Postharvest nitric oxide treatment delays the senescence of winter jujube (Zizyphus jujuba Mill. cv. Dongzao) fruit during cold storage by regulating reactive oxygen species metabolism

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Postharvest nitric oxide treatment delays the senescence of winter jujube (Zizyphus jujuba Mill. cv. Dongzao) fruit during cold storage by regulating reactive oxygen species metabolism Yating Zhaoa, Xuan Zhub, Yuanyuan Houb, Xuanyu Wanga, Xihong Lia,* a b

State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology, Ministry of Education, Tianjin 300457, China College of Food Science and Pharmacy, Xinjiang Agricultural University, Xinjiang 830052, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Winter jujube (Zizyphus jujube Mill. cv. Dongzao) Nitric oxide fumigation Senescence Reactive oxygen species

Postharvest winter jujube (Zizyphus jujuba Mill. cv. Dongzao) were treated with 20 μL L-1 nitric oxide (NO) for 3 h and then stored at 0 ± 1 °C and 90–95 % relative humidity for 75 d. The influences of NO treatment on senescence and reactive oxygen species (ROS) of harvested winter jujube were investigated during cold storage. Results indicated that NO treatment could markedly delay weight loss, the development of decay and occurrence of flesh browning of the jujube. Also, NO treatment retained higher firmness and caused lower respiration rates. The increase in total soluble solids (TSS) content and the decrease of titratable acidity (TA) in the jujube were significantly retarded by NO treatment. NO-treated jujube showed higher activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) compared with the controls. Reductions of ascorbic acid (AsA) and glutathione (GSH) contents, and the increase of superoxide anion (O2-%) production and peroxide (H2O2) contents were also delayed by NO treatment. These effects suppressed increases in malondialdehyde (MDA) contents and cell membrane permeability. The combined results suggested that NO treatment enhanced the ROS scavenging capacity diminishing ROS accumulation, which contributed to alleviation of the oxidative damage and the maintenance of the cellular membrane integrity, and thereby, delaying the senescence of winter jujube.

1. Introduction Winter jujube (Zizyphus jujuba Mill.), a member of the Rhamnaceae, is a important fruit crop native to China. Owing to its crispy, flesh, particular flavor, and abundant nutrients, winter jujube is welcomed by the consumers in the fruit market (Chen et al., 2013). As a representative variety of fresh jujube, although winter jujube is considered as a non-climacteric fruit that has low physiological activity, it suffers from rapid senescence after harvest due to respiration and some physiological disorders, resulting in severe quality deterioration, manifested as postharvest decay, dehydration, tissue softening, and flesh browning (Zhang et al., 2016). Such undesirable changes drastically restrain its storability as well as market value. Accordingly, a better grasp of senescence is necessary for preserving quality and prolonging postharvest life of winter jujube. Postharvest fruit senescence is generally considered to closely correlate with the oxidative damage to cellular macromolecules, which arises from the overproduction of reactive oxygen species (ROS), such

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as the superoxide anion (O2-%), the hydroxyl radical (%OH) and hydrogen peroxide (H2O2) (Tian et al., 2013). Normally, intercellular levels of ROS depend upon the equilibrium between their production and the ability to scavenge them (Mittler, 2002). Various enzymic antioxidants like catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), and non-enzymic antioxidant substances, including ascorbic acid (AsA), glutathione (GSH), tocopherols, carotenoids, and phenolics, participate in the process of eliminating ROS in fruit (Chiriboga et al., 2013; Czarnocka and Karpiński, 2018). However, the capacity of these antioxidant systems to scavenge ROS is weakened during fruit senescence, and the consequential excessive ROS can react with DNA, proteins, and lipids, which triggers cellular membrane lipid peroxidation and disruption of the cellular membrane structure, finally resulting in the cell death (Blokhina et al., 2003). It has been demonstrated that a positive relationship exists between the antioxidant enzyme activities and the rate of senescence in harvested fruit like plum (Singh et al., 2012), longan (Chomkitichai et al., 2014) and kiwifruit (Xia et al., 2016). In addition,

Corresponding author. E-mail addresses: [email protected] (Y. Zhao), [email protected] (X. Li).

https://doi.org/10.1016/j.scienta.2019.109009 Received 2 August 2019; Received in revised form 2 November 2019; Accepted 5 November 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Yating Zhao, et al., Scientia Horticulturae, https://doi.org/10.1016/j.scienta.2019.109009

