Article Deceleration of the cell cycle underpins a switch from proliferative to terminal divisions in plant stomatal lineage Graphical abstract Authors SoonKi Han Arvid Herrmann Jiyuan Yang Crisanto Gutierrez EunDeok Kim Keiko U Torii Correspondence ktoriiutexasedu In brief Stomata which are cellular valves in the plant epidermis differentiate via fast asymmetric divisions of a precursor followed by a single slower symmetric division Han et al identify a plantspecific cyclindependent kinase inhibitor that regulates the length of cell cycles in the stomatal lineage to enable the transition from proliferation to differentiation Highlights d During stomatal differentiation asymmetric divisions are faster than terminal divisions d Upon commitment to differentiation MUTE induces the cellcycle inhibitor SMR4 d SMR4 decelerates the asymmetric cell division cycle via selective binding to cyclin D d Regulating duration of the G1 phase is critical for epidermal cell fate specification Han et al 2022 Developmental Cell 57 569582 March 14 2022 2022 The Authors Published by Elsevier Inc httpsdoiorg101016jdevcel202201014 ll ll OPEN ACCESS Article Deceleration of the cell cycle underpins a switch from proliferative to terminal divisions in plant stomatal lineage SoonKi Han127 Arvid Herrmann34 Jiyuan Yang4 Rie Iwasaki1 Tomoaki Sakamoto5 Benedicte Desvoyes6 Seisuke Kimura5 Crisanto Gutierrez6 EunDeok Kim34 and Keiko U Torii1348 1Institute of Transformative BioMolecules WPIITbM Nagoya University Nagoya Aichi 4648601 Japan for Advanced Research IAR Nagoya University Nagoya Aichi 4648601 Japan 3Howard Hughes Medical Institute The University of Texas at Austin Austin TX 78712 USA 4Department of Molecular Biosciences The University of Texas at Austin Austin TX 78712 USA 5Department of Industrial Life Sciences and Center for Plant Sciences Kyoto Sangyo University Kyotoshi Kyoto 6038555 Japan 6Centro de Biologia Molecular Severo Ochoa Nicolas Cabrera 1 Cantoblanco 28049 Madrid Spain 7Present address Department of New Biology DGIST Daegu Gyeongbuk Institute of Science and Technology Daegu 42988 Republic of Korea 8Lead contact Correspondence ktoriiutexasedu httpsdoiorg101016jdevcel202201014 2Institute SUMMARY Differentiation of specialized cell types requires precise cellcycle control Plant stomata are generated through asymmetric divisions of a stemcelllike precursor followed by a single symmetric division that creates paired guard cells surrounding a pore The stomatallineagespecific transcription factor MUTE terminates the asymmetric divisions and commits to differentiation However the role of cellcycle machineries in this transition remains unknown We discover that the symmetric division is slower than the asymmetric division in Arabidopsis We identify a plantspecific cyclindependent kinase inhibitor SIAMESERELATED4 SMR4 as a MUTEinduced molecular brake that decelerates the cell cycle SMR4 physically and functionally associates with CYCD31 and extends the G1 phase of asymmetric divisions By contrast SMR4 fails to interact with CYCD51 a MUTEinduced G1 cyclin and permits the symmetric division Our work unravels a molecular framework of the proliferationtodifferentiation switch within the stomatal lineage and suggests that a timely proliferative cell cycle is critical for stomatallineage identity INTRODUCTION Growth and development of multicellular organisms rely on faithful cellcycle progression in which fundamental mechanism is highly conserved across the eukaryote kingdoms Elledge 1996 Harashima et al 2013 Accumulating evidence in metazoans emphasizes that cellcycle machinery is modulated during development operating distinctly in proliferating stem cells versus differentiating cells Budirahardja and Gonczy 2009 For example early embryogenesis of flies fish and frogs as well as murine embryonic stem cells execute rapid cellcycle mode due to shortened gap phases As they undergo fate specification or differentiation the duration of cellcycle increases Coronado et al 2013 Dalton 2015 Liu et al 2019 A typical eukaryotic cell cycle is composed of four distinct phases G1SG2M Cellcycle progression is driven by the oscillation of cyclindependent kinase CDK activity triggered by phasespecific cyclins which are tightly regulated by the level of synthesis and proteolysis Harashima et al 2013 Morgan 2007 CDK activity is negatively regulated by cyclindependent kinase inhibitors CKIs The G1S transition is initiated by Dtype cyclin CyclinD and CDK complex which relieve retinoblastoma Rbmediated repression on S phase gene expression Bertoli et al 2013 Desvoyes and Gutierrez 2020 Accumulating studies suggest that the G1 extension is indicative of differentiation Coronado et al 2013 Liu et al 2019 Plants possess a large number of genes encoding cyclins CDKs and CKIs Inze and De Veylder 2006 Studies have shown how specific cellcycle components are coupled to developmental patterning For example during Arabidopsis root development transcription factors SHORTROOT and SCARECROW directly induce a CyclinD CYCD61 that drives a formative cell division to create root endodermis and cortex cells Sozzani et al 2010 Another example is lateral root formation in which auxininduced formative division is modulated by CYCD21 and plant CKI KIPRELATED PROTEIN2 KRP2 also known as ICK2 Sanz et al 2011 Some highly specialized plant cell types such as epidermal pavement cells and trichomes Developmental Cell 57 569582 March 14 2022 2022 The Authors Published by Elsevier Inc 569 This is an open access article under the CC BYNCND license httpcreativecommonsorglicensesbyncnd40 ll OPEN ACCESS undergo endoreduplication at the onset of terminal differentiation Inze and De Veylder 2006 Plantspecific CKIs including SIAMESE SIM and SIAMESERELATED1 SMR1 also known as LGO regulate morphogenesis of trichomes and sepal giant cells respectively by promoting endoreduplication presumably via inhibiting CDK activity Hamdoun et al 2016 Roeder et al 2010 Walker et al 2000 It remains unclear if the modulation of cell cycle contributes to switching the cell division mode from stem cell divisions to differentiating cell divisionsin plants Development of stomata valves on the plant aerial epidermis for gas exchange and water control is an accessible model of de novo initiation and differentiation of lineagespecific stem cells In Arabidopsis birth of pores begins with the stomatal lineage fate specification of protodermal cells which forms bipotent meristemoid mother cells MMC able to become either stomata or pavement cells Han and Torii 2016 Lau and Bergmann 2012 A series of asymmetric cell division ACD follows to amplify the number of stomatal lineage precursor cells meristemoids and stomatal lineage ground cells SLGCs The meristemoid renews itself after ACD thus behaving as a transient stem cell After a few rounds of ACDs a single round of terminal symmetric cell division SCD of a guard mother cell GMC proceeds completing a stoma composed of paired guard cells Han and Torii 2016 Lau and Bergmann 2012 Figure 1A Accumulating evidence supports that masterregulatory bHLH proteins SPEECHLESS SPCH MUTE and FAMA govern cellstate transitions within the stomatal lineage in part via directly regulating the expression of cellcycle genes Adrian et al 2015 Hachez et al 2011 Han et al 2018 Lau et al 2014 Figure 1A SPCH initiates and sustains the ACDs of a meristemoid in part via upregulating CyclinDs CYCD31 and 32 MacAlister et al 2007 Vaten et al 2018 MUTE terminates proliferative cell state and drives final SCD by activating a large subset of cellcycle regulators including CYCD51 Han et al 2018 Pillitteri et al 2007 FAMA and a Myb protein FOUR LIPS are directly induced by MUTE and inhibit SCD via direct suppression of the cellcycle genes thereby ensuring that the SCD occurs just once Hachez et al 2011 Han et al 2018 Xie et al 2010 However it is not known how proliferative ACD switches to terminal SCD and whether the core cellcycle machinery contributes to this process Through timelapse imaging of stomatal development using plant cellcycle marker Plant CellCycle Indicator PlaCCI Desvoyes et al 2020 we discovered that the stomatal SCD cycle is slower than that of ACDs Subsequent transcriptomic and ChIPsequencing analyses identified that MUTE directly induces the expression of SMR4 during