Download Characterization of Cytosolic Cyclophilin from Guard Cells of Vicia

Plant Cell Physiol. 40(1): 53-59 (1999)
JSPP © 1999
Characterization of Cytosolic Cyclophilin from Guard Cells of Viciafaba L.
Toshinori Kinoshita and Ken-ichiro Shimazaki
Department of Biology, Faculty of Science, Kyushu University, Ropponmatsu, Fukuoka, 810-8560 Japan
The effect of immunosuppressant cyclosporin A (CsA)
on inward-rectifying K+-channels and biochemical analysis have indicated the presence of cyclophilin in guard cells
of Vicia faba. In this study, we identified a full-length
cDNA sequence, vcCyP, encoding cyclophilin (CyP), a
peptidyl-prolyl cis-trans isomerase of guard cell protoplasts (GCPs) from Viciafaba L. The deduced amino acid
sequence revealed that vcCyP contained 171 amino acid
residues and exhibited a strong similarity to previously described cytosolic CyP isoforms from other plants. vcCyP
had seven extra amino acid residues, which is a characteristic of the cytosolic form of plant CyPs. A complex of
recombinant vcCyP and CsA inhibited the phosphatase
activity of bovine calcineurin, a type 2B protein phosphatase, with a half-inhibitory concentration of 0.2 //M. Protein phosphatase activity was measured in the cytosolic
fraction of GCPs using a 32P-labeled myelin basic protein
(32P-MBP) and the activity was increased by a physiological concentration of Ca2+ (1/*M). This Ca2+-stimulated
phosphatase activity was inhibited by CsA, suggesting the
presence of both cytosolic CyP and calcineurin-like protein phosphatase in guard cells. Northern blot analysis
showed that the transcription level of vcCyP was much
higher in GCPs than in root and leaf tissues of Vicia.
This property of CyP has led to the extensive study towards
elucidating the cellular function of calcineurin in animals.
CyPs have peptidyl-prolyl cis-trans isomerase activity that
is inhibited by CsA and catalyze refolding of some denatured proteins indicating that CyPs are involved in protein folding and maturation in vivo (Gething and Sambrook 1992). In support of this hypothesis, evidence has
been presented that a CyP homologue, nina A, in Drosophila is required for proper maturation of a subset of
eye-specific retina proteins (Schneuwly et al. 1989). In
higher plants, the presence of cytosolic, chloroplast and
mitochondrial forms of CyPs has been reported, and
cDNAs for CyPs have been isolated from several higher
plants. The expression of CyPs is affected by environmental and stress conditions (Lippuner et al. 1994, Luan et al.
1994a, b, Marivet et al. 1994, 1995). Very recently, Arabidopsis CyPs were found to interact with both VirD2 and
VirE2 proteins, that have important functions in integrating the T-DNA into host plant cells, in the T complex of
Agrobacterium tumefaciens (Deng et al. 1998). However,
very little is known about the physiological role of CyPs in
higher plants.
Stomatal pores surrounded by guard cells regulate exchanges of CO2 and water between leaves and the atmosphere (Zeiger 1983, Assmann 1993). Since guard cells
respond to various environmental stimuli such as light, humidity, CO2, Ca 2+ , and plant hormones, guard cells have
been extensively investigated as an excellent model system
to elucidate the mechanisms for perception and transduction of signals. Opening of stomata is mediated by an accumulation of K+ through inward-rectifying K + channels
in the plasma membranes of guard cells, and K + accumulation is driven by an inside-negative electrical potential
across the plasma membrane (Assmann et al. 1985,
Shimazaki et al. 1986, Hedrich and Schroeder 1989). Inward-rectifying K+ channels have been shown to be inhibited by cytoplasmic Ca 2+ (Schroeder and Hagiwara
1989), and this Ca2+-dependent inhibition of the K +
channels is nullified by immunosuppressant CsA in guard
cell protoplasts (GCPs) from Vicia faba L. (Luan et al.
