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INTRODUCTION |
Photosystem II contains at least four plastid-encoded chlorophyll
apoproteins (D1, D2, CP47, CP43). Among these, D2 and D1 form a
heterodimer, which houses the photosystem II reaction center chlorophyll P680. D1 and D2 are relatively unstable in illuminated plants (1-5). Therefore, synthesis of D1 and D2 is selectively elevated in mature barley chloroplasts in order to maintain the levels
of these subunits and PSII function (5, 6). Maintenance of high rates
of D1 and D2 synthesis in mature barley chloroplasts is paralleled by
the retention of elevated levels of psbA and psbD
mRNAs, which encode these proteins (6-8). D1 mRNA levels remain high in mature barley chloroplasts primarily due to the high
stability of its mRNA, although transcription from psbA
is also increased by light (9-12). Maintenance of high levels of psbD mRNA results primarily from the activation of
psbD transcription by blue light combined with a small
increase in RNA stability (5, 13).
The chloroplast genome in most higher plants is circular and ranges in
size from 120 to 217 kilobase pairs (reviewed in Refs. 14-17). The
genome encodes approximately 135 genes including genes for rRNAs,
tRNAs, subunits of the plastid 70 S ribosome, subunits of an RNA
polymerase (rpoA, rpoB, rpoC1, and
rpoC2), and proteins that comprise the photosynthetic
apparatus. Transcription of the chloroplast genome is complex and
highly regulated (reviewed in Refs. 17 and 18). Plastid genes are
transcribed by at least two different RNA polymerases
(RNAPs).1 The
catalytic subunits of one RNAP are encoded by the chloroplast genes
rpoA, rpoB, and rpoC1/C2 (reviewed in
Ref. 19). This RNAP recognizes prokaryotic
10 and
35 promoter
elements (reviewed in Ref. 18). Other types of plastid promoters have
been identified. For example, the promoter for the rps16
gene contains only a
35 element (20). Other genes, such as
trnS, trnR (21), rpoB (22), rpl32 (23), and rpl23 (24) are not preceded by
typical prokaryotic promoter consensus elements. Many of these genes
are transcribed by a nucleus-encoded RNAP (Refs. 22, 25, and 26;
reviewed in Ref. 17). This polymerase is likely to be encoded by the nuclear gene rpoZ, which shows sequence similarity to the
bacteriophage T7 and SP6 RNA polymerases (27). Plastid transcription is
also regulated via multiple
-factors (28-30), which may be
phosphorylated (31, 32). Other DNA binding complexes, such as CDF2 and
AGF, have been identified, which modulate transcription of
rrn (33), and psbD-psbC (34), respectively.
In barley, psbD is located in a complex operon that also
contains psbC, psbK, psbI,
orf62, and trnG (35). The psbD operon is transcribed from at least three different promoters (13). One of the
psbD promoters is activated when plants are illuminated by
high fluence blue light but not by red or far-red illumination (5, 36).
Transcripts arising from the blue light-responsive promoter (BLRP)
become the most abundant psbD transcripts in chloroplasts of
mature barley leaves (13, 37). Light-induced accumulation of
psbD transcripts has been observed in a wide variety of
plants (37-39). A ~130-bp region surrounding the psbD
BLRP is conserved among cereals, dicots, and black pine (34, 37)
despite DNA rearrangements upstream of the psbD BLRP in some
plants (37). The conserved psbD BLRP contains sequences with
significant similarity to typical prokaryotic
10 and
35 promoter
regions (13). In addition, two conserved regions, termed the AAG-box
and PGT-box, are located upstream of the putative
35 element (34).
Previously, we showed that the AAG-box and its cognate DNA-binding
protein complex, AGF, are required for transcription from the barley
psbD BLRP in vitro (34). Furthermore, the DNA
region containing the PGT and AAG-boxes was shown to be important for
transcription from the tobacco psbD BLRP in vivo
(40). In the present study, we define a minimal DNA region required for
transcription of the barley psbD BLRP and further dissect
the architecture of the promoter using deletion, insertion, and point
mutation analyses.
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EXPERIMENTAL PROCEDURES |
Plant Growth--
Barley (Hordeum vulgare var. Morex)
seedlings were grown in controlled environmental chambers at 23 °C
as described by Kim et al. (12). Seedlings were germinated
and grown in complete darkness. After 7.5 days, the dark-grown
seedlings were either harvested or transferred to a continuously
illuminated chamber (fluorescent plus incandescent light, light
intensity 250 microeinsteins m
2 s
1) for an
additional 16 h before harvesting. Plastids were isolated from the
top 5-7 cm of primary leaves of barley seedlings by Percoll gradient
(35-75%) centrifugation (41). The concentration of plastids was
quantitated (plastids per microliter) by phase contrast microscopy
using a hemacytometer.
Preparation of Plastid Extracts for in Vitro Transcription
Experiments--
The plastid high salt extracts used for in
vitro transcription experiments in this study were prepared
according to Kim and Mullet (34). Protein extract from 5.2 × 108 plastids obtained from approximately 7.5-day-grown
barley plants was used in each in vitro transcription assay.
In Vitro Transcription and Primer Extension
Analyses--
Transcription of exogenous DNA templates in
vitro and primer extension analyses of in vitro
transcribed DNA were performed as described by Kim and Mullet (34). The
minus 40 primer was used to analyze transcripts originating from the
plasmid pLRP140 and its derivative recombinant plasmids; the T3 primer
was used for ppsbA138, prbcL216, and their derivative recombinant plasmids.
