173 1. Introduction Tomato as a plant indigenous to tropical regions is sus- ceptible to chilling injury when subjected to low tempera- ture storage (Saltveit, 2001). Chilling injury limits tomato storage life and leads to significant degradation of fruit quality and decreases the market value. It can increase membrane permeability, and a resultant increase in leakage of ions from cell membrane, surface pitting, susceptibility to decay and diseases, weight losses, abnormal ripening, change in respiration, ethylene production and senescence. Chilling injury symptoms mainly develop during shelf life following cold storage (Candan et al., 2007). Numerous attempts, such as breeding for increased chilling tolerance, genetic engineering, modifying crop management practices and application of chemicals, have been made to increase chilling tolerance and avoid chilling injury (Baninasab, 2009). Chilling alleviation in fruits and vegetables has been attributed to several factors including accumulation of polyamines, nitric oxide and proline (Agh- dam and Bodbodak, 2013). Over the years, studies have shown the involvement of polyamines (PAs) in a wide array of processes in plants, ranging from triggering organogenesis to protect- ing against stress (Walden et al., 1997). PA accumulation occurs under abiotic stresses including drought, salinity, extreme temperatures, UV-B, heavy metals, mechanical wounding and herbicide treatment (Hussain et al., 2011). Amongst different kind of PAs, the diamine putrescine (Put), triamine spermidine (Spd), and tetramine spermine (Spm) are the most common PAs in plant cells, while oth- ers are of more limited occurrence (Galston and Sawhney, 1990; Valero et al., 2002). Distribution of these biogenic amines differs between species with Put and Spd being particularly abundant and Spm the least abundant in plant cells. These amines are important for cell viability and their intracellular levels are tightly regulated, making it difficult to characterize individual effects of Put, Spd and Spm on plant growth and developmental processes (Mat- too et al., 2010). It has been reported that exogenous PA application leads to an inhibition of ethylene emission rate in the climacteric fruits (Valero et al., 2002) and delays ripening and fruit abscission (Paksasorn et al., 1995). On the other hand, PAs are precursors of many important secondary metabolites and changes in PA biosynthesis appear to have a reciproca1 effect on ethylene biosynthesis (Walden et al., 1997). Ac- cumulation of Put in tissues seems to be a general response of plants to chilling temperatures (Faust and Wang, 1992). Accordingly, we hypothesized that exogenous application of PAs may have effects on the postharvest physiology of fruits, especially on chilling tolerance. An experiment was arranged to study the effects of exogenous PA application Post-storage quality and physiological responses of tomato fruits treated with polyamines J. Javanmardi(1), M. Rahemi, M. Nasirzadeh Department of Horticultural Sciences, College of Agriculture, Shiraz University, Shiraz, Iran. Key words: chilling injury, electrolyte leakage, fruit quality, vitamin C. Abstract: Two greenhouse F1 tomato cultivars, M19 and M79, were grown hydroponically and the mature green fruits were harvested and subjected to eight polyamine (PA) treatments including 1 and 2 mM putrescine (Put), spermidine (Spd) and their combination before being placed at 3°C for 15 and 25 days. Electrolyte leakage, weight loss, fruit firm- ness, decay percentage, chilling injury index, titratable acidity, total soluble solid content and ascorbic acid content were then measured after keeping at 20°C for 3 days and compared to control. The Put:Spd (2:2 mM) treatment decreased electrolyte leakage (over 50%), chilling injury index and fruit decay percentage. Combinations of PAs caused greater total soluble solids and greater effect on decreasing weight loss during storage when compared to their sole PA applica- tion. PAs caused a net increase in fruit firmness during post-harvest life. Titratable acidity increased with increasing duration of low temperature storage for all treatments. Ascorbic acid in fruits stored at low temperature for 25 days was greater than those stored for 15 days. The effects of exogenous PAs on reducing chilling-related disorders decreased with time. Correlations among weight loss, electrolyte leakage, chilling injury, decay percentage and fruit firmness during low temperature storage were positive and significant, but they were non-significant or significantly negative when compared against ascorbic acid, titratable acidity and TSS. Adv. Hort. Sci., 2013 27(4): 173-181 (1) Corresponding author: javanm@shirazu.ac.ir Received for publication 4 January 2014 Accepted for publication 7 March 2014 174 on the level of chilling injury, ripening, shelf life and some quality factors of tomato fruit (as a model plant) after a pe- riod of low temperature storage. Another goal of the study was to determine which polyamine, concentration and/or their combination is more effective. Two greenhouse to- mato cultivars were selected to evaluate whether PAs play the same role in different cultivars or not. 