Reduced chilling injury in mango fruit by 2,4-dichlorophenoxyacetic acid and the antioxidant response

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Postharvest Biology and Technology 48 (2008) 172–181

Reduced chilling injury in mango fruit by 2,4-dichlorophenoxyacetic acid and the antioxidant response Baogang Wang a,b , Jianhui Wang a , Hao Liang a , Jianyong Yi a , Jingjing Zhang a , Lin Lin a , Yu Wu a , Xiaoyuan Feng a,b , Jiankang Cao a , Weibo Jiang a,∗ a

College of Food Science and Nutritional Engineering, China Agricultural University, PO Box 111, Qinghua Donglu No. 17, Beijing 100083, China b Institute of Forestry and Pomology, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100093, China Received 21 May 2007; accepted 8 October 2007

Abstract The aim of this study was to evaluate the effects of 2,4-dichlorophenoxyacetic acid (2,4-D) on chilling injury (CI) in mango fruit. 2,4-D treatment at 150 mg L−1 could significantly alleviate CI or disease incidence of mango fruit during 7 days storage at 4 ◦ C and an additional 14 days at 20 ◦ C (P < 0.05). Fruit quality, including increased soluble solids, soluble sugar, fruit firmness, and peel chlorophyll level, was strongly improved by 2,4-D treatment. These results indicate that 2,4-D could be used as an effective method to preserve the postharvest life of mango fruit. Endogenous H2 O2 levels in the fruit were increased by 2,4-D treatment and decreased by exogenous H2 O2 treatment. The activities of catalase, superoxide dismutase, ascorbate peroxidase (APX) and glutathione reductase (GR) in the 2,4-D-treated fruit were much higher than those in control fruit. However, exogenous H2 O2 -treated fruit showed higher CI and lower activities of APX and GR. In addition, the results showed that 2,4-D treatment could significantly enhance levels of the endogenous ABA and GA3 , and reduce IAA and zeatin riboside levels in the fruit. These results suggest that 2,4-D could regulate chilling resistance by changing the balance of endogenous hormones levels. © 2007 Elsevier B.V. All rights reserved. Keywords: 2,4-D; Chilling injury; Antioxidant enzymes; Endogenous hormones; Quality; Mango

1. Introduction 2,4-Dichlorophenoxyacetic acid (2,4-D) is one of the most widely used herbicides due to its relatively moderate toxicity and biodegradability in soil. At high concentrations, 2,4-D acts as a herbicide on dicotyledoneous plants and has been used for several decades in cereal crops (Heering and Peeper, 1991; Sterling and Hall, 1997). However, at low concentrations, 2,4-D is broadly used in in vitro plant tissue culture for induction of callus formation and to stimulate somatic embryogenesis, due to its similarity to auxins (Kitamiya et al., 2000; Ramanayake and Wanniarachchi, 2003). Less known is the fact that 2,4-D is applied pre- and postharvest in fruit production. The primary use is for controlling the pre-harvest fruit drop. Citrus trees are treated with 2,4-D in order to delay the abscission of mature fruit and prevent stem end rot development (Coggins, 1969; Cohen, 1977). When applied

∗

Corresponding author. Tel.: +86 1 62736565; fax: +86 1 62736565. E-mail address: [email protected] (W. Jiang).

0925-5214/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2007.10.005

postharvest, 2,4-D has been shown to induce healing of injuries, retard senescence and control postharvest decay (Ferguson et al., 1982; Eckert, 1990; Papadopoulou-Mourkidou, 1991). A similar approach to the control of postharvest disease in mango has been reported in the past by Johnson et al. (1998) and Kobiler et al. (2001). To date, other physiological effects of 2,4-D applied postharvest on mango fruit have not been shown. Mango (Mangifera indica, L.) fruit is susceptible to chilling injury, which leads to a storage disorder manifested mainly as dark, scald-like discoloration and pitting or sunken lesions on the peel when fruit are stored at low temperature (below 13 ◦ C) for long periods. Storage treatments, such as heat treatment, intermittent warming, modified atmosphere, and application of plant growth regulators have been developed to prevent or alleviate this storage disorder (McCollum et al., 1993; Wang, 1993; Pesis et al., 2000; Gonz´alez-Aguilar et al., 2001; Nair and Singh, 2003). In this work, the effect of 2,4-D on the chilling injury symptoms, fruit qualities, antioxidative enzymes and hormone levels of mango fruit was evaluated. On the basis of the results obtained, a possible active mechanism for the effects of 2,4-