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2.2. Determination of physicochemical quality parameters

a growing volume of evidence has demonstrated that enhancing the antioxidant capacity for eliminating overproduction of ROS can further delay senescence and help preserve the postharvest quality of peach (Gao et al., 2016), blueberry (Chen et al., 2019), and sweet cherry (Zhao et al., 2019a). These studies supply valuable evidence that ROS plays a crucial role in regulation of fruit senescence. However, studies are rarely reported on postponing senescence of jujube fruit in association with ROS metabolism. Nitric oxide (NO), a highly reactive signaling molecule, participates in various physiological processes of the plants starting from seed germination to programmed cell death (Wendehenne et al., 2004; Crawford and Guo, 2005). Postharvest employing optimum concentration of NO to horticultural products can prolong their postharvest life, enhance resistance to diseases, alleviate chilling injury, and inhibit browning (Manjunatha et al., 2010). In addition, it has been reported that exogenous application of NO can delay fruit ripening and senescence in an array of fruit, but such effects were mainly linked with delaying the respiration rate and ethylene biosynthesis. Recently, increasing studies have confirmed that NO is capable of protecting fruit against the oxidative damage through regulating enzymatic or nonenzymatic antioxidant activities that reduce ROS accumulation (Ma et al., 2019). Wu et al. (2012) found that NO could suppress the increases in the O2-% production rate and H2O2 content, and increased the activities of SOD, CAT and APX, thereby maintaining the balance between the formation and detoxification of ROS and avoiding damage. Also, work by Rabiei et al. (2019) has proved that NO lowered H2O2 accumulation through enhancing the activity of ROS scavenging enzymes including SOD, CAT, APX and GR, contributing to controlling the senescence accompanying browning. Based on these findings, it could be inferred that the NO exerts an positive influence on fruit senescence caused by ROS overproduction. To our knowledge, there is little information available on the impact of NO on ROS production-scavenging system and senescence of winter jujube. Thus, in the current study, we aimed to determine the effect of NO treatment on ROS metabolism and to understand how the NO delays senescence of postharvest winter jujube in association with fruit quality during storage. The changes induced by NO treatment, such as physiochemical quality attributes, respiration rate, cell membrane permeability, MDA content, ROS production, and activities of ROS scavenging enzymes, were investigated in harvested jujube during cold storage.

2.2.1. Decay incidence, weight loss, firmness and flesh browning index A total of 100 winter jujube fruit from each replicate were randomly selected for determining decay incidence. Fruit with visible fungal growth or bacterial lesions were considered decayed. The decay incidence was calculated as followed, decay incidence (%) = (N/T) × 100, where N is number of decayed fruit and T is total number of fruit. From each replicate, 30 jujube fruit were chose to measure weight loss. Weight loss was assayed by monitoring the weight decline of winter jujube over time and was calculated as follows: weight loss (%) = [(W0 − W1)/W0]× 100, where W0 is the initial weight and W1 is the weight measured after storage. Fruit firmness was determined using a digital hand-held fruit firmness tester (GY-4; Zhejiang Top Instrument Co., Ltd., China) fitted with a 3.5 mm cylinder probe. A total of 10 winter jujube fruit from each replicate were chose and their firmness was measured on two sides along the equatorial region. The results of the firmness were expressed as N. Flesh browning was assessed with the method proposed by Sun et al. (2007) using 30 fruit, which were horizontally cut along the equator. The degree of the flesh browning was classified based on the percentage of browning area: 0 = no browning; 1 = < 5 % browning area; 2 = 5–25 % browning area; 3 = 25–50 % browning area; 4 = > 50 % browning area. The flesh browning index was calculated by the equation:

Browning index

(%)

∑ (browning scale × number of fruit with that scale) 5 × 30 × 100% 2.2.2. Total soluble solids (TSS) and titratable acidity (TA) A total of 10 winter jujube fruit from each replicate were homogenized to assay TSS and TA. TSS was determined using a pocket refractometer (PAL-1, Atago, Japan). TA was measured using the procedure described by Wang et al. (2014). The contents of TSS and TA were expressed as percent soluble solids, and percentage of citric acid, respectively. 2.3. Determination of respiration rate

2. Materials and methods The assay was done following the method described by Fan et al. (2018) and the interval between each measurement was 15 days. Thirty fruit were sealed in 4 L glass container and kept for 1 h at 0 °C. One mL gas of the headspace gas from container was injected to a 7890A gas chromatograph (Agilent, Palo Alto, CA, USA) equipped with a stainless steel column, a hydrogen flame ionization detector (FID) and a methane conversion. For CO2 measurement, CO2 was transformed to CH4, and the amount of CO2 component was measured indirectly by the response value generated in the FID detector. Respiration rate was calculated using a CO2 calibration curve and expressed as CO2 mg kg−1 h−1. The measurement was carried out in three replicates.

