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Plant Cell Physiol. 49(2): 135–141 (2008)
doi:10.1093/pcp/pcm177, available online at www.pcp.oxfordjournals.org
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Rapid paper
The Bacterial Stringent Response, Conserved in Chloroplasts,
Controls Plant Fertilization
Shinji Masuda 1, *, Kazuki Mizusawa 1, Takakuni Narisawa 2, Yuzuru Tozawa
Ken-ichiro Takamiya 1, 5
2, 3
, Hiroyuki Ohta
4, 5
and
1
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, 226-8501 Japan
Graduate School of Science and Engineering, Ehime University, Ehime, 890-8577 Japan
3
Cell-Free Science and Technology Research Center, Ehime University, Ehime, 890-8577 Japan
4
Center for Biological Resources and Informatics, Tokyo Institute of Technology, Yokohama, 226-8501 Japan
5
Research Center for the Evolving Earth and Planets, Tokyo Institute of Technology, Yokohama, 226-8501 Japan
2
Introduction
The chloroplast, an essential organelle for plants,
performs a wide variety of metabolic processes for host
cells, which include photosynthesis as well as amino acid and
fatty acid biosynthesis. The organelle conserves many
bacterial systems in its functions, implicating its origin from
symbiosis of a photosynthetic bacterium. In bacterial cells,
the stringent response acts as a global regulatory system for
gene expression mediated by a small nucleotide, guanosine
50 -diphosphate 30 -diphosphate (ppGpp), that is necessary for
cell adaptation to diverse environmental stimuli such as amino
acid starvation. Recent studies indicated that proteins similar
to the bacterial ppGpp synthase/hydrolyase are conserved in
plants, although their precise roles are not known. Here we
show that the stringent response in chloroplasts is crucial for
normal plant fertilization. Specifically, one of the Arabidopsis
ppGpp synthase homologs, CRSH (Ca2þ-activated RelA/
SpoT homolog), exhibits calcium-dependent ppGpp synthesis
activity in vitro, and is localized in chloroplasts in vivo.
A knockdown mutation of CRSH in Arabidopsis results in
a significant reduction in silique size and seed production,
indicating that plant reproduction is under the control of
chloroplast function through a ppGpp-mediated stringent
response.
The stringent response, one of the most important
regulatory systems for bacterial gene expression, has been
extensively studied for almost half a century (Cashel et al.
1996, Magnusson et al. 2005, Braeken et al. 2006). These
studies have established that the response is mediated
through the synthesis of a second messenger, guanosine
50 -diphosphate 30 -diphosphate (ppGpp), the levels of which
are maintained in Escherichia coli cells by two enzymes,
RelA and SpoT. Both enzymes synthesize ppGpp by
phosphorylating GTP using ATP (pppGpp is initially
produced and is then converted to ppGpp) in response to
several environmental changes such as deficiencies in amino
acids, iron, nitrogen, phosphate or carbon. ppGpp controls
transcription of a large number of genes through direct
interaction with RNA polymerase (Artsimovitch et al.
2004), and also controls translation (Ochi 2007), DNA
replication (McGlynn and Lloyd 2000, Wang et al. 2007),
GTP-binding proteins (Ochi 2007, Raskin et al. 2007),
proteases (Kuroda et al. 1999, Kuroda et al. 2001) and
activity of enzymes for purine biosynthesis (Gallant et al.
1971, Hou et al. 1999), which is necessary for adaptation
to environmental stimuli. ppGpp can be hydrolyzed under
certain conditions by SpoT, but not by RelA.
Recent genome sequence data have indicated that
RelA/SpoT-like proteins are present in algae and higher
plants (van der Biezen et al. 2000, Kasai et al. 2002).
In addition, (p)ppGpp was identified in chloroplasts
(Takahashi et al. 2004), suggesting that the stringent
response is conserved in plant cells. In Arabidopsis, three
genes for RelA/SpoT-homologs (RSH1, RSH2 and RSH3)
have been identified (van der Biezen et al. 2000).
Keywords: Arabidopsis — Chloroplast — ppGpp —
RelA — SpoT — Stringent response.
