Journal of Experimental Botany, Vol. 51, No. 349, pp. 1341–1348, August 2000
Active transport of CO and bicarbonate is induced in
2
response to external CO concentration in the green alga
2
Chlorella kessleri
Gale G. Bozzo1, Brian Colman1,3 and Yusuke Matsuda2
1 Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3
2 Department of Chemistry, Kwansei-Gakuin University, 1–1-155 Uegahara, Nishinomiya, Japan 662
Received 10 January 2000; Accepted 31 March 2000
Abstract
The time-course of induction of CO and HCO− trans2
3
port has been investigated during the acclimation of
high CO -grown Chlorella kessleri cells to dissolved
2
inorganic carbon (DIC)-limited conditions. The rate of
photosynthesis of the cells in excess of the uncatalysed supply rate of CO from HCO− was taken as an
2
3
indicator of HCO− transport, while a stimulation of
3
photosynthesis on the addition of bovine carbonic
anhydrase was used as an indicator of CO transport.
2
The maximum rate of photosynthesis (P ) was similar
max
for high CO -grown and low CO -grown cells, but the
2
2
apparent whole cell affinity for DIC and CO of high
2
CO -grown cells was found to be about 30-fold greater
2
than in air-grown cells, which indicates a lower affinity
for DIC and CO . It was found that HCO− and CO
2
3
2
transport were induced in 5.5 h in cells acclimating to
air in the light and in the presence and absence of
21% O , which indicates that a change in the CO /O
2
2 2
ratio in the acclimating medium does not trigger induction of DIC transport. No active DIC transport was
detected in high CO -grown cells maintained on high
2
CO for 5.5 h in the presence of 5 mM aminooxyacet2
ate, an aminotransferase inhibitor. These results indicate no involvement of photorespiration in triggering
induction. Active DIC transport induction was inhibited
in cells treated with 5 mg ml−1 cycloheximide, but was
unaffected by chloramphenicol treatment, indicating
that the induction process requires de novo cytoplasmic protein synthesis. The total DIC concentration
eliciting the induction and repression of CO and
2
HCO− transport was higher at pH 7.5 than at pH 6.6.
3
The concentrations of external CO required for the
2
induction and repression of DIC transport were 0 and
120 mM, respectively, and was independent of the pH
of the acclimation medium. Prolonged exposure to a
critical external CO concentration elicits the induction
2
of DIC transport in C. kessleri.
Key words: Bicarbonate transport, Chlorella kessleri, CO
2
transport, induction.
Introduction
Microscopic algae and cyanobacteria respond to limitations in extracellular dissolved inorganic carbon (DIC )
by the induction of a high DIC-affinity photosynthesis,
described as a carbon concentrating mechanism (CCM ).
This results from the induction of active CO and/or
2
active bicarbonate transport systems and, in some microalgae, the induction of an extracellular carbonic anhydrase (CA) (Badger et al., 1980; Kaplan et al., 1994;
Matsuda and Colman, 1995a, b; Matsuda et al., 1998;
Beardall et al., 1998; Sültemeyer et al., 1998a, b; Moroney
and Somanchi, 1999). The acclimation of cells to DIClimited conditions is characterized by an increased capacity to accumulate intracellular DIC against a concentration gradient which elevates the CO concentration
