This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and

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1 This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier s archiving and manuscript policies are encouraged to visit:

2 Postharvest Biology and Technology 91 (2014) Contents lists available at ScienceDirect Postharvest Biology and Technology jou rn al h om epage: Postharvest treatment of polyamines maintains quality and extends shelf-life of table grapes (Vitis vinifera L.) cv. Flame Seedless W.A. Harindra Champa a,, M.I.S. Gill a, B.V.C. Mahajan b, N.K. Arora a a Department of Fruit Science, Punjab Agricultural University, Ludhiana , Punjab, India b Punjab Horticultural Post Harvest Technology Centre, Punjab Agricultural University, Ludhiana , Punjab, India a r t i c l e i n f o Article history: Received 3 September 2013 Accepted 21 December 2013 Keywords: Vitis vinifera L. Polyamines Putrescine Spermidine Quality Shelf-life a b s t r a c t Investigations were carried out to verify the potential of putrescine and spermidine as a postharvest dip treatment for maintaining quality and extending storage life of table grapes (Vitis vinifera L.) cv. Flame Seedless during the 2012 and 2013 seasons. Grape clusters were manually harvested at the commercial mature stage and were dipped in different concentrations (0.0, 0.5, 1.0 and 1.5 mm) of putrescine and spermidine, and then stored at 3 4 C, and 90 95% RH. Evaluation of physico-chemical parameters and other fruit quality attributes were made at 0 day (before treatment) and at 30, 45, 60 and 75 days of storage. Putrescine and spermidine at the lowest dose (0.5 mm) effectively maintained berry firmness, peel colour (L*, C*, h ) and stabilized anthocyanins as well as suppressing the activity of pectin methylesterase and reducing the rate of electrolyte leakage. The polyamines also retarded the degradation of TSS and TA while maintaining higher total phenol content and reduced decay incidence. Putrescine and spermidine at 1.0 mm exhibited almost similar effects with a 0.5 mm dose. The highest doses (1.5 mm) of both polyamines showed detrimental effects, especially on weight loss, decay incidence, rachis browning and organoleptic properties, as found in the control group, which was commercially acceptable only up to 45 days. Furthermore, analysis of linear regressions and correlations showed that many quality parameters were interdependent. The postharvest dip treatment of spermidine or putrescine at a dose of 0.5 mm for 5 min could be an effective means for prolonging storage and increasing shelf-life of Flame Seedless grapes Elsevier B.V. All rights reserved. 1. Introduction Grape (Vitis vinifera L.) is a non-climacteric fruit with a relatively low physiological activity. However, the perishable nature of the berry, as indicated by weight loss, softening, degradation of colour, increased berry shatter, rachis browning, decay development and degradation of organoleptic properties, impairs its long term storability. Attempts have been made to extend the storage life of table grape cultivars such as Flame Seedless by means of low temperature storage along with in-package dual release SO 2 generator pads, which helps store the fruit for up to 45 days (Mahajan et al., 2010). However, there would be value in extending the storage life further. For that, it is necessary to down-regulate accelerated Corresponding author at: Institute of Postharvest Technology, R&D Center, Jayanthi Mawatha, Anuradhapura 50000, Sri Lanka. Tel.: / addresses: harindra74@gmail.com, harindra74@hotmail.com (W.A.H. Champa), misgill@pau.edu (M.I.S. Gill), mahajanbvc@gmail.com (B.V.C. Mahajan), naresh arora@pau.edu (N.K. Arora). senescence of berries by suppressing ripening-related changes during postharvest storage. Much research evidence indicates that polyamines (PAs) in their free forms act as anti-senescent agents, reduce rate of respiration, delay ethylene production, retard colour changes, increase fruit firmness, induce mechanical resistance and reduce chilling symptoms (Valero et al., 2002). Polyamines are positively charged aliphatic amines that are ubiquitous in living organisms. Putrescine (a diamine) is the first polyamine in the biosynthetic pathway followed by spermidine (a triamine) and spermine (a tetraamine), which is formed by successive addition of aminopropyl groups derived from decarboxylated S-adenosyl methionine (dsam). The universal occurrence of putrescine, spermidine and spermine in plant organs suggests that they perform important functions in plant growth regulation. They can affect DNA and RNA synthesis and degradation, regulate rates of transcription, inhibit activities of protease, ribonuclease, peroxidase and polygalactouronase, stabilize ribosomal structure and maintain membrane integrity (Wang et al., 1993, and references therein). Postharvest application of PAs have been demonstrated to influence shelf-life and quality of various climacteric and non-climacteric fruit such as apple (Kramer et al., 1991; Wang et al., 1993), litchi (Jiang and Chen, 1995), /$ see front matter 2014 Elsevier B.V. All rights reserved.

