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Development and the cell cycle are closely coordi-nated. Cell division is usually coupled with growth, and in most cases, differentiating cells no longer di-vide. However, growth and cell division are not in-trinsically linked (John and Qi, 2008; Harashima and Schnittger, 2010), and the relationship between growth and division can be tailored to meet the needs of par-ticular developmental contexts (Tsukaya, 2008). For ex-ample, many animals have large zygotes that undergo rapid cell divisions with minimal growth to produce the many small cells of the embryo (Newport and Kirschner, 1982). In contrast, the alternative cell cycle

known as endoreplication or endoreduplication, in which DNA is replicated without subsequent division, results in cells that are highly polyploid and are often large and highly specialized (De Veylder et al., 2011; Fox and Duronio, 2013).

The SIAMESE (SIM) gene encodes a cyclin-dependent kinase (CDK) inhibitor that plays a key role in establish-ing endoreplication cycles in Arabidopsis (Arabidopsis thaliana) trichomes (Churchman et al., 2006; Kumar et al., 2015). All eukaryotes have a class of Ser/Thr protein kinases, known as CDKs, that control pro-gression through cell cycle checkpoints. CDKs are themselves regulated by cyclin-dependent kinase in-hibitors (CKIs) that act to prevent premature passage through checkpoints or to arrest cell cycle progres-sion in response to developmental or stress signals (Morgan, 2007). In turn, CKIs are typically regulated posttranscriptionally by phosphorylation, proteoly-sis, and nuclear localization (Nash et al., 2001; Liang et al., 2002; Connor et al., 2003; Lu and Hunter, 2010). In plants, CKIs regulate cell cycle responses to a variety of developmental, abiotic, and biotic cues, in addition to their role in regulating transitions in typical mitotic cycles (Kumar and Larkin, 2017).

Plant genomes encode two distinct families of CKIs, the INHIBITOR/INTERACTOR OF CDC2 KINASE/KIP-RELATED PROTEIN (ICK/KRP1) family and the SIAMESE-RELATED (SMR) family, of which SIM is the founding member (Kumar and Larkin, 2017). Mem-bers of the ICK/KRP family share a C-terminal domain of approximately 30 residues that is similar to the N- terminal domain of the mammalian CDK-INTERACTING PROTEIN/KINASE INHIBITOR PROTEIN (CIP/KIP)

Functional Analysis of Short Linear Motifs in the Plant Cyclin-Dependent Kinase Inhibitor SIAMESE1[OPEN]

Narender Kumar,2 Renee Dale, Daniel Kemboi,3 Elizabeth A. Zeringue, Naohiro Kato, and John C. Larkin4

Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803ORCID IDs: 0000-0002-2009-3158 (N.K.); 0000-0002-1674-1247 (R.D.); 0000-0002-0541-4064 (D.K.); 0000-0002-5080-431X (E.A.Z.); 0000-0002-4571-3456 (N.K.); 0000-0002-7156-508X (J.C.L.)

Endoreplication, a modified cell cycle in which DNA is replicated without subsequent cell division, plays an important but poorly understood role in plant growth and in plant responses to biotic and abiotic stress. The Arabidopsis (Arabidopsis thaliana) SIAMESE (SIM) gene encodes the first identified member of the SIAMESE-RELATED (SMR) family of cyclin-dependent kinase inhibitors. SIM controls endoreplication during trichome development, and sim mutant trichomes divide several times instead of endoreplicating their DNA. The SMR family is defined by several short linear amino acid sequence motifs of largely unknown function, and family members have little sequence similarity to any known protein functional domains. Here, we investigated the roles of the conserved motifs in SIM site-directed Arabidopsis mutants using several functional assays. We identified a potential cyclin-dependent kinase (CDK)-binding site, which bears no resemblance to other known CDK interaction motifs. We also identified a potential site of phosphorylation and two redundant nuclear localization sequences. Surprisingly, the only motif with similarity to the other family of plant CDK inhibitors, the INHIBITOR/INTERACTOR OF CDC2 KINASE/KIP- RELATED PROTEIN proteins, is not required for SIM function in vivo. Because even highly divergent members of the SMR family are able to replace SIM function in Arabidopsis trichomes, it is likely that the results obtained here for SIM will apply to other members of this plant-specific family of CDK inhibitors.

1This work was supported in part by a National Science Foundation award (MCB1615782) to J.C.L. and N.Ka.

2Current address: Department of Botany and Plant Pathology, Lilly Hall of Life Sciences, 915 West State Street, West Lafayette, IN 47907-2054.

3Current address: Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742.

4Address correspondence to jlarkin@lsu.edu.The author responsible for distribution of materials integral to

the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: John C. Larkin (jlarkin@lsu.edu).

N.Ku. and J.C.L. conceived and planned the experiments; N.Ku. performed most of the experiments and wrote the first draft of the article; R.D. and N.Ka. carried out and analyzed the split-luciferase assays; E.A.Z. carried out light and scanning electron microscopy and analyzed the data in Tables 1 to 3; D.K. and N.Ku. constructed plasmids and carried out microscopy for nuclear localization; all au-thors revised the article and approved the final version.

