全 文 ：Received 31 Mar. 2004 Accepted 19 Jul. 2004
Supported by the Chinese “863” Project (2002AA628090), the National Natural Science Foundation of China (30300050, 39830060),
Science Technology Bureau of Guangdong Province (2002C32705) and the Natural Science Foundation of Guangdong Province (032048,
04010990).
* Author for correspondence. E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (10): 1178-1185
Comparative Mechanisms of Photosynthetic Carbon Acquisition
in Hizikia fusiforme Under Submersed and Emersed Conditions
ZOU Ding-Hui1, GAO Kun-Shan1, 2*
(1. Science Center, Marine Biology Institute, Shantou University, Shantou 515063, China;
2. Institute of Hydrobiology, The Chinese Academy of Sciences, Wuhan 430072, China)
Abstract: The economic seaweed Hizikia fusiforme (Harv.) Okamura (Sargassaceae, Phaeophyta) usually
experiences periodical exposures to air at low tide. Photosynthetic carbon acquisition mechanisms were
comparatively studied under submersed and emersed conditions in order to establish a general under-
standing of its photosynthetic characteristics associated with tidal cycles. When submersed in seawater,
H. fusiforme was capable of acquiring HCO3- as a source of inorganic carbon (Ci) to drive photosynthesis,
while emersed and exposed to air, it used atmospheric CO2 for photosynthesis. The pH changes surrounding
the H. fusiforme fronds had less influence on the photosynthetic rates under emersed condition than
under submersed condition. When the pH was as high as 10.0, emersed H. fusiforme could photosynthesize
efficiently, but the submersed alga exhibited very poor photosynthesis. Extracellular carbonic anhydrase
(CA) played an important role in the photosynthetic acquisitions of exogenous Ci in water as well as in air.
Both the concentrations of dissolved inorganic carbon in general seawater and CO2 in air were
demonstrated to limit the photosynthesis of H. fusiforme, which was sensitive to O2. It appeared that the
exogenous carbon acquisition system, being dependent of external CA activity, operates in a way not
enough to raise intracellular CO2 level to prevent photorespiration. The inability of H. fusiforme to achieve
its maximum photosynthetic rate at the current ambient Ci levels under both submersed and emersed
conditions suggested that the yield of aquaculture for this economic species would respond profitably to
future increases in CO2 concentration in the sea and air.
Key words: Hizikia fusiforme ; photosynthesis; inorganic carbon; carbonic anhydrase; submersion;
emersion; tide cycle
The intertidal seaweeds spend alternatively part of their
time in atmosphere and part in seawater throughout the
day with the fluctuation of tidal level. They therefore un-
dergo two very distinct environmental conditions for pho-
tosynthesis and growth. It is of general interest to study
the physiology of intertidal seaweeds when considering
how to deal with the high frequency cycles of the aquatic
and aerial conditions (Raven, 1999; Zou and Gao, 2002a).
When the tide is high, the intertidal seaweeds are sub-
mersed in seawater and exposed to two potential sources
of exogenous carbon for photosynthesis: dissolved CO2
and bicarbonate (HCO3-). In air-equilibrium natural
seawater, at normal pH 8.2 and 20 oC, the bulk of total dis-
solved inorganic carbon (DIC) is HCO3- (ca. 2.0 mmol/L),
and CO2 (only 12 mmol/L) is less than 1 % of the total DIC.
It is reported that a large number of seaweeds have devel-
oped mechanisms that permit the acquisition of HCO3- pool
in seawater during photosynthesis (Raven, 1997; Larsson
and Axelsson, 1999; Zou and Gao, 2001). Paradoxically,
intertidal seaweeds, which are exposed to atmospheric CO2
periodically during emersion at low tide, seem to acquire
HCO3- in seawater more efficiently than those growing in
the subtidal zone (Maberly, 1990; Mercado et al., 1998). An
immediate change in the “CO2” supply for intertidal
seaweeds will take place when they get out of the water at
low tide, although a seawater film usually retains the algal
thalli surface due to their viscosity and hydrophilia. The
exact effect of this change in CO2-supply on the photosyn-
thetic rates of intertidal seaweeds is waiting for being fully
established. It appears that CO2 will become limiting for
photosynthesis more often for the seaweeds under emersed
condition than under submersed condition (Gao et al., 1999;
Raven, 1999; Zou and Gao, 2002b; Zou and Gao, 2004).
