A transcriptomic hourglass in plant embryogenesis. b K a /K s (A. thaliana A. lyrata)

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1 doi:1.138/nture11394 A trnscriptomic hourglss in plnt emryogenesis Mrcel Quint 1, Hjk-Georg Drost 2, Alexnder Gel 2, Kristin Krsten Ullrich 1, Mrkus Bönn 2,3 & Ivo Grosse 2 Animl nd plnt development strts with constituting phse clled emryogenesis, which evolved independently in oth lineges 1. Comprtive ntomy of verterte development sed on the Meckel-Serrèslw 2 nd von Ber s lws of emryology 3 from the erly nineteenth century shows tht emryos from vrious tx pper different in erly stges, converge to similr form during mid-emryogenesis, nd gin diverge in lter stges. This morphogenetic series is known s the emryonic hourglss 4,5, nd its ottleneck of high conservtion in mid-emryogenesis is referred to s the phylotypic stge 6. Recent nlyses in zerfish nd Drosophil emryos provided convincing moleculr support for the hourglss model, ecuse during the phylotypic stge the trnscriptome ws dominted y ncient genes 7 nd glol gene expression profiles were reported to e most conserved 8. Although extensively explored in nimls, n emryonic hourglss hs not een reported in plnts, which represent the second mjor kingdom in the tree of life tht evolved emryogenesis. Here we provide phylotrnscriptomic evidence for moleculr emryonic hourglss in Aridopsis thlin, using two complementry pproches. This is prticulrly significnt ecuse the possile sence of n hourglss sed on morphologicl fetures in plnts suggests tht morphologicl nd moleculr ptterns might e uncoupled. Together with the reported developmentl hourglss ptterns in nimls, these findings indicte convergent evolution of the moleculr hourglss nd conserved logic of emryogenesis cross kingdoms. In flowering plnts, emryogenesis cn e seprted into three mjor phses. The erly phs chrcterized y symmetric cell divisions to estlish picl sl polrity. In the intermedite phse, mjor orgns nd primordi re initited, which expnd in the lte phse to the mture emryo 9,1. One notle difference etween emryogenesis in nimls nd plnts concerns the estlishment of morphologicl vrition etween tx. For exmple, vertertes develop morphologicl vrition in lte emryogenesis, wheres differences etween flowering plnt tx re only estlished during post-emryonic development. Inspired y the historicl relevnce of the emryonic hourglss model in nimls, y recent trnscriptionl support from studies in zerfish 7 nd Drosophil 7,8, nd y the sence of ny reported ntomicl evidence for such pttern during plnt emryogenesis, we ssess the possile existence of trnscriptionl hourglss during emryogenesis of the plnt reference species A. thlin. Recently, genome-wide expression profiles of complete developmentl series from the zygote to the mture emryo in A. thlin were otined 11. To investigte the presence of n emryonic hourglss in plnts, we comine this trnscriptome informtion with two different mesures of evolutionry distnce: evolutionry ge nd sequence divergence. We compute two different trnscriptome indices for ech gene, the trnscriptome ge index (TAI) 7 sed on evolutionry ge, nd the trnscriptome divergence index (TDI) sed on sequence divergence. We investigte the profiles of these two trnscriptome indices cross the seven smpled emryo stges, nd sk if nd to wht degree they show n hourglss pttern similr to tht found for zerfish 7 or Drosophil 7,8. For clculting the TAI, we ssign n evolutionry ge to ech gene in the A. thlin genome y sorting ech gene into its phylostrtum, defined s the most distnt phylogenetic node contining t lest one species with detectle homologue (Methods, Supplementry Fig. 1, Supplementry Tles 1 nd 2). The resulting phylostrtigrphic mp 12 contins 13 phylostrt, PS1 PS13 (Fig. 1). PS1 includes the evolutionrily oldest genes with homologous sequences in prokryotes, nd PS13 includes the evolutionrily youngest genes with no homologue in ny other species. K /K s (A. thlin A. lyrt) K /K s (A. thlin C. ruell) Aridopsis Brssicles PS12 (29) Mlvids PS11 (1,755) PS1 Rosids (18) Core Eudicotyledons PS9 Eudicotyledons PS8 (627) Mgnoliophyt PS7 (539) Trcheophyt PS6 (884) Emryophyt PS5 (3,587) Viridiplnte PS4 (1,99) (6,271) Eukryot PS3 (1,42) Cell. org. PS1 (4,523) d PS2 (5,378) Kendll τ =.26 K /K s (A. thlin T. hlophil) c.8 A.thlin PS13 (1,245) Kendll τ =.2 e Kendll τ =.24 K /K s (A. thlin B. rp) Kendll τ =.3 Figure 1 Evolutionry ge nd sequence divergence of A. thlin genes., Phylostrtigrphic mp of A. thlin. Numers in prenthesis denote the numer of genes per phylostrtum (PS1 PS13). Cell. org., cellulr orgnisms descried y PS1. e, Sctter plots of phylostrtum versus K /K s rtios over ll genes. K /K s rtios re derived from orthologous genes etween A. thlin nd, A. lyrt, c, T. hlophil, d, C. ruell nd e, B. rp. Kendll t vlues denote the Kendll rnk correltion coefficients mesuring the ssocition etween oth prmeters. See Methods for detils. 1 Leiniz Institute of Plnt Biochemistry, Independent Junior Reserch Group, Deprtment of Moleculr Signl Processing, Weinerg 3, 612 Hlle (Sle), Germny. 2 Institute of Computer Science, Mrtin Luther University Hlle Wittenerg, 612 Hlle (Sle), Germny. 3 UFZ Helmholtz Centre for Environmentl Reserch, Deprtment of Soil Ecology, 612 Hlle (Sle), Germny. 212 Mcmilln Pulishers Limited. All rights reserved MONTH 212 VOL NATURE 1

2 RESEARCH LETTER For clculting the TDI, we determine the sequence divergence etween A. thlin nd its sister species Aridopsis lyrt or ny one of the closely relted Brssiccees, Brssic rp, Cpsell ruell nd Thellungiell hlophil, y computing the K /K s rtio (Supplementry Tle 3). Here K is the numer of non-synonymous sustitutions per non-synonymous site nd K s is the numer of synonymous sustitutions per synonymous site for ech orthologous gene pir. The K / K s rtio is n indictor of selective pressure within protein coding regions nd, thus, reflects nturl selection, one of the mjor forces driving moleculr evolution. Interestingly, evolutionry ge nd sequence divergence s quntified ove show only wek correltions (Kendll s rnk correltion coefficient rnging from.2 to.26; Fig. 1 e), indicting tht oth mesures of evolutionry distnce cn e regrded s complementry (Supplementry Note). In comintion with trnscript informtion, the TAI quntifies the men evolutionry ge of trnscriptome, where the evolutionry ge (phylostrtum) of ech gen weighted y its expression level 7. Anlogously, we define the TDI s the men sequence divergence of trnscriptome, where the sequence divergence (K /K s ) of ech gen weighted y its expression level (Methods). Figure 2 nd Supplementry Fig. 2 show the TAI nd TDI profiles cross the seven smpled emryo stges of A. thlin. We find tht trnscriptomes of erly plnt emryonic stges such s zygote nd qudrnt re evolutionrily young (high TAI), trnscriptomes of the mid-emryogenic phse rnging from the gloulr to the torpedo stge re older (low TAI), nd trnscriptomes of lter stges of emryogenesis re younger gin (Fig. 2). Qulittively, this TAI profile strikingly resemles the moleculr hourglss pttern discovered for zerfish nd Drosophil 7. Likewise, we find tht trnscriptomes of erly stges re divergent (high TDI), trnscriptomes of the midemryogenic phse re more conserved (low TDI), nd trnscriptomes of lter stges of emryogenesis re more divergent gin (Fig. 2). Remrkly, the TDI profile qulittively resemles the moleculr hourglss pttern of the gene expression divergence profile discovered for Drosophil 8 nd recently lso Cenorhditis 13. Compring oth profiles, we mke two oservtions. First, ech of the profiles shows n hourglss pttern, where the TAI reflects longterm evolutionry chnges covering 4 illion yers since the origin of life, nd the TDI reflects short-term evolutionry chnges covering Young Old Z Conserved Divergent TAI M B T H G Q Time M B T H G Q Z Figure 2 Trnscriptome indices cross A. thlin emryogenesis., The