《现代食品工程高新技术》课程教学资源（文献资料）食品保鲜新技术 The effects of modified atmosphere packaging on core browning and the expression patterns of PPO and PAL genes in ‘Yali’ pears during cold storage.pdf

The effects of modified atmosphere packaging on core browning and the expression patterns of PPO and PAL genes in ‘Yali’ pears during cold storage* Yudou Cheng a, b , Liqin Liu a, b , Guoqun Zhao c , Chengguo Shen a, b , Hongbo Yan a, b , Junfeng Guan a, b, * , Kun Yang a, b a Institute of Genetics and Physiology, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang 050051, PR China b Plant Genetic Engineering Center of Hebei Province, Shijiazhuang 050051, PR China c College of Bioscience and Bioengineering, Hebei University of Science and Technology, Shijiazhuang 050018, PR China article info Article history: Received 28 April 2013 Received in revised form 28 June 2014 Accepted 1 September 2014 Available online 16 September 2014 Keywords: MAP Core browning PAL PPO abstract Pear fruits often experience core browning when exposed to excessive CO2 during storage. Therefore, it is critical to understand the mechanism of this process to optimize atmospheric conditions during postharvest pear storage. In the present study, the browning process, phenolic content, polyphenol oxidase (PPO) activity and expression profiles of phenylalanine ammonia-lyase (PAL) and PPO genes in the core tissue of ‘Yali’ pears (Pyrus bretschneideri Rehd cv. Yali) were investigated under modified atmosphere packaging (MAP). The results showed that MAP1 (with a thickness of 10 mm) reduced core browning, retarded the peak appearance of PPO activity and phenolic content, and inhibited the expression of PbPAL1, PbPAL2, and PbPPO1 genes in core tissue relative to the control (which had no packaging), but MAP2 (with a thickness of 30 mm) exerted the opposite effects during cold storage (0 C). There was no significant relation between PbPPO4, PbPPO5 and PbPPO6 expression in core tissue and core browning. These results suggested that MAP1 was suitable for cold storage and that the PbPAL1, PbPAL2 and PbPPO1 genes might be involved in core browning under modified atmosphere storage in ‘Yali’ pears. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction The ‘Yali’ pear (Pyrus bretschneideri Rehd cv. Yali) is one of the primary pear cultivars in China, and core browning is a serious problem during postharvest storage. The avoidance of core browning in pears during storage is a major challenge. CA (control atmosphere) and MA (modified atmosphere) storage can improve fruit quality and extend storage life; however, pear fruits are sensitive to CO2 during storage. High CO2 concentrations usually lead to core browning in pears (Chen, Yu, & Zhou, 1991; Larrigaudiere, Pinto, Lentheric, & Vendrell, 2001; Maguire & MacKay, 2003; Wang et al., 2009); thus, it is essential to determine how to regulate the CO2 concentration inside the storage environment effectively. Modified atmosphere packaging (MAP) was proposed as a good preservation technology for horticultural commodities because it not only could control the appropriate ratio of CO2 to O2 within the packaging, but it could also inhibit respiration and ethylene production, retard senescence, inhibit browning and improve fruit quality under optimum conditions (Mattheis & Fellman, 2000; Sivakumar & Korsten, 2006). In this way, the gas permeability of packaging material is a vital factor in modified atmosphere storage (Al-Ati & Hotchkiss, 2003). High-density film with poor gas permeability led to the accumulation of excessive CO2 inside the packaging, which might lead to hypoxia reactions, reducing quality and accelerating browning in pears (Li, Zhang, Sun, & Liu, 2009). Therefore, the CO2 concentration under MA storage should be maintained at a low level (0e0.5%) during pear fruit storage (Li & Bi, 2010), and low-density film with high CO2 permeability could be used as an ideal packaging material to improve postharvest pear quality. Fruit browning is a quinine formation process resulting from the phenolic oxidation catalyzed by polyphenol oxidase (PPO) (Franck et al., 2007; Tomas-Barber an & Espín, 2001). Phenylalanine ammonia-lyase (PAL) was the first key enzyme in phenolic * This manuscript was presented in the international conference of “Food Innova- 2012” Hangzhou, China, December 12e14, 2012. * Corresponding author. Institute of Genetics and Physiology, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang 050051, PR China. Tel.: þ86 311 87652118; fax: þ86 311 87652335. E-mail address: junfeng-guan@263.net (J. Guan). Contents lists available at ScienceDirect LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt http://dx.doi.org/10.1016/j.lwt.2014.09.005 0023-6438/© 2014 Elsevier Ltd. All rights reserved. LWT - Food Science and Technology 60 (2015) 1243e1248

