Báo cáo khoa học: DNA binding and partial nucleoid localization of the chloroplast stromal enzyme ferredoxin:sulfite reductase pptx

DNA binding and partial nucleoid localization of the
chloroplast stromal enzyme ferredoxin:sulfite reductase
Kohsuke Sekine
1,2
, Makoto Fujiwara
2
, Masato Nakayama
3
, Toshifumi Takao
3
, Toshiharu Hase
3
and Naoki Sato
1,2
1 Department of Molecular Biology, Faculty of Science, Saitama University, Japan
2 Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Japan
3 Institute for Protein Research, Osaka University, Japan
The assimilation of sulfur is an important process for
the synthesis of various sulfur compounds such as
amino acids, sulfolipids, and coenzymes. Sulfite reduc-
tase (SiR) is a central enzyme within the sulfur assi-
milation pathway. Sulfate ions taken up by the
sulfate transporter are first activated with ATP by
ATP sulfurylase, forming adenosine-5¢-phosphosulfate.
Adenosine-5¢-phosphosulfate is further phosphoryl-
ated by adenosine-5¢-phosphosulfate kinase, forming
3¢-phosphoadenosine-5¢-phosphosulphate. 3¢-Phosphoa-
denosine-5¢-phosphosulfate is reduced to sulfite by
3¢-phosphoadenosine-5¢-phosphosulfate reductase, and
sulfite is further reduced to sulfide by SiR. The resul-
tant sulfide is fixed into cysteine by cysteine synthase

using O-acetylserine as an acceptor. SiR is localized to
chloroplasts in green leaves and to nongreen plastids
in nonphotosynthetic tissues. SiR has been identified
as one of the main constituents of plastid nucleoids in
pea [1] and soybean [2]. Chloroplast DNA was previ-
ously thought to occur dissolved in the stroma, but
recent studies have revealed that the functional form
of chloroplast DNA is a DNA–protein complex called
a nucleoid [3]. Plant SiR contains a siroheme and a
[4Fe-4S] cluster and catalyzes the six-electron reduction
of sulfite to sulfide, depending on ferredoxin as an
electron donor [4]. Plant SiR was considered to be a
Keywords
bifunctional protein; chloroplast nucleoid;
DNA-binding protein; ferredoxin:sulfite
reductase
Correspondence
N. Sato, Department of Life Sciences,
Graduate School of Arts and Sciences,
University of Tokyo, 3-8-1 Komaba,
Meguro-ku, Tokyo 153–8902, Japan
Fax: +81 3 5454699
Tel: +81 3 54546631
E-mail:
Note
Nucleotide sequence data for PsSiR are
available in the DDBJ ⁄ EMBL ⁄ GenBank
databases under accession number
AB168112
(Received 1 December 2006, revised 15

February 2007, accepted 19 February 2007)
doi:10.1111/j.1742-4658.2007.05748.x
Sulfite reductase (SiR) is an important enzyme catalyzing the reduction of
sulfite to sulfide during sulfur assimilation in plants. This enzyme is locali-
zed in plastids, including chloroplasts, and uses ferredoxin as an electron
donor. Ferredoxin-dependent SiR has been found in isolated chloroplast
nucleoids, but its localization in vivo or in intact plastids has not been
examined. Here, we report the DNA-binding properties of SiRs from pea
(PsSiR) and maize (ZmSiR) using an enzymatically active holoenzyme with
prosthetic groups. PsSiR binds to both double-stranded and single-stranded
DNA without significant sequence specificity. DNA binding did not affect
the enzymatic activity of PsSiR, suggesting that ferredoxin and sulfite are
accessible to SiR molecules within the nucleoids. Comparison of PsSiR and
ZmSiR suggests that ZmSiR does indeed have DNA-binding activity, as
was reported previously, but the DNA affinity and DNA-compacting abil-
ity are higher in PsSiR than in ZmSiR. The tight compaction of nucleoids
by PsSiR led to severe repression of transcription activity in pea nucleoids.
Indirect immunofluorescence microscopy showed that the majority of SiR
molecules colocalized with nucleoids in pea chloroplasts, whereas no parti-
cular localization to nucleoids was detected in maize chloroplasts. These
results suggest that SiR plays an essential role in compacting nucleoids in
plastids, but that the extent of association of SiR with nucleoids varies
among plant species.
Abbreviations
PsSiR, pea (Pisum sativum) sulfite reductase; SiR, sulfite reductase; ZmSiR, maize (Zea mays) sulfite reductase.
2054 FEBS Journal 274 (2007) 2054–2069 ª 2007 The Authors Journal compilation ª 2007 FEBS
stromal protein [5–7], but localization to plastid nucle-
oids provides a new aspect of this enzyme and chloro-
plast molecular biology.
Plastid nucleoids are comprised of various proteins

[3,8–13], and the protein composition changes during
plastid development. Several plastid nucleoproteins
have been studied. CND41 [14–17] is a bifunctional
protease, considered to be a negative regulator of plas-
tid gene expression. The PEND protein [18–21] is
thought to anchor nucleoids to the envelope membrane
in developing chloroplasts. MFP1 [22,23] binds to the
thylakoid membrane in mature chloroplasts and is
thought to function in a manner similar to PEND in
developing chloroplasts. In a proteomic analysis of
DNA–protein complexes, substantially similar to what
we call nucleoids here, polypeptides derived from var-
ious enzymes were reported in addition to those
involved in transcription, DNA replication, DNA
topology, and DNA binding. These were iron super-
oxide dismutase, putative thioredoxin, pfkB-type car-
bohydrate kinase family protein, and Mur ligase
family protein in Arabidopsis and mustard [24], and
proteins involved in pyruvate dehydrogenase, acetyl-
CoA carboxylase, ATP synthase, ribulose bisphosphate
carboxylase, and Calvin cycle proteins in pea [25]. Dif-
ferent researchers use different criteria to judge if
unexpected proteins are the result of contamination.
Therefore, it is important to confirm either the direct
or indirect DNA-binding properties of these putative
components of nucleoids in vitro.
In previous studies of the role of SiR in chloroplast
nucleoids, SiR was suggested to repress DNA synthesis
[26] and transcription [27] within nucleoids. Relaxing
the DNA compaction of nucleoids by release of SiR

activates transcription, whereas increasing DNA com-
paction by the addition of exogenous SiR represses
transcription. These observations led us to propose
that transcription regulation occurs through DNA
compaction by SiR in the plastid nucleoids [27]. The
localization of SiR to nucleoids was confirmed by
immunofluorescence microscopy of isolated pea and
soybean chloroplasts, showing that SiR localizes to
nucleoids within chloroplasts [2].
In our previous studies of the role of SiR in DNA
compaction in nucleoids, we used maize SiR (ZmSiR),
the sole recombinant holoenzyme successfully prepared
at that time. Studies of SiR in other plants were forced
to use recombinant, but enzymatically inactive, pro-
teins lacking prosthetic groups. However, we have
been using pea to isolate chloroplast nucleoids because
a large amount of chloroplast nucleoid is obtained effi-
ciently. Here, we isolated the cDNA of pea SiR
(PsSiR), deduced the structure of PsSiR and prepared
a recombinant holoenzyme of PsSiR. Using the active
recombinant SiR, we examined basic DNA-binding
properties of SiR and the relationship between SiR
activity and DNA-binding activity. We also examined
differences in the properties of PsSiR and ZmSiR, such
as DNA binding and localization within chloroplasts.
Results
Isolation and molecular characterization
of a cDNA clone encoding pea SiR
The k gt10 pea cDNA library was screened. One posi-
tive plaque was obtained from recombinant phages,

and the insert was cloned to pCR2.1-TOPO. The
insert, designated Seq1, had a length of 2216 bases and
contained a reading frame encoding 663 amino acids,
although the initiation codon was missing. The amino-
terminal sequence of SiR from the pea plastid nuc-
leoid, ‘VSTPAKS’ [1], was found within the deduced
amino acid sequence (Fig. 1). To determine the missing
region, the k gt11 pea cDNA library was screened.
Three clones were obtained. The longest nucleotide
sequence, designated Seq2, contained the other three
inserts. The overlapping regions of Seq1 and Seq2,
which had a length of 275 bases, matched completely.
A putative initiation codon was found in the extended
region. The sequence assembled from Seq1 and Seq2,
designated PsSiR, therefore encodes the precursor of
pea SiR consisting of 685 amino acids. Two candidates
for a poly(A) signal, ATAAA and ATAAT, were
found 12 bases and 55 bases upstream of the poly(A)
start site, respectively (data not shown).
The amino acid sequence deduced from PsSiR
exhibited significant homology with SiRs from various
other organisms. The amino acid sequences of SiRs
from angiosperms, a red alga, and two cyanobacteria
were aligned with the hemoprotein subunit (CysI) of
the Escherichia coli SiR complex, which is another type
of SiR using NADPH as an electron donor (Fig. 1).
The similarity of the putative mature PsSiR was 91%
to Nicotiana tabacum SiR, 85% to Arabidopsis thaliana
SiR, 79% to Zea mays SiR, 79% to Oryza sativa SiR,
59% to Cyanidioschyzon merolae SiRA, 57% to SiRB,

