Analysis of Chlamydomonas mutants with abnormal expression of CO2 and HCO3- uptake systems

Abstract

Eukaryotic microalgae such as Chlamydomonas reinhardtii possess an inducible CO₂ concentrating mechanism that operates as a very close interaction between pyrenoid-based Rubisco, various carbonic anhydrases (CAs), and inorganic carbon (Ci) transport systems. While external and internal CAs have been characterised to the molecular level, the biochemistry and molecular biology of Ci uptake mechanisms have not been elucidated. Both Ci species, CO₂ and HCO₃^-, are taken up by the cells and chloroplasts during steady-state photosynthesis. After acclimation to limiting Ci, CO₂ and HCO₃^- transport, measured in whole cells or chloroplasts, change their kinetic characteristics from a constitutive low-affinity state to an inducible high-affinity state. In order to learn more about the genes involved in the signal transduction pathway and in the Ci transport systems, we performed insertional mutagenesis using the arg7 gene as a selectable marker. Application of aqueous membrane inlet mass spectrometry allowed discrimination between CO₂ and HCO₃^- uptake. Data is presented on two mutants, M46 and M21, which show severe damage to the constitutive Ci uptake systems and which are unable to induce a high-affinity state. The mutations might be either in the signal transduction pathway or in the transporters themselves. In addition, we present data that shows a very close connection between high-affinity HCO₃^- uptake and high-affinity NO₃^- uptake in cells of C. reinhardtii.