Quantification of Extracellular Carbonic Anhydrase Activity in Two Marine Diatoms and Investigation of Its Role

Brian M. Hopkinson,Christof Meile,Chen Shen

doi:10.1104/pp.113.217737

Abstract

Many microalgae induce an extracellular carbonic anhydrase (eCA), associated with the cell surface, at low carbon dioxide (CO2) concentrations. This enzyme is thought to aid inorganic carbon uptake by generating CO2 at the cell surface, but alternative roles have been proposed. We developed a new approach to quantify eCA activity in which a reaction-diffusion model is fit to data on (18)O removal from inorganic carbon. In contrast to previous methods, eCA activity is treated as a surface process, allowing the effects of eCA on cell boundary-layer chemistry to be assessed. Using this approach, we measured eCA activity in two marine diatoms (Thalassiosira pseudonana and Thalassiosira weissflogii), characterized the kinetics of this enzyme, and studied its regulation as a function of culture pH and CO2 concentration. In support of a role for eCA in CO2 supply, eCA activity specifically responded to low CO2 rather than to changes in pH or HCO3(-), and the rates of eCA activity are nearly optimal for maintaining cell surface CO2 concentrations near those in the bulk solution. Although the CO2 gradients abolished by eCA are small (less than 0.5 μm concentration difference between bulk and cell surface), CO2 uptake in these diatoms is a passive process driven by small concentration gradients. Analysis of the effects of short-term and long-term eCA inhibition on photosynthesis and growth indicates that eCA provides a small energetic benefit by reducing the surface-to-bulk CO2 gradient. Alternative roles for eCA in CO2 recovery as HCO3(-) and surface pH regulation were investigated, but eCA was found to have minimal effects on these processes.

Highlights

CO2 supply, extracellular carbonic anhydrase (eCA) activity are nearly activity optimal for maintaining cell surface CO2 concentrations near those in the bulk solution
The model fits could not account for a depletion of the 13C18O16O intermediate species later in the assay (Fig. 2B). This signature suggests reduced exchange between the surface layer and the bulk solution (Silverman et al, 1981), and the fit can be improved by reducing the diffusive HCO32 flux to the cell surface
Consistent with a role for eCA in CO2 supply, we find that eCA is up-regulated at low CO2 concentrations and that induction occurs at a higher CO2 concentration in the larger diatom T. weissflogii, which is more prone to diffusive limitation

Summary

Introduction

CO2 supply, eCA activity are nearly activity optimal for maintaining cell surface CO2 concentrations near those in the bulk solution. Intracellular carbonic anhydrases (iCAs) play multiple roles in CCMs, including the conversion of accumulated HCO32 to CO2 around Rubisco and the prevention of CO2 leakage (Badger, 2003). ECA accelerates the dehydration of HCO32 to CO2 within the boundary layer, increasing the surface CO2 concentration. Support for this role has come from experiments showing that inhibition of eCA reduces photosynthetic rates and Ci accumulation in disparate microalgae, including the green alga C. reinhardtii, the dinoflagellate Prorocentrum micans, the prymnesiophyte Phaeocystis globosa, and the diatom Thalassiosira weissflogii (Moroney et al, 1985; Nimer et al, 1999; Elzenga et al, 2000; Burkhardt et al, 2001)

Methods

Results

Discussion

Conclusion