Characterization of Chloroplast Clp proteins in Arabidopsis: Localization, tissue specificity and stress responses.

Abstract

The ATP-dependent Clp protease is one of the newly identified proteolytic systems in plant organelles that incorporate the activity of molecular chaperones to target specific polypeptide substrates and avoid inadvertent degradation of others. We describe new nuclear-encoded ClpC (ClpC1) and ClpP (ClpP3-5) isomers in Arabidopsis thaliana that raise the total number of identified Clp proteins to 19. The extra Clp proteins are localized within the stroma of chloroplasts along with the ClpD, -P1 and -P6 proteins. Potential differential regulation among these Clp proteins was analysed at both the mRNA and protein level. A comparison between different tissues showed increasing amounts of all plastid Clp proteins from roots to stems to leaves suggested the greatest abundance of proteins was in chloroplasts. The increases in protein were mirrored at the mRNA level for most ClpP isomers (ClpP1, -3, -4 and -6) but not for the three Hsp100 proteins (ClpC1, -C2 and -D) and ClpP5, which exhibited little change in transcript levels, suggesting post-transcriptional/translational regulation. Potential stress induction was also tested for all chloroplast Clp proteins by a series of brief and prolonged stress conditions. Short-term moderate and severe stresses (desiccation, high salt, cold, heat, oxidation, wounding and high light) all failed to elicit significant or rapid increases in any chloroplast Clp protein. However, increases in mRNA and protein content for ClpD and several ClpP isomers did occur during long-term high light and cold acclimation of Arabidopsis plants. These results reveal the great complexity of Clp proteins within the stroma of plant chloroplasts, and that these proteins, rather than being rapidly induced stress proteins, are primarily constitutive proteins that may also be involved in plant acclimation to different physiological conditions.

Full text

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PHYSIOLOGIA PLANTARUM 114: 92–101. 2002
Printed in Denmark – all rights reserved
Copyright C Physiologia Plantarum 2002
ISSN 0031-9317
Characterization of Chloroplast Clp proteins in Arabidopsis:
Localization, tissue specificity and stress responses
Bo Zhenga, Tami Halperinb, Olga Hruskova-Heidingsfeldovac, Zach Adamband Adrian K. Clarkea,1,*
aUmeå Plant Sciences Centre, Department of Plant Physiology, Umeå University, SE-901 87 Umeå, Sweden
bDepartment of Agricultural Botany, Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot 76100, Israel
cDepartment of Biochemistry, Institute of Organic Chemistry and Biochemistry, Flemingovo na ´m.2, C2-16610 Prague 6, Czech Republic
1Present address: Botanical Institute, Göteborg University, Box 461, SE-405 30 Göteborg, Sweden
*Corresponding author, e-mail: adrian.clarke/botinst.gu.se
Received 19 June 2001; revised 31 August 2001
The ATP-dependent Clp protease is one of the newly identified
proteolytic systems in plant organelles that incorporate the ac-
tivity of molecular chaperones to target specific polypeptide
substrates and avoid inadvertent degradation of others. We de-
scribe new nuclear-encoded ClpC (ClpC1) and ClpP (ClpP3–
5) isomers in Arabidopsis thaliana that raise the total number
of identified Clp proteins to 19. The extra Clp proteins are lo-
calizedwithinthestromaofchloroplastsalongwiththeClpD,–
P1 and –P6 proteins. Potential differential regulation among
these Clp proteins was analysed at both the mRNA and protein
level.Acomparisonbetweendifferenttissuesshowedincreasing
amountsofallplastidClpproteinsfromrootstostemstoleaves
suggested the greatest abundance of proteins was in chloro-
plasts. The increases in protein were mirrored at the mRNA
level for most ClpP isomers (ClpP1, ª3, ª4 and ª6) but not
forthethreeHsp100proteins(ClpC1,–C2and–D)andClpP5,
Introduction
The protein environment inside plastids is one of the
more dynamic ones in plant cells, and yet much remains
unclear about the mechanisms by which these complex
protein interactions are regulated. Molecular chaperones
are a ubiquitous group of regulatory proteins well
known to directly influence the structure and function
of many polypeptides. They assist the folding, assembly
and translocation of numerous cellular proteins during
both normal and adverse growth conditions. Certain
chaperones also target irreversibly damaged proteins for
proteolysis, thereby preventing the accumulation of po-
tentially cytotoxic polypeptides (Parsell and Lindquist
1993). Integral to this degradative process is the direct
interaction between certain molecular chaperones and
proteases, many of which are dependent on ATP. In plas-
tids of higher plants, three such chaperone/proteolytic
Physiol. Plant. 114, 2002
92
which exhibited little change in transcript levels, suggesting
post-transcriptional/translational regulation. Potential stress
induction was also tested for all chloroplast Clp proteins by a
series of brief and prolonged stress conditions. Short-term
moderate and severe stresses (desiccation, high salt, cold, heat,
oxidation, wounding and high light) all failed to elicit signifi-
cantorrapidincreasesinanychloroplastClpprotein.However,
increases in mRNA and protein content for ClpD and several
ClpP isomers did occur during long-term high light and cold
acclimation of Arabidopsis plants. These results reveal the
great complexity of Clp proteins within the stroma of plant
chloroplasts, and that these proteins, rather than being rapidly
induced stress proteins, are primarily constitutive proteins that
may also be involved in plant acclimation to different physio-
logical conditions.
mechanisms have so far been identified, of which all are
homologous to well characterized proteases from eubac-
teria (Adam et al. 2001). The DegP protease extrinsi-
cally attached to the lumenal and stromal surfaces of
thylakoid membranes, and the FtsH proteases also
located on the stromal surface, both incorporate chap-
erone and proteolytic activities within the one polypep-
tide (Lindahl et al. 1996, Itzhaki et al. 1998, Haußühl
et al. 2001). The soluble Clp protease, however, has sep-
arate chaperone and proteolytic subunits, and like the
other two proteolytic systems, little is yet understood
about their precise functions in plastids.
Most knowledge about the Clp protease is derived
from extensive studies on the model enzyme from Esch-
erichia coli (reviewed by Porankiewicz et al. 1999). The
ATP-dependent Clp protease is a large complex of two

