Eukaryotic-type plastid nucleoid protein pTAC3 is essential for transcription by the bacterial-type plastid RNA polymerase.
ABSTRACT Plastid transcription is mediated by two distinct types of RNA polymerases (RNAPs), bacterial-type RNAP (PEP) and phage-type RNAP (NEP). Recent genomic and proteomic studies revealed that higher plants have lost most prokaryotic transcription regulators and have acquired eukaryotic-type proteins during plant evolution. However, in vivo dynamics of chloroplast RNA polymerases and eukaryotic-type plastid nucleoid proteins have not been directly characterized experimentally. Here, we examine the association of the α-subunit of PEP and eukaryotic-type protein, plastid transcriptionally active chromosome 3 (pTAC3) with transcribed regions in vivo by using chloroplast chromatin immunoprecipitation (cpChIP) assays. PEP α-subunit preferentially associates with PEP promoters of photosynthesis and rRNA genes, but not with NEP promoter regions, suggesting selective and accurate recognition of PEP promoters by PEP. The cpChIP assays further demonstrate that the peak of PEP association occurs at the promoter-proximal region and declines gradually along the transcribed region. pTAC3 is a putative DNA-binding protein that is localized to chloroplast nucleoids and is essential for PEP-dependent transcription. Density gradient and immunoprecipitation analyses of PEP revealed that pTAC3 is associated with the PEP complex. Interestingly, pTAC3 associates with the PEP complex not only during transcription initiation, but also during elongation and termination. These results suggest that pTAC3 is an essential component of the chloroplast PEP complex. In addition, we demonstrate that light-dependent chloroplast transcription is mediated by light-induced association of the PEP-pTAC3 complex with promoters. This study illustrates unique dynamics of PEP and its associated protein pTAC3 during light-dependent transcription in chloroplasts.
- SourceAvailable from: ncbi.nlm.nih.gov[show abstract] [hide abstract]
ABSTRACT: Chloroplast DNA (cpDNA) is packed into discrete structures called chloroplast nucleoids (cp-nucleoids). The structure of cpDNA is thought to be important for its maintenance and regulation. In bacteria and mitochondria, histone-like proteins (such as HU and Abf2, respectively) are abundant and play important roles in DNA organization. However, a primary structural protein has yet to be found in cp-nucleoids. Here, we identified an abundant DNA binding protein from isolated cp-nucleoids of the primitive red alga Cyanidioschyzon merolae. The purified protein had sequence homology with the bacterial histone-like protein HU, and it complemented HU-lacking Escherichia coli mutants. The protein, called HC (histone-like protein of chloroplast), was encoded by a single gene (CmhupA) in the C. merolae chloroplast genome. Using immunofluorescence and immunoelectron microscopy, we demonstrated that HC was distributed uniformly throughout the entire cp-nucleoid. The protein was expressed constitutively throughout the cell and the chloroplast division cycle, and it was able to condense DNA. These results indicate that HC, a bacteria-derived histone-like protein, primarily organizes cpDNA into the nucleoid.The Plant Cell 08/2002; 14(7):1579-89. · 9.25 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The large subunit of ribulose 1,5-bisphosphate carboxylase (rbcL) and the beta subunit of chloroplast ATP synthase (atpB) are encoded by divergently transcribed genes on the plastid genome. We have identified DNA binding factors specific for sequences located in the intergenic region between these two genes. Soluble plastid extracts from pea or whole cell extracts from maize protected a maize chloroplast DNA probe containing the 160-base pair region between the 5' ends of rbcL and atpB genes from exonuclease III digestion between positions -16 and -101 relative to the rbcL gene transcription start site. Competition assay with partial sequences from this intergenic region demonstrated that specific sequence(s) are required for the protection. The borders of the binding domain are conserved among the homologous regions of maize, tobacco, spinach, and pea chloroplast genomes. Gel filtration chromatography revealed a molecular weight of about 115,000 for the active complex involved in DNA binding. Using the exonuclease III protection assay, we have also shown that purified Escherichia coli RNA polymerase protects from +25 to -20 of the rbcL gene and from +21 to -23 of the atpB gene relative to their respective transcription start sites. These regions are analogous to open complexes found when E. coli RNA polymerase interacts with the prokaryotic promoters and are consistent with the ability of E. coli RNA polymerase to initiate transcription correctly on linear templates containing these chloroplast promoters. Possible role(s) for the chloroplast DNA binding factor in chloroplast gene expression and its regulation are discussed.Journal of Biological Chemistry 07/1988; 263(17):8288-93. · 