compounds synthesis, and it promoted the catalytic conversion of L-phenylalanine into trans-cinnamic acid. Trans-cinnamic acid was transformed into phenol by a series of enzymatic reactions (Anterola, Jeon, Davin, & Lewis, 2002). PPO was able to catalyze the oxidation of o-diphenolic compounds to quinones (Mayer, 2006; Yoruk & Marshall, 2003). Previous reports have shown that PAL and PPO were multi-gene families, and they were involved in many biotic and abiotic stress processes, such as pathogen infection (Li & Steffens, 2002), wounding (Constabel, Bergey, & Ryan, 1995; Constabel, Yip, Patton, & Christopher, 2000; Thipyapong & Steffens, 1997), and cold stress (Sanchez-Ballesta, Lafuente, Zacarias, & Granell, 2000). These genes were correlated with tissue browning in many species, such as potatoes (Coetzer, Corsini, Love, Pavek, & Tumer, 2001), apples (Kim et al., 2001; Murata et al., 2001), pineapples (Stewart, Sawyer, Bucheli, & Robinson, 2001; Zhou et al., 2003) and eggplants (Shetty, Chandrashekar, & Venkatesh, 2011). However, the molecular mechanism of PAL and PPO as involved in pear tissue browning is poorly understood. For a previous work, we cloned two PAL genes and four PPO genes in ‘Yali’ pears, and they were named PbPAL1, PbPAL2, and PbPPO1, PbPPO4, PbPPO5, and PbPPO6. It is important to study the molecular mechanism of browning in pear fruit. The primary purpose of this study is to analyze the MAP effect on the phenolic content, core browning and the expression pattern of PPO and PAL genes in the core of ‘Yali’ pears and to further to clarify the biochemical and molecular mechanism that PPO and PAL are involved in pear core browning under MAP conditions under low temperature storage. 2. Materials and methods 2.1. Materials Pear fruits (Pyrus bretschneideri Rehd cv. Yali) were harvested at commercial maturity (average weight: 215.55 ± 16.61 g, firmness: 69.78 ± 4.03 N) on September 22, 2009, at Jinzhou (E115.04, N38.03), Hebei Province, China, and then immediately transported to our laboratory within 4 h. The fruits were selected for uniformity of size and shape, and they were free from visual defects. The pears were divided into three lots. One lot was packaged in 10-mm-thick low-density polyethylene film (Product of National Engineering and Technology Research Center for Preservation of Agricultural Products, Tianjin, China) and designated as MAP1, the second lot was packaged in 30-mm-thick low-density polyethylene film (Product of National Engineering and Technology Research Center for Preservation of Agricultural Products, Tianjin, China) and designated as MAP2, and the third lot without packaging was designated as the control. Detailed film properties were listed in Table 1, and each film bag contained fifteen fruits to make one replicate. After being treated, all of the fruits were immediately stored at 0 C. This assay was performed after 0, 60, 80 and 100 days of storage, and after measuring the core browning index, the core tissues were rapidly frozen in liquid nitrogen and then stored at 80 C until analysis for phenolic content, PPO activity and PAL and PPO expression levels. 2.2. Calculating the browning index of core tissue Thirty fruits per treatment (10 from each replicate) were used to assess core browning. Browning was evaluated visually after a transverse cut through the equator of the fruit, and browning was scored on a scale of four levels according to the browning area by accounting for the percentage of the whole core area, in which 0, 1, 2 and 3 indicated no browning, less than 25%, 25%e50% and more than 50% browned, respectively. The results were expressed as a browning index that was calculated by using the following formula (Wang, Tian, & Xu, 2005): Core browning index ¼ hXðbrowning levelnumber of fruit at the browning levelÞ= ð3total number of fruitÞ i 100 2.3. Determining the O2 and CO2 contents within the package bag The O2 and CO2 concentrations within the selected MAP headspaces were measured with a handheld O2/CO2 analyzer (WITT, Witten, Germany). Approximately 10 ml of gas was extracted from the MAP bag, and 10 ml of fresh air was simultaneously injected into the same bag by using a syringe. After taking a measurement, the small holes punctured by the syringe were covered with a piece of sticky tape. All measurements were performed in three replicates. 