Impaginato 299 Adv. Hort. Sci., 2018 32(3): 299-309 DOI: 10.13128/ahs-22260 Postharvest melatonin treatment reduces chilling injury and enhances antioxidant capacity of tomato fruit during cold storage F. Azadshahraki 1 (*), B. Jamshidi 1, S. Mohebbi 2 1 Agricultural Engineering Research Institute, Agricultural Research Education and Extension Organization (AREEO), Karaj 31585-845, Iran. 2 Department of Horticultural Sciences, College of Agriculture and Natural Resources, University of Tehran, Karaj 31587-77871, Iran. Key words: enzyme activity, lycopene, melatonin, proline, tomato fruit. Abstract: In this study, tomato fruit was treated with 50, 100 or 200 µM mela- tonin and then stored at 5°C for 28 days to investigate the effect of melatonin treatment on chilling injury, nutritional quality and changes in the antioxidant system. Tomato fruit developed chilling injury, manifested as surface pitting and irregular red color development during storage. These chilling injury symp- toms, ion leakage and malondialdehyde content were significantly reduced, and proline and carotenoids contents were significantly increased by melatonin treatment. Meanwhile, melatonin substantially reduced O2 - production rate and H2O2 content, which result from significantly higher activities of superoxide dismutase, catalase, and peroxidase than control during the storage. These results suggest that melatonin treatment can effectively enhance chilling toler- ance and reduce chilling injury. The reduction in chilling injury by melatonin may be associated with enhanced enzymatic and non-enzymatic antioxidants, in favor of membrane integrity and thus low cellular and tissue damage. 1. Introduction Cold storage is one of the most effective postharvest technologies to preserve the quality of fresh produces from the time of harvest until final preparation for human consumption in food chain (Bourne, 2006). However, cold storage imposes great risk on postharvest commodities sensitive to chilling injury (CI). Tomato (Lycopersicon esculentum), as one of the most important tropical crops, is typically cold sensitive (Hong and Gross, 2006). The most common visual injury symptoms of CI depicted for tomato fruit include irregular ripening and red color development as well as surface pitting on the fruit. Furthermore, as the chilled tissues are weakened, they become prone to decay and microbial spoilage. This phe- nomenon limits postharvest life and leads to significant degradation of (*) Corresponding author: farzad_shahrakiazad@yahoo.com Citation: AZADSHAHRAKI F., JAMSHIDI B., MOHEBBI S., 2018 - Postharvest melatonin treatment reduces chilling injury and enhances antioxidant capacity of tomato fruit during cold storage. - Adv. Hort. Sci., 32(3): 299-309 Copyright: © 2018 Azadshahraki F., Jamshidi B., Mohebbi S. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 6 December 2017 Accepted for publication 31 January 2018 AHS Advances in Horticultural Science Adv. Hort. Sci., 2018 32(3): 299-309 300 produce quality (Wang, 1993). Melatonin, first discovered in tomato in 1995, accumulates in the fruits as they mature (Dubbels et al., 1995; Hattori et al., 1995). Melatonin content, as an endogenous signaling molecule, increases in response to abiotic and biotic stress, such as drought, salinity, chilling, and pathogens to protect against damage caused by them (Zhang et al., 2014; Arnao a n d H e r n á n d e z - R u i z , 2 0 1 5 ; L i u e t a l . , 2 0 1 6 ) . Accumulation of higher levels of melatonin in horti- cultural crops is beneficial not only for human health, but also for prolonging storability (Tan et al., 2012). Melatonin, a naturally occurring indoleamine, acts as endogenous elicitor and signaling molecule for plants growth and development, decreasing of biotic and abiotic stress, as well as a potent hydroxyl radical scavenger and antioxidant (Zhang et al., 2014; Zhang and Zhang, 2014; Manchester et al., 2015; Zhang et al., 2015). Melatonin contribution has been evi- denced in a semilunar rhythm in macroalgae guard- ing this plant against high temperature stress (Tal et al., 2011). Melatonin treatment decreased apoptosis chilling-induced in carrot suspension cells. Moreover, melatonin treatment alleviated chilling-induced shrinkage and disruption of carrot cell plasma mem- branes (Lei et al., 2004). It has been reported that melatonin treatment reduced chilling injury in peach fruits by enhancement of chilling tolerance and pro- voking of