Plasmid Construction of pLRP97, pLRP80, pLRP69, and
pLRP121--
Recombinant plasmids pLRP97, pLRP80, pLRP69, and pLRP121
were constructed based on PCR cloning, using pLRP185 (34) as a DNA
template. The 3'-end primers used for amplification of LRP97, LRP80,
and LRP69 were designed based on cDNA-like sequences as follows:
LRP97, 5'-TTCGCggATcCAATTTCATCTAC (+33 to +11 region of the
psbD BLRP); LRP80, 5'-TTCATCTggATCcAATTTATATA
(+19 to
4 region of the psbD BLRP); LRP69,
5'-GAATTggatccTCAGAATAGCGGA (+7 to +17 region of the
psbD BLRP). Engineered BamHI sites are
underscored, and nucleotide changes from native chloroplast sequences
are designated by lowercase letters. The 5'-end primer used for PCR
amplification of the three DNA fragments mentioned above was based on
mRNA-like sequences as follows:
5'-TCAAATCtAgaATAAAATTGGAAA (
81 to
62 region of the
psbD BLRP, engineered XbaI site underscored with nucleotide changes from native chloroplast sequences designated by
lowercase letters).
The 5'- and 3'-end primers used for PCR amplification of LRP121 are as
follows: 5'-end primer, 5'-ATTGGtctAgaCATAAAGTAAGTA (mRNA-like sequences,
68 to
45 region of the psbD
BLRP); 3'-end primer, 5'-TTCGCggATcCAATTTCATCTAC
(cDNA-like sequences, +33 to +11 region of the psbD
BLRP). Both of the engineered BamHI and XbaI
sites are underscored, and nucleotide changes from native chloroplast
sequences are shown with lowercase letters. All of the PCR products
mentioned above were digested by BamHI and XbaI, gel-purified, and ligated into BamHI and XbaI
sites of pBluescript SK+. The resulting plasmids were named pLRP97,
pLRP80, pLRP69, and pLRP121, respectively.
Plasmid Construction of pLRP140/bmt, pLRP140/bb'mt,
pLRP140/ntSwitch, pLRP140/(
)5nt DL, pLRP140/(
)10nt DL,
pLRP140/(+)3nt IN, pLRP140/(+)7nt IN, pLRP140/(+)10nt
IN--
Recombinant plasmids such as pLRP140/bmt, pLRP140/bb'mt,
pLRP140/ntSwitch, pLRP140/(
)5nt DL, pLRP140/(
)10nt DL,
pLRP140/(+)3nt IN, pLRP140/(+)7nt IN, and pLRP140/(+)10nt IN were
constructed based on PCR cloning, using pLRP140 (34) as a DNA template. The 5'-end primer used for amplification of each DNA fragment to be
cloned was designed based on mRNA-like sequences as follows: pLRP140/bmt, CCATAAAATTGGAAAGAAGCATAAAGTAAGTAGtagTGA (
76 to
38 region of the psbD BLRP); pLRP140/bb'mt,
CCATAAAATTGGAAAGAAGCATAAAGTAAGTAGtagTGtgaCC (
76 to
34 region of the
psbD BLRP); pLRP140/ntSwitch,
CCATAAAATTGGAAAGAAGCATAAAGTAAGTAGACCTGACTCgTTGAATctTGCCTgaAT (
76 to
17 region of the psbD BLRP); pLRP140/(
)5nt DL,
CCATAAAATTGGAAAGAAGCATAAAGTAAGTAGACCTGACTCC ... TGATGCCTC (
76 to
20 region of the psbD BLRP); pLRP140/(
)10nt DL,
CCATAAAATTGGAAAGAAGCATAAAGTAAGTAGACCTGACTCC ... CCTCTATCCGC (
76
to
13 region of the psbD BLRP); pLRP140/(+)3nt IN,
CCATAAAATTGGAAAGAAGCATAAAGTAAGTAGACCTGACTCCTACTTGAA (
76
to
29 region of the psbD BLRP); pLRP140/(+)7nt IN,
CCATAAAATTGGAAAGAAGCATAAAGTAAGTAGACCTGACTCCTACTTTGAATCTAGATGCC (
76 to
22 region of the psbD BLRP); pLRP140/(+)10nt IN,
CCATAAAATTGGAAAGAAGCATAAAGTAAGTAGACCTGACTCCTACTTTTGAATCTAACGATGCC (
76 to
22 region of the psbD BLRP). Nucleotide
changes from native chloroplast sequences are designated by lowercase
letters, and deleted or inserted sequences are shown by dots or
underlines. The 3'-end primer used for amplification of all of the DNA
fragments mentioned above was T7 primer, which hybridizes to the region originating from the vector sequences of pBluescript SK+ in pLRP140. All of the PCR products mentioned above were digested by
ApaI, gel-purified, and ligated into SmaI and
ApaI sites of pBluescript SK+. The nucleotide sequences of
the cloned DNA fragments were confirmed by dideoxy sequencing reactions.
