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  • 1.
    Bautista, Rocí­o
    et al.
    University of Malaga.
    Villalobos, David, P.
    University of Malaga.
    Diaz-Moreno, Sara M
    University of Malaga.
    Cantón, Francisco, R.
    University of Malaga.
    Cánovas, Francisco, M.
    University of Malaga.
    Gonzalo Claros, M.
    University of Malaga.
    Toward a Pinus pinaster bacterial artificial chromosome library2007In: Annals of Forest Science, ISSN 1286-4560, E-ISSN 1297-966X, Vol. 64, no 8, p. 855-864Article in journal (Refereed)
    Abstract [en]

    Conifers are of great economic and ecological importance, but little is known concerning their genomic organization. This study is an attempt to obtain high-quality high-molecular-weight DNA from Pinus pinaster cotyledons and the construction of a pine BAC library. The preparation incorporates modifications like low centrifugation speeds, increase of EDTA concentration for plug maintenance, use of DNase inhibitors to reduce DNA degradation, use of polyvinylpyrrolidone and ascorbate to avoid secondary metabolites, and a brief electrophoresis of the plugs prior to their use. A total of 72 192 clones with an average insert size of 107 kb, which represents an equivalent of 11X pine haploid genomes, were obtained. The proportions of clones lacking inserts or containing chloroplast DNA are both approximately 1.6%. The library was screened with cDNA probes for seven genes, and two clones containing Fd-GOGAT sequences were found, one of them seemingly functional. Ongoing projects aimed at constructing a pinebacterial artificial chromosome library may benefit from the methods described here.

  • 2.
    Canales, Javier
    et al.
    University of Malaga.
    Ávila, Concepción
    University of Malaga.
    Cantón, Francisco
    University of Malaga.
    Pacheco-Villalobos, David
    University of Malaga.
    Díaz-Moreno, Sara
    University of Malaga.
    Ariza, David
    University of Cordoba.
    Molina-Rueda, Juan
    University of Malaga.
    Navarro-Cerrillo, Rafael
    University of Cordoba.
    Claros, M.
    University of Malaga.
    Cánovas, Francisco
    University of Malaga.
    Gene expression profiling in the stem of young maritime pine trees: detection of ammonium stress-responsive genes in the apex2011In: Trees, ISSN 0931-1890, E-ISSN 1432-2285, Vol. 26, no 2, p. 609-619Article in journal (Refereed)
    Abstract [en]

    The shoots of young conifer trees represent an interesting model to study the development and growth of conifers from meristematic cells in the shoot apex to differentiated tissues at the shoot base. In this work, microarray analysis was used to monitor contrasting patterns of gene expression between the apex and the base ofmaritime pine shoots. A group of differentially expressed genes were selected and validated by examining their relative expression levels in different sections along thestem, from the top to the bottom. After validation of the microarray data, additional geneexpression analyses were also performed in the shoots of young maritime pine treesexposed to different levels of ammonium nutrition. Our results show that the apex ofmaritime pine trees is extremely sensitive to conditions of ammonium excess or deficiency, as revealed by the observed changes in the expression of stress-responsivegenes. This new knowledge may be used to precocious detection of early symptoms of nitrogen nutritional stresses, thereby increasing survival and growth rates of young treesin managed forests. 

  • 3.
    Cañas, Rafael A
    et al.
    University of Malaga.
    Villalobos, David P
    University of Malaga.
    Díaz-Moreno, Sara
    University of Malaga.
    Cánovas, Francisco M
    University of Malaga.
    Cantón, Francisco R
    University of Malaga.
    Molecular and functional analyses support a role of Ornithine-{delta}-aminotransferase in the provision of glutamate for glutamine biosynthesis during pine germination2008In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 148, no 1, p. 77-88Article in journal (Refereed)
    Abstract [en]

