Effects of different levels of CO2 on photosynthesis and cell ...

Journal of Applied Phycology 11: 455–461, 1999. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.

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Effects of different levels of CO2 on photosynthesis and cell components of the red alga Porphyra leucosticta Jesús M. Mercado∗ , F. Javier, L. Gordillo, F. Xavier Niell & Félix L. Figueroa Departamento de Ecolog´ıa, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos, E-29071, Málaga, Spain (∗ Author for correspondence; e-mail: [email protected]) Received 1 June 1999; revised 15 September 1999; accepted 16 September 1999

Key words: CO2 , inorganic carbon, macroalgae, photosynthesis, PAM

Abstract Photosynthesis and cell composition of Porphyra leucosticta discs grown at low (< 0.0001% in air), current (control) and high (1% CO2 in air) inorganic carbon (Ci ) concentrations were analyzed. Carbohydrate content in discs grown at high Ci increased (15.1 mg g−1 FW) with respect to the control (6.4 mg g FW−1 ), whereas soluble protein content decreased to one-third (5.6 to 2.1 mg g−1 FW). Carbohydrate content was unaffected and soluble protein slightly increased in discs grown at low Ci . As a consequence of these changes, a lower C/N molar ratio (8.6) was found in the discs grown at low compared to high Ci (12.4). Nitrate reductase activity increased −1 FW h−1 indicating that reduction and assimilation of at high Ci from 0.3 ± 0.2 to 1.7 ± 0.4 µmol NO− 2 g nitrate were uncoupled. The response of photosynthesis to increasing irradiance, estimated from O2 evolution vs. irradiance curves, was affected by the treatments. Maximum quantum yield (8O 2 ◦ ) and effective quantum yield (8O 2 ) at 150 µmol photon m−2 s−1 decreased by 20% and 50%, respectively, at low Ci . These differences could be due to changes in photosynthetic electron flow between PSII and PSI. Treatments also produced changes in maximal (Fv /Fm ) and effective (1F/F0m ) quantum yield for photosystem II charge separation.

Introduction It is anticipated that in the second half of the next century the atmospheric CO2 concentration will reach 700 µbar (Bowes, 1993), and that the CO2 concentration in surface seawater would double and the HCO− 3 concentration will rise by only 6% (Stumm & Morgan, 1981). It has also been suggested that this change might influence the selective advantage associated with mechanisms to improve the utilization of the seawater HCO− 3 pool by algae (Raven & Johnston, 1991). Hence, the influence of high external levels of CO2 on inorganic carbon acquisition mechanisms has been extensively studied. Low affinity to Ci and high photosynthetic sensitivity to O2 have been described in Chlamydomonas, Chlorella, Scenedesmus and Dunaliella species grown at high levels of CO2 (Badger et al., 1980; Spalding et al., 1984). When these cells were trans-

ferred to air levels of CO2 , carbonic anhydrase (CA) activity and affinity to Ci increased (Aizawa & Miyachi, 1986; Ramazanov et al., 1994). As regards macroalgae, Björk et al. (1993) demonstrated that HCO− 3 utilization in Ulva can be suppressed by growth at elevated CO2 levels. Decreases in the capacity to use HCO− 3 and CA activity have also been described for Fucus serratus, Gracilaria tenuistipitata and Porphyra leucosticta grown at high levels of CO2 (Johnston & Raven, 1990; García-Sánchez et al., 1994; Mercado et al., 1997). From these experiments, it was demonstrated that acclimation to different CO2 levels affected not only inorganic carbon acquisition mechanisms but also pigment content. Eley (1971) described a higher content of chlorophyll a in the cyanobacterium Anacystis adapted to a low CO2 concentration, and García-Sánchez et al. (1994) found that chlorophyll a and phycobili-

