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Biochemical characterization of family 43 glycosyltransferases in the Populus xylem: challenges and prospects
KTH, School of Biotechnology (BIO), Glycoscience.
KTH, School of Biotechnology (BIO), Glycoscience.
KTH, School of Biotechnology (BIO), Glycoscience.ORCID iD: 0000-0002-8576-4370
KTH, School of Biotechnology (BIO), Glycoscience.
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2010 (English)In: Plant Biotechnology, ISSN 1342-4580, Vol. 27, no 3, 283-288 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
2010. Vol. 27, no 3, 283-288 p.
Keyword [en]
GT43 glycosyltransferase, IRX9, populus xylem, xylan, xylosyltransferase.
National Category
URN: urn:nbn:se:kth:diva-14265ISI: 000280085800010ScopusID: 2-s2.0-77955322528OAI: diva2:332003
Colloquium on Green Chemistry in Plants and Microorganisms Japanese Soc Promot Sci, Stockholm, SWEDEN, MAY 25, 2009
Swedish Foundation for Strategic Research

QC 20110404

Available from: 2010-07-29 Created: 2010-07-29 Last updated: 2016-04-18Bibliographically approved
In thesis
1. Investigation of genes and proteins involved in xylan biosynthesis
Open this publication in new window or tab >>Investigation of genes and proteins involved in xylan biosynthesis
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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


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


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

Abstract [sv]

Vedbildning, eller xylogenes, är en grundläggande mekanism för så skilda områden som industri, boende och en hållbar miljö. Ved består av sekundärt xylem som är starka, stora celler med tjocka cellväggar som är lignifierade. Grunden för de starka cellerna är en avancerad komposit bestående av cellulosafibrer tvärbundna av hemicellulosa och slutligen ingjutet i lignin. Denna fiberkomposit är den sekundära cellväggen i vedartade växter. Celldelning och differentiering regleras genom att sätta igång och stänga av gener. Proteiner som kodas av dessa gener utför de viktigaste funktionerna i cellerna. De styr hela maskineriet som upprätthåller cellernas struktur och funktion, underhåller tillväxt samt tillverkar nödvändiga produkter såsom cellväggskolhydraterna.

Här beskriver vi utforskningen av gener och proteiner som är inblandade i xylanbildning liksom utvecklandet av ett modellsystem som kommer vara en hjälp i den funktionella analysen av vedbildning. Xylan är den vanligaste hemicellulosan, eller korsbindande glykanen, i lövträd och därför en av de vanligaste kolhydraterna på jorden. Vi demonstrerar att hybridaspcellkulturer i suspension kan användas som ett modellsystem för sekundär cellväggsbildning. Vi har också undersökt glykosyltransferaser från CAZy-familj 43 som tycks spela en viktig roll i bildandet av sekundär cellvägg. Vi har fokuserat på en av dessa, Pt×tGT43A, en trolig ortolog till Arabidopsis IRX9 som spelar en viktig roll i xylanbildning. Proteinet har uttryckts övergående i Nicotiana benthamiana och dess funktion och lokalisering beskrivs. Dessutom undersöker vi ett glykosidhydrolas, Pt×tXyn10A, involverad i vedbildning. Dess roll är oklar men högst sannolikt modifierar det xylan medan det inkorporeras i sekundära cellväggen efter sekretion från Golgi. Detta influerar interaktionen mellan cellulosa, hemicellulosa och lignin i den slutliga vedcellen. Vi har också klonat en transkriptionsfaktor, Pt×tMYB021, en trolig ortolog till Arabidopsis MYB46 och vi visar att den aktiverar GT43A, GT43B och Xyn10A.

Genom analys av promotorsekvenserna har vi identifierat ett CA-rikt motiv förmodat viktigt för xylemspecifika gener.Genom att bemästra proteinerna som är ansvariga för vedbildning får vi verktyg att utveckla skogsproduktsmarknaden. Xylan är en ofantligt stor outnyttjad källa till förnyelsebara kolhydrater. En vision är nya produkter som till exempel snabbväxande träd, ändrade fiberegenskaper, optimerat användande av vedkolhydrater för biobränsle och biomaterial såväl som utvecklandet av intelligenta material genom biomimetisk ingenjörskonst.

Place, publisher, year, edition, pages
Stockholm: KTH, 2010. vii, 52 p.
Trita-BIO-Report, ISSN 1654-2312 ; 2010:1
Populus, xylan biosynthesis, hemicellulose, glycosyltransferase, GT43, IRX, glycoside hydrolase, GH10, Xyn10A, CAZy, transcription factor, MYB, wood formation, secondary cell wall, Arabidopsis
National Category
Biochemistry and Molecular Biology
urn:nbn:se:kth:diva-11897 (URN)978-91-7415-538-9 (ISBN)
Public defence
2010-02-12, FB42, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00 (English)
QC20100730Available from: 2010-01-21 Created: 2010-01-21 Last updated: 2010-07-30Bibliographically approved

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