Hizikia fusiforme, belonging to Sargassaceae
(Phaeophyta), is distributed uniquely in the west-northern
parts of the coast of the Pacific. It has traditionally been
used as a food delicacy in China, Japan and Korea (Zhang
et al., 2002). Suzuki et al. (1996) showed that Hizikia
ZOU Ding-Hui et al.: Comparative Mechanisms of Photosynthetic Carbon Acquisition in Hizikia fusiforme Under Submersed
and Emersed Conditions 1179
contained higher soluble dietary fiber than other seaweeds.
The extract from this seaweed has an immunomodulating
activity on human, which might be useful for clinical appli-
cation to treat diseases (Shan et al., 1999; Katayama et al.,
2002). Additionally, H. fusiforme is an important raw mate-
rial for alginates production. It now becomes one of the
potential important species for seaweed cultivation, owing
to its high commercial value and market demand (Zhang et
al., 2002). A large number of studies have been carried out
on its life history (Park et al., 1995; Ruan and Xu, 2001) and
cultivation technique (Hwang et al., 1997; Li, 2001).
However, the photosynthetic characteristics of H. fusiforme
have been less studied (Zou et al., 2003). H. fusiforme is
distributed at lower parts of the intertidal zone, frequently
spending a part of tidal cycles in the emersed state. The aim
of this study is to compare its photosynthetic strategies
for exogenous inorganic carbon acquisition under sub-
mersed and emersed conditions, in order to establish a gen-
eral knowledge about its physiological behavior associ-
ated with tidal cycles.
1 Materials and Methods
1.1 Algal materials
Hizikia fusiforme (Harv.) Okamura was collected from
lower intertidal rocks along the coast of Nanao, Shantou,
China when the tide went out. Samples sealed in a plastic
bag with some seawater were transported to the laboratory
in an insulated cooler (ca. 5 oC) within 4 h. The material was
maintained in a glass aquarium tank containing filtered natu-
ral seawater (salinity ca. 33 ‰) under 100 mmol.m-2.s-1
(PAR, 400-700 nm) illuminated by fluorescent tubes for 14
h out of each 24 h and at room temperature (18-22 oC). The
seawater was aerated vigorously and was renewed daily.
Experiments were conducted within a period of 5-d labora-
tory maintenance for each collection, during which the al-
gal material showed stable photosynthetic activity. After
this period, the remains were abandoned and fresh material
was collected again.
1.2 Effects of pH and inhibitor on photosynthetic rates
Buffered natural seawater of varied pH values with or
without the addition of acetazolamide (AZ, 100 mmol/L of
final concentration) were prepared. Different pH values were
obtained by adding a known amount of biological buffers
(Sigma) to give final concentrations of 20 mmol/L. TRIS
was used for buffering pH 8.2 (a pH value representative of
that in natural seawater) and 9.0, and CAPS for pH 10.0. AZ
stock solutions prepared with 40 mmol/L NaOH, were added
into the buffers to the final concentrations of 100 mmol/L.
AZ is known as a relatively membrane-impermeable
inhibitor of extracellular carbonic anhydrase (CA) activity
(Axelsson et al., 1999; Moroney et al., 2001).
Photosynthetic rates of submersed plants were mea-
sured as oxygen evolution using a Biological Oxygen Moni-
tor (YSI Model 5300, USA) at 20 oC and at saturating pho-
ton flux density of 500 mmol.m-2.s-1. The oxygen elec-
trode was held in a temperature-controlled chamber. The
fronds of H. fusiforme were cut into small segments (0.5-
0.7 cm length) with a shape razor blade and incubated in
seawater under 500 mmol.m-2.s-1 and 20 oC for at least 2 h
before the measurements. This pre-treatment aimed to mini-
mize the possible effect of cutting damage (wound
respiration). Segments of H. fusiforme of about 0.3 g FW
were incubated in the reaction chamber with 10 mL of buff-
ered seawater that was magnetically stirred, and the linear
O2 evolution versus time was recorded.