456 proteins were reduced by 50% in Gracilaria tenuisitipitata at high CO2 . The photosynthetic response to irradiance was also affected. Changes in fluorescence associated with the induction of the Ci concentrating mechanism (CCM) in Chlamydomonas reinhardtii have been found (Spalding et al., 1984; Bürger et al., 1988; Palmqvist et al., 1990); Badour & Irvine (1990) reported an enhancement of noncyclic electron transport coupled with O2 uptake for generation of the ATP required for Ci accumulation in C. reinhardtii adapted to low CO2 . Little attention has been paid to the effects of CO2 on the cellular components and the metabolism of other nutrients such as nitrogen in algae. In contrast, higher plants cultured at high CO2 have decreased levels of nitrogen and soluble protein (Cave et al., 1981; Petterson & McDonald, 1992), increments of soluble carbohydrate (Sheen, 1990) and lower growth rates (Sage et al., 1989). The characteristics of the Ci acquisition mechanism in the red macroalga Porphyra leucosticta have been extensively studied (Mercado et al., 1997, 1999). These authors showed that carbonic anhydrase activity and affinity to HCO− 3 are strongly influenced by the level of CO2 applied during growth. However, no data are available on the effects of different CO2 levels on the use of light by P. leucosticta. Changes in cell composition also remain undescribed. The aim of this study was to investigate changes in photosynthesis and cell composition of Porphyra leucosticta in response to different Ci concentrations. The effect of Ci level on quantum yield of photosynthesis, growth rate, soluble proteins, carbohydrates, carbon and nitrogen contents and nitrate reductase activity were determined following growth of P. leucosticta at three different levels of CO2 .

Materials and methods Porphyra leucosticta Thuret et Le Jolis was collected from the supralittoral zone near Lagos (Málaga, Southern Spain) and maintained in natural seawater at 15 ◦ C for one week. The medium was aerated vigorously (about 3 L air min−1 ). Light was provided by daylight fluorescent lamps (F20W/DL Osram, Munich, Germany), with 12 h light per day and a photon irradiance of 60 µmol photon m−2 s−1 (Figueroa et al., 1995). Irradiance was determined by a quantum spherical PAR sensor (193SB, Li-Cor Inc., Lincoln, USA) connected to a radiometer (Li-1000, Li-Cor Inc., Lincoln, USA).

Porphyra leucosticta was cultivated at high, current and low Ci concentration for 7–8 days. Discs (9 mm diameter) taken from the thallus were cultured in Plexiglas cylinders containing 3 L seawater enriched with Provasoli medium (Starr & Zeikus, 1987) and buffered with 50 mM Tris at pH 8.1. The different Ci concentrations in the cultures were obtained by bubbling air with different CO2 concentrations (Mercado et al., 1997). Seawater was aerated with air from outside (control, 0.035% CO2 ), with air enriched with 1% CO2 (high-Ci treatment) or with air passed through a 5 N KOH solution (low-Ci, final CO2 concentration in air < 0.0001%). The pH of the cultures varied slightly from 7.4 at high Ci to 8.5 at low Ci . The concentrations of Ci in the medium were estimated to be 0.2 M (high Ci ), 2.5 mM (current Ci ) and less than 6 µM (low Ci ). They were determined from CO2 concentrations calculated according to Henry’s law. The solubility of CO2 in seawater and dissociation coefficients of carbonic acid were adopted from Riley & Chester (1977). Final pH of cultures was taken into account for the calculations. Light and temperature conditions were as described above and the experiment was repeated three times with similar results. For pigment extraction, four discs from each treatment were collected and frozen at − 20 ◦ C. Chlorophyll a (Chl a) was extracted in N,Ndimethylformamide and concentrations calculated according Inskeep & Bloom (1985). Phycoerythrin (PE) and phycocyanin (PC) were extracted at 4 ◦ C in 0.1 M phosphate buffer (pH 6.5) containing 10 mM of EDTA-Na2 and 4 mM phenylmethylsulphonylfluoride (PMSF). Biliproteins were determined spectrophotometrically using the equations of Beer & Eshel (1985). Soluble proteins were estimated by the Coomassie Blue G-250 method (Bradford, 1976). The in vivo absorbance peak due to Chl a (678) was determined with a spectrophotometer (BeckmanDU7, Fullerton, California). Algal discs were attached to microscope slides and placed next to the detector with the white side of the opal glass directed to the light source of the spectrophotometer. Absorptance (A) was obtained from absorbance (OD) according to A = 1–10−OD . Total absorptance corrected for the reflectance was calculated as published by Mercado et al. (1996). Soluble and insoluble carbohydrates were determining according to Vergara et al. (1995). Total intracellular carbon and nitrogen contents were estimated using a CHN analyzer (Perkin-Elmer 2400). Nitrate

