Current Biology 20, 452457, March 9, 2010 2010 Elsevier Ltd All
rights reserved DOI
10.1016/j.cub.2010.01.018ReportSCHIZORIZAEncodesaNuclearFactorRegulatingAsymmetryofStemCellDivisionsintheArabidopsisRootColette
A. ten Hove,1Viola Willemsen,1Wouter J. de Vries,1Anja van
Dijken,1Ben Scheres,1and Renze Heidstra1,*1Molecular Genetics,
Department of Biology,Faculty of Science, Utrecht University,
Padualaan 8,3584 CH Utrecht, The NetherlandsSummaryCell
divisionsgeneratingdaughter cellsdifferent insize,shape, identity,
andfunctionareindispensablefor manydevelopmental
processesincludingfatespecication, tis-sue patterning, and
self-renewal. In animals and
yeast,perturbationsinfactorsrequiredforwell-describedasym-metric
cell divisions generally yield cells of equal fate. Herewereport
onSCHIZORIZA(SCZ), asinglenuclear factorwith homology to heat-shock
transcription factors that con-trolsthe separationof cellfateina
setofstemcells gener-atingdifferent root tissues: root cap,
epidermis, cortex,andendodermis. Loss-of-function, expression,
andrecon-stitutionexperimentsindicatethat SCZactsmainlyfromwithin
its cortical expression domain in the stem cell
niche,exertingbothautonomousandnonautonomouseffectstospecifycortexidentityandcontrol
theseparationof cellfates in surrounding layers. Thus, SCZ denes a
novelpathway for asymmetric cell division in plants.Results and
DiscussionAsymmetric cell division is a fundamental and universal
mech-anismfor generating diversity and pattern in
multicellularorganisms[1, 2]. Theradial organizationof
theArabidopsisrootisderived
fromstereotypedasymmetriccelldivisionsofdifferent stemcells, the
initials. These cells and their daughtersproducedenedtissuelayers
withdistinct cell fates (Fig-ure1A) [3]. Thestemcellssurroundasmall
groupof rarelydividingcells, thequiescent center (QC), requiredfor
theirmaintenance. The QC itself is formed early during
embryogen-esis whenanasymmetricdivisionof thehypophyseal cellforms
the lens-shaped QC progenitor cell and future columellaroot cap
[4]. QC fate is specied in parallel by the PLETHORA(PLT),
SHORTROOT(SHR), andSCARECROW(SCR) tran-scriptionfactors[57].
SHRandSCRarealsorequiredforground tissue patterning: shr and scr
mutants lack the asym-metric periclinal division in the ground
tissue stem cell daugh-ter, resulting in a single ground tissue
layer. For shr, this layerlacks endodermal identity, but in scr,
this layer displays mixedcortical/endodermal identity [810].
Several other reports
haveappearedthatsuggestplantcellsmaypossessmixedfates[1113]. To our
knowledge, the only known example of mixedfate phenotypes in animal
development comes from extensivegenetic screening approaches in C.
elegans, which haveuncoveredmutantsinwhichasingleneuronal
fatedecisionisinappropriatelyexecuted,resultinginamixedfatepheno-type[14].
Here, wedescribetheSCHIZORIZA(SCZ) nuclearfactor, which is required
for plant cell fate separation in
severaltissues,actingbothcell-autonomouslyandnon-cell-autono-mously.
Our datahighlight anovel mechanismof cell fateseparation in plants
that is particularly relevant for asymmetriccell divisions within
stem cell areas.SCZ Encodes a Member of the Heat-Shock
TranscriptionFactor FamilyTo nd novel genes involved in QC
specication and stem cellmaintenance, we performed a QC
marker-based mutagenesisscreen. A line doubly homozygous for QC25
and QC46promoters [7] fused toERCFP andERYFP, respectively
(Figures1Band1C), was mutagenized, andtheM2progeny
wereanalyzedforalteredexpression.Fivephenotypicallyindistin-guishablemutantlinescombinedreducedQC25::ERCFPandQC46::ERYFP
activity with retarded root growth and disorgani-zationof
thestemcell niche(Figures1Dand1E; seealsobelow). Complementation
tests showed that all ve linescarriedallelicmutations.
