Tagged Mutagenesis and Gene-trap in the Moss, Physcomitrella patens by Shuttle Mutagenesis

The moss, Physcomitrella patens has been used as a useful materialin many fields, because of its simple body plan, ease of genetargeting, and other reasons. Although many mutants have beenreported, no method to isolate the corresponding genes was reported.We developed a gene tagging and gene-trap system in P. patensby using the shuttle mutagenesis technique, which has been usedin the budding yeast. In 5264 tagged lines, 203 mutants withaltered developmental or morphological phenotypes were obtained.In 129 of 4757 gene-trap lines, ß-glucuronidase (GUS)activity was detected in some tissue. Although multiple copiesof a tag were detected in many tagged lines by Southern analyses,most copies are likely integrated at the same locus accordingto PCR analyses.     

Dual-tagging gene trap of novel genes in Drosophila melanogaster

A gene-trap system is established for Drosophila. Unlike the conventional enhancer-trap system, the gene-trap system allows the recovery only of fly lines whose genes are inactivated by a P-element insertion, i.e., mutants. In the gene-trap system, the reporter gene expression reflects precisely the spatial and temporal expression pattern of the trapped gene. Flies in which gene trap occurred are identified by a two-step screening process using two independent markers, mini-w and Gal4, each indicating the integration of the vector downstream of the promoter of a gene (dual tagging). mini-w has its own promoter but lacks a polyadenylation signal. Therefore, mini-w mRNA is transcribed from its own promoter regardless of the vector integration site in the genome. However, the eyes of flies are not orange or red unless the vector is incorporated into a gene enabling mini-w to be spliced to a downstream exon of the host gene and polyadenylated at the 3' end. The promoter-less Gal4 reporter is expressed as a fusion mRNA only when it is integrated downstream of the promoter of a host gene. The exons of trapped genes can be readily cloned by vectorette RT-PCR, followed by RACE and PCR using cDNA libraries. Thus, the dual-tagging gene-trap system provides a means for (i) efficient mutagenesis, (ii) unequivocal identification of genes responsible for mutant phenotypes, (iii) precise detection of expression patterns of trapped genes, and (iv) rapid cloning of trapped genes.     

Use of an inducible reporter gene system for the analysis of auxin distribution in the moss<Emphasis Type="Italic"> Physcomitrella patens</Emphasis>

The plant hormone auxin plays a major role in a variety of growth and developmental responses, even in the more ancient plants-for example, cell differentiation in mosses. Nevertheless, almost nothing is known about the distribution of auxin during moss development. To address this question, we characterised auxin distribution in the moss Physcomitrella patens using auxin-inducible reporter gene systems. Stable transgenic Physcomitrella plants were produced expressing the beta-glucuronidase (GUS) gene driven by the auxin-inducible promoters GH3 and DR5, respectively. Both fusions showed remarkable differences with respect to auxin-induced promoter strength and expression kinetics. A detailed characterisation of the GUS expression pattern in different developmental stages revealed that the highest auxin concentrations were in dividing and ontogenetic young cells.     

Cryptochrome light signals control development to suppress auxin sensitivity in the moss Physcomitrella patens

The blue light receptors termed cryptochromes mediate photomorphological responses in seed plants. However, the mechanisms by which cryptochrome signals regulate plant development remain obscure. In this study, cryptochrome functions were analyzed using the moss Physcomitrella patens. This moss has recently become known as the only plant species in which gene replacement occurs at a high frequency by homologous recombination. Two cryptochrome genes were identified in Physcomitrella, and single and double disruptants of these genes were generated. Using these disruptants, it was revealed that cryptochrome signals regulate many steps in moss development, including induction of side branching on protonema and gametophore induction and development. In addition, the disruption of cryptochromes altered auxin responses, including the expression of auxin-inducible genes. Cryptochrome disruptants were more sensitive to external auxin than wild type in a blue light-specific manner, suggesting that cryptochrome light signals repress auxin signals to control plant development.     

Cyclin D-knockout uncouples developmental progression from sugar availability

Multicellular organisms need to modulate proliferation and differentiation in response to external conditions. An important role in these processes plays the mitogen-stimulated induction of cyclin D (cycD) gene expression. D-type cyclins have been identified as the crucial intracellular sensors for cell-cycle regulation in all eukaryotes. However, cycD deletions have been found to cause specific phenotypic alterations in animals but not yet in plants. An insertional mutation of a so far uncharacterized Arabidopsis cycD gene did not alter the plant phenotype. To gain new insights into CycD function of land plants, we generated targeted cycD gene knockouts in the moss Physcomitrella patens and observed a surprisingly limited disruption phenotype. While wild-type plants reacted to exogenous glucose sources with prolonged growth of juvenile stages and retarded differentiation, cycD knockouts exhibited developmental progression independent of sugar supply. On the other hand, growth rate, cell sizes or plant size were not affected. Thus, we conclude that Physcomitrella CycD might not be essential for cell-cycle regulation but is important for coupling the developmental progression to nutrient availability.     

