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The evolutionary divergence of the genetic networks that control flowering in distinct
species
Della Pina, S.
Link to publication
Citation for published version (APA):
Della Pina, S. (2016). The evolutionary divergence of the genetic networks that control flowering in distinct
species
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Download date: 16 Jun 2017
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Arguments!in!the!evo0devo!debate:!Say!it!with!flowers!!
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Serena!Della!Pina,!Erik!Souer,!Ronald!Koes!
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Published!in!Journal(of(Experimental(Botany!(2014),!65!(9):!2231A2242.!
S.D.P!wrote!the!article!!
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Chapter!2!
Abstract!
A! key! question! in! evolutionary! developmental! biology! is! how! DNA! sequence! changes! have! the!
evolution! of! morphological! diversity.! The! widelyAaccepted! view! was! that! morphological! changes!
resulted!from!differences!in!number!and/or!type!of!transcription!factors,!or!even!from!small!changes!
in! the! amino! acid! sequence! of! similar! proteins.! Research! over! the! last! two! decades! indicated! that!
most! of! the! developmental! and! genetic! mechanisms! that! produce! new! structures! involve! proteins!
that!are!deeply!conserved.!These!proteins!are!encoded!by!a!type!of!genes!known!as!“toolkit”!genes!
that! control! a! plethora! of! processes! essential! for! the! correct! development! of! the! organism.!
Mutations! in! these! toolkit! genes! produce! deleterious! pleiotropic! effects.! In! contrast,! alterations! in!
regulatory! regions! affect! their! expression! only! at! specific! sites! in! the! organism! by! facilitating!
morphological!change!at!tissue!and!organ!level.!However,!some!examples!from!the!animal!and!plant!
fields!indicate!that!coding!mutations!also!contributed!to!phenotypic!evolution.!Therefore,!the!main!
question! to! ask! at! this! point! is! to! what! extent! these! mechanisms! contributed! to! the! evolution! of!
morphological!diversity.!Today,!an!increasing!amount!of!data,!especially!from!the!plant!field,!implies!
that!changes!in!cisAregulatory!sequences!in!fact!played!a!major!role!in!evolution.!
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8!
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Evolution!of!morphological!diversity!
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!Introduction!
Plant! and! animal! species! both! display! wide! divergent! morphologies! regarding! for! example! the!
arrangement!of!different!organs!on!their!bodies!or!the!shape!and!size!of!various!body!parts!A!that!are!
thought! to! result! from! evolution! via! mutation! and! selection.! The! Cambrian! explosion! (550! mya)!
marks! the! appearance! of! most! animal! phyla! that! are! known! today (Wray! et! al.,! 1996),! while! land!
plants! began! their! first! diversification! 450! mya! (Kenrick! and! Crane,! 1997).! Flowering! plants!
(Angiosperms)! appeared! only! around! 90A130! mya! (Crane! et! al.,! 1995).! Compared! to! animals,! the!
evolution! of! flowering! plants! on! land! was! relatively! fast.! Plants! “invented”! and! diversified! new!
structures,! like! flowers,! over! a! ”small”! lapse! of! time,! making! these! relatively! young! events! more!
tractable! for! genetic! dissection.! Since! an! everAgrowing! number! of! plant! species! is! amenable! to!
genetic! analyses! and! transgenesis,! plants! offer! excellent! opportunities! for! research! inEvolutionary!
Developmental!Biology!(EvoADevo).!
Here,!we!summarize!our!current!knowledge!of!the!molecular!basis!of!morphological!changes!
during! the! course! of! evolution.! We! present! general! ideas! and! give! examples! of! experimental! data!
that!support!these!theories.!While!some!examples!are!taken!from!the!animal!field,!our!emphasis!is!
on!plant!development!as!this!represents!our!main!research!subject.!
Lessons!from!the!animal!field!
Although! it! is! widely! accepted! that! morphological! variation! between! organisms! arose! from! genetic!
alterations,! the! molecular! details! remain! poorly! understood.! Initially! it! was! assumed! that! species!
specific! characters! resulted! from! speciesAspecific! proteins.! However,! as! the! number! of! sequenced!
genes,!proteins!and!eventually!entire!genomes!grew,!it!became!clear!that!the!genomes!of!organisms!
with! very! different! morphologies! are! composed! mostly! of! conserved! genes! (Martin! et! al.,! 2010;!
Miyamoto!et!al.,!1987),!and!genes!controlling!development!were!no!exception.!!
Among! the! first! examples! were! the! homeotic! HOX! genes! that! specify! the! identity! of!
(para)segments!along!the!anteriorAposterior!axis!of!embryos!ranging!from!insects!to!mammals!(Mallo!
et!al.,!2010).!They!encode!conserved!homeodomain!transcription!factors!and!are!arranged!in!clusters!
with! a! conserved! gene! order! that! correlates! with! their! expression! patterns! along! the! anteroA
posterior!axis!in!early!embryos!(colinearity).!Despite!700!MY!of!evolutionary!separation,!mammalian!
HOX! proteins! can! still! functionally! replace! their! Drosophila! homologs.! For! example,! ectopic!
expression!of!the!mouse!HOXa5!protein!or!the!Drosophila!homolog!Sex!combs!reduced!(Scr)!caused!
similar!defects!in!transgenic!flies,!including!the!transformation!of!antennae!into!T1!legs!(Zhao!et!al.,!
1993),! although! HOXa5! plays! a! different! role! in! mice! (Aubin! et! al.,! 1999).! Similarly,! expression! of!
chicken! HOXb1! could! fully! rescue! the! defects! of! Drosophila! labial! mutants! (Lutz! et! al.,! 1996).!
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Chapter!2!
Comparable! findings! were! made! with! nonAHOX! genes.! For! instance,! EYELESS! of! Drosophila! and! its!
mouse! homolog! PAX6/SMALL! EYE! are! both! required! for! the! development! of! eyes.! Even! though!
mouse! and! insect! eyes! have! no! obvious! morphological! similarity,! expression! of! mouse! PAX6! can!
drive!the!development!of!ectopic!eyes!in!transgenic!flies!(Halder!et!al.,!1995).!A!similar!conservation!
was!observed!for!extracellular!proteins,!such!as!HEDGEHOG!and!WNT,!and!their!signalling!pathways,!
which!pattern!(regions!of)!embryos!and!various!organs!(De!Robertis,!2008).!
These! findings! lead! to! the! conclusion! that! the! development! of! morphologically! disparate!
animals! is! governed! by! a! “toolkit”! of! deeply! conserved! genes.! This! triggered! the! hypothesis,! which!
was! already! put! forward! early! on! (King! and! Wilson,! 1975),! that! morphological! divergence! results!
primarily! from! alterations! in! gene! expression! patterns! (Carroll,! 2000,! 2008;! Doebley! and! Lukens,!
1998).!This!idea!was!supported!by!comparative!analyses!showing,!for!example,!that!changes!in!the!
body! plan! of! vertebrate! and! invertebrate! species! correlated! with! shifts! in! HOX! gene! expression!
patterns!(Angelini!and!Kaufman,!2005;!Burke!et!al.,!1995;!Heffer!and!Pick,!2013).!
