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
Mar 17, 2016
Aroma: an integrative approach for understanding
and improving a complex quality trait
Bruno Defilippi B.
Mauricio González A.
Daniel Manríquez B.
Unidad de PostcosechaInstituto de Investigaciones Agropecuarias
Importance of Flavor Metabolites
1. Plant
Importance from a biological perspective:
- phenolic compounds (plant defense)
- volatiles (signaling molecules)
- sugars, organic acids, volatiles (aroma and taste).
Survival of the specie
2. Human behavior
- Attribute of fruit quality (sweetness, acidity, aroma)
- Acceptance of the commodity by the consumer ($)
- Nutritional (phenolic as antioxidants…)
- Postharvest biology
Postharvest-life under optimum conditions
0 20 40 60 80 100
Based on flavor and nutritional quality
Based on firmness
Based on appearance (visual quality)
(Kader, 2003)
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)
Flavor Compounds in Fruit
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.
And finally…it is a dynamic process with major changes in volatile profile during development and ripening
Fruit development
Ester biosynthesis in fruit
Membrane degradationProtein degradation
Fatty acidsAmino acids
-oxidationTransamination
DecarboxylationBranched aldehydes
Aliphatic aldehydes
Reduction
Aliphatic and branched alcohols
Acyl-CoA
Acylation
Esterification
Aliphatic and branched esters
ADH
AAT
Methionine
SAM
ACC
Receptor
ACS
ACO
Silencing, AVG, CA
1-MCP
Response Flavor compounds
?
Aroma: esters, aldehydes, alcohols
Sweetness: fructose, sucrose, glucose
Acidity: malic acid, citric acid
Astringency: phenolic compounds
Ethylene inhibition
Ethylene enhancement
C2H4
X
X
X
ETHYLENE
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
Ethylene Biosynthesis Analyses in Transgenic Lines
Methionine SAM ACS ACC ACO Ethylene
Ethylene production Enzyme activity Gene expression
Ethylene Biosynthesis Analyses in 1-MCP Treated Fruit
Methionine SAM ACS ACC ACO Ethylene
Ethylene production Enzyme activity Gene expression
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
Compounds GS 68GInitial 13 d Initial 13 d 21 d 21d + C2H4
Hexanal 670±80 702±23 844±20 755±35 605±20 650±40
(2E) Hexenal 214±20 528±23 99±10 290±34 419±15 678±40
Total aldehydes 884±100 1231±46 944±12 1044±65 1024±36 1328±79
Butanol 7±1 30±1 ND ND 5±1 14±2
2-Methylbutanol ND2 30±1 ND ND ND 18±1
Hexanol 56±8 52±10 22±4 41±4 56±1 75±5
Total alcohols 63±8 110±12 22±4 41±4 61±2 106±8
Butyl butanoate 30±8 79±8 ND 20±2 40±4 120±14
Butyl 2-methylbutanoate 21±6 95±1 ND 4±1 4±1 58±5
Hexyl butanoate 24±20 145±5 10±1 40±6 56±6 156±6
Hexyl 2-methyl butanoate
30±10 211±4 10±1 13±2 32±2 111±8
Hexyl propanoate 30±6 53±1 3±0.5 12±1 25±2 28±5
Hexyl hexanoate 17±5 52±5 11±2 9±1 20±2 31±3
Total esters 120±51 621±17 43±2 87±10 177±17 494±17
Level of volatile compounds after 21 d at 20°C
Effect of ethylene regulation on aroma production of apple
-suppression of biosynthesis ACO antisense line > 95% reduction in ethylene
production
-ethylene enhancement80 μLL-1
GS
GS
GS
Defilippi et al., JAFC 2005
Ester production is under ethylene regulation.
+ ethylene
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
What about the biosynthesis?: AAT and ADH activity levels
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.
