Plant primary productivity is at the basis of our human societies: for food, for fuel and for renewable materials. Photosynthesis is currently proposed as the final frontier for increasing biomass production. For such an ambitious undertaking, an integrated approach, starting from the molecular level and analyzing the effects of changes at increasingly organized levels is essential. Both in Europe and internationally, a number of prospective meetings have been organized to address the problem of the limitations of photosynthesis, research projects have been undertaken to tackle these questions, and a few examples of photosynthetic yield improvements in the field have been proposed. We have chosen to target cytochrome b6f (cyt b6f), which represents one of the limiting steps for photosynthesis. The cyt b6f is one of the most complex chloroplast proteins. It is central in the regulation of both electron transfer and proton transfer, thus key in the regulation of electron fluxes through the photosynthetic electron transfer chain. It is also a pivotal regulatory point for photo-protective mechanisms such as state transitions. In photosynthesis, O2 production and CO2 capture is possible because of electron transfer through two photosystems coupled in series by cyt b6f complex. This complex does not only transfer electrons, it also contributes to regulate the light distribution between Photosystem I (PSI) and Photosystem II (PSII) to optimize the quantum yield of photosynthesis. We are far from understanding exactly how it works, except that cyt b6f is a hub between the pool of quinones (binding to the Qo and Qi site of the complex) and the serine-threonine specific MAP kinase, named Stt7, that phosphorylates the light harvesting complexes 2 (LHCII) which migrate from the grana stacks rich in PSII, to PSI in the lamellae. The mechanism of activation of the kinase by cytochrome b6f was poorly understood as we were left with the idea that the kinase domain of Stt7 was located on the stromal side of the membrane (Qi site) when the activation signal was supposed to originate from the luminal side of the membrane (Qo site). We have recently deciphered a new mechanism for the regulation of photosynthesis: the activation mechanism of the kinase involved in state transitions (Dumas, Zito et al. 2017). We disclosed that the triggering of the Stt7 kinase to phosphorylate LHCII proteins occurs via a direct interaction with the chloroplast cytochrome b6f complex in the stromal compartment. This discovery broadens ours horizons because we have now spotted key regulatory residues of cyt b6f on the stromal side of the membrane. We showed that cytochrome b6f subunit IV was directly involved in the activation of the Stt7 kinase (Dumas, Zito et al. 2017). We have now lifted the main scientific and technical barrier: arginine in position 125 (Arg125) from SuIV (Arg125SuIV) is involved in a direct and pivotal interaction with Stt7. We are now making the hypothesis that Stt7 is activated by autophosphorylation, through an unknown mechanism involving Arg125SuIV. We are planning to dissect this mechanism by a series of biochemical and biophysical methods, from purified complexes and recombinant proteins up to increasingly integrated systems, native membranes and whole cells. Our research project is a structure / function project, with scientific objectives focused on the mechanisms and on the dynamic interaction between cyt b6f complex and Stt7 kinase.

Fatty acid photodecarboxylase (FAP) is a photoenzyme recently discovered and characterized by the coordinator and a partner of this proposal (Sorigué et al. 2017 Science 357:903). FAP catalyses the light-driven decarboxylation of fatty acids into hydrocarbons and CO2. It is only the third photoenzyme to be identified and thus represents a unique opportunity to deepen the understanding of light-driven catalysis. In spite of our initial characterization of FAP, 3D structures of intermediate-states and a detailed mechanistic understanding of FAP catalytic events have remained elusive. Our goal is to gain insight into the mechanistic events along the FAP photocycle, occurring from the ultra-fast time scale (femto- to picoseconds) right after photon absorption to product formation on slower time scales (nano- to milliseconds). Our central hypothesis is that photon absorption by FAD leads to product formation in FAP via a sequence of intermediate states within a photocycle that involves distinct spectroscopic and structural changes. Our objectives are the structural and spectroscopic characterization of FAD excited states on the ultra-fast time scale, the structure determination of catalytic intermediate states, the spectroscopic elucidation of electron and proton transfer steps, the structural and spectroscopic observation of the cleavage of the C-C bond leading to substrate decarboxylation, and the identification of a proton (or hydrogen atom) donor (amino acid or water molecule) whose existence has been postulated in the photocycle. The chosen methodology consists of a combination of experimental biophysical and biochemical techniques, including time-resolved crystallography at synchrotrons and X-ray free electron lasers (XFEL), FTIR and time-resolved infrared, absorption and fluorescence spectroscopy on FAP in solution and in crystals and complementary QM/MM computational methods. In particular, the project SNAPsHOTs will fully exploit the unique capabilities of the European facility for X-ray free electron laser (XFEL) that has been recently inaugurated and to which France contributed financially. Funding will be critical to maintain French leadership in a highly competitive field with potential industrial applications. Ultimately, our project will provide a molecular movie of the FAP photocycle that features the structural changes occurring during light-driven enzyme catalysis at atomic resolution. Structural and mechanistic information should also be useful to improve stability, turnover or specificity of FAP in view of biotechnological applications.

