Seawater pH,and not inorganic nitrogen source,affects pH at the blade surface of Macrocystis pyrifera: implications for responses of the giant kelp to future oceanic conditions

Ocean acidification (OA), the ongoing decline in seawater pH, is predicted to have wide‐ranging effects on marine organisms and ecosystems. For seaweeds, the pH at the thallus surface, within the diffusion boundary layer (DBL), is one of the factors controlling their response to OA. Surface pH is controlled by both the pH of the bulk seawater and by the seaweeds' metabolism: photosynthesis and respiration increase and decrease pH within the DBL (pH_DBL), respectively. However, other metabolic processes, especially the uptake of inorganic nitrogen (N_i; NO₃^? and NH₄⁺) may also affect the pH_DBL. Using Macrocystis pyrifera, we hypothesized that (1) NO₃^? uptake will increase the pH_DBL, whereas NH₄⁺ uptake will decrease it, (2) if NO₃^? is cotransported with H⁺, increases in pH_DBL would be greater under an OA treatment (pH = 7.65) than under an ambient treatment (pH = 8.00), and (3) decreases in pH_DBL will be smaller at pH 7.65 than at pH 8.00, as higher external [H⁺] might affect the strength of the diffusion gradient. Overall, N_i source did not affect the pH_DBL. However, increases in pH_DBL were greater at pH 7.65 than at pH 8.00. CO₂ uptake was higher at pH 7.65 than at pH 8.00, whereas HCO₃^? uptake was unaffected by pH. Photosynthesis and respiration control pH_DBL rather than N_i uptake. We suggest that under future OA, Macrocystis pyrifera will metabolically modify its surface microenvironment such that the physiological processes of photosynthesis and N_i uptake will not be affected by a reduced pH.     

Abiotic influences on bicarbonate use in the giant kelp,Macrocystis pyrifera,in the Monterey Bay

In the Monterey Bay region of central California, the giant kelp Macrocystis pyrifera experiences broad fluctuations in wave forces, temperature, light availability, nutrient availability, and seawater carbonate chemistry, all of which may impact their productivity. In particular, current velocities and light intensity may strongly regulate the supply and demand of inorganic carbon (Ci) as substrates for photosynthesis. Macrocystis pyrifera can acquire and utilize both CO₂ and bicarbonate (HCO₃^?) as Ci substrates for photosynthesis and growth. Given the variability in carbon delivery (due to current velocities and varying [DIC]) and demand (in the form of saturating irradiance), we hypothesized that the proportion of CO₂ and bicarbonate utilized is not constant for M. pyrifera, but a variable function of their fluctuating environment. We further hypothesized that populations acclimated to different wave exposure and irradiance habitats would display different patterns of bicarbonate uptake. To test these hypotheses, we carried out oxygen evolution trials in the laboratory to measure the proportion of bicarbonate utilized by M. pyrifera via external CA under an orthogonal cross of velocity, irradiance, and acclimation treatments. Our Monterey Bay populations of M. pyrifera exhibited proportionally higher external bicarbonate utilization in high irradiance and high flow velocity conditions than in sub‐saturating irradiance or low flow velocity conditions. However, there was no significant difference in proportional bicarbonate use between deep blades and canopy blades, nor between individuals from wave‐exposed versus wave‐protected sites. This study contributes a new field‐oriented perspective on the abiotic controls of carbon utilization physiology in macroalgae.     

