The Plant Journal 52 Transcription of plastid genes …sugita-g/pub/KabeyaTPJ(2007).pdfJournal...
Transcript of The Plant Journal 52 Transcription of plastid genes …sugita-g/pub/KabeyaTPJ(2007).pdfJournal...
Transcription of plastid genes is modulated by twonuclear-encoded a subunits of plastid RNA polymerasein the moss Physcomitrella patens
Yukihiro Kabeya†, Yuki Kobayashi‡, Hiromichi Suzuki, Jun Itoh and Mamoru Sugita*
Center for Gene Research, Nagoya University, Nagoya 464-8602, Japan
Received 15 June 2007; revised 18 July 2007; accepted 23 July 2007.
*For correspondence (fax +81 52 789 3080; e-mail [email protected]).†Present address: Miyagishima Initiative Research Unit, Frontier Research System, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.‡Present address: Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113-0032, Tokyo, Japan.
The nucleotide sequences reported in this paper have been submitted to the DDBJ/EMBL/GenBank database under accession numbers AB110071 (PpRpoA2 gene)
and AB110072 (PpRpoA2 cDNA).
Summary
In general, in higher plants, the core subunits of a bacterial-type plastid-encoded RNA polymerase (PEP) are
encoded by the plastid rpoA, rpoB, rpoC1 and rpoC2 genes. However, an rpoA gene is absent from the moss
Physcomitrella patens plastid genome, although the PpRpoA gene (renamed PpRpoA1) nuclear counterpart is
present in the nuclear genome. In this study, we identified and characterized a second gene encoding the
plastid-targeting a subunit (PpRpoA2). PpRpoA2 comprised 525 amino acids and showed 59% amino acid
identity with PpRpoA1. Two PpRpoA proteins were present in the PEP active fractions separated from the
moss chloroplast lysate, confirming that both proteins are a subunits of PEP. Northern blot analysis showed
that PpRpoA2 was highly expressed in the light, but not in the dark, whereas PpRpoA1 was constitutively
expressed. Disruption of the PpRpoA1 gene resulted in an increase in the PpRpoA2 transcript level, but most
plastid gene transcript levels were not significantly altered. This indicates that transcription of most plastid
genes depends on PpRpoA2-PEP rather than on PpRpoA1-PEP. In contrast, the transcript levels of petN, psbZ
and ycf3 were altered in the PpRpoA1 gene disruptant, suggesting that these are PpRpoA1-PEP-dependent
genes. These observations suggest that plastid genes are differentially transcribed by distinct PEP enzymes
with either PpRpoA1 or PpRpoA2.
Keywords: a subunit, chloroplast, plastid-encoded RNA polymerase, Physcomitrella, transcription.
Introduction
Plastids are semi-autonomous organelles that possess their
own genetic information. The components of photosynthe-
sis complexes and the translational and transcriptional
apparatus are encoded separately by plastid and nuclear
genomes. The nuclear-encoded components synthesized in
the cytoplasm are imported post-translationally into the
plastids and assembled with the plastid-encoded compo-
nents (Martin and Herrmann, 1998).
Plastids of higher (seed) plants contain two distinct DNA-
dependent RNA polymerases: the plastid-encoded plastid
RNA polymerase (PEP), and the nuclear-encoded plastid
RNA polymerase (NEP) (Maliga, 1998; Hess and Borner,
1999). The PEP enzyme comprises a core complex aabb¢b¢¢
encoded as plastid genes rpoA, rpoB, rpoC1 and rpoC2.
Transcription initiation by PEP is required for multiple
nuclear-encoded r factors, which recognize the bacterial-
type promoter sequence of the photosynthesis genes while
containing canonical –10 and –35 elements (Shiina et al.,
2005). PEP activity is severely inhibited by tagetitoxin, an
inhibitor of prokaryote RNA polymerase (Mathews and
Durbin, 1990). In contrast, NEP preferentially transcribes
housekeeping genes such as rpoB, rpl23 and clpP. The NEP
promoter resembles the plant mitochondrial promoter
sequence and differs completely from the PEP promoter
(Kapoor and Sugiura, 1999; Liere and Maliga, 1999). In
general, genes for non-photosynthetic components are
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The Plant Journal (2007) 52, 730–741 doi: 10.1111/j.1365-313X.2007.03270.x
transcribed by NEP during the early stage of plastid differ-
entiation and development. Subsequent transcription of the
genes for photosynthesis-related components is directed by
PEP (Hajdukiewicz et al., 1997). Several lines of evidence
indicated that a plastid-localized bacteriophage-type RNA
polymerase (RpoT) is an NEP (Lerbs-Mache, 1993; Hedtke
et al., 1997; Liere et al., 2004). NEP activity is not affected by
tagetitoxin.
We have reported previously that the rpoA gene is absent
from the plastid genome of the moss Physcomitrella patens,
and we have identified a nuclear counterpart, PpRpoA
(Sugiura et al., 2003). The loss of rpoA from the plastid
genome is a general occurrence in the arthrodontous
mosses, suggesting that the rpoA gene was lost from the
plastid genome and transferred to the nucleus during the
evolutionary history of the mosses (Sugita et al., 2004;
Goffinet et al., 2005). However, it is unclear whether the
nuclear PpRpoA gene really encodes the a subunit of PEP
and is required for the function of PEP. The RNA polymerase
core enzyme of Escherichia coli is assembled in the
sequence: a fi aa fi aab fi aabb¢, indicating that the a sub-
unit plays a key role in the assembly of the core enzyme
(Kimura and Ishihama, 1995).
In this study, we identified a second PpRpoA gene
(PpRpoA2), and showed that both PpRpoA and PpRpoA2
proteins were fractionated with PEP enzyme activity. The
two PpRpoA genes were differently expressed under differ-
ent light and dark conditions. We disrupted the PpRpoA1
gene by homologous recombination and characterized the
disruptant with respect to plastid gene expression. Disrup-
tion of the PpRpoA1 gene resulted in the alteration of several
chloroplast genes at the transcript level. We discuss this
transcription modulation, which appears to be mediated by
the two a subunits of PEP in Physcomitrella.
Results
Identification and characterization of the PpRpoA2 gene
To search for PpRpoA paralog(s) we performed a tBLAST
search using the query as the amino acid sequence of the
PpRpoA against the expressed sequence tag (EST) database
at PHYSCObase (http://moss.nibb.ac.jp) (Nishiyama et al.,
2003). End sequences of an EST clone pphf35o11 (DDBJ/
EMBL/GenBank accession nos BJ947461 and BJ958286)
were found to encode a partial coding sequence homolo-
gous to PpRpoA. We then isolated and sequenced the
cognate cDNA. The encoded protein comprised 525 amino
acid residues, which showed 59.1% identity with PpRpoA.
