Comprehensive analyses of the postharvest physiology and browning mechanism of pomegranate (Punica granatum L.) during cold storage

Comprehensive analyses of the postharvest physiology and browning mechanism of pomegranate (Punica granatum L.) during cold storage | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comprehensive analyses of the postharvest physiology and browning mechanism of pomegranate (Punica granatum L.) during cold storage Feng-xia Tian, En-ping Zhou, Zi-han Yu, Guang-ling Xu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4310848/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Pomegranate (Punica granatum L.) belongs to the Punicaceae family. Pomegranates are valued for their social, ecological, economic and aesthetic value and, more recently, for their health benefits. Tunisian pomegranate, which has soft seeds and large arils that can be easily swallowed, is particularly popular. In this study, soft seed pomegranate fruit was stored at chilling injury (CI) temperature (0 °C) and non-CI temperature (8 °C) after harvest to investigate the impacts of these temperatures on CI development and browning and the associated underlying mechanisms. Pomegranates are susceptible to CI when stored at temperatures below 7 °C. The results showed that at 0 °C, the CI index, browning degree, and chlorophyll content were greater than those at 8 °C. Furthermore, storage at 0 °C increased cell membrane permeability and increased polyphenol oxidase activity. These findings demonstrated that CI and browning in pomegranate were closely associated with the metabolism of membrane lipids and phenolics. The aim of this study was to explore environmental factors that affect fruit skin browning as well as the relationship between changes in enzyme activity and fruit skin browning. A temperature of 0 °C can cause low-temperature damage to pomegranates, while at 8 °C, the fruit hardly experiences cold damage. In the later stage of fruit storage, damage to cell membrane permeability, a gradual increase in the content of the membrane lipid peroxidation product malondialdehyde, oxidation and degradation of phenolic substances, slight ageing and browning of the fruit skin, and an acidic tissue pH were observed. Soft seed pomegranate storage temperature chilling injury browning Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Pomegranate ( Punica granatum L.) is an exotic deciduous tree belonging to the Punicaceae family, and its fruit has distinctive flavours and biologically active ingredients (Hegazi et al. 2021; Melgarejo-Sánchez et al. 2021). Because of its beautiful appearance (it has a bright red, pink or white colour), sweet and sour taste and auspicious connotation (it is a symbol of a full and happy life), the pomegranate is considered one of the favourite fruits among Chinese populations. Pomegranate is a fruit that combines food, medicine, and aesthetics. Pomegranates are rich in nutrients, such as vitamins (Opara et al. 2009) and antioxidants (Malviya et al. 2014). Pomegranates can also be good nutritional supplements and have antiaging and cosmetic effects. Additionally, regular pomegranate consumption helps lower blood pressure (Lynn et al. 2012) and cholesterol (Matthaiou et al. 2014) and improve diabetes (Banihani et al. 2014). The earliest pomegranate cultivars cultivated in China always had hard seeds. The hard testa is not easy to swallow; thus, the nutrients in the seeds are wasted. Tunisian pomegranate, a soft-seed pomegranate cultivar, was introduced to China in 1986. Its seed coat is relatively soft, edible and easily swallowed allowing the nutrients within to be ingested. After ten years of careful cultivation, Tunisian pomegranate was adapted to our country’s local environment. It is currently the best soft-seed pomegranate cultivar. In China, the pomegranate harvest season typically lasts from August to October. However, pomegranates are still highly perishable commodities along the postharvest value chain, from harvest to consumption, because of peel browning, weight loss, colour and flavour deterioration, chilling injury, and quality loss, which reduce storability and affect consumer acceptance of the fruits, leading to direct financial loss (Valdenegro et al. 2018; Pareek et al. 2015; Lufu et al. 2019). Temperature is one of the key factors affecting the growth and development of crops. Low temperature not only affects the cellular metabolism of plants, resulting in slow growth and development, but also severely restricts their geographical distribution (Sharma and Pandey 2015). Pomegranates are very sensitive to chilling injury (CI) during postharvest storage, which limits their cold storage and commercialization. Usually, such damage appears after storage at temperatures below 5-7 °C (Passafiume et al. 2019). Plants have evolved complex cold tolerance mechanisms to avoid the negative effects of low temperature and manage low-temperature stress, including osmotic regulators, protective enzyme systems and biofilm systems (Thomashow 1999). Furthermore, plants subjected to low-temperature stress can induce a range of signalling molecules, initiate or inhibit the expression of different genes to change the characteristics of cell membranes, regulate stomatal opening and closing, and regulate the synthesis and degradation of sugar and fatty acids, providing a foundation for the cultivation of cold resistance mechanisms to improve the adaptability of plants to unfavourable temperatures (Chinnusamy et al. 2007). Additionally, the development of CI and browning in fresh produce is associated with the structural integrity of cellular membranes and the metabolism of phenolics. Generally, enzymatic browning results from phenolic oxidation via peroxidase (POD) and polyphenol oxidase (PPO), but the structural compartmentalization of cell membranes in fresh produce prevents POD and PPO contact with phenolics and thus oxidation of phenolics and also restrains enzymatic browning (Lin et al. 2016). Generally, fruit quality and consumption trends depend on changes in appearance and internal quality. Moreover, fruit quality loss frequently occurs during cold storage, and husk damage and physiological disorders severely affect the storage life and marketability of pomegranates, especially CI and browning (Pareek et al. 2015). In the present work, the impacts of non-chilling temperature (8 °C) and chilling temperature (0 °C) on CI index, browning degree, and pericarp pigments involved in the metabolism of phenolics were investigated. Understanding the detailed mechanisms of CI and browning development in postharvest pomegranates is important for maintaining their quality and prolonging their storage time. The aims of this study were to elucidate the mechanisms underlying CI development and browning in pomegranate during cold storage. Pomegranate fruits may experience skin browning, loss of freshness, and diminishing flavour during storage, markedly affecting their commercial value. Due to the relatively concentrated harvest period, simple storage cannot effectively solve the above problems, resulting in considerable economic losses to fruit farmers and thus reducing their enthusiasm for planting. Therefore, postharvest physiological research on pomegranates is urgently needed. In recent years, low-temperature storage has become the main method for storing pomegranates. Although this method extends the storage period of pomegranates, it also has certain drawbacks. Therefore, this study used the locally planted soft seed pomegranate in Nanyang as an example to study changes in its physiological indicators after harvesting, providing a theoretical reference for postharvest storage technology. Materials And Methods Plant materials and treatment The experimental material was soft seed pomegranates collected from the pomegranate base in Nanyang City, Henan Province. Fruits free from pests, diseases, and mechanical damage that were uniform in size and relatively similar in maturity were selected as test materials. The sorted fruits were packaged in polyethylene plastic bags and stored in constant-temperature refrigerators at 0 °C and 8 °C for a total of 112 days. Samples were taken every 28 days to investigate relevant indicators. Assays of the fruit CI index CI symptoms were scored visually according to the methods of Fan et al. (2022) and Kong et al. (2011). One hundred individual fruits were categorized into six CI grades from 1 to 6 according to the degree of chilling injury: 1, no injury; 2, mild injury, chilling spots < 25%; 3, moderate injury, 25% ≤ chilling spots < 50%; 4, severe injury, 50% ≤ chilling spots < 75%; 5, very severe injury, 75% ≤ chilling spots < 100%; and 6, complete injury, chilling spots = 100% of the total fruit surface area. The CI index was computed according to the following formula: CI index =∑ (CI grade × the number of pomegranate fruits in each CI grade)/the total number of pomegranate fruits. Assays of fruit browning degree (BD) Pomegranate samples (1 g) were homogenized with distilled water (10 ml) and centrifuged for 20 min at 12,000 × g at 4 °C. The absorbance of the supernatant was observed at 420 nm using a Hitachi U-2001 spectrophotometer (Hitachi Ltd., Japan). The BD was calculated using the formula BD = A 420 × 10. Determination of flesh firmness Fruit flesh firmness was recorded in Newtons (N) using a penetrometer with an appropriate plunger (11 mm). Determination of photosynthetic pigments Photosynthetic pigments were extracted from 0.05 g of pomegranate samples in 10 ml of 80% aqueous acetone. The homogenate was filtered, and 1 ml of the suspension was diluted by the addition