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Chilling injury in mango fruit peel: Cultivar differences are related to the activity of phenylalanine ammonia lyase
Postharvest Biology and Technology 62 (2011) 59–63
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Chilling injury in mango fruit peel: Cultivar differences are related to the activity of phenylalanine ammonia lyase Sugunya Chidtragool a,1 , Saichol Ketsa a,b,c,∗ , Judith Bowen d , Ian B. Ferguson d , Wouter G. van Doorn e a
Department of Horticulture, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand Postharvest Technology Innovation Center, Commission on Higher Education, Bangkok 10400, Thailand c Academy of Science, The Royal Institute, Thailand d Plant and Food Research Institute, Private Bag 92169, Auckland, New Zealand e Mann Laboratory, Department of Plant Sciences, University of California, Davis 95616, CA, USA b
a r t i c l e
i n f o
Article history: Received 5 November 2010 Accepted 25 April 2011 Keywords: Browning Low temperature Peel Pulp
a b s t r a c t In mango (Mangifera indica) cv. Nam Dok Mai fruit, stored at 4 ◦ C, peel browning occurred within 9 d, while no browning was found in cv. Choke Anan fruit stored at 4 ◦ C for 30 d. During 6 d of shelf life at 27–28 ◦ C, following various periods of low temperature storage, the peel browning in cv. Nam Dok Mai (if not yet maximal) became worse, whereas little browning was observed in cv. Choke Anan fruit. The pulp of the fruit of both cultivars did not show browning during the 4 ◦ C storage, but the pulp of cv. Nam Dok Mai exhibited some browning during shelf life if the fruit had been stored at 4 ◦ C for more than 18 d. Peel and pulp color were not correlated with total free phenolics. A high correlation coefficient was observed between peel browning and PAL activity in the peel, while a very low correlation was found with peel catechol oxidase activity. The browning in the pulp was not correlated with the measured enzyme activities. The data therefore show a relation between PAL activity in the peel and low temperature-induced peel browning. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Many fruits, vegetables, and ornamentals of tropical or subtropical origin cannot be stored at low temperature. Chilling injury (CI) is often defined as the appearance of injury symptoms after a critical period of exposure to temperatures below 15–10 ◦ C, but above freezing point (Wang, 1993; Kaniuga, 2008). Produce that has been subjected to prolonged low temperature storage often still looks fine at the end of storage, but CI is rapidly manifested after transfer to warmer temperature. Depending on the commodity, the CI symptoms include surface lesions, surface discoloration, internal discoloration, water-soaking of the tissue, and failure to ripen normally (Saltveit and Morris, 1990). Low temperature-induced browning has been associated with enzymatic conversion of free phenolics to o-quinones, which then become non-enzymatically polymerized to brown pigments. Phenylalanine ammonia lyase (PAL) is an enzyme that starts the onset of the synthesis of free phenolics. In some fruit PAL activity
∗ Corresponding author at: Department of Horticulture, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand. Tel.: +66 2 5614891; fax: +66 2 5791951x112. E-mail address: [email protected] (S. Ketsa). 1 Former Ph.D. student, deceased. 0925-5214/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2011.04.011
has been suggested to be the limiting factor for low-temperatureinduced browning. In banana, for example, a correlation was found between chilling-induced peel browning and PAL activity (Nguyen et al., 2003) and in mandarin and oranges cold storage also induced PAL activity related to chilling injury symptoms (Lafuente et al., 2003). Polyphenol oxidase (PPO), or more specifically catechol oxidase, uses free phenolics as its substrate. Depending on the species, catechol oxidase activity can also be a limiting factor for tissue browning. For example, its activity correlated with the low-temperature-induced browning symptoms in some leafy vegetables (Baritaux et al., 1991; Ose et al., 1995). Loss of membrane integrity might be important in the effect of low temperature (Saruyama et al., 2005). Catechol oxidase is mainly localized in the plastids, while free phenolics are located in the vacuole (Staehelin and Newcomb, 2000). Breakdown of cellular compartments, by loss of membrane integrity, is therefore apparently required for the enzyme to react with the substrate (Veltman and Peppelenbos, 2003; Saruyama et al., 2005). Mango fruit stored at 12 ◦ C for prolonged periods showed red and green spots at the lenticels (Pesis et al., 2000). When stored below 12–13 ◦ C, the peel also showed browning (González-Aguilar et al., 2001; Nair and Singh, 2003; Wang et al., 2008). Some mango cultivars are much more chilling sensitive than others (Phakawat mongkol et al., 2004). Our preliminary experiments indicated that peel browning in low-temperature stored cv.