2.1. Fruit materials and treatments Winter jujube (Zizyphus jujuba Mill. cv. Dongzao) fruit at commercial maturity (firmness: 18−21 N; soluble solids content: 25–27 %) were picked from an orchard in Korla, Xinjiang in China. Harvested winter jujube of uniform shape, color, and no visual defects were selected and then randomly allocated into two groups of 2190 fruit each, consisting of three replicates (730 fruit per replicate). These selected fruit were treated with the following conditions: (1) for NO treatment, the jujube fruit were fumigated with NO (20 μL L−1) in a sealed plastic container (67 L) for 3 h at 0 °C following the method of Zhao et al. (2019b); (2) for the control group, the fruit were sealed the same condition without adding NO. After treatment, the fruit of both groups were stored at 0 ± 1 °C with 90–95 % relative humidity for 75 d. The jujube fruit were sampled during storage at 15 d intervals. For each group, 130 fruit selected from each replicate were used for the measurement of weight loss and decay incidence regularly. At each sampling time, a total of 80 fruit were randomly collected for assessing other physiochemical quality parameters, membrane permeability, and 20 fruit left were peeled and frozen with liquid nitrogen, then stored at -80 °C to assay ROS metabolism parameters. All of the analyses mentioned above were carried out in triplicate.

2.4. Determination of membrane permeability and malondialdehyde (MDA) Membrane permeability was assessed following the procedure of Campos et al. (2003) with slight modification: 20 disks from 9 fruit sample were immersed in 50 mL of distilled water. Subsequently, the initial electrolyte leakage (EI0) was determined after for 1 h of incubation of discs in distilled water. The eventual leakage (EI1) was evaluated after heating at 100 °C for 10 min. Relative electrolyte leakage was employed to express membrane permeability, calculated as follows: membrane permeability (%) = (EI0/EI1) × 100. MDA content was quantified in accordance with the modified 2

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day 30 and day 75, respectively. In Fig. 1B, winter jujube remained unchanged during the first 15 d of storage, and the weight loss of the control winter jujube rapidly rose as the storage period progressed. However, the increase rate in weight loss was much slower in NO-treated jujube fruit in comparison with that in control throughout the storage time. On storage day 75, the weight loss of the NO-treated winter jujube was only 30.3% of the weight loss of the control winter jujube. As shown in Fig. 1C, variation in firmness showed a downward trend during storage regardless of the treatment; however, the winter jujube subjected to NO fumigation retained firmness at a higher level, compared with the control during the entire storage period. For instance, the firmness of the NO-treated winter jujube was 12.4 % and 30.7 % higher (P < 0.05) than that of the control after 30 and 60 d of storage, respectively. Fig. 1D shows that no browning appeared in the flesh of winter jujube fruit during the first 15 d of storage. Flesh browning was observed in the control fruit on 30 d of storage; thereafter, flesh browning index showed a sharp increase as storage time progressed. In winter jujube treated with NO, the appearance of the flesh browning was retarded, and the rise in flesh browning index was suppressed. At the end of storage, the flesh browning index of NO-treated winter jujube fruit was detected 72.1 % lower than control group.

