Defilippi, B * ., Dandekar, A.M. and Kader, A.A.

1. Understanding the role of ethylene in ester biosynthesis in apples (Malus x domestica Borkh. cv. Greensleeves). Defilippi, B*., Dandekar, A.M. and Kader, A.A. Department of Pomology, University of California. One Shields Ave, Davis CA 95616, USA *email: bdefilippi@ucdavis.edu ABSTRACT The role of ethylene in ripening parameters has been investigated on apple fruits obtained from 40 lines defective in ethylene biosynthesis. The lines were transformed expressing the cDNAs of ACC-synthase (ACS) and ACC-oxidase (ACO) enzymes in either a sense or antisense pattern. In some transformed lines, more than 97% of suppression of enzyme activity and ethylene production was measured (Figure 1, Table 1). As expected, preliminary results have shown differential level of dependence of ripening parameters to ethylene biosynthesis revealing either ethylene-non associated events as acidity and soluble solids, or ethylene-associated events including flesh firmness and color changes (Fig. 2). Regarding aroma production, head space analysis showed a reduction in ester production in the ethylene-suppressed lines (Fig. 3). The information available in the scientific literature suggests that ester production in apple fruits is an ethylene-dependent process; however, the regulatory mechanisms of ethylene in ester biosynthesis have not been elucidated. This will be the focus of this research project. 14 days at 20°C MATERIALS AND METHODS Fig 2. Maturity parameters in clones of Greensleeves apple fruit. • Transgenic Greensleeves apple apple fruits defective in ethylene were obtained from different lines grown in an experimental orchard in Davis, California, and stored at 20°C for 14 days. • Evaluations: • Fruit ripening at harvest and after storage was evaluated. Among parameters considered were: firmness, peel color, soluble solids, volatile production and respiration rate. • Ethylene biosynthesis characterization: ACS activity, ACC content, ACO activity and ethylene production were determined. Fig 3. Ester concentration in Greensleeves apple fruit using a CAR/PDMS fiber. DISCUSSION AND FUTURE WORK • The availability of these transformed lines defective in ethylene biosynthesis is a good opportunity for expanding the present biochemical and molecular understanding of ester biosynthesis in apple; therefore, further experimental work will be performed. The hypotheses to be tested include (1) the mechanism of ester biosynthesis is mainly determined by the regulatory effect of ethylene on alcohol acyl CoA transferase (AAT), and (2) ethylene may be affecting substrate availability and enzymes other than AAT involved in ester biosynthesis in apple fruit. • The general objective is to identify the regulatory mechanism of ethylene in ester biosynthesis in apple fruit. The specific objectives are (a) to identify the main enzyme(s) involved in ester production, (b) to quantify the role of ACS and ACO activity in ethylene biosynthesis and their influence in ester biosynthesis, and (c) to characterize the temporal events of both ester and ethylene biosynthesis. Fig 1. Ethylene production and respiration rate in Greensleeves fruit stored 14 days at 20°C (R=ACO sense, G=ACO antisense and Y=ACS sense). Table 1. ACS activity, ACC content and ACO activity of Greensleeves apple fruit stored for 14 days at 20°C. LITERATURE REVIEWED Aharoni, A., Keizer, L.C.P., Bouwmeester, H.J., Sun, Z., Alvarez-Huerta, M., Verhoeven, H.A., Blaas, J., L. van Houwelingen, M.L., De Vos, R.C.H., van der Voet, H., Jansem, R.C., Guis, M., Mol, J., Davis, R.W., Schena, M., van Tunen, A.J. and O’Connell, A.P. 2000. Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarray. The Plant Cell 12:647-661 Dixon, J. and Hewett, E.W. 2000.  Factors affecting apple aroma/flavour volatile concentration: a review. New Zealand Journal of Crop and Horticultural Science 28(3):155-173 Fellman, J.K., T.W. Miller, D.S. Mattinson and J.P. Mattheis. 2000. Factors that influence biosynthesis of volatile flavor compound in apple fruits. HortScience 35(6): 1026-1033 Yahyaoui, F.E.L., Wongs-Aree, C., Latche, A., Hackett, R., Grierson, D., Pech, JC. 2002.  Molecular and biochemical characteristics of a gene encoding an alcohol acyl-transferase involved in the generation of aroma volatile esters during melon ripening.European Journal of Biochemistry 269(9):2359-2366