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  • 1.
    Aspeborg, Henrik
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bhalerao, Rishikeshi
    Hertzberg, Magnus
    Johansson, Karin
    Johnsson, P.
    Karlsson, Ann
    Sandberg, Göran
    Schrader, Jarmo
    Sundberg, Björn
    Teeri, Tuula
    Trygg, Johan
    Wallbäcks, Lars
    Vegetabile material, plants and a method of producing a plant having altered lignin properties2008Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    The present invention is related to a set of genes, which when modified in plants gives altered lignin properties. The invention provides DNA construct such as a vector useful in the method of the invention. Further, the invention relates to a plant cell or plant progeny of the plants and wood produced by the plants according to the invention Lower lignin levels will result in improved saccharification for bio-refining and ethanol production and improved pulp and paper. Increased lignin levels will utilise lignin properties for energy production. The genes and DNA constructs may be used for the identification of plants having altered lignin characteristics as compared to the wild-type. According to the invention genes and DNA constructs may also be used as candidate genes in marker assisted breeding.

  • 2.
    Aspeborg, Henrik
    et al.
    KTH, School of Biotechnology (BIO).
    Schrader, J.
    Coutinho, P. M.
    Stam, M.
    Kallas, A.
    Djerbi, S.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Denman, S.
    Amini, B.
    Sterky, Fredrik
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Master, E.
    Sandberg, G.
    Mellerowicz, E.
    Sundberg, B.
    Henrissat, B.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Carbohydrate-active enzymes involved in the secondary cell wall biogenesis in hybrid aspen2005In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 137, no 3, p. 983-997Article in journal (Refereed)
    Abstract [en]

    Wood formation is a fundamental biological process with significant economic interest. While lignin biosynthesis is currently relatively well understood, the pathways leading to the synthesis of the key structural carbohydrates in wood fibers remain obscure. We have used a functional genomics approach to identify enzymes involved in carbohydrate biosynthesis and remodeling during xylem development in the hybrid aspen Populus tremula x tremuloides. Microarrays containing cDNA clones from different tissue-specific libraries were hybridized with probes obtained from narrow tissue sections prepared by cryosectioning of the developing xylem. Bioinformatic analyses using the sensitive tools developed for carbohydrate-active enzymes allowed the identification of 25 xylem-specific glycosyltransferases belonging to the Carbohydrate-Active EnZYme families GT2, GT8, GT14, GT31, GT43, GT47, and GT61 and nine glycosidases (or transglycosidases) belonging to the Carbohydrate-Active EnZYme families GH9, GH10, GH16, GH17, GH19, GH28, GH35, and GH51. While no genes encoding either polysaccharide lyases or carbohydrate esterases were found among the secondary wall-specific genes, one putative O-acetyltransferase was identified. These wood-specific enzyme genes constitute a valuable resource for future development of engineered fibers with improved performance in different applications.

  • 3.
    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.

  • 4. Bollhoner, Benjamin
    et al.
    Jokipii-Lukkari, Soile
    Bygdell, Joakim
    Stael, Simon
    Adriasola, Mathilda
    KTH, School of Biotechnology (BIO).
    Muniz, Luis
    Van Breusegem, Frank
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Wingsle, Gunnar
    Tuominen, Hannele
    The function of two type II metacaspases in woody tissues of Populus trees2018In: New Phytologist, ISSN 0028-646X, E-ISSN 1469-8137, Vol. 217, no 4, p. 1551-1565Article in journal (Refereed)
    Abstract [en]

    Metacaspases (MCs) are cysteine proteases that are implicated in programmed cell death of plants. AtMC9 (Arabidopsis thaliana Metacaspase9) is a member of the Arabidopsis MC family that controls the rapid autolysis of the xylem vessel elements, but its downstream targets in xylem remain uncharacterized. PttMC13 and PttMC14 were identified as AtMC9 homologs in hybrid aspen (Populustremulaxtremuloides). A proteomic analysis was conducted in xylem tissues of transgenic hybrid aspen trees which carried either an overexpression or an RNA interference construct for PttMC13 and PttMC14. The proteomic analysis revealed modulation of levels of both previously known targets of metacaspases, such as Tudor staphylococcal nuclease, heat shock proteins and 14-3-3 proteins, as well as novel proteins, such as homologs of the PUTATIVE ASPARTIC PROTEASE3 (PASPA3) and the cysteine protease RD21 by PttMC13 and PttMC14. We identified here the pathways and processes that are modulated by PttMC13 and PttMC14 in xylem tissues. In particular, the results indicate involvement of PttMC13 and/or PttMC14 in downstream proteolytic processes and cell death of xylem elements. This work provides a valuable reference dataset on xylem-specific metacaspase functions for future functional and biochemical analyses.

  • 5. Bygdell, Joakim
    et al.
    Srivastava, Vaibhav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Obudulu, Ogonna
    Srivastava, Manoj K.
    Nilsson, Robert
    Sundberg, Bjorn
    Trygg, Johan
    Mellerowicz, Ewa J.
    Wingsle, Gunnar
    Protein expression in tension wood formation monitored at high tissue resolution in Populus2017In: Journal of Experimental Botany, ISSN 0022-0957, E-ISSN 1460-2431, Vol. 68, no 13, p. 3405-3417Article in journal (Refereed)
    Abstract [en]

    Tension wood (TW) is a specialized tissue with contractile properties that is formed by the vascular cambium in response to gravitational stimuli. We quantitatively analysed the proteomes of Populus tremula cambium and its xylem cell derivatives in stems forming normal wood (NW) and TW to reveal the mechanisms underlying TW formation. Phloem-, cambium-, and wood-forming tissues were sampled by tangential cryosectioning and pooled into nine independent samples. The proteomes of TW and NW samples were similar in the phloem and cambium samples, but diverged early during xylogenesis, demonstrating that reprogramming is an integral part of TW formation. For example, 14-3-3, reactive oxygen species, ribosomal and ATPase complex proteins were found to be up-regulated at early stages of xylem differentiation during TW formation. At later stages of xylem differentiation, proteins involved in the biosynthesis of cellulose and enzymes involved in the biosynthesis of rhamnogalacturonan-I, rhamnogalacturonan-II, arabinogalactan-II and fasciclin-like arabinogalactan proteins were up-regulated in TW. Surprisingly, two isoforms of exostosin family proteins with putative xylan xylosyl transferase function and several lignin biosynthesis proteins were also up-regulated, even though xylan and lignin are known to be less abundant in TW than in NW. These data provided new insight into the processes behind TW formation.

  • 6.
    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. 

  • 7.
    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.

  • 8. 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.

