Bryophytes as Model Systems

Bryophytes as Model Systems Author(s): Andrew J. Wood, Melvin J. Oliver, David J. Cove Source: The Bryologist, 103(1):128-133. 2000. Published By: The American Bryological and Lichenological Society, Inc. DOI: http://dx.doi.org/10.1639/0007-2745(2000)103[0128:BAMS]2.0.CO;2 URL: http://www.bioone.org/doi/full/10.1639/0007-2745%282000%29103%5B0128%3ABAMS %5D2.0.CO%3B2

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The Bryologist 103(1), pp. 128–133 Copyright q 2000 by the American Bryological and Lichenological Society, Inc.

INVITED ESSAY New Frontiers in Bryology and Lichenology Bryophytes as Model Systems ANDREW J. WOOD1 Department of Plant Biology, Southern Illinois University-Carbondale, Carbondale, IL 62901-6509, U.S.A.

MELVIN J. OLIVER Plant Stress and Water Conservation Laboratory, Plant Stress and Genome Development Unit, 3810 Fourth Street, Lubbock, TX 79415, U.S.A.

DAVID J. COVE Leeds Institute of Plant Biotechnology and Agriculture (LIBA), University of Leeds, Leeds, LS2 9JT, U.K.

Abstract. Bryophytes have been powerful experimental tools for the elucidation of complex biological processes. Analysis of organisms from these ancient clades is an active and ongoing enterprise that will provide greater insight into the development, physiology, phylogenetics, and stress-induced cellular responses of plants. To maintain their relevance as experimental models, the analysis of mosses must expand to include modern molecular tools such as a knowledge of the genome via large-scale DNA sequencing, the ability to create transgenic individuals via transformation, and the capability to create gene knock-outs by homologous recombination. The availability of these molecular tools is limited when compared to flowering plants. However, in mosses such as Physcomitrella patens, Funaria hygrometrica, Ceratodon purpureus, and Tortula ruralis these tools are rapidly being developed for the study of molecular genetics. Efficient targeted gene disruption (i.e., homologous recombination) is a well-established tool in both yeast and murine cells that until recently was unknown in any plant model system. Recently, Schaefer and Zryd (1997) demonstrated that efficient homologous recombination occurs in P. patens. The ability to perform efficient homologous recombination in P. patens is at present unique amongst all plants and represents an extremely powerful technique for the functional analysis of plant genes.

Bryophytes have been powerful experimental tools or models for the elucidation of complex biological processes in plants (Cove et al. 1997; Reski 1998; Schumaker & Dietrich 1998). Analysis of organisms from these ancient clades is an active and ongoing enterprise that will provide greater insight into the development (Cove et al. 1997), physiology (Reski 1997), phylogenetics (Mishler et al. 1994) and stress-induced cellular responses of plants (Oliver & Wood 1997). Traditionally, good model systems have exhibited several key attributes: ease of growth and maintenance, fast generation time, amenable genetics (i.e., the ability to make directed crosses), and the ability to select for mutant phenotypes. Over the past 10 years, however, several key molecular attributes have been added to this list: a knowledge of the genome via 1 To whom correspondence should be addressed: [email protected]; FAX (618) 453-3441.

large-scale DNA sequencing, the ability to create transgenic individuals via transformation, and the capability to create gene knock-outs by homologous recombination. In the past, mosses have been attractive experimental plants because they exhibit the traditional attributes with the added advantage of a haploid gametophyte that allowed developmental mutants to be recovered with relative ease. To maintain their relevance as model systems, the analysis of mosses must expand to include these molecular attributes. The availability of these molecular tools is limited when compared to flowering plants. However, in mosses such as Physcomitrella patens (Cove et al. 1997; Reski 1999), Funaria hygrometrica (Schumaker & Dietrich 1998) Ceratodon purpureus (Hofmann et al. 1999), and Tortula ruralis (Wood et al. 1999) the study of bryophyte molecular genetics is underway and these tools are rapidly being developed. These four mosses will soon become ideal experimental models for the study of many plant genetic processes.

