Chilling injury in mango (Mangifera indica) fruit peel: Relationship with ascorbic acid concentrations and antioxidant enzyme activities

Postharvest Biology and Technology 86 (2013) 409–417

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Chilling injury in mango (Mangifera indica) fruit peel: Relationship with ascorbic acid concentrations and antioxidant enzyme activities Ukrit Chongchatuporn a , Saichol Ketsa a,b,c,∗ , Wouter G. van Doorn d a

Department of Horticulture, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand Postharvest Technology Innovation Center, Commission on Higher Education, Bangkok 10400, Thailand Academy of Science, The Royal Institute, Dusit, Bangkok 10300, Thailand d Mann Laboratory, Department of Plant Sciences, University of California, Davis, CA 95616, USA b c

a r t i c l e

i n f o

Article history: Received 11 March 2013 Accepted 16 July 2013 Keywords: Chilling Browning Mango Peel Pulp

a b s t r a c t We investigated the degree of chilling injury (CI) in mango (Mangifera indica) fruit stored at 4 ◦ C or 12 ◦ C, in relation to peel ascorbic acid concentrations, total antioxidant capacity, and the activities of four antioxidative enzymes. In cv. Nam Dok Mai fruit exposed to 4 ◦ C, CI (peel browning) was found after 5 days, whilst CI in cv. Choke Anan fruit started after 10 days and did not reach the same degree. When held at 27–28 ◦ C, following various periods of exposure to 4 ◦ C, peel browning in both cultivars increased, but that in cv. Nam Dok Mai remained higher than in cv. Choke Anan. An inverse correlation was found between peel browning and ascorbic acid concentrations, and between peel browning and total antioxidant capacity, measured using the FRAP method. In cv. Nam Dok Mai, the superoxide dismutase (SOD) and catalase (CAT) activities were lower during storage at 4 ◦ C than during storage at 12 ◦ C, while such a difference was not found in cv. Choke Anan. When compared to cv. Choke Anan, lower activities of ascorbate peroxidase (APX) and of guaiacol peroxidase (POX) were found in the peel of cv. Nam Dok Mai. However, no difference was observed in APX and in POX activities in the peel of cv. Nam Dok Mai stored at 4 ◦ C or 12 ◦ C. This means that the relationships between CI and APX and POX activities were weak. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Many plants or plant parts of tropical or subtropical origin show chilling injury (CI) symptoms when exposed to temperatures lower than about 10 ◦ C but higher than freezing point (Wang, 1993). Two main hypotheses, which are not mutually exclusive, have been forwarded to explain the damaging effect of low temperature in these species. The first hypothesis suggests that chilling results in rigidification of cell membranes, which induces inhibition of the activity of membrane-bound enzymes or carriers (Wolfe, 2006; Zhang et al., 2010). This rigidification would depend on the fatty acid composition of the membranes. Some evidence in favor of the membrane rigidification hypothesis has been found (Nishida and Murata, 1996; Promyou et al., 2008), but other reports found no clear relationship between membrane fatty acid composition and

Abbreviations: APX, ascorbate peroxidase; CAT, catalase; POX, guaiacol perodixase; SOD, superoxide dismutase; TAC, total antioxidant activity. ∗ Corresponding author at: Department of Horticulture, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand. Tel.: +66 2 5790308; fax: +66 2 5791951x112. E-mail addresses: [email protected], [email protected] (S. Ketsa). 0925-5214/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2013.07.023

CI sensitivity (Prasad, 2001; Wongsheree et al., 2009; Sirikesorn et al., 2013). The second hypothesis refers to the production of active oxygen species (AOS). AOS comprise hydrogen peroxide (H2 O2 ) and the superoxide and hydroxyl radicals. The AOS, if not scavenged, rapidly react with various molecules, including DNA and proteins, and results in membrane lipid peroxidation (Rice-Evans et al., 1997). This leads to cellular damage or cell death (Blokhina et al., 2003). In the presence of transition metal ions (such as those based on Fe and Cu) hydrogen peroxide may be reduced by the superoxide radical to hydroxyl radicals. Among the primary produced radicals, the hydroxyl radicals are the most reactive and therefore the most damaging (Apel and Hirt, 2004). Evidence for the second hypothesis includes the finding of Lee et al. (2002) that the Arabidopsis frostbite1 (fro1) mutant was chilling-sensitive and accumulated AOS constitutively. Another transgenic Arabidopsis line had increased chilling tolerance and showed lower levels of AOS, in particular hydrogen peroxide, than the wild type (Moon et al., 2003). A similar finding was reported in a transgenic tomato line (Hsieh et al., 2002). Plants have two interlinked lines of defense against AOS. The first is the presence of antioxidative compounds. Antioxidant chemicals include glutathione, ascorbate (vitamin C), vitamin A,

