MLK4‐mediated phosphorylation of histone H3T3 promotes flowering by transcriptional silencing ofFLC/MAFinArabidopsis thaliana

Abstract

Casein kinase I (CK1), a ubiquitous Ser/Thr protein kinase in eukaryotes, plays a critical role in higher plant flowering. Arabidopsis CK1 family member MUT9-LIKE KINASEs, such as MLK1 and MLK3, have been shown to phosphorylate histone H3 at threonine 3 (H3T3), an evolutionarily conserved residue, and the modification is associated with the transcriptional repression of euchromatic and heterochromatic loci. This study demonstrates that mlk4-3, a T-DNA insertion mutant of MLK4, flowered late, and that overexpression of MLK4 caused early flowering. The nuclear protein MLK4 phosphorylated histone H3T3 both in vitro and in vivo, and this catalytic activity required the conserved lysine residue K175. mutation of MLK4 at K175 failed to restore the level of phosphorylated H3T3 (H3T3ph) or to complement the phenotypic defects of mlk4-3. The FLC/MAF-clade genes, including FLC, MAF4 and MAF5, were significantly upregulated in mlk4-3. The double mutant mlk4-3 flc-3 flowered earlier than mlk4-3, suggesting that functional FLC is crucial for flowering repression in mlk4-3. Chromatin immunoprecipitation assays showed that MLK4 bound to FLC/ MAF chromatin and that H3T3ph occupancy at the promoter of FLC/MAF was negatively associated with its transcriptional level. In accordance, H3T3ph accumulated at FLC/MAF in 35S::MLK4/mlk4-3 but diminished in 35S::MLK4(K175R)/mlk4-3 plants. Moreover, the amount of RNA Pol II deposited at FLC/MAF was clearly enriched in mlk4-3 relative to the wild type. Therefore, MLK4-dependent phosphorylation of H3T3 contributes to accelerating flowering by repressing the transcription of negative flowering regulator FLC/MAF. This study sheds light on the delicate control of flowering by the plant-specific CK1, MLK4, via post-translational modification of histone H3.

Phosphorylation‐mediated signalling in flowering: prospects and retrospects of phosphoproteomics in crops

“…Interruption of MLK3 caused early flowering and down-regulation of FLC transcripts, indicating that MLK3 negatively regulates flowering possibly by derepressing FLC expression (Kang et al, 2020). Wang et al (2020c) reported that the nuclear protein MLK4 phosphorylated histone H3 at Thr 3 both in vitro and in vivo. Moreover, a transfer DNA (T-DNA) insertion mutant of MLK4 flowered late, but overexpression of MLK4 caused early flowering.…”

“…Wa ng et al . (2020 c ) reported that the nuclear protein MLK4 phosphorylated histone H3 at Thr 3 both in vitro and in vivo . Moreover, a transfer DNA (T‐DNA) insertion mutant of MLK4 flowered late, but overexpression of MLK4 caused early flowering.…”

“…Moreover, a transfer DNA (T‐DNA) insertion mutant of MLK4 flowered late, but overexpression of MLK4 caused early flowering. Thus, MLK4‐dependent phosphorylation of histone H3 contributes to accelerating flowering by repressing the transcription of negative flowering regulator FLC / MA F‐clade genes (Wa ng et al ., 2020 c ). Consistently, MLK4 positively regulates flowering possibly through the circadian rhythm by phosphorylation of histone H2A at Ser 95 (Su et al ., 2017).…”

Phosphorylation of Histone H2A at Serine 95 Is Essential for Flowering Time and Development in Arabidopsis

“…Since MLK1, MLK2, and MLK4 also functioned as kinases to histone H3 at threonine 3 ( Wang et al, 2015 , 2021 ), we examined the H3T3ph levels. No obvious difference of H3T3ph levels were observed in wild type, mlk1 mlk2 mlk3 , mlk1 mlk2 mlk4 , mlk1 mlk3 mlk4 , and mlk2 mlk3 mlk4 triple mutants ( Supplementary Figure 5A ).…”

“…MLK4 interacts with CCA1, and loss of MLK4 functions resulted in late flowering and reduction of H2AS95ph and H2A.Z level at GI ( Su et al, 2017 ). A recent study showed that MLK4 deposited H3T3ph at FLC/MAF ( Wang et al, 2021 ). Phosphoproteomics showed that MLKs phosphorylated proteins in circadian clock, multiple metabolic, and signaling pathways in Arabidopsis ( Wilson et al, 2021 ).…”