of 2 ml of acetone. Chlorophyll a (chl a ), chlorophyll b (chl b ), and carotenoid contents were determined using a Hitachi U-2001 spectrophotometer (Hitachi Ltd., Japan) at 3 wavelengths (663.2, 646.8, and 470.0 nm). The concentrations of pigments (mg/g FW) were calculated using a previously reported equation (Lichtenthaler 1987). Determination of total soluble solids (TSSs) TheTSS content was determined using a digital refractometer, Atago PR-101 (Atago Co., Ltd., Tokyo, Japan), at 20 °C and was expressed as a percentage (Sayyari et al. 2011). Determination of titratable acidity (TA) TA was measured in juice samples as described in Awad et al. (2017). Determination of the ascorbic acid content The ascorbic acid (AsA) content of the pomegranates was detected by the 2,6-dichlorophenol indophenol (DCIP) titration method described by Chen et al. (2022). Approximately 0.5 g of fresh pomegranate ground with liquid nitrogen was mixed with 50 ml of 2% (m/v) oxalic acid solution. Then, 10 ml of the solution was transferred to a triangular flask (50 ml), and the DCIP solution that had been calibrated was immediately used for sample solution titration. The terminal point was recorded once a reddish appearance without fading was noted, generally at 15 s. The ascorbic acid content of each sample was determined by the consumed volume of the DCIP solution. Determination of the malondialdehyde content The malondialdehyde (MDA) level was assayed according to Quan et al. (2004). Leaves (50-100 mg) were homogenized in 5 ml of 10% trichloroacetic acid (TCA) and centrifuged at 12 000 × g for 10 min. Four millilitres of 0.6% thiobarbituric acid (TBA) in 10% TCA were added to 2 ml of the supernatant. The mixture was heated in boiling water for 15 min and then quickly cooled in an ice bath. After centrifugation at 12 000 × g for 10 min, the absorbance of the supernatant at 450, 532 and 600 nm was determined with a spectrometer. The concentration of MDA was calculated by the following formula: C (µmol l −1 ) = 6.45 (OD 532 -OD 600 )-0.56OD 450 . Determination of relative electrical conductivity Electrolyte leakage was measured according to Cao et al. (2007). Six pomegranate sample discs (0.8 cm in diameter) were placed into 10 ml of distilled water, vacuumed for 30 min, and then surged for 3 h to measure the initial electrical conductivity (S1) (25 °C). A test tube was filled with leaf discs and distilled water, and the mixture was cooked (100 °C) for 30 min and then reduced to room temperature (25 °C) to determine the final electrical conductivity (S2). The relative electrical conductivity (REC) was calculated as REC = S1 × 100/S2. Determination of the phenolic content Phenolics were quantified using a Folin–Ciocalteu assay. LMW was diluted to 4 mg/ml in methanol, and then 10 μl of each diluted extract was mixed well with 490 μl of methanol and 500 μl of 0.25 M Folin–Ciocalteu reagent. Next, 1 ml of 15% (w/v) Na 2 CO 3 was added, and the absorbance of the mixed solution was measured at 740 nm after 30 min of incubation. Determination of PPO activity Enzyme extracts were prepared following the methods of Li et al. (2019) but with several modifications. The samples (0.1 g) were homogenized with 1.5 ml of 0.2 M ice-cold sodium phosphate buffer (pH 6.8) containing 1% (w/v) polyvinylpyrrolidone (PVP). The samples were centrifuged at 12,000 × g for 20 min at 4 °C. The collected supernatant was used as an enzyme extract to determine PPO activity. PPO activity was measured with a Multiskan FC spectrophotometer (Thermo Scientific, Waltham, MA, USA). Enzyme extracts (100 μl) and 0.2 M catechol (200 μl) were placed in enzyme-linked immunosorbent assay (ELISA) plates, and the absorbance was measured at 420 nm. The reaction mixture was shaken for 20 s and measured every 10 s for 5 min at 37 °C. A blank was prepared by placing 100 μl of sodium phosphate buffer and 200 μl of catechol in the ELISA plate. One unit of enzyme activity was defined as an increase in the absorbance by 0.01 min -1 . Enzyme activity was reported as units of enzyme per kg fresh weight. Statistical analysis Data analyses were performed using SPSS 16.0 (SPSS Inc., USA) software. The data are presented as the means ± SEs of 3 replicates, and statistical significance was determined by the LSD test (P < 0.05). Results Phenotypic analysis During the storage period of pomegranates, the colour of the skin changes slowly, and the skin loses water, resulting in a wrinkled shape. Then, from the surface to the inside, separation occurs between the placenta and the inner skin. Due to water loss, the fruit quickly becomes soft and even rots. Fig. 1 shows the macroperformance of pomegranate( P. granatum L.) during cold storage. Within a storage period of 28 days, soft seed pomegranates stored at both 0 °C and 8 °C showed no decay. After 112 days of storage, the soft seed pomegranate stored at 8 °C did not rot, while the soft seed pomegranate stores at 0 °C severely rotted. As shown in Fig. 2A, after 28 days at 0 °C, the pomegranates showed cold damage affecting 1/4 of the fruit surface, yielding a cold damage index of 0.1666, while those stored at 8 °C showed no cold damage spots and therefore had a cold damage index of 0. Pomegranates stored at 8 °C had fewer cold damage spots and a lower cold damage index than those stored at 0 °C. After 56 days at 0 °C, two pomegranates had cold damage spots affecting 2/3 of the fruit surface, and one pomegranate had cold damage spanning 1/2 of the fruit surface, resulting in a cold damage index of 0.833. At 8 °C, the pomegranates had no cold damage spots and a cold damage index of 0. After 84 days at 0 °C, all three pomegranates showed cold damage affecting 2/3 of the fruit surface, yielding a cold damage index of 1. At 8 °C, the pomegranates showed no cold damage and had a cold damage index of 0. The cold damage index of the three pomegranates stored at 0 °C was 4 at 112 d, whereas those stored at 8 °C had no cold damage spots, and their cold damage index was 1. With increasing storage time, the cold damage index of the pomegranates stored at 0 °C significantly increased compared to their initial value. Pomegranates stored at 8 °C had fewer cold damage spots and a lower cold damage index than those stored at 0 °C. As shown in Fig. 2B, at 28 days, the pomegranates stored at 0 °C had browning spanning 1/4 of the fruit surface, resulting in a browning index of 1/6, while those stored at 8 °C, showed no browning, and their colour was normal; the cold damage index was 0. At 56 days, one pomegranate stored at 0 °C showed browning affecting 1/4 of the fruit surface, one pomegranate showed browning affecting 1/2 of the fruit surface, and one pomegranate showed browning affecting 3/4 of the fruit surface, yielding a browning index of 2/3. The pomegranates stored at 8 °C exhibited no browning, and their colour was normal; the browning index was 0. At 84 days, all three pomegranates stored at 0 °C showed browning affecting 1/2 of the fruit surface, with a browning index of 1. At 8 °C, one pomegranate had browning covering approximately 1/4 of the fruit surface, while the other two showed a normal colour, resulting in a browning index of 0.33. At 112 days, all three pomegranates stored at 0 °C had browning spanning 1/2 of the total area or more, with a browning index of 1. Among the pomegranates stored at 8 °C, one had browning affecting approximately 1/4 of the fruit surface, and one had a normal colour; the browning index was 0.22. With increasing storage time, the browning index of the pomegranates stored at 0 °C significantly increased compared to their initial index, while the browning index of the pomegranates stored at 8 °C did not significantly change compared to their initial index. As shown in Fig. 3, with increasing storage time, the hardness of the fruits stored at 0 °C and 8 °C decreased. The decrease in fruit hardness with storage at 0 °C was more significant after 56 days, possibly due to decreases in phenolic substances in the fruit, an increase in the fruit browning index, and reduced cell membrane permeability, ultimately leading to decreased fruit hardness. The increase in fruit hardness with storage at 0 °C for 84 days may be due to cold damage to the flesh, which leads to the formation of ice crystals in the intercellular spaces of the tissues. Overall, the hardness of the pomegranates stored at 0 °C was lower than that of the pomegranates stored at 8 °C, and the hardness of the pomegranates decreased with decreasing temperature. According to the changes in chlorophyll content shown in Fig. 4, the chlorophyll content in the fruit peel at both temperatures initially increased but gradually decreased with increasing storage time until the end of the storage period. In the early stage of storage, the chlorophyll content in the fruit peel sharply decreased, followed by a transitional stage where the reductions in chlorophyll content in the fruit peel slowed. The rate of chlorophyll reduction in the fruit peel following storage at 0 °C was greater than that following storage at 8 °C. Nutritional quality The TSS contents of the soft seed pomegranates stored at two different temperatures tended to first decrease and then increase with increasing storage time. However, the TSS contents of the soft seed pomegranates stored at 8 °C eventually reached their lowest value. As shown in Fig. 5A, during the experiment, the fruit juice of the pomegranates stored at 8 °C had a bright colour, while the fruit juice of those stored at 0 °C had a dark colour. Ascorbic acid is an important indicator of a fruit’s nutritional quality and plays an important role in the