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Group III-A XTH genes encode predominant xyloglucan endo hydrolase active in expanding tissues of Arabidopsis thaliana
KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
Visa övriga samt affilieringar
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Genetik
Identifikatorer
URN: urn:nbn:se:kth:diva-28352OAI: oai:DiVA.org:kth-28352DiVA, id: diva2:387350
Anmärkning
QC 20110114Tillgänglig från: 2011-01-14 Skapad: 2011-01-14 Senast uppdaterad: 2011-04-01Bibliografiskt granskad
Ingår i avhandling
1. In vitro and in vivo approaches in the characterization of XTH gene products
Öppna denna publikation i ny flik eller fönster >>In vitro and in vivo approaches in the characterization of XTH gene products
2011 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

ABSTRACT

The xyloglucan endo-transglycosylase/hydrolase (XTH) genes are found in all vascular and some nonvascular plants. The XTH genes encode proteins which comprise a subfamily of glycoside hydrolase (GH) family 16 in the Carbohydrate-Active enZYmes (CAZY) classification. The XTH gene products are believed to play intrinsic role in cell wall modification during growth and development throughout the lifetime of the plant. In the present investigation, biochemical and reverse genetic approaches were used to better understand the functions of individual members of the XTH gene family of two important plants: the model organism Arabidopsis thaliana and the grain crop barley (Hordeum vulgare). A phylogenetic tree of the xyloglucan-active enzymes of GH16 has previously been constructed, where enzymes with similar activities have been shown to cluster together. Several members of phylogenetic Group I/II and III-B, predicted to exhibit xyloglucan endo-transglycosylase activity (EC 2.4.1.207) and members of Group III-A, predicted to exhibit xyloglucan endo-hydrolase activity (EC 3.2.1.151), were included to analyze the functional diversity of XTH gene products. A heterologous expression system using the yeast Pichia pastoris was found to be effective for recombinant protein production with a success rate of ca. 50%. XTH gene products were obtained in soluble and active forms for subsequent biochemical characterization.

In order to be able to screen larger numbers of protein producing clones, a fast and easy method is required to identify clones expressing active protein in high enough amounts. Thus, a miniaturized XET/XEH assay for high-throughput analysis was developed, which was able to identify activities with good precision and with a reduced time and materials consumption and a reduced work load.

Enzyme kinetic analysis indicated that the XET or XEH activity of all XTH gene products characterized in the present study corresponded to predictions based on the previously revised phylogenetic clustering. To gain insight into the biological function of the predominant XEHs AtXTH31 and AtXTH32, which are highly expressed in rapidly developing tissues, a reverse genetic approach was employed using T-DNA insertion lines of the A. thaliana Columbia ecotype. Genotypic and phenotypic characterization, together with in situ assays of XET and XEH activities, in single- and double-knock-out mutants indicated that these Group III-A enzymes are active in expanding tissues of the A. thaliana roots and hypocotyl.  Although suppression of in muro XEH activity was clearly observed in the double-knock-out, no significant growth phenotype was observed, with the exception that radicle emergence appeared to be faster than in the wild type plants.

Keywords: Arabidopis thaliana, Hordeum vulgare, plant cell wall, xyloglucan, glycoside hydrolase family 16, xyloglucan endo-transglycosylase/hydrolase gene family, xyloglucan endo-transglycosylase, xyloglucan endo-hydrolase, heterologous protein expression, Pichia pastoris, T-DNA insertion, in situ XET/XEH assay, high-throughput screening

Ort, förlag, år, upplaga, sidor
Stockholm: KTH, 2011. s. x, 48
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2011: 1
Nyckelord
Arabiodopsis thaliana, Hordeum vulgare, plant cell wall, xyloglucan, glycoside hydrolase family 16, xyloglucan endo-transglycosylase/hydrolase gene family, xyloglucan endo-hydrolase, heterologous protein expression, Pichia pastoris, T-DNA insertion, in situ XET/XEH assay, high-througput screening
Nationell ämneskategori
Botanik Biokemi och molekylärbiologi Biokemi och molekylärbiologi
Forskningsämne
SRA - Molekylär biovetenskap
Identifikatorer
urn:nbn:se:kth:diva-28222 (URN)978-91-7415-848-9 (ISBN)
Disputation
2011-02-02, FB54, Roslagstullsbacken 21, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Anmärkning
QC 20110114Tillgänglig från: 2011-01-14 Skapad: 2011-01-12 Senast uppdaterad: 2011-01-14Bibliografiskt granskad
2. Plant and microbial xyloglucanases: Function, Structure and Phylogeny
Öppna denna publikation i ny flik eller fönster >>Plant and microbial xyloglucanases: Function, Structure and Phylogeny
2011 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

In this thesis, enzymes acting on the primary cell wall hemicellulose xyloglucan are studied.  Xyloglucans are ubiquitous in land plants which make them an important polysaccharide to utilise for microbes and a potentially interesting raw material for various industries.  The function of xyloglucans in plants is mainly to improve primary cell wall characteristics by coating and tethering cellulose microfibrils together.  Some plants also utilise xyloglucans as storage polysaccharides in their seeds.

In microbes, a variety of different enzymes for degrading xyloglucans have been found.  In this thesis, the structure-function relationship of three different microbial endo-xyloglucanases from glycoside hydrolase families 5, 12 and 44 are probed and reveal details of the natural diversity found in xyloglucanases.  Hopefully, a better understanding of how xyloglucanases recognise and degrade their substrate can lead to improved saccharification processes of plant matter, finding uses in for example biofuel production.

In plants, xyloglucans are modified in muro by the xyloglucan transglycosylase/hydrolase (XTH) gene products.  Interestingly, closely related XTH gene products catalyse either transglycosylation (XET activity) or hydrolysis (XEH activity) with dramatically different effects on xyloglucan and on cell wall characteristics.  The strict transglycosylases transfer xyloglucan segments between individual xyloglucan molecules while the hydrolases degrade xyloglucan into oligosaccharides.  Here, we describe and determine, a major determinant of transglycosylation versus hydrolysis in XTH gene products by solving and comparing the first 3D structure of an XEH, Tm-NXG1 and a XET, PttXET16-34.  The XEH activity was hypothesised, and later confirmed to be restricted to subset of the XTH gene products.  The in situ localisation of XEH activity in roots and hypocotyls of Arabidopsis was also visualised for the first time.  Furthermore, an evolutionary scheme for how XTH gene products developed from bacterial beta-1,3;1,4 glucanases was also presented based on the characterisation of a novel plant endo-glucanase, PtEG16-1. The EG16s are proposed to predate XTH gene products and are with activity on both xyloglucan and beta-1,3;1,4 glucans an “intermediate” in the evolution from beta-1,3;1,4 glucanases to XTH gene products.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH Royal Institute of Technology, 2011. s. 61
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2011:07
Nyckelord
xyloglucan, xyloglucanases, XTH, XET, XEH, endoglucanases, EG16
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:kth:diva-31677 (URN)978-91-7415-932-5 (ISBN)
Disputation
2011-04-15, FR4, Albanova Universitetscentrum, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Anmärkning
QC 20110401Tillgänglig från: 2011-04-01 Skapad: 2011-03-22 Senast uppdaterad: 2011-11-03Bibliografiskt granskad

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