Site-directed Mutagenesis of
10,
35, and TATA Elements in
pLRP140, prbcL216, and ppsbA138--
Site-specific base substitution
in
10,
35, and TATA elements in pLRP140, prbcL216, and ppsbA138
(34) was introduced based on a PCR-based "overlap extension
technique" described by Higuchi et al. (42) and Ho
et al. (43). Previously, prbcL216 was constructed by
inserting a PCR-amplified DNA fragment, extending from
156 to +60,
flanking the transcription initiation site of the barley rbcL (44), into BamHI and EcoRI sites
of pBluescript SK+. The inside primers, coupled with the outside
primers, to generate two overlapping primary PCR fragments, which bear
the same mutations in the region of overlap, are as follows:
pLRP140/
35mt, 5'-TGACTCCaTcAATGATGCCT for upper strand primer,
3'-ACTGAGGtAgTTACTACGGA for lower strand primer (
40 to
21 region of
the psbD BLRP, respectively); pLRP140/
10mt, 5'-TATCCGCaATTCaGATATAT for upper strand primer,
3'-ATAGGCGtTAAGtCTATATA for lower strand primer (
19 to +1 region of
the psbD BLRP, respectively); pLRP140/
35&
10mt,
5'-TCCaTcAATGATGCCTCTATCCGCaATTCaGAT for upper strand primer,
3'-AGGtAgTTACTACGGAGAATAGGCGtTAAGtCTA for lower strand primer (
36 to
4 region of the psbD BLRP, respectively); prbcL216/
35mt,
5'-ATTTGGGaTcCGCTATACCT for upper strand primer, 3'-TAAACCCtAgGCGATATGGA for lower strand primer (
41 to
22 region of
the barley rbcL, respectively); prbcL216/
10mt,
5'-CAAGAGTAaACAAaAATGATGG for upper strand primer,
3'-GTTCTCATtTGTTtTTACTACC for lower strand primer (
19 to +4 region of
the barley rbcL, respectively); ppsbA138/
35mt, 5'-TGACTTGGaTcACATTGGTATA for upper strand, 3'-ACTGAACCtAgTGTAACCATAT for lower strand primer (
45 to
19 region of the barley
psbA, respectively); ppsbA138/
10mt,
5'-GTCTATGTaATACaGTTAAATA for upper strand primer,
3'-CAGATACAtTATGtCAATTTAT for lower strand primer (
21 to +1 region of
the barley psbA, respectively (45)); ppsbA138/TATAmt, 5'-GACATTGGaAgAaAGTCTATGT for upper strand primer,
3'-CTGTAACCtTcTtTCAGATACA for lower strand primer (
35 to
14 region
of the barley psbA, respectively); ppsbA138/
35&TATAmt,
5'-TGACTTGGaTcACATTGGaAgAaAGTCTAT for upper strand primer,
3'-ACTGAACCtAgTGTAACCtTcTtTCAGATA for lower strand primer (
45 to
16
region of the barley psbA, respectively). The specific base
substitutions introduced in each primer, which create mismatch between
a primer and the individual template target sequence, are designated by
lowercase letters. T3 and T7 primers, which hybridize to the regions
originating from the vector sequences of pBluescript SK+ in pLRP140,
prbcL216, and ppsbA138, respectively, were used together with the
primers described above to generate the overlapping primary PCR
fragments. Each set of overlapping primary PCR products was
gel-separated, mixed together, denatured, and allowed to reanneal. Each
resulting extended segment was then used for the secondary
amplification of the combined sequences, using the outside T3 and T7
primers, which were employed to produce the primary fragments. After
the secondary PCR amplification, XbaI and XhoI
restriction enzyme sites were used to insert the individual DNA
fragments into pBluescript SK+.
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RESULTS |
Minimal DNA Region Required for Transcription from the psbD
BLRP--
The structure of the barley chloroplast psbD BLRP
is shown in Fig. 1A.
Comparisons of the psbD BLRP region among numerous plants
showed several stretches of sequence conservation from approximately
+30 to
100. In particular, sequences surrounding the AAG-box (
36 to
64) and PGT-box (
71 to
100) are highly conserved (34, 37).
Previous analysis of the psbD BLRP demonstrated that the
region from +64 to
76 was sufficient to activate transcription in vitro (34). In this study, sequences important for
transcription in vitro were further delineated using a
series of deletions of pLRP140 (Fig. 1A).

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Fig. 1.
In vitro transcription from the
psbD BLRP and modified psbD BLRP
domains using barley plastid extracts. A, schematic
representation of the barley chloroplast psbD BLRP and
psbD BLRP constructs used for in vitro
transcription. The boxed regions identify
conserved sequences including the AAG-box, the PGT-box, and sequences
homologous to E. coli 35 and 10 promoter elements (34).
The site of psbD transcription initiation is designated by
an arrow, and labeled as +1. Repeated sequences in the
AAG-box are underlined. pLRP140 is a recombinant plasmid
containing 140 bp of DNA ( 76 to +64) from the psbD BLRP
(34). pLRP185 was used as a template to construct recombinant
plasmids, pLRP97, pLRP80, pLRP69, and pLRP 121. B,
in vitro transcription of the recombinant plasmids shown in
A using barley plastid extracts. Transcripts were analyzed
using primer extension analysis. The arrows designate
primary transcripts produced from the recombinant plasmids. The
asterisk marks the position of a signal produced from
plastid extracts in the absence of template. DNA size markers in base
pairs are indicated to the right.
