Pyrenoid Starch Sheath Is Required for LCIB Localization and the CO₂-Concentrating Mechanism in Green Algae

Aquatic photosynthetic organisms induce a CO₂-concentrating mechanism (CCM) to overcome the difficulty of acquiring inorganic carbon under CO₂-limiting conditions. As part of the CCM, the CO₂-fixing enzyme Rubisco is enriched in the pyrenoid located in the chloroplast, and, in many green algae, several thick starch plates surround the pyrenoid to form a starch sheath. In Chlamydomonas reinhardtii, low-CO₂-inducible protein B (LCIB), which is an essential factor for the CCM, displays altered cellular localization in response to a decrease in environmental CO₂ concentration, moving from dispersed throughout the chloroplast stroma to around the pyrenoid. However, the mechanism behind LCIB migration remains poorly understood. Here, we report the characteristics of an Isoamylase1-less mutant (4-D1), which shows aberrant LCIB localization and starch sheath formation. Under very-low-CO₂ conditions, 4-D1 showed retarded growth, lower photosynthetic affinities against inorganic carbon, and a decreased accumulation level of the HCO₃ ^- transporter HLA3. The aberrant localization of LCIB was also observed in another starch-sheathless mutant sta11-1, but not in sta2-1, which possesses a thinned starch sheath. These results suggest that the starch sheath around the pyrenoid is required for the correct localization of LCIB and for the operation of CCM.