2.4. Determining the phenolic content and PPO activity The total phenolic contents of the cores were determined according to the method described by Ju (1989). Three grams of core tissue was homogenized in a mixture containing 5 ml of ethanol and 5 ml of trichloroacetic acid aqueous solution (10 g/100 ml), and it was then centrifuged at 12,000 g for 15 min after being kept at 4 C for 24 h. The supernatant was mixed with Folin-Ciocalteu reagent (Merck, Darmstadt, Germany), which contained Na2CO3 (10 g/100 ml) and NaOH (2 g/100 ml). The total phenolic content was then determined by using a UV-2100 spectrophotometer (United Products & Instruments Inc., Dayton, USA) at 750 nm. A standard curve for gallic acid (SigmaeAldrich, Shanghai, China) was used to quantify the total phenolic content. The extraction and assay of polyphenol oxidase (PPO) activity were performed as described by Serradell et al. (2000). A 2.5-g quantity of core tissue was homogenized in 10 ml of phosphate buffer (0.1 mol/L, pH 7.8) with 1 g of polyvinylpyrrolidone (PVP) (Sangon Biotech, Shanghai, China), and the solution was then centrifuged at 20,000 g for 15 min at 4 C. The supernatant was collected as a crude PPO extract. The reaction mixture contained 0.1 mol/L catechol (SigmaeAldrich, Shanghai, China) in 0.05 mol/L phosphate buffer (pH 6.0). Changes in the absorbance at 410 nm were measured. One unit of PPO activity was defined as a change of 0.01 at 410 nm in the absorbance per min. The protein content of PPO crude extract was estimated by Coomassie brilliant blue method (Okutucu, Dinçer, Habib, & Zihnioglu, 2007) with bovine serum albumin as a standard. 2.5. Quantitative RT-PCR analysis of PPO and PAL gene expression Total RNA was isolated by CTAB (hexadecyl trimethyl ammonium bromide) (Sangon Biotech, Shanghai, China) method (Gasic, Table 1 Properties of the films used for packaging ‘Yali’ pear. Film thickness Pco2 (mL/m2 $d$atm) Po2 (mL/m2 $d$atm) Permeability rate (Pco2/Po2) Size(cm2 ) 10 mm 75200.41 10955.56 6.864132 30 40 30 mm 15844.76 4766.8 3.323983 30 40 Note: Pco2 and Po2 indicate the values of CO2 and O2 permeability of films respectively. 1244 Y. Cheng et al. / LWT - Food Science and Technology 60 (2015) 1243e1248

Hernandez, & Korban, 2004). After being digested with DNaseI, 0.5 mg of total RNA was reverse-transcribed into first-strand cDNA by using a Takara RNA PCR (AMV) Version 3.0 Kit (TaKaRa Biomedicals, Dalian, China). Primer sequences were shown in Table 2. The qRT-PCR reaction was performed in a final volume of 25 mL containing 12.5 mL of SYBR Green PCR Premix Ex Taq (TaKaRa Biomedicals, Dalian, China), 1 mmol/L forward and reverse primers, and 10 ng cDNA, and the reactions were performed as follows: 10 s at 95 C, 40 cycles of 95 C for 5 s, and 60 C for 34 s. To confirm the quality and primer specificity, the melting temperature of the amplification products was analyzed in a dissociation curve by using an ABI 7500 instrument (Applied Biosystems, Foster City, USA). The relative expression level was calculated by using the comparative Ct method, and PbActin2 (PbACT2) was used to normalize the amount of gene-specific qRT-PCR products. Data were collected over three independent assays. 2.6. Statistical analysis All experiments were replicated three times. All data were subjected to an analysis of variance (ANOVA). A difference at P < 0.05 was considered to be significant. Data were presented as the means ± standard errors. 3. Results and discussion 3.1. MAP effects on core browning The core browning indexes among the three treatments were different. The fruit core became brown in the control and MAP2 at the 60th day of storage, which was 20 days earlier than that of MAP1. At the 100th day of storage, the core browning index in MAP2 was significantly higher than it was in the control, and the core browning index in MAP1 was lower (Fig. 1). These results suggested that the gas component inside different treatments might be different. The gas component in MAP1 might form an appropriate condition for ‘Yali’ pear storage, and it effectively reduced core browning. On the contrary, the MAP2 condition was inappropriate for ‘Yali’ pear storage. 