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Recombinant plasmids pLRP97, pLRP80, and pLRP69 contain a series of
3'-end deletions of the psbD BLRP (Fig. 1A). Each
of the recombinant plasmids was added to chloroplast in
vitro transcription extracts obtained from 8-day-old barley plants
that had been illuminated for 16 h. The psbD
transcripts produced from the plasmids were assayed using primer
extension analysis. No psbD transcript 5' termini were
observed when mock transcription reactions were analyzed (data not
shown). However, as shown in Fig. 1B (lanes
1-4), all of the 3' deletion recombinant plasmids and
pLRP140 were equally good templates. Similar results were observed with
plastid extracts from 7.5-day-old, dark-grown barley plants (data not shown).
Previous analyses demonstrated that the sequence AAAGTAAG (
54 to
47) in the AAG-box (see Fig. 1A) was required for
transcription from the BLRP (34). To examine the influence of sequences
upstream of this sequence, pLRP121 was constructed, which contains a 5' deletion to
57 (Fig. 1A). This deletion caused no loss of
transcription activity from the psbD BLRP (Fig.
1B, lane 5). These results indicate that the ~53-bp DNA region from
57 to
5 is sufficient for
transcription from the psbD BLRP in
vitro.
The sites of transcription initiation from the 3' deletion constructs
of pLRP140 were fine mapped using primer extension analysis (Fig.
2). The 5' termini of the psbD
transcripts produced from pLRP97 and pLRP80 mapped seven nucleotides
downstream from a potential
10 promoter element (TATTCT) at the same
site as the 5' terminus of the transcript produced from pLRP140 (34).
Similarly, transcripts produced from pLRP69, which contained a 3'
deletion to
5, mapped seven nucleotides downstream from the TATTCT
10 sequence although native nucleotides from +1 to
4 (TATAT) had
been deleted and replaced by CTAGG. This indicates that the sequences
immediately surrounding the site of transcription initiation can be
modified with minimal influence on transcription initiation.

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Fig. 2.
Fine maps of transcript 5'-ends synthesized
from the psbD BLRP with 3'-deletions.
Lanes labeled cDNA (G,
A, T, and C) refer to the
dideoxynucleotide sequencing reactions performed on recombinant
plasmids pLRP97, pLRP80, and pLRP69. A portion of the cDNA sequence
is shown to the left of each set of sequencing reactions.
Boxes indicate native psbD BLRP sequences, and
the nucleotides shown in boldface letters
indicate sequences from the cloning vector. The same primer used for
sequencing reactions was also used for the primer extension analyses
shown to the right of each set of sequencing
lanes. THe arrows indicate transcript 5' termini
revealed by this analysis.
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Analysis of Putative Prokaryotic
35 and
10 Promoter
Sequences--
The psbD BLRP contains potential prokaryotic
35 (TTGAAT) and
10 (TATTCT) promoter elements, located at positions
28 to
33 and
7 to
12, respectively (see Fig. 1A).
These sequences are separated by 15 bp. Similar prokaryotic promoter
elements, which are separated by 18 bp, have previously been identified upstream of the sites of transcription initiation in the
rbcL and psbA promoters (reviewed in Ref. 16). In
addition, a TATATA sequence located between the psbA
10
and
35 promoter elements contributes to promoter activity in mustard
(46).
The function of the putative
10 and
35 prokaryotic promoter
elements present in the psbD BLRP was analyzed by
site-directed mutagenesis. As a control, the influence of modifying the
10 and
35 elements in the rbcL and psbA
promoters was examined to ensure the in vitro transcription
extract was faithfully replicating previous results. Our general
approach was to introduce point mutations in potential
35 and
10
sequences at sites that show the highest conservation in both plastid
and bacterial promoters (
35, TTGaca;
10, TAtaaT) (Ref. 47; reviewed
in Refs. 16 and 48-51). The first and the third nucleotides (T and G)
in the potential
35 promoter element of each promoter were switched to A and C, respectively (Figs.
3A and
4A). In the case of potential
10 promoter elements, the first and the sixth nucleotides, T and T,
were both switched to A (Figs. 3A and 4A). The
point mutations described above did not create any other potential
35
or
10 promoter elements. Each of the recombinant plasmids containing the point mutations was added to plastid transcription extracts, which
were obtained from either 7.5-day-old, dark-grown barley plants, or
similar plants that had been further illuminated for 16 h.

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Fig. 3.
In vitro transcription from wild
type (WT) and modified psbA and
rbcL promoters in plastid extracts from dark- and
light-grown barley plants. A, schematic representation
of the recombinant plasmids used for in vitro transcription
experiments shown in B. Wild-type 35, TATATA, and 10
promoter sequences in ppsbA138 and prbcL216 are underlined.
The sites of transcription initiation in psbA and
rbcL are designated +1. Sequence changes in the
ppsbA138-derived recombinant plasmids, ppsbA138/ 35mt
( 35mt), ppsbA138/ 10mt ( 10mt),
ppsbA138/TATAmt (TATAmt), and ppsbA138/ 35&TATAmt
( 35&TATAmt), and in prbcL216/ 35mt ( 35mt)
and prbcL216/ 10mt ( 10mt) are shown in
boldface type. B and C,
in vitro transcription of the recombinant plasmids
represented in A using plastid extracts obtained from
dark-grown (DK) and light-grown (LT) barley
plants. Transcripts from the psbA promoter (B,
lanes 1-6) and the rbcL promoter
(C, lanes 1-4) were analyzed using
primer extension analysis. The arrow indicates the primary
transcript produced from the recombinant plasmids. DNA size markers in
base pairs are indicated to the left.