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Nam Dok Mai mangoes was much more frequent than in cv. Choke Anan. In the present study we compared the details of peel and pulp discoloration in these two cultivars, both during low temperature storage and during subsequent shelf life. We hypothesized that the changes would correlate with the levels of free phenolics (the substrate of the browning reactions), and with the activities of PAL and catechol oxidase, as both enzymes have been suggested to be involved in these reactions. 2. Materials and methods 2.1. Plant material Experiments used 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. 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 mangoes were then allowed to air dry at room temperature (24–30 ◦ C) before further use. The fruit were placed in cardboard boxes and kept at 4 ◦ C (87% RH). Fruit were randomly sampled every 3 d to determine chilling injury, total free phenolics and the activities of PAL and catechol oxidase, both in the peel and pulp. Fruit of a second group were transferred to room temperature (27–28 ◦ C, 67–69% RH) for 6 d, and the same parameters were measured at 3 d intervals. 2.2. Effect of tropolone, an inhibitor of catechol oxidase Mango fruit cv Nam Dok Mai were placed in 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 mM aqueous tropolone solutions for 6 h at 22 ◦ C, while the control fruit were placed in water at the same temperature for the same duration. All mango fruit were allowed to air dry at 25 ◦ C (72–75% RH) and were placed at 4 ◦ C (87% RH). The time to peel blackening was determined by daily inspection. 2.3. 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 = none, 1 = 10% of the peel or pulp are brown, 2 = 11–25% of peel or pulp are brown, 3 = 26–40%, 4 = 40–50%, 5 = >50%. The CI index was calculated as follows (Concellón et al., 2004):
for 1 min, which contained 3.95 mL of 0.10 M potassium phosphate buffer (pH 7) and 1 mL of the supernatant. Extraction and assay of PAL was carried out as described by Ke and Saltveit (1986). Peel tissue (1 g) was homogenized with 4 mL of 50 mM borate buffer (pH 8.5) containing 5 mM 2-mercaptoethanol and 0.4 g polyvinylpyrrolidone (PVP). The homogenate was filtered through 3 layers of cotton cloth and then centrifuged at 13,522 × g for 20 min at 4 ◦ C. PAL activity in the supernatant was assayed by adding 0.7 mL of 100 mM l-phenylalanine and 3 mL of 50 mM borate buffer (pH 8.5) to 0.3 mL of the extract. After heating to 40 ◦ C for 1 h, the reaction was stopped by adding 0.1 mL of 5 N of HCl. PAL activity was determined by measuring the absorbance of assay mixture at 290 nm for the production of cinnamic acid. Protein content in the enzyme extracts was estimated using the standard Bradford method. Specific activity of the enzyme was expressed as units per kg protein. Each treatment had five replications and each replication consisted of the whole peel from 10 to 15 fruit, pooled together. Determinations of enzyme activity were made twice in each extract. 2.5. Total free phenolics Total free phenolics in the peel and pulp was estimated colorimetrically, using a procedure that was slightly modified from the one described by Singleton et al. (1999). Five grams of peel or pulp were extracted at room temperature and in the absence of light, using pure methanol containing 1% 2,6-di-tert-butyl-4methylphenol (BHT) as an antioxidant and an ultrasonic bath. Samples were extracted twice with 10 mL of the methanol solvent for 1 h, 10 mL for 30 min, and then with 5 mL for 30 min. The extracts were combined to a final volume of 25 mL. Phenolic compounds were determined using the Folin–Ciocalteu reagent. A mixture of 0.5 mL of extract (or spectrophotometric standard), 0.5 mL of Folin–Ciocalteu reagent and 10 mL of Na2 CO3 1 M were introduced in a 25 mL volumetric flask. After reaction for 1 h, the solutions were measured at 750 nm. Ellagic acid has been reported to be a major phenolic compound in mango fruit (Schieber et al., 2000). Total free phenolics were therefore expressed as ellagic acid equivalents. 2.6. Statistical analysis In the test on visible browning scores, three replications containing 12–15 fruit each were used in each treatment. Data were compared by analysis of variance and calculation of Least Significant Difference (LSD). Regression analyses (r value) were performed between catechol oxidase activity and browning of peel and pulp. The experiments were repeated once at a later date.
CI index
=
(injury classification level × number of fruit at that level)
3. Results
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 one used for peel browning. 2.4. Catechol oxidase and PAL Catechol oxidase was extracted and assayed by the method of Yang et al. (2001). Peel tissue (1 g) was homogenized with 5 mL of 0.1 M potassium phosphate buffer (pH 7) and 0.2 g polyvinylpyrrolidone (PVP). The homogenate was filtered through 3 layers of cotton cloth and then centrifuged at 13,522 × g for 20 min at 4 ◦ C. One unit of catechol oxidase activity was determined by measuring the rate of increase in absorbance at 420 nm at 25 ◦ C
3.1. Browning, total free phenolics, PAL and catechol oxidase activities in the peel The peel of cv. Nam Dok Mai mangoes stored at 4 ◦ C did not exhibit discoloration until day 6. On day 9 the peel of several fruits had turned greyish brown and this discoloration rapidly increased in fruit stored for a longer period (Table 1). The peel of cv. Choke Anan mangoes did not show any color change (Table 1). Upon transfer to 27–28 ◦ C for 6 d (shelf life), at various intervals during cold storage, the peel of cv. Nam Dok Mai mango showed increased browning, if browning was not yet maximal (Table 1). The peel of cv. Choke Anan mango exhibited slight browning during shelf life, although only when taken out of low temperature storage on the last two sampling dates, i.e., day 27 and 30 (Table 1).
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Table 1 Browning of the peel of mango fruit cvs. Nam Dok Mai (䊉) and Choke Anan () during storage and after storage at 4 ◦ C for 6 d of shelf life at 27–28 ◦ C (B). Data are means ± SE of three replications (12–15 fruit each). Browning (scores)a
Treatment
Time in storage (d)
Choke Anan Peel (during storage) Peel (after storage) Pulp (during storage) Pulp (after storage) Nam Dok Mai Peel (during storage) Peel (after storage) Pulp (during storage) Pulp (after storage)
3
6
9
12
15
18
21
24
27
30
1.00 1.00 1.00 1.00
1.00 1.00 1.00 1.00
1.00 1.00 1.00 1.00
1.00b 1.00b 1.00b 1.00b
1.00b 1.00b 1.00b 1.00b
1.00b 1.00b 1.00b 1.00b
1.00b 1.00b 1.00b 1.00b
1.00c 1.00c 1.00c 1.00c
1.00c 1.00c 1.00c 1.00c
1.00c 1.20c 1.00c 1.00c
1.00b 1.25b 1.00b 1.00b
1.00 1.00 1.00 1.00
1.00 1.00 1.00 1.00
1.07 2.17 1.00 1.00
1.67a 2.50a 1.00b 1.00b
3.75a 3.50a 1.00b 1.00b
4.17a 4.50a 1.00b 1.00b
4.33a 4.80a 1.00b 1.00b
4.42a 5.00a 1.00c 2.00b
4.83a 5.00a 1.00c 2.50b
5.00a 5.00a 1.00c 4.17b
5.00a 5.00a 1.00b 5.00a
Values in same column, followed by different letters, are significantly different at P ≤ 0.01 (Analysis of variance and DMRT).