1993). Endogenous CyP in guard cells forms a complex
with CsA, which may be involved in Ca2+-dependent regulation of inward-rectifying K + channels by inhibiting the
calcineurin-like protein phosphatase. Calcineurin-like
phosphatase activity has been found in extracts from Vicia
epidermal peels in which guard cells are the only intact cell
type (Luan et al. 1993). However, CyPs, intracellular receptors for CsA, in guard cells have not been further
Key words: Calcineurin (EC 3.1.3.16) — Cyclophilin (EC
5.2.1.8) — Cyclosporin A — Guard cell — Stomata —
Viciafaba L.
Cyclophilin (CyP) (EC 5.2.1.8) is an abundant, highly
conserved protein present in various organisms (Schreiber
1991). CyP was first identified as a high affinity binding
protein for the immunosuppressive drug cyclosporin A
(CsA) (Handchumacher et al. 1984). The CyP-CsA complex suppresses the immune response through inhibition of
a Ca2+-dependent phosphoprotein phosphatase, calcineurin, a type 2B protein phosphatase (EC 3.1.3.16)
(Haendler et al. 1987, Liu et al. 1991, Alsh et al. 1992).
Abbreviations: CsA, cyclosporin A; CyP, cyclophilin;
EGTA, l,2-bis(2-aminophenoxy)ethylene N.N.N'.N'-tetiancetic
acid; GCPs, guard cell protoplasts; GST, glutathione S-transferase; MCPs, mesophyll cell protoplasts; OA, okadaic acid;
32
P-MBP, 32P-labeled myelin basic protein.
The nucleotide sequence reported in this paper has been
submitted to the DDBJ, EMBL, GenBank under accession number ABO 12947.
53
Cytosolic cyclophilin in guard cells
54
characterized. In this study, we determined a full-length
cDNA sequence encoding cytosolic CyP of guard cells
from Vicia faba L. and showed that the complex of cytosolic CyP and CsA inhibited the activities of both animal
calcineurin and Ca 2+ -dependent calcineurin-like protein
phosphatase in guard cells. We also showed that the transcription level of the CyP is much higher in guard cells than
in other tissues of the Vicia plant.
Materials and Methods
Plant materials and isolation of protoplasts—Vicia faba L.
(cv. Ryosai Issun) was cultured hydroponically in a green house
(Shimazaki et al. 1992). GCPs were isolated enzymatically from
the lower epidermis of 4- to 6-week-old leaves of Vicia faba as
reported previously (Gotow et al. 1984). Mesophyll cell protoplasts (MCPs) were prepared according to a method described
previously (Shimazaki et al. 1982).
Construction of the first-strand cDNA and circular cDNA
—RNA was extracted from GCPs of Vicia faba with acid
guanidinium thiocyanate-phenol-chloroform (ISOGEN; Nippon
Gene, Tokyo, Japan) according to the method reported by
Chomczynski and Sacchi (1987). The first-strand cDNA (25 ft\)
was synthesized from total RNA (10 fi%) of GCPs by AMV reverse
transcriptase (Takara, Tokyo, Japan) using Oligo(dT)i2-i8 as a
primer, and was used as a template for PCR. The circular
cDNA (25 ftl) was made from an aliquot of the first-strand
cDNA (12.5 fA) using two distinct enzymes. The 5' terminus of the
first-strand cDNA was phosphorylated with T4 polynucleotide
kinase (Takara, Tokyo, Japan) for 30 min at 37°C to increase the
efficiency of self-ligation, and the resultant 5'-phosphate of the
first-strand cDNA was self-ligated with T4 RNA ligase (Takara,
Tokyo, Japan) for 16 h at 14°C. The circular cDNA was used for
inverse PCR.
Isolation of PCR fragment and DNA sequencing—All PCR
procedures were carried out using a recombinant Taq DNA
polymerase (Takara, Tokyo, Japan) under normal conditions
(Maniatis et al. 1982). cDNA fragments of CyP were amplified
from the first-strand cDNA using degenerate oligonucleotide
primers (5-GGIGGIGARWSIATHTAYGG-3' and 5'-AARCAYGTNGTNTTYGAR-3'). PCR was carried out using 1.0/il of the
first-strand cDNA obtained as described above. Two primers (5ATTCCTGGACCGGTGTGCTT-3' and 5'-CGGAACCAACGGATCTCAGT-3') oriented in the reverse direction were designed
from the PCR-amplified fragment, and were used for inverse PCR.
