REPORTS

share several characteristics with xylem vessels Contribution of NAC Transcription from vascular plants, such as an elongated shape and an absence of cellular contents (13). How- ever, unlike xylem vessels, hydroids lack distinct Factors to Plant Adaptation to Land pits on their lateral walls, and there is an absence of lignified secondary wall (14–17). Stereid cells Bo Xu,1 Misato Ohtani,2* Masatoshi Yamaguchi,1† Kiminori Toyooka,2 Mayumi Wakazaki,2 have thickened cell walls and are hypothesized to Mayuko Sato,2 Minoru Kubo,3‡ Yoshimi Nakano,1 Ryosuke Sano,1 Yuji Hiwatashi,3§ function as supporting elements (18). Both hydroids Takashi Murata,3 Tetsuya Kurata,1 Arata Yoneda,1 Ko Kato,1 Mitsuyasu Hasebe,3,4 Taku Demura1,2* and stereids are formed mainly in the gameto- The development of cells specialized for water conduction or support is a striking innovation of plants phytic generation, unlike xylem vessels and fiber that has enabled them to colonize land. The NAC transcription factors regulate the differentiation of cells, which are formed only in the sporophytic these cells in vascular plants. However, the path by which plants with these cells have evolved from generation of vascular plants. Therefore, it re- their nonvascular ancestors is unclear. We investigated genes of the moss Physcomitrella patens that mains unclear whether hydroids and stereids share encode NAC proteins. Loss-of-function mutants formed abnormal water-conducting and supporting an evolutionary lineage with xylem vessels and cells, as well as malformed sporophyte cells, and overexpression induced ectopic differentiation of fiber cells, respectively. water-conducting–like cells. Our results show conservation oftranscriptionalregulationandcellular The moss P. patens genome has eight loci that function between moss and Arabidopsis thaliana water-conducting cells. Theconservedgeneticbasis share similarity with VND/NST/SND (19–21). We suggests roles for NAC proteins in the adaptation of plants to land. sequenced cDNA from these genes and named the gene family PpVNS [VND-, NST/SND-, SMB he acquisition of water-conducting tis- transcriptional regulatory system for secondary (SOMBRERO)-related protein], with genes 1 through sue enabled the transition of plants from wall biosynthesis has been proposed (11). 8(22)(fig.S1andtableS1).Forphylogeneticanal- Tan aqueous environment to land. To achieve In mosses, specialized cell types have been ysis, publicly available genomic and transcriptomic this, vascular plants developed xylem vessel characterized. For example, hydroid cells conduct sequence data were searched. Although we iden- elements, cells that have a characteristic thick- water internally (12, 13), and mature hydroids tified a single gene of Marchantia polymorpha, ened secondary cell wall and undergo programmed cell death at maturity. These cells provide me- chanical strength to the stem, while allowing ef- REPORTS on February 27, 2015 ficient water conduction. Recent work revealed share several characteristics with xylem vessels Contribution of NAC Transcription from vascular plants, such as an elongated shape that a group of NAC [no apical meristem and an absence of cellular contents (13). How- ever, unlike xylem vessels, hydroids lack distinct Factors to Plant Adaptation to Land pits on their lateral walls, and there is an absence (NAM), Arabidopsis transcription activation factor of lignified secondary wall (14–17). Stereid cells Bo Xu,1 Misato Ohtani,2* Masatoshi Yamaguchi,1† Kiminori Toyooka,2 Mayumi Wakazaki,2 have thickened cell walls and are hypothesized to Mayuko Sato,2 Minoru Kubo,3‡ Yoshimi Nakano,1 Ryosuke Sano,1 Yuji Hiwatashi,3§ function as supporting elements (18). Both hydroids (ATAF1/2), and cup-shaped cotyledonTakashi Murata,3 (CUC)]Tetsuya Kurata,1 Arata Yoneda,1 Ko Kato,1 Mitsuyasu Hasebe,REPORTS3,4 Taku Demura1,2* and stereids are formed mainly in the gameto- The development of cells specialized for water conduction or support is a striking innovation of plants phytic