全 文 ：Received 16 Jul. 2003 Accepted 7 Nov. 2003
Supported by the National Natural Science Foundation of China (30070536, 30371007) and the Guangdong Provincial Science Foundation,
China (2000979, 20032233).
* Author for correspondence. E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (3): 355-362
Cloning and Expression Analysis of an XET cDNA in the Peel and Pulp of
Banana Fruit Ripening and Softening
LU Wang-Jin1*, Ryohei NAKANO2, Yasutaka KUBO 2, Akitsugu INABA2, JIANG Yue-Ming3
(1. Department of Horticulture, South China Agricultural University, Guangzhou 510642, China;
2. Faculty of Agriculture, Okayama University, Tsushima, Okayama 700-8530, Japan;
3. South China Institute of Botany, The Chinese Academy of Sciences, Guangzhou 510650, China)
Abstract: Xyloglucan endotransglycosylase (XET) is thought to be involved in fruit softening through
disassembly of xyloglucan, which is the predominant hemicellulose of cell wall. To study the relationship
between fruit softening and XET during banana (Musa acuminata Colla cv. Grand Nain) fruit ripening, a full
length cDNA (1 095 bp) encoding an XET, MA-XET1, was isolated from ripening banana fruit using RT-PCR
and RACE-PCR (rapid amplification of cDNA ends) methods. Sequence analysis showed that the cDNA
contains 5 untranslated region of 66 bp, 3 untranslated region of 189 bp and ORF of 840 bp, encoding a
predicted polypeptide of 280 amino acids, including DEIDFEFL motif, which is a presumptive catalytic
domain conserved in XETs. DNA gel blot analysis demonstrated that MA-XET1 is encoded by a multi-copy
family in the banana genome. RNA gel blot analysis revealed that the level of MA-XET1 transcript in the
pulp was undetectable, increased and decreased slightly at the preclimacteric, climacteric and postclimacteric
stages, respectively. In the peel, accumulation of MA-XET1 transcript was low, increased dramatically and
then decreased rapidly, at preclimacteric, climacteric and postclimacteric stages, respectively. Treatment
of fruit with propylene, an analog of ethylene, decreased the firmness and enhanced the accumulation of
MA-XET1 transcript in the peel and pulp. These results suggest that MA-XET1 is involved in softening of
the peel and pulp during banana fruit ripening and its expression is regulated by ethylene at transcriptional
level.
Key words: xyloglucan endotransglycosylase (XET); banana fruit; ripening and softening
Fruit ripening is accompanied by significant physiologi-
cal and biochemical changes including solubilization of the
cell wall. From unripe stage to ripe stage, banana fruit be-
come gradually edible with an optimum blend of color, taste,
aroma and texture. From an economical standpoint, change
in texture is the most crucial of all as it directly affects the
shelf life and quality of the fruit.
During fruit ripening both pectic and hemicellulose poly-
mers generally undergo substantial depolymerization and
solubilization (Fisher and Bennett, 1991; Rose et al., 1998).
Molecular genetic studies have revealed that pectin degra-
dation is not a primary determinant of fruit softening and
disassembly of hemicellulose by multiple enzymes might
play an important role in the process (Rose and Bennett,
1999). Xyloglucan represents the predominant hemicellu-
lose in many fruits and xyloglucan endotransglycosylase
(XET) has been identified as a hydroxylase which modifies
xyloglucan molecule in cell wall (Schroder et al., 1998).
The XET genes have been isolated mostly from rapidly
growing plant tissues. In many cases, significant positive
correlations between XET activity and tissue elongation
have been described (Burstin, 2000; Uozu et al., 2000; Reidy
et al., 2001). However, little attention has been given to the
role of XET enzyme (Redgwell and Fry, 1993; Cutillus-
Iturralde et al., 1994) and related XET genes (Arrowsmith
and Silva, 1995; Schroder et al., 1998; Chen et al., 1999;
Catala et al., 2001; Nunan et al., 2001) with respect to fruit
ripening.
In banana fruit, the possible candidate genes and en-
zymes responsible for cell wall pectic polysaccharides me-
tabolism have been reported (Dominguez-Puigianer et al.,
1992; Dominguez-Puigianer et al., 1997; Pua et al., 2001).
Hemicellulose has been shown to be degraded during fruit
ripening in banana (Kojima et al., 1997); however, to date
there has been no report on possible candidate genes for
hemicellulose disassembly relating to fruit softening. The
objective of this study was to isolate XET cDNA from rip-
ening banana fruit, and characterize its expression in the
peel and pulp tissues in relation to fruit softening and in
response to ethylene.
