Planta (1997) 201: 319–325
Regulation of the mechanism for HCO3) use by the inorganic carbon
level in Porphyra leucosticta Thur. in Le Jolis (Rhodophyta)
Jesús M. Mercado, F. Xavier Niell, Félix L. Figueroa
Departamento do Ecologı́a, Facultad de Ciencias, Universidad de Málaga, Campus Universitario de Teatinos, E-29071 Málaga, Spain
Received: 20 April 1996 / Accepted: 5 August 1996
Abstract. The capacity for HCO3) use by Porphyra
leucosticta Thur. in Le Jolis grown at different concentrations of inorganic carbon (Ci) was investigated. The
use of HCO3) at alkaline pH by P. leucosticta was demonstrated by comparing the O2 evolution rates measured
with the O2 evolution rates theoretically supported by the
CO2 spontaneously formed from HCO3) . Both external
and internal carbonic anhydrase (CA; EC 4.2.1.1) were
implied in HCO3) use during photosynthesis because O2
evolution rates and the increasing pH during photosynthesis were inhibited in the presence of azetazolamide and
ethoxyzolamide (inhibitors for external and total CA
respectively). Both external and internal CA were regulated by the Ci level at which the algae were grown. A high
Ci level produced a reduction in total CA activity and a
low Ci level produced an increase in total CA activity. In
contrast, external CA was increased at low Ci although it
was not affected at high Ci . Parallel to the reduction in
total CA activity at high Ci is a reduction in the affinity for
Ci, as estimated from photosynthesis versus Ci curves,
was found. However, there was no evident relationship
between external CA activity and the capacity for HCO3)
use because the presence of external CA became redundant when P. leucosticta was cultivated at high Ci. Our
results suggest that the system for HCO3) use in P.
leucosticta is composed of different elements that can be
activated or inactivated separately. Two complementary
hypotheses are postulated: (i) internal CA is an absolute
requirement for a functioning Ci-accumulation mechanism; (ii) there is a CO2 transporter that works in
association with external CA.
Key words: Carbonic anhydrase – Inorganic carbon –
Macroalga – pH – Photosynthesis – Porphyra
Abbreviations: AZ = azetazolamide; CA = carbonic anhydrase;
Chl a = chlorophyll a; Ci = inorganic carbon; EZ = 6-ethoxyzolamide; gp = photosynthetic conductance; PQ′ = photosynthetic
quotient; Rubisco = ribulose-1,5-bisphosphate carboxylase/oxygenase
Correspondence to: J.M. Mercado; Fax: 34 (52) 132000
Introduction
The total concentration of inorganic carbon (Ci) in
natural seawater is approx. 2.2 mM. At equilibrium with
air, CO2 represents 8% and HCO3) 92% of the total Ci in
seawater. The CO2 diffusion rate in seawater is low and
about 10 000 times lower than in air. Since the diffusion
rate is so low, the occurrence of purely diffusive entry of
CO2 into the cell in aquatic plants imposes constraints
on the in-vivo rate of photosynthesis as a function of
external CO2 concentration, taking into account the
quantity and kinetics of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). In fact, it has been shown
that the photosynthetic rates of various macroalgae are
not saturated at the Ci concentration of seawater (Surif
and Raven 1989). However, many another macroalgae,
including the red macroalgae, show low rates of
photorespiration and very high affinity for Ci, probably
because of the presence of a carbon-concentrating
mechanism (CCM; Johnston and Raven 1987). It has
been suggested that macroalgal CCMs can be biochemical, as in terrestrial C4 plants (Reiskind and Bowes
1991) and/or biophysical (by means of active transport
of Ci ).
Carbon dioxide is the only source of Ci for some
macroalgae (Holbrook et al. 1988; Surif and Raven 1989;
Maberly 1990). However, the O2 evolution rates of many
other macroalgae in seawater are higher than the maximal rates that theoretically could be supported by the
CO2 formed in the medium by uncatalyzed spontaneous
dehydration of HCO3) . The use of HCO3) by these
macroalgae has been suggested. Several mechanisms of
HCO3) incorporation have been proposed: CO2 uptake
dependent on a dehydration of HCO3) catalyzed by an
external carbonic anhydrase (CA; EC 4.2.1.1), ATPasedependent HCO3) transport, and H+/HCO3) co-transport and OH)/HCO3) antiport systems (Lucas 1983).
