Plant casein kinases phosphorylate and destabilize a cyclin-dependent kinase inhibitor to promote cell division.

Cell cycle is one of the most fundamentally conserved biological processes of plants and mammals. Casein kinase1s (CK1s) are critical for cell proliferation in mammalian cells; however, how CK1s coordinate cell division in plants remains unknown. Through genetic and biochemical studies, here we demonstrated that plant CK1, Arabidopsis (Arabidopsis thaliana) EL1-like (AELs), regulate cell cycle/division by modulating the stability and inhibitory effects of Kip-related protein6 (KRP6) through phosphorylation. Cytological analysis showed that AELs deficiency results in suppressed cell-cycle progression mainly due to the decreased DNA replication rate at S phase and increased period of G2 phase. AELs interact with and phosphorylate KRP6 at serines 75 and 109 to stimulate KRP6's interaction with E3 ligases, thus facilitating the KRP6 degradation through the proteasome. These results demonstrate the crucial roles of CK1s/AELs in regulating cell division through modulating cell-cycle rates and elucidate how CK1s/AELs regulate cell division by destabilizing the stability of cyclin-dependent kinase inhibitor KRP6 through phosphorylation, providing insights into the plant cell-cycle regulation through CK1s-mediated posttranslational modification.

Introduction

Cell cycle is one of the most comprehensively studied biological processes, particularly given its importance for growth and development of multicellular organisms (Dewitte and Murray, 2003). The eukaryotic cell proliferation comprises G1–S–G2–M series phase (Venuto and Merla, 2019), which is regulated mainly by cyclin-dependent kinase (CDK), whose activity is regulated by other cell-cycle regulators through interaction (Morgan, 1997; Mironov et al., 1999; Meijer and Murray, 2000; Dewitte and Murray, 2003). In plants, rate and duration of cell cycle determine cell number and size that correspond with organ/tissue growth (Gonzalez et al., 2012; Sablowski and Carnier Dornelas, 2014). Inhibitors of CDK (ICKs) or Kip-related proteins (KRPs) suppress CDK’s activity (Wang et al., 1997; Pines, 1999; De Veylder et al., 2001; Zhou et al., 2002). KRP6 is a partner in plant CDK/cyclin complex that inhibits the activation of gibberellin-dependent cell cycle (Van Leene et al., 2007; Nieuwland et al., 2016) and plays essential roles in cell division inhibition by suppressing CDKA;1 activity (Dewitte and Murray, 2003; Inze and De Veylder, 2006). In addition, KRP6 regulates plant growth and development including size of rosette leaves (De Veylder et al., 2001; Cheng et al., 2013; Sizani et al., 2019), gametogenesis (Kim et al., 2008; Liu et al., 2008; Gusti et al., 2009; Zhao et al., 2012), germline development (Zhao et al., 2017), as well as cytokinesis (Vieira et al., 2014), through inhibiting cell division.

Protein phosphorylation modification plays a cardinal role in regulating multiple physiological processes. Casein kinase1 (CK1) is a Ser/Thr protein kinase that is highly conserved in eukaryotes. CK1s are involved in the regulation of many important physiological and signaling processes including cell-cycle control, circadian rhythms, vesicle trafficking, DNA repair, growth, and morphogenesis of mammals (Gross and Anderson, 1998; Knippschild et al., 2005; Lowrey and Takahashi, 2011; Knippschild et al., 2014). In addition, CK1s also play important regulatory roles in various processes of growth and development of plants. Rice (Oryza sativa) OsCK1 is involved in the regulation of root development (Liu et al., 2003) and seedling growth under low temperature (Lu et al., 2014). Arabidopsis (Arabidopsis thaliana) CK1-Like 6 (CKL6) phosphorylates and binds cortical tubules to regulate the cell growth and morphogenesis (Ben-Nissan et al., 2008). Arabidopsis CK1.3 and CK1.4 phosphorylate blue light receptor cryptochrome 2 (CRY2) to regulate blue-light-mediated photomorphogenesis (Tan et al., 2013); and CK1.8 phosphorylates and regulates the stability of 1-aminocyclopropane-1-carboxylic acid synthase 5, key enzyme of ethylene synthesis, and eventually the ethylene content of plants (Tan and Xue, 2014).

Phylogenetical analysis divides CK1 members in plants into two subgroups, CK1-like (CKL) subgroup and plant-specific CK1 subgroup (Chen et al., 2018, Kang et al., 2020). In Arabidopsis genome, there are 13 CKLs (Uehara et al., 2019) and four plant-specific CK1s, which are designated as Arabidopsis EL1-like proteins (AELs) based on their homology to rice Early Flowering1 (EL1, Dai and Xue, 2010; Chen et al., 2018). Recent studies showed that AELs are involved in the regulation of various signaling or processes including hormone signaling, photomorphogenesis, flowering time, and epigenetic regulation (Liu et al., 2017; Ni et al., 2017; Su et al., 2017; Chen et al., 2018; Zheng et al., 2018).

Whether CK1s are also involved in plant cell division regulation remains unclear yet. Considering the crucial roles of CK1s/AELs in plant growth and development, and the close connection between CK1 and cell-cycle control in mammals, it is conceivable that AELs might play important roles in cell division regulation of plants as well. Here we characterized the molecular mechanism how AELs regulate plant cell division by phosphorylating KRP6 at two specific phosphorylation sites, Ser75 and Ser109, to promote its interactions with E3s and degradation. Our study demonstrates that CK1s/AELs-mediated phosphorylation is critical for cell division regulation through phosphorylation of CDK inhibitor KRP6 and provides informative clues for elucidating the complex regulatory network of cell division in plants.

Results

Deficiency of AELs results in decreased cell number/cell division

Arabidopsis EL1-likes 1-4 (AEL1-4) were reported to suppress abscisic acid (ABA) signaling through phosphorylating ABA receptors (Chen et al., 2018). Phenotypic observation of vegetative growth showed that although there were no obvious changes in ael single or double mutants (Supplemental Figure S1), the ael triple mutants (ael123, ael124, and ael134) present much smaller rosette leaves in comparison to the wild-type (Wt) plants (Figure 1A; Supplemental Figure S1). Complementation study showed that expression of genomic fragment of AEL1, AEL2, or AEL4 recovered the phenotype of triple mutant (Supplemental Figure S2), confirming the small leaves of triple mutants were due to AELs deficiency and indicating that functionally redundant CK1s/AELs regulate organ size. Further cytological observation of cell size and number at adaxial and abaxial epidermis of rosette leaves by scanning electron microscope showed the reduced cell size, and more significantly reduced cell number in leaf epidermis of ael triple mutants (Figure 1B), indicating the reduced leaf size under AELs deficiency is mainly resulted from the suppressed cell division. In addition to the reduced leaf size, triple mutants (ael123, ael124, ael134) display aborted seeds and heterozygous quadruple mutant ael1234 is sterile (Supplemental Figure S3), indicating the possible role of AELs in plant reproductive development.