Acta Botanica Sinica 植物学报 Vol.46 No.3 2004356
1 Materials and Methods
1.1 Plant materials
Preclimacteric banana (Musa acuminata Colla cv. Grand
Nain) fruit imported from the Philippines were supplied by
a local importer in Okayama, Japan. Each banana hand was
cut into individual fingers and ripened at 22 ℃. In each
experiment, fingers from the same hand were used as a
sample group to reduce variation in ripening behaviors
(Inaba and Nakamura, 1986; Liu et al., 1999). Preclimacteric
fruit were treated with 1 000 mL/L propylene for 16 h and
thereafter ripened at 22 ℃. The experiments were conducted
in Laboratory of Postharvest Horticulture, Faculty of
Agriculture, Okayama University, Japan.
1.2 Ethylene production and firmness
Ethylene production rate was measured by placing three
fruits in an airtight container for 1 h at 25 ℃. One mL of the
headspace gas was withdrawn and injected into a gas chro-
matograph (model GC-4CMPE, Shimadzu Corp., Kyoto,
Japan) fitted with a flame ionization detector (150 ℃) and
an activated alumina column (80 ℃).
Firmness of the peel and pulp was determined at four
equational regions of the peel or peeled pulp of three fruits
using a penetrometer (model STM-T-50, Toyo Baldwin,
Tokyo, Japan), equipped with a flat probe (8-mm diameter).
1.3 RNA extraction
Total RNA was extracted from the peel and pulp tissues
by the hot borate method (Wan and Wilkins, 1994),
Poly (A+) RNA was isolated using Oligo dT(30) (TaKaRa,
Kyoto, Japan) according to the manufacturer’s protocol.
1.4 Cloning and sequencing of XET
Poly (A+) RNA extracted from ripened pulp was used as
templates for the RT-PCR. The product (first-strand cDNA)
was subjected to PCR amplification using degenerate prim-
ers designed on the alignment of XETs from Grammaceae
species; sense, 5-GA(TC)GA(AG)(AC)TIGA(TC)(AT)T
(ATC)GA(AG)TT(TC)(CA)TIGG-3, antisense, 5-(CT)
GIGTIGCCCAI (TG)(CA) (GA)T CI (GC) C(AG)T(TC)CC-3.
Reactions for the RT-PCR were subjected to 35 cycles
(95 ℃ for 1 min, 40 ℃ for 2 min, and 72 ℃ for 2 min) and one
cycle of 72 ℃ for 10 min. PCR products were cloned into
pGEM-T-Easy vector (Promega, Madison, WL).
The nucleotide sequences of the cDNA inserts were
determined using a DNA sequencer (Model DSQ-1000,
Shimadzu Corp., Kyoto, Japan) with either –21M13 or M13
sequencing primers according to the manufacturer’s instruc-
tions (Amersham, Uppsala, Sweden).
1.5 Amplification of full-length cDNA by RACE-PCR
RACE-PCR was performed using a cDNA amplification
kit (Marathon, Clontech, Palo Alto, CA) according to the
manufacturer’s protocol. To amplify 5-end and 3-end
fragments, the specific primers: 5-GCTGGCTGTTGGGGAA-
GGCGATGCC-3 for 5 -end, 5-CAATGTGTTCACCCAGG-
GAAAGGGA-3 for 3 -end, were designed on the nucle-
otide sequences of the cDNA fragments cloned by RT-PCR.
The RT-PCR products were cloned and sequenced as de-
scribed above.
1.6 Northern blotting analysis
Total RNA (10 mg) was separated on a 1.2% agarose-
formadehyde gel and capillary blotted onto Biodyne nylon
membrane (PALL, Tokyo, Japan). The membrane was blot-
dried and cross-linked with UV. DIG-labeled specific probe
of 3-end regions of MA-XET1 was made using PCR DIG
Probe Synthesis kit (Roche Applied Science, Mannheim,
Germany) using primers: sense, 5-ACTGCATCATACAG-
AAACTTCAAGG-3 and antisense 5-CTACTCGCTATGC-
TTTTGATAGCTC-3. The membrane was hybridized with
DIG-labeled probe for 16 h at 43 ℃ in high-SDS buffer (7%
SDS, 5×SSC, 50 mmol/L sodium-phosphate, pH 7.0, 2%
blocking reagent and 0.1% N-lauroylsarcosine) containing
50% deionized formamide (V/V) (Roche, Germany). Blots
were washed twice at 37 ℃ in 2×SSC and 0.1% SDS for 10
min, followed by washing twice at 58 ℃ in 0.1×SSC and
0.1% SDS for 30 min. The membranes were then subjected
to immunological detection according to the manufacturer’s
instructions (Roche Applied Science, Mannheim, Germany).
