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Plant Physiol. (1998) 116: 183-192
Fast Induction of High-Affinity
HCO3
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The induction of a high-affinity
state of the CO2-concentration mechanism was investigated
in two cyanobacterial species, Synechococcus sp. strain
PCC7002 and Synechococcus sp. strain PCC7942. Cells
grown at high CO2 concentrations were resuspended in
low-CO2 buffer and illuminated in the presence of carbonic anhydrase for 4 to 10 min until the inorganic C compensation point was
reached. Thereafter, more than 95% of a high-affinity
CO2-concentration mechanism was induced in both species.
Mass-spectrometric analysis of CO2 and
HCO3
fluxes indicated that only the
affinity of HCO3 transport increased
during the fast-induction period, whereas maximum transport activities
were not affected. The kinetic characteristics of CO2
uptake remained unchanged. Fast induction of high-affinity HCO3 transport was not inhibited by
chloramphenicol, cantharidin, or okadaic acid. In contrast, fast
induction of high-affinity HCO3
transport did not occur in the presence of K252a, staurosporine, or
genistein, which are known inhibitors of protein kinases. These results
show that induction of high-affinity
HCO3 transport can occur within
minutes of exposure to low-inorganic-C conditions and that fast
induction may involve posttranslational phosphorylation of existing
proteins rather than de novo synthesis of new protein components.
Cyanobacteria possess a CCM (Badger and Price, 1992 The efficiency of the CCM changes in response to the environmental
conditions, especially the availability of Ci (Mayo et al., 1986 Yu et al. (1994a) The molecular genetic and biochemical basis of the processes involved
in induction are poorly understood. The presence of light seems to be
an absolute requirement for complete induction (Badger and Price, 1992 High- and low-Ci-adapted cells of cyanobacteria are able to transport
both CO2 and
HCO3 This paper explores further the nature of the changes that occur to
cyanobacteria when they are adapted to growth at limiting Ci. In
contrast to previous research, we find that induction of a
high-affinity state can be very rapid, occurring within minutes, which
raises questions about the primary involvement of de novo protein
synthesis. The results show that it is
HCO3 Growth of Cyanobacteria
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
; Kaplan et
al., 1994) that functions to elevate CO2 levels
around Rubisco. This CCM is absolutely essential in cyanobacteria for
the performance of efficient photosynthesis in their aquatic
environment. A functionally active CCM in cyanobacteria consists of two
functional components: (a) a Ci-transport system that actively acquires
Ci from the surrounding medium and accumulates
HCO3 in the cell cytosol; and
(b) the carboxysome, which provides a compartment in which Rubisco and
CA are specifically co-localized, allowing the cytosolic
HCO3 to be converted to
CO2, leading to the localized elevation of CO2 in the vicinity of Rubisco (Badger and Price,
1992; Kaplan et al., 1994).
;
Badger and Gallagher, 1987). When cyanobacteria are grown at high
CO2 they develop a CCM with a reduced affinity
for external Ci and a diminished capacity to accumulate internal Ci. When cells that have been cultivated at high CO2
are transferred to limiting CO2 conditions (e.g.
20 µL L1), a high photosynthetic affinity for
external Ci is induced (Badger and Price, 1992; Kaplan et al., 1994).
The induction process at low Ci is known to include both an increase in
the number of carboxysomes and carboxysomal CA activity and an
induction of a high-affinity Ci transport system (Badger and Price,
1992; Kaplan et al., 1994). Evidence indicates that both
HCO3 and
CO2 simultaneously serve as substrates for both
low- and high-affinity Ci transport systems (Miller and Canvin, 1985;
Badger et al., 1994; Yu et al., 1994a, 1994b; Sültemeyer et al.,
1995, 1997a, 1997b; Salon et al., 1996; Tyrrell et al., 1996).
investigated the induction time course of the kinetic
changes of the CO2 and
HCO3 transport during transfer
from air levels of CO2 to 20 µL
CO2 L1. When rapidly
sparged with ambient air, air-grown cells show physiological
characteristics similar to those of high-Ci cells (Mayo et al., 1986;
Badger and Gallagher, 1987; Yu et al., 1994a). Within 24 h of
transfer to limiting Ci, the values for
K1/2(CO2) and
K1/2(HCO3)
were reduced by about 3- and 15-fold, respectively, and the induction
was close to completion after 4 h. Similar time courses for
induction of a high-affinity CCM are also reported for eukaryotic green
algae (Palmqvist et al., 1988; Sültemeyer et al., 1991; Matsuda
and Colman, 1995).
