Expression and localization of two β-carbonic anhydrases in Bienertia, a single-cell C₄ plant

Carbonic anhydrases (CAs) are ubiquitous enzymes that catalyze reversibly both the hydration and dehydration reactions of CO₂ and HCO₃-, respectively. Higher plants contain many different isoforms of CAs that can be classified into α-, β- and γ-type subfamilies. β-type CAs play a key role in the CO₂-concentrating mechanism, thereby contributing to efficient photosynthesis in the C₄ plants in addition to many other biochemical reactions in plant metabolism. Here, we characterized at the molecular, cellular and biochemical levels two β-type CAs in Bienertia sinuspersici, a plant that operates a C₄ carbon concentrating mechanism within individual cells without the Kranz anatomy. These two β-type CAs (BsCAβs), named BsCAβ1 and BsCAβ2, in Bienertia were strongly induced along with maturation of leaves. Both BsCAβ1 and BsCAβ2 existed as a dimeric form in vivo but showed differential localization. BsCAβ2 was localized exclusively to the plasma membrane in Bienertia and when expressed heterologously in the C₃ Arabidopsis. In contrast, BsCAβ1 largely localized to the cytosol together with a portion to the plasma membrane (PM) in both plants. BsCAβ2 had two cysteine residues at the N-terminal region for palmitoylation and their substitution with serine residues led to a change in the localization from the plasma membrane (PM) to the cytosol. Thus, we propose that BsCAβ2 localizes to the PM using a lipid moiety added posttranslationally plays a role in conversion of cytosolic CO₂ into HCO₃- as part of the CO₂-concentrating mechanism, thereby contributing to the single-cell C₄ photosynthesis in Bienertia.