Mol. Cells, Vol. 15, No. 3, pp. 327-332
M olecules
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KSMCB 2003
Capsicum annuum dehydrin, an Osmotic-Stress Gene in
Hot Pepper Plants
Eunsook Chung, Soo-Yong Kim, So Young Yi, and Doil Choi*
Plant Genomics Laboratory, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-600, Korea.
(Received December 24, 2002; Accepted March 10, 2003)
Osmotic stress-related genes were selected from an
EST database constructed from 7 cDNA libraries from
different tissues of the hot pepper. A full-length cDNA
of Capsicum annuum dehydrin (Cadhn), a late embryogenesis abundant (lea) gene, was selected from the 5′
single pass sequenced cDNA clones and sequenced.
The deduced polypeptide has 87% identity with potato
dehydrin C17, but very little identity with the dehydrin genes of other organisms. It contains a serinetract (S-segment) and 3 conserved lysine-rich domains
(K-segments). Southern blot analysis showed that 2
copies are present in the hot pepper genome. Cadhn
was induced by osmotic stress in leaf tissues as well as
by the application of abscisic acid. The RNA was most
abundant in green fruit. The expression of several osmotic stress-related genes was examined and Cadhn
proved to be the most abundantly expressed of these in
response to osmotic stress.
Keywords: ABA; Dehydrin; Hot Pepper; lea; Osmotic
Stress; Pathogen.
Cohen and Bray, 1992; Godoy et al., 1990).
LEA proteins are classified into a number of groups
(Dure, 1993) but share characteristics such as hydrophilicity, heat stability, and random coil structure. Dehydrin is a member of the group 2 LEA 11 proteins (Dure,
1993) characterized by the presence of a conserved Cterminal 15-mer motif, EEKKGIMDKIKELPG. Most
RAB or COR proteins also belong to this group (Nylander
et al., 2001). These proteins may function as chaperons,
or preserve protein structure during dehydration (Bray,
1993). Based on in vitro studies of the binding of purified
Zea mays DHN1 with phospholipid vesicles, it has been
suggested that dehydrins play a role in stabilization of
vesicles or membrane structures in stressed plants (Koag
et al., 2003). There are also reports that over-expression
of LEA proteins in plants and yeasts increases their resistance to osmotic stress (Duan et al., 1996; Imai et al.,
1996; Xu et al., 1996; Zhang et al., 2000). However, the
function of LEA proteins in stressed plants remains
uncertain.
In this study, we report the isolation of a hot pepper lea
gene, Cadhn, and its expression in response to various
stresses, and to ABA treatment.
Introduction
When plants are exposed to water-deficit stress, or are
developing seeds, the increased level of abscisic acid
(ABA) triggers the expression of a number of genes (reviewed by Bray, 1993). One group of differentially expressed genes is the late embryogenesis abundant (lea)
genes, whose protein levels are high in developing embryos during seed maturation (Baker et al., 1988; Galau et
al., 1986). The lea genes are also induced by application
of ABA or of stresses such as cold, dehydration or salt
treatment, to the vegetative tissues (Choi et al., 1999;
* To whom correspondence should be addressed.
Tel: 82-42-860-4340; Fax: 82-42-860-4309
E-mail: [email protected]
Material and Methods
Plant material and pathogen inoculation Hot pepper (Capsicum annuum L. cv. Bukang) seeds were germinated and grown
in a growth chamber at 25°C with a 16 h light and 8 h dark cycle. RNA was extracted from the leaves, stems, roots, open and
closed flowers, green fruits and mature dried seeds of 2 monthold healthy plants. It was immediately frozen in liquid nitrogen
and stored at –80°C.
A single colony of freshly grown Xanthomonas axonopodis
pv. glycines 8ra was inoculated into YEP media containing
rifampicin (50 mg/L) and grown overnight in a 30°C shaker.
Abbreviaiton: Cadhn, Capsicum annuum dehydrin.
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The bacteria were harvested by centrifugation and resuspended
in sterilized water at approximately 109 cells/ml (A600, 1.0). The
suspension was pressure-infiltrated into leaves with a needleless
syringe.
Isolation of Cadhn A hot pepper cDNA library was constructed,
and the cDNAs were amplified in E. coli after in vivo excision,
as previously described (Choi et al., 1996). The 5′ partial nucleotide sequences and deduced polypeptides obtained are given
in (http://plant.pdrc.re.kr/ks200201/pepper.html). This database
was then searched for osmotic-stress-related genes. To determine the full-length sequence of Cadhn, its cDNA was sequenced with T3 / T7 primers in pBluescript SK (−).