  • 9.
    Choong, Ferdinand X.
    et al.
    Karolinska Inst, Dept Neurosci, Swedish Med Nanosci Ctr, SE-17177 Stockholm, Sweden..
    Back, Marcus
    Linkoping Univ, IFM, Dept Chem, SE-58183 Linkoping, Sweden..
    Schulz, Anette
    Karolinska Inst, Dept Neurosci, Swedish Med Nanosci Ctr, SE-17177 Stockholm, Sweden..
    Nilsson, K. Peter. R.
    Linkoping Univ, IFM, Dept Chem, SE-58183 Linkoping, Sweden..
    Edlund, Ulrica
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Richter-Dahlfors, Agneta
    Karolinska Inst, Dept Neurosci, Swedish Med Nanosci Ctr, SE-17177 Stockholm, Sweden..
    Stereochemical identification of glucans by oligothiophenes enables cellulose anatomical mapping in plant tissues2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 3108Article in journal (Refereed)
    Abstract [en]

    Efficient use of plant-derived materials requires enabling technologies for non-disruptive composition analysis. The ability to identify and spatially locate polysaccharides in native plant tissues is difficult but essential. Here, we develop an optical method for cellulose identification using the structure-responsive, heptameric oligothiophene h-FTAA as molecular fluorophore. Spectrophotometric analysis of h-FTAA interacting with closely related glucans revealed an exceptional specificity for beta-linked glucans. This optical, non-disruptive method for stereochemical differentiation of glycosidic linkages was next used for in situ composition analysis in plants. Multi-laser/multi-detector analysis developed herein revealed spatial localization of cellulose and structural cell wall features such as plasmodesmata and perforated sieve plates of the phloem. Simultaneous imaging of intrinsically fluorescent components revealed the spatial relationship between cell walls and other organelles, such as chloroplasts and lignified annular thickenings of the trachea, with precision at the sub-cellular scale. Our non-destructive method for cellulose identification lays the foundation for the emergence of anatomical maps of the chemical constituents in plant tissues. This rapid and versatile method will likely benefit the plant science research fields and may serve the biorefinery industry as reporter for feedstock optimization as well as in-line monitoring of cellulose reactions during standard operations.

  • 10. Cifuentes, Carolina
    et al.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Emons, Anne Mie C.
    Biosynthesis of Callose and Cellulose by Detergent Extracts of Tobacco Cell Membranes and Quantification of the Polymers Synthesized in vitro2010In: J INTEGR PLANT BIOL, ISSN 1672-9072, Vol. 52, no 2, p. 221-233Article in journal (Refereed)
    Abstract [en]

    The conditions that favor the in vitro synthesis of cellulose from tobacco BY-2 cell extracts were determined. The procedure leading to the highest yield of cellulose consisted of incubating digitonin extracts of membranes from 11-day-old tobacco BY-2 cells in the presence of 1 mM UDP-glucose, 8 mM Ca2+ and 8 mM Mg2+. Under these conditions, up to nearly 40% of the polysaccharides synthesized in vitro corresponded to cellulose, the other polymer synthesized being callose. Transmission electron microscopy analysis revealed the occurrence of two types of structures in the synthetic reactions. The first type consisted of small aggregates with a diameter between 3 and 5 nm that associated to form fibrillar strings of a maximum length of 400 nm. These structures were sensitive to the acetic/nitric acid treatment of Updegraff and corresponded to callose. The second type of structures was resistant to the Updegraff reagent and corresponded to straight cellulose microfibrils of 2-3 nm in diameter and 200 nm to up to 5 mu m in length. In vitro reactions performed on electron microscopy grids indicated that the minimal rate of microfibril elongation in vitro is 120 nm/min. Measurements of retardance by liquid crystal polarization microscopy as a function of time showed that small groups of microfibrils increased in retardance by up to 0.047 nm/min per pixel, confirming the formation of organized structures.

  • 11.
    Clergeot, Pierre-Henri
    et al.
    Stockholm University.
    Rivetti, Claudia
    Stockholm University.
    Hamiduzzaman, M. Md.
    Stockholm University.
    Ekengren, Sophia
    Stockholm University.
    The corky root rot pathogen, Pyrenochaeta lycopersici manipulates tomato roots with molecules secreted early during their interaction2012In: Acta Agriculturae Scandinavica - Section B, ISSN 0906-4710, E-ISSN 1651-1913, Vol. 62, no 4, p. 300-310Article in journal (Refereed)
    Abstract [en]

    Corky root rot is a ubiquitous soil-borne disease of tomato caused by the pathogen Pyrenochaeta lycopersici. This filamentous fungus is found on the roots of many crops and can persist in the soil up to 15 years as microsclerotia. High prevalence of corky root rot can be partly explained by the endurance and the broad host range of P. lycopersici, but how this fungus can gain access to host roots is still poorly understood, as its competitive saprophytic ability is very low. We have combined microscopy and reporter gene techniques to investigate the tomato-P. lycopersici interaction in vitro, and discovered the pathogen secretes molecules that change the direction of root growth and induce cell necrosis specifically in the apical part of the root of tomato ( apex, elongation zone and beginning of the root hair zone). Moreover, we found that the fungus preferentially infects immature root cells that are sensitive to these secreted fungal molecules, whereas infection is blocked in mature and insensitive parts of the root. Our study sheds light on novel and important features of the biology of this pathogen, which could contribute to its fitness in the rhizosphere.

  • 12.
    Du, Xueyu
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology.
    Gellerstedt, Göran
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology.
    Li, Jiebing
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology.
    Universal fractionation of lignin-carbohydrate complexes (LCCs) from lignocellulosic biomass: an example using spruce wood2013In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 74, no 2, p. 328-338Article in journal (Refereed)
    Abstract [en]

    It is of both theoretical and practical importance to develop a universally applicable approach for the fractionation and sensitive lignin characterization of lignin-carbohydrate complexes (LCCs) from all types of lignocellulosic biomass, both natively and after various types of processing. In the present study, a previously reported fractionation approach that is applicable for eucalyptus (hardwood) and flax (non-wood) was further improved by introducing an additional step of barium hydroxide precipitation to isolate the mannan-enriched LCC (glucomannan-lignin, GML), in order to suit softwood species as well. Spruce wood was used as the softwood sample. As indicated by the recovery yield and composition analysis, all of the lignin was recovered in three LCC fractions: a glucan-enriched fraction (glucan-lignin, GL), a mannan-enriched fraction (GML) and a xylan-enriched fraction (xylan-lignin, XL). All of the LCCs had high molecular masses and were insoluble or barely soluble in a dioxane/water solution. Carbohydrate and lignin signals were observed in H-1 NMR, C-13 CP-MAS NMR and normal- or high-sensitivity 2D HSQC NMR analyses. The carbohydrate and lignin constituents in each LCC fraction are therefore believed to be chemically bonded rather than physically mixed with one another. The three LCC fractions were found to be distinctly different from each other in terms of their lignin structures, as revealed by highly sensitive analyses by thioacidolysis-GC, thioacidolysis-SEC and pyrolysis-GC.

