Plant Science 152 (2000) 123134

Expression of a Solanum tuberosum cyclophilin gene is regulated

by fungal infection and abiotic stress conditions
Andrea V. Godoy
a
, Alejandra S. Lazzaro
a
, Claudia A. Casalongue
a,
*,
Blanca San Segundo
b
a
Departamento de Biolog a, Instituto de In6estigaciones Biologicas, Uni6ersidad Nacional de Mar del Plata, Funes 3250, cc 1245,
7600 Mar del Plata, Argentina
b
Centro de In6estigacion y Desarrollo (CSIC), Barcelona, Jordi Girona 1824, 08034 Barcelona, Spain
Received 31 March 1999; received in revised form 11 October 1999; accepted 14 October 1999
Abstract
Cyclophilins (CyPs) are ubiquitous proteins with an intrinsic enzymatic activity of peptidyl-prolyl cis-trans isomerase that
catalyzes the rotation of X-Pro peptide bonds. These enzymes are believed to play a role in the folding of certain proteins. In
addition, CyPs might be important in signal transduction processes. A cDNA library was prepared from potato (Solanum
tuberosusm) tubers infected with the fungus Fusarium solani f. sp eumartii. Using a PCR-amplied subtracted cDNA probe, a
clone encoding a cytosolic form of CyP, called StCyP (S6 olanum t6uberosum CyP), was isolated. Except in tubers, StCyP is
expressed at high levels in tissues of healthy potato plants. Northern blot analyses revealed that both wounding and fungal
infection increased the level of StCyP mRNA in tubers. However, whereas wounding causes a transient accumulation of StCyP
mRNA, fungal infection results in a maintained accumulation of this transcript. StCyP mRNA accumulation is also stimulated
by the application of absicic acid (ABA) and methyl jasmonate (MeJA) in tubers. Treatment with fungal elicitor or salicilic acid
(SA) has no effect on the level of StCyP mRNA accumulation. Together these results indicate that the observed accumulation of
StCyP mRNAs in fungal-infected potato tubers might be a response to the wound produced by the penetration and colonization
of the tissue by the pathogen. Furthermore, accumulation of StCyP transcripts was also detected when the potato tubers were
exposed to heat-shock treatment. These ndings support a role for cyclophilins in the plant response to environmental stresses.
2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Cyclophilin; Fusarium solani ; Peptidyl-prolyl cis-trans isomerase; Potato; Stress responses; Wounding
www.elsevier.com/locate/plantsci
1. Introduction
Cyclophilins (CyPs) are ubiquitous proteins
with an intrinsic enzymatic activity of peptidyl-
prolyl cis-trans isomerase (PPIase or rotamase) [1].
This enzyme catalyses the cis-trans isomerization
of proline peptide bonds and accelerates the fold-
ing of certain proteins [25]. CyP appears to
participate in the protein folding process not only
as a prolyl isomerase but also as a chaperone [2].
This possibility is supported by the results of
Sykes et al. [6] who demonstrated the heat-shock-
responsive expression of cyclophilin mRNAs in
yeast. Additionally, Duina et al. [7] reported that
two Saccharomyces cere6isiae CyPs, Cpr6 and
Cpr7, form complexes with Hsp90, a protein
chaperon.
The presence of CyPs has been described in a
wide range of organisms: animals including man,
higher plants, fungi and bacteria [8,9]. The high
Abbre6iations: ABA, absicic acid; CWF, cell wall carbohydrate
fractions; CyP, cyclophilin; CsA, cyclosporin A; ENTS, sunower
rRNA cDNA fragment; F. eumartii, Fusarium solani f. sp eumartii ;
JA, jasmonic acid; MeJA, methyl jasmonate; PCR, polymerase chain
reaction; PDA, potato dextrose agar; P. infestans, Phytophthora
infestans; PKPI, Kunitz-type proteinase inhibitor; PPIase, peptidyl-
prolyl cis-trans isomerase; SA, salicilic acid; ScCyP, Solanum com-
mersonii cyclophilin; StCyP, Solanum tuberosum cyclophilin.
* Corresponding author. Tel: +54-223-4753030; fax: +54-223-
4753150.
E-mail address: casalong@mdp.edu.ar (C.A. Casalongue)
0168-9452/00/$ - see front matter 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0168- 9452( 99) 00211- 3
A.V. Godoy et al. / Plant Science 152 (2000) 123134 124
degree of conservation which is found in CyP
amino acid sequences of distantly related organ-
isms indicates a strong selective pressure for
maintenance of the structure of this protein during
evolution and suggests an important conserved
cellular function for cyclophilins. In higher plants,
CyP has been found in both dicotyledoneous and
monocotyledoneous species. Thus, cDNAs and ge-
nomic sequences encoding CyPs have been iso-
lated from maize, tomato, Brassica [3,10],
Arabidopsis thaliana [8,1113], rice [14], Solamun
commersonii [15], tobacco [16], and bean [1719].