Figure 1

Suppressed cell division under AELs deficiency. A, Phenotypic observation (left and middle, scale bar = 1 cm) and measurement (right) showed that ael triple mutants display smaller rosette leaves. Three-week-old plants were observed and eighth rosette leaves were measured. Data are presented as means ± sem (n = 20) and statistical analysis using Student’s t test revealed significant differences (***P < 0.001, compared with Wt). B, Electron microscopy observation (left, scale bar = 20 μm) and measurement of adaxial and abaxial leaf epidermal cells (right) showed that AELs deficiency resulted in decreased cell size and cell number. Fully expanded eight rosette leaves of 3-week-old Wt and ael triple mutants were analyzed. Size of epidermal cells was measured by ImageJ and cell number was calculated by dividing leaf area by average epidermal cell area. Data are presented as means ± sem (n = 15) and statistical analysis using Student’s t test revealed significant differences (*P < 0.05; **P < 0.01, compared with Wt). C, Ploidy analysis of nuclei showed prolonged time for cell division in ael triple mutants. First leaf pair of Wt and ael triple mutants at 13 DAS was used to isolate nuclei and analyzed by flow cytometry. Proportion of cells with different DNA content (left) and 2C to 4C DNA content (right) were calculated and data are presented as means ± sem (n = 15). C-value means the amount of DNA contained within a haplophasic nucleus. Statistical analysis using Student’s t test revealed significant differences (*P < 0.05; **P < 0.01, compared with Wt). D, Reduced DNA replication rate of mitosis S phase of ael triple mutants. First leaf pair at 13 DAS were treated with EdU at 0, 1, 2, 3, or 4 h and detected with flow cytometry. Ratio (newly formed cells/pre-existing cells) representing DNA replication rate were calculated and data were presented as means ± sem (n = 10). Statistical analysis using Student’s t test revealed significant differences (*P < 0.05, **P < 0.01, compared with Wt).

Leaf development is composed of cell proliferation by cell division through mitosis and cell expansion mainly by endoreplication (Gonzalez et al., 2012). To confirm the suppressed cell division in triple mutants, kinematical analysis of the first leaf pair from 5 to 25 d after stratification (DAS; Sizani et al., 2019) showed that ael triple mutants had smaller leaf blade area and cell area, fewer cell number and reduced cell division rate (Supplemental Figure S4, A–D). The fewer cell number of ael triple mutants is consistent with the much smaller leaf blade area, which is verified by the low cell division rate especially before 13 DAS (Supplemental Figure S4D). Further, we detected the cell ploidy of first leaf pair with flow cytometry in ael triple mutants (Sizani et al., 2019; Zeng et al., 2012). Being consistent with the previous study revealing diploid-predominant before 13 DAS and similar proportion of diploid and tetraploid of Wt at 13 DAS (Sizani et al., 2019), analysis of cell ploidy showed similar phenomena in Wt, while that of diploid was obviously higher than tetraploid in ael triple mutants (similar proportion was much delayed, Figure 1C; Supplemental Figure S4E), indicating that prolonged time of cell division led to the fewer cell numbers and smaller leaves and demonstrating a cell-cycle arrest of ael triple mutants.

The G1-to-S phase transition is an essential cell-cycle checkpoint (Evan and Vousden, 2001). CYCD3;1 has been proved with a specific role to dominantly drive the G1/S transition and the overexpression of CYCD3;1 results in a higher frequency of arrest at G2 phase in response to sucrose starvation (Menges et al., 2006). The examination of CYCD3;1 expression by reverse transcription quantitative polymerase chain reaction (RT-qPCR) revealed the much-increased CYCD3;1 expression in ael triple mutants in comparison to Wt (Supplemental Figure S5A), indicating a reduction of G1 phase and reduced stringency of G1 control point. KRP2 and KRP4 encode cell-cycle inhibitors (Zhiponova et al., 2013), and transcripts of them peak at S phase especially KRP2. Subsequent examination by RT-qPCR showed the increased expressions of KRP2 and KRP4 in ael triple mutants (Supplemental Figure S5B). Further comparison of DNA replication efficiency by using 5-ethynyl-2′-deoxyuridine (EdU) assay and flow cytometry quantitative analysis (Kotogány et al., 2010; Zeng et al., 2012) showed that compared with Wt, proportion of newly formed cells with EdU fluorescence signal decreased significantly in ael triple mutants at different time points (Figure 1D), indicating a decreased efficiency of DNA synthesis.

Additional RT-qPCR analysis of CYCB1;1 (marker gene transcribed specifically at G2/M phase, Colon-Carmona et al., 1999; Zhiponova et al., 2013; Chen et al., 2014) and promoter–reporter gene fusion studies showed the increased CYCB1;1 expression in ael triple mutants (Supplemental Figure S5, A and C), confirming the increased G2 phase that may be due to the stranded S phase under AELs deficiency. These results suggest that suppressed cell division of ael triple mutants is resulted from the reduced cell-cycle rates, mainly due to the decreased DNA replication rate at S phase and arrested G2 phase thus delayed activation of mitotic genes, and indicate an essential role of AELs in regulating cell-cycle progression.

AELs interact with KRP6

Considering the inhibited cell division of ael triple mutants, candidate substrates are predicted by analyzing the phosphorylation sites of key factors involving in cell-cycle regulation by Scansite 4.0 (https://scansite4.mit.edu.). Several factors are identified containing candidate CK1 site (Supplemental Table S1) and further analysis by yeast two-hybrid assay showed that AEL1 interacts with Kip-related protein6 (KRP6, Supplemental Figure S6A), a CDK inhibitor that plays important roles in inhibiting cell division by suppressing CDKA activity (Dewitte and Murray, 2003; Inze and De Veylder, 2006). Yeast-based liquid quantification assay proved that KRP6 interacts with all AELs (AEL1-4) with slightly different interaction intensities (Figure 2A; Supplemental Figure S6B), which was confirmed by fluorescence resonance energy transfer analysis using fluorescence lifetime imaging microscopy (FRET-FLIM; Supplemental Figure S6C), indicating KRP6 as a candidate substrate of AELs.

