A polycomb repressive complex 2 gene regulatesapogamy and gives evolutionary insights into earlyland plant evolutionYosuke Okanoa,1, Naoki Aonoa,1, Yuji Hiwatashia,b, Takashi Murataa,b, Tomoaki Nishiyamac,d, Takaaki Ishikawaa,Minoru Kuboc, and Mitsuyasu Hasebea,b,c,2

aNational Institute for Basic Biology, Okazaki 444-8585, Japan; bSchool of Life Science, The Graduate University for Advanced Studies, Okazaki 444-8585,Japan; cERATO, Japan Science and Technology Agency, Okazaki 444-8585, Japan; and dAdvanced Science Research Center, Kanazawa University, Kanazawa920-0934, Japan

Edited by Peter R. Crane, The University of Chicago, Chicago, IL, and approved July 27, 2009 (received for review June 22, 2009)

Land plants have distinct developmental programs in haploid(gametophyte) and diploid (sporophyte) generations. Althoughusually the two programs strictly alternate at fertilization andmeiosis, one program can be induced during the other program. Ina process called apogamy, cells of the gametophyte other than theegg cell initiate sporophyte development. Here, we report for themoss Physcomitrella patens that apogamy resulted from deletionof the gene orthologous to the Arabidopsis thaliana CURLY LEAF(PpCLF), which encodes a component of polycomb repressivecomplex 2 (PRC2). In the deletion lines, a gametophytic vegetativecell frequently gave rise to a sporophyte-like body. This body grewindeterminately from an apical cell with the character of a sporo-phytic pluripotent stem cell but did not form a sporangium.Furthermore, with continued culture, the sporophyte-like bodybranched. Sporophyte branching is almost unknown among extantbryophytes. When PpCLF was expressed in the deletion lines oncethe sporophyte-like bodies had formed, pluripotent stem cellactivity was arrested and a sporangium-like organ formed. Sup-ported by the observed pattern of PpCLF expression, these resultsdemonstrate that, in the gametophyte, PpCLF represses initiationof a sporophytic pluripotent stem cell and, in the sporophyte,represses that stem cell activity and induces reproductive organdevelopment. In land plants, branching, along with indeterminateapical growth and delayed initiation of spore-bearing reproductiveorgans, were conspicuous innovations for the evolution of adominant sporophyte plant body. Our study provides insights intothe role of PRC2 gene regulation for sustaining evolutionaryinnovation in land plants.

branching � PRC2 � protracheophytes

Development of land plants starts from a zygote in thesporophyte generation and from a spore in the gametophyte

generation. Although sporophyte development is usually pre-vented in the gametophyte until fertilization, it can occurnaturally and be induced experimentally. Female gametes (eggcells) can initiate embryogenesis with parthenogenetic develop-ment in the absence of fertilization (1, 2). Additionally, in aprocess called apogamy, somatic gametophyte cells are repro-grammed to start a sporophytic developmental program (3).While apogamy can be induced experimentally in floweringplants from synergid and antipodal cells, this is rare and notknown to happen naturally (4); in contrast, this type of asexualreproduction is more widely observed in non-seed plants, in-cluding pteridophytes (5) and bryophytes (6). Bypassing game-togenesis and fertilization, gametophyte somatic cells of non-seed plants divide several times to form a sporophyte apicalmeristem including a pluripotent stem cell. Apogamy in thesespecies can be induced with exogenous factors, such as hydration,sugars, chloral hydrate, and phytohormones (6). Even thoughapogamy has been well-studied from the viewpoints of physiol-

ogy, development, and evolution (5–7), the gene regulatorynetwork remains undeciphered.