  • 13. Escudero, Viviana
    et al.
    Jorda, Lucia
    Sopena-Torres, Sara
    Melida, Hugo
    Miedes, Eva
    Munoz-Barrios, Antonio
    Swami, Sanjay
    Alexander, Danny
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Sanchez-Vallet, Andrea
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Jones, Alan M.
    Molina, Antonio
    Alteration of cell wall xylan acetylation triggers defense responses that counterbalance the immune deficiencies of plants impaired in the beta-subunit of the heterotrimeric G-protein2017In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 92, no 3, p. 386-399Article in journal (Refereed)
    Abstract [en]

    Arabidopsis heterotrimeric G-protein complex modulates pathogen-associated molecular pattern-triggered immunity (PTI) and disease resistance responses to different types of pathogens. It also plays a role in plant cell wall integrity as mutants impaired in the G- (agb1-2) or G-subunits have an altered wall composition compared with wild-type plants. Here we performed a mutant screen to identify suppressors of agb1-2 (sgb) that restore susceptibility to pathogens to wild-type levels. Out of the four sgb mutants (sgb10-sgb13) identified, sgb11 is a new mutant allele of ESKIMO1 (ESK1), which encodes a plant-specific polysaccharide O-acetyltransferase involved in xylan acetylation. Null alleles (sgb11/esk1-7) of ESK1 restore to wild-type levels the enhanced susceptibility of agb1-2 to the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM), but not to the bacterium Pseudomonas syringae pv. tomato DC3000 or to the oomycete Hyaloperonospora arabidopsidis. The enhanced resistance to PcBMM of the agb1-2 esk1-7 double mutant was not the result of the re-activation of deficient PTI responses in agb1-2. Alteration of cell wall xylan acetylation caused by ESK1 impairment was accompanied by an enhanced accumulation of abscisic acid, the constitutive expression of genes encoding antibiotic peptides and enzymes involved in the biosynthesis of tryptophan-derived metabolites, and the accumulation of disease resistance-related secondary metabolites and different osmolites. These esk1-mediated responses counterbalance the defective PTI and PcBMM susceptibility of agb1-2 plants, and explain the enhanced drought resistance of esk1 plants. These results suggest that a deficient PTI-mediated resistance is partially compensated by the activation of specific cell-wall-triggered immune responses. Significance Statement The plant heterotrimeric G protein complex is an essential component of Pathogen Associated Molecular Pattern-triggered immunity (PTI) and of plant disease resistance to several types of pathogens. We found that modification of the degree of xylan acetylation in plant cell walls activates PTI-independent resistance responses that counterbalance the hypersusceptibility to particular pathogens of plants lacking the heterotrimeric G subunit. These data demonstrate that immune deficient response can be partially compensated by the activation of cell wall-triggered immunity that confers specific disease resistance.

  • 14.
    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.

  • 15. Geisler-Lee, J.
    et al.
    Geisler, M.
    Coutinho, P. M.
    Segerman, B.
    Nishikubo, N.
    Takahashi, J.
    Aspeborg, H.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Djerbi, S.
    Master, E.
    Andersson-Gunneras, S.
    Sundberg, B.
    Karpinski, S.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Kleczkowski, L. A.
    Henrissat, B.
    Mellerowicz, E. J.
    Poplar carbohydrate-active enzymes. Gene identification and expression analyses2006In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 140, no 3, p. 946-962Article in journal (Refereed)
    Abstract [en]

    Over 1,600 genes encoding carbohydrate-active enzymes (CAZymes) in the Populus trichocarpa (Torr.&Gray) genome were identified based on sequence homology, annotated, and grouped into families of glycosyltransferases, glycoside hydrolases, carbohydrate esterases, polysaccharide lyases, and expansins. Poplar ( Populus spp.) had approximately 1.6 times more CAZyme genes than Arabidopsis ( Arabidopsis thaliana). Whereas most families were proportionally increased, xylan and pectin-related families were underrepresented and the GT1 family of secondary metabolite-glycosylating enzymes was overrepresented in poplar. CAZyme gene expression in poplar was analyzed using a collection of 100,000 expressed sequence tags from 17 different tissues and compared to microarray data for poplar and Arabidopsis. Expression of genes involved in pectin and hemicellulose metabolism was detected in all tissues, indicating a constant maintenance of transcripts encoding enzymes remodeling the cell wall matrix. The most abundant transcripts encoded sucrose synthases that were specifically expressed in wood-forming tissues along with cellulose synthase and homologs of KORRIGAN and ELP1. Woody tissues were the richest source of various other CAZyme transcripts, demonstrating the importance of this group of enzymes for xylogenesis. In contrast, there was little expression of genes related to starch metabolism during wood formation, consistent with the preferential flux of carbon to cell wall biosynthesis. Seasonally dormant meristems of poplar showed a high prevalence of transcripts related to starch metabolism and surprisingly retained transcripts of some cell wall synthesis enzymes. The data showed profound changes in CAZyme transcriptomes in different poplar tissues and pointed to some key differences in CAZyme genes and their regulation between herbaceous and woody plants.

  • 16. Gerttula, S.
    et al.
    Zinkgraf, M.
    Muday, G. K.
    Lewis, D. R.
    Ibatullin, Farid M.
    KTH, School of Biotechnology (BIO), Glycoscience. National Research Center Kurchatov Institute, Russian Federation.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience. University of British Columbia, Canada.
    Hart, F.
    Mansfield, S. D.
    Filkov, V.
    Groover, A.
    Transcriptional and hormonal regulation of gravitropism of woody stems in populus2015In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 27, no 10, p. 2800-2813Article in journal (Refereed)
    Abstract [en]

    Angiosperm trees reorient their woody stems by asymmetrically producing a specialized xylem tissue, tension wood, which exerts a strong contractile force resulting in negative gravitropism of the stem. Here, we show, in Populus trees, that initial gravity perception and response occurs in specialized cells through sedimentation of starch-filled amyloplasts and relocalization of the auxin transport protein, PIN3. Gibberellic acid treatment stimulates the rate of tension wood formation and gravibending and enhances tissue-specific expression of an auxin-responsive reporter. Gravibending, maturation of contractile fibers, and gibberellic acid (GA) stimulation of tension wood formation are all sensitive to transcript levels of the Class I KNOX homeodomain transcription factor-encoding gene ARBORKNOX2 (ARK2). We generated genome-wide transcriptomes for trees in which gene expression was perturbed by gravistimulation, GA treatment, and modulation of ARK2 expression. These data were employed in computational analyses to model the transcriptional networks underlying wood formation, including identification and dissection of gene coexpression modules associated with wood phenotypes, GA response, and ARK2 binding to genes within modules. We propose a model for gravitropism in the woody stem in which the peripheral location of PIN3-expressing cells relative to the cambium results in auxin transport toward the cambium in the top of the stem, triggering tension wood formation, while transport away from the cambium in the bottom of the stem triggers opposite wood formation.

