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Stefan A Rensing - One of the best experts on this subject based on the ideXlab platform.
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Characterization of spa mutants in the moss Physcomitrella provides evidence for functional divergence of SPA genes during the evolution of land plants
The New phytologist, 2019Co-Authors: Oliver Artz, Stefan A Rensing, Aashish Ranjan, Stephen Dickopf, Melanie Kreiss, Elena Theres Abraham, Vanessa Boll, Ute HoeckerAbstract:The Arabidopsis COP1/SPA complex is a key repressor of photomorphogenesis that suppresses light signaling in the dark. Both COP1 and SPA proteins are essential components of this complex. Although COP1 also exists in humans, SPA genes are specific to the green lineage. To elucidate the evolution of SPA genes we analyzed SPA functions in the moss Physcomitrella patens by characterizing knockout mutants in the two Physcomitrella SPA genes PpSPAa and PpSPAb. Light-grown PpspaAB double mutants exhibit smaller gametophores than the wild-type. In the dark, PpspaAB mutant gametophores show enhanced continuation of growth but etiolate normally. Gravitropism in the dark is reduced in PpspaAB mutant protonemata. The expression of light-regulated genes is mostly not constitutive in PpspaAB mutants. PpSPA and PpCOP1 interact; PpCOP1 also interacts with the transcription factor PpHY5 and, indeed, PpHY5 is destabilized in dark-grown Physcomitrella. Degradation of PpHY5 in darkness, however, does not require PpSPAa and PpSPAb. The data suggest that COP1/SPA-mediated light signaling is only partially conserved between Arabidopsis and Physcomitrella. Whereas COP1/SPA interaction and HY5 degradation in darkness is conserved, the role of SPA proteins appears to have diverged. PpSPA genes, unlike their Arabidopsis counterparts, are only required to suppress a subset of light responses in darkness.
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a single target mitochondrial rna editing factor of funaria hygrometrica can fully reconstitute rna editing at two sites in Physcomitrella patens
Plant and Cell Physiology, 2017Co-Authors: Stefan A Rensing, Mareike Schallenbergrudinger, Bastian Oldenkott, Manuel Hiss, Phuong Le TrinhAbstract:Nuclear-encoded pentatricopeptide repeat (PPR) proteins are key factors for site-specific RNA editing, converting cytidines into uridines in plant mitochondria and chloroplasts. All editing factors in the model moss Physcomitrella patens have a C-terminal DYW domain with similarity to cytidine deaminase. However, numerous editing factors in flowering plants lack such a terminal DYW domain, questioning its immediate role in the pyrimidine base conversion process. Here we further investigate the Physcomitrella DYW-type PPR protein PPR_78, responsible for mitochondrial editing sites cox1eU755SL and rps14eU137SL. Complementation assays with truncated proteins demonstrate that the DYW domain is essential for full PPR_78 editing functionality. The DYW domain can be replaced, however, with its counterpart from another editing factor, PPR_79. The PPR_78 ortholog of the related moss Funaria hygrometrica fully complements the Physcomitrella mutant for editing at both sites, although the editing site in rps14 is lacking in Funaria. Editing factor orthologs in different taxa may thus retain editing capacity for multiple sites despite the absence of editing requirement.
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functional analysis of cop1 and spa orthologs from Physcomitrella and rice during photomorphogenesis of transgenic arabidopsis reveals distinct evolutionary conservation
BMC Plant Biology, 2014Co-Authors: Stefan A Rensing, Aashish Ranjan, Stephen Dickopf, Kristian K Ullrich, Ute HoeckerAbstract: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 r ice 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, rice and Arabidopsis have been functionally conserved during evolution, while the SPA proteins showed considerable functional divergence. This may -a t least in part -r eflect the fact thatCOP1 is a single copy gene in seed plants, while SPA proteins are encoded by a small gene family of two to four members with possibly sub- or neofunctionalized tasks.
