Science.gov

Sample records for physcomitrella patens ftsz

  1. Microscopy of Physcomitrella patens sperm cells.

    PubMed

    Horst, Nelly A; Reski, Ralf

    2017-01-01

    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.

  2. Paenibacillus physcomitrellae sp. nov., isolated from the moss Physcomitrella patens.

    PubMed

    Zhou, Xun; Nan Guo, Guan; Qi Wang, Le; Lan Bai, Su; Li Li, Chun; Yu, Rong; Hong Li, Yan

    2015-10-01

    A Gram-stain-positive, facultatively anaerobic and rod-shaped bacterium, designated strain XBT, was isolated from Physcomitrella patens growing in Beijing, China. The isolate was identified as a member of the genus Paenibacillus based on phenotypic characteristics and phylogenetic inferences. The novel strain was spore-forming, motile, catalase-negative and weakly oxidase-positive. Optimal growth of strain XBT occurred at 28°C and pH 7.0-7.5. The major polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and several unidentified components, including one phospholipid, two aminophospholipids, three glycolipids, one aminolipid and one lipid. The predominant isoprenoid quinone was MK-7. The diamino acid found in the cell-wall peptidoglycan was meso-diaminopimelic acid. The major fatty acid components (>5 %) were anteiso-C15 : 0 (51.2 %), anteiso-C17 : 0 (20.6 %), iso-C16 : 0 (8.3 %) and C16 : 0 (6.7 %). The G+C content of the genomic DNA was 53.3 mol%. Phylogenetic analysis, based on the 16S rRNA gene sequence, showed that strain XBT fell within the evolutionary distances encompassed by the genus Paenibacillus; its closest phylogenetic neighbour was Paenibacillus yonginensis DCY84T (96.6 %). Based on phenotypic, chemotaxonomic and phylogenetic properties, strain XBT is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus physcomitrellae sp. nov., is proposed. The type strain is XBT ( = CGMCC 1.15044T = DSM 29851T).

  3. Physcomitrella patens Activates Defense Responses against the Pathogen Colletotrichum gloeosporioides

    PubMed Central

    Reboledo, Guillermo; del Campo, Raquel; Alvarez, Alfonso; Montesano, Marcos; Mara, Héctor; Ponce de León, Inés

    2015-01-01

    The moss Physcomitrella patens is a suitable model plant to analyze the activation of defense mechanisms after pathogen assault. In this study, we show that Colletotrichum gloeosporioides isolated from symptomatic citrus fruit infects P. patens and cause disease symptoms evidenced by browning and maceration of tissues. After C. gloeosporioides infection, P. patens reinforces the cell wall by the incorporation of phenolic compounds and induces the expression of a Dirigent-protein-like encoding gene that could lead to the formation of lignin-like polymers. C. gloeosporioides-inoculated protonemal cells show cytoplasmic collapse, browning of chloroplasts and modifications of the cell wall. Chloroplasts relocate in cells of infected tissues toward the initially infected C. gloeosporioides cells. P. patens also induces the expression of the defense genes PAL and CHS after fungal colonization. P. patens reporter lines harboring the auxin-inducible promoter from soybean (GmGH3) fused to β-glucuronidase revealed an auxin response in protonemal tissues, cauloids and leaves of C. gloeosporioides-infected moss tissues, indicating the activation of auxin signaling. Thus, P. patens is an interesting plant to gain insight into defense mechanisms that have evolved in primitive land plants to cope with microbial pathogens. PMID:26389888

  4. Moss (Physcomitrella patens) GH3 proteins act in auxin homeostasis.

    PubMed

    Ludwig-Müller, Jutta; Jülke, Sabine; Bierfreund, Nicole M; Decker, Eva L; Reski, Ralf

    2009-01-01

    Auxins are hormones involved in many cellular, physiological and developmental processes in seed plants and in mosses such as Physcomitrella patens. Control of auxin levels is achieved in higher plants via synthesis of auxin conjugates by members of the GH3 family. The role of the two GH3-like proteins from P. patens for growth and auxin homeostasis was therefore analysed. The in vivo-function of the two P. patens GH3 genes was investigated using single and double knockout mutants. The two P. patens GH3 proteins were also heterologously expressed to determine their enzymatic activity. Both P. patens GH3 enzymes accepted the auxin indole acetic acid (IAA) as substrate, but with different preferences for the amino acid to which it is attached. Cytoplasmic localization was shown for PpGH3-1 tagged with green fluorescent protein (GFP). Targeted knock-out of either gene exhibited an increased sensitivity to auxin, resulting in growth inhibition. On plain mineral media mutants had higher levels of free IAA and less conjugated IAA than the wild type, and this effect was enhanced when auxin was supplied. The DeltaPpGH3-1/DeltaPpGH3-2 double knockout had almost no IAA amide conjugates but still synthesized ester conjugates. Taken together, these data suggest a developmentally controlled involvement of P. patens GH3 proteins in auxin homeostasis by conjugating excess of physiologically active free auxin to inactive IAA-amide conjugates.

  5. Genotoxin induced mutagenesis in the model plant Physcomitrella patens.

    PubMed

    Holá, Marcela; Kozák, Jaroslav; Vágnerová, Radka; Angelis, Karel J

    2013-01-01

    The moss Physcomitrella patens is unique for the high frequency of homologous recombination, haploid state, and filamentous growth during early stages of the vegetative growth, which makes it an excellent model plant to study DNA damage responses. We used single cell gel electrophoresis (comet) assay to determine kinetics of response to Bleomycin induced DNA oxidative damage and single and double strand breaks in wild type and mutant lig4 Physcomitrella lines. Moreover, APT gene when inactivated by induced mutations was used as selectable marker to ascertain mutational background at nucleotide level by sequencing of the APT locus. We show that extensive repair of DSBs occurs also in the absence of the functional LIG4, whereas repair of SSBs is seriously compromised. From analysis of induced mutations we conclude that their accumulation rather than remaining lesions in DNA and blocking progression through cell cycle is incompatible with normal plant growth and development and leads to sensitive phenotype.

  6. Genotoxin Induced Mutagenesis in the Model Plant Physcomitrella patens

    PubMed Central

    Holá, Marcela; Kozák, Jaroslav; Vágnerová, Radka; Angelis, Karel J.

    2013-01-01

    The moss Physcomitrella patens is unique for the high frequency of homologous recombination, haploid state, and filamentous growth during early stages of the vegetative growth, which makes it an excellent model plant to study DNA damage responses. We used single cell gel electrophoresis (comet) assay to determine kinetics of response to Bleomycin induced DNA oxidative damage and single and double strand breaks in wild type and mutant lig4 Physcomitrella lines. Moreover, APT gene when inactivated by induced mutations was used as selectable marker to ascertain mutational background at nucleotide level by sequencing of the APT locus. We show that extensive repair of DSBs occurs also in the absence of the functional LIG4, whereas repair of SSBs is seriously compromised. From analysis of induced mutations we conclude that their accumulation rather than remaining lesions in DNA and blocking progression through cell cycle is incompatible with normal plant growth and development and leads to sensitive phenotype. PMID:24383055

  7. An Innate Immunity Pathway in the Moss Physcomitrella patens.

    PubMed

    Bressendorff, Simon; Azevedo, Raquel; Kenchappa, Chandra Shekar; Ponce de León, Inés; Olsen, Jakob V; Rasmussen, Magnus Wohlfahrt; Erbs, Gitte; Newman, Mari-Anne; Petersen, Morten; Mundy, John

    2016-06-01

    MAP kinase (MPK) cascades in Arabidopsis thaliana and other vascular plants are activated by developmental cues, abiotic stress, and pathogen infection. Much less is known of MPK functions in nonvascular land plants such as the moss Physcomitrella patens Here, we provide evidence for a signaling pathway in P. patens required for immunity triggered by pathogen associated molecular patterns (PAMPs). This pathway induces rapid growth inhibition, a novel fluorescence burst, cell wall depositions, and accumulation of defense-related transcripts. Two P. patens MPKs (MPK4a and MPK4b) are phosphorylated and activated in response to PAMPs. This activation in response to the fungal PAMP chitin requires a chitin receptor and one or more MAP kinase kinase kinases and MAP kinase kinases. Knockout lines of MPK4a appear wild type but have increased susceptibility to the pathogenic fungi Botrytis cinerea and Alternaria brassisicola Both PAMPs and osmotic stress activate some of the same MPKs in Arabidopsis. In contrast, abscisic acid treatment or osmotic stress of P. patens does not activate MPK4a or any other MPK, but activates at least one SnRK2 kinase. Signaling via MPK4a may therefore be specific to immunity, and the moss relies on other pathways to respond to osmotic stress. © 2016 American Society of Plant Biologists. All rights reserved.

  8. Phototropism in gametophytic shoots of the moss Physcomitrella patens

    PubMed Central

    Bao, Liang; Yamamoto, Kotaro T; Fujita, Tomomichi

    2015-01-01

    Shoot phototropism enables plants to position their photosynthetic organs in favorable light conditions and thus benefits growth and metabolism in land plants. To understand the evolution of this response, we established an experimental system to study phototropism in gametophores of the moss Physcomitrella patens. The phototropic response of gametophores occurs slowly; a clear response takes place more than 24 hours after the onset of unilateral light irradiation, likely due to the slow growth rate of gametophores. We also found that red and far-red light can induce phototropism, with blue light being less effective. These results suggest that plants used a broad range of light wavelengths as phototropic signals during the early evolution of land plants. PMID:25848889

  9. Phototropism in gametophytic shoots of the moss Physcomitrella patens.

    PubMed

    Bao, Liang; Yamamoto, Kotaro T; Fujita, Tomomichi

    2015-01-01

    Shoot phototropism enables plants to position their photosynthetic organs in favorable light conditions and thus benefits growth and metabolism in land plants. To understand the evolution of this response, we established an experimental system to study phototropism in gametophores of the moss Physcomitrella patens. The phototropic response of gametophores occurs slowly; a clear response takes place more than 24 hours after the onset of unilateral light irradiation, likely due to the slow growth rate of gametophores. We also found that red and far-red light can induce phototropism, with blue light being less effective. These results suggest that plants used a broad range of light wavelengths as phototropic signals during the early evolution of land plants.

  10. Physcomitrella patens: a model for tip cell growth and differentiation.

    PubMed

    Vidali, Luis; Bezanilla, Magdalena

    2012-12-01

    The moss Physcomitrella patens has emerged as an excellent model system owing to its amenability to reverse genetics. The moss gametophyte has three filamentous tissues that grow by tip growth: chloronemata, caulonemata, and rhizoids. Because establishment of the moss plant relies on this form of growth, it is particularly suited for dissecting the molecular basis of tip growth. Recent studies demonstrate that a core set of actin cytoskeletal proteins is essential for tip growth. Additional actin cytoskeletal components are required for modulating growth to produce caulonemata and rhizoids. Differentiation into these cell types has previously been linked to auxin, light and nutrients. Recent studies have identified that core auxin signaling components as well as transcription factors that respond to auxin or nutrient levels are required for tip-growing cell differentiation. Future studies may establish a connection between the actin cytoskeleton and auxin or nutrient-induced cell differentiation. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Putting Physcomitrella Patens on the Tree of Life: The Evolution and Ecology of Mosses

    USDA-ARS?s Scientific Manuscript database

    Physcomitrella patens is an important model system for studies of genetics and physiology, and with its newly-sequenced genome, is perfectly placed phylogenetically to serve as a point of comparison for angiosperms. This chapter addresses three main questions. (1) How typical of a moss is P. patens...

  12. Desiccation sensitivity and tolerance in the moss Physcomitrella patens: assessing limits and damage.

    USDA-ARS?s Scientific Manuscript database

    The moss Physcomitrella patens is becoming the model of choice for functional genomic studies at the cellular level. Studies report that P. patens survives moderate osmotic and salt stress, and that desiccation tolerance can be induced by exogenous ABA. Our goal was to quantify the extent of dehydr...

  13. Role of RNA Interference (RNAi) in the Moss Physcomitrella patens

    PubMed Central

    Arif, Muhammad Asif; Frank, Wolfgang; Khraiwesh, Basel

    2013-01-01

    RNA interference (RNAi) is a mechanism that regulates genes by either transcriptional (TGS) or posttranscriptional gene silencing (PTGS), required for genome maintenance and proper development of an organism. Small non-coding RNAs are the key players in RNAi and have been intensively studied in eukaryotes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs are synthesized from a short hairpin structure while siRNAs are derived from long double-stranded RNAs (dsRNA). Both miRNA and siRNAs control the expression of cognate target RNAs by binding to reverse complementary sequences mediating cleavage or translational inhibition of the target RNA. They also act on the DNA and cause epigenetic changes such as DNA methylation and histone modifications. In the last years, the analysis of plant RNAi pathways was extended to the bryophyte Physcomitrella patens, a non-flowering, non-vascular ancient land plant that diverged from the lineage of seed plants approximately 450 million years ago. Based on a number of characteristic features and its phylogenetic key position in land plant evolution P. patens emerged as a plant model species to address basic as well as applied topics in plant biology. Here we summarize the current knowledge on the role of RNAi in P. patens that shows functional overlap with RNAi pathways from seed plants, and also unique features specific to this species. PMID:23344055

  14. A SABATH Methyltransferase from the moss Physcomitrella patens catalyzes

    SciTech Connect

    Zhao, Nan; Ferrer, Jean-Luc; Moon, Hong S; Kapteyn, Jeremy; Zhuang, Xiaofeng; Hasebe, Mitsuyasu; Stewart, Neal C.; Gang, David R.; Chen, Feng

    2012-01-01

    Known SABATH methyltransferases, all of which were identified from seed plants, catalyze methylation of either the carboxyl group of a variety of low molecular weight metabolites or the nitrogen moiety of precursors of caffeine. In this study, the SABATH family from the bryophyte Physcomitrella patens was identified and characterized. Four SABATH-like sequences (PpSABATH1, PpSABATH2, PpSABATH3, and PpSABATH4) were identified from the P. patens genome. Only PpSABATH1 and PpSABATH2 showed expression in the leafy gametophyte of P. patens. Full-length cDNAs of PpSABATH1 and PpSABATH2 were cloned and expressed in soluble form in Escherichia coli. Recombinant PpSABATH1 and PpSABATH2 were tested for methyltransferase activity with a total of 75 compounds. While showing no activity with carboxylic acids or nitrogen-containing compounds, PpSABATH1 displayed methyltransferase activity with a number of thiols. PpSABATH2 did not show activity with any of the compounds tested. Among the thiols analyzed, PpSABATH1 showed the highest level of activity with thiobenzoic acid with an apparent Km value of 95.5 lM, which is comparable to those of known SABATHs. Using thiobenzoic acid as substrate, GC MS analysis indicated that the methylation catalyzed by PpSABATH1 is on the sulfur atom. The mechanism for S-methylation of thiols catalyzed by PpSABATH1 was partially revealed by homology-based structural modeling. The expression of PpSABATH1 was induced by the treatment of thiobenzoic acid. Further transgenic studies showed that tobacco plants overexpressing PpSABATH1 exhibited enhanced tolerance to thiobenzoic acid, suggesting that PpSABATH1 have a role in the detoxification of xenobiotic thiols.

  15. Lichen secondary metabolites affect growth of Physcomitrella patens by allelopathy.

    PubMed

    Goga, Michal; Antreich, Sebastian J; Bačkor, Martin; Weckwerth, Wolfram; Lang, Ingeborg

    2017-05-01

    Lichen secondary metabolites can function as allelochemicals and affect the development and growth of neighboring bryophytes, fungi, vascular plants, microorganisms, and even other lichens. Lichen overgrowth on bryophytes is frequently observed in nature even though mosses grow faster than lichens, but there is still little information on the interactions between lichens and bryophytes.In the present study, we used extracts from six lichen thalli containing secondary metabolites like usnic acid, protocetraric acid, atranorin, lecanoric acid, nortistic acid, and thamnolic acid. To observe the influence of these metabolites on bryophytes, the moss Physcomitrella patens was cultivated for 5 weeks under laboratory conditions and treated with lichen extracts. Toxicity of natural mixtures of secondary metabolites was tested at three selected doses (0.001, 0.01, and 0.1 %). When the mixture contained substantial amounts of usnic acid, we observed growth inhibition of protonemata and reduced development of gametophores. Significant differences in cell lengths and widths were also noticed. Furthermore, usnic acid had a strong effect on cell division in protonemata suggesting a strong impact on the early stages of bryophyte development by allelochemicals contained in the lichen secondary metabolites.Biological activities of lichen secondary metabolites were confirmed in several studies such as antiviral, antibacterial, antitumor, antiherbivore, antioxidant, antipyretic, and analgetic action or photoprotection. This work aimed to expand the knowledge on allelopathic effects on bryophyte growth.

  16. Characterization of Arsenic Biotransformation by a Typical Bryophyte Physcomitrella patens.

    PubMed

    Yin, Xixiang; Wang, Lihong; Liu, Yifei; Jiang, Tenglong; Gao, Jianwei

    2017-02-01

    Arsenic (As) is a ubiquitous environmental toxin that has created catastrophic human health and environmental problems around world. Physcomitrella patens is a potential model plant for the study of environmental monitoring, which exists in all kinds of ecosystems. In this study, arsenic metabolism was investigated by this moss. When supplied with different levels of arsenate (50, 100, 200 µmol/L) for a 4-week period, the total arsenic concentrations were up to 231.4-565.4 mg/kg DW in this moss. Arsenite concentration increased with increasing external arsenate concentrations, the proportion was up to 25.1-36.8% of the total As. An arsenate reductase, PpACR2, was identified and functionally characterized. Heterologous expression of PpACR2 in an As(V)-sensitive strain WC3110 (ΔarsC) of Escherichia coli conferred As(V) resistance. Purified PpACR2 protein exhibited the arsenate reductase activity. Given its powerful As accumulation ability, the bryophyte could be exploited in bioremediation of As-contaminated environments.

  17. Biosynthesis of C9-aldehydes in the moss Physcomitrella patens.

    PubMed

    Stumpe, Michael; Bode, Julia; Göbel, Cornelia; Wichard, Thomas; Schaaf, Andreas; Frank, Wolfgang; Frank, Markus; Reski, Ralf; Pohnert, Georg; Feussner, Ivo

    2006-03-01

    After wounding, the moss Physcomitrella patens emits fatty acid derived volatiles like octenal, octenols and (2E)-nonenal. Flowering plants produce nonenal from C18-fatty acids via lipoxygenase and hydroperoxide lyase reactions, but the moss exploits the C20 precursor arachidonic acid for the formation of these oxylipins. We describe the isolation of the first cDNA (PpHPL) encoding a hydroperoxide lyase from a lower eukaryotic organism. The physiological pathway allocation and characterization of a downstream enal-isomerase gives a new picture for the formation of fatty acid derived volatiles from lower plants. Expression of a fusion protein with a yellow fluorescent protein in moss protoplasts showed that PpHPL was found in clusters in membranes of plastids. PpHPL can be classified as an unspecific hydroperoxide lyase having a substrate preference for 9-hydroperoxides of C18-fatty acids but also the predominant substrate 12-hydroperoxy arachidonic acid is accepted. Feeding experiments using arachidonic acid show an increase in the 12-hydroperoxide being metabolized to C8-aldehydes/alcohols and (3Z)-nonenal, which is rapidly isomerized to (2E)-nonenal. PpHPL knock out lines failed to emit (2E)-nonenal while formation of C8-volatiles was not affected indicating that in contrast to flowering plants, PpHPL is only involved in formation of a specific subset of volatiles.

  18. Physcomitrella patens has lipoxygenases for both eicosanoid and octadecanoid pathways.

    PubMed

    Anterola, Aldwin; Göbel, Cornelia; Hornung, Ellen; Sellhorn, George; Feussner, Ivo; Grimes, Howard

    2009-01-01

    Mosses have substantial amounts of long chain C20 polyunsaturated fatty acids, such as arachidonic and eicosapentaenoic acid, in addition to the shorter chain C18 alpha-linolenic and linoleic acids, which are typical substrates of lipoxygenases in flowering plants. To identify the fatty acid substrates used by moss lipoxygenases, eight lipoxygenase genes from Physcomitrella patens were heterologously expressed in Escherichia coli, and then analyzed for lipoxygenase activity using linoleic, alpha-linolenic and arachidonic acids as substrates. Among the eight moss lipoxygenases, only seven were found to be enzymatically active in vitro, two of which selectively used arachidonic acid as the substrate, while the other five preferred alpha-linolenic acid. Based on enzyme assays using a Clark-type oxygen electrode, all of the active lipoxygenases had an optimum pH at 7.0, except for one with highest activity at pH 5.0. HPLC analyses indicated that the two arachidonic acid lipoxygenases form (12S)-hydroperoxy eicosatetraenoic acid as the main product, while the other five lipoxygenases produce mainly (13S)-hydroperoxy octadecatrienoic acid from alpha-linolenic acid. These results suggest that mosses may have both C20 and C18 based oxylipin pathways.

  19. The Physcomitrella patens Chloroplast Proteome Changes in Response to Protoplastation

    PubMed Central

    Fesenko, Igor; Seredina, Anna; Arapidi, Georgij; Ptushenko, Vasily; Urban, Anatoly; Butenko, Ivan; Kovalchuk, Sergey; Babalyan, Konstantin; Knyazev, Andrey; Khazigaleeva, Regina; Pushkova, Elena; Anikanov, Nikolai; Ivanov, Vadim; Govorun, Vadim M.

    2016-01-01

    Plant protoplasts are widely used for genetic manipulation and functional studies in transient expression systems. However, little is known about the molecular pathways involved in a cell response to the combined stress factors resulted from protoplast generation. Plants often face more than one type of stress at a time, and how plants respond to combined stress factors is therefore of great interest. Here, we used protoplasts of the moss Physcomitrella patens as a model to study the effects of short-term stress on the chloroplast proteome. Using label-free comparative quantitative proteomic analysis (SWATH-MS), we quantified 479 chloroplast proteins, 219 of which showed a more than 1.4-fold change in abundance in protoplasts. We additionally quantified 1451 chloroplast proteins using emPAI. We observed degradation of a significant portion of the chloroplast proteome following the first hour of stress imposed by the protoplast isolation process. Electron-transport chain (ETC) components underwent the heaviest degradation, resulting in the decline of photosynthetic activity. We also compared the proteome changes to those in the transcriptional level of nuclear-encoded chloroplast genes. Globally, the levels of the quantified proteins and their corresponding mRNAs showed limited correlation. Genes involved in the biosynthesis of chlorophyll and components of the outer chloroplast membrane showed decreases in both transcript and protein abundance. However, proteins like dehydroascorbate reductase 1 and 2-cys peroxiredoxin B responsible for ROS detoxification increased in abundance. Further, genes such as thylakoid ascorbate peroxidase were induced at the transcriptional level but down-regulated at the proteomic level. Together, our results demonstrate that the initial chloroplast reaction to stress is due changes at the proteomic level. PMID:27867392

  20. The molecular and physiological responses of Physcomitrella patens to ultraviolet-B radiation.

    PubMed

    Wolf, Luise; Rizzini, Luca; Stracke, Ralf; Ulm, Roman; Rensing, Stefan A

    2010-07-01

    Ultraviolet-B (UV-B) radiation present in sunlight is an important trigger of photomorphogenic acclimation and stress responses in sessile land plants. Although numerous moss species grow in unshaded habitats, our understanding of their UV-B responses is very limited. The genome of the model moss Physcomitrella patens, which grows in sun-exposed open areas, encodes signaling and metabolic components that are implicated in the UV-B response in flowering plants. In this study, we describe the response of P. patens to UV-B radiation at the morphological and molecular levels. We find that P. patens is more capable of surviving UV-B stress than Arabidopsis (Arabidopsis thaliana) and describe the differential expression of approximately 400 moss genes in response to UV-B radiation. A comparative analysis of the UV-B response in P. patens and Arabidopsis reveals both distinct and conserved pathways.

  1. An ancient genome duplication contributed to the abundance of metabolic genes in the moss Physcomitrella patens

    PubMed Central

    Rensing, Stefan A; Ick, Julia; Fawcett, Jeffrey A; Lang, Daniel; Zimmer, Andreas; Van de Peer, Yves; Reski, Ralf

    2007-01-01

    Background: Analyses of complete genomes and large collections of gene transcripts have shown that most, if not all seed plants have undergone one or more genome duplications in their evolutionary past. Results: In this study, based on a large collection of EST sequences, we provide evidence that the haploid moss Physcomitrella patens is a paleopolyploid as well. Based on the construction of linearized phylogenetic trees we infer the genome duplication to have occurred between 30 and 60 million years ago. Gene Ontology and pathway association of the duplicated genes in P. patens reveal different biases of gene retention compared with seed plants. Conclusion: Metabolic genes seem to have been retained in excess following the genome duplication in P. patens. This might, at least partly, explain the versatility of metabolism, as described for P. patens and other mosses, in comparison to other land plants. PMID:17683536

  2. Generating Targeted Gene Knockout Lines in Physcomitrella patens to Study Evolution of Stress-Responsive Mechanisms

    PubMed Central

    Maronova, Monika; Kalyna, Maria

    2016-01-01

    The moss Physcomitrella patens possesses highly efficient homologous recombination allowing targeted gene manipulations and displays many features of the early land plants including high tolerance to abiotic stresses. It is therefore an invaluable model organism for studies of gene functions and comparative studies of evolution of stress responses in plants. Here, we describe a method for generating targeted gene knockout lines in P. patens using a polyethylene glycol-mediated transformation of protoplasts including basic in vitro growth, propagation, and maintenance techniques. PMID:26867627

  3. Effects of engineered iron nanoparticles on the bryophyte, Physcomitrella patens (Hedw.) Bruch & Schimp, after foliar exposure.

    PubMed

    Canivet, L; Dubot, P; Garçon, G; Denayer, F-O

    2015-03-01

    The effects of iron nanoparticles on bryophytes (Physcomitrella patens) were studied following foliar exposure. We used iron nanoparticles (Fe-NP) representative of industrial emissions from the metallurgical industries. After a characterization of iron nanoparticles and the validation of nanoparticle internalization in cells, the effects (cytotoxicity, oxidative stress, lipid peroxidation of membrane) of iron nanoparticles were determined through the axenic culturing of Physcomitrella patens exposed at five different concentrations (5 ng, 50 ng, 500 ng, 5 µg and 50 µg per plant). Following exposure, the plant health, measured as ATP concentrations, was not impacted. Moreover, we studied oxidative stress in three ways: through the measure of reactive oxygen species (ROS) production, through malondialdehyde (MDA) production and also through glutathione regulation. At concentrations tested over a short period, the level of ROS, MDA and glutathione were not significantly disturbed.

  4. Prenylation is required for polar cell elongation, cell adhesion, and differentiation in Physcomitrella patens.

    PubMed

    Thole, Julie M; Perroud, Pierre-Francois; Quatrano, Ralph S; Running, Mark P

    2014-05-01

    Protein prenylation is required for a variety of growth and developmental processes in flowering plants. Here we report the consequences of loss of function of all known prenylation subunits in the moss Physcomitrella patens. As in Arabidopsis, protein farnesyltransferase and protein geranylgeranyltransferase type I are not required for viability. However, protein geranylgeranyltransferase type I activity is required for cell adhesion, polar cell elongation, and cell differentiation. Loss of protein geranylgeranyltransferase activity results in colonies of round, single-celled organisms that resemble unicellular algae. The loss of protein farnesylation is not as severe but also results in polar cell elongation and differentiation defects. The complete loss of Rab geranylgeranyltransferase activity appears to be lethal in P. patens. Labeling with antibodies to cell wall components support the lack of polarity establishment and the undifferentiated state of geranylgeranyltransferase type I mutant plants. Our results show that prenylated proteins play key roles in P. patens development and differentiation processes.

  5. Role of PP2C-mediated ABA signaling in the moss Physcomitrella patens.

    PubMed

    Sakata, Yoichi; Komatsu, Kenji; Taji, Teruaki; Tanaka, Shigeo

    2009-09-01

    Plant hormone abscisic acid (ABA) is found in a wide range of land plants, from mosses to angiosperms. However, our knowledge concerning the function of ABA is limited to some angiosperm plant species. We have shown that the basal land plant Physcomitrella patens and the model plant Arabidopsis thaliana share a conserved abscisic acid (ABA) signaling pathway mediated through ABI1-related type 2C protein phosphatases (PP2Cs). Ectopic expression of Arabidopsis abi1-1, a dominant allele of ABI1 that functions as a negative regulator of ABA signaling, or targeted disruption of Physcomitrella ABI1-related gene (PpABI1A) resulted in altered ABA sensitivity and abiotic stress tolerance of Physcomitrella, as demonstrated by osmostress and freezing stress. Moreover, transgenic Physcomitrella overexpressing abi1-1 showed altered morphogenesis. These transgenic plants had longer stem lengths compared to the wild type, and continuous growth of archegonia (female organ) with few sporophytes under non-stress conditions. Our results suggest that PP2C-mediated ABA signaling is involved in both the abiotic stress responses and developmental regulation of Physcomitrella.

  6. Polyphenol oxidases in Physcomitrella: functional PPO1 knockout modulates cytokinin-dependent developmentin the moss Physcomitrella patens

    PubMed Central

    von Schwartzenberg, Klaus

    2012-01-01

    Polyphenol oxidases (PPOs) are copper-binding enzymes of the plant secondary metabolism that oxidize polyphenols to quinones. Although PPOs are nearly ubiquitous in seed plants, knowledge on their evolution and function in other plant groups is missing. This study reports on the PPO gene family in the moss Physcomitrella patens (Hedw.) B.S.G. asan example for an early divergent plant. The P. patens PPO multigene family comprises 13 paralogues. Phylogenetic analyses suggest that plant PPOs evolved with the colonization of land and that PPO duplications within the monophyletic P. patens paralogue clade occurred after the separation of the moss and seed plant lineages. PPO functionality was demonstrated for recombinant PPO6. P. patens was analysed for phenolic compounds and six substances were detected intracellularly by LC-MS analysis: 4-hydroxybenzoic acid, p-cumaric acid, protocatechuic acid, salicylic acid, caffeic acid, and an ester of caffeic acid. Targeted PPO1 knockout (d|ppo1) plants were generated and plants lacking PPO1 exhibited only ~30% of the wild-type PPO activity in the culture medium, thus suggesting extracellular localization of PPO1, which is in contrast to the mostly plastidic PPO localization in seed plants. Further, d|ppo1 lines formed significantly more gametophores with a reduced areal plant size, which could be related to an increase of endogenously produced cytokinins and indicates an impact of PPO1 on plant development. d|ppo1 plants were less tolerant towards applied 4-methylcatechol compared to the wild type, which suggests a role of extracellular PPO1 in establishing appropriate conditions by the removal of inhibitory extracellular phenolic compounds. PMID:22865913

  7. Polyphenol oxidases in Physcomitrella: functional PPO1 knockout modulates cytokinin-dependent development in the moss Physcomitrella patens.

    PubMed

    Richter, Hanna; Lieberei, Reinhard; Strnad, Miroslav; Novák, Ondrej; Gruz, Jiri; Rensing, Stefan A; von Schwartzenberg, Klaus

    2012-09-01

    Polyphenol oxidases (PPOs) are copper-binding enzymes of the plant secondary metabolism that oxidize polyphenols to quinones. Although PPOs are nearly ubiquitous in seed plants, knowledge on their evolution and function in other plant groups is missing. This study reports on the PPO gene family in the moss Physcomitrella patens (Hedw.) B.S.G. asan example for an early divergent plant. The P. patens PPO multigene family comprises 13 paralogues. Phylogenetic analyses suggest that plant PPOs evolved with the colonization of land and that PPO duplications within the monophyletic P. patens paralogue clade occurred after the separation of the moss and seed plant lineages. PPO functionality was demonstrated for recombinant PPO6. P. patens was analysed for phenolic compounds and six substances were detected intracellularly by LC-MS analysis: 4-hydroxybenzoic acid, p-cumaric acid, protocatechuic acid, salicylic acid, caffeic acid, and an ester of caffeic acid. Targeted PPO1 knockout (d|ppo1) plants were generated and plants lacking PPO1 exhibited only ~30% of the wild-type PPO activity in the culture medium, thus suggesting extracellular localization of PPO1, which is in contrast to the mostly plastidic PPO localization in seed plants. Further, d|ppo1 lines formed significantly more gametophores with a reduced areal plant size, which could be related to an increase of endogenously produced cytokinins and indicates an impact of PPO1 on plant development. d|ppo1 plants were less tolerant towards applied 4-methylcatechol compared to the wild type, which suggests a role of extracellular PPO1 in establishing appropriate conditions by the removal of inhibitory extracellular phenolic compounds.

  8. Identification and expression profile analysis of NUCLEAR FACTOR-Y families in Physcomitrella patens

    PubMed Central

    Zhang, Fang; Han, Min; Lv, Qiang; Bao, Fang; He, Yikun

    2015-01-01

    NUCLEAR FACTOR Y transcription factors belong to a multimember family and consist of NF-YA/B/C subunits. Members of the NF-Y family have been reported to regulate physiological processes in plant. In this study, we identified and annotated two NF-YA, nine NF-B, and twelve NF-YC proteins in the genome of Physcomitrella patens. Analyses of conserved domains demonstrated that PpNF-YA/B/C shared the same conserved domains with their orthologous proteins in Arabidopsis, O. sativa and mouse. Expression profiles indicated that PpNF-Ys were widely expressed in different tissues and developmental stages of P. patens throughout protonema and gametophores. The majority of PpNF-Y genes were responsive to abiotic stress via either ABA-independent or -dependent pathways. Some of ABA-regulated PpNF-Y expression were mediated by ABI3. To our knowledge, this study was the first to evaluate NF-Y families in Physcomitrella patens, and provides a foundation to dissect the function of PpNF-Ys. PMID:26347760

  9. Quantitative analysis of organelle distribution and dynamics in Physcomitrella patens protonemal cells.

    PubMed

    Furt, Fabienne; Lemoi, Kyle; Tüzel, Erkan; Vidali, Luis

    2012-05-17

    In the last decade, the moss Physcomitrella patens has emerged as a powerful plant model system, amenable for genetic manipulations not possible in any other plant. This moss is particularly well suited for plant polarized cell growth studies, as in its protonemal phase, expansion is restricted to the tip of its cells. Based on pollen tube and root hair studies, it is well known that tip growth requires active secretion and high polarization of the cellular components. However, such information is still missing in Physcomitrella patens. To gain insight into the mechanisms underlying the participation of organelle organization in tip growth, it is essential to determine the distribution and the dynamics of the organelles in moss cells. We used fluorescent protein fusions to visualize and track Golgi dictyosomes, mitochondria, and peroxisomes in live protonemal cells. We also visualized and tracked chloroplasts based on chlorophyll auto-fluorescence. We showed that in protonemata all four organelles are distributed in a gradient from the tip of the apical cell to the base of the sub-apical cell. For example, the density of Golgi dictyosomes is 4.7 and 3.4 times higher at the tip than at the base in caulonemata and chloronemata respectively. While Golgi stacks are concentrated at the extreme tip of the caulonemata, chloroplasts and peroxisomes are totally excluded. Interestingly, caulonemata, which grow faster than chloronemata, also contain significantly more Golgi dictyosomes and fewer chloroplasts than chloronemata. Moreover, the motility analysis revealed that organelles in protonemata move with low persistency and average instantaneous speeds ranging from 29 to 75 nm/s, which are at least three orders of magnitude slower than those of pollen tube or root hair organelles. To our knowledge, this study reports the first quantitative analysis of organelles in Physcomitrella patens and will make possible comparisons of the distribution and dynamics of organelles

  10. In vivo assembly of DNA-fragments in the moss, Physcomitrella patens.

    PubMed

    King, Brian Christopher; Vavitsas, Konstantinos; Ikram, Nur Kusaira Binti Khairul; Schrøder, Josephine; Scharff, Lars B; Hamberger, Björn; Jensen, Poul Erik; Simonsen, Henrik Toft

    2016-04-29

    Direct assembly of multiple linear DNA fragments via homologous recombination, a phenomenon known as in vivo assembly or transformation associated recombination, is used in biotechnology to assemble DNA constructs ranging in size from a few kilobases to full synthetic microbial genomes. It has also enabled the complete replacement of eukaryotic chromosomes with heterologous DNA. The moss Physcomitrella patens, a non-vascular and spore producing land plant (Bryophyte), has a well-established capacity for homologous recombination. Here, we demonstrate the in vivo assembly of multiple DNA fragments in P. patens with three examples of effective genome editing: we (i) efficiently deleted a genomic locus for diterpenoid metabolism yielding a biosynthetic knockout, (ii) introduced a salt inducible promoter, and (iii) re-routed endogenous metabolism into the formation of amorphadiene, a precursor of high-value therapeutics. These proof-of-principle experiments pave the way for more complex and increasingly flexible approaches for large-scale metabolic engineering in plant biotechnology.

  11. The potential of Physcomitrella patens as a platform for the production of plant-based vaccines.

    PubMed

    Rosales-Mendoza, Sergio; Orellana-Escobedo, Lucía; Romero-Maldonado, Andrea; Decker, Eva L; Reski, Ralf

    2014-02-01

    The moss Physcomitrella patens has a number of advantages for the production of biopharmaceuticals, including: i) availability of standardized conditions for cultivation in bioreactors; ii) not being part of the food chain; iii) high biosafety; iv) availability of highly efficient transformation methods; v) a haploid, fully sequenced genome providing genetic stability and uniform expression; vi) efficient gene targeting at the nuclear level allows for the generation of mutants with specific post-translational modifications (e.g., glycosylation patterns); and vii) oral formulations are a viable approach as no toxic effects are attributed to ingestion of this moss. In the light of this panorama, this opinion paper analyzes the possibilities of using P. patens for the production of oral vaccines and presents some specific cases where its use may represent significant progress in the field of plant-based vaccine development. The advantages represented by putative adjuvant effects of endogenous secondary metabolites and producing specific glycosylation patterns are highlighted.

  12. Flavodiiron proteins act as safety valve for electrons in Physcomitrella patens.

    PubMed

    Gerotto, Caterina; Alboresi, Alessandro; Meneghesso, Andrea; Jokel, Martina; Suorsa, Marjaana; Aro, Eva-Mari; Morosinotto, Tomas

    2016-10-25

    Photosynthetic organisms support cell metabolism by harvesting sunlight to fuel the photosynthetic electron transport. The flow of excitation energy and electrons in the photosynthetic apparatus needs to be continuously modulated to respond to dynamics of environmental conditions, and Flavodiiron (FLV) proteins are seminal components of this regulatory machinery in cyanobacteria. FLVs were lost during evolution by flowering plants, but are still present in nonvascular plants such as Physcomitrella patens We generated P. patens mutants depleted in FLV proteins, showing their function as an electron sink downstream of photosystem I for the first seconds after a change in light intensity. flv knock-out plants showed impaired growth and photosystem I photoinhibition when exposed to fluctuating light, demonstrating FLV's biological role as a safety valve from excess electrons on illumination changes. The lack of FLVs was partially compensated for by an increased cyclic electron transport, suggesting that in flowering plants, the FLV's role was taken by other alternative electron routes.

  13. Additional diterpenes from Physcomitrella patens synthesized by copalyl diphosphate/kaurene synthase (PpCPS/KS).

    PubMed

    Zhan, Xin; Bach, Søren Spanner; Hansen, Nikolaj Lervad; Lunde, Christina; Simonsen, Henrik Toft

    2015-11-01

    The bifunctional diterpene synthase, copalyl diphosphate/kaurene synthase from the moss Physcomitrella patens (PpCPS/KS), catalyses the formation of at least four diterpenes, including ent-beyerene, ent-sandaracopimaradiene, ent-kaur-16-ene, and 16-hydroxy-ent-kaurene. The enzymatic activity has been confirmed through generation of a targeted PpCPS/KS knock-out mutant in P. patens via homologous recombination, through transient expression of PpCPS/KS in Nicotiana benthamiana, and expression of PpCPS/KS in E. coli. GC-MS analysis of the knock-out mutant shows that it lacks the diterpenoids, supporting that all are products of PpCPS/KS as observed in N. benthamiana and E. coli. These results provide additional knowledge of the mechanism of this bifunctional diterpene synthase, and are in line with proposed reaction mechanisms in kaurene biosynthesis.

  14. Comparing copper resistance in two bryophytes: Mielichhoferia elongata Hornsch. versus Physcomitrella patens Hedw.

    PubMed

    Sassmann, Stefan; Wernitznig, Stefan; Lichtscheidl, Irene K; Lang, Ingeborg

    2010-10-01

    The bryophyte Mielichhoferia elongata is known to occur on copper-rich substrate, but the exact resistance level remained to be determined by in vitro experiments. Here, we tested its copper tolerance in graded copper solutions and compared the results to the moss Physcomitrella patens that is not known to inhabit heavy metal sites. Our results confirm the survival of M. elongata in classical resistance experiments of up to 10 mM Cu-ethylenediaminetetraacetic acid (EDTA) solution. Interestingly, P. patens is equally resistant. Cultured on copper-enriched agar plates for over 5 weeks, P. patens survived even higher copper levels of up to 100 mM Cu-EDTA and an increment of growth was detected on all concentrations tested. Obviously, P. patens is able to withstand harmfully high levels of copper in both solution and substrate. In this short communication, we give a detailed description of the growth rates and discuss the results in comparison to other moss species and heavy metals.

  15. Phototropins mediate blue and red light-induced chloroplast movements in Physcomitrella patens.

    PubMed

    Kasahara, Masahiro; Kagawa, Takatoshi; Sato, Yoshikatsu; Kiyosue, Tomohiro; Wada, Masamitsu

    2004-07-01

    Phototropin is the blue-light receptor that mediates phototropism, chloroplast movement, and stomatal opening in Arabidopsis. Blue and red light induce chloroplast movement in the moss Physcomitrella patens. To study the photoreceptors for chloroplast movement in P. patens, four phototropin genes (PHOTA1, PHOTA2, PHOTB1, and PHOTB2) were isolated by screening cDNA libraries. These genes were classified into two groups (PHOTA and PHOTB) on the basis of their deduced amino acid sequences. Then phototropin disruptants were generated by homologous recombination and used for analysis of chloroplast movement. Data revealed that blue light-induced chloroplast movement was mediated by phototropins in P. patens. Both photA and photB groups were able to mediate chloroplast avoidance, as has been reported for Arabidopsis phot2, although the photA group contributed more to the response. Red light-induced chloroplast movement was also significantly reduced in photA2photB1photB2 triple disruptants. Because the primary photoreceptor for red light-induced chloroplast movement in P. patens is phytochrome, phototropins may be downstream components of phytochromes in the signaling pathway. To our knowledge, this work is the first to show a function for the phototropin blue-light receptor in a response to wavelengths that it does not absorb.

  16. Gibberellin precursor is involved in spore germination in the moss Physcomitrella patens.

    PubMed

    Anterola, Aldwin; Shanle, Erin; Mansouri, Katayoun; Schuette, Scott; Renzaglia, Karen

    2009-03-01

    Gibberellins are ent-kaurene derived phytohormones that are involved in seed germination, stem elongation, and flower induction in seed plants, as well as in antheridia formation and spore germination in ferns. Although ubiquitous in vascular plants, the occurrence and potential function(s) of gibberellins in bryophytes have not yet been resolved. To determine the potential role of gibberellin and/or gibberellin-like compounds in mosses, the effect of AMO-1618 on spores of Physcomitrella patens (Hedw.) B.S.G. was tested. AMO-1618, which inhibited ent-kaurene and gibberellin biosynthesis in angiosperms, also inhibited the bifunctional copalyl diphosphate synthase (E.C. 5.5.1.13)/ent-kaurene synthase (E.C. 4.2.3.19) of P. patens. AMO-1618 also caused a decrease in spore germination rates of P. patens, and this inhibitory effect was less pronounced in the presence of ent-kaurene. These results suggest that ent-kaurene biosynthesis is required by P. patens spores to germinate, implying the presence of gibberellin-like phytohormones in mosses.

  17. An Innate Immunity Pathway in the Moss Physcomitrella patens[OPEN

    PubMed Central

    Bressendorff, Simon; Azevedo, Raquel; Kenchappa, Chandra Shekar; Ponce de León, Inés; Olsen, Jakob V.; Rasmussen, Magnus Wohlfahrt; Erbs, Gitte; Newman, Mari-Anne; Petersen, Morten; Mundy, John

    2016-01-01

    MAP kinase (MPK) cascades in Arabidopsis thaliana and other vascular plants are activated by developmental cues, abiotic stress, and pathogen infection. Much less is known of MPK functions in nonvascular land plants such as the moss Physcomitrella patens. Here, we provide evidence for a signaling pathway in P. patens required for immunity triggered by pathogen associated molecular patterns (PAMPs). This pathway induces rapid growth inhibition, a novel fluorescence burst, cell wall depositions, and accumulation of defense-related transcripts. Two P. patens MPKs (MPK4a and MPK4b) are phosphorylated and activated in response to PAMPs. This activation in response to the fungal PAMP chitin requires a chitin receptor and one or more MAP kinase kinase kinases and MAP kinase kinases. Knockout lines of MPK4a appear wild type but have increased susceptibility to the pathogenic fungi Botrytis cinerea and Alternaria brassisicola. Both PAMPs and osmotic stress activate some of the same MPKs in Arabidopsis. In contrast, abscisic acid treatment or osmotic stress of P. patens does not activate MPK4a or any other MPK, but activates at least one SnRK2 kinase. Signaling via MPK4a may therefore be specific to immunity, and the moss relies on other pathways to respond to osmotic stress. PMID:27268428

  18. Erwinia carotovora elicitors and Botrytis cinerea activate defense responses in Physcomitrella patens

    PubMed Central

    Ponce de León, Inés; Oliver, Juan Pablo; Castro, Alexandra; Gaggero, Carina; Bentancor, Marcel; Vidal, Sabina

    2007-01-01

    Background Vascular plants respond to pathogens by activating a diverse array of defense mechanisms. Studies with these plants have provided a wealth of information on pathogen recognition, signal transduction and the activation of defense responses. However, very little is known about the infection and defense responses of the bryophyte, Physcomitrella patens, to well-studied phytopathogens. The purpose of this study was to determine: i) whether two representative broad host range pathogens, Erwinia carotovora ssp. carotovora (E.c. carotovora) and Botrytis cinerea (B. cinerea), could infect Physcomitrella, and ii) whether B. cinerea, elicitors of a harpin (HrpN) producing E.c. carotovora strain (SCC1) or a HrpN-negative strain (SCC3193), could cause disease symptoms and induce defense responses in Physcomitrella. Results B. cinerea and E.c. carotovora were found to readily infect Physcomitrella gametophytic tissues and cause disease symptoms. Treatments with B. cinerea spores or cell-free culture filtrates from E.c. carotovoraSCC1 (CF(SCC1)), resulted in disease development with severe maceration of Physcomitrella tissues, while CF(SCC3193) produced only mild maceration. Although increased cell death was observed with either the CFs or B. cinerea, the occurrence of cytoplasmic shrinkage was only visible in Evans blue stained protonemal cells treated with CF(SCC1) or inoculated with B. cinerea. Most cells showing cytoplasmic shrinkage accumulated autofluorescent compounds and brown chloroplasts were evident in a high proportion of these cells. CF treatments and B. cinerea inoculation induced the expression of the defense-related genes: PR-1, PAL, CHS and LOX. Conclusion B. cinerea and E.c. carotovora elicitors induce a defense response in Physcomitrella, as evidenced by enhanced expression of conserved plant defense-related genes. Since cytoplasmic shrinkage is the most common morphological change observed in plant PCD, and that harpins and B. cinerea induce this

  19. In Silico and Biochemical Analysis of Physcomitrella patens Photosynthetic Antenna: Identification of Subunits which Evolved upon Land Adaptation

    PubMed Central

    Alboresi, Alessandro; Caffarri, Stefano; Nogue, Fabien; Bassi, Roberto; Morosinotto, Tomas

    2008-01-01

    Background In eukaryotes the photosynthetic antenna system is composed of subunits encoded by the light harvesting complex (Lhc) multigene family. These proteins play a key role in photosynthesis and are involved in both light harvesting and photoprotection. The moss Physcomitrella patens is a member of a lineage that diverged from seed plants early after land colonization and therefore by studying this organism, we may gain insight into adaptations to the aerial environment. Principal Findings In this study, we characterized the antenna protein multigene family in Physcomitrella patens, by sequence analysis as well as biochemical and functional investigations. Sequence identification and analysis showed that some antenna polypeptides, such as Lhcb3 and Lhcb6, are present only in land organisms, suggesting they play a role in adaptation to the sub-aerial environment. Our functional analysis which showed that photo-protective mechanisms in Physcomitrella patens are very similar to those in seed plants fits with this hypothesis. In particular, Physcomitrella patens also activates Non Photochemical Quenching upon illumination, consistent with the detection of an ortholog of the PsbS protein. As a further adaptation to terrestrial conditions, the content of Photosystem I low energy absorbing chlorophylls also increased, as demonstrated by differences in Lhca3 and Lhca4 polypeptide sequences, in vitro reconstitution experiments and low temperature fluorescence spectra. Conclusions This study highlights the role of Lhc family members in environmental adaptation and allowed proteins associated with mechanisms of stress resistance to be identified within this large family. PMID:18446222

  20. Identification of a 4-coumarate:CoA ligase gene family in the moss, Physcomitrella patens.

    PubMed

    Silber, Martina V; Meimberg, Harald; Ebel, Jürgen

    2008-10-01

    Since the early evolution of land plants from primitive green algae, phenylpropanoid compounds have played an important role. In the biosynthesis of phenylpropanoids, 4-coumarate:CoA ligase (4CL; EC 6.2.1.12) has a pivotal role at the divergence point from general phenylpropanoid metabolism to several major branch pathways. Although higher plant 4CLs have been extensively studied, little information is available on the enzymes from bryophytes. In Physcomitrella patens, we have identified a 4CL gene family consisting of four members, taking advantage of the available EST sequences and a draft sequence of the P. patens genome. The encoded proteins of three of the genes display similar substrate utilization profiles with highest catalytic efficiency towards 4-coumarate. Interestingly, the efficiency with cinnamate as substrate is in the same range as with caffeate and ferulate. The deduced proteins of the four genes share sequence identities between 78% and 86%. The intron/exon structures are pair wise similar. Pp4CL2 and Pp4CL3 each consists of four exons and three introns, whereas Pp4CL1 and Pp4CL4 are characterized each by five exons and four introns. Pp4CL1, Pp4CL2 and Pp4CL3 are expressed in both gametophore and protonema tissue of P. patens, unlike Pp4CL4 whose expression could not be demonstrated under the conditions employed. Phylogenetic analysis suggests an early evolutionary divergence of Pp4CL gene family members. Using Streptomyces coelicolor cinnamate:CoA ligase (ScCCL) as an outgroup, the P. patens 4CLs are clearly separated from the spermatophyte proteins, but are intercalated between the angiosperm 4CL class I and class II. A comparison of three P. patens subspecies from diverse geographical locations shows high sequence identities for the four 4CL isoforms.

  1. Does the core circadian clock in the moss Physcomitrella patens (Bryophyta) comprise a single loop?

    PubMed Central

    2010-01-01

    Background The endogenous circadian clock allows the organism to synchronize processes both to daily and seasonal changes. In plants, many metabolic processes such as photosynthesis, as well as photoperiodic responses, are under the control of a circadian clock. Comparative studies with the moss Physcomitrella patens provide the opportunity to study many aspects of land plant evolution. Here we present a comparative overview of clock-associated components and the circadian network in the moss P. patens. Results The moss P. patens has a set of conserved circadian core components that share genetic relationship and gene expression patterns with clock genes of vascular plants. These genes include Myb-like transcription factors PpCCA1a and PpCCA1b, pseudo-response regulators PpPRR1-4, and regulatory elements PpELF3, PpLUX and possibly PpELF4. However, the moss lacks homologs of AtTOC1, AtGI and the AtZTL-family of genes, which can be found in all vascular plants studied here. These three genes constitute essential components of two of the three integrated feed-back loops in the current model of the Arabidopsis circadian clock mechanism. Consequently, our results suggest instead a single loop circadian clock in the moss. Possibly as a result of this, temperature compensation of core clock gene expression appears to be decreased in P. patens. Conclusions This study is the first comparative overview of the circadian clock mechanism in a basal land plant, the moss P. patens. Our results indicate that the moss clock mechanism may represent an ancestral state in contrast to the more complex and partly duplicated structure of subsequent land plants. These findings may provide insights into the understanding of the evolution of circadian network topology. PMID:20550695

  2. Does the core circadian clock in the moss Physcomitrella patens (Bryophyta) comprise a single loop?

    PubMed

    Holm, Karl; Källman, Thomas; Gyllenstrand, Niclas; Hedman, Harald; Lagercrantz, Ulf

    2010-06-15

    The endogenous circadian clock allows the organism to synchronize processes both to daily and seasonal changes. In plants, many metabolic processes such as photosynthesis, as well as photoperiodic responses, are under the control of a circadian clock. Comparative studies with the moss Physcomitrella patens provide the opportunity to study many aspects of land plant evolution. Here we present a comparative overview of clock-associated components and the circadian network in the moss P. patens. The moss P. patens has a set of conserved circadian core components that share genetic relationship and gene expression patterns with clock genes of vascular plants. These genes include Myb-like transcription factors PpCCA1a and PpCCA1b, pseudo-response regulators PpPRR1-4, and regulatory elements PpELF3, PpLUX and possibly PpELF4. However, the moss lacks homologs of AtTOC1, AtGI and the AtZTL-family of genes, which can be found in all vascular plants studied here. These three genes constitute essential components of two of the three integrated feed-back loops in the current model of the Arabidopsis circadian clock mechanism. Consequently, our results suggest instead a single loop circadian clock in the moss. Possibly as a result of this, temperature compensation of core clock gene expression appears to be decreased in P. patens. This study is the first comparative overview of the circadian clock mechanism in a basal land plant, the moss P. patens. Our results indicate that the moss clock mechanism may represent an ancestral state in contrast to the more complex and partly duplicated structure of subsequent land plants. These findings may provide insights into the understanding of the evolution of circadian network topology.

  3. Hydroxyproline O-arabinosyltransferase mutants oppositely alter tip growth in Arabidopsis thaliana and Physcomitrella patens.

    PubMed

    MacAlister, Cora A; Ortiz-Ramírez, Carlos; Becker, Jörg D; Feijó, José A; Lippman, Zachary B

    2016-01-01

    Hydroxyproline O-arabinosyltransferases (HPATs) are members of a small, deeply conserved family of plant-specific glycosyltransferases that add arabinose sugars to diverse proteins including cell wall-associated extensins and small signaling peptides. Recent genetic studies in flowering plants suggest that different HPAT homologs have been co-opted to function in diverse species-specific developmental contexts. However, nothing is known about the roles of HPATs in basal plants. We show that complete loss of HPAT function in Arabidopsis thaliana and the moss Physcomitrella patens results in a shared defect in gametophytic tip cell growth. Arabidopsis hpat1/2/3 triple knockout mutants suffer from a strong male sterility defect as a consequence of pollen tubes that fail to fully elongate following pollination. Knocking out the two HPAT genes of Physcomitrella results in larger multicellular filamentous networks due to increased elongation of protonemal tip cells. Physcomitrella hpat mutants lack cell-wall associated hydroxyproline arabinosides and can be rescued with exogenous cellulose, while global expression profiling shows that cell wall-associated genes are severely misexpressed, implicating a defect in cell wall formation during tip growth. Our findings point to a major role for HPATs in influencing cell elongation during tip growth in plants.

  4. Functional cross-kingdom conservation of mammalian and moss (Physcomitrella patens) transcription, translation and secretion machineries.

    PubMed

    Gitzinger, Marc; Parsons, Juliana; Reski, Ralf; Fussenegger, Martin

    2009-01-01

    Plants and mammals are separated by a huge evolutionary distance. Consequently, biotechnology and genetics have traditionally been divided into 'green' and 'red'. Here, we provide comprehensive evidence that key components of the mammalian transcription, translation and secretion machineries are functional in the model plant Physcomitrella patens. Cross-kingdom compatibility of different expression modalities originally designed for mammalian cells, such as native and synthetic promoters and polyadenylation sites, viral and cellular internal ribosome entry sites, secretion signal peptides and secreted product proteins, and synthetic transactivators and transrepressors, was established. This mammalian expression portfolio enabled constitutive, conditional and autoregulated expression of different product genes in a multicistronic expression format, optionally adjusted by various trigger molecules, such as butyrolactones, macrolide antibiotics and ethanol. Capitalizing on a cross-kingdom-compatible expression platform, we pioneered a prototype biopharmaceutical manufacturing scenario using microencapsulated transgenic P. patens protoplasts cultivated in a Wave Bioreactor. Vascular endothelial growth factor 121 (VEGF(121)) titres matched those typically achieved by standard protonema populations grown in stirred-tank bioreactors. The full compatibility of mammalian expression systems in P. patens further promotes the use of moss as a cost-effective alternative for the manufacture of complex biopharmaceuticals, and as a valuable host system to advance synthetic biology in plants.

  5. Unexpected complexity of the Aquaporin gene family in the moss Physcomitrella patens

    PubMed Central

    Danielson, Jonas ÅH; Johanson, Urban

    2008-01-01

    Background Aquaporins, also called major intrinsic proteins (MIPs), constitute an ancient superfamily of channel proteins that facilitate the transport of water and small solutes across cell membranes. MIPs are found in almost all living organisms and are particularly abundant in plants where they form a divergent group of proteins able to transport a wide selection of substrates. Results Analyses of the whole genome of Physcomitrella patens resulted in the identification of 23 MIPs, belonging to seven different subfamilies, of which only five have been previously described. Of the newly discovered subfamilies one was only identified in P. patens (Hybrid Intrinsic Protein, HIP) whereas the other was found to be present in a wide variety of dicotyledonous plants and forms a major previously unrecognized MIP subfamily (X Intrinsic Proteins, XIPs). Surprisingly also some specific groups within subfamilies present in Arabidopsis thaliana and Zea mays could be identified in P. patens. Conclusion Our results suggest an early diversification of MIPs resulting in a large number of subfamilies already in primitive terrestrial plants. During the evolution of higher plants some of these subfamilies were subsequently lost while the remaining subfamilies expanded and in some cases diversified, resulting in the formation of more specialized groups within these subfamilies. PMID:18430224

  6. A single CMT methyltransferase homolog is involved in CHG DNA methylation and development of Physcomitrella patens.

    PubMed

    Noy-Malka, Chen; Yaari, Rafael; Itzhaki, Rachel; Mosquna, Assaf; Auerbach Gershovitz, Nitzan; Katz, Aviva; Ohad, Nir

    2014-04-01

    C-5 DNA methylation is an essential mechanism controlling gene expression and developmental programs in a variety of organisms. Though the role of DNA methylation has been intensively studied in mammals and Arabidopsis, little is known about the evolution of this mechanism. The chromomethylase (CMT) methyltransferase family is unique to plants and was found to be involved in DNA methylation in Arabidopsis, maize and tobacco. The moss Physcomitrella patens, a model for early terrestrial plants, harbors a single homolog of the CMT protein family designated as PpCMT. Our phylogenetic analysis suggested that the CMT family is unique to embryophytes and its earliest known member PpCMT belongs to the CMT3 subfamily. Thus, P. patens may serve as a model to study the ancient functions of the CMT3 family. We have generated a ΔPpcmt deletion mutant which demonstrated that PpCMT is essential for P. patens protonema and gametophore development and is involved in CHG methylation as demonstrated at four distinct genomic loci. PpCMT protein accumulation pattern correlated with proliferating cells and was sub-localized to the nucleus as predicted from its function. Taken together, our results suggested that CHG DNA methylation mediated by CMT has been employed early in land plant evolution to control developmental programs during both the vegetative and reproductive haploid phases along the plant life cycle.

  7. A novel plant major intrinsic protein in Physcomitrella patens most similar to bacterial glycerol channels.

    PubMed

    Gustavsson, Sofia; Lebrun, Anne-Sophie; Nordén, Kristina; Chaumont, François; Johanson, Urban

    2005-09-01

    A gene encoding a novel fifth type of major intrinsic protein (MIP) in plants has been identified in the moss Physcomitrella patens. Phylogenetic analyses show that this protein, GlpF-like intrinsic protein (GIP1;1), is closely related to a subclass of glycerol transporters in bacteria that in addition to glycerol are highly permeable to water. A likely explanation of the occurrence of this bacterial-like MIP in P. patens is horizontal gene transfer. The expressed P. patens GIP1;1 gene contains five introns and encodes a unique C-loop extension of approximately 110 amino acid residues that has no obvious similarity with any other known protein. Based on alignments and structural comparisons with other MIPs, GIP1;1 is suggested to have retained the permeability for glycerol but not for water. Studies on heterologously expressed GIP1;1 in Xenopus laevis oocytes confirm the predicted substrate specificity. Interestingly, proteins of one of the plant-specific subgroups of MIPs, the NOD26-like intrinsic proteins, are also facilitating the transport of glycerol and have previously been suggested to have evolved from a horizontally transferred bacterial gene. Further studies on localization and searches for GIP1;1 homologs in other plants will clarify the function and significance of this new plant MIP.

  8. Quantitative analysis of organelle distribution and dynamics in Physcomitrella patens protonemal cells

    PubMed Central

    2012-01-01

    Background In the last decade, the moss Physcomitrella patens has emerged as a powerful plant model system, amenable for genetic manipulations not possible in any other plant. This moss is particularly well suited for plant polarized cell growth studies, as in its protonemal phase, expansion is restricted to the tip of its cells. Based on pollen tube and root hair studies, it is well known that tip growth requires active secretion and high polarization of the cellular components. However, such information is still missing in Physcomitrella patens. To gain insight into the mechanisms underlying the participation of organelle organization in tip growth, it is essential to determine the distribution and the dynamics of the organelles in moss cells. Results We used fluorescent protein fusions to visualize and track Golgi dictyosomes, mitochondria, and peroxisomes in live protonemal cells. We also visualized and tracked chloroplasts based on chlorophyll auto-fluorescence. We showed that in protonemata all four organelles are distributed in a gradient from the tip of the apical cell to the base of the sub-apical cell. For example, the density of Golgi dictyosomes is 4.7 and 3.4 times higher at the tip than at the base in caulonemata and chloronemata respectively. While Golgi stacks are concentrated at the extreme tip of the caulonemata, chloroplasts and peroxisomes are totally excluded. Interestingly, caulonemata, which grow faster than chloronemata, also contain significantly more Golgi dictyosomes and fewer chloroplasts than chloronemata. Moreover, the motility analysis revealed that organelles in protonemata move with low persistency and average instantaneous speeds ranging from 29 to 75 nm/s, which are at least three orders of magnitude slower than those of pollen tube or root hair organelles. Conclusions To our knowledge, this study reports the first quantitative analysis of organelles in Physcomitrella patens and will make possible comparisons of the distribution

  9. Characterization of a PDK1 homologue from the moss Physcomitrella patens.

    PubMed

    Dittrich, Anna C Nelson; Devarenne, Timothy P

    2012-02-01

    The serine/threonine protein kinase 3-phosphoinositide-dependent protein kinase 1 (PDK1) is a highly conserved eukaryotic kinase that is a central regulator of many AGC kinase subfamily members. Through its regulation of AGC kinases, PDK1 controls many basic cellular processes, from translation to cell survival. While many of these PDK1-regulated processes are conserved across kingdoms, it is not well understood how PDK1 may have evolved within kingdoms. In order to better understand PDK1 evolution within plants, we have isolated and characterized the PDK1 gene from the moss Physcomitrella patens (PpPDK1), a nonvascular representative of early land plants. PpPDK1 is similar to other plant PDK1s in that it can functionally complement a yeast PDK1 knockout line. However, unlike PDK1 from other plants, the P. patens PDK1 protein does not bind phospholipids due to a lack of the lipid-binding pleckstrin homology domain, which is used for lipid-mediated regulation of PDK1 activity. Sequence analysis of several PDK1 proteins suggests that lipid regulation of PDK1 may not commonly occur in algae and nonvascular land plants. PpPDK1 can phosphorylate AGC kinase substrates from tomato (Solanum lycopersicum) and P. patens at the predicted PDK1 phosphorylation site, indicating that the PpPDK1 substrate phosphorylation site is conserved with higher plants. We have also identified residues within the PpPDK1 kinase domain that affect kinase activity and show that a mutant with highly reduced kinase activity can still confer cell viability in both yeast and P. patens. These studies lay the foundation for further analysis of the evolution of PDK1 within plants.

  10. Cloning and characterization of chalcone synthase from the moss, Physcomitrella patens.

    PubMed

    Jiang, Chenguang; Schommer, Clark K; Kim, Sun Young; Suh, Dae-Yeon

    2006-12-01

    Since the early evolution of land plants from primitive green algae, flavonoids have played an important role as UV protective pigments in plants. Flavonoids occur in liverworts and mosses, and the first committed step in the flavonoid biosynthesis is catalyzed by chalcone synthase (CHS). Although higher plant CHSs have been extensively studied, little information is available on the enzymes from bryophytes. Here we report the cloning and characterization of CHS from the moss, Physcomitrella patens. Taking advantage of the available P. patens EST sequences, a CHS (PpCHS) was cloned from the gametophores of P. patens, and heterologously expressed in Escherichia coli. PpCHS exhibited similar kinetic properties and substrate preference profile to those of higher plant CHS. p-Coumaroyl-CoA was the most preferred substrate, suggesting that PpCHS is a naringenin chalcone producing CHS. Consistent with the evolutionary position of the moss, phylogenetic analysis placed PpCHS at the base of the plant CHS clade, next to the microorganism CHS-like gene products. Therefore, PpCHS likely represents a modern day version of one of the oldest CHSs that appeared on earth. Further, sequence analysis of the P. patens EST and genome databases revealed the presence of a CHS multigene family in the moss as well as the 3'-end heterogeneity of a CHS gene. Of the 19 putative CHS genes, 10 genes are expressed and have corresponding ESTs in the databases. A possibility of the functional divergence of the multiple CHS genes in the moss is discussed.

  11. The Moss Physcomitrella patens as a Model System to Study Interactions between Plants and Phytopathogenic Fungi and Oomycetes

    PubMed Central

    Ponce de León, Inés

    2011-01-01

    The moss Physcomitrella patens has a great potential as a model system to perform functional studies of plant interacting with microbial pathogens. P. patens is susceptible to fungal and oomycete infection, which colonize and multiply in plant tissues generating disease symptoms. In response to infection, P. patens activates defense mechanisms similar to those induced in flowering plants, including the accumulation of reactive oxygen species, cell death with hallmarks of programmed cell death, cell wall fortification, and induction of defense-related genes like PAL, LOX, CHS, and PR-1. Functional analysis of genes with possible roles in defense can be performed due to the high rate of homologous recombination present in this plant that enables targeted gene disruption. This paper reviews the current knowledge of defense responses activated in P. patens after pathogen assault and analyzes the advantages of using this plant to gain further insight into plant defense strategies. PMID:22567339

  12. The Molecular and Physiological Responses of Physcomitrella patens to Ultraviolet-B Radiation1[W][OA

    PubMed Central

    Wolf, Luise; Rizzini, Luca; Stracke, Ralf; Ulm, Roman; Rensing, Stefan A.

    2010-01-01

    Ultraviolet-B (UV-B) radiation present in sunlight is an important trigger of photomorphogenic acclimation and stress responses in sessile land plants. Although numerous moss species grow in unshaded habitats, our understanding of their UV-B responses is very limited. The genome of the model moss Physcomitrella patens, which grows in sun-exposed open areas, encodes signaling and metabolic components that are implicated in the UV-B response in flowering plants. In this study, we describe the response of P. patens to UV-B radiation at the morphological and molecular levels. We find that P. patens is more capable of surviving UV-B stress than Arabidopsis (Arabidopsis thaliana) and describe the differential expression of approximately 400 moss genes in response to UV-B radiation. A comparative analysis of the UV-B response in P. patens and Arabidopsis reveals both distinct and conserved pathways. PMID:20427465

  13. A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae)

    DOE PAGES

    Scavuzzo-Duggan, Tess R.; Chaves, Arielle M.; Roberts, Alison W.

    2015-07-14

    Here, a method for rapid in vivo functional analysis of engineered proteins was developed using Physcomitrella patens. A complementation assay was designed for testing structure/function relationships in cellulose synthase (CESA) proteins. The components of the assay include (1) construction of test vectors that drive expression of epitope-tagged PpCESA5 carrying engineered mutations, (2) transformation of a ppcesa5 knockout line that fails to produce gametophores with test and control vectors, (3) scoring the stable transformants for gametophore production, (4) statistical analysis comparing complementation rates for test vectors to positive and negative control vectors, and (5) analysis of transgenic protein expression by Westernmore » blotting. The assay distinguished mutations that generate fully functional, nonfunctional, and partially functional proteins. In conclusion, compared with existing methods for in vivo testing of protein function, this complementation assay provides a rapid method for investigating protein structure/function relationships in plants.« less

  14. Exploring the mechanism of Physcomitrella patens desiccation tolerance through a proteomic strategy.

    PubMed

    Wang, Xiao Qin; Yang, Ping Fang; Liu, Zheng; Liu, Wei Zhong; Hu, Yong; Chen, Hui; Kuang, Ting Yun; Pei, Zhen Ming; Shen, Shi Hua; He, Yi Kun

    2009-04-01

    The moss Physcomitrella patens has been shown to tolerate abiotic stresses, including salinity, cold, and desiccation. To better understand this plant's mechanism of desiccation tolerance, we have applied cellular and proteomic analyses. Gametophores were desiccated over 1 month to 10% of their original fresh weight. We report that during the course of dehydration, several related processes are set in motion: plasmolysis, chloroplast remodeling, and microtubule depolymerization. Despite the severe desiccation, the membrane system maintains integrity. Through two-dimensional gel electrophoresis and image analysis, we identified 71 proteins as desiccation responsive. Following identification and functional categorization, we found that a majority of the desiccation-responsive proteins were involved in metabolism, cytoskeleton, defense, and signaling. Degradation of cytoskeletal proteins might result in cytoskeletal disassembly and consequent changes in the cell structure. Late embryogenesis abundant proteins and reactive oxygen species-scavenging enzymes are both prominently induced, and they might help to diminish the damage brought by desiccation.

  15. Proteome analysis of Physcomitrella patens exposed to progressive dehydration and rehydration

    PubMed Central

    Cui, Suxia; Hu, Jia; Guo, Shilei; Wang, Jie; Cheng, Yali; Dang, Xinxing; Wu, Lili; He, Yikun

    2012-01-01

    Physcomitrella patens is an extremely dehydration-tolerant moss. However, the molecular basis of its responses to loss of cellular water remains unclear. A comprehensive proteomic analysis of dehydration- and rehydration-responsive proteins has been conducted using quantitative two-dimensional difference in-gel electrophoresis (2D-DIGE), and traditional 2-D gel electrophoresis (2-DE) combined with MALDI TOF/TOF MS. Of the 216 differentially-expressed protein spots, 112 and 104 were dehydration- and rehydration-responsive proteins, respectively. The functional categories of the most differentially-expressed proteins were seed maturation, defence, protein synthesis and quality control, and energy production. Strikingly, most of the late embryogenesis abundant (LEA) proteins were expressed at a basal level under control conditions and their synthesis was strongly enhanced by dehydration, a pattern that was confirmed by RT-PCR. Actinoporins, phosphatidylethanolamine-binding protein, arabinogalactan protein, and phospholipase are the likely dominant players in the defence system. In addition, 24 proteins of unknown function were identified as novel dehydration- or rehydration-responsive proteins. Our data indicate that Physcomitrella adopts a rapid protein response mechanism to cope with dehydration in its leafy-shoot and basal expression levels of desiccation-tolerant proteins are rapidly upgraded at high levels under stress. This mechanism appears similar to that seen in angiosperm seeds. PMID:21994173

  16. PHYTOCHROME INTERACTING FACTORs from Physcomitrella patens are active in Arabidopsis and complement the pif quadruple mutant.

    PubMed

    Xu, Tengfei; Hiltbrunner, Andreas

    2017-10-06

    Phytochromes are red/far-red light receptors in plants involved in the regulation of growth and development in response to changes in the ambient environment. An important mode of action of plant phytochromes depends on their light-regulated relocation from the cytosol into the nucleus and control of gene expression; in addition, there is also evidence for a cytosolic or plasma membrane associated function of phytochromes in different species. The PHYTOCHROME INTERACTING FACTORs (PIFs) form a subgroup of the bHLH transcription factors and it is well established that PIFs are key components of phytochrome downstream signalling in the nucleus of seed plants. Recent studies identified members of the PIF family also in the liverwort Marchantia polymorpha and the moss Physcomitrella patens. Here, we show that all four potential PIF homologs from Physcomitrella have PIF function when expressed in the Arabidopsis pifQ mutant, which is deficient in multiple PIFs. We propose that PIFs are ancient components of nuclear phytochrome signalling that have emerged in the last common ancestor of today's land plants.

  17. Simple and Efficient Targeting of Multiple Genes Through CRISPR-Cas9 in Physcomitrella patens

    PubMed Central

    Lopez-Obando, Mauricio; Hoffmann, Beate; Géry, Carine; Guyon-Debast, Anouchka; Téoulé, Evelyne; Rameau, Catherine; Bonhomme, Sandrine; Nogué, Fabien

    2016-01-01

    Powerful genome editing technologies are needed for efficient gene function analysis. The CRISPR-Cas9 system has been adapted as an efficient gene-knock-out technology in a variety of species. However, in a number of situations, knocking out or modifying a single gene is not sufficient; this is particularly true for genes belonging to a common family, or for genes showing redundant functions. Like many plants, the model organism Physcomitrella patens has experienced multiple events of polyploidization during evolution that has resulted in a number of families of duplicated genes. Here, we report a robust CRISPR-Cas9 system, based on the codelivery of a CAS9 expressing cassette, multiple sgRNA vectors, and a cassette for transient transformation selection, for gene knock-out in multiple gene families. We demonstrate that CRISPR-Cas9-mediated targeting of five different genes allows the selection of a quintuple mutant, and all possible subcombinations of mutants, in one experiment, with no mutations detected in potential off-target sequences. Furthermore, we confirmed the observation that the presence of repeats in the vicinity of the cutting region favors deletion due to the alternative end joining pathway, for which induced frameshift mutations can be potentially predicted. Because the number of multiple gene families in Physcomitrella is substantial, this tool opens new perspectives to study the role of expanded gene families in the colonization of land by plants. PMID:27613750

  18. Physcomitrella patens activates reinforcement of the cell wall, programmed cell death and accumulation of evolutionary conserved defense signals...upon Botrytis cinerea infection

    USDA-ARS?s Scientific Manuscript database

    The moss Physcomitrella patens is an evolutionarily basal model system suitable to analyze plant defense responses activated after pathogen assault. Upon infection with the necrotroph Botrytis cinerea (B. cinerea), several defense mechanisms are induced in P. patens, including the fortification of t...

  19. In vivo assembly of DNA-fragments in the moss, Physcomitrella patens

    PubMed Central

    King, Brian Christopher; Vavitsas, Konstantinos; Ikram, Nur Kusaira Binti Khairul; Schrøder, Josephine; Scharff, Lars B.; Hamberger, Björn; Jensen, Poul Erik; Simonsen, Henrik Toft

    2016-01-01

    Direct assembly of multiple linear DNA fragments via homologous recombination, a phenomenon known as in vivo assembly or transformation associated recombination, is used in biotechnology to assemble DNA constructs ranging in size from a few kilobases to full synthetic microbial genomes. It has also enabled the complete replacement of eukaryotic chromosomes with heterologous DNA. The moss Physcomitrella patens, a non-vascular and spore producing land plant (Bryophyte), has a well-established capacity for homologous recombination. Here, we demonstrate the in vivo assembly of multiple DNA fragments in P. patens with three examples of effective genome editing: we (i) efficiently deleted a genomic locus for diterpenoid metabolism yielding a biosynthetic knockout, (ii) introduced a salt inducible promoter, and (iii) re-routed endogenous metabolism into the formation of amorphadiene, a precursor of high-value therapeutics. These proof-of-principle experiments pave the way for more complex and increasingly flexible approaches for large-scale metabolic engineering in plant biotechnology. PMID:27126800

  20. Giant peroxisomes in a moss (Physcomitrella patens) peroxisomal biogenesis factor 11 mutant.

    PubMed

    Kamisugi, Yasuko; Mitsuya, Shiro; El-Shami, Mahmoud; Knight, Celia D; Cuming, Andrew C; Baker, Alison

    2016-01-01

    Peroxisomal biogenesis factor 11 (PEX11) proteins are found in yeasts, mammals and plants, and play a role in peroxisome morphology and regulation of peroxisome division. The moss Physcomitrella patens has six PEX11 isoforms which fall into two subfamilies, similar to those found in monocots and dicots. We carried out targeted gene disruption of the Phypa_PEX11-1 gene and compared the morphological and cellular phenotypes of the wild-type and mutant strains. The mutant grew more slowly and the development of gametophores was retarded. Mutant chloronemal filaments contained large cellular structures which excluded all other cellular organelles. Expression of fluorescent reporter proteins revealed that the mutant strain had greatly enlarged peroxisomes up to 10 μm in diameter. Expression of a vacuolar membrane marker confirmed that the enlarged structures were not vacuoles, or peroxisomes sequestered within vacuoles as a result of pexophagy. Phypa_PEX11 targeted to peroxisome membranes could rescue the knock out phenotype and interacted with Fission1 on the peroxisome membrane. Moss PEX11 functions in peroxisome division similar to PEX11 in other organisms but the mutant phenotype is more extreme and environmentally determined, making P. patens a powerful system in which to address mechanisms of peroxisome proliferation and division. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  1. Myosin XI Is Essential for Tip Growth in Physcomitrella patens[W

    PubMed Central

    Vidali, Luis; Burkart, Graham M.; Augustine, Robert C.; Kerdavid, Erin; Tüzel, Erkan; Bezanilla, Magdalena

    2010-01-01

    Class XI myosins are plant specific and responsible for cytoplasmic streaming. Because of the large number of myosin XI genes in angiosperms, it has been difficult to determine their precise role, particularly with respect to tip growth. The moss Physcomitrella patens provides an ideal system to study myosin XI function. P. patens has only two myosin XI genes, and these genes encode proteins that are 94% identical to each other. To determine their role in tip growth, we used RNA interference to specifically silence each myosin XI gene using 5′ untranslated region sequences. We discovered that the two myosin XI genes are functionally redundant, since silencing of either gene does not affect growth or polarity. However, simultaneous silencing of both myosin XIs results in severely stunted plants composed of small rounded cells. Although similar to the phenotype resulting from silencing of other actin-associated proteins, we show that this phenotype is not due to altered actin dynamics. Consistent with a role in tip growth, we show that a functional, full-length fusion of monomeric enhanced green fluorescent protein (mEGFP) to myosin XI accumulates at a subcortical, apical region of actively growing protonemal cells. PMID:20525854

  2. Immuno and Affinity Cytochemical Analysis of Cell Wall Composition in the Moss Physcomitrella patens

    SciTech Connect

    Berry, Elizabeth A.; Tran, Mai L.; Dimos, Christos S.; Budziszek, Michael J.; Scavuzzo-Duggan, Tess R.; Roberts, Alison W.

    2016-03-08

    In contrast to homeohydric vascular plants, mosses employ a poikilohydric strategy for surviving in the dry aerial environment. A detailed understanding of the structure, composition, and development of moss cell walls can contribute to our understanding of not only the evolution of overall cell wall complexity, but also the differences that have evolved in response to selection for different survival strategies. The model moss species Physcomitrella patens has a predominantly haploid lifecycle consisting of protonemal filaments that regenerate from protoplasts and enlarge by tip growth, and leafy gametophores composed of cells that enlarge by diffuse growth and differentiate into several different types. Advantages for genetic studies include methods for efficient targeted gene modification and extensive genomic resources. Immuno and affinity cytochemical labeling were used to examine the distribution of polysaccharides and proteins in regenerated protoplasts, protonemal filaments, rhizoids, and sectioned gametophores of P. patens. The cell wall composition of regenerated protoplasts was also characterized by flow cytometry. Crystalline cellulose was abundant in the cell walls of regenerating protoplasts and protonemal cells that developed on media of high osmolarity, whereas homogalactuonan was detected in the walls of protonemal cells that developed on low osmolarity media and not in regenerating protoplasts. Mannan was the major hemicellulose detected in all tissues tested. Arabinogalactan proteins were detected in different cell types by different probes, consistent with structural heterogneity. The results reveal developmental and cell type specific differences in cell wall composition and provide a basis for analyzing cell wall phenotypes in knockout mutants.