Abbreviations: BAPTA, 1,2-bis(o-aminophenoxy)ethaneN,N,N,0 N0 -tetraacetic acid; CaMV, cauliflower mosaic virus;
CRSH, Ca2þ-activated RelA/SpoT homolog; GFP, green
fluorescent protein; GUS, b-glucuronidase; IPTG, isopropyl-bD-thiogalactopyranoside; LHCP, light-harvesting chlorophyllbinding protein; LSU, RUBISCO large subunit; (p)ppGpp,
guanosine 50 -di(tri)phosphate 30 -diphosphate; RSH, RelA/SpoT
homolog.
*Corresponding author: E-mail, [email protected]; Fax, þ81-45-924-5823.
135
136
The stringent response in chloroplasts
(a)
(d)
1
H2N
cTP
(e)
583
48
RelA-SpoT domain
CO2H
WT
ATP
EF hand
+ Vector
(b)
HD domain
+ CRSH
ppGpp
(c)
pppGpp
(f)
BAPTA (mM)
1
5
10
T S T S T S T S
0
Origin
0 1 5 10
BAPTA (mM)
Fig. 1 Calcium-dependent ppGpp synthesis by CRSH. Primary structure of Arabidopsis CRSH (a) and partial amino acid sequence
alignments between Arabidopsis RSH1, RSH2, RSH3 and CRSH, and E. coli RelA and SpoT (b, c). An arrow indicates the conserved
glycine residue necessary for ppGpp synthetase activity (see text). (d) The E. coli relA-spoT mutant harboring the CRSH expression plasmid
(þ CRSH) complemented the loss of relA/spoT function. (e, f) The putative mature form of Arabidopsis CRSH was synthesized by a cell-free
system with the indicated concentrations of BAPTA in the presence of [g-32P]ATP to monitor Ca2þ-dependent (p)ppGpp synthesis by CRSH
(e) or [14C]leucine to monitor CRSH synthesis by the cell-free system (f). The reaction mixtures (T) and the soluble fractions (S) isolated
by centrifugation were analyzed.
In addition, we recently identified another RelA/SpoT
homolog, CRSH (Ca2þ-activated RelA/SpoT homolog),
from rice (Tozawa et al. 2007). Given that CRSH genes
have only been found in the genome sequences of
monocotyledonous and dicotyledonous angiosperms,
CRSH and its paralogs may be specialized for function in
higher plants. This idea is supported by the phylogenetic
tree based on the deduced amino acid sequences of RelA/
SpoT-like domains, which indicates that CRSHs are a
distinct branch from bacterial RelA/SpoT proteins and
plant RSHs (Supplementary Fig. S1). However, the
physiological roles of CRSH are uncharacterized. In this
study, we have obtained biochemical and genetic evidence
showing that Arabidopsis CRSH, localized in chloroplasts,
is required for host plant fertilization.
Results
Arabidopsis CRSH exhibits Ca2þ-dependent ppGpp
synthetase activity
Arabidopsis has a single gene (At3g17470) encoding
CRSH that contains a putative chloroplast transit peptide
at its N-terminus, a RelA/SpoT-like domain in its central
region and two Ca2þ-binding EF-hand motifs at its
C-terminus (Fig. 1a). The RelA/SpoT domain of CRSH
has a conserved glycine residue necessary for ppGpp
synthetase activity (Wendrich and Marahiel 1997)
(Fig. 1c), but does not conserve an HD domain responsible
for ppGpp hydrolysis (Aravind and Koonin 1998) (Fig. 1b),
suggesting that CRSH possesses only ppGpp synthetase
activity, as does E. coli RelA protein. To determine whether
CRSH functions as a RelA/SpoT homolog, we expressed
the putative mature form of CRSH in an E. coli relA-spoT
double mutant. The mutant strain harboring only an empty
vector could not grow on minimal medium because of its
inability to synthesize ppGpp; however, the mutant strain
expressing CRSH could grow on minimal medium like the
wild type (Fig. 1d), indicating that CRSH possesses ppGpp
synthetase activity.
