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Plant Physiol. (1999) 119: 663-670
ADP-Dependent Phosphorylation Regulates Association of a
DNA-Binding Complex with the Barley Chloroplast psbD
Blue-Light-Responsive Promoter1
Minkyun Kim,
David A. Christopher, and
John E. Mullet*
Department of Biochemistry and Biophysics, Crop Biotechnology
Center, Texas A&M University, College Station, Texas 77843 (M.K.,
J.E.M.); Department of Molecular Biosciences and Biosystems
Engineering, University of Hawaii at Manoa, St. John 503, Honolulu,
Hawaii 96822 (D.A.C.); and Department of Agricultural Chemistry,
Division of Applied Biology and Chemistry, College of Agriculture and
Life Sciences, Seoul National University, Suwon 441-744, Republic of
Korea (M.K.)
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ABSTRACT |
The chloroplast gene
psbD encodes D2, a chlorophyll-binding protein located
in the photosystem II reaction center. Transcription of
psbD in higher plants involves at least three promoters,
one of which is regulated by blue light. The psbD
blue-light-regulated promoter (BLRP) consists of a  10 promoter
element and an activating complex, AGF, that binds immediately upstream
of 35. A second sequence-specific DNA-binding complex, PGTF, binds
upstream of AGF between 71 and 100 in the barley (Hordeum
vulgare) psbD BLRP. In this study we report that
ADP-dependent phosphorylation selectively inhibits the binding of PGTF
to the barley psbD BLRP. ATP at high concentrations
(1-5 mM) inhibits PGTF binding, but in the presence of
phosphocreatine and phosphocreatine kinase, this capacity is lost,
presumably due to scavenging of ADP. ADP inhibits PGTF binding at
relatively low concentrations (0.1 mM), whereas other
nucleotides are unable to mediate this response. ADP-mediated
inhibition of PGTF binding is reduced in the presence of the protein
kinase inhibitor K252a. This and other results suggest that
ADP-dependent phosphorylation of PGTF (or some associated protein)
inhibits binding of PGTF to the psbD BLRP and reduces transcription. ADP-dependent phosphorylation is expected to increase in
darkness in parallel with the rise in ADP levels in chloroplasts. ADP-dependent phosphorylation in chloroplasts may, therefore, in
coordination, inactivate enzymes involved in carbon assimilation, protein synthesis, and transcription during diurnal light/dark cycles.
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INTRODUCTION |
The chloroplast genes psbA and psbD encode
D1 and D2, chlorophyll-binding proteins that comprise the reaction
center of PSII. Expression of these and other plastid and nuclear genes
involved in photosynthesis increases coordinately with leaf and
chloroplast development (for review, see Mullet, 1988 , 1993). Light
regulates leaf and chloroplast development, together with overall
chloroplast gene expression, through the action of red-light
photoreceptors such as phytochrome (for review, see Pratt et al., 1997;
Quail, 1997), and blue-light photoreceptors (cryptochromes) (Cashmore, 1997). Hence, mutation of genes in the photoreceptor signal
transduction pathways, such as det1 (Chory and Peto, 1990),
modifies chloroplast gene expression, including transcription of
psbD (Christopher and Hoffer, 1998). In addition, light
regulates the initial accumulation of D1, P700, and CP43 during
development in higher plants through the light-dependent accumulation
of chlorophyll, which is needed to stabilize chlorophyll-binding
proteins (Mullet et al., 1990; Kim et al., 1994).
Once leaf and chloroplast biogenesis is complete, expression of most
plastid genes decreases to levels needed for maintenance of the
photosynthetic apparatus (Gamble et al., 1988). However, synthesis of
D1 and D2 is maintained at relatively high levels in mature
chloroplasts of developed leaves (Gamble et al., 1988; Christopher and
Mullet, 1994). Maintenance of high rates of synthesis of these proteins
is needed to replace D1 and D2 subunits that are damaged and turned
over in illuminated plants (Mattoo et al., 1984, 1989; Ohad et al.,
1985; Schuster et al., 1988; Christopher and Mullet, 1994). Elevated D1
synthesis in mature chloroplasts is paralleled by high levels of
psbA RNA (Mullet and Klein, 1987; Gamble et al., 1988;
Baumgartner et al., 1993). The relatively high abundance of
psbA mRNA is due primarily to the unusual stability of these
transcripts (Kim et al., 1993) and to a smaller extent to light-induced
transcription (Klein and Mullet, 1990). Even though psbA
mRNA levels are relatively constant in dark and light in mature
chloroplasts, D1 synthesis is light dependent and regulated at the
level of translation (Fromm et al., 1985; Malnoë et al., 1988).