Parthenogenetic development of egg cells has been observedin some alleles of loss-of-function mutants of the Arabidopsisthaliana genes FERTILIZATION-INDEPENDENT SEED 2(FIS2), MEDEA (MEA), and MULTICOPY SUPPRESSOR OFIRA 1 (MSI1) (8, 9), which encode members of the polycombrepressive complex 2 (PRC2) (8, 10, 11). The PRC2 complex wasfirst characterized in Drosophila melanogaster as a regulator ofHOX genes and includes four core proteins: Extra sex comb(ESC), Enhancer of zeste [E(Z)], Suppressor of zeste 12[SU(Z)12], and P55 (12). Orthologs of PRC2 genes were foundin a wide range of organisms including land plants and thiscomplex dynamically mediates transcriptional silencing of nu-merous genes, based on modifying trimethylation of lysine 27 onhistone H3 (12, 13). Several E(Z) and SU(Z)12 paralogs arecoded in the A. thaliana genome and at least three distinctcomplexes, with different combinations of the paralogs, havebeen implicated in plant development: the FIS complex in seeddevelopment including the prevention of parthenogenesis, theVERNALIZATION (VRN) complex in vernalization response,and the EMBRYONIC FLOWER (EMF) complex in floweringand flower development (14–16).

We sought to investigate the ancestral function and subse-quent evolution of the PRC2 complex in land plants, takingadvantage of the genomics (17), the feasibility of gene targeting,and the accessible development of the moss, Physcomitrellapatens. During the course of study, we found that deletion in P.patens of the gene orthologous to D. melanogaster E(Z) and A.thaliana CURLY LEAF (CLF)/MEA/SWINGER (SWN) (Pp-CLF) induced unusual sporophyte-like bodies. Here, we reportthe characterization of the role of the PpCLF gene in P. patensdevelopment, revealing multiple functions, including the repres-sion of apogamy in the haploid generation, that bear on theevolution of body plan in land plants.

ResultsMolecular Cloning of a CURLY LEAF Ortholog in Physcomitrella patens.A candidate cDNA sequence of PpCLF was obtained using theA. thaliana CLF amino acid sequence (18) as the query for a

Author contributions: Y.O., N.A., Y.H., and M.H. designed research; Y.O., N.A., Y.H., T.M.,T.N., T.I., and M.H. performed research; M.K. contributed new reagents/analytic tools; Y.O.,N.A., Y.H., and M.H. analyzed data; and Y.O., T.M., M.K., and M.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequences reported in this paper have been deposited in the GenBankdatabase (accession nos. AB472766).

1Y.O. and N.A. contributed equally to this work.

2To whom correspondence should be addressed. E-mail: mhasebe@nibb.ac.jp.

This article contains supporting information online at www.pnas.org/cgi/content/full/0906997106/DCSupplemental.

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TBLASTN search (19) against the P. patens subsp. patens v. 1.1genome database. The PpCLF cDNA sequence was obtained byRT-PCR using gene-specific primers. With the sequenced ge-nome (17), we identified PpCLF as the sole homolog of the E(Z)component of PRC2 (Fig. S1). This component is represented inthe A. thaliana genome by three genes, CLF, MEA, and SWN.PpCLF has the SET, CXC, and C5 domains as other E(Z)proteins have. The P. patens genome also includes homologuesof the other members of the PRC2 complex (10, 20).

Deletion of PpCLF Gene Induces Sporophyte-Like Body. Using genetargeting, we generated four deletion lines of PpCLF (ppclf-del-1to -4) (Fig. S2), and the phenotypes of the lines were indistin-guishable from each other. Following spore germination in P.patens, a cell filament forms, known as a protonema, whoseapical, or tip, cell has the characteristics of a pluripotent stemcell. When grown under red-light (21), in both wild-type anddeletion lines, the apical cell continuously produced protonemacells (Fig. 1 A and B), although the chloroplasts in the deletionlines were smaller than those of the wild type. When red-light-grown wild-type protonemata were transferred to white light, theprotonema cells formed side-branch initial cells that gave rise toprotonema apical cells, which are pluripotent (Fig. 1C). Incontrast, when red-light-grown PpCLF deletion lines weremoved into white light, although side-branch initials formed,they gave rise to tissue that differed from protonemata (Fig. 1D).This tissue had a single apical cell with two faces, each producinga row of cells, with subsequent periclinal divisions forming innerand outer cell layers (Fig. 1 E and F). The development andthree-dimensional cellular organization of this tissue is similar tothose of a young sporophyte (22). The frequency of side branchformation of whatever fate was approximately similar in wildtype and deletion lines, but while the fate of side-branchdevelopment in the wild type, as expected (23), could beeffectively changed from protonema to gametophore by cytoki-nin, the fate of the sporophyte-like side branches in the deletionline remained unchanged on cytokinin (Table S1).