trnscriptome ge index (TAI) profile., The trnscriptome divergence index (TDI) profile. Emryo stges: Z, zygote; Q, qudrnt; G, gloulr; H, hert; T, torpedo; B, ent cotyledon; M, mture. Representtive drwings (not on the sme scle) re given for ech smpled emryo stge. The lue shded re mrks the predicted phylotypic stge. The grey lines represent the stndrd error estimted y ootstrp nlysis. The overll ptterns of the TAI nd TDI profiles re highly significnt, s mesured y permuttion tests (P TAI ; P TDI ). See Methods for detils. TDI roughly 5 16 million yers since the divergence of A. thlin nd the other four Brssiccees (Supplementry Note). Second, oth profiles point to the torpedo stge s the predicted phylotypic stge, representing simultneously the stge with the oldest s well s the most conserved/lest divergent trnscriptome. An independent, ut comprle trnscriptome dtset 18,19 from A. thlin (Supplementry Fig. 3), which likewise covers emryogenesis from erly phses to the mture emryo, confirms the hourglss pttern for oth indices (Supplementry Fig. 4). Together, these oservtions suggest the possiility of convergent evolution of moleculr emryonic hourglss in nimls nd A. thlin, nd mke it tempting to conjecture its universl presence cross niml nd plnt kingdoms. Given tht developmentl processes during plnt nd niml emryogenesis cn e very different from the zygote stge on 2,nd tht the emryonic hourglss must hve evolved independently in plnts nd nimls, we wish to understnd how the torpedo stge s the on fide phylotypic stge in A. thlin reltes to niml phylotypic stges. Across different niml tx, the phylotypic stge ws defined s the stge t which ll mjor ody prts re represented t their finl positions s undifferentited cell condenstions 21. In reltion to this ontogenic progression in nimls, the mid-emryogenic trnsition from the gloulr to the hert stge my conceptully serve s the corresponding stge in flowering plnts. Here, polr xes re estlished nd shoot nd root picl meristems re initited 22. Hence, the ensuing torpedo stge t the trnsition from mid- to lte-emryogenesis mrks n ontogenic progression tht seems more dvnced thn the phylotypic stge known from nimls. Considering tht morphologicl diversity nd mny importnt orgns in flowering plnts develop post-emryogeniclly, it is possile tht the phylotypic stge my e shifted towrds the trnsition from mid- to lte-emryogenesis compred to nimls. Furthermore, the torpedo stge roughly mrks the trnsition from morphogenesis to the mturtion phse. Morphogenesis involves the estlishment of the emryo s ody pln, wheres mturtion involves cell expnsion nd ccumultion of storge mcromolecules to prepre for desicction, germintion nd erly seedling growth 23. Wheres ll lnd plnts/emryophytes (ll species from PS4 on) including lower lnd plnts pss through morphogenesis phse, only the emryogenesis of higher lnd plnts concludes with mturtion phse. Completely different signlling cscdes re involved in oth phses. One set is switched off nd the other on initited. Becuse torpedo stge emryos re in the trnsition etween these different developmentl progrms, it is conceivle tht trnscriptionl progrms re likewise reduced to conserved nd evolutionry ncient processes tht re reflected y the neck of the hourglss (Fig. 2). Encourged y these findings, we seek to understnd how the moleculr hourglss pttern of the TAI profil determined. Two simple scenrios tht would result in decrese of TAI vlues include up-regultion of old genes, or down-regultion of young genes during mid-emryogenesis. To distinguish etween oth scenrios, we compute the reltive expression levels of genes from phylostrt contining pre-emryogenesis species (PS1 PS3) versus post-emryogenesis phylostrt (lnd plnts/emryophytes from PS4 PS13, representing plnt species tht pss through emryogenesis). Wheres expression levels of old genes vry only mrginlly cross emryo stges, young genes re down-regulted towrds the torpedo stge, nd the rtio of the reltive expression levels of old nd young genes is mximized in the torpedo stge (Fig. 3, Supplementry Fig. 5). Next, we divide the genes long the medin of the K /K s rtios over ll genes nd perform n nlogous nlysis for conserved (elow medin) versus divergent (ove medin) genes. Interestingly, we find similr pttern, with divergent genes eing more down-regulted towrds the torpedo stge thn conserved genes (Fig. 3; Supplementry Figs 6 9). These results re confirmed y the independent dtset 18,19 (Supplementry Figs 1 14). 