meristemoidtoGMC transition Through lossoffunction and stomatallineagespecific overexpression of SMR4 as well as its functional interaction studies with CyclinDs we elucidate that SMR4 acts as a molecular brake to decelerate cell cycle in G1 phase to ensure termination of the ACD cycle and facilitate faithful progression to SCD Slowing down the ACD cycle resulted in skewed stomata with pavement celllike characters Taken together we reveal a molecular framework of the cell proliferationtodifferentiation switch within the stomatal lineage and suggest that a timely proliferative ACD cycle is critical for the generation of stomata with proper GC size shape and identity 570 Developmental Cell 57 569582 March 14 2022 Article RESULTS The single symmetric division of stomatal precursor is slower than amplifying asymmetric division The stomatal precursor cells execute a unique transition from amplifying ACD to a single SCD a step coordinated by the bHLH protein MUTE Figure 1A To understand if a switch from the ACDtoSCD division mode links to the cellcycle dynamics we first performed timelapse imaging of developing cotyledon epidermis by using the multicolor PlaCCI Desvoyes et al 2020 Figure 1C and examined each phase of cell cycle during asymmetric and symmetric divisions Figures 1B 1D and 1E Video S1 The average cellcycle time of ACD of meristemoid and SCD of GMC was 12 164 and 2027 373 h respectively Figure 1B Table S1 indicating that ACD is faster by 75 h than SCD that creates a pair of guard cells Measuring cell division time using a plasmamembrane GFP marker LTI6b Kurup et al 2005 yielded essentially the same results Figure S1 On the basis of these findings we conclude that the switching from ACD to SCD involves cellcycle slow down SMR4 is expressed in stomatal lineage cells and directly induced by MUTE In eukaryotic cells CKIs negatively regulate cellcycle progression To identify a factor that plays a role in the decelerating cell cycle during the ACDtoSCD transition we surveyed publicly available transcriptome data Han et al 2018 Lau et al 2014 to search for CKIs that are induced by SPCH and MUTE Among the 7 KRP and 17 SIMSMR genes Kumar and Larkin 2017 Peres et al 2007 only SMR4 exhibits marked increase by the induced MUTE overexpression iMUTE Figure 2A On the other hand a majority of SMRs and KRPs is either downregulated or unchanged upon iMUTE or iSPCH Figure 2A Subsequently we performed time course induction analysis Consistent with the transcriptome data SMR4 expression was increased by iMUTE with similar kinetics to a known direct MUTE target TMM Han et al 2018 Figure 2B In addition iMUTE and in a lesser extent iSPCH weakly induced SMR8 Figure S2 Our previous transcriptome study Han et al 2018 found that MUTE induces a suite of cellcycle and mitoticdivisionrelated genes driving the SCD of stomata To test whether these genes are indeed direct MUTE targets we performed genomewide MUTE ChIPsequencing see STAR Methods Table S2 MUTEbound genes as well as those MUTEbound genes that are induced by MUTE are highly enriched in the Gene Ontology GO categories Figure 2C Table S2 mitotic cellcycle phase 8322fold enrichment p 215e02 ACD 1891fold enrichment p 24e02 and other cellcyclemitosisrelated categories Figure 2C pink bars as well as the genes involved in stomatal development GMC differentiation 3783fold enrichment p834e04 and stomatal complex development 227fold enrichment p 175e12 Figure 2C cyan bars Strong MUTEbound peaks are detected at the 50 and 30 regions of known MUTE target loci ERL1 TMM EPF2 and CDKB11 Figure 2D Most importantly MUTE robustly bound to the 50 region of SMR4 indicating that SMR4 is a direct MUTE target As expected no MUTE binding peak was detected in SMR1 loci ll Article OPEN ACCESS Figure 1 Cellcycle duration between asymmetric cell division and symmetric cell division during stomatal development A Cartoon of the heterodimeric transcription factors specifying stomatal development A series of ACD is triggered by SPCHSCRM2 and a single symmetric cell division SCD is coordinated by MUTESCRM2 and FAMASCRM2 How the mode of cell cycle switches from ACD to SCD is not known red line and question mark MMC meristemoid mother cell M meristemoid GMC guard mother cell imGC and GC immature and mature guard cell B Duration of the cellcycle time of stomatal precursors undergoing ACD and SCD in wild type n 15 for each cell division mode Twotailed Student t test was performed p 2129e07 C PlaCCI color code Cyan CDT1aCFP signal onset of G1 phase black short period with no fluorescence signal magenta HTR13mCherry signal SG2 through late M orange CYCB11YFP signal Postmitotic referred to G1 or G0 terminal state D and E Representative timelapse confocal images of ACD D and SCD E in stomatal lineage cells from 1 to 3dayold cotyledon of Col0 expressing both PlaCCI and LTi6B green CDT1aCFP signal cyan marks the starting point 0 h at the onset of G1 phase for ACD and SCD Note that the CYCB11YFP D green nucleuschromosomes M phase is not always visible due to timelapse recordings obtained at 30 min time intervals Arrows point to nuclei with a fluorescent signal in different cell stages Scale bar 10 mm See also Figure S1 and Table S1 which is not induced by iMUTE and thus not a direct MUTE target Figures 2B and 2D We further characterized the SMR4 expression patterns using seedlings expressing nuclearlocalized GFP driven by the SMR4 promoter proSMR4nucGFP Figure 2E A strong GFP signal was detected in stomatal lineage cells with the highest expression in a late meristemoid to GMC and persisted in immature GCs Figure 2E Likewise a translational reporter of SMR4 YFP fusion protein driven by the SMR4 promoter proSMR4SMR4YFP exhibited similar accumulation patterns predominantly in the nuclei Figure 2F arrows A weak SMR4YFP signal was also detected in the cytoplasm Figure 2F asterisks which may imply the regulation of SMR4 proteins These expression patterns mirror that of MUTE Pillitteri et al 2007 Finally to address whether MUTE is required for the SMR4 expression during the meristemoidtoGMC transition we examined the SMR4 reporters in the MUTEnull mutant mute2 Pillitteri et al 2008 Figure S3 SMR4YFP was not detected in the arrested mute2 meristemoids and transcriptional reporter proSMR4nucGFP signals were diminished Figure S3 Combined our results indicate that MUTE directly promotes the SMR4 expression in stomatal precursor cells before the onset of the SCD and that MUTE is both necessary and sufficient for this boosted expression We also noted weak backgroundlevel of nucGFP signals in few meristemoids Figure S3B implying a putative role for SMR4 in a MUTE independent process The SMR4 expression suggests its distinct role from that of canonical CKIs in endorduplication Developmental Cell 57 569582 March 14 2022 571 ll OPEN ACCESS Article Figure 2 SMR4 one of the plantspecific CKIs expresses in stomatal lineage and is a direct target of MUTE A Heatmap represents the changes in expression of 24 CKIs in Arabidopsis by SPCH or MUTE induction RNAseq data adapted from Lau et al 2014 iSPCH and Han et al 2018 iMUTE Heatmap denotes log2 ratio of changes in expression compared with noninduced control B Time course expression for 12 h with 2h interval of SMR4 and SMR1 by iMUTE monitored by qRTPCR TMM was used as a positive control for a MUTE inducible gene est 10 mM estradiol treated mock nontreated control DMSO only Data are presented as mean SEM C GO categories of direct MUTE targets MUTE bound iMUTE up ranked by fold enrichment compared with background genome p 005 Pink bars cell cycle division mitotic categories blue bars stomatal categories gray bars others D IGV snapshots of ChIPseq profile of MUTE binding to the promoters of SMR4 SMR1 and known MUTE targets ERL1 TMM EPF2 and CDKB11 Han et al 2018 Qi et al 2017 No MUTE binding was detected to SMR1 loci A green arrow under the gene annotation indicates gene orientation and transcriptional start sites E and F Expression patterns of SMR4 transcriptional and translational reporters proSMR4nucGFP E and proSMR4SMR4YFP F in stomatal lineage precursor cell specific on the epidermis White arrows nuclei with GFP or YFP signal Asterisks cytoplasmic YFP signal Scale bar 10 mm See also Figures S2 and S3 and Table S2 572 Developmental Cell 57 569582 March 14 2022 ll Article OPEN