VARIABILITY IN THE PRIMARY SITE OF PHOTOSYNTHETIC DAMAGE IN SYMBIODINIUM SP. (DINOPHYCEAE) EXPOSED TO THERMAL STRESS1

Exposure to elevated temperature is known to cause photosynthetic inhibition in the coral symbiont Symbiodinium sp. Through the use of the artificial electron acceptor, methyl viologen, this study identified how reduced photosynthetic capacity occurs as a result of inhibition up‐ and/or downstream of ferredoxin in Symbiodinium sp. in hospite and in culture. Heterogeneity between coral species and symbiont clades was identified in the thermal sensitivity of photosynthesis in the symbionts of the scleractinian corals Stylophora pistillata and Pocillopora damicornis, as well as among Symbiodinium cultures of clades A, B, and C. The in hospite symbionts of S. pistillata and the cultured clade C Symbiodinium both exhibited similar patterns in that their primary site of thermal inhibition occurred downstream of ferredoxin at 32°C. In contrast, the primary site of thermal inhibition occurred upstream of ferredoxin in clades A and B at 32°C, while at 34°C, all samples showed combined up‐ and downstream inhibition. Although clade C is common to both P. damicornis and S. pistillata, the manner of thermal inhibition was not consistent when observed in hospite. Results showed that there is heterogeneity in the primal site of thermal damage in Symbiodinium among coral species and symbiont clades.     

PHOTOSYNTHESIS AND PRODUCTION OF HYDROGEN PEROXIDE BY SYMBIODINIUM (PYRRHOPHYTA) PHYLOTYPES WITH DIFFERENT THERMAL TOLERANCES1

Occurrences whereby cnidaria lose their symbiotic dinoflagellate microalgae (Symbiodinium spp.) are increasing in frequency and intensity. These so‐called bleaching events are most often related to an increase in water temperature, which is thought to limit certain Symbiodinium phylotypes from effectively dissipating absorbed excitation energy that is otherwise used for photochemistry. Here, we examined photosynthetic characteristics and hydrogen peroxide (H₂O₂) production, a possible signal involved in bleaching, from two Symbiodinium types (a thermally “tolerant” A1 and “sensitive” B1) representative of cnidaria–Symbiodinium symbioses of reef‐building Caribbean corals. Under steady‐state growth at 26°C, a higher efficiency of PSII photochemistry, rate of electron turnover, and rate of O₂ production were observed for type A1 than for B1. The two types responded very differently to a period of elevated temperature (32°C): type A1 increased light‐driven O₂ consumption but not the amount of H₂O₂ produced; in contrast, type B1 increased the amount of H₂O₂ produced without an increase in light‐driven O₂ consumption. Therefore, our results are consistent with previous suggestions that the thermal tolerance of Symbiodinium is related to adaptive constraints associated with photosynthesis and that sensitive phylotypes are more prone to H₂O₂ production. Understanding these adaptive differences in the genus Symbiodinium will be crucial if we are to interpret the response of symbiotic associations, including reef‐building corals, to environmental change.     

BICARBONATE STIMULATION OF CALCIFICATION AND PHOTOSYNTHESIS IN TWO HERMATYPIC CORALS1

A wide range of bicarbonate concentrations was used to monitor the kinetics of bicarbonate (HCO₃^?) use in both photosynthesis and calcification in two reef‐building corals, Porites porites and Acropora sp. Experiments carried out close to the P. porites collection site in Barbados showed that additions of NaHCO₃ to synthetic seawater proportionally increased the calcification rate of this coral until the concentration exceeded three times that of seawater (6 mM). Photosynthetic rates were also stimulated by HCO₃^? addition, but these became saturated at a lower concentration (4 mM). Similar experiments on aquarium‐acclimated colonies of Indo‐Pacific Acropora sp. showed that calcification and photosynthesis in this coral were enhanced to an even greater extent than P. porites, with calcification continuing to increase above 8 mM HCO₃^?, and photosynthesis saturating at 6 mM. Calcification rates of Acropora sp. were also monitored in the dark, and, although these were lower than in the light for a given HCO₃^? concentration, they still increased dramatically with HCO₃^? addition, showing that calcification in this coral is light stimulated but not light dependent.     

