LHCSR PROTEIN IN THE MOSS PHYSCOMITRELLA PATENS: IN VITRO AND IN VIVO APPROACHES FOR THE STUDY OF ITS PHOTOPROTECTIVE ROLE

Science has always had a major role to satisfy human needs. Over the next decade, the demand for energy will increase exponentially, while fossil fuels will be less available. This is a major problem for modern society and renewable energy, which today covers only 6% of the energy used, is one of the possible solutions. By what kind of magic, light energy can be used to form organic substances from water and carbon dioxide? The answer is photosynthesis. This natural process is used by photoautotrophic organisms (plants, algae, cyanobacteria), and will be the key for the production of renewable energies. The light-harvesting antenna of photosynthetic organisms regulate light utilization. They have been studied by many scientists in various disciplines: plant physiology, biochemistry and molecular biology. Recently, an ancient light-harvesting protein was discovered and called LHCSR. It prevents green algae from absorbing too much sunlight during photosynthesis and therefore controls oxidation damage. LHCSR is an activator of the fastest photoprotective process called Non Photochemical Quenching (NPQ), which consists in the thermal dissipation of excess absorbed energy. NPQ is triggered by the generation of a ΔpH gradient across thylakoid membranes. In spite of the many publications dealing with antenna proteins, the knowledge to fully understand their structure, function and regulation is still insufficient. The aim of this thesis is to describe the role of LHCSR in plant evolution. To do this, I used the moss Physcomitrella patens as a unique model organism for the study of photosynthesis and NPQ in particular. In the first part I will introduce the basis of photosynthesis as well as some description of the model organism I have been using. Three protocols widely use in our laboratory, and indispensable for the realization of my thesis, are also describe in the first part: - In vitro refolding of LHC complexes. - Transformation of the moss Physcomitrella patens. - In vivo chlorophyll fluorescence used to measure NPQ. In Chapter 2, I will introduce a review that we have written on the structural and functional properties of LHC antenna proteins of photosysthem II. You will find information on the role of antenna complexes in light harvesting and/or photoprotection. In addition, the part that deals with the evolution of LHC proteins, allow us to localize LHCSR from P. patens in the phylogenetic tree of major LHCs and understand why it has been exposed to evolutionary pressure. The experimental results will be treated in Chapter 3 and Chapter 4, depending on the approach that I used to characterize the protein, either in vitro or in vivo, respectively. In Chapter 3, I start from in silico approach to get an LHCSR structural model. LHCSR sequence is highly similar to that of LHCII proteins, and it is characterize by eight putative chlorophylls binding sites and its localization is predict to the thylakoid membranes. More detail experiments show that this protein appears to be close to PSII but not strongly bound. This first step has been followed by an in vitro procedure in order to obtain refold pigment-protein complex demonstrating that PpLHCSR can coordinate pigments. Compare to a PSII antenna protein, LHCSR1 is the protein with the highest Chl a/b ratio and strong zeaxanthin dependence. By site direct mutagenesis on the primary sequence of the protein, each hypothetical-binding site was mutated and spectroscopic properties of the refolded complexes were analysed in order to determine a major quenching site. The quenching site has not been found but an important role in the protein stability has been attributed to the Chls bound in site A1 and A4. While Chls in site A3 and B3 appeared to be strongly link and coordinating the only trace of Chl b in the protein. Chapter 3 contains the study of Lhcb5 antenna protein from Arabidopsis thaliana. Using site direct mutagenesis on chl-binding sites and monitoring both carotenoids and chlorophylls triplet formation and quenching, we demonstrate that the interaction between Car in site L2 with Chl bind to site A4 and A5 is crucial for efficient 3Chl quenching. In Chapter 4, I exploited Physcomitrella patens ability of gene targeting to express mutated form of LHCSR protein in vivo. In parallel to the reconstituted proteins, we integrated the same mutations in vivo using a specific vector for Physcomitrella patens. With our construct, the mutated sequence goes by homologous recombination under the control of the endogenous promoter exactly in the same position as the wild-type allele. We integrated the mutated forms of LHCSR1 protein in psbs-lhcsr2 KO genetic background. Among the three mutations (A1, A2 and A5) that we are able to analyze, only the mutant on site A1 has shown a strong effect on NPQ. This is in accord with the in vitro results and its role of protein stability. We have seen above that NPQ consists in the thermal dissipation of excess absorbed energy and is activated within second upon a change in light intensity, by the formation of a ∆pH through thylakoid membranes. Yet its activation relies on different gene products depending on the organisms. In vascular plants, pH transduction signal is operated by PsbS after the protonation of two glutamic acid residues (E122 and E226) exposed at the luminal site. While the green algae Chlamydomonas reinhardti requires a distinct LHC-like polypeptide called LHCSR. On the basis of sequence alignment, I produced single, double and triple mutant of the following point mutations: D117N, E232Q and E233Q in vivo, in order to make an analysis of the role of these acidic amino acids in pH sensing. This study of Physcomitrella patens LHCSR protein can provide information on the adaptation to terrestrial environment. These data could be used to optimize the photoautotrophic organisms growth, used as feedstock for future biofuels. Julien Girardon