Giventheequal allelestrength, wecontinued to use one allele, qc351,
for further analysis.Compared to wild-type, qc351 roots developed
more hairs,lacked the stereotypical pattern of alternating hair and
nonhairles, and initiated root hairs fromsubepidermal tissue
reminis-cent of the schizoriza (scz) mutant phenotype (see Figures
S1AandS1Bavailableonline)[15].Complementationanalysisre-vealed that
qc351 was allelic to scz. Accordingly, we renamedour mutant alleles
scz-2 through
scz-6.Wemolecularlycharacterizedthesczmutationviaamap-based
approach. Fine mapping located SCZ to a single locusin an area of
70 kb on chromosome 1 (Figure 1G). One of thecandidate genes that
we sequenced was the heat shock tran-scriptionfactor B4(HsfB4)
locus(At1g46264), basedonitsstemcell-enrichedinsilicoroot
expressionpattern(http://www.arexdb.org/;[1618].Comparisonwiththecorrespond-ingwild-typesequencerevealeddifferent
mutationsintheHsfB4 gene for all scz alleles, including two
additional TILLINGalleles (Figure 1G; Table S1). The identical
phenotype indicatesthat they are likely HsfB4 mutant null alleles.
For Arabidopsis,the 21 Hsfs are classied into three major groups, A
(16 mem-bers), B(4 members, including SCZ), andC(1
member),according to the different exible linkers in their HR-A/B
oligo-merizationregions(Figure1G,greenbar).
Despiteconsider-ablediversicationinsizeandsequence,thebasicstructureof
Hsfs is conserved among eukaryotes [19].SCZmRNAisrst detectedat
triangular-stageembryosintheQCprogenitor cells. Fromheart
stageonward, SCZmRNA accumulation expands into ground and vascular
tissueprogenitorsandtheirimmediatedaughters(Figures2A2C).Thisexpressionpatternismaintainedinthepostembryonicroot
with highest SCZ mRNA accumulation in QC and
groundtissuestemcellsandtheirimmediatedaughters(Figure2D).Inthescz-2mutant,
hybridizationsignal isabsent fromthesubepidermal layer
(FiguresS2AandS2B).
SCZpromoter-reporterfusionsessentiallycorroboratetheinsituhybridiza-tionexpressionpattern(FiguresS2CS2E).
Consistent withits function as a putative transcription factor, the
complement-ing35S::GFP:SCZtranslational fusion localizes tothe
cellnucleus (see below). *Correspondence: [email protected]
Asymmetric Cell Division from EmbryogenesisOnward in sczThe
reduction in root growth together with altered expressionof the QC
markers QC25::ERCFP and QC46::ERYFP in mutants(Figures 1D and 1E)
might indicate a role for SCZ in QC/stemcell specication and/or
function. However, scz-2 rootscontinue to grow in an indeterminate
manner, with root
lengthlaggingbyabouthalfbehindwild-type(Figure1F).Similarly,root
meristemsizeisreducedbut
maintainedinscz-2(Fig-ureS1C).TheseobservationsshowthatSCZisnotcriticallyrequired
for stem cell
maintenance.CellsatthepositionoftheQCandcolumellastemcellsinscz-2mutantrootsaremorphologicallyabnormal
andaccu-mulate starch granules, which marks differentiated
columellainwild-type(Figures2Fand2G). QC25, QC46, andQC184markers
are displacedfromthe QCandexpress diffuse
activityinthestarchgranule-containingcells.
WOX5alsomarkstheQC,andthegeneproductisrequiredtomaintaintheunder-lyingcolumellastemcells.
WOX5::ERGFPexpressionfadedfrom the position of the QC but did not
appear in the columella(Figures2F2I; FiguresS2FS2I). Apparently,
QCandcolu-mella fatesare presentbut notseparated inscz-2roots,
andFigure 1. Identication and Cloning of SCZ(A) Schematicviewof
theArabidopsisrootmeristem. Thefollowing abbreviations are used:
En, endodermis; Co,cortex; Ep, epidermis; QC, quiescent center;
Col, columella;LRC, lateral root cap.(BD) Four-day-old wild-type
root expressing both QC46::ERYFP (B; arrowhead indicatesQC)
andQC25::ERCFP (C)and4-day-oldqc351/scz-2root
showingQC46::ERYFPexpres-sion (D; false green).(E) Eight-day-old
wild-type and scz-2 through
scz-6seedlings.(F)Rootlength(inmm)ofwild-typeandscz-2seedlingsattheindicatednumber
of dayspostgermination(dpg). Foreachdatapoint,n R
57;errorbarsindicatestandarderrorof the mean.(G) Schematic
representation of the identication of the SCZgenebypositional
cloning. SCZ/HsfB4locationisshownrelativetoacontigof veBACclones;
theproportionofrecombinant seedlings is shown in parentheses. Boxes
