Shabana Bhatti

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 INORGANIC CARBON ACQUISITION IN AN ACID-TOLERANT, FREE-LIVING SPECIES OF COCCOMYXA (CHLOROPHYTA)1

The processes of CO2 acquisition were characterized for the acid‐tolerant, free‐living chlorophyte alga, CPCC 508. rDNA data indicate an affiliation to the genus Coccomyxa, but distinct from other known members of the genus. The alga grows over a wide range of pH from 3.0 to 9.0. External carbonic anhydrase (CA) was detected in cells grown above pH 5, with the activity increasing marginally from pH 7 to 9, but most of the CA activity was internal. The capacity for HCO3− uptake of cells treated with the CA inhibitor acetazolamide (AZA), was investigated by comparing the calculated rate of uncatalyzed CO2 formation with the rate of photosynthesis. Active bicarbonate transport occurred in cells grown in media above pH 7.0. Monitoring CO2 uptake and O2 evolution by membrane‐inlet mass spectrometry demonstrated that air‐grown cells reduced the CO2 concentration in the medium to an equilibrium concentration of 15 μM, but AZA‐treated cells caused a drop in extracellular CO2 concentration to a compensation concentration of 27 μM at pH 8.0. CO2‐pulsing experiments with cells in the light indicated that the cells do not actively take up CO2. An internal pool of unfixed inorganic carbon was not detected at the CO2 compensation concentration, probably because of the lack of active CO2 uptake, but was detectable at times before compensation point was reached. These results indicate that this free‐living Coccomyxa possesses a CO2‐concentrating mechanism (CCM) due to an active bicarbonate‐uptake system, unlike the Coccomyxa sp. occurring in symbiotic association with lichens.

ACQUISITION OF INORGANIC CARBON BY THE MARINE HAPTOPHYTE ISOCHRYSIS GALBANA (PRYMNESIOPHYCEAE)1

Inorganic carbon acquisition has been investigated in the marine haptophyte Isochrysis galbana. External carbonic anhydrase (CA) was present in air‐grown (0.034% CO2) cells but completely repressed in high (3%) CO2‐grown cells. External CA was not inhibited by 1.0 mM acetazolamide. The capacity of cells to take up bicarbonate was examined by comparing the rate of photosynthetic O2 evolution with the calculated rate of spontaneous CO2 supply; at pH 8.2 the rates of O2 evolution exceeded the CO2 supply rate 14‐fold, indicating that this alga was able to take up HCO3−. Monitoring CO2 concentrations by mass spectrometry showed that suspensions of high CO2‐grown cells caused a rapid drop in the extracellular CO2 in the light and addition of bovine CA raised the CO2 concentration by restoring the HCO3−‐CO2 equilibrium, indicating that cells were maintaining the CO2 in the medium below its equilibrium value during photosynthesis. A rapid increase in extracellular CO2 concentration occurred on darkening the cells, indicating that the cells had accumulated an internal pool of unfixed inorganic carbon. Active CO2 uptake was blocked by the photosynthetic electron transport inhibitor 3‐(3′,4′‐dichlorphenyl)‐1,1‐dimethylurea, indicating that CO2 transport was supported by photosynthetic reactions. These results demonstrate that this species has the capacity to take up HCO3− and CO2 actively as sources of substrate for photosynthesis and that inorganic carbon transport is not repressed by growth on high CO2, although external CA expression is regulated by CO2 concentration.

Inorganic carbon acquisition in some synurophyte algae.

Some characteristics of photosynthesis of three synurophyte algae, Synura petersenii, Synura uvella and Tessellaria volvocina were investigated to determine the mechanism of inorganic carbon (C(i)) uptake. All three species were found to have no external carbonic anhydrase, no capacity for direct bicarbonate uptake and a low whole-cell affinity for C(i). The internal pH of S. petersenii determined using (14)C-benzoic acid and [2-(14)C]-5,5-dimethyloxazolidine-2,4-dione was pH 7.0-7.5, over an external pH range of 5.0-7.5. Thus, the pH difference between the cell interior of S. petersenii and the external medium was large enough, over the algas growth range, to allow the accumulation of C(i) by the diffusive uptake of CO(2). Monitoring O(2) evolution and CO(2) uptake by suspensions of S. petersenii at pH 7.0 by mass spectrometry did not indicate a rapid uptake of CO(2), and the final CO(2) compensation concentration reached was 24 +/- 0.7 microM. Furthermore, when the cells were darkened, a brief burst of CO(2) occurred before a steady rate of dark respiration was established, suggesting a loss of CO(2) by photorespiration. An examination of the kinetics of ribulose-1,5-bisphosphate carboxylase/oxygenase in homogenates of cells of S. petersenii, S. uvella and Mallomonas papillosa showed that values of the K(m) (CO(2)) were 28.4, 41.8 and 18.2 microM, respectively. These species lack the characteristics of cells with a CO(2)-concentrating mechanism because the cell affinity for C(i) appears to be determined by the relatively high CO(2) affinity of the Rubisco of these algae.

