Deceleration of the cell cycle underpins a switch

from proliferative to terminal divisions in plant

stomatal lineage

Authors

SoonKi Han Arvid Herrmann

Jiyuan Yang  Crisanto Gutierrez

EunDeok Kim Keiko U Torii

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

During stomatal differentiation asymmetric divisions are

faster than terminal divisions

Upon commitment to differentiation MUTE induces the cellcycle inhibitor SMR4

SMR4 decelerates the asymmetric cell division cycle via

selective binding to cyclin 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

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

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

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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

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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

Article

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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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

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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

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

Developmental Cell 57 569582 March 14 2022 577

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

Article

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

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

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 birth amplification and

termination of a selfrenewing population Dev Cell 33 107118

Babicki S Arndt D Marcu A Liang Y Grant JR Maciejewski A and

Wishart DS 2016 Heatmapper webenabled heat mapping for all

Nucleic Acids Res 44 W147W153

Bertoli C Skotheim JM and de Bruin RA 2013 Control of cell cycle transcription during G1 and S phases Nat Rev Mol Cell Biol 14 518528

Bhosale R Boudolf V Cuevas F Lu R Eekhout T Hu Z Van Isterdael

G Lambert GM Xu F Nowack MK et al 2018 A spatiotemporal DNA

endoploidy map of the Arabidopsis Root reveals roles for the endocycle in root

development and stress adaptation Plant Cell 30 23302351

tterer J Laizet Y Hennig L and Gruissem W

Borghi L Gutzat R Fu

2010 Arabidopsis RETINOBLASTOMARELATED is required for stem cell

maintenance cell differentiation and lateral organ production Plant Cell 22

17921811

Boudolf V Barroco R de Almeida Engler J Verkest A Beeckman T

Naudts M Inze D and De Veylder L 2004 B1type cyclindependent kinases are essential for the formation of stomatal complexes in Arabidopsis

thaliana Plant Cell 16 945955

Budirahardja Y and Gonczy P 2009 Coupling the cell cycle to development Development 136 28612872

Coronado D Godet M Bourillot PY Tapponnier Y Bernat A Petit M

Afanassieff M Markossian S Malashicheva A Iacone R et al 2013 A

short G1 phase is an intrinsic determinant of naive embryonic stem cell pluripotency Stem Cell Res 10 118131

Dalton S 2015 Linking the cell cycle to cell fate decisions Trends Cell Biol

25 592600

Desvoyes B AranaEcharri A Barea MD and Gutierrez C 2020 A

comprehensive fluorescent sensor for spatiotemporal cell cycle analysis in

Arabidopsis Nat Plants 6 13301334

Hamdoun S Zhang C Gill M Kumar N Churchman M Larkin JC

Kwon A and Lu H 2016 Differential roles of two homologous cyclindependent kinase inhibitor genes in regulating cell cycle and innate immunity

in Arabidopsis Plant Physiol 170 515527

Han SK Qi X Sugihara K Dang JH Endo TA Miller KL Kim ED

Miura T and Torii KU 2018 MUTE directly orchestrates cellstate switch

and the single symmetric division to create stomata Dev Cell 45 303315e5

Han SK and Torii KU 2016 Lineagespecific stem cells signals and

asymmetries during stomatal development Development 143 12591270

Harashima H Dissmeyer N and Schnittger A 2013 Cell cycle control

across the eukaryotic kingdom Trends Cell Biol 23 345356

Houbaert A Zhang C Tiwari M Wang K de Marcos Serrano A Savatin

DV Urs MJ Zhiponova MK Gudesblat GE Vanhoutte I et al 2018

POLARguided signalling complex assembly and localization drive asymmetric cell division Nature 563 574578

Inze D and De Veylder L 2006 Cell cycle regulation in plant development

Annu Rev Genet 40 77105

James P Halladay J and Craig EA 1996 Genomic libraries and a host

strain designed for highly efficient twohybrid selection in yeast Genetics

144 14251436

Koncz C Nemeth K Redei GP and Schell J 1992 TDNA insertional

mutagenesis in Arabidopsis Plant Mol Biol 20 963976

Kumar N Harashima H Kalve S Bramsiepe J Wang K Sizani BL

Bertrand LL Johnson MC Faulk C Dale R et al 2015 Functional conservation in the SIAMESERELATED family of cyclindependent kinase inhibitors in land plants Plant Cell 27 30653080