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Fig. 4.
In vitro transcription from
wild type and modified psbD BLRPs in plastid extracts
from dark- and light-grown barley plants. A, schematic
representation of the recombinant plasmids used for in vitro
transcription experiments shown in B. Wild-type AAG-box,
35, and 10 putative promoter elements in pLRP140 are
underlined. Sequence modifications in pLRP140/ 35mt
( 35mt), pLRP140/ 10mt ( 10mt),
pLRP140/ 35 & 10mt ( 35& 10mt), and pLRP140 (nt
Switch) are shown in boldface type. Deleted
nucleotides in the ( )5nt DL and ( )10nt DL constructs are indicated
by lines. The location of sequences inserted into several
constructs are shown below constructs labeled (+)3nt
IN, (+)7nt IN, and (+)10nt IN. B,
in vitro transcription of the recombinant plasmids using
plastid extracts obtained from dark-grown (DK) and
light-grown (LT) barley plants. Transcripts were analyzed
using primer extension analysis. The arrow indicates the
primary transcript produced from the recombinant plasmids. The
asterisk identifies a signal produced in the absence of
template. DNA size markers in base pairs are indicated to the the
right.
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Transcription from the psbA and rbcL promoter
constructs is shown in Fig. 3, B and C.
Transcription from the wild type psbA or rbcL
promoters was active in extracts of etioplasts isolated from dark-grown
plants or chloroplasts isolated from illuminated plants (Fig. 3,
B and C, lanes 1 and
2). Modification of the
35 sequences in these two
promoters caused transcription to decrease to very low levels (Fig. 3,
B and C, lane 3).
Similarly, modification of the
10 sequences also caused transcription
to decrease significantly (Fig. 3, B and C,
lane 4). When the TATATA sequence located between the
35 and
10 elements in psbA was modified,
transcription was reduced although not eliminated (Fig. 3B,
lane 5). Not surprisingly, modification of both
the
35 and TATATA sequence in the psbA promoter reduced
transcription to nondetectable levels (Fig. 3B,
lane 6).
The results in Fig. 4 show the influence
of mutation of putative
10 and
35 promoter elements found in the
psbD BLRP. Mutation of the
35 sequences did not alter
transcription from the psbD BLRP (Fig. 4B,
lane 2 versus lane
3). In contrast, mutation of the prokaryotic
10 element,
TATTCT, reduced transcription from the psbD BLRP to very low
levels (Fig. 4B, lane 2 versus lane 4). As expected, point
mutations in both of the
10 and
35 sequences also abolished
transcription (Fig. 4B, lane 5). These
results indicate that the
10 sequence is required for transcription
from the psbD BLRP in vitro, whereas the
35 and
10 elements are both required for transcription from the
rbcL and psbA promoters. In addition, the
psbA TATATA sequence is important for transcription from the
psbA promoter in barley. Transcription of all constructs was
greater in extracts of plastids from illuminated plants compared with
extracts from etioplasts of dark-grown plants, although the influence
of illumination was greatest on the psbD BLRP (~6.5-fold versus 4-fold (psbA) and 2-fold (rbcL)
(Figs. 3 and 4)).
Role of the Sequence and Spacing between the AAG-box and
10
Sequence in the psbD BLRP--
In the psbA promoter, a
TATATA sequence located between the
10 and
35 elements contributes
to promoter activity. Therefore, to determine if additional motifs in
the psbD BLRP confer promoter activity, we tested the
influence of five point mutations in the sequences located between the
AAG-box and the prokaryotic
10 element (see Fig. 4A,
nt Switch). As shown in Fig. 4B, these
substitutions did not alter transcription from the psbD
BLRP, suggesting the absence of important sequence motifs between the
AAG-box and the
10 promoter element (Fig. 4B,
lane 6).
The AGF, which binds to the AAG-box, may stabilize and orient the RNA
polymerase relative to the
10 element of the psbD BLRP. Therefore, spacing between the AAG-box and the
10 element may be
important to maintain alignment of AGF and the RNA polymerase on the
same face of the psbD BLRP. The AAG-box and the
10 motif are separated by 23 bp in the psbD BLRP. In contrast, most
plastid
10 and
35 elements are separated by 18 bp (reviewed in Ref. 16). Therefore, the influence of altering the spacing between the
AAG-box and the prokaryotic
10 element was investigated. Nucleotide
deletions (5 and 10 bp) or insertions (3, 7, and 10 bp) were introduced
between the AAG-box and the
10 element in plasmid pLRP140 (Fig.
4A). When a 5-bp deletion was introduced to reduce spacing
between the AAG-box and the
10 element to 18 bp, transcription from
the psbD BLRP was undetectable (Fig. 4B, lane 7). Deletion of 10 bp, which represents one
helical turn, resulted in low but detectable levels of transcription
(Fig. 4B, lane 8). Insertion of 3, 7, or 10 bp between the AAG-box and the
10 element also reduced
transcription to very low levels (Fig. 4B, lanes
9-11). These results indicate the importance of the 23-nucleotide spacing between the AAG-box and the
10 element for
transcription from the psbD BLRP.
