Planta (2001) 213: 64±70
Non-photosynthetic enhancement of growth by high CO2 level
in the nitrophilic seaweed Ulva rigida C. Agardh (Chlorophyta)
Francisco J. L. Gordillo*, F. Xavier Niell, FeÂlix L. Figueroa
Departamento de EcologõÂ a, Facultad de Ciencias, Universidad de MaÂlaga, Campus de Teatinos, 29071 MaÂlaga, Spain
Received: 27 March 2000 / Accepted: 9 October 2000
Abstract. The e€ects of increased CO2 levels (10,000 ll
l)1) in cultures of the green nitrophilic macroalga Ulva
rigida C. Agardh were tested under conditions of N
saturation and N limitation, using nitrate as the only N
source. Enrichment with CO2 enhanced growth, while
net photosynthesis, gross photosynthesis, dark respiration rates and soluble protein content decreased. The
internal C pool remained constant at high CO2, while
the assimilated C that was released to the external
medium was less than half the values obtained under
ambient CO2 levels. This higher retention of C provided
the source for extra biomass production under N
saturation. In N-sucient thalli, nitrate-uptake rate
and the activity of nitrate reductase (EC 1.6.6.1)
increased under high CO2 levels. This did not a€ect
the N content or the internal C:N balance, implying that
the extra N-assimilation capacity led to the production
of new biomass in proportion to C. Growth enhancement by increased level of CO2 was entirely dependent
on the enhancement e€ect of CO2 on N-assimilation
rates. The increase in nitrate reductase activity at high
CO2 was not related to soluble carbohydrates or internal
C. This indicates that the regulation of N assimilation by
CO2 in U. rigida might involve a di€erent pathway from
that proposed for higher plants. The role of organic C
release as an e€ective regulatory mechanism maintaining
the internal C:N balance in response to di€erent CO2
levels is discussed.
*Present address: Aquatic Systems Group, Agriculture and
Environmental Science Division, Queen's University Belfast,
Newforge Lane, BT9 5PX Belfast, Northern Ireland, UK
Abbreviations: DIC = dissolved inorganic carbon; DOC = dissolved organic carbon; gp = conductance for DIC; K0.5 DIC = semi-saturation constant for DIC; NPS = net photosynthesis rate;
NRA = nitrate reductase activity; PFR = photon ¯uence rate;
Pmax = maximum photosynthesis rate; POC = particulate organic
carbon
Correspondence to: F. J. L. Gordillo;
E-mail: [email protected]
Key words: Carbon ± Carbon dioxide ± Nitrate
reductase ± Nitrogen ± Organic carbon release ±
Ulva (C: N balance)
Introduction
The expectation that the atmospheric CO2 concentration
will double by the end of the next century (Ramanathan
1998) has provoked much interest in the impact of
elevated CO2 on plants and ecosystems. Carbon dioxide
has generally been thought to enhance plant growth by
increasing photosynthetic carbon ®xation of species
whose photosynthetic rate is not saturated at current
inorganic C concentrations. However, the response in
macroalgae is heterogeneous with some species showing
accelerated growth (Bjork et al. 1993) and others a
severe inhibition (Mercado et al. 2000) or no response
(AndrõÂ a et al. 1999). Moreover, it is not clear that the
e€ect on photosynthesis causes a proportional response
in the growth rate.
It has been proposed that stimulation of growth by
CO2 needs adequate sinks for excess photosynthate
(Poorter 1993). The release of organic C to the external
medium as a sink for assimilated C has been proposed as
a mechanism to maintain the metabolic integrity of the
cells, avoiding the feedback e€ects of accumulated
photosynthates (Fogg 1983; Ormerod 1983). The e€ect
of high CO2 levels on the organic carbon release rate in
macroalgae is unknown. When C assimilation is enhanced and internal C and growth rate are not a€ected,
no further information on the target for the extra C ®xed
is reported. Nevertheless, the release of organic C under
high CO2 levels has been found to increase in the
unicellular green alga Dunaliella salina (Giordano et al.
