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  • 1. Derba-Maceluch, Marta
    et al.
    Awano, Tatsuya
    Takahashi, Junko
    Lucenius, Jessica
    Ratke, Christine
    Kontro, Inkeri
    Busse-Wicher, Marta
    Kosik, Ondrej
    Tanaka, Ryo
    Winzell, Anders
    KTH, School of Biotechnology (BIO), Glycoscience.
    Kallas, Åsa
    KTH, School of Biotechnology (BIO), Glycoscience.
    Lesniewska, Joanna
    Berthold, Fredrik
    Immerzeel, Peter
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Dupree, Paul
    Serimaa, Ritva
    Mellerowicz, Ewa J.
    Suppression of xylan endotransglycosylase PtxtXyn10A affects cellulose microfibril angle in secondary wall in aspen wood2015In: New Phytologist, ISSN 0028-646X, E-ISSN 1469-8137, Vol. 205, no 2, p. 666-681Article in journal (Refereed)
    Abstract [en]

    Certain xylanases from family GH10 are highly expressed during secondary wall deposition, but their function is unknown. We carried out functional analyses of the secondary-wall specific PtxtXyn10A in hybrid aspen (Populus tremulaxtremuloides).PtxtXyn10A function was analysed by expression studies, overexpression in Arabidopsis protoplasts and by downregulation in aspen.PtxtXyn10A overexpression in Arabidopsis protoplasts resulted in increased xylan endotransglycosylation rather than hydrolysis. In aspen, the enzyme was found to be proteolytically processed to a 68kDa peptide and residing in cell walls. Its downregulation resulted in a corresponding decrease in xylan endotransglycosylase activity and no change in xylanase activity. This did not alter xylan molecular weight or its branching pattern but affected the cellulose-microfibril angle in wood fibres, increased primary growth (stem elongation, leaf formation and enlargement) and reduced the tendency to form tension wood. Transcriptomes of transgenic plants showed downregulation of tension wood related genes and changes in stress-responsive genes. The data indicate that PtxtXyn10A acts as a xylan endotransglycosylase and its main function is to release tensional stresses arising during secondary wall deposition. Furthermore, they suggest that regulation of stresses in secondary walls plays a vital role in plant development.

  • 2.
    Ezcurra, Ines
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Johansson, Camilla
    KTH, School of Biotechnology (BIO), Glycoscience.
    Tamizhselvan, Prashanth
    KTH, School of Biotechnology (BIO), Glycoscience.
    Winzell, Anders
    KTH, School of Biotechnology (BIO), Glycoscience.
    Aspeborg, Henrik
    KTH, School of Biotechnology (BIO), Glycoscience.
    An AC-type element mediates transactivation of secondary cell wall carbohydrate-active enzymes by PttMYB021, the Populus MYB46 orthologue2011In: BMC Proceedings, ISSN 1753-6561, E-ISSN 1753-6561, Vol. 5Article in journal (Refereed)
  • 3. 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.

  • 4.
    Ohlsson, Anna B.
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Djerbi, Soraya
    KTH, School of Biotechnology (BIO), Glycoscience.
    Winzell, Anders
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bessueille, Laurence
    Ståldal, Veronika
    Li, Xinguo
    KTH, School of Biotechnology (BIO), Glycoscience.
    Blomqvist, Kristina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Berglund, Torkel
    KTH, School of Biotechnology (BIO), Glycoscience.
    Cell suspension cultures of Populus tremula x P. tremuloides exhibit a high level of cellulose synthase gene expression that coincides with increased in vitro cellulose synthase activity.2006In: Protoplasma, ISSN 0033-183X, E-ISSN 1615-6102, Vol. 228, no 4, p. 221-9Article in journal (Refereed)
    Abstract [en]

    Compared to wood, cell suspension cultures provide convenient model systems to study many different cellular processes in plants. Here we have established cell suspension cultures of Populus tremula L. x P. tremuloides Michx. and characterized them by determining the enzymatic activities and/or mRNA expression levels of selected cell wall-specific proteins at the different stages of growth. While enzymes and proteins typically associated with primary cell wall synthesis and expansion were detected in the exponential growth phase of the cultures, the late stationary phase showed high expression of the secondary-cell-wall-associated cellulose synthase genes. Interestingly, detergent extracts of membranes from aging cell suspension cultures exhibited high levels of in vitro cellulose synthesis. The estimated ratio of cellulose to callose was as high as 50 : 50, as opposed to the ratio of 30 : 70 so far achieved with membrane preparations extracted from other systems. The increased cellulose synthase activity was also evidenced by higher levels of Calcofluor white binding in the cell material from the stationary-phase cultures. The ease of handling cell suspension cultures and the improved capacity for in vitro cellulose synthesis suggest that these cultures offer a new basis for studying the mechanism of cellulose biosynthesis.