indi-cate coding sequence. Conserved functional
domainsaccordingto[19]areindicatedasfollows:gray,conservedDNA-binding
domain; green, HR-A/Boligomerization region;orange, nuclear
localization signal (NLS); blue, nuclearexport signal (NES);
purple, R(R/KLFGV) motif [33]. The posi-tionsof
thenucleotidesequencechangesareshownforeach mutant allele; scz-7
andscz-8 are TILLINGalleles[34]. See also Figure S1.Figure 2. SCZ
Expression and Postembryonic Mutant StemCell Defects(AD) In situ
hybridization with SCZ antisense probe in wild-type triangular-
(A), heart- (B; black arrowhead marks groundtissueaccumulation),
andtorpedo-stage(C) embryoand2-day-old seedling (D). White
arrowheads in (A)(D), (F), (K),and (L) indicate QC.(E and J)
Heart-stage wild-type (WT; E) and scz-2 (J)embryos. Inset
showsmagnicationof normal anticlinallydivided ground tissue initial
(E; green arrowhead) and aber-rant
periclinallydividedgroundtissueinitial (J; redarrow-head).(F and G)
QC184 expression (blue) in 6-day-old wild-type (F)shifts in the
columella in scz-2 (G) roots. Red arrow indicatescolumella stem
cells devoid of purple starch granules;
blackarrowindicatesaccumulationofstarchgranulesinmutantQC region.(H
and I) WOX5::ERGFP expression in 4-day-old wild-type (H)and scz-2
(I) roots.(KandL) Anilineblue-stainedwild-type(K)
andscz-2(L)matureembryos. Whitearrowindicatesendoflateral rootcap
layer, dashed line indicates root/hypocotyl boundary, #indicates
ground tissue layers in hypocotyl, *
indicatespericlinalgroundtissuedivisionsleadingto
supernumerarymutant layers in scz-2. See also Figure S2.SCHIZORIZA
Regulates Cell Fate Separation453the separation defect does not
affect stem cell niche activity.Interestingly, thisobservationof
mixedfatesisreminiscentoftheformationof roothairsfromsubepidermal
tissuesre-ported when scz mutants were rst described [15].To gain
more insight into the possible role for SCZ as a
fatedeterminationand/orseparationfactor,
wetracedthescz-2defectsbacktotheir embryonicorigin. Theinitial
defect inscz-2occursinheart-stageembryos, wheregroundtissueinitials
performan aberrant periclinal division (Figure
2J),resultingintheectopicgroundtissuelayerobservedattor-pedostage([15];
datanot shown). Theinner
groundtissuedaughtercellscontinuetoperformadditional periclinal
divi-sionsgeneratingmoreectopiclayers(Figure2L,
asterisks).Theorganizationof QC, columella, pericycle,
andvascula-tureappearsnormal throughout embryogenesis.
Inmaturewild-typeembryos, anadditional cortical layer proximal
tothe endof the lateral root capmarks the hypocotyl (Fig-ure 2K;
[20]). Similarly, in scz-2, the hypocotyl
possessesthreegroundtissuelayers(Figure2L). However, thelateralroot
cap layer contains fewer cells, and epidermal cells belowthe
root-hypocotyl junction appear long and at, morphologi-cally
reminiscent of lateral root cap cells suggestive of
mixedfates(Figure2L, proximal toarrow). TheseobservationsareFigure
3. SCZ Species Cortex and Segregates Cell Fates inthe Root NicheThe
following abbreviations are used: WT, wild-tpe; ep,epidermis; lrc,
lateral root cap; c, cortex; e, endodermis.* indicates mutant
subepidermal layer, arrowhead indicatesQC, and square bracket
indicates ectopic endodermallayers.(AH) WER[ERCFP (false red) and
SMB:GFP expression inbent cotyledon-stage wild-type (Aand B) and
scz-2 (Cand D)embryosandin4-day-oldwild-type(EandF) andscz-2(G and
H) roots. Arrows in (G) and (H) indicate WER[ERCFPand SMB:GFP
expression in distal subepidermal cells.(IL) Radial root tissue
sections of 4-day-old wild-type (I andJ) andscz-2 (Kand L) roots
stained for GL2::GUSexpression.scz-2 subepidermal cells express
GL2::GUS (* in K) and formroot hairs (# in L).(MandN) Embryonicroot
Co2::H2BYFPexpressioninlatetorpedo-stage wild-type embryo (M) is
absent in scz-2embryo (bracket in N).(OandP)
WOX5::ERGFPexpressioninlatetorpedo-stagewild-type (O) and scz-2 (P)
embryos.(QV)Four-day-oldwild-type(Q,S,andU)andscz-2(R,T,and V)
roots expressing SHR::SHR:GFP (Q and
R),SCR::H2BYFP(SandT),andCo3::H2BYFP(UandV).FadingSCR::H2BYFPexpression
in periclinally divided ground tissuestemcell daughter (arrowin S)
is maintained in scz-2 subepi-dermal cell patch(arrowsinT).