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D involving activated oxygen species (AOS) and hormones is proposed. 2. Materials and methods 2.1. Plant materials and experiments Mature green mangoes (M. indica, L. cv. Tainong) were harvested from a commercial orchard located in Hainan Province of China and transported to Beijing. Mature fruit of uniform size and free from visual symptoms of any disease or defects were used for this experiment. Mangoes were washed with 1% sodium hypochlorite, air-dried and randomly divided into four lots of 120 fruit each for 2,4-dichlorophenoxyacetic acid (2,4-D) treatment. Experiments were carried out three times. The results obtained from each experiment were similar. Each treatment comprised three replications, each of 120 fruit. Experiment I: Fruit were dipped in 100 or 150 mg L−1 2,4-D in a 150 L stainless steel vacuum container and vacuuminfiltrated under low pressure (−50 kPa) at 25 ◦ C for 5 min; thereafter, the fruit were consistently kept under air pressure for 1 h. The control fruit were treated by water under the same conditions. After the treatment, both 2,4-D-treated and control fruit were kept in trays covered with plastic films and stored at 4 or 20 ◦ C, 85–95% RH. After 7 days at 4 ◦ C, fruit were transferred for an additional 14 days at 20 ◦ C (shelf-life period). Ten fruit from each treatment and storage temperature were sampled after 3 and 7 days at 4 ◦ C or 7 and 14 days at 20 ◦ C. Fruit were evaluated for changes in firmness, chlorophyll, total soluble solids, titratable acidity (TA) and total soluble sugar (TSS). Chilling injury and decay symptoms were evaluated after 7 days at 4 ◦ C and the shelf-life period. Experiment II: Based on the results of Experiment I, to more intensively investigate the mechanisms involved in reducing the chilling injury of mango fruit by 2,4-D treatment, fruit were dipped into 150 mg L−1 2,4-D or 100 mM H2 O2 solution in a 150 L stainless steel vacuum container and vacuum-infiltrated under low pressure (−50 kPa) at 25 ◦ C for 5 min. Fruit sampled at intervals were used to evaluate active oxidative species, antioxidative enzymes and hormone levels. 2.2. Chilling injury When chilling injury (CI) symptoms visible as sunken lesions or pitting were observed the fruit were considered to have CI. CI incidence of the fruit was defined as CI-fruit/total fruit. The CI index was based on the percentage of total surface area affected by sunken lesions or pitting; 0 = no injury; 1 = slight, 2 = moderate and 3 = severe, according to Gonz´alez-Aguilar et al. (2001). The CI index was determined for each treatment by multiplying the number of fruit in each category by their scores, and then dividing this sum by total number of fruit assessed. 2.3. Malondialdehyde The malondialdehyde (MDA) content in mango peel was measured according to the method of Hodges et al. (1999). MDA

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formation was determined by measuring the absorbance of the reaction production of MDA with 2 mL 0.67% 2-thiobarbituric acid (TBA) in 2 mL supernatant at 532 nm, corrected for nonspecific turbidity by subtracting the absorbance at 600 nm and interference generated by TBA-sucrose complexes at 450 nm. The MDA formation was expressed as nmol g−1 FW. 2.4. Quality attributes Disease incidence of the fruit was recorded at intervals. When visible disease area on a fruit was more than 1 mm wide, it was recorded as a decayed fruit. Disease incidence was defined as decayed fruit/total fruit. The disease score was rated on a scale of 0–3, where 0 = no surface area affected, 1 = slight (surface area affected <1/4 total fruit surface area), 2 = moderate (1/4 to 1/2 surface area affected) and 3 = severe (>1/2 surface area affected). The disease index was determined for each treatment by multiplying the number of fruit in each category by their score, and then dividing this sum by total number of fruit assessed. Firmness, titratable acidity and total soluble solids contents in the pulp of mango were carried out as described by Jiang et al. (2004). Chlorophyll contents of mango peel were measured according to the method of Jiang et al. (2002). Total sugars were determined from fruit following the method of AOAC (1996) with some modifications. Sugars were calculated and expressed in percent. 2.5. Analysis of sensory quality Sensory quality of each replicate pulp was evaluated based on the method described by Del-Valle et al. (2005) with a few modifications. An acceptability test with a nine-point hedonic scale was used. The acceptability test was carried out using semi-structured scales, scoring one (lowest) to nine (highest). A trained panel of 16 people evaluated texture, taste, appearance and odor after 25 days of storage (almost all the control fruit were uneatable). The judges’ average response was calculated for each attribute. 2.6. Hydrogen peroxide (H2 O2 ) content Peel samples (1 g) were homogenized with 3 mL of cold acetone at 4 ◦ C. The homogenate was centrifuged at 10,000 × g at 4 ◦ C for 10 min. H2 O2 in the supernatant was estimated by forming the titanium–hydroperoxide complex according to Prochazkova et al. (2001). The H2 O2 content was expressed as mmol g−1 FW. 2.7. Antioxidant enzymes extraction and analysis Peel tissue (1 g fresh weight) was ground in liquid nitrogen with pestle and mortar and homogenized with 0.05 g PVPP (insoluble polyvinylpolypyrrolidone) in 3 mL 0.1 M phosphate buffer (pH 7.8) supplemented with 1 mM dithiothreitol, 1 mM PEG-4000 and 0.1 mM EDTA. The homogenate was centrifuged at 4 ◦ C for 20 min at 10,000 × g and the supernatant was collected for enzyme activity analysis.

