L1 Division and Differentiation Patterns Influence Shoot Apical Meristem Maintenance1

14 Pages·2006·0.65 MB·English

L1 Division and Differentiation Patterns Influence Shoot
Apical Meristem Maintenance1
Sharon Kessler2, Brad Townsley, and Neelima Sinha*
Section of Plant Biology, University of California, Davis, California 95616
Plant development requires regulation of both cell division and differentiation. The class 1 KNOTTED1-like homeobox
(KNOX) genes such as knotted1 (kn1) in maize (Zea mays) and SHOOTMERISTEMLESS in Arabidopsis (Arabidopsis thaliana)
play a role in maintaining shoot apical meristem indeterminacy, and their misexpression is sufficient to induce cell division
andmeristemformation.KNOXoverexpressionexperimentshaveshownthatthesegenesinteractwiththecytokinin,auxin,
and gibberellin pathways. The L1 layer has been shown to be necessary for the maintenance of indeterminacy in the
underlyingmeristem layers.This workexploresthepossibility thatthe L1affects meristemfunction bydisrupting hormone
transport pathways. The semidominant Extra cell layers1 (Xcl1) mutation in maize leads to the production of multiple
epidermallayersbyoverproductionofanormalgeneproduct.Meristemsizeisreducedinmutantplantsandmorecellsare
incorporated into the incipient leaf primordium. Thus, Xcl1 may provide a link between L1 division patterns, hormonal
pathways,andmeristemmaintenance.WeuseddoublemutantsbetweenXcl1anddominantKNOXmutantsandshowedthat
Xcl1suppressestheKn1phenotypebuthasasynergisticinteractionwithgnarley1androughsheath1,possiblycorrelatedwith
changes in gibberellin and auxin signaling. In addition, double mutants between Xcl1 and crinkly4 had defects in shoot
meristem maintenance. Thus, proper L1 development is essential for meristem function, and XCL1 may act to coordinate
hormonaleffects with KNOX gene function at theshoot apex.
Cell division and differentiation are tightly linked (Sinhaetal.,1993;Chucketal.,1996).Inmaize,ectopic
processes in plant development. In recent years, sev- expressionoftheKNOXgeneskn1,roughsheath1(rs1),
eralgenesthatareinvolvedincontrollingshootapical andgnarley1(gn1)indominantmutantscausesabnor-
meristem(SAM)celldivisionanddifferentiationhave mal cell divisions in the leaf (Becraft and Freeling,
been identified. The class 1 KNOTTED1-like homeo- 1994; Schneeberger et al., 1995; Foster et al., 1999).
box(KNOX)genessuchasknotted1(kn1)inmaize(Zea These results indicate that misexpression of KNOX
mays) and SHOOTMERISTEMLESS (STM) in Arabi- genes leads to similar outcomes and is sufficient to
dopsis(Arabidopsisthaliana)areexpressedthroughout induce celldivisionand meristemformation.
the SAM and down-regulated in both incipient leaf The control of cell division and differentiation by
primordia and developing leaves (Vollbrecht et al., KNOX genes probably occurs through modulation of
1990;Smithetal.,1992;Jacksonetal.,1994),indicating hormonal pathways. KNOX overexpressing tobacco
that these genes play a role in maintaining SAM leaves display a delay in senescence similar to that
indeterminacy. Loss-of-function mutations in Arabi- seeninplantswithincreasedcytokininlevels(Kusaba
dopsis STM lead to loss of shoot meristems (Barton et al., 1998). Expressing KN1 under the control of the
and Poethig, 1993; Long et al., 1996). In addition, senescence-associatedgenepromoterintobaccoleads
overexpression of KN1 in tobacco (Nicotiana tabacum) to increased levels of cytokinin and a delay in leaf
and another class 1 KNOX gene, KNAT1 (also known senescence,suggestingthatKN1actstoincreasecyto-
as BREVIPEDICELLUS [BP]), in Arabidopsis lead to kininlevels(Orietal.,1999).Intobacco,ectopicKNOX
theproductionoflobedleaveswithectopicmeristems gene expression directly down-regulatesthe gibberel-
lic acid biosynthesis gene, GA20 oxidase, through
bindingoftargetsitesinthepromoterandfirstintron
1ThisworkwassupportedbytheNationalScienceFoundation (Sakamotoetal.,2001).Furthermore,inducibleexpres-
(Plant Cell Biology Training Grant and NSF-IBN 0316877 and sion of maize KN1 in Arabidopsis leads to the down-
0344743toN.S.)andbytheUniversityofCaliforniaatDavis(Jastro regulation of GA20 oxidase (Hay et al., 2003). KNOX
Shields,ElsieStocking,andRosalindRussellfellowshipstoS.K.). genes have also been shown to coordinately regulate
2Presentaddress:InstituteforPlantBiology,UniversityofZurich, GA and cytokinin activity in Arabidopsis (Jasinski
Zollikerstrasse107,8008Zurich,Switzerland. et al., 2005; Yanai et al., 2005). The maize semaphore
*Corresponding author; e-mail [email protected]; fax 530– mutation leads to ectopic expression of KNOX genes
752–5410.
andalsodisruptspolarauxintransport(Scanlonetal.,
Theauthorresponsiblefordistributionofmaterialsintegraltothe
2002).
findings presented in this article in accordance with the policy
KNOX genes have also been implicated in cell
describedintheInstructionsforAuthors(www.plantphysiol.org)is:
differentiation pathways. For example, bp mutants
NeelimaSinha([email protected]).
