1. Regulatory mechanisms of ethylene in aroma biosynthesis of apples (Malus x domestica Borkh) cv. Greensleeves Bruno G. Defilippi 1,2, Abhaya M. Dandekar 1 and Adel A. Kader 1 1 Department of Pomology, University of California, Davis, CA 95616 2 Institute for Agricultural Research (INIA-La Platina), Santiago, Chile The role of ethylene in aroma biosynthesis was investigated in apple fruits. In order to demonstrate this role, ethylene production and action were reduced using apple trees lines transformed for suppressing ACC-synthase and ACC-oxidase enzymes activity and an ethylene action inhibitor (1-MCP), respectively. In fruits derived from these treatments, a major reduction on ethylene biosynthesis and respiration rate was measured. As expected, results showed differential level of dependence of ripening parameters to ethylene biosynthesis and action revealing either ethylene-non associated changes as acidity and soluble solids, or ethylene-associated changes including flesh firmness and aroma production. Regarding aroma production, head space analysis showed a reduction in ester production in the ethylene-suppressed lines and in the fruit treated with two concentrations of 1-MCP. On the other hand, no major differences were measured in alcohol and aldehydes volatiles. In the biosynthetic pathway of aroma compounds, alcohol acyl-CoA transferase (AAT) activity, a key enzyme in ester biosynthesis, was partially reduced when ethylene biosynthesis and action was affected. On the contrary, no differences were observed in the activity of alcohol dehydrogenase (ADH). These results suggest that the regulatory mechanisms of aroma biosynthesis in apple are under partial ethylene regulation. EXPERIMENTAL STRATEGY RESULTS FUTURE WORK -To determine the role of ethylene in the pattern of volatile components and biosynthesis in transgenic apple fruit silenced for ethylene biosynthesis. -To study at the molecular levels the role of ethylene in aroma biosynthesis. -To understand the role of ethylene in overall flavor in apples, including sugars (sweetness), organic acids (acidity) and phenolic compounds (astringency). Table 3. Quality attributes at harvest and after storage for 12 days at 20°C. * Figure 1. Ethylene production in transgenic apples (a) and in fruit treated with 1-MCP (b). (a)(b) Figure 2. Effect of ethylene (suppression and enhancement) on the biosynthesis of hexyl esters (means ± SE) in GS apples evaluated at harvest and at the end of storage (12 days at 20°C.) Figure 3. AAT and ADH activities in five lines of GS apples stored at 20°C for 12 days (a), and GS apples treated with 0, 0.5 and 1.5 µL L -1 1-MCP at harvest and stored at 20°C for 12 days (b). LineAt HarvestAfter 12 days at 20°C Firmness (Newton) Soluble solids (°Brix) Color (Hue angle) Firmness (Newton) Soluble solids (°Brix) Color (Hue angle) GS 79  2.6 1 11.6  0.9109  0.657  5.315.1  0.4 98  0.1 67G 84  4.611.1  1.4113  0.269  9.814.1  0.9111  1.0 130Y 82  4.411.2  0.6113  0.571  3.513.5  0.6110  0.7 GS + 1.5 MCP 78  1.311.6  0.8106  0.565  3.013.0  0.5105  0.4 * Headspace analysis using a PDMS/DVB SPME fiber. * No differences were observed in titratable acidity (data not presented). GS = control G = ACO antisense Y = ACS sense GS61 G 68 G 130 Y OVERVIEW LineACS activity ACC concentration ACO activity Ethylene production rate GS 100 67 G721,200 0.8 6 68 G623,200 0.4 4 130 Y10 12158 6 Table 1. Ethylene biosynthesis of transgenic apples as a percentage of that observed for the untransformed lines * * Values of control: ACS activity = 2.5 nmolmg -1 (protein) h -1, ACC concentration = 1.6 nmoles ACC g -1 (tissue), ACO activity= 205nLC 2 H 4 mg -1 (protein)h -1, ethylene production=73 uL C 2 H 4 kg -1 h -1 Table 2. Content of major volatile compounds in Greensleeves fruits derived from different lines. Fruits were evaluated after 12 days at 20°C. Compound (nL L -1 ) GS 68G 130Y 1.5 uLL -1 MCP Aldehydes Hexanal273 ± 47 398 ± 23 320 ± 35 230 ± 19 (E) 2-Hexenal420 ± 30 290 ± 40 300 ± 30 200 ± 39 Alcohols Butanol 12 ± 2 3 ± 1 8 ± 1 1 ± 0 Methyl 2-butanol 6 ± 2 ND * ND ND Hexanol 74 ± 7 54 ± 7 73 ± 6 30 ± 2 Esters Butyl butanoate 55 ± 5 45 ± 4 36 ± 5 9 ± 2 Butyl 2-methylbutanoate 40 ± 7 8 ± 2 7 ± 2 ND Hexyl acetate 12 ± 2 ND ND Traces Hexyl propanoate 41 ± 6 20 ± 4 ND ND Hexyl butanoate 340 ± 20 100 ± 25 98 ± 9 43 ± 4 AROMA COMPOUNDS AND ESTER BIOSYNTHESIS *ND=Not detected (a) (b) ETHYLENE BIOSYNTHESIS Esters Acids Alcohols Aldehydes ADH AAT Picture courtesy of New York Apple Association Precursors: Fatty acids Amino acids Ethylene ? Methionine SAM ACC Ethylene Receptor ACS ACO ETHYLENE INHIBITION Silencing (sense, antisense) 1-MCP X Picture courtesy of New York Apple Association