;
Kaplan et al., 1994), and some evidence indicates involvement of de
novo protein synthesis during the induction process. Such evidence
includes: an increase in the abundance of carboxysomes per cell by
severalfold in cells grown under Ci limitation compared with those
grown under high-Ci conditions (Turpin et al., 1984; McKay et al.,
1993); an increase in activities of carboxysomal and membrane-bound CA
by up to 10-fold during growth at low Ci (Badger and Price, 1989; Price
et al., 1992); and an increase in a 42-kD polypeptide that has been
shown to accumulate significantly within the plasma membrane fraction
in cyanobacteria upon transfer to low-CO2
conditions (Omata and Ogawa, 1985; Omata et al., 1990). Unfortunately,
specific inactivation of the structural gene (cmpA) coding
for this protein by interposon mutagenesis did not affect the ability
of the cells to acclimate to 300 µL L1
CO2 (Omata et al., 1990), indicating that the
42-kD polypeptide may not be directly involved in the adaptation of
plasma membrane Ci transport processes to air levels of
CO2. In addition to these observed protein
increases, the induction of a high-affinity state over a period of
hours rather than minutes is most consistent with protein-synthesis
processes resulting in the appearance of new polypeptide components.
species during
steady-state photosynthesis with more or less similar maximum rates (Yu
et al., 1994a, 1994b; Sültemeyer et al., 1995, 1997a, 1997b). The
main difference in transport characteristics between high- and low-Ci
cells appears to be in the affinity for substrate rather than maximum
activities, because the K1/2 values for
CO2 and
HCO3 decrease by more than 1 order of magnitude (Yu et al., 1994a; Sültemeyer et al., 1995,
1997a, 1997b). It was therefore suggested that the increase in
transport efficiency was caused by a qualitative change in the
transport system rather than a quantitative increase in transport
components (Yu et al., 1994a; Sültemeyer et al., 1995). In
considering the induction process, it is therefore interesting to
notice that changes in the pattern of phosphorylation of polypeptides with changes in the efficiency of Ci transport have been reported (Bloye et al., 1992). A correlation has been found between the appearance of phosphorylated proteins and the loss of efficiency of Ci
transport, either by transferring low-Ci cells to high-Ci conditions or
by switching to a medium containing Glc (Bloye et al., 1992),
indicating that the affinity of Ci transport may be under the control
of a protein kinase/phosphatase regulation.
and not
CO2 transport that is rapidly induced, and that
the induction processes may be primarily mediated by posttranslational
modification to existing protein components.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
2 s1) as described
previously (Yu et al., 1994; Sültemeyer et al., 1995). Cultures
were vigorously sparged with air enriched with 2%
CO2 (20,000 µL of CO2
L1; high-Ci cells) or air containing 20 µL of
CO2 L1 (low-Ci cells).
For MS measurements, the cells were collected by centrifugation
(4000g for 10 min) and washed twice with fresh CO2-free assay buffer (growth medium enriched
with 50 mm bis-tris-propane, pH 8.0 and 8.2 for the 7942 and 7002 strains, respectively). The pellet was resuspended in the same
buffer to a final Chl content of 200 to 500 µg
mL1 and kept in the dark until used for the
experiments (usually not longer than 30 min). Chl was extracted in
methanol and the content determined according to the method of Porra et
al. (1989). CO2-free assay medium was prepared by
continuously sparging the buffer with CO2-free
air, which had been passed through a carbosorb column (Dräger,
Leipzig, Germany), for a period of several days. After this time the
total Ci in the medium was less than 30 µm.
