Localization of putative carbonic anhydrases in two marine diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana

It is believed that intracellular carbonic anhydrases (CAs) are essential components of carbon concentrating mechanisms in microalgae. In this study, putative CA-encoding genes were identified in the genome sequences of the marine diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. Subsequently, the subcellular localizations of the encoded proteins were determined. Nine and thirteen CA sequences were found in the genomes of P. tricornutum and T. pseudonana, respectively. Two of the beta-CA genes in P. tricornutum corresponded to ptca1 and ptca2 identified previously. Immunostaining transmission electron microscopy of a PtCA1: YFP fusion expressed in the cells of P. tricornutum clearly showed the localization of PtCA1 within the central part of the pyrenoid structure in the chloroplast. Besides these two beta-CA genes, P. tricornutum likely contains five alpha- and two gamma-CA genes, whereas T. pseudonana has three alpha-, five gamma-, four delta-, and one zeta-CA genes. Semi-quantitative reverse transcription PCR performed on mRNA from the two diatoms grown in changing light and CO2 conditions revealed that levels of six putative alpha- and gamma-CA mRNAs in P. tricornutum did not change between cells grown in air-level CO2 and 5% CO2. However, mRNA levels of one putative alpha-CA gene, CA-VII in P. tricornutum, were reduced in the dark compared to that in the light. In T. pseudonana, mRNA accumulation levels of putative alpha-CA (CA-1), zeta-CA (CA-3) and delta-CA (CA-7) were analyzed and all levels found to be significantly reduced when cells were grown in 0.16% CO2. Intercellular localizations of eight putative CAs were analyzed by expressing GFP fusion in P. tricornutum and T. pseudonana. In P. tricornutum, CA-I and II localized in the periplastidial compartment, CA-III, VI, VII were found in the chloroplast endoplasmic reticulum, and CA-VIII was localized in the mitochondria. On the other hand, T. pseudonana CA-1 localized in the stroma and CA-3 was found in the periplasm. These results suggest that CAs are constitutively present in the four chloroplastic membrane systems in P. tricornutum and that CO2 responsive CAs occur in the pyrenoid of P. tricornutum, and in the stroma and periplasm of T. pseudonana.