method used by Hodges et al. (2004). First, 1 g of fresh winter jujube fruit tissue was homogenized with 5 mL of cold 0.1 % trichloroacetic acid (TCA). After centrifugation at 10,000 g for 20 min at 4 °C, 2 mL of the supernatant was thoroughly mixed with 2 mL of 0.67 % TCA containing 0.5 % thiobarbituric acid. The mixture was incubated at 100 °C for 20 min, immediately cooled to room temperature and centrifuged again. The absorbance levels of the supernatant were measured at 450, 532, 600 nm. MDA content was expressed on a fresh weight as μmol kg1 . 2.5. Determination of O2-% production rate and H2O2 content assay The O2-% generation rate and H2O2 content were conducted according to the method of Li et al. (2017) and Song et al. (2016), respectively. The O2-% generation rate was expressed as nmol min-1 g-1 and the H2O2 level was expressed as μmol g-1, which were all expressed on a fresh weight basis. 2.6. Determination of ROS-scavenging enzymes activities Extraction of enzymes including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) was performed by the method of Zhang et al. (2015). Activities of SOD and CAT were determined by the method of Toivonen and Sweeney (1998); Chance and Maehly (1955), respectively. One unit of SOD activity was defined as the enzyme content that caused 50 % of NBT inhibition per second, and the result was expressed in U kg-1. One unit of CAT activity was defined as an increase in absorbance of 0.01 at 240 nm per second, and the result was expressed as U kg-1. APX activity was evaluated with the method described by Nakano and Asada (1987). The APX activity unit was designed as a change of 0.01 in the absorbance of the reaction solution at 290 nm per second, and the result was expressed as U kg-1. GR activity was assayed according to the method by Smith et al. (1988). The oxidation of 1 nmol NADPH per minute was defined as one unit of GR activity, and the result was expressed as U kg-1. The activity of all enzymes was expressed on a fresh weight basis.

3.2. Effect of NO treatment on TSS and TA of winter jujube As shown in Fig. 2A, the TSS contents in the control and NO-treated fruit increased slightly at the early storage, followed by a decline after storage day 45. Although no significant differences were detected in TSS content among NO-treated fruit and the control fruit within the 45 d of storage, NO treatment delayed the increase of TSS content in the winter jujube fruit. In addition, the peak value of the NO-treated fruit was 4.0 % lower than that of the control (P < 0.05). TA contents in all samples exhibited similar decrease trends during storage from day 0 to day 30; after this period, the decline rate of the TA content of the control fruit largely varied from those of the NOtreated fruit (Fig. 2B). The TA content of the control fruit markedly decreased from day 30 till the final storage period, but it declined less noticeably in NO treatment during the aforementioned storage period. A significant difference was observed in TA content in the control and NO treatment throughout the entire storage time (P < 0.05).

2.7. Measurement of contents of ascorbic acid (AsA) and glutathione (GSH) The measurement of AsA content employed the method of Kampfenkel et al. (1995). The AsA content was expressed on a fresh weight basis as g kg-1. The method of Brehe and Burch (1976) was applied to assay GSH content. The result was expressed on a fresh weight basis as mmol kg−1.

3.3. Effect of NO treatment on the respiration rate of the winter jujube As shown in Fig. 3, the respiration rate of the control winter jujube fruit exhibited a rapid increase from 10.9 CO2 mg kg-1 h-1 (day 0) to 13.2 mg kg-1 h-1 (day 45), but underwent a slightly reduction on storage day 60, followed by a rise from day 60 to day 75 of storage. Comparing with the control, the respiration rate was significantly lower (P < 0.05) in the NO-treated fruit throughout the whole storage period, which was 9.3 % lower than in control winter jujube fruit at the end of storage.

2.8. Statistical analysis Statistical analyses were performed using IBM SPSS Statistics 20.0 software (SPSS Inc., Chicago, USA). The data were analyzed via oneway analysis of variance. Comparison of the means was performed using an independent samples t-test. Differences at P < 0.05 were considered statistically significant.

3.4. Effect of NO treatment on membrane permeability and MDA content of the winter jujube

3. Results

Fig. 4A shows that the membrane permeability of the winter jujube fruit continuously increased during the whole storage period in each treatment. This increase was much lower in NO-treated fruit than in control fruit (P < 0.05). At the end of storage, the membrane permeability of the control fruit was approximately 61.0 %; however, that of the NO-treated fruit only increased to 46.2 % on the same day, which was significantly lower than that of the control (P < 0.05). As displayed in Fig. 4B, a similar upward trend in the MDA contents of the winter jujubes was observed under all treatments. Compared with the control, the NO-treated fruit exhibited lower MDA contents.

3.1. Effect of NO treatment on physical quality of winter jujube The decay incidence of winter jujube with or without NO treatment showed a significant increase during storage, while the changes in the control were larger than those in NO-treated winter jujube (Fig. 1A). Specifically, no noticeably decayed winter jujube was observed under the NO treatment during storage for 15 d. Decay incidence was 77.3 % and 44.7 % lower in NO-treated jujube fruit than that in control fruit on 3

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Fig. 1. Effect of NO treatment on decay incidence (A), weight loss (B), firmness (C), and flesh browning index (D) of winter jujube during storage. Error bars indicate the standard error of three replicates. The different letters (a, b) represent a significant difference (P < 0.05) between NO-treated winter jujube and the control winter jujube within the same time period.

