Role of carbonic anhydrase in photosynthesis and inorganic-carbon assimilation in the red alga Gracilaria tenuistipitata

The mechanism of inorganic-carbon (C_i) accumulation in the red seaweed Gracilaria tenuistipitata Zhang et Xia has been investigated. Extracellular and intracellular carbonic-anhydrase (CA) activities have been detected. Photosynthetic O₂ evolution in thalli and protoplasts of G. tenuistipitata were higher at pH 6.5 than at pH 8.6, where HCO ₃ ^– is the predominant form of C_i. Dextran-bound sulfonamide (DBS), a specific inhibitor of extracellular CA, reduced photosynthetic O₂ evolution at pH 8.6 and did not have any effect at pH 6.5. After inhibition with DBS, O₂ evolution was similar to the rate that could be supported by CO₂ from spontaneous dehydration of HCO ₃ ^– . The rate of photosynthetic alkalization of the surrounding medium by the algal thallus was dependent on the concentration of C_i and inhibited by DBS. We suggest that the general form of C_i that enters through the plasma membrane of G. tenuistipitata is CO₂. Bicarbonate is utilized mainly by an indirect mechanism after dehydration to CO₂, and this mechanism involves extracellular CA.Abbreviations C_i inorganic carbon (CO₂ + HCO ₃ ^– ) - CA carbonic anhydrase - DIC dissolved inorganic carbon (total) - DBS dextran-bound sulfonamide - EZ ethoxyzolamide - NSW natural seawater - PPFD photosynthetic photon flux density - REA relative enzyme activity - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase This research was supported by the Deutsche Forschungsgemeinschaft (Bonn) as a programme of the Sonderforschungsbereich 251 der Universität Würzburg and by the Fonds der Chemischen Industrie (Frankfurt). Joint work in Würzburg was possible thanks to travel grants from the Chancellor of the University of Würzburg, Professor R. Günther, from the Australian National University under the auspices of its Overseas Studies Programme, and from the New Zealand — Federal Republic of Germany Scientific and Technological Exchange Programme, which are gratefully acknowledged. We thank Dr. A. Meyer and Ms. E. Kilian for untiringly conducting part of the experimental work, Ms. G. Theumer and Ms. D. Faltenbacher-Werner for their valuable assistance, and Mr. H. Walz (Walz Company, Effeltrich, FRG) for his skilled help with the calibration of our gas-exchange system for measurements with helox. The Department of Conservation, New Zealand, is thanked for permission to collect lichens.     

Photosynthetic production of hydrogen peroxide by Ulva rigida C. Ag. (Chlorophyta)

Production of hydrogen peroxide has been found in Ulva rigida (Chlorophyta). The formation of H₂O₂ was light dependent with a production of 1.2 mol·g FW^–1·h^–1 in sea water (pH 8.2) at an irradiance of 700 mol photons m^–2·s^–1. The excretion was also pH dependent: in pH 6.5 the production was not detectable (< 5 nmol·g FW^–1·h^–1) but at pH 9.0 the production was 5.0 mol·g FW^–1·h^–1. The production of H₂O₂ was totally inhibited by 3-(3,4-dichlorophenyl)-1,1 dimethylurea (DCMU). The ability of U. rigida growing in tanks (7501) under a natural light regime to excrete H₂O₂ was checked and found to be seven times higher at 08.00 hours than other times of the day. The H₂O₂ concentration in the cultivation tank (density: 2 g FW·l^–1) reached the highest value (3 M) at 11.00 hours. Photosynthesis was not influenced by H₂O₂ formation. The H₂O₂ is suggested to come from the Mehler reaction (pseudocyclic photophosphorylation). With an oxygen evolution of 120 mmol·g FW^–1·h^–1 at pH 8.2 and 90 mmol·g FW^–1·h^–1 at pH 9.0, 0.5% and 2.7% of the electrons were used for extracellular H₂O₂ production. The H₂O₂ production is sufficiently high to be of physiological and ecological significance, and is suggested to be a part of the defence against epi and endophytes.Abbreviations ACL artificial, continuous light - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - GNL greenhouse - LDC Luminol-dependent chemiluminescence - SOD Superoxide dismutase This investigation was supported by SAREC (Swedish Agency for Research Cooperation with Developing Countries), Hierta-Retzius Foundation, Marianne and Marcus Wallenberg Foundation, the Swedish Environmental Protection Board, and CICYT Spain.     