The Rhodophyta cover a large range in their ability to
use HCO3) from seawater. A small fraction of the marine
red macroalgae examined by Johnston et al. (1992) and
Maberly (1990) appeared unable to use HCO3); most of
320
these algae, which apparently use only CO2, are subtidal.
Cook et al. (1986) found that HCO3) entry is necessary
to explain the rate of photosynthesis by 15 species of
marine red algae, based on photosynthesis at a faster
rate than uncatalysed conversion of HCO3) to CO2 in
the medium and the absence of external CA; however,
these results do not conflict for any species with those of
Johnston et al. (1992) and Maberly (1990). The presence
of an external CA involved in Ci uptake has been
described in other algae of this group (Giordano and
Maberly 1989; Surif and Raven 1989; Haglund et al.
1992a; Sültemeyer et al. 1993), including Porphyra sp.,
the apparently most efficient HCO3) -user among
Rodophyceae (Axelsson et al. 1991).
The aim of this paper is to describe the role of CA in
carbon assimilation by the red alga Porphyra leucosticta.
The affinity for HCO3) and effect of the inhibitors of CA
when the plants are exposed to low and high Ci
concentrations are described.
Materials and methods
Plant material. Porphyra leucosticta Thur. in Le Jolis was collected
in the supralittoral zone near Lagos (Málaga, Southern Spain)
during winter 1995. It was maintained in natural, nutrient-poor
seawater aerated vigorously (about 3 L air · min)1) at 15 °C under
white fluorescent lamps (F20W; CW Osram, Munich, Germany), at
12 h light per day and a photon fluence rate of 60 lmol · m)2 · s)1
(Figueroa et al. 1995). The photon fluence rate was determined by
means of a quantum spherical PAR sensor (193SB; Li-Cor,
Lincoln, Neb., USA) connected to a radiometer (Li-1000; Licor).
Experimental design. P. leucosticta was cultivated at high, normal
and low Ci concentrations for 7–8 d. Discs (9 mm in diameter)
taken from the thallus were cultured in Plexiglas cylinders
containing 3 L of seawater enriched with Provasoli medium (Starr
and Zeikus 1987) and buffered with Tris 50 mM. For each culture
1.2–1.5 g of alga was used. The different Ci concentrations in the
cultures were obtained by bubbling air with different CO2 concentrations. In the control culture (normal Ci ), seawater was aerated
with air from outside (0.035% CO2). In the high-Ci treatment,
seawater was bubbled with air enriched with 1% CO2. In the low-Ci
treatment, air was passed through a 5 N KOH solution (final CO2
concentration in air less than 0.0001%). Light and temperature
conditions were as described above. Duplicate experiments gave
similar results.
Oxygen evolution and pH change. Oxygen evolution was measured
in small (8–9 ml) temperature-controlled (15–16 °C) seawater
chambers containing oxygen electrodes, at an irradiance of 250
lmol photons · m)2 · s)1. Chambers were equipped with an oxygen
probe (522l; Yellow Springs, Ohio, USA). The suitable agitation of
the medium in the chamber was obtained by a magnetic stirrer. Ten
P. leucosticta discs were transferred to the oxygen-evolution
chamber containing Ci-free synthetic seawater medium buffered
to a pH of 5.6, 8.7 or 9.2 with 50 mM buffer (final concentration).
Biological buffers 2-(N-morpholino)ethanesulfonic acid (Mes) and
Tris, respectively, were used. After a zero net O2 exchange rate,
different amounts of 200 mM NaHCO3 solution were injected into
the chambers in order to create different concentrations of Ci.
Evolution of O2 was recorded for 10–15 min. Values for Km and
Vmax were estimated from the fit to the Michaelis-Menten equation
using the KaleidaGraph program (Abelbeck Software, USA). The
goodness of fit was tested by using least-squares regression analysis.
J.M. Mercado et al.: Regulation by Ci level of CA in Porphyra
Photosynthetic conductance (gp) based on the concentration of Ci
was calculated as the initial slope of Ci saturation curves (Johnston
et al. 1992). According to Figueroa et al (1995), a value of 5.91 for
the fresh weight per unit surface area relationship (mg · FW per
cm2) was used in calculating the O2 evolution rate per surface unit.
Chlorophyll a was extracted in N,N-dimethylformamide (Inskeep
and Bloom 1985).
pH change during photosynthesis was recorded in the oxygenevolution chamber by fitting a pH electrode (Ingold micropH 2001;
Crison Instruments S.A., Barcelona, Spain) equipped with pHmeter (micropH 2001; Crison Instruments S.A.). Oxygen evolution
and pH change were measured simultaneously. The photosynthetic
quotient (PQ′ ) was estimated according to Axelsson (1988).
