Abstract. The eects of increased CO2levels (10,000 ll l)1) in cultures of the green nitrophilic macroalgaUlva
rigida C. Agardh were tested under conditions of N saturation and Nlimitation, using nitrate as the only N source. Enrichment with CO2 enhanced growth, while net photosynthesis, gross photosynthesis, dark respira-tion 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 CO2levels. This higher retention of C provided the source for extra biomass production under N saturation. In N-sucient thalli, nitrate-uptake rate and the activity of nitrate reductase (EC 1.6.6.1) increased under high CO2 levels. This did not aect the Ncontent or the internal C:Nbalance, implying that the extra N-assimilation capacity led to the production of new biomass in proportion to C. Growth enhance-ment by increased level of CO2 was entirely dependent on the enhancement eect of CO2 on N-assimilation rates. The increase in nitrate reductase activity at high CO2was not related to soluble carbohydrates or internal C. This indicates that the regulation of Nassimilation by CO2inU. rigidamight involve a dierent pathway from that proposed for higher plants. The role of organic C release as an eective regulatory mechanism maintaining the internal C:Nbalance in response to dierent CO2 levels is discussed.
Key words: Carbon ± Carbon dioxide ± Nitrate reductase ± Nitrogen ± Organic carbon release ±
Ulva(C: Nbalance)
Introduction
The expectation that the atmospheric CO2concentration will double by the end of the next century (Ramanathan 1998) has provoked much interest in the impact of elevated CO2on 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 (AndrõÂa et al. 1999). Moreover, it is not clear that the eect 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 eects of accumulated photosynthates (Fogg 1983; Ormerod 1983). The eect of high CO2levels on the organic carbon release rate in macroalgae is unknown. When C assimilation is en-hanced and internal C and growth rate are not aected, 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 algaDunaliella salina (Giordano et al. 1994), and is suggested to act as a valve controlling the internal C: Nbalance 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
*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 = dis-solved organic carbon;gp= conductance for DIC;K0.5 DIC=
se-mi-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]
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 EcologõÂa, Facultad de Ciencias, Universidad de MaÂlaga, Campus de Teatinos, 29071 MaÂlaga, Spain Received: 27 March 2000 / Accepted: 9 October 2000
availability; the response is transitory and results only in an increase in soluble carbohydrate and the C: Nratio (Loehle 1995). However, few studies have shown that high CO2 levels can aect the Nassimilation 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 GarcõÂa-SaÂnchez et al. 1994; Yunes 1995; AndrõÂa et al. 1999). The CO2-driven stimulation of nitrate reductase (NR), the main enzyme in the nitrate assim-ilatory 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(AndrõÂa et al. 1999). Thus, the eect of CO2on algal metabolism may go further than simply being a substrate for photosyn-thesis.
The aim of this work was to study the eect 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 eects of elevated CO2, and the combined eects of CO2and Nsupply 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 rigidaC. 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 100lmol m)2s)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 LiCor-1000 DataLogger radiometer.
Experimental design
The algae were cultured for 10 d under two CO2conditions:
non-manipulated air (actual atmospheric concentration, 350ll l)1) and
CO2-enriched aeration (10,000ll l)1); and under two initial N
concentrations added in batch mode: Nsuciency (5 mM NO3))
and Nlimitation (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
0.22lm) enriched with Provasoli-based medium (Provasoli 1968). Temperature, aeration, PFR and photoperiod were the same as for maintenance conditions described above. The water motion pro-duced 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 CO2enrichment eliminated any
statistically signi®cant in¯uence of pH on the results.
Carbon uptake and photosynthesis
Net photosynthesis (NPS) under saturating PFR
(600lmol m)2s)1) (P
max) and culture PFR (100lmol m)2s)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-buered (50 mM, pH 8.1)
arti®cial seawater, initially free of inorganic carbon. The anity 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 Nneeds for growth.
All samples for biochemical analyses were taken after 10 d of culture. Total internal C and Nwere 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 buer [0.1 M phosphate, 4 mM EDTA and 2 mM phenylmethylsulfonyl ¯uoride (PMSF); pH 6.5 at 4°C] and centrifuged (5,000g, 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 forU. rigidaby Corzo and Niell (1991). The in situ method has become the suitable method when the extraction procedure for in vitro determination of NRA is dicult or even impossible without the inactivation of the enzyme (Gordillo et al. 1998; Mercado et al. 2000), as is the case forUlva. In the in situ procedure the enzyme is assayed in its original cellular location; the method involves a buer, 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 10lM 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
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 Ninput 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, Bran-Luebbe, 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, CO2dialysis
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: Nelemental 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 signif-icant dierence (PLSD; 5% con®dence level).
