Journal of Applied Botany and Food Quality 93, 20 - 25 (2020), DOI:10.5073/JABFQ.2020.093.003 1Institute of Genetics and Physiology, Hebei Academy of Agriculture and Forestry Sciences, PR China 2Plant Genetic Engineering Center of Hebei Province, PR China 3College of Chemistry and Chemical Engineering, Xingtai University, PR China The involvement of phenolic metabolism in superficial scald development in ‘Wujiuxiang’ pear Shuo Zhou1,2a, Yunxiao Feng1,2a, Zhe Zhao1,3, Yudou Cheng1,2, Junfeng Guan1,2 (Submitted: August 16, 2019; Accepted: December 6, 2019) * Corresponding author a These authors contributed equally to the article. Summary Superficial scald often occurs after a long term of cold storage in apples and pears. In this study, the superficial scald index, the con- tents of major phenolic compounds, polyphenol oxidase (PPO) activity and its related genes expression in peel was investigated during cold storage period and at shelf life in ‘Wujiuxiang’ pear (Pyrus communis L. cv. Wujiuxiang) with or without 1-MCP treat- ment. It showed that arbutin, chlorogenic acid, catechin and epi- catechin were the main phenolic compounds in the peel, and 1-MCP treatment significantly inhibited scald development while altering the composition of phenolic compounds, inhibited PPO activity and the expression of phenylalanine ammonia ligase (PAL1, PAL2), cin- namate 4-hydroxylase (C4H1, C4H2) and PPO (PPO1, PPO5) and up-regulated the expression of hydroxycinnamoyl-CoA shikimate/ quinate hydroycinnamoyltransferase (HCT1), p-coumarate-3-hydro- xylase (C3H) and PPO (PPO4 and PPO6) in the peel. These results suggested that the phenolic metabolism iss closely related to the scald development, and several genes related to phenolic metabolism were involved in scald development. Keywords: superficial scald; 1-methylcyclopropene; polyphenol; polyphenol oxidase; gene expression Introduction Superficial scald, as a physiological disorder causing brown or black patches on fruit peel, often occurs after a long term of cold storage in apples and pears (Lurie and Watkins, 2012). The development of the scald was associated with α-farnesene metabolism (Yazdani et al., 2011; zhou et al., 2017), and antioxidant system (Gao et al., 2015; LarriGaudière et al., 2016, 2019; Li et al., 2016; sabban-amin et al., 2011; zhou et al., 2016), which could be dependent on ethylene action (FenG et al., 2018; Xie et al., 2016; zhi et al., 2018). However, a full understanding about the mechanism of scald development re- mains elusive. It has been showed that phenolic metabolism may be associated with the scald development (abbasi et al., 2008; busatto et al., 2018; busatto et al., 2014; Lu et al., 2014; Piretti et al., 1996). It is not only because most phenolic compounds have antioxidant activity which could resist against scald development (andrés-Lacueva et al., 2010; Lee et al., 2003), but also phenolic oxidation products catalyzed by polyphenol oxidase (PPO) most likely results in brown- ing reaction during the development of scald symptom (maYer and hareL, 1991; nishimura et al., 2003). In general, after a long term of cold storage, loss of membrane integrity allows the contacting of phenolic substrates with PPO, in which phenolic substrates could be catalyzed into oxidized forms by PPO, such as quinones, and then, quinones could react with thiol and amines groups, finally leading to the formation of brown or black pigments in peel (vaLentines et al., 2005). However, in pears few evidence confirmed the involvement of phenolic metabolism in scald development, and multiple PPO gene members existed which usually exhibited various expression patterns in the process of growth and development or in response to stress (thiPYaPonG et al., 2007), which specific PPO members might play important role in scald development is unknown. It shows that chlorogenic acid may be main substrate for PPO-cata- lyzed reaction during scald development (busatto