Article,publicationdate,andcitationinformationcanbefoundat show an increase in lignin biosynthetic gene expres-
www.plantphysiol.org/cgi/doi/10.1104/pp.105.076075. sion (Mele et al., 2003). In contrast, plants mutant for
Plant Physiology, August 2006, Vol. 141, pp. 1349–1362, www.plantphysiol.org (cid:1) 2006 American Society of Plant Biologists 1349
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Kessler et al.
therice(Oryzasativa)OSH15gene(orthologoustoBP) toderm cells divide predominantly in the anticlinal
shownomeristemdefectsbuthavereducedhypoder- plane. The Extra cell layers1 (Xcl1) mutation causes
mal sclerenchyma (Sato et al., 1999), suggesting a protoderm cells to divide in oblique periclinal orien-
difference in response between monocots and dicots. tations, which leads to the production of multiple
A genetic approach may help to dissect out these epidermallayers(Kessleretal.,2002).Dosageanalysis
diverse influences of KNOX genes and plant hor- oftheXcl1mutationshowedthatitisahypermorphic
moneson shootmorphogenesis. mutation leading to overproduction of a normal gene
HigherangiospermSAMsareorganizedintolayers, product. Increased levels of XCL1 are thought to
with the L1 (or outer layer, also known as the tunica) prolong cell division during leaf development, such
dividing predominantly in the anticlinal plane and that cells that have already perceived epidermal dif-
giving rise to the epidermis, and the internal layer(s), ferentiation signals divide in altered planes and dif-
theL2inmaizeandL2andL3inArabidopsis,making ferentiate according to lineage instead of position.
up the body (or corpus) of the meristem. Mosaic While the most apparent Xcl1 phenotype is the mul-
analysis experiments showed that in Kn1 mutants, tipleepidermallayersthatleadtothickerleaves,Xcl1
ectopicexpressionofKN1intheinnerlayersoftheleaf also affects the SAM. Xcl1 meristems are 35% shorter
was sufficientto induce periclinal divisionsin theL1, than normal sibling meristems and also recruit more
leading to knot formation (Hake and Freeling, 1986; cellsintotheincipientleafprimordia(P ;Kessleretal.,
0
Sinha and Hake, 1990). The KN1 protein has been 2002). This indicates that XCL1 may not only be
shown to move between the L2 and L1 in meristems involved in controlling protoderm division and dif-
(Lucasetal.,1995;Kimetal.,2002,2003);however,the ferentiation patterns, but also plays an earlier role in
functional significance of this intercellular transport regulating meristem size and leaf initiation and may
in meristem maintenance has yet to be determined. providealinkbetweenL1divisionpatterns,hormonal
Since knots form over major lateral veins, it has been pathways,and meristemmaintenance.
suggested that auxin signaling may play a role in In this study, we utilized double mutants between
conditioningtheknphenotype(Gelinasetal.,1969).In Xcl1 and dominant KNOX mutants to gain an under-
arecentstudyontherelationshipbetweenpolarauxin standingofthemechanismscoordinatingcelldivision
transport and leaf initiation, auxin was proposed to anddifferentiationandtotrytoteaseaparttheeffects
movefromleafprimordiatotheapicaldomethrough of these diverse KNOX genes on downstream parts
the L1 layer (Benkova et al., 2003; Reinhardt et al., of the pathway. Xcl1 suppresses the Kn1 phenotype,
2003b). The role of the L1 in meristem activity is just possibly through modulation of gibberellin (GA)
beginningtobeunderstood.Microsurgicalremovalof levels. In combination with Xcl1, both Gn1 and Rs1
theL1intomato(Lycopersiconesculentum)apicesleads displayedsynergisticphenotypesinwhichleafdevel-
to a cessation of leaf initiation and an eventual differ- opmentandmeristemmaintenancewereaffectedina
entiationofcellsintheunderlyingcentralzoneofthe mannersimilartothatseenwhenseedlingsaretreated
meristem (Reinhardt et al., 2003a). Once the L1 of the with polar auxin transport inhibitors. In addition,
meristem is removed, it never grows back. Thus, double mutants between Xcl1 and another epidermal
underlyingcelllayerscannotberespecifiedasL1cells. division and differentiation mutant, crinkly4 (cr4),
This suggests a strict lineage-based maintenance of failed to maintain a shoot meristem. Thus, proper L1
theL1andalsoindicatesthattheL1isnecessaryforthe development is essential for meristem function, and
maintenanceofindeterminacyintheunderlyingmer- XCL1mayplayaroleincoordinatinghormonaleffects
istemlayers.AdditionalevidenceforanL1roleinmeri- atthe shoot apex.
stem function comes from a study in which a double
mutant combination of two Arabidopsis L1-specific
glabra 2-like homeobox genes, protodermal factor 2 and
Arabidopsisthalianameristemlayer1,leadstotheforma- RESULTS
tion of a disorganized shoot apex during embryogen-
Xcl1
esis and problems in leaf initiation and development.
Leavesthatareinitiatedbythedoublemutantsdonot Xcl1 is a semidominant mutation that affects both
haveanepidermis,indicatingthatthesetwogenesare meristems and leaves (Kessler et al., 2002). In wild-
necessary for epidermal identity (Abe et al., 2003). typeSAMs,Plastochron3(P3)andhigherleafprimor-
These instances of L1 involvement in meristem func- dia completely encircle the SAM (Fig. 1A), while in
tion have relied on ablation of the L1 by physical or Xcl1, leaves up to P4 do not completely encircle the
genetic mechanisms. It is possible that other, more SAM (Fig. 1B), indicating that Xcl1 leaves are narrow
subtle changes involving division or differentiation fromearlyindevelopment.ByP2,wild-type(Fig.1,C
patternsintheL1couldalsoaffectmeristemfunction, and E) and Xcl1 (Fig. 1, D and F) leaf primordia look
perhapsbydisruptinghormonetransportpathways. very similar, although the apical dome is shorter in
Maize mutants defective in epidermal division and Xcl1 than in wild type. The most conspicuous Xcl1
differentiation are useful for elucidating the mecha- phenotype is the presence of multiple epidermal
nisms responsible for coordinating cell division and layers(Fig.1,GcomparedtoH)thathavebeenshown
differentiation. During epidermal development, pro- to arise from oblique divisions in the L1 early in leaf
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Epidermal Differentiation and KNOTTED1-Like Homeobox Genes
development (Kessler et al., 2002). In addition, Xcl1
mutantleaveshavefewerintermediateveins(Fig.1I).
Kn1;Xcl1
The reduction in meristem height seen in the Xcl1
singlemutants(Kessleretal.,2002)indicatesthatXcl1
may play a role in regulating the function of Kn1, a
class1KNOXgeneexpressedintheSAM.Toexamine
this possibility, double mutants were made between
Xcl1 and two different dominant alleles of Kn1,
Kn1-N2, and Kn1-O. Both of these mutations cause
ectopic expression of KN1 in leaves that leads to the
formationoflargeprotrusions,orknots,overthemajor
veins. Kn1-O is a spontaneous mutation that was
caused by a duplication at the KN1 locus leading to a
dominant, neomorphic phenotype (Veit et al., 1990).