2. Materials and Methods Plant material and polyamine treatments F1 hybrid seeds of tomato (Solanum lycopersicom L.) cultivars M19 and M79 (Tropica Seeds, IndoSem Ltd. In- dia) were sown in 30 × 30 × 60 mm/cell plastic plug trays filled with peat and perlite (1:1 v:v). Seedlings were grown for five weeks in a polycarbonate greenhouse (25/21°C and 60-70% relative humidity). Plugs were then transplanted into 12-l pots filled with a peat:perlite (60:40 v/v) mix- ture. Pots were kept in the same greenhouse with an aver- age 60% relative humidity and 25±5°C temperature until the end of the experiment. Cultural practices consisting of standard recommendations for growing tomato seedlings and plants in a hydroponic system were according to Papa- dopoulos (1991). Fruits for study were harvested at mature green stage based on the “Color Classification Require- ment in United States Standards for Grades of Fresh To- matoes” chart, published by the USDA. Eight polyamine treatments consisted of immersing same-sized, selected mature green fruits in 1 and 2 mM putrescine (Put), spermidine (Spd) and their combina- tion for 4 min as indicated by Mirdehghan et al. (2007 a). Immersion in distilled water for the same period was taken as control (Table 3). Fruits were then held at 3°C in a temperature-controlled chamber in darkness with relative humidity of 90%. After 15 and 25 days, five fruits from each treatment replicate were sampled and stored at 20°C for 3 days. Electrolyte leakage, weight loss, fruit firmness, decay percentage, chilling injury index, titratable acidity, total soluble solid content (TSS) and ascorbic acid content of fruits were measured as described below. Electrolyte leakage Electrolyte leakage was used to assess membrane per- meability. The procedure was based on Lutts et al. (1996) with slight modification. Briefly, five tomato fruit pericarp discs (10 mm diameter) per replicate from randomly chosen fruits were taken and placed in test tubes containing 10 mL of distilled water followed by three washes with distilled water to remove surface contamination. Samples were incu- bated at room temperature on a shaker for 24 h. Electrical conductivity (EC) of the bathing solution (EC1) was read after incubation. The samples were then placed in a boiling water bath for 20 min and the second reading (EC 2) was determined after cooling the solution to room temperature. The electrolyte leakage was calculated using EC1/EC2 and expressed as a percentage. All leakage data were expressed as a percentage of the total electrolyte readings. Weight loss Weight loss during postharvest storage of individual replicates was determined by subtracting sample weights on sampling dates (day 15 or 25 of low temperature stor- age) from their initial weight on day 0 and presented as percent of weight loss compared to initial weight. Fruit firmness Fruit firmness was determined according to Ben-Ye- hoshua et al. (1983). A compression tester using a 1 kg weight centered over a locule on the equatorial region of each tomato fruit was used. Full deformation was mea- sured 30 s after exerting the force on the fruit, then the weight was removed and residual deformation was mea- sured 15 s later. Lower readings denoted firmer fruit. Five fruits were measured for each treatment replicate. Firm- ness was expressed as mm deformation. Chilling injury index Chilling injury (CI) was evaluated according to Ding et al. (2002) at 20°C for 3 days, following 15- and 25- day low temperature storage period. Briefly, tomato fruit surface pitting was considered as CI symptom. The sever- ity of CI symptoms was assessed visually according to a four-stage scale: 0= no pitting; 1= a few scattered pits; 2= pitting covering up to 5% of the fruit surface; 3= extensive pitting covering 5-25% of the fruit surface, and 4= exten- sive pitting covering more than 25% of the fruit surface. The average extent of CI damage was expressed as a CI index and calculated using the following formula: CI index = ∑(injury classification level x number of fruit at that level) total number of friut at the treatment Decay percentage Extent of decay was assessed according to González- Aguilar et al. (2000), based on the area of decay and the surface area with microorganisms growing on it. Decay was rated for each replicate of treatments at the end of the 15- and 25-day cold storage periods after an additional 3 days at 20°C. The values were expressed as decay percentage. Total soluble solids, titratable acidity and ascorbic acid content Total soluble solids concentrations (TSS) of fruit juice were determined using a digital refractometer (Pal-3, ATAGO Co., Ltd. Tokyo, Japan) at 20°C and presented as °Brix. Fruit juice titratable acidity (TA) was determined by titration of 1 mL juice in 25 mL distilled water with 0.1N NaOH until the pH reached 8.1, according to El Ghaouth et al. (1992). Results were expressed as gram of citric acid equivalent per 100 g fresh weight (g CAE/100 g fw). Fruit ascorbic acid content was determined according to the method described by the AOAC (1984). Statistical analysis The experiment was arranged in a completely random- 175 ized design. Each polyamine treatment consisted of three replicates; each replicate consisted of 10 plants. Data were analyzed separately for tomato cultivars and days of low temperature incubation using one-way analysis of vari- ance. Means for each low temperature storage duration and cultivar were compared separately using the least sig- nificant differences (LSD) test at p≤0.01. All data analy- ses including correlation analysis were performed using SPSS21 (SPSS Inc., Chicago, IL) computer software for Windows. 