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The Core of Chloroplast Nucleoids Contains Architectural
SWIB Domain Proteins
W OA
Joanna Melonek,a Andrea Matros,b Mirl Trösch,a Hans-Peter Mock,b and Karin Krupinskaa,1
a Institute
b Leibniz
of Botany, Christian-Albrechts-University of Kiel, 24098 Kiel, Germany
Institute of Plant Genetics and Crop Plant Research, 06466, Gatersleben, Germany
A highly enriched fraction of the transcriptionally active chromosome from chloroplasts of spinach (Spinacia oleracea) was
analyzed by two-dimensional gel electrophoresis and mass spectrometry to identify proteins involved in structuring of the
nucleoid core. Among such plastid nucleoid-associated candidate proteins a 12-kD SWIB (SWI/SNF complex B) domain–
containing protein was identified. It belongs to a subgroup of low molecular mass SWIB domain proteins, which in
Arabidopsis thaliana has six members (SWIB-1 to SWIB-6) with predictions for localization in the two DNA-containing
organelles. Green/red fluorescent protein fusions of four of them were shown to be targeted to chloroplasts, where they
colocalize with each other as well as with the plastid envelope DNA binding protein in structures corresponding to plastid
nucleoids. For SWIB-6 and SWIB-4, a second localization in mitochondria and nucleus, respectively, could be observed.
SWIB-4 has a histone H1 motif next to the SWIB domain and was shown to bind to DNA. Moreover, the recombinant SWIB-4
protein was shown to induce compaction and condensation of nucleoids and to functionally complement a mutant of
Escherichia coli lacking the histone-like nucleoid structuring protein H-NS.
INTRODUCTION
Chloroplast function is indispensable for plant productivity.
Chloroplasts are thought to originate from ancestral cyanobacteria and hence have retained prokaryotic features in the organization of their genome and the structure of the corresponding
nucleoids. Typically, chloroplasts contain hundreds of plastid
genomes in the form of circular 120- to 180-kb molecules organized in aggregates that in analogy with bacterial nucleoids
were named plastid nucleoids (Kuroiwa et al., 1998; Lilly et al.,
2001; Sato et al., 2003). Number, position, and compactness of
nucleoids are known to parallel the changes in gene expression
occurring during chloroplast development. Two DNA binding
proteins, the plastid envelope DNA binding protein (PEND) (Sato
et al., 1998) and the matrix attachment region binding filamentlike protein1 (MFP1) (Jeong et al., 2003), are known to be
involved in redistribution of the plastid nucleoids from the envelope membrane to the thylakoids during the transition from
proplastids to chloroplasts. However, proteins involved in regulation of nucleoid morphology and compactness and corresponding
to the bacterial HU (for histone-like protein from Escherichia coli
strain U93) and HU-like proteins have not been identified hitherto
(Sato et al., 2003; Sakai et al., 2004).
Plastid nucleoids were prepared by different approaches.
Preparations shown to preserve the morphological integrity of
nucleoids involve sucrose density gradient centrifugation (Kuroiwa
1 Address
correspondence to [email protected].
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Karin Krupinska
([email protected]).
W
Online version contains Web-only data.
OA
Open Access articles can be viewed online without a subscription.
www.plantcell.org/cgi/doi/10.1105/tpc.112.099721
and Suzuki, 1981; Sato et al., 1997; Cannon et al., 1999; Majeran
et al., 2012). For enrichment of transcriptional activity, nucleoids
are released from thylakoids using the nonionic detergent Triton
X-100 and are purified by gel filtration. These so-called transcriptionally active chromosomes (TACs) (Igloi and Kössel,
1992) contain subunits of the plastid-encoded RNA polymerase
(Suck et al., 1996), which together with further DNA binding
proteins are also found in transcriptionally active soluble fractions prepared from chloroplasts (Schröter et al., 2010; Steiner
et al., 2011). TAC fractions have been prepared from chloroplasts of Euglena gracilis (Hallick et al., 1976), mustard (Sinapis
alba; Bülow et al., 1987), barley (Hordeum vulgare; Krupinska
and Falk, 1994; Suck et al., 1996), spinach (Spinacia oleracea;
Krause and Krupinska, 2000), and Arabidopsis thaliana (Pfalz
et al., 2006). The conventionally prepared TAC fraction of spinach chloroplasts (TAC-I) was shown to have a rather complex
protein composition and was further purified by precipitation
with protamine sulfate and resolubilization in the presence of heparin
followed by a second round of gel filtration. The resulting fraction,
enriched in the core components of the complex and showing
50-fold higher specific transcription activity rates, was termed
TAC-II (Krause and Krupinska, 2000).
Apart from the proteins of the transcriptional apparatus,
the TAC fraction is expected to contain a number of nucleoidassociated proteins with DNA binding properties that might be
involved in replication, recombination, and repair of DNA as well
as in structuring of nucleoids (Sakai et al., 2004). In bacteria, low
molecular mass proteins, such as the 9-kD HU and HU-like
proteins (HLPs), are responsible for formation of nucleosomelike structures (Dillon and Dorman, 2010) and affect DNAassociated processes, such as replication and transcription
(Dorman and Deighan, 2003; Kamashev et al., 2008; Dillon and
Dorman, 2010). However, genes encoding potential counterparts
of the bacterial nucleoid-associated proteins have not been found
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in the genomes of higher plants (Riechmann et al., 2000; Sato
et al., 2003). Among the few structural proteins of plastid nucleoids thus far identified is the bifunctional sulfite reductase,
which was shown to induce compaction of plastid nucleoids coinciding with a repression of transcriptional activity (Sekine et al.,
2002).
Proteome analyses performed with TAC fractions isolated
from mustard and Arabidopsis did not identify DNA binding
proteins with a molecular mass below 20 kD (Pfalz et al., 2006).
Nevertheless, several novel TAC-associated proteins (PTACs)
with DNA/RNA binding domains were identified (Pfalz et al.,
2006). PTAC1 is identical with the plastid-nucleus-located Whirly1
(Grabowski et al., 2008). Its plastidial form was shown to bind to
a subset of intron-containing RNA species (Prikryl et al., 2008;
Melonek et al., 2010). PTAC3 belongs to the SAP (for SAF A/B,
Acinus, and PIAS) domain protein family, members of which
are involved in chromosomal organization (Aravind and Koonin,
2000). The PTAC11 protein (Pfalz et al., 2006) is identical with
Whirly3, which recently was described to function together with
Whirly1 as an antirecombination protein (Maréchal et al., 2009).
PTAC14 from Arabidopsis was identified as a SET (for Suppressor of variegation 3-9 [Su(var)3-9], Enhancer of zeste
[E(Z)], and Trithorax [Trx]) domain–containing protein. Nuclear
proteins with a SET domain were shown to function as histone
methyltransferases (Thorstensen et al., 2011). PTAC14 belongs
to those plastidial proteins, which were acquired from the nucleus where they are involved in chromatin remodeling (Jacob
et al., 2009).
