Molecular cartography of leaf development — role of transcription factors
Kavitha Sarvepalli
1, Mainak Das Gupta
2, Krishna Reddy Challa
1and Utpal Nath
1Organelaborationinplantsoccursalmostexclusivelybyan increaseincellnumberandsize.Leaves,theplanarlateral appendagesofplants,arenoexception.Forwardandreverse geneticapproacheshaveidentifiedseveralgeneswhoserolein leafmorphogenesishasbeeninferredfromtheirprimaryeffect oncellnumberandsize,therebydistinguishingthemaseither promotersorinhibitorsofcellproliferationandexpansion.
Whilesuchclassificationisusefulinstudyingsizecontrol,a similarlinkbetweengenesandshapegenerationispoorly understood.Computationalmodellingcanprovidea conceptualframeworktore-evaluatetheknowngenetic informationandassignspecificmorphogeneticrolestothe transcriptionfactor-encodinggenes.Herewediscussrecent advancesinourunderstandingoftherolesoftranscription factorsintheplanargrowthofleaflaminaintwoorthogonal dimensions.
Addresses
1DepartmentofMicrobiologyandCellBiology,IndianInstituteof Science,Bangalore560012,India
2DepartmentofComparativeDevelopmentandGenetics,MaxPlanck InstituteforPlantBreedingResearch,Cologne50829,Germany Correspondingauthor:Nath,Utpal([email protected])
CurrentOpinioninPlantBiology2019,47:22–31
ThisreviewcomesfromathemedissueonGrowthanddevelopment EditedbyAdrienneHKRoederandCJillHarrison
ForacompleteoverviewseetheIssueandtheEditorial Availableonline14thSeptember2018
https://doi.org/10.1016/j.pbi.2018.08.002 1369-5266/ã2018ElsevierLtd.Allrightsreserved.
Introduction
Leavesevolvedfromancestralbranchingsystemsmulti- pletimes amongthe vascularplant lineages [1].Angio- spermleavesshareacommonphylogeneticorigin,which isreflectedintheconservedsequenceofdevelopmental eventsleadinguptotheinitiationofarod-shapedbulge called primordium on the flanks of the shoot apical meristem(SAM), havingdistinct upper andlower sides [2]. Yet, angiosperm leaves are distinguished by their tremendousarchitecturaldiversityinthefinalsize,shape, andcomplexity[2,3].Thisisattributedtothevariationin
thepost-initiationgrowthpatternsalongthe‘base-to-tip’
andthe‘middle-to-margin’axes[2,4].Thesimpleleaves of the winter annual Arabidopsis, the lobed leaves of lettuce, the compound leaves of tomato and even the intricately-designed insect-trapping leaves of the blad- derwortarisebythemodificationofthegrowthparame- ters,suchasgrowthdurationandgrowthdirection,along thesetwoperpendicularaxes.
Growthinaleafprimordiumoccursbyanincreaseincell numberandcellsize,whichisregulatedinspaceandtime primarilythroughtheactivitiesofgrowth-promotingand growth-repressingtranscriptionfactors,sometimesthem- selves expressed in gradients, as a result of their tran- scriptionalregulation and/or post-transcriptional control bytheupstreamregulatorymicroRNAs.Transcriptional output is often a modulation of cellular properties and responsetohormones,whichactasintercellularmessen- gers.Thesemolecularplayerslaydowntheblueprintfor growthpatterns in space and time.The challenge is to decipher the connection between proximal effects of gene activities and its ultimate manifestation on organ growth.Thecurrentreviewhighlightstheattemptsmade overthepastdecadeinjoiningthedotsbymakinguseof theinsightsgleanedfromcomputationalmodels.
Howdoleaves grow?
A leaf is characterized by a flat, bifacial lamina with a stalk-likebase.Laminargrowthproceedsbydivisionand expansion of cells thought to arise from a short-lived meristematicactivitylocalizedatthebaseoftheprimor- dium[5].Initially,activeproliferationoccursthroughout theprimordiumbutisquicklylimitedtothebasebythe suddenappearanceof anarrestedzoneatthedistalend [6–9]. Cells distal to the proliferation-differentiation boundary, also called arrest front, contribute to growth byundergoingexpansion andmaturation, whereas cells proximaltothearrestfrontcontinuetoincreaseinnum- ber tillthe complete cessation of proliferating activity.
