Postharvest melatonin treatment inhibited longan (Dimocarpus longan Lour.) pericarp browning by increasing ROS scavenging ability and protecting cytomembrane integrity

Abstract Postharvest melatonin treatments have been reported to improve the quality and storability, especially to inhibit browning in many fruits, but the effect had not been systematically investigated on longan fruit. In this study, the effect of 0.4 mM melatonin (MLT) dipping on the quality and pericarp browning of longan fruits stored at low temperature was investigated. The MLT treatment did not influence the TSS content of longan fruits but lead to increased lightness and h° value while decreased a* value of pericarp. More importantly, the treatment significantly delayed the increase in electrolyte leakage and malonaldehyde accumulation, inhibited the activities of polyphenol oxidase and peroxidase, and thus retarded pericarp browning. In addition, the treatment significantly inhibited the production of O2 •− and H2O2 while promoted the accumulation of glutathione, flavonoids, and phenolics at earlier storage stages in longan pericarp. Interestingly, the activities of ascorbate peroxidase (APX) and superoxide dismutase (SOD) were significantly upregulated but activities of catalase were downregulated in the MLT‐treated longan pericarp. MLT treatment effectively enhanced APX and SOD activities, increased flavonoid, phenolics, and glutathione content, protected cytomembrane integrity, inhibited the production of O2 •− and H2O2 and browning‐related enzymes, and thus delayed the longan pericarp browning.


| INTRODUC TI ON
Longan (Dimocarpus longan Lour.), one of the typical tropical and subtropical fruits with high commercial value, is a kind of globally consumed fruit but is extremely unresistant to long-term postharvest storage and transportation (Han et al., 2020;Luo, Niu, et al., 2019;Luo et al., 2021;. Longan fruits are usually harvested in summer with high temperatures and humidity. Therefore, deterioration of longan fruit characterized as pericarp browning and aril breakdown quickly occurs within a few days without low-temperature storage (Han et al., 2020;Luo, Niu, et al., 2019). Compared with the aril breakdown of longan fruits, which was an indicator of interior quality deterioration and significantly varied among cultivars, the pericarp browning is a much more common appearance index of senescence in longan pericarp . Postharvest pericarp browning of longan was proved to be related to the oxidation of peroxidase (POD) and polyphenol oxidase (PPO) on phenolics . This oxidation process might be exacerbated by the burst of reactive oxygen species , hydrogen peroxide-induced energy deficiency , and peroxidation of membrane lipid , all of which might be caused by the absence of enzymatic and nonenzymatic ability for scavenging free radicals. In addition, destroying of cellular compartmentation and integrity of cell membrane, which are possibly induced by water loss (Lin et al., 2010), pathogen infection , and other stresses, are also important factors promoting postharvest browning of longan pericarp.
However, SO 2 fumigation and fungicide treatments under residue limits were still widely used for retarding deterioration of longan fruits due to their long effective action and convenience.
Melatonin (MLT), namely N-acetyl-5-methoxytryptamine, was proved to be effective to keep the postharvest quality and health properties of fresh fruit. The documents about its application on postharvest fruit concerning the shelf-life extension and quality maintenance increased rapidly in the past decades (Yun et al., 2021). The delaying of postharvest MLT treatment on the senescence was reported on many fruits such as apple (Onik et al., 2020), citrus (Lin et al., 2019), grape , kiwifruit , mango (Rastegar et al., 2020), peach (Gao et al., 2016), pear (Liu et al., 2019), strawberry (Liu et al., 2018), sweet cherry (Xia et al., 2020), tomato , and litchi fruit (Zhang et al., 2018). The decreased physiological characters in MLT-treated fruit compared with control fruit were malonaldehyde (MDA) or/and electrolyte leakage, which were reported in apple (Onik et al., 2020), kiwifruit , peach (Gao et al., 2016), strawberry (Liu et al., 2018), sweet cherry (Xia et al., 2020), tomato , pomegranate (Jannatizadeh et al., 2019), and litchi fruit (Zhang et al., 2018). More importantly, postharvest MLT treatment resulted in significant changes in the ROS level, the content of antioxidants, and the enzymatic abilities for scavenging free radicals. Most of the literature reported that MLT treatment leads to increased content of antioxidants such as ascorbic acid, flavonoids, phenolics, and anthocyanins but decreased activity of PPO. Therefore, the postharvest MLT treatment was proved to significantly retard the browning of pomegranate (Jannatizadeh et al., 2019), sweet cherry (Xia et al., 2020), and litchi fruit (Zhang et al., 2018). However, the effect of MLT treatment on the postharvest quality and deterioration of longan fruits have not been investigated yet. In this study, the "Chuliang" longan fruits were treated with prochloraz to eliminate the influence of microorganisms on the deterioration. Then, the longan fruits were treated with dipping in 0.4 mM MLT and stored at low temperature. The contents of total soluble solids (TSS), pericarp chromatic values, browning index, membrane permeability index (MDA and electrolyte leakage), browning-related enzyme activities, the content of ROS and antioxidants, and activities of enzymes for scavenging free radicals were determined to evaluate the effect of MLT on the deterioration especially pericarp browning of longan fruits. This study was expected to clarify the mechanism for inhibition of MLT on longan pericarp browning and thus provide a theoretical basis for applying MLT in developing new preservative methods.