457 reductase activity in situ (NRA) was estimated according to Corzo & Niell (1991). Oxygen evolution was measured in 9 mL seawater temperature-controlled (15 ± 0.5 ◦ C) chambers containing oxygen electrodes (YSI 5331, Yellow Springs Instruments Co., Inc., OH, USA) at 8 different photon irradiances ranging from 10 to 1000 µmol m−2 s−1 , obtained using neutral density filters. Respiration was measured in darkness prior to exposure to light. Photon irradiances were measured with a flat sensor (192 SB, Li-Cor Inc., Lincoln, USA) connected to a Licor Li-1000 radiometer. A slide projector with a halogen lamp was used as a source of white light. Ten P. leucosticta discs from each treatment were transferred to the oxygen evolution chamber containing natural seawater buffered at pH 8.1 with 50 mM Tris-HCl (2.1 mM Ci concentration). Agitation of the medium was achieved using a magnetic stirrer. Oxygen evolution was recorded over 10–15 min period. Light compensation point (LCP), semisaturation point (Ks ) and maximal photosynthetic rate (Pmax ) were obtained from the fit of photosynthesisirradiance (P-I) curves to the equation of Edwards & Walker (1983). The goodness of fit was tested using least-squares regression analysis. P-I curves were produced in triplicate. Quantum yield at light limitation for gross photosynthesis based on absorbed light (8O 2 ◦ ) was calculated by multiplying the initial slope of P-I curve by absorptance. For this, oxygen evolution rates were expressed on an area basis. A value of 5.91 for fresh weight per unit surface area was used in calculating the O2 evolution rate per surface unit (Figueroa et al. 1995). The initial slope of the P-I curves was calculated as Pmax / Ks . Effective quantum yield (8O 2 ) was obtained by dividing the O2 evolution rate at 150 µmol photon m−2 s−1 by this irradiance. Maximum quantum yields were measured for stable charge separation at PSII using a Pulse Amplitude Modulated (PAM) fluorometer (PAM-2000, Walz, Effeltrich, Germany). A single disc was introduced into the measurement chamber containing natural seawater buffered at pH 8.1 (2.1 mM Ci concentration). The minimum fluorescence level (Fo ) was measured after dark adaptation in the absence of nonphotochemical quenching according to the protocol described by Hanelt et al. (1997) for red algae. This protocol is specific for the Rhodophyceae and reduces the state transition compound in the nonphotochemical quenching. Then, a short saturating flash was provided in order to determine maximum fluorescence level (Fm ). Variable fluorescence (Fv ) was obtained from

Table 1. Chlorophyll a (Chl a), phycoerythrin (PE) and phycocyanin (PC) concentrations of Porphyra leucosticta discs after growing for 7–8 days at different Ci concentrations. Chl a/(PE+PC) and PE/PC ratios are also provided. Values are means of three independent experiments Pigment content Low Ci (mg g−1 FW) Chl a PE PC Chl a/(PC+PE) PE/PC

1.05 ± 0.08a 2.79 ± 0.44a 0.91 ± 0.05a 0.28 ± 0.04a 3.37 ± 0.30a

Current Ci

High Ci

1.07 ± 0.06a 2.20 ± 0.13a 0.70 ± 0.09a 0.37 ± 0.03a 3.51 ± 0.39a

0.63 ± 0.03b 0.79 ± 0.27b 0.34 ± 0.09b 0.56 ± 0.18b 1.37 ± 0.42b

Within each row, means with different superscripts are significantly different at p = 0.05. Number of replicates = 12.

