Description

The Arabidopsis (Arabidopsis thaliana) MYB5 (At3g13540) transcription factor (TF) forms transcriptional regulatory (MBW) complexes with basic-Helix-Loop-Helix (bHLH) and TRANSPARENT TESTA GLABRA1 (TTG1) (WD-repeat) TF proteins. These MBW complexes regulate seed coat, trichome and root cell differentiation via a multi-tiered transcriptional mechanism (Golz et al., 2018; Li et al., 2020). MYB5 is partially redundant with MYB23 and TRANSPARENT TESTA2 (TT2) (another MYB protein, MYB123) in MBW complex formation. TTG1 is subject to site-specific phosphorylation by SHAGGY-LIKE KINASE11 (SK11) and SK12 which inhibits TTG1 interactions with TT2 (Li et al., 2018). MYB5 also possesses potential phosphorylation sites at the N-terminus (Li et al., 1996).

To identify proteins interacting with MYB5, the Matchmaker® Gold Yeast Two-hybrid system was used to screen a prey cDNA library. Three candidate bait constructs contained cDNA nucleotide sequences encoding the MYB5 full length, C-terminal and N-terminal regions (Fig 1A), respectively. The constructs containing full length MYB5 and the MYB5 C-terminal region activated MEL1 and AUR1-C reporter genes resulting in blue colonies (Fig 1B) indicating that the C-terminal region acts as an activator motif in yeast. The MYB5 N-terminal construct (pMYB5NT::GAL4BD) failed to activate the reporter genes (Fig 1B) and was therefore used as the bait for library screening. On the screening plates, five yeast colonies were identified and their prey cDNA fragments sequenced. Three of the five cDNA sequences encoded a serine/threonine protein kinase CAESIN KINASE2 BETA3 (CK2β3) (At3g60250) subunit fused in frame with the GAL4AD domain-encoding nucleotide sequence. The remaining two cDNA sequences encoded Hypothetical/HEAT RESPONSE PROTEIN (At5g10010) and PHOSPHOGLUCOSE ISOMERASE (At4g24620). The dependency of pCK2β3::GAL4AD on pMYB5NT::GAL4BD in activating four reporter genes, namely AUR1-C, HIS3, ADE2 and MEL1, was then confirmed (Fig 1C).

MYB5 directly regulates ALPHA/BETA ESTERASE/HYDROLASE1 (ABE1, At2g23550) and ABE4 (At2g23580) expression (Li et al., 2020). The electrophoretic mobility shift assay (EMSA) was used to determine in vitro binding of purified MYB5 protein to the ABE4 promoter. A truncated MYB5 protein containing the MYB DNA-binding domain was expressed in E. coli and purified. The ABE4 promoter probe (Suppl. Fig 1) contains two sequences identified as MYB5 binding sites using chromatin immunoprecipitation (Li et al., 2020). The EMSA identified two weak band shifts (Fig 1D) indicating that MYB5 binds weakly to the ABE4 promoter region in vitro and may therefore be subject to phosphorylation/activation for enhanced MYB-DNA binding in vivo.

The Arabidopsis CK2β3 subunit is part of the CK2 holoenzyme which is a tetramer consisting of two regulatory β-subunits and two catalytic α-subunits collectively implicated in a number of developmental and stress-responsive pathways (Mulekar and Huq, 2013; Wei et al., 2021). Known CK2 substrates primarily include transcription factors or regulatory proteins (Mulekar and Huq, 2013). CK2β3 is highly expressed in developing seeds and has an expression pattern overlapping that of MYB5, TTG1, ABE1 and ABE4 (Winter et al., 2007). CK2β3 localises to the nucleus and cytosol (Salinas et al., 2006) and can modulate the activity of the MYB-related CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) both by direct physical interaction in vivo and phosphorylation (Sugano et al., 1999). CK2β3 shares 75% amino acid sequence similarity with CK2β1 and 71% with CK2β2 (Sugano et al., 1998). CK2β3 and CK2β4 subunits share 87% amino acid sequence identity and 92% sequence similarity. Moreover, like CK2β3, CK2β4 has also been linked to the phosphorylation of CCA1 as over-expression of both genes leads to period-shortening of circadian clock-related gene expression (Perales et al., 2006). Although CK2β3 does not exhibit phosphorylation activity, it has been shown to facilitate CCA1 binding to the promoter of target gene CHLOROPHYLL A/B BINDING PROTEIN1 (CAB1) (Sugano et al., 1998). CK2 also interacts with the MYB-related LATE ELONGATED HYPOCOTYL (LHY) and YING YANG1 (YY1) via CK2β3 and phosphorylates LHY and YY1 proteins in vitro (Sugano et al., 1999; Wu and Li, 2017).

The transcriptional activities of Pinus taeda MYB4 (PtMYB4) and PtMYB46 are positively regulated by phosphorylation (Morse et al., 2009) although phosphorylation of TFs such as LTF1 (which regulates lignin biosynthesis in Populus) can result in its degradation (Gui et al., 2019). In contrast, DNA binding of Arabidopsis MYB15, a transcriptional repressor of cold signalling, is reduced by phosphorylation (Kim et al., 2017). While specific MYB5-CK2β3 interactions in planta are yet to be determined, CK2β3 may facilitate direct MYB5 binding to its target gene promoters or may phosphorylate MYB5 indirectly by recruiting the CK2 catalytic α-subunits. The MYB5 N-terminal region contains several threonine and serine residues which are potential phosphorylation sites (Li et al., 1996) and phosphorylation of these residues may enhance MYB5 protein-DNA binding affinity. Alternatively, as MYB5-TTG1 and/or MYB5-bHLH interactions were not detected in the initial yeast two-hybrid screening, MYB5 may also require activation to facilitate TF protein-protein binding. TTG1 is subject to site-specific phosphorylation which regulates binding with TT2 (MYB123) to form some MBW complex combinations (Li et al., 2018). MYB5-CK2B3 interactions and proposed phosphorylation may contribute to MYB5-bHLH-TTG1 (MBW) complex formation which, in turn, may further increase MYB5 protein-DNA binding affinity in vivo.

In the proposed model (Fig 1E), MYB5-bHLH-TTG1 complexes activate the expression of target genes by binding to their promoters. CK2β3 interacts with MYB5 transiently and recruits the CK2 holoenzyme to phosphorylate MYB5 (Fig 1E) resulting in more active MYB5-bHLH-TTG1 complexes thereby enhancing expression of MYB5 target genes.