  3. A CELLULOSE SYNTHASE (CESA) gene essential for gametophore morphogenesis in the moss Physcomitrella patens.

    PubMed

    Goss, Chessa A; Brockmann, Derek J; Bushoven, John T; Roberts, Alison W

    2012-06-01

    In seed plants, different groups of orthologous genes encode the CELLULOSE SYNTHASE (CESA) proteins that are responsible for cellulose biosynthesis in primary and secondary cell walls. The seven CESA sequences of the moss Physcomitrella patens (Hedw.) B. S. G. form a monophyletic sister group to seed plant CESAs, consistent with independent CESA diversification and specialization in moss and seed plant lines. The role of PpCESA5 in the development of P. patens was investigated by targeted mutagenesis. The cesa5 knockout lines were tested for cellulose deficiency using carbohydrate-binding module affinity cytochemistry and the morphology of the leafy gametophores was analyzed by 3D reconstruction of confocal images. No defects were identified in the development of the filamentous protonema or in production of bud initials that normally give rise to the leafy gametophores. However, the gametophore buds were cellulose deficient and defects in subsequent cell expansion, cytokinesis, and leaf initiation resulted in the formation of irregular cell clumps instead of leafy shoots. Analysis of the cesa5 knockout phenotype indicates that a biophysical model of organogenesis can be extended to the moss gametophore shoot apical meristem.

  4. Genome-wide transcriptomic analysis of the sporophyte of the moss Physcomitrella patens.

    PubMed

    O'Donoghue, Martin-Timothy; Chater, Caspar; Wallace, Simon; Gray, Julie E; Beerling, David J; Fleming, Andrew J

    2013-09-01

    Bryophytes, the most basal of the extant land plants, diverged at least 450 million years ago. A major feature of these plants is the biphasic alternation of generations between a dominant haploid gametophyte and a minor diploid sporophyte phase. These dramatic differences in form and function occur in a constant genetic background, raising the question of whether the switch from gametophyte-to-sporophyte development reflects major changes in the spectrum of genes being expressed or alternatively whether only limited changes in gene expression occur and the differences in plant form are due to differences in how the gene products are put together. This study performed replicated microarray analyses of RNA from several thousand dissected and developmentally staged sporophytes of the moss Physcomitrella patens, allowing analysis of the transcriptomes of the sporophyte and early gametophyte, as well as the early stages of moss sporophyte development. The data indicate that more significant changes in transcript profile occur during the switch from gametophyte to sporophyte than recently reported, with over 12% of the entire transcriptome of P. patens being altered during this major developmental transition. Analysis of the types of genes contributing to these differences supports the view of the early sporophyte being energetically and nutritionally dependent on the gametophyte, provides a profile of homologues to genes involved in angiosperm stomatal development and physiology which suggests a deeply conserved mechanism of stomatal control, and identifies a novel series of transcription factors associated with moss sporophyte development.

  5. Flavodiiron proteins act as safety valve for electrons in Physcomitrella patens

    PubMed Central

    Gerotto, Caterina; Meneghesso, Andrea; Jokel, Martina; Suorsa, Marjaana; Aro, Eva-Mari

    2016-01-01

    Photosynthetic organisms support cell metabolism by harvesting sunlight to fuel the photosynthetic electron transport. The flow of excitation energy and electrons in the photosynthetic apparatus needs to be continuously modulated to respond to dynamics of environmental conditions, and Flavodiiron (FLV) proteins are seminal components of this regulatory machinery in cyanobacteria. FLVs were lost during evolution by flowering plants, but are still present in nonvascular plants such as Physcomitrella patens. We generated P. patens mutants depleted in FLV proteins, showing their function as an electron sink downstream of photosystem I for the first seconds after a change in light intensity. flv knock-out plants showed impaired growth and photosystem I photoinhibition when exposed to fluctuating light, demonstrating FLV’s biological role as a safety valve from excess electrons on illumination changes. The lack of FLVs was partially compensated for by an increased cyclic electron transport, suggesting that in flowering plants, the FLV’s role was taken by other alternative electron routes. PMID:27791022

  6. Immuno and Affinity Cytochemical Analysis of Cell Wall Composition in the Moss Physcomitrella patens

    PubMed Central

    Berry, Elizabeth A.; Tran, Mai L.; Dimos, Christos S.; Budziszek, Michael J.; Scavuzzo-Duggan, Tess R.; Roberts, Alison W.

    2016-01-01

    In contrast to homeohydric vascular plants, mosses employ a poikilohydric strategy for surviving in the dry aerial environment. A detailed understanding of the structure, composition, and development of moss cell walls can contribute to our understanding of not only the evolution of overall cell wall complexity, but also the differences that have evolved in response to selection for different survival strategies. The model moss species Physcomitrella patens has a predominantly haploid lifecycle consisting of protonemal filaments that regenerate from protoplasts and enlarge by tip growth, and leafy gametophores composed of cells that enlarge by diffuse growth and differentiate into several different types. Advantages for genetic studies include methods for efficient targeted gene modification and extensive genomic resources. Immuno and affinity cytochemical labeling were used to examine the distribution of polysaccharides and proteins in regenerated protoplasts, protonemal filaments, rhizoids, and sectioned gametophores of P. patens. The cell wall composition of regenerated protoplasts was also characterized by flow cytometry. Crystalline cellulose was abundant in the cell walls of regenerating protoplasts and protonemal cells that developed on media of high osmolarity, whereas homogalactuonan was detected in the walls of protonemal cells that developed on low osmolarity media and not in regenerating protoplasts. Mannan was the major hemicellulose detected in all tissues tested. Arabinogalactan proteins were detected in different cell types by different probes, consistent with structural heterogneity. The results reveal developmental and cell type specific differences in cell wall composition and provide a basis for analyzing cell wall phenotypes in knockout mutants. PMID:27014284

  7. Genome-wide transcriptomic analysis of the sporophyte of the moss Physcomitrella patens

    PubMed Central

    O’Donoghue, Martin-Timothy; Chater, Caspar; Wallace, Simon; Gray, Julie E.; Beerling, David J.; Fleming, Andrew J.

    2013-01-01

    Bryophytes, the most basal of the extant land plants, diverged at least 450 million years ago. A major feature of these plants is the biphasic alternation of generations between a dominant haploid gametophyte and a minor diploid sporophyte phase. These dramatic differences in form and function occur in a constant genetic background, raising the question of whether the switch from gametophyte-to-sporophyte development reflects major changes in the spectrum of genes being expressed or alternatively whether only limited changes in gene expression occur and the differences in plant form are due to differences in how the gene products are put together. This study performed replicated microarray analyses of RNA from several thousand dissected and developmentally staged sporophytes of the moss Physcomitrella patens, allowing analysis of the transcriptomes of the sporophyte and early gametophyte, as well as the early stages of moss sporophyte development. The data indicate that more significant changes in transcript profile occur during the switch from gametophyte to sporophyte than recently reported, with over 12% of the entire transcriptome of P. patens being altered during this major developmental transition. Analysis of the types of genes contributing to these differences supports the view of the early sporophyte being energetically and nutritionally dependent on the gametophyte, provides a profile of homologues to genes involved in angiosperm stomatal development and physiology which suggests a deeply conserved mechanism of stomatal control, and identifies a novel series of transcription factors associated with moss sporophyte development. PMID:23888066

  8. Immuno and Affinity Cytochemical Analysis of Cell Wall Composition in the Moss Physcomitrella patens

    DOE PAGES

    Berry, Elizabeth A.; Tran, Mai L.; Dimos, Christos S.; ...

    2016-03-08

    In contrast to homeohydric vascular plants, mosses employ a poikilohydric strategy for surviving in the dry aerial environment. A detailed understanding of the structure, composition, and development of moss cell walls can contribute to our understanding of not only the evolution of overall cell wall complexity, but also the differences that have evolved in response to selection for different survival strategies. The model moss species Physcomitrella patens has a predominantly haploid lifecycle consisting of protonemal filaments that regenerate from protoplasts and enlarge by tip growth, and leafy gametophores composed of cells that enlarge by diffuse growth and differentiate into severalmore » different types. Advantages for genetic studies include methods for efficient targeted gene modification and extensive genomic resources. Immuno and affinity cytochemical labeling were used to examine the distribution of polysaccharides and proteins in regenerated protoplasts, protonemal filaments, rhizoids, and sectioned gametophores of P. patens. The cell wall composition of regenerated protoplasts was also characterized by flow cytometry. Crystalline cellulose was abundant in the cell walls of regenerating protoplasts and protonemal cells that developed on media of high osmolarity, whereas homogalactuonan was detected in the walls of protonemal cells that developed on low osmolarity media and not in regenerating protoplasts. Mannan was the major hemicellulose detected in all tissues tested. Arabinogalactan proteins were detected in different cell types by different probes, consistent with structural heterogneity. The results reveal developmental and cell type specific differences in cell wall composition and provide a basis for analyzing cell wall phenotypes in knockout mutants.« less

  9. Endogenous Small-Noncoding RNAs and Potential Functions in Desiccation Tolerance in Physcomitrella Patens

    PubMed Central

    Xia, Jing; Wang, Xiaoqin; Perroud, Pierre-François; He, Yikun; Quatrano, Ralph; Zhang, Weixiong

    2016-01-01

    Early land plants like moss Physcomitrella patens have developed remarkable drought tolerance. Phytohormone abscisic acid (ABA) protects seeds during water stress by activating genes through transcription factors such as ABSCISIC ACID INSENSITIVE (ABI3). Small noncoding RNA (sncRNA), including microRNAs (miRNAs) and endogenous small-interfering RNAs (endo-siRNAs), are key gene regulators in eukaryotes, playing critical roles in stress tolerance in plants. Combining next-generation sequencing and computational analysis, we profiled and characterized sncRNA species from two ABI3 deletion mutants and the wild type P. patens that were subject to ABA treatment in dehydration and rehydration stages. Small RNA profiling using deep sequencing helped identify 22 novel miRNAs and 6 genomic loci producing trans-acting siRNAs (ta-siRNAs) including TAS3a to TAS3e and TAS6. Data from degradome profiling showed that ABI3 genes (ABI3a/b/c) are potentially regulated by the plant-specific miR536 and that other ABA-relevant genes are regulated by miRNAs and ta-siRNAs. We also observed broad variations of miRNAs and ta-siRNAs expression across different stages, suggesting that they could potentially influence desiccation tolerance. This study provided evidence on the potential roles of sncRNA in mediating desiccation-responsive pathways in early land plants. PMID:27443635

  10. Defective Kernel 1 (DEK1) is required for three-dimensional growth in Physcomitrella patens

    PubMed Central

    Perroud, Pierre-François; Demko, Viktor; Johansen, Wenche; Wilson, Robert C; Olsen, Odd-Arne; Quatrano, Ralph S

    2014-01-01

    Orientation of cell division is critical for plant morphogenesis. This is evident in the formation and function of meristems and for morphogenetic transitions. Mosses undergo such transitions: from two-dimensional tip-growing filaments (protonema) to the generation of three-dimensional leaf-like structures (gametophores). The Defective Kernel 1 (DEK1) protein plays a key role in the perception of and/or response to positional cues that specify the formation and function of the epidermal layer in developing seeds of flowering plants. The moss Physcomitrella patens contains the highly conserved DEK1 gene. Using efficient gene targeting, we generated a precise PpDEK1 deletion (Δdek1), which resulted in normal filamentous growth of protonema. Two distinct mutant phenotypes were observed: an excess of buds on the protonema, and abnormal cell divisions in the emerging buds resulting in developmental arrest and the absence of three-dimensional growth. Overexpression of a complete PpDEK1 cDNA, or the calpain domain of PpDEK1 alone, successfully complements both phenotypes. These results in P. patens demonstrate the morphogenetic importance of the DEK1 protein in the control of oriented cell divisions. As it is not for protonema, it will allow dissection of the structure/function relationships of the different domains of DEK1 using gene targeting in null mutant background. PMID:24844771

  11. Characterization of the GPI-anchored lipid transfer proteins in the moss Physcomitrella patens.

    PubMed

    Edstam, Monika M; Laurila, Maiju; Höglund, Andrey; Raman, Amitha; Dahlström, Käthe M; Salminen, Tiina A; Edqvist, Johan; Blomqvist, Kristina

    2014-02-01

    The non-specific lipid transfer proteins (nsLTPs) are characterized by a compact structure with a central hydrophobic cavity very suitable for binding hydrophobic ligands, such as lipids. The nsLTPs are encoded by large gene families in all land plant lineages, but seem to be absent from green algae. The nsLTPs are classified to different types based on molecular weight, sequence similarity, intron position or spacing between the cysteine residues. The Type G nsLTPs (LTPGs) have a GPI-anchor in the C-terminal region which may attach the protein to the exterior side of the plasma membrane. Here, we present the first characterization of nsLTPs from an early diverged plant, the moss Physcomitrella patens. Moss LTPGs were heterologously produced and purified from Pichia pastoris. The purified moss LTPGs were found to be extremely heat stable and showed a binding preference for unsaturated fatty acids. Structural modeling implied that high alanine content could be important for the heat stability. Lipid profiling revealed that cutin monomers, such as C16 and C18 mono- and di-hydroxylated fatty acids, could be identified in P. patens. Expression of a moss LTPG-YFP fusion revealed localization to the plasma membrane. The expressions of many of the moss LTPGs were found to be upregulated during drought and cold treatments.

  12. Identification and functional analysis of bifunctional ent-kaurene synthase from the moss Physcomitrella patens.

    PubMed

    Hayashi, Ken-Ichiro; Kawaide, Hiroshi; Notomi, Miho; Sakigi, Yuka; Matsuo, Akihiko; Nozaki, Hiroshi

    2006-11-13

    ent-Kaurene is the key intermediate in biosynthesis of gibberellins (GAs), plant hormones. In higher plants, ent-kaurene is synthesized successively by copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) from geranylgeranyl diphosphate (GGDP). On the other hand, fungal ent-kaurene synthases are bifunctional cyclases with both CPS and KS activity in a single polypeptide. The moss Physcomitrella patens is a model organism for the study of genetics and development in an early land plant. We identified ent-kaurene synthase (PpCPS/KS) from P. patens and analyzed its function. PpCPS/KS cDNA encodes a 101-kDa polypeptide, and shows high similarity with CPSs and abietadiene synthase from higher plants. PpCPS/KS is a bifunctional cyclase and, like fungal CPS/KS, directly synthesizes the ent-kaurene skeleton from GGDP. PpCPS/KS has two aspartate-rich DVDD and DDYFD motifs observed in CPS and KS, respectively. The mutational analysis of two conserved motifs in PpCPS/KS indicated that the DVDD motif is responsible for CPS activity (GGDP to CDP) and the DDYFD motif for KS activity (CDP to ent-kaurene and ent-16alpha-hydroxykaurene).

  13. Bending of protonema cells in a plastid glycolate/glycerate transporter knockout line of Physcomitrella patens.

    PubMed

    Nakahara, Jin; Takechi, Katsuaki; Myouga, Fumiyoshi; Moriyama, Yasuko; Sato, Hiroshi; Takio, Susumu; Takano, Hiroyoshi

    2015-01-01

    Arabidopsis LrgB (synonym PLGG1) is a plastid glycolate/glycerate transporter associated with recycling of 2-phosphoglycolate generated via the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). We isolated two homologous genes (PpLrgB1 and B2) from the moss Physcomitrella patens. Phylogenetic tree analysis showed that PpLrgB1 was monophyletic with LrgB proteins of land plants, whereas PpLrgB2 was divergent from the green plant lineage. Experiments with PpLrgB-GFP fusion proteins suggested that both PpLrgB1 and B2 proteins were located in chloroplasts. We generated PpLrgB single (∆B1 and ∆B2) and double (∆B1/∆B2)-knockout lines using gene targeting of P. patens. The ∆B1 plants showed decreases in growth and photosynthetic activity, and their protonema cells were bent and accumulated glycolate. However, because ∆B2 and ∆B1/∆B2 plants showed no obvious phenotypic change relative to the wild-type or ∆B1 plants, respectively, the function of PpLrgB2 remains unclear. Arabidopsis LrgB could complement the ∆B1 phenotype, suggesting that the function of PpLrgB1 is the same as that of AtLrgB. When ∆B1 was grown under high-CO2 conditions, all novel phenotypes were suppressed. Moreover, protonema cells of wild-type plants exhibited a bending phenotype when cultured on media containing glycolate or glycerate, suggesting that accumulation of photorespiratory metabolites caused P. patens cells to bend.

  14. A multifunctional lipoxygenase with fatty acid hydroperoxide cleaving activity from the moss Physcomitrella patens.

    PubMed

    Senger, Toralf; Wichard, Thomas; Kunze, Susan; Göbel, Cornelia; Lerchl, Jens; Pohnert, Georg; Feussner, Ivo

    2005-03-04

    A complex mixture of fatty acid-derived aldehydes, ketones, and alcohols is released upon wounding of the moss Physcomitrella patens. To investigate the formation of these oxylipins at the molecular level we isolated a lipoxygenase from P. patens, which was identified in an EST library by sequence homology to lipoxygenases from plants. Sequence analysis of the cDNA showed that it exhibits a domain structure similar to that of type2 lipoxygenases from plants, harboring an N-terminal import signal for chloroplasts. The recombinant protein was identified as arachidonate 12-lipoxygenase and linoleate 13-lipoxygenase with a preference for arachidonic acid and eicosapentaenoic acid. In contrast to any other lipoxygenase cloned so far, this enzyme exhibited in addition an unusual high hydroperoxidase and also a fatty acid chain-cleaving lyase activity. Because of these unique features the pronounced formation of (2Z)-octen-1-ol, 1-octen-3-ol, the dienal (5Z,8Z,10E)-12-oxo-dodecatrienoic acid and 12-keto eicosatetraenoic acid was observed when arachidonic acid was administered as substrate. 12-Hydroperoxy eicosatetraenoic acid was found to be only a minor product. Moreover, the P. patens LOX has a relaxed substrate tolerance accepting C(18)-C(22) fatty acids giving rise to even more LOX-derived products. In contrast to other lipoxygenases a highly diverse product spectrum is formed by a single enzyme accounting for most of the observed oxylipins produced by the moss. This single enzyme might, in a fast and effective way, be involved in the formation of signal and/or defense molecules thus contributing to the broad resistance of mosses against pathogens.

  15. Two novel Physcomitrella patens fatty acid elongases (ELOs): identification and functional characterization.

    PubMed

    Eiamsa-Ard, Pradinunt; Kanjana-Opas, Akkharawit; Cahoon, Edgar B; Chodok, Pichit; Kaewsuwan, Sireewan

    2013-04-01

    The lower plant Physcomitrella patens synthesizes several long-chain polyunsaturated fatty acids (LC-PUFAs) by a series of desaturation and elongation reactions. In the present study, the full-length cDNAs for two novel fatty acid elongases designated PpELO1 and PpELO2 were isolated from P. patens using a PCR-based cloning strategy. These cDNAs encoding proteins of 335 and 280 amino acids with predicted molecular masses of 38.7 and 32.9 kDa, respectively, are predicted to contain seven transmembrane domains with a possible localization in the subcellular endoplasmic reticulum. Sequence comparisons and phylogenetic analysis revealed that they are closely related to other LC-PUFA elongases of the lower eukaryotes such as the Δ(5)- and Δ(6)-elongases of Marchantia polymorpha as well as the Δ(6)-elongase of P. patens. Heterologous expression of the PpELO1 in Saccharomyces cerevisiae led to the elongation of Δ(9)-, Δ(6)-C18, and Δ(5)-C20 LC-PUFAs, whereas only Δ(9)- and Δ(6)-C18 LC-PUFA substrates were used by PpELO2. Chimeric proteins were constructed to identify the amino acid regions most likely to be involved in the determination of the fatty acid substrate specificity. The expression of eight chimeric proteins in yeast revealed that substitution of the C-terminal 50 amino acids from PpELO1 into PpELO2 resulted in a high specificity for C20 fatty acid substrates. As a result, we suggest that the C-terminal region of PpELO1 is sufficient for C20 substrate elongation. Overall, these results provide important insights into the structural basis for substrate specificity of PUFA-generating ELO enzymes.

  16. Proteomic analysis of Physcomitrella patens treated with 12-oxo-phytodienoic acid, an important oxylipin in plants.

    PubMed

    Toshima, Erika; Nanjo, Yohei; Komatsu, Setsuko; Abe, Tatsuya; Matsuura, Hideyuki; Takahashi, Kosaku

    2014-01-01

    12-Oxo-phytodienoic acid (OPDA) is biosynthesized in the octadecanoid pathway and is considered to be a signaling molecule in plants. In Physcomitrella patens, OPDA is induced by bacterial infection and mechanical stress and is known to suppress growth; however, the functional mechanism of OPDA signaling remains elusive. In this study, we performed a proteomic analysis of P. patens treated with OPDA and found that the expression of 82 proteins was significantly altered, with approximately 80% of these proteins being downregulated by OPDA. The identified proteins were mainly categorized as being involved in photosynthesis, metabolism, and protein synthesis, and most of the proteins that were upregulated by OPDA are involved in light-dependent reactions, suggesting that OPDA regulates a function in chloroplasts. Additionally, OPDA induced the expression of an allene oxide cyclase (PpAOC1) in the octadecanoid pathway, demonstrating positive feedback regulation by OPDA in P. patens.

  17. An experimental method to facilitate the identification of hybrid sporophytes in the moss Physcomitrella patens using fluorescent tagged lines

    PubMed Central

    Perroud, Pierre-François; Cove, David J.; Quatrano, Ralph S.; McDaniel, Stuart F.

    2011-01-01

    Summary The sequencing of the Physcomitrella patens genome, combined with the high frequency of gene targeting in this species, makes it ideal for reverse genetic studies. For forward genetic studies, experimental crosses and genetic analysis of progeny are essential.Since P. patens is monoicous, producing both male and female gametes on the same gametophore, and undergoing self-fertilization at a high frequency, the identification of crossed sporophytes is difficult. Usually spores from many sporophytes from a mixed culture must be testsed for the production of recombinant progeny.Here, we describe the use of transgenic lines that express a fluorescent transgene constitutively, to provide a direct visual screen for hybrid sporophytes.We show that segregations in crosses obtained with this technique are as expected, and demonstrate its utility for the study of the rate of outcrossing between three isolates of P. patens. PMID:21366596

  18. Hormonal diterpenoids derived from ent-kaurenoic acid are involved in the blue-light avoidance response of Physcomitrella patens.

    PubMed

    Miyazaki, Sho; Nakajima, Masatoshi; Kawaide, Hiroshi

    2015-01-01

    Gibberellins (GAs) are diterpenoid hormones that regulate growth and development in flowering plants. The moss Physcomitrella patens has part of the GA biosynthetic pathway from geranylgeranyl diphosphate to ent-kaurenoic acid via ent-kaurene, but it does not produce GA. Disruption of the ent-kaurene synthase gene in P. patens suppressed caulonemal differentiation. Application of ent-kaurene or ent-kaurenoic acid restored differentiation, suggesting that derivative(s) of ent-kaurenoic acid, but not GAs, are endogenous regulator(s) of caulonemal cell differentiation. The protonemal growth of P. patens shows an avoidance response under unilateral blue light. Physiological studies using gene mutants involved in ent-kaurene biosynthesis confirmed that diterpenoid(s) regulate the blue-light response. Here, we discuss the implications of these findings, and provide data for the ent-kaurene oxidase gene-disrupted mutant.

  19. Eight types of stem cells in the life cycle of the moss Physcomitrella patens.

    PubMed

    Kofuji, Rumiko; Hasebe, Mitsuyasu

    2014-02-01

    Stem cells self-renew and produce cells that differentiate to become the source of the plant body. The moss Physcomitrella patens forms eight types of stem cells during its life cycle and serves as a useful model in which to explore the evolution of such cells. The common ancestor of land plants is inferred to have been haplontic and to have formed stem cells only in the gametophyte generation. A single stem cell would have been maintained in the ancestral gametophyte meristem, as occurs in extant basal land plants. During land plant evolution, stem cells diverged in the gametophyte generation to form different types of body parts, including the protonema and rhizoid filaments, leafy-shoot and thalloid gametophores, and gametangia formed in moss. A simplex meristem with a single stem cell was acquired in the sporophyte generation early in land plant evolution. Subsequently, sporophyte stem cells became multiple in the meristem and were elaborated further in seed plant lineages, although the evolutionary origin of niche cells, which maintain stem cells is unknown. Comparisons of gene regulatory networks are expected to give insights into the general mechanisms of stem cell formation and maintenance in land plants and provide information about their evolution. P. patens develops at least seven types of simplex meristem in the gametophyte and at least one type in the sporophyte generation and is a good material for regulatory network comparisons. In this review, we summarize recently revealed molecular mechanisms of stem cell initiation and maintenance in the moss. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. The chromatin landscape of the moss Physcomitrella patens and its dynamics during development and drought stress.

    PubMed

    Widiez, Thomas; Symeonidi, Aikaterini; Luo, Chongyuan; Lam, Eric; Lawton, Michael; Rensing, Stefan A

    2014-07-01

    The moss Physcomitrella patens is an important model organism for evo-devo studies. Here, we determined the genome-wide chromatin landscape of five important histone three (H3) modifications (H3K4me3, H3K27me3, H3K27Ac, H3K9Ac and H3K9me2) and describe the changes to these histone marks in two contrasted situations, developmental transition and abiotic (drought) stress. Integrative analysis of these histone H3 modifications revealed their preferential association into 15 chromatin states (CS) in genic regions of the P. patens genome. Synergistic relationships that influence expression levels were revealed for the three activating marks H3K4me3, H3K27Ac and H3K9Ac, while an antagonistic relationship was found between CS containing the H3K27me3 and H3K27Ac marks, suggesting that H3K27 is a key indexing residue regarding transcriptional output. Concerning the alteration of histone marks in response to developmental transition (juvenile to adult) and drought stress, the three activating marks H3K4me3, H3K27Ac and H3K9Ac show significant changes in both situations. However, changes to H3K27me3 are central only for genes differentially expressed during development. Interestingly, genes induced during drought stress show significant histone mark toggling during developmental transition. This situation suggests that drought induced adult (gametophore expressed) genes are primed to respond to this stress during the juvenile to adult transition.

  1. A Lin28 homologue reprograms differentiated cells to stem cells in the moss Physcomitrella patens

    PubMed Central

    Li, Chen; Sako, Yusuke; Imai, Akihiro; Nishiyama, Tomoaki; Thompson, Kari; Kubo, Minoru; Hiwatashi, Yuji; Kabeya, Yukiko; Karlson, Dale; Wu, Shu-Hsing; Ishikawa, Masaki; Murata, Takashi; Benfey, Philip N.; Sato, Yoshikatsu; Tamada, Yosuke; Hasebe, Mitsuyasu

    2017-01-01

    Both land plants and metazoa have the capacity to reprogram differentiated cells to stem cells. Here we show that the moss Physcomitrella patens Cold-Shock Domain Protein 1 (PpCSP1) regulates reprogramming of differentiated leaf cells to chloronema apical stem cells and shares conserved domains with the induced pluripotent stem cell factor Lin28 in mammals. PpCSP1 accumulates in the reprogramming cells and is maintained throughout the reprogramming process and in the resultant stem cells. Expression of PpCSP1 is negatively regulated by its 3′-untranslated region (3′-UTR). Removal of the 3′-UTR stabilizes PpCSP1 transcripts, results in accumulation of PpCSP1 protein and enhances reprogramming. A quadruple deletion mutant of PpCSP1 and three closely related PpCSP genes exhibits attenuated reprogramming indicating that the PpCSP genes function redundantly in cellular reprogramming. Taken together, these data demonstrate a positive role of PpCSP1 in reprogramming, which is similar to the function of mammalian Lin28. PMID:28128346

  2. Cytoplasmic Nucleation and Atypical Branching Nucleation Generate Endoplasmic Microtubules in Physcomitrella patens[OPEN

    PubMed Central

    Nakaoka, Yuki; Kimura, Akatsuki; Tani, Tomomi; Goshima, Gohta

    2015-01-01

    The mechanism underlying microtubule (MT) generation in plants has been primarily studied using the cortical MT array, in which fixed-angled branching nucleation and katanin-dependent MT severing predominate. However, little is known about MT generation in the endoplasm. Here, we explored the mechanism of endoplasmic MT generation in protonemal cells of Physcomitrella patens. We developed an assay that utilizes flow cell and oblique illumination fluorescence microscopy, which allowed visualization and quantification of individual MT dynamics. MT severing was infrequently observed, and disruption of katanin did not severely affect MT generation. Branching nucleation was observed, but it showed markedly variable branch angles and was occasionally accompanied by the transport of nucleated MTs. Cytoplasmic nucleation at seemingly random locations was most frequently observed and predominated when depolymerized MTs were regrown. The MT nucleator γ-tubulin was detected at the majority of the nucleation sites, at which a single MT was generated in random directions. When γ-tubulin was knocked down, MT generation was significantly delayed in the regrowth assay. However, nucleation occurred at a normal frequency in steady state, suggesting the presence of a γ-tubulin-independent backup mechanism. Thus, endoplasmic MTs in this cell type are generated in a less ordered manner, showing a broader spectrum of nucleation mechanisms in plants. PMID:25616870

  3. Spectroscopic analysis of moss (Ceratodon purpureus and Physcomitrella patens) recombinant non-symbiotic hemoglobins

    PubMed Central

    Vázquez-Limón, Consuelo; Castro-Bustos, Saraí; Arredondo-Peter, Raúl

    2012-01-01

    Non-symbiotic hemoglobins (nsHbs) are O2-binding proteins widely distributed in land plants, including primitive bryophytes. Little is known about the properties of bryophyte nsHbs. Here, we report the spectroscopic characterization of two moss recombinant nsHbs, CerpurnsHb of Ceratodon purpureus and PhypatnsHb of Physcomitrella patens. Spectra showed that the absorption maxima of the ferrous and ferric forms of recombinant CerpurnsHb are located at 418, 531 and 557 nm and 407, 537, 569 (shoulder) and 632 (shoulder) nm, respectively, and of PhypatnsHb are located at 422, 529 and 557 nm and 407, 531, 571 (shoulder) and 647 (shoulder) nm, respectively. These absorption maxima are similar to those of rice Hb1. Also, the absorption maxima of the oxygenated ferrous form of recombinant CerpurnsHb and PhypatnsHb are located at 412, 541 and 575 nm and 414, 541 and 574 nm, respectively, similar to those of oxygenated rice Hb1 and cowpea leghemoglobin II. This evidence indicates that CerpurnsHb and PhypatnsHb are mostly hexacoordinate and that they bind O2. PMID:23336017

  4. Strigolactones inhibit caulonema elongation and cell division in the moss Physcomitrella patens.

    PubMed

    Hoffmann, Beate; Proust, Hélène; Belcram, Katia; Labrune, Cécile; Boyer, François-Didier; Rameau, Catherine; Bonhomme, Sandrine

    2014-01-01

    In vascular plants, strigolactones (SLs) are known for their hormonal role and for their role as signal molecules in the rhizosphere. SLs are also produced by the moss Physcomitrella patens, in which they act as signaling factors for controlling filament extension and possibly interaction with neighboring individuals. To gain a better understanding of SL action at the cellular level, we investigated the effect of exogenously added molecules (SLs or analogs) in moss growth media. We used the previously characterized Ppccd8 mutant that is deficient in SL synthesis and showed that SLs affect moss protonema extension by reducing caulonema cell elongation and mainly cell division rate, both in light and dark conditions. Based on this effect, we set up bioassays to examine chemical structure requirements for SL activity in moss. The results suggest that compounds GR24, GR5, and 5-deoxystrigol are active in moss (as in pea), while other analogs that are highly active in the control of pea branching show little activity in moss. Interestingly, the karrikinolide KAR1, which shares molecular features with SLs, did not have any effect on filament growth, even though the moss genome contains several genes homologous to KAI2 (encoding the KAR1 receptor) and no canonical homologue to D14 (encoding the SL receptor). Further studies should investigate whether SL signaling pathways have been conserved during land plant evolution.

  5. AP2-type transcription factors determine stem cell identity in the moss Physcomitrella patens.

    PubMed

    Aoyama, Tsuyoshi; Hiwatashi, Yuji; Shigyo, Mikao; Kofuji, Rumiko; Kubo, Minoru; Ito, Motomi; Hasebe, Mitsuyasu

    2012-09-01

    Stem cells are formed at particular times and positions during the development of multicellular organisms. Whereas flowering plants form stem cells only in the sporophyte generation, non-seed plants form stem cells in both the sporophyte and gametophyte generations. Although the molecular mechanisms underlying stem cell formation in the sporophyte generation have been extensively studied, only a few transcription factors involved in the regulation of gametophyte stem cell formation have been reported. The moss Physcomitrella patens forms a hypha-like body (protonema) and a shoot-like body (gametophore) from a protonema apical cell and a gametophore apical cell, respectively. These apical cells have stem cell characteristics and are formed as side branches of differentiated protonema cells. Here, we show that four AP2-type transcription factors orthologous to Arabidopsis thaliana AINTEGUMENTA, PLETHORA and BABY BOOM (APB) are indispensable for the formation of gametophore apical cells from protonema cells. Quadruple disruption of all APB genes blocked gametophore formation, even in the presence of cytokinin, which enhances gametophore apical cell formation in the wild type. All APB genes were expressed in emerging gametophore apical cells, but not in protonema apical cells. Heat-shock induction of an APB4 transgene driven by a heat-shock promoter increased the number of gametophores. Expression of all APB genes was induced by auxin but not by cytokinin. Thus, the APB genes function synergistically with cytokinin signaling to determine the identity of the two types of stem cells.

  6. Cell-specific transcriptomic analyses of three-dimensional shoot development in the moss Physcomitrella patens.

    PubMed

    Frank, Margaret H; Scanlon, Michael J

    2015-08-01

    Haploid moss gametophytes harbor distinct stem cell types, including tip cells that divide in single planes to generate filamentous protonemata, and bud cells that divide in three planes to yield axial gametophore shoots. This transition from filamentous to triplanar growth occurs progressively during the moss life cycle, and is thought to mirror evolution of the first terrestrial plants from Charophycean green algal ancestors. The innovation of morphologically complex plant body plans facilitated colonization of the vertical landscape, and enabled development of complex vegetative and reproductive plant morphologies. Despite its profound evolutionary significance, the molecular programs involved in this transition from filamentous to triplanar meristematic plant growth are poorly understood. In this study, we used single-cell type transcriptomics to identify more than 4000 differentially expressed genes that distinguish uniplanar protonematal tip cells from multiplanar gametophore bud cells in the moss Physcomitrella patens. While the transcriptomes of both tip and bud cells show molecular signatures of proliferative cells, the bud cell transcriptome exhibits a wider variety of genes with significantly increased transcript abundances. Our data suggest that combined expression of genes involved in shoot patterning and asymmetric cell division accompanies the transition from uniplanar to triplanar meristematic growth in moss.

  7. Profilin is essential for tip growth in the moss Physcomitrella patens.

    PubMed

    Vidali, Luis; Augustine, Robert C; Kleinman, Ken P; Bezanilla, Magdalena

    2007-11-01

    The actin cytoskeleton is critical for tip growth in plants. Profilin is the main monomer actin binding protein in plant cells. The moss Physcomitrella patens has three profilin genes, which are monophyletic, suggesting a single ancestor for plant profilins. Here, we used RNA interference (RNAi) to determine the loss-of-function phenotype of profilin. Reduction of profilin leads to a complete loss of tip growth and a partial inhibition of cell division, resulting in plants with small rounded cells and fewer cells. We silenced all profilins by targeting their 3' untranslated region sequences, enabling complementation analyses by expression of profilin coding sequences. We show that any moss or a lily (Lilium longiflorum) profilin support tip growth. Profilin with a mutation in its actin binding site is unable to rescue profilin RNAi, while a mutation in the poly-l-proline binding site weakly rescues. We show that moss tip growing cells contain a prominent subapical cortical F-actin structure composed of parallel actin cables. Cells lacking profilin lose this structure; instead, their F-actin is disorganized and forms polarized cortical patches. Plants expressing the actin and poly-l-proline binding mutants exhibited similar F-actin disorganization. These results demonstrate that profilin and its binding to actin are essential for tip growth. Additionally, profilin is not needed for formation of F-actin, but profilin and its interactions with actin and poly-l-proline ligands are required to properly organize F-actin.

  8. An ancestral stomatal patterning module revealed in the non-vascular land plant Physcomitrella patens.

    PubMed

    Caine, Robert S; Chater, Caspar C; Kamisugi, Yasuko; Cuming, Andrew C; Beerling, David J; Gray, Julie E; Fleming, Andrew J

    2016-09-15

    The patterning of stomata plays a vital role in plant development and has emerged as a paradigm for the role of peptide signals in the spatial control of cellular differentiation. Research in Arabidopsis has identified a series of epidermal patterning factors (EPFs), which interact with an array of membrane-localised receptors and associated proteins (encoded by ERECTA and TMM genes) to control stomatal density and distribution. However, although it is well-established that stomata arose very early in the evolution of land plants, until now it has been unclear whether the established angiosperm stomatal patterning system represented by the EPF/TMM/ERECTA module reflects a conserved, universal mechanism in the plant kingdom. Here, we use molecular genetics to show that the moss Physcomitrella patens has conserved homologues of angiosperm EPF, TMM and at least one ERECTA gene that function together to permit the correct patterning of stomata and that, moreover, elements of the module retain function when transferred to Arabidopsis Our data characterise the stomatal patterning system in an evolutionarily distinct branch of plants and support the hypothesis that the EPF/TMM/ERECTA module represents an ancient patterning system. © 2016. Published by The Company of Biologists Ltd.

  9. An ancestral stomatal patterning module revealed in the non-vascular land plant Physcomitrella patens

    PubMed Central

    Chater, Caspar C.; Kamisugi, Yasuko

    2016-01-01

    The patterning of stomata plays a vital role in plant development and has emerged as a paradigm for the role of peptide signals in the spatial control of cellular differentiation. Research in Arabidopsis has identified a series of epidermal patterning factors (EPFs), which interact with an array of membrane-localised receptors and associated proteins (encoded by ERECTA and TMM genes) to control stomatal density and distribution. However, although it is well-established that stomata arose very early in the evolution of land plants, until now it has been unclear whether the established angiosperm stomatal patterning system represented by the EPF/TMM/ERECTA module reflects a conserved, universal mechanism in the plant kingdom. Here, we use molecular genetics to show that the moss Physcomitrella patens has conserved homologues of angiosperm EPF, TMM and at least one ERECTA gene that function together to permit the correct patterning of stomata and that, moreover, elements of the module retain function when transferred to Arabidopsis. Our data characterise the stomatal patterning system in an evolutionarily distinct branch of plants and support the hypothesis that the EPF/TMM/ERECTA module represents an ancient patterning system. PMID:27407102

  10. Physcomitrella patens DNA methyltransferase 2 is required for recovery from salt and osmotic stress.

    PubMed

    Arya, Deepshikha; Kapoor, Sanjay; Kapoor, Meenu

    2016-02-01

    DNA methyltransferase 2 (DNMT2) unlike other members of the cytosine DNA methyltransferase gene family has dual substrate specificity and it methylates cytosines in both the DNA and transfer RNA (tRNA). Its role in plants, however, has remained obscure to date. In this study, we demonstrate that DNMT2 from Physcomitrella patens accumulates in a temporal manner under salt and osmotic stress showing maximum accumulation during recovery, i.e. 24 h after plants are transferred to normal growth medium. Therefore, to study its role in stress tolerance, we generated PpDNMT2 targeted knockout plants (ppdnmt2ko). Mutant plants show increased sensitivity to salt and osmotic stress and are unable to recover even after 21 days of growth on optimal growth media. ppdnmt2ko, however, accumulate normal levels of dehydrin-like and small heat shock protein encoding transcripts under stress but show dramatic reduction in levels of tRNA(A) (sp-) (GUC) . The levels of tRNA(A) (sp-) (GUC) , in contrast, increase ~ 25-30-fold in ppdnmt2ko under non-stress conditions and > 1200-fold in wild-type plants under stress. The role of PpDNMT2 in modulating biogenesis/stability of tRNA(A) (sp-) (GUC) under salt and osmotic stress is discussed in the light of these observations.

  11. Chloroplast actin filaments organize meshwork on the photorelocated chloroplasts in the moss Physcomitrella patens.

    PubMed

    Yamashita, Hiroko; Sato, Yoshikatsu; Kanegae, Takeshi; Kagawa, Takatoshi; Wada, Masamitsu; Kadota, Akeo

    2011-02-01

    Cytoskeleton dynamics during phototropin-dependent chloroplast photorelocation movement was analyzed in protonemal cells of actin- and microtubule-visualized lines of Physcomitrella patens expressing GFP- or tdTomato-talin and GFP-tubulin. Using newly developed epi- and trans-microbeam irradiation systems that permit fluorescence observation of the cell under blue microbeam irradiation inducing chloroplast relocation, it was revealed that meshwork of actin filaments formed at the chloroplast-accumulating area both in the avoidance and accumulation movements. The structure disappeared soon when blue microbeam was turned off, and it was not induced under red microbeam irradiation that did not evoke chloroplast relocation movement. In contrast, no apparent change in microtubule organization was detected during the movements. The actin meshwork was composed of short actin filaments distinct from the cytoplasmic long actin cables and was present between the chloroplasts and plasma membrane. The short actin filaments emerged from around the chloroplast periphery towards the center of chloroplast. Showing highly dynamic behavior, the chloroplast actin filaments (cp-actin filaments) were rapidly organized into meshwork on the chloroplast surface facing plasma membrane. The actin filament configuration on a chloroplast led to the formation of actin meshwork area in the cell as the chloroplasts arrived at and occupied the area. After establishment of the meshwork, cp-actin filaments were still highly dynamic, showing appearance, disappearance, severing and bundling of filaments. These results indicate that the cp-actin filaments have significant roles in the chloroplast movement and positioning in the cell.

  12. Cell cycle reentry from the late S phase: implications from stem cell formation in the moss Physcomitrella patens.

    PubMed

    Ishikawa, Masaki; Hasebe, Mitsuyasu

    2015-05-01

    Differentiated cells are in a non-dividing, quiescent state, but some differentiated cells can reenter the cell cycle in response to appropriate stimuli. Quiescent cells are generally arrested at the G0/G1 phase, reenter the cell cycle, and progress to the S phase to replicate their genomic DNA. On the other hand, some types of cells are arrested at the different phase and reenter the cell cycle from there. In the moss Physcomitrella patens, the differentiated leaf cells of gametophores formed in the haploid generation contain approximately 2C DNA content, and DNA synthesis is necessary for reentry into the cell cycle, which is suggested to be arrested at late S phase. Here we review various cell-division reactivation processes in which cells reenter the cell cycle from the late S phase, and discuss possible mechanisms of such unusual cell cycle reentries with special emphasis on Physcomitrella.

  13. The X-ray crystal structure of APR-B, an atypical adenosine 5'-phosphosulfate reductase from Physcomitrella patens.

    PubMed

    Stevenson, Clare E M; Hughes, Richard K; McManus, Michael T; Lawson, David M; Kopriva, Stanislav

    2013-11-15

    Sulfonucleotide reductases catalyse the first reductive step of sulfate assimilation. Their substrate specificities generally correlate with the requirement for a [Fe4S4] cluster, where adenosine 5'-phosphosulfate (APS) reductases possess a cluster and 3'-phosphoadenosine 5'-phosphosulfate reductases do not. The exception is the APR-B isoform of APS reductase from the moss Physcomitrella patens, which lacks a cluster. The crystal structure of APR-B, the first for a plant sulfonucleotide reductase, is consistent with a preference for APS. Structural conservation with bacterial APS reductase rules out a structural role for the cluster, but supports the contention that it enhances the activity of conventional APS reductases.

  14. CHUP1 mediates actin-based light-induced chloroplast avoidance movement in the moss Physcomitrella patens.

    PubMed

    Usami, Hiroka; Maeda, Takuma; Fujii, Yusuke; Oikawa, Kazusato; Takahashi, Fumio; Kagawa, Takatoshi; Wada, Masamitsu; Kasahara, Masahiro

    2012-12-01

    Chloroplasts change their intracellular distribution in response to light intensity. CHUP1 (CHLOROPLAST UNUSUAL POSITIONING1) is indispensable for this response in Arabidopsis thaliana. However, involvement of CHUP1 in light-induced chloroplast movement is unknown in other plants. In this study, CHUP1 orthologues were isolated from a moss, Physcomitrella patens, and a fern, Adiantum capillus-veneris, by cDNA library screening and PCR cloning based on the P. patens genome sequence. Functional motifs found in CHUP1 of A. thaliana were conserved among the CHUP1 orthologues. In addition to the putative functional regions, the C-terminal regions (approximately 250 amino acids), which are unique in CHUP1s, were highly conserved. Green fluorescent protein (GFP) fusions of P. patens CHUP1s (PpCHUP1A, PpCHUP1B and PpCHUP1C) were transiently expressed in protoplast cells. All GFP fusions were localized on the chloroplasts. Light-induced chloroplast avoidance movement of chup1 disruptants of P. patens was examined in the presence of cytoskeletal inhibitors because of the utilization of both microtubules and actin filaments for the movement in P. patens. When actin filaments were disrupted by cytochalasin B, the wild type (WT) and all chup1 disruptants showed chloroplast avoidance movement. However, when microtubules were disrupted by Oryzalin, chloroplasts in ∆chup1A and ∆chup1A/B rarely moved and stayed in the strong light-irradiated area. On the other hand, WT, ∆chup1B and ∆chup1C showed chloroplast avoidance movement. These results suggest that PpCHUP1A predominantly mediates the actin-based light-induced chloroplast avoidance movement. This study reveals that CHUP1 functions on the chloroplasts and is involved in the actin-based light-induced chloroplast avoidance movement in P. patens.

  15. Physcomitrella patens auxin conjugate synthetase (GH3) double knockout mutants are more resistant to Pythium infection than wild type.

    PubMed

    Mittag, Jennifer; Šola, Ivana; Rusak, Gordana; Ludwig-Müller, Jutta

    2015-07-01

    Auxin homeostasis is involved in many different plant developmental and stress responses. The auxin amino acid conjugate synthetases belonging to the GH3 family play major roles in the regulation of free indole-3-acetic acid (IAA) levels and the moss Physcomitrella patens has two GH3 genes in its genome. A role for IAA in several angiosperm--pathogen interactions was reported, however, in a moss--oomycete pathosystem it had not been published so far. Using GH3 double knockout lines we have investigated the role of auxin homeostasis during the infection of P. patens with the two oomycete species, Pythium debaryanum and Pythium irregulare. We show that infection with P. debaryanum caused stronger disease symptoms than with P. irregulare. Also, P. patens lines harboring fusion constructs of an auxin-inducible promoter from soybean (GmGH3) with a reporter (ß-glucuronidase) showed higher promoter induction after P. debaryanum infection than after P. irregulare, indicating a differential induction of the auxin response. Free IAA was induced upon P. debaryanum infection in wild type by 1.6-fold and in two GH3 double knockout (GH3-doKO) mutants by 4- to 5-fold. All GH3-doKO lines showed a reduced disease symptom progression compared to wild type. Since P. debaryanum can be inhibited in growth on medium containing IAA, these data might indicate that endogenous high auxin levels in P. patens GH3-doKO mutants lead to higher resistance against the oomycete.

  16. Role of DNA methylation in growth and differentiation in Physcomitrella patens and characterization of cytosine DNA methyltransferases.

    PubMed

    Malik, Garima; Dangwal, Meenakshi; Kapoor, Sanjay; Kapoor, Meenu

    2012-11-01

    Epigenetic mechanisms such as DNA methylation are known to regulate important developmental processes in higher eukaryotes. However, little is known about the necessity and role of this process in early land plants. Using the methyltransferase (MTase) inhibitor zebularine (1-(β-d-ribofuranosyl)-1,2-dihydropyrimidine-2-one), the impact of loss of genome-wide methylation on the overall development in Physcomitrella patens was analyzed. It is observed that various aspects of growth and differentiation during gametophyte development become aberrant. A search for the core molecular components of methylation machinery, cytosine DNA MTases, revealed the presence of seven loci in the P. patens genome. Five of the loci code for MTases that are similar to corresponding proteins in higher plants, while two MTases appear specific to P. patens and are closely related to human DNMT3a and DNMT3b, respectively. These proteins possess all the conserved catalytic motifs characteristic of MTases and a domain of unknown function, DUF3444. Association of these highly conserved motifs with a DUF has not been reported in any of the MTases known so far. All the seven genes are differentially but ubiquitously expressed in gametophytes at low levels. Subcellular localization of GFP-fused proteins shows patterns of distribution that can be correlated with their putative cellular functions. This work bridges the knowledge of MTases in P. patens and makes this simple model plant accessible for studies on epigenetic aspects that remain intractable in higher plants.

  17. Apical myosin XI anticipates F-actin during polarized growth of Physcomitrella patens cells.

    PubMed

    Furt, Fabienne; Liu, Yen-Chun; Bibeau, Jeffrey P; Tüzel, Erkan; Vidali, Luis

    2013-02-01

    Tip growth is essential for land colonization by bryophytes, plant sexual reproduction and water and nutrient uptake. Because this specialized form of polarized cell growth requires both a dynamic actin cytoskeleton and active secretion, it has been proposed that the F-actin-associated motor myosin XI is essential for this process. Nevertheless, a spatial and temporal relationship between myosin XI and F-actin during tip growth is not known in any plant cell. Here, we use the highly polarized cells of the moss Physcomitrella patens to show that myosin XI and F-actin localize, in vivo, at the same apical domain and that both signals fluctuate. Surprisingly, phase analysis shows that increase in myosin XI anticipates that of F-actin; in contrast, myosin XI levels at the tip fluctuate in identical phase with a vesicle marker. Pharmacological analysis using a low concentration of the actin polymerization inhibitor latrunculin B showed that the F-actin at the tip can be significantly diminished while myosin XI remains elevated in this region, suggesting that a mechanism exists to cluster myosin XI-associated structures at the cell's apex. In addition, this approach uncovered a mechanism for actin polymerization-dependent motility in the moss cytoplasm, where myosin XI-associated structures seem to anticipate and organize the actin polymerization machinery. From our results, we inferred a model where the interaction between myosin XI-associated vesicular structures and F-actin polymerization-driven motility function at the cell's apex to maintain polarized cell growth. We hypothesize this is a general mechanism for the participation of myosin XI and F-actin in tip growing cells. © 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.

  18. Functional analyses of the Physcomitrella patens phytochromes in regulating chloroplast avoidance movement.

    PubMed

    Uenaka, Hidetoshi; Kadota, Akeo

    2007-09-01

    Red light-induced chloroplast movement in Physcomitrella patens (Pp) is mediated by dichroic phytochrome in the cytoplasm. To analyze the molecular function of the photoreceptor in the cytoplasm, we developed a protoplast system in which chloroplast photomovement was exclusively dependent on the expression of phytochrome cDNA constructs introduced by polyethylene glycol (PEG) transformation. YFP was fused to the phytochrome constructs and their expression was detected by fluorescence. The chloroplast avoidance response was induced in the protoplasts expressing a YFP fusion of PHY1-PHY3, but not of PHY4 or YFP alone. Phy::yfp fluorescence was detected in the cytoplasm. No change in the location of phy1::yfp or phy2::yfp was revealed before and after photomovement. When phy1::yfp and phy2::yfp were targeted to the nucleus by fusing a nuclear localization signal to the constructs, red light avoidance was not induced. To determine the domains of PHY2 essential for avoidance response, various partially-deleted PHY2::YFP constructs were tested. The N-terminal extension domain (NTE) was found to be necessary but the C-terminal histidine kinase-related domain (HKRD) was dispensable. An avoidance response was not induced under expression of phytochrome N-terminal half domain [deleting both the PAS (Per, Arnt, Sim)-related domain (PRD) and HKRD]. GUS fusion of this N-terminal half domain, reported to be fully functional in Arabidopsis for several phyA- and phyB-regulated responses was not effective in chloroplast avoidance movement. Domain requirement and GUS fusion effect were also confirmed in PHY1. These results indicate that Pp phy1-Pp phy3 in the cytoplasm mediate chloroplast avoidance movement, and that NTE and PRD, but not HKRD, are required for their function.

  19. The Transcriptional Response to DNA-Double-Strand Breaks in Physcomitrella patens

    PubMed Central

    Kamisugi, Yasuko; Whitaker, John W.

    2016-01-01

    The model bryophyte Physcomitrella patens is unique among plants in supporting the generation of mutant alleles by facile homologous recombination-mediated gene targeting (GT). Reasoning that targeted transgene integration occurs through the capture of transforming DNA by the homology-dependent pathway for DNA double-strand break (DNA-DSB) repair, we analysed the genome-wide transcriptomic response to bleomycin-induced DNA damage and generated mutants in candidate DNA repair genes. Massively parallel (Illumina) cDNA sequencing identified potential participants in gene targeting. Transcripts encoding DNA repair proteins active in multiple repair pathways were significantly up-regulated. These included Rad51, CtIP, DNA ligase 1, Replication protein A and ATR in homology-dependent repair, Xrcc4, DNA ligase 4, Ku70 and Ku80 in non-homologous end-joining and Rad1, Tebichi/polymerase theta, PARP in microhomology-mediated end-joining. Differentially regulated cell-cycle components included up-regulated Rad9 and Hus1 DNA-damage-related checkpoint proteins and down-regulated D-type cyclins and B-type CDKs, commensurate with the imposition of a checkpoint at G2 of the cell cycle characteristic of homology-dependent DNA-DSB repair. Candidate genes, including ATP-dependent chromatin remodelling helicases associated with repair and recombination, were knocked out and analysed for growth defects, hypersensitivity to DNA damage and reduced GT efficiency. Targeted knockout of PpCtIP, a cell-cycle activated mediator of homology-dependent DSB resection, resulted in bleomycin-hypersensitivity and greatly reduced GT efficiency. PMID:27537368

  20. Physcomitrella patens Has Kinase-LRR R Gene Homologs and Interacting Proteins

    PubMed Central

    Tanigaki, Yusuke; Ito, Kenji; Obuchi, Yoshiyuki; Kosaka, Akiko; Yamato, Katsuyuki T.; Okanami, Masahiro; Lehtonen, Mikko T.; Valkonen, Jari P. T.; Akita, Motomu

    2014-01-01

    Plant disease resistance gene (R gene)-like sequences were screened from the Physcomitrella patens genome. We found 603 kinase-like, 475 Nucleotide Binding Site (NBS)-like and 8594 Leucine Rich Repeat (LRR)-like sequences by homology searching using the respective domains of PpC24 (Accession No. BAD38895), which is a candidate kinase-NBS-LRR (kinase-NL) type R-like gene, as a reference. The positions of these domains in the genome were compared and 17 kinase-NLs were predicted. We also found four TIR-NBS-LRR (TIR-NL) sequences with homology to Arabidopsis TIR-NL (NM_001125847), but three out of the four TIR-NLs had tetratricopeptide repeats or a zinc finger domain in their predicted C-terminus. We also searched for kinase-LRR (KLR) type sequences by homology with rice OsXa21 and Arabidopsis thaliana FLS2. As a result, 16 KLRs with similarity to OsXa21 were found. In phylogenetic analysis of these 16 KLRs, PpKLR36, PpKLR39, PpKLR40, and PpKLR43 formed a cluster with OsXa21. These four PpKLRs had deduced transmembrane domain sequences and expression of all four was confirmed. We also found 14 homologs of rice OsXB3, which is known to interact with OsXa21 and is involved in signal transduction. Protein–protein interaction was observed between the four PpKLRs and at least two of the XB3 homologs in Y2H analysis. PMID:24748046

  1. Endogenous diterpenes derived from ent-kaurene, a common gibberellin precursor, regulate protonema differentiation of the moss Physcomitrella patens.

    PubMed

    Hayashi, Ken-ichiro; Horie, Keisuke; Hiwatashi, Yuji; Kawaide, Hiroshi; Yamaguchi, Shinjiro; Hanada, Atsushi; Nakashima, Tamotsu; Nakajima, Masatoshi; Mander, Lewis N; Yamane, Hisakazu; Hasebe, Mitsuyasu; Nozaki, Hiroshi

    2010-07-01

    Gibberellins (GAs) are a group of diterpene-type plant hormones biosynthesized from ent-kaurene via ent-kaurenoic acid. GAs are ubiquitously present in seed plants. The GA signal is perceived and transduced by the GID1 GA receptor/DELLA repressor pathway. The lycopod Selaginella moellendorffii biosynthesizes GA and has functional GID1-DELLA signaling components. In contrast, no GAs or functionally orthologous GID1-DELLA components have been found in the moss Physcomitrella patens. However, P. patens produces ent-kaurene, a common precursor for GAs, and possesses a functional ent-kaurene synthase, PpCPS/KS. To assess the biological role of ent-kaurene in P. patens, we generated a PpCPS/KS disruption mutant that does not accumulate ent-kaurene. Phenotypic analysis demonstrates that the mutant has a defect in the protonemal differentiation of the chloronemata to caulonemata. Gas chromatography-mass spectrometry analysis shows that P. patens produces ent-kaurenoic acid, an ent-kaurene metabolite in the GA biosynthesis pathway. The phenotypic defect of the disruptant was recovered by the application of ent-kaurene or ent-kaurenoic acid, suggesting that ent-kaurenoic acid, or a downstream metabolite, is involved in protonemal differentiation. Treatment with uniconazole, an inhibitor of ent-kaurene oxidase in GA biosynthesis, mimics the protonemal phenotypes of the PpCPS/KS mutant, which were also restored by ent-kaurenoic acid treatment. Interestingly, the GA(9) methyl ester, a fern antheridiogen, rescued the protonemal defect of the disruption mutant, while GA(3) and GA(4), both of which are active GAs in angiosperms, did not. Our results suggest that the moss P. patens utilizes a diterpene metabolite from ent-kaurene as an endogenous developmental regulator and provide insights into the evolution of GA functions in land plants.

  2. DICER-LIKE3 Activity in Physcomitrella patens DICER-LIKE4 Mutants Causes Severe Developmental Dysfunction and Sterility

    PubMed Central

    Arif, M. Asif; Fattash, Isam; Cho, Sung Hyun; Beike, Anna K.; Reski, Ralf; Axtell, Michael J.; Frank, Wolfgang

    2012-01-01

    Trans-acting small interfering RNAs (ta-siRNAs) are plant-specific siRNAs released from TAS precursor transcripts after microRNA-dependent cleavage, conversion into double-stranded RNA, and Dicer-dependent phased processing. Like microRNAs (miRNAs), ta-siRNAs direct site-specific cleavage of target RNAs at sites of extensive complementarity. Here, we show that the DICER-LIKE 4 protein of Physcomitrella patens (PpDCL4) is essential for the biogenesis of 21 nucleotide (nt) ta-siRNAs. In ΔPpDCL4 mutants, off-sized 23 and 24-nt ta-siRNAs accumulated as the result of PpDCL3 activity. ΔPpDCL4 mutants display severe abnormalities throughout Physcomitrella development, including sterility, that were fully reversed in ΔPpDCL3/ΔPpDCL4 double-mutant plants. Therefore, PpDCL3 activity, not loss of PpDCL4 function per se, is the cause of the ΔPpDCL4 phenotypes. Additionally, we describe several new Physcomitrella trans-acting siRNA loci, three of which belong to a new family, TAS6. TAS6 loci are typified by sliced miR156 and miR529 target sites and are in close proximity to PpTAS3 loci. PMID:22511605

  3. Microarray analysis of transcriptional responses to abscisic acid and osmotic, salt, and drought stress in the moss, Physcomitrella patens.

    PubMed

    Cuming, Andrew C; Cho, Sung Hyun; Kamisugi, Yasuko; Graham, Helen; Quatrano, Ralph S

    2007-01-01

    Dehydration tolerance was an adaptive trait necessary for the colonization of land by plants, and remains widespread among bryophytes: the nearest extant relatives of the first land plants. A genome-wide analysis was undertaken of water-stress responses in the model moss Physcomitrella patens to identify stress-responsive genes. An oligonucleotide microarray was used for transcriptomic analysis of Physcomitrella treated with abscisic acid (ABA), or subjected to osmotic, salt and drought stress. Bioinformatic analysis of the Physcomitrella genome identified the responsive genes, and a number of putative stress-related cis-regulatory elements. In protonemal tissue, 130 genes were induced by dehydration, 56 genes by ABA, but only 10 and eight genes, respectively, by osmotic and salt stress. Fifty-one genes were induced by more than one treatment. Seventy-six genes, principally encoding chloroplast proteins, were drought down-regulated. Many ABA- and drought-responsive genes are homologues of angiosperm genes expressed during drought stress and seed development. These ABA- and drought-responsive genes include those encoding a number of late embryogenesis abundant (LEA) proteins, a 'DREB' transcription factor and a Snf-related kinase homologous with the Arabidopsis ABA signal transduction component 'OPEN STOMATA 1'. Evolutionary capture of conserved stress-regulatory transcription factors by the seed developmental pathway probably accounts for the seed-specificity of desiccation tolerance among angiosperms.

  4. A specific member of the Cab multigene family can be efficiently targeted and disrupted in the moss Physcomitrella patens.

    PubMed

    Hofmann, A H; Codón, A C; Ivascu, C; Russo, V E; Knight, C; Cove, D; Schaefer, D G; Chakhparonian, M; Zrÿd, J P

    1999-02-01

    The analysis of phenotypic change resulting from gene disruption following homologous recombination provides a powerful technique for the study of gene function. This technique has so far been difficult to apply to plants because the frequency of gene disruption following transformation with constructs containing DNA homologous to genomic sequences is low (0.01 to 0.1%). It has recently been shown that high rates of gene disruption (up to 90%) can be achieved in the moss Physcomitrella patens using genomic sequences of unknown function. We have used this system to examine the specificity of gene disruption in Physcomitrella using a member of the Cab multigene family. We have employed the previously characterised Cab gene ZLAB1 and have isolated segments of 13 other closely related members of the Cab gene family. In the 199-bp stretch sequenced, the 13 new members of the Cab family show an average of 8.5% divergence from the DNA sequence of ZLAB1. We observed 304 silent substitutions and 16 substitutions that lead to a change in the amino acid sequence of the protein. We cloned 1029 bp of the coding region of ZLAB1 (including 177 of the 199 bp with high homology to the 13 new Cab genes) into a vector containing a selectable hygromycin resistance marker, and used this construct to transform P. patens. In three of nine stable transformants tested, the construct had inserted in, and disrupted, the ZLAB1 gene. There was no discernible phenotype associated with the disruption. We have therefore shown that gene disruption is reproducible in P. patens and that the requirement for sequence homology appears to be stringent, therefore allowing the role of individual members of a gene family to be analysed in land plants for the first time.

  5. Characterization of Phytochrome Interacting Factors from the Moss Physcomitrella patens Illustrates Conservation of Phytochrome Signaling Modules in Land Plants.

    PubMed

    Possart, Anja; Xu, Tengfei; Paik, Inyup; Hanke, Sebastian; Keim, Sarah; Hermann, Helen-Maria; Wolf, Luise; Hiß, Manuel; Becker, Claude; Huq, Enamul; Rensing, Stefan A; Hiltbrunner, Andreas

    2017-02-01

    Across the plant kingdom, phytochrome (PHY) photoreceptors play an important role during adaptive and developmental responses to light. In Arabidopsis thaliana, light-activated PHYs accumulate in the nucleus, where they regulate downstream signaling components, such as phytochrome interacting factors (PIFs). PIFs are transcription factors that act as repressors of photomorphogenesis; their inhibition by PHYs leads to substantial changes in gene expression. The nuclear function of PHYs, however, has so far been investigated in only a few non-seed plants. Here, we identified putative target genes of PHY signaling in the moss Physcomitrella patens and found light-regulated genes that are putative orthologs of PIF-controlled genes in Arabidopsis. Phylogenetic analyses revealed that an ancestral PIF-like gene was already present in streptophyte algae, i.e., before the water-to-land transition of plants. The PIF homologs in the genome of P. patens resemble Arabidopsis PIFs in their protein domain structure, molecular properties, and physiological effects, albeit with notable differences in the motif-dependent PHY interaction. Our results suggest that P. patens PIFs are involved in PHY signaling. The PHY-PIF signaling node that relays light signals to target genes has been largely conserved during land plant evolution, with evidence of lineage-specific diversification.

  6. Strigolactones regulate protonema branching and act as a quorum sensing-like signal in the moss Physcomitrella patens.

    PubMed

    Proust, Hélène; Hoffmann, Beate; Xie, Xiaonan; Yoneyama, Kaori; Schaefer, Didier G; Yoneyama, Koichi; Nogué, Fabien; Rameau, Catherine

    2011-04-01

    Strigolactones are a novel class of plant hormones controlling shoot branching in seed plants. They also signal host root proximity during symbiotic and parasitic interactions. To gain a better understanding of the origin of strigolactone functions, we characterised a moss mutant strongly affected in strigolactone biosynthesis following deletion of the CAROTENOID CLEAVAGE DIOXYGENASE 8 (CCD8) gene. Here, we show that wild-type Physcomitrella patens produces and releases strigolactones into the medium where they control branching of protonemal filaments and colony extension. We further show that Ppccd8 mutant colonies fail to sense the proximity of neighbouring colonies, which in wild-type plants causes the arrest of colony extension. The mutant phenotype is rescued when grown in the proximity of wild-type colonies, by exogenous supply of synthetic strigolactones or by ectopic expression of seed plant CCD8. Thus, our data demonstrate for the first time that Bryophytes (P. patens) produce strigolactones that act as signalling factors controlling developmental and potentially ecophysiological processes. We propose that in P. patens, strigolactones are reminiscent of quorum-sensing molecules used by bacteria to communicate with one another.

  7. The kinesin-like proteins, KAC1/2, regulate actin dynamics underlying chloroplast light-avoidance in Physcomitrella patens.

    PubMed

    Shen, Zhiyuan; Liu, Yen-Chen; Bibeau, Jeffrey P; Lemoi, Kyle P; Tüzel, Erkan; Vidali, Luis

    2015-01-01

    In plants, light determines chloroplast position; these organelles show avoidance and accumulation responses in high and low fluence-rate light, respectively. Chloroplast motility in response to light is driven by cytoskeletal elements. The actin cytoskeleton mediates chloroplast photorelocation responses in Arabidopsis thaliana. In contrast, in the moss Physcomitrella patens, both, actin filaments and microtubules can transport chloroplasts. Because of the surprising evidence that two kinesin-like proteins (called KACs) are important for actin-dependent chloroplast photorelocation in vascular plants, we wanted to determine the cytoskeletal system responsible for the function of these proteins in moss. We performed gene-specific silencing using RNA interference in P. patens. We confirmed existing reports using gene knockouts, that PpKAC1 and PpKAC2 are required for chloroplast dispersion under uniform white light conditions, and that the two proteins are functionally equivalent. To address the specific cytoskeletal elements responsible for motility, this loss-of-function approach was combined with cytoskeleton-targeted drug studies. We found that, in P. patens, these KACs mediate the chloroplast light-avoidance response in an actin filament-dependent, rather than a microtubule-dependent manner. Using correlation-decay analysis of cytoskeletal dynamics, we found that PpKAC stabilizes cortical actin filaments, but has no effect on microtubule dynamics.

  8. G6PDH activity highlights the operation of the cyclic electron flow around PSI in Physcomitrella patens during salt stress

    PubMed Central

    Gao, Shan; Zheng, Zhenbing; Huan, Li; Wang, Guangce

    2016-01-01

    Photosynthetic performances and glucose-6-phosphate dehydrogenase (G6PDH) activity in Physcomitrella patens changed greatly during salt stress and recovery. In P. patens, the cyclic electron flow around photosystem (PS) I was much more tolerant to high salt stress than PSII. After high salt stress, the PSII activity recovered much more slowly than that of PSI, which was rapidly restored to pretreatment levels even as PSII was almost inactivate. This result suggested that after salt stress the recovery of the cyclic electron flow around PSI was independent of PSII activity. In addition, G6PDH activity and NADPH content increased under high salt stress. When G6PDH activity was inhibited by glucosamine (Glucm, a G6PDH inhibitor), the cyclic electron flow around PSI and the NADPH content decreased significantly. Additionally, after recovery in liquid medium containing Glucm, the PSI activity was much lower than in liquid medium without Glucm. These results suggested the PSI activity was affected significantly by G6PDH activity and the NADPH content. Based on the above results, we propose that G6PDH in P. patens has a close relationship with the photosynthetic process, possibly providing NADPH for the operation of the cyclic electron flow around PSI during salt stress and promoting the restoration of PSI. PMID:26887288

  9. Interaction between the moss Physcomitrella patens and Phytophthora: a novel pathosystem for live-cell imaging of subcellular defence.

    PubMed

    Overdijk, Elysa J R; DE Keijzer, Jeroen; DE Groot, Deborah; Schoina, Charikleia; Bouwmeester, Klaas; Ketelaar, Tijs; Govers, Francine

    2016-08-01

    Live-cell imaging of plant-pathogen interactions is often hampered by the tissue complexity and multicell layered nature of the host. Here, we established a novel pathosystem with the moss Physcomitrella patens as host for Phytophthora. The tip-growing protonema cells of this moss are ideal for visualizing interactions with the pathogen over time using high-resolution microscopy. We tested four Phytophthora species for their ability to infect P. patens and showed that P. sojae and P. palmivora were only rarely capable to infect P. patens. In contrast, P. infestans and P. capsici frequently and successfully penetrated moss protonemal cells, showed intracellular hyphal growth and formed sporangia. Next to these successful invasions, many penetration attempts failed. Here the pathogen was blocked by a barrier of cell wall material deposited in papilla-like structures, a defence response that is common in higher plants. Another common response is the upregulation of defence-related genes upon infection and also in moss we observed this upregulation in tissues infected with Phytophthora. For more advanced analyses of the novel pathosystem we developed a special set-up that allowed live-cell imaging of subcellular defence processes by high-resolution microscopy. With this set-up, we revealed that Phytophthora infection of moss induces repositioning of the nucleus, accumulation of cytoplasm and rearrangement of the actin cytoskeleton, but not of microtubules.

  10. Moss Pathogenesis-Related-10 Protein Enhances Resistance to Pythium irregulare in Physcomitrella patens and Arabidopsis thaliana.

    PubMed

    Castro, Alexandra; Vidal, Sabina; Ponce de León, Inés

    2016-01-01

    Plants respond to pathogen infection by activating signaling pathways leading to the accumulation of proteins with diverse roles in defense. Here, we addressed the functional role of PpPR-10, a pathogenesis-related (PR)-10 gene, of the moss Physcomitrella patens, in response to biotic stress. PpPR-10 belongs to a multigene family and encodes a protein twice the usual size of PR-10 proteins due to the presence of two Bet v1 domains. Moss PR-10 genes are differentially regulated during development and inoculation with the fungal pathogen Botrytis cinerea. Specifically, PpPR-10 transcript levels increase significantly by treatments with elicitors of Pectobacterium carotovorum subsp. carotovorum, spores of B. cinerea, and the defense hormone salicylic acid. To characterize the role of PpPR-10 in plant defense against pathogens, we conducted overexpression analysis in P. patens and in Arabidopsis thaliana. We demonstrate that constitutive expression of PpPR-10 in moss tissues increased resistance against the oomycete Pythium irregulare. PpPR-10 overexpressing moss plants developed less symptoms and decreased mycelium growth than wild type plants. In addition, PpPR-10 overexpressing plants constitutively produced cell wall depositions in protonemal tissue. Ectopic expression of PpPR-10 in Arabidopsis resulted in increased resistance against P. irregulare as well, evidenced by smaller lesions and less cellular damage compared to wild type plants. These results indicate that PpPR-10 is functionally active in the defense against the pathogen P. irregulare, in both P. patens and Arabidopsis, two evolutionary distant plants. Thus, P. patens can serve as an interesting source of genes to improve resistance against pathogen infection in flowering plants.

  11. Phosphatase and Tensin Homolog Is a Growth Repressor of Both Rhizoid and Gametophore Development in the Moss Physcomitrella patens.

    PubMed

    Saavedra, Laura; Catarino, Rita; Heinz, Tobias; Heilmann, Ingo; Bezanilla, Magdalena; Malhó, Rui

    2015-12-01

    Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid phosphatase implicated in cellular proliferation and survival. In animal cells, loss of PTEN leads to increased levels of phosphatidylinositol (3,4,5)-trisphosphate, stimulation of glucose and lipid metabolism, cellular growth, and morphological changes (related to adaptation and survival). Intriguingly, in plants, phosphatidylinositol (3,4,5)-trisphosphate has not been detected, and the enzymes that synthesize it were never reported. In this study we performed a genetic, biochemical, and functional characterization of the moss Physcomitrella patens PTEN gene family. P. patens has four PTENs, which are ubiquitously expressed during the entire moss life cycle. Using a knock-in approach, we show that all four genes are expressed in growing tissues, namely caulonemal and rhizoid cells. At the subcellular level, PpPTEN-green fluorescent protein fusions localized to the cytosol and the nucleus. Analysis of single and double knockouts revealed no significant phenotypes at different developmental stages, indicative of functional redundancy. However, compared with wild-type triple and quadruple pten knockouts, caulonemal cells grew faster, switched from the juvenile protonemal stage to adult gametophores earlier, and produced more rhizoids. Furthermore, analysis of lipid content and quantitative real-time polymerase chain reaction data performed in quadruple mutants revealed altered phosphoinositide levels [increase in phosphatidylinositol (3,5)-bisphosphate and decrease in phosphatidylinositol 3-phosphate] and up-regulation of marker genes from the synthesis phase of the cell cycle (e.g. P. patens proliferating cell nuclear antigen, ribonucleotide reductase, and minichromosome maintenance) and of the retinoblastoma-related protein gene P. patens retinoblastoma-related protein1. Together, these results suggest that PpPTEN is a suppressor of cell growth and morphogenic development in plants.

  12. Moss Pathogenesis-Related-10 Protein Enhances Resistance to Pythium irregulare in Physcomitrella patens and Arabidopsis thaliana

    PubMed Central

    Castro, Alexandra; Vidal, Sabina; Ponce de León, Inés

    2016-01-01

    Plants respond to pathogen infection by activating signaling pathways leading to the accumulation of proteins with diverse roles in defense. Here, we addressed the functional role of PpPR-10, a pathogenesis-related (PR)-10 gene, of the moss Physcomitrella patens, in response to biotic stress. PpPR-10 belongs to a multigene family and encodes a protein twice the usual size of PR-10 proteins due to the presence of two Bet v1 domains. Moss PR-10 genes are differentially regulated during development and inoculation with the fungal pathogen Botrytis cinerea. Specifically, PpPR-10 transcript levels increase significantly by treatments with elicitors of Pectobacterium carotovorum subsp. carotovorum, spores of B. cinerea, and the defense hormone salicylic acid. To characterize the role of PpPR-10 in plant defense against pathogens, we conducted overexpression analysis in P. patens and in Arabidopsis thaliana. We demonstrate that constitutive expression of PpPR-10 in moss tissues increased resistance against the oomycete Pythium irregulare. PpPR-10 overexpressing moss plants developed less symptoms and decreased mycelium growth than wild type plants. In addition, PpPR-10 overexpressing plants constitutively produced cell wall depositions in protonemal tissue. Ectopic expression of PpPR-10 in Arabidopsis resulted in increased resistance against P. irregulare as well, evidenced by smaller lesions and less cellular damage compared to wild type plants. These results indicate that PpPR-10 is functionally active in the defense against the pathogen P. irregulare, in both P. patens and Arabidopsis, two evolutionary distant plants. Thus, P. patens can serve as an interesting source of genes to improve resistance against pathogen infection in flowering plants. PMID:27200053

  13. Light-harvesting antenna complexes in the moss Physcomitrella patens: implications for the evolutionary transition from green algae to land plants.

    PubMed

    Iwai, Masakazu; Yokono, Makio

    2017-06-01

    Plants have successfully adapted to a vast range of terrestrial environments during their evolution. To elucidate the evolutionary transition of light-harvesting antenna proteins from green algae to land plants, the moss Physcomitrella patens is ideally placed basally among land plants. Compared to the genomes of green algae and land plants, the P. patens genome codes for more diverse and redundant light-harvesting antenna proteins. It also encodes Lhcb9, which has characteristics not found in other light-harvesting antenna proteins. The unique complement of light-harvesting antenna proteins in P. patens appears to facilitate protein interactions that include those lost in both green algae and land plants with regard to stromal electron transport pathways and photoprotection mechanisms. This review will highlight unique characteristics of the P. patens light-harvesting antenna system and the resulting implications about the evolutionary transition during plant terrestrialization. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae)

    SciTech Connect

    Scavuzzo-Duggan, Tess R.; Chaves, Arielle M.; Roberts, Alison W.

    2015-07-14

    Here, a method for rapid in vivo functional analysis of engineered proteins was developed using Physcomitrella patens. A complementation assay was designed for testing structure/function relationships in cellulose synthase (CESA) proteins. The components of the assay include (1) construction of test vectors that drive expression of epitope-tagged PpCESA5 carrying engineered mutations, (2) transformation of a ppcesa5 knockout line that fails to produce gametophores with test and control vectors, (3) scoring the stable transformants for gametophore production, (4) statistical analysis comparing complementation rates for test vectors to positive and negative control vectors, and (5) analysis of transgenic protein expression by Western blotting. The assay distinguished mutations that generate fully functional, nonfunctional, and partially functional proteins. In conclusion, compared with existing methods for in vivo testing of protein function, this complementation assay provides a rapid method for investigating protein structure/function relationships in plants.

  15. MRE11 and RAD50, but not NBS1, are essential for gene targeting in the moss Physcomitrella patens

    PubMed Central

    Kamisugi, Yasuko; Schaefer, Didier G.; Kozak, Jaroslav; Charlot, Florence; Vrielynck, Nathalie; Holá, Marcela; Angelis, Karel J.; Cuming, Andrew C.; Nogué, Fabien

    2012-01-01

    The moss Physcomitrella patens is unique among plant models for the high frequency with which targeted transgene insertion occurs via homologous recombination. Transgene integration is believed to utilize existing machinery for the detection and repair of DNA double-strand breaks (DSBs). We undertook targeted knockout of the Physcomitrella genes encoding components of the principal sensor of DNA DSBs, the MRN complex. Loss of function of PpMRE11 or PpRAD50 strongly and specifically inhibited gene targeting, whilst rates of untargeted transgene integration were relatively unaffected. In contrast, disruption of the PpNBS1 gene retained the wild-type capacity to integrate transforming DNA efficiently at homologous loci. Analysis of the kinetics of DNA-DSB repair in wild-type and mutant plants by single-nucleus agarose gel electrophoresis revealed that bleomycin-induced fragmentation of genomic DNA was repaired at approximately equal rates in each genotype, although both the Ppmre11 and Pprad50 mutants exhibited severely restricted growth and development and enhanced sensitivity to UV-B and bleomycin-induced DNA damage, compared with wild-type and Ppnbs1 plants. This implies that while extensive DNA repair can occur in the absence of a functional MRN complex; this is unsupervised in nature and results in the accumulation of deleterious mutations incompatible with normal growth and development. PMID:22210882

  16. MRE11 and RAD50, but not NBS1, are essential for gene targeting in the moss Physcomitrella patens.

    PubMed

    Kamisugi, Yasuko; Schaefer, Didier G; Kozak, Jaroslav; Charlot, Florence; Vrielynck, Nathalie; Holá, Marcela; Angelis, Karel J; Cuming, Andrew C; Nogué, Fabien

    2012-04-01

    The moss Physcomitrella patens is unique among plant models for the high frequency with which targeted transgene insertion occurs via homologous recombination. Transgene integration is believed to utilize existing machinery for the detection and repair of DNA double-strand breaks (DSBs). We undertook targeted knockout of the Physcomitrella genes encoding components of the principal sensor of DNA DSBs, the MRN complex. Loss of function of PpMRE11 or PpRAD50 strongly and specifically inhibited gene targeting, whilst rates of untargeted transgene integration were relatively unaffected. In contrast, disruption of the PpNBS1 gene retained the wild-type capacity to integrate transforming DNA efficiently at homologous loci. Analysis of the kinetics of DNA-DSB repair in wild-type and mutant plants by single-nucleus agarose gel electrophoresis revealed that bleomycin-induced fragmentation of genomic DNA was repaired at approximately equal rates in each genotype, although both the Ppmre11 and Pprad50 mutants exhibited severely restricted growth and development and enhanced sensitivity to UV-B and bleomycin-induced DNA damage, compared with wild-type and Ppnbs1 plants. This implies that while extensive DNA repair can occur in the absence of a functional MRN complex; this is unsupervised in nature and results in the accumulation of deleterious mutations incompatible with normal growth and development.

  17. Functional analyses of chitinases in the moss Physcomitrella patens: chitin oligosaccharide-induced gene expression and enzymatic characterization.

    PubMed

    Kobaru, Saki; Tanaka, Ryusuke; Taira, Toki; Uchiumi, Toshiki

    2016-12-01

    Plant chitinases play diverse roles including defense against pathogenic fungi. Using reverse-transcription quantitative PCR analysis, we found that six chitinase (PpChi) genes and two genes for chitin elicitor receptor kinases (PpCERKs) are expressed at considerable levels in the moss Physcomitrella patens subsp. patens. The expressed PpChis belonged to glycoside hydrolase family 19 (class I: PpChi-Ia and -Ib; class II: PpChi-IIa and -IIc; and class IV: PpChi-IV) and to glycoside hydrolase family 18 (class V: PpChi-Vb). Treatment with chitin tetramer or hexamer increased the expression of class I and IV PpChi genes and decreased that of class II PpChi genes. Recombinant PpChi-Ia, PpChi-IV, and PpChi-Vb were characterized. PpChi-IV exhibited higher activity against chitin tetramer and pentamer than PpChi-Ia did. PpChi-Vb showed transglycosylation activity and PpChi-Ia inhibited fungal growth. These results suggest that chitinases of different classes play different roles in defense mechanism of moss plant against fungal pathogens.