To analyze further the enzymatic properties of CRSH,
we performed a ppGpp synthetase assay coupled with cellfree protein synthesis. The putative mature form of CRSH
was synthesized and assayed in the presence of [g-32P]ATP
and in the presence of different concentrations of the Ca2þspecific chelator BAPTA [1,2-bis(o-aminophenoxy)ethaneN,N,N0 ,N0 -tetraacetic acid]. Given that the reaction mixture
contained GTP, any CRSH synthesized had the potential of
producing (p)ppGpp in the reaction. We also monitored
CRSH synthesis under identical conditions except
for replacement of [g-32P]ATP with [14C]leucine.
The CRSH synthesized by the cell-free system showed
(p)ppGpp synthetase activity in the absence of BAPTA,
The stringent response in chloroplasts
(a)
(b)
GFP
137
Chl
Merge
(anti-GFP )
CRSH-GFP
75
75
60
(anti-CRSH )
CRSH-GFP
CRSH
75
50
(CBB)
(d)
(kDa)
CRSH
60
75
50
(c)
R
oo
t
100
R
os
e
C tte
au le
lin af
Si e le
liq
af
u
Fl e
ow
e
St r
em
(kDa)
Line 3
Line 3
Line 4
Col.
Line 1
Pro35S ::CRSH-GFP
(CBB )
(e)
Soluble
20
10
Insoluble
5
20
10
5
(µl)
CRSH
LSU
ProCRSH ::GUS
LHCP
Fig. 2 Subcellar and organ-specific expression of Arabidopsis CRSH. (a) Western analysis with rosette leaves of Pro35S::CRSH-GFP lines,
showing the highest expression in line 3. An equal amount of total proteins was loaded in each lane. (b) Fluorescence from GFP and
chlorophyll in a transgenic plant (line 3), indicating the localization of CRSH–GFP fusion protein in chloroplasts. (c) Western analysis with
soluble and insoluble fractions of isolated chloroplasts. Anti-LSU and anti-LHCP were used as the respective controls for soluble and
insoluble fractions. (d) Organ-specific CRSH expression examined by Western analysis, indicating the highest expression in flowers. An
equal amount of total proteins was loaded in each lane. (e) A plant expressing ProCRSH::GUS showing GUS activity in mature pistils, green
petals and immature sepals.
and 1 mM BAPTA completely inhibited the enzymatic
activity (Fig. 1e). On the other hand, CRSH synthesis by the
cell-free system was unaffected in the presence of 1 mM
BAPTA (Fig 1f), indicating that Ca2þ is required for
(p)ppGpp synthesis by CRSH. These results indicate that
Arabidopsis CRSH possesses Ca2þ-activated (p)ppGpp
synthetase activity.
Localization of Arabidopsis CRSH
We next determined the localization of CRSH in
Arabidopsis. For this purpose, we generated transgenic
plants expressing a CRSH–GFP (green fluorescent protein)
fusion protein under control of the cauliflower mosaic virus
(CaMV) 35S promoter. Western analysis in the transgenic
lines tested indicated that line 3 showed the highest
expression of the fusion protein (Fig. 2a). The CRSH
fusion protein was visible in the leaves of this transgenic
line, and GFP fluorescence was clearly merged with
chlorophyll autofluorescence (Fig. 2b), indicating that the
fusion protein is localized to chloroplasts. Subcellular
fractionation coupled with Western analysis also indicated
that immunoreactivity with anti-CRSH antiserum was
highly specific for the soluble fraction of isolated chloroplasts from Arabidopsis leaf tissues (Fig. 2c). The size of
the CRSH band (60 kDa) in Western analysis was smaller
than the calculated value for full-length CRSH (66.6 kDa),
suggesting that the N-terminal transit peptide of CRSH had
been removed during translocation across the chloroplast.
From these results, we conclude that Arabidopsis CRSH is
localized to and functions in chloroplasts.