2
around the main carboxylating enzyme, Rubisco, and
thus reduces the inhibition of CO fixation by O . During
2
2
acclimation to low CO , there is also a marked decrease
2
in the whole cell K for DIC and CO and a decrease
1/2
2
in the CO -compensation point of the cells (Matsuda and
2
Colman, 1995a, b; Matsuda et al., 1998; Colman et al.,
1998; Palmqvist et al., 1988). De novo protein synthesis
of cytoplasmic proteins encoded by the phototroph’s
3 To whom correspondence should be addressed. Fax: +1 416 736 5698. E-mail: [email protected]
© Oxford University Press 2000
1342 Bozzo et al.
nuclear genome is thought to arise during the induction
of the CCM (Shiraiwa and Miyachi, 1985; Palmqvist
et al., 1988; Matsuda and Colman, 1995a; Matsuda et al.,
1998). The regulation of de novo protein synthesis in the
CCM is important in understanding the acclimation
response to low CO . Although the signalling pathway
2
which initiates induction in green algae and cyanobacteria
is not known, it has been proposed that the induction of
high affinity photosynthesis may be in response to a buildup of photorespiratory pathway intermediates within the
cell (Marcus et al., 1983). The photorespiratory signal
model had been proposed because there is an intracellular
accumulation and release of glycolate into the external
medium during the acclimation of Chlamydomonas reinhardtii to ambient CO levels. This is thought to be the
2
result of a decrease in the CO :O ratio in the growth
2 2
medium, which would stimulate the oxygenase activity of
Rubisco, and cause an increase in photorespiratory pathway intermediates. The trigger for induction is therefore
light- and O -dependent. The requirement for light has
2
also been reported in regulating the activity of external
carbonic anhydrase, an enzyme which is thought to play
a role in the CCM of C. reinhardtii (Spalding and Ogren,
1982). For example, it was found that an increase in CA
mRNA occurred in C. reinhardtii within 2 h of acclimation to low-CO in the light; but remained unchanged
2
when cells were acclimated in darkness (Dionisio-Sese
et al., 1990). However, it was also demonstrated that
periplasmic CA transcript was made in the dark after a
lag period (Rawat and Moroney, 1995).
There is increasing evidence to suggest that cells do not
respond to an internal metabolic signal, but to a critical
concentration of dissolved CO in the external growth
2
medium (Matsuda and Colman, 1995b; Matsuda et al.,
1998). In the unicellular green alga, Chlorella ellipsoidea,
the induction of the CCM occurs when cells are acclimated to low CO in darkness (Matsuda and Colman,
2
1995b) and similarly, a decrease in the K
CO for
1/2
2
Chlorella regularis was reported during acclimation to
low CO , which was independent of photosynthesis
2
( Umino et al., 1991). These results cannot be explained
by the photorespiratory signal model. A model was
proposed in which the concentration of dissolved CO
2
molecules occupying a CO sensor at the cell surface
2
dictates the magnitude of active DIC transport: the
response is graded as opposed to being an all-or-nothing
type of response (Matsuda and Colman, 1996a).
In this report, the induction of active DIC uptake in
the unicellular green alga Chlorella kessleri, during the
acclimation of high-CO grown cells to low-CO condi2
2
tions, is described. The effect of dissolved O concentra2
tion on the induction process was also investigated and
the critical concentrations of total DIC and CO , eliciting
2
the induction of active DIC transport in C. kessleri were
determined.
Materials and methods
An axenic culture of Chlorella kessleri (Fott et Nováková,
UTEX 1808) was obtained from the University of Texas Culture
Collection. Cells were grown axenically in batch culture, in
Bold’s basal medium as described previously (Gehl et al., 1990),
under a constant light fluence (100 mmol m−2 s−1). Cultures
were aerated with 5% CO (high-CO ) or with air (0.035%
2
2
CO ); zero CO -grown cells were aerated with CO -free air or
2
2
2
at a low aeration rate (0.01 l min−1) with an ambient air
stream, to ensure that cells were maintained in suspension, both
of which gave a DIC concentration in the medium of
approximately zero.
The physiological characteristics of cells grown under the
various CO concentrations were assessed after they had been
2
harvested at mid-log growth phase (A
0.4–0.5) by centrifu730
gation at 4500 g for 3 min at room temperature. Cells were
washed twice with N -equilibrated, 50 mM Na+/K+ -phosphate
2
buffer (pH 7.8), containing less than 5 mM DIC, and resuspended in the same buffer. Photosynthetic oxygen evolution
rates at various DIC concentrations were measured in a Clarktype O electrode as described previously (Gehl and Colman,
2
1985) with a light fluence of 400 mmol m−2 s−1. The apparent
whole cell affinity (K ) for DIC and CO was determined
1/2
2
according to the method of Rotatore and Colman, with and
without the addition of bovine CA (Rotatore and Colman,
1991). The CO -compensation point of the cells was measured
2
by gas chromatography (Birmingham and Colman, 1979).
