Phylogenetic analysis showed MLK3 is closely related to MLK1, MLK2, and MLK4 (Supplementary Figure 1). To investigate the function of MLK3, we isolated the two T-DNA insertion mutants. Genotyping showed that the mlk3-1 and mlk3-2 alleles, each contain a T-DNA in the exon10 and exon12, respectively (Figures 1A,B). RT-PCR analyses revealed that mlk3-1 and mlk3-2 are null alleles (Figure 1C). The primary leaf number at bolting of mlk3-1 and mlk3-2 is more than that of the wild type (Supplementary Table 1). mlk3-1 and mlk3-2 exhibited late flowering-time phenotype under long day (LD) photoperiod, but not in short day (SD) photoperiod (Figures 1D,E and Supplementary Table 1), suggesting that MLK3 is involved in photoperiod pathway.

To investigate the redundant functions of MLK3 and MLK4, we crossed mlk3-1 into mlk4-2 and generated the mlk3-1 mlk4-2 double mutant. The flowering time of mlk3-1 mlk4-2 double mutant is later than those of mlk3-1 and mlk4-2 in LD, but not in SD (Figures 1F,G, and Supplementary Table 1). mlk3-2 mlk4-3 double mutant further supported that the functions of MLK3 and MLK4 in flowering time (Supplementary Table 1). These results suggested that MLK3 and MLK4 have unequal redundancy in flowering time.

To confirm this, we crossed the mlk3-1 into co-9 and generated mlk3-1 co-9 double mutant. The flowering time of mlk3-1 co-9 is similar to that of co-9 (Figures 1H,I and Supplementary Table 1), suggesting that MLK3 is involved in CO-dependent flowering pathway. The transcripts of CO were examined, and the transcription levels of CO were reduced in mlk3 mutants, and further reduced in mlk3 mlk4 mutants (Figure 1J and Supplementary Figure 2A), suggesting that CO is downstream of MLK3 and MLK4. We then investigated transcripts of FT and found that the transcript levels of FT were reduced in mlk3, mlk4, and mlk3 mlk4 plants, which are positively correlated with transcript levels of CO (Supplementary Figure 2B). We then investigated the transcripts of MLK3 and found that MLK3 exhibits a circadian rhythm dependent expression pattern with a peak at the end of the 16-h light period, which is similar to the expression patterns of CO and FT (Supplementary Figures 2A–C). However, the transcript levels of FLC did not show an obvious difference in mlk3, mlk4 single mutants, and mlk3 mlk4 double mutants (Supplementary Figure 2D).

Mutation in MLK1 or MLK2 did not show any flowering-time phenotype, and mlk1 mlk2 double mutant displayed the late flowering under LD, but not SD (Zheng et al., 2018; Supplementary Figures 3A,B and Supplementary Table 1). We examined whether MLK1/2 is involved in the photoperiod pathway. We crossed mlk1-3 mlk2-3 into co-9 and found that the flowering time of mlk1-3 mlk2-3 co-9 triple mutant is similar to that of co-9 (Figures 2A,B, Supplementary Figure 3C, and Supplementary Table 1), suggesting that MLK/2 is involved in the photoperiod pathway. These results were confirmed with reduced CO transcription level in mlk1-3 mlk2-3 double mutant (Supplementary Figure 3D).

To investigate the relationship between MLK3 with MLK1 and MLK2, we crossed mlk3-1 into mlk1-3 mlk2-3 double mutant and generated mlk1-3 mlk2-3 mlk3-1 triple mutant. The flowering time of mlk1-3 mlk2-3 mlk3-1 triple mutant is similar to that in mlk1-3 mlk2-3 double mutant in LD and SD (Figures 2C,D and Supplementary Table 1). The transcript levels support these results that the transcription levels of CO in mlk1 mlk2 double mutant are similar to those in mlk1 mlk2 mlk3 triple mutant (Supplementary Figure 3D).