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assigning dyw type ppr proteins to rna editing sites in the funariid mosses Physcomitrella patens and funaria hygrometrica
Plant Journal, 2011Co-Authors: Mareike Rudinger, Stefan A Rensing, Peter Szovenyi, Volker KnoopAbstract:The plant-specific pentatricopeptide repeat (PPR) proteins with variable PPR repeat lengths (PLS-type) and protein extensions up to the carboxyterminal DYW domain have received attention as specific recognition factors for the C-to-U type of RNA editing events in plant organelles. Here, we report a DYW-protein knockout in the model plant Physcomitrella patens specifically affecting mitochondrial RNA editing positions cox1eU755SL and rps14eU137SL. Assignment of DYW proteins and RNA editing sites might best be corroborated by data from a taxon with a slightly different, yet similarly manageable low number of editing sites and DYW proteins. To this end we investigated the mitochondrial editing status of the related funariid moss Funaria hygrometrica. We find that: (i) Funaria lacks three mitochondrial RNA editing positions present in Physcomitrella, (ii) that F. hygrometrica cDNA sequence data identify nine DYW proteins as clear orthologues of their P. patens counterparts, and (iii) that the 'missing' 10th DYW protein in F. hygrometrica is responsible for two mitochondrial editing sites in P. patens lacking in F. hygrometrica (nad3eU230SL, nad4eU272SL). Interestingly, the third site of RNA editing missing in F. hygrometrica (rps14eU137SL) is addressed by the DYW protein characterized here and the presence of its orthologue in F. hygrometrica is explained through its simultaneous action on site cox1eU755SL conserved in both mosses.
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RNA editing: Only eleven sites are present in the Physcomitrella patens mitochondrial transcriptome and a universal nomenclature proposal
Molecular Genetics and Genomics, 2009Co-Authors: Mareike Rüdinger, Helena T Funk, Stefan A Rensing, Uwe G. MaierAbstract:RNA editing in mitochondria and chloroplasts of land plants alters the coding content of transcripts through site-specific exchanges of cytidines into uridines and vice versa. The abundance of RNA editing in model plant species such as rice or Arabidopsis with some 500 affected sites in their organelle transcripts hinders straightforward approaches to elucidate its mechanisms. The moss Physcomitrella patens is increasingly being appreciated as an alternative plant model system, enhanced by the recent availability of its complete chloroplast, mitochondrial, and nuclear genome sequences. We here report the transcriptomic analysis of Physcomitrella mitochondrial mRNAs as a prerequisite for future studies of mitochondrial RNA editing in this moss. We find a strikingly low frequency of RNA editing affecting only eleven, albeit highly important, sites of C-to-U nucleotide modification in only nine mitochondrial genes. Partial editing was seen for two of these sites but no evidence for any silent editing sites (leaving the identity of the encoded amino acid unchanged) as commonly observed in vascular plants was found in Physcomitrella, indicating a compact and efficient organization of the editing machinery. Furthermore, we here wish to propose a unifying nomenclature to clearly identify and designate RNA editing positions and to facilitate future communication and database annotation.
Ralf Reski - One of the best experts on this subject based on the ideXlab platform.
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ppascl a moss ortholog of anther specific chalcone synthase like enzymes is a hydroxyalkylpyrone synthase involved in an evolutionarily conserved sporopollenin biosynthesis pathway
New Phytologist, 2011Co-Authors: Che C Colpitts, Carl J Douglas, Ralf Reski, Sungsoo Kim, Sarah E Posehn, Christina Jepson, Sun Young Kim, Gertrud Wiedemann, Andrew G H Wee, Daeyeon SuhAbstract:Summary • 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 a-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 sporophytespecific enzymes to provide polyhydroxylated precursors of sporopollenin in a pathway common to land plants.
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a piib type ca2 atpase is essential for stress adaptation in Physcomitrella patens
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Enas Qudeimat, Ralf Reski, Alexander M C Faltusz, Glen L Wheeler, Daniel Lang, Hauke Holtorf, Colin Brownlee, Wolfgang FrankAbstract:Transient cytosolic Ca2+ ([Ca2+]cyt) elevations are early events in plant signaling pathways including those related to abiotic stress. The restoration of [Ca2+]cyt to prestimulus levels involves ATP-driven Ca2+ pumps, but direct evidence for an essential role of a plant Ca2+-ATPase in abiotic stress adaptation is missing. Here, we report on a stress-responsive Ca2+-ATPase gene (PCA1) from the moss Physcomitrella patens. Functional analysis of PCA1 in a Ca2+ transport-deficient yeast mutant suggests that PCA1 encodes a PIIB-type Ca2+-ATPase harboring an N-terminal autoinhibitory domain. In vivo localizations identified membranes of small vacuoles as the integration site for a PCA1:GFP fusion protein. PCA1 mRNA levels are up-regulated by dehydration, NaCl, and abscisic acid, and PCA1 loss-of-function mutants (ΔPCA1) exhibit an enhanced susceptibility to salt stress. The ΔPCA1 lines show sustained elevated [Ca2+]cyt in response to salt treatment in contrast to WT that shows transient Ca2+ elevations, indicating a direct role for PCA1 in the restoration of prestimulus [Ca2+]cyt. The altered Ca2+ response of the ΔPCA1 mutant lines correlates with altered expression levels of stress-induced genes, suggesting disturbance of a stress-associated signaling pathway. We propose that PCA1 is an essential component for abiotic stress adaptation in Physcomitrella involved in the generation of a specific salt-induced Ca2+ signature.