4.65 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Chloroplast DNA (cpDNA) binds to the envelope membrane of actively dividing chloroplasts (plastids) in young pea leaves. South-western blotting was used to identify and characterize the protein involved in the binding of cpDNA to the envelope membrane. A 130 kDa protein in the inner chloroplast (plastid) envelope membrane binds specific sequences within the cpDNA. These included a 0.41 kbp sequence located upstream of the psaAB gene, a 0.57 kbp sequence located downstream of the petA gene and a 1.2 kbp sequence located within the rpoC2 gene. The protein was detected in the envelope membrane of young pea leaves in which the cpDNA had been located by fluorescence microscopy at the chloroplast periphery, whereas it was undetectable in mature leaves. We therefore propose that the 130 kDa protein is involved in the binding of cpDNA to the envelope membrane, and named it plastid envelope DNA-binding protein.The EMBO Journal 03/1993; 12(2):555-61. · 9.82 Impact Factor
Eukaryotic-type plastid nucleoid protein pTAC3 is
essential for transcription by the bacterial-type
plastid RNA polymerase
Yusuke Yagia,b,c, Yoko Ishizakic, Yoichi Nakahirac, Yuzuru Tozawad, and Takashi Shiinac,1
aFaculty of Agriculture andbInstitute of Advanced Study, Kyushu University, Fukuoka 812-8581, Japan;cGraduate School of Life and Environmental Science,
Kyoto Prefectural University, Kyoto 606-8522, Japan; anddCell-Free Science and Technology Research Center, Ehime University, Matsuyama 790-8577, Japan
Edited by André T. Jagendorf, Cornell University, Ithaca, NY, and approved April 3, 2012 (received for review November 24, 2011)
Plastid transcription is mediated by two distinct types of RNA pol-
ymerases (RNAPs), bacterial-type RNAP (PEP) and phage-type
RNAP (NEP). Recent genomic and proteomic studies revealed that
higher plants have lost most prokaryotic transcription regulators
and have acquired eukaryotic-type proteins during plant evolu-
tion. However, in vivo dynamics of chloroplast RNA polymerases
and eukaryotic-type plastid nucleoid proteins have not been di-
rectly characterized experimentally. Here, we examine the associ-
ation of the α-subunit of PEP and eukaryotic-type protein, plastid
transcriptionally active chromosome 3 (pTAC3) with transcribed
regions in vivo by using chloroplast chromatin immunoprecipita-
tion (cpChIP) assays. PEP α-subunit preferentially associates with
PEP promoters of photosynthesis and rRNA genes, but not with
NEP promoter regions, suggesting selective and accurate recogni-
tion of PEP promoters by PEP. The cpChIP assays further demon-
strate that the peak of PEP association occurs at the promoter-
proximal region and declines gradually along the transcribed
region. pTAC3 is a putative DNA-binding protein that is localized to
chloroplast nucleoids and is essential for PEP-dependent transcrip-
tion. Density gradient and immunoprecipitation analyses of PEP
revealed that pTAC3 is associated with the PEP complex. Interest-
ingly, pTAC3 associates with the PEP complex not only during
transcription initiation, but also during elongation and termina-
tion. These results suggest that pTAC3 is an essential component
of the chloroplast PEP complex. In addition, we demonstrate that
light-dependent chloroplast transcription is mediated by light-in-
duced association of the PEP–pTAC3 complex with promoters. This
study illustrates unique dynamics of PEP and its associated protein
pTAC3 during light-dependent transcription in chloroplasts.
and are thought to have originated from an ancestral cya-
nobacterial endosymbiont. Whereas cyanobacteria contain over
3,000 genes, the plastid genome in higher plants consists of small,
circular, double-stranded DNA (120–150 kbp) encoding ∼120
genes for photosynthesis and gene expression machineries (1, 2),
indicating massive transfer of chloroplast genes to nuclear ge-
nome during evolution (3). Vascular plants have evolved a com-
plex transcriptional network that is mediated by two types of
RNA polymerases (RNAPs): cyanobacterium-derived plastid-
encoded plastid RNA polymerase (PEP) and nuclear-encoded
phage-type RNA polymerase (NEP). PEP is composed of four
catalytic subunits and a promoter recognition subunit, σ-factor
(4). Genes for PEP core subunits, α, β, β′, and β′′ were retained
in plastid genomes as rpoA, rpoB, rpoC1, and rpoC2 during plant
evolution, but genes for σ-factors involved in transcription ini-
tiation, have been transferred to the nuclear genome (5), which
allows the nucleus to control PEP transcription initiation in
response to developmental and environmental cues (recently
reviewed in ref. 6). PEP is responsible for transcription of pho-
tosynthesis genes in chloroplast in response to light. On the other
hand, housekeeping genes encoding PEP core subunits and ri-
bosomal proteins are transcribed by the phage-type NEP (7, 8).
lastids are DNA-containing organelles unique to plant cells
Thus, two types of RNAP have distinct roles in chloroplast
transcription in higher plants.
