Dynamics and Adaptive Benefits of Protein Domain Emergence and Arrangements during Plant Genome Evolution.

0.78 MB·English

by  Kersting, Anna R.

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Dynamics and Adaptive Benefits of Protein Domain
Emergence and Arrangements during Plant Genome
Evolution
Anna R. Kersting, Erich Bornberg-Bauer, Andrew D. Moore*, and Sonja Grath*
EvolutionaryBioinformaticsGroup,InstituteforEvolutionandBiodiversity,UniversityofMuenster(WWU),Germany
*Correspondingauthor:E-mail:[email protected];[email protected].
Accepted:9January2012
Abstract
Plant genomes are generally very large, mostly paleopolyploid, and have numerous gene duplicates and complex genomic
featuressuchasrepeatsandtransposableelements.Manyofthesefeatureshavebeenhypothesizedtoenableplants,which
cannot easily escape environmental challenges, to rapidly adapt. Another mechanism, which has recently been well
described as a major facilitator of rapid adaptation in bacteria, animals, and fungi but not yet for plants, is modular
rearrangement of protein-coding genes. Due to the high precision of profile-based methods, rearrangements can be well
capturedattheproteinlevelbycharacterizingtheemergence,loss,andrearrangementsofproteindomains,theirstructural,
functional, and evolutionary building blocks. Here, we study the dynamics of domain rearrangements and explore their
adaptivebenefitin27plantand3algalgenomes.Weuseaphylogenomicapproachbywhichwecanexplaintheformation
of 88% of all arrangements by single-step events, such as fusion, fission, and terminal loss of domains. We find many
domains are lost along every lineage, but at least 500 domains are novel, that is, they are unique to green plants and
emergedmoreorlessrecently.Thesenoveldomainsduplicateandrearrangemorereadilywithintheirgenomesthanancient
domainsandareoverproportionallyinvolvedinstressresponseanddevelopmentalinnovations.Noveldomainsmoreoften
affectregulatoryproteinsandshowahigherdegreeofstructuraldisorderthanancientdomains.Whereasarelativelylarge
and well-conserved core set of single-domain proteins exists, long multi-domain arrangements tend to be species-specific.
Wefindthatduplicatedgenesaremoreofteninvolvedinrearrangements.Althoughfissioneventstypicallyimpactmetabolic
proteins, fusion events often create new signaling proteins essential for environmental sensing. Taken together, the high
volatility of single domains and complex arrangements in plant genomes demonstrate the importance of modularity for
environmental adaptability of plants.
Key words: plant genome evolution, modular evolution, whole-genome duplication, evolution of stress response.
Introduction speed. In contrast, the number of domain arrangements,
thatis,thecombinationofthesedomainsinproteins,con-
Thewealthofgenomicdatahasgovernedanumberofin-
tinues to rapidly grow(Levitt 2009; Yang et al. 2009). The
sightfulstudiesongenomeevolution.Todate,moststudies
studyofdomainrearrangementsacrosslargephylahaspro-
haveconcentratedongeneduplications,genefamilyexpan-
videdadetailedunderstandingofmodularproteinevolution
sionorreduction,selectivesweepsorsignalsofselectionus-
(Bjo¨rklundetal.2005;Ekmanetal.2007;Fongetal.2007;
ingsite-basedstatistics.Analternativeapproachtostudying
WangandCaetano-Anolles2009;Yangetal.2009)andhas
genome evolution utilizes the modular nature of proteins.
Most proteins are composed of one or many protein do- demonstrated that domain rearrangements, paired with
mains, which are the units of protein structure, function, the occasional formation of novel domains (Moore and
and evolution (So¨ding and Lupas 2003; Moore et al. Bornberg-Bauer2012),createanenormousdegreeofpro-
2008). The majority of proteins can be described using tein diversity (Apic et al. 2001; Levitt 2009; Yang et al.
a small set of domains, which, despite the ever-increasing 2009).Themajorityofeukaryoticproteinshavemorethan
amountofavailablesequencedata,growsatonlymoderate onedomain(Apicetal.2001;Ekmanetal.2005;Yangetal.
ªTheAuthor(s)2012.PublishedbyOxfordUniversityPressonbehalfoftheSocietyforMolecularBiologyandEvolution.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http://creativecommons.org/licenses/by-nc/
3.0),whichpermitsunrestrictednon-commercialuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
316 GenomeBiol.Evol.4(3):316–329. doi:10.1093/gbe/evs004 AdvanceAccesspublicationJanuary16,2012
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ModularEvolutioninPlants
2009), and while many domains are found infew arrange- events have created many large genomes with various de-
ments,onlyfewdomainsareversatileandformawidearray grees of ploidy within a relatively short period of time.
ofdifferentarrangements(Weineretal.2008;Cohen-Gihon 35% of all vascular plants are recent polyploids (Wood
et al. 2011). Rearrangement events at the protein level are et al. 2009). Moreover, angiosperms have undergone up to
easy to detect, and the key mechanisms are thought to be fourroundsofWGDinroughly320Myr,withoneWGDcom-
fusion, fission, and terminal deletion (Bjo¨rklund et al. montoallseedplants319MaandoneWGDcommontoall
2005; Weiner et al. 2006). These events are likely fueled angiosperms 192 Ma (van de Peer et al. 2009; Jiao et al.