generation, unlike xylem vessels and fiber transcription factors, includingthat VASCULAR- has enabled them to colonize land. The NAC transcription factors regulate the differentiationP. patens of ,cells,Selaginella which are formed moellendorffii only in the sporophytic, A. thaliana, plants (20, 21). We examined the tissue-specific ex- in tissues from protonemata and gametophytes. these cells in vascular plants. However, the path by which plants with these cells have evolvedpoplar, from andgeneration rice all of vascularhad many plants. (fig.Therefore, S2), it re- suggesting pression patterns of PpVNS by quantitative reverse In the gametophytic generation, after haploid spore RELATED NAC-DOMAIN6 (VND6)their nonvascular and ancestors VND7 is unclear. We investigated genes of the moss Physcomitrella patens that mains unclear whether hydroids and stereids share encode NAC proteins. Loss-of-function mutants formed abnormal water-conducting and supportingan early expansionan evolutionary lineage of VNS with xylemgenes vessels in and prevascular transcription polymerase chain reaction (qRT-PCR) germination, P. patens forms filamentous cells called of Arabidopsis thaliana,regulatexylemvesselcells, as well as malformed sporophyte cells, and overexpression induced ectopic differentiation of fiber cells, respectively. water-conducting–like cells. Our results show conservation oftranscriptionalregulationandcellular The moss P. patens genome has eight loci that function between moss and Arabidopsis thaliana water-conducting cells. TheconservedgeneticbasisFig. 2. Defectsshare similarity in with midribVND/NST/SND development(19–21). We in the differentiation by inducing genessuggests for roles sec for NACondary proteins in the adaptation of plants to land. sequenced cDNA from these genes and named ppvns1 ppvns6the gene family ppvns7PpVNS [VNDmutant.-, NST/SND(-,ASMBto C)Trans- he acquisition of water-conducting tis- transcriptional regulatory system for secondary (SOMBRERO)-related protein], with genes 1 through cell wall biosynthesis and xylem vesselsue enabled–specific the transition of plants from wall biosynthesis has been proposedport (11). of Evans8(22)(fig.S1andtableS1).Forphylogeneticanal- Blue dye in the leaves of wild-type www.sciencemag.org Tan aqueous environment to land. To achieveVascular Plant Innovaons (connued): In mosses, specialized cell types(WT) have been plantsysis, (A) publicly andREPORTS available in ppvns1 genomic and ppvns6 transcriptomic ppvns7 line 8 programmed cell death (1–6). NACthis, vascular plants proteins developed xylem vessel characterized. For example, hydroid cells conduct sequence data were searched. Although we iden- elements, cells that have a characteristic thick- water internally (12, 13), and mature(B) hydroids and linetified a 35 single (C) geneP. afterpatens of Marchantia, 30Selaginella min polymorpha moellendorffii of incubation., , A. thaliana, plants (20, 21). We examined the tissue-specific ex- in tissues from protonemata and gametophytes. also regulate the development of fiberened secondary cells: cell wall and scle- undergo programmedA conserved GRN for water conducon and support cells Arrowheads indicatepoplar, positions and rice all where had many Evans(fig. S2), suggesting Blue pression patterns of PpVNS by quantitative reverse In the gametophytic generation, after haploid spore cell death at maturity. These cells provide me- an early expansion of VNS genes in prevascular transcription polymerase chain reaction (qRT-PCR) germination,REPORTSP. patens forms filamentous cells called chanical strength to the stem, while allowing ef- dye was transported from the base. (D to I)Typical on February 27, 2015 renchyma cells found in vascularficient plants, water conduction. which Recent work revealed images of transmissionFig. electron 2. Defects in microscopy midrib development trans- in the that a group of NAC [no apical meristem ppvns1 ppvns6 ppvns7 mutant. (A to C)Trans- share several characteristics with xylem vessels (NAM), Arabidopsis