Physiological changes in high CO -grown cells were deter2
mined during periods of acclimation to air. High CO -grown
2
cells were harvested at mid-log phase, resuspended in Bold’s
basal medium (pH 6.6), and allowed to acclimate to air for
24 h. The DIC concentration in the medium was monitored
periodically. During the acclimation process, cells were harvested
periodically and the capacity of the cells to actively take up
HCO− was assessed by comparing the O evolution rate at
3
2
50 mM DIC, pH 7.8 and 25 °C, with the spontaneous rate of
CO formation from HCO− in the medium, calculated according
2
3
to the method of Miller and Colman (Miller and Colman,
1980). Stimulation of the O evolution rate at a DIC
2
concentration of 50 mM upon the addition of bovine CA
(10 mg ml−1), was used as a measure of active CO uptake. The
2
effect of O concentration in the medium during acclimation
2
was examined by transferring high CO -grown cells to Bold’s
2
basal medium (pH 6.6), aerated with O -free N , enriched with
2
2
0.035% CO .
2
The critical DIC conditions corresponding to the induction
of active CO and HCO− transport were determined by the
2
3
procedure of Matsuda and Colman (Matsuda and Colman,
1995b). High CO -grown cells were harvested at mid-log growth
2
phase (A
0.4), and resuspended in Bold’s basal medium
730
(phosphate-buffered at pH 6.6 or 7.5). The cell suspensions
were axenically transferred to 0.5 l cylindrical culture vessels
equipped with a sampling port plugged with a rubber serum
stopper, and aerated with defined CO concentrations, in the
2
range of zero to 0.42%. A constant dissolved CO concentration
2
in the medium was maintained by adjusting the pH to ±0.1
units, by injections of 2.0 M HCl or 2.0 M NaOH and by
controlling the inflow CO concentration. Inflow CO concentra2
2
tions and the DIC concentration of the medium were measured
by gas chromatography. Equilibrium conditions between
HCO− and CO in the culture medium were verified by
3
2
comparing the calculated concentrations of DIC at each pH
and inflow CO concentration (Buch, 1960; Stumm and
2
Morgan, 1981) with the measured concentration of DIC in the
medium. Cells were harvested after 5.5 h of acclimation to the
DIC transport induction in Chlorella
defined CO concentration and rates of photosynthetic oxygen
2
evolution at 50 mM DIC, pH 7.8 and 25 °C were determined,
with and without bovine CA.
The effect of protein synthesis inhibitors on the acclimation
of high-CO -grown cells to low-CO was determined by the
2
2
method of Matsuda and Colman (Matsuda and Colman,
1995a). High CO -grown cells were harvested and resuspended
2
in Bold’s basal medium containing 5 mg ml−1 cycloheximide or
400 mg ml−1 chloramphenicol and aerated with 0.035% CO for
2
5.5 h. Cells were assayed for active DIC transport after the
acclimation period following the method described above. The
effect of 5 mM aminooxyacetate, an aminotransferase inhibitor,
on high CO -grown cells maintained on high CO for 5.5 h was
2
2
also examined.
Results
Photosynthetic affinity
C. kessleri cells were grown under 5% CO , 0.035% CO
2
2
and 0% CO conditions, and photosynthetic oxygen
2
evolution rates measured at various DIC concentrations
at pH 7.8, once the CO compensation point of the cell
2
suspension had been reached. C. kessleri cells grown
under DIC-limited conditions demonstrated a high photosynthetic affinity (K ) for DIC and CO ( Table 1) in
1/2
2
comparison to 5% CO -grown cells. The K DIC was
2
1/2
lowest in cells grown in CO -free medium ( Table 1).
2
When bovine CA was added to algal cell suspensions
during the assay there was a further decrease in the K
1/2
DIC and K CO values under all growth conditions
1/2
2
( Table 1). The maximum rate of photosynthetic oxygen
evolution (P ) was similar for cells grown in CO max
2
enriched and CO -limited media ( Table 1). The CO
2
2
compensation point was also found to decrease when the
CO -level in the growth medium was reduced ( Table 1).