  18. Light-Harvesting Complex Stress-Related Proteins Catalyze Excess Energy Dissipation in Both Photosystems of Physcomitrella patens

    PubMed Central

    Cazzaniga, Stefano; Nevo, Reinat; Levin-Zaidman, Smadar; Reich, Ziv

    2015-01-01

    Two LHC-like proteins, Photosystem II Subunit S (PSBS) and Light-Harvesting Complex Stress-Related (LHCSR), are essential for triggering excess energy dissipation in chloroplasts of vascular plants and green algae, respectively. The mechanism of quenching was studied in Physcomitrella patens, an early divergent streptophyta (including green algae and land plants) in which both proteins are active. PSBS was localized in grana together with photosystem II (PSII), but LHCSR was located mainly in stroma-exposed membranes together with photosystem I (PSI), and its distribution did not change upon high-light treatment. The quenched conformation can be preserved by rapidly freezing the high-light-treated tissues in liquid nitrogen. When using green fluorescent protein as an internal standard, 77K fluorescence emission spectra on isolated chloroplasts allowed for independent assessment of PSI and PSII fluorescence yield. Results showed that both photosystems underwent quenching upon high-light treatment in the wild type in contrast to mutants depleted of LHCSR, which lacked PSI quenching. Due to the contribution of LHCII, P. patens had a PSI antenna size twice as large with respect to higher plants. Thus, LHCII, which is highly abundant in stroma membranes, appears to be the target of quenching by LHCSR. PMID:26508763

  19. In vitro synthesis of cellulose microfibrils by a membrane protein from protoplasts of the non-vascular plant Physcomitrella patens.

    PubMed

    Cho, Sung Hyun; Du, Juan; Sines, Ian; Poosarla, Venkata Giridhar; Vepachedu, Venkata; Kafle, Kabindra; Park, Yong Bum; Kim, Seong H; Kumar, Manish; Nixon, B Tracy

    2015-09-01

    Plant cellulose synthases (CesAs) form a family of membrane proteins that are associated with hexagonal structures in the plasma membrane called CesA complexes (CSCs). It has been difficult to purify plant CesA proteins for biochemical and structural studies. We describe CesA activity in a membrane protein preparation isolated from protoplasts of Physcomitrella patens overexpressing haemagglutinin (HA)-tagged PpCesA5. Incubating the membrane preparation with UDP-glucose predominantly produced cellulose. Negative-stain EM revealed microfibrils. Cellulase bound to and degraded these microfibrils. Vibrational sum frequency generation (SFG) spectroscopic analysis detected the presence of crystalline cellulose in the microfibrils. Putative CesA proteins were frequently observed attached to the microfibril ends. Combined cross-linking and gradient centrifugation showed bundles of cellulose microfibrils with larger particle aggregates, possibly CSCs. These results suggest that P. patens is a useful model system for biochemical and structural characterization of plant CSCs and their components.

  20. Digital gene expression profiling by 5'-end sequencing of cDNAs during reprogramming in the moss Physcomitrella patens.

    PubMed

    Nishiyama, Tomoaki; Miyawaki, Kaori; Ohshima, Masumi; Thompson, Kari; Nagashima, Akitomo; Hasebe, Mitsuyasu; Kurata, Tetsuya

    2012-01-01

    Stem cells self-renew and repeatedly produce differentiated cells during development and growth. The differentiated cells can be converted into stem cells in some metazoans and land plants with appropriate treatments. After leaves of the moss Physcomitrella patens are excised, leaf cells reenter the cell cycle and commence tip growth, which is characteristic of stem cells called chloronema apical cells. To understand the underlying molecular mechanisms, a digital gene expression profiling method using mRNA 5'-end tags (5'-DGE) was established. The 5'-DGE method produced reproducible data with a dynamic range of four orders that correlated well with qRT-PCR measurements. After the excision of leaves, the expression levels of 11% of the transcripts changed significantly within 6 h. Genes involved in stress responses and proteolysis were induced and those involved in metabolism, including photosynthesis, were reduced. The later processes of reprogramming involved photosynthesis recovery and higher macromolecule biosynthesis, including of RNA and proteins. Auxin and cytokinin signaling pathways, which are activated during stem cell formation via callus in flowering plants, are also activated during reprogramming in P. patens, although no exogenous phytohormone is applied in the moss system, suggesting that an intrinsic phytohormone regulatory system may be used in the moss.

  1. Structure and function of nucleoside hydrolases from Physcomitrella patens and maize catalyzing the hydrolysis of purine, pyrimidine, and cytokinin ribosides.

    PubMed

    Kopecná, Martina; Blaschke, Hanna; Kopecny, David; Vigouroux, Armelle; Koncitíková, Radka; Novák, Ondrej; Kotland, Ondrej; Strnad, Miroslav; Moréra, Solange; von Schwartzenberg, Klaus

    2013-12-01

    We present a comprehensive characterization of the nucleoside N-ribohydrolase (NRH) family in two model plants, Physcomitrella patens (PpNRH) and maize (Zea mays; ZmNRH), using in vitro and in planta approaches. We identified two NRH subclasses in the plant kingdom; one preferentially targets the purine ribosides inosine and xanthosine, while the other is more active toward uridine and xanthosine. Both subclasses can hydrolyze plant hormones such as cytokinin ribosides. We also solved the crystal structures of two purine NRHs, PpNRH1 and ZmNRH3. Structural analyses, site-directed mutagenesis experiments, and phylogenetic studies were conducted to identify the residues responsible for the observed differences in substrate specificity between the NRH isoforms. The presence of a tyrosine at position 249 (PpNRH1 numbering) confers high hydrolase activity for purine ribosides, while an aspartate residue in this position confers high activity for uridine. Bud formation is delayed by knocking out single NRH genes in P. patens, and under conditions of nitrogen shortage, PpNRH1-deficient plants cannot salvage adenosine-bound nitrogen. All PpNRH knockout plants display elevated levels of certain purine and pyrimidine ribosides and cytokinins that reflect the substrate preferences of the knocked out enzymes. NRH enzymes thus have functions in cytokinin conversion and activation as well as in purine and pyrimidine metabolism.

  2. Light-Harvesting Complex Stress-Related Proteins Catalyze Excess Energy Dissipation in Both Photosystems of Physcomitrella patens.

    PubMed

    Pinnola, Alberta; Cazzaniga, Stefano; Alboresi, Alessandro; Nevo, Reinat; Levin-Zaidman, Smadar; Reich, Ziv; Bassi, Roberto

    2015-11-01

    Two LHC-like proteins, Photosystem II Subunit S (PSBS) and Light-Harvesting Complex Stress-Related (LHCSR), are essential for triggering excess energy dissipation in chloroplasts of vascular plants and green algae, respectively. The mechanism of quenching was studied in Physcomitrella patens, an early divergent streptophyta (including green algae and land plants) in which both proteins are active. PSBS was localized in grana together with photosystem II (PSII), but LHCSR was located mainly in stroma-exposed membranes together with photosystem I (PSI), and its distribution did not change upon high-light treatment. The quenched conformation can be preserved by rapidly freezing the high-light-treated tissues in liquid nitrogen. When using green fluorescent protein as an internal standard, 77K fluorescence emission spectra on isolated chloroplasts allowed for independent assessment of PSI and PSII fluorescence yield. Results showed that both photosystems underwent quenching upon high-light treatment in the wild type in contrast to mutants depleted of LHCSR, which lacked PSI quenching. Due to the contribution of LHCII, P. patens had a PSI antenna size twice as large with respect to higher plants. Thus, LHCII, which is highly abundant in stroma membranes, appears to be the target of quenching by LHCSR. © 2015 American Society of Plant Biologists. All rights reserved.

  3. Dehydration stress-induced oscillations in LEA protein transcripts involves abscisic acid in the moss, Physcomitrella patens.

    PubMed

    Shinde, Suhas; Nurul Islam, M; Ng, Carl K-Y

    2012-07-01

    Physcomitrella patens is a bryophyte belonging to early diverging lineages of land plants following colonization of land in the Ordovician period. Mosses are typically found in refugial habitats and can experience rapidly fluctuating environmental conditions. The acquisition of dehydration tolerance by bryophytes is of fundamental importance as they lack water-conducting tissues and are generally one cell layer thick. • Here, we show that dehydration induced oscillations in the steady-state transcript abundances of two group 3 late embryogenesis abundant (LEA) protein genes in P. patens protonemata, and that the amplitudes of these oscillations are reflective of the severity of dehydration stress. • Dehydration stress also induced elevations in the concentrations of abscisic acid (ABA), and ABA alone can also induce dosage-dependent oscillatory increases in the steady-state abundance of LEA protein transcripts. Additionally, removal of ABA resulted in rapid attenuation of these oscillatory increases. • Our data demonstrate that dehydration stress-regulated expression of LEA protein genes is temporally dynamic and highlight the importance of oscillations as a robust mechanism for optimal responses. Our results suggest that dehydration stress-induced oscillations in the steady-state abundance of LEA protein transcripts may constitute an important cellular strategy for adaptation to life in a constantly changing environment.

  4. A SABATH Methyltransferase from the moss Physcomitrella patens catalyzes S-methylation of thiols and has a role in detoxification.

    PubMed

    Zhao, Nan; Ferrer, Jean-Luc; Moon, Hong S; Kapteyn, Jeremy; Zhuang, Xiaofeng; Hasebe, Mitsuyasu; Stewart, C Neal; Gang, David R; Chen, Feng

    2012-09-01

    Known SABATH methyltransferases, all of which were identified from seed plants, catalyze methylation of either the carboxyl group of a variety of low molecular weight metabolites or the nitrogen moiety of precursors of caffeine. In this study, the SABATH family from the bryophyte Physcomitrella patens was identified and characterized. Four SABATH-like sequences (PpSABATH1, PpSABATH2, PpSABATH3, and PpSABATH4) were identified from the P. patens genome. Only PpSABATH1 and PpSABATH2 showed expression in the leafy gametophyte of P. patens. Full-length cDNAs of PpSABATH1 and PpSABATH2 were cloned and expressed in soluble form in Escherichia coli. Recombinant PpSABATH1 and PpSABATH2 were tested for methyltransferase activity with a total of 75 compounds. While showing no activity with carboxylic acids or nitrogen-containing compounds, PpSABATH1 displayed methyltransferase activity with a number of thiols. PpSABATH2 did not show activity with any of the compounds tested. Among the thiols analyzed, PpSABATH1 showed the highest level of activity with thiobenzoic acid with an apparent Km value of 95.5μM, which is comparable to those of known SABATHs. Using thiobenzoic acid as substrate, GC-MS analysis indicated that the methylation catalyzed by PpSABATH1 is on the sulfur atom. The mechanism for S-methylation of thiols catalyzed by PpSABATH1 was partially revealed by homology-based structural modeling. The expression of PpSABATH1 was induced by the treatment of thiobenzoic acid. Further transgenic studies showed that tobacco plants overexpressing PpSABATH1 exhibited enhanced tolerance to thiobenzoic acid, suggesting that PpSABATH1 have a role in the detoxification of xenobiotic thiols. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Enhancement of non-photochemical quenching in the Bryophyte Physcomitrella patens during acclimation to salt and osmotic stress.

    PubMed

    Azzabi, Ghazi; Pinnola, Alberta; Betterle, Nico; Bassi, Roberto; Alboresi, Alessandro

    2012-10-01

    Drought and salt stress are major abiotic constraints affecting plant growth worldwide. Under these conditions, the production of reactive oxygen species (ROS) is a common phenomenon taking place mainly in chloroplasts, peroxisomes, mitochondria and apoplasts, especially when associated with high light stress. ROS are harmful because of their high reactivity to cell components, thereby leading to cytotoxicity and cell death. During the Ordovician and early Devonian period, photosynthetic organisms colonized terrestrial habitats, and the acquisition of desiccation tolerance has been a major component of their evolution. We have studied the capacity for acclimation to drought and salt stress of the moss Physcomitrella patens, a representative of the early land colonization stage. Exposure to high concentrations of NaCl and sorbitol strongly affects chloroplast development, the Chl content and the thylakoid protein composition in this moss. Under sublethal conditions (0.2 M NaCl and 0.4 M sorbitol), the photosynthetic apparatus of P. patens responds to oxidative stress by increasing non-photochemical quenching (NPQ). Surprisingly, the accumulation of PSBS and LHCSR, the two polypeptides essential for NPQ in P. patens, was not up-regulated in these conditions. Rather, an increased NPQ amplitude correlated with the overaccumulation of zeaxanthin and the presence of the enzyme violaxanthin de-epoxidase. These results suggest that the regulation of excess energy dissipation through control of PSBS and LHCSR is mainly driven by light conditions, while osmotic and salt stress act through acclimative regulation of the xanthophyll cycle. We conclude that regulation of the xanthophyll cycle is an important anticipatory strategy against photoinhibition by high light.

  6. The Chloroplast SRP Systems of Chaetosphaeridium globosum and Physcomitrella patens as Intermediates in the Evolution of SRP-Dependent Protein Transport in Higher Plants

    PubMed Central

    Ziehe, Dominik; Dünschede, Beatrix; Zenker, Mira; Funke, Silke; Nowaczyk, Marc M.; Schünemann, Danja

    2016-01-01

    The bacterial signal recognition particle (SRP) mediates the cotranslational targeting of membrane proteins and is a high affinity complex consisting of a SRP54 protein subunit (Ffh) and an SRP RNA. The chloroplast SRP (cpSRP) pathway has adapted throughout evolution to enable the posttranslational targeting of the light harvesting chlorophyll a/b binding proteins (LHCPs) to the thylakoid membrane. In spermatophytes (seed plants), the cpSRP lacks the SRP RNA and is instead formed by a high affinity interaction of the conserved 54-kD subunit (cpSRP54) with the chloroplast-specific cpSRP43 protein. This heterodimeric cpSRP recognizes LHCP and delivers it to the thylakoid membrane. However, in contrast to spermatophytes, plastid SRP RNAs were identified within all streptophyte lineages and in all chlorophyte branches. Furthermore, it was shown that cpSRP43 does not interact with cpSRP54 in chlorophytes (e.g., Chlamydomonas reinhardtii). In this study, we biochemically characterized the cpSRP system of the charophyte Chaetosphaeridium globosum and the bryophyte Physcomitrella patens. Interaction studies demonstrate low affinity binding of cpSRP54 to cpSRP43 (Kd ~10 μM) in Chaetosphaeridium globosum and Physcomitrella patens as well as relatively low affinity binding of cpSRP54 to cpSRP RNA (Kd ~1 μM) in Physcomitrella patens. CpSRP54/cpSRP43 complex formation in charophytes is supported by the finding that specific alterations in the second chromodomain of cpSRP43, that are conserved within charophytes and absent in land plants, do not interfere with cpSRP54 binding. Furthermore, our data show that the elongated apical loop structure of the Physcomitrella patens cpSRP RNA contributes to the low binding affinity between cpSRP54 and the cpSRP RNA. PMID:27861610

  7. Characterization of phosphatidylinositol phosphate kinases from the moss Physcomitrella patens: PpPIPK1 and PpPIPK2.

    PubMed

    Saavedra, Laura; Balbi, Virginia; Dove, Stephen K; Hiwatashi, Yuji; Mikami, Koji; Sommarin, Marianne

    2009-03-01

    Phosphoinositides (PIs) play a major role in eukaryotic cells, despite being a minor component of most membranes. This is the first report on PI metabolism in a bryophyte, the moss Physcomitrella patens. Moss PI composition is similar to that of other land plants growing under normal conditions. In contrast to the large number of PIPK genes present in flowering plants, the P. patens genome encodes only two type I/II PIPK genes, PpPIPK1 and PpPIPK2, which are very similar at both the nucleotide and protein product levels. However, the expression of the two genes is differentially regulated, and in vitro biochemical characterization shows that the resulting enzymes have different substrate specificities. PpPIPK1 uses PtdIns4P and PtdIns3P with similar preference and also metabolizes PtdIns(3,4)P(2) to produce PtdIns(3,4,5)P(3), a PI not yet detected in intact plant cells. PpPIPK2 prefers PtdIns as substrate and is much less active towards PtdIns4P and PtdIns3P. Thus, PpPIPK2 shows properties reminiscent of both PtdInsP-kinase and PtdIns-kinases. Moreover, a substitution of glutamic acid by alanine in the activation loop drastically reduced PpPIPK1 activity and altered the substrate specificity to PtdIns5P being the preferred substrate compared with PtdIns4P and PtdIns3P. These findings demonstrate that the substrate specificity of plant PIPKs is determined in a plant-specific manner, which provides new insights into the regulatory modes of PIPK activity in plants.

  8. The DEK1 Calpain Linker Functions in Three-Dimensional Body Patterning in Physcomitrella patens1[OPEN

    PubMed Central

    Demko, Viktor; Mekhlif, Ahmed Khaleel

    2016-01-01

    The DEFECTIVE KERNEL1 (DEK1) calpain is a conserved 240-kD key regulator of three-dimensional body patterning in land plants acting via mitotic cell plane positioning. The activity of the cytosolic C-terminal calpain protease is regulated by the membrane-anchored DEK1 MEM, which is connected to the calpain via the 600-amino acid residue Linker. Similar to the calpain and MEM domains, the Linker is highly conserved in the land plant lineage, the similarity dropping sharply compared with orthologous charophyte sequences. Using site-directed mutagenesis, we studied the effect on Physcomitrella patens development by deleting the Linker and two conserved Linker motifs. The results show that removal of the Linker has nearly the same effect as removal of the entire DEK1 gene. In contrast, deletion of the conserved Laminin_G3 (LG3) domain had a milder effect, perturbing leafy gametophore patterning and archegonia development. The LG3 domain from Marchantia polymorpha is fully functional in P. patens, whereas angiosperm sequences are not functional. Deletion of a C-terminal Linker subsegment containing a potential calpain autolytic site severely disturbs gametophore development. Finally, changing one of the three calpain active-site amino acid residues results in the same phenotype as deleting the entire DEK1 gene. Based on the conserved nature of animal and DEK1 calpains, we propose that the DEK1 MEM-Linker complex inactivates the calpain by forcing apart the two calpain subunits carrying the three amino acids of the active site. PMID:27506240

  9. Both the transglycosylase and transpeptidase functions in plastid penicillin-binding protein are essential for plastid division in Physcomitrella patens

    PubMed Central

    TAKAHASHI, Yoshiko; TAKECHI, Katsuaki; TAKIO, Susumu; TAKANO, Hiroyoshi

    2016-01-01

    Class A penicillin-binding proteins (PBPs) are active in the final step of bacterial peptidoglycan biosynthesis. They possess a transglycosylase (TG) domain to polymerize the glycan chains and a transpeptidase (TP) domain to catalyze peptide cross-linking. We reported that knockout of the Pbp gene in the moss Physcomitrella patens (ΔPpPbp) results in a macrochloroplast phenotype by affecting plastid division. Here, expression of PpPBP-GFP in ΔPpPbp restored the wild-type phenotype and GFP fluorescence was observed mainly in the periphery of each chloroplast. Stable transformants expressing Anabaena PBP with the plastid-targeting sequence, or PpPBP replacing the Anabaena TP domain exhibited partial recovery, while chloroplast number was recovered to that of wild-type plants in the transformant expressing PpPBP replacing the Anabaena TG domain. Transient expression experiments with site-directed mutagenized PpPBP showed that mutations in the conserved amino acids in both domains interfered with phenotype recovery. These results suggest that both TG and TP functions are essential for function of PpPBP in moss chloroplast division. PMID:27941308

  10. Quantitative analysis of the mitochondrial and plastid proteomes of the moss Physcomitrella patens reveals protein macrocompartmentation and microcompartmentation.

    PubMed

    Mueller, Stefanie J; Lang, Daniel; Hoernstein, Sebastian N W; Lang, Erika G E; Schuessele, Christian; Schmidt, Anton; Fluck, Melanie; Leisibach, Desirée; Niegl, Christina; Zimmer, Andreas D; Schlosser, Andreas; Reski, Ralf

    2014-04-01

    Extant eukaryotes are highly compartmentalized and have integrated endosymbionts as organelles, namely mitochondria and plastids in plants. During evolution, organellar proteomes are modified by gene gain and loss, by gene subfunctionalization and neofunctionalization, and by changes in protein targeting. To date, proteomics data for plastids and mitochondria are available for only a few plant model species, and evolutionary analyses of high-throughput data are scarce. We combined quantitative proteomics, cross-species comparative analysis of metabolic pathways, and localizations by fluorescent proteins in the model plant Physcomitrella patens in order to assess evolutionary changes in mitochondrial and plastid proteomes. This study implements data-mining methodology to classify and reliably reconstruct subcellular proteomes, to map metabolic pathways, and to study the effects of postendosymbiotic evolution on organellar pathway partitioning. Our results indicate that, although plant morphologies changed substantially during plant evolution, metabolic integration of organelles is largely conserved, with exceptions in amino acid and carbon metabolism. Retargeting or regulatory subfunctionalization are common in the studied nucleus-encoded gene families of organelle-targeted proteins. Moreover, complementing the proteomic analysis, fluorescent protein fusions revealed novel proteins at organelle interfaces such as plastid stromules (stroma-filled tubules) and highlight microcompartments as well as intercellular and intracellular heterogeneity of mitochondria and plastids. Thus, we establish a comprehensive data set for mitochondrial and plastid proteomes in moss, present a novel multilevel approach to organelle biology in plants, and place our findings into an evolutionary context.

  11. Enhancing the growth of Physcomitrella patens by combination of monochromatic red and blue light - a kinetic study.

    PubMed

    Cerff, Martin; Posten, Clemens

    2012-04-01

    In the current work we demonstrate the relevance of monochromatic light conditions in moss plant cell culture. Light intensity and illumination wavelength are important cultivation parameters due to their impact on growth and chlorophyll formation kinetics of the moss Physcomitrella patens. This moss was chosen as a model organism due to its capability to produce complex recombinant pharmaceutical proteins. Filamentous moss cells were cultivated in mineral medium in shaking flasks. The flasks were illuminated by light emitting diodes (LED) providing nearly monochromatic red and blue light as well as white light as a reference. A maximum growth rate of 0.78 day((1) was achieved under additional CO(2) aeration and no growth inhibition was observed under high light illumination. The application of dual red and blue light is the most effective way to reach high growth and chlorophyll formation rates while minimizing energy consumption of the LEDs. These observations are discussed as effects of photo sensory pigments in the moss. The combination of monochromatic red and blue light should be considered when a large scale process is set up.

  12. BRICK1 is required for apical cell growth in filaments of the moss Physcomitrella patens but not for gametophore morphology.

    PubMed

    Perroud, Pierre-François; Quatrano, Ralph S

    2008-02-01

    When BRK1, a member of the Wave/SCAR complex, is deleted in Physcomitrella patens (Deltabrk1), we report a striking reduction of filament growth resulting in smaller and fewer cells with misplaced cross walls compared with the normal protonemal cells. Using an inducible green fluorescent protein-talin to detect actin in living tissue, a characteristic broad accumulation of actin is observed at the tip of wild-type apical cells, whereas in Deltabrk1, smaller, more distinct foci of actin are present. Insertion of brk1-yfp into Deltabrk1 rescues the mutant phenotype and results in BRK1 being localized only in the tip of apical cells, the exclusive site of cell extension and division in the filament. Like BRK1, ARPC4 and At RABA4d are normally localized at the tip of apical cells and their localization is correlated with rapid tip growth in filaments. However, neither marker accumulates in apical cells of Deltabrk1 filaments. Although the Deltabrk1 phenotypes in protonema are severe, the leafy shoots or gametophores are normally shaped but stunted. These and other results suggest that BRK1 functions directly or indirectly in the selective accumulation/stabilization of actin and other proteins required for polar cell growth of filaments but not for the basic structure of the gametophore.

  13. Kinesins have a dual function in organizing microtubules during both tip growth and cytokinesis in Physcomitrella patens.

    PubMed

    Hiwatashi, Yuji; Sato, Yoshikatsu; Doonan, John H

    2014-03-01

    Microtubules (MTs) play a crucial role in the anisotropic deposition of cell wall material, thereby affecting the direction of growth. A wide range of tip-growing cells display highly polarized cell growth, and MTs have been implicated in regulating directionality and expansion. However, the molecular machinery underlying MT dynamics in tip-growing plant cells remains unclear. Here, we show that highly dynamic MT bundles form cyclically in the polarized expansion zone of the moss Physcomitrella patens caulonemal cells through the coalescence of growing MT plus ends. Furthermore, the plant-specific kinesins (KINID1) that are is essential for the proper MT organization at cytokinesis also regulate the turnover of the tip MT bundles as well as the directionality and rate of cell growth. The plus ends of MTs grow toward the expansion zone, and KINID1 is necessary for the stability of a single coherent focus of MTs in the center of the zone, whose formation coincides with the accumulation of KINID1. We propose that KINID-dependent MT bundling is essential for the correct directionality of growth as well as for promoting growth per se. Our findings indicate that two localized cell wall deposition processes, tip growth and cytokinesis, previously believed to be functionally and evolutionarily distinct, share common and plant-specific MT regulatory components.

  14. Kinesins Have a Dual Function in Organizing Microtubules during Both Tip Growth and Cytokinesis in Physcomitrella patens[W][OPEN

    PubMed Central

    Hiwatashi, Yuji; Sato, Yoshikatsu; Doonan, John H.

    2014-01-01

    Microtubules (MTs) play a crucial role in the anisotropic deposition of cell wall material, thereby affecting the direction of growth. A wide range of tip-growing cells display highly polarized cell growth, and MTs have been implicated in regulating directionality and expansion. However, the molecular machinery underlying MT dynamics in tip-growing plant cells remains unclear. Here, we show that highly dynamic MT bundles form cyclically in the polarized expansion zone of the moss Physcomitrella patens caulonemal cells through the coalescence of growing MT plus ends. Furthermore, the plant-specific kinesins (KINID1) that are is essential for the proper MT organization at cytokinesis also regulate the turnover of the tip MT bundles as well as the directionality and rate of cell growth. The plus ends of MTs grow toward the expansion zone, and KINID1 is necessary for the stability of a single coherent focus of MTs in the center of the zone, whose formation coincides with the accumulation of KINID1. We propose that KINID-dependent MT bundling is essential for the correct directionality of growth as well as for promoting growth per se. Our findings indicate that two localized cell wall deposition processes, tip growth and cytokinesis, previously believed to be functionally and evolutionarily distinct, share common and plant-specific MT regulatory components. PMID:24642939

  15. Zeaxanthin Binds to Light-Harvesting Complex Stress-Related Protein to Enhance Nonphotochemical Quenching in Physcomitrella patens[W

    PubMed Central

    Pinnola, Alberta; Dall’Osto, Luca; Gerotto, Caterina; Morosinotto, Tomas; Bassi, Roberto; Alboresi, Alessandro

    2013-01-01

    Nonphotochemical quenching (NPQ) dissipates excess energy to protect the photosynthetic apparatus from excess light. The moss Physcomitrella patens exhibits strong NPQ by both algal-type light-harvesting complex stress-related (LHCSR)–dependent and plant-type S subunit of Photosystem II (PSBS)-dependent mechanisms. In this work, we studied the dependence of NPQ reactions on zeaxanthin, which is synthesized under light stress by violaxanthin deepoxidase (VDE) from preexisting violaxanthin. We produced vde knockout (KO) plants and showed they underwent a dramatic reduction in thermal dissipation ability and enhanced photoinhibition in excess light conditions. Multiple mutants (vde lhcsr KO and vde psbs KO) showed that zeaxanthin had a major influence on LHCSR-dependent NPQ, in contrast with previous reports in Chlamydomonas reinhardtii. The PSBS-dependent component of quenching was less dependent on zeaxanthin, despite the near-complete violaxanthin to zeaxanthin exchange in LHC proteins. Consistent with this, we provide biochemical evidence that native LHCSR protein binds zeaxanthin upon excess light stress. These findings suggest that zeaxanthin played an important role in the adaptation of modern plants to the enhanced levels of oxygen and excess light intensity of land environments. PMID:24014548

  16. System for stable β-estradiol-inducible gene expression in the moss Physcomitrella patens.

    PubMed

    Kubo, Minoru; Imai, Akihiro; Nishiyama, Tomoaki; Ishikawa, Masaki; Sato, Yoshikatsu; Kurata, Tetsuya; Hiwatashi, Yuji; Reski, Ralf; Hasebe, Mitsuyasu

    2013-01-01

    Inducible transgene expression provides a useful tool to analyze gene function. The moss Physcomitrellapatens is a model basal land plant with well-developed research tools, including a high efficiency of gene targeting and substantial genomics resources. However, current systems for controlled transgene expression remain limited. Here we report the development of an estrogen receptor mediated inducible gene expression system, based on the system used in flowering plants. After identifying the appropriate promoters to drive the chimeric transducer, we succeeded in inducing transcription over 1,000-fold after 24 h incubation with β-estradiol. The P. patens system was also effective for high-level long-term induction of gene expression; transcript levels of the activated gene were maintained for at least seven days on medium containing β-estradiol. We also established two potentially neutral targeting sites and a set of vectors for reproducible expression of two transgenes. This β-estradiol-dependent system will be useful to test genes individually or in combination, allowing stable, inducible transgenic expression in P. patens.

  17. The CYP701B1 of Physcomitrella patens is an ent-kaurene oxidase that resists inhibition by uniconazole-P.

    PubMed

    Miyazaki, Sho; Katsumata, Takumi; Natsume, Masahiro; Kawaide, Hiroshi

    2011-06-23

    The moss Physcomitrella patens produces both ent-kaurene and ent-kaurenoic acid, which are intermediates of gibberellin biosynthesis in flowering plants. The CYP701 superfamily of cytochrome P450s functions as ent-kaurene oxidases in the biosynthesis of ent-kaurenoic acid. A candidate gene encoding ent-kaurene oxidase in P. patens, CYP701B1, was cloned and heterologously expressed in yeast to examine enzyme activities in vitro. The recombinant CYP701B1 protein catalyzed the oxidation reaction from ent-kaurene to ent-kaurenoic acid. CYP701B1 activity was highly resistant to the ent-kaurene oxidase inhibitor uniconazole-P (IC(50) 64 μM), even though the activity of Arabidopsis ent-kaurene oxidase (CYP701A3) was sensitive (IC(50) 0.26 μM).

  18. Two ancient classes of MIKC-type MADS-box genes are present in the moss Physcomitrella patens.

    PubMed

    Henschel, Katrin; Kofuji, Rumiko; Hasebe, Mitsuyasu; Saedler, Heinz; Münster, Thomas; Theissen, Günter

    2002-06-01

    Characterization of seven MADS-box genes, termed PPM1-PPM4 and PpMADS1-PpMADS3, from the moss model species Physcomitrella patens is reported. Phylogeny reconstructions and comparison of exon-intron structures revealed that the genes described here represent two different classes of homologous, yet distinct, MIKC-type MADS-box genes, termed MIKC(c)-type genes-"(c)" stands for "classic"-(PPM1, PPM2, PpMADS1) and MIKC(*)-type genes (PPM3, PPM4, PpMADS2, PpMADS3). The two gene classes deviate from each other in a characteristic way, especially in a sequence stretch termed intervening region. MIKC(c)-type genes are abundantly present in all land plants which have been investigated in this respect, and give rise to well-known gene types such as floral meristem and organ identity genes. In contrast, LAMB1 from the clubmoss Lycopodium annotinum was identified as the only other MIKC(*)-type gene published so far. Our findings strongly suggest that the most recent common ancestor of mosses and vascular plants contained at least one MIKC(c)-type and one MIKC(*)-type gene. Our studies thus reveal an ancient duplication of an MIKC-type gene that occurred before the separation of the lineages that led to extant mosses and vascular plants more than about 450 MYA. The identification of bona fide K-domains in both MIKC(*)-type and MIKC(c)-type proteins suggests that the K-domain is more ancient than is suggested by a recent alternative hypothesis. MIKC(*)-type genes may have escaped identification in ferns and seed plants so far. It seems more likely, however, that they represent a class of genes which has been lost in the lineage which led to extant ferns and seed plants. The high number of P. patens MADS-box genes and the presence of a K-box in the coding region and of some potential binding sites for MADS-domain proteins and other transcription factors in the putative promoter regions of these genes suggest that MADS-box genes in mosses are involved in complex gene regulatory

  19. Blue-light irradiation up-regulates the ent-kaurene synthase gene and affects the avoidance response of protonemal growth in Physcomitrella patens.

    PubMed

    Miyazaki, Sho; Toyoshima, Hikaru; Natsume, Masahiro; Nakajima, Masatoshi; Kawaide, Hiroshi

    2014-07-01

    We report a novel physiological response to blue light in the moss Physcomitrella patens . Blue light regulates ent -kaurene biosynthesis and avoidance response to protonemal growth. Gibberellins (GAs) are a group of diterpene-type plant hormones biosynthesized from ent-kaurenoic acid via ent-kaurene. While the moss Physcomitrella patens has part of the GA biosynthetic pathway, from geranylgeranyl diphosphate to ent-kaurenoic acid, no GA is found in this species. Caulonemal differentiation in a P. patens mutant with a disrupted bifunctional ent-copalyl diphosphate synthase/ent-kaurene synthase (PpCPS/KS) gene is suppressed under red light, and is recovered by application of ent-kaurene and ent-kaurenoic acid. This indicates that derivatives of ent-kaurenoic acid, not GAs, might act as endogenous developmental regulators. Here, we found unique responses in the protonemal growth of P. patens under unilateral blue light, and these regulators were involved in the responses. When protonemata of the wild type were incubated under blue light, the chloronemal filaments grew in the opposite direction to the light source. Although this avoidance was not observed in the ent-kaurene deficient mutant, chloronemal growth toward a blue-light source in the mutant was suppressed by application of ent-kaurenoic acid, and the growth was rescued to that in the wild type. Expression analysis of the PpCPS/KS gene showed that the mRNA level under blue light was rapidly increased and was five times higher than under red light. These results suggest that regulators derived from ent-kaurenoic acid are strongly involved not only in the growth regulation of caulonemal differentiation under red light, but also in the light avoidance response of chloronemal growth under blue light. In particular, growth under blue light is regulated via the PpCPS/KS gene.

  20. Functional divergence of the glutathione S-transferase supergene family in Physcomitrella patens reveals complex patterns of large gene family evolution in land plants.

    PubMed

    Liu, Yan-Jing; Han, Xue-Min; Ren, Lin-Ling; Yang, Hai-Ling; Zeng, Qing-Yin

    2013-02-01

    Plant glutathione S-transferases (GSTs) are multifunctional proteins encoded by a large gene family that play major roles in the detoxification of xenobiotics and oxidative stress metabolism. To date, studies on the GST gene family have focused mainly on vascular plants (particularly agricultural plants). In contrast, little information is available on the molecular characteristics of this large gene family in nonvascular plants. In addition, the evolutionary patterns of this family in land plants remain unclear. In this study, we identified 37 GST genes from the whole genome of the moss Physcomitrella patens, a nonvascular representative of early land plants. The 37 P. patens GSTs were divided into 10 classes, including two new classes (hemerythrin and iota). However, no tau GSTs were identified, which represent the largest class among vascular plants. P. patens GST gene family members showed extensive functional divergence in their gene structures, gene expression responses to abiotic stressors, enzymatic characteristics, and the subcellular locations of the encoded proteins. A joint phylogenetic analysis of GSTs from P. patens and other higher vascular plants showed that different class GSTs had distinct duplication patterns during the evolution of land plants. By examining multiple characteristics, this study revealed complex patterns of evolutionary divergence among the GST gene family in land plants.

  1. Evolutionary conservation of structure and function of the UVR8 photoreceptor from the liverwort Marchantia polymorpha and the moss Physcomitrella patens.

    PubMed

    Soriano, Gonzalo; Cloix, Catherine; Heilmann, Monika; Núñez-Olivera, Encarnación; Martínez-Abaigar, Javier; Jenkins, Gareth I

    2017-09-11

    The ultraviolet-B (UV-B) photoreceptor UV RESISTANCE LOCUS 8 (UVR8) mediates photomorphogenic responses to UV-B in Arabidopsis through differential gene expression, but little is known about UVR8 in other species. Bryophyte lineages were the earliest diverging embryophytes, thus being the first plants facing the UV-B regime typical of land. We therefore examined whether liverwort and moss species have functional UVR8 proteins and whether they are regulated similarly to Arabidopsis UVR8. We examined the expression, dimer/monomer status, cellular localisation and function of Marchantia polymorpha and Physcomitrella patens UVR8 in experiments with bryophyte tissue and expression of green fluorescent protein (GFP)-UVR8 fusions in Nicotiana leaves and transgenic Arabidopsis. P. patens expresses two UVR8 genes that encode functional proteins, whereas the single M. polymorpha UVR8 gene expresses two transcripts by alternative splicing that encode functional UVR8 variants. P. patens UVR8 proteins form dimers that monomerise and accumulate in the nucleus following UV-B exposure, similar to Arabidopsis UVR8, but M. polymorpha UVR8 has weaker dimers and the proteins appear more constitutively nuclear. We conclude that liverwort and moss species produce functional UVR8 proteins. Although there are differences in expression and regulation of P. patens and M. polymorpha UVR8, the mechanism of UVR8 action is strongly conserved in evolution. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  2. Heavy Metal Absorption Efficiency of two Species of Mosses (Physcomitrella patens and Funaria hygrometrica) Studied in Mercury Treated Culture under Laboratory Condition

    NASA Astrophysics Data System (ADS)

    Pradhan, Abanti; Kumari, Sony; Dash, Saktisradha; Prasad Biswal, Durga; Kishore Dash, Aditya; Panigrahi, Kishore C. S.

    2017-08-01

    As an important component of ecosystems, mosses have a strong influence on the cycling of water, energy and nutrient. Given their sensitivity to environmental change, mosses can be used as bioindicators of water quality, air pollution, metal accumulation and climate change. In the present study, the growth, differentiation and heavy metal (Hg) absorption of two species of mosses like Physcomitrella patens and Funariahygrometrica were studied in solid cultures under laboratory conditions. It was observed that, the number of gametophores developed from single inoculated gametophores after 45 days of growth of F. hygrometrica was 11±2.0 in control where as it has decreased at higher concentrations, 4±1.5 in 1ppm of mercury treatment. P. patens also shows a similar trend. The heavy metal uptake of both the species of mosses was studied. It was observed that Hg content in pseudo leaves of P. patens ranged from 0.98 ppm to 2.76 ppm at different Hg treatment (0.1-1 ppm), whereas in F. hygrometrica it ranged from 0.78 ppm to 2.43 ppm under the same treatment condition. Comparing between the Hg content in pseudo-leaves and rhizoids of P. patens and F. hygrometrica, it was observed that the Hg content was elevated about 60-64% in rhizoids than that of pseudo-leaves at 0.1% treatment level, whereas it was increased almost up to 50% in other treatment level.

  3. Comprehensive Annotation of Physcomitrella patens Small RNA Loci Reveals That the Heterochromatic Short Interfering RNA Pathway Is Largely Conserved in Land Plants[OPEN

    PubMed Central

    Coruh, Ceyda; Cho, Sung Hyun; Shahid, Saima; Liu, Qikun; Wierzbicki, Andrzej; Axtell, Michael J.

    2015-01-01

    Many plant small RNAs are sequence-specific negative regulators of target mRNAs and/or chromatin. In angiosperms, the two most abundant endogenous small RNA populations are usually 21-nucleotide microRNAs (miRNAs) and 24-nucleotide heterochromatic short interfering RNAs (siRNAs). Heterochromatic siRNAs are derived from repetitive regions and reinforce DNA methylation at targeted loci. The existence and extent of heterochromatic siRNAs in other land plant lineages has been unclear. Using small RNA-sequencing (RNA-seq) of the moss Physcomitrella patens, we identified 1090 loci that produce mostly 23- to 24-nucleotide siRNAs. These loci are mostly in intergenic regions with dense DNA methylation. Accumulation of siRNAs from these loci depends upon P. patens homologs of DICER-LIKE3 (DCL3), RNA-DEPENDENT RNA POLYMERASE2, and the largest subunit of DNA-DEPENDENT RNA POLYMERASE IV, with the largest subunit of a Pol V homolog contributing to expression at a smaller subset of the loci. A MINIMAL DICER-LIKE (mDCL) gene, which lacks the N-terminal helicase domain typical of DCL proteins, is specifically required for 23-nucleotide siRNA accumulation. We conclude that heterochromatic siRNAs, and their biogenesis pathways, are largely identical between angiosperms and P. patens, with the notable exception of the P. patens-specific use of mDCL to produce 23-nucleotide siRNAs. PMID:26209555

  4. Molecular Characterization of Three PRORP Proteins in the Moss Physcomitrella patens: Nuclear PRORP Protein Is Not Essential for Moss Viability

    PubMed Central

    Tanaka, Korechika; Kometani, Kazuki; Satoh, Hiroyuki; Sugita, Mamoru

    2014-01-01

    RNase P is a ubiquitous endonuclease that removes the 5′ leader sequence from pre-tRNAs in all organisms. In Arabidopsis thaliana, RNA-free proteinaceous RNase Ps (PRORPs) seem to be enzyme(s) for pre-tRNA 5′-end processing in organelles and the nucleus and are thought to have replaced the ribonucleoprotein RNase P variant. However, the evolution and function of plant PRORPs are not fully understood. Here, we identified and characterized three PRORP-like proteins, PpPPR_63, 67, and 104, in the basal land plant, the moss Physcomitrella patens. PpPPR_63 localizes to the nucleus, while PpPPR_67 and PpPPR_104 are found in both the mitochondria and chloroplasts. The three proteins displayed pre-tRNA 5′-end processing activity in vitro. Mutants with knockout (KO) of the PpPPR_63 gene displayed growth retardation of protonemal colonies, indicating that, unlike Arabidopsis nuclear RPORPs, the moss nuclear PpPPR_63 is not essential for viability. In the KO mutant, nuclear-encoded tRNAAsp (GUC) levels were slightly decreased, whereas most nuclear-encoded tRNA levels were not altered. This indicated that most of the cytosolic mature tRNAs were produced normally without proteinaceous RNase P-like PpPPR_63. Single PpPPR_67 or 104 gene KO mutants displayed different phenotypes of protonemal growth and chloroplast tRNAArg (ACG) accumulation. However, the levels of all other tRNAs were not altered in the KO mutants. In addition, in vitro RNase P assays showed that PpPPR_67 and PpPPR_104 efficiently cleaved chloroplast pre-tRNAArg (CCG) and pre-tRNAArg (UCU) but they cleaved pre-tRNAArg (ACG) with different efficiency. This suggests that the two proteins have overlapping function but their substrate specificity is not identical. PMID:25272157

  5. Specific pools of endogenous peptides are present in gametophore, protonema, and protoplast cells of the moss Physcomitrella patens.

    PubMed

    Fesenko, Igor A; Arapidi, Georgij P; Skripnikov, Alexander Yu; Alexeev, Dmitry G; Kostryukova, Elena S; Manolov, Alexander I; Altukhov, Ilya A; Khazigaleeva, Regina A; Seredina, Anna V; Kovalchuk, Sergey I; Ziganshin, Rustam H; Zgoda, Viktor G; Novikova, Svetlana E; Semashko, Tatiana A; Slizhikova, Darya K; Ptushenko, Vasilij V; Gorbachev, Alexey Y; Govorun, Vadim M; Ivanov, Vadim T

    2015-03-15

    Protein degradation is a basic cell process that operates in general protein turnover or to produce bioactive peptides. However, very little is known about the qualitative and quantitative composition of a plant cell peptidome, the actual result of this degradation. In this study we comprehensively analyzed a plant cell peptidome and systematically analyzed the peptide generation process. We thoroughly analyzed native peptide pools of Physcomitrella patens moss in two developmental stages as well as in protoplasts. Peptidomic analysis was supplemented by transcriptional profiling and quantitative analysis of precursor proteins. In total, over 20,000 unique endogenous peptides, ranging in size from 5 to 78 amino acid residues, were identified. We showed that in both the protonema and protoplast states, plastid proteins served as the main source of peptides and that their major fraction formed outside of chloroplasts. However, in general, the composition of peptide pools was very different between these cell types. In gametophores, stress-related proteins, e.g., late embryogenesis abundant proteins, were among the most productive precursors. The Driselase-mediated protonema conversion to protoplasts led to a peptide generation "burst", with a several-fold increase in the number of components in the latter. Degradation of plastid proteins in protoplasts was accompanied by suppression of photosynthetic activity. We suggest that peptide pools in plant cells are not merely a product of waste protein degradation, but may serve as important functional components for plant metabolism. We assume that the peptide "burst" is a form of biotic stress response that might produce peptides with antimicrobial activity from originally functional proteins. Potential functions of peptides in different developmental stages are discussed.

  6. Crystal structures of Physcomitrella patens AOC1 and AOC2: insights into the enzyme mechanism and differences in substrate specificity.

    PubMed

    Neumann, Piotr; Brodhun, Florian; Sauer, Kristin; Herrfurth, Cornelia; Hamberg, Mats; Brinkmann, Jens; Scholz, Julia; Dickmanns, Achim; Feussner, Ivo; Ficner, Ralf

    2012-11-01

    In plants, oxylipins regulate developmental processes and defense responses. The first specific step in the biosynthesis of the cyclopentanone class of oxylipins is catalyzed by allene oxide cyclase (AOC) that forms cis(+)-12-oxo-phytodienoic acid. The moss Physcomitrella patens has two AOCs (PpAOC1 and PpAOC2) with different substrate specificities for C₁₈- and C₂₀-derived substrates, respectively. To better understand AOC's catalytic mechanism and to elucidate the structural properties that explain the differences in substrate specificity, we solved and analyzed the crystal structures of 36 monomers of both apo and ligand complexes of PpAOC1 and PpAOC2. From these data, we propose the following intermediates in AOC catalysis: (1) a resting state of the apo enzyme with a closed conformation, (2) a first shallow binding mode, followed by (3) a tight binding of the substrate accompanied by conformational changes in the binding pocket, and (4) initiation of the catalytic cycle by opening of the epoxide ring. As expected, the substrate dihydro analog cis-12,13S-epoxy-9Z,15Z-octadecadienoic acid did not cyclize in the presence of PpAOC1; however, when bound to the enzyme, it underwent isomerization into the corresponding trans-epoxide. By comparing complex structures of the C₁₈ substrate analog with in silico modeling of the C₂₀ substrate analog bound to the enzyme allowed us to identify three major molecular determinants responsible for the different substrate specificities (i.e. larger active site diameter, an elongated cavity of PpAOC2, and two nonidentical residues at the entrance of the active site).

  7. Molecular characterization of three PRORP proteins in the moss Physcomitrella patens: nuclear PRORP protein is not essential for moss viability.

    PubMed

    Sugita, Chieko; Komura, Yoshihiro; Tanaka, Korechika; Kometani, Kazuki; Satoh, Hiroyuki; Sugita, Mamoru

    2014-01-01

    RNase P is a ubiquitous endonuclease that removes the 5' leader sequence from pre-tRNAs in all organisms. In Arabidopsis thaliana, RNA-free proteinaceous RNase Ps (PRORPs) seem to be enzyme(s) for pre-tRNA 5'-end processing in organelles and the nucleus and are thought to have replaced the ribonucleoprotein RNase P variant. However, the evolution and function of plant PRORPs are not fully understood. Here, we identified and characterized three PRORP-like proteins, PpPPR_63, 67, and 104, in the basal land plant, the moss Physcomitrella patens. PpPPR_63 localizes to the nucleus, while PpPPR_67 and PpPPR_104 are found in both the mitochondria and chloroplasts. The three proteins displayed pre-tRNA 5'-end processing activity in vitro. Mutants with knockout (KO) of the PpPPR_63 gene displayed growth retardation of protonemal colonies, indicating that, unlike Arabidopsis nuclear RPORPs, the moss nuclear PpPPR_63 is not essential for viability. In the KO mutant, nuclear-encoded tRNAAsp (GUC) levels were slightly decreased, whereas most nuclear-encoded tRNA levels were not altered. This indicated that most of the cytosolic mature tRNAs were produced normally without proteinaceous RNase P-like PpPPR_63. Single PpPPR_67 or 104 gene KO mutants displayed different phenotypes of protonemal growth and chloroplast tRNA(Arg) (ACG) accumulation. However, the levels of all other tRNAs were not altered in the KO mutants. In addition, in vitro RNase P assays showed that PpPPR_67 and PpPPR_104 efficiently cleaved chloroplast pre-tRNA(Arg) (CCG) and pre-tRNA(Arg) (UCU) but they cleaved pre-tRNA(Arg) (ACG) with different efficiency. This suggests that the two proteins have overlapping function but their substrate specificity is not identical.

  8. A stromal heat shock protein 70 system functions in protein import into chloroplasts in the moss Physcomitrella patens.

    PubMed

    Shi, Lan-Xin; Theg, Steven M

    2010-01-01

    Heat shock protein 70s (Hsp70s) are encoded by a multigene family and are located in different cellular compartments. They have broad-ranging functions, including involvement in protein trafficking, prevention of protein aggregation, and assistance in protein folding. Hsp70s work together with their cochaperones, J domain proteins and nucleotide exchange factors (e.g., GrpEs), in a functional cycle of substrate binding and release accompanied by ATP hydrolysis. We have taken advantage of the gene targeting capability of the moss Physcomitrella patens to investigate the functions of chloroplast Hsp70s. We identified four Hsp70 genes and two GrpE cochaperone homolog genes (CGE) in moss that encode chloroplast proteins. Disruption of one of the Hsp70 genes, that for Hsp70-2, caused lethality, and protein import into heat-shocked chloroplasts isolated from temperature-sensitive hsp70-2 mutants was appreciably impaired. Whereas the double cge null mutant was not viable, we recovered a cge1 null/cge2 knock down mutant in which Hsp70-2 was upregulated. Chloroplasts isolated from this mutant demonstrated a defect in protein import. In addition, two different precursors staged as early import intermediates could be immunoprecipitated with an Hsp70-2-specific antibody. This immunoprecipitate also contained Hsp93 and Tic40, indicating that it represents a precursor still in the Toc/Tic translocon. Together, these data indicate that a stromal Hsp70 system plays a crucial role in protein import into chloroplasts.

  9. CHASE domain-containing receptors play an essential role in the cytokinin response of the moss Physcomitrella patens

    PubMed Central

    von Schwartzenberg, Klaus; Lindner, Ann-Cathrin; Gruhn, Njuscha; Šimura, Jan; Novák, Ondřej; Strnad, Miroslav; Gonneau, Martine; Nogué, Fabien; Heyl, Alexander

    2016-01-01

    While the molecular basis for cytokinin action is quite well understood in flowering plants, little is known about the cytokinin signal transduction in early diverging land plants. The genome of the bryophyte Physcomitrella patens (Hedw.) B.S. encodes three classical cytokinin receptors, the CHASE domain-containing histidine kinases, CHK1, CHK2, and CHK3. In a complementation assay with protoplasts of receptor-deficient Arabidopsis thaliana as well as in cytokinin binding assays, we found evidence that CHK1 and CHK2 receptors can function in cytokinin perception. Using gene targeting, we generated a collection of CHK knockout mutants comprising single (Δchk1, Δchk2, Δchk3), double (Δchk1,2, Δchk1,3, Δchk2,3), and triple (Δchk1,2,3) mutants. Mutants were characterized for their cytokinin response and differentiation capacities. While the wild type did not grow on high doses of cytokinin (1 µM benzyladenine), the Δchk1,2,3 mutant exhibited normal protonema growth. Bud induction assays showed that all three cytokinin receptors contribute to the triggering of budding, albeit to different extents. Furthermore, while the triple mutant showed no response in this bioassay, the remaining mutants displayed budding responses in a diverse manner to different types and concentrations of cytokinins. Determination of cytokinin levels in mutants showed no drastic changes for any of the cytokinins; thus, in contrast to Arabidopsis, revealing only small impacts of cytokinin signaling on homeostasis. In summary, our study provides a first insight into the molecular action of cytokinin in an early diverging land plant and demonstrates that CHK receptors play an essential role in bud induction and gametophore development. PMID:26596764

  10. Protein encoding genes in an ancient plant: analysis of codon usage, retained genes and splice sites in a moss, Physcomitrella patens

    PubMed Central

    Rensing, Stefan A; Fritzowsky, Dana; Lang, Daniel; Reski, Ralf

    2005-01-01

    Background The moss Physcomitrella patens is an emerging plant model system due to its high rate of homologous recombination, haploidy, simple body plan, physiological properties as well as phylogenetic position. Available EST data was clustered and assembled, and provided the basis for a genome-wide analysis of protein encoding genes. Results We have clustered and assembled Physcomitrella patens EST and CDS data in order to represent the transcriptome of this non-seed plant. Clustering of the publicly available data and subsequent prediction resulted in a total of 19,081 non-redundant ORF. Of these putative transcripts, approximately 30% have a homolog in both rice and Arabidopsis transcriptome. More than 130 transcripts are not present in seed plants but can be found in other kingdoms. These potential "retained genes" might have been lost during seed plant evolution. Functional annotation of these genes reveals unequal distribution among taxonomic groups and intriguing putative functions such as cytotoxicity and nucleic acid repair. Whereas introns in the moss are larger on average than in the seed plant Arabidopsis thaliana, position and amount of introns are approximately the same. Contrary to Arabidopsis, where CDS contain on average 44% G/C, in Physcomitrella the average G/C content is 50%. Interestingly, moss orthologs of Arabidopsis genes show a significant drift of codon fraction usage, towards the seed plant. While averaged codon bias is the same in Physcomitrella and Arabidopsis, the distribution pattern is different, with 15% of moss genes being unbiased. Species-specific, sensitive and selective splice site prediction for Physcomitrella has been developed using a dataset of 368 donor and acceptor sites, utilizing a support vector machine. The prediction accuracy is better than those achieved with tools trained on Arabidopsis data. Conclusion Analysis of the moss transcriptome displays differences in gene structure, codon and splice site usage in

  11. The nitric oxide production in the moss Physcomitrella patens is mediated by nitrate reductase.

    PubMed

    Medina-Andrés, Rigoberto; Solano-Peralta, Alejandro; Saucedo-Vázquez, Juan Pablo; Napsucialy-Mendivil, Selene; Pimentel-Cabrera, Jaime Arturo; Sosa-Torres, Martha Elena; Dubrovsky, Joseph G; Lira-Ruan, Verónica

    2015-01-01

    During the last 20 years multiple roles of the nitric oxide gas (•NO) have been uncovered in plant growth, development and many physiological processes. In seed plants the enzymatic synthesis of •NO is mediated by a nitric oxide synthase (NOS)-like activity performed by a still unknown enzyme(s) and nitrate reductase (NR). In green algae the •NO production has been linked only to NR activity, although a NOS gene was reported for Ostreococcus tauri and O. lucimarinus, no other Viridiplantae species has such gene. As there is no information about •NO synthesis neither for non-vascular plants nor for non-seed vascular plants, the interesting question regarding the evolution of the enzymatic •NO production systems during land plant natural history remains open. To address this issue the endogenous •NO production by protonema was demonstrated using Electron Paramagnetic Resonance (EPR). The •NO signal was almost eliminated in plants treated with sodium tungstate, which also reduced the NR activity, demonstrating that in P. patens NR activity is the main source for •NO production. The analysis with confocal laser scanning microscopy (CLSM) confirmed endogenous NO production and showed that •NO signal is accumulated in the cytoplasm of protonema cells. The results presented here show for the first time the •NO production in a non-vascular plant and demonstrate that the NR-dependent enzymatic synthesis of •NO is common for embryophytes and green algae.

  12. The Nitric Oxide Production in the Moss Physcomitrella patens Is Mediated by Nitrate Reductase

    PubMed Central

    Medina-Andrés, Rigoberto; Solano-Peralta, Alejandro; Saucedo-Vázquez, Juan Pablo; Napsucialy-Mendivil, Selene; Pimentel-Cabrera, Jaime Arturo; Sosa-Torres, Martha Elena; Dubrovsky, Joseph G.; Lira-Ruan, Verónica

    2015-01-01

    During the last 20 years multiple roles of the nitric oxide gas (•NO) have been uncovered in plant growth, development and many physiological processes. In seed plants the enzymatic synthesis of •NO is mediated by a nitric oxide synthase (NOS)-like activity performed by a still unknown enzyme(s) and nitrate reductase (NR). In green algae the •NO production has been linked only to NR activity, although a NOS gene was reported for Ostreococcus tauri and O. lucimarinus, no other Viridiplantae species has such gene. As there is no information about •NO synthesis neither for non-vascular plants nor for non-seed vascular plants, the interesting question regarding the evolution of the enzymatic •NO production systems during land plant natural history remains open. To address this issue the endogenous •NO production by protonema was demonstrated using Electron Paramagnetic Resonance (EPR). The •NO signal was almost eliminated in plants treated with sodium tungstate, which also reduced the NR activity, demonstrating that in P. patens NR activity is the main source for •NO production. The analysis with confocal laser scanning microscopy (CLSM) confirmed endogenous NO production and showed that •NO signal is accumulated in the cytoplasm of protonema cells. The results presented here show for the first time the •NO production in a non-vascular plant and demonstrate that the NR-dependent enzymatic synthesis of •NO is common for embryophytes and green algae. PMID:25742644

  13. Sliced microRNA targets and precise loop-first processing of MIR319 hairpins revealed by analysis of the Physcomitrella patens degradome

    PubMed Central

    Addo-Quaye, Charles; Snyder, Jo Ann; Park, Yong Bum; Li, Yong-Fang; Sunkar, Ramanjulu; Axtell, Michael J.

    2009-01-01

    Expression profiling of the 5′ ends of uncapped mRNAs (“degradome” sequencing) can be used to empirically catalog microRNA (miRNA) targets, to probe patterns of miRNA hairpin processing, to examine mRNA decay, and to analyze accumulation of endogenous short interfering RNA (siRNA) precursors. We sequenced and analyzed the degradome of the moss Physcomitrella patens, an important model system for functional genomic analyses in plant evolution. A total of 52 target mRNAs of 27 different Physcomitrella miRNA families were identified. Many targets of both more conserved and less conserved miRNA families encoded putative regulatory proteins. Remnants of MIRNA hairpin processing also populated the degradome data and indicated an unusual “loop-first” mode of precise processing for the MIR319 gene family. Precise loop-first processing was confirmed for native Physcomitrella, rice, and Arabidopsis MIR319 hairpins, as well as an Arabidopsis artificial MIRNA (aMIRNA) based upon a MIR319 backbone. MIR319 is thus a conserved exception to the general rule of loop-last processing of MIRNA hairpins. Loop-first MIR319 processing may contribute to the high efficacy of a widely used MIR319-based strategy for aMIRNA production in plants. PMID:19850910

  14. Structural modelling and transcriptional responses highlight a clade of PpKAI2-LIKE genes as candidate receptors for strigolactones in Physcomitrella patens.

    PubMed

    Lopez-Obando, Mauricio; Conn, Caitlin E; Hoffmann, Beate; Bythell-Douglas, Rohan; Nelson, David C; Rameau, Catherine; Bonhomme, Sandrine

    2016-06-01

    A set of PpKAI2 - LIKE paralogs that may encode strigolactone receptors in Physcomitrella patens were identified through evolutionary, structural, and transcriptional analyses, suggesting that strigolactone perception may have evolved independently in basal land plants in a similar manner as spermatophytes. Carotenoid-derived compounds known as strigolactones are a new class of plant hormones that modulate development and interactions with parasitic plants and arbuscular mycorrhizal fungi. The strigolactone receptor protein DWARF14 (D14) belongs to the α/β hydrolase family. D14 is closely related to KARRIKIN INSENSITIVE2 (KAI2), a receptor of smoke-derived germination stimulants called karrikins. Strigolactone and karrikin structures share a butenolide ring that is necessary for bioactivity. Charophyte algae and basal land plants produce strigolactones that influence their development. However phylogenetic studies suggest that D14 is absent from algae, moss, and liverwort genomes, raising the question of how these basal plants perceive strigolactones. Strigolactone perception during seed germination putatively evolved in parasitic plants through gene duplication and neofunctionalization of KAI2 paralogs. The moss Physcomitrella patens shows an increase in KAI2 gene copy number, similar to parasitic plants. In this study we investigated whether P. patens KAI2-LIKE (PpKAI2L) genes may contribute to strigolactone perception. Based on phylogenetic analyses and homology modelling, we predict that a clade of PpKAI2L proteins have enlarged ligand-binding cavities, similar to D14. We observed that some PpKAI2L genes have transcriptional responses to the synthetic strigolactone GR24 racemate or its enantiomers. These responses were influenced by light and dark conditions. Moreover, (+)-GR24 seems to be the active enantiomer that induces the transcriptional responses of PpKAI2L genes. We hypothesize that members of specific PpKAI2L clades are candidate strigolactone

  15. Overexpression of RelA/SpoT homologs, PpRSH2a and PpRSH2b, induces the growth suppression of the moss Physcomitrella patens.

    PubMed

    Sato, Michio; Takahashi, Tomohiro; Ochi, Kozo; Matsuura, Hideyuki; Nabeta, Kensuke; Takahashi, Kosaku

    2015-01-01

    Two genes encoding RelA/SpoT homologs, PpRSH2a and PpRSH2b, which are involved in the synthesis of bacterial alarmone guanosine 5'-diphosphate 3'-diphosphate (ppGpp) for the stringent response, were isolated from the moss, Physcomitrella patens. A complementary analysis of PpRSH2a and PpRSH2b in Escherichia coli showed that these genes had ppGpp biosynthetic activity. The recombinant PpRSH2a and PpRSH2b were also shown to synthesize ppGpp in vitro. Both proteins were localized to the chloroplasts of P. patens. Expression of the PpRSH genes was induced upon treatment with abscisic acid or abiotic stresses, such as dehydration and UV irradiation. Overexpression of PpRSH2a and PpRSH2b caused suppression of the growth in response to 1% (w/v) of glucose. The present study suggests the existence of a mechanism to regulate the growth of P. patens, which is governed by plant RSH in chloroplasts.

  16. The knock-out of ARP3a gene affects F-actin cytoskeleton organization altering cellular tip growth, morphology and development in moss Physcomitrella patens.

    PubMed

    Finka, Andrija; Saidi, Younousse; Goloubinoff, Pierre; Neuhaus, Jean-Marc; Zrÿd, Jean-Pierre; Schaefer, Didier G

    2008-10-01

    The seven subunit Arp2/3 complex is a highly conserved nucleation factor of actin microfilaments. We have isolated the genomic sequence encoding a putative Arp3a protein of the moss Physcomitrella patens. The disruption of this ARP3A gene by allele replacement has generated loss-of-function mutants displaying a complex developmental phenotype. The loss-of function of ARP3A gene results in shortened, almost cubic chloronemal cells displaying affected tip growth and lacking differentiation to caulonemal cells. In moss arp3a mutants, buds differentiate directly from chloronemata to form stunted leafy shoots having differentiated leaves similar to wild type. Yet, rhizoids never differentiate from stem epidermal cells. To characterize the F-actin organization in the arp3a-mutated cells, we disrupted ARP3A gene in the previously described HGT1 strain expressing conditionally the GFP-talin marker. In vivo observation of the F-actin cytoskeleton during P. patens development demonstrated that loss-of-function of Arp3a is associated with the disappearance of specific F-actin cortical structures associated with the establishment of localized cellular growth domains. Finally, we show that constitutive expression of the P. patens Arp3a and its Arabidopsis thaliana orthologs efficiently complement the mutated phenotype indicating a high degree of evolutionary conservation of the Arp3 function in land plants.

  17. Involvement of the CYP78A subfamily of cytochrome P450 monooxygenases in protonema growth and gametophore formation in the moss Physcomitrella patens.

    PubMed

    Katsumata, Takumi; Fukazawa, Jutarou; Magome, Hiroshi; Jikumaru, Yusuke; Kamiya, Yuji; Natsume, Masahiro; Kawaide, Hiroshi; Yamaguchi, Shinjiro

    2011-01-01

    CYP78 is a plant-specific family of cytochrome P450 monooxygenases, some members of which regulate the plastochron length and organ size in angiosperms. The CYP78 family appears to be highly conserved in land plants, but there have been no reports on the role of CYP78s in bryophytes. The moss, Physcomitrella patens, possesses two CYP78As, CYP78A27 and CYP78A28. We produced single and double mutants and overexpression lines for CYP78A27 and CYP78A28 by gene targeting to investigate the function of CYP78As in P. patens. Neither the cyp78a27 nor cyp78a28 single mutant showed any obvious phenotype, while the double mutant exhibited severely retarded protonemal growth and gametophore development. The endogenous levels of some plant hormones were also altered in the double mutant. Transgenic lines that overexpressed CYP78A27 or CYP78A28 showed delayed and reduced bud formation. Our results suggest that CYP78As participate in the synthesis of a critical growth regulator in P. patens.

  18. Large-scale gene expression profiling data for the model moss Physcomitrella patens aid understanding of developmental progression, culture and stress conditions.

    PubMed

    Hiss, Manuel; Laule, Oliver; Meskauskiene, Rasa M; Arif, Muhammad A; Decker, Eva L; Erxleben, Anika; Frank, Wolfgang; Hanke, Sebastian T; Lang, Daniel; Martin, Anja; Neu, Christina; Reski, Ralf; Richardt, Sandra; Schallenberg-Rüdinger, Mareike; Szövényi, Peter; Tiko, Theodhor; Wiedemann, Gertrud; Wolf, Luise; Zimmermann, Philip; Rensing, Stefan A

    2014-08-01

    The moss Physcomitrella patens is an important model organism for studying plant evolution, development, physiology and biotechnology. Here we have generated microarray gene expression data covering the principal developmental stages, culture forms and some environmental/stress conditions. Example analyses of developmental stages and growth conditions as well as abiotic stress treatments demonstrate that (i) growth stage is dominant over culture conditions, (ii) liquid culture is not stressful for the plant, (iii) low pH might aid protoplastation by reduced expression of cell wall structure genes, (iv) largely the same gene pool mediates response to dehydration and rehydration, and (v) AP2/EREBP transcription factors play important roles in stress response reactions. With regard to the AP2 gene family, phylogenetic analysis and comparison with Arabidopsis thaliana shows commonalities as well as uniquely expressed family members under drought, light perturbations and protoplastation. Gene expression profiles for P. patens are available for the scientific community via the easy-to-use tool at https://www.genevestigator.com. By providing large-scale expression profiles, the usability of this model organism is further enhanced, for example by enabling selection of control genes for quantitative real-time PCR. Now, gene expression levels across a broad range of conditions can be accessed online for P. patens.

  19. Comprehensive Annotation of Physcomitrella patens Small RNA Loci Reveals That the Heterochromatic Short Interfering RNA Pathway Is Largely Conserved in Land Plants.

    PubMed

    Coruh, Ceyda; Cho, Sung Hyun; Shahid, Saima; Liu, Qikun; Wierzbicki, Andrzej; Axtell, Michael J

    2015-08-01

    Many plant small RNAs are sequence-specific negative regulators of target mRNAs and/or chromatin. In angiosperms, the two most abundant endogenous small RNA populations are usually 21-nucleotide microRNAs (miRNAs) and 24-nucleotide heterochromatic short interfering RNAs (siRNAs). Heterochromatic siRNAs are derived from repetitive regions and reinforce DNA methylation at targeted loci. The existence and extent of heterochromatic siRNAs in other land plant lineages has been unclear. Using small RNA-sequencing (RNA-seq) of the moss Physcomitrella patens, we identified 1090 loci that produce mostly 23- to 24-nucleotide siRNAs. These loci are mostly in intergenic regions with dense DNA methylation. Accumulation of siRNAs from these loci depends upon P. patens homologs of DICER-LIKE3 (DCL3), RNA-DEPENDENT RNA POLYMERASE2, and the largest subunit of DNA-DEPENDENT RNA POLYMERASE IV, with the largest subunit of a Pol V homolog contributing to expression at a smaller subset of the loci. A MINIMAL DICER-LIKE (mDCL) gene, which lacks the N-terminal helicase domain typical of DCL proteins, is specifically required for 23-nucleotide siRNA accumulation. We conclude that heterochromatic siRNAs, and their biogenesis pathways, are largely identical between angiosperms and P. patens, with the notable exception of the P. patens-specific use of mDCL to produce 23-nucleotide siRNAs. © 2015 American Society of Plant Biologists. All rights reserved.

  20. The peptidoglycan biosynthesis genes MurA and MraY are related to chloroplast division in the moss Physcomitrella patens.

    PubMed

    Homi, Shoko; Takechi, Katsuaki; Tanidokoro, Koji; Sato, Hiroshi; Takio, Susumu; Takano, Hiroyoshi

    2009-12-01

    In the moss Physcomitrella patens, 10 Mur genes involved in peptidoglycan biosynthesis were found, and the MurE and Pbp genes are related to plastid division. Although the MraY and MurG genes were missing in our previous expressed sequence tag screening, they were discovered in the P. patens genome in this study, indicating that P. patens has a full set of genes capable of synthesizing peptidoglycan. In addition, a second MurA gene (PpMurA2) was found. Whereas Northern analyses indicated that PpMurA1, PpMurG and PpMraY were expressed, transcripts of PpMurA2 were detected only when RT-PCR was employed. Whereas GFP fusion proteins with either PpMurA1 or PpMraY were detected in chloroplasts, the PpMurA2 fusion proteins were located in the cytoplasm. Protonema cells in the wild-type plants had an average of 46 chloroplasts. PpMurA1 gene-disrupted lines had <10 chloroplasts, whereas approximately 30 chloroplasts existed in the PpMurA2 knockout lines. The PpMurA1/A2 double-knockout lines had only a few macrochloroplasts, suggesting a redundant function for these two genes. Disruption of the PpMraY gene in P. patens resulted in the appearance of macrochloroplasts. Anabaena MraY fused to the N-terminal region of PpMraY and A. thaliana MraY could complement the macrochloroplast phenotype in the PpMraY knockout line. Electron microscopic observations showed no obvious differences in the shape or stacking of thylakoid membranes between all knockout transformants and wild-type plants, suggesting that these Mur genes are related only to plastid division in moss.

  1. Phylogenetic analysis of pectin-related gene families in Physcomitrella patens and nine other plant species yields evolutionary insights into cell walls

    PubMed Central

    2014-01-01

    Background Pectins are acidic sugar-containing polysaccharides that are universally conserved components of the primary cell walls of plants and modulate both tip and diffuse cell growth. However, many of their specific functions and the evolution of the genes responsible for producing and modifying them are incompletely understood. The moss Physcomitrella patens is emerging as a powerful model system for the study of plant cell walls. To identify deeply conserved pectin-related genes in Physcomitrella, we generated phylogenetic trees for 16 pectin-related gene families using sequences from ten plant genomes and analyzed the evolutionary relationships within these families. Results Contrary to our initial hypothesis that a single ancestral gene was present for each pectin-related gene family in the common ancestor of land plants, five of the 16 gene families, including homogalacturonan galacturonosyltransferases, polygalacturonases, pectin methylesterases, homogalacturonan methyltransferases, and pectate lyase-like proteins, show evidence of multiple members in the early land plant that gave rise to the mosses and vascular plants. Seven of the gene families, the UDP-rhamnose synthases, UDP-glucuronic acid epimerases, homogalacturonan galacturonosyltransferase-like proteins, β-1,4-galactan β-1,4-galactosyltransferases, rhamnogalacturonan II xylosyltransferases, and pectin acetylesterases appear to have had a single member in the common ancestor of land plants. We detected no Physcomitrella members in the xylogalacturonan xylosyltransferase, rhamnogalacturonan I arabinosyltransferase, pectin methylesterase inhibitor, or polygalacturonase inhibitor protein families. Conclusions Several gene families related to the production and modification of pectins in plants appear to have multiple members that are conserved as far back as the common ancestor of mosses and vascular plants. The presence of multiple members of these families even before the divergence of other

  2. Exclusion of Na+ via Sodium ATPase (PpENA1) Ensures Normal Growth of Physcomitrella patens under Moderate Salt Stress1

    PubMed Central

    Lunde, Christina; Drew, Damian P.; Jacobs, Andrew K.; Tester, Mark

    2007-01-01

    The bryophyte Physcomitrella patens is unlike any other plant identified to date in that it possesses a gene that encodes an ENA-type Na+-ATPase. To complement previous work in yeast (Saccharomyces cerevisiae), we determined the importance of having a Na+-ATPase in planta by conducting physiological analyses of PpENA1 in Physcomitrella. Expression studies showed that PpENA1 is up-regulated by NaCl and, to a lesser degree, by osmotic stress. Maximal induction is obtained after 8 h at 60 mm NaCl or above. No other abiotic stress tested led to significant increases in PpENA1 expression. In the gametophyte, strong expression was confined to the rhizoids, stem, and the basal part of the leaf. In the protonemata, expression was ubiquitous with a few filaments showing stronger expression. At 100 mm NaCl, wild-type plants were able to maintain a higher K+-to-Na+ ratio than the PpENA1 (ena1) knockout gene, but at higher NaCl concentrations no difference was observed. Although no difference in chlorophyll content was observed between ena1 and wild type at 100 mm NaCl, the impaired Na+ exclusion in ena1 plants led to an approximately 40% decrease in growth. PMID:17556514

  3. WOX13-like genes are required for reprogramming of leaf and protoplast cells into stem cells in the moss Physcomitrella patens.

    PubMed

    Sakakibara, Keiko; Reisewitz, Pascal; Aoyama, Tsuyoshi; Friedrich, Thomas; Ando, Sayuri; Sato, Yoshikatsu; Tamada, Yosuke; Nishiyama, Tomoaki; Hiwatashi, Yuji; Kurata, Tetsuya; Ishikawa, Masaki; Deguchi, Hironori; Rensing, Stefan A; Werr, Wolfgang; Murata, Takashi; Hasebe, Mitsuyasu; Laux, Thomas

    2014-04-01

    Many differentiated plant cells can dedifferentiate into stem cells, reflecting the remarkable developmental plasticity of plants. In the moss Physcomitrella patens, cells at the wound margin of detached leaves become reprogrammed into stem cells. Here, we report that two paralogous P. patens WUSCHEL-related homeobox 13-like (PpWOX13L) genes, homologs of stem cell regulators in flowering plants, are transiently upregulated and required for the initiation of cell growth during stem cell formation. Concordantly, Δppwox13l deletion mutants fail to upregulate genes encoding homologs of cell wall loosening factors during this process. During the moss life cycle, most of the Δppwox13l mutant zygotes fail to expand and initiate an apical stem cell to form the embryo. Our data show that PpWOX13L genes are required for the initiation of cell growth specifically during stem cell formation, in analogy to WOX stem cell functions in seed plants, but using a different cellular mechanism.

  4. Circadian expression of the PpLhcb2 gene encoding a major light-harvesting chlorophyll a/b-binding protein in the moss Physcomitrella patens.

    PubMed

    Aoki, Setsuyuki; Kato, Seiji; Ichikawa, Kazuhiro; Shimizu, Masashi

    2004-01-01

    Circadian clocks control the expression of Lhcb genes encoding the chlorophyll a/b-binding proteins broadly in seed plants. We show here that this regulation is also conserved in the primitive moss Physcomitrella patens. Northern blotting analyses revealed a robust daily oscillation of Lhcb mRNA levels in protonema cells in 12-h : 12-h light-dark cycles (12 : 12LD) that damped rapidly in continuous darkness (DD). In continuous light (LL), by contrast with typical profiles in higher plants, Lhcb mRNA levels only peaked during the first day and thereafter it showed constant levels. Reverse transcription (RT)-PCR analyses showed similar patterns of expression in LL for three distinct Lhcb genes (PpLhcb1, PpLhcb2 and Zlab1). Moreover, transgenic reporter strains expressing luciferase under the control of the PpLhcb2 promoter showed bioluminescence patterns consistent with the Northern and RT-PCR data. At a higher concentration (4.5%) of glucose in the medium, the reporter strain showed self-sustained rhythms in DD, which was entrained to a differently phased 12 : 12LD, revealing a circadian regulation on the transcription. Kinetics of bioluminescent peaks in 12 : 12LD from gametophore was different to those from protonema, indicating a developmental regulation on PpLhcb2. Together, the regulatory link between the clock and Lhcb genes in P. patens shows characteristics that appear to differ from those in higher plants.

  5. Insights from the cold transcriptome of Physcomitrella patens: global specialization pattern of conserved transcriptional regulators and identification of orphan genes involved in cold acclimation

    PubMed Central

    Beike, Anna K; Lang, Daniel; Zimmer, Andreas D; Wüst, Florian; Trautmann, Danika; Wiedemann, Gertrud; Beyer, Peter; Decker, Eva L; Reski, Ralf

    2015-01-01

    The whole-genome transcriptomic cold stress response of the moss Physcomitrella patens was analyzed and correlated with phenotypic and metabolic changes. Based on time-series microarray experiments and quantitative real-time polymerase chain reaction, we characterized the transcriptomic changes related to early stress signaling and the initiation of cold acclimation. Transcription-associated protein (TAP)-encoding genes of P. patens and Arabidopsis thaliana were classified using generalized linear models. Physiological responses were monitored with pulse-amplitude-modulated fluorometry, high-performance liquid chromatography and targeted high-performance mass spectrometry. The transcript levels of 3220 genes were significantly affected by cold. Comparative classification revealed a global specialization of TAP families, a transcript accumulation of transcriptional regulators of the stimulus/stress response and a transcript decline of developmental regulators. Although transcripts of the intermediate to later response are from evolutionarily conserved genes, the early response is dominated by species-specific genes. These orphan genes may encode as yet unknown acclimation processes. PMID:25209349

  6. Structure and Function of Nucleoside Hydrolases from Physcomitrella patens and Maize Catalyzing the Hydrolysis of Purine, Pyrimidine, and Cytokinin Ribosides1[W

    PubMed Central

    Kopečná, Martina; Blaschke, Hanna; Kopečný, David; Vigouroux, Armelle; Končitíková, Radka; Novák, Ondřej; Kotland, Ondřej; Strnad, Miroslav; Moréra, Solange; von Schwartzenberg, Klaus

    2013-01-01

    We present a comprehensive characterization of the nucleoside N-ribohydrolase (NRH) family in two model plants, Physcomitrella patens (PpNRH) and maize (Zea mays; ZmNRH), using in vitro and in planta approaches. We identified two NRH subclasses in the plant kingdom; one preferentially targets the purine ribosides inosine and xanthosine, while the other is more active toward uridine and xanthosine. Both subclasses can hydrolyze plant hormones such as cytokinin ribosides. We also solved the crystal structures of two purine NRHs, PpNRH1 and ZmNRH3. Structural analyses, site-directed mutagenesis experiments, and phylogenetic studies were conducted to identify the residues responsible for the observed differences in substrate specificity between the NRH isoforms. The presence of a tyrosine at position 249 (PpNRH1 numbering) confers high hydrolase activity for purine ribosides, while an aspartate residue in this position confers high activity for uridine. Bud formation is delayed by knocking out single NRH genes in P. patens, and under conditions of nitrogen shortage, PpNRH1-deficient plants cannot salvage adenosine-bound nitrogen. All PpNRH knockout plants display elevated levels of certain purine and pyrimidine ribosides and cytokinins that reflect the substrate preferences of the knocked out enzymes. NRH enzymes thus have functions in cytokinin conversion and activation as well as in purine and pyrimidine metabolism. PMID:24170203

  7. Abiotic stress-induced oscillations in steady-state transcript levels of Group 3 LEA protein genes in the moss, Physcomitrella patens.

    PubMed

    Shinde, Suhas; Shinde, Rupali; Downey, Frances; Ng, Carl K-Y

    2013-01-01

    The moss, Physcomitrella patens is a non-seed land plant belonging to early diverging lineages of land plants following colonization of land in the Ordovician period in Earth's history. Evidence suggests that mosses can be highly tolerant of abiotic stress. We showed previously that dehydration stress and abscisic acid treatments induced oscillations in steady-state levels of LEA (Late Embryogenesis Abundant) protein transcripts, and that removal of ABA resulted in rapid attenuation of oscillatory increases in transcript levels. Here, we show that other abiotic stresses like salt and osmotic stresses also induced oscillations in steady-state transcript levels and that the amplitudes of the oscillatory increases in steady-state transcript levels are reflective of the severity of the abiotic stress treatment. Together, our results suggest that oscillatory increases in transcript levels in response to abiotic stresses may be a general phenomenon in P. patens and that temporally dynamic increases in steady-state transcript levels may be important for adaptation to life in constantly fluctuating environmental conditions.

  8. Sequence analysis of expressed sequence tags from an ABA-treated cDNA library identifies stress response genes in the moss Physcomitrella patens.

