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  • 1.
    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.

  • 2. Bhalerao, R.
    et al.
    Keskitalo, J.
    Sterky, Fredrik
    KTH, Superseded Departments, Biotechnology.
    Erlandsson, R.
    Bjorkbacka, H.
    Birve, S. J.
    Karlsson, J.
    Gardestrom, P.
    Gustafsson, P.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Jansson, S.
    Gene expression in autumn leaves2003In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 131, no 2, p. 430-442Article in journal (Refereed)
    Abstract [en]

    Two cDNA libraries were prepared, one from leaves of a field-grown aspen (Populus tremula) tree, harvested just before any visible sign of leaf senescence in the autumn, and one from young but fully expanded leaves of greenhouse-grown aspen (Populus tremula X tremuloides). Expressed sequence tags (ESTs; 5,128 and 4,841, respectively) were obtained from the two libraries. A semiautomatic method of annotation and functional classification of the ESTs, according to a modified Munich Institute of Protein Sequences classification scheme, was developed, utilizing information from three different databases. The patterns of gene expression in the two libraries were strikingly different. In the autumn leaf library, ESTs encoding metallothionein, early light-inducible proteins, and cysteine proteases were most abundant. Clones encoding other proteases and proteins involved in respiration and breakdown of lipids and pigments, as well as stress-related genes, were also well represented. We identified homologs to many known senescence-associated genes, as well as seven different genes encoding cysteine proteases, two encoding aspartic proteases, five encoding metallothioneins, and 35 additional genes that were up-regulated in autumn leaves. We also indirectly estimated the rate of plastid protein synthesis in the autumn leaves to be less that 10% of that in young leaves.

  • 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.
    Eklöf, Jens M.
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    The XTH Gene Family: An Update on Enzyme Structure, Function, and Phylogeny in Xyloglucan Remodeling2010In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 153, no 2, p. 456-466Article in journal (Refereed)
  • 6. 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.

  • 7. Gray-Mitsumune, Madoka
    et al.
    Mellerowicz, Ewa J.
    Abe, Hisashi
    Schrader, Jarmo
    Winzell, Anders
    KTH, Superseded Departments, Biotechnology.
    Sterky, Fredrik
    KTH, Superseded Departments, Biotechnology.
    Blomqvist, Kristina
    KTH, Superseded Departments, Biotechnology.
    McQueen-Mason, Simon
    Teeri, Tuula T.
    KTH, Superseded Departments, Biotechnology.
    Sundberg, Björn
    Expansins abundant in secondary xylem belong to subgroup a of the alpha-expansin gene family (1 w )2004In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 135, no 3, p. 1552-1564Article in journal (Refereed)
    Abstract [en]

    Differentiation of xylem cells in dicotyledonous plants involves expansion of the radial primary cell walls and intrusive tip growth of cambial derivative cells prior to the deposition of a thick secondary wall essential for xylem function. Expansins are cell wall-residing proteins that have an ability to plasticize the cellulose-hemicellulose network of primary walls. We found expansin activity in proteins extracted from the cambial region of mature stems in a model tree species hybrid aspen (Populus tremula X Populus tremuloides Michx). We identified three a-expansin genes (PttEXP1, PttEXP2, and PttEXP8) and one beta-expansin gene (PttEXPB1) in a cambial region expressed sequence tag library, among which PttEXP1 was most abundantly represented. Northern-blot analyses in aspen vegetative organs and tissues showed that PttEXP1 was specifically expressed in mature stems exhibiting secondary growth, where it was present in the cambium and in the radial expansion zone. By contrast, PttEXP2 was mostly expressed in developing leaves. In situ reverse transcription-PCR provided evidence for accumulation of mRNA of PttEXP1 along with ribosomal rRNA at the tips of intrusively growing xylem fibers, suggesting that PttEXP1 protein has a role in intrusive tip growth. An examination of tension wood and leaf cDNA libraries identified another expansin, PttEXP5, very similar to PttEXP1, as the major expansin in developing tension wood, while PttEXP3 was the major expansin expressed in developing leaves. Comparative analysis of expansins expressed in woody stems in aspen, Arabidopsis, and pine showed that the most abundantly expressed expansins share sequence similarities, belonging to the subfamily A of alpha-expansins and having two conserved motifs at the beginning and end of the mature protein, RIPVG and KNFRV, respectively. This conservation suggests that these genes may share a specialized, not yet identified function.