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specific gene silencing by artificial micrornas in Physcomitrella patens an alternative to targeted gene knockouts
Plant Physiology, 2008Co-Authors: Basel Khraiwesh, Ralf Reski, Stephan Ossowski, Detlef Weigel, Wolfgang FrankAbstract:MicroRNAs (miRNAs) are approximately 21-nucleotide-long RNAs processed from nuclear-encoded transcripts, which include a characteristic hairpin-like structure. MiRNAs control the expression of target transcripts by binding to reverse complementary sequences directing cleavage or translational inhibition of the target RNA. Artificial miRNAs (amiRNAs) can be generated by exchanging the miRNA/miRNA* sequence within miRNA precursor genes, while maintaining the pattern of matches and mismatches in the foldback. Thus, for functional gene analysis, amiRNAs can be designed to target any gene of interest. The moss Physcomitrella patens exhibits the unique feature of a highly efficient homologous recombination mechanism, which allows for the generation of targeted gene knockout lines. However, the completion of the Physcomitrella genome necessitates the development of alternative techniques to speed up reverse genetics analyses and to allow for more flexible inactivation of genes. To prove the adaptability of amiRNA expression in Physcomitrella, we designed two amiRNAs, targeting the gene PpFtsZ2-1, which is indispensable for chloroplast division, and the gene PpGNT1 encoding an N-acetylglucosaminyltransferase. Both amiRNAs were expressed from the Arabidopsis (Arabidopsis thaliana) miR319a precursor fused to a constitutive promoter. Transgenic Physcomitrella lines harboring the overexpression constructs showed precise processing of the amiRNAs and an efficient knock down of the cognate target mRNAs. Furthermore, chloroplast division was impeded in PpFtsZ2-1-amiRNA lines that phenocopied PpFtsZ2-1 knockout mutants. We also provide evidence for the amplification of the initial amiRNA signal by secondary transitive small interfering RNAs, although these small interfering RNAs do not seem to have a major effect on sequence-related mRNAs, confirming specificity of the amiRNA approach.
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the mechanism of gene targeting in Physcomitrella patens homologous recombination concatenation and multiple integration
Nucleic Acids Research, 2006Co-Authors: Yasuko Kamisugi, Stefan A Rensing, David J. Cove, Ralf Reski, Andrew C Cuming, Katja Schlink, Gabriele Schween, Mark Von StackelbergAbstract:The model bryophyte Physcomitrella patens exhibits high frequencies of gene targeting when transformed with DNA constructs containing sequences homologous with genomic loci. ‘Targeted gene replacement’ (TGR) resulting from homologous recombination (HR) between each end of a targeting construct and the targeted locus occurs when either single or multiple targeting vectors are delivered. In the latter instance simultaneous, multiple, independent integration of different transgenes occurs at the targeted loci. In both single gene and ‘batch’ transformations, DNA can also be found to undergo ‘targeted insertion’ (TI), integrating at one end of the targeted locus by HR with one flanking sequence of the vector accompanied by an apparent non-homologous end-joining (NHEJ) event at the other. Untargeted integration at nonhomologous sites also occurs, but at a lower frequency. Molecular analysis of TI at a single locus shows that this occurs as a consequence of concatenation of the transforming DNA, in planta, prior to integration, followed by HR between a single site in the genomic target and two of its repeated homologues in the concatenated vector. This reinforces the view that HR is the major pathway by which transforming DNA is integrated in Physcomitrella.