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family of CKIs (Wang et al., 1997; De Veylder et al., 2001). In both the plant and animal proteins, this do-main interacts with CDKs (Russo et al., 1996; Wang et al., 1998). Other motifs present in various family members have been shown to be involved in protein degradation, cyclin binding, and nuclear localization (Wang et al., 1998; Jakoby et al., 2006). ICK/KRP pro-teins inhibit CDK activity in vitro and interact with cy-clins as well as CDKs (Wang et al., 1998; De Veylder et al., 2001). Moderate levels of ICK/KRP expression preferentially suppress mitosis and promote endorep-lication, while high-level expression inhibits DNA rep-lication as well and can result in cell death (De Veylder et al., 2001; Schnittger et al., 2003; Verkest et al., 2005; Roeder et al., 2010). ICK/KRP family members also play a key role in the G1/S checkpoint of the cell cycle, maintaining the inhibition of S-phase CDK activity un-til they are degraded by a SKIP1-CULLIN1_F-BOX E3 ubiquitin ligase complex containing the FBL17 F-box protein (Kim et al., 2008; Zhao et al., 2012; Noir et al., 2015).

In contrast, members of the SMR family have no similarity to any nonplant CKIs and share with the ICK/KRP family only one short motif implicated in cyclin binding (Churchman et al., 2006; Peres et al., 2007). All land plant genomes contain multiple SMR family members, with 17 SMRs found in the Arabidop-sis genome (Kumar et al., 2015). SMR1/LGO regulates endoreplication in giant cells of the sepal epidermis as well as during leaf development (Roeder et al., 2010), and SMR2 plays a role in the timing of the switch from cell division to endoreplication early in leaf devel-opment (Kumar et al., 2015), emphasizing the role of SMRs in controlling endoreplication. SMRs also have been implicated in blocking cell division in response to DNA damage or drought stress and in responses to pathogens (Wang et al., 2014; Yi et al., 2014; Hamdoun et al., 2016; Schwarz and Roeder, 2016; Dubois et al., 2018). Multiple SMRs from Arabidopsis, as well as other plants, can complement the sim trichome phe-notype, in which cells divide several times instead of endoreplicating their DNA, indicating that there is an underlying functional similarity among all members of the family tested (Peres et al., 2007; Kumar et al., 2015).

SIM was identified by recessive loss-of-function mutations that result in cell division in Arabidopsis trichomes, in contrast to the endoreplication found in the unicellular trichomes of wild-type plants (Walker et al., 2000). Constitutive overexpression of SIM re-sults in small plants with giant, highly endoreplicated leaf epidermal cells (Churchman et al., 2006); together, these two observations demonstrate that SIM encodes a mitotic inhibitor that is a key regulator of endorep-lication. SIM binds to CDKs and inhibits the activity of both CDKA;1 and the mitotic CDKB1;1 in vitro, demonstrating that SIM is a true CKI (Kumar et al., 2015). Although SIM can suppress the S-phase kinase CDKA;1 in vitro, overexpression of SMRs in transgenic plants only suppresses mitosis without suppressing DNA replication, even when SIM is expressed from a

strong promoter (Churchman et al., 2006). This is in contrast to what is seen with strong ICK/KRP expres-sion, which suppresses both division and DNA rep-lication and can result in cell death (Schnittger et al., 2003). These observations suggest that the inhibition of S-phase CDK activity may be prevented by posttrans-lational regulation of SIM, although no such mecha-nism has been identified.

In contrast to proteins whose functions are carried out by discrete folded secondary structure domains, SIM and other proteins of the SMR family are recogniz-able by the presence of a series of short linear motifs. Although such motifs are typically found to be sites of protein interaction, phosphorylation sites, localization signals, and similar functions (Neduva and Russell, 2005), for the most part no clear functions have been assigned to the motifs in the SMR family. Here, we in-vestigated the functions of the various conserved short linear motifs in SIM via the ability of mutated SIM transgenes to complement the sim mutant phenotype. We identified SIM motif A as a potential CDK inter-action motif and have located two redundant nuclear localization sequences (NLSs). Surprisingly, we found that the putative cyclin interaction motif that is con-served between SMRs and ICK/KRPs is not absolutely required for SIM function. We also present evidence that a conserved Thr in motif A is a potential phos-phorylation site, suggesting a possible mechanism for the posttranslational regulation of SIM. These results provide important insights into the mechanism of ac-tion and regulation of the SMR family of CKIs.

RESULTS

Motifs A and B, But Not Motif C, Are Essential for SIM Function

The SMR family is defined by a series of short lin-ear motifs that occur in the same order in all family members, with few exceptions (Fig. 1; Churchman et al., 2006; Peres et al., 2007; Kumar et al., 2015). While motif C of SIM resembles motif 2 of the ICK/KRP class of CKIs and has been implicated in interaction with cyclins (Churchman et al., 2006; Peres et al., 2007), the sequences of motifs A and B do not suggest a specific function. Two sequences resembling NLSs also are in-dicated in Figure 1.

Wild-type trichomes are unicellular, while sim mu-tant trichomes are multicellular (Fig. 2; Table 1). Fifteen different site-directed substitution mutants in these motifs were generated and assayed for SIM function by introduction into sim mutant plants and examined for their ability to suppress the sim multicellular tri-chome phenotype. These constructs were expressed under the control of the GLABRA2 (GL2) promoter, GL2pro, because the native SIM promoter is not fully defined at present. GL2pro, like SIM itself, is under the direct control of the trichome initiation factor GL3 (Morohashi and Grotewold, 2009), and the expression

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of SIM under the control of GL2pro complements the sim trichome phenotype (Churchman et al., 2006). A list of the site-directed mutants generated and their qual-itative abilities to suppress the sim phenotype are given in Supplemental Table S1. Most notably, muta-tions affecting motifs A and B eliminated the ability of the transgene to restore the wild-type unicellular tri-chome phenotype, while mutations in the other puta-tive conserved motifs had little apparent effect on SIM function.

