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Induction of apple scald by anaerobiosis has similar characteristics to naturally occurring superficial scald in ‘Granny Smith’ apple fruit
Postharvest Biology and Technology 16 (1999) 9 – 14
Induction of apple scald by anaerobiosis has similar characteristics to naturally occurring superficial scald in ‘Granny Smith’ apple fruit Anne D. Bauchot, Suzanne J. Reid, Gavin S. Ross, Douglas M. Burmeister * HortResearch, Pri6ate Bag 92 169, Auckland, New Zealand Received 18 August 1998; received in revised form 3 December 1998; accepted 7 December 1998
Abstract Scald, which can be induced by anaerobiosis (N2-induced), may involve similar physiological and biochemical mechanisms to those which contribute to naturally occurring superficial scald. To investigate this hypothesis, Granny Smith apples were harvested weekly from four orchards and stored in air for 16 weeks at 0°C, before being ventilated with N2 for up to 4 days, or held in air at 20°C. Following N2 treatment, scald developed within a few minutes of transfer to air and its severity was positively correlated with duration of N2 treatment. Susceptibility to induction of scald with N2 decreased through the harvest season and increased with length of refrigerated storage. Following N2 treatment, the levels of a-farnesene, conjugated triene (CTH) and OD200 (Abs200) were significantly lower than in fruit held continuously in air. This effect was more apparent in the early than late harvest fruit. RNA gel blot analysis of polyphenol oxidase (PPO) was conducted on selected samples. Expression of PPO was very low while fruit were held in N2, suggesting that the regulation of PPO gene expression is dependent on oxygen. Once fruit were removed to air, browning occurred almost immediately, too rapidly for the initial development of browning symptoms in scald to be attributed to increased PPO gene expression. We conclude that anaerobically induced scald development has similar characteristics of naturally occurring superficial scald and in both cases elevated PPO gene expression may not be associated with the initial development of symptoms. © 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Malus domestica Borkh.; Superficial scald; Physiological disorders; Anaerobiosis; Polyphenol oxidase
1. Introduction
* Corresponding author. Tel.: +64-9-8154200; fax: +64-98154202. E-mail address: [email protected] (D.M. Burmeister)
Superficial scald is a disorder of apples and pears that occurs after long-term storage. Superficial scald is characterised by a browning of the hypodermal layers of the skin. The disorder is
0925-5214/99/$ - see front matter © 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 5 2 1 4 ( 9 9 ) 0 0 0 0 2 - 2
10
A.D. Bauchot et al. / Posthar6est Biology and Technology 16 (1999) 9–14
manifested when fruit are transferred from storage to ambient temperatures, but can also develop in storage after prolonged periods. Fruit with the disorder may still be utilised for processing but the dessert quality is destroyed. Fruit that are immature at harvest are prone to scald (Huelin and Coggiola, 1968). Susceptibility has been inversely related to preharvest chilling temperatures (B 10°C; Watkins and Bramlage, 1994), although other preharvest factors, such as fruit nutrition and size, may also affect development (Emongor et al., 1994). Control of the disorder is accomplished by application of the general antioxidants diphenylamine (DPA) or ethoxyquin. Controlled atmosphere (CA) storage has also been shown to be effective in delaying or preventing the onset of the disorder (Lau, 1990) and other treatments such as heat (Klein and Lurie, 1992), intermittent warming (Watkins et al., 1995), food compatible antioxidants (Bauchot et al., 1995) and ethanol vapours (Scott et al., 1995) have shown promising results. Scald research has been hindered by the lack of an experimental system or treatment which can reliably induce the disorder. Nevertheless, superficial scald-like symptoms can be induced by anaerobic treatment at 20°C following storage (Dilley et al., 1963). This phenomenon appears to consist of two phases, an anaerobic (induction) and an aerobic (development) phase (Dilley et al., 1963). The induction of scald is inversely proportional to O2 concentration, whereas the development occurs readily at O2 concentrations above 5% (Dilley et al., 1965; Burmeister and Dilley, 1995). The incidence and severity of the anaerobically-induced scald symptoms were also directly proportional to the duration of the anaerobic treatment and are reduced by pre-storage treatment with DPA (Dilley et al., 1965). The enzyme polyphenol oxidase (PPO) is known to have a role in browning reactions of fruit tissue. In tissue showing superficial scald, PPO gene expression is up-regulated (Boss et al., 1995), although the significance of this change as a possible cause of the disorder is unclear. As browning in superficial scald can occur over several days, it is difficult to correlate the precise timing of symptoms with PPO gene expression.