antioxidant, anti-ageing, and anti-browning properties of fruits. The ascorbic acid contents of the soft seed pomegranates stored at different temperatures decreased continuously with increasing storage time. The rate of reduction in the ascorbic acid contents of the soft seed pomegranates stored at 8 °C was lower than that of the soft seed pomegranates stored at 0 °C, indicating that 8 °C is more suitable for storing soft seed pomegranates (Fig. 5B). According to Fig. 5C, the TA contents of the soft seed pomegranates stored at different temperatures showed a similar trend with increasing storage time—first decreasing, then increasing, and finally decreasing. During the same storage period, little difference in TA contents was noted between the soft seed pomegranates stored at the two temperatures, indicating that different temperatures have little effect on the TA contents of soft seed pomegranates and that temperature is therefore not an important factor in TA content changes. However, as the storage time increased, the flavour of the soft seed pomegranates changed significantly, and the red seeds also showed dullness and a lack of lustre during ageing. Changes in soft seed pomegranates were more significant when stored at 0 °C, indicating that storage at 8 °C was more effective. Changes in the Membrane Lipid Peroxide Product MDA and Cell Membrane Permeability As shown in Fig. 6A, the accumulation of the membrane lipid peroxidation product MDA reflects the degree of damage to the cell membrane system. MDA accumulation in pomegranate peels stored at 0 °C was greater than that in pomegranate peels stored at 8 °C. At 56 days, the MDA content with storage at 0 °C was 52% greater than the prestorage value. At this time, the membrane permeability of the fruit peel cells increased sharply, indicating that the membrane system may have been severely damaged. MDA accumulation in the early stage of storage at 8 °C was relatively slow. In the later stage, with the ageing and browning of the fruit, the degree of membrane lipid peroxidation gradually increased, and the MDA content also increased. During fruit ageing or cold injury, the fluidity and integrity of cell membranes are disrupted, leading to increased membrane permeability. The degree of membrane permeability is generally expressed by relative conductivity. During storage, the relative conductivity of the pomegranate peels gradually increased at the different storage temperatures. As shown in Figure 5, after 28 days of storage at 0 °C, the relative conductivity of the fruits increased rapidly, and membrane permeability increased. At this time, the low temperature of 0 °C may have caused damage to the cell membrane system of the fruits. At 8 °C, the relative conductivity of the fruits also showed an overall upward trend, with a significant increase in the later stages of storage. Changes in phenolic substances and polyphenol oxidase (PPO) As shown in Fig. 7A, the trend of changes in phenolic substances in the flesh of postharvest pomegranates stored under different temperatures was the same, but the amplitude of changes in phenolic substances in the flesh of the soft seed pomegranates stored at 8 °C was greater than that in the soft seed pomegranates stored at 0 °C. Phenolic substances play a major role in the enzymatic browning of fruit peels, and the browning index of fruit peels is negatively correlated with phenolic substance levels. The content of phenolic substances varied at different temperatures. Storage at 8 °C had a relatively small impact on the browning index of the fruit peel and appears to be better for long-term pomegranate preservation. As shown in Fig. 7B, the PPO activity of the pomegranates stored at 0 °C and 8 °C showed a similar trend. PPO activity increased before storage for 84 days, peaked at 84 days, and decreased after 84 days until the end of the storage period. However, the reasons for the changes in enzyme activity at the two temperatures are different. The change at 8 °C was caused by ageing, while the change at 0 °C was caused by low-temperature cold damage. Compared with 0 °C, 8 °C is more conducive to maintaining the freshness of pomegranates and reducing the probability of browning. Phenolic substances are substrates for the enzymatic browning of fruits and vegetables. Discussion CI development in postharvest pomegranates during chilling storage Pulp browning and cell membrane injury, which are induced by chilling stress, are the main CI symptoms in fresh produce (Kong et al. 2012; Kong et al. 2018). Previous work reported pericarp browning and enhanced permeability of cell membranes in postharvest litchi fruit after long-term storage at a low temperature (Liu et al. 2011). Nukuntornprakit (2015) reported an increased rate of electrolyte leakage that was consistent with CI development in the pulp of pineapple during cold storage. Purwanto et al. (2013) reported that 8 °C-stored mangoes displayed a greater ion leakage rate and more severe CI symptoms than 13 °C-stored mangoes during storage. Additionally, treatment with oxalic acid for peaches (Jin et al. 2014) or treatment with glycine betaine for peach fruit (Shan et al. 2016) could effectively increase the energy charge and ATP, which was beneficial for preventing cell membrane injury and enhancing the cold tolerance of fruits during cold storage. In this work, CI symptoms developed in the pomegranates during low-temperature storage. The CI index tended to increase with prolonged storage. The CI index in chilling-stored (0 °C) fruit increased markedly after 28 d of storage and was greater than that in non-chilling-stored (8 °C) fruit after 28-112 d (Fig. 2A). Similarly, the degree of pulp browning increased rapidly in both groups, and a clearly greater degree of pulp browning was observed in chilling-stored (0 °C) fruits than in non-chilling-stored (8 °C) fruits after 28-112 d of storage (Fig. 2B). Correlation analysis revealed that the increases in the CI index and pulp browning degree were positively correlated with the MDA content and cell membrane permeability (Fig. 5). Furthermore, the CI index and pulp browning degree in both groups were negatively related to fruit hardness and chlorophyll content (Figs. 2, 3). Consequently, one of the symptoms of CI in pomegranate is browning of the pulp and pericarp, with the pulp showing an increased browning degree and the pericarp showing discolouration caused by chlorophyll degradation. More importantly, these findings demonstrated that CI development in pomegranate, which is characterized by increases in the pulp browning degree and cell membrane permeability, discolouration, and pigment variations, was stimulated during storage at 0 °C. Nutritional quality Total soluble solids are an important quality metric indicating a fruit’s maturity level. Because the amount of TSS in a fruit increases as it matures and ripens, the soluble solids content of the fruit can serve as a useful indicator of maturity or ripeness stage. Moreover, the accumulation of anthocyanins and SSC in arils during cold storage was also reported by Selcuk and Erkan, Aghdam et al., and Amiri et al., which may be attributed to rapid moisture loss in juicy fruits that increases with storage duration. Vitamin C is considered one of the most important antioxidants required for plant growth and defence (Foyer and Noctor 2011). Fruit firmness is an important factor in determining a fruit’s postharvest life and quality. Chilling storage induced changes in phenolic metabolism in relation to the development of chilling injury and browning in pomegranate PPO is an important phenolase that is involved in the degradation of enzymatic browning substances (phenolics) in fresh postharvest produce (Gao et al. 2018; Lin et al. 2016). Under stress conditions, cell compartmentalization damage and compromised cell membrane integrity lead to contact between phenolase and enzymatic browning substances (phenolics and flavonoids), which promotes enzymatic browning, thus aggravating browning occurrence and CI symptoms (Ge et al. 2019; Wang et al. 2018). In this study, during storage, the increase in PPO activity (Fig. 4A) in the pulp of chilling-stored (0 °C) and non-chilling-stored (8 °C) Chinese olives coincided with the increases in the CI index (Fig. 1A), the pulp browning degree (Fig. 1B) and cell membrane permeability (Fig. 1C); however, the total phenolic content exhibited the opposite trend—the total phenolic content decreased (Fig. 4C–E). Correlation analysis (Fig. 5) demonstrated that during storage, the decrease in the total phenolic content was negatively associated with increased PPO activity and increases in the CI index, pulp browning degree and cell membrane permeability in the pulp of cold-stored (2 °C) and non-chilling-stored (8 °C) Chinese olives. Additionally, the increased PPO activity presented positive relationships with the CI index, the degree of pulp browning, and cell membrane permeability (Fig. 5). Furthermore, compared with fruits stored at 8 °C, fruits stored at 2 °C exhibited greater CI indexes (Fig. 1A), pulp browning degrees (Fig. 1B), cell membrane permeability (Fig. 1C), PPO activity (Fig. 4A–B), and total phenolic contents (Fig. 4C–E). These data indicated that CI development and browning in chilling-stored (0 °C) Chinese olive fruits resulted from cellular membrane disorders and enhanced oxidation of enzymatic browning substances (total phenolics, tannins and flavonoids) through increased activity of phenolases (PPOs). Conclusion The present study revealed that chilling temperature storage at 0 °C enhanced CI index, the pulp browning degree, and cell membrane permeability but