Largeknots protrudefromtheabaxial side ofthe leaf
at the blade-sheath junction and sheath identity is
pushed up into the blade (Fig. 2A). Ligule displace-
mentintothebladeandectopicligulesontheadaxial
sideoftheblademidribarealsoprominentfeaturesof
Kn1-O leaves (Gelinas et al., 1969; Hake and Freeling,
1986; Sinha and Hake, 1994). These phenotypes are
mostevidentinadultleaves,althoughliguledisplace-
ment can alsobe seen in juvenile leaves.
Xcl1;Kn1-O double mutants have a suppressed
Kn1-O phenotype. Instead of a huge mass of knotted
tissue at the blade-sheath boundary (Fig. 2A), one
largeprotrusionwasseenineveryleafnearthetopof
the sheath in double mutants (Fig. 2B). However,
ectopic ligules were still seen on the adaxial blade
midrib, indicating that this aspect of the Kn1-O phe-
notype was not affected by the presence of the Xcl1
mutation(Fig.2B,arrow).Thenumberofknotsinthe
sheathandatthebaseofthebladeweredeterminedin
Kn1-OsinglemutantsandKn1-O;Xcl1doublemutants
attwodifferentpositionsontheplant(Fig.2,EandF).
Both the third and sixth leaf from the tassel had
significantly fewer knots when Xcl1 was present, in-
dicating that the effect of Xcl1 on knot formation was
similar in all adult leaves.
While the Kn1-O allele primarily affects the blade/
sheath junction of adult leaves, the Kn1-N2 allele
causes knots to form primarily on leaf blades (Fig.
2C). In Kn1-N2, both juvenile and adult leaf blades
have a severe knotted phenotype. Since the timing of
Kn1-N2 knot formation is different than in Kn1-O,
crosses were made with Xcl1 to see if there was a
Figure1. Wild-type(A,C,E,andG)andXcl1(B,D,F,andH)meristem differenceinknotsuppression.InKn1-N2;Xcl1double
and leaf phenotypes. A, Wild-type meristem with P3 completely mutants, knot development in adult leaf blades was
encircling the meristem and margins overlapping. B, Xcl1 meristem also significantly inhibited (Fig. 2D). Seedling leaves
withP3marginsthatdonotoverlap.C,Dissectedwild-typemeristemat showed a similar effect in that double mutants had
theP1/P2stage.D,DissectedXcl1meristematP1/P2showingshorter onlyonetothreeknotsonleaves1and2,whileKn1-N2
apicaldome.E,Medianlongitudinalsectionofawild-typemeristem
singlemutantshadmorethan10knotsontheseleaves.
showing vascular traces (*) into P2 and P3. F, Median longitudinal
It is interesting to note that while both Kn1-N2 and
section of an Xcl1 meristem showing vascular traces (*) extending
Kn1-Omutantleavesarewiderthanwild-typeorXcl1
higherintothemeristem.G,Transversesectionofawild-typeleaf.H,
leaves,doublemutantKn1;Xcl1leavesarenarrowlike
TransversesectionofanXcl1leafshowingmultipleepidermallayers.I,
Averagenumberofveinsatthreedifferentpositionsinwild-type(solid Xcl1leaves(Fig.2,A–D).SegregationanalysisonanF2
bars)andXcl1(stripedbars)leaves.Scalebars5100mm. Kn1-N2;Xcl1 family showed that while most of the
Plant Physiol. Vol. 141, 2006 1351
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Copyright © 2006 American Society of Plant Biologists. All rights reserved.
Kessler et al.
viduals were genotyped based on the presence of an
rDotted transposable element inserted in the fourth
intron of Kn1-N2 (Sinha, 1990; N. Sinha, unpublished
data).Primersthatspannedexon/exonjunctionswere
used to specifically amplify cDNAs to examine the
expression of KN1 in expanded leaf 1 of each of the
mutant classes (Fig. 3A) by semiquantitative reverse
transcription (RT)-PCR with GLYCERALDEHYDE-
3-PHOSPHATE DEHYDROGENASE (ZmGAPDH) as
the constitutively expressed control (Richert et al.,
1996).Asexpected,inwild-typeandXcl1individuals,
wedidnotdetectKN1transcriptinleaves,butdouble
mutant individuals with knots had KN1 transcript at
similar levels to that seen in Kn1-N2 leaves. In seed-
lings that were homozygous for Xcl1 and heterozy-
gousforKn1-N2butdidnothaveanyvisibleknotson
leaf1,KN1transcriptwasdetectedatsimilarlevelsto
thatseeninindividualswithknots(Fig.3B),indicating
that Xcl1 suppression of the Kn1-N2 phenotype in
leaves isnotatthe transcriptional level.
GAlevelshavebeenshowntobereducedintobacco
leavesthatoverexpressNicotianatabacumhomeobox15
(NTH15; Tamaoki et al., 1997), and NTH15 was sub-
sequently shown to directly suppress the GA biosyn-
thesis gene GA20 oxidase (Sakamoto et al., 2001). In
addition,inducibleexpressionofmaizeKN1inArabi-
dopsis led to down-regulation of GA20 oxidase (Hay
etal.,2003).GAlevelshavebeenshowntobelowinthe
SAMasaresultoftheactivityofGA2oxidase,andthis
enzymewasproposedtobeKNOXregulated(Jasinski
et al., 2005; Yanai et al., 2005). This data led us to hy-
pothesizethatchangingGAlevelswouldalsoaffectthe
severity of the Kn1 phenotype. Applying GA to seed-
lings suppressed the Kn1-N2 phenotype, while the
presenceoftheGAbiosynthesisinhibitor,uniconazole,
enhancedtheKn1-N2phenotype(Fig.3D).Theresults
indicatethatthesuppressionofKn1-N2byXcl1could
Figure2. Xcl1suppressesKnottedmutations.A,Kn1-Oadultleafwith
beatthelevelofGAbiosynthesisorsignaling(Jasinski
knotsattheblade-sheathjunction.B,Kn1-O;Xcl1doublemutantwith
et al., 2005; Yanai et al., 2005). We identified maize
suppressed knots at the blade-sheath junction. Ectopic ligule on the
expressed sequence tags that encode a GA20 oxidase
adaxialsideofleafbladeisindicatedbyanarrow.C,Kn1-N2adultleaf
hasknotsprimarilyonthebladeandnotthesheath.D,Kn1-N2;Xcl1 geneanda GA2oxidase gene.Levels of theZmGA20
adultleafhasareducednumberofknotsontheblade.E,Comparison oxidaseandZmGA2oxidasegeneswereexaminedby
ofthenumberofknotsinKn1-O(blue)andKn1-O;Xcl1(pink)leaf3
(fromthetassel),blade(solid),andsheath(striped).F,Comparisonof
thenumberofknotsonleaf6(fromtassel).
