A Polycomb Repressive Complex 2 Gene Regulates Apogamy and Gives Evolutionary Insights Into Early Land Plant Evolution

A polycomb repressive complex 2 gene regulates apogamy and gives evolutionary insights into early land plant evolution

Yosuke 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, Kanazawa 920-0934, 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 ogy, development, and evolution (5–7), the gene regulatory (gametophyte) and diploid (sporophyte) generations. Although network remains undeciphered. usually the two programs strictly alternate at fertilization and Parthenogenetic development of egg cells has been observed meiosis, one program can be induced during the other program. In in some alleles of loss-of-function mutants of the Arabidopsis a process called apogamy, cells of the gametophyte other than the thaliana genes FERTILIZATION-INDEPENDENT SEED 2 egg cell initiate sporophyte development. Here, we report for the (FIS2), MEDEA (MEA), and MULTICOPY SUPPRESSOR OF moss Physcomitrella patens that apogamy resulted from deletion IRA 1 (MSI1) (8, 9), which encode members of the polycomb of the gene orthologous to the Arabidopsis thaliana CURLY LEAF repressive complex 2 (PRC2) (8, 10, 11). The PRC2 complex was (PpCLF), which encodes a component of polycomb repressive first characterized in Drosophila melanogaster as a regulator of complex 2 (PRC2). In the deletion lines, a gametophytic vegetative HOX genes and includes four core proteins: Extra sex comb cell frequently gave rise to a sporophyte-like body. This body grew (ESC), Enhancer of zeste [E(Z)], Suppressor of zeste 12 indeterminately from an apical cell with the character of a sporo- [SU(Z)12], and P55 (12). Orthologs of PRC2 genes were found EVOLUTION phytic pluripotent stem cell but did not form a sporangium. in a wide range of organisms including land plants and this Furthermore, with continued culture, the sporophyte-like body complex dynamically mediates transcriptional silencing of nu- branched. Sporophyte branching is almost unknown among extant merous genes, based on modifying trimethylation of lysine 27 on bryophytes. When PpCLF was expressed in the deletion lines once histone H3 (12, 13). Several E(Z) and SU(Z)12 paralogs are the sporophyte-like bodies had formed, pluripotent stem cell coded in the A. thaliana genome and at least three distinct activity was arrested and a sporangium-like organ formed. Sup- complexes, with different combinations of the paralogs, have been implicated in plant development: the FIS complex in seed ported by the observed pattern of PpCLF expression, these results development including the prevention of parthenogenesis, the demonstrate that, in the gametophyte, PpCLF represses initiation VERNALIZATION (VRN) complex in vernalization response, of a sporophytic pluripotent stem cell and, in the sporophyte, and the EMBRYONIC FLOWER (EMF) complex in flowering represses that stem cell activity and induces reproductive organ and flower development (14–16). development. In land plants, branching, along with indeterminate We sought to investigate the ancestral function and subse- apical growth and delayed initiation of spore-bearing reproductive quent evolution of the PRC2 complex in land plants, taking organs, were conspicuous innovations for the evolution of a advantage of the genomics (17), the feasibility of gene targeting, dominant sporophyte plant body. Our study provides insights into and the accessible development of the moss, Physcomitrella the role of PRC2 gene regulation for sustaining evolutionary patens. During the course of study, we found that deletion in P. innovation in land plants. patens of the gene orthologous to D. melanogaster E(Z) and A. thaliana CURLY LEAF (CLF)/MEA/SWINGER (SWN)(Pp- branching ͉ PRC2 ͉ protracheophytes CLF) induced unusual sporophyte-like bodies. Here, we report the characterization of the role of the PpCLF gene in P. patens evelopment of land plants starts from a zygote in the development, revealing multiple functions, including the repres- Dsporophyte generation and from a spore in the gametophyte sion of apogamy in the haploid generation, that bear on the generation. Although sporophyte development is usually pre- evolution of body plan in land plants. vented in the gametophyte until fertilization, it can occur naturally and be induced experimentally. Female gametes (egg Results cells) can initiate embryogenesis with parthenogenetic develop- Molecular Cloning of a CURLY LEAF Ortholog in Physcomitrella patens. ment in the absence of fertilization (1, 2). Additionally, in a A candidate cDNA sequence of PpCLF was obtained using the process called apogamy, somatic gametophyte cells are repro- A. thaliana CLF amino acid sequence (18) as the query for a grammed to start a sporophytic developmental program (3). While apogamy can be induced experimentally in flowering Author contributions: Y.O., N.A., Y.H., and M.H. designed research; Y.O., N.A., Y.H., T.M., plants from synergid and antipodal cells, this is rare and not T.N., T.I., and M.H. performed research; M.K. contributed new reagents/analytic tools; Y.O., known to happen naturally (4); in contrast, this type of asexual N.A., Y.H., and M.H. analyzed data; and Y.O., T.M., M.K., and M.H. wrote the paper. reproduction is more widely observed in non-seed plants, in- The authors declare no conﬂict of interest. cluding pteridophytes (5) and bryophytes (6). Bypassing game- This article is a PNAS Direct Submission. togenesis and fertilization, gametophyte somatic cells of non- Data deposition: The sequences reported in this paper have been deposited in the GenBank seed plants divide several times to form a sporophyte apical database (accession nos. AB472766). meristem including a pluripotent stem cell. Apogamy in these 1Y.O. and N.A. contributed equally to this work. species can be induced with exogenous factors, such as hydration, 2To whom correspondence should be addressed. E-mail: [email protected]. sugars, chloral hydrate, and phytohormones (6). Even though This article contains supporting information online at www.pnas.org/cgi/content/full/ apogamy has been well-studied from the viewpoints of physiol- 0906997106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0906997106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 25, 2021 TBLASTN search (19) against the P. patens subsp. patens v. 1.1 genome database. The PpCLF cDNA sequence was obtained by RT-PCR using gene-specific primers. With the sequenced genome (17), we identified PpCLF as the sole homolog of the E(Z) component of PRC2 (Fig. S1). This component is represented in the 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 homologues of the other members of the PRC2 complex (10, 20).