3. Result and Discussions One-way analysis of variance showed significant dif- ferences for all measured characteristics in both cultivars except for ascorbic acid content in cv. M79 after 15 days of low temperature storage (Table 1 a, b, and Table 2 a, b). Electrolyte leakage Application of PAs decreased electrolyte leakage of low-temperature incubated tomato fruits. The highest Table 1 a - One way analysis of variance for characteristics of polyamine-treated tomato cultivar M19 after 15 and 25 days storage at 3°C SOV df Mean Squares Electrolyte leakage Weight loss Fruit firmness Chilling injury 15 25 15 25 15 25 15 25 Polyamines 8 88.692** 163.37** 5.193** 3.055** 0.755** 0.87** 114.593** 65.167** Error 18 3.751 3.731 0.161 0.283 0.051 0.047 3.625 16.759 Total 26 29.887 52.848 1.709 1.136 0.268 0.3 37.767 31.654 ns, *, ** non-significant and significant at 0.05 and 0.01, respectively. Table 1 b - One-way analysis of variance for characteristics of polyamine-treated tomato cultivar M19 after 15 and 25 days storage at 3°C SOV df Mean Squares Decay percentage TSS Titratable acidity Ascorbic acid 15 25 15 25 15 25 15 25 Polyamines 8 370.37 ** 902.759 ** 0.832 ** 0.592 ** 0.028 ** 0.033 ** 1.697 ** 15.319 ** Error 18 37.037 65.852 0.167 0.079 0.002 0.003 0.185 0.999 Total 26 139.601 323.362 0.369 0.236 0.01 0.012 0.650 5.406 ns, *, ** non-significant and significant at 0.05 and 0.01, respectively. Table 2 a - One-way analysis of variance for characteristics of polyamine-treated tomato cultivar M79 after 15 and 25 days storage at 3°C SOV df Mean Squares Electrolyte leakage Weight loss Fruit firmness Chilling injury 15 25 15 25 15 25 15 25 Polyamines 8 48.792** 101.07** 4.593** 4.708** 0.564** 0.749** 80.624** 135.995** Error 18 0.948 0.673 0.034 0.087 0.017 0.046 5.701 10.417 Total 26 15.669 31.564 1.437 1.509 0.185 0.262 28.754 49.056 ns, *, ** non-significant and significant at 0.05 and 0.01, respectively. Table 2 b - One-way analysis of variance for characteristics of polyamine-treated tomato cultivar M79 after 15 and 25 days storage at 3°C SOV df Mean Squares Decay percentage TSS Titratable acidity Ascorbic acid 15 25 15 25 15 25 15 25 Polyamines 8 193.667** 424.833** 0.431** 0.951** 0.021** 0.029** 1.043ns 3.987** Error 18 13.852 29.667 0.026 0.029 0.003 0.002 0.989 0.822 Total 26 69.179 151.256 0.150 0.313 0.009 0.01 1.005 1.795 ns, *, ** non-significant and significant at 0.05 and 0.01, respectively. 176 electrolyte leakage was observed in control treatments as almost 100% greater than those treated with the highest PA concentrations in combined treatments on both culti- vars (Table 3). Pretreatment of cucumber plants with PAs diminished the increased electrolyte leakage caused by chilling in the leaves (Gill and Tuteja, 2010). The exoge- nous application of polyamines on pomegranate (Punica granatum L.) protected the membrane lipid from being converted from liquid crystalline to a solid-gel state (in- duced by chilling) through preventing lipid peroxidation (Mirdehghan et al., 2007 b). Previously, Put has been re- ported to act as protective toward cold stress in tomato plants, since reduced cold-induced electrolyte leakage in leaves due to its application was observed (Kim et al., 2002). In addition, PA application induced cold ac- climation through maintenance of membrane fluidity at low temperatures and reduced electrolyte leakage, skin browning, and thus the severity of CI symptoms (Mird- ehghan et al., 2007 a). In general, the effect of Spd on decreasing electrolyte leakage was significantly greater than Put but the differ- ences were not significant when they were applied in com- binations (Table 3). In agreement with our results, Gill and Tuteja (2010) found different patterns of Put and Spd ac- tion in different cucumber cultivars. Accordingly, it seems the mode of action of PAs may differ within species. A large amount of evidence showed that exogenous application of PAs plays a role in stabilizing plant cell membranes and protecting them from damage under stress conditions (Liu et al., 2007; He et al., 2008; Gill and Tuteja, 2010). PAs in their free forms have been de- scribed as anti-senescence agents (Valero et al., 2002) due to their capacity to preserve membrane stability, which is crucial in plant adaptation to temperature stress- es (Oufir et al., 2008). Their attachment to membranes by way of phospholipids results in altered patterns of solute permeation through those membranes and decreased flu- idity of membrane components (Galston and Sawhney, 1990). They are involved in the regulation of many basic cellular processes, including cellular cation-anion bal- ance and membrane stability (Gill and Tuteja, 2010). In the present experiment, tomato cv. M19 showed