Therefore, the newly identified sequence was designated
as PpRpoA2 and the previously identified PpRpoA as
PpRpoA1. No other homologous sequences were found
in this analysis. Comparison of the PpRpoA2 cDNA and
the corresponding genomic sequences in PHYSCObase
(gnl|ti|870055012, gnl|ti|870076393, gnl|ti|86233862,
gnl|ti|713796403, gnl|ti|692455034 and gnl|ti|846052148)
revealed that the PpRpoA2 gene comprises seven exons
and six introns (Figure 1a). The first and sixth introns are
located in the 5¢- and 3¢-untranslated regions, respectively.
The intron insertion positions are not conserved between
PpRpoA1 and PpRpoA2 genes (Figure 1c). As shown in
Figure 1b, PpRpoA2 protein has a sequence that is homo-
logous to a part of HSP70 at the N-terminal region. PpRpoA2
is highly homologous to the sequences of known plastid-
encoded RpoAs and a cyanobacterium RpoA. Functional
amino acid residues were determined in the a subunit of
E. coli (Kimura and Ishihama, 1995). These are involved in
the dimerization of a subunits (45Arg at residue 45), in the
assembly of aab (48Leu) and in the core complex formation
of aabb¢ (86Lys and 173 Val). The two PpRpoA proteins also
have these conserved amino acid residues (Figure 1c), and
therefore are predicted be able to assemble the core PEP
enzyme.
PpRpoA2 has the N-terminal extension sequence, which is
predicted to specify plastid targeting (a score of 0.863) by the
TARGETP program for protein sorting (Emanuelsson et al.,
2000). To examine the cellular localization of PpRpoA2, we
constructed the plasmid PpRpoA2-gfp encoding a chimeric
protein of the N-terminal 125 amino acid residues fused to
sGFP and introduced it to the moss protonemal protoplasts.
Green fluorescence of PpRpoA2-GFP was localized in the
chloroplasts (Figure 1d, panel a) as was that of PpRpoA1-
GFP (Figure 1d, panel c). This clearly indicates that PpRpoA2
is a plastid-localized protein. This strongly suggests that the
two PpRpoA proteins function as an a subunit of the PEP
enzyme in the Physcomitrella plastids.
PpRpoA1 and PpRpoA2 proteins are components
of PEP enzyme
Amino acid sequence identity and the domain structures of
PpRpoA proteins strongly suggested that PpRpoA1 and
PpRpoA2 are the a subunit of PEP. To confirm this, we
investigated whether PpRpoA1 and PpRpoA2 proteins are
contained in PEP active fractions separated by anion
exchange column chromatography. As shown in Figure 2a
and Figure S1, transcriptional active fractions 21–26 were
eluted with about 0.4 M KCl, and these transcriptional
activities were severely inhibited by the addition of tageti-
toxin, an inhibitor of PEP transcription activity. Western blot
analysis showed that PpRpoA1 and PpRpoA2 are detected as
40-kDa and 50-kDa bands, respectively, in the PEP active
fraction 25, but not in inactive fractions 11 and 31
(Figure 2b). The cross-reaction of the antibodies against the
respective PpRpoA proteins with the recombinant proteins
was estimated to be less than 5% (Figure 2c). This result
indicates that both PpRpoA1 and PpRpoA2 proteins are
bona fide components of the functional PEP enzyme.
Nuclear-encoded a subunits of PEP in moss 731
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(a) (b)
(c)
(d)
732 Yukihiro Kabeya et al.
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PpRpoA1 and PpRpoA2 genes are differentially
expressed in the protonemata
To investigate the transcript levels of PpRpoA1 and
PpRpoA2 in the protonemata, we performed Northern blot
analysis. The transcript levels of PpRpoA1 accumulated at
substantial levels and showed low-amplitude fluctuation for
all RNA samples (Figure 3), as reported previously (Ichikawa
et al., 2004). The transcript level of PpRpoA2 decreased
significantly after 24 h in the dark, and its transcript declined
to 20% of its level in constant light (Figure 3, 4 L versus 3LD).
Upon transfer of the moss protonemata back into light, these
transcripts accumulated until they were restored to the
control level (Figure 3, 3LDL). This profile was similar to that
of Lhcb2 (encoding light-harvesting chlorophyll a/b binding
protein), a known light-responsive gene. This result indi-
cates that expression of the PpRpoA2 gene is differentially
regulated in a light-dependent manner.
Targeted disruption of the PpRpoA1 gene
To investigate further the precise role of PpRpoA1 and
PpRpoA2 gene products, we attempted to generate either
(a)
(b)
(c)
Figure 2. Preparation of plastid-encoded RNA polymerase active fractions
from the isolated chloroplasts and immunoblot detection of PpRpoA1 and
PpRpoA2.
(a) Fractionation of transcription activity by anion exchange chromatography.
Fractions were eluted with a linear KCl gradient. Transcription activities of
each fraction were measured as incorporation of [a-32P]UTP into the
synthesized RNA. The broken line indicates the concentration of KCl. Squares
indicate transcription activity without tagetitoxin and closed circles indicate
transcription activity with tagetitoxin.
(b) Immunoblot detection of PpRpoA1 and PpRpoA2 in the eluted fractions.
Aliquots (10 ll) of fractions 11 (lanes 1 and 4), 25 (lanes 2 and 5), and 35 (lanes
3 and 6), were separated using 10% PAGE, and then subjected to
immunodetection using anti-PpRpoA1 or anti-PpRpoA2 antisera.
(c) Evaluation of the cross-reaction of PpRpoA1 and PpRpoA2 with the
respective anti-PpRpoA antisera. A series of diluted (100, 25 or 5 ng) Thio-
PpRpoA1 or His-PpRpoA2 was loaded in 10% PAGE and then subjected to
immunodetection.
Figure 3. Transcript levels of the PpRpoA1 and PpRpoA2 genes in the moss
protonemata. Total RNA (15 lg) was separated on a 1.2% formaldehyde-
containing agarose gel and subjected to northern blot analysis. RNA was
extracted from 4-day-old protonemata grown under continuous illumination
(4 L), from 4-day-old protonemata treated for 1 day in darkness before
harvesting (3LD) and 3LD protonemata treated for a further 1 day under light
conditions (3LDL). Ethidium bromide-staining gel was shown as a loading
control (rRNA). Sizes of detected transcripts are indicated on the right of the
panels.
