Identification and characterization of genes differentially expressed during cold storage of cherimoya (Annona cherimola Mill).

Mauricio González-Agüero*, Bruno G. Defilippi, Orianne Gudenschwager, Carlos Muñoz, Reinaldo Campos-Vargas.

*Institute of Agricultural Research (INIA-La Platina), Santiago, Chile. maugonzalez@inia.cl

Experimental design

Results

Conclusions

Cherimoya (Annona cherimola Mill.) is a valuable fruit of the Annonaceae family. However, along with its desirable delicate taste, the high susceptibility to oxidative events makes it difficult to handle during cold storage and ripening. The isolation of mRNA transcripts encoding proteins associated with the ripening process is a powerful tool to understand the changes that occur on postharvest. To isolate differentially expressed genes during cherimoya ripening, a forward suppression subtractive hybridization (SSH) cDNA library was constructed. SSH was performed with cDNA from fruit at harvest and after 12 days in cold storage (0 °C). A total of 120 differentially expressed clones in the SSH library were identified and sequenced; 75 of them are non-redundant expressed cDNAs. Blastx analysis revealed that a 79% of cDNAs had significant sequence homologies with known sequences in the NCBI database. The identified cDNAs encoded proteins involved in diverse processes such as protein synthesis and modification, signal transduction, endomembrane traffic, transcription and post-transcription, primary metabolism, and other metabolisms. To further characterize differentially expressed genes in the SSH library, RACE-PCRs to obtained full length cDNAs were conducted. In addition fruit specific genes were identified. Real-time PCR analysis for selected genes, which are understood not to be related with cold storage, demonstrated that all genes were expressed highly during ripening. The information generated in this study provides new clues to understand the cherimoya ripening process.

1. Fruit material. Cherimoyas were cross-sectional sliced (fresh-cut) and stored for 12 days at 0 °C. Samples were taken after 0 (T0), 6 (T6) and 12 days (T12) of cold storage. After each evaluation, samples were frozen with liquid nitrogen and kept at -80 °C for further analyses. The appearance of physiological disorders was measured through a visual evaluation (A), and browning development was determined by means of differential activity of PPO enzyme (B) (Prieto et al., 2007)1.

2. Construction and characterization of a specific subtraction library of cherimoya under cold storage. A subtracted cDNA library was made using the PCR-select cDNA subtraction kit (Clontech). Individual transformants carrying cDNA fragments were manually selected and the presence of an unique insert (400 → 2,000 bp) was determined by colony PCR amplification colonies (A). From these, only 5% contained more than one insert (line 45 and 47 in panel A). In B a current summary of the characterized library is shown.

3. Identification and characterization of new A. cherimola genes. To date we have identified close to 100 genes with good alignments (E<10-10) with other plant species. From these, we analyzed and compared the expression levels of 12 selected genes and 4 control genes in cDNAs from different cherimoya tissues, including growing fruits (GF), leaves (L), flowers (F), mature fruit rind (RMF) and mature fruit flesh (FMF), and with the non-subtracted cDNA (NS) and subtracted (S, used to elaborate the library).

4. Gene expression analysis for six A. cherimola genes within control and treated fruit. Expression patterns for six transcripts were characterized by qPCR in 4 fruits for control (T0) and treated (T12) samples. Amplification assays were performed three times. Gene expression was normalized considering a housekeeping gene (18sRibosomal), and expressed as a percentage of the highest value of relative abundance.

Funded by PBCT PSD03

A B

St 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 St 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

St 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 St 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72

Summary of subtraction library:

- Positive clones ~ 250 clones

- Sequenced 200

- Average insert size 700 bp

- Redundancy of sequens. ~ 30%

- Sequences analyzed 105

A B

- We have identified several genes related to cold storage of cherimoya, some of them with a specific expression in fruit. This is a very important feature for using them in genetic breeding as possible markers, or as candidates for transgenesis.

- In the future our work will add more than 70 new genes for A. cherimola, contributing to duplicate the current data available for this specie.