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vitamin E, phenolics, and anthocyanins (Blokhina et al., 2003; Tsantili et al., 2010; Cai et al., 2011). The total anti-oxidative capacity (TAC) is a measure of the total activity of these chemicals (Katalinic et al., 2006; Dudonné et al., 2009). The second line of defense against AOS is the activity of antioxidative enzymes, which react with AOS, in some cases using antioxidative compounds as the other substrate (Prasad, 1997; Huang et al., 2008; Li et al., 2011). In the absence of stress, the synchronous action of antioxidant enzymes is able to control the AOS concentrations (Noctor and Foyer, 1998; Shigeoka et al., 2002; Apel and Hirt, 2004; Huang et al., 2008; Li et al., 2011). Upon exposure to low temperatures or other types of stress the activities of these enzymes might become inadequate, leading to damage (Apel and Hirt, 2004). As far as is now known, there are no known scavengers of hydroxyl radicals. The only way to avoid the extensive damage that can be induced by this free radical, therefore, is to control the levels of superoxide and H2 O2 (Apel and Hirt, 2004). As far as is known, four main enzymes are involved in this control. The conversion of superoxide radicals to H2 O2 is carried out by superoxide dismutases (SOD). At least three enzymes carry out the subsequent conversion of H2 O2 to water: catalase (CAT), ascorbate peroxidase (APX), and glutathione peroxidase (GPX). APX is part of the ascorbate peroxidase cycle, using ascorbate as a substrate. GPX is part of the glutathione peroxidase cycle. It uses glutathione as a substrate (Apel and Hirt, 2004). The activity of peroxidases (PODs) has also been thought to be related to the prevention of chilling damage, as they also use H2 O2 . One group of these peroxidases is generally assessed by using guaiacol as a substrate, but these enzymes can also use other phenolic substrates such as p-phenylenediamine (Anderson et al., 1995). This enzyme activity will here be called POX. Isozymes of POX have been found in cell walls, the cytosol, the endoplasmatic reticulum + Golgi, mitochondria, chloroplasts, and the vacuole (Ranier et al., 2001). The increase in resistance of plants that had become acclimatized to low temperature was correlated with increased POX activity (Prasad et al., 1994; Kang and Saltveit, 2002; Li et al., 2011). Treatments with jasmonate or salicylate can affect the antioxidant system and increase resistance to chilling injury in various fruit (Ding et al., 2002; Wang et al., 2006), including mango (González-Aguilar et al., 2001; Ding et al., 2007). For example, treatment of mango with salicylate resulted in significantly higher reduction states of ascorbate and glutathione, lower superoxide anion content, higher hydrogen peroxide content, and higher activities of superoxide dismutase, catalase, guaiacol peroxidase, ascorbate peroxidise, and glutathione reductase (Ding et al., 2007). This suggests that the antioxidant system is involved in determining the chilling symptoms in mango fruit. Apart from inducing damage, at relatively high levels, OAS are also thought to act as messengers, although at relatively low levels. The above-mentioned antioxidants and antioxidative enzymes may be important in controlling the strength of these messages (Dat et al., 2000; Apel and Hirt, 2004; Suzuki and Mittler, 2006). Plants or plant parts that have been exposed to low temperature often still look undamaged at the end of the exposure period, but CI may be rapidly manifested after transfer to warmer temperatures. In other species, damage is observed during exposure to low temperature but the symptoms are exacerbated upon transfer to warmer temperature (Wang, 1993). It is not clear what the physiological mechanism is behind these effects. It is possible that accumulated toxic chemicals cannot do as much harm during exposure to low temperature, as the metabolic rate is low, compared to subsequent exposure to higher temperatures which results in an increase in metabolic rate.