We next individually examined the MLK1 and MLK2 in contribution to flowering time in mlk3 mlk4 double mutant. We introduced mlk1-3 and mlk2-3 into mlk3-1 mlk4-2, and generated mlk1-3 mlk3-1 mlk4-2 and mlk2-3 mlk3-1 mlk4-2 triple mutant, respectively. The flowering time of mlk1-3 mlk3-1 mlk4-2 and mlk2-3 mlk3-1 mlk4-2 is similar to that of mlk3-1 mlk4-2 under LD (Figures 3A–D and Supplementary Table 1). These observations suggested that mutations of MLK1 or MLK2 in mlk3 mlk4 double mutant failed to reinforce the late-flowering phenotype of mlk3 mlk4 under LD. Collecting the flowering-time of mlk3, mlk4, mlk3 mlk4, mlk1 mlk3 mlk4, and mlk2 mlk3 mlk4 mutants (Figures 1F,G, 2C,D, 3A–D), we concluded that MLK4 played critical roles in flowering time in MLK family.

The functions of MLK1/2 and MLK4 were then investigated. The mlk1-3 mlk2-3 displayed semidwarf phenotype, whereas mlk1-3 mlk2-3 mlk4-2 exhibited severe dwarf phenotype with short petiole, sharp, and curly leaf (Figures 3E,F and Supplementary Figure 3E); suggesting that MLK4 is involved in plant development with MLK1/2. The flowering-time of mlk1-3 mlk2-3 mlk4-2 is early than that of mlk4-2 under LD (Figures 3G,H). One possibility is due to strong growth defects in mlk1-3 mlk2-3 mlk4-2 (Figures 3E,F and Supplementary Figure 3E).

We investigated the MLK3 phosphorylation activity and found that MLK3 exhibited high activity to histone H2A (Figure 4A). MLK3 prefer to phosphorylate the serine of H2A and the six serines of H2A are located at amino acid positions 9, 17, 19, 20, 95, and 124. The H2A histones containing serine-to-alanine substitutions at 9, 17, 19, and 20 were strongly phosphorylated, but this phosphorylation activity was impaired when serine 95 and serine 124 were substituted with alanine (Figures 4B,C). Further study showed that serine 95 substituted by alanine primarily abolished the MLK3 activity (Figure 4C). Similar results were observed in MLK1/2 phosphorylation activity assays (Supplementary Figures 4A–F).

The global levels of H2AS95ph were examined and no obvious changes were observed in mlk1, mlk2, mlk3, and mlk4 single mutants and mlk1 mlk2 double mutants (Figures 4D,E). However, mutations in MLK3 or MLK4 in mlk1 mlk2 double mutant reduced global H2AS95ph levels (Figures 4D,E), suggesting that MLK3 and MLK4 are crucial in H2AS95ph deposition. These results confirmed that reduction of H2AS95ph levels in mlk3 mlk4 double mutants was observed (Figures 4F,G). In addition, H2AS95ph levels in mlk1 mlk3 mlk4 and mlk2 mlk3 mlk4 triple mutants are similar to those of mlk3 mlk4 double mutants (Figures 4F,G). These observations indicated that the downregulated H2AS95ph levels were correlated with late-flowering time phenotype in mlk3 mlk4, mlk1 mlk3 mlk4, and mlk2 mlk3 mlk4 plants. In addition to flowering time, MLK1 and MLK2 were also involved in plant development and osmotic stress resistance (Wang et al., 2015; Zheng et al., 2018), suggesting that MLK1 and MLK2 might function independently with MLK3 and MLK4.

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). These results suggested that MLKs are predominately involved in H2AS95 phosphorylation, but not H3T3 phosphorylation.