Total free phenolics levels in the peel of both cv. Nam Dok Mai and cv. Choke Anan mangoes were initially high, then decreased (Fig. 1A). A lower level of total free phenolics was correlated with a higher score of peel browning during cold storage (nonlinear correlation coefficient, r2 = 0.86; Fig. 4A). After 6 d of transfer the fruit from cold storage to 27–28 ◦ C the total free phenolics levels in the peel of cv. Nam Dok Mai mango were higher than that in cv. Choke Anan peel (Fig. 1B). During cold storage the PAL activity increased, more so in the peel of cv. Nam Dok Mai mango than that of cv. Choke Anan (Fig. 2A). The PAL activity in the peel of cv. Nam Dok Mai was higher than in cv. Choke Anan, on day 21–30 (Fig. 2A). The nonlinear correlation coefficient between PAL activity in the peel and peel browning in cold-stored cv. Nam Dok Mai was high (r2 = 0.99; Fig. 4B). Upon transfer of the fruit, at various intervals of cold storage, to 27–28 ◦ C, the PAL activity in both cultivars was low (Fig. 2B). A low correlation coefficient was found between the activity of PAL in the peel and peel browning during shelf life (data not shown). The catechol oxidase activity in the peel of cv. Nam Dok Mai mango was higher than that of cv. Choke Anan, both during storage
6
3.0
5
2.5
4
2.0
1.2
1.5
0.9
1.0
0.6
0.5
0.3
-1
2
10
1
0
0
B LSD
0.05
LSD
= 7.72
= 0.68
D
0.05
6 5
40
4 30 3 20
2
10
1
0
0 0 3 6 9 12 15 18 21 24 27 30
0 3 6 9 12 15 18 21 24 27 30
LSD = 0.27 0.05
1.8 1.5
0.0 2.5
B LSD = 0.44 0.05
D LSD = 0.05 0.05
0.0 1.5
2.0
1.2
1.5
0.9
1.0
0.6
0.5
0.3
0.0
0 3 6 9 12 15 18 21 24 27 30
0 3 6 9 12 15 18 21 24 27 30
-1
3 20
LSD = 0.53 0.05
C
-3
30
A
PAL activity x 10 (units kg )
0.05
-1
= 7.63
40
Total free phenolics (g kg )
During cold storage of both cv. Nam Dok Mai and cv. Choke Anan mango fruit no browning or other discoloration was found in the pulp. Six days after transfer to 27–28 ◦ C of cv. Nam Dok Mai fruit, pulp browning was only observed in fruit that had been stored at the low temperature for 21 d or longer. Browning was higher in fruit that had been stored for a longer period. In contrast, during shelf life the pulp of cv. Choke Anan mangoes did not show browning, even if the fruit had been stored in the cold for 29 d (data not shown).
-3
0.05
C
-1
LSD
= 0.66
LSD
Total free phenolics (g kg )
A
50
3.2. Browning, total free phenolics, PAL and catechol oxidase activities in the pulp
7
60 50
at 4 ◦ C (Fig. 3A) and after transfer to 27–28 ◦ C (Fig. 3B). Six days after transfer of the fruit to 27–28 ◦ C, the catechol oxidase activity in the peel of both cultivars showed a maximum on day 12 of taking the fruit out of cold storage (Fig. 3B). No obvious relation was observed between the catechol oxidase activity in the peel and peel browning (Fig. 4B). A low correlation coefficient was also found between the activity of catechol oxidase in the peel and peel browning during shelf life (data not shown).
PAL activity x 10 (units kg )...
a
0
0.0
Time in storage (d)
Time in storage (d) Fig. 1. Total free phenolics of the peel (A and B) and pulp (C and D) of mango fruit cvs. Nam Dok Mai (䊉) and Choke Anan () during storage at 4 ◦ C (A and C) and after storage at 4 ◦ C and 6 d of shelf life at room temperature (27–28 ◦ C; B and D). Data in B and D refer to the date the fruit was transferred from cold storage to room temperature. Data are means ± SE of three replications (12–15 fruit each).
Fig. 2. Phenylalanine ammonia lyase (PAL) activity of the peel (A and B) and peel (C and D) of mango fruit cvs. Nam Dok Mai (䊉) and Choke Anan () during storage at 4 ◦ C (A and C) and after storage at 4 ◦ C and 6 d of shelf life at room temperature (27–28 ◦ C; B and D). Data in B and D refer to the date the fruit was transferred from cold storage to room temperature. Data are means ± SE of three replications (12–15 fruit each).
S. Chidtragool et al. / Postharvest Biology and Technology 62 (2011) 59–63
5 LSD
0.05
4
LSD
C
= 0.66
0.05
5 4
3
3
2
2
1
1
0
0
B
5
= 0.71
LSD
D LSD
0.05
= 0.44
0.05
5
4
4
3
3
2
2
1
1
0
-3
-3
-1
-1
Catechol oxidase activity x 10 (units kg )
A
= 0.59
Catechol oxidase activity x 10 (units kg )
62
0 0 3 6 9 12 15 18 21 24 27 30
0 3 6 9 12 15 18 21 24 27 30
During cold storage the catechol oxidase activity in the pulp of cv. Nam Dok Mai mango was considerably higher than that of cv. Choke Anan fruit (Fig. 3B). Six days after transfer to 27–28 ◦ C, the catechol oxidase activities in the pulp of both cultivars were very low (Fig. 3D). Very low linear correlation coefficients were found between pulp browning and the activities of the two measured enzymes (data not shown). 3.3. Effect of tropolone on browning Peel browning of the control mango fruit was similar to that in mango fruit treated with 0.1 or 0.2 mM tropolone, hence there was no effect of low tropolone concentrations. This was in contrast to the negative effect of higher tropolone concentrations. Compared to the controls, the peel of mango fruit treated with 0.4–3.2 mM tropolone turned more brown during storage at 4 ◦ C. The higher the concentration used, the more peel browning was found. In contrast, the tropolone treatments did not affect pulp browning (results not shown).
Time in storage (d) 4. Discussion Fig. 3. Catechol oxidase activity of the peel (A and B) and pulp (C and D) of mango fruit cvs. Nam Dok Mai (䊉) and Choke Anan () during storage at 4 ◦ C (A) and after storage at 4 ◦ C for 6 d of shelf life at 27–28 ◦ C (B). Data in B and D refer to the date the fruit was transferred from cold storage to room temperature. Data are means ± SE of three replications (12–15 fruit each).