  • 5. Takahashi, Junko
    et al.
    Awano, Tatsuya
    Winzell, Anders
    KTH, School of Biotechnology (BIO), Glycoscience.
    Kallas, Åsa
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ratke, Christine
    Gorzsas, Andras
    Lesniewska, Joanna
    Anne, Gouget
    Berthold, Fredrik
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Glycoscience.
    Sundberg, Björn
    Mellerowicz, Eva
    Suppression of wood expressed xylanase affects cell expansion and secondary wall compositionManuscript (preprint) (Other academic)
  • 6.
    Winzell, Anders
    KTH, School of Biotechnology (BIO).
    Analysis of Genes and Proteins Involved in Wood Formation2007Licentiate thesis, comprehensive summary (Other scientific)
    Abstract [en]

    ABSTRACT

    Wood formation or xylogenesis is a fundamental process for so diverse issues as industry, shelter and a sustainable environment. Xylogenesis originates from the vascular cambium where organised cell divisions are followed by differentiation into specialised wood cells. Wood is comprised of secondary xylem, rigid large cells with thick cell walls that are lignified. These cells transport water and nutrients as well as give the tree its strength. The basis for the sturdy cells is an advanced composite made up of cellulose fibers cross-linked by hemicelluloses and finally embedded in lignin. This fiber-composite is the secondary cell walls of woody plants. Cell division and differentiation is regulated by switching on and off genes. Proteins encoded by these genes execute the major functions in the cells. They steer the entire machinery operating the structure and function of the cells, maintaining growth and synthesising essential products such as the cell wall carbohydrates.

    The Wood Biotechnology research group has been part of a massive undertaking of identifying proteins responsible for the key processes of xylogenesis. This has been achieved by monitoring the gene expression at different time points of wood cell maturation in Populus tremula x tremuloides. Here we describe investigation of proteins involved in cell expansion and secondary cell wall formation as well as the development of a model system that will aid the functional analysis of cellulose synthesis. Expansin is a protein involved in cell expansion in all tissues of the plant. We have studied one expansin, PttEXPA1, found to be especially abundant in cells undergoing wood formation. Also, we have examined glycosyltransferases that seemingly play a part in secondary cell wall formation. One of these, PttGT43A is expressed in the yeast Pichia pastoris as well as in baculovirus infected insect cells. However, protein levels were too low for biochemical characterisation. We have also demonstrated that hybrid aspen cell suspension cultures can be used as a model system for cellulose synthesis.

    By mastering proteins involved in xylogenesis we acquire the tools to improve and develop the wood product market. New products envisioned include e.g. faster growing trees, changed fiber characteristics, production of wood carbohydrates as well as invention of intelligent materials by biomimetic engineering.

  • 7.
    Winzell, Anders
    KTH, School of Biotechnology (BIO), Glycoscience.
    Investigation of genes and proteins involved in xylan biosynthesis2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Wood formation or xylogenesis is a fundamental process for so diverse issues as industry, shelter and a sustainable environment. Wood is comprised of secondary xylem, rigid large cells with thick cell walls that are lignified. The basis for the sturdy cells is an advanced composite made up of cellulose fibers cross-linked by hemicelluloses and finally embedded in lignin. This fiber-composite is the secondary cell walls of woody plants. Cell division and differentiation is regulated by switching on and off genes. Proteins encoded by these genes execute the major functions in the cells. They steer the entire machinery operating the structure and function of the cells, maintaining growth and synthesising essential products such as the cell wall carbohydrates.

     

    Here we describe the investigation of genes and proteins involved in xylan formation as well as the development of a model system that will aid the functional analysis of wood formation. Xylan is the main hemicellulose or cross linking glycan in dicot wood and thereby one of the most abundant carbohydrates on earth. We demonstrate that hybrid aspen cell suspension cultures can be used as a model system for secondary cell wall formation. We have also examined glycosyltransferases from CAZy family 43 that play a part in secondary cell wall formation. We have focused on one of these, Pt×tGT43A, a likely ortholog of Arabidopsis IRX9, which plays a crucial role in xylan formation. The protein was transiently expressed in Nicotiana benthamiana and its function and localization is described. Also, we investigate a glycoside hydrolase, Pt×tXyn10A, involved in wood formation. Its role is not clear but it most likely modifies xylan as it gets incorporated into the secondary cell wall after secretion from the Golgi. This influences the interaction between cellulose, xylan and lignin in the finished wood cell. We have also cloned a transcription factor, Pt×tMYB021, a likely ortholog of Arabidopsis MYB46 and we show that it activates GT43A, GT43B and Xyn10A. By analysis of the promoter sequences we identify a CA-rich motif putatively important for xylem-specific genes.

     

    By mastering proteins involved in xylogenesis we will acquire the tools to improve and develop the wood product market. Xylan is an immense unexploited source of renewable carbohydrate. New products envisioned include e.g. faster growing trees, changed fiber characteristics, optimised utilization of wood carbohydrates for biofuels and biomaterials as well as invention of intelligent materials by biomimetic engineering.