Expression Patterns of MKN4 and PpLFY2 in the Sporophyte-Like BodyAre Similar to Those of the Wild-Type Sporophyte. To examinewhether side-branch initial cells are fated to form sporophyteapical cells instead of protonema or gametophore apical cells, wedeleted the PpCLF gene in the MKN4-GUS-3 (22) and PpLFY2-GUS-1 (24) lines (Fig. S3). In the parental MKN4-GUS-3 line,the MKN4-GUS fusion protein is detected specifically in sporo-phyte apical cells but not in gametophyte apical cells (22). In aPpCLF deletion background, the MKN4-GUS signal was de-tected only in the apical cells of sporophyte-like tissue (Fig. 1G).In the PpLFY2-GUS-1 line, GUS signal is present throughoutthe young sporophyte (24), similar to its appearance in thesporophyte-like tissue in the PpCLF deletion background (Fig.1H). Despite sporophyte-like morphology and marker expres-sion, the DNA content of the sporophyte-like body, measuredwith flow cytometry, indicated a haploid DNA content (Fig. S4).Evidently, the deletion of PpCLF converted side-branch initialsto sporophyte initials, while retaining haploid DNA content.

Sporophyte-like bodies instead of gametophores were alsoformed in the deletion lines of PpFIE (Fig. S5), which is anothercomponent of the PRC2 complex and directly interacts with PpCLF(25). We did not observe any gametophores including their initials,which were reported in the insertion mutant lines (25).

Expression Patterns of PpCLF-Citrine Fusion Protein in Wild Type. Tocharacterize the spatial and temporal expression patterns ofPpCLF, the yellow fluorescent protein, Citrine (26), was recom-bined in frame with the endogenous gene (Fig. S6). The mor-phology of the knock-in plants was indistinguishable from thatof the wild type, indicating that the fusion protein is functional.

The PpCLF-Citrine signal was detected in the nucleus of pro-tonema apical cells (Fig. 2 A and A�), and expression wasmaintained in differentiated protonema cells (Fig. 2 B and B�).When side-branch initial cells formed, the signal was retained inthe new protonema apical cells (Fig. 2 C and C�) and in the newgametophore apical cells (Fig. 2 D and D�). During gametophoredevelopment, signal was detected in gametophore apical cellsand young leaves (Fig. 2 E and E�). Signals in nuclei were alsodetected in gametangia including spermatogenous cells (Fig. 2Fand F�) and sperm (Fig. 2 G and G�). The signal in sperm nuclei

Fig. 1. A PpCLF deletion line (ppclf-del-3) forms sporophyte-like bodies asside branches. (A and B) Wild-type (A) and ppclf-del-3 (B) protonemata grownunder red light for 7 days. Asterisks and arrows indicate apical cells and septa,respectively. (C and D) Protonema of the wild type (C) and ppclf-del-3 (D)grown in white light for 2 days after 7 days of culture in red light. (E and F)Sporophyte-like body formed on the ppclf-del-3 protonema grown underwhite light for 7 days without (E) or with (F) clearing. Arrows indicate theapical cell. (G and H) GUS activity in sporophyte-like tissue of the ppclf/MKN4-GUS-3–1 (G) and ppclf/PpLFY2-GUS-1–1 (H) lines. (I) Sporophyte-like tissue ofppclf-del-3 grown in white light for 14 days after 7 days in red light. [Scale bars,(A–D) 100 �m; (E–I) 50 �m.]

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was weaker than that in spermatogenous cells. In the femalegametangia, Citrine signal was detected in unfertilized egg cells(Fig. 2 H and H�), as well as in surrounding archegonium cells.