byaseriesofunderlyinggeneticeventssuchasnonallelicho- 2011).Althoughpolyploidyeventsposeagenomicchallenge
mologous recombination, exon-shuffling, nonhomologous to their host and most polyploidy events are considered
end joining or transposition (Babushok et al. 2007; Buljan a‘‘deadend’’forevolution(Mayroseetal.2011),ithasbeen
et al. 2010). However, with few exceptions (e.g., Oshima suggestedthatpolyploidy,beittheresultofautopolyploidyor
etal.2010),tracesofthegeneticmechanismsofrearrange- allopolyploidy, may occasionally provide a starting point for
mentswiftlydecay.Buljanetal.(2010)exploredthegenetic evolutionary innovation (Freeling et al. 2006; van de Peer
eventsthatfacilitatedomaingaineventstoexistingarrange- et al. 2009). The benefit of an increased amount of genetic
ments. Their results provide support to the notion that material might be to allow for swift adaptation to extreme
domainsaretypicallyaddedateitherterminus.Thekeymech- environments(vandePeeretal.2009).Forexample,thein-
anism for such domain gain events involves the joining of creasedheterozygosityresultingfrompolyploidyimpactsthe
exonsbetweengenesorterminalexonextension.Thestudy wiringofsignalingcascadesandcanfacilitatestrongvariation
ofdomaincontentevolutionineukaryoteshasillustratedthat in gene expression (Osborn et al. 2003). Numerous studies
domain loss and gain are frequent events (Moore and have also explicitly explored the impact of WGD in plants
Bornberg-Bauer2011; ZmasekandGodzik 2011).Whereas atthegenomiclevel,forexample,byexploringduplicatere-
lostdomainstendtobeofcatalyticnature,gaineddomains tention rates (Hanada et al. 2008; Tang et al. 2008; Zheng
tend to be regulatory. Despite the diverse studies that have et al. 2009), gene dosage effects (Freeling et al. 2006;
explored modular evolution across many species as well as Misook et al. 2007; Bekaert et al. 2011), or recombination
in restricted clades, to date no study has quantitatively rates(Akhunovetal.2003).WGDsmayenhancethepotential
addressed the topic of modularity in a set of plant species. fordiversificationandspeciation(vandePeeretal.2009),yet
However,modularevolutionmaybeofparticularimportance thedetailsremainpoorly understood.
forplants,astheyfaceachallengethatmanyotherspeciesdo Asgenomicstabilityislargelyinfluencedbygenomesize
not—they cannot easily evade environmental changes andrepeat content (Bennetzen2005),onemight speculate
because of their sessile nature. In particular the fusion of thatplantshavehighratesofrecombinationandhenceex-
genes, and consequently of domain arrangements, allows hibitahighnumberofdomainrearrangements.Indeed,com-
for‘‘jumps’’inproteinevolutionandmaygoverntrulynovel parative studies have illustrated that angiosperms exhibit
geneticphenotypes.Hencesuchfusionproteinsmayexhibit higher recombination rates than vertebrates (Kejnovsky
greatadaptivepotential.Indeed,recentfindingssuggestthat et al. 2009). However, to date, no study has explored the
chimeric genes formed by gene fusion can be found in extent of modular proteinevolution inplants.
regions of selectivesweeps (Rogers and Hartl 2012). Giventheirlargegenomesize,higherrecombinationrates,
Fusion events have been shown to be associated with and the inability to flee upon environmental challenges, it
regulatory proteins such as the metazoan bHLH transcrip- seemslikelythatplantsmayutilizetheirabundantgenomic
tion factors (Amoutzias et al. 2005) or the MIKC-type material to facilitate rapid evolutionary innovation. Conse-
MADS-box transcription factor proteins in plants (Veron quently, the benefits of modular domain rearrangements
et al. 2007; Shan et al. 2009). Innovation of transcription mightbeparticularlypronounced,sincetheabilityofmodular
factor families is often the result of duplication events, evolutiontoswiftlyimplementchangestotheproteinreper-
whichmayoccurinchromosomalregionswithhighrecom- toiremaybeakeyprocessinbothexploitingexistingandcre-
binationrates.Furthermore,ithasbeenillustratedthatdu- ating functionalities. So far, all studies on the evolutionary
plication events in combination with high recombination dynamics and the adaptive potential of domain rearrange-
rates are strong forces in genome evolution (Lang et al. mentshavebeenreportedforbacteria(EnrightandOuzounis
2010). 2001),metazoa(Ekmanetal.2007),orfungi(Cohen-Gihon
Duplications have been more frequently described for etal. 2011),butnonefor plants.
plantsthanelsewhereandplantgenomeevolutionisspecial Inthisreport,weexplorethenatureofmodularevolution
inseveralaspects.First,plantgenomesarerepeat-richand in 29 green plant species (Viridiplantae) with taxa ranging
transposableelementshaveaparticularlyprominentrolein from green algae to liliopsida and eudicotyledons. Our aim
creating retrocopies of genes, for example, in monocots is tounderstand the evolutionary dynamics by studying the
(Bennetzen 2005; Baucom et al. 2009; Baucom, Estill et al. frequencyofindividualmodulareventssuchasfusion,fission,
2009). Second, several whole-genome duplication (WGD) or terminal loss. We apply a maximum parsimony-based
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Kerstingetal.
approachtoreconstructeventsplacingthisstudyintoaphy- For the annotation of Pfam-A domains, we used the
logenomicframeworkandquantitativelyaddresstheroleof model-definedgatheringthresholdandquerysequenceswere
domain emergence and domain rearrangements. Further- requiredtomatchatleast30%ofthedefiningmodel(Buljan
more, we explore the speed with which new domains, etal.2010).Pfam-BdomainswereannotatedusinganEvalue
and their arrangements, are gained and lost; how many of cutoffof0.001(Ekmanetal.2007).Pfam-Adomainswithclan
theseeventsarecladeorspecies-specificandwhetherevent membershipweremappedtotheirclansanddomainsoftype
‘‘hotspots’’ can be found amongst the phylogenies of the ‘‘repeat’’ or ‘‘motif’’ were collapsed into one large domain
considered species. Finally, we employ several functional instance(Ekmanet al. 2005; Forslundetal.2007).
analyses based on the Gene Ontology (GO) classification
(Ashburner and Lewis 2002) to shed light on the potential
Reconstruction of the Ancestral Domain State; Domain
adaptivebenefitsofdomainemergenceandrearrangements
Gain, Loss, and Emergence
during plantgenomeevolution.
Wereconstructedancestraldomaincontentsusingamaxi-
mumparsimonyapproachasfollows:thetree(seefig.1B)
Materials and Methods wastraversedtwice,firstfromleavestorootthenfromroot
toleaves.Domainpresenceorabsenceisdeterminedbyma-
Proteomes and Domain Annotation
jorityrule.Duringfirsttraversal(leaves/root),thestateof
Comparativeanalysesofproteindomainsandtheirarrange- domaindissettopresentatanoden,ifdispresentinthe
ments were performed on the following 29 plant genomes: majority of leaves of the subtree rooted in n (leaves of n).
