Final Report Summary - SRPAS (Structural response of photosynthetic apparatus to stress) In nature, plants are continuously exposed to varying and often stressful environmental conditions. Therefore, during the course of evolution they developed a wide range of adaptive mechanisms, which control their life and propagation cycle. Their photosynthetic performance is delicately regulated at the level of the chloroplast and the photosynthetic apparatus associated with the thylakoid membrane. Major components of the photosynthetic apparatus are Photosystem II (PSII) and Photosystem I (PSI). They mediate together with other integral membrane proteins, such as the light harvesting complex (LHC) and cytochrome b6/f (Cytb6f) complex, a light-driven linear electron transport across the thylakoid membrane, which ultimately leads to reduction of NADP+ to NADPH. In addition, there are other, less abundant components such as the Proton Gradient Regulation5 (PGR5) and PGR5-LIKE1 (PGRL1) complex and NAD(P)H dehydrogenase (NDH) complex, which mediate cyclic electron transport. The cyclic electron transport is highly relevant for plants especially under stress conditions, as it helps to balance the ATP/NADPH ratio and to protect the photosynthetic apparatus against photo-inhibition and oxidative stress. Both types of electron transport pathways are coupled with the translocation of protons across the membrane, which leads to the generation of a transmembrane ΔpH gradient utilized by ATP synthase to produce ATP. Our current knowledge indicates that the regulatory mechanisms and stress induced response of photosynthetic apparatus involve, beside others, structural modifications of photosynthetic apparatus at the level of individual proteins and/or their organization in the thylakoid membrane. Further, the response involves a reversible formation of a large variety of specific protein-protein complexes, supercomplexes or even larger assemblies as megacomplexes. Revealing their structures is crucial for our better understanding of their function and relevance in photosynthesis under stress conditions and to grasp aspects of their formation. However, structural characterization of large protein assemblies is often a very challenging task, because they are formed only transiently in the membrane and they are very fragile. Single particle electron microscopy (EM) is very suitable technique, which allows solving structures of such large and fragile proteins. Because the technique is also fast and can reveal the low-medium resolution structures, the impact of its application is growing rapidly.We have successfully implemented with the support of Marie Curie Career Integration Grant the method of single particle EM at Department of Biophysics (Palacky University, Olomouc, Czech Republic), which we used in a structural characterization of photosynthetic protein supercomeplxes. Introduction of this technique broadens a wide range of biophysical and biochemical techniques at the Department of Biophysics, which are available also to other groups at Palacky University. Using the single particle EM we solved several structures of PSI-containing supercomplexes, which play an important role under stress conditions. Both PSI-NDH supercomplex and PSI-Cytb6f supercomplex, which operate in the cyclic electron transport, were structurally characterized for the first time. We found that one copy of the NDH complex associates preferentially with two copies of PSI complexes, but even larger supercomplexes with up to four PSI complexes per one NDH complex were also observed. We established the binding position of the dimeric Cytb6f complex at the antenna side of PSI. Our structural data indicate that the organization of PSI, either in a complex with NDH or with Cytb6f complex, may improve regulation of electron transport by the control of binding partners and distances in small domains.Structural analysis of plant PSII supercomplexes also revealed several novel aspects of their architecture. We found a remarkable ability of PSII to associate into large variable PSII megacomplexes formed by two PSII supercomplexes. The presence of PSII megacomplexes was also detected on the level of a native thylakoid membrane, which is an evidence of their native origin and a physiological relevance under specific light conditions. Structural analysis of PSII supercomplexes from Norway spruce revealed another unexpected feature. We proofed the absence of two light harvesting proteins, Lhcb3 and Lhcb6, in the spruce genome (family Pinaceae) and the sister family Gnetales, which leads to a structural difference of spruce PSII supercomplex with respect to other representatives of land plants. These two subunits have evolved during transition of plants from water to land and were considered to be characteristic for all land plants. Their absence in these plant groups breaks the current evolutionary dogma and modifies PSII supercomplex in such a way that it resembles PSII from evolutionary older organism, alga Chlamydomonas reinhardtii. We suggest that the loss of these proteins might be associated with long-term high-light conditions during the evolution of their ancestor.