Here, we report on identification of a low molecular mass
SWIB domain–containing plastid nucleoid-associated protein
(ptNAP) in the proteome of the TAC-II fraction that is highly
enriched in nucleoid core proteins from spinach chloroplasts.
SWIB domain–containing proteins are subunits of nuclear ATPdependent chromatin-remodeling complexes of the SWI/SNF
type that facilitate transcriptional activation (Bennett-Lovsey
et al., 2002). In silico searches showed that the genome of
Arabidopsis encodes six low molecular mass SWIB domain
proteins. By fusion with green fluorescent protein (GFP), four of
them were shown to be targeted to chloroplasts where they
colocalize in speckles, indicating association with nucleoids.
One of these having a histone H1-like domain (SWIB-4) was
shown to bind to DNA. Recombinant SWIB-4 protein induced
condensation of E. coli nucleoids in vivo and functionally complemented an E. coli mutant lacking the gene encoding the
histone-like nucleoid protein H-NS. Our results indicate that the
low molecular mass SWIB domain proteins in chloroplasts might
be functional equivalents of the bacterial nucleoid-associated
proteins (NAPs) involved in shaping of nucleoid architecture.
RESULTS
Proteomic Identification of Chloroplast
Nucleoid-Associated Proteins
To identify novel proteins involved in shaping of chloroplast
nucleoids, a highly enriched fraction of the TAC from spinach
chloroplasts was used for proteome analysis. For our studies,
we used chloroplasts isolated from spinach leaves, which provide excellent material for biochemical studies requiring highly
purified and functional organelles (Gruissem et al., 1986;
Babujee et al., 2010) that cannot easily be prepared from
Arabidopsis (Bayer et al., 2011). To obtain a highly enriched
fraction (TAC-II), a conventional TAC fraction (TAC-I) prepared
from 20 kg of spinach leaves was subjected to a second round of
gel filtration resulting in TAC-II (Figure 1A). The protein compositions of both fractions were analyzed by two-dimensional gel
electrophoresis (2-DE). For isoelectric focusing, pH gradients from
3 to 10 were chosen. After second-dimension denaturing gel
electrophoresis, proteins were visualized by staining with Coomassie blue (Figures 1B and 1C). Image analysis allowed the
detection of 132 spots on the 2-DE gel of TAC-I, whereas on the
2-DE gel of TAC-II, only 85 protein spots were clearly discernible
(Figure 1D). These 85 proteins were manually excised from the gel
and digested with trypsin (see Supplemental Figure 1 online). By
Figure 1. Two-Dimensional Separation of TAC-I and TAC-II Proteins.
(A) Scheme of the purification protocol of TAC-I and TAC-II fractions
from spinach chloroplasts (Krause and Krupinska, 2000). After digestion
with DNase I and RNase A, the proteins were separated by isoelectric
focusing in a pH gradient from 3 to 10 and by 2-DE.
(B) and (C) Spot patterns obtained on gels after staining with Coomassie
blue of TAC-I (B) and TAC-II (C) proteins, respectively, are shown. Image
analysis of the 2-DE gels revealed 132 protein spots on the TAC-I and 85
on TAC-II gel.
(D) Relative spot intensity of proteins detected in TAC-II compared with
the intensities of corresponding proteins in the TAC-I fraction as determined with Progenesis PG240 software.
SWIB Domain Proteins of the Chloroplast Nucleoid Core
means of mass spectrometry, 53 TAC-II proteins with protein
scores of 60 or more and individual peptide scores of 39 or more
could be identified (see Supplemental Data Set 1 online). Thirteen of them were identified by homology with protein sequences of spinach, 15 by homologies with protein sequences
from Arabidopsis, and 14 by homology with sequences from rice
(Oryza sativa), and single proteins were identified by homologies
with other plant species (see Supplemental Data Set 1 online).
Despite precipitation with protamine sulfate and the second
round of gel filtration used for preparation of TAC-II (Krause and
Krupinska, 2000), the fraction contains several contaminants as
it has previously been reported for other preparations enriched
in nucleoid-associated proteins (Schröter et al., 2010; Majeran
et al., 2012). For example, the two polygalacturonases (spots 7
and 51) are likely contaminants. These proteins are synthesized
as precursors at the endoplasmic reticulum from where they
are transported in vesicles to their destinations in the plasma
membrane and cell wall. It is likely that these proteins in the TAC
fraction are derived from the endoplasmic reticulum, which is
known to be physically tightly associated with chloroplast envelope membranes (Andersson et al., 2007).
Among the identified proteins in TAC-II are several putative
nucleoid core components with putative roles in transcription,
replication, maintenance, and structuring of DNA. As expected,
subunits of the plastid-encoded RNA polymerase, namely,
RpoC1 (spot 13) and RpoC2 (spot 18), were detected in TAC-II.
The RpoA subunit was identified with a score under 60 and
therefore was not included in Supplemental Data Set 1 online.
Six of the identified TAC proteins are putative architectural
ptNAPs with DNA binding properties that are able to alter the
DNA compaction through bending, wrapping, or bridging (Table
1; see Supplemental Data Set 1 online, spots 10, 17, 32, 37, 39,
and 53).
Previous biochemical investigations revealed low molecular
mass DNA binding proteins in nucleoid fractions, which were
assigned as candidates for functional replacement of the missing
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HU-like proteins in plants (Briat et al., 1984). Hitherto, an identification of such basic low molecular mass proteins was not
possible. Purification of the TAC fraction from spinach chloroplasts enabled the identification of a SWIB domain protein in
spot 37 of the 2-DE gel (see Supplemental Figure 1 online). The
peptide masses determined for the spinach protein matched
with a 12-kD SWIB domain–containing protein encoded by the
At2g35605 gene (Table 1). The low molecular mass SWIB domain protein was found to have a high Lys content and a high
isoelectric point, thereby resembling bacterial NAP (Dillon and
Dorman, 2010) (Table 1).
The smallest putative ptNAP identified in spot 37 belongs to
the family of SWIB domain proteins that has 20 members in
Arabidopsis as identified by The Arabidopsis Information Resource (TAIR; www.arabidopsis.org) database analyses (Figure
2A). Phylogenetic analysis with MEGA5 software (Tamura et al.,
2011) revealed that based on their amino acid sequences, plant
SWIB proteins can be subdivided into four major groups (Figure
2A; see Supplemental Data Set 2 online). The two group 4
proteins with a molecular mass of ;50 kD have high similarity
with the yeast SWP73 protein (Jerzmanowski, 2007), which is
a component of the SWI/SNF complex with a functional role in
transcriptional activation by modulation of the chromatin structure (Cairns et al., 1996). Proteins of groups 2 and 3 have, in
addition to the SWIB domain, other domains, such as Really
Interesting New Gene, zinc-finger, plus-3, and GYF (for Gly-TyrPhe) or the DEK domain. All members of these two groups are
predicted to be involved in histone modification and regulation
of transcription in the nucleus (TAIR) (Swarbreck et al., 2008).