1 cDNA GF: Growing fruits

2 cDNA L: Leaves

3 cDNA F: Flowers

4 cDNA RMF: Mature fruit rind

5 cDNA FMF: Mature fruit flesh

6 cDNA NS: Non-subtracted cDNA

7 cDNA S: Subtracted cDNA

A clone 006ssh, clathrin-coat assembly protein-like; B clone 027ssh, putative annexin; C clon 032ssh, putative adaptin; D clone 004ssh, putative syntaxin of plant 71; E clone 041ssh, putative 4-alpha-glucanotransferase; F clone 003ssh, UDP-

glucose pyrophosphorylase-like; G clone 015ssh, putative beta-ketoacyl-acyl carrier protein synthase III; H clone 019ssh, putative lyase; I clone 020ssh, metallothionein-like protein; J clone 030ssh, actin-related protein C4; K clone 072ssh,

putative enolase; L clone 101ssh, ubiquitin-conjugating enzyme-like; M Actin (annotation in progress); N ACC synthase (ACS), GenBank AF443280; O Polyphenol Oxidase (PPO), GenBank DQ990911; P 18s ribosomal protein, GenBank

AY819054. * New actin isoform only expressed in subtracted cDNAs samples

1 2 3 4 5 6 7363 bp

267 bp

296 bp

230 bp

253 bp

234 bp

A

B

C

D

E

F

298 bpH

252 bpG

1 2 3 4 5 6 7363 bp

267 bp

296 bp

230 bp

253 bp

234 bp

A

B

C

D

E

F

298 bpH

252 bpG

253 bpP

1 2 3 4 5 6 7199 bp

261 bp

348 bp

343 bp

318 bp

I

J

K

L

M

399 bp

346 bp

N

O

*

253 bpP

1 2 3 4 5 6 7199 bp

261 bp

348 bp

343 bp

318 bp

I

J

K

L

M

399 bp

346 bp

N

O

*

a

b0

40

80

120

T0 T12

% d

el m

áxim

o

a

b

0

40

80

120

T0 T12

% d

el m

áxim

o

a

b

0

40

80

120

T0 T12

% d

el m

áxim

o

a

a

0

40

80

120

T0 T12

% d

el m

áxim

o

b

a

0

40

80

120

T0 T12

% d

el m

áxim

o

a

a

0

40

80

120

T0 T12

% d

el m

áxim

o

A clone 027ssh, putative annexin

B clone 004ssh, putative syntaxin of

plant 71

C clone 003ssh, UDP-glucose

pyrophosphorylase-like

E clone 072ssh, putative enolase

F clone 101ssh, ubiquitin-conjugating

enzyme-like

D ACC synthase (ACS), GenBank

AF443280.

A B

D

C

EF

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

Cherimoya cv. Concha Lisa

Harvest

→ 7 days at 20°C

Control fruit (T0)

Gene characterization

Search of orthologous sequences

Sequencing and analysis

Specific transcripts of treated condition

Expression patterns of genes differentially expressed in

treated condition

Visual and analytic evaluation of disorders

Treated fruit (T6-T12)

12 days at 0°C

0 days at 0°C

RNA extraction, cDNA synthesis

Suppression subtractive hybridization (SSH)

Clone selection

Generation of subtraction library

PoolPool

RACE-PCRExpression levels

Fresh cut

0 d (T0) 6 d (T6) 12 d (T12)

Cold storage (days)aa

a

0

5

10

15

20

T0 T6 T12

Ab

s /

min

/ m

g

1Prieto, H, Utz, D, Castro, A, Aguirre, C, González-Agúero, M, Valdés, H, Cifuentes, N, Defilippi BG, Zamora, P, Zúñiga, G and Campos-Vargas, R. 2007. Browning in Annona cherimola fruit: role of polyphenol oxidase (PPO) and characterization of a coding sequence of the enzyme. J. Agric. Food. Chem. 55, 9208-9218.