Arabidopsis thaliana v9.0 (The Arabidopsis Initiative 2000); Similarly,dissettoabsentatn,ifdisabsentinthemajority
Arabidopsis lyrata v1.0 (Hu et al. 2011); Carica papaya v1.0 ofleavesofn.Ifthereisnostatemajorityfordinthechild
(Mingetal.2008);Citrussinensisv1.0(SweetOrangeGenome nodesofn(i.e.,thereisanidenticalproportionofpresence
Project 2010); Citrus clementine v0.9 (Haploid Clementine andabsencestatesfordintheleaves),thestateofdatnis
Genome International Citrus Genome Consortium 2011); settounknown.Astraversalcontinuestowardtheroot,dis
Eucalyptus grandis v1.0 (Eucalyptus grandis Genome Project settopresent(absent)atnassoonasthemajorityofleaves
2010);Mimulusguttatusv1.1;Aquilegiacoerulea;Theobroma ofnexhibitthepresent(absent)state.Ergo,presentandun-
cacao v1.0 (Argout et al. 2011); Glycine max v1.0 (Schmutz knownareresolvedtopresent,whileunknownandabsent
et al. 2010); Medicago truncatula v3.0 (Young et al. 2005); areresolved toabsent. Thefirsttraversalterminatesatthe
Lotusjaponicav1.0(Youngetal.2005);Populustrichocarpa root node. All unknown states at the root node are set to
v2.0(Tuskanetal.2006);Ricinuscommunisv1.0(Chanetal. present(notethatthisrootincludestheoutgroups).During
2010); Manihot esculenta v1.1; Malus domestica (Velasco the second traversal (root / leaves), unknown states are
etal.2010);Prunuspersicav1.0(InternationalPeachGenome resolved by setting them to the state of their ancestor.
Initiative2010);Cucumussativav1.0(Huangetal.2009);Vitis We used a combination of custom-made python scripts
vinifera v1.0 (Jaillon et al. 2007); Setaria italica v2.0 (Setaria and the ETE2 package (Huerta-Cepas et al. 2010) for tree
italica Genome Sequencing Project 2011); Zea mays v4a.53 traversal.Branchlengthsofthetree(Soltisetal.2002;Choi
(Schnable et al. 2009); Sorghum bicolor v1.4 (Dubchak et al. 2004; Magallo´n and Sanderson 2005; Hedges et al.
etal.2009);Oryzasativav6.1(Goetal.2002);Brachypodium 2006; Cartwright and Collins 2007; Anderson and Janßen
distachyon v1.0 (Vogel et al. 2010); Phoenix dactylifera v2.0 2009; Bhattacharya et al. 2009; Bremer et al. 2009;
(Al-Dous et al. 2011); Selaginella moellendorffii v1.0 (Banks Forest and Chase 2009; Herron et al. 2009; Wang and
et al. 2011); Physcomitrella patens v1.5 (Rensing et al. Caetano-Anolles 2009; Lang et al. 2010; Reineke et al.
2008); Chlamydomonas reinhardtii v4.0 (Merchant et al. 2011) and whole-genome duplication events (Blanc and
2007); Ostreococcus lucimarinus v2.0 (Palenik et al. 2007); Wolfe 2004; Schnable et al. 2009; van de Peer et al.
andMicromonaspusillav3.0(Wordenet al. 2009). 2009; Jiao et al. 2011) were extracted from the literature.
Werootedthetree;1.700MabyincludingTrichoplaxad- We performed a Blast (Altschul et al. 1997) search to
haerens v1.0 (Srivastava et al. 2008), Rhizopus oryzae (Ma identifyrecentlyduplicatedproteins.Proteinswithasimilar-
et al. 2009) and Drosophila melanogaster v5.11 (Adams ityof75%ormoreandanEvalue(cid:1)10(cid:2)20wereconsidered
et al. 2000). Phylogenetic relationships for all 32 species to be paralogs. We employed a synteny analysis to distin-
(29 plants and 3 outgroups) used for this study are given guish between tandem and segmental duplications. Two
in supplementary figure 1 (Supplementary Material online). geneswereconsideredtobetandemduplicatesiftheywere
If several splice variants were present for one protein, we fiveorlessgenesapart.Paralogswithmorethanfivegenes
excluded all but the longest transcript. All proteomes were betweenthemwereconsideredtobearesultofasegmental
scanned for domains with the pfam_scan utility and duplication event (Hanada et al. 2008).
HMMER3.0 (Eddy 2011) against the Pfam-A and Pfam-B Domaingainandlosseventsalongbrancheswerecalcu-
models obtained from Pfam (v.24) (Finn et al. 2008). latedbycomparisonofdomaincontentatagivennodewith
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FIG. 1.—Domaingain,loss,emergenceandproteomecoverageof26plantgenomes.(A)Correlationofdomaingainandlosswithbranchlength.
Bothgainandlosscorrelatesignificantlywithbranchlength(gain:q50.6,P,0.001;loss:q50.63,P,0.001).(B)Phylogeneticrelationshipofall
speciesusedinthisstudy.Foreachbranch,thesizeofthegreencirclecorrespondstothenumberofdomainemergenceeventsalongthebranch.
Branchescoloredinredindicatethatthegainand/orlossatthisbranchishigherthantheaveragegainand/orlossrates.Exactvaluesfordomaingain,
loss,andemergencearegiveninsupplementarytable2(SupplementaryMaterialonline).(C)Domaincoverageforproteins.Theloweraxis(percentage
ofproteinswithdomains)displaystheproportionofproteinswithonlyPfam-Adomains(red),onlyPfam-Bdomains(darkblue),bothPfam-Aand
Pfam-Bdomains(lightblue),andwithoutanyproteindomainannotation(yellow).Theupperaxisdisplaysproteomesizeindicatedasverticalblackline
foreachspecies.Statisticsforthreespecies(Setariaitalica,Prunuspersica,andMimulusguttatus)thatarestillunderFortLauderdalerestrictionarenot
provided.
thedomaincontentofitsancestor.Wedistinguishbetween wasusedtodetectstructuraldisorderindomainsequences.