The 12-kD SWIB domain protein found in TAC-II belongs to
group 1, consisting of so-called stand-alone SWIB domain
proteins, where the SWIB domain is found as an isolated protein
and is not part of a larger protein. In prokaryotes, such SWIB
domain proteins were found exclusively in Chlamydia, which as
parasitic bacteria probably have acquired the SWIB domain
gene from a mammalian host (Bennett-Lovsey et al., 2002).
Table 1. Identified Putative Plastid Nucleoid-Associated Proteins in the TAC-II Fraction
No.
(Spot No.)a
1 (37)
2 (39)
3 (32)
4 (17)
5 (53)
6 (10)
a
Identified ptNAP Proteins
SWIB complex BAF60b domaincontaining protein
Myb-related transcription factor
SANT superfamily
Jasmonate- and ethylene-responsive
factor3 (JERF3)
Hypothetical protein, armadillo/b-catenin
repeat family protein
Myb-related transcription factor
SANT superfamily
Unknown protein, RecF/RecN/SMC
N-terminal domain
AGI
Accession
Numberb
At2g35605
At2g02060
At3g14230
At2g27430
At5g02320
Conserved Domains/
Motifs/Regions
kD
pI
Lys
Content
SWIB/MDM2 domain,
low complexity region
N-CoR and TFIIIB DNA
binding domains
AP2 DNA binding
Chromatin remodeling
12
10
9%
DNA binding, regulation
of transcription
Regulation of transcription
29
7
6%
36
9
8%
Armadillo-type fold,
armadillo-like helical
N-CoR and TFIIIB DNA
binding domains
Nucleic acid binding/protein
binding
DNA binding, regulation of
transcription
49
8
8%
62
8
10%
66
5
11%
RecF/RecN/SMC
N-terminal domain
Chromatin and DNA
dynamics/DNA metabolism
and recombination
At5g36780
Spot number on the TAC-II 2-DE gel (see Supplemental Data Set 1 online).
AGI, Arabidopsis Genome Initiative.
b
Function
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of the AP2/EREBP subfamily in organelles (Schwacke et al.,
2007). Moreover, for the At2g44940 protein, a dual targeting to
chloroplasts and nucleus could be shown (Schwacke et al., 2007).
The protein identified in spot 10 represents a structural maintenance of chromosomes (SMC) protein. SMC proteins belong to
a ubiquitous protein family present in almost all prokaryotic and
eukaryotic organisms and have functions in chromosome condensation, segregation, cohesion, and DNA recombination and
repair (Graumann and Knust, 2009). In E. coli, SMC proteins were
shown to induce negative supercoiling of DNA in vivo, indicating
their involvement in organization and compaction of nucleoids
(Graumann and Knust, 2009). An SMC (At-SMC1) as well as an
armadillo/b-catenin protein were previously identified in the proteome of chloroplasts from Arabidopsis (Kleffmann et al., 2004;
Zybailov et al., 2008). The protein identified in spot 17 has similarity in sequence with the Arabidopsis armadillo/b-catenin protein. Armadillo repeat proteins form a large group of 108 proteins
in Arabidopsis. Two further putative ptNAPs identified in TAC-II
belong to the family of the MYB/SANT domain transcription factors having a helix-turn-helix (HTH) DNA binding domain (spots
39 and 53). Animal and fungal proteins containing SANT domains
(for SWI3, ADA2, N-Cor, TFIIB) were found to be associated with
histone modifying enzymes and ATP-dependent chromatin remodeling proteins (Boyer et al., 2004; Jerzmanowski, 2007).
Subcellular Localization of the Low Molecular Mass SWIB
Proteins of Group 1
Figure 2. Phylogenetic Analysis of 20 SWIB Domain–Containing Proteins of Arabidopsis and Characterization of the Putative Organellar
SWIB Proteins.
(A) The phylogenetic tree was constructed using MEGA5 with the
neighbor-joining method. Bootstrap values calculated from 1000 trials
are shown at each node. The evolutionary distances were computed
using the Poisson correction method and are in units of the number of
amino acid substitutions per site. The multiple sequence alignment was
performed with ClustalW algorithm and is available as Supplemental
Data Set 2 online. The proteins could be subdivided in four groups.
Members of group 1 have molecular masses ranging from 10 to 20 kD.
Bar indicates number of substitutions per site.
(B) Table presenting the nomenclature and molecular properties of the
six proteins of group 1. AGI, Arabidopsis gene identifier; Mw, molecular
mass; H1, histone H1-like motif. TargetP localization predictions: M,
mitochondria; C, chloroplasts; TP, targeting peptide length (aa, amino
acids). The proteins were ordered according to their molecular mass.
The other TAC-II proteins possessing DNA binding domains
have molecular masses higher than 20 kD (Table 1). The protein identified in spot 32 is a member of the APETALA2 (AP2)
subfamily of the AP2/EREBP (for ethylene-responsive element
binding proteins) family of transcription factors unique to plants
(Okamuro et al., 1997). The AP2/EREBP domain consists of 60
to 70 amino acids and is involved in binding to DNA (Weigel,
1995). In silico analyses predicted localization for some members
Proteome analysis of TAC-II allowed identification of a 12-kD
SWIB domain protein. Masses of peptides obtained by trypsin
digestion fitted best to the 12-kD SWIB-6 protein, which has the
lowest molecular mass of the six members of group 1 (Figure 2).
All SWIB proteins designated to this group are predicted to be
targeted to either chloroplasts or mitochondria (TargetP; Figure
2B) (Emanuelsson et al., 2000). To investigate the subcellular
localization of the proteins, GFP fusion constructs were prepared
and employed for transient transformation assays with tobacco
(Nicotiana tabacum) protoplasts. Microscopy inspections revealed
that apart from SWIB-1, which was observed in the cytoplasm
when fused to GFP, GFP fusion proteins of all group 1 SWIBs were
targeted to chloroplasts (SWIB-2, SWIB-3, and SWIB-4), to mitochondria (SWIB-5), or to both organelles (SWIB-6) (Figure 3). All
GFP chimeric proteins located in chloroplasts showed punctuate
patterns resembling the distribution of nucleoids visualized by the
PEND:GFP construct (Terasawa and Sato, 2005) on the red
background of the chlorophyll autofluorescence (Figure 3). The
mitochondrial localization of SWIB-5:GFP and SWIB-6:GFP
proteins could be confirmed by labeling with MitoTracker Orange
(see Supplemental Figure 2 online). As a control a construct encoding GFP alone is shown (Figure 3).
