Control of flowering time and vernalization in Arabidopsis thaliana by the N-end rule pathway

The N-end rule pathway of proteolysis targets proteins for destruction based on the nature of their N-terminus. I have shown that the N-end rule pathway in Arabidopsis regulates the ‘Methionine-Cysteine (MC)-initiating’ protein VERNALIZATION2 (VRN2). VRN2 functions to coordinate cold-responsive flowering and has several other key developmental roles. VRN2 is one of three plant homologues of the Drosophila protein SUPPRESSOR OF ZESTE12 (Su(z)12), which functions as part of the polycomb repressive complex2 (PRC2), a conserved eukaryotic complex that regulates the epigenetic silencing of genes through depositing the Histone 3 Lysine 27 tri- methylation (H3K27me3) repressive mark to chromatin. Here I provide in vitro and in vivo evidence that VRN2 is a physiological substrate of the N-end rule pathway. VRN2 is stabilised under hypoxia and NO-limited conditions and post-translational accumulation of VRN2 during vernalization is linked to its regulation by the N-end rule. One hypothesis to explain VRN2 stabilisation is that cold-induced VERNALIZATION INSENSITIVE3 (VIN3) shields the MC terminus to prevent it being targeted for degradation by the E3 ligase PROTEOLYSIS6 (PRT6). However, in vitro and via inducible VIN3 transgenic lines VRN2 is still degraded in the presence of VIN3. Additionally, this project demonstrates that the destabilising N-terminus of VRN2 likely arose following gene duplication and N-terminal truncation of an ancient homologue of EMBRYONIC FLOWER2 (EMF2), providing new insight into how proteins can become co-opted to the N-end rule pathway during evolution to provide new functions. Finally, I have found that EMF2c in Barley is also a substrate of the N-end rule pathway and may represent a functional homologue of VRN2