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oligomeric subunits: a serine-type protease (ClpP) and a
regulatory ATPase (ClpA or –X). In E.coli, the Clp pro-
tease has a central core comprised of two face-to-face
heptameric rings of ClpP, which are flanked at one or
both sides by a single hexameric ring of ClpA or –X
(Grimaud et al. 1998). In an ATP-dependent fashion,
the regulatory subunits selectively bind and unfold the
polypeptide targeted for degradation and then translo-
cate it into the central cavity of the barrel-like ClpP
complex (Wang et al. 1997, Singh et al. 2000). Within
this proteolytic chamber, ClpP rapidly degrades the
polypeptide into smaller fragments that then diffuse out.
Because of the different protein specificities of the regu-
latory subunits, the ClpAP and ClpXP complexes can
be considered as distinct proteases.
ClpA and ClpX are members of a new family of mol-
ecular chaperones known as Hsp100/Clp. The family can
be separated into two broad groups, with proteins in the
first (ClpA-E) having two different ATP-binding do-
mains (ATP-1 and ATP-2) while those in the second
(ClpX, –Y) have only one such domain (Schrimer et al.
1996). The five types of proteins in the first group are
distinguished by conserved amino acid sequences, length
of the spacer region separating the ATP-1 and -2 do-
mains, and the organism they originate from. Hsp100
proteins are thought to function with a common mech-
anism of dismantling oligomeric protein complexes, as
has been shown for E.coli ClpA and –X (Wickner et al.
1994, Levchenko et al. 1995). When associated with
ClpP, certain Hsp100 proteins perform dual regulatory/
chaperone activities, thereby influencing the eventual
fate of selected protein substrates.
Accompanying the detailed biochemical/structural
analysis of Clp proteases in E.coli has been an increasing
number of studies of Clp proteins in many different bac-
teria and eukaryotes, and studies of their importance
for vital processes such as sporulation, DNA replication,
protein turnover, stress tolerance and acclimation, and
regulation of gene expression (Porankiewicz et al. 1999).
In particular, plants as shown in the model species Arab-
idopsis thaliana possess by far the greatest number of
Clp proteins, with isomeric forms of ClpP and most
Hsp100 members. Of the Hsp100 proteins, several nu-
clear-encoded varieties are present in plants, with ClpB
proteins located in the cytosol (Schirmer et al. 1994),
ClpX in mitochondria (Halperin et al. 2001) and ClpC
and –D in chloroplasts (Moore and Keegstra 1993,
Weaver et al. 1999). In comparison, six distinct ClpP iso-
mers have now been identified in Arabidopsis (ClpP1–6,
Adam et al. 2001), all of which are nuclear-encoded ex-
cept for the plastomic ClpP1 protein. Arabidopsis also
has four additional genes encoding proteins with overall
sequence similarity to ClpP but which all lack the pro-
teolytic active site characteristic of ClpP (i.e. Ser-His-
Asp catalytic triad), and as such they have been dis-
tinguished as ClpR (Clarke 1999, Adam et al. 2001).
Because of their recent discovery and great complexity,
much remains unknown about the regulation, structure
and function of Clp proteins in Arabidopsis, especially
Physiol. Plant. 114, 2002
93
those localized in chloroplasts. Genetic evidence to date
has shown chloroplastic ClpC and ClpP1 are essential
constitutive proteins (Shanklin et al. 1995, Shikanai
et al. 2001). The chloroplast Clp proteins reported so
far (ClpC, –D, –P1 and –P6) are localized in the stroma
(Moore and Keegstra 1993, Shanklin et al. 1995, Soko-
lenko et al. 1998, Weaver et al. 1999), with possible as-
sociations between ClpC and ClpP1/P6 (Desimone et al.
1997, Sokolenko et al. 1998). In this study we describe
the identification of additional isomeric forms of ClpC
and ClpP in Arabidopsis, and the localization of these
proteins in the chloroplast stroma. We also detail the
first concerted approach to determine simultaneously
the expression and synthesis characteristics of most
chloroplast Clp proteins in various tissues and under a
range of physiological conditions.
Materials and methods
Cloning, sequencing and data analysis of Arabidopsis clp
genes
A computer search of the Arabidopsis EST database
using known ClpC and ClpP sequences from other plant
species was performed to identify cDNA clones for
novel clp genes in Arabidopsis. Partially sequenced
clones coding for a single ClpC and four ClpP polypep-
tides were identified and obtained from Dr Amie Frank-
lin and the Arabidopsis Resource Center (Ohio State
University, OH, USA), respectively. The most intact
cDNA clones for each gene were then completely se-
quenced using the Taq DyeDeoxy terminator cycle se-
quencing kit (DYEnamicTM, Amersham Pharmacia Bi-
otech, Uppsala, Sweden) and analysed on an automated
sequencer (ABI377, Perkin Elmer, Foster City, CA,
USA). Sequences were viewed with the AutoAssembler
(PE Applied Biosystems, Foster City, CA, USA) com-
puter software and then analysed with the Genetics
Computer Group (GCG Wisconsin Package, Accelrys
Inc., CA, USA) program. Predictions of intracellular
locations and transit peptide processing sites were done
using TargetP version 1.01 (Emanuelsson et al. 2000)
and ChloroP version 1.01 (Emanuelsson et al. 1999).
Production of specific antibodies
Specific polyclonal antibodies were generated against
Arabidopsis Clp proteins using either fusion proteins or
synthetic peptides. For ClpD, –P1 and –P6, C-terminal
fusion proteins with the maltose-binding protein (MBP)
were over-expressed in E.coli using the inducible pMAL-
c2 vector (New England Biolabs, Beverly, MA, USA).
For ClpD, the 510 bp 31region downstream of the ATP-
2 domain was PCR amplified with the high fidelity pfu
DNA polymerase (Stratagene, La Jolla, CA, USA). The
clpD fragment was ligated in-frame to the 31-end of the
malE gene coding for MBP in the pMAL-c2 plasmid
and transformed into E.coli DH5a. Similar fusion con-
structs with MBP were made for the 51exon of clpP1