Association of SWIB-2, SWIB-3, SWIB-4, and SWIB-6 with
Chloroplast Nucleoids
To investigate whether the four chloroplast-located SWIB domain proteins (SWIB-2, SWIB-3, SWIB-4, and SWIB-6) might be
components of the same subplastidial structures, their potential
colocalization was examined by simultaneous transformation of
SWIB Domain Proteins of the Chloroplast Nucleoid Core
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protoplasts with GFP and red fluorescent protein (RFP) fusion
constructs. Microscopy analyses showed that SWIB-2:GFP colocalized with SWIB-4:RFP, SWIB-3:GFP with SWIB-6:RFP, and
SWIB-4:GFP with SWIB-6:RFP (Figure 4A). In all four GFP/RFP
combinations tested, fluorescence signals frequently overlapped,
indicating an association of all four SWIB proteins with the same
subplastidial structures. To examine whether these structures
could represent nucleoids, SWIB-4:GFP and SWIB-6:GFP constructs were transiently cotransformed with a PEND:dsRED
construct in tobacco protoplasts. The PEND protein is known
to bind to plastid DNA and to be suited for visualization of
plastid nucleoids (Terasawa and Sato, 2005). The overlay of
green and red fluorescence signals within the chloroplasts revealed that both SWIB-4 and SWIB-6 colocalize with the PEND
protein (Figure 4A).
Sequence Homology, Conserved Regions, and Potential
DNA Binding Domains of Plastidial SWIB Proteins
The amino acid sequences of the four SWIB domain proteins
targeted to chloroplast nucleoids have high similarity due to
the highly conserved SWIB domain (Figure 4B). One of the
masses determined by mass spectrometry belongs to a theoretical peptide derived from the SWIB domain of SWIB-6 as revealed by theoretical digestion with trypsin using the program
ExPASy (Expert Protein Analysis System; http://www.expasy.
ch/; Gasteiger et al., 2003). Masses of five further peptides could
correspond to peptides obtained by partial digestion of SWIB-6
or to modified peptides. When the theoretical masses of peptides obtained by digestion with trypsin from the four different
proteins were compared, it became obvious that SWIB-2 and
SWIB-3 are very similar sharing four identical peptides with
masses above 500 D. SWIB-4 and SWIB-6 were found to share
two identical peptides with masses above 500 D but not to
share peptides with SWIB-2 and SWIB-3. Besides the SWIB
domain, SWIB-2, -3, and -4 have low compositional complexity
regions (LCRs) in their sequences (Figure 4B). So far no universal function for such LCRs could be elucidated (Coletta
et al., 2010). The LCR region (amino acids 31 to 59; Figure 4B)
at the N terminus of the SWIB-4 protein shows high similarity
to a part of the C-terminal domain of the histone H1 of tobacco
(see Supplemental Figure 3A online). Moreover, in silico analysis revealed that this region of SWIB-4, which is extremely
rich in Lys, is highly similar to an analogous region found at the
N terminus of the CND41 (for chloroplast nucleoid DNA binding
protein 41 kD) protein from tobacco (see Supplemental Figure
3A online) that was described to function as a DNA binding
domain (Nakano et al., 1997; Murakami et al., 2000). This part
of the SWIB-4 protein might represent a domain for binding to
DNA. No such motifs could be found in the sequences of
SWIB-2, -3, and -6, respectively.
Figure 3. Subcellular Distribution of SWIB:GFP Chimeric Proteins.
The six low molecular mass SWIB domain proteins of Arabidopsis predicted to be located in organelles were fused with GFP at their C termini
and transiently expressed in tobacco protoplasts. As controls, constructs encoding PEND:GFP and GFP alone were used. Bars = 7.5 or
4 mm (as indicated by an asterisk).
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SWIB-4 in TAC-II Binds to DNA as Revealed by
Immunological Analysis
For immunological analyses, the peptide sequence KSDSPAKKTPRSTG representing the residues 50 to 63 of SWIB-4
was chosen for production of a SWIB-4–specific antibody (Figure
4B). Protein fractions derived from spinach chloroplasts and
subfractions thereof were analyzed by immunoblotting. The predominant form of the protein in chloroplasts had a molecular
mass of 17 kD (Figure 5), which is consistent with the molecular
mass of the processed form of the in vitro translation product
imported into chloroplasts (see Supplemental Figure 3B online).
A second higher molecular mass form of the protein could result
from posttranslational modification (Figure 5A). Two plastidial and
two higher molecular mass nuclear forms of the SWIB-4 protein
also could be detected in protein fractions isolated from chloroplasts and nuclei of Arabidopsis leaves (see Supplemental Figure
4 online).
We further tested the presence of SWIB-4 in TAC-I and TACII. When TAC-I and TAC-II fractions, having the same amount of
protein, were analyzed, the level of immunoreactive protein
detected by the SWIB-4 antibody was higher in TAC-II than in
TAC-I, indicating an enrichment of the SWIB-4 protein during
purification of TAC. In comparison, the relative level of the nucleoid-associated protein MFP1 decreased during purification of
TAC-I as shown previously with corresponding fractions from
barley chloroplasts (Melonek et al., 2010). SWIB-4 binds to DNA
when it is not altered by posttranslational modification because
TAC fractions contained only the lower molecular mass form of
SWIB-4 (Figure 5A).
Next, we examined whether SWIB-4 in TAC-II binds to DNA.
For this purpose, TAC-II proteins after transfer onto nitrocellulose membranes were renatured. By incubation with a radiolabeled 16S rDNA probe, three low molecular mass protein
bands were detected (Figure 5B, lane 1). When the membrane
prior to incubation with DNA was incubated with the SWIB-4
antibody, the intensity of the band corresponding to a DNA
binding protein of ;17 kD was diminished (Figure 5B, lane 2).
This result indicates that SWIB-4 might represent one of the low
molecular mass DNA binding proteins of TAC fractions detected
previously (Bülow et al., 1987).
Remodeling of Nucleoid Architecture by SWIB-4
To investigate whether SWIB-4 might have an architectural function in nucleoids, we overexpressed the gene encoding SWIB-4 in
E. coli. DNA staining with 49,6-diamidino-2-phenylindole (DAPI)
showed that after induction with isopropyl-b-D-thiogalactopyranoside (IPTG), cells accumulating the recombinant SWIB-4
Figure 4. Colocalization and Sequence Analysis of Plastidial SWIB Proteins.
(A) Colocalization of the plastidial SWIB proteins as well as SWIB-4:GFP
and SWIB-6:GFP with the PEND:dsRED protein in chloroplast nucleoids.
For better contrast, red fluorescence of chlorophyll was converted into
blue. Bar = 2 µm.
(B) Sequence alignment of four chloroplast located stand-alone SWIB
domain containing proteins. The SWIB domain is framed. The Lys-rich
region similar to the part of histone H1 is written in red color. The
alignment was performed with ClustalW (http://www.ebi.ac.uk/Tools/
clustalw2/index.html) and depicted using The Sequence Manipulation
Suite (http://www.bioinformatics.org/sms2/).