    We report the molecular characterization and functional analysis of a gene (PsdeltaOAT) from Scots pine (Pinus sylvestris) encoding Orn-delta-aminotransferase (delta-OAT; EC 2.6.1.13), an enzyme of arginine metabolism. The deduced amino acid sequence contains a putative N-terminal signal peptide for mitochondrial targeting. The polypeptide is similar to other delta-OATs from plants, yeast, and mammals and encoded by a single-copy gene in pine. PsdeltaOAT encodes a functional delta-OAT as determined by expression of the recombinant protein in Escherichia coli and analysis of the active enzyme. The expression of PsdeltaOAT was undetectable in the embryo, but highly induced at early stages of germination and seedling development in all different organs. Transcript levels decreased in later developmental stages, although an increase was observed in lignified stems of 90-d-old plants. An increase of delta-OAT activity was observed in germinating embryos and seedlings and appears to mirror the observed alterations in PsdeltaOAT transcript levels. Similar expression patterns were also observed for genes encoding arginase and isocitrate dehydrogenase. Transcripts of PsdeltaOAT and the arginase gene were found widely distributed in different cell types of pine organs. Consistent with these results a metabolic pathway is proposed for the nitrogen flow from the megagametophyte to the developing seedling, which is also supported by the relative abundance of free amino acids in embryos and seedlings. Taken together, our data support that delta-OAT plays an important role in this process providing glutamate for glutamine biosynthesis during early pine growth.

  • 4. Cho, Sung Hyun
    et al.
    Purushotham, Pallinti
    Fang, Chao
    Maranas, Cassandra
    Diaz-Moreno, Sara M
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Zimmer, Jochen
    Kumar, Manish
    Nixon, B. Tracy
    Synthesis and Self-Assembly of Cellulose Microfibrils from Reconstituted Cellulose Synthase2017In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 175, no 1, p. 146-156Article in journal (Refereed)
    Abstract [en]

    Cellulose, the major component of plant cell walls, can be converted to bioethanol and is thus highly studied. In plants, cellulose is produced by cellulose synthase, a processive family-2 glycosyltransferase. In plant cell walls, individual beta-1,4-glucan chains polymerized by CesA are assembled into microfibrils that are frequently bundled into macrofibrils. An in vitro system in which cellulose is synthesized and assembled into fibrils would facilitate detailed study of this process. Here, we report the heterologous expression and partial purification of His-tagged CesA5 from Physcomitrella patens. Immunoblot analysis and mass spectrometry confirmed enrichment of PpCesA5. The recombinant protein was functional when reconstituted into liposomes made from yeast total lipid extract. The functional studies included incorporation of radiolabeled Glc, linkage analysis, and imaging of cellulose microfibril formation using transmission electron microscopy. Several microfibrils were observed either inside or on the outer surface of proteoliposomes, and strikingly, several thinner fibrils formed ordered bundles that either covered the surfaces of proteoliposomes or were spawned from liposome surfaces. We also report this arrangement of fibrils made by proteoliposomes bearing CesA8 from hybrid aspen. These observations describe minimal systems of membrane-reconstituted CesAs that polymerize beta-1,4-glucan chains that coalesce to form microfibrils and higher-ordered macrofibrils. How these micro-and macrofibrils relate to those found in primary and secondary plant cell walls is uncertain, but their presence enables further study of the mechanisms that govern the formation and assembly of fibrillar cellulosic structures and cell wall composites during or after the polymerization process controlled by CesA proteins.

  • 5.
    Fernández-Pozo, Noé
    et al.
    University of Malaga.
    Canales, Javier
    University of Malaga.
    Guerrero-Fernández, Darío
    University of Malaga.
    Villalobos, David P
    University of Malaga.
    Díaz-Moreno, Sara M
    University of Malaga.
    Bautista, Rocío
    University of Malaga.
    Flores-Monterroso, Arantxa
    University of Malaga.
    Guevara, M Ángeles
    CIFOR-UNIA.
    Perdiguero, Pedro
    Universidad Politecnica de Madrid.
    Collada, Carmen
    Universidad Politecnica de Madrid.
    Cervera, M Teresa
    Universidad Politecnica de Madrid.
    Soto, Alvaro
    Universidad Politecnica de Madrid.
    Ordás, Ricardo
    University of Oviedo.
    Cantón, Francisco R
    University of Malaga.
    Avila, Concepción
    University of Malaga.
    Cánovas, Francisco M
    University of Malaga.
    Claros, M Gonzalo
    University of Malaga.
    EuroPineDB: a high-coverage web database for maritime pine transcriptome2011In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 12, p. 366-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Pinus pinaster is an economically and ecologically important species that is becoming a woody gymnosperm model. Its enormous genome size makes whole-genome sequencing approaches are hard to apply. Therefore, the expressed portion of the genome has to be characterised and the results and annotations have to be stored in dedicated databases.

