Materials and methods Directory UMM :Data Elmu:jurnal:P:Postharvest Biology and Technology:Vol20.Issue3.Nov2000:

long-term cold storage Ingle and D’Souza, 1989. Largely on the basis of correlative data, the sesquiterpene a-farnesene, and more directly its conjugated triene CT oxidation products, have long been thought to play a central role in scald induction Huelin and Coggiola, 1968, 1970a; Anet and Coggiola, 1974; Whitaker et al., 1998. How- ever, the biochemical mechanism of the disorder has not been elucidated. Recent findings are consistent with the proposal that a volatile end-product of a -farnesene oxidation, 6-methyl-5-hepten-2-one MHO, causes the discoloration and death of hypodermal cells which lead to development of scald symptoms Mir and Beaudry, 1999; Mir et al., 1999. Exogenous MHO induced scald-like brown- ing in peel tissue from fruit of scald-susceptible apple cultivars Mir and Beaudry, 1999, and a poststorage burst of MHO evolution was associated with intensification of scald symptoms in ‘Cortland’ apples Mir et al., 1999. Moreover, it was recently shown that the CTs which accumulate in the skin and epicuticular wax of apple fruit during storage, mainly 7E,9E and 7E,9Z isomers of 2,6,10- trimethydodeca-2,7,9,11-tetraen-6-ol conjugated trienols in a ratio of 9:1 Rowan et al., 1995; Whitaker et al., 1997, autoxidize at 20°C yielding MHO as a major product Whitaker and Saftner, 2000. Regardless of the role of a-farnesene oxidation products, scald development very likely involves the adverse effects of oxidative stress that occurs with prolonged storage at chilling temperatures Du and Bramlage, 1995; Shewfelt and Purvis, 1995; Watkins et al., 1995; Rao et al., 1998. Huelin and Coggiola 1970b and Anet 1972 first proposed that scald susceptibility or resistance in different apple cultivars is, at least in part, determined by the efficiency of their natural antioxidant defenses. Anet 1974 subsequently examined the levels of endogenous lipophilic antioxidants in the cuticle of 16 apple cultivars and found a correlation between scald resistance and the presence of antioxidant levels sufficient to curtail a-farnesene oxidation during storage. Meir and Bramlage 1988 also showed a negative correlation between scald suscep- tibility and high levels of unidentified lipophilic antioxidants with an absorbance maximum at 200 nm in the cuticle of ‘Cortland’ apples. More recent investigations have compared the accumula- tion of peroxides and lipid peroxidation products, and the activities of enzymes that detoxify active oxygen species AOS, in peel tissue of scald-suscep- tible and -resistant apple fruit stored at 0 – 1°C in air Du and Bramlage, 1994a, 1995; Rao et al., 1998. Du and Bramlage 1994a, 1995, compared apple cultivars with wide variation in scald susceptibility ‘Cortland’, ‘Delicious’ and ‘Empire’ and found no marked changes in peroxidation or activities of antioxidative defense enzymes related to scald de- velopment. In contrast, Rao et al. 1998 found a close correlation between increasing levels of H 2 O 2 and lipid peroxidation products, declining perox- idase and catalase activities, and the occurrence and severity of scald symptoms in fruit of susceptible and resistant ‘White Angel’בRome Beauty’ hybrid selections grown at the same location under identi- cal conditions. In their study of the ‘White Angel’בRome Beauty’ selections, Rao et al. 1998 assessed changes in a-farnesene and CT levels spectrophoto- metrically using hexane- dip extracts of individual fruit. Although a-farnesene and CT concentrations appeared somewhat higher in scald-susceptible than in scald-resistant lines early in storage, a-farnesene synthesis and oxidation were not elevated in apples that eventually developed scald symptoms. In addi- tion, Rupasinghe et al. 1998 determined that a -farnesene synthase activity was about threefold lower in scald-developing compared with scald-free ‘Delicious’ apple peel tissue. These findings chal- lenge the hypothesis that oxidation of a-farnesene is directly linked with scald induction. Thus, the present study was undertaken to rigorously examine a -farnesene synthesis and oxidation in individual red- and yellow-pigmented ‘White Angel’בRome Beauty’ selections during storage using HPLC – UV analysis Whitaker et al., 1997, and to assess the relationship of a-farnesene metabolism to the inci- dence and severity of scald after storage.