Fast Induction of High-Affinity
HCO3 Transport
; Sültemeyer et al.,
1995). Four milliliters of fresh CO2-free assay
buffer (see above) was pipetted into the chamber and the temperature was adjusted to 30°C (strain 7942) or 33°C (strain 7002). To obtain "fast-induced" cells, aliquots from concentrated high-Ci cells of
Synechococcus sp. strain 7002 or Synechococcus
sp. strain 7942 were injected into CO2-free assay
medium in the cuvette to yield a final Chl content of 10 µg
mL1. The chamber was then closed with a
plexiglass stopper and the cells were kept in the dark for up to 3 min
to allow for temperature equilibration. When necessary, CA (100 Wilbur-Anderson units mL1) or chloramphenicol
(200 µg mL1) was added during the dark
period. After 2 to 3 min of darkness, the light (750 µmol photons
m2 s1) was switched on
and the cells were allowed to consume the remaining Ci in the cuvette.
Changes in the concentration of CO2
(m/z = 44) and O2
(m/z = 32) were simultaneously followed by MS (see Fig.
1). The initial Ci concentration before the onset of illumination was
between 50 and 100 µm in all experiments, depending on
the amount of Ci that was carried over from the concentrated cell suspensions. After 4 to 10 min in the light, the cells were
darkened again for 2 to 3 min. During this second dark period the
cells were prepared for measurements of O2
evolution, CO2 uptake, and HCO3 transport.
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Flux Measurements of O2 Evolution, CO2
Uptake, and HCO3 Transport
1 was removed,
centrifuged, resuspended in 4 mL of CO2-free
assay buffer, and placed into the reaction chamber attached to the mass spectrometer. Rates of O2 evolution,
CO2 uptake, and
HCO3 transport were determined
during steady-state photosynthesis using a MS disequilibrium technique
(Badger et al., 1994). Application of this method is only possible if
extracellular CA activity is absent, because the method relies on the
occurrence of only the relatively slow and uncatalyzed spontaneous
hydration and dehydration of CO2 and
HCO3, respectively. Therefore,
after dilution of the fast-induced cells we added 50 µm
AZA to the suspension. Separate measurements of
18O exchange from doubly labeled
CO2
(13C18O2;
Badger and Price, 1989) have shown that this amount of AZA was
sufficient enough to inhibit all remaining external CA activity (data
not shown).
uptake) were calculated
using the previously published formulas (Badger et al., 1994). The
equations also permit the estimation of the actual
CO2 and
HCO3 concentrations at the
time the measurements are made. The pseudo-first-order rate constant
k2
(HCO3 to
CO2) was obtained by injection of a known amount
of HCO3 into the reaction
chamber under experimental conditions (Sültemeyer et al., 1995).
The initial slope of CO2 evolution from
HCO3 was a direct measure for
k2, and was determined as 4.89 × 101 min1. Using the
separately measured
HCO3:CO2
ratio of 41 (at pH 8.0 and 30°C) and 78 (at pH 8.2 and 33°C), the
k1 rate constants were calculated as 3.05 and 3.82 min1, respectively. Initial rates of
CO2 uptake were obtained from the slope of the
CO2 uptake traces within the first 10 to 15 s of illumination according to the method of Sültemeyer et al. (1997a, 1997b). Comparative analysis of Ci fluxes revealed that 50 µm AZA had no effect on the kinetics of
CO2 and
HCO3 uptake in high- or low-Ci
cells (data not shown).
Chemicals
CA (EC 4.2.1.1) from bovine erythrocytes was purchased from Sigma (C-3934). Cantharidin, okadaic acid, K252a, staurosporine, and genistein were from Biomol (Hamburg, Germany).| |
RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Fast Induction of High-Ci Cells
Our first indications that acclimation to low-Ci conditions could occur within a few minutes was obtained from experiments similar to those shown in Figure 1. In Figure 1A, the experimental protocol for the fast induction of high-affinity cells from cyanobacteria is shown. The assay mixture contained Chl (10 µg mL1), CA (100 Wilbur-Anderson units
mL1), and a low initial Ci concentration
(50-100 µm). After a short time in the dark, the light
was turned on and the cells were allowed to deplete external Ci to the
compensation point. O2 evolution was initially
high but declined as Ci became limiting. After about 10 min the Ci
compensation point was reached, as indicated by no further changes in
the O2 and CO2
concentrations. Under these conditions, the total Ci concentration in
the medium was almost 0. After reaching the Ci compensation point, the
cell suspension was darkened again for up to 3 min. This second dark
period was applied to provide the cells with Ci that was released
mainly by respiratory processes.