2 NATURE VOL MONTH Mcmilln Pulishers Limited. All rights reserved

3 RESEARCH 1. PS1 PS3 PS4 PS13 3 Multicellulrity Emryogenesis Animls Men reltive expression level Men reltive expression level K /K s elow medin K /K s ove medin Hence, the emryonic hourglss in A. thlin seems to e coordinted y the quntittive down-regultion of young/divergent genes or, qulittively, y the expression of fewer young/divergent genes towrds the torpedo stge. This is in notle greement with oservtions from the niml kingdom 7,8,24,25. As only frction of these downregulted young genes in A. thlin disply n hourglss shped expression profile cross the smpled emryo stges themselves, the hourglss pttern is most proly cused y different sets of young genes. One set is involved in morphogenesis (up-regulted efore the torpedo stge nd down-regulted therefter) nd one set is involved in mturtion (down-regulted efore the torpedo stge nd up-regulted therefter; Supplementry Figs 15 nd 16). In ddition, we find tht significntly enriched gene ontology terms mong young plnt genes down-regulted in the torpedo stge compred to erly- or lte emryogenesis descrie signlling processes, such s responses to endogenous stimuli nd hormones (Supplementry *** Zygote Qudrnt Gloulr Hert Torpedo Bent Mture P = *** Zygote Qudrnt Gloulr Hert Torpedo Bent Mture P =.197 Figure 3 Reltive expression levels over emryo stges., Left xis, men reltive expression levels of genes in PS1 PS3 (open rs) nd PS4 PS13 (shded rs); reltive expression levels rnge from to 1. Right xis, rtio of men reltive expression levels etween PS1 PS3 nd PS4 PS13, dt points connected y dshed line., Anlogously to, genes were divided long the medin of the K /K s rtios over ll genes. Open nd shded rs show K /K s vlues respectively elow nd ove the medin; dt points connected y dshed line show the rtio of low to high K /K s vlues. Error rs, stndrd error. Asterisks denote significnt differences etween PS1 PS3 nd PS4 PS13 vlues () nd conserved (elow medin) versus divergent (ove medin) genes t the torpedo stge (); *P,.5; ***P, PS1 PS3/PS4 PS13 rtio (Low K /K s )/(high K /K s ) rtio Single-celled common ncestor Multicellulrity Tles 5 nd 6). This indictes tht signlling processes controlling trnscription of reltively recently evolved genes re down-regulted during the predicted phylotypic stge of A. thlin emryogenesis. Using phylotrnscriptomic pproch sed on two complementry mesures of evolutionry distnce nd two independent dtsets, we hve oserved moleculr emryonic hourglss in plnts, which seems to e predominntly cused y down-regultion of young nd divergent genes towrds the torpedo stge (Fig. 4). This oservtion is surprising for two resons. First, morphologicl diversity during emryogenesis of flowering plnts is negligile, so the increse of oth trnscriptome indices in lte emryogenesis precedes the morphologicl differences estlished only during post-emryonic development. Second, convergent evolution of moleculr hourglss pttern in nimls nd plnts suggests opertion of fundmentl developmentl profile controlling the expression of evolutionrily young or rpidly evolving genes cross kingdoms. We speculte tht such mechnism my e required for enling sptio-temporl orgniztion nd differentition of complex multicellulr life. METHODS SUMMARY Following ref. 7, 1,459 species with completely sequenced genomes (Supplementry Tle 1) were sorted into 13 phylostrt (Supplementry Fig. 