  • 8. Harris, Darby
    et al.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ding, Shi-You
    DeBolt, Seth
    Tools for Cellulose Analysis in Plant Cell Walls2010In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 153, no 2, p. 420-426Article in journal (Refereed)
  • 9.
    Ibatullin, Farid M.
    et al.
    KTH, School of Biotechnology (BIO).
    Banasiak, Alicja
    Baumann, Martin J.
    KTH, School of Biotechnology (BIO).
    Greffe, Lionel
    KTH, School of Biotechnology (BIO).
    Takahashi, Junko
    Mellerowicz, Ewa J.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    A Real-Time Fluorogenic Assay for the Visualization of Glycoside Hydrolase Activity in Planta2009In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 151, no 4, p. 1741-1750Article in journal (Refereed)
    Abstract [en]

    There currently exists a diverse array of molecular probes for the in situ localization of polysaccharides, nucleic acids, and proteins in plant cells, including reporter enzyme strategies (e. g. protein-glucuronidase fusions). In contrast, however, there is a paucity of methods for the direct analysis of endogenous glycoside hydrolases and transglycosidases responsible for cell wall remodeling. To exemplify the potential of fluorogenic resorufin glycosides to address this issue, a resorufin beta-glycoside of a xylogluco-oligosaccharide (XXXG-beta-Res) was synthesized as a specific substrate for in planta analysis of XEH activity. The resorufin aglycone is particularly distinguished for high sensitivity in muro assays due to a low pK(a) (5.8) and large extinction coefficient (epsilon 62,000 M-1 cm(-1)), long-wavelength fluorescence (excitation 571 nm/emission 585 nm), and high quantum yield (0.74) of the corresponding anion. In vitro analyses demonstrated that XXXG-beta-Res is hydrolyzed by the archetypal plant XEH, nasturtium (Tropaeolum majus) NXG1, with classical Michaelis-Menten substrate saturation kinetics and a linear dependence on both enzyme concentration and incubation time. Further, XEH activity could be visualized in real time by observing the localized increase in fluorescence in germinating nasturtium seeds and Arabidopsis (Arabidopsis thaliana) inflorescent stems by confocal microscopy. Importantly, this new in situ XEH assay provides an essential complement to the in situ xyloglucan endotransglycosylase assay, thus allowing delineation of the disparate activities encoded by xyloglucan endotransglycosylase/hydrolase genes directly in plant tissues. The observation that XXXG-beta-Res is also hydrolyzed by diverse microbial XEHs indicates that this substrate, and resorufin glycosides in general, may find broad applicability for the analysis of wall restructuring by polysaccharide hydrolases during morphogenesis and plant-microbe interactions.

  • 10.
    Kaewthai, Nomchit
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Gendre, Delphine
    Eklöf, Jens M.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Ibatullin, Farid M.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Bhalerao, Rishikesh P
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Group III-A XTH Genes of Arabidopsis Encode Predominant Xyloglucan Endohydrolases That Are Dispensable for Normal Growth2013In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 161, no 1, p. 440-454Article in journal (Refereed)
    Abstract [en]

