Plant and Cell Physiology Advance Access published July 29, 2015
Kimitsune Ishizaki1,*, Ryuichi Nishihama2, Katsuyuki T. Yamato3 and Takayuki Kohchi2 1
Graduate School of Science, Kobe University, Kobe 657-8501 Japan Graduate School of Biostudies, Kyoto University, Kyoto, 606-8502 Japan 3 Faculty of Biology-Oriented Science and Technology, Kinki University, Wakayama, 649-6493 Japan 2
*Corresponding author: E-mail, [email protected]; Fax, +81-78-803-5727. (Received March 7, 2015; Accepted June 18, 2015)
Keywords: Bryophytes Evolution Evo-devo Molecular genetics. Abbreviations: CaMV, Cauliflower mosaic virus; Cas9, CRISPR-associated protein 9; CRISPR, clustered regulatory interspaced short palindromic repeats; FR, far-red light; GFP, green fluorescent protein; GUS, b-glucuronidase; HR, homologous recombination; MpEF1a, Marchantia polymorpha ELONGATION FACTOR 1a; R, red light.
Introduction Land plants comprise a monophyletic group that emerged from freshwater green algae around 500 million years ago. Bryophytes, consisting of liverworts, mosses and hornworts, represent the earliest diverging group of land plants.
As embryophyta, bryophytes have multicellular bodies in the sporophyte generation and undergo alternation of generations. Both the gametophyte and sporophyte generations of land plants have undergone major morphological and physiological changes during the course of evolution. Bryophytes, as basal land plants, have features distinct from those of the other land plant lineages, such as the lack of a vascular system, the absence of a lignified cell wall, the use of motile sperm for fertilization and dominance of the haploid gametophyte generation over the diploid sporophyte generation during their life cycle. When and how did land plants acquire the genetic systems that led to such substantial changes? Recent advances of genome analyses in various lineages of land plants, including angiosperms, gymnosperms, lycophytes and mosses, have revealed that many of their regulatory gene families are conserved (Floyd and Bowman 2007, Rensing et al. 2008, Banks et al. 2011, Nystedt et al. 2013). Recently, it was suggested that many of the fundamental features of land plants appeared in the gametophytes of bryophytes first, and were then co-opted to the sporophytes in vascular plants (Ligrone et al. 2012, Pires and Dolan 2012). Therefore, the molecular biology of basal plant lineages has received more attention, especially to determine the fundamental mechanisms and principles common to land plants, as well as characteristics specific to bryophytes. In bryophytes, while the phylogenic relationship between liverworts, mosses and hornworts is still debated, the critical position of liverworts as one of the earliest land plant lineages is unequivocal (Qiu et al. 2006, Chang and Graham 2011, Wicket et al. 2014). Therefore, along with mosses and hornworts, liverworts are a key group in comparative genomics to address fundamental questions in plant biology, such as the genetic basis of the key innovations that allowed land plants to evolve from aquatic ancestors and to adapt to life on land, the developmental genetic changes responsible for the multicellularity, and the alterations in body plan within land plants (Bowman et al. 2007). Marchantia polymorpha is a widespread liverwort species, whose taxonomy and morphology are described in a review article by Shimamura (2015). Marchantia polymorpha has a long history as an experimental organism, and knowledge of its anatomy and physiological properties has accumulated over a few centuries (Bowman 2015). Burgeff (1943) characterized various morphological mutants, and described the development and genetics of
Plant Cell Physiol. 0(0): 1–9 doi:10.1093/pcp/pcv097, Advance Access publication on 26 June 2015, available online at www.pcp.oxfordjournals.org ! The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]
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Liverworts occupy a basal position in the evolution of land plants, and are a key group to address a wide variety of questions in plant biology. Marchantia polymorpha is a common, easily cultivated, dioecious liverwort species, and is emerging as an experimental model organism. The haploid gametophytic generation dominates the diploid sporophytic generation in its life cycle. Genetically homogeneous lines in the gametophyte generation can be established easily and propagated through asexual reproduction, which aids genetic and biochemical experiments. Owing to its dioecy, male and female sexual organs are formed in separate individuals, which enables crossing in a fully controlled manner. Reproductive growth can be induced at the desired times under laboratory conditions, which helps genetic analysis. The developmental process from a single-celled spore to a multicellular body can be observed directly in detail. As a model organism, molecular techniques for M. polymorpha are well developed; for example, simple and efficient protocols of Agrobacterium-mediated transformation have been established. Based on them, various strategies for molecular genetics, such as introduction of reporter constructs, overexpression, gene silencing and targeted gene modification, are available. Herein, we describe the technologies and resources for reverse and forward genetics in M. polymorpha, which offer an excellent experimental platform to study the evolution and diversity of regulatory systems in land plants.
Special Focus Issue – Mini Review
Molecular Genetic Tools and Techniques for Marchantia polymorpha Research
K. Ishizaki et al. | Tools for Marchantia research
Accessions The wild-type male and female accessions, Takaragaike-1 (Tak-1) and Takaragaike-2 (Tak-2), respectively, were isolated in Kyoto, Japan (Okada et al. 2000, Ishizaki et al. 2008), and have been used in recent molecular genetic studies. The Y chromosome and some loci in the X chromosome from Tak-1 and Tak-2, respectively, have been sequenced (Yamato et al. 2007). The ongoing whole-genome project at DOE-JGI has adopted a female line that is inferred to have the autosomes from Tak-1 and the X chromosome from Tak-2. Marchantia polymorpha is distributed worldwide, from tropical to arctic climates, and natural accessions other than Tak-1 and Tak-2 have been collected for experimental research in various continents. A comprehensive collection of such accessions will be a valuable resource to investigate complex genetic interactions underlying responses to the environment and the diversity of morphological traits.
Culture and Crossing in the Laboratory Certain features of the life cycle of M. polymorpha are beneficial for genetic and biochemical analyses. First, the haploid dominance of its life cycle eliminates the possibility of heterozygosity 2
in the gametophyte generation. Phenotypes of transformants and mutants can be observed in their isolated generation. Genetic analysis can be simpler and more rapid in M. polymorpha than in diploid plants. Secondly, a gametophyte developed from a single spore or a gemma comprises isogenic cells, and such genetically homogeneous individuals can be established rapidly. Thirdly, isogenic individuals can be easily propagated from vegetative tissues in an asexual manner. The life cycle of M. polymorpha is shown in Fig. 1, and described in detail below. The gametophyte generation (sporeling, thallus, gemma/gemmaling and gametangiophore) can be maintained under axenic conditions using synthetic growth media, such as Gamborg’s B5 basal medium, without vitamin supplements.
