Carbonic anhydrase activity and photosynthesis in marine diatoms

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1 Eur. J. Phycol., (2007), 42(3): Carbonic anhydrase activity and photosynthesis in marine diatoms ANNICK MORANT-MANCEAU, THI LE NHUNG NGUYEN, ELISABETH PRADIER AND GERARD TREMBLIN Faculte des Sciences et Techniques, Laboratoire de Physiologie et Biochimie ve ge tales, EA 2663, Universite du Maine, Avenue Olivier Messiaen, Le Mans cedex 9, France (Received 21 November 2006; accepted 23 April 2007) The photosynthesis and carbonic anhydrase activity of four marine diatoms currently found in oyster-ponds near the French Atlantic coast, Haslea ostrearia, Navicula phyllepta, Entomoneis paludosa and Amphora coffeaeformis, were investigated. Photosynthetic parameters determined from photosynthesis versus irradiance curves showed that A. coffeaeformis (a benthic species) had lower maximum net photosynthesis but a higher light utilization coefficient than the other species studied. Carbonic anhydrase (CA) activity was measured using intact cells (external CA) and extracts (total CA) of these four species. In all four diatoms, the internal CA activity accounted for between 56 and 63% of the total CA activity. Two or three active forms of CA were separated by polyacrylamide gel electrophoresis. Two enzymatic bands of low molecular mass (4.5 and 6.5 kda) were found in all four diatoms. A third enzymatic band was detected at 33 kda in H. ostrearia and 11.7 kda in E. paludosa. Acetazolamide, an inhibitor of external CA, reduced the net photosynthesis in H. ostrearia, E. paludosa and A. coffeaeformis by about 13%, but could not be tested on N. phyllepta as it is permeable to this substance. Direct uptake of HCO 3 was estimated using the anion-exchange inhibitor 4,40 -diisothiocyanatostilbene-2,2 0 -disulphonate. Bicarbonate accounted for about 50% of fixed inorganic carbon in E. paludosa, and for about 60% in the other species. The higher affinity (K 1/2 ) for dissolved inorganic carbon in H. ostrearia (24.5 mm) than in E. paludosa (38.2 mm) seemed to be related to greater external CA activity, and the presence of a more efficient anion exchange protein in the plasmalemma. Key words: bicarbonate, carbon-concentrating mechanism, carbonic anhydrase, diatom, photosynthesis Introduction Marine diatoms are one of the major primary producers in the oceans, and are thought to contribute more than 25% of the earth s total carbon fixation (Tre guer et al., 1995; Falkowski & Raven, 1997; Granum et al., 2005). In diatoms, the carboxylating enzyme RuBisCO requires CO 2 as a substrate (Beardall et al., 1976; Morel et al., 2002). In seawater, algae are CO 2 -limited because dissolved inorganic carbon (DIC) is mainly in the form of HCO 3 at alkaline ph, when the system is in equilibrium with atmospheric CO 2. A significant fraction of algal photosynthesis obtains CO 2 as a result of its active transport to RuBisCO (Rotatore et al., 1995; Korb et al., 1998; Raven & Beardall, 2003), but many photosynthetic organisms are able to use HCO 3 as well as CO 2 (for review, see Badger et al., 1998; Kaplan & Reinhold, 1999; Raven & Beardall, 2003). Bicarbonate can be used Correspondence to: Annick Morant-Manceau. Tel.: þ Fax: þ annick.manceau@univlemans.fr after either direct active uptake or dehydration catalysed by carbonic anhydrase (CA) outside the plasmalemma. In these carbon concentrating mechanisms in cells, CA (EC ) plays an important role in ensuring the supply of inorganic carbon (Ci) to RuBisCO and to phosphoenolpyruvate carboxylase, but also for other biological processes, such as respiration and lipid synthesis (Badger & Price, 1994; Raven, 1995; Granum et al., 2005). Despite the significance of diatoms in marine productivity, the enzymatic activity of CA in marine diatoms has so far been studied in only very few species (Tanaka et al., 2005). In this study, four diatoms found off the French Atlantic coast were investigated: Haslea ostrearia, Navicula phyllepta, Entomoneis paludosa and Amphora coffeaeformis. These species also live in oysterponds, where a very varied diatom community (about 43 genera and 100 species) displays seasonal fluctuations (Rince, 1978). One of these, H. ostrearia, is a peculiar diatom that synthesizes a blue pigment, marennine, which is responsible for ISSN print/issn online/07/ ß 2007 British Phycological Society DOI: /