    The molecular basis of primary wall extension endures as one of the central enigmas in plant cell morphogenesis. Classical cell wall models suggest that xyloglucan endo-transglycosylase activity is the primary catalyst (together with expansins) of controlled cell wall loosening through the transient cleavage and religation of xyloglucan-cellulose cross links. The genome of Arabidopsis (Arabidopsis thaliana) contains 33 phylogenetically diverse XYLOGLUCAN ENDO-TRANSGLYCOSYLASE/HYDROLASE (XTH) gene products, two of which were predicted to be predominant xyloglucan endohydrolases due to clustering into group III-A. Enzyme kinetic analysis of recombinant AtXTH31 confirmed this prediction and indicated that this enzyme had similar catalytic properties to the nasturtium (Tropaeolum majus) xyloglucanase1 responsible for storage xyloglucan hydrolysis during germination. Global analysis of Genevestigator data indicated that AtXTH31 and the paralogous AtXTH32 were abundantly expressed in expanding tissues. Microscopy analysis, utilizing the resorufin beta-glycoside of the xyloglucan oligosaccharide XXXG as an in situ probe, indicated significant xyloglucan endohydrolase activity in specific regions of both roots and hypocotyls, in good correlation with transcriptomic data. Moreover, this hydrolytic activity was essentially completely eliminated in AtXTH31/AtXTH32 double knockout lines. However, single and double knockout lines, as well as individual overexpressing lines, of AtXTH31 and AtXTH32 did not demonstrate significant growth or developmental phenotypes. These results suggest that although xyloglucan polysaccharide hydrolysis occurs in parallel with primary wall expansion, morphological effects are subtle or may be compensated by other mechanisms. We hypothesize that there is likely to be an interplay between these xyloglucan endohydrolases and recently discovered apoplastic exo-glycosidases in the hydrolytic modification of matrix xyloglucans.

  • 11. Kaida, Rumi
    et al.
    Satoh, Yumi
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Yamada, Yohko
    Kaku, Tomomi
    Hayashi, Takahisa
    Kaneko, Takako S.
    Activation of beta-Glucan Synthases by Wall-Bound Purple Acid Phosphatase in Tobacco Cells2009In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 150, no 4, p. 1822-1830Article in journal (Refereed)
    Abstract [en]

    Wall-bound purple acid phosphatases have been shown to be potentially involved in the regulation of plant cell growth. The aim of this work was to further investigate the function of one of these phosphatases in tobacco (Nicotiana tabacum), NtPAP12, using transgenic cells overexpressing the enzyme. The transgenic cells exhibited a higher level of phosphatase activity in their walls. The corresponding protoplasts regenerating a cell wall exhibited a higher rate of beta-glucan synthesis and cellulose deposition was increased in the walls of the transgenic cells. A higher level of plasma membrane glucan synthase activities was also measured in detergent extracts of membrane fractions from the transgenic line, while no activation of Golgi-bound glycan synthases was detected. Enzymatic hydrolysis and methylation analysis were performed on the products synthesized in vitro by the plasma membrane enzymes from the wild-type and transgenic lines extracted with digitonin and incubated with radioactive UDP-glucose. The data showed that the glucans consisted of callose and cellulose and that the amount of each glucan synthesized by the enzyme preparation from the transgenic cells was significantly higher than in the case of the wildtype cells. The demonstration that callose and cellulose synthases are activated in cells overexpressing the wall-bound phosphatase NtPAP12 suggests a regulation of these carbohydrate synthases by a phosphorylation/dephosphorylation process, as well as a role of wall-bound phosphatases in the regulation of cell wall biosynthesis.

  • 12.
    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.

  • 13. Nishikubo, Nobuyuki
    et al.
    Takahashi, Junko
    Roos, Alexandra A.
    Derba-Maceluch, Marta
    Piens, Kathleen
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Stalbrand, Henrik
    Mellerowicz, Ewa J.
    Xyloglucan endo-Transglycosylase-Mediated Xyloglucan Rearrangements in Developing Wood of Hybrid Aspen2011In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 155, no 1, p. 399-413Article in journal (Refereed)
    Abstract [en]

