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David J. Cove - One of the best experts on this subject based on the ideXlab platform.

  • Chemical and UV mutagenesis of spores and protonemal tissue from the moss Physcomitrella patens.
    Cold Spring Harbor protocols, 2009
    Co-Authors: David J. Cove, Pierre-françois Perroud, Audra J. Charron, Stuart F. Mcdaniel, Abha Khandelwal, Ralph S. Quatrano
    Abstract:

    This protocol describes how to mutagenize spores and protonemal tissue from moss (Physcomitrella patens) using chemicals or ultraviolet (UV) light. Spores are mutagenized using the alkylating agents N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and ethyl methanesulfonate (EMS), and protonemal tissue is mutagenized with NTG and UV light. Compared to alkylating agents, UV is less effective as a mutagen, but it may be advantageous because it is less hazardous and may not lead to clustered lesions.

  • Transformation of the moss Physcomitrella patens using direct DNA uptake by protoplasts.
    Cold Spring Harbor protocols, 2009
    Co-Authors: David J. Cove, Pierre-françois Perroud, Audra J. Charron, Stuart F. Mcdaniel, Abha Khandelwal, Ralph S. Quatrano
    Abstract:

    This protocol describes how to transform moss (Physcomitrella patens) protoplasts using polyethylene glycol (PEG)-mediated DNA uptake. The transformation rates for direct uptake by protoplasts of DNA with and without genomic sequence (a targeting construct) are typically 10 and 10, respectively. (These are the frequencies of stable transformants among regenerants surviving the transformation procedure.)

  • Physcomitrella patens: mosses enter the genomic age.
    Current opinion in plant biology, 2007
    Co-Authors: Ralph S. Quatrano, Pierre-françois Perroud, Stuart F. Mcdaniel, Abha Khandelwal, David J. Cove
    Abstract:

    The sequenced genome of the moss Physcomitrella patens provides a powerful tool for comparative analyses of land plant genomes. In parallel, several tools for studying gene function have been developed in P. patens, including RNA interference, inducible promoters and gene targeting, a unique attribute of this plant system. The results of these initiatives are now being realized. For example, transcriptomic analyses illustrate commonalities among plant lineages in gene content, structure, and regulation. Transgenic studies show that the regulatory factors ABSCISIC ACID INSENSITIVE3 (ABI3) and LEAFY (LFY) have molecular functions that are conserved between moss and angiosperms, in spite of the fact that they function in non-homologous tissues. Future work in P. patens will contribute to our understanding of the molecular basis of plant development and evolution.

  • the moss Physcomitrella patens
    Annual Review of Genetics, 2005
    Co-Authors: David J. Cove
    Abstract:

    AbstractThe moss Physcomitrella patens, like seed plants, shows alternation of generations, but its gametophyte, the haploid phase of the life cycle, is dominant, making it ideal for genetic studies. Crosses show direct segregations, so F2 or test crosses are unnecessary. Mutagenesis yields mutants, the phenotype of which is directly evident. Haploid tissue can be propagated vegetatively, allowing the maintenance of mutants blocked early in development. Protoplasts, isolated from filamentous gametophytic tissue, regenerate directly into filamentous tissue, providing an abundant supply of single haploid cells for transformation. Recombination occurs at a high frequency between genomic sequences in transforming DNA and the corresponding chromosomal sequences, allowing precise inactivation or modification of genes. RNAi technology allows the inactivation of the expression of gene families and the partial knockdown of essential genes. Over 100,000 ESTs have been sequenced and annotated, and sequencing of the ...