PHYSIOLOGICAL CHARACTERISTICS OF PHOTOSYNTHESIS IN PORPHYRIDIUM CRUENTUM: EVIDENCE FOR BICARBONATE TRANSPORT IN A UNICELLULAR RED ALGA1

Various physiological characteristics of photosynthesis in the unicellular red alga Porphyridium cruentum Naegeli have been investigated. The rate of photosynthesis was optimal at 25° C and pH 7.5 and was not inhibited by 21% oxygen over a temperature range of 5 to 35° C. Kinetics of whole cell photosynthesis as a function of substrate concentration gave a K_1/2, (CO₂) of 0.3 μM. CO₂ compensation point, measured in a closed system at pH 7.5, was a constant 6.7 m?L · L^?1 over the temperature range 15 to 30° C and was unaffected by O₂ concentration. Whole cell photosynthesis, measured in a closed system at alkaline pH, showed that the rates of oxygen evolution were greatly in excess of the rate of CO₂ supply from the spontaneous dehydration of HCO₃^? in the medium. This indicates that bicarbonate is utilized by the cell to support this photosynthetic rate. These physiological characteristics of Porphyridium cruentum are consistent with the hypothesis that this alga transports bicarbonate across the plasmalemma.     

PHOTOSYNTHETIC UTILIZATION OF INORGANIC CARBON IN THE ECONOMIC BROWN ALGA,HIZIKIA FUSIFORME (SARGASSACEAE) FROM THE SOUTH CHINA SEA1

The mechanism of inorganic carbon (C_i) acquisition by the economic brown macroalga, Hizikia fusiforme (Harv.) Okamura (Sargassaceae), was investigated to characterize its photosynthetic physiology. Both intracellular and extracellular carbonic anhydrase (CA) were detected, with the external CA activity accounting for about 5% of the total. Hizikia fusiforme showed higher rates of photosynthetic oxygen evolution at alkaline pH than those theoretically derived from the rates of uncatalyzed CO₂ production from bicarbonate and exhibited a high pH compensation point (pH 9.66). The external CA inhibitor, acetazolamide, significantly depressed the photosynthetic oxygen evolution, whereas the anion‐exchanger inhibitor 4,4′‐diisothiocyano‐stilbene‐2,2′‐disulfonate had no inhibitory effect on it, implying the alga was capable of using HCO₃^? as a source of C_i for its photosynthesis via the mediation of the external CA. CO₂ concentrations in the culture media affected its photosynthetic properties. A high level of CO₂ (10,000 ppmv) resulted in a decrease in the external CA activity; however, a low CO₂ level (20 ppmv) led to no changes in the external CA activity but raised the intracellular CA activity. Parallel to the reduction in the external CA activity at the high CO₂ was a reduction in the photosynthetic CO₂ affinity. Decreased activity of the external CA in the high CO₂ grown samples led to reduced sensitiveness of photosynthesis to the addition of acetazolamide at alkaline pH. It was clearly indicated that H. fusiforme, which showed CO₂‐limited photosynthesis with the half‐saturating concentration of C_i exceeding that of seawater, did not operate active HCO₃^? uptake but used it via the extracellular CA for its photosynthetic carbon fixation.     

