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  • 1. Alvarez, Francisco J.
    et al.
    Ryman, Kicki
    Hooijmaijers, Cornelis
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ljungdahl, Per O.
    Diverse Nitrogen Sources in Seminal Fluid Act in Synergy To Induce Filamentous Growth of Candida albicans2015In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 81, no 8, 2770-2780 p.Article in journal (Refereed)
    Abstract [en]

    The pathogenic fungus Candida albicans is the leading cause of vulvovaginal candidiasis (VVC). VVC represents a major quality- of-life issue for women during their reproductive years, a stage of life where the vaginal epithelium is subject to periodic hormonally induced changes associated with menstruation and concomitant exposure to serum as well as potential intermittent contact with seminal fluid. Seminal fluid potently triggers Candida albicans to switch from yeastlike to filamentous modes of growth, a developmental response tightly linked to virulence. Conversely, vaginal fluid inhibits filamentation. Here, we used artificial formulations of seminal and vaginal fluids that faithfully mimic genuine fluids to assess the contribution of individual components within these fluids to filamentation. The high levels of albumin, amino acids, and N-acetylglucosamine in seminal fluid act synergistically as potent inducers of filamentous growth, even at atmospheric levels of CO2 and reduced temperatures (30 degrees C). Using a simplified in vitro model that mimics the natural introduction of seminal fluid into the vulvovaginal environment, a pulse of artificial seminal fluid (ASF) was found to exert an enduring potential to overcome the inhibitory efficacy of artificial vaginal fluid (AVF) on filamentation. These findings suggest that a transient but substantial change in the nutrient levels within the vulvovaginal environment during unprotected coitus can induce resident C. albicans cells to engage developmental programs associated with virulent growth.

  • 2. Badreddine, Ilham
    et al.
    Lafitte, Claude
    Heux, Laurent
    Skandalis, Nicholas
    Spanou, Zacharoula
    Martinez, Yves
    Esquerre-Tugaye, Marie-Therese
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Dumas, Bernard
    Bottin, Arnaud
    Cell Wall Chitosaccharides Are Essential Components and Exposed Patterns of the Phytopathogenic Oomycete Aphanomyces euteiches2008In: Eukaryotic Cell, ISSN 1535-9778, E-ISSN 1535-9786, Vol. 7, no 11, 1980-1993 p.Article in journal (Refereed)
    Abstract [en]

    Chitin is an essential component of fungal cell walls, where it forms a crystalline scaffold, and chitooligosaccharides derived from it are signaling molecules recognized by the hosts of pathogenic fungi. Oomycetes are cellulosic fungus-like microorganisms which most often lack chitin in their cell walls. Here we present the first study of the cell wall of the oomycete Aphanomyces euteiches, a major parasite of legume plants. Biochemical analyses demonstrated the presence of ca. 10% N-acetyl-D-glucosamine (GlcNAc) in the cell wall. Further characterization of the GlcNAc-containing material revealed that it corresponds to noncrystalline chitosaccharides associated with glucans, rather than to chitin per se. Two putative chitin synthase (CHS) genes were identified by data mining of an A. euteiches expressed sequence tag collection and Southern blot analysis, and full-length cDNA sequences of both genes were obtained. Phylogeny analysis indicated that oomycete CHS diversification occurred before the divergence of the major oomycete lineages. Remarkably, lectin labeling showed that the Aphanomyces euteiches chitosaccharides are exposed at the cell wall surface, and study of the effect of the CHS inhibitor nikkomycin Z demonstrated that they are involved in cell wall function. These data open new perspectives for the development of antioomycete drugs and further studies of the molecular mechanisms involved in the recognition of pathogenic oomycetes by the host plants.

  • 3. Belmonte, Rodrigo
    et al.
    Wang, Tiehui
    Duncan, Gary J.
    Skaar, Ida
    Melida, Hugo
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    van West, Pieter
    Secombes, Christopher J.
    Role of Pathogen-Derived Cell Wall Carbohydrates and Prostaglandin E-2 in Immune Response and Suppression of Fish Immunity by the Oomycete Saprolegnia parasitica2014In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 82, no 11, 4518-4529 p.Article in journal (Refereed)
    Abstract [en]

    Saprolegnia parasitica is a freshwater oomycete that is capable of infecting several species of fin fish. Saprolegniosis, the disease caused by this microbe, has a substantial impact on Atlantic salmon aquaculture. No sustainable treatment against saprolegniosis is available, and little is known regarding the host response. In this study, we examined the immune response of Atlantic salmon to S. parasitica infection and to its cell wall carbohydrates. Saprolegnia triggers a strong inflammatory response in its host (i. e., induction of interleukin-1 beta(1) [IL-1 beta(1)], IL-6, and tumor necrosis factor alpha), while severely suppressing the expression of genes associated with adaptive immunity in fish, through downregulation of T-helper cell cytokines, antigen presentation machinery, and immunoglobulins. Oomycete cell wall carbohydrates were recognized by fish leukocytes, triggering upregulation of genes involved in the inflammatory response, similar to what is observed during infection. Our data suggest that S. parasitica is capable of producing prostaglanding E-2 (PGE(2)) in vitro, a metabolite not previously shown to be produced by oomycetes, and two proteins with homology to vertebrate enzymes known to play a role in prostaglandin biosynthesis have been identified in the oomycete genome. Exogenous PGE(2) was shown to increase the inflammatory response in fish leukocytes incubated with cell wall carbohydrates while suppressing genes involved in cellular immunity (gamma interferon [IFN-gamma] and the IFN-gamma-inducible protein [gamma-IP]). Inhibition of S. parasitica zoospore germination and mycelial growth by two cyclooxygenase inhibitors (aspirin and indomethacin) also suggests that prostaglandins may be involved in oomycete development.

  • 4. Bessueille, Laurence
    et al.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    A survey of cellulose biosynthesis in higher plants2008In: PLANT BIOTECHNOLOGY, ISSN 1342-4580, Vol. 25, no 3, 315-322 p.Article in journal (Refereed)
    Abstract [en]

    Cellulose plays a central role in plant development and its biosynthesis represents one of the most important biochemical processes in plant biology. However, the corresponding molecular mechanisms are not well understood, despite the progress made in the past years in the identification of genes that code for the catalytic subunits of the cellulose synthases and other proteins potentially involved in cellulose formation. A major bottleneck is the high instability of the cellulose synthase complexes and their location in the plasma membrane. Additional efforts are currently being made to unravel the mechanisms of cellulose biosynthesis. Indeed, understanding how cellulose is formed and how its crystallinity is achieved is relevant not only for studying plant development, but also for improving the digestibility of the plant biomass, which is foreseen as an alternative to fossil fuels for the production of energy. This review summarizes the major unanswered questions related to the process of cellulose biosynthesis, and describes the recent progress that has been made in the area through the combination of biochemical approaches and molecular genetics.

