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  • 1. 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.

  • 2. Dalman, Kerstin
    et al.
    Wind, Julia Johanna
    KTH, School of Biotechnology (BIO).
    Nemesio-Gorriz, Miguel
    Hammerbacher, Almuth
    Lunden, Karl
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Elfstrand, Malin
    Overexpression of PaNAC03, a stress induced NAC gene family transcription factor in Norway spruce leads to reduced flavonol biosynthesis and aberrant embryo development2017In: BMC Plant Biology, ISSN 1471-2229, E-ISSN 1471-2229, Vol. 17, article id 6Article in journal (Refereed)
    Abstract [en]

    Background: The NAC family of transcription factors is one of the largest gene families of transcription factors in plants and the conifer NAC gene family is at least as large, or possibly larger, as in Arabidopsis. These transcription factors control both developmental and stress induced processes in plants. Yet, conifer NACs controlling stress induced processes has received relatively little attention. This study investigates NAC family transcription factors involved in the responses to the pathogen Heterobasidion annosum (Fr.) Bref. sensu lato. Results: The phylogeny and domain structure in the NAC proteins can be used to organize functional specificities, several well characterized stress-related NAC proteins are found in III-3 in Arabidopsis (Jensen et al. Biochem J 426: 183-196, 2010). The Norway spruce genome contain seven genes with similarity to subgroup III-3 NACs. Based on the expression pattern PaNAC03 was selected for detailed analyses. Norway spruce lines overexpressing PaNAC03 exhibited aberrant embryo development in response to maturation initiation and 482 misregulated genes were identified in proliferating cultures. Three key genes in the flavonoid biosynthesis pathway: a CHS, a F3'H and PaLAR3 were consistently down regulated in the overexpression lines. In accordance, the overexpression lines showed reduced levels of specific flavonoids, suggesting that PaNAC03 act as a repressor of this pathway, possibly by directly interacting with the promoter of the repressed genes. However, transactivation studies of PaNAC03 and PaLAR3 in Nicotiana benthamiana showed that PaNAC03 activated PaLAR3A, suggesting that PaNAC03 does not act as an independent negative regulator of flavan-3-ol production through direct interaction with the target flavonoid biosynthetic genes. Conclusions: PaNAC03 and its orthologs form a sister group to well characterized stress-related angiosperm NAC genes and at least PaNAC03 is responsive to biotic stress and appear to act in the control of defence associated secondary metabolite production.

  • 3. 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.

  • 4. Eklund, D. Magnus
    et al.
    Staldal, Veronika
    Valsecchi, Isabel
    Cierlik, Izabela
    Eriksson, Caitriona
    Hiratsu, Keiichiro
    Ohme-Takagi, Masaru
    Sundstrom, Jens F.
    Thelander, Mattias
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Glycoscience.
    Sundberg, Eva
    The Arabidopsis thaliana STYLISH1 Protein Acts as a Transcriptional Activator Regulating Auxin Biosynthesis2010In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 22, no 2, p. 349-363Article in journal (Refereed)
    Abstract [en]

    The establishment and maintenance of auxin maxima in vascular plants is regulated by auxin biosynthesis and polar intercellular auxin flow. The disruption of normal auxin biosynthesis in mouse-ear cress ( Arabidopsis thaliana) leads to severe abnormalities, suggesting that spatiotemporal regulation of auxin biosynthesis is fundamental for normal growth and development. We have shown previously that the induction of the SHORT-INTERNODES/STYLISH (SHI/STY) family member STY1 results in increased transcript levels of the YUCCA (YUC) family member YUC4 and also higher auxin levels and auxin biosynthesis rates in Arabidopsis seedlings. We have also shown previously that SHI/STY family members redundantly affect development of flowers and leaves. Here, we further examine the function of STY1 by analyzing its DNA and protein binding properties. Our results suggest that STY1, and most likely other SHI/STY members, are DNA binding transcriptional activators that target genes encoding proteins mediating auxin biosynthesis. This suggests that the SHI/STY family members are essential regulators of auxin-mediated leaf and flower development. Furthermore, the lack of a shoot apical meristem in seedlings carrying a fusion construct between STY1 and a repressor domain, SRDX, suggests that STY1, and other SHI/STY members, has a role in the formation and/or maintenance of the shoot apical meristem, possibly by regulating auxin levels in the embryo.