SWIB Domain Proteins of the Chloroplast Nucleoid Core
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To investigate whether SWIB-4 could be a functional analog
of an abundant histone-like protein termed H-NS, we aimed at
complementation of an E. coli mutant lacking this NAP (Spassky
et al., 1984; Spurio et al., 1992). The H-NS protein was shown to
induce compaction of nucleoids and to affect expression of
;5% of the E. coli genes (Hommais et al., 2001). Among the
genes repressed by H-NS is the cryptic b-glucoside (bgl) operon, which encodes all gene products necessary for uptake and
fermentation of the aryl-b,D-glucosides, such as salicin or arbutin (Higgins et al., 1988; Schnetz, 1995; Caramel and Schnetz,
1998). Dhns mutant cells that can metabolize salicin appear as
red colonies on salicin-containing indicator plates, whereas colonies unable to ferment salicin are colorless (Schnetz et al., 1987).
To test whether the Arabidopsis SWIB-4 gene can functionally
substitute for the E. coli hns gene, cells of the Dhns mutant strain
were transformed with the pQE30 plasmid containing SWIB-4.
After 2 d of growth on indicator plates, colonies of the hns
mutant and the hns mutant transformed with pQE30 plasmid
were red (Figure 6D), whereas colonies of the Dhns mutant strain
transformed with a control pQE30+SWIB-4 plasmid stayed
colorless as colonies of the wild type (Figure 6D). This result
showed that SWIB-4 in vivo can functionally replace the E. coli
H-NS protein.
The T-DNA insertion mutants so far available have insertions
either in 59 untranslated region (swib-4-1 and -2) or in the intron
(swib-4-3) of the SWIB-4 gene (see Supplemental Figure 5A and
Supplemental Table 1 online). The 20-fold to fivefold enhanced
transcript levels in some of the mutants were found to coincide
with retarded postgermination development of seedlings (see
Supplemental Figures 5B and 5C online).
Figure 5. Immunological Detection of the SWIB-4 Protein in Chloroplast
Subfractions and in TAC and DNA Binding Analysis.
(A) Immunoreactions were performed with total protein (TP), chloroplast
protein (C), membrane (M), and stroma (S) fractions extracted from
spinach leaves as well as TAC-I and TAC-II protein extracts (10 µg), respectively. To demonstrate the purity of the chloroplast subfractions,
immunoblots decorated with antibodies directed toward cytochrome
b559 apoprotein A (PsbE) and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RbcL) are shown. In addition, the
level of MFP1 in the TAC-I and TAC-II fraction was determined. For
comparison, the Coomassie blue staining of the protein fractions is
shown (CBB).
(B) Protein/DNA gel blot analysis with TAC-II proteins. After SDS-PAGE
and blotting, TAC-II proteins were renatured and incubated with a radioactively labeled 16S rDNA probe. The sample shown in lane 1 was
directly incubated with DNA, while the samples shown in lanes 2 and 3
prior to incubation with DNA were incubated with the anti-SWIB-4 antibody or with BSA, respectively. The protein band preventing binding to
DNA in the presence of the SWIB-4 antibody is indicated by an arrow.
For comparison, silver staining of the fraction is shown in lane 4.
protein had highly condensed nucleoids, while control cells
showed a uniform distribution of DNA (Figure 6A). Accumulation
of SWIB-4 (Figure 6B) coincided with growth arrest of the cells
for several hours shortly after addition of IPTG (Figure 6C).
A similar effect on growth of E. coli cells and compaction of
nucleoids had been observed previously when the abundant
histone-like protein H-NS was overproduced (Spassky et al.,
1984; Spurio et al., 1992).
DISCUSSION
Our proteome analysis of a fraction highly enriched in transcriptionally active chromosomes prepared from spinach chloroplasts
(TAC-II) revealed the presence of architectural nucleoid-associated
proteins of plastids (ptNAP) possessing domains typically found
in proteins with functions in chromatin organization, such as
SMC, Armadillo, SANT, and SWIB. The ptNAPs identified in
TAC-II have not been detected in TAC fractions from mustard
and Arabidopsis (Pfalz et al., 2006), indicating that a second
round of gel filtration after precipitation of conventionally prepared TAC-I with protamine sulfate (Krause and Krupinska,
2000) is suited to enrich proteins having low abundance in the
conventional TAC preparations. On the other hand, due to limitations in the resolution of highly charged proteins by isoelectric
focusing, several DNA binding proteins of the core nucleoids
might have escaped detection. As described previously, the
specific transcriptional activity of the TAC-II fraction was enhanced by more than 50-fold compared with TAC-I (Krause and
Krupinska, 2000). During preparation of TAC-II from TAC-I,
several proteins most probably loosely attached to DNA were
lost, for example, MFP1 and PTAC1, which is identical with
Whirly1 (Melonek et al., 2010). It is likely that the TAC-II fraction is
enriched in proteins belonging to the nucleoid core, which has
been described as central body by electron microscopy (Herrmann
et al., 1974; Yoshida et al., 1978). Also for nucleoids of human
mitochondria, a layered structure has been proposed (Bogenhagen
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The Plant Cell
Figure 6. Impact of Chloroplast SWIB-4 on Compactness of Bacterial Nucleoids and Complementation of the H-NS–Deficient Mutant.
(A) The M15 E. coli cells were transformed either with the pQE30 vector containing the full coding sequence of SWIB-4 or with the empty pQE30 vector.
Cells were cultivated at 37°C and the IPTG was added at an OD600 ranging from 0.7 to 1. For visualization of the nucleoids, the E. coli cells were stained
with DAPI. Bar = 10 µm.
(B) Immunological analysis with the SWIB-4 antibody. Proteins of bacterial cultures were resolved by denaturing gel electrophoresis on a 14% (w/v)
polyacrylamide gel and either transferred onto nitrocellulose membrane or stained with Coomassie blue. The arrow indicates the protein band representing SWIB-4. Ctrl, control (uninduced E. coli cells).
(C) Effect of nucleoid condensation mediated by SWIB-4 on E. coli cell proliferation. E. coli cells expressing either the empty pQE30 vector or the pQE30
vector containing the SWIB-4 coding sequence were grown at 37°C, and after addition of IPTG, the OD600 was measured at different time points.
(D) Complementation of the H-NS–deficient mutant by SWIB-4. E. coli Dhns::FRT mutant cells grown on MacConkey agar plates supplemented with
salicin, Dhns::FRT+pQE30 mutant cells transformed with the empty plasmid, Dhns::FRT+pQE30+SWIB-4 mutant cells transformed with plasmid encoding the Arabidopsis SWIB-4 gene, as well as E. coli wild-type (WT) cells are shown. Arabidopsis SWIB-4 can functionally replace the E. coli H-NS
protein in vivo as demonstrated by the colorless colonies formed by the Dhns::FRT+pQE30+SWIB-4 cells.
et al., 2008). In this model of mitochondrial nucleoid architecture,
replication and transcription were suggested to occur in the central
core, whereas translation and complex assembly occur in peripheral regions (Bogenhagen et al., 2008).