3.2. O2 and CO2 contents within the MAP bag The CO2 and O2 concentrations inside the MAP treatments were detected to define the gas components within. The results showed that the CO2 concentration accumulated up to 1.6% in MAP2, which was much higher than that of the control, and this concentration exceeded the CO2 tolerance for ‘Yali’ pears (Chen et al., 1991; Li & Bi, 2010), which might in turn account for the distinct core browning under MAP2. However, the CO2 concentration under MAP1 only accumulated up to 0.3% during storage, which was lower than the tolerated CO2 concentration in ‘Yali’ pears (Chen et al., 1991; Li & Bi, 2010), and the O2 concentration in MAP1 decreased to approximately 19.5% (Fig. 2). The CO2 and O2 concentrations inside the MAP1 package was suitable for ‘Yali’ pear storage, and this storage condition might effectively induce core browning by influencing the respiration and ethylene production of ‘Yali’ pears (Mangaraj & Goswami, 2009). 3.3. MAP effects on the total phenolic contents of core tissue As the substrate of PPO, phenolics were closely linked with tissue browning (Tomas-Barber an & Espín, 2001). In this work, the total phenolic content of the core increased rapidly in MAP2 and was higher than that of the control at the 60th day, and this product might be the result of excess CO2-induced injury (Chen et al., 1991; Ding, Chachin, Ueda, Imahori, & Kurooka, 1999). However, the phenolic content of cores stored under MAP1 showed a slow increasing trend and was maintained at a high level at the late stage (Fig. 3), which was consistent with the results observed in bananas (Nguyen, Ketsa, & Van Doorn, 2004) and bamboo shoots (Shen, Kong, & Wang, 2006). We hypothesized that the appropriate ratio of CO2 to O2 under MAP1 delayed the core browning process, thereby putting off the synthesis of phenolics, or retarding their oxidation process (Jones & Saxena, 2013). 3.4. MAP influence on PAL gene expression The expression patterns of PbPAL1 and PbPAL2 in the core were correlated with changes in phenolic contents in all treatments (Fig. 3). Both the PbPAL1 and PbPAL2 mRNA increased quickly on the 60th day and then decreased in MAP2, and the PbPAL1 and PbPAL2 levels were much higher than that of the control over the entire storage (Fig. 4A and B), which was similar to the expression patterns of PALs described in citrus chilling injury (Lafuente, Zacarias, Martínez-Tellez, Sanchez-Ballesta, & Granell, 2003). The PbPAL1 expression level under MAP1 was lower on the 60th and 80th days than that of the control, and PbPAL2 was lower over the whole storage stage (Fig. 4A and B). PAL has been shown to have a close Table 2 Primers for quantitative PCR analysis of expression of PPO and PAL. Gene Accession NO. Forward Reverse PbPPO1 HQ729709 50 -TCCCTACTCACAAAGCCCAAG-30 50 -GACCTCCAAGACCAAGAAGCA-30 PbPPO4 GU906265 50 -AAGGTGACAATGATAACCAAGAC-30 50 -TGCCGCACCGTAGAGACC-30 PbPPO5 GU906266 50 -ACCAAAACAAAAACCATTCCAC-30 50 -CAGCCACTCCACCATACAGG-30 PbPPO6 GU906267 50 -AGAAGGCGGAACGAGAGGA-30 50 -ACTCTGGCTGGGCTGACTT-30 PbPAL1 GU906268 50 -GCAAAGAGGACTTTAACAACTGG-30 50 -TACTCCCTATCGACAACTTTAAGC-30 PbPAL2 GU906269 50 -CCGGGAAATAACCAAACC-30 50 -ATGGCTCCCTTTCATTGC-30 PbACT2 GU830959 50 -GGACATTCAACCCCTCGTCT-30 50 -ATCCTTCTGACCCATACCAACC-30 Fig. 1. The core browning index of cold-stored ‘Yali’ pears. (▫) Control; ( ) MAP1 (10 mm); and (-) MAP2 (30 mm). The values are the means ± SE of three replicates. The star represents a significant difference (P < 0.05). Y. Cheng et al. / LWT - Food Science and Technology 60 (2015) 1243e1248 1245

relation to fruit browning (Chen et al., 2008; Chidtragool, Ketsa, Bowen, Ferguson, & van Doorn, 2011; Lafuente et al., 2003; Nguyen, Ketsa, & Van Doorn, 2003, 2004); thus, PbPAL1 and PbPAL2 might have participated in ‘Yali’ pear core browning during MAP storage. 3.5. MAP effects on the PPO activity of core tissue A high concentration of CO2 increased PPO activity and induced tissue browning, and a low CO2 concentration decreased the PPO activity and inhibited tissue browning in pears (Chen et al., 1991). In this work, the PPO activity of the core was clearly higher under MAP2 than that of the control over the entire storage period (Fig. 5), which was similar to earlier findings by Zhou, Shu, and Wu (1993). However, the PPO activity under MAP1 was inhibited at first, and the peak of this activity was delayed (Fig. 5). In all treatments, the PPO activity clearly increased at the early stage of core browning, indicating that PPO also participated in the core browning of ‘Yali’ pears. 