The emersed photosynthetic rates were determined as
CO2 uptake with an infrared gas analyzer (LCA-4, Analyti-
cal Development Company Ltd., UK) in an open circuit un-
der the same light/temperature conditions as for the sub-
mersed samples. Before introducing into the photosynthetic
leaf chamber, the samples were respectively immersed in
above seawater buffers for 30 min, aiming to adjust the pH
value within the surface water film surrounding the fronds
when the algal samples were emersed. The buffered
seawaters used were in equilibrium with atmosphere in terms
of CO2. Thus, in our photosynthesis-determining system,
the difference of CO2 concentration between the inlet and
outlet of the assimilation chamber was due to CO2 uptake
by the algal photosynthesis. The rate of CO2 uptake (Pn)
(mmol CO2.g-1FW.h-1) was calculated as follows: Pn = DC
×F×60×273 / ((273+T)×22.4×FW), where DC is the
difference in CO2 concentration (mL/L) between the inlet
and outlet air; F, the gas flow rate (L/min); T, temperature
(oC); FW, fresh weight (g).
1.3 Inorganic carbon-dependent photosynthetic rates
DIC-free seawater was prepared by removing inorganic
carbon (Ci) from the natural seawater by lowering pH to
less than 4.0 with 0.5 mol/L HCl and sparging with pure N2
gas for 2 h at least. A known amount of TRIS (Sigma) was
added to give a final concentration of 20 mmol/L, and the
pH was then adjusted to 8.2 with freshly prepared 0.5 mol/
L NaOH and 0.5 mol/L HCl. All manipulations were carried
out under N2. Segments of H. fusiforme of about 0.3 g FW
were incubated in the reaction chamber with 10 mL of buff-
ered DIC-free seawater. The algae were left to photosyn-
thesize to deplete the possible Ci present in the medium
and in the algal cells till no further O2 evolved, which took
about 20 min. Aliquots of NaHCO3 stock solution were then
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041180
injected into the chamber in order to create the appropriate
final concentrations of Ci in the reaction medium. O2 evolu-
tion was recorded after addition of NaHCO3. Additionally,
the Ci-dependent O2 evolution (i.e. P-C response curve)
was carried out with the presence of AZ (100 mmol/L).
The P-C response curve was also determined in air
(under emersed condition). Samples were pretreated in buff-
ered seawater (Tris 20 mmol/L, pH 8.2) with or without the
addition of AZ (100 mmol/L of final concentration) for 30
min. Photosynthetic CO2 uptake was then determined at
different CO2 concentrations (over the range of 2.6-62.4
mmol/L). CO2 in the ambient air was removed to varied de-
grees by pumping it through a soda lime column to obtain
lower concentrations of CO2. Concentrations of CO2 higher
than ambient air were obtained by injecting pure CO2 be-
fore pumping ambient air into an air bag (1 m3). The air bags
were used to maintain constant CO2 supply.
1.4 Oxygen sensitivity
Photosynthetic O2 evolution rate of submersed samples
was respectively measured at two levels of O2, i.e. less than
30% of air-equilibrated level of O2 (low O2) which was
achieved by bubbling N2 gas into the reaction chamber,
and 100% of air-equilibration concentration of O2 (ambient
O2). Similarly, the emersed photosynthetic CO2 uptake was
respectively examined under normal atmosphere (ambient
O2, 21% of O2 concentration) and under atmosphere with
low O2 concentration (< 6%) which was obtained by pump-
ing air through a Na2S2O3 solution. The O2 concentration
in air and in water were examined by the Infrared Gas Ana-
lyzer (CGT-7000, Shimadzu Corporation, Japan) and the Bio-
logical Oxygen Monitor (YSI Model 5300, USA)
respectively.
1.5 Calculations of the photosynthetic parameters and
theoretical photosynthetic rates
Ci-saturated maximal rate of photosynthesis (Vmax) and
half-saturation constant (K0.5, the inorganic carbon con-
centration required to give half of Vmax) were estimated by
fitting the P-C curve to the Michaelis-Menten equation.