2 A. Morant-Manceau et al. 264 the greening of the oyster gills (Robert, 1983). Rince (1978) observed that H. ostrearia colonizes oyster-ponds just weeks after these shallow ponds have been filled up. Navicula is among the best represented genera, especially in the sediment, E. paludosa disappears between April and September, and A. coffeaeformis is present but not very abundant. Under laboratory conditions, the photosynthetic parameters of the four species were determined by measuring O 2 photosynthetic exchanges in relation to irradiance. The effects of CA and HCO 3 transport inhibitors on photosynthesis were measured in order to investigate the importance of these mechanisms in photosynthetic activity. External and total CA activities were measured, and the molecular mass of the native CA extracted from the four species was determined by polyacrylamide gel electrophoresis. The utilization of dissolved inorganic carbon was investigated in H. ostrearia and E. paludosa, which have high and low CA activities, respectively. The physiological features of these diatoms are discussed in terms of various environmental conditions in oyster-ponds. Materials and methods Algae and culture conditions Haslea ostrearia (Bory) Simonsen, N. phyllepta Ku tzing, A. coffeaeformis (Agardh) Ku tzing and E. paludosa (W. Smith) Reimer supplied by the Laboratoire de Biologie Marine of Nantes University (France) were cultured in 250-ml Erlenmeyer flasks containing artificial seawater ph 8.5 (Harrison et al., 1980) at 15 C, at an irradiance of 100 mmol m 2 s 1 (Li-Cor quantum meter Li-189) supplied by cool-white fluorescent tubes (Philips TLD 18 W) in a 14 : 10 h light dark cycle. The calcium concentration was reduced from 9.4 mm to 0.25 mm for the A. coffeaeformis culture in order to avoid adhesion of the cells to the flasks (Cooksey, 1981). Axenic cultures were maintained in the exponential growth phase by diluting with fresh medium every 2 or 3 days. To avoid CO 2 fluctuation between the different cultures, the culture media were changed the day before the experiments. Before each experiment, the cell density was determined using a Nageotte haemocytometer for H. ostrearia, and a Neubauer haemocytometer for the other species. Photosynthetic O 2 evolution Oxygen evolution versus irradiance was measured in a chamber containing 1.5 ml of algal suspension at 15 C with a Clark-type oxygen electrode (Hansatech Instruments Ltd., Norfolk, UK). The diatoms (about 0.3 mg Chl a ml 1 ) were illuminated with white actinic light provided via an optic fibre from a cold light source (Volpi AG, Switzerland). Oxygen evolution in the four species versus irradiance was determined with the algae in their culture medium supplemented with 2 mm NaHCO 3 (Mouget et al., 1999). Respiration was measured after placing the microalgae in darkness. Equations and descriptive parameters were estimated by fitting the hyperbolic tangent equation proposed by Jassby & Platt (1976) to the experimental data (nonlinear regression, MacCurves Fit 1.1). Under saturating irradiance (300 mmol m 2 s 1 ), net photosynthesis was also measured for 15 min after injecting increasing concentrations of inhibitors of carbon-uptake mechanisms. The inhibitors used were acetazolamide (AZ) for periplasmic CA, ethoxyzolamide (EZ) for total CA (Moroney et al., 1985), and 4,4 0 -diisothiocyanato-stilbene-2,2 0 -disulphonate (DIDS) for an anion exchange protein (Larsson & Axelsson, 1999; Herfort et al., 2002). Stock solutions of AZ and EZ were prepared in 0.05 M NaOH (Mercado et al., 1998), and DIDS was dissolved in artificial seawater. Determination of K 1/2 for DIC The photosynthetic response of H. ostrearia and E. paludosa to increasing DIC concentrations was measured at saturating irradiance (300 mmol m 2 s 1 ). Diatoms grown at 100 mmol m 2 s 1 in artificial seawater (Harrison et al., 