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A major limitation in the study of molecular genetics for any organism is the ability to assign the proper function to a nucleotide sequence. Assessing gene function in vivo requires a variety of genetic approaches that can be divided into two broad classes 1) forward genetics and 2) reverse genetics. Forward genetics encompasses the traditional isolation of mutants and the analysis of overexpressed and antisensed transgenes. Reverse genetics encompasses the perturbation of gene function by gene disruption. Efficient targeted gene disruption (i.e., homologous recombination) is a well-established tool in both yeast and murine cells that until recently was unknown in plant model systems. Schaefer and Zryd (1997) demonstrated that efficient homologous recombination occurs in P. patens (see below and Table 1). The ability to perform efficient homologous recombination (i.e., gene knock-outs) in P. patens is at present unique amongst all plants and represents an extremely powerful technique for the functional analysis of many plant genes. MOLECULAR ATTRIBUTES

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I. Large-scale DNA sequencing.—In the late 1980’s genome projects were established for several eukaryotic and prokaryotic organisms, most notably the Human Genome Project. The goal of these projects was the structural analysis of the respective genomes by determining the complete chromosomal DNA sequences. A genome project provides a complete description of the genome (i.e., both the coding and non-coding portions). For researchers interested in the expressed portion of the genome, or those lacking the resources to establish a genome project using their model organism, the analysis of large numbers of cDNA clones representing all transcripts present within the cell at any one time provides an alternative strategy. The structural analysis of genomes by large scale single-pass sequencing of randomly selected cDNA clones was pioneered using human brain tissue (Adams et al. 1991) and has subsequently been applied to a number of model vascular plants. The analysis of these randomly selected cDNA clones or expressed sequence tags (ESTs), given this name because they represent only genes expressed at a particular time or under a particular circumstance, has been an important technique for the discovery of new genes (Boguski 1995). This powerful analytical technique has recently been applied to P. patens treated with abscisic acid (Machuka et al. 1999) or cytokinin (Reski et al. 1998), desiccated T. ruralis (Wood et al. 1999), and untreated C. purpureus (D. J. Cove & R. S. Quatrano, unpublished results) (see Table 1). The published moss EST databases are relative-

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TABLE 1. Key molecular and genetic attributes of selected model bryophyte systems. I. STRUCTURAL GENOMICS A. Generation of Expressed Sequnce Tags (ESTs) Species Reference Physcomitrella patens Machuka et al. 1999; Reski et al. 1998 Tortula ruralis Wood et al. 1999 II. FUNCTIONAL GENOMICS A. Stable, PEG-mediated Species Physcomitrella patens Ceratodon purpureus

Transformation Reference Schaefer et al. 1991 Thummler et al. 1992; Zeidler et al. 1999 Funaria hygrometrica Schumaker, personal communication to the authors

B. Homologous Recombination Species Reference Physcomitrella patens Girod et al. 1999; Hofmann et al. 1999; Schaefer & Zyrd 1997; Strepp et al. 1998

ly small as yet, 253 ESTs derived from P. patens and 152 ESTs from T. ruralis with the majority of the ESTs (52% and 71%, respectively) having no significant similarity to previously characterized genes. We postulate several reasons why we obtain such a large number of novel EST clones in bryophytes 1) the under-representation of plant DNA sequences in the databases, 2) the near absence of bryophyte DNA sequences in the databases, and 3) the unique nature of the plant material (i.e., moss gametophytes). The continued generation of bryophyte EST databases represents a vital experimental tool. They aid in the identification of bryophyte genes homologous to previously characterized genes (i.e., those already deposited to the various molecular databases), and in bryophytes with unique phenotypes, such as the desiccation-tolerant T. ruralis (Wood et al. 1999; Wood & Oliver 1999), they will allow the identification of novel plant genes that are associated with important phenotypes. Detailed evolutionary studies will also be possible as the EST databases expand, both in number and species diversity. Mosses, and P. patens in particular, will be key model systems for the analysis of homologous gene function in plants. Finally, a large catalog of bryophyte genes, identified as ESTs, will be increasingly important for the assessment of gene function via homologous recombination (see below). II. Transformation.—The ability to introduce exogenous DNA into the genome of an organism, thereby creating a transgenic individual, is one of the core techniques of plant molecular genetics. A number of protocols have been developed and op-