; Miller
et al., 1988, 1991; Badger et al., 1994; Sültemeyer et al., 1995,
1997b). After about 10 min the CO2 concentration
was almost 0; however, the addition of CA in the light caused a sudden
increase in the CO2 concentration, indicating
that a considerable amount of
HCO3 (approximately 56 µm) was still present. These measurements show that CA is
necessary to enable high-Ci cells to rapidly deplete Ci to the
compensation point so that rapid-induction experiments at very low Ci
can be conducted.
Effect of Fast Induction on Photosynthetic Affinity
The extent of acclimation during the fast-induction period shown in Figure 1 is examined further in Table I and Figure 2. Synechococcus sp. strain PCC7002 cells that were continuously grown on 2% CO2 (high-Ci cells) or 20 µL of CO2 L1 (low-Ci cells)
exhibited similar maximum rates of O2 evolution but had vastly different affinities for external Ci. High-Ci cells had
a K1/2(Ci) of 161 µm, whereas
low-Ci cells showed a value of 13 µm (Fig. 2; Table I),
indicating that the latter are more than 10 times more effective at
using small amounts of external Ci. Compared with these two acclimation
states, fast-induced cells showed a decline in
K1/2(Ci) to 20 µm, indicating
that about 95% of the high-affinity state seen with low-Ci cells had
been induced within 10 min of exposure to very low Ci (Fig. 2; Table
I). The ability of high-Ci cells to rapidly induce a high-affinity
state within the 10-min period is facilitated by the addition of active extracellular CA. In the absence of CA, high-Ci cells were not able to
induce a high-affinity state, with these cells showing almost the same
K1/2(Ci) as high-Ci cells (167 µm) (Fig. 2; Table I). Similarly, when CA was inhibited
with 50 µm AZA at the start of the fast-induction
experiment the photosynthetic affinity for Ci remained almost unchanged
(Fig. 2).
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CO2 and HCO3 Uptake
under Steady-State Photosynthesis
serve as substrates for
the transport systems in cyanobacteria (Badger and Andrews, 1982; Miller and Canvin, 1985; Yu et al., 1994a, 1994b; Sültemeyer et
al., 1995, 1997a, 1997b; Salon et al., 1996; Tyrrell et al., 1996). To
determine how various components of Ci transport are affected after
fast induction we analyzed CO2 and
HCO3 uptake in fast-induced
cells from Synechococcus sp. strain PCC7002 and compared the
kinetics with those of high- and low-Ci cells (Fig.
3; Table
II). Regardless of the
CO2 concentration during growth and induction
treatment, all types of cells exhibited similar maximum transport
activities during steady-state photosynthesis; maximum rates varied
from 461 to 521 µmol mg1 Chl
h1 for
HCO3 transport, and from 161 to 176 µmol mg1 Chl
h1 for CO2 uptake.
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and CO2. Although the
K1/2(HCO3)
and K1/2(CO2) were
167 and 3.8 µm in high-Ci cells, respectively, the
corresponding values for low-Ci cells were 9.0 and 0.5 µm (Fig. 3; Table II). After a very short induction period of only 4 min, the
K1/2(HCO3)
decreased to 27.5 µm in fast-induced cells (Fig. 3; Table
II). If CA was omitted during the 4-min depletion of Ci, there was almost no change in the affinity of
HCO3 transport for its
substrate. After the same 4-min period of induction, the kinetics of
CO2 uptake were only slightly affected,
regardless of the presence or absence of CA (Fig. 3; Table II).
)
of HCO3 transport decreased
from 248 µm (high-Ci cells) to 15.5 µm
(fast-induced cells), and was approaching the
K1/2(HCO3)
value of low-Ci cells (9 µm
HCO3). No dramatic changes in
the maximum activities of HCO3
uptake were found regardless of the treatment (Table II). Again, the
affinity of CO2 uptake remained unchanged after
the short induction period, but was about 7-fold higher in low-Ci than
in high-Ci cells (Table II).
