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Proc Natl Acad Sci U S A. Sep 22, 2009; 106(38): 16321–16326.
Published online Sep 9, 2009. doi:  10.1073/pnas.0906997106
PMCID: PMC2752547
Evolution

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

Abstract

Land plants have distinct developmental programs in haploid (gametophyte) and diploid (sporophyte) generations. Although usually the two programs strictly alternate at fertilization and meiosis, one program can be induced during the other program. In a process called apogamy, cells of the gametophyte other than the egg cell initiate sporophyte development. Here, we report for the moss Physcomitrella patens that apogamy resulted from deletion of the gene orthologous to the Arabidopsis thaliana CURLY LEAF (PpCLF), which encodes a component of polycomb repressive complex 2 (PRC2). In the deletion lines, a gametophytic vegetative cell frequently gave rise to a sporophyte-like body. This body grew indeterminately from an apical cell with the character of a sporophytic pluripotent stem cell but did not form a sporangium. Furthermore, with continued culture, the sporophyte-like body branched. Sporophyte branching is almost unknown among extant bryophytes. When PpCLF was expressed in the deletion lines once the sporophyte-like bodies had formed, pluripotent stem cell activity was arrested and a sporangium-like organ formed. Supported by the observed pattern of PpCLF expression, these results demonstrate that, in the gametophyte, PpCLF represses initiation of a sporophytic pluripotent stem cell and, in the sporophyte, represses that stem cell activity and induces reproductive organ development. In land plants, branching, along with indeterminate apical growth and delayed initiation of spore-bearing reproductive organs, were conspicuous innovations for the evolution of a dominant sporophyte plant body. Our study provides insights into the role of PRC2 gene regulation for sustaining evolutionary innovation in land plants.

Keywords: branching, PRC2, protracheophytes

Development of land plants starts from a zygote in the sporophyte generation and from a spore in the gametophyte generation. Although sporophyte development is usually prevented in the gametophyte until fertilization, it can occur naturally and be induced experimentally. Female gametes (egg cells) can initiate embryogenesis with parthenogenetic development in the absence of fertilization (1, 2). Additionally, in a process called apogamy, somatic gametophyte cells are reprogrammed to start a sporophytic developmental program (3). While apogamy can be induced experimentally in flowering plants from synergid and antipodal cells, this is rare and not known to happen naturally (4); in contrast, this type of asexual reproduction is more widely observed in non-seed plants, including pteridophytes (5) and bryophytes (6). Bypassing gametogenesis and fertilization, gametophyte somatic cells of non-seed plants divide several times to form a sporophyte apical meristem including a pluripotent stem cell. Apogamy in these species can be induced with exogenous factors, such as hydration, sugars, chloral hydrate, and phytohormones (6). Even though apogamy has been well-studied from the viewpoints of physiology, development, and evolution (57), the gene regulatory network remains undeciphered.

Parthenogenetic development of egg cells has been observed in some alleles of loss-of-function mutants of the Arabidopsis thaliana genes FERTILIZATION-INDEPENDENT SEED 2 (FIS2), MEDEA (MEA), and MULTICOPY SUPPRESSOR OF IRA 1 (MSI1) (8, 9), which encode members of the polycomb repressive complex 2 (PRC2) (8, 10, 11). The PRC2 complex was first characterized in Drosophila melanogaster as a regulator of HOX 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 found in a wide range of organisms including land plants and this complex dynamically mediates transcriptional silencing of numerous genes, based on modifying trimethylation of lysine 27 on histone H3 (12, 13). Several E(Z) and SU(Z)12 paralogs are coded in the A. thaliana genome and at least three distinct complexes, with different combinations of the paralogs, have been implicated in plant development: the FIS complex in seed development including the prevention of parthenogenesis, the VERNALIZATION (VRN) complex in vernalization response, and the EMBRYONIC FLOWER (EMF) complex in flowering and flower development (1416).

