Condensation of Rubisco into a proto-pyrenoid in higher plant chloroplasts

Summary Photosynthetic CO 2 fixation in plants is limited by the inefficiency of the CO 2 -assimilating enzyme Rubisco (D-ribulose-1,5-bisphosphate carboxylase/ oxygenase) 1–3 . In plants possessing the C3 pathway, which includes most major staple crops, Rubisco is typically evenly distributed throughout the chloroplast stroma. However, in almost all eukaryotic algae Rubisco aggregates within a microcompartment known as the pyrenoid, in association with a CO 2 -concentrating mechanism that improves photosynthetic operating efficiency under conditions of low inorganic carbon 4 . Recent work has shown that the pyrenoid matrix is a phase-separated, liquid-like condensate 5 . In the alga Chlamydomonas reinhardtii , condensation is mediated by two components: Rubisco and the linker protein EPYC1 (Essential Pyrenoid Component 1) 6,7 . Here we show that expression of mature EPYC1 and a plant-algal hybrid Rubisco leads to spontaneous condensation of Rubisco into a single phase-separated compartment in Arabidopsis chloroplasts, with liquid-like properties similar to a pyrenoid matrix. The condensate displaces the thylakoid membranes and is enriched in hybrid Rubisco containing the algal Rubisco small subunit required for phase separation. Promisingly, photosynthetic CO 2 fixation and growth is not impaired in stable transformants compared to azygous segregants. These observations represent a significant initial step towards enhancing photosynthesis in higher plants by introducing an algal CO 2 -concentrating mechanism, which is predicted to significantly increase the efficiency of photosynthetic CO 2 uptake 8,9 .