MLK3 with conserved kinase domains belongs to a separate subgroup of CK1s specific to plant kingdom
Previously, we have identified four plant-specific casein kinase 1 encoding genes, namely MLK1-4 from Arabidopsis [10]. The four MLKs shared sequence identity of 67.9% - 91.1%. Homology analysis of MLKs and the homologs from several crops demonstrated that the plant-specific CK1s were classified into two main branches (I and II). MLK3, distinct from its paralogs, was grouped into a separate branch (Additional file 1, Figure S1). Consistently, the phylogenetic analysis showed that MLK3 was relatively distant from the other three MLKs (Fig. 1a). Sequence alignment showed that MLK3 shared the conserved CK1 functional domains including substrate recognition region, kinase catalytic loop, ATP binding site and a predicted nuclear localization signal [4] (Additional file 2: Figure S2). The predicted isoelectric point of MLKs ranges from 9.09 to 9.66 (Additional file 3: Table S1), suggesting the preference to acidic substrates, such as serine and threonine residues. Thus, albeit divergent from other MLKs in Arabidopsis, MLK3 possessed the common features of CK1, implying its enzymatic activity as a kinase in phosphorylating target protein(s).
MLK3 was ubiquitously expressed and the MLK3-GFP recombinant protein was localized in the nucleus
Three MLKs (MLK1, 2 and 4) have been functionally identified [17-19]. To investigate the biological role of MLK3, the most divergent MLK, we first examined its spatial and temporal expression patterns using semi-quantitative RT-PCR. As shown in Figure 1b, MLK3 was expressed in roots, stems, leaves and flowers, which is in agreement with the results from eFP Browser (http://bbc.botany.utoronto.ca/efp) [20]. Comparison of the absolute transcription level demonstrated that MLK3 transcript is the lowest, while MLK4 is the highest, which is about 2.8-fold of MLK3 (Additional file 4:Figure S3).
Considering that MLK3 has a predicted nuclear localization signal (Additional file 2: Figure S2) [4, 10], the subcellular localization of MLK3-GFP recombinant protein was examined. As expected, when transiently expressed in tobacco leaves by infiltration, the green signal of the MLK3-GFP fusion protein was observed exclusively in the nucleus of the leaf epidermal cells as indicated by DAPI staining, while the signal of 35S::GFP control displayed a universal distribution in the epidermal cells of the tobacco leaves (Fig. 1c). Hence, consistent with the previous findings in tobacco and Arabidopsis protoplasts [8, 11, 12], MLK3, like its paralogs, is a nuclear protein, implying its potential role in histone modification.
MLK3 phosphorylated histone H3 at threonine 3 in vitro
Given that nuclear protein MLK3 shares the canonical features of CK1, we tested whether MLK3 functions as protein kinase. First, MLK3 was fused with maltose binding protein (MBP) and expressed in E. coli BL21 (DE3) strain. The purified MLK3-MBP recombinant protein was then incubated with the phosphoryl donor ATP and substrate before being dotted on membrane. Finally, immuno-blotting was performed using an antibody specifically against phosphorylated H3T3 [4]. Our results showed that strong immune-signal (bottom row) was detected with substrate H3T3ph peptide (a phosphor-histone H3 (Thr3) peptide) (Fig. 2a), which was used as positive control, confirming the specificity of the antibody. For substrate of unmodified histone H3 peptide (H3), anti-H3T3ph signal was detected when both ATP and the recombinant protein MLK3-MBP were present (middle row), while anti-H3T3ph signal was undetectable in the absence of MLK3-MBP fusion protein (upper row) (Fig. 2a). The detection of the phosphorylated histone H3 at threonine 3 indicated that with ATP as phosphoryl donor, the recombinant protein MLK3-MBP catalyzed in vitro phosphorylation of unmodified histone H3 peptide substrate. Hence, MLK3 phosphorylated histone H3 at threonine 3 (H3T3ph), a predominant target of the plant-specific kinase Mut9 and MLK1 in Chlamydomonas and Arabidopsis, respectively [4, 10].
