Abstract

Braeburn apples grown in New Zealand are susceptible to the development of a internal browning disorder called Braeburn browning disorder (BBD). BBD develops during the first 2-4 weeks of storage and incidence and severity are increased when fruit are held under elevated CO₂ and low O₂ storage conditions (Elgar et al. 1998). Other factors contributing to predisposition of Braeburn to BBD and similarities with other CO₂-related injuries are discussed in Watkins et al. (1998). After a delay in air prior to controlled atmosphere (CA) storage fruit become acclimated and do not develop BBD (Burmeister et al., 1998). Late harvested fruit are also more susceptible. In the following experiments susceptibility of Braeburn to BBD was varied by harvesting early vs late or by application of CA immediately vs holding fruit for 14d in air storage at 0^oC. BBD development was also evaluated in response to pretreatment with C₂H₄ , DACP, or DPA.

Materials and methods

Analysis. Ethanol and acetaldehyde was measured by placing cortical tissue plugs in a 100 X 15 mm test tube sealed with a rubber septum, equilibrating for 1 hour in a 25^oC water bath, and measuring the headspace concentrations by gas chromatography. Fruit were sampled at 0, 4, 7, 12, 18, and 21d after application of CA.

Ascorbate was measured by analysis of 0.1-0.2g of freeze dried tissue by the methods of Barden and Bramlage, (1994). Data points in Figures 1-3 are mean ±SEM of analysis of two 1 cm length X 0.9 diameter tissue plugs taken from opposite sides of the mid cortical region of 10 individual fruit.

Pretreatments. Fruit were placed in a static chamber with »200 µl l^-1 C₂H₄ , or 10ml of pre-irradiated DACP for 12 hours at 20^oC. Satchets of hydrated lime (0.2 Kg per 20 Kg fruit) were included in each chamber to scrub CO₂.

Diphenylamine (DPA) treatment was by submersion of fruit for 3 min in 1.5 mg l^-1 commercial formulation, no scald-DPA then allowing to dry at 20^oC for 4 hours. Following pretreatments fruit were cooled overnight and placed in CA or air for 4 weeks.

Results and Discussion

Figure 1. BBD Score (incidence x severity) during CA storage of early and late harvested fruit placed in CA after 1d or after 14d in 0^oC air.

Acetaldehyde and ethanol accumulation. In both harvests when fruit were placed in CA immediately acetaldehyde accumulated to a maximum level after 4-6 days. Then levels remained relatively constant except in late harvested fruit held in air for 14d prior to CA where levels declined to below that of the air control. There was not as much difference between CA and air stored fruit in the late vs the early harvest. The most ethanol accumulation occurred in fruit from early harvest that was placed in CA immediately (Fig. 2). If CA application was delayed the amount of ethanol accumulation was reduced in both harvests.

Figure 2. Acetaldehyde accumulation in cortical tissue plugs during development of BBD.

Acetaldehyde and/or ethanol accumulation has often been associated with injury to fruit tissues. More recent thinking has considered that these compounds may actually have some protective role especially in the prevention of chilling injury (Scott et al., 1995). In other studies, we have not been able to show reduction in BBD by ventilation of fruit under CA with 10,000 µl l^-1 ethanol vapours (data not shown). No consistent pattern of acetaldehyde or ethanol accumulation appeared to be associated with susceptibility of fruit to BBD.

Ascorbate content. Ascorbate levels increased during storage for both harvests except for the late harvested fruit placed in CA immediately where ascorbate levels declined and remained low. Ascorbate levels were almost always lower in the CA than air stored fruit (Fig. 3). This suggests that there is some differences in response to CA between harvests and delay treatments. Ascorbate can reduce free radicals either directly or can act as substrate for ascorbate peroxidase in the reduction of the reduced O₂ species, H₂O₂ to H₂O (Winston, 1990). Agar et al., (1997) has suggested that oxidation of ascorbate is stimulated and/or reduction of mono- or dehydroascorbic acid is inhibited under high CO₂ conditions in berry fruits. Reduction in levels of ascorbate could render fruit tissue susceptible to injury.

Figure 3. Ethanol accumulation in cortical tissue plugs during development of BBD.

C₂H₄ and DACP. C₂H₄ treatment reduced BBD incidence and severity by about 50% and 66%, respectively while DACP treatment had no effect. This indicates that C₂H₄ exerts its effect through C₂H₄ action and not the receptor. It could be hypothesised that an C₂H₄ induced respiration increase would result in damaging internal O₂ and CO₂ partial pressures. We suggest that C₂H₄ could be enhancing the processes involved in acclimation.