Gene! expression! patterns! are! governed! by! complex! gene! regulatory! networks! (GRNs)!
consisting! of! transAacting! (transcription)! factors! that! bind! to! specific! cisAacting! DNA! sequences! in!
downstream! genes! to! promote! or! inhibit! their! transcription.! Binding! sites! usually! cluster! in! small!
regions,! known! as! enhancers,! which! promote! transcription! in! specific! cells.! Genes! often! contain!
multiple! enhancers! A! for! expression! on! different! sites! A! that! can! lie! many! kilobases! away! from! the!
coding!sequence.!Genes!that!control!body!architecture,!like!HOX!genes,!are!highly!connected!nodes.!
They!are!expressed!throughout!development!in!complex!patterns!via!an!array!of!distinct!enhancers!
and!transcription!factors!and!in!turn!they!regulate!a!vast!number!of!subordinate!genes!involved!in!a!
plethora! of! developmental! processes.! By! contrast,! transcription! factors! involved! in! terminal!
differentiation!processes,!or!“physiology”,!(”inputAoutput!genes”)!tend!to!form!less!connected!nodes.!!
The! modification! of! a! gene! expression! pattern! requires! (some)! rewiring! of! GRNs! through!
changes! in! CREs! or! upstream! transcription! factor! proteins! (Fig.! 1).! Alterations! in! highly! connected!
transcription! factors! will! simultaneously! affect! many! other! genes! and! developmental! processes!
resulting! in! mostly! deleterious! pleiotropic! effects.! This! may! be! one! reason! for! the! amazing!
conservation!of!insect!and!vertebrate!toolkit!proteins!after!>!700!MY.!of!separation.!Changes!in!less!
connected!“inputAoutput”!transcription!factors,!are!more!likely!to!be!fixed,!because!they!cause!less!
pleiotropic! effects,! and! are! thought! to! account! for! the! subtle! phenotypic! variation! at! the! species!
level!(Bhardwaj!et!al.,!2010;!Carrera!et!al.,!2009).!The!complex!expression!patterns!of!toolkit!genes!
governing!development!are!often!controlled!by!multiple!enhancers!that!are!active!on!different!sites:!
alterations!in!an!individual!enhancer!may!alter!the!expression!of!the!toolkit!gene!on!one!site,!without!
affecting! expression! on! other! sites,! and! thus! have! less! pleiotropic! defects! than! alterations! in! the!
encoded! protein.! Based! on! such! theoretical! considerations! Carroll! argued! that! the! evolution! of!
10!
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Evolution!of!morphological!diversity!
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“anatomy”!and!“physiology”!is!essentially!different!and!that!alterations!in!CREs,!rather!than!protein!
coding!sequences!must!be!the!major!contributor!to!the!evolution!of!form!(Carroll,!2005,!2008).!!
Although! the! idea! of! morphological! diversification! via! evolution! of! CREs! is! attractive,! others!
criticized! it! for! being! premature! and! based! on! insufficient! evidence! (Hoekstra! and! Coyne,! 2007).!
Moreover,!there!is!also!evidence!for!morphological!changes!via!evolution!of!transcription!factors.!In!
Arthropods,! for! example,! the! appearance! of! insects! with! only! six! (thoracic)! legs! but! no! legs! on! the!
abdomen! is! associated! with! a! change! in! the! HOX! protein! ULTRABITHORAX! (UBX).! This! difference!
enables! UBX! to! repress! the! expression! of! DISTALLESS! in! the! abdomen,! which! is! necessary! for! the!
development! of! appendages! (Galant! and! Carroll,! 2002;! Ronshaugen! et! al.,! 2002).! Similarly,! the!
mammalian!HOX11a!protein!acquired!the!ability!to!activate!certain!target!genes,!possibly!in!relation!
to! the! evolution! of! extraAembryonic! membrane! required! for! embryogenesis! in! utero! (Lynch! et! al.,!
2008).! Thus,! studies! with! animals! support! the! evolution! of! body! architecture! both! via! changes! in!
CREs! and! changes! in! the! protein! sequence.! However,! a! very! limited! number! of! studies! exist! which!
give! solid! support! to! either! one! or! the! other! hypothesis.! As! illustrated! in! the! following! sections,!
plants!offer!excellent!tools!in!order!to!study!the!molecular!processes!that!drive!the!evolution!of!body!
architecture!and!shape.!
Lessons!from!the!plant!field!
During! evolution! Angiosperms! developed! specialized! organs! and! tissues! that! have! contributed! to!
their! diversity! and! success.! According! to! fossil! records,! flowerAlike! structures! originated! 160–147!
MYA!(Frohlich,!2006).!Already!in!1970,!Ohno!and!colleagues!hypothesized!a!significant!role!for!gene!
duplication! in! the! evolution! of! biological! complexity! (Ohno,! 1970).! Land! plants! underwent! several!
Whole! Genome! Duplications! (WGD)! (Jiao! et! al.,! 2011)! that! facilitated! their! evolution! by! providing!
with! multiple! copies! of! all! the! preAexisting! genes.! Duplicated! genes! were! released! from! the!
constraint! of! maintaining! their! original! function! and! could! therefore! evolve! in! different! ways,!
through!mutations!in!either!regulatory!or!coding!regions.!In!the!following!paragraphs!we!will!review!
data! on! the! importance! of! CRE! and! protein! changes! in! the! evolution! of! flowering! plants,! taking!
examples!from!a!variety!of!stages!of!the!plant!lifeAcycle.!!
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Chapter!2!
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Fig.!1.!Schematic!representation!of!a!gene!with!its!CREs!(CisARegulatory!Elements)!and!the!potential!
mutations!that!can!affect!transcriptional!processes.!
(A)! Functional! CREs! (AABAC)! together! with! their! respective! Transcription! Factors! (TFs)! allow!
expression!of!a!gene!in!a!specific!organ!(or!tissue).!
(B)! Mutation! in! one! CRE! (binding! site! of! A! in! this! case! became! of! D)! leads! to! loss! of! expression! in!
sepal!but!the!gene!acquires!expression!in!leaf.!!
(C)! Mutation! in! a! TF! (A! in! this! example)! leads! to! lack! of! activation! of! the! gene! in! a! specific! organ!
(sepal!in!this!case).!
12!
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Evolution!of!morphological!diversity!
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Before!flowering:!Architecture!of!the!vegetative!plant!body!
Angiosperms!show!relatively!little!diversity!during!embryogenesis!(Weijers!and!Jurgens,!2005),!apart!
from!the!typical!differences!between!monocots!and!dicots;!most!architectural!differences!arise!later!
in!development.!After!germination!the!shoot!apical!meristem!(SAM)!grows!indeterminately!and!leaf!
primordia! initiate! at! its! periphery.! Leaves! can! be! simple,! consisting! of! a! single! blade! (Fig.! 2A),! or!
compound,!consisting!of!several!leaflets!on!the!same!petiole!(Fig.!2B).!SAM!cells!that!are!located!at!
the! periphery! of! the! meristem! downAregulate! KNOX! transcription! factors! to! enable! differentiation!