A COMPARISON OF PHOTOSYNTHETIC ELECTRON TRANSPORT RATES IN MACROALGAE MEASURED BY PULSE AMPLITUDE MODULATED CHLOROPHYLL FLUOROMETRY AND MASS SPECTROMETRY

The relationship between whole chain photosynthetic electron transport and PSII activity was investigated in Porphyra columbina (Montagne) (Rhodophyta), Ulva australis (Areschoug) (Chlorophyta), and Zonaria crenata ( J. Agardh) (Phaeophyta). Mass spectrometric measurements of gross O₂ evolution and gross O₂ uptake were combined with simultaneous measurement of pulse-modulated chl fluorescence under a range of irradiances and inorganic carbon (C_i) concentrations. At light-limiting irradiance, a good correlation between gross O₂ evolution and the electron transport rate (ETR) calculated from chl fluorescence ((F_m′− F_s)/F_m′) was found in the optically thin species (Ulva and Porphyra). The calculated ETR was equivalent to the theoretical electron requirement in these species but overestimated gross O₂ evolution in the thicker species Zonaria. In saturating light, especially when C_i availability was low, ETR overestimated gross O₂ evolution in all species. Excess electron flow could not be accounted for by an increase in gross O₂ uptake; thus neither Mehler-ascorbate-peroxidase reaction nor the photosynthetic carbon oxidation cycle were enhanced at high irradiance or low C _i. Alternative explanations for the loss of correlation include cyclic electron flow around PSII that may be engaged under these conditions or nonphotochemical energy quenching within PSII centers. The loss of correlation between ETR and linear photosynthetic electron flow as irradiance increased from limiting to saturating or at low C_i availability and in the case of optically thick thalli limits the application of this technique for measuring photosynthesis in macroalgae.     

The determination of activity of the enzyme Rubisco in cell extracts of the dinoflagellate alga Symbiodinium sp. by manganese chemiluminescence and its response to short‐term thermal stress of the alga

The dinoflagellate alga Symbiodinium sp., living in symbiosis with corals, clams and other invertebrates, is a primary producer in coral reefs and other marine ecosystems. The function of the carbon‐fixing enzyme ribulose 1,5‐bisphosphate carboxylase/oxygenase (Rubisco) in dinoflagellates is difficult to study because its activity is rapidly lost after extraction from the cell. We report procedures for the extraction of Rubisco from Symbiodinium cells and for stable storage. We describe a continuous assay for Rubisco activity in these crude cell extracts using the Mn²⁺ chemiluminescence of Rubisco oxygenase. Chemiluminescence time courses exhibited initial transients resembling bacterial Form II Rubisco, followed by several minutes of linearly decreasing activity. The initial activity was determined from extrapolation of this linear section of the time course. The activity of fast‐frozen cell extracts was stable at ?80 °C and, after thawing and storage on ice, remained stable for up to 1 h before declining non‐linearly. Crude cell extracts bound [¹⁴C] 2‐carboxy‐D‐arabitinol 1,5‐bisphosphate to a high molecular mass fraction separable by gel filtration chromatography. After pre‐treatment of Symbiodinium cell cultures in darkness at temperatures above 30 °C, the extracted Rubisco activities decreased, with almost complete loss of activity above 36 °C. The implications for the sensitivity to elevated temperature of Symbiodinium photosynthesis are assessed.     

Effect of Root Cooling on Photosynthesis of Artemisia tridentata Seedlings under Different Light Levels

Root chilling has been shown to inhibit shoot photosynthesis yet the mechanism for such an action is not clearly understood. A study was designed to elucidate the mechanism by which root cooling may affect net photosynthesis. Roots of Artemisia tridentata seedlings were cooled from 20°C to 5°C while their shoot temperature remained at 20°C. This was conducted at two light levels (700 and 1300 μmol m^?2 s^?1). The time course of shoot net photosynthesis (A), stomatal conductance to water vapor (g_s), intercellular CO₂ concentration (C_i) and root respiration (R_s) were determined on a whole-plant basis. Root cooling caused a 25% reduction in A at high PPFD, which was preceded by more than 50% reduction of g_s and about 10% reduction in C_i. A versus C_i curves for single branches showed no difference between cold and warm soil temperatures, although stomatal conductance was lower for the lower soil temperature. This suggests that a stomatal limitation may have been involved in the inhibition of A. Furthermore, a concomitant decrease of as much as 23% in leaf relative water content (RWC) indicated that root cooling affected stomatal closure due to decreased water supply to the foliage. At lower PPFD, root cooling did not cause a decrease in A of the whole plant despite a moderate drop in g_s, C_i and RWC. Cold soil also led to a substantial and rapid reduction in root respiration rate (R_s) regardless of the light level.     