transcription activation factor verse sections of theport tip of [(D) Evans and Blue dye (G)], in the middle leaves of wild-type [(E) from vascular plants, such as an elongated shape are also characterized by a thickened secondaryContribution of NAC Transcription(WT) plants (A) and in ppvns1 ppvns6 ppvns7 line 8 (ATAF1/2), and cup-shaped cotyledon (CUC)] and (H)], and basal [(F) and ( I)] regions of leaves of and an absence of cellular contents (13). How- transcription factors, including VASCULAR- (B) and line 35 (C) after 30 min of incubation. Arrowheads indicate positions where Evans Blue wall (7–10). In A. thaliana,NACSECONDARYRELATED NAC-DOMAIN6 (VND6) and VND7 wild-type plants [(D) to (F)] and ppvns1 ppvns6 ever, unlike xylem vessels, hydroids lack distinct Factors to Plant Adaptationdye was transported to from theLand base. (D to I)Typical of Arabidopsis thaliana,regulatexylemvessel ppvns7 [(G) to (I)]. h,images e, and of transmission s indicate electron content-free microscopy trans- pits on their lateral walls, and there is an absence differentiation by inducing genes for secondary WALL THICKENING PROMOTING FACTOR 1 2 1 verse sections of2 the tip [(D) and (G)], middle2 [(E) of lignified secondary wall (14–17). Stereid cells cell wall biosynthesis and xylem vesselBo–specific Xu, Misato Ohtani, * Masatoshihydroid Yamaguchi, cells† withKiminori thin Toyooka, cell walls,Mayumi epidermal Wakazaki, cells and (H)], and basal [(F) and ( I)] regions ofwww.sciencemag.org leaves of have thickened cell walls and are hypothesized to programmed cell death (1–6). NACMayuko proteins Sato,2 Minoru Kubo,3‡ Yoshimiwith Nakano, developed1 Ryosuke chloroplasts,wild-type Sano,1 plantsYuji and [(D) Hiwatashi, stereid to (F)] and cells3ppvns1§ with ppvns6 REPORTS (NST)/SECONDARY WALL-ASSOCIATEDalso regulate the development of fiber cells: scle- ppvns7 [(G) to (I)]. h, e, and s indicate content-free function as supporting elements (18). Both hydroids Takashi Murata,3 Tetsuya Kurata,1 Aratathicker Yoneda, cell1 Ko walls, Kato,1 respectively.Mitsuyasu Hasebe, (J to3,4L)ImagesofTaku Demura1,2* renchyma cells found in vascular plants, which hydroid cells with thinprotonemata, cell walls, from epidermal which cellsleafy gametophoresand stereids de- that are all PpVNS formedgenes mainly were preferentially in the gameto- expressed of differentiated cells, including hydroids and are also characterized by a thickened secondary with developed chloroplasts, and stereid cells with NAC DOMAIN PROTEIN (SND) proteins, con- wild-type plants (J) and mutants [(K)velop. and Although (L)] the incu- expression of PpVNS3 was in gametophores, except for PpVNS3-GUS,whose stereids (Fig. 1 and figs. S3 and S5 to S7), sug- Downloaded from wall (7–10). In A. thaliana,NACSECONDARYThe development of cells specialized for water conduction or supportthicker is cell a strikingwalls, respectively.barely innovation detected (J to inL ofthe)Imagesof plantstested tissues, thephytic other sev- generation,signal was unlike too low xylem to be detected vessels in and any tissue fiber gesting that PpVNS genes function in midrib de- bated for 8 hours underwild-type the plants low-humidity (J) and mutants [(K) and condi- (L)] incu- stituting a sister group to VND,WALL regulate THICKENING PROMOTING fiberthat FACTOR has enabled them to colonize land. The NAC transcription factors regulateen thePpVNS differentiationgenes were allof expressed,cells, and theywhich(Fig. are 1, formed A to BB, and only fig. in S3). the PpVNS-GUS sporophytic ac- velopment. PpVNS1, PpVNS6,andPpVNS7 were (NST)/SECONDARY WALL-ASSOCIATED bated for 8 hours underwere tmorehe low-humidity abundant in condi- gametophores than in tivity was prominent in the newly emerged leaf preferentially expressed in these regions (table S2), these cells in vascular plants. However, thetion. path Wiltedby which leaves plants weretion. with Wilted these observed leaves cells were have in observed the evolved mutant.in the from mutant. generation of vascular plants. Therefore, it re-