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Catalase (CAT) activity was measured following the decrease of H2 O2 content (Havir and McHale, 1987). Superoxide dismutase (SOD) activity was assayed by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) according to Gay and Tuzun (2000). One unit of SOD was considered to be the amount of enzyme that inhibited NBT reduction by 50%. Ascorbate peroxidase (APX) activity was determined spectrophotometrically (Nakano and Asada, 1981). GR activity was assayed according to Foyer and Halliwell (1976). The units of all enzyme activities were defined as the change in absorbance of 0.001 g−1 fresh weight min−1 . Each experiment was repeated three times. 2.8. Determination of endogenous hormones of mango peel Mango peel (1 g) was ground in liquid nitrogen using a mortar and pestle, extracted with ice-cold 80% methanol (v/v) containing 1 mM butylated hydroxytoluene to prevent oxidation, and then stored for 4 h at 4 ◦ C. The extracts were then centrifuged at 10,000 × g for 20 min at 4 ◦ C. The residues were suspended in the same ice-cold extraction solution and stored at 4 ◦ C for 1 h, then centrifuged again at 10,000 × g for 20 min at 4 ◦ C. The two resulting supernatants were combined and passed through a C18 Sep-Pak cartridge (Waters, Milford, MA, USA). The effluent was collected and dried in N2 . The residues were then dissolved in 0.01 M pH 7.5 phosphate buffer and concentrations of indole-3-acetic acid (IAA), gibberellic acid (GA3 ), abscisic acid (ABA) and zeatin riboside (ZR) were determined in an enzyme-linked immunosorbent assay (ELISA) according to the methods described previously (He, 1993).

Fig. 1. Effect of 2,4-D treatment on chilling injury of mango fruit. Mango fruit were treated with 100 or 150 mg L−1 2,4-D and vacuum-infiltrated under low pressure (−50 kPa) at 25 ◦ C for 5 min. After treatment, all fruit were stored at 4 ◦ C for 7 days, and then transferred to 20 ◦ C to observe chilling injury (CI). The CI index and CI incidence were evaluated at 4 ◦ C for 7 days and then 20 ◦ C for 7 days (shelf-life period). Each value is the mean for three replicates and vertical bars indicate the standard deviation.

2.9. Statistical analysis Data were analyzed by analysis of variance (ANOVA) with SPSS 11.0 statistical software. Significant differences were performed by a least significant difference method (LSD test, P ≤ 0.05).

the fruit treated with 150 mg L−1 2,4-D were assayed during storage. 3.2. Effect of 2,4-D treatment on disease development of mango fruit during cold storage

3. Results 3.1. Effect of 2,4-D treatment on chilling injury of mango fruit during cold storage The CI index was significantly (P < 0.05) reduced with the 2,4-D vacuum infiltration treatment compared with the control. As shown in Fig. 1A, the CI index of the fruit treated with 100 or 150 mg L−1 2,4-D were 61.3 or 63.9% lower than that of the control at 4 ◦ C for 7 days, respectively. After 7 days at 20 ◦ C, more severe CI symptoms were observed on all fruit; however, no significant difference was observed between 100 mg L−1 and 150 mg L−1 2,4-D treatment. In addition, as shown in Fig. 1B, the CI was significantly reduced by 2,4D treatment and the higher concentration of 2,4-D showed lower CI than other treatments when the fruit were transferred to 20 ◦ C for 7 days. Therefore, the following attributes of

2,4-D treatment significantly reduced the decay of mangoes (Fig. 2). After 7 days at 4 ◦ C and 14 days at 20 ◦ C, control fruit showed more severe symptoms of decay than those treated with 2,4-D. The disease index in fruit treated with 2,4-D was 30.8% lower than that in the control (Fig. 2A), and similarly, disease incidence in treated fruit was 33.3% lower than that in control fruit (Fig. 2B). 3.3. Effect of 2,4-D treatment on firmness of mango fruit during cold storage As shown in Fig. 3, firmness in the mango fruit decreased during storage, but 2,4-D treatment delayed this reduction. The firmness in the fruit treated with 2,4-D was 22.0 or 192.3% higher (P < 0.05) than that in the control on the day 7 and 21 during the whole storage period, respectively. However, firmness