  • The MossPhyscomitrella patens - The Moss Physcomitrella patens
    Annual review of genetics, 2005
    Co-Authors: David J. Cove
    Abstract:

    The moss Physcomitrella patens, like seed plants, shows alternation of generations, but its gametophyte, the haploid phase of the life cycle, is dominant, making it ideal for genetic studies. Crosses show direct segregations, so F2 or test crosses are unnecessary. Mutagenesis yields mutants, the phenotype of which is directly evident. Haploid tissue can be propagated vegetatively, allowing the maintenance of mutants blocked early in development. Protoplasts, isolated from filamentous gametophytic tissue, regenerate directly into filamentous tissue, providing an abundant supply of single haploid cells for transformation. Recombination occurs at a high frequency between genomic sequences in transforming DNA and the corresponding chromosomal sequences, allowing precise inactivation or modification of genes. RNAi technology allows the inactivation of the expression of gene families and the partial knockdown of essential genes. Over 100,000 ESTs have been sequenced and annotated, and sequencing of the genome should be completed by the end of 2005.

Ralph S. Quatrano - One of the best experts on this subject based on the ideXlab platform.

  • Phospho-proteomic analysis of developmental reprogramming in the moss Physcomitrella patens
    Journal of proteomics, 2014
    Co-Authors: Xiaoqin Wang, Zhou, Lu Chen, Ralph S. Quatrano
    Abstract:

    Abstract One of the most common post-translational modifications is protein phosphorylation, which controls many activities of plant life. However, its role in the reprogramming of developmental pathways of plant cells remains elusive. Here, using Physcomitrella patens , we characterize the phospho-proteome for protonemata, protoplasts made therefrom, and protoplasts regenerated for 2 d. Through a titanium dioxide (TiO 2 )-based phospho-peptide enrichment method and liquid chromatography–tandem mass spectrometry (LC–MS/MS), more than 2000 phospho-proteins were identified. Among the 519 proteins with functional annotation in fresh protoplasts and protoplasts regenerated for 2 d, proteins involved in epigenetic modification, post-transcriptional gene regulation, hormone signal transduction, and meristem maintenance have been previously reported to be important for developmental reprogramming. Several novel transcription factors including SWI/SNF complex protein, SNF2 family protein and MADS-domain transcription factor appear to be important in developmental reprogramming plant cells. Phosphorylation of marker proteins such as somatic embryogenesis receptor kinase and NAC transcription factor, suggests that this post-translational modification is vital for the cell's ability to adjust its developmental program. Together, our study presents a more complete understanding of the plant cell's developmental reprogramming. Biological significance Protoplast regeneration is an ideal model system for investigating developmental reprogramming in plants. Here, for Physcomitrella patens , we characterize the phospho-proteome for protonemata, protoplasts made therefrom, and for protonemata regenerated from the protoplasts for 2 d. Among the 519 proteins with functional annotation in fresh protoplasts and protoplasts regenerated for 2 d, proteins involved in epigenetic modification, post-transcriptional gene regulation, hormone signal transduction, and meristem maintenance have been reported to be important for expression of developmental reprogramming. Together, our study presents a more complete understanding of the plant cell's developmental reprogramming.

  • Transformation of the moss Physcomitrella patens using direct DNA uptake by protoplasts.
    Cold Spring Harbor protocols, 2009
    Co-Authors: David J. Cove, Pierre-françois Perroud, Audra J. Charron, Stuart F. Mcdaniel, Abha Khandelwal, Ralph S. Quatrano
    Abstract:

    This protocol describes how to transform moss (Physcomitrella patens) protoplasts using polyethylene glycol (PEG)-mediated DNA uptake. The transformation rates for direct uptake by protoplasts of DNA with and without genomic sequence (a targeting construct) are typically 10 and 10, respectively. (These are the frequencies of stable transformants among regenerants surviving the transformation procedure.)

  • Chemical and UV mutagenesis of spores and protonemal tissue from the moss Physcomitrella patens.
    Cold Spring Harbor protocols, 2009
    Co-Authors: David J. Cove, Pierre-françois Perroud, Audra J. Charron, Stuart F. Mcdaniel, Abha Khandelwal, Ralph S. Quatrano
    Abstract:

    This protocol describes how to mutagenize spores and protonemal tissue from moss (Physcomitrella patens) using chemicals or ultraviolet (UV) light. Spores are mutagenized using the alkylating agents N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and ethyl methanesulfonate (EMS), and protonemal tissue is mutagenized with NTG and UV light. Compared to alkylating agents, UV is less effective as a mutagen, but it may be advantageous because it is less hazardous and may not lead to clustered lesions.