NAC DOMAIN PROTEIN (SND) proteins, con- protonemata (fig. S3,Downloaded from A and B). The PpVNS- central region (which is thought to subsequent- whereas the other PpVNS genes were also ex- Scale bars, 200 mmin(A)to(C);5mmin(D)to(I); their nonvascular ancestors is unclear. WeScale investigated bars, 200 genesmmin(A)to(C);5 of the moss PhyscomitrellaGUSmmin(D)to(I);(b-glucuronidase) patens that reporter linesmains (22)(fig. unclearly develop whether into hydroids the midrib) and and in stereids the developing share pressed in other regions of the leaf (fig. S5 and cell differentiation, suggesting anstituting evoluti a sister grouponary to VND, regulate fiber and 500 mmin(J)to(L). cell differentiation, suggesting an evolutiencodeonary NAC proteins. Loss-of-function mutantsand 500 formedmmin(J)to(L). abnormal water-conductingS4), in and agreement supporting with qRT-PCR results,an evolutionary showed leaf midrib, lineage which with is composed xylem of vessels several types and table S2). GUS signals were also detected in stems relationship between vessels andrelationship fibers between ( vessels7, 8 and). fibers (7, 8). fiber cells, respectively. Thus, conservation of a VND/NST/SND-basedcells, as well as malformed sporophyte cells, and overexpression induced ectopic differentiation of water-conducting–like cells. Our results show conservation oftranscriptionalregulationandcellularFig. 3. Defects in stem and sporophyte devel- The moss P. patens genome has eight loci that opment in the mutant. A Thus, conservation of a VND/NST/SND-basedfunction between moss and Arabidopsis thaliana water-conducting cells. Theconservedgeneticbasisppvns4 ( )Transportof share similarity with VND/NST/SND (19–21). We 1 Evans Blue dye through stems of wild-type (WT) Graduate School of Biological Sciences, Nara Institute of Sci- plants and ppvns4 deletion mutants after 30 min sequenced cDNA from these genes and named ence and Technology, Ikoma, Nara 630-0192,suggests Japan. 2RIKEN roles for NAC proteins in the adaptationFig. 3. Defects of plants to in land. stem and sporophyte devel- Center for Sustainable Resource Science, Yokohama, Kanagawa opment in the ppvns4of incubation.mutant. Black arrowheads(A)Transportof indicate positions the gene family PpVNS [VND-, NST/SND-, SMB 230-0045, Japan. 3National Institute for Basic Biology, Okazaki, where Evans Blue dye was transported from the base. 4 he acquisition of water-conducting tis- transcriptional regulatory system for secondary (SOMBRERO)-related protein], with genes 1 through Aich 444-8585, Japan. School of Life Science, Graduate Univer- Evans Blue dye through(B to E)Microscopicimagesofstemsectionsofwild- stems of wild-type (WT) sity for Advanced Studies, Okazaki, Aich 444-8585, Japan. sue enabled the transition of plants from wall biosynthesistype plants has [(B) beenand (D)] proposed and ppvns4 (mutants11). [(C) 8(22)(fig.S1andtableS1).Forphylogeneticanal- 1 *Corresponding author. E-mail: [email protected] (T.D.);an aqueous environment to land. Toplants achieve and ppvns4In mosses,deletionand (E)]. specialized (D) mutantsand (E) Magnified cell after types views 30 are have shown min been in ysis, publicly available genomic and transcriptomic Graduate School of Biological Sciences, [email protected] Institute (M.O.) of Sci-T of incubation. Black(B) arrowheads and (C), respectively. indicate The h’sin(D)indicatecy- positions †Present address: Institute for2 Environmental Sciencethis, and vascularTech- plants developed xylem vessel characterized.toplasmic For contentexample,–free hydroid cells cells with a conduct thin cell sequence data were searched. Although we iden- ence and Technology, Ikoma, Nara 630-0192,nology, Saitama Japan. University, SaitamaRIKEN 338-8570,elements, Japan; and cells that have a characteristicwhere thick- Evanswater Blue dyeinternallywall was in the transported (12 central, 13 region), and of from wild-type mature the plants. hydroidsbase. (F)Typ- tified a single gene of Marchantia polymorpha, Japan Science and Technology Agency, PRESTO, Kawaguchi, Fig. 1. Expression of PpVNS genes in P. patens tissues. GUS expressionical pattern images of ofPpVNS1-GUS wild-type plants(A to and ppvns4 mutants Saitama 332-0012, Japan. ened secondaryG), PpVNS6-GUS cell wall and(H to undergoN), PpVNS7-GUS prog(Brammed(Ototo UE),)Microscopicimagesofstemsectionsofwild- and PpVNS4-GUS (V toincubatedBB)reporterlines.Thecentral for 8 hours under the low-humidity con- Center for Sustainable Resource Science, Yokohama,‡Present address: Center Kanagawa for Frontier Science and Technology, region of young leaves [(B), (I), (P), and (W)], midribs [(C), (J), (Q), and (X)],dition. rhizoids Wilted [(D), leaves (K), (R), were and observed (Y), in ppvns4.