Effect of inorganic carbon supply on the photosynthetic physiology of Gracilaria tenuistipitata

Gracilaria tenuistipitata Zhang et Xia was cultured for 15 d at low, normal and high inorganic carbon concentrations under constant light, temperature and nutrient conditons. Carbonic anhydrase (CA; EC 4.2.1.1.) activity, ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco; EC 4.1.1.39) content, pigment content and C/N ratio were measured, and also the photosynthesis and growth rates. Both Rubisco content and CA activity increased under conditions of low inorganic carbon (C_i) but decreased at high C_i with respect to the control. The amount of pigments declined considerably at high C_i and was slightly higher at low C_i. The maximum rate of photosynthesis and the photosynthetic efficiency increased in low C_i and the opposite was found at high C_i concentration. The effects of C_i concentration on maximum rate of photosynthesis and photosynthetic efficiency are discussed in relation to the variation in pigment and Rubisco contents and CA activity. The data indicate that C_i may be an important factor controlling the photosynthetic physiology of G. tenuistipitata with regard, not only to the enzymes of C_i metabolism, but also to the pigment content.Abbreviations APS_max maximum apparent photosynthetic rate - CA carbonic anhydrase - Chl chlorophyll - C_i inorganic carbon - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase This work has been supported by grants No. PB91-0962 and No. MAR90-0365 from Spanish Direction for Science and Technology (DIGICYT). M.J. G-S holds a fellowship from the DIGICYT.     

A COMPARISON OF AIR AND WATER AS ENVIRONMENTS FOR PHOTOSYNTHESIS BY THE INTERTIDAL ALGA FUCUS SPIRALIS (PHAEOPHYTA)1

The response of photosynthesis and respiration of the intertidal brown alga Fucus spiralis L. to light and temperature at ambient and elevated concentrations of inorganic carbon was investigated. The light-saturated rate of photosynthesis was greater in air at 15° C and 20° C, but greater in water at 10° C. Light compensation point and I_k was about 50% lower under submerged relative to emerged conditions, whereas the initial slope of photosynthesis versus irradiance was higher, except at 20° C. Under both submerged and emerged conditions light-saturated photosynthesis was limited to a similar degree (78%, and 65%, respectively) by the availability of inorganic carbon at naturally occuring concentrations. In air, slight desiccation at tissue water contents of about 96% to 92% caused a stimulation in the rate of net photosynthesis to 110–148% of fully hydrated fronds. At lower water contents the rate of net photosynthesis declined linearly with decreasing water content and became zero at a water content of about 15%. Dark respiration declined linearly with tissue water content and remained positive to a water content of 8%. Upon reimmersion the fronds showed a complete recovery within 35 min following desiccation to a water content of 20–30%. Thus F. spiralis seems to be very tolerant to desiccation. Since F. spiralis photosynthesizes effectively in air, even at a higher rate than in water as long as it has not lost a large proportion of its water in desiccation, the alternating exposure to air may be beneficial by increasing the daily carbon gain compared to a fully submerged situation.     

Photosynthesis of Ectocarpus siliculosus in red light and after pulses of blue light at high pH – evidence for bicarbonate uptake