67% to Anabaena sp. PCC7120 SiR, 66% to Synecho-
cystis sp. PCC6803 SiR, and 48% to E. coli CysI. The
three-dimensional structure of E. coli CysI determined
using X-ray crystallography [28] revealed that the
siroheme and the [4Fe-4S] cluster are retained within
the active site of the enzyme through four cysteine lig-
ands, Cys434, Cys440, Cys479, and Cys483, and that
four basic residues, Arg83, Arg153, Lys215, and
Lys217, are involved in the substrate coordination to
K. Sekine et al. DNA binding of sulfite reductase
FEBS Journal 274 (2007) 2054–2069 ª 2007 The Authors Journal compilation ª 2007 FEBS 2055
Fig. 1. Comparison of the amino acid sequences of different SiRs. The precursor sequence of PsSiR is compared with those predicted from the cDNA of N. tabacum (NtSiR; GenBank acces-
sion number D83583), A. thaliana (GenBank accession number BT000593), Z. mays (ZmSiR; GenBank accession number D50679), O. sativa (OsSiR; GenBank accession number AK103289),
C. merolae SiRA (CmSiRA), SiRB (CmSiRB), Anabaena sp. PCC7120 (AnSiR; CyanoBase accession number alr1348), Synechocystis sp. PCC6803 (CyanoBase accession number slr0963), and
CysI, the hemoprotein of E. coli NADPH-SiR (EcCysI; GenBank accession number M23007). Residues similar to PsSiR are indicated by white letters on a black background. The N-terminal
amino acid sequence ‘VSTPAKS’ is marked by an arrow. The amino acid residues that bind to the substrate are indicated by R and K. The cysteine residues predicted to be ligands for the
[4Fe-4S] cluster and the siroheme are indicated by C.
DNA binding of sulfite reductase K. Sekine et al.
2056 FEBS Journal 274 (2007) 2054–2069 ª 2007 The Authors Journal compilation ª 2007 FEBS
siroheme. These residues are completely conserved in
all SiRs.
To estimate the molecular phylogeny of the SiRs, we
constructed phylogenetic trees using the maximum
likelihood method, using treefinder. Essentially sim-
ilar trees were obtained by the neighbor-joining and
maximum parsimony methods. All SiRs of flowering
plants were monophyletic and the SiRs of plants ori-
ginated from cyanobacterial SiR (Fig. 2). The reliabil-
ity of the tree was confirmed by bootstrapping. The
important nodes for the above statement were suppor-

ted with high confidence levels.
Basic characterization of the PsSiR gene
cDNA gel blot analysis was carried out with pea
genomic DNA restricted with EcoRI or HindIII. A sin-
gle band was detected in both of the digests (Fig. 3D),
suggesting a single PsSiR gene per genome. To exam-
ine the expression level of pea SiR, RNA gel blot
(Fig. 3A,B) and immunoblot (Fig. 3C) analyses were
carried out using leaves, stems, and roots. Comparable
amounts of SiR transcripts and proteins were detected
in the three organs (Fig. 3A,C). The transcript expres-
sion level was not significantly different in light- and
dark-grown leaves (Fig. 3B). These results suggest that
the expression of SiR is constitutive in pea, in agree-
ment with previous studies in tobacco [29], maize [30],
and A. thaliana [31].
The presence of SiR in chloroplast nucleoids was
detected previously using immunological and enzymo-
logical methods. To confirm that the cloned PsSiR was
actually present within the chloroplast nucleoid, the
band corresponding to PsSiR on the SDS ⁄ PAGE of
chloroplast nucleoids from pea leaves was analyzed by
MALDI-TOF ⁄ MS after in-gel digestion with lysyl
endopeptidase or endoproteinase Asp-N (AspN)
(Table S1). In lysyl endopeptidase digestion, 13 peptides
corresponding to the predicted digestion products of
PsSiR were detected. The sequence coverage was 30.3%
of the mature protein. In AspN digestion, six pep-
tides were detected with 13.9% sequence coverage.
Importantly, the AspN digestion gave peptides corres-

ponding to the first 18 residues of the N terminus and
the last 16 residues of the C terminus of the predicted
mature protein. No peptide corresponding to the predic-
ted transit peptide sequence was detected. Because
AspN cleaves peptide bonds at the N-terminal side of
aspartic and glutamic acids under our conditions, the
18-mer peptide beginning with the N-terminal valine
should not have resulted from enzymatic digestion with
AspN. This result clearly indicates that PsSiR exists in
chloroplast nucleoids and that it consists of the full-
length mature polypeptide beginning with the valine
residue.
Preparation of recombinant SiR and its
DNA-binding activities
ZmSiR was overproduced in E. coli cells under co-
expression of siroheme synthase and purified by a com-
bination of three successive chromatographies on
ion-exchange, hydrophobic, and ferredoxin-affinity
resin, as described previously [32]. PsSiR was also
produced in a similar way and could be purified by
ferredoxin-affinity chromatography only. The final
Fig. 2. Phylogenetic tree of SiRs based on
amino acids sequences. The tree was con-
structed using the maximum likelihood
method. The three numbers on each branch
show confidence levels for the maximum
likelihood ⁄ neighbor-joining ⁄ maximum parsi-
mony analyses, estimated by bootstrap ana-
lysis with 1000 replicates.
K. Sekine et al. DNA binding of sulfite reductase

FEBS Journal 274 (2007) 2054–2069 ª 2007 The Authors Journal compilation ª 2007 FEBS 2057
preparation of PsSiR showed a UV-visible absorption
spectrum characteristic of siroheme-containing proteins
and gave a single major band of around 70 kDa in
SDS ⁄ PAGE (Fig. 4A,B).
This preparation was used to examine the DNA-bind-
ing activity of SiR. A gel-mobility shift assay was car-
ried out using radiolabeled 40-mer or 20-mer synthetic
dsDNA as a probe (Fig. 5A,B). Various amounts of
recombinant PsSiR and 40-mer or 20-mer dsDNA were
mixed, and then the mixtures were electrophoresed. The
intensity of the shifted bands (complexes of DNA and
PsSiR) increased with the amount of PsSiR in both
experiments. The most retarded band was very close to
the origin, indicating that a large complex was formed
(arrowheads in Fig. 5). The band could represent
insoluble materials stuck on the upper edge of the gel.
But the materials did enter agarose gels (Fig. 6B,C) and
are not insoluble materials. The apparent dissociation
constants (K
d
) of 40-mer and 20-mer dsDNA were
about 55 nm and 142 nm, respectively, which indicates a
high affinity of PsSiR for long DNA. The recombinant
PsSiR also shifted ssDNA (Fig. 5C).
The sequence specificity of SiR during DNA binding
was examined. Non-labeled poly(dI-dC)Æpoly(dI-dC)
was added to the mixture of labeled 20-mer dsDNA
and PsSiR as a competitor, and then the mixture was
electrophoresed (Fig. 6A). The densities of shifted

band signals at two-, five- and 10-fold excess of com-
petitor were 34, 17 and 12%, respectively, of that with-
out a competitor, indicating comparable affinities of
SiR for the 20-mer dsDNA and the poly(dI-dC). The
k DNA digested with StyI and pea chloroplast DNA
digested with XbaI were mixed with PsSiR and electro-
phoresed (Fig. 6B). All fragments were shifted and
concentrated in a few slowly migrating bands in the
vicinity of the wells. These results suggest that SiR
binds to DNA with low sequence specificity.
Effects of DNA binding on sulfite reductase
activity
We measured the sulfite reductase activity of the
recombinant PsSiR by a cysteine synthase-coupled
system using recombinant maize ferredoxin I as an
electron donor for SiR and dithionite as a reductant
for ferredoxin I (Fig. 7). The Michaelis constant (K
m
)
of PsSiR for maize ferredoxin I was about 18 lm,
300 400 500 600
0.4
0.3
0.2
0.5
0.1
0
Absorbance
nm
175

47.5
62
83
KDa
12
AB
Fig. 4. (A) UV-visible absorption spectrum and (B) SDS ⁄ PAGE ana-
lysis of purified recombinant PsSiR. (A) Absorption maxima at 389
and 580 nm (arrows) indicate the presence of a siroheme-contain-
ing prosthetic group. (B) A single band (arrowhead) was observed
by staining with Coomassie brilliant blue.
Leaves
Stems
Roots
PsSiR
28S rRNA
PsTUB1
Light
Dark
PsSiR
AB
Leaves
Stems
Roots
α-PsSiR
12
5
2
kbp
HindIII

