MOLECULAR AND PHYSIOLOGICAL BASES OF AROMA BIOSYNTHESIS IN APRICOT FRUIT (Prunus armeniaca L.)

Bruno G. Defilippi1*, Mauricio González-Agüero1, Sebastián Troncoso2, Orianne Gudenschwager1, Alejandra Moya-León3, Reinaldo Campos-Vargas1.

1 Laboratorio de Postcosecha, Instituto de Investigaciones Agropecuarias, CRI La Platina. 2 Facultad de Química y Biología, U. de Santiago de Chile.3 Laboratorio de Fisiología Vegetal, IBVB, Universidad de Talca, Chile *bdefilip@inia.cl

Experimental design

Results

Conclusions

A salient genetic attribute of tree fruits is the unique blend of sugar, acid, phenolic and volatile components that determine their flavor. This complex genetic trait is manifested in ripe fruit through a complex interaction of metabolic pathways and regulatory circuits that results in the unique fruit flavor composition, a key to fruit consumption. Loss of flavor, particularly the aroma attribute, is a limiting factor in apricot quality. In spite of its significance, very little is known at the molecular, genetic and biochemical level of the genes and pathways that are responsible for the synthesis, accumulation and regulation of volatile compounds. In order to understand the biological basis of aroma biosynthesis we characterized and differentiated four stages in terms of maturity parameters, aroma-related volatile compounds, and gene expression levels. We cloned and quantified by qPCR the genes encoding: alcohol acyl transferase (AAT), alcohol dehydrogenase (ADH), lipoxygenase (LOX) and pyruvate decarboxylase (PDC), key enzymes involved in alcohol, aldehyde and ester synthesis. As fruit ripening progressed, we observed an increase in adh and aat transcript levels simultaneously with an increase in esters (hexyl acetate) and a decrease in aldehydes (i.e. hexanal and (E)-2-hexenal) and alcohols (i.e. 1-hexanol). We think that further studies to be performed in terms of identifying and characterizing these genes in P. armeniaca will contribute to understand overall aroma development during fruit ripening.

Apricot cv. Modesto

Maturity stages

Evaluation of quality attributes

Genes analyzed: aat, adh, lox, pdc

Search of ortholog sequences

Full length coding sequences (RACE-PCR)

Primers design for qPCR

Gene expression analyses of adh, lox, pdc

Real Time PCR (qPCR)

RNA extraction, cDNA synthesis

1. Characterization of maturity stages: Maturity parameters analyzed during maturity and ripening of apricots (cv Modesto) included: fruit firmness, total soluble solids (TSS), titratable acidity (TA), ethylene and CO2 production rates. After evaluation we identified 4 maturity stages:

Maturity stage Weight(g)

Firmness(Kgf)

TSS(%)

TA(% Malic acid)

Ethylene(µL C2H4 / k*h)

CO2

(mL CO2 /k*h)

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

2. Identification and quantification of volatiles: six key aroma volatile compounds were identified by using GC-MS. Quantification was performed by GC considering internal standards for each compound.

3. Identification, cloning and characterization of aat, adh, lox and pdc genes in P. armeniaca: For each gene analyzed we obtained the full length sequence by RACE-PCR. (A) Amino acid sequence comparison between the peptides of the four aroma related genes with proteins from others species. (B) shows the schematic representation of predicted structure and the multiple alignment with closely related sequences using a Clustal software and manually alignment of selected motifs of each protein.

(A) (B)

4. Gene expression analyses for aat, adh, lox and pdc within maturity stages: Expression patterns for the four transcripts were characterized by qPCR in 4 fruits for each maturity stage (M1 to M4). Amplification assays were performed three times. Gene expression was normalized considering an external control (Gene dap from Bacillus subtilis), and expressed as a percentage of the highest value of relative abundance.

This work was funded by Fondecyt 1060179.

Glycolysis β-oxidation

transamination

Pyruvate

AldehydesAcetaldehyde

ADH

PDC

ADHAlcohol

AATEsters

-Cte

-

+

Cte

Up-regulated expression geneNon-changes in gene expression

-+

Detected volatile compound level

Changes detected between ripening stages

* Bars followed by different small letter are significantly different at p<0,05

* Different letters represent significant differences at P < 0.05 by LSD test.

Lipids

Fatty acids

(linoleic, linolenic)

β-oxidation Lipoxigenase

Acyl-CoAs

Butyl esters

Hexanal

Hexenal

Hexanol

LOX

a

b b b

0

25

50

75

100

b b b

a

0

25

50

75

100

aaa

a

0

25

50

75

100 a

bb c

c0

25

50

75

100

adh

loxpdc

aat%

of M

axim

um

M1 M2 M3 M4Maturity stages

M1 M2 M3 M4

aa

bb

0

8

16

24

a a

bb

0

40

80

120

c

a

bc

0

6

12

18

bb

aa

0

20

40

60

ccbc

a

0

250

500

750

a

b

bcbc

0

300

600

900

Con

cent

ratio

n (n

g . K

g -1

)

M1 M2 M3 M4Maturity stages

M1 M2 M3 M4 M1 M2 M3 M4

hexanal

(E)-2-hexenalhexyl acetate linalool

1-hexanol ethyl octanoate

adh

lox

pdc

aat Protein

Name Size Organism Accession number

Amino acid identity (%) a

Pa-AAT 448 Prunus armeniaca N.A. ----------

Pc-AAT 442 Pyrus communis AAS48090 58

Md-AAT 459 Malus x domestica AAS79797 58

Vv-AAT 451 Vitis vinifera CAO66728 52 Alc

ohol

acy

l tr

ansf

eras

e (A

AT

)

Cs-AAT 456 Citrus sinensis ABW81204 50

Pa-ADH 267 Prunus armeniaca EU395433 ----------

Pm-ADH 267 Prunus mume BAE48662 99

Vv-ADH 266 Vitis vinifera CAO49038 74

Cm-ADH 266 Cucumis melo ABC02082 71

Alc

ohol

de

shydr

ogen

ase

(AD

H)

At-ADH 266 Arabidopsis thaliana AAM65725 57

Pa-PDC 605 Prunus armeniaca EU395434 ----------

Fa-PDC 605 Fragaria x ananassa AAG13131 91

Lc-PDC 606 Lotus corniculatus AAO72533 87

At-PDC 603 Arabidopsis thaliana NP_195752 85

Pyru

vate

dec

arbo

xyla

se

(PD

C)

St-PDC 592 Solanum tuberosum BAC23043 86

Pa-LOX 590 * Prunus armeniaca EU439430 ----------

Ca-LOX 873 Corylus avellana CAD10740 75

Vv-LOX 864 Vitis vinifera CAO17594 72

St-LOX 862 Solanum tuberosum AAB67865 71 Lip

oxyg

enas

e

(LO

X)

Pd-LOX 862 Prunus dulcis CAB94852 71

1