  • 5.
    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)
  • 6.
    Ezcurra, Inés
    et al.
    KTH, Superseded Departments, Biotechnology.
    Wycliffe, P.
    Nehlin, L.
    Ellerstrom, M.
    Rask, L.
    Transactivation of the Brassica napus napin promoter by ABI3 requires interaction of the conserved B2 and B3 domains of ABI3 with different cis-elements: B2 mediates activation through an ABRE, whereas B3 interacts with an RY/G-box2000In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 24, no 1, p. 57-66Article in journal (Refereed)
    Abstract [en]

    The transcriptional activator ABI3 is a key regulator of gene expression during embryo maturation in crucifers. In monocots, the related VP1 protein regulates the Em promoter synergistically with abscisic acid (ABA). We identified cis-elements in the Brassica napus napin napA promoter mediating regulation by ABI3 and ABA, by analyzing substitution mutation constructs of napA in transgenic tobacco plantlets ectopically expressing ABI3. In transient analysis using particle bombardment of tobacco leaf sections, a tetramer of the distB ABRE (abscisic acid-responsive element) mediated transactivation by ABI3 and ABI3-dependent response to ABA, whereas a tetramer of the composite RY/G complex, containing RY repeats and a G-box, mediated only ABA-independent transactivation by ABI3. Deletion of the conserved B2 and B3 domains of ABI3 abolished transactivation of napA by ABI3. The two domains of ABI3 interact with different cis-elements: B2 is necessary for ABA-independent and ABA-dependent activations through the distB ABRE, whereas B3 interacts with the RY/G complex. Thus B2 mediates the interaction of ABI3 with the protein complex at the ABRE. The regulation of napA by ABI3 differs from Em regulation by VP1, in that the B3 domain of ABI3 is essential for the ABA-dependent regulation of napA.

  • 7.
    Fugelstad, Johanna
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bouzenzana, Jamel
    Djerbi, Soraya
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ezcurra, Inés
    KTH, School of Biotechnology (BIO), Glycoscience.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Arvestad, Lars
    KTH, School of Computer Science and Communication (CSC), Numerical Analysis and Computer Science, NADA.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    A novel family of cellulose synthase genes from the Oomycete Saprolegnia monoica: functional characterization using cellulose synthesis inhibitorsManuscript (Other academic)
  • 8.
    Fugelstad, Johanna
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bouzenzana, Jamel
    Djerbi, Soraya
    Guerriero, Gea
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ezcurra, Inés
    KTH, School of Biotechnology (BIO), Glycoscience.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Arvestad, Lars
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Identification of the cellulose synthase genes from the Oomycete Saprolegnia monoica and effect of cellulose synthesis inhibitors on gene expression and enzyme activity2009In: Fungal Genetics and Biology, ISSN 1087-1845, E-ISSN 1096-0937, Vol. 46, no 10, p. 759-767Article in journal (Refereed)
    Abstract [en]

    Cellulose biosynthesis is a vital but yet poorly understood biochemical process in Oomycetes. Here, we report the identification and characterization of the cellulose synthase genes (CesA) from Saprolegnia monoica. Southern blot experiments revealed the occurrence of three CesA homologues in this species and phylogenetic analyses confirmed that Oomycete CesAs form a clade of their own. All gene products contained the D,D,D,QXXRW signature of most processive glycosyltransferases, including cellulose synthases. However, their N-terminal ends exhibited Oomycete-specific domains, i.e. Pleckstrin Homology domains, or conserved domains of an unknown function together with additional putative transmembrane domains. Mycelial growth was inhibited in the presence of the cellulose biosynthesis inhibitors 2,6-dichlorobenzonitrile or Congo Red. This inhibition was accompanied by a higher expression of all CesA genes in the mycelium and increased in vitro glucan synthase activities. Altogether, our data strongly suggest a direct involvement of the identified CesA genes in cellulose biosynthesis.