Mango fruit exposed to temperatures below 12 ◦ C show peel browning (Wang et al., 2008), whereby some cultivars are much more sensitive than others (Phakawatmongkol et al., 2004). For example, when held at 4 ◦ C, cv. Nam Dok Mai mangoes showed more peel browning than cv. Choke Anan fruit (Chidtragool et al., 2011). In the present study we tested the hypotheses that the degree of CI in these two cultivars would correlate with the levels of ascorbate total antioxidant capacity (TAC) and the activities of CAT, APX, and POX. We studied these hypotheses both during exposure to low temperature and during the subsequent period at higher temperature. 2. Materials and methods 2.1. Plant material Experiments were carried out with cvs. Nam Dok Mai and Choke Anan mango fruit (Mangifera indica L.). A batch, consisting of 12–15 fruit, was the unit of replication. Fruit were harvested at commercial maturity from a plantation in Uthaithani province (Northern Thailand). The fruit were transported to the laboratory in plastic baskets, and arrived within 6 h of harvest by an air-conditioned truck (25 ◦ C). In the laboratory, the fruit were selected for uniformity of size and color, were cleaned in a solution of 0.1 g L−1 hypochlorite and then dipped in 0.3 g L−1 prochloraz solution for 2–3 min to control fruit rot. The mango fruit were then allowed to air-dry at room temperature (24–30 ◦ C) before further use. The fruit were placed in cardboard boxes and the boxes were kept at 4 ◦ C or 12 ◦ C and about 87% RH. Fruit were randomly sampled every 3 d to determine chilling injury, total antioxidant capacity (TAC), catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX) and guaiacol peroxidase (POX) activities in the peel. Fruit of a second group were transferred, at 5 day storage intervals, to room temperature (27–28 ◦ C, 67–69% RH). The same parameters were measured on d 3 and d 6 of placement at room temperature. 2.2. Chilling injury Mango fruit were randomly sampled for determination of chilling injury using a scale of 1–5. The degree of browning visually assessed using the following scale: 0 = no browning, 1 = up to 10% of the peel or pulp is brown, 2 = 11–25% of the peel or pulp is brown, 3 = 26–40%, 4 = 40–55%, 5 = >55%. The CI index was calculated as follows



CI index =

(Injury classification level × Number of fruit at that level) Total number of fruit in the treatment

Browning of the pulp was examined after longitudinally cutting the pulp close to the endocarp, using a scale similar to the one used for peel browning. 2.3. Electrolyte leakage Electrolyte leakage (EL) of the peel was measured according to Campos et al. (2003), with slight modification. Peel segments (0.5 cm2 ) were excised with a razor blade and washed in deionized water. Half a gram FW was placed in an Erlenmeyer flask containing 30 mL of 0.3 M mannitol. The flasks were shaken at 100 rpm for 1 h. The solution electric conductivity was measured using a Consort model C831 (Turnhout, Belgium). Maximum conductivity was measured after incubating flasks in an autoclave at 121 ◦ C for 30 min and allowed to cool to 25 ◦ C. Data were expressed as a percentage of maximum conductivity.