Initial Rates of CO2 Uptake
Kinetic analysis of CO2 and HCO3 transport in fast-induced
cells (Fig. 3; Table II) indicates that high-affinity
CO2 transport is not rapidly induced during
initial exposure to low Ci. To further investigate whether any changes
occurred to CO2 uptake processes we measured
CO2 uptake under nonsteady-state photosynthetic
conditions. Nonsteady-state CO2 uptake occurs
during the initial phase of illumination in the absence of net
O2 evolution and even in the presence of
inhibitors that block photosynthetic electron flow (Badger and Andrews,
1982; Miller et al., 1991; Sültemeyer et al., 1991, 1997b). To
investigate the effect of fast induction on initial
CO2 transport we measured the rates of this
process at various CO2 concentrations (Fig.
4). In all cases the initial rates of
CO2 transport were strongly dependent on the
CO2 concentration in the assay medium.
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1 Chl h1,
respectively. When the cells were continuously grown on 20 µL of
CO2 L1 the maximum rate
of initial CO2 transport increased to 533 µmol mg1 Chl h1. In addition
to the quantitative differences in the rates of initial
CO2 uptake, the apparent affinities of
CO2 transport for its substrate increased in
low-Ci cells, indicated by a decrease in
K1/2(CO2) from 8.5 µm in high-Ci cells to 1.6 µm in low-Ci cells. Similar changes in the maximum activity and the
CO2 affinity of CO2
transport have previously been reported after acclimation to low
CO2 (Sültemeyer et al., 1997a, 1997b). In
contrast, 4-min fast-induced cells exhibited a
K1/2(CO2) of 8.1 µm CO2, which was not further
improved even after a prolonged induction period of 15 min (data not
shown).
Mediation of Fast Induction by Protein Synthesis or Protein Modification
The ability of the high-Ci cells to rapidly develop high-affinity HCO3 transport within 4 min
may indicate that protein synthesis is not involved in this
fast-acclimation process. To test this hypothesis the 4-min
fast-induction experiment (Fig. 3) was repeated with high-Ci cells to
which chloramphenicol (200 µg mL1) was added
during the first dark period (Fig. 5).
After fast induction in the presence of chloramphenicol, the cells
showed a
K1/2(HCO3)
for net O2 evolution and
HCO3 transport of 27.5 and
21.0 µm, respectively (Fig. 5), both being much lower
than for the corresponding high-Ci cells (Table II). As expected, the
affinity for CO2 uptake remained unaffected by chloramphenicol (data not shown). However, it is noteworthy that the
maximum rates of O2 evolution and
HCO3 and
CO2 transport were inhibited by about 20% by
chloramphenicol treatment (Fig. 5).
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transport was insensitive
to chloramphenicol, it is reasonable to conclude that its regulation
does not depend on de novo synthesis of proteins. Among cyanobacteria,
posttranslational modifications of proteins by
phosphorylation/dephosphorylation are common (Mann, 1994). In
addition to His protein kinases, which are part of the two-component regulatory systems, eukaryote-like Ser/Thr protein kinases have been
found in cyanobacteria (Mann, 1994; Kaneko et al., 1996). A Ser/Thr
protein kinase from a gram-negative bacterium, Mycobacterium tuberculosis, was inhibited by staurosporine (Peirs et al., 1997), which is thought to act on the ATP-binding site of the catalytic domain
common to eukaryotic and prokaryotic Ser/Thr protein kinases (Meggio et
al., 1995). In addition, indications for a protein Tyr phosphorylation
have been reported (McCartney et al., 1997), and a low-molecular-weight
protein Tyr phosphatase has been identified based on sequence
similarities to eukaryotic protein phosphatases (Kaneko et al., 1996).