Figure 2

AELs interact with KRP6 protein. A, Yeast two-hybrid assay showed that KRP6 interacts with AEL1-4. AELs-AD (GAL4 DNA-activation domain) were coexpressed with KRP6-BD (GAL4 DNA-binding domain) fusion protein in AH109 yeast cells and positive clones were grown on synthetic dropout (–Leu–Trp–His) medium supplemented with 3-amino-1,2,4-triazole (3-AT, 2.5 mM) and 5-bromo-4-chloro-3-indoxyl-α-d-galactopyranoside (X-α-Gal, 0.5 mg·mL−1), or synthetic dropout (–Leu–Trp–His-Ade) medium. B, BiFC assay demonstrated AEL1 interacts with KRP6 in vivo. Nicotiana benthamiana leaves are infiltrated with agrobacteria containing different construct combinations. YFP fluorescence was observed in cells cotransfected by KRP6-nYFP and AEL1-cYFP. Scale bar = 50 μm. C, CoIP analysis revealed the AEL1-KRP6 interaction in vivo. Total proteins were extracted from 7-d-old Arabidopsis seedlings expressing KRP6-HA (KPR6ox), AEL1-Flag (AEL1ox) or coexpressing AEL1-Flag and KRP6-HA (KPR6ox in AEL1ox), and incubated with anti-Flag M2 magnetic beads. Input or IP fractions were analyzed using anti-Flag or anti-HA antibodies, respectively.

Consistent with the previous studies (Bird et al., 2007), subcellular localization studies verified that KRP6 localized in nucleus (Supplemental Figure S6D), same as AELs (Chen et al., 2018). In addition, bimolecular fluorescence complementation (BiFC; Figure 2B), FRET-FLIM (Supplemental Figure S6E), as well as coimmunoprecipitation (CoIP) analysis using transgenic Arabidopsis coexpressing KRP6 and AEL1 (Figure 2C) confirmed their interaction in vivo.

AELs-mediated phosphorylation is crucial for KRP6 degradation and protein stability

Phos-tag gel shift assay showed that KRP6 protein can be directly phosphorylated by AEL1 (Figure 3A). Considering the importance of phosphorylation in regulating stability, besides activity and subcellular localization, of target proteins (Luan, 2003), we first examined whether AELs affect the degradation/stability of KRP6 through phosphorylation. An in vitro cell-free assay (Wang et al., 2009) was performed and results revealed that compared with in Wt, KRP6 degradation is significantly suppressed in ael123 and ael124 mutants (Figure 3B). Further analysis by crossing Wt with ael123 lines expressing KRP6 confirmed the suppressed KRP6 degradation in ael123 in vivo (Figure 3C), demonstrating the crucial roles of AELs-mediated phosphorylation in regulating degradation and stability of KRP6, which is consistent with the negative effects of KRP6 on cell cycle and inhibited cell division of ael triple mutants.

Figure 3

AELs-mediated phosphorylation promotes KRP6 protein degradation and is essential for KRP6 effects. A, Phos-tag gel shift assay showed that AEL1 directly phosphorylates KRP6. Recombinant His-AEL1 (1 µg) and His-KRP6 (10 µg) proteins from E. coli and casein (10 µg) were used for analysis. Red arrowheads indicated the KRP6 proteins and the upper arrowheads indicated the phosphorylated KRP6. Phosphorylated proteins were separated on a Phos-tag gel (10% SDS–PAGE gel containing 50-µM Phos-tag Acrylamade reagent and 100-µM MnCl2). Load proteins were shown in the PAGE gel (bottom). B, Suppressed degradation of KRP6 in ael123 or ael124 triple mutants. Stability of purified His-KRP6 fusion protein was examined by in vitro cell-free analysis using anti-His antibody (left). CBB staining showed equal loading of total proteins. Band density is measured by Image J and relative density was calculated by setting KRP6 intensity at 0 min as 1.0. Data are presented as means ± sem (n = 3, right). C, Western blot analysis showed that AELs deficiency resulted in reduced KRP6 protein degradation. Wt lines overexpressing KRP6 (KRP6ox in AEL123/ael123) were generated by crossing homozygous ael123 lines expressing HA-KRP6 (KRP6ox in ael123/ael123) with Wt. Seven-day-old seedlings were pretreated with MG132 (50 μM) for 1 h and then treated with protein translation inhibitor CHX (100 µM) for different times (0, 15, 30, or 60 min). Total proteins were extracted and detected with HA antibody (left). CBB staining showed equal loading of total proteins (below). Band density is measured by Image J and relative density was calculated by setting KRP6 intensity at 0 min as 1.0. Data are presented as means ± sem (n = 3, right). D–G, Phenotypic observation (left) and measurement (right) of eight rosette leaves showed that KRP6 overexpression resulted in more decreased leaf areas under AELs deficiency, while no effect under AELs overexpression. Three-week-old seedlings overexpressing KRP6 in Wt (D) and ael123 (E), KRP6 in AEL1ox (F), KRP6 in AEL3ox (G) background were analyzed. Data are presented as means ± sem (n = 15) and statistical analysis using Student’s t test revealed significant differences (**P < 0.01; ***P < 0.001, compared with transgenic background plants). Scale bar = 1 cm.

There are seven members of Arabidopsis KRP family and krp6 null mutant was indistinguishable from Wt (Supplemental Figure S7), possibly due to the functional redundancy or compensation between KRP members (only downregulation of more than three members can promote cell proliferation, leading to enlarged organs and seeds of Arabidopsis; Cheng et al., 2013; Sizani et al., 2019). KRP6 overexpression led to decreased cell number and reduced size of rosette leaves due to arrested cell division (De Veylder et al., 2001; Le Foll et al., 2008; Guerinier et al., 2013). To explore the physiological function of AELs-mediated phosphorylation on KRP6, transgenic plants expressing similar level of KRP6 in different backgrounds were generated (Supplemental Figures S8, A and D; S9). Compared to reduced leaf areas of Wt overexpressing KRP6 (Figure 3D), more significant reduction is observed in ael123 overexpressing KRP6 (Figure 3E). However, no obvious difference of germination time, growth rate at different periods, and leaf areas was observed in AELox lines overexpressing KRP6 (Figure 3, F and G), indicating that overexpressed KRP6 in AELox lines may degrade more quickly while the protein is more stable in ael123 background, confirming the crucial roles of AELs-mediated phosphorylation on KRP6 stability and functions.