| Longan fruits, treatments, and storage
Commercial mature "Chuliang" longan fruits were harvested in an orchard at the Hezhou Academy of Agricultural Sciences (Guangxi province, China) and immediately transported to the laboratory.
More than 1,200 fruits with no disease and no damage were selected and dipped in 500 mg/L prochloraz solution for 3 min.
The fruits were then dried at room temperature for 30 min. After that, 600 fruits were dipped in 0.4 mM MLT (98% HPLC purity, Macklin, Shanghai Macklin Biochemical Co., Ltd) for 3 min (abbreviated to 0.4 mM MLT), and the other 600 fruits were dipped in clean water for 3 min (CK). The fruits were naturally dried for 60 min, and every 20 fruits were packed into one polyethylene bag (0.03 mm thick). The fruits were then stored at 4 ± 1°C and 85% relative humidity.

| Determination of chromatic value
The fruit color was measured, respectively, at 0, 8, 16, 24, and 32 days after storage (DAS) using a color analyzer (KONICA MINOLTA CR-300). Ten fruits were randomly selected and the L*, a*, b*, c*, and h° values on equatorial plane of each fruit were subjected to three detections. The color index (CI) was calculated by the equation 1 according to Luo et al. (2015):

| Sampling and determination of TSS content
The longan fruits were, respectively, sampled at 0, 8, 16, 24, and 32 DAS. The pericarp and aril of each longan fruit were separated. The collected pericarp of treated or CK fruits was immediately frozen in liquid nitrogen, ground and stored at −80°C until be used. The separated aril from each fruit was used for juicing and determination of TSS content by a brix refractometer (PR-32α, ATAGO). Sampling and TSS analysis were performed by three times (one bag per time).

| Analysis of pericarp relative electrolytic leakage
The cell membrane permeability of pericarp was measured according to a previously reported method (Zhang et al., 2018).

| Determination of MDA content in pericarp
The MDA content was determined using the thiobarbituric acid (TBA) method with some adjustments (Zhang et al., 2018). A 1.0 g sample of powder was fully mixed with 8 ml precooled 10% TCA solution and shaken for 30 s. The sample was extracted for 10 min in an ice-bath. After a centrifugation for 20 min at 5,000 × g and 4°C, 2 ml supernatant was moved out to be mixed with 2 ml TBA and then heated in boiling water for 20 min. After be cooled and centrifuged at 5,000 × g for 5 min, the absorbance of supernatant at 532 nm was recorded and the value for nonspecific absorption at 600 nm and 450 nm was subtracted. Mixture of 2 ml 10% TCA and 2 ml TBA was set as a blank sample. All samples were subjected to three repeats.

| Determination of polyphenol oxidase (PPO) and peroxidase (POD) enzymatic activities
Polyphenol oxidase activity was determined according to Zhang et al. (2018) with some modification. A 1.0 g sample of powder was added into a precooled tube containing 8 ml 50 mM PBS (pH 5.5) and 0.2 g PVP, and then fully mixed. After a centrifugation at 12,000 × g and 4°C for 20 min, the supernatant was removed to a new tube. The precipitate was washed by another 6 ml 50 mM PBS (pH 5.5) and centrifuged at 12,000 × g and 4°C for 20 min. The supernatants of two centrifugations were merged into one tube and the volume was filled to constant 20 ml by deionized water. The reaction system containing 0.1 ml enzymatic extraction, 3.9 ml 10 mM acetic acid buffer (pH 5.5), and 1 ml 0.1 M catechol was kept at 35°C for 10 min. After that, the solution was cooled by ice-bath and mixed with 2 ml 30% TCA to stop the reaction. The enzymatic extraction inactivated by boiling water was set as a control sample. The increase of 0.01 OD 525nm per minute was recorded as one enzyme activity unit (U). The result was expressed as U g -1 FW.
The activity of POD in pericarp was assayed by a change of absorbance at 470 nm caused by production of tetraguaiacol from guaiacol in the presence of H 2 O 2 (Zhang et al., 2018). A change of 0.01 in the absorbance per minute was recorded as one unit of POD enzyme activity. The result was expressed as U g -1 FW.