  • 9. 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.

  • 10.
    Guerriero, Gea
    et al.
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Martin, Nathalie
    Golovko, Anna
    Sundstrom, Jens F.
    Rask, Lars
    Ezcurra, Inés
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    The RY/Sph element mediates transcriptional repression of maturation genes from late maturation to early seedling growth2009In: New Phytologist, ISSN 0028-646X, E-ISSN 1469-8137, Vol. 184, no 3, p. 552-565Article in journal (Refereed)
    Abstract [en]

    P>In orthodox seeds, the transcriptional activator ABI3 regulates two major stages in embryo maturation: a mid-maturation (MAT) stage leading to accumulation of storage compounds, and a late maturation (LEA) stage leading to quiescence and desiccation tolerance. Our aim was to elucidate mechanisms for transcriptional shutdown of MAT genes during late maturation, to better understand phase transition between MAT and LEA stages. Using transgenic and transient approaches in Nicotiana, we examined activities of two ABI3-dependent reporter genes driven by multimeric RY and abscisic acid response elements (ABREs) from a Brassica napus napin gene, termed RY and ABRE, where the RY reporter requires ABI3 DNA binding. Expression of RY peaks during mid-maturation and drops during late maturation, mimicking the MAT gene program, and in Arabidopsis thaliana RY elements are over-represented in MAT, but not in LEA, genes. The ABI3 transactivation of RY is inhibited by staurosporine, by a PP2C phosphatase, and by a repressor of maturation genes, VAL1/HSI2. The RY element mediates repression of MAT genes, and we propose that transcriptional shutdown of the MAT program during late maturation involves inhibition of ABI3 DNA binding by dephosphorylation. Later, during seedling growth, VAL1/HSI2 family repressors silence MAT genes by binding RY elements. New Phytologist (2009)doi: 10.1111/j.1469-8137.2009.02977.x.

  • 11. Guerriero, Gea
    et al.
    Silvestrini, Lucia
    Obersriebnig, Michael
    Hausman, Jean-Francois
    Strauss, Joseph
    Ezcurra, Inés
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    A WDR gene is a conserved member of a chitin synthase gene cluster and influences the cell wall in Aspergillus nidulans2016In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 17, no 7, p. 1031-Article in journal (Refereed)
    Abstract [en]

    WD40 repeat (WDR) proteins are pleiotropic molecular hubs. We identify a WDR gene that is a conserved genomic neighbor of a chitin synthase gene in Ascomycetes. The WDR gene is unique to fungi and plants, and was called Fungal Plant WD (FPWD). FPWD is within a cell wall metabolism gene cluster in the Ascomycetes (Pezizomycotina) comprising chsD, a Chs activator and a GH17 glucanase. The FPWD, AN1556.2 locus was deleted in Aspergillus nidulans strain SAA.111 by gene replacement and only heterokaryon transformants were obtained. The re-annotation of Aspergilli genomes shows that AN1556.2 consists of two tightly linked separate genes, i.e., the WDR gene and a putative beta-flanking gene of unknown function. The WDR and the beta-flanking genes are conserved genomic neighbors localized within a recently identified metabolic cell wall gene cluster in genomes of Aspergilli. The heterokaryons displayed increased susceptibility to drugs affecting the cell wall, and their phenotypes, observed by optical, confocal, scanning electron and atomic force microscopy, suggest cell wall alterations. Quantitative real-time PCR shows altered expression of some cell wall-related genes. The possible implications on cell wall biosynthesis are discussed.

