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
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.
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).
Experimental design
Expression patterns of genes
differentially expressed in
treated condition
4
5
6
363 bp
Pool
C
RNA extraction,
cDNA synthesis
234 bp
F
252 bp
G
298 bp
H
Results
B
2 cDNA L: Leaves
261 bp
3 cDNA F: Flowers
348 bp
*
12 d (T12)
15
N
O
253 bp
P
7 cDNA S: Subtracted cDNA
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.
10
A
a
120
80
40
E clone 072ssh, putative enolase
5
F clone 101ssh, ubiquitin-conjugating
T6
enzyme-like
T12
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.
B
St 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Summary of subtraction library:
- Positive clones
- Sequenced
St 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72
120
C
80
40
b
a
120
80
40
b
b
0
0
T0
F
T12
a
120
80
0
T0
- Average insert size
T12
T0
T12
40
0
a
120
80
40
b
0
T12
D
a
120
a
80
40
0
T0
T12
T0
T12
AF443280.
* Different letters represent significant differences at P < 0.05 by LSD test.
Conclusions
- 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.
~ 250 clones
200
700 bp
- Redundancy of sequens. ~ 30%
- Sequences analyzed
E
a
T0
A
B
a
pyrophosphorylase-like
a
D ACC synthase (ACS), GenBank
St 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
6 cDNA NS: Non-subtracted
cDNA
346 bp
C clone 003ssh, UDP-glucose
T0
6 7 8 9 10 11 12 13 14 15 16 17 18
5 cDNA FMF: Mature fruit
flesh
318 bp
399 bp
plant 71
a
a
0
St 1 2 3 4 5
4 cDNA RMF: Mature fruit
rind
L
B clone 004ssh, putative syntaxin of
20
Abs / min / mg
6 d (T6)
199 bp
343 bp
A clone 027ssh, putative annexin
0 d (T0)
1 cDNA GF: Growing fruits
* New actin isoform only expressed in subtracted cDNAs samples
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.
Cold storage (days)
7
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, UDPglucose 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.
Suppression subtractive
hybridization (SSH)
A
6
253 bp M
Clone selection
Specific transcripts of
treated condition
5
J
230 bp
D
Generation of subtraction
library
4
% del máximo
Pool
3
296 bp K
E
Visual and analytic evaluation of
disorders
2
I
267 bp
B
Sequencing
and analysis
1
7
% del máximo
Treated fruit
(T6-T12)
3
A
Search of orthologous
sequences
12 days
at 0°C
2
% del máximo
Control
fruit (T0)
Expression
levels
1
Fresh cut
0 days
at 0°C
Gene
characterization
% del máximo
RACE-PCR
% del máximo
Harvest
→ 7 days at 20°C
% del máximo
Cherimoya
cv. Concha Lisa
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.
105
Funded by PBCT PSD03