It has been proposed that light-dependent initiation of tran-
scription by PEP is controlled by light-induced expression of
nuclear-encoded plastid σ-factors (6). Contrarily, several evi-
dences suggest that phosphorylated PEP may tightly bind to the
promoter region to arrest transcription in the dark (9–11). Dark-
induced phosphorylation of PEP and/or σ-factors by redox-reg-
ulated plastid transcription kinase (PTK) may be a key step in
light-dependent plastid gene transcription. Thus, molecular
mechanism of light-dependent transcription in chloroplasts still
Plastid DNAs are densely packed into protein–DNA com-
plexes called “plastid nucleoids,” as well as bacterial nucleoids.
However, higher plants and moss have lost prokaryotic major
nucleoid proteins including Hu during evolution (12, 13),
whereas several eukaryotic-type proteins such as PEND (14),
MFP1 (15), SiR (16), and CND41 (17) have been identified as
major components of nucleoids in higher plants. Furthermore,
recent proteome analysis identified eukaryotic-type chloroplast
nucleoid proteins including putative DNA/RNA-binding pro-
teins as components of a chloroplast-derived DNA–protein
complex termed pTAC (plastid transcriptionally active chromo-
some) (18) and a blue native (BN)-PAGE separated basic PEP
complex (19). These findings suggest that chloroplasts have lost
most of prokaryotic nucleoid proteins involved in DNA packag-
ing, replication, transcription, and translation and acquired
eukaryotic-type chloroplast nucleoid proteins during evolution
(20). Because vascular plants lack prokaryotic transcription reg-
ulators such as DNA-binding proteins and transcription elonga-
tion factors except for σ-factors, chloroplast transcription might
be mediated by a unique hybrid system of prokaryotic-type RNA
polymerase and eukaryotic-type accessory factors, However, the
role of the nonprokryotic nucleoid proteins in plastid transcrip-
tion remains largely unknown.
Earlier studies on pTAC proteins and PEP-associated proteins
(PAPs) have focused mainly on in planta analyses using mutant
and transgenic plants [e.g., pTAC2, pTAC6, and pTAC12 (18);
PAP3/pTAC10, PAP6/FLN1, and PAP7/pTAC14 (19); ET1 (21);
Trx-z (22)]. However, mutations in plastid transcription-related
genes occasionally give rise to drastic pleiotropic phenotypes
such as albino or pale green plants due to reduced accumulation
of plastid rRNA and tRNAs, which are mainly transcribed by
PEP, and impaired translation of chloroplast-encoded essential
proteins. Thus, it is sometimes difficult to characterize the
Author contributions: Y.Y., Y.N., and T.S. designed research; Y.Y., Y.I., Y.N., and Y.T.
performed research; Y.Y. analyzed data; and Y.Y. and T.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| May 8, 2012
| vol. 109
| no. 19
molecular function of pTAC proteins in chloroplast transcription
using null mutants. Chromatin immunoprecipitation (ChIP)
assays have been widely used to study protein–DNA interactions
and map the localization of proteins to specific DNA sequences
in the cell nucleus and bacterial nucleoids. However, very little
work has been done using ChIP to analyze the association of
PEP and its accessory proteins with transcribed regions in
chloroplast genomes. Here, we examine the association of the
α-subunit of PEP and pTAC3 with transcribed regions in vivo
by using chloroplast chromatin immunoprecipitation (cpChIP)
assays. A pTAC3 has been identified in various PEP-containing
chloroplast fractions (18, 19, 23, 24). The pTAC3 contains a SAP
DNA-binding domain (Pfam 02037), which is found in matrix
attachment region binding protein (25) (Fig. 1A). In this study,
cpChIP assays demonstrate that pTAC3 associates with the PEP
complex not only during transcription initiation, but also during
elongation and termination. In addition, we demonstrate light-
dependent association of the PEP–pTAC3 complex with PEP
promoters. This study provides unique insight into the role of
the eukaryotic-type PEP accessory protein pTAC3 in PEP-
dependent transcription in chloroplasts.
pTAC3 Is a Land Plant-Specific Protein That Is Localized to Plastid
Nucleoids. To investigate whether pTAC3 is an evolutionally ac-
quired component of plastid transcription, we examined its evo-
lutionary history. BLAST and position-specific iterative (PSI)-
BLAST searches revealed that pTAC3 is conserved among land
plants including moss and higher plants, but not in cyanobacteria
and algae (Fig. 1B and Fig. S1), suggesting a functional conser-
vation in embryophytes. TargetP and PSORT, subcellular locali-
zation prediction programs, suggest that AtpTAC3 (At3g04260)
contains a putative transit peptide of 29 amino acid residues at the
N terminus.To examine the subcellularlocalization of pTAC3, we
expressed GFP-tagged pTAC3 in Arabidopsis. Full-length Atp-
TAC3 cDNA was fused to the GFP coding region and transiently
expressed under the control of the constitutive CaMV35S pro-
moter in protoplasts prepared from Arabidopsis mesophyll cells
(Fig. 1C). It has been reported that GFP-fused plastid DNA-
binding proteins such as PEND localized to plastid nucleoids
(26). As shown in Fig. 1D, GFP-tagged AtpTAC3 appeared in
small dot-like structures that were observed throughout chlor-
oplasts and colocalized with DAPI-stained nucleoid DNA, sug-
gesting that pTAC3 is a chloroplast nucleoid-associated protein.
