Aroma: an integrative approach for understanding and improving a complex quality trait

1. Aroma: an integrative approach for understanding and improving a complex quality trait Bruno Defilippi B. Mauricio González A. Daniel Manríquez B. Unidad de Postcosecha Instituto de Investigaciones Agropecuarias

2. Importance of Flavor Metabolites • Plant • Importance from a biological perspective: • phenolic compounds (plant defense) • volatiles (signaling molecules) • sugars, organic acids, volatiles (aroma and taste). Survival of the specie

3. 2. Human behavior - Attribute of fruit quality (sweetness, acidity, aroma) - Acceptance of the commodity by the consumer ($) - Nutritional (phenolic as antioxidants…) - Postharvest biology (Kader, 2003)

4. Flavor Compounds in Fruit Sweetness Glucose Fructose Sucrose Sorbitol Pathways: Starch and sucrose metabolism. Acidity Malic acid Citric acid Others Pathways: Energy metabolism Smell Esters Aldehydes Alcohols Pathways: Lipid and amino acids metabolism. Astringency Bitterness Phenolic compounds Pathways: Secondary metabolites (flavonoids)

5. Why should be aroma considered a complex quality attribute? Defilippi et al., ABR 2009

6. Broad number of compounds (>400 in apple!) Aldehydes Alcohols Esters Lactones Others (acids, ketones, phenols) Present in very small amounts (ppb) Aroma is due to a mixture of a small number compounds (Character Impact Compounds). Ex: hexanal (maturity stage) Ex: ethyl-2-methylbutanoate and butyl acetate (ripening stage) A few characteristics…with a major impact.

7. And finally…it is a dynamic process with major changes in volatile profile during development and ripening Fruit development

8. What have we learned about aroma in fruit?

9. ADH AAT Ester biosynthesis in fruit Protein degradation Membrane degradation Fatty acids Amino acids Transamination Decarboxylation b-oxidation Aliphatic aldehydes Branched aldehydes Reduction Aliphatic and branched alcohols Acyl-CoA Acylation Esterification Aliphatic and branched esters

10. Ethylene inhibition Methionine SAM ACC Receptor X ACS Silencing, AVG, CA X Aroma: esters, aldehydes, alcohols Sweetness: fructose, sucrose, glucose Acidity: malic acid, citric acid Astringency: phenolic compounds ACO X C2H4 1-MCP ETHYLENE Response Flavor compounds ? Ethylene enhancement

11. Silencing of Apple Trees • cv. Greensleeves (Golden Delicious with James Grieve). • Binary vector that express the cDNAs of ACC-synthase (ACS) and ACC oxidase (ACO) enzymes in either a sense or antisense orientation. Dandekar et al., TR 2004

12. Dandekar et al., TR 2004

13. Methionine SAM ACSACCACOEthylene Ethylene Biosynthesis Analyses in Transgenic Lines Ethylene production Enzyme activity Gene expression

14. Methionine SAM ACSACCACOEthylene Ethylene Biosynthesis Analyses in 1-MCP Treated Fruit Ethylene production Enzyme activity Gene expression

15. Phenotype: Delay in ripening Delay in softening Retention of green color Reduction in loss of titratable acidity Delay in total soluble solids accumulation Reduced overall aroma Dandekar et al., TR 2004

16. Level of volatile compounds after 21 d at 20°C

17. Effect of ethylene regulation on aroma production of apple + ethylene GS • -suppression of biosynthesis • ACO antisense line • > 95% reduction in ethylene production GS • -ethylene enhancement • 80 μLL-1 GS Ester production is under ethylene regulation. Defilippi et al., JAFC 2005

18. What about the biosynthesis?: AAT and ADH activity levels • AAT activity levels were concomitant with both climacteric peak and changes in ester accumulation. • Activity levels responded to ethylene regulation. • Levels of AAT activity higher in the peel than the flesh. • Similar pattern between epidermal and cortical tissues C2H4 peel flesh • Reduction in ADH activity levels (peel). Not concomitant with alcohol accumulation. • Partial or no response to ethylene regulation. • Levels of ADH activity higher in the peel than the flesh. Defilippi et al., JAFC 2005

19. Cloning of AAT and ADH genes by RT-PCR from Greensleeves apple and gene expression analysis by real-time PCR Suppression of Ethylene Biosynthesis Defilippi et al., PSc. 2005

20. Met Methionine ATP SAM S-adenosylmethionine ACS ACC synthase ACC 1-aminocyclopropane -1-carboxylic acid ACO ACC oxidase WT AS 15 days @ 25°C Antisense ACO Ethylene CH2=CH2 Ethylene biosynthesis and AS melon