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2.4. Vitamin C concentrations Determination followed the method described by AOAC (1990). Briefly, 2 mL of mango juice was mixed with 5 mL of 0.24 M oxalic acid, then titrated with solution of 0.86 mM 2,6dinitrochlorophenol until the end point (pink color). 2.5. Total antioxidant capacity (TAC) An extract was made of 0.5 g frozen peel tissue using a homogenizer and 10 mL of cold phosphate buffer (pH 7.0) with 2% (w/v) polyvinypolypyrrolidone (PVPP). The homogenate was filtered through 4 layers of miracloth and then centrifuged at 9170 × g for 30 min at 4 ◦ C. TAC activity was assayed using the Ferric Reducing Ability of Plasma (FRAP) assay, following the method described by Benzie and Strain (1996). The assay mixture consisted of 2.4 mL reagent (300 mM acetate buffer: 2,4,6-tripyridyl-s-triazine: pure iron (II) chloride hexahydrate 10:1:1) and 0.2 mL of tissue extract. The color intensity after incubation for 5 min was measured as absorbance at 593 nm. 2.6. Enzyme activities Enzymes were extracted from 0.5 g of frozen tissue, using a homogenizer, after adding 10 mL of 0.1 M phosphate buffer (pH 7.0). The homogenate was filtered through 4 layers of miracloth and then centrifuged at 17,000 × g for 20 min at 4 ◦ C. The supernatant was used as a crude essay extract. SOD activity was assayed using the method of Ukeda et al. (1997). The reaction mixture contained 50 mM phosphate buffer (pH 8.0), 3 mM xanthine, 3 mM EDTA, 0.75 mM XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium5carboxanilide), and 0.14 units of xanthine oxidase. Activity was determined by monitoring the reduction rate of XTT at 470 nm. The protein content was determined by the standard Bradford method, using bovine serum albumin as a standard. CAT activity was assayed using a modification of the method of Aebi (1983). It was determined by monitoring the decomposition of H2 O2 at 420 nm. The reaction mixture contained 50 mM phosphate buffer (pH 7.0), 30 mM H2 O2 and enzyme extract. For assessment of APX activity (Ali et al., 2005), the reaction mixture contained 0.5 mM ascorbic acid in 0.1 M phosphate buffer (pH 7.0), 100 ␮L of crude extract and a final concentration of 0.1 mM H2 O2 . Activity was determined by monitoring ascorbate oxidation at 290 nm. POX activity was assayed using a modification of the method of Wang et al. (2005a). The reaction mixture contained 0.1 M phosphate buffer (pH 7.0), 24 mM H2 O2 , 8 mM guaiacol and enzyme extract. POX activity was determined by measuring the absorbance at 470 nm due to the production of cinnamic acid. One unit of activity was defined as 0.001 per min increase in absorbance at 470 nm. The protein content was determined by the standard Bradford method, using bovine serum albumin as a standard. 2.7. Statistical analysis The measurements of visible peel browning (CI), electrolyte leakage, ascorbic acid concentration, total antioxidant capacity, and enzyme activities used three biological replicates per treatment and storage period (day 0, 5, 10, 15 and 20 of storage, and after 3 and 6 days at room temperature, following each of these storage periods). Each replication contained 12–15 fruit, pooled together. Data were compared by analysis of variance (ANOVA) and calculation of Least Significant Difference (LSD). Wherever possible, three-way ANOVA analysis was applied. Regression analyses were applied to the relations between peel browning scores and TAC, and

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between peel browning scores and enzyme activities. The experiments were repeated once at a later date with essentially the same results. 3. Results 3.1. Peel browning and changes in pulp color The peel of mango fruit is green at harvest and becomes yellow afterwards. In the present experiments the fruit remained green during exposure to 4◦ or 12 ◦ C, but became yellow during ripening at room temperature, after the exposure to lower temperatures. In fruit exposed to 12 ◦ C for up to 20 days, no peel browning was found, in either cultivar (Fig. 1A). During exposure to 4 ◦ C, cv. Nam Dok Mai fruit showed the beginning of peel browning by day 5. The browning increased with time until it was about maximal by day 15 (Fig. 1A). Cv. Choke Anan mango fruit exposed to 4 ◦ C showed the beginning of peel browning by day 10. By day 15 browning was similar to that in cv. Nam Dok Mai fruit on day 10 (Fig. 1A). Browning in Cv. Choke Anan did not reach the same level as in cv. Nam Dok Mai, during the 20 d of the experiment (Fig. 1A). Peel browning was also observed after transfer of the fruit to 27–28 ◦ C, after 0, 5, 10, 15, or 20 d of exposure to 4 ◦ C or 12 ◦ C. Three days after transfer of cv. Nam Dok Mai fruit to room temperature an increase in peel browning was found if the browning was not already close to its maximum during storage (Fig. 1B). An increase in peel browning was also found in cv. Choke Anan fruit, but the browning stayed lower than in cv. Nam Dok Mai fruit (Fig. 1B). Three-way ANOVA analysis showed a significant effect of cultivar and temperature (and of their interaction), throughout storage (Supplementary Table 1). During subsequent placement at room temperature, the effect of temperature was significant throughout the various storage periods, and the effect of cultivar was significant except with fruit taken out of storage on day 10. Interaction was also significant except day 10 (Supplementary Table 2). No pulp browning was observed during exposure to 12 or 4 ◦ C, in either of the cultivars studied (data not shown). Three days after transfer of cv. Nam Dok Mai fruit from 4 ◦ C to room temperature, the pulp exhibited a little browning, although only if the fruit had been exposed to 4 ◦ C for 20 d. The pulp of cv. Nam Dom Mai fruit showed increased browning by day 6 of placement at room temperature, compared to day 3. In contrast, the pulp of cv. Choke Anan mangoes did not show browning when held for 3 or 6 d at room temperature, even if the fruit had been exposed to 4 ◦ C for 20 d (data not shown). However, when exposed to 4 ◦ C for 25 or more days its pulp became whitish during subsequent placement at room temperature, in contrast to the normal yellow pulp color of fruit that had ripened without previous exposure to low temperature. The white pulp was considered not suitable for consumption. If no pulp discoloration was observed, the pulp was ready to eat (being yellow and sweet) a few days after transfer of the fruit from 4 ◦ C or 12 ◦ C to room temperature (27–28 ◦ C). After transfer from 12 ◦ C, readiness to eat took 2 days in cv. Choke Anan and 3–4 days in cv. Nam Dok Mai. After transfer from 4 ◦ C, readiness to eat took 2–6 days, independent of the cultivar. 3.2. Electrolyte leakage In fruit stored at 12 ◦ C, no clear increase in peel electrolyte leakage was observed, in either cultivar (Fig. 2A). During storage at 4 ◦ C electrolyte leakage of the peel increased, in both cultivars studied (Fig. 2A). Difference between the cultivars existed already on day 0. During storage, no difference between the cultivars was observed. No clear further changes occurred in electrolyte leakage of fruit that