Thereafter, growth is propelled by post-mitotic cell expansion alone till the mature size is achieved. This lineargradient of cell divisionand expansionalong the proximo-distalaxis,thoughcommontomostmonocotand dicotmodelspeciesis,however,notuniversalandseveral othertypesof growthgradient exist,atleast amongthe eudicotspecies(Box1,Figure3)[10,11].Nevertheless, whereasthelinkbetweenthebase-to-tipgrowthgradient andfinalleafsizehasbeenwell-studied[12,13],howthe
patternsofcelldivisionandexpansiongeneratespecies- specific shapesisstillanenigma.
Classicalstudiestrackinglaminargrowthbyobservingthe movementofartificiallandmarksduringdevelopmentor by clonalanalysis in fig, tobaccoand cottonleaves, had alreadyestablishedthatthegrowthofaprimordiumdoes notoccurbyuniformenlargementoftheprimordialbulge [14–18].Rather,differentregionsoftheprimordiumgrow atdifferentratesandorientationsthatcanbequantified using computational tools [19,20]. Growth orientations diverge at the leaf base and converge towards the tip [21]. Not only do the growth rates progressively decrease frombaseto tipas reflectedin thepatternsof cell division and cell expansion, they differ along the medio-lateralaxisaswell,withmoregrowthinthelateral than in the medial domain [21]. Recently, tracking lamina growth influorescently-labelled Arabidopsisleaf cells hasrevealedthatthegrowthpatternisestablished earlyonduringtheprimordiumdevelopment[21].
Although suchstudies providearoadmap for describing growthpatterns,themajorchallengeistounderstandhow theyarespecifiedatthegeneticlevel[4,22].Geneexpres- sion patterns in space and time, when combined with mutantphenotypes,informushowagenespecifiesgrowth rateand/ororientationlocally.However,cellsinatissueare mechanically constrained by being connected to their neighbours and a specified growth pattern of cells in a regionmayconflictwiththatintheneighbouringregions leadingto bucklingof thetissueoutofplanegenerating curvatureorbending[4].Thus,a‘resultant’growthpattern is an emergent property of any anisotropically-growing
organandmaynotbeintuitedfromstudyinggenemutant phenotypesand/orexpressiondomainsinisolation[4].
Modelling leafgrowth predicts a molecular toolkit forshapespecification
Time-lapsegrowthanalysesincombinationwithcompu- tational modelling can reveal coherent links between gene activity and the resultant growth patterns [8,21,23,24,25]. While elegant reports described modelling growth in the petals or leaf margin [24,25], Kuchen et al built a model to simulate the observedleafshapeand growthpatternsinArabidopsis byincorporatingtwokeysystems—anetworkoffactors specifyingtherateofgrowthandafactorfordetermining theorientationofgrowth alongthetwoorthogonallami- nar axes [21] (Figures 1a and 2 a). Two growth-pro- motingfactors,PGRADthatisexpressedinadecreasing gradient from base to tip, and LAM that is expressed uniformly,promotegrowthalongtheproximo-distaland the medio-lateral axes, respectively. In addition, two growth-inhibitory factors were introduced into the model to account for the cessation of leaf growth—a uniformly-distributed late-acting factor LATE and a midline-restrictedfactorMIDthatrepressgrowthalong the proximo-distal and the medio-lateral axes, respec- tively.Finally,atissuepolarityorganizer,expressedina decreasingbase-to-tipgradient,wasincludedthatdeter- minedthedirection ofgrowth (Figure1a).Theregula- tion of growth rate was thus uncoupled from that of growth orientation. Further, the distribution of these factorschangedinspaceandtimewithgrowth,allowing afeedbackfromtissuedeformationto specifiedgrowth pattern. This ‘deforming growth-orientation organizer’
model could accurately reproduce shape changes and growthpatternsobservedexperimentallyinawild-type Arabidopsis leaf. Varying the parameters ofthe model generated avariety ofshapesthat are observed among thesimple-leavedspecieswithsmoothmargins,indicat- ing that the specification of growth patterns through interactions of growth-modulating and polarity-deter- mining factors along the orthogonal axes underlies diverse simpleleafforms.Anindependentstudy incor- porated the directional growth of veins as the major determinant ofthe orientation ofspecified growth and the resulting model accounted for the generation of diversity in complex leaf shapes, including serrations, lobesand leaflets [26].
