Plant Cell Physiol. 45(8): 960–967 (2004)
JSPP © 2004
Rapid Paper
ARC3, a Chloroplast Division Factor, is a Chimera of Prokaryotic FtsZ and
Part of Eukaryotic Phosphatidylinositol-4-phosphate 5-kinase
Hiroshi Shimada 1, Masato Koizumi, Kouta Kuroki, Mariko Mochizuki, Hitoshi Fujimoto, Hiroyuki Ohta,
Tatsuru Masuda and Ken-ichiro Takamiya
Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259, Nagatsuta-cho,
Midori-ku, Yokohama, Japan
;
The arc3 (accumulation and replication of chloroplast)
mutant of Arabidopsis thaliana has a small number of
abnormally large chloroplasts in the cell, suggesting that
chloroplast division is arrested in the mutant and ARC3
has an important role in the initiation of chloroplast division. To elucidate the role of ARC3, first we identified the
ARC3 gene, and determined the location of ARC3 protein
during chloroplast division because the localization and
spatial orientation of such division factors are vital for correct chloroplast division. Sequencing analysis showed that
ARC3 was a fusion of the prokaryotic FtsZ and part of the
eukaryotic phosphatidylinositol-4-phosphate 5-kinase (PIP5K)
genes. The PIP5K-homologous region of ARC3 had no catalytic domain but a membrane-occupation-and-recognitionnexus (MORN) repeat motif. Immunofluorescence microscopy, Western blotting analysis and in vitro chloroplast
import and protease protection assays revealed that ARC3
protein was soluble, and located on the outer surface of the
chloroplast in a ring-like structure at the early stage of
chloroplast division. Prokaryotes have one FtsZ as a gene
for division but have no ARC3 counterparts, the chimera of
FtsZ and PIP5K, suggesting that the ARC3 gene might have
been generated from FtsZ as another division factor during
the evolution of chloroplast by endosymbiosis.
dark-grown leaves), amyloplast (in seed and tuber), chromoplast (in fruits and flowers) and leucoplast (in petals) (Kirk and
Tilney-Bassett 1978). The plastid is involved in a variety of
important processes such as photosynthesis, fatty acid synthesis
and the fixation of nitrogen and sulphur (Joyard et al. 1998). In
Arabidopsis thaliana, ecotype Landsberg erecta, the mature
mesophyll cell contains about 120 chloroplasts (Pyke and
Leech 1992). The number of chloroplasts per cell is strictly
regulated by a variety of environmental factors. Light microscopy revealed that chloroplast division is accompanied by a
lengthening and narrowing of the chloroplast, and at late
stages, a narrow, twisted isthmus joining the two daughter chloroplasts is observed (Leech et al. 1981). Electron microscopy
revealed that formation of the central constriction is frequently
associated with the appearance of an electron-dense annular
structure termed the plastid-dividing (PD) ring (Mita and
Kuroiwa 1988, Kuroiwa et al. 1998). PD rings consist of an
outer ring on the cytoplasmic face of the chloroplast outer
membrane and an inner ring on the stromal face of the chloroplast inner membrane. Previous studies have identified the factors that control the different phases of chloroplast division.
The first protein identified as responsible for the division of
chloroplasts was FtsZ (Osteryoung and Vierling 1995). In
plants, FtsZ genes are encoded in the nucleus (Osteryoung and
Vierling 1995, Miyagishima et al. 2003). FtsZ is a GTPase, and
polymerizes to a ring (Z-ring) during constriction division of
bacterial cells (Bi and Lutkenhaus 1991, Errington et al. 2003).
Immunoelectron microscopy showed that the FtsZ of plants
appears in the inner stromal ring and not in the outer cytosolic
ring (Vitha et al. 2001, Mori et al. 2001, Kuroiwa et al. 2002).
Most bacteria have a single FtsZ gene, but plants have multiple
FtsZ genes that group into two families, FtsZ1 and FtsZ2. Inhibition of the expression of either A. thaliana FtsZ gene
(AtFtsZ1-1 or AtFtsZ2-1) with antisense technology in transgenic plants reduced the number of chloroplasts in mature leaf
cells from 100 to one (Osteryoung et al. 1998). The result suggested that FtsZ1 and FtsZ2 were not functionally redundant
with regard to their roles in chloroplast division.
