Structure and structural dynamics of Photosystem II supercomplex in higher plants

Photosynthesis is indisputably the primary biological process to introduce chemical energy and biomass into ecosystems by oxidizing water and reducing carbon dioxide into organic compounds. Photosystem II (PSII) is a unique protein complex, present in thylakoid membranes of all oxygenic photosynthetic organisms, able to catalyze the water-splitting reaction using sunlight as driving force, thus being responsible for the generation of all the molecular oxygen accumulated in the atmosphere for over three billion years. Although its catalytic core has been extremely conserved throughout evolution, from cyanobacteria to higher plants, the necessity of different photosynthetic organisms to cope with ever-changing environmental light conditions led to the emergence of a great variability among its peripheral antenna systems, differentiating in extrinsic phycobilisomes in cyanobacteria and intrinsic light harvesting complexes (LHCII) in green algae and higher plants. LHCII are integral membrane proteins that occur as heterotrimers of Lhcb1-2-3 subunits and monomeric Lhcb4-5-6 polypeptides and associate peripherally with the PSII core in variable numbers, thus forming large supramolecular assemblies called PSII‐LHCII supercomplexes. The minimal functional unit, found in all light conditions, consists of a dimeric PSII core (C2) with two strongly bound LHCII trimers (S2), made of Lhcb1 and Lhcb2, connected by two monomeric Lhcb4 and Lhcb5 subunits, and is called C2S2. In limiting light conditions, the C2S2 can further associate with one or two moderately bound LHCII trimers (M2) which consist of Lhcb1, Lhcb2 and Lhcb3 proteins connected by the monomeric Lhcb6, a peculiar subunit found only in higher plants, originating supercomplexes of type C2S2M1-2. A further supramolecular organization is due to the lateral association of PSII-LHCII supercomplexes within the thylakoid membrane plane, forming PSII-LHCII megacomplexes, or even higher ordered arrays. The LHCII fulfill a dual role by either quenching the excess light energy, often occurring in natural environments, or optimizing its harvesting in ecosystems where there is competition and mutual shading. The rearrangement of the PSII’s modular antenna system through its dynamic interaction with the PSII core, therefore, appears to be a key process in light harvesting regulation. Moreover, plant’s PSII and LHCII are spatially and functionally segregated into piled discs of thylakoid membranes (grana), where they occupy 80% of the surface. Their structural arrangement into PSII-LHCII supercomplexes interacting dynamically with each other appears to be critical in determining the overall membrane architecture and ultimately the efficiency of photosynthesis. Although the overall structure of the basic C2S2 supercomplex in plants has been recently resolved at nearly atomic resolution, there is still a lack of knowledge regarding its structural rearrangement in different light conditions as well as its specific interaction within the membrane plane and between adjacent membranes. During this thesis’ work we have been able to isolate pure PSII-LHCII super- and megacomplexes from pea plants grown in moderate light by mild solubilization of stacked thylakoid membranes. In order to assess their overall functional architecture, the full biochemical characterization of isolated PSII-LHCII supercomplexes, comprehensive of accurate proteomic analyses, was coupled with structural studies. Their structural characterization, performed by transmission electron microscopy (TEM) in cryogenic conditions (cryo-EM) and subsequent single particle analysis, led to a novel 3D structure at about 14 Å resolution of the supercomplexes of type C2S2M. The obtained electron density map revealed that under normal light conditions most of the supercomplexes within the grana are of type C2S2M and occur as paired supercomplexes, whose interactions are mediated by physical connections across the stromal gap of adjacent membranes. The specific overlapping of LHCII trimers facing each other in paired supercomplexes, as already observed in other studies, suggests that this conformation might be representative of their native state within the membranes. The physical connections observed across the stromal gap might be attributable to the mutual interaction between the long N-terminal loops of the monomeric Lhcb4 subunits. These subunits occupy a pivotal position in the 3D map of the paired supercomplexes and are clearly bridged across the stromal gap by electron densities attributable to these loops. In addition, despite the its structural flexibility, the remarkable sequence conservation of this region, even in distant phylogenetic photosynthetic organisms, may suggest its major involvement in structural dynamics. The specific interaction observed in paired supercomplexes seems to be mediated by cations present within the chloroplast in relatively low concentrations as their depletion from buffers used for isolation leads to the dissociation of the paired supercomplexes into single ones. Moreover, this evidence was also strongly supported by the decrease in the PSII excitonic connectivity measured in-vivo. The paired behavior has also been observed in higher oligomerization forms of isolated PSII-LHCII supercomplexes in which two paired supercomplexes laterally interact with each other in the membrane plane, thus forming paired megacomplexes. This novel structure has been obtained by EM and 2D reconstruction of negatively stained particles and, despite its low resolution, reveals how PSII-LHCII supercomplexes may laterally and stromally interact with each other in different ways. The observation of the potential overlapping of LHCII trimers in megacomplexes facing each other, as well as the occurrence of different geometries of interaction between supercomplexes within the membrane plane and between megacomplexes in adjacent membranes, provide intriguing insights on how PSII and LHCII might interact in a very stable manner within the thylakoid membrane and between different discs in the grana. In order to study the PSII-LHCII supercomplex remodeling in the context of ever-changing light environmental conditions, PSII-LHCII supercomplexes have been isolated from pea plants grown at different light intensities: low (LL), moderate (CL) and high light (HL). The accurate profiling and quantitation of the LHCII subunits in the isolated supercomplexes and in the native thylakoids, achieved by using a mass-spectrometry based proteomic approach, was coupled with the evaluation in-vivo of their functional antenna size (ASII). At increasing light intensities, the structural remodeling of the modular PSII’s antenna system led to the reduction of the amount of LHCII M-trimers in the isolated complexes, attested by the decreased level of Lhcb3 and Lhcb6. This specific remodeling does not occur at the same rate in the entire thylakoid membrane. The whole LHCII pool is downregulated only in plants grown in HL, suggesting the occurrence of different acclimation strategies. The remarkable decrease of the ASII observed in HL acclimated plants, when compared to LL plants, can be attributed to the significant increase of the Lhcb4 specific isoform Lhcb4.3, occurring both in isolated supercomplexes and in thylakoid membranes. Unlike isoforms Lhcb4.1-2, the Lhcb4.3 isoform, whose transcription is enhanced upon HL exposure, interestingly has a truncated C-terminus that is located at the binding interface with Lhcb6 within the supercomplex structure. The incorporation of Lhcb4.3 in the PSII-LHCII supercomplex might play a major role in decreasing its functional antenna size by reducing its affinity to bind additional M-trimers, thus regulating its light harvesting efficiency even at moderate light intensities. Conversely, the exposure to HL induces the decrease of the PSII antenna cross-section in isolated supercomplexes and the partial depletion of the whole antenna system of PSII in the thylakoid membranes, thus constitutively preventing damages to the reaction center when light continuously exceeds its energy-processing capacity. These results aim at broadening the current knowledge on how the light harvesting antenna system associated with the PSII core is finely regulated upon plants’ long term acclimation to different light intensities. The flexibility of the PSII’s modular antenna system, accompanied by its finely tuned structural interaction with the core complex, pivotal for the 3D organization of plant thylakoid membranes, certainly played a key role in determining its remarkable evolutionary outcome. Taken together, these results may provide new research directions while certainly broadening the knowledge on how PSII-LHCII assemblies and their supramolecular interaction contribute to maintain the complex architecture of thylakoid membranes and the overall efficiency of photosynthesis in ever changing environmental conditions