Co3::H2BYFPexpressionislost fromthemutant subepidermal layer
inscz-2(* inV).See also Figure S3.consistent with a role for SCZ in
cell fate separa-tion from heart-stage embryogenesis
onward.Non-Cell-Autonomous Regulationof Epidermis/Lateral Root Cap
Fate SeparationToprobetheidentityofthescz-2epidermis,weanalyzed the
promoter activity of the WEREWOLFgene (WER; [21]) and accumulation
ofSOMBRERO(SMB):GFPprotein[22]
inembryosandroots.WERhasaroleinepidermal cell
fatespecication,andWER[ERCFPisexpressedinlateral root cap and
epidermis, including thestemcell inwild-type (Figures 3A, 3B, 3E,
and3F). SMBrepresses stem cell-like divisions in the root cap, and
accord-ingly, SMB::SMB:GFP is expressed in nuclei of root cap
stemcelldaughtersandmaturingrootcaplayers(Figures3A,3B,3E, and 3F).
In scz-2 embryos, SMB:GFP is also expressed inepidermal cells, and
both markers even extend to the subepi-dermal cells (Figures3C and
3D). In the distal root meristem,theepidermal
expressionoverlapismaintained,butsubepi-dermal
expressionisobservedonlyinthedistalmost
cells,includingthestemcells(Figures3Gand3H, arrows). Next,we
combined GLABRA2 promoter expression marking devel-oping nonhair
cells (GL2::GUS; [23]) with transverse rootsectionstoexaminecell
numberandshapetypical
fortheirdifferentiationstatus(Figures3I3L).Cross-sectionsofscz-2root
meristemsreveal that vascular tissuesarenormal butthat adoublelayer
of endodermis-likecellsispresent (Fig-ure3K). Epidermal cell number
isreducedfromthenormalw22 rectangular-shaped cells to w12
ellipsoidal cells resem-bling the underlying cortex-like cells
(Figures 3I and 3K).An additional layer with lateral root cap
morphology surroundstheepidermis. GL2::GUSisweaklyexpressedinalmost
allepidermal and in fewsubepidermal cells (Figure 3K).
Strikingly,whereas epidermal cell les are tightly connected in
theCurrent Biology Vol 20 No 5454wild-type, they separate as
tissues mature in scz-2, a featurethat is restricted to root cap
cells in the wild-type (Figure 3L).Our data indicate that scz-2
root epidermal and subepidermaltissues are compromisedin fate
segregation. Importantly,thenearesttissueexpressingSCZisthecortex,
suggestingthat SCZ acts non-cell-autonomously in
epidermis/lateralroot cap fate separation.SCZ Is Required for
Cortex Fate SpecicationInitial
characterizationofsczrevealedectopicexpressionofepidermalmarkers
into the groundtissue and misexpressionof a ground tissue marker
[15]. To further investigate the fate
ofthemutantgroundtissuelayers, weanalyzedexpressionofendodermal
markers. SHR::SHR:GFPis expressedinstelecells,
andtheproteinmovestoaccumulateinthenuclei ofQC, ground tissue stem
cells, and endodermis, where it
acti-vatesitstarget,SCR(Figure3Q;[10]).Uponpericlinalasym-metricdivisionofthegroundtissuestemcelldaughter,SCRpromoter
activity is rapidly shut down in the outer cortex
cell(Figure3S,arrow;[24]).InlinewiththeQCdefectsbywhichwe selected
scz mutants, SHR:GFP and SCR::H2BYFP expres-sion is absent fromthe
QC region (Figures 3R and 3T,arrowhead). In the scz-2 ground
tissue, SHR:GFPis onlyobserved in the innermost layer adjacent to
the stele(Figure3R), whereasSCR::H2BYFPexpressionextendsintothenext
layer (Figure3T). Takingthesedatatogether withthecell
morphologycharacteristics, weconcludethattheselayersrepresent
endodermis. Inaddition, SCR::H2BYFP, likeFigure4. All
SCZFateSeparationFunctionsAreEnabledfrom the Cortex Expression
DomainArrowhead indicates QC.(AD) Five-day-old 35S::WOX5 (A) and
scz-2 35S::WOX5 (B)roots expressingQC46::ERYFP, and4-day-oldsmb-3
(C)andscz-2smb-3(D) roots. Introductionof
35S::WOX5orsmb-3correctlysegregatesQC(BandD) andcolumellastem cell
(blue arrow in D) fates.(EH) Four-day-old35S::SCZroots
expressingQC25 (E),SMB::SMB:GFP(F), WER[ERCFP(G), andCo2::H2BYFP(H;
false green).(I) Four-day-old 35S::GFP:SCZ roots showing nuclear
accu-mulationof GFP:SCZfusionprotein.