3 58 W.A.H. Champa et al. / Postharvest Biology and Technology 91 (2014) lemon (Valero et al., 1998), kiwifruit (Petkou et al., 2004), mango (Malik and Singh, 2005), pomegranate (Mirdehghnan et al., 2007; Ramezanian and Rahemi, 2010), strawberry (Khosroshahi et al., 2007), peach (Martinez-Romero et al., 2000), plums (Perez Vincente et al., 2002; Serrano et al., 2003; Khan et al., 2007) and apricot (Martinez-Romero et al., 2002; Khosroshahi and Esna-ashari, 2007). Therefore, the present study was conducted to investigate the effects of postharvest treatments of putrescine and spermidine at concentrations of 0.0, 0.5, 1.0 and 1.5 mm, on maintaining quality and extending the postharvest life of grapes cv. Flame Seedless in cold storage (3 4 C and 90 95% RH). 2. Materials and methods 2.1. Experimental procedure The experiment was carried out in the postharvest laboratory of the Department of Fruit Science, Punjab Agricultural University, Ludhiana, Punjab, India, during two consecutive cropping seasons of 2012 and Table grape (V. vinifera L.) cv. Flame Seedless was obtained from the vineyard of the department. Bunches were harvested manually at commercial maturity (TSS 16%), packed in plastic crates of kg capacity, and transported to the laboratory immediately. Well coloured, properly formed bunches free from any visible defects, were selected and 6 kg of grapes was kept separately for day 0 (before treatment) analysis. The remaining grape bunches were divided into 7 lots each containing 24 kg of grapes (per experimental unit). Each lot of grapes was dipped in an aqueous solutions of polyamines (diamine putrescine and triamine spermidine, Sigma Aldrich Co., USA) at different concentrations: 0.0 (control), 0.5, 1.0 and 1.5 mm for 5 min. The surfactant Tween 20 (polyoxy ethylenesorbitan monolaurate polyethylene glycol, Sigma Aldrich Co., USA) at 0.1% was also added to enhance infiltration of the solutions. After dip treatment, the bunches were air-dried and 2 kg of grapes were weighed into low density polyethylene film (LDPE, 37.5 m) packages of 3 kg capacity, each containing an in-package dual release SO 2 generator pad (Grapage sodium metabisulphite 40%, JK Enterprises, Pune, India). These primary packages were put into 2 kg capacity CFB boxes and kept in the cold room at 3 4 C and RH of 90 95%. Observations were repeated at 30, 45, 60 and 75 days of storage Colour, firmness and membrane electrolyte leakage Peel colour of berries was measured using the Hunter lab colour difference meter (ColorFlex EZ, USA). The values of L*, a*, b* were recorded and hue angle (h ) arc tan (b*/a*) and chroma (C*) [(a*) 2 + (b*) 2 ] ½ were calculated. Berry firmness was measured using a Texture Analyzer (TA + HDi Stable Micro Systems, UK) equipped with a HDP/90 platform and 5 kg load cell. The measurement was made on the equatorial position of the berry with 4 mm probe at a test speed of 1 mm/s to a constant compression distance of 1 mm. The readings were expressed in N (Rolle et al., 2011). Membrane electrolyte leakage was measured as described by Jiang et al. (2001) by using a conductivity meter (ELICO CM 180, cell constant 1.04) and the results were expressed as S/100 g fresh weight (FW) Total anthocyanins, phenols and enzymatic activity of pectin methylesterase Total anthocyanin content of berries was determined as described by Ranganna (1986) with ethanolic HCL as the extraction solvent and absorbance was measured at 535 nm using a spectrophotometer (Spectronic 200 +, Thermo Scientific). Total phenols were determined by the Folin Ciocalteu method, based on colourimetric oxidation/reduction reactions of phenols (Slinkard and Singleton, 1977). Phenol extraction was carried out with 80% ethanol and the absorbance was measured at 765 nm against a blank using a spectrophotometer (Spectronic 200 +, Thermo Scientific). The results were expressed as mg of gallic acid equivalents/100 g FW using a gallic acid standard curve. The enzyme activity of pectin methylesterase was measured with 5 g of tissue sample as per the AOAC (2005). The volume of 0.02 N NaOH consumed to adjust the ph to 7.5 was recorded and the results were expressed as g/g FW Determination of other chemical parameters For analysis of other chemical parameters, 50 berries from each replicate were squeezed and the juice obtained was filtered through a cheese cloth. TSS was measured by a temperature