Vegetative growth The haploid life of M. polymorpha starts from a single-celled spore, which is generated by meiosis of a spore mother cell formed in the diploid sporophyte (Fig. 1). Spores of M. polymorpha can remain dormant for long periods and are revived by imbibing water, which promotes Chl biosynthesis (Nakazato et al. 1999). The subsequent germination of a spore is induced by photosynthesis-derived sugars (Nakazato et al. 1999). After germination, the spore undergoes mitosis to develop into the thallus via the sporeling. Sporeling refers to a group of cells at the developmental stage immediately following spore germination (Fig. 1). It has been reported that red light (R) represses cell elongation and promotes cell proliferation in sporelings through the action of the R and far-red light (FR) receptor phytochrome (Nishihama et al. 2015b). Thus, elongated protonema-like cells can be obtained by modulating the light conditions (Fig. 1). The developmental process from a spore to a multicellular body can be readily observed in detail at this stage, which is an experimental advantage over seed plants. The thallus, the main gametophyte body, of M. polymorpha permits flexible strategies for cultivation. Clonal propagules, called gemmae, are developed from single epidermal cells at the bottom of gemma-cups, which are formed periodically on the dorsal side of a thallus (Barnes and Land 1908). There are over a hundred gemmae in a fully developed gemma-cup. The frequency of gemma-cup formation is influenced by environmental factors, such as light and nutrients (Voth and Hamner 1940, Voth 1941, Benson-Evans 1964). Under laboratory conditions, the addition of 1% sucrose to the growth medium promotes the formation of the gemma-cup. A gemma develops from a single cell solely by mitotic cell divisions; therefore, a non-chimeric line comprising isogenic cells can be established through subculture of a gemma, and the established line can be propagated through subculture of the gemmae (Fig. 2). The M. polymorpha thallus per se can be maintained and propagated asexually by transferring excised fragments. An apical explant of thallus, containing the meristem at the apical notch, continues to propagate apically through bifurcation of the meristem. Furthermore, M. polymorpha has a large regenerative ability (Vo¨chting 1885). A basal thallus explant regenerates meristems without the application of growth regulators and develops into an intact thallus (Kubota et al. 2013,
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M. polymorpha. In the molecular era, the genomes of a plastid (Ohyama et al. 1986) and a mitochondrion (Oda et al. 1992) of Marchantia cultured cells were the first to be fully sequenced in plants. The Y chromosome, as well as a part of the X chromosome, of M. polymorpha was also sequenced, providing insights into the evolution of sex chromosomes in organisms with haploid genomes (Yamato et al. 2007). In view of its critical evolutionary position, whole-genome analysis of M. polymorpha has been initiated under the Community Sequencing Program at the Joint Genome Institute (DOE-JGI: http://jgi.doe.gov/whysequence-a-liverwort/), which has revealed that most of the genes that regulate growth and development in other land plants are conserved, but show less redundancy, in the M. polymorpha genome. Another bryophyte model, Physcomitrella patens, has an increased number of genes in its genome because of large-scale genome duplication during the evolution of mosses (Rensing et al. 2007, Rensing et al. 2008). Over the last two decades, plant molecular biology has focused mainly on a few angiosperm model species, as well as the moss P. patens, because of the availability of molecular techniques, and it was not until recently that molecular genetic studies became popular in M. polymorpha. Today, a wide range of molecular genetic tools, including transformation technologies (Ishizaki et al. 2008, Kubota et al. 2013, Tsuboyama and Kodama 2014, Tsuboyama-Tanaka and Kodama 2015) and targeted genome modification (Ishizaki et al. 2013a, Sugano et al. 2014), are available for M. polymorpha, turning this plant into a versatile model that occupies a basal evolutionary position, making it suitable to address evolutionary, molecular, cellular and developmental questions. In this review, we introduce characters of M. polymorpha as an experimental model, focusing on the tools available for molecular genetic studies.
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Fig. 1 Life cycle of Marchantia polymorpha. A haploid spore germinates and develops into a thallus. A group of cells developing from a spore is called a sporeling. The morphology of the sporeling is influenced by light conditions. While a sporeling grows into a sphere-like cell mass under white light, it develops into an elongated protonema-like structure under low red light (R) conditions. A thallus is a multilayered gametophyte body with dorsoventrality that has a growing point at the apical notch of each lobe, which undergoes periodical bifurcations. In the vegetative growth phase, dozens of clonal progeny, gemmae, are produced in gemma-cups, which are formed repeatedly on the dorsal side of a thallus. A mature gemma has two distinct growing points, but remains dormant while inside the gemma-cup. After dispersion and water uptake, each gemma develops into an individual thallus. Marchantia polymorpha transits from vegetative to reproductive growth under a long-day condition supplemented with far-red light (FR), and develops gametangiophores on the thallus. The male gametangiophore is called an antheridiophore and contains antheridia, which produce flagellated sperm (photo courtesy of Dr. Masaki Shimamura of Hiroshima University). The female gametangiophore is called an archegoniophore and has archegonia holding an egg below the lobes. Sperms released into water swim up the neck of the archegonium and fertilize the eggs. The zygote undergoes mitotic divisions, and develops into a multicellular diploid sporophyte. Nutrients are supplied to the developing sporophyte from the surrounding gametophyte tissues. Meiosis occurs inside the sporangium, and hundreds of thousands of haploid spores are produced per sporangium. Inside a sporangium, spores are attached to elaters, which are tubular with spiral cell wall thickenings, and function in spore dispersal.
Nishihama et al. 2015b). Therefore, the thallus itself can be either maintained through subculture of the apical explant without regeneration, or propagated effectively through the regeneration of the basal explant. The regeneration of the thallus is promoted by an R signal mediated by a phytochrome, which is rate limited by sucrose availability (Nishihama et al. 2015b).
Reproductive growth The sexual reproduction of M. polymorpha can be fully managed under laboratory conditions, which aids genetic analysis. It has been reported that M. polymorpha undergoes a transition from vegetative to reproductive phase under
long-day conditions, and initiates the formation of gametangiophores, which are archegoniophores in female individuals and antheridiophores in male individuals (Wann 1925, Benson-Evans 1961). To date, the GIGANTEA (GI) and FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1) complexmediated regulatory machinery of the photoperiodic growth phase transition, which is analogous to the regulatory mechanism for photoperiodic control of flowering in angiosperms, has been demonstrated to operate in M. polymorpha (Kubota et al. 2014). Knockout of either the GI or the FKF1 ortholog completely abolished the long-day-dependent growth-phase transition, and overexpression of either gene promoted growth phase transition, even under short-day 3
K. Ishizaki et al. | Tools for Marchantia research
conditions in M. polymorpha, indicating the critical role of the GI–FKF complex in the transition from the vegetative to reproductive growth phase. In addition, FR is indispensable to induce the transition in M. polymorpha (Chiyoda et al. 2008), suggesting the involvement of a phytochrome in the regulatory process. While natural light (sunlight) contains FR, regular white fluorescent tubes lack FR, and, therefore, FR must be supplemented by an extra light source, such as an FR-LED, for the induction of gametangiophores under laboratory conditions. These findings have allowed us to cross M. polymorpha at any time in the laboratory, which helps routine preparation of spores and genetic analyses. Motile sperm generated in the antheridia fertilize eggs in the archegonia (Fig. 1), and water is indispensable in this process. During crossing under laboratory conditions, sperm are released into a water droplet put on an antheridiophore, collected with a pipette and applied to archegoniophores that have closed or partially opened finger-like lobes. Water triggers the release of most sperm present in mature antheridia. Sperm can be collected later again from newly maturing antheridia in the same antheridiophore. Repetitive application of sperm every few days ensures and increases the production of sporangia, because archegonia at various developmental stages are present in an archegoniophore. After fertilization, a zygote develops into a multicellular sporophyte, and spore mother cells formed in the mature sporophyte undergo meiosis to generate spores (Durand 1908), each of which is genetically different. There are hundreds of thousands of spores generated in a sporangium of M. polymorpha. Linkage analysis is achieved using an F1 population of the individuals developed from spores. Double mutants can be isolated directly from an F1 population generated by crossing parental mutant strains. 4
Generation time and preservation Having a rapid life cycle is an important feature of a model organism. It takes 3–4 weeks for a spore to develop into a mature thallus that is competent for the induction of gametangiophores. Under the inducing condition described above, it usually takes about 2 weeks for a mature thallus to form visible gametangiophores. In the case of male plants, a further couple of weeks are required to develop fertile sperm. After fertilization, it takes about 3–4 weeks to obtain mature sporangia that contain spores. Thus, the minimum time required for the completion of the whole sexual life cycle from spore to spore is about 3 months in M. polymorpha. Meanwhile, for asexual reproduction, it takes 2–3 weeks for a gemmaling to grow into a thallus with newly formed gemmae ready for subculture and experimentation. Spores and gemmae are dormant in M. polymorpha. Dried spores in mature sporangia can be stored in a deep freezer (80 C) for years and used as needed. Although thalli can be maintained by subculture, it should be noted that continuous subculture might result in the accumulation of spontaneous mutations. Axenic thalli and gemmae can be preserved for 6 months and several years, respectively, on agar medium containing 1% sucrose at 4 C in dim light. Furthermore, ultra-low temperature storage protocols for gemmae have become available (Tanaka et al. 2015); thus, experimental resources, such as wild-type accessions and transgenics, can be stored stably for years.