Pyke and Leech isolated arc (accumulation and replication of chloroplast) mutants of A. thaliana (Pyke and Leech
Keywords: Arabidopsis thaliana — ARC3 — FtsZ — Phosphatidylinositol-4-phosphate 5-kinase — Plastid division.
Abbreviations: ARC, accumulation and replication of chloroplast; Fts, filamentous temperature sensitive; PIP5K, phosphatidylinositol-4-phosphate 5-kinase; MBS, m-maleimidobenzoyl-Nhydroxysuccinimide ester; MORN, membrane-occupation-and-recognition-nexus; PD ring, plastid-dividing ring.
The sequence of the cDNA reported here has been deposited in
the DNA database under accession nos. AB094044 and AB159444.
Introduction
The plastid is an important organelle in plant cells and can
transform into a chloroplast (in green tissues), etioplast (in
1
Corresponding author: E-mail, [email protected]; Fax, +81-45-924-5823.
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Chloroplast division factor, ARC3
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Fig. 1 Chloroplast phenotypic characteristics of arc3 mutants. Photomicrographs of chloroplast autofluorescence
in mesophyll cells from wild type (a),
arc3-1 (b), SALK_057144 (c) and
arc3-1 complemented with the ARC3
genomic DNA clone (d). The magnification of (a) to (d) is the same. Bar,
30 µm. Electron microscopy of chloroplasts in mesophyll cells from wild
type (e) and arc3-1 (f). Bar, 5 µm.
1992, Pyke and Leech 1994, Pyke 1997). Their results showed
that the mesophyll chloroplasts in these mutants differ considerably from those in the wild type with regard to number, size
and shape. The number of chloroplasts in wild-type mesophyll
cells was 80–120, but the numbers in arc3, arc5 and arc6
were 16, 13 and 2, respectively. The mean chloroplast plan area
in the wild type was about 50 µm2, but in arc3, arc5 and arc6,
the value was, 294, 312 and 1,081 µm2, respectively. Many
chloroplasts of the arc5 mutant showed some degree of permanent central constriction. ARC5 was the first gene identified
among arc mutants, and ARC5 is a cytosolic dynamin-like
protein of the chloroplast division machinery (Gao et al. 2003,
Osteryoung and Nunnari 2003). The recent identification of a
new plastid division gene, ARC6, suggests that a molecular
chaperone functions in chloroplast division (Vitha et al. 2003).
It is thought that ARC3 plays an important role in the initiation
of chloroplast division, since the number of chloroplasts in an
arc3 mutant cell is the same as the final proplastid number, i.e.,
no chloroplast division occurs (Marrison et al. 1999). It is also
thought that the expansion of young chloroplasts in the arc3
mutant is not stopped, and the chloroplasts become too large to
divide at the time of chloroplast division (Marrison et al. 1999).
Here we report that ARC3 is a chimera of FtsZ and an Nterminal region of phosphatidylinositol-4-phosphate 5-kinase,
the membrane-occupation-and-recognition-nexus (MORN) repeat motif.
Results
Phenotype of arc3 mutant
Leaf mesophyll cells in the arc3 mutant contain fewer and
larger chloroplasts than those of the wild type (Fig. 1a, b). The
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Chloroplast division factor, ARC3
Fig. 2 Summary of the positional cloning of the ARC3 gene. A partial genetic
map of A. thaliana chromosome I
between 27.5 Mb and 28.0 Mb with several BAC clones is shown (bold lines).
Open reading frames (ORF) of F9E10
predicted by GENESCAN analysis are
shown by arrows. The structure of ARC3
and molecular nature of the mutations
found in arc3-1 and SALK_057144 are
shown. Boxes and lines between boxes
show exons and introns, respectively.
numbers of chloroplasts in mesophyll cells of the wild type and
arc3 mutant were 116±11 and 15±4 (n = 20), respectively. The
shape of chloroplasts in the wild type was globular but that in
the arc3 mutant was irregularly globular. For further characterization of the arc3 mutant, we examined the ultrastructure of
chloroplasts under a transmission electron microscope. The
chloroplasts in mesophyll cells of the wild type were crescentshaped, and thylakoid membranes including stroma thylakoids
and grana thylakoids were observed in the chloroplasts (Fig.