Since MLK3 is involved in photoperiod pathway, we tested whether MLK3 interacts with any of the factors in the circadian rhythm pathway. A yeast two-hybrid result showed that MLK3 directly interacts with CCA1, but not with the controls (Figure 5A). These results were confirmed with pull-down and BiFC. Beads containing His fused with CCA1 (His-CCA1), but not His, bound to soluble GST fused with MLK3 (GST-MLK3) (Figure 5B). Reciprocally, beads containing GST-MLK3 were bound to soluble His-CCA1 (Figure 5C). These results were validated by BiFC, and reconstituted YFP fluorescence was observed in the nucleus with coexpressing MLK3 fused to the YFP N-terminus (MLK3-YFP^N) and CCA1 fused to the YFP C-terminus (CCA1-YFP^C) (Figure 5D). The co-immunoprecipitation (Co-IP) supported our finding that MLK3 directly binds to CCA1. HA fused with MLK3 (HA-MLK3) and FLAG fused with CCA1 (FLAG-CCA1) were coexpressed in Arabidopsis protoplasts and immunoprecipitated with anti-FLAG antibody. MLK3, but not control, could be coimmunoprecipitated with CCA1 (Figure 5E).

The mlk3 cca1 double mutant was then generated by crossing mlk3-1 into cca1-22. Mutations in MLK3 reversed the early-flowering time of cca1-22, and the flowering time of mlk3-1 cca1-22 is similar to mlk3-1 plants (Figures 5F,G). Since CCA1 could bind to the promoter of GI (Su et al., 2017), we then investigated the transcripts of GI and found transcription levels of GI in mlk3-1 cca1-22 are similar to those in mlk3-1 plants (Figure 5H).

To investigate the relationship between MLK3 and GI, we crossed gi-1 into mlk3-1 and generated the mlk3-1 gi-1 double mutant. The flowering time of mlk3-1 gi-1 is similar to gi-1 (Figures 6A,B and Supplementary Table 1). To confirm these, we generated mlk3-1 mlk4-2 gi-1 triple mutant and found that the flowering time of mlk3-1 mlk4-2 gi-1 is similar to that of gi-1 under LD (Figures 6C,D and Supplementary Table 1). These results suggested that MLK3 and MLK4 are redundantly involved in GI-dependent flowering time pathway.

We then examined the transcription levels of GI. The decreased GI transcripts were further reduced in mlk3 mlk4 plants (Figure 6E and Supplementary Figure 6A). However, mutations of MLK1 or MLK2 in mlk3 mlk4 double mutants could not further reduce the attenuated GI transcripts in mlk3 mlk4 plants (Figure 6F). These results are consistent with the flowering time phenotype of mlk3 mlk4, mlk1 mlk3 mlk4, and mlk2 mlk3 mlk4 mutants.

We investigated the distribution of H2AS95ph by chromatin immunoprecipitation (ChIP), followed by the quantitative PCR measurement of DNA enrichment. The H2AS95ph was highly enriched at the promoter and the gene body at GI in wild type. The H2AS95ph enrichment was reduced in mlk3 and mlk4 single mutant, and further reduced in mlk3 mlk4 double mutant (Figures 6G–I and Supplementary Figure 6B). We then examined the H2AS95ph levels in triple mutants and found that the H2AS95ph levels in mlk134 and mlk234 triple mutants are similar to those in mlk3 mlk4 double mutant (Figures 6H,I), suggesting that MLK3 and MLK4 primarily deposited H2AS95ph at GI. These observations indicated that the transcription levels of GI are positively associated with the levels of H2AS95ph.

The Arabidopsis thaliana ecotype Col-0 was grown at 22°C under a LD photoperiod in a 16-h-light/8-h-dark cycle and light intensity of 140 μmol m^–2 s^–1 or a SD photoperiod with 8-h-light/16-h dark. The mutant strains obtained from the SALK collection were as follows: mlk1-2, Salkseq_132455; mlk1-3, SALK_039903; mlk2-2, SALK_149222; mlk2-3, SALK_064333; mlk3-1, SALK_017102; mlk3-2, SAIL_1151_E03; mlk4-2, SALK_201615c; mlk4-3, SAILseq_317_D02.1; cca1-22, SALKseq_120169; gi-1, CS3123; and co-9, CS870084.

The plasmids were constructed with the DNA primers and protocols described in Supplementary Data Set 1. All cloned DNAs were confirmed by DNA sequencing.