Conserved lysine (K) 146 of MLK3 is essential for MLK3-modulated in vitro phosphorylation of histone H3T3
It has been reported that the conserved lysine residue (K174 for Mut9p, K175 for MLK4/PPK1) was essential for the catalytic activity of the kinases [4, 11, 21]. To test whether the counterpart lysine (K146) of MLK3 is critical for phosphorylation of histone H3T3, the conserved K146 was point mutated to arginine (R). The MBP-fused MLK3 (K146R) was purified from E. coli., then the kinase activity was examined by dot blotting as mentioned above and the reaction without enzyme was served as negative control. Expectedly, H3T3ph peptide, the positive control, showed anti-H3T3ph signal in the immuno-analysis (bottom row) (Fig. 2b). No signal was detected for unmodified histone H3 peptide substrate with ATP served as phosphoryl donor, no matter the mutated recombinant protein MLK3 (K146R)-MBP was supplied or not (Fig. 2b). These results indicated that unlike MLK3, the point-mutated MLK3 (K146R) was catalytically inactive. Therefore, an intact lysine at the conserved position is crucial for the substrate phosphorylation mediated by the plant-specific CK1.
MLK3 affected leaf growth and flowering time
To address the biological function of MLK3, a T-DNA insertion line (SALK_017102) was obtained [10]. PCR analysis revealed that the T-DNA was integrated into the 12th exon of MLK3, resulting in a truncated peptide of 498 amino acid residues (Fig. 3a, b). In homozygous mlk3 mutant, MLK3 transcript flanking the insertion site was undetectable by RT-PCR, while a transcript upstream of the insertion site was detected (Fig. 3c), suggesting the partial expression of MLK3. Hereafter, the primers flanking the T-DNA insertion site were used to analyze the expression of MLK3. Morphologically, during vegetative stage mlk3 was slightly smaller than wild type under LD (Fig. 3d). Measurement of the rosette leaf numbers showed that mlk3 had 1.8, 2.4 and 3.2 fewer leaves on average than wild type in Week-2, 3 and 4, respectively (P<0.05) (Fig. 3e), suggesting the progressive retardance of leaf growth in mlk3. Our calculation of leaf area (the 5th leaf) demonstrated that the fifth leaf of mlk3 was about 2.4-5.0 mm2 smaller than that of wild type during the three weeks (P<0.05) (Fig. 3f).
For flowering time, Huang et al., [12] revealed that in terms of the days to inflorescence at one centimeter, statistically mlk3 had a minor fewer number of days than wild type, indicating mlk3 flowered slightly earlier. Consistently, we observed that under LD, mlk3 flowered at 19.2 days after germination (DAG), while wild type flowered at 22.3 DAG (P<0.05) (Fig. 4a, b). Consequently, at five-week old mlk3 displayed more siliques (16/plant vs 8/plant, P<0.01) than wild type at the same stage (Fig. 4c). No abnormal flowering time was observed under short day (SD) conditions (Additional file 5:Table S2). Therefore, truncation of MLK3 altered leaf growth and flowering time simultaneously under LD.
The negative role of MLK3 in flowering regulation required the intact lysine (K) 146
To confirm the role of MLK3 in flowering regulation, MLK3 CDS driven by the 35S promoter was introduced into mlk3 mutant. The transcriptional analysis of MLK3 using semi-quantitative RT-PCR showed that distinct from mlk3 mutant, MLK3 transcript was detected in the transgenic mlk3 plants expressing 35S::MLK3 (e.g. Line 8) (Fig. 5a), indicating the expression of MLK3 in the transgenic plants. For flowering time, different from mlk3, the two independent transgenic lines (Lines 4 and 8) flowered at a similar time to wild type (Fig. 5b). In addition, the transgenic plants possessed a similar number of rosette leaves to that of wild type (Additional file 6: Figure S4). These results indicated that constitutive expression of MLK3 rescued the morphologic abnormalities of mlk3 in both leaf growth and flowering time.