In order to obtain the pH compensation point, pH-drift
experiments were conducted in the photosynthesis chambers: pH
was measured until there was not further increase (after about
6 h).
The maximum rate of dehydration of HCO3) to CO2 was
calculated for a given salinity, temperature and pH according to
Johnson (1982). The apparent dissociation constants of carbonic
acid in seawater were taken from Riley and Chester (1977); K1 and
K2 at 15 °C were 9.12 ·10)7 and 6.17 · 10)10, respectively. Theoretical O2 production was calculated assuming that algae consumed
CO2 at a rate causing the CO2 concentration to approach zero.
Values for PQ′ estimated from the simultaneous measurements of
change of pH and O2 evolution were used.
Preparation of inhibitors and assay of CA (EC 4.2.1.1) in discs.
6-Ethoxyzolamide (EZ) and azetazolamide (AZ) were used for
these experiments (Sigma-Aldrich Quı́mica S.A., Madrid, Spain). It
is generally assumed that AZ cannot penetrate into the cell and
only inhibits the extracellular CA. The EZ penetrates into the cell
and inhibits external and internal CA. Carbon dioxide-free stock
solutions of the inhibitors were prepared in 0.05 N NaOH to a final
concentration of 50 mM.
Carbonic anhydrase activity was measured potentiometrically
at 0–2 °C by determining the time taken for a linear drop of 1.0 unit
of pH range 8.5–7.5. according to Haglund et al. (1992a). A cuvette
containing 3 ml of buffer (50 mM Tris, 25 mM isoascorbic acid, 25
mM mercaptoethanol, 5 mM EDTA) was used. External CA
activity was measured using seven to ten algal discs (60–90 mg)
transferred directly from the culture to the cuvette. In order to
estimate total CA activity, 10–20 mg of sample was extracted in the
buffer described above and assayed immediately. The reaction was
started by introducing 1 ml of ice-cold CO2 distilled water. One unit
of relative enzyme activity (REA) was defined as (to / tc) ) 1, where
to and tc were the time for pH change of the nonenzymatic (buffer)
and enzymatic reactions, respectively.
Extraction and determination of the amount of Rubisco (EC
4.1.1.39). Samples of 0.2 g FW were powdered in liquid nitrogen
and 1.2 mL of extraction buffer (0.1 M Na2HPO4, 4 mM
Na2EDTA and 2 mM phenylmethylsulfonyl fluoride (PMSF), pH
6.5) were added. The extract was centrifuged at 20 000 · g for 15 min
at 4 °C. The supernatants were subjected to SDS-polyacrylamide
gel electrophoresis (10% separating gel; 5% stacking gel) using the
discontinuous buffer system of Laemmli (1970). All the reagents
and molecular-weight markers were from Sigma-Aldrich Quı́mica
S.A. Purified Rubisco of spinach was used as a standard. Gel
staining and destaining were carried out according to Rintamaki
et al. (1988).
Statistics. The results were expressed as the mean value ± standard deviation (SD). Statistical significances of means were tested
with a model 1 one-way analysis of variance (ANOVA) followed by
a multirange test using Fisher’s protected least significant difference
(Sokal and Rohlf 1981).
J.M. Mercado et al.: Regulation by Ci level of CA in Porphyra
Results
The PQ′ of normal-Ci-grown discs, estimated from
measurements of O2 evolution and change of pH, was
1.04. This value was used to calculate theoretical O2
evolution rates supported by CO2 spontaneously formed
from HCO3). The maximum theoretical rate of CO2
formation in 9 ml of seawater at 15 °C at pH 8.7 was
98.37 nmol O2 · min)1, and at pH 9.2 it was 46.68 nmol
O2 · min)1. O2 evolution rates of discs cultivated under
normal and low Ci were five times higher than the
theoretical CO2-dependent O2 evolution rates at pH 8.7.
At pH 9.2, measured O2 evolution exceeded the theoretical CO2-dependent O2 evolution by six and eight
times in discs grown at normal and low Ci, respectively.
In discs cultivated under high Ci, the ratio was approx. 1
at pH 8.7 and lower than 1 at pH 9.2.