1e). In arc3 mutant plants, although the chloroplast was elongate and thin, stroma thylakoids and grana thylakoids were still
retained (Fig. 1f). The plant shape and growth rate of the
mutant were almost the same as those of the wild type (data not
shown).
Cloning of ARC3 gene and characterization of ARC3 protein
Chromosome mapping of the ARC3 gene was performed,
and ARC3 was located between 27.5 Mb and 28.0 Mb of chromosome I (Fig. 2). Based on an analysis of 1,000 F2 plants
(2,000 chromosomes), we located the ARC3 locus within a 97kb region of the BAC clone, F9E10, which was completely
sequenced by the Arabidopsis Genome Initiative (GenBank
accession no. AC013258). Database searches revealed that
At1g75010 has a region homologous to FtsZ. 3′-RACE and 5′RACE were used to obtain a full-length cDNA for ARC3 (wild
type) and arc3-1. Sequencing of At1g75010 for the wild type
and arc3-1 mutant revealed a single base-pair mutation; the
2001st base guanine is converted to adenine, thereby converting the tryptophan of position 667 of ARC3 (wild type) to a
stop codon in arc3-1.
To confirm that the ARC3 gene is At1g75010, we utilized
T-DNA insertional lines of the Salk Institute Genome Analysis
Laboratory (http://signal.salk.edu/cgi-bin/tdnaexpress). The
database showed that a T-DNA insertional line, SALK_057144,
had T-DNA in the At1g75010 gene region. We obtained the
line from ABRC (Arabidopsis Biological Resource Center),
analyzed the chloroplast phenotype and checked the position of
the T-DNA insertion. The plant shape and growth rate of the TDNA line were almost the same as those of the wild type (data
not shown). However, the T-DNA line has abnormally large
chloroplasts, and the number of chloroplasts per mesophyll cell
was 11±3 (n = 20), thus, we concluded that the chloroplast phenotype is the same as that of arc3-1 (Fig. 1c). The T-DNA
insertion site was checked by PCR and sequencing. In the TDNA line, T-DNA was inserted into the 13th intron of
At1g75010 (Fig. 2). In addition, the wild-type DNA fragment
of the ARC3 gene was introduced into the arc3-1 mutant for
complement assays. The chloroplast phenotype of the transgenic plant was similar with respect to chloroplast size and
number (98±13) in cells (Fig. 1d). Based on a forward and
reverse genetic analysis, we confirmed that the ARC3 gene was
At1g75010.
The ARC3 protein is composed of 742 amino acids with a
molecular weight of 82,600 and has a region homologous to
FtsZ at the N-terminal and phosphatidylinositol-4-phosphate 5kinase (PIP5K) at the C-terminal (Fig. 3a). The identity and
similarity of the FtsZ-homologous region (between the 1st and
419th amino acid) of ARC3 with AtFtsZ2-1 are 23% and 40%,
respectively. The identity and similarity of the PIP5K-homologous region of ARC3 (between the 603rd and 730th amino
acid) with AtPIP5K1 are 38% and 52%, respectively. Although
Arabidopsis FtsZs (AtFtsZs) have a transit peptide at the Nterminus, the ARC3 protein does not. The FtsZ-homologous
region of ARC3 does not have complete GTP-binding and
hydrolysis motifs that are highly conserved among bacteria and
archaea. The AtPIP5K-homologous region of ARC3 has no
catalytic domain of PIP5K and had a MORN repeat motif (Fig.
3b, c). This motif is discussed below.
Chloroplast division factor, ARC3
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Fig. 3 Structure and alignment of
ARC3. (a) Structure of ARC3 protein.
Gray, black and hatched boxes represent
FtsZ- and PIP5K-homologous regions and
the MORN repeat, respectively. Alignment between ARC3 and AtFtsZ2-1 (b) or
AtPIP5K (c). AtPIP5K and AtFtsZ2-1 are
At1g60890 and At2g36250, respectively.
Black and shaded boxes show identical
and similar amino acids, respectively.
Boxes show the MORN repeat motif.
Expression of ARC3
We analyzed the mRNA levels of the ARC3 gene in 5-dayold wild type and arc3 mutant plants. Since the ARC3 transcript was not detectable by Northern hybridization, non-saturated conditions for RT-PCR were used in the quantitative
assays. The PCR amplified the ARC3 cDNA between –35 and
+1,257 (numbered from the translation start site). As shown in
Fig. 4a, the amount of arc3 transcript in arc3-1 and in
SALK_057144 was lower than that of ARC3 transcript in the
wild type.