    Xyloglucan endo-transglycosylases (XETs) encoded by xyloglucan endo-transglycosylases/hydrolase (XTH) genes modify the xyloglucan-cellulose framework of plant cell walls, thereby regulating their expansion and strength. To evaluate the importance of XET in wood development, we studied xyloglucan dynamics and XTH gene expression in developing wood and modified XET activity in hybrid aspen (Populus tremula X tremuloides) by overexpressing PtxtXET16-34. We show that developmental modifications during xylem differentiation include changes from loosely to tightly bound forms of xyloglucan and increases in the abundance of fucosylated xyloglucan epitope recognized by the CCRC-M1 antibody. We found that at least 16 Populus XTH genes, all likely encoding XETs, are expressed in developing wood. Five genes were highly and ubiquitously expressed, whereas PtxtXET16-34 was expressed more weakly but specifically in developing wood. Transgenic up-regulation of XET activity induced changes in cell wall xyloglucan, but its effects were dependent on developmental stage. For instance, XET overexpression increased abundance of the CCRC-M1 epitope in cambial cells and xylem cells in early stages of differentiation but not in mature xylem. Correspondingly, an increase in tightly bound xyloglucan content was observed in primary-walled xylem but a decrease was seen in secondary-walled xylem. Thus, in young xylem cells, XET activity limits xyloglucan incorporation into the tightly bound wall network but removes it from cell walls in older cells. XET overexpression promoted vessel element growth but not fiber expansion. We suggest that the amount of nascent xyloglucan relative to XET is an important determinant of whether XET strengthens or loosens the cell wall.

  • 14. 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.

  • 15.
    Rajangam, Alex S.
    et al.
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Kumar, Manoj
    Aspeborg, Henrik
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Guerriero, Gea
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Arvestad, Lars
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB.
    Pansri, Podjamas
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Brown, Christian J. L.
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Blomqvist, Kristina
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Ezcurra, Inés
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Mellerowicz, Ewa
    Sundberg, Bjorn
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    MAP20, a Microtubule-Associated Protein in the Secondary Cell Walls of Hybrid Aspen, Is a Target of the Cellulose Synthesis Inhibitor 2,6-Dichlorobenzonitrile2008In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 148, no 3, p. 1283-1294Article in journal (Refereed)
    Abstract [en]

    We have identified a gene, denoted PttMAP20, which is strongly up-regulated during secondary cell wall synthesis and tightly coregulated with the secondary wall-associated CESA genes in hybrid aspen (Populus tremula x tremuloides). Immunolocalization studies with affinity-purified antibodies specific for PttMAP20 revealed that the protein is found in all cell types in developing xylem and that it is most abundant in cells forming secondary cell walls. This PttMAP20 protein sequence contains a highly conserved TPX2 domain first identified in a microtubule-associated protein (MAP) in Xenopus laevis. Overexpression of PttMAP20 in Arabidopsis (Arabidopsis thaliana) leads to helical twisting of epidermal cells, frequently associated with MAPs. In addition, a PttMAP20-yellow fluorescent protein fusion protein expressed in tobacco (Nicotiana tabacum) leaves localizes to microtubules in leaf epidermal pavement cells. Recombinant PttMAP20 expressed in Escherichia coli also binds specifically to in vitro-assembled, taxol-stabilized bovine microtubules. Finally, the herbicide 2,6-dichlorobenzonitrile, which inhibits cellulose synthesis in plants, was found to bind specifically to PttMAP20. Together with the known function of cortical microtubules in orienting cellulose microfibrils, these observations suggest that PttMAP20 has a role in cellulose biosynthesis.