Transformation of the Nuclear Genome Physical DNA delivery (particle bombardment) As an essential component of functional genomics, transformation techniques have been developed for M. polymorpha. The
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Fig. 2 Establishment of isogenic lines using gemmae. A transformant after the first screening could be chimeric. A gemma develops from a single cell; therefore, an isogenic line (G1) could be established by selecting a single gemma out of those formed on transformants after the first selection. Once established, multiple isogenic lines could be used for experiments by subculturing G2 gemmae formed on the G1 plant. The G1 line could be maintained by subculturing thallus fragments, as described in the text.
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genetic transformation of the M. polymorpha nuclear genome was achieved by particle bombardment using 2-week-old thalli grown from gemmae (Takenaka et al. 2000). The use of sporelings significantly improved the performance of particle bombardment-based transformation (Chiyoda et al. 2008). Particle bombardment-based transformation was used for the functional analyses of genes by overexpression, gene silencing and mutagenesis (Kajikawa et al. 2003, Kajikawa et al. 2008, Yamaoka et al. 2004). However, physical DNA delivery often results in a large number of independent insertions and extremely complex transgene rearrangements (Kohli et al. 2003), which complicates further genetic analysis of such mutants.
Agrobacterium-mediated transformation
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Agrobacterium-mediated genetic transformation has significant advantages over physical methods of transformation such as particle bombardment, including integration of intact T-DNA fragments flanked by the right and left border sequences, reduced frequency of genomic rearrangement, introduction of fewer copies of a transgene into the host genome, transfer of larger segments of DNA and easier manipulation (Kohli et al. 2003). A practical Agrobacterium-mediated transformation procedure for M. polymorpha was first achieved using sporelings (Ishizaki et al. 2008). In this method, spores are germinated and grown into sporelings for 5–7 d in liquid culture, co-cultivated with Agrobacterium for 2 d and transferred directly to selective agar medium after washing. Hundreds of transformants can be obtained using a single sporangium as early as 2 weeks after transfer to selection medium. DNA analyses demonstrated random integrations of 1–5 copies of intact TDNAs with the right and the left borders into the M. polymorpha genome in seven transformants examined (Ishizaki et al. 2008). Sporeling transformation is particularly useful for experiments requiring a large number of transformants, such as screening of T-DNA-tagged mutants (Ishizaki et al. 2013b) and homologous recombination (HR)-mediated gene targeting (Ishizaki et al. 2013a). A simpler version of this protocol, Agarutilized Transformation with Pouring Solutions (AgarTrap), is also available, which generates a sufficient number of independent transformants for molecular analysis (Tsuboyama and Kodama 2014). In the AgarTrap protocol, sporelings grown and fixed on agar media are co-cultured with Agrobacterium, washed, and selected in the same Petri dish. However, sporelings generated from parental lines with different genetic backgrounds are genetically heterogeneous because of recombination during meiosis, which might affect the consistency among the resulting transgenic plants. Recently, alternative Agrobacterium-mediated transformation protocols have been developed either using a tissue regenerated from a thallus from which the apical regions have been surgically removed (Kubota et al. 2013), or using gemmalings (Tsuboyama-Tanaka and Kodama 2015). The important advantage of these protocols over sporeling transformation is the ability to obtain transformants with an identical genetic background. In addition, these protocols use thalli that can be propagated quickly through asexual reproduction, and thus
eliminate the need for spore preparation, which includes the induction of reproductive organs, crossing, spore maturation and the collection of sporangia, followed by appropriate desiccation. Thallus transformation should be the first choice for the generation of just dozens of transgenic lines. This protocol is also useful for the secondary transformation of existing transgenic lines, e.g. the introduction of a reporter fusion construct into existing transgenics, and genetic complementation of existing mutants obtained by T-DNA tagging or a gene targeting strategy (Ishizaki et al. 2013a, Komatsu et al. 2014, Kubota et al. 2014). Multiple transformation events can occur in a multicellular tissue; therefore, a regenerant from transformants of a sporeling, a thallus explant or a gemmaling can be chimeric after the first selection. Subculture of a gemma from the initial transformant is a reliable strategy to avoid the potential chimerism (Fig. 2). In these Agrobacterium-mediated transformation protocols, it takes 2–3 weeks for a transgenic plant to appear on the first selection plate, and a further 2–3 weeks to establish isogenic G1 lines (Fig. 2).
Agrobacterium strains and vectors These Agrobacterium-mediated transformation protocols in M. polymorpha are based on the use of popular Agrobacterium strains with the C58 chromosomal background, including GV2260 and GV3101. Common reporter genes such as b-glucuronidase (GUS) and fluorescent proteins have been used successfully to investigate gene functions in M. polymorpha (Ishizaki et al. 2012, Ishizaki et al. 2013b, Komatsu et al. 2014, Kubota et al. 2014). However, it should be noted that green fluorescent protein (GFP) has a toxic effect for some unknown reason when it is localized in the cytosol, and thus other fluorescent proteins, such as Citrine (Shaner et al. 2005), are recommended for cytosolic expression. It has been reported recently that overexpression and/or a weak dimerizing nature of GFP or Citrine results in artificial formation of bulb-like structures in vacuoles in M. polymorpha, and the use of monomeric fluorescent proteins, such as mCitrine, should be the choice to create a fusion protein localized in the vacuolar membrane (Kanazawa et al. 2015). Common binary vectors developed for other model plant systems, such as pCAMBIA (http://www.cambia.org/daisy/ cambia/585), pPZP (Hajdukiewicz et al. 1994) and pGWBs (Nakagawa et al. 2007a) can be used for M. polymorpha. However, it should be noted that the nopaline synthase gene promoter, which has been used to drive marker genes in some binary vectors (Karimi et al. 2002, Nakagawa et al. 2007b), does not show sufficient promoter activity in M. polymorpha. A series of Gateway binary vectors, pMpGWBs, containing four different marker genes, have been developed and are available for studies using M. polymorpha. These marker genes are the hygromycin phosphotransferase gene, the gentamicin 30 -acetyltransferase gene, the mutated acetolactate synthase gene and the neomycin phosphotransferase II gene, for selection with hygromycin, gentamicin, chlorsulfuron and G418, respectively. The marker genes are driven by a double-enhancer version of the Cauliflower mosaic virus (CaMV) 35S promoter. 5
K. Ishizaki et al. | Tools for Marchantia research
Gene Targeting and Reverse Genetics HR-mediated gene targeting Gene targeting mediated by HR is a powerful tool for functional analysis in reverse genetics. Although exceptionally efficient in P. patens (Schaefer and Zryd 1997), gene targeting by HR is difficult in other plants. A reproducible gene targeting procedure developed for the monocot rice, using a positive (hygromycin resistance gene)/negative (diphtheria toxin A fragment gene) selection system (Terada et al. 2002), was successfully applied to M. polymorpha (Ishizaki et al. 2013a). The high frequency of the Agrobacterium-mediated sporeling transformation is advantageous for a gene targeting strategy based on the positive/negative selection system. In the case of the targeted knockout of the NOPPERABO1 (NOP1) gene, about 2% of transformants that passed the positive/negative selection showed the expected morphological phenotype, and the NOP1 locus in these plants was successfully disrupted by HR. This procedure has been applied to the targeted disruption of other genetic loci, including MpGI, MpFKF (Kubota et al. 2014), MpPHOT (Komatsu et al. 2014) and MpTAA (Eklund et al. 2015). To date, the statistics suggest that screening of fewer than 400 candidate transformants is sufficient to isolate at least one knockout clone for most non-essential genes. Gene targeting mediated by HR should be useful not only to generate knockouts but also to modify targeted sequences and fusion tags by knock-in. The tissue to be transformed is haploid; therefore, it is theoretically impossible to generate knockout plants for essential genes in M. polymorpha. To overcome this problem, conditional gene knockout strategies would be useful. Recently, Nishihama et al. (2015a) developed a heat- and dexamethasone-controllable gene expression/deletion system in M. polymorpha by expressing the P1-phage Cre site-specific recombinase fused with the ligand-binding site of the rat 6
glucocorticoid receptor, under the control of an endogenous heat-inducible promoter. This system could be used to generate conditional knockout mutants for essential genes in M. polymorpha. As an alternative, an inducible artificial microRNAmediated knockdown strategy is available in M. polymorpha, which provides a useful tool to study gene function, particularly if null mutations are potentially lethal (Flores-Sandoval et al. 2015a).
