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 (TpαCA1), was localized in the chloroplast stroma; the second, a zeta-type CA (TpζCA1), was localized to the periplasmic space. In the present study, cloning and localization of the remaining CAs were carried out. TpγCA2, TpγCA3, TpγCA4, TpγCA5, TpδCA1, TpδCA2, TpδCA3, and TpζCA1 were responsive to CO2 availability at the transcriptional level, being significantly reduced in cells grown at 0.4 % CO2, whereas TpαCA1, TpαCA2, TpαCA3, TpγCA1, and TpδCA4 transcript levels were constitutive with respect to CO2 concentration. Full-length cDNAs for TpγCA1, TpγCA2, TpγCA3, TpγCA4, TpδCA1, and TpδCA2 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 δ-type TpδCA1 was localized on the periphery of the cell, strongly suggesting localization to the periplasmic space or the frustule. The δ-type TpδCA3 and the γ-type TpγCA2 were, respectively, localized in a periplastidal compartment and the cytosol. The δ-type TpδCA2, and the γ-types TpγCA1, 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.