  • 5. Bessueille, Laurence
    et al.
    Sindt, Nicolas
    Guichardant, Michel
    Djerbi, Soraya
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Plasma membrane microdomains from hybrid aspen cells are involved in cell wall polysaccharide biosynthesis2009In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 420, 93-103 p.Article in journal (Refereed)
    Abstract [en]

    Detergent-resistant plasma membrane microdomains [DRMs (detergent-resistant membranes)] were isolated recently from several plant species. As for animal cells, a large range of cellular functions, such as signal transduction, endocytosis and protein trafficking, have been attributed to plant lipid rafts and DRMs. The data available are essentially based on protcomics and more approaches need to be undertaken to elucidate the precise function of individual populations of DRMs in plants. We report here the first isolation of DRMs from purified plasma membranes of a tree species, the hybrid aspen Populus tremula x tremuloides, and their biochemical characterization. Plasma membranes were solubilized with Triton X-100 and the resulting DRMs were isolated by flotation in sucrose density gradients. The DRMs were enriched in sterols, sphingolipids and glycosylphosphatidylinositol-anchored proteins and thus exhibited similar properties to DRMs from other species. However, they contained key carbohydrate synthases involved in cell wall polysaccharide biosynthesis, namely callose [(1 -> 3)-beta-D-glucan] and cellulose synthases. The association of these enzymes with DRMs was demonstrated using specific glucan synthase assays and antibodies, as well as biochemical and chemical approaches for the characterization of the polysaccharides synthesized in vitro by the isolated DRMs. More than 70% of the total glucan synthase activities present in the original plasma membranes was associated with the DRM fraction. In addition to shedding light on the lipid environment of callose and cellulose synthases, our results demonstrate the involvement of DRMs in the biosynthesis of important cell wall polysaccharides. This novel concept suggests a function of plant membrane microdomains in cell growth and morphogenesis.

  • 6. Bottin, Armaud
    et al.
    Brown, Christian
    KTH, School of Biotechnology (BIO), Glycoscience.
    Mélida, Hugo
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    The phytopathogenic oomycete Aphanomyces euteiches contains two distinct N-acetylglucosaminyltransferase activities that form chitin-like saccharides in vitroManuscript (preprint) (Other academic)
  • 7. Bouzenzana, Jamel
    et al.
    Pelosi, Ludovic
    Briolay, Anne
    Briolay, Jerome
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Identification of the first Oomycete annexin as a (1 -> 3)-beta-D-glucan synthase activator2006In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 62, no 2, 552-565 p.Article in journal (Refereed)
    Abstract [en]

    (1 -> 3)-beta-D-Glucans are major components of the cell walls of Oomycetes and as such they play an essential role in the morphogenesis and growth of these microorganisms. Despite the biological importance of (1 -> 3)-beta-D-glucans, their mechanisms of biosynthesis are poorly understood. Previous studies on (1 -> 3)-beta-D-glucan synthases from Saprolegnia monoica have shown that three protein bands of an apparent molecular weight of 34, 48 and 50 kDa co-purify with enzyme activity. However, none of the corresponding proteins have been identified. Here we have identified, purified, sequenced and characterized a protein from the 34 kDa band and clearly shown that it has all the biochemical properties of proteins from the annexin family. In addition, we have unequivocally demonstrated that the purified protein is an activator of (1 -> 3)-beta-D-glucan synthase. This represents a new type of function for proteins belonging to the annexin family. Two other proteins from the 48 and 50 kDa bands were identified as ATP synthase subunits, which most likely arise from contaminations by mitochondria during membrane preparation. The results, which are discussed in relation with the possible regulation mechanisms of (1 -> 3)-beta-D-glucan synthases, represent a first step towards a better understanding of cell wall polysaccharide biosynthesis in Oomycetes.

  • 8. Briolay, Anne
    et al.
    Bouzenzana, Jamel
    Guichardant, Michel
    Deshayes, Christian
    Sindt, Nicolas
    Bessueille, Laurence
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Cell Wall Polysaccharide Synthases Are Located in Detergent-Resistant Membrane Microdomains in Oomycetes2009In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 75, no 7, 1938-1949 p.Article in journal (Refereed)
    Abstract [en]

    The pathways responsible for cell wall polysaccharide biosynthesis are vital in eukaryotic microorganisms. The corresponding synthases are potential targets of inhibitors such as fungicides. Despite their fundamental and economical importance, most polysaccharide synthases are not well characterized, and their molecular mechanisms are poorly understood. With the example of Saprolegnia monoica as a model organism, we show that chitin and (1 -> 3)-beta-D-glucan synthases are located in detergent-resistant membrane microdomains (DRMs) in oomycetes, a phylum that comprises some of the most devastating microorganisms in the agriculture and aquaculture industries. Interestingly, no cellulose synthase activity was detected in the DRMs. The purified DRMs exhibited similar biochemical features as lipid rafts from animal, plant, and yeast cells, although they contained some species-specific lipids. This report sheds light on the lipid environment of the (1 -> 3)-beta-D-glucan and chitin synthases, as well as on the sterol biosynthetic pathways in oomycetes. The results presented here are consistent with a function of lipid rafts in cell polarization and as platforms for sorting specific sets of proteins targeted to the plasma membrane, such as carbohydrate synthases. The involvement of DRMs in the biosynthesis of major cell wall polysaccharides in eukaryotic microorganisms suggests a function of lipid rafts in hyphal morphogenesis and tip growth.

  • 9.
    Brown, Christian
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Leijon, Felicia
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    Radiometric and spectrophotometric in vitro assays of glycosyltransferases involved in plant cell wall carbohydrate biosynthesis2012In: Nature Protocols, ISSN 1754-2189, Vol. 7, no 9, 1634-1650 p.Article in journal (Refereed)
    Abstract [en]

    Most of the glycosyltransferases (GTs) that catalyze the formation of plant cell wall carbohydrates remain to be biochemically characterized. This can be achieved only if specific assays are available for these enzymes. Here we present a protocol for in vitro assays of processive and nonprocessive membrane-bound GTs. The assays are either based on the use of radioactive nucleotide sugars (NDP sugars; e.g., UDP-[U-C-14] glucose) and the quantification of the radiolabeled monosaccharides incorporated into soluble or insoluble carbohydrates, or on the coupling of the GT reaction with that of pyruvate kinase (PK) and the oxidation of NADH by lactate dehydrogenase (LDH). The radiometric assays are more suitable for exploratory work on poorly characterized enzymes, whereas the spectrophotometric assays require the availability of highly enriched GTs. Both assays can be performed within 1 d, depending on the number of fractions to be assayed or reaction mixtures to be tested.