  • 16.
    Srivastava, Vaibhav
    et al.
    Umeå Plant Science Centre.
    Srivastava, Manoj Kumar
    Chibani, Kamel
    Nilsson, Robert
    Rouhier, Nicolas
    Melzer, Michael
    Wingsle, Gunnar
    Alternative splicing studies of the reactive oxygen species gene network in Populus reveal two isoforms of high-isoelectric-point superoxide dismutase2009In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 149, no 4, p. 1848-1859Article in journal (Refereed)
    Abstract [en]

    Recent evidence has shown that alternative splicing (AS) is widely involved in the regulation of gene expression, substantially extending the diversity of numerous proteins. In this study, a subset of expressed sequence tags representing members of the reactive oxygen species gene network was selected from the PopulusDB database to investigate AS mechanisms in Populus. Examples of all known types of AS were detected, but intron retention was the most common. Interestingly, the closest Arabidopsis (Arabidopsis thaliana) homologs of half of the AS genes identified in Populus are not reportedly alternatively spliced. Two genes encoding the protein of most interest in our study (high-isoelectric-point superoxide dismutase [hipI-SOD]) have been found in black cottonwood (Populus trichocarpa), designated PthipI-SODC1 and PthipI-SODC2. Analysis of the expressed sequence tag libraries has indicated the presence of two transcripts of PthipI-SODC1 (hipI-SODC1b and hipI-SODC1s). Alignment of these sequences with the PthipI-SODC1 gene showed that hipI-SODC1b was 69 bp longer than hipI-SODC1s due to an AS event involving the use of an alternative donor splice site in the sixth intron. Transcript analysis showed that the splice variant hipI-SODC1b was differentially expressed, being clearly expressed in cambial and xylem, but not phloem, regions. In addition, immunolocalization and mass spectrometric data confirmed the presence of hipI-SOD proteins in vascular tissue. The functionalities of the spliced gene products were assessed by expressing recombinant hipI-SOD proteins and in vitro SOD activity assays.

  • 17. Sun, Qi
    et al.
    Emanuelsson, Olof
    Stockholms universitet.
    van Wijk, Klaas J.
    Analysis of curated and predicted plastid subproteomes of Arabidopsis. Subcellular compartmentalization leads to distinctive proteome properties2004In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 135, no 2, p. 723-734Article in journal (Refereed)
    Abstract [en]

    Carefully curated proteomes of the inner envelope membrane, the thylakoid membrane, and the thylakoid lumen of chloroplasts from Arabidopsis were assembled based on published, well-documented localizations. These curated proteomes were evaluated for distribution of physical-chemical parameters, with the goal of extracting parameters for improved subcellular prediction and subsequent identification of additional (low abundant) components of each membrane system. The assembly of rigorously curated subcellular proteomes is in itself also important as a parts list for plant and systems biology. Transmembrane and subcellular prediction strategies were evaluated using the curated data sets. The three curated proteomes differ strongly in average isoelectric point and protein size, as well as transmembrane distribution. Removal of the cleavable, N-terminal transit peptide sequences greatly affected isoelectric point and size distribution. Unexpectedly, the Cys content was much lower for the thylakoid proteomes than for the inner envelope. This likely relates to the role of the thylakoid membrane in light-driven electron transport and helps to avoid unwanted oxidation-reduction reactions. A rule of thumb for discriminating between the predicted integral inner envelope membrane and integral thylakoid membrane proteins is suggested. Using a combination of predictors and experimentally derived parameters, four plastid subproteomes were predicted from the fully annotated Arabidopsis genome. These predicted subproteomes were analyzed for their properties and compared to the curated proteomes. The sensitivity and accuracy of the prediction strategies are discussed. Data can be extracted from the new plastid proteome database (http://ppdb.tc.cornell.edu).

  • 18. Uddenberg, Daniel
    et al.
    Reimegård, Johan
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Clapham, David
    Almqvist, Curt
    von Arnold, Sara
    Emanuelsson, Olof
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sundström, Jens F.
    Early Cone Setting in Picea abies acrocona Is Associated with Increased Transcriptional Activity of a MADS Box Transcription Factor2013In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 161, no 2, p. 813-823Article in journal (Refereed)
    Abstract [en]