1), nd mino cid sequences of A. thlin were compred with the mino cid sequences of these species using Blst. If no Blst hit ws identified, the corresponding gene of A. thlin ws ssigned to PS13; otherwise, it ws ssigned to the phylogeneticlly most distnt phylostrtum contining t lest one Blst hit. This procedure resulted in the phylostrtigrphic mp shown in Fig. 1. Orthologous gene pirs of A. thlin nd A. lyrt, T. hlophil, C. ruell, or B. rp were determined with the method of est hits using Blstp. Amino cid sequence lignments of ech pir generted with MAFFT 26 (L-INS-i option) were used for codon lignments generted with PAL2NAL 27 to compute sequence divergence levels (K /K s ) with GESTIMATOR 28. Gene pirs with K,.5, K s, 5 nd K /K s rtios, 2 were retined. Introduced in ref. 7, the TAI of developmentl stge s is the weighted men of the phylostrtum ps i of gene i weighted y the expression level of gene i t developmentl stge s TAI s ~ ps i Young or divergent genes Emryogenesis Plnts Time Figure 4 Convergent evolution of moleculr hourglss in niml nd plnt emryogenesis. Originting from single-celled common ncestor, niml nd plnt lineges evolved oth multicellulrity nd emryogenesis independently. For the coordinted progression of the orgnisms through emryogenesis, the trnscriptomes hve to follow n hourglss pttern with mximlly ncient nd conserved trnscriptomes during the phylotypic stge. where n is the totl numer of genes nlysed. Low PS vlues correspond to evolutionrily old genes, so low TAI vlues correspond to evolutionrily old trnscriptomes. Likewise, high PS vlues correspond to evolutionrily young genes, so high TAI vlues correspond to evolutionrily young trnscriptomes. Anlogously, we introduce the TDI of developmentl stge s y replcing ps i in eqution (1) y the K /K s rtio of gene i:? ð1þ 212 Mcmilln Pulishers Limited. All rights reserved MONTH 212 VOL NATURE 3

4 RESEARCH LETTER TDI s ~ Ki e Ksi is Here, low/high K /K s rtios correspond to conserved/divergent genes, so low/high TDI vlues correspond to conserved/divergent trnscriptomes. Full Methods nd ny ssocited references re ville in the online version of the pper. Received 26 Jnury; ccepted 3 July 212. Pulished online 5 Septemer Meyerowitz, E. M. Plnts compred to nimls: the rodest comprtive study of development. Science 295, (22). 2. Meckel, J. F. Beyträge zur vergleichenden Antomie (Reclm, Leipzig, 1811). 3. von Ber, K. E. Üer Entwicklungsgeschichte der Thiere: Beochtung und Reflexion (Gerüder Bornträger, Königserg, 1828). 4. Duoule, D. Temporl colinerity nd the phylotypic progression: sis for the stility of verterte Bupln nd the evolution of morphologies through heterochrony. Dev., Suppl. 1994, (1994). 5. Rff, R. A. The Shpe of Life: Genes, Development nd the Evolution of Animl Form (Univ. Chicgo Press, 1996). 6. Snder, K. in Development nd Evolution (eds Goodwin, B. C., Holder, N. & Wylie, C. C.) (Cmridge Univ. Press, 1983). 7. Domzet-Lošo, T. & Tutz, D. A phylogeneticlly sed trnscriptome ge index mirrors ontogenetic divergence ptterns. Nture 468, (21). 8. Klink, A. T. et l. Gene expression divergence recpitultes the developmentl hourglss model. Nture 468, (21). 9. De Smet, I., Lu, S., Myer, U. & Jürgens, G. Emryogenesis the humle eginnings of plnt life. Plnt J. 61, (21). 1. Peris, C. I., Rdemcher, E. H. & Weijers, D. Green eginnings pttern formtion in the erly plnt emryo. Curr. Top. Dev. Biol. 91, 1 27 (21). 11. Xing, D. et l. Genome-wide nlysis revels gene expression nd metolic network dynmics during emryo development in Aridopsis. Plnt Physiol. 156, (211). 12. Domzet-Lošo, T., Brjković, J. & Tutz, D. A phylostrtigrphy pproch to uncover the genomic history of mjor dpttions in metzon lineges. Trends Genet. 23, (27). 13. Levin, M., Hshimshony, T., Wgner, F. & Yni, I. Developmentl milestones punctute gene expression in the Cenorhditis emryo. Dev. Cell 22, (212). 