3.5. Effect of NO treatment on the rate of O2-% production and H2O2 content of the winter jujube

Specifically, during the entire storage period, the level of the MDA content in the control fruit markedly increased from 3.5 μmol kg-1 to 9.9 μmol kg-1; whereas, the MDA content in the NO-treated fruit slowly increased and only peaked at 7.7 μmol kg-1 at the end of the storage period.

The O2-% production rate and H2O2 content in winter jujube increased continuously regardless to the given treatment and no significant differences were found between the control and NO-treated winter jujube within the first 15 d of storage period (Fig. 5A–B).

Fig. 2. Effect of NO treatment on TSS (A) and TA (B) of winter jujube during storage. Error bars indicate the standard error of three replicates. The different letters (a, b) represent a significant difference (P < 0.05) between NO-treated winter jujube and the control winter jujube within the same time period. 4

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winter jujube decreased slowly during the same storage time. In comparison with the control winter jujube, there were noticeably higher CAT activity in the NO-treated winter jujube during storage day 30 to day 75 (P < 0.05). The APX activities in both control winter jujube and NO-treated winter jujube tended to increase during the first 30 d of the storage period and, afterwards, dropped until the end of the storage (Fig. 6C), but NO treatment retained higher APX activity as compared to the control group throughout the entire storage time. For instance, the APX activity in the NO-treated winter jujube was about 32.0 % higher than that in the control winter jujube after 60 d of storage (P < 0.05). The GR activity increased steadily in all winter jujube samples within the initial 30 d of storage, and rose sharply from 30 d till 45 d of storage, then declined quickly in the following storage days (Fig. 6D). However, NO treatment promoted GR activity, as dramatically higher GR activity was found in NO-treated winter jujube as compared to the control winter jujube from 30 d to 75 d of storage (P < 0.05). Specifically, the GR activity was 35.7 % and 74.7 % higher in the NO-treated fruit than that of the control fruit on 45 d and 75 d of storage, respectively. Fig. 3. Effect of NO treatment on the respiration rate of the winter jujube during storage. Error bars indicate the standard error of three replicates. The different letters (a, b) represent a significant difference (P < 0.05) between NO-treated winter jujube and the control winter jujube within the same time period.

3.7. Effect of NO treatment on the contents of AsA and GSH of the winter jujube There was a continuous reduction in the AsA content during the postharvest storage, and the decline was retarded by the NO treatment (Fig. 7A). The AsA concentration of the NO-treated winter jujube was 1.3-fold higher after 45 d and 1.4-fold higher after 75 d as compared to that of the controls. A significantly higher difference (P < 0.05) in AsA content was found by statistical analysis between the NO-treated winter jujube and the control winter jujube during during the entire storage period. As demonstrated in Fig. 7B, the GSH content in both the control winter jujube and the NO-treated winter jujube declined as time progressed. The GSH content in the control winter jujube decreased from 103.5 mmoL kg-1 on 0 d of storage to 57.6 mmoL kg-1 on 75 d of storage. However, higher GSH content value with slower reduction were observed in the NO-treated winter jujube during storage period. At the end of the storage time, the GSH content in NO-treated winter jujube only decreased to 77.5 mmoL kg-1.

Afterwards, both the O2-% generation and H2O2 content increased quickly, but NO treatment remarkably diminished and delayed O2-% generation rates and H2O2 contents in NO-treated fruit were 28.0 % and 32.8 % lower than those in control fruit on the 75 day of storage, respectively. 3.6. Effect of NO treatment on ROS-scavenging enzymes of the winter jujube fruit As shown in Fig. 6A, the SOD activity in winter jujube under both treatments went up quickly during the first 15 d of storage. Thereafter, the SOD activity in the control winter jujube increased progressively and peaked at storage day 60, then decreased in the following storage days. The SOD activity in the NO-treated winter jujube increased at a much faster rate and reached 37.3 U kg-1 on storage day 45, which was 12.7 % higher than that of control winter jujube at the same storage period, then decreased from day 45 to day 75 of storage. The CAT activity rose from storage day 0 to day 30 for both the control and NO-treated winter jujube (Fig. 6B). The CAT activity of the control winter jujube declined rapidly from 153.8 U kg-1 (day 30) to 125.5 U kg-1 (day 75) after thirty storage days, while the NO-treated