Ocean acidification and kelp development: Reduced pH has no negative effects on meiospore germination and gametophyte development of Macrocystis pyrifera and Undaria pinnatifida

The absorption of anthropogenic CO₂ by the oceans is causing a reduction in the pH of the surface waters termed ocean acidification (OA). This could have substantial effects on marine coastal environments where fleshy (non‐calcareous) macroalgae are dominant primary producers and ecosystem engineers. Few OA studies have focused on the early life stages of large macroalgae such as kelps. This study evaluated the effects of seawater pH on the ontogenic development of meiospores of the native kelp Macrocystis pyrifera and the invasive kelp Undaria pinnatifida, in south‐eastern New Zealand. Meiospores of both kelps were released into four seawater pH treatments (pH_T 7.20, extreme OA predicted for 2300; pH_T 7.65, OA predicted for 2100; pH_T 8.01, ambient pH; and pH_T 8.40, pre‐industrial pH) and cultured for 15 d. Meiospore germination, germling growth rate, and gametophyte size and sex ratio were monitored and measured. Exposure to reduced pH_T (7.20 and 7.65) had positive effects on germling growth rate and gametophyte size in both M. pyrifera and U. pinnatifida, whereas, higher pH_T (8.01 and 8.40) reduced the gametophyte size in both kelps. Sex ratio of gametophytes of both kelps was biased toward females under all pH_T treatments, except for U. pinnatifida at pH_T 7.65. Germling growth rate under OA was significantly higher in M. pyrifera compared to U. pinnatifida but gametophyte development was equal for both kelps under all seawater pH_T treatments, indicating that the microscopic stages of the native M. pyrifera and the invasive U. pinnatifida will respond similarly to OA.     

The induction of inorganic carbon transport and external carbonic anhydrase in Chlamydomonas reinhardtii is regulated by external CO2 concentration

Induction of the carbon concentrating mechanism (CCM) has been investigated during the acclimation of 5% CO₂‐grown Chlamydomonas reinhardtii 2137 mt + cells to well‐defined dissolved inorganic carbon (C_i) limited conditions. The CCM components investigated were active HCO₃^? transport, active CO₂ transport and extracellular carbonic anhydrase (CA_ext) activity. The CA_ext activity increased 10‐fold within 6 h of acclimation to 0·035% CO₂ and there was a further slight increase over the next 18 h. The CA_ext activity also increased substantially after an 8 h lag period during acclimation to air in darkness. Active CO₂ and HCO₃^? uptake by C. reinhardtii cells were induced within 2 h of acclimation to air, but active CO₂ transport was induced prior to active HCO₃^? transport. Similar results were obtained during acclimation to air in darkness. The critical C_i concentrations effecting the induction of active C_i transport and CA_ext activity were determined by allowing cells to acclimate to various inflow CO₂ concentrations in the range 0·035–0·84% at constant pH. The total C_i concentration eliciting the induction and repression of active C_i transport was higher during acclimation at pH 7·5 than at pH 5·5, but the external CO₂ concentration was the same at both pHs of acclimation. The concentration of external CO₂ required for the full induction and repression of C_i transport and CA_ext activity were 10 and 100 μM , respectively. The induction of CA_ext and active C_i transport are not correlated temporally, but are regulated by the same critical CO₂ concentration in the medium.     

Saturating light and not increased carbon dioxide under ocean acidification drives photosynthesis and growth in Ulva rigida (Chlorophyta)

Carbon physiology of a genetically identified Ulva rigida was investigated under different CO_2(aq) and light levels. The study was designed to answer whether (1) light or exogenous inorganic carbon (Ci) pool is driving growth; and (2) elevated CO_2(aq) concentration under ocean acidification (OA) will downregulate CA_ext-mediated dehydration and alter the stable carbon isotope (δ¹³C) signatures toward more CO₂ use to support higher growth rate. At pH_T 9.0 where CO_2(aq) is <1 μmol L⁻¹, inhibition of the known use mechanisms, that is, direct uptake through the AE port and CA_ext-mediated dehydration decreased net photosynthesis (NPS) by only 56–83%, leaving the carbon uptake mechanism for the remaining 17–44% of the NPS unaccounted. An in silico search for carbon-concentrating mechanism elements in expressed sequence tag libraries of Ulva found putative light-dependent transporters to which the remaining NPS can be attributed. The shift in δ¹³C signatures from –22‰ toward –10‰ under saturating light but not under elevated CO_2(aq) suggest preference and substantial use to support photosynthesis and growth. U. rigida is Ci saturated, and growth was primarily controlled by light. Therefore, increased levels of CO_2(aq) predicted for the future will not, in isolation, stimulate Ulva blooms.     