ACCESS Figure 3 smr4 CRISPR knockout mutants produce smaller cells the phenotype is enhanced by smr8 A Abaxial cotyledon epidermis from 4dayold seedlings of wild type smr41cr smr42cr smr81 smr82 and smr41cr smr81double mutant Epidermal cells size is color coded as a color scale at bottom GCs are marked in black Scale bar 100 mm B Bar graphs showing the percentage of each category of cell area rightmost from the images for the genotype presented in A GCs are not included in the category of cell area CE Density of stomatal precursor cells meristemoidGMC C stomata D and total epidermal cell E 10 mm2 area for the genotypes shown in A Oneway ANOVA followed by Tukeys post hoc test was performed for comparing all genotypes Different letter denotes significant difference Double letter denotes insignificance p 005 or p 001 The number of plants from each genotype WT n 10 smr41cr n 6 smr42cr n 6 smr81 n 7 smr82 n 8 smr41cr smr81 n 10 See also Figure S4 SMR4 suppresses cell proliferation in part with SMR8 To understand the role of SMR4 in stomatal development we next sought to characterize its lossoffunction phenotypes Because no TDNA insertion line is available for SMR4 presumably owing to its short coding sequence 219 bp we employed CRISPRCas9 system Tsutsui and Higashiyama 2017 see STAR Methods A guide RNA targeting to SMR4 yielded either a basepair deletion smr41cr or insertion smr42cr at 80 bp from the translation start site which leads to a frameshift and premature stop codon Figures S4A and S4B A quantitative analysis of segmented epidermal cells see STAR Methods revealed that smr4cr epidermis is increased in small cells 50 mm2 and concomitantly decreased in large pavement cells 4000 mm2 Figures 3A 3B and 3E Stomatal precursor cell meristemoid and GMC density is also increased in smr4cr alleles Figure 3C On the other hand stomatal density was not significantly changed in smr4cr Figure 3D suggesting that SMR4 primarily restricts the divisions of early stomatal precursor cells Introduction of functional SMR4 transgene proSMR4HASMR4 fully rescued the phenotypes of smr41cr Figures S4DS4G indicating that increased numbers of stomatal precursor cells in smr4 mutant are due to the loss of function of SMR4 Because SMR8 expression was marginally increased by iMUTE Figure S2 we further characterized the lossoffunction phenotypes of SMR8 Two TDNA insertion lines smr81 and smr82 accumulate a reducedlevel of SMR8 transcripts Figure S4C Like smr4 smr8 mutants conferred an increase in small epidermal and stomatal precursor cells Figures 3AC and 3E and the total epidermal cell numbers become most exaggerated in the smr4 smr8 double mutant Figure 3E Therefore SMR4 plays a role in repressing ACD in part redundantly with SMR8 Stomatallineagespecific expression of CKIs reveals their unique functions Our study revealed that SMR4 is a direct MUTE target and expresses during the transition from proliferating meristemoid state Developmental Cell 57 569582 March 14 2022 573 ll OPEN ACCESS Article Figure 4 Stomatal lineage overexpression phenotype of a suite of CKIs reveal their unique activities AE Epidermal phenotype of abaxial cotyledons from 4dayold wild type A proPOLARSMR4 B proPOLARSMR8 C proPOLARSMR1 D and proPOLARKRP1 E Scale bars 50 mm Insets enlarged mature guard cell and precursor cells from each genotype Scale bars 20 mm Orange asterisks undivided singlecelled stomata Pink brackets skewed stomata FI Quantification of epidermal cell number of abaxial cotyledon from 4dayold wildtype and transgenic plants Stomatal index F stomatal density G total epidermal cells H and fraction of normal light green skewed purple and singlecelled stomata pink found on each genotype I in 10mm2 area Oneway ANOVA with Tukeys post hoc test was performed to compare all genotypes The number of plants from each genotype WT n 6 proPOLARSMR4 n 16 proPOLARSMR8 n 19 proPOLARSMR1 n 13 proPOLARKRP1 n 14 See also Figure S5 to differentiating GMC state SMR proteins are known to promote endoreduplication in trichomes pavement cells and sepal giant cells Hamdoun et al 2016 Kumar and Larkin 2017 Roeder et al 2010 Walker et al 2000 However unlike trichomes and pavement cells stomatal lineage cells do not undergo endoreduplication Melaragno et al 1993 We thus hypothesized that SMR4 may function differently from the other canonical SMRs To address this SMR4 SMR8 and SMR1 along with KRP1 are ectopically expressed in the stomatal lineage cells MMC meristemoids and SLGC by using POLAR promoter Pillitteri et al 2011 Figures 4 and S5 Unlike SMR1 or KRP1 POLARpromoterdriven SMR4 and SMR8 did not significantly changed stomatal index number of stomatanumber of stomata and nonstomatal epidermal cells 3100 Figure 4F reflecting reduction in the number of both stomata and epidermal cells Figures 4G and 4H Whereas GMCs of POLARpromoterdriven SMR4 executed SCD they occasionally formed stomata 574 Developmental Cell 57 569582 March 14 2022 composed of skewed guard cells Figure 4B pink bracket Figures 4I and S5 This suggests that SMR4 does not inhibit the final SCD per se Similar deformed stomata were also observed in proPOLARSMR8 Figure 4C pink bracket Figures 4I and S5 Unlike SMR4 ectopic stomatal lineage expression of SMR1 and KRP1 displayed cell division defects with unique consequences proPOLARSMR1 produced abnormally large undivided GMClike cells Figure 4D asterisks Figure S5 which constitute over 60 of the all stomata Figure 4I This result is consistent with the known role of SMR1 in suppressing the activity of CDKB11 thereby promoting endoreduplication Kumar et al 2015 Finally proPOLARKRP1 severely inhibited the ACDs resulting in epidermis vastly consisted of pavement cells with low stomatal index Figures 4E and 4F resembling spch mutant MacAlister et al 2007 Pillitteri et al 2007 Among those proPOLARKRP1 stomata approximately onethird were deformed Figure 4I ll OPEN ACCESS Article Figure 5 Stomatal lineage overexpression of SMR4 reduces proliferative activity of meristemoids A and B A proTMMGUSGFP abaxial cotyledon from 4day postgermination stage seedling 4 dpg B proTMMGUSGFP in proPOLARSMR4 4dpg Scale bar 20 mm Insets zoomed stomatal lineage cells expressing GFP Scale bar 10 mm C Size distribution versus circularity of the stomatal lineage precursor cells expressing proTMMGUSGFP in wildtype green dots and proPOLARSMR4 purple dots plants DI Confocal images of representative stomata wildtype stoma D mixed fate stoma developed proPOLARSMR4 E proMUTEnYFP in proPOLARSMR4 F mature GC marker E994 in wild type G and proPOLARSMR4 H and I Cyan arrowheads division site of GCs Scale bars 20 mm legend continued on next page Developmental Cell 57 569582 March 14 2022 575 ll OPEN ACCESS The phenotype of proPOLARSMR4 and SMR8 is consistent with the diminished proliferative activity of meristemoids To uncouple formative step from differentiation we introduced proPOLARSMR4 into mute mutant in which a number of amplifying ACDs are increased and meristemoids arrest Pillitteri et al 2007 Figure S5 Indeed proPOLARSMR4 significantly reduced the number of ACDs resulted in low number of larger meristemoids Figures S5JS5N Since MUTE is absolutely required for GMC identity Pillitteri et al 2007 these enlarged meristemoids never became stomata Taken together we conclude that SMR4 to some extent SMR8 possesses a unique feature different from canonical CKIs to specifically terminate but not fully inhibit ACDs of meristemoids but allow final SCD to proceed in GMC Furthermore the formation of skewed irregularshaped stomata some resembling pavement cells Figure 4B implies that excessive SMR4 activity disrupts guard cell morphogenesis SMR4 balances between cell proliferation and differentiation To further understand the identity of abnormally shaped stomata observed in proPOLARSMR4 stomatal lineage markers were introduced In wild type stomatal lineage precursor marker proTMMGUSGFP Nadeau and Sack 2002 was detected in stomatal lineage cells with the brightest signal in triangular shaped meristemoids Figure 5A Surprisingly both stomatal lineage cells and enlarged pavement celllike cells in proPOLARSMR4 plants expressed proTMMGUSGFP Figures 5B and S6 Some of these GFPexpressing cells in proPOLARSMR4 plants divide symmetrically even without ACD or after a single round of ACD with significantly