Performance limitations from delay in human and mechanical motor control

We discuss natural limitations on motor performance caused by the time delay required for feedback signals to propagate within the human body or mechanical control systems. By considering a very simple delayed linear servomechanism model, we show there exists a best possible speed-accuracy trade-off similar to Fitts’ law that cannot be exceeded when delay is present. This is strictly a delay effect and does not occur for the ideal case of instantaneous feedback. We then examine the performance of the vector integration to endpoint (VITE) circuit as a model of human movement and show that when this circuit is generalized to include delayed feedback the performance may not exceed that of the servomechanism with an equal delay. We suggest the existence of such a limitation may be a ubiquitous consequence of delay in motor control with the implication that the index of performance in Fitts’ law cannot arbitrarily large.

Inorganic carbon acquisition in two species of marine prymnesiophytes

Inorganic carbon acquisition has been investigated in the marine prymnesiophytes Dicrateria inornata and Ochrosphaera neapolitana. The presence of external and internal carbonic anhydrase (CA) was detected in air-grown cells of D. inornata by potentiometric assay and corroborated by mass spectrometry. Growth on 3% CO2 repressed external CA activity. Extracts of air-grown cells of O. neapolitana showed considerable internal CA activity, but no external CA activity was detected by mass spectrometry. Illumination of air-grown cells of D. inornata incubated with 100 μM H13CO3 and treated with the CA inhibitor acetazolamide caused a rapid drop in the extracellular CO2 concentration to a level below its equilibrium concentration indicating that cells were actively taking up CO2 from the medium. The rate of O2 evolution at the CO: compensation concentration was higher than could be supported by the uncatalysed formation of CO2 from HCO3, . demonstrating that the cells were taking up HCO3 −directly. Similar results were obtained with air-grown cells of O. neapolitana. Active CO2 transport in both species was not repressed by growth under high levels of CO2. The photosynthetic electron transport inhibitor 3-(3′ 4′-dichlorophenyl)-l, 1-dimethylurea (DCMU) inhibited CO2 uptake in air-grown cells of both species, demonstrating that the energy to support active CO2 transport was derived from photosynthetic linear electron transport. These results show that the physiological characteristics of the CO2 concentrating mechanism in related species do not appear to be correlated with the availability of CO2 in the surrounding medium.

Characterization of the CO2-concentrating mechanism in the unicellular alga Eustigmatos vischeri

The presence and characteristics of the CO2-concentrating mechanism in the freshwater eustigmatophyte Eustigmatos vischeri was investigated. The capacity of cells to remove CO2 actively from the medium was established by mass spectrometry; illumination of a cell suspension incubated with inorganic carbon (Ci) caused a rapid drop in the extracellular CO2 concentration to levels below its equilibrium value with bicarbonate. No activity of external carbonic anhydrase was detected in these experiments. The occurrence of the active uptake of CO2 was corroborated when cells were pulsed with pure CO2 in the light and in the dark and the rates of CO2 disappearance were faster than those measured in a buffer solution. CO2 transport in the light and in the dark was inhibited by low concentrations (100 μM) of the mitochondrial respiration inhibitor cyanide, and cells treated with azide, which effectively blocks mitochondrial complex IV, also lost their capacity to remove CO2. The ability of cells to take up bicarbonate was also examined by comparing the rate of photosynthetic O2 evolution with the calculated rate of spontaneous CO2 supply; at pH 8.0, the rates of O2 evolution exceeded the CO2 supply rate 4-fold, indicating that this alga was able to take up . Cyanide and azide also inhibited the uptake of . These results demonstrate that E. vischeri uses both CO2 and as external carbon sources for photosynthesis and indicate a respiratory control of the Ci acquisition process.