Effects of jasmonates on production of aroma volatile compounds by 'Tsugaru' apples IMalus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.I at harvest, and by Delicious' apples after harvest were examined. Among volatiles classified as alcohols, esters, ketones, aldehydes, acetic acid, and a-farnesene, esters were the most prevalent compounds, followed by alcohols. Jasmonate treatment at pre-climacteric stage increased the production of esters (butyl propanoate, butyl butyrate, and propyl butyrate) and of anthocyanin. In addition, jasmonate treatment stimulated 1-aminocyclopropane-1-carboxylate oxidase 1 (ACO 1) mRNA transcript and thylene production at 3 days after treatment. This result suggests that jasmonates may influence ACC oxidase activity. The impact of jasmonate application after harvest on volatile production differed with cultivar. The combination of ethephon with jasmonates reduced volatile production by 'Delicious' compared with ethephon only. The effect of jasmonates on volatile production was related to the effect of jasmonates on internal ethylene concentration. The results show that the effect of jasmonates on aroma volatiles in apples may be mediated by ethylene. Furthermore, the effect of jasmonates on aroma volatiles may depend on the fruit development stage when treated with jasmonates. INTRODUCTION Aroma volatile compounds are one of the factors that determine apple fruit quality. Aroma production increases with ripening and is associated with ethylene production (Song and Bangerth, 1996; Lalel et al., 2003b). Methyl jasmonate (MeJA) also influences the production of volatile compounds, but the effect can vary with volatile compound chemical class (Fan et al., 1997). Although many types of aroma volatiles are synthesized in apple fruit, the relative abundance of each type differs among cultivars (Fan et al., 1997; Mattheis et al., 1998). For most cultivars, esters are qualitatively and quantitatively predominant (Rowan et al.. 1999). Aroma volatiles are primarily synthesized in the skin (Knee and Hatfield, 1981), and Kondo et al. (2001) showed that jasmonates effectively stimulate anthocyanin biosynthesis and that the effect is independent of ethylene. Therefore, Jasnionates have been applied prior to harvest in the field for the promotion of red color development in Japan. However, the effect ofjasmonate applied prior to harvest on aroma volatiles is unclear. In this study, effects ofjasmonates and ethephon, alone or in combination, on aroma volatile and ethylene production during apple fruit ripening were examined. MATERIALS AND METHODS Chemicals n-Propyl dihydrojasmonate (PDJ. a.i. 5.0 %) was a gift from Nippon Zeon Co. (Tokyo). 2-Chloroethyl phosphonic acid (Ethephon, a. i. 21.7 %) was purchased from Rhone-Poulenc Co. (Research Triangle Park, NC). Methyl jasmonate (MeJA. 95 %) was Purchased from Sigma-Aldrich Co. (Milwaukee, WI). Proc IV IC Oo MQU1C 381 Eds. A.C. I'm is et al. Acta i-Ion. 712, IS! IS 2006 Plant Material Six randomly selected 17-year-old 'Tsugaru' apple trees, grafted onto Mailing 9 (M. 9) rootstocks, growing in an open field at Prefectural University of Hiroshima were used in 2005. Each tree was trained as a central leader and planted in a single row from east to west with spacing of 3.0 m x 4,0 in. Furthermore, for 'Tsugaru', PDJ solution of 0.39 mM was applied by spraying whole trees 107 days after full bloom (DAFB). Thirty fruit (10 from each tree) were sampled at 7 days intervals. For 'Delicious' fruit were harvested 179 DAFB in 2003. Immediately after harvest, 304 fruit were randomly separated to 4 groups of 76 fruit for the following treatments 1) MeJA, 2) Ethephon, 3) Ethephon + MeJA, 4) Untreated control. Group I and 3 fruit were dipped for 5 mm in a solution of 0.177 % (v/v) Tween® 20 with 5 mM MeJA or the same solution in combination with 400 a. i. mg'L' ethephon, respectively. Group 4 fruit were dipped for 5 min in a solution of the same concentration of Twecn 20 in deionized water (untreated control). After treatment, apples were placed in the dark at 20 °C and sampled at 7 (lays intervals for analysis of volatile compounds, ethylene production, and jasmonate concentrations. Analysis of Aroma Volatile Compounds and Ethylene Analysis of volatile compounds was performed with a modification of the method previously described by Mattheis et al. (1991). Four apples per treatment (three replications) were placed in four 1 glass jars and the jars purged with air that had been passed through activated charcoal and molecular sieve. Air flowed through the jars at 50 ml min ' for 3 h before collection of volatile compounds onto glass traps containing 50 mg Tenax GC (AlItch Assoc., Deerfield, IL). The absorbed contents in the trap were desorbed into the injection port of gas chromatograph with a mass selective detector ((iCMSD) (HP 5890, 5971A, Hewlett Packard, Avondale, PA). The column oven temperature was held at 35°C for 3 mm, then increased from 35°C to 225°C at a rate of 8°C niin ' . For Tsugaru'. hue value and ethylene production from fruit were measured according to the previous report (Kondo and Takano, 2000 Kondo et al., 2001). For Delicious', internal ethylene in the core was analyzed, using a HP5890 gas chromatograph (Hewlett-Packard, Avondale, PA) equipped with a glass column (610 mm x 3.2 mm i.d.) packed with Porapak Q (80-100 mesh) (Supelco. Bellefonte, PA) and flame ionization detector. Extraction and Analysis of Jasmonates Extraction and quantification of MeJA were carried out as described previousl y by Kondo et al. (2000). The deuterium-labeled MeJA [(11 2)-MeJA: methyl (+)-[9. 10--H2] jasinonate] used for the internal standard was prepared according to the method of Seto et al. (1996). Lyophilized skinless gulp samples (5 g dry weight (DW)); three replications) were homogenized with I tg (-H 2) MeJA in 50 nil diethyl ether containing 11.3 1iM hutylated hydroxytoluene as an antioxidant, 20 ml saturated NaCl solution, and I ml I M citric acid. The MeJA in the samples was derivatized with pentafluorobenzyl (PFB) bromide. The amount of PFB-MeJA was analyzed by GC-MS selected ion monitoring (SIM) (QP 5000 Shimadzu, Kyoto, Japan). Northern Blot Hybridization eDNA of ACC oxidase (ACO 1) was a gift from Dr. T. Ilarada at Hirosaki university. The fragment used for probes was from bp 3558 to bp 3780 of the ACO I with accession number AF030859. The Northern blot analysis was carried out using previously reported method (kondo et al., 2002). Statistical Analysis The SAS ANOVA procedure was used to determine treatment effects, and mean separation was analyzed by Fisher's least significant difference (p 0.05)(SAS, Cary, NC).