2
The time-course of acclimation
Suspensions of high CO -grown cells were allowed to
2
acclimate to air or to O -free nitrogen supplemented with
2
0.035% CO for 24 h. The DIC concentration in the
2
medium was initially about 5.0 mM at pH 6.6 and
decreased to 30 mM at pH 6.6 after 2 h of acclimation.
1343
Samples of the cell suspension were taken at intervals
over the 24 h period for the determination of photosynthetic rates.
Photosynthetic O evolution rates for cells acclimating
2
to 0.035% CO in the presence and absence of 21%
2
dissolved O were measured at 50 mM DIC, pH 7.8 and
2
25 °C. Under these conditions, the calculated maximum
dehydration rate of HCO− is 5.39 nmol CO ml−1 min−1,
3
2
following the method of Miller and Colman (Miller and
Colman, 1980). Within 2 h of acclimation, in the presence
and absence of O , O evolution rates measured in the
2 2
absence of CA were significantly greater than the calculated maximum rate of CO supply and, assuming a
2
photosynthetic quotient of unity, this indicates that active
HCO− transport was induced in cells within 2 h. The
3
addition of CA during this measurement stimulated the
O evolution rate 1.5-fold ( Fig. 1), indicating active CO
2
2
transport, since the addition of excess CA maintains the
CO supply available to the cells. In both O -free acclim2
2
ated and air-acclimated cells, O evolution rates of cells
2
acclimating for 6 h and measured without added CA,
were 1.5-fold greater than those of cells harvested at 2 h.
The O evolution rates measured with added CA were
2
approximately 2-fold greater than cells harvested at 2 h.
Active HCO− and CO transport were fully induced after
3
2
6 h of acclimation to air, regardless of the concentration
of dissolved O in the medium (Fig. 1).
2
Critical external DIC concentration during acclimation
Cell suspensions were aerated with various inflow CO
2
concentrations, in the range of zero to 0.42%, at pH 6.6
and pH 7.5, and allowed to acclimate for a 5.5 h period.
The rate of HCO− and CO transport at 50 mM DIC,
3
2
pH 7.8 and 25 °C was assayed at the end of the acclimation
period. At pH 6.6, O evolution rates measured in the
2
absence of CA, indicated that HCO− transport was
3
repressed at approximately 240 mM DIC ( Fig. 2), when
the O evolution rate was compared to the maximum rate
2
of uncatalysed CO supply, whereas at pH 7.5, the DIC
2
concentration in the external medium that repressed
Table 1. Photosynthetic characteristics of Chlorella kessleri cells grown in high CO or low CO conditions
2
2
Values were determined using cell suspensions of 40 mg Chl ml−1 at pH 7.8 and 25 °C, with and without added CA.
Growth type
CO -free
2
Air
High CO
2
CPa
0.0
12.38±6.9
48.45±3.1
−CA
K d
1/2
P b
max
K c
1/2
102.5±4.3
111.8±6.8
138.7±15
16.4±4.3
51.9±14.5
1887±452
0.6±0.1
1.8±0.5
64.2±15
CPa
0.0
13.1±5.7
32.5±10
+CA
K d
1/2
P b
max
K c
1/2
109.6±7.5
107.9±6.3
117.4±14
20.8±8.3
35.5±10
850±294
0.7±0.3
1.2±0.3
29.2±10
aThe CO -compensation point concentration (mM ) is the mean±SD of five separate experiments.
2
bThe maximum rate of photosynthesis (mmol O mg−1 Chl h−1) is the mean±SD of five separate experiments.
2
cThe apparent whole cells affinity for DIC, K [DIC ] in mM, was measured according to the method of Rotatore and Colman (Rotatore and
1/2
Colman, 1991). Values are the means±SD of five separate experiments.
dK [CO ] is reported in mM, and was calculated based on the premise that DIC in the O evolution rate experiments is in equilibrium. At
1/2
2
2
pH 7.8, 3.4% of the total DIC is dissolved CO . Values are means ±SD of five separate experiments.