Recent studies suggest that light regulates D1 synthesis by
ADP-dependent phosphorylation (Danon and Mayfield, 1994a) and redox-regulated association of an RNA-binding protein complex with the
psbA RNA 5 -untranslated region (Danon and Mayfield, 1994b).
It is interesting that ADP-dependent phosphorylation was found much
earlier to modulate the activity of enzymes involved in carbon
assimilation in chloroplasts (Ashton and Hatch, 1983). For example,
ADP-dependent phosphorylation of PPDK inactivates this enzyme when
plants are transferred to darkness (Budde et al., 1986; Smith et al.,
1994). Similarly, redox state is well known to regulate the
organization of the light-harvesting chlorophyll proteins (for review,
see Bennett, 1991; Allen and Nilsson, 1997; Gal et al., 1997). In this
case, a redox-responsive protein kinase associated with the chloroplast
thylakoid membrane phosphorylates PSII proteins to modify light
harvesting and energy transfer among the photosystems.
The capacity for high rates of D2 synthesis is retained in mature
chloroplasts primarily due to a psbD BLRP (Gamble and
Mullet, 1989; Sexton et al., 1990). Transcription from this promoter
requires a prokaryotic 10 promoter element but not a 35 element
(Kim et al., 1999). The function of the 35 element is replaced
by an activating complex, AGF, which binds to an AAG-box motif
immediately upstream of 35 (Kim and Mullet, 1995). An additional DNA
region located between 71 and 100 has also been shown to contribute to promoter activity in vivo (Allison and Maliga, 1995). Sequences in
this region resemble the GT-motif found in light-regulated nuclear
genes and the region was therefore named the PGT-box (Kim and Mullet,
1995). In this study we further investigate a protein complex that
binds to the PGT-box and report that binding is inhibited by
ADP-dependent phosphorylation. These data suggest that ADP-dependent phosphorylation in higher-plant chloroplasts modulates transcription in
addition to translation and enzyme activity.
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MATERIALS AND METHODS |
Plant Growth and Plastid Isolation
Barley (Hordeum vulgare L. var Morex) seedlings
were grown in controlled environmental chambers at 23°C, as described
by Kim et al. (1993). Seedlings were germinated and grown in complete darkness. After 4.5 d the dark-grown seedlings were harvested under a dim-green safelight. Plastids were isolated from the top 4 cm
of the primary leaves of barley seedlings by Percoll gradient (35%-75%) centrifugation (Klein and Mullet, 1986). The concentration of plastids was quantitated (plastids per microliter) by phase-contrast microscopy using a hemacytometer.
Preparation of Radiolabeled DNA Probes, Competitor Fragments, and
Plastid Extracts
The plastid extracts and DNA fragments used as either radiolabeled
probes or competitor fragments were prepared as described by Kim and
Mullet (1995).
Gel-Retardation Assay
Experiments were done as described by Kim and Mullet (1995). Some
of the binding reactions were performed after preincubation of plastid
extracts with various chemicals or enzymes described in the figure
legends (Ap5A was obtained from Boehringer Mannheim, K252a was from
Calbiochem, and all of the other chemicals were from Sigma) for 10 min
at room temperature. However, preincubation of plastid extracts with
K252a was done in complete darkness due to the instability of K252a in
the light. The concentration of the chemicals used is specified in the
figure legends. Phosphocreatine kinase (type III) was diluted with DEAE
buffer supplemented with 10 mM MgCl2
and 0.1% albumin, according to the supplier's instructions.
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RESULTS |
Analysis of psbD PGT-Box Binding Proteins
A diagram of the psbD BLRP appears in Figure
1A. We have previously shown that protein
complexes bind specifically to the AAG-box and PGT-box sequences
present in the psbD BRLP (Kim and Mullet, 1995). The protein
complex that bound to the AAG-box, labeled AGF, was required for in
vitro transcription from the psbD BLRP (Kim and Mullet,
1995). This was one of five protein complexes formed when LRP136, which
contained the 30 to 165 region of the psbD BLRP, was
radiolabeled and used in gel-shift assays (Fig. 1B, lane 2, [AGF
corresponds to complex D]). All of the gel-shift bands were removed by
protease treatment, indicating that they were formed by protein-DNA
interactions (data not shown).