The maximum rates of CO2 supply derived from spontane-
ous (uncatalysed) dehydration of HCO3- in seawater were
calculated according to Matsuda et al. (2001) as the theo-
retical rates of CO2 supply for photosynthesis. The pre-
sumption was adopted that the algal samples consumed
CO2 at a rate causing the CO2 concentration in seawater
surrounding the algae to approach zero. This gave a theo-
retical maximal rate of uncatalysed conversion of CO2 from
HCO3- in seawater. The flux of CO2 across the surface sea-
water film under emersed condition was not considered.
The volume of the surface water film surrounding the algal
samples was estimated as a mass difference before and
after blotting off the superficial water. The value obtained
from this procedure was (0.6± 0.2) mL per gram fresh
weight of alga sample. The theoretical rate of CO2 supply
(d(CO2)/dt) was calculated by the following equations: d
(CO2)/dt=K1×(DIC)/A+K3×(DIC)×[H+]/KH2CO3/A, and
A=1+ [H+]/K1+K2/[H+], where [DIC] is the concentration of
dissolved inorganic carbon in seawater. K1 and K3 are the
rate constants of reactions HCO3-→ CO2＋ OH- and
H2CO3→CO2＋H2O, respectively. KH2CO3 and K2 repre-
sent respectively the dissociation constants of the reac-
tions H＋＋ HCO3- ←→ H2CO3 and H＋＋ CO32-←→
HCO3-. The values of K1, K3, KH2CO3 and K2 are according
to Johnson (1982) and Stumm and Morgan (1996). Photo-
synthetic rates based on O2 evolution or CO2 uptake were
compared by assuming the photosynthetic quotient of 1.0.
1.6 Statistics
The data were expressed as the mean values ± SE (n
≥ 3). Statistical significance of the data was tested with
ANOVA or t-test at P<0.05.
2 Results
2.1 Effects of pH and AZ on the photosynthetic rates
under submersed and emersed conditions
Figure 1 shows the effects of pH and AZ on photosyn-
thetic rates of H. fusiforme fronds under submersed and
Fig.1. Effects of pH and acetazolamide (AZ) on net photosyn-
thetic rates (Pn) of Hizikia fusiforme under submersed and emersed
conditions.
ZOU Ding-Hui et al.: Comparative Mechanisms of Photosynthetic Carbon Acquisition in Hizikia fusiforme Under Submersed
and Emersed Conditions 1181
emersed conditions. Photosynthetic O2 evolution of sub-
mersed plants was reduced drastically as the pH in the
seawater increased from 8.2 to 10.0. O2 evolution at pH 9.0
and 10.0 was reduced by 54.4 % and 90.5 %, respectively,
compared to that at pH 8.2. By contrast, there was only a
slight decrease in photosynthetic CO2 uptake of emersed
plants with the increasing pH in the surface water film sur-
rounding H. fusiforme fronds. There was no significant dif-
ference (P > 0.1) in CO2 uptake between pH at 8.2 and 9.0.
CO2 uptake at 10.0 was reduced by 44.5% compared to that
at pH 8.2. It was shown that the photosynthetic rate was
significantly (P < 0.01) greater at pH 8.2, but was conspicu-
ously (P < 0.01) lower at pH 10.0, in submersed plants than
in emersed plants. AZ remarkably (P < 0.01) inhibited the
photosynthetic rate at all the pH values tested for both
submersed plants and emersed plants. The inhibitory ef-
fect of AZ ranged from 76 % to 82 % in water, but from 87 %
to 68 % in air, when the pH value raised from 8.2 to 10.0.
In order to test whether or not the CO2 supplies derived
from uncatalysed spontaneous dehydration of HCO3- in
seawater medium surrounding the algal fronds were enough
to support the measured photosynthetic rates of O2 evolu-
tion by emersed plants or CO2 uptake by emersed plants,
respectively, the observed rates of photosynthesis were
compared with the theoretical rates (Table 1). The observed
rates at all the pHs tested for the submersed plants ex-
ceeded (P < 0.01) those supported solely by the spontane-
ous CO2 formation. This indicated that submersed H.
fusiforme frond was capable of using external HCO3- as a
source of Ci to drive photosynthetic O2 evolution. However,
the theoretical rate was fast enough to account for the ob-
served rate of photosynthetic O2 evolution at pH 8.2 with
the presence of AZ. Under emersed condition, the ratios of
observed to theoretical rates were much higher than 1.0,
and those increased by two orders of magnitude with in-
creasing pH from 8.2 to 10.0. The ratios in case of AZ were
also much greater than 1.0 and dramatically increased with
increasing pH, though the values of these ratios were
reduced compared to the controls. As the CO2 fluxes from
the atmosphere to the fronds were not taken into account
in calculating the theoretical values, it could be inferred
that the atmospheric CO2 was the predominant source of Ci
driving the photosynthesis of H. fusiforme fronds under
emersed condition.
