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

Abstract

Introduction

As sessile organisms, land plants are able to perceive and adapt to constantly changing external conditions, such as temperature, light and photoperiod. This ability enables them to adjust their developmental transitions, such as flowering time, in response to diel rhythms or seasonal changes (Mouradov et al., 2002; Creux & Harmer, 2019; Bao et al., 2020). Premature flowering affects the overall fitness of plants, thereby negatively influencing crop yield and productivity (Gaudinier & Blackman, 2020). To avoid this, plants employ a series of complex regulatory networks. To date, our knowledge of flowering rests largely at the transcriptional level, with numerous transcription factors having been identified to fine-tune flowering time. Ultimately, these networks culminate with FLOWERING LOCUS C (FLC), a major flowering repressor, directly regulating the expression of floral integrators, FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), to suppress premature flowering (Lee & Lee, 2010; Deng et al., 2011; Bao et al., 2020).

Post-translational modifications (PTMs), such as protein phosphorylation and ubiquitination, work synchronously with transcriptional changes to control flowering time (Linden & Callis, 2020). Protein phosphorylation is directed by protein kinases, which transfer the phosphate group from adenosine triphosphate (ATP) to Ser, Thr and Tyr residues of a protein substrate (Ubersax & Ferrell, 2007), resulting in a multitude of outcomes, including altered enzymatic activity, protein stability and subcellular localization (Humphrey et al., 2015). For instance, CALCIUM-DEPENDENT PROTEIN KINASE28 (CPK28) directly phosphorylates PLANT U-BOX 25 (PUB25) and PUB26 to upregulate its ubiquitin ligase activity and ensure immune homeostasis (J. Wang et al., 2018). So far, several kinases have been related to flowering time control (Ogiso et al., 2010; Chen et al., 2020; Li et al., 2022; Sanagi et al., 2021), including SHAGGY-LIKE KINASE 12 (SK12), which was recently resolved to phosphorylate the core flowering regulator CONSTANS (CO) causing rapid degradation of CO by the 26S proteasome, thereby repressing flowering (Chen et al., 2020).

In addition to phosphorylation, protein ubiquitination is also associated with flowering. Protein ubiquitination is mediated by a multi-enzymatic cascade. This involves E1 enzymes, which activate ubiquitin molecules that are then passed to E2 enzymes to form an E2-ubiquitin intermediate. Lastly, E3 enzymes direct the transfer of an ubiquitin moiety from E2 enzymes to specifically recognized protein targets, leading to the ubiquitination of substrates (Vierstra, 2009). Interestingly, a number of E2s and E3s have been shown to possess regulatory functions in flowering development. For example, the interaction of RING-type ubiquitin ligases UBIQUITIN-CONJUGATING ENZYME 1/2 (UBC1/2) with HISTONE MONO-UBIQUITINATION1 (HUB1) and HUB2 is to mono-ubiquitinate histone B2 (H2B) proteins and regulate flowering time (Cao et al., 2008; Gu et al., 2009). The absence of ubiquitin-modified H2B (H2Bub1) has been suggested to decrease FLC levels, leading to early flowering phenotypes in hub1 and hub2 mutants (Cao et al., 2008; Gu et al., 2009). H2Bub1 is also known to modulate the circadian expression of genes (Himanen et al., 2012; Woloszynska et al., 2019) and to induce light-responsive genes (Bourbousse et al., 2012), in addition to modulating the expression of auxin biosynthesis genes (Zhang et al., 2021). Most recently, HUB1 and 2 have been shown to interact with SPLIT ENDS 3 (SPEN3) and KH DOMAIN-CONTAINING PROTEIN1 (KHD1) to influence the formation of anti-sense COOLAIR transcripts (Woloszynska et al., 2019). Collectively, these findings indicate that HUB2 maintains a complex, multifaceted connection to flowering time. Despite HUB2 being extensively linked to multiple biological processes in plants, how HUB2 is modulated in plants remains unknown. Interestingly, the human orthologs of HUB1/2, RING FINGER PROTEIN 20/40 (RNF20/40), are found to be phosphorylated by the ATAXIA-TELANGIECTASIA MUTATED protein kinase, which modulates H2Bub1 levels, suggesting that phosphorylation may affect the ubiquitin ligase activity of HUB2 (Moyal et al., 2011).

Recently, a subfamily of Arabidopsis B4 Raf-like MAPKKKs (RAF18, RAF20 and RAF24) was reported to phosphorylate SUCROSE NONFERMENTING-1-RELATED KINASES (SnRK2s) (MAPK Group, 2002; Lozano-Juste et al., 2020). Upon osmotic stress conditions, RAF18, RAF20 and RAF24 strongly phosphorylate subclass I SnRK2s, which then phosphorylate and activate VARICOSE (VCS) to modulate the mRNA population and plant stress response (Fàbregas et al., 2020; Lin et al., 2020; Soma et al., 2020). In fact, Lin et al. (2020) also found that RAF24 was able to weakly phosphorylate SnRK2.6, one of the members from subclass III. On the other hand, some RAFs have been found to be involved in plant growth and fitness. For example, CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1)/RAF1 was characterized as a negative regulator of ethylene hormone signaling (Ju et al., 2012), while ENHANCED DISEASE RESISTANCE 1 (EDR1)/RAF2 has been implicated in fine-tuning plant immunity (Zhao et al., 2014). In addition, RAF22 and RAF36 were reported to negatively regulate abiotic stress responses and BLUE LIGHT-DEPENDENT H⁺-ATPASE PHOSPHORYLATION (BHP)/RAF27 was shown to be a regulator of stomatal opening (Hayashi et al., 2017; Kamiyama et al., 2021; Sun et al., 2022).

Although RAF18, RAF20 and RAF24 have been assigned roles in osmotic stress response, other biological roles for these RAFs remain unknown. In this study, we report that RAF24 functions as a flowering time repressor that operates independently of its close relatives: RAF18 and RAF20. Our combined use of genetic, molecular and biochemical analyses coupled with quantitative phosphoproteomics reveals that RAF24, through select subclass I and III SnRKs, regulates HUB2 to influence flowering time. Moreover, we reveal that the ability of RAF24 to fine-tune flowering seems to be dependent on FLC and FT, but not SOC1. We also find that loss of RAF24 results in changes in both HUB2 ubiquitin ligase activity and its protein interactome. Finally, using a combination of phospho-mimetic (HUB2^S314D) and phospho-ablative (HUB2^S314A) mutations, we show that phosphorylation of HUB2 at S³¹⁴ suppresses the early flowering phenotype of the raf24-2 mutant. Taken together, we reveal RAF24 to be a protein kinase whose function is to repress flowering by influencing HUB2 ligase activity and its interactions with other proteins.