Brian Colman

The diversity of inorganic carbon acquisition mechanisms in eukaryotic microalgae

Eukaryotic microalgae have developed CO2concentrating mechanisms to maximise the concentration of CO2 at the active site of Rubisco in response to the low CO2 concentrations in the external aquatic medium. In these organisms, the modes of inorganic carbon (Ci) uptake are diverse, ranging from diffusive CO2 uptake to the active transport of HCO3 -and CO2 and many have an external carbonic anhydrase to facilitate HCO3- use. There is unequivocal evidence for the mechanisms of Ci uptake in only about 25 species of microalgae of the chlorophyte, haptophyte, rhodophyte, diatom, and eustigmatophyte groups. Most of these species take up both CO2 and HCO3-, but the rates of uptake of each of these substrates varies with the algal species. A few species take up only one of the two forms of Ci, an adaptation that is not necessarily correlated with their ecological distribution. Evidence is presented for the active uptake of HCO3- and CO2 in two marine haptophytes,Isochrysis galbana Parke and Dicrateria inornata Parke, and for active transport of CO2 but lack of HCO3- uptake in two marine dinoflagellates, Amphidinium carteraeHulburt and Heterocapsa oceanica Stein.

Photosynthetic carbon assimilation and the suppression of photorespiration in the cyanobacteria

Abstract The physiology and biochemistry of photosynthetic carbon assimilation in cyanobacteria is discussed in relation to its apparent suppression of photorespiration in these organisms. Experimental evidence indicates that CO 2 fixation in cyanobacteria is mediated by the C 3 pathway and although C 4 acids are among the initial products of photosynthesis, these organisms do not have the enzymatic capacity for the initial products of photosynthesis, these organisms of not have for the biosynthesis of amino acidsm necessitated by the absence of a complete TCA cycle in these organisms. The ribulose-1,5-bisphosphate carboxylase isolated from cyanobacteria has a low CO 2 affinity and is markedly inhibited by O 2 but it may be protected from O 2 inhibition by sequestration in carboxylasomes. There is no evidence that glycolate, the photo-respiratory substrate, is produced in air-grown cyanobacteria and a glycolate oxidation pathway, similar to that C 3 plants, appears to be lacking in these organisms. Excretion of glycolate by cyanobacteria is an artifact of growth on high CO 2 concentrations, or of experimental manipulation, and is not a normal consequence of photosynthesis. Cyanobacterial photosynthesis is not inhibited by O 2 and the cells have a high apparent affinity for CO 2 and very low CO 2 compensation points. No photorespiratory CO 2 release has been detected in cyanobacterial cells maintained at CO 2 concentrations above the CO 2 compensation point. Cyanobacteria take up extracellular HCO 3 - by a Na + and energy-dependent active transport mechanism which results in the intracellular accumulation of inorganic carbon 500–1000-fold the concentration outside the cells. There is also some evidence of a small component of active CO 2 uptake by cyanobacterial cells. The active intracellular accumulation of inorganic carbon may be sufficient to inhibit ribulose-1,5-bisphosphate oxygenase activity in cyanobacterial cells and therefore to suppress photorespiration. The evidence for bicarbonate uptake by cyanobacteria in nature is sparse. Laboratory studies suggest, however, that cyanobacteria growing under conditions of DIC depletion and high O 2 tension have the ability to take up bicarbonate and that photorespiration is suppressed under these conditions.