Ser75 and Ser109 of KRP6 mediate the phosphorylation effect of AELs on KRP6

To investigate the relevant regulatory mechanism, we identified serine (Ser, S) 75 and 109 of KRP6 as candidate phosphosites of CK1 by analysis using Scansite (Figure 4A; Supplemental Table S1). The two phosphosites were further verified by liquid chromatography–mass spectrometry (LC–MS) analysis with purified KRP6 protein that is phosphorylated by AEL1 in vitro. Phosphosites were detected at four serine and three threonine (Thr, T) residues (Figure 4B; Supplemental Figure S10). Among the candidate residues, S109 was identified both in prediction and LC–MS analysis. S75 was not detected by LC–MS; however, phosphorylation signal of peptide containing S75 is statistically significant. Considering the high possibility of two phosphosites, kinase assay was performed and results showed that mutation at S75 or S109 (Ser to alanine, A; KRP6S75A, KRP6S109A) reduced the phosphorylation signal, and double mutation (KRP6S75AS109A) significantly suppressed phosphorylation by AEL1 (Figure 4C), indicating that Ser75 and Ser109 are the major phosphorylation sites of KRP6 by AELs.

Figure 4

AELs phosphorylate KRP6 at Ser75 and Ser109. A, Prediction of candidate phosphosites of KRP6 by different kinases using software Scansite4.0. S75 and S109 are potential phosphosites of CK1s. B, Detected phosphosites (S/T) of KRP6 by LC/MS are highlighted in red (numbers showed position of amino acids, upper). Experiments were biologically repeated and representative phosphopeptides and phosphosites are shown (bottom). The maximum probability for phosphosite is calculated by Andromeda algorithm integrated in MaxQuant. C, Kinase assay by 32P-γ-ATP autoradiograph showed that mutation of phosphosites (serine to alanine, S to A) resulted in significantly reduced phosphorylation of KRP6. Recombinant His-AEL1 (1 μg), His-KRP6 with mutation (S75A, S109A, S75AS109A, 10 μg) were analyzed. Band density is measured by Image J and data are presented as means ± sem (n = 3, right). Statistical analysis using Student’s t test revealed significant differences (***P < 0.001, compared with normal KRP6). D, Western blot analysis confirmed that mimicking nonphosphorylation mutations led to reduced phosphorylation of KRP6 in vivo. Total proteins were extracted from 7-d-old Wt or ael123 seedlings expressing KRP6 (KRP6ox in AEL123/ael123, KRP6ox in ael123/ael123) or KRP6 (KRP6 in AEL123/ael123, KRP6 in ael123/ael123) and incubated with anti-HA magnetic beads. KRP6 or KRP6S75AS109A in IP fractions and phosphorylation of KRP6 or KRP6S75AS109A were analyzed using anti-HA or anti-phos (S/T) antibody respectively. Wt lines expressing KRP6 or KRP6S75AS109A were generated by crossing homozygous ael123 lines expressing HA-KRP6 or HA-KRP6 with Wt. Band density is measured by Image J and relative phosphorylation level was calculated by dividing HA-KRP6 by Phos-KRP6. Data are presented as means ± sem (n = 3) and statistical analysis using Student’s t test revealed significant differences (*P < 0.05; **P < 0.01, ***P < 0.001).

To confirm the correctness of identified phosphosites in vivo, phosphorylation level of simulated nonphosphorylated mutation KRP6S75AS109A and normal KRP6 was analyzed using Arabidopsis Wt and ael123 seedlings expressing HA-KRP6 and HA-KRP6S75AS109A fusion proteins. Western blot analysis using serine phosphorylation antibody showed that phosphorylation level of KRP6 was significantly lower in ael123 compared to Wt, and that of KRP6S75AS109A is much lower than KRP6 in Wt and ael123 (Figure 4D), again confirming the important role of AELs on KRP6 phosphorylation and S75 and S109 are major phosphorylation sites by AELs in plants. In addition, even lower phosphorylation of KRP6S75AS109A than KRP6 in ael123 suggested that AEL4 or other kinases may involve in modulation of KRP6 phosphorylation status, which may play distinct regulatory roles on KRP6 protein and functions.

Ser75 and Ser109 are crucial for KRP6 stability and function

Mutated KRP6 mimicking nonphosphorylation or phosphorylation was applied to study the roles of AEL1-mediated phosphorylation on KRP6. Cell-free analysis showed that compared to normal KRP6, nonphosphorylation version KRP6S75AS109A presented suppressed degradation, while phosphorylation-mimic KRP6S75DS109D presented enhanced degradation in both Wt and ael123 (Figure 5, A and B), confirming that AELs promote KRP6 degradation through phosphorylation. Further, plants expressing KRP6 or KRP6 in different backgrounds were generated (Supplemental Figure S8, B, C, E, and F) and significantly enhanced stability of KRP6S75AS109A in both Wt and ael123 in vivo (Figure 5, C–F) indicated the essential roles of AELs-mediated phosphorylation on promoting KRP6 degradation.

Figure 5

Crucial roles of Ser75 and Ser109 in regulating KRP6 stability through phosphorylation. A, B, Altered stability of KRP6 protein with point mutation. Stability of purified His-KRP6 fusion protein and mutated KRP6 (KRP6S75AS109A and KRP6S75DS109D) in Wt (A) or ael123 triple mutant (B) background were examined by in vitro cell-free analysis using anti-His antibody (left). CBB staining showed equal loading of total proteins. Band density is measured by Image J and relative density was calculated by setting intensity of KRP6 (or KRP6S75AS109A, KRP6S75DS109D) at 0 min as 1.0. Data are presented as means ± sem (n = 3, right). C–F, Western blot analysis confirmed that reduced phosphorylation resulted in enhanced protein stability of KRP6 in vivo. Wt lines expressing KRP6 or KRP6S75AS109A (KRP6ox in AEL123/ael123, KRP6 in AEL123/ael123) were generated by crossing homozygous ael123 lines expressing HA-KRP6 or HA-KRP6 (KRP6ox in ael123/ael123, KRP6 in ael123/ael123) with Wt, and seedling with similar expressed protein level were used for analysis. Seven-day-old seedlings were pretreated with MG132 (50 μM) for 1 h and then treated with protein translation inhibitor CHX (100 µM) for different times (0, 15, 30, or 60 min). Total proteins were extracted and detected by using HA antibody (C, D). CBB staining of Rubisco showed equal loading of proteins. Band density is measured by Image J and relative density was calculated by setting intensity of KRP6 (or KRP6S75AS109A) at 0 min as 1.0 (E, F corresponding to C, D, respectively). Data are presented as means ± sem (n = 3). G, Phenotypic observation (left) and measurement (right) of eight rosette leaves showed that expression of KRP6 mimicking nonphosphorylation (KRP6S75AS109A) led to more significantly reduced leaf areas in both Wt and ael123 background. Three-week-old Wt and ael123 seedling expressing KRP6 or KRP6 were analyzed. Wt expressing KRP6 or KRP6 (KRP6 in AEL123/ael123, KRP6 in AEL123/ael123) were generated by crossing homozygous ael123 lines expressing HA-KRP6 or HA-KRP6 (KRP6 in ael123/ael123, KRP6 in ael123/ael123) with Wt. Data were presented as means ± sem (n = 15) and statistical analysis using Student’s t test revealed significant differences (***P < 0.001, compared with Wt or ael123). Scale bar = 1 cm.