  • 17. Greger, M.
    et al.
    Bergqvist, C.
    Sandhi, Arifin
    KTH, School of Architecture and the Built Environment (ABE), Sustainable development, Environmental science and Engineering, Land and Water Resources Engineering. Stockholm University, Sweden.
    Landberg, T.
    Influence of silicon on arsenic uptake and toxicity in lettuce2015In: Journal of Applied Botany and Food Quality / Angewandte Botanik, ISSN 1613-9216, E-ISSN 1439-040X, Vol. 88, p. 234-240Article in journal (Refereed)
    Abstract [en]

    Lettuce grown in soil is found to contain high concentrations of arsenic (As). This paper investigates the uptake and speciation of As in lettuce as well as the influence of silicon (Si) on As uptake, since Si may decrease it. Lettuce plants were cultivated in nutrient solution containing arsenite or arsenate with or without silicate. The uptake and distribution of As between roots and shoots, As accu-mulation in cell walls, As speciation, and toxic effects on growth were analysed. Results indicate that arsenite was more toxic to lettuce than was arsenate. Silicate decreased arsenate toxicity but had little effect on arsenite toxicity. In contrast, Si decreased arsenite uptake more than arsenate uptake. The concentration of arsenate was higher than that of arsenite in the plants independent of the As species added. When arsenate was added, the As concentration in shoots was half of that in the roots and this distribution did not change with Si addition. When arsenite was added, approximately 10% of As was found in the shoots and 90% in the roots; this pattern changed in the presence of Si, and As became evenly distributed in the plant. In both roots and shoots, approximately 40% of the As was found in the cell wall fraction; when arsenite was added, the presence of Si increased this fraction to 47%, but only in the shoots. The extraction efficiency when analysing the As species was lower in shoots than in roots, especially in the presence of arsenite and Si. The opposite was found for As concentration in pellets after extraction. This indicated variation in the binding strength of arsenite and arsenate between roots and shoots and between Si-and non-Si-treated plants.

  • 18. Guerriero, Gea
    et al.
    Hausman, Jean-Francois
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    WD4O-Repeat Proteins in Plant Cell Wall Formation: Current Evidence and Research Prospects2015In: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462X, Vol. 6, article id 1112Article in journal (Refereed)
    Abstract [en]

    The metabolic complexity of living organisms relies on supramolecular protein structures which ensure vital processes, such as signal transduction, transcription, translation and cell wall synthesis. In eukaryotes WD40-repeat (WDR) proteins often function as molecular "hubs" mediating supramolecular interactions. WDR proteins may display a variety of interacting partners and participate in the assembly of complexes involved in distinct cellular functions. In plants, the formation of lignocellulosic biomass involves extensive synthesis of cell wall polysaccharides, a process that requires the assembly of large transmembrane enzyme complexes, intensive vesicle trafficking, interactions with the cytoskeleton, and coordinated gene expression. Because of their function as supramolecular hubs, WDR proteins could participate in each or any of these steps, although to date only few WDR proteins have been linked to the cell wall by experimental evidence. Nevertheless, several potential cell wall-related WDR proteins were recently identified using in silico approaches, such as analyses of co-expression, interactome and conserved gene neighborhood. Notably, some WDR genes are frequently genomic neighbors of genes coding for GT2-family polysaccharide synthases in eukaryotes, and this WDR-GT2 collinear microsynteny is detected in diverse taxa. In angiosperms, two WDR genes are collinear to cellulose synthase genes, CesAs, whereas in ascomycetous fungi several WDR genes are adjacent to chitin synthase genes, chs. In this Perspective we summarize and discuss experimental and in silico studies on the possible involvement of WDR proteins in plant cell wall formation. The prospects of biotechnological engineering for enhanced biomass production are discussed.

  • 19.
    Henriksson, Maria
    KTH, School of Biotechnology (BIO).
    Production and engineering of a xyloglucan endo-transglycosylase from Populus tremula x tremuloides2007Licentiate thesis, comprehensive summary (Other scientific)
    Abstract [en]

    The aim of this work was to develop a production process for the enzyme xyloglucan endo-transglycosylase from Populus tremula x tremuloides (PttXET16-34). The natural transglycosylating activity of this enzyme has previously been employed in a XET-Technology. This chemo enzymatic method is useful for biomimetic modification of cellulose surfaces and holds great potential for industrial applications. Thus, it requires that the XET-enzyme can be produced in larger scale.

    This work also shows how the wildtype PttXET16-34 was modified into a glycosynthase. By mutation of the catalytic nucleophile into an alanine, glycine or serine residue, enzymes capable of synthesising defined xyloglucan fragments were obtained. These defined compounds are very valuable for further detailed studies of xyloglucan active-enzymes, but are also useful in molecular studies of the structurally important xyloglucan-cellulose interaction.

    A heterologous production system for PttXET16-34 was previously developed in the methylotrophic yeast Pichia pastoris. A methanol-limited fed-batch process was also previously established, but the yield of active XET was low due to proteolysis problems and low productivity. Therefore, two alternative fed-batch techniques were investigated for the production of PttXET16-34: a temperature-limited fed-batch (TLFB) and an oxygen-limited high-pressure fed-batch (OLHPFB).

    For the initial recovery of XET after the fermentation process, two different downstream processes were investigated: expanded bed adsorption (EBA) and cross-flow filtration (CFF).

  • 20.
    Henriksson, Maria
    et al.
    KTH, School of Biotechnology (BIO).
    Teeri, Tuula
    KTH, School of Biotechnology (BIO).
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO).
    Jahic, Mehmedalija
    KTH, School of Biotechnology (BIO).
    Improvements of Pichia pastoris fermentation technique for production of recombinant proteinsManuscript (Other academic)
  • 21. Huang, Fang
    et al.
    Zhang, XP
    Szigyarto, Cristina
    Glaser, Elzbieta
    Import and processing of nuclear-encoded mitochondrial proteins during development of pea plants: Plant Mitochondria from Gene to Function1998Conference paper (Refereed)
  • 22. Just, K.
    et al.
    Arif, U.
    KTH, School of Biotechnology (BIO), Glycoscience. Swedish University of Agricultural Sciences, Sweden.
    Luik, A.
    Kvarnheden, A.
    Monitoring infection of tomato fruit by Tomato yellow leaf curl virus2016In: Plant Pathology, ISSN 0032-0862, E-ISSN 1365-3059Article in journal (Refereed)
    Abstract [en]

    Fruit constitutes a strong sink organ and thus accumulates infecting viruses, but there is limited information about the infection process of viruses in fruit. Tomato yellow leaf curl virus (TYLCV, genus Begomovirus, family Geminiviridae) is one of the most important viruses affecting the production of tomato fruit. Using real-time quantitative PCR, TYLCV was shown to accumulate with increasing titres in early developing tomato fruit tissues from anthesis until 21 days post-anthesis. In situ hybridization demonstrated that TYLCV DNA and transcripts of the coat protein gene localized specifically to the phloem tissue of young fruit as well as sepals and petals. Embryos of developing seeds were also found to be infected. Expression of a host histone H4 gene was used as a marker for S-phase and the gene was also found to be expressed in phloem cells of young tomato fruit. The results indicate that TYLCV is transported to developing tomato fruit, where the virus titre gradually increases because of movement and probably also due to virus replication. In this study, the accumulation and localization of TYLCV in early developing tomato fruit are monitored for the first time.