2
1344 Bozzo et al.
Fig. 1. Changes in the photosynthetic O evolution rate in high CO -grown cells of Chlorella kessleri during acclimation to air and O -free air. O
2
2
2
2
evolution rates were measured at 50 mM DIC, pH 7.8 and 25 °C, at approximately 40 mg Chl ml−1. O evolution rates in cells: acclimating to air
2
assayed with (%) and without (1) added CA; and acclimating to O -free N supplemented with 0.035% CO assayed with ($) and without (+)
2
2
2
added CA. The dashed line represents the rate at which the spontaneous formation of CO is maximum at 50 mM DIC and pH 7.8. Values are the
2
means ±SE of three separate experiments.
Fig. 2. Acclimation of high CO -grown Chlorella kessleri cells to various
2
external concentrations of DIC and CO (inset) at pH 6.6 for 5.5 h. O
2
2
evolution rates were determined at 50 mM DIC, pH 7.8 and 25 °C with
(%) and without ($) added CA. The dashed line represents the
calculated maximum rate of CO formation from 50 mM HCO− at
2
3
pH 7.8 and 25 °C.
active HCO− transport was 1300 mM (Fig. 3). At both
3
pH 6.6 and 7.5 during the 5.5 h acclimation period, the
dissolved CO concentration eliciting the fully-repressed
2
responses were similar, being 86.4 and 86.0 mM,
respectively.
The DIC concentration corresponding to the O evolu2
tion rate at which half the maximum HCO− transport is
3
induced was greater in cells acclimating at pH 7.5 than
in cells adapting at pH 6.6 ( Table 2). The dissolved
Fig. 3. Acclimation of high CO -grown Chlorella kessleri cells to various
2
external concentrations of DIC and CO (inset) at pH 7.5 for 5.5 h. O
2
2
evolution rates were determined at 50 mM DIC, pH 7.8 and 25 °C with
(%) and without ($) added CA. The dashed line represents the
calculated maximum rate of CO formation from 50 mM HCO− at
2
3
pH 7.8 and 25 °C.
external CO concentration corresponding to the induc2
tion of the half maximum HCO− transport was approxi3
mately 10 mM at both pH values ( Table 2). The total
DIC concentration in the external medium corresponding
to repression of active DIC transport in C. kessleri cells
was approximately 5.5-fold greater in cells acclimating at
pH 7.5, in comparison to cells acclimating at pH 6.6;
whereas the external CO concentration eliciting the same
2
response was 120 mM at both pH values. The presence of
CA in the measurement of O evolution rates in C.
2
DIC transport induction in Chlorella
1345
Table 2. CO and DIC concentrations eliciting induction of active
2
CO and HCO− transport in high CO -grown cells acclimating
2
3
2
at two different pH values for 5.5 h
DIC concentration
(mM ) at acclimation
pH of
6.6
Concentration at which 60%
of maximum DIC transport is induced
Total [DIC ]
Total [CO ]
2
Concentration at which half of maximum
HCO− transport is induced
3
Total [DIC ]
Total [CO ]
2
Concentration at which bicarbonate
transport is first repressed
Total [DIC ]
Total [CO ]
2
7.5
58
21
348
23
28
10
181
12
240
86.4
1300
86
kessleri cells acclimating to defined external CO concen2
trations indicated that at 120 mM CO , the rate of photo2
synthetic O evolution was comparable to rates measured
2
in 5% CO -grown cells ( Figs 2, 3). High CO -grown C.
2
2
kessleri cells acclimating to external CO concentrations
2
greater than 120 mM, showed no significant difference in
O evolution rates measured at 50 mM DIC, pH 7.8 and
2
25 °C with and without added CA (data not shown)
indicating that the cells had a greatly reduced capacity to
transport CO when acclimated at CO concentrations
2
2
greater than 120 mM.