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in the control fruit stored at 20 ◦ C could not be measured on day 21. 3.4. Effect of 2,4-D treatment on chemical parameters of mango fruit during cold storage

Fig. 2. Effect of 2,4-D treatment on disease development of mango fruit. Mango fruit were treated with 150 mg L−1 2,4-D and vacuum-infiltrated under low pressure (−50 kPa) at 25 ◦ C for 5 min. After treatment, all fruit were stored at 4 ◦ C for 7 days, and then transferred to 20 ◦ C for 14 days (shelf-life period) to observe the disease symptoms. The disease index and disease incidence were evaluated at 20 ◦ C for 7 and 14 days. Each value is the mean for three replicates and vertical bars indicate the standard deviation.

Slight differences in soluble solids contents were found between control and 2,4-D-treated fruit (Table 1). 2,4-D treatment increased the soluble solids contents during storage. Contents in the fruit stored directly at 20 ◦ C rapidly increased and reached a peak (10.7%) on day 14 of storage, however, those in 2,4-D-treated fruit were 10.2% up until day 21 of storage. Total sugar content gradually increased in all mango fruit during storage, and this increase was accelerated by 2,4-D treatment (Table 1). This was particularly so in fruit treated with 2,4-D at 4 ◦ C, which was 35.1 or 26.5% higher than that in control fruit on the day 3 and 7 of storage (Table 1). No differences in total sugar content were observed in 2,4-D-treated fruit and the fruit stored directly at 20 ◦ C. Low-temperature storage was more favorable with regard to higher titratable acidity (TA) content than storage at 20 ◦ C (Table 1). Decline of TA in mango fruit during ripening was significantly inhibited by 2,4-D (P < 0.05). The rate of chlorophyll degradation in peel was inhibited by 2,4-D treatment (Table 1) whether fruit were stored at 4 ◦ C, and then transferred 20 ◦ C or solely at 20 ◦ C. The level of chlorophyll in 2,4-D-treated fruit peel at 4 or 20 ◦ C was 60.1 or 114.5% higher than that in control fruit peel on day 7 of storage. Sensory analysis revealed that the control fruit on day 25 of storage were uneatable because of uneven ripening or decay, while the fruit treated with 2,4-D were acceptable. The mango fruit treated with 2,4-D were evaluated with a higher score for texture, taste and odor than those of control fruit (Table 2). All the fruit stored directly at 20 ◦ C were uneatable because of overripening or decay on day 25 of storage. 3.5. Effect of 2,4-D treatment on malondialdehyde of mango fruit during cold storage Malondialdehyde (MDA) in control fruit slightly increased during the initial 7 days of storage at 4 ◦ C, and was markedly reduced by the 2,4-D treatment. As shown in Fig. 4, MDA content in the fruit treated with 2,4-D was 19.6% lower (P < 0.05) than that in control fruit on day 7 of storage. After being transferred to 20 ◦ C for storage, the fruit treated with 2,4-D also had lower MDA levels than control fruit. 3.6. Effect of 2,4-D treatment on H2 O2 levels of mango fruit peel during cold storage

Fig. 3. Effect of 2,4-D treatment on firmness of mango fruit. Mango fruit were treated with 150 mg L−1 2,4-D and vacuum-infiltrated under low pressure (−50 kPa) at 25 ◦ C for 5 min. After treatment, control and treated-fruit were stored at 4 ◦ C for 7 days of storage, and then transferred to 20 ◦ C for 14 days (shelf-life period). Additional fruit including control and 2,4-D treatedfruit were stored directly at 20 ◦ C. Fruit firmness was evaluated during storage. Each value is the mean for three replicates and vertical bars indicate the standard deviation.

2,4-D treatment remarkably enhanced the accumulation of H2 O2 in fruit during cold storage. As shown in Fig. 5, the H2 O2 level in fruit of the 2,4-D treatment was 7.7 or 47.1% higher than that in control fruit 3 or 7 days after storage at 4 ◦ C, respectively, and then gradually declined after the fruit were stored at 20 ◦ C for ripening. However, lower H2 O2 levels were observed in the

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Table 1 Effect of 2,4-D treatment on chemical parameters of mango fruit during cold storage Days of storage

Treatments

SSC (%)

TSC (%)

TA (%)

Chlorophyll (mg per 100 g FW)