We sought to investigate the ancestral function and subsequent evolution of the PRC2 complex in land plants, taking advantage of the genomics (17), the feasibility of gene targeting, and the accessible development of the moss, Physcomitrella patens. 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) (PpCLF) induced unusual sporophyte-like bodies. Here, we report the characterization of the role of the PpCLF gene in P. patens development, revealing multiple functions, including the repression of apogamy in the haploid generation, that bear on the evolution of body plan in land plants.

Results

Molecular Cloning of a CURLY LEAF Ortholog in Physcomitrella patens.

A candidate cDNA sequence of PpCLF was obtained using the A. thaliana CLF amino acid sequence (18) as the query for a 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).

Fig. 1.
A PpCLF deletion line (ppclf-del-3) forms sporophyte-like bodies as side branches. (A and B) Wild-type (A) and ppclf-del-3 (B) protonemata grown under red light for 7 days. Asterisks and arrows indicate apical cells and septa, respectively. (C and D) ...

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 PpCLF deletion background, the MKN4-GUS signal was detected only in the apical cells of sporophyte-like tissue (Fig. 1G). In the PpLFY2-GUS-1 line, GUS signal is present throughout the young sporophyte (24), similar to its appearance in the sporophyte-like tissue in the PpCLF deletion background (Fig. 1H). Despite sporophyte-like morphology and marker expression, the DNA content of the sporophyte-like body, measured with flow cytometry, indicated a haploid DNA content (Fig. S4). Evidently, the deletion of PpCLF converted side-branch initials 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 (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.

To characterize the spatial and temporal expression patterns of PpCLF, the yellow fluorescent protein, Citrine (26), was recombined in frame with the endogenous gene (Fig. S6). The morphology of the knock-in plants was indistinguishable from that of the wild type, indicating that the fusion protein is functional. The PpCLF-Citrine signal was detected in the nucleus of protonema apical cells (Fig. 2 A and A′), and expression was maintained in differentiated protonema cells (Fig. 2 B and B′). When side-branch initial cells formed, the signal was retained in the new protonema apical cells (Fig. 2 C and C′) and in the new gametophore apical cells (Fig. 2 D and D′). During gametophore development, signal was detected in gametophore apical cells and young leaves (Fig. 2 E and E′). Signals in nuclei were also detected in gametangia including spermatogenous cells (Fig. 2 F and F′) and sperm (Fig. 2 G and G′). The signal in sperm nuclei was weaker than that in spermatogenous cells. In the female gametangia, Citrine signal was detected in unfertilized egg cells (Fig. 2 H and H′), as well as in surrounding archegonium cells.

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 ...

However, a Citrine signal was detected neither in the zygote (Fig. 2 I and I′) nor in sporophyte cells while the apical cell actively divides (Fig. 2 J–K′). At this stage, the signal in archegonium cells was absent (Fig. 2J′). After several divisions, the sporophyte apical cell stops dividing whereas the derived cells continue dividing to form the sporangium (22). Around the time when the sporophyte apical cell stops dividing, a PpCLF-Citrine signal was detected in all sporophyte nuclei, including the sporophyte apical cell (Fig. 2 L and L′), and remained detectable for 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 apical cells (Fig. 2 S and S′).

These expression patterns are consistent with the hypothesized function of PpCLF to repress the formation of sporophyte apical cells in the gametophyte generation. In addition, the expression pattern suggests that PpCLF is involved in the arrest of the division of sporophyte apical cells and in promoting the subsequent development of the sporangium. This is consistent with the PpCLF deletion phenotype, in which a sporophyte-like apical cell continuously divides, forming an extended sporophyte-like body, with no development of a sporangium (Fig. 1I).

Induction of PpCLF in the ppclf Deletion Line.