TableI. SegregationofKn1-N2;Xcl1family
InanF Kn1-N2;Xcl1family,twoclasses(bold)deviatedsignificantly
phenotypic classes were represented in the expected 2
fromthenumbersexpectedforanadditiveinteraction.FewerKn1-N2/?;
proportions,twoclassesdeviatedsignificantlyfromthe Xcl1/Xcl1(strongXcl1phenotype,verymildknots)andmoreKn1-N2/
numbersexpectedforanadditiveinteraction(TableI). kn1-N2;Xcl1/Xcl1(strongXcl1phenotype,noknots)individualsthan
Fewer Kn1-N2/?;Xcl1/Xcl1 (strong Xcl1 phenotype, expected were found. The x2 value rejects the segregation ratio/null
verymild knots) thanexpected were found, and more hypothesis.Sx2518.1;criticalvalue516.75(P50.005,5degreesof
Kn1-N2/Kn1-N2;Xcl1/Xcl1 (strong Xcl1 phenotype, no freedom).
knots) individuals than expected were found. This re- Expected Expected Observed
Genotype x2
sultindicatesthatinsomehomozygousXcl1seedlings, Ratio No. No.
the Kn1-N2 mutation was present, but knot formation kn1/kn1;xcl1/xcl1 1/16 3.75 5 0.42
wascompletelysuppressed. kn1/kn1;Xcl1/xcl1 2/16 7.5 12 2.7
The suppression of Kn1 leaf phenotypes when Xcl1 kn1/kn1;Xcl1/Xcl1 1/16 3.75 10 10.4
is present suggests that Xcl1 may affect the ectopic Kn1/?;xcl1/xcl1 3/16 11.25 10 0.14
expressionofKN1indoublemutants.Thishypothesis Kn1/?;Xcl1/xcl1 6/16 22.5 18 0.9
Kn1/?;Xcl1/Xcl1 3/16 11.25 5 3.5
was tested in a Kn1-N2;Xcl1 segregating family. Indi-
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Epidermal Differentiation and KNOTTED1-Like Homeobox Genes
Figure 3. Kn1-N2;Xcl1 seedling pheno-
types.A,Thenumberofknotsonseedling
leavesisreducedwhenXcl1ispresent.B,
Semiquantitative RT-PCR of KN1 expres-
sion reveals that KN1 RNA is present in
double mutant leaves with suppressed
knots.Numbersinbracketsindicatenum-
berofPCRcycles.ControlRT-PCRforthe
constitutively expressed ZmGAPDH. C,
GA20 oxidase levels are increased when
Xcl1ispresentanddecreasedinKn1-N2
single mutants. D, Knots per leaf on the
third leaf of Kn1-N2 heterozygote seed-
lings treated with Tween 20 (control),
Uniconazole,orGA.Differencesbetween
Uniconazole and GA treatments were
foundtobestatisticallysignificantatP5
0.01anddifferencesbetweencontroland
GAatP50.05usingANOVA.Errorbars
areSEs,n53.E,DetectionofGA2oxidase
RNA levels with QRT-PCR in wild-type
(wt),Xcl1,Kn1-N2(Kn1),andXcl1/Kn1-N2
seedlings. The data represents two repli-
cateruns.RNAlevelswerenormalizedto
ZmGAPDHmRNAandareshownrelative
towild-typelevels.BarsrepresentSEsover
fourtechnicalreplicates.
RT-PCRinKn1-N2;Xcl1segregantstodetermineifXcl1 levelsduringleafdevelopment,allowingforsuppression
isregulatingKNOXactivitybyalteringGAlevels(Fig. oftheKn1-N2phenotypeinleaves.
3C).WhilenoapparentalterationwasseenintheGA20
oxidaselevels,quantitativeRT-PCR(QRT-PCR)revealed
Gn1;Xcl1 and Rs1;Xcl1 Double Mutants
highinductionofGA2oxidaseintheKn1-N2genotype,
andthepresenceoftheXcl1mutationdrasticallyreduced Gn1 and Rs1 aredominant mutations in duplicated
theGA2oxidasetranscriptlevels(Fig.3E)anddecreased KNOXgenes(BecraftandFreeling,1994;Schneeberger
thenumberofknots,indicatingthatXcl1maychangeGA etal.,1995;Fosteretal.,1999).Bothofthesemutations
Plant Physiol. Vol. 141, 2006 1353
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Copyright © 2006 American Society of Plant Biologists. All rights reserved.