We next determined the organ specificity of CRSH
expression. Western analysis indicated that Arabidopsis
CRSH is not expressed in roots, but is expressed in all shoot
tissues tested, with the highest expression in flowers
(Fig. 2d). To analyze its expression pattern further, we
constructed transgenic Arabidopsis expressing a reporter
gene fusion of the CRSH promoter and the b-glucuronidase
gene (ProCRSH::GUS). In the flowering stage of the
transformants, intense specific staining was observed in
mature pistils, green petals and immature sepals (Fig. 2e),
suggesting that CRSH has a role in pistil maturation.
Knockdown mutation of CRSH results in the unusual silique
formation
To gain insight into the physiological importance of
CRSH function, especially during the flowering stage, we
constructed transgenic lines expressing CRSH cDNA under
the control of the CaMV 35S promoter. In T1 transgenic
lines, a variety of CRSH expression levels could be observed
(Fig. 3a). Some lines showed very low CRSH expression,
138
O
X1
0
O
X1
5
O
X1
9
CRSH
(kDa)
75
CRSH
LSU
50
(b)
(d)
C
ol
.
O
X1
9
O -a
X1
9b
(c)
Pro35S::CRSH
C
ol
.
O
X3
O
X4
O
X7
O
X8
(a)
The stringent response in chloroplasts
anti-CRSH
Col.
OX19-a
OX19-b
Ponso
(e)
Col.
OX19
Col.
OX19-a
Fig. 3 Phenotype caused by a CRSH knockdown mutation in Arabidopsis. (a) Western analysis with rosette leaves of T1 transgenic plants
expressing Pro35S::CRSH showing no detectable CRSH expression in OX19. An equal amount of total proteins was loaded in each lane.
Protein bands of LSU were detected as loading controls. (b) An OX19 plant showing abnormal silique formation. (c) Western analysis with
rosette leaves of two T2 lines derived from an OX19 T1 plant, OX19-a and OX19-b. An equal amount of total proteins was loaded on
each lane. (d, e) Overall growth of the wild type (Col.), OX19-a and OX19-b did not differ significantly; however, OX19-a, but not OX19-b,
had abnormal siliques. Plants were 45 d old. Bars are 1 cm.
perhaps due to co-suppression of the introduced CRSH
cDNA with the endogenous CRSH. These transgenic lines
showed no difference in overall growth profiles; however, at
the reproductive stage, one of the transgenic lines (OX19)
with no detectable CRSH expression (Fig. 3a) formed
abnormal siliques that were significantly smaller than those
of the wild type (Fig. 3b). In the OX19 line, a few mature
seeds sometimes formed, with only 100 T2 seeds reaching
maturity, although 430,000 mature seeds were obtained
from the wild type under identical conditions. This aberrant
phenotype was not observed in other transgenic lines tested,
including OX7 and OX8, which retained low levels of
CRSH expression (Fig. 3a).
We next tested whether the phenotype of OX19 was
observable in later generations. Western analysis indicated
that lack of CRSH expression observed in OX19 T2 plants
was unstable; CRSH expression was recovered in T2 plants,
with one exception showing almost no CRSH expression,
like the OX19 T1 plant. We selected two lines, OX19-a and
OX19-b, that respectively represented almost no and a high
expression level of CRSH from the T2 plants (Fig. 3c).
The overall growth profiles of OX19-a and OX19-b were
the same as that of wild-type plants; however, at the
reproductive phase, siliques of OX19-a, but not those of
OX19-b, were significantly smaller than those of wild-type
plants (Fig. 3d, e). The phenotype of OX19-a was stable
even in further generations (T3 plants). These results
indicate that CRSH has an important role in normal silique
development and seed production.