Role of intracellular carbonic anhydrase in inorganic-carbon assimilation by Porphyridium purpureum

Air-grown cells of Porphyridium purpurem contain appreciable carbonic-anhydrase activity, comparable to that in air-grown Chlamydomonas reinhardtii, but activity is repressed in CO₂-grown cells. Assay of carbonic-anhydrase activity in intact cells and cell extracts shows all activity to be intracellular in Porphyridium. Measurement of inorganic-carbon-dependent photosynthetic O₂ evolution shows that sodium ions increase the affinity of Porphyridium cells for HCO ₃ ^- . Acetazolamide and ethoxyzolamide were potent inhibitors of carbonic anhydrase in cell extracts but at pH 5.0 both acetazolamide and ethoxyzolamide had little effect upon the concentration of inorganic carbon required for the half-maximal rate of photosynthetic O₂ evolution (K_0.5[CO₂]). At pH 8.0, where HCO ₃ ^- is the predominant species of inorganic carbon, the K_0.5 (CO₂) was increased from 50 M to 950 M in the presence of ethoxyzolamide. It is concluded that in air-grown cells of Porphyridium. HCO ₃ ^- is transported across the plasmalemma and intracellular carbonic anhydrase increases the steady-state flux of CO₂ from inside the plasmalemma to ribulose-1,5-bisphosphate carboxylase-oxygenase by catalysing the interconversion of HCO ₃ ^- and CO₂ within the cell.Abbreviations AZ acetazolamide - EZ ethoxyzolamide - K_0.5[CO₂] half-maximal rate of photosynthetic O₂ evolution     

Role of external carbonic anhydrase in light-dependent alkalization byFucus serratus L. andLaminaria saccharina (L.) Lamour. (Phaeophyta)

It has been proposed that many marine macroalgae are able to utilize HCO ₃ ^– for photosynthesis and growth, and that energy-dependent ion pumping is involved in this process. We have therefore studied the light-dependent alkalization of the surrounding medium by two species of marine macroscopic brown algae,Fucus serratus L. andLaminaria saccharina (L.) Lamour. with the aim of investigating the role of extracellular carbonic anhydrase (EC 4.2.1.1.) in the assimilation of inorganic carbon from the seawater medium. In particular, the influence of membrane-impermeable or slowly permeable carbonic-anhydrase inhibitors on the rate of alkalization of the seawater has been investigated. Inhibition of the alkalization rate occurred in both species at an alkaline pH (pH 8.0) but no inhibition was observed at an acidic pH (pH 6.0). The alkalization was found to be light-dependent and inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea and, thus, correlated with photosynthesis. Alkalization by macroalgae has previously been shown to be proportional to inorganiccarbon uptake. We suggest that alkalization of the medium at alkaline pH in both of the species examined is mainly the consequence of an extracellular reaction. The reaction is catalyzed by extracellular carbonic anhydrase which converts HCO ₃ ^– to OH^– and CO₂; CO₂ is then taken up through the plasmalemma. However, we do not exclude the involvement of other mechanisms of inorganic-carbon uptake.Abbreviations AZ acetazolamide - CA carbonic anhydrase - CAext extracellular carbonic anhydrase - C_i inorganic carbon - DBS dextran-bound sulfonamide - DCMU 3-(3,4-dichloro-phenyl)-1,1-dimethylurea - PPFD photosynthetic photon flux density This study was carried out with financial support by SAREC (Swedish Agency for Research Cooperation with Developing Countries), Carl Trygger's Fund for Scientific Research (Sweden), SJFR (Swedish Council for Forestry and Agricultural Research) and CICYT (Spain). Z. Ramazanov is an invited professor of Ministerio de Educación y Ciencia, Spain.     

Photon- and carbon-use efficiency in<Emphasis Type="Italic"> Ulva rigida</Emphasis> at different CO<Subscript>2</Subscript> and N levels

The seaweed Ulva rigida C. Agardh (Chlorophyta) was cultured under two CO₂ conditions supplied through the air bubbling system: non-manipulated air and 1% CO₂-enriched aeration. These were also combined with N sufficiency and N limitation, using nitrate as the only N source. High CO₂ in U. rigida led to higher growth rates without increasing the C fixed through photosynthesis under N sufficiency. Quantum yields for charge separation at photosystem II (PSII) reaction centres (_PSII) and for oxygen evolution (_O2) decreased at high CO₂ even in N-sufficient thalli. Cyclic electron flow around PSII as part of a photoprotection strategy accompanied by decreased antennae size was suspected. The new re-arrangement of the photosynthetic energy at high CO₂ included reduced investment in processes other than C fixation, as well as in carbon diverted to respiration. As a result, quantum yield for new biomass-C production (^growth) increased. The calculation of the individual quantum yields for the different processes involved allowed the completion of the energy flow scheme through the cell from incident light to biomass production for each of the CO₂ and N-supply conditions studied.Abbreviations A total thallus absorptance - A_pig absorptance due to pigments - A_str Absorptance due to non-pigmented structures - a* spectrally averaged in vivo absorption cross-section of chlorophyll a - CCM carbon-concentrating mechanism - Chl chlorophyll - DOC dissolved organic carbon - ETR electron transport rate - F_v/F_m optimum quantum yield for PSII charge separation - GP gross O₂ evolution rate - k_pig specific light absorption coefficient for pigments - k_str specific light absorption coefficient for non-pigmented structures - OP optimum O₂ evolution rate - PFR photon fluence rate - POC particulate organic carbon - PS photosystem - q_N non-photochemical quenching - q_P photochemical quenching - ^growth quantum yield for new biomass-C production - _O2 quantum yield for gross O₂ evolution - _PSII quantum yield for PSII charge separation     