Kessler et al.
affect cell division at the blade-sheath junction of
TableII. SegregationofGn1;Xcl1F family
leaves. Since Gn1 and Rs1 have similar phenotypes 2
FourF phenotypicclasseswereobservedinaGn1;Xcl1F popu-
to Kn1, we wanted to determine if Xcl1 would also 2 2
lationina9:3:3:1ratio.Thelargestphenotypicclassofseedling-lethal
suppress these phenotypes. When an F2 Gn1;Xcl1 individualsdidnotresemblesingleXcl1orGn1mutants.Thex2value
population was examined, four phenotypic classes failstorejectthesegregationratio/nullhypothesis.Sx254.2;critical
wereobservedina9:3:3:1ratio(Fig.4A;TableII).The value57.81(P50.05).
largest phenotypic class was composed of seedlings
withseverelyreducedandagravitropicshootandroot Genotype Expected Expected Observed x2
Ratio No. No.
development. This group of seedling-lethal individ-
gn1/gn1;xcl1/xcl1 1/16 5.8 8 0.5
ualsdidnotresemblesingleXcl1orGn1mutantsand
Gn1/?;xcl1/xcl1 3/16 17 10 2.8
represented genotypes that were either homozygous
gn1/gn1;Xcl1/? 3/16 17 18 0.1
or heterozygous for both Gn1 and Xcl1. Rs1;Xcl1 Gn1/?;Xcl1/? 9/16 52 57 0.8
double mutants had similar seedling phenotypes to
Gn1;Xcl1 seedlings (Fig. 4, A and B). The seedling-
lethal phenotypes of Gn1;Xcl1 and Rs1;Xcl1 double stages of development (Fig. 4, C and D compared to
mutants suggest that meristem maintenance may be Fig. 1, A and C). However, Gn1;Xcl1 meristems are
affected in the double mutants. Scanning electron strikingly different than either Gn1 or Xcl1 single
microscopy (SEM) was used to examine SAMs in mutantmeristems.Indoublemutants,theshootapices
each of the phenotypic classes. Gn1 meristems are were2to3timeslargerthanapicesofeithersinglemu-
very similar to wild-type meristems at comparable tant at similar stages of development (Fig. 4, E and
F). When dissected down to P4, Gn1;Xcl1 meristems
are very exposed, none of the leaves completely en-
circle the meristem, and the older leaves are often
bifurcated (Fig. 4F). In addition, extremely reduced
leaves with radial tips and aberrant phyllotaxy were
sometimes observed in double mutants (Fig. 4, E and
G). Rs1;Xcl1 meristems were very similar in appear-
ance to the Gn1;Xcl1 meristems. SEMs showed that
Rs1;Xcl1apiceswereextremelylargeandleafprimor-
diadidnotencirclethemeristem(Fig.4H).Inaddition,
somedoublemutantmeristemsfailedtoinitiateleaves
along one face of the meristem (Fig. 4I). The apical
domeoftenended in an acute tip, andtherewas con-
siderableshootexpansionbetweensuccessiveleafini-
tiation events (Fig. 4, H and I). These results indicate
thatthepresenceofbothGn1orRs1andXcl1leadsto
unique problems in normal meristem maintenance
andleaf initiation.
Polyclonal antibodies against KN1 and RS1 recog-
nize multiple class 1 KNOX genes and have been
used as markers for meristem identity in maize and
other species (Smith et al., 1995; Scanlon et al., 1996;
Bharathan et al., 2002). We performed immunolocali-
zations with a polyclonal KN1 antibody to analyze
meristem identity in single and double mutants. In
normal and Xcl1 meristems, KNOX proteins were
localizedtothestemandSAMandwerenotdetected
Figure4. SeedlingandmeristemphenotypesofGn1;Xcl1andRs1;Xcl1
in developing leaves (Fig. 5, A and B). In Gn1;Xcl1
doublemutants.A,Gn1seedlingshaveligulardisplacementleadingto
erect leaves 1 and 2 compared to wild-type and Xcl1 seedlings. doublemutants,theregionofsmall,denselycytoplas-
Gn1;Xcl1doublemutants(atright)haveextremelyreducedleaves,are mic cells expressing KNOX proteins normally associ-
agravitropic,anddonotinitiatelateralroots.B,Rs1seedlingsaresimilar ated with meristematic identity was nearly absent
toGn1.Alessseveredoublemutantwithoneleafisshownattheright.C, at the pointed shoot apex, and the internodes were
Gn1meristemattheP3stageisverysimilartowildtype(Fig.1A).D,Gn1 greatly expanded (Fig. 5, E and F). In contrast to Gn1
meristem dissected to P1/P2 stage. E, Gn1;Xcl1 apex appears much single mutants (Fig. 5, C and D), KNOX protein was
largerthanGn1andleavesdonotencirclethemeristem.Adelayedleaf
not observed in double mutant leaves (Fig. 5E), indi-
withradialtipisalsopresent(arrow).F,RotatedviewofmeristeminE
cating that Xcl1 may suppress the ectopic expression
revealsabifurcatedleafprimordium.G,DissectedGn1;Xcl1meristem
ofGN1andotherKNOXproteinsinleaves.Thus,not
(same as E) with aberrant phyllotaxy and leaf shape. H, Rs1;Xcl1
only does Xcl1 reduce ectopic KNOX protein expres-
meristemwithaberrantphyllotaxyandelongatedinternodes.I,Naked
Rs1;Xcl1meristemthathasfailedtoinitiateleafprimordiaatregular sion in Xcl1;Gn1 leaves, but it also suppresses KNOX
intervals.Scalebarsforallsections5200mm. expression in double mutant meristems, leading to a
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Epidermal Differentiation and KNOTTED1-Like Homeobox Genes
failure of meristem maintenance. Kn1-N2;Xcl1 double
mutant meristems had no problems in maintaining
meristemidentity,andKNOXexpressionwasdetected
in leaves (Fig. 3B).
GN1 and RS1 transcripts were absent from ex-
panded Xcl1 seedling leaves and present at normal
levels in Xcl1 meristems (Fig. 5G). This indicates that
Xcl1 does not directly affect GN1 and RS1 transcript
levels. The similarity of the Xcl1;Gn1 and Xcl1;Rs1
doublemutantmeristemstoArabidopsismutationsin
the PIN1 auxin efflux carrier and to Arabidopsis and
tomato meristems treated with polar auxin transport
inhibitors(Galweileretal.,1998;Reinhardtetal.,2000;
Stieger et al., 2002) indicates that auxin signaling
pathways may be compromised in these double mu-
tant seedlings. The transcript levels of the PIN family
of polar auxin transport genes have been shown to
correlatewithchangesinproteinlevelsinresponseto
perturbations in auxin levels (Benkova et al., 2003;
Furutani et al.,2004;Peeretal., 2004). Weidentified a
maizePIN1orthologinthepublicdatabasesandused
RT-PCR to examine PIN1 levels in mutant leaves and
meristems. ZmPIN1 transcript levels were reduced in
Xcl1 plants (Fig. 5I) and in Xcl1;Gn1 meristems (Fig.