Kumar N and Larkin JC 2017 Why do plants need so many cyclindependent kinase inhibitors Plant Signal Behav 12 e1282021

Kurup S Runions J Kohler U Laplaze L Hodge S and Haseloff J

2005 Marking cell lineages in living tissues Plant J 42 444453

Langmead B and Salzberg SL 2012 Fast gappedread alignment with

Bowtie 2 Nat Methods 9 357359

Lau OS and Bergmann DC 2012 Stomatal development a plants

perspective on cell polarity cell fate transitions and intercellular communication Development 139 36833692

Lau OS Davies KA Chang J Adrian J Rowe MH Ballenger CE and

Bergmann DC 2014 Direct roles of SPEECHLESS in the specification of

stomatal selfrenewing cells Science 345 16051609

Li H Handsaker B Wysoker A Fennell T Ruan J Homer N Marth G

Abecasis G and Durbin R 1000 Genome Project Data Processing

Subgroup 2009 The Sequence AlignmentMap format and SAMtools

Bioinformatics 25 20782079

Liu L Michowski W Kolodziejczyk A and Sicinski P 2019 The cell cycle

in stem cell proliferation pluripotency and differentiation Nat Cell Biol 21

10601067

Desvoyes B and Gutierrez C 2020 Roles of plant retinoblastoma protein

cell cycle and beyond EMBO J 39 e105802

MacAlister CA OhashiIto K and Bergmann DC 2007 Transcription factor control of asymmetric cell divisions that establish the stomatal lineage

Nature 445 537540

Dewitte W Scofield S Alcasabas AA Maughan SC Menges M Braun

N Collins C Nieuwland J Prinsen E Sundaresan V and Murray JAH

2007 Arabidopsis CYCD3 Dtype cyclins link cell proliferation and endocycles and are ratelimiting for cytokinin responses Proc Natl Acad Sci

USA 104 1453714542

Maruyama D Hamamura Y Takeuchi H Susaki D Nishimaki M

Kurihara D Kasahara RD and Higashiyama T 2013 Independent control

by each female gamete prevents the attraction of multiple pollen tubes Dev

Cell 25 317323

Dong J MacAlister CA and Bergmann DC 2009 BASL controls asymmetric cell division in Arabidopsis Cell 137 13201330

Elledge SJ 1996 Cell cycle checkpoints preventing an identity crisis

Science 274 16641672

Matos JL Lau OS Hachez C CruzRamrez A Scheres B and

Bergmann DC 2014 Irreversible fate commitment in the Arabidopsis stomatal lineage requires a FAMA and RETINOBLASTOMARELATED module

Elife 3 e03271

Feng J Liu T Qin B Zhang Y and Liu XS 2012 Identifying ChIPseq

enrichment using MACS Nat Protoc 7 17281740

Melaragno JE Mehrotra B and Coleman AW 1993 Relationship between endopolyploidy and cell size in epidermal tissue of Arabidopsis Plant

Cell 5 16611668

Hachez C OhashiIto K Dong J and Bergmann DC 2011

Differentiation of Arabidopsis guard cells analysis of the networks incorporating the basic helixloophelix transcription factor FAMA Plant Physiol

155 14581472

Menges M de Jager SM Gruissem W and Murray JA 2005 Global

analysis of the core cell cycle regulators of Arabidopsis identifies novel genes

reveals multiple and highly specific profiles of expression and provides a

coherent model for plant cell cycle control Plant J 41 546566

Developmental Cell 57 569582 March 14 2022 581

Mi H Muruganujan A Ebert D Huang X and Thomas PD 2019

PANTHER version 14 more genomes a new PANTHER GOslim and improvements in enrichment analysis tools Nucleic Acids Res 47 D419D426