  • 23.
    Kaewthai, Nomchit
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Harvey, Andrew J.
    Hrmova, Maria
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Glycoscience.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Fincher, Geoffrey B.
    Heterologous expression of diverse barley XTH genes in the yeast Pichia pastoris2010In: PLANT BIOTECHNOLOGY, ISSN 1342-4580, Vol. 27, no 3, p. 251-258Article in journal (Refereed)
    Abstract [en]

    Heterologous expression of plant genes, particularly those encoding carbohydrate-active enzymes such as glycoside hydrolases and glycosyl transferases, continues to be a major hurdle in the functional analysis of plant proteomes. Presently, there are few convenient systems for the production of recombinant plant enzymes in active form and at adequate levels for biochemical and structural characterization. The methylotrophic yeast Pichia pastoris is an attractive expression host due to its ease of manipulation and its capacity to perform post-translational protein modifications, such as N-glycosylation [Daly and Hearn (2005) J Mol Recognit 18: 119-138]. Here, we demonstrate the utility of the P. pastoris SMD1168H/pPICZ-alpha C system for the expression of a range of xyloglucan endo-transglycosylase/hydrolase (XTH) cDNAs from barley (Hordeum vulgare). Although stable transformants were readily obtained by positive selection for vector-induced antibiotic resistance for all of the nine constructs tested, only five isoforms were secreted as soluble proteins into the culture medium, four in active form. Furthermore, production levels of these five isoforms were found to be variable, depending on the transformant, which further underscores the necessity of screening multiple clones for expression of active enzyme. Failure to express certain XTH isoforms in P. pastoris could not be correlated with any conserved gene or protein sequence properties, and this precluded using rational sequence engineering to enhance heterologous expression of the cDNAs. Thus, while significant advances are reported here, systems for the heterologous production of plant proteins require further development.

  • 24.
    Martinez-Abad, Antonio
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Berglund, Jennie
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Toriz, Guillermo
    Gatenholm, Paul
    Henriksson, Gunnar
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Lindström, Mikael
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Wohlert, Jakob
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Vilaplana, Francisco
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Regular Motifs in Xylan Modulate Molecular Flexibility and Interactions with Cellulose Surfaces2017In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 175, no 4, p. 1579-1592Article in journal (Refereed)
    Abstract [en]

    Xylan is tightly associated with cellulose and lignin in secondary plant cell walls, contributing to its rigidity and structural integrity in vascular plants. However, the molecular features and the nanoscale forces that control the interactions among cellulose microfibrils, hemicelluloses, and lignin are still not well understood. Here, we combine comprehensive mass spectrometric glycan sequencing and molecular dynamics simulations to elucidate the substitution pattern in softwood xylans and to investigate the effect of distinct intramolecular motifs on xylan conformation and on the interaction with cellulose surfaces in Norway spruce (Picea abies). We confirm the presence of motifs with evenly spaced glycosyl decorations on the xylan backbone, together with minor motifs with consecutive glucuronation. These domains are differently enriched in xylan fractions extracted by alkali and subcritical water, which indicates their preferential positioning in the secondary plant cell wall ultrastructure. The flexibility of the 3-fold screw conformation of xylan in solution is enhanced by the presence of arabinofuranosyl decorations. Additionally, molecular dynamic simulations suggest that the glycosyl substitutions in xylan are not only sterically tolerated by the cellulose surfaces but that they increase the affinity for cellulose and favor the stabilization of the 2-fold screw conformation. This effect is more significant for the hydrophobic surface compared with the hydrophilic ones, which demonstrates the importance of nonpolar driving forces on the structural integrity of secondary plant cell walls. These novel molecular insights contribute to an improved understanding of the supramolecular architecture of plant secondary cell walls and have fundamental implications for overcoming lignocellulose recalcitrance and for the design of advanced wood-based materials.

  • 25.
    McKee, Lauren
    et al.
    Newcastle University, United Kingdom; University of Georgia, United States.
    Pena, Maria J.
    Rogowski, Artur
    Jackson, Adam
    Lewis, Richard J.
    York, William S.
    Krogh, Kristian B. R. M.
    Vikso-Nielsen, Anders
    Skjot, Michael
    Gilbert, Harry J.
    Marles-Wright, Jon
    Introducing endo-xylanase activity into an exo-acting arabinofuranosidase that targets side chains2012In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 17Article in journal (Refereed)
    Abstract [en]

    The degradation of the plant cell wall by glycoside hydrolases is central to environmentally sustainable industries. The major polysaccharides of the plant cell wall are cellulose and xylan, a highly decorated beta-1,4-xylopyranose polymer. Glycoside hydrolases displaying multiple catalytic functions may simplify the enzymes required to degrade plant cell walls, increasing the industrial potential of these composite structures. Here we test the hypothesis that glycoside hydrolase family 43 (GH43) provides a suitable scaffold for introducing additional catalytic functions into enzymes that target complex structures in the plant cell wall. We report the crystal structure of Humicola insolens AXHd3 (HiAXHd3), a GH43 arabinofuranosidase that hydrolyses O3-linked arabinose of doubly substituted xylans, a feature of the polysaccharide that is recalcitrant to degradation. HiAXHd3 displays an N-terminal five-bladed beta-propeller domain and a C-terminal beta-sandwich domain. The interface between the domains comprises a xylan binding cleft that houses the active site pocket. Substrate specificity is conferred by a shallow arabinose binding pocket adjacent to the deep active site pocket, and through the orientation of the xylan backbone. Modification of the rim of the active site introduces endo-xylanase activity, whereas the resultant enzyme variant, Y166A, retains arabinofuranosidase activity. These data show that the active site of HiAXHd3 is tuned to hydrolyse arabinofuranosyl or xylosyl linkages, and it is the topology of the distal regions of the substrate binding surface that confers specificity. This report demonstrates that GH43 provides a platform for generating bespoke multifunctional enzymes that target industrially significant complex substrates, exemplified by the plant cell wall.