    Conifers normally go through a long juvenile period, for Norway spruce (Picea abies) around 20 to 25 years, before developing male and female cones. We have grown plants from inbred crosses of a naturally occurring spruce mutant (acrocona). One-fourth of the segregating acrocona plants initiate cones already in their second growth cycle, suggesting control by a single locus. The early cone-setting properties of the acrocona mutant were utilized to identify candidate genes involved in vegetative-to-reproductive phase change in Norway spruce. Poly(A(+)) RNA samples from apical and basal shoots of cone-setting and non-cone-setting plants were subjected to high-throughput sequencing (RNA-seq). We assembled and investigated 33,383 expressed putative protein-coding acrocona transcripts. Eight transcripts were differentially expressed between selected sample pairs. One of these (Acr42124_1) was significantly up-regulated in apical shoot samples from cone-setting acrocona plants, and the encoded protein belongs to the MADS box gene family of transcription factors. Using quantitative real-time polymerase chain reaction with independently derived plant material, we confirmed that the MADS box gene is up-regulated in both needles and buds of cone-inducing shoots when reproductive identity is determined. Our results constitute important steps for the development of a rapid cycling model system that can be used to study gene function in conifers. In addition, our data suggest the involvement of a MADS box transcription factor in the vegetative-to-reproductive phase change in Norway spruce.

  • 19. Urbanowicz, B. R.
    et al.
    Bennett, A. B.
    Del Campillo, E.
    Catalá, C.
    Hayashi, T.
    Henrissat, B.
    Höfte, H.
    McQueen-Mason, S. J.
    Patterson, S. E.
    Shoseyov, O.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Rose, J. K. C.
    Structural organization and a standardized nomenclature for plant endo-1,4-β-glucanases (cellulases) of glycosyl hydrolase family 92007In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 144, no 4, p. 1693-1696Article in journal (Refereed)
  • 20. Vanholme, Bartel
    et al.
    Vanholme, Ruben
    Turumtay, Halbay
    Goeminne, Geert
    Cesarino, Igor
    Goubet, Florence
    Morreel, Kris
    Rencoret, Jorge
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Hooijmaijers, Cortwa
    KTH, School of Biotechnology (BIO), Glycoscience.
    De Rycke, Riet
    Gheysen, Godelieve
    Ralph, John
    De Block, Marc
    Meulewaeter, Frank
    Boerjan, Wout
    Accumulation of N-Acetylglucosamine Oligomers in the Plant Cell Wall Affects Plant Architecture in a Dose-Dependent and Conditional Manner2014In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 165, no 1, p. 290-308Article in journal (Refereed)
    Abstract [en]

    To study the effect of short N-acetylglucosamine (GlcNAc) oligosaccharides on the physiology of plants, N-ACETYLGLUCOSAMINYLTRANSFERASE (NodC) of Azorhizobium caulinodans was expressed in Arabidopsis (Arabidopsis thaliana). The corresponding enzyme catalyzes the polymerization of GlcNAc and, accordingly, beta-1,4-GlcNAc oligomers accumulated in the plant. A phenotype characterized by difficulties in developing an inflorescence stem was visible when plants were grown for several weeks under short-day conditions before transfer to long-day conditions. In addition, a positive correlation between the oligomer concentration and the penetrance of the phenotype was demonstrated. Although NodC overexpression lines produced less cell wall compared with wildtype plants under nonpermissive conditions, no indications were found for changes in the amount of the major cell wall polymers. The effect on the cell wall was reflected at the transcriptome level. In addition to genes encoding cell wall-modifying enzymes, a whole set of genes encoding membrane- coupled receptor-like kinases were differentially expressed upon GlcNAc accumulation, many of which encoded proteins with an extracellular Domain of Unknown Function26. Although stress-related genes were also differentially expressed, the observed response differed from that of a classical chitin response. This is in line with the fact that the produced chitin oligomers were too small to activate the chitin receptor-mediated signal cascade. Based on our observations, we propose a model in which the oligosaccharides modify the architecture of the cell wall by acting as competitors in carbohydrate-carbohydrate or carbohydrate-protein interactions, thereby affecting noncovalent interactions in the cell wall or at the interface between the cell wall and the plasma membrane.

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