Jesús M. Mercado

External carbonic anhydrase and affinity for inorganic carbon in intertidal macroalgae

Abstract Sixteen intertidal macroalga species from the Gibraltar Strait (Southern Spain) have been examined for both their external carbonic anhydrase activity (CA; 4.2.1.1.) and their affinity for inorganic carbon (C i ). Information on the ability to use HCO 3 − was obtained by measuring the effect of increasing seawater pH on the photosynthesis and by examining the O 2 evolution curves vs. C i concentration. The species exhibited a range of characteristics of C i utilisation: the green macroalgae were saturated at seawater C i concentration with K m ranging from 250.0 to 493.3 mmol m −3 and the photosynthetic O 2 production, expressed as conductance for CO 2 ranging from 432.8 to 16.1×10 −6 m s −1 . Only three out of the eleven tested red and brown macroalgae were saturated at C i concentration of seawater and for these algae HCO 3 − use was clearly indicated by the high initial slope of the O 2 evolution rate vs. C i curve. The most efficient HCO 3 − users were species from the high intertidal habitats and rockpools. Two methods were used to infer the presence of external CA: standard method (direct enzyme assay) and use of acetazolamide (AZ), a non-permeant inhibitor of CA, in assays of photosynthetic rate. Some discrepancies between the two methods were found and we obtained indications that the use of AZ could be a more sensitive method to detect external CA. It so, we suggest that external CA might be present in all the macroalgae examined. There appeared to be no correlation between the ability to use HCO 3 − and the presence of external CA. Therefore, the presence of external CA was not a strong indication for an efficient use of HCO 3 − , although some dependence between the requirement for external CA and habitats was found.

Utilization of HCO3 − at high pH by the brown macroalga Laminaria saccharina

The brown macroalga Laminaria saccharina exhibits a type of HCO3 − utilization that could be almost completely inhibited either by proton buffers or by acetazolamide, an inhibitor of extracellularly operating carbonic anhydrase. This means of HCO3 − utilization featured properties similar to direct HCO3 − uptake in that photosynthetic rates were proportional to the HCO3 − concentration of the seawater over a wide pH range (pH 7.0−9.5). Despite this, it must be characterized as carbonic anhydrase-catalysed external HCO3 − dehydration and not as direct HCO3 − uptake. A mechanism is suggested involving a CO2-concentrating capability located at the cell membrane. This mechanism, which might be common in brown algae, is suggested to have an adaptational advantage in colder regions of the sea (as compared with the direct HCO3 − uptake of green macroalgae). This means of HCO3 − utilization is inhibited even by fairly low concentrations of buffer, with consequences for the interpretation of earlier experimental studies on L. saccharina (and possibly other brown algae). These consequences relate both to ecology (e.g. determination of inorganic C affinity) and physiology (e.g. assessing mechanisms for inorganic C uptake).

Effects of different levels of CO2 on photosynthesis and cell components of the red alga Porphyra leucosticta

Photosynthesis and cell composition of Porphyraleucosticta 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 to2.1 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 at high Ci from 0.3 ± 0.2 to 1.7 ± 0.4 μmolNO2- g-1 FW h-1indicating that reduction and assimilation of nitrate were uncoupled. The response of photosynthesis to increasing irradiance, estimated from O2evolution vs. irradiance curves, was affected by the treatments. Maximum quantum yield (Φ O2°) and effective quantum yield (Φ O2) at 150 μmol photon m-2s-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 (ΔF/Fm′)quantum yield for photosystem II charge separation.

Regulation of the mechanism for HCO3 use by the inorganic carbon level in Porphyra leucosticta Thür, in Le Jolis (Rhodophyta)

