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Functional genomics of wood degradation and biosynthesis
KTH, School of Biotechnology (BIO).
2005 (English)Licentiate thesis, comprehensive summary (Other scientific)
Abstract [en]

Forest biotechnology is a fast emerging field of research. The application of biotechnological tools will enhance the quality of the forest products. The resultant value added and environmentally sustainable products are an absolute necessity in the future. The study of wood biosynthesis and degradation will result in enormous knowledge resources, which can be used for exploiting wood properties. This thesis addresses questions representing both wood degradation and biosynthesis.

The wood degrading fungus Phanerochaete chrysosporium is expression profiled with the microarray technology. The objective is to understand the expression pattern of the extracellular carbohydrate active enzymes (CAZymes) secreted by the organism. The data obtained increases our understanding of gene expression upon growth on cellulose.

Wood biosynthesis is studied with the model wood forming tree species, Populus. The plentiful data resources from the expression profiling during wood formation in Populus are used as the platform of this work. One of the wood specific genes, PttMAP20, previously with an unknown function is studied in this thesis. The immunolocalisation of PttMAP20 with specific antibodies is demonstrated. The putative microtubule-targeting domain of the protein is demonstrated microscopically and by using a biochemical binding assay.

Place, publisher, year, edition, pages
Stockholm: KTH , 2005. , 42 p.
Keyword [en]
populus, xylogenesis, secondary cell wall, cellulose, hemicellulose, microarrays, transcript profiling
National Category
Other Biological Topics
Identifiers
URN: urn:nbn:se:kth:diva-592ISBN: 91-7178-250-8 (print)OAI: oai:DiVA.org:kth-592DiVA: diva2:14542
Presentation
2006-01-30, Sal FB54, AlbaNova, Roslagstullsbacken, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20101217Available from: 2006-01-16 Created: 2006-01-16 Last updated: 2011-11-23Bibliographically approved
List of papers
1. The Phanerochaete chrysosporium secretome: database predictions and initial mass spectrometry peptide identifications in cellulose-grown medium
Open this publication in new window or tab >>The Phanerochaete chrysosporium secretome: database predictions and initial mass spectrometry peptide identifications in cellulose-grown medium
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2005 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 118, 17-34 p.Article in journal (Refereed) Published
Abstract [en]

The white rot basidiomycete, Phanerochaete chrysosporium, employs an array of extracellular enzymes to completely degrade the major polymers of wood: cellulose, hemicellulose and lignin. Towards the identification of participating enzymes, 268 likely secreted proteins were predicted using SignalP and TargetP algorithms. To assess the reliability of secretome predictions and to evaluate the usefulness of the current database, we performed shotgun LC-MS/MS on cultures grown on standard cellulose-containing medium. A total of 182 unique peptide sequences were matched to 50 specific genes, of which 24 were among the secretome subset. Underscoring the rich genetic diversity of R chrysosporium, identifications included 32 glycosyl hydrolases. Functionally interconnected enzyme groups were recognized. For example, the multiple endoglucanases and processive exocellobiohydrolases observed quite probably attack cellulose in a synergistic manner. In addition, a hemicellulolytic system included endoxylanases, alpha-galactosidase, acetyl xylan esterase, and alpha-L-arabinofuranosidase. Glucose and cellobiose metabolism likely involves cellobiose dehydrogenase, glucose oxidase, and various inverting glycoside hydrolases, all perhaps enhanced by an epimerase. To evaluate the completeness of the current database, mass spectroscopy analysis was performed on a larger and more inclusive dataset containing all possible ORFs. This allowed identification of a previously undetected hypothetical protein and a putative acid phosphatase. The expression of several genes was supported by RT-PCR amplification of their cDNAs.

Keyword
Phanerochaete chrysosporium, genome, proteome, secretome, glycosyl hydrolase, cellulase
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-9010 (URN)10.1016/j.jbiotec.2005.03.010 (DOI)000230328500003 ()2-s2.0-20444468865 (Scopus ID)
Note
QC 20101217Available from: 2006-01-16 Created: 2006-01-16 Last updated: 2017-12-14Bibliographically approved
2. MAP20, a Microtubule-Associated Protein in the Secondary Cell Walls of Hybrid Aspen, Is a Target of the Cellulose Synthesis Inhibitor 2,6-Dichlorobenzonitrile
Open this publication in new window or tab >>MAP20, a Microtubule-Associated Protein in the Secondary Cell Walls of Hybrid Aspen, Is a Target of the Cellulose Synthesis Inhibitor 2,6-Dichlorobenzonitrile
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2008 (English)In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 148, no 3, 1283-1294 p.Article in journal (Refereed) Published
Abstract [en]

We have identified a gene, denoted PttMAP20, which is strongly up-regulated during secondary cell wall synthesis and tightly coregulated with the secondary wall-associated CESA genes in hybrid aspen (Populus tremula x tremuloides). Immunolocalization studies with affinity-purified antibodies specific for PttMAP20 revealed that the protein is found in all cell types in developing xylem and that it is most abundant in cells forming secondary cell walls. This PttMAP20 protein sequence contains a highly conserved TPX2 domain first identified in a microtubule-associated protein (MAP) in Xenopus laevis. Overexpression of PttMAP20 in Arabidopsis (Arabidopsis thaliana) leads to helical twisting of epidermal cells, frequently associated with MAPs. In addition, a PttMAP20-yellow fluorescent protein fusion protein expressed in tobacco (Nicotiana tabacum) leaves localizes to microtubules in leaf epidermal pavement cells. Recombinant PttMAP20 expressed in Escherichia coli also binds specifically to in vitro-assembled, taxol-stabilized bovine microtubules. Finally, the herbicide 2,6-dichlorobenzonitrile, which inhibits cellulose synthesis in plants, was found to bind specifically to PttMAP20. Together with the known function of cortical microtubules in orienting cellulose microfibrils, these observations suggest that PttMAP20 has a role in cellulose biosynthesis.

Keyword
carbohydrate-active enzymes, plant functional genomics, kinesin-like, protein, cortical microtubules, plasma-membrane, wood formation, arabidopsis-thaliana, conifer tracheids, angiosperm trees, urea solution
National Category
Natural Sciences
Identifiers
urn:nbn:se:kth:diva-17951 (URN)10.1104/pp.108.121913 (DOI)000260719500010 ()2-s2.0-57749101097 (Scopus ID)
Note

QC 20100525

Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved

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