However, a Citrine signal was detected neither in the zygote(Fig. 2 I and I�) nor in sporophyte cells while the apical cellactively divides (Fig. 2 J–K�). At this stage, the signal in arche-gonium cells was absent (Fig. 2 J�). After several divisions, thesporophyte apical cell stops dividing whereas the derived cellscontinue dividing to form the sporangium (22). Around the timewhen the sporophyte apical cell stops dividing, a PpCLF-Citrinesignal was detected in all sporophyte nuclei, including thesporophyte apical cell (Fig. 2 L and L�), and remained detectablefor the duration of sporophyte development (Fig. 2 M–Q),except that signal was absent in mature spores (Fig. 2 R and R�).Right after germination, signal reappeared in protonema apicalcells (Fig. 2 S and S�).

These expression patterns are consistent with the hypothe-sized function of PpCLF to repress the formation of sporophyteapical cells in the gametophyte generation. In addition, theexpression pattern suggests that PpCLF is involved in the arrestof the division of sporophyte apical cells and in promoting the

subsequent development of the sporangium. This is consistentwith the PpCLF deletion phenotype, in which a sporophyte-likeapical cell continuously divides, forming an extended sporo-phyte-like body, with no development of a sporangium (Fig. 1I).

Induction of PpCLF in the ppclf Deletion Line. To examine further thefunction of PpCLF, we expressed a PpCLF cDNA in the PpCLFdeletion background. To monitor expression, the cDNA wasfused to the coding sequence of the cyan fluorescent protein,Cerulean (27); to control expression, the cDNA was driven by aheat-shock promoter (28) (HSP-PpCLF-Cerulean/ppclf-del-1and -2 lines; Figs. S7 and S8). Heat shock was not continuous butinstead was given as a 1-hour exposure to 37 °C every 12 h. In theabsence of heat shock, the line was indistinguishable from theparental PpCLF deletion line; in contrast, during cultivation ofthe line under white light at 25 °C with heat-shock treatment forten days, colony morphology became similar to that of the wildtype (Fig. 3 A–D). For detailed examination, protonemata of thelines were cultivated with heat-shock for 7 days under unilateralred light, and then transferred to white light for 2 days, with heatshock. Few of the side branch initials formed sporophyte-like

Fig. 2. PpCLF-Citrine is expressed in protonema and gametophore apical cells but not in sporophyte apical cells. (A and A�) Protonema apical cell. (B and B�)The third protonema cell from the apical cell. (C and C�) Side-branch initial cell. (D and D�) Gametophore stem cell. (E and E�) Gametophore tip. An arrow indicatesthe gametophore apical cell. (F and F�) Young antheridium. (G and G�) Mature antheridium including sperm. (H and H�) Unopened archegonium and an egg cell.(I and I�) Zygote in an opened archegonium. (J–K�) Sporophytes with an active sporophyte apical cell after the first (J and J�) and fifth (K and K�) zygotic cell division.(L–O) Sporophyte once its apical cell had stopped division and sister cells commenced division. (P–Q) Sporangium with archesporial cells. (R and R�) Spore. (S andS�) Germinating spore. (N, O, and Q) show the boxed regions in (M� and P�). Arrows (A�, B�, C�, and H�) indicate a nucleus. For each image pair, left-hand panelis bright-field, right-hand panel is fluorescence, with the citrine signal in green and chloroplasts in magenta or white. Fluorescence of chloroplasts was detectedeven with the barrier filter because the fluorescence of PpCLF-Citrine was relatively weak and the exposure time needed was relatively long. [Scale bars, (A–D�,F–L�, N, O, and Q–S�) 25 �m; (E, E�, M, M�, P, and P�) 100 �m.]

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bodies but instead formed protonemata or gametophores (Fig. 3 Eand F and Table S2). It is noteworthy that gametophores wereinduced in addition to protonemata, whereas only protonemata

formed in the wild type under the same conditions. This indicatesthat PpCLF has an inductive role of gametophore in wild type.

To examine PpCLF functions in gametophores, heat shocktreatment was used to allow gametophores to form, which were thencultivated for several days without further heat shock. When heatshock ceased at an early stage, before leaf formation (Fig. 3F),gametophore development was arrested and several sporophyte-like bodies were formed (Fig. 3G). When heat shock ceased at alater stage, sporophyte-like bodies formed at the gametophore tipbut not from differentiated leaves or stems (Fig. 3H).