    DESCRIPTION: EuroPineDB is the largest sequence collection available for a single pine species, Pinus pinaster (maritime pine), since it comprises 951 641 raw sequence reads obtained from non-normalised cDNA libraries and high-throughput sequencing from adult (xylem, phloem, roots, stem, needles, cones, strobili) and embryonic (germinated embryos, buds, callus) maritime pine tissues. Using open-source tools, sequences were optimally pre-processed, assembled, and extensively annotated (GO, EC and KEGG terms, descriptions, SNPs, SSRs, ORFs and InterPro codes). As a result, a 10.5× P. pinaster genome was covered and assembled in 55 322 UniGenes. A total of 32 919 (59.5%) of P. pinaster UniGenes were annotated with at least one description, revealing at least 18 466 different genes. The complete database, which is designed to be scalable, maintainable, and expandable, is freely available at: http://www.scbi.uma.es/pindb/. It can be retrieved by gene libraries, pine species, annotations, UniGenes and microarrays (i.e., the sequences are distributed in two-colour microarrays; this is the only conifer database that provides this information) and will be periodically updated. Small assemblies can be viewed using a dedicated visualisation tool that connects them with SNPs. Any sequence or annotation set shown on-screen can be downloaded. Retrieval mechanisms for sequences and gene annotations are provided.

    CONCLUSIONS: The EuroPineDB with its integrated information can be used to reveal new knowledge, offers an easy-to-use collection of information to directly support experimental work (including microarray hybridisation), and provides deeper knowledge on the maritime pine transcriptome.

  • 6. Jiang, Rays H. Y.
    et al.
    de Bruijn, Irene
    Haas, Brian J.
    Belmonte, Rodrigo
    Loebach, Lars
    Christie, James
    van den Ackerveken, Guido
    Bottin, Arnaud
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Diaz-Moreno, Sara M.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Dumas, Bernard
    Fan, Lin
    Gaulin, Elodie
    Govers, Francine
    Grenville-Briggs, Laura J.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Horner, Neil R.
    Levin, Joshua Z.
    Mammella, Marco
    Meijer, Harold J. G.
    Morris, Paul
    Nusbaum, Chad
    Oome, Stan
    Phillips, Andrew J.
    van Rooyen, David
    Rzeszutek, Elzbieta
    KTH, School of Biotechnology (BIO), Glycoscience.
    Saraiva, Marcia
    Secombes, Chris J.
    Seidl, Michael F.
    Snel, Berend
    Stassen, Joost H. M.
    Sykes, Sean
    Tripathy, Sucheta
    van den Berg, Herbert
    Vega-Arreguin, Julio C.
    Wawra, Stephan
    Young, Sarah K.
    Zeng, Qiandong
    Dieguez-Uribeondo, Javier
    Russ, Carsten
    Tyler, Brett M.
    van West, Pieter
    Distinctive Expansion of Potential Virulence Genes in the Genome of the Oomycete Fish Pathogen Saprolegnia parasitica2013In: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 9, no 6, p. e1003272-Article in journal (Refereed)
    Abstract [en]

    Oomycetes in the class Saprolegniomycetidae of the Eukaryotic kingdom Stramenopila have evolved as severe pathogens of amphibians, crustaceans, fish and insects, resulting in major losses in aquaculture and damage to aquatic ecosystems. We have sequenced the 63 Mb genome of the fresh water fish pathogen, Saprolegnia parasitica. Approximately 1/3 of the assembled genome exhibits loss of heterozygosity, indicating an efficient mechanism for revealing new variation. Comparison of S. parasitica with plant pathogenic oomycetes suggests that during evolution the host cellular environment has driven distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens. S. parasitica possesses one of the largest repertoires of proteases (270) among eukaryotes that are deployed in waves at different points during infection as determined from RNA-Seq data. In contrast, despite being capable of living saprotrophically, parasitism has led to loss of inorganic nitrogen and sulfur assimilation pathways, strikingly similar to losses in obligate plant pathogenic oomycetes and fungi. The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S. parasitica, including those encoding RXLR effectors, Crinkler's, and Necrosis Inducing-Like Proteins (NLP). S. parasitica also has a very large kinome of 543 kinases, 10% of which is induced upon infection. Moreover, S. parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes, including disintegrins and galactose-binding lectins, whose expression and evolutionary origins implicate horizontal gene transfer in the evolution of animal pathogenesis in S. parasitica.