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.     

The role of external carbonic anhydrase in photosynthesis during growth of the marine diatom Chaetoceros muelleri

Carbon dioxide concentrating mechanisms (CCMs) act to improve the supply of CO₂ at the active site of ribulose‐1,5‐bisphosphate carboxylase/oxygenase. There is substantial evidence that in some microalgal species CCMs involve an external carbonic anhydrase (CA_ext) and that CA_ext activity is induced by low CO₂ concentrations in the growth medium. However, much of this work has been conducted on cells adapted to air‐equilibrium concentrations of CO₂, rather than to changing CO₂ conditions caused by growing microalgal populations. We investigated the role of CA_ext in inorganic carbon (C_i) acquisition and photosynthesis at three sampling points during the growth cycle of the cosmopolitan marine diatom Chaetoceros muelleri. We observed that CA_ext activity increased with decreasing C_i, particularly CO₂, concentration, supporting the idea that CA_ext is modulated by external CO₂ concentration. Additionally, we found that the contribution of CA_ext activity to carbon acquisition for photosynthesis varies over time, increasing between the first and second sampling points before decreasing at the last sampling point, where external pH was high. Lastly, decreases in maximum quantum yield of photosystem II (F_v/F_m), chlorophyll, maximum relative electron transport rate, light harvesting efficiency (α) and maximum rates of C_i‐ saturated photosynthesis (V_max) were observed over time. Despite this decrease in photosynthetic capacity an up‐regulation of CCM activity, indicated by a decreasing half‐saturation constant for CO₂ (K_0.5CO₂), occurred over time. The flexibility of the CCM during the course of growth in C. muelleri may contribute to the reported dominance and persistence of this species in phytoplankton blooms.     

The role of extracellular carbonic anhydrase for accumulation of inorganic carbon in the green alga Chlamydomonas reinhardtii. A comparison between wild-type and cell-wall-less mutant cells

The role of extracellular carbonic anhydrase (CA_ex) for dissolved inorganic carbon (DIC) accumulation in the green alga Chlamydomonas reinhardtii was investigated. It was found that when algal cells were bubbled with ambient air, cell-wall-less mutant cells exhibited the same high photosynthetic affinity for CO₂ as wild-type cells despite a 10 times lower activity of CA^ex. It was also found that the affinity for CO₂ was further increased when the total DIC concentration of the algal medium was reduced from that in equilibrium with ambient air to even lower levels. This increased affinity was not correlated with any further increase in the CA_ex activity. Dextran-bound sulfonamide (DBS. 100 μM bound ligand) completely inhibited the activity of CA_ex in intact, low-DIC grown, wild-type cells, while photosynthesis at <2 μM CO₂(aq) proceeded at a far greater rate than could be maintained by CO₂ supplied from the spontaneous dehydration of HCO^?₃. DBS-inhibition of CA_ex, during the induction of the DIC-accumulating mechanism in previously high-DIC grown cells, only caused a 50% inhibition of photosynthesis at 10 μM CO₂(aq) after 1 h of low-DIC acclimation. It was also shown that 50 μM acetazolamide (AZ) inhibited photosynthesis at low DIC concentrations to a relatively higher degree than DBS, suggesting that AZ inhibited intracellular CA as well. Taken together, these results suggest that low-DIC grown cells of C. reinhardtii have the ability to transport HCO^?₃ across the plasma membrane in addition to the CA_ex-mediated, facilitated diffusion and/or transport of CO₂. It is also suggested that the relative importance of these two fluxes (CO₂ or HCO^?₃) is dependent on the growth and experimental conditions. Facilitated CO₂ uptake seems to be most prevalent, supported by HCO^?₃-transport under more or less extreme situations, such as a reduction of CO₂ to extremely low concentrations, leakage of CA_ex to the medium as in cultures of cell-wall-less mutant cells or when the activity of CA_ex has been artificially inhibited.     