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timized for the efficient transformation of plants: Agrobacterium tumefaciens-mediated transfer, particle bombardment (i.e., biolistics), electroporation, and PEG-mediated uptake of DNA into protoplasts (see Hansen & Wright 1999). Physcomitrella patens, the first successfully transformed moss species, was initially transformed via PEG-mediated uptake of DNA by protoplasts (Schaefer et al. 1991) and has subsequently been transformed using the biolistic procedure (Sawahel et al. 1992). For reasons not clearly understood, P. patens has been recalcitrant to A. tumefaciens-mediated transformation (C. D. Knight, University of Leeds, personal communication to DJC). Using the PEG-mediated protoplast delivery system optimized in P. patens, transformation procedures are currently being developed in C. purpureus (Thummler et al. 1992; Zeidler et al. 1999), T. ruralis (M. J. Oliver, unpublished results), and F. hygrometrica (K. S. Schumaker, University of Arizona, personal communication to AJW) (see Table 1). The foreign plasmid DNA used to transform plants, which includes the exogenous transgene(s) of interest, usually has no homology to the target genome. When such non-homologous DNA is introduced into an organism via one of the above transformation protocols, the plasmid DNA is inserted into the genome in an essentially random fashion. As a result, the expression of both the introduced gene and the selectable marker (i.e., antibiotic resistance) is often variable. This variation in gene expression is termed ‘‘position effect’’ and is hypothesized to reflect differences in the genomic environment at the site of integration which impact, either positively or negatively, transcription of the transgene. Nevertheless, this procedure is still a very useful tool in both the analysis of gene function and the control of gene expression by both cis and trans active gene promoter sequences. III. Homologous recombination.—Homologous recombination is the targeting and insertion of an exogenous DNA sequence to the corresponding homologous genomic sequence (Hofmann et al. 1999; Reski 1998, 1999). In stark contrast to the random DNA insertion that leads to position effects (see above), homologous recombination leads to the specific alteration of the target locus thus ensuring that expression is predictable and, if not negated, reflects the levels normal to the native gene. Gene targeting, or gene replacement, by homologous recombination is a well-characterized phenomena in yeast and murine cells (see Hofmann et al. 1999 and references therein). Homologous recombination does occur in flowering plants such as Arabidopsis (Reski 1998); however, it is an inefficient process that cannot be effectively exploited. Efficient homologous recombination in plants was first