(G 3 Nara Institute of Science and Technology, Ikoma,cell Nara death 630- atyellow maturity. arrowheads)], These protonemata cells [(E),providetype (G), (L), plantsme- (N), (S), (U), [(B) (Z), and and (BB)], (D)] and sporophytes and ppvns4 [(F), (M),mutants (T), [(C) 0192, Japan. to N)Maturedsporophytesofwild-typeplants[(G) 230-0045, Japan. National Institute for Basic Biology, Okazaki,chanical strengthand (ZZ), to early the stages; stem, (G), while(N), (U), andallowing (BB), late ef- stages] are shown. Scale bars, 5 mm in (A), (H), (O), and §Present address: National Plant Phenomics Centre, Institute and (E)]. (D) and (E)to Magnified (J)] and ppvns4 viewsmutants are[(K) to shown (N)]. (H) and in (L) on February 27, 2015 4 (V); 100 mmin(B)to(E),(I)to(L),(P)to(S),and(W)to(Z);and200mmin(F),(G),(M),(N),(T),(U),(AA), Aich 444-8585, Japan. School of Life Science,of Biological, Graduate Environmental Univer- and Rural Sciences,ficient Aberystwyth water conduction. Recent work(B) revealed and (C), respectively.Magnified The views h’sin(D)indicatecy- of the regions enclosed in black University, Aberystwyth SY23 3EB, UK. that a groupand (BB). of NAC [no apical meristem squares in (G) and (K), respectively. Red arrowheads toplasmic content–freeindicate hydroid the boundary cells between with a the thin seta and cell the sity for Advanced Studies, Okazaki, Aich 444-8585, Japan. (NAM), Arabidopsis transcription activation factor foot. Thin solid lines and dotted lines in (H) and (L) (ATAF1/2),www.sciencemag.org and cup-shapedSCIENCE cotyledonVOL 343wall (CUC)] 28 in MARCH the central 2014 regionindicate of the wild-type position of transverse plants.1505 section (F)Typ- shown in (I) and (M), and (J) and (N), respectively. t indicates *Corresponding author. E-mail: [email protected] (T.D.);transcription factors, including VASCULAR-ical images of wild-typetransfer plants cells. Asterisks and indicateppvns4 malformedmutants vacuoles RELATED NAC-DOMAIN6 (VND6) andincubated VND7 for 8 hours(N). under Archegonium the low-humiditycells surrounding a sporophyte con- [email protected] (M.O.) remain in (G) to (J). Scale bars, 1 mm in (A); 20 mm of Arabidopsis thaliana,regulatexylemvesseldition. Wilted leavesin were (B) to (E), observed (H) to (J), and in (L) toppvns4 (N); 500 mmin(F);.(G †Present address: Institute for Environmental Science and Tech-differentiation by inducing genes for secto ondaryN)Maturedsporophytesofwild-typeplants[(G)and 80 mmin(G)and(K). cell wall biosynthesis and xylem vessel–specific nology, Saitama University, Saitama 338-8570, Japan; and to (J)] and ppvns4 mutants [(K) to (N)]. (H) and (L) www.sciencemag.org programmed cell death (1–6). NAC proteins Japan Science and Technology Agency, PRESTO, Kawaguchi,also regulateFig. the 1. development Expression of fiber cells:Magnified of scle-PpVNS views of thegenes regions in enclosedP. patens in black tissues. GUS expression pattern of PpVNS1-GUS (A to Saitama 332-0012, Japan. renchyma cells found in vascular plants,squares which in (G) and (K), respectively. Red arrowheads are alsoG characterized), PpVNS6-GUS by a thickened( secondaryindicateH to N the), boundaryPpVNS7-GUS between the(O setato andU), the and PpVNS4-GUS (V to BB)reporterlines.Thecentral ‡Present address: Center for Frontier Science and Technology,wall (7region–10). In A. thaliana of young,NACSECONDARY leavesfoot. Thin [(B), solid lines(I), and(P), dotted and lines (W)], in (H) midribs and (L) [(C), (J), (Q), and (X)], rhizoids [(D), (K), (R), and (Y), Nara Institute of Science and Technology, Ikoma, Nara 630-WALL THICKENING PROMOTING FACTORindicate the position of transverse section shown in (NST)/SECONDARYyellow arrowheads)], WALL-ASSOCIATED(I) and protonemata (M), and (J) and (N), [(E), respectively. (G), (L), t indicates (N), (S), (U), (Z), and (BB)], and sporophytes [(F), (M), (T),