‘‘gained’’domains,whicharealldomainsfoundpresentat Emergeddomainsweredividedintofourbins(Viridiplantae,
a node while absent in its ancestor, and ‘‘emerged’’ do- Embryophyta, Tracheophyta, and Magnoliophyta), corre-
mains,whicharegaineddomainswhichcanonlybefound spondingtotheiremergencenodes.Domainsthatemerged
withinViridiplantae.Ergo,emergeddomainsareasubsetof after the Magnoliophyta node were pooled into one ‘‘RE-
thegaineddomains.Emergeddomainsweredeterminedby CENT’’bin.Tocomparedisorderofemergeddomainswith
scanninggaineddomainswithHMMER3.0againstNCBINR old domains (i.e., domains that exist at the root), a bin
andIntegr8(Kerseyetal.2005).Gaineddomains,whichare ‘‘OLD’’wasconstructedconsistingof500randomlypicked
not present in the outgroups were also scanned against domainsoccurringintheroot.Inaddition,weconstructed
NCBINRtodeterminethekingdomswherethesedomains a ‘‘RANDOM’’ bin consisting of 100 randomly selected do-
arepresent(supplementarytable6,SupplementaryMaterial mains,whichexistattheroot.Toaccountforsamplingbias,
online).Domaineventrates(gainandloss)werecalculated werepeatedtherandomselection100times.Statisticalinfer-
bydividing thenumberofeventspredicted tooccuralong encewasconductedwiththekruskalmctestoftheRpackage
a given branch by the branch length (in million years). pgirmess (Siegel and Castellan 1988; R Development Core
Giventheevidencethatnoveldomainsarefrequentlyen- Team 2008).
richedinstructuraldisorder(Buljanetal.2010;Mooreand Wequantifieddomainemergenceandexploredasetof
Bornberg-Bauer 2012), we predicted disorder in domains attributes (Moore and Bornberg-Bauer 2012). Domain
classified as emerging. VSL2.0 (Obradovic et al. 2005) frequency, d(f), is defined as the absolute frequency of
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Kerstingetal.
adomainacrossallplantgenomesusedfortheanalysis.The etal.2006;Weineretal.2006;Buljanetal.2010).Theal-
domainratex(d)ofdomaindisdefinedasthedomainfre- gorithmassignsafusioneventwhentwoancestralarrange-
quencydividedbythenumberofplantsinwhichdoccurs. ments can be fused to form the gained arrangement. A
Thedomainsuccessratecorrespondstothedomainratedi- gainedarrangementisconsideredtobetheresultoffission
videdbythenodeage(inmillionyears)atwhichthedomain if an ancestral arrangement can be split to give rise to the
firstemerged.TheprevalenceP(d)ofadomaindisthenum- newarrangement;bothproductsofthesplitarerequiredto
berofplantswithddividedbythenumberofplantswiththe bepresentinthecurrentnode.Incontrast,forterminalde-
emergence node of d as an ancestor. letion, only one product of the split (the gained arrange-
ment) may be present in the current node (the other
Functional Analysis of Domains productisconsideredtobelost).Thealgorithmcountsado-
main addition event when the newly gained arrangement
Where available, GO (Ashburner and Lewis 2002) annota-
contains a domain that is absent in the ancestral node.
tion of proteomes was obtained from PLAZA 2.0 (Proost
Note that in general, any new arrangement can be ex-
et al. 2009); Blast2GO (Go¨tz et al. 2008) with default set-
plained by a sufficiently large ‘‘chain’’ of events. However,
tingswasusedtofunctionallyannotatetheremainingpro-
since the likelihood of events is not available, we make
teomes. Comparative functional analyses were performed
noassumptionsabouttherelativecostsofeachmechanism
by assessing GO-term overrepresentation (overrepresenta-
and therefore are not able to determine the most likely
tionanalysis,ORA)intwoseparatesteps.First,foremerging
chain. Instead, we focus on single-step solutions, that is,
domains,weperformedthefunctionalanalysisindirectlyby
on cases where a newly gained arrangement can be ex-
using the GO annotation of arrangements that harbor at
plainedbyasingleevent.Usingthisstrategy,wecandiffer-
leastoneemergingdomain,similartoapreviousapproach
entiatebetweenarrangementswithexactsolution(i.e.,the
(MooreandBornberg-Bauer2012).Statisticalinferencewas
formationcanbeexplainedbyexactlyonemechanism),ar-
conductedusingtheRpackageTopGo(Alexaetal.2006).
rangements with nonambiguous solution (i.e., only one
As universe, we used the GO annotation of all proteins in
mechanismexplainsthearrangementbutthereareseveral
our data set; the sample consisted of arrangements with
eventspossible)andarrangementswithambiguoussolution
emerging domains. Second, for assessing functional over-
(i.e., conflicting solutions of different types). All arrange-
representation of arrangements in events (such as fusion
ments with solution are referred to as ‘‘simple gains,’’
orfission),weagainconductedanORAusingtheproteins
whereas all other arrangements are considered to be
GOannotation,however,oursampleherewasthearrange-
‘‘complex gains.’’
ment setthat resultsfrom a specific event (e.g.,all gained
arrangements explainable by a fusion event). P value
transformed TermClouds were created by logarithmic
Results
transformation of the False Discovery Rate (FDR)-corrected
(BenjaminiandHochberg1995)Pvalueobtainedfromthe
Domain Coverage
ORA,suchthattermsizerepresentsthesignificanceofthe
Inplants,onaverage,50%oftheproteomeresidueswere
GO term. Visualization was created using Wordle (http://
foundtobecoveredbydomainannotation;theresiduecov-
www.wordle.net/)withthetransformedPvalueasacustom
eragerangesfrom30%to70%(supplementarytable1and
scaling factor.
fig. 2, Supplementary Material online). For an average of
35% of the residues, for each plant, a Pfam-A domain
Reconstruction of the Ancestral Domain Arrangements
can be detected, whereas Pfam-B domains affect 15% of
State, Arrangement Gain, and Loss
allresidues.Residuecoveragelevelsforallspeciesaregiven
We defined domain arrangements as ordered sets of
in supplementary table 1 (Supplementary Material online).
domainsforeachprotein.Fortheanalysisofarrangements
Attheproteinlevel,thecoveragedistributionismoredi-
in this study, only Pfam-A domains were used. Ancestral
verse (supplementary table 1 and fig. 2, Supplementary
states for arrangements were reconstructed as previously
Material online). On average, 70% of the proteins for one
described. Similarly, arrangement gain and loss was
plant species have at least one Pfam-A or Pfam-B domain.
determined by comparing current and ancestral states.