[SALK110045 (ptac3-1)] displayed an albino phenotype (Fig. 2A
: Transit peptide
: SAP domain
Chlorophyll GFP Marge
tion of pTAC3 proteins. Black bar corresponds to 100 aa in length. Blue and
red boxes represent the chloroplast transit peptide and SAP domain, re-
spectively. (B) Phylogenic tree of pTAC3 homolog proteins. Phylogenetic
analysis was conducted using ClustalW based on amino acid sequences of
pTAC3 proteins including At, Arabidopsis thaliana; Rc, Ricinus communis;
Gm, Glycine max; Vv, Vitis vinifera; Pt, Populus trichocarpa; Os, Oryza sativa;
Bd, Brachypodium distachyon; Pp, Physcomitrella patens. (C) Localization
analysis of pTAC3–GFP in a protoplast transient expression system. Proto-
plasts prepared from wild-type A. thaliana leaves were transformed with
expression plasmids harboring the GFP gene (control) or the full-length
AtpTAC3 fused to GFP at its N terminus under the control of the CaMV 35S
promoter. GFP fluorescence and chlorophyll autofluorescence of trans-
formed protoplasts were observed by confocal microscopy. (Scale bar, 5 μm.)
(D) DAPI staining of protoplasts expressing pTAC3–GFP. Protoplasts were
disrupted by osmotic pressure, stained with DAPI, and observed by confocal
microscopy. (Scale bar, 5 μm.)
pTAC3 is a chloroplast nucleoid protein. (A) Schematic representa-
WT (a) and ptac3-1 (b) were grown on 1/2 MS medium containing 1%
(wt/vol) sucrose for 8 d. (B) RNA gel blotting analysis of plastid gene ex-
pression in WT and ptac3-1. Five micrograms of RNA extracted from 8-d-
old WT (a) or ptac3-1 (b) was separated on 1% (wt/vol) agarose gels, and
blots were hybridized with
position of 16S, 18S, mature 23S, and 25S ribodomal RNA, which corre-
spond to 1.4, 1.8, 2.8, and 3.3 kbp in length, respectively. (C) RNA gel
blotting (Left) and Western blotting analysis (Right) of pTAC3 expression
during greening of etiolated seedlings. Wild-type Arabidopsis plants were
grown in continuous darkness for 4 d, and then the etiolated plants were
exposed to light for 1, 3, or 6 h. Total RNA and protein were extracted and
analyzed by RNA gel blotting and Western blotting, respectively. EtBr and
CBB staining image are shown below. psbD light-dependent expression is
Analysis of pTAC3-deficient mutants. (A) Phenotype of ptac3-1.
32P-labeled probes. Sizes determined by the
| www.pnas.org/cgi/doi/10.1073/pnas.1119403109Yagi et al.
and Fig. S2 A and B). To analyze the role of pTAC3 in plastid
transcription, we examined global plastid gene expression pat-
terns in ptac3-1 by using a plastid DNA macroarray. Expression
of PEP-dependent photosynthesis genes, which are actively
transcribed in chloroplasts, is significantly reduced in 8-d-old
ptac3-1 mutant plants (Fig. S3). By contrast, knockout of pTAC3
resulted in increased accumulation of several low abundance
gene transcripts, including NEP-dependent genes such as accD
and PEP core subunit genes (rpoA, and the rpoB-C1-C2 operon).
RNA gel blot analysis confirmed the reduced accumulation of
PEP-dependent photosynthesis and rrn transcripts, and the up-
regulation of NEP-dependent transcripts in ptac3-1 (Fig. 2B).