Effect of metabolic inhibitors
High CO -grown cells were allowed to acclimate to
2
0.035% CO at pH 6.6 for 5.5 h in the presence of protein
2
synthesis inhibitors. Treatment with a cytoplasmic protein
synthesis inhibitor, cycloheximide (5 mg ml−1), inhibited
the induction of active HCO− and CO transport, the
3
2
activities of which remained comparable to those of cells
maintained on 5% CO for 5.5 h. Treatment with the
2
chloroplastic protein synthesis inhibitor, chloramphenicol
(400 mg ml−1), did not inhibit the induction of active
DIC transport in C. kessleri cells acclimating to low CO ,
2
and the O evolution rates measured in the presence and
2
absence of CA were similar to cells acclimating to low
CO with no inhibitor ( Fig. 4).
2
It has been suggested that the accumulation of intermediates of the photorespiratory pathway, possibly phosphoglycolate (Marcus et al., 1983; Suzuki et al., 1990) or
glycolate could act as triggers for the induction of the
CCM in algae. In order to test this hypothesis, high CO 2
grown cells, maintained on high CO , were treated with
2
the photorespiratory pathway inhibitors, 5 mM AOA and
10 mM isonicotinyl hydrazide for 5.5 h. Neither AOA
(Fig. 4) or INH (data not shown) had a stimulatory effect
Fig. 4. The effect of inhibitors of protein synthesis and inhibition of
aminotransferases on the induction of active DIC transport in high
CO -grown Chlorella kessleri cells acclimating for 5.5 h. O evolution
2
2
rates were determined at 50 mM DIC, pH 7.8, and 25 °C with (&) and
without (%) added CA. Values are means ±SE of three to five
experiments. High CO -grown cells were also acclimated to air and to
2
high CO as controls. The dashed line represents the calculated
2
maximum rate of CO formation from 50 mM HCO− at pH 7.8
2
3
and 25 °C.
on the induction of active DIC transport of C. kessleri
cells.
Discussion
C. kessleri cells induce a CCM during acclimation to low
CO in response to a critical dissolved CO concentration
2
2
in the external medium during the acclimation process.
Cells grown under a 5% CO aeration, exhibit a low
2
affinity for DIC and for CO in comparison to low CO 2
2
grown cells, although there is no difference in the maximum rate of photosynthesis between the two growth
conditions ( Table 1). High affinity photosynthesis in low
CO -grown cells was similar to that in other green alga
2
(Matsuda and Colman, 1995a; Sültemeyer et al., 1991;
Mayo et al., 1986; Badger et al., 1980) and to cases where
the CCM is constitutively expressed under all CO concen2
trations as in Chlorella saccharophila and CO -insensitive
2
C. ellipsoidea mutants (Matsuda and Colman, 1996a, b).
Air-grown C. kessleri cells have a high affinity for dissolved CO ( Table 1) and there was an increase in affinity
2
in CO -free-grown cells, indicating that a fully inducible
2
CCM may be responding to an external CO concentra2
tion between ambient and CO -free conditions.
2
Air-grown cells had a high affinity for CO as indicated
2
by the low K CO in the presence of bovine CA which
1/2
2
would maintain a constant CO concentration in the
2
medium ( Table 1). The CO affinity of air-grown and
2
CO -free-grown cells is high, but the increase in CO
2
2
affinity of high-CO cells on the addition of CA indicates
2
the presence of some CO transport activity in these cells
2
( Table 1). In all growth conditions the cells display a
higher affinity for CO than for HCO− ( Table 1). It has
2
3
been reported that air-grown C. ellipsoidea and C. saccharophila had higher affinities for CO in comparison to
2
that for HCO− (Matsuda et al., 1999). C. kessleri showed
3
the same phenomenon, but had affinities that were signi-
1346 Bozzo et al.
ficantly higher for both DIC species (Matsuda et al.,
1999). It appears that C. kessleri utilizes available inorganic carbon better than other Chlorella spp (Matsuda
et al., 1999).