植物分子生物学研究极具前景的模式系统--小立碗藓

小立碗藓作为植物分子生物学研究极具前景的模式系统已日益受到人们的重视,它的生活史周期短,易于培养,转基因植株易于分析,核基因组容易和外源DNA发生同源重组,这些特点使它成为研究基因功能的良好材料.一些成功的基因敲除和基因破坏已经在小立碗藓中实现,这些基因的功能也通过小立碗藓转化植株的特点得以证实.小立碗藓标签突变文库已经建立,其应用为小立碗藓基因的进一步研究打下了基础.关于小立碗藓的ESTs数据库已经建立,已有67 000条ESTs信息.     

Moss systems biology en route: phytohormones in Physcomitrella development

The moss Physcomitrella patens has become a powerful model system in modern plant biology. Highly standardized cell culture techniques, as well as the necessary tools for computational biology, functional genomics and proteomics have been established. Large EST collections are available and the complete moss genome will be released soon. A simple body plan and the small number of different cell types in Physcomitrella facilitate the study of developmental processes. In the filamentous juvenile moss tissue, developmental decisions rely on the differentiation of single cells. Developmental steps are controlled by distinct phytohormones and integration of environmental signals. Especially the phytohormones auxin, cytokinin, and abscisic acid have distinct effects on early moss development. In this article, we review current knowledge about phytohormone influences on early moss development in an attempt to fully unravel the complex regulatory signal transduction networks underlying the developmental decisions of single plant cells in a holistic systems biology approach.     

Plant water uptake by hard red winter wheat (Triticum aestivum L.) genotypes at 2°C and low light intensity

Background

The moss Physcomitrella patens is an attractive model system for plant biology and functional genome analysis. It shares many biological features with higher plants but has the unique advantage of an efficient homologous recombination system for its nuclear DNA. This allows precise genetic manipulations and targeted knockouts to study gene function, an approach that due to the very low frequency of targeted recombination events is not routinely possible in any higher plant.

Results

As an important prerequisite for a large-scale gene/function correlation study in this plant, we are establishing a collection of Physcomitrella patens transformants with insertion mutations in most expressed genes. A low-redundancy moss cDNA library was mutagenised in E. coli using a derivative of the transposon Tn1000. The resulting gene-disruption library was then used to transform Physcomitrella. Homologous recombination of the mutagenised cDNA with genomic coding sequences is expected to target insertion events preferentially to expressed genes. An immediate phenotypic analysis of transformants is made possible by the predominance of the haploid gametophytic state in the life cycle of the moss. Among the first 16,203 transformants analysed so far, we observed 2636 plants ( = 16.2%) that differed from the wild-type in a variety of developmental, morphological and physiological characteristics.

Conclusions

The high proportion of phenotypic deviations and the wide range of abnormalities observed among the transformants suggests that mutagenesis by gene-disruption library transformation is a useful strategy to establish a highly diverse population of Physcomitrella patens mutants for functional genome analysis.     

植物分子生物学研究极具前景的模式系统——小立碗藓

小立碗藓作为植物分子生物学研究极具前景的模式系统已日益受到人们的重视,它的生活史周期短,易于培养,转基因植株易于分析,核基因组容易和外源DNA 发生同源重组,这些特点使它成为研究基因功能的良好材料。一些成功的基因敲除和基因破坏已经在小立碗藓中实现,这些基因的功能也通过小立碗藓转化植株的特点得以证实。小立碗藓标签突变文库已经建立,其应用为小立碗藓基因的进一步研究打下了基础。关于小立碗藓的ESTs 数据库已经建立,已有67 000 条ESTs 信息。     

苔藓植物小立碗藓，功能基因组学研究新的模式系统

苔藓植物具有相对简单的发育模式,单倍体的配子体在其生活史中占主导地位,作为研究植物生物学过程的模式系统具有诸多的优越性。苔藓植物小立碗藓能够高效地通过同源重组的方式将外源核酸整合到其核DNA,这就使得基因打靶在此物种中就像在小鼠胚胎干细胞和酵母中一样成为一个非常便利的技术。另外由于小立碗藓与高等植物在生物特征上有很大相似之处加之其有其他诸多优越性,它有望成为一个诱人的植物生物学和功能基因组学研究的模式系统。Abstract：The potential of moss as a model system to study plant biological process is associated with their relatively simple developmental pattern and the dominance of the haploid gametophyte in the life cycle. The moss Physcomitrella patens exhibits a very high rate of homologous recombination in its nuclear DNA, making gene targeting approaches in this plant as convenient as in yeast or in ES cells of mice. Sharing many biological features with higher plants and having many other advantages, the moss Physcomitrella patens will be an attractive model system for plant biology and functional genome analysis.     