-1
(g kg )...
50 45 40 35 30 25 20 15 10 5 0
A R² = 0.86
R² = 0.99
3.5
B
3.0
-1
(units kg )...
Activity of catechol oxidase and PAL x 10
-3
Total free phenolics
Total free phenolics in the pulp of both cv. Nam Dok Mai and cv. Choke Anan mangoes were similar and did not change consistently during storage at 4 ◦ C (Fig. 1C) and upon transfer to 27–28 ◦ C (Fig. 1D). Total free phenolics in the pulp of both cultivars were about 9–10 times lower than in the peel, throughout low temperature storage and shelf life (Fig. 1). PAL activities in the pulp of both cv. Nam Dok Mai and cv. Choke Anan mangoes were much lower than that of the peel (Fig. 2). The PAL activity was generally higher in Nam Dok Mai than in Choke Anan (Fig. 2B). The activity was very low 6 d after transfer to 27–28 ◦ C (Fig. 2D).
2.5 2.0 1.5 1.0 0.5 0.0 0
1
2
3
4
5
6
Browning (scores) Fig. 4. Relations between peel browning, total phenolics, and the activities of PAL and catechol oxidase in the peel of mango fruit cv. Nam Dok Mai stored at 4 C. (A) Relationship with total phenolics concentrations. (B) Relationship with the activity of PAL (solid symbol) and catechol oxidase (open symbol).
The data show that cv. Nam Dok Mai mango fruit showed peel damage during as little as 9 d of storage at 4 ◦ C, while in cv. Choke Anan fruit no peel browning was found during the 30 d of such cold storage. Even during the subsequent 6 d of shelf life at 27–28 ◦ C cv. Choke Anan fruit showed no peel browning if the fruit had been stored at 4 ◦ C for as long as 24 d. The level of total free phenolics in the peel was similar in both cultivars. However, a rather high nonlinear inverse correlation between total phenolics and peel browning of ‘Nam Dok Mai’ was observed. This suggests that total free phenolic levels were ratelimiting in the peel browning reactions. A similar effect has been found in banana (Nguyen et al., 2003). Low temperature storage had less effect on pulp color than on peel color, but again cv. Nam Dok Mai was more sensitive. Browning compounds are synthesized from free phenolic acids through PAL (Camm and Towers, 1977). PAL activity in the peel was much higher than that of the pulp, which was correlated with the much higher level of total free phenolics in the peel compared with the pulp. During low temperature storage the PAL activity in the peel of both cultivars increased. In cv. Nam Dok Mai the increase was higher, from day 18 of storage. However, browning of the peel was already found from day 9 of cold storage. The measured in vitro PAL activity, therefore, did not explain the early peel browning in cold-stored Nam Dok Mai fruit. Nonetheless, a correlation was found at later points in time. When expressing the data of peel browning against PAL activity, both measured every 3 d, a high nonlinear correlation was observed. In citrus fruit, Lafuente et al. (2003) and banana, Nguyen et al. (2003) similarly found that low temperature induced an increase in PAL activity in the peel. No relation was found between PAL activity in the pulp of mango and pulp browning. During low temperature storage the in vitro catechol oxidase in the peel of cv. Nam Dok Mai was considerably higher than in cv. Choke Anan. However, when expressing the data of peel browning against catechol oxidase activity, no obvious relation was observed. This suggests that catechol oxidase was not rate liming for peel browning. Tropolone is a rather specific inhibitor of catechol oxidase (Richardson et al., 1997). In our tests it had no effect at the relatively low concentrations used (0.1 and 0.2 mM for 6 h) and was toxic at higher concentrations. Tropolone thus did not reduce low temperature-induced browning, also indicating that catechol oxidase activity was not a limiting factor.
S. Chidtragool et al. / Postharvest Biology and Technology 62 (2011) 59–63
It is concluded that peel browning rather than pulp browning limited the cold storage period of the two mango cultivars tested. A high nonlinear correlation coefficient was found between peel browning and PAL activity, while the correlation coefficient with catechol oxidase was very low. No obvious relation was found between pulp browning and the measured enzyme activities. Acknowledgements The work was financially supported by the Thailand Research Fund (TRF), Postharvest Technology Innovation Center (PHTIC), Commission on Higher Education (CHE), Ministry of Education, and Kasetsart University Research and Development (KURDI). This study was part of the planned Ph.D. dissertation of Sugunya Chidtragool. Regrettably, she passed away while working at HortResearch in Auckland, NZ. This paper is dedicated to her memory. References Baritaux, O., Amiot, M.J., Nicolas, J., 1991. Enzymatic browning of basil (Ocimum basilicum L.): studies on phenolic compounds and polyphenol oxidase. Sci. Aliment. 11, 49–62. Camm, E.L., Towers, G.H.N., 1977. Phenylalanine ammonia lyase. Phytochemistry 12, 961–973. ˜ Concellón, A., Anón, M.C., Chaves, A.R., 2004. Characterization and changes in polyphenol oxidase from eggplant fruit (Solanum melongena L.) during storage at low temperature. Food Chem. 88, 17–24. González-Aguilar, G.A., Buta, J.G., Wang, C.Y., 2001. Methyl jasmonate reduces chilling injury symptoms and enhances colour development of ‘Kent’ mangoes. J. Sci. Food Agric. 81, 1244–1249. Kaniuga, Z., 2008. Chilling response of plants: importance of galactolipase, free fatty acids and free radicals. Plant Biol. 10, 171–184. Ke, D., Saltveit, M.E., 1986. Effects of calcium and auxin on russet spotting and phenylalanine ammonia-lyase activity in iceberg lettuce. Hortscience 21, 1169–1171. Lafuente, M.T., Zacarias, L., Martinez-Tellez, M.A., Sanchez-Ballesta, M.T., Granell, A., 2003. Phenylalanine ammonia-lyase and ethylene in relation to chilling injury as affected by fruit age in citrus. Postharvest Biol. Technol. 29, 308–311. Nair, S., Singh, Z., 2003. Pre-storage Ethrel dip reduces chilling injury, enhances respiration rate, ethylene production and improves fruit quality of ‘Kensington’ mango. Food Agric. Environ. 1, 93–97.