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and the entire clpP6 gene. Over-expression of all fusion
constructs from the tac promoter was induced by ad-
dition of IPTG to exponentially growing cells. Fusion
proteins were then purified according to the method by
Riggs (1990). For the remaining ClpP isomers, specific
antibodies were made to KLH-or BSA-conjugated syn-
thetic peptides corresponding to unique amino acid
sequences: ClpP3, KVEGTKKDNTNLPSERSMTQ;
ClpP4, FEELDTTNMLLRQRI; and ClpP5, DIDI-
QANEMLHHKANLNGYL. All Clp fusion proteins
and conjugated synthetic peptides were injected into rab-
bits intramuscularly and subcutaneously (AgriSera,
Hällnäs, Sweden).
Plant material, stress treatments and Chl fluorescence
measurements
Arabidopsis thaliana plants (ecotype: Columbia) were
grown in soil under controlled conditions: day/night
temperatures 23/18æC, 8 h photoperiod, 62% humidity
and 150 mmol mª2sª1irradiance. Six-week-old plants
used for all experiments were sampled 2 h into the
photoperiod. For each stress, at least three replicate
leaves were taken for each time point.
To test the effects of short-term severe stresses on the
expression and synthesis of Clp proteins, detached Arab-
idopsis leaves were treated to different stress regimes. For
each stress, all other growth conditions were kept con-
stant unless stated. Desiccation stress was done by de-
hydrating leaves on Whatman 3 mm paper (Whatman
Inc., NJ, USA) for 1 or 2 h at room temperature at an
irradiance of 10 mmol mª2sª1. For salt and oxidative
stress, leaves were floated on 200 or 400 mM NaCl, or
2% H2O2solutions for 2 h, respectively. Wounding was
done by rolling a metal brush across the detached leaves
once (light) or several times (heavy) and then floated on
water for 2 h. Cold and heat shock treatments were done
by floating leaves on prechilled or preheated water inside
growth cabinets at 5æC for 4 h and 37æC for 2 h, respec-
tively. Extreme high light stress was applied to detached
leaves for 4 h at 1500 mmol mª2sª1as described by Hed-
dad and Adamska (2000). Control leaves were also de-
tached and floated on water under standard growth con-
ditions for 2 or 4 h to test for changes in Clp proteins
resulting from leaf excision.
For the longer, continuous high light treatment, whole
plants were transferred from the standard irradiance of
150 mmol mª2sª1to 850 mmol mª2sª1for 1 d. Leaves
were collected just prior to the shift (time 0 control), and
then at 4, 8, 12 and 24 h of high light. Leaves from
plants kept for 24 h at the standard irradiance of 150
mmol mª2sª1were taken at the same time points as con-
trols. For the prolonged high light, plants were shifted
for 7 d to 850 mmol mª2sª1but with the same photo-
period (8 h day/16 h night).
For cold acclimation, plants were transferred to day/
night temperatures of 5/5æC, with the photoperiod and
irradiance kept constant. As controls, several plants were
kept at 23/18æC for the duration of the cold treatment.
Physiol. Plant. 114, 2002
94
Leaves developed at 23æC were taken 0 (control), 3, 7,
15 and 21 d after the cold shift; while fully expanded
leaves from the new rosette developed at 5æC were also
taken as the cold acclimated samples. Photochemical ef-
ficiency of PSII in leaves was measured for the high light
and cold experiments as the FV/FMratio as previously
described (Ottander and Öquist 1991).
Leaf cellular protein extraction and chloroplast
subfractionation
Total cell protein samples were isolated from leaves by
grinding in liquid N2and transferring the frozen powder
to 400 ml of solubilization buffer (100 mM sucrose, 50
mM Tris/Cl [pH8.0], 1 mM EDTA, 1 mM Pefabloc, 20
mM DTT, 2% lithium dodecyl sulphate). After mixing,
samples were heated at 75æC for 5 min and then centri-
fuged at approximately 14 000 g for 15 min at 4æC. Insol-
uble pellets were discarded, while supernatants contain-
ing solubilized proteins were kept for immunoblot analy-
sis. Chl contents were determined spectrophotomet-
rically (Porra et al. 1989). For the tissue specificity
analysis, solubilized protein extracts were first precipi-
tated in 80% acetone to remove Chl and then resus-
pended in solubilization buffer. Protein concentrations
were determined by the Bradford method according to
the manufacturer’s protocol (Pierce). Isolation and
subfractionation of intact chloroplasts from leaves were
achieved on Percoll gradients as previously described
(Clarke and Critchley 1992, Kunst 1998).
Polyacrylamide gel electrophoresis and immunoblot
analysis
Denatured protein samples containing equal Chl (1 mg)
were separated on precast 3–8% gradient Tris-Acetate
(ClpC/D) or 12% linear Bis-Tris (ClpP) NuPAGE gels
(Novex, San Diego, CA, USA). After separation, pro-
teins were transferred electrophoretically to a poly-
vinylidenne difluoride filter (Immobilon, Millipore,
Bedford, MA, USA). Arabidopsis Clp proteins were de-
tected using specific polyclonal antibodies as described
above, except for ClpC, which is described earlier (Por-
ankiewicz and Clarke 1997). Primary antibodies were
detected with the horseradish peroxidase-linked, anti-
rabbit secondary antibody from donkey (Amersham
Pharmacia) and visualized by enhanced chemilumi-
nescence (Amersham Pharmacia) on X-ray film.
RNA preparation and analysis
For analysis of transcript levels, total RNA was isolated
using the TrizolTMreagent (Life Technologies, CA,
USA) and treated with RNase-free DNase (Promega).
RT-PCR was performed with the SuperScriptTMOne-
step RT-PCR kit Life Technologies, USA) using 4 ng of
RNA for clpP1, 20 ng for clpP3–6 and clpC1, and 100
ng for clpC2 and clpD. These RNA concentrations were
found in preliminary experiments to be within the linear