5.92 ± 0.43

3.79 ± 0.75

2.43 ± 0.02

19.91 ± 1.42

3

Control at Control 2,4-D

20 ◦ C

6.15 ± 0.26 a 6.13 ± 0.14 a 5.97 ± 0.21 a

4.36 ± 0.27 a 3.99 ± 0.41 b 5.39 ± 0.36 c

1.89 ± 0.16 a 2.29 ± 0.04 b 2.53 ± 0.03 c

10.15 ± 0.62 a 13.18 ± 0.97 b 16.99 ± 0.23 c

7

Control at 20 ◦ C Control 2,4-D

8.18 ± 0.08 a 6.40 ± 0.17 b 7.70 ± 0.01 c

5.14 ± 0.14 a 4.49 ± 0.25 b 5.68 ± 0.35 c

1.79 ± 0.17 a 2.25 ± 0.11 b 2.43 ± 0.10 c

7.48 ± 0.55 a 10.01 ± 0.94 b 16.04 ± 0.68 c

14

Control at 20 ◦ C Control 2,4-D

10.72 ± 0.16 a 7.98 ± 0.03 b 8.63 ± 0.29 c

7.37 ± 0.29 a 4.92 ± 0.33 b 6.66 ± 0.49 c

1.14 ± 0.14 a 2.39 ± 0.04 b 2.66 ± 0.07 c

3.92 ± 0.41 a 5.31 ± 0.25 b 7.59 ± 0.55 c

21

Control at 20 ◦ C Control 2,4-D

9.73 ± 0.25 a 9.75 ± 0.01 a 10.17 ± 0.25 a

8.17 ± 0.41 a 6.85 ± 0.89 b 8.14 ± 0.58 a

0.27 ± 0.04 a 0.77 ± 0.01 b 1.38 ± 0.02 c

3.91 ± 0.79 a 3.38 ± 0.32b 4.62 ± 0.34 a

0

Fruit were treated with 150 mg L−1 2,4-D and vacuum-infiltrated under low pressure (−50 kPa) at 25 ◦ C for 5 min. After treatment, control and treated-fruit were stored at 4 ◦ C for 7 days of storage, and then transferred to 20 ◦ C for 14 days (shelf-life period). Additional fruit were stored directly at 20 ◦ C. The total sugar (TSC), soluble solids (SSC), chlorophyll contents and titratable acidity (TA) were evaluated during storage. Each value is the mean for three replicates and vertical bars indicate the standard deviation.

Table 2 Effect of 2,4-D treatment on sensory quality of mango fruit

fruit treated by exogenous H2 O2 than those in control fruit during the whole of storage.

Treatments

Appearance

Taste

Texture

Odor

Control 2,4-D

0.83 5.17

3.58 6.75

4.83 6.33

4.67 5.83

Fruit were treated with 150 mg L−1 2,4-D and vacuum-infiltrated under low pressure (−50 kPa) at 25 ◦ C for 5 min. After treatment control and treated-fruit were stored at 4 ◦ C for 7 days of storage, and then transferred to 20 ◦ C for 21 days (shelf-life period). Then general appearance, texture, odor and taste of the fruit were evaluated by a trained panel. Scores were given between 1 (lowest) and 9 (highest). The average response was calculated for each attribute.

Fig. 4. Effect of 2,4-D treatment on malondialdehyde of mango fruit. Mango fruit were treated with 150 mg L−1 2,4-D solution and vacuum-infiltrated under low pressure (−50 kPa) at 25 ◦ C for 5 min. After treatment, all fruit were stored at 4 ◦ C for 7 days of storage, and then transferred to 20 ◦ C for 14 days (shelf-life period). The MDA content was evaluated at 20 ◦ C for 7 and 14 days. Each value is the mean for three replicates and vertical bars indicate the standard deviation.

3.7. Effect of 2,4-D treatment on antioxidant enzymes activities of mango fruit peel during cold storage As shown in Fig. 6, activities of CAT, SOD, APX and GR in fruit were notably enhanced by 2,4-D treatment during the whole storage. CAT, SOD, APX and APX activities in 2,4-Dtreated fruit were 308.3, 10.9, 292.7 and 30.1% higher than those in the controls on day 7 of storage at 4 ◦ C. Activities of all the enzymes in 2,4-D-treated mango fruit were still significantly (P < 0.05) higher than those in control fruit after the fruit were

Fig. 5. Effect of 2,4-D treatment on H2 O2 level of mango fruit peel. Mango fruit were treated with 150 mg L−1 2,4-D or 100 mM H2 O2 and vacuum-infiltrated under low pressure (−50 kPa) at 25 ◦ C for 5 min. After treatment, control and treated-fruit were stored at 4 ◦ C for 7 days of storage, and then transferred to 20 ◦ C for 14 days (shelf-life period).