Pulses of blue light cause stimulation of red light saturated photosynthesis in Ectocarpus siliculosus, because blue light activates the operation of a pathway for inorganic carbon (C_i) acquisition by inducing the mobilization of CO₂ from an intermediate metabolite. In the absence of exogenous C_i, photosynthetic rates roughly equal those of CO₂ release by respiration. In seawater of pH 9·5 (2·3 mol m^–3 total C_i, but concentrations of free CO₂ below 0·2 mmol m^–3), photosynthesis was clearly above these rates, although they were only ≈ 30% of those in normal seawater (≈ pH 8). The degree and the time course of the stimulations of photosynthesis by pulses of blue light were unaltered at high pH. Essentially the same characteristics were found after buffering or in the presence of acetazolamide, an inhibitor of extracellular carbonic anhydrase activity. Therefore, it is concluded that Ectocarpus is able to directly take up HCO₃^– in addition to CO₂ (uptake of CO₃^2– cannot be excluded). The dependence of photosynthesis on C_i at pH 9·5 was biphasic, with C_i below 0·2 mol m^–3 having no effect at all. In C_i-free seawater, the shapes of the stimulations after blue light pulses differed for pH 6, pH 8 and pH 9·5. At low pH, only the fast peak (maximum ≈ 5 min after blue light) was detected, whereas at high pH mainly the slow peak (maximum ≈ 20 min after blue light) was observed. At the intermediate pH 8, both peaks were present. As inhibition of total carbonic anhydrase by ethoxyzolamide brought out the fast peak of the stimulations at pH 9·5 it is concluded that the fast component was due to a transient disequilibrium of an intracellular pool of C_i which, after blue light, was fed by CO₂ released from the postulated storage intermediate.     

EFFECTS OF LIGHT AND TEMPERATURE ON NET PHOTOSYNTHESIS AND DARK RESPIRATION OF GAMETOPHYTES AND EMBRYONIC SPOROPHYTES OF MACROCYSTIS PYRIFERA1

The rates of net photosynthesis as a function of irradiance and temperature were determined for gametophytes and embryonic sporophytes of the kelp, Macrocystis pyrifera (L.) C. Ag. Gametophytes exhibited higher net photosynthetic rates based on oxygen and pH measurements than their derived embryonic sporophytes, but reached light saturation at comparable irradiance levels. The net photosynthesis of gametophytes reached a maximum of 66.4 mg O₂ g dry wt^?1 h^?1 (86.5 mg CO₂ g dry wt^?1 h^?1), a value approximately seven times the rate reported previously for the adult sporophyte blades. Gametophytes were light saturated at 70 μE m^?2 s^?1 and exhibited a significant decline in photosynthetic performance at irradiances 140 μE m^?1 s^?1. Embryonic sporophytes revealed a maximum photosynthetic capacity of 20.6 mg O₂ g dry wt^?1 h^?1 (25.3 mg CO₂ g dry wt^?1 h^?1), a rate about twice that reported for adult sporophyte blades. Embryonic sporophytes also became light saturated at 70 μE m^?2 s^?1, but unlike their parental gametophytes, failed to exhibit lesser photosynthetic rates at the highest irradiance levels studied; light compensation occurred at 2.8 μE m^?2 s^?1. Light-saturated net photosynthetic rates of gametophytes and embryonic sporophytes varied significantly with temperature. Gametophytes exhibited maximal photosynthesis at 15° to 20° C, whereas embryonic sporophytes maintained comparable rates between 10° and 20° C. Both gametophytes and embryonic sporophytes declined in photosynthetic capacity at 30° C. Dark respiration of gametophytes was uniform from 10° to 25° C, but increased six-fold at 30° C; the rates for embryonic sporophytes were comparable over the entire range of temperatures examined. The broader light and temperature tolerances of the embryonic sporophytes suggest that this stage in the life history of M. pyrifera is well suited for the subtidal benthic environment and for the conditions in the upper levels of the water column.     

Symbiodinium mitigate the combined effects of hypoxia and acidification on a noncalcifying cnidarian