The capacity for HCO3− use by Porphyra leucosticta Thur. in Le Jolis grown at different concentrations of inorganic carbon (Ci) was investigated. The use of HCO3− at alkaline pH by P. leucosticta was demonstrated by comparing the O2 evolution rates measured with the O2 evolution rates theoretically supported by the CO2 spontaneously formed from HCO3−. Both external and internal carbonic anhydrase (CA; EC 4.2.1.1) were implied in HCO3− use during photosynthesis because O2 evolution rates and the increasing pH during photosynthesis were inhibited in the presence of azetazolamide and ethoxyzolamide (inhibitors for external and total CA respectively). Both external and internal CA were regulated by the Ci level at which the algae were grown. A high Ci level produced a reduction in total CA activity and a low Ci level produced an increase in total CA activity. In contrast, external CA was increased at low Ci although it was not affected at high Ci. Parallel to the reduction in total CA activity at high Ci is a reduction in the affinity for Ci, as estimated from photosynthesis versus Ci curves, was found. However, there was no evident relationship between external CA activity and the capacity for HCO3− use because the presence of external CA became redundant when P. leucosticta was cultivated at high Ci. Our results suggest that the system for HCO3− use in P. leucosticta is composed of different elements that can be activated or inactivated separately. Two complementary hypotheses are postulated: (i) internal CA is an absolute requirement for a functioning Ci-accumulation mechanism; (ii) there is a CO2 transporter that works in association with external CA.

Use of light and inorganic carbon acquisition by two morphotypes of Zostera noltii Hornem

The affinity for dissolved inorganic carbon (DIC) and the mechanisms to use HCO3 as a source of DIC for photosynthesis were investigated in two morphotypes of Zostera noltii Hornem. Both morphotypes were collected at Ria Formosa lagoon (Southern Portugal) at two different levels in the intertidal. Affinity for DIC at saturating photon fluence rate (PFR), estimated as photosynthetic conductance for CO2 (gp(CO2)), was reduced by 75% in the Z. noltii plants adapted to shade conditions (lower intertidal) in comparison to the sun morphotype (45 � 10 � 6 and 182 � 10 � 6 ms � 1 , respectively), indicating that the plants acclimated to sun conditions (higher intertidal) had a higher capacity to use HCO3 as DIC source for photosynthesis. Since external carbonic anhydrase activity was negligible and a large inhibitory effect was produced by Tris buffer addition, this HCO3 use was attributed to the operation of H + ATPases creating low pH zones in periplasmic space. The photosynthetic CO2-flux supported for this mechanism was calculated to be 53 Amol O2 m � 2 s � 1 in sun morphotype, about 80% out of maximum photosynthesis rate. In order to determine the possible photosynthetic energy cost of the HCO3 use, the effect of decreasing light on photosynthetic rates and gp(CO2) was estimated. Photosynthetic conductance decreased in both morphotypes at nonsaturating PFR. This dependence of gp(CO2) on PFR indicated the existence of a positive interactive effect between DIC and PFR which was more pronounced in the shade morphotype since the ascending slope of O2 evolution vs. PFR curves at limiting PFRs was reduced from 7.2 to 2.3 mmol O2 mol photon � 1 at 4 and 0.5 mol m � 3 of DIC, respectively.

Photosynthetic oxygen production and PAM fluorescence in the brown algaPadina pavonica measured in the field under solar radiation

The effects of solar radiation on photosynthetic oxygen production, pulse amplitude-modulated (PAM) fluorescence and pigmentation were measured in the Mediterranean brown macroalgaPadina pavonica (Linnaeus) Lamouroux under field conditions and natural sunlight. Exposure of thalli to solar radiation for 1 h caused a dramatic decrease of their photosynthetic quantum yield, which recovered to initial levels after they had been placed in the shade for 3 h. Photoinhibition also occurred at the natural growth site ofP. pavonica during the hours of maximal solar irradiance. Photosynthetic oxygen production was also affected by high levels of solar radiation both in algae harvested from the surface and from 6 m depth; oxygen production started to decrease after a few minutes of exposure, and negative values were found after 1 h of solar exposure. Chlorophylla content inP. pavonica also decreased during the hours of maximal solar irradiation. These results suggest that photoinhibition ofP. pavonica occurs during part of a typical summer day on Mediterranean coasts.