To more carefully investigate the functions of these three motifs, enhanced YELLOW FLUORESCENT PROTEIN (eYFP) was fused to the N terminus of wild-type SIM and several representative mutant genes, and these constructs were expressed in sim mutant plants using the GL2 promoter to test their functions in vivo. For motif A, a mutant gene in which residue T35, the most conserved residue in the SMR family, was replaced by Ala fused to eYFP (referred to as eYFP:motifA). The T35 residue is immediately followed by a Pro, matching the minimum consensus for a CDK phosphorylation site, which made this site additionally interesting. For motifs B and C, blocks of residues representing the core of the conserved motif sequence were substituted with Ala. A motif B mutant, in which Pro residues P51, P52, P53, and P54 were replaced by Ala, was fused to eYFP (referred to as eYFP:motifB), and a motif C mutant, in which the six core residues of the motif, E91, I92, E93, R94, F95, and F96, were replaced by Ala, was fused to eYFP (referred to as eYFP:motifC). A mutant rice (Oryza sativa) SMR in which the six residues equivalent to MotifC of SIM were substituted with Ala was unable to bind to a rice d-type cyclin in a yeast two-hybrid assay (Peres et al., 2007).

GL2pro:eYFP:SIM fully complements the sim mutant (Fig. 2C; Table 1), restoring unicellular trichomes. As predicted by our initial results, GL2pro:eYFP:motifA and GL2pro:eYFP:motifB fail to suppress division in sim mutant trichomes (Fig. 2, E and G; Table 1), while GL2pro:eYFP:motifC complements the mutant phenotype completely (Fig. 2I; Table 1). The GL2pro:eYFP:motifA and GL2pro:eYFP:motifC plants show nucleus-localized YFP fluorescence in their trichomes equivalent to that seen in GL2pro:eYFP:SIM plants, indicating that the fusion proteins are stable and correctly localized (Fig. 2, D, F, and J). In contrast, we detected YFP fluores-cence in only two of more than 40 independent GL2pro:

eYFP:motifB lines examined, and in both cases the fluo-rescence was much fainter than that observed with the other constructs (Fig. 2H), although the fluorescence was localized to the nucleus.

The Conserved and Essential T35 Residue Is a Potential Phosphorylation Site

The motif A Thr residue at position 35 and the fol-lowing Pro at position 36 are the most conserved amino acid residues in the SMR family, and the results pre-sented above (Fig. 2, E and F; Table 1) confirm that T35 is essential for SIM function. A Thr or Ser followed by a Pro (TP or SP) is the minimal consensus site for phosphorylation by CDKs, suggesting that phosphor-ylation of T35 may be required for SIM function. To test this hypothesis, we created and introduced into sim plants SIM genes in which T35 was replaced by either Asp (T35D) or Glu (T35E), negatively charged amino acids that can sometimes function as phosphomimics (Fig. 3). While the nonphosphorylatable GL2pro:T35A construct as well as the GL2pro:T35E transgenes were nonfunctional (Fig. 3, C and E; Supplemental Table S1), the GL2pro:T35D construct was functional, as shown by its ability to suppress cell division in sim trichomes (Fig. 3D; Supplemental Table S1). To further explore the function of the T35 residue, we constructed YFP fu-sions of these mutant genes and introduced them into sim mutant plants under the control of the GL2 pro-moter. As expected, while the nonphosphorylatable GL2pro:eYFP:T35A construct was unable to suppress cell division in sim trichomes (Table 2), the GL2pro:eYF-P:T35D transgene was functional, as shown by its abil-ity to suppress cell division in sim trichomes (Table 2). Both constructs resulted in nucleus-localized fluores-cence equivalent to that of sim GL2pro:eYFP:SIM (Fig. 4), indicating that these transgenes produced similar amounts of correctly localized fusion proteins.

CDK substrates often are phosphorylated on multi-ple SP or TP sites. While the predicted SIM sequence includes no SP sites, residues T50 and T63 are each fol-lowed by a Pro residue. However, when expressed in sim mutant plants, a T50A T63A double mutant was able to suppress the sim phenotype, indicating that neither of these Thr residues is essential for SIM func-tion (Supplemental Fig. S1). As expected, the triple mutant T35A T50A T63A fails to suppress division in

Figure 1. SIM amino acid sequence, starting at residue 20, indicating motif locations and consensus sequences. Deletion mutants are named for the amino acids that are removed (e.g. Δ66-127 lacks amino acids 66 through 127, retaining only the N-terminal 65 amino acids). In the consensus sequences, x indicates any amino acid residue and ϕ indicates any hydrophobic residue. The motif consensus sequences shown here are simplified from those presented by Kumar et al. (2015).

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sim trichomes (Supplemental Fig. S1). Thus, the T35 residue is the only potential candidate for a CDK phos-phorylation site in SIM.