Phenotypic observation showed that transgenic plants expressing KRP6 presented most significantly reduced leaf areas, whereas there was no significant difference in plants expressing KRP6 compared to Wt (Figure 5G), which is consistent with the altered stability of mutated KRP6 forms and confirms the accuracy and importance of two phosphosites of KPR6.

AELs-mediated phosphorylation stimulates interaction between KRP6 and E3s

Phosphorylation frequently stimulates ubiquitination and thus proteasome-dependent degradation of target proteins (Tan et al., 2013; Tan and Xue, 2014; Ni et al., 2017; Chen et al., 2018). The examination of the degradation mode of KPR6 protein by applying MG132 showed the accumulated KPR6 and suggested the KRP6 degradation is proteasome-dependent (Supplemental Figure S11), which is consistent with the previous report (Kim et al., 2008). Accumulation of KRP6 protein under MG132 treatment was observed in both AEL1ox and ael123 seedlings (Supplemental Figure S11), indicating that phosphorylation does not change the degradation mode of KPR6. Considering E3 ligases recognize and interact with the target protein for ubiquitination and degradation, whether AELs-mediated phosphorylation affects the E3–KRP6 interaction is then investigated.

E3s F-box-like 17 (FBL17) and RING-H2 group F 1a (RHF1a) interact with KRP6 to promote its degradation (Liu et al., 2008; Kim et al., 2008; Gusti et al., 2009). Yeast two-hybrid analysis and liquid quantification showed the explicit interaction between KRP6 and these two E3 ligases; however, the interactions were suppressed with nonphosphorylated forms (KRP6S75A, KRP6S109A, KRP6S75AS109A), while significantly stimulated with phosphorylated forms (KRP6S75D, KRP6S109D, and KRP6S75DS109D; Figure 6A; Supplemental Figure S12). Further in vitro protein pull-down assay confirmed the altered interactions between GST–RHF1a or GST–FBL17 with various KRP6 forms (Figure 6B), indicating the importance of AELs-mediated phosphorylation on KRP6–E3s interaction.

Figure 6

AEL1s-mediated phosphorylation promoted interaction of KRP6 with E3 ligases. A, Y2H assay revealed that phosphorylation promoted KRP6 interaction with FBL17 or RHF1a. Positive AH109 yeast strains expressing various AD and BD fusion proteins were used to examine the interaction intensity by liquid quantification through enzyme labeling apparatus. Experiments were repeated three times. Data are presented as means ± sem (n = 3) and statistical analysis using Student’s t test revealed significant differences (*P < 0.05; **P < 0.01; ***P < 0.001, compared with vector control). B, GST pull-down assay revealed the enhanced binding of phosphorylated KRP6 to RHF1a or FBL17. Recombinant expressed and purified GST, GST–FBL17, and GST–RHF1a were used as bait to detect the binding to KRP6 and various versions. GST and His antibodies were used to detect the fusion proteins. C, CK1s/AELs phosphorylate and promote the KRP6 degradation, hence stimulate cell division. Phosphorylation at S75 and S109 by CK1s/AELs promotes KRP6 interaction with E3s FBL17 and RHF1a, resulting in an enhanced degradation of KRP6 through proteasomal pathway. As an inhibitor of cyclin-dependent protein kinase CDKA;1, accumulated KRP6 under CK1s/AELs deficiency deactivates CDKA;1 activity on G1/S and S phase to arrest cell cycle, thereby inhibiting mitosis/proliferation of cells.

Discussion

Although CK1s have profound impacts on numerous processes, whether and how they affect cell division especially in plants remains largely unknown. KRP6 is one of the essential cell-cycle regulators that inhibits cell proliferation by attenuating the CDKA;1 activity (De Veylder et al., 2001). We here showed that CK1s/AELs phosphorylate CDK inhibitor KRP6 to promote interaction with E3 ligases and hence degradation, thus to coordinate cell division in plants (Figure 6C). These results elucidate how CK1s/AELs-mediated phosphorylation regulates cell division by regulating KRP6 stability. Phosphorylation at serine 75 and 109 of KRP6 greatly enhances the interaction of KRP6 with two E3 ligases, RHF1a and FBL17, to promote KRP6 degradation through ubiquitin–proteasome pathway. Our studies confirm the pivotal roles of CK1s/AELs-mediated phosphorylation in regulating cell division and help to elucidate the complex regulatory network of cell division.

Proliferative cell division is a conserved and complex process, and many regulators are involved via distinct mechanisms. Arabidopsis plants deficiency of AELs presented reduced leaf area due to suppressed cell division caused by stranded cell cycle at G1/S phase, confirming the crucial roles of CK1s in regulating cell division. Plants KRPs are crucial for cell-cycle control and KRP6 overexpression leads to retarded leaf growth with reduced cell proliferation (Kim et al., 2006; Nieuwland et al., 2016). We identified the function of AELs and AELs–KRP6–E3s (FBL17/RHF1a) regulatory module in regulating plant cell division, and analysis of the pleiotropic defect of aels mutants indicates that AELs may involve in the regulation of various organ development by phosphorylating KRP6 and promoting cell division. Besides the retarded growth and smaller rosette leaves, similar defects of lateral root, leaf primordia, petal size, and seed size were observed in both aels mutants and KRP6 overexpression lines. In addition, aborted seeds of ael mutants (Supplemental Figure S3) phenocopy the KRP6 overexpression, as well as a reduced fertility and aborted seeds of fbl17 and aborted ovules of rhf1a rhf2a double mutant (Zhou et al., 2002; Liu et al., 2008; Gusti et al., 2009).

Cell division and cell expansion are the two decisive factors of leaf development. Although cell division and expansion are regulated independently under distinct signals (De Vos et al., 2017), it is worth to mention that altered KRPs expressions have opposite effects on division (cell number) and endoreplication (cell size; De Veylder et al., 2001; Sizani et al., 2019), which is different from the observation in ael triple mutants with both decreased cell number and size. We attributed the decreased cell number (arrested cell cycle) to AELs-mediated phosphorylation of KRP6, while regulation on cell expansion/size is independent, which can be explained by the fact that phosphosites Ser75 and Ser109 are presented only in KRP6 and KRP7 (Supplemental Figure S13), resulting in a specific regulation by AELs. The protein interaction assay showed that KRP7 does not interact with AEL1 (Supplemental Figure S6A), suggesting that AELs specifically interact with and phosphorylate KRP6 to regulate cell cycle. CK1s/AELs regulate KRP6 stability through phosphorylation and hence coordinate cell division in plants, providing important clues to cell division regulation in eukaryotes and expanding the knowledge of CK1s functions in modulating cell cycle.