0192, Japan. NAC DOMAIN PROTEIN (SND) proteins,transfer con- cells. Asterisks indicate malformed vacuoles Downloaded from §Present address: National Plant Phenomics Centre, Institutestitutingand a sister (ZZ), group early to VND, stages; regulate fiber (G), (N), (U), and (BB), late stages] are shown. Scale bars, 5 mm in (A), (H), (O), and cell differentiation, suggesting an evoluti(N).onary Archegonium cells surrounding a sporophyte 1506 28 MARCH 2014 VOL 343 SCIENCE www.sciencemag.org of Biological, Environmental and Rural Sciences, Aberystwythrelationship(V); between 100 m vesselsmin(B)to(E),(I)to(L),(P)to(S),and(W)to(Z);and200 and fibersremain (7, 8). in (G) to (J). Scale bars, 1 mm in (A); 20 mm mmin(F),(G),(M),(N),(T),(U),(AA), Thus, conservation of a VND/NST/SND-basedin (B) to (E), (H) to (J), and (L) to (N); 500 mmin(F); University, Aberystwyth SY23 3EB, UK. and (BB). and 80 mmin(G)and(K). Fig. 4. Effects of estrogen-induced overexpression of PpVNS7 genes in number of genes belonging to the category. Colored nodes represent GO terms P. patens and A. thaliana. Phenotypes shown in protonemata and game- that are significantly overrepresented (corrected P value <0.05), and the color 1Graduate School of Biological Sciences, Nara Institute of Sci- 2 tophores of P. patens (A to D, F to I)andA. thaliana leaves (E and J)treated scale indicates increasingly higher statistical significance. Overrepresented GO ence and Technology, Ikoma, Nara 630-0192, Japan. RIKEN with 1 mM17-b-estradiol (ER), wild-typeER-WT[(A)to(E)],andthePpVNS7 terms detected commonly between P. patens and A. thaliana are indicated in Center for Sustainable Resource Science, Yokohama, Kanagawa overexpressor ER-PpVNS7 [(F) to (J)]. (B) and (G) Magnified views shown in (A) red characters (left). (M)ChangesintheexpressionlevelofP. patens genes that www.sciencemag.org230-0045, Japan. 3National Institute for BasicSCIENCE Biology, Okazaki, VOL 343and (F), respectively. 28 MARCH (D) and (I) Leaves from gametophores 2014 shown in (C) and are putatively orthologous to well-characterized A. thaliana genes involved in 1505 Aich 444-8585, Japan. 4School of Life Science, Graduate Univer- (H), respectively. Yellow arrowheads indicate ectopic secondary cell wall depo- cell wall biosynthesis and cell death. The color scale indicates fold changes of sity for Advanced Studies, Okazaki, Aich 444-8585, Japan. sition. (K and L) Enriched gene ontology (GO) terms for the up-regulated gene expression (on a log 2 scale). Asterisks indicate statistically significant 2910 P. patens genes in the PpVNS7 overexpressor (K) and the reported 448 changes. Scale bars, 500 mmin(A)and(F);100mmin(B),(D),(G),and(I); *Corresponding author. E-mail: [email protected] (T.D.); VND/NST/SND direct target genes in A. thaliana (L). Node size reflects the 1mmin(C)and(H);and50mmin(E)and(J). [email protected] (M.O.) †Present address: Institute for Environmental Science and Tech- nology, Saitama University, Saitama 338-8570, Japan; and www.sciencemag.org SCIENCE VOL 343 28 MARCH 2014 1507 Japan Science and Technology Agency, PRESTO, Kawaguchi, Fig. 1. Expression of PpVNS genes in P. patens tissues. GUS expression pattern of PpVNS1-GUS (A to Saitama 332-0012, Japan. G), PpVNS6-GUS (H to N), PpVNS7-GUS (O to U), and PpVNS4-GUS (V to BB)reporterlines.Thecentral ‡Present address: Center for Frontier Science and Technology, region of young leaves [(B), (I), (P), and (W)], midribs [(C), (J), (Q), and (X)], rhizoids [(D), (K), (R), and (Y), Nara Institute of Science and Technology, Ikoma, Nara 630- yellow arrowheads)], protonemata [(E), (G), (L), (N), (S), (U), (Z), and (BB)], and sporophytes [(F), (M), (T), 0192, Japan. §Present address: National Plant Phenomics Centre, Institute and (ZZ), early stages; (G), (N), (U), and (BB), late stages] are shown. Scale bars, 5 mm in (A), (H), (O), and of Biological, Environmental and Rural Sciences, Aberystwyth (V); 100 mmin(B)to(E),(I)to(L),(P)to(S),and(W)to(Z);and200mmin(F),(G),(M),(N),(T),(U),(AA), University, Aberystwyth SY23 3EB, UK. and (BB).