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identified by Schaefer and Zyrd (1997) using P. patens (Table 1). The most common experimental use of homologous recombination is to target a gene for inactivation in order to investigate its normal function, a type of genetic ablation in some respects. There are two pathways for gene inactivation following homologous recombination between homologous exogenous transforming DNA and target moss genomic DNA, both of which are depicted in Figure 1. The majority of gene inactivation data generated using homologous recombination in P. patens has involved circular plasmid DNA and transformation via the PEG-mediated protoplast delivery system (Reski 1999). In this scenario, an internal homologous fragment of the targeted gene (b, c, d) is cloned in the plasmid adjacent to the selective cassette. A single cross-over between the circular plasmid and the genomic locus yields two incomplete copies of the target gene separated by plasmid DNA, resulting in its inactivation (Fig. 1A). The gene is inactivated as neither copy of the target gene is complete, one copy representing a 39 deletion and the other a 59 deletion. A second scenario involves a linear DNA fragment in which the center of the homologous exogenous sequence has been substituted for a plasmid-derived selective cassette (Fig. 1B). A double cross-over between a transformed genomic fragment and the targeted genomic locus (c) will result in a stable transgenic organism with an interrupted and thus inactive gene containing the plasmid-derived selective cassette. Recently, several genes have been disrupted in P. patens by homologous recombination in order to analyze the resulting phenotypic changes: the Cab multigene family (Hofmann et al. 1999), the multiubiquitin chain binding protein RNP10 (Girod et al. 1999), a D-6-acyl-lipid desaturase (Girke et al. 1998) and a moss homologue of the bacterial cell division protein ftsZ (Strepp et al. 1998). To more clearly illustrate the technique, we will detail the experiment involving the bacterial cell division protein ftsZ. Reski and his colleagues isolated a 1775 bp P. patens cDNA homologue of the ftsZ protein using a PCR-based strategy, designated PpFtsZ (Strepp et al. 1998). A linear DNA fragment was created for homologous recombination by flanking the nptII selective cassette with 247 bp of 59 cDNA sequence and 685 bp of 39 cDNA sequence from the PpFtsZ cDNA. Following transformation via the PEG-mediated protoplast delivery system, 51 independently stably transformed plants were isolated and subsequent analysis determined that 14% of the transgenic plants were the result of homologous recombination. Disruption of the P. patens PpftsZ gene produced plants with chloroplasts which were incapable of proper plastid division,

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FIGURE 1. Two pathways for gene inactivation following homologous recombination between transforming DNA and bryophyte genomic DNA. — A. Gene inactivation by a single cross-over between a circular plasmid and the genomic locus. An internal fragment of the targeted gene (b, c, d) is cloned adjacent to the selective cassette. A single cross-over yields two incomplete copies of the gene separated by plasmid DNA. Gene function is lost as neither copy of the gene is complete, one is a 39 deletion and the other a 59 deletion. — B. Gene inactivation by a double crossover between a transformed genomic fragment and the genomic locus. A selective cassette has been substituted for a central portion of the gene to be targeted, and has been used for transformation. A cross-over on both sides of the selective cassette will result in a stable transgenic organism with an inactive gene. For clarity, the chromosomal locus has been arbitrarily labeled to consist of five sub-regions, a, b, c, d & e.

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and so had cells which each contained a single large chloroplast. Homologous recombination clearly established the role of this previously unknown eukaryotic gene in chloroplast development. CONCLUDING REMARKS.—Homologous recombination is a powerful technique for the functional analysis of gene function in vivo. Targeted gene replacement (i.e., reverse genetics) by homologous recombination will allow geneticists to ‘‘engineer alleles’’ (rather than simple gene inactivation) and thereby study the role of specific domains, sub-domains, codons, or single base-pair substitutions in gene and/or enzyme function. Over the next few months, the complete sequence of the Arabidopsis genome will have been determined and made available to the scientific community. Bioinformatic analysis of this sequence database predicts that the Arabidopsis genome contains approximately 20,000 genes; however, less than 500 of these genes have been identified as visible mutations and placed on the genetic map. As the only land plant in which efficient homologous recombination is known to occur, P. patens will become an increasingly important model system for plant molecular genetics. In our opinion, P. patens will be exhaustively studied for two main purposes 1) as a model for homologous recombination in plants with the longterm goal of exporting the technology to angiosperm models such as Arabidopsis and 2) as a platform for the detailed molecular study of a wide variety of plant genes which are either difficult to analyze in their native systems or have no easily discernable phenotype. With respect to other mosses, investigating the capability of F. hygrometrica, C. purpureus, and T. ruralis to undergo homologous recombination will only add to their respective utilities as experimental models and may help elucidate the complex biochemical nuances of this and other crucial processes. ACKNOWLEDGMENTS The authors thank Barbara Crandall-Stotler (Southern Illinois University, Carbondale, IL) for critical comments upon the manuscript. This review was supported in part by a grant from USDA, National Research Initiative-Competitive Grants Program to AJ Wood (grant #9735100).

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