Figure 1. (a) Diagrams of the PpRpoA1 and PpRpoA2 genes. Boxes indicate exons.
(b) Diagrams of the PpRpoA1 and PpRpoA2 proteins. The black boxes indicate the C-terminal domain of the a subunit (aCTD), the gray boxes indicate the N-terminal
domain of the a subunit (aNTD) and the white box indicates a part of the HSP70 protein.
(c) Alignment of a subunit homologs of PpRpoA2 (AB293564) with PpRpoA1 (AB110072), Glycine max RpoA (Gm_rpoA; DQ317523), Nicotiana sylvestris (Ns_rpoA;
NC007500), Oryza sativa (Os_rpoA; X15901), fern Adiantum capillus-veneris (Ac_rpoA; NC004766), liverwort Marchantia polymorpha (Mp_rpoA; X04465), red alga
Cyanidiaschyzon merolae (Cm_rpoA; NC004799) and cyanobacterium Synechococcus elongatus PCC 6301(sy_rpoA; AP008231). The black and white arrowheads
indicate the positions of introns in PpRpoA1 and PpRpoA2, respectively. The asterisks indicate the functional amino acid residues, which were identified in the E. coli
a subunit (Kimura and Ishihama, 1995).
(d) Localization of PpRpoA-GFP fusion proteins. Physcomitrella protoplasts were transformed with PpRpoA2-GFP fusion plasmids PpRpoA2-gfp (a and b) and
PpRpoA1-gfp (c and d). a and c, fluorescence of GFP (green) using the cube U-MNIBA (Olympus, http://www.olympus-global.com); b and c, fluorescence of
chlorophyll (red) using the cube U-MWIG.
Nuclear-encoded a subunits of PEP in moss 733
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PpRpoA1 or PpRpoA2 knock-out mosses. Targeted disrup-
tion of the PpRpoA1 gene was achieved by inserting an nptII
cassette into the HindIII site within exon 2 of the plasmid
(Figure 4a). As shown in Figure 4b, we isolated eight G418-
resistant mosses and performed Southern blot analysis to
verify the targeted disruption. Probing with PpRpoA1 cDNA
detected the predicted 6.1-kb EcoRV signal in the wild-type
moss. In contrast, an 8.1-kb signal appeared in the G418-
resistant moss lines (#14, #22, #23 and #24), corresponding
to the 2.0-kb nptII cassette integrated into the PpRpoA1
locus. A uniform population of the transformed moss gen-
ome in the transgenic moss was verified further by PCR
analysis (Figure 4b). In contrast, all transformants for con-
struction of the PpRpoA2 disruptant possessed both the
wild-type and the nptII cassette-inserted PpRpoA2 gene,
representing heteroplasmic moss lines (Figure S2). Among
(a)
(b)
(e)
(c)
(d)
Figure 4. Generation of the PpRpoA1 gene disruptant and phenotype of the disruptant.
(a) The genomic structure of the wild-type and the PpRpoA1 disruptant. The expected fragment sizes after EcoRV digestion of genomic DNA for the DNA-blot
analyzed are shown. Primers (P-F, forward and P-R, reverse) and the expected fragment sizes for PCR analysis are also shown.
(b) DNA-blot analysis of G418-resistant mosses. Total DNA from wild-type (WT) and eight independent G418-resistant mosses (#11 to #24) were digested with EcoRV
and hybridized with the PpRpoA1 cDNA or ntpII probes. PCR analysis showed that 1.9-kb and 3.9-kb fragments were derived from the wild-type and G418-resistant
mosses, respectively.
(c) Total RNA (15 lg) from 4-day-old protonemata of the wild type or transgenic line #22 was subjected to Northern blot analysis with the PpRpoA1 cDNA probe.
(d) Total cellular protein (50 lg) from wild type and line #22 was subjected to Western blot analysis. To control for loading, antiserum detected against tobacco
chloroplast RNA-binding protein cp28 was used. The PpRpoA1 protein (40 kDa) was detected in WT but not in line #22.
(e) Morphology of the wild type and the PpRpoA1 disruptant protonemal colonies under continuous light conditions. Scale bars = 1 cm (protonema colony) and
1 mm (leafy shoot). Colonies were grown for 18 days and average diameters and SD of 10 colonies are plotted. The length of the leafy shoots from the wild type and
disruptant #22 was also measured and the SD are shown.
734 Yukihiro Kabeya et al.
ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 730–741
the PpRpoA1-disruptant mosses, the #22 transgenic moss
was selected as the representative PpRpoA1 disruptant and
was characterized further.
Both the transcript and the gene product of PpRpoA1 were
not detected by RNA-blot and immunoblot analyses in the
#22 transgenic moss (Figures 4c,d). This result clearly indi-
cates that PpRpoA1 protein is absent from the PpRpoA1
disruptant. The PpRpoA1 disruptant displayed the green
phenotype like the wild-type mosses, but showed slightly
retarded growth (Figures 4d,e). In the continuous light
condition, the colony size was smaller in the PpRpoA1
disruptant than in the wild type. The mean length of the leafy
shoot of the PpRpoA1 disruptant was the same as that of
wild type until the 34-day-old adult gametophore stage.
Thereafter, the disruptant leafy shoots grew slowly and were
somewhat smaller than those of the wild-type moss. Thus,
the phenotypic characters did not differ significantly
between the disruptant and wild-type mosses.
Effect of PpRpoA1 disruption on the plastid gene expression
To examine the effect of PpRpoA1 disruption on the
expression of PpRpoA2, the transcript level of PpRpoA2 was
measured by Northern blot analysis (Figure 5a). The
PpRpoA2 transcript level in the PpRpoA1 disruptant was
twice that in the wild type. To further examine the steady-
state transcript levels of the plastid genes in the PpRpoA1
disruptant, we performed plastid DNA microarray analysis.
In the 4-day-old protonemata grown under constant light
conditions, most plastid genes including psaA, psbA, psbD
and rrn16 were expressed at similar levels in the wild type
and in the PpRpoA1 disruptant, but some tRNA levels
increased in the disruptant (Table 1).