defense response during cold storage (Cao et al., 2016). Soleimani Aghdam and Rezapour Fard (2017) reported that melatonin treatment at 100 µM decreased strawberry fungal decay resulting from higher superoxide dismutase (SOD) activity, associat- ed with lower catalase (CAT) and ascorbate peroxi- dase (APX) activities as well as higher phenylalanine ammonia lyase (PAL) enzyme activity leading to high- er total phenols and anthocyanins accumulation a l o n g w i t h h i g h e r D P P H s c a v e n g i n g c a p a c i t y . Likewise, marssonina apple blotch caused by fungus Diplocarpon mali decreased by melatonin treatment at 0.1 mM which is caused by higher H2O2 accumula- tion leading to enhancing pathogenesis related (PR) proteins accumulation such as peroxidase, chitinase and b-1,3-glucanase, and triggering phenylpropanoid pathway by enhancing phenylalanine ammonia lyase (PAL) enzyme activity (Yin et al., 2013). It has been reported that the attenuating of postharvest physio- logical deterioration in cassava roots by melatonin treatment, obtained by lower H2O2 accumulation as a result of increasing antioxidant enzymes; SOD, CAT and GR activities causing higher membrane integrity indicated by lower malondialdehyde (MDA) accumu- lation (Ma et al., 2016). Gao et al. (2016) reported that lower O-2 and H2O2 accumulation in melatonin treated peach fruits resulted from higher antioxidant enzymes SOD, CAT, APX activities, concurrent with lower lipoxygenase (LOX) enzyme activity leading to higher membrane integrity indicated by lower MDA accumulation. In addition to antifungal and antioxidant activities, melatonin is useful in increasing postharvest sensory and nutritional quality of fresh produces (Meng et al., 2015; Cao et al., 2016; Gao et al., 2016; Liu et al., 2016; Ma et al., 2016). It has been reported that pre- veraison melatonin-treated grape berries showed higher endogenous melatonin accumulation, which not only enhances berry size and weight, indicated by higher sugars accumulation and higher endogenous hormones GA/ABA ratio, but also enhances syn- chronicity of berry ripening (Meng et al., 2015). It has been observed that preharvest melatonin-treated tomato fruits showed higher fruits weight caused by higher sugars accumulation, as well as higher organic acids accumulation results in tomato fruits with favorable flavor. Moreover, higher lycopene and ascorbic acid contents were observed in preharvest melatonin-treated tomato fruits (Liu et al., 2016). Since there is a lack of knowledge about the influ- ence of melatonin on chilling tolerance of tomato fruit during low temperature storage, the present work was initiated to determine the efficacy of postharvest melatonin treatment on chilling demon- strations and enhanced fruits visual and nutritional qualities through augmenting antioxidant capacity of tomato fruits frequently encountered under cold storage. 2. Materials and Methods Fruit and treatment Tomato fruit (Lycopersicon esculentum Mill. cv Banemi) were harvested at the mature green stage (i.e., liquefying locular tissue, seeds not cut with a knife) (Saltveit, 1991) from a local producer in Mohammad Shahr, Karaj (Iran) and then immediately transported to Karaj Agricultural Engineering and Engineering Research Institute Laboratory. The green stage of maturity with homogeneous size and ran- domly allotted into three groups (100 fruits per grop) for treatment in triplicate by dipping of fruits at 0 (control), 50, 100 and 200 µM melatonin solution for Azadshahraki et al. - Postharvest melatonin treatment in tomato fruit during cold storage 301 5 min at 20°C. The selected concentrations were based on published effects of these compounds on peach, strawberries and cherry tomatoes (Sun et al., 2015; Cao et al., 2016; Soleimani Aghdam and Rezapour Fard, 2017). Following immersion, the fruits were dried for 1 h at room temperature. The tomato fruits were then put in plastic baskets, cov- ered with a perforated plastic bag to retard weight loss and stored at 5°C with 80-85% relative humidity for 4 weeks. The seven-day intervals during storage at 5°C followed by shelf life at 20°C for 1 and 3 days, the development of chilling injury and ripening char- acteristics as well as enhanced fruits nutritional qual- ity through augmenting antioxidant capacity were measured, respectively (Ding et al., 2002). Measurements of chilling injury and ripening charac- teristics Chilling injury (CI) of fruits was evaluated at 20°C