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| Figure 1.
Schematic representation of the barley
chloroplast psbD BLRP. A, The top portion of the figure
shows the location of the AAG-box and PGT-box present upstream of the
psbD transcription initiation site (marked by an open
arrow and designated +1). The psbD open reading frame is
shown at the far right. The double-headed arrows represent DNA
fragments from the psbD light-responsive promoter used
for gel-retardation and competition-binding experiments. GT30 and LRP20
represent four (4X) and three (3X) tandem copies of each region
indicated in the figure. B, Gel-shift assays using a GT30 tetramer
(lane 1) or the LRP136 DNA fragment (lane 2). All gel-shift assays
contained 1 µg of poly (dI-dC)·(dI-dC). Gel-shift complexes A to
E and I to III are noted. Band D was previously identified as an AGF,
which binds specifically to the AAG-box shown in A. C, Gel-shift assays
using the radiolabeled tetramer of GT30 and various amounts (100-200
ng) of unlabeled LRP136, GT30, or LRP20 DNA. The migration of complexes
I, II, and III and the free probe is indicated. No Comp., No
competitor.
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In a previous study we noted that the formation of complex A with
LRP136 could be prevented by the addition of a tetramer of the sequence
from 71 to 100 (Kim and Mullet, 1995). This 30-bp DNA sequence
corresponds to the PGT-box and is referred to as GT30. Results in
Figure 1B show that three protein complexes were formed when a tetramer
of GT30 was added to plastid extracts from 4.5-d-old, dark-grown barley
plants (Fig. 1B, lane 1). The addition of unlabeled LRP136 or GT30
before the gel-shift assays effectively competed for the binding of
complex I and the GT30 tetramer (Fig. 1C, lanes 2-5). A nonspecific
competitor DNA, LRP20, did not reduce the abundance of complex I (Fig.
1C, lanes 6 and 7). When a similar experiment was carried out using
radiolabeled LRP136, GT30 and LRP136 were able to compete for binding
to complex A (Kim and Mullet, 1995). Based on these results, we
concluded that complex A formed with LRP136 and complex I formed with
GT30 contained similar DNA-binding proteins that allowed specific
binding to the PGT-box. The protein-binding complex that interacted
with the PGT-box is labeled as PGTF in Figure 1A. Figure 1C also
indicates that unlabeled LRP136 and GT30 did not significantly reduce
the abundance of complex II or III, suggesting that binding in these complexes was nonspecific.
ADP Inhibits PGTF Binding to the PGT-Box
We tested the modulation of protein association with the
psbD BLRP by protein phosphorylation or nucleotide binding
by the addition of ATP and other ribonucleotides to plastid extracts before the gel-shift assays. We incubated plastid high-salt extracts obtained from 4.5-d-old, dark-grown barley with 5.0 mM ATP, UTP, GTP, or CTP for 10 min at room
temperature, and then carried out gel-retardation assays using LRP136
as the DNA probe (Fig. 2A). Preincubation
of plastid extracts with 5.0 mM ATP reduced the abundance of complex A (PGTF) and led to the appearance of a new complex that migrated just below complex B (Fig. 2A, lane 2). Incubation with UTP, GTP, and CTP had little effect on the abundance of
complex A (Fig. 2A, lanes 3-5). Preincubation of the protein extracts
with 1.0 mM ADP also caused a loss of complex A
(Fig. 2B, lane 2). Other ribonucleoside diphosphates and AMP had little effect on the abundance of complex A (Fig. 2B, lanes 3-6).
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| Figure 2.
Gel-retardation experiments with plastid extracts
preincubated with various nucleotides. Experiments in A and B were done
with radiolabeled LRP136 DNA fragments and plastid extracts that were
preincubated with either 5.0 mM ATP, UTP, GTP, or CTP (A,
lanes 2-5, respectively) or 1.0 mM ADP, UDP, GDP, CDP, or
AMP (B, lanes 2-6, respectively). Plastid extracts were obtained from
4.5-d-old, dark-grown barley plants. The major complexes observed with
the preincubation of plastid extracts without any exogenous nucleotides
(control reaction, lanes 1) are labeled A, B, C, D, and E. Migration of
the free probe (FP) is indicated.
