Localization of putative carbonic anhydrases in the marine diatom, Thalassiosira pseudonana

Thirteen putative carbonic anhydrase (CA) genes have been identified in the marine multipolar centric diatom, Thalassiosira pseudonana, and two of these CAs have been localized previously. The first, an alpha CA (Tp alpha CA1), was localized in the chloroplast stroma; the second, a zeta-type CA (Tp zeta CA1), was localized to the periplasmic space. In the present study, cloning and localization of the remaining CAs were carried out. Tp gamma CA2, Tp gamma CA3, Tp gamma CA4, Tp gamma CA5, Tp delta CA1, Tp delta CA2, Tp delta CA3, and Tp zeta CA1 were responsive to CO2 availability at the transcriptional level, being significantly reduced in cells grown at 0.4 % CO2, whereas Tp alpha CA1, Tp alpha CA2, Tp alpha CA3, Tp gamma CA1, and Tp delta CA4 transcript levels were constitutive with respect to CO2 concentration. Full-length cDNAs for Tp gamma CA1, Tp gamma CA2, Tp gamma CA3, Tp gamma CA4, Tp delta CA1, and Tp delta CA2 were isolated and fused with the enhanced-green fluorescent gene at their 3' termini. These GFP-fusion constructs were transformed into T. pseudonana, and the resulting GFP fusion products were localized using fluorescence microscopy. The delta-type Tp delta CA1 was localized on the periphery of the cell, strongly suggesting localization to the periplasmic space or the frustule. The delta-type Tp delta CA3 and the gamma-type Tp gamma CA2 were, respectively, localized in a periplastidal compartment and the cytosol. The delta-type Tp delta CA2, and the gamma-types Tp gamma CA1, 3, and 4 were localized in the mitochondria. The distribution of CAs in T. pseudonana contrasts notably with that of the raphid pennate diatom P. tricornutum, with likely consequences for CCM function including modes of CO2 acquisition, regions in which DIC is accumulated, and needs for minimizing CO2 leakage from the chloroplast.