Thesis presented December 11, 2023

Abstract:
During chloroplast biogenesis, the assembly of the photosynthetic apparatus is under transcriptional regulations occurring in the nucleus and plastids. Yet, the activity of the Plastid-encoded RNA Polymerase (PEP) is coupled to nuclear transcription for the coordinated expression of Photosynthesis-associated-plastid genes (PhAPGs) and Photosynthesis-associated-nuclear genes (PhANGs). The PhAPGs are specifically transcribed by the light-activated PEP complex that comprises four catalytic subunits (α2, β, β′, β″), 12-PEP-associated proteins (PAPs) and fleeting interactors, such as redox-associated proteins. Among the 12 PAPs, PAP4 (FSD2) and PAP9 (FSD3) are Fe-superoxide dismutases found only in chloroplasts and protect the PEP complex from oxidative stress. These proteins are essential for facing the surge in reactive oxygen species (ROS) that occurs during the first photosynthetic reactions. A strategy of PAP8 proximity labelling led to the identification of another pivotal redox protein PRIN2 (Plastid Redox Insensitive 2) previously reported to interact with the thioredoxin PAP10 and the RNA-binding protein CSP41b (Chloroplast Stem-loop binding Protein). All These proteins are essential to the PEP activity as attested by their photosynthetically-deficient mutant phenotypes . . The thesis investigates the superoxide dismutase activity of purified PAP4 and PAP9 proteins. The question of PAP4 and PAP9’s role to be structural or catalytic in the PEP complex was aimed to be answered. The structure of CSP41b was characterised by cryo-EM at 3.4 Å resolution. The study aimed to identify the interactions between PRIN2 and CSP41b by biophysical techniques such as size-exclusion chromatography, isothermal titration calorimetry, mass spectrometry and cryo-EM. This interaction and others were tested in onion epidermal cells using bimolecular fluorescence complementation assay. For the unbiased fishing of PRIN2 interactors, a proximity labelling strategy was designed. The genetic constructions were cloned and tested in transient experiments proving its feasability. The study presented here is part of a broader project that aims to highlight the functional innovations around plastid transcription specific to Angiosperms.

Keywords:
Redox signaling, Transcription, Chloroplast biogenesis, RNA Polymerase, Photosynthesis