EcoRI
C
D
95 kDa
Fig. 3. Basic characterization of PsSiR. RNA blot analyses of the
expression of PsSiR in (A) various organs of pea plants and (B) pea
leaves grown in light or dark. Total RNA (10 lg) prepared from
green leaves, stems, or roots, and polyA + RNAs (3 lg) prepared
from leaves grown in light or dark were electrophoresed on 1.2%
agarose gel. The blots were probed with a DIG-labeled DNA frag-
ment corresponding to the second exon of PsSiR. Lower panels
indicate the staining of (A) 28S rRNA and (B) blotted mRNA of pea
b-tubulin 1 as controls. (C) Immunoblot analysis of the distribution
of PsSiR in various organs. Total extracts of 5 mg fresh weight of
green leaves, stems, or roots in NaCl ⁄ P
i
were separated on 10%
gel by SDS ⁄ PAGE. The blots were probed with antibodies raised
against PsSiR. As a loading control, the staining pattern of a 95-kDa
major band (putative heat shock protein) is shown below. (D) DNA
blot analysis of the pea genome. Genomic DNA of pea was diges-
ted with EcoRI and HindIII and separated on 0.8% agarose gel. The
blots were probed with the same probe used for RNA blotting.
DNA binding of sulfite reductase K. Sekine et al.
2058 FEBS Journal 274 (2007) 2054–2069 ª 2007 The Authors Journal compilation ª 2007 FEBS
higher than that of ZmSiR [33], which is about 4 lm.
Unlike PsSiR, the activity of ZmSiR was assayed by
measuring the increase in NADP
+
oxidized by reduc-

tion of ferredoxin III donating electrons to SiR as des-
cribed by Yonekura-Sakakibara et al. [30]. The
difference in K
m
may be due to the differences in the
methods used to measure activity.
To examine the effects of DNA binding on sulfite
reductase activity, recombinant PsSiR was mixed with
DNA to form a DNA–SiR complex, and then enzy-
matic activity was measured (Fig. 7). There was no sig-
nificant difference in activity of DNA-bound and
DNA-free PsSiR. This indicates that sulfite reductase
remains functional when it is bound to DNA. Binding
of PsSiR to DNA in the reaction mixture during the
measurement of activity was confirmed by an experi-
ment of coprecipitation of SiR with DNA-cellulose in
the reaction medium (data not shown).
Characteristics of pea SiR with respect to maize
SiR
We previously used recombinant ZmSiR in studies of
the DNA binding of sulfite reductase [1,27]. Here, we
compared the DNA-binding activity of ZmSiR and
PsSiR (Fig. 5, right). An increase in the intensity of
the shifted bands was found as the concentration
of ZmSiR increased with 40-mer ds-, 20-mer ds- or
40-mer ssDNA. However, the apparent K
d
values of
ZmSiR were higher than those of PsSiR, suggesting
that the binding of ZmSiR to DNA is weaker than

that of PsSiR. It should be noted that very slowly
migrating bands, as detected with PsSiR, were scarcely
detected with ZmSiR. This result suggests that ZmSiR
has a lower ability to compact DNA than PsSiR. An
additional rapidly migrating band was detected with
ssDNA and PsSiR (asterisk in Fig. 5C).
Differences in the ability to compact DNA were also
examined for PsSiR and ZmSiR. Changes over time
were examined by fluorescence microscopy after mix-
ing DNA with PsSiR or ZmSiR. Immediately after
mixing, a number of blurred particles were formed
upon addition of PsSiR, whereas either ZmSiR or
98765432110
FD
K
d

≅
55 nM K
d

≅
165 nM
A
PsSiR ZmSiR
K
d

≅
142 nM K

d

≅
294 nM
FD
B
PsSiR ZmSiR
98765432110
FD
K
d

≅
115 nM K
d

≅
266 nM
C
PsSiR ZmSiR
98765432110
Fig. 5. DNA binding activity of SiRs. The
32
P-labeled (A) 40-mer
dsDNA, (B) 20-mer dsDNA, and (C) 40-mer ssDNA were incubated
without (lanes 1 and 10) or with 25, 50, 100, and 200 n
M PsSiR
(lanes 2–5) or ZmSiR (lanes 6–9) before electrophoresis in 6% poly-
acrylamide gel. FD indicates free DNA. The bands very close to
wells, which suggest large complexes, are indicated by arrow-

heads. The slightly shifted bands detected in ssDNA with PsSiR
are indicated by an asterisk. The apparent dissociation constants
(K
d
) are shown below the lane numbers.
K. Sekine et al. DNA binding of sulfite reductase
FEBS Journal 274 (2007) 2054–2069 ª 2007 The Authors Journal compilation ª 2007 FEBS 2059
buffer alone did not engender such particles (Fig. 8).
After incubation for >2 h, bright and structurally
well-defined particles were formed with PsSiR. Some
particles increased in brightness over time, indicating
that the quantity of DNA per particle increased
and ⁄ or DNA became more tightly compacted. After
incubation for more than 6 h, blurred particles similar
to those at 0 h in PsSiR were formed in ZmSiR. These
results suggest that the DNA-compacting activity of
ZmSiR is weaker than that of PsSiR.
We previously performed an in vitro transcription
assay using isolated chloroplast nucleoids and showed
that UTP incorporation into RNA was repressed by
the addition of recombinant ZmSiR [27]. We expected
that PsSiR is more active in repressing the transcrip-
tional activity of chloroplast nucleoids. Isolated pea
chloroplast nucleoids were mixed with various con-
centrations of recombinant PsSiR or ZmSiR and
incubated for 30 min on ice before the addition of
radiolabeled UTP to initiate run-on transcription
(Fig. 9, closed and open circles, respectively). The
incorporation of radioactive UTP into the high
molecular weight fraction was used as a measure of

transcriptional activity. In Fig. 9, the activity is
expressed in percentage of the control activity in the
presence of 100 lgÆmL
)1
heparin. In a previous paper
[27], we showed that addition of heparin releases the
endogenous SiR from nucleoids, and the full transcrip-
tional activity is obtained. When ZmSiR was used,
transcriptional activity gradually decreased as ZmSiR
concentration increased. The activity decreased by
about one-half at a ZmSiR concentration of 1.6 lm
(not shown). This result is consistent with the results
of our previous experiment [27]. When PsSiR was
added at a concentration of 0.2 lm, the transcriptional
activity of the nucleoids was reduced dramatically to
about 20% of that in the absence of exogenous SiR.
Therefore, PsSiR more strongly repressed transcription
than did ZmSiR. This is consistent with the higher
affinity of PsSiR for DNA.
0
100
200
300
400
0 5 10 15 20
Ferredoxin (µ
M)

Cys (mol/min/mol SiR)
Fig. 7. Measurement of enzymatic activity of DNA-bound PsSiR.

PsSiR was incubated without (open circles with solid thin line) or
with 10 lgÆmL
)1
(filled squares with bold solid line) or 20 lgÆmL
)1
(filled triangles with dashed line) HindIII digested k DNA before
measurement. Various concentrations of recombinant maize ferre-
doxin I were added as an electron donor to SiR. The amount of cys-
teine produced per minute per mole of SiR was used as a measure
of enzymatic activity.
54321
poly(dI-dC)
A
2x 5x 10x
987654321
10 11 141312
B
PsSiR
ZmSiR
PsSiR
ZmSiR
Fig. 6. Sequence specificity for DNA-binding by SiRs. (A) Competition for DNA-SiR complex formation with poly(dI-dC)Æpoly(dI-dC). The
32
P-labeled 20-mer dsDNA and none, two-, five-, or 10-fold mass excess of nonlabeled poly(dI-dC)poly(dI-dC) (lanes 2–5, respectively) were
mixed with 100 n
M PsSiR prior to electrophoresis in 6% polyacrylamide gel. The bands shifted by PsSiR and faded by the competitor are
indicated by arrowheads. Lane 1 is DNA alone. (B) Binding of SiR to StyI-digested k phase DNA (lanes 1–7) and XbaI-digested chloroplast
DNA (lanes 8–14). Each digested DNA was incubated without (lanes 1 and 8) or with 200, 400, and 800 n
M PsSiR (lanes 2–4 and 9–11) or
ZmSiR (lanes 5–7 and 12–14) prior to electrophoresis in 1% agarose gel.