INDUCIBLE MECHANISMS FOR HCO3– UTILIZATION AND REPRESSION OF PHOTORESPIRATION IN PROTOPLASTS AND THALLI OF THREE SPECIES OF ULVA (CHLOROPHYTA)1

Thalli of Ulva reticulata Forskaal, Ulva rigida C. Ag., and Ulva pulchra Jaasund were incubated at different concentrations of dissolved CO₂. Incubation at a high CO₂ concentration resulted in decreased oxygen evolution rate and lower affinity for inorganic carbon at high pH conditions, i.e. the ability to use HCO₃^– as a carbon source was reduced. This effect was reversible, and plants regained this HCO₃^– uptake capacity when transferred to air concentrations of CO₂. The phytosynthetic oxygen evolution rate of plants grown at high CO₂ concentration was reduced by high O₂ concentrations, whereas thalli and protoplasts from cultures grown at air concentration were not affected. This is interpreted as a deactivation of the carbon-concentrating mechanism during conditions of high CO₂ resulting in high photorespiration when plants are exposed to high O₂ concentrations. Protoplasts were not affected by high O₂ to the same extent and were not able to utilize HCO₃^– from the medium. The algae were able to grow at very low CO₂ concentrations, but growth was suppressed when an inhibitor of external carbonic anhydrase was present. Assay of carbonic anhydrase activities showed that external and internal CA activities were lower in plants grown at a high CO₂ concentration compared to plants grown at a low concentration of CO₂. Possible mechanisms for HCO₃^– utilization in these Ulva species are discussed.     

Inorganic-carbon transport in some marine eukaryotic microalgae

Inorganic-carbon transport was investigated in the eukaryotic marine microalgaeStichococcus minor, Nannochloropsis oculata and aMonallantus sp. Photosynthetic O₂ evolution at constant inorganic-carbon concentration but varying pH showed thatS. minor had a greater capacity for CO₂ rather than HCO ₃ ^– utilization but forN. oculata andMonallantus HCO ₃ ^– was the preferred source of inorganic carbon. All three microalgae had a low affinity for CO₂ as shown by the measurement of inorganic-carbon-dependent photosynthetic O₂ evolution at pH 5.0. At pH 8.3, where HCO ₃ ^– is the predominant form of inorganic carbon, the concentration of inorganic carbon required for half-maximal rate of photosynthetic O₂ evolution [K _0.5 (CO₂)] was 53 M forMonallantus sp. and 125 M forN. oculata, values compatible with HCO ₃ ^– transport. Neither extra- nor intracellular carbonic anhydrase was detected in these three microalgal species. It is concluded that these microalgae lack a specific transport system for CO₂ but that HCO ₃ ^– transport occurs inN. oculata andMonallantus, and in the absence of intracellular carbonic anhydrase the conversion of HCO ₃ ^– to CO₂ may be facilitated by the internal pH of the cell.     

The relationship of arginine groups to photosynthetic HCO 3 - uptake inUlva sp. mediated by a putative anion exchanger