A NEW METHOD FOR ESTIMATING EXTERNAL, CARBONIC ANHYDRASE ACTMTY IN MACROALGAE1,2

The effects of external carbonic anhydrase (CA, E.C. 4.2.1.1) on HCO−3and CO2use under disequilibnum conditions were examined in 14 species of macroalgae. CO2 was added to the algae in synthetic seawater free from inorganic carbon and buffered to pH 8.7. This resulted in a transient O2 evolution, which was similar for most of the species tested: an initial rapid increase in the rate was followed by a gradual decrease, approaching a steady state within 10 min. The initial high rate of O2evolution was attributed to diffusive entry of CO2into the cells and the steady state to use of HCO−3. An enhancement of CO2diffusive entry was obtained with acetazolamide, an inhibitor of external CA activity. Using this enhancement, a variable was developed to quantify the degree to which CO2entry into the cell was prevented by the external CA. This variable, CA (%), was used as a measure of the external CA activity of the alga. Comparative measurements of the external CA activity using a potentiomtric method revealed that the new method was able to detect low levels of external CA activity, where the potentiometric method failed. These two different methods could be used together to increase the reliability of the measurements. The new method was useful for red and brown macroalgae and for those green macroalgae that lacked direct HCO3uptake. Thus prominent external CA activity was found for Valonia utricularis (green), Halopteris scoparia (brown), and Osmunda pinnatifida (red), where the potentiometric method failed, or nearly failed, to indicate any external CA activity. Direct uptake of HCO−3interfered with the new method and the degree of this interference was dependent on the magnitude of the uptake.

EFFECTS OF SOLAR RADIATION ON PHOTOSYNTHESIS AND PHOTOINHIBITION IN RED MACROPHYTES FROM AN INTERTIDAL SYSTEM OF SOUTHERN SPAIN

The effects of solar radiation on photosynthesis, chlorophyll content and photoinhibition of the red macrophytes Asparagopsis armata, Gelidium sesquipedale, Plocamium cartilagineum and Feldmannophycus rayssiae from an intertidal system of southern Spain were estimated by means of pulse amplitude modulated fluorometer (PAM), by measurement of the O2 exchange, and by quantification of the chlorophyll content of the thalli. The effective quantum yield ( / ^) decreased in all the experimental organisms after 60 min of exposure to solar radiation; moreover, photoinhibition seemed to be more pronounced and long-lasting in shade-type plants. Secondly, all macrophytes from this study suffered more or less pronounced photoinhibition during some hours of the day at their natural living site. Photoinhibition was maximal around noon and the early afternoon, but almost complete recovery of photosynthesis was achieved by dusk. Similarly, oxygen production and chlorophyll a content were minimal around noon. The ratio (l-qP)/qN was lower in shade than in sun algae indicating a lower electron flow rate, and a lower rate of protective energy dissipation in shade than in sun-grown algae. This ratio is suggested as a good indicator of light stress, to compare different algal species with different pigment content, i. e. sunand shade-type algae.

Inorganic carbon acquisition in algal communities: are the laboratory data relevant to the natural ecosystems?

Most of the experimental work on the effects of ocean acidification on the photosynthesis of algae has been performed in the laboratory using monospecific cultures. It is frequently assumed that the information obtained from these cultures can be used to predict the acclimation response in the natural environment. CO2 concentration is known to regulate the expression and functioning of the CCMs in the natural communities; however, ambient CO2 can become quite variable in the marine ecosystems even in the short- to mid-term. We propose that the degree of saturation of the photosynthesis for a given algal community should be defined in relation to the particular characteristics of its habitat, and not only in relation to its taxonomic composition. The convenience of high CO2 experiments to infer the degree of photosynthesis saturation by CO2 in the natural algal communities under the present ocean conditions, as well as its trend in a coming future is discussed taking into account other factors such as the availability of light and nutrients, and seasonality.

Relationship between bio-optical characteristics and photoinhibition of phytoplankton

The relationship between the bio-optical properties of different microalgae and photoinhibition after short-term exposure (15 and 30 min) to solar radiation was analyzed. Photoinhibition was determined as the decrease in oxygen production and in the in vivo-induced chlorophyll fluorescence. Microalgae with different chlorophyll concentrations, cell size and volume were used. Both photoinhibition and recovery of oxygen production and quantum yield were higher after 30 than after 15 min exposure to solar radiation. Photoinhibition was reduced when UV-A and UV-B radiations were eliminated from the solar radiation. The decrease of effective quantum yield and oxygen production was not dependent on cell size, biovolume or chlorophyll concentration in the algal cultures. It was, however, related to the bio-optical property of the cultures. manifested as the specific attenuation coefficient (Kc). As a general response, the inhibition of