Motif A May Be a CDK Interaction Motif

We and others previously demonstrated interac-tions of the SIM protein with CDKs using a variety of assays, including a split-luciferase complementation assay in Arabidopsis protoplasts (Churchman et al., 2006; Van Leene et al., 2010; Kumar et al., 2015). Using the split-luciferase assay, we tested the ability of the motif A, B, and C mutants fused to the C-terminal por-tion of luciferase (CLuc:SIM mutants) to interact with CDKA;1 fused to the N-terminal portion of luciferase (NLuc:CDKA;1). The interaction of histones H2A and H2B served as a positive control, and interaction with the transcription factor PERIANTHIA (PAN) fused to the N-terminal portion of luciferase (NLuc:PAN) served as a negative control. Interaction with CDKA;1 was seen with the T34AT35AP36A motif A mutant (CLuc:motifA-3As/NLucCDKA;1), the motif B mu-tant (Cluc:motifB/Nluc:CDKA;1), and the motif C mutant (Cluc:motifC/Nluc:CDKA;1) as well as with the Cluc:SIM positive control (Fig. 5). However, a mu-tant in which 10 motif A residues were replaced by Ala (T35 through P44; CLuc:motifA-10As) interacted with NLuc:CDKA;1 no better than it interacted with the NLuc:PAN negative control (Fig. 5), thus implicating motif A as a potential CDK-binding site.

SIM Contains Redundant NLSs

As indicated in Figure 1, the C-terminal half of SIM contains two short stretches of basic amino acids that resemble NLSs, although NLS1 was initially proposed as a possible cyclin interaction sequence due to its sim-ilarity to the Cy motif, which is found in some cyclin- binding proteins (Adams et al., 1996; Wohlschlegel et al., 2001; Churchman et al., 2006). The presence of putative NLSs was somewhat surprising because the 14-kD protein encoded by SIM is well under the ∼40-kD limit below which proteins would be expected to freely diffuse into the nucleus. To further explore the role of conserved motifs in the C-terminal half of SIM, a

Figure 2. Mutations in conserved residues of motifs A and B, but not motif C, disrupt SIM function. The ability of YFP fusions of SIM and SIM motif mutants expressed from the GL2 promoter to functionally complement the multicellular trichome phenotype of the sim mutant was tested. Scanning electron micrographs are shown for wild-type Columbia-0 (Col-0) unicellular trichomes (A), sim mutant multicellular trichomes (B), trichomes of sim GL2pro:YFP:SIM (C), trichomes of sim GL2pro:YFP:motifA (E), trichomes of sim GL2pro:YFP:motifB (G), and trichomes of sim GL2pro:YFP:motifC (I). YFP fluorescence in developing trichomes of the indicated genotypes is shown in D, F, H and J. Bars for = 100 μm (A–C, E, G, and I) and 50 μm (D, F, H, and J).

Table 1. Complementation of a sim mutant by transgenic constructs with mutations in various motifs of SIM

The multicellular trichome phenotype of a complementation line homozygous for a single T-DNA insert for each of the indicated SMRs was assessed by counting the number of 4,6-diamidino-2-phenylin-dole (DAPI)-stained nuclei at each trichome initiation site (TIS) for each genotype. All values followed by the letter a have significantly fewer nuclei per TIS than the sim mutant (P < 1 × 10−11 in a one-tailed Student’s t test, after applying a Bonferroni correction for multiple tests). For each transgenic genotype, at least two additional indepen-dent homozygous T3 lines were obtained having a phenotype qualita-tively equivalent to the line shown here.

Genotype No. of Nuclei per TIS No. of TIS

Col-0 1.00 ± 0.00 a 50Sim 2.16 ± 0.98 50sim GL2pro:YFP:SIM 1.03 ± 0.18 a 50sim GL2pro:YFP:motifA 1.98 ± 0.80 50sim GL2pro:YFP:motifB 1.96 ± 0.67 50sim GL2pro:YFP:motifC 1.02 ± 0.14 a 50

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series of C-terminal deletions of SIM were constructed that sequentially removed these motifs (Figs. 1 and 6). The Δ104-127 construct lacking NLS2 and the Δ82-127 construct lacking NLS2 and motif C were both capable of complementing the sim trichome phenotype when expressed from the GL2 promoter (Fig. 6, A and B; Ta-ble 3). However, the construct with the longest dele-tion, Δ66-127, lacking all three C-terminal motifs, was unable to complement the sim mutant (Fig. 6C; Table 3), suggesting that, at a minimum, NLS1 might be re-quired for SIM function. All three of these C-terminal deletions were able to interact with CDKA;1 in split- luciferase complementation assays (Fig. 7), consistent with sequences in the C-terminal half of the protein being the primary site of CDK interaction. To test the

functional requirement for the NLSs, mutant SIM constructs were generated in which the basic resi-dues of NLS1, NLS2, or both were replaced by Ala (GL2pro:nls1, GL2pro:nls2, and GL2pro:nls1nls2, respec-tively), and these constructs were introduced into the sim mutant plants. Both individual nls mutants were functional (Fig. 6, D and E). Only the GL2pro:nls1nls2 double motif mutant failed to complement the sim phenotype (Fig. 6F; Table 3).