Fifty percent of the proteins contain only Pfam-A domains,
14% contain only Pfam-B domains, and 6% contain both
Determination of Arrangement Rates
Pfam-AandPfam-Bdomains(fig.1C).Allproteincoverage
For each gained arrangement, we applied a search algo- values are given in supplementary table 1 (Supplementary
rithm to determine the possible mechanism that led to its Material online). The total number of proteins containing
formation.Weconsideredthefourmostimportantmecha- Pfam-A and Pfam-B domains is highly variable between
nismsofmodularrearrangements—fusion,fission,terminal the different proteomes (fig. 1C, supplementary table 1,
deletion,anddomainaddition(Bjo¨rklundetal.2005;Pasek Supplementary Material online).
320 GenomeBiol.Evol.4(3):316–329. doi:10.1093/gbe/evs004 AdvanceAccesspublicationJanuary16,2012
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FIG. 2.—GeneOntology(GO)termsassociatedwithemergingdomains.GOtermsaffectedbyemergenceweretestedforoverrepresentation
usingtheTopGOpackageandalltermspresentinplantsasuniverse(fordetails,seeMaterialsandMethods).Thefontsizecorrespondstothevalueof
significanceobtainedforthisterm.SignificancewasdeterminedaftercorrectionformultipletestingusingFDR(BenjaminiandHochberg1995)correction
atP,0.01.ThevastmajorityofGOtermsisrelatedtostimulusresponse,development,reproduction,regulation,andplant-specificmetabolicprocesses.
Domain Emergence unknownfunction(DUFs)orbelongtothesetofpoorlyan-
notatedPfam-Bdomains.Weassessedfunctionaloverrepre-
Toinvestigatedomaingain,loss,andemergenceacrossthe
sentationusingthefunctionofproteinsthatobtainemerging
considered plants, we reconstructed the ancestral domain
domains—wearehencenotexploringwhichfunctionalmod-
contentateachinternalnodeofthetree(seealsoMaterials
ulesemergebutratherwhichproteinfunctionalitiesundergo
andMethods;supplementaryfig.1,SupplementaryMaterial
innovation (by the addition of an emerging domain).
online).Intotal,545domainsemergedintheplantkingdom,
Thereisincreasingevidencethatyoungdomainscanex-
thatis,thesedomainsareexclusivelyfoundinViridiplantae.
hibithigherlevelsofstructuraldisorderthanestablisheddo-
The largest amount of domain emergence within plants
mains (Buljan et al. 2010; Moore and Bornberg-Bauer
occursalongthebranchleadingtoEmbryophyta,whichsees
2012). We examined the degree of structural disorder in
theemergenceof262domains(fig.1B).Atotalof114and
emerging domains. The results indicate that emerging do-
66 domains emerge along the branches to Magnoliophyta
mains are significantly enriched in intrinsic disorder, more
and Tracheophyta, respectively. Fifty-one domains emerged
thaninrandomlychosendomains(seeMaterialsandMeth-
prior to the split of Embryophyta and the green algae and
ods;supplementaryfig.3,SupplementaryMaterialonline).
52 domains are the result of recent emergence events and
Furthermore,theyoungeradomain,thehigherthedegree
canonlybefoundwithinMagnoliophyta(seealsoDiscussion
of disorder.
below) (fig. 1B).
Domain Gain and Loss
Radiation and Functional Impact of Emerging Domains
Domaingainandlossarefrequenteventsinplantevolution,
Next, we assessed whether emerged domains confer spe-
and thereis a strongvariation between differentbranches
cificfunctionalitiesandwhetherthesemightprovideadap-
(fig.1A).Nevertheless,bothgainandlossratescorrelatesig-
tive benefit. We assessed functional overrepresentation
nificantly with branch length (Spearman rank correlation,
using GO categories and TopGO (Alexa et al. 2006) (see
gain: q 5 0.6, P , 0.001; loss: q 5 0.63, P , 0.001). On
MaterialsandMethodsfordetails).WefindthatGOterms
average, plants have a domain gain rate of 6.64/Myr and
prefixed by response_to are overrepresented along with
a domain loss rate of 6.11/Myr (fig. 1A, supplementary
functionalities related to reproduction, developmental
table 2 and fig. 9, Supplementary Material online). In
mechanisms, and metabolic processes (fig. 2).
monocots, the average domain gain rate (6.7/Myr) is
Webinnedemergingdomainsaccordingtotheirpointof
lowerthanthedomainlossrate(7.4/Myr),whereasineu-
emergence (for details, see Materials and Methods) and
dicotsthesituationisreversed;eudicotsshowalossrate
rankedthembytheirfrequencyd(f).The5%highestranked
of 7.4/Myr and a gain rate of 8.3/Myr (supplementary
domainsfromeachagebin(supplementarytable3,Supple-
table2andfig.9,SupplementaryMaterialonline).Some
mentaryMaterialonline)weresubjecttofurtherinvestigation
branches exhibit very high loss rates, such as the branch
as these can be considered to be particularly ‘‘successful’’
leadingtoP.dactylifera,thebranchestothetwoFabaceae
emerging domains. Among these, we find domains with
M. truncatula and L. japonica, and the branches to the
plant-specificfunctionssuchasfloweringcontrol,auxinreg-
two Andropogoneae Z. mays and S. bicolor (fig. 1B).
ulation,fruitdevelopment,cellwalldevelopment,andplant
organelle recognition. Furthermore, we detected domains
Gain, Loss, and Distribution of Arrangements
related to the F-box protein family, to transcription factors
andtoDNAbinding.Forthemajorityofemergingdomains, We next explored the dynamics of arrangement gain and
directfunctionalannotationisdifficult—thelargestpropor- loss. After determining the presence/absence of arrange-
tion(85%)ofallemergingdomainsinplantsaredomainsof ments at ancestral nodes (for details, see Materials and
GenomeBiol.Evol.4(3):316–329. doi:10.1093/gbe/evs004 AdvanceAccesspublicationJanuary16,2012 321
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Kerstingetal.