Genome editing by the CRISPR/Cas9 system Recently, a simple genome editing technology, the CRISPR (clustered regulatory interspaced short palindromic repeats)/ Cas9 (CRISPR-associated protein 9) system, which is based on a prokaryote-specific adaptive immune system (Wiedenheft et al. 2012), has been developed for eukaryotic model organisms, including plants (Belhaj et al. 2014). CRISPR/Cas9-based targeted mutagenesis has been demonstrated in M. polymorpha (Sugano et al. 2014). Multiple mutant alleles that showed the expected auxin-resistant phenotype were directly established in the gametophyte generation, using the endogenous U6 promoter-driven guide RNA designed to disrupt the gene encoding AUXIN RESPONSE FACTOR1 (ARF1) and the Cas9 gene under control either of the CaMV 35S or MpEF1a promoter (Sugano et al. 2014). The CRISPR/Cas9 system should allow simple and rapid genome modification of target loci in M. polymorpha.
Mutagenesis and Forward Genetics Forward genetics is a powerful approach to link genes to a particular phenotype, and has been used to obtain information about gene function in plants. Physcomitrella patens is suited for reverse genetic analysis by virtue of its ability to undertake targeted transgene integration by homologous recombination (Schaefer and Zryd 1997). However, the forward genetic approach has been limited in the moss because of large-scale genome duplication events (Strotbek et al. 2013). Marchantia polymorpha has a significant potential as a model haploid plant for forward genetics, because of its low genetic redundancy. A T-DNA tagging strategy to generate mutants has been successful in M. polymorpha (Ishizaki et al. 2013b, Ueda et al. 2013), using the efficient Agrobacterium-mediated transformation protocol (Ishizaki et al. 2008), and the genes affected were readily identified. A morphological mutant showing impaired air-chamber formation, nop1, was isolated by screening the T1 generation from 10,000 T-DNA-tagged lines (Ishizaki et al. 2013b). Linkage analysis of the nopperabo1 (nop1) phenotype and the inserted T-DNA, as hygromycin resistance, was carried out using an F1 population generated by crossing of nop1 and a wild-type accession. The responsible gene, NOP1, was isolated by identifying flanking sequences of the T-DNA by PCR and confirmed by genetic complementation by the Agrobacteriummediated transformation protocol using regenerating thalli (Ishizaki et al. 2013b). Assuming that 1–5 copies of T-DNAs are randomly inserted to the nuclear genome of 280 Mb in size, approximately 200,000 independent transformants are estimated to be sufficient
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In M. polymorpha, two constitutive promoters, the CaMV 35S promoter and the endogenous ELONGATION FACTOR 1a (MpEF1a) promoter, have been characterized (Althoff et al. 2014) and used for both gene knockout and overexpression studies (Kajikawa et al. 2003, Kubota et al. 2014, Sugano et al. 2014, Eklund et al. 2015, Flores-Sadoval et al. 2015b, Kato et al. 2015). The CaMV 35S and MpEF1a promoters are both capable of driving strong expression in M. polymorpha, but there are significant differences in terms of their spatial distributions. The CaMV 35S promoter has a weak activity in the meristematic zones, but strong in the other part of thallus, while the MpEF1a promoter is strong at the meristematic zone in thallus and generally ubiquitous in the other tissues, such as gemmae and gametangiophores (Althoff et al. 2014). Furthermore, it was reported that the CaMV 35S promoter is not active in oil body cells of M. polymorpha, which contain liverwort-specific organelles, termed oil bodies, which are filled with specific isoprenoids, phenolics and bisbenzyl compounds (Kanazawa et al. 2015). Thus, the MpEF1a promoter would be preferable over the CaMV 35S promoter for overexpression of transgenes in the thallus, gemma and gametangiophore.
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Plastid Transformation Methods for plastid transformation have been established in several land plant species, including tobacco and P. patens, and have been used widely in basic research and for biotechnological applications (Day and Goldschmidt-Clermont 2011, Bock 2014). In M. polymorpha, efficient plastid transformation protocols have been established for suspension-cultured cells, as well as for sporelings (Chiyoda et al. 2007, Chiyoda et al. 2014). This strategy was used successfully for the targeted disruption of the plastid-encoded ndhB gene via insertion of the aadA cassette into the 30 exon (Ueda et al. 2012). Despite multiple copies of the plastid genome, homoplasmic transformants of thalli, where all copies of the plastid genome are transformed, could be obtained immediately after primary selection by recovering gemmae from transformed sporelings, whereas it takes 12–16 weeks of repeated subculture to obtain homoplasmic transformants from suspension-cultured cells (Chiyoda et al. 2014). In combination with the transformation system for the nuclear genome, this efficient strategy for plastid genome transformation should aid basic research on plastids, such as replication and gene expression, as well as various applications to plant biotechnology.
Conclusion and Perspective Despite the large evolutionary distance and the difference in life cycle between vascular plants and bryophytes, accumulating evidence indicates that many regulatory pathways in vascular plants are also found in bryophytes. This supports a model of plant evolution whereby many features found in vascular plants were developed by harnessing gene regulatory networks that had already been acquired in the common ancestor of bryophytes and vascular plants (Pires and Dolan 2012, Kubota et al. 2014, Flores-Sandoval et al. 2015b, Kato et al. 2015). Thus, comparative molecular characterization of the regulatory
machineries in bryophytes and vascular plants should not only provide in-depth insights into plant evolution, but also increase our understanding of the principal regulatory modules in land plants. The liverwort M. polymorpha has substantial potential as a model system for plant biology because of its critical phylogenetic position in the evolution of land plants; its conserved, yet less complex, developmental pattern and responses to plant growth factors and environmental stimuli; and its gametophyte-dominant life cycle, which renders genetic analyses less complicated. Tools and resources for reverse and forward genetics should accelerate the functional analysis of genes in M. polymorpha and reveal the evolution and diversity of regulatory systems in land plants. Downloaded from http://pcp.oxfordjournals.org/ at University of Cambridge on August 18, 2015
for saturation mutagenesis to cover >95% of the genes in M. polymorpha, and it is feasible to isolate a number of transformants using about 200–300 sporangia for transformation. The T-DNA tagging strategy in M. polymorpha, powered by the high transformation efficiency and ongoing whole-genome sequencing project, will promote forward genetics in this basal land plant. Marchantia polymorpha can also be chemically or physically mutagenized. For example, Millar et al. (1962) isolated and characterized 12 nutritionally deficient mutants of M. polymorpha from X-ray-irradiated gemmae. However, there has been no report of the successful identification of underlying mutations causing the phenotypic variations to date. The use of nextgeneration sequencing technology will provide a high-throughput platform to identify responsible genes because of the haploidy and small genome size of M. polymorpha. The forward genetic approach in M. polymorpha should contribute to our understanding of molecular mechanisms that are difficult to uncover in other model plants, where such an approach is challenging because of genetic redundancy.
Funding This work was supported by the Ministry of Education, Culture, Sports, Science and Technology [Grant-in-Aid for Scientific Research on Innovative Area (No. 25113009 to T.K.; 25119711 and 15H01233 to K.I.)]; the Japan Society for the Promotion of Science [Grant-in-Aid for Scientific Research (B) (15H04391 to K.I.); Scientific Research (C) (24570048 to R.N.)]; Challenging Exploratory Research [26650095 to T.K.]; the Asahi Glass Foundation [to K.I.]; the SUNTORY Foundation [to K.I.].
Acknowledgments The authors thank Hideyuki Takami for preparation of the illustrations for this manuscript.