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Chilling injury in mango fruit peel: Cultivar differences are related to the activity of phenylalanine ammonia lyase Sugunya Chidtragool a,1 , Saichol Ketsa a,b,c,∗ , Judith Bowen d , Ian B. Ferguson d , Wouter G. van Doorn e a
Department of Horticulture, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand Postharvest Technology Innovation Center, Commission on Higher Education, Bangkok 10400, Thailand c Academy of Science, The Royal Institute, Thailand d Plant and Food Research Institute, Private Bag 92169, Auckland, New Zealand e Mann Laboratory, Department of Plant Sciences, University of California, Davis 95616, CA, USA b
a r t i c l e
i n f o
Article history: Received 5 November 2010 Accepted 25 April 2011 Keywords: Browning Low temperature Peel Pulp
a b s t r a c t In mango (Mangifera indica) cv. Nam Dok Mai fruit, stored at 4 ◦ C, peel browning occurred within 9 d, while no browning was found in cv. Choke Anan fruit stored at 4 ◦ C for 30 d. During 6 d of shelf life at 27–28 ◦ C, following various periods of low temperature storage, the peel browning in cv. Nam Dok Mai (if not yet maximal) became worse, whereas little browning was observed in cv. Choke Anan fruit. The pulp of the fruit of both cultivars did not show browning during the 4 ◦ C storage, but the pulp of cv. Nam Dok Mai exhibited some browning during shelf life if the fruit had been stored at 4 ◦ C for more than 18 d. Peel and pulp color were not correlated with total free phenolics. A high correlation coefficient was observed between peel browning and PAL activity in the peel, while a very low correlation was found with peel catechol oxidase activity. The browning in the pulp was not correlated with the measured enzyme activities. The data therefore show a relation between PAL activity in the peel and low temperature-induced peel browning. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Many fruits, vegetables, and ornamentals of tropical or subtropical origin cannot be stored at low temperature. Chilling injury (CI) is often defined as the appearance of injury symptoms after a critical period of exposure to temperatures below 15–10 ◦ C, but above freezing point (Wang, 1993; Kaniuga, 2008). Produce that has been subjected to prolonged low temperature storage often still looks fine at the end of storage, but CI is rapidly manifested after transfer to warmer temperature. Depending on the commodity, the CI symptoms include surface lesions, surface discoloration, internal discoloration, water-soaking of the tissue, and failure to ripen normally (Saltveit and Morris, 1990). Low temperature-induced browning has been associated with enzymatic conversion of free phenolics to o-quinones, which then become non-enzymatically polymerized to brown pigments. Phenylalanine ammonia lyase (PAL) is an enzyme that starts the onset of the synthesis of free phenolics. In some fruit PAL activity
∗ Corresponding author at: Department of Horticulture, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand. Tel.: +66 2 5614891; fax: +66 2 5791951x112. E-mail address: [email protected] (S. Ketsa). 1 Former Ph.D. student, deceased. 0925-5214/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2011.04.011
has been suggested to be the limiting factor for low-temperatureinduced browning. In banana, for example, a correlation was found between chilling-induced peel browning and PAL activity (Nguyen et al., 2003) and in mandarin and oranges cold storage also induced PAL activity related to chilling injury symptoms (Lafuente et al., 2003). Polyphenol oxidase (PPO), or more specifically catechol oxidase, uses free phenolics as its substrate. Depending on the species, catechol oxidase activity can also be a limiting factor for tissue browning. For example, its activity correlated with the low-temperature-induced browning symptoms in some leafy vegetables (Baritaux et al., 1991; Ose et al., 1995). Loss of membrane integrity might be important in the effect of low temperature (Saruyama et al., 2005). Catechol oxidase is mainly localized in the plastids, while free phenolics are located in the vacuole (Staehelin and Newcomb, 2000). Breakdown of cellular compartments, by loss of membrane integrity, is therefore apparently required for the enzyme to react with the substrate (Veltman and Peppelenbos, 2003; Saruyama et al., 2005). Mango fruit stored at 12 ◦ C for prolonged periods showed red and green spots at the lenticels (Pesis et al., 2000). When stored below 12–13 ◦ C, the peel also showed browning (González-Aguilar et al., 2001; Nair and Singh, 2003; Wang et al., 2008). Some mango cultivars are much more chilling sensitive than others (Phakawat mongkol et al., 2004). Our preliminary experiments indicated that peel browning in low-temperature stored cv.