Protein phosphorylation is widely accepted as a predominant posttranslational modification of proteins and plays crucial roles in various signaling pathways. CK1s/AELs-mediated phosphorylation suppresses KRP6 stability by promoting its interaction with E3s, FBL17, or RHF1a, which is consistent with altered phosphorylation status by substituting phosphosites Ser75 and Ser109. Phosphorylation finally results in enhanced ubiquitination and degradation of KRP6. In addition, identification of specific phosphosites supplies a new discipline that links posttranslational modifications to perturbations of physiological activity precisely.

As the most fundamentally biological processes, key regulators and machinery determining cell cycle have received increasing attentions; however, how the basic cell-cycle machinery integrates with growth and development in different organisms needs further investigations. Our studies elucidated the mechanism of CK1s in regulating cell division, suggesting a central role of protein phosphorylation in controlling cell proliferation by integrating the relevant signaling networks. Further investigations on upstream regulation of CK1s/AELs are needed as CK1s can also be phosphorylated by other kinases and regulated through posttranslational modifications. In addition, the heterogeneity of lipid organization in membranes and phosphoinositide second messengers may function upstream of CK1s/AELs to involve in cell-cycle control.

Materials and methods

Plant materials and growth conditions

Arabidopsis (A. thaliana, Columbia-0 ecotype, Col-0) plants were used as the Wt control. T-DNA insertion line krp6 (At3g19150, SALK_142997) was obtained from Arabidopsis Biological Resource Center. Sterilized seeds of Wt and various mutants were stratified at 4°C for 2 d, then sown on half-strength Murashige and Skoog medium (1/2 MS, Duchefa) and germinated under 22°C ± 1°C with a 16-h-light/8-h-dark photoperiod. Seven-day-old seedlings were transferred to soil and seedlings were grown under same conditions in all experiments.

Generation of various transgenic lines

Full-length cDNA sequences of KRP6 were obtained from TAIR (http://www.arabidopsis.org/) and open reading frame (ORF) was amplified by PCR using corresponding gene-specific primers (Supplemental Table S2), sequenced to confirm the correctness, and subcloned into pHB vector, which drives the expressions of KRP6 and mutated KRP6 forms under CaMV35S promoter and introduces a sextuple HA epitope at N terminus of expressed protein. Resultant binary constructs (p35S::6×HA-KRP6, p35S::6×HA-KRP6S75AS109A, and p35S::6×HA-KRP6S75DS109D) were transformed into Arabidopsis by floral dip method (Clough and Bent, 1998) and transgenic plants were screened by antibiotic resistance and confirmed by western blotting.

For complementation study, full-length cDNA sequences of AEL1-4 were obtained from TAIR and ORFs were amplified by PCR using corresponding gene-specific primers (Supplemental Table S2) and subcloned into pCambia 1306 vector, which drives the expressions of AELs under CaMV35S promoter and introduces a flag tag at C terminus of expressed protein. Resultant binary constructs were transformed into triple mutants ael123 and ael124 by the floral dip method, and transgenic plants were screened by antibiotic resistance and confirmed by western blotting.

Microscopic observations and phenotypic analysis

To measure leaf areas and cell size, the eight fully expanded leaves of 3-week-old seedlings were fixed with a formalin/acetic acid/alcohol solution and dehydrated with gradient ethanol (50%, 70%, 80%, 90%, 95%, 100%, v/v, 3 min for each gradient). Leaves and cells were observed using a scanning electron microscope (JEOL6360LV). Cell number was determined as average by dividing leaf area by average cell area of 15 leaves. Statistical analysis was conducted using a two-tailed Student’s t test.

Kinematic analysis was performed according to previous report (Sizani et al., 2019). Briefly, cellular measurements were performed on six pairs of first leaf pair from 5 until 25 DAS with scanning electron microscope (JEOL6360LV). Abaxial epidermal cells were photographed and analyzed with ImageJ software (http://imagej.nih.gov/ij/) to calculate the average cell area and number of cells per leaf by dividing leaf blade area by average cell area. Relative rate of cell number increase in each hour was regarded as cell division rate represented by the first derivative of a five-point quadratic function fitted by logarithmic values of mean of cell number.

Analysis of ploidy and S phage DNA replication efficiency

Approximately 10 pairs of first leaf pair were chopped with a razor blade in Nuclei isolation and staining solution (NPE Analyzer, NPE 731085) on ice. The suspension was filtered through a 40-μm nylon mesh (Sysmex Cell Trics) after incubation at room temperature for 5–10 min. Nuclei were analyzed with a FACSCalibur flow cytometer (Moflo XDP 217) and submitted to 5.2 software. At least 20,000 nuclei were counted per sample. Each experiment was repeated at least three times.

To detect the S phage DNA replication efficiency, ∼15 first leaf pair were chopped with a razor blade in 500-μL 1/2 MS liquid medium and filtered through a 40-μm nylon mesh. Liquid medium was replaced with EdU medium (diluting EdU solution with 1/2 MS medium at a ratio of 1,000:1) and incubated for 0, 1, 2, 3, or 4 h, respectively. Cells were collected into a flow tube, centrifuged at 1,500 rpm for 5 min and resuspended with PBS after discarding the supernatant. Cells were then centrifuged at 1,500 rpm for 5 min again and fixed with 4% paraformaldehyde for 30 min, neutralized with 2-mg·mL−1 glycine for 5 min, and washed with PBS before incubating with 0.5% tritonX-100 at room temperature for 10 min and washing with PBS. Next, 300-μL 1X Apollo dyeing buffer was added and the solution was incubated at room temperature under dark for 10 min. Flow detection was performed with FACSCalibur flow cytometer (Moflo XDP 217) immediately after cleaning with Triton X-100 penetrant twice and resuspended in PBS. Submit5.2 software was used to analyze the cell proportion containing EDU signal.

Histochemical analysis of GUS activities

aels triple mutants were crossed with transgenic lines expressing GUS reporter genes driven by CYCB1;1 promoter and positive offsprings were used for analysis. First leaf pair at different developmental stages (5, 7, 9, 11 d) were incubated in GUS staining buffer (1-mM K3Fe[CN]6, 0.1% Triton X-100, 10-mM EDTA, 100-mM phosphate buffer [NaPO4, pH 7.0], and 2-mM X-Gluc) at 37°C for 16 h and nonspecific staining was removed with 50% ethanol. Samples were observed using Nikon SMZ1500.