Osmotic stress and ABA treatment For controls, pepper leaves
were placed in distilled water for 24 h. For water-deficit stress,
leaves were laid on a paper towel for up to 24 h at room temperature; for cold treatment, they were placed in distilled water
and kept in 4°C cold room under dim light condition for 8 or 24
h and for salt stress they were incubated in 0.25 M NaCl for 24
h. ABA stock solution was prepared by dissolving ABA [(±) cis,
trans-ABA; Sigma, USA] in small aliquots of 1 N NaOH. The
stock was diluted to 10−3 M with distilled water and adjusted to
pH 6.0 with 0.1 N HCl. 10−4 and 10−5 M ABA solutions were
made by further dilution. The ABA solutions were applied to
detached leaves through their petiole.
RNA extraction and RNA blot analysis Total RNA was extracted from stored tissues by the LiCl-phenol method (Prescott
and Martin, 1987). It was size-fractionated on 1% (w/v) denaturing formaldehyde agarose gels according to Sambrook et al.
(1989), and transferred to Hybond-NX membranes (Amersham
Pharmacia Biotech, UK) with 20× SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0) as transfer solution. It was cross-linked to
the membranes by UV irradiation at 300 nm for 3 min. RNA
blots were pre-hybridized overnight at 42°C in hybridization
buffer: 50% formamide, 5× SSPE, 5× Denhardt’s, 0.1% SDS.
PCR products corresponding to partial Cadhn cDNA (KS01072
D04; Fig. 1A), and osmotin or ERD15 cDNAs were labeled with
[α-32P] dCTP by random priming (Prime-a-gene kit; Promega,
USA). All membranes were hybridized at 42°C with hybridization buffer containing labeled DNA probe. They were washed
with 2× SSC/0.2% (w/v) SDS at room temperature for 5 min,
followed by two washes with 1× SSC/0.2% (w/v) SDS at 42°C
for 30 min, and two more washes with 0.1× SSC/0.2% (w/v)
SDS at 65ºC for 60 min. Dried filters were placed on X-ray film
at –80°C for 1 d and developed.
Southern hybridization Genomic DNA was prepared according to Dellaporta et al. (1983). Twenty micrograms of total
DNA was digested with HindIII, EcoRI, or XbaI, and the digested DNAs were separated by size on 0.8% (w/v) agarose gels.
Southern transfer was carried out by the standard method (Sambrook et al., 1989), and hybridization and washes were performed according to Church and Gilbert (1984). Membranes
A
B
Fig. 1. Schematic diagram of Cadhn EST cDNA contigs (A),
and phylogenetic cladogram of plant dehydrins (B). A. Restriction maps of partial and full-length Cadhn EST cDNAs. B. Tree
based on deduced amino acid sequences of dehydrins constructed using the PhyloDraw program: (http://pearl.cs.pusan.
ac.kr/phylodraw). Dehydrins are: Ecpp44 (Dacus carota; AB
010898), Bcbdn1 (Boea crassifolia; AF190474), ScDhn2 (Solanum commersonii; AF386075), Cadhn (C. annuum), StCI7 (S.
tuberosum; T07779), TaCOR410 (Triticum aestivum; L29152),
Hvdhn8 (Hordeum vulgare; AF181458), AtLTI30 (Arabidopsis
thaliana; X77613), Psdhn1 (Pisum sativum; X63061), AtRAB18
(A. thaliana; AF428458), Zmdhn1 (Zea mays; X15290),
Hvdhn9 (H. vulgare; X15289), AtERD14 (A. thaliana; AF
326904), AtCOR47 (A. thaliana; X90959), and AtLTI29 (A.
thaliana; X90958).
were hybridized with a 32P-labeled fragment of the PCR product
of the partial Cadhn cDNA (KS01072D04; Fig. 1A) in a buffer
consisting of 1% BSA /1 mM EDTA/0.5 M NaHPO4, pH 7.2/7%
SDS at 65°C overnight and washed in 0.5% BSA/1 mM
EDTA/40 mM NaHPO4, pH 7.2/5% SDS at room temperature
for 5 min. The blots were then washed three times with high
stringency wash buffer (1 mM EDTA/40 mM NaHPO4, pH
7.2/5% SDS) at 65°C. Dried blots were placed on X-ray film at
−80°C for a week and developed.
Results and Discussion
Determination of Cadhn sequences and phylogenetic
analysis of the deduced dehydrin polypeptides A hot
Eunsook Chung et al.