Disclosures The authors have no conflicts of interest to declare.
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Kimitsune Ishizaki1,*, Ryuichi Nishihama2, Katsuyuki T. Yamato3 and Takayuki Kohchi2 1
Graduate School of Science, Kobe University, Kobe 657-8501 Japan Graduate School of Biostudies, Kyoto University, Kyoto, 606-8502 Japan 3 Faculty of Biology-Oriented Science and Technology, Kinki University, Wakayama, 649-6493 Japan 2
*Corresponding author: E-mail, [email protected]; Fax, +81-78-803-5727. (Received March 7, 2015; Accepted June 18, 2015)
Keywords: Bryophytes Evolution Evo-devo Molecular genetics. Abbreviations: CaMV, Cauliflower mosaic virus; Cas9, CRISPR-associated protein 9; CRISPR, clustered regulatory interspaced short palindromic repeats; FR, far-red light; GFP, green fluorescent protein; GUS, b-glucuronidase; HR, homologous recombination; MpEF1a, Marchantia polymorpha ELONGATION FACTOR 1a; R, red light.
Introduction Land plants comprise a monophyletic group that emerged from freshwater green algae around 500 million years ago. Bryophytes, consisting of liverworts, mosses and hornworts, represent the earliest diverging group of land plants.
As embryophyta, bryophytes have multicellular bodies in the sporophyte generation and undergo alternation of generations. Both the gametophyte and sporophyte generations of land plants have undergone major morphological and physiological changes during the course of evolution. Bryophytes, as basal land plants, have features distinct from those of the other land plant lineages, such as the lack of a vascular system, the absence of a lignified cell wall, the use of motile sperm for fertilization and dominance of the haploid gametophyte generation over the diploid sporophyte generation during their life cycle. When and how did land plants acquire the genetic systems that led to such substantial changes? Recent advances of genome analyses in various lineages of land plants, including angiosperms, gymnosperms, lycophytes and mosses, have revealed that many of their regulatory gene families are conserved (Floyd and Bowman 2007, Rensing et al. 2008, Banks et al. 2011, Nystedt et al. 2013). Recently, it was suggested that many of the fundamental features of land plants appeared in the gametophytes of bryophytes first, and were then co-opted to the sporophytes in vascular plants (Ligrone et al. 2012, Pires and Dolan 2012). Therefore, the molecular biology of basal plant lineages has received more attention, especially to determine the fundamental mechanisms and principles common to land plants, as well as characteristics specific to bryophytes. In bryophytes, while the phylogenic relationship between liverworts, mosses and hornworts is still debated, the critical position of liverworts as one of the earliest land plant lineages is unequivocal (Qiu et al. 2006, Chang and Graham 2011, Wicket et al. 2014). Therefore, along with mosses and hornworts, liverworts are a key group in comparative genomics to address fundamental questions in plant biology, such as the genetic basis of the key innovations that allowed land plants to evolve from aquatic ancestors and to adapt to life on land, the developmental genetic changes responsible for the multicellularity, and the alterations in body plan within land plants (Bowman et al. 2007). Marchantia polymorpha is a widespread liverwort species, whose taxonomy and morphology are described in a review article by Shimamura (2015). Marchantia polymorpha has a long history as an experimental organism, and knowledge of its anatomy and physiological properties has accumulated over a few centuries (Bowman 2015). Burgeff (1943) characterized various morphological mutants, and described the development and genetics of
Plant Cell Physiol. 0(0): 1–9 doi:10.1093/pcp/pcv097, Advance Access publication on 26 June 2015, available online at www.pcp.oxfordjournals.org ! The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]
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Liverworts occupy a basal position in the evolution of land plants, and are a key group to address a wide variety of questions in plant biology. Marchantia polymorpha is a common, easily cultivated, dioecious liverwort species, and is emerging as an experimental model organism. The haploid gametophytic generation dominates the diploid sporophytic generation in its life cycle. Genetically homogeneous lines in the gametophyte generation can be established easily and propagated through asexual reproduction, which aids genetic and biochemical experiments. Owing to its dioecy, male and female sexual organs are formed in separate individuals, which enables crossing in a fully controlled manner. Reproductive growth can be induced at the desired times under laboratory conditions, which helps genetic analysis. The developmental process from a single-celled spore to a multicellular body can be observed directly in detail. As a model organism, molecular techniques for M. polymorpha are well developed; for example, simple and efficient protocols of Agrobacterium-mediated transformation have been established. Based on them, various strategies for molecular genetics, such as introduction of reporter constructs, overexpression, gene silencing and targeted gene modification, are available. Herein, we describe the technologies and resources for reverse and forward genetics in M. polymorpha, which offer an excellent experimental platform to study the evolution and diversity of regulatory systems in land plants.
Special Focus Issue – Mini Review
Molecular Genetic Tools and Techniques for Marchantia polymorpha Research
K. Ishizaki et al. | Tools for Marchantia research
Accessions The wild-type male and female accessions, Takaragaike-1 (Tak-1) and Takaragaike-2 (Tak-2), respectively, were isolated in Kyoto, Japan (Okada et al. 2000, Ishizaki et al. 2008), and have been used in recent molecular genetic studies. The Y chromosome and some loci in the X chromosome from Tak-1 and Tak-2, respectively, have been sequenced (Yamato et al. 2007). The ongoing whole-genome project at DOE-JGI has adopted a female line that is inferred to have the autosomes from Tak-1 and the X chromosome from Tak-2. Marchantia polymorpha is distributed worldwide, from tropical to arctic climates, and natural accessions other than Tak-1 and Tak-2 have been collected for experimental research in various continents. A comprehensive collection of such accessions will be a valuable resource to investigate complex genetic interactions underlying responses to the environment and the diversity of morphological traits.
Culture and Crossing in the Laboratory Certain features of the life cycle of M. polymorpha are beneficial for genetic and biochemical analyses. First, the haploid dominance of its life cycle eliminates the possibility of heterozygosity 2
in the gametophyte generation. Phenotypes of transformants and mutants can be observed in their isolated generation. Genetic analysis can be simpler and more rapid in M. polymorpha than in diploid plants. Secondly, a gametophyte developed from a single spore or a gemma comprises isogenic cells, and such genetically homogeneous individuals can be established rapidly. Thirdly, isogenic individuals can be easily propagated from vegetative tissues in an asexual manner. The life cycle of M. polymorpha is shown in Fig. 1, and described in detail below. The gametophyte generation (sporeling, thallus, gemma/gemmaling and gametangiophore) can be maintained under axenic conditions using synthetic growth media, such as Gamborg’s B5 basal medium, without vitamin supplements.