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Fig. 6. Effect of 2,4-D treatment on antioxidant enzymes activities of mango fruit peel. Mango fruit were treated with 150 mg L−1 2,4-D or 100 mM H2 O2 and vacuum-infiltrated under low pressure (−50 kPa) at 25 ◦ C for 5 min. After treated, control and treated-fruit were stored at 4 ◦ C for 7 days of storage, and then transferred to 20 ◦ C for 14 days (shelf-life period).

Fig. 7. Effect of 2,4-D treatment on endogenous hormones of mango fruit peel. Mango fruit were treated with 150 mg L−1 2,4-D or 100 mM H2 O2 and vacuuminfiltrated under low pressure (−50 kPa) at 25 ◦ C for 5 min. After treatment, control and treated-fruit were stored at 4 ◦ C for 7 days of storage, and then transferred to 20 ◦ C for 14 days (shelf-life period).

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transferred to 20 ◦ C for ripening. Like the 2,4-D treatment, H2 O2 also increased CAT and SOD activities (Fig. 6A and B). CAT and SOD activity in H2 O2 -treated fruit was 108.9 and 14.7% higher, respectively than that in the controls on day 7 of storage at 4 ◦ C. However, activities of antioxidant enzymes including APX and GR in fruit were significantly reduced by the H2 O2 treatment. The APX and GR activities in fruit treated with H2 O2 were 10.1 and 40.6% lower, respectively than those in controls on day 7 of storage at 4 ◦ C (Fig. 6C and D). 3.8. Effect of 2,4-D treatment on endogenous hormones of mango fruit peel during cold storage As shown in Fig. 7, levels of endogenous abscisic acid (ABA) and gibberellin (GA3 ) in fruit were significantly (P < 0.05) enhanced by 2,4-D treatment during storage. ABA and GA3 levels in 2,4-D-treated fruit were 40.2 and 72.6% higher, respectively than those in controls on day 7 of storage at 4 ◦ C (Fig. 7A and C). However, ABA levels in H2 O2 -treated fruit were not different from the control. Levels of ABA and GA3 in 2,4-D-treated fruit were still markedly higher than those in control fruit after the fruit were transferred to 20 ◦ C for ripening. Unlike ABA or GA3 , indole-3-acetic acid (IAA) and zeatin riboside (ZR) levels in the fruit were reduced by the 2,4-D treatment. As shown in Fig. 7B and D, IAA and ZR levels in 2,4-D-treated fruit were 16.9 and 18.1% lower, respectively than those in controls on day 7 of storage at 4 ◦ C. However, H2 O2 treatment increased the IAA levels in the fruit (Fig. 7B); in H2 O2 -treated fruit they were 4.2 or 21.9% higher, respectively than that in controls on day 3 or 7 during storage at 4 ◦ C. In accordance with 2,4-D, H2 O2 treatment also reduced ZR activity (Fig. 7D). 4. Discussion Mangoes, like many other tropical and subtropical fruit, are susceptible to CI when held below some critical minimum temperature (Lizada, 1991; Pesis et al., 2000; Gonz´alez-Aguilar et al., 2001). The CI symptoms observed in this work included skin darkening, development of poor color and uneven ripening which were similar to the CI symptoms reported earlier in mango fruit (Chaplin et al., 1991; Lederman et al., 1997). Various methods have been reported with a limited success in reducing the CI symptoms in mango fruit, including modified atmospheres (Pesis et al., 2000), heat treatment (McCollum et al., 1993) and methyl jasmonate (Gonz´alez-Aguilar et al., 2001). Our results showed that treatment with 2,4-D prior to low-temperature storage significantly reduced CI symptoms in mango fruit (P < 0.01). The previous studies have showed that 2,4-D treatment could control the development of postharvest disease in mangoes under normal temperatures (Johnson et al., 1998; Kobiler et al., 2001). Similar results were observed in this study, which could result from inhibiting the incidence of fungi, slowing the ripening of fruit or preventing the decrease in antifungal compounds (Coggins, 1969; Prusky and Keen, 1993; Kobiler et al., 2001). Further studies are necessary, however, to elucidate the mechanisms by which 2,4-D reduces CI and pathogen invasion in mango fruit.