1980) were collected by centrifuging gently (15 min, 900 g), and were then resuspended in sterile artificial seawater, at ph 8.2, with no added NaHCO 3. Before the measurement, suspensions of H. ostrearia ( cell ml 1 ) and E. paludosa ( cell ml 1 ) were illuminated until a zero net O 2 exchange rate was reached. The algal suspension (1.5 ml) was then poured into the O 2 -free chamber surrounded by N 2 atmosphere. After few minutes, a zero net O 2 exchange was attained. Known amounts of NaHCO 3 were then injected into the chamber in order to create various DIC concentrations. Different diatom samples were used for each NaHCO 3 concentration tested. The affinity of the cells for DIC (K 1/2 [DIC]) was determined by calculating the concentration of DIC required to produce half of the maximum rate of net photosynthesis. The fit was assessed using a Lineweaver-Burke curve. Chlorophyll a content Total chlorophyll a (Chl a) was measured in a spectrophotometer after filtering (Whatman GF/C) an aliquot of the algal suspensions and carrying out an extraction with dimethylformamide according to Speziale et al. (1984). Carbonic anhydrase activity The carbonic anhydrase activity was assayed using the potentiometric method. The time required for the ph to fall 0.4 units between ph was measured at 0 2 C after adding 3 ml CO 2 -saturated water to 3 ml of 20 mm veronal-hcl buffer ph 8.3 supplemented with 0.48 mm NaCl. Enzyme activity was calculated in enzyme units, EU ¼ (T/T s ) 1, where T and T s represent the times required for the ph to change without and with an algal sample, respectively (Haglund et al., 1992). The external

3 New XML Template (2006) {TANDF_REV}TEJP/TEJP_I_42_03/TEJP_A_ d [ :33pm] (TEJP) [ ] [Revised Proof] 265 Carbonic anhydrase in marine diatoms CA activity (CAext) was determined using intact cells, and the total CA activity (CAtot) was measured from crude extracts of cells homogenized in liquid nitrogen with 20 mm veronal-hcl ph 8.3 buffer. Internal CA activity was calculated as the difference between CAtot and CAext. In order to determine the concentration of AZ necessary to inhibit CAext completely, cells of H. ostrearia, cells of E. paludosa and cells of N. phyllepta and A. coffeaeformis suspended in veronal-hcl buffer were exposed for 5 min to increasing concentrations of AZ before measuring the CA activity. Determination of the molecular mass of CA Proteins were extracted by grinding pellets of diatoms in a mortar with liquid nitrogen and electrophoresis buffer (Tris-glycine) containing 20% glycerol. Crude extracts were centrifuged at 5000 g for 5 min. The molecular mass (MM) of CA was determined by nondenaturing electrophoresis (2.5 h at 150 V) in gels containing a 10 to 20% polyacrylamide gradient (BioRad, CA, USA). CA in crude diatom extracts and protein standards migrated in parallel in gel that was cut longitudinally into two halves after electrophoresis. CA activity was detected by blowing CO2 over gel stained with cold Bromothymol Blue and then put into ice-cold CO2 saturated water (Beuf et al., 2000). Protein standards (Human serum albumin, soybean trypsin inhibitor and insulin) were stained with Coomassie Blue R-250 dye. Results Photosynthesis Oxygen exchange rates versus irradiance curves (P/E) were plotted as net photosynthesis per mg Chl a (Fig. 1). The photosynthetic parameters estimated from the fitted curves are shown in Table 1. P/E curves of H. ostrearia and E. paludosa had similar shapes and features. The maximum net photosynthetic rates (Pnm ) of these two species were higher than those of N. phyllepta and A. coffeaeformis, but the highest light utilization coefficient ( ) was found for A. coffeaeformis. From 1000 mmol m 2 s 1, photoinhibition was observed in N. phyllepta alone. The light saturating index (Ek) and the compensation irradiance (Ec) were lower for A. coffeaeformis than for the other three species. Respiratory intensity was higher in N. phyllepta and E. paludosa than in the other species. Fig. 1. Net photosynthesis versus irradiance curves for H. ostrearia, N. phyllepta, E. paludosa and A. coffeaeformis grown under 100 mmol m 2 s 1.