Fm -Fo . Quantum yields for maximum (Fv /Fm ) charge separation was calculated according to the equation: Fv /Fm = (Fm – Fo )/ Fm

(1)

The sample was then exposed to 150 µmol photon m−2 s−1 of irradiance provided by light emitting diodes (maximum emission at 655 nm). Steady-state (Ft ), maximum (F0m ) and minimum (F0o ) fluorescence were determined following the method of Schreiber et al. (1986). Quantum yield for effective (1F/F0m ) charge separation at 150 µmol photon m−2 s−1 was calculated according to the equation: 1F/F0m = (F0m – Ft )/F0m

(2)

The results are expressed as mean ± standard deviation (SD). Statistical significance of means was tested with a model 1 one-way least significance difference (LSD) following Sokal & Rohlf (1981). Results The inorganic carbon concentration affected the pigment content and the spectral light absorption of Porphyra leucosticta (Table 1). Chlorophyll a, PE and PC concentrations decreased by 40%, 65% and 52% respectively in discs grown at high Ci . Pigment contents in discs grown at low and current Ci were not significantly different (p > 0.05). Chlorophyll a/(PC+PE) and PC/PE ratios were affected by the treatments; the proportion of Chl a to phycobiliproteins increased in high-Ci-grown discs and decreased slightly in lowCi -grown discs. Differences in the pigment content were also evident from in vivo measurements of the absorptance peaks. Concomitant changes in the absorption spectrum occurred with changes in pigment content of high-Ci -grown discs.

458 Table 2. Growth rate (r) and content of soluble proteins (SP), soluble carbohydrates (SCH), insoluble carbohydrates (ICH), total carbon and total nitrogen expressed as mg per g fresh weight and carbon/nitrogen molar ratio (C/N). The results are the means from three experiments. Nitrate reductase activity (NRA: µmol −1 FW h−1 ) estimated in a single experiment (n = 3) is NO− 2 g also indicated

r (% day−1 ) SP SCH ICH C N C/N NRA

Low Ci

Current Ci

High Ci

0.64 ± 0.02 7.5 ± 2.9a 1.5 ± 0.3a 4.8 ± 1.8a 105.4 ± 2.1a 14.3 ± 0.5a 8.6 ± 0.1a 0.3 ± 0.2a

5.8 ± 1.39 5.6 ± 1.1a 1.3 ± 0.3a 4.0 ± 1.4a 54.0 ± 9.5b 9.0 ± 1.3b 6.9 ± 0.2b 0.4 ± 0.1a

0.91 ± 0.11 2.1 ± 1.6b 2.1 ± 0.9a 13.0 ± 5.4b 45.3 ± 3.2b 5.2 ± 0.3c 12.4 ± 0.7c 1.7 ± 0.4b

Within each row, means with different superscripts are significantly different at p = 0.01. Number of replicates = 9 (with exception of NRA).

Figure 1. Oxygen evolution vs. irradiance curves obtained by incubation of discs of P. leucosticta in natural seawater buffered at pH 8.1.

Table 3. Respiration rate (R), maximal gross photosynthetic rates (Pmax : µmol O2 g−1 FW h−1 ), half saturation constant (Ks : µmol photon m−2 s−1 ), light compensation point (LCP: µmol photon m−2 s−1 ) and maximum quantum yield (8O 2 ◦ :mol O2 mol photon−1 ) and effective quantum yield at 150 µmol photon m−2 s−1 (8O 2 : mol O2 mol photon−1 ) for oxygen evolution. The results are the means of three P-I curves analyzed separately. Similar results were obtained from the other two experimental series

Table 4. Maximum quantum yield and effective quantum yield at 150 µmol photon m−2 s−1 for charge separation estimated from measurements of the fluorescence of PS II. All measurements were taken within the experimental chamber containing natural seawater buffered at pH 8.1 (2.1 mM Ci concentration). The results are the mean values from the one experiment described in Table 3. Similar results were obtained from the other two experimental series

