GoKawiilNative plant photosystem supercomplexes
In vascular plants, PSII and PSI assemble into modular supercomplexes comprising a core, containing the reaction centre, surrounded by multiple LHC antenna proteins, as reported by biochemical and spectroscopic studies21,40,48. However, structures obtained from purified plant PSII have predominantly resolved C 2 S 2 M 2 -type supercomplexes, and lack several LHCII trimers inferred from in vivo measurements, suggesting the loss of weakly associated antenna subunits during purification. Using our in situ approach, we resolved a C 2 S 2 M 2 L 4 -type PSII–LHCII supercomplex at high resolution, identifying four previously uncharacterized loosely bound LHCII trimers and thereby revealing a substantially larger antenna than observed in purified samples. Of note, these L-LHCII trimers exhibit substantial flexibility, as reflected by the weaker density and lower local resolution in these regions relative to the PSII core, as well as the S-LHCII and M-LHCII trimers. Under physiological conditions, L-LHCII is inherently dynamic and participates in state transitions49 and light adaptation processes21,22,40 to regulate excitation energy distribution. Although an average position for these LHCII trimers was defined from the composite maps, their precise orientations and distances are expected to vary in the native membrane. Under the in situ sample conditions used here, a detailed characterization of this conformational heterogeneity is inherently limited—their association is relatively weak and highly dynamic, making it difficult to capture a stable, single conformation. However, EET calculations based on this supercomplex closely match in vivo measured trapping times (Fig. 5), which indicates that although some heterogeneity can be expected in the L-LHCII trimers regions, the current C 2 S 2 M 2 L 4 organization is a genuine representation of PSII in the membrane. PSI similarly exists in at least two native forms: PSI–LHCI and PSI–LHCI–LHCII.
Higher-order PSII assembly in thylakoid
Beyond individual photosystems, our in situ experiments reveal how these supercomplexes associate within and between thylakoid layers. In the resolved maps, additional densities adjacent to PSII–LHCII indicate the presence of interacting partner proteins. Extensive cross-classification identifies multiple dimeric, trimeric and tetrameric assemblies formed through side-by-side, trans-lumenal and trans-stromal interactions. On the basis of these observations, we propose that PSII–LHCII supercomplexes form a large-scale skeleton structure, which shapes thylakoid architecture through lumenal and lateral contacts, and stromal contacts mediate the stacking of multiple thylakoids to form grana. These interactions are weak and are therefore disrupted during isolation, highlighting the power of the in situ approach to capture native organizations. The side-by-side, trans-lumenal and trans-stromal organizations identified here provide insight into how photosystem assemblies contribute to thylakoid membrane architecture. Higher-order PSII–LHCII associations are likely to influence local membrane curvature and lumenal spacing, and the native geometry of the thylakoid system in turn constrains the relative orientation, distribution and conformations of individual supercomplexes. SS dimers, which lack strong intermolecular contacts, may primarily optimize packing within the crowded membrane. By contrast, TL and TS dimers adopt ‘close yet separate’ geometries stabilized by electrostatic interaction and localized hydrogen-bond networks involving PsbO, CP43 and PsbR (Fig. 3f,i, Extended Data Fig. 4e,f and Supplementary Fig. 15). We propose that these defined spatial arrangements contribute to the emergence and maintenance of thylakoid membrane morphology.
Some regions of the reconstructed higher-order PSII–LHCII supercomplexes exhibit anisotropic resolution, probably arising from preferential particle orientation inherent to the sample preparation, relative movements among different components and the intrinsic flexibility of the lipid membrane. Nonetheless, the well-resolved overall architectures, particularly in their core regions, provide strict positional constraints for subsequent rigid-body docking, enabling the establishment of reliable models for analysing the stacking modes of PSII–LHCII supercomplexes within the native thylakoid membrane layers.
The molecular basis of grana stacking has long been debated, with LHCII and other antenna complexes proposed to have central roles50,51. However, the in situ structures reported here show no direct interactions across the stromal gap involving the N-terminal regions or stromal loops of LHCII or CP29. Notably, these stromal loops adopt conformations similar to those observed in purified complexes2. By contrast, the stromal-side interactions observed here involve the stromal loop of the PsbR subunit and are compatible with cross-linking experiments performed in native membranes32. Together, these observations indicate that the stacking interactions do not involve direct contacts between stromal antenna regions but instead involve interactions between PSII–LHCII supercomplex cores.
Functional implications for EET
The calculated trapping times for the C 2 S 2 M 2 and C 2 S 2 M 2 L 4 complexes closely match in vivo measurements from Arabidopsis thaliana grown under different light conditions, including those in which four LHCII trimers are associated per PSII22. This agreement indicates that the C 2 S 2 M 2 L 4 arrangement resolved in O. sativa is likely to represent a general PSII organization in higher plants. The slow excitation exchange involving L-LHCII trimers is consistent with their peripheral position and with spectroscopic data showing that they slow down excitation migration more strongly than S-LHCII and M-LHCII22,40. Together with the observation that L-LHCII trimers are also structurally only weakly associated with C 2 S 2 M 2 and that plants regulate the expression and degradation of Lhcb1 and Lhcb2 during light acclimatization52, these findings suggest that L-LHCII trimers are the primary components that are modulated during light acclimation.
Furthermore, the weak connectivity that we observe between adjacent PSII dimers indicates that excitation sharing is efficient only within individual supercomplexes. Moreover, the large stromal separations between trans-stromal complexes preclude energy transfer across grana layers, in agreement with spectroscopic observations42. Together, these results highlight how structural organization at different hierarchical levels shapes the functional antenna size and overall photochemical efficiency.
Perspectives
... continue reading