Microsensor studies on Padina from a natural CO2 seep: implications of morphology on acclimation to low pH

Low seawater pH can be harmful to many calcifying marine organisms, but the calcifying macroalgae Padina spp. flourish at natural submarine carbon dioxide seeps where seawater pH is low. We show that the microenvironment created by the rolled thallus margin of Padina australis facilitates supersaturation of CaCO₃ and calcifi‐cation via photosynthesis‐induced elevated pH. Using microsensors to investigate oxygen and pH dynamics in the microenvironment of P. australis at a shallow CO₂ seep, we found that, under saturating light, the pH inside the microenvironment (pH_ME) was higher than the external seawater (pH_SW) at all pH_SW levels investigated, and the difference (i.e., pH_ME ? pH_SW) increased with decreasing pH_SW (0.9 units at pH_SW 7.0). Gross photosynthesis (P_g) inside the microenvironment increased with decreasing pH_SW, but algae from the control site reached a threshold at pH 6.5. Seep algae showed no pH threshold with respect to P_g within the pH_SW range investigated. The external carbonic anhydrase (CA) inhibitor, acetazolamide, strongly inhibited P_g of P. australis at pH_SW 8.2, but the effect was diminished under low pH_SW (6.4–7.5), suggesting a greater dependence on membrane‐bound CA for the dehydration of HCO₃^? ions during dissolved inorganic carbon uptake at the higher pH_SW. In comparison, a calcifying green alga, Halimeda cuneata f. digitata, was not inhibited by AZ, suggesting efficient bicarbonate transport. The ability of P. australis to elevate pH_ME at the site of calcification and its strong dependence on CA may explain why it can thrive at low pH_SW.     

Carbonate ions appear to neither inhibit nor stimulate use of bicarbonate ions in photosynthesis by Ulva lactuca

Rates of photosynthesis by the marine macroalga Ulva lactuca were measured in a factorial experiment at five concentrations of HCO₃^? and CO₃^2- between 0·20 and 1·26 mol m^?3, but very low concentrations of CO₂. The results demonstrated that HCO₃^? was available for use, but an analysis of variance showed that CO₃^2- had neither an inhibiting nor a stimulating effect on rates of photosynthesis over this concentration range. Over the experiment, pH varied from 8·46 to 10·06 and this also had no significant effect on rates of photosynthesis. The lack of a stimulatory effect of high concentrations of CO₃^2- on the rate of photosynthesis at low concentrations of HCO₃^? was taken as circumstantial evidence for direct uptake of HCO₃^? rather than proton extrusion and external production of CO₂. In the rockpools in which U. lactuca grows, pH values up to 10·35 have been recorded, and for much of the time, CO₃^2- was the major form of inorganic carbon available. The apparent lack of an ability to use CO₃^2- under these conditions suggests that direct use of CO₃^2- as a source of inorganic carbon for photosynthesis is unlikely to be widespread.     

