The Moss Physcomitrella patens, Now and Then

Mosses (Musci, Bryophytaea) are one of the oldest groups of land plants present among the earth's flora. They originated 500 million years ago (for a recent discussion of the time scale involved, see Heckman et al., 2001) and are currently represented by approximately 10,000 species that colonize diverse habitats that range from high mountains to deep forests and from Antarctica to deserts. Evolutionary studies support the monophyletic origin of land plants and indicate that bryophytes may form a sister clade with tracheophytes, although the exact relationship between bryophytes (hornworts, liverworts, and mosses) and vascular plants is still a matter of debate (Kenrick and Crane, 1997; Nickrent et al., 2000). This nevertheless places mosses at an evolutionary position that is ideal for comparative studies of the evolution of biological processes in land plants. Their life cycle is dominated by a photoautotrophic haploid gametophytic generation that supports a relatively simple and mainly heterotrophic diploid sporophyte. The haploid gametophyte itself is characterized by two distinct developmental stages: the protonema, a filamentous network of chloronemal and caulonemal cells, which develop by apical growth and cell division of apical and subapical cells; and the gametophore or leafy shoot, which differentiates by caulinary growth from a simple apical meristem (the bud). The latter is made up of a photosynthetic non-vascularized stem, which carries the leaves and the reproductive organs and of filamentous rhizoids that arise from the base of the stem (for review, see Reski, 1998). A, The life cycle of P. patens. (1) Spores, (2)light-dependent spore germination generating primary chloronemata,(3) 15-d-old protonemal colony, (4) branching chloronema, (5) caulonema cells characterized by an oblique cell wall and a small number of chloroplasts, (6)filamentous two-dimensional structure switches to three dimensions with the appearance of young bud, (7) young bud developing to form the leafy shoot of the gametophore, (8) a fully developed moss leafy gametophyte (4 weeks old), (9) an archegonium (female structure) dissected out from a gametophore,(10) two antheridia (male structures) dissected out from a gametophore. P. patens being a monoecious species both structures are present on the same plant and appears after a cold treatment (15°C for 3 weeks), (11) after fertilization by swimming spermatozoids (under water), the egg cell develops into a small diploid sporophyte and within its capsule meiosis occurs leading to spore formation (approximately 5,000 spores per capsule). The whole cycle can be achieved under optimal conditions in less than 12 weeks. B, Developmentally regulated expression of the GUS gene in gene- and enhancer-trap lines of P. patens (by courtesy of Yuji Hiwatashi and Mitsuyatsu Hasebe). A, Chloronema cells of a gene-trap line YH261. B, A gametophore of a gene-trap line YH206 showing the rhizoid cells predominantly stained. C, A gametophore of an enhancer-trap line ET41. The apex of the gametophore is stained. D, A gametophore of an enhancer-trap line ET326. Leaf blades are stained. E, An archegonium of a gene-trap line YH126; the ventral cell and egg cell are stained. The bars in A, B, C, and E = 50 μm; in D = 500 μm. The modern history of P. patens goes back to a seminal paper (Engel, 1968) describing the successful isolation and genetic analysis of auxotrophic mutants for thiamine, nicotinic acid, and para-aminobenzoate. This work prompted the groups of David Cove in Cambridge and later in Leeds and of Wolfgang Abel at the University of Hamburg to further develop P. patens as a model genetic system. Their work led to the isolation and biological characterization of several biochemical and developmental mutants generated by chemical mutagenesis (Ashton and Cove, 1977; Ashton et al., 1979; Abel et al., 1989; Cove et al., 1997). Techniques for genetic analysis were also developed; P. patensis self fertile and test crosses were found to be facilitated by the use of self-sterile but cross-fertile auxotrophic mutants (Courtice et al., 1978). One critical step was achieved with the successful isolation and efficient regeneration of protoplasts from young chloronemal filaments (Grimsley et al., 1977). The simultaneous development of polyethylene-glycol-mediated protoplast fusion made genetic dominance studies and complementation analysis of sterile mutants possible (Grimsley et al., 1977). These initial studies indicated that P. patens could be a useful model system for studying developmental genetics in plants (Cove, 1992). In the early eighties the major focus in plant research was the development of plant transformation methods using eitherAgrobacterium tumefaciens or polyethylene glycol (PEG)-mediated direct DNA transfer to protoplasts. These methods coupled with the newly discovered 35S promoter allowed a variety of selectable expression cassettes to be introduced into plants. The huge potential of these approaches also attracted a handful of scientists who realized their potential for studies on P. patens. As mosses do not interact with A. tumefaciens, the main focus was on direct transformation of protoplasts through various methods with an emphasis on PEG-mediated transformation. The first successful transformation was achieved by PEG-mediated DNA transfer into protoplasts only 10 years later using 35S-driven plasmids carrying antibiotic resistance markers (Schaefer et al., 