compensated digital refractometer (Atago PAL-1, model 3810, Japan) and expressed as percent soluble solids. TA was determined as per AOAC (2005) and expressed as grams of tartaric acid equivalents per 100 ml of juice Weight loss, berry shatter, rachis browning and decay incidence Weight loss was recorded by subtracting final weight from the initial weight of the clusters and then expressed as percent weight loss with reference to the initial weight. Percent berry shatter was calculated for each box by dividing the weight of free berries from the total weight of boxed grapes. Rachis condition was rated according to Crisosto et al. (2002), as: 1 = healthy, entire rachis including the pedicels are green and healthy, 2 = slight, rachis in good condition, but noticeable browning of pedicels, 3 = moderate, browning of pedicels and secondary rachis, 4 = severe, pedicels, secondary and primary rachis completely brown. Decay incidence was expressed as the proportion (by weight) of rotten berries relative to total weight of berries within each box. Isolation and identification of types of pathogens were carried out as described by Franck et al. (2005) with a slight modification, i.e. potato dextrose agar (PDA) was used as the culture medium instead of acidified PDA Evaluation of organoleptic quality The berries were rated by an in-house panel of 30 judges on the basis of colour, texture, flavour and overall acceptability. A nine point hedonic scale described by Amerine et al. (1965) was used Experimental design and analysis The treatments were distributed according to a complete randomized design (CRD) with three replicates. Data were analyzed for variance by using the SAS (V 9.3, SAS Institute Inc., USA) package. When interactions between treatments were significant (P 0.05), the effect of each treatment was determined separating the means by least significant difference (LSD). Linear regressions were performed for some selected parameters. Data of rachis browning and organoleptic quality was analyzed by the Kruskal Wallis test and Friedman test using MINITAB 15 software (Minitab Inc., USA). 3. Results 3.1. Firmness and enzymatic activity of pectin methylesterase Berry firmness declined during storage both in treated and control fruit (Fig. 1A). However, the loss of firmness was significantly (P < 0.001) reduced by PA treatment although the rate of reduction was different among two PAs and between concentrations examined. Grape clusters dipped in 0.5 mm

4 W.A.H. Champa et al. / Postharvest Biology and Technology 91 (2014) Fig. 1. Variation in firmness (A), enzyme activity of pectin methylesterase (PME, B), total anthocyanin content (TAC, C) and total phenol content (TPC, D) of grape cv. Flame Seedless during cold storage (3 4 C, 90 95% RH) in relation to postharvest treatment with different concentrations of putrescine and spermidine. Vertical bars represent ±S.E. of pooled mean, C: control (0.0 mm), P: putrescine (mm), S: spermidine (mm), T: treatment, D: days. Values at harvest (0 day): Firmness (N/mm) = 1.66 ± 0.05, PME ( g/g FW) = ± 0.012, TAC (mg/100 g FW) = ± 6.63, TPC (mg GAE/100 g FW) = ± spermidine maintained a significantly higher firmness than with higher doses (1.0 and 1.5 mm), and a similar pattern was showed by putrescine; grape clusters dipped in 0.5 mm maintained comparatively higher firmness than observed in the other two doses (1.0 and 1.5 mm). There was no significant difference observed between 0.5 mm putrescine and 0.5 mm spermidine up to 60 days of storage, and thereafter, firmness of berries treated with 0.5 mm putrescine displayed a rapid reduction in comparison to berries treated with 0.5 mm spermidine. Pectin methylesterase (PME) activity of the berries increased gradually up to 45 days and thereafter exhibited a sharp rise coming to a peak at 60 days of storage, then showed a sharp fall during the next 15 days (Fig. 1B). However, clusters treated with PAs showed a significantly lower (P < 0.05) PME activity than the control. Among the two PAs examined, spermidine was found to be more effective in suppressing PME activity than putrescine. However, the lower doses of spermidine (0.5 and 1.0 mm) effectively suppressed PME activity up to 60 days of storage. The highest dose of spermidine (1.5 mm) resulted in a higher PME activity and showed no significant difference from the control up to 45 days of storage. Grape clusters treated