Ectopic PpCLF Arrests Proliferation of the Sporophyte-Like Apical Celland Induces a Sporangium-Like Organ. To examine the involvementof PpCLF in the formation of a reproductive organ, the sporan-gium, we isolated sporophyte-like bodies, from the HSP-PpCLF-Cerulean/ppclf-del-1 line, formed at 25 °C and cultivated themsubsequently with heat-shock. The apical cell of the sporophyte-like body stopped dividing and the tissue began to expand (Fig.3I), forming a structure whose outer morphology was similar tothat of the wild-type sporophyte (Fig. 3J). However, the innertissue structure was different from wild type, although cyto-plasm-rich cells similar to archesporial cells were observed (Fig.3 K–N). The heat-shocked material formed neither operculum,columella, nor stomata. Although the growth of the sporophyte-like body ceased, the archesporium-like cells did not entermeiosis or form spores.

Formation of Branched Sporophyte-Like Body. When we continuedthe cultivation of PpCLF deletion lines at 25 °C for severalweeks, new sporophyte-like apical cells initiated below theoriginal apical cell of the sporophyte-like body and grew out asbranches, with MKN4-GUS staining in the apical cells (Fig. 3O).Repeated formation of branches led a bushy morphology ofthese sporophyte-like bodies (Fig. 3P).

DiscussionPpCLF Represses the Initiation of Sporophyte Development in theHaploid Generation and Regulates Apogamy. In the PpCLF deletionlines, sporophyte-like bodies formed as protonemal sidebranches. The apical cell of these bodies had several features thatresembled young wild-type sporophytes, including two cuttingfaces (Fig. 1 E and F), cellular organization (Fig. 1 E and F), andthe expression patterns of MKN4 and PpLFY2 genes (Fig. 1 Gand H). Sporophyte-like bodies also formed on gametophoresafter PpCLF-Cerulean expression was decreased or eliminated(Fig. 3 G and H). In wild-type plants, PpCLF-Citrine fusionprotein was detected in both side branch initial cells andgametophore tips (Fig. 2 C� and E�). Taken together, theseresults indicate that PpCLF functions to repress the earlysporophyte developmental program in these gametophyte cellsand that the loss of PpCLF induces the apogamy.

It appears that PpCLF has other functions in gametophytedevelopment. PpCLF-Citrine was expressed throughout theprotonema, not only in the apical cell, and the average size ofchloroplasts was reduced visibly in the deletion lines (Fig. 1 Aand B). That PpCLF plays an inductive role promoting gameto-phore apical cell fate in side-branch initial cells is indicated bythe inability of cytokinin to induce gametophores in the deletionlines (Table S1) and the increased proportion of gametophoresamong side branches formed on protonemata expressing Pp-CLF-Cerulean (Table S2).

PpCLF Represses the Activity of Sporophyte Apical Cells and InducesReproductive Organ Development. In wild type, the sporophyteapical cell is initiated at the first zygotic cell division and dividesapproximately 12 times (22); derived cells subsequently proliferateand differentiate to form the mature sporophyte. In contrast, theapical cell of the sporophyte-like bodies did not stop dividing and