1994), and is suggested to act as a valve controlling the
internal C: N balance in the cyanobacterium Spirulina
platensis (Gordillo et al. 1999).
In higher plants, while growth can be initially
enhanced by elevated CO2, it is usually limited by N
F.J.L. Gordillo et al.: E€ects of CO2 on Ulva rigida
availability; the response is transitory and results only in
an increase in soluble carbohydrate and the C: N ratio
(Loehle 1995). However, few studies have shown that
high CO2 levels can a€ect the N assimilation rate in
algae, despite the fact that nitrogen is considered the
limiting nutrient for most macroalgae, and is responsible
for their massive growth in contaminated coastal areas
(but see GarcõÂ a-SaÂnchez et al. 1994; Yunes 1995; AndrõÂ a
et al. 1999). The CO2-driven stimulation of nitrate
reductase (NR), the main enzyme in the nitrate assimilatory pathway (Berges 1997), has previously been
reported (Larsson et al. 1985; Fonseca et al. 1997;
Mercado et al. 2000). In some cases, CO2 enrichment
led to the re-allocation of internal N (AndrõÂ a et al.
1999). Thus, the e€ect of CO2 on algal metabolism may
go further than simply being a substrate for photosynthesis.
The aim of this work was to study the e€ect of
increased levels of CO2 on C sources and sinks in
relation to nitrogen metabolism in the nitrophilic alga
Ulva rigida under both N-saturating and N-limiting
conditions. This species has been reported to have its
photosynthesis saturated at the current inorganic carbon
concentration of seawater (Bjork et al. 1993; Mercado
et al. 1998), but shows increased growth rates at high
CO2 (Bjork et al. 1993). However, no explanation for
the source of C for extra growth has been given. Here,
especial emphasis is put on nitrate reductase activity
(NRA) and organic carbon release. Data presented are
relevant since non-photosynthetic e€ects of elevated
CO2, and the combined e€ects of CO2 and N supply in
macroalgae, are scarcely considered compared to other
plant groups, despite the fact that macroalgae often
control the biogeochemical ¯uxes in coastal areas and
become costly weed problems (Bowes 1993; Rivers and
Peckol 1995).
Materials and methods
Plant cultivation
Ulva rigida C. Agardh was collected in an intertidal rocky shore in
MaÂlaga (Mediterranean Sea, Southern Spain). Healthy thalli free of
macroscopic epibiota were selected and kept for 3±4 d in ®ltered
(Whatman GF/F) and enriched sea water (Provasoli 1968) at
25 °C, bubbled with air at 1 l min)1, a photoperiod of 12 h light:
12 h darkness and a photon ¯uence rate (PFR) of
100 lmol m)2 s)1 provided by white ¯uorescent lamps (Osram
daylight L 20 W/10 S). Light in the PAR range was measured by
means of a spherical sensor (LiCor 193 SB) connected to a LiCor1000 DataLogger radiometer.
Experimental design
The algae were cultured for 10 d under two CO2 conditions: nonmanipulated air (actual atmospheric concentration, 350 ll l)1) and
CO2-enriched aeration (10,000 ll l)1); and under two initial N
concentrations added in batch mode: N suciency (5 mM NO3))
and N limitation (0.25 mM NO3), net uptake ceased after 4 d). For
cultivation under these treatments, thalli were cut into discs of 2 cm
in diameter. The cultures started with 1.5 g FW placed in an
Erlenmeyer ¯ask containing 1 l ®ltered seawater (Millipore
65
0.22 lm) enriched with Provasoli-based medium (Provasoli 1968).
Temperature, aeration, PFR and photoperiod were the same as for
maintenance conditions described above. The water motion produced by the aeration allowed the discs to move softly without
tumbling.
The pH in CO2 enriched cultures was never below 7.7. A
control culture at pH 7.7 without CO2 enrichment eliminated any
statistically signi®cant in¯uence of pH on the results.