  • 12. Guerriero, Gea
    et al.
    Spadiut, Oliver
    Kerschbamer, Christine
    Giorno, Filomena
    Baric, Sanja
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Glycoscience.
    Analysis of cellulose synthase genes from domesticated apple identifies collinear genes WDR53 and CesA8A: Partial co-expression, bicistronic mRNA, and alternative splicing of CESA8A2012In: Journal of Experimental Botany, ISSN 0022-0957, E-ISSN 1460-2431, Vol. 63, no 16, p. 6045-6056Article in journal (Refereed)
    Abstract [en]

    Cellulose synthase (CesA) genes constitute a complex multigene family with six major phylogenetic clades in angiosperms. The recently sequenced genome of domestic apple, Malus-domestica was mined for CesA genes, by blasting full-length cellulose synthase protein (CESA) sequences annotated in the apple genome against protein databases from the plant models Arabidopsis thaliana and Populus trichocarpa. Thirteen genes belonging to the six angiosperm CesA clades and coding for proteins with conserved residues typical of processive glycosyltransferases from family 2 were detected. Based on their phylogenetic relationship to Arabidopsis CESAs, as well as expression patterns, a nomenclature is proposed to facilitate further studies. Examination of their genomic organization revealed that MdCesA8-A is closely linked and co-oriented with WDR53, a gene coding for a WD40 repeat protein. The WDR53 and CesA8 genes display conserved collinearity in dicots and are partially co-expressed in the apple xylem. Interestingly, the presence of a bicistronic WDR53-CesA8A transcript was detected in phytoplasma-infected phloem tissues of apple. The bicistronic transcript contains a spliced intergenic sequence that is predicted to fold into hairpin structures typical of internal ribosome entry sites, suggesting its potential cap-independent translation. Surprisingly, the CesA8A cistron is alternatively spliced and lacks the zinc-binding domain. The possible roles of WDR53 and the alternatively spliced CESA8 variant during cellulose biosynthesis in M.-xdomestica are discussed.

  • 13. Hrmova, Maria
    et al.
    Farkas, Vladimir
    Harvey, Andrew J.
    Lahnstein, Jelle
    Wischmann, Bente
    Kaewthai, Nomchit
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ezcurra, Inés
    KTH, School of Biotechnology (BIO), Glycoscience.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Fincher, Geoffrey B.
    Substrate specificity and catalytic mechanism of a xyloglucan xyloglucosyl transferase HvXET6 from barley (Hordeum vulgare L.)2009In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 2, p. 437-456Article in journal (Refereed)
    Abstract [en]

    A family 16 glycoside hydrolase, xyloglucan xyloglucosyl transferase (EC 2.4.1.207), also known as xyloglucan endotransglycosylase (XET), and designated isoenzyme HvXET6, was purified approximately 400-fold from extracts of young barley seedlings. The complete amino acid sequence of HvXET6 was deduced from the nucleotide sequence of a near full-length cDNA, in combination with tryptic peptide mapping. An additional five to six isoforms or post-translationally modified XET enzymes were detected in crude seedling extracts of barley. The HvXET6 isoenzyme was expressed in Pichia pastoris, characterized and compared with the previously purified native HvXET5 isoform. Barley HvXET6 has a similar apparent molecular mass of 33-35 kDa to the previously purified HvXET5 isoenzyme, but the two isoenzymes differ in their isoelectric points, pH optima, kinetic properties and substrate specificities. The HvXET6 isoenzyme catalyses transfer reactions between xyloglucans and soluble cellulosic substrates, using oligo-xyloglucosides as acceptors, but at rates that are significantly different from those observed for HvXET5. No hydrolytic activity could be detected with either isoenzyme. Comparisons of the reaction rates using xyloglucan or hydroxyethyl cellulose as donors and a series of cellodextrins as acceptors indicated that the acceptor site of HvXET can accommodate five glucosyl residues. Molecular modelling supported this conclusion and further confirmed the ability of the enzyme's active site to accommodate xyloglucan and cellulosic substrates. The two HvXETs followed a ping-pong (Bi, Bi) rather than a sequential reaction mechanism.

  • 14. Kaewthai, Nomchit
    et al.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Glycoscience.
    Development of a high-throughput assay for screening xyloglucan endo-transglycosylase and endo-xyloglucanase expression in crude microbial supernatantsManuscript (preprint) (Other academic)
  • 15.
    Kaewthai, Nomchit
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Gendre, Delphine
    Eklöf, Jens M.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ibatullin, Farid M.
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bhalerao, Rishikesh P.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Group III-A XTH genes encode predominant xyloglucan endo hydrolase active in expanding tissues of Arabidopsis thalianaManuscript (preprint) (Other academic)
  • 16.
    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.