FIG. 3.—Arrangementssharedbetweenspecies.Thedashedline represents thenumberofarrangementssharedby thedifferentnumbersof
species(rightaxis).Thedistributionofuniquearrangementsisroughlybimodalwiththemajorityofarrangementssharedbyeitherfeworallspecies.
Theleftaxisandbarplotsdisplaythefrequencyofarrangementswithacertainlength(one,two,three,four,five,six,andsevenormoredomains).
Althoughsingle-domainarrangementstendtooccurinallspecies,longerarrangementsareoftenspecies-specific.
Methods),wecomparedarrangementcontentateachnode Modular Rearrangements
with the content at the corresponding parent node to de-
Usingasimplemodelofmodularrearrangement(fordetails,
terminearrangementgainandloss.Asexpected,bothgain
seeMaterialsandMethods),wenext exploredthemecha-
and loss rates correlate significantly with branch length
nisms that can facilitate the formation of novel arrange-
(Spearmanrankcorrelation,gain:q50:56,P,0.001;loss:
ments. For this, we considered fusion, fission, terminal
q50.38,P50.003,supplementaryfig.5,Supplementary
deletion, and domain addition. The results illustrate that
Material online). Overall, arrangement gain rate is higher
70%ofallgainedarrangementscanbeexplainedbyexactly
than arrangement loss rate. However, both rates correlate onesolution(exactsolutions).Ofthegainedarrangements,
significantly with each other (q 5 0.56, P , 0.001). By 14%canbeexplainedbyoneparticularmechanism,how-
far,thelargestamountofarrangementgain(2,814arrange- ever,withanumberofdifferentpossiblesolutions(nonam-
ments) occurs along the branch to M. domestica followed biguous solutions); only 4% have conflicting solutions
by the branch to R. communis (1,018). Large amounts of (ambiguous solutions). The remaining 12% of all new ar-
arrangementlosscanbefoundalongthebranchestoP.dac- rangements are complex gains that likely arose by a chain
tylifera (1,028) and L. japonica (680); both plants also ofevents(seeMaterialsandMethods;fig.4).Thedifferent
showed a high amount of domain loss. All values for ar- events were found to occur with different frequencies
rangement gain and loss are given in supplementary table (table 1). Fusion events makeup the largest proportion of
4 (Supplementary Material online). exact solutions, followed by domain addition, fission, and
Weinvestigatedtheamountofarrangementssharedby terminal deletion. Fusion events occur with a frequency
differentplantsspecies(fig.3).Thedistributionisbimodal, of 4.59/Myr, followed by fission with 1.98/Myr, and gain
withthelargestnumberofarrangementsbeingeitherspe- with 1.89/Myr. Domain deletion events can be split in
cifictoonespecies(;7,000)orsharedbyall(;1,000);only C-terminal and N-terminal domain deletion; both events
a very small amount of arrangements is shared by 10–20 haveafrequencyof0.7/Myr.Allrateswereaveragedacross
species. Although by far the largest proportion of arrange- allbranches.Wefurtherexploredeventfrequenciesacross
mentssharedbyallspeciesconsistsofsingle-domainproteins, different age bins. At the Embryophyta node, 68% of
thecontraryistrueforspecies-specificarrangements. Here, new arrangements are affected by domain addition and
thelargestnumberofarrangementstendstobecomposed 26% by fusion. Domain deletion (4%) and fission (3%)
ofmorethanonedomain,withalargeproportioncontain- are less prevalent at this node. Over time, the frequency
ingsevenormoredomains.Thisindicatesthatthelongeran of domain deletion and fission increases up to 13% and
arrangement is, the more likely it is species-specific. 21% in recent rearrangements, whereas domain additions
322 GenomeBiol.Evol.4(3):316–329. doi:10.1093/gbe/evs004 AdvanceAccesspublicationJanuary16,2012
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ModularEvolutioninPlants
Table1 emergenceofnewdomainscanprovideanimportantstrat-
ContributionofFusion,Fission,C-TerminalDeletion,N-Terminal egy for evolving stress response. More than 500 domains
DeletionandDomainAdditiontoSimpleArrangementGains emerged within Viridiplantae of which more than 100 do-
Fusion Fission C-Del N-Del Add mains are unique for Tracheophyta (fig. 1). We recently as-
Totalnumber 9,669 4,073 1,283 1,424 4,848 sessed the impact of domain emergence in a set of
Averagenumber/Myr 4.59 1.98 0.7 0.7 1.89 insects,whereonly30domainsemergedwithin19insectge-
nomes spanning roughly 300 Myr of evolution (Moore and
NOTE.—Del,deletion;Add,addition.