  • 26. Nilsson, Anders K.
    et al.
    Johansson, Oskar N.
    Fahlberg, Per
    Kommuri, Murali
    Topel, Mats
    Bodin, Lovisa J.
    Sikora, Per
    Modarres, Masoomeh
    Ekengren, Sophia
    KTH, School of Biotechnology (BIO), Glycoscience.
    Nguyen, Chi T.
    Farmer, Edward E.
    Olsson, Olof
    Ellerstrom, Mats
    Andersson, Mats X.
    Acylated monogalactosyl diacylglycerol: prevalence in the plant kingdom and identification of an enzyme catalyzing galactolipid head group acylation in Arabidopsis thaliana2015In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 84, no 6, p. 1152-1166Article in journal (Refereed)
    Abstract [en]

    The lipid phase of the thylakoid membrane is mainly composed of the galactolipids mono-and digalactosyl diacylglycerol (MGDG and DGDG, respectively). It has been known since the late 1960s that MGDG can be acylated with a third fatty acid to the galactose head group (acyl-MGDG) in plant leaf homogenates. In certain brassicaceous plants like Arabidopsis thaliana, the acyl-MGDG frequently incorporates oxidized fatty acids in the form of the jasmonic acid precursor 12-oxo-phytodienoic acid (OPDA). In the present study we further investigated the distribution of acylated and OPDA-containing galactolipids in the plant kingdom. While acyl-MGDG was found to be ubiquitous in green tissue of plants ranging from non-vascular plants to angiosperms, OPDA-containing galactolipids were only present in plants from a few genera. A candidate protein responsible for the acyl transfer was identified in Avena sativa (oat) leaf tissue using biochemical fractionation and proteomics. Knockout of the orthologous gene in A. thaliana resulted in an almost total elimination of the ability to form both non-oxidized and OPDA-containing acyl-MGDG. In addition, heterologous expression of the A. thaliana gene in E. coli demonstrated that the protein catalyzed acylation of MGDG. We thus demonstrate that a phylogenetically conserved enzyme is responsible for the accumulation of acyl-MGDG in A. thaliana. The activity of this enzyme in vivo is strongly enhanced by freezing damage and the hypersensitive response.

  • 27. Pawar, Prashant Mohan-Anupama
    et al.
    Ratke, Christine
    Balasubramanian, Vimal K.
    Chong, Sun-Li
    Gandla, Madhavi Latha
    Adriasola, Mathilda
    KTH, School of Biotechnology (BIO).
    Sparrman, Tobias
    Hedenström, Mattias
    Szwaj, Klaudia
    Derba-Maceluch, Marta
    Gaertner, Cyril
    Mouille, Gregory
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Tenkanen, Maija
    Jönsson, Leif J.
    Mellerowicz, Ewa J.
    Downregulation of RWA genes in hybrid aspen affects xylan acetylation and wood saccharification2017In: New Phytologist, ISSN 0028-646X, E-ISSN 1469-8137, Vol. 214, no 4, p. 1491-1505Article in journal (Refereed)
    Abstract [en]

    High acetylation of angiosperm wood hinders its conversion to sugars by glycoside hydrolases, subsequent ethanol fermentation and (hence) its use for biofuel production. We studied the REDUCED WALL ACETYLATION (RWA) gene family of the hardwood model Populus to evaluate its potential for improving saccharification. The family has two clades, AB and CD, containing two genes each. All four genes are expressed in developing wood but only RWA-A and -B are activated by master switches of the secondary cell wall PtNST1 and PtMYB21. Histochemical analysis of promoter:: GUS lines in hybrid aspen (Populus tremula x tremuloides) showed activation of RWA-A and -B promoters in the secondary wall formation zone, while RWA-C and -D promoter activity was diffuse. Ectopic downregulation of either clade reduced wood xylan and xyloglucan acetylation. Suppressing both clades simultaneously using the wood-specific promoter reduced wood acetylation by 25% and decreased acetylation at position 2 of Xylp in the dimethyl sulfoxide-extracted xylan. This did not affect plant growth but decreased xylose and increased glucose contents in the noncellulosic monosaccharide fraction, and increased glucose and xylose yields of wood enzymatic hydrolysis without pretreatment. Both RWA clades regulate wood xylan acetylation in aspen and are promising targets to improve wood saccharification.

  • 28. Roberts, Alison W.
    et al.
    Lahnstein, Jelle
    Hsieh, Yves S. Y.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Xing, Xiaohui
    Yap, Kuok
    Chaves, Arielle M
    Scavuzzo-Duggan, Tess R
    Dimitroff, George
    Lonsdale, Andrew
    Roberts, Eric M.
    Bulone, Vincent
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Fincher, Geoffrey B
    Doblin, Monika Susanne
    Bacic, Antony
    Burton, Rachel A
    Functional Characterization of a Glycosyltransferase from the Moss Physcomitrella patens Involved in the Biosynthesis of a Novel Cell Wall Arabinoglucan2018In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 30, no 6, p. 1293-1308Article in journal (Refereed)
    Abstract [en]

    Mixed-linkage (1,3;1,4)-β-glucan (MLG), an abundant cell wall polysaccharide in the Poaceae, has been detected in ascomycetes, algae, and seedless vascular plants, but not in eudicots. Although MLG has not been reported in bryophytes, a predicted glycosyltransferase from the moss Physcomitrella patens (Pp3c12_24670) is similar to a bona fide ascomycete MLG synthase. We tested whether Pp3c12_24670 encodes an MLG synthase by expressing it in wild tobacco (Nicotiana benthamiana) and testing for release of diagnostic oligosaccharides from the cell walls by either lichenase or (1,4)-β-glucan endohydrolase. Lichenase, an MLG-specific endohydrolase, showed no activity against cell walls from transformed N. benthamiana, but (1,4)-β-glucan endohydrolase released oligosaccharides that were distinct from oligosaccharides released from MLG by this enzyme. Further analysis revealed that these oligosaccharides were derived from a novel unbranched, unsubstituted arabinoglucan (AGlc) polysaccharide. We identified sequences similar to the P. patens AGlc synthase from algae, bryophytes, lycophytes, and monilophytes, raising the possibility that other early divergent plants synthesize AGlc. Similarity of P. patens AGlc synthase to MLG synthases from ascomycetes, but not those from Poaceae, suggests that AGlc and MLG have a common evolutionary history that includes loss in seed plants, followed by a more recent independent origin of MLG within the monocots.