1991). Biolistic delivery of genes has since been used with partial success (Knight et al., 1995). When sequential transformation of P. patens was attempted (for example by retransforming a transgenic strain already resistant to kanamycin with the same transformation plasmid containing instead the hygromycin resistance marker) it was observed that the resulting transformed plants almost always displayed a very close genetic linkage of the two resistance transgenes. These genetic data suggested that the second plasmid integrated at the previously generated artificial locus by homologous recombination, providing the first evidence for efficient gene targeting in P. patens (Schaefer, 1994; Kammerer and Cove 1996). Transformation experiments using cloned P. patensgenomic sequences confirmed this hypothesis leading to the conclusion that, contrary to other plants studied to-date, the integration of foreign DNA sequences into the genome occurs predominantly at targeted locations by homologous recombination (Schaefer and Zrÿd, 1997). These studies opened the door for high efficiency targeted mutagenesis in a plant species (see Schaefer [2001] for a detailed account of gene targeting in P. patens and other eukaryotes). Targeted mutagenesis by gene targeting is the ultimate method for studying gene function in biological systems as it enables the direct generation of loss-of-function and point mutations in the gene under study. It is used as a routine method for functional genomic studies in bacteria and yeast since transfected DNA integrates essentially at targeted locations by homologous recombination. Yet, in multicellular eukaryotes, this methodology is not accessible since integration of foreign DNA sequences occurs at random locations in the genome by illegitimate recombination with a frequency that is orders of magnitude higher than that observed for homologous recombination. The only notable exception is in mice where embryonic stem cells transformation is used to generate predetermined mutations in the mouse genome (Müller, 1999). In the whole plant kingdom, gene targeting is still unfeasible as a routine procedure (Vergunst and Hooykaas, 1999) with the sole exception of P. patens. The potential of P. patens was soon demonstrated by the publication of several key papers characterizing specific gene disruptions. Strepp and coworkers (1998) disrupted theftsZ1 gene, a moss homolog of a bacterial protein that shares structural features with tubulin and is an essential component of the prokaryotic cell division machinery. Cells of ftsZ1knock-out P. patens strains are characterized by the presence of a single huge chloroplast per cell instead of the approximately 50 chloroplasts found in normal cells. This phenotype resulted from dysfunctional chloroplast division and provided functional evidence for the involvement of FtsZ protein in the process. Another group interested in the metabolism of unsaturated fatty acids was able to show that disruption of a Δ-6 desaturase gene was responsible for a severe alteration of the lipid profile ofP. patens (Girke et al., 1998). The exquisite specificity of gene targeting was assessed in a successful experiment designed to disrupt one specific member of the highly conserved chlorophyll a/b-binding protein (Cab) multigene family (Hofmann et al., 1999). In a study of the proteasome-ubiquitin-mediated proteolytic pathway of P. patens, Girod and coworkers (1999) successfully knocked-out the mcb1 gene. The Mcb1 protein is a component of the 19S regulatory complex of the highly conserved 26S proteasome present in all eukaryotes and its function remains to be elucidated. Mcb1 knock-out performed in yeast did not reveal any strong phenotype, except an increased sensitivity to amino acid analogs, whereas it led to embryonic lethality in mouse (Kawahara et al., 2000). Remarkably, the P. patens knockout displayed a developmental phenotype characterized by impaired bud differentiation. This study illustrates out how critical it is that different multicellular organisms are used to study the biological functions of proteins involved in complex regulatory pathways. The success and the potential of gene targeting in P. patens has also prompted heavy private investment from the German agrochemical company BASF, which has developed a large expressed sequence tag (EST) database (more than 110,000 entries to date representing more than 20,000 genes). In the public domain, the EST program involving the University of Leeds (UK) in collaboration with Washington University (St. Louis, MO) has totaled 14,000 entries to-date (Quatrano et al., 1999). Other groups, notably in Japan, have recently reported their commitment to develop new EST databases. The genome size of P. patens is estimated to be around 460 Mb distributed among 27 chromosomes, which corresponds to the size of the rice genome (Reski, 1999), and preliminary analyses of ESTs and of genomic sequences clearly indicate that P. patens and other land plant genes are highly similar, at the level of both intron-exon structure and codon usage. We have mentioned only the first published papers from a selection of the 160 to 170 papers dedicated to P. patens until today (a list is available athttp://www.unil.ch/lpc/docs/physco1.html). Recently, there has been a rapid growth of P. patensresearch as was clearly demonstrated during the last international MOSS meetings held in Switzerland (MOSS 2000 Abstracts can be found athttp://www.unil.ch/lpc/docs/moss2000.pdf) and in Japan (MOSS 2001 