Furthermore, the presence of cytosolic and chloro-
plast forms of CyP has been described in various
higher plant species [12,17]. In Arabidopsis, at least
six CyP genes have being identied, and ve of
them appear to produce cytosolic proteins [8].
Plant CyP genes are stress-responsive as their
expression can be induced by abiotic stresses,
namely, treatment with chemical agents, heat-
shock, salt stress or low temperature [10,15,17
20]. The expression of Arabidopsis CyP genes has
also been shown to be regulated by light and
wounding [8], although differences in the timing
and regulation degree of the individual Arabidopsis
CyP genes were observed. Together, these observa-
tions support the involvement of plant CyPs in
biological processes during stress conditions.
There is a permanent interest in elucidating the
events underlying the mechanisms by which plants
respond to pathogenic microorganisms, particu-
larly fungal pathogens. Even though fungi repre-
sent the most harmful phytopathogens of the
world, the mechanisms of the intricate relation-
ships between plant and fungal pathogen remain
unknown. We are investigating the extent to which
the expression of defense-related genes and
proteins occurs in potato plants in response to
fungal infection. In order to identify new genes
involved in the response of a potato commercial
cultivar, Solanum tuberosum subp tuberosum cv
Spunta (S. tuberosum), to a potential pathogen,
Fusarium solani f. sp eumartii (F. eumartii ), differ-
ential screening of a fungal-infected potato tuber
cDNA library was carried out, using a subtracted
and a control cDNA probe. Following this strat-
egy, we isolated a potato CyP cDNA which has
been named StCyP (for S6 olanum t6uberosum CyP).
We present data on the effects of different stresses,
such as wounding, fungal infection and heat-
shock, and of different agents, such as fungal
elicitors salicilic acid (SA), absicic acid (ABA), and
methyl jasmonate (MeJA), on the level of StCyP
mRNAs.
2. Materials and methods
2.1. Plant and fungal material
Potato tubers from S. tuberosum were harvested
in the late summer and stored at 4C in the dark
for 4 months. The potato plants were cultivated in
a growth chamber at 25C with uorescent light
(250 mmol/m
2
/s), under a 14 h photoperiod. Four
to ve week-old plants at the vegetative stage were
analysed. For this purpose, stems, fully expanded
leaves (mature leaves) and developing leaves
(young leaves) were harvested, frozen in liquid
nitrogen and stored at 80C. Potato buds from
previously induced sprouting tubers were also
collected.
F. eumartii was obtained from the INTA Collec-
tion, Balcarce, Argentina. The fungus was grown
at 25C on potato dextrose agar (PDA) in Petri
dishes for 3 weeks with uorescent light (150
mmol/m
2
/s) under a 14 h photoperiod.
2.2. Fungal cell wall preparation
Fungal cell wall carbohydrate fractions (CWF)
were obtained as described by Ayers et al. [21].
After extraction the cell walls were exhaustively
washed with deionized water and organic solvents,
lyophilised and stored at 80C. Approximately
100 mg of the lyophilised CWF were suspended in
1 ml sterile water before used.
2.3. Fungal infection, wounding and treatment
with different agents of potato tubers
The tubers were kept at 25C in the dark for 24
h before treatments. Acclimatized tubers were
washed with water, surface-sterilized by immersion
in 0.5% sodium hypochlorite for 5 min and then
rinsed with sterile water. Fungal inoculation of
potato tubers and treatments with all the agents
mentioned in this section were performed at 25C
in the dark, using the hollow punch method de-
scribed by Radtke and Escande [22]. This method
causes mechanical injury to the tissue (wounding).
A.V. Godoy et al. / Plant Science 152 (2000) 123134 125
Initially, the effect of fungal infection and
wounding was tested. For fungal infection,
mycelium and spores of F. eumartii grown on
PDA (0.5 cm disks) were used to inoculate potato
tubers. Controls were made by placing sterile PDA
disks in potato tubers (wounded, but non-infected
tubers). At different times tissue samples (0.5 cm
around the inoculation site) were collected, frozen
in liquid nitrogen and stored at 80C.
For F. eumartii elicitor treatment, potato tubers
were inoculated with 10 mg of CWF. Controls
were prepared by inoculating potato tubers with
sterile water. Tissue samples were collected 15 h
after the onset of treatment.