The inverse PCR was carried out using 2.0 fA of the circular cDNA
obtained as described above. Two primers (5'-GTTCTCGTCAGCGAACTTGG-3' and 5-AGACTGACTGGCTCGACGG-3)
oriented in the reverse direction were designed from the PCRamplified fragment and were used for the nested PCR. Nested
PCR was carried out using 1.0 fA of the product of inverse PCR.
PCR products were cloned into a pCRII vector (Invitrogen, San
Diego, CA, U.S.A.). Sequences were determined from both
strands of the cDNA using A.L.F. red DNA sequencer (Pharmacia Biotech, Tokyo, Japan). Nucleotide and amino acid sequences were analyzed using the GENETYX software system
(Software Development Co., Tokyo, Japan).
Expression of recombinant VcCyP protein in Escherichia
coli—The cDNA fragment encoding the mature protein of the
VcCyP (47-562 bp) was amplified by PCR and cloned in frame
with glutathione S-transferase (GST) into the pGEX-2T plasmid
vector (Pharmacia Biotech, Tokyo, Japan). E. coli (JM109) cells
transformed with the plasmid having the fusion protein construct
were grown at 30°C in 2xYTA medium. When the culture
showed a turbidity of A6Oonm=0.8, isopropyl-6-D-thiogalactopyranoside at a final concentration of 0.1 mM was added to induce recombinant vcCyP. Cells were harvested 4 h after the induction and resuspended in PBS buffer. Purification of the
recombinant protein was performed according to the method of
Bulk and RediPack GST purification modules (Pharmacia Biotech, Tokyo, Japan).
Calcineurin assay—Activity of calcineurin was determined
using 32P-labeled myelin basic protein (32P-MBP) as a substrate.
MBP was phosphorylated with [y-32P]ATP by agarose-conjugated p44 MAP kinase (ERK1, UBI, New York, NY, U.S.A.),
and the phosphorylated product was prepared as reported previously (Chajry et al. 1996). The assay mixture (50/ul) contained 50
mM Tris-HCl (pH 7.5), 0.2 mM CaCl2, 6 mM MgCl2, 0.5 mM
dithiothreitol, 100//gmP 1 bovine serum albumin, 0.1/uM
calmodulin from bovine (Sigma), 0.1 ftM calcineurin from bovine
(Boehringer Mannheim, Tokyo, Japan), 2.5 fig 32P-MBP (6,000
cpm /Ug~'), and various concentrations of the recombinant vcCyP
and CsA complex. The recombinant vcCyP protein and CsA were
premixed at a molar ratio of 1 : 5 and incubated at 4°C for 2 h
before the assay. The reaction was allowed to proceed at 30°C for
10 min and was terminated by the addition of 200 fA of 20% trichloroacetic acid to the mixture. After centrifugation at 10,000 x g
for 10 min at 4°C, the released 32P; in the supernatant was determined on a Beckman LS65O0 system.
Protein phosphatase assay in guard cell extract—A cytosolic fraction of GCPs was obtained according to the previous
method with slight modifications (Kinoshita et al. 1993). Protoplast suspensions (1 mgmP 1 protein) were mixed with the same
volume of 100 mM HEPES-KOH (pH 7.4), 4 mM l,2-bis(2aminophenoxy)ethylene N,N,N,./V-tetraacetic acid (EGTA), 2
mM dithiothreitol, 200/ugml~1 bovine serum albumin, 20 fig
ml" 1 leupeptin, 1 mM phenylmethylsulfonyl fluoride. After keeping these suspensions on ice for 2 min, the protoplasts were ruptured using a Teflon hand-held homogenizer. Homogenized
protoplasts were centrifuged at 10,000 x g for 10 min. The resulting supernatant as a cytosolic fraction was used for protein
phosphatase assay. The supernatants (20 /il) were preincubated in
the presence and absence of okadaic acid (OA), Ca 2+ , and CsA at
different concentrations for 10 min at 24° C, and then dephosphorylation was started by addition of 20(A of 0.5 mgml" 1 32 PMBP (6,000 cpm fig'1). Ca 2+ concentration was calculated using
the dissociation constants of EGTA chelates of Ca 2+ and Mg 2+
(Sillen and Martell 1971). The reaction and determination of activity were the same as described above.