decreased the level of chlorophyll, which disrupted the integrity of cell membrane structures, resulting in browning due to contact between PPO and enzymatic browning substances (total phenolics) and thus CI and browning development in harvested Chinese olive fruits. Thus, our study demonstrated that CI to and browning of postharvest Chinese olive fruits induced by chilling temperature storage are involved in membrane lipid and phenolic metabolism, but the underlying molecular mechanism requires further investigation. Declarations Acknowledgements This project was financially supported by the Henan Province Artemisiae argyi Development and Utilization Engineering Technology Research Center. Author contributions FXT: conceptualization, writing of the original draft, and funding acquisition. EPZ: conceptualization and methodology development. GLX investigation and methodology. ZHY: investigation and writing. Funding This research was funded by the National Natural Science Foundation of China (No. 32301615), the Education Department of Henan Province (No. 24A180021), the Open Fund of the State Key Laboratory of Tree Genetics and Breeding (Chinese Academy of Forestry) (No. TGB2021010), and the Henan Students' Platform for Innovation and Entrepreneurship Training Program (202310481023). Data availability The datasets analysed during the current study are available from the corresponding author upon reasonable request. Declarations Conflict of interest: All authors have read and approved this version of the article, and due care has been taken to ensure the integrity of this work. The authors declare that they have no conflicts of interest. References Aghdam MS, Luo ZS, Li L, Jannatizadeh A, Fard JR, Pirzad F (2020) Melatonin treatment maintains nutraceutical properties of pomegranate fruits during cold storage. 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NFS J 16: 9–14. https://doi.org/10.1016/j.nfs.2019.06.001 Purwanto YA, Okvitasari H, Mardjan SS, Ahmad U, Makino Y, Oshita S, Kawagoe Y (2013) Chilling injury in green mature ‘Gedong Gincu’ mango fruits based on the changes in ion leakage. Acta Hortic 1011:219–226. https://doi. org/10.17660/ActaHortic.2013.1011.26 Quan RD, Shang M, Zhang H, Zhao YX, Zhang JR (2004) Improved chilling tolerance by transformation with betA gene for the enhancement of glycinebetaine synthesis in maize. Plant Sci 166(1):141-149. https://doi.org/10.1016/j.plantsci.2003.08.018 Sayyari M, Castillo S, Valero D, Díaz-Mula HM, Serrano M (2011) Acetyl salicylic acid alleviates chilling injury and maintains nutritive and bioactive compounds and antioxidant activity during postharvest storage of pomegranates. Postharvest Biol Technol 60:136–142. https://doi.org/10.1016/j.postharvbio.2010.12.012 Selcuk N, Erka M (2015) Changes in phenolic compounds and antioxidant activity of sour–sweet pomegranates cv. ‘Hicaznar’ during long-term storage under modified atmosphere packaging. Postharvest Biol Technol 109:30–9. https://doi.org/10.1016/j.postharvbio.2015.05.018 Shan TM, Jin P, Zhang Y, Huang YP, Wang X L, Zheng YH (2016) Exogenous glycine betaine treatment enhances chilling tolerance of peach fruit during cold storage. Postharvest Biol Technol 114: 104–110. https://doi. org/10.1016/j.postharvbio.2015.12.005 Sharma M, Pandey GK (2015) Elucidation of abiotic stress signaling in plants. New York, USA: Springer 95–117. https://doi. org/10.1007/978-1-4939-2211-6 Thomashow MF (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Biol 50(1):571–599. https://doi.org/ 10.1146/annurev.arplant.50.1.571 Valdenegro M, Huidobro C, Monsalve L, Bernales M, Fuentes L, Simpson R (2018) Effects of ethrel, 1-MCP and modified atmosphere packaging on the quality of ‘Wonderful’ pomegranates during cold storage. J Sci Food Agric 98:4854–4865. https://doi.org/10.1002/jsfa.9015 Wang H, Chen YH, Sun JZ, Lin YF, Lin YX, Lin MS, Hung Y, Ritenour M, Lin HT (2018) The changes in metabolisms of membrane lipids and phenolics induced by Phomopsis longanae Chi infection in association with pericarp browning and disease occurrence of postharvest longan fruit. J Agric Food Chem 66:12794–12804. https://doi.org/10.1021/acs.jafc.8b04616 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4310848","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":298250746,"identity":"43894079-923a-47c5-8848-b90e28b88ac5","order_by":0,"name":"Feng-xia Tian","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYDACZijNxsDY+OADjw0PP38Dfh08CC3MzYYzZNJkJGccIKAFwWRvk+axOWxj0JCAX4s9O/OzxzwVdxL7JBLbJGfknOcxYDjA+OFjDj6HsZkb85x5Zswmkdhs8eHMbR5z5gZmyZnb8PrFTDq37bAcUEvjzZk9t3ksGw6wMfPi1cL+TTr332EeoJYGad5/53gMDiQQ0sIDtKUBbEuTNA/PASK0HOYpk/5z7LAxG89DYCDzJPNIzjjYjNcv7P3Ht0nOqDmcOL89/SEwKu3s+fmbD374iEcLNsDYQJr6UTAKRsEoGAUYAADbiEvk7K8P9AAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-9107-6251","institution":"Nanyang Normal University","correspondingAuthor":true,"prefix":"","firstName":"Feng-xia","middleName":"","lastName":"Tian","suffix":""},{"id":298250747,"identity":"43e4a9f2-797b-4c6d-b2f8-1ce1889822d2","order_by":1,"name":"En-ping Zhou","email":"","orcid":"","institution":"Nanyang Normal University","correspondingAuthor":false,"prefix":"","firstName":"En-ping","middleName":"","lastName":"Zhou","suffix":""},{"id":298250748,"identity":"bf0a1be4-39a2-4743-9297-8762c601608e","order_by":2,"name":"Zi-han Yu","email":"","orcid":"","institution":"Nanyang Normal University","correspondingAuthor":false,"prefix":"","firstName":"Zi-han","middleName":"","lastName":"Yu","suffix":""},{"id":298250749,"identity":"168e0b27-9625-4f63-b340-4e28695f73be","order_by":3,"name":"Guang-ling Xu","email":"","orcid":"","institution":"Nanyang Normal University","correspondingAuthor":false,"prefix":"","firstName":"Guang-ling","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2024-04-23 09:22:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4310848/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4310848/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56202326,"identity":"5428cc71-f093-4f6c-9fee-31c73a6774c4","added_by":"auto","created_at":"2024-05-09 19:43:36","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":34445,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CI temperature (0 °C) and non-CI temperature (8 °C) on changes in the appearance of pomegranate fruits during storage.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4310848/v1/9898b8d68022f5c8272496e6.jpg"},{"id":56202328,"identity":"393e8e4c-d481-4cc5-9b26-4dcf7d660c27","added_by":"auto","created_at":"2024-05-09 19:43:38","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":226054,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CI temperature (0 °C) and non-CI temperature (8 °C) on the CI index (A) and browning degree (B) of pomegranate during storage. The values are the means ± SEs of three biological replicates. * and ** indicate significant differences at the \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 and \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 levels, respectively.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4310848/v1/a552e28e6f98137fdaa887b0.jpg"},{"id":56202523,"identity":"be71a211-0f29-4f7b-af81-8be463638b2d","added_by":"auto","created_at":"2024-05-09 19:51:38","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":55636,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CI temperature (0 °C) and non-CI temperature (8 °C) on the flesh firmness of pomegranate during storage. The values are the means ± SEs of three biological replicates. * and ** indicate significant differences at the P \u0026lt; 0.05 and P \u0026lt; 0.01 levels, respectively.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4310848/v1/b62e29f947176b33dedcf1c9.jpg"},{"id":56202520,"identity":"269efdf7-3c03-4904-924f-2342fb0aeb2a","added_by":"auto","created_at":"2024-05-09 19:51:37","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":62792,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CI temperature (0 °C) and non-CI temperature (8 °C) on the chlorophyll content of pomegranate during storage. The values are the means ± SEs of three biological replicates. * and ** indicate significant differences at the \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 and \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 levels, respectively.\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4310848/v1/ca5b377c6f8f98fb7e5e5503.jpg"},{"id":56202331,"identity":"84a0d99e-63ee-4ce6-b314-25139599443a","added_by":"auto","created_at":"2024-05-09 19:43:38","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":234067,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CI temperature (0 °C) and non-CI temperature (8 °C) on the TSS content (A), titratable acidity (B), and ascorbic acid (AsA) content (C) of pomegranate during storage. The values are the means ± SEs of three biological replicates. * and ** indicate significant differences at the \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 and \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 levels, respectively.\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4310848/v1/f1f5f7ba3183b9abbff02dc7.jpg"},{"id":56202956,"identity":"a54a9828-26d8-48b8-b7ae-b692667291ee","added_by":"auto","created_at":"2024-05-09 20:07:39","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":258883,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CI temperature (0 °C) and non-CI temperature (8 °C) on the malondialdehyde content (A) and relative electrical conductivity (B) of pomegranate during storage. The values are the means ± SEs of three biological replicates. * and ** indicate significant differences at the \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 and \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 levels, respectively.