| Measurement of contents of ascorbic acid and glutathione (GSH) in pericarp
The supernatant for determining the content of ascorbic acid and GSH was extracted according to the same process used for measurement of MDA. One milliliter supernatant was mixed with 1 ml 5% TCA and 1 ml ethanol. After a full mixture by shaking, 0.5 ml 0.4% phosphoric acid ethanol buffer, 1 ml 0.5% bathophenanthroline-ethanol, and 0.5 ml 0.03% FeCl 3 -ethanol were added and the reaction were kept at 30°C for 90 min. The absorbance of the solution at 543 nm was recorded. The ascorbic acid content was calculated according to a standard curve and expressed as μmol/g FW.
For measuring GSH content, 0.5 ml supernatant was mixed with 2 ml 0.1 M PBS buffer (pH 7.8) and 0.5 ml 5,5'-Dithiobis-2-nitrobenzoic acid (4 mM). The solution was kept at 30°C for 20 min. The absorbance of the solution at 412 nm was recorded. The GSH content was determined according to a standard curve and expressed as μmol/g FW.

| Detection of the total flavonoid and total phenolic acid contents in pericarp
The extraction for analyzing total phenolics and total flavonoid content was same to the previous method (Luo, Niu, et al., 2019). The absorbance of extract was firstly assayed at 325 nm for flavonoid determination and then at 280 nm for determining content of total phenolics. The total phenolics and total flavonoid content were calculated and expressed as OD 325nm g -1 FW or OD 280nm g -1 FW.

| Assays of superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) activities
One gram pericarp powder was fully mixed with 8 ml precooled 0.1 M phosphate buffer (pH 7.8, containing 5 mM DTT and 2% PVP, w/v) by shaking for 30 s. The sample was extracted for 10 min in an ice-bath. After be centrifuged at 8,000 × g and 4°C for 15 min, the supernatant was used for enzymatic activity measurement of SOD (Zhang et al., 2018) and CAT .

| Statistical analysis
The variance of data was determined by SPSS software package release 17.0 (SPSS Inc.). Multiple comparisons were performed by oneway ANOVA based on Duncan's multiple range tests.

| Effect of MLT treatment on the TSS content and exterior quality of low-temperature stored longan fruits
The TSS contents in both of the CK and MLT-treated longan fruits were found to be quickly decreased (from 23.80% to 22.71%) in the first 8 days and then slowly decreased to be about 22.36% at 16 DAS and showed a stable level in the next 24 days. However, no significant difference of TSS content was found between CK and MLT-treated longan fruits at each time point (Figure 1a). Significantly higher chromatic L* and h° value but lower chromatic a*, b* value and CI value were observed on the MLT-treated longan pericarp at the two storage stages (Figure 1b-f). The above results indicated that the MLT treatment did not significantly influenced the TSS content, but inhibited the deterioration of longan fruits' appearance at the later stages of low-temperature storage.

| Effect of MLT treatment on the browning, relative electrolytic leakage, and MDA content in longan pericarp
As shown in Figure

| Effect of MLT treatment on the PPO enzymatic activity, superoxide anion, and H 2 O 2 content in longan pericarp
As shown in Figure 3a, the enzymatic activity of PPO in the CK pericarp was observed to be significantly higher than that of PPO in the  (Figure 3a). In total, the POD activity in the MLT-treated longan pericarp was lower than that in the CK pericarp during the whole storage although the POD activity in the MLT-treated longan pericarp was only found to be significantly lower at 16 and 32 DAS (Figure 3b).

| Effect of MLT treatment on the contents of ascorbic acid, glutathione, flavonoid, and phenolics in longan pericarp
No significant difference of ascorbic acid content was found be-