Northern blot analysis—Northern blot hybridization was
performed according to the standard procedure of a Digoxigenin
Luminescent Detection Kit (Boehringer Mannheim, Tokyo,
Japan). Total RNA was extracted from GCPs, MCPs, leaves and
roots of Vicia faba with ISOGEN (Nippon Gene, Tokyo, Japan).
A cDNA from 14 bp to 856 bp of VcCyP was obtained by PCR
using a cDNA-containing adapter primer and was used as a
probe. The cDNA-containing adapter primer was synthesized
from the mRNA of GCPs using Oligo(dT)-containing adapter
primer (GIBCO BRL, MD, U.S.A.).
Results
Isolation of cDNA fragments of CyP—We designed
two degenerate oligonucleotide primers from conserved
Cytosolic cyclophilin in guard cells
amino acid sequences (GGESIYG and KHVVFG) of CyPs
(Fig. 1). These primers were used for amplification of a
first-strand cDNA template which was made from Vicia
GCPs, by PCR. We obtained PCR-amplified fragments of
170 bp in length, and sequenced twenty of these products. All fragments had identical nucleotide sequences and
contained a CyP-related sequence.
Determination of a full-length sequence of CyP by
inverse PCR—A full-length cDNA could not be isolated by
55
screening of the AZAPII cDNA library from Vicia GCPs
with the amplified fragment of 170 bp as a probe. Thus, a
circular cDNA was made from the first strand cDNA, and
a cDNA encoding a 5'- and 3'-region of CyP was obtained from the circular cDNA by the inverse PCR (see
Methods). Four primers in the reverse direction of usual
orientation were designed on the basis of a 170 bp PCRamplified fragment for the inverse and nested PCRs
(Fig. 1). We obtained a PCR product of 765 bp in length.
TTTTACCCATTACGATCTGATTCAACCACTTCTCAAACCCTAAGCCATGTCAAACCCCAA
---------------------M S N P K
60
5
AGTTTTCTTCGATATGACCGTCGGCGGCCAAAACGCTGGACGCATCATCTTTGAGCTTTT
V F F D M T V G G Q N A G R I I F E L F
120
25
TGCCGATGTCACTCCCAGAACCGCTGAGAATTTCCGTGCTCTCTGCACCGGCGAGAAAGG
A D V T P R T A E N F R A L C T G E K G
180
45
AGTCGGTCGTAGCGGCAAGCCACTCCACTTCAAGGGATCCTCCTTCCACCGTGTGATCCC
V G R S G K P L H F K G S S F H R V I P .
240
65
TAACTTCATGTGCCAGGGAGGTGACTTCACCGCCGGAAACGGCACCGGAGGAGAATCGAT
N F M C Q G G D F T A G N G T
|G G E S I
300
85
CTACGGTTCCAAGTTCGCTGACGAGAACTTCATCAAGAAGCACACCGGTCCAGGAATCTT
Y G| S K F A D E N F I K K H T G P G I L
360
105
ATCCATGGCGAACGCTGGACCCGGAACCAACGGATCTCAGTTCTTCATCTGCACTGCCAA
S M A N A G P G T N G S Q F F I C T A K
420
125
GACTGACTGGCTCGACGGGAAACACGTCGTGTTCGGTCAGGTTGTCGATGGATTGAACGT
T D W L D G |K H V V F (f| Q V V D G L N V
480
145
TGTGAGGGATATTGAGAAGGTTGGATCTGGCTCTGGCAAGACCTCTAAGCCTGTTGTGAT
V R D I E K V G S G S G K T S K P V V I
540
165
CGCCAATTGTGGACAACTGTAGATCATACTGTTTTGGCGTTTTTTAAACTGGTGGCTCTG
A N C G Q L *
600
171
ATTTTTAATTTGATCTCAAATTCTAGTTGTGTTTTTCTGATTCGCGTCGTTTTACTTTTC
660
TCGTGTTAGGGTTTGTGGTTGTGGTGCTCTATGGATCCTATTGAACCCTCCCTTTTTAAT
720
TTTCTGTTACTGTTACTATCCGTATGAAATTAGTAGTTGAGTGAGTTGAGTCTTGTTATG
780
CTGAACATTAAAATGATGAGCTACCTTCAATAAATTAGATTTTATTCCTAAAAAAAAAAA
840
AAAAAAAAAAAAAAAA
856
Fig. 1 Nucleotide (upper) and deduced amino acid sequence (lower) of vcCyP. In frame termination codon within the 5'-untranslated sequence is double underlined. Asterisk shows termination codon. The amino acid sequences corresponding to degenerate oligonucleotide primers and nucleotide sequences corresponding to primers for the inverse and nested PCRs are boxed and underlined,
respectively. Nucleotide sequences corresponding to primers for PCR using cDNA-adapter primer (see Methods) are dash underlined. Within the 3'-untranslated sequence, a direct repeat is indicated by bold letters.