Carbon uptake and photosynthesis
Net
photosynthesis
(NPS)
under
saturating
PFR
(600 lmol m)2 s)1) (Pmax) and culture PFR (100 lmol m)2 s)1),
as well as dark respiration rates were estimated by oxygen
evolution using a Clark-type oxygen electrode (5331; Yellow
Spring Instruments, Ohio, USA) in 9-ml custom-made transparent
Plexiglas chambers at 25 ‹ 0.2 °C. Rate measurements were made
at 10-min intervals. Conductance for inorganic carbon (gp) was
calculated from the initial slope of O2 evolution vs. inorganic
carbon concentration plots. These plots were made by adding
known quantities of HCO3) to Tris-bu€ered (50 mM, pH 8.1)
arti®cial seawater, initially free of inorganic carbon. The anity
constant for inorganic carbon (K0.5 DIC) was also calculated from
these plots by ®tting a Michaelis-Menten-type equation.
Growth and biochemical composition
Growth rate was calculated using the exponential model for the
increase in biomass measured as fresh weight at day 10. For
cultures labelled )N, calculations of growth rate were made
between days 4 and 10, since this is the period of time they can be
considered N-limited as net uptake of N from the medium was
below the N needs for growth.
All samples for biochemical analyses were taken after 10 d of
culture. Total internal C and N were measured with a C:H:N
elemental analyser (Perkin-Elmer 2400CHN) after drying three
discs from each culture overnight at 80 °C. For the extraction of
soluble carbohydrates and soluble proteins, six discs from each
culture were ground in a mortar with extraction bu€er [0.1 M
phosphate, 4 mM EDTA and 2 mM phenylmethylsulfonyl ¯uoride
(PMSF); pH 6.5 at 4 °C] and centrifuged (5,000 g, 15 min, 4 °C).
Soluble carbohydrates (shown as glucose equivalents, Kochert
1978) and soluble proteins (Bradford 1976) were estimated from the
supernatant.
Nitrate reductase activity
Nitrate reductase activity (NRA) was measured using fresh
material directly from the cultures according to the in situ
method improved for U. rigida by Corzo and Niell (1991). The in
situ method has become the suitable method when the extraction
procedure for in vitro determination of NRA is dicult or even
impossible without the inactivation of the enzyme (Gordillo et al.
1998; Mercado et al. 2000), as is the case for Ulva. In the in situ
procedure the enzyme is assayed in its original cellular location;
the method involves a bu€er, a compound able to permeabilise
the membrane, nitrate in excess and a source of reducing power.
Thus, the reaction mixture contained 0.1 M sodium phosphate,
0.5 mM EDTA, 0.1% propanol (v/v), 30 mM nitrate and 10 lM
glucose, in a ®nal volume of 5 ml, at pH 8. The test tubes
contained 0.16 g FW and were incubated at 30 °C for 30 min in
darkness and under anaerobic conditions. Nitrite concentration
was determined as described below. The observed activity is a
potential estimate of the NRA of the cell under the conditions
prior to the assay. As this enzyme usually shows circadian
periodicity, reaching a maximum during the light period and a
minimum in darkness (Velasco et al. 1989; Deng et al. 1991), the
F.J.L. Gordillo et al.: E€ects of CO2 on Ulva rigida
66
activity was measured in the middle of the light period and in the
middle of the dark period on day 10. The response of NRA to an
N input in N-limited thalli was monitored for 6 h following the
addition of 0.5 mM NO3) to the culture medium in the middle of
the light period.
Determination of nitrate, nitrite and organic carbon
Samples for the determination of nitrate, nitrite and dissolved
organic carbon (DOC) present in the growth medium were taken
every 2 d and analysed by an automated system (Traacs 800, BranLuebbe, Germany) according to the manufacturer's protocols after
®ltration (Whatman GF/F). Nitrate and nitrite determination
protocols were based on Wood et al. (1967) and Snell and Snell
(1949), respectively. The DOC was estimated by means of the
persulfate oxidation method including UV radiation, CO2 dialysis
and colorimetric determination (Koprivnjak et al. 1995). The ®lter
was dried overnight at 80 °C and used for determination of
particulate carbon (POC) using a C: H: N elemental analyser
(Perkin-Elmer 2400CHN).