2.2 The dependence to exogenous inorganic carbon for
photosynthetic rates under submersed and emersed con-
ditions
Effects of external inorganic carbon concentrations on
net photosynthetic rate with or without AZ are shown in
Fig.2 for the submersed H. fusiforme fronds and in Fig.3 for
the emersed fronds. The photosynthesis was far from satu-
rated with ambient Ci levels under both submersed and
emersed conditions. This was in accordance with the high
K0.5 values in water as well as in air (Table 2). Saturating Ci
level seemed to be reached at about 8.8 mmol/L for the
submersed plants. It appeared that the emersed photosyn-
thetic rate was not to be saturated over the range of CO2 in
air (2.6-62.4 mmol/L) used in the experiments. Though the
Table 1 Ratios of measured to theoretically calculated photo-
synthetic rates at different pH values for Hizikia fusiforme under
submersed and emersed conditions
pH 8.2 pH 9.0 pH 10.0
Water
Control 1.5± 0.2 10.0± 1.5 55.3± 6.2
+AZ 0.4± 0.1 1.9± 0.2 9.8± 12.8
Air
Control 36.3± 2.4 424.0± 60.6 7 699.0± 2 005.6
+AZ 4.9± 0.2 75.3± 8.0 2 450.0± 506.2
AZ, aeetazola mide.
Fig.2. Photosynthetic oxygen evolution rate as a function of
dissolved inorganic carbon (DIC) concentration in seawater for
the submersed Hizikia fusiforme with or without the presence of
acetazolamide (AZ).
Table 2 The inorganic carbon-saturated maximum photosyn-
thetic rates (Vmax) and the apparent half-saturation constant
(K0.5) in Hizikia fusiforme under submersed and emersed conditions
Vmax K0.5 (DIC) K0.5 (CO2)
(mmol O2 or CO2.g-1 (mmol/L) (mmol/L)
FW.h -1)
Water
Control 44.7± 13.1 2.72± 0.91 14.8± 5.0
+AZ 11.2± 1.3 3.88± 1.76 21.2± 9.6
Air
Control 49.7± 15.5 - 51.0± 14.7
+AZ 13.6± 5.3 - 41.2± 21.4
AZ, acetazolamide; DIC, dissolved inorganic carbon.
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041182
net photosynthetic rates at ambient Ci were significantly
higher in submersed plants than in emersed plants, the Ci-
saturated maximum photosynthetic rates (Vmax) were simi-
lar (P > 0.1) in both submersed and emersed plants (Table
2). AZ strongly depressed photosynthetic activities of H.
fusiforme fronds at all Ci levels of the measurements under
submersed condition as well as emersed condition (Figs.2,
3). However, the K0.5 values showed no significantly (P >
0.05) difference between with and without the presence of
AZ (Table 2).
2.3 Sensitivity to O2 concentration for photosynthetic
rates under submersed and emersed conditions
Effect of O2 concentration on photosynthetic rates un-
der submersed and emersed conditions are shown in Fig. 4.
Photosynthetic rates is significantly inhibited (P < 0.05) by
O2 at ambient O2 concentration in comparison with low O2
concentration for H. fusiforme fronds under both submersed
and emersed conditions, indicating a C3-like photosynthetic
gas exchange physiology.