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Nam Dok Mai mangoes was much more frequent than in cv. Choke Anan. In the present study we compared the details of peel and pulp discoloration in these two cultivars, both during low temperature storage and during subsequent shelf life. We hypothesized that the changes would correlate with the levels of free phenolics (the substrate of the browning reactions), and with the activities of PAL and catechol oxidase, as both enzymes have been suggested to be involved in these reactions. 2. Materials and methods 2.1. Plant material Experiments used 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. 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 mangoes were then allowed to air dry at room temperature (24–30 ◦ C) before further use. The fruit were placed in cardboard boxes and kept at 4 ◦ C (87% RH). Fruit were randomly sampled every 3 d to determine chilling injury, total free phenolics and the activities of PAL and catechol oxidase, both in the peel and pulp. Fruit of a second group were transferred to room temperature (27–28 ◦ C, 67–69% RH) for 6 d, and the same parameters were measured at 3 d intervals. 2.2. Effect of tropolone, an inhibitor of catechol oxidase Mango fruit cv Nam Dok Mai were placed in 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 mM aqueous tropolone solutions for 6 h at 22 ◦ C, while the control fruit were placed in water at the same temperature for the same duration. All mango fruit were allowed to air dry at 25 ◦ C (72–75% RH) and were placed at 4 ◦ C (87% RH). The time to peel blackening was determined by daily inspection. 2.3. 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 = none, 1 = 10% of the peel or pulp are brown, 2 = 11–25% of peel or pulp are brown, 3 = 26–40%, 4 = 40–50%, 5 = >50%. The CI index was calculated as follows (Concellón et al., 2004):
for 1 min, which contained 3.95 mL of 0.10 M potassium phosphate buffer (pH 7) and 1 mL of the supernatant. Extraction and assay of PAL was carried out as described by Ke and Saltveit (1986). Peel tissue (1 g) was homogenized with 4 mL of 50 mM borate buffer (pH 8.5) containing 5 mM 2-mercaptoethanol and 0.4 g polyvinylpyrrolidone (PVP). The homogenate was filtered through 3 layers of cotton cloth and then centrifuged at 13,522 × g for 20 min at 4 ◦ C. PAL activity in the supernatant was assayed by adding 0.7 mL of 100 mM l-phenylalanine and 3 mL of 50 mM borate buffer (pH 8.5) to 0.3 mL of the extract. After heating to 40 ◦ C for 1 h, the reaction was stopped by adding 0.1 mL of 5 N of HCl. PAL activity was determined by measuring the absorbance of assay mixture at 290 nm for the production of cinnamic acid. Protein content in the enzyme extracts was estimated using the standard Bradford method. Specific activity of the enzyme was expressed as units per kg protein. Each treatment had five replications and each replication consisted of the whole peel from 10 to 15 fruit, pooled together. Determinations of enzyme activity were made twice in each extract. 2.5. Total free phenolics Total free phenolics in the peel and pulp was estimated colorimetrically, using a procedure that was slightly modified from the one described by Singleton et al. (1999). Five grams of peel or pulp were extracted at room temperature and in the absence of light, using pure methanol containing 1% 2,6-di-tert-butyl-4methylphenol (BHT) as an antioxidant and an ultrasonic bath. Samples were extracted twice with 10 mL of the methanol solvent for 1 h, 10 mL for 30 min, and then with 5 mL for 30 min. The extracts were combined to a final volume of 25 mL. Phenolic compounds were determined using the Folin–Ciocalteu reagent. A mixture of 0.5 mL of extract (or spectrophotometric standard), 0.5 mL of Folin–Ciocalteu reagent and 10 mL of Na2 CO3 1 M were introduced in a 25 mL volumetric flask. After reaction for 1 h, the solutions were measured at 750 nm. Ellagic acid has been reported to be a major phenolic compound in mango fruit (Schieber et al., 2000). Total free phenolics were therefore expressed as ellagic acid equivalents. 2.6. Statistical analysis In the test on visible browning scores, three replications containing 12–15 fruit each were used in each treatment. Data were compared by analysis of variance and calculation of Least Significant Difference (LSD). Regression analyses (r value) were performed between catechol oxidase activity and browning of peel and pulp. The experiments were repeated once at a later date.
CI index
=
(injury classification level × number of fruit at that level)
3. Results
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 one used for peel browning. 2.4. Catechol oxidase and PAL Catechol oxidase was extracted and assayed by the method of Yang et al. (2001). Peel tissue (1 g) was homogenized with 5 mL of 0.1 M potassium phosphate buffer (pH 7) and 0.2 g polyvinylpyrrolidone (PVP). The homogenate was filtered through 3 layers of cotton cloth and then centrifuged at 13,522 × g for 20 min at 4 ◦ C. One unit of catechol oxidase activity was determined by measuring the rate of increase in absorbance at 420 nm at 25 ◦ C
3.1. Browning, total free phenolics, PAL and catechol oxidase activities in the peel The peel of cv. Nam Dok Mai mangoes stored at 4 ◦ C did not exhibit discoloration until day 6. On day 9 the peel of several fruits had turned greyish brown and this discoloration rapidly increased in fruit stored for a longer period (Table 1). The peel of cv. Choke Anan mangoes did not show any color change (Table 1). Upon transfer to 27–28 ◦ C for 6 d (shelf life), at various intervals during cold storage, the peel of cv. Nam Dok Mai mango showed increased browning, if browning was not yet maximal (Table 1). The peel of cv. Choke Anan mango exhibited slight browning during shelf life, although only when taken out of low temperature storage on the last two sampling dates, i.e., day 27 and 30 (Table 1).
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Table 1 Browning of the peel of mango fruit cvs. Nam Dok Mai (䊉) and Choke Anan () during storage and after storage at 4 ◦ C for 6 d of shelf life at 27–28 ◦ C (B). Data are means ± SE of three replications (12–15 fruit each). Browning (scores)a
Treatment
Time in storage (d)
Choke Anan Peel (during storage) Peel (after storage) Pulp (during storage) Pulp (after storage) Nam Dok Mai Peel (during storage) Peel (after storage) Pulp (during storage) Pulp (after storage)
3
6
9
12
15
18
21
24
27
30
1.00 1.00 1.00 1.00
1.00 1.00 1.00 1.00
1.00 1.00 1.00 1.00
1.00b 1.00b 1.00b 1.00b
1.00b 1.00b 1.00b 1.00b
1.00b 1.00b 1.00b 1.00b
1.00b 1.00b 1.00b 1.00b
1.00c 1.00c 1.00c 1.00c
1.00c 1.00c 1.00c 1.00c
1.00c 1.20c 1.00c 1.00c
1.00b 1.25b 1.00b 1.00b
1.00 1.00 1.00 1.00
1.00 1.00 1.00 1.00
1.07 2.17 1.00 1.00
1.67a 2.50a 1.00b 1.00b
3.75a 3.50a 1.00b 1.00b
4.17a 4.50a 1.00b 1.00b
4.33a 4.80a 1.00b 1.00b
4.42a 5.00a 1.00c 2.00b
4.83a 5.00a 1.00c 2.50b
5.00a 5.00a 1.00c 4.17b
5.00a 5.00a 1.00b 5.00a
Values in same column, followed by different letters, are significantly different at P ≤ 0.01 (Analysis of variance and DMRT).
