Molecular architecture of thylakoid membranes within intact spinach chloroplasts

Thylakoid membranes coordinate the light reactions of photosynthesis across multiple scales, coupling the architecture of an elaborate membrane network to the spatial organization of individual protein complexes embedded within this network. Previously, we used in situ cryo-electron tomography (cryo-ET) to reveal the native thylakoid architecture of the green alga <jats:italic>Chlamydomonas reinhardtii</jats:italic> (Engel et al., 2015) and then map the molecular organization of these thylakoids with single-molecule precision (Wietrzynski et al., 2020). However, it remains to be shown how generalizable this green algal blueprint is to the thylakoids of vascular plants, which possess distinct membrane architecture subdivided into grana stacks interconnected by non-stacked stromal lamellae. Here, we continue our cryo-ET investigation to reveal the molecular architecture of thylakoids within intact chloroplasts isolated from spinach (<jats:italic>Spinacia oleracea</jats:italic>). We visualize the fine ultrastructural details of grana membranes, as well as interactions between thylakoids and plastoglobules. We apply AI-based computational approaches (Lamm et al., 2024) to quantify the organization of photosynthetic complexes within the plane of the thylakoid membrane and across adjacent stacked membranes. Our analysis reveals that the molecular organization of thylakoid membranes in vascular plants and green algae is strikingly similar. We find that PSII organization is non-crystalline and has uniform concentration both within the membrane plane and across stacked grana membranes. Similar to <jats:italic>C. reinhardtii</jats:italic>, we observe strict lateral heterogeneity of PSII and PSI at the boundary between appressed and non-appressed thylakoid domains, with no evidence for a distinct grana margin region where these complexes have been proposed to intermix. Based on these measurements, we support a simple two-domain model for the molecular organization of thylakoid membranes in both green algae and plants.