Vegetative growth The haploid life of M. polymorpha starts from a single-celled spore, which is generated by meiosis of a spore mother cell formed in the diploid sporophyte (Fig. 1). Spores of M. polymorpha can remain dormant for long periods and are revived by imbibing water, which promotes Chl biosynthesis (Nakazato et al. 1999). The subsequent germination of a spore is induced by photosynthesis-derived sugars (Nakazato et al. 1999). After germination, the spore undergoes mitosis to develop into the thallus via the sporeling. Sporeling refers to a group of cells at the developmental stage immediately following spore germination (Fig. 1). It has been reported that red light (R) represses cell elongation and promotes cell proliferation in sporelings through the action of the R and far-red light (FR) receptor phytochrome (Nishihama et al. 2015b). Thus, elongated protonema-like cells can be obtained by modulating the light conditions (Fig. 1). The developmental process from a spore to a multicellular body can be readily observed in detail at this stage, which is an experimental advantage over seed plants. The thallus, the main gametophyte body, of M. polymorpha permits flexible strategies for cultivation. Clonal propagules, called gemmae, are developed from single epidermal cells at the bottom of gemma-cups, which are formed periodically on the dorsal side of a thallus (Barnes and Land 1908). There are over a hundred gemmae in a fully developed gemma-cup. The frequency of gemma-cup formation is influenced by environmental factors, such as light and nutrients (Voth and Hamner 1940, Voth 1941, Benson-Evans 1964). Under laboratory conditions, the addition of 1% sucrose to the growth medium promotes the formation of the gemma-cup. A gemma develops from a single cell solely by mitotic cell divisions; therefore, a non-chimeric line comprising isogenic cells can be established through subculture of a gemma, and the established line can be propagated through subculture of the gemmae (Fig. 2). The M. polymorpha thallus per se can be maintained and propagated asexually by transferring excised fragments. An apical explant of thallus, containing the meristem at the apical notch, continues to propagate apically through bifurcation of the meristem. Furthermore, M. polymorpha has a large regenerative ability (Vo¨chting 1885). A basal thallus explant regenerates meristems without the application of growth regulators and develops into an intact thallus (Kubota et al. 2013,
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M. polymorpha. In the molecular era, the genomes of a plastid (Ohyama et al. 1986) and a mitochondrion (Oda et al. 1992) of Marchantia cultured cells were the first to be fully sequenced in plants. The Y chromosome, as well as a part of the X chromosome, of M. polymorpha was also sequenced, providing insights into the evolution of sex chromosomes in organisms with haploid genomes (Yamato et al. 2007). In view of its critical evolutionary position, whole-genome analysis of M. polymorpha has been initiated under the Community Sequencing Program at the Joint Genome Institute (DOE-JGI: http://jgi.doe.gov/whysequence-a-liverwort/), which has revealed that most of the genes that regulate growth and development in other land plants are conserved, but show less redundancy, in the M. polymorpha genome. Another bryophyte model, Physcomitrella patens, has an increased number of genes in its genome because of large-scale genome duplication during the evolution of mosses (Rensing et al. 2007, Rensing et al. 2008). Over the last two decades, plant molecular biology has focused mainly on a few angiosperm model species, as well as the moss P. patens, because of the availability of molecular techniques, and it was not until recently that molecular genetic studies became popular in M. polymorpha. Today, a wide range of molecular genetic tools, including transformation technologies (Ishizaki et al. 2008, Kubota et al. 2013, Tsuboyama and Kodama 2014, Tsuboyama-Tanaka and Kodama 2015) and targeted genome modification (Ishizaki et al. 2013a, Sugano et al. 2014), are available for M. polymorpha, turning this plant into a versatile model that occupies a basal evolutionary position, making it suitable to address evolutionary, molecular, cellular and developmental questions. In this review, we introduce characters of M. polymorpha as an experimental model, focusing on the tools available for molecular genetic studies.
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Fig. 1 Life cycle of Marchantia polymorpha. A haploid spore germinates and develops into a thallus. A group of cells developing from a spore is called a sporeling. The morphology of the sporeling is influenced by light conditions. While a sporeling grows into a sphere-like cell mass under white light, it develops into an elongated protonema-like structure under low red light (R) conditions. A thallus is a multilayered gametophyte body with dorsoventrality that has a growing point at the apical notch of each lobe, which undergoes periodical bifurcations. In the vegetative growth phase, dozens of clonal progeny, gemmae, are produced in gemma-cups, which are formed repeatedly on the dorsal side of a thallus. A mature gemma has two distinct growing points, but remains dormant while inside the gemma-cup. After dispersion and water uptake, each gemma develops into an individual thallus. Marchantia polymorpha transits from vegetative to reproductive growth under a long-day condition supplemented with far-red light (FR), and develops gametangiophores on the thallus. The male gametangiophore is called an antheridiophore and contains antheridia, which produce flagellated sperm (photo courtesy of Dr. Masaki Shimamura of Hiroshima University). The female gametangiophore is called an archegoniophore and has archegonia holding an egg below the lobes. Sperms released into water swim up the neck of the archegonium and fertilize the eggs. The zygote undergoes mitotic divisions, and develops into a multicellular diploid sporophyte. Nutrients are supplied to the developing sporophyte from the surrounding gametophyte tissues. Meiosis occurs inside the sporangium, and hundreds of thousands of haploid spores are produced per sporangium. Inside a sporangium, spores are attached to elaters, which are tubular with spiral cell wall thickenings, and function in spore dispersal.
Nishihama et al. 2015b). Therefore, the thallus itself can be either maintained through subculture of the apical explant without regeneration, or propagated effectively through the regeneration of the basal explant. The regeneration of the thallus is promoted by an R signal mediated by a phytochrome, which is rate limited by sucrose availability (Nishihama et al. 2015b).
Reproductive growth The sexual reproduction of M. polymorpha can be fully managed under laboratory conditions, which aids genetic analysis. It has been reported that M. polymorpha undergoes a transition from vegetative to reproductive phase under
long-day conditions, and initiates the formation of gametangiophores, which are archegoniophores in female individuals and antheridiophores in male individuals (Wann 1925, Benson-Evans 1961). To date, the GIGANTEA (GI) and FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1) complexmediated regulatory machinery of the photoperiodic growth phase transition, which is analogous to the regulatory mechanism for photoperiodic control of flowering in angiosperms, has been demonstrated to operate in M. polymorpha (Kubota et al. 2014). Knockout of either the GI or the FKF1 ortholog completely abolished the long-day-dependent growth-phase transition, and overexpression of either gene promoted growth phase transition, even under short-day 3
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conditions in M. polymorpha, indicating the critical role of the GI–FKF complex in the transition from the vegetative to reproductive growth phase. In addition, FR is indispensable to induce the transition in M. polymorpha (Chiyoda et al. 2008), suggesting the involvement of a phytochrome in the regulatory process. While natural light (sunlight) contains FR, regular white fluorescent tubes lack FR, and, therefore, FR must be supplemented by an extra light source, such as an FR-LED, for the induction of gametangiophores under laboratory conditions. These findings have allowed us to cross M. polymorpha at any time in the laboratory, which helps routine preparation of spores and genetic analyses. Motile sperm generated in the antheridia fertilize eggs in the archegonia (Fig. 1), and water is indispensable in this process. During crossing under laboratory conditions, sperm are released into a water droplet put on an antheridiophore, collected with a pipette and applied to archegoniophores that have closed or partially opened finger-like lobes. Water triggers the release of most sperm present in mature antheridia. Sperm can be collected later again from newly maturing antheridia in the same antheridiophore. Repetitive application of sperm every few days ensures and increases the production of sporangia, because archegonia at various developmental stages are present in an archegoniophore. After fertilization, a zygote develops into a multicellular sporophyte, and spore mother cells formed in the mature sporophyte undergo meiosis to generate spores (Durand 1908), each of which is genetically different. There are hundreds of thousands of spores generated in a sporangium of M. polymorpha. Linkage analysis is achieved using an F1 population of the individuals developed from spores. Double mutants can be isolated directly from an F1 population generated by crossing parental mutant strains. 4
Generation time and preservation Having a rapid life cycle is an important feature of a model organism. It takes 3–4 weeks for a spore to develop into a mature thallus that is competent for the induction of gametangiophores. Under the inducing condition described above, it usually takes about 2 weeks for a mature thallus to form visible gametangiophores. In the case of male plants, a further couple of weeks are required to develop fertile sperm. After fertilization, it takes about 3–4 weeks to obtain mature sporangia that contain spores. Thus, the minimum time required for the completion of the whole sexual life cycle from spore to spore is about 3 months in M. polymorpha. Meanwhile, for asexual reproduction, it takes 2–3 weeks for a gemmaling to grow into a thallus with newly formed gemmae ready for subculture and experimentation. Spores and gemmae are dormant in M. polymorpha. Dried spores in mature sporangia can be stored in a deep freezer (80 C) for years and used as needed. Although thalli can be maintained by subculture, it should be noted that continuous subculture might result in the accumulation of spontaneous mutations. Axenic thalli and gemmae can be preserved for 6 months and several years, respectively, on agar medium containing 1% sucrose at 4 C in dim light. Furthermore, ultra-low temperature storage protocols for gemmae have become available (Tanaka et al. 2015); thus, experimental resources, such as wild-type accessions and transgenics, can be stored stably for years.