The smallest ptNAP identified in the TAC-II proteome has a
SWIB domain. SWIB domain–containing proteins were also
found in the proteome of TAC-II fractions isolated from barley
plastids (data not shown) and therefore seem to be essential
components of plastid nucleoids. In a proteome study on rice
etioplasts, a low molecular mass homolog of Arabidopsis SWIB
proteins was identified (von Zychlinski et al., 2005). SWIB-3 was
recently identified in a comprehensive proteome analysis of
maize (Zea mays) chloroplast nucleoids prepared by sucrose
gradient centrifugation (Majeran et al., 2012). In the TAC-II
fraction, only SWIB-6 was detectable and none of the other
three putative plastidial SWIB proteins. This could be due to
unusual high isoelectric points of the spinach SWIB proteins
whose sequences are not known so far, likely preventing their
resolution by isoelectric focusing.
Database analyses showed that the SWIB domain protein
identified by our study of a nucleoid subproteome belongs to
a group of low molecular mass SWIB domain proteins predicted
to be targeted to either chloroplasts or mitochondria. By fusion
with GFP, four members of this group could be observed to be
targeted to chloroplasts, where they were shown to have a
speckled distribution resembling the distribution of the nucleoids. Colocalization with GFP and RFP fusion proteins showed
that all four members of the group colocalize in the nucleoids.
For SWIB-4 and SWIB-6, the nucleoid localization was furthermore shown by cotransformation with a PEND:dsRED construct.
In addition, SWIB-6:GFP also was found to be targeted to mitochondria, whereas SWIB-4:GFP also was shown to be located in
the nucleus. The nuclear localization could be confirmed by immunological analysis of isolated nuclei. Several nuclear regulatory
DNA binding proteins are predicted to be dually targeted to both
organelles or one organelle and the nucleus (Small et al., 1998;
Schwacke et al., 2007; Krause and Krupinska, 2009). Such
proteins might have roles in coordination of the nuclear genome
SWIB Domain Proteins of the Chloroplast Nucleoid Core
and the plastid nucleoids, which together with the chondriome
constitute the integrated genetic system of the plant cell (Herrmann
et al., 2003). Most of the already described proteins targeted to two
or even three of the DNA containing compartments have
functions in the maintenance of DNA, telomere structuring, or
in regulation of gene expression (Krause and Krupinska, 2009;
Hammani et al., 2011).
Immunological analyses showed that SWIB-4 is indeed
a component of TAC-II. When the same amounts of protein were
loaded onto an SDS gel, the level of SWIB-4 detected in TAC-II
was higher than that in the TAC-I, indicating that during enrichment of the TAC core components, the SWIB-4 protein also
got enriched. SWIB-4 was shown to colocalize with the other
three low molecular SWIB proteins targeted to chloroplasts. Its
enrichment in TAC-II suggests that all plastidial SWIB proteins
are core components of the nucleoids.
Among the four SWIB proteins shown to be targeted to
chloroplast nucleoids when fused to GFP, SWIB-4 is the only
one having in addition to the SWIB domain a histone-H1-like
domain, making it an excellent candidate for functional replacement of the original bacterial HLPs in the plastid nucleoid.
If SWIB-4 is indeed functionally related to bacterial architectural proteins, it should influence the shape of chloroplast
nucleoids. The structure of plastid nucleoids and DNA topology were proposed to have important consequences for gene
expression (Bogorad, 1991; Salvador et al., 1998), which is known
to undergo dramatic changes during chloroplast development
(Nemoto et al., 1988, 1989; Baumgartner et al., 1989; Krupinska
and Falk, 1994).
We propose that the SWIB-4 protein, which has a low molecular mass, high isoelectric point, and a Lys-rich region, is a
plastid-located NAP. Bacterial NAPs (e.g., the 9-kD HU, 11-kD
Fis [for factor for inversion stimulation] and 15 kD H-NS proteins)
have in common a low molecular mass, a high Lys content, and
nonspecific binding to DNA (Luijsterburg et al., 2006; Dillon and
Dorman, 2010) and to other nucleic acids (Balandina et al., 2002;
Brescia et al., 2004; Kamashev et al., 2008). SWIB-4 has a histonelike domain that is likely to be involved in binding to DNA as well
as RNA and single-stranded DNA as also shown for histones
(Spelsberg et al., 1970).
Overexpression in E. coli revealed the architectural activity of
chloroplast SWIB-4. This result confirmed that bacteria and eukaryotes use functionally conserved basic low molecular mass
proteins for shaping and remodeling of their genomes (Luijsterburg
et al., 2006). Complementation of an E. coli mutant lacking H-NS
suggests that SWIB-4 is functionally equivalent to this most
abundant NAP that when overproduced was shown to condense
nucleoids resulting in death of the E. coli cells (Spassky et al.,
1984; Spurio et al., 1992). For mutants having up to 20-fold enhanced level of the SWIB-4 transcript, a delayed postgermination
development could be observed (see Supplemental Figure 5 online). Whether induced overexpression of the gene in plants would
lead to a change in plastid nucleoid architecture and an arrest of
growth remains to be demonstrated.
When we compared the expression of the genes encoding the
four SWIB proteins of Arabidopsis at different developmental
stages using the data of Genevestigator (Zimmermann et al.,
2004), it became obvious that the gene encoding SWIB-4 has
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the lowest and almost constitutive level of expression, while the
expression profiles of genes encoding SWIB-2, -3, and -6 show
fluctuations (see Supplemental Figure 6 online). Expression of
the gene encoding SWIB-4 is enhanced in plants at the time of
bolting and in siliques. This pattern of expression is in accordance with a function of SWIB-4 in meristematic tissue where it
might silence transcriptional activity in plastids by compaction
of nucleoids when it is overexpressed.
The Lys-rich N-terminal sequence of the mature chloroplast
SWIB-4 has high similarity with the sequence of the histoneH1-like protein (Hc1) from Chlamydophila felis, a chlamydial
bacterium possessing the only two SWIB domain proteins
described for prokaryotes. One is a low molecular stand-alone
SWIB protein, and in the second the SWIB domain is fused to
the COOH terminus of topoisomerase I (Barry et al., 1992). It
has been demonstrated that this H1-like protein together with
the SWIB domain protein functions in remodeling of the nucleoid during the chlamydial life cycle (Hackstadt et al., 1991;
Barry et al., 1992). In eukaryotes, histone H1 is acting as a
DNA bridging protein (Hendzel et al., 2004). Dinoflagellates,
despite being eukaryotes, are known to lack histones and corresponding nucleosome structures. Three histone-like proteins,
HCc1, HCc2, and HCc3, found in dinoflagellates were shown
to share homology with both eukaryotic histone H1 and prokaryotic HU (Wong et al., 2003). While HCc1 and HCc2 were
shown to complement mutants lacking HU, HCc3 did not complement the phenotype of HU-deficient cells and is thus not functionally equivalent to HU (Chang et al., 1999). Similarly to HCc3,
SWIB-4 could not reconstitute mutants lacking HU protein (data not
shown).