\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4310848/v1/593b21c716420f6c4fdf9aca.jpg"},{"id":56202719,"identity":"a4440630-d316-4253-916d-b1f2a92acce1","added_by":"auto","created_at":"2024-05-09 19:59:38","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":268705,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CI temperature (0 °C) and non-CI temperature (8 °C) on the total phenolic content (A) and PPO activity (B) of pomegranate during storage. The values are the means ± SEs of three biological replicates. * and ** indicate significant differences at the \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 and \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 levels, respectively.\u003c/p\u003e","description":"","filename":"Fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4310848/v1/9ee6c9a533d86d1776b40e25.jpg"},{"id":58316000,"identity":"1712beaf-28b7-4042-b8d6-46e754afd510","added_by":"auto","created_at":"2024-06-13 21:00:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1574009,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4310848/v1/1815b8ab-e6b9-4676-b4bf-8cccc5804bd1.pdf"}],"financialInterests":"","formattedTitle":"Comprehensive analyses of the postharvest physiology and browning mechanism of pomegranate (Punica granatum L.) during cold storage","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePomegranate (\u003cem\u003ePunica granatum\u003c/em\u003e L.) is an exotic deciduous tree belonging to the Punicaceae family, and its fruit has distinctive flavours and biologically active ingredients (Hegazi\u0026nbsp;et al. 2021; Melgarejo-S\u0026aacute;nchez et al. 2021). Because of its beautiful appearance (it has a bright red, pink or white colour), sweet and sour taste and auspicious connotation (it is a symbol of a full and happy life), the pomegranate is considered one of the favourite fruits among Chinese populations. Pomegranate is a fruit that combines food, medicine, and aesthetics. Pomegranates are rich in nutrients, such as vitamins (Opara et al. 2009) and antioxidants (Malviya et al. 2014). Pomegranates can also be good nutritional supplements and have antiaging and cosmetic effects. Additionally, regular pomegranate consumption helps lower blood pressure (Lynn et al. 2012) and cholesterol (Matthaiou et al. 2014) and improve diabetes (Banihani et al. 2014). The earliest pomegranate cultivars cultivated in China always had hard seeds. The hard testa is not easy to swallow; thus, the nutrients in the seeds are wasted. Tunisian pomegranate, a soft-seed pomegranate cultivar, was introduced to China in 1986. Its seed coat is relatively soft, edible and easily swallowed allowing the nutrients within to be ingested. After ten years of careful cultivation, Tunisian pomegranate was adapted to our country\u0026rsquo;s local environment. It is currently the best soft-seed pomegranate cultivar.\u003c/p\u003e\n\u003cp\u003eIn China, the pomegranate harvest season typically lasts from August to October. However, pomegranates are still highly perishable commodities along the postharvest value chain, from harvest to consumption, because of peel browning, weight loss, colour and flavour deterioration, chilling injury, and quality loss, which reduce storability and affect consumer acceptance of the fruits, leading to direct financial loss (Valdenegro\u0026nbsp;et al. 2018; Pareek et al. 2015; Lufu et al. 2019).\u0026nbsp;Temperature is one of the key factors affecting the growth and development of crops. Low temperature not only affects the cellular metabolism of plants, resulting in slow growth and development, but also severely restricts their geographical distribution (Sharma and Pandey 2015). Pomegranates are very sensitive to chilling injury (CI) during postharvest storage, which limits their cold storage and commercialization. Usually, such damage appears after storage at\u0026nbsp;temperatures below 5-7 \u0026deg;C (Passafiume et al. 2019).\u003c/p\u003e\n\u003cp\u003ePlants have evolved complex cold tolerance mechanisms to avoid the negative effects of low temperature and manage low-temperature stress, including osmotic regulators, protective enzyme systems and biofilm systems (Thomashow 1999). Furthermore, plants subjected to low-temperature stress can induce a range of signalling molecules, initiate or inhibit the expression of different genes to change the characteristics of cell membranes, regulate stomatal opening and closing, and regulate the synthesis and degradation of sugar and fatty acids, providing a foundation for the cultivation of cold resistance mechanisms to improve the adaptability of plants to unfavourable temperatures (Chinnusamy et al. 2007). Additionally, the development of CI and browning in fresh produce is associated with the structural integrity of cellular membranes and the metabolism of phenolics. Generally, enzymatic browning results from phenolic oxidation via peroxidase (POD) and polyphenol oxidase (PPO), but the structural compartmentalization of cell membranes in fresh produce prevents POD and PPO contact with phenolics and thus oxidation of phenolics and also restrains enzymatic browning (Lin et al. 2016).\u003c/p\u003e\n\u003cp\u003eGenerally, fruit quality and consumption trends depend on changes in appearance and internal quality. Moreover, fruit quality loss frequently occurs during cold storage, and husk damage and physiological disorders severely affect the storage life and marketability of pomegranates, especially\u0026nbsp;CI\u0026nbsp;and browning (Pareek et al. 2015).\u003c/p\u003e\n\u003cp\u003eIn the present work, the impacts of non-chilling temperature (8 \u0026deg;C) and chilling temperature (0 \u0026deg;C) on CI index, browning degree, and pericarp pigments involved in the metabolism of phenolics were investigated. Understanding the detailed mechanisms of CI and browning development in postharvest pomegranates is important for maintaining their quality and prolonging their storage time. The aims of this study were to elucidate the mechanisms underlying CI development and browning in pomegranate during cold storage.\u003c/p\u003e\n\u003cp\u003ePomegranate fruits may experience skin browning, loss of freshness, and diminishing flavour during storage, markedly affecting their commercial value. Due to the relatively concentrated harvest period, simple storage cannot effectively solve the above problems, resulting in considerable economic losses to fruit farmers and thus reducing their enthusiasm for planting. Therefore, postharvest physiological research on pomegranates is urgently needed. In recent years, low-temperature storage has become the main method for storing pomegranates. Although this method extends the storage period of pomegranates, it also has certain drawbacks. Therefore, this study used the locally planted soft seed pomegranate in Nanyang as an example to study changes in its physiological indicators after harvesting, providing a theoretical reference for postharvest storage technology.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePlant materials and treatment\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental material was soft seed pomegranates collected from the pomegranate base in Nanyang City, Henan Province. Fruits free from pests, diseases, and mechanical damage that were uniform in size and relatively similar in maturity were selected as test materials. The sorted fruits were packaged in polyethylene plastic bags and stored in constant-temperature refrigerators at 0 \u0026deg;C and 8 \u0026deg;C for a total of 112 days. Samples were taken every 28 days to investigate relevant indicators.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAssays of the fruit CI index\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCI symptoms were scored visually according to the methods of Fan et al. (2022) and Kong et al. (2011). One hundred individual fruits were categorized into six CI grades from 1 to 6 according to the degree of chilling injury: 1, no injury; 2, mild injury, chilling spots \u003cem\u003e\u0026lt;\u0026nbsp;\u003c/em\u003e25%; 3, moderate injury, 25% \u0026le; chilling spots \u003cem\u003e\u0026lt;\u0026nbsp;\u003c/em\u003e50%; 4, severe injury, 50% \u0026le; chilling spots \u003cem\u003e\u0026lt;\u0026nbsp;\u003c/em\u003e75%; 5, very severe injury, 75% \u0026le; chilling spots \u003cem\u003e\u0026lt;\u0026nbsp;\u003c/em\u003e100%; and 6, complete injury, chilling spots = 100% of the total fruit surface area. The CI index was computed according to the following formula: CI index =\u0026sum; (CI grade \u0026times; the number of pomegranate fruits in each CI grade)/the total number of pomegranate fruits.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAssays of fruit browning degree (BD)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePomegranate samples (1 g) were homogenized with distilled water (10 ml) and centrifuged for 20 min at 12,000 \u0026times; g at 4 \u0026deg;C. The absorbance of the supernatant was observed at 420 nm using a Hitachi U-2001 spectrophotometer (Hitachi Ltd., Japan). The BD was calculated using the formula BD = A\u003csub\u003e420\u003c/sub\u003e \u0026times; 10.