Phytophthora infestans inoculation was carry
out using 45-week-old potato plants (see Section
2.1). Whole plants were inoculated by spraying
with a suspension containing 210
3
sporangia/ml
of P. infestans using a ne glass atomiser. Control
plants were water-sprayed. The plants were kept at
18C in a moist chamber for different times. Then
the 4th, 5th and 6th leaves beginning from the
youngest leaf on each plant, were harvested frozen
in liquid nitrogen and stored at 80C.
For heat-shock treatment, the tubers were trans-
ferred to 42C and kept for 4 and 8 h, in the dark.
Medullar tissue samples just below the periderm
were collected.
For MeJA and ABA treatments, tubers were
inoculated with the solution of the agent to be
tested and at different concentrations. Racemic
cis-trans ABA (Sigma) was dissolved in 30% (v/v)
ethanol to make a 100 mM stock solution. Tubers
were inoculated with 100 ml ABA solution at a
nal concentration of 10, 50 or 100 mM. MeJA
(Sigma) was dissolved in N,N-dimethylformamide
(100 mM stock solution). For treatments of potato
tubers, 100 ml sterile water MeJA dilutions at nal
concentrations of 20, 50 or 100 mM were used. To
test the effect of SA (Droguer a Internacional),
100 ml of a 10 mM SA solution prepared in sterile
water just before use were applied. Control tubers
were treated with 100 ml sterile water. At different
times tissue samples were collected, frozen in liq-
uid nitrogen and stored at 80C.
2.4. Preparation and differential screening of the
cDNA library
Total and poly(A)
+
RNAs were isolated using
the RNAgents total RNA and the PolyATtract
mRNA isolation systems, respectively (Promega),
following the manufacturers instructions. A direc-
tional cDNA library was constructed in the UNI-
ZAP XR cloning vector (ZAP-cDNA Gigapack II
Gold Cloning kit, Stratagene) using 3 mg poly(A)
+
RNA from 24 h F. eumartii -infected potato
tubers.
Differential screening of the cDNA library with
a polymerase chain reaction (PCR)-amplied con-
trol and subtracted probe was carried out. The
preparation of the probes was performed basically
according to Gamas et al. [23]. For preparation of
the subtracted probe, 3 mg poly(A)
+
RNA from 24
h F. eumartii inoculated tubers were reversed-tran-
scribed using a mixture of oligodT
1218
and ran-
dom hexamers to prime the cDNA synthesis. The
poly(A)
+
RNA (10 mg) obtained from control
tubers (tubers in which PDA sterile disks were
kept for 24 h) was photobiotinylated. Next, a
subtracted hybridization step between biotinylated
poly(A)
+
RNA prepared from control tubers and
the single-stranded cDNA prepared from fungal-
infected tubers was performed. Finally, the sub-
tracted cDNA was converted to double strand
molecules, ligated to linkers [24] and amplied by
PCR. A control cDNA probe was also prepared
following the same procedure, starting from 3 mg
poly(A)
+
RNA from 24 h PDA sterile disks-
treated tubers, and avoiding the subtractive step.
Both cDNA probes were labeled with [h-
32
P]dCTP
by random priming [25], and used directly for the
screening of the potato tuber cDNA library.
About 500 000 plaques at a density of approxi-
mately 50 000 plaques/15 cm plate were screened.
Hybridizations were carried out according to
Gamas et al. [23]. Positive recombinant clones
were isolated by plaque purication using the
same hybridization conditions. In vivo excision
was carried out from the selected Uni-ZAP XR
cDNA clones, following the manufacturers in-
structions (Stratagene) and recombinant pBlue-
script SK() containing colonies were obtained
from phagemid infections of XL1-Blue strain
(Stratagene).
2.5. RNA isolation and Northern blot analysis
Frozen tissue (2 g fresh mass) was ground to a
ne powder in liquid nitrogen using a prechilled
mortar and pestle. Total RNA was isolated from
different potato samples using the guanidineHCl
A.V. Godoy et al. / Plant Science 152 (2000) 123134 126
extraction and LiCl precipitation described by
Laxalt et al. [26]. RNA samples (10 mg) were
electrophoresed on 1.2% formaldehydeagarose
gels and transferred onto nylon membranes (Hy-
bond N, Amersham) following standard proce-
dures [27]. Filters were hybridized to the
32
P-labeled insert isolated from the cDNA clone
containing the StCyP nucleotide sequence. A
sunower rRNA cDNA fragment (ENTS) [28] was
used as a probe to check whether different samples
have been loaded and transferred in equivalent
amounts. Additionally, the cDNA sequence of the
Kunitz-type proteinase inhibitor (PKPI) gene from
S. tuberosum was used as a control probe for the
experiments with MeJA and ABA treatments [29].