Fast Induction of High-Affinity
HCO3). As shown in Table
III, the presence of either of these
inhibitors had almost no effect on the rapid induction of high-affinity
O2 evolution and
HCO3 transport. However, when
high-Ci cells were fast induced in the presence of K252a or
staurosporine, which are widely used inhibitors for eukaryotic Ser/Thr
protein kinases (MacKintosh and MacKintosh, 1994), no high-affinity
O2 evolution or
HCO3 transport was induced
(Table III). Even genistein, which inhibits some Tyr kinases
(MacKintosh and MacKintosh, 1994), caused almost complete repression of
the fast-inducible high-affinity state (Table III). Unlike
chloramphenicol, treatment of the cells with these inhibitors had
basically no effect on the maximum rates of O2
evolution or HCO3 transport
(data not shown).
View this table:
Table III.
Protein kinase inhibitors prevent fast induction
of high- affinity HCO3 transport
The effect of inhibitors of protein phosphatases (cantharidin, okadaic
acid) and kinases (K252a, staurosporine, genistein) on the
K1/2 values for O2 evolution,
HCO3 transport, and CO2 uptake after fast
induction in Synechococcus sp. strain PCC7002. Fast-induced
cells were obtained from high-Ci cells (10 µg mL1), which
were allowed to run out of Ci for 4 to 8 min in the presence of CA (100 Wilbur-Anderson units mL1) and the desired inhibitor. During
the second dark period, an aliquot of the cells (8-12 µg of Chl) was
centrifuged and resuspended in 4 mL of CO2-free assay buffer to
enable mass spectrometric flux measurements. The data represent the
mean ± sd from three independent experiments.
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
Transport
transport that is
dramatically increased during this period rather than any change in the
CO2-transport kinetics of the cells (Fig. 3;
Table II). The rapid nature of the acclimation and the fact that fast
induction of high-affinity
HCO3 transport is not
inhibited by chloramphenicol (Fig. 5), but is inhibited by protein
kinase inhibitors such as K252a, staurosporine, and genistein,
indicates that posttranslational modification rather than de novo
protein synthesis mediates these dramatic kinetic changes to
HCO3 transport. To our
knowledge, this is the first report of fast modulation of Ci transport
that is potentially mediated by phosphorylation of a protein component
upon transfer of cells to limiting Ci concentrations.
transport and with an
enhanced capacity to accumulate Ci (Omata and Ogawa, 1985; Badger and
Price, 1992; Kaplan et al., 1994; Yu et al., 1994a, 1994b;
Sültemeyer et al., 1995, 1997a, 1997b). Therefore, we were
surprised to see that only the affinity of HCO3 transport was
dramatically increased within 4 to 10 min of fast induction (Table II;
Fig. 3), whereas no significant changes in the kinetics of
CO2 uptake occurred, regardless of whether
CO2 transport was observed under steady-state
(Fig. 3; Table II) or nonsteady-state conditions (Fig. 4). Apparently,
a longer adaptation period is required to induce the high-affinity
CO2 transport system, suggesting that induction
of the high-affinity CO2 and
HCO3 transporters is regulated
differently. A similar conclusion has been previously reported (Yu et
al., 1994a).
The Requirement for External CA
Our experimental system for fast induction relies on the cells being rapidly exposed to very low initial Ci concentrations (Figs. 1 and 2). To achieve this, the cells must photosynthesize in the presence of CA, which allows the Ci compensation point to be obtained within minutes of illumination in a low-Ci reaction buffer. The lower the initial [Ci] the faster the Ci compensation point is reached and the more rapidly kinetic changes for HCO3 transport are observed.
With an initial [Ci] of around 50 µm the cells run out
of external Ci within 4 min (Figs. 3-5), whereas it took about 10 min
when the initial [Ci] was 100 µm (Figs. 1 and 2).
transport increased
within these short time periods (Figs. 1-3; Tables I and II). The fact
that the photosynthetic affinity for Ci remained unchanged when CA was
inhibited by AZA (Fig. 2) indicates that the catalytic properties of CA
rather than the physical presence of a foreign protein is important for
fast induction. The requirement for CA can be simply interpreted as
allowing both CO2 and
HCO3 transport processes to
contribute to Ci uptake at low Ci and alkaline pH (8.0-8.2), rather
than being chiefly dependent on CO2 transport, as
occurs under conditions of slow interconversion between
CO2 and
HCO3 when CA is absent (Price
and Badger, 1989). Consumption of external Ci by both transport systems
thus allows the compensation [Ci] (close to 0) to be reached within 4 to 10 min of illumination (Fig. 1A). This was not achieved in the
absence of CA (Fig. 1B).