(Long! et! al.,! 1996;! Vollbrecht! et! al.,! 1991).! In! Arabidopsis,! maize! and! Antirrhinum! KNOX! genes! are!
repressed! in! the! developing! simple! leaves! by! orthologous! MYB! proteins! encoded! by! ASYMMETRIC!
LEAVES1,! ROUGH! SHEATH2! and! PHANTASTICA! (ARP! genes),! respectively.! In! species! with! lobed! or!
compound! leaves,! such! as! Arabidopsis! lyrata! and! Cardamine! hirsuta,! KNOX! genes! are! reactivated!
during! leaf! development!(Hay! and! Tsiantis,! 2006;! Piazza! et! al.,! 2010).! Ectopic! ZmKN1! expression! in!
tobacco!causes!severely!lobed!leaves!(Sinha!et!al.,!1993),!while!in!tomato!it!causes!iterations!of!the!
compound! pattern,! resulting! in! the! formation! of! superAcompound! leaves! bearing! thousands! of!
leaflets! (Hareven! et! al.,! 1996).! In! Arabidopsis! ectopic! expression! of! distinct! class! 1! KNOX! genes!
resulted!in!lobed!leaves!in!all!cases!(Hay!and!Tsiantis,!2010),!while!silencing!of!the!KNOX!gene!SHOOT!
MERISTEMLESS! (STM)! inhibited! the! lobing! of! leaves! of! Arabidopsis! suecica! (Piazza! et! al.,! 2010).!
Swapping! KNOX! genes! or! promoters! between! Arabidopsis! thaliana! and! relatives! with! lobed! leaves!
revealed!that!the!divergent!KNOX!expression!patterns!result!from!differences!in!CREs!(Piazza!et!al.,!
2010).! In! addition,! promoter! swaps! between! Arabidopsis! and! Cardamine! hirsuta! suggested! that! a!
change!in!the!promoter!of!STM!may!be!sufficient!to!determine!the!leaf!complexity!in!the!latter!(Hay!
and!Tsiantis,!2006).!Taken!together,!these!data!show!that!class!1!KNOX!proteins!of!different!species!
are! functionally! similar! and! that! changes! in! the! CREs! of! KNOX! genes! were! a! key! factor! in! the!
divergence!of!leaf!shape.!
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Chapter!2!
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Fig.2.!Morphological!variation!between!Angiosperms.!!
(A)! Variation! in! leaf! margin! of! Arabidopsis! thaliana! (top)! and! Arabidopsis! lyrata! (bottom)! and,! (B)!
from!left!to!right,!compounds!leaves!of!Cardamine!hirsuta,!Pisum!sativus!and!Solanum!lycopersicum.!
(C)! Variation! in! inflorescence! architecture:! from! left! to! right,! solitary! flower,! cyme,! raceme! and!
compounds!inflorescences.!
(DAK):! Variation! in! pigmentation! and! flower! shape/size:! (D)! Fritillaria! Persica,! (E)! Leucospermum!
cordifolium,!(F)!Tulipa,!fringed!hybrids,!(G)!Agapanthus,!(H)!Phalaenopsis,!(I)!Passiflora,!!(J)!Cosmos!
bipinnatus!and!(K)!Heliconia!Caribaea.!
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A!subclade!of!species!within!the!Fabaceae!forms!compound!leaves!that!do!not!express!KNOX1!
genes! (Champagne! et! al.,! 2007).! Leaf! complexity! is! established! in! these! species! by! distinct! genes,!
such!as!UNIFOLIATA!(UNI)!and!STAMINA!PETALOIDA!(STP)!from!pea!(Hofer!et!al.,!1997;!Taylor!et!al.,!
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Evolution!of!morphological!diversity!
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2001)!and!SINGLE!LEAFLET1!(SGL1)!from!alfalfa!(Wang!et!al.,!2008).!UNI!and!SGL1!are!orthologs!of!
LEAFY!(LFY)!of!Arabidopsis,!which!is!a!transcription!factor!that!determines!floral!meristem!identity!in!
Angiosperms!(Krizek!and!Fletcher,!2005).!STP!is!the!ortholog!of!UNUSUAL!FLORAL!ORGANS,!which!is!
an!FABox!protein!that!binds!to!and!activates!LFY!(Chae!et!al.,!2008;!Souer!et!al.,!2008).!Hence,!STP!
presumably!controls!leaf!through!activation!of!UNI.!
Several! findings! suggest! that! the! role! of! LFY! as! a! promoter! of! meristem! proliferation/!
outgrowth! in! the! development! of! compound! leaves! is! more! ancient! than! its! role! in! flower!
development.! First,! in! primitive! plants! lacking! flowers,! like! the! moss! Physcomitrella,! LFY! promotes!
cell!divisions!at!different!stages!of!development!(Tanahashi!et!al.,!2005).!Second,!tomato!(falsiflora)!
and! Lotus! (proliferating! floral! meristem)! mutations! in! LFY! orthologs! also! reduce! leaf! complexity!
(MolineroARosales! et! al.,! 1999;! Wang! et! al.,! 2013),! albeit! mildly! because! these! leafs! also! express!
KNOX1! genes.! Third,! mutation! of! the! rice! homolog! reduces! outgrowth! of! tillers! (Rao! et! al.,! 2008).!
Fourth,! recent! work! revealed! that! the! auxin! response! factor! MONOPTEROUS! promotes! LFY!
expression! in! floral! primordia! and! that! LFY! induces! auxinAsensing! and! growth/proliferation! of! the!
meristem,! which! is! evident! in! certain! double! mutant! combinations! but! not! in! single! lfy! mutants!
(Chahtane!et!al.,!2013;!Li!et!al.,!2013;!Yamaguchi!et!al.,!2013).!This!suggests!that!in!early!land!plants!
LFY! regulated! cell! division! and! with! the! appearance! of! flowers! acquired! a! new! role! in! the!
specification!of!floral!meristem!identity.!Functional!and!structural!comparisons!of!LFY!proteins!from!
algae!to!angiosperms!indicated!that!the!acquisition!of!floral!function(s)!was!associated!with!changes!
in! its! DNA! binding! domain,! possibly! in! conjunction! with! alterations! in! CREs! of! subordinate! genes.!
Interestingly,!LFY!evolution!apparently!involved!a!promiscuous!intermediate!form,!found!today!in!a!
hornworts,! with! both! the! old! and! new! DNA! binding! specificities,! which! may! have! helped! to!
circumvent!(initial)!deleterious!pleiotropic!effects!(Maizel!et!al.,!2005;!Sayou!et!al.,!2014).!
