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Plant Science 152 (2000) 123134

Expression of a Solanum tuberosum cyclophilin gene is regulatedby 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 Biologa, Instituto de Inestigaciones Biologicas, Uniersidad Nacional de Mar del Plata, Funes 3250, cc 1245,

7600Mar del Plata, Argentinab Centro de Inestigacion y Desarrollo (CSIC), Barcelona, Jordi Girona 1824, 08034Barcelona, 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-amplified subtracted cDNA probe, a

clone encoding a cytosolic form of CyP, called StCyP (Solanum tuberosum 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 ofStCyP 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 findings 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 proteinswith an intrinsic enzymatic activity of peptidyl-

prolylcis -transisomerase (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 cereisiae 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

Abbreiations: ABA, absicic acid; CWF, cell wall carbohydrate

fractions; CyP, cyclophilin; CsA, cyclosporin A; ENTS, sunflower

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-

mersoniicyclophilin; StCyP, Solanum tuberosum cyclophilin.

* Corresponding author. Tel: +54-223-4753030; fax: +54-223-

4753150.

E-mail address:[email protected] (C.A. Casalongue)

0168-9452/00/$ - see front matter 2000 Elsevier Science Ireland Ltd. All rights reserved.

P I I : S 0 1 6 8 - 9 4 5 2 ( 9 9 ) 0 0 2 1 1 - 3


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A.V. Godoy et al./Plant Science 152 (2000) 123134124

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 identified, and five of

them appear to produce cytosolic proteins [8].

Plant CyP genes are stress-responsive as theirexpression 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 ArabidopsisCyP 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 theevents 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 solanif. 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 Solanum tuberosum 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. tuberosumwere 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 fluorescent light

(250 mol/m2/s), under a 14 h photoperiod. Four

to five 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 liquidnitrogen and stored at 80C. Potato buds from

previously induced sprouting tubers were also

collected.

F.eumartiiwas 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 fluorescent light (150

mol/m2/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 treatmentwith 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 methodcauses mechanical injury to the tissue (wounding).


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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, frozenin liquid nitrogen and stored at 80C.

For F. eumartiielicitor 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 2103 sporangia/ml

ofP. infestansusing a fine 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 wereinoculated 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 l ABA solution at a

final concentration of 10, 50 or 100 M. MeJA

(Sigma) was dissolved in N,N-dimethylformamide

(100 mM stock solution). For treatments of potato

tubers, 100 l sterile water MeJA dilutions at final

concentrations of 20, 50 or 100 M were used. To

test the effect of SA (Droguera Internacional),100 l of a 10 mM SA solution prepared in sterile

water just before use were applied. Control tubers

were treated with 100 l 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 usingthe 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 g poly(A)+

RNA from 24 h F. eumartii-infected potato

tubers.

Differential screening of the cDNA library witha polymerase chain reaction (PCR)-amplified 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 g poly(A)+ RNA from 24

h F.eumartiiinoculated tubers were reversed-tran-

scribed using a mixture of oligodT1218 and ran-

dom hexamers to prime the cDNA synthesis. The

poly(A)+ RNA (10 g) 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 amplified by

PCR. A control cDNA probe was also prepared

following the same procedure, starting from 3 g

poly(A)+ RNA from 24 h PDA sterile disks-

treated tubers, and avoiding the subtractive step.Both cDNA probes were labeled with [-32P]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 purification 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

fine powder in liquid nitrogen using a prechilled

mortar and pestle. Total RNA was isolated fromdifferent potato samples using the guanidineHCl


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extraction and LiCl precipitation described by

Laxalt et al. [26]. RNA samples (10 g) were

electrophoresed on 1.2% formaldehyde agarose

gels and transferred onto nylon membranes (Hy-

bond N, Amersham) following standard proce-

dures [27]. Filters were hybridized to the32P-labeled insert isolated from the cDNA clone

containing the StCyP nucleotide sequence. Asunflower 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 [-32P]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 NH2PO4, and 1m M EDTA (pH 7.7)), 5

Denhardts solution, 0.5% sodium dodecyl sul-

phate (SDS) and 50 g/l 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 film). 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 P0.05 was considered significant.