RNA extraction and RT-qPCR analysis

Total RNAs were extracted from 7-d-old seedlings using Universal Plant RNA Extraction Kit (TIANGEN) and reversely transcribed using PrimeScript 1st Strand cDNA Synthesis Kit (Takara). RT-qPCR was performed using a CFX Connect TM real-time thermal cycler (BioRad) with the SYBR Green Real-time PCR Master Mix (Toyobo). Gene-specific primers were listed in Supplemental Table S2.

Subcellular localization and BiFC assay

For BiFC assay, ORFs of KRP6 and AEL1 were amplified and subcloned into vector p35S::nYFP or p35S::cYFP. Nicotiana benthamiana leaves were infiltrated with Agrobacteria containing different construct combinations and observed after infiltration for 2 d by confocal laser scanning microscopy (Olympus FV10i) with an argon laser excitation wavelength of 488 nm and emission wavelengths of 520–550 nm.

For localization of KRP6, construct p35S::KRP6-nYFP was transformed into N. benthamiana leaves with Agrobacteria and observed by confocal laser scanning microscopy (Olympus FV10i) after infiltration for 2 d. The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI, 100 μM) for 10 min.

Expression of recombinant protein and western blotting analysis

ORFs of AEL1 or KRP6 were subcloned into pET51b vector (Novagen) and expressed in Escherichia coli strain BL21 (DE3). Construct pET51b-KRP6 was used to generate the single amino acid mutation with Fast Mutagenesis System (FM111-01, TransGen Biotech). Resultant constructs were used to generate double amino acids mutation through same protocol. Primers were listed in Supplemental Table S2.

Recombinant proteins were purified according to manufacturer’s protocol (Novagen). For western blotting analysis, protein samples were separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) gel (Life) and then transferred onto polyvinylidene difluoride membranes (Perkin-Elmer), which were incubated with a primary mouse antibody and then with a goat anti-mouse immunoglobulin G AP-conjugated secondary antibody (Santa Cruz Biotechnology). AP activity was detected by BCIP/NBT kit (Invitrogen) according to the supplier’s instructions. The brand density was measured and calculated by Image J.

Yeast two-hybrid analysis

Yeast two-hybrid analysis and α-galactosidase activity measurement were performed according to Yeast Protocol Handbook (Clontech, CA, USA). Constructs expressing fusion proteins RBR1-BD, CYCA2;3-BD, KRP7-BD, KRP6-BD, KAC2-BD, AEL1-AD, AEL2-AD, AEL3-AD, AEL4-AD, KRP6S75A-BD, KRP6S109A-BD, KRP6S75AS109A-BD, KRP6S75D-BD, KRP6S109D-BD, KRP6S75DS109D-BD, FBL17-AD, and RHF1a-AD were generated by subcloning amplified DNA fragments of corresponding full-length cDNAs into appropriate vectors (pGBKT7 or pGADT7). Confirmed constructs were transformed into yeast strain AH109 and yeast growth was observed. Primers used were listed in Supplemental Table S2.

In vitro kinase activity assay

Kinase activity was measured in the presence of 32P-γ-ATP (10 mCi, NEC902A; Perkin-Elmer) in a total volume of 20-µL kinase activity buffer (50-mM Tris–HCl, pH 8.0, 10-mM MgCl2, 1-mM DTT, 10-mM ATP, 1-µg kinase, and 10-µg substrate proteins). Reactions were initiated by incubation at 25°C for 1 h and terminated by adding 2× SDS loading buffer. After boiling for 5 min, the reaction products were fractionated by SDS–PAGE. The gel was examined by autoradiography according to manufacturer instruction (Fujifilm FLA 9000 plus DAGE) and band density was analyzed by Image J.

In vitro Phos-tag gel shift assay

For kinase activity, purified substrate protein was incubated in a total volume of 20-µL kinase activity buffer (50-mM Tris–HCl, pH 8.0, 10-mM MgCl2, 1-mM DTT, 10-mM ATP, 1-µg kinase, and 10-µg substrate proteins). Reactions were initiated by incubation at 25°C for 1 h and terminated by adding 2× SDS loading buffer and incubated at 95°C for 5 min.

For phosphatase treatment, purified protein (KRP6) was incubated with Lambda Protein Phosphatase (λ-PPase; NEB, Cat. No. P0753S) at 30°C for 90 min and subsequently added 2× SDS sample buffer for 15 min on ice and incubated at 95°C for 5 min.

Proteins were separated on an 8% SDS–PAGE gel containing 50-µM Phos-tag reagent (Wako, Cat. No. 304-93525) and 100-µM MnCl2. Separated proteins were then detected by western blot with anti-His antibody.

In vitro degradation assay of KRP6 by cell-free analysis

Seven-day-old seedlings were harvested and ground into fine powder in liquid nitrogen. Total proteins were extracted in buffer (25-mM Tris–HCl, pH 7.5, 10-mM NaCl, 10-mM MgCl2, 4-mM PMSF, 5-mM DTT, and 10-mM ATP) and cell debris was removed by two centrifugations (12,000 rpm, 10 min, 4°C) to collect the supernatants. Protein concentration was determined by Pierce BCA Protein Assay Kit (ThermoFisher Scientific). For degradation assay of KRP6, purified His-KRP6 fusion proteins were incubated in total proteins (150 µL, ∼500 µg) extracted from same amount seedlings of Wt, ael123 or ael124 triple mutants.

For degradation assay of KRP6, KRP6S75AS109A, and KRP6S75DS109D, equal amounts (∼1 µg) of purified His-KRP6 or mutated forms were incubated in total proteins (∼500 µg) extracted from same amount seedlings of Wt or ael123 triple mutants. The mixtures were incubated at 22°C and analyzed at indicated intervals after preincubation for 1 h. Protein abundances of KRP6 and mutated forms were determined by immunoblot using anti-His antibody (sc-8036, Santa Cruz Biotechnology).

In vivo degradation assay of KRP6

Isogenic plants expressing HA-KRP6 fusion protein were generated by crossing homozygous transgenic ael123 plants expressing HA-KRP6 with Wt. Transgenic plants with similar levels of HA-KRP6 or HA-KRP6S75AS109A were selected for in vivo degradation assay. Seven-day-old seedlings were incubated in liquid MS medium supplemented with 50-μM MG132 for 1 h to preaccumulate KRP6 protein and then incubated in MS medium supplemented with 100-µM CHX (C7698, Sigma-Aldrich) to block proteins synthesis. After CHX treatment for different times (0, 15, 30, or 60 min), seedlings were collected and frozen in liquid nitrogen immediately. Proteins were extracted with extraction buffer (PEB, 20-mM Tris–HCl, pH 7.5, 150-mM NaCl, 0.5% Tween-20, 1-mM EDTA, 1-mM DTT) containing a protease inhibitor cocktail “Complete” (04693116001, Roche) and phosphatase inhibitor cocktail “PhosSTOP” (04906845001, Roche), and abundance of HA-KRP6 fusion protein was determined using anti-HA antibody (H9658, Sigma) by western blotting analysis.