DNA binding of sulfite reductase K. Sekine et al.
2060 FEBS Journal 274 (2007) 2054–2069 ª 2007 The Authors Journal compilation ª 2007 FEBS
Figure 9 also shows comparison of nucleoids from
mature (14-day-old) and developing (6-day-old) leaves.
As previously reported [10,18], the leaves of 6-day-old
seedlings are small buds that are pale green and are
not yet open. The developing chloroplasts in such
leaves contain a higher amount of chloroplast DNA,
but are not active in photosynthesis. The results in
Fig. 9 show that the nucleoids from developing leaves
(closed square at zero SiR concentration) are less act-
ive in transcription than those from mature leaves
(closed circle) due to stronger repression by endo-
genous SiR. This is consistent with the previous results
of immunoblot [10]. The effects of exogenous SiR,
either from pea or maize, were similar in both mature
and developing nucleoids.
Intrachloroplast localization of SiR
To examine the intrachloroplast location of SiR, indi-
rect immunofluorescence microscopy of isolated chloro-
plasts was performed (Fig. 10). Isolated pea or maize
chloroplasts fixed in paraformaldehyde were incubated
with an antibody raised against PsSiR and then with
an AlexaFluor-tagged secondary antibody. The chloro-
plasts were also counterstained with 4¢,6-diamidino-2-
phenylindole to visualize chloroplast DNA. In pea
chloroplasts, the AlexaFluor signal was detected non-
uniformly within the chloroplast. The spots that were
densely stained with AlexaFluor coincided with the
areas of 4¢,6-diamidino-2-phenylindole staining. These

results indicate that PsSiR exists within nucleoids, as
well as in the stroma. In maize chloroplasts, the Alexa-
Fluor signal was located uniformly throughout the
whole chloroplast and no dense AlexaFluor signal was
colocalized with a 4¢,6-diamidino-2-phenylindole signal.
This indicates that ZmSiR is not particularly concen-
trated in nucleoids.
Discussion
Comparative aspects of SiR
The sequence alignment (Fig. 1) indicates that PsSiR
has the archetypal molecular structural characteristics
0
1
6
2
20
PsSiR ZmSiR
h
co 0 h co 20 h
A
F
EJ
D
I
C
H
B
G
K
L

Fig. 8. Compaction of chloroplast DNA by SiRs. Isolated pea chloro-
plast DNA was incubated with recombinant PsSiR (A, B, C, D, E)
or ZmSiR (F, G, H, I, J) on ice. The mixture was stained with 4¢,6-di-
amidino-2-phenylindole and examined by fluorescence microscopy
immediately (A, F) or at 1 h (B, G), 2 h (C, H), 6 h (D, I), or 20 h (E, J)
after mixing. Chloroplast DNA incubated in buffer alone was also
examined immediately (K) or at 20 h (L) after mixing as a control.
K. Sekine et al. DNA binding of sulfite reductase
FEBS Journal 274 (2007) 2054–2069 ª 2007 The Authors Journal compilation ª 2007 FEBS 2061
of plant ferredoxin-dependent SiRs, i.e. four cysteines
as ligands for the prosthetic groups and two lysines and
two arginines involved in the substrate coordination to
siroheme are completely conserved. Region A in Fig. 1
indicates the common insertion in ferredoxin-dependent
SiRs and ferredoxin-dependent nitrite reductases, with
respect to E. coli CysI, and is reported as a candidate
for an interaction site with ferredoxin [29]. PsSiR reta-
ins this insertion from Ala234 to Phe261 with high
homology, as expected. However, region A sequences
in C. merolae SiRA and SiRB are poorly conserved,
)%( ytivitca lanoitpircsnarT
SiR (µ
M
)
40
20
0
0.40.20
A
less active active

developing
mature
B
SiR
DNA
Fig. 9. Effects of SiRs on the transcriptional activity of nucleoids
from mature and developing chloroplasts. (A) The isolated pea
chloroplast nucleoids were incubated with various concentrations
of PsSiR (solid line) or ZmSiR (dashed line) on ice for 30 min and
then added to the reaction mixture. After preincubation at 25 °C for
30 min,
3
H-labeled UTP was added to start the reaction and the
mixture was incubated at 25 °C for 30 min. The count rate (cpm) of
radioactive UTP incorporated into the high-molecular weight fraction
was measured. The transcriptional activity is expressed as a per-
centage of the activity in the presence of 100 lgÆmL
)1
heparin,
which releases all endogenous SiR and makes the nucleoids fully
active in transcription. We used such measure because the amount
of cpDNA, as estimated by Southern blotting with rbcL as a probe,
may not be very accurate for comparing different samples. The act-
ual UTP incorporation in the nucleoids from mature and developing
chloroplasts was 601 ± 83 and 824 ± 113 cpm Æ ng
)1
cpDNA,
respectively. s,d, nucleoids from mature pea chloroplasts (14-day-
old leaves); h,j, nucleoids from developing pea chloroplasts (6-
day-old leaf buds).s,h, addition of maize SiR; d,j, addition of pea

SiR. (B) A schematic view on the compaction status of nucleoids in
developing and mature chloroplasts. In developing chloroplasts, a
large amount of SiR is bound to the nucleoids and represses the
transcription severely. In mature chloroplasts, the amount of SiR is
reduced and the nucleoids are more active in transcription.
DAPI
AlexaFluor
maize pea
A
B
C
D
EF
GH
Fig. 10. Intrachloroplast localization of SiR. Isolated pea (A, B, C, D)
or maize (E, F, G, H) chloroplasts were fixed with paraformalde-
hyde, probed with antibodies raised against PsSiR (A, B, E, F) or
preimmunized serum (C, D, G, H) and AlexaFluor488-tagged secon-
dary antibodies, and stained with 4¢,6-diamidino-2-phenylindole.
Left-hand panels (A, C, E, G) display 4¢,6-diamidino-2-phenylindole
and chlorophyll fluorescence. Right-hand panels (B, D, F, H) display
AlexaFluor488 fluorescence. The bar indicates 10 lm.
DNA binding of sulfite reductase K. Sekine et al.
2062 FEBS Journal 274 (2007) 2054–2069 ª 2007 The Authors Journal compilation ª 2007 FEBS
with an additional insertion: YWK(R ⁄ K)(D ⁄ E)(I ⁄ L). It
will be interesting to determine whether SiRA and
SiRB have affinity for ferredoxin.
The phylogenetic tree (Fig. 2) indicates that the SiRs
of cyanobacteria and plants (using ferredoxin as an
electron donor) originate from bacterial SiRs (using

NADPH as an electron donor via the flavin subunit).
The plant SiRs originate from cyanobacterial SiRs,
with Gloeobacter as the root. However, cyanobacterial
SiRs are divided into two clades, one consisting of
Synechococcus and Prochlorococcus, and the other
consisting of Anabaena–Nostoc, Synechocystis, and
Thermosynechococcus. These two clades correspond to
the two major lineages of cyanobacteria [34]. Phylo-
genetic analysis with plastid-encoded protein genes
suggested that plastids originate from the Anabaena–
Synechocystis clade. However, plant SiRs are asso-
ciated with the Synechococcus–Prochlorococcus clade,
although the confidence level of the branches near
Synechococcus sp. PCC 6301 is low (Fig. 2). The
angiosperm SiRs form a monophyletic cluster distinct
from the SiRs of cyanobacteria or C. merolae. The
C. merolae SiRs and Thalassiosira (diatom) SiR are
monophyletic, which suggests that the SiR gene was
transferred from a red algal endosymbiont during sec-
ondary endosymbiosis. In C. merolae, an ORF (CMR
440 C) homologous to the a-component (flavoprotein)
of NADPH-dependent SiR is also found [35]. One of
the SiRs in C. merolae (possibly SiRB) could function
with this flavoprotein, rather than ferredoxin.
The genomic DNA blot analysis (Fig. 3D) suggests
that there is a single SiR gene in the haploid pea
genome. A single SiR gene is also found in A. thaliana
[36], rice [37], and N. tabacum [29]. Another copy must
be present in tobacco because it is an amphidiploid.
Except in tobacco, the SiR gene occurs as a single-

copy gene in all known flowering plants. SiR was
known as a stromal enzyme before it was found locali-
zed to plastid nucleoids [1,2]. The nucleoid localization
of SiR could have been explained by an isozyme enco-
ded by a different gene, but the copy number analysis
indicates that this is not likely.
Reductase activity and DNA binding
Here, the large-scale production of enzymatically act-
ive recombinant SiR containing prosthetic groups
enabled detailed experiments on the relationship
between enzymatic activity and DNA-binding activity.
The DNA gel-mobility shift assay using recombinant
SiR revealed that SiR is an authentic DNA-binding
protein, with high DNA-binding affinity. Our data
demonstrate that SiR directly binds to dsDNA, as well
as to ssDNA (Fig. 5). This suggests that SiR binds to
DNA during replication, which may cause the repres-
sion of DNA synthesis by SiR in isolated nucleoids
[26]. Radiolabeled DNA and poly(dI-dC)Æpoly(dI-dC)
competed comparably for binding to SiR, and all the
restriction fragments of both k and chloroplast DNA
were shifted by SiR binding (Fig. 6). This shows that
SiR binds to DNA without notable sequence specificity
and supports our argument that SiR is a global regula-
tor of nucleoid functions such as transcription [27] and
replication [26].
Intrachloroplast localization
Indirect immunofluorescence microscopy of isolated
chloroplasts demonstrated the presence of PsSiR in the
nucleoids of pea chloroplasts. Our data are basically