Nuclear localization of these NLS motif mutants was tested via Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana as GFP fusions (Fig. 8). Wild-type GFP::SIM was found exclusively in the nucleus (Fig. 8A). A GFP fusion of a cytoplasmic carbonic anhydrase (DiMario et al., 2016) served as a control for cytoplasmic localization (Fig. 8B). For the two shortest deletion constructs, Δ104-127 and Δ82-127, GFP fluorescence was observed primarily in the nu-cleus, although the Δ82-127 construct did show some cytoplasmic GFP signal (Fig. 8, C and D). By contrast, GFP fluorescence resulting from the construct with the greatest C-terminal deletion, Δ66-127, was distributed equally between the nucleus and the cytoplasm. When constructs in which only one of the two NLS motifs had been replaced by Ala were expressed transiently in N. benthamiana leaves, GFP fluorescence was detected almost exclusively in the nucleus (Fig. 8, F and G). In contrast, the nls1nls2:GFP construct transformants ex-hibited substantial cytoplasmic fluorescence (Fig. 8H). However, it was apparent that leaves expressing the Δ66-127:GFP construct (Fig. 8H) exhibited greater cyto-plasmic localization in proportion to its degree of nu-clear localization than did the nls1nls2:GFP construct

Figure 3. T35 is necessary for SIM function and is a potential phosphorylation site. Scanning electron micrographs are shown for wild-type Col-0 trichomes (A), multicellular sim mutant trichomes (B), trichomes of sim GL2pro:eYFP:T35A (C), trichomes of sim GL2pro:eYFP:T35D (D), and trichomes of sim GL2pro:eYFP:T35E (E). Bars = 200 μm.

Table 2. Substitution of the T35 residue of SIM with Asp (T35D) can restore function to the SIM T35A mutant

The multicellular trichome phenotype of a complementation line homozygous for a single T-DNA insert for each of the indicated SMRs was assessed by counting the number of DAPI-stained nuclei at each trichome initiation site (TIS) for each genotype. All values followed by the letter a have significantly fewer nuclei per TIS than the sim mutant (P < 1 × 10−6 in a one-tailed Student’s t test, after applying a Bonfer-roni correction for multiple tests). For each transgenic genotype, at least two additional independent homozygous T3 lines were obtained having a phenotype qualitatively equivalent to the line shown here.

Genotype No. of Nuclei per TIS No. of TIS

Col-0 1.02 ± 0.02 a 50sim 2.24 ± 3.00 50sim GL2pro:YFP:SIM 1.00 ± 0.00 a 50sim GL2pro:YFP:T35A 2.16 ± 1.20 50sim GL2pro:YFP:T35D 1.08 ± 0.27 a 50

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(Fig. 8H). This may indicate that C-terminal sequences other than NLS1 or NLS2 located between residues 66 and 104 also contribute to SIM nuclear localization.

DISCUSSION

Motif A in SIM Is a Potential Novel CDK Interaction Motif

Previous work showed that SIM binds to CDKs (Churchman et al., 2006; Van Leene et al., 2010; Kumar et al., 2015), but the region of SIM and other SMRs in-volved in CDK binding has not been identified. Unlike ICK/KRPs, which contain a CDK-binding motif that is conserved across both the animal and plant kingdoms, none of the conserved motifs found in the SMRs resem-ble the sequence of any known CDK-binding motif. The work presented here demonstrates that the ami-no acid residues in motif A are essential for SIM func-tion (Fig. 2E; Table 1; Supplemental Table S1) and that

mutation of the core residues of motif A disrupts CDK binding in a split-luciferase assay, while mutations in motifs B and C have little effect on the ability of SIM to bind CDKA;1 (Fig. 5). Furthermore, a construct lacking all conserved sequence motifs downstream of motifs A and B is still capable of binding to CDKA;1 (Figs. 1 and 7), consistent with motif A being involved directly in CDK binding. Motif A also is the most strongly conserved region of the SMR family, consistent with a crucial role in CKI function. Taken together, these results suggest that motif A potentially is a novel CDK-binding motif essential to the CDK-inhibitory function of the SMR family of CKIs. However, muta-tion of three highly conserved residues at the N ter-minus of motif A did not disrupt the interaction with CDKA, and it was only when all 10 residues of motif A were substituted by Ala that the interaction was disrupted. The recent results of Dubois et al. (2018) indicate that a C-terminal region of SMR1/LGO lack-ing motifs A and B also may be involved in binding to CDKA;1. Perhaps multiple regions of SIM contribute to its binding to CDK. It is clear that more work will need to be done to understand how SIM and other SMRs interact with CDKs.

The role of motif B is less clear. This Pro-rich mo-tif is located close to the C-terminal end of motif A in most SMRs, although with somewhat variable spac-ing between the two motifs (Kumar et al., 2015). Our results show that replacing the initial four successive

Figure 4. SIM T35 substitution mutants produce stable nucleus-localized proteins in developing trichomes. Nucleus-localized eYFP fluores-cence is shown for developing trichomes of sim GL2pro:eYFP:SIM (A), sim GL2pro:eYFP:T35A (B), and sim GL2pro:eYFP:T35D (C) plants. Bars = 10 μm.

Figure 5. A motif A mutant disrupts interactions between SIM and CDKA;1 in a split-luciferase complementation assay. Relative lumi-nescence is shown for the interaction of CLuc:SIM or CLuc fused to various SIM motif mutants with Nluc:CDKA;1 in Arabidopsis proto-plasts. The CLuc:motifA-3As construct contains the substitution T34AT-35AP36A, and the CLuc:motifA-10As construct replaces the motif A residues T35 through P44 with Ala. Interaction of NLuc:Histone2A (NLuc:H2A) and CLuc:Histone2B (CLuc:H2B) was used as a positive control; interactions with fusions of the transcription factor PERIAN-THA (PAN), NLuc:PAN and CLuc:PAN, were used as negative controls. Data are shown for two biological replicates of the experiment. For each biological replicate, n = 4, and the error bars indicate se.