Further Analysis of Sequences in the AAG-box--
The AAG-box,
shown in Fig. 5A, was defined
in past experiments as a 22-bp DNA region (
36 to
57) containing two
motifs designated aa' and bb' (37). We have previously shown by point
mutation analyses that the aa' motif is important for both AGF binding and transcription from the psbD BLRP (34). To test the
importance of the bb' motif for transcription from the psbD
BLRP, we introduced point mutations in this sequence (GACCTGACT) in
plasmid pLRP140 (see Fig. 5A). Transcription analysis showed
that mutation of GACC to GTAG inhibited transcription from the
psbD BLRP (Fig. 5B, lane 1 versus lane 2 and lane
4 versus lane 5).
Furthermore, mutations of GACCTGACT to GTAGTGTGA abolished
transcription from the psbD BLRP (Fig. 5B, bb'
mt, lanes 3 and 6). To determine
whether the bb' sequences within the AAG-box also influence binding of AGF, gel retardation and competition binding experiments were carried
out (Fig. 5C). As observed previously (34), AGF binds to
radiolabeled pLRP140 in the absence of specific competitor DNA
fragments (Fig. 5C, lane 1). The
addition of unlabeled pLRP140 to the binding assays greatly reduces the
amount of AGF gel shift complex (Fig. 5C, lane
2). As described previously, LRP140 DNA fragments containing
modified aa' sequences (AAAGTAAG to AAATTCAT) do not compete well with
native LRP140 (lane 3) (34). Modification of bb'
sequences in pLRP140 (b mt and bb' mt) reduces the ability of the
resulting DNAs to bind AGF to some extent (Fig. 5C,
lanes 4 and 5), indicating that the
bb' sequence contributes to AGF binding either directly or
indirectly.

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Fig. 5.
In vitro transcription and gel
retardation competition assays involving the psbD BLRP
containing mutations in the AAG-box sequence. A,
diagram of pLRP140, the wild-type (WT) psbD-psbC
blue light promoter construct. Two conserved sequence motifs within the
AAG-box, designated aa' and bb', are underlined. Modified
pLRP140 constructs, b mt and bb' mt, contain mutations shown in
boldface letters, in the bb' sequence.
B, primer extension analysis of RNAs synthesized from
plasmids containing the psbD-psbC blue light promoter in
plastid extracts obtained from dark grown (DK) and light
grown (LT) barley plants. The arrow indicates the
primary transcript produced from the recombinant plasmids. The
asterisk indicates a signal produced from plastid extracts
in the absence of template. DNA size markers in base pairs are
indicated to the right. C, competition binding
assays carried out using radiolabeled LRP136, which contains the entire
psbD-psbC blue light-responsive promoter, using high salt
extracts from plastids isolated from 4.5-day-old dark-grown barley.
Competiton reactions were carried out with 200 ng of competitor DNA in
the presence of 1 µg of poly(dI-dC)·(dI-dC). Competitor DNAs
included the native sequence (pLRP140; labeled 140), mt2
(LRP140 containing a mutation in the aa' sequence) (34), b mt, or bb'
mt DNAs shown in A. A control binding assay in the absence of competitor DNA is shown
in lane 1 (No Comp.). The
arrow designates the AGF-binding complex consistent with
previous analysis (34). The migration of free probe is indicated.
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DISCUSSION |
Delineation of a 53-bp Core psbD BLRP Promoter Domain--
The
psbD BLRP is located approximately 570 bp upstream of the
psbD translational start codon in cereals and even further
upstream of the psbD open reading frame in dicots (37). In
higher plants, a DNA region of approximately 130 bp surrounding the
site of transcription initiation from the psbD BLRP is
highly conserved (~60%) relative to sequences more than 100 bp
upstream of the promoter or sequences between the promoter and the
psbD open reading frame (9%) (37). At least 25 bp of the
conserved region extends downstream of the site of transcription
initiation. In this study, we determined that deletion of sequences
from
5 to +64, relative to the site of transcription initiation, had
no influence on transcription from the psbD BLRP in
vitro. This result indicates that the conserved sequences
downstream of the initiation site are probably not important for
transcription. Previous analysis of changes in psbD
transcription and RNA levels during leaf and chloroplast development
indicated that psbD transcripts become more stable during
light-mediated leaf maturation (8, 13). Therefore, the conserved
sequences immediately downstream from the site of transcription
initiation, which are present in the 5'-untranslated region of
transcripts produced from the psbD BLRP, may be important
for RNA stability.
The 100-bp DNA region immediately upstream of the psbD BLRP
initiation site contains several stretches of sequence that are conserved among psbD genes from higher plants (37). Deletion of sequences from
107 to
55 in the tobacco psbD BLRP
reduced transcription activity in vivo ~5-fold without
altering light-stimulated transcription following dark adaptation of
plants (40). In barley, this region of the psbD BLRP
specifically binds a protein complex (PGTF) present in chloroplasts
(34). In the current study, however, deletion of sequences upstream of
57 in the psbD BLRP had minimal effect on in
vitro transcription. This suggests that this region of the
psbD BLRP and the PGTF complex that binds in this region are
not modulating transcription from the psbD BLRP in
vitro. Mutation of sequences immediately downstream of
57 (34)
or upstream of
5 (Fig. 5) reduce transcription from the
psbD BLRP. These experiments define a 53-bp region that is
required for transcription from the psbD BLRP in
vitro.
