Decreasing postharvest chilling injury of guava fruit by using melatonin treatment

: Guava fruit is a tropical fruit thus sensitive to the chilling injury. In this study the eﬀects of melatonin (known to protect membrane integrity and to help to face abiotic and biotic stress) is evaluated for reduction of chilling injury during postharvest. Guava fruits were dipped into 10, 100 and 1000 μmol L ­1 melatonin solutions, then kept at cold storage (10±1°C and 90% relative humidity) for 21 days. Several parameters including chilling injury, malondi­ aldehyde content, electrolyte leakage and increased total phenolic compounds and antioxidant activity, phospholipase D and lipoxygenase activity were mea­ sured after treatment. Measurements were made every 7 days during the stor­ age. Results showed that melatonin decreased chilling injury, malondialdehyde content, electrolyte leakage and increased total phenolic compounds and antioxidant activity compared to the control. Also, results indicated that chill­ ing injury of guava fruit by using melatonin decreased through increasing integrity of membrane and reducing phospholipase D and lipoxygenase activity. Thus, melatonin can be a useful treatment for decreasing postharvest chilling disorder of guava fruit.


Introduction
Guava (Psidium guajava L.) is one of the most important fruits of tropical and subtropical regions in the world. The fruits are delicious, rich in vitamin C and minerals (Deepthi et al., 2016). There is a great demand of guava fruits in both domestic and international markets for fresh and processing purposes.
Cold storage is one of postharvest technologies for maintaining quality of horticultural crops until human consumption. However, guava is sensitive to chilling disorder of cold storage (temperature of below 12°C). Signs of chilling injury include irregular ripening and surface pitting on the fruit which decreases quality of fruit (Etemadipoor et al., 2020). Resistance to the chilling temperature related to several factors. One of the most important factors is maintaining membrane integrity (Wongsheree et al., 2009). Membrane integrity can be measured using leakage, malondialdehyde con tent, lipoxygenase and phospholipase D (Aghdam et al., 2014). Several methods used to decrease chilling injury symptoms of fruit rely on the use of hot water and UVC (Pongprasert et al., 2011).
Melatonin plays in fruit ripening and senescence and membrane integrity and protection against abiotic and biotic stresses (Rastegar et al., 2020).
However, melatonin effects on reducing chilling injury of guava fruit have not been evaluated during cold storage. Therefore, the purpose of this study was to investigate melatonin effects on chilling injury reduction.

Materials and Methods
The guava fruits (green stage maturity) were bought from a commercial orchard in Hormozgan province, Iran and the uniform sized fruits were transferred to the laboratory. Twelve fruits per four replications were dipped into 10 (T2), 100 (T3) and 1000 (T4) μmol L −1 melatonin solutions for 10 min. Distilled water was used as the control (T1). Fruit were kept at cold storage (10±1°C and 90% relative humidity) for 21 days. Several parameters were mea sured soon after treatment, and then the measure ments were made every 7 days during the storage. Finally, the parameters were checked again one day after exposing to the ambient temperature (25±1°C). The measurements included chilling injury index assessment, percentage of ion leakage, malondialde hyde content, weight loss, titratable acidity, soluble solids concentration (SSC), ascorbic acid, total pheno lic content (TPC), antioxidant activity, phospholipase D and lipoxygenase activity.
The MDA content was determined based on the method described by Wang et al. (2015). The content of MDA was expressed as nmol g 1 FW.
Weight loss, Soluble solids concentration (SSC), Titratable acidity (TA), Ascorbic acid, Total phenolic content (TPC) and Total Antioxidant activity Weight loss was measured based on initial and final experiment at 7day intervals during storage using a digital balance, and results were expressed as percentage.
Soluble solids concentration (SSC) and TA of pulp tissues were measured by using the method of Ali et al. (2011) and the results were expressed as %SSC and %TA, respectively. Ascorbic acid was measured using dye method described by Ranggana (1986) and results were expressed as mg 100 g 1 fresh weight (FW).
TPC were assayed using FolinCiocalteu procedure (Singleton and Rossi, 1965). Results were expressed as mg of gallic acid equivalents (GAE) per gram of fresh weight (mg GAE g 1 FW). The DPPH assay was measured according to the method described by Mirshekari et al. (2019). Results were expressed as percentage.

Phospholipase D and Lipoxygenase activity
Phospholipase D and lipoxygenase assay was determined based on the method described by Aghdam and Mohammadkhani (2014). One unit of Phospholipase D was defined as the amount of enzyme that catalyzed the formation of 1 nmol D nitrophenol h 1 . One unit of lipoxygenase was defined as the amount of enzyme which causes an increase in absorption of 0.01 min 1 at 234 nm and 25°C when linoleic acid was used as the substrate. Protein con tent was estimated according to Bradford (1976). Enzymes activities were expressed as units per mil ligram of protein.

Statistical analysis
Experiments were carried out using completely randomized design. Four replications per treatment used for this study. Data were analyzed using (SAS) version 8.2 (SAS Institute Inc., Cary, NC, USA). Variation Sources were storage days and treatments and means were compared with Duncan's Multiple Range Test (DMRT) at significance level of 0.05.