Figure 1 shows the increase in pH in the medium
during photosynthesis by discs of P. leucosticta cultivated at normal Ci. The increase in pH ceased in the dark
and after the addition of AZ, an inhibitor of external
CA. Ethoxyzolamide, an inhibitor of total CA, had a
similar effect (data not shown). Similar patterns were
found for discs grown at low and high Ci.
Table 1 contains the values for external and total CA
activity and pH compensation point. The results show
Fig. 1. Influence of light and AZ, an inhibitor of external CA, on pH
change in the medium caused by photosynthesis of P. leucosticta in
natural seawater. Vertical arrows indicate when the light was turned
off and on, and the addition of AZ Azetazolamide
321
the presence of extracellular and intracellular CA in
P. leucosticta. The external CA activity was significantly
higher (P < 0.001) in the discs cultivated at low Ci.
External CA activities in normal and high Ci did not
differ significantly (P > 0.01). At high Ci, total CA
activity decreased by 50%. In contrast, at low Ci, total
CA activity increased by 90%. The pH compensation
point obtained after 6 h was modified by the concentration of Ci. The lowest pH compensation point was
obtained for discs grown at high Ci. An appreciable
increase in this parameter was obtained at low Ci.
The effect of AZ on oxygen evolution rates at high pH
values is shown in Fig. 2. The photosynthetic rates were
expressed on chlorophyll a (Chl a) basis. The lowest Chl
a content was obtained for discs grown at high Ci
(0.75 ± 0.20 mg Chl a · (gFW))1). The Chl a contents for
discs grown at normal and low Ci were 1.40 ± 0.05
and 1.09 ± 0.05 mg Chl a · (gFW))1, respectively. The
change in pH from 8.7 to 9.2 did not affect photosynthesis by discs grown at low and normal Ci. However, O2
evolution rates by high-Ci-grown discs were reduced at
pH 9.2. Oxygen evolution rates by high-Ci-grown discs
were lower than O2 evolution rates by normal and lowCi-grown discs (P < 0.01) at pH 8.7 as well as at pH 9.2.
The inhibition of photosynthesis at pH 8.7 by AZ was
nearly 80% in normal and low Ci, and almost 50% in
high Ci. A similar effect was found at pH 9.2. 6Ethoxyzolamide completely inhibited photosynthesis in
all the treatments. No effect by AZ or EZ was found at
pH 5.6 (data not shown).
At pH 8.7, the HCO3) concentration is 99% of total
inorganic carbon (TIC) and the CO2 concentration is 10
times the HCO3) concentration. The curves of Cisaturation at pH 8.7 and 5.6 are shown in Fig. 3. The
Km(TIC) values for Ci (Table 2) at pH 8.7 were not
significantly different (P > 0.01) in discs grown at the
different Ci levels. At pH 5.6, the Km(TIC) obtained for
high-Ci-grown discs was lower than that obtained for
normal- and low-Ci-grown-discs. The value for Vmax was
higher for low- and normal-Ci-grown discs than for cells
grown at high Ci (P < 0.01) at pH 8.7 as well as at pH
5.6. The photosynthetic conductance (gp) based on the
concentration of Ci estimated from the initial slope of
Ci-O2 evolution curves at pH 8.7 was increased at low Ci
and decreased at high Ci (P < 0.01). The gp values
estimated from saturating Ci curves at pH 5.6 were not
significantly different (P > 0.01).
Figure 4 shows an electrophoretic analysis of soluble
proteins from homogenates of P. leucosticta cells grown
Table 1. Carbonic anhydrase activity of P. leucosticta discs grown at different levels of Ci. Measurements were performed on either intact
cells (to obtain activity of external CA) or on homogenate of cells (to obtain the total CA activity). Data are mean values (± SD) for n = 8
from two independent experimental series. The pH compensation point was obtained by incubation of discs in the photosynthesis chambers
in natural unbuffered seawater for 6 h. REA = relative enzyme activity
Culture
Low Ci
Normal Ci
High Ci
External CA
activity
(REA · (gFW)
14.41 ± 3.66
6.05 ± 1.57
4.93 ± 0.77
)1
Total CA activity
(REA · (gFW) )1)
pH compensation
point
76.52 ± 11.57
40.09 ± 5.64
20.41 ± 4.10
9.45
9.25
8.88
)
J.M. Mercado et al.: Regulation by Ci level of CA in Porphyra
322
Fig. 2. Oxygen evolution rates in discs of P. leucosticta grown at
different Ci concentrations. Oxygen evolution was measured in natural
seawater buffered at pH 8.7 and 9.2. The effects of AZ and EZ were
determined. Inhibitor concentration was 100 lM. 6-Ethoxyzolamide
inhibited photosynthesis by 100% (data not shown). Values are means
of five to six measurements. Numbers in parentheses give percentage
inhibition by AZ
at low, normal and high Ci concentrations. Each
homogenate was obtained from 200 mg of plant material. The soluble protein concentration varied among the
treatments (data not shown) with a similar pattern to
that of Rubisco. It should be noted that the intensity of
the Rubisco band decreased considerably at high Ci and
was similar for normal and low Ci. These results indicate
that the Rubisco concentration is reduced at high Ci.