14. Koch, M. A., Huold, B. & Mitchell-Olds, T. Comprtive evolutionry nlysis of chlcone synthse nd lcohol dehydrogense loci in Aridopsis, Aris, nd relted gener (Brssiccee). Mol. Biol. Evol. 17, (2). 15. Arkki, M. et l. Contemporneous nd recent rditions of the world s mjor succulent plnt lineges. Proc. Ntl Acd. Sci. USA 18, (211). ð2þ 16. Koch, M. A. & Kiefer, M. Genome evolution mong cruciferous plnts: lecture from the comprison of the genetic mps of three diploid species Cpsell ruell, Aridopsis lyrt susp. petre, nda. thlin. Am. J. Bot. 92, (25). 17. Oh, D.-H. et l. Genome structures nd hlophyte-specific gene expression of the extremophile Thellungiell prvul in comprison with Thellungiell slsugine (Thellungiell hlophil) nd Aridopsis. Plnt Physiol. 154, (21). 18. Zuer, H. et l. Theseed composition of Aridopsis mutntsfor the group 3 sulfte trnsporters indictes role in sulfte trnsloction within developing seeds. Plnt Physiol. 154, (21). 19. Le, B. H. et l. Glol nlysis of gene ctivity during Aridopsis seed development nd identifiction of seed-specific trnscription fctors. Proc. Ntl Acd. Sci. USA 17, (21). 2. Nodine, M. D. & Brtel, D. P. Mternl nd pternl genomes contriute eqully to the trnscriptome of erly plnt emryos. Nture 482, (212). 21. Slck, J. M., Hollnd, P. W. & Grhm, C. F. The zootype nd the phylotypic stge. Nture 361, (1993). 22. Lu, S., Slne, D., Herud, O., Kong, J. & Jürgens, G. Erly emryogenesis in flowering plnts: setting up the sic ody pttern. Annu. Rev. Plnt Biol. 63, (212). 23. Prk, S. & Hrd, J. J. Aridopsis emryogenesis. Methods Mol. Biol. 427, 3 16 (28). 24. Irie, N. & Kurtni, S. Comprtive trnscriptome nlysis revels verterte phylotypic period during orgnogenesis. Nture Commun 2, 248 (211). 25. Irie, N. & Sehr-Fujisw, A. The verterte phylotypic stge nd n erly ilterin-relted stge in mouse emryogenesis defined y genomic informtion. BMC Biol. 5, 1(27). 26. Ktoh, K., Kum, K., Toh, H. & Miyt, T. MAFFT version 5: improvementin ccurcy of multiple sequence lignment. Nucleic Acids Res. 33, (25). 27. Suym, M., Torrents, D. & Bork, P. PAL2NAL: roust conversion of protein sequencelignments intothecorrespondingcodonlignments. Nucleic Acids Res. 34, W69 W612 (26). 28. Thornton, K. Lisequence: C11 clss lirry for evolutionry genetic nlysis. Bioinformtics 19, (23). Supplementry Informtion is ville in the online version of the pper. Acknowledgements We re grteful to S. Ael, L. I. A. Clderón Villloos, C. Delker, T. Gre, D. Gru, J. J. Hrd, D. Jckson, R. Pxton, A. Soro nd C. Wsternck for discussions, to S. Neumnn for support with the IPB-cluster, nd the Exzellenznetzwerk für Biowissenschften of the Federl Stte of Schsen-Anhlt, Germny, for finncil support of M.Q. Author Contriutions M.Q. conceived the study. M.Q. nd I.G. supervised the project. H.-G.D., A.G., K.K.U. nd M.B. nlysed the dt. M.Q., H.-G.D., A.G., K.K.U., M.B. nd I.G. interpreted the results. M.Q. nd I.G. wrote the mnuscript. Author Informtion Reprints nd permissions informtion is ville t The uthors declre no competing finncil interests. Reders re welcome to comment on the online version of the pper. Correspondence nd requests for mterils should e ddressed to M.Q. (mquint@ip-hlle.de). 4 NATURE VOL MONTH Mcmilln Pulishers Limited. All rights reserved

5 RESEARCH METHODS Phylostrtigrphic procedure. A full ccount of the procedure of constructing phylostrtigrphic mp hs een presented previously 7,12. Adpted to A. thlin, the following procedure ws used in this study. The phylogeny of A. thlin hs een ssigned ccording to the NCBI txonomy dtse. For ech of the 13 phylostrt shown in Supplementry Fig. 1, ll 6,617,32 mino cid sequences of ll 1,459 species with completely sequenced genomes were extrcted from Phytozome 29 nd the dtses listed in Supplementry Tle 1. A dtse of these sequences ws generted, nd ech of the 27,258 mino cid sequences of A. thlin (TAIR9) with minimum length of 3 mino cids ws lsted ginst this dtse using lstp (BLAST version ) with n E-vlue cut-off of If no lst hit ws