Unsuccessful pollination of the CRSH knockdown mutant
Given that CRSH is specifically expressed in pistils
(Fig. 2e), a knockdown mutation of CRSH in an OX19-a
line could affect pistil maturation. To test this hypothesis, we analyzed a series of flowers from wild-type and
OX19-a lines (Fig. 4); these stages of flower development
have been described previously (Smyth et al. 1990,
Sanders et al. 1999). In wild-type plants, the filaments
and petals had elongated to a position above the stigma
at the time of flower opening (stages 12 and 13), and
pollen release occurred before extension of the stigma
above the anthers (stage 13L). Following successful
pollination, the pistil elongated to generate a silique
(stage 14). In contrast, OX19-a pistils were already very
long at the time of flower opening, and the filaments
could not position the anthers in line with the stigma,
which was fully expanded before flower opening (stages
13 and 13L). As a result, pollination of OX19-a failed
(stage 14). Anther dehiscence was also delayed in OX19-a,
such that mutant anthers did not dehisce at the time of
flower opening (stage 13) or release pollen grains at the
initiation of flower senescence (stage 14). The pollen
grains germinated similarly to those of the wild type
in vitro (stage 14; inset), and artificial pollination with
OX19-a pistils and pollen grains resulted in normal silique
formation (Fig. 5). From these observations, we conclude
that CRSH has a crucial role in adjusting the timing of
pistil and pollen maturation that is required for successful
pollination.
The stringent response in chloroplasts
Stage 12
Stage 13
Stage 13L
139
Stage 14
Col.
OX19-a
Fig. 4 CRSH knockdown mutation results in unusual flower
development. A developmental series of flowers from the wild type
(Col.) and a CRSH knockdown mutant (OX19-a) were photographed using a digital microscope. These stages were defined
previously (Smyth et al. 1990, Sanders et al. 1999). Inset:
microscopic images of germinated pollen. White and black bars
are 0.5 mm and 100 mm, respectively.
Discussion
In this study, we have shown that Ca2þdependent ppGpp synthesis by CRSH in chloroplasts is
crucial for higher plant fertilization. This is the first report
showing a physiological role for plant RelA/SpoT-like
proteins.
Arabidopsis has three other RelA/SpoT homologs,
RSH1, RSH2 and RSH3 (van der Biezen et al. 2000,
Givens et al. 2004). RSH2 and RSH3 show very high
similarity (80%), and seem to have both ppGpp synthetase and hydrolyase activities, because they have a
conserved glycine residue necessary for ppGpp synthetase
activity and the HD domain responsible for ppGpp
hydrolyase activity (Fig. 1b, c). On the other hand, RSH1
does not have a conserved glycine residue (but does
conserve the HD domain), suggesting that it lacks ppGpp
synthetase activity (Fig. 1c). Thus, RSH1, RSH2/3 and
CRSH may differentially control ppGpp levels to modulate
plastid function. We recently isolated Arabidopsis RSH1,
RSH2 and RSH3 mutants generated by T-DNA insertion;
pollination in the mutant lines was like that in the wild
type (S. Masuda, K. Mizusawa and H. Ohta, unpublished
data), indicating that CRSH acts as a specific ppGpp
generator to control plastid function during flower
development. The observation that CRSH has been found
only in flowering land plants (Supplementary Fig. S1)
supports this idea. ppGpp levels in chloroplasts are
Fig. 5 An OX19-a inflorescent flower was artificially pollinated
with OX19-a anthers releasing pollen grains. The flower was
marked with purple thread. After 6 d, the silique was found at the
specific position of the developing inflorescence (indicated by a
white arrow).
markedly increased in response to environmental stresses,
such as pathogens, wounding, heat shock, cold shock,
drought and plant hormonal treatments (Takahashi et al.
2004). Because expression of Nicotiana tabacum RSH2
is activated by jasmonate treatment (Givens et al. 2004),
the elevated levels of ppGpp in response to such stresses
are dependent on RSH2, RSH3 or both, although the
functions of the homologs have not yet been established
in planta.
How CRSH controls pollination is open to speculation. Given that Ca2þ signaling has been shown to modulate
plant fertilization in multiple sequential steps (Dumas and
Gaude 2006), ppGpp-dependent control of plastid function
may be involved in the signaling network. One possible role
for ppGpp is control of nucleotide, amino acid and fatty
acid biosynthesis as in bacterial cells, which would affect the
homeostatic levels of plant hormones such as auxin,
cytokinin and jasmonates that are synthesized from their
respective precursors tryptophan, ATP/ADP and linolenic
acid (Turner et al. 2002, Farmer et al. 2003, Woodward and
Bartel 2005, Sakakibara 2006). Clearly, gaining further
insight into how CRSH works in this complex signaling
network will be of great interest in understanding cell
signaling in plants. Because some genes for plastid proteins
are encoded in the nucleus, host cells have been thought to
control plastid function exclusively. However, the results
from the present study suggest that plastids have maintained the ability to produce ppGpp during evolution,
which may have served the purpose of controlling their
reproduction, and effected the successful spread of the
organelle in nature.