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response range of the PCR amplification for each set of
gene-specific primers. For all RT-PCR reactions, an
extra control reaction was performed without reverse
transcriptase but with Taq polymerase to detect possible
DNA contamination. RT-PCR products were separated
on 1.0–1.2% agarose gels and viewed with a ChemiImag-
erTM4000 Low Light Imaging System (Alpha Innotech
Corp., San Leandro, CA, USA). The identities of all RT-
PCR products were confirmed by DNA sequencing.
Results
Identification of ClpC and ClpP homologues in
Arabidopsis
The major Hsp100 protein in plant chloroplasts is ClpC,
with two distinct, but closely related isomers in tomato,
both predicted as being located in chloroplasts (Gottes-
man et al. 1990). A search of the Arabidopsis EST data-
base revealed a cDNA coding for a ClpC-like protein,
which we later obtained and completely sequenced. The
cDNA contained a 2784-bp ORF encoding a 928-amino
acid polypeptide. Prediction analysis of the N-terminus
revealed a 92-amino acid chloroplast transit peptide. The
complete protein has the two distinct ATP-binding do-
mains characteristic of large Hsp100 proteins, as well
as the spacer region of intermediate length and the two
conserved N-terminal domains representative of the
ClpC class (Schrimer et al. 1996). The putative mature
Arabidopsis protein has high sequence identity to ClpC
in other plants (90–94%) and cyanobacteria (approxi-
mately 80%), whereas the match to ClpC in Gram posi-
tive bacteria (e.g. B.subtilis, 60%) and ClpA in Gram
negative bacteria (e.g. E.coli, 41%) is much lower. Arabi-
dopsis ClpC is also dissimilar to the other chloroplast-
localized Hsp100 protein (ClpD, Kiyosue et al. 1993),
being only 50% identical. Recently, a second ClpC iso-
mer was identified in Arabidopsis (87% identity; Naka-
bayashi et al. 1999), and which has since been dis-
tinguished from the protein in this study (hereafter re-
ferred to as ClpC1) as ClpC2 (Adam et al. 2001).
AsearchoftheArabidopsisESTandgenomicdatabases
was also done for homologues of the ClpP proteolytic
subunit. Genes coding for six putative ClpP proteins were
subsequently identified, with the isomers recently desig-
nated as ClpP1–6 (Adam et al. 2001). Four extra genes
coding for ClpP-like proteins were also identified in the
Arabidopsis genome, but all four predicted polypeptides
lacked the highly conserved catalytic triad characteristic
of ClpP proteins (i.e., Ser-His-Asp). Because of this dis-
tinction, these four additional polypeptides have been
designated as ClpR1–4 (Clarke 1999, Adam et al. 2001).
Six other Clp-like sequences were also identified but all
have features too divergent to classify as either ClpP/R or
Hsp100. A detailed analysis of these sequences has re-
cently been reported (Peltier et al. 2001).
At the time of our study, only partially sequenced
cDNA clones were available for ClpP3–6. We obtained
these cDNA clones and completed their DNA sequenc-
Physiol. Plant. 114, 2002
95
ing. The residues, Ser (194), His (219) and Asp (269),
forming the proteolytic active site of the ClpP protease,
were found in all six Arabidopsis ClpP proteins. Except
for the plastid-encoded ClpP1, the five nuclear-encoded
ClpP proteins all have N-terminal extensions that are
strongly reminiscent of organellar transit peptides. Ac-
cording to predictions, ClpP3–6 with high probability
possess transit peptides targeted to plastids. In contrast,
the transit peptide for ClpP2 is predicted as mitochon-
drial, a localization we have since confirmed experimen-
tally (Halperin et al. 2001).
Antibodies specific to plastid-localized Clp proteins
To further study the various Clp proteins in Arabidopsis
predicted to be localized in plastids, we prepared a suite
of specific polyclonal antibodies. All sera produced were
tested against cell extracts from Arabidopsis leaves to de-
termine specificity. An antibody was already available
for ClpC, made to the N-terminal domain of the cyano-
bacterial ClpC protein from Synechococcus (Porankiew-
icz and Clarke 1997). In leaf extracts, a single 98 kDa
protein was detected using the ClpC antibody (Fig.1A).
However, because of the highly conserved nature of this
N-terminal domain among ClpC homologues, and the
near identity between ClpC1 and ª2 in this region, the
antibody detects both Arabidopsis isomers without dis-
tinction. For the other plastidic Hsp100 protein, ClpD,
an antibody was made to the C-terminal domain down-
stream of the second highly conserved ATP-binding do-
main. The resultant antibody detected a protein
matching the predicted mature size of ClpD (approxi-
mately 102 kDa) in Arabidopsis leaf cell extracts. The
antibody also weakly detected a slightly smaller protein
corresponding to the size of ClpC (Fig.1A) likely due to
slight cross-reaction to the more abundant ClpC protein.
Besides the Hsp100 proteins, polyclonal antibodies
were made for each of the Arabidopsis ClpP isomers.
Each of the antibodies detected a single ClpP protein of
the expected molecular mass (ClpP1, 20.5 kDa; ClpP3,
28.5 kDa; ClpP4, 27 kDa; ClpP5, 22.5 kDa; ClpP6, 21.5
kDa) without observable cross-reaction to any of the
other ClpP isomers (Fig.1B). In the case of ClpP3 and -
P5, slight cross-reaction to proteins larger than 35 kDa
was observed, although these did not interfere with the
detection of the ClpP isomer in this study.
Localization of Clp proteins in chloroplast stroma
With the identification of new ClpP isomers in Arabi-
dopsis, we investigated whether they were localized in
chloroplasts as was predicted by their transit peptide se-
quences. Intact chloroplasts were purified from young
Arabidopsis leaves and fractionated into stromal and
thylakoid membrane/lumenal proteins. Using our speci-
fic antibodies, all investigated Clp proteins were detected
in intact chloroplasts in amounts similar to whole leaf
extracts (Fig.2), indicating chloroplast localization for
each Clp protein. Within chloroplasts, all Clp proteins