  • 10.
    Brown, Christian
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Szpryngie, Scarlett
    Kuang, Guanglin
    Srivastava, Vaibhav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ye, Weihua
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Tu, Yaoquan
    Mäler, Lena
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Structural and functional characterization of the “Microtubule Interacting and Trafficking": domains of two oomycetes chitin synthasesManuscript (preprint) (Other academic)
  • 11.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Analysis of 1,3-beta-glucans and cellulose synthesized in vitro: a key step towards the characterization of glucan synthases2007In: Cellulose: molecular and structural biology / [ed] R.M. Brown Jr; I.M. Saxena, Springer, 2007, 123-145 p.Chapter in book (Refereed)
  • 12.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bacterial cellulose-based biomimetic composites2010In: Biopolymers / [ed] M. Elnashar, Sciyo, 2010, 345-368 p.Chapter in book (Refereed)
  • 13.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Biosynthetic enzymes for (1,3)-β-glucans and (1,3;1,6)-β-glucans in protozoans and chromistans. biochemical characterization and molecular biology2009In: Chemistry, biochemistry and biology of 1,3-beta-glucans and related polysaccharides / [ed] A. Bacic, B.A. Stone and G.B. Fincher, Elsevier, 2009, 233-258 p.Chapter in book (Refereed)
    Abstract [en]

    This chapter discusses the biochemical characterization and molecular biology of biosynthetic enzymes for (1,3)-β-glucans and (1,3:1,6)-β-Glucans in Protozoans and Chromistans. The biosynthesis of linear β-glucans is generally considered to involve several steps. The process starts with the synthesis of an initiator of polymerization or primer, followed by the transfer of the primer to a membrane-bound synthase. The latter catalyses the repetitive transfer of glucosyl units from an activated sugar donor to the acceptor-i.e. the primer or the elongating chain-until the polymerization stops. Most molecular events occurring during the biosynthesis of (1,3)-β-glucans and (1,3;1,6)-β-Glucans in Protozoans and Chromistans are not well understood. The process for which most biochemical data have been accumulated is the polymerization of linear (1,3)-β-glucan chains. The ability to synthesize β-glucans in vitro represents a very useful tool for assaying glucan synthases during enzyme purification. This is also useful in identifying the type of effectors that might influence enzyme activity in vivo and thus regulate the biosynthesis of the polysaccharides.

  • 14.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Cellulose biosynthesis in higher plants2011In: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 241Article in journal (Other academic)
  • 15.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO).
    Oligo-beta-1,3-glucans having an allergic reaction inhibiting effect, and applications thereof2006Patent (Other (popular science, discussion, etc.))
  • 16.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Unraveling the secrets of cellulose biosynthesis in plants using biochemical approaches: prospects for the future2006In: The science and lore of the plant cell wall: Biosynthesis, structure and function / [ed] Takahisa Hayashi, BrownWalker Press, 2006, 87-96 p.Chapter in book (Refereed)
  • 17.
    Bulone, Vincent
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Guerriero, Gea
    De Block, Marck
    Meulewaeter, Frank
    Methods for manufacturing plant cell walls comprising chitin2010Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    Methods and means are provided for the modification of the reactivity of plant secondary cell walls, particularly in cotton cell walls found in cotton fibers. This can be conveniently achieved by expressing a chimeric gene encoding a Saprolegnia monoica chitin synthase in cotton plants.

  • 18.
    Bulone, Vincent
    et al.
    KTH, Superseded Departments, Biotechnology.
    Rademaker, G. J.
    Pergantis, S.
    Krogstad-Johnsen, T.
    Smedstad-Paulsen, B.
    Thomas-Oates, J.
    Characterisation of horse dander allergen glycoproteins using amino acid and glycan structure analyses - A mass spectrometric method for glycan chain analysis of glycoproteins separated by two-dimensional electrophoresis2000In: International Archives of Allergy and Immunology, ISSN 1018-2438, E-ISSN 1423-0097, Vol. 123, no 3, 220-227 p.Article in journal (Refereed)
    Abstract [en]

    Separation of horse dander allergens using two-dimensional PAGE resulted in the identification of 16 proteins that react with allergic patient sera. A sensitive method has been developed for analysing the structures of the glycan chains of individual glycoprotein allergens transferred to blots following two-dimensional PAGE, and has allowed the structural identification of the glycan chains of the most abundant isoforms of Equ c 1, a glycosylated horse dander major allergen. The method involves separation of the allergens by two-dimensional PAGE, transfer to polyvinylidene difluoride membranes, release of the glycan chains using peptide N-glycosidase F, permethylation and mass spectrometric analysis of the derivatised glycans. The amino acid compositions of the 16 horse dander allergens separated by two-dimensional PAGE have been determined, allowing the identification of the various isoforms of Equ c 1. These results also confirmed that the two non-glycosylated major allergens, Equ c 2.0101 and Equ c 2.0102, belong to the lipocalin family, and support the idea that these two allergens are most probably isoforms of the same protein. The glycan structures identified using the mass spectrometric method are common biantennary and triantennary glycan chains. These carbohydrate moieties may have a role in the binding of IgE; however, it is more likely that the overall glycoprotein structure involving both the glycan and protein moieties, rather than the structure of the glycan chains alone, is responsible for eliciting allergic responses.

  • 19. Butchosa, Nria
    et al.
    Brown, Christian
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Berglund, Lars A.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Antimicrobial activity of biocomposites based on bacterial cellulose and chitin nanoparticles2012In: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 243Article in journal (Other academic)
  • 20.
    Butchosa, Nuria
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Brown, Christian
    KTH, School of Biotechnology (BIO), Glycoscience.
    Larsson, Per Tomas
    KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Berglund, Lars A.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Nanocomposites of bacterial cellulose nanofibers and chitin nanocrystals: fabrication, characterization and bactericidal activity2013In: Green Chemistry, ISSN 1463-9262, E-ISSN 1463-9270, Vol. 15, no 12, 3404-3413 p.Article in journal (Refereed)
    Abstract [en]

    An environmentally friendly approach was implemented for the production of nanocomposites with bactericidal activity, using bacterial cellulose (BC) nanofibers and chitin nanocrystals (ChNCs). The antibacterial activity of ChNCs prepared by acid hydrolysis, TEMPO-mediated oxidation or partial deacetylation of a-chitin powder was assessed and the structure of the ChNC nanoparticles was characterized by X-ray diffraction, atomic force microscopy, and solid-state C-13-NMR. The partially deacetylated ChNCs (D-ChNC) showed the strongest antibacterial activity, with 99 +/- 1% inhibition of bacterial growth compared to control samples. Nanocomposites were prepared from BC nanofibers and D-ChNC by (i) in situ biosynthesis with the addition of D-ChNC nanoparticles in the culture medium of Acetobacter aceti, and (ii) post-modification by mixing D-ChNC with disintegrated BC in an aqueous suspension. The structure and mechanical properties of the BC/D-ChNC nanocomposites were characterized by Fourier transform infrared spectroscopy, elemental analysis, field-emission scanning electron microscopy, and an Instron universal testing machine. The bactericidal activity of the nanocomposites increased with the D-ChNC content, with a reduction in bacterial growth by 3.0 log units when the D-ChNC content was 50%. D-ChNC nanoparticles have great potential as substitutes for unfriendly antimicrobial compounds such as heavy metal nanoparticles and synthetic polymers to introduce antibacterial properties to cellulosic materials.

  • 21.
    Butchosa, Nuria
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Leijon, Felicia
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Enhancing toughness of cellulose nanofibrils through the expression of cellulose-binding modules in plantManuscript (preprint) (Other academic)
  • 22. 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, 146-156 p.Article 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.