56
Cytosolic cyclophilin in guard cells
Sequence analysis revealed that the 765 bp fragment contained a 5'- and 3'-region of CyP. From these sequences,
vcCyP (cDNA sequence for cytosolic CyP from Viciafaba)
was determined (Fig. 1). vcCyP was 856 bp in length and
contained a 513 bp open reading frame, encoding a putative polypeptide of 171 amino acids with a predicted molecular mass of 18,065. The deduced amino acid sequence
encoded by vcCyP had a strong similarity to the previously described cytosolic CyP isoforms of other plants. vcCyP
is closely related to cytosolic CyPs from Phaseolus vulgaris, Digitalis lanata, and Arabidopsis thaliana (84-89%
identity). However, VcCyP shares only 70% and 58% se-
vcCyP
P. vulgaris
D. lanata
A. thaliana
Human
pCyP B
quence identities respectively with human cytosolic CyP
and pCyP B, a chloroplast-localized CyP of Viciafaba L.
(Fig. 2). VcCyP contained a seven amino acid insertion
(position 48-54) that appears to be a characteristic of plant
cytosolic CyP (Lippuner et al. 1994, Buchholz et al. 1994).
On the basis of sequence similarities we conclude that
vcCyP encodes a cytosolic form of CyP in guard cells.
Inhibition of calcineurin by vcCyP and CsA complex—We expressed GST-vcCyP fusion protein in E. coli
cells and purified recombinant vcCyP. Purified recombinant vcCyP showed a single band (19 kDa) on Coomassie
Brilliant Blue (CBB)-stained SDS-polyacrylamide gel
1:-MSNPKVFFDMTVGGQNAGRIIFELFADVTPRTAENFRALCTGEK
• -L • • • AT • <
T
. .V..
PC . .VM. Y
V.K.
1:MAT
Y.
. .VM.i Y • • T • • E • • •
KV.K
1:-.V..T... I A . D . E P L . .VS
• V S . . ...KV.K...
S. .EK
VIG..G.AV.K.V...KT.S..AK
78:AKVTS.I...IEI..ES
**
* * ***
**
* **
vcCyP
P. vulgaris
4 5 rGVGRSGKPLHFKGSSFHRVIPNFMCQGGDFTAGNGTGGESIYGSK
45:
V
I
A.
U m
4 j t a « « J ! \ l « * « « « x « t «
.£ cz/icz Ccz
rx O • • _L • X * y •
Human
pCyP B
45: .F.
123:.Y.* *
A • • • • • •
\ 3• • • • • • • • • • • • • • • • • • • • •
A .•
• • • • . ] . • • • • • • • • • • & • • • • • • • • • • • • • • • • • • • * • • •
Y. ..C...I..G..
RH
K....E.
YQ..F...I
I
E
V
** *** ** ** ****** ***** **** *
vcCyP
90:FADENFIKKHTGPGILSMANAGPGTNGSQFFICTAKTDWLDGKHV
P. vulgaris 90:
V
T..E
D. lanata
90:
V
E..S
A • tho.HB.ns 9 1 t . K » . • • • • • • • • • • • • • • • • • • A N • • • • • • • • • • E * * o « « * * « « *
Hu.ni3.ri
pCyP B
oo5»E«»*»»Li«»»»»»*« •••••••£<• ••••••••••••£«•••••••
1 6 1 : . E . . S . D L . . V . . .V
*
vcCyP
P. vulgaris
D. lanata
A. thaliana
Human
pCyP B
** *
** *** * * * * * * *
N
**********
VP.P
NR. .