CARBON‐USE STRATEGIES IN MACROALGAE: DIFFERENTIAL RESPONSES TO LOWERED PH AND IMPLICATIONS FOR OCEAN ACIDIFICATION1

Ocean acidification (OA) is a reduction in oceanic pH due to increased absorption of anthropogenically produced CO₂. This change alters the seawater concentrations of inorganic carbon species that are utilized by macroalgae for photosynthesis and calcification: CO₂ and HCO₃^? increase; CO₃^2? decreases. Two common methods of experimentally reducing seawater pH differentially alter other aspects of carbonate chemistry: the addition of CO₂ gas mimics changes predicted due to OA, while the addition of HCl results in a comparatively lower [HCO₃^?]. We measured the short‐term photosynthetic responses of five macroalgal species with various carbon‐use strategies in one of three seawater pH treatments: pH 7.5 lowered by bubbling CO₂ gas, pH 7.5 lowered by HCl, and ambient pH 7.9. There was no difference in photosynthetic rates between the CO₂, HCl, or pH 7.9 treatments for any of the species examined. However, the ability of macroalgae to raise the pH of the surrounding seawater through carbon uptake was greatest in the pH 7.5 treatments. Modeling of pH change due to carbon assimilation indicated that macroalgal species that could utilize HCO₃^? increased their use of CO₂ in the pH 7.5 treatments compared to pH 7.9 treatments. Species only capable of using CO₂ did so exclusively in all treatments. Although CO₂ is not likely to be limiting for photosynthesis for the macroalgal species examined, the diffusive uptake of CO₂ is less energetically expensive than active HCO₃^? uptake, and so HCO₃^?‐using macroalgae may benefit in future seawater with elevated CO₂.     

The active uptake of carbon dioxide by the marine diatoms Phaeodactylum ticornutum and Cyclotella sp.

Mass spectromelry has been used to investigate the uptake of CO₂ by two marine diatoms, Phaeodactylum tricornutum and Cyclotella sp. The time course of CO₂ formation in the dark after addition of 100 mmol m^?3 dissolved inorganic carbon (DIC) to cell suspensions showed that external carbonic anhydrase (CA) was not present in cells of P. tricornutum but was present in Cyclotella sp. In the absence of external CA, or when it was inhibited by 5 mmol m^?3 acetazolamide, cells of both species preincubated with 100 mmol m^?3 DIG rapidly depleted almost all of the free CO₂ (3·2mmol m^?31 at pH7·5) from the suspending medium within seconds of illumination and prior to the onset of steady-state photosynthesis. Addition of bovine CA quickly restored the HCO₃^?–CO₂ equilibrium in the medium, indicating that the initial depletion of CO₂ resulted from the selective uptake of CO₂ rather than uptake of all DIG species. Transfer of cells to the dark caused a rapid increase in the CO₂ concentration in the medium, largely as a result of the efflux of unfixed inorganic carbon from the cells. The measured CO₂ uptake rates for both species accounted for 50% of the total DIG uptake at HCO₃^?–CO₂ equilibrium, indicating that HCOHCO₃^? was also being taken up. These results indicate that both Phaeodactylum tricornutum and Cyclotella sp. have the capacity to transport CO₂ actively against concentration and pH gradients.     

Affinity, capacity and oxygen sensitivity of two different mechanisms for bicarbonate utilization in Ulva lactuca L. (Chlorophyta)

Ulva lactuca, collected on the west coast of Sweden at the end of May, was able to utilize the HCO₃ ^? pool of seawater only through extracellular dehydration via carbonic anhydrase, followed by uptake of the CO₂ formed. A decrease in the CO₂ supply via this mechanism resulted in the gradual development of an additional method of HCO₃ ^? utilization, namely a direct uptake of HCO₃ ^? . Photosynthesis could then be supported by either a ‘HCO₃ ^? dehydration mechanism’ or a ‘HCO₃ ^? uptake mechanism’. Through selective inhibition of either of these mechanisms, the physiological properties of the other could be assessed. These properties suggest that the HCO₃ ^? uptake mechanism of U. lactuca is important under conditions when low concentrations of inorganic C, high pH and high external O₂ concentrations would limit photosynthesis supported by the HCO₃ ^? dehydration mechanism. Such conditions may occur during intense irradiation of the alga in rockpools or in shallow bays with low rates of water exchange. The results are discussed in relation to a possible coupling between mechanisms for inorganic C acquisition and cell structure (or even morphology) of green macroalgae. They also illustrate some necessary precautions when using Michaelis–Menten kinetics for estimations of V_max and K_1/2 values.     