The probes were generated by random primed
labelling with [h-
32
P]dCTP [25].
Prehybridization (2 h) and hybridization
(overnight) steps were conducted at 42C in 50%
formamide, 5 SSPE (1 SSPE is 0.18 M NaCl,
10 mM NH
2
PO
4
, and 1m M EDTA (pH 7.7)), 5
Denhardts solution, 0.5% sodium dodecyl sul-
phate (SDS) and 50 mg/ml salmon sperm DNA.
The membranes were washed in 0.5 SSPE, 0.1%
SDS at 42C. The membranes hybridized with the
ENTS probe were washed in 0.1 SSPE, 0.1%
SDS at 50C. Autoradiographies were carried out
at 80C (AGFA Curix lm). Autoradiograms
were scanned on a Genius color-page HR5 scan-
ner and densitometric analysis was performed with
the TN-image Analysis Software 2.13 version.
Statistic analyses was performed by ANOVA fol-
lowed by Dunnett multiple comparison tests. A
value of PB0.05 was considered signicant.
2.6. DNA isolation and Southern blot analysis
Frozen tissue (2 g fresh mass) from potato
leaves was ground to a ne powder in liquid
nitrogen using a prechilled mortar and pestle. Ge-
nomic DNA was isolated essentially according to
Dellaporta et al. [30], digested with BamHI,
EcoRI, EcoRV or SacI, fractionated on 0.8%
agarose gels and transferred onto nylon mem-
branes (Hybond N, Amersham) according to stan-
dard procedures [27]. DNAs were hybridized
overnight to StCyP probe in 0.25 M NaH
2
PO
4
,
7% SDS, 1 mM EDTA, 1% bovine seroalbumin
and 10% dextran sulphate, at 65C. After washing
in 20 mM NaH
2
PO
4
, 1% SDS and 1 mM EDTA,
at 65C (three times, 20 min each) the lter was
exposed for 6 days at 80C on AGFA Curix
lm.
2.7. cDNA sequencing and analysis
The nucleotide sequence was determined on
both strands by the dideoxynucleotide chain termi-
nation method [31] using the universal pBluescript
primers on a automated laser uorescent sequenc-
ing apparatus (Pharmacia, LKB). Sequence data
were analyzed using University of Wisconsin Ge-
netic Computer Group Software (Program Man-
ual for the Wisconsin Package Version 8, Genetics
Computer Group, Madison, WI). Homology
searches in databases by the BLAST program
were carried out through the National Center for
Biotechnology Information web site (http://
www.ncbi.nlm.nih.gov).
3. Results
3.1. Isolation of subtracti6e cDNA clones and
DNA sequence analysis
A cDNA library was prepared from poly(A)
+
RNA extracted from 24 h fungal-infected potato
tubers. Of the clones 98.6% contained a cDNA
insert. The average size of the cDNA inserts was
approximately 1000 bp indicating that the library
constitutes a good source to select cDNAs. Differ-
ential screening (about 500 000 phage plaques were
screened) using PCR-amplied subtracted and
control cDNA probes yielded approximately 300
recombinant clones which strongly hybridized
with the subtracted cDNA probe. These clones
either gave no hybridization signal, or weakly
hybridized with the control cDNA probe. Twenty
positive clones were randomly selected for the
second and third selection rounds. Among them,
12 cDNA clones repeatedly showed the differential
hybridization pattern. After in vivo excision of the
pBluescript SK() plasmid from the Uni-ZAP
XR vector, one clone was subjected to DNA
sequence analysis (Fig. 1). The potato cDNA se-
quence contained a single open reading frame of
513 bp encoding a putative polypeptide of 171
amino acids which exhibited extensive homology
to previously described plant CyP. The predicted
amino acid sequence corresponds to a protein of
molecular weight 18 kDa. In addition to the open
A.V. Godoy et al. / Plant Science 152 (2000) 123134 127
reading frame, the cDNA sequence also contains a
28 bp of 5%-, and a 237 pb of 3%-untranslated
sequences (Fig. 1).