et al., 2014). Previous researches indicated that chlorogenic acid is synthesized via phenylpropanoid pathway, which is initiated from the deamina- tion of phenylalanine to cinnamic acid by phenylalanine ammonia ligase (PAL), followed by hydroxylation of cinnamic acid by cinna- mate 4-hydroxylase (C4H) and methylation by 4-hydroxycinnamoyl- CoA ligase (4CL), respectively, to produce p-coumaric acid, and then enters hydroxycinnamoyl-CoA shikimate/quinate hydroycinnamoyl- transferase (HCT/HQT) pathway, finally leading to chlorogenic acid synthesis, which was hydroxylated by p-coumarate-3-hydroxylase (C3H), (mahesh et al., 2007; niGGeWeG et al., 2004; zhao et al., 2013). However, the involvement and regulation of chlorogenic acid synthesis in scald development was not fully understood in pears. Therefore, the objective of this study is to reveal the involvement and regulation of phenolic metabolism in scald development, and which genes may function in scald development in ‘Wujiuxiang’ pear (Pyrus communis L. cv. Wujiuxiang). Materials and methods Materials and treatments ‘Wujiuxiang’ pears were harvested in a commercial orchard in Jinzhou County (Hebei, China) at the commercial maturity stage (September 2, 2012). Fruit were transported to the laboratory in 2 h. Uniform fruit (average weight each fruit: 289.0 g) without any visual defects were selected and randomly divided into two lots of 150 fruit each. One lot was exposed to 1.0 μL/L 1-MCP (Rohm and Haas China Inc., Beijing) for 24 h at 25 ± 2 °C, and the other lot was exposed to air as control. After treatment, all fruit were stored at 0 °C. After 90 and 120 days of cold storage, fruit were removed for biochemical measurements and gene expression analysis. After 120 days of cold storage, all fruit left were transferred to 20 ± 2 °C for 7 days of shelf life. Each treatment contained three replicates of 10 fruit each at each sampling time. Scald index measurement The scald index was measured based on the percentage of the fruit surface area affected (zaneLLa, 2003), where no scald = 0, <25% = 1, 25-50% = 2, and >50% = 3. The scald index = ∑ (Score level × number of fruit at the level) / [3× (total number of fruit)]. Phenolic compounds extraction and content analysis Phenolic compound was determined as described by aWad et al. (2000) with some modifications. A 2.0 g ground frozen peel tissue Phenolic metabolism involved in superficial scald 21 were used to extract phenolic compound with 10 mL of 80% (v/v) methanol for 20 min under ultrasonic condition, and then centri- fuged at 10,000 g for 10 min at 4 °C. A 1.0 mL aliquot of superna- tant was first filtered through a C18-SPE (Bonna-Agela Technologies Inc., Tianjin, China) and then filtered via a 0.45 μm microporous membrane before being injected into the high performance liquid chromatograph (HPLC, Hitachi L-2000 system, Hitachi, Tokyo, Japan), which consisted of a Hitachi pump L-2130, a Hitachi auto- matic sample injector L-2200, and a Hitachi UV detector L-2400 equipped with a C18 column. The flow rate was maintained at 1 mL min-1 and the injection volume was 10 μL. The phenolic content was determined at wavelength of 280 nm. The integrated peaks were cal- culated by comparison with standard solutions of different known phenolic compounds. Data are expressed in mg g-1 FW. Extraction and assay of PPO activity The extraction and assay of PPO activity were performed as de- scribed by serradeLL et al. (2000). A 1.0 g ground frozen peel was homogenized in 3 ml of phosphate buffer (0.1 mol/L, pH 7.8) with 1% polyvinylpyrrolidone (PVP), and then centrifuged at 20,000 g for 15 min at 4 °C. The supernatant was collected as a crude PPO ex- tract. The reaction mixture contained 0.1 mol/L catechol containing 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. RNA isolation and quantitative RT-PCR analysis Total RNA was isolated by CTAB (cetyltrimethylammonium bro- mide) (Sangon Biotech, Shanghai, China) method (Gasic et al., 2004). After isolation, 1.0 μg of total RNA was reverse-transcribed into first-strand cDNA using PrimeScriptTM RT Reagent Kit with gDNA Eraser (TaKaRa Biomedicals). Quantitative RT-PCR was per- formed using the TB Green® Premix Ex TaqTM (Tli RNaseH Plus) Kit (TaKaRa Biomedicals) with the 7500 Real-Time PCR System (Applied Biosystems, USA). The PCR primers were designed using primer premier 6.0. The gene name, GenBank accession number, forward and reverse primers were shown in Tab. 1. The qRT-PCR reaction was performed in a final volume of 20 μL, containing 10 μL of TB Green® Premix Ex TaqTM mix, 0.4 μL of ROX II dye, 0.4 μL of forward and reverse primer each, and cDNA equivalent to 10 ng of RNA. The reaction conditions were carried out as follows: 10 s at 95 °C, 40 cycles of 95 °C for 5 s and 60 °C for 34 s. The melting tem- perature of the amplification products was analyzed using a dissocia- tion curve to confirm the specificity of amplification. All quantitative RT-PCR reactions were normalized using a Ct value corresponding to the PcActin gene. The amplification efficiency of primers was between 95 and 105%, which was calculated by serial dilutions of cDNA samples. The relative expression levels of target genes were calculated with the formula 2-ΔΔCT (Livak and schmittGen, 2001), with samples harvested at day 0 used as a calibrator (assigned an arbitrary value of 1.0). Statistical analysis All values are expressed as the means ± standard errors (SE) of three replicates. The significance of the differences between means was calculated by Student t test using SPSS software (Version 19.0, SPSS Inc., Chicago, IL, USA). Differences were considered to be signifi- cant at P < 0.05. Results Scald development during cold Storage and shelf life In the control fruit, scald symptom was appeared after 120 days of cold storage, and after 7 days of shelf life, scald developed more se- verely, while no scald was found in 1-MCP-treated fruit during all of cold storage and shelf life (Fig. 1). Tab. 1: Primers for quantitative RT-PCR analysis. Gene GenBank NO. Forward (5’-3’) Reverse (5’-3’) PcPAL1 GU906268 GCAAAGAGGACTTTAACAACTGG TACTCCCTATCGACAACTTTAAGC PcPAL2 GU906269 CCGGGAAATAACCAAACC ATGGCTCCCTTTCATTGC PcC4H1 XM_009376113 AACTTCGAGCTTCTGCCTCC CCCCAAGCATCAATCTACGC PcC4H2 XM_009356593 CAAGCACACGGGCTACAAC GATCGACCACAACGTGGTTT PcHCT1 JQ280303 CCCCCTCCAGTCTGACCA CCAATGAAAACACAAACACGTC PcC3H XM_009357051 GGTGCAACAAAAGGCTCAAG TTAGTGGTGTTGGAGGGTGC PcPPO1 HQ729709 TCCCTACTCACAAAGCCCAAG GACCTCCAAGACCAAGAAGCA PcPPO4 GU906265 AAGGTGACAATGATAACCCAGAC TGCCGCACCGTAGAGACC PcPPO5 GU906266 ACCAAAATAAAAACCCTTCCAC CAGCCACTCCACCATACAGG PcPPO6 GU906267 AGAAGGCGGAACGAGAGGA GGTCTGGCTGGGCTGACTT PcActin AB190176.1 GCTGAGAGATTCCGGTGCCC TTGACCCACCACTGAGCACG Fig. 1: Scald index in ‘Wujiuxiang’ pear during cold storage and shelf life. The error bars represent SE of the means. The asterisk “*” indicates a significant difference (P<0.05). The content of phenolic compounds during scald development The phenolic compounds, including arbutin, chlorogenic acid, gallic acid, catechin, epicatechin, coumaric acid, and caffeic acid, were detected in peel after 90, 120 days of cold storage and 7 days of shelf life. It showed that arbutin, chlorogenic acid, catechin, and epi- catechin were the main phenolic compounds in the peel of ‘Wujiux- 22 S. Zhou, Y. Feng, Z. Zhao, Y. Cheng, J. Guan iang’ pear. In general, the content of arbutin, chlorogenic acid, gallic acid, epicatechin, and caffeic acid were increased during cold stor- age and shelf life, while catechin and coumaric acid were decreased. For arbutin, chlorogenic acid, gallic acid, epicatechin, and caffeic acid, 1-MCP treatment could significantly inhibit the accumulation of these phenolic compounds, especially after 7 days of shelf life (Fig. 2). PPO activity during scald development The PPO activity in peel showed no significant changes after 90 and 120 days of cold