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Pro residues of motif B with Ala abolishes SIM func-tion (Fig. 2G; Table 1), but this mutation also appears to reduce the amount of stable protein produced. In most SMRs, the motif B Pro residues are arranged such that there are one or more instances of the sequence PXXP, followed by one or more basic residues. This pattern is seen in the poly-Pro helix motifs that are found in ligands of SH3 and WW domain-containing proteins (Kay et al., 2000), suggesting that this region may be a protein interaction motif. However, the motifB mu-tant still interacts with CDKA;1 in the split-luciferase assay, so these Pro residues are not necessary for that interaction.

The Conserved Motif C Is Not Essential for SIM Function

Surprisingly, motif C is not essential for SIM func-tion, as assayed by complementation of the sim tri-chome cell division phenotype. This motif is conserved among SMRs, and between SMRs and KRPs, and was shown previously to be required for the binding of a

rice SMR to a d-type cyclin and for inhibition of the kinase activity of CDK complexes from cell extracts (Churchman et al., 2006; Peres et al., 2007). Nonethe-less, both a mutation replacing the six key residues of motif C with Ala (Fig. 2I; Table 1) and a C-terminal de-letion mutant that completely lacks the entire motif C (Fig. 6B; Table 3) are fully able to complement the sim phenotype. Perhaps this motif plays an important role in the interaction of SMRs with cyclins when SMRs are present at low concentrations, but it is unnecessary when SIM is expressed from the strong GL2 promoter. Regardless, our results show that motif C is not abso-lutely required for SIM function despite its evolutionary conservation.

SIM Requires an NLS for Its Function

Our results show that SIM contains two NLSs and that at least one of these NLSs must be present for the biological function and complete nuclear localization of SIM. While NLS2 fits the consensus KR(K/R)R for

Figure 6. Complementation of the sim mutant trichome phenotype by the transgenic SIM C-terminal deletion mu-tants and nls mutants. Scanning electron micrographs are shown for GL2pro: SIMΔ104-127 (A), GL2pro:SIMΔ82-127 (B), GL2pro:SIMΔ66-127 (C), GL2pro: nls1 (D), GL2pro:nls2 (E), and GL2pro: nls1nls2 (F). Bars = 100 μm.

Table 3. Redundant NLSs in the C-terminal end of SIM are required for its full functionThe multicellular trichome phenotype of a complementation line homozygous for a single T-DNA in-

sert for each of the indicated SMRs was assessed by counting the number of DAPI-stained nuclei at each trichome initiation site (TIS) for each genotype. All genotypes followed by the letter a have significantly fewer nuclei per TIS than the sim mutant (P < 1 × 10−9 in a one-tailed Student’s t test, after applying a Bonferroni correction for multiple tests). The genotype followed by the letter b has significantly fewer nuclei than the sim mutant (P < 0.01 in a one-tailed Student’s t test, after applying a Bonferroni correction for multiple tests) but significantly more nuclei per TIS than Col-0 (P < 1 × 10−6 in a one-tailed Student’s t test, after applying a Bonferroni correction for multiple tests). For each transgenic genotype, at least two additional independent homozygous T3 lines were obtained having a phenotype qualitatively equivalent to the line shown here.

Genotype No. of Nuclei per TIS No. of TIS

Col-0 1.00 ± 0.00 a 50sim 2.16 ± 0.98 50sim GL2pro:Δ104-127 1.08 ± 0.27 a 50sim GL2pro:Δ82-127 1.02 ± 0.14 a 50sim GL2pro:Δ66-127 1.84 ± 0.71 50sim GL2pro:nls1 1.04 ± 0.20 a 50sim GL2pro:nls2 1.02 ± 0.14 a 50sim GL2pro:nls1 nls2 1.50 ± 0.74 b 50

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the canonical type 1 monopartite NLSs that bind the major binding site on α-importin, NLS1 resembles a class 4 NLS, which binds to the minor binding site on α-importin (Kosugi et al., 2009). Although NLS1 was originally proposed to be a Cy-type cyclin-binding motif (Churchman et al., 2006), our results (Fig. 6D; Table 3) clearly show that mutating the basic residues of this motif has no effect on the in vivo function of SIM. Furthermore, SIM genes containing several different mutations of individual or multiple residues within this motif retained their function (C72A, R74A, L76A, R74A L76A, C72A R74A L76A, K73A, and K74A; Sup-plemental Table S1). Therefore, our evidence indicates that the NLS1 motif most likely functions primarily or solely in nuclear localization.

The requirement for an NLS to localize SIM to the nucleus is somewhat surprising, given the 14-kD size of the protein, which would be expected to diffuse freely into the nucleus. Indeed, in the GFP::nls1nls2 line, fluorescence is present in both the nucleus and the cytoplasm. It may be that efficient nuclear local-ization is required to achieve sufficient intranuclear concentrations of SIM for its function as a CDK in-hibitor. However, recent work has indicated that SIM has direct interactions with components of the nucle-ar pore complex that play a specific role in pathogen- defense-related functions of SMRs (Wang et al., 2014; Gu et al., 2016). Thus, we cannot rule out the possibility that there is a more specific function for NLSs in SIM

beyond just increasing the protein concentration in the nucleus.

