998 J. AMER. SOC. HORT. SCI. 127(6):998–1005. 2002. J. AMER. SOC. HORT. SCI. 127(6):998–1005. 2002. Temporal Relationship between Ester Biosynthesis and Ripening Events in Bananas Sastry Jayanty, 1 Jun Song, 2 Nicole M. Rubinstein, 3 Andrés Chong, 4 and Randolph M. Beaudry 5 Department of Horticulture, Michigan State University, East Lansing, MI 48824 ADDITIONAL INDEX WORDS. Musa spp., respiration, ethylene, aroma, chlorophyll fluorescence, AAT, ACC oxidase ABSTRACT. The temporal relationship between changes in ethylene production, respiration, skin color, chlorophyll fluores- cence, volatile ester biosynthesis, and expression of ACC oxidase (ACO) and alcohol acyl-CoA transferase (AAT) in ripening banana (Musa L. spp., AAA group, Cavendish subgroup. ‘Valery’) fruit was investigated at 22 ° C. Ethylene production rose to a peak a few hours after the onset of its logarithmic phase; the peak in production coincided with maximal ACO expression. The respiratory rise began as ethylene production increased, reaching its maximum ≈ 30 to 40 hours after ethylene production had peaked. Green skin coloration and photochemical efficiency, as measured by chlorophyll fluorescence, declined simultaneously after the peak in ethylene biosynthesis. Natural ester biosynthesis began 40 to 50 hours after the peak in ethylene biosynthesis, reaching maximal levels 3 to 4 days later. While AAT expression was detected throughout, the maximum level of expression was detected at the onset of natural ester biosynthesis. The synthesis of unsaturated esters began 100 hours after the peak in ethylene and increased with time, suggesting the lipoxygenase pathway be a source of ester substrates late in ripening. Incorporation of exogenously supplied ester precursors (1-butanol, butyric acid , and 3-methyl- 1-butanol) in the vapor phase into esters was maturity-dependent. The pattern of induced esters and expression data for AAT suggested that banana fruit have the capacity to synthesize esters over 100 hours before the onset of natural ester biosynthesis. We hypothesize the primary limiting factor in ester biosynthesis before natural production is precursor availability, but, as ester biosynthesis is engaged, the activity of alcohol acyl-CoA transferase the enzyme responsible for ester biosynthesis, exerts a major influence. respiration, and volatile biosynthesis has not been reported. Esters are derived from amino acid and fatty acid metabolism (Myers et al., 1970; Tressl et al., 1970). Alcohol acyl-CoA trans- ferase (AAT, EC 2.3.1.84) combines alcohols and CoA derivatives of short to medium chain length fatty acids to form esters. AAT has been identified and partially purified in ripe banana fruit (Harada et al., 1985; Ueda and Ogata, 1977) and its gene identified originally in strawberry (Fragaria ×ananassa Duchesne) (Aharoni et al., 2000) and subsequently in banana (Aharoni et al., 2001). AAT activity increases during banana fruit ripening for fruit induced to ripen by exposure to exogenous ethylene (Harada et al., 1985). In the study by Harada et al. (1985), in vivo AAT activity was estimated by supplying tissue samples with 3-methyl-1-butanol and measur- ing the increase in the production of 3-methylbutyl acetate. Gene expression for AAT has not been reported for banana, but has been shown to increase for strawberry as they ripen (Aharoni et al., 2000). The primary objective of this study was to characterize the temporal relationship between changes in ripening indices and aroma-related volatile biosynthesis. Particular emphasis was placed on temporal changes in the capacity of banana fruit to synthesize esters in vivo from exogenously supplied alcohols, acids, and their combination. In our study, whole banana fruit were enclosed in respiratory chambers at room temperature (22 °C) and changes in skin hue, chlorophyll fluorescence, CO 2 production, and C 2 H 4 biosynthesis were determined relative to the synthesis of aroma- related volatile compounds. In addition, whole fruit and tissue plugs of peel and pulp were excised at three stages of fruit development (before the ethylene climacteric, at the peak in ethylene synthesis, and at the peak in ester formation) and their capacity to incorporate alcohols and acids into esters was determined by measuring net changes in ester production. We focused on the metabolism of 3- methyl-1-butanol, 1-butanol, and butyrate and the important aroma impact compounds 3-methylbutyl acetate and 3-methylbutyl butanoate. Finally, to understand the relation between changes in the production of ethylene and esters, we performed Northern analysis for ACC oxidase (ACO) and AAT, respectively. Received for publication 27 Dec. 2000. Accepted for publication 28 Aug. 2002. Research supported in part by the High School Honors Science Program at MSU. We acknowledge Gail Richmond for her efforts in this program. We thank Asaph Aharoni for kindly providing the AAT clone for banana and Michael Lay-Yee for the ACO clone for apple. Mention of a trademark does not imply endorsement of the product. 1 Research associate. 2 Research associate. Currently Research Scientist, Agriculture and Agri-Food Canada, Kentville Research Centre, 32 Main St., Kentville, NS B4N 1J5, Canada. 3 Student, Roslyn High School, Andover, N.Y. Currently Dept. Appl. Econ. Mgt., Cornell Univ. Ithaca, N.Y. 4 Graduate student. 5 Professor and corresponding author; e-mail beaudry@msu.edu. A temporal relationship has been established between ethylene production and several physiological changes including those re- lated to skin color (Peacock, 1972; Seymour et al., 1987), respira- tion, and starch conversion (Beaudry et al., 1989). Although ripen- ing is associated with a decline in chlorophyll fluorescence (Smillie et al., 1987), the temporal relationship between chlorophyll fluores- cence and natural ethylene synthesis has not been described. Simi- larly, aroma biosynthesis is known to be associated with ethylene synthesis and action (Golding et al., 1998; Peacock, 1972); how- ever, its temporal relationship to ethylene production and other ripeness stage indices such as respiration, color, and chlorophyll fluorescence has not been studied for individual fruit. The aroma of banana fruit (Musa spp. AAA group, Cavendish subgroup. ‘Valery’) is an important eating quality criteria that influences consumer acceptability. More than 250 volatile com- ponents have been identified in banana (Macku and Jennings, 1987; Shiota, 1993; Tressl et al., 1970). By combining analytical chemistry with sensory measurements, several volatile com- pounds have been determined to be ‘impact compounds’, which contribute significantly to aroma and confer typical aroma per- ceptions. Penten-2-one and the 3-methylbutyl and 2-methylpropyl esters of acetate and butanoate, have been found to be the major contributors to banana fruit aroma (Berger et al., 1986). The temporal relationship between endogenous ethylene production,