Reducing External Chilling Injury in Stored `Hass' Avocados with Dry Heat Treatments

'Hass' avocados (Persea americana Mill.) were heated in air at 25 to 46 °C for 0.5 to 24 hours and stored at 0, 2, or 6 °C. After storage, fruit were ripened at 20 °C and their quality was evaluated. In unheated fruit, external chilling injury occurred in fruit stored at 0 or 2 °C, but not 6 °C. Chilling injury was also evident after storage at 2 °C in fruit heated at 34 °C, and to a lesser extent in fruit heated at 36 °C. A heat treatment (HT) of 38 °C for 3, 6, or 10 hours and 40 °C for 0.5 hour further reduced external chilling injury induced by storage at 2 °C. These HT’s did not reduce internal fruit quality and resulted in more marketable fruit than unheated fruit stored at 6 °C. Low-temperature storage and HT slowed avocado ripening, resulting in longer shelf life after storage. In flesh tissue sampled directly after selected HT’s, the levels of mRNA homologous to cDNA probes for two plant heat-shock protein (HSP) genes (HSP17 and HSP70) increased to a maximum at 40 °C and declined at higher temperatures. These increases in gene expression coincided with the extent to which HT’s prevented chilling injury. Hotair HT’s confer significant protection against low-temperature damage to avocados. Long-distance transport and marketing of avocados is limited by the fruit's relatively short storage and shelf life. Although low-temperature storage reduces ripening rate and may be a means of disinfestation (Kerbel et al., 1987; Sanxter et al., 1994), avocados are susceptible to chilling injury below 6 °C. The symptoms are primarily a blackening of the skin's surface. Internal injury is less clearly defined. Flesh symptoms such as browning or graying of the mesocarp have been described (Zauberman et al., 1985), although these symptoms may not be solely a low-temperature response (Bower and Cutting, 1988). Heat treatments (HT’s) are receiving increasing attention as a means of reducing chilling injury in some fruit. Hot air or hot water HT’s reduce chilling injury in mango (McCollum et al., 1993), tomato (Lurie and Klein, 1991), and orange (Wild and Hood, 1989). HT’s can also be used as a disinfestation technique and to address storage problems (Paull, 1990). Sanxter et al. (1994) have reported beneficial effects of air HT’s on quality of 'Sharwil' avocados stored at <6 °C, and preliminary work with 'Hass' suggests similar results (Florissen et al., 1994; Woolf et al., 1994). Reported effects of HT’s on the physiology of various fruit include reduced ethylene production (Klein and Lurie, 1991; Paull, 1990), reduced gene expression of ACC oxidase (Picton and Grierson, 1988), lower levels of ion leakage (Saltveit, 1991), and slower rates of flesh softening and pectin solubilization (Klein et al., 1990). The effect of HT’s in reducing chilling injury may be related to some of the above responses. However, chilling injury is manifested in a variety of ways in different fruit, such as skin blackening in avocado (Sanxter et al., 1994), flesh gelling in persimmon (MacRae, 1987), and inhibition of ripening in apple (Klein and Lurie, 1990) and tomato (Lurie and Klein, 1991). Thus, the range of chilling symptoms suggests an array of effects from HT’s. Responses of plant tissues to high temperatures often include reduced protein synthesis, with induction of specific heat-shock protein (HSP) genes. It has been proposed that HSP’s confer thermotolerance by protecting other proteins from breakdown through a chaperone-like function or by removing heat-denatured proteins in the cell (Howarth and Ougham, 1993). HSP’s have been found in heated papaya (Paull, 1990), apples (Lurie and Klein, 1990), and tomatoes (Lurie and Klein, 1991), and HSP gene expression is enhanced in heated tomatoes (Lurie et al., 1993). Many beneficial effects of HT’s occur at temperatures where HSP gene expression is high (e.g., >36 °C), and HSP’s have been implicated in reduced chilling injury after HT (Lafuente et al., 1991). We investigated the response of 'Hass' avocados to dry air HT’s [nonsaturating relative humidities (RH’s)], particularly with respect to reducing chilling injury during subsequent low-temperature storage. We used a range of durations at each of several temperatures to define an optimal HT to reduce chilling injury. Using response surface analysis, resulting in contour plots, has helped define optimal treatments. We also examined HSP gene expression in relation to HT’s and the subsequent development of chilling injury in storage. Materials and Methods FRUIT HANDLING. Export-quality 'Hass' avocados were obtained from a commercial packhouse in the Bay of Plenty, New Zealand. Fruit were graded for uniformity of size (205 ± 15 g) and freedom from blemishes and placed in a 20 °C room (RH 65% ± 10%) overnight before HT. EXPERIMENTS. Three experiments were carried out involving HT’s of a range of temperatures and durations. In Expt. 1, fruit were treated at 25, 34, 38, or 42 °C for 6 or 24 h and stored at either 0 or 6 °C for 3 weeks. In Expt. 2, fruit were treated at 34, 36, 38, 40, or 42 °C for 3, 6, 10, or 24 h. In Expt. 3, fruit were treated at 40, 42, 44, or 46 °C for 0.5, 1.5, 3, or 6 h. In Expts. 