Abstracts can be found athttp://www.nibb.ac.jp/%7Emhasebe/MOSS2001/index.html). The complete sequence of the Arabidopsis genome (The Arabidopsis Genome Initiative, 2000) and the million of sequences from other plants deposited in the databases provides ample scope for functional analysis of plant genes. Sophisticated molecular genetic tools that have been developed in model plant systems, such as gene tagging and gene trapping approaches or collections of insertional mutants (for a discussion, see Bouchez and Hofte, 1998), provide an extremely valuable set of methods for deciphering plant gene functions. What is the additional potential of the P. patensmodel system and how can it be used by the biological community for functional genomic studies? Sequencing full or partial genomes is only the first part of a greater challenge confronting biology today. The next step is to decipher the function of genes and to unravel the complex interactions governing genetic networks. Global approaches aimed at describing the expression levels of sets of genes under specific experimental conditions (transcriptomic), and the protein patterns that follow (proteomic) provide extremely valuable information on the genetic networks controlling specific biological processes. Yet a precise understanding of how these proteins function and interact with each other in a cellular context also requires the ability to introduce precise alterations within specific components of these networks. In this respect P. patens is poised to fill a gap in the tools that are presently available for studying the function of proteins in vivo. Typical vector design used in P. patens transformation. A, The insertion vector carries a genomic fragment (dark gray) beside a selectable Targeted integration is characterized by the insertion of one or several of the vector through homologous recombination with the genomic The resulting is a gene by B, A vector carries a selectable between two genomic occurs through two homologous recombination The is a function through is the of such a and could be designed to point Sophisticated tools only to systems have been used recently in P. patens by the group of in to genes et al., Hiwatashi et al., mutagenesis et al., was used to generate of moss genomic sequences by the insertion of a bacterial carrying a gene- or enhancer-trap GUS were at of and for P. patens transformed with gene- and enhancer-trap This is 10 higher than of trapping observed in Arabidopsis by illegitimate different resulting from such an each for the expression of the GUS gene. of lines at different developmental primary to illustrates that the whole life cycle of P. patens is accessible with this high these tools in it is possible to use P. patens to biological that be studied using other plants the of both systems for the analysis of protein function in and in a for a or a critical protein and promoter at the of a gene to a new or the function of plant genes through are of can only be in P. patens. research on the development of large collections of knock-out the involved and the time to a transgenic mouse with the of use and the time to a targeted in P. patens, plant patens are at the of functional genomic of genome structure and to functional genomic approaches in Arabidopsis and P. patens in the of the studied not in the of the studied at locations or with of genome structure and to functional genomic approaches in Arabidopsis and P. patens in the of the studied not in the of the studied at locations or with do bryophytes and to each other and how have these groups the molecular the of biological processes in various organisms is one of the in in this understanding the between function and structure of genes is Evolutionary developmental genetics is a that could from more studies on P. patens developmental biology and from the of large EST databases. are to by gene by a with in the whole of the it is of to study such gene in plant groups other than and to molecular with developmental We the with two The first example a family of DNA proteins present in plants and proteins are characterized by the presence of three whereas plant proteins have only two genes). Recently, genes have been patens and Arabidopsis et al., 2000). The amino acid sequences of their show a high to of and less to the proteins from plants. This that proteins containing three the of the and plant genes may have a conserved function in eukaryotes, whereas the plant genes predominantly which during plant and have been in P. patens and functional studies by targeted mutagenesis could on their in plant The second example with proteins involved in the of the of the genes that and reproductive development to the and genes and P. patens have been and with the higher plant genes and and et al., these it that the of (a family of containing the of the from the leading to vascular plants. the other the of genes observed in may have after the of both since genes patens the function of the or genes patens may to the of the moss developmental that to the shoot apical meristem or meristem that are essential in the development of vascular plants. this information with that available from plants may a model that can account for the evolution of development in land plants et al., In both reported the of analyses of the expression and of the knockout phenotype of the genes. In the the ability patens to introduce specific point mutations in these genes to the of of specific protein In more can that the functional of moss genes