SCZoverexpressioninducesformationof additional
columellastemcells(bluearrows in E and F), an ectopic epidermal
layer (* in EI), andectopic Co2::H2BYFP expression (white arrow in
H) inprogenyofsupernumeraryepidermis/lateral rootcapstemcells
(white arrow in GI).(J) Wild-type N9094
root.(KandL)N9094[SCZ-complementedscz-2rootsdisplaynormal
tissuearrangementandmarkerexpression(K) andcorrectly segregated QC
and columella stem cell fates (L).(M) Role of SCZ in asymmetric
division. See text for details.gtc indicates ground tissue stem
cell. See also Figure S4.WER[ERCFPandSMB:GFP,evenextendsintothe
distal subepidermal tissue (Figure 3T, arrows).Apparently,
SCR::H2BYFP fails to segregate to theendodermis in a timely manner
and is maintainedindependentlyof SHRpresence. Thissuggeststhat
SCRpromoter
downregulationinwild-typeisaidedbycorticallyexpressedSCZ.
Similarly,the ectopic endodermis division can be explainedby
coexpression of SHRand SCRresulting in
divi-sionofthegroundtissue,asinwild-type,butintheabsenceof SCZ,
theouter cell lefailstoadopt cortex
fate.TofurthersubstantiateacausallinkbetweenSCZexpres-sion and
cortex fate determination, we analyzed Co2::H2BYFPandCo3::H2BYFP,
whicharehighlyexpressedinthecortexbut excluded from the QC and
undivided ground tissue stemcellsfromembryogenesisonward(Figures3U;
FigureS3A).Occasional weak expression in endodermal cells is
observedinthewild-type. Importantly, inscz-2seedlingroots,
bothmarkers are rarely expressed, and if so, onlythe
weakendo-dermal expression is observed (Figure 3V; Figure
S3B).Already during embryogenesis, Co2::H2BYFPexpression isexcluded
from the root ground tissue but remains expressedin the hypocotyl
ground tissue, consistent with the
root-specicdefectsinscz-2(Figure3N). WeconcludethatSCZis necessary
for the specication of root cortex cell
identity.TotestwhetherSCZissufcienttodeterminecortexfate,weectopicallyexpressedSCZfromthe35Spromoter.DistaltotheQCin35S::SCZroots,
thepresenceof anadditionalcolumella stem cell layer is apparent by
the absence of starchgranulesandlackof
SMB::SMB:GFPexpressionthatmarksdifferentiatedcolumella(Figures4Eand4F).
Examinationofradial tissue markers reveals the formation of an
ectopicepidermallayerthatshowsactivityoftheWER[ERCFPandGL2[ERGFP
epidermal markers (Figures 4G and 4H; FiguresS4AandS4B).
Cross-sectionsreveal occasional misexpres-sionof GL2[ERGFP(Figure
S4C). Inaddition, the
lateralrootcaplayerdoesnotsloughoffintheproximal
meristem(FiguresS4EandS4F). WeconcludethatoverexpressionofSCZ
introduces new cell fate separation defects.SCHIZORIZA Regulates
Cell Fate Separation455Analysis of SCZ protein expression in
35S::GFP:SCZ
overex-pressionlinesrevealsrelativelyhighlevelsinthecolumella,lateral
root cap, and distal epidermal tissues correlatingtoregionsof
ectopiccell division(Figure4I).
Interestingly,WER[ERCFPexpressionislost
fromtheepidermis/lateralrootcapstemcellregionwhereitisnormallyobserved(Fig-ure
4G; compare to Figures 3E and 3F), suggesting
inhibitionofepidermal/lateralrootcapfate.WER[ERCFPandGL2[ERGFP are
(re)expressed in outer layers proximally (Figure
4G;FigureS4B),suggestingtransientinhibitionofepidermalfatebyhighlevelsof
SCZor, alternatively, higher sensitivityofthe stemcell
regiontotheSCZeffect. Strikingly, ectopicexpressionof
Co2::H2BYFPisobservedinthesehigh-GFP:SCZ-expressing root cap cells
(Figure 4H), corroborating
thatSCZexpressionissufcienttoinducecortexfateandinhibitepidermal
and lateral root cap fates.Cortex-Expressed SCZ Rescues Mutant
DefectsThe lack of distal QC and columella fate separation
promptedus to examine embryonic QC25, QC46, QC184, SCR::H2BYFP,and
WOX5::ERGFP marker expression. Surprisingly,
allmarkerswereappropriatelyexpressedinscz-2throughoutembryogenesis,
indicating that cell identities are correctly
setupbutcannotbemaintained,whichcorrelateswithreducedstem cell
activity and root growth (Figures 3O and 3P;
FiguresS3CandS3D;datanotshown).Wetestedwhethercompro-misedstemcell
activitymight causethefailuretoseparatecell fates by crossingscz-2
toWOX5-overexpressingandsmb-3knockout mutantsthat
displayincreaseddistal stemcell activityandnumbers[22, 25].