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The nature of the new complex migrating just below complex B, which
forms with LRP136 when PGTF (complex A) binding is reduced, is
presently unknown (Fig. 2, lane 2). Kim and Mullet (1995) observed this
complex previously, when the abundance of complex A was reduced by the
addition of DNA fragments (GT30 or LRP136) that competed for PGTF
binding. Previous studies also showed that this complex could not
compete with unlabeled LRP136 or GT30, at least with normal levels of
DNA used in competition assays. This suggests that the new complex was
formed by either an abundant specific-binding complex (i.e. RNA
polymerase) or nonspecific-binding proteins. Similar complexes were not
formed when ATP, ADP, or competitor DNAs induced a similar reduction in
the abundance of complex I formed with GT30 (Figs. 1C and 3B; Kim and
Mullet [1995]). This indicates that the complex binding to LRP136
when PGTF binding was reduced must have interacted with a region of
LRP136 outside of the GT30 domain.
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| Figure 3.
Gel-retardation experiments with plastid extracts
preincubated with various concentrations of either ATP or ADP.
Experiments were done with either radiolabeled LRP136 (A) or GT30 (B)
DNA fragments and plastid extracts that were preincubated with ATP (A,
B, lanes 2-4) or ADP (A, B, lanes 5-7). The concentration of ATP and
ADP used is indicated above the lanes. Plastid extracts were obtained
from 4.5-d-old, dark-grown barley seedlings. The major complexes
observed in plastid extracts without exogenous nucleotides (control
reaction, lanes 1) are labeled A, B, C, D, and E (A) and I, II, and III
(B). Migration of the free probe (FP) is indicated and the binding
activity of nonspecific-binding proteins to the GT30 probe is
designated NS.
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The concentration dependence of ATP and ADP-induced loss of complex
A (probe LRP136) and complex I (probe GT30) appears in Figure
3. We preincubated the plastid extracts
obtained from 4.5-d-old, dark-grown barley with concentrations of
either ATP or ADP (0.1-5.0 mM), and then carried out
gel-retardation assays. The abundance of complex A and complex I
decreased when extracts were incubated with 0.1 mM ADP;
however, 1.0 mM ATP was required to cause a similar decrease in abundance of either complex A or complex I (Fig. 3). The
ATP or ADP-induced reduction of complex I abundance (GT30) resulted in
an increase in complex II (Fig. 3B). In addition, at 5 mM
ADP the abundance of complex B also decreased (Fig. 3A, lane 7). The
abundance of the other protein complexes formed with LRP136 and GT30
was not altered by ATP or ADP, showing that the response to nucleotides
was selective.
Plastid extracts probably have endogenous activity that converts
nucleoside triphosphates to nucleoside diphosphates. Therefore, the
ATP-induced loss of complex A binding could be due to ADP formed from
ATP. We tested this possibility by adding phosphocreatine and
phosphocreatine kinase to extracts to convert ADP into ATP. As shown in
Figure 4, the addition of ATP to extracts
in the presence of phosphocreatine and phosphocreatine kinase greatly
reduced the ability of ATP to inhibit formation of complex I (Fig. 4, lane 2 versus lane 3). Preincubation of extracts with ADP in the presence of phosphocreatine and phosphocreatine kinase also reduced the ability of ADP to inhibit formation of complex I, presumably because ADP was converted into ATP. In addition, ADP-induced
inhibition of complex A formation did not change when plastid extracts
were preincubated with ADP in the presence of the adenylate kinase inhibitor, Ap5A (Fig. 4B). This inhibitor would have slowed the conversion of ADP to ATP by the endogenous adenylate kinase in plastid
extracts. These results suggest that ADP rather than ATP was required
to inhibit formation of complex A and complex I.
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| Figure 4.