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

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

  • 24.
    Dahlin, Paul
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. Stockholm Univ, Sweden.
    Müller, Marion C.
    KTH, School of Biotechnology (BIO), Glycoscience. Stockholm Univ, Sweden.
    Ekengren, Sophia
    KTH, School of Biotechnology (BIO), Glycoscience. Stockholm Univ, Sweden.
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. Univ Adelaide, Australia.
    The Impact of Steroidal Glycoalkaloids on the Physiology of Phytophthora infestans, the Causative Agent of Potato Late Blight2017In: Molecular Plant-Microbe Interactions, ISSN 0894-0282, E-ISSN 1943-7706, Vol. 30, no 7, 531-542 p.Article in journal (Refereed)
    Abstract [en]

    Steroidal glycoalkaloids (SGAs) are plant secondary metabolites known to be toxic to animals and humans and that have putative roles in defense against pests. The proposed mechanisms of SGA toxicity are sterol-mediated disruption of membranes and inhibition of cholinesterase activity in neurons. It has been suggested that phytopathogenic microorganisms can overcome SGA toxicity by enzymatic deglycosylation of SGAs. Here, we have explored SGA-mediated toxicity toward the invasive oomycete Phytophthora infestans, the causative agent of the late blight disease in potato and tomato, as well as the potential for SGA deglycosylation by this species. Our growth studies indicate that solanidine, the nonglycosylated precursor of the potato SGAs a-chaconine and a-solanine, has a greater physiological impact than its glycosylated forms. All of these compounds were incorporated into the mycelium, but only solanidine could strongly inhibit the growth of P. infestans in liquid culture. Genes encoding several glycoside hydrolases with potential activity on SGAs were identified in the genome of P. infestans and were shown to be expressed. However, we found no indication that deglycosylation of SGAs takes place. We present additional evidence for apparent host-specific adaptation to potato SGAs and assess all results in terms of future pathogen management strategies.

  • 25.
    Dahlin, Paul
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. Stockholm University, Sweden.
    Srivastava, Vaibhav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. University of Adelaide, Australia.
    Mckee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    The Oxidosqualene Cyclase from the Oomycete Saprolegnia parasitica Synthesizes Lanosterol as a Single Product2016In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 7, 1802Article in journal (Refereed)
    Abstract [en]

    The first committed step of sterol biosynthesis is the cyclisation of 2,3-oxidosqualene to form either lanosterol (LA) or cycloartenol (CA). This is catalyzed by an oxidosqualene cyclase (OSC). LA and CA are subsequently converted into various sterols by a series of enzyme reactions. The specificity of the OSC therefore determines the final composition of the end sterols of an organism. Despite the functional importance of OSCs, the determinants of their specificity are not well understood. In sterol-synthesizing oomycetes, recent bioinformatics, and metabolite analysis suggest that LA is produced. However, this catalytic activity has never been experimentally demonstrated. Here, we show that the OSC of the oomycete Saprolegnia parasitica, a severe pathogen of salmonid fish, has an uncommon sequence in a conserved motif important for specificity. We present phylogenetic analysis revealing that this sequence is common to sterol-synthesizing oomycetes, as well as some plants, and hypothesize as to the evolutionary origin of some microbial sequences. We also demonstrate for the first time that a recombinant form of the OSC from S. parasitica produces LA exclusively. Our data pave the way for a detailed structural characterization of the protein and the possible development of specific inhibitors of oomycete OSCs for disease control in aquaculture.

  • 26.
    Dahlin, Paul
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Srivastava, Vaibhav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ekengren, Sophia
    KTH, School of Biotechnology (BIO), Glycoscience.
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Comparative analysis of sterol acquisition in the oomycetes Saprolegnia parasitica and Phytophthora infestans2017In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 2, e0170873Article in journal (Refereed)
    Abstract [en]

    The oomycete class includes pathogens of animals and plants which are responsible for some of the most significant global losses in agriculture and aquaculture. There is a need to replace traditional chemical means of controlling oomycete growth with more targeted approaches, and the inhibition of sterol synthesis is one promising area. To better direct these efforts, we have studied sterol acquisition in two model organisms: the sterol-autotrophic Saprolegnia parasitica, and the sterol-heterotrophic Phytophthora infestans. We first present a comprehensive reconstruction of a likely sterol synthesis pathway for S. parasitica, causative agent of the disease saprolegniasis in fish. This pathway shows multiple potential routes of sterol synthesis, and draws on several avenues of new evidence: bioinformatic mining for genes with sterol-related functions, expression analysis of these genes, and analysis of the sterol profiles in mycelium grown in different media. Additionally, we explore the extent to which P. infestans, which causes the late blight in potato, can modify exogenously provided sterols. We consider whether the two very different approaches to sterol acquisition taken by these pathogens represent any specific survival advantages or potential drug targets.

  • 27. Douchkov, D.
    et al.
    Lueck, S.
    Hensel, G.
    Kumlehn, J.
    Rajaraman, J.
    Johrde, A.
    Doblin, M. S.
    Beahan, C. T.
    Kopischke, M.
    Fuchs, R.
    Lipka, V.
    Niks, R. E.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. Univ Adelaide, Australia.
    Chowdhury, J.
    Little, A.
    Burton, R. A.
    Bacic, A.
    Fincher, G. B.
    Schweizer, P.
    The barley (Hordeum vulgare) cellulose synthase-like D2 gene (HvCslD2) mediates penetration resistance to host-adapted and nonhost isolates of the powdery mildew fungus2016In: New Phytologist, ISSN 0028-646X, E-ISSN 1469-8137, Vol. 212, no 2, 421-433 p.Article in journal (Refereed)
    Abstract [en]

    Cell walls and cellular turgor pressure shape and suspend the bodies of all vascular plants. In response to attack by fungal and oomycete pathogens, which usually breach their host's cell walls by mechanical force or by secreting lytic enzymes, plants often form local cell wall appositions (papillae) as an important first line of defence. The involvement of cell wall biosynthetic enzymes in the formation of these papillae is still poorly understood, especially in cereal crops. To investigate the role in plant defence of a candidate gene from barley (Hordeum vulgare) encoding cellulose synthase-like D2 (HvCslD2), we generated transgenic barley plants in which HvCslD2 was silenced through RNA interference (RNAi). The transgenic plants showed no growth defects but their papillae were more successfully penetrated by host-adapted, virulent as well as avirulent nonhost isolates of the powdery mildew fungus Blumeria graminis. Papilla penetration was associated with lower contents of cellulose in epidermal cell walls and increased digestion by fungal cell wall degrading enzymes. The results suggest that HvCslD2-mediated cell wall changes in the epidermal layer represent an important defence reaction both for nonhost and for quantitative host resistance against nonadapted wheat and host-adapted barley powdery mildew pathogens, respectively.

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

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

  • 29. Faijes, M.
    et al.
    Ima, T.
    Bulone, Vincent
    KTH, Superseded Departments, Biotechnology.
    Planas, A.
    In vitro synthesis of a crystalline (1 -> 3,1 -> 4)-beta-D-glucan by a mutated (1 -> 3,1 -> 4)-beta-D-glucanase from Bacillus2004In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 380, 635-641 p.Article in journal (Refereed)
    Abstract [en]

