Chloroplast biogenesis: function, regulation and structure of the plastid-encoded RNA-polymerase complex and its associated proteins PAPs – PEPRegulChloro3D

The major source of renewable bioenergy on Earth comes from the ability of plants to convert sunlight into chemical energy in a chloroplast-dependent process designated as photosynthesis. During this energy conversion, there are consumption of carbon dioxide and release of di-oxygen impacting the Earth’s atmosphere. Plants are therefore not only the major source of food on Earth (either directly through the grocery market, or indirectly through animals feed), but also the best recycling factory for animal waste products and human modern activities. Plants also offer fibers for construction, clothing, biofuels and all sorts of secondary compounds used in industry and pharmaceuticals. We support the idea that our future will depend on the ability to use photosynthesis of the green lineage to solve great challenges of mankind such as food scarcity and quality, climate change, and environmental pollution. This requires a deep understanding of the chloroplast biology, notably its biogenesis and maintenance in photosynthetic cells. The chloroplast is a semi-autonomous organelle containing its own DNA (plastome) encoding about 75-80 proteins among which the four rpo plastid-encoded polymerase subunits. Therefore most of the 3500-4000 membrane and soluble chloroplastic proteins are synthesized from nuclear genes. Hence chloroplast biogenesis requires a coordinated expression of the plastome and nuclear genome, requiring bi-directional communication between the two cellular compartments. Genetic signals coming from the nucleus are called anterograde and correspond mostly to the nuclear-encoded proteins traveling through the import machinery of the chloroplast envelope thanks to their chloroplastic transit peptides (cTP). In the other direction, the retrograde signals and their genetic components remain poorly understood despite over 30 years of research. Chloroplast biogenesis depends on the assembly of the plastid-encoded RNA-polymerase complex (PEP, 1.1 MDa). While the core components (4 rpo subunits) are encoded in the plastids, 12 major PEP-associated proteins (PAPs) are encoded in the nucleus. Mutations in pap genes yield albino plants incapable of photosynthesis, indicating that the PEP is a keystone of chloroplast biogenesis. At least four PAPs are localized both in plastids and the nucleus where they could form a sub-complex involved in the retrograde signals. These PAPs have a nuclear localization sequence (NLS) in addition to the cTP. This may reveal unforeseen signals traveling from the chloroplast to the nucleus thanks to the bi-localized proteins. Since little is known about the formation, regulation, and dynamics of the PEP and the PAPs, we aim to develop a multi-scale integrated approach to assess their function in the whole organism and to reveal their structure at the atomic level. The following institutes IBS (partner 1), BIG/LPCV (partner 2 designed hereafter as BIG) and IGBMC-CERBM (partner 3) built a partnership that will use many complementary approaches to work on the chloroplast biogenesis. Using biochemistry and molecular biology we will produce and purify PEP and PAPs for their biophysical characterization. Using plant genetics, we will study PEP and PAP mutants, their respective function in different cell types and organelles, and the signaling between the nucleus and chloroplast. By combining mass spectrometry, cryo-electron microscopy, X-ray crystallography and NMR, we will solve the 3D structures of PAPs and PEP. This project will bring basic knowledge that can potentially be applied in biotechnology and agriculture to better face environmental, social and economic challenges of the 21st Century.