Statistics
Data presented are the mean of three independent experiments,
each consisting of two cultures running in parallel for each
treatment. Three sub-samples were taken from each culture vessel
and the mean used as a replicate, unless otherwise indicated.
Treatments were compared by two-way analyses of variance
(ANOVA) followed by multirange Fisher's protected least significant di€erence (PLSD; 5% con®dence level).
Results
Extra CO2 in the cultures increased the growth rate
under N suciency to more than double that observed
in air (Fig. 1). Nitrogen limitation implied a decrease in
growth rate and only a slight e€ect of CO2 was observed.
The total internal C pool was not a€ected by CO2
and slightly decreased under N limitation (Fig. 1B). The
increase in CO2 levels did not a€ect the internal N, and
the C: N ratio remained constant under N saturation
(Fig. 1C,D). Nitrogen limitation led to the lowest values
of internal N regardless of the CO2 level, while the
highest C: N ratio was reached in high CO2 under N
limitation. The internal pool of soluble carbohydrates
increased only in cultures at high CO2 and N limitation
with respect to the control (non-enriched air and N
saturation) (Fig. 1E). The soluble protein content was
very sensitive to the culture conditions, decreasing to
25% at high CO2 levels in N limitation with respect to
non-enriched air and N-replete medium (Fig. 1F).
Uptake of dissolved inorganic carbon (DIC) was
measured as the anity constant (K0.5 DIC), and the
conductance (gp) from plots of O2-evolution rate vs.
inorganic C concentration (Table 1). The K0.5 DIC
increased as a result of both CO2 enrichment and N
limitation. In N-sucient thalli, the increase in CO2
provoked a decrease in gp, while N limitation led to the
lowest values regardless the CO2 level.
Fig. 1. Speci®c growth rate (A), total
internal C (B) and N (C) and C:N ratio
(D), soluble carbohydrates (E) and soluble protein content (F) of Ulva rigida
grown under normal (350 ll l)1; Air) and
high (10,000 ll l)1; +CO2) CO2, and
under N suciency (+N) or N limitation
()N). Mean values ‹ SD (n ˆ 6). Different letters indicate signi®cant di€erences (P<0.05)
F.J.L. Gordillo et al.: E€ects of CO2 on Ulva rigida
67
Net photosynthesis rates measured at saturating
irradiance (Pmax) and growth irradiance (NPS), as well
as dark respiration rates, were measured as O2-exchange
rates (Table 1). Nitrogen-limited thalli showed a severe
decrease in Pmax and NPS for both CO2 conditions.
Under N suciency, Pmax, NPS and dark respiration
rates were decreased by high CO2, indicating that the
inhibitory e€ect of CO2 on gross photosynthesis was
higher than during dark respiration. Therefore, the e€ect
of high CO2 on photosynthesis did not agree with the
response in growth rate (Fig. 1A).
A fraction of the carbon ®xed by photosynthesis was
released to the external medium. The dissolved (DOC)
and particulate (POC) fractions were measured and
referred as a percentage of the total primary production
(Fig. 2). This percentage was calculated as
…POC ‡ DOC†=…POC ‡ DOC ‡ C in new biomass†
100. Nitrogen limitation led to similar percentages at
both CO2 levels, but in N-sucient cultures at high CO2
the percentage of the primary production released
decreased to 14%.
The nitrate-uptake rate following the addition of
250 lM NO3) at the beginning of the experiment was
higher in CO2-enriched cultures during the ®rst 2 d
(Fig. 3). As nitrate was consumed in the external
medium, cultures became N-limited (only cultures )N),
resulting in a higher nitrate-uptake rate under nonenriched air with respect to CO2-enriched in the middle
of the culture time, and values close to zero after 6 d of
culture for both CO2 conditions.