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDetermination of flesh firmness\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFruit flesh firmness was recorded in Newtons (N) using a penetrometer with an appropriate plunger (11 mm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDetermination of photosynthetic pigments\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhotosynthetic pigments were extracted from 0.05 g of pomegranate samples in 10 ml of 80% aqueous acetone. The homogenate was filtered, and 1 ml of the suspension was diluted by the addition of 2 ml of acetone. Chlorophyll \u003cem\u003ea\u003c/em\u003e (chl \u003cem\u003ea\u003c/em\u003e), chlorophyll \u003cem\u003eb\u003c/em\u003e (chl \u003cem\u003eb\u003c/em\u003e), and carotenoid contents were determined using a Hitachi U-2001 spectrophotometer (Hitachi Ltd., Japan) at 3 wavelengths (663.2, 646.8, and 470.0 nm). The concentrations of pigments (mg/g FW) were calculated using a previously reported equation (Lichtenthaler 1987).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDetermination of total soluble solids (TSSs)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTheTSS content was determined using a digital refractometer, Atago PR-101 (Atago Co., Ltd., Tokyo, Japan), at 20 \u0026deg;C and was expressed as a percentage (Sayyari\u0026nbsp;et al. 2011).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDetermination of titratable acidity (TA)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTA was measured in juice samples as described in Awad et al. (2017).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDetermination of the\u0026nbsp;ascorbic acid content\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ascorbic acid (AsA) content of the pomegranates was detected by the 2,6-dichlorophenol indophenol (DCIP) titration method described by Chen et al. (2022). Approximately 0.5 g of fresh pomegranate ground with liquid nitrogen was mixed with 50 ml of 2% (m/v) oxalic acid solution. Then, 10 ml of the solution was transferred to a triangular flask (50 ml), and the DCIP solution that had been calibrated was immediately used for sample solution titration. The terminal point was recorded once a reddish appearance without fading was noted, generally at 15 s. The ascorbic acid content of each sample was determined by the consumed volume of the DCIP solution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDetermination of the malondialdehyde content\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe malondialdehyde (MDA) level was assayed according to Quan et al. (2004).\u0026nbsp;Leaves (50-100 mg) were homogenized in 5 ml of 10% trichloroacetic acid (TCA) and centrifuged at 12 000 \u0026times; g for 10 min. Four millilitres of 0.6% thiobarbituric acid (TBA) in 10% TCA were added to 2 ml of the supernatant. The mixture was heated in boiling water for 15 min and then quickly cooled in an ice bath. After centrifugation at 12 000 \u0026times; g for 10 min, the absorbance of the supernatant at 450, 532 and 600 nm was determined with a spectrometer. The concentration of MDA was calculated by the following formula: C (\u0026micro;mol l\u003csup\u003e\u0026minus;1\u003c/sup\u003e) = 6.45 (OD\u003csub\u003e532\u003c/sub\u003e-OD\u003csub\u003e600\u003c/sub\u003e)-0.56OD\u003csub\u003e450\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDetermination of relative electrical conductivity\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eElectrolyte leakage was measured according to Cao et al. (2007). Six pomegranate sample discs (0.8 cm in diameter) were placed into 10 ml of distilled water, vacuumed for 30 min, and then surged for 3 h to measure the initial electrical conductivity (S1) (25 \u0026deg;C). A test tube was filled with leaf discs and distilled water, and the mixture was cooked (100 \u0026deg;C) for 30 min and then reduced to room temperature (25 \u0026deg;C) to determine the final electrical conductivity (S2). The relative electrical conductivity (REC) was calculated as REC = S1 \u0026times; 100/S2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDetermination of the phenolic content\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhenolics were quantified using a Folin\u0026ndash;Ciocalteu assay. LMW was diluted to 4 mg/ml in methanol, and then 10 \u0026mu;l of each diluted extract was mixed well with 490 \u0026mu;l of methanol and 500 \u0026mu;l of 0.25 M Folin\u0026ndash;Ciocalteu reagent. Next, 1 ml of 15% (w/v) Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e was added, and the absorbance of the mixed solution was measured at 740 nm after 30 min of incubation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDetermination of PPO activity\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEnzyme extracts were prepared following the methods of Li et al. (2019) but with several modifications. The samples (0.1 g) were homogenized with 1.5 ml of 0.2 M ice-cold sodium phosphate buffer (pH 6.8) containing 1% (w/v) polyvinylpyrrolidone (PVP). The samples were centrifuged at 12,000 \u0026times; g for 20 min at 4 \u0026deg;C. The collected supernatant was used as an enzyme extract to determine PPO activity.\u003c/p\u003e\n\u003cp\u003ePPO activity was measured with a Multiskan FC spectrophotometer (Thermo Scientific, Waltham, MA, USA). Enzyme extracts (100 \u0026mu;l) and 0.2 M catechol (200 \u0026mu;l) were placed in enzyme-linked immunosorbent assay (ELISA) plates, and the absorbance was measured at 420 nm. The reaction mixture was shaken for 20 s and measured every 10 s for 5 min at 37 \u0026deg;C. A blank was prepared by placing 100 \u0026mu;l of sodium phosphate buffer and 200 \u0026mu;l of catechol in the ELISA plate. One unit of enzyme activity was defined as an increase in the absorbance by 0.01 min\u003csup\u003e-1\u003c/sup\u003e. Enzyme activity was reported as units of enzyme per kg fresh weight.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData analyses were performed using SPSS 16.0 (SPSS Inc., USA) software. The data are presented as the means \u0026plusmn; SEs of 3 replicates, and statistical significance was determined by the LSD test (P \u0026lt; 0.05).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003ePhenotypic analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDuring the storage period of pomegranates, the colour of the skin changes slowly, and the skin loses water, resulting in a wrinkled shape. Then, from the surface to the inside, separation occurs between the placenta and the inner skin. Due to water loss, the fruit quickly becomes soft and even rots.\u0026nbsp;Fig. 1 shows the macroperformance of pomegranate(\u003cem\u003eP. granatum\u003c/em\u003e L.) during cold storage. Within a storage period of 28 days, soft seed pomegranates stored at both 0 \u0026deg;C and 8 \u0026deg;C showed no decay. After 112 days of storage, the soft seed pomegranate stored at 8 \u0026deg;C did not rot, while the soft seed pomegranate stores at 0 \u0026deg;C severely rotted.\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. 2A, after 28 days at 0 \u0026deg;C, the pomegranates showed cold damage affecting 1/4 of the fruit surface, yielding a cold damage index of 0.1666, while those stored at 8 \u0026deg;C showed no cold damage spots and therefore had a cold damage index of 0. Pomegranates stored at 8 \u0026deg;C had fewer cold damage spots and a lower cold damage index than those stored at 0 \u0026deg;C. After 56 days at 0 \u0026deg;C, two pomegranates had cold damage spots affecting 2/3 of the fruit surface, and one pomegranate had cold damage spanning 1/2 of the fruit surface, resulting in a cold damage index of 0.833. At 8 \u0026deg;C, the pomegranates had no cold damage spots and a cold damage index of 0. After 84 days at 0 \u0026deg;C, all three pomegranates showed cold damage affecting 2/3 of the fruit surface, yielding a cold damage index of 1. At 8 \u0026deg;C, the pomegranates showed no cold damage and had a cold damage index of 0. The cold damage index of the three pomegranates stored at 0 \u0026deg;C was 4 at 112 d, whereas those stored at 8 \u0026deg;C had no cold damage spots, and their cold damage index was 1. With increasing storage time, the cold damage index of the pomegranates stored at 0 \u0026deg;C significantly increased compared to their initial value. Pomegranates stored at 8 \u0026deg;C had fewer cold damage spots and a lower cold damage index than those stored at 0 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. 2B, at 28 days, the pomegranates stored at 0 \u0026deg;C had \u0026nbsp;browning spanning 1/4 of the fruit surface, resulting in a browning index of 1/6, while those stored at 8 \u0026deg;C, showed no browning, and their colour was normal; the cold damage index was 0. At 56 days, one pomegranate stored at 0 \u0026deg;C showed browning affecting 1/4 of the fruit surface, one pomegranate showed browning affecting 1/2 of the fruit surface, and one pomegranate showed browning affecting 3/4 of the fruit surface, yielding a browning index of 2/3. The pomegranates stored at 8 \u0026deg;C exhibited no browning, and their colour was normal; the browning index was 0. At 84 days, all three pomegranates stored at 0 \u0026deg;C showed browning affecting 1/2 of the fruit surface, with a browning index of 1. At 8 \u0026deg;C, one pomegranate had browning covering approximately 1/4 of the fruit surface, while the other two showed a normal colour, resulting in a browning index of 0.33. At 112 days, all three pomegranates stored at 0 \u0026deg;C had browning spanning 1/2 of the total area or more, with a browning index of 1. Among the pomegranates stored at 8 \u0026deg;C, one had browning affecting approximately 1/4 of the fruit surface, and one had a normal colour; the browning index was 0.22. With increasing storage time, the browning index of the pomegranates stored at 0 \u0026deg;C significantly increased compared to their initial index, while the browning index of the pomegranates stored at 8 \u0026deg;C did not significantly change compared to their initial index.