh (Fig. 9) which was consistent with the obvious browning inhibition. This implied that 1-MCP plays an important role in the integrity maintaining of membrane lipid in the stem-end flesh of ‘Red Fuji’ apple. Conclusion Postharvest 1-MCP (1.0 μL L-1) treatment can effectively improve the quality of ‘Red Fuji’ apple after long-term cold storage, and keep a low respiration rate and minimum ethylene production rate. Moreover, 1-MCP treatment markedly reduce the stem-end flesh browning by down-regulating the expression of MdPAL1, MdHCT3, MdPPO1 and MdLOX, and then inhibiting the increase of chlorogenic acid content and PPO activity. In conclusion, 1-MCP based technology shows positive effects on quality control and reducing storage browning in ‘Red Fuji’ apple. Acknowledgements This research was funded by the HAAFS Agriculture Science and Technology Innovation Project (2019-2-1), Modern Agricultural Industry Technology Research System of Hebei Province (HBCT2018100207), and The Young Talents Fund of Hebei pro- vince. Conflicts of interest No potential conflict of interest was reported by the authors. References AGHDAM, M.S., BODBODAK, S., 2013: Physiological and biochemical mecha- nisms regulating chilling tolerance in fruits and vegetables under post- harvest salicylates and jasmonates treatments. Sci. Hortic. 156, 73-85. 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DOI: 10.1016/j.fpsl.2018.03.010 CHENG, Y.D., LIU, L.Q., ZHAO, G.Q., SHEN, C.G., GUAN, J.F., 2015: The effects of modified atmosphere packaging on core browning and the Tab. 2: Correlation coefficient of flesh browning rate with chlorogenic acid content, PPO activity and the expression level of relative genes in ‘Red Fuji’ apple Browning Chlorogenic PPO MdERS1 MdPAL1 MdPAL2 Md4CL2 MdHCT3 MdPPO1 MdPPO5 MdLOX Rate Acid Content Activity Browning Rate 1.000 Chlorogenic 0.962** 1.000 Acid Content PPO Activity 0.915** 0.865** 1.000 MdERS1 0.465* 0.516* 0.334 1.000 MdPAL1 0.572** 0.500* 0.683** -0.192 1.000 MdPAL2 -0.184 -0.153 -0.167 0.666** -0.601** 1.000 Md4CL2 -0.020 -0.046 0.149 0.225 0.110 0.255 1.000 MdHCT3 0.926** 0.904** 0.889** 0.397 0.483* -0.100 -0.225 1.000 MdPPO1 0.743** 0.720** 0.841** 0.396 0.619** -0.017 0.347 0.683** 1.000 MdPPO5 0.097 0.081 0.208 0.484* -0.356 0.784** 0.077 0.273 0.247 1.000 MdLOX 0.595** 0.551** 0.658** 0.581** 0.487* 0.179 0.679** 0.404 0.818** 0.161 1.000 ** Significant correlation at 0.01 level (both sides); * Significant correlation at 0.05 level (both sides) http://dx.doi.org/10.1016/j.scienta.2013.03.028 http://dx.doi.org/10.1073/pnas.0438070100 http://dx.doi.org/10.1016/S0925-5214(02)00246-6 http://dx.doi.org/10.1016/j.fpsl.2018.03.010 138 J. 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Gene 534(2), 408-416. DOI: 10.1016/j.gene.2012.09.051 ZHAO, S.C., TUAN, P.A., LI, X.H., KIM, Y.B., KIM, H., PARK, C.G., YANG, J.L., LI, C.H., PARK, S.U., 2013: Identification of phenylpropanoid bio- synthetic genes and phenylpropanoid accumulation by transcriptome analysis of Lycium chinense. BMC Genomics. 14(1), 802. DOI: 10.1186/1471-2164-14-802 ORCID Jingang He https://orcid.org/0000-0001-9986-8720 Yunxiao Feng https://orcid.org/0000-0003-0117-7062 Yudou Cheng https://orcid.org/0000-0002-5990-0041 Shen Wang https://orcid.org/0000-0002-1932-4641 Junfeng Guan https://orcid.org/0000-0001-8570-9219 Address of the corresponding author: Junfeng Guan, Institute of Genetics and Physiology, Hebei Academy of Agri- culture and Forestry Sciences, Shijiazhuang, China E-Mail: junfeng-guan@263.net © The Author(s) 2021. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creative- commons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1016/j.gene.2012.09.051 http://dx.doi.org/10.1186/1471-2164-14-802 https://orcid.org/0000-0001-9986-8720 https://orcid.org/0000-0003-0117-7062 https://orcid.org/0000-0002-5990-0041 https://orcid.org/0000-0002-1932-4641 https://orcid.org/0000-0001-8570-9219