Potential NRA was measured in situ in the middle of
the light period and the middle of the dark period after
10 d of culture. High CO2 enhanced NRA in the light to
almost double the values in non-enriched air. This
increase was only observed in light and under N
saturation (Fig. 4A). Nitrogen limitation led to low
NRA values for both CO2 conditions. In darkness,
NRA values were lower than in light for all the
treatments (Fig. 4B).
The amount of N provided by nitrate reduction
through NRA matched the amount of N needed for
growth under steady-state conditions in the control
treatment (normal air, N-suciency; Table 2). The
increase in CO2 in the cultures resulted in extra N needs
that were not covered by the measured NRA despite the
increase observed during the light period (Fig. 4A). The
degradation of soluble protein experienced after changing the culture conditions from the control (Fig. 1F) can
be assumed to serve as an additional N source. Considering that protein contains 16% N in weight, the
degradation of initially excessive soluble proteins at
high CO2 covered most of the remaining N needs.
Nitrogen limitation implied higher potential NRA than
actually used by the cell due to the lack of substrate,
regardless of the CO2 level, indicating a minimum
constitutive value for NRA in this species.
When 500 lM of nitrate were added to N-limited
thalli in light, NRA increased 400% in 30 min. under
high CO2, while the increase was only 45% in nonenriched air (Fig. 5). After 2.5 h of N addition,
maximum NRA values were found for both CO2
conditions. Values close to those found in N-sucient
Fig. 2. Organic C released as a percentage of the primary production
[…DOC ‡ POC†=…DOC ‡ POC ‡ C in new biomass† 100] in U.
rigida after 10 d of culture under di€erent CO2 and N conditions.
Mean values ‹ SD (n ˆ 6). Di€erent letters indicate signi®cant
di€erences (P<0.05)
Fig. 3. Nitrate uptake rate after the addition of 250 lM NO3) in
cultures of U. rigida under normal (hashed columns) and high CO2
(black columns). Mean values ‹ SD (n ˆ 6)
Table 1. Photosynthetic parameters of Ulva rigida after 10 d of
culture under di€erent CO2 and di€erent N-supply conditions.
Conductance (gp) and anity constant for inorganic carbon (K0.5
DIC) were obtained from O2 evolution vs. DIC concentration plots
(at saturating PFR). NPS Net photosynthesis at culture PFR
(100 lmol m)2 s)1); Pmax net photosynthesis at saturating PFR
(600 lmol m)2 s)1). Standard deviations in parentheses (n = 6).
Di€erent superscripts indicate signi®cant di€erences (P<0.05)
Air
+CO2
+N
NPS
(mmol O2 m)2 h)1)
Pmax
(mmol O2 m)2 h)1)
Dark respiration
(mmol O2 m)2 h)1)
gp (10)6 m s)1)
K0.5 DIC (lM DIC)
)N
a
6.2 (0.5)
+N
b
1.1 (0.1)
)N
c
5.2 (0.5)
1.0 (0.2)b
10.4 (0.3)a 1.6 (0.2)b 7.9 (1.7)c 1.7 (0.3)b
2.1 (0.5)a 2.1 (0.4)a 1.2 (0.2)b 2.5 (0.6)a
5.7 (0.9)a 1.9 (0.5)b 4.1 (0.6)c 1.9 (0.3)b
144 (30)a 520 (15)b 444 (39)b 571 (246)b
F.J.L. Gordillo et al.: E€ects of CO2 on Ulva rigida
68
suppressed at high CO2, is involved in the utilisation of
HCO3). The gp values shown in this work add evidence
for a lower capability of HCO3) use at high CO2. The
decrease in soluble protein content experienced at high
CO2 could well indicate a decrease in the main soluble
protein, Rubisco (AndrõÂ a et al. 1999). This, together
with the inactivation of CCMs would result in the
decrease in photosynthetic rate observed (Table 1).