In accordance with the presence of histone-like proteins in
plastids, in recent years, evidence has accumulated that counterparts of proteins known to be involved in histone modifications
in the nucleus do also occur in plastids. One example is the
Arabidopsis SET domain–containing Trithorax-related protein 5
(ATXR5) acting as a monomethyltransferase in the nucleus
(Raynaud et al., 2006; Jacob et al., 2009), which was found by
fusion with GFP to be dually targeted to plastids and the nucleus
(Raynaud et al., 2006). Another example are two rice histone
deacetylases (OsHDAC10 and 6) (Chung et al., 2009).
Overproduction of SWIB-4 of Arabidopsis as well as HCc3
from dinoflagellates in E. coli resulted in increased compactness
of nucleoids. The condensing effect of these proteins on nucleoid structure is in accordance with a function as DNA bridger.
The major DNA-bridging protein in bacterial nucleoids is H-NS
(Bertin et al., 2001; Dame et al., 2005; Luijsterburg et al., 2008).
Complementation of a mutant lacking H-NS by chloroplast
SWIB-4 indicates that the two proteins are functionally equivalent (Bertin et al., 2001; Dame et al., 2005). Recently, it has been
reported that the architectural activity of H-NS can switch between bridging and stiffening and that the switch depends on
pH and divalent cations (Barber, 1986; Liu et al., 2010). In
chloroplasts, divalent cations are counterions of protons forming
gradients above the thylakoid membrane (Izawa and Good,
1966; Barber, 1986). It will be an exciting question for future
research whether SWIB-4 is involved in the adjustment of the
plastid nucleoid architecture and function in response to photosynthetic performance of chloroplasts.
10 of 14
The Plant Cell
METHODS
Plant Materials
Spinach (Spinacia oleracea cv Deutscher Frisee) leaves used for chloroplast
preparation were purchased from the local market. Tobacco (Nicotiana
tabacum cv Xanthi) plants used for protoplast transformation were grown
for 5 to 6 weeks on sterilized Murashige and Skoog medium at 24°C with
a 16-h photoperiod.
Isolation of TAC Fractions
For isolation of TAC fractions, chloroplasts were prepared from spinach
leaves by an established procedure (Douce et al., 1973). TAC extracts
from spinach chloroplasts were prepared as described previously (Krause
and Krupinska, 2000). To examine the transcriptional activity, aliquots of
TAC fractions were incubated at 30°C with radioactively labeled a-32UTP (Hartmann Analytic) as described (Krupinska and Falk, 1994).
Two-Dimensional Separation of TAC Proteins and
Mass Spectrometry
Prior to 2-DE, TAC protein fractions were treated with DNase I and RNase A
(MBI Fermentas) and precipitated with chloroform/methanol following the
protocol of Wessel and Flügge (1984). Protein concentration was estimated
with the Plus One 2D-Quant Kit (GE Healthcare). 2-DE and protein staining
were performed according to Witzel et al. (2009), with the following modifications: 50 µg of TAC proteins were loaded on immobilized pH gradient
dry gel strips (7 cm, pH gradient 3 to 10; GE Healthcare). The second dimension was performed on 11.25% (w/v) polyacrylamide gels containing
0.1% (w/v) of SDS. Separated proteins were stained with colloidal Coomassie Brilliant Blue (Gel Code Blue; Thermo Fisher Scientific). Protein spots
were manually excised and trypsin digested (Porcine Sequencing Grade;
Promega) as described (Witzel et al., 2009). Acquisition of peptide mass
fingerprint data and corresponding LIFT spectra was performed using an
ultrafleXtreme matrix-assisted laser desorption/ionization time-of-flight
device (Bruker Daltonics) equipped with a Smartbeam-II laser with a repetition rate of 1000 Hz. The spectra were calibrated using external calibration
and subsequent internal mass correction. For databank searching, Biotools
3.2 software (Bruker Daltonics) with the implemented MASCOT search
engine (Matrix Science) was used, searching for Viridiplantae in the nonredundant National Center for Biotechnology Information database (26/07/
2010, Green Plants 284943 sequences). Search parameters were as follows:
monoisotopic mass accuracy; 50 to 100 ppm tolerance; fragment tolerance
of 0.3 D; missed cleavages 1; and the allowed variable modifications were
oxidation (Met), propionamide (Cys), and carbamidomethyl (Cys).
amplified from Arabidopsis total cDNA by iProof High-Fidelity DNA polymerase (Bio-Rad) using the following specific forward and reverse primers:
At3g03590for, 59-caccatgtcttccgttgca-39; At3g03590rev, 59-agcagtcttcacaaagtgc-39; At2g14880for, 59-caccatggcggtttcttct-39; At2g14880rev,
59-gaggaagtgaggaccgat-39; At4g34290for, 59-caccatggctctttcttctg-39;
At4g34290rev, 59-aaagtgaggaccaatgag-39; At2g35605for, 59-caccatgtcgagggttttc-39; and At2g35605rev, 59-agcagacttaggaaaatgttg-39. The forward primers contained a CACC overhang for cloning into pENTR/D/
TOPO vector (Invitrogen). pENTR plasmids encoding the particular SWIB
genes were used for site-specific recombination into binary Gateway expression vector pB7FWG2,0 or pB7RWG2 (Invitrogen). The PEND:GFP and
PEND:dsRED constructs were prepared as described (Terasawa and Sato,
2005; Terasawa et al., 2005). As a control, the coding sequence for the
green fluorescent protein was cloned into the pBluescript vector. All constructs were under control of the 35S cauliflower mosaic virus promoter.
Transient Transformation Assays
Tobacco protoplasts used for transient transformation assay were
prepared as described (Nagy and Maliga, 1976). Polyethylene glycol–
mediated transformation was performed as described (Negrutiu et al.,
1987). Briefly, 50 µg of plasmid DNA was gently mixed with 2 3 106
protoplasts and incubated in 40% (w/v) polyethylene glycol 1500 (Merck).
In colocalization experiments, protoplasts were transformed with two
plasmids simultaneously. After 24 h incubation, protoplasts were inspected
by fluorescence microscopy (Leica TCS SP5; Leica Microsystems). For
detection of GFP fluorescence and chlorophyll autofluorescence, the
samples have been excited by the 488- and 458-nm lines of an argon
laser, respectively.