Anthropogenic nutrient inputs enhance microbial respiration within many coastal ecosystems, driving concurrent hypoxia and acidification. During photosynthesis, Symbiodinium spp., the microalgal endosymbionts of cnidarians and other marine phyla, produce O₂ and assimilate CO₂ and thus potentially mitigate the exposure of the host to these stresses. However, such a role for Symbiodinium remains untested for noncalcifying cnidarians. We therefore contrasted the fitness of symbiotic and aposymbiotic polyps of a model host jellyfish (Cassiopea sp.) under reduced O₂ (~2.09 mg/L) and pH (~ 7.63) scenarios in a full‐factorial experiment. Host fitness was characterized as asexual reproduction and their ability to regulate internal pH and Symbiodinium performance characterized by maximum photochemical efficiency, chla content and cell density. Acidification alone resulted in 58% more asexual reproduction of symbiotic polyps than aposymbiotic polyps (and enhanced Symbiodinium cell density) suggesting Cassiopea sp. fitness was enhanced by CO₂‐stimulated Symbiodinium photosynthetic activity. Indeed, greater CO₂ drawdown (elevated pH) was observed within host tissues of symbiotic polyps under acidification regardless of O₂ conditions. Hypoxia alone produced 22% fewer polyps than ambient conditions regardless of acidification and symbiont status, suggesting Symbiodinium photosynthetic activity did not mitigate its effects. Combined hypoxia and acidification, however, produced similar numbers of symbiotic polyps compared with aposymbiotic kept under ambient conditions, demonstrating that the presence of Symbiodinium was key for mitigating the combined effects of hypoxia and acidification on asexual reproduction. We hypothesize that this mitigation occurred because of reduced photorespiration under elevated CO₂ conditions where increased net O₂ production ameliorates oxygen debt. We show that Symbiodinium play an important role in facilitating enhanced fitness of Cassiopea sp. polyps, and perhaps also other noncalcifying cnidarian hosts, to the ubiquitous effects of ocean acidification. Importantly we highlight that symbiotic, noncalcifying cnidarians may be particularly advantaged in productive coastal waters that are subject to simultaneous hypoxia and acidification.     

CORRESPONDENCE BETWEEN COLD TOLERANCE AND TEMPERATE BIOGEOGRAPHY IN A WESTERN ATLANTIC SYMBIODINIUM (DINOPHYTA) LINEAGE1

Many corals form obligate symbioses with photosynthetic dinoflagellates of the genus Symbiodinium Freudenthal (1962). These symbionts vary genotypically, with their geographical distribution and abundance dependent upon host specificity and tolerance to temperature and light variation. Despite the importance of these mutualistic relationships, the physiology and ecology of Symbiodinium spp. remain poorly characterized. Here, we report that rDNA internal transcribed spacer region 2 (ITS2) defined Symbiodinium type B2 associates with the cnidarian hosts Astrangia poculata and Oculina arbuscula from northerly habitats of the western Atlantic. Using pulse‐amplitude‐modulated (PAM) fluorometry, we compared maximum photochemical efficiency of PSII of type B2 to that of common tropical Symbiodinium lineages (types A3, B1, and C2) under cold‐stress conditions. Symbiont cultures were gradually cooled from 26°C to 10°C to simulate seasonal temperature declines. Cold stress decreased the maximum photochemical efficiency of PSII and likely the photosynthetic potential for all Symbiodinium clades tested. Cultures were then maintained at 10°C for a 2‐week period and gradually returned to initial conditions. Subsequent to low temperature stress, only type B2 displayed rapid and full recovery of PSII photochemical efficiency, whereas other symbiont phylotypes remained nonfunctional. These findings indicate that the distribution and abundance of Symbiodinium spp., and by extension their cnidarian hosts, in temperate climates correspond significantly with the photosynthetic cold tolerance of these symbiotic algae.     

PURIFICATION OF RIBULOSE 1,5-BISPHOSPHATE CARBOXYLASE AND CARBON ISOTOPE FRACTIONATION BY WHOLE CELLS AND CARBOXYLASE FROM CYLINDROTHECA SP. (BACILLARIOPHYCEAE.)1,2,3