for 1 day after the 7-, 14- 21 or 28-day cold-storage periods. Symptoms of tomato fruit chilling injury were manifested as surface pitting and large green patches or blotchy yellow areas resulting from loss of full red color development ability (Wang, 1993). The severity of the symptoms was assessed visually according to the following four-stage scale: 0= no pit- ting; 1= pitting covering <25% of the fruit surface; 2= pitting covering <50%, but >25% of surface; 3= pit- ting covering <75%, but >50% of surface and 4= pit- ting covering >75% of surface. The average extent of chilling-injury damage was expressed as a chilling- injury (CI) index, which was calculated using the fol- lowing formula: CI index (%) = {[(CI level) × (number of fruit at the CI level)]/(total number of fruits) × 4} × 100. For determining the effect of different treatments on ripening, fruits following 28 days storage, were incubated in diffused light at 20°C for 3 days to full r e d c o l o r d e v e l o p m e n t ( D i n g e t a l . , 2 0 0 2 ) . Measurement of full red color development in terms o f c a r o t e n o i d s a c c u m u l a t i o n w a s c o n d u c t e d . Lycopene and β-carotene were determined by the method described by Nagata and Yamashita (1992). The amount of 0.1 g of fruit tissue was mixed with 20 mL of hexane:acetone solution (3:2). An aliquot was taken from the supernatant and measured at 453, 505, 645, and 663 nm in a spectrophotometer. The content of lycopene and β-carotene was estimated using the following equations: Lycopene = -0.0458 A 663 + 0.204 A 645 + 0.372 A 505 - 0.0806 A 453 β-carotene = -0.216 A 663 - 1.220 A 645 + 0.304 A 505 + 0.452 A 453 The results were expressed in milligrams per 100 g fresh weight (mg 100 g-1 FW). Measurements of ion leakage and malondialdehyde content Ion leakage was measured at 20̊°C for 3 days after the 7-, 14-, 21- or 28- day cold-storage period accord- ing to the method of Zhao et al. (2009). 3 mm thick of mesocarp tissues were excised from equator part of 5 fruits. Disks were put into aqueous 0.1 M mannitol and shaken at 100 cycles/min for 2 h. The conductivi- ty of the solution (L1) was measured with a conduc- tivity meter. Solutions were boiled for 10 min and then cooled to 20°C. The conductivity of tissues (L2) was measured. Ion leakage was calculated as the ratio of L1 to L2. Malondialdehyde (MDA) content was measured at 20°C for 3 days after the 7-, 14-, 21- or 28- day cold-storage period using the thiobarbi- turic acid method described by Zhao et al. (2009) w i t h m o d i f i c a t i o n . A b s o r b a n c e a t 5 3 2 n m w a s recorded and corrected for nonspecific absorbance at 600 nm. The amount of MDA expressed as μmol MDA per gram of pulp. Measurement of proline content Proline content was measured at 20°C for 3 days after the 7-, 14-, 21- or 28- day cold-storage period using the acid ninhydrin method described by Shan et al. (2007). Proline in tissues was extracted with 30 mL L-1 sulfosalicylic acid at 100°C for 10 min with shaking. The extract was mixed with an equal volume of glacial acetic acid and acid ninhydrin reagent and boiled for 30 min. After cooling, the reaction mix was partitioned against toluene and the absorbance of the organic phase was recorded at 520 nm. The resulting values were compared with a standard curve constructed using known amounts of proline and expressed as μg proline g−1 fresh weight (FW). Measurements of O2 - production rate and H2O2 con- tent The O2- production rate and H2O2 content were measured at 20°C for 3 days after the 7-, 14-, 21- or 28- day cold-storage period. O2- production was mea- sured using the method of Elstner (1976) with modifi- cation. 4 g of fruit tissue was homogenized with 5 ml of 50 mM phosphate buffer (pH 7.8) and then cen- trifuged at 8000×g for 20 min at 4°C. The supernatant was used for the determination of O2- production and expressed as nmol g-1 FW min-1. For H2O2 measurement, 2 g of fruit tissue was homogenized with 5 ml of cold acetone and then centrifuged for 15 min at 8000×g at 4°C, the super- natant was collected immediately for H2O2 analysis according to the method of Patterson et al. (1984). Adv. Hort. Sci., 2018 32(3): 299-309 302 H2O2 content was expressed as nmol g -1 FW. Enzyme extraction and analysis Enzyme activities were measured at 20°C for 3 days after the 7-, 14-, 21- or 28- day cold-storage period. 