    PubMed

    Machuka, J; Bashiardes, S; Ruben, E; Spooner, K; Cuming, A; Knight, C; Cove, D

    1999-04-01

    Partial cDNA sequencing was used to obtain 169 expressed sequence tags (ESTs) in the moss, Physcomitrella patens. The source of ESTs was a random cDNA library constructed from 7 day-old protonemata following treatment with 10(-4) M abscisic acid (ABA). Analysis of the ESTs identified 69% with homology to known sequences, 61% of which had significant homology to sequences of plant origin. More importantly, at least 11 ESTs had significant similarities to genes which are implicated in plant stress-responses, including responses which may involve ABA. These included a cDNA associated with desiccation tolerance, two heat shock protein genes, one cold acclimation protein cDNA and five others that may be involved in either oxidative or chemical stress or both, i.e., Zn/Cu-superoxide dismutase, NADPH protochlorophyllide oxidoreductase (PorB), selenium binding protein, glutathione peroxidase and glutathione S transferase. Analysis of codon usage between P. patens and seed plants indicated that although mosses and higher plants are to a large extent similar, minor variations also exists that may represent the distinctiveness of each group.

  9. CYP710A genes encoding sterol C22-desaturase in Physcomitrella patens as molecular evidence for the evolutionary conservation of a sterol biosynthetic pathway in plants.

    PubMed

    Morikawa, Tomomi; Saga, Hirohisa; Hashizume, Hiroko; Ohta, Daisaku

    2009-05-01

    We have characterized cytochromes P450, CYP710A13, and CYP710A14, as the sterol C22-desaturase in the moss Physcomitrella patens. GC-MS analyses demonstrated that P. patens accumulated stigmasterol as the major sterol (56-60% of total sterol) and sitosterol to a lesser extent (8-12%); this sterol profile contrasts with those in higher plants accumulating stigmasterol as a minor component. Recombinant CYP710A13 and CYP710A14 proteins prepared using a baculovirus/insect cell system exhibited the C22-desaturase activity with beta-sitosterol to produce stigmasterol, while campesterol and 24-epi-campesterol were not accepted as the substrates. The K(m) values for beta-sitosterol of CYP710A13 (1.0 +/- 0.043 microM) and CYP710A14 (2.1 +/- 0.17 microM) were at comparable levels of those reported with higher plant CYP710A proteins. In Arabidopsis T87 cells over-expressing CYP710A14, stigmasterol contents reached a level 20- to 72-fold higher than those in the basal level of T87 cells, confirming the C22-desaturase activity of this P450 enzyme. The occurrence of the end-products together with the enzymes involved in the last step of the pathway substantiated the presence of an entire sterol biosynthetic pathway in P. patens, providing evidence for the conservation of the sterol biosynthetic pathway through the evolutionary process of land plants.

  10. The GID1-Mediated Gibberellin Perception Mechanism Is Conserved in the Lycophyte Selaginella moellendorffii but Not in the Bryophyte Physcomitrella patens[W

    PubMed Central

    Hirano, Ko; Nakajima, Masatoshi; Asano, Kenji; Nishiyama, Tomoaki; Sakakibara, Hitoshi; Kojima, Mikiko; Katoh, Etsuko; Xiang, Hongyu; Tanahashi, Takako; Hasebe, Mitsuyasu; Banks, Jo Ann; Ashikari, Motoyuki; Kitano, Hidemi; Ueguchi-Tanaka, Miyako; Matsuoka, Makoto

    2007-01-01

    In rice (Oryza sativa) and Arabidopsis thaliana, gibberellin (GA) signaling is mediated by GIBBERELLIN-INSENSITIVE DWARF1 (GID1) and DELLA proteins in collaboration with a GA-specific F-box protein. To explore when plants evolved the ability to perceive GA by the GID1/DELLA pathway, we examined these GA signaling components in the lycophyte Selaginella moellendorffii and the bryophyte Physcomitrella patens. An in silico search identified several homologs of GID1, DELLA, and GID2, a GA-specific F-box protein in rice, in both species. Sm GID1a and Sm GID1b, GID1 proteins from S. moellendorffii, showed GA binding activity in vitro and interacted with DELLA proteins from S. moellendorffii in a GA-dependent manner in yeast. Introduction of constitutively expressed Sm GID1a, Sm G1D1b, and Sm GID2a transgenes rescued the dwarf phenotype of rice gid1 and gid2 mutants. Furthermore, treatment with GA4, a major GA in S. moellendorffii, caused downregulation of Sm GID1b, Sm GA20 oxidase, and Sm GA3 oxidase and degradation of the Sm DELLA1 protein. These results demonstrate that the homologs of GID1, DELLA, and GID2 work in a similar manner in S. moellendorffii and in flowering plants. Biochemical studies revealed that Sm GID1s have different GA binding properties from GID1s in flowering plants. No evidence was found for the functional conservation of these genes in P. patens, indicating that GID1/DELLA-mediated GA signaling, if present, differs from that in vascular plants. Our results suggest that GID1/DELLA-mediated GA signaling appeared after the divergence of vascular plants from the moss lineage. PMID:17965273

  11. DNA METHYLTRANSFERASE 1 is involved in (m)CG and (m)CCG DNA methylation and is essential for sporophyte development in Physcomitrella patens.

    PubMed

    Yaari, Rafael; Noy-Malka, Chen; Wiedemann, Gertrud; Auerbach Gershovitz, Nitzan; Reski, Ralf; Katz, Aviva; Ohad, Nir

    2015-07-01

    DNA methylation has a crucial role in plant development regulating gene expression and silencing of transposable elements. Maintenance DNA methylation in plants occurs at symmetrical (m)CG and (m)CHG contexts ((m) = methylated) and is maintained by DNA METHYLTRANSFERASE 1 (MET1) and CHROMOMETHYLASE (CMT) DNA methyltransferase protein families, respectively. While angiosperm genomes encode for several members of MET1 and CMT families, the moss Physcomitrella patens, serving as a model for early divergent land plants, carries a single member of each family. To determine the function of P. patens PpMET we generated ΔPpmet deletion mutant which lost (m)CG and unexpectedly (m)CCG methylation at loci tested. In order to evaluate the extent of (m)CCG methylation by MET1, we reexamined the Arabidopsis thaliana Atmet1 mutant methylome and found a similar pattern of methylation loss, suggesting that maintenance of DNA methylation by MET1 is conserved through land plant evolution. While ΔPpmet displayed no phenotypic alterations during its gametophytic phase, it failed to develop sporophytes, indicating that PpMET plays a role in gametogenesis or early sporophyte development. Expression array analysis revealed that the deletion of PpMET resulted in upregulation of two genes and multiple repetitive sequences. In parallel, expression analysis of the previously reported ΔPpcmt mutant showed that lack of PpCMT triggers overexpression of genes. This overexpression combined with loss of (m)CHG and its pleiotropic phenotype, implies that PpCMT has an essential evolutionary conserved role in the epigenetic control of gene expression. Collectively, our results suggest functional conservation of MET1 and CMT families during land plant evolution. A model describing the relationship between MET1 and CMT in CCG methylation is presented.

  12. The Polycomb group protein CLF emerges as a specific tri-methylase of H3K27 regulating gene expression and development in Physcomitrella patens.

    PubMed

    Pereman, Idan; Mosquna, Assaf; Katz, Aviva; Wiedemann, Gertrud; Lang, Daniel; Decker, Eva L; Tamada, Yosuke; Ishikawa, Takaaki; Nishiyama, Tomoaki; Hasebe, Mitsuyasu; Reski, Ralf; Ohad, Nir

    2016-07-01

    Packaging of eukaryotic DNA largely depends on histone modifications that affect the accessibility of DNA to transcriptional regulators, thus controlling gene expression. The Polycomb group (PcG) chromatin remodeling complex deposits a methyl group on lysine 27 of histone 3 leading to repressed gene expression. Plants encode homologs of the Enhancer of zeste (E(z)), a component of the PcG complex from Drosophila, one of which is a SET domain protein designated CURLY LEAF (CLF). Although this SET domain protein exhibits a strong correlation with the presence of the H3K27me3 mark in plants, the methyl-transferase activity and specificity of its SET domain have not been directly tested in-vivo. Using the evolutionary early-diverged land plant model species Physcomitrella patens we show that abolishment of a single copy gene PpCLF, as well as an additional member of the PcG complex, FERTILIZATION-INDEPENDENT ENDOSPERM (PpFIE), results in a specific loss of tri-methylation of H3K27. Using site-directed mutagenesis of key residues, we revealed that H3K27 tri-methylation is mediated by the SET domain of the CLF protein. Moreover, the abolishment of H3K27me3 led to enhanced expression of transcription factor genes. This in turn led to the development of fertilization-independent sporophyte-like structures, as observed in PpCLF and PpFIE null mutants. Overall, our results demonstrate the role of PpCLF as a SET protein in tri-methylation of H3K27 in-vivo and the importance of this modification in regulating the expression of transcription factor genes involved in developmental programs of P. patens.

  13. Genetic Analysis of DEFECTIVE KERNEL1 Loop Function in Three-Dimensional Body Patterning in Physcomitrella patens1[C][W][OPEN

    PubMed Central

    Demko, Viktor; Perroud, Pierre-François; Johansen, Wenche; Delwiche, Charles F.; Cooper, Endymion D.; Remme, Pål; Ako, Ako Eugene; Kugler, Karl G.; Mayer, Klaus F.X.; Quatrano, Ralph; Olsen, Odd-Arne

    2014-01-01

    DEFECTIVE KERNEL1 (DEK1) of higher plants plays an essential role in position-dependent signaling and consists of a large transmembrane domain (MEM) linked to a protease catalytic domain and a regulatory domain. Here, we show that the postulated sensory Loop of the MEM domain plays an important role in the developmental regulation of DEK1 activity in the moss Physcomitrella patens. Compared with P. patens lacking DEK1 (∆dek1), the dek1∆loop mutant correctly positions the division plane in the bud apical cell. In contrast with an early developmental arrest of ∆dek1 buds, dek1∆loop develops aberrant gametophores lacking expanded phyllids resulting from misregulation of mitotic activity. In contrast with the highly conserved sequence of the protease catalytic domain, the Loop is highly variable in land plants. Functionally, the sequence from Marchantia polymorpha fully complements the dek1∆loop phenotype, whereas sequences from maize (Zea mays) and Arabidopsis (Arabidopsis thaliana) give phenotypes with retarded growth and affected phyllid development. Bioinformatic analysis identifies MEM as a member of the Major Facilitator Superfamily, membrane transporters reacting to stimuli from the external environment. Transcriptome analysis comparing wild-type and ∆dek1 tissues identifies an effect on two groups of transcripts connected to dek1 mutant phenotypes: transcripts related to cell wall remodeling and regulation of the AINTEGUMENTA, PLETHORA, and BABY BOOM2 (APB2) and APB3 transcription factors known to regulate bud initiation. Finally, sequence data support the hypothesis that the advanced charophyte algae that evolved into ancestral land plants lost cytosolic calpains, retaining DEK1 as the sole calpain in the evolving land plant lineage. PMID:25185121

  14. Reannotation and extended community resources for the genome of the non-seed plant Physcomitrella patens provide insights into the evolution of plant gene structures and functions

    PubMed Central

    2013-01-01

    Background The moss Physcomitrella patens as a model species provides an important reference for early-diverging lineages of plants and the release of the genome in 2008 opened the doors to genome-wide studies. The usability of a reference genome greatly depends on the quality of the annotation and the availability of centralized community resources. Therefore, in the light of accumulating evidence for missing genes, fragmentary gene structures, false annotations and a low rate of functional annotations on the original release, we decided to improve the moss genome annotation. Results Here, we report the complete moss genome re-annotation (designated V1.6) incorporating the increased transcript availability from a multitude of developmental stages and tissue types. We demonstrate the utility of the improved P. patens genome annotation for comparative genomics and new extensions to the cosmoss.org resource as a central repository for this plant “flagship” genome. The structural annotation of 32,275 protein-coding genes results in 8387 additional loci including 1456 loci with known protein domains or homologs in Plantae. This is the first release to include information on transcript isoforms, suggesting alternative splicing events for at least 10.8% of the loci. Furthermore, this release now also provides information on non-protein-coding loci. Functional annotations were improved regarding quality and coverage, resulting in 58% annotated loci (previously: 41%) that comprise also 7200 additional loci with GO annotations. Access and manual curation of the functional and structural genome annotation is provided via the http://www.cosmoss.org model organism database. Conclusions Comparative analysis of gene structure evolution along the green plant lineage provides novel insights, such as a comparatively high number of loci with 5’-UTR introns in the moss. Comparative analysis of functional annotations reveals expansions of moss house-keeping and metabolic genes

  15. Cytokinins in the bryophyte Physcomitrella patens: analyses of activity, distribution, and cytokinin oxidase/dehydrogenase overexpression reveal the role of extracellular cytokinins.

    PubMed

    von Schwartzenberg, Klaus; Núñez, Marta Fernández; Blaschke, Hanna; Dobrev, Petre I; Novák, Ondrej; Motyka, Václav; Strnad, Miroslav

    2007-11-01

    Ultra-performance liquid chromatography-tandem mass spectrometry was used to establish the cytokinin profile of the bryophyte Physcomitrella patens (Hedw.) B.S.G.; of 40 analyzed cytokinins, 20 were detected. cis-Zeatin-riboside-O-glucoside, N(6)-(Delta(2)-isopentenyl)adenosine-5'-monophosphate (iPRMP), and trans-zeatin-riboside-O-glucoside were the most abundant intracellular cytokinins. In addition, the aromatic cytokinins N(6)-benzyladenosine (BAR), N(6)-benzyladenine, meta-, and ortho-topolin were detected. Unexpectedly, the most abundant extracellular cytokinin was the nucleotide iPRMP, and its identity was confirmed by quadrupole time-of-flight mass spectrometry. The effects of overexpressing a heterologous cytokinin oxidase/dehydrogenase (CKX; EC 1.4.3.18/1.5.99.12) gene (AtCKX2 from Arabidopsis [Arabidopsis thaliana]) on the intracellular and extracellular distribution of cytokinins was assessed. In cultures of CKX-transformed plants, ultra-performance liquid chromatography-tandem mass spectrometry measurements showed that there were pronounced reductions in the extracellular concentrations of N(6)-(Delta(2)-isopentenyl)adenine (iP) and N(6)-(Delta(2)-isopentenyl)adenosine (iPR), but their intracellular cytokinin concentrations were only slightly affected. In vitro and in vivo measured CKX activity was shown to be strongly increased in the transformants. Major phenotypic changes observed in the CKX-overexpressing plants included reduced and retarded budding, absence of sexual reproduction, and abnormal protonema cells. In bud-induction bioassays with wild-type Physcomitrella, the nucleotides iPRMP, trans-zeatin-riboside-5'-monophosphate, BAR monophosphate, and the cis-zeatin forms cZ and cZR had no detectable effects, while the activities displayed by other selected cytokinins were in the following order: iP > tZ > N(6)-benzyladenine > BAR > iPR > tZR > meta-topolin > dihydrozeatin > ortho-topolin. The results on wild type and CKX transgenics suggest that

  16. Physcomitrella patens DCL3 Is Required for 22–24 nt siRNA Accumulation, Suppression of Retrotransposon-Derived Transcripts, and Normal Development

    PubMed Central

    Cho, Sung Hyun; Addo-Quaye, Charles; Coruh, Ceyda; Arif, M. Asif; Ma, Zhaorong; Frank, Wolfgang; Axtell, Michael J.

    2008-01-01

    Endogenous 24 nt short interfering RNAs (siRNAs), derived mostly from intergenic and repetitive genomic regions, constitute a major class of endogenous small RNAs in flowering plants. Accumulation of Arabidopsis thaliana 24 nt siRNAs requires the Dicer family member DCL3, and clear homologs of DCL3 exist in both flowering and non-flowering plants. However, the absence of a conspicuous 24 nt peak in the total RNA populations of several non-flowering plants has raised the question of whether this class of siRNAs might, in contrast to the ancient 21 nt microRNAs (miRNAs) and 21–22 nt trans-acting siRNAs (tasiRNAs), be an angiosperm-specific innovation. Analysis of non-miRNA, non-tasiRNA hotspots of small RNA production within the genome of the moss Physcomitrella patens revealed multiple loci that consistently produced a mixture of 21–24 nt siRNAs with a peak at 23 nt. These Pp23SR loci were significantly enriched in transposon content, depleted in overlap with annotated genes, and typified by dense concentrations of the 5-methyl cytosine (5 mC) DNA modification. Deep sequencing of small RNAs from two independent Ppdcl3 mutants showed that the P. patens DCL3 homolog is required for the accumulation of 22–24 nt siRNAs, but not 21 nt siRNAs, at Pp23SR loci. The 21 nt component of Pp23SR-derived siRNAs was also unaffected by a mutation in the RNA-dependent RNA polymerase mutant Pprdr6. Transcriptome-wide, Ppdcl3 mutants failed to accumulate 22–24 nt small RNAs from repetitive regions while transcripts from two abundant families of long terminal repeat (LTR) retrotransposon-associated reverse transcriptases were up-regulated. Ppdcl3 mutants also displayed an acceleration of leafy gametophore production, suggesting that repetitive siRNAs may play a role in the development of P. patens. We conclude that intergenic/repeat-derived siRNAs are indeed a broadly conserved, distinct class of small regulatory RNAs within land plants. PMID:19096705

  17. Knockout of GH3 genes in the moss Physcomitrella patens leads to increased IAA levels at elevated temperature and in darkness.

    PubMed

    Mittag, Jennifer; Gabrielyan, Anastasia; Ludwig-Müller, Jutta

    2015-12-01

    Two proteins of the GRETCHEN HAGEN3 (GH3) family of acyl acid amido synthetases from the moss Physcomitrella patens conjugate indole-3-acetic acid (IAA) to a series of amino acids. The possible function of altered auxin levels in the moss in response to two different growth perturbations, elevated temperatures and darkness, was analyzed using a) the recently described double knockout lines in both P. patens GH3 genes (GH3-doKO) and b) a previously characterized line harboring an auxin-inducible soybean GH3 promoter::reporter fused to β-glucuronidase (G1-GUS). The GUS activity as marker of the auxin response increased at higher temperatures and after cultivation in the darkness for a period of up to four weeks. Generally, the double knockout plants grew more slowly than the wild type (WT). The altered growth conditions influenced the phenotypes of the double knockout lines differently from that of WT moss. Higher temperatures negatively affected GH3-doKO plants compared to WT which was shown by stronger loss of chlorophyll. On the other hand, a positive effect was found on the concentrations of free IAA which increased at 28 °C in the GH3-doKO lines compared to WT plants. A different factor, namely darkness vs. a light/dark cycle caused the adverse phenotype concerning chlorophyll concentrations. Mutant moss plants showed higher chlorophyll concentrations than WT and these correlated with higher free IAA in the plant population that was classified as green. Our data show that growth perturbations result in higher free IAA levels in the GH3-doKO mutants, but in one case - growth in darkness - the mutants could cope better with the condition, whereas at elevated temperatures the mutants were more sensitive than WT. Thus, GH3 function in P. patens WT could lie in the regulation of IAA concentrations under unfavorable environmental conditions. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  18. Recovery from heat, salt and osmotic stress in Physcomitrella patens requires a functional small heat shock protein PpHsp16.4

    PubMed Central

    2013-01-01

    Background Plant small heat shock proteins (sHsps) accumulate in response to various environmental stresses, including heat, drought, salt and oxidative stress. Numerous studies suggest a role for these proteins in stress tolerance by preventing stress-induced protein aggregation as well as by facilitating protein refolding by other chaperones. However, in vivo evidence for the involvement of sHsps in tolerance to different stress factors is still missing, mainly due to the lack of appropriate mutants in specific sHsp genes. Results In this study we characterized the function of a sHsp in abiotic stress tolerance in the moss Physcomitrella patens, a model for primitive land plants. Using suppression subtractive hybridization, we isolated an abscisic acid-upregulated gene from P. patens encoding a 16.4 kDa cytosolic class II sHsp. PpHsp16.4 was also induced by salicylic acid, dithiothreitol (DTT) and by exposure to various stimuli, including osmotic and salt stress, but not by oxidative stress-inducing compounds. Expression of the gene was maintained upon stress relief, suggesting a role for this protein in the recovery stage. PpHsp16.4 is encoded by two identical genes arranged in tandem in the genome. Targeted disruption of both genes resulted in the inability of plants to recover from heat, salt and osmotic stress. In vivo localization studies revealed that PpHsp16.4 localized in cytosolic granules in the vicinity of chloroplasts under non stress conditions, suggesting possible distinct roles for this protein under stress and optimal growth. Conclusions We identified a member of the class II sHsp family that showed hormonal and abiotic stress gene regulation. Induction of the gene by DTT treatment suggests that damaged proteins may act as signals for the stress-induction of PpHsp16.4. The product of this gene was shown to localize in cytosolic granules near the chloroplasts, suggesting a role for the protein in association with these organelles. Our study

  19. An ATP Binding Cassette Transporter Is Required for Cuticular Wax Deposition and Desiccation Tolerance in the Moss Physcomitrella patens[W

    PubMed Central

    Buda, Gregory J.; Barnes, William J.; Fich, Eric A.; Park, Sungjin; Yeats, Trevor H.; Zhao, Lingxia; Domozych, David S.; Rose, Jocelyn K.C.

    2013-01-01

    The plant cuticle is thought to be a critical evolutionary adaptation that allowed the first plants to colonize land, because of its key roles in regulating plant water status and providing protection from biotic and abiotic stresses. Much has been learned about cuticle composition and structure through genetic and biochemical studies of angiosperms, as well as underlying genetic pathways, but little is known about the cuticles of early diverging plant lineages. Here, we demonstrate that the moss Physcomitrella patens, an extant relative of the earliest terrestrial plants, has a cuticle that is analogous in both structure and chemical composition to those of angiosperms. To test whether the underlying cuticle biosynthetic pathways were also shared among distant plant lineages, we generated a genetic knockout of the moss ATP binding cassette subfamily G (ABCG) transporter Pp-ABCG7, a putative ortholog of Arabidopsis thaliana ABCG transporters involved in cuticle precursor trafficking. We show that this mutant is severely deficient in cuticular wax accumulation and has a reduced tolerance of desiccation stress compared with the wild type. This work provides evidence that the cuticle was an adaptive feature present in the first terrestrial plants and that the genes involved in their formation have been functionally conserved for over 450 million years. PMID:24163310

  20. Homologues of the Arabidopsis thaliana SHI/STY/LRP1 genes control auxin biosynthesis and affect growth and development in the moss Physcomitrella patens.

    PubMed

    Eklund, D Magnus; Thelander, Mattias; Landberg, Katarina; Ståldal, Veronika; Nilsson, Anders; Johansson, Monika; Valsecchi, Isabel; Pederson, Eric R A; Kowalczyk, Mariusz; Ljung, Karin; Ronne, Hans; Sundberg, Eva

    2010-04-01

    The plant hormone auxin plays fundamental roles in vascular plants. Although exogenous auxin also stimulates developmental transitions and growth in non-vascular plants, the effects of manipulating endogenous auxin levels have thus far not been reported. Here, we have altered the levels and sites of auxin production and accumulation in the moss Physcomitrella patens by changing the expression level of homologues of the Arabidopsis SHI/STY family proteins, which are positive regulators of auxin biosynthesis genes. Constitutive expression of PpSHI1 resulted in elevated auxin levels, increased and ectopic expression of the auxin response reporter GmGH3pro:GUS, and in an increased caulonema/chloronema ratio, an effect also induced by exogenous auxin application. In addition, we observed premature ageing and necrosis in cells ectopically expressing PpSHI1. Knockout of either of the two PpSHI genes resulted in reduced auxin levels and auxin biosynthesis rates in leafy shoots, reduced internode elongation, delayed ageing, a decreased caulonema/chloronema ratio and an increased number of axillary hairs, which constitute potential auxin biosynthesis sites. Some of the identified auxin functions appear to be analogous in vascular and non-vascular plants. Furthermore, the spatiotemporal expression of the PpSHI genes and GmGH3pro:GUS strongly overlap, suggesting that local auxin biosynthesis is important for the regulation of auxin peak formation in non-vascular plants.

  1. Auxin promotes the transition from chloronema to caulonema in moss protonema by positively regulating PpRSL1and PpRSL2 in Physcomitrella patens.

    PubMed

    Jang, Geupil; Dolan, Liam

    2011-10-01

    Protonemata are multicellular filamentous networks that develop following the germination of a haploid moss spore and comprise two different cell types - chloronema and caulonema. The ROOT HAIR DEFECTIVE SIX-LIKE1 (PpRSL1) and PpRSL2 basic helix-loop-helix transcription factors and auxin promote the development of caulonema in Physcomitrella patens but the mechanism by which these regulators interact during development is unknown. We characterized the role of auxin in regulating the function of PpRSL1 and PpRSL2 in the chloronema-to-caulonema transition during protonema development. Here, we showed that a gradient of cell identity developed along protonemal filaments; cells were chloronemal in proximal regions near the site of spore germination becoming progressively more caulonemal distally as filaments elongated. Auxin controlled this transition by positively regulating the expression of PpRSL1 and PpRSL2 genes. Auxin did not induce caulonemal development in Pprsl1 Pprsl2 double mutants that lack PpRSL1 and PpRSL2 gene activity while constitutive co-expression of PpRSL1 and PpRSL2 in the absence of auxin was sufficient to program constitutive caulonema development. Together, these data indicate that auxin positively regulates PpRSL1 and PpRSL2 whose expression is sufficient to promote caulonema differentiation in moss protonema.

  2. Immunofluorescence microscopy of microtubules in intact cell lineages of the moss, Physcomitrella patens. I. Normal and CIPC-treated tip cells.

    PubMed

    Doonan, J H; Cove, D J; Lloyd, C W

    1985-04-01

    Monoclonal antibodies to yeast tubulin have been used to visualize the distribution of microtubules in the intact filamentous protonemata of the moss Physcomitrella patens. Protonemata were prepared for immunofluorescence by fixation in formaldehyde and cells were made permeable with Driselase. Extensive cell files were preserved by 'blotting' the moss onto glutaraldehyde-derivatized coverslips. Problems due to fluorescence from chloroplasts were obviated by extraction with dimethyl sulphoxide and the non-ionic detergent, Nonidet NP40. These improvements allowed us to determine that microtubules were present throughout the cell cycle in the apical dome of caulonemal tip cells, that was a pronounced association of microtubules with the nucleus, that 'astral' microtubules were associated with the mitotic spindle and during anaphase may be involved in reorientation of the spindle before an oblique cytokinesis in caulonemata and that the cytokinetic phragmoplast appeared identical to the structure described for higher plants. Microtubules appeared to converge at the very tip of apical caulonemal cells and this was studied further by treating cells with CIPC--a drug that is known to produce multiple microtubule-organizing centres--and which here produces multiple foci for microtubules at the tip. These observations emphasize the involvement of microtubules in tip growth, alignment of the cell plate and nuclear migration--processes that are fundamental to the morphogenesis of filamentous organisms.

  3. Genetic analysis of Physcomitrella patens identifies ABSCISIC ACID NON-RESPONSIVE, a regulator of ABA responses unique to basal land plants and required for desiccation tolerance

    DOE PAGES

    Stevenson, Sean Ross; Kamisugi, Yasuko; Trinh, Chi H.; ...

    2016-05-18

    The anatomically simple plants that first colonized land must have acquired molecular and biochemical adaptations to drought stress. Abscisic acid (ABA) coordinates responses leading to desiccation tolerance in all land plants. We identified ABA nonresponsive mutants in the model bryophyte Physcomitrella patens and genotyped a segregating population to map and identify the ABA NON-RESPONSIVE (ANR) gene encoding a modular protein kinase comprising an N-terminal PAS domain, a central EDR domain, and a C-terminal MAPKKK-like domain. anr mutants fail to accumulate dehydration tolerance-associated gene products in response to drought, ABA, or osmotic stress and do not acquire ABA-dependent desiccation tolerance. Themore » crystal structure of the PAS domain, determined to 1.7-Å resolution, shows a conserved PAS-fold that dimerizes through a weak dimerization interface. Targeted mutagenesis of a conserved tryptophan residue within the PAS domain generates plants with ABA nonresponsive growth and strongly attenuated ABA-responsive gene expression, whereas deleting this domain retains a fully ABA-responsive phenotype. ANR orthologs are found in early-diverging land plant lineages and aquatic algae but are absent from more recently diverged vascular plants. Lastly, we propose that ANR genes represent an ancestral adaptation that enabled drought stress survival of the first terrestrial colonizers but were lost during land plant evolution.« less

  4. Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens.

    PubMed

    Decker, Eva L; Alder, Adrian; Hunn, Stefan; Ferguson, Jenny; Lehtonen, Mikko T; Scheler, Bjoern; Kerres, Klaus L; Wiedemann, Gertrud; Safavi-Rizi, Vajiheh; Nordzieke, Steffen; Balakrishna, Aparna; Baz, Lina; Avalos, Javier; Valkonen, Jari P T; Reski, Ralf; Al-Babili, Salim

    2017-03-06

    In seed plants, strigolactones (SLs) regulate architecture and induce mycorrhizal symbiosis in response to environmental cues. SLs are formed by combined activity of the carotenoid cleavage dioxygenases (CCDs) 7 and 8 from 9-cis-β-carotene, leading to carlactone that is converted by cytochromes P450 (clade 711; MAX1 in Arabidopsis) into various SLs. As Physcomitrella patens possesses CCD7 and CCD8 homologs but lacks MAX1, we investigated if PpCCD7 together with PpCCD8 form carlactone and how deletion of these enzymes influences growth and interactions with the environment. We investigated the enzymatic activity of PpCCD7 and PpCCD8 in vitro, identified the formed products by high performance liquid chromatography (HPLC) and LC-MS, and generated and analysed ΔCCD7 and ΔCCD8 mutants. We defined enzymatic activity of PpCCD7 as a stereospecific 9-cis-CCD and PpCCD8 as a carlactone synthase. ΔCCD7 and ΔCCD8 lines showed enhanced caulonema growth, which was revertible by adding the SL analogue GR24 or carlactone. Wild-type (WT) exudates induced seed germination in Orobanche ramosa. This activity was increased upon phosphate starvation and abolished in exudates of both mutants. Furthermore, both mutants showed increased susceptibility to phytopathogenic fungi. Our study reveals the deep evolutionary conservation of SL biosynthesis, SL function, and its regulation by biotic and abiotic cues.

  5. MICROTUBULE-ASSOCIATED PROTEIN65 is essential for maintenance of phragmoplast bipolarity and formation of the cell plate in Physcomitrella patens.

    PubMed

    Kosetsu, Ken; de Keijzer, Jeroen; Janson, Marcel E; Goshima, Gohta

    2013-11-01

    The phragmoplast, a plant-specific apparatus that mediates cytokinesis, mainly consists of microtubules (MTs) arranged in a bipolar fashion, such that their plus ends interdigitate at the equator. Membrane vesicles are thought to move along the MTs toward the equator and fuse to form the cell plate. Although several genes required for phragmoplast MT organization have been identified, the mechanisms that maintain the bipolarity of phragmoplasts remain poorly understood. Here, we show that engaging phragmoplast MTs in a bipolar fashion in protonemal cells of the moss Physcomitrella patens requires the conserved MT cross-linking protein MICROTUBULE-ASSOCIATED PROTEIN65 (MAP65). Simultaneous knockdown of the three MAP65s expressed in those cells severely compromised MT interdigitation at the phragmoplast equator after anaphase onset, resulting in the collapse of the phragmoplast in telophase. Cytokinetic vesicles initially localized to the anaphase midzone as normal but failed to further accumulate in the next several minutes, although the bipolarity of the MT array was preserved. Our data indicate that the presence of bipolar MT arrays is insufficient for vesicle accumulation at the equator and further suggest that MAP65-mediated MT interdigitation is a prerequisite for maintenance of bipolarity of the phragmoplast and accumulation and/or fusion of cell plate-destined vesicles at the equatorial plane.

  6. The moss Physcomitrella patens contains cyclopentenones but no jasmonates: mutations in allene oxide cyclase lead to reduced fertility and altered sporophyte morphology.

    PubMed

    Stumpe, Michael; Göbel, Cornelia; Faltin, Bernd; Beike, Anna K; Hause, Bettina; Himmelsbach, Kiyoshi; Bode, Julia; Kramell, Robert; Wasternack, Claus; Frank, Wolfgang; Reski, Ralf; Feussner, Ivo

    2010-11-01

    • Two cDNAs encoding allene oxide cyclases (PpAOC1, PpAOC2), key enzymes in the formation of jasmonic acid (JA) and its precursor (9S,13S)-12-oxo-phytodienoic acid (cis-(+)-OPDA), were isolated from the moss Physcomitrella patens. • Recombinant PpAOC1 and PpAOC2 show substrate specificity against the allene oxide derived from 13-hydroperoxy linolenic acid (13-HPOTE); PpAOC2 also shows substrate specificity against the allene oxide derived from 12-hydroperoxy arachidonic acid (12-HPETE). • In protonema and gametophores the occurrence of cis-(+)-OPDA, but neither JA nor the isoleucine conjugate of JA nor that of cis-(+)-OPDA was detected. • Targeted knockout mutants for PpAOC1 and for PpAOC2 were generated, while double mutants could not be obtained. The ΔPpAOC1 and ΔPpAOC2 mutants showed reduced fertility, aberrant sporophyte morphology and interrupted sporogenesis.

  7. Cloning and functional characterisation of an enzyme involved in the elongation of Delta6-polyunsaturated fatty acids from the moss Physcomitrella patens.

    PubMed

    Zank, Thorsten K; Zähringer, Ulrich; Beckmann, Christoph; Pohnert, Georg; Boland, Wilhelm; Holtorf, Hauke; Reski, Ralf; Lerchl, Jens; Heinz, Ernst

    2002-08-01

    The moss Physcomitrella patens contains high proportions of polyunsaturated very-long-chain fatty acids with up to 20 carbon atoms. Starting from preformed C18 polyunsaturated fatty acids, their biosynthesis involves a sequence of Delta6-desaturation, Delta6-elongation and Delta5-desaturation. In this report we describe for the first time the characterisation of a cDNA (PSE1) of plant origin with homology to the ELO-genes from Saccharomyces cerevisiae, encoding a component of the Delta6-elongase. Functional expression of PSE1 in S. cerevisiae led to the elongation of exogenously supplied Delta6-polyunsaturated fatty acids. By feeding experiments with different trienoic fatty acids of natural and synthetic origin, both substrate specificity and substrate selectivity of the enzyme were investigated. The activity of Pse1, when expressed in yeast, was not sensitive to the antibiotic cerulenin, which is an effective inhibitor of fatty acid synthesis and elongation. Furthermore, the PSE1 gene was disrupted in the moss by homologous recombination. This led to a complete loss of all C20 polyunsaturated fatty acids providing additional evidence for the function of the cDNA as coding for a component of the Delta6-elongase. The elimination of the elongase was not accompanied by a visible alteration in the phenotype, indicating that C20-PUFAs are not essential for viability of the moss under phytotron conditions.

  8. ATP requirement for chloroplast protein import is set by the Km for ATP hydrolysis of stromal Hsp70 in Physcomitrella patens.

    PubMed

    Liu, Li; McNeilage, Robert T; Shi, Lan-Xin; Theg, Steven M

    2014-03-01

    The 70-kD family of heat shock proteins (Hsp70s) is involved in a number of seemingly disparate cellular functions, including folding of nascent proteins, breakup of misfolded protein aggregates, and translocation of proteins across membranes. They act through the binding and release of substrate proteins, accompanied by hydrolysis of ATP. Chloroplast stromal Hsp70 plays a crucial role in the import of proteins into plastids. Mutations of an ATP binding domain Thr were previously reported to result in an increase in the Km for ATP and a decrease in the enzyme's kcat. To ask which chloroplast stromal chaperone, Hsp70 or Hsp93, both of which are ATPases, dominates the energetics of the motor responsible for protein import, we made transgenic moss (Physcomitrella patens) harboring the Km-altering mutation in the essential stromal Hsp70-2 and measured the effect on the amount of ATP required for protein import into chloroplasts. Here, we report that increasing the Km for ATP hydrolysis of Hsp70 translated into an increased Km for ATP usage by chloroplasts for protein import. This thus directly demonstrates that the ATP-derived energy long known to be required for chloroplast protein import is delivered via the Hsp70 chaperones and that the chaperone's ATPase activity dominates the energetics of the reaction.

  9. CRISPR-Cas9-mediated efficient directed mutagenesis and RAD51-dependent and RAD51-independent gene targeting in the moss Physcomitrella patens.

    PubMed

    Collonnier, Cécile; Epert, Aline; Mara, Kostlend; Maclot, François; Guyon-Debast, Anouchka; Charlot, Florence; White, Charles; Schaefer, Didier G; Nogué, Fabien

    2017-01-01

    The ability to address the CRISPR-Cas9 nuclease complex to any target DNA using customizable single-guide RNAs has now permitted genome engineering in many species. Here, we report its first successful use in a nonvascular plant, the moss Physcomitrella patens. Single-guide RNAs (sgRNAs) were designed to target an endogenous reporter gene, PpAPT, whose inactivation confers resistance to 2-fluoroadenine. Transformation of moss protoplasts with these sgRNAs and the Cas9 coding sequence from Streptococcus pyogenes triggered mutagenesis at the PpAPT target in about 2% of the regenerated plants. Mainly, deletions were observed, most of them resulting from alternative end-joining (alt-EJ)-driven repair. We further demonstrate that, in the presence of a donor DNA sharing sequence homology with the PpAPT gene, most transgene integration events occur by homology-driven repair (HDR) at the target locus but also that Cas9-induced double-strand breaks are repaired with almost equal frequencies by mutagenic illegitimate recombination. Finally, we establish that a significant fraction of HDR-mediated gene targeting events (30%) is still possible in the absence of PpRAD51 protein, indicating that CRISPR-induced HDR is only partially mediated by the classical homologous recombination pathway. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  10. Genetic Analysis of Physcomitrella patens Identifies ABSCISIC ACID NON-RESPONSIVE, a Regulator of ABA Responses Unique to Basal Land Plants and Required for Desiccation Tolerance.

    PubMed

    Stevenson, Sean R; Kamisugi, Yasuko; Trinh, Chi H; Schmutz, Jeremy; Jenkins, Jerry W; Grimwood, Jane; Muchero, Wellington; Tuskan, Gerald A; Rensing, Stefan A; Lang, Daniel; Reski, Ralf; Melkonian, Michael; Rothfels, Carl J; Li, Fay-Wei; Larsson, Anders; Wong, Gane K-S; Edwards, Thomas A; Cuming, Andrew C

    2016-06-01

    The anatomically simple plants that first colonized land must have acquired molecular and biochemical adaptations to drought stress. Abscisic acid (ABA) coordinates responses leading to desiccation tolerance in all land plants. We identified ABA nonresponsive mutants in the model bryophyte Physcomitrella patens and genotyped a segregating population to map and identify the ABA NON-RESPONSIVE (ANR) gene encoding a modular protein kinase comprising an N-terminal PAS domain, a central EDR domain, and a C-terminal MAPKKK-like domain. anr mutants fail to accumulate dehydration tolerance-associated gene products in response to drought, ABA, or osmotic stress and do not acquire ABA-dependent desiccation tolerance. The crystal structure of the PAS domain, determined to 1.7-Å resolution, shows a conserved PAS-fold that dimerizes through a weak dimerization interface. Targeted mutagenesis of a conserved tryptophan residue within the PAS domain generates plants with ABA nonresponsive growth and strongly attenuated ABA-responsive gene expression, whereas deleting this domain retains a fully ABA-responsive phenotype. ANR orthologs are found in early-diverging land plant lineages and aquatic algae but are absent from more recently diverged vascular plants. We propose that ANR genes represent an ancestral adaptation that enabled drought stress survival of the first terrestrial colonizers but were lost during land plant evolution.

  11. Involvement of PpDof1 transcriptional repressor in the nutrient condition-dependent growth control of protonemal filaments in Physcomitrella patens

    PubMed Central

    Sugiyama, Takumi; Ishida, Tetsuya; Tabei, Nobumitsu; Shigyo, Mikao; Konishi, Mineko; Yoneyama, Tadakatsu; Yanagisawa, Shuichi

    2012-01-01

    In higher plants, the Dof transcription factors that harbour a conserved plant-specific DNA-binding domain function in the regulation of diverse biological processes that are unique to plants. Although these factors are present in both higher and lower plants, they have not yet been characterized in lower plants. Here six genes encoding Dof transcription factors in the moss Physcomitrella patens are characterized and two of these genes, PpDof1 and PpDof2, are functionally analysed. The targeted disruption of PpDof1 caused delayed or reduced gametophore formation, accompanied by an effect on development of the caulonema from the chloronema. Furthermore, the ppdof1 disruptants were found to form smaller colonies with a reduced frequency of branching of protonemal filaments, depending on the nutrients in the media. Most of these phenotypes were not apparent in the ppdof2 disruptant, although the ppdof2 disruptants also formed smaller colonies on a particular medium. Transcriptional repressor activity of PpDof1 and PpDof2 and modified expression of a number of genes in the ppdof disruptant lines were also shown. These results thus suggest that the PpDof1 transcriptional repressor has a role in controlling nutrient-dependent filament growth. PMID:22345635

  12. Physcomitrella patens activates reinforcement of the cell wall, programmed cell death and accumulation of evolutionary conserved defence signals, such as salicylic acid and 12-oxo-phytodienoic acid, but not jasmonic acid, upon Botrytis cinerea infection.

    PubMed

    Ponce De León, Inés; Schmelz, Eric A; Gaggero, Carina; Castro, Alexandra; Álvarez, Alfonso; Montesano, Marcos

    2012-10-01

    The moss Physcomitrella patens is an evolutionarily basal model system suitable for the analysis of plant defence responses activated after pathogen assault. Upon infection with the necrotroph Botrytis cinerea, several defence mechanisms are induced in P. patens, including the fortification of the plant cell wall by the incorporation of phenolic compounds and the induced expression of related genes. Botrytis cinerea infection also activates the accumulation of reactive oxygen species and cell death with hallmarks of programmed cell death in moss tissues. Salicylic acid (SA) levels also increase after fungal infection, and treatment with SA enhances transcript accumulation of the defence gene phenylalanine ammonia-lyase (PAL) in P. patens colonies. The expression levels of the genes involved in 12-oxo-phytodienoic acid (OPDA) synthesis, including lipoxygenase (LOX) and allene oxide synthase (AOS), increase in P. patens gametophytes after pathogen assault, together with a rise in free linolenic acid and OPDA concentrations. However, jasmonic acid (JA) could not be detected in healthy or infected tissues of this plant. Our results suggest that, although conserved defence signals, such as SA and OPDA, are synthesized and are probably involved in the defence response of P. patens against B. cinerea infection, JA production appears to be missing. Interestingly, P. patens responds to OPDA and methyl jasmonate by reducing moss colony growth and rhizoid length, suggesting that jasmonate perception is present in mosses. Thus, P. patens can provide clues with regard to the evolution of different defence pathways in plants, including signalling and perception of OPDA and jasmonates in nonflowering and flowering plants.

  13. Comparison of the chloroplast peroxidase system in the chlorophyte Chlamydomonas reinhardtii, the bryophyte Physcomitrella patens, the lycophyte Selaginella moellendorffii and the seed plant Arabidopsis thaliana

    PubMed Central

    2010-01-01

    Background Oxygenic photosynthesis is accompanied by the formation of reactive oxygen species (ROS), which damage proteins, lipids, DNA and finally limit plant yield. The enzymes of the chloroplast antioxidant system are exclusively nuclear encoded. During evolution, plastid and mitochondrial genes were post-endosymbiotically transferred to the nucleus, adapted for eukaryotic gene expression and post-translational protein targeting and supplemented with genes of eukaryotic origin. Results Here, the genomes of the green alga Chlamydomonas reinhardtii, the moss Physcomitrella patens, the lycophyte Selaginella moellendorffii and the seed plant Arabidopsis thaliana were screened for ORFs encoding chloroplast peroxidases. The identified genes were compared for their amino acid sequence similarities and gene structures. Stromal and thylakoid-bound ascorbate peroxidases (APx) share common splice sites demonstrating that they evolved from a common ancestral gene. In contrast to most cormophytes, our results predict that chloroplast APx activity is restricted to the stroma in Chlamydomonas and to thylakoids in Physcomitrella. The moss gene is of retrotransposonal origin. The exon-intron-structures of 2CP genes differ between chlorophytes and streptophytes indicating an independent evolution. According to amino acid sequence characteristics only the A-isoform of Chlamydomonas 2CP may be functionally equivalent to streptophyte 2CP, while the weakly expressed B- and C-isoforms show chlorophyte specific surfaces and amino acid sequence characteristics. The amino acid sequences of chloroplast PrxII are widely conserved between the investigated species. In the analyzed streptophytes, the genes are unspliced, but accumulated four introns in Chlamydomonas. A conserved splice site indicates also a common origin of chlorobiont PrxQ. The similarity of splice sites also demonstrates that streptophyte glutathione peroxidases (GPx) are of common origin. Besides a less related cysteine

  14. Comparison of the chloroplast peroxidase system in the chlorophyte Chlamydomonas reinhardtii, the bryophyte Physcomitrella patens, the lycophyte Selaginella moellendorffii and the seed plant Arabidopsis thaliana.

    PubMed

    Pitsch, Nicola T; Witsch, Benjamin; Baier, Margarete

    2010-06-28

    Oxygenic photosynthesis is accompanied by the formation of reactive oxygen species (ROS), which damage proteins, lipids, DNA and finally limit plant yield. The enzymes of the chloroplast antioxidant system are exclusively nuclear encoded. During evolution, plastid and mitochondrial genes were post-endosymbiotically transferred to the nucleus, adapted for eukaryotic gene expression and post-translational protein targeting and supplemented with genes of eukaryotic origin. Here, the genomes of the green alga Chlamydomonas reinhardtii, the moss Physcomitrella patens, the lycophyte Selaginella moellendorffii and the seed plant Arabidopsis thaliana were screened for ORFs encoding chloroplast peroxidases. The identified genes were compared for their amino acid sequence similarities and gene structures. Stromal and thylakoid-bound ascorbate peroxidases (APx) share common splice sites demonstrating that they evolved from a common ancestral gene. In contrast to most cormophytes, our results predict that chloroplast APx activity is restricted to the stroma in Chlamydomonas and to thylakoids in Physcomitrella. The moss gene is of retrotransposonal origin.The exon-intron-structures of 2CP genes differ between chlorophytes and streptophytes indicating an independent evolution. According to amino acid sequence characteristics only the A-isoform of Chlamydomonas 2CP may be functionally equivalent to streptophyte 2CP, while the weakly expressed B- and C-isoforms show chlorophyte specific surfaces and amino acid sequence characteristics. The amino acid sequences of chloroplast PrxII are widely conserved between the investigated species. In the analyzed streptophytes, the genes are unspliced, but accumulated four introns in Chlamydomonas. A conserved splice site indicates also a common origin of chlorobiont PrxQ.The similarity of splice sites also demonstrates that streptophyte glutathione peroxidases (GPx) are of common origin. Besides a less related cysteine-type GPx, Chlamydomonas

  15. Large-scale proteome analysis of abscisic acid and ABSCISIC ACID INSENSITIVE3-dependent proteins related to desiccation tolerance in Physcomitrella patens.

    PubMed

    Yotsui, Izumi; Serada, Satoshi; Naka, Tetsuji; Saruhashi, Masashi; Taji, Teruaki; Hayashi, Takahisa; Quatrano, Ralph S; Sakata, Yoichi

    2016-03-18

    Desiccation tolerance is an ancestral feature of land plants and is still retained in non-vascular plants such as bryophytes and some vascular plants. However, except for seeds and spores, this trait is absent in vegetative tissues of vascular plants. Although many studies have focused on understanding the molecular basis underlying desiccation tolerance using transcriptome and proteome approaches, the critical molecular differences between desiccation tolerant plants and non-desiccation plants are still not clear. The moss Physcomitrella patens cannot survive rapid desiccation under laboratory conditions, but if cells of the protonemata are treated by the phytohormone abscisic acid (ABA) prior to desiccation, it can survive 24 h exposure to desiccation and regrow after rehydration. The desiccation tolerance induced by ABA (AiDT) is specific to this hormone, but also depends on a plant transcription factor ABSCISIC ACID INSENSITIVE3 (ABI3). Here we report the comparative proteomic analysis of AiDT between wild type and ABI3 deleted mutant (Δabi3) of P. patens using iTRAQ (Isobaric Tags for Relative and Absolute Quantification). From a total of 1980 unique proteins that we identified, only 16 proteins are significantly altered in Δabi3 compared to wild type after desiccation following ABA treatment. Among this group, three of the four proteins that were severely affected in Δabi3 tissue were Arabidopsis orthologous genes, which were expressed in maturing seeds under the regulation of ABI3. These included a Group 1 late embryogenesis abundant (LEA) protein, a short-chain dehydrogenase, and a desiccation-related protein. Our results suggest that at least three of these proteins expressed in desiccation tolerant cells of both Arabidopsis and the moss are very likely to play important roles in acquisition of desiccation tolerance in land plants. Furthermore, our results suggest that the regulatory machinery of ABA- and ABI3-mediated gene expression for desiccation

  16. Large-scale proteome analysis of abscisic acid and ABSCISIC ACID INSENSITIVE3-dependent proteins related to desiccation tolerance in Physcomitrella patens

    SciTech Connect

    Yotsui, Izumi; Serada, Satoshi; Naka, Tetsuji; Saruhashi, Masashi; Taji, Teruaki; Hayashi, Takahisa; Quatrano, Ralph S.; Sakata, Yoichi

    2016-03-18

    Desiccation tolerance is an ancestral feature of land plants and is still retained in non-vascular plants such as bryophytes and some vascular plants. However, except for seeds and spores, this trait is absent in vegetative tissues of vascular plants. Although many studies have focused on understanding the molecular basis underlying desiccation tolerance using transcriptome and proteome approaches, the critical molecular differences between desiccation tolerant plants and non-desiccation plants are still not clear. The moss Physcomitrella patens cannot survive rapid desiccation under laboratory conditions, but if cells of the protonemata are treated by the phytohormone abscisic acid (ABA) prior to desiccation, it can survive 24 h exposure to desiccation and regrow after rehydration. The desiccation tolerance induced by ABA (AiDT) is specific to this hormone, but also depends on a plant transcription factor ABSCISIC ACID INSENSITIVE3 (ABI3). Here we report the comparative proteomic analysis of AiDT between wild type and ABI3 deleted mutant (Δabi3) of P. patens using iTRAQ (Isobaric Tags for Relative and Absolute Quantification). From a total of 1980 unique proteins that we identified, only 16 proteins are significantly altered in Δabi3 compared to wild type after desiccation following ABA treatment. Among this group, three of the four proteins that were severely affected in Δabi3 tissue were Arabidopsis orthologous genes, which were expressed in maturing seeds under the regulation of ABI3. These included a Group 1 late embryogenesis abundant (LEA) protein, a short-chain dehydrogenase, and a desiccation-related protein. Our results suggest that at least three of these proteins expressed in desiccation tolerant cells of both Arabidopsis and the moss are very likely to play important roles in acquisition of desiccation tolerance in land plants. Furthermore, our results suggest that the regulatory machinery of ABA- and ABI3-mediated gene expression for desiccation

  17. Identification of the single amino acid involved in quenching the ent-kauranyl cation by a water molecule in ent-kaurene synthase of Physcomitrella patens.

    PubMed

    Kawaide, Hiroshi; Hayashi, Ken-ichiro; Kawanabe, Ryo; Sakigi, Yuka; Matsuo, Akihiko; Natsume, Masahiro; Nozaki, Hiroshi

    2011-01-01

    ent-Kaurene is a tetracyclic diterpene hydrocarbon and a biosynthetic intermediate of the plant hormone gibberellins. In flowering plants, ent-kaurene is biosynthesized from geranylgeranyl diphosphate (GGDP) by two distinct cyclases, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). Recently, the moss Physcomitrella patens ent-kaurene biosynthetic gene was cloned and functionally characterized. The bifunctional ent-kaurene synthase [P. patens CPS/KS (PpCPS/KS)] produces both ent-kaurene and 16α-hydroxy-ent-kaurane from GGDP via ent-copalyl diphosphate. Here, we cloned and analyzed the function of a cDNA encoding bifunctional ent-kaurene synthase from the liverwort Jungermannia subulata [J. subulata CPS/KS (JsCPS/KS)]. JsCPS/KS catalyzes the cyclization reaction of GGDP to produce ent-kaurene but not 16α-hydroxy-ent-kaurane, even though the PpCPS/KS (881 amino acids) and JsCPS/KS (886 amino acids) sequences share 60% identity. To determine the regions and amino acids involved in 16α-hydroxy-ent-kaurane formation, we analyzed the enzymic functions of JsCPS/KS and PpCPS/KS chimeric proteins. When the C-terminal region of PpCPS/KS was exchanged with the JsCPS/KS C-terminal region, the chimeric cyclases produced only ent-kaurene. The replacement of PpCPS/KS Ala710 with Met or Phe produced a JsCPS/KS-type cyclase that converted GGDP to ent-kaurene as the sole product. In contrast, replacing Ala710 with Gly, Cys or Ser did not affect the PpCPS/KS product profile as much as replacement of Cys of JsCPS/KS by Ala. Thus, the hydrophobicity and size of the side chain residue at the PpCPS/KS amino acid 710 is responsible for quenching the ent-kauranyl cation by the addition of a water molecule.

  18. Pseudo-response regulator (PRR) homologues of the moss Physcomitrella patens: insights into the evolution of the PRR family in land plants.

    PubMed

    Satbhai, Santosh B; Yamashino, Takafumi; Okada, Ryo; Nomoto, Yuji; Mizuno, Takeshi; Tezuka, Yuki; Itoh, Tomonori; Tomita, Mitsuru; Otsuki, Susumu; Aoki, Setsuyuki

    2011-02-01

    The pseudo-response regulators (PRRs) are the circadian clock component proteins in the model dicot Arabidopsis thaliana. They contain a receiver-like domain (RLD) similar to the receiver domains of the RRs in the His-Asp phosphorelay system, but the RLDs lack the phosphoacceptor aspartic acid residue invariably conserved in the receiver domains. To study the evolution of PRR genes in plants, here we characterize their homologue genes, PpPRR1, PpPRR2, PpPRR3 and PpPRR4, from the moss Physcomitrella patens. In the phylogenetic analysis, PpPRRs cluster together, sister to an angiosperm PRR gene subfamily, illustrating their close relationships with the angiosperm PRRs. However, distinct from the angiosperm sequences, the RLDs of PpPRR2/3/4 exhibit a potential phosphoacceptor aspartic acid-aspartic acid-lysine (DDK) motif. Consistently, the PpPRR2 RLD had phosphotransfer ability in vitro, suggesting that PpPRR2 functions as an RR. The PpPRR1 RLD, on the other hand, shows a partially diverged DDK motif, and it did not show phosphotransfer ability. All PpPRRs were expressed in a circadian and light-dependent manner, with differential regulation between PpPRR2/4 and PpPRR1/3. Altogether, our results illustrate that PRRs originated from an RR(s) and that there are intraspecific divergences among PpPRRs. Finally, we offer scenarios for the evolution of the PRR family in land plants.

  19. Genome-wide transcriptomic analysis of the effects of sub-ambient atmospheric oxygen and elevated atmospheric carbon dioxide levels on gametophytes of the moss, Physcomitrella patens.

    PubMed

    Shinde, Suhas; Behpouri, Ali; McElwain, Jennifer C; Ng, Carl K-Y

    2015-07-01

    It is widely accepted that atmospheric O2 has played a key role in the development of life on Earth, as evident from the coincidence between the rise of atmospheric O2 concentrations in the Precambrian and biological evolution. Additionally, it has also been suggested that low atmospheric O2 is one of the major drivers for at least two of the five mass-extinction events in the Phanerozoic. At the molecular level, our understanding of the responses of plants to sub-ambient O2 concentrations is largely confined to studies of the responses of underground organs, e.g. roots to hypoxic conditions. Oxygen deprivation often results in elevated CO2 levels, particularly under waterlogged conditions, due to slower gas diffusion in water compared to air. In this study, changes in the transcriptome of gametophytes of the moss Physcomitrella patens arising from exposure to sub-ambient O2 of 13% (oxygen deprivation) and elevated CO2 (1500 ppmV) were examined to further our understanding of the responses of lower plants to changes in atmospheric gaseous composition. Microarray analyses revealed that the expression of a large number of genes was affected under elevated CO2 (814 genes) and sub-ambient O2 conditions (576 genes). Intriguingly, the expression of comparatively fewer numbers of genes (411 genes) was affected under a combination of both sub-ambient O2 and elevated CO2 condition (low O2-high CO2). Overall, the results point towards the effects of atmospheric changes in CO2 and O2 on transcriptional reprogramming, photosynthetic regulation, carbon metabolism, and stress responses. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  20. The Physcomitrella patens exocyst subunit EXO70.3d has distinct roles in growth and development, and is essential for completion of the moss life cycle.

    PubMed

    Rawat, Anamika; Brejšková, Lucie; Hála, Michal; Cvrčková, Fatima; Žárský, Viktor

    2017-04-11

    The exocyst, an evolutionarily conserved secretory vesicle-tethering complex, spatially controls exocytosis and membrane turnover in fungi, metazoans and plants. The exocyst subunit EXO70 exists in multiple paralogs in land plants, forming three conserved clades with assumed distinct roles. Here we report functional analysis of the first moss exocyst subunit to be studied, Physcomitrella patens PpEXO70.3d (Pp1s97_91V6), from the, as yet, poorly characterized EXO70.3 clade. Following phylogenetic analysis to confirm the presence of three ancestral land plant EXO70 clades outside angiosperms, we prepared and phenotypically characterized loss-of-function Ppexo70.3d mutants and localized PpEXO70.3d in vivo using green fluorescent protein-tagged protein expression. Disruption of PpEXO70.3d caused pleiotropic cell elongation and differentiation defects in protonemata, altered response towards exogenous auxin, increased endogenous IAA concentrations, along with defects in bud and gametophore development. During mid-archegonia development, an abnormal egg cell is formed and subsequently collapses, resulting in mutant sterility. Mutants exhibited altered cell wall and cuticle deposition, as well as compromised cytokinesis, consistent with the protein localization to the cell plate. Despite some functional redundancy allowing survival of moss lacking PpEXO70.3d, this subunit has an essential role in the moss life cycle, indicating sub-functionalization within the moss EXO70 family. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  1. Crystal Structures of Physcomitrella patens AOC1 and AOC2: Insights into the Enzyme Mechanism and Differences in Substrate Specificity1[W][OA

    PubMed Central

    Neumann, Piotr; Brodhun, Florian; Sauer, Kristin; Herrfurth, Cornelia; Hamberg, Mats; Brinkmann, Jens; Scholz, Julia; Dickmanns, Achim; Feussner, Ivo; Ficner, Ralf

    2012-01-01

    In plants, oxylipins regulate developmental processes and defense responses. The first specific step in the biosynthesis of the cyclopentanone class of oxylipins is catalyzed by allene oxide cyclase (AOC) that forms cis(+)-12-oxo-phytodienoic acid. The moss Physcomitrella patens has two AOCs (PpAOC1 and PpAOC2) with different substrate specificities for C18- and C20-derived substrates, respectively. To better understand AOC’s catalytic mechanism and to elucidate the structural properties that explain the differences in substrate specificity, we solved and analyzed the crystal structures of 36 monomers of both apo and ligand complexes of PpAOC1 and PpAOC2. From these data, we propose the following intermediates in AOC catalysis: (1) a resting state of the apo enzyme with a closed conformation, (2) a first shallow binding mode, followed by (3) a tight binding of the substrate accompanied by conformational changes in the binding pocket, and (4) initiation of the catalytic cycle by opening of the epoxide ring. As expected, the substrate dihydro analog cis-12,13S-epoxy-9Z,15Z-octadecadienoic acid did not cyclize in the presence of PpAOC1; however, when bound to the enzyme, it underwent isomerization into the corresponding trans-epoxide. By comparing complex structures of the C18 substrate analog with in silico modeling of the C20 substrate analog bound to the enzyme allowed us to identify three major molecular determinants responsible for the different substrate specificities (i.e. larger active site diameter, an elongated cavity of PpAOC2, and two nonidentical residues at the entrance of the active site). PMID:22987885

  2. Genome-wide transcriptomic analysis of the effects of sub-ambient atmospheric oxygen and elevated atmospheric carbon dioxide levels on gametophytes of the moss, Physcomitrella patens

    PubMed Central

    Shinde, Suhas; Behpouri, Ali; McElwain, Jennifer C.; Ng, Carl K.-Y.

    2015-01-01

    It is widely accepted that atmospheric O2 has played a key role in the development of life on Earth, as evident from the coincidence between the rise of atmospheric O2 concentrations in the Precambrian and biological evolution. Additionally, it has also been suggested that low atmospheric O2 is one of the major drivers for at least two of the five mass-extinction events in the Phanerozoic. At the molecular level, our understanding of the responses of plants to sub-ambient O2 concentrations is largely confined to studies of the responses of underground organs, e.g. roots to hypoxic conditions. Oxygen deprivation often results in elevated CO2 levels, particularly under waterlogged conditions, due to slower gas diffusion in water compared to air. In this study, changes in the transcriptome of gametophytes of the moss Physcomitrella patens arising from exposure to sub-ambient O2 of 13% (oxygen deprivation) and elevated CO2 (1500 ppmV) were examined to further our understanding of the responses of lower plants to changes in atmospheric gaseous composition. Microarray analyses revealed that the expression of a large number of genes was affected under elevated CO2 (814 genes) and sub-ambient O2 conditions (576 genes). Intriguingly, the expression of comparatively fewer numbers of genes (411 genes) was affected under a combination of both sub-ambient O2 and elevated CO2 condition (low O2–high CO2). Overall, the results point towards the effects of atmospheric changes in CO2 and O2 on transcriptional reprogramming, photosynthetic regulation, carbon metabolism, and stress responses. PMID:25948702

  3. The PpCMT chromomethylase affects cell growth and interacts with the homolog of LIKE HETEROCHROMATIN PROTEIN 1 in the moss Physcomitrella patens.

    PubMed

    Dangwal, Meenakshi; Kapoor, Sanjay; Kapoor, Meenu

    2014-02-01

    Chromomethylases (CMTs) are plant-specific cytosine DNA methyltransferases that are involved in maintenance of CpNpG methylation. In seed plants, histone methylation and interaction of CMT with LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) is essential for recruitment of CMT to target sites. LHP1 has been characterized as a putative component of the POLYCOMB REPRESSIVE COMPLEX1 (PRC1) in plants, and functions downstream of PRC2 to maintain genes in repressed state for orchestrated development. In the present study, we show that targeted disruption of PpCMT results in an approximately 50% reduction in global cytosine methylation levels. This affects growth of apical cells, predominantly growth of side branch initials emerging from chloronema cells. In some places, these cells develop thick walls with plasmolyzed cellular contents. Transcript accumulation patterns of genes involved in apical cell extension and metabolism of hemicelluloses, such as xyloglucans, in the primary cell walls decreased many fold in ppcmt mutant lines, as determined by real-time PCR. Using yeast two-hybrid method and bimolecular fluorescence complementation assay, we show that PpCMT and PpLHP1 interact through their chromo domains, while PpLHP1 homodimerizes through its chromo shadow domain. The results presented in this study provide insight into the role of the single chromomethylase, PpCMT, in proliferation of protonema filaments, and shed light on the evolutionary conservation of proteins interacting with these methylases in the early land plant, Physcomitrella patens. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  4. The MOSS Physcomitrella patens reproductive organ development is highly organized, affected by the two SHI/STY genes and by the level of active auxin in the SHI/STY expression domain.

    PubMed

    Landberg, Katarina; Pederson, Eric R A; Viaene, Tom; Bozorg, Behruz; Friml, Jirí; Jönsson, Henrik; Thelander, Mattias; Sundberg, Eva

    2013-07-01

    In order to establish a reference for analysis of the function of auxin and the auxin biosynthesis regulators SHORT INTERNODE/STYLISH (SHI/STY) during Physcomitrella patens reproductive development, we have described male (antheridial) and female(archegonial) development in detail, including temporal and positional information of organ initiation. This has allowed us to define discrete stages of organ morphogenesis and to show that reproductive organ development in P. patens is highly organized and that organ phyllotaxis differs between vegetative and reproductive development. Using the PpSHI1 and PpSHI2 reporter and knockout lines, the auxin reporters GmGH3(pro):GUS and PpPINA(pro):GFP-GUS, and the auxin-conjugating transgene PpSHI2(pro):IAAL, we could show that the PpSHI genes, and by inference also auxin, play important roles for reproductive organ development in moss. The PpSHI genes are required for the apical opening of the reproductive organs, the final differentiation of the egg cell, and the progression of canal cells into a cell death program. The apical cells of the archegonium, the canal cells, and the egg cell are also sites of auxin responsiveness and are affected by reduced levels of active auxin, suggesting that auxin mediates PpSHI function in the reproductive organs.

  5. PpASCL, the Physcomitrella patens Anther-Specific Chalcone Synthase-Like Enzyme Implicated in Sporopollenin Biosynthesis, Is Needed for Integrity of the Moss Spore Wall and Spore Viability

    PubMed Central

    Daku, Rhys M.; Rabbi, Fazle; Buttigieg, Josef; Coulson, Ian M.; Horne, Derrick; Martens, Garnet; Ashton, Neil W.; Suh, Dae-Yeon

    2016-01-01

    Sporopollenin is the main constituent of the exine layer of spore and pollen walls. The anther-specific chalcone synthase-like (ASCL) enzyme of Physcomitrella patens, PpASCL, has previously been implicated in the biosynthesis of sporopollenin, the main constituent of exine and perine, the two outermost layers of the moss spore cell wall. We made targeted knockouts of the corresponding gene, PpASCL, and phenotypically characterized ascl sporophytes and spores at different developmental stages. Ascl plants developed normally until late in sporophytic development, when the spores produced were structurally aberrant and inviable. The development of the ascl spore cell wall appeared to be arrested early in microspore development, resulting in small, collapsed spores with altered surface morphology. The typical stratification of the spore cell wall was absent with only an abnormal perine recognisable above an amorphous layer possibly representing remnants of compromised intine and/or exine. Equivalent resistance of the spore walls of ascl mutants and the control strain to acetolysis suggests the presence of chemically inert, defective sporopollenin in the mutants. Anatomical abnormalities of late-stage ascl sporophytes include a persistent large columella and an air space incompletely filled with spores. Our results indicate that the evolutionarily conserved PpASCL gene is needed for proper construction of the spore wall and for normal maturation and viability of moss spores. PMID:26752629

  6. PpASCL, the Physcomitrella patens Anther-Specific Chalcone Synthase-Like Enzyme Implicated in Sporopollenin Biosynthesis, Is Needed for Integrity of the Moss Spore Wall and Spore Viability.

    PubMed

    Daku, Rhys M; Rabbi, Fazle; Buttigieg, Josef; Coulson, Ian M; Horne, Derrick; Martens, Garnet; Ashton, Neil W; Suh, Dae-Yeon

    2016-01-01

    Sporopollenin is the main constituent of the exine layer of spore and pollen walls. The anther-specific chalcone synthase-like (ASCL) enzyme of Physcomitrella patens, PpASCL, has previously been implicated in the biosynthesis of sporopollenin, the main constituent of exine and perine, the two outermost layers of the moss spore cell wall. We made targeted knockouts of the corresponding gene, PpASCL, and phenotypically characterized ascl sporophytes and spores at different developmental stages. Ascl plants developed normally until late in sporophytic development, when the spores produced were structurally aberrant and inviable. The development of the ascl spore cell wall appeared to be arrested early in microspore development, resulting in small, collapsed spores with altered surface morphology. The typical stratification of the spore cell wall was absent with only an abnormal perine recognisable above an amorphous layer possibly representing remnants of compromised intine and/or exine. Equivalent resistance of the spore walls of ascl mutants and the control strain to acetolysis suggests the presence of chemically inert, defective sporopollenin in the mutants. Anatomical abnormalities of late-stage ascl sporophytes include a persistent large columella and an air space incompletely filled with spores. Our results indicate that the evolutionarily conserved PpASCL gene is needed for proper construction of the spore wall and for normal maturation and viability of moss spores.

  7. Photoperiod-dependent regulation of cell growth by PpCCA1a and PpCCA1b genes encoding single-myb clock proteins in the moss Physcomitrella patens.

    PubMed

    Okada, Ryo; Satbhai, Santosh B; Aoki, Setsuyuki

    2009-10-01

    The PpCCA1a and PpCCA1b genes of the moss Physcomitrella patens are functional homologs of the Arabidopsis thaliana circadian clock genes CCA1/LHY. We made use of disruptant lines for PpCCA1a and/or PpCCA1b to elucidate the physiological significance of these genes in the growth of moss protonemal tissue under alternating day/night cycles. Protonemal cells of the double disruptant line, carrying neither of the two genes, grew faster than those of the wild-type plant (WT) in long days (LD), whereas no difference in the growth rate was detected between them in short days (SD). The double disruptant line also showed day length-dependent phenotypic changes in the PpCCA1b promoter activity: the diurnal profile of bioluminescence from the P(CCA1b)::LUC+ reporter strain was more significantly affected in LD than in SD. These observations are the first demonstration of a physiological function of the circadian clock in non-angiosperm land plants, and are consistent with recent findings that the clock controls hypocotyl elongation of A. thaliana in a photoperiod-dependent manner.

  8. Genetic analysis of Physcomitrella patens identifies ABSCISIC ACID NON-RESPONSIVE, a regulator of ABA responses unique to basal land plants and required for desiccation tolerance

    SciTech Connect

    Stevenson, Sean Ross; Kamisugi, Yasuko; Trinh, Chi H.; Schmutz, Jeremy; Jenkins, Jerry W.; Grimwood, Jane; Muchero, Wellington; Tuskan, Gerald A.; Rensing, Stefan A.; Lang, Daniel; Reski, Ralf; Melkonian, Michael; Rothfels, Carl J.; Li, Fay -Wei; Larsson, Anders; Wong, Gane Ka-Shu; Edwards, Thomas A.; Cuming, Andrew C.

    2016-05-18

    The anatomically simple plants that first colonized land must have acquired molecular and biochemical adaptations to drought stress. Abscisic acid (ABA) coordinates responses leading to desiccation tolerance in all land plants. We identified ABA nonresponsive mutants in the model bryophyte Physcomitrella patens and genotyped a segregating population to map and identify the ABA NON-RESPONSIVE (ANR) gene encoding a modular protein kinase comprising an N-terminal PAS domain, a central EDR domain, and a C-terminal MAPKKK-like domain. anr mutants fail to accumulate dehydration tolerance-associated gene products in response to drought, ABA, or osmotic stress and do not acquire ABA-dependent desiccation tolerance. The crystal structure of the PAS domain, determined to 1.7-Å resolution, shows a conserved PAS-fold that dimerizes through a weak dimerization interface. Targeted mutagenesis of a conserved tryptophan residue within the PAS domain generates plants with ABA nonresponsive growth and strongly attenuated ABA-responsive gene expression, whereas deleting this domain retains a fully ABA-responsive phenotype. ANR orthologs are found in early-diverging land plant lineages and aquatic algae but are absent from more recently diverged vascular plants. Lastly, we propose that ANR genes represent an ancestral adaptation that enabled drought stress survival of the first terrestrial colonizers but were lost during land plant evolution.

  9. Genetic analysis of Physcomitrella patens identifies ABSCISIC ACID NON-RESPONSIVE, a regulator of ABA responses unique to basal land plants and required for desiccation tolerance

    SciTech Connect

    Stevenson, Sean Ross; Kamisugi, Yasuko; Trinh, Chi H.; Schmutz, Jeremy; Jenkins, Jerry W.; Grimwood, Jane; Muchero, Wellington; Tuskan, Gerald A.; Rensing, Stefan A.; Lang, Daniel; Reski, Ralf; Melkonian, Michael; Rothfels, Carl J.; Li, Fay -Wei; Larsson, Anders; Wong, Gane Ka-Shu; Edwards, Thomas A.; Cuming, Andrew C.

    2016-05-18

    The anatomically simple plants that first colonized land must have acquired molecular and biochemical adaptations to drought stress. Abscisic acid (ABA) coordinates responses leading to desiccation tolerance in all land plants. We identified ABA nonresponsive mutants in the model bryophyte Physcomitrella patens and genotyped a segregating population to map and identify the ABA NON-RESPONSIVE (ANR) gene encoding a modular protein kinase comprising an N-terminal PAS domain, a central EDR domain, and a C-terminal MAPKKK-like domain. anr mutants fail to accumulate dehydration tolerance-associated gene products in response to drought, ABA, or osmotic stress and do not acquire ABA-dependent desiccation tolerance. The crystal structure of the PAS domain, determined to 1.7-Å resolution, shows a conserved PAS-fold that dimerizes through a weak dimerization interface. Targeted mutagenesis of a conserved tryptophan residue within the PAS domain generates plants with ABA nonresponsive growth and strongly attenuated ABA-responsive gene expression, whereas deleting this domain retains a fully ABA-responsive phenotype. ANR orthologs are found in early-diverging land plant lineages and aquatic algae but are absent from more recently diverged vascular plants. Lastly, we propose that ANR genes represent an ancestral adaptation that enabled drought stress survival of the first terrestrial colonizers but were lost during land plant evolution.

  10. Molecular evidence for convergent evolution and allopolyploid speciation within the Physcomitrium-Physcomitrella species complex

    PubMed Central

    2014-01-01

    Background The moss Physcomitrella patens (Hedw.) Bruch & Schimp. is an important experimental model system for evolutionary-developmental studies. In order to shed light on the evolutionary history of Physcomitrella and related species within the Funariaceae, we analyzed the natural genetic diversity of the Physcomitrium-Physcomitrella species complex. Results Molecular analysis of the nuclear single copy gene BRK1 reveals that three Physcomitrium species feature larger genome sizes than Physcomitrella patens and encode two expressed BRK1 homeologs (polyploidization-derived paralogs), indicating that they may be allopolyploid hybrids. Phylogenetic analyses of BRK1 as well as microsatellite simple sequence repeat (SSR) data confirm a polyphyletic origin for three Physcomitrella lineages. Differences in the conservation of mitochondrial editing sites further support hybridization and cryptic speciation within the Physcomitrium-Physcomitrella species complex. Conclusions We propose a revised classification of the previously described four subspecies of Physcomitrella patens into three distinct species, namely Physcomitrella patens, Physcomitrella readeri and Physcomitrella magdalenae. We argue that secondary reduction of sporophyte complexity in these species is due to the establishment of an ecological niche, namely spores resting in mud and possible spore dispersal by migratory birds. Besides the Physcomitrium-Physcomitrella species complex, the Funariaceae are host to their type species, Funaria hygrometrica, featuring a sporophyte morphology which is more complex. Their considerable developmental variation among closely related lineages and remarkable trait evolution render the Funariaceae an interesting group for evolutionary and genetic research. PMID:25015729

  11. Two DYW subclass PPR proteins are involved in RNA editing of ccmFc and atp9 transcripts in the moss Physcomitrella patens: first complete set of PPR editing factors in plant mitochondria.

    PubMed

    Ichinose, Mizuho; Sugita, Chieko; Yagi, Yusuke; Nakamura, Takahiro; Sugita, Mamoru

    2013-11-01

    The moss Physcomitrella patens has 11 RNA editing sites in mitochondrial transcripts. We previously identified six DYW subclass pentatricopeptide repeat (PPR) proteins as RNA editing factors for nine out of 11 sites. In this study, we identified two novel DYW subclass PPR proteins, PpPPR_65 and PpPPR_98, as RNA editing factors. Disruption of the PpPPR_65 gene resulted in a complete loss of RNA editing at two neighboring sites, ccmFc-C103 and ccmFc-C122, in the mitochondrial ccmFc transcript. To confirm this result, we further generated PpPPR_65 knockdown (KD) mutants by an inducible RNA interference (RNAi) system. The generated RNAi lines displayed reduced levels of RNA editing at both ccmFc-C103 and ccmFc-C122 sites. Next, we characterized the function of PpPPR_98 by constructing a KD mutant of PpPPR_98 expression. The KD mutant showed a 30% reduction in the level of atp9-C92 editing. When PpPPR_98 cDNA was introduced into the KD mutant, RNA editing levels were restored to the wild-type level. This indicates that PpPPR_98 is an editing factor for the atp9-C92 site. The recombinant PpPPR_98 protein bound to the upstream sequence of the editing site that was created by splicing of atp9 transcript. This suggests that atp9 RNA editing occurs after splicing of atp9 transcript. Our present and previous data provide the first evidence that all 11 known editing events require at least eight DYW subclass PPR proteins in the moss mitochondria.

  12. Moss systems biology en route: phytohormones in Physcomitrella development.

    PubMed

    Decker, E L; Frank, W; Sarnighausen, E; Reski, R

    2006-05-01

    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.

  13. The Physcomitrella genome reveals evolutionary insights into the conquest of land by plants

    SciTech Connect

    Rensing, Stefan A.; Lang, Daniel; Zimmer, Andreas D.; Terry, Astrid; Salamov, Asaf; Shapiro, Harris; Nishiyama, Tomaoki; Perroud, Pierre-Francois; Lindquist, Erika A.; Kamisugi, Yasuko; Tanahashi, Takako; Sakakibara, Keiko; Fujita, Tomomichi; Oishi, Kazuko; Shin, Tadasu; Kuroki, Yoko; Toyoda, Atsushi; Suzuki, Yutaka; Hashimoto, Shin-ichi; Yamaguchi, Kazuo; Sugano, Sumio; Kohara, Yuji; Fujiyama, Asao; Anterola, Aldwin; Aoki, Setsuyuki; Ashton, Neil; Barbazuk, W. Brad; Barker, Elizabeth; Bennetzen, Jeffrey L.; Blankenship, Robert; Cho, Sung Hyun; Dutcher, Susan K.; Estelle, Mark; Fawcett, Jeffrey A.; Gundlach, Heidrum; Hanada, Kousuke; Melkozernov, Alexander; Murata, Takashi; Nelson, David R.; Pils, Birgit; Prigge, Michael; Reiss, Bernd; Renner, Tanya; Rombauts, Stephane; Rushton, Paul J.; Sanderfoot, Anton; Schween, Gabriele; Shiu, Shin-Han; Stueber, Kurt; Theodoulou, Frederica L.; Tu, Hank; Van de Peer, Yves; Verrier, Paul J.; Waters, Elizabeth; Wood, Andrew; Yang, Lixing; Cove, David; Cuming, Andrew C.; Hasebe, Mitsayasu; Lucas, Susan; Mishler, Brent D.; Reski, Ralf; Grigoriev, Igor V.; Quatrano, Rakph S.; Boore, Jeffrey L.

    2007-09-18

    We report the draft genome sequence of the model moss Physcomitrella patens and compare its features with those of flowering plants, from which it is separated by more than 400 million years, and unicellular aquatic algae. This comparison reveals genomic changes concomitant with the evolutionary movement to land, including a general increase in gene family complexity; loss of genes associated with aquatic environments (e.g., flagellar arms); acquisition of genes for tolerating terrestrial stresses (e.g., variation in temperature and water availability); and the development of the auxin and abscisic acid signaling pathways for coordinating multicellular growth and dehydration response. The Physcomitrella genome provides a resource for phylogenetic inferences about gene function and for experimental analysis of plant processes through this plant's unique facility for reverse genetics.

  14. Physcomitrella HMGA-type proteins display structural differences compared to their higher plant counterparts

    SciTech Connect

    Lyngaard, Carina; Stemmer, Christian; Stensballe, Allan; Graf, Manuela; Gorr, Gilbert; Decker, Eva; Grasser, Klaus D.

    2008-10-03

    High mobility group (HMG) proteins of the HMGA family are chromatin-associated proteins that act as architectural factors in nucleoprotein structures involved in gene transcription. To date, HMGA-type proteins have been studied in various higher plant species, but not in lower plants. We have identified two HMGA-type proteins, HMGA1 and HMGA2, encoded in the genome of the moss model Physcomitrella patens. Compared to higher plant HMGA proteins, the two Physcomitrella proteins display some structural differences. Thus, the moss HMGA proteins have six (rather than four) AT-hook DNA-binding motifs and their N-terminal domain lacks similarity to linker histone H1. HMGA2 is expressed in moss protonema and it localises to the cell nucleus. Typical of HMGA proteins, HMGA2 interacts preferentially with A/T-rich DNA, when compared with G/C-rich DNA. In cotransformation assays in Physcomitrella protoplasts, HMGA2 stimulated reporter gene expression. In summary, our data show that functional HMGA-type proteins occur in Physcomitrella.

