**5. Carbonic anhydrase**

CAs are metalloenzymes that catalyse the reversible interconversion of CO2 and HCO3 <sup>−</sup> [73]. They are encoded by six evolutionary divergent gene families and the corresponding enzymes are designated as α, β, γ, δ, ε and ζ-CA [39]. These six types of CAs share no sequence similarity in their primary amino acid sequences and seem to have evolved independently [26, 74]. In macroalgae, almost all known CAs belong to α, β and γ classes, with the β class predominating [26, 39]. The δ, ε and ζ classes of CA are found only in some diatoms [75], bacteria [76] and marine protists [77, 78]. The active site of CA contains a zinc ion (Zn2+), which plays a critical role in the catalytic activity of the enzyme. The ζ and γ classes of CAs represent exceptions to this rule since they can use cadmium (ζ), iron (γ) or cobalt (γ) as cofactors [79–81]. CA plays an important role in photosynthesis by supplying either CO2 to RuBPCO or HCO3 − to PEPC. They also participate in some other physiological reactions such as respiration, pH homeosta‐ sis, ion transport and catalysis of key steps in the pathways for the biosynthesis of physiolog‐ ically important metabolites [41]. The CA synthesis in the cytoplasm [82] is located in the periplasmic space, mitochondria, chloroplast stroma and chloroplast thylakoid lumen, carboxysome and pyrenoid [66, 70, 83, 84]. Different subcellular localizations make different CA functions in CCM. Periplasmic CA (CAext) can catalyse the conversion of HCO3 − into CO2 to promote the diffusion of CO2 at the cell surface across the plasma membrane [85, 86]. Therefore, CAext has been postulated to be part of the CCM in most macroalgae. The cytoplasm CA stores Ci in the form of HCO3 − to avoid leakage of CO2 and to regulate the pH value of cytoplasm by maintaining the equilibrium of different forms of Ci, which is important for algal CCM [39]. CAs on the chloroplast membrane and in the stroma mainly provide CO2 for RuBisCo [26, 38, 87]. In cyanobacteria, CAs in the carboxysomal shell function to convert accumulated HCO3 − into CO2 and pass it to RuBisCo inside the cytoxysome [88]. CA in the thylakoid lumen was proposed to function to create an efficient CO2 supply to RuBisCo by taking advantage of the acidity of the lumenal compartment [69]. Stromal CA is also thought to operate by converting leaking CO2 into HCO3 <sup>−</sup> [70]. Recently, data provided by various genome sequencing studies have revealed the multiplicity of CA isoforms in algae. For example, in the model microalga *C. reinhardtii*, there are at least 12 genes that encode CA isoforms, including three α, six β and three γ or γ-like CAs [39]. For marine diatom, nine and thirteen CA sequences were found in the genomes of *P. tricornutum* and *Thalassiosira pseudo‐ nana*, respectively [89]. *P. tricornutum* contains two β-CA genes, five α and two γ CA genes, whereas *T. pseudonana* has three α-, five γ-, four δ- and one ζ-CA genes [89]. As for macroalgae, CA genes have only been reported in few species. Six full-length CA of *P. haitanensis* (PhCA) genes were reported, which include two α-CAs, three β-CAs and one γ-CA [90]. Besides, one β-CA and one α-CA were reported in *P. yezoensis* [91] and *S. japonica* [92, 93]. Otherwise, although the activity of CAext and CAint has been detected in many macroalgae, the subcellular localization and functions of CAext and CAint remain unclear [71, 93].

Conclusively, CAs, including CAext and CAint (Figure 1), play an important role in the trans‐ portation or concentration process of the Ci . And as for C3 and C4 metablisms have different carboxylase, CAs might play different roles in CCMs of macroalgae with different photosyn‐ thetic mode. Thus, isolating of the *CA* genes, studies on their expression levels in different CO2 concentrations, in different life phase, and under different environmental stress, as well as studies on subcellular locations of CAs should be conducted in macroalgae to help reveal their Ci assimilation processes.