\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. 3, with increasing storage time, the hardness of the fruits stored at 0 \u0026deg;C and 8 \u0026deg;C decreased. The decrease in fruit hardness with storage at 0 \u0026deg;C was more significant after 56 days, possibly due to decreases in phenolic substances in the fruit, an increase in the fruit browning index, and reduced cell membrane permeability, ultimately leading to decreased fruit hardness. The increase in fruit hardness with storage at 0 \u0026deg;C for 84 days may be due to cold damage to the flesh, which leads to the formation of ice crystals in the intercellular spaces of the tissues. Overall, the hardness of the pomegranates stored at 0 \u0026deg;C was lower than that of the pomegranates stored at 8 \u0026deg;C, and the hardness of the pomegranates decreased with decreasing temperature.\u003c/p\u003e\n\u003cp\u003eAccording to the changes in chlorophyll content shown in Fig. 4, the chlorophyll content in the fruit peel at both temperatures initially increased but gradually decreased with increasing storage time until the end of the storage period. In the early stage of storage, the chlorophyll content in the fruit peel sharply decreased, followed by a transitional stage where the reductions in chlorophyll content in the fruit peel slowed. The rate of chlorophyll reduction in the fruit peel following storage at 0 \u0026deg;C was greater than that following storage at 8 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNutritional quality\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe TSS contents of the soft seed pomegranates stored at two different temperatures tended to first decrease and then increase with increasing storage time. However, the TSS contents of the soft seed pomegranates stored at 8 \u0026deg;C eventually reached their lowest value. As shown in Fig. 5A, during the experiment, the fruit juice of the pomegranates stored at 8 \u0026deg;C had a bright colour, while the fruit juice of those stored at 0 \u0026deg;C had a dark colour. Ascorbic acid is an important indicator of a fruit\u0026rsquo;s nutritional quality and plays an important role in the antioxidant, anti-ageing, and anti-browning properties of fruits. The ascorbic acid contents of the soft seed pomegranates stored at different temperatures decreased continuously with increasing storage time. The rate of reduction in the ascorbic acid contents of the soft seed pomegranates stored at 8 \u0026deg;C was lower than that of the soft seed pomegranates stored at 0 \u0026deg;C, indicating that 8 \u0026deg;C is more suitable for storing soft seed pomegranates (Fig. 5B). According to Fig. 5C, the TA contents of the soft seed pomegranates stored at different temperatures showed a similar trend with increasing storage time\u0026mdash;first decreasing, then increasing, and finally decreasing. During the same storage period, little difference in TA contents was noted between the soft seed pomegranates stored at the two temperatures, indicating that different temperatures have little effect on the TA contents of soft seed pomegranates and that temperature is therefore not an important factor in TA content changes. However, as the storage time increased, the flavour of the soft seed pomegranates changed significantly, and the red seeds also showed dullness and a lack of lustre during ageing. Changes in soft seed pomegranates were more significant when stored at 0 \u0026deg;C, indicating that storage at 8 \u0026deg;C was more effective.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eChanges in the Membrane Lipid Peroxide Product MDA and Cell Membrane Permeability\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. 6A, the accumulation of the membrane lipid peroxidation product MDA reflects the degree of damage to the cell membrane system. MDA accumulation in pomegranate peels stored at 0 \u0026deg;C was greater than that in pomegranate peels stored at 8 \u0026deg;C. At 56 days, the MDA content with storage at 0 \u0026deg;C was 52% greater than the prestorage value. At this time, the membrane permeability of the fruit peel cells increased sharply, indicating that the membrane system may have been severely damaged. MDA accumulation in the early stage of storage at 8 \u0026deg;C was relatively slow. In the later stage, with the ageing and browning of the fruit, the degree of membrane lipid peroxidation gradually increased, and the MDA content also increased. During fruit ageing or cold injury, the fluidity and integrity of cell membranes are disrupted, leading to increased membrane permeability. The degree of membrane permeability is generally expressed by relative conductivity. During storage, the relative conductivity of the pomegranate peels gradually increased at the different storage temperatures. As shown in Figure 5, after 28 days of storage at 0 \u0026deg;C, the relative conductivity of the fruits increased rapidly, and membrane permeability increased. At this time, the low temperature of 0 \u0026deg;C may have caused damage to the cell membrane system of the fruits. At 8 \u0026deg;C, the relative conductivity of the fruits also showed an overall upward trend, with a significant increase in the later stages of storage.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eChanges in phenolic substances and polyphenol oxidase (PPO)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. 7A, the trend of changes in phenolic substances in the flesh of postharvest pomegranates stored under different temperatures was the same, but the amplitude of changes in phenolic substances in the flesh of the soft seed pomegranates stored at 8 \u0026deg;C was greater than that in the soft seed pomegranates stored at 0 \u0026deg;C. Phenolic substances play a major role in the enzymatic browning of fruit peels, and the browning index of fruit peels is negatively correlated with phenolic substance levels. The content of phenolic substances varied at different temperatures. Storage at 8 \u0026deg;C had a relatively small impact on the browning index of the fruit peel and appears to be better for long-term pomegranate preservation. As shown in Fig. 7B, the PPO activity of the pomegranates stored at 0 \u0026deg;C and 8 \u0026deg;C showed a similar trend. PPO activity increased before storage for 84 days, peaked at 84 days, and decreased after 84 days until the end of the storage period. However, the reasons for the changes in enzyme activity at the two temperatures are different. The change at 8 \u0026deg;C was caused by ageing, while the change at 0 \u0026deg;C was caused by low-temperature cold damage. Compared with 0 \u0026deg;C, 8 \u0026deg;C is more conducive to maintaining the freshness of pomegranates and reducing the probability of browning. Phenolic substances are substrates for the enzymatic browning of fruits and vegetables.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cem\u003eCI development in postharvest pomegranates during chilling storage\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePulp browning and cell membrane injury, which are induced by chilling stress, are the main CI symptoms in fresh produce (Kong et al. 2012; Kong et al. 2018). Previous work reported pericarp browning and enhanced permeability of cell membranes in postharvest litchi fruit after long-term storage at a low temperature (Liu et al. 2011). Nukuntornprakit (2015) reported an increased rate of electrolyte leakage that was consistent with CI development in the pulp of pineapple during cold storage. Purwanto et al. (2013) reported that 8 \u0026deg;C-stored mangoes displayed a greater ion leakage rate and more severe CI symptoms than 13 \u0026deg;C-stored mangoes during storage. Additionally, treatment with oxalic acid for peaches (Jin et al. 2014) or treatment with glycine betaine for peach fruit (Shan et al. 2016) could effectively increase the energy charge and ATP, which was beneficial for preventing cell membrane injury and enhancing the cold tolerance of fruits during cold storage.\u003c/p\u003e\n\u003cp\u003eIn this work, CI symptoms \u0026nbsp;developed\u0026nbsp;in the pomegranates\u0026nbsp;during low-temperature storage. The CI index tended to increase with prolonged storage. The CI index in chilling-stored (0 \u0026deg;C) fruit increased markedly after 28 d of storage and was greater than that in non-chilling-stored (8 \u0026deg;C) fruit after 28-112 d (Fig. 2A). Similarly, the degree of pulp browning increased rapidly in both groups, and a clearly greater degree of pulp browning was observed in chilling-stored (0 \u0026deg;C) fruits than in non-chilling-stored (8 \u0026deg;C) fruits after 28-112 d of storage (Fig. 2B).\u003c/p\u003e\n\u003cp\u003eCorrelation analysis revealed that the increases in the CI index and pulp browning degree were positively correlated with the MDA content and cell membrane permeability (Fig. 5). Furthermore, the CI index and pulp browning degree in both groups were negatively related to fruit hardness and chlorophyll content (Figs. 2, 3). Consequently, one of the symptoms of CI in pomegranate is browning of the pulp and pericarp, with the pulp showing an increased browning degree and the pericarp showing discolouration caused by chlorophyll degradation. More importantly, these findings demonstrated that CI development in pomegranate, which is characterized by increases in the pulp browning degree and cell membrane permeability, discolouration, and pigment variations, was stimulated during storage at 0 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNutritional quality\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTotal soluble solids are an important quality metric indicating a fruit\u0026rsquo;s maturity level. Because the amount of TSS in a fruit increases as it matures and ripens, the soluble solids content of the fruit can serve as a useful indicator of maturity or ripeness stage. Moreover, the accumulation of anthocyanins and SSC in arils during cold storage was also reported by Selcuk and Erkan, Aghdam et al., and Amiri et al., which may be attributed to rapid moisture loss in juicy fruits that increases with storage duration. Vitamin C is considered one of the most important antioxidants required for plant growth and defence (Foyer and Noctor 2011). Fruit firmness is an important factor in determining a fruit\u0026rsquo;s postharvest life and quality.