In this paper, we present biochemical and molecular data in support of the hypothesis that anaerobically (N2)-induced scald involves similar physiological and biochemical mechanisms to those which contribute to naturally occurring superficial scald. As N2-induced scald has a short time-frame for development, we also use this as a system to investigate whether PPO expression is upregulated in advance of scald symptoms.
2. Materials and methods
2.1. Fruit and treatments ‘Granny Smith’ apples were harvested weekly from four commercial orchards in the Hawkes Bay region of New Zealand and air-freighted overnight to the Mt. Albert Research Centre, Auckland, NZ. Fruit were stored for 16 weeks in air at 0°C. Immediately after removal from storage, fruit were placed into 20-l chambers and ventilated with 100% N2 at a flow rate 0.5 l min − 1 at 20°C for 0, 2 or 4 days. In addition, fruit were held in air at 20°C for 2 days, then placed in N2 for 4 days.
2.2. Scald assessment and analysis Fruit were visually assessed for scald according to the percentage of scalded surface on a scale of 0–4 (0: no scald; 1: 510%; 2: 10–25%; 3: 25– 50%, 4: \ 50%). Fruit were assessed immediately upon removal from storage and N2 treatment, and after a shelf-life of 4 days. This shelf-life period allowed for maximal expression of symptoms. For each apple, ten peel discs of 1 cm2 were randomly excised and dipped in 10 ml HPLC grade hexane for 10 min. The UV absorption between 190 and 300 nm were recorded. a-Farnesene and conjugated triene (CTH) levels were calculated with E232 = 29 000 and E281 – 290 = 25 000, respectively and expressed as 1 nmole cm − 2 of surface area (Anet, 1972; Meir and Bramlage, 1988). The peak at 200 nm was expressed as OD200 1000. We measured a-farnesene, CTH and OD200nm in both air and N2 treated fruit, to determine whether these levels changed in
A.D. Bauchot et al. / Posthar6est Biology and Technology 16 (1999) 9–14
a similar manner in both N2-induced and superficial scald. Measurements were also made in fruit from two harvests with differing incidences of scald (April 11th and 27th; Fig. 1).
2.3. RNA gel blot analysis of PPO gene expression Samples of fruit apple peel were taken after 2 or 4 days in air or N2 and after 4 days in air following N2 treatment. RNA was extracted using methods described by Boss et al. (1995). RNA samples (20 mg) were denatured and fractionated on a 1.2% agarose gel containing 0.66 M formaldehyde and transferred to Hybond N + membrane by capillary transfer in 50 mM NaOH. RNA on the membrane was hybridised with a 32 P-labelled cDNA probe which is homologous to apple PPO (Boss et al., 1995). Following hybridisation, the blot was washed with 1 ×SSC (150 mM NaCl, 15 mM tri-sodium citrate), 0.1% (w/v) sodium dodecyl sulfate at 65°C for 15 min (twice) and exposed to X-ray film. To correct for differences in RNA loadings, following autoradiography, the membranes were stripped in boiling 0.5% SDS and hybridised with an 18 S ribosomal probe (Simon and Weeden, 1992). All autoradiographs were processed using densitometry software (Sigmascan™/Image, Jandel Scientific Software) to quantify the observed homologous signal.