| Effect of MLT treatment on activities of SOD, APX, and CAT in longan pericarp
The SOD activity in the MLT-treated longan pericarp quickly decreased from 2,710.37 to 3,500.84 U g -1 at 8 DAS and then kept at high level at the later three stages, which was parallelly higher than that in the CK pericarp during the whole storage (Figure 5a).
Similarly, the APX activity in the MLT-treated longan pericarp increased from 10.00 to 20.00 U g -1 from 0 DAS to 16 DAS and then slowly decreased to be 16.00 U g -1 , which was found to be lower at 8 DAS but higher at 16 DAS and 24 DAS than the APX activity in the CK pericarp (Figure 5b). It was worthy to note that the CAT activity in the MLT-treated pericarp were lower than that in the CK pericarp at each time from 8 DAS to 32 DAS (Figure 5c). The CAT activity in the CK pericarp increased from 39.67 to 59.80 U g -1 during the first 24 days and then decreased to be the initial level. The CAT activity in the MLT-treated pericarp did not increase during the first 24 days and then significantly decreased from 41.93 to 27.53 U g -1 . These results suggested that MLT might induce some enzymatic activities (i.e., SOD and APX) for scavenging free radical while reduce the induction of another enzymatic activities for scavenging free radical (i.e., CAT and POD) due to its direct scavenging on free radicals.

F I G U R E 4
The content of ascorbic acid (a), GSH (b), total flavonoid (c), and total phenolics (d) in the pericarp of the CK and MLT-treated longan fruits during low-temperature storage. Note: different lowercase letters indicated a significant difference, p < .05

| Effect of MLT treatment on the quality and postharvest enzymatic pericarp browning of longan fruits
Postharvest application of MLT had been reported to effectively improve the storage performance in many fruits. Onik et al. reported that 1 mM MLT treatment significantly reduced the weight loss and MDA content, maintained better skin structure of apple during storage at 1°C for 56 days (Onik et al., 2020). Wang et al. found that immersing in 200 μM MLT significantly reduced berry abscission and rotten index of table grape stored at 4 ± 0.5°C . Hu et al. (2018) reported that 100 μM MLT lead to delayed decrease of firmness, starch contents and color L* value, as well as lower mass loss rate and decay incidence in kiwifruit. MLT application was also reported to do not influence the TSS content, titratable acidity and color factors of mango fruits, but 1,000 μM MLT was able to reserve the firmness of mango during storage (Rastegar et al., 2020). Gao et al. (2016) found that 100 μM MLT treatment for 10 min reduced weight loss and decay incidence, maintained firmness and TSS content, significantly decreased the MDA content in peach fruits stored at 25-28°C.
Treatment with 100 μM MLT was found to reduce rates of respiration, inhibit the softening and ripening, and delay the senescence of European pear fruit (Liu et al., 2019). Application of 0.1 or 1 mmol/L MLT on strawberry fruit was reported to effectively reduce decay and weight loss, delay senescence by maintaining the color, firmness, TSS content, and titratable acidity (Liu et al., 2018).

| Effect of MLT treatment on the antioxidants and nonenzymatic scavenging of ROS
The imbalance between scavenging ability and production of oxygen free radicals might lead to the burst of ROS , hydrogen peroxide-induced energy deficiency , and peroxidation of membrane lipid  and further accelerate fruit senescence and browning caused by PPO and POD in longan fruit . Wang et al. (2020) reported that 200 μM MLT immersion greatly enhanced amino acids accumulation and significantly increased phenolics content of "Kyoho" grape stored at 4 ± 0.5°C. Hu et al. (2018) reported that 100 μM MLT significantly increased the contents of ascorbic acid and glutathione in kiwifruit stored at 0 ± 0.5°C. Rastegar et al. (2020) found that 1 mM MLT application significantly inhibited the decrease in phenolics, flavonoids, and antioxidants of mango during storage at 15 ± 1°C. In peach fruits stored at 25-28°C, 100 μM MLT treatment for 10 min was observed to maintain ascorbic acid and reduce the production rate of O 2 • − and H 2 O 2 content (Gao et al., 2016). Application of 0.1 or 1 mmol/L MLT was notably effective to increase the total phenolics and flavonoid contents, which resulted in the higher antioxidant capacity in strawberry fruit but had a negative impact on the ascorbic acid content (Liu et al., 2018 (Gao et al., 2016). Hu et al., reported that 100 μM MLT maintained higher SOD, APX, and glutathione reductase (GR) activity, increased the peak of catalase in kiwifruit stored at 0 ± 0.5°C .
Rastegar et al., found that 1 mM MLT application significantly increased the activity of the CAT and POD enzymes in mango during storage at 15 ± 1°C (Rastegar et al., 2020). In peach fruits stored at 25-28°C, 100 μM MLT treatment for 10 min was proved to significantly enhanced the activities of SOD, CAT, POD, and APX in both cultivars ("Shahong" and "Qinmi") (Gao et al., 2016). Jannatizadeh re-

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L A PPROVA L
The study does not involve any human or animal testing.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author, upon reasonable request.