The Trigger for Induction
Although it is still unclear what external factor primarily triggers the fast induction of high-affinity HCO3 transport, we can draw
some clues from the experiments shown in Figure 1. First, because the
CO2 concentration decreased to almost 0 in both
experiments, but fast induction was observed only when total Ci was
close to 0 in the presence of CA (Figs. 1A and 2), it seems unlikely
that the CO2 concentration in the external medium
is the primary signal to which the cells respond. Previous experiments
have hinted at a similar conclusion (Mayo et al., 1986; Badger and
Gallagher, 1987). Second, it has been suggested that the
CO2:O2 ratio in the medium
regulates the induction of the high-affinity CCM (Kaplan et al., 1994),
indicating that a metabolite in the photorespiratory cycle might be
involved in the signal transduction pathway. However, in the presence
of CA (Fig. 1A), the O2 concentration during the
fast-induction procedure increased from 18 to 28% and similar changes
in O2 content were observed when CA was omitted
(Fig. 1B, and data not shown). Because in both experiments the
CO2 concentration was close to 0, a similar CO2:O2 ratio in the
different assays and, consequently, a similar degree of induction would
be expected if the CO2:O2
ratio were responsible for induction. This was clearly not the case
(Figs. 1 and 2) and, therefore, we conclude that the
CO2:O2 ratio is not crucial
for fast induction.
concentration
in the medium after 10 min of illumination. Because fast induction of a
high-photosynthetic affinity for Ci occurred only when total Ci and,
therefore, HCO3 was almost 0 (Fig. 1A), it seems reasonable to assume that
HCO3 is involved as a primary
signal. This conclusion is consistent with previous suggestions for
cyanobacteria (Mayo et al., 1986; Badger and Gallagher, 1987) and green
algae (Matsuda and Colman, 1995). So far, however, the threshold
concentration for HCO3 below
which the high-affinity state of the CCM is induced is unknown, as is
what components HCO3 is
interacting with to initiate the induction changes.
Induction in Synechococcus sp. Strain PCC7002 versus PCC7942
Most of the data presented in this paper have been obtained with the marine cyanobacterium Synechococcus sp. strain PCC7002, but the ability for fast induction of the high-affinity CCM appears not to be restricted to one particular species. A comparison of the fast-induction characteristics between the marine species and the freshwater species, Synechococcus sp. strain PCC7942, revealed a high degree of similarity: almost the same time period for fast induction, a similar net increase in photosynthetic affinity for Ci (Table I), and fast induction being specific to high-affinity HCO3 transport without
changing the kinetics of the CO2-uptake system (Table II). These similarities in the induction characteristics between
two species acclimated to different habitats may be indicative of a
common regulatory pathway of fast-inducible, high-affinity HCO3 transport among
cyanobacteria. This possibility needs further investigation.
Posttranslational Modification of an Existing Protein
Several lines of evidence indicate that de novo protein synthesis is not responsible for the rapid change in the kinetic characteristics of photosynthetic O2 evolution and HCO3 transport. Within 4 min
of illumination more than 90% of the high-affinity
HCO3 uptake system was induced
compared with low-Ci cells (Fig. 3; Table II). At a concentration of
200 µg mL1, chloramphenicol did not inhibit
fast induction of high-affinity HCO3 transport (Fig. 5). The
observation that the maximum rate of O2 evolution
was reduced by about 20% in the presence of the drug (Fig. 5)
indicates that it had entered the cells and, therefore, should have
affected protein synthesis. It is interesting to note in this context
that we added a four-times-higher concentration of chloramphenicol than
other authors used to inhibit protein synthesis in a short time in
intact cells of Anacystis nidulans (Herrero et al., 1984).
transport (Table III).
The effects of these inhibitors on cyanobacterial protein kinases have
not been investigated in detail. However, there are eight putative,
eukaryotic-like protein kinases represented in the genome database from
Synechocystis sp. strain PCC6803 (Kaneko et al., 1996).