consistent with previous results [2]. In the previous
study, however, the fluorescence signal of SiR clearly
coincides with DNA in pea chloroplast nucleoids, and
essentially no fluorescence was detected in the stroma
[2]. We found an SiR signal throughout the whole
chloroplast and in dense patches that coincided with
the nucleoids. Chi-Ham et al. [2] fixed chloroplasts in
a buffer containing formaldehyde and then dehydrated
them with ethanol and acetone on slides, whereas we
used formaldehyde fixation without dehydration and
performed immunoreaction and 4¢,6-diamidino-2-phen-
ylindole staining in a test tube. We suspect that the
stromal components were washed away during the
washing and dehydration process in the experiments of
Chi-Ham et al. [2]. The mild processing in our experi-
ments made the localization of SiR slightly obscure,
but this indicates that SiR is not confined to the nucle-
oids and is also present in the stroma.
Comparison of PsSiR and ZmSiR
In the gel-mobility shift assay of PsSiR, shifted bands
that remained very close to the origin were detected.
These bands represent potentially large DNA–SiR
complexes formed by the intermolecular aggregation of
DNA fragments. The large complexes are only found
with PsSiR, indicating high DNA-compacting ability.
In contrast, ZmSiR formed no such complex, indica-
ting that the DNA-compacting ability of ZmSiR is
inferior to that of PsSiR.
The difference in DNA-compacting ability between
PsSiR and ZmSiR was clearly demonstrated by the

in vitro compaction assay (Fig. 8). Previously, we
showed that nucleoid-like particles were formed only
several hours after the mixing of recombinant ZmSiR
and pea chloroplast DNA [1]. Our current results repro-
K. Sekine et al. DNA binding of sulfite reductase
FEBS Journal 274 (2007) 2054–2069 ª 2007 The Authors Journal compilation ª 2007 FEBS 2063
duced the previous findings, but the compaction by
ZmSiR was not as tight as that found in the previous
study because we did not add spermidine in the present
study. Nevertheless, PsSiR did form blurred particles
immediately after mixing, and well-defined particles
were eventually formed, demonstrating the even higher
DNA-compaction ability of PsSiR than of ZmSiR.
The two SiRs had different effects on the transcrip-
tional activity of nucleoids. PsSiR repressed the trans-
criptional activity of chloroplast nucleoids more
strongly than did ZmSiR (Fig. 9). We previously
reported that ZmSiR reversibly repressed transcrip-
tional activity by enhancing DNA compaction. How-
ever, PsSiR had far stronger transcriptional repression
than ZmSiR. Based on the results of the DNA-binding
study, the tighter compaction of DNA induced by
PsSiR was a result of its stronger binding to DNA.
The results of immunofluorescence microscopy need
additional explanation. In the isolated maize chloro-
plasts, SiR was detected throughout the whole chloro-
plast and the distribution was almost uniform. This
result was different from that in pea chloroplasts. In
developing soybean chloroplasts [2], similarly diffuse
SiR-indicative fluorescence was observed. This was

explained by the differentiation status of cells because
fluorescence coincided with less-defined nucleoids.
However, our result cannot be explained by the same
reasoning because the maize chloroplasts contained
well-defined nucleoids. Because ZmSiR clearly has
DNA-binding activity, SiR may partly associate with
nucleoids in maize chloroplasts. This association may
not be detected clearly by immunofluorescence because
of the presence of SiR within the stroma. In proteomic
mass spectroscopy, a number of peptides, including
SiR, were identified from the Triton-insoluble fraction
prepared from pea chloroplasts [25], whereas only a few
peptides that do not completely satisfy identification
criteria were identified from transcriptionally active
chromosomes purified from Arabidopsis and mustard
chloroplasts [24]. The nucleoid fraction, which was pre-
pared from Arabidopsis chloroplasts in the same way,
did not contain a detectable amount of SiR (unpub-
lished results). This could be due to treatment with a
high concentration of detergent during nucleoid prepar-
ation. These different results in different plants can be
explained by differences in the affinity of SiRs to DNA.
Relationship between DNA binding and
enzymatic activity of SiR
What is the role of SiR within the nucleoids? Here, the
enzymatic activity assay using recombinant ferredox-
in demonstrated that the reductase activity of PsSiR is
not affected by its DNA binding. Although there is cur-
rently no decisive evidence for a physiological advant-
age of the association of SiR with nucleoids, one

probable explanation is that the nucleoid SiR, contain-
ing a siroheme and a [4Fe-4S] cluster as catalytically
active redox centers, could act as a sensor of the redox
state of the chloroplast because the expression of some
chloroplast genes is regulated by the chloroplast redox
state [38,39]. It is intriguing that FrxB, a subunit of
NAD(P)H dehydrogenase containing a [4Fe-4S] cluster,
was isolated as a plastid DNA-binding protein [40].
This provides a reasonable hypothesis for the redox
control of the functional and ⁄ or morphological state of
chloroplast nucleoids. Various metabolic enzymes,
which do not have obvious activity related to nucleoid
functions such as DNA replication, DNA maintenance,
or transcription, constitute mitochondrial nucleoids in
baker’s yeast [41–44], Xenopus laevis [41,45], and
human HeLa cells [46]. Interestingly, aconitase, a citric
acid cycle enzyme, is essential for the stability of mit-
ochondrial DNA in yeast [42,47]. Aconitase also con-
tains a [4Fe-4S] cluster that is essential for its catalytic
activity [48]. Shadel [49] showed that the disassembly
or oxidation of the aconitase iron–sulfur cluster in-
duced by reactive oxygen species generated by oxidative
phosphorylation results in its reallocation from
the matrix to mitochondrial nucleoids, stabilizing mito-
chondrial DNA under oxidative stress. In contrast,
Chen and Butow [41] suggested that, in respiratory
conditions, an increased level of aconitase might substi-
tute for Abf2, which tightly packages mitochondrial
DNA and changes the structure of nucleoids into a
metabolically favorable conformation and protects

DNA in the remodeled conformation. In chloroplasts,
various reactive oxygen species are produced during
photosynthesis [50,51]. We hypothesize that the DNA
binding of SiR might be dependent on the metabolic
state, e.g. photosynthesis, to optimize nucleoid confor-
mation for metabolic activity by regulating gene expres-
sion and ⁄ or for maintenance of chloroplast DNA by
protecting it from harmful agents.
Experimental procedures
Plant materials and growth conditions
Seeds of pea (Pisum sativum L. cv. ‘Alaska’) and maize
(Zea mays L. cv. ‘Golden Cross Bantam’) were soaked in
tap water overnight at room temperature and then sown on
moist vermiculite and allowed to germinate at about 25 °C
for pea and 32 °C for maize. The seedlings were grown
under white fluorescent lamps at a fluence rate of about
50 lmolÆm
)2
Æs
)1
.
DNA binding of sulfite reductase K. Sekine et al.
2064 FEBS Journal 274 (2007) 2054–2069 ª 2007 The Authors Journal compilation ª 2007 FEBS
Preparation of chloroplasts and nucleoids
Pea chloroplasts were prepared from the leaf buds of epico-
tyls of 6- or 7-day-old seedlings or the mature leaves of
14-day-old plants as described previously [10]. Maize chlo-
roplasts were isolated from the cotyledons of 6-day-old
plants in a similar way. Nucleoids were prepared as des-
cribed previously [10] using TAN buffer (20 mm Tris ⁄ HCl,