with 0.5 mm putrescine also maintained a significant lower PME activity throughout the storage period in comparison to 1.0 and 1.5 mm putrescine, though it was relatively higher than in clusters receiving 0.5 mm spermidine Anthocyanins and phenols Total anthocyanin content (TAC) of grape berries initially showed an increasing trend up to 45 days of storage (Fig. 1C) except in the clusters dipped in 0.5 and 1.0 mm spermidine, where they displayed an increasing trend up to 30 days of storage and then declined. Reduction in TAC at a rapid rate after 45 days of storage was prominent in control grapes compared to putrescine or spermidine treated samples, where both treatments were equally effective in inhibiting the rate of reduction. By the end of 75 days of low temperature storage, berries treated with 0.5 and 1.0 mm putrescine and 0.5 mm spermidine maintained TAC levels of 55.82, and mg/100 g FW, respectively, while the control had a TAC of 32.5 mg/100 g FW. Total phenol content (TPC) of all grape samples with or without PA treatment, first increased and reached a peak value at 45 day, then decreased gradually during the next 30 days of cold storage (Fig. 1D). Both putrescine and spermidine affected TPC in a dose-dependent manner, where the higher concentrations of phenols were maintained by the highest doses of PAs Colour At the optimum harvest stage, Flame Seedless berries are characterized by a reddish purple skin colour, with CIE lab parameters of ± 1.51 (L*), 4.38 ± 0.36 (C*) and ± 3.48 (h ). During 75 days of cold storage, lightness (L*) and hue angle (h ) declined progressively, while saturation (C*) showed an increasing trend up to 30 days, then declining irrespective of treatment (Table 1). The greatest lightness was observed in berries treated with 1.0 mm spermidine, whereas the highest hue angle up to 45 days of storage was maintained by berries dipped in 0.5 mm spermidine. After 60 days of low temperature storage, a sharp decline in hue angle was observed in all treated and control samples, except with berries treated with 0.5 and 1.0 mm spermidine. With reference to saturation, a distinct deviation from control samples was not observed with the treated samples, except with 0.5 mm spermidine, in which a significant higher saturation was observed on 30 and 45 days of storage TSS and TA TSS and TA were significantly affected by treatment with PAs (Table 2). TSS exhibited an increasing trend up to 45 days of storage, where the control berries showed a greater increase in contrast to berries treated with PAs. After this point, TSS declined in both treated and control berries from 45 to 60 days; the reduction of TSS in control berries was 1.4% while reduction in putrescine 0.5, 1.0 and 1.5 mm treated berries was 1.1%, 0.3% and 0.4%, respectively. Parallel to putrescine, spermidine at the doses of 1.0 and 1.5 mm also showed lower reduction in TSS during the same period and the reduction was 0.9% and 0.2%, respectively. TA was only slightly changed during the whole storage period, although a reduction was observed on day 45, which might be due to the biological variation among samples. Otherwise, treated fruit maintained significantly higher levels of TA in contrast to the control. Among the two PAs, spermidine retained higher levels of TA in comparison to putrescine, and among concentrations, 0.5 mm putrescine and 1.0 and 1.5 mm spermidine maintained higher TA in comparison to other doses Weight loss, berry shatter, decay incidence and types of pathogens Weight loss increased during storage, although it was significantly higher in the control than in treated grape bunches (Fig. 2A). Spermidine showed higher efficacy in reducing weight loss in comparison to putrescine. With regard to different doses, spermidine 0.5 mm and putrescine 0.5 mm maintained a significantly lower rate of weight loss during the storage period. By the end of 75 days of cold storage,

