B4 Raf-like MAPKKK RAF24 regulates Arabidopsis thaliana flowering time through HISTONE MONO-UBIQUITINATION 2

The timing of flowering is a critical agronomic trait governed by an extensive and sophisticated regulatory network. To date, limited understanding of how posttranslational modifications regulate flowering time exists. Here, using Arabidopsis, we resolve a role for the B4 Raf-like MAPKKK protein kinase RAF24 in regulating flowering time. Loss of RAPIDLY ACCELERATED FIBROSARCOMA24 (RAF24) led to premature flowering time through altered expression of FLC and FT. Comparative phosphoproteomic analysis of raf24 and wild-type plants revealed a list of known flowering-related phosphoproteins from distinct flowering pathways displaying downregulated phosphorylation. Of these, the RING-type ubiquitin ligase HISTONE MONO-UBIQUITINATION 2 (HUB2) lacked phosphorylation in the absence of RAF24. Absence of RAF24 induced H2Bub1 overaccumulation, with protein-protein interactome analysis of HUB2 in the presence and absence of RAF24 influencing HUB2 protein interaction partners, such as H2B. HUB2 was also found to physically interact with SUCROSE NONFERMENTING KINASE 2.4 (SnRK2.4) and SnRK2.6, known substrates of RAF24. Using phospho-mimetic and phospho-ablative plant lines, we then validated the importance of HUB2 phosphorylation at serine 314 (S314) in maintaining appropriate flowering time. Our findings uncovered a novel biological role of RAF24, as a higher-order flowering regulator, while further implicating HUB2 as a centerpiece of flowering time regulation.