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

Why should be aroma considered a complex quality attribute?

Defilippi et al., ABR 2009

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

What have we learned about aroma in fruit?

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

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

Ethylene production and ADH gene

expression

Manríquez et al., PMB 2006

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

Ethylene production and Cm-AATs gene

expression

Manríquez et al., PMB 2005

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.

Do we have the same behavior in all climacteric fruit?

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

Free amino acids accumulation:

Defilippi et al., PSc. 2005

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

“The smellys”