Table 2. Rates of oxygen evolution at Ci
saturation (Vmax) and half-saturated rate of
photosynthesis for total inorganic carbon
(Km (TIC)) and CO2 (Km (CO2)) at pH 8.7 and
5.6. Conductance for Ci (gp) was obtained
as the initial slope of photosynthesis-Ci
curves with units of mol · m)2 · s)1 and
mol · m)3, respectively
Fig. 3. Effect of Ci on O2 evolution rates of P. leucosticta discs grown
in normal, low and high Ci. The experiment was performed at pH 8.7
and 5.6. Synthetic seawater free of Ci was buffered with Tris and Mes
(50 mM final concentration) and different concentrations of Ci were
achieved by injecting solutions of 200 mM NaHCO3 into the
chambers. The photon fluence rate was 250 lmol photons · m)2s)1
(solid circles, cells grown in normal Ci open circles, cells grown in low
Ci open squares, cells grown in high Ci)
Discussion
The use of HCO3) by P. leucosticta. Implication of
external CA in photosynthesis at alkaline pH. From our
results the use of HCO3) by P. leucosticta can be inferred.
The value of the pH compensation point obtained with
air-grown discs of P. leucosticta also supports this
conclusion. Based on data obtained using the pH-drift
Km (TIC)
(lM)
Km (CO2)
(lM)
Vmax
(lmol O2 · mg Chl a))1 · h)1)
gp
(10)6m · s)1)
pH 8.7
Low Ci
Normal Ci
High Ci
240 ± 50
610 ± 90
277 ± 40
1.21 ± 0.13
3.28 ± 0.05
1.50 ± 0.22
125.75 ± 15.85
165.50 ± 9.70
35.89 ± 1.45
8.60
4.45
2.12
pH 5.6
Low Ci
Normal Ci
High Ci
24.31 ± 4.37
26.87 ± 4.83
5.51 ± 0.58
21.18 ± 3.10
23.41 ± 3.43
4.80 ± 0.50
152.63 ± 20.67
242.02 ± 24.24
29.29 ± 0.46
147.86
103.08
87.30
J.M. Mercado et al.: Regulation by Ci level of CA in Porphyra
Fig. 4. Protein analysis of homogenates of P. leucosticta grown at
normal low and high Ci. The extracts were subjected to SDSpolyacrylamide gel electrophoresis (10% separating gel; 5% stacking
gel) using the discontinuous buffer system of Laemmli (1970) and gels
were stained with Coomassie blue. Lane 1, homogenate from high-Cigrown discs; lane 2, homogenate from low-Ci-grown discs; lane 3,
homogenate from normal-Ci-grown discs; lane 4, purified Rubisco of
spinach that was used as a standard; Lane 5, molecular-weight
markers. The arrowhead indicates the Rubisco band
technique, several papers have reported the ability of
marine macroalgae to use HCO3) (Axelsson and
Uusitalo 1988; Surif and Raven 1989). Maberly (1990)
found that the most effective HCO3)-user algae increased
the pH of the medium to over 10.5 and depleted the
concentration of Ci to less than 50%. In contrast, those
species restricted to utilising CO2 did not increase the pH
above 9.0. The value of the pH-compensation point for
normal-Ci-grown P. leucosticta estimated in these
experiments demonstrates that this alga can use HCO3)
as a source of Ci. As mentioned above, evidence for the
ability to use HCO3) has been reported among Rhodophyta with the exception of a few species (Cook et
al.1986; Giordano and Maberly 1989; Maberly 1990;
Johnston et al. 1992). Both the ability to use HCO3) and
external CA activity have been described in different
species of Porphyra (with the exception of P. schizophylla
according to Jolliffe and Tregunna 1970). To our
knowledge, there have not been previous reports about
the presence of external CA activity in P. leucosticta
Both AZ and EZ inhibited O2 evolution by air-grown
P. leucosticta discs at pH 8.7 but not at pH 5.6. This
provides evidence that external CA has a role in HCO3)
acquisition at alkaline pH in P. leucosticta. This
hypothesis is supported by our observation that the
photosynthetic increase of pH is inhibited by AZ. The
pH change in natural seawater by P. leucosticta thalli
depends on photosynthesis because this increase was
observed in the light and was stopped in darkness.