Large-scale screening of Arabidopsis enhancer-trap lines for seed germination-associated genes

Enhancer trap is a powerful approach for identifying tissue- and stage-specific gene expression in plants and animals. For Arabidopsis research, beta-glucuronidase (GUS) enhancer-trap lines have been created and successfully used to identify tissue-specific gene expression in many plant organs. However, limited applications of these lines for seed germination research have been reported. This is probably due to the impermeability of the testa to the GUS substrate. By focusing on the stages between testa and endosperm rupture, we were able to circumvent the testa barrier to the GUS substrate and observe diverse tissue-specific gene expression during germination sensu stricto. One hundred and twenty-one positive subpools of 10 lines out of 1130 were isolated. Approximately 4500 plants from these subpools were grown in a greenhouse and one to seven individual plants exhibiting GUS expression in seeds were isolated for each subpool. This library of the Arabidopsis seed enhancer-trap lines is an efficient tool for identifying seed germination-associated genes. The individual lines from this library will be provided to the international seed biology research community. International collaboration to identify the trapped genes using genome-walking PCR and to characterize the gene functions using knockout plants will significantly enhance our understanding of the molecular mechanisms of seed germination.     

Phytochrome evolution: Phytochrome genes in ferns and mosses

We have isolated phytochrome genes from the moss Physcomitrella , the fern Psilotum and PCR-generated phytochrome sequences from a few other ferns. The phytochrome gene of the moss Physcomitrella turned out not to contain the aberrant C-terminal third of the phytochrome from the moss Ceratodon , but the transmitter module-like sequences found in other phytochromes. A series of different phytochrome genes was detected in Psilotum . Differences between the amino acid sequences derived from them ranged from about 5 to more than 22%. Some of these genes are likely pseudogenes. Analysis by phylogenetic tree constructions revealed that higher and lower plant phytochromes evolved with different velocities. Lower plant phytochromes form a separate family characterized by a high degree of similarity. The amino acid differences between phytochrome types detected in a single species of higher plants are about two-fold higher than the differences between phytochromes of species of lower plants belonging to different divisions ( Physcomitrella and Selaginella ). Future studies on phytochrome sequences may eventually also throw light on the significance of Psilotum in the evolution of vascular plants.     

RNA interference in the moss Physcomitrella patens

The moss Physcomitrella patens performs efficient homologous recombination, which allows for the study of individual gene function by generating gene disruptions. Yet, if the gene of study is essential, gene disruptions cannot be isolated in the predominantly haploid P. patens. Additionally, disruption of a gene does not always generate observable phenotypes due to redundant functions from related genes. However, RNA interference (RNAi) can provide mutants for both of these situations. We show that RNAi disrupts gene expression in P. patens, adding a significant tool for the study of plant gene function. To assay for RNAi in moss, we constructed a line (NLS-4) expressing a nuclearly localized green fluorescent protein (GFP):beta-glucuronidase (GUS) fusion reporter protein. We targeted the reporter protein with two RNAi constructs, GUS-RNAi and GFP-RNAi, expressed transiently by particle bombardment. Transformed protonemal cells are marked by cobombardment with dsRed2, which diffuses between the nucleus and cytoplasm. Cells transformed with control constructs have nuclear/cytoplasmic red fluorescence and nuclear green fluorescence. In cells transformed with GUS-RNAi or GFP-RNAi constructs, the nuclear green fluorescence was reduced on average 9-fold as soon as 48 h after transformation. Moreover, isolated lines of NLS-4 stably transformed with GUS-RNAi construct have silenced nuclear GFP, indicating that RNAi is propagated stably. Thus, RNAi adds a powerful tool for functional analysis of plant genes in moss.     

Quantitative promoter analysis in <Emphasis Type="Italic">Physcomitrella patens</Emphasis>: a set of plant vectors activating gene expression within three orders of magnitude

Background  

In addition to studies of plant gene function and developmental analyses, plant biotechnological use is largely dependent upon transgenic technologies. The moss Physcomitrella patens has become an exciting model system for studying plant molecular processes due to an exceptionally high rate of nuclear gene targeting by homologous recombination compared with other plants. However, its use in transgenic approaches requires expression vectors that incorporate sufficiently strong promoters. To satisfy this requirement, a set of plant expression vectors was constructed and equipped with either heterologous or endogenous promoters.     

A novel gene family in moss (Physcomitrella patens) shows sequence homology and a phylogenetic relationship with the TIR-NBS class of plant disease resistance genes

Plant disease resistance (R) genes encode proteins in which several motifs of the nucleotide-binding region (NBS) are highly conserved. Using degenerate primers designed according to the kinase 1 (P-loop) and hydrophobic (HD) motifs of the R gene NBS domains, homologous sequences were cloned from moss (Physcomitrella patens; phylum Bryophyta) representing an ancient nonvascular plant. A novel gene family (PpC) with at least eight homologous members was found. Expression of five members was detected. The level of expression was dependent on the developmental stage of moss, being higher in the gametophyte tissue than in the protonema tissue. The PpCs contained the conserved motifs characteristic of the NBS regions of R genes, and a kinase domain was found upstream from the NBS region. Phylogenetic analysis using the deduced NBS amino acid sequences of the PpCs and the plant genes available in databanks indicated that the PpCs show the closest relationship with the TIR-NBS class of R genes. No significant similarity to plant genes other than R genes was observed. These findings shed novel light on the evolutionary history of the R gene families, suggesting that the NBS region characteristic of the TIR-NBS class of R-like genes evolved prior to the evolutionary differentiation of vascular and nonvascular plants.