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eChilling storage induced changes in phenolic metabolism in relation to the development of chilling injury and browning in pomegranate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePPO is an important phenolase that is involved in the degradation of enzymatic browning substances (phenolics) in fresh postharvest produce (Gao et al. 2018; Lin et al. 2016). Under stress conditions, cell compartmentalization damage and compromised cell membrane integrity lead to contact between phenolase and enzymatic browning substances (phenolics and flavonoids), which promotes enzymatic browning, thus aggravating browning occurrence and CI symptoms (Ge et al. 2019; Wang et al. 2018).\u003c/p\u003e\n\u003cp\u003eIn this study, during storage, the increase in PPO activity (Fig. 4A) in the pulp of chilling-stored (0 \u0026deg;C) and non-chilling-stored (8 \u0026deg;C) Chinese olives coincided with the increases in the CI index (Fig. 1A), the pulp browning degree (Fig. 1B) and cell membrane permeability (Fig. 1C); however, the total phenolic content exhibited the opposite trend\u0026mdash;the total phenolic content decreased (Fig. 4C\u0026ndash;E). Correlation analysis (Fig. 5) demonstrated that during storage, the decrease in the total phenolic content was negatively associated with increased PPO activity and increases in the CI index, pulp browning degree and cell membrane permeability in the pulp of cold-stored (2 \u0026deg;C) and non-chilling-stored (8 \u0026deg;C) Chinese olives.\u003c/p\u003e\n\u003cp\u003eAdditionally, the increased PPO activity presented positive relationships with the CI index, the degree of pulp browning, and cell membrane permeability (Fig. 5). Furthermore, compared with fruits stored at 8 \u0026deg;C, fruits stored at 2 \u0026deg;C exhibited greater CI indexes (Fig. 1A), pulp browning degrees (Fig. 1B), cell membrane permeability (Fig. 1C), PPO activity (Fig. 4A\u0026ndash;B), and total phenolic contents (Fig. 4C\u0026ndash;E).\u003c/p\u003e\n\u003cp\u003eThese data indicated that CI development and browning in chilling-stored (0 \u0026deg;C) Chinese olive fruits resulted from cellular membrane disorders and enhanced oxidation of enzymatic browning substances (total phenolics, tannins and flavonoids) through increased activity of phenolases (PPOs).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present study revealed that chilling temperature storage at 0 \u0026deg;C enhanced CI index, the pulp browning degree, and cell membrane permeability but decreased the level of chlorophyll, which disrupted the integrity of cell membrane structures, resulting in browning due to contact between PPO and enzymatic browning substances (total phenolics) and thus CI and browning development in harvested Chinese olive fruits. Thus, our study demonstrated that CI to and browning of postharvest Chinese olive fruits induced by chilling temperature storage are involved in membrane lipid and phenolic metabolism, but the underlying molecular mechanism requires further investigation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was financially supported by\u0026nbsp;the\u0026nbsp;Henan Province Artemisiae argyi Development and Utilization Engineering Technology Research Center.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFXT: conceptualization,\u0026nbsp;writing\u0026nbsp;of the\u0026nbsp;original draft, and funding acquisition. EPZ: conceptualization\u0026nbsp;and\u0026nbsp;methodology development. GLX investigation\u0026nbsp;and\u0026nbsp;methodology. ZHY:\u0026nbsp;investigation and writing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by\u0026nbsp;the\u0026nbsp;National Natural Science Foundation of China (No. 32301615), the Education Department of Henan Province (No. 24A180021), the Open Fund of\u0026nbsp;the\u0026nbsp;State Key Laboratory of Tree Genetics and Breeding (Chinese Academy of Forestry) (No. TGB2021010),\u0026nbsp;and the\u0026nbsp;Henan Students\u0026apos; Platform for\u0026nbsp;Innovation and Entrepreneurship Training Program\u0026nbsp;(202310481023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets analysed during the current study are available from the corresponding author\u0026nbsp;upon\u0026nbsp;reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConflict of interest: All authors have read and approved this version of the article, and due care has been taken to ensure the integrity of this work. The authors declare that they have no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAghdam MS, Luo ZS, Li L, Jannatizadeh A, Fard JR, Pirzad F (2020) Melatonin treatment maintains nutraceutical properties of pomegranate fruits during cold storage. Food Chem 303:125385. https://doi.org/: 10.1016/j.foodchem.2019. 125385\u003c/li\u003e\n\u003cli\u003eAmiri A, Ramezanian A, Ramezanian SMH, Hosseini SMA (2020) Ultrasonic potential in maintaining the quality and reducing the microbial load of minimally processed pomegranate. Ultrason Sonochem 70:105302. https://doi.org/10.1016/j.ultsonch.2020.105302\u003c/li\u003e\n\u003cli\u003eAwad MA, Al-Qurashi AD, Mohamed SA, El-Shishtawy RM and Ali MA (2017) Postharvest chitosan, gallic acid and chitosan gallate treatments effects on shelf life quality, antioxidant compounds, free radical scavenging capacity and enzymes activities of \u0026lsquo;Sukkari\u0026rsquo; bananas. 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Postharvest Biol Technol 114: 104\u0026ndash;110. https://doi. org/10.1016/j.postharvbio.2015.12.005\u003c/li\u003e\n\u003cli\u003eSharma M, Pandey GK (2015) Elucidation of abiotic stress signaling in plants. New York, USA: Springer 95\u0026ndash;117. https://doi. org/10.1007/978-1-4939-2211-6\u003c/li\u003e\n\u003cli\u003eThomashow MF (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Biol 50(1):571\u0026ndash;599. https://doi.org/ 10.1146/annurev.arplant.50.1.571\u003c/li\u003e\n\u003cli\u003eValdenegro M, Huidobro C, Monsalve L, Bernales M, Fuentes L, Simpson R (2018) Effects of ethrel, 1-MCP and modified atmosphere packaging on the quality of \u0026lsquo;Wonderful\u0026rsquo; pomegranates during cold storage. 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J Agric Food Chem 66:12794\u0026ndash;12804. https://doi.org/10.1021/acs.jafc.8b04616\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Soft seed pomegranate, storage temperature, chilling injury, browning","lastPublishedDoi":"10.21203/rs.3.rs-4310848/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4310848/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Pomegranate (Punica granatum L.) belongs to the Punicaceae family. Pomegranates are valued for their social, ecological, economic and aesthetic value and, more recently, for their health benefits. Tunisian pomegranate, which has soft seeds and large arils that can be easily swallowed, is particularly popular. In this study, soft seed pomegranate fruit was stored at chilling injury (CI) temperature (0 °C) and non-CI temperature (8 °C) after harvest to investigate the impacts of these temperatures on CI development and browning and the associated underlying mechanisms. Pomegranates are susceptible to CI when stored at temperatures below 7 °C. The results showed that at 0 °C, the CI index, browning degree, and chlorophyll content were greater than those at 8 °C. Furthermore, storage at 0 °C increased cell membrane permeability and increased polyphenol oxidase activity. These findings demonstrated that CI and browning in pomegranate were closely associated with the metabolism of membrane lipids and phenolics. The aim of this study was to explore environmental factors that affect fruit skin browning as well as the relationship between changes in enzyme activity and fruit skin browning. A temperature of 0 °C can cause low-temperature damage to pomegranates, while at 8 °C, the fruit hardly experiences cold damage. In the later stage of fruit storage, damage to cell membrane permeability, a gradual increase in the content of the membrane lipid peroxidation product malondialdehyde, oxidation and degradation of phenolic substances, slight ageing and browning of the fruit skin, and an acidic tissue pH were observed.","manuscriptTitle":"Comprehensive analyses of the postharvest physiology and browning mechanism of pomegranate (Punica granatum L.) during cold storage","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-09 19:43:26","doi":"10.21203/rs.3.rs-4310848/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cc490fc6-5060-4e5c-b0c5-39c17e86f638","owner":[],"postedDate":"May 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-13T20:52:45+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-09 19:43:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4310848","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4310848","identity":"rs-4310848","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}