Moreover, our results also showed that 2,4-D could maintain quality of fruit during prolonged storage. This result agrees with that reported previously for ‘Tommy Atkins’ mangoes (Kobiler et al., 2001). Effects of 2,4-D treatment on other parameters, including delaying the decline in TA and chlorophyll contents, also showed that the shelf-life of mango fruit was prolonged by 2,4-D. According to previous studies, mango fruit with severe chilling cannot ripen normally and not reach normal total soluble sugar (TSS) levels (Lederman et al., 1997; Gonz´alez-Aguilar et al., 2001). We showed that the fruit ripening after storage at 4 ◦ C was partially inhibited. However, the 2,4-D-treated fruit could ripen normally after storage at 4 ◦ C for 7 days, as indicated by TSS and SSC levels. As well, sensory analysis revealed that the fruit treated with 2,4-D had good eating qualities. These results suggest that 2,4-D treatment should have a high potential for practical application in mango fruit. An increasing amount of evidence suggests that oxidative stress is an early response of fruit and vegetables to chilling injury since it might initiate membrane degradation, causing lipid peroxidation (Wise and Naylor, 1987; Hariyadi and Parkin, 1991; Dat et al., 2000; Shewfelt and del Rosario, 2000). In this study we showed that the increase of malondialdehyde (MDA) level in the fruit stored at low temperature, which could represent the extent of membrane lipid peroxidation and membrane integrity, had been effectively inhibited by 2,4-D treatment. A direct result of stress-induced cellular changes is the enhanced accumulation of toxic compounds in cells that include active oxygen species (AOS). Hydrogen peroxide (H2 O2 ), as a stable molecular AOS species, has been considered as a signal molecule in the environment stress response (Foyer et al., 1997). A transient increase in H2 O2 was suggested to signal activation of protective mechanisms for acclimation to chilling (Prasad et al., 1994). Our results showed that 2,4-D treatment stimulated endogenous H2 O2 production and alleviated the CI symptoms during cold storage; however, exogenous H2 O2 treatment inhibited the endogenous H2 O2 levels and aggravated the CI symptoms. These results suggest that the CI incidence in mango fruit, to some extent, seems to be negatively correlated to the level of endogenous H2 O2 . However, some studies reported that excess H2 O2 production related to lipid peroxidation is detrimental to plant tissue (Asada, 1992; Lacan and Baccou, 1998; Larrigaudi`ere et al., 2004). A possible hypothesis is that the equilibrium between H2 O2 production and defence mechanisms in favor of H2 O2 could be established. This balance between the formation and detoxification of activated oxygen species is critical to cell survival during cold storage (Zhang et al., 1995). Chilling stress, along with many other stresses, shifts the AOS balance of the cell and, increases the levels of AOS (Foyer et al., 1994; Hodges, 2003) and thus the level of oxidative injury experienced by the cell. Plants have evolved efficient antioxidant systems to scavenge AOS. SOD and CAT are part of the primary defence system against AOS (Davies, 1986; Mittler, 2002). SOD plays a key role in H2 O2 biosynthesis, converting it from O2 •− , and CAT is involved in the degradation of H2 O2 . Sala (1998) has reported that higher CAT and SOD activities were related to the

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chilling tolerance of mandarin fruit. Our results showed that the activities of these antioxidant enzymes in the mango fruit were enhanced by 2,4-D treatment. Similarly, H2 O2 treatment also induced the activities of CAT and SOD, although more severe chilling injury was observed in H2 O2 -treated fruit, which could be explained by exogenous H2 O2 treatment directly leading to fruit damage and subsequent CI symptoms. Induction of SOD and CAT activities could not counteract the effects of exogenous H2 O2 . In plant cells, APX is also an important antioxidant enzyme in scavenging or utilizing H2 O2 (Asada, 1992). GR is known to act in conjunction with APX to metabolism H2 O2 to water through an AsA-GSH cycle (Asada, 1992). In our experiments, 2,4-D treatment-induced APX and GR activities, which could be involved in inhibition of AOS stress. However, APX and GR activities were suppressed by exogenous H2 O2 , which could result in AOS damage. Similar results also showed that increasing APX and GR activities were involved with alleviating chilling injury of peach fruit by SA (Wang et al., 2006). Wang (1996) also reported that the enhancement of chilling resistance in squash could be related to the increase in APX activity. Therefore, we concluded that the enzymes APX and GR might be more necessary to protect mango fruit from chilling injury during cold storage than SOD and CAT. This is in agreement with Foyer et al. (1994), who suggested that an increase in several components of the antioxidative defence system (SOD, APX and GR activities) simultaneously would be necessary to increase stress tolerance of plants. The involvement of endogenous hormones in chilling sensitivity of mango fruit remains unclear. Many previous studies have shown that endogenous ABA levels are associated with chilling susceptibility of plants. Endogenous ABA levels and chilling tolerance are increased by low-temperature hardening (12 ◦ C) in courgette squash (Wang, 1991) and by cold acclimation in other plant species (Ryu and Li, 1994). Kawada (1980) observed parallel variations between seasonal resistance to CI of grapefruit and levels of ABA at time of harvest. Raghavan et al. (2006) has shown that 2,4-D could induce gene expression associated with ABA pathways and biosynthesis in Arabidopsis. In this study, 2,4-D but not H2 O2 treatment induced an increase in endogenous ABA levels, which suggests that endogenous ABA might act as protection from CI in mango fruit. Nevertheless, the potential role of this hormone as a protective stress signal in mango fruit is still controversial. It has been demonstrated in ‘Fortune’ citrus that treatments which increased ABA levels favored development of CI (Lafuente et al., 1997; Gosalbes et al., 2004). Therefore, the regulation of CI observed in mango fruit in response to 2,4-D could be a direct result of stress-induced ABA or by a more general stress response in the fruit. Another notable difference between 2,4-D-treated fruit and H2 O2 -treated fruit was represented in endogenous indole-3-acetic acid (IAA) levels. Frankel and Dyck (1973) showed that the decrease of IAA in pear was necessary for the onset of fruit ripening. Therefore, decreasing the IAA levels might be beneficial to increase resistance to chilling stress. Higher levels of gibberellin acid (GA3 ) in the fruit treated with 2,4-D than in control and H2 O2 -treated fruit could also be available for alleviating CI of mango fruit.