Fig. 1. Severity of scald in ‘Granny Smith’ apples harvested on four dates, stored for 16 weeks at 1°C, transferred to either air or 100% N2 and assessed after 4 days at 20°C. For N2-treated fruit, scald developed within several minutes of removal to air; scald in air-stored took several days to develop. Mean comparison with harvest dates at P50.05.
11
3. Results and discussion Fruit showed no symptoms of superficial scald while in air storage at 1°C but developed symptoms within 4 days at 20°C (Fig. 1). Fruit that had received a N2 treatment, developed scald symptoms within a few minutes of transfer to air (data not shown). With progressive harvests, there was a reduced incidence of scald after 4 days at 20°C in all treatments (Fig. 1). This is consistent with the well-known relationship between harvest maturity and severity of superficial scald (Emongor et al., 1994). Anaerobically induced scald showed some dose-response characteristics. The 4-day N2 treatment induced more scald than the 2-day N2 treatment, which in turn induced more scald than the levels observed in the air control (Fig. 1). When fruit from the earliest harvest were held at 20°C prior to nitrogen treatment, the incidence of scald was less than in the air control. In fruit from later harvests N2-induced scald was harvest reduced to near the level seen in the air control. This indicates that the development of N2-induced scald is dependent on a concurrent shift in temperature from 0 to 20°C, i.e. once fruit have equilibrated to 20°C they are no longer susceptible to N2-induced scald. These data are consistent with the concept that superficial scald is a chilling injury, where physiological dysfunction occurs and injury symptoms are expressed once fruit are removed from refrigerated storage (Watkins et al., 1995). There was also some evidence for an orchard effect of scald severity (Table 1), with considerable variation between orchards. The orchard with fruit with the lowest incidence of naturally occurring superficial scald, also produced fruit which showed little N2-induced scald. However, this correlation was not consistent across other orchards. Susceptibility to N2-induced scald developed with increasing storage duration, in that fruit which were removed to 20°C after 16 weeks were susceptible to N2-induced scald, whereas earlier samples (12, 14 weeks) were not susceptible (data not shown). These observations are what we would expect if the mechanisms of N2-induced scald were similar to superficial scald that develops during storage.
12 A.D. Bauchot et al. / Posthar6est Biology and Technology 16 (1999) 9–14 Fig. 2. The effect of 100% N2 treatment for 4 days on a-farnesene, CTH-281 and OD200 concentrations of ‘Granny Smith’ apple peel for fruit harvested April 11th or 27th. Data are averages of four orchards of ten fruit each. Mean comparison between treatments for each harvest at P5 0.05.
A.D. Bauchot et al. / Posthar6est Biology and Technology 16 (1999) 9–14 Table 1 Effect of nitrogen induction of scald on fruit from different orchardsa Orchard
1 2 3 4
Scald index Control
Nitrogen induction
1.2bb 1.0b 2.8a 1.5b
3.9a 1.2c 3.4b 4.0a
a Fruit were harvested on 11th April and stored at 0°C for 16 weeks. Nitrogen was applied 4 days after removal from cold store. Scald was assessed 8 days after transfer to 20°C. b Means within a column and followed by the same letter are not significantly different (PB0.05).