DPA. Treatment with commercial formulation of the general antioxidant DPA dramatically reduced the incidence and severity of BBD (Table 1). This indicates that oxidative processes may be involved in the development of tissue injury. DPA reduced internal CO₂-injury in ‘Bramley’s seedling’ apples (Johnson et al., 1998). It is hypothesised that DPA would quench reduced O₂ species that would damage proteins and other macromolecules leading to tissue injury (Burmeister and Dilley, 1995).

Although BBD has many characteristics in common with other CO₂ related disorders (Elgar et al., 1998), the physiological and biochemical mechanisms by which ‘Braeburn’ develop the disorder are not known. That there is no clear relationship of acetaldehyde and ethanol accumulation and that C₂H₄ pretreatment reduced rather than increased BBD indicates that more than an accumulation of toxic metabolites due to increased respiration is involved. C₂H₄ pretreatment has been demonstrated to promote ascorbate peroxidase activity and provide protection from H₂O₂ (Mehlhorn, 1990). That DPA reduced the disorder indicates that production of free radical species may be involved be involved in BBD development. We suggest that C₂H₄ could be exerting its effect by upregulation of free-radical scavenging enzymes.

Succinate has been observed to accumulate in fruit held in high CO₂ atmospheres (Hulme, 1956). It has been suggested that a-ketoacids such as pyruvate and succinate may have a role in regulation of reduced O₂ species (Purvis, 1997). If these organic acids of the Kreb’s cycle are acting as antioxidants, this could explain how respiratory metabolism is involved in BBD development.

To understand BBD it may be useful to understand of how fruit become acclimated during air storage and tolerant of subsequent CA. We speculate that the mechanisms of the CO₂-injury, BBD and acclimation are complex and may involve pathways in both respiratory and oxidative metabolism.

Figure 4. Ascorbate content of Braeburn during BBD development.

Literature Cited

Barden, C.L. and W.J. Bramlage. 1994. Accumulation of Antioxidants in Apple Peel as Related to Preharvest Factors and Superficial Scald Susceptibility of the Fruit. J. Amer. Soc. Hort. Sci. 119: 264-269.

Burmeister, D.M., Yearsley, C.W. and H.J. Elgar. 1998. Acclimation of ‘Braeburn’ apples in air at 0^oC, 10^oC, and 20^oC prior to storage in high CO₂ and low O₂ reduces susceptibility to ‘Braeburn’ browning disorder. Postharvest Biology and Technology (in press).

Elgar, H.J., D.M. Burmeister, D.M. and C.B. Watkins. 1998. Storage and handling effects on a CO₂ -related internal browning disorder in ‘Braeburn’ apples. HortScience (in press).

Hulme, A.C. 1956. Carbon dioxide injury and the presence of succinic acid in apples. Nature, London. 178, 218-219.

Johnson, D.S., C.J. Dover and R.J. Colgan. 1998. Effect of rate of cooling and establishment of CA conditions on the development of CO2 injury in ‘Bramley’s Seedling’ apples. Acta Hort. (in press).

Mehlhorn, H. 1990. Ethylene-promoted ascorbate peroxidase activity protects plants against hydrogen peroxide, ozone and paraquat. 13: 971-976.

Purvis, A.C. 1997. Role of pyruvate in regulating reactive oxygen levels in plant tissues. Acta Hort. (in press).

Scott, K.J.; Yuen, C.M.C.; Ghahramani, F. 1995: Ethanol vapour - a new anti-scald treatment for apple. Postharvest Biology and Technology 6: 201-208.

Watkins, C.B. Burmeister, D.M. and H.J. Elgar. 1998. A comparison of Two Carbon Dioxide-Related Injuries of Apple Fruit. In: A. Kader (Ed.) Proceedings of the Seventh Controlled Atmosphere Research Conference. U.C. Davis, California. in press.

Wilkinson, K.L. and J.C. Fidler. 1973. Injuries caused by incorrect concentrations of carbon dioxide and/or oxygen. p. 81-87. In: Fidler, J.C., Wilkinson, B.G., Edney, K.L. and Sharples, R.O. (Eds.). The biology of apple and pear storage. Res. Rev. 3, Commonwealth Bureau of Horticulture and Plantation Crops, East Malling, England.

Winston, G.W. 1990. Physiochemical basis for free radical formation in cells: Production and defenses. In: R.G. Alscher amd J.R. Cummmings (eds.). Stress Responses in Plants: Adaptation and Acclimation Mechanisms. Wiley-Liss, New York. pp. 57-86.