To determine whether an intact K146 is critical for MLK3-mediated flowering, 35S::MLK3 (K146R) was introduced into mlk3 plants. RT-PCR showed that a similar intensity of MLK3 transcript was detected in the transgenic plant and wild type (e.g. Line 6) (Fig. 5a). In contrast, the flowering time analysis of the two independent lines (Lines 3 and 6) demonstrated that similar to mlk3, DAG of the both lines was significantly fewer than wild type (P<0.05) (Fig. 5b), suggesting that the transgenic lines flowered earlier. Consistently, the leaf number of the transgenic mlk3 ectopically expressing MLK3 (K146R) did not significantly differ from that of mlk3 (Additional file 6: Figure S4). These results indicated that unlike MLK3, which successfully restored the early-flowering phenotype of mlk3, the catalytically inactive MLK3 (K146R) expressed constitutively did not alter either flowering time or leaf growth. Therefore, the conserved lysine K146, which is essential for phosphorylation of H3T3, is indispensable for MLK3-mediated flowering repression.
Truncation of MLK3 did not significantly affect the transcriptional level of the major flowering regulators nor the global intensity of phosphorylated H3T3
To profile the transcriptome of mlk3, RNA-sequencing was carried out. Consistent with the truncation of MLK3, the unique reads of mlk3 matching the exons downstream of the T-DNA insertion site was clearly depleted compared with wild type, while no significant difference was monitored upstream of the T-DNA interruption (Additional file 7: Figure S5). By the criteria of │Log2FC│≥1 and P<0.01, a total of 425 genes were differentially expressed with 133 up-regulated and 292 down-regulated in mlk3 relative to wild type (Additional file 8: Figure S6a). None of the transcript of MLK3 paralogs was significantly changed in mlk3, implying no clear compensation of other MLKs. Based on gene annotation, no flowering regulator was differentially expressed in mlk3 (Additional file 9: Table S3), suggesting that truncation of MLK3 may not significantly affect the known flowering signaling components at the transcriptional level. Functional categorization of the DEGs based on gene ontology (GO) annotations revealed that the genes categorized to “negative regulation process”, such as “developmental process” and “multicellular organismal process”, were up-regulated, while the genes categorized to “positive regulation process” of the above two biological processes were down-regulated (Additional file 8: Figure S6b).
To compare the global level of H3T3ph between mlk3 and wild type, western blot was performed with anti-H3T3ph antibody. The intensity of H3T3ph in mlk3 was not notably different from that of wild type (Additional file 10: Figure S7), suggesting the functional redundancy of other MLKs, especially MLK1 and MLK2 [10]. Therefore, truncation of MLK3 caused no significant alteration on either the transcriptional level of the main components of flowering pathway or the intensity of H3T3ph globally.
MLK3 acts antagonistically to MLK4 in regulating flowering time
It has been documented that the loss-of-function mutant of MLK4, the closest paralog of MLK3, flowered late [12, 17]. To investigate the genetic relationship between MLK3 and MLK4 in flowering regulation, we generated the mlk3 mlk4 double mutant by crossing the two single mutants (Fig. 6a). In homozygous mlk3 mlk4, the expression level of both MLK3 and MLK4 was eliminated to that of in the corresponding single mutants (Fig. 6b). Given that FT transcript was reduced significantly in mlk4 mutant [17], the expression level of FT in mlk3 mlk4 was tested by RT-qPCR. As shown in Fig. 6b, FT in mlk3 mlk4 was about 60% of wild type, while in mlk3 and mlk4, it was about 135% and 52% of wild type, respectively (Fig. 6b), indicating a compromised level of FT in mlk3 mlk4 relative to its parental lines. Statistical analysis of the average DAG showed that mlk3 mlk4 flowered at 30.2 DAG, while the two parental lines flowered at 19.7 DAG for mlk3 and 32.8 DAG for mlk4, respectively (Fig. 6c), indicating that mlk3 mlk4 flowered later than mlk3 but earlier than mlk4. These results suggested that MLK3 acted antagonistically to MLK4 in regulating flowering time.