extended duration Figures 5B 5E and 5F cyan arrows Figure S6 pink arrows Video S2 Further quantitative analysis shows that compared with wild type these proTMMGUSGFPpositive cells in proPOLARSMR4 are greater in size range 50800 mm2 and in addition have low circularity Figure 5C This might reflect mixed cell fate pavement cell shape with stomatal identity Some of these cells express stomatal fate commitment marker proMUTEnucYFP Figure 5F and finally differentiate into mature guard cells Figures 5G5I exhibiting large wavy and skewed shape Figures 5H and 5I but expressing a mature guard cell marker E994 Thus the large skewed GCs in proPOLARSMR4 originate from the enlarged stomatal lineage precursors caused by delayed and fewer ACD cycles Figure S6 Video S2 To address whether these enlarged GCs undergo endoreduplication process when SMR4 is ectopically expressed the DNA content was measured using DAPI fluorescence Figures 5J5L Half of GC populations exhibited the fluorescence similar to the wild type the median value is 15 mm2 in wild type and 20 mm2 in proPOLARSMR4 plants whereas some showed fluorescence values nearly doubled in proPOLARSMR4 plants Figures 5JL Likewise the GC nuclei size measurements using H2BGFP Maruyama et al 2013 are consistent with the DAPI Article measurements Figures S7AS7D The difference in nuclei size was more pronounced in pavement cells Furthermore none of the GCs expresses the endoreduplication marker proSMR1nlsGFPGUS Bhosale et al 2018 regardless of the GC size in proPOLARSMR4 plants whereas pavement cells where endoreduplication normally occurs express GFP signal Figure 5M Combined these results suggest that stomatal lineage overexpression of SMR4 may confer doubled DNA content probably due to cellcycle arrest in G2 after the S phase Unlike SMR1 however SMR4 does not trigger the endoreduplication cycle in the stomatal lineage This feature distinguishes SMR4 from the known SIMSMRfamily of CKIs The unique noncanonical activity of SMR4 is also supported by systematic stomatal lineage overexpression of selected CKIs where only SMR1 generated huge undivided GMC cells Figures 4D and S5 Indeed quantitative analysis showed that the nuclear size of proPOLARSMR1 GCs is 10 times larger than that of the control wildtype plants Figures S7ES7H consistent with the known role of SMR1 in triggering endoreduplication Hamdoun et al 2016 Kumar et al 2015 Schwarz and Roeder 2016 SMR4 decelerates cellcycle progression by G1 phase extension We elucidated that stomatal lineage ACDs are faster than the final SCD Figures 1B and 1C What is the ramification of stomatal lineage overexpression or lossoffunction mutation of SMR4 on cellcycle duration of ACDs and SCD To address this question we introduced PlaCCI to proPOLARSMR4 and smr41cr mutant plants and performed timelapse imaging Figure 6 Videos S3 and S4 The stomatal precursor cells meristemoids in proPOLARSMR4 seedlings showed extended ACD cycle duration from 1200 to 1847 h Figures 6A 6B and 6G Table S1 This made the ACD cycle duration statistically nonsignificant from that of the SCD Figure 6A Further analysis of cellcycle phase emphasized the striking extension of G1 phase as determined by the time window from the onset of CDT1aCFP expression to HTR13mCherry expression Desvoyes et al 2020 Figures 6B and 6C from average of 373 to 797 h Figure 6I Video S3 Table S1 In contrast the cellcycle duration of the SCD was not significantly affected by proPOLARSMR4 1957 h Figures 6A and 6C Video S3 Table S1 During the ACD in smr41cr mutant the G1 phase became shortened by 1 h compared with wild type Figures 6E and 6I Table S1 Video S4 WT 373 h versus smr41cr 273 h while the S G2 and M phases remained unchanged Table S1 indicating that in the absence of SMR4 the ACD cell cycle becomes accelerated Again the cellcycle duration or the G1 phase duration of SCD was not significantly changed by the smr41cr mutation Figures 6D 6F 6H and 6J Video S4 Table S1 Taken together the results highlight that SMR4 is both necessary and sufficient to slow down cellcycle progression by G1 phase extension to prevent the further occurrence of ACDs once stomatal differentiation has been committed J and K DAPIstained nuclei in mature GCs from wildtype J and proPOLARSMR4 plants K L Quantitative analysis of DAPIstained nuclear area in wildtype and proPOLARSMR4 GCs Twotailed Students t test was performed p 312e32 M Endoreduplication marker proSMR1GFPGUS expression in proPOLARSMR4 plants Cyan arrows indicate enlarged GCs with no GFP expression Scale bar 50 mm See also Figures S6 and S7 576 Developmental Cell 57 569582 March 14 2022 ll OPEN ACCESS Article Figure 6 SMR4 slows down the cellcycle progression of ACD through G1 extension A and D Cellcycle duration of ACD and SCD measured by PlaCCI in proPOLARSMR4 A and smr41cr D n 15 for each cell division mode B C E and F Representative timelapse confocal images of ACD and SCD in stomatal lineage cells from 1 to 3dayold cotyledon of proPOLARSMR4 B and C and smr41cr E and F expressing PlaCCI Cell outlines green for proPOLARSMR4 B and C were handdrawn based on digital overexposure of confocal legend continued on next page Developmental Cell 57 569582 March 14 2022 577 ll OPEN ACCESS Physical and functional interactions of SMR4 with Dtype cyclins underscore the switch from ACD to SCD SIM is known to interact with CYCA23 to promote endoreduplication Wang et al 2020 Unlike SIM stomatal lineage overexpression of SMR4 can extend G1 cycles of ACD but allow execution of the final SCD Figures 4 5 and 6 We thus predict that SMR4 regulates the G1S transition via associating with CyclinDs To understand the mode of action of SMR4 we first surveyed publicly available proteinprotein interactome data Szklarczyk et al 2019 including a largescale Arabidopsis in vivo massspectrometrybased interactome profiling of cellcycle components through tandemaffinity purificationbased technology Van Leene et al 2010 The known SMR4 interactors include major components of cellcycle progression CSK1 CKS2 CDKA1 CDC2 CYCD21 CYCD31 and CYCD71 Van Leene et al 2010 Figure 7A Among them CYCD31 activity rises when cell reenter the cell cycle RiouKhamlichi et al 1999 and is highly induced by SPCH Adrian et al 2015 whereas CYCD71 is involved in the SCD of GMC Weimer et al 2018 Our yeast twohybrid Y2H analysis shows that consistent with the interactome data Figure 7A SMR4 associates with CYCD31 and CYCD71 Figure 7B In contrast no interaction was observed for SMR4 and CYCD51 a direct MUTE target initiating the single SCD Han et al 2018 Figure 7B We also did not observe the direct interaction of SMR4 with CDKA1 or CDKB11 Figure 7B Next to address the biological significance of SMR4 interactions with CYCD31 and CYCD71 but not with CYC51 we examined the effects conferred by stomatal lineage overexpression of three CyclinDs in the presence or absence of functional SMR4 As shown in Figures 7C and 7D in the absence of SMR4 POLARpromoterdriven expression of CYCD31 and CYCD71 exaggerated the ACDs resulting in significant increase in the density of stomatal precursor cells Importantly proPOLARCYCD31 did not influence the stomatal precursor cell density in wild type which carries functional SMR4 whereas the ratio of the precursor cell density between wild type and smr41cr became greater in the presence of proPOLARCYCD31 124 to 165 These results suggest that increase of stomatal precursors by CYCD31 requires the absence of SMR4 Figure 7D By contrast proPOLARCYCD51 increased the stomatal precursor cells regardless of the presence or absence of SMR4 indicating that CYCD51 activity is SMR4independent Figures 7C and 7D On the basis of these findings we conclude that SMR4 can suppress the stomatal lineage divisions by direct association with CYCD31 and possibly with CYCD71 but not with CYCD51 and this differential interaction with CyclinDs underscores the transition from proliferative ACDs to final SCD Figure 7E DISCUSSION In this study we discovered that proliferative ACDs has faster cellcycle duration than the single terminal SCD within the sto Article matal cell lineages A subsequent genomewide profiling of MUTE targets followed by phenotypic and functional characterizations identified SMR4 as a noncanonical CKI that sets a cellcycle brake to facilitate transition from ACD to SCD SMR4 is a direct