This is a typical plastid gene expression pattern of mutant plants
with impaired PEP transcription. Furthermore, we analyzed
the developmental expression of AtpTAC3 during greening of
etiolated seedlings. Wild-type Arabidopsis plants were grown in
continuous darkness for 4 d and subsequently exposed to light
for 1, 3, or 6 h. Both AtpTAC3 mRNA and AtpTAC3 protein are
induced by light within 1 h. (Fig. 2C). The transcript levels of
psbD, which are a marker for light-dependent plastid transcrip-
tion, are also increased by light. These data suggest that pTAC3
may play a crucial role in PEP-dependent transcription and be
essential for light-induced chloroplast development.
cpChIP Analysis of pTAC3 and PEP Association with Wheat Chloroplast
DNA. To understand the role of pTAC3 in PEP-dependent
transcription in chloroplasts, we designed a modified cpChIP
assay using wheat chloroplasts. First we evaluated the association
of PEP α-subunit with specific regions of plastid DNA, including
PEP promoters (psbA, rbcL, psaA, rrn16, psbDLRP, and trnEY),
a NEP promoter (rpoB), the coding region of a NEP-dependent
gene (rpoA), and the noncoding region between rps12 and
rrn16 (Spacer) (Fig. S4 and Table S1). The immunoprecipitated
DNA was analyzed by quantitative PCR and quantified using
standard curves based on a dilution series of input samples. The
data were analyzed as a percentage of the input sample (details
in Materials and Methods). The cpChIP assay showed binding of
PEP α-subunit to the promoter regions of PEP-dependent pho-
tosynthesis genes and the rrn operon, but not to those of NEP-
dependent genes or the spacer region in vivo (Fig. 3A). By
contrast, the cpChIP assay using an antibody against the non–
DNA-binding chloroplast protein, glutamime synthetase 2 (GS2)
resulted in a lower cpChIP-QPCR signal at the psbA promoter
comparable to that at the rpoB promoter (Fig. S5). These results
indicate that the cpChIP analysis can be used to detect the
specific association of DNA-binding protein with chloroplast
DNA. Furthermore, cpChIP signal of PEP α-subunit character-
istics vary markedly among PEP promoters, suggesting that the
relative association of PEP with promoters may be dependent on
promoter architecture. The highest signal was detected at the
psbA promoter, which is known as the most active promoter in
the chloroplast genome. We further examined the distribution of
PEP α-subunit along the psbA gene including the promoter,
coding, and termination regions (Fig. 3B). The peak association
with PEP α-subunit was localized in the promoter region, and the
level decreased slightly toward the termination region of psbA,
whereas the signal in the trnK-matK region, which is located
upstream of psbA, was significantly lower. Less intensive ChIP
signals in the coding region compared with the promoter region
were also observed in rbcL and the rrn operon (Fig. S6), sug-
gesting that PEP density decreases gradually along the tran-
scription unit. This cpChIP assay provides the first direct
evidence that PEP exclusively recognizes PEP promoters and
initiates transcription in vivo, suggesting that PEP–σ complexes
have high binding affinity to PEP promoters.
We also examined the association of pTAC3 with chloroplast
DNA via cpChIP assays. As in the case of PEP α-subunit, pTAC3
preferentially binds to promoter regions of PEP-dependent
genes, but not of NEP-dependent genes, suggesting a role for
pTAC3 in the PEP complex (Fig. 3A and Fig. S3). We further
examined the local patterns of spatial association of pTAC3 with
the psbA transcription unit (Fig. 3B). We found that pTAC3
binds not only to the promoter region but also the transcription
elongation region during transcription, suggesting that pTAC3
as well as PEP is associated with chloroplast DNA along the
psbA transcription unit. Thus, it is unlikely that pTAC3 binds to
specific sequences in the psbA promoter; rather it may be an
important component of the PEP complex in chloroplasts.
To examine further the interaction between pTAC3 and PEP,
we isolated the PEP complex by glycerol density gradient cen-
trifugation and probed for the presence of pTAC3 by Western
blotting. The molecular weight of the PEP complex from wheat
chloroplasts was estimated as around 700 kDa, as reported
previously in mustard (27). pTAC3 was detected in two fractions,
corresponding to a peak containing PEP α-subunit (fraction 8)
and a lower molecular weight fraction (roughly estimated to 200–
400 kDa; fractions 4 and 5) (Fig. 4A). These results suggest that
pTAC3 in wheat chloroplasts is associated mainly with the PEP
complex, although a portion of the pTAC3 may form another
complex without PEP. Furthermore, immunoprecipitation assays
using pTAC3 antibody with wheat chloroplast extracts demon-
strated the presence of α-subunit in the pTAC3 immunopreci-
pitated complex (Fig. 4B). These results demonstrate that
pTAC3 has a direct function in PEP transcription.
Association of PEP α-subunit and pTAC3 with PEP promoter regions (PpsbA,
PrbcL, PpsaA, Prrn16, psbDLRP, PtrnEY), a NEP promoter region (PrpoB), the
coding region of the rpoA gene (rpoA), and a noncoding spacer region lo-
cated between rps12 and rrn16 (Spacer) was analyzed by ChIP assay.
Chloroplasts were prepared from wheat seedlings grown for 5 d in the light
and subjected to ChIP assays using antibodies against PEP α-subunit and
pTAC3. NoAb, no antibody control. Enriched DNA was quantified by qPCR.