4 A. Morant-Manceau et al. 266 Table 1. Parameters of the photosynthesis versus irradiance curves (Fig. 1) of H. ostrearia, N. phyllepta, E. paludosa and A. coffeaeformis grown under 100 mmol m 2 s 1 Species P n m E k E c (m mol O 2 h 1 mg 1 (mmol Chl a (m mol m 2 s 1 ) 1 ) m 2 s 1 ) (m mol O 2 h 1 mg 1 Chl a) (m mol m 2 s 1 ) H. ostrearia N. phyllepta E. paludosa A. coffeaeformis Table 2. Carbonic anhydrase (CA) activity in H. ostrearia, N. phyllepta, E. paludosa and A. coffeaeformis grown under 100 mmol m 2 s 1, acetazolamide (AZ) concentration necessary to inhibit external CA activity and the percentage of net photosynthesis inhibition in presence of AZ. Mean SE (n ¼ 3) CA activity (EU mg 1 Chl a) Species Internal External Total AZ conc. (mm) % Inhibition H. ostrearia N. phyllepta E. paludosa A. coffeaeformis Carbonic anhydrase External and internal CA activities were detected in the four species tested, H. ostrearia having the highest CA tot activity and E. paludosa the lowest (Table 2). In the four diatoms studied, CA int corresponded to between 56 and 63% of the total CA activity (CA tot ). The AZ concentration necessary to inhibit completely the CA ext activity of each algal species was determined (Table 2). It was noticeable that the higher CA ext activity, the higher the concentration of AZ required. Under non-denaturing polyacrylamide gel electrophoresis, several different bands with CA activity were detected in soluble protein extracts of all four diatoms (Table 3). Two low molecularmass enzyme bands (6.5 and 4.4 kda) were present in all four species. A third band was also visualized on the gels for H. ostrearia and E. paludosa, corresponding to molecular masses of 33 and 11.7 kda, respectively. Effect of inhibitors on photosynthetic O 2 exchange A concentration of 200 mm of EZ, which penetrates into the cells, was required to produce rapid inhibition of photosynthesis in H. ostrearia, N. phyllepta and A. coffeaeformis. A higher concentration was necessary to inhibit photosynthesis completely in E. paludosa (Fig. 2A). The inhibition of CA ext activity by AZ (at the concentrations previously determined, see Table 2) Table 3. Molecular mass of native carbonic anhydrase (CA1, CA2 and CA3) in H. ostrearia, N. phyllepta, E. paludosa and A. coffeaeformis determined by electrophoresis on polyacrylamide gradient gel. Mean SE (n ¼ 6) Molecular mass (kda) Species CA1 CA2 CA3 H. ostrearia N. phyllepta E. paludosa A. coffeaeformis greatly reduced the net photosynthesis of N. phyllepta (by about 83%). In contrast, in the other three species, inhibiting CA ext activity induced a reduction of about 13% in the maximum net photosynthesis. We checked that higher AZ concentrations did not significantly alter the percentage of net photosynthesis inhibition except in A. coffeaeformis, where % inhibition was obtained with 400 mm of AZ. Rising concentrations of DIDS (from 300 to 900 mm) reduced the photosynthetic activity of all four diatoms (Fig. 2B), indicating that an anion transport was involved in supplying Ci. Entomoneis paludosa was the species least sensitive to this inhibitor. A higher concentration did not significantly change the percentage inhibition.

5 Carbonic anhydrase in marine diatoms 267 Determination of K 1/2 for DIC Haslea ostrearia and E. paludosa use bicarbonate as a carbon source for photosynthesis; net photosynthesis versus DIC concentration curves were hyperbolic (Fig. 3). The maximum net photosynthesis was very similar in these two diatoms, between 40 and 45 mmol O 2 h 1 mg 1 Chl a, whereas the K 1/2 for DIC was 24.5 mm for H. ostrearia and 38.2 mm for E. paludosa. Fig. 2. Inhibition of net photosynthesis (% of the control) in H. ostrearia, N. phyllepta, E. paludosa and A. coffeaeformis by (A) ethoxyzolamide (EZ) and (B) 4,4 0 -diisothiocyanato-stilbene-2,2 0 -disulphonate (DIDS). Mean SE (n ¼ 3). Discussion The photosynthesis versus irradiance (P/E ) curves previously used in photoacclimation studies in H. ostrearia (Mouget et al., 1999) made it possible to compare different photosynthetic parameters in several marine diatoms acclimated here to low irradiance. The two planktonic species, H. ostrearia and E. paludosa, had higher maximum net photosynthetic rates than the two benthic species, N. phyllepta and A. coffeaeformis. The light utilization coefficient () ofa. coffeaeformis was clearly higher than in the other three species, and N. phyllepta clearly absorbed light less efficiently. The E k parameter gives an indication of the irradiance at which photosynthesis begins to be saturated. E k was low in A. coffeaeformis, which also had a high light utilization coefficient. Mouget et al. (1999) and Tremblin et al. (2000) have shown that the photosynthetic parameters of H. ostrearia and Skeletonema costatum depended on irradiance and light quality, respectively. Our results are consistent with the usual observation that benthic Fig. 3. Net photosynthesis in H. ostrearia and E. paludosa in relation to increasing dissolved inorganic carbon (DIC) concentration at ph 8.2. Mean SE (n ¼ 3).