C. kessleri cells fully induce and repress active DIC
transport at critical dissolved CO concentrations which
2
are pH independent (Figs 2, 3). After 5.5 h, DIC transport
is fully induced at about 120 mM dissolved CO and
2
induction reached its maximum at about 0 mM CO . The
2
inflow CO concentrations required to achieve these crit2
ical CO concentrations were 0.4% and 0% CO , respect2
2
ively. Active CO transport in C. kessleri does not seem
2
to be completely repressed under high CO conditions
2
and is similar to the low affinity CO uptake that has
2
been shown to occur in high CO -grown C. reinhardtii
2
cells (Sültemeyer et al., 1989). The low affinity CO
2
transport system is not a characteristic of all green algae,
however; for example, a basal level of CO uptake is
2
absent in high CO -grown C. ellipsoidea cells (Matsuda
2
and Colman, 1995a).
There is a marked difference in the CO concentration
2
triggering the induction of the two uptake systems. In C.
kessleri, bicarbonate transport is induced at a lower CO
2
concentration in comparison to active CO transport.
2
Although high affinity CO transport is induced at 120 mM
2
CO , active bicarbonate uptake remains repressed (Figs
2
2, 3). In C. kessleri cells acclimating to an inflow CO
2
concentration of 0.2% CO , which corresponds to a
2
dissolved CO concentration of 60 mM, active bicarbonate
2
transport is first observed ( Table 2). At this external CO
2
concentration, active CO transport is 30% induced. The
2
derepression of the bicarbonate transport system occurred
at a similar external CO concentration in C. ellipsoidea
2
cells (Matsuda and Colman, 1995b). In comparing the
induction of active DIC transport in C. ellipsoidea with
that in C. kessleri cells, active DIC transport is fully
induced at 35 mM CO in C. ellipsoidea, and this critical
2
CO concentration corresponds to 50% of fully induced
2
active DIC transport in C. kessleri ( Figs 2, 3). The CO
2
concentration range corresponding to the full repression
to the full induction of active DIC transport is wider in
low CO -acclimating C. kessleri, than in C. ellipsoidea
2
cells (Matsuda and Colman, 1995b).
There is also a temporal separation in the induction of
the two active uptake systems. High CO -grown cells
2
transferred rapidly to a medium with a low partial CO
2
pressure derepress active CO transport prior to derepres2
sion of active HCO− transport; high CO -grown C.
3
2
kessleri cells acclimating to air-induced active CO trans2
port within 1 h, whereas active bicarbonate transport
remained repressed ( Fig. 1). A similar phenomenon has
been reported in high CO -grown C. ellipsoidea cells
2
acclimating to air, where active CO transport was
2
induced within 2 h, and active HCO− transport remained
3
repressed (Matsuda and Colman, 1995b).
It has been postulated that the induction of active DIC
transport in microalgae and cyanobacteria is triggered by
the intracellular accumulation of an intermediate of the
photorespiratory pathway (Marcus et al., 1983; Suzuki
et al., 1990; Coleman, 1991; Marek and Spalding, 1991;
Kaplan et al., 1994). This theory is consistent with the
large decrease in CO concentration and increase in O
2
2
concentration which occur when high-CO -grown cells
2
are transferred to air. However, if this mechanism were
pivotal to the induction of active DIC transport during
acclimation to low CO , the induction of the CCM in C.
2
kessleri should only occur in the light. The induction of
active HCO− and CO transport was apparent during
3
2
acclimation to 0.035% CO in darkness (Matsuda et al.,
2
1998; Colman et al., 1998), although the maximum O
2
evolution rate in the bicarbonate and CO transport
2
assays was higher in cells adapting in the presence of
light. The difference in the degree of active transport
between acclimation in the light and in darkness, may be
a modification of the energy-coupled reactions, that might
accompany active DIC transport.