  • 17.
    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.

  • 18. Kim, H. U.
    et al.
    Cotter, R.
    Johnson, S.
    Senda, M.
    Dodds, P.
    Kulikauskas, R.
    Tang, W. H.
    Ezcurra, Inés
    KTH, Superseded Departments, Biotechnology.
    Herzmark, P.
    McCormick, S.
    New pollen-specific receptor kinases identified in tomato, maize and Arabidopsis: the tomato kinases show overlapping but distinct localization patterns on pollen tubes2002In: Plant Molecular Biology, ISSN 0167-4412, E-ISSN 1573-5028, Vol. 50, no 1, p. 1-16Article in journal (Refereed)
    Abstract [en]

    We previously characterized LePRK1 and LePRK2, pollen-specific receptor kinases from tomato (Muschietti et al., 1998). Here we identify a similar receptor kinase from maize, ZmPRK1, that is also specifically expressed late in pollen development, and a third pollen receptor kinase from tomato, LePRK3. LePRK3 is less similar to LePRK1 and LePRK2 than either is to each other. We used immunolocalization to show that all three LePRKs localize to the pollen tube wall, in partially overlapping but distinct patterns. We used RT-PCR and degenerate primers to clone homologues of the tomato kinases from other Solanaceae. We deduced features diagnostic of pollen receptor kinases and used these criteria to identify family members in the Arabidopsis database. RT-PCR confirmed pollen expression for five of these Arabidopsis candidates; two of these are clearly homologues of LePRK3. Our results reveal the existence of a distinct pollen-specific receptor kinase gene family whose members are likely to be involved in perceiving extracellular cues during pollen tube growth.

  • 19.
    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.

  • 20. Ratke, Christine
    et al.
    Pawar, Prashant Mohan-Anupama
    Balasubramanian, Vimal K.
    Naumann, Marcel
    Duncranz, Mathilda Lönnäs
    Derba-Maceluch, Marta
    Gorzsas, Andras
    Endo, Satoshi
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Mellerowicz, Ewa J.
    Populus GT43 family members group into distinct sets required for primary and secondary wall xylan biosynthesis and include useful promoters for wood modification2015In: Plant Biotechnology Journal, ISSN 1467-7644, E-ISSN 1467-7652, Vol. 13, no 1, p. 26-37Article in journal (Refereed)
    Abstract [en]

    The plant GT43 protein family includes xylosyltransferases that are known to be required for xylan backbone biosynthesis, but have incompletely understood specificities. RT-qPCR and histochemical (GUS) analyses of expression patterns of GT43 members in hybrid aspen, reported here, revealed that three clades of the family have markedly differing specificity towards secondary wall-forming cells (wood and extraxylary fibres). Intriguingly, GT43A and B genes (corresponding to the Arabidopsis IRX9 clade) showed higher specificity for secondary-walled cells than GT43C and D genes (IRX14 clade), although both IRX9 and IRX14 are required for xylosyltransferase activity. The remaining genes, GT43E, F and G (IRX9-L clade), showed broad expression patterns. Transient transactivation analyses of GT43A and B reporters demonstrated that they are activated by PtxtMYB021 and PNAC085 (master secondary wall switches), mediated in PtxtMYB021 activation by an AC element. The high observed secondary cell wall specificity of GT43B expression prompted tests of the efficiency of its promoter (pGT43B), relative to the CaMV 35S (35S) promoter, for overexpressing a xylan acetyl esterase (CE5) or downregulating REDUCED WALL ACETYLATION (RWA) family genes and thus engineering wood acetylation. CE5 expression was weaker when driven by pGT43B, but it reduced wood acetyl content substantially more efficiently than the 35S promoter. RNAi silencing of the RWA family, which was ineffective using 35S, was achieved when using GT43B promoter. These results show the utility of the GT43B promoter for genetically engineering properties of wood and fibres.