  • 8.
    Winzell, Anders
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Aspeborg, Henrik
    KTH, School of Biotechnology (BIO), Glycoscience.
    Wang, Yucheng
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Glycoscience.
    Conserved CA-rich motifs in gene promoters of Pt x tMYB021-responsive secondary cell wall carbohydrate-active enzymes in Populus2010In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 394, no 3, p. 848-853Article in journal (Refereed)
    Abstract [en]

    In order to understand gene regulation during wood formation, we cloned a MYB46-like gene in hybrid aspen. Populus tremula x tremuloides, called PtxtMYB021 Phylogenetic and paired identity analysis of MYB46-like genes in Populus and Arabidopsis reveals relationships between paralogous pairs of Populus MYB46-like proteins and their Arabidopsis counterparts MYB46 and MYB83, and suggest that PtxtMYB021 is the ortholog of MYB46 Ptxt-MYB021 is expressed mainly in xylem tissues, and transiently expressed PtxtMYB46 transactivates gene promoters of xylan-active CAZymes GT43A, GT43B and Xyn10A Analysis of conserved motifs within these promoters identify the sequence CCACCAAC, called ACTYP, which is similar to the AC elements mediating transactivation by MYB transcription factors during lignin biosynthesis Further analysis by Motif Finder identifies four 6 bp CA-rich motifs overlapping ACTYP, and we show that these motifs are enriched in xylem-specific promoters We propose that AC-type regulatory elements mediate xylem-specific MYB46-dependent expression of secondary cell wall carbohydrate-active enzymes (CAZymes), besides activating gene expression of lignin biosynthesis enzymes.

  • 9.
    Winzell, Anders
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Aspeborg, Henrik
    KTH, School of Biotechnology (BIO), Glycoscience.
    Wang, Yucheng
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Glycoscience.
    Molecular cloning and functional characterization of Pt×tMYB021, a MYB46-like transcription factor in PopulusManuscript (preprint) (Other academic)
  • 10.
    Winzell, Anders
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Guerriero, Gea
    KTH, School of Biotechnology (BIO), Glycoscience.
    Aspeborg, Henrik
    KTH, School of Biotechnology (BIO), Glycoscience.
    Wang, Yiqiang
    KTH, School of Biotechnology (BIO), Glycoscience.
    Rajangam, Alex S.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ezcurra, Inés
    KTH, School of Biotechnology (BIO), Glycoscience.
    Biochemical characterization of family 43 glycosyltransferases in the Populus xylem: challenges and prospects2010In: Plant Biotechnology, ISSN 1342-4580, Vol. 27, no 3, p. 283-288Article in journal (Refereed)
    Abstract [en]

    Wood formation is a biological process of great economical importance. Genes active during the secondary cellwall formation of wood fibers from Populus tremulatremuloides were previously identified by expression profilingthrough microarray analyses. A number of these genes encode glycosyltransferases (GTs) with unknown substratespecificities. Here we report heterologous expression of one of these enzymes, PttGT43A, a putative IRREGULARXYLEM9 (IRX9) homologue. Expression trials in Pichia pastoris and insect cells revealed very low levels of accumulationof immunoreactive PttGT43A, whereas transient expression in Nicotiana benthamiana leaves by Agrobacterium infiltration(agroinfiltration) using a viral vector produced substantial amounts of protein that mostly precipitated in the crude pellet.Agroinfiltration induced weak endogenous xylosyltransferase activity in microsomal extracts, and transient PttGT43Aexpression further increased this activity, albeit only to low levels. PttGT43A may be inactive as an individual subunit,requiring complex formation with unknown partners to display enzymatic activity. Our results suggest that transient coexpressionin leaves of candidate subunit GTs may provide a viable approach for formation of an active xylanxylosyltransferase enzymatic complex.

  • 11.
    Winzell, Anders
    et al.
    KTH, School of Biotechnology (BIO).
    Rajangam, Alex
    KTH, School of Biotechnology (BIO).
    Arvestad, Lars
    KTH, School of Computer Science and Communication (CSC).
    Filling, Charlotta
    KTH, School of Biotechnology (BIO).
    Divine, Christina
    KTH, School of Biotechnology (BIO).
    Aspeborg, Henrik
    KTH, School of Biotechnology (BIO).
    Master, Emma R.
    KTH, School of Biotechnology (BIO).
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO).
    Sequence Analysis and Recombinant Expression of Family 43 GlycosyltransferasesManuscript (preprint) (Other academic)
  • 12.
    Winzell, Anders
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ratke, Christine
    Naumann, Marcel
    Wang, Yucheng
    KTH, School of Biotechnology (BIO), Glycoscience.
    Aspeborg, Henrik
    KTH, School of Biotechnology (BIO), Glycoscience.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Mellerowicz, Ewa
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Glycoscience.
    Family 43 glycosyltransferases in Populus and Arabidopsis: phylogeny and expression analysisManuscript (preprint) (Other academic)
1 - 12 of 12
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