Gel-retardation experiments showing the effects of
the preincubation of plastid extracts with either ATP, ADP,
phosphocreatine and phosphocreatine kinase, and/or an inhibitor of
adenylate kinase. A, Gel-shift assays were done using radiolabeled GT30
DNA fragments and plastid extracts, which were preincubated with either
5.0 mM ATP or 1.0 mM ADP in the presence of 5.0 mM phosphocreatine (PC) and 2 units of phosphocreatine
kinase (PC Kin) (lanes 3 and 5, respectively). Binding reactions using
plastid extracts preincubated with 5.0 mM ATP or 1.0 mM ADP alone are shown in lanes 2 and 4, respectively.
Plastid extracts were obtained from 4.5-d-old, dark-grown barley
plants. The major complexes observed using the extracts without any
additions (control reaction, lane 1) are labeled I, II, and III.
Migration of the free probe (FP) is indicated. B, Gel-retardation
experiments using radiolabeled LRP136 DNA probe testing the effect of
preincubation of plastid extracts with ADP in the presence of the
adenylate kinase inhibitor Ap5A. Plastid extracts were preincubated,
before binding reactions, with 1.0 mM Ap5A in the presence
(1.0 mM, lane 3) or absence (lane 2) of ADP. The major
complexes observed in plastid extracts without additions are labeled A,
B, C, D, and E (Control, lane 1). Migration of the free probe (FP) is
indicated.
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The nonhydrolyzable analog of ATP, AMP-PNP, did not inhibit formation
of complex A (Fig. 5, lanes 3-5). In
contrast, formation of complex A was reduced in the presence of
ATP- -S and ADP- -S (Fig. 5, lanes 6 and 7, and lanes 9 and 10, respectively). These results are consistent with a previous study that
showed that these ATP/ADP analogs could be used as the substrates for
ADP-dependent phosphorylation of the psbA RNA-binding
complex (Danon and Mayfield, 1994a).
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| Figure 5.
Influence of ADP and ATP analogs on protein
binding to the psbD BLRP. Plastid extracts were
preincubated with ATP (lane 1), AMP-PNP (lanes 3-5), ATP--S (lanes
6 and 7), ADP (lane 8), or ADP--S (lanes 9 and 10) prior to
gel-shift assays using LRP136 as a probe. The concentration of the
chemical used is indicated above the lanes. Binding experiments were
done with plastid extracts obtained from 4.5-d-old, dark-grown barley
plants. The major complexes observed without additions are labeled A,
B, C, D, and E (control reaction, lane 1). Migration of the free probe
(FP) is indicated.
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An ADP-Dependent Protein Kinase Inhibits Formation of Complex A
ADP could have inhibited complex A and complex I formation by
binding or by the kinase-mediated phosphorylation of a protein that may
have been associated with the complex. Dialysis of plastid extracts
after incubation with ADP did not reverse the loss of complex A
binding, suggesting that ADP was not acting by binding to complex A
(data not shown). We tested the involvement of protein kinases by
incubating plastid extracts with the general protein kinase inhibitor
K252a. This inhibitor acts on various protein kinases, including
Ser/Thr kinases, protein kinase C, cAMP- and cGMP-dependent protein
kinases, and casein kinase II (Hashimoto, 1988). Treatment with
inhibitor in the absence of ADP had no influence on the formation of
complex A (Fig. 6, lane 2). However,
K252a inhibited ADP-induced inhibition of complex A formation in
plastid extracts (Fig. 6, lane 3 versus lane 4). This result indicates that an ADP-dependent protein kinase inhibited the formation of complex
A.
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| Figure 6.
Gel-retardation experiments showing the effects of
preincubation of plastid extracts with K252a. Gel-shift assays were
carried out using radiolabeled LRP136 DNA fragment and plastid extracts
obtained from 4.5-d-old, dark-grown barley plants. Before binding
reactions, plastid extracts were preincubated either in the presence
(lane 3) or absence (lane 4) of K252a (10 µM, final
concentration), then further incubated with 1.0 mM ADP. The
effect of the incubation of the plastid extracts with K252a alone was
tested (lane 2). The major complexes observed with the preincubation of
the extracts without additions are labeled A, B, C, D, and E (Control,
lane 1). Migration of the free probe (FP) is indicated.