140
The stringent response in chloroplasts
Materials and Methods
Plant materials and generation of transgenic plants
All plants were the Columbia ecotype of Arabidopsis thaliana.
A cDNA clone for AtCRSH was kindly provided by RIKEN
Bioresource Center. The cDNA was first cloned into plasmid
pDONR/ZEO (Invitrogen, California, USA) and then inserted
into the pGWB2 and pGWB5 vectors (kindly provided by Dr.
Nakagawa at Shimane University) using the Gateway system
(Invitrogen, California, USA); the resulting plasmids were used for
creation of transgenic Pro35S::CRSH and Pro35S::CRSH-GFP
lines, respectively. A region 1.5 kb upstream from the start
codon of AtCRSH was amplified by PCR with forward
(50 -GGCATGCTGGAACTGGGATTGAGATAC-30 ) and reverse
(50 -GGGATCCCACAGACATTTGAGAATTTGAG-30 ) primers,
and the fragment was cloned into pBI101 (Clontech, California,
USA). The resulting plasmid was used for creation of a transgenic
ProCRSH::GUS line. These plasmid constructs were separately
introduced into Agrobacterium tumefaciens strain GV310 and
transferred into plants by infiltration of flowers. Plants were grown
at 228C on Murashige–Skoog medium or soil at about 50 mmol
photons m2 s1.
Complementation analysis of E. coli
Escherichia coli wild-type strain (W3110) and CF1678 (relA,
spoT) (Xiao et al. 1991) were used for the complementation
analysis. These strains were lysogenized with -DE3 phage (Merck,
Nottingham, UK) in order to confer isopropyl-b-D-thiogalactopyranoside (IPTG)-inducible gene expression from the T7 promoter. For construction of an expression vector for CRSH, the
insert DNA of pETCRSH (see below) was cut out by digestion
with NdeI and NotI, and the fragment was inserted into the
pET21a vector (Merck, Nottingham, UK). The resulting plasmid
was used to transform CF1678(DE3). As controls, pET21a was
transferred into W3110(DE3) and CF1678(DE3). These cells were
grown at 378C on an agar plate of modified MOPS medium
(Masuda and Bauer 2004) containing 0.1 mM IPTG and 100 mg l1
ampicillin.
Assay of translation-coupled (p)ppGpp synthetase activity
A cDNA encoding putative mature forms of AtCRSH was
constructed by PCR with a plasmid harboring the full-length
cDNA as template and a gene-specific forward primer (50 -ATCA
TATGTCGGAGGTGGAGGATACTGC-30 ) and reverse primer
The
(50 -ATCTAGATTAATGGGTTGAGAGACGATCC-30 ).
amplified fragments were digested with NdeI and XbaI, and then
cloned into the corresponding sites of pEU3NH, which is a version
of pEU3b (Cell-Free Sciences, Yokohama, Japan) modified by
introduction of an NdeI linker into the original multiple cloning
site. The resulting plasmid, designated pEACRSHD39, was used as
template for in vitro transcription with SP6 RNA polymerase
(Madin et al. 2000).
The synthesized mRNA for the putative mature forms of
AtCRSH was synthesized with a cell-free system according to a
batch method (Madin et al. 2000) in the presence of [g-32P]ATP
(3,000 Ci mmol1) or [14C]leucine (0.074 mCi ml1) (GE Healthcare, Buckinghamshire, UK). Reaction mixtures (25 ml) in the
presence or absence of BAPTA were incubated at 268C for 2.5 h,
and the reaction was stopped by the addition of 1 ml of 88% formic
acid. After the further addition of 12.5 ml of phenol : chloroform : isoamyl alcohol (25 : 24 : 1, by vol.), the mixture was agitated and
then centrifuged at 10,000g for 5 min at 48C. The aqueous phase
was transferred to another tube, and a 3 ml portion was spotted
onto a polyethyleneimine–cellulose thin-layer sheet (Merck,
Nottingham, UK). For one-dimensional analysis, 1.5 M KH2PO4
was used as the chromatographic solvent (Kasai et al. 2004). The
proteins synthesized in the presence of [14C]leucine were assessed
by SDS–PAGE. The chromatogram was exposed to a BAS-III
imaging plate, and the associated radioactivity was detected with a
BAS-2500 analyzer and quantitated with Image Gauge version
3.41software (Fujifilm, Tokyo, Japan).