A comparison of the deduced amino acid se-
quence of StCyP with those of other plant CyPs is
presented in Fig. 2. The StCyP has its highest
sequence identity with cytosolic CyPs from
Solanum commersonii (96%) [15] and Lycopersicon
esculentum (95%) [3] (Fig. 2). Less identity exists
with other CyPs from Phaseolus 6ulgaris (83%)
[18] and with ROC1 (81%) [12] and ROC3 (81%)
[8] from A. thaliana. The high degree of identity
found among the various plant species supports
the idea that plant CyPs are highly conserved
through the vegetal kingdom.
3.2. Genomic Southern blot analysis
The copy number of the StCyP gene was esti-
mated by genomic Southern blot analysis. Potato
genomic DNA was digested with SacI, EcoRI,
BamHI and EcoRV, probed with the StCyP
cDNA and then washed under stringent condi-
tions. The hybridization pattern is shown in Fig. 3.
In the case of the EcoRV- and BamHI-digested
DNA, at least two and three fragments were re-
spectively hybridized to the cDNA probe suggest-
ing that more than one CyP gene might be present
in the potato genome. Due to the presence of
internal SacI and EcoRI sites in the cDNA probe,
the number of bands increased in the SacI- and
EcoRI-digested genomic DNA. In addition, the
presence of weak bands detected for EcoRI-,
BamHI- and Eco RV-digested DNA indicates the
existence of other CyP-related sequences. All this
evidences suggests that there is a StCyP gene
family in potato.
3.3. Expression of StCyP mRNA in tissues of
healthy potato plants
Northern blot analysis was performed to deter-
mine the pattern of StCyP gene expression in
tissues of healthy potato plants. For this total
RNA was extracted from young leaves, mature
leaves, stems, tuber buds and tubers. After hy-
bridizing with the StCyP cDNA probe, a single
transcript of approximately 1 kb was detected in
all the tissues assayed here (Fig. 4, upper panel).
This analysis revealed that StCyP mRNA accumu-
lated at very high levels in photosynthetic organs,
such as leaves and stems, and in developing or-
Fig. 1. Nucleotide sequence of the StCyP cDNA. The coding region is shown with the translated amino acid sequence. In the
3%-untranslated region, the putative polyadenylation signal is underlined. Brackets indicate restriction enzyme sites for SacI and
EcoRI.
A.V. Godoy et al. / Plant Science 152 (2000) 123134 128
Fig. 2. Alignment of the deduced amino acid sequences of the StCyP protein (this study) and other selected cytosolic CyP of
higher plant species. Identical amino acids in different plant CyP are indicated by asterisks (*). Dots (.) indicate conserved
substitutions. Gaps introduced to optimise alignments are indicated by dashes (-). Gene or EMBL Bank accession numbers are
shown in parenthesis for each sequence: S. commersonii, SSCCYP, (U92087), L. esculentum, TOMCYP (M55019), P. 6ulgaris,
PVCYCGNA, (X74403), A. thaliana, ROC3 (U40399), A. thaliana, ROC1 (L14844), A. thaliana (U32186) Brassica napus
(M55018). The comparison was carried out using the default parameters of the CLUSTALW program of the GCG package.
gans, such as tuber buds. A low level of StCyP
mRNA was, however, observed in the tubers.
These results are in agreement with those reported
by several other authors on the expression of CyP
genes from various plant species [21,29].
3.4. Effect of wounding and fungal infection on
the le6el of StCyP mRNA accumulation
The method used for infection of potato tubers
with the fungus F. eumartii involves wounding of
the inoculated tissue, therefore, in order to deter-
mine whether the expression of the StCyP gene is
at least partly associated with the response to
fungal infection and/or mechanical wounding
StCyP mRNA accumulation was compared in
both wounded tubers treated with sterile PDA
disk and in wounded plus infected potato tubers,
at different times after the onset of treatment (Fig.
5). The StCyP mRNA is barely detectable in
non-wound-non-inoculated tubers (Fig. 5B, lane
0). After wounding the level increases, reaching a
maximum at 24 h after wounding and then de-
creases (Fig. 5A, dashed bars and Fig. 5B, W). By
72 h after wounding, StCyP mRNA accumulation
was close to that initially observed at 4 h after
wounding, the shortest time analysed here.
Next, the effect of infection was analysed. In
contrast with what was observed in wounded tu-
bers, StCyP mRNA accumulation increased by
infection, its level being maintained up to the
latest time analyzed here (72 h after initial treat-
ment) (Fig. 5A, black bars and Fig. 5B, W+F).
Furthermore, StCyP transcript accumulation is
signicantly higher in wounded plus infected than
in wounded plus sterile PDA treated tubers at 14,
48 and 72 h after treatments (Fig. 5A and B).