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DISCUSSION |
The results described here show that ADP-dependent phosphorylation
inhibited the binding of PGTF to the chloroplast psbD BLRP in vitro. Inhibition of PGTF binding was caused by ADP rather than by
ATP. This conclusion is based on several lines of evidence. First, ADP
was able to reduce PGTF binding at a much lower concentration than ATP
(threshold of 0.1 versus 1.0 mM). Second, the
addition of phosphocreatine and phosphocreatine kinase, an
ADP-scavenging system, to extracts in the presence of ATP inhibited the
ability of ATP to modify PGTF binding. Third, ADP was able to inhibit PGTF binding in the presence of Ap5A, an inhibitor of adenylate kinase.
Other ribonucleotides were unable to modify PGTF binding. Moreover, ADP
only inhibited PGTF binding, suggesting that the modification was
selective.
The ability of ADP to modify PGTF binding was inhibited by the protein
kinase inhibitor K252a. This suggests that ADP-dependent protein
phosphorylation modified PGTF binding either directly or indirectly.
ADP-dependent phosphorylation of an active-site Thr was previously
reported to have inactivated the stromal enzyme PPDK (Smith et al.,
1994). In addition, ADP-dependent Thr phosphorylation inhibited the
association of an RNA-binding complex with the psbA RNA
5-untranslated region (Danon and Mayfield, 1994a). The psbA RNA-binding complex was modified by ADP--S and ATP--S, but not by
AMP-PNP (Danon and Mayfield, 1994a). Similarly, ADP--S and ATP--S
were both able to inhibit PGTF binding, whereas AMP-PNP was not (Fig.
5). It seems likely that in our plastid extracts ATP--S is having
its effect on PGTF binding after conversion to ADP. Danon and Mayfield
(1994a) showed that a 60-kD protein in the psbA RNA-binding
complex was phosphorylated on a Thr residue. Unfortunately, at
present we do not have a method to isolate the PGTF and therefore have
not been able to investigate the site and type of phosphorylation
involved in the modulation of PGTF binding.
Diurnal regulation of PPDK activity was a consequence of ADP-dependent
phosphorylation (inactivation) and phosphate-dependent dephosphorylation (activation) by a bifunctional kinase/phosphatase (Smith et al., 1994). Phosphorylation of PPDK increased in darkness in
parallel with an increase in ADP concentration in chloroplasts, and
dephosphorylation occurred upon plant illumination (Heber and
Santarius, 1965; Hampp et al., 1982; Roeske and Chollet, 1989). Danon
and Mayfield (1994a) also proposed that dark-induced, ADP-dependent phosphorylation of the psbA RNA-binding protein contributed
to the inactivation of D1 translation in chloroplasts. In a similar way
it seems likely that ADP-dependent phosphorylation of PGTF increased in
darkness and caused a decrease in binding of this complex to the
psbD BLRP. Deletion of the PGT-box in transgenic tobacco
reduced the transcription from psbD BLRP 5-fold (Allison and
Maliga, 1995). Therefore, it is reasonable to conclude that PGTF
stimulated transcription in illuminated plants and ADP-dependent phosphorylation reduced binding and transcription from the
psbD BLRP in darkness. The ability of K252a, an inhibitor of
ADP-dependent phosphorylation, to stimulate transcription of
psbD in dark-grown plants is consistent with this hypothesis
(Christopher et al., 1997). Although this hypothesis is consistent with
available information, we have not yet obtained consistent differences
in the abundance of PGTF binding in extracts of dark-treated versus
illuminated plants that confirm this idea. The action of kinases,
phosphatases, and other factors during preparation of extracts or
during binding assays may make this analysis subject to experimental
variation. In the current study extracts from 4.5-d-old,
dark-grown barley plants were used because they provided a relatively
consistent amount of PGTF binding.
We expect that the ADP-dependent phosphorylation of PGTF increased in
darkness, when levels of ADP were elevated (0.3-0.6 mM),
relative to illuminated chloroplasts. The concentration of ADP in
chloroplasts of dark-adapted plants (Heber and Santarius, 1965; Hampp
et al., 1982) was sufficient to inhibit PGTF binding based on our in
vitro analyses, which show that 0.1 mM ADP was required
(Fig. 3). However, it is not clear that the 2-fold change in ADP
concentration that normally occurs in chloroplasts during light/dark
transitions is sufficient by itself to modulate PGTF binding in vivo. A
similar question was raised concerning regulation of PPDK activity by
ADP-dependent phosphorylation (Roeske and Chollet, 1989). Other factors
such as metabolites, pH, redox potential, and other phosphorylation
events may have modulated the susceptibility of PGTF to
phosphorylation.