  • Physcomitrella patens: mosses enter the genomic age.
    Current opinion in plant biology, 2007
    Co-Authors: Ralph S. Quatrano, Pierre-françois Perroud, Stuart F. Mcdaniel, Abha Khandelwal, David J. Cove
    Abstract:

    The sequenced genome of the moss Physcomitrella patens provides a powerful tool for comparative analyses of land plant genomes. In parallel, several tools for studying gene function have been developed in P. patens, including RNA interference, inducible promoters and gene targeting, a unique attribute of this plant system. The results of these initiatives are now being realized. For example, transcriptomic analyses illustrate commonalities among plant lineages in gene content, structure, and regulation. Transgenic studies show that the regulatory factors ABSCISIC ACID INSENSITIVE3 (ABI3) and LEAFY (LFY) have molecular functions that are conserved between moss and angiosperms, in spite of the fact that they function in non-homologous tissues. Future work in P. patens will contribute to our understanding of the molecular basis of plant development and evolution.

  • Transgenesis of Physcomitrella patens
    2007
    Co-Authors: Sung Hyun Cho, Ralph S. Quatrano, Jeong Sheop Shin
    Abstract:

    The moss Physcomitrella patens has a simple life cycle, relatively few cell types during gametophytic growth, similar responses to plant growth regulators and environmental factors as seed plants, high regeneration capacity after homogenization, and dominant haploid generation, and as such, is an excellent experimental system. In addition to being able to use most molecular and biochemical methodologies, one can apply efficient gene targeting techniques to study gene function. For transformation, polyethyleneglycol-mediated protoplast transformation and particle bombardment-mediated biolistic delivery method are both efficient methods to introduce genes into gametophytic tissue. Finally, the genome of P. patens genome has been sequenced and assembled.

Ralf Reski - One of the best experts on this subject based on the ideXlab platform.

  • Microscopy of Physcomitrella patens sperm cells.
    Plant methods, 2017
    Co-Authors: Nelly A. Horst, Ralf Reski
    Abstract:

    Archegoniates (bryophytes, ferns and gymnosperms), such as the moss Physcomitrella patens, possess freely motile sperm cells (spermatozoids) which reach the egg cell via surface water. Although these motile flagellated sperm cells are a traditional botanical subject, they have not been thoroughly analysed in the flagship non-seed plant model species P. patens. Protocols are required to determine the behaviour of wild type sperms as a prerequisite for future research such as the characterization of mutants or factors that influence sperm number, morphology, viability and motility. Here, we present protocols for the observation of fixed, as well as live sperms utilizing a standard microscope at intermediate magnifications. Fixed samples can be used for the fast assessment of sperm number and morphology. To determine functionality, the observation of live sperms is required. Protocols for determining both sperm motility and viability are provided, allowing both parameters to be distinguished. These step-by-step protocols are particularly useful for researchers so far not familiar with the analysis of motile gametes and are meant to aid the establishment and improvement of these analyses in order to stimulate research on spermatogenesis in the moss model species P. patens.

  • The mitochondrial proteome of the moss Physcomitrella patens
    Mitochondrion, 2016
    Co-Authors: Stefanie J. Mueller, Sebastian N. W. Hoernstein, Ralf Reski
    Abstract:

    Extant basal land plants are routinely used to trace plant evolution and to track strategies for high abiotic stress resistance. Whereas the structure of mitochondrial genomes and RNA editing are already well studied, mitochondrial proteome research is restricted to a few data sets. While the mitochondrial proteome of the model moss Physcomitrella patens is covered to an estimated 15-25% by proteomic evidence to date, the available data have already provided insights into the evolution of metabolic compartmentation, dual targeting and mitochondrial heterogeneity. This review summarizes the current knowledge about the mitochondrial proteome of P. patens, and gives a perspective on its use as a mitochondrial model system. Its amenability to gene editing, metabolic labelling as well as fluorescence microscopy provides a unique platform to study open questions in mitochondrial biology, such as regulation of protein stability, responses to stress and connectivity to other organelles. Future challenges will include improving the proteomic resources for P. patens, and to link protein inventories and modifications as well as evolutionary differences to the functional level.

