SMR4 publication.txt

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
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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

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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
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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

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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
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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
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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
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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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582 Developmental Cell 57 569582 March 14 2022

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OPEN ACCESS

Article
STARMETHODS
KEY RESOURCES TABLE

Reagent or resource

Source

Identifier

Abcam

Abcam Cat ab290
Lot GR3184251 RRID AB_303395

Koncz et al 1992

na

Antibodies
AntiGFP antibody  ChIP Grade
Bacterial and virus strains
Argrobacterium GV3101pMP90
Chemicals peptides and recombinant proteins
Propidium iodide

SigmaAldrich

P4170

FM464

Invitrogen

T13320

DAPI

SigmaAldrich

D9542

cOmplete Mini Protease Inhibitor
Cocktail

Roche

11836153001

bEstradiol

SigmaAldrich

E2758

3amino124triazole

SigmaAldrich

A8056

Deposited data
Raw and processed MUTE ChIPseq data

This study

GEO GSE173338

iMUTE RNAseq data

Han et al 2018

GEO GSE107018

iSPCH RNAseq data

Lau et al 2014

GEO GSE57953

TAIR10 Arabidopsis annotation

TAIR

ftpftparabidopsisorghometairGenes
TAIR10_genome_release

Experimental models Organismsstrains
Arabidopsis thaliana Wild type Col0

ABRC

CS1093

Arabidopsis thaliana mute

Pillitteri et al 2007

na

Arabidopsis thaliana mute2

Pillitteri et al 2008

na

Arabidopsis thaliana iMUTE

Han et al 2018

na

Arabidopsis thaliana iSPCH

Han et al 2018

na

Arabidopsis thaliana proMUTEMUTEGFP scrmD

Qi et al 2017 Han et al 2018

na

Arabidopsis thaliana proSMR4nucGFP
nls3xGFP

This study

na

Arabidopsis thaliana proSMR4nucGFP
mute2

This study

na

Arabidopsis thaliana proSMR4SMR4YFP

This study

na

Arabidopsis thaliana proSMR4SMR4YFP
mute2

This study

na

Arabidopsis thaliana proSMR4HASMR4
smr41cr

This study

na

Arabidopsis thaliana smr41cr

This study

na

Arabidopsis thaliana smr42cr

This study

na

Arabidopsis thaliana smr81

ABRC

SALK_126253

Arabidopsis thaliana smr82

ABRC

SALK_074523

Arabidopsis thaliana smr41cr smr82

This study

na

Arabidopsis thaliana proPOLARSMR4

This study

na

Arabidopsis thaliana proPOLARSMR8

This study

na

Arabidopsis thaliana proPOLARSMR1

This study

na

Arabidopsis thaliana proPOLARKRP1

This study

na

Arabidopsis thaliana proTMMGUSGFP

ABRC Nadeau and Sack 2002

CS65759
Continued on next page

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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
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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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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

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