greater electrolyte leakage for all PA and low temperature stor- age treatments compared to cv. M79 (Table 3). Possibly the PAs pattern of action in the studied tomato cultivars was at different rates, as previously found for cucumber cultivars (Gill and Tuteja, 2010). Increasing the duration of low temperature storage from 15 to 25 days increased electrolyte leakage in both cultivars (Table 3). This could be an indicator that the ef- fects of exogenous PAs on lowering electrolyte leakage decrease with time. Weight loss It has been reported that tomatoes at room temperature showed greater weight loss than those stored in cold stor- age (Javanmardi and Kubota, 2006). At least 50% greater weight loss was found in control treatments compared to those with PA applied in both cultivars and low tempera- ture durations (Table 3). Transpiration has been considered the main cause of weight loss during tomato storage (Ja- vanmardi and Kubota, 2006). Reduction in weight loss and respiration rate due to Put and Spd application in mango has been reported (Malik and Singh, 2005). The differences between combined PA treatments were not significant in the studied cultivars for the two low tem- perature durations, however they showed less weight loss when PAs were applied singularly (Table 3). It has been reported that chilled fruits had a greater weight loss rate than non-chilled fruits after transfer to non-chilling conditions. This is due to the development of microscopic cracks in peel tissue (Cohen et al., 1994), cellular breakdown and loss of membrane integrity which have an important role in water exchange through the rind (González-Aguilar et al., 2000). Storage conditions or treatments that reduce fruit water loss have been shown Table 3 - Effect of polyamine application and duration (15 and 25 days) of low-temperature (3°C) storage on electrolyte leakage and fruit weight loss in tomato cultivars M19 and M79 Treatment Electrolyte leakage Weight loss M 19 M 79 M 19 M 79 15 25 15 25 15 25 15 25 Control 30.06 a 42.43 a 24.40 a 35.64 a 5.48 a 5.22 a 5.27 a 5.87 a Put 1 mM 19.55 b 24.96 b 15.43 b 20.13 b 3.13 b 3.27 b 2.52 b 3.22 b Put 2 mM 18.50 b 22.77 bc 14.20 b 19.78 b 2.54 bc 2.90 bcd 2.40 b 2.86 bc Spd 1 mM 16.43 bc 22.98 bc 14.20 b 19.47 bc 2.40 cd 3.17 bc 2.14 bc 2.96 b Spd 2 mM 14.93 bc 21.83 bc 13.91 b 17.73 bc 1.92 cde 2.48 bcd 1.90 cd 2.40 cd Put 1 mM + Spd 1 mM 15.56 bc 21.31 bc 13.13 bc 18.81 bc 1.69 de 2.23 cd 1.59 de 2.20 de Put 1 mM + Spd 2 mM 14.98 bc 21.99 bc 12.97 bc 17.74 bc 1.61 de 2.13 d 1.61 de 2.00 de Put 2 mM + Spd 1 mM 11.45 c 18.19 c 10.32 c 17.31 c 1.19 e 2.17 cd 1.19 e 1.93 de Put 2 mM + Spd 2 mM 13.01 c 18.05 c 11.41 c 17.30 c 1.40 e 1.97 d 1.27 e 1.79 e LSD value 0.01 4.55 4.54 2.29 1.92 0.94 1.25 0.43 0.69 Means in columns followed by the same letter are not significantly different, P ≤ 0.05, LSD test. Means for each column were compared separately. 177 to alleviate CI (Wang, 1993). In our experiment, PA appli- cation resulted in less membrane permeability (less elec- trolyte leakage) and therefore less water loss than control fruits. Also storage duration affected weight loss: the lon- ger fruits remained in low-temperature storage, the greater their weight loss. Fruit firmness Fruit firmness was affected by PA application (Table 4). All PA-treated fruits (except for Put 1 mM after 15 days of storage in M19) showed firmer fruit (less com- pression) than control fruits (Table 4). The differences between combined PA treatments in each low tempera- ture storage duration were not significant. However, when compared to the control they showed at least 43 and 69% greater firmness in M19 tomato, and 80 and 45% in M79 tomato for 15 and 25 days storage, respec- tively. It has been shown that fruits and vegetables in- filtrated with PAs had a net increase in firmness during post-harvest life. This effect of PAs on fruit firmness has been attributed to the cross linking to the COO- group of the pectic substance and changes in polygalacturonic acids in the cell wall (Valero et al., 2002). Retarded fruit softening due to Put and Spd application in mango has been reported (Malik and Singh, 2005). Although it is believed that the overall softening process results from a number of changes in turgor pressure, cell wall and mem- brane composition and degradation, but cell wall modifi- cations have been implicated to be the major determinant of fruit softening (Smith et al., 2002). Put and Spd have anti-senescence properties (Saftner and Baldi, 1990), and are able to retard the maturation process (including soft- ening) in a wide range of climacteric and non-climacteric fruits (Valero et al., 2002). The ethylene production in tomato fruits, enhances softening, but its effect may de- crease due to increased polyamine level (Tiecher et al., 2013). Increased duration of low temperature storage decreased fruit firmness for all treatments. Fruit firm- ness of control plants M19 and M79 showed 52 and 56% decrease, respectively, when 15 days of low temperature storage values were compared width 25 days. The values obtained from 2.2 mM, Put:Spd tratment were 108 and 148% for M19 and M79, respectively (Table 4). PAs with higher number of