To analyze the amount of ARC3 protein, we prepared a
rabbit antibody against recombinant ARC3 protein and performed Western blotting. Total protein of 5-day-old plants
grown under light was separated by SDS-PAGE, and reacted
with the antibody. The anti-ARC3 antibody specifically recognized a single band with a molecular weight of approximately
80,000 (Fig. 4b, lane 1). The band was detected in the sample
of the wild-type plant, but not in the samples of arc3-1 and
SALK_057144. These results indicated that the abnormal chloroplast phenotype of arc3-1 and SALK_057144 was caused by
loss of the ARC3 protein.
Observation of the behavior of ARC3 protein in chloroplasts
using immunofluorescence microscopy
To analyze the subcellular localization of ARC3 protein in
detail, we examined its localization under the immunofluorescence microscope (Fig. 5). In 5-day-old cotyledon cells, many
chloroplasts were undergoing binary division, and various
stages of division were discerned. ARC3 protein was visualized
with green fluorescence and chloroplasts were visualized with
red chlorophyll autofluorescence. ARC3 protein was located at
the site of chloroplast division in a ring-shaped structure at the
early and middle stages of the process (Fig. 5a, c) but was not
detectable at the late stage of the division (data not shown).
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Chloroplast division factor, ARC3
Fig. 4 Analysis of transcripts and protein of ARC3 in wild type, arc31 and SALK_057144. Analysis of ARC3 transcripts by RT-PCR (a). The
number of PCR cycles was 19 and the reaction was not saturated. Fiveday-old wild type (lane 1), arc3-1 (lane 2) and SALK_057144 (lane 3)
plants grown under light. Western blotting analysis of ARC3 protein in
wild type, arc3-1 and SALK_057144. (b). Twenty µg of total protein of
5-day-old wild type (lane 1), arc3-1 (lane 2) and SALK_057144 (lane
3) plants grown under light.
When pre-immune serum was used in this experiment, no signals were detected in dividing chloroplasts (Fig. 5b, d).
Localization of ARC3 protein in A. thaliana
To determine the distribution of ARC3 protein in A. thaliana chloroplasts, we isolated chloroplasts by two-step Percoll
gradient centrifugation (Aronsson and Jarvis 2002) and then
purified the soluble and insoluble fractions by osmotic disrup-
Fig. 6 Subcellular localization of ARC3 protein. (a) Western blotting analysis of chloroplast proteins probed with anti-ARC3 and control proteins (Rubisco-L and LHCP). Purified chloroplasts (lane 1)
were lysed and the insoluble (lane 2) and soluble (lane 3) fractions
were separated by 12.5% SDS-PAGE. One µg of protein of all fractions was used for the assay. Anti-ARC3 proteins were detected with
the ECL system. (b) Chloroplast import assay. In vitro-synthesized,
radiolabeled proteins were incubated with isolated pea chloroplasts
(lane 1). Chloroplasts were then incubated with thermolysin (lane 2) or
trypsin (lane 3) for 30 min on ice.
tion, and used anti-ARC3 for immunoblot analysis. A strong
signal was detected in the soluble fraction of chloroplasts (Fig.
6a).
To further define the topology of the ARC3 protein in
chloroplasts, we examined in vitro the import of ARC3 protein
into chloroplasts and effect of protease treatment on the
import. A radiolabeled translation product corresponding to
the full-length ARC3 was generated by coupled transcription/
translation, and then incubated with isolated pea chloroplasts
(Fig. 6b). We used two proteases with established differences in
Fig. 5 Immunofluorescence images of ARC3 protein. Red and
green indicate chlorophyll autofluorescence and the secondary antibody of goat anti-rabbit IgG conjugated with Alexa Flour 488.
Anti-ARC3 antibodies (a and c) or pre-immune serum (b and d)
were applied in this assay. (a) and (b) show the early stage of chloroplast division, while (c) and (d) show the mid-stages.