pH 7.5, 0.5 mm EDTA, 0.5 m sucrose, 7 mm 2-mercapto-
ethanol, 0.4 mm phenylmethylsulfonyl fluoride, 1.2 mm
spermidine) and then stored in the presence of 33% glycerol
at )80 °C until use.
Screening cDNA Libraries
PCR was carried out with genomic DNA (laboratory stock)
using the degenerate primers 5¢-ATCAAGTTYCAYGG
WAGCTA-3¢ and 5¢-AYACGACCAYTRTCWACRTG-3¢,
which were designed based on the highly conserved regions
of SiR. The PCR products were cloned into pCR2.1-TOPO
(Invitrogen, Carlsbad, CA, USA). DNA sequencing and a
subsequent homology search in GenBank were used to iden-
tify the PCR products as a portion of an SiR gene. This frag-
ment was used as a probe to screen two types of cDNA
libraries [19], a cDNA library in k gt10, which was primed
with oligo-dT, and a cDNA library in k gt11, which was
primed with random hexamers. The details of the cloning are
described in the Results section. The inserts in the isolated
k clones were amplified by PCR using vector primers, and the
products were then cloned into pCR2.1-TOPO. DNA
sequencing was performed by the dideoxy chain-termination
method using a DNA sequencing kit, Big-Dye Terminator 3
(Applied Biosystems, Foster City, CA, USA), with a DNA
sequencer (Model 377; Applied Biosystems). The DNA
sequence was assembled with AutoAssembler (Applied Bio-
systems) and manipulated with genetyx (Software Develop-
ment, Tokyo, Japan).
Phylogenetic analysis
Various SiR sequences were retrieved from the GenomeNet
website (). Database sequences and

alignment files were manipulated using siseq [52]. Amino
acid sequences were aligned with muscle version 6.5 [53]
using the default settings. After alignment, highly variable
sites (> 20%) as well as both termini were removed. The
phylogenetic tree was constructed by the maximum likeli-
hood method using treefinder (version May 2006) [54]. A
neighbor-joining tree was calculated using mega (version 3.1)
[55] with the jtt model and gamma value of 2.0. A maximum
parsimony tree was calculated using paup (version 4b10, Si-
nauer Associates, Sunderland, Massachusetts, USA) with a
heuristic search. A graphical representation of the phylo-
genetic tree was made using the njplot [56].
DNA and RNA blot hybridization
Genomic DNA (4 lg) was digested with EcoRI and
HindIII and the DNA fragments were resolved on 0.8%
agarose gel. The restriction fragments were transferred to a
nylon membrane (Hybond-N
+
GE Healthcare, Piscataway,
NJ, USA) according to the protocol recommended by the
manufacturer. After prehybridization, hybridization was
carried out in a hybridization mixture containing 50%
formamide with digoxigenin (DIG)-labeled probes for the
second exon of the pea SiR gene. Other details of DNA
blot hybridization, including chemiluminescent detection,
have been described previously [19].
RNA blot analysis was performed essentially as des-
cribed previously [19]. Briefly, 10 lg of total RNA or
poly (A) + RNA was electrophoresed in 1.2% agarose gel
and then transferred to a nylon membrane (Biodyne A;

Pall, New York, USA). The probe as described for DNA
blotting was used for hybridization.
Immunoblot analysis
Various plant tissues stored at )80 °C were ground with a
mortar and pestle under liquid nitrogen. The powder was
transferred into a micro tube and homogenized with a
pestle in two volumes of 50 mm Tris ⁄ HCl (pH 7.5),
50 mm NaCl, 1 mm MgCl
2
,1mm EDTA, and 1 mm
phenylmethylsulfonyl fluoride on ice. The homogenate was
centrifuged at 2000 g for 1 min. The crude extract was
mixed with SDS to a final concentration of 1% and
boiled for 5 min. Electrophoresis and immunoblotting
were performed with a 10% polyacrylamide gel as des-
cribed previously [18].
MALDI-TOF ⁄ MS
The total proteins associated with chloroplast nucleoids
isolated from pea leaves were reduced and carboxymeth-
ylated essentially as described previously [57]. Modified
samples were dialyzed against 50 mm NH
4
HCO
3
and
lyophilized, then dissolved in 2 mm NaOH and separated
by SDS ⁄ PAGE. Proteins were stained with a Gel-Negat-
ive Stain Kit (Nacalai Tesque, Kyoto, Japan). The negat-
ively stained bands were manually excised from the gel
and destained according to the manufacturer’s instruc-

tions. Gel pieces were ground, dried in vacuo, and resus-
pended in 100 lL MilliQ water containing 1 milliunit
activity Achromobacter lysyl endopeptidase (Wako,
Osaka, Japan) or endoproteinase Asp-N (Roche, Basel,
Switzerland) for digestion over 12 h at room temperature.
Peptides were sequentially extracted from the gel in 0,
10, 50, and 100% acetonitrile, all in 0.1% trifluoroacetic
acid. Mass spectrometry was performed as described pre-
viously [58].
K. Sekine et al. DNA binding of sulfite reductase
FEBS Journal 274 (2007) 2054–2069 ª 2007 The Authors Journal compilation ª 2007 FEBS 2065
Preparation of recombinant SiR
Recombinant PsSiR and ZmSiR were over-expressed in
E. coli cells and purified with ferredoxin-affinity column
chromatography as described in Ideguchi et al. [32]. To
facilitate the production of enzymatically active recombin-
ant SiRs, coexpression of the E. coli cysG gene (GenBank
accession number X14202) encoding siroheme synthase was
attempted. In the purification of PsSiR, the three column-
chromatography steps in [32] (anion exchange, gel filtration,
and hydrophobic chromatographies) were not used.
Instead, desalting after ammonium sulfate fractionation
was performed by dialysis, and the precipitates were
removed by centrifugation. This step was effective in
removing major contaminants.
Gel-mobility shift assay
A double-stranded 40-mer DNA probe was prepared by
annealing the oligonucleotide 5¢-AGTCTAGACTGCAG
TTGAGTCCTTGCTAGGACGGATCCCT-3¢ and its com-
plementary strand. A 20-mer dsDNA probe was similarly

prepared by annealing 5¢-AGTCTAGACTGCAGTTGA
GT-3¢ and its complementary strand. The oligonucleotide
described above for 40-mer dsDNA was also used as a sin-
gle-stranded DNA probe. These probes were 5¢-end labeled.
The labeled DNA probes were incubated with recombinant
SiR in binding buffer (4 mm Tris ⁄ HCl, pH 8.0, 5% glycerol)
on ice for 30 min, and then the binding mixtures were elec-
trophoresed in TEA buffer (6.7 mm Tris ⁄ HCl, pH 7.9, 1 mm
EDTA, 3.3 mm sodium acetate). The gel was fixed with 10%
acetic acid, 10% methanol and dried under vacuum. The
radioactive bands were detected by autoradiography with
X-ray film (BioMax; Kodak, Rochester, NY, USA). The
SiR concentration at one-half of the maximum concentra-
tion of DNA associated with SiR was taken as the apparent
dissociation constant. The experiments in Fig. 6(B) were per-
formed according to the method of Sasaki et al. [59], using
XbaI-digested chloroplast DNA or StyI-digested k phage
DNA (Toyobo, Osaka, Japan) as probes. In this assay, the
mixtures were electrophoresed in 1% agarose gel in TEA
buffer. After electrophoresis, the gel was stained with ethi-
dium bromide for the detection of DNA bands.
Measurement of SiR enzymatic activity
The enzymatic activity of SiR was assayed by measuring
the amount of cysteine formed from S
2–
by cysteine syn-
thase added to the system [60], as described previously [29].
O-Acetylserine was added as the sulfide acceptor. The reac-
tion was started by the addition of sodium dithionite to a
mixture containing recombinant maize ferredoxin I (labor-

atory stock [61]) as an electron donor for SiR. Recombin-
ant PsSiR was incubated with or without HindIII-digested
k DNA (New England Biolabs, Beverly, MA, USA) at
room temperature for 30 min to form DNA–SiR complexes
before the reductase activity measurement. The SiR alone
or DNA–SiR complex was added to the SiR assay mixture
to a final concentration of 0.5 lm SiR.
In vitro compaction of DNA
Pea chloroplast DNA was prepared as described previously
[18]. Chloroplast DNA (2 lg) was mixed with PsSiR or
ZmSiR (35 pmol) in 50 lLof50mm Tris ⁄ HCl, pH 7.5, and
incubated at 4 °C. Aliquots of 4 l L were taken from the mix-
ture, mixed with 4 lL of 1% glutaraldehyde, and then
stained with 4 lLof1lgÆmL
)1
4¢,6-diamidino-2-phenylin-
dole. All reagents contained 50 mm Tris ⁄ HCl, pH 7.5. The
specimens were examined under a fluorescence microscope
(BX-60, Olympus, Tokyo, Japan) equipped with a WU cube.
In vitro transcription assay
The in vitro transcription assay was performed essentially
according to the method of Sakai et al. [62], with some modi-
fication. The reaction mixture contained 40 mm Tris ⁄ HCl
(pH 7.6), 7 mm MgCl
2
,24lm (NH
4
)
2
SO

