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	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

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	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

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	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

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	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
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	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

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	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
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	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

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	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

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	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

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	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

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	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

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	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

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	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
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	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

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	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

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	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

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	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

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	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

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	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
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	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