Thevalueofcomputationalmodellingliesinitstestable predictionsaboutgeneactioninmorphogenesis,leadingto new insights.Forexample,modellingthedevelopmentofa petalprimordiumthatshowsdivergentgrowthpatternatits tip relativeto itsbase,incontrast totheleaf,ledto the predictionoftheexistenceofadistally-expressedpolarity organizerinadditiontoaproximally-expressedorganizer [24].Basedonitsexpressiondomainandmutantpheno- type whichmatched therequirement of themodel, the
Box1Divergentgrowthpolaritypatternsandtheirevolution Leavesofallmodelplantsstudiedsofardisplayacommon
‘basipetal’patternofprogressionofthecell-proliferationarrestfrom thedistaltiptowardstheproximalbase[6,7,15,76].Eventhough suchagrowthpatternwasinitiallyassumedtobeuniversalinplants, abroaderstudyusingalargenumberofdicotspeciesrevealedthree othertypesofproximo-distalgradient:firstly,acropetalpattern wherethearrestfrontprogressesinthebase-to-tipdirection,sec- ondly,bi-directionalpatternwheretwosimultaneousarrestfronts progressfrombase-to-tipandtip-to-baseandthirdly,diffusegrowth wherethereisnoprogressionofthegrowtharrestandcells throughoutthelaminaproliferateanddifferentiatesimultaneously (Figure3)[11].Thesedivergentpatternsarealsocloselyassociated withtheexpressionofgrowthpromotingtranscriptionfactors(such astheGRFs)andgrowthrepressinggenes(suchasmiR396)(Fig- ure3)[11].Aphylogeneticanalysisshowedthatsomeofthese growthpatternsevolvedindependentlyinseveralplantlineages [32].Ithasbeenspeculatedthatthedirectionofthegrowthpattern isunderthecontrolofaconservedgeneregulatorymodule,which wasco-optedtonovelexpressiondomainsduringevolutiontocre- atediversegrowthgradients,eitherbymutationintheproximal- regulatorygenes(e.g.CIN-TCPsthatregulatemiR396andhence GRFexpression)orintheregulatorysequencesoftheindividual genes(e.g.GRFs)[11].
JAGGED gene encodinga Zn-finger transcription factor wasidentifiedandvalidatedasthedistalpolarityorganizer inadditionto itspreviously-knownfunctioninpetalcell division[24].Likewise,themodelproposedbyKuchen et al. suggests candidate transcription factor-encoding genes that couldfunction as the ‘leaf architects’, based on their known expression patterns and developmental
roles[21](Figures1band2b).Followingisadiscussion onsuchfactorsandtheirmodesofaction.
Growth-promotingfactors alongtheproximo- distalaxis
GROWTH-REGULATINGFACTORS(GRFs)—Theseare aconserved groupofplant-specific transcriptionfactors that
Figure2
(a) (b)
LAM CIN-TCP
CIN-TCP miR319
GRF-AN3
YABBY
GRF-AN3
WOX
NGA miR396
MID LATE
PGRAD
Current Opinion in Plant Biology
Growth-regulatorynetworkconsistingofpositiveandnegativeregulatoryfactorsintheearlyleafprimordiumashypothesizedbyKuchenetal.(a) andasdiscussedinthetext(b).Theproximo-distalandthemedio-lateralaxesarerepresentedbytheorthogonallyintersectingbrokengreylines, alongwhichgrowthisregulatedbytranscriptionfactorsasindicated.