Active transport of CO2 by three species of marine microalgae

The occurrence of an active CO2 transport system and of carbonic anhydrase (CA) has been investigated by mass spectrometry in the marine, unicellular rhodophyte Porphyridium cruentum (S.F. Gray) Naegeli and two marine chlorophytes Nannochloris atomus Butcher and Nannochloris maculata Butcher. Illumination of darkened cells incubated with 100 μM H13CO3− caused a rapid initial drop, followed by a slower decline in the extracellular CO2 concentration. Addition of bovine CA to the medium raised the CO2 concentration by restoring the HCO3−–CO2 equilibrium, indicating that cells were taking up CO2 and were maintaining the CO2 concentration in the medium below its equilibrium value during photosynthesis. Darkening the cell suspensions caused a rapid increase in the extracellular CO2 concentration in all three species, indicating that the cells had accumulated an internal pool of unfixed inorganic carbon. CA activity was detected by monitoring the rate of exchange of 18O from 13C18O2 into water. Exchange of 18O was rapid in darkened cell suspensions, but was not inhibited by 500 μM acetazolamide, a membrane‐impermeable inhibitor of CA, indicating that external CA activity was not present in any of these species. In all three species, the rate of exchange was completely inhibited by 500 μM ethoxyzolamide, a membrane‐permeable CA‐inhibitor, showing that an intracellular CA was present. These results demonstrate that the three species are capable of CO2 uptake by active transport for use as a carbon source for photosynthesis.

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 K1/2, (CO2) of 0.3 μM. CO2 compensation point, measured in a closed system at pH 7.5, was a constant 6.7 m̈L · 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.

Measurements of photorespiration in some microscopic algae.

SummaryThe rate of photorespiration in three green algae and four blue-green algae was determined by the measurement of the rate of loss of photosynthetically fixed 14CO2 in light in CO2-free air at 25°. In all algae studied, CO2 evolution in light was considerably less than that in the dark, except for Chlamydomonas reinhardii which released slightly more CO2 in the light. Raising the temperature to 35° had little effect on the ratio of light to dark 14CO2 release. Blue-green algae showed the lowest photorespiration rate of the algae studied.

THE UPTAKE AND OXIDATION OF GLYCOLIC ACID BY BLUE‐GREEN ALGAE1

Two species of blue‐green algae Anabaena flosaquae and Oscillatoria sp. were shown to assimilate glycolic acid. In the presence of DCMU in light, approximately 50% of it wax oxidized to carbon dioxide; 90% was oxidized in the dark. Glycolate assimilation was increased fivefold by lowering the pH of the medium from 9.0 to 5.0, and the rate of uptake increased with increasing concentration of exogenous glycolate up to a saturation concentration of 12–14 mM. α‐Hydroxysulfonates markedly inhibited glycolate uptake and oxidation but iso‐nicotinyl hydrazide had little effect. These results indicate that glycolate oxidation occurs in vivo, but that the glycolate pathway in these algae differs some‐what from that of higher plants.

Source of inorganic carbon for photosynthesis in two marine dinoflagellates

Inorganic carbon uptake was investigated in two marine dinoflagellates, Amphidinium carterae Hulburt and Heterocapsa oceanica Stein. Mass spectrometric and potentiometric assays indicated that both species lacked external carbonic anhydrase (CA). The presence of internal CA was demonstrated by potentiometric assay and by the inhibition of photosynthesis upon the addition of 500 μM ethoxyzolamide a membrane‐permeable inhibitor of CA. The capacity for bicarbonate transport was investigated by comparing the calculated rate of spontaneous CO2 formation at pH 8.2 and 25°C with the rate of photosynthesis after the addition of 100 μM NaHCO3. Both species appeared to have a very limited capacity for direct bicarbonate uptake. Monitoring of CO2 and O2 fluxes in both species by mass spectrometry demonstrated a rapid uptake of CO2 on illumination, to concentrations below the CO2 equilibrium concentration, indicating an effective selective uptake of CO2. This dependence of photosynthesis on free CO2 alone suggests that these species are CO2 limited in their natural environment because the CO2 concentration of seawater is very low.

An investigation of glycolate excretion in two species of blue-green algae.

SummaryThe amount of 14C-glycolate excreted by Oscillatoria sp. and Anabaena flos-aquae is less than 1% of the 14C fixed by the algae during photosynthesis. Transfer of cells grown on 5% CO2 in air to a medium of low bicarbonate concentration or treatment of the cells with isonicotinyl hydrazide (INH) during photosynthesis, caused little increase in glycolate excretion. α-Hydroxysulfonates failed to stimulate massive excretion of glycolate. Although these blue-green algae excreted little glycolate, a significant proportion of the photosynthetically fixed carbon was excreted in the form of basic, neutral and acidic compounds, and such excretion was greater in 5% CO2-grown cells than in air-grown cells.