In vivo phosphorylation assay of KRP6 and KRP6S75AS109A

To detect the phosphorylation of KRP6 in vivo, Wt lines expressing KRP6 and KRP6S75AS109A were generated by crossing homozygous ael123 lines expressing KRP6 and KRP6S75AS109A with Wt respectively. Seven-day-old seedlings were treated with 50-µM MG132 for 6 h, then ground into powder in liquid nitrogen, and dissolved with protein extraction buffer (50-mM Tris–HCl, pH 7.4, 150-mM NaCl, 1-mM EDTA, 1-mM DDT, 0.1% [v/v] Triton X-100, and 1× protease inhibitor cocktail from Sigma plus 1-mM PMSF). Amounts of KRP6 and KRP6S75AS109A in immunoprecipitated (IP) fraction were detected using anti-HA antibody and adjusted to equal amount. Phosphorylation level was detected using anti-Ser antibody (Ab9334 Abcam).

FRET-FLIM analysis

FRET-FLIM analysis was performed using an inverted Leica TCS SP8 FALCON microscope equipped with a pulsed White Light Laser (WLL, Leica). Excitation (488 nm) was achieved by WLL laser at a repetition rate of 80 MHz, via a water immersion objective (×60, numerical aperture = 1.2). Emitted light was detected at 488 nm by a Hyd-SMD (Leica) detector. Data were collected and analyzed using LAS X software (Leica).

CoIP assay

Seven-day-old Arabidopsis seedlings (∼1.0 g) coexpressing KRP6 and AEL1 (see above) were harvested and ground into powder in liquid nitrogen. Proteins were extracted by adding extraction buffer (50-mM Tris–HCl, pH 7.4, 150-mM NaCl, 1-mM EDTA, 1-mM DDT, 0.1% [v/v] Triton X-100, and 1× protease inhibitor cocktail from Sigma plus 1-mM PMSF) for 30 min (on ice), then centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was incubated with prebalanced 20-μL monoclonal Flag M2 Magnetic Beads (Sigma) at 4°C for at least 4 h with gentle rotation. Beads were washed at least four times with lysis buffer and boiled in 50-μL of 2× SDS loading buffer for 5 min. Input or IP fractions were subjected to Western blots and detected with anti-Flag or anti-HA antibodies, respectively.

LC–MS/MS analysis for phosphosites of KRP6

To identify the phosphorylated amino acid residues, KRP6 was phosphorylated by AEL1 in vitro (see above), subjected to in-gel Chymotrypsin digestion, and resultant peptides were analyzed by Orbitrap Fusion LC–MS/MS analysis using an Easy-nLC 1000 LC system (Thermo Fisher Scientific) connected to an LTQ Orbitrap mass spectrometer (Thermo Electron, Bremen, Germany) equipped with a nanoelectrospray ion source (Thermo Fisher Scientific). Chymotryptic peptides were dissolved with loading buffer (5% acetonitrile and 0.1% formic acid, phase A) and then injected into homemade 2 cm × 150 μm ID, C18 trap-column and subsequently eluted and separated on 12 cm × 150 μm ID column (C18, 1.9 μm, 120 Å) with a series of adjusted linear gradients of phase B (95% acetonitrile in 0.1% formic acid) according to the hydrophobicity of fractions, and then injected into the mass spectrometer at a constant column-tip flow rate of 500 nL·min−1. Eluted peptides were analyzed by MS and data-dependent MS/MS acquisition. Scan range was set from m/z 300 to m/z 1,400. The dynamic exclusion of previously acquired precursor ions was enabled at 18 s. Spectral data were searched against UniProt-Arabidopsis database with protein sequence of KRP6 (UniProt KB-A0A1I9LM10) to achieve a false discovery rate of <1%. Mass tolerance was set to be 20 ppm for precursor, and it was set 0.1 Da for the tolerance of product ions. Oxidation (M), Phosph (ST), and Phosph (Y) were chosen as variable modifications.

Protein pull-down assay

Recombinant proteins were expressed and purified from E. coli according to manufacturer’s protocol. Prey proteins of interest were incubated with the immobilized bait protein for 2 h at 4°C. After binding in vitro, unbound prey proteins were washed with 1× PBS buffer for three times and eluted proteins were boiled in 2× SDS loading dye. Protein samples were then separated by SDS–PAGE.

Software and database

Protein sequence of KRP family members were obtained from TAIR (https://www.arabidopsis.org/) and these protein sequence alignments were conducted using DNAMAN.

Protein phosphorylation sites were predicted using Scansite4.0 website (https://scansite4.mit.edu).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Accession numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: AEL1 (AT2G25760), AEL2 (AT3G13670), AEL3 (AT3G03940), AEL4 (AT5G18190), KRP6 (AT3G19150), KRP7 (AT1G49620), KRP2 (AT3G50630), KRP4 (AT2G32710), CYCA2;3 (AT1G15570), CYCD3;1 (AT4G34160), CYCB1;1 (AT4G37490), RBR1 (AT3G12280), CDKA1/KAC2 (AT5G65460), CSLCO4 (AT3G28180), CDKC2 (AT5G64960), DAWDLE (AT3G20550), FBL17 (AT3G54650), and RHF1a (AT4G14220).

Supplemental data

The following materials are available in the online version of this article.

. Prediction of CK1 phosphorylation sites of cell-cycle related regulators.

. Sequences of the primers used in this study.

. Arabidopsis ael single and double mutants do not present altered leaf areas.

. Recovery of the smaller leaves of ael123 and ael124 triple mutants by complemented expression of AELs genomic fragments.

. Reduced fertility and aborted seeds of ael triple mutants (ael123, ael124, ael134) and sterile seeds of heterozygous ael1234.

. Suppressed cell division under deficiencies of AELs.

. Expressions of cell-cycle-related genes of aels triple mutants.

. AEL1-4 interact with KRP6.

. Identification and phenotypic observation of krp6 mutant.

. Expressions of KRP6 and mutated forms in transgenic plants.

. Expressions of KRP6 in AEL1-4 overexpression lines.

. Detection of KRP6 phosphorylation site using LC–MS.

. KRP6 is degraded through proteasomal pathway.

. Phosphorylation-mimicking form KRP6S75DS109D interacts with FBL17 or RHF1a more definitely.

. Phosphosites S75 and S109 are not conserved in Arabidopsis KRP members.

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