* ***
**
135 : VFGQWDGLNWRDIEKVGSGSGKTSKPWIANCGQL
135:
E..D..K
AR. .A. .D. . . .S
135:
E.MD. . .A
Q. . . .A
D. . .IC
136:
E
D. .R
D.. .IS
128: . . .K.KE.M. I.EAM.RF. .RN
KIT. .D
E
206:...H.IE..D..KQL.SQETSKLDN.PKKPCKIAKSGELPLDG
*** * * • *
*
Fig. 2 Sequence alignment of CyP protein sequences. The amino acid sequence of vcCyP deduced from the corresponding cDNA
sequence is aligned with CyPs of Phaseolus vulgaris (X74403), Digitalis lanata (Y0832O), ROC3 from Arabidopsis thaliana (Chou and
Gasser 1997), human (P05092), and pCyP B from Viciafaba L. (Luan et al. 1994b). Dots indicate amino acids which are identical to
the vcCyP sequence, and dashes indicate gaps introduced to allow for optimal alignment of the sequences. Asterisks show those
positions where all six sequences are identical. A computer-assisted homology search of vcCyP was performed with a DDBJ homology system using the program BLAST version 1.4.9 (Altschul et al. 1990).
Cytosolic cyclophilin in guard cells
(Fig. 3A). A complex of recombinant vcCyP and immunosuppressant CsA was produced by preincubation for a
sufficient time, and the complex inhibited bovine calcineurin activity at a half-inhibitory concentration of 0.2 fjM
(Fig. 3B). This value is comparable to that in inhibition of
calcineurin by the complexes of CsA with CyPs of mammalians and plants (Haendler et al. 1987, Liu et al. 1991,
Alsh et al. 1992, Luan et al. 1994b). The recombinant
vcCyP protein and CsA alone had no effect on the calcineurin activity.
Inhibition of Ca2*-dependent protein phosphatase
activity in guard cells by CsA—Protein phosphatase activity was determined using 32P-MBP as a substrate in the
cytosolic extract from Vicia GCPs. We tested the effect of
OA, an inhibitor of type 1 and type 2A protein phosphatase, on this phosphatase activity. The phosphatase activity was inhibited by OA at 0.1 /xM and the extent of inhibition was the same at a higher concentration of OA
(Fig.4A). The OA-sensitive protein phosphatase activity
comprised 65% of the total phosphatase activity in this
experimental condition. The phosphatase activity was increased by a physiological concentration of Ca 2+ (1 fiM)
in the presence of OA, and this Ca2+-stimulated, OAinsensitive phosphatase activity was suppressed by CsA
(Fig.4B). This indicates that a cytosolic fraction of GCPs
contains Ca2+-dependent calcineurin-like protein phosphatase, and that endogenous cytosolic CyP has the ability
to inhibit calcineurin-like protein phosphatase activity in
the presence of CsA. CsA had no effect on the phospha-
94
67
43
«.
""ITT.'
B
12
°
T
1
3
LI
§
2
o ol control)
1
kDa
20.1
14.4
200
T
!
fso 1
(0
ta
3%
li
1
L
.
8
1
i
6 01"
40;
'.
T
T
ri
i—i
i
i
•
;
©
o;
0
•:
0
0.00
I
•m
[
i 20-s
I!
g 100
0.01
0.1
I
i
CsA
-
-
+
+
Fig. 4 Protein phosphatase activity in guard cells. (A) OAinhibition of protein phosphatase activity in cytosolic fraction of
GCPs. Phosphatase activity was determined using 32P-MBP as a
substrate in the presence and absence of different concentrations
of OA. Relative activity is presented as a percentage of control
activity. Vertical bars represent standard errors of three separate experiments. (B) Effect of Ca 2+ and CsA on the OA-insensitive protein phosphatase activity. Reaction mixture contained OA
at 1 ftM. +Ca 2 + , 1 fiM; +CsA, 5 ftM. Other conditions were the
same as in (A).
tase activity without Ca 2+ .