Another!trait!that!diversifies!the!vegetative!plant!body!is!the!formation!of!sidebranches!from!
meristems! in! leaf! axils.! The! outgrowth! (or! dormancy)! of! these! axillary! meristems! is! regulated! by!
signals! originating! from! the! apex! (auxin)! and! the! basal! part! (strigolactones).! Auxin! produced! in! the!
apex!moves!downward!via!the!stem!and!inhibits!the!activity!of!axillary!buds,!a!process!that!is!known!
as! apical! dominance.! The! absence! of! lateral! shoots! (tillers)! in! modern! maize,! for! example,! results!
from! a! strong! apical! dominance.! This! architecture! evolved! during! its! domestication! from! its!
progenitor!Teosinte,!which!displays!less!apical!dominance!and!is!highly!branched!and!could!be!traced!
to! the! altered! expression! of! TEOSINTE! BRANCHED! (TB1),! which! encodes! a! transcription! factor! that!
supresses!meristem!outgrowth!(Doebley!et!al.,!1997).!In!maize!TB1!is!highly!expressed!and!branching!
is! repressed,! whereas! in! teosinte! TB1! expression! is! relatively! low,! resulting! in! more! extensive!
branching.! The! elevated! expression! of! TB1! in! maize! correlates! with! the! insertion! of! a! HOPSCOTCH!
!
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Chapter!2!
retrotransposon! at! approximately! 60! kb! upstream! of! the! TB1! coding! sequence,! thereby! changing!
gene!regulation!and!architectural!morphology!(Studer!et!al.,!2011).!
Evolution!of!flowering!time!response!(or!mechanism)!!
Angiosperms!display!an!amazing!variation!regarding!the!moment!when!they!switch!from!vegetative!
growth!to!flowering!(flowering!time).!Environmental!and!endogenous!signals!determine!the!moment!
that!a!plant!undergoes!this!transition.!Temperature!and!day!length!are!the!principal!cues!that!trigger!
the! onset! of! flowering! in! a! particular! season.! Because! these! requirements! are! speciesAdependent,!
some! plants! need! a! particular! day! length! (photoperiod)! in! order! to! flower,! while! others! are!
photoperiod! insensitive.! In! addition,! some! species! require! exposure! to! low! winter! temperatures!
(vernalization),! while! summerAannual! plants! do! not.! External! and! internal! stimuli! converge! in! the!
regulation! of! the! main! components! of! the! longAdistance! florigen! signal,! FLOWERING! LOCUS! T! (FT)!
and!its!paralog!TWIN!SISTER!OF!FT!(TSF)!(Andres!and!Coupland,!2012;!Song!et!al.,!2013).!After!their!
induction! in! leaves,! FT! and! TSF! proteins! are! loaded! into! the! phloem! and! translocated! to! the! apex!
where!they!interact!with!a!bZIP!transcription!factor,!FLOWERING!LOCUS!D!(FD)!via!a!14A3A3!protein!
(Abe! et! al.,! 2005;! Taoka! et! al.,! 2011;! Wigge! et! al.,! 2005).! These! FT–FD! and! TSFAFD! transcriptional!
complexes!activate!the!expression!of!several!floral!pathway!integrators:!SUPPRESSOR!OF!CONSTANS1!
(SOC1),! SQUAMOSA! PROMOTER! BINDING! PROTEIN! LIKE! (SPL)! and! Flower! Meristem! Identity! genes!
(FMI)! to! promote! flowering! (Pose! et! al.,! 2012).! Once! FMI! genes! are! transcribed,! the! plants! is!
irreversibly! committed! to! floral! initiation,! and! FT! and! TSF! become! unnecessary! (Corbesier! et! al.,!
2007).!
In! the! long! day! (LD)! plant! Arabidopsis,! FT! is! activated! by! the! zincAfinger! transcription! factor!
CONSTANS!(CO).!CO!activity!is!regulated!transcriptionally!by!light!and!the!circadian!clock,!and!postA
translationally!by!light,!in!such!a!way!that!during!short!days!(SD)!CO!protein!is!degraded!whereas!in!
LD,! CO! is! stabilized! and! FT! transcription! activated! (Turck! et! al.,! 2008).! CO! has! an! ancient! origin! as!
homologs! that! can! functionally! replace! CO! in! Arabidopsis! can! be! found! even! in! the! alga!
Chlamydomonas! reinhardtii.! Interestingly,! CrCO! is! also! regulated! by! dayAlength! and! the! clock! in!
Chlamydomonas!and!involved!in!rhythmic!output!processes!(Serrano!et!al.,!2009).!
Clearly!FTAlike!proteins!represent!a!universal!flowering!signal!conserved!during!plant!evolution.!
The! diversification! of! seasonal! control! of! flowering! appears! to! be! due! to! rewiring! of! the! upstream!
network!that!controls!FT!expression!and/or!the!downstream!network!for!the!output!of!FTAsignal.!In!
rice,!a!SD!plant,!the!COAhomolog!HEADING!DATE!1!(HD1)!activates!the!FT‑like!genes!HD3A!and!RICE!
FT‑LIKE1!but! only! in! SD! conditions.!The! difference! with! Arabidopsis! is! that!HD1!is! in!LDs!converted!
from!an!activator!into!a!repressor!of!HD3A!by!a!pathway!that!is!controlled!by!phytochrome!B,!which!
confers! a! robust! regulation! by! day! length! and! light! quality! (Ishikawa! et! al.,! 2009;! Ishikawa! et! al.,!
16!
!
Evolution!of!morphological!diversity!
!
2005).!In!addition,!EARLY!HEADING!DATE!1!(EHD1),!a!BAtype!response!regulator,!induces!HD3A!in!SDs!
independently!of!HD1.!(Doi!et!al.,!2004).!In!another!SD!plant,!Fragaria!vesca!(strawberry),!LDs!induce!
expression! of! FvFT! and! (subsequently)! FvSOC1,! as! in! Arabidopsis.! However,! in! strawberry! plants!
FvSOC1! activates! a! repressor! of! flowering! that! is! homologous! to! TERMINAL! FLOWER1! from!
Arabidopsis!(Mouhu!et!al.,!2013).!!
Sugar!beet,!a!biennial!that!requires!vernalization!and!long!days!for!flowering,!contains!two!FT!
paralogs.!One!of!these!(BvFT2)!has!a!similar!function!as!FT!in!Arabidopsis,!whereas,!the!other!(BvFT1)!
is! a! repressor! of! flowering! that! is! thought! to! be! involved! in! the! vernalization! response! (Pin! et! al.,!
2010).!The!divergence!of!BvFT1!and!BvFT2!function!involved!changes!in!their!CREs,!because!they!are!
differentially! expressed! in! beet,! and! changes! in! the! encoded! proteins,! because! BvFT2! induces! and!
BvFT1!represses!flowering!when!expressed!in!Arabidopsis!(Pin!et!al.,!2010).!!
In! other! species! the! CO/FT! system! acquired! new! roles! in! entirely! different! developmental!
processes.! In! Populus! trees,! which! are! perennials,! FT! promotes! the! onset! of! flowering,! as! in!
Arabidopsis,! and! in! addition! supresses! the! growth! cessation! and! bud! set! in! the! fall! when! days! are!
shortening! (Bohlenius! et! al.,! 2006).! Interestingly,! the! critical! day! length! for! downAregulation! of! FT,!
growth! cessation! and! bud! set! is! shorter! for! varieties! growing! at! moderate! latitudes! compared! to!
those! from! northern! latitudes.! This! adaptive! change,! which! enables! the! timely! preparation! for!
winter,!is!associated!with!an!altered!CO!regulation,!because!of!which!CO!mRNA!peaks!several!hours!
earlier!after!dawn!in!trees!from!southern!populations!compared!to!northern!populations!(Bohlenius!
et!al.,!2006).!The!genetic!changes!that!caused!the!divergent!CO!expression!remain,!however,!to!be!
established.!!