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A

Chilling injury index (scores)

5

B LSD0.05 = 0.10

LSD0.05 = 0.17

4 3 2 1 0 0

5

10

15

20

0

5

10

15

20

Time in storage (d) Fig. 1. Chilling injury (CI) index in the peel of cvs. Nam Dok Mai (--, -䊉-) and Choke Anan (--, --) mango fruit stored at 4 (--, --) and 12 (-䊉-, --) ◦ C. (A) CI during storage and (B) after 5 d intervals of storage (indicated on the X-axis) and subsequent 3 d placement at room temperature. Data are means of three replications (12–15 fruit each).

had been held for 5, 10, 15, or 20 d at 4 ◦ C or 12 ◦ C and then placed for 3 d at room temperature (Fig. 2B). Three-way ANOVA showed an effect of cultivar and temperature, throughout storage (Supplementary Table 3). After transfer to room temperature at various intervals of storage there was only an effect of the cultivar, throughout storage (Supplementary Table 4).

3.4. Total antioxidant capacity (FRAP method) The TAC values, determined using the FRAP method, in cv. Nam Dok Mai peel were considerably higher than those in cv. Choke Anan peel, throughout the 20 d of exposure to 4 ◦ C or 12 ◦ C (Fig. 4A). No differences were found between fruit exposed to 4 ◦ C or exposed to 12 ◦ C. In both cultivars and at both temperatures the TAC values only slightly changed (Fig. 4A). TAC values were also determined three d after transfer of the fruit to 27–28 ◦ C, after 0, 5, 10, 15, or 20 days of exposure to 4 ◦ C. The values stayed higher in cv. Nam Dok Mai peel than in cv. Choke Anan peel (Fig. 4B). Three-way ANOVA showed an effect of the cultivar, both during storage and during transfer to room temperature after storage (Supplementary Tables 7 and 8).

3.3. Vitamin C concentration in the peel The concentration of ascorbic acid (vitamin C) in the peel decreased during the first 5 days of storage, at both storage temperatures (Fig. 3A). During storage at 12 ◦ C the concentrations remained similar (Fig. 3A). During storage at 4 ◦ C the concentration increased between day 5 and 10 in cv. Nam Dok Mai. An increase, although less than in cv. Nam Dok Mai, was found in cv. Choke Anan stored at 4 ◦ C, between day 10 and day 15 (Fig. 3A). Fruit were held for 5, 10, 15, or 20 d at 4 ◦ C or 12 ◦ C and then placed for 3 d at 27–28 ◦ C. Concentrations were similar after both storage temperatures, and were low from day 5 of storage (Fig. 3B). Three-way ANOVA showed that the effect of temperature was significant from day 15 of storage (Supplementary Table 5). After transfer to room temperature an effect of temperature was also found from day 15 of storage (Supplementary Tables).

3.5. SOD and CAT activity Both the SOD and the CAT activities in cv. Nam Dok Mai peel tended to be higher than that in the peel of cv. Choke Anan (Fig. 5A and C). During storage at 12 ◦ C peel SOD activities in cv. Choke Anan did not show a clear change, but increased during day 0–10 in cv. Nam Dok Mai (Fig. 5A). Storage at 4 ◦ C did not change the peel SOD

30

B

Electrolyte leakage (%)

A LSD0.05 = 1.22

LSD0.05 = 1.11

20

10

0 0

5

10

15

20

0

5

10

15

20

Time in storage (d) Fig. 2. Electrolyte leakage (EL) in the peel of cvs. Nam Dok Mai (--, -䊉-) and Choke Anan (--, --) mango fruit stored at 4 (--, --) and 12 (-䊉-, --) ◦ C. (A) EL during storage and (B) after 5 d intervals of storage (indicated on the X-axis) and subsequent 3 d placement at room temperature. Data are means of three replications (12–15 fruit each).