available cations have greater effect on fruit firmness, as Spm+4>Spd+3>Put+2 (Valero et al., 2002). According to our results, the PA concentration would also affect the fruit firmness. Chilling injury index Chilling injury index drastically increased with time of low temperature storage in both cultivars (Table 4). The increased percentages of 25 days compared to 15 days of low temperature storage for the control treatment were 64 and 102% in M19 and M79, respectively. The combined PA treatments resulted in greater CI index (but less than control) after 25 days of low temperature stor- age than 15 days, especially when 2:2 mM Put:Spd was used (Table 4). The effects of PA on 15 days low temper- ature stored tomatoes showed significant decrease in CI index, but its impact for a longer period (25 days) was not significant, except for 1 mM Put (Table 4). The lowest CI index after 15 days of low temperature storage was found in Put:Spm (2:2 mM) treated tomatoes in both cultivars (Table 4). It is possible that the impact of PAs on lower- ing CI index in tomato are time as well as species depen- dent. Pre-storage application of PAs improved shelf-life of pomegranate (Punica granatum L.) stored at chilling temperature by increasing endogenous polyamine levels (Mirdehghan et al., 2007 a). Although increased endog- enous Put in tomato due to low temperature storage is considered to act as protective toward cold stress, the reported mechanism is unclear (Gonzalez-Aguilar et al., 1998). The involvement of polyamines in reducing chill- ing injury has been related to reducing oxidative damage via increases of antioxidant or reducing the activity of oxidative enzyme (Oufir et al., 2008). Table 4 - Effect of polyamine application and duration (15 and 25 days) of low-temperature (3°C) storage on fruit firmness and chilling injury index in tomato cultivars M19 and M79 Treatment Fruit firmness (mm deformation) Chilling injury index (%) M 19 M 79 M 19 M 79 15 25 15 25 15 25 15 25 Control 2.41 a 3.67 a 2.19 a 3.43 a 19.17 a 31.50 a 16.42 a 33.33 a Put 1 mM 2.04 ab 2.95 b 1.69 b 2.85 b 16.67 ab 25.67 ab 13.33 ab 20.00 b Put 2 mM 1.82 b 2.48 c 1.45 c 2.52 bc 15.50 b 24.17 abc 9.17 bc 16.67 bcd Spd 1 mM 1.88 b 2.49 c 1.54 bc 2.79 b 9.17 c 21.67 bc 10.00 bc 17.50 bcd Spd 2 mM 1.68bc 2.24 cd 1.40 c 2.37 cd 5.83 c 19.18 bc 7.67 cd 14.17 bcd Put 1 mM + Spd 1 mM 1.68bc 2.16 cd 1.21 d 2.35 cd 6.67 c 22.50 bc 4.17 def 15.00 bcd Put 1 mM + Spd 2 mM 1.16 c 1.94 d 1.03 d 2.04 de 1.75 d 15.83 c 2.50 ef 11.67 cd Put 2 mM + Spd 1 mM 1.18c 1.99 d 1.04 d 2.19 cde 0.75 d 21.33 bc 6.67 cde 14.17 bcd Put 2 mM + Spd 2 mM 0.92 c 1.92 d 0.78 d 1.94 e 0.42 d 17.67 bc 0.0f 10.83 d LSD value 0.01 0.53 0.49 0.31 0.37 4.47 9.62 5.61 7.85 Means in columns followed by the same letter are not significantly different, P ≤ 0.05, LSD test. Means for each column were compared separately 178 Decay percentage Decay incidence in all control treatments was signifi- cantly greater than in PA-treated fruits (Table 5). Prolong- ing low temperature storage from 15 to 25 days resulted in greater decay incidence. Although the differences between PA-treated fruits were not significant, the combined PA treatments could be considered more effective since 0% decay decreases possible further contamination and decay. As a susceptible crop to chilling injury, tomato shows in- creased susceptibility to decay when stored at low tem- peratures after harvest (Ding et al., 2002). In the present study, treatment with Put:Spd (2:2 mM) was very effective in alleviating chilling injury and decreasing the incidence of decay in tomato fruits (Tables 4, 5). This result suggest that PA application enhances the natural resistance of the fruits to chilling injury and decaying agents. Taken togeth- er, results obtained in this study indicate that the higher levels of PAs reduce CI and decay of tomato fruit. The reduction in CI symptoms by PAs has been related to their capacity to preserve membrane integrity, both by lowering the membrane phase-transition temperature fluidity and by retarding lipid peroxidation, resulting in increased cell viability (González-Aguilar et al., 2000). Total soluble solids and titratable acidity In all cases the highest values for TSS were observed in Put:Spd (2:2 mM), however the differences among oth- er PA combinations were also not significant (Table 5). The differences in TSS between 15 and 25 days of low temperature storage were not significant in the two culti- vars (data not shown). The earlier experiment showed no significant changes in TSS between room temperature and low temperature stored tomatoes (Javanmardi and Kubota, 2006). It has been reported that TSS remains unchanged after chilling and reconditioning (Luengwilai and Beckles, 2010), however it is possible that individual sugars and the sugar-acid balance may be adversely affected (Beckles, 2012). Harvesting riper fruit (i.e. those with already well- developed sugar profiles) would reduce the harm caused by chilling injury due to lower temperature (Beckles, 2012). Titratable acidity in combined PA treatments for fruits stored 15 days at low temperature were significantly great- er than other treatments in both cultivars. Treated