  • 29.
    Sandhi, Arifin
    et al.
    KTH, School of Architecture and the Built Environment (ABE), Sustainable development, Environmental science and Engineering, Land and Water Resources Engineering. KTH, School of Architecture and the Built Environment (ABE), Sustainable development, Environmental science and Engineering, Water and Environmental Engineering. KTH, School of Architecture and the Built Environment (ABE), Sustainable development, Environmental science and Engineering, Sustainability and Environmental Engineering. Department of Ecology, Environment and Plant Sciences, Stockholm University, SE 10691 Stockholm, Sweden.
    Landberg, Tommy
    Department of Ecology, Environment & Plant Sciences, Stockholm University, SE-10691 Stockholm, Sweden.
    Greger, Maria
    Department of Ecology, Environment & Plant Sciences, Stockholm University, SE-10691 Stockholm, Sweden.
    Effect of pH, temperature, and oxygenation on arsenic phytofiltration by aquatic moss (Warnstorfia fluitans)2018In: Journal of Environmental Chemical Engineering, ISSN 2160-6544, E-ISSN 2213-3437, Vol. 6, no 4, p. 3918-3925Article in journal (Refereed)
    Abstract [en]

    Phytofiltration of arsenic (As)-contaminated water could reduce As in irrigation and surface water. In a previous study, we found that the aquatic moss Warnstorfia fluitans efficiently removes arsenic from water contaminated with arsenate and arsenite. This work investigates how factors such as pH, temperature, and oxygenation influence As removal, since these factors vary in the environment. Plants were grown in a medium with 5 or 10 μM arsenite or arsenate and: 1) a pH of 2.5, 6.5, or 9.5; 2) a temperature of 12, 20, or 30 °C; and 3) oxygenation of <2 or 13 mg O2 L−1. Removal of As was monitored over 48–96 h, and the content and speciation of As were analysed in moss plants at the termination of the experiments. Results indicate that As removal was faster in arsenite than arsenate solutions. Arsenic removal from arsenite solution was the fastest, i.e., 80–90% within 2 h, at pH 6.5 and 9.5 and at 20 and 30 °C. At pH 2.5, plants were stressed and the net removal was low throughout the treatment period. Arsenic removal was more efficient at low than high oxygenation levels. Besides this, no As net efflux process was seen in the water system except after 48 h in arsenate-treated medium in high-temperature (30 °C) regimes. Regardless of As species added, usually only arsenite was found in the plants after treatment. Most internal As, i.e., 95% in the arsenate and 85% in the arsenite treatments, was firmly bound to the tissue. The study found that at 20 °C, neutral pH, and low oxygenation, this aquatic moss has great potential for As phytofiltration.

  • 30.
    Sandhi, Arifin
    et al.
    KTH, School of Architecture and the Built Environment (ABE), Sustainable development, Environmental science and Engineering, Land and Water Resources Engineering. Department of Ecology, Environment and Plant Sciences, Stockholm University, SE 10691 Stockholm, Sweden.
    Landberg, Tommy
    Department of Ecology, Environment & Plant Sciences, Stockholm University, SE-10691 Stockholm, Sweden.
    Greger, Maria
    Department of Ecology, Environment & Plant Sciences, Stockholm University, SE-10691 Stockholm, Sweden.
    Phytofiltration of arsenic by aquatic moss (Warnstorfia fluitans)2017In: Environmental Pollution, ISSN 0269-7491, E-ISSN 1873-6424Article in journal (Refereed)
    Abstract [en]

    This work investigates whether aquatic moss (Warnstorfia fluitans) originating from an arsenic (As)-contaminated wetland close to a mine tailings impoundment may be used for phytofiltration of As. The aim was to elucidate the capacity of W. fluitans to remove As from arsenite and arsenate contaminated water, how nutrients affect the As uptake and the proportion of As adsorption and absorption by the moss plant, which consists of dead and living parts.

    Arsenic removal from 0, 1, or 10% Hoagland nutrient solution containing 0–100 μM arsenate was followed over 192 h, and the total As in aquatic moss after treatment was analysed. The uptake and speciation of As in moss cultivated in water containing 10 μM arsenate or arsenite were examined as As uptake in living (absorption + adsorption) and dead (adsorption) plant parts.

    Results indicated that W. fluitans removed up to 82% of As from the water within one hour when 1 μM arsenate was added in the absence of nutrients. The removal time increased with greater nutrient and As concentrations. Up to 100 μM As had no toxic effect on the plant biomass. Both arsenite and arsenate were removed from the solution to similar extents and, independent of the As species added, more arsenate than arsenite was found in the plant. Of the As taken up, over 90% was firmly bound to the tissue, a possible mechanism for resisting high As concentrations. Arsenic was both absorbed and adsorbed by the moss, and twice as much As was found in living parts as in dead moss tissue. This study revealed that W. fluitans has potential to serve as a phytofilter for removing As from As-contaminated water without displaying any toxic effects of the metalloid.

  • 31. Schrick, Kathrin
    et al.
    DeBolt, Seth
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Deciphering the molecular functions of sterols in cellulose biosynthesis2012In: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462X, Vol. 3, p. 84-Article in journal (Refereed)
    Abstract [en]

    Sterols play vital roles in plant growth and development, as components of membranes and as precursors to steroid hormones. Analysis of Arabidopsis mutants indicates that sterol composition is crucial for cellulose biosynthesis. Sterols are widespread in the plasma membrane (PM), suggesting a possible link between sterols and the multimeric cellulose synthase complex. In one possible scenario, molecular interactions in sterol-rich PM microdomains or another form of sterol-dependent membrane scaffolding may be critical for maintaining the correct subcellular localization, structural integrity and/or activity of the cellulose synthase machinery. Another possible link may be through steryl glucosides, which could act as primers for the attachment of glucose monomers during the synthesis of beta - (1 -> 4) glucan chains that form the cellulose microfibrils. This mini-review examines genetic and biochemical data supporting the link between sterols and cellulose biosynthesis in cell wall formation and explores potential approaches to elucidate the mechanism of this association.

  • 32. Sweetlove, Lee J.
    et al.
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fernie, Alisdair R.
    Engineering central metabolism - a grand challenge for plant biologists2017In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 90, no 4, p. 749-763Article in journal (Refereed)
    Abstract [en]

    The goal of increasing crop productivity and nutrient-use efficiency is being addressed by a number of ambitious research projects seeking to re-engineer photosynthetic biochemistry. Many of these projects will require the engineering of substantial changes in fluxes of central metabolism. However, as has been amply demonstrated in simpler systems such as microbes, central metabolism is extremely difficult to rationally engineer. This is because of multiple layers of regulation that operate to maintain metabolic steady state and because of the highly connected nature of central metabolism. In this review we discuss new approaches for metabolic engineering that have the potential to address these problems and dramatically improve the success with which we can rationally engineer central metabolism in plants. In particular, we advocate the adoption of an iterative 'design-build-test-learn' cycle using fast-to-transform model plants as test beds. This approach can be realised by coupling new molecular tools to incorporate multiple transgenes in nuclear and plastid genomes with computational modelling to design the engineering strategy and to understand the metabolic phenotype of the engineered organism. We also envisage that mutagenesis could be used to fine-tune the balance between the endogenous metabolic network and the introduced enzymes. Finally, we emphasise the importance of considering the plant as a whole system and not isolated organs: the greatest increase in crop productivity will be achieved if both source and sink metabolism are engineered.