  • 21. Reidt, W.
    et al.
    Wohlfarth, T.
    Ellerstrom, M.
    Czihal, A.
    Tewes, A.
    Ezcurra, Inés
    KTH, Superseded Departments, Biotechnology.
    Rask, L.
    Baumlein, H.
    Gene regulation during late embryogenesis: the RY motif of maturation-specific gene promoters is a direct target of the FUS3 gene product2000In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 21, no 5, p. 401-408Article in journal (Refereed)
    Abstract [en]

    The Arabidopsis mutants fus3 and abi3 show pleiotropic effects during embryogenesis including reduced levels of transcripts encoding embryo-specific seed proteins. To investigate the interaction between the BO-domain-containing transcription factors FUSS and ABI3 with the RY cis-motif, conserved in many seed-specific promoters, a promoter analysis as well as band-shift experiments were performed. The analysis of promoter mutants revealed the structural requirements for the function of the RY cis-element. It is shown that both the nucleotide sequence and the alternation of purin and pyrimidin nucleotides (RY character) are essential for the activity of the motif. Further, it was shown that FUSS and ABI3 can act independently of each other in controlling promoter activity and that the RY cis-motif is a target for both transcription factors. For FUSS, which is so far the smallest known member of the B3-domain family, a physical interaction with the RY motif was established. The functional and biochemical data demonstrate that the regulators FUSS and ABI3 are essential components of a regulatory network acting in concert through the RY-promoter element to control gene expression during late embryogenesis and seed development.

  • 22. 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)
  • 23. Tang, W. H.
    et al.
    Ezcurra, Inés
    KTH, Superseded Departments, Biotechnology.
    Muschietti, J.
    McCormick, S.
    A cysteine-rich extracellular protein, LAT52, interacts with the extracellular domain of the pollen receptor kinase LePRK22002In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 14, no 9, p. 2277-2287Article in journal (Refereed)
    Abstract [en]

    Pollen germination and pollen tube growth are thought to require extracellular cues, but how these cues are perceived and transduced remains largely unknown. Pollen receptor kinases are plausible candidates for this role; they might bind extracellular ligands and thereby mediate cytoplasmic events required for pollen germination and pollen tube growth. To search for pollen-expressed ligands for pollen receptor kinases, we used the extracellular domains of three pollen-specific receptor kinases of tomato (LePRK1, LePRK2, and LePRK3) as baits in a yeast two-hybrid screen. We identified numerous secreted or plasma membrane-bound candidate ligands. One of these, the Cys-rich protein LAT52, was known to be essential during pollen hydration and pollen tube growth. We used in vivo coimmunoprecipitation to demonstrate that LAT52 was capable of forming a complex with LePRK2 in pollen and to show that the extracellular domain of LePRK2 was sufficient for the interaction. Soluble LAT52 can exist in differently sized forms, but only the larger form can interact with LePRK2. We propose that LAT52 might be a ligand for LePRK2.

  • 24. Tang, W. H.
    et al.
    Kelley, D.
    Ezcurra, Inés
    KTH, Superseded Departments, Biotechnology.
    Cotter, R.
    McCormick, S.
    LeSTIG1, an extracellular binding partner for the pollen receptor kinases LePRK1 and LePRK2, promotes pollen tube growth in vitro2004In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 39, no 3, p. 343-353Article in journal (Refereed)
    Abstract [en]

    As pollen tubes grow through the pistil they are thought to perceive and respond to diverse signals. The tomato pollen-specific receptor kinases LePRK1 and LePRK2 might participate in signaling during pollen tube growth. We previously showed that the extracellular domain of LePRK2 interacts with a pollen protein, LAT52, before but not after pollen germination. To determine whether LePRK2 might have different binding partner(s) after pollen germination, we characterized two more proteins that, like LAT52, were identified in yeast two-hybrid screens using the extracellular domains of LePRK1 and LePRK2 as baits. We show that LeSHY, a leucine-rich repeat protein from pollen, and LeSTIG1, a small cysteine-rich protein from pistil, can bind the extracellular domains of both LePRK1 and LePRK2 in vitro. In vitro binding assays with the extracellular domain of LePRK2 suggested that LeSTIG1 could displace binding of LAT52, consistent with the idea that LePRK1 and LePRK2 might interact with different ligands at different stages of pollen tube growth. Exogenous LeSTIG1 promotes pollen tube growth in vitro. The interaction of these pollen kinases with LeSTIG1 supports the notion that LePRK1 and LePRK2 are involved in mediating pollen-pistil interactions.