  • Biosynthesis of allene oxides in Physcomitrella patens.
    BMC plant biology, 2012
    Co-Authors: Julia Scholz, Florian Brodhun, Ellen Hornung, Cornelia Herrfurth, Michael Stumpe, Anna K. Beike, Bernd Faltin, Wolfgang Frank, Ralf Reski, Ivo Feussner
    Abstract:

    Background The moss Physcomitrella patens contains C18- as well as C20-polyunsaturated fatty acids that can be metabolized by different enzymes to form oxylipins such as the cyclopentenone cis(+)-12-oxo phytodienoic acid. Mutants defective in the biosynthesis of cyclopentenones showed reduced fertility, aberrant sporophyte morphology and interrupted sporogenesis. The initial step in this biosynthetic route is the conversion of a fatty acid hydroperoxide to an allene oxide. This reaction is catalyzed by allene oxide synthase (AOS) belonging as hydroperoxide lyase (HPL) to the cytochrome P450 family Cyp74. In this study we characterized two AOS from P. patens, PpAOS1 and PpAOS2.

  • Identification and characterization of NAGNAG alternative splicing in the moss Physcomitrella patens
    BMC plant biology, 2010
    Co-Authors: Rileen Sinha, Daniel Lang, Ralf Reski, Stefan A. Rensing, Andreas Zimmer, Kathrin Bolte, Matthias Platzer, Rolf Backofen
    Abstract:

    Background Alternative splicing (AS) involving tandem acceptors that are separated by three nucleotides (NAGNAG) is an evolutionarily widespread class of AS, which is well studied in Homo sapiens (human) and Mus musculus (mouse). It has also been shown to be common in the model seed plants Arabidopsis thaliana and Oryza sativa (rice). In one of the first studies involving sequence-based prediction of AS in plants, we performed a genome-wide identification and characterization of NAGNAG AS in the model plant Physcomitrella patens, a moss.

  • Molecular tools to study Physcomitrella patens.
    Plant biology (Stuttgart Germany), 2005
    Co-Authors: Wolfgang Frank, Eva L. Decker, Ralf Reski
    Abstract:

    Abstract: The moss Physcomitrella patens has become a suitable model plant system for the analysis of diverse aspects of modern plant biology. The research strategies have been influenced by the implementation of state-of-the-art cell culture and molecular biology techniques. The forthcoming completion of the Physcomitrella genome sequencing project will generate many open questions, the examination of which will rely on a diverse set of molecular tools. Within this article, we intend to introduce the essential cell culture and molecular biology techniques which have been adopted in recent years to make Physcomitrella amenable to a wide range of genetic analyses. Many research groups have made valuable contributions to improve the methodology for the study of Physcomitrella. We would like to apologise to all colleagues whose important contributions could not be cited within this manuscript.

Pierre-françois Perroud - One of the best experts on this subject based on the ideXlab platform.

  • PEATmoss (Physcomitrella Expression Atlas Tool): a unified gene expression atlas for the model plant Physcomitrella patens.
    The Plant journal : for cell and molecular biology, 2020
    Co-Authors: Noe Fernandez-pozo, Pierre-françois Perroud, Rabea Meyberg, Fabian B. Haas, Kristian K. Ullrich, Manuel Hiss, Sebastian Hanke, Viktor Kratz, Adrian F. Powell, Eleanor F. Vesty
    Abstract:

    Physcomitrella patens is a bryophyte model plant that is often used to study plant evolution and development. Its resources are of great importance for comparative genomics and evo-devo approaches. However, expression data from Physcomitrella patens were so far generated using different gene annotation versions and three different platforms: CombiMatrix and NimbleGen expression microarrays and RNA sequencing. The currently available P. patens expression data are distributed across three tools with different visualization methods to access the data. Here, we introduce an interactive expression atlas, Physcomitrella Expression Atlas Tool (PEATmoss), that unifies publicly available expression data for P. patens and provides multiple visualization methods to query the data in a single web-based tool. Moreover, PEATmoss includes 35 expression experiments not previously available in any other expression atlas. To facilitate gene expression queries across different gene annotation versions, and to access P. patens annotations and related resources, a lookup database and web tool linked to PEATmoss was implemented. PEATmoss can be accessed at https://peatmoss.online.uni-marburg.de.