5 60 W.A.H. Champa et al. / Postharvest Biology and Technology 91 (2014) Table 1 Luminosity (L*), saturation (C*) and hue angle of Flame Seedless berries subjected to various treatments of putrescine and spermidine during cold storage (3 4 C, 90 95% RH). Parameter At harvest Time (days of storage) L* Control ± ± 0.40 c ± 0.22 c ± 0.36 c ± 0.43 e Putrescine (mm) ± 0.44 c ± 0.33 c ± 0.52 c ± 0.16 bc ± 0.89 c ± 0.56 b ± 0.31 ab ± 0.35 c ± 0.57 b ± 0.33 b ± 1.03 b ± 0.21 d Spermidine (mm) ± 0.33 c ± 0.17 c ± 0.46 d ± 0.72 a ± 0.42 a ± 0.31 a ± 0.27 a ± 0.15 ab ± 0.49 c ± 0.22 b ± 0.46 d ± 0.12 e LSD (P < 0.05) C* Control 4.38 ± ± 1.84 c 7.27 ± 0.21 bc 7.31 ± 0.28 a 6.72 ± 0.12 a Putrescine (mm) ± 0.09 bc 8.51 ± 1.23 b 5.14 ± 0.02 b 5.66 ± 0.35 bc ± 1.08 c 6.88 ± 0.55 c 5.75 ± 0.40 b 5.11 ± 0.13 c ± 0.25 e 5.15 ± 0.38 e 7.36 ± 1.45 a 6.13 ± 0.12 ab Spermidine (mm) ± 0.60 a ± 0.71 a 3.95 ± 0.13 c 4.40 ± 0.17 d ± 0.23 b 6.47 ± 1.06 cd 4.94 ± 0.67 bc 5.69 ± 0.73 bc ± 0.10 d 5.54 ± 0.38 ed 4.65 ± 0.13 bc 5.63 ± 0.27 bc LSD (P < 0.05) h Control ± ± 5.29 c ± 1.26 d ± 0.73 ab 4.77 ± 0.62 c Putrescine (mm) ± 2.56 a ± 1.66 b ± 0.44 a 7.5 ± 1.84 cd ± 4.27 cd ± 2.13 c ± 1.43 a 7.45 ± 1.94 cd ± 2.07 d ± 2.17 c ± 4.66 a 7.29 ± 1.51 cd Spermidine (mm) ± 2.46 a ± 1.64 a 6.62 ± 1.00 cd 2.43 ± 1.08 b ± 2.44 b ± 3.41 b 8.58 ± 2.40 bc 7.38 ± 2.50 a ± 0.94 cd ± 2.91 d 3.54 ± 2.76 d 9.84 ± 1.48 d LSD (P < 0.05) Means in a column with the same letter are not significantly different (at P 0.05) according to LSD. L*: luminosity, C*: saturation, h : hue angle. Each value represents pooled mean of 2 years (2012 and 2013) (n = 30). cumulative weight loss was 4.35% in control berries, while this percentage in 0.5 mm putrescine treated clusters were almost halved (2.03%) and in clusters treated with 0.5 mm spermidine was even less (1.76%). The percent berry shatter was significantly affected (P < ) by PA treatments (Fig. 2B). Among the two PAs studied, the lowest doses (0.5 mm putrescine and 0.5 mm spermidine) displayed significantly lower levels of berry shatter, with 0.5 mm putrescine the most effective. Similarly to weight loss, clusters which received the highest doses of PAs (1.5 mm putrescine and 1.5 mm spermidine) exhibited significantly higher shatter percentages over the control at 30 days of storage, and after this point these two treatment showed no significant differences with the control throughout the storage period. At the end of the experiment, the cumulative berry shatter levels of control, 0.5 mm putrescine and 0.5 mm spermidine samples were 27.92%, 12.36% and 17.31%, respectively. The rate and severity of decay development varied, among the treatments and the control (Fig. 2C). All three concentrations of putrescine and 0.5 and 1.0 mm spermidine suppressed decay development significantly up to 45 days of storage, and there was no significant difference observed among them during this period. In contrast, grape clusters treated by 1.5 mm spermidine exhibited a higher incidence of decay; however, the occurrence of decay was still lower than in the control. At the end of experiment, the occurrence of decay was over 50% in control grapes, while this percentage was 25% in treatments of 0.5 and 1.0 mm putrescine and 0.5 mm spermidine. Macroscopic and microscopic observations of isolated cultures showed infections of gray mold caused by Botrytis cinerea and Rhizopus rot caused by Rhizopus stolonifer Membrane electrolyte leakage All PA-treated grape clusters exhibited a significantly lower membrane electrolyte leakage rate compared to the control (Fig. 2D). Putrescine showed a higher potential of inhibiting electrolyte leakage compared to spermidine. The lowest membrane electrolyte leakage rate was found in berries treated with 0.5 mm putrescine followed by 0.5 mm spermidine, 1.0 and 1.5 mm putrescine. Clusters dipped in 1.0 and 1.5 mm spermidine as well as the control group displayed membrane electrolyte leakage rates of >18 S/100 g FW at 60 days of storage, and after this point, the electrolyte leakage rate of these two treatments and the control increased dramatically, showing a similar trend Rachis browning and organoleptic rating Grape clusters dipped in putrescine and spermidine maintained a healthy green rachis in comparison to higher doses (1.5 mm) and the control (Table 3, supplementary data). Both 0.5 mm putrescine and spermidine exhibited healthy green rachis up to 60 days of storage even though there were some signs of browning in pedicels and secondary rachis, in contrast to the control and the other two treatments, where they were commercially unacceptable at this point. Supplementary material related to this article can be found, in the online version, at Samples treated by 0.5 and 1.0 mm putrescine and 0.5 mm spermidine were assigned higher overall acceptability ratings by the taste panel in contrast to the control (Table 