Impact of suppression of ethylene biosynthesis or action on flavor metabolites in apple fruitBruno G. Defilippi, Sandra L. Uratsu, Betty Hess-Pierce, Gianni Teo, Abhaya M. Dandekar and Adel A. Kader

cv. Greensleeves (Golden Delicious with James Grieve). From Dandekars labBinary vector that express the cDNAs of ACC-synthase (ACS) and ACC oxidase (ACO) enzymes in either a sense or antisense pattern.Experimental approach: Reduction and enhancement of ethylene biosynthesis and action

ReductionEnhancementGenetic approachChemical approachChemical approachLines with a high suppression of ethylene0.5 uLL-1 1-MCP1.5 uLL-1 1-MCP20 uLL-1 Ethylene100 uLL-1 Ethylene

Table. Classification of clones according to ethylene production after 12-14 days at 20C.R=ACO sense, G=ACO antisense, Y=ACS sense, B=ACS antisense

Ethylene production (uLC2H4Kg-1h-1)Very low1008R9R11R25R33R61G67G68G75G80G103Y105Y130Y150Y35R51G54G55G112Y115Y190B13R40R102Y107Y114Y119Y125Y140Y20R113Y129Y133Y166B21R48R49R110Y174Y174B175BGS (control)

Figure. Ethylene production and biosynthesis in Greensleeves fruits harvested from different lines and stored at 20C for 12 days.Ethylene biosynthesis:

LineRelative activity (%)ACS activityACC contentACO activityGS67G68G103Y130Y10014572571008381300294910022160160

Figure. Ethylene evolution and respiration rate in Greensleeves fruits harvested from fruit treated with 1-MCP or ethylene.

GS105Y68GApples cv. Greensleeves14 days at 20CGS103Y68GGS1 month after harvestACO Silenced (61-G)ACO Silenced (68-G)ACS Silenced (130-Y)3 months after harvestGSACO Silenced

Aldehydes AlcoholsAcidsAAT EstersFatty acidsAmino acids PrecursorsAroma biosynthesis in apples:ADHFigure. Total aroma composition in Greensleeves fruits derived from different clones.

Table. Content of major volatile compounds in Greensleeves fruits derived from different lines. Fruits were evaluated after 12 days at 20C.Compound (nL L-1) GS 68G 130Y 1.5 uLL-1 MCP Aldehydes Hexanal273 47 298 23 320 35 230 19(E) 2-Hexenal420 30 290 40 300 30 200 39 AlcoholsButanol 12 2 3 1 8 1 1 0Methyl 2-butanol 6 2 ND ND NDHexanol 74 7 54 17 73 6 30 2 EstersButyl butanoate 55 5 35 4 36 5 9 2Butyl 2-methylbutanoate 40 7 8 2 7 2 NDHexyl acetate 12 2 ND ND TracesHexyl propanoate 41 6 20 4 ND NDHexyl butanoate 340 20 100 25 98 9 43 4

Figure. Effect of ethylene (suppression and enhancement) in the biosynthesis of hexyl esters in Greensleeves fruits. Fruits were evaluated at harvest and after 12 days at 20C (means SE)

Figure. Alcohol acyl transferase (AAT) and alcohol dehydrogenase (ADH) activity in Greensleeves fruits harvested from different and stored at 20C for 12 days (a), and in fruit treated with 1-MCP (b). ab

20 uLL-1 ethyleneFigure. AAT and ADH activity in Greensleeves fruits harvested from different and stored at 20C for 12 days, and in fruit treated with ethylene. With ethylene

Do we need a threshold of ethylene?- 100 uLL-1 ethylene- whole fruit vs. pulpEthylene (%)

100117

LinesAAT activity(nmoles min-1 mg-1 (protein))Variation (%)At harvestEnd of storageWhole fruitGS 461 381648 47 c+4168G227 27247 35 a +968 G plus ethylene 536 39 bc+72PulpGS 340 221 505 44 b+4868G 371 33362 23 a -268 G plus ethylene 441 19 b+20

Ethylene (%)

100410Figure. AAT activity in Greensleeves fruit treated with 1-MCP and ethylene, and stored at 20C for 14 days

Days at 20CTreatmentsAt harvestEnd of storageVariation%1-MCPEthyleneWhole fruit0236 15114NoNo354 18 b +50YesNo 192 9 a -18YesYes318 15 b +34Pulp0240 3014NoNo 324 35 b+35YesNo 222 30 a -8YesYes 275 27 a +15

Aroma volatiles: - Ester biosynthesis is an ethylene dependent process. - It seems that AAT is an important control point under ethylene regulation. - Substrate availability (aldehydes) is playing a significant role in ester biosynthesis. Upstream substrates (fatty acids)? (F. Bangerth). - What about other enzymes (esterases and Lox for example)Ethylene:

- Differential tissue response to ethylene action suppression