To confirm the microarray analysis results, we performed
RNA blot hybridization (Figure 5). The transcripts of psaA,
psbA, psbD, chlN, atpF, ycf4, trnL-UAG, trnfM-CAU and
rrn16 accumulated at similar levels in the wild type and in
the PpRpoA1 disruptant under both light and dark condi-
tions (Figure 5b). This result was consistent with that of the
array analysis. In addition, the transcripts of six genes (atpB,
ycf2, matK, rpoC1, chlB and psaM) accumulated greatly and
at similar levels in the wild type and in the disruptant grown
under constant light, whereas their transcripts declined to
faint levels in dark conditions (Figure 5c). In contrast, petN
and ycf3 transcript levels decreased to 40% and 20% of the
wild-type level, respectively, under constant light conditions
(Figure 5d, 4L lanes). In addition, psbZ transcript level in the
disruptant decreased to 25% of wild-type level under dark
conditions (Figure 5d, 3LD lanes), although it was
unchanged under constant light conditions (4 L lanes). The
six tRNAs accumulated at 2–10-fold higher levels in the
disruptant than in the wild-type protonemata grown under
(a)
(d)
(e)
(b) (c)
Figure 5. Transcript levels of the PpRpoA2 gene and plastid genes in the wild type and the PpRpoA1 disruptant. Total RNA was extracted from the wild type (WT) or
the PpRpoA1 disruptant (DA1) of 4-day-old protonemata grown under continuous illumination (4 L) and 4-day-old protonemata treated for 1 day in darkness before
harvesting (3LD).
(a) Transcripts of three nuclear genes, PpRpoA1, PpRpoA2 and Lhcb2 were detected by Northern blot analysis.
(b)–(e) Transcripts of plastid genes were detected using plastid gene-specific probes. Positions of RNA markers were indicated on the right of panels.
Nuclear-encoded a subunits of PEP in moss 735
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Table 1 Genes whose expression was affected by the PpRpoA1 disruptant
4L 3LD 4L 3LD
Gene Name log2(DA1/WT)* log2(DA1/WT)* Gene Name log2(DA1/WT)* log2(DA1/WT)*
rps18/psaJ 0.08 � 0.04 )0.21 + 0.29 rpoB-a )0.11 + 0.08 0.76 + 1.613’-rps12 0.16 + 0.04 )0.10 + 0.23 rpoB-b )0.14 + 0.07 )0.27 + 0.645’-rps12 0.00 + 0.04 )0.03 + 0.31 rpoC1-e2 )0.08 + 0.09 1.11 + 0.59aadA 0.62 + 1.05 ND rpoC2-a )0.06 + 0.10 0.56 + 0.59accD )0.10 + 0.08 )0.46 + 0.33 rpoC2-b )0.34 + 0.08 NDActin )0.96 + 0.18 ND rps11/rps8 )0.57 + 0.05 0.91 + 0.43atpA 0.02 + 0.08 )0.55 + 0.23 rps14 )0.57 + 0.09 )0.21 + 0.55atpB )0.08 + 0.02 1.52 + 0.20 rps15 )0.79 + 0.07 )0.35 + 1.22atpE 0.08 + 0.05 1.42 + 0.39 rps2 )0.61 + 0.03 1.46 + 0.55atpF 0.01 + 0.01 )0.93 + 0.30 rps3/rps19 )0.51 + 0.02 0.39 + 0.13atpH 0.26 + 0.06 )0.55 + 0.32 rps4 )0.93 + 0.11 0.67 + 0.19atpI )0.84 + 0.03 )0.24 + 0.42 rps7 0.07 + 0.10 )0.34 + 0.19cemA (ycf10) )0.86 + 0.03 0.54 + 0.32 rrn )2.26 + 0.07 )0.89 + 0.32chlB 0.07 + 0.09 1.02 + 0.29 rrn16 1.24 + 0.04 0.00 + 0.57chlL )0.30 + 0.09 )0.16 + 0.78 rrn23 )0.25 + 0.07 0.28 + 0.70chlN )0.05 + 0.05 1.03 + 0.38 rrn4.5/rrn5 0.59 + 0.11 )0.25 + 0.49clpP )0.06 + 0.06 )0.56 + 0.23 spacer+ORF19 0.28 + 0.07 )0.36 + 0.78matK 0.25 + 0.08 1.49 + 0.23 trnA-UGC )0.20 + 0.02 0.98 + 0.17ndhA )0.71 + 0.06 )0.51 + 0.29 trnC-GCA 0.17 + 0.12 NDndhB 0.01 + 0.06 0.23 + 0.22 trnE-Y-D ND NDndhC )0.02 + 0.13 0.26 + 0.55 trnF-GAA 1.33 + 0.49 )1.72 + 0.67ndhD )0.53 + 0.10 )0.94 + 0.31 trnfM-CAU )0.91 + 0.40 NDndhE/ndhI )0.36 + 0.07 )0.39 + 0.13 trnG-UCC 1.71 + 0.09 NDndhF )0.45 + 0.04 )0.67 + 0.50 trnG-GCC 0.55 + 0.05 NDndhH )0.83 + 0.08 )0.55 + 0.16 trnH-GUG 0.35 + 0.82 NDndhJ )0.02 + 0.10 0.30 + 0.60 trnI-GAU 0.18 + 0.07 0.66 + 0.32ndhK 0.02 + 0.04 0.46 + 0.35 trnI-CAU ND NDnptII 0.37 + 0.60 4.33 + 0.19 trnK )0.52 + 0.14 )0.02 + 0.37ORF40/ORF197 0.79 + 0.04 ND trnL-UAA 0.25 + 0.11 NDpBS 0.82 + 0.82 ND trnL-CAA ND )0.14 + 0.74petA )0.40 + 0.05 0.59 + 0.32 trnL-UAG )0.74 + 1.24 NDpetB )0.15 + 0.06 )0.68 + 0.16 trnM-CAU 0.29 + 0.06 0.03 + 0.59petD 0.08 + 0.09 )0.60 + 0.13 trnN-GUU )1.50 + 5.00 NDpetG/petL 0.37 + 0.08 )1.16 + 0.27 trnP-UGG/W-CCA 2.36 + 0.02 0.24 + 0.08psaA )0.70 + 0.07 0.12 + 0.56 trnQ-UUG 2.66 + 0.04 )0.74 + 0.40psaB )0.25 + 0.05 )0.18 + 0.14 trnR-ACG 0.16 + 0.29 )2.50 + 1.78psaC 0.50 + 0.11 )0.28 + 1.11 trnR-CCG ND )0.74 + 0.21psaI 0.00 + 0.11 )0.52 + 0.17 trnS-GCU 0.65 + 0.03 )0.40 + 0.50psaM 1.41 + 0.22 )0.70 + 0.31 trnS-GUA 0.57 + 0.05 )0.62 + 0.93psbA 0.11 + 0.08 )0.56 + 0.21 trnS-UGA 2.64 + 0.10 )0.79 + 0.20psbB )0.05 + 0.09 )0.61 + 0.26 trnT-GGU ND )1.89 + 1.42psbC )0.22 + 0.09 )0.38 + 0.12 trnT-UGU )0.80 + 0.62 NDpsbD )0.16 + 0.08 )0.56 + 0.16 trnV-UAC )0.18 + 0.05 )0.02 + 0.40psbE/F/L/J 0.13 + 0.02 )0.28 + 0.24 trnV-GAC )2.11 + 1.15 NDpsbH/T )0.02 + 0.04 )0.49 + 0.20 ycf12 1.07 + 0.11 NDpsbK/psbI 0.60 + 0.04 )0.27 + 0.26 ycf1-a )0.88 + 0.03 0.63 + 2.02psbM 0.76 + 0.11 )0.70 + 0.31 ycf1-b )0.79 + 0.05 )0.12 + 1.37pseudotrnV ND )1.79 + 1.20 ycf2-a 1.01 + 0.06 1.40 + 0.87rbcL 0.08 + 0.04 )0.50 + 0.46 ycf2-b 1.31 + 0.06 )0.10 + 0.69rpl14/rps16 )0.61 + 0.03 0.38 + 1.20 ycf3 )0.74 + 0.07 0.34 + 0.19rpl2/rpl23 )0.87 + 0.08 0.57 + 0.46 ycf4 )0.86 + 0.04 1.09 + 0.36rpl20 0.77 + 0.05 0.37 + 0.84 ycf6 (petN) 0.57 + 0.16 )1.88 + 0.12rpl21/rpl32 )0.57 + 0.19 0.44 + 1.45 ycf66 )0.06 + 0.07 )0.46 + 0.40PpRpoA1 )0.87 + 0.24 ND ycf9 (psbZ) 0.17 + 0.07 )1.44 + 0.66