  • 7.
    Lara, Antonio J.
    et al.
    University of Malaga.
    Perez-Trabado, Guillermo
    University of Malaga.
    Villalobos, David P.
    University of Malaga.
    Diaz-Moreno, Sara M.
    University of Malaga.
    Canton, Francisco R.
    University of Malaga.
    Claros, M. Gonzalo
    University of Malaga.
    A Web Tool to Discover Full-Length Sequences: Full-Lengther2007In: Innovations in Hybrid Intelligent Systems / [ed] Emilio Corchado, Juan M Corchado, Ajith Abraham, Springer Berlin/Heidelberg, 2007, p. 361-368Chapter in book (Refereed)
  • 8. Pacheco-Villalobos, David
    et al.
    Diaz-Moreno, Sara M.
    KTH, School of Biotechnology (BIO), Glycoscience.
    van der Schuren, Alja
    Tamaki, Takayuki
    Kang, Yeon Hee
    Gujas, Bojan
    Novak, Ondrej
    Jaspert, Nina
    Li, Zhenni
    Wolf, Sebastian
    Oecking, Claudia
    Ljung, Karin
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Hardtke, Christian S.
    The Effects of High Steady State Auxin Levels on Root Cell Elongation in Brachypodium2016In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 28, no 5, p. 1009-1024Article in journal (Refereed)
    Abstract [en]

    The long-standing Acid Growth Theory of plant cell elongation posits that auxin promotes cell elongation by stimulating cell wall acidification and thus expansin action. To date, the paucity of pertinent genetic materials has precluded thorough analysis of the importance of this concept in roots. The recent isolation of mutants of the model grass species Brachypodium distachyon with dramatically enhanced root cell elongation due to increased cellular auxin levels has allowed us to address this question. We found that the primary transcriptomic effect associated with elevated steady state auxin concentration in elongating root cells is upregulation of cell wall remodeling factors, notably expansins, while plant hormone signaling pathways maintain remarkable homeostasis. These changes are specifically accompanied by reduced cell wall arabinogalactan complexity but not by increased proton excretion. On the contrary, we observed a tendency for decreased rather than increased proton extrusion from root elongation zones with higher cellular auxin levels. Moreover, similar to Brachypodium, root cell elongation is, in general, robustly buffered against external pH fluctuation in Arabidopsis thaliana. However, forced acidification through artificial proton pump activation inhibits root cell elongation. Thus, the interplay between auxin, proton pump activation, and expansin action may be more flexible in roots than in shoots.

  • 9.
    Pallinti, Purushotham Naidu
    et al.
    Univ Virginia, Charlottesville, VA USA..
    Cho, Sung Hyun
    Penn State, Biochem & Mol Biol, University Pk, PA USA..
    Diaz-Moreno, Sara M.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Kumar, Manish
    Penn State, Dept Chem Engn, University Pk, PA USA..
    Nixon, B. Tracy
    Penn State, Biochem & Mol Biol, University Pk, PA USA..
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. Royal Inst Technol, Div Glycosci, Stockholm, Sweden.;Australian Res Council Ctr Excellence Plant Cell, Sch Agr Food & Wine, Urrbrae, SA, Australia..
    Zimmer, Jochen
    Univ Virginia, Charlottesville, VA USA..
    Cellulose microfibril formation in vitro by a single heterologously expressed plant cellulose synthase isoform2017In: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 253Article in journal (Other academic)
  • 10. Park, Eunsook
    et al.
    Diaz-Moreno, Sara M.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Davis, Destiny J.
    Wilkop, Thomas E.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Drakakaki, Georgia
    Endosidin 7 Specifically Arrests Late Cytokinesis and Inhibits Callose Biosynthesis, Revealing Distinct Trafficking Events during Cell Plate Maturation2014In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 165, no 3, p. 1019-1034Article in journal (Refereed)
    Abstract [en]