5 g of fruits tissue were homogenized with 50 mmol/L phosphate buffer (pH 7) containing 0.2 mmol/L EDTA and 2% PVP. The homogenate was cen- trifuged at 12,000×g for 20 min at 4°C and the super- natant was used. SOD (EC 1.15.1.1) activity was determined according to Giannopolitis and Ries (1977) with modification. One unit of SOD activity was defined as enzyme that caused 50% inhibition of nitro blue tetrazolium reduction by recording the absorbance at 560 nm. According to Zhang et al. (2013) with modification, 1 unit of CAT (EC 1.11.1.6) activity was defined as 0.01 decrease in absorbance at 240 nm per min. POD (EC 1.11.1.7) activity was determined according to Maehly and Chance (1954) with modification. One unit of POD was defined as 0.01 increase of absorbance at 470 nm as a result of guaiacol oxidation. Statistical analysis Experiments were performed using a completely randomized design. All statistical analyses were per- formed with SAS 9.2 software package. Data were analyzed by one-way analysis of variance (ANOVA). Mean comparisons were performed using HSD in Tukey’s test for comparing treatment group at level of 1% (P<0.01) on three biological replicates. 3. Results Chilling injury and ripening characteristics The chilling injury (CI) symptoms were expressed on control group as surface pitting and large green patches or blotchy yellow areas resulting from loss of full red color development ability (Wang, 1993), only after 7-day of cold storage and following shelf life at 20°C for 1 and 3 days, respectively (Fig. 1). No signifi- cant difference in CI was observed between the control and 50 μM melatonin-treated fruit. Whereas, melatonin-treated groups with 100 or 200 μM under- went normal ripening at 20°C and only few visual chilling-injury symptoms were observed after 14 days storage at 5°C (Fig. 1). Fruits treated with 100 or 200 μM melatonin maintained the same quality as before chilling-temperature storage except for developing a slight yellow pigmentation, and the effect of the used formulas increased with increasing their concentra- tions (Fig. 2A). The results indicate that a 14-day stor- age was the maximum that could be tolerated by untreated mature green fruit. In this experiment, treatments with higher concentrations (100 or 200 μM) of melatonin were more effective in protecting against chilling injury than lower concentration (50 μM). For examining the effect of melatonin treatment on color development of fruit after cold storage, mature green tomatoes were transferred to 20°C for 3 days for ripening. Treatment with 100 or 200 μM melatonin, prior to 5°C storage, was effective at alle- viating chilling injury; this treatment category result- ed in normally fruit ripening and uniform red color development caused by significantly (P<0.01) more lycopene and β-carotene accumulations, and the effect of the used formulas on fruit ripening and eventually visual quality increased with increasing their concentrations (Fig. 2). However, control and melatonin-treated group with 50 µM failed to devel- op the normal red color, with lower lycopene and β- carotene values, demonstrated by large green patch- es or blotchy yellow areas. Interaction effects and time of storage had no meaningful influence on these traits. Ion leakage, malondialdehyde content Ion leakage and MDA, as a consequence of mem- brane damage, are credible parameters for CI devel- opment and degree for postharvest tomato fruit Fig. 1 - Chilling injury (CI) index (%) of tomato fruits treated with 100 and 200 µM during storage at 5°C and after 1 day of shelf life. All data are presented as a mean of three biolo- gical replicates, and vertical bars indicated the standard errors. Different letters indicate significant differences at P<0.01. Azadshahraki et al. - Postharvest melatonin treatment in tomato fruit during cold storage 303 (Zhao et al., 2009). In this experiment, significantly the highest ion leakage was detected in control group (P<0.01) (Fig. 3A). However, no significant dif- ferences were statistically found in the ion leakage incidence between melatonin-treated groups with 100 or 200 μM. As shown in figure 2B, MDA content showed a similar pattern of change during storage. MDA content was significantly (P<0.01) lower in melatonin-treated groups with 100 or 200 μM com- pared with control at the same time of cold storage, and the highest level observed about 14 to 21 days (Fig. 3B). Proline content There was a peak of proline content appearing in 14-day in all groups, which suggested that low tem- perature induced the