MUTE target specifically induced by MUTE but not by SPCH Figure 2 thus highlighting the orchestration of cellstate switch from proliferation meristemoids to differentiation stomata at the control of cellcycle duration This view is further supported by the findings that prolonged G1 phase specifically during the proliferative ACDs by stomatal lineage overexpression of SMR4 causes misspecification of guard cells Figures 4 and 5 In contrast of SIM and SMR1 known regulators of endoreduplication Roeder et al 2010 Wang et al 2020 we found that SMR4 delays the G1S transition during stomatal ACDs Figure 6 It has been shown that SIM associates with CYCA23 but not with CYCD31 Wang et al 2020 Assuming that SMR1 functions similarly to SIM the enlarged singlecelled stomata conferred by the stomatal lineage overexpression of SMR1 Figure 4 can be attributed to the direct inhibition of CYCA23CDKB1 complex by SMR1 Indeed higherorder mutations in CYCA2s cycA21 22 23 triple mutant as well as the dominantnegative inhibition of CDKB11 exhibit the identical singlecelled stomata phenotypes Boudolf et al 2004 Vanneste et al 2011 By contrast we found that SMR4 functionally associates with CyclinDs Figure 7 Thus distinct functions among SIMSMRs lie in their unique interaction potential with different cyclinCDK complexes During mammalian cell cycle a series of CKIs exhibit inhibitory roles during G1S transition via associating with CyclinD123CDK46 and then with CyclinECDK2 complexes Sherr and Roberts 2004 Among them p27KIP1 can bind with multiple cyclinCDK complexes and exert different regulatory effects on each complex Sherr and Roberts 2004 Plants lack CyclinE but the previous largescale expression analysis of cellcycle genes suggests that the plant CYCDs adopt both metazoan CyclinD and CyclinE functions Menges et al 2005 That SMR4 binds with different CyclinDs to negatively regulate G1S phase therefore echoes its functional parallel to metazoan CKI p27KIP1 How could SMR4 decelerate cell cycle in proliferative ACDs but not in terminal SCD Our results suggest that the specificity lies on preferential association of SMR4 with different CyclinDs each with a unique expression pattern within the stomatal cell lineages For example CYCD31 and CYCD32 are induced by SPCH and promote ACDs Adrian et al 2015 Dewitte et al 2007 Han et al 2018 Lau et al 2014 By contrast CYCD51 is directly induced by MUTE to drive the terminal SCD Han et al 2018 CYCD71 is likely involved in the terminal SCD however its expression starts later and persists longer than CYCD51 Han et al 2018 Weimer et al 2018 Based on the physical and functional associations of SMR4 with CYCD31 and CYCD71 but not with CYCD51 we propose the following model of regulatory circuit driving the asymmetrictosymmetric images LTi6B green are introduced into smr41cr E and F For the color code and time setting see Figure 1 legends White arrows indicate the nuclei with fluorescent signal Pink arrows indicate the nucleus of a sister cell from the prior round of ACD Scale bar 10 mm G and H Cellcycle duration of ACD G and SCD H among WT smr41cr and proPOLARSMR4 I and J G1 phase duration of ACD I and SCD J among WT smr41cr and proPOLARSMR4 A D G H I and J Twotailed Students t test was performed p values were indicated on top of each boxplot See also Table S1 and Videos S3 and S4 578 Developmental Cell 57 569582 March 14 2022 ll Article OPEN ACCESS Figure 7 SMR4 decelerates the cell cycle via direct interactions with a selected set of Dtype cyclins A SMR4 interacting proteins from in vivo interactome Van Leene et al 2010 visualized by cytoscape B Yeast twohybrid assays Bait the DNAbinding domain BD alone or fused to SMR4 Prey the activation domain alone AD or fused to CYCD31 CYCD51 CYCD71 CDKA1 and CDKB11 Transformed yeast were spotted in 10fold serial dilutions on appropriate selection media C Transgenic plants harboring CYCD31 CYCD51 and CYCD71 driven by the POLAR promoter in wild type WT and smr41cr in comparison with wild type and smr41cr Orange brackets stomatal lineage precursors Scale bars 50 mm D Quantification of stomatal precursor cells in 10 mm2 area from 7dayold seedlings MannWhitney test was performed p values were marked on top of the boxplot Independent T1 transgenic plants were analyzed The number of plants used WT n 11 smr41cr n 12 proPOLARCYCD31 n 17 proPOLARCYCD31 smr41cr n 16 proPOLARCYCD51 n 12 proPOLARCYCD51 smr41cr n 12 proPOLARCYCD71 n 9 proPOLARCYCD71 smr41cr n 12 E Schematic model SPCHSCRM2 initiate and sustain ACD and MUTESCRM2 trigger SCD gray arrows by transcriptionally activating CYCD31 and CYCD51 shaded blue arrows respectively MUTE directly upregulates SMR4 transcription Blue arrow SMR4 and SMR8 in part suppress the activity of CYCD31 and possibly CYCD71 complexed with CDKs red line but not CYCD51 to terminate the ACD mode and ensure faithful progression of SCD Question marks and dotted line indicate the possible roles of SMR8 in termination of ACD and SMR4 with CYCD71 in symmetric cell division respectively division switch Figure 7E First SPCH initiates and sustains the fast and reiterative ACDs of a meristemoid During the meristemoidtoGMC transition MUTE directly induces SMR4 which directly associates with CYCD31 and likely with CYCD32 and inhibit CYCD31CKDA1 complex to terminate amplifying ACDs At the same time MUTE directly induces CYCD51 Because CYCD51 is not directly inhibited by SMR4 the final SCD can start even in the presence of SMR4 SMR4 may finetune the SCD by being able to inhibit the laterexpressed CYCD71 which is likely complexed with CDKA1 The endogenous expression of SMR4 disappears immediately after the execution of SCD Figure 2F hence the robust differentiation of stomata ensured SMR8 has partially redundant role with SMR4 and is weakly induced by both SPCH and MUTE Figures 2 and S2 as such SMR8 is likely participating in finetuning of this transition Unlike CycD3s and CycD71 CycD51 lacks part of the core domain Strzalka et al 2015 which may explain the differential SMR4 binding Developmental Cell 57 569582 March 14 2022 579 ll OPEN ACCESS In addition to CKIs Rb protein negatively regulates G1S transition Bertoli et al 2013 The plant RETINOBLASTOMA RELATED RBR protein functions as key cellcycle regulators during stomatal development and its reduced expression confers excessive proliferative ACDs within the stomatal cell lineages in part due to dysregulated SPCH expression Borghi et al 2010 Weimer et al 2012 Whereas both CYCD31 and CYCD71 possess LxCxE RBRbinding motif CYCD51 bears a variant motif which may compromise the RBR association Vandepoele et al 2002 Thus CYCD51s unique activity to execute the single SCD might involve the lack of negative regulation by RBR Interestingly RBR regulates the expression and activities of stomatal bHLH proteins SPCH and FAMA respectively Matos et al 2014 Weimer et al 2012 but not MUTE Thus the commitment to differentiation by MUTEorchestrated network may be inherently resilient to inhibition at G1S transition Our study showed that extended G1 phase by stomatal lineage overexpression of SMR4 conferred irregularshaped meristemoids and eventual differentiation of stomata with skewed guard cells Some guard cells exhibit a jigsawpuzzled shape which is indicative of pavement celllike characteristics Figures 4 and 5 Thus without timely execution of an ACD the stomatal precursor cell can adopt hybrid identity of a guard cell and pavement cell An intrinsic polarity protein BASL ensures that only one of the two daughter cells the meristemoid maintains high SPCH levels thereby able to reiterate proliferative ACDs Dong et al 2009 The remaining daughter cell readily loses SPCH protein and differentiate into a pavement cell This process involves a dynamic subcellular relocalization of BASL protein between the nucleus and polarly localized cell cortex the latter activates MAP kinase cascade that inhibits SPCH protein accumulation via phosphorylation Zhang et al 2015 2016 It is not known whether the cellcycle phase influences BASL behaviors but our work implies that it could be the case The SMR4mediated cellcycle deceleration during the meristemoidtoGMC transition mirrors the fundamental importance of G1phase extension for cell fate decision and differentiation during development Dalton 2015 Liu et al 2019 During