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In vitro synthesis of (1→3)-β-D-glucan (callose) and cellulose by detergent extracts of membranes from cell suspension cultures of hybrid aspen
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
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2004 (English)In: Cellulose (London), ISSN 0969-0239, E-ISSN 1572-882X, Vol. 11, no 3-4, 313-327 p.Article in journal (Refereed) Published
Abstract [en]

The aim of this work was to optimize the conditions for in vitro synthesis of (1 --> 3)-beta-D-glucan (callose) and cellulose, using detergent extracts of membranes from hybrid aspen (Populus tremula x tremuloides) cells grown as suspension cultures. Callose was the only product synthesized when CHAPS extracts were used as a source of enzyme. The optimal reaction mixture for callose synthesis contained 100 mM Mops buffer pH 7.0, 1 mM UDP-glucose, 8 mM Ca2+, and 20 mM cellobiose. The use of digitonin to extract the membrane-bound proteins was required for cellulose synthesis. Yields as high as 50% of the total in vitro products were obtained when cells were harvested in the stationary phase of the growth curve, callose being the other product. The optimal mixture for cellulose synthesis consisted of 100 mM Mops buffer pH 7.0, 1 mM UDP-glucose, 1 mM Ca2+, 8 mM Mg2+, and 20 mM cellobiose. The in vitro beta-glucans were identified by hydrolysis of radioactive products, using specific enzymes. C-13-Nuclear magnetic resonance spectroscopy and transmission electron microscopy were also used for callose characterization. The (1-->3)-beta-D-glucan systematically had a microfibrillar morphology, but the size and organization of the microfibrils were affected by the nature of the detergent used for enzyme extraction. The discussion of the results is included in a short review of the field that also compares the data obtained with those available in the literature. The results presented show that the hybrid aspen is a promising model for in vitro studies on callose and cellulose synthesis.

Place, publisher, year, edition, pages
2004. Vol. 11, no 3-4, 313-327 p.
Keyword [en]
(1 -> 3)-beta-D-glucan (callose) and cellulose synthases, hybrid aspen (Populus tremula x tremuloides), in vitro synthesis of callose and cellulose, plant cell walls, suspension cultures
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-6247DOI: 10.1023/B:CELL.0000046404.25406.19ISI: 000225573000005OAI: oai:DiVA.org:kth-6247DiVA: diva2:10903
Note
QC 20100917Available from: 2005-09-13 Created: 2005-09-13 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Cellulose synthases in Populus- identification, expression analyses and in vitro synthesis
Open this publication in new window or tab >>Cellulose synthases in Populus- identification, expression analyses and in vitro synthesis
2005 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Cellulose is a biopolymer of great relevance in the plant cell walls, where it constitutes the most important skeletal component. Cellulose is also an important raw material in the pulp- and paper, forest, and textile industries, among others. Cellulose biosynthesis in particular, and xylogenesis in general are processes which are currently poorly understood. Yet, research in cellulose synthesis is progressing and different applications of cellulose, mainly cellulose derivatives for e.g. pharmaceuticals and coatings, are constantly emerging. This thesis depicts how cellulose synthase (CesA) genes in Populus were identified and characterized by gene expression- and bioinformatics analyses. Within an EST database of more than 100,000 clones from wood forming tissues of three different Populus taxa, ten CesA genes were identified in Populus tremula x tremuloides. Subsequent gene expression analyses by using microarrays and real-time PCR experiments in woody tissues, revealed distinct regulation patterns among the genes of interest. This enabled proper classification and characterization of the secondary cell wall related CesA genes, in particular. Bioinformatic analyses of the genome sequence of Populus trichocarpa further provided a complete picture of the number of putative CesA genes retained after several duplication events during tree evolution. In contrast to the previously reported set of ten 'true' CesA genes in many other plant species, the genome of P. trichocarpa encodes 18 putative proteins, which could be assembled into nine groups according to their sequence similarities. Interestingly, studies in the EST database suggested that paralogs within at least two groups have corresponding orthologs in P. tremula x tremuloides, which are furthermore transcribed. This implies that at least some of the duplicated genes have remained functional, or may have acquired a modified function.

By focusing on the CesA genes associated with secondary cell wall formation, cellulose synthesis was also studied in poplar cell suspension cultures. Selection of CesA enriched material was performed by determining expression intensities of the CesA genes using RT-PCR, whereupon membrane protein extraction was initiated. CesA proteins are part of large cellulose synthesizing complexes in the plasma membrane. Subsequent proteomic approaches comprised partial purification of these cellulose synthesizing complexes from protein enriched culture material and in vitro cellulose synthesis experiments. De novo synthesized material was successfully characterized and the acquired yields were as high as 50% cellulose (compared to previously reported yields of 30% in other plant systems) of the total in vitro synthesized product. Elevated CesA gene expression levels can thus be correlated to increased protein activity in poplar cell suspension cultures. In addition, antibodies raised against CesA antigens were used in Western blot analyses comprising samples along the protein extraction- and purification procedure. Proteins with corresponding molecular weight to the theoretical 120kDa of CesA proteins were recognized by a range of different specific antibodies. The study demonstrates that poplar cell suspension cultures can provide a valuable model system for studies of cellulose synthesis and different aspects of xylogenesis.

Place, publisher, year, edition, pages
Stockholm: KTH, 2005. 79 p.
Keyword
cellulose synthase, Populus, secondary cell wall, gene upregulation, cell suspension culture, stationary phase
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-414 (URN)91-7178-109-9 (ISBN)
Public defence
2005-09-23, Svedbergssalen, FD5, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:00
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Note
QC 20101005Available from: 2005-09-13 Created: 2005-09-13 Last updated: 2010-10-05Bibliographically approved

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