proline synthesis mechanism in fruits (Zhao et al., 2009). However, proline accumula- tion was about 2 times high in the melatonin-treated groups with 100 and 200 µM compared to control from 14-day to the end of storage period, and the effect of the used formulas on proline content increased with increasing their concentrations (Fig. 4). No significant difference in proline content was observed between the control and 50 μM melatonin- treated fruit. Fig. 2 - Fruit ripening uniformity (%), lycopene (A) and β-carote- ne (B) contents of tomato fruits treated with 100 and 200 µM during storage at 5°C and after 3 days of shelf life. All data are presented as a mean of three biological replicates, and vertical bars indicated the standard errors. Different letters indicate significant differences at P<0.01. Fig. 3 - Ion leakage (%) (A) and MDA (B) content of tomato fruits treated with 100 and 200 µM melatonin during storage at 5°C and after 3 days of shelf life. All data are presen- ted as a mean of three biological replicates, and vertical bars indicated the standard errors. Different letters indi- cate significant differences at P<0.01. 304 Adv. Hort. Sci., 2018 32(3): 299-309 O2 - production and H2O2 content In figure 5, the measured levels of O2- and H2O2 were shown as an influence of low temperature to ROS generation in fruits exposure to chilling stress. C o n t e n t s o f O 2 - a n d H 2O 2 r e m a i n e d r e l a t i v e l y unchanged in control and melatonin-treated groups within the first 14 days of cold storage. Thereafter, both O2- and H2O2 contents increased rapidly, treat- ment with 100 and 200 µM melatonin significantly (P<0.01) restrained the enhancement of O2- and H2O2 contents, and again the effect of the used formulas on O2- production rate and H2O2 content decreased with increasing their concentrations (Fig. 4). No sig- nificant difference in O2- and H2O2 contents was observed between the control and 50 μM melatonin- treated fruit. SOD, CAT, POD activities As depicted in figure 6A, the SOD activity in both control and melatonin-treated groups steadily increased during storage, nonetheless significantly the highest SOD activity was observed in melatonin treated groups with 200 and 100 µM throughout the storage, respectively (P<0.01). The changes of CAT and POD activities in tomato fruit showed a similar pattern during the cold storage. The activities of both enzymes in control and melatonin-treated groups oscillatory increased with storage time. Melatonin treatment significantly promoted the increases in activities of CAT and POD, the activities of both enzymes were significantly higher (P<0.01) in these groups than those in control group during the whole storage (Fig. 6). 4. Discussion and Conclusions Little information is available on melatonin treat- ment of horticultural commodities, even though there are many reports suggesting that melatonin is an endogenous signaling molecule for the activation of certain plant defense responses and the onset of the tolerance has often been correlated with the accumulation of defense-related enzymes and com- pounds (Zhang et al., 2014; Arnao and Hernández- Fig. 4 - Prolin content of tomato fruits treated with 100 and 200 µM melatonin during storage at 5°C and after 3 days of shelf life. All data are presented as a mean of three bio- logical replicates, and vertical bars indicated the stan- dard errors. Different letters indicate significant differen- ces at P<0.01. Fig. 5 - O2- Production (A) and H2O2 (B) content of tomato fruits treated with 100 and 200 µM during storage at 5°C and after 3 days of shelf life. All data are presented as a mean of three biological replicates, and vertical bars indicated the standard errors. Different letters indicate significant differences at P<0.01. 305 Azadshahraki et al. - Postharvest melatonin treatment in tomato fruit during cold storage Ruiz, 2015; Liu et al., 2016). Exogenous melatonin application has been shown to result in an improved chilling tolerance and reduced incidence of chilling injury in peach and strawberry fruits (Cao et al., 2016; Soleimani Aghdam and Rezapour Fard, 2017). In this experiment, we found that melatonin treat- ment could effectively not only reduce development of surface pitting on the fruit and irregular ripening and full red color development (large green patches or blotchy yellow areas), the typical chilling injury symptoms in tomato fruit, but also enhance fruits nutritional quality. This