storage in both control and 1-MCP-treated fruit. However, it increased in control at shelf life, while it was still stable and significantly lower than control in 1-MCP-treated fruit (Fig. 3). Fig. 2: The content of phenolic compounds during cold storage and shelf life with and without 1-MCP treatment in ‘Wujiuxiang’ pear: arbutin (A), chloro- genic acid (B), gallic acid (C), catechin (D), epicatechin (E), coumaric acid (F), and caffeic acid (G). The error bars represent SE of the means. The asterisk “*” indicates a significant difference (P<0.05). The expression of genes associated with phenolic compounds synthesis during scald development In this study, the expression levels of phenolic compounds synthesis- related genes, including PcPAL1, PcPAL2, PcC4H1, PcC4H2, PcHCT1, and PcC3H were detected during cold storage and shelf life with and without 1-MCP treatment in ‘Wujiuxiang’ pear. The expression levels of PcPAL1 and PcPAL2 increased constantly dur- ing cold storage and shelf life in both control and 1-MCP-treated fruit, while 1-MCP treatment significantly inhibited their expression (Fig. 4A, B). The expression of PcC4H1 was slightly increased after 90 days of cold storage, and then declined, while 1-MCP-treatment inhibited the expression except for day 120 (Fig. 4C). However, no significant change was observed in the expression of PcC4H2 (Fig. 4D). Phenolic metabolism involved in superficial scald 23 Fig. 3: PPO activities in peel of ‘Wujiuxiang’ pear during cold storage and shelf life. The error bars represent SE of the means. The asterisk “*” indicates a significant difference (P<0.05). Fig. 4: Expression levels of chlorogenic acid synthesis-related genes during cold storage and shelf life with and without 1-MCP treatment in ‘Wujiuxiang’ pear: PcPAL1 (A), PcPAL2 (B), PcC4H1 (C), PcC4H2 (D), PcHCT1 (E), and PcC3H (F). The error bars represent SE of the means. The asterisk “*” indicates a significant difference (P<0.05). The expression of PcHCT1 and PcC3H were slightly inhibited dur- ing cold storage in both control and 1-MCP-treated fruit, while it was increased to a higher level after 7 days of shelf life in 1-MCP-treated fruit (Fig. 4E, F). The expression of PPO gene members during scald development The expression of PcPPO1 and PcPPO5 increased dramatically during cold storage and afterwards declined at shelf life in control, whereas they were significantly suppressed by 1-MCP treatment (Fig. 5A, C). The expression levels of PcPPO4 and PcPPO6 were decreased during cold storage and shelf life in control, while 1-MCP- treatment could up-regulate their expression. (Fig. 5B, D). Discussion Superficial scald could result in severe fruit quality loss in apples and pears, and many postharvest methods have been established to inhibit scald development (Lurie and Watkins, 2012). In this study, 1-MCP treatment significantly inhibited scald development in ‘Wu- jiuxiang’ pear (Fig. 1), in good agreement with our previous reports (Gao et al., 2015; zhou et al., 2016, 2017), confirming the crucial role of ethylene action during scald development in ‘Wujiuxiang’ pear. Phenolic compounds are particularly important in fruit, not only because they contribute to color and flavor, but also they could be antioxidant function in fruit, which may involve in multiple stress and senescence-related processes (andrés-Lacueva et al., 2010). In this study, it was found that arbutin, chlorogenic acid, catechin, and epicatechin were main phenolic compounds in peel of ‘Wujiuxiang’ pear (Fig. 2). Rich and varied phenolic compounds make ‘Wujiu- 24 S. Zhou, Y. Feng, Z. Zhao, Y. Cheng, J. Guan conclusion that after a long term cold storage, phenolic compounds released from the vacuole can get in contact with PPO enzyme and activate it, being oxidized which results in tissue browning (abbasi et al., 2008). The expression levels of PcPPO1 and PcPPO5 showed a significant up-regulation in control during cold storage, and were