Interestingly, scz-235S::WOX5andscz-2smb-3double-mutant
rootsdisplaywild-typeQCmorphologyandpositioningasvisualizedbystrongre-expressionof
QC46::ERYFPandSCR::H2BYFPintheQC(Figures4Aand4B; FigureS4D). Lackof
starchstaininginscz-2 smb-3 reveals presence of columella stem
cells, indica-tive of improved QC function (Figure 4D). However,
the radialcell fateseparationdefectsremainandroot
growthisnotrescuedinthesedouble-mutant combinations (Figures
4Band4D;FigureS4G). Ourdataindicatethatmixedcell fatesarenot
dependent onQCfunction. Theremaininggrowthdefect is reminiscent of
that observed when scr mutantsarecomplementedby QC-expressedSCR,
whichrestoresafunctional stemcell nichebutnotcell
fateseparationandgrowth [7].TodeterminewhereSCZactstopromotecell
fatesep-aration, we reintroducedthe gene in specic tissues
andexaminedcomplementationof thescz-2mutantdefect.
TheGAL4VP16-UAStransactivationsystem[26]
wasadoptedtodriveexpressionofSCZandtheERGFPreportervia(1)WOL(stele),
WOX5(QC), andGL2(epidermis) promotersinscz-2and (2) scz-2 N9135
(endodermis and QC) and N9094 (cortex,endodermis, and occasional
QC) enhancer trap crosses. Strik-ingly, thescz-2mutant
couldbecompletelyrescuedintheN9094[SCZ scz-2 line, with restoration
of growth, QC func-tion, groundtissue, andepidermis(Figures 4J-4L;
FiguresS4G and S4H). None of the other drivers was able to
comple-mentscz-2(FiguresS4IS4L),
indicatingthatSCZactivityisrequiredinthecortextoexert itseffect
onthecorrect fatesegregation in surrounding
tissues.OurstudiesindicatethatSCZactsasafatedeterminationandseparationfactor(Figure4M).
SCZactioninthegroundtissueinitial
determinesitsfatefromembryogenesisonwardandsuppressesepidermisandlateral
root capfateinthegroundtissue. Non-cell-autonomous
SCZactionmaintainsQC fate and suppresses columella fate in the QC
and in addi-tion segregates epidermis and lateral root cap fate,
putativelythrough a ground tissue-derived factor X. After the
SHR/SCR-induced periclinal division of the ground tissue
stemcelldaughtertakesplace, SHRpromotesendodermal fate. SCZaction
promotes cortex fate and suppresses endodermalfate,
possiblybydownregulationof
SHR/SCRexpressioninthematuregroundtissue. Furthermore, SCZcontinues
itsnon-cell-autonomous suppressionof epidermis andlateralroot
capfate in the mature ground tissue. The ability to
expressdifferentiatedcharacteristicsof epidermis, lateral root
cap,QC, andcolumellacell typesimpliesthat
thedifferentiationpathwaysforthesetissuesarestill
intactanddonotrequirea functional SCZ gene.Although SCZ belongs to
the Hsf family, diverse microarrayanalyses show that SCZ hardly
responds during stress situa-tions, suggestingthat it might
beintegratedintosignalingpathways not directly relatedtothe
heat-shock response[2729]. The shepherdmutant that harbors a
mutation oftheER-specicHSP90producesoral andshoot
meristemphenotypes closely resembling those of the clavata
(clv)mutants,showingmore diverse rolesfor Hsfsand Hspsthansolely in
stress signaling [30]. In addition to their role in
adap-tationtostress, yeastandanimal
HsfsandHspshavebeendemonstratedtobeinvolvedindifferentiationanddevelop-ment
(reviewedin[31]. Our discoverythat theArabidopsisSCZgeneiscrucial
forcellfateseparationsuggestsanovelmechanismof asymmetriccell
divisioncontrol byHsfs, inwhich key determinants that are
sequestered into bothdaughter cellsaredifferentiallydegraded.
Recent workonthe membrane protein BASL in stomatal precursors
cellsalsoindicatesthat noncanonical mechanismscontrol
plantasymmetric cell division [32]. Future work will have to
establishwhetherandhowsuchnovel
factorscanbeintegratedwithwell-established mechanistic frameworks
for asymmetric celldivision in other kingdoms of life.Supplemental
InformationSupplemental Informationincludesfour gures, twotables,
andSupple-mentalExperimentalProceduresandcanbefoundwiththisarticleonlineat
doi:10.1016/j.cub.2010.01.018.AcknowledgmentsWe are grateful to M.