A B C D

E F

G H I J

K L M N

O P

Fig. 3. A sporangium-like organ formed following to exogenous PpCLF induc-tion inthePpCLFdeletion line. (A–D)Coloniesofwild type (A),ppclf-del-3 (B), andHSP-PpCLF-Cerulean/ppclf-del-2 (C and D) grown in white light for 10 days with(A–C) or without (D) heat-shock treatment. (E) Incipient protonema (arrow) andgametophores (arrowheads) intheHSP-PpCLF-Cerulean/ppclf-del-2 linegrowninwhite light for 2 days after 7 days in red light with heat-shock treatment. (F) Ayoung gametophore before forming leaves in the HSP-PpCLF-Cerulean/ppclf-del-2 line,grownwithheat-shocktreatmentfor7days inred lightandthen3daysin white light. (G and H). Sporophyte-like bodies (arrows) formed in the HSP-PpCLF-Cerulean/ppclf-del-2 line on heat-shock induced gametophores 8 daysafter cessation of heat-shock treatment. A young gametophore without leaves(G) and a leafy gametophore (H). (I) Sporangium-like organ of the HSP-PpCLF-Cerulean/ppclf-del-2 line grown in white light for 10 days with heat-shock treat-ment. (J) Wild-type sporangium. (K–N) Toluidine blue-stained sections of a spo-rangium-like organ of the HSP-PpCLF-Cerulean/ppclf-del-2 line (K and L) and ofwild-type sporangium (M and N). (L and N) show the boxed regions in (K) and (M),respectively. (O) GUS activity in the branched sporophyte-like bodies in theppclf/MKN4-GUS-3–1 line. (P) Sporophyte-like body with numerous branches(ppclf-del-3). [Scale bars, (A–D) 1 mm; (E, F, G, H, K, M, and O) 100 �m; (I and J) 200�m; (L and N) 25 �m; (P) 0.5 mm.]

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thus formed a cylindrical thallus, an indeterminate structure neverobserved in wild type (Fig. 1I). PpCLF-Citrine expression wasdetected neither in zygotes nor in young sporophytes with an activeapical cell, whereas signal was detected concomitantly with thearrest of apical cell proliferation (Fig. 2 L and L�). The induction ofPpCLF-Cerulean in the deletion line arrested the division of theapical cells of the sporophyte-like body (Fig. 3I). Evidently, PpCLFrepresses sporophyte apical cell activity.

When PpCLF-Cerulean was induced in the sporophyte-like bodyof the PpCLF deletion lines and the apical cell was arrested, asporangium-like organ differentiated (Fig. 3I). Given the expres-sion of PpCLF-Citrine during sporangium formation (Fig. 2),PpCLF appears to regulate the change in the sporophyte from anon-reproductive to a reproductive phase, in which meiosis andspore formation proceed. The reason is unclear why the inducedsporangium-like structure ceased developing before meiosis. Oneexplanation is haploidy of the organ, insofar as experimentallyinduced apogamous sporophytes on haploid gametophytes seldomform spores, whereas diploid apogamous sporophytes on apos-porous diploid gametophytes typically do form spores (6). Anotherexplanation is the lack of connection between the sporophyte-likebody and a subtending gametophore, which occurs during wild-typesporophyte development (29).

Involvement of the PRC2 complex in developmental phasechange has also been reported for A. thaliana, in which the VRNand EMF complexes regulate the expression of several transcriptionfactors involved in flowering (30). The PRC2 complex acts as arepressor rather than as an activator in flowering. Future studies ongene networks of the PRC2 complex will give insights on the generaland diversified functions of this gene family.

Evolution of PRC2 and Land Plant Body Plan. Morphological inno-vation is essential to the diversification and adaptation of livingorganisms (31, 32). A sporophyte with long-lasting apical growthand branching is dominant in extant vascular plants, a habit thatevolved from a subordinate, short-lived, and unbranched sporo-phyte, present among the earliest land colonizers (33). However,the morphological intermediates and genetic basis for thisevolutionary process are largely unknown.

The formation of branched body in the PpCLF deletion line(Fig. 3 O and P) gives us evolutionary implications on the earlyevolution of land plants. In mosses, branched sporophytes havebeen reported rarely (34). The presence of a long-lived branchingbody without secondarily thickened xylem (Fig. 3K) is concor-dant with the diagnostic characters of protracheophytes, whichinclude extinct taxa only and are placed between bryophytes andvascular plants (tracheophytes) (34). Branching in the PpCLFdeletion lines prompts us to hypothesize that regulatory net-works among PpCLF and other PRC2-family genes acted on thelongevity of sporophyte apical cells and, at an early stage ofvascular plant evolution, allowed an autonomously branchedsporophyte to form without additional mutations. To verify thishypothesis, studies on the regulatory mechanisms of the branch-ing in the mutant and on PRC2 functions in pteridophytes will

be worthwhile, as will be a comparison of the branching patternsand stem anatomy between the deletion lines and fossil plants.