In!potato!SDs!activate!two!FT!paralogs;!one!(StSP3A)!is!involved!in!the!transition!to!flowering,!
and! the! other! (StSP6A)! in! the! transition! to! tuberization! (Navarro! et! al.,! 2011).! Since! StSP6A! can!
functionally!replace!FT!in!Arabidopsis!and!rice!Hd3A!promotes!both!flowering!and!tuberization!when!
expressed!in!potato,!the!functional!divergence!of!StSP3A!and!StSP6A!seems!mostly!due!to!changes!in!
their!transcriptional!regulation!and!CREs,!rather!than!the!encoded!proteins!(Navarro!et!al.,!2011).!
Say!it!with!flowers:!the!evolution!of!inflorescences.!
After! transition! to! flowering,! flowers! emerge! in! specific! positions! on! the! plant! body.! Some! species!
form!a!single!(solitary)!flower,!while!others!generate!inflorescence!branches!that!bear!many!flowers!
in! a! variety! of! arrays! (Fig.! 2C).! In! cymes! flowers! develop! from! apical! meristems! and! inflorescence!
growth! continues! from! a! secondary! lateral! (sympodial)! meristem! formed! at! its! flank,! whereas! in!
racemes,! the! apical! meristem! maintains! its! indeterminacy! and! flowers! develop! from! lateral!
meristems!(Krizek!and!Fletcher,!2005).!!
!
!
17!
Chapter!2!
In!Arabidopsis!the!expression!of!AP1!and!LFY!is!largely!restricted!to!floral!meristems,!though!
LFY!is!also!expressed!at!low!level!during!vegetative!growths.!These!genes!are!strongly!expressed!in!
lateral!(floral)!meristems,!while!in!the!apical!(inflorescence)!meristem!their!expression!is!prevented!
by!TERMINAL!FLOWER!1!(TFL1),!in!Arabidopsis,!and!CENTRORADIALIS!in!Antirrhinum!(Bradley!et!al.,!
1996;! Bradley! et! al.,! 1997).! Constitutive! expression! of! either! LFY! or! AP1,! which! positively! regulate!
each!other!in!a!regulatory!loop,!causes!precocious!flowering!and!converts!the!apical!meristem!into!a!
flower,!and,!consequently,!the!racemose!inflorescence!into!a!solitary!flower!(Mandel!and!Yanofsky,!
1995;! Weigel! and! Nilsson,! 1995).! Hence,! the! spatioAtemporal! regulation! of! LFY! and/or! AP1!
transcription! is! the! major! factor! determining! when! and! where! flowers! form! in! Arabidopsis.! Similar!
results!were!obtained!with!other!species,!including!trees!such!as!aspen!and!citrus!(Pena!et!al.,!2001;!
Weigel!and!Nilsson,!1995).!However!in!Petunia,!a!cyme,!constitutive!expression!of!LFY!or!the!Petunia!
homolog! ABERRANT! LEAF! AND! FLOWER! (ALF)! does! not! affect! flowering! time! or! inflorescence!
architecture.!This!indicates!that!the!transcription!of!ALF,!though!required!for!floral!identity,!is!not!the!
limiting!factor!that!restricts!flower!formation!in!time!or!space.!Instead,!the!limiting!factor!is!another!
FMI!gene:!DOUBLE!TOP!(DOT)!(Souer!et!al.,!2008).!DOT!is!the!homolog!of!UNUSUAL!FLORAL!ORGANS!
(UFO)!from!Arabidopsis.!DOT!and!UFO!are!functionally!interchangeable!FAbox!protein!components!of!
an! SCFAtype! ubiquitin! ligase! complex! that! binds! to! ALF/LFY! to! promote! its! transcription! activation!
potential!(Chae!et!al.,!2008;!Souer!et!al.,!2008).!While!expression!of!DOT!is!restricted!to!(incipient)!
flowers,!UFO!is!expressed!in!many!meristems,!including!the!SAM!in!embryos!and!seedlings.!Indeed,!
constitutive! expression! of! DOT! or! UFO! in! Petunia! causes! precocious! flowering! and! changes! the!
cymose!inflorescence!into!a!solitary!flower,!while!in!Arabidopsis!no!such!effects!are!seen.!Transgenic!
experiments! in! which! promoterAreporter! genes! were! swapped! between! Petunia! and! Arabidopsis!
indicated! that! the! diversification! of! DOT! and! UFO! expression! was! caused! at! least! in! part! through!
differences!in!their!CREs!(Kusters,!2011).!
Interestingly! LFY! expression! patterns! vary! even! between! species! with! similar! inflorescence!
architectures.!Brassicaceae!like!Ionopsidium!acaule!(violet!cress,!Iac)!and!Laevenworthia!crassa!(Lcr)!
bear! flowers! in! the! axils! of! rosette! leaves! (“rosette! flowering”)! and! can! be! seen! as! racemes! with!
reduced! internodes! (Shu! et! al.,! 2000).! When! introduced! in! Arabidopsis! a! LcrLFY! transgene,! or! a!
fusion!of!the!LcrLFY!promoter!(LcrLFYp)!to!the!AtLFY!coding!sequence,!rectifies!the!lfy!phenotype,!and!
in! addition! converts! the! apical! meristem! into! a! terminal! flower,! which! fits! with! the! finding! that!
LcrLFYp! is! active! in! the! IM! (Sliwinski! et! al.,! 2006;! Yoon! and! Baum,! 2004).! Even! though! IacLFY! is!
expressed!in!floral!mersitems!and!the!apical!meristem!in!I.!acaule,!IacLFYp!is!not!active!in!the!apical!
meristem! of! Arabidopsis,! and! a! IacLFY! transgene! does! not! affect! the! development! the! apical!
meristem! in! Arabidopsis! (Shu! et! al.,! 2000;! Yoon! and! Baum,! 2004).! These! findings! imply! that! LFY!
expression!patterns!varied!even!between!closely!related!species,!through!alterations!in!CREs!within!
18!
!
Evolution!of!morphological!diversity!
!
their!promoters!or!in!the!upstream!network,!without!any!obvious!alterations!in!architecture.!It!may!
be!that!the!apical!IM!of!L.crassa!and!I.acaule!does!not!acquire!floral!identity,!even!when!it!expresses!
LFY,! because! it! lacks/lost! expression! of! an! essential! partner,! such! as! the! UFO/DOT! homolog.! The!
picture!that!emerges!is!that!expression!patterns!of!distinct!FMI!genes!diverged!substantially!between!
angiosperms! and! thus! created! new! patterns! where! essential! partners! are! coAexpressed! and! floral!
identity!is!established.!!
Flowers!of!all!hues:!variations!in!flower!architecture!