    Oligo- and poly-saccharides have a large number of important biological functions, and they occur in natural composite materials, such as plant cell walls, where they self-assemble during biosynthesis in a poorly understood manner. They can also be used for the formation of artificial composite materials with industrial applications. Fundamental and applied research in biology and nanobiotechnology would benefit from the possibility of synthesizing tailor-made oligo-/poly-saccharides. In the present paper, we demonstrate that such syntheses are possible using genetically modified glycoside hydrolases, i.e. glycosynthases. The ability of the endoglycosynthase derived from Bacillus ( 1 --> 3,1 --> 4)-beta-D-glucanase to catalyse self-condensation of sugar donors was exploited for the in vitro synthesis of a regular polysaccharicle. The specificity of the enzyme allowed the polymerization of alpha-laminaribiosyl fluoride via the formation of (1 --> 4)-beta-linkages to yield a new linear crystalline (1 --> 3,1 --> 4)-beta-D-glucan with a repeating 4betaG3betaG unit. MS and methylation analyses indicated that the in vitro product consisted of a mixture of oligosaccharides, the one having a degree of polymerization of 12 being the most abundant. Morphological characterization revealed that the (1 --> 3,1 --> 4)-beta-D-glucan forms spherulites which are composed of platelet crystals. X-ray and electron diffraction analyses allowed the proposition of a putative crystallographic structure which corresponds to a monoclinic unit cell with a = 0.834 nm, b = 0.825 nm, c = 2.04 nm and gamma = 90.5degrees. The dimensions of the ab plane are similar to those of cellulose I-beta, but the length of the c-axis is nearly twice that of cellulose I. It is proposed that four glucose residues are present in an extended conformation along the c-axis of the unit cell. The data presented show that glycosynthases represent promising enzymic systems for the synthesis of novel polysaccharides with specific and controlled structures, and for the analysis in vitro of the mechanisms of polymerization and crystallization of potysaccharides.

  • 30. Fairweather, J. K.
    et al.
    Him, J. L. K.
    Heux, L.
    Driguez, H.
    Bulone, Vincent
    KTH, Superseded Departments, Biotechnology.
    Structural characterization by C-13-NMR spectroscopy of products synthesized in vitro by polysaccharide synthases using C-13-enriched glycosyl donors: application to a UDP-glucose:(1 -> 3)-beta-D-glucan synthase from blackberry (Rubus fruticosus)2004In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 14, no 9, 775-781 p.Article in journal (Refereed)
    Abstract [en]

    A simple and sensitive method for the characterization of products synthesized in vitro by polysaccharide synthases is described. it relies on the use of C-13-enriched nucleotide sugars as substrates and on the analysis of the newly synthesized polysaccharides by C-13-nuclear magnetic resonance (NMR) spectroscopy. The method was validated with a (1-->3)-beta-D-glucan synthase from blackberry, but it may be applied to the study of any glycosyltransferase. The chemical synthesis of UDP-D-[U-C-13]glucose was achieved in a classical procedure with an overall yield of 50%. A uniformly labeled (1-->3)-beta-D-glucan was synthesized from this substrate, using detergent extracts of blackberry cell membranes as a source of synthase. One hundred micrograms of product was sufficient for liquid and solid-state C-13-NMR spectroscopy analyses. The method is at least 100 times more sensitive than in the case of non-enriched polysaccharides. It allows the unequivocal identification and direct structural characterization of the products synthesized in vitro, as opposed to conventional methods that rely on the use of radioactive substrates and enzymatic hydrolysis of the polysaccharides with specific glycoside hydrolases. The method proves that the glycan analyzed was synthesized de novo because the final product is enriched in C-13. Information on the 3D organization of the polymer may also be obtained by solid-state NMR spectroscopy.

  • 31.
    Fernandes, Susana C. M.
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. University of the Basque Country, Spain.
    Alonso-Varona, Ana
    Palomares, Teodoro
    Zubillaga, Veronica
    Labidi, Jalel
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. University of Adelaide, Australia.
    Exploiting Mycosporines as Natural Molecular Sunscreens for the Fabrication of UV-Absorbing Green Materials2015In: ACS Applied Materials and Interfaces, ISSN 1944-8244, E-ISSN 1944-8252, Vol. 7, no 30, 16558-16564 p.Article in journal (Refereed)
    Abstract [en]

    Ultraviolet radiations have many detrimental effects in living organisms that challenge the stability and function of cellular structures. UV exposure also alters the properties and durability of materials and affects their lifetime. It is becoming increasingly important to develop new biocompatible and environmentally friendly materials to address these issues. Inspired by the strategy developed by fish, algae, and microorganisms exposed to UV radiations in confined ecosystems, we have constructed novel UV-protective materials that exclusively consist of natural compounds. Chitosan was chosen as the matrix for grafting mycosporines and mycosporine-like amino acids as the functional components of the active materials. Here, we show that these materials are biocompatible, photoresistant, and thermoresistant, and exhibit a highly efficient absorption of both UV-A and UV-B radiations. Thus, they have the potential to provide an efficient protection against both types of UV radiations and overcome several shortfalls of the current UV-protective products. In practice, the same concept can be applied to other biopolymers than chitosan and used to produce multifunctional materials. Therefore, it has a great potential to be exploited in a broad range of applications in living organisms and nonliving systems.

  • 32. Fonteyne, Margot
    et al.
    Correia, Ana
    De Plecker, Sofie
    Vercruysse, Jurgen
    Ilic, Ilija
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience.
    Veryaet, Chris
    Remon, Jean Paul
    Onofre, Fernanda
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    De Beer, Thomas
    Impact of microcrystalline cellulose material attributes: A case study on continuous twin screw granulation2015In: International Journal of Pharmaceutics, ISSN 0378-5173, E-ISSN 1873-3476, Vol. 478, no 2, 705-717 p.Article in journal (Refereed)
    Abstract [en]

    The International Conference on Harmonisation (ICH) states in its Q8 'Pharmaceutical Development' guideline that the manufacturer of pharmaceuticals should have an enhanced knowledge of the product performance over a range of material attributes, manufacturing process options and process parameters. The present case study evaluates the effect of unspecified variability of raw material properties upon the quality attributes of granules; produced using a continuous from-powder-to-tablet wet granulation line (ConsiGma (TM) 25). The impact of different material attributes of six samples of microcrystalline cellulose (MCC) was investigated. During a blind study the different samples of MCC were used separately and the resulting granules were evaluated in order to identify the differences between the six samples. Variation in size distribution due to varying water binding capacity of the MCC samples was observed. The cause of this different water binding capacity was investigated and was caused by a different degree of crystallinity. Afterwards, an experimental design was conducted in order to evaluate the effect of both product and process variability upon the granule size distribution. This model was used in order to calculate the required process parameters to obtain a preset granule size distribution regardless of the type of MCC used. The difference in water binding capacity and its effect on granular properties was still present when combining the MCC grades with different binders.

  • 33. Ford, Kristina L.
    et al.
    Chin, Tony
    Srivastava, Vaibhav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Zeng, Wei
    Doblin, Monika S.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bacic, Antony
    Comparative "Golgi" Proteome Study of Lolium multiflorum and Populus trichocarpa2016In: PROTEOMES, ISSN 2227-7382, Vol. 4, no 3, 23Article in journal (Refereed)
    Abstract [en]

    The Golgi apparatus (GA) is a crucial organelle in the biosynthesis of non-cellulosic polysaccharides, glycoproteins and proteoglycans that are primarily destined for secretion to the cell surface (plasma membrane, cell wall and apoplast). Only a small proportion of the proteins involved in these processes have been identified in plants, with the majority of their functions still unknown. The availability of a GA proteome would greatly assist plant biochemists, cell and molecular biologists in determining the precise function of the cell wall-related proteins. There has been some progress towards defining the GA proteome in the model plant system Arabidopsis thaliana, yet in commercially important species, such as either the cereals or woody species there has been relatively less progress. In this study, we applied discontinuous sucrose gradient centrifugation to partially enrich GA from suspension cell cultures (SCCs) and combined this with stable isotope labelling (iTRAQ) to determine protein sub-cellular locations. Results from a representative grass species, Italian ryegrass (Lolium multiflorum) and a dicot species, black cottonwood (Populus trichocarpa) are compared. The results confirm that membrane fractionation approaches that provide effective GA-enriched fractions for proteomic analyses in Arabidopsis are much less effective in the species examined here and highlight the complexity of the GA, both within and between species.