3 Discussion
H. fusiforme, which normally grows in low intertidal zone,
will be exposed to air when the tide goes out, especially
during a spring tidal cycle. However, field desiccation sel-
dom occurs due to the continuous waves, sea spray and
extensive shingle-overlapping. The morphology of
coarsely-branched fronds of H. fusiforme further reduces
the probability of desiccation. Thus, H. fusiforme could
often maintain lengthy hydrated statue while exposed. The
present work showed that H. fusiforme exhibited different
photosynthetic activity under emersed condition compared
to submersed condition, which might result from different
availability of exogenous inorganic carbon for
photosynthesis, and/or the mechanism of carbon acquisi-
tion between in and out of water. For example, the concen-
tration of DIC in seawater is about 140 times greater in
water than in air (2.2 mmol/L vs 15.6 mmol/L), but the diffu-
sion rate of CO2 in air is 10 000 times higher than in water.
Oates (1985) and Romaine et al. (1997) guessed that the
difficulty in absorbing CO2 in its molecular form or block-
age of its movement into the cells might result in the lower
photosynthetic rate in air. On the other hand, the carbon-
saturated maximum photosynthetic rate of H. fusiforme was
similar between in water and in air (Table 2), implying the
comparable carboxylatory capacity of Rubisco in both the
environmental conditions.
Rates of theoretical CO2 supply lower than the observed
rates of photosynthetic O2 evolution could be considered
as evidence for the capacity of submersed H. fusiforme to
acquire external HCO3- in seawater to drive photosynthesis,
as reported in some other algae for their abilities of using
HCO3- (Johnston et al., 1992; Gao and Zou, 2001). The
inhibition of photosynthetic O2 evolution by AZ addition
indicated that extracellular CA activity acted as an essen-
tial part of HCO3- acquisition by submersed H. fusiforme. It
has been previously shown that extracellular CA evidently
occurred in H. fusiforme (Zou et al., 2003). In some inter-
tidal seaweeds, there seems a poor correlation between ex-
ternal CA activity and the capacity of HCO3- acquisition,
and the main cause is that those seaweeds possessed the
mechanism of direct HCO3- uptake (Mercado et al., 1998).
However, the experiment of culturing H. fusiforme under
different CO2 concentrations was in support that the exter-
nal CA activity was closely linked to the ability of acquiring
HCO3- in seawater (Zou et al., 2003). Therefore, when
Hizikia was submersed in seawater, external CA catalyzed
Fig.3. Photosynthetic CO2 uptake rate as a function of CO2
concentration in air for the emersed Hizikia fusiforme with or
without the presence of acetazolamide (AZ).
Fig.4. Comparison of photosynthetic rates measured at low O2
concentration and those measured at ambient O2 concentration
for Hizikia fusiforme under submersed and emersed conditions.
ZOU Ding-Hui et al.: Comparative Mechanisms of Photosynthetic Carbon Acquisition in Hizikia fusiforme Under Submersed
and Emersed Conditions 1183
the dehydration of HCO3- externally, and the CO2 formed
was the species of Ci that crossed the plasma membrane.
However, although the rate of conversion between HCO3-
and CO2 catalyzed by extracellular CA activity is almost
instantaneous, the resulting equilibrium CO2 concentration
is rather low at high pH (Raven, 1997; Axelsson et al., 1999;
Zou and Gao, 2001). As a consequence of that, the extracel-
lular CA-mediated conversion of HCO3- to CO2 is much
less efficient at high pH values. This gave the physiologi-
cal explanation for the results that submersed photosyn-
thetic rate of H. fusiforme was conspicuously reduced with
increasing pH (Fig.1). On the other hand, the relationship
between the photosynthetic rate and pH under emersed
condition differed from that under submersed condition, in
which the emersed photosynthetic rate did not significantly
decrease as pH rose. Such discrepancy of photosynthetic
performances in H. fusiforme between in water and in air
could be ascribed to the different source of Ci for
photosynthesis, as suggested by Mercado and Niell (2000).
Though submersed H. fusiforme mainly used HCO3- pool
in seawater for photosynthesis, emersed H. fusiforme ac-
quired CO2 in air as a principal source for photosynthesis.
The CO2 uptake rate substantially exceeded the theoretical
CO2 flux derived from the spontaneous conversion of
HCO3- in the surface seawater film surrounding the fronds
of H. fusiforme, supplying further evidence that the main
source of Ci for emersed photosynthesis came from atmo-
spheric CO2.