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Zeatin riboside (ZR) are cytokinins that play an essential role in regulating plant growth and development (Silverman et al., 1998). Additional work has shown that elevated ZR levels in plants were related to improved cold stress resistance (Bouza et al., 1994; Barna et al., 1997; Sz´ek´acs et al., 2000). However, in our study, both 2,4-D and H2 O2 inhibited the endogenous ZR levels in mango peel, the reasons for which are still unknown. Few studies have shown a good relation among the hormones, enzymes and physical parameters and the possible development of CI symptoms in fruit. The increase in enzyme activities could be a hormone-induced response, which is only stimulated in mango fruit under chilling temperatures. A similar study has shown that salicylic acid, as a hormonal substance, could alleviate the chilling injury of peach fruit, this being attributed to its ability to induce antioxidant systems (Wang et al., 2006). Hormones and endogenous H2 O2 , as signal molecules, may play a critical role in inducing the expression of anti-cold genes involved with reducing CI (Wang et al., 2007). The induction of antioxidant enzymes by 2,4-D in mango fruit during lowtemperature storage may be regulated at transcriptional and translational levels, and is related to reduction in CI symptoms. In conclusion, 2,4-D can alleviate chilling injury and maintain postharvest qualities of mango fruit during cold storage and shelf-life. In addition, application of 2,4-D to mango fruit can enhance activities of antioxidant enzymes, levels of endogenous ABA/GA3 and decrease IAA levels. Acknowledgements This research was supported by the 11th Five-Year Key Technologies R&D Program of China (2006BAD22B03; 2006BAD22B04). References AOAC, 1996. Official Methods of Analysis of AOAC International, 16th ed. AOAC International (Computer file), Gaithersburg, MD. Asada, K., 1992. Ascorbate peroxidase—a hydrogen-scavenging enzyme in plants. Physiol. Plant. 85, 235–241. Barna, B., Ad´am, A.L., Kir´aly, Z., 1997. Increased levels of cytokinin induce tolerance to necrotic diseases and various oxidative stress-causing agents in plants. Phyton 37, 25–31. Bouza, L., Jacques, M., Sotta, B., Miginiac, E., 1994. The reactivation of tree peony (Paeonia suffruticosa Andr.) vitroplants by chilling is correlated with modifications of abscisic acid, auxin and cytokinin levels. Plant Sci. 97, 153–160. Chaplin, G.R., Cole, S.P., Landrigan, M., Nuevo, P.A., Lam, P.A., Graham, D., 1991. Chilling injury and storage of mango (Mangifera indica L.) fruit held under low temperatures. Acta Hortic. 291, 461–471. Coggins, C.W., 1969. Use of growth regulators to delay maturity and prolong shelf-life of citrus. Acta Hortic. 34, 469–472. Cohen, E., 1977. Some physiological aspects of citrus fruit degreening. Proc. Int. Soc. Citricult. 1, 215–219. Dat, J., Vandenabeele, E., Vranova, M., Van Montagu, M., Inze, D., Van Breusegem, F., 2000. Dual action of the active oxygen species during plant stress responses. Cell. Mol. Life Sci. 57, 779–795. Davies, K.J., 1986. Intracellular proteolytic systems may function as secondary anti-oxidant defense: a hypothesis. Free Radi. Biol. Med. 2, 155–173. Del-Valle, V., Hern´andez-Mu˜noz, P., Guarda, A., Galotto, M.J., 2005. Development of a cactus-mucilage edible coating and its application to extend strawberry shelf-life. Food Chem. 91, 751–756.

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