*Values shown are means � SD of data from six spots.Hybridization signals for the wild type (WT) and the PpRpoA1 disruptant (DA1) were measured to estimate the signal intensity (log10 [D1*WT]) andratio of disruptant to wild type (log2 [D1/WT]). The hybridization was repeated twice with Cy3 or Cy5 dye swapping. The reliability of thehybridization signals was evaluated by the intensity of the signal (log10 [D1*WT] >4), and the dispersion among six spots on two slide glasses (SDof log2 [D1/WT] should be <0.5). Genes whose expression was judged to be altered are shaded in gray. 4L, four-day-old protonemata grown underconstant light condition. 3LD, four-day-old protonemata treated for one day in darkness before harvesting. ND, Not detected.
736 Yukihiro Kabeya et al.
ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 730–741
constant light conditions (Figure 5e). In dark-treated proto-
nemata, however, their transcript levels were the same in the
disruptant and in the wild type (Figure 5e).
Disruption of PpRpoA1 does not affect the
expression of plastid r factor genes
The effect of PpRpoA1 gene disruption on the plastid gene
expression may be caused by the modulation of the
expression of plastid r factors. To investigate this possibil-
ity, the transcript levels of three genes, PpSig1, PpSig2 and
PpSig5, encoding the plastid r factor were measured by
reverse transcriptase-polymerase chain reaction (RT-PCR) in
the wild type and the PpRpoA1 disruptant (Figure 6). The
three r factor genes were highly expressed in the light (4 L
and 3LDL) and were low in the dark (3LD). This expression
profile was similar to those of the PpRpoA1 and PpRpoA2
genes. Although there could be other differences at the
protein level, at least the transcript levels of the three PpSig
genes were not significantly different between the wild type
and the PpRpoA1 disruptant.
Discussion
In this study, we identified the second nuclear gene
PpRpoA2 encoding the PEP a subunit. Both PpRpoA2 and
PpRpoA1 proteins were detected immunologically in the
tagetitoxin-sensitive PEP active fractions. This biochemical
property confirms that the nuclear-encoded RpoA consti-
tutes the core PEP enzyme.
Northern blot analysis (Figure 3) indicated that expression
of the PpRpoA2 gene is regulated tightly in a light-depen-
dent manner, as is the light-responsive gene Lhcb2, whereas
PpRpoA1 is expressed constitutively in both light and dark
conditions. This suggests that the two PpRpoA proteins play
different roles in the transcription of plastid genes. Although
disruption of the PpRpoA1 gene resulted in the slightly
retarded growth of protonemal colonies, expression of most
plastid genes, including psbA, rrn16 or psaA, was not
affected by disruption of PpRpoA1. This implies that
PpRpoA1 is dispensable to plastid function and that
PpRpoA2 plays a central role in plastid transcription. An
alternative possibility is that PpRpoA2 merely complements
the loss of PpRpoA1 function. We prefer the first suggestion
because PpRpoA1 disruptants were obtained easily, but
PpRpoA2 was not disrupted.
The most interesting finding is that the expression of
petN, psbZ, ycf3 and several tRNA genes was altered in the
PpRpoA1 disruptant (Figure 5). Of these genes, three (petN,
psbZ and ycf3) can be categorized as PpRpoA1-PEP-depen-
dent genes. Thus the modulation of transcription may be
mediated by two a subunits of PEP in the moss Physcomit-
rella. In the wild-type moss chloroplasts the PEP enzyme
comprises PpRpoA1 (a1 subunit) or PpRpoA2 (a2 subunit), or
both, together with bb¢b¢¢, and may exist as three isoforms
(a1a1bb¢b¢¢, a1a2bb¢b¢¢ and a2a2bb¢b¢¢). In contrast, in the
PpRpoA1 disruptant, PEP exists presumably as a uniform
complex of a2a2bb¢b¢¢. The lower petN, psbZ and ycf3
transcript levels in the PpRpoA1 disruptant indicates that
the three genes are transcribed predominantly by PpRpoA1-
PEP (a1a1bb¢b¢¢). Interestingly, transcript levels of some tRNA
genes were very low in the light and high in the dark
(Figure 5e). The transcript levels of those tRNA genes were
enhanced significantly by PpRpoA1 disruption even under
light conditions (Figure 5e). This might be caused by over-
expression of PpRpoA2. Perhaps their transcript levels may
be modulated by different stability under diurnal day and
night control in the disruptant.