The amount of immunoprecipitated DNA in each sample is presented as
a percentage of the total input chromatin. Mean values and SDs of three
independent experiments are shown. (B) Spatial association of α-subunit and
pTAC3 along the psbA transcription unit. Data are shown as in A. Schematic
gene map of the matK-psbA region is shown below. Horizontal black bar
represents 200 bp. Arrow indicates the transcription start site of the psbA
gene and the direction of transcription. DNA regions corresponding to the
psbA promoter (P), coding region (C), terminator (T), and two units (a and b)
in loci upstream of psbA are shown.
Association of PEP α-subunit and pTAC3 with chloroplast DNA. (A)
Yagi et al. PNAS
| May 8, 2012
| vol. 109
| no. 19
Light-Dependent Association of the PEP–pTAC3 Complex with Chlo-
roplast DNA. PEP transcription activity is greatly stimulated by
light in mature wheat chloroplasts (28). We therefore examined
the light-dependent association of PEP with promoter regions of
several plastid genes in vivo. Chloroplasts were prepared from
wheat seedlings grown for 5 d in the light and then dark adapted
for 24 h or seedlings reilluminated for 6 h after the 24-h dark
adaptation. Immunoblot analysis revealed the constitutive accu-
mulation of both PEP α-subunit and pTAC3 upon light illumi-
nation in wheat seedlings (Fig. S7), suggesting that the expression
level of the PEP complex is not affected by light. On the other
hand, the cpChIP assays showed that the relative amount of PEP
α-subunit associated with the promoter region of photosynthesis
genes including psbA, psbD, psaA, and rbcL, and ribosomal RNA
rrn16, was two- to fivefold higher in the illuminated chloroplasts
than in the dark-adapted chloroplasts (Fig. 5A). Amounts of
chloroplast DNA in input samples between both conditions were
not significantly different (Fig. S8). Thus, the light-dependent
cpChIP signals would be indicative of light-dependent association
of PEP α-subunit and pTAC3 to chloroplast DNA. Moreover, we
could not detect tight binding of PEP α-subunit to the promoter
region of photosynthesis genes in the dark-adapted chloroplasts,
suggesting a limited role for transcription arrest mediated by
dark-induced phosphorylation of PEP subunits and σ-factors. In
addition to promoter regions, we also detected light-dependent
association of PEP α-subunit with coding and termination regions
of psbA (Fig. 5B). These results demonstrate that recruitment of
PEP to its target promoters is dependent on light. On the other
hand, light-dependent association of PEP with NEP-dependent
genes including rpoA and rpoB and the spacer region was not
detected. Furthermore, ChIP analysis of pTAC3 showed that
light accelerated the association of pTAC3 with not only the PEP
promoter region but also the coding region in the psbA tran-
scription unit (Fig. 5 A and B) similar to the distribution pattern
of PEP α-subunit, suggesting that pTAC3 associates with PEP-
dependent transcribed regions as a component of a large PEP
complex in a light-dependent manner.
This study reveals light-dependent associations of PEP and
pTAC3 with chloroplast DNA in vivo using cpChIP assays. ChIP
assays have been widely used to detect specific binding sites
for transcription factors (29), distribution patterns of several
123456789 10 11 12 13
complex by glycerol density gradient centrifugation. Total chloroplast pro-
teins prepared from wheat grown under continuous light for 6 d were
loaded onto a 10–30% (vol/vol) glycerol density gradient and separated
by centrifugation. Thirteen fractions were collected from top to bottom and
analyzed by immunoblotting with anti-PEP α-subunit or pTAC3 antibodies.
Gel staining with CBB is also shown. (B) Immunoprecipitation analysis of the
pTAC3 complex. Total wheat chloroplast extracts were subjected to immu-
noprecipitation with pTAC3 antibody or without (NoAb) and analyzed by
immunoblotting with anti-PEP α-subunit and pTAC3 antibodies.
Analysis of the pTAC3 protein complex. (A) Separation of the PEP
with chloroplast DNA. (A) Association of PEP α-subunit and pTAC3 with
chloroplast DNA in response to light. Chloroplasts were prepared from
wheat seedlings grown for 5 d in the light and then dark adapted for 24 h
(dark) or seedlings reilluminated for 6 h after the 24-h dark adaptation
(light). ChIP was performed to determine the association level of PEP
α-subunit and pTAC3 with PEP promoter regions (PpsbA, PrbcL, PpsaA,
Prrn16, psbDLRP, and PtrnEY), a NEP promoter (PrpoB), the coding region of
the rpoA gene (rpoA), and a noncoding region located between rps12 and
rrn16 (Spacer), using anti-PEP α-subunit or pTAC3 antibodies or no antibody
(NoAb). The immunoprecipitated DNA was analyzed by quantitative PCR
and quantified via standard curves based on a dilution series of input sam-
ples. The amount of immunoprecipitated DNA in each sample is presented as
a percentage of the total input chromatin. Mean values and SDs of three
independent experiments are shown. (B) Association level of PEP α-subunit
and pTAC3 with regions of the psbA transcription unit in response to light.