  15. Holophytochrome-Interacting Proteins in Physcomitrella: Putative Actors in Phytochrome Cytoplasmic Signaling

    PubMed Central

    Ermert, Anna Lena; Mailliet, Katharina; Hughes, Jon

    2016-01-01

    Phytochromes are the principle photoreceptors in light-regulated plant development, primarily acting via translocation of the light-activated photoreceptor into the nucleus and subsequent gene regulation. However, several independent lines of evidence indicate unambiguously that an additional cytoplasmic signaling mechanism must exist. Directional responses in filament tip cells of the moss Physcomitrella patens are steered by phy4 which has been shown to interact physically with the blue light receptor phototropin at the plasma membrane. This complex might perceive and transduce vectorial information leading to cytoskeleton reorganization and finally a directional growth response. We developed yeast two-hybrid procedures using photochemically functional, full-length phy4 as bait in Physcomitrella cDNA library screens and growth assays under different light conditions, revealing Pfr-dependent interactions possibly associated with phytochrome cytoplasmic signaling. Candidate proteins were then expressed in planta with fluorescent protein tags to determine their intracellular localization in darkness and red light. Of 14 candidates, 12 were confirmed to interact with phy4 in planta using bimolecular fluorescence complementation. We also used database information to study their expression patterns relative to those of phy4. We discuss the likely functional characteristics of these holophytochrome-interacting proteins (HIP’s) and their possible roles in signaling. PMID:27242820

  16. The Mechanics of FtsZ Fibers

    PubMed Central

    Turner, Daniel J.; Portman, Ian; Dafforn, Timothy R.; Rodger, Alison; Roper, David I.; Smith, Corinne J.; Turner, Matthew S.

    2012-01-01

    Inhibition of the Fts family of proteins causes the growth of long filamentous cells, indicating that they play some role in cell division. FtsZ polymerizes into protofilaments and assembles into the Z-ring at the future site of the septum of cell division. We analyze the rigidity of GTP-bound FtsZ protofilaments by using cryoelectron microscopy to sample their bending fluctuations. We find that the FtsZ-GTP filament rigidity is κ = 4.7±1.0×10 − 27κ=4.7±1.0×10−27 Nm2, with a corresponding thermal persistence length of lp = 1.15±0.25lp=1.15±0.25μm, much higher than previous estimates. In conjunction with other model studies, our new higher estimate for FtsZ rigidity suggests that contraction of the Z-ring may generate sufficient force to facilitate cell division. The good agreement between the measured mode amplitudes and that predicted by equipartition of energy supports our use of a simple mechanical model for FtsZ fibers. The study also provides evidence that the fibers have no intrinsic global or local curvatures, such as might be caused by partial hydrolysis of the GTP. PMID:22385843

  17. Enzymatic Properties and Mutational Studies of Chalcone Synthase from Physcomitrella patens

    PubMed Central

    Rahman, Raja Noor Zaliha Raja Abdul; Zakaria, Iffah Izzati; Salleh, Abu Bakar; Basri, Mahiran

    2012-01-01

    PpCHS is a member of the type III polyketide synthase family and catalyses the synthesis of the flavonoid precursor naringenin chalcone from p-coumaroyl-CoA. Recent research reports the production of pyrone derivatives using either hexanoyl-CoA or butyryl-CoA as starter molecule. The Cys-His-Asn catalytic triad found in other plant chalcone synthase predicted polypeptides is conserved in PpCHS. Site directed mutagenesis involving these amino acids residing in the active-site cavity revealed that the cavity volume of the active-site plays a significant role in the selection of starter molecules as well as product formation. Substitutions of Cys 170 with Arg and Ser amino acids decreased the ability of the PpCHS to utilize hexanoyl-CoA as a starter molecule, which directly effected the production of pyrone derivatives (products). These substitutions are believed to have a restricted number of elongations of the growing polypeptide chain due to the smaller cavity volume of the mutant’s active site. PMID:22949824

  18. Both chloronemal and caulonemal cells expand by tip growth in the moss Physcomitrella patens.

    PubMed

    Menand, Benoît; Calder, Grant; Dolan, Liam

    2007-01-01

    Tip growth is a mode of cell expansion in which all growth is restricted to a small area that forms a tip in an elongating cell. In green plants, tip growth has been shown to occur in root hairs, pollen tubes, rhizoids, and caulonema. Each of these cell types has a longitudinally elongated shape, longitudinally oriented microtubules and actin microfilaments, and a characteristic cytoplasmic organization at the growing tip which is required for growth. Chloronema are elongated cylindrical shaped cells that form during the development of the moss protonema. Since there are no published reports on the precise mode of chloronema elongation and conflicting interpretations of its cytology, the mechanism of cell growth has remained unclear. To determine if chloronema elongate by tip or diffuse growth, time-lapse light microscopy was employed to follow the movement of fluorescent microspheres attached to the surface of growing cells. It is shown here that chloronemal cells elongate by a form of tip growth. However, the slower growth of chloronema compared with caulonema is probably the result of differences in cytological organization of the growing tip.

  19. Orbital experiment ``Gravisensor'': phototropic reactions of the moss Physcomitrella patens to different types of LED lighting.

    NASA Astrophysics Data System (ADS)

    Nikitin, Vladimir; Berkovich, Yuliy A.; Skripnikov, Alexander; Zyablova, Natalya; Mukhoyan, Makar; Emelianov, Grigory

    The experiment was conducted on Russian Biological Satelite Bion-M #1 19.04-19.05 2013. Five transparent plastic cultural flasks were placed in five light isolated sections of Biocont-B2 cylindrical container with inner diameter of 120 mm and height of 230 mm. In four sections the flasks could be illuminated by top or side LED with wavelength of 458 nm, 630 nm, 730 nm, and white (color temperature 5000º К, peaks 453, 559 nm). Photon flux in each variant was 15 umol/(m2c). In the fifth section the flask with the shoots was in conditions of constant dark. Each section was equipped with its own video camera module. Cameras, video recorder and lighting were managed by micro controller. 12 days before launch, 5 tips of the moss shoots were explanted at each of the five flasks on the agar medium with nutrient components and were cultivated under white fluorescent lamps at 12 hour photo period till the launch. After entering the orbit and during next 14 days of flight top LEDs were turned on above the flasks. Then for the following 14 days of flight the side LEDs of similar wavelength were turned on. The moss gametophores were cultivated at 12-h photoperiod. During the experiment on an hourly basis a video recording of the moss was performed. Similar equipment was used for ground control. After the experiment video files were used to produce separate time-lapse films for each flask using AviSynth program. In flight the shoots demonstrated the maximum growth speed with far red lighting and slower speed with white lighting. With blue and red lighting after switching to side light stimuli the growth of shoots almost stopped. In the dark the shoots continued to grow until the 13 day after launch of the satellite, then their growth stopped. In ground control the relation of growth rate with various LEDs remained basically the same, with the exception of side blue lighting, where the shoots demonstrated considerable vertical growth. In flight the angle of inclination towards the light source was maximal (about 90º) with white lighting, and somewhat smaller with 730 nm. Under red and blue light the angle of phototropic inclination was difficult to measure due to poor growth of the shoots.In ground control the growth rate under blue light was several times higher, than in flight and final degree of inclination of the shoot tip came to about 10º. In ground control under side red lighting the growth was weak, while demonstrating a pronounced phototropic bend of 90º. In ground control in the dark a vertical growth of one shoot was observed with the rate somewhat larger, than in flight variant. Data on the dynamics of inclination of experimental and control plants are presented. The acquired data will be used to analyse the mechanisms of phototropic growth changes of moss shoots.

  20. Enzymatic properties and mutational studies of chalcone synthase from Physcomitrella patens.

    PubMed

    Rahman, Raja Noor Zaliha Raja Abdul; Zakaria, Iffah Izzati; Salleh, Abu Bakar; Basri, Mahiran

    2012-01-01

    PpCHS is a member of the type III polyketide synthase family and catalyses the synthesis of the flavonoid precursor naringenin chalcone from p-coumaroyl-CoA. Recent research reports the production of pyrone derivatives using either hexanoyl-CoA or butyryl-CoA as starter molecule. The Cys-His-Asn catalytic triad found in other plant chalcone synthase predicted polypeptides is conserved in PpCHS. Site directed mutagenesis involving these amino acids residing in the active-site cavity revealed that the cavity volume of the active-site plays a significant role in the selection of starter molecules as well as product formation. Substitutions of Cys 170 with Arg and Ser amino acids decreased the ability of the PpCHS to utilize hexanoyl-CoA as a starter molecule, which directly effected the production of pyrone derivatives (products). These substitutions are believed to have a restricted number of elongations of the growing polypeptide chain due to the smaller cavity volume of the mutant's active site.

  1. FtsZ polymerization assays: simple protocols and considerations.

    PubMed

    Król, Ewa; Scheffers, Dirk-Jan

    2013-11-16

    During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long filaments in the presence of GTP in vitro, and polymerization is regulated by several accessory proteins. FtsZ polymerization has been extensively studied in vitro using basic methods including light scattering, sedimentation, GTP hydrolysis assays and electron microscopy. Buffer conditions influence both the polymerization properties of FtsZ, and the ability of FtsZ to interact with regulatory proteins. Here, we describe protocols for FtsZ polymerization studies and validate conditions and controls using Escherichia coli and Bacillus subtilis FtsZ as model proteins. A low speed sedimentation assay is introduced that allows the study of the interaction of FtsZ with proteins that bundle or tubulate FtsZ polymers. An improved GTPase assay protocol is described that allows testing of GTP hydrolysis over time using various conditions in a 96-well plate setup, with standardized incubation times that abolish variation in color development in the phosphate detection reaction. The preparation of samples for light scattering studies and electron microscopy is described. Several buffers are used to establish suitable buffer pH and salt concentration for FtsZ polymerization studies. A high concentration of KCl is the best for most of the experiments. Our methods provide a starting point for the in vitro characterization of FtsZ, not only from E. coli and B. subtilis but from any other bacterium. As such, the methods can be used for studies of the interaction of FtsZ with regulatory proteins or the testing of antibacterial drugs which may affect FtsZ polymerization.

  2. FtsZ Polymerization Assays: Simple Protocols and Considerations

    PubMed Central

    Król, Ewa; Scheffers, Dirk-Jan

    2013-01-01

    During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long filaments in the presence of GTP in vitro, and polymerization is regulated by several accessory proteins. FtsZ polymerization has been extensively studied in vitro using basic methods including light scattering, sedimentation, GTP hydrolysis assays and electron microscopy. Buffer conditions influence both the polymerization properties of FtsZ, and the ability of FtsZ to interact with regulatory proteins. Here, we describe protocols for FtsZ polymerization studies and validate conditions and controls using Escherichia coli and Bacillus subtilis FtsZ as model proteins. A low speed sedimentation assay is introduced that allows the study of the interaction of FtsZ with proteins that bundle or tubulate FtsZ polymers. An improved GTPase assay protocol is described that allows testing of GTP hydrolysis over time using various conditions in a 96-well plate setup, with standardized incubation times that abolish variation in color development in the phosphate detection reaction. The preparation of samples for light scattering studies and electron microscopy is described. Several buffers are used to establish suitable buffer pH and salt concentration for FtsZ polymerization studies. A high concentration of KCl is the best for most of the experiments. Our methods provide a starting point for the in vitro characterization of FtsZ, not only from E. coli and B. subtilis but from any other bacterium. As such, the methods can be used for studies of the interaction of FtsZ with regulatory proteins or the testing of antibacterial drugs which may affect FtsZ polymerization. PMID:24300445

  3. Targeting the Bacterial Division Protein FtsZ.

    PubMed

    Hurley, Katherine A; Santos, Thiago M A; Nepomuceno, Gabriella M; Huynh, Valerie; Shaw, Jared T; Weibel, Douglas B

    2016-08-11

    Similar to its eukaryotic counterpart, the prokaryotic cytoskeleton is essential for the structural and mechanical properties of bacterial cells. The essential protein FtsZ is a central player in the cytoskeletal family, forms a cytokinetic ring at mid-cell, and recruits the division machinery to orchestrate cell division. Cells depleted of or lacking functional FtsZ do not divide and grow into long filaments that eventually lyse. FtsZ has been studied extensively as a target for antibacterial development. In this Perspective, we review the structural and biochemical properties of FtsZ, its role in cell biochemistry and physiology, the different mechanisms of inhibiting FtsZ, small molecule antagonists (including some misconceptions about mechanisms of action), and their discovery strategies. This collective information will inform chemists on different aspects of FtsZ that can be (and have been) used to develop successful strategies for devising new families of cell division inhibitors.

  4. Isopentenyltransferase-1 (IPT1) knockout in Physcomitrella together with phylogenetic analyses of IPTs provide insights into evolution of plant cytokinin biosynthesis

    PubMed Central

    von Schwartzenberg, Klaus

    2014-01-01

    The moss Physcomitrella patens is part of an early divergent clade of land plants utilizing the plant hormone cytokinin for growth control. The rate-limiting step of cytokinin biosynthesis is mediated by isopentenyltransferases (IPTs), found in land plants either as adenylate-IPTs or as tRNA-IPTs. Although a dominant part of cytokinins in flowering plants are synthesized by adenylate-IPTs, the Physcomitrella genome only encodes homologues of tRNA-IPTs. This study therefore looked into the question of whether cytokinins in moss derive from tRNA exclusively. Targeted gene knockout of ipt1 (d|ipt1) along with localization studies revealed that the chloroplast-bound IPT1 was almost exclusively responsible for the A37 prenylation of tRNA in Physcomitrella. Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS)-based cytokinin profiling demonstrated that the total amount of all free cytokinins in tissue was almost unaffected. However, the knockout plants showed increased levels of the N 6-isopentenyladenine (iP)- and trans-zeatin (tZ)-type cytokinins, considered to provide active forms, while cis-zeatin (cZ)-type cytokinins were reduced. The data provide evidence for an additional and unexpected tRNA-independent cytokinin biosynthetic pathway in moss. Comprehensive phylogenetic analysis indicates a diversification of tRNA-IPT-like genes in bryophytes probably related to additional functions. PMID:24692654

  5. FtsZ Placement in Nucleoid-Free Bacteria

    PubMed Central

    Pazos, Manuel; Casanova, Mercedes; Palacios, Pilar; Margolin, William; Natale, Paolo; Vicente, Miguel

    2014-01-01

    We describe the placement of the cytoplasmic FtsZ protein, an essential component of the division septum, in nucleoid-free Escherichia coli maxicells. The absence of the nucleoid is accompanied in maxicells by degradation of the SlmA protein. This protein, together with the nucleoid, prevents the placement of the septum in the regions occupied by the chromosome by a mechanism called nucleoid occlusion (NO). A second septum placement mechanism, the MinCDE system (Min) involving a pole-to-pole oscillation of three proteins, nonetheless remains active in maxicells. Both Min and NO act on the polymerization of FtsZ, preventing its assembly into an FtsZ-ring except at midcell. Our results show that even in the total absence of NO, Min oscillations can direct placement of FtsZ in maxicells. Deletion of the FtsZ carboxyl terminal domain (FtsZ*), a central hub that receives signals from a variety of proteins including MinC, FtsA and ZipA, produces a Min-insensitive form of FtsZ unable to interact with the membrane-anchoring FtsA and ZipA proteins. This protein produces a totally disorganized pattern of FtsZ localization inside the maxicell cytoplasm. In contrast, FtsZ*-VM, an artificially cytoplasmic membrane-anchored variant of FtsZ*, forms helical or repetitive ring structures distributed along the entire length of maxicells even in the absence of NO. These results show that membrane anchoring is needed to organize FtsZ into rings and underscore the role of the C-terminal hub of FtsZ for their correct placement. PMID:24638110

  6. FtsZ placement in nucleoid-free bacteria.

    PubMed

    Pazos, Manuel; Casanova, Mercedes; Palacios, Pilar; Margolin, William; Natale, Paolo; Vicente, Miguel

    2014-01-01

    We describe the placement of the cytoplasmic FtsZ protein, an essential component of the division septum, in nucleoid-free Escherichia coli maxicells. The absence of the nucleoid is accompanied in maxicells by degradation of the SlmA protein. This protein, together with the nucleoid, prevents the placement of the septum in the regions occupied by the chromosome by a mechanism called nucleoid occlusion (NO). A second septum placement mechanism, the MinCDE system (Min) involving a pole-to-pole oscillation of three proteins, nonetheless remains active in maxicells. Both Min and NO act on the polymerization of FtsZ, preventing its assembly into an FtsZ-ring except at midcell. Our results show that even in the total absence of NO, Min oscillations can direct placement of FtsZ in maxicells. Deletion of the FtsZ carboxyl terminal domain (FtsZ*), a central hub that receives signals from a variety of proteins including MinC, FtsA and ZipA, produces a Min-insensitive form of FtsZ unable to interact with the membrane-anchoring FtsA and ZipA proteins. This protein produces a totally disorganized pattern of FtsZ localization inside the maxicell cytoplasm. In contrast, FtsZ*-VM, an artificially cytoplasmic membrane-anchored variant of FtsZ*, forms helical or repetitive ring structures distributed along the entire length of maxicells even in the absence of NO. These results show that membrane anchoring is needed to organize FtsZ into rings and underscore the role of the C-terminal hub of FtsZ for their correct placement.

  7. Oil Bodies and Oleosins in Physcomitrella Possess Characteristics Representative of Early Trends in Evolution1[W][OA

    PubMed Central

    Huang, Chien-Yu; Chung, Chun-I; Lin, Yao-Cheng; Hsing, Yue-Ie Caroline; Huang, Anthony H.C.

    2009-01-01

    Searches of sequenced genomes of diverse organisms revealed that the moss Physcomitrella patens is the most primitive organism possessing oleosin genes. Microscopy examination of Physcomitrella revealed that oil bodies (OBs) were abundant in the photosynthetic vegetative gametophyte and the reproductive spore. Chromatography illustrated the neutral lipids in OBs isolated from the gametophyte to be largely steryl esters and triacylglycerols, and SDS-PAGE showed the major proteins to be oleosins. Reverse transcription-PCR revealed the expression of all three oleosin genes to be tissue specific. This tissue specificity was greatly altered via alternative splicing, a control mechanism of oleosin gene expression unknown in higher plants. During the production of sex organs at the tips of gametophyte branches, the number of OBs in the top gametophyte tissue decreased concomitant with increases in the number of peroxisomes and level of transcripts encoding the glyoxylate cycle enzymes; thus, the OBs are food reserves for gluconeogenesis. In spores during germination, peroxisomes adjacent to OBs, along with transcripts encoding the glyoxylate cycle enzymes, appeared; thus, the spore OBs are food reserves for gluconeogenesis and equivalent to seed OBs. The one-cell-layer gametophyte could be observed easily with confocal microscopy for the subcellular OBs and other structures. Transient expression of various gene constructs transformed into gametophyte cells revealed that all OBs were linked to the endoplasmic reticulum (ER), that oleosins were synthesized in extended regions of the ER, and that two different oleosins were colocated in all OBs. PMID:19420327

  8. Functional analysis of COP1 and SPA orthologs from Physcomitrella and rice during photomorphogenesis of transgenic Arabidopsis reveals distinct evolutionary conservation

    PubMed Central

    2014-01-01

    Background Plants have evolved light sensing mechanisms to optimally adapt their growth and development to the ambient light environment. The COP1/SPA complex is a key negative regulator of light signaling in the well-studied dicot Arabidopsis thaliana. COP1 and members of the four SPA proteins are part of an E3 ubiquitin ligase that acts in darkness to ubiquitinate several transcription factors involved in light responses, thereby targeting them for degradation by the proteasome. While COP1 is also found in humans, SPA proteins appear specific to plants. Here, we have functionally addressed evolutionary conservation of COP1 and SPA orthologs from the moss Physcomitrella, the monocot rice and the dicot Arabidopsis. Results To this end, we analyzed the activities of COP1- and SPA-like proteins from Physcomitrella patens and rice when expressed in Arabidopsis. Expression of rice COP1 and Physcomitrella COP1 protein sequences predominantly complemented all phenotypic aspects of the viable, hypomorphic cop1-4 mutant and the null, seedling-lethal cop1-5 mutant of Arabidopsis: rice COP1 fully rescued the constitutive-photomorphogenesis phenotype in darkness and the leaf expansion defect of cop1 mutants, while it partially restored normal photoperiodic flowering in cop1. Physcomitrella COP1 partially restored normal seedling growth and flowering time, while it fully restored normal leaf expansion in the cop1 mutants. In contrast, expression of a SPA ortholog from Physcomitrella (PpSPAb) in Arabidopsis spa mutants did not rescue any facet of the spa mutant phenotype, suggesting that the PpSPAb protein is not functionally conserved or that the Arabidopsis function evolved after the split of mosses and seed plants. The SPA1 ortholog from rice (OsSPA1) rescued the spa mutant phenotype in dark-grown seedlings, but did not complement any spa mutant phenotype in light-grown seedlings or in adult plants. Conclusion Our results show that COP1 protein sequences from Physcomitrella

  9. Functional analysis of COP1 and SPA orthologs from Physcomitrella and rice during photomorphogenesis of transgenic Arabidopsis reveals distinct evolutionary conservation.

    PubMed

    Ranjan, Aashish; Dickopf, Stephen; Ullrich, Kristian K; Rensing, Stefan A; Hoecker, Ute

    2014-07-01

    Plants have evolved light sensing mechanisms to optimally adapt their growth and development to the ambient light environment. The COP1/SPA complex is a key negative regulator of light signaling in the well-studied dicot Arabidopsis thaliana. COP1 and members of the four SPA proteins are part of an E3 ubiquitin ligase that acts in darkness to ubiquitinate several transcription factors involved in light responses, thereby targeting them for degradation by the proteasome. While COP1 is also found in humans, SPA proteins appear specific to plants. Here, we have functionally addressed evolutionary conservation of COP1 and SPA orthologs from the moss Physcomitrella, the monocot rice and the dicot Arabidopsis. To this end, we analyzed the activities of COP1- and SPA-like proteins from Physcomitrella patens and rice when expressed in Arabidopsis. Expression of rice COP1 and Physcomitrella COP1 protein sequences predominantly complemented all phenotypic aspects of the viable, hypomorphic cop1-4 mutant and the null, seedling-lethal cop1-5 mutant of Arabidopsis: rice COP1 fully rescued the constitutive-photomorphogenesis phenotype in darkness and the leaf expansion defect of cop1 mutants, while it partially restored normal photoperiodic flowering in cop1. Physcomitrella COP1 partially restored normal seedling growth and flowering time, while it fully restored normal leaf expansion in the cop1 mutants. In contrast, expression of a SPA ortholog from Physcomitrella (PpSPAb) in Arabidopsis spa mutants did not rescue any facet of the spa mutant phenotype, suggesting that the PpSPAb protein is not functionally conserved or that the Arabidopsis function evolved after the split of mosses and seed plants. The SPA1 ortholog from rice (OsSPA1) rescued the spa mutant phenotype in dark-grown seedlings, but did not complement any spa mutant phenotype in light-grown seedlings or in adult plants. Our results show that COP1 protein sequences from Physcomitrella, rice and Arabidopsis have

  10. Reconstitution of Contractile FtsZ Rings in Liposomes

    PubMed Central

    Osawa, Masaki; Anderson, David E.; Erickson, Harold P.

    2009-01-01

    FtsZ is a tubulin homolog and the major cytoskeletal protein in bacterial cell division. It assembles into the Z ring, which contains FtsZ and a dozen other division proteins, and constricts to divide the cell. We have constructed a membrane-targeted FtsZ (FtsZ-mts) by splicing an amphipathic helix to its C terminus. When mixed with lipid vesicles, FtsZ-mts was incorporated into the interior of some tubular vesicles. There it formed multiple Z rings that could move laterally in both directions along the length of the liposome and coalesce into brighter Z rings. Brighter Z rings produced visible constrictions in the liposome, suggesting that FtsZ itself can assemble the Z ring and generate a force. No other proteins were needed for assembly and force generation. PMID:18420899

  11. Ruthenium red-induced bundling of bacterial cell division protein, FtsZ.

    PubMed

    Santra, Manas Kumar; Beuria, Tushar K; Banerjee, Abhijit; Panda, Dulal

    2004-06-18

    The assembly of FtsZ plays a major role in bacterial cell division, and it is thought that the assembly dynamics of FtsZ is a finely regulated process. Here, we show that ruthenium red is able to modulate FtsZ assembly in vitro. In contrast to the inhibitory effects of ruthenium red on microtubule polymerization, we found that a substoichiometric concentration of ruthenium red strongly increased the light-scattering signal of FtsZ assembly. Further, sedimentable polymer mass was increased by 1.5- and 2-fold in the presence of 2 and 10 microm ruthenium red, respectively. In addition, ruthenium red strongly reduced the GTPase activity and prevented dilution-induced disassembly of FtsZ polymers. Electron microscopic analysis showed that 4-10 microm of ruthenium red produced thick bundles of FtsZ polymers. The significant increase in the light-scattering signal and pelletable polymer mass in the presence of ruthenium red seemed to be due to the bundling of FtsZ protofilaments into larger polymers rather than the actual increase in the level of polymeric FtsZ. Furthermore, ruthenium red was found to copolymerize with FtsZ, and the copolymerization of substoichiometric amounts of ruthenium red with FtsZ polymers promoted cooperative assembly of FtsZ that produced large bundles. Calcium inhibited the binding of ruthenium red to FtsZ. However, a concentration of calcium 1000-fold higher than that of ruthenium red was required to produce similar effects on FtsZ assembly. Ruthenium red strongly modulated FtsZ polymerization, suggesting the presence of an important regulatory site on FtsZ and suggesting that a natural ligand, which mimics the action of ruthenium red, may regulate the assembly of FtsZ in bacteria.

  12. Arabidopsis FtsZ2-1 and FtsZ2-2 are functionally redundant, but FtsZ-based plastid division is not essential for chloroplast partitioning or plant growth and development.

    PubMed

    Schmitz, Aaron J; Glynn, Jonathan M; Olson, Bradley J S C; Stokes, Kevin D; Osteryoung, Katherine W

    2009-11-01

    FtsZ1 and FtsZ2 are phylogenetically distinct families of FtsZ in plants that co-localize to mid-plastid rings and facilitate division of chloroplasts. In plants, altered levels of either FtsZ1 or FtsZ2 cause dose-dependent defects in chloroplast division; thus, studies on the functional relationship between FtsZ genes require careful manipulation of FtsZ levels in vivo. To define the functional relationship between the two FtsZ2 genes in Arabidopsis thaliana, FtsZ2-1 and FtsZ2-2, we expressed FtsZ2-1 in an ftsZ2-2 null mutant, and vice versa, and determined whether the chloroplast division defects were rescued in plants expressing different total levels of FtsZ2. Full rescue was observed when either the FtsZ2-1 or FtsZ2-2 level approximated total FtsZ2 levels in wild-type (WT). Additionally, FtsZ2-2 interacts with ARC6, as shown previously for FtsZ2-1. These data indicate that FtsZ2-1 and FtsZ2-2 are functionally redundant for chloroplast division in Arabidopsis. To rigorously validate the requirement of each FtsZ family for chloroplast division, we replaced FtsZ1 with FtsZ2 in vivo, and vice versa, while maintaining the FtsZ level in the transgenic plants equal to that of the total level in WT. Chloroplast division defects were not rescued, demonstrating conclusively that FtsZ1 and FtsZ2 are non-redundant for maintenance of WT chloroplast numbers. Finally, we generated ftsZ triple null mutants and show that plants completely devoid of FtsZ protein are viable and fertile. As plastids are presumably essential organelles, these findings suggest that an FtsZ-independent mode of plastid partitioning may occur in higher plants.

  13. Cationic lipid enhances assembly of bacterial cell division protein FtsZ: a possible role of bacterial membrane in FtsZ assembly dynamics.

    PubMed

    Kuchibhatla, Anuradha; Bellare, Jayesh; Panda, Dulal

    2011-11-01

    The assembly of FtsZ plays an important role in bacterial cell division. Lipids in the bacterial cell membrane have been suggested to play a role in directing the site of FtsZ assembly. Using lipid monolayer and bilayer (liposome) systems, we directly examined the effects of cationic lipids on FtsZ assembly. We found that cationic lipids enhanced the assembly of FtsZ in association with an increase in the GTPase activity of FtsZ. The system consisting of lipid monolayer and bilayer (liposome) may mimic the bacterial membrane and therefore, the data might indicate the influence of bacterial membrane on the assembly of FtsZ protofilaments.

  14. Polymer Stability Plays an Important Role in the Positional Regulation of FtsZ

    PubMed Central

    Levin, Petra Anne; Schwartz, Rachel L.; Grossman, Alan D.

    2001-01-01

    We conducted a series of experiments examining the effect of polymer stability on FtsZ localization dynamics in Bacillus subtilis. A loss-of-function mutation in ezrA, a putative polymer-destabilizing factor, suppresses the defects in FtsZ polymer stability associated with minCD overexpression. In addition, a mutation that is predicted to stabilize the FtsZ polymer leads to the formation of polar FtsZ rings. These data support the hypothesis that carefully balanced polymer stability is important for the assembly and localization of FtsZ during the bacterial cell cycle. PMID:11514533

  15. Curcumin inhibits FtsZ assembly: an attractive mechanism for its antibacterial activity.

    PubMed

    Rai, Dipti; Singh, Jay Kumar; Roy, Nilanjan; Panda, Dulal

    2008-02-15

    The assembly and stability of FtsZ protofilaments have been shown to play critical roles in bacterial cytokinesis. Recent evidence suggests that FtsZ may be considered as an important antibacterial drug target. Curcumin, a dietary polyphenolic compound, has been shown to have a potent antibacterial activity against a number of pathogenic bacteria including Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus. We found that curcumin induced filamentation in the Bacillus subtilis 168, suggesting that it inhibits bacterial cytokinesis. Further, curcumin strongly inhibited the formation of the cytokinetic Z-ring in B. subtilis 168 without detectably affecting the segregation and organization of the nucleoids. Since the assembly dynamics of FtsZ protofilaments plays a major role in the formation and functioning of the Z-ring, we analysed the effects of curcumin on the assembly of FtsZ protofilaments. Curcumin inhibited the assembly of FtsZ protofilaments and also increased the GTPase activity of FtsZ. Electron microscopic analysis showed that curcumin reduced the bundling of FtsZ protofilaments in vitro. Further, curcumin was found to bind to FtsZ in vitro with a dissociation constant of 7.3+/-1.8 microM and the agent also perturbed the secondary structure of FtsZ. The results indicate that the perturbation of the GTPase activity of FtsZ assembly is lethal to bacteria and suggest that curcumin inhibits bacterial cell proliferation by inhibiting the assembly dynamics of FtsZ in the Z-ring.

  16. Tubulin-like protofilaments in Ca2+-induced FtsZ sheets.

    PubMed Central

    Löwe, J; Amos, L A

    1999-01-01

    The 40 kDa protein FtsZ is a major septum-forming component of bacterial cell division. Early during cytokinesis at midcell, FtsZ forms a cytokinetic ring that constricts as septation progresses. FtsZ has a high propensity to polymerize in vitro into various structures, including sheets and filaments, in a GTP-dependent manner. Together with limited sequence homology, the occurrence of the tubulin signature motif in FtsZ and a similar three-dimensional structure, this leads to the conclusion that FtsZ is the bacterial tubulin homologue. We have polymerized FtsZ1 from Methanococcus jannaschii in the presence of millimolar concentrations of Ca2+ ions to produce two-dimensional crystals of plane group P2221. Most of the protein precipitates and forms filaments approximately 23.0 nm in diameter. A three-dimensional reconstruction of tilted micrographs of FtsZ sheets in negative stain between 0 and 60 degrees shows protofilaments of FtsZ running along the sheet axis. Pairs of parallel FtsZ protofilaments associate in an antiparallel fashion to form a two-dimensional sheet. The antiparallel arrangement is believed to generate flat sheets instead of the curved filaments seen in other FtsZ polymers. Together with the subunit spacing along the protofilament axis, a fitting of the FtsZ crystal structure into the reconstruction suggests a protofilamant structure very similar to that of tubulin protofilaments. PMID:10228151

  17. FtsZ filament capping by MciZ, a developmental regulator of bacterial division

    PubMed Central

    Bisson-Filho, Alexandre W.; Discola, Karen F.; Castellen, Patrícia; Blasios, Valdir; Martins, Alexandre; Sforça, Maurício L.; Garcia, Wanius; Zeri, Ana Carolina M.; Erickson, Harold P.; Dessen, Andréa; Gueiros-Filho, Frederico J.

    2015-01-01

    Cytoskeletal structures are dynamically remodeled with the aid of regulatory proteins. FtsZ (filamentation temperature-sensitive Z) is the bacterial homolog of tubulin that polymerizes into rings localized to cell-division sites, and the constriction of these rings drives cytokinesis. Here we investigate the mechanism by which the Bacillus subtilis cell-division inhibitor, MciZ (mother cell inhibitor of FtsZ), blocks assembly of FtsZ. The X-ray crystal structure reveals that MciZ binds to the C-terminal polymerization interface of FtsZ, the equivalent of the minus end of tubulin. Using in vivo and in vitro assays and microscopy, we show that MciZ, at substoichiometric levels to FtsZ, causes shortening of protofilaments and blocks the assembly of higher-order FtsZ structures. The findings demonstrate an unanticipated capping-based regulatory mechanism for FtsZ. PMID:25848052

  18. FtsZ filament capping by MciZ, a developmental regulator of bacterial division.

    PubMed

    Bisson-Filho, Alexandre W; Discola, Karen F; Castellen, Patrícia; Blasios, Valdir; Martins, Alexandre; Sforça, Maurício L; Garcia, Wanius; Zeri, Ana Carolina M; Erickson, Harold P; Dessen, Andréa; Gueiros-Filho, Frederico J

    2015-04-28

    Cytoskeletal structures are dynamically remodeled with the aid of regulatory proteins. FtsZ (filamentation temperature-sensitive Z) is the bacterial homolog of tubulin that polymerizes into rings localized to cell-division sites, and the constriction of these rings drives cytokinesis. Here we investigate the mechanism by which the Bacillus subtilis cell-division inhibitor, MciZ (mother cell inhibitor of FtsZ), blocks assembly of FtsZ. The X-ray crystal structure reveals that MciZ binds to the C-terminal polymerization interface of FtsZ, the equivalent of the minus end of tubulin. Using in vivo and in vitro assays and microscopy, we show that MciZ, at substoichiometric levels to FtsZ, causes shortening of protofilaments and blocks the assembly of higher-order FtsZ structures. The findings demonstrate an unanticipated capping-based regulatory mechanism for FtsZ.

  19. Organization of FtsZ filaments in the bacterial division ring measured from polarized fluorescence microscopy.

    PubMed

    Si, Fangwei; Busiek, Kimberly; Margolin, William; Sun, Sean X

    2013-11-05

    Cytokinesis in bacteria is accomplished by a ring-shaped cell-division complex (the Z-ring). The primary component of the Z-ring is FtsZ, a filamentous tubulin homolog that serves as a scaffold for the recruitment of other cell-division-related proteins. FtsZ forms filaments and bundles. In the cell, it has been suggested that FtsZ filaments form the arcs of the ring and are aligned in the cell-circumferential direction. Using polarized fluorescence microscopy in live Escherichia coli cells, we measure the structural organization of FtsZ filaments in the Z-ring. The data suggest a disordered organization: a substantial portion of FtsZ filaments are aligned in the cell-axis direction. FtsZ organization in the Z-ring also appears to depend on the bacterial species. Taken together, the unique arrangement of FtsZ suggests novel unexplored mechanisms in bacterial cell division.

  20. Preferential cytoplasmic location of FtsZ, a protein essential for Escherichia coli septation.

    PubMed

    Pla, J; Sánchez, M; Palacios, P; Vicente, M; Aldea, M

    1991-07-01

    An ftsZ thermonull mutant has been constructed in which the ftsZ gene has been deleted from the Escherichia coli chromosome while maintaining a wild-type copy of the gene in a thermosensitive plasmid. Under conditions in which the ftsZ+ allele is unable to be replicated at the same pace as the chromosome, the cells become non-viable and grow as filaments, indicating that, contrary to other reports, FtsZ performs a function essential for cell survival. Antibodies raised against FtsZ have been used to detect the cellular location of FtsZ and its contents per cell. Fractionation experiments indicate that most of the total FtsZ present in the cell stays in the cytoplasm.

  1. Studies of Physcomitrella patens reveal that ethylene-mediated submergence responses arose relatively early in land-plant evolution.

    PubMed

    Yasumura, Yuki; Pierik, Ronald; Fricker, Mark D; Voesenek, Laurentius A C J; Harberd, Nicholas P

    2012-12-01

    Colonization of the land by multicellular green plants was a fundamental step in the evolution of life on earth. Land plants evolved from fresh-water aquatic algae, and the transition to a terrestrial environment required the acquisition of developmental plasticity appropriate to the conditions of water availability, ranging from drought to flood. Here we show that extant bryophytes exhibit submergence-induced developmental plasticity, suggesting that submergence responses evolved relatively early in the evolution of land plants. We also show that a major component of the bryophyte submergence response is controlled by the phytohormone ethylene, using a perception mechanism that has subsequently been conserved throughout the evolution of land plants. Thus a plant environmental response mechanism with major ecological and agricultural importance probably had its origins in the very earliest stages of the colonization of the land. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

  2. Localization of an evolutionarily conserved protein proton pyrophosphatase in evolutionarily distant plants oryza sativa and physcomitrella patens

    USDA-ARS?s Scientific Manuscript database

    Proton Pyrophosphatase (H+-PPase) is a highly evolutionarily conserved protein that is prevalent in the plant kingdom. One of the salient features of H+-PPase expression pattern, at least in vascular plants like Arabidopsis, is its conspicuous localization in both actively dividing cells and the phl...

  3. FtsZ Condensates: An In Vitro Electron Microscopy Study

    PubMed Central

    Popp, David; Iwasa, Mitsusada; Narita, Akihiro; Erickson, Harold P.; Maéda, Yuichiro

    2009-01-01

    In vivo cell division protein FtsZ from E. coli forms rings and spirals which have only been observed by low resolution light microscopy. We show that these suprastructures are likely formed by molecular crowding which is a predominant factor in prokaryotic cells and enhances the weak lateral bonds between proto-filaments. Although FtsZ assembles into single proto-filaments in dilute aqueous buffer, with crowding agents above a critical concentration, it forms polymorphic supramolecular structures including rings and toroids (with multiple protofilaments) about 200 nm in diameter, similar in appearance to DNA toroids, and helices with pitches of several hundred nm as well as long, linear bundles. Helices resemble those observed in vivo, whereas the rings and toroids may represent a novel energy minimized state of FtsZ, at a later stage of Z-ring constriction. We shed light on the molecular arrangement of FtsZ filaments within these suprastructures using high resolution electron microscopy. PMID:19137575

  4. NDK Interacts with FtsZ and Converts GDP to GTP to Trigger FtsZ Polymerisation - A Novel Role for NDK

    PubMed Central

    Mishra, Saurabh; Jakkala, Kishor; Srinivasan, Ramanujam; Arumugam, Muthu; Ranjeri, Raghavendra; Gupta, Prabuddha; Rajeswari, Haryadi; Ajitkumar, Parthasarathi

    2015-01-01

    Introduction Nucleoside diphosphate kinase (NDK), conserved across bacteria to humans, synthesises NTP from NDP and ATP. The eukaryotic homologue, the NDPK, uses ATP to phosphorylate the tubulin-bound GDP to GTP for tubulin polymerisation. The bacterial cytokinetic protein FtsZ, which is the tubulin homologue, also uses GTP for polymerisation. Therefore, we examined whether NDK can interact with FtsZ to convert FtsZ-bound GDP and/or free GDP to GTP to trigger FtsZ polymerisation. Methods Recombinant and native NDK and FtsZ proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis were used as the experimental samples. FtsZ polymersation was monitored using 90° light scattering and FtsZ polymer pelleting assays. The γ32P-GTP synthesised by NDK from GDP and γ32P-ATP was detected using thin layer chromatography and quantitated using phosphorimager. The FtsZ bound 32P-GTP was quantitated using phosphorimager, after UV-crosslinking, followed by SDS-PAGE. The NDK-FtsZ interaction was determined using Ni2+-NTA-pulldown assay and co-immunoprecipitation of the recombinant and native proteins in vitro and ex vivo, respectively. Results NDK triggered instantaneous polymerisation of GDP-precharged recombinant FtsZ in the presence of ATP, similar to the polymerisation of recombinant FtsZ (not GDP-precharged) upon the direct addition of GTP. Similarly, NDK triggered polymerisation of recombinant FtsZ (not GDP-precharged) in the presence of free GDP and ATP as well. Mutant NDK, partially deficient in GTP synthesis from ATP and GDP, triggered low level of polymerisation of MsFtsZ, but not of MtFtsZ. As characteristic of NDK’s NTP substrate non-specificity, it used CTP, TTP, and UTP also to convert GDP to GTP, to trigger FtsZ polymerisation. The NDK of one mycobacterial species could trigger the polymerisation of the FtsZ of another mycobacterial species. Both the recombinant and the native NDK and FtsZ showed interaction with each other in vitro and ex vivo, alluding

  5. NDK Interacts with FtsZ and Converts GDP to GTP to Trigger FtsZ Polymerisation--A Novel Role for NDK.

    PubMed

    Mishra, Saurabh; Jakkala, Kishor; Srinivasan, Ramanujam; Arumugam, Muthu; Ranjeri, Raghavendra; Gupta, Prabuddha; Rajeswari, Haryadi; Ajitkumar, Parthasarathi

    2015-01-01

    Nucleoside diphosphate kinase (NDK), conserved across bacteria to humans, synthesises NTP from NDP and ATP. The eukaryotic homologue, the NDPK, uses ATP to phosphorylate the tubulin-bound GDP to GTP for tubulin polymerisation. The bacterial cytokinetic protein FtsZ, which is the tubulin homologue, also uses GTP for polymerisation. Therefore, we examined whether NDK can interact with FtsZ to convert FtsZ-bound GDP and/or free GDP to GTP to trigger FtsZ polymerisation. Recombinant and native NDK and FtsZ proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis were used as the experimental samples. FtsZ polymersation was monitored using 90° light scattering and FtsZ polymer pelleting assays. The γ32P-GTP synthesised by NDK from GDP and γ32P-ATP was detected using thin layer chromatography and quantitated using phosphorimager. The FtsZ bound 32P-GTP was quantitated using phosphorimager, after UV-crosslinking, followed by SDS-PAGE. The NDK-FtsZ interaction was determined using Ni2+-NTA-pulldown assay and co-immunoprecipitation of the recombinant and native proteins in vitro and ex vivo, respectively. NDK triggered instantaneous polymerisation of GDP-precharged recombinant FtsZ in the presence of ATP, similar to the polymerisation of recombinant FtsZ (not GDP-precharged) upon the direct addition of GTP. Similarly, NDK triggered polymerisation of recombinant FtsZ (not GDP-precharged) in the presence of free GDP and ATP as well. Mutant NDK, partially deficient in GTP synthesis from ATP and GDP, triggered low level of polymerisation of MsFtsZ, but not of MtFtsZ. As characteristic of NDK's NTP substrate non-specificity, it used CTP, TTP, and UTP also to convert GDP to GTP, to trigger FtsZ polymerisation. The NDK of one mycobacterial species could trigger the polymerisation of the FtsZ of another mycobacterial species. Both the recombinant and the native NDK and FtsZ showed interaction with each other in vitro and ex vivo, alluding to the possibility of direct

  6. Energetics and Geometry of FtsZ Polymers: Nucleated Self-Assembly of Single Protofilaments☆

    PubMed Central

    Huecas, Sonia; Llorca, Oscar; Boskovic, Jasminka; Martín-Benito, Jaime; Valpuesta, José María; Andreu, José Manuel

    2008-01-01

    Essential cell division protein FtsZ is an assembling GTPase which directs the cytokinetic ring formation in dividing bacterial cells. FtsZ shares the structural fold of eukaryotic tubulin and assembles forming tubulin-like protofilaments, but does not form microtubules. Two puzzling problems in FtsZ assembly are the nature of protofilament association and a possible mechanism for nucleated self-assembly of single-stranded protofilaments above a critical FtsZ concentration. We assembled two-dimensional arrays of FtsZ on carbon supports, studied linear polymers of FtsZ with cryo-electron microscopy of vitrified unsupported solutions, and formulated possible polymerization models. Nucleated self-assembly of FtsZ from Escherichia coli with GTP and magnesium produces flexible filaments 4–6 nm-wide, only compatible with a single protofilament. This agrees with previous scanning transmission electron microscopy results and is supported by recent cryo-electron tomography studies of two bacterial cells. Observations of double-stranded FtsZ filaments in negative stain may come from protofilament accretion on the carbon support. Preferential protofilament cyclization does not apply to FtsZ assembly. The apparently cooperative polymerization of a single protofilament with identical intermonomer contacts is explained by the switching of one inactive monomer into the active structure preceding association of the next, creating a dimer nucleus. FtsZ behaves as a cooperative linear assembly machine. PMID:18024502

  7. Inhibition of bacterial cell division protein FtsZ by cinnamaldehyde.

    PubMed

    Domadia, Prerna; Swarup, Sanjay; Bhunia, Anirban; Sivaraman, J; Dasgupta, Debjani

    2007-09-15

    Cinnamaldehyde is a natural product from spices that inhibits cell separation in Bacillus cereus. Cell division is regulated by FtsZ, a prokaryotic homolog of tubulin. FtsZ assembles into the Z-ring at the site of cell division. Here, we report the effect of cinnamaldehyde on FtsZ and hence on the cell division apparatus. Cinnamaldehyde decreases the in vitro assembly reaction and bundling of FtsZ. It is found that cinnamaldehyde perturbs the Z-ring morphology in vivo and reduces the frequency of the Z ring per unit cell length of Escherichia coli. In addition, GTP dependent FtsZ polymerization is inhibited by cinnamaldehyde. Cinnamaldehyde inhibits the rate of GTP hydrolysis and binds FtsZ with an affinity constant of 1.0+/-0.2 microM(-1). Isothermal titration calorimetry reveals that binding of cinnamaldehyde to FtsZ is driven by favorable enthalpic interactions. Further, we map the cinnamaldehyde binding region of FtsZ, using the saturation transfer difference-nuclear magnetic resonance and an in silico docking model. Both predict the cinnamaldehyde binding pocket at the C terminal region involving the T7 loop of FtsZ. Our results show that cinnamaldehyde binds FtsZ, perturbs the cytokinetic Z-ring formation and inhibits its assembly dynamics. This suggests that cinnamaldehyde, a small molecule of plant origin, is a potential lead compound that can be developed as an anti-FtsZ agent towards drug design.

  8. Energetics and geometry of FtsZ polymers: nucleated self-assembly of single protofilaments.

    PubMed

    Huecas, Sonia; Llorca, Oscar; Boskovic, Jasminka; Martín-Benito, Jaime; Valpuesta, José María; Andreu, José Manuel

    2008-03-01

    Essential cell division protein FtsZ is an assembling GTPase which directs the cytokinetic ring formation in dividing bacterial cells. FtsZ shares the structural fold of eukaryotic tubulin and assembles forming tubulin-like protofilaments, but does not form microtubules. Two puzzling problems in FtsZ assembly are the nature of protofilament association and a possible mechanism for nucleated self-assembly of single-stranded protofilaments above a critical FtsZ concentration. We assembled two-dimensional arrays of FtsZ on carbon supports, studied linear polymers of FtsZ with cryo-electron microscopy of vitrified unsupported solutions, and formulated possible polymerization models. Nucleated self-assembly of FtsZ from Escherichia coli with GTP and magnesium produces flexible filaments 4-6 nm-wide, only compatible with a single protofilament. This agrees with previous scanning transmission electron microscopy results and is supported by recent cryo-electron tomography studies of two bacterial cells. Observations of double-stranded FtsZ filaments in negative stain may come from protofilament accretion on the carbon support. Preferential protofilament cyclization does not apply to FtsZ assembly. The apparently cooperative polymerization of a single protofilament with identical intermonomer contacts is explained by the switching of one inactive monomer into the active structure preceding association of the next, creating a dimer nucleus. FtsZ behaves as a cooperative linear assembly machine.

  9. Two types of FtsZ proteins in mitochondria and red-lineage chloroplasts: the duplication of FtsZ is implicated in endosymbiosis.

    PubMed

    Miyagishima, Shin-ya; Nozaki, Hisayoshi; Nishida, Keishin; Nishida, Keiji; Matsuzaki, Motomichi; Kuroiwa, Tsuneyoshi

    2004-03-01

    The ancestors of plastids and mitochondria were once free-living bacteria that became organelles as a result of endosymbiosis. According to this theory, a key bacterial division protein, FtsZ, plays a role in plastid division in algae and plants as well as in mitochondrial division in lower eukaryotes. Recent studies have shown that organelle division is a process that combines features derived from the bacterial division system with features contributed by host eukaryotic cells. Two nonredundant versions of FtsZ, FtsZ1 and FtsZ2, have been identified in green-lineage plastids, whereas most bacteria have a single ftsZ gene. To examine whether there is also more than one type of FtsZ in red-lineage chloroplasts (red algal chloroplasts and chloroplasts that originated from the secondary endosymbiosis of red algae) and in mitochondria, we obtained FtsZ sequences from the complete sequence of the primitive red alga Cyanidioschyzon merolae and the draft sequence of the stramenopile (heterokont) Thalassiosira pseudonana. Phylogenetic analyses that included known FtsZ proteins identified two types of chloroplast FtsZ in red algae (FtsZA and FtsZB) and stramenopiles (FtsZA and FtsZC). These analyses also showed that FtsZB emerged after the red and green lineages diverged, while FtsZC arose by the duplication of an ftsZA gene that in turn descended from a red alga engulfed by the ancestor of stramenopiles. A comparison of the predicted proteins showed that like bacterial FtsZ and green-lineage FtsZ2, FtsZA has a short conserved C-termmal sequence (the C-terminal core domain), whereas FtsZB and FtsZC, like the green-lineage FtsZ1, lack this sequence. In addition, the Cyanidioschyzon and Dictyostelium genomes encode two types of mitochondrial FtsZ proteins, one of which lacks the C-terminal variable domain. These results suggest that the acquisition of an additional FtsZ protein with a modified C terminus was common to the primary and secondary endosymbioses that produced

  10. The development of FtsZ inhibitors as potential antibacterial agents.

    PubMed

    Ma, Siti; Ma, Shutao

    2012-07-01

    The emergence and prevalence of bacterial resistance has resulted in a clear demand for novel antibacterial drugs. As a tubulin homologue, FtsZ is an essential cell-division protein in prokaryotic organisms and is showing increasing promise as a target for antibacterial drug discovery. This review describes the role of FtsZ in bacterial cytokinesis and various FtsZ inhibitors, with particular focus on their discovery, antibacterial activities, mechanisms of action, synthetic methods, and representative analogues.

  11. Super-resolution imaging of the bacterial cytokinetic protein FtsZ.

    PubMed

    Jennings, Phoebe C; Cox, Guy C; Monahan, Leigh G; Harry, Elizabeth J

    2011-06-01

    The idea of a bacterial cytoskeleton arose just 10 years ago with the identification of the cell division protein, FtsZ, as a tubulin homolog. FtsZ plays a pivotal role in bacterial division, and is present in virtually all prokaryotes and in some eukaryotic organelles. The earliest stage of bacterial cell division is the assembly of FtsZ into a Z ring at the division site, which subsequently constricts during cytokinesis. FtsZ also assembles into dynamic helical structures along the bacterial cell, which are thought to act as precursors to the Z ring via a cell cycle-mediated FtsZ polymer remodelling. The fine structures of the FtsZ helix and ring are unknown but crucial for identifying the molecular details of Z ring assembly and its regulation. We now reveal using STED microscopy that the FtsZ helical structure in cells of the gram positive bacterium, Bacillus subtilis, is a highly irregular and discontinuous helix of FtsZ; very different to the smooth cable-like appearance observed by conventional fluorescence optics. STED also identifies a novel FtsZ helical structure of smaller pitch that is invisible to standard optical methods, identifying a possible third intermediate in the pathway to Z ring assembly, which commits bacterial cells to divide.

  12. Identification and Characterization of ZapC, a Stabilizer of the FtsZ Ring in Escherichia coli▿ †

    PubMed Central

    Durand-Heredia, Jorge M.; Yu, Helen H.; De Carlo, Sacha; Lesser, Cammie F.; Janakiraman, Anuradha

    2011-01-01

    In Escherichia coli, spatiotemporal control of cell division occurs at the level of the assembly/disassembly process of the essential cytoskeletal protein FtsZ. A number of regulators interact with FtsZ and modulate the dynamics of the assembled FtsZ ring at the midcell division site. In this article, we report the identification of an FtsZ stabilizer, ZapC (Z-associated protein C), in a protein localization screen conducted with E. coli. ZapC colocalizes with FtsZ at midcell and interacts directly with FtsZ, as determined by a protein-protein interaction assay in yeast. Cells lacking or overexpressing ZapC are slightly elongated and have aberrant FtsZ ring morphologies indicative of a role for ZapC in FtsZ regulation. We also demonstrate the ability of purified ZapC to promote lateral bundling of FtsZ in a sedimentation reaction visualized by transmission electron microscopy. While ZapC lacks sequence similarity with other nonessential FtsZ regulators, ZapA and ZapB, all three Zap proteins appear to play an important role in FtsZ regulation during rapid growth. Taken together, our results suggest a key role for lateral bundling of the midcell FtsZ polymers in maintaining FtsZ ring stability during division. PMID:21216995

  13. Torsion and curvature of FtsZ filaments.

    PubMed

    González de Prado Salas, Pablo; Hörger, Ines; Martín-García, Fernando; Mendieta, Jesús; Alonso, Álvaro; Encinar, Mario; Gómez-Puertas, Paulino; Vélez, Marisela; Tarazona, Pedro

    2014-03-28

    FtsZ filaments participate in bacterial cell division, but it is still not clear how their dynamic polymerization and shape exert force on the underlying membrane. We present a theoretical description of individual filaments that incorporates information from molecular dynamic simulations. The structure of the crystallized Methanococcus jannaschii FtsZ dimer was used to model a FtsZ pentamer that showed a curvature and a twist. The estimated bending and torsion angles between monomers and their fluctuations were included in the theoretical description. The MD data also permitted positioning the curvature with respect to the protein coordinates and allowed us to explore the effect of the relative orientation of the preferred curvature with respect to the surface plane. We find that maximum tension is attained when filaments are firmly attached and oriented with their curvature perpendicular to the surface and that the twist serves as a valve to release or to tighten the tension exerted by the curved filaments on the membrane. The theoretical model also shows that the presence of torsion can explain the shape distribution of short filaments observed by Atomic Force Microscopy in previously published experiments. New experiments with FtsZ covalently attached to lipid membranes show that the filament on-plane curvature depends on lipid head charge, confirming the predicted monomer orientation effects. This new model underlines the fact that the combination of the three elements, filament curvature, twist and the strength and orientation of its surface attachment, can modulate the force exerted on the membrane during cell division.

  14. Structure of the Z Ring-associated Protein, ZapD, Bound to the C-terminal Domain of the Tubulin-like Protein, FtsZ, Suggests Mechanism of Z Ring Stabilization through FtsZ Cross-linking.

    PubMed

    Schumacher, Maria A; Huang, Kuo-Hsiang; Zeng, Wenjie; Janakiraman, Anuradha

    2017-03-03

    Cell division in most bacteria is mediated by the tubulin-like FtsZ protein, which polymerizes in a GTP-dependent manner to form the cytokinetic Z ring. A diverse repertoire of FtsZ-binding proteins affects FtsZ localization and polymerization to ensure correct Z ring formation. Many of these proteins bind the C-terminal domain (CTD) of FtsZ, which serves as a hub for FtsZ regulation. FtsZ ring-associated proteins, ZapA-D (Zaps), are important FtsZ regulatory proteins that stabilize FtsZ assembly and enhance Z ring formation by increasing lateral assembly of FtsZ protofilaments, which then form the Z ring. There are no structures of a Zap protein bound to FtsZ; therefore, how these proteins affect FtsZ polymerization has been unclear. Recent data showed ZapD binds specifically to the FtsZ CTD. Thus, to obtain insight into the ZapD-CTD interaction and how it may mediate FtsZ protofilament assembly, we determined the Escherichia coli ZapD-FtsZ CTD structure to 2.67 Å resolution. The structure shows that the CTD docks within a hydrophobic cleft in the ZapD helical domain and adopts an unusual structure composed of two turns of helix separated by a proline kink. FtsZ CTD residue Phe-377 inserts into the ZapD pocket, anchoring the CTD in place and permitting hydrophobic contacts between FtsZ residues Ile-374, Pro-375, and Leu-378 with ZapD residues Leu-74, Trp-77, Leu-91, and Leu-174. The structural findings were supported by mutagenesis coupled with biochemical and in vivo studies. The combined data suggest that ZapD acts as a molecular cross-linking reagent between FtsZ protofilaments to enhance FtsZ assembly.

  15. The GTPase Activity of Escherichia coli FtsZ Determines the Magnitude of the FtsZ Polymer Bundling by ZapA in Vitro†

    PubMed Central

    2009-01-01

    FtsZ polymerizes in a ring-like structure at mid cell to initiate cell division in Escherichia coli. The ring is stabilized by a number of proteins among which the widely conserved ZapA protein. Using antibodies against ZapA, we found surprisingly that the cellular concentration of ZapA is approximately equal to that of FtsZ. This raised the question of how the cell can prevent their interaction and thereby the premature stabilization of FtsZ protofilaments in nondividing cells. Therefore, we studied the FtsZ−ZapA interaction at the physiological pH of 7.5 instead of pH 6.5 (the optimal pH for FtsZ polymerization), under conditions that stimulate protofilament formation (5 mM MgCl2) and under conditions that stimulate and stabilize protofilaments (10 mM MgCl2). Using pelleting, light scattering, and GTPase assays, it was found that stabilization and bundling of FtsZ polymers by ZapA was inversely correlated to the GTPase activity of FtsZ. As GTP hydrolysis is the rate-limiting factor for depolymerization of FtsZ, we propose that ZapA will only enhance the cooperativity of polymer association during the transition from helical filament to mid cell ring and will not stabilize the short single protofilaments in the cytoplasm. All thus far published in vitro data on the interaction between FtsZ and ZapA have been obtained with His-ZapA. We found that in our case the presence of a His tag fused to ZapA prevented the protein to complement a ΔzapA strain in vivo and that it affected the interaction between FtsZ and ZapA in vitro. PMID:19842714

  16. Cytological Profile of Antibacterial FtsZ Inhibitors and Synthetic Peptide MciZ

    PubMed Central

    Araújo-Bazán, Lidia; Ruiz-Avila, Laura B.; Andreu, David; Huecas, Sonia; Andreu, José M.

    2016-01-01

    Cell division protein FtsZ is the organizer of the cytokinetic ring in almost all bacteria and a target for the discovery of new antibacterial agents that are needed to counter widespread antibiotic resistance. Bacterial cytological profiling, using quantitative microscopy, is a powerful approach for identifying the mechanism of action of antibacterial molecules affecting different cellular pathways. We have determined the cytological profile on Bacillus subtilis cells of a selection of small molecule inhibitors targeting FtsZ on different binding sites. FtsZ inhibitors lead to long undivided cells, impair the normal assembly of FtsZ into the midcell Z-rings, induce aberrant ring distributions, punctate FtsZ foci, membrane spots and also modify nucleoid length. Quantitative analysis of cell and nucleoid length combined, or the Z-ring distribution, allows categorizing FtsZ inhibitors and to distinguish them from antibiotics with other mechanisms of action, which should be useful for identifying new antibacterial FtsZ inhibitors. Biochemical assays of FtsZ polymerization and GTPase activity combined explain the cellular effects of the FtsZ polymer stabilizing agent PC190723 and its fragments. MciZ is a 40-aminoacid endogenous inhibitor of cell division normally expressed during sporulation in B. subtilis. Using FtsZ cytological profiling we have determined that exogenous synthetic MciZ is an effective inhibitor of B. subtilis cell division, Z-ring formation and localization. This finding supports our cell-based approach to screen for FtsZ inhibitors and opens new possibilities for peptide inhibitors of bacterial cell division. PMID:27752253

  17. Imaging-based identification of a critical regulator of FtsZ protofilament curvature in Caulobacter

    PubMed Central

    Goley, Erin D.; Dye, Natalie A.; Werner, John N.; Gitai, Zemer; Shapiro, Lucy

    2010-01-01

    SUMMARY FtsZ is an essential bacterial GTPase that polymerizes at midcell, recruits the division machinery, and may generate constrictive forces necessary for cytokinesis. However, many of the mechanistic details underlying these functions are unknown. We sought to identify FtsZ-binding proteins that influence FtsZ function in Caulobacter crescentus. Here, we present a microscopy-based screen through which we discovered two FtsZ-binding proteins, FzlA and FzlC. FzlA is conserved in α-proteobacteria and was found to be functionally critical for cell division in Caulobacter. FzlA altered FtsZ structure both in vivo and in vitro, forming stable higher order structures that were resistant to depolymerization by MipZ, a spatial determinant of FtsZ assembly. Electron microscopy revealed that FzlA organizes FtsZ protofilaments into striking helical bundles. The degree of curvature induced by FzlA depended on the nucleotide bound to FtsZ. Induction of FtsZ curvature by FzlA carries implications for regulating FtsZ function by modulating its superstructure. PMID:20864042

  18. Bacterial Division: FtsZ Treadmills to Build a Beautiful Wall.

    PubMed

    Schoenemann, Kara M; Margolin, William

    2017-04-24

    The tubulin-like FtsZ protein polymerizes into a contractile ring structure required for cytokinesis in most bacteria. Two new studies report that FtsZ polymers move around the ring by treadmilling, which guides and regulates the inward growth of the septal wall. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Synthesis and Evaluation of Quinazolines as Inhibitors of the Bacterial Cell Division Protein FtsZ.

    PubMed

    Nepomuceno, Gabriella M; Chan, Katie M; Huynh, Valerie; Martin, Kevin S; Moore, Jared T; O'Brien, Terrence E; Pollo, Luiz A E; Sarabia, Francisco J; Tadeus, Clarissa; Yao, Zi; Anderson, David E; Ames, James B; Shaw, Jared T

    2015-03-12

    The bacterial cell division protein FtsZ is one of many potential targets for the development of novel antibiotics. Recently, zantrin Z3 was shown to be a cross-species inhibitor of FtsZ; however, its specific interactions with the protein are still unknown. Herein we report the synthesis of analogues that contain a more tractable core structure and an analogue with single-digit micromolar inhibition of FtsZ's GTPase activity, which represents the most potent inhibitor of Escherichia coli FtsZ reported to date. In addition, the zantrin Z3 core has been converted to two potential photo-cross-linking reagents for proteomic studies that could shed light on the molecular interactions between FtsZ and molecules related to zantrin Z3.

  20. Cloning and characterization of ftsZ and pyrF from the archaeon Thermoplasma acidophilum.

    PubMed

    Yaoi, T; Laksanalamai, P; Jiemjit, A; Kagawa, H K; Alton, T; Trent, J D

    2000-09-07

    To characterize cytoskeletal components of archaea, the ftsZ gene from Thermoplasma acidophilum was cloned and sequenced. In T. acidophilum ftsZ, which is involved in cell division, was found to be in an operon with the pyrF gene, which encodes orotidine-5'-monophosphate decarboxylase (ODC), an essential enzyme in pyrimidine biosynthesis. Both ftsZ and pyrF from T. acidophilum were expressed in Escherichia coli and formed functional proteins. FtsZ expression in wild-type E. coli resulted in the filamentous phenotype characteristic of ftsZ mutants. T. acidophilum pyrF expression in an E. coli mutant lacking pyrF complemented the mutation and rescued the strain. Sequence alignments of ODCs from archaea, bacteria, and eukarya reveal five conserved regions, two of which have homology to 3-hexulose-6-phosphate synthase (HPS), suggesting a common substrate recognition and binding motif. Copyright 2000 Academic Press.

  1. Cloning and characterization of ftsZ and pyrF from the archaeon Thermoplasma acidophilum

    NASA Technical Reports Server (NTRS)

    Yaoi, T.; Laksanalamai, P.; Jiemjit, A.; Kagawa, H. K.; Alton, T.; Trent, J. D.

    2000-01-01

    To characterize cytoskeletal components of archaea, the ftsZ gene from Thermoplasma acidophilum was cloned and sequenced. In T. acidophilum ftsZ, which is involved in cell division, was found to be in an operon with the pyrF gene, which encodes orotidine-5'-monophosphate decarboxylase (ODC), an essential enzyme in pyrimidine biosynthesis. Both ftsZ and pyrF from T. acidophilum were expressed in Escherichia coli and formed functional proteins. FtsZ expression in wild-type E. coli resulted in the filamentous phenotype characteristic of ftsZ mutants. T. acidophilum pyrF expression in an E. coli mutant lacking pyrF complemented the mutation and rescued the strain. Sequence alignments of ODCs from archaea, bacteria, and eukarya reveal five conserved regions, two of which have homology to 3-hexulose-6-phosphate synthase (HPS), suggesting a common substrate recognition and binding motif. Copyright 2000 Academic Press.

  2. Sequence Variation in the ftsZ Gene of Bartonella henselae Isolates and Clinical Samples

    PubMed Central

    Ehrenborg, C.; Wesslén, L.; Jakobson, Å.; Friman, G.; Holmberg, M.

    2000-01-01

    In a search for methods for subtyping of Bartonella henselae in clinical samples, we amplified and sequenced a 701-bp region in the 3′ end of the ftsZ gene in 15 B. henselae isolates derived from cats and humans in the United States and Europe. The ftsZ sequence variants that were discovered were designated variants Bh ftsZ 1, 2, and 3 and were compared with 16S rRNA genotypes I and II of the same isolates. There was no ftsZ gene variation in the strains of 16S rRNA type I, all of which were Bh ftsZ 1. The type II strains constituted two groups, with nucleotide sequence variation in the ftsZ gene resulting in amino acid substitutions at three positions, one of which was shared by the two groups. One 16S rRNA type II isolate had an ftsZ gene sequence identical to those of the type I strains. Variants Bh ftsZ 1 and 2 were detected in tissue specimens from seven Swedish patients with diagnoses such as chronic multifocal osteomyelitis, cardiomyopathy, and lymphadenopathy. Patients with similar clinical entities displayed either Bh ftsZ variant. The etiological role of B. henselae in these patients was supported by positive Bartonella antibody titers and/or amplification and sequencing of a part of the B. henselae gltA gene. B. henselae ftsZ gene sequence variation may be useful in providing knowledge about the epidemiology of various B. henselae strains in clinical samples, especially when isolation attempts have failed. This report also describes manifestations of atypical Bartonella infections in Sweden. PMID:10655367

  3. Doxorubicin inhibits E. coli division by interacting at a novel site in FtsZ.

    PubMed

    Panda, Pragnya; Taviti, Ashoka Chary; Satpati, Suresh; Kar, Mitali Madhusmita; Dixit, Anshuman; Beuria, Tushar Kant

    2015-11-01

    The increase in antibiotic resistance has become a major health concern in recent times. It is therefore essential to identify novel antibacterial targets as well as discover and develop new antibacterial agents. FtsZ, a highly conserved bacterial protein, is responsible for the initiation of cell division in bacteria. The functions of FtsZ inside cells are tightly regulated and any perturbation in its functions leads to inhibition of bacterial division. Recent reports indicate that small molecules targeting the functions of FtsZ may be used as leads to develop new antibacterial agents. To identify small molecules targeting FtsZ and inhibiting bacterial division, we screened a U.S. FDA (Food and Drug Administration)-approved drug library of 800 molecules using an independent computational, biochemical and microbial approach. From this screen, we identified doxorubicin, an anthracycline molecule that inhibits Escherichia coli division and forms filamentous cells. A fluorescence-binding assay shows that doxorubicin interacts strongly with FtsZ. A detailed biochemical analysis demonstrated that doxorubicin inhibits FtsZ assembly and its GTPase activity through binding to a site other than the GTP-binding site. Furthermore, using molecular docking, we identified a probable doxorubicin-binding site in FtsZ. A number of single amino acid mutations at the identified binding site in FtsZ resulted in a severalfold decrease in the affinity of FtsZ for doxorubicin, indicating the importance of this site for doxorubicin interaction. The present study suggests the presence of a novel binding site in FtsZ that interacts with the small molecules and can be targeted for the screening and development of new antibacterial agents.

  4. Decomposition of saltmeadow cordgrass (Spartina patens) in Louisiana coastal marshes

    USGS Publications Warehouse

    Foote, A.L.; Reynolds, K.A.

    1997-01-01

    In Louisiana, plant production rates and associated decomposition rates may be important in offsetting high rates of land loss and subsidence in organic marsh soils. Decomposition of Spartina patens shoot and leaf material was studied by using litter bags in mesohaline marshes in the Barataria and Terrebonne basins of coastal Louisiana. Spartina patens decomposed very slowly with an average decay constant of 0.0007, and approximately 50% of the material remained after 2 years in the field. Material at the Barataria site decomposed faster than did Terrebonne material with trend differences apparent during the first 150 days. This difference might be explained by the higher content of phosphorus in the Barataria material or a flooding period experienced by the Barataria bags during their first 10 days of deployment. Nitrogen and carbon content of the plant material studied did not differ between the two basins. We detected no consistent significant differences in decomposition above, at, or below sediment/water level. Because S. patens is the dominant plant in these marshes, and because it is so slow to decompose, we believe that S. patens shoots are an important addition to vertical accretion and, therefore, marsh elevation.

  5. FtsZ rings and helices: physical mechanisms for the dynamic alignment of biopolymers in rod-shaped bacteria

    NASA Astrophysics Data System (ADS)

    Fischer-Friedrich, Elisabeth; Friedrich, Benjamin M.; Gov, Nir S.

    2012-02-01

    In many bacterial species, the protein FtsZ forms a cytoskeletal ring that marks the future division site and scaffolds the division machinery. In rod-shaped bacteria, most frequently membrane-attached FtsZ rings or ring fragments are reported and occasionally helices. By contrast, axial FtsZ clusters have never been reported. In this paper, we investigate theoretically how dynamic FtsZ aggregates align in rod-shaped bacteria. We study systematically different physical mechanisms that affect the alignment of FtsZ polymers using a computational model that relies on autocatalytic aggregation of FtsZ filaments at the membrane. Our study identifies a general tool kit of physical and geometrical mechanisms by which rod-shaped cells align biopolymer aggregates. Our analysis compares the relative impact of each mechanism on the circumferential alignment of FtsZ as observed in rod-shaped bacteria. We determine spontaneous curvature of FtsZ polymers and axial confinement of FtsZ on the membrane as the strongest factors. Including Min oscillations in our model, we find that these stabilize axial and helical clusters on short time scales, but promote the formation of an FtsZ ring at the cell middle at longer times. This effect could provide an explanation to the long standing puzzle of transiently observed oscillating FtsZ helices in Escherichia coli cells prior to cell division.

  6. Organization of FtsZ Filaments in the Bacterial Division Ring Measured from Polarized Fluorescence Microscopy

    PubMed Central

    Si, Fangwei; Busiek, Kimberly; Margolin, William; Sun, Sean X.

    2013-01-01

    Cytokinesis in bacteria is accomplished by a ring-shaped cell-division complex (the Z-ring). The primary component of the Z-ring is FtsZ, a filamentous tubulin homolog that serves as a scaffold for the recruitment of other cell-division-related proteins. FtsZ forms filaments and bundles. In the cell, it has been suggested that FtsZ filaments form the arcs of the ring and are aligned in the cell-circumferential direction. Using polarized fluorescence microscopy in live Escherichia coli cells, we measure the structural organization of FtsZ filaments in the Z-ring. The data suggest a disordered organization: a substantial portion of FtsZ filaments are aligned in the cell-axis direction. FtsZ organization in the Z-ring also appears to depend on the bacterial species. Taken together, the unique arrangement of FtsZ suggests novel unexplored mechanisms in bacterial cell division. PMID:24209842

  7. Transcription of ftsZ oscillates during the cell cycle of Escherichia coli.

    PubMed

    Garrido, T; Sánchez, M; Palacios, P; Aldea, M; Vicente, M

    1993-10-01

    The FtsZ protein is a key element controlling cell division in Escherichia coli. A powerful transcription titration assay was used to quantify the ftsZ mRNA present in synchronously dividing cells. The ftsZ mRNA levels oscillate during the cell cycle reaching a maximum at about the time DNA replication initiates. This cell cycle dependency is specifically due to the two proximal ftsZ promoters. A strain was constructed in which expression of ftsZ could be modulated by an exogenous inducer. In this strain cell size and cell division frequency were sensitive to the cellular FtsZ contents, demonstrating the rate-limiting role of this protein in cell division. Transcriptional activity of the ftsZ promoters was found to be independent of DnaA, indicating that DNA replication and cell division may be independently controlled at the time when new rounds of DNA replication are initiated. This suggests a parallelism between the prokaryotic cell cycle signals and the START point of eukaryotic cell cycles.

  8. Transcription of ftsZ oscillates during the cell cycle of Escherichia coli.

    PubMed Central

    Garrido, T; Sánchez, M; Palacios, P; Aldea, M; Vicente, M

    1993-01-01

    The FtsZ protein is a key element controlling cell division in Escherichia coli. A powerful transcription titration assay was used to quantify the ftsZ mRNA present in synchronously dividing cells. The ftsZ mRNA levels oscillate during the cell cycle reaching a maximum at about the time DNA replication initiates. This cell cycle dependency is specifically due to the two proximal ftsZ promoters. A strain was constructed in which expression of ftsZ could be modulated by an exogenous inducer. In this strain cell size and cell division frequency were sensitive to the cellular FtsZ contents, demonstrating the rate-limiting role of this protein in cell division. Transcriptional activity of the ftsZ promoters was found to be independent of DnaA, indicating that DNA replication and cell division may be independently controlled at the time when new rounds of DNA replication are initiated. This suggests a parallelism between the prokaryotic cell cycle signals and the START point of eukaryotic cell cycles. Images PMID:8404863

  9. Effects of rhodomyrtone on Gram-positive bacterial tubulin homologue FtsZ

    PubMed Central

    Saeloh, Dennapa; Wenzel, Michaela; Rungrotmongkol, Thanyada; Hamoen, Leendert Willem

    2017-01-01

    Rhodomyrtone, a natural antimicrobial compound, displays potent activity against many Gram-positive pathogenic bacteria, comparable to last-defence antibiotics including vancomycin and daptomycin. Our previous studies pointed towards effects of rhodomyrtone on the bacterial membrane and cell wall. In addition, a recent molecular docking study suggested that the compound could competitively bind to the main bacterial cell division protein FtsZ. In this study, we applied a computational approach (in silico), in vitro, and in vivo experiments to investigate molecular interactions of rhodomyrtone with FtsZ. Using molecular simulation, FtsZ conformational changes were observed in both (S)- and (R)-rhodomyrtone binding states, compared with the three natural states of FtsZ (ligand-free, GDP-, and GTP-binding states). Calculations of free binding energy showed a higher affinity of FtsZ to (S)-rhodomyrtone (−35.92 ± 0.36 kcal mol−1) than the GDP substrate (−23.47 ± 0.25 kcal mol−1) while less affinity was observed in the case of (R)-rhodomyrtone (−18.11 ± 0.11 kcal mol−1). In vitro experiments further revealed that rhodomyrtone reduced FtsZ polymerization by 36% and inhibited GTPase activity by up to 45%. However, the compound had no effect on FtsZ localization in Bacillus subtilis at inhibitory concentrations and cells also did not elongate after treatment. Higher concentrations of rhodomyrtone did affect localization of FtsZ and also affected localization of its membrane anchor proteins FtsA and SepF, showing that the compound did not specifically inhibit FtsZ but rather impaired multiple divisome proteins. Furthermore, a number of cells adopted a bean-like shape suggesting that rhodomyrtone possibly possesses further targets involved in cell envelope synthesis and/or maintenance. PMID:28168121

  10. Synthetic inhibitors of bacterial cell division targeting the GTP-binding site of FtsZ.

    PubMed

    Ruiz-Avila, Laura B; Huecas, Sonia; Artola, Marta; Vergoñós, Albert; Ramírez-Aportela, Erney; Cercenado, Emilia; Barasoain, Isabel; Vázquez-Villa, Henar; Martín-Fontecha, Mar; Chacón, Pablo; López-Rodríguez, María L; Andreu, José M

    2013-09-20

    Cell division protein FtsZ is the organizer of the cytokinetic Z-ring in most bacteria and a target for new antibiotics. FtsZ assembles with GTP into filaments that hydrolyze the nucleotide at the association interface between monomers and then disassemble. We have replaced FtsZ's GTP with non-nucleotide synthetic inhibitors of bacterial division. We searched for these small molecules among compounds from the literature, from virtual screening (VS), and from our in-house synthetic library (UCM), employing a fluorescence anisotropy primary assay. From these screens we have identified the polyhydroxy aromatic compound UCM05 and its simplified analogue UCM44 that specifically bind to Bacillus subtilis FtsZ monomers with micromolar affinities and perturb normal assembly, as examined with light scattering, polymer sedimentation, and negative stain electron microscopy. On the other hand, these ligands induce the cooperative assembly of nucleotide-devoid archaeal FtsZ into distinct well-ordered polymers, different from GTP-induced filaments. These FtsZ inhibitors impair localization of FtsZ into the Z-ring and inhibit bacterial cell division. The chlorinated analogue UCM53 inhibits the growth of clinical isolates of antibiotic-resistant Staphylococcus aureus and Enterococcus faecalis. We suggest that these interfacial inhibitors recapitulate binding and some assembly-inducing effects of GTP but impair the correct structural dynamics of FtsZ filaments and thus inhibit bacterial division, possibly by binding to a small fraction of the FtsZ molecules in a bacterial cell, which opens a new approach to FtsZ-based antibacterial drug discovery.

  11. A Carbocyclic Curcumin Inhibits Proliferation of Gram-Positive Bacteria by Targeting FtsZ.

    PubMed

    Groundwater, Paul W; Narlawar, Rajeshwar; Liao, Vivian Wan Yu; Bhattacharya, Anusri; Srivastava, Shalini; Kunal, Kishore; Doddareddy, Munikumar; Oza, Pratik M; Mamidi, Ramesh; Marrs, Emma C L; Perry, John D; Hibbs, David E; Panda, Dulal

    2017-01-24

    Inhibition of FtsZ assembly has been found to stall bacterial cell division. Here, we report the identification of a potent carbocyclic curcumin analogue (2d) that inhibits Bacillus subtilis 168 cell proliferation by targeting the assembly of FtsZ. 2d also showed potent inhibitory activity (minimum inhibitory concentrations of 2-4 mg/L) against several clinically important species of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. In addition, 2d displayed a significantly reduced inhibitory effect on human cervical cancer cells in comparison to its effect on bacterial cells. Using live cell imaging of GFP-FtsZ by confocal microscopy, 2d was found to rapidly perturb the cytokinetic FtsZ rings in Bacillus subtilis cells. The immunofluorescence imaging of FtsZ also showed that 2d destroyed the Z-ring in bacteria within 5 min. Prolonged treatment with 2d produced filamentous bacteria, but 2d had no detectable effect either on the nucleoids or on the membrane potential of bacteria. 2d inhibited FtsZ assembly in vitro, whereas it had minimal effects on tubulin assembly. Interestingly, 2d strongly enhanced the GTPase activity of FtsZ and reduced the GTPase activity of tubulin. Furthermore, 2d bound to purified FtsZ with a dissociation constant of 4.0 ± 1.1 μM, and the binding of 2d altered the secondary structures of FtsZ. The results together suggested that the non-natural curcumin analogue 2d possesses powerful antibacterial activity against important pathogenic bacteria, and the evidence indicates that 2d inhibits bacterial proliferation by targeting FtsZ.

