A CO2-limitation-induced cytosolic repressor enables shutdown of the algal CO2-concentrating mechanism

Aquatic photosynthetic organisms face limited CO2 availability because CO2 diffuses slowly in water and most dissolved inorganic carbon (Ci) exists as HCO3- at physiological pH. To overcome this limitation, aquatic photoautotrophs operate CO2-concentrating mechanisms (CCMs) that elevate CO2 around Rubisco and sustain carbon fixation. Because CCM operation consumes energy, it must be suppressed when CO2 becomes abundant, but how this shutdown occurs remains poorly understood. In Chlamydomonas reinhardtii, the nuclear protein CBP1 was identified as a CCM repressor, but its loss causes only partial derepression under high CO2, indicating that an additional mechanism is required for complete shutdown. Here, we identify High-Affinity CCM Repressor 1 (HCR1), a cytosolic protein related to CBP1, as a second repressor. Under high CO2, hcr1 mutants retained high affinity for Ci and derepressed CCM and photoacclimation genes. Combined disruption of HCR1 and CBP1 further increased Ci affinity, approaching that of wild-type cells with a fully induced CCM under CO2 limitation, and promoted the accumulation of Ci transporters. HCR1 loss also prevented redistribution of the chloroplast regulator CAS away from the pyrenoid and was accompanied by retention of a pyrenoid starch sheath. In contrast, LCIB, a chloroplast CO2-recapture protein, relocated normally. Unexpectedly, HCR1 accumulated during CO2 limitation and declined after transfer to high CO2. These results show that CCM shutdown is an active transition rather than the passive reversal of induction. We propose that CBP1 restrains CCM1-dependent transcription, while HCR1 is preloaded during CO2 limitation to terminate the CAS-associated, starch-sheathed, high-affinity state when CO2 becomes replete.