Solar-driven hydrogen production from the abundant and cheap electron source water is a promising way to produce renewable energy. Plants and cyanobacteria have developed a water splitting enzyme which is able to oxidize water into molecular oxygen, protons and electrons using visible light energy within the membrane protein photosystem II. The heart of the enzyme is a Mn4CaO5 cluster at which water oxidation takes place following four sequential light-induced steps. Reactions at the Mn4CaO5 cluster consist of concerted electron and proton transfer, and form intermediate states that minimize the activation energy necessary for the water oxidation process. Photosystem II is thus a paradigm for engineering bio-inspired solar energy converting applications. A recent high-resolution three-dimensional structure of photosystem II gave a precise arrangement of the Mn-Ca cluster necessary for water oxidation. In addition, the combination of theoretical catalytic models with experimental data from numerous state-of-the-art spectroscopic techniques have given a possible view of Mn oxidation states during water oxidation, of water fixation steps, have revealed the importance of a set of amino acids in the catalytic mechanism, and given hints on proton transfer reactions involving extended hydrogen bonding networks. Despite these remarkable progresses in recent years, key questions remain opened. They concern the position of reactive molecules, the formation mechanisms of the oxygen molecule itself, and relaxation processes at the Mn4CaO5 cluster involving spin-state transitions and concerted electron and proton transfer. The PS2FIR project will contribute to answer these questions. We will gather the complementary expertise of three research teams: Team 1, R. Hienerwadel & C. Berthomieu, UMR 7265; Team 2, A. Boussac UMR 9198; and Team 3, J.B. Brubach & P. Roy, Synchrotron SOLEIL, to probe light- and near infared (NIR)- induced transitions and spin conversions at the Mn4CaO5 cluster, using state-of-the-art far-infrared FTIR difference spectroscopy. Vibrational modes in the far-infrared down to 10 cm-1 will allow probing the valence state of the Mn ions, cluster conformation, and Mn-O/Ca-O interactions. Of particular interest will be the identification of libration and connectivity modes of water molecules associated to the cluster below 300 cm-1 during the reaction cycle. To overcome the challenge of exploiting small-bands in the Far-infrared domain, setups will be optimized to probe different samples in parallel and to optimize NIR-induced spin-state transitions by controlled temperature jumps. We will also benefit from the brilliance of the synchrotron AILES beamline at SOLEIL. Highly resistant photosystem II from Thermosynechoccocus elongatus prepared to precisely select within heterogeneous oxidation or spin states of photosystem II will allow to decipher the molecular origin of different Mn4CaO5 cluster conformations, and ultimately to contribute to our understanding of water oxidation and O-O bond formation.

Plant pathogens such as Ralstonia, Xylella or Xanthomonas are responsible for devastating vascular systemic infections in crops. The genetic basis of plant vascular immunity are poorly understood thus limiting the design of resistant or tolerant crops. With a focus on black rot disease of Brassicaceae caused by the devastating crop pest Xanthomonas campestris (Xc), the NEPHRON project aims to identify and characterize different layers of plant immunity at the hydathodes which are natural entry points for Xc. Hydathodes are plant organs located at the leaf margin and where guttation occurs. The NEPHRON project will establish the genetics of hydathode differentiation and physiology and its importance for the susceptibility to black rot disease. The results acquired in Arabidopsis and in different elite Brassica varieties will help to design knowledge-based strategies to better control vascular pathogens.

Synthetic microbiology is among the most promising approaches for getting more at lower cost and in the respect of the environment. Directed evolution is recognized as a key approach to obtain biobricks for synthetic biology. In this context there is a considerable interest in the development of continuous systems for directed evolution of biomolecules based on “orthogonal” evolution vector on which accumulation of mutations can be uncoupled from accumulation of mutations on the host genome. This project aims at developing such a system for the gram-positive bacterium Bacillus subtilis. An important step towards biotechnological applications will also be made by using the proposed system for: the evolution of new transcription factors for genetic circuit engineering in B. subtilis; and the evolution of new proteins binding inorganic ions such as heavy metals that might serve as biosensors and in bioextraction systems. The work program decomposes into three work-packages : development of a system for directed evolution in B. subtilis ; in silico analyses for the optimization of the system ; application to biobrick production. B. subtilis is a totally harmless bacterium of considerable biotechnological interest: it stands as the second model bacterium after Escherichia coli and is as such a natural chassis for synthetic biology; it is also a soil dweller (and probably a normal gut commensal in humans) with highly diverse physiological capabilities, and an ability to survive extreme conditions in the form of spores. B. subtilis and several of its close relatives of the Bacillus genus (notably B. licheniformis and B. amyloliquefaciens) exhibit a remarkable capacity of biological compound production that can be scaled-up to industrial levels are widely used in the industry for enzyme production.