Deletion of PpCLF Gene Induces Sporophyte-Like Body. Using gene targeting, we generated four deletion lines of PpCLF (ppclf-del-1 to -4) (Fig. S2), and the phenotypes of the lines were indistinguishable from each other. Following spore germination in P. patens, a cell filament forms, known as a protonema, whose apical, or tip, cell has the characteristics of a pluripotent stem cell. When grown under red-light (21), in both wild-type and deletion lines, the apical cell continuously produced protonema cells (Fig. 1 A and B), although the chloroplasts in the deletion lines were smaller than those of the wild type. When red-light- grown wild-type protonemata were transferred to white light, the protonema cells formed side-branch initial cells that gave rise to protonema apical cells, which are pluripotent (Fig. 1C). In contrast, when red-light-grown PpCLF deletion lines were moved 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 producing a row of cells, with subsequent periclinal divisions forming inner and outer cell layers (Fig. 1 E and F). The development and three-dimensional cellular organization of this tissue is similar to those of a young sporophyte (22). The frequency of side branch formation of whatever fate was approximately similar in wild type and deletion lines, but while the fate of side-branch development in the wild type, as expected (23), could be effectively changed from protonema to gametophore by cytokinin, the fate of the sporophyte-like side branches in the deletion line remained unchanged on cytokinin (Table S1).