Page 5

were localized in the stromal fraction, with no detectable
signal in the thylakoid membrane fraction. Polyclonal
antibodies raised against Lhcb2, a known thylakoid
membrane protein, were used as a control to confirm
the integrity of the thylakoid subfraction. These results
demonstrate that the three newly identified nuclear-en-
coded ClpP isomers (ClpP3–5) are transported into
chloroplasts and localized in the stroma along with
ClpC, –D, –P1 and –P6.
Tissue-specific expression and synthesis of Clp proteins
To study the regulatory characteristics of the chloroplast
Clp proteins mentioned above, tissue from roots, stems
and leaves were collected to test for potential differential
gene expression and protein synthesis. Levels of Arabi-
dopsis clp-specific mRNA and protein were analysed by
RT-PCR and immunoblotting, respectively. Similar tran-
script levels were observed for the hsp100 genes (clpC1, –
Fig.1. Specific polyclonal antibodies against Arabidopsis Clp pro-
teins. Cell extracts from Arabidopsis leaves were separated by dena-
turingPAGEoptimizedforlargeHsp100protein(A)orsmallerClpP
isomers (B). Immunoblot analysis was then performed with each
serum on replicate samples to test the specificity of each individual
antibody. Specificity of the antibodies is shown at the bottom of each
lane, with molecular mass standards in kDa shown on the left.
Physiol. Plant. 114, 2002
96
C2 and –D) in the three tissue types, whereas the corre-
sponding protein contents were relatively low in roots
but steadily increased from stems to leaves (Fig.3). This
discrepancy between mRNA and protein levels suggests
either post-transcriptional regulation of hsp100 tran-
scripts reducing protein levels in root and stem cell plas-
tids, or increased stability of chloroplast Hsp100 pro-
teins in leaf cells. In contrast, transcript and protein
levels for most Arabidopsis clpP genes were both low in
roots and increased steadily from stems to leaves (Fig.
3), suggesting little if any post-transcriptional regula-
tion. The one exception was clpP5, for which transcript
levels changed less dramatically in the different tissues
than protein content.
Levels of Clp proteins under stress conditions
Clp proteins in different bacteria are involved in many
stress responses, such as heat, high salt, low temperature,
and UV light, and often strongly induced after brief ex-
posures (Porankiewicz et al. 1999). Given the many Clp
Fig.2. Chloroplast localization of Clp proteins in Arabidopsis. The
intracellular location of the ClpP3–5 proteins was compared to that
of the previously studied ClpC, –D, –P1 and –P6 proteins. Proteins
from whole cell extracts (lane 1), intact chloroplasts (lane 2), thy-
lakoid membranes (lane 3) and soluble stroma (lane 4) are isolated
and fractionated from Arabidopsis leaves. Samples were separated
by denaturing PAGE on the basis of equal Chl content (1 mg, lanes
1–3) or equivalent protein content (5 mg, lane 4), and then analysed
by immunoblotting using antibodies specific for each Clp protein
as indicated on the left. Also included was the known thylakoid
membrane, Lhcb2, as a control for the chloroplast subfractionation.