Figure1
(a)
PGRAD
GRF YABBY WOX CIN-TCP CUC
LAM LATE MID POLARITY
ORGANIZER (b)
Current Opinion in Plant Biology
Spatialexpressiondomainsofthegrowth-regulatingfactorsalongthelongitudinalaxisoftheearlyleafprimordiarepresentedasrod-shaped structures.BoththehypotheticalfactorsofthegrowingpolarizedtissueframeworkmodelinKuchenetal.(a)andtheproposedcounterparts discussedinthetext(b)areshown.Theexpressiondomainsdepictedin(b)areapproximatedfrompublishedstudiesonGRF,[30];YABBY, [45];WOX,[63];CIN-TCP,[7];CUC,[25].Theprimordiumdepictedin(b)isassumedtobeatanearlygrowthstage(upto2.0mminlength), reflectingthedynamicexpressionofthegrowth-regulatorygenesdescribedhere.
promotelateralorgangrowth[27].Arabidopsisgrfmutants have smaller leaveswith reducedsize and cell number, pointing towards a role in enhancing cell proliferation, although some family members also enhance cell size [28,29].GRF expression is strongly associated with the base-anchoredproliferationzoneinthelamina[30]andis down-regulatedindifferentiatedcells atthe tipdue,atleast inpart,totheactivationofmiR396thattargetssevenofthe nineGRFsinArabidopsis[30,31].Theexpressiongradient ofthemiR396-GRFmodulealongtheproximo-distalaxis isconservedinotherspecieswithbasipetalgrowthpattern, suggestingthatthismoduleisaprimaryfactorinpromoting growth in thisaxis, similarto the PGRADfactor in the Kuchenetal.model[21](Figures1band2b).However, grfmutantshavenarrowerleaves, indicatingthatgrowth alongthemedio-lateralaxis(perpendiculartomidveinin theKuchenetal.model)isalsoaffected,leadingtoachange in shape. Remarkably a recent study has identified a correlationbetweenthepolarityofmiR396-GRFexpres- siongradientandthatoftheleafgrowthgradient(Box1) (Figure3)[11,32].
HowGRFs stimulateproliferation directlyis atpresent unclear. They seem to enhance the duration of cell proliferation andthenumberof cellsundergoing prolif- eration[33].GRFsareproposedtoregulatetheirtarget genes through a conserved interaction with GRF- INTERACTING FACTORs (GIFs) (Figure 2b). Loss ofGIFsalsoresultsinnarrowerleaveswithreducedcell
number [34–36]. They function as transcriptional co- activators that lack a DNA-binding domain [34,35], though GIF1/ANGUSTIFOLIA3 (AN3) interacts and co-regulates its target genes with several components oftheSWI/SNFchromatinremodellingcomplexcontain- ing BRAHMA or SPLAYED ATPases [37]. Further- more,AN3transcriptisspecificallyenrichedinthemeso- phyllcellsanditsproteinproductmovestotheepidermis, coordinatingcellproliferationacrosstheclonallydistinct celltypes[38].Transcriptomeprofilingofthe35S:AN3- GR transgenic line revealed up and down-regulation of transcriptsthatareinverselyregulatedduringthetransi- tionfromproliferation-driventoexpansion-drivengrowth phases, supporting a role for AN3-GRF in balancing proliferation versus differentiation in developing leaves [12,37].Inaddition,AN3promotesitsowntranscription andthatofGRF3/5/6,therebyprovidingamolecularbasis for synergistic effectof simultaneous overexpressionof AN3andGRFinleafdevelopment[29,37].Interestingly, in maize, ZmAN3 is associated preferentially with ZmGRF1inproximaldivisionzoneandwithZmGRF10 inthedistalexpansionzone;thispreferencereflectsthe mRNAandproteinabundanceoftherespectiveZmGRF partners [39]. Because ZmGRF1 stimulates and ZmGRF10 limits cell proliferation, it is hypothesized that the competition for ZmAN3 binding by different GRFsalongtheproximo-distalaxisdeterminestheposi- tionof thetransitionzonebetweenthat ofdivisionand expansionin growingleaves[39,40].
