Identification and Characterization of a Periplasmic α‐Carbonic Anhydrase (CA) in the Gametophytes of <i>Saccharina japonica</i> (Phaeophyceae)

Abstract

Periplasmic or external carbonic anhydrases (CAs) have been well accepted as playing a crucial role in the acquisition of dissolved inorganic carbon; however, no cytological evidence or molecular information on these enzymes has been reported in seaweeds to date. In this study, the full-length cDNA sequence coding for a putative periplasmic Sjα-CA2 was cloned from the gametophytes of Saccharina japonica, an industrial brown seaweed. It was 1,728 bp in length and included a 263-bp 5'-untranslated region (UTR), a 577-bp 3'-UTR, and an 888-bp open reading frame encoding a protein precursor consisting of 295 amino acids. The mature protein, after removal of a predicted 28-residue signal peptide, was composed of 267 amino acids with a relative molecular weight of 29.27 kDa. Multisequence alignment and phylogenetic analysis indicated that it was a member of the α-CA family. Enzyme activity assays showed that the recombinant Sjα-CA2 in Escherichia coli possessed CO₂ hydration and esterase activities, thus identifying this gene Sjα-CA2 in function. Immunogold electron microscopic observations with the prepared anti-Sjα-CA2 polyclonal antibody illustrated that Sjα-CA2 was located in periplasmic space of the kelp gametophyte cells. Quantitative real-time PCR results revealed that the transcription of Sjα-CA2 was induced by elevated <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msubsup><mml:mtext>HCO</mml:mtext><mml:mn>3</mml:mn><mml:mo>-</mml:mo></mml:msubsup></mml:math> levels, but it was little changed while the kelp gametophytes were subjected to elevated CO₂ concentrations. This study suggests that the periplasmic Sjα-CA2 might play a role in adapting to elevated environmental levels of <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msubsup><mml:mtext>HCO</mml:mtext><mml:mn>3</mml:mn><mml:mo>-</mml:mo></mml:msubsup></mml:math> by dehydration of <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msubsup><mml:mtext>HCO</mml:mtext><mml:mn>3</mml:mn><mml:mo>-</mml:mo></mml:msubsup></mml:math> to generate CO₂ , which could be readily taken up by S. japonica gametophytes.