Northern blot analysis of vcCyP—To determine the
transcription level of vcCyP in mature plants, a Northern
blot analysis of total RNA from GCPs, roots, MCPs and
leaves was conducted with cDNA of vcCyP as a probe. As
shown in Figure 5, VcCyP hybridized to a single mRNA
band of 850 bp in length in each lane. The transcription
level of vcCyP was much higher in GCPs than in roots and
T
r
.-
i
. j
I" 60
30
B
100 »
[
i
i~
14
I °
2
8 °
850 b p i
i
1
j
1
{
i |
ir
0 0.05 0.1 0.2 0.3 0.4 0.5 1.0 %.
[vcOyP - CsA| iM
'i
•&
u
im
GCP
Root
MCP
Leaf
Fig. 3 (A) Purification of recombinant vcCyP protein from
E. coli cells. The over-expression of GST fusion protein in E. coli
(43 kDa, lane 2), 0.5 /Jg of purified recombinant vcCyP protein
Fig. 5 Northern blot hybridized with vcCyP cDNA clone. Each
(19 kDa, lane 3), and molecular mass marker proteins (lane 1)
lane contained an equal amount (20^g) of total RNA isolated
were analyzed by SDS-PAGE and detected by CBB staining.
from GCPs, roots, MCPs and leaves. (A), blot hybridized with a
(B) Inhibition of calcineurin activity by a CyP-CsA complex.
842 bp of the vcCyP cDNA clone. (B) shows the amount of RNA
32
Calcineurin activity was determined using P-MBP as a subin each lane that has been transferred onto membrane. The gel
strate in the presence and absence of different concentrations of
was stained with ethidium bromide, and was visualized with a UV
the complex of recombinant vcCyP and CsA. vcCyP and CsA
illuminator. (C) shows the relative amount of transcript. Level of
individually added to the reaction mixture at 1 ftM and 5/^M, re- transcript was determined densitometrically. Relative values are
spectively. Relative activity is presented as a percentage of control
presented as the percentages of the transcript level in GCPs. Exactivity. Vertical bars represent standard errors of three sepaperiments repeated two times on different occasions gave similar
rate experiments.
results.
Cytosolic cyclophilin in guard cells
58
leaves. The amount of vcCyP mRNA in GCPs was approximately five- and fifteen-fold larger than those in root
and leaf tissues, respectively (Fig. 5c). The transcription of
pCyP B, a chloroplast-localized CyP with a molecular
mass of 21 kDa in Vicia leaf, is induced by heat shock at
37°C for 4 h (Luan et al. 1994 b). The same heat treatment had no effect on the transcription of vcCyP in Vicia
leaves (data not shown).
Discussion
In this study, we determined a full-length cDNA sequence, vcCyP, encoding CyP in guard cells of Vicia faba
L. Since all twenty CyP fragments, obtained by PCR using
degenerate oligonucleotide primers, contained identical sequences, vcCyP is most likely to be the major CyP in guard
cells. The primary sequence revealed that vcCyP encoded
the cytosolic form of CyP in guard cells, and the deduced
molecular mass of putative protein was 18 kDa. Previous
studies using the CsA affinity matrix have shown that
CyPs, with molecular masses of 18 and 21 kDa proteins,
are present in the leaf extracts of Vicia faba L. (Luan et al.
1993, 1994a, 1994b). The vcCyP identified in this study in
guard cells is very similar to the 18 kDa CyP found biochemically in Vicia leaf tissue. By contrast, the 21 kDa CyP
in Vicia leaf tissue is localized in chloroplasts, and was induced by heat shock (Luan et al. 1994b).
To isolate the 5 - and 3'-regions of CyP, we carried out
the inverse PCR using the circular first-strand cDNA since
our cDNA library from Vicia GCPs had no full-length
cDNA of CyP. In general, both 5'- and 3'-rapid amplification of cDNA ends are needed to isolate the 5'- and.3regions of a cDNA fragment. However, the inverse PCR
made it possible to obtain both 5'- and 3'-regions of
cDNA at the same time, and the circular cDNA could easily
be made from an aliquot of the first-strand cDNA. This
method is convenient and useful for cells and tissues that
are difficult to isolate in sufficient amounts such as guard
cells.