  12. Escherichia coli FtsA forms lipid-bound minirings that antagonize lateral interactions between FtsZ protofilaments

    PubMed Central

    Krupka, Marcin; Rowlett, Veronica W.; Morado, Dustin; Vitrac, Heidi; Schoenemann, Kara; Liu, Jun; Margolin, William

    2017-01-01

    Most bacteria divide using a protein machine called the divisome that spans the cytoplasmic membrane. Key divisome proteins on the membrane’s cytoplasmic side include tubulin-like FtsZ, which forms GTP-dependent protofilaments, and actin-like FtsA, which tethers FtsZ to the membrane. Here we present genetic evidence that in Escherichia coli, FtsA antagonizes FtsZ protofilament bundling in vivo. We then show that purified FtsA does not form straight polymers on lipid monolayers as expected, but instead assembles into dodecameric minirings, often in hexameric arrays. When coassembled with FtsZ on lipid monolayers, these FtsA minirings appear to guide FtsZ to form long, often parallel, but unbundled protofilaments, whereas a mutant of FtsZ (FtsZ*) with stronger lateral interactions remains bundled. In contrast, a hypermorphic mutant of FtsA (FtsA*) forms mainly arcs instead of minirings and enhances lateral interactions between FtsZ protofilaments. Based on these results, we propose that FtsA antagonizes lateral interactions between FtsZ protofilaments, and that the oligomeric state of FtsA may influence FtsZ higher-order structure and divisome function. PMID:28695917

  13. Targeting cell division: Small-molecule inhibitors of FtsZ GTPase perturb cytokinetic ring assembly and induce bacterial lethality

    PubMed Central

    Margalit, Danielle N.; Romberg, Laura; Mets, Rebecca B.; Hebert, Alan M.; Mitchison, Timothy J.; Kirschner, Marc W.; RayChaudhuri, Debabrata

    2004-01-01

    FtsZ, the ancestral homolog of eukaryotic tubulins, is a GTPase that assembles into a cytokinetic ring structure essential for cell division in prokaryotic cells. Similar to tubulin, purified FtsZ polymerizes into dynamic protofilaments in the presence of GTP; polymer assembly is accompanied by GTP hydrolysis. We used a high-throughput protein-based chemical screen to identify small molecules that target assembly-dependent GTPase activity of FtsZ. Here, we report the identification of five structurally diverse compounds, named Zantrins, which inhibit FtsZ GTPase either by destabilizing the FtsZ protofilaments or by inducing filament hyperstability through increased lateral association. These two classes of FtsZ inhibitors are reminiscent of the antitubulin drugs colchicine and Taxol, respectively. We also show that Zantrins perturb FtsZ ring assembly in Escherichia coli cells and cause lethality to a variety of bacteria in broth cultures, indicating that FtsZ antagonists may serve as chemical leads for the development of new broad-spectrum antibacterial agents. Our results illustrate the utility of small-molecule chemical probes to study FtsZ polymerization dynamics and the feasibility of FtsZ as a novel therapeutic target. PMID:15289600

  14. Synthesis and Evaluation of Quinazolines as Inhibitors of the Bacterial Cell Division Protein FtsZ

    PubMed Central

    2015-01-01

    The bacterial cell division protein FtsZ is one of many potential targets for the development of novel antibiotics. Recently, zantrin Z3 was shown to be a cross-species inhibitor of FtsZ; however, its specific interactions with the protein are still unknown. Herein we report the synthesis of analogues that contain a more tractable core structure and an analogue with single-digit micromolar inhibition of FtsZ’s GTPase activity, which represents the most potent inhibitor of Escherichia coli FtsZ reported to date. In addition, the zantrin Z3 core has been converted to two potential photo-cross-linking reagents for proteomic studies that could shed light on the molecular interactions between FtsZ and molecules related to zantrin Z3. PMID:25815151

  15. SB-RA-2001 Inhibits Bacterial Proliferation by Targeting FtsZ Assembly

    PubMed Central

    2015-01-01

    FtsZ has been recognized as a promising antimicrobial drug target because of its vital role in bacterial cell division. In this work, we found that a taxane SB-RA-2001 inhibited the proliferation of Bacillus subtilis 168 and Mycobacterium smegmatis cells with minimal inhibitory concentrations of 38 and 60 μM, respectively. Cell lengths of these microorganisms increased remarkably in the presence of SB-RA-2001, indicating that it inhibits bacterial cytokinesis. SB-RA-2001 perturbed the formation of the FtsZ ring in B. subtilis 168 cells and also affected the localization of the late cell division protein, DivIVA, at the midcell position. Flow cytometric analysis of the SB-RA-2001-treated cells indicated that the compound did not affect the duplication of DNA in B. subtilis 168 cells. Further, SB-RA-2001 treatment did not affect the localization of the chromosomal partitioning protein, Spo0J, along the two ends of the nucleoids and also had no discernible effect on the nucleoid segregation in B. subtilis 168 cells. The agent also did not appear to perturb the membrane potential of B. subtilis 168 cells. In vitro, SB-RA-2001 bound to FtsZ with modest affinity, promoted the assembly and bundling of FtsZ protofilaments, and reduced the GTPase activity of FtsZ. GTP did not inhibit the binding of SB-RA-2001 to FtsZ, suggesting that it does not bind to the GTP binding site on FtsZ. A computational analysis indicated that SB-RA-2001 binds to FtsZ in the cleft region between the C-terminal domain and helix H7, and the binding site of SB-RA-2001 on FtsZ resembled that of PC190723, a well-characterized inhibitor of FtsZ. The findings collectively suggested that SB-RA-2001 inhibits bacterial proliferation by targeting the assembly dynamics of FtsZ, and this can be exploited further to develop potent FtsZ-targeted antimicrobials. PMID:24749867

  16. SB-RA-2001 inhibits bacterial proliferation by targeting FtsZ assembly.

    PubMed

    Singh, Dipty; Bhattacharya, Anusri; Rai, Ankit; Dhaked, Hemendra Pal Singh; Awasthi, Divya; Ojima, Iwao; Panda, Dulal

    2014-05-13

    FtsZ has been recognized as a promising antimicrobial drug target because of its vital role in bacterial cell division. In this work, we found that a taxane SB-RA-2001 inhibited the proliferation of Bacillus subtilis 168 and Mycobacterium smegmatis cells with minimal inhibitory concentrations of 38 and 60 μM, respectively. Cell lengths of these microorganisms increased remarkably in the presence of SB-RA-2001, indicating that it inhibits bacterial cytokinesis. SB-RA-2001 perturbed the formation of the FtsZ ring in B. subtilis 168 cells and also affected the localization of the late cell division protein, DivIVA, at the midcell position. Flow cytometric analysis of the SB-RA-2001-treated cells indicated that the compound did not affect the duplication of DNA in B. subtilis 168 cells. Further, SB-RA-2001 treatment did not affect the localization of the chromosomal partitioning protein, Spo0J, along the two ends of the nucleoids and also had no discernible effect on the nucleoid segregation in B. subtilis 168 cells. The agent also did not appear to perturb the membrane potential of B. subtilis 168 cells. In vitro, SB-RA-2001 bound to FtsZ with modest affinity, promoted the assembly and bundling of FtsZ protofilaments, and reduced the GTPase activity of FtsZ. GTP did not inhibit the binding of SB-RA-2001 to FtsZ, suggesting that it does not bind to the GTP binding site on FtsZ. A computational analysis indicated that SB-RA-2001 binds to FtsZ in the cleft region between the C-terminal domain and helix H7, and the binding site of SB-RA-2001 on FtsZ resembled that of PC190723, a well-characterized inhibitor of FtsZ. The findings collectively suggested that SB-RA-2001 inhibits bacterial proliferation by targeting the assembly dynamics of FtsZ, and this can be exploited further to develop potent FtsZ-targeted antimicrobials.

  17. FtsZ Directs a Second Mode of Peptidoglycan Synthesis in Escherichia coli▿

    PubMed Central

    Varma, Archana; de Pedro, Miguel A.; Young, Kevin D.

    2007-01-01

    Certain penicillin binding protein mutants of Escherichia coli grow with spirillum-like morphologies when the FtsZ protein is inhibited, suggesting that FtsZ might govern aspects of cell wall growth other than those strictly associated with septation. While investigating the mechanism of spiral cell formation, we discovered conditions for visualizing this second function of FtsZ. Normally, inhibiting the cytoskeleton protein MreB forces E. coli cells to grow as smoothly enlarging spheres from which the poles disappear, yielding coccoid or lemon-shaped forms. However, when FtsZ and MreB were inhibited simultaneously in a strain lacking PBP 5 and PBP 7, the resulting cells ballooned outward but retained conspicuous rod-shaped extensions at sites representing the original poles. This visual phenotype was paralleled by the biochemistry of sacculus growth. Muropeptides are usually inserted homogeneously into the lateral cell walls, but when FtsZ polymerization was inhibited, the incorporation of new material occurred mainly in the central regions of cells and was significantly lower in those portions of side walls abutting a pole. Thus, reduced precursor incorporation into side walls near the poles explained why these regions retained their rod-like morphology while the rest of the cell grew spherically. Also, inhibiting FtsZ increased the amount of pentapeptides in sacculi by about one-third. Finally, the MreB protein directed the helical or diagonal incorporation of new peptidoglycan into the wall, but the location of that incorporation depended on whether FtsZ was active. In sum, the results indicate that in addition to nucleating cell septation in E. coli, FtsZ can direct the insertion of new peptidoglycan into portions of the lateral wall. PMID:17513471

  18. FtsZ and the division of prokaryotic cells and organelles.

    PubMed

    Margolin, William

    2005-11-01

    Binary fission of many prokaryotes as well as some eukaryotic organelles depends on the FtsZ protein, which self-assembles into a membrane-associated ring structure early in the division process. FtsZ is homologous to tubulin, the building block of the microtubule cytoskeleton in eukaryotes. Recent advances in genomics and cell-imaging techniques have paved the way for the remarkable progress in our understanding of fission in bacteria and organelles.

  19. Genetic Evidence for Inhibition of Bacterial Division Protein FtsZ by Berberine

    PubMed Central

    Boberek, Jaroslaw M.; Stach, Jem; Good, Liam

    2010-01-01

    Background Berberine is a plant alkaloid that is widely used as an anti-infective in traditional medicine. Escherichia coli exposed to berberine form filaments, suggesting an antibacterial mechanism that involves inhibition of cell division. Berberine is a DNA ligand and may induce filamentation through induction of the SOS response. Also, there is biochemical evidence for berberine inhibition of the cell division protein FtsZ. Here we aimed to assess possible berberine mechanism(s) of action in growing bacteria using genetics tools. Methodology/Principal Findings First, we tested whether berberine inhibits bacterial growth through DNA damage and induction of the SOS response. The SOS response induced by berberine was much lower compared to that induced by mitomycin C in an SOS response reporter strain. Also, cell filamentation was observed in an SOS-negative E. coli strain. To test whether berberine inhibits FtsZ, we assessed its effects on formation of the cell division Z-rings, and observed a dramatic reduction in Z-rings in the presence of berberine. We next used two different strategies for RNA silencing of ftsZ and both resulted in sensitisation of bacteria to berberine, visible as a drop in the Minimum Inhibitory Concentration (MIC). Furthermore, Fractional Inhibitory Concentration Indices (FICIs) showed a high level of synergy between ftsZ silencing and berberine treatment (FICI values of 0.23 and 0.25 for peptide nucleic acid- and expressed antisense RNA-based silencing of ftsZ, respectively). Finally, over-expression of ftsZ led to a mild rescue effect in berberine-treated cells. Conclusions The results argue against DNA binding as the primary mechanism of action of berberine and support the hypothesis that its antibacterial properties are due to inhibition of the cell division protein FtsZ. In addition, the genetic approach used here provides a means to rapidly test the activity of other putative FtsZ inhibitors. PMID:21060782

  20. Transcription of the ftsZ gene and cell division in Escherichia coli.

    PubMed Central

    Robin, A; Joseleau-Petit, D; D'Ari, R

    1990-01-01

    The ftsZ gene of Escherichia coli, which lies in a cluster of cell division genes at 2 min on the genetic map, codes for a protein which is thought to play a key role in triggering cell division. Using an ftsZ::lacZ operon fusion, we have studied the transcription of the ftsZ gene under conditions in which cell division was either inhibited or synchronized in the bacterial population. In ftsZ, ftsA, ftsQ, and ftsI (or pbpB) mutants, there was no change in the differential rate of expression of the ftsZ gene in nonpermissive conditions, when cell division was completely blocked. Although the FtsZ protein is thought to be limiting for cell division, in synchronized cultures the ftsZ gene was expressed not only at the moment of septation initiation but throughout the cell cycle. Its expression, however, was not exponential but linear, with a rapid doubling in rate at a specific cell age; this age, about 20 min after division in a 60-min cycle, was different from the age at which the ftsZ::lacZ operon was duplicated. However, it was close to the age at which replication initiated and at which the rate of phospholipid synthesis doubled. During the transient division inhibition after a nutritional shift-up, ftsZ transcription again became linear, with two doublings in rate at intervals equal to the mass doubling time in the rich medium; it adopted the exponential rate typical of rich medium about 60 min after the shift-up, just before the bacterial population resumed cell division. The doubling in the rate of ftsZ transcription once per cycle in synchronized cultures and once per mass doubling time during the transition period after a nutritional shift-up reflects a new cell cycle event. PMID:2106510

  1. Charged Molecules Modulate the Volume Exclusion Effects Exerted by Crowders on FtsZ Polymerization

    PubMed Central

    Monterroso, Begoña; Reija, Belén; Jiménez, Mercedes; Zorrilla, Silvia; Rivas, Germán

    2016-01-01

    We have studied the influence of protein crowders, either combined or individually, on the GTP-induced FtsZ cooperative assembly, crucial for the formation of the dynamic septal ring and, hence, for bacterial division. It was earlier demonstrated that high concentrations of inert polymers like Ficoll 70, used to mimic the crowded cellular interior, favor the assembly of FtsZ into bundles with slow depolymerization. We have found, by fluorescence anisotropy together with light scattering measurements, that the presence of protein crowders increases the tendency of FtsZ to polymerize at micromolar magnesium concentration, being the effect larger with ovomucoid, a negatively charged protein. Neutral polymers and a positively charged protein also diminished the critical concentration of assembly, the extent of the effect being compatible with that expected according to pure volume exclusion models. FtsZ polymerization was also observed to be strongly promoted by a negatively charged polymer, DNA, and by some unrelated polymers like PEGs at concentrations below the crowding regime. The influence of mixed crowders mimicking the heterogeneity of the intracellular environment on the tendency of FtsZ to assemble was also studied and nonadditive effects were found to prevail. Far from exactly reproducing the bacterial cytoplasm environment, this approach serves as a simplified model illustrating how its intrinsically crowded and heterogeneous nature may modulate FtsZ assembly into a functional Z-ring. PMID:26870947

  2. Novel Trisubstituted Benzimidazoles, Targeting Mtb FtsZ, As A New Class of Antitubercular Agents

    PubMed Central

    Kumar, Kunal; Awasthi, Divya; Lee, Seung-Yub; Zanardi, Ilaria; Ruzsicska, Bela; Knudson, Susan; Tonge, Peter J.; Slayden, Richard A.; Ojima, Iwao

    2010-01-01

    Libraries of novel trisubstituted benzimidazoles were created through rational drug design. A good number of these benzimidazoles exhibited promising MIC values in the range of 0.5-6 μg/mL (2-15 μM) for their antibacterial activity against Mtb H37Rv strain. Moreover, five of the lead compounds also exhibited excellent activity against clinical Mtb strains with different drug-resistance profiles. All lead compounds do not show appreciable cytotoxicity (IC50 >200 μM) against Vero cells, which inhibit Mtb FtsZ assembly in a dose dependent manner. The two lead compounds unexpectedly showed enhancement of the GTPase activity of Mtb FtsZ. The result strongly suggests that the increased GTPase activity destabilizes FtsZ assembly leading to efficient inhibition of FtsZ polymerization and filament formation. The TEM and SEM analyses of Mtb FtsZ and Mtb cells, respectively, treated with a lead compound strongly suggest that lead benzimidazoles have a novel mechanism of action on the inhibition of Mtb FtsZ assembly and Z-ring formation. PMID:21126020

  3. FtsZ does not initiate membrane constriction at the onset of division

    PubMed Central

    Daley, Daniel O.; Skoglund, Ulf; Söderström, Bill

    2016-01-01

    The source of constriction required for division of a bacterial cell remains enigmatic. FtsZ is widely believed to be a key player, because in vitro experiments indicate that it can deform liposomes when membrane tethered. However in vivo evidence for such a role has remained elusive as it has been challenging to distinguish the contribution of FtsZ from that of peptidoglycan-ingrowth. To differentiate between these two possibilities we studied the early stages of division in Escherichia coli, when FtsZ is present at the division site but peptidoglycan synthesizing enzymes such as FtsI and FtsN are not. Our approach was to use correlative cryo-fluorescence and cryo-electron microscopy (cryo-CLEM) to monitor the localization of fluorescently labeled FtsZ, FtsI or FtsN correlated with the septal ultra-structural geometry in the same cell. We noted that the presence of FtsZ at the division septum is not sufficient to deform membranes. This observation suggests that, although FtsZ can provide a constrictive force, the force is not substantial at the onset of division. Conversely, the presence of FtsN always correlated with membrane invagination, indicating that allosteric activation of peptidoglycan ingrowth is the trigger for constriction of the cell envelope during cell division in E. coli. PMID:27609565

  4. FtsZ Protofilament Curvature Is the Opposite of Tubulin Rings.

    PubMed

    Housman, Max; Milam, Sara L; Moore, Desmond A; Osawa, Masaki; Erickson, Harold P

    2016-07-26

    FtsZ protofilaments (pfs) form the bacterial cytokinetic Z ring. Previous work suggested that a conformational change from straight to curved pfs generated the constriction force. In the simplest model, the C-terminal membrane tether is on the outside of the curved pf, facing the membrane. Tubulin, a homologue of FtsZ, also forms pfs with a curved conformation. However, it is well-established that tubulin rings have the C terminus on the inside of the ring. Could FtsZ and tubulin rings have the opposite curvature? In this study, we explored the FtsZ curvature direction by fusing large protein tags to the FtsZ termini. Thin section electron microscopy showed that the C-terminal tag was on the outside, consistent with the bending pf model. This has interesting implications for the evolution of tubulin. Tubulin likely began with the curvature of FtsZ, but evolution managed to reverse direction to produce outward-curving rings, which are useful for pulling chromosomes.

  5. Three rings for the evolution of plastid shape: a tale of land plant FtsZ.

    PubMed

    Grosche, Christopher; Rensing, Stefan A

    2017-03-03

    Nuclear-encoded plant FtsZ genes are derived from endosymbiotic gene transfer of cyanobacteria-like genes. The green lineage (Chloroplastida) and red lineage (Rhodophyta) feature FtsZ1 and FtsZ2 or FtsZB and FtsZA, respectively, which are involved in plastid division. These two proteins show slight differences and seem to heteropolymerize to build the essential inner plastid division ring. A third gene, encoding FtsZ3, is present in glaucophyte and charophyte algae, as well as in land plants except ferns and angiosperms. This gene was probably present in the last common ancestor of the organisms united by having a primary plastid (Archaeplastida) and was lost during vascular plant evolution as well as in the red and green algae. The presence/absence pattern of FtsZ3 mirrors that of a full set of Mur genes and the peptidoglycan wall encoded by them. Based on these findings, we discuss a role for FtsZ3 in the establishment or maintenance of plastid peptidoglycan shells.

  6. Modeling FtsZ ring formation in the bacterial cell—anisotropic aggregation via mutual interactions of polymer rods

    NASA Astrophysics Data System (ADS)

    Fischer-Friedrich, Elisabeth; Gov, Nir

    2011-04-01

    The cytoskeletal protein FtsZ polymerizes to a ring structure (Z ring) at the inner cytoplasmic membrane that marks the future division site and scaffolds the division machinery in many bacterial species. FtsZ is known to polymerize in the presence of GTP into single-stranded protofilaments. In vivo, FtsZ polymers become associated with the cytoplasmic membrane via interaction with the membrane-binding proteins FtsA and ZipA. The FtsZ ring structure is highly dynamic and undergoes constantly polymerization and depolymerization processes and exchange with the cytoplasmic pool. In this theoretical study, we consider a scenario of Z ring self-organization via self-enhanced attachment of FtsZ polymers due to end-to-end interactions and lateral interactions of FtsZ polymers on the membrane. With the assumption of exclusively circumferential polymer orientations, we derive coarse-grained equations for the dynamics of the pool of cytoplasmic and membrane-bound FtsZ. To capture stochastic effects expected in the system due to low particle numbers, we simulate our computational model using a Gillespie-type algorithm. We obtain ring- and arc-shaped aggregations of FtsZ polymers on the membrane as a function of monomer numbers in the cell. In particular, our model predicts the number of FtsZ rings forming in the cell as a function of cell geometry and FtsZ concentration. We also calculate the time of FtsZ ring localization to the midplane in the presence of Min oscillations. Finally, we demonstrate that the assumptions and results of our model are confirmed by 3D reconstructions of fluorescently-labeled FtsZ structures in E. coli that we obtained.

  7. A novel membrane anchor for FtsZ is linked to cell wall hydrolysis in Caulobacter crescentus.

    PubMed

    Meier, Elizabeth L; Razavi, Shiva; Inoue, Takanari; Goley, Erin D

    2016-07-01

    In most bacteria, the tubulin-like GTPase FtsZ forms an annulus at midcell (the Z-ring) which recruits the division machinery and regulates cell wall remodeling. Although both activities require membrane attachment of FtsZ, few membrane anchors have been characterized. FtsA is considered to be the primary membrane tether for FtsZ in bacteria, however in Caulobacter crescentus, FtsA arrives at midcell after stable Z-ring assembly and early FtsZ-directed cell wall synthesis. We hypothesized that additional proteins tether FtsZ to the membrane and demonstrate that in C. crescentus, FzlC is one such membrane anchor. FzlC associates with membranes directly in vivo and in vitro and recruits FtsZ to membranes in vitro. As for most known membrane anchors, the C-terminal peptide of FtsZ is required for its recruitment to membranes by FzlC in vitro and midcell recruitment of FzlC in cells. In vivo, overproduction of FzlC causes cytokinesis defects whereas deletion of fzlC causes synthetic defects with dipM, ftsE and amiC mutants, implicating FzlC in cell wall hydrolysis. Our characterization of FzlC as a novel membrane anchor for FtsZ expands our understanding of FtsZ regulators and establishes a role for membrane-anchored FtsZ in the regulation of cell wall hydrolysis.

  8. Borrelia burgdorferi ftsZ Plays a Role in Cell Division

    PubMed Central

    Dubytska, Lydia; Godfrey, Henry P.; Cabello, Felipe C.

    2006-01-01

    ftsZ is essential for cell division in many microorganisms. In Escherichia coli and Bacillus subtilis, FtsZ plays a role in ring formation at the leading edge of the cell division septum. An ftsZ homologue is present in the Borrelia burgdorferi genome (ftsZBbu). Its gene product (FtsZBbu) is strongly homologous to other bacterial FtsZ proteins, but its function has not been established. Because loss-of-function mutants of ftsZBbu might be lethal, the tetR/tetO system was adapted for regulated control of this gene in B. burgdorferi. Sixty-two nucleotides of an ftsZBbu antisense DNA sequence under the control of a tetracycline-responsive modified hybrid borrelial promoter were cloned into pKFSS1. This construct was electroporated into a B. burgdorferi host strain carrying a chromosomally located tetR under the control of the B. burgdorferi flaB promoter. After induction by anhydrotetracycline, expression of antisense ftsZ RNA resulted in generation of filamentous B. burgdorferi that were unable to divide and grew more slowly than uninduced cells. To determine whether FtsZBbu could interfere with the function of E. coli FtsZ, ftsZBbu was amplified from chromosomal DNA and placed under the control of the tetracycline-regulated hybrid promoter. After introduction of the construct into E. coli and induction with anhydrotetracycline, overexpression of ftsZBbu generated a filamentous phenotype. This suggested interference of ftsZBbu with E. coli FtsZ function and confirmed the role of ftsZBbu in cell division. This is the first report of the generation of a B. burgdorferi conditional lethal mutant equivalent by tetracycline-controlled expression of antisense RNA. PMID:16484209

  9. BT-benzo-29 inhibits bacterial cell proliferation by perturbing FtsZ assembly.

    PubMed

    Ray, Shashikant; Jindal, Bhavya; Kunal, Kishore; Surolia, Avadhesha; Panda, Dulal

    2015-10-01

    We have identified a potent antibacterial agent N-(4-sec-butylphenyl)-2-(thiophen-2-yl)-1H-benzo[d]imidazole-4-carboxamide (BT-benzo-29) from a library of benzimidazole derivatives that stalled bacterial division by inhibiting FtsZ assembly. A short (5 min) exposure of BT-benzo-29 disassembled the cytokinetic Z-ring in Bacillus subtilis cells without affecting the cell length and nucleoids. BT-benzo-29 also perturbed the localization of early and late division proteins such as FtsA, ZapA and SepF at the mid-cell. Further, BT-benzo-29 bound to FtsZ with a dissociation constant of 24 ± 3 μm and inhibited the assembly and GTPase activity of purified FtsZ. A docking analysis suggested that BT-benzo-29 may bind to FtsZ at the C-terminal domain near the T7 loop. BT-benzo-29 displayed significantly weaker inhibitory effects on the assembly and GTPase activity of two mutants (L272A and V275A) of FtsZ supporting the prediction of the docking analysis. Further, BT-benzo-29 did not appear to inhibit DNA duplication and nucleoid segregation and it did not perturb the membrane potential of B. subtilis cells. The results suggested that BT-benzo-29 exerts its potent antibacterial activity by inhibiting FtsZ assembly. Interestingly, BT-benzo-29 did not affect the membrane integrity of mammalian red blood cells. BT-benzo-29 bound to tubulin with a much weaker affinity than FtsZ and exerted significantly weaker effects on mammalian cells than on the bacterial cells indicating that the compound may have a strong antibacterial potential.

  10. Chloroplast Division Protein ARC3 Regulates Chloroplast FtsZ-Ring Assembly and Positioning in Arabidopsis through Interaction with FtsZ2[C][W

    PubMed Central

    Zhang, Min; Schmitz, Aaron J.; Kadirjan-Kalbach, Deena K.; TerBush, Allan D.; Osteryoung, Katherine W.

    2013-01-01

    Chloroplast division is initiated by assembly of a mid-chloroplast FtsZ (Z) ring comprising two cytoskeletal proteins, FtsZ1 and FtsZ2. The division-site regulators ACCUMULATION AND REPLICATION OF CHLOROPLASTS3 (ARC3), MinD1, and MinE1 restrict division to the mid-plastid, but their roles are poorly understood. Using genetic analyses in Arabidopsis thaliana, we show that ARC3 mediates division-site placement by inhibiting Z-ring assembly, and MinD1 and MinE1 function through ARC3. ftsZ1 null mutants exhibited some mid-plastid FtsZ2 rings and constrictions, whereas neither constrictions nor FtsZ1 rings were observed in mutants lacking FtsZ2, suggesting FtsZ2 is the primary determinant of Z-ring assembly in vivo. arc3 ftsZ1 double mutants exhibited multiple parallel but no mid-plastid FtsZ2 rings, resembling the Z-ring phenotype in arc3 single mutants and showing that ARC3 affects positioning of FtsZ2 rings as well as Z rings. ARC3 overexpression in the wild type and ftsZ1 inhibited Z-ring and FtsZ2-ring assembly, respectively. Consistent with its effects in vivo, ARC3 interacted with FtsZ2 in two-hybrid assays and inhibited FtsZ2 assembly in a heterologous system. Our studies are consistent with a model wherein ARC3 directly inhibits Z-ring assembly in vivo primarily through interaction with FtsZ2 in heteropolymers and suggest that ARC3 activity is spatially regulated by MinD1 and MinE1 to permit Z-ring assembly at the mid-plastid. PMID:23715471

  11. Ca2+-mediated GTP-dependent dynamic assembly of bacterial cell division protein FtsZ into asters and polymer networks in vitro.

    PubMed Central

    Yu, X C; Margolin, W

    1997-01-01

    FtsZ, a tubulin-like GTPase that forms a dynamic ring marking the division plane of prokaryotic cells, is essential for cytokinesis. It is not known what triggers FtsZ ring assembly. In this work, we use a FtsZ-green fluorescent protein (Gfp) chimera to assay FtsZ assembly over time by using fluorescence microscopy. We show that FtsZ polymers can assemble dynamically in solution in a GTP-dependent manner. Initially, FtsZ nucleation centers grow into aster-like structures that dramatically resemble microtubule organizing centers. As assembly proceeds further, protofilament bundles emanating from different asters interconnect, mimicking the closure of the FtsZ ring in vivo. Surprisingly, millimolar levels of Ca2+ promote FtsZ dynamic assembly. FtsZ can undergo repeated GTP-dependent assembly and disassembly in solution by sequential addition and removal of Ca2+. In addition, GTP binding and hydrolysis by FtsZ are regulated by Ca2+ concentration. Although the concentration of Ca2+ required for FtsZ assembly in vitro is high, its clear and specific effect on FtsZ dynamics suggests the possibility that Ca2+ may have a role in regulating FtsZ ring assembly in the cell. PMID:9312004

  12. Phenylpropanoids inhibit protofilament formation of Escherichia coli cell division protein FtsZ.

    PubMed

    Hemaiswarya, Shanmugam; Soudaminikkutty, Rohini; Narasumani, Mohana Lakshmi; Doble, Mukesh

    2011-09-01

    The earliest step in cell division in bacteria is the assembly of FtsZ, an essential cell division protein, into a ring at the division site. FtsZ has GTPase activity and can assemble in vitro to form protein filaments. The present work involved the study of eight phenylpropanoids (cinnamic, p-coumaric, caffeic, chlorogenic, ferulic, 3,4-dimethoxycinnamic and 2,4,5-trimethoxycinnamic acids and eugenol) as inhibitors of Escherichia coli FtsZ. Phenylpropanoids make up the majority of our diet and act as antibacterial agents. Polymerization and GTPase inhibition assays showed that chlorogenic and caffeic acids were the most active amongst these (IC(50) of 70 and 106 µM, respectively). Circular dichroism studies indicated that chlorogenic acid perturbed the protein conformation and electron microscopy showed distorted filaments. Bacillus subtilis 168 cells treated with the phenylpropanoids were longer when compared to the control. The highest binding energy was observed between chlorogenic acid and the homology modelled E. coli FtsZ, which was consistent with the experimental results. A strong negative correlation was observed between binding energy and inhibition of the polymerization ability. 3D-Quantitative structure-activity relationship studies using GTPase activity indicated that the presence of more hydrophilic groups around the 3'- and 4'-carbon increased the activity. The effect of stress-induced formation of cell filamentation has to be understood before confirming the role of phenylpropanoids as FtsZ inhibitors.

  13. Condensation of FtsZ filaments can drive bacterial cell division.

    PubMed

    Lan, Ganhui; Daniels, Brian R; Dobrowsky, Terrence M; Wirtz, Denis; Sun, Sean X

    2009-01-06

    Forces are important in biological systems for accomplishing key cell functions, such as motility, organelle transport, and cell division. Currently, known force generation mechanisms typically involve motor proteins. In bacterial cells, no known motor proteins are involved in cell division. Instead, a division ring (Z-ring) consists of mostly FtsZ, FtsA, and ZipA is used to exerting a contractile force. The mechanism of force generation in bacterial cell division is unknown. Using computational modeling, we show that Z-ring formation results from the colocalization of FtsZ and FtsA mediated by the favorable alignment of FtsZ polymers. The model predicts that the Z-ring undergoes a condensation transition from a low-density state to a high-density state and generates a sufficient contractile force to achieve division. FtsZ GTP hydrolysis facilitates monomer turnover during the condensation transition, but does not directly generate forces. In vivo fluorescence measurements show that FtsZ density increases during division, in accord with model results. The mechanism is akin to van der Waals picture of gas-liquid condensation, and shows that organisms can exploit microphase transitions to generate mechanical forces.

  14. Structural reorganization of the bacterial cell-division protein FtsZ from Staphylococcus aureus.

    PubMed

    Matsui, Takashi; Yamane, Junji; Mogi, Nobuyuki; Yamaguchi, Hiroto; Takemoto, Hiroshi; Yao, Min; Tanaka, Isao

    2012-09-01

    FtsZ is a key molecule in bacterial cell division. In the presence of GTP, it polymerizes into tubulin-like protofilaments by head-to-tail association. Protofilaments of FtsZ seem to adopt a straight or a curved conformation in relation to the bound nucleotide. However, although several bacterial and archaeal FtsZ structures have been determined, all of the structures reported previously are considered to have a curved conformation. In this study, structures of FtsZ from Staphylococcus aureus (SaFtsZ) were determined in apo, GDP-bound and inhibitor-complex forms and it was found that SaFtsZ undergoes marked conformational changes. The accumulated evidence suggests that the GDP-bound structure has the features of the straight form. The structural change between the curved and straight forms shows intriguing similarity to the eukaryotic cytoskeletal protein tubulin. Furthermore, the structure of the apo form showed an unexpectedly large conformational change in the core region. FtsZ has also been recognized as a novel target for antibacterial drugs. The structure of the complex with the inhibitor PC190723, which has potent and selective antistaphylococcal activity, indicated that the inhibitor binds at the cleft between the two subdomains.

  15. Antioxidant and hepatoprotective activity of Hamelia patens extracts.

    PubMed

    Perez-Meseguer, Jonathan; Delgado-Montemayor, Cecilia; Ortíz-Torres, Tania; Salazar-Aranda, Ricardo; Cordero-Perez, Paula; de Torres, Noemí Waksman

    2016-01-01

    Hamelia patens is widely used in the traditional medicine of Mexico and Central America for the treatment of illnesses associated with inflammatory processes. In this study, antioxidant and hepatoprotective activity were assayed on the methanolic crude (ME), hexane (HE), ethyl acetate (AE), and butanol (BE) extracts of H. patens. The total phenolic content (TPC) as mg of gallic acid equivalents per g of dry extract was determined by Folin-Ciocalteu's method (ME=141.58±11.99, HE=33.96±1.13, AE=375.18±13.09, BE=132.08±3.62), and antioxidant activity by 2,2-diphenyl-1-picryl-hydrazyl (DPPH) free radical-scavenging method (EC(50) ME=77.87±5.67, HE=236.64±26.32, AE=45.87±2.24, BE=50.97±0.85μg/mL). Hepatoprotective activity was evaluated through AST activity on HepG2 cells subjected to damage with CCl(4) (ME=62.5±3.41, HE=72.25±2.87, AE=63.50±4.20, BE=43.74±4.03). BE showed the greater hepatoprotective activity and a good antioxidant capacity, while HE did not show hepatoprotective or antioxidant activity. Cytotoxicity was evaluated on Vero cells cultures; none showed significant toxicity.

  16. Recent advances in the discovery and development of antibacterial agents targeting the cell-division protein FtsZ.

    PubMed

    Haranahalli, Krupanandan; Tong, Simon; Ojima, Iwao

    2016-12-15

    With the emergence of multidrug-resistant bacterial strains, there is a dire need for new drug targets for antibacterial drug discovery and development. Filamentous temperature sensitive protein Z (FtsZ), is a GTP-dependent prokaryotic cell division protein, sharing less than 10% sequence identity with the eukaryotic cell division protein, tubulin. FtsZ forms a dynamic Z-ring in the middle of the cell, leading to septation and subsequent cell division. Inhibition of the Z-ring blocks cell division, thus making FtsZ a highly attractive target. Various groups have been working on natural products and synthetic small molecules as inhibitors of FtsZ. This review summarizes the recent advances in the development of FtsZ inhibitors, focusing on those in the last 5years, but also includes significant findings in previous years. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Treadmilling by FtsZ filaments drives peptidoglycan synthesis and bacterial cell division.

    PubMed

    Bisson-Filho, Alexandre W; Hsu, Yen-Pang; Squyres, Georgia R; Kuru, Erkin; Wu, Fabai; Jukes, Calum; Sun, Yingjie; Dekker, Cees; Holden, Seamus; VanNieuwenhze, Michael S; Brun, Yves V; Garner, Ethan C

    2017-02-17

    The mechanism by which bacteria divide is not well understood. Cell division is mediated by filaments of FtsZ and FtsA (FtsAZ) that recruit septal peptidoglycan-synthesizing enzymes to the division site. To understand how these components coordinate to divide cells, we visualized their movements relative to the dynamics of cell wall synthesis during cytokinesis. We found that the division septum was built at discrete sites that moved around the division plane. FtsAZ filaments treadmilled circumferentially around the division ring and drove the motions of the peptidoglycan-synthesizing enzymes. The FtsZ treadmilling rate controlled both the rate of peptidoglycan synthesis and cell division. Thus, FtsZ treadmilling guides the progressive insertion of new cell wall by building increasingly smaller concentric rings of peptidoglycan to divide the cell.

  18. Discovery of chlamydial peptidoglycan reveals bacteria with murein sacculi but without FtsZ

    PubMed Central

    Biboy, Jacob; Gray, Joe; Kuru, Erkin; Hall, Edward; Brun, Yves V.; VanNieuwenhze, Michael S.; Vollmer, Waldemar; Horn, Matthias; Jensen, Grant J.

    2013-01-01

    Chlamydiae are important pathogens and symbionts, with unique cell biology features. They lack the cell-division protein FtsZ, which functions in maintaining cell shape and orchestrating cell division in almost all other bacteria. In addition, the existence of peptidoglycan (PG) in chlamydial cell envelopes has been highly controversial. Using electron cryotomography, mass spectrometry and fluorescent labeling dyes, here we show that some environmental chlamydiae have cell-wall sacculi consisting of an unusual PG type. Treatment with fosfomycin (a PG synthesis inhibitor) leads to lower infection rates and aberrant cell shapes, suggesting that PG synthesis is crucial for the chlamydial life cycle. Our findings demonstrate for the first time the presence of PG in a member of the Chlamydiae. They also present a unique example of a bacterium with a PG sacculus but without FtsZ, challenging the current hypothesis that it is the absence of a cell wall that renders FtsZ non-essential. PMID:24292151

  19. Evidence That Bacteriophage λ Kil Peptide Inhibits Bacterial Cell Division by Disrupting FtsZ Protofilaments and Sequestering Protein Subunits*

    PubMed Central

    Hernández-Rocamora, Víctor M.; Alfonso, Carlos; Margolin, William; Zorrilla, Silvia; Rivas, Germán

    2015-01-01

    The effects of Kil peptide from bacteriophage λ on the assembly of Escherichia coli FtsZ into one subunit thick protofilaments were studied using combined biophysical and biochemical methods. Kil peptide has recently been identified as the factor from bacteriophage λ responsible for the inhibition of bacterial cell division during lytic cycle, targeting FtsZ polymerization. Here, we show that this antagonist blocks FtsZ assembly into GTP-dependent protofilaments, producing a wide distribution of smaller oligomers compared with the average size of the intact protofilaments. The shortening of FtsZ protofilaments by Kil is detectable at concentrations of the peptide in the low micromolar range, the mid-point of the inhibition being close to its apparent affinity for GDP-bound FtsZ. This antagonist not only interferes with FtsZ assembly but also reverses the polymerization reaction. The negative regulation by Kil significantly reduces the GTPase activity of FtsZ protofilaments, and FtsZ polymers assembled in guanosine-5′-[(α,β)-methyleno]triphosphate are considerably less sensitive to Kil. Our results suggest that, at high concentrations, Kil may use an inhibition mechanism involving the sequestration of FtsZ subunits, similar to that described for other inhibitors like the SOS response protein SulA or the moonlighting enzyme OpgH. This mechanism is different from those employed by the division site selection antagonists MinC and SlmA. This work provides new insight into the inhibition of FtsZ assembly by phages, considered potential tools against bacterial infection. PMID:26124275

  20. Evidence That Bacteriophage λ Kil Peptide Inhibits Bacterial Cell Division by Disrupting FtsZ Protofilaments and Sequestering Protein Subunits.

    PubMed

    Hernández-Rocamora, Víctor M; Alfonso, Carlos; Margolin, William; Zorrilla, Silvia; Rivas, Germán

    2015-08-14

    The effects of Kil peptide from bacteriophage λ on the assembly of Escherichia coli FtsZ into one subunit thick protofilaments were studied using combined biophysical and biochemical methods. Kil peptide has recently been identified as the factor from bacteriophage λ responsible for the inhibition of bacterial cell division during lytic cycle, targeting FtsZ polymerization. Here, we show that this antagonist blocks FtsZ assembly into GTP-dependent protofilaments, producing a wide distribution of smaller oligomers compared with the average size of the intact protofilaments. The shortening of FtsZ protofilaments by Kil is detectable at concentrations of the peptide in the low micromolar range, the mid-point of the inhibition being close to its apparent affinity for GDP-bound FtsZ. This antagonist not only interferes with FtsZ assembly but also reverses the polymerization reaction. The negative regulation by Kil significantly reduces the GTPase activity of FtsZ protofilaments, and FtsZ polymers assembled in guanosine-5'-[(α,β)-methyleno]triphosphate are considerably less sensitive to Kil. Our results suggest that, at high concentrations, Kil may use an inhibition mechanism involving the sequestration of FtsZ subunits, similar to that described for other inhibitors like the SOS response protein SulA or the moonlighting enzyme OpgH. This mechanism is different from those employed by the division site selection antagonists MinC and SlmA. This work provides new insight into the inhibition of FtsZ assembly by phages, considered potential tools against bacterial infection.

  1. Chrysophaentins are competitive inhibitors of FtsZ and inhibit Z-ring formation in live bacteria.

    PubMed

    Keffer, Jessica L; Huecas, Sonia; Hammill, Jared T; Wipf, Peter; Andreu, Jose M; Bewley, Carole A

    2013-09-15

    The bacterial cell division protein FtsZ polymerizes in a GTP-dependent manner to form a Z-ring that marks the plane of division. As a validated antimicrobial target, considerable efforts have been devoted to identify small molecule FtsZ inhibitors. We recently discovered the chrysophaentins, a novel suite of marine natural products that inhibit FtsZ activity in vitro. These natural products along with a synthetic hemi-chrysophaentin exhibit strong antimicrobial activity toward a broad spectrum of Gram-positive pathogens. To define their mechanisms of FtsZ inhibition and determine their in vivo effects in live bacteria, we used GTPase assays and fluorescence anisotropy to show that hemi-chrysophaentin competitively inhibits FtsZ activity. Furthermore, we developed a model system using a permeable Escherichia coli strain, envA1, together with an inducible FtsZ-yellow fluorescent protein construct to show by fluorescence microscopy that both chrysophaentin A and hemi-chrysophaentin disrupt Z-rings in live bacteria. We tested the E. coli system further by reproducing phenotypes observed for zantrins Z1 and Z3, and demonstrate that the alkaloid berberine, a reported FtsZ inhibitor, exhibits auto-fluorescence, making it incompatible with systems that employ GFP or YFP tagged FtsZ. These studies describe unique examples of nonnucleotide, competitive FtsZ inhibitors that disrupt FtsZ in vivo, together with a model system that should be useful for in vivo testing of FtsZ inhibitor leads that have been identified through in vitro screens but are unable to penetrate the Gram-negative outer membrane. Published by Elsevier Ltd.

  2. MinD directly interacting with FtsZ at the H10 helix suggests a model for robust activation of MinC to destabilize FtsZ polymers.

    PubMed

    Taviti, Ashoka Chary; Beuria, Tushar Kant

    2017-09-07

    Cell division in bacteria is a highly controlled and regulated process. FtsZ, a bacterial cytoskeletal protein, forms a ring-like structure known as the Z-ring and recruits more than a dozen other cell division proteins. The Min system oscillates between the poles and inhibits the Z-ring formation at the poles by perturbing FtsZ assembly. This leads to an increase in the FtsZ concentration at the mid-cell and helps in Z-ring positioning. MinC, the effector protein, interferes with Z-ring formation through two different mechanisms mediated by its two domains with the help of MinD. However, the mechanism by which MinD triggers MinC activity is not yet known. We showed that MinD directly interacts with FtsZ with an affinity stronger than the reported MinC-FtsZ interaction. We determined the MinD-binding site of FtsZ using computational, mutational and biochemical analyses. Our study showed that MinD binds to the H10 helix of FtsZ. Single-point mutations at the charged residues in the H10 helix resulted in a decrease in the FtsZ affinity towards MinD. Based on our findings, we propose a novel model for MinCD-FtsZ interaction, where MinD through its direct interaction with FtsZ would trigger MinC activity to inhibit FtsZ functions. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  3. The Kil Peptide of Bacteriophage λ Blocks Escherichia coli Cytokinesis via ZipA-Dependent Inhibition of FtsZ Assembly

    PubMed Central

    Haeusser, Daniel P.; Hoashi, Marina; Weaver, Anna; Brown, Nathan; Pan, James; Sawitzke, James A.; Thomason, Lynn C.; Court, Donald L.; Margolin, William

    2014-01-01

    Assembly of the essential, tubulin-like FtsZ protein into a ring-shaped structure at the nascent division site determines the timing and position of cytokinesis in most bacteria and serves as a scaffold for recruitment of the cell division machinery. Here we report that expression of bacteriophage λ kil, either from a resident phage or from a plasmid, induces filamentation of Escherichia coli cells by rapid inhibition of FtsZ ring formation. Mutant alleles of ftsZ resistant to the Kil protein map to the FtsZ polymer subunit interface, stabilize FtsZ ring assembly, and confer increased resistance to endogenous FtsZ inhibitors, consistent with Kil inhibiting FtsZ assembly. Cells with the normally essential cell division gene zipA deleted (in a modified background) display normal FtsZ rings after kil expression, suggesting that ZipA is required for Kil-mediated inhibition of FtsZ rings in vivo. In support of this model, point mutations in the C-terminal FtsZ-interaction domain of ZipA abrogate Kil activity without discernibly altering FtsZ-ZipA interactions. An affinity-tagged-Kil derivative interacts with both FtsZ and ZipA, and inhibits sedimentation of FtsZ filament bundles in vitro. Together, these data inspire a model in which Kil interacts with FtsZ and ZipA in the cell to prevent FtsZ assembly into a coherent, division-competent ring structure. Phage growth assays show that kil+ phage lyse ∼30% later than kil mutant phage, suggesting that Kil delays lysis, perhaps via its interaction with FtsZ and ZipA. PMID:24651041

  4. The Kil peptide of bacteriophage λ blocks Escherichia coli cytokinesis via ZipA-dependent inhibition of FtsZ assembly.

    PubMed

    Haeusser, Daniel P; Hoashi, Marina; Weaver, Anna; Brown, Nathan; Pan, James; Sawitzke, James A; Thomason, Lynn C; Court, Donald L; Margolin, William

    2014-03-01

    Assembly of the essential, tubulin-like FtsZ protein into a ring-shaped structure at the nascent division site determines the timing and position of cytokinesis in most bacteria and serves as a scaffold for recruitment of the cell division machinery. Here we report that expression of bacteriophage λ kil, either from a resident phage or from a plasmid, induces filamentation of Escherichia coli cells by rapid inhibition of FtsZ ring formation. Mutant alleles of ftsZ resistant to the Kil protein map to the FtsZ polymer subunit interface, stabilize FtsZ ring assembly, and confer increased resistance to endogenous FtsZ inhibitors, consistent with Kil inhibiting FtsZ assembly. Cells with the normally essential cell division gene zipA deleted (in a modified background) display normal FtsZ rings after kil expression, suggesting that ZipA is required for Kil-mediated inhibition of FtsZ rings in vivo. In support of this model, point mutations in the C-terminal FtsZ-interaction domain of ZipA abrogate Kil activity without discernibly altering FtsZ-ZipA interactions. An affinity-tagged-Kil derivative interacts with both FtsZ and ZipA, and inhibits sedimentation of FtsZ filament bundles in vitro. Together, these data inspire a model in which Kil interacts with FtsZ and ZipA in the cell to prevent FtsZ assembly into a coherent, division-competent ring structure. Phage growth assays show that kil+ phage lyse ∼30% later than kil mutant phage, suggesting that Kil delays lysis, perhaps via its interaction with FtsZ and ZipA.

  5. Discovery of chlamydial peptidoglycan reveals bacteria with murein sacculi but without FtsZ

    NASA Astrophysics Data System (ADS)

    Pilhofer, Martin; Aistleitner, Karin; Biboy, Jacob; Gray, Joe; Kuru, Erkin; Hall, Edward; Brun, Yves V.; Vannieuwenhze, Michael S.; Vollmer, Waldemar; Horn, Matthias; Jensen, Grant J.

    2013-12-01

    Chlamydiae are important pathogens and symbionts with unique cell biological features. They lack the cell-division protein FtsZ, and the existence of peptidoglycan (PG) in their cell wall has been highly controversial. FtsZ and PG together function in orchestrating cell division and maintaining cell shape in almost all other bacteria. Using electron cryotomography, mass spectrometry and fluorescent labelling dyes, here we show that some environmental chlamydiae have cell wall sacculi consisting of a novel PG type. Treatment with fosfomycin (a PG synthesis inhibitor) leads to lower infection rates and aberrant cell shapes, suggesting that PG synthesis is crucial for the chlamydial life cycle. Our findings demonstrate for the first time the presence of PG in a member of the Chlamydiae. They also present a unique example of a bacterium with a PG sacculus but without FtsZ, challenging the current hypothesis that it is the absence of a cell wall that renders FtsZ non-essential.

  6. Screening and Development of New Inhibitors of FtsZ from M. Tuberculosis

    PubMed Central

    Mathew, Bini; Ross, Larry; Connelly, Michele C.; Lofton, Hava; Rajagopalan, Malini; Guy, R. Kiplin; Reynolds, Robert C.

    2016-01-01

    A variety of commercial analogs and a newer series of Sulindac derivatives were screened for inhibition of M. tuberculosis (Mtb) in vitro and specifically as inhibitors of the essential mycobacterial tubulin homolog, FtsZ. Due to the ease of preparing diverse analogs and a favorable in vivo pharmacokinetic and toxicity profile of a representative analog, the Sulindac scaffold may be useful for further development against Mtb with respect to in vitro bacterial growth inhibition and selective activity for Mtb FtsZ versus mammalian tubulin. Further discovery efforts will require separating reported mammalian cell activity from both antibacterial activity and inhibition of Mtb FtsZ. Modeling studies suggest that these analogs bind in a specific region of the Mtb FtsZ polymer that differs from human tubulin and, in combination with a pharmacophore model presented herein, future hybrid analogs of the reported active molecules that more efficiently bind in this pocket may improve antibacterial activity while improving other drug characteristics. PMID:27768711

  7. Structure and Mutational Analyses of Escherichia coli ZapD Reveal Charged Residues Involved in FtsZ Filament Bundling

    PubMed Central

    Roach, Elyse J.; Wroblewski, Charles; Seidel, Laura; Berezuk, Alison M.; Brewer, Dyanne; Kimber, Matthew S.

    2016-01-01

    ABSTRACT Bacterial cell division is an essential and highly coordinated process. It requires the polymerization of the tubulin homologue FtsZ to form a dynamic ring (Z-ring) at midcell. Z-ring formation relies on a group of FtsZ-associated proteins (Zap) for stability throughout the process of division. In Escherichia coli, there are currently five Zap proteins (ZapA through ZapE), of which four (ZapA, ZapB, ZapC, and ZapD) are small soluble proteins that act to bind and bundle FtsZ filaments. In particular, ZapD forms a functional dimer and interacts with the C-terminal tail of FtsZ, but little is known about its structure and mechanism of action. Here, we present the crystal structure of Escherichia coli ZapD and show it forms a symmetrical dimer with centrally located α-helices flanked by β-sheet domains. Based on the structure of ZapD and its chemical cross-linking to FtsZ, we targeted nine charged ZapD residues for modification by site-directed mutagenesis. Using in vitro FtsZ sedimentation assays, we show that residues R56, R221, and R225 are important for bundling FtsZ filaments, while transmission electron microscopy revealed that altering these residues results in different FtsZ bundle morphology compared to those of filaments bundled with wild-type ZapD. ZapD residue R116 also showed altered FtsZ bundle morphology but levels of FtsZ bundling similar to that of wild-type ZapD. Together, these results reveal that ZapD residues R116, R221, and R225 likely participate in forming a positively charged binding pocket that is critical for bundling FtsZ filaments. IMPORTANCE Z-ring assembly underpins the formation of the essential cell division complex known as the divisome and is required for recruitment of downstream cell division proteins. ZapD is one of several proteins in E. coli that associates with the Z-ring to promote FtsZ bundling and aids in the overall fitness of the division process. In the present study, we describe the dimeric structure of E. coli

  8. [Localization of the division protein FtsZ in mycoplasma cells Mycoplasma hominis].

    PubMed

    Vishniakov, I E; Borkhsenius, S N; Basovskiĭ, Iu I; Levitskiĭ, S A; Lazarev, V N; Snigirevskaia, E S; Komissarchik, Ia Iu

    2009-01-01

    Localization of the protein FtsZ in Mycoplasma hominis cells was determined. Ultra thin sections were treated by rabbit polyclonal antibodies against FtsZ M. hominis: a conjugate of protein A with colloidal gold particles was used instead of secondary antibodies. Considerable polymorphism of cells was seen on electron microscopy pictures of M. hominis cells, which is typical for mycoplasmas. Among a wide variety of cell shapes we distinguished dumbbell-shaped dividing cells, and the cells connected with each other with the aid of thin membrane tubules (former constrictions). Dominants distribution of the label in the constriction area of dividing M. hominis cells and in the area of the thin membrane tubules was observed. We revealed the cross septum in the mycoplasma cells for the first time, as well as the gold labeling of this structure. Furthermore, in some rounded and oval cells colloidal gold particles labeled the whole plasma membrane in ring-shaped manner. Probably, the label in these cases marks a submembrane contractile ring (Z-ring). The facts mentioned above confirm that FtsZ of M. hominis plays an active role in the mycoplasma cytokinesis. In a series of cases spiral-like distribution of gold particles was observed. Probably, FtsZ protofilaments in M. hominis cells can form spiral structures similar to Z-spirals of Bacillus subtilis and Escherichia coli. Its presence in mycoplasma cells may be considered as an important argument in favour of model of Z-ring assembling through reorganization of Z-spirals. FtsZ also may participate in maintenance of mycoplasma cell shape (membrane localization).

  9. MapZ marks the division sites and positions FtsZ rings in Streptococcus pneumoniae.

    PubMed

    Fleurie, Aurore; Lesterlin, Christian; Manuse, Sylvie; Zhao, Chao; Cluzel, Caroline; Lavergne, Jean-Pierre; Franz-Wachtel, Mirita; Macek, Boris; Combet, Christophe; Kuru, Erkin; VanNieuwenhze, Michael S; Brun, Yves V; Sherratt, David; Grangeasse, Christophe

    2014-12-11

    In every living organism, cell division requires accurate identification of the division site and placement of the division machinery. In bacteria, this process is traditionally considered to begin with the polymerization of the highly conserved tubulin-like protein FtsZ into a ring that locates precisely at mid-cell. Over the past decades, several systems have been reported to regulate the spatiotemporal assembly and placement of the FtsZ ring. However, the human pathogen Streptococcus pneumoniae, in common with many other organisms, is devoid of these canonical systems and the mechanisms of positioning the division machinery remain unknown. Here we characterize a novel factor that locates at the division site before FtsZ and guides septum positioning in pneumococcus. Mid-cell-anchored protein Z (MapZ) forms ring structures at the cell equator and moves apart as the cell elongates, therefore behaving as a permanent beacon of division sites. MapZ then positions the FtsZ ring through direct protein-protein interactions. MapZ-mediated control differs from previously described systems mostly on the basis of negative regulation of FtsZ assembly. Furthermore, MapZ is an endogenous target of the Ser/Thr kinase StkP, which was recently shown to have a central role in cytokinesis and morphogenesis of S. pneumoniae. We show that both phosphorylated and non-phosphorylated forms of MapZ are required for proper Z-ring formation and dynamics. Altogether, this work uncovers a new mechanism for bacterial cell division that is regulated by phosphorylation and illustrates that nature has evolved a diversity of cell division mechanisms adapted to the different bacterial clades.

  10. Mechanism of regulation of prokaryotic tubulin-like GTPase FtsZ by membrane protein EzrA.

    PubMed

    Chung, Kuei-Min; Hsu, Hsin-Hsien; Yeh, Hsin-Yi; Chang, Ban-Yang

    2007-05-18

    At initiation of cell division, FtsZ, a tubulin-like GTPase, assembles into a so-called Z-ring structure at the site of division. The formation of Z ring is negatively regulated by EzrA, which ensures only one ring at the midcell per cell cycle. The mechanism leading to the negative regulation of Z-ring formation by EzrA has been analyzed. Our data reveal that the interaction between EzrA and FtsZ not only reduces the GTP-binding ability of FtsZ but also accelerates the rate of GTP hydrolysis, both of which are unfavorable for the polymerization of FtsZ. Moreover, the acceleration in rate of GTP hydrolysis by EzrA is attributed to stabilization of the transition state for GTP hydrolysis and reduction in the affinity of GDP for FtsZ. Clearly, EzrA is able to modify the GTP hydrolysis cycle of FtsZ. On the basis of these results, a model for how EzrA acts to negatively regulate Z-ring formation is proposed.

  11. An analysis of FtsZ assembly using small angle X-ray scattering and electron microscopy.

    PubMed

    Kuchibhatla, Anuradha; Abdul Rasheed, A S; Narayanan, Janaky; Bellare, Jayesh; Panda, Dulal

    2009-04-09

    Small angle X-ray scattering (SAXS) was used for the first time to study the self-assembly of the bacterial cell division protein, FtsZ, with three different additives: calcium chloride, monosodium glutamate and DEAE-dextran hydrochloride in solution. The SAXS data were analyzed assuming a model form factor and also by a model-independent analysis using the pair distance distribution function. Transmission electron microscopy (TEM) was used for direct observation of the FtsZ filaments. By sectioning and negative staining with glow discharged grids, very high bundling as well as low bundling polymers were observed under different assembly conditions. FtsZ polymers formed different structures in the presence of different additives and these additives were found to increase the bundling of FtsZ protofilaments by different mechanisms. The combined use of SAXS and TEM provided us a significant insight of the assembly of FtsZ and microstructures of the assembled FtsZ polymers.

  12. The bacterial tubulin FtsZ requires its intrinsically disordered linker to direct robust cell wall construction

    PubMed Central

    Sundararajan, Kousik; Miguel, Amanda; Desmarais, Samantha M.; Meier, Elizabeth L.; Huang, Kerwyn Casey; Goley, Erin D.

    2015-01-01

    The bacterial GTPase FtsZ forms a cytokinetic ring at midcell, recruits the division machinery, and orchestrates membrane and peptidoglycan cell wall invagination. However, the mechanism for FtsZ regulation of peptidoglycan metabolism is unknown. The FtsZ GTPase domain is separated from its membrane-anchoring C-terminal conserved (CTC) peptide by a disordered C-terminal linker (CTL). Here, we investigate CTL function in Caulobacter crescentus. Strikingly, production of FtsZ lacking the CTL (ΔCTL) is lethal: cells become filamentous, form envelope bulges, and lyse, resembling treatment with β-lactam antibiotics. This phenotype is produced by FtsZ polymers bearing the CTC and a CTL shorter than 14 residues. Peptidoglycan synthesis still occurs downstream of ΔCTL, however cells expressing ΔCTL exhibit reduced peptidoglycan crosslinking and longer glycan strands than wildtype. Importantly, midcell proteins are still recruited to sites of ΔCTL assembly. We propose that FtsZ regulates peptidoglycan metabolism through a CTL-dependent mechanism that extends beyond simple protein recruitment. PMID:26099469

  13. Antibacterial activity of alkyl gallates is a combination of direct targeting of FtsZ and permeabilization of bacterial membranes

    PubMed Central

    Król, Ewa; de Sousa Borges, Anabela; da Silva, Isabel; Polaquini, Carlos R.; Regasini, Luis O.; Ferreira, Henrique; Scheffers, Dirk-Jan

    2015-01-01

    Alkyl gallates are compounds with reported antibacterial activity. One of the modes of action is binding of the alkyl gallates to the bacterial membrane and interference with membrane integrity. However, alkyl gallates also cause cell elongation and disruption of cell division in the important plant pathogen Xanthomonas citri subsp. citri, suggesting that cell division proteins may be targeted by alkyl gallates. Here, we use Bacillus subtilis and purified B. subtilis FtsZ to demonstrate that FtsZ is a direct target of alkyl gallates. Alkyl gallates disrupt the FtsZ-ring in vivo, and cause cell elongation. In vitro, alkyl gallates bind with high affinity to FtsZ, causing it to cluster and lose its capacity to polymerize. The activities of a homologous series of alkyl gallates with alkyl side chain lengths ranging from five to eight carbons (C5–C8) were compared and heptyl gallate was found to be the most potent FtsZ inhibitor. Next to the direct effect on FtsZ, alkyl gallates also target B. subtilis membrane integrity—however the observed anti-FtsZ activity is not a secondary effect of the disruption of membrane integrity. We propose that both modes of action, membrane disruption and anti-FtsZ activity, contribute to the antibacterial activity of the alkyl gallates. We propose that heptyl gallate is a promising hit for the further development of antibacterials that specifically target FtsZ. PMID:25972861

  14. Chloroplast division in higher plants requires members of two functionally divergent gene families with homology to bacterial ftsZ.

    PubMed Central

    Osteryoung, K W; Stokes, K D; Rutherford, S M; Percival, A L; Lee, W Y

    1998-01-01

    The division of plastids is critical for viability in photosynthetic eukaryotes, but the mechanisms associated with this process are still poorly understood. We previously identified a nuclear gene from Arabidopsis encoding a chloroplast-localized homolog of the bacterial cell division protein FtsZ, an essential cytoskeletal component of the prokaryotic cell division apparatus. Here, we report the identification of a second nuclear-encoded FtsZ-type protein from Arabidopsis that does not contain a chloroplast targeting sequence or other obvious sorting signals and is not imported into isolated chloroplasts, which strongly suggests that it is localized in the cytosol. We further demonstrate using antisense technology that inhibiting expression of either Arabidopsis FtsZ gene (AtFtsZ1-1 or AtFtsZ2-1) in transgenic plants reduces the number of chloroplasts in mature leaf cells from 100 to one, indicating that both genes are essential for division of higher plant chloroplasts but that each plays a distinct role in the process. Analysis of currently available plant FtsZ sequences further suggests that two functionally divergent FtsZ gene families encoding differentially localized products participate in chloroplast division. Our results provide evidence that both chloroplastic and cytosolic forms of FtsZ are involved in chloroplast division in higher plants and imply that important differences exist between chloroplasts and prokaryotes with regard to the roles played by FtsZ proteins in the division process. PMID:9836740

  15. Dependency of Escherichia coli cell-division size, and independency of nucleoid segregation on the mode and level of ftsZ expression.

    PubMed

    Palacios, P; Vicente, M; Sánchez, M

    1996-06-01

    Expression of ftsZ in strain VIP205 is dissociated from its natural promoters, and is under the control of an inducible tac promoter. This abolishes the oscillation in ftsZ transcription observed in the wild type, allowing different levels of ftsZ expression. We demonstrate that this construction does not affect the expression of other genes, and has no effects on replication or nucleoid segregation. A shift in IPTG from 30 microM, that supports division at wild-type sizes, to lower (6 microM) or higher (100 microM) concentrations, indicates that VIP205 cells can divide within a broad range of FtsZ concentrations. Analysis of the morphological parameters during the transition from one IPTG concentration to another suggests that the correct timing of ftsZ expression, and the correct FtsZ concentration, are required for division to occur at normal cell sizes. After a transient division delay during the transition to lower IPTG concentrations, cells in which ftsZ is expressed continuously (yielding 80% of the wild-type FtsZ levels) divide with the same division time as the wild type, but at the expense of becoming 1.5 times larger. A precise control of ftsZ expression is required for normal division, but the existence of additional regulators to maintain the correct timing during the cell cycle cannot be ruled out.

  16. GTP hydrolysis of cell division protein FtsZ: evidence that the active site is formed by the association of monomers.

    PubMed

    Scheffers, Dirk-Jan; de Wit, Janny G; den Blaauwen, Tanneke; Driessen, Arnold J M

    2002-01-15

    The essential prokaryotic cell division protein FtsZ is a tubulin homologue that forms a ring at the division site. FtsZ forms polymers in a GTP-dependent manner. Recent biochemical evidence has shown that FtsZ forms multimeric structures in vitro and in vivo and functions as a self-activating GTPase. Structural analysis of FtsZ points to an important role for the highly conserved tubulin-like loop 7 (T7-loop) in the self-activation of GTP hydrolysis. The T7-loop was postulated to form the active site together with the nucleotide-binding site on an adjacent FtsZ monomer. To characterize the role of the T7-loop of Escherichia coli FtsZ, we have mutagenized residues M206, N207, D209, D212, and R214. All the mutant proteins, except the R214 mutant, are severely affected in polymerization and GTP hydrolysis. Charged residues D209 and D212 cannot be substituted with a glutamate residue. All mutants interact with wild-type FtsZ in vitro, indicating that the T7-loop mutations do not abolish FtsZ self-association. Strikingly, in mixtures of wild-type and mutant proteins, most mutants are capable of inhibiting wild-type GTP hydrolysis. We conclude that the T7-loop is part of the active site for GTP hydrolysis, formed by the association of two FtsZ monomers.

  17. FtsZ Bacterial Cytoskeletal Polymers on Curved Surfaces: The Importance of Lateral Interactions

    PubMed Central

    Hörger, Ines; Velasco, Enrique; Rivas, Germán; Vélez, Marisela; Tarazona, Pedro

    2008-01-01

    A recent theoretical article provided a mechanical explanation for the formation of cytoskeletal rings and helices in bacteria assuming that these shapes arise, at least in part, from the interaction of the inherent mechanical properties of the protein polymers and the constraints imposed by the curved cell membrane (Andrews, S., and A. P. Arkin. 2007. Biophys. J. 93:1872–1884). Due to the lack of experimental data regarding the bending rigidity and preferential bond angles of bacterial polymers, the authors explored their model over wide ranges of preferred curvature values. In this letter, we present the shape diagram of the FtsZ bacterial polymer on a curved surface but now including recent experimental data on the in vitro formed FtsZ polymers. The lateral interactions between filaments observed experimentally change qualitatively the shape diagram and indicate that the formation of rings over spirals is more energetically favored than estimated in the above-mentioned article. PMID:18359798

  18. Dimethyl sulphoxide and Ca2+ stimulate assembly of Vibrio cholerae FtsZ.

    PubMed

    Chatterjee, Abhisek; Chakrabarti, Gopal

    2014-10-01

    We cloned, overexpressed and purified Vibrio cholerae FtsZ protein for the first time. We used several complementary techniques to probe and compare the comparative assembly properties of recombinant Vibrio cholerae FtsZ (VcFtsZ) and Escherichia coli FtsZ (EcFtsZ). We observed that VcFtsZ polymerized at a slower rate than EcFtsZ and interestingly its polymerization was highly dependent on the presence of Ca(2+) ion. Furthermore, DMSO specifically modulated the polymerization of VcFtsZ, promoted polymer bundling and increased the stability of the VcFtsZ protofilaments. Whereas DMSO showed no significant stimulatory effect on the assembly and bundling of EcFtsZ. Transmission electron microscopy experiments demonstrated that in presence of 8% DMSO the average thickness of the VcFtsZ polymers were increased significantly. DMSO specifically stabilized the VcFtsZ polymers against dilution induced disassembly and it reduced the GTPase activity of VcFtsZ. These results collectively suggested that despite lot of sequence homology, the assembly of VcFtsZ and EcFtsZ are differently regulated processes. We expect to use this knowledge of assembly properties of VcFtsZ for screening of small molecules against VcFtsZ for development of anti-cholera agent. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  19. Architecture of the ring formed by the tubulin homologue FtsZ in bacterial cell division.

    PubMed

    Szwedziak, Piotr; Wang, Qing; Bharat, Tanmay A M; Tsim, Matthew; Löwe, Jan

    2014-12-09

    Membrane constriction is a prerequisite for cell division. The most common membrane constriction system in prokaryotes is based on the tubulin homologue FtsZ, whose filaments in E. coli are anchored to the membrane by FtsA and enable the formation of the Z-ring and divisome. The precise architecture of the FtsZ ring has remained enigmatic. In this study, we report three-dimensional arrangements of FtsZ and FtsA filaments in C. crescentus and E. coli cells and inside constricting liposomes by means of electron cryomicroscopy and cryotomography. In vivo and in vitro, the Z-ring is composed of a small, single-layered band of filaments parallel to the membrane, creating a continuous ring through lateral filament contacts. Visualisation of the in vitro reconstituted constrictions as well as a complete tracing of the helical paths of the filaments with a molecular model favour a mechanism of FtsZ-based membrane constriction that is likely to be accompanied by filament sliding.

  20. Estimating the bending modulus of a FtsZ bacterial-division protein filament

    NASA Astrophysics Data System (ADS)

    Cytrynbaum, Eric N.; Li, Yongnan Devin; Allard, Jun F.; Mehrabian, Hadi

    2012-01-01

    FtsZ, a cytoskeletal protein homologous to tubulin, is the principle constituent of the division ring in bacterial cells. It is known to have force-generating capacity in vitro and has been conjectured to be the source of the constriction force in vivo. Several models have been proposed to explain the generation of force by the Z ring. Here we re-examine data from in vitro experiments in which Z rings formed and constricted inside tubular liposomes, and we carry out image analysis on previously published data with which to better estimate important model parameters that have proven difficult to measure by direct means. We introduce a membrane-energy-based model for the dynamics of multiple Z rings moving and colliding inside a tubular liposome and a fluid model for the drag of a Z ring as it moves through the tube. Using this model, we estimate an effective membrane bending modulus of 500-700 pNnm. If we assume that FtsZ force generation is driven by hydrolysis into a highly curved conformation, we estimate the FtsZ filament bending modulus to be 310-390 pNnm2. If we assume instead that force is generated by the non-hydrolysis-dependent intermediate curvature conformation, we find that Bf>1400pNnm2. The former value sits at the lower end of the range of previously estimated values and, if correct, may raise challenges for models that rely on filament bending to generate force.

  1. Curved FtsZ protofilaments generate bending forces on liposome membranes

    PubMed Central

    Osawa, Masaki; Anderson, David E; Erickson, Harold P

    2009-01-01

    We have created FtsZ-YFP-mts where an amphipathic helix on the C-terminus tethers FtsZ to the membrane. When incorporated inside multi-lamellar tubular liposomes, FtsZ-YFP-mts can assemble Z rings that generate a constriction force. When added to the outside of liposomes, FtsZ-YFP-mts bound and produced concave depressions, bending the membrane in the same direction as the Z ring inside liposomes. Prominent membrane tubules were then extruded at the intersections of concave depressions. We tested the effect of moving the membrane-targeting sequence (mts) from the C-terminus to the N-terminus, which is approximately 180 degrees from the C-terminal tether. When mts-FtsZ-YFP was applied to the outside of liposomes, it generated convex bulges, bending the membrane in the direction opposite to the concave depressions. We conclude that FtsZ protofilaments have a fixed direction of curvature, and the direction of membrane bending depends on which side of the bent protofilament the mts is attached to. This supports models in which the FtsZ constriction force is generated by protofilament bending. PMID:19779463

  2. Variations in the binding pocket of an inhibitor of the bacterial division protein FtsZ across genotypes and species.

    PubMed

    Miguel, Amanda; Hsin, Jen; Liu, Tianyun; Tang, Grace; Altman, Russ B; Huang, Kerwyn Casey

    2015-03-01

    The recent increase in antibiotic resistance in pathogenic bacteria calls for new approaches to drug-target selection and drug development. Targeting the mechanisms of action of proteins involved in bacterial cell division bypasses problems associated with increasingly ineffective variants of older antibiotics; to this end, the essential bacterial cytoskeletal protein FtsZ is a promising target. Recent work on its allosteric inhibitor, PC190723, revealed in vitro activity on Staphylococcus aureus FtsZ and in vivo antimicrobial activities. However, the mechanism of drug action and its effect on FtsZ in other bacterial species are unclear. Here, we examine the structural environment of the PC190723 binding pocket using PocketFEATURE, a statistical method that scores the similarity between pairs of small-molecule binding sites based on 3D structure information about the local microenvironment, and molecular dynamics (MD) simulations. We observed that species and nucleotide-binding state have significant impacts on the structural properties of the binding site, with substantially disparate microenvironments for bacterial species not from the Staphylococcus genus. Based on PocketFEATURE analysis of MD simulations of S. aureus FtsZ bound to GTP or with mutations that are known to confer PC190723 resistance, we predict that PC190723 strongly prefers to bind Staphylococcus FtsZ in the nucleotide-bound state. Furthermore, MD simulations of an FtsZ dimer indicated that polymerization may enhance PC190723 binding. Taken together, our results demonstrate that a drug-binding pocket can vary significantly across species, genetic perturbations, and in different polymerization states, yielding important information for the further development of FtsZ inhibitors.

  3. The bacterial cell division proteins FtsA and FtsZ self-organize into dynamic cytoskeletal patterns.

    PubMed

    Loose, Martin; Mitchison, Timothy J

    2014-01-01

    Bacterial cytokinesis is commonly initiated by the Z-ring, a cytoskeletal structure that assembles at the site of division. Its primary component is FtsZ, a tubulin superfamily GTPase, which is recruited to the membrane by the actin-related protein FtsA. Both proteins are required for the formation of the Z-ring, but if and how they influence each other's assembly dynamics is not known. Here, we reconstituted FtsA-dependent recruitment of FtsZ polymers to supported membranes, where both proteins self-organize into complex patterns, such as fast-moving filament bundles and chirally rotating rings. Using fluorescence microscopy and biochemical perturbations, we found that these large-scale rearrangements of FtsZ emerge from its polymerization dynamics and a dual, antagonistic role of FtsA: recruitment of FtsZ filaments to the membrane and negative regulation of FtsZ organization. Our findings provide a model for the initial steps of bacterial cell division and illustrate how dynamic polymers can self-organize into large-scale structures.

  4. Simple modeling of FtsZ polymers on flat and curved surfaces: correlation with experimental in vitro observations

    PubMed Central

    Paez, Alfonso; Mateos-Gil, Pablo; Hörger, Ines; Mingorance, Jesús; Rivas, Germán; Vicente, Miguel; Vélez, Marisela; Tarazona, Pedro

    2009-01-01

    FtsZ is a GTPase that assembles at midcell into a dynamic ring that constricts the membrane to induce cell division in the majority of bacteria, in many archea and several organelles. In vitro, FtsZ polymerizes in a GTP-dependent manner forming a variety of filamentous flexible structures. Based on data derived from the measurement of the in vitro polymerization of Escherichia coli FtsZ cell division protein we have formulated a model in which the fine balance between curvature, flexibility and lateral interactions accounts for structural and dynamic properties of the FtsZ polymers observed with AFM. The experimental results have been used by the model to calibrate the interaction energies and the values obtained indicate that the filaments are very plastic. The extension of the model to explore filament behavior on a cylindrical surface has shown that the FtsZ condensates promoted by lateral interactions can easily form ring structures through minor modulations of either filament curvature or longitudinal bond energies. The condensation of short, monomer exchanging filaments into rings is shown to produce enough force to induce membrane deformations. PACS codes: 87.15.ak, 87.16.ka, 87.17.Ee PMID:19849848

  5. Studies on the Dissociation and Urea-Induced Unfolding of FtsZ Support the Dimer Nucleus Polymerization Mechanism

    PubMed Central

    Montecinos-Franjola, Felipe; Ross, Justin A.; Sánchez, Susana A.; Brunet, Juan E.; Lagos, Rosalba; Jameson, David M.; Monasterio, Octavio

    2012-01-01

    FtsZ is a major protein in bacterial cytokinesis that polymerizes into single filaments. A dimer has been proposed to be the nucleating species in FtsZ polymerization. To investigate the influence of the self-assembly of FtsZ on its unfolding pathway, we characterized its oligomerization and unfolding thermodynamics. We studied the assembly using size-exclusion chromatography and fluorescence spectroscopy, and the unfolding using circular dichroism and two-photon fluorescence correlation spectroscopy. The chromatographic analysis demonstrated the presence of monomers, dimers, and tetramers with populations dependent on protein concentration. Dilution experiments using fluorescent conjugates revealed dimer-to-monomer and tetramer-to-dimer dissociation constants in the micromolar range. Measurements of fluorescence lifetimes and rotational correlation times of the conjugates supported the presence of tetramers at high protein concentrations and monomers at low protein concentrations. The unfolding study demonstrated that the three-state unfolding of FtsZ was due to the mainly dimeric state of the protein, and that the monomer unfolds through a two-state mechanism. The monomer-to-dimer equilibrium characterized here (Kd = 9 μM) indicates a significant fraction (∼10%) of stable dimers at the critical concentration for polymerization, supporting a role of the dimeric species in the first steps of FtsZ polymerization. PMID:22824282

  6. Microenvironments created by liquid-liquid phase transition control the dynamic distribution of bacterial division FtsZ protein

    PubMed Central

    Monterroso, Begoña; Zorrilla, Silvia; Sobrinos-Sanguino, Marta; Keating, Christine D.; Rivas, Germán

    2016-01-01

    The influence of membrane-free microcompartments resulting from crowding-induced liquid/liquid phase separation (LLPS) on the dynamic spatial organization of FtsZ, the main component of the bacterial division machinery, has been studied using several LLPS systems. The GTP-dependent assembly cycle of FtsZ is thought to be crucial for the formation of the septal ring, which is highly regulated in time and space. We found that FtsZ accumulates in one of the phases and/or at the interface, depending on the system composition and on the oligomerization state of the protein. These results were observed both in bulk LLPS and in lipid-stabilized, phase-separated aqueous microdroplets. The visualization of the droplets revealed that both the location and structural arrangement of FtsZ filaments is determined by the nature of the LLPS. Relocation upon depolymerization of the dynamic filaments suggests the protein may shift among microenvironments in response to changes in its association state. The existence of these dynamic compartments driven by phase transitions can alter the local composition and reactivity of FtsZ during its life cycle acting as a nonspecific modulating factor of cell function. PMID:27725777

  7. Variations in the Life Cycle of Anemone patens L. (Ranunculaceae) in Wild Populations of Canada.

    PubMed

    Kricsfalusy, Vladimir

    2016-06-30

    Based on a study of a perennial herb Anemone patens L. (Ranunculaceae) in a variety of natural habitats in Saskatchewan, Canada, eight life stages (seed, seedling, juvenile, immature, vegetative, generative, subsenile, and senile) are distinguished and characterized in detail. The species ontogenetic growth patterns are investigated. A. patens has a long life cycle that may last for several decades which leads to the formation of compact clumps. The distribution and age of clumps vary substantially in different environments with different levels of disturbance. The plant ontogeny includes the regular cycle with reproduction occurring through seeds. There is an optional subsenile vegetative disintegration at the end of the life span. The following variations in the life cycle of A. patens are identified: with slower development in young age, with an accelerated development, with omission of the generative stage, with retrogression to previous life stages in mature age, and with vegetative dormancy. The range of variations in the life cycle of A. patens may play an important role in maintaining population stability in different environmental conditions and management regimes.

  8. Variations in the Life Cycle of Anemone patens L. (Ranunculaceae) in Wild Populations of Canada

    PubMed Central

    Kricsfalusy, Vladimir

    2016-01-01

    Based on a study of a perennial herb Anemone patens L. (Ranunculaceae) in a variety of natural habitats in Saskatchewan, Canada, eight life stages (seed, seedling, juvenile, immature, vegetative, generative, subsenile, and senile) are distinguished and characterized in detail. The species ontogenetic growth patterns are investigated. A. patens has a long life cycle that may last for several decades which leads to the formation of compact clumps. The distribution and age of clumps vary substantially in different environments with different levels of disturbance. The plant ontogeny includes the regular cycle with reproduction occurring through seeds. There is an optional subsenile vegetative disintegration at the end of the life span. The following variations in the life cycle of A. patens are identified: with slower development in young age, with an accelerated development, with omission of the generative stage, with retrogression to previous life stages in mature age, and with vegetative dormancy. The range of variations in the life cycle of A. patens may play an important role in maintaining population stability in different environmental conditions and management regimes. PMID:27376340

  9. Contribution of individual promoters in the ddlB-ftsZ region to the transcription of the essential cell-division gene ftsZ in Escherichia coli.

    PubMed

    Flärdh, K; Garrido, T; Vicente, M

    1997-06-01

    The essential cell-division gene ftsZ is transcribed in Escherichia coli from at least six promoters found within the coding regions of the upstream ddlB, ftsQ, and ftsA genes. The contribution of each one to the final yield of ftsZ transcription has been estimated using transcriptional lacZ fusions. The most proximal promoter, ftsZ2p, contributes less than 5% of the total transcription from the region that reaches ftsZ. The ftsZ4p and ftsZ3p promoters, both located inside ftsA, produce almost 37% of the transcription. An ftsAp promoter within the ftsQ gene yields nearly 12% of total transcription from the region. A large proportion of transcription (approximately 46%) derives from promoters ftsQ2p and ftsQ1p, which are located inside the upstream ddlB gene. Thus, the ftsQAZ genes are to a large extent transcribed as a polycistronic mRNA. However, we find that the ftsZ proximal region is necessary for full expression, which is in agreement with a recent report that mRNA cleavage by RNase E at the end of the ftsA cistron has a significant role in the contol of ftsZ expression.

  10. Ammonium and Urea Transporter Inventory of the Selaginella and Physcomitrella Genomes

    PubMed Central

    De Michele, Roberto; Loqué, Dominique; Lalonde, Sylvie; Frommer, Wolf B.

    2012-01-01

    Ammonium and urea are important nitrogen sources for autotrophic organisms. Plant genomes encode several families of specific transporters for these molecules, plus other uptake mechanisms such as aquaporins and ABC transporters. Selaginella and Physcomitrella are representatives of lycophytes and bryophytes, respectively, and the recent completion of their genome sequences provided us with an opportunity for comparative genome studies, with special emphasis on the adaptive processes that accompanied the conquest of dry land and the evolution of a vascular system. Our phylogenetic analysis revealed that the number of genes encoding urea transporters underwent a progressive reduction during evolution, eventually down to a single copy in vascular plants. Conversely, no clear evolutionary pattern was found for ammonium transporters, and their number and distribution in families varies between species. In particular Selaginella, similar to rice, favors the AMT2/MEP family of ammonium transporters over the plant-specific AMT1 type. In comparison, Physcomitrella presents several members belonging to both families. PMID:22639655

  11. Ammonium and urea transporter inventory of the selaginella and physcomitrella genomes.

    PubMed

    De Michele, Roberto; Loqué, Dominique; Lalonde, Sylvie; Frommer, Wolf B

    2012-01-01

    Ammonium and urea are important nitrogen sources for autotrophic organisms. Plant genomes encode several families of specific transporters for these molecules, plus other uptake mechanisms such as aquaporins and ABC transporters. Selaginella and Physcomitrella are representatives of lycophytes and bryophytes, respectively, and the recent completion of their genome sequences provided us with an opportunity for comparative genome studies, with special emphasis on the adaptive processes that accompanied the conquest of dry land and the evolution of a vascular system. Our phylogenetic analysis revealed that the number of genes encoding urea transporters underwent a progressive reduction during evolution, eventually down to a single copy in vascular plants. Conversely, no clear evolutionary pattern was found for ammonium transporters, and their number and distribution in families varies between species. In particular Selaginella, similar to rice, favors the AMT2/MEP family of ammonium transporters over the plant-specific AMT1 type. In comparison, Physcomitrella presents several members belonging to both families.