2.6. DNA isolation and Southern blot analysis

Frozen tissue (2 g fresh mass) from potato

leaves was ground to a fine powder in liquidnitrogen 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 NaH2PO4,

7% SDS, 1 mM EDTA, 1% bovine seroalbumin

and 10% dextran sulphate, at 65C. After washing

in 20 mM NaH2PO4, 1% SDS and 1 mM EDTA,at 65C (three times, 20 min each) the filter was

exposed for 6 days at 80C on AGFA Curix

film.

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 fluorescent 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 subtractie 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-amplified 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 thesecond 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 ofmolecular weight 18 kDa. In addition to the open


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reading frame, the cDNA sequence also contains a

2 8 b p o f 5-, and a 237 pb of 3-untranslated

sequences (Fig. 1).

A comparison of the deduced amino acid se-

quence ofStCyPwith 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 Lycopersiconesculentum (95%) [3] (Fig. 2). Less identity exists

with other CyPs from Phaseolus ulgaris (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 Eco RV, 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

internalSacI and Eco RI 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 Eco RI-,

BamHI- and Eco RV-digested DNA indicates theexistence 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 totalRNA 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 StCyPmRNA 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 forSac I andEcoRI.


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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. ulgaris,

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 leel 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 innon-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

significantly 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 inpotato tubers. For this experiment it is important


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to add that we did not find 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 ofStCyP 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 atP0.05. 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 1week 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 the32P-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 weightof StCyP is indicated.


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6(C) confirm 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 theStCyP gene in potato tubers. Wound tubers

were inoculated with H2O, MeJA or ABA at the indicated

concentrations. Total RNAs (10g/lane) were separated on a

denaturing formaldehyde agarose gel, transferred onto a ny-

lon membrane, and hybridized with the StCyP probe (upperpanel). The same blot was hybridized with the cDNA probe

PKPI (middle panel). Hybridization with the rDNA probe

was used to confirm 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 ofthe StCyP mRNAs. Total RNA was isolated from wound

potato tubers inoculated with sterile water (H2O) 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 (H2O)- 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

(2103 sporangia/ml) or with sterile water as control. Total

RNA was isolated at the indicated times after inoculation.

Membranes were hybridized with theStCyP probe (A, B and

C, upper panels), or with the rDNA probe (A, B and C, lowerpanels).

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 finding 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 StCyPmRNAs.

Towards this end, Northern blot analysis of RNAs

obtained from tubers treated with increasing con-

centrations of MeJA (20100 M) and ABA (10100 M) 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 withthe cDNA sequence corresponding to a Kunitz-


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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 afterheat-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 first identified in 1984 as a

protein from mammalian thymocytes that specifi-

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-prolylcis-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 wasreported that the CsACyP complex inhibits the

activity of calcineurin, a Ca2+-calmodulin protein

phosphatase, thereby blocking a Ca2+-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 acytosolic CyP of a S. tuberosum commercial culti-

var (cv Spunta). The StCyPgenes 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. Inwounded 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. eumartiiCWF 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 significant 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 elicitoror SA treatment allows us to conclude that the

Fig. 8. Effect of heat-shock on the accumulation of theStCyP

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 therDNA probe (lower panel).


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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 profiles 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 bepostulated 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 reflect the exis-

tence of different CyP genes which are differen-

tially regulated under stress conditions in tissues ofthe 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 ofdefense-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 preparationsfrom P. infestans stressed leaves. This work was

supported by the International Foundation for

Science (IFS, Suecia), Consejo Nacional de Inves-

tigaciones Cientficas y Tecnicas (CONICET),

Fundacion Antorchas, UNMDP-Argentina, and

Grant No. BIO97-0710 from the Comision Inter-

ministerial de Ciencia y Tecnologa to BSS. AVG

and ASL were recipients of a fellowship from

UNMDP and CIC, respectively.

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