From these results it is concluded that wounding
stimulates StCyP mRNA accumulation in potato
tubers and that fungal infection results in addi-
tional accumulation of the StCyP mRNA in
potato tubers. For this experiment it is important
A.V. Godoy et al. / Plant Science 152 (2000) 123134 129
to add that we did not nd differences in the
StCyP mRNA accumulation pattern between just
wounded and wounded plus PDA-treated tubers
(results not shown).
At this point it was interesting to investigate the
ability of F. eumartii CWF to induce StCyP
mRNA accumulation. Results of treatment with
this elicitor on the level of StCyP mRNA are
presented in Fig. 6(A). The ability of this prepara-
tion to induce potato defense responses has al-
Fig. 5. Time course of StCyP mRNA accumulation in potato
tubers in response to wounding and fungal infection. (A) The
histogram represents the average of at least three independent
measurements performed with different RNA preparations.
Dashed bars: wounded plus PDA-treated tubers, black bars:
wounded plus F. eumartii. Error bars represent the S.D.
Shared lower case letters (a and b) among bars indicate
statistically indistinguishable values at PB0.05. The amount
of hybridizing RNA measured in non-wound-non-inoculated
tubers has been taken as the 100% value. (B) Autoradiograms
from a single Northern blot experiment. The membrane was
hybridized with the StCyP cDNA probe and exposed for 1
week at 80C (upper panel). Next, the membrane was
washed and rehybridized with the rDNA probe to test equal
loading of total RNA/lane (lower panel). Lane 0, non-wound-
non-inoculated tubers. W, wounded plus PDA-treated tubers;
W+F, wounded plus F. eumartii.
Fig. 3. Genomic southern blot analysis of the StCyP gene.
Genomic DNA from S. tuberosum cv Spunta (a cultivated
tetraploide) was digested with SacI, EcoRI, BamHI and
EcoRV. Digested DNA was subjected to electrophoresis,
transferred to a nylon membrane and hybridized with the
32
P-labeled insert from the StCyP cDNA clone.
ready been reported [32]. As is shown in Fig. 6(A),
the treatment of potato tubers with the fungal
CWF preparations did not result in an StCyP
mRNA accumulation.
Considering that SA plays a central role in the
plant defense response to fungal attack [33], we
tested its effect on StCyP expression. As is shown
in Fig. 6(B), no increase in the amount of this
transcript was observed in SA-treated potato tu-
bers. However, expression of CyP genes from
other plant species has been reported to be in-
duced by SA treatments [10,15].
During the course of this work, StCyP gene
expression in the interaction of potato plants with
another fungal pathogen was analysed. We tested
the effect of sporangia suspension of P. infestans,
race 0, on potato leaves. Results presented in Fig.
Fig. 4. RNA blot analysis of the StCyP gene in different
tissues of healthy potato plants. Total RNAs were isolated
from tubers (T), young leaves (YL) mature leaves (ML),
stems (S) and tuber buds (B) of healthy plants, and hybridized
with the StCyP cDNA (upper panel). An rDNA probe was
used as loading control (lower panel). The molecular weight
of StCyP is indicated.
A.V. Godoy et al. / Plant Science 152 (2000) 123134 130
6(C) conrm the high constitutive level of StCyP
mRNAs previously observed in potato leaves (Fig.
4, lanes YL and ML). Inoculation with P. infes-
tans sporangia increased the StCyP mRNA levels,
although to a lesser extent than infection of tubers
with F. eumartii. From these results, it appears
that StCyP gene expression can be stimulated by
at least two fungal potato pathogens, F. eumartii
Fig. 7. Effect of treatment with MeJA or ABA on the
expression of the StCyP gene in potato tubers. Wound tubers
were inoculated with H
2
O, MeJA or ABA at the indicated
concentrations. Total RNAs (10 mg/lane) were separated on a
denaturing formaldehyde agarose gel, transferred onto a ny-
lon membrane, and hybridized with the StCyP probe (upper
panel). The same blot was hybridized with the cDNA probe
PKPI (middle panel). Hybridization with the rDNA probe
was used to conrm equal loading in all lanes (lower panel).
Fig. 6. Accumulation of StCyP mRNA in response to F.
eumartii CWF, treatment with SA and inoculation with P.
infestans. (A) Effect of fungal CWF on the accumulation of
the StCyP mRNAs. Total RNA was isolated from wound
potato tubers inoculated with sterile water (H
2
O) or F. eu-
martii CWF (CFW) at 15 h after treatment. (B) Effect of SA
treatment on potato tubers. Total RNA was isolated either
from wounded plus water (H
2
O)- or wounded plus SA-treated
(SA) tubers at the indicated times. (C) StCyP mRNA accu-
mulation in S. tuberosum leaves after inoculation with P.
infestans, race 0. Leaves were non-inoculated (lane 0) or
inoculated either with a P. infestans sporangia suspension
(210
3
sporangia/ml) or with sterile water as control. Total
RNA was isolated at the indicated times after inoculation.