Expression Patterns of MKN4 and PpLFY2 in the Sporophyte-Like Body Are Similar to Those of the Wild-Type Sporophyte. To examine whether side-branch initial cells are fated to form sporophyte apical cells instead of protonema or gametophore apical cells, we deleted 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 sporophyte apical cells but not in gametophyte apical cells (22). In a Fig. 1. A PpCLF deletion line (ppclf-del-3) forms sporophyte-like bodies as PpCLF deletion background, the MKN4-GUS signal was de- side branches. (A and B) Wild-type (A) and ppclf-del-3 (B) protonemata grown tected only in the apical cells of sporophyte-like tissue (Fig. 1G). under red light for 7 days. Asterisks and arrows indicate apical cells and septa, In the PpLFY2-GUS-1 line, GUS signal is present throughout respectively. (C and D) Protonema of the wild type (C) and ppclf-del-3 (D) the young sporophyte (24), similar to its appearance in the grown in white light for 2 days after 7 days of culture in red light. (E and F) sporophyte-like tissue in the PpCLF deletion background (Fig. Sporophyte-like body formed on the ppclf-del-3 protonema grown under 1H). Despite sporophyte-like morphology and marker expres- white light for 7 days without (E) or with (F) clearing. Arrows indicate the sion, the DNA content of the sporophyte-like body, measured apical cell. (G and H) GUS activity in sporophyte-like tissue of the ppclf/MKN4- with flow cytometry, indicated a haploid DNA content (Fig. S4). GUS-3–1 (G) and ppclf/PpLFY2-GUS-1–1 (H) lines. (I) Sporophyte-like tissue of ppclf-del-3 grown in white light for 14 days after 7 days in red light. [Scale bars, Evidently, the deletion of PpCLF converted side-branch initials (A–D) 100 ␮m; (E–I)50␮m.] to sporophyte initials, while retaining haploid DNA content. Sporophyte-like bodies instead of gametophores were also formed in the deletion lines of PpFIE (Fig. S5), which is another component of the PRC2 complex and directly interacts with PpCLF The PpCLF-Citrine signal was detected in the nucleus of protonema apical cells (Fig. 2 A and AЈ), and expression was (25). We did not observe any gametophores including their initials, Ј which were reported in the insertion mutant lines (25). maintained in differentiated protonema cells (Fig. 2 B and B ). When side-branch initial cells formed, the signal was retained in Ј Expression Patterns of PpCLF-Citrine Fusion Protein in Wild Type. To the new protonema apical cells (Fig. 2 C and C ) and in the new characterize the spatial and temporal expression patterns of gametophore apical cells (Fig. 2 D and DЈ). During gametophore PpCLF, the yellow fluorescent protein, Citrine (26), was recom- development, signal was detected in gametophore apical cells bined in frame with the endogenous gene (Fig. S6). The mor- and young leaves (Fig. 2 E and EЈ). Signals in nuclei were also phology of the knock-in plants was indistinguishable from that detected in gametangia including spermatogenous cells (Fig. 2F of the wild type, indicating that the fusion protein is functional. and FЈ) and sperm (Fig. 2 G and GЈ). The signal in sperm nuclei

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0906997106 Okano et al. Downloaded by guest on September 25, 2021 EVOLUTION

Fig. 2. PpCLF-Citrine is expressed in protonema and gametophore apical cells but not in sporophyte apical cells. (A and AЈ) Protonema apical cell. (B and BЈ) The third protonema cell from the apical cell. (C and CЈ) Side-branch initial cell. (D and DЈ) Gametophore stem cell. (E and EЈ) Gametophore tip. An arrow indicates the gametophore apical cell. (F and FЈ) Young antheridium. (G and GЈ) Mature antheridium including sperm. (H and HЈ) Unopened archegonium and an egg cell. (I and IЈ) Zygote in an opened archegonium. (J–KЈ) Sporophytes with an active sporophyte apical cell after the first (J and JЈ) and fifth (K and KЈ) 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 RЈ) Spore. (S and SЈ) Germinating spore. (N, O, and Q) show the boxed regions in (MЈ and PЈ). Arrows (AЈ, BЈ, CЈ, and HЈ) indicate a nucleus. For each image pair, left-hand panel is bright-field, right-hand panel is fluorescence, with the citrine signal in green and chloroplasts in magenta or white. Fluorescence of chloroplasts was detected even with the barrier filter because the fluorescence of PpCLF-Citrine was relatively weak and the exposure time needed was relatively long. [Scale bars, (A–DЈ, F–LЈ, N, O, and Q–SЈ)25␮m; (E, EЈ, M, MЈ, P, and PЈ) 100 ␮m.]

was weaker than that in spermatogenous cells. In the female subsequent development of the sporangium. This is consistent gametangia, Citrine signal was detected in unfertilized egg cells with the PpCLF deletion phenotype, in which a sporophyte-like (Fig. 2 H and HЈ), as well as in surrounding archegonium cells. apical cell continuously divides, forming an extended sporo- However, a Citrine signal was detected neither in the zygote phyte-like body, with no development of a sporangium (Fig. 1I). (Fig. 2 I and IЈ) nor in sporophyte cells while the apical cell actively divides (Fig. 2 J–KЈ). At this stage, the signal in arche- Induction of PpCLF in the ppclf Deletion Line. To examine further the gonium cells was absent (Fig. 2 JЈ). After several divisions, the function of PpCLF, we expressed a PpCLF cDNA in the PpCLF sporophyte apical cell stops dividing whereas the derived cells deletion background. To monitor expression, the cDNA was continue dividing to form the sporangium (22). Around the time fused to the coding sequence of the cyan fluorescent protein, when the sporophyte apical cell stops dividing, a PpCLF-Citrine Cerulean (27); to control expression, the cDNA was driven by a signal was detected in all sporophyte nuclei, including the heat-shock promoter (28) (HSP-PpCLF-Cerulean/ppclf-del-1 sporophyte apical cell (Fig. 2 L and LЈ), and remained detectable and -2 lines; Figs. S7 and S8). Heat shock was not continuous but for the duration of sporophyte development (Fig. 2 M–Q), instead was given as a 1-hour exposure to 37 °C every 12 h. In the except that signal was absent in mature spores (Fig. 2 R and RЈ). absence of heat shock, the line was indistinguishable from the Right after germination, signal reappeared in protonema apical parental PpCLF deletion line; in contrast, during cultivation of cells (Fig. 2 S and SЈ). the line under white light at 25 °C with heat-shock treatment for These expression patterns are consistent with the hypothe- ten days, colony morphology became similar to that of the wild sized function of PpCLF to repress the formation of sporophyte type (Fig. 3 A–D). For detailed examination, protonemata of the apical cells in the gametophyte generation. In addition, the lines were cultivated with heat-shock for 7 days under unilateral expression pattern suggests that PpCLF is involved in the arrest red light, and then transferred to white light for 2 days, with heat of the division of sporophyte apical cells and in promoting the shock. Few of the side branch initials formed sporophyte-like