  12. Characterization of the FtsZ C-Terminal Variable (CTV) Region in Z-Ring Assembly and Interaction with the Z-Ring Stabilizer ZapD in E. coli Cytokinesis

    PubMed Central

    Huang, Kuo-Hsiang; Mychack, Aaron; Tchorzewski, Lukasz; Janakiraman, Anuradha

    2016-01-01

    Polymerization of a ring-like cytoskeletal structure, the Z-ring, at midcell is a highly conserved feature in virtually all bacteria. The Z-ring is composed of short protofilaments of the tubulin homolog FtsZ, randomly arranged and held together through lateral interactions. In vitro, lateral associations between FtsZ protofilaments are stabilized by crowding agents, high concentrations of divalent cations, or in some cases, low pH. In vivo, the last 4–10 amino acid residues at the C-terminus of FtsZ (the C-terminal variable region, CTV) have been implicated in mediating lateral associations between FtsZ protofilaments through charge shielding. Multiple Z-ring associated proteins (Zaps), also promote lateral interactions between FtsZ protofilaments to stabilize the FtsZ ring in vivo. Here we characterize the complementary role/s of the CTV of E. coli FtsZ and the FtsZ-ring stabilizing protein ZapD, in FtsZ assembly. We show that the net charge of the FtsZ CTV not only affects FtsZ protofilament bundling, confirming earlier observations, but likely also the length of the FtsZ protofilaments in vitro. The CTV residues also have important consequences for Z-ring assembly and interaction with ZapD in the cell. ZapD requires the FtsZ CTV region for interaction with FtsZ in vitro and for localization to midcell in vivo. Our data suggest a mechanism in which the CTV residues, particularly K380, facilitate a conformation for the conserved carboxy-terminal residues in FtsZ, that lie immediately N-terminal to the CTV, to enable optimal contact with ZapD. Further, phylogenetic analyses suggest a correlation between the nature of FtsZ CTV residues and the presence of ZapD in the β- γ-proteobacterial species. PMID:27088231

  13. Characterization of the FtsZ C-Terminal Variable (CTV) Region in Z-Ring Assembly and Interaction with the Z-Ring Stabilizer ZapD in E. coli Cytokinesis.

    PubMed

    Huang, Kuo-Hsiang; Mychack, Aaron; Tchorzewski, Lukasz; Janakiraman, Anuradha

    2016-01-01

    Polymerization of a ring-like cytoskeletal structure, the Z-ring, at midcell is a highly conserved feature in virtually all bacteria. The Z-ring is composed of short protofilaments of the tubulin homolog FtsZ, randomly arranged and held together through lateral interactions. In vitro, lateral associations between FtsZ protofilaments are stabilized by crowding agents, high concentrations of divalent cations, or in some cases, low pH. In vivo, the last 4-10 amino acid residues at the C-terminus of FtsZ (the C-terminal variable region, CTV) have been implicated in mediating lateral associations between FtsZ protofilaments through charge shielding. Multiple Z-ring associated proteins (Zaps), also promote lateral interactions between FtsZ protofilaments to stabilize the FtsZ ring in vivo. Here we characterize the complementary role/s of the CTV of E. coli FtsZ and the FtsZ-ring stabilizing protein ZapD, in FtsZ assembly. We show that the net charge of the FtsZ CTV not only affects FtsZ protofilament bundling, confirming earlier observations, but likely also the length of the FtsZ protofilaments in vitro. The CTV residues also have important consequences for Z-ring assembly and interaction with ZapD in the cell. ZapD requires the FtsZ CTV region for interaction with FtsZ in vitro and for localization to midcell in vivo. Our data suggest a mechanism in which the CTV residues, particularly K380, facilitate a conformation for the conserved carboxy-terminal residues in FtsZ, that lie immediately N-terminal to the CTV, to enable optimal contact with ZapD. Further, phylogenetic analyses suggest a correlation between the nature of FtsZ CTV residues and the presence of ZapD in the β- γ-proteobacterial species.

  14. Design, synthesis and structure-activity relationships of substituted oxazole-benzamide antibacterial inhibitors of FtsZ.

    PubMed

    Stokes, Neil R; Baker, Nicola; Bennett, James M; Chauhan, Pramod K; Collins, Ian; Davies, David T; Gavade, Maruti; Kumar, Dushyant; Lancett, Paul; Macdonald, Rebecca; Macleod, Leanne; Mahajan, Anu; Mitchell, Jeffrey P; Nayal, Narendra; Nayal, Yashodanand Nandan; Pitt, Gary R W; Singh, Mahipal; Yadav, Anju; Srivastava, Anil; Czaplewski, Lloyd G; Haydon, David J

    2014-01-01

    The design, synthesis and structure-activity relationships of a series of oxazole-benzamide inhibitors of the essential bacterial cell division protein FtsZ are described. Compounds had potent anti-staphylococcal activity and inhibited the cytokinesis of the clinically-significant bacterial pathogen Staphylococcus aureus. Selected analogues possessing a 5-halo oxazole also inhibited a strain of S. aureus harbouring the glycine-to-alanine amino acid substitution at residue 196 of FtsZ which conferred resistance to previously reported inhibitors in the series. Substitutions to the pseudo-benzylic carbon of the scaffold improved the pharmacokinetic properties by increasing metabolic stability and provided a mechanism for creating pro-drugs. Combining multiple substitutions based on the findings reported in this study has provided small-molecule inhibitors of FtsZ with enhanced in vitro and in vivo antibacterial efficacy.

  15. Advances in the discovery of novel antimicrobials targeting the assembly of bacterial cell division protein FtsZ.

    PubMed

    Li, Xin; Ma, Shutao

    2015-05-05

    Currently, wide-spread antimicrobials resistance among bacterial pathogens continues being a dramatically increasing and serious threat to public health, and thus there is a pressing need to develop new antimicrobials to keep pace with the bacterial resistance. Filamentous temperature-sensitive protein Z (FtsZ), a prokaryotic cytoskeleton protein, plays an important role in bacterial cell division. It as a very new and promising target, garners special attention in the antibacterial research in the recent years. This review describes not only the function and dynamic behaviors of FtsZ, but also the known natural and synthetic inhibitors of FtsZ. In particular, the small molecules recently developed and the future directions of ideal candidates are highlighted.

  16. 4',6-Diamidino-2-phenylindole (DAPI) induces bundling of Escherichia coli FtsZ polymers inhibiting the GTPase activity.

    PubMed

    Nova, Esteban; Montecinos, Felipe; Brunet, Juan E; Lagos, Rosalba; Monasterio, Octavio

    2007-09-15

    FtsZ (Filamentous temperature sensitivity Z) cell division protein from Escherichia coli binds the fluorescence probe DAPI. Bundling of FtsZ was facilitated in the presence of DAPI, and the polymers in solution remained polymerized longer time than the protofilaments formed in the absence of DAPI. DAPI decreased both the maximal velocity of the GTPase activity and the Michaelis-Menten constant for GTP, indicating that behaves like an uncompetitive inhibitor of the GTPase activity favoring the GTP form of FtsZ in the polymers. The results presented in this work support a cooperative polymerization mechanism in which the binding of DAPI favors protofilament lateral interactions and the stability of the resulting polymers.

  17. Bacterial division proteins FtsZ and ZipA induce vesicle shrinkage and cell membrane invagination.

    PubMed

    Cabré, Elisa J; Sánchez-Gorostiaga, Alicia; Carrara, Paolo; Ropero, Noelia; Casanova, Mercedes; Palacios, Pilar; Stano, Pasquale; Jiménez, Mercedes; Rivas, Germán; Vicente, Miguel

    2013-09-13

    Permeable vesicles containing the proto-ring anchoring ZipA protein shrink when FtsZ, the main cell division protein, polymerizes in the presence of GTP. Shrinkage, resembling the constriction of the cytoplasmic membrane, occurs at ZipA densities higher than those found in the cell and is modulated by the dynamics of the FtsZ polymer. In vivo, an excess of ZipA generates multilayered membrane inclusions within the cytoplasm and causes the loss of the membrane function as a permeability barrier. Overproduction of ZipA at levels that block septation is accompanied by the displacement of FtsZ and two additional division proteins, FtsA and FtsN, from potential septation sites to clusters that colocalize with ZipA near the membrane. The results show that elementary constriction events mediated by defined elements involved in cell division can be evidenced both in bacteria and in vesicles.

  18. Bacterial Division Proteins FtsZ and ZipA Induce Vesicle Shrinkage and Cell Membrane Invagination*

    PubMed Central

    Cabré, Elisa J.; Sánchez-Gorostiaga, Alicia; Carrara, Paolo; Ropero, Noelia; Casanova, Mercedes; Palacios, Pilar; Stano, Pasquale; Jiménez, Mercedes; Rivas, Germán; Vicente, Miguel

    2013-01-01

    Permeable vesicles containing the proto-ring anchoring ZipA protein shrink when FtsZ, the main cell division protein, polymerizes in the presence of GTP. Shrinkage, resembling the constriction of the cytoplasmic membrane, occurs at ZipA densities higher than those found in the cell and is modulated by the dynamics of the FtsZ polymer. In vivo, an excess of ZipA generates multilayered membrane inclusions within the cytoplasm and causes the loss of the membrane function as a permeability barrier. Overproduction of ZipA at levels that block septation is accompanied by the displacement of FtsZ and two additional division proteins, FtsA and FtsN, from potential septation sites to clusters that colocalize with ZipA near the membrane. The results show that elementary constriction events mediated by defined elements involved in cell division can be evidenced both in bacteria and in vesicles. PMID:23921390

  19. [Effect of cinnamon and lavender oils on FtsZ gene expression in the Staphylococus aureus ATCC 29213].

    PubMed

    2013-01-01

    This study was designed to determine the effect of lavender and cinnamon oils on FtsZ gene expression in Staphylococcus aureus ATCC 29213. The cinnamon and lavender oils at least partially results from the inhibition of FtsZ transcription and disruption of cell division process at the level of the septum synthesis, what is similar to mechanisms of drug action used in anti-staphylococcal therapies. The presented results could be an important background for the further detailed research, which is needed to clarify the effect of essential oils on FtsZ synthesis at the posttranscriptional level and other stages of cell division process of S. aureus and other pathogenic bacteria.

  20. Promoting assembly and bundling of FtsZ as a strategy to inhibit bacterial cell division: a new approach for developing novel antibacterial drugs.

    PubMed

    Beuria, Tushar K; Singh, Parminder; Surolia, Avadhesha; Panda, Dulal

    2009-09-14

    FtsZ plays an essential role in bacterial cell division. We have used the assembly of FtsZ as a screen to find antibacterial agents with a novel mechanism of action. The effects of 81 compounds of 29 different structural scaffolds on FtsZ assembly in vitro were examined using a sedimentation assay. Out of these 81 compounds, OTBA (3-{5-[4-oxo-2-thioxo-3-(3-trifluoromethyl-phenyl)-thiazolidin-5-ylidenemethyl]-furan-2-yl}-benzoic acid) was found to promote FtsZ assembly in vitro. OTBA increased the assembly of FtsZ, caused bundling of FtsZ protofilaments, prevented dilution-induced disassembly of FtsZ protofilaments and decreased the GTPase activity in vitro. It bound to FtsZ with an apparent dissociation constant of 15+/-1.5 microM. Furthermore, OTBA inhibited the proliferation of Bacillus subtilis 168 cells with an MIC (minimum inhibitory concentration) of 2 microM, whereas it exerted minimal effects on mammalian cell proliferation, indicating that it might have a potential use as an antibacterial drug. In the effective proliferation inhibitory concentration range, OTBA induced filamentation in bacteria and also perturbed the formation of the cytokinetic Z-rings in bacteria. However, the agent neither perturbed the membrane structures nor affected the nucleoid segregation in B. subtilis cells. The results suggested that the OTBA inhibited bacterial cytokinesis by perturbing the formation and functioning of the Z-ring via altering FtsZ assembly dynamics. The antibacterial mechanism of action of OTBA is similar to that of the widely used anticancer drug paclitaxel, which inhibits cancer cell proliferation by promoting the assembly of tubulin, a eukaryotic homologue of FtsZ.

  1. FtsZ Filament Dynamics at Steady State: Subunit Exchange with and without Nucleotide Hydrolysis†

    PubMed Central

    Chen, Yaodong; Erickson, Harold P.

    2009-01-01

    We have measured three aspects of FtsZ filament dynamics at steady state: rates of GTP hydrolysis, subunit exchange between protofilaments, and disassembly induced by dilution or excess GDP. All three reactions were slowed with an increase in the potassium concentration from 100 to 500 mM, via replacement of potassium with rubidium, or with an increase in the magnesium concentration from 5 to 20 mM. Electron microscopy showed that the polymers assembled under the conditions of fastest assembly were predominantly short, one-stranded protofilaments, whereas under conditions of slower dynamics, the protofilaments tended to associate into long, thin bundles. We suggest that exchange of subunits between protofilaments at steady state involves two separate mechanisms: (1) fragmentation or dissociation of subunits from protofilament ends following GTP hydrolysis and (2) reversible association and dissociation of subunits from protofilament ends independent of hydrolysis. Exchange of nucleotides on these recycling subunits could give the appearance of exchange directly into the polymer. Several of our observations suggest that exchange of nucleotide can take place on these recycling subunits, but not directly into the FtsZ polymer. Annealing of protofilaments was demonstrated for the L68W mutant in EDTA buffer but not in Mg buffer, where rapid cycling of subunits may obscure the effect of annealing. We also reinvestigated the nucleotide composition of FtsZ polymers at steady state. We found that the GDP:GTP ratio was 50:50 for concentrations of GTP > 100 μM, significantly higher than the 20:80 ratio previously reported at 20 μM GTP. PMID:19527070

  2. Estimating the bending modulus of a FtsZ bacterial-division protein filament.

    PubMed

    Cytrynbaum, Eric N; Li, Yongnan Devin; Allard, Jun F; Mehrabian, Hadi

    2012-01-01

    FtsZ, a cytoskeletal protein homologous to tubulin, is the principle constituent of the division ring in bacterial cells. It is known to have force-generating capacity in vitro and has been conjectured to be the source of the constriction force in vivo. Several models have been proposed to explain the generation of force by the Z ring. Here we re-examine data from in vitro experiments in which Z rings formed and constricted inside tubular liposomes, and we carry out image analysis on previously published data with which to better estimate important model parameters that have proven difficult to measure by direct means. We introduce a membrane-energy-based model for the dynamics of multiple Z rings moving and colliding inside a tubular liposome and a fluid model for the drag of a Z ring as it moves through the tube. Using this model, we estimate an effective membrane bending modulus of 500-700 pN nm. If we assume that FtsZ force generation is driven by hydrolysis into a highly curved conformation, we estimate the FtsZ filament bending modulus to be 310-390 pN nm(2). If we assume instead that force is generated by the non-hydrolysis-dependent intermediate curvature conformation, we find that B(f)>1400 pN nm(2). The former value sits at the lower end of the range of previously estimated values and, if correct, may raise challenges for models that rely on filament bending to generate force. © 2012 American Physical Society

  3. Visualization of plastids in pollen grains: involvement of FtsZ1 in pollen plastid division.

    PubMed

    Tang, Lay Yin; Nagata, Noriko; Matsushima, Ryo; Chen, Yuling; Yoshioka, Yasushi; Sakamoto, Wataru

    2009-04-01

    Visualizing organelles in living cells is a powerful method to analyze their intrinsic mechanisms. Easy observation of chlorophyll facilitates the study of the underlying mechanisms in chloroplasts, but not in other plastid types. Here, we constructed a transgenic plant enabling visualization of plastids in pollen grains. Combination of a plastid-targeted fluorescent protein with a pollen-specific promoter allowed us to observe the precise number, size and morphology of plastids in pollen grains of the wild type and the ftsZ1 mutant, whose responsible gene plays a central role in chloroplast division. The transgenic material presented in this work is useful for studying the division mechanism of pollen plastids.

  4. The bacterial cell-division protein ZipA and its interaction with an FtsZ fragment revealed by X-ray crystallography

    PubMed Central

    Mosyak, Lidia; Zhang, Yan; Glasfeld, Elizabeth; Haney, Steve; Stahl, Mark; Seehra, Jasbir; Somers, William S.

    2000-01-01

    In Escherichia coli, FtsZ, a homologue of eukaryotic tubulins, and ZipA, a membrane-anchored protein that binds to FtsZ, are two essential components of the septal ring structure that mediates cell division. Recent data indicate that ZipA is involved in the assembly of the ring by linking FtsZ to the cytoplasmic membrane and that the ZipA–FtsZ interaction is mediated by their C-terminal domains. We present the X-ray crystal structures of the C-terminal FtsZ-binding domain of ZipA and a complex between this domain and a C-terminal fragment of FtsZ. The ZipA domain is a six-stranded β-sheet packed against three α-helices and contains the split β–α–β motif found in many RNA-binding proteins. The uncovered side of the sheet incorporates a shallow hydrophobic cavity exposed to solvent. In the complex, the 17-residue FtsZ fragment occupies this entire cavity of ZipA and binds as an extended β-strand followed by α-helix. An alanine-scanning mutagenesis analysis of the FtsZ fragment was also performed, which shows that only a small cluster of the buried FtsZ side chains is critical in binding to ZipA. PMID:10880432

  5. Differential Regulation of ftsZ Transcription during Septation of Streptomyces griseus

    PubMed Central

    Kwak, Jangyul; Dharmatilake, Amitha J.; Jiang, Hao; Kendrick, Kathleen E.

    2001-01-01

    Streptomyces has been known to form two types of septa. The data in this research demonstrated that Streptomyces griseus forms another type of septum near the base of sporogenic hyphae (basal septum). To understand the regulation of the septation machinery in S. griseus, we investigated the expression of the ftsZ gene. S1 nuclease protection assays revealed that four ftsZ transcripts were differentially expressed during morphological differentiation. The vegetative transcript (emanating from Pveg) is present at a moderate level during vegetative growth, but is switched off within the first 2 h of sporulation. Two sporulation-specific transcripts predominantly accumulated, and the levels increased by approximately fivefold together shortly before sporulation septa begin to form. Consistently, the sporulation-specific transcripts were expressed much earlier and more abundantly in a group of nonsporulating mutants that form their sporulation septa prematurely. Promoter-probe studies with two different reporter systems confirmed the activities of the putative promoters identified from the 5′ end point of the transcripts. The levels and expression timing of promoter activities were consistent with the results of nuclease protection assays. The aseptate phenotype of the Pspo mutant indicated that the increased transcription from Pspo is required for sporulation septation, but not for vegetative or basal septum formation. PMID:11489862

  6. Tubulin secondary structure analysis, limited proteolysis sites, and homology to FtsZ.

    PubMed

    de Pereda, J M; Leynadier, D; Evangelio, J A; Chacón, P; Andreu, J M

    1996-11-12

    The far-ultraviolet circular dichroism spectrum of the alpha beta-tubulin dimer analyzed by six different methods indicates an average content of approximately 33% alpha helix, 21% beta sheet, and 45% other secondary structure. Deconvolution of Fourier transform infrared spectra indicates 24% sheet, 37% (maximum) helix, and 38% (minimum) other structure. Separate alignments of 75 alpha-tubulin, 106 beta-tubulin, and 14 gamma-tubulin sequences and 12 sequences of the bacterial cell division protein FtsZ have been employed to predict their secondary structures with the multiple-sequence method PHD [Rost, B., & Sander, C. (1993a) J. Mol. Biol. 232, 584-599]. The predicted secondary structures average of 33% alpha helix, 24% beta sheet, and 43% loop for the alpha beta dimer. The predictions have been compared with sites of limited proteolysis by 12 proteases at the surfaces of the heterodimer and taxol-induced microtubules [de Pereda, J. M., & Andreu, J. M. (1996) Biochemistry 35, 14184-14202]. From 24 experimentally determined nicking sites, 18 are at predicted loops or at the extremes of secondary structure elements. Proteolysis zone A (including acetylable Lys40 and probably Lys60 in alpha-tubulin and Gly93 in beta-tubulin) and proteolysis zone B (extending between residues 167 and 183 in both chains) are accessible in microtubules. Proteolysis zone C, between residues 278 and 295, becomes partially occluded in microtubules. The alpha-tubulin nicking site Arg339-Ser340 is at a loop following a predicted alpha helix in proteolysis zone D. This site is protected in taxol microtubules; however, a new tryptic site appears which is probably located at the N-terminal end of the same helix. Zone D also contains beta-tubulin Cys354, which is accessible in microtubules. Proteolysis zone E includes the C-terminal hypervariable loops (10-20 residues) of each tubulin chain. These follow the two larger predicted helical zones (residues 372-395 and 405-432 in beta-tubulin), which

  7. Preparation and characteristics of bio-oil from the marine brown alga Sargassum patens C. Agardh.

    PubMed

    Li, Demao; Chen, Limei; Xu, Dong; Zhang, Xiaowen; Ye, Naihao; Chen, Fangjian; Chen, Shulin

    2012-01-01

    The marine brown alga, Sargassum patens C. Agardh, floating on the Yellow Sea, was collected and converted to bio-oil through hydrothermal liquefaction with a modified reactor. A maximum yield of 32.1±0.2 wt.% bio-oil was obtained after 15 min at 340 °C, at a feedstock concentration of 15 g biomass/150 ml water, without using a catalyst. The bio-oil had a heating value of 27.1MJ/kg and contained water, lipid, alcohol, phenol, esters, ethers and aromatic compounds. The solid residue obtained had a high ash and oxygen content. The results suggest that S. patens C. Agardh has potential as biomass feedstock for fuel and chemical products.

  8. Short FtsZ filaments can drive asymmetric cell envelope constriction at the onset of bacterial cytokinesis.

    PubMed

    Yao, Qing; Jewett, Andrew I; Chang, Yi-Wei; Oikonomou, Catherine M; Beeby, Morgan; Iancu, Cristina V; Briegel, Ariane; Ghosal, Debnath; Jensen, Grant J

    2017-06-01

    FtsZ, the bacterial homologue of eukaryotic tubulin, plays a central role in cell division in nearly all bacteria and many archaea. It forms filaments under the cytoplasmic membrane at the division site where, together with other proteins it recruits, it drives peptidoglycan synthesis and constricts the cell. Despite extensive study, the arrangement of FtsZ filaments and their role in division continue to be debated. Here, we apply electron cryotomography to image the native structure of intact dividing cells and show that constriction in a variety of Gram-negative bacterial cells, including Proteus mirabilis and Caulobacter crescentus, initiates asymmetrically, accompanied by asymmetric peptidoglycan incorporation and short FtsZ-like filament formation. These results show that a complete ring of FtsZ is not required for constriction and lead us to propose a model for FtsZ-driven division in which short dynamic FtsZ filaments can drive initial peptidoglycan synthesis and envelope constriction at the onset of cytokinesis, later increasing in length and number to encircle the division plane and complete constriction. © 2017 The Authors.

  9. A canonical FtsZ protein in Verrucomicrobium spinosum, a member of the Bacterial phylum Verrucomicrobia that also includes tubulin-producing Prosthecobacter species

    PubMed Central

    Yee, Benjamin; Lafi, Feras F; Oakley, Brian; Staley, James T; Fuerst, John A

    2007-01-01

    Background The origin and evolution of the homologous GTP-binding cytoskeletal proteins FtsZ typical of Bacteria and tubulin characteristic of eukaryotes is a major question in molecular evolutionary biology. Both FtsZ and tubulin are central to key cell biology processes – bacterial septation and cell division in the case of FtsZ and in the case of tubulins the function of microtubules necessary for mitosis and other key cytoskeleton-dependent processes in eukaryotes. The origin of tubulin in particular is of significance to models for eukaryote origins. Most members of domain Bacteria possess FtsZ, but bacteria in genus Prosthecobacter of the phylum Verrucomicrobia form a key exception, possessing tubulin homologs BtubA and BtubB. It is therefore of interest to know whether other members of phylum Verrucomicrobia possess FtsZ or tubulin as their FtsZ-tubulin gene family representative. Results Verrucomicrobium spinosum, a member of Phylum Verrucomicrobia of domain Bacteria, has been found to possess a gene for a protein homologous to the cytoskeletal protein FtsZ. The deduced amino acid sequence has sequence signatures and predicted secondary structure characteristic for FtsZ rather than tubulin, but phylogenetic trees and sequence analysis indicate that it is divergent from all other known FtsZ sequences in members of domain Bacteria. The FtsZ gene of V. spinosum is located within a dcw gene cluster exhibiting gene order conservation known to contribute to the divisome in other Bacteria and comparable to these clusters in other Bacteria, suggesting a similar functional role. Conclusion Verrucomicrobium spinosum has been found to possess a gene for a protein homologous to the cytoskeletal protein FtsZ. The results suggest the functional as well as structural homology of the V. spinosum FtsZ to the FtsZs of other Bacteria implying its involvement in cell septum formation during division. Thus, both bacteria-like FtsZ and eukaryote-like tubulin cytoskeletal

  10. Kinetic modeling of the assembly, dynamic steady state, and contraction of the FtsZ ring in prokaryotic cytokinesis.

    PubMed

    Surovtsev, Ivan V; Morgan, Jeffrey J; Lindahl, Paul A

    2008-07-04

    Cytokinesis in prokaryotes involves the assembly of a polymeric ring composed of FtsZ protein monomeric units. The Z ring forms at the division plane and is attached to the membrane. After assembly, it maintains a stable yet dynamic steady state. Once induced, the ring contracts and the membrane constricts. In this work, we present a computational deterministic biochemical model exhibiting this behavior. The model is based on biochemical features of FtsZ known from in vitro studies, and it quantitatively reproduces relevant in vitro data. An essential part of the model is a consideration of interfacial reactions involving the cytosol volume, where monomeric FtsZ is dispersed, and the membrane surface in the cell's mid-zone where the ring is assembled. This approach allows the same chemical model to simulate either in vitro or in vivo conditions by adjusting only two geometrical parameters. The model includes minimal reactions, components, and assumptions, yet is able to reproduce sought-after in vivo behavior, including the rapid assembly of the ring via FtsZ-polymerization, the formation of a dynamic steady state in which GTP hydrolysis leads to the exchange of monomeric subunits between cytoplasm and the ring, and finally the induced contraction of the ring. The model gives a quantitative estimate for coupling between the rate of GTP hydrolysis and of FtsZ subunit turnover between the assembled ring and the cytoplasmic pool as observed. Membrane constriction is chemically driven by the strong tendency of GTP-bound FtsZ to self-assembly. The model suggests a possible mechanism of membrane contraction without a motor protein. The portion of the free energy of GTP hydrolysis released in cyclization is indirectly used in this energetically unfavorable process. The model provides a limit to the mechanistic complexity required to mimic ring behavior, and it highlights the importance of parallel in vitro and in vivo modeling.

  11. Synthesis of Antimicrobial Natural Products Targeting FtsZ: (±)-Dichamanetin and (±)-2″′-Hydroxy-5″-benzylisouvarinol-B

    PubMed Central

    Urgaonkar, Sameer; La Pierre, Henry S.; Meir, Israel; Lund, Henrik; RayChaudhuri, Debabrata; Shaw, Jared T.

    2008-01-01

    Two natural products have been synthesized using a ZnCl2-mediated benzylic coupling reaction. Both are potent inhibitors of the GTPase activity of FtsZ, a protein that is essential for bacterial cytokinesis. PMID:16321003

  12. Roles of Arabidopsis PARC6 in Coordination of the Chloroplast Division Complex and Negative Regulation of FtsZ Assembly1[OPEN

    PubMed Central

    Chen, Cheng; Froehlich, John E.; TerBush, Allan D.

    2016-01-01

    Chloroplast division is driven by the simultaneous constriction of the inner FtsZ ring (Z ring) and the outer DRP5B ring. The assembly and constriction of these rings in Arabidopsis (Arabidopsis thaliana) are coordinated partly through the inner envelope membrane protein ACCUMULATION AND REPLICATION OF CHLOROPLASTS6 (ARC6). Previously, we showed that PARC6 (PARALOG OF ARC6), also in the inner envelope membrane, negatively regulates FtsZ assembly and acts downstream of ARC6 to position the outer envelope membrane protein PLASTID DIVISION1 (PDV1), which functions together with its paralog PDV2 to recruit DYNAMIN-RELATED PROTEIN 5B (DRP5B) from a cytosolic pool to the outer envelope membrane. However, whether PARC6, like ARC6, also functions in coordination of the chloroplast division contractile complexes was unknown. Here, we report a detailed topological analysis of Arabidopsis PARC6, which shows that PARC6 has a single transmembrane domain and a topology resembling that of ARC6. The newly identified stromal region of PARC6 interacts not only with ARC3, a direct inhibitor of Z-ring assembly, but also with the Z-ring protein FtsZ2. Overexpression of PARC6 inhibits FtsZ assembly in Arabidopsis but not in a heterologous yeast system (Schizosaccharomyces pombe), suggesting that the negative regulation of FtsZ assembly by PARC6 is a consequence of its interaction with ARC3. A conserved carboxyl-terminal peptide in FtsZ2 mediates FtsZ2 interaction with both PARC6 and ARC6. Consistent with its role in the positioning of PDV1, the intermembrane space regions of PARC6 and PDV1 interact. These findings provide new insights into the functions of PARC6 and suggest that PARC6 coordinates the inner Z ring and outer DRP5B ring through interaction with FtsZ2 and PDV1 during chloroplast division. PMID:26527658

  13. Structures of the nucleoid occlusion protein SlmA bound to DNA and the C-terminal domain of the cytoskeletal protein FtsZ

    PubMed Central

    Schumacher, Maria A.; Zeng, Wenjie

    2016-01-01

    Cell division in most prokaryotes is mediated by FtsZ, which polymerizes to create the cytokinetic Z ring. Multiple FtsZ-binding proteins regulate FtsZ polymerization to ensure the proper spatiotemporal formation of the Z ring at the division site. The DNA-binding protein SlmA binds to FtsZ and prevents Z-ring formation through the nucleoid in a process called “nucleoid occlusion” (NO). As do most FtsZ-accessory proteins, SlmA interacts with the conserved C-terminal domain (CTD) that is connected to the FtsZ core by a long, flexible linker. However, SlmA is distinct from other regulatory factors in that it must be DNA-bound to interact with the FtsZ CTD. Few structures of FtsZ regulator–CTD complexes are available, but all reveal the CTD bound as a helix. To deduce the molecular basis for the unique SlmA–DNA–FtsZ CTD regulatory interaction and provide insight into FtsZ–regulator protein complex formation, we determined structures of Escherichia coli, Vibrio cholera, and Klebsiella pneumonia SlmA–DNA–FtsZ CTD ternary complexes. Strikingly, the FtsZ CTD does not interact with SlmA as a helix but binds as an extended conformation in a narrow, surface-exposed pocket formed only in the DNA-bound state of SlmA and located at the junction between the DNA-binding and C-terminal dimer domains. Binding studies are consistent with the structure and underscore key interactions in complex formation. Combined, these data reveal the molecular basis for the SlmA–DNA–FtsZ interaction with implications for SlmA’s NO function and underscore the ability of the FtsZ CTD to adopt a wide range of conformations, explaining its ability to bind diverse regulatory proteins. PMID:27091999

  14. Design, synthesis and antibacterial activity of isatin derivatives as FtsZ inhibitors

    NASA Astrophysics Data System (ADS)

    Lian, Zhi-Min; Sun, Juan; Zhu, Hai-Liang

    2016-08-01

    Seven isatin derivatives have been designed, and their chemical structures were characterized by single crystal X-ray diffraction studies, 1H NMR, MS, and elemental analysis. Structural stabilization followed by intramolecular as well as intermolecular H-bonds makes these molecules as perfect examples in molecular recognition with self-complementary donor and acceptor units within a single molecule. These compounds were evaluated for antimicrobial activities. Docking simulations have been performed to position compounds into the FtsZ active site to determine their probable binding models. All of the compounds exhibited better antibacterial activities. Interestingly, compound 5c and 5d exhibited better antibacterial activities with IC50 values of 0.03 and 0.05 μmol/mL against Staphylococcus aureus, respectively. Compound 5g displays antibacterial activity with IC50 values of 0.672 and 0.830 μmol/mL against Escherichia coli and Pseudomonas aeruginosa, respectively.

  15. WhmD promotes the assembly of Mycobacterium smegmatis FtsZ: A possible role of WhmD in bacterial cell division.

    PubMed

    Bhattacharya, Dipanwita; Kumar, Ashutosh; Panda, Dulal

    2017-02-01

    WhmD is considered to have a role in the septation and division of Mycobacterium smegmatis cells. Since FtsZ is the central protein of the septum, we determined the effect of WhmD on the assembly of Mycobacterium smegmatis FtsZ (MsFtsZ) in vitro. WhmD increased both the rate and extent of the assembly of MsFtsZ in vitro. WhmD also increased the amount of polymerized MsFtsZ as evident from a sedimentation assay. Further, the assembly promoting activity of WhmD occurred in the presence of GTP. MsFtsZ polymerized to form thin filaments in the absence of WhmD while MsFtsZ formed thick filaments in the presence of WhmD suggesting that WhmD enhanced the bundling of MsFtsZ filaments. Interestingly, WhmD neither suppressed the dilution-induced disassembly of FtsZ filaments nor significantly altered the GTPase activity of FtsZ. Using size exclusion chromatography, circular dichroism and fluorescence spectroscopy, WhmD was found to bind to MsFtsZ in vitro. The results showed that WhmD can promote the assembly of FtsZ and indicated that WhmD may play a role in the division of M. smegmatis cells by assisting the polymerization of FtsZ.

  16. Comparison of small molecule inhibitors of the bacterial cell division protein FtsZ and identification of a reliable cross-species inhibitor.

    PubMed

    Anderson, David E; Kim, Michelle B; Moore, Jared T; O'Brien, Terrence E; Sorto, Nohemy A; Grove, Charles I; Lackner, Laura L; Ames, James B; Shaw, Jared T

    2012-11-16

    FtsZ is a guanosine triphosphatase (GTPase) that mediates cytokinesis in bacteria. FtsZ is homologous in structure to eukaryotic tubulin and polymerizes in a similar head-to-tail fashion. The study of tubulin's function in eukaryotic cells has benefited greatly from specific and potent small molecule inhibitors, including colchicine and taxol. Although many small molecule inhibitors of FtsZ have been reported, none has emerged as a generally useful probe for modulating bacterial cell division. With the goal of establishing a useful and reliable small molecule inhibitor of FtsZ, a broad biochemical cross-comparison of reported FtsZ inhibitors was undertaken. Several of these molecules, including phenolic natural products, are unselective inhibitors that seem to derive their activity from the formation of microscopic colloids or aggregates. Other compounds, including the natural product viriditoxin and the drug development candidate PC190723, exhibit no inhibition of GTPase activity using protocols in this work or under published conditions. Of the compounds studied, only zantrin Z3 exhibits good levels of inhibition, maintains activity under conditions that disrupt small molecule aggregates, and provides a platform for exploration of structure-activity relationships (SAR). Preliminary SAR studies have identified slight modifications to the two side chains of this structure that modulate the inhibitory activity of zantrin Z3. Collectively, these studies will help focus future investigations toward the establishment of probes for FtsZ that fill the roles of colchicine and taxol in studies of tubulin.

  17. Identification of Escherichia coli ZapC (YcbW) as a Component of the Division Apparatus That Binds and Bundles FtsZ Polymers▿ †

    PubMed Central

    Hale, Cynthia A.; Shiomi, Daisuke; Liu, Bing; Bernhardt, Thomas G.; Margolin, William; Niki, Hironori; de Boer, Piet A. J.

    2011-01-01

    Assembly of the cell division apparatus in bacteria starts with formation of the Z ring on the cytoplasmic face of the membrane. This process involves the accumulation of FtsZ polymers at midcell and their interaction with several FtsZ-binding proteins that collectively organize the polymers into a membrane-associated ring-like configuration. Three such proteins, FtsA, ZipA, and ZapA, have previously been identified in Escherichia coli. FtsA and ZipA are essential membrane-associated division proteins that help connect FtsZ polymers with the inner membrane. ZapA is a cytoplasmic protein that is not required for the fission process per se but contributes to its efficiency, likely by promoting lateral interactions between FtsZ protofilaments. We report the identification of YcbW (ZapC) as a fourth FtsZ-binding component of the Z ring in E. coli. Binding of ZapC promotes lateral interactions between FtsZ polymers and suppresses FtsZ GTPase activity. This and additional evidence indicate that, like ZapA, ZapC is a nonessential Z-ring component that contributes to the efficiency of the division process by stabilizing the polymeric form of FtsZ. PMID:21216997

  18. Proteolysis-Dependent Remodeling of the Tubulin Homolog FtsZ at the Division Septum in Escherichia coli

    PubMed Central

    Viola, Marissa G.; LaBreck, Christopher J.; Conti, Joseph; Camberg, Jodi L.

    2017-01-01

    During bacterial cell division a dynamic protein structure called the Z-ring assembles at the septum. The major protein in the Z-ring in Escherichia coli is FtsZ, a tubulin homolog that polymerizes with GTP. FtsZ is degraded by the two-component ATP-dependent protease ClpXP. Two regions of FtsZ, located outside of the polymerization domain in the unstructured linker and at the C-terminus, are important for specific recognition and degradation by ClpXP. We engineered a synthetic substrate containing green fluorescent protein (Gfp) fused to an extended FtsZ C-terminal tail (residues 317–383), including the unstructured linker and the C-terminal conserved region, but not the polymerization domain, and showed that it is sufficient to target a non-native substrate for degradation in vitro. To determine if FtsZ degradation regulates Z-ring assembly during division, we expressed a full length Gfp-FtsZ fusion protein in wild type and clp deficient strains and monitored fluorescent Z-rings. In cells deleted for clpX or clpP, or cells expressing protease-defective mutant protein ClpP(S97A), Z-rings appear normal; however, after photobleaching a region of the Z-ring, fluorescence recovers ~70% more slowly in cells without functional ClpXP than in wild type cells. Gfp-FtsZ(R379E), which is defective for degradation by ClpXP, also assembles into Z-rings that recover fluorescence ~2-fold more slowly than Z-rings containing Gfp-FtsZ. In vitro, ClpXP cooperatively degrades and disassembles FtsZ polymers. These results demonstrate that ClpXP is a regulator of Z-ring dynamics and that the regulation is proteolysis-dependent. Our results further show that FtsZ-interacting proteins in E. coli fine-tune Z-ring dynamics. PMID:28114338

  19. Genetic Diversity and Population Structure of the Rare and Endangered Plant Species Pulsatilla patens (L.) Mill in East Central Europe.

    PubMed

    Szczecińska, Monika; Sramko, Gabor; Wołosz, Katarzyna; Sawicki, Jakub

    2016-01-01

    Pulsatilla patens s.s. is a one of the most endangered plant species in Europe. The present range of this species in Europe is highly fragmented and the size of the populations has been dramatically reduced in the past 50 years. The rapid disappearance of P. patens localities in Europe has prompted the European Commission to initiate active protection of this critically endangered species. The aim of this study was to estimate the degree and distribution of genetic diversity within European populations of this endangered species. We screened 29 populations of P. patens using a set of six microsatellite primers. The results of our study indicate that the analyzed populations are characterized by low levels of genetic diversity (Ho = 0.005) and very high levels of inbreeding (FIS = 0.90). These results suggest that genetic erosion could be partially responsible for the lower fitness in smaller populations of this species. Private allelic richness was very low, being as low as 0.00 for most populations. Average genetic diversity over loci and mean number of alleles in P. patens populations were significantly correlated with population size, suggesting severe genetic drift. The results of AMOVA point to higher levels of variation within populations than between populations.The results of Structure and PCoA analyses suggest that the genetic structure of the studied P. patens populations fall into three clusters corresponding to geographical regions. The most isolated populations (mostly from Romania) formed a separate group with a homogeneous gene pool located at the southern, steppic part of the distribution range. Baltic, mostly Polish, populations fall into two genetic groups which were not fully compatible with their geographic distribution.Our results indicate the serious genetic depauperation of P. patens in the western part of its range, even hinting at an ongoing extinction vortex. Therefore, special conservation attention is required to maintain the populations

  20. Genetic Diversity and Population Structure of the Rare and Endangered Plant Species Pulsatilla patens (L.) Mill in East Central Europe

    PubMed Central

    Szczecińska, Monika; Sramko, Gabor; Wołosz, Katarzyna; Sawicki, Jakub

    2016-01-01

    Pulsatilla patens s.s. is a one of the most endangered plant species in Europe. The present range of this species in Europe is highly fragmented and the size of the populations has been dramatically reduced in the past 50 years. The rapid disappearance of P. patens localities in Europe has prompted the European Commission to initiate active protection of this critically endangered species. The aim of this study was to estimate the degree and distribution of genetic diversity within European populations of this endangered species. We screened 29 populations of P. patens using a set of six microsatellite primers. The results of our study indicate that the analyzed populations are characterized by low levels of genetic diversity (Ho = 0.005) and very high levels of inbreeding (FIS = 0.90). These results suggest that genetic erosion could be partially responsible for the lower fitness in smaller populations of this species. Private allelic richness was very low, being as low as 0.00 for most populations. Average genetic diversity over loci and mean number of alleles in P. patens populations were significantly correlated with population size, suggesting severe genetic drift. The results of AMOVA point to higher levels of variation within populations than between populations.The results of Structure and PCoA analyses suggest that the genetic structure of the studied P. patens populations fall into three clusters corresponding to geographical regions. The most isolated populations (mostly from Romania) formed a separate group with a homogeneous gene pool located at the southern, steppic part of the distribution range. Baltic, mostly Polish, populations fall into two genetic groups which were not fully compatible with their geographic distribution.Our results indicate the serious genetic depauperation of P. patens in the western part of its range, even hinting at an ongoing extinction vortex. Therefore, special conservation attention is required to maintain the populations

  1. Transcriptome Analysis of Hamelia patens (Rubiaceae) Anthers Reveals Candidate Genes for Tapetum and Pollen Wall Development

    PubMed Central

    Yue, Lin; Twell, David; Kuang, Yanfeng; Liao, Jingping; Zhou, Xianqiang

    2017-01-01

    Studies of the anther transcriptome on non-model plants without a known genome are surprisingly scarce. RNA-Seq and digital gene expression (DGE) profiling provides a comprehensive approach to identify candidate genes contributing to developmental processes in non-model species. Here we built a transcriptome library of developing anthers of Hamelia patens and analyzed DGE profiles from each stage to identify genes that regulate tapetum and pollen development. In total 7,720 putative differentially expressed genes across four anther stages were identified. The number of putative stage-specific genes was: 776 at microspore mother cell stage, 807 at tetrad stage, 322 at uninucleate microspore stage, and the highest number (1,864) at bicellular pollen stage. GO enrichment analysis revealed 243 differentially expressed and 108 stage-specific genes that are potentially related to tapetum development, sporopollenin synthesis, and pollen wall. The number of expressed genes, their function and expression profiles were all significantly correlated with anther developmental processes. Overall comparisons of anther and pollen transcriptomes with those of rice and Arabidopsis together with the expression profiles of homologs of known anther-expressed genes, revealed conserved patterns and also divergence. The divergence may reflect taxon-specific differences in gene expression, the use RNA-seq as a more sensitive methodology, variation in tissue composition and sampling strategies. Given the lack of genomic sequence, this study succeeded in assigning putative identity to a significant proportion of anther-expressed genes and genes relevant to tapetum and pollen development in H. patens. The anther transcriptome revealed a molecular distinction between developmental stages, serving as a resource to unravel the functions of genes involved in anther development in H. patens and informing the analysis of other members of the Rubiaceae. PMID:28119704

  2. Considering the Different Roles of Ammophila breviligulata and Spartina patens in Coastal Foredune Formation and Growth

    NASA Astrophysics Data System (ADS)

    Jass, T. L.; Moore, L. J.; Young, D. R.; Bruno, J. F.; Duran Vinent, O.; Goldstein, E. B.

    2014-12-01

    Coastal foredunes arise from interactions between vegetation and aeolian sand transport. Two of the most important grasses that influence dune growth and formation along the barrier islands of the U.S. mid-Atlantic coast are Ammophila breviligulata and Spartina patens. Variations in topography, distance to the shoreline and the associated biotic and abiotic stressors (e.g., salt spray, sand burial, soil chemistry, and competition from other plants) affect the survival of both species. Additionally, differences between the grasses in response to these stressors may directly control foredune morphology. Although a relationship between the cross-shore vegetation limit and maximum dune height has been developed (Durán and Moore 2013) we do not yet quantitatively understand how each grass affects dune formation and shape. We carried out a field experiment on Hog Island, VA and complementary model experiments to address gaps in our understanding of species control on vegetated foredunes. We transplanted 120 plants of each species along two cross-shore transects (from foredune crest to the shoreline) to investigate the effects of topography and distance to the shoreline on vegetation growth. Throughout the growing season, we monitored the longest leaf of each transplant, the frontal and basal area of each transplant, and elevation at each transplant site to quantify vegetative growth and aeolian accretion along the each transect. We used these data to improve the coastal dune model presented by Durán and Moore (2013) so that it includes two grasses which more closely represent the growth characteristics of A. breviligulata and S. patens, specifically, rather than a generic dune-building grass species. These additions allow us to conduct simulations of dune development under varying initial abundances of morphologically important species, ranging from a dune populated only with A. breviligulata to one populated only with S. patens. Results from this coupled field and

  3. Domain folding and flexibility of Escherichia coli FtsZ determined by tryptophan site-directed mutagenesis

    PubMed Central

    Díaz-Espinoza, Rodrigo; Garcés, Andrea P.; Arbildua, José J.; Montecinos, Felipe; Brunet, Juan E.; Lagos, Rosalba; Monasterio, Octavio

    2007-01-01

    FtsZ has two domains, the amino GTPase domain with a Rossmann fold, and the carboxyl domain that resembles the chorismate mutase fold. Bioinformatics analyses suggest that the interdomain interaction is stronger than the interaction of the protofilament longitudinal interfaces. Crystal B factor analysis of FtsZ and detected conformational changes suggest a connection between these domains. The unfolding/folding characteristics of each domain of FtsZ were tested by introducing tryptophans into the flexible region of the amino (F135W) and the carboxyl (F275W and I294W) domains. As a control, the mutation F40W was introduced in a more rigid part of the amino domain. These mutants showed a native-like structure with denaturation and renaturation curves similar to wild type. However, the I294W mutant showed a strong loss of functionality, both in vivo and in vitro when compared to the other mutants. The functionality was recovered with the double mutant I294W/F275A, which showed full in vivo complementation with a slight increment of in vitro GTPase activity with respect to the single mutant. The formation of a stabilizing aromatic interaction involving a stacking between the tryptophan introduced at position 294 and phenylalanine 275 could account for these results. Folding/unfolding of these mutants induced by guanidinium chloride was compatible with a mechanism in which both domains within the protein show the same stability during FtsZ denaturation and renaturation, probably because of strong interface interactions. PMID:17656575

  4. Constitutive expression of ftsZ overrides the whi developmental genes to initiate sporulation of Streptomyces coelicolor.

    PubMed

    Willemse, Joost; Mommaas, A Mieke; van Wezel, Gilles P

    2012-03-01

    The filamentous soil bacteria Streptomyces undergo a highly complex developmental programme. Before streptomycetes commit themselves to sporulation, distinct morphological checkpoints are passed in the aerial hyphae that are subject to multi-level control by the whi sporulation genes. Here we show that whi-independent expression of FtsZ restores sporulation to the early sporulation mutants whiA, whiB, whiG, whiH, whiI and whiJ. Viability, stress resistance and high-resolution electron microscopy underlined that viable spores were formed. However, spores from sporulation-restored whiA and whiG mutants showed defects in DNA segregation/condensation, while spores from the complemented whiB mutant had increased stress sensitivity, perhaps as a result of changes in the spore sheath. In contrast to the whi mutants, normal sporulation of ssgB null mutants-which fail to properly localise FtsZ-could not be restored by enhancing FtsZ protein levels, forming spore-like bodies that lack spore walls. Our data strongly suggest that the whi genes control a decisive event towards sporulation of streptomycetes, namely the correct timing of developmental ftsZ transcription. The biological significance may be to ensure that sporulation-specific cell division will only start once sufficient aerial mycelium biomass has been generated. Our data shed new light on the longstanding question as to how whi genes control sporulation, which has intrigued scientists for four decades.

  5. Mutations on FtsZ lateral helix H3 that disrupt cell viability hamper reorganization of polymers on lipid surfaces.

    PubMed

    Márquez, Ileana F; Mateos-Gil, Pablo; Shin, Jae Yen; Lagos, Rosalba; Monasterio, Octavio; Vélez, Marisela

    2017-10-01

    FtsZ filaments localize at the middle of the bacterial cell and participate in the formation of a contractile ring responsible for cell division. Previous studies demonstrated that the highly conserved negative charge of glutamate 83 and the positive charge of arginine 85 located in the lateral helix H3 bend of Escherichia coli FtsZ are required for in vivo cell division. In order to understand how these lateral mutations impair the formation of a contractile ring,we extend previous in vitro characterization of these mutants in solution to study their behavior on lipid modified surfaces. We study their interaction with ZipAand look at their reorganization on the surface. We found that the dynamic bundling capacity of the mutant proteins is deficient, and this impairment increases the more the composition and spatial arrangement of the reconstituted system resembles the situation inside the cell: mutant proteins completely fail to reorganize to form higher order aggregates when bound to an E.coli lipid surface through oriented ZipA.We conclude that these surface lateral point mutations affect the dynamic reorganization of FtsZ filaments into bundles on the cell membrane, suggesting that this event is relevant for generating force and completing bacterial division. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. The Escherichia coli cell division protein ZipA forms homodimers prior to association with FtsZ.

    PubMed

    Skoog, Karl; Daley, Daniel O

    2012-02-21

    ZipA is an essential component of the cell division machinery in E. coli and other closely related bacteria. It is an integral membrane protein that binds to FtsZ, tethering it to the inner membrane. ZipA also induces bundling of FtsZ protofilaments and may play a role in regulating FtsA activity; however, the molecular details behind these observations are not clear. In this study we have analyzed the oligomeric state of ZipA in vivo, by chemical cross-linking, and in vitro, by native gel electrophoresis (BN-PAGE). Our data indicate that ZipA can self-associate as a homodimer and that this self-interaction is not dependent on the FtsZ-binding domain. This observation rules out the possibility that FtsZ polymers mediate the ZipA self-interaction. Given this observation, it is possible that a certain population of ZipA is recruited to the division septum in a homodimeric form.

  7. Influence of FtsZ GTPase activity and concentration on nanoscale Z-ring structure in vivo revealed by three-dimensional Superresolution imaging.

    PubMed

    Lyu, Zhixin; Coltharp, Carla; Yang, Xinxing; Xiao, Jie

    2016-10-01

    FtsZ is an essential bacterial cytoskeletal protein that assembles into a ring-like structure (Z-ring) at midcell to carry out cytokinesis. In vitro, FtsZ exhibits polymorphism in polymerizing into different forms of filaments based on its GTPase activity, concentration, and buffer condition. In vivo, the Z-ring appeared to be punctate and heterogeneously organized, although continuous, homogenous Z-ring structures have also been observed. Understanding how the Z-ring is organized in vivo is important because it provides a structural basis for the functional role of the Z-ring in cytokinesis. Here, we assess the effects of both GTPase activity and FtsZ concentration on the organization of the Z-ring in vivo using three-dimensional (3D) superresolution microscopy. We found that the Z-ring became more homogenous when assembled in the presence of a GTPase-deficient mutant, and upon overexpression of either wt or mutant FtsZ. These results suggest that the in vivo organization of the Z-ring is largely dependent on the intrinsic polymerization properties of FtsZ, which are significantly influenced by the GTPase activity and concentration of FtsZ. Our work provides a unifying theme to reconcile previous observations of different Z-ring structures, and supports a model in which the wt Z-ring comprises loosely associated, heterogeneously distributed FtsZ clusters. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 725-734, 2016. © 2016 Wiley Periodicals, Inc.

  8. Transcription of the Escherichia coli dcw cluster: evidence for distal upstream transcripts being involved in the expression of the downstream ftsZ gene.

    PubMed

    de la Fuente, A; Palacios, P; Vicente, M

    2001-01-01

    Escherichia coli strains VIP596 and VIP597 have been constructed to compare the amount of transcription of the ftsZ gene derived from proximal promoters in the ddlB-ftsZ region with that originating in the upstream regions of the dcw cluster. Both strains have in common a beta-galactosidase reporter fusion located at the ddlB locus, but differ in that VIP597 has a transcription terminator Omega interposon located downstream from lacZ. In addition, these strains have the ddlB, ftsQ, ftsA and ftsZ genes under the control of the IPTG-inducible promoter P(tac), allowing to control artificially ftsZ expression for normal cell division to take place. When beta-galactosidase activity was measured in VIP596 and VIP597 and compared to the levels measured in strain VIP407, in which the lacZ reporter fusion is located in the ftsZ gene, they were found to account for nearly 66% of the total transcription entering into ftsZ. This result indicates that the reduction in ftsZ transcription observed when the promoters in the ddlB-ftsA region are disconnected from the upstream sequences of the dcw cluster (as observed by Flärdh et al., Mol. Microbiol. 30 (1998) 305-316) in strain VIP490) is the direct consequence of the interruption in the transcription originated upstream and not due to the effect of such sequences on the promoters proximal to ftsZ.

  9. Methanol extracts of Hamelia patens containing oxindole alkaloids relax KCl-induced contraction in rat myometrium.

    PubMed

    Reyes-Chilpa, Ricardo; Rivera, Jesús; Oropeza, Martha; Mendoza, Pilar; Amekraz, Badia; Jankowski, Christopher; Campos, Maria

    2004-10-01

    Hamelia patens JAQC. (Rubiaceae) is a medicinal bush widely distributed in tropical areas of the American continent. It is used in Mexican Traditional Medicine for the treatment of menstrual disorders, therefore suggesting that its chemical constituents may have some effect on myometrium contractility. Physiological effects might differ due to quantitative variations in the content of alkaloids arising from its wide geographical distribution. To test this hypothesis, the content of oxindole alkaloids in methanol extracts of five different samples collected in Mexico was quantified by GC-MS. Each extract was assayed on contractility of estrogen-primed rat myometrium. Variations in the content of alkaloids were observed among the different samples. All samples relaxed in a concentration-dependent manner the high KCl-induced contraction in rat myometrium. Those which lack rumberine and/or maruquine displayed a higher relaxant effect than samples containing them, suggesting that these alkaloids might counteract the effects of isopteropodine. However, in contrast with verapamil, Hamelia patens metanol extracts are poor relaxants.

  10. Chemical variability of Cleistopholis patens (Benth.) Engl. et Diels leaf oil from ivory coast.

    PubMed

    Ouattara, Zana Adama; Boti, Jean Brice; Attioua, Koffi Barthelemy; Ahibo, Antoine Coffy; Casanova, Joseph; Tomi, Félix; Bighelli, Ange

    2013-11-01

    The chemical composition of 48 leaf oil samples isolated from individual plants of Cleistopholis patens (Benth.) Engl. et Diels harvested in four Ivoirian forests was investigated by GC-FID (determination of retention indices), GC/MS, and (13) C-NMR analyses. The main components identified were β-pinene (traces-59.1%), sabinene (traces-54.2%), (E)-β-caryophyllene (0.3-39.3%), linalool (0.1-38.5%), (E)-β-ocimene (0.1-33.2%), germacrene D (0.0-33.1%), α-pinene (0.1-32.3%), and germacrene B (0-21.2%). The 48 oil compositions were submitted to hierarchical clustering and principal components analyses, which allowed the distinction of three groups within the oil samples. The oil composition of the major group (GroupI, 33 samples) was dominated by (E)-β-caryophyllene and linalool. The oils of Group II (eight samples) contained mainly β-pinene and α-pinene, while those of Group III (seven samples) were dominated by sabinene, limonene, and β-phellandrene. Moreover, the compositions of the Ivoirian C. patens leaf oils differed from those of Nigerian and Cameroonian origins. Copyright © 2013 Verlag Helvetica Chimica Acta AG, Zürich.

  11. In Escherichia coli, MreB and FtsZ direct the synthesis of lateral cell wall via independent pathways that require PBP 2.

    PubMed

    Varma, Archana; Young, Kevin D

    2009-06-01

    In Escherichia coli, the cytoplasmic proteins MreB and FtsZ play crucial roles in ensuring that new muropeptide subunits are inserted into the cell wall in a spatially correct way during elongation and division. In particular, to retain a constant diameter and overall shape, new material must be inserted into the wall uniformly around the cell's perimeter. Current thinking is that MreB accomplishes this feat through intermediary proteins that tether peptidoglycan synthases to the outer face of the inner membrane. We tested this idea in E. coli by using a DD-carboxypeptidase mutant that accumulates pentapeptides in its peptidoglycan, allowing us to visualize new muropeptide incorporation. Surprisingly, inhibiting MreB with the antibiotic A22 did not result in uneven insertion of new wall, although the cells bulged and lost their rod shapes. Instead, uneven (clustered) incorporation occurred only if MreB and FtsZ were inactivated simultaneously, providing the first evidence in E. coli that FtsZ can direct murein incorporation into the lateral cell wall independently of MreB. Inhibiting penicillin binding protein 2 (PBP 2) alone produced the same clustered phenotype, implying that MreB and FtsZ tether peptidoglycan synthases via a common mechanism that includes PBP 2. However, cell shape was determined only by the presence or absence of MreB and not by the even distribution of new wall material as directed by FtsZ.

  12. Drug discovery targeting cell division proteins, microtubules and FtsZ.

    PubMed

    Ojima, Iwao; Kumar, Kunal; Awasthi, Divya; Vineberg, Jacob G

    2014-09-15

    Eukaryotic cell division or cytokinesis has been a major target for anticancer drug discovery. After the huge success of paclitaxel and docetaxel, microtubule-stabilizing agents (MSAs) appear to have gained a premier status in the discovery of next-generation anticancer agents. However, the drug resistance caused by MDR, point mutations, and overexpression of tubulin subtypes, etc., is a serious issue associated with these agents. Accordingly, the discovery and development of new-generation MSAs that can obviate various drug resistances has a significant meaning. In sharp contrast, prokaryotic cell division has been largely unexploited for the discovery and development of antibacterial drugs. However, recent studies on the mechanism of bacterial cytokinesis revealed that the most abundant and highly conserved cell division protein, FtsZ, would be an excellent new target for the drug discovery of next-generation antibacterial agents that can circumvent drug-resistances to the commonly used drugs for tuberculosis, MRSA and other infections. This review describes an account of our research on these two fronts in drug discovery, targeting eukaryotic as well as prokaryotic cell division.

  13. Phytochemicals as inhibitors of bacterial cell division protein FtsZ: coumarins are promising candidates.

    PubMed

    Duggirala, Sridevi; Nankar, Rakesh P; Rajendran, Selvakumar; Doble, Mukesh

    2014-09-01

    Naturally occurring phytochemicals with reported antibacterial activity were screened for their ability to inhibit the bacterial cell division protein Escherichia coli FtsZ. Among the representative compounds, coumarins inhibit the GTPase and polymerization activities of this protein effectively. Further screening with ten coumarin analogs we identified two promising candidates, scopoletin and daphnetin. The former is found to inhibit the GTPase activity of the protein in a noncompetitive manner. Docking of these coumarins with the modeled protein indicate that they bind to T7 loop, which is different from the GTP-binding site (active site), thereby supporting the experimental data. Lowest binding energy is obtained with scopoletin. 3D QSAR indicates the need for groups such as hydroxyl, diethyl, or dimethyl amino in the 7th carbon for enhanced activity. None of the coumarins exhibited cytotoxicity against NIH/3T3 and human embryonic kidney cell lines. The length of Bacillus subtilis increases in the presence of these compounds probably due to the lack of septum formation. Results of this study indicate the role of coumarins in halting the first step of bacterial cell division process.

  14. Depolymerization dynamics of individual filaments of bacterial cytoskeletal protein FtsZ

    PubMed Central

    Mateos-Gil, Pablo; Paez, Alfonso; Hörger, Ines; Rivas, Germán; Vicente, Miguel; Tarazona, Pedro; Vélez, Marisela

    2012-01-01

    We report observation and analysis of the depolymerization filaments of the bacterial cytoskeletal protein FtsZ (filament temperature-sensitive Z) formed on a mica surface. At low concentration, proteins adsorbed on the surface polymerize forming curved filaments that close into rings that remain stable for some time before opening irreversibly and fully depolymerizing. The distribution of ring lifetimes (T) as a function of length (N), shows that the rate of ring aperture correlates with filament length. If this ring lifetime is expressed as a bond survival time, (Tb ≡ NT), this correlation is abolished, indicating that these rupture events occur randomly and independently at each monomer interface. After rings open irreversibly, depolymerization of the remaining filaments is fast, but can be slowed down and followed using a nonhydrolyzing GTP analogue. The histogram of depolymerization velocities of individual filaments has an asymmetric distribution that can be fit with a computer model that assumes two rupture rates, a slow one similar to the one observed for ring aperture, affecting monomers in the central part of the filaments, and a faster one affecting monomers closer to the open ends. From the quantitative analysis, we conclude that the depolymerization rate is affected both by nucleotide hydrolysis rate and by its exchange along the filament, that all monomer interfaces are equally competent for hydrolysis, although depolymerization is faster at the open ends than in central filament regions, and that all monomer–monomer interactions, regardless of the nucleotide present, can adopt a curved configuration. PMID:22566654

  15. Structural and Biochemical Studies Reveal a Putative FtsZ Recognition Site on the Z-ring Stabilizer ZapD

    PubMed Central

    Choi, Hwajung; Min, Kyungjin; Mikami, Bunzo; Yoon, Hye-Jin; Lee, Hyung Ho

    2016-01-01

    FtsZ, a tubulin homologue, is an essential protein of the Z-ring assembly in bacterial cell division. It consists of two domains, the N-terminal and C-terminal core domains, and has a conserved C-terminal tail region. Lateral interactions between FtsZ protofilaments and several Z-ring associated proteins (Zaps) are necessary for modulating Z-ring formation. ZapD, one of the positive regulators of Z-ring assembly, directly binds to the C-terminal tail of FtsZ and promotes stable Z-ring formation during cytokinesis. To gain structural and functional insights into how ZapD interacts with the C-terminal tail of FtsZ, we solved two crystal structures of ZapD proteins from Salmonella typhimurium (StZapD) and Escherichia coli (EcZapD) at a 2.6 and 3.1 Å resolution, respectively. Several conserved residues are clustered on the concave sides of the StZapD and EcZapD dimers, the suggested FtsZ binding site. Modeled structures of EcZapD-EcFtsZ and subsequent binding studies using bio-layer interferometry also identified the EcFtsZ binding site on EcZapD. The structural insights and the results of bio-layer interferometry assays suggest that the two FtsZ binding sites of ZapD dimer might be responsible for the binding of ZapD dimer to two protofilaments to hold them together. PMID:27871169

  16. E93R Substitution of Escherichia coli FtsZ Induces Bundling of Protofilaments, Reduces GTPase Activity, and Impairs Bacterial Cytokinesis*

    PubMed Central

    Jaiswal, Richa; Patel, Ronak Y.; Asthana, Jayant; Jindal, Bhavya; Balaji, Petety V.; Panda, Dulal

    2010-01-01

    Recently, we found that divalent calcium has no detectable effect on the assembly of Mycobacterium tuberculosis FtsZ (MtbFtsZ), whereas it strongly promoted the assembly of Escherichia coli FtsZ (EcFtsZ). While looking for potential calcium binding residues in EcFtsZ, we found a mutation (E93R) that strongly promoted the assembly of EcFtsZ. The mutation increased the stability and bundling of the FtsZ protofilaments and produced a dominating effect on the assembly of the wild type FtsZ (WT-FtsZ). Although E93R-FtsZ was found to bind to GTP similarly to the WT-FtsZ, it displayed lower GTPase activity than the WT-FtsZ. E93R-FtsZ complemented for its wild type counterpart as observed by a complementation test using JKD7–1/pKD3 cells. However, the bacterial cells became elongated upon overexpression of the mutant allele. We modeled the structure of E93R-FtsZ using the structures of MtbFtsZ/Methanococcus jannaschi FtsZ (MjFtsZ) dimers as templates. The MtbFtsZ-based structure suggests that the Arg93-Glu138 salt bridge provides the additional stability, whereas the effect of mutation appears to be indirect (allosteric) if the EcFtsZ dimer is similar to that of MjFtsZ. The data presented in this study suggest that an increase in the stability of the FtsZ protofilaments is detrimental for the bacterial cytokinesis. PMID:20667825

  17. 3D-SIM super resolution microscopy reveals a bead-like arrangement for FtsZ and the division machinery: implications for triggering cytokinesis.

    PubMed

    Strauss, Michael P; Liew, Andrew T F; Turnbull, Lynne; Whitchurch, Cynthia B; Monahan, Leigh G; Harry, Elizabeth J

    2012-01-01

    FtsZ is a tubulin-like GTPase that is the major cytoskeletal protein in bacterial cell division. It polymerizes into a ring, called the Z ring, at the division site and acts as a scaffold to recruit other division proteins to this site as well as providing a contractile force for cytokinesis. To understand how FtsZ performs these functions, the in vivo architecture of the Z ring needs to be established, as well as how this structure constricts to enable cytokinesis. Conventional wide-field fluorescence microscopy depicts the Z ring as a continuous structure of uniform density. Here we use a form of super resolution microscopy, known as 3D-structured illumination microscopy (3D-SIM), to examine the architecture of the Z ring in cells of two Gram-positive organisms that have different cell shapes: the rod-shaped Bacillus subtilis and the coccoid Staphylococcus aureus. We show that in both organisms the Z ring is composed of a heterogeneous distribution of FtsZ. In addition, gaps of fluorescence were evident, which suggest that it is a discontinuous structure. Time-lapse studies using an advanced form of fast live 3D-SIM (Blaze) support a model of FtsZ localization within the Z ring that is dynamic and remains distributed in a heterogeneous manner. However, FtsZ dynamics alone do not trigger the constriction of the Z ring to allow cytokinesis. Lastly, we visualize other components of the divisome and show that they also adopt a bead-like localization pattern at the future division site. Our data lead us to propose that FtsZ guides the divisome to adopt a similar localization pattern to ensure Z ring constriction only proceeds following the assembly of a mature divisome.

  18. 3D-SIM Super Resolution Microscopy Reveals a Bead-Like Arrangement for FtsZ and the Division Machinery: Implications for Triggering Cytokinesis

    PubMed Central

    Strauss, Michael P.; Liew, Andrew T. F.; Turnbull, Lynne; Whitchurch, Cynthia B.; Monahan, Leigh G.; Harry, Elizabeth J.

    2012-01-01

    FtsZ is a tubulin-like GTPase that is the major cytoskeletal protein in bacterial cell division. It polymerizes into a ring, called the Z ring, at the division site and acts as a scaffold to recruit other division proteins to this site as well as providing a contractile force for cytokinesis. To understand how FtsZ performs these functions, the in vivo architecture of the Z ring needs to be established, as well as how this structure constricts to enable cytokinesis. Conventional wide-field fluorescence microscopy depicts the Z ring as a continuous structure of uniform density. Here we use a form of super resolution microscopy, known as 3D-structured illumination microscopy (3D-SIM), to examine the architecture of the Z ring in cells of two Gram-positive organisms that have different cell shapes: the rod-shaped Bacillus subtilis and the coccoid Staphylococcus aureus. We show that in both organisms the Z ring is composed of a heterogeneous distribution of FtsZ. In addition, gaps of fluorescence were evident, which suggest that it is a discontinuous structure. Time-lapse studies using an advanced form of fast live 3D-SIM (Blaze) support a model of FtsZ localization within the Z ring that is dynamic and remains distributed in a heterogeneous manner. However, FtsZ dynamics alone do not trigger the constriction of the Z ring to allow cytokinesis. Lastly, we visualize other components of the divisome and show that they also adopt a bead-like localization pattern at the future division site. Our data lead us to propose that FtsZ guides the divisome to adopt a similar localization pattern to ensure Z ring constriction only proceeds following the assembly of a mature divisome. PMID:22984350

  19. Characterization of the ftsZ cell division gene of Neisseria gonorrhoeae: expression in Escherichia coli and N. gonorrhoeae.

    PubMed

    Salimnia, H; Radia, A; Bernatchez, S; Beveridge, T J; Dillon, J R

    2000-01-01

    We cloned the cell division gene ftsZ of the gram-negative coccus Neisseria gonorrhoeae (Ng) strain CH811, characterized it genetically and phenotypically, and studied its localization in N. gonorrhoeae and Escherichia coli (Ec). The 1,179-bp ORF of ftsZ(Ng) encodes a protein with a predicted molecular mass of 41.5 kDa. Protein sequence alignments indicate that FtsZ(Ng) is similar to other FtsZ proteins and contains the conserved GTP binding motif. FtsZ homologues were identified in several N. gonorrhoeae strains and in Neisseria lactamica, Neisseria sicca, Neisseria polysaccharae and Neisseria cinerea either by Western blot or by PCR-Southern blot analysis. Attempts to inactivate the ftsZ(Ng) on the chromosome failed, indicating that it is essential for gonococcal growth. FtsZ(Ng) was synthesized in an in vitro transcription/translation system and was shown to be 43 kDa, the same size as in Western blots. Expression of the ftsZ(Ng) gene from nongonococcal promoters resulted in a filamentous phenotype in E. coli. Under controlled expression, the FtsZ(Ng)-GFP fusion protein localized at the mid-cell division site in E. coli. E. coli expressing high levels of the FtsZ(Ng)-GFP fusion protein formed filaments and exhibited different fluorescent structures including helices, spiral tubules extending from pole to pole, and regularly spaced dots or bands that did not localize at the middle of the cell. Expression of the FtsZ(Ng)-GFP fusion protein in N. gonorrhoeae resulted in abnormal cell division as shown by electron microscopy. FtsZ(Ng)-GFP fusions were also expressed in a gonococcal background using a unique shuttle vector.

  20. Probing for Binding Regions of the FtsZ Protein Surface through Site-Directed Insertions: Discovery of Fully Functional FtsZ-Fluorescent Proteins

    PubMed Central

    Moore, Desmond A.; Whatley, Zakiya N.; Joshi, Chandra P.; Osawa, Masaki

    2016-01-01

    ABSTRACT FtsZ, a bacterial tubulin homologue, is a cytoskeletal protein that assembles into protofilaments that are one subunit thick. These protofilaments assemble further to form a “Z ring” at the center of prokaryotic cells. The Z ring generates a constriction force on the inner membrane and also serves as a scaffold to recruit cell wall remodeling proteins for complete cell division in vivo. One model of the Z ring proposes that protofilaments associate via lateral bonds to form ribbons; however, lateral bonds are still only hypothetical. To explore potential lateral bonding sites, we probed the surface of Escherichia coli FtsZ by inserting either small peptides or whole fluorescent proteins (FPs). Among the four lateral surfaces on FtsZ protofilaments, we obtained inserts on the front and back surfaces that were functional for cell division. We concluded that these faces are not sites of essential interactions. Inserts at two sites, G124 and R174, located on the left and right surfaces, completely blocked function, and these sites were identified as possible sites for essential lateral interactions. However, the insert at R174 did not interfere with association of protofilaments into sheets and bundles in vitro. Another goal was to find a location within FtsZ that supported insertion of FP reporter proteins while allowing the FtsZ-FPs to function as the sole source of FtsZ. We discovered one internal site, G55-Q56, where several different FPs could be inserted without impairing function. These FtsZ-FPs may provide advances for imaging Z-ring structure by superresolution techniques. IMPORTANCE One model for the Z-ring structure proposes that protofilaments are assembled into ribbons by lateral bonds between FtsZ subunits. Our study excluded the involvement of the front and back faces of the protofilament in essential interactions in vivo but pointed to two potential lateral bond sites, on the right and left sides. We also identified an FtsZ loop where

  1. Network theory inspired analysis of time-resolved expression data reveals key players guiding P. patens stem cell development.

    PubMed

    Busch, Hauke; Boerries, Melanie; Bao, Jie; Hanke, Sebastian T; Hiss, Manuel; Tiko, Theodhor; Rensing, Stefan A

    2013-01-01

    Transcription factors (TFs) often trigger developmental decisions, yet, their transcripts are often only moderately regulated and thus not easily detected by conventional statistics on expression data. Here we present a method that allows to determine such genes based on trajectory analysis of time-resolved transcriptome data. As a proof of principle, we have analysed apical stem cells of filamentous moss (P. patens) protonemata that develop from leaflets upon their detachment from the plant. By our novel correlation analysis of the post detachment transcriptome kinetics we predict five out of 1,058 TFs to be involved in the signaling leading to the establishment of pluripotency. Among the predicted regulators is the basic helix loop helix TF PpRSL1, which we show to be involved in the establishment of apical stem cells in P. patens. Our methodology is expected to aid analysis of key players of developmental decisions in complex plant and animal systems.

  2. SAR Studies on Trisubstituted Benzimidazoles as Inhibitors of Mtb FtsZ for the Development of Novel Antitubercular Agents

    PubMed Central

    Awasthi, Divya; Kumar, Kunal; Knudson, Susan E.; Slayden, Richard A.; Ojima, Iwao

    2014-01-01

    FtsZ, an essential protein for bacterial cell division, is a highly promising therapeutic target, especially for the discovery and development of new-generation anti-TB agents. Following up the identification of two lead 2,5,6-trisubstituted benzimidazoles, 1 and 2, targeting Mtb-FtsZ in our previous study, an extensive SAR study for optimization of these lead compounds was performed through systematic modification of the 5 and 6 positions. This study has successfully led to the discovery of a highly potent advanced lead 5f (MIC 0.06 µg/mL) and several other compounds with comparable potencies. These advanced lead compounds possess a dimethylamino group at the 6 position. The functional groups at the 5 position exhibit substantial effects on the antibacterial activity as well. In vitro experiments such as the FtsZ polymerization inhibitory assay and TEM analysis of Mtb-FtsZ treated with 5f and others indicate that Mtb-FtsZ is the molecular target for their antibacterial activity. PMID:24266862

  3. Metal-dependent SpoIIE oligomerization stabilizes FtsZ during asymmetric division in Bacillus subtilis

    PubMed Central

    Król, Ewa; de Sousa Borges, Anabela; Kopacz, Malgorzata

    2017-01-01

    SpoIIE is a bifunctional protein involved in asymmetric septum formation and in activation of the forespore compartment-specific transcription factor σF through dephosphorylation of SpoIIAA-P. The phosphatase activity of SpoIIE requires Mn2+ as a metal cofactor. Here, we show that the presence of a metal cofactor also influences SpoIIE oligomerization and asymmetric septum formation. Absence of Mn2+ from sporulation medium results in a delay of the formation of polar FtsZ-rings, similar to a spoIIE null mutant. We purified the entire cytoplasmic part of the SpoIIE protein, and show that the protein copurifies with bound metals. Metal binding both stimulates SpoIIE oligomerization, and results in the formation of larger oligomeric structures. The presence of SpoIIE oligomers reduces FtsZ GTP hydrolysis activity and stabilizes FtsZ polymers in a light scattering assay. Combined, these results indicate that metal binding is not just required for SpoIIE phosphatase activity but also is important for SpoIIE's role in asymmetric septum formation. PMID:28358838

  4. Ecogeomorphology of Spartina patens-dominated tidal marshes: Soil organic matter accumulation, marsh elevation dynamics, and disturbance

    USGS Publications Warehouse

    Cahoon, D.R.; Ford, M.A.; Hensel, P.F.; Fagherazzi, Sergio; Marani, Marco; Blum, Linda K.

    2004-01-01

    Marsh soil development and vertical accretion in Spartina patens (Aiton) Muhl.-dominated tidal marshes is largely dependent on soil organic matter accumulation from root-rhizome production and litter deposition. Yet there are few quantitative data sets on belowground production and the relationship between soil organic matter accumulation and soil elevation dynamics for this marsh type. Spartina patens marshes are subject to numerous stressors, including sea-level rise, water level manipulations (i.e., flooding and draining) by impoundments, and prescribed burning. These stressors could influence long-term marsh sustainability by their effect on root production, soil organic matter accumulation, and soil elevation dynamics. In this review, we summarize current knowledge on the interactions among vegetative production, soil organic matter accumulation and marsh elevation dynamics, or the ecogeomorphology, of Spartina patens-dominated tidal marshes. Additional studies are needed of belowground production/decomposition and soil elevation change (measured simultaneously) to better understand the links among soil organic matter accumulation, soil elevation change, and disturbance in this marsh type. From a management perspective, we need to better understand the impacts of disturbance stressors, both lethal and sub-lethal, and the interactive effect of multiple stressors on soil elevation dynamics in order to develop better management practices to safeguard marsh sustainability as sea level rises.

  5. E. coli low molecular weight penicillin binding proteins help orient septal FtsZ, and their absence leads to asymmetric cell division and branching

    PubMed Central

    Potluri, Lakshmi-Prasad; de Pedro, Miguel; Young, Kevin D.

    2012-01-01

    Escherichia coli cells lacking low molecular weight penicillin binding proteins (LMW PBPs) exhibit morphological alterations that also appear when the septal protein FtsZ is mislocalized, suggesting that peptidoglycan modification and division may work together to produce cell shape. We found that in strains lacking PBP5 and other LMW PBPs, higher FtsZ concentrations increased the frequency of branched cells and incorrectly oriented Z rings by 10- to 15-fold. Invagination of these rings produced improperly oriented septa, which in turn gave rise to asymmetric cell poles that eventually elongated into branches. Branches always originated from the remnants of abnormal septation events, cementing the relationship between aberrant cell division and branch formation. In the absence of PBP5, PBP6 and DacD localized to nascent septa, suggesting that these PBPs can partially substitute for the loss of PBP5. We propose that branching begins when mislocalized FtsZ triggers the insertion of inert peptidoglycan at unusual positions during cell division. Only later, after normal cell wall elongation separates the patches, do branches become visible. Thus, a relationship between the LMW PBPs and cytoplasmic FtsZ ultimately affects cell division and overall shape. PMID:22390731

  6. ARC3, a chloroplast division factor, is a chimera of prokaryotic FtsZ and part of eukaryotic phosphatidylinositol-4-phosphate 5-kinase.

    PubMed

    Shimada, Hiroshi; Koizumi, Masato; Kuroki, Kouta; Mochizuki, Mariko; Fujimoto, Hitoshi; Ohta, Hiroyuki; Masuda, Tatsuru; Takamiya, Ken-ichiro

    2004-08-01

    The arc3 (accumulation and replication of chloroplast) mutant of Arabidopsis thaliana has a small number of abnormally large chloroplasts in the cell, suggesting that chloroplast division is arrested in the mutant and ARC3 has an important role in the initiation of chloroplast division. To elucidate the role of ARC3, first we identified the ARC3 gene, and determined the location of ARC3 protein during chloroplast division because the localization and spatial orientation of such division factors are vital for correct chloroplast division. Sequencing analysis showed that ARC3 was a fusion of the prokaryotic FtsZ and part of the eukaryotic phosphatidylinositol-4-phosphate 5-kinase (PIP5K) genes. The PIP5K-homologous region of ARC3 had no catalytic domain but a membrane-occupation-and-recognition-nexus (MORN) repeat motif. Immunofluorescence microscopy, Western blotting analysis and in vitro chloroplast import and protease protection assays revealed that ARC3 protein was soluble, and located on the outer surface of the chloroplast in a ring-like structure at the early stage of chloroplast division. Prokaryotes have one FtsZ as a gene for division but have no ARC3 counterparts, the chimera of FtsZ and PIP5K, suggesting that the ARC3 gene might have been generated from FtsZ as another division factor during the evolution of chloroplast by endosymbiosis.

  7. Creating an antibacterial with in vivo efficacy: synthesis and characterization of potent inhibitors of the bacterial cell division protein FtsZ with improved pharmaceutical properties.

    PubMed

    Haydon, David J; Bennett, James M; Brown, David; Collins, Ian; Galbraith, Greta; Lancett, Paul; Macdonald, Rebecca; Stokes, Neil R; Chauhan, Pramod K; Sutariya, Jignesh K; Nayal, Narendra; Srivastava, Anil; Beanland, Joy; Hall, Robin; Henstock, Vincent; Noula, Caterina; Rockley, Chris; Czaplewski, Lloyd

    2010-05-27

    3-Methoxybenzamide (1) is a weak inhibitor of the essential bacterial cell division protein FtsZ. Alkyl derivatives of 1 are potent antistaphylococcal compounds with suboptimal drug-like properties. Exploration of the structure-activity relationships of analogues of these inhibitors led to the identification of potent antistaphylococcal compounds with improved pharmaceutical properties.