Effects of pH on growth, photosynthesis, respiration, and copper tolerance of three Scenedesmus strains

Abstract Our objective was to investigate the sensitivity of growth to acid pH, and to compare acid-sensitivity of growth, photosynthesis, and respiration of copper-tolerant (B-4, X-Cu) and copper-intolerant (X-72) strains of Scenedesmus acutus f. alternans . Further, we wanted to assess the effects of pH on copper toxicity. We hypothesized that B-4 and X-Cu will be more acid tolerant than X-72, and that growth will be more acid-sensitive than photosynthesis and respiration. Optimal growth of the three strains occurred at circumneutral pHs where growth rates were similar. Growth rates responded differently to decreasing pHs. In B-4, the most acid-tolerant strain, growth was reduced by 50% at pH 4.42. X-Cu was less tolerant since it did not grow at pH 4.5, and showed 50% growth reduction at pH 4.81. X-72 was not acid-tolerant. Growth was reduced by 50% at pH 5.05, and no growth occurred at pH 4.8 or less. Photosynthesis was less inhibited by acid pHs than growth but the strain ranking was the same: B-4 was most acid-tolerant, followed by X-Cu and X-72. A reduction in respiration with declining pH was observed in X-72 and B-4 but not in X-Cu, where respiration was unaffected by pH. CO 2 compensation points for photosynthesis in all three strains showed a general decrease with pH of growth, and were lower in B-4 and X-Cu than in X-72. The inhibitory effect of copper on photosynthesis in X-72 and B-4 was reduced at lower pHs; copper toxicity in X-Cu was unaffected by pH. These findings suggest that algae such as X-Cu with a metal-exclusion mechanism can also exclude protons. In such algae, high H + concentration in the medium does not ameliorate copper toxicity. In contrast, in non-tolerant species (e.g. X-72) and in species that accumulate copper (e.g. B-4) high H + concentrations in the range suitable for growth ameliorate copper toxicity, possibly by competing with the metal for binding sites on the cell surface thereby decreasing copper uptake.

The intracellular pH of isolated, photosynthetically active Asparagus mesophyll cells.

The intracellular pH of isolated, photosynthetically active mesophyll cells of Asparagus sprengeri Regel has been determined, in the light and dark, by the distribution of the weak acid 5,5-dimethyl-[2-14C]oxazolidine-2,4-dione ([14C]DMO) between the cells and the liquid medium. [14C]DMO was taken up rapidly, reaching equilibrium in 7–10 min of incubation, but was not metabolized by the cells, and intracellular binding of the compound was minimal. The intracellular pH, measured at saturating light fluence and 1.5 mM sodium bicarbonate, was found to remain relatively constant at 6.95–7.21 over the external pH range of 5.5–7.2. Illumination of the cells increased the intracellular pH compared to dark controls. The pH of the cytoplasm, excluding and including the chloroplasts (“cytoplasmic” and “bulk cytoplasmic”, respectively) was calculated from the experimentally derived intracellular [14C]DMO concentration and estimates of the vacuolar, chloroplastic and cytoplasmic volumes. The calculated cytoplasmic pH was similar in the light and dark, being 7.75 and 7.74, respectively, while the calculated pH of bulk cytoplasm was 7.85 in the light and 7.49 in the dark. Theoretical analysis indicated that intracellular pH is a good indicator of changes in the bulk cytoplasmic pH but insensitive to changes in vacuolar pH. The external pH optimum for photosynthesis (O2 evolution) of isolated Asparagus cells was pH 7.2. At pH 8.0 photosynthesis was inhibited by 30% and at pH 5.25 by 45%. Inhibition at alkaline pH may be the result of a decrease in the pH gradient between the cells and the medium, causing CO2 limitation in the cell. At acid pH, decrease in internal pH caused by substantial accumulation of inorganic carbon may account for the loss in photosynthetic activity.