Page 6

proteins in Arabidopsis chloroplasts, we examined if any
could be involved in similar stress responses. For this we
studied the potential stress regulation of chloroplast Clp
proteins at different levels; possible rapid protein induc-
tion upon moderate or severe stress treatments, and in-
duction during longer acclimation to high light and cold.
Several moderate and severe stresses including dehy-
dration, high salt concentration, heat and cold shock,
oxidation, wounding and high light intensity were briefly
applied to detached Arabidopsis leaves. In addition to
the time zero control, controls detached for 2 and 4 h
were also included to exclude possible increases in Clp
protein synthesis resulting solely from leaf excision.
Under certain stress conditions, decreased Clp protein
content was observed, especially for ClpP3 after
wounding and high light treatments (Fig.4). However,
no significant and reproducible induction of any chloro-
plast Clp protein occurred during the various stresses,
suggesting most if not all, are mainly constitutive pro-
teins in Arabidopsis.
High light stress response
Because of the lack of any obvious rapid induction of
chloroplast Clp proteins in response to brief but severe
Fig.3. Tissue specificity of mRNA and protein levels of plastid-
localized Clp proteins in Arabidopsis. Comparative transcript levels
for each clp gene in Arabidopsis roots, stems and leaves were deter-
mined by RT-PCR from equal amounts of total RNA. RT-PCR
products were separated on agarose gels and visualized by ethidium
bromide staining. Equivalent amounts of total rRNA for each set
of RT-PCR reactions were confirmed by staining of 25S and 18S
rRNA. Comparative Clp protein levels in root, stem and leaf tissues
were determined by immunoblotting using the specific polyclonal
antibodies. For each tissue, whole cell proteins were extracted and
separated by denaturing PAGE on the basis of equal protein (10
mg). The figure shows results representative of three replicates.
Physiol. Plant. 114, 2002
97
stresses, we investigated more prolonged moderate
stresses. High light intensity was chosen as the first
model stress since we had previously shown strong in-
duction of Clp proteins in cyanobacteria during ex-
tended photoinhibitory conditions (Clarke et al. 1998).
Initially we tested for possible diurnal expression of clp
genes in Arabidopsis to reveal any changes over the 24 h
time period chosen for the first high light treatment. Di-
urnal expression was studied for ClpP1–6, ClpC1–2 and
ClpD, but no significant changes in both transcript and
protein contents were observed throughout the 24-h
period (data not shown), indicating no significant diur-
nal rhythms for these clp genes. For the first high light
experiments, Arabidopsis plants grown at 150 mmol mª2
sª1were shifted to 850 mmol mª2sª1for 24 h, with all
other growth conditions kept constant. Several Arabi-
dopsis plants were also kept at 150 mmol mª2sª1for
24 h as a control, from which later analysis showed no
significant change in Clp protein levels throughout the
time course. The degree of high light stress in leaves was
monitored by the Chl fluorescence parameter FV/FMfor
photosynthetic efficiency. Prior to the high light shift,
control leaves had an FV/FMof 0.843 (∫0.003 SE, nΩ
7), which then fell steadily to 0.711 (∫0.012 SE, nΩ12)
after 8 h of high light, and remained at this low level for
Fig.4. Levels of chloroplast Clp proteins in Arabidopsis exposed to
short-term severe stresses. Detached leaves from six-week-old plants
were treated with different severe stresses for brief periods: 1. at-
tached control, 2. control detached (2 h), 3. control detached (4 h),
4. desiccation (1 h), 5. desiccation (2 h), 6. 200 mM NaCl (2 h), 7.
400 mM NaCl (2 h), 8. 5æC (4 h), 9. 42æC (2 h), 10. 2% H2O2(2 h),
11. light wounding (2 h), 12. heavy wounding (2 h), 13. high light
(4 h). Cellular proteins were extracted after each treatment and sep-
arated by denaturing PAGE on the basis of equal Chl (1 mg). Indi-
vidual Clp proteins were detected by immunoblotting with specific
antibodies. The figure shows results representative of three indepen-
dent replicates.

Page 7

the remaining treatment. ClpC, ClpP1, ª3, ª5 and ª6
content did not significantly change during the 24 h high
light. However, levels of ClpD and –P4 proteins both
declined (approximately 50%) after the high light shift,
but then completely recovered within the 24-h period
(data not shown).
The second high light treatment was a shift in day ir-
radiance from 150 to 850 mmol mª2sª1for 7 d, with
photoperiod and other conditions unchanged. FV/FM
values showed that plants had recovered from the early
photoinhibitory stress by 3 d, which then remained un-
changed by 7 d. Chl and fresh wt also remained constant
throughout the high light treatment. The amount of
ClpC, –P3, –P4 and –P6 did not significantly change in
leaves during the 7 d high light regime (Fig.5). In con-
trast, leaf ClpP1 content increased several fold from 3
to 7 d, while ClpP5 levels rose steadily in both the 3
and 7 d samples. ClpD protein content was also strongly
induced after 3 d at 850 mmol mª2sª1, but this returned
to control levels by 7 d (Fig.5).
Cold acclimation
Because of the induction of certain Clp proteins during
the prolonged high light treatment, we analysed their
levels during acclimation of plants to another moderate
stress condition. Cold acclimation was selected, again
because of earlier observed Clp protein induction during
cold acclimation in cyanobacteria (Porankiewicz and
Fig.5. Level of chloroplast Clp proteins in Arabidopsis leaves ex-
posed to prolonged high light. Six-week-old Arabidopsis plants were
shifted from 150 to 850 mmol mª2sª1, with the same photoperiod,
temperature and humidity. Leaves from the same rosette were taken
just prior to (0 h control) and 3 and 7 d after the change in ir-
radiance. Individual Clp proteins were detected by immunoblotting
with specific antibodies and compared on the basis of equal Chl (1
mg) content. The figure shows results representative of three inde-
pendent replicates.
Physiol. Plant. 114, 2002
98
Clarke 1997, Porankiewicz et al. 1998). Six-week-old Ar-
abidopsis plants were shifted from 23/18æC to 5/5æC day/
nights with unchanged photoperiod, irradiance and hu-
midity. Leaves were collected just prior to the shift (0 h
control) and then after 3, 7, 15, 21 and 42 d at 5æC.
Leaves from another set of plants kept at 23/18æC were
also taken at these times as extra controls, except at 42
d when leaves from the second rosette dominated and
shaded the mature, first rosette underneath. The degree
of cold stress in plants shifted to 5æC was monitored
throughout by FV/FM. In leaves developed at 23æC prior
to the cold shift (warm-developed), FV/FMdropped from
control values of 0.841 (∫0.004 SE, nΩ6) to 0.792 (∫
0.007 SE, nΩ9) after 1 d at 5æC, and remained at this
level until day 7, after which it slowly recovered to 0.820
(∫0.005 SE, nΩ12) after 21 d. At 42 d, FV/FMof new
leaves developed at 5æC from the second rosette (0.848
∫0.004 SE, nΩ9) was the same as for warm-developed
control leaves, indicating the shifted plants had ac-
climated to the low temperature.
Potential changes in clp gene expression and protein
content were analysed in Arabidopsis leaves during the
cold acclimation (Fig.6). Of the hsp100 genes, there were
neither significant changes in transcript levels for clpC1
or -C2, nor any change in total ClpC protein content.
In contrast, levels of clpD mRNA and protein in warm-
developed leaves increased markedly from 3 to 7 d at
5æC, and remained high at 15 and 21 d. The clpD mRNA
and protein also remained at this elevated level in the
cold-developed leaves at 42 d, suggesting a role for ClpD
during cold acclimation. For the clpP genes, there were
no significant changes in mRNA levels during the cold
treatment except for clpP3. Transcripts of clpP3 in-
creased 2- to 3-fold in warm-developed leaves in the first
3 d and stayed at this level until 21 d, although in cold-
developed leaves (42 d) it was slightly lower. This in-
crease in transcript level was followed by an increase in
ClpP3 protein after 7 d, which rose further by 21 d and
in cold-developed leaves at 42 d. For the other ClpP iso-
mers, the amounts of ClpP1, ª3, ª5 and ª6 protein
significantly increased after 7 or 15 d at 5æC, despite no
obvious changes in mRNA level, and all remained at this
elevated level after 21 d and in cold-developed leaves at
42 d. Only ClpP4 protein content remained unaffected
by the cold treatment, which mirrored its mRNA profile
(Fig.6).
Discussion
In this study we describe several new clp genes in Arabi-
dopsis, one coding for a ClpC homologue (ClpC1) and
three for distinct ClpP isomers (ClpP3–5). The addition
of these genes now increases the total number of con-
firmed clp genes in Arabidopsis to 19, by far the greatest
number as yet found in any organism. Moreover, six
other Clp-like sequences have also recently been iden-
tified in Arabidopsis (Peltier et al. 2001) from the re-
cently completed genome sequence, thereby possibly
further increasing the complexity of plant Clp proteins.