In E. coli the a subunit is required for transcription
activation by protein factors, and for interaction with DNA-
activation elements (Kimura and Ishihama, 1995). Sigma
factors are the most important determinants for the selec-
tion and initiation of transcription of plastid genes (Shiina
et al., 2005), and recognize the promoter consisting of –10
and –35 elements. PpRpoA proteins must interact with some
r factor bound to the promoter. To compare the promoter
sequences of the PpRpoA1-dependent or -independent
genes, we used a primer extension experiment to identify
putative transcription initiation sites of the moss plastid
genes (Figure S3). As shown in Figure 7, PpRpoA1-indepen-
dent genes have canonical –35 and –10-like elements, and
PpRpoA1-dependent genes (petN and ycf3) also have a
canonical –35 element and an extended –10 sequence,
GAT(G/A)TATATA(T/A)AT. The other putative PpRpoA1-
dependent gene, psbZ, has a sequence TCGGCCA that is
also found in the upstream region of the ycf3 ) 253. Among
the three Physcomitrella plastid r factor genes, the PpSig1
Figure 6. Semi-quantitative reverse transcriptase-polymerase chain reaction
analysis of PpRpoA1, PpRpoA2 and plastid r factor transcript levels in the wild
type and the PpRpoA1 disruptant. Total RNA was extracted from the wild type
or the PpRpoA1 disruptant of 4-day-old protonemata grown under continuous
illumination (4 L), 4-day-old protonemata treated for 1 day in darkness before
harvesting (3LD) and 3LD protonemata treated for a further 1 day under light
conditions (3LDL), reverse-transcribed, and amplified by PCR using a primer
set specific to each PpRpoA, PpSig or PpActin3 gene.
Nuclear-encoded a subunits of PEP in moss 737
ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 730–741
and PpSig2 genes are expressed throughout the day, and
their fluctuations suggest very low amplitude diurnal
rhythms of mRNA levels. In contrast, PpSig5 mRNA showed
a very high amplitude diurnal rhythm with peaks observed in
the light phases (Ichikawa et al., 2004). Although similar
results were also observed in this study (Figure 6), these
plastid r factors are unlikely to be responsible for the
alteration of plastid gene expression in the disruptant.
However, we cannot exclude the possibility that alternation
of protein levels or phosphorylation states of PpSigs influ-
ences the steady-state transcript levels of plastid genes in
the disruptant. Therefore, we hypothesize that different
combinations of the two a subunits with a certain r factor
(rather than PpSig1, PpSig2 and PpSig5), or that some
transcription factors interact with an upstream element or
promoter of each plastid gene to modulate the strength of
transcription activity. PpRpoA2 possesses a portion of DnaK/
HSP70, which may provide an additive function to PpRpoA2
as an a subunit. We speculate that PpRpoA2 protein is able
to facilitate or interfere with the r or other transcription
factors.
Previous studies of tobacco plants have demonstrated
that photosynthesis genes are transcribed by PEP, that some
plastid genes such as atpB and rrn16 are transcribed by both
PEP and NEP, and that most housekeeping genes are
transcribed by NEP (Hajdukiewicz et al., 1997). A relative
increase in NEP activity was observed in PEP-deficient
tobacco (Krause et al., 2000; Legen et al., 2002). We did not
construct PEP-deficient mosses in this study, and therefore
we cannot conclude whether NEP exists in the P. patens
chloroplasts. Double knock-out mutants of PpRpoA1 and
PpRpoA2 are needed to address this question. Alternatively,
disruption of plastid genes rpoB, rpoC1 or rpoC2 may also
help address this issue. We attempted to construct plastid
rpo gene knock-out mosses, but we obtained only hetero-
transplastomic lines, which possessed both the wild-type
plastomes and the rpo gene-disrupted plastome (unpub-
lished data). In the moss P. patens, the haploid gametophyte
dominates the life cycle, represented by the filamentous
protonema (juvenile gametophytes) and the leafy moss
plant (adult gametophyte). Plastid ontogeny in mosses
differs distinctly from that in vascular plants (Reski, 1998).
This may be the reason why this moss developed such a
unique system for plastid transcription that is unlike higher
plants.
Experimental procedures
Plant material
Physcomitrella patens (Hedew.) Bruch & Schimp subsp. patensTan was grown at 25�C under continuous illumination at30 lmol m)2 sec)1 on the minimal medium (BCD medium) sup-plemented with 0.5% glucose, 1 mM CaCl2 and 5 mM diammonium(+)-tartrate agar plate as described previously (Sugiura and Sugita,2004).
Isolation and sequence analysis of cDNA
cDNA encoding PpRpoA2 was prepared using primers cA2.F(5¢-TCTCTCCTGCAGGCCTCTTCACCTCTAC-3¢) and cA2.R(5¢-GCCTGTCAGGCTCCATCTCTAAGTGGTTTC-3¢) designed fromsequences of an EST clone (pphf35o11). Sequencing was per-formed with an ABI PRISM 3100 sequencer and the DYEnamicET Terminator Cycle Sequencing Kit (GE Healthcare, http://www.gehealthcare.com) using appropriate sequencing primers.Alignments of amino acid sequences were constructed by theCLUSTALX program, version 1.81 (Thompson et al., 1994).
Figure 7. Alignment of nucleotide sequences of
the region upstream of the 5¢-ends of PpRpoA1-
independent and -dependent gene transcripts.
The underlines indicate the position correspond-
ing to the 5¢-end of the major transcript. The
arrowheads indicate the initiation nucleotide of
mature tRNAs. The r70-type promoter in Escher-
ichia coli comprising TTGACA (–35 element) and
TATAAT (–10 element) are indicated by the gray
box. The conserved element specific to the
PpRpoA1-dependent promoter is boxed.
738 Yukihiro Kabeya et al.
ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 730–741
Construction of the PpRpoA2-GFP fusion gene
and moss transformation
A DNA fragment encoding the N-terminal 125 amino acid residuesof PpRpoA2 was amplified from the cDNA as above with primersA2GFP.F (5¢-CCCGTCGACCACCATGGCAACTGTCATGGGCGC-3¢)and A2GFP.R (5¢-ACGTGTCGACGGCCTTTTCTGCAGCTTCTGTAA-3¢). The PCR product was digested with SalI and inserted into theSalI-cleaved CaMV35S-sGFP(S65T)-nos3¢ (Chiu et al., 1996) tocreate the PpRpoA2-gfp construct. The reporter construct wasintroduced into the protoplasts prepared from the 3-day-old proto-nemata. As a positive control, the PpRpoA-gfp construct was alsoused for transformation (Sugiura et al., 2003).