Figure3
Cell differentiation miR396 expression
Basipetal gradient
Acropetal gradient
Bidirectional gradient
No gradient Cell proliferation GRF expression
Current Opinion in Plant Biology
Multiplegrowthpolaritiesineudicotleaves.Theyoungleafprimordiashowcellproliferationthroughouttheorganatinception.Apatternofcell differentiationislatersuperposedonthisproliferatingsheetofcells.Thedirectionoftheprogressionofthearrestfront(blackarrow)determines leafgrowthpolarity.Thearrestfrontprogressesfromtip-to-base(basipetalgradient),base-to-tip(acropetalgradient)orfrombothendstothe middle(bidirectionalgradient).Insomecases,cellsthroughouttheleafexitproliferationsimultaneously,therebyshowingnogrowthpolarity.The differentiatingregionsoftheleavesshowastrongcorrelationwiththeexpressionofmiR396whiletheproliferatingregionshowsagradientofGRF expression.
GRFsmaypromotelaminaroutgrowthbyotherindirect mechanisms.AstudysuggeststhatGRFsrepressclassI KNOX genes in both monocots and dicots [41]. Over- expressionofGRF5inArabidopsisandGRF2inBrassica napusleadstoanincreasedchloroplastdivisionandchlo- rophyllaccumulationwithstronginductionofthePORA geneencodinganenzymeinthetetrapyrrolepathwayfor chlorophyllbiosynthesis[33,42].Enhanced chlorophyll contentislinkedtothepromotionof cell-cycleprogres- sionastheintermediatesofthechlorophyllbiosynthesis activatecyclin-dependentkinasesthroughchloroplast-to- nucleussignalling,suggestingthatGRFslinkchloroplast division with cell division [33,43]. Further, AtGRF7 represses several stress-response genes under normal conditions which would otherwise be detrimental to growth[44].
Growth-promotingfactors alongthe medio- lateralaxis
The YABBY and WUSCHEL RELATED HOMEOBOX (WOX) transcription factors—These proteins promote lamina outgrowth downstream to the adaxial-abaxial polarity establishment [45,46]. YABBYs encode small proteins with zinc-finger and helix-loop-helix domains, conservedamongallseedplants[47].Arabidopsisplants mutatedforfourabaxially-expressed,vegetativeYABBY genesformleaveswithseveretomoderatelossoflamina andmarginaltissues withpolaritydefects[45].Asub- groupofWOXgenesencodingtranscriptionalrepressors, comprisingof thePRESSEDFLOWERS(PRS)andthe MAEWEST/WOX1subclades,regulatelaminarexpansion specifically along the medio-lateral axis [46,48,49].
These factors are expressed in the so-called middle domain between the adaxial and the abaxial domains [49,50]. The prs wox1 double mutantin Arabidopsis producesnarrowleaveswithperturbedpolaritybutunal- teredlength[49];similarphenotypeisassociatedwith thelossofWOX1homologuesinotherdicotsandthatof PRShomologues inmaize [46,48,51].
Given theirlamina-wide expressionpatternand role as
‘lamina identifiers’, the YABBY and the WOX genes qualifyastheLAMfactorthatdeterminesgrowthalong themedio-lateral axis inthe Kuchen etalmodel (Fig- ure1).YABBYactivitymaybeupstreamtothatofWOX, astheYABBYmemberFILAMENTOUSFLOWER(FIL) wasshowntoup-regulateWOX1expression[49].Veg- etativeYABBYsregulateabroadlamina-specificgenetic program involving therepression of SAMidentity and maintenancegenes(WUSCHELandKNOXI),promoting expressionofthepolarityandlaminamaturationmarkers [45,52]. The YABBY factors may act inconcert with AINTEGUMENTA(encoding anAP2/ERF familytran- scriptionfactor), as the filant and the yab3ant double mutants have reduced lamina growth compared tothe single mutants [53]. Whereas, the WOX genes mainly control cell proliferation along the medio-lateral axis,
possibly by recruiting transcriptional repressors like TOPLESStoitstargets,resultingintheindirectactiva- tionofgrowth-promotingtranscriptionfactors(SCARE- CROW-like),enzymes(KLU/CYP78A5),cell-cyclefac- tors(D-typecyclins)andmetabolicpathwaysleadingto auxinbiosynthesisandcytokininsignalling[46,49,54].