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Quantitative promoter analysis in Physcomitrella patens: a set of plant vectors activating gene expression within three orders of magnitude
BMC Biotechnology, 2004Co-Authors: Verena Horstmann, Ralf Reski, Claudia M Huether, Wolfgang Jost, Eva L DeckerAbstract:Background In addition to studies of plant gene function and developmental analyses, plant biotechnological use is largely dependent upon transgenic technologies. The moss Physcomitrella patens has become an exciting model system for studying plant molecular processes due to an exceptionally high rate of nuclear gene targeting by homologous recombination compared with other plants. However, its use in transgenic approaches requires expression vectors that incorporate sufficiently strong promoters. To satisfy this requirement, a set of plant expression vectors was constructed and equipped with either heterologous or endogenous promoters. Results Promoter activity was quantified using the dual-luciferase reporter assay system. The eight different heterologous promoter constructs tested exhibited expression levels spanning three orders of magnitude. Of these, the complete rice actin1 gene promoter showed the highest activity in Physcomitrella , followed by a truncated version of this promoter and three different versions of the cauliflower mosaic virus 35S promoter. In contrast, the Agrobacterium tumefaciens nopaline synthase promoter induced transcription rather weakly. Constructs including promoters commonly used in mammalian expression systems also proved to be functional in Physcomitrella . In addition, the 5' -regions of two Physcomitrella glycosyltransferases (i.e. α1,3- fucosyltransferase and β1,2- xylosyltransferase ) were identified and functionally characterised in comparison to the heterologous promoters. Furthermore, motifs responsible for enhancement of translation efficiency – such as the TMV omega element and a modified sequence directly prior the start codon – were tested in this model. Conclusion We developed a vector set that enables gene expression studies, both in lower and higher land plants, thus providing valuable tools applicable in both basic and applied molecular research.
Miroslav Strnad - One of the best experts on this subject based on the ideXlab platform.
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cytokinins in the bryophyte Physcomitrella patens analyses of activity distribution and cytokinin oxidase dehydrogenase overexpression reveal the role of extracellular cytokinins
Plant Physiology, 2007Co-Authors: Klaus Von Schwartzenberg, Václav Motyka, Petre I Dobrev, Ondřej Novak, Marta Fernandez Nunez, Hanna Blaschke, Miroslav StrnadAbstract: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 -(D 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 orthotopolin 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 -(D 2 -isopentenyl)adenine (iP) and N 6 -(D 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 extracellular iP and iPR are the main cytokinins responsible for inducing buds in the bryophyte Physcomitrella. Cytokinin profile is discussed regarding the evolution of cytokinin biosynthetic pathways.
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Cytokinins in the Bryophyte Physcomitrella patens: Analyses of Activity, Distribution, and Cytokinin Oxidase/Dehydrogenase Overexpression Reveal the Role of Extracellular Cytokinins
Plant physiology, 2007Co-Authors: Klaus Von Schwartzenberg, Václav Motyka, Petre I Dobrev, Ondřej Novak, Marta Fernandez Nunez, Hanna Blaschke, Miroslav StrnadAbstract: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 -(D 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 orthotopolin 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 -(D 2 -isopentenyl)adenine (iP) and N 6 -(D 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 extracellular iP and iPR are the main cytokinins responsible for inducing buds in the bryophyte Physcomitrella. Cytokinin profile is discussed regarding the evolution of cytokinin biosynthetic pathways.
Klaus Von Schwartzenberg - One of the best experts on this subject based on the ideXlab platform.
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cytokinins in the bryophyte Physcomitrella patens analyses of activity distribution and cytokinin oxidase dehydrogenase overexpression reveal the role of extracellular cytokinins
Plant Physiology, 2007Co-Authors: Klaus Von Schwartzenberg, Václav Motyka, Petre I Dobrev, Ondřej Novak, Marta Fernandez Nunez, Hanna Blaschke, Miroslav StrnadAbstract: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 -(D 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 orthotopolin 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 -(D 2 -isopentenyl)adenine (iP) and N 6 -(D 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 extracellular iP and iPR are the main cytokinins responsible for inducing buds in the bryophyte Physcomitrella. Cytokinin profile is discussed regarding the evolution of cytokinin biosynthetic pathways.
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Cytokinins in the Bryophyte Physcomitrella patens: Analyses of Activity, Distribution, and Cytokinin Oxidase/Dehydrogenase Overexpression Reveal the Role of Extracellular Cytokinins
Plant physiology, 2007Co-Authors: Klaus Von Schwartzenberg, Václav Motyka, Petre I Dobrev, Ondřej Novak, Marta Fernandez Nunez, Hanna Blaschke, Miroslav StrnadAbstract: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 -(D 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 orthotopolin 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 -(D 2 -isopentenyl)adenine (iP) and N 6 -(D 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 extracellular iP and iPR are the main cytokinins responsible for inducing buds in the bryophyte Physcomitrella. Cytokinin profile is discussed regarding the evolution of cytokinin biosynthetic pathways.
