1/23/2018 The taste and aroma of fruit The New Zealand Institute for Plant & Food Research Limited Non-volatile compounds • Sugars – sweetness • Acids – tartness, sourness • Tannins – bitterness/astringency Volatile compounds • Terpenes – floral notes • Aldehydes – green, grassy notes The genetics of ester and phenylpropene • Furans – caramel, berry notes • Esters volatile production in ripe apple fruit • Phenylpropenes Ross Atkinson The New Zealand Institute for Plant & Food Research Limited Esters Biosynthesis of esters • Esters are found in many ripe fruit. CH CH3 They give ‘fruity’ aromas not only From fatty acid 3 + acetyl-CoA to apples, but also to pears, degradation or O CH3 bananas, melons and kiwifruit amino acid CH OH precursors 3 AAT CH3 O • Aromatic apples produce high 2-methylbutanol 2-methylbutyl levels of esters such as 2-methyl- acetate butyl acetate (2-MBA), hexyl acetate and butyl acetate • Final step in ester biosynthesis is catalysed by alcohol acyl transferases (AATs) • AATs catalyse the transfer of an acyl group from a CoA donor to an alcohol acceptor The New Zealand Institute for Plant & Food Research Limited The New Zealand Institute for Plant & Food Research Limited Genetics of ester production in apple AAT1 knockdown plants produce reduced esters ‘Royal Gala’ x ‘Granny Smith’ • Esters are abundant in aromatic ‘Royal Gala’ but not ‘Granny Smith’ • Major QTL for 35 esters was located on the upper part of LG2 • QTL co-located with the MdAAT1 gene • MdAAT1 allele from RG efficiently makes 2-MBA, hexyl and butyl • Knockdowns of MdAAT1 accumulate much lower levels of acetate, whilst the allele from GS makes these esters poorly esters especially acetate esters such as 2-MBA • The fruit are perceived as less fruity in sensory trials Souleyre et al 2014, Plant J, 78: 903-915 The New Zealand Institute for Plant & Food Research Limited The New Zealand Institute for Plant & Food Research Limited 1 1/23/2018 Phenylpropenes Biosynthesis of phenylpropenes Phenylpropene Phenylpropene • Phenylpropenes are key Phenylpropanoid reductases O-methyltransferases pathway aroma compounds in many CH2OAc spices and herbs. PAL OH OCH3 Isochavicol t-Anethole C4H • Phenylpropenes such as Anethole 4CL OH estragole and chavicol are Coumaryl acetate CHS produced in apples and OH OCH3 contribute spicy/aromatic p-hydroxycinnamyl Chavicol Estragole CHI flavour notes acetates F3H CH2OAc F3’H OCH3 OCH3 OH OCH3 DFR Isoeugenol Methyl isoeugenol Eugenol ANS OCH3 OH AGT Coniferyl acetate OCH3 OCH3 OH OCH3 Anthocyanins Eugenol Methyl eugenol The New Zealand Institute for Plant & Food Research Limited The New Zealand Institute for Plant & Food Research Limited Genetics of phenylpropene production – RG x GS OMT1 knockdown plants produce reduced estragole 'Royal Gala' LG1 'Royal Gala' LG2 0.0 124604 0.0 1270705 Line Estragole Methyl Methyl Chavicol Eugenol Isoeugenol 1.5 4746500 1.5 1979331 QTL 2 -1 -1 -1 -1 (ng g ) eugenol isoeugenol (ng g ) (ng g ) (ng g ) 5.2 3273690 24.8% of variation 7.5 4411200 (ng g-1) (ng g-1) 9.7 12390423 from the RG parent Control 406.6±81.8 14.4±6.5 1.5±0.9 303.4±203.1 41.2±11.1 14.1±3.8 18.8 18093819 19.0 7255188 T646 60.0±16.4* 0±0 0±0 187.0±59.6 106.0±50.0 20.0±9.9 21.1 18695269 T924 23.0±3.2* 0±0 0±0 126.0±6.9 33.0±1.2 14.0±1.2 25.3 11969851 26.6 23134240 T930 9.2±0.5* 0±0 0±0 1089.3±159.1 110.3±8.4 55.7±25.8 QTL1 34.1 26729463 33.8 16803165 35.6 27060614 9.2% of variation 37.7 18848241 from the RG parent • Knockdowns of MdoOMT1 accumulate much lower levels 44.6 30246914 45.4 30846147 46.1 27327055 of estragole 49.1 34510440 48.4 33257933 50.6 35422934 • The fruit are perceived as more floral in sensory trials 55.9 36182763 58.2 35939850 59.8 39549680 • Gene responsible for QTL1 co-locates with an O-methyltransferase Yauk et al 2015, Plant J, 82: 937-950 • MdoOMT1 converts chavicol to estragole in vitro The New Zealand Institute for Plant & Food Research Limited The New Zealand Institute for Plant & Food Research Limited QTL re-analysis in RG x GS Pathways have an acylation step