  • 34.
    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)
  • 35.
    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, 759-767 p.Article 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.

  • 36.
    Fugelstad, Johanna
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brown, Christian
    KTH, School of Biotechnology (BIO), Glycoscience.
    Hukasova, Elvira
    Sundqvist, Gustav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Lindqvist, Arne
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Functional characterization of the pleckstrin homology domain of a cellulose synthase from the Oomycete Saprolegnia monoica2012In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 417, no 4, 1248-1253 p.Article in journal (Refereed)
    Abstract [en]

    Some oomycetes, for instance Saprolegnia parasitica, are severe fish pathogens that cause important economic losses worldwide. Cellulose biosynthesis is a vital process for this class of microorganisms, but the corresponding molecular mechanisms are poorly understood. Of all cellulose synthesizing enzymes known, only some oomycete cellulose synthases contain a pleckstrin homology (PH) domain. Some human PH domains bind specifically to phosphoinositides, but most PH domains bind phospholipids in a non-specific manner. In addition, some PH domains interact with various proteins. Here we have investigated the function of the PH domain of cellulose synthase 2 from the oomycete Saprolegnia monoica (SmCesA2), a species closely related to S. parasitica. The SmCesA2 PH domain is similar to the C-terminal PH domain of the human protein TAPP1. It binds in vitro to phosphoinositides, F-actin and microtubules, and co-localizes with F-actin in vivo. Our results suggest a role of the SmCesA2 PH domain in the regulation, trafficking and/or targeting of the cell wall synthesizing enzyme.

  • 37.
    Giacomello, Stefania
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Stockholm University, Sweden.
    Salmén, Fredrik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Terebieniec, B. K.
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Navarro, J. F.
    Alexeyenko, A.
    Reimegård, J.
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Mannapperuma, C.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. University of Adelaide, Australia.
    Ståhl, P. L.
    Sundström, J. F.
    Street, N. R.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Spatially resolved transcriptome profiling in model plant species2017In: Nature Plants, ISSN 2055-0278, Vol. 3, 17061Article in journal (Refereed)
    Abstract [en]

    Understanding complex biological systems requires functional characterization of specialized tissue domains. However, existing strategies for generating and analysing high-throughput spatial expression profiles were developed for a limited range of organisms, primarily mammals. Here we present the first available approach to generate and study high-resolution, spatially resolved functional profiles in a broad range of model plant systems. Our process includes high-throughput spatial transcriptome profiling followed by spatial gene and pathway analyses. We first demonstrate the feasibility of the technique by generating spatial transcriptome profiles from model angiosperms and gymnosperms microsections. In Arabidopsis thaliana we use the spatial data to identify differences in expression levels of 141 genes and 189 pathways in eight inflorescence tissue domains. Our combined approach of spatial transcriptomics and functional profiling offers a powerful new strategy that can be applied to a broad range of plant species, and is an approach that will be pivotal to answering fundamental questions in developmental and evolutionary biology.

  • 38. Greffe, L.
    et al.
    Bessueille, L.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Synthesis, preliminary characterization, and application of novel surfactants from highly branched xyloglucan oligosaccharides2005In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 15, no 4, 437-445 p.Article in journal (Refereed)
    Abstract [en]

    A novel class of nonionic, carbohydrate-based surfactants has been synthesized from the plant polysaccharide xyloglucan. Enzymatic hydrolysis of xyloglucan yielded a series of well-defined, highly branched oligosaccharides that, following reductive amination, were readily conjugated with fatty acids bearing C-8 to C-18 chains under mild conditions. The critical micelle concentration, determined by tensiometry and dye-inclusion measurements, showed a typical dependence on acyl chain length and was sensitive to the degree of galactosylation of the head group. Several compounds from this new group of surfactants, especially those with C-14 and C-16 chains, were useful for the extraction of membrane-bound enzyme markers from different plant cell compartments in catalytically active form.

  • 39.
    Grenville-Briggs, Laura J.
    et al.
    Aberdeen Oomycete Group, University of Aberdeen, Institute of Medical Sciences.
    Anderson, Victoria L.
    Aberdeen Oomycete Group, University of Aberdeen, Institute of Medical Sciences.
    Fugelstad, Johanna
    KTH, School of Biotechnology (BIO), Glycoscience.
    Avrova, Anna O.
    Plant-Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee.
    Bouzenzana, Jamel
    Organisation et Dynamique des Membranes Biologiques, Unité Mixte de Recherche 5246, Université Lyon I.
    Williams, Alison
    Aberdeen Oomycete Group, University of Aberdeen, Institute of Medical Sciences.
    Wawra, Stephan
    Aberdeen Oomycete Group, University of Aberdeen, Institute of Medical Sciences.
    Whisson, Stephen C.
    Plant-Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee.
    Birch, Paul R. J.
    Plant-Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    van West, Pieter
    Aberdeen Oomycete Group, University of Aberdeen, Institute of Medical Sciences.
    Cellulose synthesis in Phytophthora infestans is required for normal appressorium formation and successful infection of potato2008In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 20, no 3, 720-738 p.Article in journal (Refereed)
    Abstract [en]

    Cellulose, the important structural compound of cell walls, provides strength and rigidity to cells of numerous organisms. Here, we functionally characterize four cellulose synthase genes (CesA) in the oomycete plant pathogen Phytophthora infestans, the causal agent of potato (Solanum tuberosum) late blight. Three members of this new protein family contain Pleckstrin homology domains and form a distinct phylogenetic group most closely related to the cellulose synthases of cyanobacteria. Expression of all four genes is coordinately upregulated during pre- and early infection stages of potato. Inhibition of cellulose synthesis by 2,6-dichlorobenzonitrile leads to a dramatic reduction in the number of normal germ tubes with appressoria, severe disruption of the cell wall in the preinfection structures, and a complete loss of pathogenicity. Silencing of the entire gene family in P. infestans with RNA interference leads to a similar disruption of the cell wall surrounding appressoria and an inability to form typical functional appressoria. In addition, the cellulose content of the cell walls of the silenced lines is >50% lower than in the walls of the nonsilenced lines. Our data demonstrate that the isolated genes are involved in cellulose biosynthesis and that cellulose synthesis is essential for infection by P. infestans.