Data are presented as in A. Schematic gene map of the matK-psbA region is
shown as in Fig. 3B.
Analysis of light-dependent association of PEP α-subunit and pTAC3
| www.pnas.org/cgi/doi/10.1073/pnas.1119403109Yagi et al.
modified histones (30), and trafficking of RNAP and its associ-
ated proteins on genomic DNA (31). In chloroplasts, a few
studies have shown the association of endogenous (Whirly1) and
recombinant (LacI) transcription factors with chloroplast pro-
moters in vivo by using ChIP assays (32, 33). However, in vivo
dynamics of chloroplast RNA polymerases and/or their associ-
ated proteins have not been directly characterized experimen-
tally. Unlike cyanobacteria, the chloroplasts of higher plants
have two types of RNA polymerase, PEP and NEP. Transcrip-
tome analyses of PEP- or NEP-deficient mutants and in vitro
transcription analyses using isolated PEP and NEP have shown
that PEP and NEP preferentially initiate transcription from
bacterial-type and phage-type promoters, respectively. Here, we
provide direct evidence that PEP exclusively associates in vivo
with PEP promoters of photosynthesis genes and the rRNA
operon, but not with NEP promoters by using cpChIP assay (Fig.
3), suggesting selective and accurate recognition of PEP pro-
moters by PEP.
It has been shown that several PEP promoters including psbA,
psbD LRP, rbcL, psaA, and rrn P1 are regulated by unique cis-
elements, termed as the TATA-box, PGT-box, and AAG-box,
CDF1-binding site, region U, and RUA, respectively, which are
located upstream of or within the core promoter (28, 34–37) and
recognized by promoter-specific transcription factors. However,
sequence specific DNA-binding proteins have not been identified
and characterized in chloroplasts of higher plants. Chloroplast
protein pTAC3 contains a putative DNA-binding SAP domain.
Here we show that pTAC3 is localized in chloroplast nucleoids
and is essential for PEP-dependent transcription, suggesting that
pTAC3 may be a chloroplast transcription factor that regulates
PEP-dependent transcription. Thus, we further searched for
pTAC3-binding regions on plastid DNA by using cpChIP assays.
We found that pTAC3 binds to the transcribed regions of all
PEP-dependent genes examined (psbA, psaA, psbD, rbcL, and
rrn promoters), but not to a specific cis-element in a particular
promoter, suggesting that pTAC3 is a PEP-associated general,
rather than sequence-specific transcription factor. In contrast to
PEP-dependent transcribed loci, pTAC3 associates weakly with
NEP-dependent transcribed loci, suggesting that pTAC3 does
not associate with the NEP transcription complex. However,
chloroplasts exhibit very low NEP-dependent transcription ac-
tivity. NEP proteins are thought to be present at very low levels
in leaf chloroplasts, because they were not detected in the pro-
teomic analysis of whole chloroplast proteins and pTAC frac-
tions. Thus, there remains a possibility that the cpChIP assay
could not detect the association of pTAC3 with NEP-dependent
transcribed loci, due to the low levels of accumulation of NEP
In bacteria, it is proposed that RNAP accessory proteins mod-
ulate the promoter accessibility to RNAP by altering the 3D
structure of the nucleoid. The nucleoid proteins factor for in-
version stimulation (FIS) and histone-like nucleoid structuring
DNA at many regions (38). Furthermore, FIS and H-NS were
copurified with RNAP subunits and ribosomes in Escherichia coli
(39). Similarly, cpChIP assays demonstrated that pTAC3 asso-
ciates with PEP-dependent transcribed regions of photosynthesis
and rRNA genes together with PEP α-subunit. Density gradient
and immunoprecipitation analyses of PEP demonstrate that
pTAC3 is an essential component of PEP in chloroplasts. Fur-
thermore, pTAC3 is crucial for PEP activity and its expression
increases dramatically during chloroplast development, as PEP
activity increases. Taken together, these results suggest that
pTAC3 is an essential component of the PEP complex and criti-
cally involved in chloroplast differentiation from immature plas-
tids. Considering the eukaryotic origin of the SAP domain as
a DNA-binding motif, our findings suggest that pTAC3 was
acquired from host cells during plant evolution to enhance and/or
maintain transcription of photosynthesis genes in higher plants.