Immunoblotting and histochemical GUS analyses
AtCRSH antibody was prepared as follows: The cDNA for
the putative mature form of AtCRSH was amplified by PCR using
a forward (50 -GGGGGGGCATATGACGGCTCGGTCTCCGG
AG-30 ) and a reverse primer (50 -GGCTGCAGATGGGTTGAG
AGACGATCC-30 ), and the amplified fragment was inserted into
the pZErO-2 vector (Invitrogen, California, USA) at its EcoRV
site. The inserted fragment was excised by digestion with NdeI and
NotI, and then cloned into the pET28a vector (Merck,
Nottingham, UK). The resulting plasmid was named pETCRSH.
His-tagged AtCRSH was overexpressed in E. coli strain
BL21(DE3)/pETCRSH in LB medium at 168C for at least 16 h.
The expressed protein was purified from inclusion bodies using
His-bind resin according to the methods provided by the
manufacturer (Merck, Nottingham, UK). A rabbit was immunized
with 0.5 mg of the purified His-tagged CRSH, and the antiserum
obtained was used for Western analysis.
Plants were homogenized in buffer A (20 mM Tris–HCl,
pH 7.5, and 10 mM NaCl), the homogenate was centrifuged
at 5,000g for 10 min, and the supernatant was used as total
protein. The intact chloroplasts isolated (Douce and Joyard 1982)
were disrupted by five freeze–thaw cycles in buffer A, and then
centrifuged at 15,000g for 10 min. The supernatant and
precipitate were used as soluble and insoluble fractions, respectively. The insoluble fraction was further washed three times
with buffer A. An equal amount of total protein or proteins
of chloroplast fractions (5–15 mg) was separated on an SDS–
polyacrylamide gel and electroblotted onto a polyvinylidene
difluoride (PVDF) membrane. The bands immunoreactive against
anti-CRSH, anti-GFP (Living Colors; Clontech, California, USA),
anti-LSU (Rubisco large subunit; Agrisera AB, Vannas, Sweden)
and anti-LHCP (light-harvesting chlorophyll-binding protein;
Agrisera AB, Vannas, Sweden) were detected using the
enhanced chemiluminescence (ECL) system (GE Healthcare,
Buckinghamshire, UK).
Detection of GUS expression was performed as follows.
Tissue samples were soaked at 378C for 1 d in a buffer
containing 1 mM 5-bromo-4-chloro-3-indolylglucuronide, 0.5 mM
K3Fe(CN)6, 0.5 mM K4Fe(CN)6, 0.3% (v/v) Triton X-100, 20%
(v/v) methanol and 50 mM phosphate-buffered saline. Then, the
samples were soaked in 70% (w/v) ethanol to stop the reaction.
In vitro pollen grain germination
Pollen grain germination was conducted as described
previously (Fan et al. 2001).
Supplementary material
Supplementary material mentioned in the article is available
to online subscribers at the journal website www.pcp.
oxfordjournals.org.
The stringent response in chloroplasts
Funding
The Sumitomo Foundation and the Research
Foundation for Opto-Science & Technology (to S.M.);
the Ministry of Education, Culture, Sports, Science and
Technology of Japan (to S.M., Y.T. and H.O.).
Acknowledgments
We dedicate this paper to Ken-ichiro Takamiya, who passed
away in an unfortunate traffic accident while this study was being
conducted. We thank Hiroshi Shimada and Mie Shimojima for
helpful suggestions during the study.
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(Received November 30, 2007; Accepted December 18, 2007)