Wojciech Wietrzynski

Thylakoid membrane remodeling regulates photosynthetic activity in response to changing light, but it is unknown whether such membrane-scale control exists in cyanobacteria, which lack the spatially segregated membrane domains of green algae and plants. Using cryo-electron tomography guided by single-cell hyperspectral correlative imaging, we link spectral acclimation state to thylakoid architecture in far-red light adapting cyanobacteria. We determine the native structures of phycobilisome-photosystem II supercomplexes at subnanometer resolution and show that light-dependent molecular changes propagate across scales to restructure the photosynthetic machinery throughout the thylakoids. We further reveal persistent membrane heterogeneity after prolonged far-red light acclimation and identify a low abundance phycobilisome-photosystem I supercomplex in far-red light conditions. Together, our results frame cyanobacterial thylakoids as plastic membranes that remodel antenna-photosystem structure and organization to regulate photosynthesis.

Photosynthetic electron transfer relies on the coordinated function and spatial organization of large protein complexes within the thylakoid membrane. The cytochrome b6f complex (b6f) functionally interconnects photosystem (PS) II and PSI in photosynthetic electron transfer and is equally distributed between appressed and non-appressed thylakoid membranes. Here, we investigate the functional link between the lateral distribution of b6f and efficient photosynthetic electron flow in Chlamydomonas reinhardtii. We engineered strains with stromal fusions between PetA of b6f and two fluorescent proteins (FPs) of different molecular mass: Clover and ATeam. Under oxic conditions, these strains exhibited significantly slower electron transfer rates (ETR), lower PSII quantum yields, and increased donor-side limitation of PSI. State transitions were diminished in the fusion strains, accompanied by a strong impairment of STT7-dependent function, suggesting that the presence of fused FPs at b6f interfere with STT7 function. Yet, ETR phenotypes were STT7-independent and FP fusion did not impact intrinsic b6f function. In situ cryogenic electron tomography revealed a significant depletion of b6f from appressed thylakoid membranes in the ATeam strains, while the overall membrane protein concentration remained unchanged. Overall, our data indicate that a balanced distribution of b6f between appressed and non-appressed thylakoid membranes is essential for regulating photosynthetic electron transfer, highlighting the functional importance of thylakoid molecular architecture in vivo.

Across kingdoms, cells fold their membranes into precise shapes closely linked to their functions. In mature land-plant chloroplasts, the photosynthetic membranes have been viewed as strictly lamellar and it is unknown whether they can take on a different structure while remaining functional. Here, we show that mature Arabidopsis thaliana chloroplasts can transform this network into a gyroid-type cubic membrane, which we call the gyrobody. The gyrobody forms reversibly during the night and preserves photosystem II photochemistry. A decrease in stromal side thylakoid surface charge, caused by lower protein phosphorylation, triggers the lamellar-to-gyroid transition which the curvature-inducing lipid MGDG facilitates. This shows that the mature plant thylakoid network is not locked into its lamellar form, revealing unexpected structural flexibility of this system.

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 Chlamydomonas reinhardtii (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 (Spinacia oleracea). 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 C. reinhardtii, 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.

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 Chlamydomonas reinhardtii [1] and then map the molecular organization of these thylakoids with single-molecule precision [2]. 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 (Spinacia oleracea). We visualize the fine ultrastructural details of grana membranes, as well as interactions between thylakoids and plastoglobules. We apply and further develop AI-based computational approaches for automated membrane segmentation and membrane protein picking [3], enabling us to quantify the organization of photosynthetic complexes within the plane of the thylakoid membrane and across adjacent stacked membranes. Our analysis reveals that, despite different 3D architecture, the molecular organization of thylakoid membranes in vascular plants and green algae is strikingly similar. In contrast to isolated plant thylakoids, where semi-crystalline arrays of photosystem II (PSII) appear to hold some membranes together, we find in intact chloroplasts that PSII is non-crystalline and has uniform concentration both within the membrane plane and across stacked grana membranes. Similar to C. reinhardtii, 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.

In situ cryo-electron tomography (cryo-ET) has emerged as the method of choice to investigate structures of biomolecules in their native context. However, challenges remain in the efficient production of large-scale cryo-ET datasets, as well as the community sharing of this information-rich data. Here, we applied a cryogenic plasma-based focused ion beam (cryo-PFIB) instrument for high-throughput milling of the green alga Chlamydomonas reinhardtii , a useful model organism for in situ visualization of numerous fundamental cellular processes. Combining cryo-PFIB sample preparation with recent advances in cryo-ET data acquisition and processing, we generated a dataset of 1829 reconstructed and annotated tomograms, which we provide as a community resource to drive method development and inspire biological discovery. To assay the quality of this dataset, we performed subtomogram averaging (STA) of both soluble and membrane-bound complexes ranging in size from >3 MDa to ∼200 kDa, including 80S ribosomes, Rubisco, nucleosomes, microtubules, clathrin, photosystem II, and mitochondrial ATP synthase. The majority of these density maps reached sub-nanometer resolution, demonstrating the potential of this C. reinhardtii dataset, as well as the promise of modern cryo-ET workflows and open data sharing towards visual proteomics.

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 Chlamydomonas reinhardtii [1] and then map the molecular organization of these thylakoids with single-molecule precision [2]. 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 ( Spinacia oleracea ). We visualize the fine ultrastructural details of grana membranes, as well as interactions between thylakoids and plastoglobules. We apply and further develop AI-based computational approaches for automated membrane segmentation and membrane protein picking [3], enabling us to quantify the organization of photosynthetic complexes within the plane of the thylakoid membrane and across adjacent stacked membranes. Our analysis reveals that, despite different 3D architecture, the molecular organization of thylakoid membranes in vascular plants and green algae is strikingly similar. In contrast to isolated plant thylakoids, where semi-crystalline arrays of photosystem II (PSII) appear to hold some membranes together, we find in intact chloroplasts that PSII is non-crystalline and has uniform concentration both within the membrane plane and across stacked grana membranes. Similar to C. reinhardtii , 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.

EMPIAR, the Electron Microscopy Public Image Archive centered at EMBL-EBI, is a public resource for raw electron microscopy images related to EMDB, contains micrographs, particle sets and tilt-series.

Oxygenic photosynthesis evolved billions of years ago, becoming Earth's main source of biologically available carbon and atmospheric oxygen. Since then, phototrophic organisms have diversified from prokaryotic cyanobacteria into several distinct clades of eukaryotic algae and plants through endosymbiosis events. This diversity can be seen in the thylakoid membranes, complex networks of lipids, proteins, and pigments that perform the light-dependent reactions of photosynthesis. In this review, we highlight the structural diversity of thylakoids, following the evolutionary history of phototrophic species. We begin with a molecular inventory of different thylakoid components and then illustrate how these building blocks are integrated to form membrane networks with diverse architectures. We conclude with an outlook on understanding how thylakoids remodel their architecture and molecular organization during dynamic processes such as biogenesis, repair, and environmental adaptation.

Photosynthesis—the conversion of energy from sunlight into chemical energy—is essential for life on Earth. Yet there is much we do not understand about photosynthetic energy conversion on a fundamental level: how it evolved and the extent of its diversity, its dynamics, and all the components and connections involved in its regulation. In this commentary, researchers working on fundamental aspects of photosynthesis including the light-dependent reactions, photorespiration, and C4 photosynthetic metabolism pose and discuss what they view as the most compelling open questions in their areas of research.