3, supplementary data). The berries in the control group gained lower scores mainly because of poor texture, while perception of off-flavours developed by treatments of 1.5 mm putrescine and 1.0 and 1.5 mm spermidine was the reason for assigning them lower scores Correlations The results from linear regressions between some selected parameters are shown in the Table 4 (supplementary data). There were highly negative relationships between firmness vs. membrane electrolyte leakage, firmness vs. enzyme activity of pectin methylesterase (PME), and firmness vs. weight loss. In addition, we observed that both weight loss and PME were positively correlated with membrane electrolyte leakage. Berry shatter and decay incidence displayed highly significant positive correlations with weight loss. A negative correlation was found between decay incidence and total phenols, while the relationship between chromaticity (C*) and anthocyanins was positive. Supplementary material related to this article can be found, in the online version, at 4. Discussion Firmness is one of the main indicators in judging the quality of table grapes for fresh consumption. Grapes lose their firmness by loss of water and/or by changes in their structure/composition during postharvest storage (Bermstein and Lustig, 1981). In the present study, reduced firmness with prolonged storage was prominent in control fruit compared to fruit treated with putrescine or spermidine (Fig. 1A). Being positively charged, PAs strengthen cell walls by cross linking to the carboxyl ( COO ) group of the pectic substances in the cell wall, resulting in wall rigidity. This binding also

6 W.A.H. Champa et al. / Postharvest Biology and Technology 91 (2014) Table 2 TSS and TA of Flame Seedless berries subjected to various treatments of putrescine and spermidine during cold storage (3 4 C, 90 95% RH). Parameter At harvest Time (days of storage) TSS (%) Control ± ± 0.06 b ± 0.08 a ± 0.06 b ± 0.06 a Putrescine (mm) ± 0.06 c ± 0.06 c ± 0.06 d ± 0.06 c ± 0.07 b ± 0.06 b ± 0.06 a ± 0.06 a ± 0.08 f ± 0.07 c ± 0.11 b ± 0.07 b Spermidine (mm) ± 0.06 d ± 0.06 d ± 0.06 e ± 0.06 e ± 0.06 a ± 0.10 f ± 0.06 f ± 0.06 d ± 0.06 e ± 0.06 e ± 0.10 c ± 0.06 c LSD (P 0.05) TA (g of tartaric acid equiv./100 ml juice) Control 0.53 ± ± 0.01 b 0.37 ± c 0.45 ± 0.01 d 0.58 ± d Puttrescine (mm) ± b 0.46 ± 0.02 ab 0.48 ± 0.01 bc 0.60 ± c ± a 0.49 ± 0.04 a 0.51 ± 0.02 b 0.59 ± d ± b 0.49 ± a 0.48 ± 0.02 dc 0.59 ± d Sppermidine (mm) ± b 0.45 ± b 0.49 ± 0.01 bc 0.64 ± b ± a 0.48 ± 0.02 ab 0.55 ± 0.02 a 0.66 ± a ± 0.02 a 0.49 ± 0.01 a 0.55 ± 0.02 a 0.67 ± a LSD (P 0.05) Means in a column with the same letter are not significantly different (at P 0.05) according to LSD. Each value represents pooled mean of 2 years (2012 and 2013) (n = 50). blocks the access of cell wall degrading enzymes, including pectin methyl esterase (PME), pectin esterase (PE) and polygalactouronase (PG), reducing the rate of softening during storage (Valero et al., 2002). Evidently, the same concentrations that enhanced firmness of grape berries exhibited lower activity of PME (Fig. 1B), and we observed that there was a significant negative correlation (r = 0.69) between these two parameters (Table 4, supplementary data). The role of PAs in maintaining firmness, while suppressing fruit softening, is widely documented for many climacteric and non climacteric fruit, both (Martinez-Romero et al., 2000; Perez Vincente et al., 2002; Serrano et al., 2003; Petkou et al., 2004; Khan et al., 2007; Khosroshahi et al., 2007; Khosroshahi and Esna-ashari, 2007; Mirdehghnan et al., 2007). Lower pectin methylesterase (PME) activity was found in fruit treated with putrescine or spermidine (Fig. 1B). It is well known that changes in the pectin matrix affect cell wall structure during fruit ripening and senescence (Deytieux-Belleau et al., 2008). In this context, PME activity constitutes a key control point for both the assembly and disassembly of pectin networks. The modifications of pectin chains by the action of PME determine the accessibility of the galacturonans to degradation by polygalacturonases (Barnavon et al., 2001). During the first 45 days of storage, no significant increase in PME activity was detected. However, a surge of PME activity was observed after this point which may be considered as a signal for induction of rapid softening/accelerated ripening in this non-climacteric fruit. Reduced activity of fruit softening enzymes Fig. 2. Variation in weight loss (A), berry shatter (B), decay incidence (C) and membrane electrolyte leakage (MEL, D) of grape cv. Flame Seedless during cold storage (3 4 C, 90 95% RH) in relation to postharvest treatment with different concentrations of putrescine and spermidine. Vertical bars represent ± S.E. of pooled mean. C: control (0.0 mm), P: putrescine (mm), S: spermidine (mm), T: treatment, D: days.