1.7 Southern blotting analysis
Genomic DNA was isolated from young banana leaves
by the method of Murray and Thompson (1980). For South-
ern analysis, 5 mg DNA was digested overnight with differ-
ent endo-nuclease (EcoRⅠ, EcoRⅤ, HindⅢ and XbaⅠ),
then separated on 0.8% (W/V) agarose gel and transferred
DNA to Biodyne nylon membrane (PALL, Tokyo, Japan) as
described above. The blots were hybridized with the DIG-
labeled MA-XET1 specific probe.
2 Results
2.1 Isolation and sequence analysis of full length XET
cDNA in banana fruit
A fragment (294 bp) was cloned from mRNA of ripe pulp
by RT-PCR. BLAST search of GenBank revealed that this
fragment shared 82% identity with that of Hv-XEA
(accession number X93173) and was, therefore, considered
to be a cDNA fragment of XET. The full length of the cDNA
was obtained using RACE-PCR. Sequence analysis showed
that the MA-XET1 cDNA consists of 5 untranslated region
of 66 bp, 3 untranslated region of 189 bp, and an ORF of
840 bp that encodes a predicted polypeptides of 280 amino
LU Wang-Jin et al.: Cloning and Expression Analysis of an XET cDNA in the Peel and Pulp of Banana Fruit Ripening and Softening 357
acids (Fig.1). The deduced amino acid sequence of MA-
XET1 contains the features conserved in known XET genes,
including hydrophobic signal peptide region, a central
DEIDFEFLG sequence (dashed line in Fig.1) shared with b-
glucanases (Campbell and Braam, 1999), the putative N-
linked glycosylation consensus site (N-X-S/T) (the bold-
face region in Fig.1) adjacent to the conserved DEIDFEFLG
motif and two pairs of cysteines in the more variable car-
boxyl terminal region (the shaded “C” in Fig.1). The de-
duced amino acid sequence of MA-XET1 showed 49.6%
identity with AdXET5 (accession number L46792) ex-
pressed in ripening kiwifruit (Schroder et al., 1998) and
46.1% identity with LeEXT (accession number D16456),
expressed in rapidly growing tomato fruit (Catala et al.,
2000) (Fig.2).
2.2 Southern analysis of MA-XET1
To estimate the copy numbers of MA-XET, Southern
blotting analysis was performed (Fig.3). Genomic DNA di-
gested with EcoRⅠ, EcoRⅤ, HindⅢ and XbaⅠ yielded
three restriction bands (two strong bands and one faint
Fig.1. Full length of nucleotide sequence and its deduced amino acid sequence of MA-XET1 cDNA. The predicted amino acid sequence
of MA-XET1 is shown below the DNA sequence; a single dashed line denoted the catalytic domain near N-terminal; the boldface “NPS”
adjacent to the DEIDFEFLG motif indicated the N-linked C-terminal glycosylation regions; two pairs of shaded “C” denoted the C-
terminal conserved regions.
Acta Botanica Sinica 植物学报 Vol.46 No.3 2004358
band) on the blot, indicating that MA-XET1 is likely
encoded by three isogenes.
2.3 Ethylene production, softening and MA-XET1
expression in naturally ripened fruit
Figure 4 shows the changes in ethylene production of
intact fruit (A), firmness of peel (B) and pulp (C) during fruit
Fig.2. Multiple alignment of the deduced amino acid sequences of full length MA-XET1 cDNA from different species, Le-XET4
(AF186777), tXET-B1 (X82685), Ad-XET5 (L46792), Le-EXT (D16456) and MA-XET1 were aligned using the online Clustal W method
( http://www.ddbj.nig.ac.jp ); the boxed “DEIDFEFLG” and shaded “C” mean the same as described in Fig.1.
LU Wang-Jin et al.: Cloning and Expression Analysis of an XET cDNA in the Peel and Pulp of Banana Fruit Ripening and Softening 359
ripening at 22 ℃. Ethylene production commenced on day
11, reached a sharp peak on the next day and then de-
creased (Fig.4A). The pulp firmness declined sharply with
commencement of ethylene production. In contrast, the peel
firmness declined steadily during ripening (Fig. 4B,C ).
MA-XET1 exhibited a typical ripening-associated expres-
sion (Fig.5). The MA-XET1 transcript in pulp was unde-
tectable at preclimacteric stage (3-11.5 d), appeared with
commencement of ethylene production (12 d), peaked on
12.5 d, decreased thereafter (13 d). The expression pattern
was consistent with that of increase in ethylene produc-
tion and decrease in pulp firmness. In the peel, accumula-
tion of MA-XET1 transcript was detected at low level dur-
ing preclimacteric stage (6, 8 or 11 d), increased dramati-
cally at climacteric stage (11.5-12 d) and then decreased
gradually at postclimacteric stage (12.5-13 d).