21. (*) 50 ml*l-1 ethylene WT AS AS+Ethylene Role of ethylene in aroma biosynthesis Hexylacetate Hexanol Butylacetate 8 ) -1 7 6 5 4 Concentration (mmol*kg 3 2 1 0 Flores et al. 2002

22. Ethylene production and ADH gene expression Manríquez et al., PMB 2006

23. Cm-ADH1 Cm-ADH2 Aldehydes NADH NADPH NADH NADPH Acetaldehyde 473.5 + 95.5 2.216.3 + 227.8 487.2 + 38.2 79.5 + 5.6 Butyraldehyde 315.5 + 40.7 980.0 + 32.6 272.8 + 40.5 59.6 + 8.4 Capronaldehyde 284.5 + 56.2 977.9 + 63.0 240.5 + 35.6 57.3 + 1.1 2-methylbutyraldehyde 23.8 + 12.6 40.5 + 4.6 7.2 + 0.4 ND 3-methylbutyraldehyde 129.2 + 29.7 483.3 + 31.5 14.0 + 3.2 TR 2-methylpropionaldehyde 76.3 + 6.0 88.2 + 10.9 16.1 + 1.9 TR Benzaldehyde 14.9 + 2.1 30.0 + 1.7 6.3 + 1.9 ND Substrate specificity of the recombinant ADHs Manríquez et al., PMB 2006

24. Ethylene production and Cm-AATs gene expression Manríquez et al., PMB 2005

25. Cm-AAT1 Cm-AAT3 Cm-AAT4 Substrate specificity of the recombinantprotein Cm-AATs Cm-AAT1 E-2-hexene-ol + acetyl-CoA Cm-AAT3 benzyl alcohol + acetyl-CoA Cm-AAT 4 cinnamyl alcohol and acetyl-CoA Acetates Propan. Hexan.

26. Do we have the same behavior in all climacteric fruit?

27. Apricot as a model for studying flavor loss after harvest… González-Agüero et al., PBB 2009

28. Increase in ethylene is concomitant with an increase in aat and adh expression… aat adh pdc lox Cloning of volatile-related genes by RT-PCR from apricot and gene expression analysis by real-time PCR González-Agüero et al., PBB 2009

29. But in terms of volatile production…. Hexanal 1-Hexanol Ethyl octanoate Hexyl acetate Linalool E-2-Hexenal González-Agüero et al., PBB 2009

30. b a a a b b a a a Ethylene inhibitors (1-MCP and AVG) 20°C Harvest 2 days 4 days Ripe fruit

31. Lipids Fatty acids (linoleic, linolenic) ß-oxidation Lipoxygenase LOX HPL ISO acyl-CoAs Butyl esters Hexanal (2E) Hexenal Hexanol Hexyl esters Precursor availability for volatile production Amino acids valine isoleucine leucine 2-methylbutanoic 2-methylbutanol 2-methyl butanoate

32. Fatty acids accumulation: • levels peel > flesh • changes before volatile accumulation • partially affected by ethylene C2H4 Hexanal C2H4 (2E)-Hexenal Defilippi et al., PSc. 2005

33. Free amino acids accumulation: Defilippi et al., PSc. 2005

34. Ethylene - Agronomic practices Early harvest 1-MCP AVG CA storage Pre-harvest - Mechanism 2 Mechanism 1 Respiration - - Amino acids Fatty acids ? Aldehydes Alcohols Acids - AAT Esters

35. 1. Study the role of other signals in modulating aroma, especially in non climacteric fruit. Special research needs in aroma….”whishing list” 2. Go for other pathways…aroma is more than C6 compounds. 3. Include sensory analysis in order to establish the actual role of a especific compound. 4. Establish a metabolomic platform for pursuing studies. 5. Back to the field….”Postharvest Ecophysiology” We DO really need more people involved in flavor

36. 5. Include flavor attribute as a key trait in breeding programs. • 6. Develope quantitative approaches suitable for the industry. • Gene base • E-nose systems (Defilippi et al., 2009)

37. Team work Apple (UCDavis) B. Defilippi A. Kader A. Dandekar Funding Fondecyt 1060179 Fondecyt 11090098 The Plant Cell Biotechnology Nucleus Fundación Andes Washington Tree Fruit Research Commission Beca Doctoral (DM) Conicyt Apricot (INIA) Postharvest Unit, INIA R. Campos, UNAB A. Moya, U. Talca R. Infante, UCH J. Sánchez, UCH O. Gudenschawer, INIA S. Troncoso, USACH P. Rubio, UCH M. Pizarro, UCH H. Valdés, INIA W. San Juan, UCH A. Aballay, UCH Melon (ENSAT) D. Manríquez J.C. Pech A. Latchè Cherimoya (INIA) Postharvest Unit, INIA

38. “The smellys”