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pepper cDNA library was constructed from 7 different
plant tissues: leaves infiltrated with X. axonopodis pv.
glycines 8ra suspension cells, main and axillary root,
flower, fruit, placenta and anther. An EST database was
then generated by single pass sequencing of 5′ cDNA
termini (http://plant.pdrc.re.kr/ks200201/pepper.html).
To detect a potential osmotic-stress marker gene, we
examined the mRNA expression patterns of several genes
related to osmotic-stress, such as dehydrin, lea 5 and
GRAM (Glucosyltransferases, Rab-like GTPase Activators
and Myotubulins). Dehydrin mRNA showed the greatest
increase in response to osmotic stress and ABA treatment
(data not shown).
A number of partial and full-length dehydrin EST
cDNAs were found in the database. The overlapping dehydrin cDNA contigs turned out to be derived from the
same transcript (Fig. 1A). A full-length dehydrin cDNA
(KS09037C02) is described in this study, and the corresponding gene is abbreviated as Cadhn (Capsicum annuum dehydrin). The evolutionary relationship between
the deduced amino acid sequences of fifteen dehydrin
genes, including Cadhn, was analyzed (Fig. 1B). The deduced amino acid sequence of Cadhn has 87% identity
with the potato (Solanum tuberosum) dehydrin homolog,
CI7 (U69633), that is induced in tubers by cold stress
(van Berkel et al., 1992), and 52% identity with Solanum
commersonii dehydrin 2 (AF386075), induced by low
temperature. The acidic SK-type Arabidopsis dehydrins
AtCOR47, AtLTI29 and AtERD14 are grouped with
Cadhn, but have only 40% identity with it, and the K-type
dehydrins, AtLTI30 and Psdhn1, were even more divergnt.
YSK-type dehydrins, such as AtRAB18, Letas14,
Zmdhn1 and Hvdhn9, differ the most from SK-type dehydrins. We conclude that only Cadhn and potato CI7 are
closely related.
The deduced amino acid sequence of Cadhn is shown
in Fig. 2. The highly hydrophilic polypeptide is composed
of 219 amino acids (M.W.; 24.6 kDa) and is acidic (pI;
5.41). It has a serine tract (S-segment) consisting of 9
serine residues (Fig. 2) and the corresponding residues of
RAB17 and TAS14 are phosphorylated (Goday et al.,
1994a; 1994b; Jensen et al., 1998). There is a putative
nuclear localization signal (NLS) consisting of 6 lysine
residues (KKKKKK), and 3 lysine-rich consensus motifs
(EKKGIMDKIKEKLPG; K-segments), which are predicted to form amphipathic α-helices. This consensus
motif is found in all dehydrins and is thought to enable
them to interact with membranes, and to stabilize membrane structures in stressed plants (Koag et al., 2003).
Cadhn lacks the Y motif, (V/T)DEYGNP (Close, 1996),
found in other dehydrins. On the basis of its conserved
domains, Cadhn should be classified as an SK dehydrin.
Gel blot analysis of genomic DNA Genomic DNA was
digested with 3 different restriction enzymes (Fig. 3), and
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Fig. 2. Nucleotide and deduced amino acid sequence of Cadhn
(GenBank accession number AY225438). Predicted amino acids
are in one letter code. Putative NLS is boxed, serine segment (S)
is underlined (
); three conserved lysine-rich repeats (K)
are shaded.
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the probe used was a 400-bp PCR product corresponding
to the N-terminal region of the Cadhn cDNA clone (Fig.
1A; KS01072D04). All restriction enzyme digestions
gave two hybridizing bands, suggesting that 2 copies of
Cadhn gene are present in the hot pepper genome. The
Dhn genes of plants such as barley (Choi et al., 1999) and
Arabidopsis (Nylander et al., 2001) are multigene families.
Expression of Cadhn during water deficit and pathogen infection We performed a Northern blot analysis of
Cadhn expression during water deficit and X. axonopodis
pv glycines 8ra infection (Fig. 4A). Cadhn mRNA accumulated strongly 2 h after the onset of water deficit; it
was maximal after 4 to 8 h and decreased slightly by 24 h.
Cadhn mRNA was transiently induced 2 to 4 h after X.
axonopodis pv glycines 8ra inoculation and was undetectable after 8 h (Fig. 4A). It is possible that infiltration onto
the plants of a dense microbial suspension (O.D. = 1.0)
causes a transitory increase of osmotic pressure that in-
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Dehydrin in Pepper
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A
B
Fig. 3. Gel blot analysis of genomic DNA. Genomic DNA was
digested with EcoRI (E), indIII (H) or XbaI (X), fractionated on
0.8% agarose gels, and transferred to nylon membranes. The
membranes were hybridized with a 32P-labeled fragment of PCR
product containing the partial Cadhn cDNA (KS01072D04; Fig.