The psbD BLRP contains a 10 promoter element and an
AAG-box sequence that binds the transcription activator, AGF (Kim and Mullet, 1995; Kim et al., 1999). This minimal 53-bp promoter
domain was sufficient to drive transcription from the psbD
BLRP in vitro. Sequences upstream of the AAG-box, including the
PGT-box, had little influence on transcription from the psbD
BLRP in vitro. The lack of influence of the PGT-box on transcription in
vitro may have been due to the fact that binding of PGTF can be
modulated by phosphorylation and other factors (i.e. phosphatases in
the extracts) that are difficult to control under standard conditions used for in vitro transcription assays and during preparation of
extracts. In addition, the influence of PGTF on transcription has only
been assayed in extracts isolated from plants grown under a limited
number of conditions.
In barley the PGT-box is located between 71 and 100 relative to the
site of transcription initiation. The PGT-box sequence resembles
regions (GT motifs) of several nuclear genes that are involved
in light and circadian regulation (box II, box III, and I-box) (e.g.
Green et al., 1988; Lam and Chua, 1990; Schindler and Cashmore, 1990;
Borello et al., 1993; Kusnetsov et al., 1996; for review, see Gilmartin
et al., 1990, 1991). However, nuclear GT motifs (i.e. box II; Green et
al., 1988) and related sequences such as as-2, ga-1, and the
CGF-binding elements (Gilmartin et al., 1991; Anderson et al., 1994)
did not compete efficiently for PGTF binding to the PGT-box (data not
shown).
Deletion of the 30-bp sequence corresponding to the PGT-box in tobacco
(labeled A-rich) decreased the output from the psbD BLRP
promoter 5-fold in vivo in plants grown several days in cycling light
conditions (16-h light:8-h dark) (Allison and Maliga, 1995). In
contrast, the same deletion had only a small effect on white light (24 h) reactivation of psbD transcription in vivo in plants placed in darkness for 3 d. These results indicate that the
PGT-box played an important role in regulating transcription from the psbD BLRP, but that the physiological role and optimal
conditions to assay the influence of this element have not yet been
established. It is possible that PGTF acts by blocking read-through
transcription from the upstream psbD promoter that is active
in dark-grown plants (Sexton et al., 1990). Similarly, PGTF may
decrease transcription from trnS, which is located
immediately upstream of the psbD BLRP, and read from the opposite DNA
strand (Sexton et al., 1990). It is also possible that PGTF represents
a more global regulator of chloroplast transcription that couples
transcription of genes encoding proteins involved in photosynthesis in
mature chloroplasts with the higher energy state present in illuminated
plants. This activity may have complemented phosphorylation
(inactivation) of sigma factors by an ATP-dependent kinase that
occurred in dark-grown mustard seedlings (Tiller and Link, 1993). If
PGTF is involved in global regulation, we would expect additional
binding sites for PGTF in the chloroplast genome. This aspect of
PGTF function is currently being analyzed through site-directed
mutagenesis to better define the binding site and by searching other
promoters for protein complexes that are modulated by ADP-dependent
phosphorylation.
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FOOTNOTES |
1
This research was supported by the National
Institutes of Health (grant no. GM 37987 to J.E.M.) and by the Texas
Agricultural Experiment Station.
*
Corresponding author; jmullet{at}tamu.edu; fax
1-409-862-4718.
Received June 30, 1998;
accepted November 6, 1998.
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ABBREVIATIONS |
Abbreviations:
AMP-PNP, adenylyl-imidodiphosphate.
Ap5A, P1,P5-Di(adenosine-5)pentaphosphate.
ATP--S
and ADP--S, and forms of adenosine
5-O-(3-thiotriphosphate), respectively.
BLRP, blue-light-regulated
promoter.
PPDK, pyruvate orthophosphate dikinase.
| |
ACKNOWLEDGMENTS |
The authors thank Ueli Klahre in Dr. Nam-Hai Chua's laboratory
and Dr. Steve Kay for sending recombinant plasmids containing multimers
of box II, as-2, ga-1, and CGF-binding elements.
| |
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Top
Abstract
Introduction