Both YABBY and WOX factors regulate lamina out- growthnon-cell autonomously,affecting differentiation oftissueslackingtheirexpression[45,49,52,55,56].It ispossiblethattheygenerateamobilesignal,suchasthe phytohormones.YABBYs havebeen postulatedtocon- trolauxin response and flux along the leafmargin; the quadrupleyabbymutantinArabidopsisshowsperturbed venation and lack of marginal structures [45]. The WOX1homologueinNicotianasylvestris,LAM1,promotes auxinbiosynthesisandco-applicationofauxinandcyto- kinin can rescue lamina growth defect in the lam1 mutant[46].
Growth-repressingfactors—common to proximo-distaland medio-lateral axes?
CINCINNATA-LIKE TEOSINTE BRANCHED1/
CYCLOIDEA/PROLIFERATING CELL FACTORS (CIN-TCP) transcription factors—These belong to the plant-specific, non-canonical,bHLH domain-containing TCPfamily thatredundantly repress growth[57]. Five ArabidopsisCIN-TCPsarepost-transcriptionallyco-reg- ulatedbymicroRNA319(miR319)[58].Perturbation of theconservedmiR319-TCP moduleeither bymutation ofCIN-TCPsorbyectopicexpressionofmiR319resultsin largerleaveswithalteredshapeandlossofflatnessdueto prolongedcellproliferationphasemoretowardsthemar- gin[7,58,59].Ontheotherhand,prematureorincreased activation of these factors leads to precocious cellular, organandorganismmaturation,suggestingthattheyare heterochronicregulatorsof morphogenesis[60–62].
CIN-TCPexpressionoccursinadynamicspatio-tempo- ralgradient during the primordiumdevelopment,as an outputoftheirtranscriptionalandthemiR319-mediated post-transcriptionalcontrol[63](Figure1b).Initially,the CIN expression in snapdragon and TCP4 expression in Arabidopsisstartsatthetipandlatergetsrestrictedtothe leafbase, overlappingwiththebase-anchored prolifera- tion zone, before disappearing with the cessation of mitotic activity [7,63]. Given the relatively late onset ofCIN-TCPgrowth-repressoractivity,compared tothe growth-promoterslikeYABBYs,CIN-TCPs couldserve as the late-acting growth-inhibitor LATE (Figure 2b).
However,LATEisproposedtobeuniformlydistributed and retardsgrowth along proximo-distal axis whilepro- motinggrowthalongmedio-lateralaxis.Thisisinconsis- tent with the expression pattern and activity of CIN- TCPs which restrict growth along both the axes, as evident from phenotypes of their gain-of-function and loss-of-functionmutants[61].
CIN-TCPsarepostulatedtomainlypromotetheonsetof cellmaturationprogram,therebyindirectlyinhibitingcell proliferation[61];althoughsomestudiessuggestadirect linkwithcell-cyclesuppression [64,65].CIN-TCPs also activate miR396 expression leading to temporal and spatial decline in its cognate GRF target levels [30,65]
(seeBox1).Theyalsodown-regulateGRF5/6 andAN3 expression independentofmiR396[30],thus efficiently repressing the overall growth-promoting activity of the GRF-AN3complexalongtheproximo-distalaxis,similar to the antagonistic activities proposed for the PGRAD and LATE factors (Figure 2). In addition, CIN-TCPs regulate the level and/or response to several growth- regulatingphytohormonessuchasauxin,cytokinin,gib- berellicacidandjasmonicacidamongothers[66,67].