  • 40.
    Guerriero, Gea
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Avino, Mariano
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience.
    Fugelstad, Johanna
    KTH, School of Biotechnology (BIO), Glycoscience.
    Clergeot, Pierre-Henri
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Chitin Synthases from Saprolegnia Are Involved in Tip Growth and Represent a Potential Target for Anti-Oomycete Drugs2010In: PLOS PATHOG, ISSN 1553-7366, Vol. 6, no 8, e1001070- p.Article in journal (Refereed)
    Abstract [en]

    Oomycetes represent some of the most devastating plant and animal pathogens. Typical examples are Phytophthora infestans, which causes potato and tomato late blight, and Saprolegnia parasitica, responsible for fish diseases. Despite the economical and environmental importance of oomycete diseases, their control is difficult, particularly in the aquaculture industry. Carbohydrate synthases are vital for hyphal growth and represent interesting targets for tackling the pathogens. The existence of 2 different chitin synthase genes (SmChs1 and SmChs2) in Saprolegnia monoica was demonstrated using bioinformatics and molecular biology approaches. The function of SmCHS2 was unequivocally demonstrated by showing its catalytic activity in vitro after expression in Pichia pastoris. The recombinant SmCHS1 protein did not exhibit any activity in vitro, suggesting that it requires other partners or effectors to be active, or that it is involved in a different process than chitin biosynthesis. Both proteins contained N-terminal Microtubule Interacting and Trafficking domains, which have never been reported in any other known carbohydrate synthases. These domains are involved in protein recycling by endocytosis. Enzyme kinetics revealed that Saprolegnia chitin synthases are competitively inhibited by nikkomycin Z and quantitative PCR showed that their expression is higher in presence of the inhibitor. The use of nikkomycin Z combined with microscopy showed that chitin synthases are active essentially at the hyphal tips, which burst in the presence of the inhibitor, leading to cell death. S. parasitica was more sensitive to nikkomycin Z than S. monoica. In conclusion, chitin synthases with species-specific characteristics are involved in tip growth in Saprolegnia species and chitin is vital for the micro-organisms despite its very low abundance in the cell walls. Chitin is most likely synthesized transiently at the apex of the cells before cellulose, the major cell wall component in oomycetes. Our results provide important fundamental information on cell wall biogenesis in economically important species, and demonstrate the potential of targeting oomycete chitin synthases for disease control.

  • 41.
    Guerriero, Gea
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Fugelstad, Johanna
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    What Do We Really Know about Cellulose Biosynthesis in Higher Plants?2010In: JOURNAL OF INTEGRATIVE PLANT BIOLOGY, ISSN 1672-9072, Vol. 52, no 2, 161-175 p.Article, review/survey (Refereed)
    Abstract [en]

    Cellulose biosynthesis is one of the most important biochemical processes in plant biology. Despite the considerable progress made during the last decade, numerous fundamental questions related to this key process in plant development are outstanding. Numerous models have been proposed through the years to explain the detailed molecular events of cellulose biosynthesis. Almost all models integrate solid experimental data with hypotheses on several of the steps involved in the process. Speculative models are most useful to stimulate further research investigations and bring new exciting ideas to the field. However, it is important to keep their hypothetical nature in mind and be aware of the risk that some undemonstrated hypotheses may progressively become admitted. In this review, we discuss the different steps required for cellulose formation and crystallization, and highlight the most important specific aspects that are supported by solid experimental data.

  • 42. Haas, Brian J.
    et al.
    Kamoun, Sophien
    Zody, Michael C.
    Jiang, Rays H. Y.
    Handsaker, Robert E.
    Cano, Liliana M.
    Grabherr, Manfred
    Kodira, Chinnappa D.
    Raffaele, Sylvain
    Torto-Alalibo, Trudy
    Bozkurt, Tolga O.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Fugelstad, Johanna
    KTH, School of Biotechnology (BIO), Glycoscience.
    Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans2009In: Nature, ISSN 0028-0836, Vol. 461, no 7262, 393-398 p.Article in journal (Refereed)
    Abstract [en]

    Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on human history, resulting in famine and population displacement(1). To this day, it affects world agriculture by causing the most destructive disease of potato, the fourth largest food crop and a critical alternative to the major cereal crops for feeding the world's population(1). Current annual worldwide potato crop losses due to late blight are conservatively estimated at $6.7 billion(2). Management of this devastating pathogen is challenged by its remarkable speed of adaptation to control strategies such as genetically resistant cultivars(3,4). Here we report the sequence of the P. infestans genome, which at similar to 240 megabases (Mb) is by far the largest and most complex genome sequenced so far in the chromalveolates. Its expansion results from a proliferation of repetitive DNA accounting for similar to 74% of the genome. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection or are predicted to have activities that alter host physiology. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the P. infestans genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential.

  • 43. 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, 420-426 p.Article in journal (Refereed)
  • 44. Henriksson, Marielle
    et al.
    Berglund, Lars A.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience.
    Method of producing and the use of microfibrillated paper2009Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    The present invention relates to a method of producing a cellulose based paper, the paper itself and the use thereof where the paper exhibits enhanced mechanical properties. The method involves providing a suspension of well dispersed modified cellulose at a low concentration. The properties and the chemical structure of the paper make it suitable for in vivo applications such as implant material.

  • 45.
    Henriksson, Marielle
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Berglund, Lars
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience.
    Producing paper, useful as e.g. filter paper, speaker membrane and suture, comprises providing modified nanofibrils of cellulose, providing suspension of modified nanofibrils, and filtering, dewatering and drying the nanofibrils2009Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    The present invention relates to a method of producing a cellulose based paper, the paper itself and the use thereof where the paper exhibits enhanced mechanical properties. The method involves providing a suspension of well dispersed modified cellulose at a low concentration. The properties and the chemical structure of the paper make it suitable for in vivo applications such as implant material.

  • 46. Him, J. L. K.
    et al.
    Chanzy, H.
    Pelosi, L.
    Putaux, J. L.
    Bulone, Vincent
    KTH, Superseded Departments, Biotechnology.
    Recent developments in the field of in vitro biosynthesis of plant beta-glucans2003In: American Chemical Society Symposium Series (ACS), ISSN 0097-6156, Vol. 840, 65-77 p.Article, review/survey (Refereed)
    Abstract [en]

    This chapter reports the use of various detergents for the preparation of vesicles and micelles from plant plasma membranes bearing beta-glucan synthases. The effects of these detergents and other parameters on the in vitro activity of cellulose and (1-->3)-beta-glucan synthases are described. Morphological and structural aspects of the vesicles and micelles; are presented together with the characterization of the products synthesized in vitro by the detergent-extracted beta-glucan synthases. The possible involvement of porins for extrusion of cell wall glucans through plasma membranes as well as future developments for the study of in vitro biosynthesis of these polysaccharides are discussed in relation to these results.