  • 33. Szigyarto, Cristina
    et al.
    Glaser, Elzbieta
    Membrane-associated ATP-dependent degradation of mitochondrial precursor proteins: Proteolysis in Cell Functions. V K Hopsu-Havu, M Jarvinen, Heidrun Kirschke1997In: / [ed] Hopsu-Havu V K, Järvinen M, Kirschke H, Amsterdam, Netherlands: IOS Press, 1997, p. 298-306Conference paper (Refereed)
  • 34. Szigyarto, Cristina
    et al.
    Salvagnac, Virginie
    Glaser, Elzbieta
    Proteolysis of Newly imported precursor proteins1998In: / [ed] I.M. Mφller, P. Gardeström, K. Glimelius and E. Glaser, 1998, p. 287-290Conference paper (Refereed)
  • 35. Verhertbruggen, Y
    et al.
    Marcus, S.E.
    Haeger, A.
    Verhoef, R.
    Schols, H.A.
    McCleary, B.V.
    McKee, Lauren
    Newcastle University, United Kingdom.
    Gilbert, H.J.
    Knox, J.P.
    Developmental complexity of arabinan polysaccharides and their processing in plant cell walls2009In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 59, no 3Article in journal (Refereed)
    Abstract [en]

    Plant cell walls are constructed from a diversity of polysaccharide components. Molecular probes directed to structural elements of these polymers are required to assay polysaccharide structures in situ, and to determine polymer roles in the context of cell wall biology. Here, we report on the isolation and the characterization of three rat monoclonal antibodies that are directed to 1,5-linked arabinans and related polymers. LM13, LM16 and LM17, together with LM6, constitute a set of antibodies that can detect differing aspects of arabinan structures within cell walls. Each of these antibodies binds strongly to isolated sugar beet arabinan samples in ELISAs. Competitive-inhibition ELISAs indicate the antibodies bind differentially to arabinans with the binding of LM6 and LM17 being effectively inhibited by short oligoarabinosides. LM13 binds preferentially to longer oligoarabinosides, and its binding is highly sensitive to arabinanase action, indicating the recognition of a longer linearized arabinan epitope. In contrast, the binding of LM16 to branched arabinan and to cell walls is increased by arabinofuranosidase action. The presence of all epitopes can be differentially modulated in vitro using glycoside hydrolase family 43 and family 51 arabinofuranosidases. In addition, the LM16 epitope is sensitive to the action of β-galactosidase. Immunofluorescence microscopy indicates that the antibodies can be used to detect epitopes in cell walls, and that the four antibodies reveal complex patterns of epitope occurrence that vary between organs and species, and relate both to the probable processing of arabinan structural elements and the differing mechanical properties of cell walls.

  • 36. Vildova, A.
    et al.
    Poborilova, Z.
    Berglund, T.
    Ohlsson, Anna B.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Lindstrom, A.
    A new sustainable strategy for medicinal plants protection against stress?2014In: Planta Medica, ISSN 0032-0943, E-ISSN 1439-0221, Vol. 80, no 16, p. 1532-1532Article in journal (Refereed)
  • 37.
    Wang, Yang
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Discovery and investigation of glycoside hydrolase family 5 enzymes with potential use in biomass conversion2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Glycoside hydrolases (GHs) cleave glycosidic bonds in glycoconjugates, oligosaccharides and polysaccharides such as cellulose and various hemicelluloses. Mannan is a major group of hemicelluloses. In higher plants, they usually serve as storage carbohydrates in seeds and tubers or as structural polysaccharides cross-linking with cellulose/lignin in cell walls. In industrial fields, this renewable biomass component can be used in various areas such as production of biofuels and health-benefit manno-oligosaccharides; and mannan degrading enzymes, especially mannanases, are important molecular tools for controlling mannan polysaccharides properties in biomass conversion. In this thesis, the evolution, substrate specificity and subfamily classification of the most important GH family, i.e., glycoside hydrolase family 5 (GH5), are presented providing a powerful tool for exploring GH5 enzymes in search for enzymes with interesting properties for sustainable biomass conversion. Additionally, three GH5_7 mannanases from Arabidopsis thaliana (AtMan5-1, AtMan5-2 and AtMan5-6) were investigated in the present study. Bioinformatics tools, heterologous expression, and enzymology were applied in order to reveal the catalytic properties of the target enzymes, increase understanding of plant mannanase evolution, and evaluate their potential use in biomass conversion. This approach revealed: (1) AtMan5-1 exhibits mannan hydrolase/transglycosylase activity (MHT), (2) AtMan5-2 preferably degrades mannans with a glucomannan backbone, and (3) AtMan5-6 is a relatively thermotolerant enzyme showing high catalytic efficiency for conversion of glucomannan and galactomannan making this plant mannanase an interesting candidate for biotechnological applications of digesting various mannans. Moreover, these studies suggest an evolutionary diversification of plant mannanase enzymatic function.

  • 38.
    Wang, Yang
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Azhar, Shoaib
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Gandini, Rosaria
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Aspeborg, Henrik
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Biochemical characterization of the novel endo-β-mannanase AtMan5-2 from Arabidopsis thalianaManuscript (preprint) (Other academic)
  • 39.
    Wang, Yang
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Azhar, Shoaib
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Gandini, Rosaria
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Aspeborg, Henrik
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Investigating the function and biochemical properties of Arabidopsis mannanase 5-6Manuscript (preprint) (Other academic)
  • 40.
    Wang, Yang
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Vilaplana, Francisco
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Aspeborg, Henrik
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Enzymatic characterization of a glycoside hydrolase family 5 subfamily 7 (GH5_7) mannanase from Arabidopsis thaliana2014In: Planta, ISSN 0032-0935, E-ISSN 1432-2048, Vol. 239, no 3, p. 653-665Article in journal (Refereed)
    Abstract [en]

    Each plant genome contains a repertoire of beta-mannanase genes belonging to glycoside hydrolase family 5 subfamily 7 (GH5_7), putatively involved in the degradation and modification of various plant mannan polysaccharides, but very few have been characterized at the gene product level. The current study presents recombinant production and in vitro characterization of AtMan5-1 as a first step towards the exploration of the catalytic capacity of Arabidopsis thaliana beta-mannanase. The target enzyme was expressed in both E. coli (AtMan5-1e) and P. pastoris (AtMan5-1p). The main difference between the two forms was a higher observed thermal stability for AtMan5-1p, presumably due to glycosylation of that particular variant. AtMan5-1 displayed optimal activity at pH 5 and 35 A degrees C and hydrolyzed polymeric carob galactomannan, konjac glucomannan, and spruce galactoglucomannan as well as oligomeric mannopentaose and mannohexaose. However, the galactose-rich and highly branched guar gum was not as efficiently degraded. AtMan5-1 activity was enhanced by Co2+ and inhibited by Mn2+. The catalytic efficiency values for carob galactomannan were 426.8 and 368.1 min(-1) mg(-1) mL for AtMan5-1e and AtMan5-1p, respectively. Product analysis of AtMan5-1p suggested that at least five substrate-binding sites were required for manno-oligosaccharide hydrolysis, and that the enzyme also can act as a transglycosylase.

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