6 A. Morant-Manceau et al. 268 species tend to be adapted to low irradiance levels and pelagic species to higher ones. Studies have shown microalgae have a CO 2 concentrating mechanism (Aisawa & Miyachi, 1986; Badger & Price, 1994; Moroney et al., 2001; Raven & Beardall, 2003) in which CA activity plays an important role. The potentiometric method we used, although it has poor accuracy (Williams & Turpin, 1987), did allow us to measure CA ext and CA tot activities in all four species. Haslea ostrearia had the highest CA tot activity, and E. paludosa the lowest. Our results show no correlation between P n m and CA tot activity in the four diatoms studied. Satoh et al. (2001) reported that external CA is absent in Phaeodactylum tricornutum. In other strains of P. tricornutum (Iglesias-Rodriguez & Merrett, 1997; Szabo & Colman, 2007) and in S. costatum (Nimer et al., 1999), CA ext activity is not detected when carbon is not limiting, but a rapid development of CA ext is observed in response to carbon limitation. Our results are consistent with those of Patel & Merrett (1986), who showed that CA ext activity accounts for 36 67% of CA tot activity in some marine microalgae. CA activity varies according to species and culture conditions (Dionisio et al., 1989; Geib et al., 1996; Hobson et al., 2001), and especially to the CO 2 concentration. When ph increases, CO 2 concentration decreases, but no significant difference in CA activity was found in either H. ostrearia or E. paludosa grown in buffered ASW at ph 8.1 or 8.8 (data not shown). The molecular mass (MM) of the CA of some photosynthetic organisms has been determined by various methods, but few data are available for marine diatoms. Roberts et al. (1997) have isolated a 27-kDa intracellular CA from Thalassiosira weissflogii, and Satoh et al. (2001) a -CA of 28 kda from P. tricornutum. Under non-denaturing conditions, Bromothymol Blue can be used on electrophoresis gels to visualize the CA activity of the species studied, which separates into two or three bands depending on the species. In our study, only H. ostrearia had a CA band with a MM ( kda) similar to those of T. weissflogii and P. tricornutum. Two enzymatic bands with a low MM (4.4 and 6.5 kda) were found in all four species studied. These two bands could be subunits of a dimer corresponding to the third band of 11.7 kda in E. paludosa. These subunits could also form a heterohexamer corresponding to the 33-kDa band in H. ostrearia. A small 4.2-kDa subunit was also identified in Chlamydomonas reinhardtii (Rawat & Moroney, 1991; Tachiki et al., 1992), which formed a periplasmic CA with a larger subunit (38 kda). Two CA inhibitors, EZ and AZ, were used with intact diatoms. Schmid & Dring (1996) used 0.3 mm DMSO for AZ and EZ stock solutions. They observed that these DMSO solutions had the same effects on the photosynthesis of Ectocarpus silicosus as when NaOH was used as a solvent. We observed inhibition of CA ext activity in H. ostrearia by 0.3 mm DMSO alone. However, adding 0.05 M NaOH had no effect on the activity of this enzyme. Ethoxyzolamide penetrated rapidly (within a few min) into the diatoms, since adding this CA inhibitor to algal suspensions completely inhibited the emission of photosynthetic O 2. This implied that CA activity is essential for photosynthesis. In order to determine the role of CA ext in photosynthesis and in the carbon concentrating mechanism, acetazolamide was used to inhibit CA ext activity, even though it has been reported that algal cell membranes are weakly permeable to this inhibitor (Moroney et al., 1985) and low concentrations of AZ (175 nm) inhibit the uptake of HCO 3 in Chlorella saccharophila (Pollock & Colman, 2001). Chen & Gao (2004a, 2004b) reported that AZ at 200 mm had little effect on the photosynthesis of Skeletonema costatum. In contrast, Beuf et al. (2000) found that a concentration of 1 mm was necessary to inhibit completely the CA ext of the marine species C. saccharophila without affecting its