Materials and MethodsMaterials and Growth Conditions. Physcomitrella patens Bruch & Schimp subsp.patens was cultured on BCDATG or BCDAT medium at 25 °C in white light (35)or unilateral red light (21). For observation of gametangia and sporophytes,protonemata were inoculated onto sterile peat pellets (22).

Generation of Transgenic Plants. Transgenic plants were generated by PEG-mediated transformation according to a published protocol (35). All transfor-mants obtained by gene targeting were verified by DNA gel-blot analyses.

Construction of Plasmids for Heat-Shock Induction. To introduce an exogenousDNA fragment, we searched for genomic regions with no significant similarityto known genes and where neither putative gene models (17) nor ESTs hadbeen assigned. The regions were designated as the P. patens intergenic (PIG)regions. We selected one of the PIG regions (PIG1) located on the scaffold116:413741–411764 of the P. patens subsp. patens v. 1.1 genome (http://genome.jgi-psf.org/Phypa1�1/Phypa1�1.home.html). With genomic DNA astemplate, we amplified two adjacent DNA fragments (PIG1bL and PIG1bR)located in PIG1 using the KSP-PIG1bLf1 and Xh-PIG1Lr1 primers (Table S3 ), andthe Xb-PIG1Rf1 and SSP-PIG1bRr1 primers, respectively. The amplified frag-ments were inserted into pBluescript II SK(�) (Agilent Technologies) at theXhoI and XbaI sites, respectively. The soybean heat-shock inducibleGmhsp17.3B promoter (28), Cerulean (27), the pea rbcS terminator (TrbcS;X01104), and the hygromycin resistance cassette (aphIV cassette: pTN86,AB267705) were inserted into the cloning sites between XhoI and XbaI. Thisplasmid was designated pPIG1HGC (accession number AB472846).

Microscopy. Sporophyte-like tissue was fixed in ethanol/acetic acid and thendehydrated in a graded ethanol series (36). The tissue was examined afterclearing with Hoyer’s solution (36) and observed using a Leica DMLB micro-scope. Histochemical detection of GUS activity was performed as describedpreviously (35). For examining histology, tissue was fixed in 2% glutaralde-hyde and 2.5% paraformaldehyde in 0.1 M phosphate buffer (pH 7.0) for 2 h.The tissue was then dehydrated in a graded ethanol series and embedded inTechnovit 7100 (Heraeus Kulzer). Sections (1.5 �m) were examined afterstaining with 1% toluidine blue in 0.1 M sodium borate (6) and observed usinga DMLB microscope (Leica). The fluorescence of PpCLF-Citrine fusion proteinswas observed using an IX70 microscope (Olympus) equipped with a CSU21spinning disk confocal unit (Yokogawa) and a 488-nm excitation laser. Chlo-rophyll fluorescence was reduced with an additional barrier filter. Imageswere captured with a Cool SNAP HQ camera (Roper Scientific) controlled byMeta Morph ver. 7.1.1.0 software (Molecular Devices). To observe developinggametophores and sporangia, excised tissue was mounted in agar medium ina glass-bottom Petri dish and observed using a FV1000-MPE two-photonmicroscope (Olympus) with a 25� (NA 1.05) water-immersion lens. The inci-dent wavelength was 950 nm. Emission between 495–540 nm and between570–625 nm was separated by a dichroic mirror with band pass filters anddetected by independent detectors. PpCLF-Cerulean fusion protein was ob-served with a BM60 fluorescence microscope (Olympus) using a CFP filter.

ACKNOWLEDGMENTS. We thank S. Nonaka for help with two-photon mi-croscopy; the National Institute of Basic Biology (NIBB) Center for AnalyticalInstruments for DNA sequencing; Futamura Chemical Industries Co., Ltd forthe cellophane; Kyowa Hakko Kogyo Co., Ltd. for driselase; R. Tsien for Citrine;M. Obara, Y. Oguri, S. Wakazuki for technical suggestions and help; T. Kurataand Y. Sato for comments on the manuscript; and T. Baskin for English editing.Computations were done in part at the Computer Lab of NIBB. This researchwas partly supported by grants from the Ministry of Education, Culture,Sports, Science, and Technology of Japan (to T.M., T.N., and M.H.).

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