2. Materials and methods

2 . 1 . Plant material and fruit source As in the previous study by Rao et al. 1998, the fruit used were harvested from a family of apple trees derived from a ‘White Angel’ and ‘Rome Beauty’ cross growing at the New York State Agricultural Experiment Station in Geneva, NY Hemmat et al., 1994; Lawson et al., 1995. Trees derived from this cross segregate for red- and yellow-skinned fruit Cheng et al., 1996 with wide variation in scald susceptibility Weeden, 1993. 2 . 2 . Tissue sampling, storage conditions, and scald assessment Over two harveststorage seasons 199798 and 199899, fruit from 16 selections, eight red- and eight yellow-skinned, were sampled and exam- ined. Among these, three red-fruited R-44, R-48 and R-85 and five yellow-fruited Y-38, Y-40, Y-55, Y-65 and Y-67 lines exhibited at least mild scald symptoms after storage, whereas the remaining lines were completely free of scald R- 01, R-03, R-16, R-20, R-22, Y-07, Y-26 and Y- 28. Five red-skinned R-03, R-20, R-22, R-48 and R-85 and three yellow-skinned Y-26, Y-55 and Y-65 selections were harvested, stored, and analyzed in both years. All apples were picked on 15 October in 1997, and on 14 October in 1998. Ten fruit from each selection were used to determine the internal ethylene concentration IEC as described by Rao et al. 1998. Thirty fruit per selection were sampled at harvest and after 7, 14 and 21 weeks of air storage at 0.5°C in perforated plastic bags. The skin and outer 2 – 3 mm of cortical tissue were peeled manually, frozen immediately in liquid N 2 , and stored at − 80°C. All fruit were peeled within 30 min of removal from 0.5°C storage. Samples of the pooled peel tissue from each selection × duration of storage 40 – 50 g were sealed in individual ziplock bags, packed in dry ice, and shipped overnight to Beltsville, MD for HPLC analysis of a-farnesene and CTols. When received, the samples were stored at − 80°C until analyzed. After 21 weeks at 0.5°C plus 1 week at 20°C, at least 100 fruit of each selection were visually assessed for scald, except for R-20 27 fruit, R-44 62 fruit and Y-28 39 fruit in the 199798 season, which had a high inci- dence of decay. On fruit showing scal sym- ptoms, scald severity was rated on a scale of 0 – 4 based on the percentage of the sur- face area affected, where 0 = no scald, 1 = 1 – 10, 2 = 11 – 33, 3 = 34 – 66 and 4 = 67 – 100. 2 . 3 . Preparation of samples and HPLC analysis Frozen peel samples 3 g were pulverized in liquid N 2 and transferred to 50-ml screw-cap culture tubes containing 9 ml of HPLC-grade hexane. The tubes were flushed with N 2 , sealed, and agitated at 5°C for 1.5 h. Extracts were vacuum filtered through glass fiber disks and re- stored to 9 ml total volume. Aliquots 1.5 ml were transferred to 2-ml vials and the hexane evaporated under a gentle stream of N 2 without heating. The residue was dissolved in 400 ml of HPLC-grade methanol and filtered through a 0.45 mm PTFE membrane prior to HPLC analy- sis. Samples 80 ml were injected manually into a Waters 600MS HPLC system fitted with a 4.6 × 250 mm, Luna C 18 column Phenomenex, Tor- rence, CA. The mobile phase was meth- anolacetonitrilewater 90:7.5:2.5 pumped at 0.8 ml min − 1 . Absorbance at 232 nm a-farnesene and 269 nm CTols was monitored by a Waters 490 programmable wavelength detector and data were gathered and processed using the Waters Baseline 810 program in a 286 PC. a- Farnesene gave a single peak that eluted at 11.9 min and CTols gave a prominent peak at 5.8 min with a small shoulder at 6.0 min. Cal- culations of a-farnesene and CTol concentrations were based on their molar extinction coefficients as previously described Whitaker et al., 1997. A second aliquot of the hexane extracts was used for spectrophotometric estimation of a-farnesene and CTol concentrations according to the method of Rao et al. 1998. Absorbance at 232 nm a-farnesene and 281 – 290 nm CTols was recorded using a Shimadzu UV-160 spectro- photometer. Samples were diluted as required to maintain A 232 nm in the linear range 5 1.2 AU. Fig. 1. a-Farnesene concentration in peel tissue of ‘White Angel’ × ‘Rome Beauty’ selections at harvest as a function of the log of the internal ethylene concentration IEC. Data points represent both red- and yellow-skinned lines from the 1997 and 1998 harvests. The polynomial equation derived from regression analysis was Y = 12.77 + 10.54X + 2.25X 2 , with an R 2 value of 0.478.

3. Results