3.6. MAP influence on PPO gene expression To define the relation between PPO genes and core browning in ‘Yali’ pears, BLAST searches were applied to analyze the homology of others species PPOs in which the functions had been studied. The result indicated that PbPPO1 protein was 95%, 92% and 91% identical to APO5, PPO3 and PPO7 in apples, respectively (GeneBank Accession Nos. P43309.1, BAA21676.1 and BAA21677.1). PbPPO4 shared 91% of MD-PPO2 in apple (GeneBank Accession No. AAK56323.1). PbPPO5 was 66% identical to APO5 and 60% to PINPPO1 in pineapple (GeneBank Accession No. AAO16865.1). PPO6 Fig. 2. Changes in the O2 (A) and CO2 (B) concentrations inside film packages. (▫) Control; ( ) MAP1 (10 mm); and (-) MAP2 (30 mm). The values are the means ± SE of three replicates. The star represents a significant difference (P < 0.05). Fig. 3. Changes in the total phenolic contents of ‘Yali’ pear cores during cold storage. (▫) Control; ( ) MAP1 (10 mm); and (-) MAP2 (30 mm). The values are the means ± SE for three replicates. The star represents a significant difference (P < 0.05). Fig. 4. The expression levels of PAL genes in ‘Yali’ pear cores during cold storage. (A): PAL1. (B): PAL2. (▫) Control; ( ) MAP1 (10 mm); and (-) MAP2 (30 mm). The values are the means ± SE of three replicates. The star represents a significant difference (P < 0.05). 1246 Y. Cheng et al. / LWT - Food Science and Technology 60 (2015) 1243e1248

was 51% identical to the MD-PPO2 of apple and 50% identical to the PINPPO1 of pineapple. PbPPO1 gene expression in MAP1-treated fruits was apparently lower during storage, and it was higher under MAP2 treatment than that of the control at the 60th and 80th days of storage (Fig. 6A). The PbPPO1 expression pattern under MAP2 was similar to that of APO5 and Md-PPO in apples, which reportedly participated in tissue browning (Boss, Gardner, Janssen, & Rose, 1995; Di Guardo et al., 2013; Kim et al., 2001); thus, this finding suggested that PbPPO1 was involved in the core browning process. PbPPO4 gene expression showed a significant downward trend in all treatments during storage, and the declining rate was the slowest in MAP1 (Fig. 6B). However, the pattern of PbPPO4 expression was not in accordance with that of the core browning (Fig. 1) and PPO activity (Fig. 5). This result was similar to that of the MD-PPO2 gene expression pattern in apples (Kim et al., 2001), indicating that PbPPO4 might not be involved in core browning. The PbPPO5 expression pattern in core tissue showed a rapid increase at first, and then a slight decrease. The mRNA levels of PbPPO5 under MAP1 and MAP2 were both significantly higher than that of the control at the 60th day (Fig. 6C). This expression pattern was clearly not synchronous with the core browning process (Fig. 1). However, the PbPPO5 expression level significantly increased after cold storage, which was similar to that of PINPPO1 in pineapple under chilling injury (Stewart et al., 2001; Zhou et al., 2003). We hypothesized that the PbPPO5 expression pattern might be influenced by chilling or other factors during the core browning process, and further study on PbPPO5 function was needed. PbPPO6 gene expression showed a slight increasing trend from the 60th day to the 100th day under MAP2; in addition, the mRNA level was higher than that of the control at the 60th and 100th days of storage. There was a marked peak in the PbPPO6 mRNA level under MAP1 at the 80th day (Fig. 6D). The PbPPO6 expression patterns were not correlated with PPO activity and tissue browning in MAP1 and MAP2; thus, they were not involved in the core browning process of ‘Yali’ pears under MAP storage. 4. Conclusions The O2 and CO2 concentrations in MAP1 were appropriate for storing ‘Yali’ pear, and this treatment effectively inhibited core Fig. 5. Changes in the PPO activity of ‘Yali’ pear cores during cold storage. (▫) Control; ( ) MAP1 (10 mm); and (-) MAP2 (30 mm).The values are the means ± SE of three replicates. The star represents a significant difference (P < 0.05). Fig. 6. The expression levels of PPO genes in ‘Yali’ pear cores during cold storage. (A): PPO1. (B): PPO4. (C): PPO5. (D): PPO6. (▫) Control; ( ) MAP1 (10 mm); and (-) MAP2 (30 mm). The values are the means ± SE of three replicates. The star represents a significant difference (P < 0.05). Y. Cheng et al. / LWT - Food Science and Technology 60 (2015) 1243e1248 1247