Finally, we noted that the construct Δ66-127 contain-ing a C-terminal deletion, lacking both NLSs as well as motif C, consistently gave a much stronger interaction signal with CDKA;1 than the wild-type protein (Fig. 7), giving a stronger signal than even our histone-pos-itive control. Unlike SIM, CDKA;1 localizes to both the nucleus and cytoplasm (Boruc et al., 2010), and it may be that the stronger signal is due to interaction in the cytoplasm in SIM lacking the NLSs. It is also possible that the C terminus of the protein contains sequences that affect protein stability and that protein concentra-tion is increased in the deletion mutant.

Amino Acid Residue T35 Is a Potential Phosphorylation Site

Kinases drive many of the key cell cycle transitions, and a large fraction of the proteins involved in the cell cycle are regulated by phosphorylation. Thus, the re-semblance of the most conserved residues in the SMR family, represented in SIM by T35 and P36, to a CDK phosphorylation target site was of great interest. Our finding that substitution of T35 by Ala eliminates SIM function (T35A; Fig. 3C) while replacing T35 with Asp

Figure 7. The N-terminal half of SIM remaining in the SIMΔ66-127 construct interacts strongly with CDKA;1 in a split-luciferase comple-mentation assay. Relative luminescence is shown for the interaction of CLuc:SIM or CLuc:SIMΔ66-127 with Nluc:CDKA;1 in Arabidopsis protoplasts. Interaction of NLuc:Histone2A (NLuc:H2A) and CLuc:His-tone2B (CLuc:H2B) was used as a positive control; interactions with fusions of the transcription factor PERIANTHA (PAN), NLuc:PAN and CLuc:PAN, were used as negative controls. Data are shown for two biological replicates of the experiment. For each biological replicate, n = 4, and the error bars indicate se.

Figure 8. Subcellular localization of eGFP:SIM mutant fusion pro-teins transiently expressed in N. benthamiana leaves. A, eGFP:SIM, previously shown to be nucleus localized. B, βCA2:eGFP, known to be cytoplasmically localized. C, eGFP:SIM-Δ104-127 mutant. D, eGFP:SIM-Δ82-127 mutant. E, eGFP:SIM-Δ66-127 mutant. F, eGFP:SIM- nls1 mutant. G, eGFP:SIM-nls2 mutant. H, eGFP:SIM-nls1nls2 mutant. Bars = 50 μm.

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preserves its function (T35D; Fig. 3D) suggests that phosphorylation of this residue may play an import-ant role in SIM function. In principle, phosphoryla-tion could affect SIM function in several ways, such as regulating protein stability, protein conformation, or protein-protein interactions. However, because the motifA-3As mutant, which replaces the three residues T34, T35, and P36 with Ala, is still able to interact with CDKA;1 in the split-luciferase assay (Fig. 5), it is un-likely that phosphorylation of T35 is required for the interaction of SIM with CDKs.

Although T35 is followed by a Pro, suggesting that this residue may be the target of CDK phosphorylation, our previously published work with several Arabidop-sis cyclin-CDK combinations in vitro did not provide evidence that any of these CYC/CDK combinations could phosphorylate SIM (Kumar et al., 2015). However, given that different Arabidopsis CYC/CDK combi-nations vary in their substrate specificity (Harashima and Schnittger, 2012; Nowack et al., 2012) and that a large number of cyclin-CDK combinations have been demonstrated among the 30 cyclins and five CDKs considered central to the cell cycle (Van Leene et al., 2010), it remains possible that SIM is a CDK phosphor-ylation target. Alternatively, SIM may be phosphory-lated either by a noncanonical CYC/CDK combination (Torres Acosta et al., 2004) or by another Thr-Pro- directed kinase such as a MAP kinase (Lu et al., 2002).

One other potential phosphorylation site in SIM also was investigated. In a study published prior to the recognition of the SMR family, it was proposed that a tomato (Solanum lycopersicum) SMR homolog known as SELF-PRUNING-INTERACTING PROTEIN4 was phosphorylated by a NEVER IN MITOSIS A-like ki-nase on the conserved Ser represented in SIM by S120, which immediately follows NLS2 (Pnueli et al., 2001). However, a SIM gene in which S120 was changed to Ala is still functional (Supplemental Table S1). While we cannot rule out the phosphorylation of this residue in vivo, it is clear that phosphorylation of this residue is not essential for SIM function.

CONCLUSION

We identified essential functional regions of SIM, in-cluding the region that may interact with CDKs, and have shown that this region contains a potential phos-phorylation site. We also identified the required NLSs and found that a motif indicated previously as a cyclin interaction motif is less important for SIM function than expected. Many questions remain. It is not clear whether SIM is actually phosphorylated in vivo, and the actual function of the conserved motif C remains to be determined. However, because even distantly related SMRs are able to functionally replace SIM in trichome development (Kumar et al., 2015), it is likely that the results obtained here will apply to the func-tions of these motifs throughout the SMR family.