Morgan DO 2007 The Cell Cycle  Principles of Control New

Science Press

Nadeau JA and Sack FD 2002 Control of stomatal distribution on the

Arabidopsis leaf surface Science 296 16971700

Nakagawa T Nakamura S Tanaka K Kawamukai M Suzuki T

Nakamura K Kimura T and Ishiguro S 2008 Development of R4 gateway

binary vectors R4pGWB enabling highthroughput promoter swapping for

plant research Biosci Biotechnol Biochem 72 624629

Nakagawa T Suzuki T Murata S Nakamura S Hino T Maeo K

Tabata R Kawai T Tanaka K Niwa Y et al 2007 Improved Gateway binary vectors highperformance vectors for creation of fusion constructs in

transgenic analysis of plants Biosci Biotechnol Biochem 71 20952100

Peres A Churchman ML Hariharan S Himanen K Verkest A

Vandepoele K Magyar Z Hatzfeld Y Van Der Schueren E Beemster

GT et al 2007 Novel plantspecific cyclindependent kinase inhibitors

induced by biotic and abiotic stresses J Biol Chem 282 2558825596

Peterson KM and Torii KU 2012 Longterm highresolution confocal

time lapse imaging of Arabidopsis cotyledon epidermis during germination

J Vis Exp 70 4426

Pillitteri LJ Bogenschutz NL and Torii KU 2008 The bHLH protein

MUTE controls differentiation of stomata and the hydathode pore in

Arabidopsis Plant Cell Physiol 49 934943

Pillitteri LJ Peterson KM Horst RJ and Torii KU 2011 Molecular

profiling of stomatal meristemoids reveals new component of asymmetric

cell division and commonalities among stem cell populations in Arabidopsis

Plant Cell 23 32603275

Pillitteri LJ Sloan DB Bogenschutz NL and Torii KU 2007

Termination of asymmetric cell division and differentiation of stomata

Nature 445 501505

Putarjunan A Ruble J Srivastava A Zhao C Rychel AL Hofstetter

AK Tang X Zhu JK Tama F Zheng N and Torii KU 2019

Bipartite anchoring of SCREAM enforces stomatal initiation by coupling

MAP kinases to SPEECHLESS Nat Plants 5 742754

Qi X Han SK Dang JH Garrick JM Ito M Hofstetter AK and Torii

KU 2017 Autocrine regulation of stomatal differentiation potential by EPF1

and ERECTALIKE1 ligandreceptor signaling Elife 6 e24102

RiouKhamlichi C Huntley R Jacqmard A and Murray JA 1999

Cytokinin activation of Arabidopsis cell division through a Dtype cyclin

Science 283 15411544

Robinson JT Thorvaldsdottir H Winckler W Guttman M Lander ES

Getz G and Mesirov JP 2011 Integrative genomics viewer Nat

Biotechnol 29 2426

Roeder AH Chickarmane V Cunha A Obara B Manjunath BS and

Meyerowitz EM 2010 Variability in the control of cell division underlies

sepal epidermal patterning in Arabidopsis thaliana PLoS Biol 8 e1000367

Sanz L Dewitte W Forzani C Patell F Nieuwland J Wen B Quelhas

P De Jager S Titmus C Campilho A et al 2011 The Arabidopsis Dtype

cyclin CYCD21 and the inhibitor ICK2KRP2 modulate auxininduced lateral

root formation Plant Cell 23 641660

Schindelin J ArgandaCarreras I Frise E Kaynig V Longair M Pietzsch

T Preibisch S Rueden C Saalfeld S Schmid B et al 2012 Fiji an

opensource platform for biologicalimage analysis Nat Methods 9 676682

Schwarz EM and Roeder AH 2016 Transcriptomic effects of the cell cycle regulator LGO in Arabidopsis Sepals Front Plant Sci 7 1744

Article

Spitzer M Wildenhain J Rappsilber J and Tyers M 2014 BoxPlotR a

web tool for generation of box plots Nat Methods 11 121122

Stemmer M Thumberger T Del Sol Keyer M Wittbrodt J and Mateo JL

2015 CCTop an intuitive flexible and reliable CRISPRCas9 target prediction tool PLoS One 10 e0124633

Strzalka WK Aggarwal C Krzeszowiec W Jakubowska A Sztatelman

O and Banas AK 2015 Arabidopsis PCNAs form complexes with selected

Dtype cyclins Front Plant Sci 6 516

Szklarczyk D Gable AL Lyon D Junge A Wyder S HuertaCepas J

Simonovic M Doncheva NT Morris JH Bork P et al 2019 STRING

v11 proteinprotein association networks with increased coverage supporting functional discovery in genomewide experimental datasets Nucleic