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kinetics of cytokinin production and bud formation in Physcomitrella analysis of wild type a developmental mutant and two of its ipt transgenics
Journal of Plant Physiology, 2000Co-Authors: Ralf Reski, Klaus Von Schwartzenberg, Peter Schulz, R Maldiney, Michel LaloueAbstract:Summary Cytokinins that are known to induce bud formation in mosses were quantified during the course of development of Physcomitrella patens (Hedw.) B.S.G. Analyses were carried out on wild type, the developmental mutant PC22 and two ipt -transgenic strains of PC22. The major cytokinins detected were isopentenyladenine (iP) and isopentenyladenosine ([9R]iP). The cytokinin overproducing ipt -strains released large amounts of iP into the culture medium (up to 32 nmol/L). For Physcomitrella wild type an iP maximum at day 9 preceded bud formation, which occurred at day 13. In the developmental mutant PC22 iP maxima were found at day 9 and at day 21; however, bud formation was not observed within this time. Two transgenics of this mutant, carrying the Agrobacterium ipt gene under control of its own promoter, released up to 34 and 372-fold more iP into the culture medium and continuously produced malformed buds beginning from the first days of culture. The time courses correlating the onset of bud formation with extracellular iP show for all 4 genotypes that iP concentration does not continuously increase but is fluctuating.
Andreas Weihe - One of the best experts on this subject based on the ideXlab platform.
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evolution of phage type rna polymerases in higher plants characterization of the single phage type rna polymerase gene from selaginella moellendorffii
Journal of Molecular Evolution, 2009Co-Authors: Chang Yin, Uwe Richter, Thomas Borner, Andreas WeiheAbstract:Selaginella moellendorfii (spikemoss) sequence trace data encoding a polypeptide highly similar to angiosperm and moss phage-type organelle RNA polymerases (RpoTs) were used to isolate a BAC clone containing the full-length gene SmRpoT as well as the corresponding cDNA. The SmRpoT mRNA comprises 3452 nt with an open reading frame of 3006 nt, encoding a putative protein of 1002 amino acids with a molecular mass of 113 kDa. The SmRpoT gene comprises 19 exons and 18 introns, conserved in their position with those of the angiosperm and Physcomitrella RpoT genes. In phylogenetic analyses, the Selaginella RpoT polymerase is in a sister position to all other phage-type polymerases of angiosperms. However, according to its conserved exon–intron structure, the Selaginella RpoT gene is representative of the molecular evolutionary lineage giving rise to the RpoT gene family of flowering plants. The N-terminal transit peptide of SmRpoT is shown to confer targeting of green fluorescent protein exclusively to mitochondria after transient expression in Arabidopsis and Selaginella protoplasts. Angiosperms and the moss P. patens possess small gene families encoding RpoTs, which include mitochondrial- and chloroplast-targeted RNA polymerases. In striking contrast, the Selaginella RpoT gene is shown to be single-copy, although Selaginella, as a lycophyte, has a phylogenetic position between Physcomitrella and angiosperms. Thus, there is no evidence that Selaginella may contain a nuclear-encoded phage-type chloroplast RNA polymerase.
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two rpot genes of Physcomitrella patens encode phage type rna polymerases with dual targeting to mitochondria and plastids
Gene, 2002Co-Authors: Uwe Richter, Thomas Borner, Ralf Reski, Eva L Decker, Justine Kiessling, Boris Hedtke, Andreas WeiheAbstract:Angiosperms possess a small family of phage-type RNA polymerase genes that arose by gene duplication from an ancestral gene encoding the mitochondrial RNA polymerase. We have isolated and sequenced the genes and cDNAs encoding two phage-type RNA polymerases, PpRpoT1 and PpRpoT2, from the moss Physcomitrella patens. PpRpoT1 comprises 19 exons and 18 introns, PpRpoT2 contains two additional introns. The N-terminal transit peptides of both polymerases are shown to confer dual-targeting of green fluorescent protein fusions to mitochondria and plastids. In vitro translation of the cDNAs revealed initiation of translation at two in-frame AUG start codons. Translation from the first methionine gives rise to a plastid-targeted polymerase, whereas initiation from the second methionine results in exclusively mitochondrial-targeted protein. Thus, dual-targeting of Physcomitrella RpoT is caused by and might be regulated by multiple translational starts. In phylogenetic analyses, the Physcomitrella RpoT polymerases form a sister group to all other phage-type polymerases of land plants. The two genes result from a gene duplication event that occurred independently from the one which led to the organellar polymerases with mitochondrial or plastid targeting properties in angiosperms. Yet, according to their conserved exon-intron structures they are representatives of the molecular evolutionary line leading to the RpoT genes of higher land plants.