Three of these have both the ATP-binding and catalytic signatures for
Ser/Thr protein kinases present as perfect matches to the Prosite
signatures (namely SLR0599, SLR0152, and SLL0776), whereas one has only
the conserved catalytic site (SLL1575), and another possesses only the
ATP-binding site (SLR1697).
),
and it is therefore likely that they act similarly against cyanobacterial homologs of protein kinases. In fact, staurosporine has already been demonstrated to inhibit a protein kinase from the
gram-negative bacterium M. tuberculosis (Peirs et al.,
1997). Taken together, our results are consistent with the view that a
constitutive low-affinity HCO3
transporter (present in high-Ci cells) is posttranslationally modified
into a high-affinity HCO3
uptake system, and that this step may involve phosphorylation of a
polypeptide(s) by a protein kinase(s).
; Mann, 1994). For
instance, Bloye et al. (1992) reported a significant increase in the
extent of phosphorylation of several polypeptides during 1 to 2 h
when low-Ci cells of Synechocystis sp. strain PCC6803 were
shifted to high-Ci conditions or supplied with Glc, which causes a
decline in the ability to accumulate Ci. This indicates that a protein
kinase is involved in the down-regulation of the efficiency of the CCM.
At this stage we have no explanation for the apparent differences
between their results and ours, except that different time scales were
used in the different experiments and different processes were being
examined. However, in this context it is interesting to note that
Goosney and Miller (1997) were unable to repeat the work of Bloye et
al. (1992) with respect to the repression of
HCO3 uptake by growth of
Synechocystis sp. strain PCC6803 on Glc. Current work in our
laboratory is aimed at identifying the polypeptide(s) that is
phosphorylated during the fast-induction period.
Short-Term and Long-Term Acclimation at Low Ci
In this paper we have reported on a short-term (4-10 min) acclimation to limiting Ci in two cyanobacterial species, in which the properties of HCO3 transport
in particular appear modified by posttranslational phosphorylation.
This does not exclude the involvement of de novo protein synthesis
during long-term acclimation to limiting Ci. Increasing numbers of
carboxysomes as well as the proteins located within these structures,
such as Rubisco and CA, are almost certainly changed via protein
biosynthesis (Price et al., 1992; McKay et al., 1993), as is the case
for plasma membrane proteins such as the 42-kD polypeptide (Omata and
Ogawa, 1985). The slower induction of high-affinity
CO2 transport may indicate that this is also achieved via protein synthesis, but this remains to be determined. As a
consequence, the pathway for full induction of the high-affinity CCM
may proceed in two steps: the first step may consist of
posttranslational modification of a constitutive
HCO3 transporter to provide a
rapid response to limiting Ci concentrations, and the second would
require a longer time period and depend on protein biosynthesis to
allow full optimization of the acclimation response.
| |
FOOTNOTES |
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Received May 27, 1997;
accepted September 16, 1997.
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ABBREVIATIONS |
|---|
Abbreviations:
AZA, acetazolamide.
CA, carbonic anhydrase.
CCM, CO2-concentrating mechanism.
Chl, chlorophyll.
Ci, inorganic carbon.
K1/2(HCO3), K1/2(CO2), concentration of
HCO3 and CO2,
respectively, required for half-maximal activity.
| |
ACKNOWLEDGMENT |
|---|
We thank Dr. Uwe Conrath (Fachbereich Biologie, Universität Kaiserslautern, Kaiserslautern, Germany) for providing us with the inhibitors for protein phosphatases and kinases.
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LITERATURE CITED |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Transport in
Cyanobacteria
, Cells grown
continuously on 20 µL of CO2 L
, cells grown continuously on 2% CO2 (high-Ci cells); 
, high-Ci cells subjected to the fast-induction protocol for
10 min in the presence of CA; 
, high-Ci cells subjected to the
fast-induction protocol for 10 min in the absence of CA; 
, high-Ci
cells subjected to the fast-induction protocol for 10 min in the
presence of CA and 50 µm AZA; 
, high-Ci cells that were not fast induced but tested in the presence of CA. Fast-induced cells were obtained in experiments similar to those shown in Figure 