photosynthesis. In preliminary trials, we determined the concentration of AZ necessary to inhibit the CA ext of the four diatoms we were investigating (Table 2). The different values obtained for the different species could have been due to the levels of enzymatic activity and the cell concentrations used to detect CA activity. Navicula phyllepta photosynthesis was reduced by 83% within 15 min after AZ injection, then O 2 emission was completely inhibited. In the light of this finding, and the other results obtained with DIDS (Fig. 2), the plasmalemma of N. phyllepta was thought to be permeable to AZ. In the three other diatoms, AZ did not penetrate into the cells during the experiments. If AZ is a specific inhibitor of CA ext in marine diatoms, our results show that the CA ext supplied about 13% of the fixed Ci. The possibility that the direct uptake of HCO 3 occurs by anion-exchange type mechanism was investigated using DIDS, which is currently used in animal cell physiology. Forster et al. (1998) reported that DIDS also reduces the CO 2 permeability of red blood cells through its action on a membrane transport protein. With algae, this inhibitor is generally used at a concentration of 300 mm (Larsson & Axelsson, 1999) or 500 mm (Merrett et al., 1996; Elzenga & Prins, 2000) but, for the four diatoms tested, we found that a higher concentration

7 Carbonic anhydrase in marine diatoms 269 (900 mm) was necessary to inhibit net photosynthesis (Fig. 2). If DIDS had blocked all the HCO 3 transport proteins and had no effect on the other Ci uptake mechanisms, this source of Ci would correspond to about 50% of fixed Ci in E. paludosa, and about 60% in the other three species. Herfort et al. (2002) also used DIDS (0 to 300 mm) to show that Emiliana huxleyi acquires bicarbonate mainly by an anion exchange protein. On the other hand, the photosynthesis of S. costatum was unaffected by DIDS at 200 mm indicating that this diatom mainly uses free CO 2 from the medium (Chen & Gao, 2004c). DIDS may be a useful probe for the quantitative determination of the relative contribution of HCO 3 influx to the total photosynthetic carbon fixation under experimental conditions. We investigated the relationship between external CA activity and the use of HCO 3 in H. ostrearia, which had the highest CA ext activity, and E. paludosa, which had the lowest CA ext activity and was insensitive to 100 or 200 mm AZ. It is clear that H. ostrearia had greater affinity toward HCO 3 (K 1/2 ¼ 24.5 mm DIC) than E. paludosa (K 1/2 ¼ 38.2 mm DIC). These values are lower than those obtained by Merrett (1991) and Merrett et al. (1996) for different marine microalgae (55 to 300 mm DIC), and higher than that obtained for the freshwater diatom N. pellicosa (2.3 mm; Colman & Rotatore, 1988). Finally, in E. paludosa, half of the fixed Ci originates from the direct uptake of HCO 3 ions at the plasmalemma level (Fig. 2B), and the other half consists of dissolved CO 2 (about 38%), since CA ext activity played a minor role in the photosynthesis of this species (12%). In H. ostrearia, on the other hand, despite the presence of high CA ext activity that increased the cells affinity for HCO 3 in alkaline medium, 60% of the fixed Ci could originate from direct HCO 3 uptake. In these two diatoms, the affinity for HCO 3 was not correlated with the P m n values. The high affinity in H. ostrearia, measured here under laboratory conditions, could explain the abundance of this species, and the disappearance of E. paludosa in oyster ponds in summer, when the ph, temperature and salinity all increase (Rince, 1978). Moreover, we had shown previously that H. ostrearia is also well adapted to high irradiance (Mouget et al., 1999), and is more resistant to UV radiation than E. paludosa (Rech et al., 2005). All these characteristics could account for the dominance of H. ostrearia in oyster-ponds, which is economically important for oyster aquaculture on the French Atlantic coast. 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