Chloroplast division factor, ARC3
their abilities to penetrate the outer envelope membrane and the
inner envelope membrane. Thermolysin cannot penetrate the
outer envelope membrane (Cline et al. 1984), while trypsin can
penetrate the outer but not the inner envelope membrane
(Jackson et al. 1998). Parallel assays were conducted with
marker proteins whose topologies with respect to the outer and
inner envelope membranes had been well established. After
thermolysin or trypsin treatment, the ARC3 translation product
was fully degraded (Fig. 6b, lanes 2 and 3). The outer envelope
protein, Toc33, was fully degraded, too. In contrast, the inner
envelope protein, Tic22, was not degraded by thermolysin but
was degraded by trypsin, and the stroma protein, Rubisco small
subunit, was not degraded by these proteases. These results
indicated that the ARC3 protein was located on the cytosolic
surface of the outer chloroplast envelope membrane.
Discussion
In this study, we showed that ARC3 was a chimera of a
prokaryotic gene, FtsZ, and eukaryotic gene, PIP5K. Rice, a
monocotyledon, had an ARC3 homolog (accession no.
AB159444). The identity and similarity of the FtsZhomologous region of ARC3 in A. thaliana with the ARC3
homolog in rice are 32.4% and 47.7%, respectively. The
identity and similarity of the PIP5K-homologous region of
ARC3 in A. thaliana with the ARC3 homolog in rice are 62.8%
and 75.7%, respectively. The high identity in the PIP5Khomologous region of ARC3 in A. thaliana and rice suggested
that the region might be important to the function of ARC3. No
other plants have been reported to have the ARC3 homolog.
Recently, the complete genome sequence of a unicellular red
alga, Cyanidioschyzon merolae 10D, was reported (Matsuzaki
et al. 2004), but this alga has no ARC3 homolog. This may be
because the red alga has only one chloroplast per cell, whereas
A. thaliana and rice have multiple chloroplasts in the cell with
regard to dissynchronous plastid division, and the regulatory
mechanisms of chloroplast division may not be the same
between the red alga and higher plants.
PIP5K phosphorylates phosphatidylinositol-4-phosphate
to produce phosphatidylinositol-4,5-bisphosphate, PtdIns(4,5)P2
(Westergren et al. 2001). In plants, phosphoinositides are
involved in the opening of stomatal guard cells (Parmar and
Brearlye 1993), and salt stress signal transduction (Dove et al.
1997, Meijer et al. 1999). We analyzed the kinase activity of
PIP5K for various forms of ARC3 protein (full-length ARC3,
putative mature ARC3 protein between the 42nd and 742nd
amino acid and PIP5K-homologous region of ARC3 protein
between the 501st and 742nd amino acid); however, no kinase
activity was detected (data not shown). The PIP5K-homologous region of ARC3 has a MORN repeat motif. The MORN
repeat motif has been identified in proteins called junctophilins
in animal cells (Takeshima et al. 2000). These proteins are
components of the junctional complexes present between the
plasma membrane and the endoplasmic reticulum. The MORN
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motif is necessary for the binding of junctophilin-1 to the
plasma membrane. The target of the MORN domain in the
plasma membrane is thought to be phospholipids. From these
reports and our present results, the MORN domain might have
an important role in the attachment of ARC3 protein to the
outer envelope membrane of the chloroplast. The arc3-1
mutant protein is expected to be smaller than wild-type ARC3
protein because arc3-1 has a single base-pair mutation, thereby
converting tryptophan 667 of ARC3 (wild type) to a stop
codon. Since the mutant had no such small ARC3 protein (see
Fig. 4b), the MORN repeat motif might affect the stability of
ARC3 protein. These possibilities will have to be examined in
the future.
Immunocytochemical studies indicated that plant FtsZ
proteins form ring structures at sites of chloroplast division in
the inner stroma (Vitha et al. 2001, Mori et al. 2001, Kuroiwa
et al. 2002), but ARC3 was located in the outer envelope of the
chloroplast (Fig. 6b). Miyagishima et al. (2001) suggested that
the outer PD ring filaments were composed of an unidentified
polypeptide of 56 kDa, which is considerably smaller than
ARC3 (approximately 80 kDa). The outer PD ring is thought to
contract plastids, and the main component of the PD ring
should be a motor protein that consumes energy such as GTP
or ATP. Thus, ARC3 may be another component of the outer
PD ring filaments.