The marine macroalgaUlva sp. can take up HCO ₃ ^- via a process which chemically resembles that of anion exchange in red blood cells (Drechsler et al. 1993, Planta191, 34–40). In this work we explore the possibility that high-pK amino-acid residues could be functionally involved in the binding/transport of HCO ₃ ^- . It was found that the specific arginyl-reacting agents phenylglyoxal and 2,3-butanedione inhibited photosynthesis ofUlva competitively with inorganic carbon at pH 8.2–8.4 (which is close to the pH of normal seawater), where HCO ₃ ^- was the predominant inorganic carbon form taken up. The inhibition by phenylglyoxal was irreversible at 32°C and high pH values, while that of butanedione became irreversible in the presence of borate. These interactions, as well as the protection of the irreversible phenylglyoxal-inhibition by inorganic carbon and by the membrane-impermeant agents 4,4-diisothiocyanostilbene 2,2-disulfonate and 4,4-dinitrostilbene-2,2-disulfonate indicate that arginine (and possibly also lysine) are involved in the HCO ₃ ^- uptake process, probably at the plasmalemma level. The photosynthetic affinity ofUlva to external inorganic carbon gradually decreased with increasing pH from 8.2 to 10.5, and this decrease parallels the decline in protonation of amino acids with a pK of around 10. Based on this information, as well as the inhibition studies, it is suggested that arginine and lysine residues are essential proteinaceous constituents involved in anionic inorganic carbon (HCO ₃ ^- and possibly also CO ₃ ^2- ) uptake into theUlva cells.Abbreviations AE1 anion exchanger 1 (of red blood cells) - BD 2,3-butanedione - CA carbonic anhydrase - CI inorganic carbon - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonate - DNDS 4,4-dinitrostilbene-2,2-disulfonate - PG phenylglyoxal This paper is in partial fulfillment of a Ph.D. study by R. Sharkia. Supported by the Israel Academy of Sciences, grant 441/93 (to S.B.), and by the Fund for Encouragement of Research, Histadrut, Israel (to R.S.).     

Nutrient and iron limitation to <Emphasis Type="Italic">Ulva</Emphasis> blooms in a eutrophic coastal lagoon (Sacca di Goro,Italy)

Growth patterns and bloom formation of the green seaweed Ulva rigida were analysed in the eutrophic Sacca di Goro lagoon (Po River Delta, Italy). Variations of standing biomasses and elemental composition of Ulva were analysed through an annual cycle with respect to nitrogen, phosphorus and iron. Growth rates, nutrient and iron uptake and nitrate storage by macroalgal thalli were also assessed with field experiments during the formation of a spring bloom. The control of Ulva growth and the bloom formation depended on multiple factors, especially on nitrogen availability and iron deficiency. In the nitrate rich waters of the Sacca di Goro lagoon, nitrate accumulation in Ulva thalli was inversely related with Fe uptake, indicating an influence of Fe limitation on N acquisition. Since length and magnitude of nitrate luxury uptake are inversely related to the size of the intracellular nitrate pools, in nitrate rich waters the fast growing Ulva may face risk of N-limitation not only when exposed to low N concentrations or at high biomass levels, but also when exposed to pulsed dissolved nitrate concentrations at low iron availability. The potential Fe limitation could be affected by processes controlled by geochemical reactions and by macroalgal growth and decomposition. Both Fe oxidation during the active macroalgal growth and the formation of insoluble FeS and FeS₂ during bloom collapse can result in a drastic decrease of soluble iron. Thus, a potential limitation of Fe to macroalgae can occur, determining positive feedbacks and potentially controlling the extent of bloom development and persistence.     

Evidence against H+−HCO 3 − symport as the mechanism for HCO 3 − transport in the cyanobacteriumAnabaena variabilis

Summary The rate of inorganic carbon uptake and its steadystate accumulation ratio (intracellular/extracellular concentration) was determined in the cyanobacteriumAnabaena variabilis as a function of extracellular pH. The free energy of protons ( ) across the plasmalemma was calculated from determinations of membrane potential, and intracellular pH, as a function of the extracellular pH. While inward proton motive force decreased with increasing extracellular pH from 6.5 to 9.5, rate of HCO ₃ ^– influx and its accumulation ration increased. The latter is several times larger than would be expected should HCO ₃ ^– influx be driven by . It is concluded that HCO ₃ ^– transport in cyanobacteria is not driven by the proton motive force.     

Growth of the seaweed Ulva rigida C. Agardh in relation to biomass densities, internal nutrient pools and external nutrient supply in the Sacca di Goro lagoon (Northern Italy)