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413

30

Vitamin C concentration (mg/100 g FW)

A

B

LSD0.05 = 2.37

LSD0.05 = 3.46

20

10

0 0

5

10

15

20

0

5

10

15

20

Time in storage (d) Fig. 3. Vitamin C concentration in the peel of cvs. Nam Dok Mai (--, -䊉-) and Choke Anan (--, --) mango fruit stored at 4 (--, --) and 12 (-䊉-, --) ◦ C. (A) Concentration during storage and (B) after 5 d intervals of storage (indicated on the X-axis) and subsequent 3 d placement at room temperature. Data are means of three replications (12–15 fruit each).

activity in cv. Choke Anan, compared to storage at 12 ◦ C, but peel SOD activity in cv. Nam Dok Mai was lower at 4 ◦ C than at 12 ◦ C (Fig. 5A). Similarly, storage at 4 ◦ C did not change the peel CAT activity in cv. Choke Anan, compared to storage at 12 ◦ C, but peel CAT activity in cv. Nam Dok Mai was lower at 4 ◦ C than at 12 ◦ C (Fig. 5C). After placement at 27–28 ◦ C, following various periods at 4 or 12 ◦ C, no difference was found between at 4 and 12 ◦ C, in cv. Choke Anan SOD and CAT activities. In cv. Nam Dok Mai, the SOD activity after storage at 4 ◦ C was lower than that after storage at 12 ◦ C (Fig. 5B) while no difference was found in CAT activity (Fig. 5D). The correlation coefficients between CI in the peel and peel SOD activities, during exposure to 4 ◦ C, were 0.22 and 0.03 for cv. Nam Dok Mai and cv. Choke Anan, respectively. The correlation coefficients between CI in the peel and peel CAT activities, during exposure to 4 ◦ C, were 0.46 and 0.16 for cv. Nam Dok Mai and cv. Chok Anan, respectively (data not shown). Three-way ANOVA of the SOD data revealed a significant effect of the cultivar, throughout storage, while there was an effect temperature from day 5 of storage (although day 15 was just not significant, Supplementary Table 9). After transfer to room temperature there was an effect of the cultivar, throughout storage (Supplementary Table 10). Three-way ANOVA of the CAT data showed an effect of cultivar throughout storage, and an effect of temperature also throughout storage, except on day 10

(Supplementary Table 11). After transfer to room temperature an effect of cultivar was observed, throughout storage (Supplementary Table 12). 3.6. APX and POX activity In contrast with SOD and CAT activities, both the APX and POX activities were higher in cv. Choke Anan than in cv. Nam Dok Mai, throughout storage (Fig. 6A and C), and at 3 days of room temperature after storage (Fig. 6B and D). Such higher activity was found already on day 0 before exposure to 4 or 12 ◦ C (Fig. 6A and C). In cv. Nam Dok Mai an increase in APX activity was observed between day 5 and day 10 of exposure to 12 and 4 ◦ C (Fig. 6A). Such an increase was also found in cv. Nam Dok Mai POX activity, between day 0 and 15 of storage at 12 and 4 ◦ C (Fig. 6C). In cv. Choke Anan no differences were found in APX activity and POX activities during storage at 12 or 4 ◦ C (Fig. 6A and C). When taken out of storage and held at room temperature for 3 d no differences were found in APX activity and POX activities, when comparing storage at 12 or 4 ◦ C. This was true for both cultivars (Fig. 6B and D). In both cultivars, the peel APX and POX activities during and after storage at 4 ◦ C showed relatively low values both when the fruit showed little and when the fruit showed intense browning,

TAC (FRAP value, mmol/l)

50

A

40

B

LSD0.05 = 1.71

LSD0.05 = 1.69

30 20 10 0 0

5

10

15

20

0

5

10

15

20

Time in storage (d) Fig. 4. Total antioxidant capacity (TAC) in the peel of cvs. Nam Dok Mai (--, -䊉-) and Choke Anan (--, --) mango fruit stored at 4 (--, --) and 12 (-䊉-, --) ◦ C. (A) TAC during storage and (B) after 5 d intervals of storage (indicated on the X-axis) and subsequent 3 d placement at room temperature. Data are means of three replications (12–15 fruit each).