5H),indicatingthatauxintransportpathwaysmaybe
compromised in the double mutants.
Vascular Defects in Xcl1
The synergistic interactions between Xcl1 and Gn1
or Rs1 indicate that Xcl1 itself may exhibit auxin
transportdefects.Auxinflowisknowntobeinvolved
in vascularpatterning (Berleth et al., 2000). Wild-type
maize leaves have up to 30 minor veins between the
major lateral veins (Freeling and Lane, 1994). In con-
trast,Xcl1leaveshavesignificantlyfewerminorveins
between the major lateral veins when compared to
Figure5. AnalysisofgeneexpressioninGn1/Xcl1doublemutants.A
wild type (Fig. 1I). When vascular development was
toF,Immunolocalizationswithanti-KN1antibodiesonseedlingSAMs.
examined in Xcl1 apices, departures from normal
A,Wild-typemeristemshowingnormalKNOXexpressionintheSAM
vascularpatterningwereseen.Innormalmaizeapices,
andunexpandedstemanddown-regulationintheP anddeveloping
0
leaves.Theprovascularstrandclosesttothemeristem(arrowhead)is the most recently initiated vascular traces in the stem
nearP3.B,Xcl1/Xcl1meristemshowingnormalKNOXexpression.The arebetween the second and third primordia (Figs. 1E
provascularstrandclosesttothemeristem(arrowhead)isnearP1.C, and5A;Sharman,1942),whileinXcl1apices,themost
Gn1 meristem with KNOX localized in the SAM and in developing recently initiated vascular traces are between P1 and
leaves.D,HighermagnificationofP3inCshowingKNOXexpression P2(Figs.1Fand5B).Sincehighlevelsofauxinareas-
(arrow).E,Gn1;Xcl1meristematsamemagnificationasAtoC.The sociated with vascular development, this indicates
stem is greatlyexpandedbetweensuccessive leaf primordiaand the
that higher levels of auxin may be present in this
oldestleafpictured(asterisks)isabortedandlooksmorelikestem.F,
region of Xcl1 apices. Interestingly, KNOX protein is
HighermagnificationofP2inE.G,SemiquantitativeRT-PCRofGN1
excludedfromthesedevelopingvascularstrands(Fig.
andRS1inB73versusXcl1meristemsshowsnormalexpressioninXcl1
5, A and B), suggesting that high levels of auxin may
meristemsand noexpressionin Xcl1 leaves.Numberedlanescorre-
spondtonumberofPCRcycles(1530,2535,3540forGN1and becorrelated with reducedKNOX expression.
RS1; 1 5 22, 2 5 26, 3 5 30 for GAP controls). H, PIN1 RT-PCR
showinglowerexpressioninGn1;Xcl1meristems.I,DetectionofPIN1 Xcl1;cr4 Double Mutants
RNA levelswithQRT-PCR inwild-type(wt) andXcl1seedlings.The
RNA levels were normalized to ZmGAPDH mRNA and are shown In Arabidopsis, the auxin efflux carrier PIN1 is lo-
relative to wild-type levels. Bars represent SEs over four technical calized in the L1 layer of the meristem, and auxin
replicates.B,B73(wildtype);X,Xcl1;G,Gn1;GX,Gn1;Xcl1;L,leaf; transport throughtheL1 hasbeen proposed toplaya
M,meristem.Scalebars5200mm. roleindeterminingphyllotaxy(Reinhardtetal.,2003b).
Therefore, changes to the L1 layer of the meristem
wouldbepredictedtoinfluencemeristemfunction.To
Plant Physiol. Vol. 141, 2006 1355
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Kessler et al.
address this hypothesis, double mutants were made phenotypes; a consistent extra epidermal layer was
betweenXcl1andcr4.MutationsinCR4,agenesimilar presentbelowtheabnormalouterepidermis (Fig. 6G),
to the human tumor necrosis factor receptor, lead to andSEMoftheadaxialsurfacerevealedcellsthatwere
aberrant epidermal differentiation and division pat- squareindimensionlikeXcl1butalsohadcrenulations
terns(Becraftetal.,1996,2001;Jinetal.,2000).Whenan onthesurface(Fig.6H).F3analysisoftheseindividuals
F populationofXcl1crossedtothecr4-6143alleleofcr4 indicated that they were heterozygous for Xcl1 and
2
was grown in the field for observation, three mutant homozygous for cr4. Thus, double homozygous mu-
phenotypic classes were observed. In comparison to tants were predicted to be seedling lethal. The same
wild-typeleaveswithsingleepidermallayers(Fig.6A) F families were germinated in flats with a light soil.
2
and rectangular, crenulated epidermal cells (Fig. 6B), Representatives from each of the genotypic classes are
Xcl1singlemutantshadmultipleepidermallayersand showninFigure7.Thesegregationforthisfamilywas
square,uncrenulatedepidermalcells(Fig.6,CandD). 3/16 xcl1/xcl1;Cr4/? (Fig. 7A); 1/16 xcl1/xcl1;cr4/cr4
cr4 single mutant leaves had epidermal cells that di- (Fig. 7B); 9/16 Xcl1/?;Cr4/? (Fig. 7C); 2/16 Xcl/xcl;
videdinabnormalplanes(Fig.6E)and,whenobserved cr4/cr4; and 1/16 had reduced leaves or no leaves
with SEM, had crenulations on the surface of cells in- produced and were seedling lethal (Fig. 7, D–F) and
stead of between cells (Fig. 6F). The third class of therefore inferred to be double mutants (Xcl1/Xcl1;
mutants seemed to be additive of the Xcl1 and cr4 cr4/cr4;TableIII).
The reduction in leaves in the double mutants
suggested that there was a problem in leaf initiation
and/or meristem maintenance that may be similar to
that seen in Xcl1;Gn1 and Xcl1;Rs1 double mutants.
Whencomparedtocr4singlemutants(Fig.8,AandB),
cr4;Xcl1doublemutantswithreducedleaves(Fig.7,D
and F) showed a range of meristem phenotypes. The
lessseveredoublemutantshadnormallookingSAMs
by SEM (Fig. 8, D and E) but reduced KNOX expres-
sioncomparedtonormal,cr4,andXcl1siblings(Fig.8,
C and F compared to Fig. 5, A and B), indicating a
gradual loss of meristem identity. Double mutant
seedlings with the most severe phenotypes did not
have a SAM (data notshown).