Haglund et al. (1992b) found that the inhibition of
external CA reduced the photosynthetic increase of pH
at alkaline pH in F. serratus and L. Saccharina.
According to these authors, the inhibition of photosynthetic alkalinization by AZ is evidence that an external
CA is involved in the mechanism for Ci uptake in
P. leucosticta.
The role external CA in Ci use. Two functions of external
CA can be presumed in macroalgae: (i) the conversion of
HCO3) to CO2 that is taken up by the macroalga
323
(Haglund et al 1992b); (ii) participation in active
transport of HCO3) across the plasma membrane by
conversion of CO2 to HCO3). A way of determining the
true role of the CA-system in acquisition of Ci by algae is
to check the effect of Ci level on both CA activity and
capacity for HCO3) use. A regulation of both external
and internal CA by the level of external CO2 has been
reported in microalgae (Nelson et al. 1969; Muñoz and
Merrett 1988; Ramazanov and Cárdenas 1992; Sültemeyer et al. 1993). Similar results have been described for
macroalgae (Johnston and Raven 1990; Björk et al.
1993) although equivalent data for red macroalgae are
relatively sparse (Garcı́a-Sánchez et al. 1994). In general,
a correlation between external CA activity and HCO3)
use was found by these authors. In our report it is shown
that external CA activity P. leucosticta was increased at
low Ci but that there was no reduction in external CA
activity under high Ci. The mechanism for using HCO3)
in P. leucosticta is activated at low and normal Ci levels
but, in contrast, is partially inactivated at high Ci.
Photosynthetic conductance based on the concentration
of Ci, the pH compensation point and photosynthetic
rates at pH 9.2 were dramatically reduced at high Ci and
these were increased at low Ci. It might look paradoxical
that the Km(TIC) at pH 8.7 was not reduced at high Ci.
However, it must be noted that the suitability of the halfsaturation constant as a measurement of the ability to
extract Ci from seawater can be questioned. According
to Raven and Johnston (1991), the Ci kinetic can be
viewed as two limiting steps: (i) the maximum velocity
limited by the rate of electron transport which controls
the rate of resupply of Rubisco for carboxylation and (ii)
the initial slope controlled by the activity and CO2
affinity of Rubisco and the supply of Ci. A decreasing
Rubisco concentration can produce a change in the Km
for TIC without a change in the affinity for Ci. In that
sense, we have found that the different treatments
produced changes in Rubisco concentration. The decreasing Rubisco concentration at high Ci can explain
the low values of Vmax and Km(TIC) obtained. The
inherent problems in interpreting the Km(TIC) values can
be overcome using the initial slope of photosynthesis-Ci
curves (gp).
From our results, there is no evident relationship
between external CA activity and the capacity for using
HCO3) in P. leucosticta, although this enzyme is
necessary for HCO3) use. A similar result was found
by Palmqvist et al. (1990) for cells of the microalga
Chlamydomonas reinhardii which showed a higher Ciaffinity when grown at low Ci than cells grown at normal
Ci although the external CA was equal in cells from the
two growth conditions. The increased affinity was
interpreted as resulting from the activation of a plasma-membrane-located HCO3)-pump under conditions
where external-CA-mediated diffusion of CO2 was
insufficient. In that sense, our results suggest that the
system for HCO3) concentrating in P. leucosticta is
composed of different elements that could be activated
or inactivated independently. Although HCO3) use is
linked to external CA in P. leucosticta, other components are essential requirements for a HCO3)-concen-
324
trating system. We have no evidence for postulating a
HCO3) transporter in P. leucosticta because photosynthesis by low-Ci-grown discs was almost completely
inhibited by AZ. Two complementary hypotheses can be
postulated.
(i) Internal CA is an absolute requirement for a
functioning Ci-accumulation mechanism. The reduction
in internal CA activity can explain the loss of capacity of
HCO3) use at high Ci. However, if internal CA was
really important for photosynthesis, an inhibitory effect
of EZ would be expected even under acidic conditions.