To examine further the function of PpCLF, we expressed a PpCLF cDNA in the PpCLF deletion background. To monitor expression, the cDNA was fused to the coding sequence of the cyan fluorescent protein, Cerulean (27); to control expression, the cDNA was driven by a heat-shock promoter (28) (HSP-PpCLF-Cerulean/ppclf-del-1 and -2 lines; Figs. S7 and S8). Heat shock was not continuous but instead was given as a 1-hour exposure to 37 °C every 12 h. In the absence of heat shock, the line was indistinguishable from the parental PpCLF deletion line; in contrast, during cultivation of the line under white light at 25 °C with heat-shock treatment for ten days, colony morphology became similar to that of the wild type (Fig. 3 A–D). For detailed examination, protonemata of the lines were cultivated with heat-shock for 7 days under unilateral red light, and then transferred to white light for 2 days, with heat shock. Few of the side branch initials formed sporophyte-like bodies but instead formed protonemata or gametophores (Fig. 3 E and F and Table S2). It is noteworthy that gametophores were induced in addition to protonemata, whereas only protonemata formed in the wild type under the same conditions. This indicates that PpCLF has an inductive role of gametophore in wild type.

Fig. 3.
A sporangium-like organ formed following to exogenous PpCLF induction in the PpCLF deletion line. (A–D) Colonies of wild type (A), ppclf-del-3 (B), and HSP-PpCLF-Cerulean/ppclf-del-2 (C and D) grown in white light for 10 days with (A–C ...

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), 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 sporangium, 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 cytoplasm-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 enter 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).

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 G and H). In wild-type plants, PpCLF-Citrine fusion protein was detected in both side branch initial cells and gametophore tips (Fig. 2 C′ and E′). Taken together, these results indicate that PpCLF functions to repress the early sporophyte developmental program in these gametophyte cells and that the loss of PpCLF induces the apogamy.

It appears that PpCLF has other functions in gametophyte development. PpCLF-Citrine was expressed throughout the protonema, not only in the apical cell, and the average size of chloroplasts was reduced visibly in the deletion lines (Fig. 1 A and B). That PpCLF plays an inductive role promoting gametophore apical cell fate in side-branch initial cells is indicated by the inability of cytokinin to induce gametophores in the deletion lines (Table S1) and the increased proportion of gametophores among side branches formed on protonemata expressing PpCLF-Cerulean (Table S2).

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 approximately 12 times (22); derived cells subsequently proliferate and differentiate to form the mature sporophyte. In contrast, the apical cell of the sporophyte-like bodies did not stop dividing and thus formed a cylindrical thallus, an indeterminate structure never observed in wild type (Fig. 1I). PpCLF-Citrine expression was detected neither in zygotes nor in young sporophytes with an active apical cell, whereas signal was detected concomitantly with the arrest of apical cell proliferation (Fig. 2 L and L′). The induction of PpCLF-Cerulean in the deletion line arrested the division of the apical cells of the sporophyte-like body (Fig. 3I). Evidently, PpCLF represses sporophyte apical cell activity.

When PpCLF-Cerulean was induced in the sporophyte-like body of the PpCLF deletion lines and the apical cell was arrested, a sporangium-like organ differentiated (Fig. 3I). Given the expression of PpCLF-Citrine during sporangium formation (Fig. 2), PpCLF appears to regulate the change in the sporophyte from a non-reproductive to a reproductive phase, in which meiosis and spore formation proceed. The reason is unclear why the induced sporangium-like structure ceased developing before meiosis. One explanation is haploidy of the organ, insofar as experimentally induced apogamous sporophytes on haploid gametophytes seldom form spores, whereas diploid apogamous sporophytes on aposporous diploid gametophytes typically do form spores (6). Another explanation is the lack of connection between the sporophyte-like body and a subtending gametophore, which occurs during wild-type sporophyte development (29).

Involvement of the PRC2 complex in developmental phase change has also been reported for A. thaliana, in which the VRN and EMF complexes regulate the expression of several transcription factors involved in flowering (30). The PRC2 complex acts as a repressor rather than as an activator in flowering. Future studies on gene networks of the PRC2 complex will give insights on the general and diversified functions of this gene family.

Evolution of PRC2 and Land Plant Body Plan.