7 62 W.A.H. Champa et al. / Postharvest Biology and Technology 91 (2014) by postharvest dip treatments of PAs has been reported for plum (Khan et al., 2007). Anthocyanins and phenolic compounds are important constituents in human health and there is rising awareness among consumers about their functional properties. Table grapes are a good source of health promoting compounds and being a coloured variety, the cv. Flame Seedless is also rich in these important antioxidants. As observed in the study, the levels of total anthocyanins and phenols remained significantly higher in PA-treated fruit (Fig. 1C and D), in contrast to the control in which a significant loss of these compounds was detected. Similar results have been reported in pomegranates and the mechanisms by which putrescine and spermidine induce these effects are still unknown (Mirdehghnan et al., 2007). Treatments with PAs significantly affected peel colour of berries and spermidine showed higher efficacy in this aspect than putrescine. This is in agreement with Valero et al. (2002) who reported that the effects of PAs on colour preservation of fruit is in the order of spermine 4+ < spermidine 3+ < putrescine 2+, following the order of their available number of cations. Similar results have been observed in apricots (Martinez-Romero et al., 2002), plums (Serrano et al., 2003) and lemon fruit (Valero et al., 1998). In the present study, TSS increased over 45 days of storage and then showed a slight declining trend. Increase in TSS from harvest to 45 days of storage is mainly due to reduction in water transport through the xylem to the berry once detached from the vine and also subsequent back flow of water from the berry to the rachis. This occurs due to differential rates of evaporation, i.e. rachis contains vascular bundles and active stomata in contrast to the berry which is covered by an exocarp that contains cuticular wax (3 m cuticle m wax), thus inhibits water loss through the berry (Chervin et al., 2012). Moreover, hydrolysis of sucrose into reducing sugars (glucose and fructose) can also be a reason. After 45 days, TSS declined due to gradual decline in stored carbohydrates as these are utilized in metabolic activities. PAs applied exogenously increase levels of endogenous PAs and reduce the rates of respiration (Perez Vincente et al., 2002; Valero et al., 2002) and ethylene production (Serrano et al., 2003; Khosroshahi et al., 2007; Barman et al., 2011) thus inhibiting ripening-related changes within the berries resulting in lower degradation of TSS in treated samples. High rates of weight loss, berry shatter and decay incidence were observed in control fruit during storage. These changes, which are associated with acceleration of senescence, were significantly suppressed by exogenous treatment of putrescine or spermidine. The lower weight loss in putrescine or spermidine treated fruit (Fig. 2A) could be attributed to stabilization and consolidation of both cell integrity and permeability of the tissues. This is further evidenced by the strong negative correlation between firmness vs. weight loss and by the highly positive relationship between weight loss vs. membrane electrolyte leakage (Table 4, supplementary data). PAs can decrease respiration through inhibition of ethylene biosynthesis or reducing rates of metabolism, leading to significant reduction in weight loss. Parallel to lower weight loss, the same treatments displayed reduced berry shatter (Fig. 2B) and there was a strong positive correlation between these two parameters (r = 0.88). The action of PAs on reducing berry shatter might result from PA ethylene interaction, because it is well established that ethylene, in parallel with falling auxin levels, induces the formation of an abscission zone in many fruit including grapes, thus stimulating berry drop (Lydakis and Amed, 2003). PAs influence ethylene formation in fruit by preventing transcription, synthesis