Chloroplast isolation and anion
exchange chromatography
Intact moss chloroplasts were isolated from 4-day-old protonemalcells as described previously (Kabeya and Sato, 2005). To preparethe chloroplast lysate, intact chloroplasts were resuspended inbuffer 1 (10 mM Tris–HCl, pH 8.0, 1 mM EDTA, 5 mM DTT, 1 M KCl)and incubated for 15 min on ice. An equal volume of buffer 2 (50 mM
Tris–HCl, pH 8.0, 10 mM MgCl2, 2 mM DTT, 20% sucrose, 50% glyc-erol) was added to the lysed plastids, and (NH4)2SO4 was added tothe plastid lysates to a final concentration of 10%. The mixture wasstirred for 30 min on ice and then centrifuged at 50 000 g for 1 h.(NH4)2SO4 was added to the supernatant to a final concentration of60%. The pellet was resuspended in buffer 3 (20 mM HEPES, pH 8.0,100 mM KCl, 12.5 mM MgCl2, 2 mM EDTA) and dialyzed with 10volumes of buffer 3 containing protease inhibitor cocktail (Roche,http://www.roche.com) for 16 h. The supernatant was loaded onto aMini-Q FPLC column (GE Healthcare). The column was washed withbuffer 3, eluted with a 30-ml liner gradient of 0.1–0.4 M KCl, and thento 1.0 M KCl, and fractions (1 ml) were collected.
Measurement of transcription activity
Transcription activity was measured as incorporation of [a-32P]UTP.The reactions were carried out in a total volume of 60 ll containingtranscription buffer (60 mM Tris–HCl, pH 8.0, 10 mM MgCl2), 80 URNase inhibitor (Takara, http://www.takara-bio.com), 2% glycerol,2 mM DTT, 0.5 mM ATP, 0.5 mM CTP, 0.5 mM GTP, 5 lM [a-32P]UTP(at a specific radioactivity 0.16 GBq lmol)1), 6 lg denatured calfthymus DNA as a template and 10 ll dialyzed supernatants of eachfraction, as described above. The reaction mixtures were incubatedin the presence or absence of tagetitoxin (10 lM) for 30 min at 25�Cand then 10-ll aliquots were spotted onto DEAE paper (DE-81;Whatman, http://www.whatman.com). After successive washingwith 5% Na2PO4, water and ethanol, the radioactivity was deter-mined by scintillation counting.
Preparation of recombinant PpRpoA proteins
and Western blot analysis
A 1068-bp DNA fragment encoding the amino acid residues 90–450of PpRpoA1 were amplified from the PpRpoA1 cDNA (Sugiura et al.,2003) using specific primers rA1.F (5¢-GACGTACTAGCTT-GGACAAAAGCT-3¢) and rA1.R (5¢-ATTGCATAATGGATTGTTCT-CAG-3¢). The PCR product was inserted into the expression vectorpBAD/Thio-TOPO (Invitrogen, http://www.invitrogen.com) and theresultant plasmid pBAD-A1 was obtained. A 1146-bp DNA fragment
encoding the amino acid residues 143–525 of PpRpoA2 was ampli-fied from the PpRpoA2 cDNA (this study) using specific primersrA2.F (5¢-AGTAGATCTACTACCACTGCGGACGGACCCATG-3¢) andrA2.R (5¢-AGTGTCGACCTACTACGTCCTGCAGTGACTTTGCAG-3¢).The PCR product was digested with SalI and BglII, and inserted intoBamHI and SalI sites of the expression vector pQE-30 (Qiagen, http://www.qiagen.com) and the resultant plasmid pQE-A2 was gener-ated. pBAD-A1 and pQE-A2 was transformed into E. coli LMG194and M15/pRep4 cells, respectively. The overexpression and purifi-cation of thioredoxin-tagged PpRpoA1 protein (Thio-PpRpoA1) orHis-tagged PpRpoA2 protein (His-PpRpoA2) with Ni-NTA agarosewas carried out according to the manufacturer’s instructions. Puri-fied recombinant proteins were used to immunize rabbits. Poly-clonal antisera were obtained and used in the immunoblot analysis.For immunoblot analysis, sodium dodecylsulphate–polyacryl-amide-gel electrophoresis and blotting were carried out using a 10%polyacrylamide gel as described previously (Kabeya et al., 2002).Anti-tobacco chloroplast RNA-binding protein cp28 antibody wasused as the control (Nakamura et al., 1999).
Isolation and gel-blot analysis of DNA and RNA
Total DNA and RNA were isolated from the protonemata asdescribed previously (Hattori et al., 2007). RNA was extracted fromthe protonemata grown under different light and dark conditions, asindicated. For Southern blot analysis, DNA was digested withrestriction enzymes, separated on 1% agaraose gel, and blottedonto a Hybond N+ membrane (GE Healthcare). The membrane washybridized for 15 h at 65�C with a [32P]-labeled DNA probe andwashed at 65�C. For RNA gel-blot analysis, total RNA (15 lg) wassubjected to electrophoresis in 1.2% formaldehyde-containingagarose gel, and transferred to Hybond N+ membranes. Hybridiza-tion and detection were carried out as described using digoxigenin-labeled DNA probes (Kabeya et al., 2002). As plastid gene-specificDNA probes, DNA fragments spotted on the moss plastid DNAmicroarray (Nakamura et al., 2005) were used. A PpRpoA1 cDNAprobe (a NotI-digested 1414-bp 5¢ RACE-cDNA clone; Sugiura et al.,2003) and an nptII gene cassette probe (a HindIII-digested fragmentfrom pMBL6, a gift from Dr Jesse Machuka of the PhyscomitrellaEST Programme) were used. A PpRpoA2 probe was amplified withthe primers rA2.F and rA2.R, and a Lhcb2 probe was prepared usingPCR with primers Lhcb2.F (5¢-TAACGGTGAGTTCGCTGGTGAC-3¢)and Lhcb2.R (5¢-GTTCATGTCAATAGTCTAGTTC-3¢).