4. Discussion Fruit senescence in winter jujube is commonly involves weight loss, rotting, flesh browning and a decrease in firmness and AsA (Li et al., 2004). Water losses, which usually arise from evaporation and respiration, could affect the sensory quality of winter jujube and lead to Fig. 4. Effect of NO treatment on membrane permeability (A) and MDA (B) of the winter jujube during storage. Error bars indicate the standard error of three replicates. The different letters (a, b) represent a significant difference (P < 0.05) between NO-treated winter jujube and the control winter jujube within the same time period.

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Fig. 5. Effect of NO treatment on the rate of O2-% production (A) and H2O2 content (B) of winter jujube during storage. Error bars indicate the standard error of three replicates. The different letters (a, b) represent a significant difference (P < 0.05) between NOtreated winter jujube and the control winter jujube within the same time period.

retaining firmness of winter jujube during cold storage (Fig. 1A–D), postponing quality deterioration and prolonging the storage time of winter jujube. These results were in line with previous studies employing NO treatment to impede the development of decay and softening, maintain postharvest quality in tomato (Lai et al., 2011), grape (Ghorbani et al., 2017), apple (Pristijono et al., 2006), and mango (Hu et al., 2014). TSS and TA are major chemical properties that are also considered

losses of nutrition and flavor (Zhang et al., 2019a). Fruit decay is the major factor of postharvest losses in winter jujube (Fu et al., 2019). Firmness is an essential quality attribute in determining storablity and consumer preference of winter jujube (Zhang et al., 2019a). Flesh browning, one of typical storage disorders in winter jujube, reflects a degree of winter jujube senescence to a certain extent (Sun et al., 2007). The results of the current study indicated that postharvest NO treatment reduced weight loss, decay incidence, and flesh browning, as well as

Fig. 6. Effect of NO treatment on the activities of the SOD (A), CAT (B), APX (C), and GR (D) in the winter jujube during storage. Error bars indicate the standard error of three replicates. The different letters (a, b) represent a significant difference (P < 0.05) between NO-treated winter jujube and the control winter jujube within the same time period. 6

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Fig. 7. Effect of NO treatment on the contents of the AsA (A) and GSH (B) of the winter jujube during storage. Error bars indicate the standard error of three replicates. The different letters (a, b) represent a significant difference (P < 0.05) between NO-treated winter jujube and the control winter jujube within the same time period.

in assessing the flavor and nutritive quality of jujube fruit (Hernández et al., 2015). In this study, The TSS content increased slightly and then decline in postharvest storage of winter jujube, while the TA content progressively decreased (Fig. 2A–B). Exogenous NO retarded the increase in TSS content as well as the decline of TA (Fig. 2A–B). In agreement with our finding, Li et al (2014) have found a delay in the increase in TSS and TA reduction in papaya fruit treated with 60 μL L-1 NO during ripening. Respiration, involving a set of oxidation-reduction reactions with the consumption of sugar and organic acid as substrates, directly determines the rate of senescence of postharvest fruit through affecting the energy state and redox state (Chumyam et al., 2017). In our study, respiration rate in winter jujube generally exhibited an upward trend throughout the entire period (Fig. 3). This result was consistent with the report that winter jujube show a non-climacteric respiratory pattern during storage (Sheng et al., 2003). However, the increase in respiration rate was retarded by NO treatment compared with control (Fig. 3), coinciding with the lower weight loss (Fig. 1B) and TA reduction (Fig. 2B), which suggested that the capacity of NO to delay fruit senescence can be linked with suppression of respiration rate. Similar results have also been reported in peach (Flores et al., 2008), plum (Singh et al., 2009) and mango (Zaharah and Singh, 2011). These findings mentioned above collectively demonstrated that NO postponed the development of physio-chemical quality deterioration and the rate of senescence of winter jujube during cold storage. The loss of cellular membrane integrity and function, a consequence of lipid peroxidation, is regarded as the main factor causing postharvest fruit senescence (Tian et al., 2013). It can promote fruit decay and aggravate other metabolic disorders (Shewfelt and del Rosario, 2000). The structural/functional membrane dysfunction under senescence stress is associated with excessive ROS accumulation and the MDA content (Gao et al., 2016). MDA, as the major byproduct of cellular membrane lipid peroxidation, indicates the degree of oxidative damage of the membrane (Sharma et al., 2012). The present study revealed that application of NO efficiently restrained the increase in membrane permeability and MDA content (Fig. 4A–B) as well as in the production of ROS like O2-% generation rate and H2O2 content in winter jujube during storage (Fig. 5A–B). The reduced ROS accumulation in NOtreated winter jujube coincided with the lower membrane permeability and MDA content, implying that NO treatment could prevent the membrane from peroxidation by quenching overproduction of ROS. Our results were consistent with the previous study by Zhu et al. (2008) and Iakimova and Woltering (2015), who claimed that the reduction of membrane oxidative damage owing to the diminished ROS accumulation by NO treatment in kiwifruit and fresh-cut lettuce was benefit of delaying senescence and quality deterioration. Plants possess ROS-scavenging systems comprising of enzymatic and nonenzymatic substances (Sharma et al., 2012). SOD, CAT, APX,