Total free phenolics levels in the peel of both cv. Nam Dok Mai and cv. Choke Anan mangoes were initially high, then decreased (Fig. 1A). A lower level of total free phenolics was correlated with a higher score of peel browning during cold storage (nonlinear correlation coefficient, r2 = 0.86; Fig. 4A). After 6 d of transfer the fruit from cold storage to 27–28 ◦ C the total free phenolics levels in the peel of cv. Nam Dok Mai mango were higher than that in cv. Choke Anan peel (Fig. 1B). During cold storage the PAL activity increased, more so in the peel of cv. Nam Dok Mai mango than that of cv. Choke Anan (Fig. 2A). The PAL activity in the peel of cv. Nam Dok Mai was higher than in cv. Choke Anan, on day 21–30 (Fig. 2A). The nonlinear correlation coefficient between PAL activity in the peel and peel browning in cold-stored cv. Nam Dok Mai was high (r2 = 0.99; Fig. 4B). Upon transfer of the fruit, at various intervals of cold storage, to 27–28 ◦ C, the PAL activity in both cultivars was low (Fig. 2B). A low correlation coefficient was found between the activity of PAL in the peel and peel browning during shelf life (data not shown). The catechol oxidase activity in the peel of cv. Nam Dok Mai mango was higher than that of cv. Choke Anan, both during storage
6
3.0
5
2.5
4
2.0
1.2
1.5
0.9
1.0
0.6
0.5
0.3
-1
2
10
1
0
0
B LSD
0.05
LSD
= 7.72
= 0.68
D
0.05
6 5
40
4 30 3 20
2
10
1
0
0 0 3 6 9 12 15 18 21 24 27 30
0 3 6 9 12 15 18 21 24 27 30
LSD = 0.27 0.05
1.8 1.5
0.0 2.5
B LSD = 0.44 0.05
D LSD = 0.05 0.05
0.0 1.5
2.0
1.2
1.5
0.9
1.0
0.6
0.5
0.3
0.0
0 3 6 9 12 15 18 21 24 27 30
0 3 6 9 12 15 18 21 24 27 30
-1
3 20
LSD = 0.53 0.05
C
-3
30
A
PAL activity x 10 (units kg )
0.05
-1
= 7.63
40
Total free phenolics (g kg )
During cold storage of both cv. Nam Dok Mai and cv. Choke Anan mango fruit no browning or other discoloration was found in the pulp. Six days after transfer to 27–28 ◦ C of cv. Nam Dok Mai fruit, pulp browning was only observed in fruit that had been stored at the low temperature for 21 d or longer. Browning was higher in fruit that had been stored for a longer period. In contrast, during shelf life the pulp of cv. Choke Anan mangoes did not show browning, even if the fruit had been stored in the cold for 29 d (data not shown).
-3
0.05
C
-1
LSD
= 0.66
LSD
Total free phenolics (g kg )
A
50
3.2. Browning, total free phenolics, PAL and catechol oxidase activities in the pulp
7
60 50
at 4 ◦ C (Fig. 3A) and after transfer to 27–28 ◦ C (Fig. 3B). Six days after transfer of the fruit to 27–28 ◦ C, the catechol oxidase activity in the peel of both cultivars showed a maximum on day 12 of taking the fruit out of cold storage (Fig. 3B). No obvious relation was observed between the catechol oxidase activity in the peel and peel browning (Fig. 4B). A low correlation coefficient was also found between the activity of catechol oxidase in the peel and peel browning during shelf life (data not shown).
PAL activity x 10 (units kg )...
a
0
0.0
Time in storage (d)
Time in storage (d) Fig. 1. Total free phenolics of the peel (A and B) and pulp (C and D) of mango fruit cvs. Nam Dok Mai (䊉) and Choke Anan () during storage at 4 ◦ C (A and C) and after storage at 4 ◦ C and 6 d of shelf life at room temperature (27–28 ◦ C; B and D). Data in B and D refer to the date the fruit was transferred from cold storage to room temperature. Data are means ± SE of three replications (12–15 fruit each).
Fig. 2. Phenylalanine ammonia lyase (PAL) activity of the peel (A and B) and peel (C and D) of mango fruit cvs. Nam Dok Mai (䊉) and Choke Anan () during storage at 4 ◦ C (A and C) and after storage at 4 ◦ C and 6 d of shelf life at room temperature (27–28 ◦ C; B and D). Data in B and D refer to the date the fruit was transferred from cold storage to room temperature. Data are means ± SE of three replications (12–15 fruit each).
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5 LSD
0.05
4
LSD
C
= 0.66
0.05
5 4
3
3
2
2
1
1
0
0
B
5
= 0.71
LSD
D LSD
0.05
= 0.44
0.05
5
4
4
3
3
2
2
1
1
0
-3
-3
-1
-1
Catechol oxidase activity x 10 (units kg )
A
= 0.59
Catechol oxidase activity x 10 (units kg )
62
0 0 3 6 9 12 15 18 21 24 27 30
0 3 6 9 12 15 18 21 24 27 30
During cold storage the catechol oxidase activity in the pulp of cv. Nam Dok Mai mango was considerably higher than that of cv. Choke Anan fruit (Fig. 3B). Six days after transfer to 27–28 ◦ C, the catechol oxidase activities in the pulp of both cultivars were very low (Fig. 3D). Very low linear correlation coefficients were found between pulp browning and the activities of the two measured enzymes (data not shown). 3.3. Effect of tropolone on browning Peel browning of the control mango fruit was similar to that in mango fruit treated with 0.1 or 0.2 mM tropolone, hence there was no effect of low tropolone concentrations. This was in contrast to the negative effect of higher tropolone concentrations. Compared to the controls, the peel of mango fruit treated with 0.4–3.2 mM tropolone turned more brown during storage at 4 ◦ C. The higher the concentration used, the more peel browning was found. In contrast, the tropolone treatments did not affect pulp browning (results not shown).
Time in storage (d) 4. Discussion Fig. 3. Catechol oxidase activity of the peel (A and B) and pulp (C and D) of mango fruit cvs. Nam Dok Mai (䊉) and Choke Anan () during storage at 4 ◦ C (A) and after storage at 4 ◦ C for 6 d of shelf life at 27–28 ◦ C (B). Data in B and D refer to the date the fruit was transferred from cold storage to room temperature. Data are means ± SE of three replications (12–15 fruit each).