The oxidation of a-farnesene into CTH has been the focus of much of the research on superficial scald (Emongor et al., 1994). The OD200nm has been negatively correlated with scald susceptibility and thought to be an estimate of total antioxidant activity (Meir and Bramlage, 1988). In both air-stored and N2-treated fruit from two harvests, the levels of a-farnesene decreased during 4 days in air (Fig. 2). Levels of a-farnesene were lower in fruit from the April 11th harvest, 4 days in air after N2 treatment. Fruit from this treatment also showed the highest scald index (Fig. 1). CTH levels in fruit from the first harvest were higher than in fruit from the later harvest. Both CTH levels and OD200 in N2-treated fruit were significantly lower than in fruit that had experienced only air-storage. The decline in these readings was more pronounced in the April 11th harvest. There was no significant change in the levels of these compounds beyond 4 days in air in any of the treatments (data not shown). Expression of PPO was very low while fruit were held in nitrogen, suggesting that the regulation of PPO gene expression is dependent on oxygen. Once fruit were removed to air, browning occurred almost immediately, such that tissue which was peeled and frozen immediately after removal from N2 was already browning (Fig. 3, lanes 2, 3). This time-frame is too rapid for the browning to be attributed to elevated PPO gene expression and suggests that the initial development of browning symptoms is not dependent on
13
increased expression of PPO mRNA. Over the 4 days after removal from N2 or air, PPO mRNA accumulated to high levels (Fig. 3, lanes 4, 5), but may be a consequence of other events associated with cell damage. These data suggest that the initial browning reactions in scald are unlikely to be attributed to elevated levels of PPO mRNA. However, altered PPO mRNA stability or activity of the enzyme may still be important. It has been proposed that upon shifting of tissue from anaerobic to aerobic conditions, active O2 species such as O2− are produced (Crawford and Braendle, 1996). The role of these species in causing tissue injury in plants is widely accepted (Winston, 1990). We speculate that N2-induction of scald involves the same mechanism, with cells experiencing post-anoxic injury. During N2 treatment of apples, there is also a decline in the levels of the antioxidant ascorbate (Burmeister and Dilley, 1995). Less ascorbate may then be available to quench active oxygen species produced upon return to air. The observation that natural superficial scald symptoms also do not usually develop in storage but appear during shelf-life (Bauchot et al., 1995), invites speculation that
Fig. 3. Expressed apple PPO mRNA in relation to N2-induced scald. Samples were taken from fruit held at 20°C for 0 days (lane 1), 2 or 4 days in the absence of oxygen (lanes 2 and 3), 4 days in air (lane 4) and from fruit held for 4 days in 100% N2 followed by 4 days in air (lane 5). Total mRNA (20 mg) was loaded on each lane. Bars indicate the relative intensity of the PPO mRNA signal as adjusted for RNA loadings.
14
A.D. Bauchot et al. / Posthar6est Biology and Technology 16 (1999) 9–14
superficial scald may also result from the production of active O2 species during the shift of apples from storage to ambient temperatures via the same mechanism. Nitrogen-induced scald represents a useful experimental system which may be used to confirm this and investigate other browning reactions in apples.
References Anet, E.F.L.J., 1972. Superficial scald, a functional disorder of stored apples. IX. Effect of maturity and ventilation. J. Sci. Food Agr. 23, 763 – 769. Bauchot, A.D., John, P., Soria, Y., Recasens, I., 1995. Sucrose ester-based coatings formulated with food-compatible antioxidants in the prevention of superficial scald in stored apples. J. Am. Soc. Hort. Sci. 120 (3), 491–496. Boss, P.K., Gardner, R.C., Janssen, B.J., Ross, G.S., 1995. An apple polyphenol oxidase cDNA is up-regulated in wounded tissues. Plant Mol. Biol. 27, 429–433. Burmeister, D.M., Dilley, D.R., 1995. A ‘scald-like’ controlled atmosphere storage disorder of Empire apples—a chilling injury induced by CO2. Postharvest Biol. Technol. 6, 1–7. Crawford, R.M.M., Braendle, R., 1996. Oxygen deprivation stress in a changing environment. J. Exp. Botany 47 (295), 145 – 159. Dilley, D.R., MacLead, D.C., Dewey, D.H., 1963. Apple scald induction by anaerobiosis. Nature 200, 1229–1230. Dilley, D.R., Dedolph, R.R., MacLead, D.C., Dewey, D.H., 1965. Physiological basis for apple scald (Abstr.). Proceedings of the Sixty-Second Annual Meeting of the American Society for Horticultural Sciences, University of Illinois, Urbana, IL, p. 20.
.