(ii) There is a CO2 transporter which works in
association with external CA. This transporter could be
inactivated at high Ci. Our data indicated that external
CA catalyzes the conversion HCO3) to CO2 at the surface
of the plasmalemma. Carbon dioxide could penetrate
into cells both by diffusion (Gutknecht et al. 1977) and by
an active CO2-transport mechanism (Sültemeyer et al.
1989). The presence of this enzyme could become
redundant when the CO2-pump is inactivated. The
simultaneous presence of external CA and direct uptake
of HCO3) and/or CO2 has been proposed in microalgae
(Badger and Price 1994; Palmqvist et al. 1994).
A way to investigate the presence of a CO2-pump in
P. leucosticta would be to determine the energy cost of
the CO2 accumulation at alkaline pH. If photosynthesis
depends on both the CO2 gradient created by external
CA and the CO2 entries by difusion, then the energy cost
could be lower than if an additional pump was activated.
An indication of the energy cost in the mechanism for
CO2 accumulation at alkaline pH in P. leucosticta is that
the Ci-saturated photosynthesis by low- and normal-Cigrown discs at acidic pH was higher than at alkaline pH.
This preference for CO2 as a carbon source was also
pointed out by Haglund et al. (1992a) for Gracilaria
tenuistipitata.
In conclusion, we suggest that external CA catalyzes the
external dehydration of HCO3) followed by CO2
uptake. Uptake of CO2 could be a transporter-mediated
process that is regulated by Ci level. The activation of
both CA and a hypothetical CO2-pump could explain
the high affinity for Ci in P. leucosticta grown at low Ci.
Further experiments are necessary in order to validate
this hypothesis.
This work was financed by the Ministry of Education and Science
of Spain (MESS). J.M. Mercado is supported by a grant from MESS.
References
Axelsson L (1988) Changes in pH as a measure of photosynthesis
by marine macroalgae. Mar Biol 97: 287–294
Axelsson L, Uusitalo J (1988) Carbon acquisition strategies for
marine macroalgae. I. Utilization of proton exchanges visualized during photosynthesis in a closed system. Mar Biol 97:
295–300
Axelsson L, Uusitalo J, Ryberg H (1991) Mechanisms for concentrating and storage of inorganic carbon in marine macroalgae
In: Garcı́a-Reina G, Pedersén M (eds) Seaweed cellular
biotechnology. Universided de las Palmas de Gran Canaria,
pp 185–198
J.M. Mercado et al.: Regulation by Ci level of CA in Porphyra
Badger MR, Price GD (1994) The role of carbonic anhydrase in
photosynthesis. Annu Rev Plant Physiol Plant Mol Biol 45:
369–392
Björk M, Haglund K, Ramazanov Z, Pedersén M (1993) Inducible
mechanisms for HCO3) utilization and repression of photorespiration in protoplast and thalli of three species of Ulva
(Chlorophyta). J Phycol 29: 166–173
Cook CM, Lanaras T, Colman B (1986) Evidence for bicarbonate
transport in species of red and brown macrophytic marine
algae. J Exp Bot 37: 977–984
Figueroa FL, Aguilera J, Niell FX (1995) Red and blue light
regulation of growth and photosynthetic metabolism in
porphyra umbilicalist (Bangiales, Rhodophyta). Eur J Phycol
30: 11–18
Garcı́a-Sánchez MJ, Fernández JA, Niell X (1994) Effect of
inorganic carbon supply on the photosynthetic physiology of
Gracilaria tenuistipitata. Planta 194: 55–61
Giordano M, Maberly SC (1989) Distribution of carbonic anhydrase in British marine macroalgae. Oecologia 81: 534–539
Gutknecht J, Bisson MA, Tosteson FC (1977) Diffusion of carbon
dioxide through lipid bilayer membranes. Effects of carbonic
anhydrase, bicarbonate and unstirred layers. J Gen Physiol 69:
779–794
Haglund K, Björk M, Ramazanov Z, Garcı́a-Reina G, Pedersén M
(1992a) Role of carbonic anhydrase in photosynthesis and
inorganic-carbon assimilation in the red alga Gracilaria tenuistipitata. Planta 187: 275–281
Haglund K, Ramazanov Z, Mtolera M, Pedersén M (1992b) Role
of external carbonic anhydrase in light-dependent alkalization
by Fucus serratus L. and Laminaria saccharina (L.) Lamour.