Parameter

Low Ci

Current Ci

High Ci

R Pmax Ks LCP 8O 2 ◦ 8O 2

15.1 ± 0.3a

20.4 ± 1.2b 290 ± 30b

29.7 ± 9.1b

227 ± 23a 208 ± 66a 15.1 ± 11.5a 0.040 ± 0.004a 0.018 ± 0.002a

100 ± 51b 10.6 ± 12.5a 0.091 ± 0.009b 0.025 ± 0.002b

335 ± 17b 317 ± 46c 29.6 ± 6.2a 0.051 ± 0.003c 0.035 ± 0.003c


Cellular composition was also affected by the treatments. The soluble protein content was reduced at high Ci and only slightly increased at low Ci (Table 2). In contrast, carbohydrates associated with structural parts (insoluble) increased three-fold in high-C igrown discs, while soluble carbohydrates were unchanged. As a result, a higher carbon to nitrogen ratio was found in high-Ci -grown discs than in current-Cigrown discs. It is interesting to note that total carbon content did not change significantly but total nitrogen content decreased in high-Ci -grown discs compared to current-Ci-grown discs. Nitrate reductase activity in situ increased four-fold at high Ci .

Fv /Fm 1F/F0m

Low Ci

Current Ci

High Ci

0.61 ± 0.03a 0.52 ± 0.07a

0.59 ± 0.05a 0.54 ± 0.06a

0.41 ± 0.06b 0.35 ± 0.05b


Photosynthesis vs. irradiance (P-I) curves are presented in Figure 1 and Table 3. Dark respiration rate decreased by 20–30% at low Ci but was not affected at high Ci . Light compensating point (LCP) was unaffected by the treatments but Pmax was (p < 0.01). The highest 8O 2 ◦ was obtained for current-Cigrown discs and was reduced by 40% and 60% in highand low-Ci -grown discs, respectively. The effective quantum yield of O2 production (8O 2 ) at 150 µmol photon m−2 s−1 was lower than 8O 2 ◦ . There was no correlation between 8O 2 values and 8O 2 ◦ (p > 0.05). Table 4 shows the maximal quantum yield (Fv /Fm ) and effective quantum yield (1F/F0m ) at 150 µmol photon m−2 s−1 for stable charge separation. It must be noted that the variation pattern for Fv /Fm was different from 8O 2 ◦ . In fact, the lowest 8O 2 ◦ was

459 obtained for low-Ci -grown discs but the lowest Fv /Fm was obtained for discs grown at high Ci . The lowest 1F/F0m at 150 µmol photon m−2 s−1 was obtained from high-Ci-grown discs.

Discussion The highest growth rates of P. leucosticta were obtained under current (0.035% CO2 ) conditions of Ci supply. Mercado et al. (1997) found a relationship between Ci growth level and affinity to HCO− 3 and CA activity in P. leucosticta. The results reported imply that the whole metabolism of P. leucosticta, and not just the mechanism involved in HCO− 3 utilization, was affected by the level of CO2 . From our data, it can be said that carbon vs. nitrogen metabolism was enhanced at high Ci since both carbohydrate content and C/N ratio increased and soluble protein content decreased. Similar changes have been described for the red macroalga Gracilaria tenuistipitata (GarcíaSánchez et al., 1994). The treatments produced slight changes in the pH of the medium which partly explain the differences found. However, the lower soluble protein content appears to be a general response of higher plants cultivated under high CO2 (Spencer & Bowes, 1986; van Oosten et al., 1992; Sicher et al., 1994), whereas different effects on carbon content have been found (Poorter et al., 1992). Van Oosten et al. (1992) suggested that the reduction in soluble protein is due to the increment of soluble carbohydrates since they reduce the expression of genes related to the photosynthetic pathway (Sheen, 1990). However, soluble carbohydrates were not accumulated at high Ci in P. leucosticta and therefore, the lower soluble protein content is more likely due to (1) a lower uptake of − NO− 3 and/or (2) its reduction into NO2 by means of nitrate reductase (Lara et al., 1987). In discs of P. leucosticta grown at high Ci , NO− 3 uptake could be affected, since nitrate reductase activity increased. A similar result has been reported for Fucus serratus (Axelsson et al., 1991) and Ulva ridiga (Magnusson et al., 1996) subjected to similar treatments. These results indicate that NO− 3 uptake and reduction are uncoupled when algae are grown at high CO2 (Corzo & Niell, 1991). Absorption characteristics and photosynthetic responses to increasing irradiances of P. leucosticta were also strongly affected by exposure to high Ci levels. These changes paralleled those of chlorophyll a and biliprotein concentration, although changes were dis-