Membranes were hybridized with the StCyP probe (A, B and
C, upper panels), or with the rDNA probe (A, B and C, lower
panels).
and P. infestans, and in different potato tissues,
tubers and leaves, respectively.
3.5. StCyP mRNA accumulation is induced by
MeJA and ABA
A role of the hormones ABA and jasmonic acid
(JA), and its derivative MeJA, as wounding signals
in potato plants has been demonstrated [3436].
Due to the nding that the expression of the
StCyP gene was induced in response to mechani-
cal wounding in potato tubers, it was important to
determine the effect of the application of ABA and
MeJA on the accumulation of the StCyP mRNAs.
Towards this end, Northern blot analysis of RNAs
obtained from tubers treated with increasing con-
centrations of MeJA (20100 mM) and ABA (10
100 mM) were performed. The results shown in
Fig. 7 (upper panel) indicate that both com-
pounds, MeJA and ABA, induce the accumulation
of StCyP mRNA in potato tubers. Higher levels
of StCyP mRNAs were found with increasing
hormone concentrations. The maximum concen-
trations of the diluents used for ABA and MeJA
applications (0.03% (v/v) ethanol and 0.1% (v/v)
DMF, respectively) did not affect the StCyP
mRNA levels (data not shown).
As a control, the same blot was hybridized with
the cDNA sequence corresponding to a Kunitz-
A.V. Godoy et al. / Plant Science 152 (2000) 123134 131
type proteinase inhibitor gene, the PKPI gene, for
which JA inducibility in potato tuber disks has
been described [29]. As is shown in Fig. 7 (middle
panel), MeJA- and ABA-treated potato tubers
showed increased levels of PKPI mRNA when
compared with sterile water-treated tubers.
3.6. Accumulation of StCyP mRNAs after
heat-shock treatment
To investigate the effect of other types of abiotic
stress on StCyP gene expression, tubers were ex-
posed to thermic stress. The effect of heat-shock
treatment on the StCyP mRNA level is shown in
Fig. 8. Heat-shock stress, over 4 and 8 h (Fig. 8,
lanes 2 and 3, respectively) at 42C, resulted in
accumulation of the StCyP transcript. Enhance-
ment of the level of StCyP mRNA was observed
in tubers kept for only 4 h at 42C. When potato
tubers stored at 4C were transferred and accli-
mated at 25C for 24 or 48 h however, no accumu-
lation of StCyP mRNA was observed (results not
shown). From these results it appears that expo-
sure to high temperature increases StCyP mRNA
levels in potato tubers.
4. Discussion
Cyclophilin was rst identied in 1984 as a
protein from mammalian thymocytes that speci-
cally binds to the immunosupressive cyclic unde-
capeptide cyclosporin A (CsA) [37]. Later in 1989,
it was reported that CyP is identical to the previ-
ously described peptidyl-prolyl cis-trans isomerase
or rotamase [1,38]. In view of their ability to
catalyse cis-trans isomerization of peptidyl-prolyl
bonds, it is not surprising that CyPs participate in
some stage of protein folding, i.e. by accelerating
the rate at which proteins fold into their native
conformation. Protein folding studies with car-
bonic anhydrase support the idea that CyPs can
function as chaperones [2]. Additionally, it was
reported that the CsACyP complex inhibits the
activity of calcineurin, a Ca
2+
-calmodulin protein
phosphatase, thereby blocking a Ca
2+
-dependent
signal transduction pathway in a variety of cells,
including human T lymphocytes [39,40], yeast [41]
and plant guard cells [42]. Thus, in addition to
their role in protein folding, CyPs might be impor-
tant in signal transduction processes.
Here we report the isolation and molecular
characterization of a cDNA clone encoding for a
cytosolic CyP of a S. tuberosum commercial culti-
var (cv Spunta). The StCyP genes represent a gene
family in the S. tuberosum genome. Except in
tubers, StCyP is expressed at high levels in all the
tissues of healthy potato plants examined here.