Page 8

Of the recognized polypeptides, nine are members of the
Hsp100/Clp family of molecular chaperones (4 ClpB, 2
ClpC, 1 ClpD and 2 ClpX), six are homologues of the
ClpP proteolytic subunit, and four are ClpP-like pro-
teins now termed ClpR. To clarify this situation, we have
recently proposed a standardized nomenclature for most
of these Clp proteins (Adam et al. 2001), which we have
conformed to in this study.
Most of the Clp proteins in Arabidopsis are localized
in chloroplasts. We were able to demonstrate the stromal
localization of the new ClpP isomers, ClpP3–5, and con-
firm similar localizations for ClpC, -D, -P1 and –P6
(Shanklin et al. 1995, Nakabayashi et al. 1999). Of the
Fig.6. Arabidopsis chloroplast Clp proteins during cold acclim-
ation. Plants growing at 23/18æC were shifted to 5/5æC with the same
photoperiod and irradiance. Plants were chilled at 5æC until cold-
acclimated leaves from the second rosette were fully expanded.
Leaves from the same rosette developed at 23æC were taken prior to
(0 h control) and 3, 7, 15 and 21 d after the cold shift. Mature
leaves from the new rosette developed at 5æC were also harvested as
the acclimated samples (Dev). Levels of mRNA and protein for
each Clp protein were determined by RT-PCR and immunoblotting
on the basis of equal total RNA (10 mg) or Chl (1 mg) content.
Leaves from plants kept under standard conditions for the first 21
d of cold were also examined for possible changes in protein content
during senescence. The figure shows results representative of three
independent replicates.
Physiol. Plant. 114, 2002
99
remaining Clp proteins, ClpR1–3 also appear to be lo-
calized in chloroplasts (Nakabayashi et al. 1999, Peltier
et al. 2001), while ClpX1, –X2 and –P2 are found in
mitochondria (Halperin et al. 2001). The intracellular
location of ClpR4 remains unknown. All the stromal
Clp proteins in this study almost certainly function in
ATP-dependent Clp proteases although direct evidence
for such remains absent. It is equally plausible that the
chloroplastic Hsp100 proteins also perform chaperone
roles independent of ClpP proteolysis, as do the Hsp100
homologues in other organisms (Wickner et al. 1994,
Levchencko et al. 1995), with an involvement in chloro-
plast protein import being one such possible function
(Nielsen et al. 1997). With the addition of ClpP3–5 pro-
teins, however, there are now at least five potential pro-
teolytic subunits for stromal Clp proteases. Why so
many ClpP isomers occur in chloroplasts and whether
they form homogeneous or heterogeneous ClpP com-
plexes remains unclear. Since it is the Hsp100 chaperone
subunit that confers substrate specificity to the Clp pro-
tease and regulates its activity, there seems no obvious
explanation why plants possess more than one ClpP in
chloroplasts. Based on structural comparisons of the
three ClpP isomers in cyanobacteria (Porankiewicz et al.
1999), we proposed they might have different affinities
for different Hsp100 partners. If true for chloroplast
ClpP proteins, then more than one isomer is likely to
associate with each Hsp100 protein since only three
probable Clp regulatory subunits exist in chloroplasts
(ClpC1, –C2 and –D). An alternative possibility is that
some chloroplast ClpP proteins form proteolytic com-
plexes with other chaperones like Hsp60, thereby in-
creasing the number of potential regulatory partners
and broadening the range of protein substrates. The bac-
terial Hsp60 (GroEL) is known to activate the proteo-
lytic activity of ClpP in E.coli in the absence of Hsp100
proteins (Kandror et al. 1994), and Hsp60 is one of the
major molecular chaperones in the chloroplast stroma.
An equally important issue to resolve is the role of ClpR
polypeptides in chloroplasts and whether they interact
with ClpP isomers or Hsp100 proteins.
Before this study it was unknown whether the numer-
ous Clp proteins in chloroplasts exhibited differential
gene expression/protein synthesis under different physio-
logical conditions. Given that such variations in the syn-
thesis of each Clp protein type would greatly affect the
composition and substrate specificity of potential
chloroplast Clp proteases, it was crucial to perform a
more comprehensive analysis than had been previously
attempted. For this we combined the powerful tech-
niques of RT-PCR and immunoblotting to compare
mRNA and protein levels for each Clp protein in differ-
ent tissues and growth regimes. In terms of tissue speci-
ficity, all the studied plastid Clp proteins showed similar
protein profiles, being most abundant in leaves and rela-
tively low in roots. This suggests that all plastid localized
Clp proteins are most abundant in chloroplasts, with
levels highest in tissues with the greatest chloroplast den-
sity, as seen when comparing stems and leaves. In the