Transcription from the psbD BLRP Requires a Prokaryotic
10
Element but Not a
35 Promoter Element or the psbA TATATA
Element--
The psbD BLRP contains the sequence TATTCT,
located between
7 and
12, which resembles a prokaryotic
10
promoter element. Mutation of this sequence to AATTCA reduced
transcription from the psbD BLRP to very low levels.
Similarly, mutation of
10 sequences found in the psbA
(TATACT to AATACA) and rbcL (TACAAT to AACAAA) promoters
rendered these promoters inactive. In Escherichia coli,
10
promoter elements are recognized via interaction with
-factors that
are associated with the RNAP (reviewed in Refs. 52-54). These results
are consistent with in vitro transcription of the
psbD BLRP by a chloroplast RNAP containing a
-like
subunit that interacts with the
10 promoter element (29, 31, 55-57).
Transcription from mustard psbA is stimulated by a TATATA
sequence located between the
10 and
35 promoter elements (46). The
TATATA sequence might be involved in the recruitment of RNA polymerase
or in the isomerization from the "closed" to "open" complex
formation (Refs. 58 and 59; reviewed in Refs. 60 and 61). Moreover, in mustard, this sequence may allow transcription in dark-grown plants that is not dependent on a
35 element from the psbA
promoter (31, 46). Mutation of a similar sequence present in the barley psbA promoter decreased transcription in plastid extracts
from dark-grown and illuminated plants (Fig. 3). In contrast, the
psbD BLRP lacks the TATATA sequence, and mutation of
sequences located between
10 and
35 in the psbD BLRP had
little influence on transcription activity.
The chloroplast-encoded RNAP's ability to transcribe rbcL
and psbA depends on a prokaryotic
35 promoter element
(Figs. 3 and 4) (reviewed in Refs. 16 and 49). In contrast, mutation of
the
35 sequence in the psbD BLRP had little effect on
transcription in vitro (Fig. 5). The function of the
35
sequence in the psbD BLRP appears to be replaced by the
action of AGF, an activating complex that binds immediately upstream of
the
35 sequence (Ref. 34; see below).
Two Different Sequences in the AAG-box Are Involved in psbD BLRP
Transcription--
The sequence from
36 to
64 in the
psbD BLRP was previously reported to be required for
transcription from the psbD BLRP in vitro (34).
In the current study, this region was further truncated to
57 without
loss of activity. The corresponding sequence in the tobacco
psbD BLRP was also found to be important for activity in vivo (40). The region from
36 to
57, termed the
AAG-box, was previously reported to contain two conserved motifs (aa'
and bb') (37). A protein complex, designated AGF, was found to
specifically interact with sequences within the AAG-box. Footprint
analysis indicated that AGF binding protected sequences from at least
40 to
63 (34). In a previous study, site-directed mutagenesis of
the aa' sequence (AAAGTAAGT to AAATTCAT) caused loss of AGF binding and
eliminated transcription from the psbD BLRP (34). In the
current study, site-directed mutagenesis of the bb' sequence located
immediately downstream from the aa' motif and upstream of
35 caused a
reduction in transcription as well as a reduction in the ability of DNA
in this region to bind to AGF (Fig. 5). These results suggest that
proteins in AGF interact with the bb' sequence. It is also possible
that some other currently undetected protein binds to the bb' sequence
and that this modifies AGF binding. In tobacco, proteins also bind
specifically to the bb' sequence (40). Unfortunately, the relationship
between the barley and tobacco AAG-box binding complexes could not be established.
Model for AGF Activation of the psbD BLRP--
A model of the
barley psbD BLRP is shown in Fig.
6 along with diagrams of the
rbcL and psbA promoters. All three genes are shown being transcribed by the chloroplast-encoded RNAP with an associated
-factor. This is consistent with several lines of evidence. First, light-induced transcription from the psbD
BLRP in vivo is inhibited if plants are pretreated with
tagetitoxin (13). The chloroplast-encoded RNAP and E. coli
RNAP are sensitive to tagetitoxin, whereas the chloroplast-localized,
nucleus-encoded RNAP and the homologous bacteriophage RNA polymerases,
T7 or SP6, are not inhibited by tagetitoxin (62, 63). Second, plants that lack the chloroplast-encoded RNAPs do not accumulate transcripts from the psbD BLRP (or from rbcL, psbA), although
they accumulate transcripts from many genes involved in transcription
and translation that lack prokaryotic
10 and
35 promoter elements
(22, 64). Third, mutation of sequences surrounding the psbD
BLRP site of transcription initiation (*) from TTCTGATATAT*AAAT
to TTCTGAGGATC*CCCC had no influence on transcription in
vitro (Figs. 1 and 2). The nucleus-encoded chloroplast RNAP has
been proposed to use a promoter sequence located in the 10 bases
immediately adjacent to the site of transcription initiation (64).