In snapdragon, CIN expression shows a strong medio- lateralgradient;moreexpressionatthemarginthanatthe centreoflamina.Thisisconsistentwithmorede-repres- sion of growth and mitotic marker expression at the margin than atthecentrein the cinmutant, suggesting that CIN-TCPs also function as a component of the proposed MID that inhibits growth along the medio- lateral axis, though the observed CIN-TCP expression pattern is in contrast to that hypothesized for MID [7,21]. Two recent studies have shed light on the relevanceofCIN-TCPactivityinthemedio-lateralaxis totheregulationofleafmorphogenesis[68,69].Tran- scriptomeprofilingofyoungwild-typesnapdragonandcin mutant leaves allowed the identification of AmHISTI- DINE KINASE4 (encoding a homolog of Arabidopsis cytokininreceptor)andAmIAA3/SHY2(encodingahomo- log of the Arabidopsis AUX/IAA repressors of auxin signalling), as the direct downstream targets of CIN [68].Interestingly,thesetwogenesareexpressedmore stronglyatthemarginsthanatthemedialregion,similar toCIN,raisingthepossibilitythatCINregulatesgrowth suppression at the margins by modulating the balance betweenauxin andcytokininsignals [70].Onthe other hand, direct identification of the margin and centre- enrichedgenesinArabidopsisenabledananalysisoftheir differential regulation in the tcp2/3/4/10 quadruple mutant [69]. The margin-enriched genes (genes expressedmore inthemarginsthaninthecentre)were more down-regulated in the cin-tcp mutant than the centre-enrichedgenesandincludedseveraltranscription factorsknowntocontrolmargindevelopment,suchasthe NGATHA,STYLISHandeventhemiR319-resistantCIN- TCPfamilymembers.Otherdifferentiationmarkerssuch as photosynthesis-relatedgenes, shownto beexpressed duringtheproliferation-to-expansiontransition[9],were moredown-regulatedatthemarginsthanatthecentreof cin-tcp mutant; in contrast,the mitosis markersand the growth-promotingtranscriptionfactorssuchasANT and WOX1 were up-regulated [69]. These studies have established CIN-TCPs as the major growth repressors notonlyalongtheproximo-distalaxisbutalsoalongthe
medio-lateral axis; thus CIN-TCPs could serve as the LATEandMIDfactorsthatinhibitgrowth(Figure2b).
AnotherstudyhasrevealedanovelroleofCIN-TCPsand NGA genes in regulatingdeterminate leaf growth[63].
Simultaneous down-regulationof miR319-targetedCIN- TCPsandfourNGAgenes,eitherthroughoutthelamina oronlyatthemargin,resultsinadramaticindeterminate growthwithsustaineddenovoorganogenesisatthemar- gin,whosemolecularsignatureresemblesthatof undif- ferentiatedinitiatingleafprimordia.Strikingly,therewas noectopicexpressionofSAM-specificgenesatthemar- gin; the authors proposed that the phenotype rather results from the de-repression of a short-lived bonafide
‘leafmeristem’,whichisnormallyactiveonlytransiently at the leaf base and is kept suppressed in the distal marginsbythecoordinatedCIN-TCPandNGAactivity [63]. CIN-TCPs have been shownto directly activate NGA genes;however, thephenotypeof theircombined down-regulation, absent from individual cin-tcp and nga mutants, suggests a synergistic relationship between thesetwofamilymembers[63,71].Possibly,CIN-TCPs interactwithNGAtoco-regulatemargin-enrichedgenes for determinategrowth[69](Figure 2b).
Growth orientation-determiningfactors CUP-SHAPED COTYLEDON transcription factors— These are the NAC (NAM, ATAF1/2, CUPULIFOR- MIS) domain-containingtranscriptionfactors,character- ized by their roles in organ boundary formation and serration development [72,73]. CUC2 and CUC3 are expressed attheleaf baseand margin,demarcating the boundaries of incipient serrations [73]. Anisotropic growthrequires amechanismfor thecellsto determine theorientationofgrowthinresponsetogrowthregulatory factors. In the Kuchen et al. model, this information is derived from a polarity organizer expressed at the leaf base andgrowthoccursalong theaxesparalleland per- pendiculartoitsproximo-distalgradient(Figure1a).The CUCgenescouldserveasthecandidateorganizerbased ontheirexpressionpatternandfunction[25](Figure1b).
