EPOS1 is required for normal pyrenoid starch sheath formation and efficiency of the CO2-concentrating mechanism in Chlamydomonas reinhardtii

Pyrenoid-based CO 2-concentrating mechanisms (pCCMs) boost photosynthesis by delivering elevated levels of CO 2 to Ribulose-1,5-bisphosphate carboxylase/oxygenase. Pyrenoids are often surrounded by a starch sheath thought to enhance pCCM efficiency, but little is known about how the sheath is formed. Here, we have assessed the role of the pyrenoid-associated protein EPOS1 in starch sheath formation in Chlamydomonas reinhardtii. Using an epos1 mutant, we assessed the role of EPOS1 in starch sheath morphology by fluorescence and electron microscopy, and its impact on pCCM function by CO 2 uptake and growth assays. We determined potential interactors of EPOS1 via TurboID proximity labelling. In the absence of EPOS1, cells fail to correctly assemble the starch sheath, have lower affinity for inorganic carbon and grow slower under CO 2-limiting conditions. EPOS1 localises to the starch sheath and is physically close to a range of starch metabolism enzymes and other potential scaffolding proteins. Finally, we show that EPOS1 associates with starch in the land plant Arabidopsis thaliana. EPOS1 may act as a pyrenoid-specific regulator or scaffold for starch metabolism enzymes. EPOS1 could contribute to ongoing efforts to build a functional pyrenoid in a land plant.