mammalian adipogenesis commitment of proliferating precursors to terminal differentiation is governed by the molecular competition of mitogens and differentiation stimuli at the G1phase in which timing determines the final numbers of adipocytes Zhao et al 2020 During plant stomatal differentiation we found that the timing of G1phase is not only critical for the commitment to differentiation but also maintaining the shape and size of stomatal lineage cells In any event the finetuning of the G1 phase duration may be the universal mechanism for proper celltype differentiation in multicellular organisms The direct role of MUTE to execute both termination of proliferative asymmetric divisions and orchestration of the single terminal symmetric division occurs through interwoven regulation of core cellcycle drivers and a braker Understanding how cellcycle machineries in turn regulate the precise expression of MUTE which likely involves epigenetic state changes will provide a full picture of cellcycle control of cell fate specification in plants Limitations of the study We use CDT1aCFP loading as a proxy for the G1 phase duration We noticed that in stomatal lineage cells CDF1aCFP does not 580 Developmental Cell 57 569582 March 14 2022 Article accumulate for the entire G1 phase Nevertheless it is clear that ACD is faster than SCD based on both the quantitative analysis of the actual cell division time and the observed shorter duration of CDT1aCFP signals in ACDs In addition the timelapse imaging was performed using cotyledons grown under the microscope and thus it may not represent the exact cellcycle time of stomatal precursors in vegetative leaves from fieldgrown plants STARMETHODS Detailed methods are provided in the online version of this paper and include the following d d d d d KEY RESOURCES TABLE RESOURCE AVAILABILITY B Lead contact B Materials availability B Data and code availability EXPERIMENTAL MODEL AND SUBJECT DETAILS METHOD DETAILS B Plasmid construction and generation of CRISPRbased mutant alleles B Plant growth condition and estradiol treatment B Confocal microscopy B Quantitative analysis of epidermal phenotype B cDNA preparation and qRTPCR B Chromatin immunoprecipitation sequencing ChIPseq B Measurement of DNA content and nuclei size B Yeast two hybrid assay QUANTIFICATION AND STATISTICAL ANALYSIS SUPPLEMENTAL INFORMATION Supplemental information can be found online at httpsdoiorg101016j devcel202201014 ACKNOWLEDGMENTS We thank Lieven De Veylder for proSMR1nlsGFPGUS Daisuke Kurihara for proRPS5AH2BGFP ABRC for GFPLti6B and SMR8 TDNA lines and James Green Machiko Arakawa and Ayami Nakagawa for assistance in plant care This work is supported by MEXT KAKENHI GrantinAid for Scientific Research on Innovative Areas 17H06476 WPIITbM and the startup funds from the UT Austin to KUT grant RTI2018094793BI00 from Spanish Ministry of Science and Innovation and grant 2018AdG_833617 from European Research Council to CG KUT acknowledges the support from Howard Hughes Medical Institute and Johnson Johnson Centennial Chair in Plant Cell Biology at the UT Austin SKH was supported by the Young Leader Cultivation Program from Nagoya University AH is supported by the Walter Benjamin Program Deutsche Forschungsgemeinschaft 447617898 AUTHOR CONTRIBUTIONS Conceptualization SKH and KUT experimental design SKH EDK and KUT performance of experiments SKH AH JY RI and TS bioinformatics analysis SKH SK EDK and KUT visualization SKH AH JY and KUT essential materials and tools BD and CG Writing original draft SKH and KUT Writing review editing SKH AH JY BD CG EDK and KUT project administration KUT funding acquisition CG and KUT ll Article DECLARATION OF INTERESTS The authors declare no competing interests INCLUSION AND DIVERSITY One or more of the authors of this paper selfidentifies as a member of the LGBTQ community Received April 27 2021 Revised November 19 2021 Accepted January 19 2022 Published February 10 2022 REFERENCES Adrian J Chang J Ballenger CE Bargmann BO Alassimone J Davies KA Lau OS Matos JL Hachez C Lanctot A et al 2015 Transcriptome dynamics of the stomatal lineage 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Arabidopsis Plant Cell 24 40834095 Wickham H 2016 ggplot2 elegant graphics for data analysis In Use R Springer p 1 online resource XVI 260 pages 232 illustrations 140 illustrations in color Xie Z Lee E Lucas JR Morohashi K Li D Murray JA Sack FD and Grotewold E 2010 Regulation of cell proliferation in the stomatal lineage by the Arabidopsis MYB four LIPS via direct targeting of core cell cycle genes Plant Cell 22 23062321 Zhang Y Guo X and Dong J 2016 Phosphorylation of the polarity protein BASL differentiates asymmetric cell fate through MAPKs and SPCH Curr Biol 26 29572965 Sherr CJ and Roberts JM 2004 Living with or without cyclins and cyclindependent kinases Genes Dev 18 26992711 Zhang Y Wang P Shao W Zhu JK and Dong J 2015 The BASL polarity protein controls a MAPK signaling feedback loop in asymmetric cell division Dev Cell 33 136149 Sozzani R Cui H MorenoRisueno MA Busch W Van Norman JM Vernoux T Brady SM Dewitte W Murray JA and Benfey PN 2010 Spatiotemporal regulation of cellcycle genes by SHORTROOT links patterning and growth Nature 466 128132 Zhao ML Rabiee A Kovary KM BahramiNejad Z Taylor B and Teruel MN 2020 Molecular competition in G1 controls when cells simultaneously commit to terminally differentiate and exit the cell cycle Cell Rep 31 107769 582 Developmental Cell 57 569582 March 14 2022 ll OPEN ACCESS Article STARMETHODS KEY RESOURCES TABLE Reagent or resource Source Identifier Abcam Abcam Cat ab290 Lot GR3184251 RRID AB_303395 Koncz et al 1992 na Antibodies AntiGFP antibody ChIP Grade Bacterial and virus strains Argrobacterium GV3101pMP90 Chemicals peptides and recombinant proteins Propidium iodide SigmaAldrich P4170 FM464 Invitrogen T13320 DAPI SigmaAldrich D9542 cOmplete Mini Protease Inhibitor Cocktail Roche 11836153001 bEstradiol SigmaAldrich E2758 3amino124triazole SigmaAldrich A8056 Deposited data Raw and processed MUTE ChIPseq data This study GEO GSE173338 iMUTE RNAseq data Han et al 2018 GEO GSE107018 iSPCH RNAseq data Lau et al 2014 GEO GSE57953 TAIR10 Arabidopsis annotation TAIR ftpftparabidopsisorghometairGenes TAIR10_genome_release Experimental models Organismsstrains Arabidopsis thaliana Wild type Col0 ABRC CS1093 Arabidopsis thaliana mute Pillitteri et al 2007 na Arabidopsis thaliana mute2 Pillitteri et al 2008 na Arabidopsis thaliana iMUTE Han et al 2018 na Arabidopsis thaliana iSPCH Han et al 2018 na Arabidopsis thaliana proMUTEMUTEGFP scrmD Qi et al 2017 Han et al 2018 na Arabidopsis thaliana proSMR4nucGFP nls3xGFP This study na Arabidopsis thaliana proSMR4nucGFP mute2 This study na Arabidopsis thaliana proSMR4SMR4YFP This study na Arabidopsis thaliana proSMR4SMR4YFP mute2 This study na Arabidopsis thaliana proSMR4HASMR4 smr41cr This study na Arabidopsis thaliana smr41cr This study na Arabidopsis thaliana smr42cr This study na Arabidopsis thaliana smr81 ABRC SALK_126253 Arabidopsis thaliana smr82 ABRC SALK_074523 Arabidopsis thaliana smr41cr smr82 This study na Arabidopsis thaliana proPOLARSMR4 This study na Arabidopsis thaliana proPOLARSMR8 This study na Arabidopsis thaliana proPOLARSMR1 This study na Arabidopsis thaliana proPOLARKRP1 This study na Arabidopsis thaliana proTMMGUSGFP ABRC Nadeau and Sack 2002 CS65759 Continued on next page Developmental Cell 57 569582e1e6 March 14 2022 e1 ll OPEN ACCESS Article Continued Reagent or resource Source Identifier Arabidopsis thaliana proPOLARSMR4 proTMMGUSGFP This study na Arabidopsis thaliana proMUTEnucYFP Qi et al 2017 na Arabidopsis thaliana proPOLARSMR4 pMUTEnucYFP This study na Arabidopsis thaliana E994 ABRC Pillitteri et al 2007 CS70070 Arabidopsis thaliana proPOLARSMR4 E994 This study na Arabidopsis thaliana proSMR1nlsGFPGUS Bhosale et al 2018 na Arabidopsis thaliana proPOLARSMR4 proSMR1nlsGFPGUS This study na Arabidopsis thaliana PlaCCI Desvoyes et al 2020 na Arabidopsis thaliana LTI6bGFP Kurup et al 2005 ABRC CS84726 Arabidopsis thaliana PlaCCI Lti6bGFP This study na Arabidopsis thaliana PlaCCI Lti6bGFP proPOLARSMR4 This study na Arabidopsis thaliana proPOLARSMR4 smr41cr This study na Arabidopsis thaliana proPOLARCYCD31 This study na Arabidopsis thaliana proPOLARCYCD31 smr41cr This study na Arabidopsis