MATERIALS AND METHODS

Plant Growth, Genotyping, and Phenotyping

Arabidopsis (Arabidopsis thaliana) plants were grown on soil as described previously (Larkin et al., 1999). Col-0 was used as the wild type, and the sim-1 allele, which was isolated in a Col-0 background, has been described previ-ously (Churchman et al., 2006). Mutant sim-1 homozygotes were transformed by the floral dip method (Clough and Bent, 1998). Qualitative assessment of the ability of various mutant transgenes to complement sim-1 when expressed from the GL2 promoter was determined by inspection of a minimum of 12 T2 families per construct for trichome clustering. These T2 lines were screened on soil for lines containing a single BASTA-resistant insert, and the three most strongly complementing lines were used to produce homozygous T3 lines. The presence of the genomic sim-1 point mutation in all transgene complemen-tation experiments was confirmed by PCR genotyping as described previously (Kumar et al., 2015). For the quantitative assessment of complementation, nu-clei per trichome initiation site were counted on first leaves stained with DAPI as described previously (Walker et al., 2000), using a Leica DM6 B upright mi-croscope equipped with differential interference contrast and epifluorescence optics, using the 10× or 20× objective. Fluorescence micrographs in Figure 2 were obtained with a Leica TCS SP8 confocal microscope using an HC PL APO 20×/0.75 objective, with identical settings for laser power and detector gain for all images. Fluorescence micrographs in Figure 4 were obtained using a Leica DM RX2A microscope equipped with differential interference contrast and epifluorescence optics using identical exposure settings for all images. Scanning electron microscopy was carried out on fresh first leaves in high vac-uum mode in a JEOL JSM 6610LV scanning electron microscope as described previously (Kumar et al., 2015).

Construction of Motif Mutants and Transformation Constructs

Site-directed mutants of SIM were created either using a QuickChange II site-directed mutagenesis kit following the manufacturer’s instructions (Agilent) or were ordered as synthetic coding regions (Integrated DNA Technologies). Mutant SIM coding regions were PCR amplified and recloned in pENTR/ D-TOPO (Thermo Fisher). For all constructs, error-free entry clones were confirmed by DNA sequencing prior to inserting genes into Gateway desti-nation vectors via LR Clonase reactions. For complementation experiments, the binary T-DNA destination vector pAMPAT-PROGL2 was used. This vector expresses inserted genes from the GL2 promoter, which is trichome specific in developing leaves (Weinl et al., 2005). For complementation experiments using YFP:SIM fusions, the eYFP coding region was PCR amplified from the vector pSAT6-EYFP-C1 flanked by attB5 and attB2 sites and inserted into the MultiSite Gateway vector pDONR221 P5-P2. SIM mutant coding regions were amplified flanked by attB1 and attB5r sites and inserted into pDONR221 P1-P5r. These donor clones were then introduced into the destination vector pAMPAT-PROGL2 as described by the manufacturer (Thermo Fisher).

Split-Luciferase Protein Interaction Assay

SIM mutants were introduced into the expression vector pDuExDn6 (Fujikawa and Kato, 2007) via Gateway cloning (Thermo Fisher), which creates a fusion with the C-terminal portion of the Renilla reniforms luciferase coding region (Cluc; amino acids 230–311) at the N-terminal end of the SIM gene to create a series of Cluc:SIM mutant fusions. Proper orientation and correct se-quence of the inserts in all constructs were confirmed by sequence analysis. The Nluc:CDKA;1 construct in the vector pDuExAn6, consisting of a fusion of the N-terminal 229 codons of the R. reniforms luciferase coding region (Nluc) to the N terminus of the CDKA;1 coding region, has been described previously (Kumar et al., 2015). Split-luciferase assays using these plasmids were carried out in Arabidopsis protoplasts as described previously (Fujikawa and Kato, 2007; Kumar et al., 2015).

Transient Expression in Nicotiana benthamiana

Transient expression of the constructs of interest in N. benthamiana leaves was enhanced by coexpression with the p19 protein of the Tomato bushy stunt virus. N. benthamiana plants were grown on soil at 21°C with continuous light, and approximately 1-month-old plants were used for transient expression. SIM site-directed mutants and deletions were introduced into the pK7WGF2

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vector (Karimi et al., 2002) via Gateway cloning (Thermo Fisher), and these plasmids were used to transform Agrobacterium tumefaciens strain GV3101 by electroporation. A. tumefaciens cultures of these constructs, along with cultures of A. tumefaciens containing the p19 expression plasmid p19:pKYLX7 (Schardl et al., 1987; Fontenot et al., 2015), were grown to OD600 ∼ 1. Cultures were combined at a ratio of 1 construct:0.5 p19:pKYLX7, centrifuged, washed with 10 mm MgCl2, and resuspended in 2 mL of an infiltration solution (10 mm MES, pH 5.9, 10 mm MgCl2, and 15 m acetosyringone). Leaves were infiltrated through the abaxial side with 1 mL of the suspension, and infiltrated plants were grown for 2 to 3 d prior to microscopy. Images were captured at 40× using a Leica DM RXA2 upright microscope equipped with differential inter-ference contrast and epifluorescence optics.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: Arabidopsis SIM (At5g04470), Arabidopsis CDKA;1 (At3g48750), H2A (At4g27230), H2B (At5g22880), and PAN (At1g68640).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Mutating the potential phosphorylation sites T50 and T63 to Ala does not affect SIM function.

Supplemental Table S1. Mutant constructs tested for functional complementation.

ACKNOWLEDGMENTS

We thank Robert Dimario for the gift of the βCA2:eGFP plasmid, Maheshi Dassanayake for critical reading of the article, and the staff of the Socolofsky Microscopy Facility, part of the Shared Instrumentation Facility at Louisiana State University, for assistance with microscopy. This work is dedicated to the memory of L. Alice Simmons, who provided expert technical assistance during the first part of this work and passed away before the work was completed.

Received February 6, 2018; accepted June 5, 2018; published June 14, 2018.

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