Transformation of the Nuclear Genome Physical DNA delivery (particle bombardment) As an essential component of functional genomics, transformation techniques have been developed for M. polymorpha. The
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Fig. 2 Establishment of isogenic lines using gemmae. A transformant after the first screening could be chimeric. A gemma develops from a single cell; therefore, an isogenic line (G1) could be established by selecting a single gemma out of those formed on transformants after the first selection. Once established, multiple isogenic lines could be used for experiments by subculturing G2 gemmae formed on the G1 plant. The G1 line could be maintained by subculturing thallus fragments, as described in the text.
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genetic transformation of the M. polymorpha nuclear genome was achieved by particle bombardment using 2-week-old thalli grown from gemmae (Takenaka et al. 2000). The use of sporelings significantly improved the performance of particle bombardment-based transformation (Chiyoda et al. 2008). Particle bombardment-based transformation was used for the functional analyses of genes by overexpression, gene silencing and mutagenesis (Kajikawa et al. 2003, Kajikawa et al. 2008, Yamaoka et al. 2004). However, physical DNA delivery often results in a large number of independent insertions and extremely complex transgene rearrangements (Kohli et al. 2003), which complicates further genetic analysis of such mutants.
Agrobacterium-mediated transformation
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Agrobacterium-mediated genetic transformation has significant advantages over physical methods of transformation such as particle bombardment, including integration of intact T-DNA fragments flanked by the right and left border sequences, reduced frequency of genomic rearrangement, introduction of fewer copies of a transgene into the host genome, transfer of larger segments of DNA and easier manipulation (Kohli et al. 2003). A practical Agrobacterium-mediated transformation procedure for M. polymorpha was first achieved using sporelings (Ishizaki et al. 2008). In this method, spores are germinated and grown into sporelings for 5–7 d in liquid culture, co-cultivated with Agrobacterium for 2 d and transferred directly to selective agar medium after washing. Hundreds of transformants can be obtained using a single sporangium as early as 2 weeks after transfer to selection medium. DNA analyses demonstrated random integrations of 1–5 copies of intact TDNAs with the right and the left borders into the M. polymorpha genome in seven transformants examined (Ishizaki et al. 2008). Sporeling transformation is particularly useful for experiments requiring a large number of transformants, such as screening of T-DNA-tagged mutants (Ishizaki et al. 2013b) and homologous recombination (HR)-mediated gene targeting (Ishizaki et al. 2013a). A simpler version of this protocol, Agarutilized Transformation with Pouring Solutions (AgarTrap), is also available, which generates a sufficient number of independent transformants for molecular analysis (Tsuboyama and Kodama 2014). In the AgarTrap protocol, sporelings grown and fixed on agar media are co-cultured with Agrobacterium, washed, and selected in the same Petri dish. However, sporelings generated from parental lines with different genetic backgrounds are genetically heterogeneous because of recombination during meiosis, which might affect the consistency among the resulting transgenic plants. Recently, alternative Agrobacterium-mediated transformation protocols have been developed either using a tissue regenerated from a thallus from which the apical regions have been surgically removed (Kubota et al. 2013), or using gemmalings (Tsuboyama-Tanaka and Kodama 2015). The important advantage of these protocols over sporeling transformation is the ability to obtain transformants with an identical genetic background. In addition, these protocols use thalli that can be propagated quickly through asexual reproduction, and thus
eliminate the need for spore preparation, which includes the induction of reproductive organs, crossing, spore maturation and the collection of sporangia, followed by appropriate desiccation. Thallus transformation should be the first choice for the generation of just dozens of transgenic lines. This protocol is also useful for the secondary transformation of existing transgenic lines, e.g. the introduction of a reporter fusion construct into existing transgenics, and genetic complementation of existing mutants obtained by T-DNA tagging or a gene targeting strategy (Ishizaki et al. 2013a, Komatsu et al. 2014, Kubota et al. 2014). Multiple transformation events can occur in a multicellular tissue; therefore, a regenerant from transformants of a sporeling, a thallus explant or a gemmaling can be chimeric after the first selection. Subculture of a gemma from the initial transformant is a reliable strategy to avoid the potential chimerism (Fig. 2). In these Agrobacterium-mediated transformation protocols, it takes 2–3 weeks for a transgenic plant to appear on the first selection plate, and a further 2–3 weeks to establish isogenic G1 lines (Fig. 2).
Agrobacterium strains and vectors These Agrobacterium-mediated transformation protocols in M. polymorpha are based on the use of popular Agrobacterium strains with the C58 chromosomal background, including GV2260 and GV3101. Common reporter genes such as b-glucuronidase (GUS) and fluorescent proteins have been used successfully to investigate gene functions in M. polymorpha (Ishizaki et al. 2012, Ishizaki et al. 2013b, Komatsu et al. 2014, Kubota et al. 2014). However, it should be noted that green fluorescent protein (GFP) has a toxic effect for some unknown reason when it is localized in the cytosol, and thus other fluorescent proteins, such as Citrine (Shaner et al. 2005), are recommended for cytosolic expression. It has been reported recently that overexpression and/or a weak dimerizing nature of GFP or Citrine results in artificial formation of bulb-like structures in vacuoles in M. polymorpha, and the use of monomeric fluorescent proteins, such as mCitrine, should be the choice to create a fusion protein localized in the vacuolar membrane (Kanazawa et al. 2015). Common binary vectors developed for other model plant systems, such as pCAMBIA (http://www.cambia.org/daisy/ cambia/585), pPZP (Hajdukiewicz et al. 1994) and pGWBs (Nakagawa et al. 2007a) can be used for M. polymorpha. However, it should be noted that the nopaline synthase gene promoter, which has been used to drive marker genes in some binary vectors (Karimi et al. 2002, Nakagawa et al. 2007b), does not show sufficient promoter activity in M. polymorpha. A series of Gateway binary vectors, pMpGWBs, containing four different marker genes, have been developed and are available for studies using M. polymorpha. These marker genes are the hygromycin phosphotransferase gene, the gentamicin 30 -acetyltransferase gene, the mutated acetolactate synthase gene and the neomycin phosphotransferase II gene, for selection with hygromycin, gentamicin, chlorsulfuron and G418, respectively. The marker genes are driven by a double-enhancer version of the Cauliflower mosaic virus (CaMV) 35S promoter. 5
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Gene Targeting and Reverse Genetics HR-mediated gene targeting Gene targeting mediated by HR is a powerful tool for functional analysis in reverse genetics. Although exceptionally efficient in P. patens (Schaefer and Zryd 1997), gene targeting by HR is difficult in other plants. A reproducible gene targeting procedure developed for the monocot rice, using a positive (hygromycin resistance gene)/negative (diphtheria toxin A fragment gene) selection system (Terada et al. 2002), was successfully applied to M. polymorpha (Ishizaki et al. 2013a). The high frequency of the Agrobacterium-mediated sporeling transformation is advantageous for a gene targeting strategy based on the positive/negative selection system. In the case of the targeted knockout of the NOPPERABO1 (NOP1) gene, about 2% of transformants that passed the positive/negative selection showed the expected morphological phenotype, and the NOP1 locus in these plants was successfully disrupted by HR. This procedure has been applied to the targeted disruption of other genetic loci, including MpGI, MpFKF (Kubota et al. 2014), MpPHOT (Komatsu et al. 2014) and MpTAA (Eklund et al. 2015). To date, the statistics suggest that screening of fewer than 400 candidate transformants is sufficient to isolate at least one knockout clone for most non-essential genes. Gene targeting mediated by HR should be useful not only to generate knockouts but also to modify targeted sequences and fusion tags by knock-in. The tissue to be transformed is haploid; therefore, it is theoretically impossible to generate knockout plants for essential genes in M. polymorpha. To overcome this problem, conditional gene knockout strategies would be useful. Recently, Nishihama et al. (2015a) developed a heat- and dexamethasone-controllable gene expression/deletion system in M. polymorpha by expressing the P1-phage Cre site-specific recombinase fused with the ligand-binding site of the rat 6
glucocorticoid receptor, under the control of an endogenous heat-inducible promoter. This system could be used to generate conditional knockout mutants for essential genes in M. polymorpha. As an alternative, an inducible artificial microRNAmediated knockdown strategy is available in M. polymorpha, which provides a useful tool to study gene function, particularly if null mutations are potentially lethal (Flores-Sandoval et al. 2015a).