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2.0

A

B LSD0.05 = 0.13

SOD activity (units/g protein)

LSD0.05 = 0.15

1.5

1.0

0.5

0.0

4.0

CAT activity (units/mg protein)

D

C LSD0.05 = 1.01

LSD0.05 = 0.61

3.0 2.0 1.0 0.0 0

5

10

15

0

20

5

10

15

20

Time in storage (d) Fig. 5. Superoxide dismutase (SOD) and catalase (CAT) activity in the peel of cvs. Nam Dok Mai (--, -䊉-) and Choke Anan (--, --) mango fruit stored at 4 (--, --) and 12 (-䊉-, --) ◦ C. (A) SOD and (C) CAT during storage, (B) SOD and (D) CAT after 5 d intervals of storage (indicated on the X-axis) and subsequent 3 d placement at room temperature. Data are means of three replications (12–15 fruit each).

1.0

A

0.8

APX activity (units/g protein)

B

LSD0.05 = 0.15

LSD0.05 = 0.06

0.6 0.4 0.2 0.0 LSD0.05 = 0.11

0.8

POX activity (units/g protein)

D

C LSD0.05 = 0.09

0.6 0.4 0.2 0.0 0

5

10

15

20

0

5

10

15

20

Time in storage (d) Fig. 6. Ascorbate peroxidase guaiacol (APX) and peroxidase (POX) activity in the peel of cvs. Nam Dok Mai (--, -䊉-) and Choke Anan (--, --) mango fruit stored at 4 (--, --) and 12 (-䊉-, --) ◦ C. (A) APX and (C) POX during storage, (B) APX and (D) POX after 5 d intervals of storage (indicated on the X-axis) and subsequent 3 d placement at room temperature. Data are means of three replications (12–15 fruit each).

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while high activities were found at intermediate levels of peel browning. The (nonlinear) correlation coefficients of browning and APX activity were 0.61 for cv. Choke Anan and 0.62 for cv. Nam Dok Mai. Those of browning and POX activity were 0.65 for cv. Choke Anan and 0.56 for cv. Nam Dok Mai (data not shown). Three-way ANOVA of the APX data showed a significant effect of the cultivar throughout storage (Supplementary Table 13), and an effect of temperature from day 10 of storage. After transfer to room temperature an effect was observed of both the cultivar and the temperature, throughout storage (Supplementary Table 14). Three-way ANOVA of the POX data showed an effect of cultivar throughout storage, except on day 10 which was just not significant, while there was an effect of temperature throughout storage (Supplementary Table 15). After transfer to room temperature an effect of cultivar was observed, throughout storage except day 20 which was just not significant, as well as an effect of temperature throughout storage (Supplementary Table 16).

4. Discussion In the present study we tested the hypotheses that peel CI in mango fruit would correlate with (a) concentrations of ascorbic acid in the peel, (b) total antioxidant capacity (TAC) in the peel, and peel activities of (c) SOD, (d) CAT, (e) APX and (f) POX. The vitamin C concentration in the peel increased more and earlier during 4 ◦ C storage in cv. Nam Dok Mai than in cv. Choke Anan. This is inversely correlated with the induction of chilling injury. We previously reported the concentration of another group of antioxidants, relatively simple water-soluble phenols (called free phenolics), in the peel of these two cultivars. A small decrease was found during storage at 4 or 12 ◦ C but no difference was observed between the two cultivars. After 6 d of transfer to room temperature, following 3 d storage intervals, the concentrations tended to be higher in cv. Nam Dok Mai than in cv. Choke Anan (Chidtragool et al., 2011). These data suggest that vitamin C or free phenolics are not limiting factors in the development of CI in these two cultivars. We found that the total antioxidant capacity (TAC) of cv. Nam Dok Mai peel was considerably higher than that of cv. Choke Anan peel. This inverse correlation with CI sensitivity again suggests that in these fruit the presence of antioxidant chemicals, before exposure to low temperature or as a response to exposure to low temperature, was not a limiting factor in the defense against CI. However, it should be borne in mind that FRAP method here used does not assay antioxidants dissolved in lipids, such as vitamin E and also does not measure SH-group-containing antioxidants (Somogyi et al., 2007). The data thus only suggest that water-soluble anti-oxidative chemicals are not limiting in protecting the fruit studied against CI. The SOD and CAT activities in cv. Nam Dok Mai peel were considerably higher than those in cv. Choke Anan peel. This was inversely correlated with the induction of CI during exposure to low temperature. This might suggest that the activities of SOD and CAT were not limiting the induction of chilling injury. However, it was found that in cv. Nam Dok Mai the SOD and CAT activities were lower during storage at 4 ◦ C than during storage at 12 ◦ C, while such a difference was not found in cv. Choke Anan. So if we ignore the absolute enzyme activities and focus on the difference between the storage temperatures, there is a good relationship between CI and the activities of SOD and CAT. Overexpression of SOD genes in transgenic tobacco plants increased chilling tolerance of the plants (Sen Gupta et al., 1993;