-1
(g kg )...
50 45 40 35 30 25 20 15 10 5 0
A R² = 0.86
R² = 0.99
3.5
B
3.0
-1
(units kg )...
Activity of catechol oxidase and PAL x 10
-3
Total free phenolics
Total free phenolics in the pulp of both cv. Nam Dok Mai and cv. Choke Anan mangoes were similar and did not change consistently during storage at 4 ◦ C (Fig. 1C) and upon transfer to 27–28 ◦ C (Fig. 1D). Total free phenolics in the pulp of both cultivars were about 9–10 times lower than in the peel, throughout low temperature storage and shelf life (Fig. 1). PAL activities in the pulp of both cv. Nam Dok Mai and cv. Choke Anan mangoes were much lower than that of the peel (Fig. 2). The PAL activity was generally higher in Nam Dok Mai than in Choke Anan (Fig. 2B). The activity was very low 6 d after transfer to 27–28 ◦ C (Fig. 2D).
2.5 2.0 1.5 1.0 0.5 0.0 0
1
2
3
4
5
6
Browning (scores) Fig. 4. Relations between peel browning, total phenolics, and the activities of PAL and catechol oxidase in the peel of mango fruit cv. Nam Dok Mai stored at 4 C. (A) Relationship with total phenolics concentrations. (B) Relationship with the activity of PAL (solid symbol) and catechol oxidase (open symbol).
The data show that cv. Nam Dok Mai mango fruit showed peel damage during as little as 9 d of storage at 4 ◦ C, while in cv. Choke Anan fruit no peel browning was found during the 30 d of such cold storage. Even during the subsequent 6 d of shelf life at 27–28 ◦ C cv. Choke Anan fruit showed no peel browning if the fruit had been stored at 4 ◦ C for as long as 24 d. The level of total free phenolics in the peel was similar in both cultivars. However, a rather high nonlinear inverse correlation between total phenolics and peel browning of ‘Nam Dok Mai’ was observed. This suggests that total free phenolic levels were ratelimiting in the peel browning reactions. A similar effect has been found in banana (Nguyen et al., 2003). Low temperature storage had less effect on pulp color than on peel color, but again cv. Nam Dok Mai was more sensitive. Browning compounds are synthesized from free phenolic acids through PAL (Camm and Towers, 1977). PAL activity in the peel was much higher than that of the pulp, which was correlated with the much higher level of total free phenolics in the peel compared with the pulp. During low temperature storage the PAL activity in the peel of both cultivars increased. In cv. Nam Dok Mai the increase was higher, from day 18 of storage. However, browning of the peel was already found from day 9 of cold storage. The measured in vitro PAL activity, therefore, did not explain the early peel browning in cold-stored Nam Dok Mai fruit. Nonetheless, a correlation was found at later points in time. When expressing the data of peel browning against PAL activity, both measured every 3 d, a high nonlinear correlation was observed. In citrus fruit, Lafuente et al. (2003) and banana, Nguyen et al. (2003) similarly found that low temperature induced an increase in PAL activity in the peel. No relation was found between PAL activity in the pulp of mango and pulp browning. During low temperature storage the in vitro catechol oxidase in the peel of cv. Nam Dok Mai was considerably higher than in cv. Choke Anan. However, when expressing the data of peel browning against catechol oxidase activity, no obvious relation was observed. This suggests that catechol oxidase was not rate liming for peel browning. Tropolone is a rather specific inhibitor of catechol oxidase (Richardson et al., 1997). In our tests it had no effect at the relatively low concentrations used (0.1 and 0.2 mM for 6 h) and was toxic at higher concentrations. Tropolone thus did not reduce low temperature-induced browning, also indicating that catechol oxidase activity was not a limiting factor.
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It is concluded that peel browning rather than pulp browning limited the cold storage period of the two mango cultivars tested. A high nonlinear correlation coefficient was found between peel browning and PAL activity, while the correlation coefficient with catechol oxidase was very low. No obvious relation was found between pulp browning and the measured enzyme activities. Acknowledgements The work was financially supported by the Thailand Research Fund (TRF), Postharvest Technology Innovation Center (PHTIC), Commission on Higher Education (CHE), Ministry of Education, and Kasetsart University Research and Development (KURDI). This study was part of the planned Ph.D. dissertation of Sugunya Chidtragool. Regrettably, she passed away while working at HortResearch in Auckland, NZ. This paper is dedicated to her memory. References Baritaux, O., Amiot, M.J., Nicolas, J., 1991. Enzymatic browning of basil (Ocimum basilicum L.): studies on phenolic compounds and polyphenol oxidase. Sci. Aliment. 11, 49–62. Camm, E.L., Towers, G.H.N., 1977. Phenylalanine ammonia lyase. Phytochemistry 12, 961–973. ˜ Concellón, A., Anón, M.C., Chaves, A.R., 2004. Characterization and changes in polyphenol oxidase from eggplant fruit (Solanum melongena L.) during storage at low temperature. Food Chem. 88, 17–24. González-Aguilar, G.A., Buta, J.G., Wang, C.Y., 2001. Methyl jasmonate reduces chilling injury symptoms and enhances colour development of ‘Kent’ mangoes. J. Sci. Food Agric. 81, 1244–1249. Kaniuga, Z., 2008. Chilling response of plants: importance of galactolipase, free fatty acids and free radicals. Plant Biol. 10, 171–184. Ke, D., Saltveit, M.E., 1986. Effects of calcium and auxin on russet spotting and phenylalanine ammonia-lyase activity in iceberg lettuce. Hortscience 21, 1169–1171. Lafuente, M.T., Zacarias, L., Martinez-Tellez, M.A., Sanchez-Ballesta, M.T., Granell, A., 2003. Phenylalanine ammonia-lyase and ethylene in relation to chilling injury as affected by fruit age in citrus. Postharvest Biol. Technol. 29, 308–311. Nair, S., Singh, Z., 2003. Pre-storage Ethrel dip reduces chilling injury, enhances respiration rate, ethylene production and improves fruit quality of ‘Kensington’ mango. Food Agric. Environ. 1, 93–97.
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