effectively inhibited by 1-MCP treatment (Fig. 5A, C). It suggests that PcPPO1 and PcPPO5 may involve in browning reactions during scald development. In contrast, the expression levels of PcPPO4 and PcPPO6 were down-regulated in control, while they were up-regu- lated slightly in 1-MCP-treated fruit (Fig. 5B, D), implying PcPPO4 and PcPPO6 may play positive role in cold acclimation and senes- cence process in fruits. Our results are analogous to other reports, which showed MdPPO was linked with scald development in apple (busatto et al., 2018; busatto et al., 2014; sabban-amin et al., 2011). However, only one PPO gene member was analyzed in their reports. Our results indicate that different member of PPO genes may have different functions during scald development in pears. In summary, these results showed the involvement of phenolic me- tabolism in the scald development in pears, in which arbutin, chloro- genic acid, and epicatechin might be the main phenolic compounds, and PcPAL1, PcPAL2, PcPPO1 and PcPPO5 were the key genes re- lated with scald development. Meanwhile, 1-MCP inhibited the scald development by regulating expression of above phenolic metabolism- related genes. Acknowledgments This study was funded by the Earmarked Fund for Modern Pear- Industry Technology Research System of China (CARS-29-20), the Key R & D Project of Hebei Province (19227111D), and the HAAFS Agriculture Science and Technology Innovation Project (2019-2-01). Conflict of interest No potential conflict of interest was reported by the authors. References abbasi, n., kushad, m., haFiz, i., maqbooL, m., 2008: Relationship of superficial scald related fruit maturity with polyphenoloxidase and su- Fig. 5: Expression levels of PPO genes during cold storage and at shelf life with and without 1-MCP treatment in ‘Wujiuxiang’ pear: PcPPO1 (A), PcPPO4 (B), PcPPO5 (C), and PcPPO6 (D). The error bars represent SE of the means. The asterisk “*” indicates a significant difference (P<0.05). xiang’ pear more nutrient, however, they may also contribute to high scald incidence (busatto et al., 2014; Piretti et al., 1996). Some studies have suggested that the phenolic metabolism could be involved in scald development and could be altered by 1-MCP treatment in apples (abbasi et al., 2008; busatto et al., 2014; Pesis et al., 2007). In this study, the content of arbutin, chlorogenic acid, and epicatechin increased constantly in control fruit during cold storage and shelf life (Fig. 2A, B, E), while the scald symptom ap- peared and developed (Fig. 1). It suggested the involvement of phe- nolic compounds in cell senescence after a long cold storage and providing more substrates for PPO-catalyzed reaction during scald development. In contrast, 1-MCP effectively inhibited the accumula- tion of most phenolic compounds during cold storage and shelf life (Fig. 2A, B, E), while retarding the scald development (Fig. 1). These results suggested that arbutin, chlorogenic acid, and epicatechin may be associated with scald development in ‘Wujiuxiang’ pear. It was in agreement with the results in apples, which implied the accumulation of chlorogenic acid was positively correlated with the scald develop- ment (busatto et al., 2014). Phenolic biosynthesis pathway is initiated by PAL, in which by converting the phenylalanine to cinnamic acid. The expression of PcPAL1 and PcPAL2 dramatically increased during scald develop- ment and was effectively inhibited by 1-MCP treatment (Fig. 4A, B), confirming the involvement of phenolic metabolism in scald de- velopment. However, other phenolic synthesis-related genes, such as PcC4H1, showed a slight up-regulation (Fig. 4C), and PcHCT1 and PcC3H, showed down-regulated expression pattern (Fig. 4E, F), while PcC4H2 expression had no significant change during cold stor- age and shelf life in control (Fig. 4D), which were not coincident with scald development (Fig. 1). 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