Okano et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 25, 2021 ABCD formed in the wild type under the same conditions. This indicates that PpCLF has an inductive role of gametophore in wild type. To examine PpCLF functions in gametophores, heat shock treatment was used to allow gametophores to form, which were then cultivated for several days without further heat shock. When heat shock ceased at an early stage, before leaf formation (Fig. 3F), E F gametophore development was arrested and several sporophyte- like bodies were formed (Fig. 3G). When heat shock ceased at a later stage, sporophyte-like bodies formed at the gametophore tip but not from differentiated leaves or stems (Fig. 3H).

Ectopic PpCLF Arrests Proliferation of the Sporophyte-Like Apical Cell and Induces a Sporangium-Like Organ. To examine the involvement of PpCLF in the formation of a reproductive organ, the sporan- G H I J gium, we isolated sporophyte-like bodies, from the HSP-PpCLF- Cerulean/ppclf-del-1 line, formed at 25 °C and cultivated them subsequently 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 to that of the wild-type sporophyte (Fig. 3J). However, the inner tissue 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- L N like body ceased, the archesporium-like cells did not enter K M meiosis or form spores.

Formation of Branched Sporophyte-Like Body. When we continued the cultivation of PpCLF deletion lines at 25 °C for several weeks, new sporophyte-like apical cells initiated below the original apical cell of the sporophyte-like body and grew out as branches, with MKN4-GUS staining in the apical cells (Fig. 3O). Repeated formation of branches led a bushy morphology of these sporophyte-like bodies (Fig. 3P). O P Discussion PpCLF Represses the Initiation of Sporophyte Development in the Haploid Generation and Regulates Apogamy. In the PpCLF deletion lines, sporophyte-like bodies formed as protonemal side branches. The apical cell of these bodies had several features that resembled young wild-type sporophytes, including two cutting faces (Fig. 1 E and F), cellular organization (Fig. 1 E and F), and the expression patterns of MKN4 and PpLFY2 genes (Fig. 1 G and H). Sporophyte-like bodies also formed on gametophores after PpCLF-Cerulean expression was decreased or eliminated Fig. 3. A sporangium-like organ formed following to exogenous PpCLF induc- (Fig. 3 G and H). In wild-type plants, PpCLF-Citrine fusion tion in the PpCLF deletion line. (A–D) Colonies of wild type (A), ppclf-del-3 (B), and protein was detected in both side branch initial cells and HSP-PpCLF-Cerulean/ppclf-del-2 (C and D) grown in white light for 10 days with gametophore tips (Fig. 2 CЈ and EЈ). Taken together, these (A–C) or without (D) heat-shock treatment. (E) Incipient protonema (arrow) and results indicate that PpCLF functions to repress the early gametophores (arrowheads) in the HSP-PpCLF-Cerulean/ppclf-del-2 line grown in sporophyte developmental program in these gametophyte cells white light for 2 days after 7 days in red light with heat-shock treatment. (F)A and that the loss of PpCLF induces the apogamy. young gametophore before forming leaves in the HSP-PpCLF-Cerulean/ppclf- del-2 line, grown with heat-shock treatment for 7 days in red light and then 3 days It appears that PpCLF has other functions in gametophyte in white light. (G and H). Sporophyte-like bodies (arrows) formed in the HSP- development. PpCLF-Citrine was expressed throughout the PpCLF-Cerulean/ppclf-del-2 line on heat-shock induced gametophores 8 days protonema, not only in the apical cell, and the average size of after cessation of heat-shock treatment. A young gametophore without leaves chloroplasts was reduced visibly in the deletion lines (Fig. 1 A (G) and a leafy gametophore (H). (I) Sporangium-like organ of the HSP-PpCLF- and B). That PpCLF plays an inductive role promoting gameto- Cerulean/ppclf-del-2 line grown in white light for 10 days with heat-shock treat- phore apical cell fate in side-branch initial cells is indicated by ment. (J) Wild-type sporangium. (K–N) Toluidine blue-stained sections of a spo- the inability of cytokinin to induce gametophores in the deletion rangium-like organ of the HSP-PpCLF-Cerulean/ppclf-del-2 line (K and L) and of lines (Table S1) and the increased proportion of gametophores wild-type sporangium (M and N). (L and N) show the boxed regions in (K) and (M), among side branches formed on protonemata expressing Pp- respectively. (O) GUS activity in the branched sporophyte-like bodies in the ppclf/MKN4-GUS-3–1 line. (P) Sporophyte-like body with numerous branches CLF-Cerulean (Table S2). (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.] PpCLF Represses the Activity of Sporophyte Apical Cells and Induces Reproductive Organ Development. In wild type, the sporophyte apical cell is initiated at the first zygotic cell division and divides bodies but instead formed protonemata or gametophores (Fig. 3 E approximately 12 times (22); derived cells subsequently proliferate and F and Table S2). It is noteworthy that gametophores were and differentiate to form the mature sporophyte. In contrast, the induced in addition to protonemata, whereas only protonemata apical cell of the sporophyte-like bodies did not stop dividing and