  • 25.
    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)
  • 26.
    Wang, Yang
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Azhar, Shoaib
    KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center. KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Gandini, Rosaria
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Karolinska Institutet, Stockholm.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Karolinska Institutet, Stockholm.
    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 thaliana2015In: Plant Science, ISSN 0168-9452, E-ISSN 1873-2259, Vol. 241, p. 151-163Article in journal (Refereed)
    Abstract [en]

    Plant mannanases are enzymes that carry out fundamentally important functions in cell wall metabolism during plant growth and development by digesting manno-polysaccharides. In this work, the Arabidopsis mannanase 5-2 (AtMan5-2) from a previously uncharacterized subclade of glycoside hydrolase family 5 subfamily 7 (GH5_7) has been heterologously produced in Pichia pastoris. Purified recombinant AtMan5-2 is a glycosylated protein with an apparent molecular mass of 50 kDa, a pH optimum of 5.5-6.0 and a temperature optimum of 25 degrees C. The enzyme exhibits high substrate affinity and catalytic efficiency on mannan substrates with main chains containing both glucose and mannose units such as konjac glucomannan and spruce galactoglucomannan. Product analysis of manno-oligosaccharide hydrolysis shows that AtMan5-2 requires at least six substrate-binding subsites. No transglycosylation activity for the recombinant enzyme was detected in the present study. Our results demonstrate diversification of catalytic function among members in the Arabidopsis GH5_7 subfamily.

  • 27.
    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)
  • 28.
    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.

  • 29.
    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)
  • 30.
    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.

  • 31.
    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)
  • 32. Wycliffe, P.
    et al.
    Sitbon, F.
    Wernersson, J.
    Ezcurra, Inés
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ellerstrom, M.
    Rask, L.
    Continuous expression in tobacco leaves of a Brassica napus PEND homologue blocks differentiation of plastids and development of palisade cells2005In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 44, no 1, p. 1-15Article in journal (Refereed)
    Abstract [en]

    Brassica napus complementary deoxyribonucleic acid (cDNA) clones encoding a DNA-binding protein, BnPEND, were isolated by Southwestern screening. A distinctive feature of the protein was a bZIP-like sequence in the amino-terminal portion, which, after expression in Escherichia coli, bound DNA. BnPEND transcripts were present in B. napus roots and flower buds, and to a lesser extent in stems, flowers and young leaves. Treatment in the dark for 72 h markedly increased the amount of BnPEND transcript in leaves of all ages. Sequence comparison showed that BnPEND was similar to a presumed transcription factor from B. napus, GSBF1, a protein deduced from an Arabidopsis thaliana cDNA (BX825084) and the PEND protein from Pisum sativum, believed to anchor the plastid DNA to the envelope early during plastid development. Homology to expressed sequence tag (EST) sequences from additional species suggested that BnPEND homologues are widespread among the angiosperms. Transient expression of BnPEND fused with green fluorescent protein (GFP) in Nicotiana benthamiana epidermal cells showed that BnPEND is a plastid protein, and that the 15 amino acids at the amino-terminal contain information about plastid targeting. Expression of BnPEND in Nicotiana tabacum from the Cauliflower Mosaic Virus 35S promoter gave stable transformants with different extents of white to light-green areas in the leaves, and even albino plants. In the white areas, but not in adjacent green tissue, the development of palisade cells and chloroplasts was disrupted. Our data demonstrate that the BnPEND protein, when over-expressed at an inappropriate stage, functionally blocks the development of plastids and leads to altered leaf anatomy, possibly by preventing the release of plastid DNA from the envelope.

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