Protein Isolation and Immunoblot Analysis
For extraction of total protein fractions, spinach leaves were ground
in liquid nitrogen. Protein concentrations were determined using RotiNanoquant according to the manufacturer’s instructions (Roth) based on
the method of Bradford (1976). For SDS-PAGE, the buffer system of
Laemmli (1970) was used. For an efficient separation of proteins having
low molecular masses, 16% (w/v) polyacrylamide gels were used and
proteins were blotted onto nitrocellulose (Schleicher and Schuell). For
immunoblot analysis, the polyclonal anti SWIB-4 antiserum was diluted
1:1000 with buffer consisting of 50 mM Tris/HCl, pH 7.4, and 150 mM
NaCl. The antibody was directed toward the peptide KSDSPKKTPRSTG
(residues 50 to 63 of the protein) and was raised in rabbits by Biogenes.
Further primary antibodies used in our study were directed toward cytochrome b559 apoprotein A (PsbE; Vallon et al., 1987), large subunit of
ribulose-1,5-bis-phosphate carboxylase/oxygenase (RbcL; Agrisera), and
MFP1 (Jeong et al., 2003).
Phylogenetic Analysis
The amino acid sequences of 20 Arabidopsis SWIB domain–containing
proteins were obtained from TAIR (www.arabidopsis.org). The multiple
sequence alignment was performed with ClustalW (Larkin et al., 2007)
and is available as Supplemental Data Set 1 online. The phylogenetic tree
was constructed using MEGA5 software (Tamura et al., 2011) based on
the neighbor-joining method (Saitou and Nei, 1987) with parameters of
Poisson correction model, complete deletion of the gaps, and 1000
bootstrap replicates. The tree is drawn to scale, with branch lengths in
the same units as those of the evolutionary distances used to infer the
phylogenetic tree.
Cloning of GFP Fusion Constructs
For preparation of GFP constructs, the complete coding sequences of
the At2g14880, At4g34290, At3g03590, and At2g356050 genes were
DNA Binding Blot Analysis of TAC Proteins
DNA binding assays with TAC proteins were performed as described (Bülow
et al., 1987) with minor modifications. About 5 µg of total TAC proteins from
spinach were separated under denaturing conditions in 14% (w/v) polyacrylamide gels (Laemmli, 1970) and subsequently transferred onto a nitrocellulose membrane employing a transfer buffer consisting of 50 mM
NaCl, 2 mM EDTA, 0.1 mM DTT, and 10 mM Tris/HCl, pH 7.0. Renaturation
of the proteins took place overnight in renaturation buffer consisting of
2 mM EDTA, 10 mM Tris/HCl, pH 7.0, 0.02% BSA, 1% (w/v) low-fat milk
powder, and 0.02% (w/v) polyvinylpyrrolidone. The membrane was cut into
two halves, and one half was incubated with the SWIB-4–specific antibody
diluted 1:50 with buffer consisting of 50 mM Tris/HCl, pH 7.4, and 150 mM
NaCl. The other half of the membrane was incubated with the buffer only.
After washing in transfer buffer, the membrane was incubated with an
[a-32P]-labeled 16S rDNA probe in renaturation buffer. After hybridization,
SWIB Domain Proteins of the Chloroplast Nucleoid Core
the membranes were washed three times for 15 min with renaturation
buffer.
DNA Condensation Assays in Escherichia coli Cells
The full coding sequence of the Arabidopsis SWIB-4 gene was cloned into
the pQE30 vector (Qiagen) for the transformation in E. coli M15 cells. The
cultures were grown in Luria-Bertani medium at 37°C for several hours
and IPTG was added at OD600 0.7-1 to a final concentration of 1 mM. After
staining with DAPI, the cells were examined by differential interference
contrast and fluorescence microscopy with a Zeiss Axiophot microscope
(Carl Zeiss).
Complementation of the E. coli hns Mutant
The E. coli strain hns::FRT, which carries a precise deletion of hns
(Venkatesh et al., 2010), was transformed with the pQE30 plasmid encoding the Arabidopsis SWIB-4 gene and with the empty pQE30 plasmid
as a control. The bacteria were streaked on MacConkey-salicin indicator
plates, which were prepared from BD Difco MacConkey Agar Base (BD
Diagnostics) and salicin (Sigma-Aldrich) as described (Schnetz et al.,
1987). When required, the medium was supplemented with ampicillin
(100 mg/ml) and IPTG (1 mM).
11 of 14
Christian-Albrechts-University of Kiel) for providing the confocal microscope and Christine Desel (Christian-Albrechts-University of Kiel) for expert
assistance and advice. We also thank Iris Meier (The Ohio State University,
Columbus, OH) for providing the antibody directed towards MFP1 and
René Lorbiecke (Hamburg University, Germany) for providing the PEND:
GFP and PEND:dsRED plasmids. We thank Annegret Wolf (IPK Gatersleben, Germany) and Andreas Prescher (Christian-Albrechts-University of
Kiel) for technical assistance and Kirsten Krause (University of Tromsø,
Norway) for stimulating discussions. This work was supported by a grant
from the Deutsche Forschungsgemeinschaft (Kr1350/8).
AUTHOR CONTRIBUTIONS
K.K. and H.-P.M. designed the research. J.M., A.M., and M.T. performed
research. J.M. and A.M. analyzed data. J.M. and K.K. wrote the article.
Received April 20, 2012; revised June 14, 2012; accepted June 26, 2012;
published July 12, 2012.
REFERENCES
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
numbers: At3g48600 (SWIB-1), At2g14880 (SWIB-2), At4g34290 (SWIB-3),
At3g03590 (SWIB-4), At1g34760 (SWIB-5), and At2g35605 (SWIB-6).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Two-Dimensional Separation of the Highly
Enriched Transcriptionally Active Chromosome (TAC-II) Isolated from
Spinach Chloroplasts.
Supplemental Figure 2. MitoTracker Orange Labeling of Onion
Epidermal Cells Transiently Expressing SWIB-5:GFP and SWIB-6:
GFP Constructs.
Supplemental Figure 3. Sequence Alignment of the Histone H1 Motif of
the SWIB-4 Protein and in Vitro Import Assays with Pea Chloroplasts.
Supplemental Figure 4. Immunological Detection of SWIB-4 in
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Supplemental Figure 5. Molecular Characterization of SWIB-4 T-DNA
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Supplemental Figure 6. Development-Dependent Expression of
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Domain Family of Arabidopsis.
Supplemental Table 1. List of Primers Used for Molecular Characterization of swib-4 Mutants.
Supplemental Data Set 1. Proteins Identified in the Spots of the TACII 2-DE Gel Shown in Supplemental Figure 1.
Supplemental Data Set 2. Amino Acid Alignment Used to Generate
Figure 2A.
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We thank Karin Schnetz (University of Cologne, Germany) for expert
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The Core of Chloroplast Nucleoids Contains Architectural SWIB Domain Proteins
Joanna Melonek, Andrea Matros, Mirl Trösch, Hans-Peter Mock and Karin Krupinska
Plant Cell; originally published online July 12, 2012;
DOI 10.1105/tpc.112.099721
This information is current as of June 15, 2017
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