Ribulose 1,5-biphosphate carboxylase has been purified to homogeneity from extracts of Cylindrotheca sp. (strain N-1), a marine, pennate diatom. The carboxylase has a molecular weight and structural composition similar to the enzyme from higher plants. When assayed in the presence of 1 mM NaHCO₃ the enzyme was stimulated nearly 40% by 1 mM aspartate and over 20% by 1 mM malate, and was inhibited to over 60% by 1 mM phosphoenolpyruvate. Similar experiments, using spinach carboxylase, failed to show activation by these metabolites. When assayed in the presence of 20 mM NaHCO₃, 6-phosphogluconate (1 mM) inhibited activity of ribulose bisphosphate carboxylase from Cylindrotheca by 60%, and higher concentrations of maiate (10 mM) inhibited activity by 25% Carbon isotope fractionation by ribulose bisphosphate carboxylase was -32.6% (ppt) when measured under N₂ using homogeneous enzyme, whereas maximum carbon isotope fractionation by the whole alga grown in 1% -C0₂-in air averaged - 16.8%. Carbon isotope fractionation by the whole alga varied with the density of the culture and was maximum at a low cell density (1.7 ± 10⁶ cellslml). At higher densities, the fractionation decreased by 4.0%. Carbon isotope fractionation has been used previously to determine the pathway of carbon metabolism in other organisms; the results of this investigation seem to indicate that this strain uses both the reductive pentose phosphate pathway and the C₄ carbon pathway for primary CO₂ fixation.     

PHOTOSYNTHESIS AND CALCIFICATION BY EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) AS A FUNCTION OF INORGANIC CARBON SPECIES

To test the possibility of inorganic carbon limitation of the marine unicellular alga Emiliania huxleyi (Lohmann) Hay and Mohler, its carbon acquisition was measured as a function of the different chemical species of inorganic carbon present in the medium. Because these different species are interdependent and covary in any experiment in which the speciation is changed, a set of experiments was performed to produce a multidimensional carbon uptake scheme for photosynthesis and calcification. This scheme shows that CO₂ that is used for photosynthesis comes from two sources. The CO₂ in seawater supports a modest rate of photosynthesis. The HCO is the major substrate for photosynthesis by intracellular production of CO₂ (HCO+ H⁺→ CO₂+ H₂O → CH₂O + O₂). This use of HCO is possible because of the simultaneous calcification using a second HCO, which provides the required proton (HCO+ Ca²⁺→ CaCO₃+ H⁺). The HCO is the only substrate for calcification. By distinguishing the two sources of CO₂ used in photosynthesis, it was shown that E. huxleyi has a K_½ for external CO₂ of “only” 1.9 ± 0.5 μM (and a V_max of 2.4 ± 0.1 pmol·cell⁻¹·d⁻¹). Thus, in seawater that is in equilibrium with the atmosphere ([CO₂]= 14 μM, [HCO]= 1920 μM, at fCO₂= 360 μatm, pH = 8, T = 15° C), photosynthesis is 90% saturated with external CO₂. Under the same conditions, the rate of photosynthesis is doubled by the calcification route of CO₂ supply (from 2.1 to 4.5 pmol·cell⁻¹·d⁻¹). However, photosynthesis is not fully saturated, as calcification has a K_½ for HCO of 3256 ± 1402 μM and a V_max of 6.4 ± 1.8 pmol·cell⁻¹·d⁻¹. The H⁺ that is produced during calcification is used with an efficiency of 0.97 ± 0.08, leading to the conclusion that it is used intracellularly. A maximum efficiency of 0.88 can be expected, as NO uptake generates a H⁺ sink (OH⁻ source) for the cell. The success of E. huxleyi as a coccolithophorid may be related to the efficient coupling between H⁺ generation in calcification and CO₂ fixation in photosynthesis.     