identified, the corresponding gene of A. thlin ws ssigned to phylostrtum 13 (PS13). Otherwise, the corresponding gene of A. thlin ws ssigned to the phylogeneticlly most distnt (oldest) phylostrtum contining t lest one species with t lest one lst hit (Supplementry Tle 2). This procedure resulted in the phylostrtigrphic mp shown in Fig. 1. To investigte the dependence of the results on the lstp stringency, the entire procedure ws repeted for vrying E-vlue cut-offs of lstp rnging from 1 21 to 1 22, resulting in the TAI profiles shown in Supplementry Fig. 17. K /K s rtios. Orthologous gene pirs of A. thlin nd A. lyrt or the closely relted Brssiccees T. hlophil, C. ruell or B. rp were determined with the method of est hits using lstp. Amino cid sequence lignments of ech pir generted with MAFFT 26 (L-INS-i option) were used for codon lignments generted with PAL2NAL 27 to compute sequence divergence levels (K /K s ) with GESTIMATOR 28. Gene pirs with K,.5, K s, 5 nd K /K s rtios, 2 were retined (Supplementry Tles 3 nd 4). TAI nd TDI. Expression levels for the first dtset were extrcted from ref. 11, nd fter outlier detection nd ID mpping, 25,158 genes represented on the microrrys were included in susequent nlyses. The TAI nd the TDI re weighted mens of evolutionry ge nd sequence divergence, respectively, nd defined s follows. Introduced in ref. 7, the trnscriptome ge index TAI s of developmentl stge s (s 5 zygote, qudrnt, gloulr, hert, torpedo, ent cotyledon, or mture) is the weighted men of the evolutionry ge (phylostrtum) ps i of gene i weighted y the expression level of gene i t developmentl stge s, TAI s ~ ps i where n is the totl numer of genes nlysed. Low PS vlues correspond to evolutionrily old genes, so low TAI vlues correspond to evolutionrily old trnscriptomes. Likewise, high PS vlues correspond to evolutionrily young genes, so high TAI vlues correspond to evolutionrily young trnscriptomes. Anlogously, we introduce the trnscriptome divergence index TDI s of developmentl stge s simply y replcing ps i in the ove eqution y the K / K s rtio of gene i, TDI s ~ Ki e Ksi is Hence, low/high K /K s rtios correspond to conserved/divergent genes, so low/ high TDI vlues correspond to conserved/divergent trnscriptomes. The sme procedure ws repeted for the second independent dtset covering the emryo propers of A. thlin emryo stges pre-gloulr, gloulr, hert, liner cotyledon/torpedo, nd mture 18,19 (GEO ccession numer GSE1244). We normlized this dtset using the GCRMA pckge (version 2.) from the Bioconductor project with defult prmeter settings 3. For ech proe set we computed the stge-wise rithmetic men of the replictes to get representtive expression vlues for ech stge. Here, 2,31 genes represented on the microrrys were included in the nlyses. Sttisticl significnce of TAI nd TDI profiles. To determine the sttisticl significnce of the TAI nd TDI profiles, the following permuttion test ws performed. The vrince V TAI of the seven vlues of TAI s (for s 5 zygote, qudrnt,, mture) ws computed s test sttistic. For determining the null distriution of V TAI, ll PS vlues within ech developmentl stge s were rndomly permuted, seven surrogte vlues of TAI s were computed from this permuted dtset, nd surrogte vlue of V TAI ws computed from these seven surrogte vlues of TAI s. This procedure ws repeted 1, times, yielding histogrm of 1, vlues of V TAI, which cn e pproximted y gmm distriution. The two prmeters of the gmm distriution were estimted y the method of moments, the fitted gmm distriution ws considered the null distriution of V TAI, nd the P-vlue of the oserved vlue of V TAI ws computed from this null distriution. The sme procedure ws repeted for the seven vlues of TDI s, yielding P-vlue of the TDI profile. Likewise, the second dtset 18,19 ws nlysed ccordingly. Reltive expression levels for phylostrt. Reltive expression levels were computed s descried previously 7. In rief, the men expression