Inward-rectifying K + channels have been shown to act
as a major pathway for K + uptake during stomatal opening
and can be inhibited by cytoplasmic Ca 2+ (Schroeder and
Hagiwara 1989). This Ca 2+ -dependent inhibition of the
K + channels is abolished by immunosuppressant CsA in
GCPs from Vicia faba L. (Luan et al. 1993), indicating that
endogenous CyP in guard cells forms a complex with CsA
and the complex inhibits inward-rectifying K + channels by
inhibiting the calcineurin-like protein phosphatase. We
first investigated an inhibitory effect of the complex on
calcineurin activity in vitro (Fig. 3). Recombinant vcCyP
was generated in E. coli as a fusion protein of GSTvcCyP using an isolated cDNA fragment encoding a mature protein of vcCyP, and purified. The complex of
recombinant vcCyP and CsA was shown to inhibit animal
calcineurin (Fig. 3). Calcineurin-like activity in epidermal
guard cell extract has been found in the presence of 1 mM
Ca 2+ (Luan et al. 1993). We confirmed the calcineurinlike activity in guard cells at a physiological concentration of Ca 2+ at 1 jiM in this study. This Ca2+-stimulated
phosphatase activity was suppressed by the sole addition of
CsA to the cytosolic fraction of GCPs, indicating that
GCPs contain cytosolic CyP and the complex of endogenous CyP and exogenous CsA inhibits the calcineurinlike phosphatase activity in guard cells (Fig. 4).
A transcript of cytosolic CyP was distributed in mesophyll cells, guard cells and root cells. However, the transcript was found in a much larger amount in guard cells
than in root and mesophyll cells (Fig. 5). The significantly higher amount of the transcript of cytosolic CyP in
GCPs is due possibly to the induction of the transcript
during the isolation procedure of GCPs by cellulolytic enzymes under a high osmotic pressure, because various types
of stress treatments including heat, cold, salts, chemical
and wounding treatments induce the gene expression of
CyPs (Lippuner et al. 1994, Luan et al. 1994a, b, Marivet
et al. 1994, 1995). However, this may not be the case. The
amount of vcCyP transcription products was similar in
MCPs and leaf (Fig. 5) although MCPs were isolated with
cellulase under high mannitol concentration. In general, a
large amount of CyP mRNA is found in developing and
young tissues of plants (Marivet et al. 1994, 1995). The
much higher content of CyP mRNA in guard cells may be
related to protein degradation and synthesis that proceed
very actively in these cells, and that the CyP acts as a
chaperone-like molecule in the folding and maturation of
proteins synthesized de novo. Interestingly, Fukuda et al.
(1998) have found that microtubules in mature guard cells
increase in number and orient in a radial manner in the
daytime and decompose from evening to midnight, andthey suggested that these diurnal dynamic changes are
similar to the typical pattern observed in the development and differentiation of immature guard cells. Furthermore, guard cells seem to have a higher activity of protein synthesis than mesophyll cells in the yellow leaves in
particular, because the photosynthetic electron transport
system in guard cells remains active in falling leaves of
Ginkgo biloba even if the mesophyll counterpart has lost
its chlorophyll (Zeiger and Schwartz 1982).
From these observations, we conclude that vcCyP encodes the major type of cytosolic CyP in guard cells and
that CyP can inhibit both animal calcineurin and plant
calcineurin-like protein phosphatase in the presence of
CsA. This should help elucidate the functional role of
calcineurin, a type 2B protein phosphatase, using the immunosuppressant CsA in guard cells, although the physiological role of CyPs in plant cells remains to be clarified.
Cytosolic cyclophilin in guard cells
We thank Drs. J. Iwashita and K. Kusumi, Kyushu University, for their technical advice on the phosphatase assay and
Northern blot analysis. This work was supported in part by a
grant from the Ministry of Education, Science, Sports and Culture of Japan, No. 10740371 to T.K. and a Grant-in-Aid for
Scientific Research Priority Areas (No. 10170224) to K.S.
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(Received August 18, 1998; Accepted October 29, 1998)