peel
flesh
Defilippi et al., JAFC 2005
C2H4
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
Ethylene biosynthesis and AS melon
ACS
ACC synthase
ATP
ACO
ACC oxidase
Met
Methionine
SAM
S-adenosylmethionine
Ethylene
CH2=CH2
ACC
1-aminocyclopropane
-1-carboxylic acid
Antisense ACO
0
10
20
30
40
50
60
70
80
90
0 5 10 15 20 25 30 35 40 45
Days after pollination
Ethy
lene
conc
entr
atio
n(p
pm)
WTAS
0
10
20
30
40
50
60
70
80
90
0 5 10 15 20 25 30 35 40 45
Days after pollination
Ethy
lene
conc
entr
atio
n(p
pm)
WTAS
WT AS
15 days @ 25°C
Role of ethylene in aroma biosynthesis
0
1
2
3
4
5
6
7
8
WT AS AS+Ethylene
Con
cent
ratio
n (m
mol
*kg
-1)
Hexylacetate Hexanol Butylacetate
(*) 50 l*l-1 ethylene
Flores et al. 2002
ND6.3 + 1.930.0 + 1.714.9 + 2.1
Benzaldehyde
TR16.1 + 1.988.2 + 10.976.3 + 6.02-methylpropionaldehyde
TR14.0 + 3.2483.3 + 31.5129.2 + 29.73-methylbutyraldehyde
ND7.2 + 0.440.5 + 4.623.8 + 12.62-methylbutyraldehyde
57.3 + 1.1240.5 + 35.6977.9 + 63.0284.5 + 56.2Capronaldehyde
59.6 + 8.4272.8 + 40.5980.0 + 32.6315.5 + 40.7Butyraldehyde
79.5 + 5.6487.2 + 38.22.216.3 + 227.8473.5 + 95.5Acetaldehyde
NADPHNADHNADPHNADHAldehydes
Cm-ADH2Cm-ADH1
Substrate specificity of the recombinant ADHs
Manríquez et al., PMB 2006
Substrate specificity of the recombinantprotein Cm-AATs
Rel
ativ
e A
AT
activ
ity (%
)
0
20
40
60
80
100
Rel
ativ
e A
AT
activ
ity (%
)
0
20
40
60
80
100
ESTERS1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Rel
ativ
e A
AT
activ
ity (%
)
0
20
40
60
80
100
A
C
B
Cm-AAT1
Cm-AAT4
Cm-AAT3
Cm-AAT1E-2-hexene-ol + acetyl-CoA
Cm-AAT3benzyl alcohol + acetyl-CoA
Cm-AAT 4cinnamyl alcohol and acetyl-CoA
Acetates Propan. Hexan.
González-Agüero et al., PBB 2009
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
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
Increase in ethylene is concomitant with an increase in aat and adh expression…
Hexanal 1-Hexanol
Ethyl octanoate Hexyl acetate
LinaloolE-2-Hexenal
González-Agüero et al., PBB 2009
But in terms of volatile production….
Harvest 2 days 4 days Ripe fruit
20°C20°CEthylene Ethylene
inhibitorsinhibitors
(1-MCP and AVG)(1-MCP and AVG)
a a a bab
b
a a
Precursor availability for volatile production
Amino acids
valine isoleucine leucine
2-methylbutanoic2-methylbutanol2-methyl butanoate
Lipids
ß-oxidation Lipoxygenase
Fatty acids(linoleic, linolenic)
Hexanal(2E) HexenalHexanolHexyl esters
acyl-CoAsButyl esters
LOX
HPL
ISO
Fatty acids accumulation:
- levels peel > flesh- changes before volatile accumulation- partially affected by ethylene
Hexanal
(2E)-Hexenal
Defilippi et al., PSc. 2005
C2H4
C2H4
Aldehydes
Acids
AAT
Esters
Amino acids
Alcohols
Ethylene Agronomic practices
Early harvest
1-MCP
AVG
CA storage
Pre-harvest
Respiration
Fatty acids
-
-
-
?
-
Mechanism 1Mechanism 2
-
1. Study the role of other signals in modulating aroma, especially in non climacteric fruit.
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.
Special research needs in aroma….”whishing list”
We DO really need more people involved in flavor
5. Back to the field….”Postharvest Ecophysiology”
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)
Team work
FundingFondecyt 1060179
Fondecyt 11090098
The Plant Cell Biotechnology Nucleus
Fundación Andes
Washington Tree Fruit Research Commission
Beca Doctoral (DM) Conicyt
Apple (UCDavis)B. DefilippiA. KaderA. Dandekar
Apricot (INIA)Postharvest Unit, INIAR. Campos, UNABA. Moya, U. TalcaR. Infante, UCHJ. Sánchez, UCHO. Gudenschawer, INIAS. Troncoso, USACHP. Rubio, UCHM. Pizarro, UCHH. Valdés, INIAW. San Juan, UCHA. Aballay, UCH
Melon (ENSAT)D. ManríquezJ.C. PechA. Latchè
Cherimoya (INIA)Postharvest Unit, INIA