level e js of phylostrtum j nd developmentl stge s ws computed for ech j nd s s the rithmetic men of expression levels of ll genes i elonging to phylostrtum j. The men expression levels e js were linerly trnsformed to the intervl [,1] ccording to f js ~ e js{e jmin e jmx {e jmin where e jmin /e jmx is the minimum/mximum men expression level of phylostrtum j over the seven developmentl stges s. This liner trnsformtion corresponds to shift y e jmin nd susequent shrinkge y e jmx e jmin. As result, the reltive expression level f js of developmentl stge s with minimum e js is, the reltive expression level f js of the developmentl stge s with mximum e js is 1, nd the reltive expression levels f js of ll other stges s rnge etween nd 1, ccordingly. Next, reltive expression levels were grouped into two PS clsses, where the first PS clss consists of reltive expression levels of genes elonging to the three oldest phylostrt PS1 PS3, nd where the second PS clss consists of reltive expression levels of genes elonging to the younger phylostrt PS4 PS13. This grouping ws chosen to distinguish phylostrt of plnts tht pss through emryogenesis (PS4 PS13) from the remining phylostrt (PS1 PS3), in which the vst mjority of species did not evolve emryogenesis. For ech developmentl stge s nd ech PS clss, the men vlue nd stndrd error of the reltive expression levels were computed. In ddition, the rtio (foldchnge) of the two reltive expression levels ws computed for ech developmentl stge s, nd Welch s two-smple t-test ws performed. Reltive expression levels for K /K s quntiles. In contrst to PS vlues, which re discrete, K /K s rtios re continuous. For computing reltive expression levels of genes elonging to different K /K s groups, continuous K /K s rtios were grouped into deciles (1% quntiles). Reltive expression levels of these ten K /K s groups were computed in nlogy to the computtion of reltive expression levels of the 13 phylostrt. Likewise, reltive expression levels were grouped into two K /K s clsses, where the first K /K s clss consists of reltive expression levels of genes elonging to the first five K /K s groups (K /K s rtios elow medin, conserved genes), nd where the second K /K s clss consists of reltive expression levels of genes elonging to the remining five K /K s groups (K /K s rtios ove medin, divergent genes). This grouping ws chosen ecuse the medin is nturl choice, mking oth K /K s clsses eqully lrge, nd ecuse the grouping of genes into different PS clsses lso resulted in two PS clsses of roughly similr sizes (first dtset: 1,695 genes in PS1 PS3 nd 14,463 genes in PS4 PS13; second dtset: 9,28 nd 11,3 genes, respectively). The computtion of men vlues, stndrd errors, fold-chnges nd P-vlues of Welch s two-smple t-test were performed s descried in the previous section. To investigte the dependence of the results on the grouping into two K /K s clsses, the entire nlysis ws repeted for the following six pirs of K /K s clsses: two deciles/eight deciles, three deciles/seven deciles,, seven deciles/three deciles. The six resulting plots of mens, stndrd errors, fold-chnges nd P-vlues re presented in Supplementry Figs 6 9, Gene ontology nlysis. Enrichment nlysis of gene ontology terms ws performed on genes from PS4 PS13 tht were down-regulted t lest twofold in the torpedo stge compred to t lest one of the developmentl stges zygote/qudrnt/gloulr/hert/ent cotyledon/mture (first dtset), or pre-gloulr/gloulr/hert/mture (second dtset). Gene ontology term enrichment ws nlysed using AmiGO 31 (filter options: TAIR nd A. thlin, P-vlue ). 29. Goodstein, D. M, et l. Phytozome: comprtive pltform for green plnt genomics. Nucleic Acids Res. 4, D (212). 3. Wu, Z. et l. A model-sed ckground djustment for oligonucleotide expression rrys. J. Am. Stt. Assoc. 99, (24). 31. Cron, S, et l. AmiGO: online ccess to ontology nd nnottion dt. Bioinformtics 25, (29). 212 Mcmilln Pulishers Limited. All rights reserved