  8. New insights into FtsZ rearrangements during the cell division of Escherichia coli from single-molecule localization microscopy of fixed cells.

    PubMed

    Vedyaykin, Alexey D; Vishnyakov, Innokentii E; Polinovskaya, Vasilisa S; Khodorkovskii, Mikhail A; Sabantsev, Anton V

    2016-06-01

    FtsZ - a prokaryotic tubulin homolog - is one of the central components of bacterial division machinery. At the early stage of cytokinesis FtsZ forms the so-called Z-ring at mid-cell that guides septum formation. Many approaches were used to resolve the structure of the Z-ring, however, researchers are still far from consensus on this question. We utilized single-molecule localization microscopy (SMLM) in combination with immunofluorescence staining to visualize FtsZ in Esherichia coli fixed cells that were grown under slow and fast growth conditions. This approach allowed us to obtain images of FtsZ structures at different stages of cell division and accurately measure Z-ring dimensions. Analysis of these images demonstrated that Z-ring thickness increases during constriction, starting at about 70 nm at the beginning of division and increasing by approximately 25% half-way through constriction. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  9. FtsZ-less prokaryotic cell division as well as FtsZ- and dynamin-less chloroplast and non-photosynthetic plastid division

    PubMed Central

    Miyagishima, Shin-ya; Nakamura, Mami; Uzuka, Akihiro; Era, Atsuko

    2014-01-01

    The chloroplast division machinery is a mixture of a stromal FtsZ-based complex descended from a cyanobacterial ancestor of chloroplasts and a cytosolic dynamin-related protein (DRP) 5B-based complex derived from the eukaryotic host. Molecular genetic studies have shown that each component of the division machinery is normally essential for normal chloroplast division. However, several exceptions have been found. In the absence of the FtsZ ring, non-photosynthetic plastids are able to proliferate, likely by elongation and budding. Depletion of DRP5B impairs, but does not stop chloroplast division. Chloroplasts in glaucophytes, which possesses a peptidoglycan (PG) layer, divide without DRP5B. Certain parasitic eukaryotes possess non-photosynthetic plastids of secondary endosymbiotic origin, but neither FtsZ nor DRP5B is encoded in their genomes. Elucidation of the FtsZ- and/or DRP5B-less chloroplast division mechanism will lead to a better understanding of the function and evolution of the chloroplast division machinery and the finding of the as-yet-unknown mechanism that is likely involved in chloroplast division. Recent studies have shown that FtsZ was lost from a variety of prokaryotes, many of which lost PG by regressive evolution. In addition, even some of the FtsZ-bearing bacteria are able to divide when FtsZ and PG are depleted experimentally. In some cases, alternative mechanisms for cell division, such as budding by an increase of the cell surface-to-volume ratio, are proposed. Although PG is believed to have been lost from chloroplasts other than in glaucophytes, there is some indirect evidence for the existence of PG in chloroplasts. Such information is also useful for understanding how non-photosynthetic plastids are able to divide in FtsZ-depleted cells and the reason for the retention of FtsZ in chloroplast division. Here we summarize information to facilitate analyses of FtsZ- and/or DRP5B-less chloroplast and non-photosynthetic plastid division. PMID

  10. Influence of GTP/GDP and magnesium ion on the solvated structure of the protein FtsZ: a molecular dynamics study.

    PubMed

    Jamous, Carla; Basdevant, Nathalie; Ha-Duong, Tap

    2014-01-01

    We present here a structural analysis of ten extensive all-atom molecular dynamics simulations of the monomeric protein FtsZ in various binding states. Since the polymerization and GTPase activities of FtsZ depend on the nature of a bound nucleotide as well as on the presence of a magnesium ion, we studied the structural differences between the average conformations of the following five systems: FtsZ-Apo, FtsZ-GTP, FtsZ-GDP, FtsZ-GTP-Mg, and FtsZ-GDP-Mg. The in silico solvated average structure of FtsZ-Apo significantly differs from the crystallographic structure 1W59 of FtsZ which was crystallized in a dimeric form without nucleotide and magnesium. The simulated Apo form of the protein also clearly differs from the FtsZ structures when it is bound to its ligand, the most important discrepancies being located in the loops surrounding the nucleotide binding pocket. The three average structures of FtsZ-GTP, FtsZ-GDP, and FtsZ-GTP-Mg are overall similar, except for the loop T7 located at the opposite side of the binding pocket and whose conformation in FtsZ-GDP notably differs from the one in FtsZ-GTP and FtsZ-GTP-Mg. The presence of a magnesium ion in the binding pocket has no impact on the FtsZ conformation when it is bound to GTP. In contrast, when the protein is bound to GDP, the divalent cation causes a translation of the nucleotide outwards the pocket, inducing a significant conformational change of the loop H6-H7 and the top of helix H7.

  11. Control of ftsZ Expression, Cell Division, and Glutamine Metabolism in Luria-Bertani Medium by the Alarmone ppGpp in Escherichia coli

    PubMed Central

    Powell, Bradford S.; Court, Donald L.

    1998-01-01

    Inactivation of transcription factor ς54, encoded by rpoN (glnF), restores high-temperature growth in Luria-Bertani (LB) medium to strains containing the heat-sensitive cell division mutation ftsZ84. Mutational defects in three other genes involved in general nitrogen control (glnD, glnG, and glnL) also suppress lethal filamentation. Since addition of glutamine to LB medium fully blocks suppression by each mutation, the underlying cause of suppression likely derives from a stringent response to the limitation of glutamine. This model is supported by several observations. The glnL mutation requires RelA-directed synthesis of the nutrient alarmone ppGpp to suppress filamentation. Artificially elevated levels of ppGpp suppress ftsZ84, as do RNA polymerase mutations that reproduce global effects of the ppGpp-induced state. Both the glnF null mutation and an elevated copy number of the relA gene similarly affect transcription from the upstream (pQ) promoters of the ftsQAZ operon, and both of these genetic conditions increase the steady-state level of the FtsZ84 protein. Physiological suppression of ftsZ84 by a high salt concentration was also shown to involve RelA. Additionally, we found that the growth of a glnF or glnD strain on LB medium depends on RelA or supplemental glutamine in the absence of RelA function. These data expand the roles for ppGpp in the regulation of glutamine metabolism and the expression of FtsZ during cell division. PMID:9495742

  12. Design, synthesis and antibacterial activity of cinnamaldehyde derivatives as inhibitors of the bacterial cell division protein FtsZ.

    PubMed

    Li, Xin; Sheng, Juzheng; Huang, Guihua; Ma, Ruixin; Yin, Fengxin; Song, Di; Zhao, Can; Ma, Shutao

    2015-06-05

    In an attempt to discover potential antibacterial agents against the increasing bacterial resistance, novel cinnamaldehyde derivatives as FtsZ inhibitors were designed, synthesized and evaluated for their antibacterial activity against nine significant pathogens using broth microdilution method, and their cell division inhibitory activity against four representative strains. In the in vitro antibacterial activity, the newly synthesized compounds generally displayed better efficacy against Staphylococcus aureus ATCC25923 than the others. In particular, compounds 3, 8 and 10 exerted superior or comparable activity to all the reference drugs. In the cell division inhibitory activity, all the compounds showed the same trend as their in vitro antibacterial activity, exhibiting better activity against S. aureus ATCC25923 than the other strains. Additionally, compounds 3, 6, 7 and 8 displayed potent cell division inhibitory activity with an MIC value of below 1 μg/mL, over 256-fold better than all the reference drugs.

  13. A Method for DNA Extraction from the Desert Cyanobacterium Chroococcidiopsis and Its Application to Identification of ftsZ

    PubMed Central

    Billi, Daniela; Grilli Caiola, Maria; Paolozzi, Luciano; Ghelardini, Patrizia

    1998-01-01

    A method was developed for extraction of DNA from Chroococcidiopsis that overcomes obstacles posed by bacterial contamination and the presence of a thick envelope surrounding the cyanobacterial cells. The method is based on the resistance of Chroococcidiopsis to lysozyme and consists of a lysozyme treatment followed by osmotic shock that reduces the bacterial contamination by 3 orders of magnitude. Then DNase treatment is performed to eliminate DNA from the bacterial lysate. Lysis of Chroococcidiopsis cells is achieved by grinding with glass beads in the presence of hot phenol. Extracted DNA is further purified by cesium-chloride density gradient ultracentrifugation. This method permitted the first molecular approach to the study of Chroococcidiopsis, and a 570-bp fragment of the gene ftsZ was cloned and sequenced. PMID:9758840

  14. Design, synthesis and evaluation of novel 2,5,6-trisubstituted benzimidazoles targeting FtsZ as antitubercular agents.

    PubMed

    Park, Bora; Awasthi, Divya; Chowdhury, Soumya R; Melief, Eduard H; Kumar, Kunal; Knudson, Susan E; Slayden, Richard A; Ojima, Iwao

    2014-05-01

    Filamenting temperature-sensitive protein Z (FtsZ), an essential cell division protein, is a promising target for the drug discovery of new-generation antibacterial agents against various bacterial pathogens. As a part of SAR studies on benzimidazoles, we have synthesized a library of 376 novel 2,5,6-trisubstituted benzimidazoles, bearing ether or thioether linkage at the 6-position. In a preliminary HTP screening against Mtb H37Rv, 108 compounds were identified as hits at a cut off concentration of 5 μg/mL. Among those hits, 10 compounds exhibited MIC values in the range of 0.63-12.5 μg/mL. Light scattering assay and TEM analysis with the most potent compound 5a clearly indicate that its molecular target is Mtb-FtsZ. Also, the Kd of 5a with Mtb-FtsZ was determined to be 1.32 μM.

  15. FtsZ inhibition and redox modulation with one chemical scaffold: Potential use of dihydroquinolines against mycobacteria.

    PubMed

    Duggirala, Sridevi; Napoleon, John Victor; Nankar, Rakesh P; Senu Adeeba, V; Manheri, Muraleedharan K; Doble, Mukesh

    2016-11-10

    The dual effect of FtsZ inhibition and oxidative stress by a group of 1,2-dihydroquinolines that culminate in bactericidal effect on mycobacterium strains is demonstrated. They inhibited the non-pathogenic Mycobacterium smegmatis mc(2) 155 with MIC as low as 0.9 μg/mL and induced filamentation. Detailed studies revealed their ability to inhibit polymerization and GTPase activity of MtbFtsZ (Mycobacterial filamentous temperature sensitive Z) with an IC50 value of ∼40 μM. In addition to such target specific effects, these compounds exerted a global cellular effect by causing redox-imbalance that was evident from overproduction of ROS in treated cells. Such multi-targeting effect with one chemical scaffold has considerable significance in this era of emerging drug resistance and could offer promise in the development of new therapeutic agents against tuberculosis. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  16. Essential protein SepF of mycobacteria interacts with FtsZ and MurG to regulate cell growth and division.

    PubMed

    Gupta, Shamba; Banerjee, Srijon Kaushik; Chatterjee, Ayan; Sharma, Arun Kumar; Kundu, Manikuntala; Basu, Joyoti

    2015-08-01

    Coordinated bacterial cell septation and cell wall biosynthesis require formation of protein complexes at the sites of division and elongation, in a temporally controlled manner. The protein players in these complexes remain incompletely understood in mycobacteria. Using in vitro and in vivo assays, we showed that Rv2147c (or SepF) of Mycobacterium tuberculosis interacts with the principal driver of cytokinesis, FtsZ. SepF also interacts with itself both in vitro and in vivo. Amino acid residues 189A, 190K and 215F are required for FtsZ-SepF interaction, and are conserved across Gram-positive bacteria. Using Mycobacterium smegmatis as a surrogate system, we confirmed that sepFMSMEG is essential. Knockdown of SepF led to cell elongation, defective growth and failure of FtsZ to localize to the site of division, suggesting that SepF assists FtsZ localization at the site of division. Furthermore, SepF interacted with MurG, a peptidoglycan-synthesizing enzyme, both in vitro and in vivo, suggesting that SepF could serve as a link between cell division and peptidoglycan synthesis. SepF emerges as a newly identified essential component of the cell division complex in mycobacteria.

  17. Direct interaction of FtsZ and MreB is required for septum synthesis and cell division in Escherichia coli.

    PubMed

    Fenton, Andrew K; Gerdes, Kenn

    2013-07-03

    How bacteria coordinate cell growth with division is not well understood. Bacterial cell elongation is controlled by actin-MreB while cell division is governed by tubulin-FtsZ. A ring-like structure containing FtsZ (the Z ring) at mid-cell attracts other cell division proteins to form the divisome, an essential protein assembly required for septum synthesis and cell separation. The Z ring exists at mid-cell during a major part of the cell cycle without contracting. Here, we show that MreB and FtsZ of Escherichia coli interact directly and that this interaction is required for Z ring contraction. We further show that the MreB-FtsZ interaction is required for transfer of cell-wall biosynthetic enzymes from the lateral to the mature divisome, allowing cells to synthesise the septum. Our observations show that bacterial cell division is coupled to cell elongation via a direct and essential interaction between FtsZ and MreB.

  18. Expression of Brassica oleracea FtsZ1-1 and MinD alters chloroplast division in Nicotiana tabacum generating macro- and mini-chloroplasts.

    PubMed

    Chikkala, Veera R N; Nugent, Gregory D; Stalker, David M; Mouradov, Aidyn; Stevenson, Trevor W

    2012-05-01

    FtsZ1-1 and MinD plastid division-related genes were identified and cloned from Brassica oleracea var. botrytis. Transgenic tobacco plants expressing BoFtsZ1-1 or BoMinD exhibited cells with either fewer but abnormally large chloroplasts or more but smaller chloroplasts relative to wild-type tobacco plants. An abnormal chloroplast phenotype in guard cells was found in BoMinD transgenic tobacco plants but not in BoFtsZ1-1 transgenic tobacco plants. Transgenic tobacco plants bearing the macro-chloroplast phenotype had 10 to 20-fold increased levels of total FtsZ1-1 or MinD, whilst the transgenic tobacco plants bearing the mini-chloroplast phenotype had lower increased FtsZ1-1 or absence of detectable MinD. We also described for the first time, plastid transformation of macro-chloroplast bearing tobacco shoots with a gene cassette allowing for expression of green fluorescent protein (GFP). Homoplasmic plastid transformants from normal chloroplast and macro-chloroplast tobacco plants expressing GFP were obtained. Both types of transformants accumulated GFP at ~6% of total soluble protein, thus indicating that cells containing macro-chloroplasts can regenerate shoots in tissue culture and can stably integrate and express a foreign gene to similar levels as plant cells containing a normal chloroplast size and number.

  19. Comparative phylogenomics of the CBL-CIPK calcium-decoding network in the moss Physcomitrella, Arabidopsis, and other green lineages.

    PubMed

    Kleist, Thomas J; Spencley, Andrew L; Luan, Sheng

    2014-01-01

    Land plants have evolved a host of anatomical and molecular adaptations for terrestrial growth. Many of these adaptations are believed to be elaborations of features that were present in their algal-like progenitors. In the model plant Arabidopsis, 10 Calcineurin B-Like proteins (CBLs) function as calcium sensors and modulate the activity of 26 CBL-Interacting Protein Kinases (CIPKs). The CBL-CIPK network coordinates environmental responses and helps maintain proper ion balances, especially during abiotic stress. We identified and analyzed CBL and CIPK homologs in green lineages, including CBLs and CIPKs from charophyte green algae, the closest living relatives of land plants. Phylogenomic evidence suggests that the network expanded from a small module, likely a single CBL-CIPK pair, present in the ancestor of modern plants and algae. Extreme conservation of the NAF motif, which mediates CBL-CIPK physical interactions, among all identified CIPKs supports the interpretation of CBL and CIPK homologs in green algae and early diverging land plants as functionally linked network components. We identified the full complement of CBL and CIPK loci in the genome of Physcomitrella, a model moss. These analyses demonstrate the strong effects of a recent moss whole genome duplication: CBL and CIPK loci appear in cognate pairs, some of which appear to be pseudogenes, with high sequence similarity. We cloned all full-length transcripts from these loci and performed yeast two-hybrid analyses to demonstrate CBL-CIPK interactions and identify specific connections within the network. Using phylogenomics, we have identified three ancient types of CBLs that are discernible by N-terminal localization motifs and a "green algal-type" clade of CIPKs with members from Physcomitrella and Arabidopsis.

  20. Dissolved inorganic nitrogen pools and surface flux under different brackish marsh vegetation types, common reed (Phragmites australis) and salt hay (Spartina patens)

    USGS Publications Warehouse

    Windham-Myers, L.

    2005-01-01

    The current expansion of Phragmites australis into the high marsh shortgrass (Spartina patens, Distichlis spicata) communities of eastern U.S. salt marshes provided an opportunity to identify the influence of vegetation types on pools and fluxes of dissolved inorganic nitrogen (DIN). Two brackish tidal marshes of the National Estuarine Research Reserve system were examined, Piermont Marsh of the Hudson River NERR in New York and Hog Island in the Jacques Coustaeu NERR of New Jersey. Pools of DIN in porewater and rates of DIN surface flux were compared in replicated pairs of recently-expanded P. australis and neighboring S. patens-dominated patches on the high marsh surface. Both marshes generally imported nitrate (NO3-) and exported ammonium (NH4+), such that overall DIN was exported. No differences in surface exchange of NO3- or NH4+ were observed between vegetation types. Depth-averaged porewater NH4+ concentrations over the entire growing season were 56% lower under P. australis than under S. patens (average 1.4 vs. 3.2 mg NH4+ L-1) with the most profound differences in November. Porewater profiles showed an accumulation of NH4+ at depth in S. patens and constant low concentrations in P. australis from the soil surface to 50 cm depth, with no significant differences in porewater salinity. Despite these profound differences in porewater, NH 4+ diffusion from soils of P. australis and S. patens were not measurably different, were similar to other published rates, and were well below estimated rates based on passive diffusion alone. Rapid adsorption and uptake by litter and microbes in surface soils of both communities may buffer NH4+ loss to flooding tides in both communities, thereby reducing the impact of P. australis invasion on NH4+ flux to flooding waters. ?? Springer 2005.

  1. Synthesis and antibacterial activity of novel 4-bromo-1H-indazole derivatives as FtsZ inhibitors.

    PubMed

    Wang, Yi; Yan, Mi; Ma, Ruixin; Ma, Shutao

    2015-04-01

    A series of novel 4-bromo-1H-indazole derivatives as filamentous temperature-sensitive protein Z (FtsZ) inhibitors were designed, synthesized, and assayed for their in vitro antibacterial activity against various phenotypes of Gram-positive and Gram-negative bacteria and their cell division inhibitory activity. The results indicated that this series showed better antibacterial activity against Staphylococcus epidermidis and penicillin-susceptible Streptococcus pyogenes than the other tested strains. Among them, compounds 12 and 18 exhibited 256-fold and 256-fold more potent activity than 3-methoxybenzamide (3-MBA) against penicillin-resistant Staphylococcus aureus, and compound 18 showed 64-fold better activity than 3-MBA but 4-fold weaker activity than ciprofloxacin in the inhibition of S. aureus ATCC29213. Particularly, compound 9 presented the best activity (4 µg/mL) against S. pyogenes PS, being 32-fold, 32-fold, and 2-fold more active than 3-MBA, curcumin, and ciprofloxacin, respectively, but it was four times less active than oxacillin sodium. In addition, some synthesized compounds displayed moderate inhibition of cell division against S. aureus ATCC25923, Escherichia coli ATCC25922, and Pseudomonas aeruginosa ATCC27853, sharing a minimum cell division concentration of 128 µg/mL.

  2. Defining restoration targets for water depth and salinity in wind-dominated Spartina patens (Ait.) Muhl. coastal marshes

    USGS Publications Warehouse

    Nyman, J.A.; La Peyre, M.K.; Caldwell, A.; Piazza, S.; Thom, C.; Winslow, C.

    2009-01-01

    Coastal wetlands provide valued ecosystem functions but the sustainability of those functions often is threatened by artificial hydrologic conditions. It is widely recognized that increased flooding and salinity can stress emergent plants, but there are few measurements to guide restoration, management, and mitigation. Marsh flooding can be estimated over large areas with few data where winds have little effect on water levels, but quantifying flooding requires hourly measurements over long time periods where tides are wind-dominated such as the northern Gulf of Mexico. Estimating salinity of flood water requires direct daily measurements because coastal marshes are characterized by dynamic salinity gradients. We analyzed 399,772 hourly observations of water depth and 521,561 hourly observations of water salinity from 14 sites in Louisiana coastal marshes dominated by Spartina patens (Ait.) Muhl. Unlike predicted water levels, observed water levels varied monthly and annually. We attributed those observed variations to variations in river runoff and winds. In stable marshes with slow wetland loss rates, we found that marsh elevation averaged 1 cm above mean high water, 15 cm above mean water, and 32 cm above mean low water levels. Water salinity averaged 3.7 ppt during April, May, and June, and 5.4 ppt during July, August, and September. The daily, seasonal, and annual variation in water levels and salinity that were evident would support the contention that such variation be retained when designing and operating coastal wetland management and restoration projects. Our findings might be of interest to scientists, engineers, and managers involved in restoration, management, and restoration in other regions where S. patens or similar species are common but local data are unavailable. ?? 2009 Elsevier B.V.

  3. Defining restoration targets for water depth and salinity in wind-dominated Spartina patens (Ait.) Muhl. coastal marshes

    USGS Publications Warehouse

    Nyman, J.A.; LaPeyre, Megan K.; Caldwell, Andral W.; Piazza, Sarai C.; Thom, C.; Winslow, C.

    2009-01-01

    Coastal wetlands provide valued ecosystem functions but the sustainability of those functions often is threatened by artificial hydrologic conditions. It is widely recognized that increased flooding and salinity can stress emergent plants, but there are few measurements to guide restoration, management, and mitigation. Marsh flooding can be estimated over large areas with few data where winds have little effect on water levels, but quantifying flooding requires hourly measurements over long time periods where tides are wind-dominated such as the northern Gulf of Mexico. Estimating salinity of flood water requires direct daily measurements because coastal marshes are characterized by dynamic salinity gradients. We analyzed 399,772 hourly observations of water depth and 521,561 hourly observations of water salinity from 14 sites in Louisiana coastal marshes dominated by Spartina patens (Ait.) Muhl. Unlike predicted water levels, observed water levels varied monthly and annually. We attributed those observed variations to variations in river runoff and winds. In stable marshes with slow wetland loss rates, we found that marsh elevation averaged 1 cm above mean high water, 15 cm above mean water, and 32 cm above mean low water levels. Water salinity averaged 3.7 ppt during April, May, and June, and 5.4 ppt during July, August, and September. The daily, seasonal, and annual variation in water levels and salinity that were evident would support the contention that such variation be retained when designing and operating coastal wetland management and restoration projects. Our findings might be of interest to scientists, engineers, and managers involved in restoration, management, and restoration in other regions where S. patens or similar species are common but local data are unavailable.

  4. ZapE is a novel cell division protein interacting with FtsZ and modulating the Z-ring dynamics.

    PubMed

    Marteyn, Benoit S; Karimova, Gouzel; Fenton, Andrew K; Gazi, Anastasia D; West, Nicholas; Touqui, Lhousseine; Prevost, Marie-Christine; Betton, Jean-Michel; Poyraz, Oemer; Ladant, Daniel; Gerdes, Kenn; Sansonetti, Philippe J; Tang, Christoph M

    2014-03-04

    Bacterial cell division requires the formation of a mature divisome complex positioned at the midcell. The localization of the divisome complex is determined by the correct positioning, assembly, and constriction of the FtsZ ring (Z-ring). Z-ring constriction control remains poorly understood and (to some extent) controversial, probably due to the fact that this phenomenon is transient and controlled by numerous factors. Here, we characterize ZapE, a novel ATPase found in Gram-negative bacteria, which is required for growth under conditions of low oxygen, while loss of zapE results in temperature-dependent elongation of cell shape. We found that ZapE is recruited to the Z-ring during late stages of the cell division process and correlates with constriction of the Z-ring. Overexpression or inactivation of zapE leads to elongation of Escherichia coli and affects the dynamics of the Z-ring during division. In vitro, ZapE destabilizes FtsZ polymers in an ATP-dependent manner. IMPORTANCE Bacterial cell division has mainly been characterized in vitro. In this report, we could identify ZapE as a novel cell division protein which is not essential in vitro but is required during an infectious process. The bacterial cell division process relies on the assembly, positioning, and constriction of FtsZ ring (the so-called Z-ring). Among nonessential cell division proteins recently identified, ZapE is the first in which detection at the Z-ring correlates with its constriction. We demonstrate that ZapE abundance has to be tightly regulated to allow cell division to occur; absence or overexpression of ZapE leads to bacterial filamentation. As zapE is not essential, we speculate that additional Z-ring destabilizing proteins transiently recruited during late cell division process might be identified in the future.

  5. The Arabidopsis minE mutation causes new plastid and FtsZ1 localization phenotypes in the leaf epidermis

    PubMed Central

    Fujiwara, Makoto T.; Kojo, Kei H.; Kazama, Yusuke; Sasaki, Shun; Abe, Tomoko; Itoh, Ryuuichi D.

    2015-01-01

    Plastids in the leaf epidermal cells of plants are regarded as immature chloroplasts that, like mesophyll chloroplasts, undergo binary fission. While mesophyll chloroplasts have generally been used to study plastid division, recent studies have suggested the presence of tissue- or plastid type-dependent regulation of plastid division. Here, we report the detailed morphology of plastids and their stromules, and the intraplastidic localization of the chloroplast division-related protein AtFtsZ1-1, in the leaf epidermis of an Arabidopsis mutant that harbors a mutation in the chloroplast division site determinant gene AtMinE1. In atminE1, the size and shape of epidermal plastids varied widely, which contrasts with the plastid phenotype observed in atminE1 mesophyll cells. In particular, atminE1 epidermal plastids occasionally displayed grape-like morphology, a novel phenotype induced by a plastid division mutation. Observation of an atminE1 transgenic line harboring an AtMinE1 promoter::AtMinE1-yellow fluorescent protein fusion gene confirmed the expression and plastidic localization of AtMinE1 in the leaf epidermis. Further examination revealed that constriction of plastids and stromules mediated by the FtsZ1 ring contributed to the plastid pleomorphism in the atminE1 epidermis. These results illustrate that a single plastid division mutation can have dramatic consequences for epidermal plastid morphology, thereby implying that plastid division and morphogenesis are differentially regulated in epidermal and mesophyll plastids. PMID:26500667

  6. Biological activity of Pinus nigra terpenes--evaluation of FtsZ inhibition by selected compounds as contribution to their antimicrobial activity.

    PubMed

    Sarac, Zorica; Matejić, Jelena S; Stojanović-Radić, Zorica Z; Veselinović, Jovana B; Džamić, Ana M; Bojović, Srdjan; Marin, Petar D

    2014-11-01

    In the current work, in vitro antioxidant, antibacterial, and antifungal activites of the needle terpenes of three taxa of Pinus nigra from Serbia (ssp. nigra, ssp. pallasiana, and var. banatica) were analyzed. The black pine essential oils showed generally weak antioxidative properties tested by two methods (DPPH and ABTS scavenging assays), where the highest activity was identified in P. nigra var. banatica (IC50=25.08 mg/mL and VitC=0.67 mg (vitamin C)/g when tested with the DPPH and ABTS reagents, respectively). In the antimicrobial assays, one fungal (Aspergilus niger) and two bacterial strains (Staphylococcus aureus and Bacillus cereus) showed sensitivity against essential oils of all three P. nigra taxa. The tested oils have been shown to possess inhibitory action in the range from 20.00 to 0.62 mg/mL, where var. banatica exhibited the highest and ssp. nigra the lowest antimicrobial action. In order to determine potential compounds that are responsible for alternative mode of action, molecular docking simulations inside FtsZ (a prokaryotic homolog of tubulin) were performed. Tested compounds were the most abundant terpenoid (germacrene D-4-ol) and its structurally similar terpene (germacrene D), both present in all three essential oils. It was determined that the oxygenated form of the molecule creates stable bonds with investigated enzyme FtsZ, and that this compound, through this mechanism of action participates in the antimicrobial activity. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Concentration and Assembly of the Division Ring Proteins FtsZ, FtsA, and ZipA during the Escherichia coli Cell Cycle

    PubMed Central

    Rueda, Sonsoles; Vicente, Miguel; Mingorance, Jesús

    2003-01-01

    The concentration of the cell division proteins FtsZ, FtsA, and ZipA and their assembly into a division ring during the Escherichia coli B/r K cell cycle have been measured in synchronous cultures obtained by the membrane elution technique. Immunostaining of the three proteins revealed no organized structure in newly born cells. In a culture with a doubling time of 49 min, assembly of the Z ring started around minute 25 and was detected first as a two-dot structure that became a sharp band before cell constriction. FtsA and ZipA localized into a division ring following the same pattern and time course as FtsZ. The concentration (amount relative to total mass) of the three proteins remained constant during one complete cell cycle, showing that assembly of a division ring is not driven by changes in the concentration of these proteins. Maintenance of the Z ring during the process of septation is a dynamic energy-dependent event, as evidenced by its disappearance in cells treated with sodium azide. PMID:12754232

  8. ATG5-knockout mutants of Physcomitrella provide a platform for analyzing the involvement of autophagy in senescence processes in plant cells.

    PubMed

    Mukae, Kyosuke; Inoue, Yuko; Moriyasu, Yuji

    2015-01-01

    Autophagy is a pathway in which a cell degrades part of its cytoplasm in vacuoles or lysosomes. To identify the physiological functions of autophagy in plants, we disrupted ATG5, an autophagy-related gene, in Physcomitrella, and confirmed that atg5 mutants are deficient in the process of autophagy. On carbon or nitrogen starvation medium, atg5 colonies turned yellow earlier than the wild-type (WT) colonies, showing that Physcomitrella atg5 mutants, like yeast and Arabidopsis, are sensitive to nutrient starvation. In the dark, even under nutrient-sufficient conditions, colonies turned yellow and the net degradation of chlorophyll and Rubisco protein occurred together with the upregulation of several senescence-associated genes. Yellowing reactions were inhibited by the protein synthesis inhibitor cycloheximide, suggesting that protonemal colonies undergo dark-induced senescence like the green leaves of higher plants. Such senescence responses in the dark occurred earlier in atg5 colonies than WT colonies. The sugar content was almost the same between WT and atg5 colonies, indicating that the early-senescence phenotype of atg5 is not explained by sugar deficiency. However, the levels of 7 amino acids showed significantly different alteration between atg5 and WT in the dark: 6 amino acids, particularly arginine and alanine, were much more deficient in the atg5 mutants, irrespective of the early degradation of Rubisco protein. On nutrient-sufficient medium supplemented with casamino acids, the early-senescence phenotype was slightly moderated. We propose that the early-senescence phenotype in atg5 mutants is partly explained by amino acid imbalance because of the lack of cytoplasmic degradation by autophagy in Physcomitrella.

  9. Comparison of the effectiveness of ISJ and SSR markers and detection of outlier loci in conservation genetics of Pulsatilla patens populations

    PubMed Central

    Szczecińska, Monika

    2016-01-01

    Background Research into the protection of rare and endangered plant species involves genetic analyses to determine their genetic variation and genetic structure. Various categories of genetic markers are used for this purpose. Microsatellites, also known as simple sequence repeats (SSR), are the most popular category of markers in population genetics research. In most cases, microsatellites account for a large part of the noncoding DNA and exert a neutral effect on the genome. Neutrality is a desirable feature in evaluations of genetic differences between populations, but it does not support analyses of a population’s ability to adapt to a given environment or its evolutionary potential. Despite the numerous advantages of microsatellites, non-neutral markers may supply important information in conservation genetics research. They are used to evaluate adaptation to specific environmental conditions and a population’s adaptive potential. The aim of this study was to compare the level of genetic variation in Pulsatilla patens populations revealed by neutral SSR markers and putatively adaptive ISJ markers (intron-exon splice junction). Methods The experiment was conducted on 14 Polish populations of P. patens and three P. patens populations from the nearby region of Vitebsk in Belarus. A total of 345 individuals were examined. Analyses were performed with the use of eight SSR primers specific to P. patens and three ISJ primers. Results SSR markers revealed a higher level of genetic variation than ISJ markers (He = 0.609, He = 0.145, respectively). An analysis of molecular variance (AMOVA) revealed that, the overall genetic diversity between the analyzed populations defined by parameters FST and ΦPT for SSR (20%) and ΦPT for ISJ (21%) markers was similar. Analysis conducted in the Structure program divided analyzed populations into two groups (SSR loci) and three groups (ISJ markers). Mantel test revealed correlations between the geographic distance and genetic

  10. Species and population variation to salinity stress in Panicum hemitomon, Spartina patens, and Spartina alterniflora: Morphological and physiological constraints

    USGS Publications Warehouse

    Hester, M.W.; Mendelssohn, I.A.; McKee, K.L.

    2001-01-01

    Panicum hemitomon, Spartina patens, and Spartina alterniflora are wide-spread dominant grasses of fresh, brackish, and salt marsh plant communities, respectively. Our previous research identified significant intraspecific variation in salt tolerance and morphology among populations within each species. In this study our objectives were to determine shorter-term physiological/biochemical responses to salinity stress and identify potential indicators of salt tolerance, with the ultimate goal of discerning similarities and differences in the mechanisms of salinity stress resistance. We subjected a subset of six populations within each species, ranging from high to low salt tolerance, to sublethal salinity levels (4, 20, and 30 ppt, respectively, for species) and monitored physiological and growth responses after 1 week (early harvest) and 5 weeks (late harvest). In all three species sublethal salinity levels generally resulted in significantly reduced net CO2 assimilation, leaf expansion, midday leaf xylem pressure, water use efficiency, and live and total biomass; and significantly increased leaf Na+/K+ ratio, leaf proline, leaf glycine betaine, leaf sucrose, root-to-shoot ratio, and dead:total aboveground biomass ratio. All three species displayed significant population (intraspecific) variation in net CO2 assimilation, leaf expansion, water use efficiency, midday leaf xylem pressure, leaf proline, leaf glycine betaine (except Panicum, where it could not be accurately determined), leaf Na+/K+ ratio, leaf sucrose, total plant biomass, dead:total aboveground biomass ratio, and root-to-shoot ratio. General indicators of salt tolerance (regardless of species) included high net CO2 assimilation rates and water use efficiencies, and low ratios of root-to-shoot and dead:total aboveground biomass. Factor analysis and a-priori linear contrasts revealed some unique differences between species in terms of the relative importance of morphology and physiology in explaining

  11. Composition and Chemical Variability of Cleistopholis patens Trunk Bark Oil from Côte d'Ivoire.

    PubMed

    Ouattara, Zana A; Boti, Jean Brice; Ahibo, Coffy Antoine; Bekro, Yves-Alain; Janat, Mamyrbekova; Casanova, Joseph; Tomi, Félix; Bighelli, Ange

    2017-02-01

    The chemical composition of trunk bark oil from Cleistopholis patens (Benth.) Engl. & Diels, growing wild in Côte d'Ivoire, has been investigated by GC (FID) in combination with retention indices, GC/MS and (13) C-NMR. Moreover, one oil sample has been subjected to CC and all the fractions analyzed by GC (RI) and (13) C-NMR. In total, 61 components have been identified, including various sesquiterpene esters scarcely found in essential oils. (13) C-NMR was particularly efficient for the identification of a component not eluted on GC and for the quantification of heat-sensitive compounds. Then, 36 oil samples, isolated from trunk bark harvested in six Ivoirian forests have been analyzed. The content of the main components varied drastically from sample to sample: (E)-β-caryophyllene (0.4 - 69.1%), β-pinene (0 - 57%), α-phellandrene (0 - 33.2%), α-pinene (0.1 - 30.6%), β-elemol (0.1 - 29.9%), germacrene D (0 - 25.4%), juvenile hormone III (0 - 22.9%), germacrene B (0 - 20.6%) and sabinene (tr-20.3%). Statistical analysis, hierarchical clustering and principal components analysis, carried out on the 36 compositions evidenced a fair chemical variability of the stem bark oil of this species. Indeed, three clusters have been distinguished: the composition of group I (ten samples) was dominated by β-pinene and α-pinene, group II (nine samples) was represented by α-phellandrene and p-cymene and group III (16 samples) by β-elemol. A sample displayed an atypical composition dominated by (E)-β-caryophyllene.

  12. Pythium infection activates conserved plant defense responses in mosses

    USDA-ARS?s Scientific Manuscript database

    The moss Physcomitrella patens (P. patens) is a useful model to study abiotic stress responses since it is highly tolerant to drought, salt and osmotic stress. However, little is known about the defense mechanisms activated in this moss after pathogen assault. Here the induction of defense responses...

  13. SlmA, a Nucleoid-Associated, FtsZ Binding Protein Required for Blocking Septal Ring Assembly over Chromosomes in E. coli

    PubMed Central

    Bernhardt, Thomas G.; de Boer, Piet A.J.

    2010-01-01

    Summary Cell division in Escherichia coli begins with assembly of the tubulin-like FtsZ protein into a ring structure just underneath the cell membrane. Spatial control over Z ring assembly is achieved by two partially redundant negative regulatory systems, the Min system and nucleoid occlusion (NO), which cooperate to position the division site at midcell. In contrast to the well-studied Min system, almost nothing is known about how Z ring assembly is blocked in the vicinity of nucleoids to effect NO. Reasoning that Min function might become essential in cells impaired for NO, we screened for mutations synthetically lethal with a defective Min system (slm mutants). By using this approach, we identified SlmA (Ttk) as the first NO factor in E. coli. Our combined genetic, cytological, and bio-chemical results suggest that SlmA is a DNA-associated division inhibitor that is directly involved in preventing Z ring assembly on portions of the membrane surrounding the nucleoid. PMID:15916962

  14. The peroxyl radical-induced oxidation of Escherichia coli FtsZ and its single tryptophan mutant (Y222W) modifies specific side-chains, generates protein cross-links and affects biological function.

    PubMed

    Escobar-Álvarez, Elizabeth; Leinisch, Fabian; Araya, Gissela; Monasterio, Octavio; Lorentzen, Lasse G; Silva, Eduardo; Davies, Michael J; López-Alarcón, Camilo

    2017-11-01

    FtsZ (filamenting temperature-sensitive mutant Z) is a key protein in bacteria cell division. The wild-type Escherichia coli FtsZ sequence (FtsZwt) contains three tyrosine (Tyr, Y) and sixteen methionine (Met, M) residues. The Tyr at position 222 is a key residue for FtsZ polymerization. Mutation of this residue to tryptophan (Trp, W; mutant Y222W) inhibits GTPase activity resulting in an extended time in the polymerized state compared to FtsZwt. Protein oxidation has been highlighted as a determinant process for bacteria resistance and consequently oxidation of FtsZwt and the Y222W mutant, by peroxyl radicals (ROO•) generated from AAPH (2,2'-azobis(2-methylpropionamidine) dihydrochloride) was studied. The non-oxidized proteins showed differences in their polymerization behavior, with this favored by the presence of Trp at position 222. AAPH-treatment of the proteins inhibited polymerization. Protein integrity studies using SDS-PAGE revealed the presence of both monomers and oligomers (dimers, trimers and high mass material) on oxidation. Western blotting indicated the presence of significant levels of protein carbonyls. Amino acid analysis showed that Tyr, Trp (in the Y222W mutant), and Met were consumed by ROO•. Quantification of the number of moles of amino acid consumed per mole of ROO• shows that most of the initial oxidant can be accounted for at low radical fluxes, with Met being a major target. Western blotting provided evidence for di-tyrosine cross-links in the dimeric and trimeric proteins, confirming that oxidation of Tyr residues, at positions 339 and/or 371, are critical to ROO•-mediated crosslinking of both the FtsZwt and Y222W mutant protein. These findings are in agreement with di-tyrosine, N-formyl kynurenine, and kynurenine quantification assessed by UPLC, and with LC-MS data obtained for AAPH-treated protein samples. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Targeting of (D)MinC/MinD and (D)MinC/DicB complexes to septal rings in Escherichia coli suggests a multistep mechanism for MinC-mediated destruction of nascent FtsZ rings.

    PubMed

    Johnson, Jay E; Lackner, Laura L; de Boer, Piet A J

    2002-06-01

    The MinC protein is an important determinant of septal ring positioning in Escherichia coli. The N-terminal domain ((Z)MinC) suppresses septal ring formation by interfering with FtsZ polymerization, whereas the C-terminal domain ((D)MinC) is required for dimerization as well as for interaction with the MinD protein. MinD oscillates between the membrane of both cell halves in a MinE-dependent fashion. MinC oscillates along with MinD such that the time-integrated concentration of (Z)MinC at the membrane is minimal, and hence the stability of FtsZ polymers is maximal, at the cell center. MinC is cytoplasmic and fails to block FtsZ assembly in the absence of MinD, indicating that recruitment of MinC by MinD to the membrane enhances (Z)MinC function. Here, we present evidence that the binding of (D)MinC to MinD endows the MinC/MinD complex with a more specific affinity for a septal ring-associated target in vivo. Thus, MinD does not merely attract MinC to the membrane but also aids MinC in specifically binding to, or in close proximity to, the substrate of its (Z)MinC domain. MinC-mediated division inhibition can also be activated in a MinD-independent fashion by the DicB protein of cryptic prophage Kim. DicB shows little homology to MinD, and how it stimulates MinC function has been unclear. Similar to the results obtained with MinD, we find that DicB interacts directly with (D)MinC, that the (D)MinC/DicB complex has a high affinity for some septal ring target(s), and that MinC/DicB interferes with the assembly and/or integrity of FtsZ rings in vivo. The results suggest a multistep mechanism for the activation of MinC-mediated division inhibition by either MinD or DicB and further expand the number of properties that can be ascribed to the Min proteins.

  16. Dynamic FtsA and FtsZ localization and outer membrane alterations during polar growth and cell division in Agrobacterium tumefaciens.

    PubMed

    Zupan, John R; Cameron, Todd A; Anderson-Furgeson, James; Zambryski, Patricia C

    2013-05-28

    Growth and cell division in rod-shaped bacteria have been primarily studied in species that grow predominantly by peptidoglycan (PG) synthesis along the length of the cell. Rhizobiales species, however, predominantly grow by PG synthesis at a single pole. Here we characterize the dynamic localization of several Agrobacterium tumefaciens components during the cell cycle. First, the lipophilic dye FM 4-64 predominantly stains the outer membranes of old poles versus growing poles. In cells about to divide, however, both poles are equally labeled with FM 4-64, but the constriction site is not. Second, the cell-division protein FtsA alternates from unipolar foci in the shortest cells to unipolar and midcell localization in cells of intermediate length, to strictly midcell localization in the longest cells undergoing septation. Third, the cell division protein FtsZ localizes in a cell-cycle pattern similar to, but more complex than, FtsA. Finally, because PG synthesis is spatially and temporally regulated during the cell cycle, we treated cells with sublethal concentrations of carbenicillin (Cb) to assess the role of penicillin-binding proteins in growth and cell division. Cb-treated cells formed midcell circumferential bulges, suggesting that interrupted PG synthesis destabilizes the septum. Midcell bulges contained bands or foci of FtsA-GFP and FtsZ-GFP and no FM 4-64 label, as in untreated cells. There were no abnormal morphologies at the growth poles in Cb-treated cells, suggesting unipolar growth uses Cb-insensitive PG synthesis enzymes.

  17. Inundation and salinity impacts to above- and belowground productivity in Spartina patens and Spartina alterniflora in the Mississippi River Deltaic Plain: implications for using river diversions as restoration tools

    USGS Publications Warehouse

    Snedden, Gregg A.; Cretini, Kari Foster; Patton, Brett

    2015-01-01

    Inundation and salinity directly affect plant productivity and processes that regulate vertical accretion in coastal wetlands, and are expected to increase as sea level continues to rise. In the Mississippi River deltaic plain, river diversions, which are being implemented as ecosystem restoration tools, can also strongly increase inundation in coastal wetlands. We used an in situ mesocosm approach to examine how varying salinity (two levels) and inundation rates (six levels) influenced end-of-season above- and belowground biomass of Spartina patens and Spartina alterniflora during the growing season (March–October) in 2011. Above- and belowground biomass was highest in both species at higher elevations when inundation was minimal, and decreased exponentially with decreased elevation and increased flood duration. This negative biomass response to flooding was more pronounced in S. patens than in S. alterniflora, and S. patens also showed stronger biomass reductions at higher salinities. This salinity effect was absent for belowground biomass in S. alterniflora. These findings suggest that even subtle increases in sea level may lead to substantial reductions in productivity and organic accretion, and also illustrate the importance of considering the inundation tolerance of co-dominant species in receiving areas when utilizing river diversions for delta restoration.

  18. Visualisation of plastids in endosperm, pollen and roots of transgenic wheat expressing modified GFP fused to transit peptides from wheat SSU RubisCO, rice FtsZ and maize ferredoxin III proteins.

    PubMed

    Primavesi, Lucia F; Wu, Huixia; Mudd, Elisabeth A; Day, Anil; Jones, Huw D

    2008-08-01

    The ability to target marker proteins to specific subcellular compartments is a powerful research tool to study the structure and development of organelles. Here transit sequences from nuclear-encoded, plastid proteins, namely rice FtsZ, maize non-photosynthetic ferredoxin III (FdIII) and the small subunit of RubisCO were used to target a modified synthetic GFP (S65G, S72A) to plastids. The localisations of the fusion proteins expressed in transgenic wheat plants and under the control of the rice actin promoter were compared to an untargeted GFP control. GFP fluorescence was localised to non-green plastids in pollen, roots and seed endosperm and detected in isolated leaf chloroplasts using a GFP-specific antibody. Transit peptides appeared to influence the relative fluorescence intensities of plastids in different tissues. This is consistent with differential targeting and/or turnover of GFP fusion proteins in different plastid types. Replacement of GFP sequences with alternative coding regions enables immediate applications of our vectors for academic research and commercial applications.

  19. Multi-modal digital holographic microscopy for wide-field fluorescence and 3D phase imaging

    NASA Astrophysics Data System (ADS)

    Quan, Xiangyu; Xia, Peng; Matoba, Osamu; Nitta, Koichi; Awatsuji, Yasuhiro

    2016-03-01

    Multi-modal digital holographic microscopy is a combination of epifluorescence microscopy and digital holographic microscopy, the main function of which is to obtain images from fluorescence intensity and quantified phase contrasts, simultaneously. The proposed system is mostly beneficial to biological studies, with the reason that often the studies are depending on fluorescent labeling techniques to detect certain intracellular molecules, while phase information reflecting properties of unstained transparent elements. This paper is presenting our latest researches on applications such as randomly moving micro-fluorescent beads and living cells of Physcomitrella patens. The experiments are succeeded on obtaining a succession of wide-field fluorescent images and holograms from micro-beads, and different depths focusing is realized via numerical reconstruction. Living cells of Physcomitrella patens are recorded in the static manner, the reconstruction distance indicates thickness of cellular structure. These results are implementing practical applications toward many biomedical science researches.

  20. PpASCL, a moss ortholog of anther-specific chalcone synthase-like enzymes, is a hydroxyalkylpyrone synthase involved in an evolutionarily conserved sporopollenin biosynthesis pathway.

    PubMed

    Colpitts, Che C; Kim, Sung Soo; Posehn, Sarah E; Jepson, Christina; Kim, Sun Young; Wiedemann, Gertrud; Reski, Ralf; Wee, Andrew G H; Douglas, Carl J; Suh, Dae-Yeon

    2011-12-01

    Sporopollenin is the main constituent of the exine layer of spore and pollen walls. Recently, several Arabidopsis genes, including polyketide synthase A (PKSA), which encodes an anther-specific chalcone synthase-like enzyme (ASCL), have been shown to be involved in sporopollenin biosynthesis. The genome of the moss Physcomitrella patens contains putative orthologs of the Arabidopsis sporopollenin biosynthesis genes. We analyzed available P.patens expressed sequence tag (EST) data for putative moss orthologs of the Arabidopsis genes of sporopollenin biosynthesis and studied the enzymatic properties and reaction mechanism of recombinant PpASCL, the P.patens ortholog of Arabidopsis PKSA. We also generated structure models of PpASCL and Arabidopsis PKSA to study their substrate specificity. Physcomitrella patens orthologs of Arabidopsis genes for sporopollenin biosynthesis were found to be expressed in the sporophyte generation. Similarly to Arabidopsis PKSA, PpASCL condenses hydroxy fatty acyl-CoA esters with malonyl-CoA and produces hydroxyalkyl α-pyrones that probably serve as building blocks of sporopollenin. The ASCL-specific set of Gly-Gly-Ala residues predicted by the models to be located at the floor of the putative active site is proposed to serve as the opening of an acyl-binding tunnel in ASCL. These results suggest that ASCL functions together with other sporophyte-specific enzymes to provide polyhydroxylated precursors of sporopollenin in a pathway common to land plants.

  1. Moss cell walls: structure and biosynthesis

    PubMed Central

    Roberts, Alison W.; Roberts, Eric M.; Haigler, Candace H.

    2012-01-01

    The genome sequence of the moss Physcomitrella patens has stimulated new research examining the cell wall polysaccharides of mosses and the glycosyl transferases that synthesize them as a means to understand fundamental processes of cell wall biosynthesis and plant cell wall evolution. The cell walls of mosses and vascular plants are composed of the same classes of polysaccharides, but with differences in side chain composition and structure. Similarly, the genomes of P. patens and angiosperms encode the same families of cell wall glycosyl transferases, yet, in many cases these families have diversified independently in each lineage. Our understanding of land plant evolution could be enhanced by more complete knowledge of the relationships among glycosyl transferase functional diversification, cell wall structural and biochemical specialization, and the roles of cell walls in plant adaptation. As a foundation for these studies, we review the features of P. patens as an experimental system, analyses of cell wall composition in various moss species, recent studies that elucidate the structure and biosynthesis of cell wall polysaccharides in P. patens, and phylogenetic analysis of P. patens genes potentially involved in cell wall biosynthesis. PMID:22833752

  2. Sequence analysis of the Hsp70 family in moss and evaluation of their functions in abiotic stress responses

    PubMed Central

    Tang, Ting; Yu, Anmin; Li, Ping; Yang, Hong; Liu, Gaojing; Liu, Li

    2016-01-01

    The 70-kD heat shock proteins (Hsp70s) are highly conserved molecular chaperones that play essential roles in cellular processes including abiotic stress responses. Physcomitrella patens serves as a representative of the first terrestrial plants and can recover from serious dehydration. To assess the possible relationship between P. patens Hsp70s and dehydration tolerance, we analyzed the P. patens genome and found at least 21 genes encoding Hsp70s. Gene structure and motif composition were relatively conserved in each subfamily. The intron-exon structure of PpcpHsp70-2 was different from that of other PpcpHsp70s; this gene exhibits several forms of intron retention, indicating that introns may play important roles in regulating gene expression. We observed expansion of Hsp70s in P. patens, which may reflect adaptations related to development and dehydration tolerance, and results mainly from tandem and segmental duplications. Expression profiles of rice, Arabidopsis and P. patens Hsp70 genes revealed that more than half of the Hsp70 genes were responsive to ABA, salt and drought. The presence of overrepresented cis-elements (DOFCOREZM and GCCCORE) among stress-responsive Hsp70s suggests that they share a common regulatory pathway. Moss plants overexpressing PpcpHsp70-2 showed salt and dehydration tolerance, further supporting a role in adaptation to land. This work highlights directions for future functional analyses of Hsp70s. PMID:27644410

  3. Sporophyte Formation and Life Cycle Completion in Moss Requires Heterotrimeric G-Proteins1[OPEN

    PubMed Central

    Hackenberg, Dieter; Quatrano, Ralph

    2016-01-01

    In this study, we report the functional characterization of heterotrimeric G-proteins from a nonvascular plant, the moss Physcomitrella patens. In plants, G-proteins have been characterized from only a few angiosperms to date, where their involvement has been shown during regulation of multiple signaling and developmental pathways affecting overall plant fitness. In addition to its unparalleled evolutionary position in the plant lineages, the P. patens genome also codes for a unique assortment of G-protein components, which includes two copies of Gβ and Gγ genes, but no canonical Gα. Instead, a single gene encoding an extra-large Gα (XLG) protein exists in the P. patens genome. Here, we demonstrate that in P. patens the canonical Gα is biochemically and functionally replaced by an XLG protein, which works in the same genetic pathway as one of the Gβ proteins to control its development. Furthermore, the specific G-protein subunits in P. patens are essential for its life cycle completion. Deletion of the genomic locus of PpXLG or PpGβ2 results in smaller, slower growing gametophores. Normal reproductive structures develop on these gametophores, but they are unable to form any sporophyte, the only diploid stage in the moss life cycle. Finally, the mutant phenotypes of ΔPpXLG and ΔPpGβ2 can be complemented by the homologous genes from Arabidopsis, AtXLG2 and AtAGB1, respectively, suggesting an overall conservation of their function throughout the plant evolution. PMID:27550997

  4. Chloroplast genome sequence of the moss Tortula ruralis: gene content, polymorphism, and structural arrangement relative to other green plant chloroplast genomes

    PubMed Central

    2010-01-01

    Background Tortula ruralis, a widely distributed species in the moss family Pottiaceae, is increasingly used as a model organism for the study of desiccation tolerance and mechanisms of cellular repair. In this paper, we present the chloroplast genome sequence of T. ruralis, only the second published chloroplast genome for a moss, and the first for a vegetatively desiccation-tolerant plant. Results The Tortula chloroplast genome is ~123,500 bp, and differs in a number of ways from that of Physcomitrella patens, the first published moss chloroplast genome. For example, Tortula lacks the ~71 kb inversion found in the large single copy region of the Physcomitrella genome and other members of the Funariales. Also, the Tortula chloroplast genome lacks petN, a gene found in all known land plant plastid genomes. In addition, an unusual case of nucleotide polymorphism was discovered. Conclusions Although the chloroplast genome of Tortula ruralis differs from that of the only other sequenced moss, Physcomitrella patens, we have yet to determine the biological significance of the differences. The polymorphisms we have uncovered in the sequencing of the genome offer a rare possibility (for mosses) of the generation of DNA markers for fine-level phylogenetic studies, or to investigate individual variation within populations. PMID:20187961

  5. Current achievements in the production of complex biopharmaceuticals with moss bioreactors.

    PubMed

    Decker, Eva L; Reski, Ralf

    2008-01-01

    Transgenic plants are promising alternatives for the low-cost and safe pathogen-free production of complex recombinant pharmaceutical proteins (molecular farming). Plants as higher eukaryotes perform posttranslational modifications similar to those of mammalian cells. However, plant-specific protein N-glycosylation was shown to be immunogenic, a fact that represents a drawback for many plant systems in biopharmaceutical production. The moss Physcomitrella patens offers unique properties as a contained system for protein production. It is grown in the predominant haploid gametophytic stage as tissue suspension cultures in photobioreactors. Efficient secretory signals and a transient transfection system allow the secretion of freshly synthesized proteins to the surrounding medium. The key advantage of Physcomitrella compared to other plant systems is the feasibility of targeted gene replacements. By this means, moss strains with non-immunogenic humanized glycan patterns were created. Here we present an overview of the relevant aspects for establishing moss as a production system for recombinant biopharmaceuticals.

  6. Chloroplast movement: dissection of events downstream of photo- and mechano-perception.

    PubMed

    Sato, Yoshikatsu; Kadota, Akeo; Wada, Masamitsu

    2003-02-01

    The study of chloroplast photorelocation movement is progressing rapidly now that mutants for chloroplast movement have become available in Arabidopsis thaliana. However, mechanistic approaches in cell biology still stand to elucidate the mechanisms and regulations of such movement. The fern Adiantum capillus-veneris and the moss Physcomitrella patens are particularly suitable materials for analyzing the kinetics of intracellular chloroplast movement. In these plants, chloroplast movement is induced by red light as well as blue light, mediated by phytochrome and blue light receptor, respectively. In this paper, we review the unique force-generating system for chloroplast motility in P. patens. In addition to light-induced chloroplast movement, we also summarize mechanically induced chloroplast movement in these plants and the motility systems involved. Finally, the different dependency of mechano- and photo-relocation movement on external Ca(2+) is discussed.

  7. Plant genomes enclose footprints of past infections by giant virus relatives.

    PubMed

    Maumus, Florian; Epert, Aline; Nogué, Fabien; Blanc, Guillaume

    2014-06-27

    Nucleocytoplasmic large DNA viruses (NCLDVs) are eukaryotic viruses with large genomes (100 kb-2.5 Mb), which include giant Mimivirus, Megavirus and Pandoravirus. NCLDVs are known to infect animals, protists and phytoplankton but were never described as pathogens of land plants. Here, we show that the bryophyte Physcomitrella patens and the lycophyte Selaginella moellendorffii have open reading frames (ORFs) with high phylogenetic affinities to NCLDV homologues. The P. patens genes are clustered in DNA stretches (up to 13 kb) containing up to 16 NCLDV-like ORFs. Molecular evolution analysis suggests that the NCLDV-like regions were acquired by horizontal gene transfer from distinct but closely related viruses that possibly define a new family of NCLDVs. Transcriptomics and DNA methylation data indicate that the NCLDV-like regions are transcriptionally inactive and are highly cytosine methylated through a mechanism not relying on small RNAs. Altogether, our data show that members of NCLDV have infected land plants.

  8. Simultaneous isolation of pure and intact chloroplasts and mitochondria from moss as the basis for sub-cellular proteomics.

    PubMed

    Lang, Erika G E; Mueller, Stefanie J; Hoernstein, Sebastian N W; Porankiewicz-Asplund, Joanna; Vervliet-Scheebaum, Marco; Reski, Ralf

    2011-02-01

    The moss Physcomitrella patens is increasingly being used as a model for plant systems biology studies. While genomic and transcriptomic resources are in place, tools and experimental conditions for proteomic studies need to be developed. In the present study we describe a rapid and efficient protocol for the simultaneous isolation of chloroplasts and mitochondria from moss protonema. Routinely, 60-100 μg mitochondrial and 3-5 mg chloroplast proteins, respectively, were obtained from 20 g fresh weight of green moss tissue. Using 14 plant compartment marker antibodies derived from seed plant and algal protein sequences, respectively, the evolutionary conservation of the compartment marker proteins in the moss was demonstrated and purity and intactness of the extracted organelles confirmed. This isolation protocol and these validated compartment markers may serve as basis for sub-cellular proteomics in P. patens and other mosses.

  9. A single homeobox gene triggers phase transition, embryogenesis and asexual reproduction.

    PubMed

    Horst, Nelly A; Katz, Aviva; Pereman, Idan; Decker, Eva L; Ohad, Nir; Reski, Ralf

    2016-01-18

    Plants characteristically alternate between haploid gametophytic and diploid sporophytic stages. Meiosis and fertilization respectively initiate these two different ontogenies(1). Genes triggering ectopic embryo development on vegetative sporophytic tissues are well described(2,3); however, a genetic control of embryo development from gametophytic tissues remains elusive. Here, in the moss Physcomitrella patens we show that ectopic overexpression of the homeobox gene BELL1 induces embryo formation and subsequently reproductive diploid sporophytes from specific gametophytic cells without fertilization. In line with this, BELL1 loss-of-function mutants have a wild-type phenotype, except that their egg cells are bigger and unable to form embryos. Our results identify BELL1 as a master regulator for the gametophyte-to-sporophyte transition in P. patens and provide mechanistic insights into the evolution of embryos that can generate multicellular diploid sporophytes. This developmental innovation facilitated the colonization of land by plants about 500 million years ago(4) and thus shaped our current ecosystems.

  10. Evolutionary processes during the formation of the plant-specific Dof transcription factor family.

    PubMed

    Shigyo, Mikao; Tabei, Nobumitsu; Yoneyama, Tadakatsu; Yanagisawa, Shuichi

    2007-01-01

    We found 19 putative genes for plant-specific Dof transcription factors in the moss Physcomitrella patens and one Dof gene in the green alga Chlamydomonas reinhardtii, but no identifiable Dof gene in the red alga Cyanidioschyzon merolae and the diatom Thalassiosira pseudonana, suggesting that the origin of the Dof transcription factors pre-dates the divergence of the green algae and the ancestors of terrestrial plants. The phylogenetic analyses contended that the Dof family in angiosperms formed through a series of evolutionary processes, including intensive duplications of a specific ancestral gene after the divergence of the moss and the angiosperm lineages.

  11. RPK2 functions in diverged CLE signaling.

    PubMed

    Sawa, Shinichiro; Tabata, Ryo

    2011-01-01

    Shoot apical meristem is a well organized undifferentiated tissue which produces plant body. CLV3 peptide hormone regulates SAM homeostasis, and is perceived by several receptor complexes, CLV1, CLV2-SOL2/CRN, and RPK2. CLV1 homologues are encoded in various plants genome. However CLV2 and SOL2/CRN homologues are found only in higher plants. Here we show that the RPK2 homologues were found not only in moss, Physcomitrella patens, but also in liverwort, Marchantia polymorpha. Although CLV2-SOL2/CRN might have specific function in SAM homeostasis, CLV1 and RPK2 may regulate various plant physiological events during plant evolution.

  12. RPK2 functions in diverged CLE signaling

    PubMed Central

    Tabata, Ryo

    2011-01-01

    Shoot apical meristem is a well organized undifferentiated tissue which produces plant body. CLV3 peptide hormone regulates SAM homeostasis, and is perceived by several receptor complexes, CLV1, CLV2-SOL2/CRN and RPK2. CLV1 homologues are encoded in various plants genome. However CLV2 and SOL2/CRN homologues are found only in higher plants. Here we show that the RPK2 homologues were found not only in moss, Physcomitrella patens, but also in liverwort, Marchantia polymorpha. Although CLV2-SOL2/CRN might have specific function in SAM homeostasis, CLV1 and RPK2 may regulate various plant physiological events during plant evolution. PMID:21301217

  13. Adaptation Mechanisms in the Evolution of Moss Defenses to Microbes.

    PubMed

    Ponce de León, Inés; Montesano, Marcos

    2017-01-01

    Bryophytes, including mosses, liverworts and hornworts are early land plants that have evolved key adaptation mechanisms to cope with abiotic stresses and microorganisms. Microbial symbioses facilitated plant colonization of land by enhancing nutrient uptake leading to improved plant growth and fitness. In addition, early land plants acquired novel defense mechanisms to protect plant tissues from pre-existing microbial pathogens. Due to its evolutionary stage linking unicellular green algae to vascular plants, the non-vascular moss Physcomitrella patens is an interesting organism to explore the adaptation mechanisms developed in the evolution of plant defenses to microbes. Cellular and biochemical approaches, gene expression profiles, and functional analysis of genes by targeted gene disruption have revealed that several defense mechanisms against microbial pathogens are conserved between mosses and flowering plants. P. patens perceives pathogen associated molecular patterns by plasma membrane receptor(s) and transduces the signal through a MAP kinase (MAPK) cascade leading to the activation of cell wall associated defenses and expression of genes that encode proteins with different roles in plant resistance. After pathogen assault, P. patens also activates the production of ROS, induces a HR-like reaction and increases levels of some hormones. Furthermore, alternative metabolic pathways are present in P. patens leading to the production of a distinct metabolic scenario than flowering plants that could contribute to defense. P. patens has acquired genes by horizontal transfer from prokaryotes and fungi, and some of them could represent adaptive benefits for resistance to biotic stress. In this review, the current knowledge related to the evolution of plant defense responses against pathogens will be discussed, focusing on the latest advances made in the model plant P. patens.

  14. Adaptation Mechanisms in the Evolution of Moss Defenses to Microbes

    PubMed Central

    Ponce de León, Inés; Montesano, Marcos

    2017-01-01

    Bryophytes, including mosses, liverworts and hornworts are early land plants that have evolved key adaptation mechanisms to cope with abiotic stresses and microorganisms. Microbial symbioses facilitated plant colonization of land by enhancing nutrient uptake leading to improved plant growth and fitness. In addition, early land plants acquired novel defense mechanisms to protect plant tissues from pre-existing microbial pathogens. Due to its evolutionary stage linking unicellular green algae to vascular plants, the non-vascular moss Physcomitrella patens is an interesting organism to explore the adaptation mechanisms developed in the evolution of plant defenses to microbes. Cellular and biochemical approaches, gene expression profiles, and functional analysis of genes by targeted gene disruption have revealed that several defense mechanisms against microbial pathogens are conserved between mosses and flowering plants. P. patens perceives pathogen associated molecular patterns by plasma membrane receptor(s) and transduces the signal through a MAP kinase (MAPK) cascade leading to the activation of cell wall associated defenses and expression of genes that encode proteins with different roles in plant resistance. After pathogen assault, P. patens also activates the production of ROS, induces a HR-like reaction and increases levels of some hormones. Furthermore, alternative metabolic pathways are present in P. patens leading to the production of a distinct metabolic scenario than flowering plants that could contribute to defense. P. patens has acquired genes by horizontal transfer from prokaryotes and fungi, and some of them could represent adaptive benefits for resistance to biotic stress. In this review, the current knowledge related to the evolution of plant defense responses against pathogens will be discussed, focusing on the latest advances made in the model plant P. patens. PMID:28360923

  15. Remote Sensing in Archeology: Classifying Bajos of the Paten, Guatemala

    NASA Technical Reports Server (NTRS)

    Lowry, James D., Jr.

    1998-01-01

    This project focuses on the adaptation of human populations to their environments from prehistoric times to the present. It emphasizes interdisciplinary research to develop ecological baselines through the use of remotely sensed imagery, in situ field work, and the modeling of human population dynamics. It utilizes cultural and biological data from dated archaeological sites to assess the subsistence and settlement patterns of human societies in response to changing climatic and environmental conditions. The utilization of remote sensing techniques in archaeology is relatively new, exciting, and opens many doors.

  16. Evolutionary conservation of plant gibberellin signalling pathway components

    PubMed Central

    Vandenbussche, Filip; Fierro, Ana C; Wiedemann, Gertrud; Reski, Ralf; Van Der Straeten, Dominique

    2007-01-01

    Background: Gibberellins (GA) are plant hormones that can regulate germination, elongation growth, and sex determination. They ubiquitously occur in seed plants. The discovery of gibberellin receptors, together with advances in understanding the function of key components of GA signalling in Arabidopsis and rice, reveal a fairly short GA signal transduction route. The pathway essentially consists of GID1 gibberellin receptors that interact with F-box proteins, which in turn regulate degradation of downstream DELLA proteins, suppressors of GA-controlled responses. Results: Arabidopsis sequences of the gibberellin signalling compounds were used to screen databases from a variety of plants, including protists, for homologues, providing indications for the degree of conservation of the pathway. The pathway as such appears completely absent in protists, the moss Physcomitrella patens shares only a limited homology with the Arabidopsis proteins, thus lacking essential characteristics of the classical GA signalling pathway, while the lycophyte Selaginella moellendorffii contains a possible ortholog for each component. The occurrence of classical GA responses can as yet not be linked with the presence of homologues of the signalling pathway. Alignments and display in neighbour joining trees of the GA signalling components confirm the close relationship of gymnosperms, monocotyledonous and dicotyledonous plants, as suggested from previous studies. Conclusion: Homologues of the GA-signalling pathway were mainly found in vascular plants. The GA signalling system may have its evolutionary molecular onset in Physcomitrella patens, where GAs at higher concentrations affect gravitropism and elongation growth. PMID:18047669

  17. Moonlighting activity of presenilin in plants is independent of γ-secretase and evolutionarily conserved

    PubMed Central

    Khandelwal, Abha; Chandu, Dilip; Roe, Catherine M.; Kopan, Raphael; Quatrano, Ralph S.

    2007-01-01

    Presenilins (PS) provide the catalytic activity for γ-secretase, which cleaves physiologically relevant substrates including Notch, ErbB4, and APP. Recent genetic studies indicated that the contribution of PS1 to mouse development includes γ-secretase-independent functions that cannot be easily explained by any of the demonstrated or hypothesized functions of this protein. To begin a nonbiased analysis of PS1 activity unencumbered by the dominant effect stemming from loss of Notch function, we characterized PS functions in the early land plant Physcomitrella patens, which lacks Notch, ErbB4, and APP. Removal of P. patens PS resulted in phenotypic abnormalities. Further assays performed to delineate the defective pathways in PS-deficient P. patens implicated improper function of the cytoskeletal network. Importantly, this characterization of a nonmetazoan PS uncovered a previously undescribed, evolutionarily conserved function (human PS1 can rescue the growth and light responses) that is γ-secretase-independent (mutants with substitutions of the catalytic aspartyl residues retain the activity). Introduction of PpPS into PS-deficient mouse embryonic fibroblasts rescues normal growth rates, demonstrating that at least some metazoan functions of PS are evolutionarily conserved. PMID:17684101

  18. Role of ENA ATPase in Na(+) efflux at high pH in bryophytes.

    PubMed

    Fraile-Escanciano, Ana; Garciadeblás, Blanca; Rodríguez-Navarro, Alonso; Benito, Begoña

    2009-12-01

    Potassium or Na(+) efflux ATPases, ENA ATPases, are present in all fungi and play a central role in Na(+) efflux and Na(+) tolerance. Flowering plants lack ENA ATPases but two ENA ATPases have been identified in the moss Physcomitrella patens, PpENA1 and PpENA2. PpENA1 mediates Na(+) efflux in Saccharomyces cerevisiae. To propose a general function of ENA ATPases in bryophytes it was necessary to demonstrate that these ATPases mediate Na(+) efflux in planta and that they exist in more bryophytes than P. patens. For these demonstrations (1) we cloned a third ATPase from P. patens, PpENA3, and studied the expression pattern of the three PpENA genes; (2) we constructed and studied the single and double Deltappena1 and Deltappena2 mutants; and (3) we cloned two ENA ATPases from the liverwort Marchantia polymorpha, MpENA1 and MpENA2, and expressed them in S. cerevisiae. The results from the first two approaches revealed that the expression of ENA ATPases was greatly enhanced at high pH and that Na(+) efflux at high pH depended on PpENA1. The ENA1 ATPase of M. polymorpha suppressed the defective growth of a S. cerevisiae mutant at high K(+) or Na(+) concentrations, especially at high K(+).

  19. Long-Term Growth of Moss in Microfluidic Devices Enables Subcellular Studies in Development1[OPEN

    PubMed Central

    2016-01-01

    Key developmental processes that occur on the subcellular and cellular level or occur in occluded tissues are difficult to access, let alone image and analyze. Recently, culturing living samples within polydimethylsiloxane (PDMS) microfluidic devices has facilitated the study of hard-to-reach developmental events. Here, we show that an early diverging land plant, Physcomitrella patens, can be continuously cultured within PDMS microfluidic chambers. Because the PDMS chambers are bonded to a coverslip, it is possible to image P. patens development at high resolution over long time periods. Using PDMS chambers, we report that wild-type protonemal tissue grows at the same rate as previously reported for growth on solid medium. Using long-term imaging, we highlight key developmental events, demonstrate compatibility with high-resolution confocal microscopy, and obtain growth rates for a slow-growing mutant. By coupling the powerful genetic tools available to P. patens with long-term growth and imaging provided by PDMS microfluidic chambers, we demonstrate the capability to study cellular and subcellular developmental events in plants directly and in real time. PMID:27406170

  20. Activation of Defense Mechanisms against Pathogens in Mosses and Flowering Plants

    PubMed Central

    de León, Inés Ponce; Montesano, Marcos

    2013-01-01

    During evolution, plants have developed mechanisms to cope with and adapt to different types of stress, including microbial infection. Once the stress is sensed, signaling pathways are activated, leading to the induced expression of genes with different roles in defense. Mosses (Bryophytes) are non-vascular plants that diverged from flowering plants more than 450 million years ago, allowing comparative studies of the evolution of defense-related genes and defensive metabolites produced after microbial infection. The ancestral position among land plants, the sequenced genome and the feasibility of generating targeted knock-out mutants by homologous recombination has made the moss Physcomitrella patens an attractive model to perform functional studies of plant genes involved in stress responses. This paper reviews the current knowledge of inducible defense mechanisms in P. patens and compares them to those activated in flowering plants after pathogen assault, including the reinforcement of the cell wall, ROS production, programmed cell death, activation of defense genes and synthesis of secondary metabolites and defense hormones. The knowledge generated in P. patens together with comparative studies in flowering plants will help to identify key components in plant defense responses and to design novel strategies to enhance resistance to biotic stress. PMID:23380962

  1. Nonflowering Plants Possess a Unique Folate-Dependent Phenylalanine Hydroxylase That Is Localized in Chloroplasts[W

    PubMed Central

    Pribat, Anne; Noiriel, Alexandre; Morse, Alison M.; Davis, John M.; Fouquet, Romain; Loizeau, Karen; Ravanel, Stéphane; Frank, Wolfgang; Haas, Richard; Reski, Ralf; Bedair, Mohamed; Sumner, Lloyd W.; Hanson, Andrew D.

    2010-01-01

    Tetrahydropterin-dependent aromatic amino acid hydroxylases (AAHs) are known from animals and microbes but not plants. A survey of genomes and ESTs revealed AAH-like sequences in gymnosperms, mosses, and algae. Analysis of full-length AAH cDNAs from Pinus taeda, Physcomitrella patens, and Chlamydomonas reinhardtii indicated that the encoded proteins form a distinct clade within the AAH family. These proteins were shown to have Phe hydroxylase activity by functional complementation of an Escherichia coli Tyr auxotroph and by enzyme assays. The P. taeda and P. patens AAHs were specific for Phe, required iron, showed Michaelian kinetics, and were active as monomers. Uniquely, they preferred 10-formyltetrahydrofolate to any physiological tetrahydropterin as cofactor and, consistent with preferring a folate cofactor, retained activity in complementation tests with tetrahydropterin-depleted E. coli host strains. Targeting assays in Arabidopsis thaliana mesophyll protoplasts using green fluorescent protein fusions, and import assays with purified Pisum sativum chloroplasts, indicated chloroplastic localization. Targeting assays further indicated that pterin-4a-carbinolamine dehydratase, which regenerates the AAH cofactor, is also chloroplastic. Ablating the single AAH gene in P. patens caused accumulation of Phe and caffeic acid esters. These data show that nonflowering plants have functional plastidial AAHs, establish an unprecedented electron donor role for a folate, and uncover a novel link between folate and aromatic metabolism. PMID:20959559

  2. Long-Term Growth of Moss in Microfluidic Devices Enables Subcellular Studies in Development.

    PubMed

    Bascom, Carlisle S; Wu, Shu-Zon; Nelson, Katherine; Oakey, John; Bezanilla, Magdalena

    2016-09-01

    Key developmental processes that occur on the subcellular and cellular level or occur in occluded tissues are difficult to access, let alone image and analyze. Recently, culturing living samples within polydimethylsiloxane (PDMS) microfluidic devices has facilitated the study of hard-to-reach developmental events. Here, we show that an early diverging land plant, Physcomitrella patens, can be continuously cultured within PDMS microfluidic chambers. Because the PDMS chambers are bonded to a coverslip, it is possible to image P. patens development at high resolution over long time periods. Using PDMS chambers, we report that wild-type protonemal tissue grows at the same rate as previously reported for growth on solid medium. Using long-term imaging, we highlight key developmental events, demonstrate compatibility with high-resolution confocal microscopy, and obtain growth rates for a slow-growing mutant. By coupling the powerful genetic tools available to P. patens with long-term growth and imaging provided by PDMS microfluidic chambers, we demonstrate the capability to study cellular and subcellular developmental events in plants directly and in real time.

  3. Spatio-temporal patterning of arginyl-tRNA protein transferase (ATE) contributes to gametophytic development in a moss.

    PubMed

    Schuessele, Christian; Hoernstein, Sebastian N W; Mueller, Stefanie J; Rodriguez-Franco, Marta; Lorenz, Timo; Lang, Daniel; Igloi, Gabor L; Reski, Ralf

    2016-02-01

    The importance of the arginyl-tRNA protein transferase (ATE), the enzyme mediating post-translation arginylation of proteins in the N-end rule degradation (NERD) pathway of protein stability, was analysed in Physcomitrella patens and compared to its known functions in other eukaryotes. We characterize ATE:GUS reporter lines as well as ATE mutants in P. patens to study the impact and function of arginylation on moss development and physiology. ATE protein abundance is spatially and temporally regulated in P. patens by hormones and light and is highly abundant in meristematic cells. Further, the amount of ATE transcript is regulated during abscisic acid signalling and downstream of auxin signalling. Loss-of-function mutants exhibit defects at various levels, most severely in developing gametophores, in chloroplast starch accumulation and senescence. Thus, arginylation is necessary for moss gametophyte development, in contrast to the situation in flowering plants. Our analysis further substantiates the conservation of the N-end rule pathway components in land plants and highlights lineage-specific features. We introduce moss as a model system to characterize the role of the NERD pathway as an additional layer of complexity in eukaryotic development.

  4. Activation of Defense Mechanisms against Pathogens in Mosses and Flowering Plants.

    PubMed

    Ponce de León, Inés; Montesano, Marcos

    2013-02-04

    During evolution, plants have developed mechanisms to cope with and adapt to different types of stress, including microbial infection. Once the stress is sensed, signaling pathways are activated, leading to the induced expression of genes with different roles in defense. Mosses (Bryophytes) are non-vascular plants that diverged from flowering plants more than 450 million years ago, allowing comparative studies of the evolution of defense-related genes and defensive metabolites produced after microbial infection. The ancestral position among land plants, the sequenced genome and the feasibility of generating targeted knock-out mutants by homologous recombination has made the moss Physcomitrella patens an attractive model to perform functional studies of plant genes involved in stress responses. This paper reviews the current knowledge of inducible defense mechanisms in P. patens and compares them to those activated in flowering plants after pathogen assault, including the reinforcement of the cell wall, ROS production, programmed cell death, activation of defense genes and synthesis of secondary metabolites and defense hormones. The knowledge generated in P. patens together with comparative studies in flowering plants will help to identify key components in plant defense responses and to design novel strategies to enhance resistance to biotic stress.

  5. Acquisition, Conservation, and Loss of Dual-Targeted Proteins in Land Plants1[W][OA

    PubMed Central

    Xu, Lin; Carrie, Chris; Law, Simon R.; Murcha, Monika W.; Whelan, James

    2013-01-01

    The dual-targeting ability of a variety of proteins from Physcomitrella patens, rice (Oryza sativa), and Arabidopsis (Arabidopsis thaliana) was tested to determine when dual targeting arose and to what extent it was conserved in land plants. Overall, the targeting ability of over 80 different proteins from rice and P. patens, representing 42 dual-targeted proteins in Arabidopsis, was tested. We found that dual targeting arose early in land plant evolution, as it was evident in many cases with P. patens proteins that were conserved in rice and Arabidopsis. Furthermore, we found that the acquisition of dual-targeting ability is still occurring, evident in P. patens as well as rice and Arabidopsis. The loss of dual-targeting ability appears to be rare, but does occur. Ascorbate peroxidase represents such an example. After gene duplication in rice, individual genes encode proteins that are targeted to a single organelle. Although we found that dual targeting was generally conserved, the ability to detect dual-targeted proteins differed depending on the cell types used. Furthermore, it appears that small changes in the targeting signal can result in a loss (or gain) of dual-targeting ability. Overall, examination of the targeting signals within this study did not reveal any clear patterns that would predict dual-targeting ability. The acquisition of dual-targeting ability also appears to be coordinated between proteins. Mitochondrial intermembrane space import and assembly protein40, a protein involved in oxidative folding in mitochondria and peroxisomes, provides an example where acquisition of dual targeting is accompanied by the dual targeting of substrate proteins. PMID:23257241

  6. Acquisition, conservation, and loss of dual-targeted proteins in land plants.

    PubMed

    Xu, Lin; Carrie, Chris; Law, Simon R; Murcha, Monika W; Whelan, James

    2013-02-01

    The dual-targeting ability of a variety of proteins from Physcomitrella patens, rice (Oryza sativa), and Arabidopsis (Arabidopsis thaliana) was tested to determine when dual targeting arose and to what extent it was conserved in land plants. Overall, the targeting ability of over 80 different proteins from rice and P. patens, representing 42 dual-targeted proteins in Arabidopsis, was tested. We found that dual targeting arose early in land plant evolution, as it was evident in many cases with P. patens proteins that were conserved in rice and Arabidopsis. Furthermore, we found that the acquisition of dual-targeting ability is still occurring, evident in P. patens as well as rice and Arabidopsis. The loss of dual-targeting ability appears to be rare, but does occur. Ascorbate peroxidase represents such an example. After gene duplication in rice, individual genes encode proteins that are targeted to a single organelle. Although we found that dual targeting was generally conserved, the ability to detect dual-targeted proteins differed depending on the cell types used. Furthermore, it appears that small changes in the targeting signal can result in a loss (or gain) of dual-targeting ability. Overall, examination of the targeting signals within this study did not reveal any clear patterns that would predict dual-targeting ability. The acquisition of dual-targeting ability also appears to be co