Spatiotemporal biogenesis of thylakoid membranes in the green alga Chlamydomonas reinhardtii

Thylakoid membranes are essential for oxygenic photosynthesis, yet the mechanisms underlying their spatial and temporal biogenesis remain poorly understood. Here, using a light-induced membrane regeneration system in Chlamydomonas reinhardtii, we generate a time-resolved map of thylakoid formation and photosynthetic complex assembly at unprecedented spatial and temporal resolution by integrating super-resolution fluorescence microscopy, cryo-electron tomography, proteomics, and spectroscopy. We show that thylakoid biogenesis is globally distributed and multipolar, with new membrane formation occurring at multiple sites across the chloroplast, including regions adjacent to the inner envelope membranes in both basal and lobe regions. We identify F-ATP synthase storage membranes and map the redistribution of translating ribosomes from T-zone enrichment to a chloroplast-wide distribution at early stages of thylakoid formation, consistent with decentralized synthesis of photosynthetic complexes. We further delineate the hierarchical assembly of photosystem supercomplexes, starting with formation of reaction centers. Together, these findings refine the spatial organization of photosynthetic membrane biogenesis and establish a unified spatiotemporal framework linking thylakoid formation, translational activation, and functional maturation, providing a mechanistic basis for plastid engineering to enhance photosynthetic efficiency.