Based on comparative alignments, a rather variable promoter consensus
sequence, ATAGAAT(A/G)AA, has been proposed for this polymerase (24,
64). This sequence is somewhat different from both the native and
mutated psbD BLRP promoters that are active in
vitro. Fourth, mutation of the prokaryotic
10 element, located
between
7 and
12, dramatically reduced transcription from this
promoter. Finally, the chloroplast-encoded RNAP preferentially
transcribes genes encoding proteins involved in photosynthesis;
therefore, transcription from the psbD BLRP is consistent
with this tendency. However, further biochemical analysis of the
nucleus-encoded RNAP will be needed to definitively eliminate a role
for this RNAP in psbD BLRP transcription.

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Fig. 6.
Models of transcription complexes associated
with the psbD BLRP, rbcL and
psbA promoters. Arrows (+1) indicate the site and
direction of transcription initiation. Important transcription
cis-elements (-10, -35, TATA, and aa'/bb') are boxed and the
sequences and spacing between elements is indicated. A chloroplast RNAP
and an associated sigma factor is shown interacting with each promoter.
In addition, the AGF/BB' complex, which binds to the AAG-box sequences
aa'/bb', is shown interacting with the RNAP to promote transcription
from the psbD BLRP.
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The RNAPs in Fig. 6 are shown associated with a generic
-factor.
However, there are several reasons to think that the
-factor involved in transcription of the psbD promoter may be
different from
-factors involved in transcribing rbcL and
psbA. First, in the case of the rbcL and
psbA promoters,
-factors are likely to interact with both
10 and
35 promoter elements, based on analysis of bacterial
-factor binding (reviewed in Refs. 52 and 65). An additional
interaction may occur between the
-factor and the TATATA sequence in
the psbA promoter. In contrast, the psbD BLRP
lacks functional
35 and TATATA elements, and the sequence of its
10
element differs from those of rbcL and psbA.
Second, the psbD AAG-box did not activate transcription when
fused upstream of a derivative of the rbcL promoter shown in
Fig. 3, which lacks an active
35 element (data not shown). This could
mean that AGF interacts with an RNA polymerase containing a
-factor
that is incompatible with the rbcL promoter. Third,
utilization of a different
-factor for transcription of the
psbD BLRP would allow blue light-specific regulation of this
promoter via the
-factor. Recently, genes encoding three chloroplast
-factors have been cloned (29, 30). Moreover, the expression of at
least one
-factor gene is regulated by light (56, 66), and previous
work showed that these factors are the target of light-mediated
regulation of chloroplast transcription (31).
The function of the
35 promoter element in the psbD BLRP
is likely to be replaced by an activating complex bound to the AAG-box (Fig. 6, AGF/BB'). The AAG-box contains two binding domains, aa' and
bb', which bind AGF. The AGF, unlike
-factors, binds to DNA in the
absence of the RNAP (34). A subunit of AGF or perhaps a separate
protein, noted in Fig. 6 as BB', binds specifically to the bb' motif.
The AGF/BB' could activate the psbD BLRP by recruiting the
RNA polymerase to the psbD BLRP, by stabilizing the binding
of the RNAP to the BLRP, or by changing RNAP recognition of the
10
element, thus promoting transcription (reviewed in Ref. 67).
The structure of the psbD BLRP shown in Fig. 6 resembles a
class of bacterial promoters that use activating proteins to stimulate transcription (reviewed in Refs. 52 and 65). The activating sequences
in one class of these promoters (type I; i.e. cAMP receptor protein binding site in lacP1) can be moved various
distances upstream of the promoter (68). In type II promoters such as galP1, the site of activator binding must be immediately
upstream of
35 (68). In both cases, the
-subunit of RNAP interacts with the activating complex (Refs. 68 and 69; reviewed in Ref. 70),
although in different ways (71). In this regard, the psbD
BLRP is similar to a type II bacterial promoter. The addition of 3, 7, or 10 bp between the
10 element and the AAG-box dramatically
inhibited transcription, indicating that the AGF factor needs to be
approximately 23 bp from the
10 element. Moving the AAG-box closer to
the
10 element by removal of five nucleotides between the
10 and
AAG-box also inhibited transcription. However, constructs with deletion
of 10 bp still showed a low level of activity. Deletion of 10 bp, or
one helical turn, would keep the AAG-box and the
10 element in the
same relative orientation along the DNA helix. Therefore, a low level
of transcription from this template is possible, although packing of
the RNAP and AGF on the template must be tight.
Regulation of the psbD BLRP--
Illumination of 7.5-day-old,
dark-grown barley with white light caused a 10-fold increase in
transcription from the psbD BLRP and a 4-fold increase in
transcription from rbcL in vivo (72). Surprisingly, in
vitro transcription of the 53-bp psbD BLRP in plastid
extracts from 7.5-day-old, dark-grown plants that had been illuminated
for 16 h, was approximately 6.5-fold higher than in extracts of
dark-grown plants (Fig. 4). Transcription from the rbcL
promoter was also approximately 2-fold greater in extracts from
illuminated plants (Fig. 3). This suggests that light-induced modifications that activate transcription in vivo are
retained in vitro. Light could induce the accumulation of a
transcription factor and/or cause modification of the RNAP, a
-factor, or the AGF during the illumination period. Inhibitor
studies have implicated the involvement of protein kinases and
phosphatases in blue light modulation of transcription from the
psbD BLRP (73). Future experiments will be directed toward
identification of the potential targets of these protein
kinases/phosphatases and an understanding of their role in blue light
modulation of the psbD BLRP.