Growth of the seaweed Ulva rigida C. Agardh was investigated in relation to biomass densities, internal nutrient pools and external nutrient supply. Research was carried out from 23 March to 5 July 1994 in the Sacca di Goro (Po Delta, Northern Italy), whose south-eastern part was covered by extensive mats of Ulva rigida. Two types of field experiments were conducted by incubating Ulva thalli inside large cages. In the first experiment, beginning on 23 March, 100 g of wet thalli were placed into the cages, allowed to grow for two weeks, then collected and replaced. This procedure was repeated 8 times over the study period. In the second experiment, Ulva thalli were left inside the cages and collected at selected time intervals (14, 27, 41, 64 and 76 days) in order to simulate the effects of increased density on growth and nutrient storage.We recorded specific growth rates (NGR) ranging from 0.025 to 0.081 d^–1 for a period up to two months in the repeated short-term experiments performed at relatively low initial algal densities (300–500 g AFDW m^–3). These NGR resulted significantly related to dissolved inorganic nitrogen (DIN) in the water column. Tissue concentrations of total Kjeldahl nitrogen (TN) were almost constant, while extractable nitrate decreased in a similar manner to DIN in the water column. Total phosphorus showed considerable variation, probably linked to pulsed freshwater inflow.In the long-term incubation experiment, NGR of Ulva was inversely related to density. Internal concentrations of both total P and TN reached maximum values after one month; thereafter P concentration remained almost constant, while TN decreased below 2% w/w (by dry weight). The TN decrease was also accompanied by an abrupt decrease in nitrate tissue concentration. The biomass incubated over the two month period suffered a progressive N limitation as shown by a decreasing NY ratio (49.4 to 14.6). The reciprocal control of Ulva against biogeochemical environment and viceversa is a key factor in explaining both resource competition and successional stages in primary producer communities dominated by Ulva. However, when the biomass exceeds a critical threshold level, approximately 1 kg AFDW m^–3, the macroalgal community switches from active production to rapid decomposition, probably as a result of selfshading, biomass density and development of anaerobic conditions within the macroalgal beds.     

Two modes of bicarbonate utilization in the marine green macroalga Ulva lactuca

The green marine macroalga Ulva lactuca L. was found to be able to utilize HCO₃^? from sea water in two ways. When grown in flowing natural sea water at 16°C under constant dim irradiance, photosynthesis at pH8.4 was suppressed by acetazolamide but unaffected by 4,4′-diisothiocyanostilbene-2,2′-disulphonate. These responses indicate that photosynthetic HCO₃^? utilization was via extracellular carbonic anhydrase (CA) -mediated dehydration followed by CO₂ uptake. The algae were therefore described as being in a ‘CA state’. If treated for more than 10 h in a sea water flow-through system at pH9.8, these thalli became insensitive to acetazolamide but sensitive to 4,4′-diisothiocyanostilbene-2,2′-disulphonate. This suggests the involvement of an anion exchanger (AE) in the direct uptake of HCO₃^?, and these plants were accordingly described as being in an ‘AE state’. Such thalli showed an approximately 10-fold higher apparent affinity for HCO₃^? (at pH9.4) than those in the ‘CA state’, while thalli of both states showed a very high apparent affinity for CO₂. These results suggest that the two modes of HCO₃^? utilization constitute two ways in which inorganic carbon may enter the Ulva lactuca cells, with the direct entry of HCO₃^?, characterizing the ‘AE state’, being inducible and possibly functioning as a complementary uptake system at high external pH values (e.g. under conditions conducive to high photosynthetic rates). Both mechanisms of entry appear to be connected to concentrating CO₂ inside the cell, probably via a separate mechanism operating intracellularly.     

Relations between electron transport rates determined by pulse amplitude modulated chlorophyll fluorescence and oxygen evolution in macroalgae under different light conditions

The relationship between O₂-based gross photosynthesis (GP) and in vivo chlorophyll fluorescence of Photosystem II-based electron transport rate (ETR) as well as the relationship between effective quantum yield of fluorescence (Φ_PSII) and quantum yield of oxygen evolution (Φ_{O_2}) were examined in the green algae Ulva rotundata and Ulva olivascens and the red alga Porphyra leucosticta collected from the field and incubated for 3 days at 100 μmol m⁻² s⁻¹ in nutrient enriched seawater. Maximal GP was twice as high in Ulva species than that measured in P. leucosticta. In all species ETR was saturated at much higher irradiance than GP. The initial slope of ETR versus absorbed irradiance was higher than that of GP versus absorbed irradiance. Only under absorbed irradiances below saturation or at values of GP <2 μmol O₂ m⁻² s⁻¹ a linear relationship was observed. In the linear phase, calculated O₂ evolved /ETR molar ratios were closed to the theoretical value of 0.25 in Ulva species. In P. leucosticta, the estimated GP was associated to the estimated ETR only at high irradiances. ETR was determined under white light, red light emitting by diodes and solar radiation. In Ulva species the maximal ETR was reached under red light and solar radiation whereas in P. leucosticta the maximal ETR was reached under white light and minimal under red light. These results are in agreement with the known action spectra for photosynthesis in these species. In the case of P. leucosticta, GP and ETR were additionally determined under saturating irradiance in algae pre-incubated for one week under white light at different irradiances and at white light (100 μmol m⁻² s⁻¹) enriched with far-red light. GP and growth rate increased at a growth irradiance of 500 μmol m⁻² s⁻¹ becoming photoinhibited at higher irradiances, while ETR increased when algae were exposed to the highest growth irradiance applied (2000 μmol m⁻² s⁻¹). The calculated O₂ evolved /ETR molar ratios were close to the theoretical value of 0.25 when algae were pre-incubated under 500–1000 μmol m⁻² s⁻¹. The enrichment by FR light provoked a decrease in both GP and ETR and an increase of nonphotochemical quenching although the irradiance of PAR was maintained at a constant level. In addition to C assimilation, other electron sinks, such as nitrogen assimilation, affected the GP–ETR relationship. The slopes of GP versus ETR or Φ_PSII versus Φ_{O_2} were lower in the algae with the highest N assimilation capacity, estimated as nitrate reductase activity and internal nitrogen contents, i.e., Ulva rotundata and Porphyra leucosticta, than that observed in U. olivascens. The possible mechanisms to explain this discrepancy between GP and ETR are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of CO2 and nitrogen supply on the biochemical composition of Ulva rigida with especial emphasis on lipid class analysis