  • An intragenic mutagenesis strategy in Physcomitrella patens to preserve intron splicing.
    Scientific reports, 2017
    Co-Authors: Ako Eugene Ako, Pierre-françois Perroud, Joseph Innocent, Viktor Demko, Odd-arne Olsen, Wenche Johansen
    Abstract:

    Gene targeting is a powerful reverse genetics technique for site-specific genome modification. Intrinsic homologous recombination in the moss Physcomitrella patens permits highly effective gene targeting, a characteristic that makes this organism a valuable model for functional genetics. Functional characterization of domains located within a multi-domain protein depends on the ability to generate mutants harboring genetic modifications at internal gene positions while maintaining the reading-frames of the flanking exons. In this study, we designed and evaluated different gene targeting constructs for targeted gene manipulation of sequences corresponding to internal domains of the DEFECTIVE KERNEL1 protein in Physcomitrella patens. Our results show that gene targeting-associated mutagenesis of introns can have adverse effects on splicing, corrupting the normal reading frame of the transcript. We show that successful genetic modification of internal sequences of multi-exon genes depends on gene-targeting strategies which insert the selection marker cassette into the 5' end of the intron and preserve the nucleotide sequence of the targeted intron.

  • Production of taxa-4(5),11(12)-diene by transgenic Physcomitrella patens
    Transgenic Research, 2009
    Co-Authors: Aldwin Anterola, Erin Shanle, Pierre-françois Perroud, Ralph Quatrano
    Abstract:

    Taxadiene synthase gene from Taxus brevifolia was constitutively expressed in the moss Physcomitrella patens using a ubiquitin promoter to produce taxa-4(5),11(12)-diene, the precursor of the anticancer drug paclitaxel. In stable moss transformants, taxa-4(5),11(12)-diene was produced up to 0.05% fresh weight of tissue, without significantly affecting the amounts of the endogenous diterpenoids ( ent -kaurene and 16-hydroxykaurane). Unlike higher plants that had been genetically modified to produce taxa-4(5),11(12)-diene, transgenic P. patens did not exhibit growth inhibition due to alteration of diterpenoid metabolic pools. Thus we propose that P. patens is a promising alternative host for the biotechnological production of paclitaxel and its precursors.

  • Transformation of the moss Physcomitrella patens using direct DNA uptake by protoplasts.
    Cold Spring Harbor protocols, 2009
    Co-Authors: David J. Cove, Pierre-françois Perroud, Audra J. Charron, Stuart F. Mcdaniel, Abha Khandelwal, Ralph S. Quatrano
    Abstract:

    This protocol describes how to transform moss (Physcomitrella patens) protoplasts using polyethylene glycol (PEG)-mediated DNA uptake. The transformation rates for direct uptake by protoplasts of DNA with and without genomic sequence (a targeting construct) are typically 10 and 10, respectively. (These are the frequencies of stable transformants among regenerants surviving the transformation procedure.)

  • Chemical and UV mutagenesis of spores and protonemal tissue from the moss Physcomitrella patens.
    Cold Spring Harbor protocols, 2009
    Co-Authors: David J. Cove, Pierre-françois Perroud, Audra J. Charron, Stuart F. Mcdaniel, Abha Khandelwal, Ralph S. Quatrano
    Abstract:

    This protocol describes how to mutagenize spores and protonemal tissue from moss (Physcomitrella patens) using chemicals or ultraviolet (UV) light. Spores are mutagenized using the alkylating agents N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and ethyl methanesulfonate (EMS), and protonemal tissue is mutagenized with NTG and UV light. Compared to alkylating agents, UV is less effective as a mutagen, but it may be advantageous because it is less hazardous and may not lead to clustered lesions.