2.4 Effect of propylene on expression of MA-XET1 in
peel and pulp
To examine the relationship between expression of MA-
XET1 and softening in banana fruit and its regulation by
ethylene, banana fruit at preclimacteric stage were treated
with 1 000 mL/L propylene for 16 h at 22 ℃. The pulp firm-
ness declined sharply from day 1 to day 3 while peel firm-
ness decreased steadily (Fig.6A, B). The accumulation of
MA-XET1 transcript in pulp was undetectable at the end of
propylene treatment, day 1, then increased rapidly with a
sharp decline in firmness at day 2, and decreased to trace
level until day 6 (over-ripe stage). On the other hand, the
accumulation of MA-XET1 transcript in the peel was in-
duced at day 1, remained at high level until day 3 and then
decreased slightly.
Fig.3. DNA gel blot analysis of MA-XET1. Five mg genomic
DNA was digested with EcoRⅠ, EcoRⅤ, Hind Ⅲ and XbaⅠ,
separated by 0.8% agarose gel electrophoresis and hybridized
with DIG-labeled gene-specific MA-XET1 probes.
Fig.4. Changes in ethylene production rate (A), firmness of peel
(B) and pulp (C) of banana fruit ripened naturally.
Fig.5. Changes in mRNA accumulation of MA-XET1 in the peel
and pulp of naturally ripened banana. The above number shows
days at 22 ℃. On the sampling day, ethylene production was
determined in the 3 fruit, and the peel and pulp from the same 3
fruit were used for determination of firmness and RNA extraction
for Northern analysis. the lower panel shows ethidium bromide-
stained rRNA to indicate the relative RNA loading of each lane.
Acta Botanica Sinica 植物学报 Vol.46 No.3 2004360
3 Discussion
To our knowledge, there has been no report on the iso-
lation and expression of XET genes in banana fruit. In this
study, we isolated MA-XET1 as a homologue of XETs and
the deduced amino acid sequence of the protein shared
common features with XETs, which have been obtained
from plants.
The plant primary cell wall is a complete network of cel-
lulose microfibrils that are interwoven by two classes of
matrix polymers, hemicellulose and pectin (Rose and
Bennett, 1999). Xyloglucan is the major hemicellulose in
dicotyledons and is thought to be degraded during ripen-
ing and softening. In banana, a monocotyledon, only a few
studies have been done to investigate the component of
cell wall polysaccharides and their changes during ripen-
ing and softening. The analysis of cell wall disassembly in
banana fruit showed that hemicellulose is degraded signifi-
cantly during fruit ripening (Kojima et al., 1997; Prabha and
Bhagyalakshmi, 1998). Endo-b-1,4-glucanases (EGase) and
XETs are candidate enzymes, which are responsible for the
reorganization of the cellulose-xyloglucan framework in
plant. The activity of XET has been shown to increase
during ripening in kiwifruit (Redgwell and Fry, 1993) and
tomato fruit (Maclachlan and Brady, 1994), indicating that
it is involved in fruit ripening and softening.
The results presented have shown that the expression
of MA-XET1 coincides well with pulp softening in both
fruit ripened naturally and fruit treated with propylene. In
addition to pectin and starch degradation (Kojima et al.,
1997), modification of hemicellulose associated with MA-
XET1 may also be involved in pulp softening. In peel, the
expression of MA-XET1 correlated well with changes in
firmness. However, peel firmness declined steadily during
all the ripening stages while accumulation of MA-XET1
decreased only at postclimacteric stage. Thus, MA-XET1
may not be the sole factor contributing to peel firmness
and other factors such as expansin, PG and EGase, may
also play an important role in cell wall modification in ba-
nana fruit peel. As peel texture changes during fruit ripening,
the peel becomes more susceptible to fungi, such as
Fursaium sp. and Colletotrium musae, resulting in devel-
opment of lesion and rot on fruit. In addition to its involve-
ment in peel softening, MA-XET1 may contribute to in-
creased susceptibility of banana peel to fungi during fruit
ripening.
The expression pattern of MA-XET1 in both peel and
pulp in naturally ripened fruit was correlated well with the
changes in ethylene production and propylene treatment
enhanced the accumulation of MA-XET1 transcript. These
observations suggest that MA-XET1 is regulated by ethyl-
ene at transcriptional level.
Acknowledgements: We thank Dr. Francies M. Mathooko
of Jomo Kenyatta University of Agriculture and
Technology, Kenya for his careful reading and valuable
comments on this manuscript.
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