Because the FtsZ-homologous region of ARC3 does not
have complete GTP-binding and hydrolysis motifs which are
highly conserved among bacteria and archaea, ARC3 is not
expected to have the same function as FtsZ. As mentioned
above, both Arabidopsis of dicotyledoneae and rice of monocotyledoneae have ARC3, but prokaryotes have no ARC3
counterparts. If we assume that most of the higher plants have
an ARC3 homolog, during the generation of chloroplasts by
endosymbiosis, ARC3 might be generated by fusion of the
prokaryotic FtsZ and part of the eukaryotic PIP5K. Marrison et
al. (1999) suggested that ARC3 plays an important role in the
initiation of chloroplast division. The FtsZ-homologous region
of ARC3 may be important for the localization in chloroplasts,
and the PIP5K-homologous region may be important for the
regulation by the nucleus. This hypothesis remains to be tested.
Recently, ARC5 and ARC6 were cloned and characterized
(Gao et al. 2003, Vitha et al. 2003). ARC5 is a cytosolic
dynamin-like protein of the chloroplast division machinery. An
ARC5-GFP fusion protein localized to a ring at the chloroplast
division site. Chloroplast import and protease protection assays
indicated that the ARC5 ring is positioned on the outer surface
of the chloroplast. ARC6 is a chloroplast-targeted DnaJ-like
protein localized to the plastid envelope membrane. An ARC6GFP fusion protein was localized to a ring at the center of the
chloroplast. However, the molecular mechanisms by which
these ARC proteins functions have not been elucidated. It was
reported that chloroplast division requires FtsZ1, FtsZ2, MinD,
MinE, and ARTEMIS (see review by Osteryoung and Nunnari
2003). But the molecular actions and cooperative nature of
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Chloroplast division factor, ARC3
these ARCs, FtsZs, Mins and ARTEMIS in chloroplast division are still not clear. Further study not only of ARC3 but also
of these proteins and chloroplast division is required.
Materials and Methods
Plant material
A. thaliana, Landsberg erecta, Columbia and arc3 (Landsberg
eracta background) (Pyke and Leech 1994), and SALK_057144
(ABRC) were used for all experiments.
Fine mapping of ARC3
To fine map ARC3, an F2 population was generated from a cross
between arc3 and Columbia wild type. Among 5,000 F2 plants, 1,000
mutants were identified by microscopy. PCR primers for PCR-based
mapping are F1M20.3-5, 5′-ACGATGAGACTGAACACCTTGACCTGTTCC-3′; F1M20.3-3, 5′-AAGGCTCAAAGCTGTAAAACAACATTGACC-3′; F25A4.6-5, 5′-AGAATTTATATTCTTACAGGTCCATTACTC-3′; F25A4.6-3, 5′-TTATTTTTCATTGAAATCATTTTATCTCGC-3′; F25A4.4-5, 5′-ACTTTCCCGAGAGAAAACAATGGCTCTCAG-3′; F25A4.4-3, 5′-CTATCATCAACGGCAAGAACATGTAACTCC-3′; F10A5-5, 5′-TACTTACCTAGTAACCATGTCTACCTCGTC-3′; F10A5-3, 5′-GAGTGAACCGGATTCAAAGACCTCTTTGAG3′; F9E10.Cla-5, 5′-GACCAACTATTCCTTCAATAGACCTAATCG3′; F9E10.Cla-3, 5′-TTTCTCACTGTTTTGAAGAGTACCTTACTG3′. F9E10.Cla is a CAPS (cleaved-amplified polymorphic sequence)
marker. After PCR, the products were digested with ClaI. The other
marker is an INDEL (Insert-Deletion) marker. A GENESCAN analysis (genes.mit.edu/GENESCAN.html) of the BAC clone predicted the
number of open reading frames (ORF) (http://mips.gsf.de/cgi-bin/proj/
thal/bac_cosmid?F9E10).
RNA expression analyzed by RT-PCR
ARC3 gene expression levels were assessed by a kinetic RT-PCR
approach. 5′-GTATTCTAAGAAGTGGTTCCGTAGAAGCGG-3′ and
5′-TCTCCAAGTCCAATATTTGTTTCTTTTTGG-3′ were used as
ARC3-specific primers, and 5′-CTTAGGTATTGCAGACCGTATGAGC-3′ and 5′-GTTTTTATCCGAGTTTGAAGAGGCT-3′, as a control for constant expression (Awai et al. 2001). All genes tested are
unique in the A. thaliana genome and show the same linear amplification rate from 13 to 21 cycles (data not shown). Single-strand DNA
was synthesized from total RNA (0.25 µg) by using avian myeloblastosis virus RT (TaKaRa, Kyoto, Japan) in the presence of oligo (dT)
primer at 55°C for 30 min. PCR aliquots (19th cycle) were analyzed
on agarose gels, blotted, and radio-labelled with specific probes corresponding to the amplified fragments.