CO2 in air must first be dissolved into and across
through the surface water film surrounding the fronds of H.
fusiforme before it reached the plasmolemma and was avail-
able for photosynthesis. The present results showed that
AZ had a considerable inhibitory effect on photosynthetic
CO2 uptake rate of H. fusiforme under emersed condition,
indicating that the external CA facilitated the atmospheric
CO2 acquisition. Firstly, external CA catalysed the conver-
sion of dissolved CO2 into HCO3- in the water film, which
allowed a CO2 gradient and produced a driving force facili-
tating the dissolving of gaseous CO2 into water film (Portielj
and Lijklema, 1995). Secondly, as CO2 was the species of Ci
that entering into cells, extracellular HCO3- must be dehy-
drated to form CO2 before acquired by the algal cells. This
process was also mediated by external CA. Therefore, ex-
ternal CA simultaneously catalyzed hydration and dehy-
dration reactions in the surface water film. However, those
two adverse reactions must be spatially separated from each
other. It might be proposed that the role of extracellular CA
in H. fusiforme could be regarded as a facilitating under
emersion condition, whereas that as a qualitatively
essential mechanism under submersion condition, as de-
scribed in some other intertidal seaweeds (Raven, 1997;
Mercado and Niell, 2000; Zou and Gao, 2004).
It was noted that at high pH value (10.0), the photosyn-
thetic rate of H.fusiforme under submersed condition was
very low, indicating the acquisition of HCO3- mediated by
external CA activity was not function well in seawater un-
der such high pH. By contrast, when exposed to air, H.
fusiforme could still photosynthesize efficiently when the
pH value of the surface water film covering the fronds was
as high as 10.0. Such high pH in the water film could accel-
erate the conversion of CO2 into HCO3- and then raise the
CO2 flux across the air-water interface (Portielje and
Lijklema, 1995). In case of high standing stock or low sea-
water motion, the pH of seawater close to H. fusiforme may
rise due to the photosynthetic acquisition of HCO3-, and
consequently the photosynthesis could be depressed.
However, when the tide goes out, the fronds of H. fusiforme
retain surface seawater film with high pH and they could
still photosynthesize efficiently. Such emersed photosyn-
thetic performance could confer H. fusiforme with ecologi-
cal significance of increasing the daily carbon gain.
Marine seaweeds usually assimilate CO2 via the C3 bio-
chemical pathway with ribulose-1,5-biophosphate carboxy-
lase/oxygenase (Rubisco) as a carboxylating enzyme (Kerby
and Raven, 1985; Raven, 1997). The carboxylase function
of Rubisco can be competitively inhibited by O2, and a
high intracellular O2:CO2 ratio is favorable for oxygenase
activity and the photorespiration pathway. However, O2
tension had hardly effect on photosynthetic rates for many
seaweeds (Kerby and Raven, 1985; Raven, 1997), i.e. they
exhibited C4-like photosynthetic gas exchange physiology.
The common explanation is that they possessed a CO2-
concentraing mechanism (CCM) maintaining elevated CO2
level intracellularlly, which was based on the active HCO3-
utilization system (Beer, 1994), as the well-established CCM
in microalgae (Kaplan and Reinhold, 1999). The O2 sensi-
tivity obtained in H. fusiforme was consistent with a C3-like
photosynthetic gas exchange physiology, albeit this spe-
cies had the ability of HCO3- use. It was proposed that the
external CA activity and the associated exogenous carbon
acquisition mechanism in H. fusiforme were not enough to
maintain elevated CO2 intracellularly and to prevent
photorespiration. The present results showed that the pho-
tosynthesis of H. fusiforme was not saturated with the cur-
rent ambient Ci levels under submersed condition as well
as under emersed condition, and substantially increased
rates of photosynthesis could be gained by addition of
DIC in seawater or CO2 in air. It is generally believed that
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041184
the atmospheric CO2 rises mainly due to anthropogenic
effect (combustion of fossil fuels; deforestation), and con-
sequently neat-shore marine dissolved CO2 levels may also
increase (Bowes, 1993; Stumm and Morgan, 1996). Such an
increase of CO2 in air and/or in seawater would no doubt
enhance the photosynthetic rate of H. fusiforme under both
submersed and emersed conditions, which thereby would
enhance the growth and production of aquaculture for this
cultivated crop.
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