(Phaeophyta). Planta 188: 1–6
Holbrook GP, Berr S, Spencer WE, Reiskind JB, Davis JS, Bowes
G (1988) Photosynthesis in marine macroalgae: evidence for
carbon limitation. Can J Bot 66: 577–582
Inskeep W, Bloom PR (1985) Extinction coefficients of Chlorophyll
a and b in N,N-dimethylformamide and 80% acetone. Plant
Physiol 77: 483–485
Johnson KS (1982) Carbon dioxide hydration and dehydration
kinetics in seawater. Limnol Oceanogr 27: 849–855
Johnston AM, Raven JA (1987) The C4-like characteristics of the
intertidal macroalga Ascophyllum nodosum (L.) Le Jolis (Fucales, Phaephyta). Phycologia 26: 159–166
Johnston AM, Raven JA (1990) Effects of culture in high CO2 on
the photosynthetic physiology of Fucus serratus. Bryo phycol J
25: 75–82
Johnston AM, Maberly SC, Raven JA (1992) The acquisition of
inorganic carbon by four red macroalgae. Oecologia 92: 317–
326
Jolliffe EA, Tregunna EB (1970) Studies on HCO3) ion uptake
during photosynthesis in benthic marine algae. Phycologia 9:
293–303
Laemmli UK (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4, Nature 277: 680–685
Lucas WJ (1983) Photosynthetic assimilation of exogenous HCO3)
by aquatic plants. Annu Rev Plant Physiol 34: 71–104
Maberly SC (1990) Exogenous sources of inorganic carbon for
photosynthesis by marine macroalgae. J Phycol 26: 439–449
Muñoz J, Merret MJ (1988) Inorganic carbon uptake in a smallcelled eukaryotic microalga. In: Rogers D, Gallon J (eds)
Biochemistry of the algae and cyanobacteria (Proc Phytochem
Soc Europe, vol 28, abstr.). Oxford University Press, Oxford,
p 43
Nelson EB, Celedella A, Tolbert NE (1969) Carbonic anhydrase
levels in Chlamydomonas. Phytochemistry 8: 2305–2306
Palmqvist K, Ramazanov Z, Samuelsson G (1990) The role of
extracellular carbonic anhydrase for accumulation of inorganic
carbon in the green alga Chlamydomonas reinhardii. A comparison between wild-type and cell-wall-less mutant cells. Physiol
Plant 80: 267–276
Palmqvist K, Yu J-W, Badger MR (1994) Carbonic anhydrase
activity and inorganic carbon fluxes in low- and high-Ci cells of
J.M. Mercado et al.: Regulation by Ci level of CA in Porphyra
Chlamydomonas reinhardtii and Scenedesmus obliquus. Physiol
Plant 90: 537–547
Ramazanov Z, Cárdenas J (1992) Inorganic carbon transport
across cell compartments of the halotolerant alga Dunaliella
salina Physiol Plant 85: 121–128
Raven JA, Johnston AM (1991) Mechanisms of inorganic-carbon
acquisition in marine phytoplankton and their implications for
the use of other resources. Limnol Oceanogr 36: 1701–1714
Reiskind JB, Bowes G (1991) The role of phosphoenolpyruvate
carboxykinase in a marine macroalga with C4-like photosynthetic characteristics. Proc Natl Acad Sci USA 88: 2883–2887
Riley JP, Chester R (1977) Introduction to marine chemistry.
Academic Press London, New York
Rintamaki EA, Keys AJ, Parry MA (1988) Comparison of the
specific activity of ribulose-1,5-bis-phospate carboxylase/oxygenase from some C3 and C4 plants. Physiol Plant 74: 326–331
325
Sokal PR, Rohlf FJ (1981) Biometry: the principles and practice of
statistics in biological research, 2nd edn. W.H. Freeman San
Francisco
Starr R, Zeikus JA (1987) UTEX: the culture collection of algae
at the University of Texas at Austin. J Phycol 23 [Suppl]: 1–
47
Sültemeyer D, Miller AG, Espie GS, Fock HP, Canvin DT (1989)
Active CO2 transport by the green alga Chlamydomonas
reinhardtii. Plant Physiol 89: 1213–1219
Sültemeyer D, Schmidt C, Heinrich PF (1993) Carbonic anhydrase
in higher plants and aquatic microorganisms. Physiol Plant 88:
179–190
Surif MB, Raven JA (1989) Exogenous inorganic carbon sources
for photosynthesis in seawater by members of the Fucales and
the Laminariales (Phaeophyta): ecological and taxonomic
implications. Oecologia 78: 97–105