Northern blot experiments revealed that both
wounding and fungal infection stimulate StCyP
mRNA accumulation in potato tubers. However,
whereas wounding causes a transient accumulation
of StCyP mRNAs, fungal infection resulted in a
maintained accumulation of these transcripts. In
wounded tubers the full induction of the transcript
was found 24 h after wounding. Then, its accumu-
lation decreased to return to a basal level. Con-
trary to this, the accumulation of the StCyP
transcript progressively increased in fungal-in-
fected tubers (up to 72 h after infection, the latest
time studied here). Interestingly, neither treatment
with F. eumartii CWF nor treatment with SA had
any effect on the level of StCyP mRNA.
On the other hand, it is well known that JA and
ABA act as endogenous signals in the plant
wound response. Indeed, these compounds usually
accumulate after pest attack or mechanical
wounding, inducing the synthesis of defense-re-
lated proteins [3436]. The results presented here
indicate that exogenously applied MeJA or ABA
led to a signicant accumulation of StCyP mR-
NAs in potato tubers. Taken together, the ob-
served response of the StCyP gene after wounding
(or exposure to ABA or MeJA, compounds that
act as wound signals), fungal infection, and elicitor
or SA treatment allows us to conclude that the
Fig. 8. Effect of heat-shock on the accumulation of the StCyP
mRNA. Total RNA was prepared from control tubers (tubers
kept at 25C) (lane 1), and tubers that had been transferred to
42C for 4 and 8 h (lanes 2 and 3, respectively).The blot was
probed with the StCyP cDNA and autoradiographed at
80C (upper panel). The same blot was hybridized with the
rDNA probe (lower panel).
A.V. Godoy et al. / Plant Science 152 (2000) 123134 132
observed accumulation of StCyP mRNA in fun-
gal-infected potato tubers is dependent upon, and
a response to the wound produced during the
process of penetration of the pathogen in the host
tissue. This would explain the differences in
mRNA accumulation proles that are observed
after wounding or after wounding plus fungal
infection.
Clearly, CyP gene expression may be differen-
tially regulated by different stimuli, and in differ-
ent tissues of a given plant. Various types of
stresses, such as wounding, chemical treatment,
salinity and biotic stress produced by a virus have
been shown to induce CyP gene expression in
higher plant species [8,15,19,20]. Low temperature-
and heat-shock inducible CyPs have also been
described [19,20]. The results presented here indi-
cate that in addition to wounding or fungal infec-
tion, heat-shock treatment stimulates the
accumulation of StCyP transcripts. It is well
known that the expression of other types of stress-
related genes, the pathogenesis-related genes, is
induced by a wide range of environmental stresses
including biotic and abiotic stresses [4346]. Fur-
thermore, the phenomenon of induced resistance,
or systemic acquired resistance has been correlated
with the expression of this family of plant defense
genes. This induced resistance is effective against a
broad range of pathogens. Similarly, it can be
postulated that plants previously exposed to one
stress might have accumulated different stress-re-
lated proteins, including CyP, and in turn, they
might gain cross-protection against another type
of stress. The phytohormone ABA which plays a
central role in many physiological responses to
environmental conditions such as wounding,
drought and temperature [35,36,4749], could be
the common signal for the activation of various
stress responses in higher plants.
Recently, it has been reported that transcripts
corresponding to a CyP gene from a wild potato
species (the ScCyP gene from S. commersonii )
accumulate in wounded leaves [15]. No informa-
tion is available on ScCyP gene expression in
tubers. Interestingly, whereas ScCyp gene expres-
sion is stimulated in response to SA in leaves, SA
treatment does not affect StCyP gene expression
in tubers. These differences may reect the exis-
tence of different CyP genes which are differen-
tially regulated under stress conditions in tissues of
the potato plant.
To conclude, it appears that the expression of
CyP genes can be regulated by various environ-
mental stresses in higher plants, their expression
being differentially regulated in the different plant
tissues. Since higher plants require a variety of
proteins to carry out a series of processes in
response to the various stress conditions, CyPs
may well be involved in the correct folding of
defense-related proteins in each stress response.
Acknowledgements
We thank P. Heizmann (University of Lyon 1,
France) for providing the rDNA clone. We are
also grateful to K. Yamagishi and Y. Kikuta for
supplying the PKPI clone. Thanks also go to Dr
Lamattinas laboratory for RNA preparations
from P. infestans stressed leaves. This work was
supported by the International Foundation for
Science (IFS, Suecia), Consejo Nacional de Inves-
tigaciones Cient cas y Tecnicas (CONICET),
Fundacio n Antorchas, UNMDP-Argentina, and
Grant No. BIO97-0710 from the Comisio n Inter-
ministerial de Ciencia y Tecnolog a to BSS. AVG
and ASL were recipients of a fellowship from
UNMDP and CIC, respectively.
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