1A).
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duces ABA accumulation.
We investigated the expression of other osmotic- and
pathogen-related genes during water deficit and X. axonopodis pv glycines 8ra infection (Fig. 4B). The level of
osmotin mRNA increased during both water deficit and
pathogen infection, as we expected since the osmotin gene,
PR-5 (pathogenesis-related protein 5), is regulated by
osmotic stress and pathogen infection (Kononowicz et al.,
1992; Zhu et al., 1995). Overexpression of the osmotin
gene in potato confers tolerance to fungal infection and
freezing stress (Zhu et al., 1996). A number of other
genes induced by pathogen inoculation, such as CaAPX1
encoding ascorbate peroxidase, are responsive to osmotic
stress (Yoo et al., 2002). Surprisingly the mRNA of a
homologue of Arabidopsis ERD15 (Early Responsive to
Dehydration gene 15; Kiyosue et al., 1994) was undetectable in water-deficit samples but accumulated strongly 24
h after pathogen infection. ERD15 is induced by the
growth-promoting rhizobacterium Paenibacillus polymyxa
(Timmusk and Wagner, 1999). We conclude that Cadhn is
more involved in responses to osmotic stress than to
pathogen infection.
Cadhn expression in relation to osmotic stress, exogenous ABA and developmental signals Further Northern
blot analyses were carried out to investigate if Cadhn is
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Fig. 4. Expression patterns of osmotin, ERD15 and Cadhn during water-deficit stress and pathogen infection. D, Detached hot
pepper leaves; NS, control for water-deficit stress; A, control for
pathogen infection. Twenty microgram samples of total RNA
were fractionated on 1% denaturing agarose gels. Membranes
were hybridized with 32P-labeled fragments of PCR products
containing partial Cadhn (KS01072D04), osmotin and ERD15
cDNA clones. See Materials and Methods.
induced by other stresses (Fig. 5A). Salt stress (0.25 M
NaCl) did induce Cadhn, while cold treatment was only
slightly effective. Water-deficit treatment resulted in the
highest level of Cadhn expression. The degree of responsiveness to different stresses may vary among the dehydrin genes (Choi et al., 1999). Application of ABA also
resulted in induction of Cadhn (Fig. 5A). Environmental
stresses are commonly associated with increased ABA
levels and result in the induction of ABA-responsive
genes. However, some genes are regulated in an ABAindependent manner (reviewed by Shinozaki and Yamaguchi-Shinozaki, 1996). The expression of Cadhn was
also examined in various organs (Fig. 5B). Its expression
was not restricted to stressed leaves; it was also detected
in non-stressed leaves, roots, flowers, young fruit and
mature seeds. Young fruit bearing developing seeds had
the highest level of Cadhn transcripts, and the high level
of Cadhn transcripts in green fruit may be due to the fact
that green fruits contain developing seeds; developing
seeds also have high levels of ABA, and of the mRNA
and protein products of a number of lea genes (Baker et
al., 1988; Bray, 1993).
In conclusion, we have demonstrated that Cadhn, a lea
Eunsook Chung et al.
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A
B
Fig. 5. Expression patterns of Cadhn upon exposure to various
stresses, and to ABA; in detached hot pepper leaves (A); in
other tissues (B). A. RNA blot analysis of Cadhn. NS (no stress),
WD (water-deficit), CS (cold stress), NaCl and ABA (abscisic
acid) B. RNA blot analysis of Cadhn in L (leaves), St (stem), R
(root), FL (flower), FR (green fruit) and S (dried seeds). Twenty
microgram samples of total RNA were fractionated on 1% denaturing agarose gels. Membranes were hybridized with 32Plabeled fragments of PCR products containing a partial Cadhn
(KS01072D04) cDNA clone.
gene, is regulated by various types of stress in hot pepper
plants. In addition, Cadhn is responsive to ABA treatment,
and its transcripts are abundant in green pepper fruit.
Acknowledgments This research was supported by grants from
the Plant Diversity Research Center (PF003301), the Crop Functional Genomics Center (CG1221), the 21st Century Frontier
Research Program funded by the Ministry of Science and Technology of the Korean government and PMGBRC through the
Korea Science and Engineering Foundation.
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