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0906997106 Okano et al. Downloaded by guest on September 25, 2021 thus formed a cylindrical thallus, an indeterminate structure never be worthwhile, as will be a comparison of the branching patterns observed in wild type (Fig. 1I). PpCLF-Citrine expression was and stem anatomy between the deletion lines and fossil plants. detected neither in zygotes nor in young sporophytes with an active apical cell, whereas signal was detected concomitantly with the Materials and Methods arrest of apical cell proliferation (Fig. 2 L and LЈ). The induction of Materials and Growth Conditions. Physcomitrella patens Bruch & Schimp subsp. PpCLF-Cerulean in the deletion line arrested the division of the patens was cultured on BCDATG or BCDAT medium at 25 °C in white light (35) I or unilateral red light (21). For observation of gametangia and sporophytes, apical cells of the sporophyte-like body (Fig. 3 ). Evidently, PpCLF protonemata were inoculated onto sterile peat pellets (22). represses sporophyte apical cell activity. When PpCLF-Cerulean was induced in the sporophyte-like body Generation of Transgenic Plants. Transgenic plants were generated by PEG- of the PpCLF deletion lines and the apical cell was arrested, a mediated transformation according to a published protocol (35). All transfor- sporangium-like organ differentiated (Fig. 3I). Given the expres- mants obtained by gene targeting were verified by DNA gel-blot analyses. sion of PpCLF-Citrine during sporangium formation (Fig. 2), PpCLF appears to regulate the change in the sporophyte from a Construction of Plasmids for Heat-Shock Induction. To introduce an exogenous non-reproductive to a reproductive phase, in which meiosis and DNA fragment, we searched for genomic regions with no significant similarity spore formation proceed. The reason is unclear why the induced to known genes and where neither putative gene models (17) nor ESTs had been assigned. The regions were designated as the P. patens intergenic (PIG) sporangium-like structure ceased developing before meiosis. One regions. We selected one of the PIG regions (PIG1) located on the scaffold explanation is haploidy of the organ, insofar as experimentally 116:413741–411764 of the P. patens subsp. patens v. 1.1 genome (http:// induced apogamous sporophytes on haploid gametophytes seldom genome.jgi-psf.org/Phypa1࿝1/Phypa1࿝1.home.html). With genomic DNA as form spores, whereas diploid apogamous sporophytes on apos- template, we amplified two adjacent DNA fragments (PIG1bL and PIG1bR) porous diploid gametophytes typically do form spores (6). Another located in PIG1 using the KSP-PIG1bLf1 and Xh-PIG1Lr1 primers (Table S3 ), and the Xb-PIG1Rf1 and SSP-PIG1bRr1 primers, respectively. The amplified frag- explanation is the lack of connection between the sporophyte-like ϩ body and a subtending gametophore, which occurs during wild-type ments were inserted into pBluescript II SK( ) (Agilent Technologies) at the XhoI and XbaI sites, respectively. The soybean heat-shock inducible sporophyte development (29). Gmhsp17.3B promoter (28), Cerulean (27), the pea rbcS terminator (TrbcS; Involvement of the PRC2 complex in developmental phase X01104), and the hygromycin resistance cassette (aphIV cassette: pTN86, change has also been reported for A. thaliana, in which the VRN AB267705) were inserted into the cloning sites between XhoI and XbaI. This and EMF complexes regulate the expression of several transcription plasmid was designated pPIG1HGC (accession number AB472846).