The yeast two-hybrid assay was performed according to the manufacturer’s protocol (Clontech; user manual 630489). Briefly, the Saccharomyces cerevisiae strain Y190 was transformed with the bait constructs pGBKT-MLK3 and pGADT7-CCA1. The yeast was scored for protein interaction based on their ability to grow on synthetic-defined medium lacking Trp, Leu, His, and adenine. The primers used to generate the constructs are shown in Supplementary Data Set 1.

The phosphorylation reaction assay was performed as described (Su et al., 2017). Briefly, 2–3 μg purified protein was incubated with 2.5 μCi γ^–32 P ATP in a reaction buffer (50 mM Tris–HCl, pH 7.4, 10 mM MgCl2, 50 mM NaCl, 1 mM DTT, 2 mMEDTA, and 50 mM ATP) at 30°C for 1 h. The reaction products were separated by SDS-PAGE and autoradiographed with Storage Phosphor System.

For the pull-down assays, 3 μg bait and prey proteins were incubated overnight at 4°C. The beads were washed with a solution containing 20 mM Tris (pH 7.4), 150 mM NaCl, and 0.05% Tween 20 separated on a sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and analyzed by immunoblotting using an anti-GST antibody (GenScript, Nanjing, China; A00866-100, lot: 18A001413) or an anti-His antibody (Abmart, Shanghai, China; M30111M, lot: 293871). Co-IP was performed as described previously (Su et al., 2017). Briefly, 1 × 10⁶ protoplasts were lysed with PEN-140 buffer (140 mM NaCl, 2.7 mM KCl, 25 mM Na2HPO4, 1.5 mM KH2PO4, 0.01 mM EDTA and 0.05% CA-630). The supernatant was precipitated with anti-FLAG (Sigma-Aldrich; H8592, lot: SLBV3799) antibodies, followed by washes with PEN-400 buffer (400 mM NaCl, 2.7 mM KCl, 25 mM Na2HPO4, 1.5 mM KH2PO4, 0.01 mM EDTA and 0.05% CA-630). The samples were analyzed by immunoblotting using anti-HA (Roche; 11867423001, lot: 13500600) and anti-FLAG antibodies.

Total RNA was isolated from 7-day seedlings grown under a LD photoperiod. RT-PCR analysis was performed with a CFX real-time PCR instrument (Bio-Rad) and SYBR Green mixture (Roche). The relative expression of the genes was quantified with the 2^–ΔΔCT calculation using Ubiquitin 10 as the reference housekeeping gene for the expression analyses. The enrichment of DNA at specific genes was quantified with the 2^–ΔΔCT calculation using Ubiquitin 10 as the reference housekeeping gene for ChIP assays. The gene-specific primers are shown in Supplementary Data Set 1.

Chromatin immunoprecipitation assay was performed as described previously (Su et al., 2017). Briefly, 7-day seedlings grown under a LD photoperiod were fixed with formaldehyde and quenched in glycine. The grounds were extracted with buffer I (0.4 M sucrose, 10 mM Tris (pH 8.0), 5 mM β-mercaptoethanol, 0.1 mM phenylmethylsulfonylfluoride (PMSF), and protease inhibitor cocktail), buffer II (0.25 M sucrose, 10 mM Tris (pH 8.0), 10 mM MgCl2, 1% Triton X-100, 5 mM β-mercaptoethanol, 0.1 mM PMSF and protease inhibitor cocktail), and buffer III (1.7 M sucrose, 10 mM Tris (pH 8.0), 10 mM MgCl2, 1% Triton X-100, 5 mM β-mercaptoethanol, 0.1 mM PMSF, and protease inhibitor cocktail). The specific antibodies anti-H2AS95ph were generated as described previously (Su et al., 2017), or control IgG serum, was added to the precleared supernatants for an overnight incubation at 4°C. The immunoprecipitated sample was extracted and analyzed by RT-PCR with the gene-specific primers shown in Supplementary Data Set 1.

Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: MLK1 (At5g18190), MLK2 (At3g03940), MLK3 (At2g25760), and MLK4 (At3g13670).