Our ChIP analysis of PEP α-subunit shows that PEP preferen-
tially associates with the promoter region rather than the coding
region (Fig. 3B and Fig. S3), suggesting that the PEP complex
specific antibodies for RNAP core subunits revealed that there is
also preferential association of the RNAP core with the promoter-
proximal region of transcribed genes and decreased RNAP-binding
density downstream from the promoter (31). The accumulation of
RNAP at the promoter-proximal region in E. coli is controlled
mainly by interactions between the elongating complex of RNAP
and elongation regulators, such as NusA, NusG, and Rho. The
Arabidopsis genome encodes only one putative bacterial-type elon-
gation factor, pTAC13 (At3g09210) containing a KOW domain,
Furthermore, maize ET1, which resembles the eukaryotic elonga-
tion factor TFIIS, was identified as a component of the chloroplast
transcriptionally active fraction, and plants deficient in ET1 showed
an aberrant chloroplast development phenotype and decreased
PEP-dependent transcription (21), suggesting that bacterial-type
pTAC13 and eukaryotic-type ET1 may be involved in PEP elon-
gation. In this study, we demonstrate that pTAC3 is associated
with PEP through transcription initiation and elongation steps,
suggesting a possible role for pTAC3 in the regulation of PEP
transcription elongation in chloroplasts.
Light-dependent activation of PEP transcription may be con-
trolled by PTK and plastid redox signaling. It has been proposed
that phosphorylated PEP and/or σ-like factors tightly bind to
promoters to arrest transcription under dark conditions (11). If
PEP is trapped at promoter regions in the dark, ChIP signals at
PEP promoters would not decrease in dark-adapted leaves. The
present data, however, showed that ChIP signals at both pro-
moters and coding regions of PEP-dependent photosynthesis
and rRNA genes were reduced in dark-adapted wheat seedlings,
suggesting that PEP dissociates from chloroplast genomic DNA
in the dark. Thus, it is likely that light regulates the association of
the PEP–pTAC3 complex with the promoter region possibly
through the light-dependent expression of σ-factors, rather than
via regulation of σ-factor phosphorylation by PTK.
In conclusion, here we present a characterization of in vivo
dynamics of chloroplast RNA polymerase PEP and eukaryotic-
type nucleoid protein pTAC3 with chloroplast DNA by using
cpChIP analysis. We show that PEP α-subunit preferentially
associates with the promoter-proximal regions of PEP-dependent
photosynthesis and rRNA genes in a light-dependent manner, but
not to NEP promoters, suggesting that light-dependent chloro-
plast transcription is mediated by accurate recognition of PEP
eukaryotic-type nucleoid protein pTAC3 is essential for PEP
activity and chloroplast development and associates with the PEP
complex not only during transcription initiation, but also during
elongation and termination. As pTAC3 has been acquired early
during land plant evolution, understanding the molecular func-
tion of pTAC3 should not only provide insights into the mecha-
nisms enabling developmental regulation of plastid transcription
but also offer a unique way of understanding plant evolution.
Materials and Methods
Plant Material. The Arabidopsis ptac3-1 (SALK_110045) mutant (Columbia
ecotype) was identified from the T-DNA insertion mutant line collection
generated at the Salk Institute. For expression analysis, wild-type (Columbia)
and ptac3-1 were grown on half-strength MS medium containing 1%
(wt/vol) sucrose under 16-h light and 8-h dark conditions (normal light; 50–
60 μmol · m−2s−1). Wheat (Triticum aestivum) seeds were grown on vermic-
ulite at 25 °C under continuous white light (50 μmol · m−2s−1) for 5–6 d.
Details regarding the identification of the mutant allele of pTAC3 are in-
cluded in SI Materials and Methods.
Yagi et al.PNAS
| May 8, 2012
| vol. 109
| no. 19
Chloroplast ChIP Assay. Detailed procedures for cpChIP assays are included in
SI Materials and Methods.
Plastid Gene Expression Analysis. Details for RNA extraction, RNA gel blotting
analysis, and construction and analysis of plastid DNA tiling macroarrays are
included in SI Materials and Methods.
Localization Analysis by Transient Expression in Protoplasts. Protoplast tran-
sient expression assays were performed as described previously (41). Details
for construction of pTAC3–GFP expression plasmids are included in SI
Materials and Methods.
Protein Analysis. Details regarding antibody materials, protein extraction
from isolated chloroplasts, protein complex separation via glycerol density
gradient centrifugation, immunoprecipitation, and immunoblotting analysis
are included in SI Materials and Methods.
ACKNOWLEDGMENTS. We thank Dr. M. H. Sato for helpful discussions, Mr.
Y. Motomura for technical assistance, and the Salk Institute for providing
Arabidopsis insertion mutants. This work was supported by Ministry of Ed-
ucation, Culture, Sports, Science and Technology Grants-in-Aid 21007485 (to
Y.Y.), 22004564 (to T.S.), and 2020060 (to Y.N.), and by the Private University
Strategic Research Foundation Support Program.
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| www.pnas.org/cgi/doi/10.1073/pnas.1119403109Yagi et al.