governing and or developmental processes with of the genes be highly for plant are with major of to the evolution of land the of the of plants are very in this respect in that the of the evolutionary group are characterized on the one by bryophytes with a haploid gametophytic generation and on the other by modern which a diploid first it is not are the evolutionary the appearance of a diploid in plants. with diploid genomes provides in the which the from the of but it also the time of and this the of of the of and recombination of specific sets of genes in bryophytes and on this to the last is the of homologous recombination and the of the of homologous DNA have been to the homologous recombination observed transformation ofP. patens (for further discussion, see the first is to the transformation per the second is to possible specific of the of the haploid gametophyte and the with in protoplasts. The large of DNA that are to protoplasts in the presence of high be an the between homologous and illegitimate integration but there is evidence available that transformation methods with gene targeting The that the haploid gametophyte a for homologous DNA integration with the sporophyte has to be in mosses and plants. to the gametophyte is transformation of the of or during be one but possible not provide evidence this this is as can that in the diploid that homologous recombination that to genetic between the other in a haploid such are not whereas homologous DNA be a critical for the of the the hypothesis from the that P. are highly cells at the during the transformation this is gene targeting efficiency is not and further (Reski, 1999). experimental approaches the characterization of the and proteins involved in and homologous recombination which have been conserved through We already a from and yeast studies and can that mutations homologous recombination or in may on this that genes responsible for efficient gene targeting be for for gene targeting in other We not the whole of that has the years on this (for further 1997; Reski, 1998), but instead on more recent P. patens, is a life in an other it is on for its but can of P. patens have an for for their whereas the and the leafy gametophore are to and to and to or An example of study in in the early filamentous growth of a moss subapical chloronemal cell of a P. patens transgenic plant showing the of is are between bryophytes and and 1997). and acid among have been studied in P. patens by isolation (Cove, 1992). P. patens a in development from a two structure apical growth (the to a three structure (the which further into the leafy shoot by caulinary This is one of the main that can be by and have been to a critical in the of cell and in the branching and of the cells et al., 1997). The to growth occurs in a initial cell from a subapical caulonema are to the the that occurs in approximately of the occurs in of cells and This provides a experimental to study the of of et al., 1998). In this P. patens has a in that cell can be from the spore through the and all the to the more complex leafy shoot and reproductive This is among model systems in developmental biology and for the success of the as model in rhizoids from the part of the these are organs that can be as the functional of the system are also The blades are on a the stem conditions of leading to the formation in the apical part of the shoot of the reproductive organs (the antheridia and the When is the from the antheridia to the and the single egg cell to a sporophyte that through meiosis spores within a It is that development of the sporophyte to be Mosses are to interact with and other organisms and has been reported the ofP. patens to This which is not very different from the of Arabidopsis years the of research in and also the of the plant in its A number of be used to this among the moss with highly or or using higher plant or to specific It is that several of plant resistance genes can be found in the EST molecular and genetic approaches but new information is at a rapid and lipid metabolism genes have been and characterized (Girke et al., and EST collections a large number of sequences to primary these data can already that P. patens appears be very to the of other land plants. We can that deciphering the of critical in photosynthetic organisms can be up in by using high efficiency targeted mutagenesis in P. patens. P. patens is an plant to work with and requires large of the tools for high mutagenesis have been on this are found to and are efficient gene targeting in P. the precise mutagenesis of specific sequences in their in a plant can be to and other as as to sequences that genes of targeting also enables the direct tagging or trapping of genes to their function in vivo. As by the of yeast and mouse embryonic stem cells, efficient gene targeting is essential an is to be developed as a model system. In the of efficient gene targeting coupled with a fully and genome has allowed to and functional to simple gene expression P. patens with the same to gene functions in plants. gene targeting in P. patens provides a new for plant research and can that it be used during the this it is highly that an genome be developed for P. its genome size of 460 and the whole genome could be in in less than are only with the to develop and a new to studying biological not the provided by P. patens. We Yuji Hiwatashi and for for providing with of gene- and enhancer-trap P. patens for providing with the of the chloronemal and for critical of the