in common Ester biosynthesis CH CH3 3 O CH Amino acid + acetyl-CoA 3 precursors Estragole QTL2 - shares CH3 OH MdAAT1 the same marker as the CH3 O 2-methylbutanol 2-methylbutyl 2MBA ester QTL !! acetate Phenylpropene CH2OH CH2OAc biosynthesis + NADPH isochavicol + SAM anethole Estragole QTL1 = + acetyl-CoA MdoOMT1 MdAAT1 PhR chavicol OMT estragole OH OH p-coumaryl p-coumaryl Phenylpropanoid alcohol acetate pathway CH2OH CH2OAc methyl- + NADPH isoeugenol + SAM isoeugenol + acetyl-CoA PhR eugenol OMT methyl- OCH3 MdAAT1 OCH3 eugenol OH OH coniferyl coniferyl acetate alcohol The New Zealand Institute for Plant & Food Research Limited The New Zealand Institute for Plant & Food Research Limited 2 1/23/2018 Biochemical analysis Validation in transgenic plants - 1 Esters Substrate 1 Substrate 2 MdAAT1-RGa MdAAT1-GSa Prediction: MdAAT1 knockdown Line Estragole (ng/g) hexanol acetyl-CoA 100% ±1.4% 2.94%±0.16% lines will produce reduced levels Control 218±43 butanol 63.8±3.0 1.03±0.06 AS70 16±3 2-methylbutanol 77.6±3.3 0.26±0.02 of phenylpropenes (and esters) AS1996 120±47 compared to the controls AS2002 35±23 Phenylpropenes AS2004 18±2 Substrate 1 Substrate 2 MdAAT1-RGa MdAAT1-GSa p-coumaryl alcohol acetyl-CoA 1.2%±0.2% 0.11%±0.01 coniferyl alcohol 0.3±0.02 not detected CH2OH CH2OAc • RG AAT1 makes volatile acetate esters efficiently (64-100%) + NADPH isochavicol + SAM anethole • RG AAT1 can use p-coumaryl and coniferyl alcohols to make p- + acetyl-CoA chavicol OMT estragole hydroxycinnamyl acetates – but not very efficiently (1.2%) AAT PhR OH x OH • The RG AAT1 allele is more efficient than the GS allele for both p-coumaryl p-coumaryl phenylpropene and ester biosynthesis – hence the QTL in RG for alcohol acetate esters and phenylpropenes. The New Zealand Institute for Plant & Food Research Limited The New Zealand Institute for Plant & Food Research Limited Validation in transgenic plants - 2 Validation in transgenic plants - 3 Prediction: MdCHS knockdown Line Total phenylpropenes ng/g • MdMyb10 is a TF that upregulates the Control-1 1971±634 whole phenylpropanoid pathway lines will produce more CHS-A5 2628±639 phenylpropenes than controls due CHS-A7.1 12798±3207 • MdMyb10 overexpressing lines produce to redirection of flux from the Control-2 837±104 more phenylpropenes than controls phenylpropanoid pathway CHS-A2 4145±270 • Effect is due to redirection of flux, not CHS-A6 1416±418 4CL CHS-A7.2 2878±490 transcriptional up-regulation CCR CAD SCoA p-coumaryl-CoA p-coumaraldehyde p-coumaryl alcohol xCHS AAT1 Anthocyanins, dihydrochalcones phenylpropenes The New Zealand Institute for Plant & Food Research Limited The New Zealand Institute for Plant & Food Research Limited Are AATs a common control point in other fruit? Summary • Strawberry and tomato make lots of eugenol • Contain fruit ripening induced AATs • MdAAT1 is a key gene for production of esters in ripe apple fruit • Transient analysis in tobacco indicated they • MdoOMT1 is a key gene for production of estragole in ripe apple fruit can make p-hydroxycinnamyl acetates • Production of esters and phenylpropenes is linked through MdAAT1 (which are converted to phenylpropenes by in ripe apple fruit MdoPhR5 in coupled assays) • Manipulation of phenylpropanoid pathway flux can dramatically alter phenylpropene levels Experiment Chavicol Eugenol • AAT genes are likely to link the two pathways in other fruit p-Coumaryl alcohol Strawberry SAAT 5018±318 4.7±2.2 • Our current focus is on how glycosylation can influence free Tomato SlAAT1 1192±95 0.4±0.4 phenylpropene volatile levels GUS 138±68 0±0 Yauk et al 2017, Plant J, 91: 292-305 The New Zealand Institute for Plant & Food Research Limited The New Zealand Institute for Plant & Food Research Limited 3 1/23/2018 Acknowledgements Yar-Khing Yauk Edwige Souleyre David Chagné, Xiuyin Chen, Sumathi Tomes, Mindy Wang, Martin Hunt, Ratnasiri Maddumage, Adam Matich, Daryl Rowan NZ Ministry of Business, Innovation and Employment Contact: [email protected] The New Zealand Institute for Plant & Food Research Limited 4.