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
A few characteristicswith a major impact.

• Broad number of compounds (gt400 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)

7
And finallyit 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
Ester biosynthesis in fruit
Protein degradation
Membrane degradation
Fatty acids
Amino acids
Transamination Decarboxylation
b-oxidation
Branched aldehydes
Aliphatic 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
Aroma esters, aldehydes, alcohols Sweetness
fructose, sucrose, glucose Acidity malic acid,
citric acid Astringency phenolic compounds
X
ACO
X
1-MCP
C2H4
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
Ethylene Biosynthesis Analyses in Transgenic Lines
Ethylene production
Enzyme activity
Gene expression
14
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 20C
Compounds GS GS 68G 68G 68G 68G
Initial 13 d Initial 13 d 21 d 21d C2H4
Hexanal 67080 70223 84420 84420 75535 60520 65040
(2E) Hexenal 21420 52823 9910 9910 29034 41915 67840
Total aldehydes 884100 123146 94412 94412 104465 102436 132879
Butanol 71 301 ND ND ND 51 142
2-Methylbutanol ND2 301 ND ND ND ND 181
Hexanol 568 5210 224 224 414 561 755
Total alcohols 638 11012 224 224 414 612 1068
Butyl butanoate 308 798 ND ND 202 404 12014
Butyl 2-methylbutanoate 216 951 ND ND 41 41 585
Hexyl butanoate 2420 1455 101 101 406 566 1566
Hexyl 2-methyl butanoate 3010 2114 101 101 132 322 1118
Hexyl propanoate 306 531 30.5 30.5 121 252 285
Hexyl hexanoate 175 525 112 112 91 202 313
Total esters 12051 62117 432 432 8710 17717 49417
17
Effect of ethylene regulation on aroma production
of apple
ethylene
GS

• -suppression of biosynthesis
• ACO antisense line
• gt 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
Ethylene biosynthesis and AS melon
21
Role of ethylene in aroma biosynthesis
Hexylacetate
Hexanol
Butylacetate
8
)
-1
7
6
5
4
Concentration (mmolkg
3
2
1
0
Flores et al. 2002
22
Ethylene production and ADH gene expression
Manríquez et al., PMB 2006
23
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
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
Maturity stage Weight (g) Firmness (kg-f) TSS () TA (g.L-1) Ethylene production (µL C2H4.kg-1.h-1) Respiration rate (mL CO2.kg-1.h-1)
M1 31.2 c 2.9 a 10.1 c 2.2 a 0.0 b 60.2 b
M2 40.5 b 1.9 b 14.9 b 1.9 a 0.0 b 70.1 a
M3 45.1 a 2.0 b 16.9 b 1.5 b 1.4 b 58.1 b
M4 46.2 a 0.4 c 21.3 a 0.8 c 29.5 a 55.3 b
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
Ethylene inhibitors (1-MCP and AVG)
20C
Harvest
2 days
4 days
Ripe fruit
31
Precursor availability for volatile production
Amino acids valine isoleucine leucine
2-methylbutanoic 2-methylbutanol 2-methyl
butanoate
32

• Fatty acids accumulation

• levels peel gt 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 1
Mechanism 2
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 pathwaysaroma 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