fruits with Put:Spd (2:2 mM) showed the highest titratable acid- ity after 25 days of storage (Table 6). Titratable acidity in- creased with increasing duration of low temperature stor- age for all treatments (Table 6). It is reported that increased endogenous Pas, spermine and spermidine, through transgenic manipulation of to- mato showed similar levels of juice TSS, pH and titratable acidity in transgenic, azygous, and wild-type fruits (Mehta et al., 2002). Application of Put and Spd on apricot did not affect TSS and TA (Koushesh Saba et al., 2012). Ascorbic acid content All treated fruits showed greater ascorbic acid in fruits stored for 25 days than 15-day low temperature storage group (Table 6). The pattern of changing ascorbic acid level in tomato fruit varies with the physiological ripen- ing stages as it increases slowly reaching a maximum and then declines slowly coinciding with the initiation of rip- ening, as indicated by color change, and an increase in the activity of ascorbate oxidase (Yahia et al., 2001). In this experiment the mature green fruits treated and stored at low temperature showed the same increasing pattern until 25 days of storage. At that time the color had not started to change. Most likely PA application does not change the ascorbic acid pathway at least until ripening symptoms (color change) appear. It is reported that during the ripening process, the levels of Spd and Spm decline Table 5 - Effect of polyamine application and duration (15 and 25 days) of low-temperature (3°C) storage on fruit decay percentage and total soluble solid content in tomato cultivars M19 and M79 Treatment Decay percentage Total soluble solid content (°Brix) M 19 M 79 M 19 M 79 15 25 15 25 15 25 15 25 Control 33.33 a 60.00 a 25.00 a 40.00 a 4.01 b 4.28 b 4.80 b 4.13 c Put 1 mM 13.33 b 16.33 b 9.67 b 9.67 b 4.50 b 4.85 ab 5.18 ab 4.73 bc Put 2 mM 6.67 b 11.37 b 3.33 b 4.00 b 5.43 ab 5.13 ab 5.43 ab 5.20 b Spd 1 mM 3.33 b 12.33 b 3.33 b 6.33 b 5.35 ab 5.08 ab 5.25 ab 5.00 bc Spd 2 mM 0.00 b 10.00 b 2.33 b 5.00 b 5.55 a 5.25 ab 5.33 ab 5.55 ab Put 1 mM + Spd 1 mM 0.00 b 6.00 b 0.00 b 3.33 b 5.35 ab 5.30 a 5.35 ab 5.50 ab Put 1 mM + Spd 2 mM 0.00 b 5.00 b 0.00 b 1.67 b 5.60 a 5.50 a 5.73 a 5.63 ab Put 2 mM + Spd 1 mM 0.00 b 6.67 b 0.00 b 6.67 b 5.28 ab 5.48 a 5.68 a 5.60 ab Put 2 mM + Spd 2 mM 0.00 b 5.67 b 0.00 b 3.33 b 5.75 a 5.68 a 5.88 a 5.85 a LSD value 0.01 14.3 19.07 8.74 12.78 0.96 0.66 0.38 0.40 Means in columns followed by the same letter are not significantly different, P≤ 0.05, LSD test. Means for each column were compared separately. 179 in fruits (Mattoo and Handa, 2008). The ripening process has been shown to be delayed in tomato fruits by infusing them with Put (Saftner and Baldi, 1990). Polyamines and ethylene are known to have opposite effects in relation to fruit ripening and senescence (Saftner and Baldi, 1990). Free polyamines inhibit ethylene production in a variety of tissues (Suttle, 1981) and the elevated level of free poly- amines may be responsible for the reduction in both eth- ylene production and ripening processes of tomato fruits (Saftner and Baldi, 1990). Elevated levels of polyamines help maintain cellular vitality and longer life of ripening tomato (Mattoo et al., 2010). Correlations among fruit characteristics The correlations among weight loss, electrolyte leak- age, chilling injury, decay percentage and fruit firmness for 15 and 25 days of low temperature storage in both cul- tivars were positive and significant, but they were either non-significant or significantly negative when analyzed against ascorbic acid, titratable acidity and TSS (Tables 7-10). The greatest impact of electrolyte leakage was found on weight loss and decay percentage for the studied Table 6 - Effect of polyamine application and duration (15 and 25 days) of low-temperature (3°C) storage on fruit juice acidity and ascorbic acid content in tomato cultivars M19 and M79 Treatment Titratable acidity (g CAE/100 g fw) Ascorbic acid (mg/100ml) M 19 M 79 M 19 M 79 15 25 15 25 15 25 15 25 Control 0.34 b 0.43 c 0.37 bc 0.47 b 9.09 b 11.01 c 11.88 a 12.07 c Put 1 mM 0.35 b 0.41 c 0.31 c 0.37 c 9.74 b 11.97 bc 10.53 a 12.12 c Put 2 mM 0.41 b 0.44 c 0.31 c 0.42 c 9.81 b 11.02 c 11.35 a 13.19 bc Spd 1 mM 0.37 b 0.43 c 0.36 bc 0.39 c 9.60 b 11.97 bc 11.25 a 12.25 c Spd 2 mM 0.39 b 0.50 b 0.40 bc 0.44 bc 11.01 a 12.44 bc 10.63 a 13.07 bc Put 1 mM + Spd 1 mM 0.52 a 0.56 b 0.48 ab 0.53 b 10.40 a 13.47 b 11.39 a 15.00 a Put 1 mM + Spd 2 mM 0.57 a 0.63 a 0.43 abc 0.69 a 11.32 a 17.91 a 12.40 a 15.60 a Put 2 mM + Spd 1 mM 0.54 a 0.60 a 0.46 ab 0.54 b 11.05 a 12.06 bc 11.88 a 13.62 abc Put 2 mM + Spd 2 mM 0.58 a 0.70 a 0.56 a 0.65 a 11.80 a 13.60 b 12.15 a 14.03 ab LSD value 0.01 0.11 0.13 0.13 0.10 1.01 2.35 2.29 2.13 Means in columns followed by the same letter are not significantly different, P ≤ 0.05, LSD test. Means for each column were compared separately. Table 7 - Correlation analysis between fruit characteristics of tomato cv. M19 treated with different polyamines after 15 days storage at 3°C Weight loss Electrolyte leakage Chilling injury Decay percentage Fruit firmness Ascorbic acid Titratable acidity Electrolyte leakage 0.927 ** Chilling injury 0.745 ** 0.623 ** Decay percentage 0.769 ** 0.803 ** 0.694 ** Firmness 0.753 ** 0.761 ** 0.741 ** 0.699 ** Ascorbic acid -0.332 ns -0.367 ns -0.258 ns -0.340 ns -0.380 ns Titratable acidity -0.679 ** -0.629 ** -0.680 ** -0.555 ** -0.726 ** 0.377 ns TSS -0.701 ** -0.719 ** -0.642 ** -0.701 ** -0.618 ** 0.083 ns 0.421 ** ns, *, ** non-significant, significant at 0.05 and 0.01, respectively. Table 8 - Correlation analysis between fruit characteristics of tomato cv. M79 treated with different polyamines after 15 days storage at 3°C Weight loss Electrolyte leakage Chilling injury Decay percentage Fruit firmness Ascorbic acid Titratable acidity Electrolyte leakage 0.950 ** Chilling injury 0.716 ** 0.737 ** Decay percentage 0.894 ** 0.909 ** 0.735 ** Firmness 0.861 ** 0.825 ** 0.806 ** 0.815 ** Ascorbic acid -0.018 ns 0.023 ns -0.162 ns 0.003 ns -0.166 ns Titratable acidity -0.353 ns -0.378 ns -0.664 ** -0.314 ns -0.617 ** 0.291 ns TSS -0.642 ** -0.633 ** -0.632 ** -0.624 ** -0.806 ** 0.415 * 0.548 ** ns, *, ** non-significant, significant at 0.05 and 0.01, respectively. 180 cultivars and low temperature durations. 4. Conclusions The results of this experiment indicate that exogenous Put and Spd application on tomato fruit could maintain or even improve fruit quality during low temperature storage. The combined application of Put with Spd (2:2 mM) could be recommended for low temperature and long duration storage of tomato fruits. References AOAC, 1984 - Official methods of analysis. - Association of Of- ficial Agricultural Chemists, 14th ed., Washington, DC, USA. AGHDAM M.S., BODBODAK S., 2013 - Physiological and biochemical mechanisms regulating chilling tolerance in fruits and vegetables under postharvest salicylates and jas- monates treatments. - Scientia Horticulturae, 156: 73-85. BANINASAB B., 2009 - Amelioration of chilling stress by pa- clobutrazol in watermelon seedlings. - Scientia Horticul- turae, 121: 144-148. BECKLES D.M., 2012 - Factors affecting the postharvest sol- uble solids and sugar content of tomato (Solanum lycoper- sicum L.) fruit. - Postharvest Biology and Technology, 63: 129-140. BEN-YEHOSHUA S., SHAPIRO B., CHEN Z.E., LURIE S., 1983 - Mode of action of plastic film in extending life of lemon and bell pepper fruits by alleviation of water stress. - Plant Physiology, 73: 87-93. CANDAN A.P., GRAELL J., LARRIGAUDIÈRE C., 2007 - Chilling injury as related to climacteric behaviour in plums, pp. 431-436. - In: RAMINA A., C. CHANG, J. GIOVANNONI, H. KLEE, P. PERATA, and E. WOLTER- ING (eds.) Advances in plant ethylene research. Springer, The Netherlands. COHEN E., SHAPIRO B., SHALOM Y., KLEIN J., 1994 - Wa- ter loss: a nondestructive indicator of enhanced cell mem- brane permeability of chilling-injured citrus fruit. - J. Amer. Soc. for Hortic. Sci., 119: 983-986. DING C.-K., WANG C., GROSS K., SMITH D., 2002 - Jasmo- nate and salicylate induce the expression of pathogenesis-re- lated-protein genes and increase resistance to chilling injury in tomato fruit. - Planta, 214: 895-901. EL GHAOUTH A., PONNAMPALAM R., CASTAIGNE F., ARUL J., 1992 - Chitosan coating to extend the storage life of tomatoes. - HortScience, 27: 1016-1018. FAUST M., WANG S.Y., 1992 - Polyamines in horticulturally important plants. - Horticultural Reviews, 14: 333-356. GALSTON A.W., SAWHNEY R.K., 1990 - Polyamines in plant physiology. - Plant Physiology, 94: 406-410. GILL S.S., TUTEJA N., 2010 - Polyamines and abiotic stress tolerance in plants. - Plant Signaling & Behavior, 5: 26-33. GONZÁLEZ-AGUILAR G., ZACARIAS L., LAFUENTE M., 1998 - Ripening affects high-temperature-induced polyamines and their changes during cold storage of hybrid Fortune man- darins. - J. of Agric. and Food Chem., 46: 3503-3508. GONZÁLEZ-AGUILAR G.A., GAYOSSO L., CRUZ R., FOR- TIZ J., BÁEZ R., WANG C.Y., 2000 - Polyamines induced Table 9 - Correlation analysis between fruit characteristics of tomato cv. M19 treated with different polyamines after 25 days storage at 3°C Weight loss Electrolyte leakage Chilling injury Decay percentage Fruit firmness Ascorbic acid Titratable acidity Electrolyte leakage 0.954 ** Chilling injury 0.625 ** 0.714 ** Decay percentage 0.912 ** 0.859 ** 0.536 ** Firmness 0.849 ** 0.845 ** 0.721 ** 0.731 ** Ascorbic acid -0.346 ns -0.287 ns -0.539 ** -0.400 * -0.576 ** Titratable acidity -0.485 * -0.532 ** -0.600 ** -0.464 * -0.664 ** 0.545 ** TSS -0.837 ** -0.806 ** -0.624 ** -0.710 ** -0.821 ** 0.428 * 0.583 ** ns, *, ** non-significant, significant at 0.05 and 0.01, respectively. Table 10 - Correlation analysis between fruit characteristics of tomato cv. M79 treated with different polyamines after 25 days storage at 3°C Weight loss Electrolyte leakage Chilling injury Decay percentage Fruit firmness Ascorbic acid Titratable acidity Electrolyte leakage 0.946 ** Chilling injury 0.884 ** 0.884 ** Decay percentage 0.860 ** 0.912 ** 0.764 ** Firmness 0.839 ** 0.787 ** 0.793 ** 0.752 ** Ascorbic acid -0.429 * -0.433 * -0.518 ** -0.415 * -0.636 ** Titratable acidity -0.331 ns -0.232 ns -0.344 ns -0.237 ns -0.604 ** -0.549 ** TSS -0.846 ** -0.795 ** -0.807 ** -0.717 ** -0.862 ** 0.651 ** 0.605 ** ns, *, ** non-significant, significant at 0.05 and 0.01, respectively. 