bioRxiv2023
BIG (also known as DOC1 and TIR3) is an 0.5 MDa protein that has been associated with multiple important functions in signalling and development through forward genetic screens in Arabidopsis thaliana. However, the biochemical function(s) of BIG are unknown. Here, we investigated whether BIG plays a role in the Arg/N-degron pathways, protein regulatory mechanisms in which substrate protein fate is influenced by the N-terminal (Nt) residue. In Arabidopsis, PROTEOLYSIS1 (PRT1) is an E3 ligase with specificity for aromatic amino acids, whereas PROTEOLYSIS6 (PRT6) targets basic N-terminal residues. We crossed a big loss-of-function allele to prt6 and prt1 mutants and examined the stability of protein substrates. Stability of model N-degron pathway substrates was enhanced in prt6-1 big-2 and prt1-1 big-2 relative to the respective single mutants. Abundance of the PRT6 physiological substrates, HYPOXIA RESPONSIVE ERF (HRE)2 and VERNALIZATION (VRN)2 was similarly increased in prt6 big double mutants, without increase in transcripts. Accordingly, hypoxia marker expression was enhanced in prt6 big double mutants, in a manner requiring arginyltransferase activity and RAP-type ERFVII transcription factors. Transcriptomic analysis of roots not only demonstrated synergistically increased expression of a plethora of hypoxia responsive genes in the double mutant relative to prt6 but also revealed other roles for PRT6 and BIG, including regulation of suberin deposition through both ERFVII-dependent and independent mechanisms, respectively. Our results show that BIG acts together with PRT6 to regulate the hypoxia response and wider processes. Significance StatementThe N-degron pathways are a group of protein regulatory mechanisms that play important roles in plant growth, development, and response to biotic and abiotic stresses. Despite rapid progress in the last decade, key enzymatic components of the pathways remain to be identified. BIG (also known as DOC1 and TIR3) is a protein of approximately 0.5 MDa, associated with multiple, distinct roles in plants but the precise biochemical functions of this protein have remained enigmatic until now. Here we identify BIG as a new component of plant N-degron pathways that acts together with the N-recognin E3 ligase PROTEOLYSIS6 (PRT6) to control the hypoxia response and other functions in Arabidopsis thaliana.