Regulation of the induction of bicarbonate uptake by dissolved CO2 in the marine diatom, Phaeodactylum tricornutum

Physiological properties of photosynthesis were determined in the marine diatom, Phaeodactylum tricornutum UTEX640, during acclimation from 5% CO₂ to air and related to H₂CO₃ dissociation kinetics and equilibria in artificial seawater. The concentration of dissolved inorganic carbon at half maximum rate of photosynthesis (K_0·5[DIC]) value in high CO₂‐grown cells was 1009 mmol m ^{? 3} but was reduced three‐fold by the addition of bovine carbonic anhydrase (CA), whereas in air‐grown cells K_0·5[DIC] was 71 mmol m ^{? 3}, irrespective of the presence of CA. The maximum rate of photosynthesis (P_max) values varied between 300 and 500 μ mol O₂ mg Chl ^{? 1} h ^{? 1} regardless of growth pCO₂. Bicarbonate dehydration kinetics in artificial seawater were re‐examined to evaluate the direct HCO₃ ^? uptake as a substrate for photosynthesis. The uncatalysed CO₂ formation rate in artificial seawater of 31·65°/_oo of salinity at pH 8·2 and 25 °C was found to be 0·6 mmol m ^{? 3} min ^{? 1} at 100 mmol m ^{? 3} DIC, which is 53·5 and 7·3 times slower than the rates of photosynthesis exhibited in air‐ and high CO₂‐grown cells, respectively. These data indicate that even high CO₂‐grown cells of P. tricornutum can take up both CO₂ and HCO₃ ^? as substrates for photosynthesis and HCO₃ ^? use improves dramatically when the cells are grown in air. Detailed time courses were obtained of changes in affinity for DIC during the acclimation of high CO₂‐grown cells to air. The development of high‐affinity photosynthesis started after a 2–5 h lag period, followed by a steady increase over the next 15 h. This acclimation time course is the slowest to be described so far. High CO₂‐grown cells were transferred to controlled DIC conditions, at which the concentrations of each DIC species could be defined, and were allowed to acclimate for more than 36 h. The K_0·5[DIC] values in acclimated cells appeared to be correlated only with [CO_2(aq)] in the medium but not to HCO₃ ^? , CO₃^{2 ?} , total [DIC] or the pH of the medium and indicate that the critical signal regulating the affinity of cells for DIC in the marine diatom, P. tricornutum, is [CO_2(aq)] in the medium.     

UPTAKE OF INORGANIC CARBON BY CLADOPHORA GLOMERATA (CHLOROPHYTA) FROM THE BALTIC SEA1

Carbon uptake in the green macroalga Cladophora glomerata (L.) Kütz. from the brackish Baltic Sea was studied by recording changes in pH, alkalinity, and inorganic carbon concentration of the seawater medium during photosynthesis. The use of specific inhibitors identified three uptake mechanisms: 1) dehydration of HCO₃ ^? into CO₂ by periplasmic carbonic anhydrase, followed by diffusion of CO₂ into the cell; 2) direct uptake of HCO₃ ^? via a 4,4′‐diisothiocyanato‐stilbene‐2,2′‐disulfonate‐sensitive mechanism; and 3) uptake of inorganic carbon by the involvement of a vanadate‐sensitive P‐type H ⁺ ‐ATPase (proton pump). A decrease in the alkalinity of the seawater medium during carbon uptake, except when treated with vanadate, indicated a net uptake of the ionic species contributing to alkalinity (i.e. HCO₃ ^? , CO₃^{2 ?} , and OH ^? ) from the medium, where OH ^? influx is equivalent to H ⁺ efflux. This would suggest that the proton pump is involved in HCO₃ ^? transport. We also show that the proton pump can be induced by carbon limitation. The inducibility of carbon uptake in C. glomerata may partly explain why this species is so successful in the upper littoral zone of the Baltic Sea. Usually, carbon limitation is not a problem in the upper littoral of the sea. However, it may occur frequently within dense Cladophora belts with high photosynthetic rates that create high pH and low carbon concentrations in the alga's microenvironment.     