  • 47. Him, J. L. K.
    et al.
    Pelosi, L.
    Chanzy, H.
    Putaux, J. L.
    Bulone, Vincent
    KTH, Superseded Departments, Biotechnology.
    Biosynthesis of (1 -> 3)-beta-D-glucan (callose) by detergent extracts of a microsomal fraction from Arabidopsis thaliana2001In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 268, no 17, 4628-4638 p.Article in journal (Refereed)
    Abstract [en]

    The aim of this work was to develop a biochemical approach to study (1-->3)-beta -D-glucan (callose) biosynthesis using suspension cultures of Arabidopsis thaliana. Optimal conditions for in vitro synthesis of callose corresponded to an assay mixture containing 50 mM Mops buffer, pH6.8, 1 mM UDP-glucose, 8 mM Ca2+ and 20 mM cellobiose. The enzyme was Ca2+-dependent, and addition of Mg2+ to the reaction mixture did not favour cellulose biosynthesis. Enzyme kinetics suggested the existence of positive. homotropic cooperativity of (1-->3)-beta -D-glucan synthase for the substrate UDP-glucose, in agreement with the hypothesis that callose synthase consists of a multimeric complex containing several catalytic subunits. Detergents belonging to different families were tested for their ability to extract and preserve membrane-bound (1-->3)-beta -D-glucan synthase activity. Cryo-transmission electron microscopy experiments showed that n-octyl-beta -D-glucopyranoside allowed the production of micelle-like structures, whereas vesicles were obtained with Chaps and Zwittergent 3-12. The morphology and size of the (1-->3)-beta -D-glucans synthesized in vitro by fractions obtained with different detergents were affected by the nature of the detergent tested. These data suggest that the general organization of the glucan synthase complexes and the properties of them in vitro products are influenced by the detergent used for protein extraction. The reaction products synthesized by different detergent extracts were characterized by infrared spectroscopy, methylation analysis, C-13-N-MR spectroscopy, electron microscopy and X-ray diffraction. These products were identified as linear (1-->3)-beta -D-glucans having a degree of polymerization higher than 100, a microfibrillar structure, and a low degree of crystallinity.

  • 48. Hrmova, M.
    et al.
    Imai, T.
    Rutten, S. J.
    Fairweather, J. K.
    Pelosi, L.
    Bulone, Vincent
    KTH, Superseded Departments, Biotechnology.
    Driguez, H.
    Fincher, G. B.
    Mutated barley (1,3)-beta-D-glucan endohydrolases synthesize crystalline (1,3)-beta-D-glucans2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 33, 30102-30111 p.Article in journal (Refereed)
    Abstract [en]

    Barley (1,3)-beta-D-glucan endohydrolases (EC 3.2.1.39), inactivated by site-directed mutagenesis of their catalytic nucleophiles, show autocondensation glucosynthetic activity with alpha-laminaribiosyl fluoride and heterocondensation glycosynthetic activity with a-laminaribiosyl fluoride and 4'-nitrophenyl beta-D-glucopyranoside. The native enzyme is a retaining endohydrolase of the family 17 group and catalyzes glycosyl transfer reactions at high substrate concentrations. Catalytic efficiencies (k(cat) K-m(-1)) of mutants E231G, E231S, and E231A as glycosynthases are 28.9, 0.9, and 0.5 x 10(-4) M-1 s(-1), respectively. Glycosynthase reactions appear to be processive and proceed with pH optima of 6-8 and yields of up to 75%. Insoluble products formed during the glycosynthase reaction appear as lamellar, hexagonal crystals when observed by electron microscopy. Methylation, NMR, and matrix-assisted laser desorption ionization time-of-flight analyses show that the reaction products are linear (1,3)-beta-D-glucans with a degree of polymerization of 30-34, whereas electron and x-ray diffraction patterns indicate that these (1,3)-beta-D-glucan chains adopt a parallel, triple helical conformation. The (1,3)-beta-D-glucan triple helices are orientated perpendicularly to the plane of the lamellar crystals. The barley (1,3)-beta-D-glucan glycosynthases have considerable potential for tailored and high efficiency synthesis of (1,3)-beta-D-linked oligo- and polysaccharides, some of which could have immunomodulating activity, or for the coupling of (1,3)-beta-D-linked glucosyl residues onto other oligosaccharides or glycoproteins.

  • 49.
    Hsieh, Yves S. Y.
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. Univ Adelaide, Australia.
    Zhang, Qisen
    Yap, Kuok
    Shirley, Neil J.
    Lahnstein, Jelle
    Nelson, Clark J.
    Burton, Rachel A.
    Millar, A. Harvey
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. Univ Adelaide, Australia.
    Fincher, Geoffrey B.
    Genetics, Transcriptional Profiles, and Catalytic Properties of the UDP-Arabinose Mutase Family from Barley2016In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 55, no 2, 322-334 p.Article in journal (Refereed)
    Abstract [en]

    Four members of the UDP-Ara mutase (UAM) gene family from barley have been isolated and characterized, and their map positions on chromosomes 2H, 3H, and 4H have been defined. When the genes are expressed in Escherichia coli, the corresponding HvUAM1, HvUAM2, and HvUAM3 proteins exhibit UAM activity, and the kinetic properties of the enzymes have been determined, including K-m, K-cat, and catalytic efficiencies. However, the expressed HvUAM4 protein shows no mutase activity against UDP-Ara or against a broad range of other nucleotide sugars and related molecules. The enzymic data indicate therefore that the HvUAM4 protein may not be a mutase. However, the HvUAM4 gene is transcribed at high levels in all the barley tissues examined, and its transcript abundance is correlated with transcript levels for other genes involved in cell wall biosynthesis. The UDP-L-Arap -> UDP-L-Araf reaction, which is essential for the generation of the UDP-Araf substrate for arabinoxylan, arabinogalactan protein, and pectic polysaccharide biosynthesis, is thermodynamically unfavorable and has an equilibrium constant of 0.02. Nevertheless, the incorporation of Araf residues into nascent polysaccharides clearly occurs at biologically appropriate rates. The characterization of the HvUAM genes opens the way for the manipulation of both the amounts and fine structures of heteroxylans in cereals, grasses, and other crop plants, with a view toward enhancing their value in human health and nutrition, and in renewable biofuel production.

  • 50. Jaeger, Diana
    et al.
    Ndi, Chi P.
    Crocoll, Christoph
    Simpson, Bradley S.
    Khakimov, Bekzod
    Guzman-Genuino, Ruth Marian
    Hayball, John D.
    Xing, Xiaohui
    KTH, School of Biotechnology (BIO), Glycoscience. School of Biotechnology, AlbaNova University Centre, Stockholm, SE-10691, Sweden; ARC Centre of Excellence in Plant Cell Walls, School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, Urrbrae, 5064, Australia.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. School of Biotechnology, AlbaNova University Centre, Stockholm, SE-10691, Sweden; ARC Centre of Excellence in Plant Cell Walls, School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, Urrbrae, 5064, Australia.
    Weinstein, Philip
    Moller, Birger L.
    Semple, Susan J.
    Isolation and Structural Characterization of Echinocystic Acid Triterpenoid Saponins from the Australian Medicinal and Food Plant Acacia ligulata2017In: Journal of natural products (Print), ISSN 0163-3864, E-ISSN 1520-6025, Vol. 80, no 10, 2692-2698 p.Article in journal (Refereed)
    Abstract [en]

    The Australian plant Acacia ligulata has a number of traditional food and medicinal uses by Australian Aboriginal people, although no bioactive compounds have previously been isolated from this species. Bioassay-guided fractionation of an ethanolic extract of the mature pods of A. ligulata led to the isolation of the two new echinocystic acid triterpenoid saponins, ligulatasides A (1) and B (2), which differ in the fine structure of their glycan substituents. Their structures were elucidated on the basis of 1D and 2D NMR, GC-MS, LC-MS/MS, and saccharide linkage analysis. These are the first isolated compounds from A. ligulata and the first fully elucidated structures of triterpenoid saponins from Acacia sensu stricto having echinocystic acid reported as the aglycone. Compounds 1 and 2 were evaluated for cytotoxic activity against a human melanoma cancer cell line (SK-MEL28) and a diploid fibroblast cell line (HFF), but showed only weak activity.

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