Didier G. Schaefer - One of the best experts on this subject based on the ideXlab platform.

  • Usefulness of Physcomitrella patens for Studying Plant Organogenesis
    Methods of Molecular Biology, 2012
    Co-Authors: Sandrine Bonhomme, Catherine Rameau, Fabien Nogué, Didier G. Schaefer
    Abstract:

    In this chapter, we review the main organogenesis features and associated regulation processes of the moss Physcomitrella patens (P. patens) , the model plant for the Bryophytes. We highlight how the study of this descendant of the earliest plant species that colonized earth, brings useful keys to understand the mechanisms that determine and control both vascular and non vascular plants organogenesis. Despite its simple morphogenesis pattern, P. patens still requires the fi ne tuning of organogenesis regulators, including hormone signalling, common to the whole plant kingdom, and which study is facilitated by a high number of molecular tools, among which the powerful possibility of gene targeting/replacement. The recent discovery of moss cells reprogramming capacity completes the picture of an excellent model for studying plant organogenesis.

  • New Frontiers in Bryology - Physioloy, Molecular Biology and Functional Genomics - Principles of Targeted Mutagenesis in the Moss Physcomitrella patens
    New Frontiers in Bryology, 2004
    Co-Authors: Didier G. Schaefer, Jean-pierre Zryd
    Abstract:

    Highly efficient gene targeting is a unique feature of the moss Physcomitrella patens. It is now possible, in a multicellular eukaryote, to use the gene replacement technology which has been so successful in yeast. Careful design of transforming vector is nevertheless a key to successful generation of targeted plants.

  • A new moss genetics: targeted mutagenesis in Physcomitrella patens.
    Annual review of plant biology, 2002
    Co-Authors: Didier G. Schaefer
    Abstract:

    The potential of moss as a model system to study plant biology is associated with their relatively simple developmental pattern that nevertheless resembles the basic organization of the body plan of land plants, the direct access to cell-lineage analysis, their similar responses to plant growth factors and environmental stimuli as those observed in other land plants, and the dominance of the gametophyte in the life cycle that facilitates genetic approaches. Transformation studies in the moss Physcomitrella patens have revealed a totally unique feature for plants, i.e., that foreign DNA sequences integrate in the genome preferentially at targeted locations by homologous recombination, enabling for the first time in plants the application of the powerful molecular genetic approaches used routinely in bacteria, yeast, and since 1989, the mouse embryonic stem cells. This article reviews our current knowledge of Physcomitrella patens transformation and its unique suitability for functional genomic studies.

  • a new moss genetics targeted mutagenesis in Physcomitrella patens
    Annual Review of Plant Biology, 2002
    Co-Authors: Didier G. Schaefer
    Abstract:

    ▪ Abstract The potential of moss as a model system to study plant biology is associated with their relatively simple developmental pattern that nevertheless resembles the basic organization of the body plan of land plants, the direct access to cell-lineage analysis, their similar responses to plant growth factors and environmental stimuli as those observed in other land plants, and the dominance of the gametophyte in the life cycle that facilitates genetic approaches. Transformation studies in the moss Physcomitrella patens have revealed a totally unique feature for plants, i.e., that foreign DNA sequences integrate in the genome preferentially at targeted locations by homologous recombination, enabling for the first time in plants the application of the powerful molecular genetic approaches used routinely in bacteria, yeast, and since 1989, the mouse embryonic stem cells. This article reviews our current knowledge of Physcomitrella patens transformation and its unique suitability for functional genomic s...

  • Gene targeting in Physcomitrella patens
    Current opinion in plant biology, 2001
    Co-Authors: Didier G. Schaefer
    Abstract:

    Gene-targeting efficiency in the land plant Physcomitrella patens (Bryophyta) can only be compared with that observed in Saccharomyces cerevisiae. Sequencing programs and microbiological molecular genetic approaches are now being developed to unravel the precise function of plant genes. Physcomitrella patens, as the new 'green yeast', might well become a major tool for functional genomic studies of multicellular eukaryotes.