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.     

Mechanisms of inorganic carbon acquisition in Gracilaria gaditana nom. prov. (Rhodophyta)

The mechanisms for acquisition of dissolved inorganic carbon (DIC) in the red macroalga Gracilaria gaditana nom. prov. have been investigated. The capacity for HCO₃ ⁻ use by an extracellular carbonic anhydrase (CA; EC 4.2.1.1), and by an anion exchanger with similar properties to that of red blood cells (AE1), has been quantified. It was illustrated by comparing O₂ evolution rates with those theoretically supported by CO₂, as well as by photosynthesis-pH curves. Both external and internal CA, and a direct uptake were involved in HCO₃ ⁻ use, since photosynthesis and pH evolution were affected by acetazolamide, 6-ethoxyzolamide (inhibitors of external and total CA, respectively) and 4,4′-diisothiocyanatostilbene-2,2′-disulfonate, (DIDS; an inhibitor of HCO₃ ⁻ exchanger protein). The activity of the external CA was detected by a potentiometric method and by an alternative method based on the study of O₂ evolution after addition of CO₂ and acetazolamide. The latter method showed a residual photosynthetic rate due to direct HCO₃ ⁻ use. Inhibitors caused a reduction in the pH compensation points in pH-drift experiments. The CO₂ compensation points for photosynthesis increased when the inhibitors were applied, indicating a suppresion of the pathways involved in the carbon-concentrating mechanism. The net photosynthesis rates as a function of DIC concentration displayed a biphasic pattern that could be supported by the occurrence of the two mechanisms of HCO₃ ⁻ use. The potential contribution to HCO₃ ⁻ acquisition by the DIDS-sensitive mechanism was higher after culturing at a high pH. Our results suggest that the HCO₃ ⁻ use by Gracilaria gaditana is carried out by the two DIC uptake mechanisms. These operate simultaneously with different affinities for DIC, the indirect HCO₃ ⁻ use by an external CA activity being the main pathway. The presence of a carbon-concentrating mechanism confers eco-physiological advantages in a fluctuating ecosystem subjected daily to high pHs and low DIC concentrations. Received: 3 July 1998 / Accepted: 30 November 1998     

Photosynthetic inorganic carbon uptake and accumulation in two marine diatoms

Some physiological characteristics of photosynthetic inorganic carbon uptake have been examined in the marine diatoms Phaeodactylum tricornutum and Cyclotella sp. Both species demonstrated a high affinity for inorganic carbon in photosynthesis at pH7.5, having K_1/2(CO₂) in the range 1.0 to 4.0mmol m^?3 and O_2? and temperature-insensitive CO₂ compensation concentrations in the range 10.8 to 17.6 cm³ m^?3. Intracellular accumulation of inorganic carbon was found to occur in the light; at an external pH of 7.5 the concentration in P. tricornutum was twice, and that in Cyclotella 3.5 times, the concentration in the suspending medium. Carbonic anhydrase (CA) was detected in intact Cyclotella cells but not in P. tricornutum, although internal CA was detected in both species. The rates of photosynthesis at pH 8.0 of P. tricornutum cells and Cyclotella cells treated with 0.1 mol m^?3 acetazolamide, a CA inhibitor, were 1.5- to 5-fold the rate of CO₂ supply, indicating that both species have the capacity to take up HCO₃^? as a source of substrate for photosynthesis. No Na⁺ dependence for HCO₃^? could be detected in either species. These results indicate that these two marine diatoms have the capacity to accumulate inorganic carbon in the light as a consequence, in part, of the active uptake of bicarbonate.     