    Although cytokinesis is vital for plant growth and development, our mechanistic understanding of the highly regulated membrane and cargo transport mechanisms in relation to polysaccharide deposition during this process is limited. Here, we present an in-depth characterization of the small molecule endosidin 7 (ES7) inhibiting callose synthase activity and arresting late cytokinesis both in vitro and in vivo in Arabidopsis (Arabidopsis thaliana). ES7 is a specific inhibitor for plant callose deposition during cytokinesis that does not affect endomembrane trafficking during interphase or cytoskeletal organization. The specificity of ES7 was demonstrated (1) by comparing its action with that of known inhibitors such as caffeine, flufenacet, and concanamycin A and (2) across kingdoms with a comparison in yeast. The interplay between cell plate-specific post-Golgi vesicle traffic and callose accumulation was analyzed using ES7, and it revealed unique and temporal contributions of secretory and endosomal vesicles in cell plate maturation. While RABA2A-labeled vesicles, which accumulate at the early stage of cell plate formation, were not affected by ES7, KNOLLE was differentially altered by the small molecule. In addition, the presence of clathrin-coated vesicles in cells containing elevated levels of callose and their reduction under ES7 treatment further support the role of endocytic membrane remodeling in the maturing cell plate while the plate is stabilized by callose. Taken together, these data show the essential role of callose during the late stages of cell plate maturation and establish the temporal relationship between vesicles and regulatory proteins at the cell plate assembly matrix during polysaccharide deposition.

  • 11. Purushotham, P.
    et al.
    Cho, S. H.
    Díaz-Moreno, Sara M
    KTH, School of Biotechnology (BIO), Glycoscience.
    Kumar, M.
    Nixon, B. T.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. Univ Adelaide, Australia.
    Zimmer, J.
    A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro2016In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 40, p. 11360-11365Article in journal (Refereed)
    Abstract [en]

    Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme's N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils.

  • 12. Villalobos, David P.
    et al.
    Díaz-Moreno, Sara M.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Said, El-Sayed S.
    Canas, Rafael A.
    Osuna, Daniel
    Van Kerckhoven, Sonia H. E.
    Bautista, Rocio
    Gonzalo Claros, Manuel
    Canovas, Francisco M.
    Canton, Francisco R.
    Reprogramming of gene expression during compression wood formation in pine: Coordinated modulation of S-adenosylmethionine, lignin and lignan related genes2012In: BMC Plant Biology, ISSN 1471-2229, E-ISSN 1471-2229, Vol. 12, p. 100-Article in journal (Refereed)
    Abstract [en]

    Background: Transcript profiling of differentiating secondary xylem has allowed us to draw a general picture of the genes involved in wood formation. However, our knowledge is still limited about the regulatory mechanisms that coordinate and modulate the different pathways providing substrates during xylogenesis. The development of compression wood in conifers constitutes an exceptional model for these studies. Although differential expression of a few genes in differentiating compression wood compared to normal or opposite wood has been reported, the broad range of features that distinguish this reaction wood suggest that the expression of a larger set of genes would be modified. Results: By combining the construction of different cDNA libraries with microarray analyses we have identified a total of 496 genes in maritime pine (Pinus pinaster, Ait.) that change in expression during differentiation of compression wood (331 up-regulated and 165 down-regulated compared to opposite wood). Samples from different provenances collected in different years and geographic locations were integrated into the analyses to mitigate the effects of multiple sources of variability. This strategy allowed us to define a group of genes that are consistently associated with compression wood formation. Correlating with the deposition of a thicker secondary cell wall that characterizes compression wood development, the expression of a number of genes involved in synthesis of cellulose, hemicellulose, lignin and lignans was up-regulated. Further analysis of a set of these genes involved in S-adenosylmethionine metabolism, ammonium recycling, and lignin and lignans biosynthesis showed changes in expression levels in parallel to the levels of lignin accumulation in cells undergoing xylogenesis in vivo and in vitro. Conclusions: The comparative transcriptomic analysis reported here have revealed a broad spectrum of coordinated transcriptional modulation of genes involved in biosynthesis of different cell wall polymers associated with within-tree variations in pine wood structure and composition. In particular, we demonstrate the coordinated modulation at transcriptional level of a gene set involved in S-adenosylmethionine synthesis and ammonium assimilation with increased demand for coniferyl alcohol for lignin and lignan synthesis, enabling a better understanding of the metabolic requirements in cells undergoing lignification.

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