Lipid class composition was analysed in the green macroalga Ulva rigida grown under normal (350 ppm) and high (10,000 ppm) CO₂ levels, and in nitrate saturated and nitrogen limited conditions. A new protocol for the extraction of lipids has been defined. Culture conditions altered the fate of assimilated carbon, and significant changes were observed in protein and total lipid content in particular. A CO₂-enriched atmosphere conditioned the effects of nitrogen limitation on lipid class composition, revealing deep qualitative changes in carbon metabolism. Triglycerides accumulated at high CO₂ and under nitrogen limitation, while chloroplast-related lipids showed an inverse response. Changes in phospholipids could be related to carbon availability as they did not respond to nitrogen limitation. The ratio sterols/acetone-mobile polar lipids followed a negative linear relation with the optimum quantum yield for photosynthetic electron transport (F_v/F_m), and was considered as an index of the «light status» of the cell. The specificity of the response of lipid classes to growth conditions in U. rigida emphasizes the potential role of lipid class analyses as a diagnostic tool for environmental stress.     

Hypoxia and sulphide influence gamete production in Ulva sp.

Gamete production after exposure to hypoxia or sulphide was studied in the marine macroalga Ulva sp. collected in the Sacca di Goro, Italy. Experiments were carried out on discs (12 mm diameter) of thalli cultured in artificial sea water in laboratory at 20 ± 1 °C, 152 μmol m⁻² s⁻¹, 16 h photoperiod and 30‰ salinity. Dehydration of thallus was used as inducer of gametogenesis and growth and gamete release during recovery after 10, 20, 30 or 40 min dehydration (20 ± 1 °C, 25% humidity) were analysed. Unlike non-dehydrated thalli the dehydrated ones produced gametes. Thallus discs, non-dehydrated or subjected to 30 min dehydration, were exposed to hypoxia (1.78–4.02 μmol O₂ L⁻¹) or sulphide (1 mM) for 3, 5, or 7 days at 20 °C in the dark. Non-dehydrated and dehydrated thalli maintained in normoxic conditions in the dark were the controls. Gamete density was checked by counting at the end of the incubation period and during the subsequent 7 days of recovery under 16 h photoperiod in normoxic conditions. Non-dehydrated thalli maintained in normoxic conditions in the dark released gametes when returned to light suggesting that dark constitutes a stimulus to gamete production. The presence of gametes at the end of 3 days incubation of dehydrated thalli in normoxia demonstrated that gametogenesis can occur even in the dark. However, gametes were not present at the end of incubation in hypoxic and sulphidic conditions. Actually, during hypoxic incubation oxygen consumption in D-thalli was very low, only 0.117 × 10⁻³ μmol O₂ mg⁻¹ h⁻¹ compared to 5.93 × 10⁻³ μmol O₂ mg⁻¹ h⁻¹ in normoxia, denoting a reduction of the metabolic rate that could not sustain gametogenesis. During recovery after incubation in normoxic, hypoxic or sulphidic conditions densities of gametes from dehydrated thalli showed significant differences and resulted after hypoxia > after normoxia > after sulphide. Differences in non-dehydrated thalli were not significant. Dehydrated thalli, still green at the end of the incubation period, underwent blanching in the course of recovery in parallel to gamete production, while non-dehydrated thalli maintained their green colour even after exposure to sulphide. Our findings suggest that macroalga Ulva sp. can survive exposure to darkness, severe hypoxia and high sulphide levels and can maintain gamete production even when the exposure to these stress conditions is joined to dehydration.     