Page 9

case of clpP1, the higher plastome copy number in
chloroplasts may also contribute to greater amounts of
ClpP1 protein in leaves relative to roots. Despite their
low concentrations, however, all plastid Clp proteins
were present in roots, thereby excluding a role for Clp
proteases exclusive to photosynthetic tissues. This sup-
ports earlier proposals that Clp proteases in plants may
at one level perform housekeeping duties within plastids
and be involved in the turnover of proteins with func-
tions unrelated to photosynthesis (Clarke 1999).
The tissue specificity study also revealed distinct regu-
latory differences between the plastid Hsp100 and most
ClpP proteins. For the Hsp100 proteins, the transcript
levels for clpC1, –C2 and –D were the same in all three
tissues, despite the considerably higher protein levels in
stems and leaves. This suggests significant post-tran-
scriptional and/or translational regulation exists in Ara-
bidopsis that restricts the synthesis of Hsp100 proteins
in root cells. In contrast, the mRNA profiles for the clpP
genes closely matched the protein profiles, indicating
that ClpP protein contents in plastids is regulated pri-
marily at the transcriptional level in Arabidopsis. The
one exception to this was clpP5, which exhibited tran-
script levels similar to those for the hsp100 genes, also
suggesting a degree of post-transcriptional regulation.
At this stage the reason for such differential regulation
of clp gene expression in Arabidopsis remains unclear.
Besides being abundant in leaves, the constitutive level
of chloroplast Clp proteins did not dramatically change
during transient severe and moderate stresses. Although
not exhaustive, many different severe stress conditions
were examined. Under all these conditions, however, no
significant induction occurred for any of the chloroplast
Clp proteins. This contrasts with studies in various eub-
acteria, in which ClpC or –P proteins are rapidly in-
duced by the same stress regimes used in this study (Por-
ankiewicz et al. 1999). In plants, a similar absence of
stress induction as shown in this study was also observed
for ClpP1 and ClpC during short-term heat shock, chill-
ing and dehydration (Shanklin et al. 1995, Ostersetzer
and Adam 1996). In the case of ClpC, this lack of stress
induction also occurred for the closely related homolo-
gue in cyanobacteria (Clarke and Eriksson 1996). For
ClpD, previous studies have reported rapid induction of
clpD gene expression in Arabidopsis leaves during severe
desiccation and high salt stress, and during natural sen-
escence and dark-induced etiolation (Kiyosue et al.
1993, Nakashima et al. 1997), although in our study
such stresses failed to significantly increase amounts of
ClpD protein. This apparent discrepancy between
mRNA and protein levels has recently been observed for
ClpD during senescence, in which levels of ClpD protein
actually decreased in Arabidopsis leaves concomitant to
increased clpD transcripts (Weaver et al. 1999), sug-
gesting that considerable post-transcriptional regulation
exists for this clp gene like that observed in Arabidopsis
roots. Overall, the lack of major increases in levels of
chloroplast Clp proteins suggests none of the tested pro-
teins are rapidly induced stress proteins in leaves. It must
Physiol. Plant. 114, 2002
100
be noted, however, that in other tissues like roots with
low constitutive levels of Clp protein, the possibility re-
mains of stress induction for one or more isomers.
In contrast to the short-term stresses, prolonged high
light and cold treatment did result in significantly in-
creased levels of certain chloroplast Clp proteins, includ-
ing ClpD and several ClpP isomers. In the case of ClpP,
both ClpP1 and –P5 were induced during the latter
stages of both high light and cold shifts, suggesting the
two proteins may be involved in acclimation to each new
growth condition. It should be noted that for the high
light shift the acclimation was to a more desirable light
intensity whereas the cold shift was to a less favourable
temperature. ClpD would also appear involved in these
acclimation processes, being the earliest induced Clp
protein under both conditions and at levels highest in
leaves recovering from the initial stress period. As leaves
acclimatise to the new condition, ClpD content declines
as after 7 d at high light and 21 d at 5æC. In the case
of cold, however, ClpD content remained high in leaves
developed at 5æC, suggesting a more prolonged involve-
ment by this chloroplast protein in cold hardiness. This
induction of ClpD during cold acclimation is the first
report where ClpD levels rise concomitantly with clpD
mRNA in response to an altered growth condition or
developmental transition. Given that the induced ClpD
and ClpP proteins likely function in Clp proteolytic
complexes,their accumulation
chloroplast protein turnover is associated with both high
light and cold acclimation.
The nucleotide sequences reported in this paper
have been submitted to the GenBank/EBI Data Bank
with accession numbers AF016621, AF022909 and
AJ012278.
suggestsincreased
Acknowledgements – Our sincere thanks to Stefan Jansson for the
antibody against Arabidopsis Lhcb2 protein, Amie Franklin for the
Arabidopsis clpC cDNA clone, and Åsa Strand and Vaughan
Hurry for assistance of setting up growth conditions and expert
advice. This work was supported in part by grants from Swedish
Agricultural and Forestry Resource Council, Carl Tryggers Found-
ation for Scientific Research, Swedish Foundation for International
Cooperation in Research and Higher Education (A.K.C.); the Israel
Science Foundation, BARD – US-Israel Binational Science Found-
ation (Z.A.); and Federation of European Biochemical Societies
(OH).
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