proportional since Chl a/BP increased by 50% at highCi compared to current-Ci. It is interesting to note that these changes resembled those produced by P. leucosticta in response to different irradiance qualities (Figueroa et al., 1995). Pmax decreased in low-Ci grown discs compared with current-Ci-grown discs. Similar results have been described for the red macroalga Gracilaria tenuistipitata (García-Sánchez et al., 1994) and the brown macroalga Fucus serratus (Johnston & Raven, 1990). Dubinsky et al. (1986) proposed that Pmax is related empirically to the concentration of photosynthetic units and their minimal turnover time. Turnover times of the photosynthetic apparatus change as a result of alterations in electron flow between PSII and PSI and/or the rate of carbon fixation. Mercado et al. (1997) found no limitation in photosynthesis by rate of carbon fixation in discs of P. leucosticta grown at low and current Ci when photosynthesis was measured under the same conditions as reported here. Furthermore, no reduction in Rubisco concentration was found. Thus, changes in photosynthetic flow between PSII and PSI could explain the decrease in Pmax at low Ci . Two other results support this hypothesis. Firstly, maximum quantum yield for O2 evolution (estimated from initial slope of P-I curves) was not constant among treatments. According to Mercado et al. (1997), there was no photorespiration in discs of P. leucosticta when cultured at current and low Ci but this process might become important at high Ci . This could explain the lower 8O 2 ◦ compared to current Ci . Secondly, variations in photosynthetic quantum yield of charge separation at PSII indicates that the amount of photon necessary to produce 1 mol of stable charge separation at PSII was higher in high-Ci-grown discs than in low- and current-Ci-grown discs. Two processes can contribute to adjustments in the electron flow from PSII to PSI: the rate of cyclic electron transport around PSI (Kroon et al., 1993) and the rate of non-cyclic electron transport coupled with O2 uptake called the Mehler reaction (Raven & Lucas, 1985). Changes in the chemical energy flow from PSII to PSI in parallel to de-activation of the CCM have been described (Badour & Irvine, 1990; Palmqvist et al., 1990). According to these authors, the adjustments would be required to compensate for the ATP-dependent uptake of Ci . The amount of energy used by the HCO− 3 utilization mechanism in P. leucosticta must be higher in discs grown at low and current Ci than in the discs grown at high Ci . Therefore, the changes in the response to light of P. leucosticta cultivated at different Ci concentrations

460 can be explained by considering the energetic requirement of the mechanism for using HCO− 3 . Furthermore, some alteration in the production of ATP relative to NADPH might also explain the imbalance between nitrate reductase activity and assimilation of nitrogen found in discs grown at high Ci . In conclusion, we propose that changes to the CO2 levels applied during growth produce readjustments of the light dependent photosynthetic reactions in P. leucosticta. Carbon and nitrogen metabolism are integrally coupled and both require ATP and NADPH generated from photosynthesis (Turpin, 1991). Therefore, the alteration in the production of ATP relative to NADPH could explain the imbalance between C- and N-metabolism found in discs grown at low and high Ci . As a further consequence, altered cell composition and growth could result.

Acknowledgements The work was financed by CICYT Project AMB960782 and AMB97-1021-CO2-O2. JMM and FJLG were supported by a doctoral fellowship of the Spanish Ministry of Education and Science.

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