Characterisation of carbon dioxide and bicarbonate transport during steady-state photosynthesis in the marine cyanobacterium Synechococcus strain PCC7002

Net O₂ evolution, gross CO₂ uptake and net HCO _inf3 ^su– uptake during steady-state photosynthesis were investigated by a recently developed mass-spectrometric technique for disequilibrium flux analysis with cells of the marine cyanobacterium Synechococcus PCC7002 grown at different CO₂ concentrations. Regardless of the CO₂ concentration during growth, all cells had the capacity to transport both CO₂ and HCO _inf3 ^su– ; however, the activity of HCO _inf3 ^su– transport was more than twofold higher than CO₂ transport even in cyanobacteria grown at high concentration of inorganic carbon (C_i = CO₂ + HCO _inf3 ^su– ). In low-C_i cells, the affinities of CO₂ and HCO _inf3 ^su– transport for their substrates were about 5 (CO₂ uptake) and 10 (HCO _inf3 ^su– uptake) times higher than in high-C_i cells, while air-grown cells formed an intermediate state. For the same cells, the intracellular accumulated C_i pool reached 18, 32 and 55 mM in high-C_i, air-grown and low-C_i cells, respectively, when measured at 1 mM external C_i. Photosynthetic O₂ evolution, maximal CO₂ and HCO _inf3 ^su– transport activities, and consequently their relative contribution to photosynthesis, were largely unaffected by the CO₂ provided during growth. When the cells were adapted to freshwater medium, results similar to those for artificial seawater were obtained for all CO₂ concentrations. Transport studies with high-C_i cells revealed that CO₂ and HCO _inf3 ^su– uptake were equally inhibited when CO₂ fixation was reduced by the addition of glycolaldehyde. In contrast, in low-C_i cells steady-state CO₂ transport was preferably reduced by the same inhibitor. The inhibitor of carbonic anhydrase ethoxyzolamide inhibited both CO₂ and HCO _inf3 ^su– uptake as well as O₂ evolution in both cell types. In high-C_i cells, the degree of inhibition was similar for HCO _inf3 ^su– transport and O₂ evolution with 50% inhibition occurring at around 1 mM ethoxyzolamide. However, the uptake of CO₂ was much more sensitive to the inhibitor than HCO _inf3 ^su– transport, with an apparent I₅₀ value of around 250 M ethoxyzolamide for CO₂ uptake. The implications of our results are discussed with respect to C_i utilisation in the marine Synechococcus strain.Abbreviations Chl chlorophyll - C_i inorganic carbon (CO₂ + HCO _inf3 ^su– ) - CA carbonic anhydrase - CCM CO₂-concentrating mechanism - EZA ethoxyzolamide - GA glycolaldehyde - K_1/2 concentration required for half-maximal response - Rubisco ribulose-1,5,-bisphosphate carboxylase-oxygenase D.S. is a recipient of a research fellowship from the Deutsche Forschungsgemeinschaft (D.F.G.). In addition, we are grateful to Donald A. Bryant, Department of Molecular and Cell Biology and Center of Biomolecular Structure Function, Pennsylvania State University, USA, for sending us the wild-type strain of Synechococcus PCC7002.     

PHOTOSYNTHETIC PROPERTIES OF PROTOPLASTS,AS COMPARED WITH THALLI,OF ULVA FASCIATA (CHLOROPHYTA)1

Protoplasts were prepared from Ulva fasciata Delile, and their photosynthetic performance was measured and compared with that of thalli discs. These protoplasts maintained maximal rates of photosynthesis as high as those of thalli (up to 300 μmol O₂·mg chlorophyll^?1·h^?1) for several hours after preparation and were therefore considered suitable for kinetic studies of inorganic carbon utilization. The photosynthetic K_1/2(inorganic carbon) at pH 6.1 was 3.8 μM and increased to 67, 158, and 1410 μM at the pH values 7.0, 7.9, and 8.9, respectively. Compared with these protoplasts, thalli had a much lower affinity for CO₂ but approximately the same affinity for HCO₃^?. Comparisons between rates of photosynthesis and the spontaneous dehydration of HCO₃^? (at 50 μM inorganic carbon) revealed that photosynthesis of both protoplasts (which lacked apparent activity of extracellular/surface-bound carbonic anhydrase) and thalli (which were only 25% inhibited by the external carbonic anhydrase inhibitor acetazolamide) could not be supported by CO₂ formation in the medium at the higher pH values, indicating HCO₃^? uptake. Since both protoplasts and thalli were sensitive to 4,4′-diisothiocyanostilbene-2,2′-disulfonate, we suggest that HCO₃^? transport was facilitated by the membrane-located anion exchange protein recently reported to function in certain Ulva thalli. These findings suggest that the presence of a cell wall may constitute a diffusion barrier for CO₂, but not for HCO₃^?, utilization under natural seawater conditions.     