thaliana proPOLARCYCD51 This study na Arabidopsis thaliana proPOLARCYCD51 smr41cr This study na Arabidopsis thaliana proPOLARCYCD71 This study na Arabidopsis thaliana proPOLARCYCD71 smr41cr This study na Arabidopsis thaliana proSMR4HASMR4 smr41cr This study na Arabidopsis thaliana proPOLARSMR4 mute This study na Arabidopsis thaliana proRPS5aH2BGFP Maruyama et al 2013 na Arabidopsis thaliana proPOLARSMR4 proRPS5aH2BGFP This study na Saccharomyces cerevisiae AH109 strain Clontech James et al 1996 na AH109 pGBKT7SMR4 pGADT7 This study na AH109 pGBKT7SMR4 pGADT7CYCD31 This study na AH109 pGBKT7SMR4 pGADT7CYCD51 This study na AH109 pGBKT7SMR4 pGADT7CYCD71 This study na AH109 pGBKT7SMR4 pGADT7CDKA1 This study na AH109 pGBKT7SMR4 pGADT7CDKB11 This study na AH109 pGBKT7 pGADT7CYCD31 This study na AH109 pGBKT7 pGADT7CYCD51 This study na AH109 pGBKT7 pGADT7CYCD71 This study na AH109 pGBKT7 pGADT7CDKA1 This study na AH109 pGBKT7 pGADT7CDKB11 This study na Continued on next page e2 Developmental Cell 57 569582e1e6 March 14 2022 ll OPEN ACCESS Article Continued Reagent or resource Source Identifier Oligonucleotides Plasmid construction Table S3 this paper na Single guide RNA for smr4 CRISPRing Table S3 this paper na qRTPCR primer genotyping Table S3 this paper na Recombinant DNA pKI11R Tsutsui and Higashiyama 2017 Addgene 85808 pGWB440 Nakagawa et al 2007 Addgene 74826 R4pGWB501 Nakagawa et al 2008 na Other recombinant DNAs generated in this study Table S3 this paper na Heatmapper Babicki et al 2016 httpwwwheatmapperca CCTop CRISPRCas9 target online predictor Stemmer et al 2015 httpscctopcosuniheidelbergde8043 R ver 402 R Development Core Team 2008 httpswwwrprojectorg R ggplot2 package Wickham 2016 na BoxPlotR Spitzer et al 2014 httpshinychemgridorgboxplotr COLORBREWER 20 Penn State Univ httpwwwColorBrewerorg FIJIImageJ Schindelin et al 2012 httpsimagejnetFiji Bowtie2 Langmead and Salzberg 2012 httpbowtiebiosourceforgenetbowtie2 indexshtml Software and algorithms Samtools Li et al 2009 httpsamtoolssourceforgenet MACS version 21020140616 Feng et al 2012 httpliulabdfciharvardeduMACS PANTHER Mi et al 2019 httpgeneontologyorg iGV Robinson et al 2011 httpssoftwarebroadinstituteorg softwareigv Other RNeasy Plant mini kit Qiagen 74904 ACCELNGS 2S PLUS DNA LIBRARY KIT with 2S Set A MID Indexing Kit Swift bioscience 21024 26148 ChIP DNA Clean Concentrator Zymo Research D5205 ReverTra Ace qPCR RT Master Mix with gDNA Remover TOYOBO FSQ301 KAPA SYBR FAST for LightCycle 480 KAPA Biosystems KK4611 DynabeadsTM Protein G invitrogen 1004D SPRIselect BECKMAN COULTER B23317 NEBuilder HiFi DNA Assembly Master Mix NEB E2621 RESOURCE AVAILABILITY Lead contact Further information and requests for resources and reagents should be directed and will be fulfilled by the Lead Contact Keiko U Torii ktoriiutexasedu Materials availability Plasmids and transgenic plants generated in this study will be available from the lead contact upon request Data and code availability d The ChIPseq data generated in this study have been deposited at the NCBI Gene Expression Omnibus GEO GSE173338 and are publicly available All data reported in this paper will be shared by the lead contact upon request d This paper does not report original code Developmental Cell 57 569582e1e6 March 14 2022 e3 ll OPEN ACCESS d Article Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request EXPERIMENTAL MODEL AND SUBJECT DETAILS The Arabidopsis Columbia Col accession was used for wild type The lossof function mutants complementation and reporter transgenic lines were reported listed in the key resources table The TDNA insertion mutants were obtained from the Arabidopsis Biological Resource Center ABRC at Ohio State Univ CRISPR gene editing was performed to obtain SMR4 knockout mutants Transgenic lines were introduced into respective mutant backgrounds by genetic crosses or Agrobacterium GV3101 pMP90 strainmediated transformation see method details and key resources table for the lines generated and genotypes were confirmed by PCR or Sanger sequencing All recombinant DNAs for transgenes introduced to Arabidopsis are listed in Table S3 For sequence of genotyping primers sequencing and cloning see Table S3 Seedlings and plants were grown in a longday or constant light condition at 22 C For yeast two hybrid analysis AH109 strain was used and resulting transformants were grown at 30 C METHOD DETAILS Plasmid construction and generation of CRISPRbased mutant alleles For a detailed information of constructs generated in this study see Table S3 Primers used for plasmid constructs were listed in Table S3 For generation of transgenic Arabidopsis plasmid constructs were electroporated into Agrobacterium GV3101pMP90 and subsequently transformed by floral dipping Lossoffunction mutant of SMR4 was generated by CRISPR by using pKAMAITACHI Vector Addgene 85808 as described previously Tsutsui and Higashiyama 2017 Briefly primers for sgRNA were designed by the CCTop CRISPRCas9 target online predictor Stemmer et al 2015 Primers were annealed and inserted into pKI11R vector cut with AarI Resulting construct was introduced into wildtype Col0 plants T1 plants were screened by Hygromycin resistance Six T1 lines were selected and sequenced to check whether mutations were introduced One of the two sgRNAs was successful for generating mutations In T2 generation seeds that do not show OLE1RFP signal were selected to exclude plants harboring transgene in the genome We established two independent homozygous lines that contain 1 bp deletion smr41cr or 1 bp insertion smr42cr at SMR4 gene Figure S4 Primers used single guide RNA sgRNA for SMR4 were listed in Table S3 Plant growth condition and estradiol treatment Arabidopsis accession Columbia Col0 was used as wildtype The following mutantstransgenic lines are reported elsewhere mute2 Pillitteri et al 2008 Lti6b Kurup et al 2005 PlaCCI Desvoyes et al 2020 TDNA insertion mutants of SMR8 smr81 SALK_126253 smr82 SALK_074523 were obtained from ABRC Their genotype and transcript reduction were confirmed The following higherorder mutantsmarker lines were generated by genetic crosses smr41cr smr81 proPOLARSMR4 mute2 proSMR4SMR4YFP mute2 proSMR4nucGFP mute2 PlaCCI Lti6b proPOLARSMR4 PlaCCI and smr41cr PlaCCI Lti6b The presence of transgenesmutant alleles were confirmed by genotyping All plant materials used in this study were listed in key resources table Sterilized seeds were grown on half strength of Murashige and Skoog MS media with 1 sucrose at 22 C under continuous light and 1014dayold seedlings grown on MS media were transplanted to soil to harvest seeds For phenotyping of smr mutants cotyledons at 4day post germination stage were imaged For phenotyping of transgenic plants of proPOLARCKIs T2 plants were grown on MS agar media containing hygromycin 15 mgml and imaged at day 4 and day 8 For proPOLARCYCD transgenic plants multiple independent T1 plants were selected from MS agar media containing hygromycin 15 mgml and imaged at day 7 For the complementation test of SMR4 T3 homozygous plants of proSMR4HASMR4 were germinated on MS agar media were imaged at 5day post germination dpg Confocal microscopy For confocal microscopy cell peripheries of seedlings were visualized with either propidium iodide Sigma P4170 or FM464 Invitrogen T13320 Images were acquired using LSM800 Zeiss or SP5WLL Leica using a 63x water lens The Zeiss LSM800 was used to image the GFP and RFP reporter with excitation at 488 nm and an emission filter of 490 to 546 nm and with excitation at 555 nm and 583617 nm emission range respectively PlaCCI lines Desvoyes et al 2020 were imaged using SP5WLL with the following conditions CFP excitation at 458 nm and emission from 468 to 600 nm GFP excitation at 488 nm and emission from 490 to 546 nm YFP excitation at 514 nm and emission from 524 to 650 nm mCherry excitation at 560 nm and emission from 590 to 650 nm Signals were visualized sequentially using separate HyD detectors DIC images were taken to delineate the cell outlines shown in magenta Raw data were collected with 1024 x 1024 pixel image and imported into FijiImageJ to generate CYMK images using the channel merge function The timelapse were collected at 30min intervals using a 20x lens Peterson and Torii 2012 Qi et al 2017 Raw images were imported into FijiImageJ to generate time