4
, 0.01% (w ⁄ v)
Nonidet P40, 180 lm ATP, 180 lm GTP, 180 lm CTP, 5 lm
[5,6-
3
H]UTP (about 0.16 TBqÆmmol
)1
), and chloroplast
nucleoids (30 lg of proteinÆmL
)1
). Before the addition of
radiolabeled UTP to start the reaction, we incubated the
mixture at 25 °C for 30 min. The reaction was carried out at
25 °C. After the reaction, an aliquot (10 lL) was spotted on
a small disk of DEAE paper (DE81; Whatman, Maidstone,
UK). The paper was washed successively in 5% Na
2
HPO
4
,
water, and ethanol, and then finally dried. The incorporation
of [5,6-
3
H]UTP radioactivity into the DEAE paper-bound
fraction was determined by liquid scintillation counting.
Indirect immunofluorescence microscopy
Isolated chloroplasts were fixed by resuspension in grinding
buffer containing 2% (w ⁄ v) paraformaldehyde. Fixed
chloroplasts were washed with NaCl ⁄ P
i

(137 mm NaCl,
2.7 mm KCl, 7.9 mm Na
2
HPO
4
, 1.3 mm NaH
2
PO
4
, pH 7.2)
and then incubated in NaCl ⁄ P
i
containing 0.05% (v ⁄ v) Tri-
ton X-100 at room temperature for 10 min. Permeabilized
chloroplasts were washed with NaCl ⁄ P
i
, incubated with dilu-
ted anti-PsSiR antibody in NaCl ⁄ P
i
containing 1% (w ⁄ v)
blocking reagent (Roche) for 1 h at 37 °C, and then washed
with NaCl ⁄ P
i
. After the reaction with the primary antibody,
chloroplasts were incubated with diluted AlexaFluor 488
goat antiguinea pig IgG (Invitrogen) in NaCl ⁄ P
i
containing
1% blocking reagent for 1 h at 37 °C, and then washed with
NaCl ⁄ P

i
. Chloroplasts were incubated in NaCl ⁄ P
i
containing
4 lgÆmL
)1
4¢,6-diamidino-2-phenylindole at room tempera-
ture for 15 min and then washed with NaCl ⁄ P
i
. The doubly
DNA binding of sulfite reductase K. Sekine et al.
2066 FEBS Journal 274 (2007) 2054–2069 ª 2007 The Authors Journal compilation ª 2007 FEBS
stained chloroplasts were examined under a fluorescence
microscope with an NIB cube for AlexaFluor 488 and a WU
cube for 4¢,6-diamidino-2-phenylindole.
Acknowledgements
This work was supported in part by a Grant-in-Aid
for JSPS Fellows (No. 1505869) to KS and a Grant-in-
Aid for Scientific Research (No. 15370017, 18017005)
to NS from the Japanese Society of for the Promotion
of Science, and by the Cooperative Research Program
of Institute for Protein Research, Osaka University
(2003–06).
References
1 Sato N, Nakayama M & Hase T (2001) The 70-kDa
major DNA-compacting protein of the chloroplast
nucleoid is sulfite reductase. FEBS Lett 487, 347–
350.
2 Chi-Ham CL, Keaton MA, Cannon GC & Heinhorst S
(2002) The DNA-compacting protein DCP68 from soy-

bean chloroplasts is ferredoxin: sulfite reductase and
co-localizes with the organellar nucleoid. Plant Mol Biol
49, 621–630.
3 Kuroiwa T (1991) The replication, differentiation, and
inheritance of plastids with emphasis on the concept of
prganelle nuclei. Int Rev Cytol 128, 1–62.
4 Nakayama M, Akashi T & Hase T (2000) Plant sulfite
reductase: molecular structure, catalytic function and
interaction with ferredoxin. J Inorg Biochem 82, 27–32.
5 Brunold C & Suter M (1989) Localization of enzymes
of assimilatory sulfate reduction in pea roots. Planta
179, 228–234.
6 Saito K (2000) Regulation of sulfate transport and syn-
thesis of sulfur-containing amino acids. Curr Opin Plant
Biol 3, 188–195.
7 Winter H, Robinson DG & Heldt HW (1994) Subcellu-
lar volumes and metabolite concentrations in spinach
leaves. Planta 193, 530–535.
8 Hashimoto H (1985) Changes in distribution of nucle-
oids in developing and dividing chloroplasts and etio-
plasts of Arena sativa. Protoplasma 127, 119–127.
9 Sato N (2001) Was the evolution of plastid genetic
machinery discontinuous? Trends Plant Sci 6, 151–155.
10 Sato N, Misumi O, Shinada Y, Sasaki M & Yoine M
(1997) Dynamics of localization and protein composi-
tion of plastid nucleoids in light-grown pea seedlings.
Protoplasma 200, 163–173.
11 Sato N, Rolland N, Block MA & Joyard J (1999) Do
plastid envelope membranes play a role in the expres-
sion of the plastid genome? Biochimie 81 , 619–629.

12 Sato N, Sekine K, Kabeya Y, Ehira S, Onuma M &
Ohta N (2004) Discontinuous evolution of plastid
genomic machinery: Radical replacement of major
DNA-binding proteins. Endocytobiosis Cell Res 15,
286–293.
13 Sato N, Terasawa K, Miyajima K & Kabeya Y (2003)
Organization, developmental dynamics, and evolution
of plastid nucleoids. Int Rev Cytol 232, 217–262.
14 Kato Y, Murakami S, Yamamoto Y, Chatani H,
Kondo Y, Nakano T, Yokota A & Sato F (2004) The
DNA-binding protease, CND41, and the degradation of
ribulose-1,5-bisphosphate carboxylase ⁄ oxygenase in sen-
escent leaves of tobacco. Planta 220, 97–104.
15 Kato Y, Yamamoto Y, Murakami S & Sato F (2005)
Post-translational regulation of CND41 protease activ-
ity in senescent tobacco leaves. Planta 222, 643–651.
16 Murakami S, Kondo Y, Nakano T & Sato F (2000)
Protease activity of CND41, a chloroplast nucleoid
DNA-binding protein, isolated from cultured tobacco
cells. FEBS Lett 468, 15–18.
17 Nakano T, Murakami S, Shoji T, Yoshida S, Yamada
Y & Sato F (1997) A novel protein with DNA binding
activity from tobacco chloroplast nucleoids. Plant Cell
9, 1673–1682.
18 Sato N, Albrieux C, Joyard J, Douce R & Kuroiwa T
(1993) Detection and characterization of a plastid envel-
ope DNA-binding protein which may anchor plastid
nucleoids. EMBO J 12, 555–561.
19 Sato N, Ohshima K, Watanabe A, Ohta N, Nishiyama Y,
Joyard J & Douce R (1998) Molecular characterization of

the PEND protein, a novel bZIP protein present in the
envelope membrane that is the site of nucleoid replication
in developing plastids. Plant Cell 10, 859–872.
20 Sato N & Ohta N (2001) DNA-binding specificity and
dimerization of the DNA-binding domain of the PEND
protein in the chloroplast envelope membrane. Nucleic
Acids Res 29, 2244–2250.
21 Terasawa K & Sato N (2005) Occurrence and character-
ization of PEND proteins in angiosperms. J Plant Res
118, 111–119.
22 Jeong SY, Peffer N & Meier I (2004) Phosphorylation
by protein kinase CKII modulates the DNA-binding
activity of a chloroplast nucleoid-associated protein.
Planta 219, 298–302.
23 Jeong SY, Rose A & Meier I (2003) MFP1 is a thyla-
koid-associated, nucleoid-binding protein with a coiled-
coil structure. Nucleic Acids Res 31, 5175–5185.
24 Pfalz J, Liere K, Kandlbinder A, Dietz K-J & Oelmu
¨
ller
R (2005) pTAC2-6, and -12 are components of the
transcriptionally active plastid chromosome that are
required for plastid gene expression. Plant Cell 18,
176–197.
25 Phinney BS & Thelen JJ (2005) Proteomic characteriza-
tion of a triton-insoluble fraction from chloroplasts
defines a novel group of proteins associated with macro-
molecular structures. J Proteome Res 4, 497–506.
K. Sekine et al. DNA binding of sulfite reductase
FEBS Journal 274 (2007) 2054–2069 ª 2007 The Authors Journal compilation ª 2007 FEBS 2067