Bornberg-Bauer2011).Hence,itwouldseemthatplantsex-
decreasetoafrequencyof24%.Thelargestfractionofre- hibitalargeamountofdomaininnovation.Onemightspec-
cently gained arrangements (49%) can be explained by fu- ulate that plants at least partly address the challenge of
sionevents(fig.4). asessilelifestylebymeansofdomaininnovation.Theinves-
tigationofGOtermsofproteinscontainingemergeddomains
Discussion furthersupportsthisnotion.Alargenumberoftermsarere-
latedtoplant-specificprocesses,suchasmegagametogenesis
Domain Emergence
anddevelopmentofplant-specificorgans.Thisisnotsurpris-
Theincreasingavailabilityofplantgenomeshasallowedus ingasthereproductivesystemandmorphologyofplantsnot
toconductacomparativedomainanalysisbetweenasetof onlydifferstronglyfromotherkingdomsbutarealsohighly
diverse plant species. Here, we reconstruct the ancestral variablebetweenplantspecies(Endress2001;Bennici2005;
statesofdomaincontentandarrangementandinvestigate Williams 2008; Kawakita and Kato 2009). Besides these
thefunctionalimpactofdomainemergenceanddomainre- plant-specific functions, a number of overrepresented GO
arrangements across a comprehensive set of 29 genomes termscorrespondtoresponse_tocategoriesandtosecondary
datingback;800Myr.However,theconsideredcladestill metabolitepathwaysrelatedtostressresponse,suchasauxin
containsanumberofspeciesforwhichgenomesequences andjasmonic acid. Such secondary metabolites are strongly
aremissing,suchasthegymnospermsorthecharophyta.As related to the defense and response mechanisms in plants
these genomes become available, a more comprehensive (GraceandLogan2000;PaterakiandKanellis2010;Kerchev
picture of modular evolution in plants will emerge. et al. 2012). As the composition of these compounds is
In contrast to animals, plants are sessile organisms that variable between plant species and also within species
areunabletoescapestrongenvironmentalshiftsandmust (Kroymann 2011), such secondary metabolites may provide
ratheradapttosuchvariation.Hence,plants,moresothan astrongflexiblebasisforimprovingadaptationanddefense.
animals,arerequiredtoevolvemechanismsinordertodeal Functionallinkstophotosynthesisarenotfoundamongst
withbioticandabioticstresses.Here,weillustratethatthe emerged domains (fig. 2). This is likely explained by
FIG. 4.—Mechanismsofrearrangementacrossdifferentclades.Weappliedasearchalgorithmtoassessthemechanismsthatmightaccountfor
newlygainedarrangements(fordetails,seeMaterialsandMethods).Only12%ofallgainedarrangementscannotbeexplainedbyaone-stepevent
(complexgains).Theremaining88%ofsimplegainscanbefurtherdifferentiatedintoexactsolutionswhereonlyoneparticularmechanism(fusion,
fission, terminal deletion, or domain addition) was necessary to explain the arrangement gain event (70%). All proteomes were divided into four
differentagebins:Embryophyta,Tracheophyta,Magnoliophyta,andRecentNodes.Thefrequenciesoffusion,fission,andterminaldeletionincrease
overtime,whereasthefrequencyofdomainadditiondecreases.
GenomeBiol.Evol.4(3):316–329. doi:10.1093/gbe/evs004 AdvanceAccesspublicationJanuary16,2012 323
GBE
Kerstingetal.
photosynthesis not being unique to plants; photosynthetic SupplementaryMaterialonline).Incontrast,proteinsformed
processescanbefoundinalgaeandinmanyspeciesofbac- byfissionmainlyplayaroleinmetabolicandbiosynthesispro-
teria (Olson 1970, 2001). Indeed, photosynthesis-related cesses(supplementaryfig.7,SupplementaryMaterialonline).
GOtermscanbedetectedbyinvestigatinggaineddomains Proteinsshapedbydomaindeletionaremainlyrelatedtoba-
which are absent in the outgroups (supplementary fig. 6, sicfunctionssuchastheprimarymetabolism,andonlyami-
Supplementary Material online), as well as response_to nor part of these proteins are stress–response related
termsandanumberofplant-specificfunctionalitiesrelated (supplementaryfig. 7, SupplementaryMaterialonline).
to development, similar to those terms found in proteins Ourresultsprovidefurtherevidencethatduplicationim-
containing emerged domains. pactsratesofmodularrearrangement(BuljanandBateman
Emergeddomainsseemtobeevolutionarilyimportantas 2009). We find that proteins affected by rearrangement
they have ahigh prevalence of 0.9–1, indicating that their eventsareoverrepresentedinduplicatedgenes(supplemen-
occurrence is strongly conserved. Besides their widespread tarytable 6, SupplementaryMaterial online).Furthermore,
occurrenceinnearlyallleaves,suchemergeddomainsoften wefindindicationthatspecieswithrecentWGDhavehigh-
occur in high copy numbers (supplementary table 3, erratesoffusionandfissionincomparisontospecieswith-
Supplementary Material online). out recent WGD (supplementary table 7, Supplementary
Investigating the most successful emerged domains un- Material online). In general, duplicates are thought to un-
coversconnectionstokeyfunctionalcategoriessuchastran- dergooneofthreedifferentscenarios:subfunctionalization,
scription factors, binding-related processes,and secondary where the two duplicates share ancestral gene function;
metabolites,includingauxinandjasmonicacid(supplemen- neofunctionalization,whereonecopyretainstheancestral
tarytable3,SupplementaryMaterialonline).Indeed,aburst functionandtheothercopydivergestowardanovelfunc-
of transcription factors and their constituent domains, tion; and pseudogenization, where one copy is not ex-
which are assumed to be correlated with increasing com- pressed and is subsequently lost (Walsh 2003). One
plexityinplantevolution(Langetal.2010),hasbeenfound explanation for sub- or neofunctionalization is the loss or
in angiosperms. The increase of plant complexity with du- change of regulatory regions (Ganko et al. 2007). As the
plicationevents(Freelingetal.2006)maypartlybetheresult conservationofnoncodingsequencesfollowsanexponen-
of duplication facilitating increasingly complex regulatory tial decay rate (Reineke et al. 2011), the retention of both
networks (Veron et al. 2007). duplicatesmightbetheresultofthechangeofoneofthe
Emergingdomainsexhibitanincreasedamountofintrin- gene’s regulatory region under relaxed selectional con-
sicdisorder;themorerecenttheemergenceevent,themore straints.Thehighretentionrateofproteinsthatresultfrom
likelythedomaininquestionexhibitsintrinsicdisorder.Dis- afusioneventmightbeexplainedbytheconservationofat
orderedsequencesmayincreasethebindingaffinityofpro- least one regulatory element in the upstream region,
teins(DysonandWright2005).Highintrinsicdisorderpaired whereasafterfission,onearisingproteinmaylosearegula-
withthefactthatemergeddomainsaresignificantlyunder- tory region and undergo pseudogenization followed by
representedinsingle-domainproteins(hypergeometrictest, gene loss. A further reason for sub- and neofunctionaliza-
P , 0.01), leads us to the speculation that emerging do- tion after duplication might be domain rearrangements in
mainsmayhavehigherinteractionpotential,whichinturn oneparalogordifferentialdomainloss(Buljanetal.2010).
