Chloroplast-mediated regulation of CO₂-concentrating mechanism by Ca²⁺-binding protein CAS in the green alga Chlamydomonas reinhardtii

Aquatic photosynthetic organisms, including the green alga Chlamydomonas reinhardtii, induce a CO₂-concentrating mechanism (CCM) to maintain photosynthetic activity in CO₂-limiting conditions by sensing environmental CO₂ and light availability. Previously, a novel high-CO₂-requiring mutant, H82, defective in the induction of the CCM, was isolated. A homolog of calcium (Ca²⁺)-binding protein CAS, originally found in Arabidopsis thaliana, was disrupted in H82 cells. Although Arabidopsis CAS is reported to be associated with stomatal closure or immune responses via a chloroplast-mediated retrograde signal, the relationship between a Ca²⁺ signal and the CCM associated with the function of CAS in an aquatic environment is still unclear. In this study, the introduction of an intact CAS gene into H82 cells restored photosynthetic affinity for inorganic carbon, and RNAseq analyses revealed that CAS could function in maintaining the expression levels of nuclear-encoded CO₂-limiting-inducible genes, including the HCO3 - transporters high-light activated 3 (HLA3) and low-CO₂-inducible gene A (LCIA). CAS changed its localization from dispersed across the thylakoid membrane in high-CO₂ conditions or in the dark to being associated with tubule-like structures in the pyrenoid in CO₂-limiting conditions, along with a significant increase of the fluorescent signals of the Ca²⁺ indicator in the pyrenoid. Chlamydomonas CAS had Ca²⁺-binding activity, and the perturbation of intracellular Ca²⁺ homeostasis by a Ca²⁺-chelator or calmodulin antagonist impaired the accumulation of HLA3 and LCIA. These results suggest that Chlamydomonas CAS is a Ca²⁺-mediated regulator of CCM-related genes via a retrograde signal from the pyrenoid in the chloroplast to the nucleus.