Construction for PpRpoA gene disruption
and moss transformation
A 2511-bp region containing the PpRpoA1 gene was amplifiedfrom P. patens genomic DNA with A1_KO.F (5¢-GTTAACAAAA-CATACAATGTAAAG-3¢) and A1_KO.R (5¢-AATGCGGTGG-TAAACTGGTCTCTG-3¢), and cloned into the pGEM-T Easy(Promega, http://www.promega.com) to generate pYK-DA1. Thechimeric nptII gene cassette from pMBL6, which consists of thecauliflower mosaic virus 35S promoter, the nptII coding region andthe 35S terminator, was excised as a 1961-bp HindIII fragment. ThenptII cassette was inserted into the HindIII site in the exon 2 of thePpRpoA1 gene in pYK-DA1, either with the same (pYK-DA1-1) oropposite (pYK-DA1-2) direction of transcription as PpRpoA1. Forconstruction of the PpRpoA2 disruptant, a 4440-bp DNA fragment(AB293563) was amplified with A2_KO.F (5¢-ATGCGGCCGCTTGT-AGATGATAATACCTCAATTCCGA-3¢) and A2_KO.R (5¢-ATGCGGCC-GCGCAAATAGTAAGACGTCCAGTAAGA-3¢). The PCR fragment
Nuclear-encoded a subunits of PEP in moss 739
ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 730–741
was digested with NotI and cloned into the NotI site of pBlueScriptIISK+ to generate pYK-DA2. The nptII cassette was inserted into theNcoI sites in the exon 2 to exon 4 of the PpRpoA2 gene in pYK-DA2.
Transformation was performed essentially according to theprocedure of Nishiyama et al. (2000). NotI-BstXI-digested pYKD-1or NotI-digested pYKD-2 (30 lg) was incubated at 45�C for 5 minwith protonemal protoplasts in polyethleneglycol (PEG) 6000. PEG-treated protoplasts were incubated at 25�C in the dark, and then inprotoplast regeneration medium for 3 days under continuous light.Regenerated protoplasts were transferred to BCDATG mediumcontaining 50 lg ml)1 geneticin (G418) to select transformants. Forverification of PpRpoA1 disruptants, PCR was performed using P-F(5¢-GTGAGAGGATTGAGACTGGTG-3¢) and P-R (5¢-TAGCCATAGA-TCAATAAAACAACC-3¢). For verification of PpRpoA2 disruption,PCR was performed using A2C.F (5¢-TAAGAGGAATTCGACTGTAG-TTGCG-3¢) and A2C.R (5¢-CGTTTGTGTGATCAATCATCCACG-3¢).
Microarray analysis
Plastid DNA microarray analysis was performed as described pre-viously (Nakamura et al., 2005). Fluorescence cDNA probes weregenerated by direct incorporation of Cy3- or Cy5-dUTP (GE Health-care) during reverse transcription. Briefly, 10 lg of plastid RNA fromwild-type and PpRpoA1 disruptant mosses was annealed with amixture of 216 plastid gene primers (1 pmol each) at 70�C for 5 min.Reverse transcription, purification of cDNA probes, hybridizationand washing were performed as described by Nakamura et al.(2005). Fluorescent images were visualized and analyzed withGENEPIX 4000 and accompanying software (Axon Instruments,http://www.axon.com).
Semi-quantitative RT-PCR
DNA-free total cellular RNA (5 lg) was reverse-transcribed usingSuperScript II (Invitrogen) with oligo(dT)15 primer, and first-strandcDNA was synthesized as previously described (Aoki et al., 2004).PCR was performed using the first-strand cDNA and appropriateprimer set as follows: A1-RT.F (5¢-TGGTCTCTGCTATAGAAGGT-TCGAATTCTA-3¢) and A1-RT.R (5¢-CTCATCAGAGTCACTGCGGAT-CACAAGCTC-3¢) for PpRpoA1, A2-RT.F (5¢-TGAAGGACAGTCAA-TCCGTACTGAGGCTTA-3¢) and A2-RT.R (5¢-AATGGAGATACGGC-AAATCGAGCGTAATGC-3¢) for PpRpoA2. The primers for PCRanalysis of PpSig (Ichikawa et al., 2004) were as follows:5¢-AAATCCGGCAGTCCGTCTGCTCGT-3¢ and 5¢-ACTGATGCTCTCT-AGTGACA-3¢ for PpSig1, 5¢-GTTGAATTGGATACAGAGGCT-3¢ and5¢-GCTCCTGAACCAGCATTCGCTTTG-3¢ for PpSig2, 5¢-CAAGTGGC-TGAGGATCAGCAAGT-3¢ and 5¢-TTGGCGCGTTGGATATTCACTCT-3¢ for PpSig5. As a control, an actin3 gene sequence was amplifiedusing primers (5¢-CGGAGAGGAAGTACAGTGTGTGGA-3¢ and5¢-ACCAGCCGTTAGAATTGAGCCCAG-3¢, Aoki et al., 2004). A cycleof PCR consisted of 30-s denaturation at 94�C, 30-s annealing at 55�Cand 40-s extension at 72�C. The optimal cycle number of PCR was30 cycles for PpRpoA1 and PpRpoA2, 32 for PpSig1, 35 for PpSig2and PpSig5, and 26 for PpActin3. PCR products were subjected to2% agarose gel electrophoresis.
Acknowledgements
We thank Dr Jesse Machuka for pMBL6 as part of the PhyscomitrellaEST Programme at the University of Leeds and Washington Uni-versity (St Louis, MO, USA). We also thank Dr Shin-ya Miyagishimafor kindly supporting this work, and Dr Takahiro Nakamura and
Dr Setsuyuki Aoki for valuable discussions. This work wassupported by a Grant-in-aid from the Ministry of Agriculture,Forestry and Fisheries (Bio-Design Project), and by a Research Grantfrom the DAIKO FOUNDATION (Nagoya). YK was a recipient ofa Japan Society for Promotion of Science (JSPS) Post-doctoralFellowship.
Supplementary Material
The following supplementary material is available for this articleonline:Figure S1. Chromatography of the chloroplast lysate on MiniQcolumn.Figure S2. Generation of the PpRpoA2 gene disruptant.Figure S3. Mapping of the 5¢-ends of plastid gene transcripts.This material is available as part of the online article from http://www.blackwell-synergy.com.
Supplementary Reference
Sambrook, J., Fritsch, E.F., and Manniatis, T. (1989) MolecularCloning: A Laboratory Mannual, 2nd ed. NY: Cold Spring HarborLaboratory Press.
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