When the e€ect of CO2 is only considered as an
increase in the substrate for photosynthesis it becomes
dicult to explain how CO2 enrichment increases the
growth rate in a species where photosynthesis is already
saturated at normal DIC level as in U. rigida (Mercado
et al. 1998). In the present study, growth is considered as
a balance between carbon sources and sinks. In higher
plants, the stimulation of the growth rate is often a
transitory response caused by an increase in soluble
carbohydrates that is re¯ected in the C: N ratio (Webber
et al. 1994; Fonseca et al. 1997), probably because of
limited N availability (Loehle 1995). The stimulation of
the growth rate in U. rigida by CO2 enrichment under N
saturation did not involve the accumulation of soluble
carbohydrates, and the total internal C pool and C: N
ratio remained unchanged. Thus, the source of carbon
for the extra biomass production could be the reduction
Fig. 4. Nitrate reductase activity (NRA) measured after 6 h of light
(A) and after 6 h of darkness (B). Photoperiod was 12 h light: 12 h
darkness. Mean values ‹ SD (n ˆ 6). Di€erent letters indicate
signi®cant di€erences (P<0.05)
thalli (Fig. 3A) were reached after 6 h of N addition
(Fig. 5).
Discussion
The inactivation of the carbon-concentrating mechanisms (CCMs) is the most commonly observed response
in the acclimation of algae to high CO2. In U. rigida, the
inactivation of CCMs was evidenced by low values of gp
and K0.5 DIC (Table 1). Bjork et al. (1993) reported an
increase in K0.5 DIC in parallel with a decrease in external
carbonic anhydrase (CAext) activity in U. rigida grown
under 50,000 ll l)1 CO2. The K0.5 DIC values reported
here are in the same range as those reported by Bjork
et al. These authors proposed that CAext, which was
Table 2. Nitrogen needs for
biomass production, and N
supplied via NRA and protein
degradation. nd, not
determined
Fig. 5. Nitrate reductase activity (NRA) after the addition of 500 lM
NO3) to N-limited U. rigida grown under normal (350 ll l)1, open
symbols) and high (10,000 ll l)1, black symbols) CO2. Mean values ‹ SD (n ˆ 6)
Air
N needs (lmol (g DW))1 d)1)
N supplied by NRAa (lmol (g DW))1 d)1)
N supplied by protein degradationb (lmol (g DW))1 d)1)
% N needs explained by NRA
% N needs explained by protein degradation
a
+CO2
+N
)N
+N
)N
46.8
47.9
0
102
0
6.4
23.5
nd
370
nd
113
68.0
44.6
60
39
7.9
21.4
nd
270
nd
Calculations assume a constant NRA during the light period equivalent to that measured in Fig. 4A,
and similarly for the dark period from Fig. 4B
b
Calculated as the di€erence between initial and ®nal soluble protein level assuming the initial level to be
the same as the control (normal air and N-suciency)
F.J.L. Gordillo et al.: E€ects of CO2 on Ulva rigida
in C losses rather than an increase in photosynthesis.
This reduction was achieved by:
(i) The decrease in the amount of C ®xed by photosynthesis that is diverted to be used in respiratory
processes (Table 1). Previous evidence of the decrease
in respiration rate by high CO2 has been reported
(Bunce and Caul®eld 1991; AzcoÂn-Bieto et al. 1994).
(ii) The reduction in the percentage of assimilated
carbon released to the external medium (Fig. 2).
Organic carbon release has been proposed as a
mechanism able to respond to the environment, maintaining the metabolic integrity of the cell (Fogg 1983;
Ormerod 1983). According to Wood and Van Valen
(1990), organic carbon release would protect the photosynthetic apparatus from an overload of products that
cannot be used in growth or stored. In U. rigida, this
mechanism seems to be repressed in response to a high
environmental CO2 level, thus maintaining the internal
C:N balance (Fig. 1D) and leaving more substrate
available for growth. However, the regulation of organic
carbon release was not e€ective under N limitation. A
similar regulatory role has been recently found in the
cyanobacterium Spirulina platensis (Gordillo et al.