Effects of pH and Repellent Tactic Stimuli on Protein Methylation Levels in Escherichia coli

Intracellular pH (pH_int) and extracellular pH (pH_ext) of Escherichia coli were measured at 12-s time resolution by ³¹P-nuclear magnetic resonance: a sudden neutral-to-acid shift in pH_ext (e.g., from 7.0 to 5.6) caused a transient failure of homeostasis, with pH_int decreasing by about 0.4 unit in ca. 30 s and then returning to its original value (ca. 7.5) over a period of several minutes. Membrane proton conductance was estimated to be 20 pmol s⁻¹ cm⁻² pH unit⁻¹. Addition of the membrane-permeant weak acid benzoate at constant pH_ext also caused a lowering of pH_int; at high concentrations it generated an inverted transmembrane pH gradient (ΔpH). The buffering capacity of the cells was estimated by such experiments to be ca. 50 mM per pH unit. Effects of pH-related stimuli on the methyl-accepting chemotaxis proteins (MCPs) were examined: the steady-state methylation of MCP I was found to decrease when pH_int was lowered by weak acid addition or when pH_ext was lowered. The extent of demethylation in the latter case was too great to be explained by imperfect steady-state homeostasis; a small but reproducible undershoot in methylation level correlated with the observed short-term homeostatic failure. MCP II underwent smaller and more complex changes than MCP I, in response to pH-related stimuli. The methylation level of MCP I could not, by any condition tested, be driven below a limit of ca. 15% of the control level (unstimulated cells at pH_ext 7.0). The weak-acid concentration needed to reach that limit was dependent on pH_ext, as would be expected on the basis of ΔpH-driven concentrative effects. The potency ranking of weak acids was the same with respect to lowering pH_int, demethylating MCP I, and causing repellent behavioral responses. The data are consistent with a model whereby MCP I and hence tactic behavior are sensitive to both pH_int and pH_ext. Evidence is presented that pH_int may also have a direct (non-MCP-related) effect on motor function. Comparison of methyl-³H- and ³⁵S-labeled MCP I revealed that in both unstimulated and repellent-stimulated cells the major species did not carry methyl label, yet it had an electrophoretic mobility that indicated that it was more positively charged than the unmethylated form observed in methyltransferase mutants, and it was susceptible to base hydrolysis. This suggests that a substantial fraction of MCP I molecules is methylated or otherwise modified but neither exchanges methyl label nor undergoes reverse modification by repellent stimuli.     

Influx and efflux of inorganic carbon in Synechococcus UTEX625

The CO₂ and HCO₃^? fluxes in air-grown cells of Synechococcus UTEX 625 al pH 8-0 were measured during dark to light and light to dark transitions using a mass spectrometer and sampling of the reaction medium. The kinetic parameters for initial uptake of CO₂ and HCO₃^? were determined during the initial period of illumination. The development of the internal C_i pool was followed up to steady-state photosynthesis, which occurred when the size of the internal inorganic carbon pool remained apparently constant for a limited period. The experimental procedure confirmed that only CO₂ transport occurred with 100mmolm^?3 Na⁺ and that both CO₂ and HCO^?3 transport occurred with 25molm^?3 Na⁺. The K_1/2 values of initial CO₂ and HCO₃ uptake were 0.7 and 17.2 mmolm^?3respectively and agreed closely with the K_1/2 values of net CO₂ and HCO₃^? transport during steady-state photosynthesis, which were 0.66 and 17.1 mmolm^?3 respectively. Maximum rates of CO₂and HCO₃^? transport were 423 and 219mmolh^?1 g^?1 Chl. Maximum CO₂ efflux observed upon darkening was 118mmolh^?1 g^?1 Chl. A permeability coefficient of the cell for CO₂ of 3 × 10^?8 m s^?1 was determined from the dark CO₂ efflux assuming an internal pH of 7.2 in the dark. Following the initial CO₂ uptake in the light, the extracellular [CO₂] steadily declined when only CO₂ transport was allowed, but an increase in the extracellular [CO₂] when HCO₃^? transport was allowed to proceed suggested that an enhanced CO₂ efflux occurred as a result of the larger size of the intracellular C_i pool.