1506 www.sciencemag.org SCIENCE VOL28 343 MARCH 28 MARCH 2014 2014 VOL 343 SCIENCE www.sciencemag.org1505 Ferns

Leptosporangiate

hp://131.230.176.4/users/pelserpb/6_18_11/18jun11a/botrychiumdaucifolium.jpg Ferns are Euphyllophytes (they have megaphylls)

LAND PLANTS NON-VASCULAR PLANTS Hepaticophyta (liverworts)

Bryophyta (mosses)

Anthocerophyta (hornworts)

VASCULAR PLANTS SEEDLESS PLANTS Lycophyta (lycophytes)

Psilotophyta (whisk ferns)

Equisetophyta (horsetails)

Pteridophyta (ferns) SEED PLANTS GYMNOSPERMS Cycadophyta (cycads)

Ginkgophyta (ginkgo)

ConifersRedwood group (junipers et al.)

Pinophyta (pines et al.)

Gnetophyta (gnetophytes)

ANGIOSPERMS Anthophyta (angiosperms)

Simpson 2010 Freeman 2010 Comparave reproducve development of ferns Homosporous ferns: Two types of sporangium development Leptosporangiate ferns Eusporangiate ferns (Maraa) Synangia: Fused sporangia

Cronk 2010

Maraa Fern (homosporous lepto) Sporophyte: Sporangia in sori

hp://131.230.176.4/cgi-bin/dol/dol_terminal.pl?order=Polypodiales Heterosporous “water” ferns (leptosporangiate)

Gametophyte development Homosporous ferns and horsetails

(Steven J. Baskauf 2002)

Heterosporous “water” ferns Spore dance movie The Plant Cell, Vol. 27: 1567–1578, June 2015, www.plantcell.org ã 2015 American Society of Plant Biologists. All rights reserved.

Horsetails Are Ancient Polyploids: Evidence from Equisetum giganteumOPEN

Kevin Vanneste,a,b Lieven Sterck,a,b Alexander Andrew Myburg,c,d Yves Van de Peer,a,b,d,1 and Eshchar Mizrachic,d,1 a Department of Plant Systems Biology, VIB, Ghent B-9052, Belgium b Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent B-9052, Belgium c Department of Genetics, Forestry, and Agricultural Biotechnology Institute, University of Pretoria, Pretoria 0028, South Africa d Department of Genetics, Genomics Research Institute, University of Pretoria, Pretoria 0028, South Africa ORCID ID: 0000-0001-7116-4000 (L.S.)

Horsetails represent an enigmatic clade within the land plants. Despite consisting only of one genus (Equisetum)thatcontains15 species, they are thought to represent the oldest extant genus within the vascular plants dating back possibly as far as the Triassic. Horsetails have retained several ancient features and are also characterized by a particularly high chromosome count (n =108). Whole-genome duplications (WGDs) have been uncovered in many angiosperm clades and have been associated with the success of angiosperms, both in terms of species richness and biomass dominance, but remain understudied in nonangiosperm clades. Here, we report unambiguous evidence of an ancient WGD in thefernlinage,basedonsequencinganddenovoassemblyofan expressed gene catalog (transcriptome) from the giant horsetail (Equisetum giganteum). We demonstrate that horsetails underwent an independent paleopolyploidy during the Late Cretaceous prior to the diversification of the genus but did not experience any recent polyploidizations that could account for their high chromosomeAncient Genome number. Duplication We in the also Horsetails discuss 1571 the specific retention of genes following the WGD and how this may be linked to their long-term survival.