The change in the CO /O concentration ratio in the
2 2
external medium might also provide a signal for CCM
induction, since more substrate will be available for the
oxygenase activity of Rubisco, and hence there will be an
increase in phosphoglycolate and other photorespiratory
pathway intermediates (Marcus et al., 1983). In C. kessleri
cells adapting to 0.035% CO , in the absence of O , there
2
2
is no effect on the degree and rate of induction of CO
2
or HCO− transport (Fig. 1), which suggests that a change
3
in the CO /O ratio in the external medium does not play
2 2
a role in induction of the CCM. The lack of induction
by AOA and INH under high-CO conditions, which
2
allow for an intracellular increase in photorespiratory
pathway intermediates, also demonstrated that a buildup of phosphoglycolate does not serve as the inducing
signal in C. kessleri (Fig. 4). The rationale for using AOA
during acclimation to high CO -grown conditions, is that
2
the inhibition of aminotransferase activity results in an
increase in phosphoglycolate, and this inhibition is independent of the external CO concentration for growth.
2
The induction of an alanine-ketoglutarate aminotransferase in C. reinhardtii cells during acclimation to low CO
2
was reported, but the inhibition of this protein by AOA
did not change the whole cell affinity for CO (Chen
2
et al., 1996).
The inhibition of CCM induction in high CO -grown
2
C. kessleri cells acclimating to low CO in the presence
2
of protein synthesis inhibitors, demonstrates that new
cytoplasmic proteins are made during the induction of
active DIC transport ( Fig. 4). Lack of inhibition by
chloramphenicol, a chloroplastic protein synthesis inhibitor revealed that the induction of a CCM is not chloroplast based. Some researchers have pointed to protein
modification playing a pivotal role in CCM induction.
DIC transport induction in Chlorella
The inhibition of protein kinases reported during the
acclimation of cyanobacteria to low CO , prevented a
2
rapid induction phenomenon (Sültemeyer et al., 1998a, b).
The level of active DIC transport is directly related to
the concentration of external CO , and there is a gradient
2
of active transport activity from fully repressed to fully
induced when comparing cells acclimated to various
external CO concentrations ( Figs 2, 3). The induction
2
of DIC transport is therefore not an all-or-nothing
response as would occur in the case of an internal
metabolic signal, but an induction of transport activity
proportional to an external signal. A continuous exposure
to such a signal is also required to accomplish full
induction of the CCM since it has been reported that
DIC transport is repressed in C. ellipsoidea cells acclimating to low CO when they are transferred back to 5%
2
CO conditions (Matsuda and Colman, 1995b).
2
The induction of the CCM in C. kessleri appears to
occur in response to external CO concentration, and fits
2
the model proposed previously (Matsuda and Colman,
1996a), in which a CO sensor at the Chlorella cell surface
2
surrounded by a high concentration of CO , transduces
2
a signal to repress active DIC transport; and during
acclimation to low CO , there is a signal to derepress
2
active CO and DIC transport. The CO sensor is thought
2
2
to be absent from C. saccharophila UTEX 2469 and some
mutants of C. ellipsoidea (Matsuda and Colman, 1996a),
since they express a constitutive CCM, irrespective of the
dissolved CO concentration in the growth medium
2
(Matsuda and Colman, 1996b).
The presence of a CO sensor on the plasma membrane
2
is not without precedent. A number of haem-based sensors
have been identified in bacteria, which regulate physiological processes in response to the presence of an extracellular inorganic compound. For example, in Bradyrhizobium
japonicum, FixL proteins not bound by O at the haem2
binding domain promote kinase activity during acclimation to hypoxic conditions (Gong et al., 1998). A similar
phenomenon has also been characterized in the photosynthetic bacterium, Rhodospirillum rubrum, which involves
binding of CO to the haem-domain of CooA protein,
promoting oxidation of CO (Shelver et al., 1997). It is
possible that a similar sensor may exist in Chlorella spp,
but bind CO rather than O . It is apparent, however,
2
2
that C. ellipsoidea and C. kessleri cells, limited by the
amount of dissolved CO in the external growth medium,
2
fully induce a CCM in response to a critical concentration
of CO , independent of pH, which requires de novo
2
cytoplasmic protein synthesis.
Acknowledgements
This work was supported by grants from the Natural Sciences
and Engineering Research Council of Canada.
1347
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