projections using the Stacks function For higher quality timelapse imaging of PlaCCI x LTi6b lines in different genetic backgrounds we used Leica Stellaris 8 FALCON with the following conditions CFP excitation at 458 nm and emission from 464 to 510 nm YFP excitation at 514 nm and emission from 520 to 560 nm mCherry excitation at 561 nm and emission from 570 to 620 nm Signals were visualized sequentially using separate HyD detectors HyDXHyDS in TauSeparation mode The timelapses were collected at 30min intervals using a 63x oillens zoom e4 Developmental Cell 57 569582e1e6 March 14 2022 ll Article OPEN ACCESS factor 15 Raw data were collected with 512 x 512 pixel image and imported into FijiImageJ v180_66 to generate RGB imageszstacks using the channel merge function To correct for drift of multichannel zstacks the StackReg plugin was applied Quantitative analysis of epidermal phenotype For quantitative analysis of abaxial leaf epidermis of smr mutants and transgenic plants confocal images were taken at the days after germination as indicated in the Figure legends Preparation of images was done as described previously Houbaert et al 2018 For counting epidermal cell types stomatal density stomatal precursor cells meristemoids and GMC total epidermal cells stomata meristemoids GMC and pavement cells and stomata index number of stomata number of total epidermal cells x100 were calculated by counting cell types in an area of 045 mm2 067 mm x 067 mm at the developmental stage indicated with the cell counter plugin in Fiji and plotted as per mm2 The epidermal cell areas of smr mutants were colorcodedcoded depending on the area calculated using ROI_Color_Coder with a range of minmax 04000 in Fiji The epidermal cells were subdivided into 9 groups according to their size One representative image from each genotype was analyzed and the cell size distribution was then calculated from 499 cells for Col0 plants 659 cells for smr41 662 cells for smr42 601 cells form smr81 611 cells form smr81 and 755 cells for smr41cr smr81 double mutant Guard cells were excluded for the cell size measurement For cell size and circularity measurement of stomatal lineage precursors images of proTMMGUSGFP were set to grayscale and GFP expressing cells were colored in black while other cell area in white by photoshop then the images were imported to Fiji Imported images were subjected to Images Threshold Analyze analyze particle Shape descriptors box has to be checked in Set measurement under the Analyze tab to get circularity values from the selected cell area For the meristemoid size in mute mutant background was measured by the same methods cDNA preparation and qRTPCR For chemical treatment plants were grown on media containing either 10uM bestradiol Sigma E8875 or DMSO For timecourse induction estradiolinducible MUTE or SPCH seeds were sown on 12 MS media and subjected to stratification at 4 C for two to three days then grown for the four to five days under continuous light Subsequent steps were performed as described Han et al 2018 Han et al 2018 RNA was isolated using RNeasy Plant Mini Kit Qiagen 74904 05 mg of RNA was converted to cDNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover TOYOBO FSQ301 according to the instructions of the manufacturer qRTPCR was performed as described in Han et al 2018 Han et al 2018 using KAPA SYBR FAST qPCR Kit Master Mix on LightCycler 96 instrument Roche Relative expression was calculated by dividing ACTIN2 gene expression over the specificgene expression and the fold change was calculated by dividing estradiol expression over DMSO mock expression at each time point indicated See Table S3 for primer sequences used for qRTPCR Chromatin immunoprecipitation sequencing ChIPseq For MUTE ChIPseq experiments transgenic plants proMUTEMUTEGFP scrmD were prepared as described previously Han et al 2018 with following modifications To shear the DNA Bioruptor Diagnode was used 30 sec on and 30 sec off cycle 1518 times Immunoprecipitation against GFP was performed using antiGFP antibody Abcam ab290 Lot GR3184251 DNAs from the immunocomplex was purified by kit Zymo Research D5205 The half of the purified DNA was subjected to library preparation using ACCELNGS 2S PLUS DNA LIBRARY KIT with 2S Set A MID Indexing Kit Swift bioscience 21024 26148 for next generation sequencing Quantitative PCR qPCR was carried out using gene specific primers Table S3 to confirm the library construction The qPCR was run using KAPA SYBR FAST qPCR Kit Master Mix on LightCycler 96 instrument Roche as previously described Han et al 2018 Three biological replicates were used for MUTE ChIPseq experiments Size distribution of the libraries was validated by 2100 Bioanalyzer Agilent The prepared libraries Col input and IP MUTEGFP input and IP with three replicates were sequenced 35 bp pairedend in length with 30million coverage per sample on Illumina NextSeq 500 system ChIP mapping and peak calling were performed as described in Feng et al 2012 Feng et al 2012 Output reads were mapped to the TAIR10 genome assembly using bowtie2 and resulting bam files were sorted and indexed via samtool The sorted bam files were subjected for MACS peak calling version 21020140616 Table S2 Bedgraph file was generated and visualized in igv browser Gene Ontology enrichment analysis was performed using GENE ONTOLOGY httpgeneontologyorg combined with manual curation to remove redundant terms Genes increased by MUTE more than Log2 FC Fold Change 04 and targeted by MUTE were tested Table S2 Following multiple hypothesis testing correction Bonferronicorrection GO term with FDR 005 were called significantly enriched Table S2 The ChIPseq data generated in this study are deposited to the NCBI with an accession number GEO GSE173338 Measurement of DNA content and nuclei size 1st true leaves were harvested from 16day old plants and fixed in a solution of 91 vv ethanol acetic acid for overnight For DAPI staining tissues were rehydrated with ethanol series DAPI 46diamidino2phenylindole staining was done in 5mgml final concentration for 15 minutes in dark Nuclear DAPI fluorescence was excited at 405 nm captured with 410 470 nm emission range DAPI stained nuclei area from the guard cells was selected and measured by using FIJI software Wildtype and two independent T2 proPOLARSMR4 transgenic lines were used The number of guard cells measured is 129 WT and 234 proPOLARSMR4 10 or 11dayold cotyledons from proRPS5AH2BGFP Maruyama et al 2013 transgenic plants were also imaged to measure the nuclei size of guard and pavement cells The area of nuclei GFP was selected and measured from the zstack Developmental Cell 57 569582e1e6 March 14 2022 e5 ll OPEN ACCESS Article projection images using FIJI software Number of guard cells and pavement cells measured 155 and 108 wild type 191 and 103 proPOLARSMR4 proRPS5AH2BGFP DAPIstained nuclear area of singlecelled GCs in proPOLARSMR1 n 24 and normal GCs in wild type n 102 in 12day old true leaves was measured Yeast two hybrid assay Y2H assays were performed using the MatchmakerTM TwoHybrid System Clontech Bait pGBKT7 and prey pGADT7 constructs were cotransformed into the yeast strain AH109 according to manufactural instruction Clontech The resulting transformants were spotted on SD Leu Trp and SD Trp Leu His selection media containing different concentration of 3amino124triazole Sigma A8056 as previously reported Putarjunan et al 2019 All constructs and primer information are listed in the Table S3 QUANTIFICATION AND STATISTICAL ANALYSIS A series of Zstack confocal images were taken to obtain images covering the epidermis and capturing GFP signal from the reporter lines The area and the number of epidermal cells were quantified by using FIJIImageJ Statistical analyses were performed using R ver 402 For the multiple sample comparison oneway ANOVA with posthoc Tukey HSD test was performed For the twosample comparison either student ttest or MannWhitney U test were performed Graphs were generated using R ggplot2 package BoxPlotR or Microsoft Excel Listed in key resources table The color of Boxplot graph was based on ColorBrewerorg The value of n the number of each experiment or samples means of error bars and how statistical significance was defined are indicated in a relevant figure legend e6 Developmental Cell 57 569582e1e6 March 14 2022