DEFINING THE GUN1-FTSH INTERACTIONS IN CHLOROPLAST BIOGENESIS IN ARABIDOPSIS THALIANA

The GENOMES UNCOUPLED 1 (GUN1) gene has been reported to encode a chloroplast-localized pentatricopeptide-repeat protein (PPR), which acts to integrate multiple indicators of plastid developmental stage and altered plastid function, as part of chloroplast-to-nucleus retrograde communication. However, the molecular mechanisms underlying the GUN1-dependent signal integration have remained elusive, until the recent identification of a set of GUN1- interacting proteins, by co-immunoprecipitation and mass-spectrometric analyses, as well as protein\u2013protein interaction assays. Within the list of GUN1 interactors the FtsH1, FtsH2, FtsH5 and FtsH8 subunits, that make up the thylakoid FTSH protease complex, were identified. The FtsH subunits are part of the ATP-dependent metalloprotease family and they play a fundamental role in the maintenance of the chloroplast protein homeostasis. In this thesis, I show that FtsH2 and FtsH5 subunits have a really important genetic interaction with GUN1 during the chloroplast biogenesis process in Arabidopsis cotyledons. In particular, I observed that GUN1 is important for the accumulation of the FtsH subunits in the thylakoid membranes, and for the import of several plastid precursor proteins that, in the absence of GUN1 protein, accumulate in the cytosol, with the consequent increase in total protein ubiquitination and cytosolic chaperone abundance. In the attempt to give a molecular explanation to the observation reported above, I was able to demonstrate that in condition of plastid protein homeostasis alteration, GUN1 controls the accumulation of Nuclear encoded RNA polymerase (NEP)-dependent transcripts and, indirectly, affects the chloroplast protein import apparatus, since a major component of the 1MDa TIC complex (Translocon at the Inner membrane of the Chloroplasts), i.e. Tic214, is encoded by the plastid genome. Such a defect in Tic214 accumulation appears to destabilize the entire plastid import machinery both in terms of protein amount and post-translation modification, as shown by the high ubiquitination levels of Toc34 subunit. Strikingly, such chloroplast alterations are sensed by nuclear transcription factors, as shown by the negative effect on the accumulation of HY5, a main transcription factor involved in retrograde signaling and chloroplast biogenesis. Overall, during this three year as Ph.D. student I believed to have discovered the primary function of GUN1 protein, shining a new light on the intricate network of chloroplast-nucleus communication