Pernas and L. Dolan for sharing results prior to publi-cation. We
thank A. van Aelst of the Wageningen University Electron
Micros-copy Center for help with scanning electron microscopy.This
project wasconancedbytheCentreforBioSystemsGenomics,
whichispartoftheNetherlands Genomics Initiative/Netherlands
Organisation for ScienticResearch.Received: November 25,
2009Revised: January 1, 2010Accepted: January 6, 2010Published
online: February 18, 2010References1. Ten Hove, C.A., and Heidstra,
R. (2008). Who begets whom? Plant
cellfatedeterminationbyasymmetriccell
division.Curr.Opin.PlantBiol.11, 3441.2. Knoblich, J.A. (2008).
Mechanisms of asymmetric stemcell division. Cell132, 583597.3.
Benfey, P.N., and Scheres, B. (2000). Root development. Curr. Biol.
10,R813R815.4. Ju rgens, G., and Mayer, U. (1994). Arabidopsis. In
Embryos: Color Atlasof Development, J. Bard, ed. (London:
Mosby-Year Book).Current Biology Vol 20 No 54565. Aida, M., Beis,
D., Heidstra, R., Willemsen, V., Blilou, I., Galinha, C.,Nussaume,
L., Noh, Y.S., Amasino, R., andScheres, B. (2004).
ThePLETHORAgenesmediatepatterningof theArabidopsisroot stemcell
niche. Cell 119, 109120.6. Galinha, C., Hofhuis, H., Luijten, M.,
Willemsen, V., Blilou, I., Heidstra, R.,and Scheres, B. (2007).
PLETHORAproteins as dose-dependent masterregulators of Arabidopsis
root development. Nature 449, 10531057.7. Sabatini, S., Heidstra,
R., Wildwater, M., and Scheres, B. (2003). SCARE-CROW is involved
in positioning the stem cell niche in the Arabidopsisroot meristem.
Genes Dev. 17, 354358.8. Di Laurenzio, L.,Wysocka-Diller,J.,
Malamy, J.E.,Pysh,L.,Helariutta,Y., Freshour, G., Hahn, M.G.,
Feldmann, K.A., and Benfey, P.N.
(1996).TheSCARECROWgeneregulatesanasymmetriccell
divisionthatisessential for generating the radial organization of
the Arabidopsisroot. Cell 86, 423433.9. Helariutta, Y., Fukaki, H.,
Wysocka-Diller, J., Nakajima, K., Jung, J.,Sena, G., Hauser, M.T.,
andBenfey, P.N. (2000). TheSHORT-ROOTgenecontrolsradial
patterningoftheArabidopsisrootthroughradialsignaling. Cell 101,
555567.10. Nakajima, K., Sena, G., Nawy, T., and Benfey, P.N.
(2001).
IntercellularmovementoftheputativetranscriptionfactorSHRinrootpatterning.Nature
413, 307311.11. Schnittger, A., Ju rgens, G., and Hu lskamp, M.
(1998). Tissue layer andorganspecicity of
trichomeformationareregulatedby GLABRA1and TRIPTYCHON in
Arabidopsis. Development 125, 22832289.12. Szymanski, D.B., and
Marks, M.D. (1998). GLABROUS1 overexpressionand TRIPTYCHONalter the
cell cycle and trichome cell fate in Arabidop-sis. Plant Cell 10,
20472062.13. Nodine, M.D., Yadegari, R., and Tax, F.E. (2007). RPK1
and TOAD2 aretwo receptor-like kinases redundantly required for
Arabidopsis embry-onic pattern formation. Dev. Cell 12, 943956.14.
Sarin, S., OMeara, M.M., Flowers, E.B., Antonio, C., Poole,
R.J.,Didiano, D.,Johnston,R.J.,Jr.,Chang, S., Narula,S., andHobert,
O.(2007).GeneticscreensforCaenorhabditiselegansmutantsdefectivein
left/right asymmetric neuronal fate specication. Genetics
176,21092130.15. Mylona, P., Linstead, P., Martienssen, R., and
Dolan, L. (2002). SCHIZO-RIZA controls an asymmetric cell division
and restricts epidermal iden-tity in the Arabidopsis root.
Development 129, 43274334.16. Birnbaum, K., Shasha, D.E., Wang,
J.Y., Jung, J.W., Lambert, G.M., Gal-braith, D.W., and Benfey, P.N.