Theextremelyreducedseedlingphenotypesseenin
cr4;Xcl1 double mutants indicated that the two genes
may act in the same pathway to influence both epi-
dermal and meristem development. To address this,
CR4transcriptlevelswereexaminedinwild-typeand
Xcl1leavesandmeristemsbyRT-PCR(Fig.8G).When
comparedtoB73,CR4levelsweregreatlydecreasedin
Xcl1 mutant SAMs and leaves, indicating that Xcl1
may regulate CR4 transcription. GA20ox and PIN1
levelswerealsoexaminedinthemutantstodetermine
if the effects of these mutations on meristem mainte-
nance are linked to the hormones that have been
shown to be involved in KNOX-mediated develop-
mental pathways. PIN1 levels were decreased in cr4
leaves (Fig. 8H), while GA20 oxidase levels were in-
creasedin cr4 leaves (Fig.8I).
Figure 6. Phenotypes of adult leaves in a cr4;Xcl1 double mutant
family.A,Transversesectionofawild-typeleaf.B,Wild-typeepidermis
withrectangularpavementcellsandevenlyspacedstomata.C,Trans-
versesectionofanXcl1singlemutantleafshowsmultipleepidermal
DISCUSSION
layers,buttheouterepidermisisflat.D,SEMoftheadaxialepidermisof
Xcl1.Pavementcellsaresquareindimension.E,Transversesectionofa The mutants described in this study all affect cell
cr4 single mutant leaf. Multiple epidermal-like layers are in smaller division and differentiation patterns in maize devel-
patches and extend out from the plane of the leaf. F, SEM of a cr4 opment. Kn1, Rs1, and Gn1 are all dominant KNOX
adaxial epidermis. Pavement cells are rectangular in shape but have
mutationsthatshowectopicgeneexpressioninleaves.
crenulations on the surface instead of between cells. G, Transverse
This ectopic gene expression leads to periclinal divi-
sectionofacr4/cr4;Xcl1/xcl1leafshowinganadditivephenotype.A
sionsthatproduceknotsand/orextracelllayersinthe
complete extra epidermal layer is present under a cr4-like outer
leaf blade and sheath (Vollbrecht et al., 1990; Smith
epidermis.H,SEMofacr4/cr4;Xcl1/xcl1adaxialepidermis.Cellsare
squareinshapelikeXcl1buthavecrenulationsonthesurfacelikecr4. et al., 1992). According to the maturation schedule
Scalebars5100mm. hypothesis,ectopicKNOXexpressioninleavesretards
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Copyright © 2006 American Society of Plant Biologists. All rights reserved.
Epidermal Differentiation and KNOTTED1-Like Homeobox Genes
maintain KNOX suppression in developing leaves in
Antirrhinum(PHANTASTICA),maize(rs2),andArabi-
dopsis (ASYMMETRIC LEAVES1; Timmermans et al.,
1999;Tsiantis et al.,1999b; Byrneetal.,2000; Orietal.,
2000).Inmaize,rs2mutantsphenocopydominant,gain-
of-function mutations in several KNOX genes, includ-
ing Kn1, Rs1, andGn1 , indicating that RS2 functions
to down-regulate these genes in leaves (Schneeberger
et al., 1998; Timmermans et al., 1999; Tsiantis et al.,
1999b).
Suppressors of the dominant KNOX phenotypes
wouldbeexpectedtorepresentdownstreamelements
necessary for KNOX activity. Xcl1 is a semidominant
mutationthatpartiallysuppressestheKn1phenotype
inleaves.Xcl1couldacteitherdirectlyorindirectlyon
Figure 7. Range of seedling phenotypes in a cr4;Xcl1 F family. A, theKN1pathwaytosuppressKn1mutantphenotypes
2
Wild-type seedling. B, cr4/cr4 seedling showing leaf adherence. C, andthuspresentsuswithawaytofurtherdissectthe
Xcl1/Xcl1seedlingwithnarrow,shinyleaves.DtoF,Rangeofreduced KNOX pathway.
leafphenotypesseenindoublemutants. Dosage analysis revealed that Xcl1 is a hypermor-
phicmutationresultingfromincreasedexpressionofa
the acquisition of competence for distal cell fates, normal geneproduct (Kessleret al., 2002). Kn1-Oand
leading to a proximalization of the leaf, or sheath Kn1-N2areneomorphicdominantmutationsinwhich
identityintheblade(Freeling,1992).Thecr4mutation KN1 is expressed ectopically in developing leaves.
also leads to changes in cell division and differentia- One modelfor the suppression ofKn1 phenotypesby
tion in the maize leaf. Loss-of-function cr4 mutants Xcl1isabiophysicalmodel.Xcl1leaveshaveaberrant
have problems in epidermal differentiation and also oblique, periclinal divisions that lead to the produc-
havepericlinaldivisionsleadingtomultiplecelllayers tionofmultipleepidermallayers.InKn1,knotforma-
(Becraftetal.,1996;Jinetal.,2000),indicatingthatthe tion requires signals from underlying mesophyll cells
CR4 protein is important for properepidermal devel- (Hake and Freeling, 1986; Sinha and Hake, 1990). In
opment. Finally, Xcl1 mutants have extra epidermal Xcl1;Kn1 double mutant leaves, it is possible that the
cell layers produced by aberrant oblique periclinal suppression of knots is merely a reflection of de-
divisions that occur late in leaf development and creased signaling through the extra epidermal layers
lead to differentiation by lineage instead of position conditioned by Xcl1. This hypothesis seems unlikely,
(Kessleretal.,2002).Becauseallofthesemutantslead because knots in Kn1 mutants form primarily over
to cell proliferation through periclinal divisions, we lateral veins (Gelinas et al., 1969; Hake and Freeling,
constructed double mutants in the hopes of gaining 1986;SinhaandHake,1990).InXcl1,multipleepider-
insight into the pathways involved in controlling cell mallayersariseaftertheinitiationoflateralveins,and
divisionduringleafdevelopment.Thesurprisingmer- therefore, the epidermis over lateral veins is always
istem arrest phenotypes seen in some of the double single layered(Kessleret al., 2002).
mutant combinations point to a more basic role for AsecondmodelforregulationoftheKN1pathway
these genes in meristemmaintenance. by Xcl1 is that in normal development, XCL1 acts to
reduce KN1 function in the P and causes periclinal
0
divisions that lead to leaf primordium outgrowth.