Emongor, V.E., Murr, D.P., Lougheed, E.C., 1994. Preharvest factors that predispose apples to superficial scald. Postharvest Biol. Technol. 4, 289 – 300. Huelin, F.E., Coggiola, I.M., 1968. Superficial scald a functional disorder of stored apples. IX, Effects of variety, maturity, oil wraps and diphenylamine on the concentration of a-farnesene in the fruit. J. Sci. Food Agri. 19, 297 – 301. Lau, O.L., 1990. Efficacy of diphenylamine, ultra-low O2 and ethylene synthesizing scrubbing on scald control in ‘Red Delicious’ apples. J. Am. Soc. Hort. Sci. 115, 959 – 961. Klein, J.D., Lurie, S., 1992. Prestorage heating of apple fruit for enhanced postharvest quality: Interaction of time and temperature. HortScience 27, 326 – 328. Meir, S., Bramlage, W.J., 1988. Antioxidant activity in ‘Cortland’ apple peel and susceptibility to superficial scald after storage. J. Am. Soc. Hort. Sci. 113, 412 – 418. Scott, K.J., Yuen, C.M.C., Ghahramani, F., 1995. Ethanol vapour — a new anti-scald treatment for apple. Postharvest Biol. Technol. 6, 201 – 208. Simon, C.J., Weeden, N.F., 1992. Molecular analysis and cloning of Malus ribosomal DNA. J. Am. Soc. Hort. Sci. 117, 164 – 168. Watkins, C.B., Bramlage, W.J., Cregoe, B.A., 1995. Superficial scald of ‘Granny Smith’ apples is expressed as a typical chilling injury. J. Am. Soc. Hort. Sci. 120 (1), 88 – 94. Watkins, C.B., Bramlage, W.J., 1994. Influences of preharvest temperature and harvest maturity on susceptibility of New Zealand and North American apples to superficial scald. New Zealand J. Crop Hort. Sci. 22, 69 – 79. Winston, G.W., 1990. Physiochemical basis for free radical formation in cells: Production and defenses. In: Alscher, R.G., Cummmings, J.R. (Eds.), Stress responses in plants: adaptation and acclimation mechanisms. Wiley-Liss, New York, pp. 57 – 86.
Induction of apple scald by anaerobiosis has similar characteristics to naturally occurring superficial scald in ‘Granny Smith’ apple fruit Anne D. Bauchot, Suzanne J. Reid, Gavin S. Ross, Douglas M. Burmeister * HortResearch, Pri6ate Bag 92 169, Auckland, New Zealand Received 18 August 1998; received in revised form 3 December 1998; accepted 7 December 1998
Abstract Scald, which can be induced by anaerobiosis (N2-induced), may involve similar physiological and biochemical mechanisms to those which contribute to naturally occurring superficial scald. To investigate this hypothesis, Granny Smith apples were harvested weekly from four orchards and stored in air for 16 weeks at 0°C, before being ventilated with N2 for up to 4 days, or held in air at 20°C. Following N2 treatment, scald developed within a few minutes of transfer to air and its severity was positively correlated with duration of N2 treatment. Susceptibility to induction of scald with N2 decreased through the harvest season and increased with length of refrigerated storage. Following N2 treatment, the levels of a-farnesene, conjugated triene (CTH) and OD200 (Abs200) were significantly lower than in fruit held continuously in air. This effect was more apparent in the early than late harvest fruit. RNA gel blot analysis of polyphenol oxidase (PPO) was conducted on selected samples. Expression of PPO was very low while fruit were held in N2, suggesting that the regulation of PPO gene expression is dependent on oxygen. Once fruit were removed to air, browning occurred almost immediately, too rapidly for the initial development of browning symptoms in scald to be attributed to increased PPO gene expression. We conclude that anaerobically induced scald development has similar characteristics of naturally occurring superficial scald and in both cases elevated PPO gene expression may not be associated with the initial development of symptoms. © 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Malus domestica Borkh.; Superficial scald; Physiological disorders; Anaerobiosis; Polyphenol oxidase
1. Introduction
* Corresponding author. Tel.: +64-9-8154200; fax: +64-98154202. E-mail address: [email protected] (D.M. Burmeister)
Superficial scald is a disorder of apples and pears that occurs after long-term storage. Superficial scald is characterised by a browning of the hypodermal layers of the skin. The disorder is
0925-5214/99/$ - see front matter © 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 5 2 1 4 ( 9 9 ) 0 0 0 0 2 - 2
10
A.D. Bauchot et al. / Posthar6est Biology and Technology 16 (1999) 9–14