indicates that postharvest treatment with melatonin increased chilling toler- ance in tomato fruit. Since melatonin treatment is easy to set up, inexpensive and safe, even if higher amounts are accumulated in the plant (Tan et al., 2012), it could be a functional method to decrease chilling injury, maintain quality and prolong shelf life of tomato fruit. Carotenoids, highly characteristic phytochemicals, known to be potent ROS scavengers and antioxi- dants, act as a cell proliferation inhibitor and hinder- ing of cancer cell growing (Tijskens and Evelo, 1994; Levi et al., 2001; Giovannucci et al., 2002; Stahl and Sies, 2005). During maturation/ripening, the green pigment chlorophyll degrades and carotenoids are synthesized. Carotenoids, particularly lycopene and β-carotene, represent the primary components of ripe fruit pigmentation in tomato pericarp and are responsible for the characteristic color of ripe toma- toes, conferring deep red and orange colors, respec- tively. These carotenoids largely influence flavor and nutritional qualities as well as commercial value and enhances consumer acceptance of fresh tomato fruit (Tijskens and Evelo, 1994). In this study, higher accu- mulation of lycopene and β-carotene in melatonin treated groups with 100 and 200 µM not only con- tributed to alleviate chilling injury to fruit, but also lead to normally fruit ripening with uniform red color development (panels B and C of Fig. 2). It has been reported that in tomatoes, the contents of lycopene and β-carotene increase from the green to the fully ripe stage (Fraser et al., 1994). Melatonin may affect directly or indirectly other carotenoid genes and/or enzymes in tomato fruit. This could be the case for example of lycopene cyclases, which is responsible for the formation of β-carotene from lycopene, which its accumulation is a ripening-related event in tomato (Giovannoni, 2001). The higher levels of these compounds in melatonin-treated red ripe fruits may be associated with a general acceleration in ripening and with some of the associated transcrip- tional events, leading to the color change of tomato fruit (Guo, 2015; Sun et al., 2015). Therefore, the improved capability of full red color development in chilling-faced melatonin-treated group is one of the most important outcome of this study for the quality of tomato. Proline, an important amino acid, has been con- sidered as a cellular osmotic regulator, protein stabi- lizer, free-radical scavenger, and lipid peroxidation inhibitor in plant (Sun et al., 2015). The elevated level Fig. 6 - Superoxide dismutase (A), Catalase (B) and peroxidase activities (C) of tomato fruits treated with 100 and 200 µM during storage at 5°C and after 3 days of shelf life. All data are presented as a mean of three biological replica- tes, and vertical bars indicated the standard errors. Different letters indicate significant differences at P<0.01. 306 Adv. Hort. Sci., 2018 32(3): 299-309 of proline found to be associated with improved cold tolerance in chilling-sensitive plants (Zhao et al., 2009; Shang et al., 2011; Zhang et al., 2013; Cao et al., 2016). Our findings are in agreement with the above reports (Zhang et al., 2010), because a signifi- c a n t l y ( P < 0 . 0 1 ) h i g h e r c o n t e n t o f p r o l i n e w a s observed in melatonin-treated tomato fruits with 100 or 200 µM during the whole storage period along with the reduced CI incidence (Fig. 4). Cao et al. (2016) reported that higher transcripts of PpP5CS and PpOA were observed in melatonin-treated peach fruits which provokes proline accumulation. Zhao et al. (2009) claimed that proline levels in a tissue may be an effective indicator for CI analysis in postharvest tomato fruits. The reduction of cell energy and/or induction of alterations in membrane integrity are occurred in chilling-sensitive horticultural commodities at low temperatures. Reducing scavenging potency through such factors as chilling-related inactivation of antioxi- dants and/or obstructed antioxidant turnover may result in the enhanced ROS generation. Chilling tem- peratures destroy the balance between ROS forma- tion and defense mechanisms which cause oxidative- ly chilling injury and consequent cellular damage (Hodges et al., 2004). It is figured that antioxidant enzymes, SOD, CAT, and POD are the primary enzy- matic scavenging mechanism of ROS that contribute to attenuate chilling injury to fruit (Sala and Lafuente, 2000; Mondal et al., 2004; Ding et