26 Cannon GC, Ward LN, Case CI & Heinhorst S (1999)
The 68 kDa DNA compacting nucleoid protein from
soybean chloroplasts inhibits DNA synthesis in vitro.
Plant Mol Biol 39, 835–845.
27 Sekine K, Hase T & Sato N (2002) Reversible DNA
compaction by sulfite reductase regulates transcriptional
activity of chloroplast nucleoids. J Biol Chem 277,
24399–24404.
28 Crane B, Siegel L & Getzoff E (1995) Sulfite reductase
structure at 1.6 A
˚
: evolution and catalysis for reduction
of inorganic anions. Science 270, 59–67.
29 Yonekura-Sakakibara K, Ashikari T, Tanaka Y,
Kusumi T-A & Hase T (1998) Molecular characteriza-
tion of tobacco sulfite reductase: enzyme purification,
gene cloning, and gene expression analysis. J Biochem
(Tokyo) 124, 615–621.
30 Yonekura-Sakakibara K, Onda Y, Ashikari T, Tanaka
Y, Kusumi T & Hase T (2000) Analysis of reductant
supply systems for ferredoxin-dependent sulfite reduc-
tase in photosynthetic and nonphotosynthetic organs of
maize. Plant Physiol 122, 887–894.
31 Borka C, Schwennb JD & Hell R (1998) Isolation and
characterization of a gene for assimilatory sulfite reduc-
tase from Arabidopsis thaliana. Gene 212, 147–153.
32 Ideguchi T, Akashi T, Onda Y & Hase T (1995) cDNA
cloning and functional expression of ferredoxin-depen-
dent sulfite reductase from maize in E. coli cells. In
Photosynthesis: from Light to Biosphere, Vol II (Mathis

P, ed), pp. 713–716. Kluwer Academic Publishers,
Dordrecht.
33 Saitoh T, Ikegami T, Nakayama M, Teshima K, Akutsu
H & Hase T (2006) NMR study of the electron transfer
complex of plant ferredoxin and sulfite reductase: map-
ping the interaction sites of ferredoxin. J Biol Chem
281, 10482–10488.
34 Sato N (2006) Origin and evolution of plastids: genomic
view on the unification and diversity of plastids. In The
Structure and Function of Plastids) (Wise RR & Hoober
JK, eds), pp. 75–102. Springer, Dordrecht.
35 Matsuzaki M, Misumi O, Shin-I T, Maruyama S,
Takahara M, Miyagishima S-y, Mori T, Nishida K,
Yagisawa F, Nishida K, Yoshida Y et al. (2004)
Genome sequence of the ultrasmall unicellular red alga
Cyanidioschyzon merolae 10D. Nature 428, 653–657.
36 Arabidopsis Genome Initiative (2000) Analysis of the
genome sequence of the flowering plant Arabidopsis
thaliana. Nature 408, 796–815.
37 International Rice Genome Sequencing Project (2005)
The map-based sequence of the rice genome. Nature
436, 793–800.
38 Link G (2003) Redox regulation of chloroplast trans-
cription. Antioxid Redox Signal 5, 79–87.
39 Pfannschmidt T, Nilsson A & Allen JF (1999) Photo-
synthetic control of chloroplast gene expression. Nature
397, 625–628.
40 Wu M, Nie ZQ & Yang J (1989) The 18-kD protein
that binds to the chloroplast DNA replicative origin is
an iron–sulfur protein related to a subunit of NADH

dehydrogenase. Plant Cell 1, 551–557.
41 Chen XJ & Butow RA (2005) The organization and
inheritance of the mitochondrial genome. Nat Rev Genet
6, 815–825.
42 Chen XJ, Wang X, Kaufman BA & Butow RA (2005)
Aconitase couples metabokic regulation to mitochon-
drial DNA maintenamce. Science 307, 714–717.
43 Kaufman BA, Newman SM, Hallberg RL, Slaughter
CA, Perlman PS & Butow RA (2000) In organello for-
maldehyde crosslinking of proteins to mtDNA: identifi-
cation of bifunctional proteins. Proc Natl Acad Sci USA
97, 7772–7777.
44 Sato H, Tachifuji A, Tamura M & Miyakawa I (2002)
Identification of the YMN-1 antigen protein and bio-
chemical analyses of protein components in the mito-
chondrial nucleoid fraction of the yeast Saccharomyces
cerevisiae. Protoplasma 219, 51–58.
45 Bogenhagen DF, Wang Y, Shen EL & Kobayashi R
(2003) Protein components of mitochondrial DNA
nucleoids in higher eukaryotes. Mol Cell Proteomics 2,
1205–1216.
46 Wang Y & Bogenhagen DF (2006) Human mitochon-
drial DNA nucleoids are linked to protein folding
machinery and metabolic enzymes at the
mitochondrial inner membrane. J Biol Chem 281,
25791–25802.
47 McCammon MT, Epstein CB, Przybyla-Zawislak B,
McAlister-Henn L & Butow RA (2003) Global trans-
cription analysis of Krebs tricarboxylic acid cycle
mutants reveals an alternating pattern of gene expres-

sion and effects on hypoxic and oxidative genes. Mol
Biol Cell 14, 958–972.
48 Hirling H, Henderson BR & Ku
¨
hn LC (1994) Muta-
tional analysis of the [4Fe-4S]-cluster converting iron
regulatory factor from its RNA-binding form to cyto-
plasmic aconitase. EMBO J 13, 453–461.
49 Shadel GS (2005) Mitochondrial DNA, aconitase
‘wraps’ it up. Trends Biochem Sci 30, 294–296.
50 Foyer CH & Noctor G (2003) Redox sensing and sig-
nalling associated with reactive oxygen in chloroplasts,
peroxisomes and mitochondria. Physiologia Plantarum
119, 355–364.
51 Laloi C, Przybyla D & Apel K (2006) A genetic
approach towards elucidating the biological activity of
different reactive oxygen species in Arabidopsis thaliana.
J Exp Bot 57, 1719–1724.
52 Sato N (2000) siseq: manipulation of multiple sequence
and large database files for common platforms. Bio-
informatics 16, 180–181.
53 Edgar RC (2004) muscle: multiple sequence alignment
with high accuracy and high throughput. Nucleic Acids
Res 32, 1792–1797.
DNA binding of sulfite reductase K. Sekine et al.
2068 FEBS Journal 274 (2007) 2054–2069 ª 2007 The Authors Journal compilation ª 2007 FEBS
54 Jobb G, von Haeseler A & Strimmer K (2004) treefin-
der: a powerful graphical analysis environment for
molecular phylogenetics. BMC Evol Biol 4, 18.
55 Kumar S, Tamara K & Nei M (2004) mega3: inte-

grated software for molecular evolutionary genetics
analysis and sequence alignment. Brief Bioinform 5,
150–163.
56 Perrie
`
re G & Gouy M (1996) www-query: an on-line
retrieval system for biological sequence banks. Biochimie
78, 368–369.
57 Crestfield AM, Moore S & Stein WH (1963) The
preparation and enzymatic hydrolysis of reduced and
S-carboxymethylated proteins. J Biol Chem 238,
622–627.
58 Hanke GT, Okutani S, Satomi Y, Takao T, Suzuki A &
Hase T (2005) Multiple iso-proteins of FNR in Arabi-
dopsis: evidence for different contributions to chloro-
plast function and nitrogen assimilation. Plant Cell
Environ 28, 1146–1157.
59 Sasaki N, Kuroiwa H, Nishitani C, Takano H,
Higashiyama T, Kobayashi T, Shirai Y, Sakai A,
Kawano S, Murakami-Murofushi K et al. (2003) Glom
is a novel mitochondrial DNA packaging protein in
Physarum polycephalum and causes intense chromatin
condensation without suppressing DNA functions. Mol
Biol Cell 14, 4758–4769.
60 von Arb C & Brunold C (1983) Measurement of ferre-
doxin-dependent sulfite reductase activity in crude
extracts from leaves using O-acetyl-l-serine sulfhydry-
lase in a coupled assay system to measure the sulfide
formed. Anal Biochem 131, 198–204.
61 Matsumura T, Sakakibara H, Nakano R, Kimata Y,

Sugiyama T & Hase T (1997) A nitrate-inducible
ferredoxin in maize roots: genomic organization
and differential expression of two nonphoto-
synthetic ferredoxin isoproteins. Plant Physiol 114,
653–660.
62 Sakai A, Saito C, Inada N & Kuroiwa T (1998) Tran-
scriptional activities of the chloroplast-nuclei and pro-
plastid-nuclei isolated from tobacco exhibit different
sensitivities to tagetitoxin: implication of the presence
of distinct RNA polymerases. Plant Cell Physiol 39,
928–934.
Supplementary material
The following supplementary material is available
online:
Fig. S1. The band pattern of nucleoid proteins on
SDS ⁄ PAGE. The gel piece containing a band corres-
ponding to SiR is indicated by a square.
Fig. S2. Gel mobility shift assay using PsSiR, ZmSiR
and BSA. Radio-labeled 20-mer dsDNA was used as a
probe. Concentrations of all proteins mixed with probe
DNA were 200 n
M.
Table S1. Identification of PsSiR from isolated chloro-
plast nucleoids.
This material is available as part of the online article
from
Please note: Blackwell Publishing is not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corres-

ponding author for the article.
K. Sekine et al. DNA binding of sulfite reductase
FEBS Journal 274 (2007) 2054–2069 ª 2007 The Authors Journal compilation ª 2007 FEBS 2069