Strong seasonal patterns of DOC release by a temperate seaweed community: Implications for the coastal ocean carbon cycle

Release of dissolved organic carbon (DOC) by seaweed underpins the microbial food web and is crucial for the coastal ocean carbon cycle. However, we know relatively little of seasonal DOC release patterns in temperate regions of the southern hemisphere. Strong seasonal changes in inorganic nitrogen availability, irradiance, and temperature regulate the growth of seaweeds on temperate reefs and influence DOC release. We seasonally surveyed and sampled seaweed at Coal Point, Tasmania, over 1 year. Dominant species with or without carbon dioxide (CO₂) concentrating mechanisms (CCMs) were collected for laboratory experiments to determine seasonal rates of DOC release. During spring and summer, substantial DOC release (10.06–33.54 μmol C · g DW⁻¹ · h⁻¹) was observed for all species, between 3 and 27 times greater than during autumn and winter. Our results suggest that inorganic carbon (C_i) uptake strategy does not regulate DOC release. Seasonal patterns of DOC release were likely a result of photosynthetic overflow during periods of high gross photosynthesis indicated by variations in tissue C:N ratios. For each season, we calculated a reef-scale net DOC release for seaweed at Coal Point of 7.84–12.9 g C · m⁻² · d⁻¹ in spring and summer, which was ~16 times greater than in autumn and winter (0.2–1.0 g C · m⁻² · d⁻¹). Phyllospora comosa, which dominated the biomass, contributed the most DOC to the coastal ocean, up to ~14 times more than Ecklonia radiata and the understory assemblage combined. Reef-scale DOC release was driven by seasonal changes in seaweed physiology rather than seaweed biomass.     

INFLUENCE OF SALINITY AND TEMPERATURE ON GROWTH AND PHOTOSYNTHESIS IN THE EXTREMOPHILIC CHLOROPHYTE,NANNOCHLORIS SP.

Major, K. M. & Henley, W. J. Department of Botany, Oklahoma State University, Stillwater, OK 74078-3013 USA Preliminary data suggest Nannochloris sp., isolated from the Great Salt Plains National Wildlife Refuge, is a true extremophile. This alga is able to withstand salinities ranging from 0 to 150 ç and temperatures up to 45°C. To test the hypothesis that acclimation to high salinity confers tolerance to high temperature, experimental cultures were acclimated to salinities of 25 and 100 ç and/or temperatures of 23 and 38°C; irradiance (500 mol photons m^-2 s^-1) was saturating for both growth and photosynthesis. Cells acclimated to low salt and low temperature exhibited high photosynthetic performance in terms of both light-saturated photosynthesis (P_max; 45.0 fmol O₂ cell^-1 h^-1) and light-harvesting efficiency (0.103 fmol O₂ cell^-1 h^-1/mol photons m^-2 s^-1). However, high-salinity cells exhibited values for net P_max (18.1 fmol O₂ cell^-1 h^-1), (0.107 fmol O₂ cell^-1 h^-1/mol photons m^-2 s^-1) and growth rates (ca. 0.4 d^-1) that were equal to, or higher than, those of low-salinity cells when acclimated to high temperature. Both the amount of light required to achieve net photosynthesis (I_c) and that required to achieve light-saturated photosynthesis (I_k) were lower in high-salinity cells than those exhibited by low-salinity cells grown at high temperature; reductions in I_c and I_k were primarily due to increases in light-harvesting efficiency. We propose that an increase in growth temperature might release Nannochloris sp. from energy constraints associated with osmolyte production and low-temperature effects on enzyme activity. These data are consistent with effects of short-term temperature stress on Chl a fluorescence kinetics in this alga.     

Regulation of the mechanism for HCO 3 − use by the inorganic carbon level in Porphyra leucosticta Thur. in Le Jolis (Rhodophyta)

The capacity for HCO₃ ⁻ use by Porphyra leucosticta Thur. in Le Jolis grown at different concentrations of inorganic carbon (C_i) 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 C_i level at which the algae were grown. A high C_i level produced a reduction in total CA activity and a low C_i level produced an increase in total CA activity. In contrast, external CA was increased at low C_i although it was not affected at high C_i . Parallel to the reduction in total CA activity at high C_i is a reduction in the affinity for C_i, as estimated from photosynthesis versus C_i 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 C_i. 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 C_i-accumulation mechanism; (ii) there is a CO₂ transporter that works in association with external CA. Received: 20 April 1996 / Accepted: 5 August 1996