Genome editing by the CRISPR/Cas9 system Recently, a simple genome editing technology, the CRISPR (clustered regulatory interspaced short palindromic repeats)/ Cas9 (CRISPR-associated protein 9) system, which is based on a prokaryote-specific adaptive immune system (Wiedenheft et al. 2012), has been developed for eukaryotic model organisms, including plants (Belhaj et al. 2014). CRISPR/Cas9-based targeted mutagenesis has been demonstrated in M. polymorpha (Sugano et al. 2014). Multiple mutant alleles that showed the expected auxin-resistant phenotype were directly established in the gametophyte generation, using the endogenous U6 promoter-driven guide RNA designed to disrupt the gene encoding AUXIN RESPONSE FACTOR1 (ARF1) and the Cas9 gene under control either of the CaMV 35S or MpEF1a promoter (Sugano et al. 2014). The CRISPR/Cas9 system should allow simple and rapid genome modification of target loci in M. polymorpha.
Mutagenesis and Forward Genetics Forward genetics is a powerful approach to link genes to a particular phenotype, and has been used to obtain information about gene function in plants. Physcomitrella patens is suited for reverse genetic analysis by virtue of its ability to undertake targeted transgene integration by homologous recombination (Schaefer and Zryd 1997). However, the forward genetic approach has been limited in the moss because of large-scale genome duplication events (Strotbek et al. 2013). Marchantia polymorpha has a significant potential as a model haploid plant for forward genetics, because of its low genetic redundancy. A T-DNA tagging strategy to generate mutants has been successful in M. polymorpha (Ishizaki et al. 2013b, Ueda et al. 2013), using the efficient Agrobacterium-mediated transformation protocol (Ishizaki et al. 2008), and the genes affected were readily identified. A morphological mutant showing impaired air-chamber formation, nop1, was isolated by screening the T1 generation from 10,000 T-DNA-tagged lines (Ishizaki et al. 2013b). Linkage analysis of the nopperabo1 (nop1) phenotype and the inserted T-DNA, as hygromycin resistance, was carried out using an F1 population generated by crossing of nop1 and a wild-type accession. The responsible gene, NOP1, was isolated by identifying flanking sequences of the T-DNA by PCR and confirmed by genetic complementation by the Agrobacteriummediated transformation protocol using regenerating thalli (Ishizaki et al. 2013b). Assuming that 1–5 copies of T-DNAs are randomly inserted to the nuclear genome of 280 Mb in size, approximately 200,000 independent transformants are estimated to be sufficient
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In M. polymorpha, two constitutive promoters, the CaMV 35S promoter and the endogenous ELONGATION FACTOR 1a (MpEF1a) promoter, have been characterized (Althoff et al. 2014) and used for both gene knockout and overexpression studies (Kajikawa et al. 2003, Kubota et al. 2014, Sugano et al. 2014, Eklund et al. 2015, Flores-Sadoval et al. 2015b, Kato et al. 2015). The CaMV 35S and MpEF1a promoters are both capable of driving strong expression in M. polymorpha, but there are significant differences in terms of their spatial distributions. The CaMV 35S promoter has a weak activity in the meristematic zones, but strong in the other part of thallus, while the MpEF1a promoter is strong at the meristematic zone in thallus and generally ubiquitous in the other tissues, such as gemmae and gametangiophores (Althoff et al. 2014). Furthermore, it was reported that the CaMV 35S promoter is not active in oil body cells of M. polymorpha, which contain liverwort-specific organelles, termed oil bodies, which are filled with specific isoprenoids, phenolics and bisbenzyl compounds (Kanazawa et al. 2015). Thus, the MpEF1a promoter would be preferable over the CaMV 35S promoter for overexpression of transgenes in the thallus, gemma and gametangiophore.
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Plastid Transformation Methods for plastid transformation have been established in several land plant species, including tobacco and P. patens, and have been used widely in basic research and for biotechnological applications (Day and Goldschmidt-Clermont 2011, Bock 2014). In M. polymorpha, efficient plastid transformation protocols have been established for suspension-cultured cells, as well as for sporelings (Chiyoda et al. 2007, Chiyoda et al. 2014). This strategy was used successfully for the targeted disruption of the plastid-encoded ndhB gene via insertion of the aadA cassette into the 30 exon (Ueda et al. 2012). Despite multiple copies of the plastid genome, homoplasmic transformants of thalli, where all copies of the plastid genome are transformed, could be obtained immediately after primary selection by recovering gemmae from transformed sporelings, whereas it takes 12–16 weeks of repeated subculture to obtain homoplasmic transformants from suspension-cultured cells (Chiyoda et al. 2014). In combination with the transformation system for the nuclear genome, this efficient strategy for plastid genome transformation should aid basic research on plastids, such as replication and gene expression, as well as various applications to plant biotechnology.
Conclusion and Perspective Despite the large evolutionary distance and the difference in life cycle between vascular plants and bryophytes, accumulating evidence indicates that many regulatory pathways in vascular plants are also found in bryophytes. This supports a model of plant evolution whereby many features found in vascular plants were developed by harnessing gene regulatory networks that had already been acquired in the common ancestor of bryophytes and vascular plants (Pires and Dolan 2012, Kubota et al. 2014, Flores-Sandoval et al. 2015b, Kato et al. 2015). Thus, comparative molecular characterization of the regulatory
machineries in bryophytes and vascular plants should not only provide in-depth insights into plant evolution, but also increase our understanding of the principal regulatory modules in land plants. The liverwort M. polymorpha has substantial potential as a model system for plant biology because of its critical phylogenetic position in the evolution of land plants; its conserved, yet less complex, developmental pattern and responses to plant growth factors and environmental stimuli; and its gametophyte-dominant life cycle, which renders genetic analyses less complicated. Tools and resources for reverse and forward genetics should accelerate the functional analysis of genes in M. polymorpha and reveal the evolution and diversity of regulatory systems in land plants. Downloaded from http://pcp.oxfordjournals.org/ at University of Cambridge on August 18, 2015
for saturation mutagenesis to cover >95% of the genes in M. polymorpha, and it is feasible to isolate a number of transformants using about 200–300 sporangia for transformation. The T-DNA tagging strategy in M. polymorpha, powered by the high transformation efficiency and ongoing whole-genome sequencing project, will promote forward genetics in this basal land plant. Marchantia polymorpha can also be chemically or physically mutagenized. For example, Millar et al. (1962) isolated and characterized 12 nutritionally deficient mutants of M. polymorpha from X-ray-irradiated gemmae. However, there has been no report of the successful identification of underlying mutations causing the phenotypic variations to date. The use of nextgeneration sequencing technology will provide a high-throughput platform to identify responsible genes because of the haploidy and small genome size of M. polymorpha. The forward genetic approach in M. polymorpha should contribute to our understanding of molecular mechanisms that are difficult to uncover in other model plants, where such an approach is challenging because of genetic redundancy.
Funding This work was supported by the Ministry of Education, Culture, Sports, Science and Technology [Grant-in-Aid for Scientific Research on Innovative Area (No. 25113009 to T.K.; 25119711 and 15H01233 to K.I.)]; the Japan Society for the Promotion of Science [Grant-in-Aid for Scientific Research (B) (15H04391 to K.I.); Scientific Research (C) (24570048 to R.N.)]; Challenging Exploratory Research [26650095 to T.K.]; the Asahi Glass Foundation [to K.I.]; the SUNTORY Foundation [to K.I.].
Acknowledgments The authors thank Hideyuki Takami for preparation of the illustrations for this manuscript.
Disclosures The authors have no conflicts of interest to declare.
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