Results
Extra CO2 in the cultures increased the growth rate under Nsuciency to more than double that observed in air (Fig. 1). Nitrogen limitation implied a decrease in growth rate and only a slight eect of CO2was observed. The total internal C pool was not aected by CO2 and slightly decreased under Nlimitation (Fig. 1B). The increase in CO2levels did not aect the internal N, and the C: Nratio remained constant under Nsaturation (Fig. 1C,D). Nitrogen limitation led to the lowest values of internal Nregardless of the CO2 level, while the highest C: Nratio was reached in high CO2 under N limitation. The internal pool of soluble carbohydrates increased only in cultures at high CO2and Nlimitation 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 Nlimitation with respect to non-enriched air and N-replete medium (Fig. 1F).
Uptake of dissolved inorganic carbon (DIC) was measured as the anity 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-sucient thalli, the increase in CO2 provoked a decrease ingp, while Nlimitation led to the lowest values regardless the CO2level.
Fig. 1. Speci®c growth rate (A), total internal C (B) and N(C) and C:Nratio (D), soluble carbohydrates (E) and solu-ble protein content (F) ofUlva rigida
grown under normal (350ll l)1;Air) and
high (10,000ll l)1;+CO
2) CO2, and
under Nsuciency (+N) or Nlimitation ()N). Mean values SD (n6). Dif-ferent letters indicate signi®cant dier-ences (P<0.05)
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 Nsuciency, Pmax, NPS and dark respiration rates were decreased by high CO2, indicating that the inhibitory eect of CO2 on gross photosynthesis was higher than during dark respiration. Therefore, the eect of high CO2on 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
POCDOC= POCDOCC in new biomass 100. Nitrogen limitation led to similar percentages at both CO2levels, but in N-sucient cultures at high CO2 the percentage of the primary production released decreased to 14%.
The nitrate-uptake rate following the addition of 250lM N O3) 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 non-enriched air with respect to CO2-non-enriched in the middle of the culture time, and values close to zero after 6 d of culture for both CO2conditions.
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 CO2enhanced 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 Nprovided by nitrate reduction through NRA matched the amount of N needed for growth under steady-state conditions in the control treatment (normal air, N-suciency; Table 2). The increase in CO2in the cultures resulted in extra Nneeds 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 chang-ing the culture conditions from the control (Fig. 1F) can be assumed to serve as an additional Nsource. Consid-ering that protein contains 16% Nin weight, the degradation of initially excessive soluble proteins at high CO2 covered most of the remaining Nneeds. 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 500lM 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 non-enriched air (Fig. 5). After 2.5 h of Naddition, maximum NRA values were found for both CO2 conditions. Values close to those found in N-sucient
Fig. 2. Organic C released as a percentage of the primary production [ DOCPOC= DOCPOCC in new biomass 100] inU. rigida after 10 d of culture under dierent CO2and Nconditions.
Mean values SD (n6). Dierent letters indicate signi®cant dierences (P<0.05)
Fig. 3. Nitrate uptake rate after the addition of 250lM N O3) in
cultures ofU. rigidaunder normal (hashed columns) and high CO2
(black columns). Mean values SD (n6)
Table 1. Photosynthetic parameters of Ulva rigida after 10 d of culture under dierent CO2 and dierent N-supply conditions.
Conductance (gp) and anity constant for inorganic carbon (K0.5 DIC) were obtained from O2evolution vs. DIC concentration plots
(at saturating PFR). NPS Net photosynthesis at culture PFR (100lmol m)2s)1); P
max net photosynthesis at saturating PFR
(600lmol m)2s)1). Standard deviations in parentheses (n= 6).
Dierent superscripts indicate signi®cant dierences (P<0.05)
Air +CO2
+N )N+N)N
NPS
(mmol O2m)2h)1)
6.2 (0.5)a 1.1 (0.1)b 5.2 (0.5)c 1.0 (0.2)b
Pmax
(mmol O2m)2h)1)
10.4 (0.3)a1.6 (0.2)b 7.9 (1.7)c 1.7 (0.3)b
Dark respiration (mmol O2m)2h)1)
2.1 (0.5)a 2.1 (0.4)a 1.2 (0.2)b 2.5 (0.6)a
gp(10)6m s)1) 5.7 (0.9)a 1.9 (0.5)b 4.1 (0.6)c 1.9 (0.3)b
thalli (Fig. 3A) were reached after 6 h of Naddition (Fig. 5).
Discussion
The inactivation of the carbon-concentrating mecha-nisms (CCMs) is the most commonly observed response in the acclimation of algae to high CO2. InU. rigida, the inactivation of CCMs was evidenced by low values ofgp and K0.5 DIC(Table 1). Bjork et al. (1993) reported an increase inK0.5 DICin parallel with a decrease in external carbonic anhydrase (CAext) activity in U. rigida grown under 50,000 ll l)1 CO2. The K
0.5 DIC values reported here are in the same range as those reported by Bjork et al. These authors proposed that CAext, which was
suppressed at high CO2,is involved in the utilisation of HCO3). Theg
pvalues 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 (AndrõÂa et al. 1999). This, together with the inactivation of CCMs would result in the decrease in photosynthetic rate observed (Table 1).