and activity of 1-aminocyclopropane-1-carboxylic-synthase (ACS), which is a key regulatory enzyme in the ethylene biosynthetic pathway (Valero et al., 2002). The suppression of formation of ethylene, which is the primary trigger of abscission (Taiz and Zeiger, 2010), might cause a reduction in berry shatter. Reduced berry shatter in grape clusters when treated with exogenous putrescine has been reported by Shiri et al. (2012). Significantly lower decay incidence (Fig. 2C) was observed in putrescine or spermidine treated fruit than the control and the results are in line with those of Khosroshahi et al. (2007) and Shiri et al. (2012). The anti-pathogenic function of polyamines has been reviewed by Walters (2003). Polyamines conjugate to phenolic compounds and hydroxycinnamic acid amides (HCAAs) and have shown to have a good correlation between accumulation of HCAAs and pathogen resistance. Furthermore, it has been shown that substantial increases in PAs induce PR-proteins (pathogenesis related) in response to powdery mildew in barley and TMV in tobacco (Walters, 2003). In the present study, we observed a highly negative correlation between decay incidence vs. total phenols (Table 4, supplementary data). One of the common features accompanying chilling and senescence is increased membrane permeability, expressed as increasing leakage of ions which is used as an indicator of membrane damage. The rate of ion leakage, reduced significantly with putrescine and spermidine in contrast to the control (Fig. 2D), demonstrating the protective role of the PAs in maintaining membrane integrity. Similar results have been reported for litchi (Jiang and Chen, 1995) and pomegranate (Mirdehghnan et al., 2007; Ramezanian and Rahemi, 2010). Rachis browning is a common problem that affects the quality and marketing of table grape clusters. Rachis browning in cold storage has been commonly associated with water loss (Lichter et al., 2011) and oxidation processes (Carvajal-Millan et al., 2001), but senescence has also been proposed as additional component (Balic et al., 2012). As has been already described, putrescine and spermidine significantly reduced water loss and other authors have reported suppression of rates of respiration (Valero et al., 2002) and ethylene production (Serrano et al., 2003; Khosroshahi et al., 2007; Barman et al., 2011; Koushesh saba et al., 2012) and subsequent delay of senescence. Moreover, PAs influence reduction of browning, peroxide levels and ion leakage (Jiang and Chen, 1995) as well as educing polyphenol oxidase activity (Koushesh saba et al., 2012). Positive effect of putrescine and spermidine on these factors might be the reason for maintaining healthy rachis in treated clusters (Table 3, supplementary data). Improved rachis quality when treated with putrescine has been observed by Shiri et al. (2012). Postharvest dip treatments of putrescine and spermidine resulted in better organoleptic quality, especially in terms of flesh firmness, appearance, colour and taste (Table 3, supplementary data). Maintaining higher organoleptic qualities by putrescine has been observed in grapes (Shiri et al., 2012), mango (Jawandha et al., 2012) and in strawberry (Khosroshahi et al., 2007). Finally, in the present investigation, lower doses of both putrescine and spermidine (0.5 mm) had positive effects on maintaining quality of table grapes in contrast to higher doses (1.5 mm), which showed detrimental effects on most of the quality parameters studied. The results are in accordance with work reported by others (Kramer et al., 1991; Petkou et al., 2004). Grape berries develop as clusters with each berry attached to the bunch stem (rachis and branches) via a pedicel which contains vascular bundles. Thus, penetration and absorption take place not only through the thin exocarp of the berry but also via bunch stems, which enhances infiltration of applied chemicals. Therefore, it is highly likely that higher concentrations of exogenously applied putrescine and spermidine become phytotoxic in this highly perishable fruit. As has been observed in our experiment, in many other fruit such as peach (Martinez-Romero et al., 2000), plum (Serrano et al., 2003) pomegranate (Mirdehghnan et al., 2007), etc., lower doses of polyamines ( 1 mm) were found to be effective in maintaining storage quality.

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