manifested when fruit are transferred from storage to ambient temperatures, but can also develop in storage after prolonged periods. Fruit with the disorder may still be utilised for processing but the dessert quality is destroyed. Fruit that are immature at harvest are prone to scald (Huelin and Coggiola, 1968). Susceptibility has been inversely related to preharvest chilling temperatures (B 10°C; Watkins and Bramlage, 1994), although other preharvest factors, such as fruit nutrition and size, may also affect development (Emongor et al., 1994). Control of the disorder is accomplished by application of the general antioxidants diphenylamine (DPA) or ethoxyquin. Controlled atmosphere (CA) storage has also been shown to be effective in delaying or preventing the onset of the disorder (Lau, 1990) and other treatments such as heat (Klein and Lurie, 1992), intermittent warming (Watkins et al., 1995), food compatible antioxidants (Bauchot et al., 1995) and ethanol vapours (Scott et al., 1995) have shown promising results. Scald research has been hindered by the lack of an experimental system or treatment which can reliably induce the disorder. Nevertheless, superficial scald-like symptoms can be induced by anaerobic treatment at 20°C following storage (Dilley et al., 1963). This phenomenon appears to consist of two phases, an anaerobic (induction) and an aerobic (development) phase (Dilley et al., 1963). The induction of scald is inversely proportional to O2 concentration, whereas the development occurs readily at O2 concentrations above 5% (Dilley et al., 1965; Burmeister and Dilley, 1995). The incidence and severity of the anaerobically-induced scald symptoms were also directly proportional to the duration of the anaerobic treatment and are reduced by pre-storage treatment with DPA (Dilley et al., 1965). The enzyme polyphenol oxidase (PPO) is known to have a role in browning reactions of fruit tissue. In tissue showing superficial scald, PPO gene expression is up-regulated (Boss et al., 1995), although the significance of this change as a possible cause of the disorder is unclear. As browning in superficial scald can occur over several days, it is difficult to correlate the precise timing of symptoms with PPO gene expression.
In this paper, we present biochemical and molecular data in support of the hypothesis that anaerobically (N2)-induced scald involves similar physiological and biochemical mechanisms to those which contribute to naturally occurring superficial scald. As N2-induced scald has a short time-frame for development, we also use this as a system to investigate whether PPO expression is upregulated in advance of scald symptoms.
2. Materials and methods
2.1. Fruit and treatments ‘Granny Smith’ apples were harvested weekly from four commercial orchards in the Hawkes Bay region of New Zealand and air-freighted overnight to the Mt. Albert Research Centre, Auckland, NZ. Fruit were stored for 16 weeks in air at 0°C. Immediately after removal from storage, fruit were placed into 20-l chambers and ventilated with 100% N2 at a flow rate 0.5 l min − 1 at 20°C for 0, 2 or 4 days. In addition, fruit were held in air at 20°C for 2 days, then placed in N2 for 4 days.
2.2. Scald assessment and analysis Fruit were visually assessed for scald according to the percentage of scalded surface on a scale of 0–4 (0: no scald; 1: 510%; 2: 10–25%; 3: 25– 50%, 4: \ 50%). Fruit were assessed immediately upon removal from storage and N2 treatment, and after a shelf-life of 4 days. This shelf-life period allowed for maximal expression of symptoms. For each apple, ten peel discs of 1 cm2 were randomly excised and dipped in 10 ml HPLC grade hexane for 10 min. The UV absorption between 190 and 300 nm were recorded. a-Farnesene and conjugated triene (CTH) levels were calculated with E232 = 29 000 and E281 – 290 = 25 000, respectively and expressed as 1 nmole cm − 2 of surface area (Anet, 1972; Meir and Bramlage, 1988). The peak at 200 nm was expressed as OD200 1000. We measured a-farnesene, CTH and OD200nm in both air and N2 treated fruit, to determine whether these levels changed in
A.D. Bauchot et al. / Posthar6est Biology and Technology 16 (1999) 9–14
a similar manner in both N2-induced and superficial scald. Measurements were also made in fruit from two harvests with differing incidences of scald (April 11th and 27th; Fig. 1).