In! Angiosperms! LFY! activates! several! genes! involved! in! floral! morphogenesis,! including! homeotic!
floral!organ!identity!genes.!Many!of!these!encode!MADS!box!transcription!factors!which!specify!the!
fate! of! emerging! organ! primordia.! According! to! the! ABC! model,! which! is! based! on! phenotypes! of!
homeotic!Arabidopsis!thaliana!and!Antirrhinum!majus! mutants,!three!classes!of!genes!(A,!B!and!C)!
specify!the!identity!of!floral!organ!primordia!(Coen!and!Meyerowitz,!1991).!A!function!specifies!sepal!
identity!in!the!outermost!whorl!(whorl!1),!coAexpression!of!A!and!B!function!specifies!petal!identity!in!
whorl! 2,! B! and! C! genes! together! control! stamen! identity! in! whorl! 3,! while! the! CAfunction! alone!
specifies! carpel! identity! in! whorl! 4.! In! addition,! the! A! and! C! functions! antagonize! each! other.! E!
function!genes!are!a!later!addition!to!the!model!and!act!as!coAregulators!in!controlling!the!four!floral!
organ!identities!(Theissen,!2001).!Nowadays!a!significant!amount!of!data!is!available!relating!to!the!
conservation/evolution!of!these!TFs.!!
Flowers! present! an! enormous! variation! in! organ! number,! size! and! shape! (Fig.! 2DAK).! In!
gymnosperm,!male!and!female!sex!organs!develop!on!separate!structures!(cones)!that!lack!most!of!
the!features!seen!in!flowers.!Putative!BA!and!CAfunction!orthologs!can!be!found!in!gymnosperms!as!
well:! BAclass! genes! are! maleAspecific,! while! C! genes! are! expressed! in! both! male! and! female! organs!
(Tandre! et! al.,! 1998).! These! genes! still! show! a! significant! level! of! functional! conservation! between!
gymnosperm! and! angiosperm! despite! millions! of! years! of! separation! (Rutledge! et! al.,! 1998).! This!
finding!has!been!used!as!starting!point!to!explain!flower!origin.!According!to!the!outAofAmale!(or!outA
ofAfemale)!theory!(Theissen!and!Becker,!2004),!class!B!genes!worked!as!a!“switch”!to!control!female!
(or!male)!organ!development!in!male!(or!female)!cones.!The!formation!of!an!apicalAbasal!dissimilarity!
in!B!gene!expression!within!the!cone!could!have!led!to!the!hermaphroditic!precursors!of!flowers.!It!is!
conceivable! that! both! trans! and! CREs! alterations! are! responsible! for! alterations! in! the! expression!
pattern!of!these!B!genes.!Further!experiments!are!necessary!to!elucidate!their!evolution,!however,!
these! studies! are! hampered! by! the! fact! that! basal! angiosperms! are! not! amenable! to! genetic!
dissection! and! ancestral! plants! bearing! hermaphroditic! precursors! of! flowers! are! unfortunately!
extinct.!!
!
!
19!
Chapter!2!
APETALA3! (AP3)! together! with! PISTILLATA! belong! to! the! BAtype! MADS! box! genes! that! are!
involved! in! the! regulation! of! petal! identity! specification! in! Arabidopsis! (Jenik! and! Irish,! 2001).! In!
Ranunculaceae! the! AP3! lineage! has! undergone! many! duplication! events,! giving! rise! to! three!
paralogous!AP3!lineages!(AP3Z1,!AP3Z2!and!AP3Z3),!which!are!found!throughout!the!family!(Kramer!
et! al.,! 2003).! Species! bearing! petals!express! AP3Z3! orthologs! specifically! in! petal! primordia! and!
developing! petals,! while! species! lacking! petals! lack! AP3Z3! expression! (Zhang! et! al.,! 2013).! AP3A3!
expression!seems!to!have!been!lost!several!times!independently!by!either!deletions!or!insertions!in!
the! coding,! promoter,! or! intronic! regions.! The! independent! occurrence! of! apetalous! species! in! the!
Ranunculaceae! reflects! a! possible! advantage:! formation! of! petals! and! nectaries! requires! energy,!
which! explains! why! many! taxa! have! evolved! general! pollination! systems! with! sepals,! stamens,! or!
filaments!which!are!attractive!to!insects.!
Another! gene! duplication,! followed! by! a! frameshift! mutation! in! the! CAterminal! region! of! the!
AP3! gene,! formed! two! new! lineages! in! the! core! eudicots:! euAP3! and! TM6! (Vandenbussche! et! al.,!
2003).! While! Arabidopsis! and! Antirrhinum! have! lost! their! TM6! copy! and! maintained! only! euAP3!
(AtAP3!and!AmDEFICIENS),!Petunia!hybrida!maintained!both!AP3!(PhDEF)!and!TM6!genes.!Mutation!
in!PhDEF!shows!a!conversion!of!petals!to!sepals,!but!the!stamens!are!still!made!due!to!the!fact!that!
TM6!works!redundantly!in!their!specification!indeed,!the!double!mutant!transforms!petals!to!sepals!
and! stamens! to! carpels! (Rijpkema! et! al.,! 2006).! TM6! shows! a! third! and! fourth! whorl! expression!
pattern,!while!PhDEF!is!expressed!in!the!second!and!third!whorl.!Interestingly,!the!divergence!of!the!
DEF/TM6!expression!patterns!correlates!with!the!loss!of!a!conserved!promoter!element!in!the!TM6!
lineage!that!might!be!responsible!for!the!subfunctionalization!of!these!genes.!Ectopic!expression!of!
TM6!is!capable!of!inducing!petal!development!in!a!def!mutant!background,!which!shows!that!protein!
function!is!identical!and!that!subfunctionalization!results!from!changes!in!TM6!regulation!(Rijpkema!
et!al.,!2006;!Vandenbussche!et!al.,!2004).!!
Heterotopic! expression! of! BAfunction! genes! in! whorl! one! of! Tulipa! explains! the! presence! of!
petals!in!the!outer!whorl,!also!known!as!tepals!(Kanno!et!al.,!2003).!Several!species!of!the!Solanaceae!
family! also! show! morphological! alterations! of! whorl! 1! too:! the! inflated! calyx! syndrome! (ICS)! is! an!
amazing! floral! morphological! novelty! where,! after! pollination,! the! growth! of! sepals! restarts! thus!
giving! rise! to! a! balloonAlike! structure! that! encapsulates! the! mature! berry.! Research! in! Physalis! has!
suggested! that! a! change! in! the! promoter! region! of! MPF2! –! a! member! of! the! StMADS16! clade! A!
accounts! for! the! alteration! in! its! expression,! which! is! responsible! for! ICS! in! this! species! (He! and!
Saedler,!2005).!
An!interesting!exception!to!the!conserved!floral!ground!plan!of!eudicots!is!found!in!Lacandonia!
schismatica! where! stamens! occur! in! the! centre! of! the! flower! and! are! surrounded! by! carpels.! This!
different!order!in!flower!organs!appears!to!be!due!to!different!spatioAtemporal!expression!patterns!
20!
!
Evolution!of!morphological!diversity!