Morphological innovation is essential to the diversification and adaptation of living organisms (31, 32). A sporophyte with long-lasting apical growth and branching is dominant in extant vascular plants, a habit that evolved from a subordinate, short-lived, and unbranched sporophyte, present among the earliest land colonizers (33). However, the morphological intermediates and genetic basis for this evolutionary 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 early evolution of land plants. In mosses, branched sporophytes have been reported rarely (34). The presence of a long-lived branching body without secondarily thickened xylem (Fig. 3K) is concordant with the diagnostic characters of protracheophytes, which include extinct taxa only and are placed between bryophytes and vascular plants (tracheophytes) (34). Branching in the PpCLF deletion lines prompts us to hypothesize that regulatory networks among PpCLF and other PRC2-family genes acted on the longevity of sporophyte apical cells and, at an early stage of vascular plant evolution, allowed an autonomously branched sporophyte to form without additional mutations. To verify this hypothesis, studies on the regulatory mechanisms of the branching in the mutant and on PRC2 functions in pteridophytes will be worthwhile, as will be a comparison of the branching patterns and stem anatomy between the deletion lines and fossil plants.

Materials and Methods

Materials 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 transformants obtained by gene targeting were verified by DNA gel-blot analyses.

Construction of Plasmids for Heat-Shock Induction.

To introduce an exogenous DNA fragment, we searched for genomic regions with no significant similarity 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) regions. We selected one of the PIG regions (PIG1) located on the scaffold 116:413741–411764 of the P. patens subsp. patens v. 1.1 genome (http://genome.jgi-psf.org/Phypa1_1/Phypa1_1.home.html). With genomic DNA as template, we amplified two adjacent DNA fragments (PIG1bL and PIG1bR) located in PIG1 using the KSP-PIG1bLf1 and Xh-PIG1Lr1 primers (TableS3), and the Xb-PIG1Rf1 and SSP-PIG1bRr1 primers, respectively. The amplified fragments were inserted into pBluescript II SK(+) (Agilent Technologies) at the XhoI and XbaI sites, respectively. The soybean heat-shock inducible Gmhsp17.3B 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. This plasmid was designated pPIG1HGC (accession number AB472846).

Microscopy.

Sporophyte-like tissue was fixed in ethanol/acetic acid and then dehydrated in a graded ethanol series (36). The tissue was examined after clearing with Hoyer's solution (36) and observed using a Leica DMLB microscope. Histochemical detection of GUS activity was performed as described previously (35). For examining histology, tissue was fixed in 2% glutaraldehyde 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 in Technovit 7100 (Heraeus Kulzer). Sections (1.5 μm) were examined after staining with 1% toluidine blue in 0.1 M sodium borate (6) and observed using a DMLB microscope (Leica). The fluorescence of PpCLF-Citrine fusion proteins was observed using an IX70 microscope (Olympus) equipped with a CSU21 spinning disk confocal unit (Yokogawa) and a 488-nm excitation laser. Chlorophyll fluorescence was reduced with an additional barrier filter. Images were captured with a Cool SNAP HQ camera (Roper Scientific) controlled by Meta Morph ver. 7.1.1.0 software (Molecular Devices). To observe developing gametophores and sporangia, excised tissue was mounted in agar medium in a glass-bottom Petri dish and observed using a FV1000-MPE two-photon microscope (Olympus) with a 25× (NA 1.05) water-immersion lens. The incident wavelength was 950 nm. Emission between 495–540 nm and between 570–625 nm was separated by a dichroic mirror with band pass filters and detected by independent detectors. PpCLF-Cerulean fusion protein was observed with a BM60 fluorescence microscope (Olympus) using a CFP filter.

Supplementary Material

Supporting Information:

Acknowledgments.

We thank S. Nonaka for help with two-photon microscopy; the National Institute of Basic Biology (NIBB) Center for Analytical Instruments for DNA sequencing; Futamura Chemical Industries Co., Ltd for the cellophane; Kyowa Hakko Kogyo Co., Ltd. for driselase; R. Tsien for Citrine; M. Obara, Y. Oguri, S. Wakazuki for technical suggestions and help; T. Kurata 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 was partly supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to T.M., T.N., and M.H.).

Footnotes

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 GenBank database (accession nos. AB472766).

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

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