Chilling injury (CI), Electrolyte leakage (EL), and Malondialdehyde (MDA)
In the present study, melatonin treatment reduced chilling injury index of guava fruit after 7 days of chilling storage when compared with the con trol (Table1). Moreover, CI increased with storage time (Table 1). At the end of storage day control (T1) had the severe chilling injury index (4.8) with highest pitting signs, while T4 had the lowest chilling injury index of 3.2. Usually, CI happens at the cell mem brane, and maintaining its integrity reduces CI (Mirdehghan et al., 2007;Mirshekari et al., 2020).
Accordingly, Electrolyte leakage has been used as an indicator of membrane damage. In this study, EL increased during storage for control and treated melatonin fruits (Table 1). However, at the end of storage day EL was lower in T3 and T4 compared to the T2 and T1 (Table 1). These results showed the role of melatonin in maintenance of membrane integrity. Comparable results have been stated for sapota fruit by Mirshekari et al. (2020). Researches have shown that melatonin treatments affect elec tron flow acceleration in mitochondria to maintain membrane integrity (Tan et al., 2013).
As shown in Table 1, MDA of control fruit increased during storage. Also, lower MDA content were observed in all melatonin treated fruits com pared to the (T1) after 21 days of chilling storage ( Table 1). One of the first events in the CI is mem brane lipid peroxidation. MDA is the final product of lipid peroxidation (Imahori et al., 2008). Lower tem peratures are the main inducers of oxidative damage which produce higher ROS and change the ratio of unsaturated fatty acids to saturated forms (Antunes and Sfakiotakis, 2008). Melatonin treatments low ered MDA accumulation of sapota fruit (Mirshekari et al., 2020).

Weight loss, SSC, TA, Ascorbic acid, TPC and Total Antioxidant activity
Weight loss was at the highest rate (14.3 %) in the control fruits (T1) after 21 days. Treated fruit (T4) showed lower weight loss (6.7%) compared to the T1 and T2 at the end of storage day (Table 2). Weight loss is an index for assessing quality of fruits during storage (Yaman and Bayonidirli, 2002). Skin strength properties of fruit by using melatonin treatment might lower weight loss. Our results are comparable with Rastegar et al. (2020) who indicated that the weight loss was decreased by using melatonin treatment in mango.
SSC and TA concentration are main factors for fruit quality judgment. The initial SSC value of this study was 5.2% in control fruits (T1) and increased Table 1 Melatonin treatments (0, 10, 100 and 1000 μmol L −1 ) effects on chilling injury index (CI), electrolyte leakage (EL) and malondi aldehyde (MDA) content in guava fruit stored at 10°C for up to 21 days (z) Small and capital letters show significant differences by DMRT at P= 0.05 between treatments in columns, and storage time for each parameter, respectively.  Table 2). One of the most signifi cant signs of the nutrient value of fruits is ascorbic acid. The ascorbic acid reduction during storage can be related ascorbic acid oxidase (Choudhary et al., 2016). Melatonin treatment increases oxidative stress resistance by increasing ascorbic acid (Gao et al., 2016). Our results are comparable with Gao et al. (2016) who stated that melatonin treatment main (z) Small and capital letters show significant differences by DMRT at P= 0.05 between treatments in columns, and storage time for each parameter, respectively.  (Table 2). However, at the end of stor age, T3 and T4 melatonin treatments decreased SSC content compared to untreated (T1) and T2 treat ments. Lower SSC content in this research is in agree ment with results of Liu et al. (2018) who showed that SSC decreased with melatonin treatments during storage. Lower amounts of SSC might be due to the slower respiration rate and a weaker metabolic activ ity due to the reduced rate of carbohydrate hydroly sis. Table 2 shows TA content of treated and control fruits. TA content was the highest at harvest day (0.9%). However, TA content decreased during stor age. At the end of storage T3 and T4 showed higher tained ascorbic acid content of peach.
TPC and antioxidant activity decreased during chilling storage. However, melatonin treated fruit had higher TPC and antioxidant capacity compared with control (Table 2). It has been shown that mela tonin treatment increased TPC by regulating gene expression in phenyl propanoid pathway (Zhang et al., 2016). Moreover, Liu et al. (2018) stated that melatonin treatment increased TPC and DPPH scav enging capacity of strawberry. This indicated that melatonin showed positive effect on antioxidant activity of guava fruit.

Phospholipase D and Lipoxygenase activity
Phospholipase D and lipoxygenase activity increased during chilling storage. However, mela tonin treatment decreased their activities during storage (Table 3). Similarly, melatonin treatment decreased CI signs and inhibited Phospholipase D and lipoxygenase activity of sapota fruit (Mirshekari et al., 2020). Studies indicated that CI was achieved by the activities of membranous lipolytic enzymes like Phospholipase D and lipoxygenase which catalyse peroxidation of polyunsaturated fatty acids and are believed to be major contributors to CI in plant tissue (Aghdam and Mohammadkhani, 2014).

Conclusions
Results of this study showed that melatonin treat ments during cold storage of guava fruit decreased chilling injury, soluble solids concentration, malondi aldehyde content, electrolyte leakage, phospholipase D and lipoxygenase activity and increased titratable acidity, ascorbic acid, total phenolic compounds and antioxidant activity compared to the control. Accordingly, we found that melatonin application reduced CI of guava fruit with enhancing membrane integrity and decreasing phospholipase D and lipoxy genase activity.
Concerning the different treatments, we found out that while 10 μmol L 1 produced results which were not significantly different from control treat ment, 100 μmol L 1 and 1000 μmol L 1 were signifi cantly different for CI, weight loss, soluble solids con centration, total phenolic compounds and phospholi pase D activity at the end of storage. In conclusion, melatonin application at 1000 μmol L 1 can be recom mended to be used to decrease CI in guava fruit under cold storage. Employing exogenous melatonin applying confers chilling tolerance in tomato fruits by upregulating ZAT2/6/12 giving rise to promoting endogenous polyamines, proline, and nitric oxide accumulation by Table 3 Melatonin treatments (0, 10, 100 and 1000 μmol L 1 ) effects on phospholipase D (PLD) and lipoxygenase (LOX) in guava fruit during storage at 10°C for up to 21 days (z) Small and capital letters show significant differences by DMRT at P= 0.05 between treatments in columns, and storage time for each parameter, respectively.