Photosynthetic response of Ulva rotundata to light and temperature during emersion on an intertidal sand flat

Summary We have investigated the diurnal response of photosynthesis and variable photosystem II (PSII) chlorophyll fluorescence at 77 K for thalli of the chlorophyte macroalga, Ulva rotundata, grown in outdoor culture and transplanted to an intertidal sand flat in different seasons. The physiological response in summer indicated synergistic effects of high PFD and aerial exposure, the latter probably attributable to temperature, which usually increased by 8 to 10° C during midday emersion. Except at extreme emersed temperatures in summer (38° C), the light-saturated photosynthesis rate (P_m) did not decline at midday. In contrast, light-limited quantum yield of photosynthetic O₂ exchange () and the ratio of variable to maximum fluorescence yield (F_v/F_m) reversibly declined during midday low tides in all seasons. Shade-grown thalli exhibited a fluorescence response suggestive of greater photodamage to PSII, whereas sun-grown thalli had greater photoprotective capacity. The fluorescence decline was smaller when high tide occurred at midday, and was delayed during morning cloudiness. These results suggest that the diurnal response to PFD in this shallow water species is modified by tidal and meteorological factors. U. rotundata has a great capacity for photoprotection which allows it to tolerate and even thrive in the harsh intertidal environment.Abbreviations F_o instantaneous yield of chlorophyll fluorescence - F_m maximum yield of fluorescence - F_v variable yield (F_m–F_o) of fluorescence - PFD photon flux density (400–700 nm) - P_m light-saturated rate of photosynthesis - PSH photosystem II - Q_A electron acceptor of PSII - light-limited quantum yield of photosynthesis     

Inorganic carbon transport across cell compartments of the halotolerant alga Dunaliella salina

The inorganic carbon (C_i) accumulation and the intracellular location of carbonic anhydrase (CA, EC 4.2.1.1) in the halotolerant unicellular alga Dunaliella salina have been investigated. The rate of HCO₃ -dependent O₂ evolution was determined by growth conditions. Algae grown under high CO₂ conditions (5% CO₂ in air, v/v; high C_i cells) had a very low affinity for HCO₃^? at pH 7.0 and 8.2, whereas algae grown under low CO₂ conditions (0.03% CO₂ in air; low C_i cells) showed a high affinity for HCO₃^? at both pH values and were sensitive to Dextran-bound sulfonamide (DBS), an inhibitor of extracellular CA. The photosynthetic rate or HCO₄^? dependent O₂ evolution was always higher at pH 7.0 than at pH 8.2. Ethoxyzolamide (EZ), an inhibitor of total (extacellular plus intracellular) CA activity, strongly inhibited photosynthesis at both pH values. During adaptation from high to low CO₂ conditions CA activity increased in chloroplasts in a process dependent on the novo protein synthesis. Carbonic anhydrase activity was found in the supernatant and pellet fractions of chloroplast homogenates. The rate of photosynthesis of chloroplasts from low C_i cells was higher at pH 7.0 than at pH 8.2. The alkalinization of the growth medium, which took place only in the presence of C_i, was partially inhibited by DBS and completely by EZ. We suggest that in D. salina CO₂ is the general form of C_i transported across the plasma membrane and the chloroplast envelope and that bicarbonate enters the cell mainly, although not entirely, by an ‘indirect’ mechanism after dehydration to CO₂.     

The effect of water flow on photosynthetic processes of the alga Ulva lactuca L.

Photosynthetic rates of aquatic macrophytes are affected by the diffusion of carbon through the boundary layer which decreases with increasing flow velocities. This is shown by a significant increase in photosynthetic rates of Ulva lactuca fronds exposed to friction velocities (u _*) between 0.0 and 0.3 cm s^–1. No further increase in photosynthetic rates is observed at u _* values between 0.3 and 1.0 cm s^–1. Stagnating flow conditions at saturating light levels caused a 29% reduction in photosynthetic rates. Experiments in a closed system indicate that Ulva may utilize internal carbon sources during periods of stagnation. On a subtidal rock directly exposed to wave action, Ulva lactuca is not exposed to such conditions while in a wave flushed intertidal pool stagnation occurs during very short time intervals based on in situ measurements of u _* levels on fronds of this species using a microprocessor-controlled, autonomous recorder and hot-film sensors.