!
in! BA! and! CAfunction! genes! (AlvarezABuylla! et! al.,! 2010),! but! further! experiments! are! necessary! in!
order!to!elucidate!whether!the!new!expression!patterns!arose!via!mutations!in!CREs!of!B!and!C!genes!
or!in!the!upstream!regulatory!network,!or!in!both.!
Flowers! can! display! radial! symmetry! (Fig.! 2IAJ),! if! all! petals! develop! and! fuse! equally,! or!
bilateral!symmetry!(zygomorphy)!(Fig.!2H),!if!their!petals!develop!in!an!unequal!way.!This!difference!
in! flower! symmetry! evolved! multiples! times! during! angiosperm! evolution! and! it! involves! the!
expression! of! TEOSINTE! BRANCHED1/CYCLOIDEA/PCF! (TCP)! genes! along! the! dorsoAventral! axis.! In!
radial! asymmetric! or! zygomorphic! flowers! these! genes! are! strongly! expressed! in! the! dorsal! petals,!
while! in! symmetric! or! actinomorphic! flowers! they! are! present! at! too! low! level! in! order! to! effect!
flower!symmetry.!Yang!and!colleagues!(Yang!et!al.,!2012)!found!that!the!promoters!of!two!genes!of!
the! CYCLOIDEA2! (CYC)! clade,! CYC1C! and! CYC1D! of! Primulina! heterotricha,! have! consensus! CYC!
binding!sites,!resulting!in!positive!autoregulation!and!crossAregulation!of!each!others!expression.!This!
feedback! loop! seems! to! operate! in! many! species! which! bear! zygomorphic! flowers! since! consensus!
CYC!binding!sites!were!identified!in!the!promoters!of!their!CYC2!genes.!In!contrast,!the!promoters!of!
CYC2! genes! in! the! actinomorphic! lineage! lack! these! CYC! binding! sites,! and! consequently! the!
autoregulatory! loop! necessary! to! maintain! their! expression! during! flower! development.! It! thus!
seems!that!a!similar!change!in!the!CREs!of!CYC2!genes!arose!multiple!times!in!angiosperms,!which!
could!explain!the!independent!origin!of!floral!zygomorphy.!!
Terminal!differentiation!of!epidermal!cells.!
Changes! in! the! expression! pattern! also! drove! the! evolution! of! metabolic! pathways,! such! as! plant!
pigmentation.! Anthocyanins! are! responsible! for! most! of! the! orange,! red! and! purple! coloration! in!
Angiosperms! (Fig.! 2DAK)! and! serve! a! variety! of! (pleiotropic)! functions:! in! vegetative! tissues! they!
protect!from!high!light!intensities,!while!in!flowers!and!fruits!they!attract!animals!that!aid!pollination!
and!seed!dispersal!(Buer!et!al.,!2010).!
Structural! anthocyanin! genes! encoding! the! enzymes! of! the! pathway! are! activated! by! a!
conserved!complex!(MBW),!consisting!of!a!MYB,!a!basicAhelixAloopAhelix!(HLH)!and!a!WD40!protein!
(Grotewold,! 2006;! Koes! et! al.,! 2005).! Although! the! MBW! complex! is! conserved! between! monocot!
and! dicot! species,! its! function! has! diverged! substantially,! in! terms! of! downstream! genes! and!
processes!that!are!activated.!The!MBW!activates!all!structural!genes!from!the!anthocyanin!pathway!
in! maize,! but! only! a! subset! of! structural! genes! in! most! dicots.! Exchanging! either! MYB! and! HLH!
regulators! or! gene! promoters! between! species! showed! that! this! is! due! to! differences! in! the!
structural!gene!promoters,!and!not!in!the!MYB!and!HLH!proteins!(Koes!et!al.,!1994;!Quattrocchio!et!
al.,!1998).!
!
!
21!
Chapter!2!
Phenotypes! of! Arabidopsis! and! Petunia! mutants! revealed! that! the! divergence! of! MBW!
function!extends!to!several!other!processes!involved!in!the!differentiation!of!epidermal!cells!(Ramsay!
and! Glover,! 2005).! In! Petunia! these! include! the! division! and! morphogenesis! of! seed! coat! cells! and!
several!vacuolar!processes!that!affect!petal!coloration,!such!as!vacuolar!acidification!and!stabilization!
of!vacuolar!anthocyanins.!In!Arabidopsis!MBW!complexes!specify!the!identity!of!incipient!trichomes!
in! stems! and! leaves,! nonAhair! cells! in! the! root! epidermis! and! the! production! of! mucilage! by! seeds.!
Interestingly,!in!Petunia!or!maize!no!trichome!defects!are!seen!in!the!gain!or!loss!of!function!mutants!
for! MBW! components,! indicating! that! in! these! species! the! MBW! complex! has! no! role! in! trichome!
development.! Nevertheless,! the! maize! WD40! and! HLH! proteins! PALE! ALEURONE! COLOR! (PAC)! and!
RED!(R)!can!drive!trichome!development!when!expressed!in!Arabidopsis!(Carey!et!al.,!2004;!Lloyd!et!
al.,!1992).!This!indicates!that!Arabidopsis!MBW!genes!acquired!their!role!in!trichome!development!
(and! possibly! noAhair! cells! in! the! root! epidermis)! via! changes! in! the! CREs! of! downstream! trichome!
genes!that!brought!them!under!MBW!control.!Genome!wide!analysis!recently!identified!some!20A40!
target! genes! that! are! bound! and! regulated! by! GLABRA1! (GL1)! and/or! GL3,! the! MYB! and! HLH!
components! of! the! Arabidopsis! MBW! complex! (Morohashi! and! Grotewold,! 2009).! Comparative!
analysis! with! other! species,! like! Petunia! or! maize! may! shed! light! on! how! these! “old! genes! learned!
new!tricks”.!
Because!pigmentation!is!an!easy!readAout!of!gene!expression,!it!is!a!convenient!system!to!use!
to!study!(adaptive)!changes!in!gene!expression!patterns.!The!pigmentation!of!floral!tissues!together!
with! other! features,! such! as! scent! and! flower! morphology,! attracts! visits! of! specific! animals! (bees,!
moths,! birds,! bats)! for! pollination.! Changes! in! any! of! these! floral! features! (pollination! syndrome),!
might! lead! to! attraction! of! different! pollinators! and! may,! in! theory,! result! in! genetic! isolation! and!
ultimately! speciation! (Wessinger! and! Rausher,! 2012).! Petunia! integrifolia,! for! example,! has! colored!
flowers!with!a!short!wide!tube!that!are!visited!by!bumblebees,!whereas!P.!axilaris!has!white!scented!
flowers!with!a!long!narrow!tube!that!are!visited!by!nocturnal!hawkmoths.!Inactivation!in!P.!axillaris!
of!the!ANTHOCYANIN2!gene,!a!MYB!regulator,!caused!different!petal!pigmentation!(Hoballah!et!al.,!