Xcl1 Suppresses the Kn1 Phenotype
When Xcl1 is expressed at higher levels than normal,
KNOX genes play an important role in the mainte- KN1 activity would be down-regulated even more,
nance of meristem identity. In simple-leafed model andmorecellswouldberecruitedintotheP ,thereby
0
species such as maize and Arabidopsis, KNOX genes
aredown-regulatedintheP andremainoffthrough-
0
out leaf development (Hake et al., 2004). However, TableIII. Segregationofarepresentativecr4;Xcl1F family
2
tomato, whichhas compound leaves, displays KNOX Thedoublehomozygotescr4/cr4;Xcl1/Xcl1wereseedlinglethal.The
expression in developing leaf primordia (Hareven x2valuefailstorejectthesegregationratio/nullhypothesis.Sx253.06;
et al., 1996; Chen et al., 1997). This expression pattern criticalvalue516.75(P50.005,5degreesoffreedom).
is seen in compound and secondarily simple-leafed Expected Expected Observed
Genotype x2
species with independent origins throughout nature, Ratio No. No.
and thus represents an evolutionarily conserved de- Cr4/?;xcl1/xcl1 3/16 8.8 9 0.01
velopmental pathway (Bharathan et al., 2002). How Cr4/?;Xcl1/xcl1 6/16 17.6 12 1.78
KNOX gene expression is regulated to bring about Cr4/?;Xcl1/Xcl1 3/16 8.8 11 0.55
different developmental programs is an unanswered cr4/cr4;xcl1/xcl1 1/16 2.9 5 1.52
question.Inrecentyears,thePHANTASTICAgroupof cr4/cr4;Xcl1/1 2/16 5.9 7 0.20
cr4/cr4;Xcl1/Xcl1 1/16 2.9 3 0.003
Myb-domain transcription factors has been shown to
Plant Physiol. Vol. 141, 2006 1357
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Kessler et al.
Figure8. Analysisofcr4andcr4/Xcl1meristemsand
geneexpression.A,cr4/cr4singlemutantapexdis-
sectedtoP3.B,cr4/cr4apexinAwithP3removed.
Leafinitiationappearsnormal.C,KNOXexpression
incr4mutantsissimilartowildtypeandXcl1(Fig.5).
D,cr4/Xcl1doublehomozygousmutantapexisvery
similartoA,excepttheP3primordiaarenarrower.E,
cr4;Xcl1apexinBwithP3removed.Leafprimordia
seem to be initiated normally, but the dome of the
meristem is shorter than in cr4. F, KNOX protein
levels are reduced in cr4;Xcl1 meristems. G, CR4
transcriptsarereducedinXcl1meristemsandleaves.
H, PIN1 transcripts are decreased in Xcl1 and cr4
leaves.I,GA20oxidasetranscriptsareincreasedincr4
leaves.B,B73(wild-type);X,Xcl1;C,cr4;M,meri-
stem; U, unexpanded leaf; L, expanded leaf. Scale
bars5100mm.
affecting meristem maintenance. This model is sup- P , complementary to KNOX expression patterns
0
portedbythefactthatXcl1meristemsareshorterthan (Sakamoto et al., 2001). Ourexperiments showed that
normal and recruit more cells into P (Kessler et al., GA treatment suppressed the Kn1 phenotype, and
0
2002). Additional evidence for XCL1 regulation of inhibiting GA biosynthesis with uniconazole en-
KN1 activity comes from the other double mutants. hancedtheKn1phenotype.WhileGA20oxidaselevels
Even though Gn1 or Rs1 gain-of-function mutants are not appreciably altered in Xcl1;Kn1-N2 double
ectopically express the proteins only in leaves, Xcl1 mutants,GA2oxidaselevelsaredecreased,indicating
suppressesKNOXexpressioninGn1;Xcl1SAMs,lead- that XCL1 overexpression may lead to higher GA
ing to loss of meristem integrity. Construction of levels,whichinturnsuppressestheformationofknots
double mutant between Xcl1 and loss-of-function kn1 in double mutant leaves. Thus, decreased GA levels
mutants (Vollbrecht et al., 2000) may also help to seem to be necessary for knot formation in dominant
definethe role of XCL1 in regulating KN1 activity. neomorphic Kn1 mutants. Increased XCL1 product
ThesuppressionofknotformationinKn1leavesby may bypass KNOX regulation of the GA pathway by
Xcl1,eventhoughectopicKN1transcriptwaspresent independently decreasing the GA metabolizing en-
in leaves, suggests that XCL1 might regulate KN1 zyme GA2 oxidase.
posttranscriptionally or could differentially regulate
genes downstream of KN1. One candidate pathway
Xcl1 Interactions with Gn1, Rs1, and cr4 Lead to Loss
for regulation by both XCL1 and KN1 is the GA
of Meristem Function
signalingpathway.WhenKN1ortobaccoKNOXgenes
areoverexpressedintobacco,leafshapeisalteredand Since dominant Gn1 and Rs1 mutants have very
ectopicmeristemsformonleaves.GAlevelshavebeen similar phenotypes to Kn1 mutants, we expected a
shown to be reduced in tobacco leaves that overex- similarsuppressionoftheKNOXoverexpressionphe-
press NTH15 (Tamaoki et al., 1997), and NTH15 was notype by Xcl1. Instead, both Gn1;Xcl1 and Rs1;Xcl1
subsequently shown to directly suppress the GA bio- doublemutantsdisplayedsynergisticphenotypesthat
synthesis gene, GA20 oxidase (Sakamoto et al., 2001). ledtomeristemarrestatearlystagesofseedlingdevel-
In addition, GA20 oxidase in tobacco is expressed in opment.Thedoublemutantseedlingshadagravitropic
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