Expression and purification of truncated ARC3 proteins
The cDNA of ARC3 protein was amplified by PCR using a
primer with inserted NdeI and HindIII restriction sites at the 5′ and 3′
ends, respectively. The amplified fragment was digested and ligated
into the expression vector pET-24a(+) (Novagen, Madison, WI, U.S.A.).
The sequence of the amplified fragment was confirmed. Expression
and purification were performed as described previously (Westergren
et al. 2001). Escherichia coli strain BL21(DE3) was used for the
expression of the recombinant fusion proteins of 6× His with the truncated ARC3. Overnight cultured BL21(DE3) harboring the truncated
ARC3 gene was diluted 1 : 10 with fresh culture medium and further
grown at 37°C for 1 h. Then, isopropyl β-D-thiogalactoside (IPTG) was
added to a final concentration of 1 mM, and the cells were further cultured at 25°C overnight. The cells were centrifuged for 10 min at 5,000
×g and 4°C. The cell pellets were suspended in a suspension buffer
[10 mM Tris-HCl (pH 8.0), 140 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), and 1 mM
PMSF]. The crude extracts were sonicated and centrifuged for 10 min
at 12,000×g and 4°C. The pellets were suspended in a suspension
buffer supplemented with 6 M urea and sonicated. Triton X-100 was
added to the samples at a final concentration of 1% (W/V) and incubated on ice for 30 min. Then, the samples were dialyzed with the dialyzing buffer [10 mM Tris-HCl (pH 8.0), 140 mM NaCl, 1 mM DTT,
and 1% (W/V) Triton X-100]. The samples were centrifuged for
10 min at 12,000×g and 4°C, and the supernatant was incubated overnight with Ni NTA agarose (Qiagen, Hilden, Germany) at 4°C. The
agarose was washed with the dialyzing buffer. Proteins bound to the
agarose were eluted with the dialyzing buffer containing 0.2 M imidazole. The purified ARC3 protein was injected into rabbits to stimulate
antibody production.
Western blotting analysis
Rabbit antiserum to recombinant ARC3 protein was prepared
according to the conventional method. Intact chloroplasts were isolated
according to the methods of Aronsson and Jarvis (2002). The isolated
chloroplasts were lysed osmotically by suspending them with 50 mM
Tris-HCl (pH 7.5) and vortexed vigorously, and then centrifuged at
30,000×g for 30 min. The bands of immunoreacted proteins were
detected using Alkaline Phosphatase Substrate Kit II (Vector Laboratories) or ECL Advance Western Blotting Detection Kit (Amersham Biosciences).
Immunofluorescence microscopy
Immunofluorescent staining of ARC3 and fluorescent microscopy were performed according to the methods of Mori et al. (2001),
with minor modifications. Five-day-old Arabidopsis was used for
immunofluorescent analysis. Before their fixation in paraformaldehyde,
the tissues were pretreated with 250 µM MBS (m-maleimidobenzoylN-hydroxysuccinimide ester) and 0.5% Triton X-100 in MSB buffer.
The secondary antibody reaction was performed with goat anti-rabbit
IgG conjugated with Alexa Flour 488 (Molecular Probes.)
In vitro chloroplast import assay
The synthesis in vitro of the ARC3 protein, chloroplast import
assays with isolated pea (Pisum sativum) chloroplasts, and protease
treatment with trypsin and thermolysin were performed as described
previously (McAndrew et al. 2001).
Acknowledgments
The authors thank Kevin Pyke, University of Nottingham, and
the Arabidopsis Biological Resource Center for providing arc3-1 and
SALK_057144 seeds. They also thank Dr. Haruko Kuroiwa, Rikkyo
University, for advice on immunofluorescence microscopy. A part of
this work was supported by a Grant of the 21st Century COE Program, Ministry of Education, Culture, Sports, Science and Technology, and a grant from the Ministry of Education, Culture, Sports,
Science and Technology (16770030).
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(Received April 14, 2004; Accepted May 25, 2004)