al., 2007; Imahori et al., 2008). Thus, this balance between the genera- tion and scavenging of ROS is crucial to cell survival during cold storage and is thought to be a major mechanism of resistance to chilling stress. It has been reported that in harvested commodities enhanced e n z y m a t i c a n t i o x i d a n t a c t i v i t i e s r e s u l t i n t h e improved chilling tolerance. A higher antioxidant enzyme activity was indicated in the chilling-tolerant mandarins compared with the chilling sensitive culti- vars (Sala, 1998). In many other studies, enhance- ment of antioxidant enzyme activity through a num- ber of postharvest treatments (e.g. heat shock, low temperature conditioning and superatmospheric oxy- gen treatment) provoked chilling tolerance and alle- viated chilling injury to fruit (Wang, 1995; Sala and Lafuente, 2000; Zheng et al., 2008). Neutralizing of the O2- by SOD is the initial step of cell defense against free radicals (Bowler et al., 1992). CAT is one o f t h e e n z y m e s t h a t p r o t e c t c e l l s a g a i n s t R O S because it catalyzes the decomposition of H2O2 to form O2- and H2O2 (Imahori et al., 2008). POD cat- alyzes H2O2 dependent oxidation of substrate (Fu and Huang, 2001). In the present work, the higher increases in activities of SOD, CAT, and POD concur- rent with reduced O2- and H2O2 content in melatonin- treated groups than those in control group were indi- cated (Fig. 6). While, the levels of H2O2 and O2- signifi- cantly increased during the development of irre- versible chilling injury symptoms surface pitting and irregular ripening and full color development in con- trol group. Treatment with melatonin significantly alleviated these chilling-induced damages and increased the activities of SOD, CAT, and POD under cold stress. The increased SOD activity could enhance the ability of the fruit to dismutate superoxide radi- cals, while the increases in CAT and POD activities would contribute to the stronger omitting of hydro- gen peroxide (Lukatkin, 2002), which may give an explanation for the lower levels of O2- and H2O2 observed in melatonin-treated groups. These results suggest that effect of melatonin in reducing the inci- dence of chilling injury was correlated to enhanced enzymatic scavenging mechanism of ROS. In mela- tonin-treated tomato fruits the continues functions of SOD, CAT, and POD may be associated with higher stress resistance and eventually extended shelf life. Membrane lipid peroxidation may be one of the first events in the manifestation of CI, in which phase MDA as a final product of polyunsaturated fatty acid oxidation was produced and damaged to cell mem- brane, resulted in ion leakage (Lukatkin, 2002; Imahori et al., 2008). As depicted in panels A and B of figure 3, the increase in ion leakage and MDA from 14-day of storage period indicates that cold storage caused a distinct deterioration of membrane integrity and activation of lipid peroxidation in the non-treat- ed control group, which could be attributed to the decreases in SOD and CAT activities as well as in antioxidant compounds including lycopene, β- carotene and proline. These reductions induced by chilling stress favor accumulation of O2- and H2O2, which can result in lipid peroxidation. Ion leakage and MDA content may be a reflection of CI develop- ment and fruit cold tolerance (Zhao et al., 2009). Furthermore, Posmyk et al. (2005) reported that ion leakage intensity and MDA content in a tissue can be a reliable indicators of the structural integrity of the membranes of plants exposed to low temperature. Given to these results, prevention of MDA accumula- tion and subsequent ion leakage by melatonin treat- ment could be related to a low degree of lipid peroxi- dation, which could result from the maintenance of high enzymatic and non-enzymatic antioxidants. It has been reported that melatonin efficiently con- 307 Azadshahraki et al. - Postharvest melatonin treatment in tomato fruit during cold storage tributes to membrane integrity maintenance, and in turn, alleviates symptoms and severity of CI (Cao et al., 2016; Soleimani Aghdam and Rezapour Fard, 2017). Treatment with melatonin attenuated chilling induced shrinkage and disruption of carrot cell plas- ma (Lei et al., 2004). As a whole, the results of this study show that melatonin treatment can effectively enhance chilling tolerance and reduce chilling injury of tomato fruit. 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