Acids Res 47 D607D613

Tsutsui H and Higashiyama T 2017 pKAMAITACHI vectors for highly efficient CRISPRCas9mediated gene knockout in Arabidopsis thaliana Plant

Cell Physiol 58 4656

Van Leene J Hollunder J Eeckhout D Persiau G Van De Slijke E Stals

H Van Isterdael G Verkest A Neirynck S Buffel Y et al 2010 Targeted

interactomics reveals a complex core cell cycle machinery in Arabidopsis thaliana Mol Syst Biol 6 397

Vandepoele K Raes J De Veylder L Rouze P Rombauts S and Inze D

2002 Genomewide analysis of core cell cycle genes in Arabidopsis Plant

Cell 14 903916

Vanneste S Coppens F Lee E Donner TJ Xie Z Van Isterdael G

Dhondt S De Winter F De Rybel B Vuylsteke M et al 2011

Developmental regulation of CYCA2s contributes to tissuespecific proliferation in Arabidopsis EMBO J 30 34303441

Vaten A Soyars CL Tarr PT Nimchuk ZL and Bergmann DC 2018

Modulation of asymmetric division diversity through cytokinin and

SPEECHLESS regulatory interactions in the Arabidopsis stomatal lineage

Dev Cell 47 5366e5

Walker JD Oppenheimer DG Concienne J and Larkin JC 2000

SIAMESE a gene controlling the endoreduplication cell cycle in Arabidopsis

thaliana trichomes Development 127 39313940

Wang K Ndathe RW Kumar N Zeringue EA Kato N and Larkin JC

2020 The CDK inhibitor SIAMESE targets both CDKA1 and CDKB1 complexes to establish endoreplication in trichomes Plant Physiol 184 165175

Weimer AK Matos JL Sharma N Patell F Murray JAH Dewitte W

and Bergmann DC 2018 Lineage and stagespecific expressed CYCD71

coordinates the single symmetric division that creates stomatal guard cells

Development 145 dev160671

Weimer AK Nowack MK Bouyer D Zhao X Harashima H Naseer S

De Winter F Dissmeyer N Geldner N and Schnittger A 2012

Retinoblastoma related1 regulates asymmetric cell divisions in 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

Article

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

D9542

cOmplete Mini Protease Inhibitor

Cocktail

Roche

11836153001

bEstradiol

E2758

3amino124triazole

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

na

Arabidopsis thaliana iSPCH

na

Arabidopsis thaliana proMUTEMUTEGFP scrmD

Qi et al 2017 Han et al 2018

na

Arabidopsis thaliana proSMR4nucGFP

nls3xGFP

This study

na

mute2

This study

na

Arabidopsis thaliana proSMR4SMR4YFP

This study

na

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

Article

Continued

Source

proTMMGUSGFP

This study

na

Arabidopsis thaliana proMUTEnucYFP

Qi et al 2017

na

pMUTEnucYFP

This study

na

Arabidopsis thaliana E994

ABRC Pillitteri et al 2007

CS70070

Arabidopsis thaliana

proPOLARSMR4 E994

This study

na

proSMR1nlsGFPGUS

Bhosale et al 2018

na

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

PlaCCI Lti6bGFP proPOLARSMR4

This study

na

smr41cr

This study

na

Arabidopsis thaliana proPOLARCYCD31

This study

na

smr41cr

This study

na

Arabidopsis thaliana proPOLARCYCD51

This study

na

smr41cr

This study

na

Arabidopsis thaliana proPOLARCYCD71

This study

na

smr41cr

This study

na

This study

na

smr41cr

This study

na

This study

na

smr41cr

This study

na

smr41cr

This study

na

proPOLARSMR4 mute

This study

na

Arabidopsis thaliana proRPS5aH2BGFP

Maruyama et al 2013

na

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

pGADT7CYCD51

This study

na

pGADT7CYCD71

This study

na

AH109 pGBKT7SMR4 pGADT7CDKA1

This study

na

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

e2 Developmental Cell 57 569582e1e6 March 14 2022

ll

Article

Continued

Source

Oligonucleotides

Plasmid construction

Table S3 this paper

na

Single guide RNA for smr4 CRISPRing

na

qRTPCR primer genotyping

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

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

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

d

Article

Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request

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

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

Developmental Cell 57 569582e1e6 March 14 2022 e5

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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

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