When the eect of CO2 is only considered as an increase in the substrate for photosynthesis it becomes dicult to explain how CO2 enrichment increases the growth rate in a species where photosynthesis is already saturated at normal DIC level as inU. 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: Nratio (Webber et al. 1994; Fonseca et al. 1997), probably because of limited Navailability (Loehle 1995). The stimulation of the growth rate inU. rigidaby CO2enrichment 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. 5. Nitrate reductase activity (NRA) after the addition of 500lM NO3) to N-limitedU. rigida grown under normal (350ll l)1, open
symbols) and high (10,000ll l)1, black symbols) CO
2. Mean
val-ues SD (n6)
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 (n6). Dierent letters indicate signi®cant dierences (P<0.05)
Table 2. Nitrogen needs for biomass production, and N supplied via NRA and protein degradation. nd, not
determined
Air +CO2
+N )N+N)N
Nneeds (lmol (g DW))1d)1) 46.8 6.4 113 7.9
Nsupplied by NRAa(lmol (g DW))1d)1) 47.9 23.5 68.0 21.4
Nsupplied by protein degradationb(lmol (g DW))1d)1) 0 nd 44.6 nd
% Nneeds explained by NRA 102 370 60 270
% Nneeds explained by protein degradation 0 nd 39 nd
aCalculations assume a constant NRA during the light period equivalent to that measured in Fig. 4A,
and similarly for the dark period from Fig. 4B
bCalculated as the dierence between initial and ®nal soluble protein level assuming the initial level to be
in C losses rather than an increase in photosynthesis. This reduction was achieved by:
(i) The decrease in the amount of C ®xed by photosyn-thesis 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, main-taining the metabolic integrity of the cell (Fogg 1983; Ormerod 1983). According to Wood and Van Valen (1990), organic carbon release would protect the photo-synthetic 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 CO2level, thus maintaining the internal C:Nbalance (Fig. 1D) and leaving more substrate available for growth. However, the regulation of organic carbon release was not eective under Nlimitation. 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 microalgaDunaliella salina, where CO2enrichment led to higher rates of release as well as growth and photosynthesis (Giordano et al. 1994).
The eect of high CO2on Nassimilation in algae is heterogeneous. Stimulation of Nassimilation has been previously reported in the cyanobacterium Anabaena variabilis (Yunes 1995), while a decrease in uptake rate was found in Gracilaria tenuistipitata (GarcõÂa-SaÂnchez et al. 1994) and Gracilariasp. (AndrõÂa et al. 1999). The accelerated NO3) uptake (Fig. 3) and the increase in NRA caused by CO2 (Fig. 4A) evidenced that CO2 enrichment enhanced Nassimilation inU. rigida. Under control conditions (normal air and Nsuciency) the measured NRA quantitatively predicted the rate of N demanded for growth (Table 2). This has been previ-ously observed in steady-state conditions in Dunaliella viridis (Gordillo et al. 1998). At high CO2 and N saturation, Nneeds could not be totally predicted from NRA. In vivo nitrate assimilation has been previously observed not to be exclusively dependent on the poten-tial NRA measured inU. rigidaunder certain conditions (Corzo and Niell 1994). In this case (high CO2 and N suciency), the calculated percentage of Nneeds for biomass production covered by the degradation of soluble proteins was sucient to explain the remaining Nneeds not covered by NRA (Table 2). Therefore, under external Nsuciency, the growth rate is governed by Nassimilation rate, under both normal-air and high-CO2 conditions. The potential NRA remaining in N-limited thalli is far in excess with respect to the amount of Ndiverted to growth. This would indicate the constitutive level of NRA, which is independent of the CO2and Nlevel (Fig. 4A).
In higher plants, it has been proposed that CO2 controls Nassimilation indirectly through the amount of stored soluble carbohydrates (Webber et al. 1994;
Fons-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 aect the internal N content, and the growth rate remained unchanged (And-rõÂa et al. 1999). High CO2did not produce a signi®cant increase in soluble carbohydrates inU. rigida (Fig. 1E) while Nassimilation 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 inP. leucostictahad the same response as inU. rigida, i.e. increasing with CO2(Mercado et al. 2000). This suggests that the regulation pattern of Nassimilation, and more speci®cally NRA by CO2, could be dierent 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 CO2following 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-sucientU. rigida
was entirely governed by Nassimilation regardless of the CO2level. The process of organic C release would act as a valve balancing the C: Nratio in response to dierent CO2 levels, reducing losses and providing more C for extra biomass production when Nassimilation is enhanced by high CO2. The activation of NR at high CO2seemed to follow a dierent 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 nitro-gen 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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