(2003). Agene expression map of the Ara-bidopsis root. Science 302,
19561960.17. Nawy, T., Lee, J.Y., Colinas, J., Wang, J.Y.,
Thongrod, S.C., Malamy,J.E., Birnbaum, K., andBenfey, P.N. (2005).
Transcriptional proleofthe Arabidopsis root quiescent center. Plant
Cell 17, 19081925.18. Brady, S.M., Orlando, D.A., Lee, J.Y., Wang,
J.Y., Koch, J., Dinneny,J.R., Mace, D., Ohler, U., andBenfey, P.N.
(2007). Ahigh-resolutionroot spatiotemporal map reveals dominant
expression patterns.Science 318, 801806.19. Nover,L.,Bharti,K.,Do
ring,P.,Mishra,S.K.,Ganguli,A.,andScharf,K.D. (2001). Arabidopsis
and the heat stress transcription factor
world:Howmanyheatstresstranscriptionfactorsdoweneed?Cell
StressChaperones 6, 177189.20. Dolan, L., Janmaat, K., Willemsen,
V., Linstead, P., Poethig, S., Roberts,K., and Scheres, B. (1993).
Cellular organisation of the Arabidopsis thali-ana root.
Development 119, 7184.21. Lee, M.M., andSchiefelbein, J. (1999).
WEREWOLF, aMYB-relatedproteinin Arabidopsis, is a
position-dependent regulatorofepidermalcell patterning. Cell 99,
473483.22. Willemsen, V., Bauch, M., Bennett, T., Campilho, A.,
Wolkenfelt, H.,
Xu,J.,Haseloff,J.,andScheres,B.(2008).TheNACdomaintranscriptionfactorsFEZandSOMBREROcontrol
theorientationof cell divisionplane in Arabidopsis root stem cells.
Dev. Cell 15, 913922.23. Masucci, J.D., Rerie, W.G., Foreman, D.R.,
Zhang, M., Galway, M.E.,Marks, M.D., and Schiefelbein, J.W. (1996).
The homeobox
geneGLABRA2isrequiredforposition-dependentcelldifferentiationintheroot
epidermis of Arabidopsis thaliana. Development 122, 12531260.24.
Wysocka-Diller, J.W., Helariutta, Y., Fukaki, H., Malamy, J.E., and
Ben-fey, P.N. (2000). MolecularanalysisofSCARECROWfunctionrevealsa
radial patterning mechanismcommon to root and shoot.
Development127, 595603.25. Sarkar, A.K., Luijten, M., Miyashima,
S., Lenhard, M., Hashimoto, T.,Nakajima, K., Scheres, B., Heidstra,
R., and Laux, T. (2007). Conservedfactors regulate signalling in
Arabidopsis thaliana shoot and root stemcell organizers. Nature
446, 811814.26. Haseloff, J. (1999). GFP variants for multispectral
imaging of living cells.Methods Cell Biol. 58, 139151.27. Miller,
G., and Mittler, R. (2006). Could heat shock transcription
factorsfunction as hydrogen peroxide sensors in plants? Ann. Bot.
(Lond.) 98,279288.28. Swindell, W.R., Huebner, M., andWeber, A.P.
(2007).
TranscriptionalprolingofArabidopsisheatshockproteinsandtranscriptionfactorsreveals
extensive overlap between heat and non-heat stress
responsepathways. BMC Genomics 8, 125.29. von Koskull-Do ring, P.,
Scharf, K.D., and Nover, L. (2007). The diversityof plant heat
stress transcription factors. Trends Plant Sci. 12, 452457.30.
Ishiguro, S., Watanabe, Y., Ito, N., Nonaka, H., Takeda, N., Sakai,
T., Ka-naya,H.,andOkada,K.(2002).SHEPHERDis
theArabidopsisGRP94responsiblefortheformationoffunctional
CLAVATAproteins. EMBOJ. 21, 898908.31. Morange, M. (2006). HSFs in
development. Handb Exp Pharmacol (172):153169.32.
Dong,J.,MacAlister,C.A.,andBergmann,D.C.(2009).BASLcontrolsasymmetric
cell division in Arabidopsis. Cell 137, 13201330.33. Ikeda, M., and
Ohme-Takagi, M. (2009). A novel group of transcriptionalrepressors
in Arabidopsis. Plant Cell Physiol. 50, 970975.34. Till, B.J.,
Reynolds, S.H., Greene, E.A., Codomo, C.A., Enns, L.C., John-son,
J.E., Burtner, C., Odden, A.R., Young, K., Taylor, N.E., et al.
(2003).Large-scale discovery of induced point mutations with
high-throughputTILLING. Genome Res. 13, 524530.SCHIZORIZA Regulates
Cell Fate Separation457