2.3. RNA gel blot analysis of PPO gene expression Samples of fruit apple peel were taken after 2 or 4 days in air or N2 and after 4 days in air following N2 treatment. RNA was extracted using methods described by Boss et al. (1995). RNA samples (20 mg) were denatured and fractionated on a 1.2% agarose gel containing 0.66 M formaldehyde and transferred to Hybond N + membrane by capillary transfer in 50 mM NaOH. RNA on the membrane was hybridised with a 32 P-labelled cDNA probe which is homologous to apple PPO (Boss et al., 1995). Following hybridisation, the blot was washed with 1 ×SSC (150 mM NaCl, 15 mM tri-sodium citrate), 0.1% (w/v) sodium dodecyl sulfate at 65°C for 15 min (twice) and exposed to X-ray film. To correct for differences in RNA loadings, following autoradiography, the membranes were stripped in boiling 0.5% SDS and hybridised with an 18 S ribosomal probe (Simon and Weeden, 1992). All autoradiographs were processed using densitometry software (Sigmascan™/Image, Jandel Scientific Software) to quantify the observed homologous signal.
Fig. 1. Severity of scald in ‘Granny Smith’ apples harvested on four dates, stored for 16 weeks at 1°C, transferred to either air or 100% N2 and assessed after 4 days at 20°C. For N2-treated fruit, scald developed within several minutes of removal to air; scald in air-stored took several days to develop. Mean comparison with harvest dates at P50.05.
11
3. Results and discussion Fruit showed no symptoms of superficial scald while in air storage at 1°C but developed symptoms within 4 days at 20°C (Fig. 1). Fruit that had received a N2 treatment, developed scald symptoms within a few minutes of transfer to air (data not shown). With progressive harvests, there was a reduced incidence of scald after 4 days at 20°C in all treatments (Fig. 1). This is consistent with the well-known relationship between harvest maturity and severity of superficial scald (Emongor et al., 1994). Anaerobically induced scald showed some dose-response characteristics. The 4-day N2 treatment induced more scald than the 2-day N2 treatment, which in turn induced more scald than the levels observed in the air control (Fig. 1). When fruit from the earliest harvest were held at 20°C prior to nitrogen treatment, the incidence of scald was less than in the air control. In fruit from later harvests N2-induced scald was harvest reduced to near the level seen in the air control. This indicates that the development of N2-induced scald is dependent on a concurrent shift in temperature from 0 to 20°C, i.e. once fruit have equilibrated to 20°C they are no longer susceptible to N2-induced scald. These data are consistent with the concept that superficial scald is a chilling injury, where physiological dysfunction occurs and injury symptoms are expressed once fruit are removed from refrigerated storage (Watkins et al., 1995). There was also some evidence for an orchard effect of scald severity (Table 1), with considerable variation between orchards. The orchard with fruit with the lowest incidence of naturally occurring superficial scald, also produced fruit which showed little N2-induced scald. However, this correlation was not consistent across other orchards. Susceptibility to N2-induced scald developed with increasing storage duration, in that fruit which were removed to 20°C after 16 weeks were susceptible to N2-induced scald, whereas earlier samples (12, 14 weeks) were not susceptible (data not shown). These observations are what we would expect if the mechanisms of N2-induced scald were similar to superficial scald that develops during storage.