2007;!Quattrocchio!et!al.,!1999).!Interestingly!an2!mutations!were!fixed!in!P.!axillaris!at!least!5!times!
independently,!but!mutations!in!other!anthocyanin!genes!were!not.!Presumably!that!is!because!AN2!
expression!is!confined!to!the!petal!limb,!while!other!anthocyanin!genes!are!also!expressed!on!many!
other!sites,!which!is!the!reason!why!mutations!in!AN2!are!the!least!pleiotropic!(Quattrocchio!et!al.,!
1999).!A!shift!from!bee!to!hummingbird!pollination!is!associated!with!transition!from!blueApurple!to!
red! colored! flowers,! together! with! other! morphological! alterations.! The! most! frequent! cause! of!
these! color! changes! is! a! reduced! expression! of! Flavonoid! 3’ZHydroxylase! (F3’H)! and/or! F3’5’H!
(Wessinger! and! Rausher,! 2012).! Expression! of! F3’5’H,! which! is! primarily! required! for! flower!
pigmentation,! was! abolished! by! mutations! in! either! the! coding! sequence! or! a! CRE.! By! contrast! in!
22!
!
Evolution!of!morphological!diversity!
!
F3’H,! which! is! involved! in! other! processes! besides! flower! pigmentation,! mutations! occurred!
predominantly!in!its!CREs,!thus!reducing!F3’H!expression!mainly!in!flowers!(Wessinger!and!Rausher,!
2012).!!
The! cases! summarized! above! concern! loss! of! function! mutations! that! reduce! or! eliminate!
pigmentation! in! certain! tissues.! They! all! have! an! impact! on! elements! that! cause! relatively! few!
pleiotropic! effects,! either! a! CRE! or! the! coding! sequence! of! a! paralog! with! a! restricted! expression!
pattern.!Cases!in!which!novel!pigmentation!patterns!were!acquired!are!more!scarcely!documented.!
That!may!be!because!such!mutations!are!not!favored!and/or!they!are!more!difficult!to!prove!without!
knowing! the! ancestral! state.! A! major! part! of! the! color! difference! between! pale! red! bumblebeeA
pollinated! flowers! of! Mimulus! lewisii! and! the! bright! red! hummingbirdApollinated! flowers! of! M.!
cardinalis! is! due! to! the! difference! in! expression! level! of! a! MYB! protein,! ROSE! INTENSITY! 1,! that!
inhibits!the!activity!of!the!MBW!complex!(Yuan!et!al.,!2013).!The!different!expression!levels!appear!
to!be!due!to!an!alteration!in!RO1!CREs,!but!since!the!ancestral!state!is!unknown!it!is!unclear!whether!
this!involved!the!loss!of!an!activating!CRE!in!M.!cardinalis,!or!a!gain!in!M.!lewisii.!!
The! creation! of! new! pigmentation! patterns! by! gain! of! function! mutations! has! been!
documented! in! a! few! domesticated! crops.! In! all! species! analyzed! the! WD40! partner! of! the! MBW!
complex!is!more!or!less!ubiquitously!expressed!and!the!pigmentation!pattern!is!largely!dictated!by!
the!expression!patterns!of!the!MYB!and!bHLH!partners!(Koes!et!al.,!2005).!Ectopic!expression!of!the!
MYB!protein!is!sufficient!to!establish!the!pigmentation!of!new!tissues!(Nesi!et!al.,!2001;!Spelt!et!al.,!
2000).! The! appearance! of! varieties! with! new! pigmentation! patterns! was! indeed! associated! with!
alterations!in!the!regulatory!region!of!the!MYB!anthocyanin!regulators.!For!instance!the!appearance!
of!blood!oranges!with!anthocyanins!in!the!flesh!of!the!fruit!happened!independently!on!at!least!two!
occasion;! in! both! cases! new! CREs! originated! from! a! retrotransposon! insertion! into! the! RUBY! gene,!
which! encode! a! MYB! component! of! the! MBW! complex! (Butelli! et! al.,! 2012).! The! color! difference!
between! red! and! green! apples! can! also! be! traced! to! a! CRE! in! the! MYB10! allele! of! red! apples! that!
results! in! enhanced! MYB10! expression! (Espley! et! al.,! 2007).! This! novel! CRE! originates! from! a!
microsatellite! and! creates! a! new! binding! site! for! MYB10! itself! resulting! in! an! autoAregulatory! loop!
(Espley!et!al.,!2009).!
Conclusion!
In! this! review! we! have! tried! to! elucidate! some! of! the! general! mechanisms! that! have! been! at! work!
throughout! plant! and! animal! evolution.! The! appearance! of! novel! structures! and! their! subsequent!
diversification! relied! primarily! on! alterations! in! spatioAtemporal! gene! expression! patterns,! rather!
than!the!sudden!appearance!of!completely!new!genes.!Indeed,!in!most!cases!morphological!novelty!
originates! from! changes! in! the! timing,! rates! or! pattern! of! expression! of! preAexisting! genes.! To! our!
!
!
23!
Chapter!2!
knowledge! very! few! cases! have! been! documented! where! a! new! gene! expression! pattern! could! be!
attributed!to!an!alteration!in!a!transcription!factor!protein.!That!might!be!because!such!events!are!
rarely!fixed!during!evolution,!due!to!their!pleiotropic!defects,!and/or!because!such!events!are!more!
difficult!to!identify!and!prove!experimentally.!However,!in!a!substantial!number!of!cases!the!cause!of!
an!altered!gene!expression!pattern!was!shown!to!result!from!changes!in!gene!promoters,!although!
the! specific! CREs! and! DNA! changes! that! were! involved! were! traced! down! in! only! a! few! cases.! This!
may! be! due! to! the! fact! that! CREs! can! lie! many! kilobases! away! from! the! coding! sequence! and! are!
difficult! to! recognize! since! single! nucleotide! changes! may! be! sufficient! to! create! or! remove!
transcription! factor! binding! sites.! The! general! picture! is! that! most! changes,! including! the! few! in!
transcription!factor!proteins,!seem!to!have!been!selected!for!minimum!pleiotropic!effects.!!
One!could!envisage!several!other!mechanisms,!besides!the!modification!of!tissueAspecific!CREs!
involved! in! transcription! control,! to! rewire! GRNs! with! minimal! pleiotropic! effects.! For! example,!
alternative!splicing!(Syed!et!al.,!2012)!or!polyadenylation!may!lead!to!the!expression!of!novel!forms!
of! a! protein,! with! new! functions,! while! leaving! the! old! function! unharmed.! Alterations! in! (transA
acting)!miRNAs,!(cisAacting)!complementary!sequences!within!mRNAs!(Kosik,!2009),!DNA!methylation!
(Cortijo! et! al.,! 2014)! and! histone! modifications! (Turck! and! Coupland,! 2013)! could,! theoretically,!
modify! gene! expression! patterns! in! a! similar! subtle! ways! as! changes! CREs.! To! what! extent! such!
mechanisms!contribute!to!morphological!diversification!remains!to!be!determined.!
Acknowledgements!
This! work! was! supported! by! a! grant! from! the! Netherlands! Organization! for! Scientific! Research!
(NWO).! We! apologize! for! not! including! many! of! the! exciting! studies! and! references! due! to! space!
limitations!
!
24!
!
!
Evolution!of!morphological!diversity!
!
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