Cellularanisotropymayresultfromunequalorpolarized distribution of molecules within the cytoplasm (e.g.
microtubule cytoskeleton) or at the plasma membrane (e.g. receptors) [4].CUC activityis associated with the generation ofmembraneanisotropy. CUC2promotesre- orientation of the plasma-membrane-localized auxin effluxcarrierproteinPINFORMED1(PIN1)intheepi- dermis to form PIN1 convergence points thatresult in auxin maxima formationat the marginsand the subse- quentserrationoutgrowth[25].WhetherasimilarCUC activity regulates growth polarity during laminar out- growthrequires furtherexperimentalvalidation.
Interestingly,CUCactivityisrepressedbymiR319-reg- ulatedCIN-TCPs.CIN-TCPsactivatethetranscription
of miR164 that targets CUC2 transcript for degradation [74].Inaddition,TCP4interactswithandinhibitsCUC2- CUC3 dimerization and dampens their transactivation potential.Thiseffectisamelioratedbythesequestration ofTCP4bythemiR156-targetedSPLtranscriptionfac- tors in the adult vegetative-phase leaves, causing age- dependentchangesintheleafmarginshape[75].Thus, thepolarityorganizer activitymaybesubjectto regula- tionbygrowth-regulatory factors.
Concludingremarks
Computational models provide a useful prism through whichacomplexbiologicalphenomenoncanbeviewed inordertorevealitsunderlyingcomponentnetwork(s)of interacting molecules.Onthe otherhand, mutantanal- ysesprovidevaluablemechanisticdetailsthatmaynotbe predictedbymodellingalone.Forexample,theyabbyand thewox1mutants(inArabidopsisandMedicagotruncatula, respectively)show down-regulationofCIN-TCP expres- sion, suggestingthat theinductionof growth-repressors bythegrowth-promotersisrequiredforbalanceddeter- minategrowth[45,46];althoughKuchenetal.hypoth- esized that the LATE factor enhances the extent to whichLAM promotesmedio-lateralgrowth, thereisno evidence that CIN-TCPs directly regulateYABBY and WOXgenes.Likewise,inhibitionofCUCsbyCIN-TCPs indicates that the determination of growth orientation may notbe independent of the growth regulatory net- work.Itwouldbeinterestingtomodeltheseinteractions andobservetheimpactonthesimulatedleafshape.
Leafformisacomplextraitthatrequiresseveralgenetic regulators[77],ofwhichonlyafewhavebeendiscussed in this review. Transcription factors such as ANT, STRUWWELPETER(acomponent of RNA Polymer- ase II-associated Mediator complex), AUXIN RESPONSE FACTOR2 and SPATULA control the durationofproliferationorthenumberofcellsundergo- ingproliferation;mostofthesemutantsshowalterationin size but not shape, suggesting that they are general regulatorsofgrowth[78–81].ThePEAPODtranscription factors,ontheotherhand,controltheonsetofasecondary proliferation arrest of the dispersed meristematic cells, moreatthecentrethanthemargins[82].Thus,theycan formacomponentoftheputativeLATEorMIDfactors.
AnothercandidatepolarityorganizerisJAGGED,which servesasimilarfunctioninpetalgrowth[24].Detailed spatio-temporal analysis of these genes coupled with time-lapsegrowthanalysisoftheirmutantsshouldclarify theirmorphogeneticrole.
Though modelling and experimental approaches together explain how diverse and complex leaf shapes can be generated, many questions still remain. Firstly, what is the evolutionary significance of this diversity.
Indeed, the existence of any strong selection pressure onleafshapeinaspecificenvironmentisstilldebated[2].
Secondly, what is the advantage of evolving diverse growth gradients, as many species grow leaves without any gradient [11]. It hasbeen suggested that specific growthpatternsconferadaptiveadvantagedependingon theecologicalnicheoftheplantspecies[32].Adetailed
‘eco-evo-devo’approachwillberequiredto gaindeeper insights.
Conflictofintereststatement Nothingdeclared.
Acknowledgements
UNacknowledgesDST-FIST,UGCCentreforAdvancedStudy,MHRD- IIScandDBT-IIScPartnershipProgramforfundingandinfrastructure support.KSacknowledgesDSTWOS-Afellowship.Weapologiseto colleagueswhoseworkcouldnotbeincludedduetoseverespaceconstraint.
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