181 by hot water treatments reduce chilling injury and decay in pepper fruit. - Postharvest Biol. and Techn., 18: 19-26. HE L., BAN Y., INOUE H., MATSUDA N., LIU J., MORIGU- CHI T., 2008 - Enhancement of spermidine content and an- tioxidant capacity in transgenic pear shoots overexpressing apple spermidine synthase in response to salinity and hyper- osmosis. - Phytochemistry, 69: 2133-2141. HUSSAIN S.S., ALI M., AHMAD M., SIDDIQUE K.H., 2011 - Polyamines: natural and engineered abiotic and biotic stress tolerance in plants. - Biotechnology Advances, 29: 300-311. JAVANMARDI J., KUBOTA C., 2006 - Variation of lycopene, antioxidant activity, total soluble solids and weight loss of tomato during postharvest storage. - Postharvest Biol. and Techn., 41: 151-155. KIM T.E., KIM S.-K., HAN T.J., LEE J.S., CHANG S.C., 2002 - ABA and polyamines act independently in primary leaves of cold-stressed tomato (Lycopersicon esculentum). - Phys- iologia Plantarum, 115: 370-376. KOUSHESH SABA M., ARZANI K., BARZEGAR M., 2012 - Postharvest polyamine application alleviates chilling injury and affects apricot storage ability. - J. of Agric. and Food Chem., 60: 8947-8953. LIU J.-H., KITASHIBA H., WANG J., BAN Y., MORIGUCHI T., 2007 - Polyamines and their ability to provide environ- mental stress tolerance to plants. - Plant Biotechnology, 24: 117-126. LUENGWILAI K., BECKLES D.M., 2010 - Climacteric eth- ylene is not essential for initiating chilling injury in tomato (Solanum lycopersicum) cv. Ailsa Craig. - J. of Stored Prod- ucts and Postharvest Res., 1: 1-8. LUTTS S., KINET J., BOUHARMONT J., 1996 - NaCl-induced senescence in leaves of rice (Oryza sativa L.) cultivars differ- ing in salinity resistance. - Annals of Botany, 78: 389-398. MALIK A., SINGH Z., 2005 - Pre-storage application of poly- amines improves shelf-life and fruit quality of mango. - J. of Hortic. Sci. & Biotech., 80: 363-369. MATTOO A.K., HANDA A.K., 2008 - Higher polyamines restore and enhance metabolic memory in ripening fruit. - Plant Science, 174: 386-393. MATTOO A.K., MINOCHA S.C., MINOCHA R., HANDA A.K., 2010 - Polyamines and cellular metabolism in plants: transgenic approaches reveal different responses to diamine putrescine versus higher polyamines spermidine and sperm- ine. - Amino Acids, 38: 405-413. MEHTA R.A., CASSOL T., LI N., ALI N., HANDA A.K., MATTOO A.K., 2002 - Engineered polyamine accumulation in tomato enhances phytonutrient content, juice quality, and vine life. - Nature Biotechnology, 20: 613-618. MIRDEHGHAN S., RAHEMI M., CASTILLO S., MAR- TíNEZ-ROMERO D., SERRANO M., VALERO D., 2007 a - Pre-storage application of polyamines by pressure or im- mersion improves shelf-life of pomegranate stored at chilling temperature by increasing endogenous polyamine levels. - Postharvest Biol. and Techn., 44: 26-33. MIRDEHGHAN S.H., RAHEMI M., SERRANO M., GUIL- LÉN F., MARTíNEZ-ROMERO D., VALERO D., 2007 b - The application of polyamines by pressure or immersion as a tool to maintain functional properties in stored pomegranate arils. - J. of Agric. and Food Chem., 55: 760. OUFIR M., LEGAY S., NICOT N., VAN MOER K., HOFF- MANN L., RENAUT J., HAUSMAN J.-F., EVERS D., 2008 - Gene expression in potato during cold exposure: changes in carbohydrate and polyamine metabolisms. - Plant Sci- ence, 175: 839-852. PAKSASORN A., HAYASAKA T., MATSUI H., OHARA H., HIRATA N., 1995 - Relationship of polyamine content to ACC content and ethylene evolution in Japanese apricot (Prunus mume) fruit. - J. of the Japan. Soc. for Hortic. Sci., 63: 761-766. PAPADOPOULOS A.P., 1991 - Growing greenhouse tomatoes in soil and in soilless media. - Agriculture Canada Publica- tion, Ottawa, Canada. SAFTNER R.A., BALDI B.G., 1990 - Polyamine levels and to- mato fruit development: possible interaction with ethylene. - Plant Physiology, 92: 547-550. SALTVEIT M.E., 2001 - Chilling injury is reduced in cucum- ber and rice seedlings and in tomato pericarp discs by heat- shocks applied after chilling. - Postharvest Biol. and Techn., 21: 169-177. SMITH D.L., ABBOTT J.A., GROSS K.C., 2002 - Down-regu- lation of tomato β-galactosidase 4 results in decreased fruit softening. - Plant Physiology, 129: 1755-1762. SUTTLE J.C., 1981 - Effect of polyamines on ethylene produc- tion. - Phytochemistry, 20: 1477-1480. TIECHER A., DE PAULA L.A., CHAVES F.C., ROMBALDI C.V., 2013 - UV-C effect on ethylene, polyamines and the regulation of tomato fruit ripening. - Postharvest Biol. and Techn., 86: 230-239. VALERO D., MARTíNEZ-ROMERO D., SERRANO M.A., 2002 - The role of polyamines in the improvement of the shelf life of fruit. - Trends in Food Sci. & Techn., 13: 228-234. WALDEN R., CORDEIRO A., TIBURCIO A.F., 1997 - Poly- amines: small molecules triggering pathways in plant growth and development. - Plant Physiology, 113: 1009. WANG C.Y., 1993 - Approaches to reduce chilling injury of fruits and vegetables. - Horticultural Reviews, 15: 63-95. YAHIA E.M., CONTRERAS-PADILLA M., GONZÁLEZ- AGUILAR G., 2001 - Ascorbic acid content in relation to ascorbic acid oxidase activity and polyamine content in to- mato and bell pepper fruits during development, maturation and senescence. - LWT. Food Science and Technology, 34: 452-457.