1999). Changes in organic carbon release have also been
observed in the green microalga Dunaliella salina, where
CO2 enrichment led to higher rates of release as well as
growth and photosynthesis (Giordano et al. 1994).
The e€ect of high CO2 on N assimilation in algae is
heterogeneous. Stimulation of N assimilation has been
previously reported in the cyanobacterium Anabaena
variabilis (Yunes 1995), while a decrease in uptake rate
was found in Gracilaria tenuistipitata (GarcõÂ a-SaÂnchez
et al. 1994) and Gracilaria sp. (AndrõÂ a et al. 1999). The
accelerated NO3) uptake (Fig. 3) and the increase in
NRA caused by CO2 (Fig. 4A) evidenced that CO2
enrichment enhanced N assimilation in U. rigida. Under
control conditions (normal air and N suciency) the
measured NRA quantitatively predicted the rate of N
demanded for growth (Table 2). This has been previously observed in steady-state conditions in Dunaliella
viridis (Gordillo et al. 1998). At high CO2 and N
saturation, N needs could not be totally predicted from
NRA. In vivo nitrate assimilation has been previously
observed not to be exclusively dependent on the potential NRA measured in U. rigida under certain conditions
(Corzo and Niell 1994). In this case (high CO2 and N
suciency), the calculated percentage of N needs for
biomass production covered by the degradation of
soluble proteins was sucient to explain the remaining
N needs not covered by NRA (Table 2). Therefore,
under external N suciency, the growth rate is governed
by N assimilation rate, under both normal-air and highCO2 conditions. The potential NRA remaining in Nlimited thalli is far in excess with respect to the amount
of N diverted to growth. This would indicate the
constitutive level of NRA, which is independent of the
CO2 and N level (Fig. 4A).
In higher plants, it has been proposed that CO2
controls N assimilation indirectly through the amount of
stored soluble carbohydrates (Webber et al. 1994; Fons-
69
eca et al. 1997). This does not seem to be the case in algae.
In Gracilaria sp. the increase in soluble carbohydrates
observed at high CO2 did not a€ect the internal N
content, and the growth rate remained unchanged (AndrõÂ a et al. 1999). High CO2 did not produce a signi®cant
increase in soluble carbohydrates in U. rigida (Fig. 1E)
while N assimilation was enhanced (as discussed above).
Porphyra leucosticta showed only slight changes in
soluble carbohydrates and, unlike U. rigida, high CO2
resulted in a strong inhibition of growth; however, NRA
in P. leucosticta had the same response as in U. rigida, i.e.
increasing with CO2 (Mercado et al. 2000). This suggests
that the regulation pattern of N assimilation, and more
speci®cally NRA by CO2, could be di€erent from that of
higher plants, probably occurring through a direct action
on de novo synthesis of the enzyme, rather than through
physiological consequences in C metabolism. The fast
activation of NR under high CO2 following the addition
of NO3) to N-limited U. rigida (Fig. 5) would support
this statement. Nevertheless, the regulation of algal NR
genes expression by CO2 needs to be more precisely
evaluated, and is beyond the scope of this work.
In conclusion, the growth rate in N-sucient U. rigida
was entirely governed by N assimilation regardless of the
CO2 level. The process of organic C release would act as a
valve balancing the C: N ratio in response to di€erent CO2
levels, reducing losses and providing more C for extra
biomass production when N assimilation is enhanced by
high CO2. The activation of NR at high CO2 seemed to
follow a di€erent pattern from that proposed for higher
plants. Our results suggest that under a hypothetical
atmosphere enriched in CO2, the presence of U. rigida
in coastal systems would be more dependent on nitrogen availability than under current atmospheric CO2
levels.
This work was supported by the CICYT projects AMB99-1088 and
AMB97-1021±C02)01. F.J.L. Gordillo was supported by a grant
from the Spanish Ministry of Education and Culture.
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