factors involved in flowering (30). The PRC2 complex acts as a EVOLUTION repressor rather than as an activator in flowering. Future studies on Microscopy. Sporophyte-like tissue was fixed in ethanol/acetic acid and then gene networks of the PRC2 complex will give insights on the general dehydrated in a graded ethanol series (36). The tissue was examined after clearing with Hoyer’s solution (36) and observed using a Leica DMLB micro- and diversified functions of this gene family. scope. Histochemical detection of GUS activity was performed as described previously (35). For examining histology, tissue was fixed in 2% glutaralde- Evolution of PRC2 and Land Plant Body Plan. Morphological inno- hyde and 2.5% paraformaldehyde in 0.1 M phosphate buffer (pH 7.0) for 2 h. vation is essential to the diversification and adaptation of living The tissue was then dehydrated in a graded ethanol series and embedded in organisms (31, 32). A sporophyte with long-lasting apical growth Technovit 7100 (Heraeus Kulzer). Sections (1.5 ␮m) were examined after and branching is dominant in extant vascular plants, a habit that staining with 1% toluidine blue in 0.1 M sodium borate (6) and observed using evolved from a subordinate, short-lived, and unbranched sporo- a DMLB microscope (Leica). The fluorescence of PpCLF-Citrine fusion proteins was observed using an IX70 microscope (Olympus) equipped with a CSU21 phyte, present among the earliest land colonizers (33). However, spinning disk confocal unit (Yokogawa) and a 488-nm excitation laser. Chlo- the morphological intermediates and genetic basis for this rophyll fluorescence was reduced with an additional barrier filter. Images evolutionary process are largely unknown. were captured with a Cool SNAP HQ camera (Roper Scientific) controlled by The formation of branched body in the PpCLF deletion line Meta Morph ver. 7.1.1.0 software (Molecular Devices). To observe developing (Fig. 3 O and P) gives us evolutionary implications on the early gametophores and sporangia, excised tissue was mounted in agar medium in evolution of land plants. In mosses, branched sporophytes have a glass-bottom Petri dish and observed using a FV1000-MPE two-photon microscope (Olympus) with a 25ϫ (NA 1.05) water-immersion lens. The inci- been reported rarely (34). The presence of a long-lived branching dent wavelength was 950 nm. Emission between 495–540 nm and between body without secondarily thickened xylem (Fig. 3K) is concor- 570–625 nm was separated by a dichroic mirror with band pass filters and dant with the diagnostic characters of protracheophytes, which detected by independent detectors. PpCLF-Cerulean fusion protein was ob- include extinct taxa only and are placed between bryophytes and served with a BM60 fluorescence microscope (Olympus) using a CFP filter. vascular plants (tracheophytes) (34). Branching in the PpCLF deletion lines prompts us to hypothesize that regulatory net- ACKNOWLEDGMENTS. We thank S. Nonaka for help with two-photon microscopy; the National Institute of Basic Biology (NIBB) Center for Analytical works among PpCLF and other PRC2-family genes acted on the Instruments for DNA sequencing; Futamura Chemical Industries Co., Ltd for longevity of sporophyte apical cells and, at an early stage of the cellophane; Kyowa Hakko Kogyo Co., Ltd. for driselase; R. Tsien for Citrine; vascular plant evolution, allowed an autonomously branched M. Obara, Y. Oguri, S. Wakazuki for technical suggestions and help; T. Kurata sporophyte to form without additional mutations. To verify this and 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 research hypothesis, studies on the regulatory mechanisms of the branch- was partly supported by grants from the Ministry of Education, Culture, ing in the mutant and on PRC2 functions in pteridophytes will Sports, Science, and Technology of Japan (to T.M., T.N., and M.H.).

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