12 A.D. Bauchot et al. / Posthar6est Biology and Technology 16 (1999) 9–14 Fig. 2. The effect of 100% N2 treatment for 4 days on a-farnesene, CTH-281 and OD200 concentrations of ‘Granny Smith’ apple peel for fruit harvested April 11th or 27th. Data are averages of four orchards of ten fruit each. Mean comparison between treatments for each harvest at P5 0.05.
A.D. Bauchot et al. / Posthar6est Biology and Technology 16 (1999) 9–14 Table 1 Effect of nitrogen induction of scald on fruit from different orchardsa Orchard
1 2 3 4
Scald index Control
Nitrogen induction
1.2bb 1.0b 2.8a 1.5b
3.9a 1.2c 3.4b 4.0a
a Fruit were harvested on 11th April and stored at 0°C for 16 weeks. Nitrogen was applied 4 days after removal from cold store. Scald was assessed 8 days after transfer to 20°C. b Means within a column and followed by the same letter are not significantly different (PB0.05).
The oxidation of a-farnesene into CTH has been the focus of much of the research on superficial scald (Emongor et al., 1994). The OD200nm has been negatively correlated with scald susceptibility and thought to be an estimate of total antioxidant activity (Meir and Bramlage, 1988). In both air-stored and N2-treated fruit from two harvests, the levels of a-farnesene decreased during 4 days in air (Fig. 2). Levels of a-farnesene were lower in fruit from the April 11th harvest, 4 days in air after N2 treatment. Fruit from this treatment also showed the highest scald index (Fig. 1). CTH levels in fruit from the first harvest were higher than in fruit from the later harvest. Both CTH levels and OD200 in N2-treated fruit were significantly lower than in fruit that had experienced only air-storage. The decline in these readings was more pronounced in the April 11th harvest. There was no significant change in the levels of these compounds beyond 4 days in air in any of the treatments (data not shown). Expression of PPO was very low while fruit were held in nitrogen, suggesting that the regulation of PPO gene expression is dependent on oxygen. Once fruit were removed to air, browning occurred almost immediately, such that tissue which was peeled and frozen immediately after removal from N2 was already browning (Fig. 3, lanes 2, 3). This time-frame is too rapid for the browning to be attributed to elevated PPO gene expression and suggests that the initial development of browning symptoms is not dependent on
13
increased expression of PPO mRNA. Over the 4 days after removal from N2 or air, PPO mRNA accumulated to high levels (Fig. 3, lanes 4, 5), but may be a consequence of other events associated with cell damage. These data suggest that the initial browning reactions in scald are unlikely to be attributed to elevated levels of PPO mRNA. However, altered PPO mRNA stability or activity of the enzyme may still be important. It has been proposed that upon shifting of tissue from anaerobic to aerobic conditions, active O2 species such as O2− are produced (Crawford and Braendle, 1996). The role of these species in causing tissue injury in plants is widely accepted (Winston, 1990). We speculate that N2-induction of scald involves the same mechanism, with cells experiencing post-anoxic injury. During N2 treatment of apples, there is also a decline in the levels of the antioxidant ascorbate (Burmeister and Dilley, 1995). Less ascorbate may then be available to quench active oxygen species produced upon return to air. The observation that natural superficial scald symptoms also do not usually develop in storage but appear during shelf-life (Bauchot et al., 1995), invites speculation that
Fig. 3. Expressed apple PPO mRNA in relation to N2-induced scald. Samples were taken from fruit held at 20°C for 0 days (lane 1), 2 or 4 days in the absence of oxygen (lanes 2 and 3), 4 days in air (lane 4) and from fruit held for 4 days in 100% N2 followed by 4 days in air (lane 5). Total mRNA (20 mg) was loaded on each lane. Bars indicate the relative intensity of the PPO mRNA signal as adjusted for RNA loadings.
14
A.D. Bauchot et al. / Posthar6est Biology and Technology 16 (1999) 9–14
superficial scald may also result from the production of active O2 species during the shift of apples from storage to ambient temperatures via the same mechanism. Nitrogen-induced scald represents a useful experimental system which may be used to confirm this and investigate other browning reactions in apples.
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