may increase their viability and result in higher prevalence Wefurtherillustrate the impact of protein domain rear-
andfrequency.Indeed,someofthemostsuccessfulemerg- rangementsonanorganism’sproteinrepertoire(fig.5).The
ing domains have links to binding-related processes. emerging domains PAN_2 (emerged in the Tracheophyta)
andS_locus_glycop(Embryophyta)oftenco-occurtogether
withtheB-lectindomain.Arrangementscontainingthetwo
Arrangement Mechanisms
emergeddomainsS_locus_glycopandPAN_2arefrequently
In plants, roughly 70% of the domain-containing proteins rearrangedwithinparalogousgenes(fig.5)andobtainacat-
are single domain (supplementary fig. 4, Supplementary alyticfunctionthroughtheadditionofkinasedomains.Pro-
Materialonline).Thishighpercentageofsingle-domainpro- teinsthatconsistofarrangementswiththesetwoemerged
teinscanbeanartifactoflowdomaincoverageor‘‘eroded- domains have GO functions related to the recognition of
domains,’’ which have diverged beyond detection (Weiner pollen, protein phosphorylation, and cell recognition. Al-
etal.2006).Recentrearrangementscanmostlybeexplained though we observed fusion events in tandemly duplicated
by the fusion of two single or two domain proteins. The genesinourcasestudy,fusioneventsarenotgenerallyover-
relative rates of fusion and fission are similar to previously represented in tandemly duplicated genes (supplementary
reported rates (Kummerfeld and Teichmann 2005). GO table5,SupplementaryMaterialonline).Afterfusion,dupli-
terms overrepresented in proteins, which arose via fusion, catesmightbedifficulttorecognizeasparalogs.Onemight
are stress-, defense-, and adaptation-related as well as therefore speculate that in tandemly duplicated proteins
related to the reproduction system (supplementary fig. 7, fused arrangements are harder to detect. The increased
324 GenomeBiol.Evol.4(3):316–329. doi:10.1093/gbe/evs004 AdvanceAccesspublicationJanuary16,2012
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ModularEvolutioninPlants
FIG. 5.—Example of two emergent domains at the Tracheophyta node (PAN_2) and Embryophyta node (S_locus_glycope). The evolution of
example arrangements over time is shown in five different species (Arabidopsis thaliana [AT], Oryza sativa [OS], Populus trichocarpa [PT], Ricinus
communis[RC],Vitisvinifera[VV]).Theobservablediversityinarrangementswithinthisfamilyisexplainablebysimpleone-stepeventsoffusion,fission,
terminaldeletion,anddomaingain.
rates of events along more recent branches might be ex- asubsetofbetween5and24proteomes,innate_immune_r-
plained by WGD which have taken place in angiosperms esponse is significantly overrepresented, suggesting that
(De Bodt et al. 2005; Freeling et al. 2006; Shoemaker there might be different pathogens affecting different sub-
et al. 2006; van de Peer et al. 2009; Paterson et al. clades.ProteinswithGOtermsrelatedtoreproduction,signal
2010).Indeed,inapairwisecomparisonoffusionandfission transduction, and prefixed with response_to are overrepre-
ratesbetweenplantpairs,whichdifferbyonerecentWGD, sented in species-specific arrangements or those shared by
we find increased rates in plants with more recent WGD only few species. The high number of species-specific ar-
(supplementary table 7, Supplementary Material online). rangementsobservedhereisinaccordancewiththeobserva-
Roughly one-third of all vascular plants have undergone tion that, within a set of five angiosperm species, around
recent WGDs (Wood et al. 2009). 20% of proteins do not align toanorthologous group (Pa-
terson et al. 2010). The high amount of species-specific ar-
Arrangement Distribution rangements and genes might also be a consequence of
frequent duplication events followed by lineage-specific re-
We investigated the distribution of shared arrangements
tention (Paterson et al. 2010). This supports the hypothesis
among theplantspecies. Themajorityofdomain arrange-
that plants have many flexible genetic mechanisms to pro-
ments are either species-specific or universal (fig. 3). This
ducespecies-specific adaptation (Bomblies 2010).
bimodaldistributionisevenstrongerwhenweconsideronly
a well-annotated subset of our species and exclude the
Gain and Loss of Domains and Arrangements
greenalgae(supplementaryfig.8,SupplementaryMaterial
online).Inparticular,proteinswithtwoorthreedomainsare Weinvestigatedgainandlossatthelevelsofdomainsand
oftenspecies-specific.Incombinationwiththeobservation domainarrangementsbyreconstructingtheancestralstates
that roughly 70% of all domain-containing proteins are based on maximum parsimony. We observe that gain and
single-domain proteins (supplementary fig. 4, Supplemen- losscanfrequentlybefoundinallcladesinplantevolution
tary Material online), this can lead to the assumption that atbothdomainandarrangementlevels.Thisisinagreement
the fusion of single-domain proteins is a powerful mecha- withBuljanandBateman(2009),whofoundanequalevent
nismtoobtainspecies-specificproteinswithnewfunction- distributionafterspeciationandduplicationwithinanimals
alities. This distribution suggests that only very few long andahighamountofchangeinarrangementsafterdupli-
arrangements are highly conserved; long arrangements cationevents.Asweheredonotconductadirectcompar-
are possibly more often affected by fission events. Proteins ison of paralogs, but instead compare presence/absence
with arrangements shared by several but not all species are patterns of domains and their arrangements across pro-
overrepresented in GO terms related to basic functions such teomes, our results only support the notion that domain
asprimarymetabolism,cellulosebiosyntheticprocess,andcell gainandlosscanbefoundalongallbranchesandthatboth
wall organization. In proteins with arrangements shared by have a significant correlation with each other and with
GenomeBiol.Evol.4(3):316–329. doi:10.1093/gbe/evs004 AdvanceAccesspublicationJanuary16,2012 325