INTRODUCTION to angiosperms (n = 15.99 on average) and homosporous ferns where the chromosome count is more than 3 times higher (n = Although ferns (monilophytes) represent the most diverse plant 57.05 on average; Klekowski and Baker, 1966). This early work lineage on Earth second only to angiosperms, almost all of these investigated a limited number of homosporous ferns and con- species are contained within the monophyletic (leptosporan- cluded that selfing was a common mechanism of reproduction giate) polypod fern lineage that contains 267 genera and ;9000 (Klekowski and Baker, 1966). Combined with later observations of species. The remaining 20% of species lie outside this derived unusually high numbers of chromosomes in homosporous ferns group and represent earlier diverging lineages (Schuettpelz and (and the resultant assumption that this was due to polyploidy), the Pryer, 2007). Ferns were the dominant plant clade on Earth both hypothesis emerged that polyploidy would present a plausible in terms of species richness and biomass during the Paleozoic mechanism allowing the generation of novel genetic material to but had to compete with the more derived angiosperms during avoid homozygous inbreeding depression by pairing of homoe- the Mesozoic. Originally it was thought that ferns experienced ologous rather than homologous chromosomes during meiosis a drastic decline (Crane, 1987), although latter work readjusted (Klekowski, 1973). This view was later challenged and revised this view by demonstrating that polypod ferns, in particular, di- through findings that species having the lowest chromosome fi versi ed during the Cretaceous in the wake of novel ecological number within each genus exhibit diploid genetic expression opportunities that opened up by the rise to dominance of an- (Haufler and Soltis, 1986) and that these genetically diploid spe- giosperms (Schneider et al., 2004). cies experience selfing rates near zero (Soltis and Soltis, 1990) Over half a century ago, the high chromosome counts of through breeding systems such as temporal separation between homosporous fern genomes caught the attention of researchers Figure 3. Absolute Age Distribution for the Dated Peak-Based Duplicatessperm Representing and egg the Paleopolyploidy release (Soltis in E. giganteum and Soltis,. 1992). It was therefore (Manton, 1950). Ferns have especially high chromosome num- The solid black line represents the kernel density estimate of the datedproposed peak-based duplicates, that a while few the verticalancient dashed cycles black line of represents polyploidy its peak, followed by bers, with Ophioglossumused as reticulatum the WGD age estimate.possessing Gray lines therepresent highest the density estimatesgene for silencing the 1000 bootstrap and maintenance replicates, and the of vertical chromosomes black dotted lines could explain known chromosome countrepresent among the corresponding extant 90% eukaryotes confidence intervals (n > for 600; the WGD age estimate. The original raw distribution of dated peak-based duplicates is also indicated by open circles. The mode used as an estimate for the consensusthe high WGD age chromosome is found at 92.42 mya counts with lower of and homosporous upper 90% confidence ferns (Haufler, Khandelwal, 1990). However,interval boundaries a distinction at 75.16 and should 112.53 mya, be respectively. made be- 1987) or alternatively that the ancestor of ferns and seed plants tween heterosporous ferns that have chromosome counts similar had a high chromosome number that was retained only in ferns DISCUSSION number. This affirms what had been observed already before (Soltisbased and on Soltis, the analysis 1987). of gel Hence, electrophoresis the paradox banding patterns between the high The E. giganteum Transcriptome Confirms That High chromosome(Haufler and number Soltis, 1986; of ferns Haufler, and 1987), their namely, diploid that state high remains an 1 Address correspondence to [email protected] or eshchar. Chromosome Counts Did Not Evolve through Repeated intriguingchromosome open counts question in homosporous (Haufler, 2002, ferns did 2014). not evolve by [email protected]. Rounds of (Recent) Polyploidy repeated rounds of recent polyploidy, at least not within the The authors responsible for distribution of materials integral to the Amonghorsetails. the However, monilophytes, this does leave horsetails open the in question particular whether have received findings presented in thisHere, article we in present accordance unambiguous with the evidence policy for described a paleopolyploidymuchthe attention. observed chromosome They consist count of is the asingle result of extant a high chro- genus (Equise- event within the horsetails based on a large-scale transcriptome in the Instructions for Authors (www.plantcell.org) are: Yves Van de Peer tum),mosome which number is the in only the ancestor remaining of both representative ferns and seed plants from the more ([email protected])assembly generated and Eshchar by next-generation Mizrachi sequencing (eshchar. of E. gi- (Soltis and Soltis, 1987), or rather the result of the paleo- ganteum. Although transcriptome data cover only genes that areancient Sphenophyta that once were a very abundant and diverse [email protected]). polyploidy in E. giganteum coupled with a slow loss of genetic actively being transcribed, these are ideally suited for explor- fl OPEN Articles can be viewed online without a subscription. cladematerial (Husby, (Hau 2013).er, 1987). The Equisetopsida are estimated to have atory analysis of the gene space (Matasci et al., 2014), and It is important to note that a putative paleopolyploidy has also www.plantcell.org/cgi/doi/10.1105/tpc.15.00157 diverged from other monilophytes somewhere around 354 million (partial) EST-based gene catalogs have been used extensively in been reported in the ancestor of the polypod fern lineage, esti-;

the past for the discovery of WGD features in KS-based age mated to be ;180 million years old, based on a WGD signature

distributions (Blanc and Wolfe, 2004; Schlueter et al., 2004; peak found in a KS-based age distribution of duplicate pairs in Sterck et al., 2005; Cui et al., 2006). Our assembly contained partial next-generation sequencing data sets of Ceratopteris ri- more than 90% of core eukaryotic genes (Parra et al., 2007), chardii and Adiantum capillus-veneris (Barker, 2009; Barker and which suggests it is complete enough to rely on the use of Wolf, 2010). The latter was interpreted in favor of ferns having mixture modeling techniques to identify WGD signature peaks undergone a limited set of paleopolyploidies that resulted in high

before a KS threshold of 2.0 where KS saturation and stochas- chromosome counts because fern genomes have retained more ticity can become problematic (Vanneste et al., 2013). genetic material from these rare events compared with angio- Despite the high chromosome count of E. giganteum (n = 108; sperms (Barker, 2013). The low gene density of active genes in Leitch and Leitch, 2013), recent polyploidizations were not de- some fern genomes (Rabinowicz et al., 2005) and their strong tected and therefore could not have contributed to this high correlation between genome size and chromosome number Nave Leptosporangiate Ferns All present in Lake 22 trail

deer fern licorice fern

lady fern maidenhair fern Cenozoic radiation of ferns in an angiosperm- dominated canopy

Angiosperms

(Schuettpelz & Pryer 2009) Vocabulary Megaphyll Euphyllophytes Planaon and webbing Leptosporangium, eusporangium, synangium Sporocarp antheridiogen Sorus/sori Indusium Epiphyc

Ceratopteris (C-Fern) Reproducon Hermaphrodic gametophyte

male gametophyte

Are gametophytes endosporic? Is this fern homosporous or heterosporous?