and GR are the major enzymes for scavenging ROS in plants (Gill and Tuteja, 2010). SOD is responsible for dismutating O2-% to H2O2 (Sharma et al., 2012). An enhancement of SOD activity could lead to the reduction of O2-% generation rate (Lin et al., 2015). In the current study, winter jujube treated with NO retained higher SOD activity (Fig. 6A) concomitant with lower O2-% production rates compared with the control winter jujube (Fig. 5A). The degradation of H2O2 to H2O can be achieved by CAT, APX, and GR in diverse routes: CAT can directly decompose H2O2 (Sharma et al., 2012); APX and GR eliminate excessive H2O2 level through oxidizing AsA to monodehydroascorbic acid and modulating reduced glutathione to oxidized glutathione (Noctor and Foyer, 1998). Higher activities of antioxidant enzymes correlated with delayed senescence has been demonstrated in jujube fruit treated with chitosan film (Yu et al., 2012) and a combined treatment of UV irradiated + chitosan (Zhang et al., 2014). In this study, activities of CAT, APX, and GR in NO-treated winter jujube was remarkably higher than those in control fruit (Fig. 6B–C), resulting in the lower H2O2 content (Fig. 5B). The results confirmed that NO treatment was instrumental in improving ROS scavenging capacity via maintaining higher levels of activities of ROS scavenging-associated enzymes like SOD, CAT, APX and GR (Fig. 6), which contributed to inhibiting membrane lipids peroxidation, retarding loss of membrane function and eventually postponing the process of fruit senescence. These result seem to agree with the findings of Rabiei et al. (2019) in cornelian cherry and Zhang et al. (2019b) in table grape. Ascorbic acid is not only an important nutrient but also an antioxidant that eliminates ROS in fruit, exerting particular influence in delaying harvested fruit senescence (Kou et al., 2019). AsA, as a substrate for APX, participates in catalyzing the decomposition of H2O2 to H2O and O2, and the dehydroascorbic acid generated can be further reduced to AsA in the presence of GSH (Meyer, 2008). The contents of AsA and GSH in this study decreased constantly with progressive storage, implying that the quality of winter jujube continuously declined during storage. But NO treatment caused a significant delay in the reduction of AsA as well as GSH (Fig. 7A–B), corresponding to lower H2O2 content in NO-treated winter jujube. Hence, it could be inferred that NO treatment was efficient in retaining higher concentrations of endogenous antioxidant components, leading to less accumulation of ROS and a delay of senescence of winter jujube, which was accordance with the report by Ma et al. (2019). 5. Conclusion In summary, postharvest fumigation with NO effectively delayed the development of decay, weight loss and the appearance of flesh browning, maintained high firmness and TA content, but lower TSS and respiration in winter jujube fruit. NO treatment also retained higher 7

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concentrations of AA and GSH, enhanced activities of SOD, CAT, APX, and GR, decreased ROS accumulation, and alleviated the degree of membrane lipid peroxidation and membrane permeability. Therefore, the mechanism by which NO may counteract senescence to conserve the physio-chemical quality attributes of the winter jujube fruit could be associated with maintaining the balance of ROS metabolism by enhancing the antioxidant enzyme activity, reducing ROS production, and alleviating lipid peroxidation.

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