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Foyer et al., 1994). In one experiment tobacco plants with suppressed CAT activity showed no change in CI tolerance (Willekens et al., 1997). However, in other tests, tomato plants expressing an antisense catalase gene resulted in a 2–8 fold reduction in total catalase activity. These plants did not survive exposure to 4 ◦ C while the wild-type plants did (Kerdnaimongkol and Woodson, 1999). Additionally, rice plants overexpressing catalase showed improved CI tolerance (Matsumura et al., 2002). Cv. Choke Anan peel had considerably higher APX and POX activities than cv. Nam Dok Mai peel, already before exposure to low temperature and throughout the period of storage. It might be suggested that the higher activities of these enzymes in cv. Choke Anan might provide a mechanism of counteracting the rise in AOS, and that these enzymes might therefore be responsible for a 5 d delay in chilling injury symptoms during exposure to 4 ◦ C. However, when comparing fruit stored at 4 ◦ C or 12 ◦ C, no difference was observed in peel APX and POX activities in cv. Nam Dok Mai. The difference in CI between storage of this cultivar at 4 ◦ C or 12 ◦ C was thus not explained by APX or POX activity. This means that the relationships between CI and APX and POX activities were weak. A relationship between CI and activities of APX and POX has been reported. Pinhero et al. (1997) found higher APX activity during exposure to low temperature, in intact plants of a chilling-tolerant maize cultivar, compared with those of a chilling-sensitive cultivar. Over-expression of ascorbate peroxidase conferred tolerance to chilling stress in rice plants (Sato et al., 2011). In what is apparently the only report on the role of antioxidative enzymes in a transgenic fruit, the overexpression of a cytosolic ascorbate peroxidase in tomato fruit increased chilling tolerance (Wang et al., 2005b). An increase in POX activity was observed during exposure of intact plants to low temperature (Queiroz et al., 1998; Lee and Lee, 2000; Kang and Saltveit, 2002; Xu et al., 2008). A correlation was found between POX activity prior to exposure to low temperature and CI during this exposure (Prasad et al., 1994; Kang and Saltveit, 2002; Li et al., 2011). It is concluded that during exposure to low temperature the fruit peel of two mango cultivars showed CI (browning). In the present study we tested the hypotheses that peel CI would correlate with (a) peel concentrations of ascorbic acid, (b) total antioxidant capacity (TAC), and activities of (c) SOD, (d) CAT, (e) APX and (f) POX. The above hypotheses a and b were not corroborated. Evidence was found for the truth of hypothesis c and d, while the hypotheses e and f were not supported by the present findings. The relationship of CI with enzyme activities was relatively complex. No correlation was found with overall activities of SOD and CAT. Nonetheless, in the CI sensitive cultivar, the SOD and CAT activities were lower during storage at 4 ◦ C than during storage at 12 ◦ C, while such a difference was not found in the CI tolerant cultivar. The data also showed a correlation between CI and overall APX and POX activities. However, as no difference was observed in APX and in POX activities in the peel of cv. Nam Dok Mai stored at 4 ◦ C or 12 ◦ C, the relationships between CI and APX and POX activities were not consistent. The data show that it is hard to decide if the overall activities of anti-oxidant enzymes are most important or that the differences in activities between exposure to 4 ◦ C or 12 ◦ C are most relevant.

Acknowledgements The work was financially supported by the Thailand Research Fund (TRF), Postharvest Technology Innovation Center (PHTIC), the Commission on Higher Education (CHE), the Ministry of Education, and the Kasetsart University Research and Development Institute (KURDI).

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