2 and 3 fruit were stored at 2 °C for 4.5 weeks and these experiments were undertaken twice. HT SYSTEM. HT’s were carried out simultaneously in six identical 147-liter chambers using a computer-controlled 340-W heater and fan, which circulated air at a rate of 10 m-s through a single layer of fruit on a 45 x 70-cm plastic mesh grid. Air and internal fruit temperatures were measured and logged every 2 min using Squirrel data loggers (model 1206; Grant, Cambridge). To measure the internal temperature, a thermistor temperature probe (CM-UU-V5-1; Grant) was inserted at the stem end to the seed surface of one fruit per chamber. Timing of the HT duration was initiated when the internal temperature had reached the target temperature (2 ± 0.25 h from the commencement of heating). After reaching the target temperature, fruit within a given chamber varied by ± 0.25 °C over time and ± 0.5 °C spatially. RH was not controlled, but three 0.5-liter containers of water were placed in each chamber. RH dropped to 40% + 5% during the initial 2-h heating period and stabilized at 60% ± 10% for the rest of the HT. HT OF FRUIT AND MEASUREMENTS. Forty-eight fruit were weighed and placed in each chamber at a given temperature. For each treatment duration, 12 fruit were removed randomly during the HT period. After removal from HT, weight loss was determined on individual fruit. All heated fruit were placed in conditions that induced chilling injury (Expt. 1, O °C; Expts. 2 and 3, 2 °C) and additional unheated (20 °C) control fruit were held at 6 °C, the New Zealand industry standard for avocado storage. In Expt. 1, heated fruit was also stored at 6 °C. A replicate of 12 unheated fruit were held at 20 °C directly after harvest and internal fruit quality was examined when fruit were ripe (see below). FRUIT QUALITY. Immediately after removal from storage, external injury (blackening, pitting of the skin surface) due to chilling injury or heat damage was rated on a relative scale (0 to 3 in 0.5 intervals; 0 = no occurrence, 3 = blackening of >90% of the fruit surface). Fruit quality after ripening at 20 C was evaluated as each fruit reached a ripeto-eat or optimum-eating-ripeness stage of maturity, which was determined by gently squeezing fruit daily, and the number of days to reach ripeness was recorded (shelf life). Several fruit quality factors were rated, including uneven ripening (uneven flesh softening such that flesh tissue adhered to the seed when the fruit was cut in half), internal rots (stem-end and body rots), tissue breakdown (presence of green fruit tissue adhering to the skin when peeled away from the flesh), vascular browning (browning of the vascular strands running vertically through the fruit tissue), and flesh browning (browning of the fruit tissue not due to disease or vascular browning). Each factor was rated on a scale of 0 to 3 in intervals of 1 (Hopkirk et al., 1994), where 0 = no occurrence; 1 = slight; 2 = a level at which the consumer would notice and possibly reject the fruit; 3 = extreme. Informal taste tests were also carried out on selected HT’s and compared with unheated fruit stored at 6 C. ETHYLENE PRODUCTION AND RESPIRATION RATE. Ethylene production and respiration rates were measured daily on fruit from selected treatments held at 20 °C after removal from storage. Five fruit per treatment were sealed individually in 1-liter containers and, after l h, l-ml gas samples were removed by syringe and ethylene and carbon dioxide concentrations were measured by gas chromatography using flame-ionization and thermal-conductivity detectors, respectively (Philips PU4500; Pye Unicam, Cambridge). RNA EXTRACTION AND NORTHERN ANALYSIS. Fruit of selected treatments were sampled immediately after HT’s in Expt. 2. A series of samples was also taken from fruit that had been heat-treated at 38 °C for 6 h and subsequently held in storage (2 °C) for 6 weeks. Fruit flesh to a depth of 10 mm was removed with a 10-mm (ID) cork borer from the equator of the fruit, and 1-g samples (with the skin removed) were immediately frozen in liquid nitrogen and held at -80 °C. Total RNA was extracted, denatured, fractionated (30 μg/ lane) and transferred to a Hybond-N+ (Amersham) membrane as described by Veierskov et al. (1992). The inserts from pMON9575 (petunia HSP70 cDNA; Winter et al., 1988) and pFS1968 (soy bean HSP17 cDNA; Schoffl et al., 1984) were labeled with P using a random primers DNA labeling system (BRL). Hybridization was carried out according to the Amersham Hybond-N+ protocol. After hybridization, the membrane was washed twice for 10 min in 2x SSC and 0.1 % SDS at room temperature, then once in 1 x SSC and 0.1% SDS at 65 °C for 15 min. The membrane was exposed to Kodak XAR film with intensifying screens at -80 °C. STATISTICAL ANALYSES. Experiment 1 was an unre