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Cellulose synthases in Populus- identification, expression analyses and in vitro synthesis
KTH, School of Biotechnology (BIO), Glycoscience.
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 [en]
cellulose synthase, Populus, secondary cell wall, gene upregulation, cell suspension culture, stationary phase
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-414ISBN: 91-7178-109-9 (print)OAI: oai:DiVA.org:kth-414DiVA: diva2:10904
Public defence
2005-09-23, Svedbergssalen, FD5, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20101005Available from: 2005-09-13 Created: 2005-09-13 Last updated: 2010-10-05Bibliographically approved
List of papers
1. Identification and expression analysis of genes encoding putative cellulose synthases (CesA) in the hybrid aspen, Populus tremula (L.) × P. tremuloides (Michx.)
Open this publication in new window or tab >>Identification and expression analysis of genes encoding putative cellulose synthases (CesA) in the hybrid aspen, Populus tremula (L.) × P. tremuloides (Michx.)
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2004 (English)In: Cellulose (London), ISSN 0969-0239, E-ISSN 1572-882X, Vol. 11, no 3-4, 301-312 p.Article in journal (Refereed) Published
Abstract [en]

Cellulose is synthesized in plant cell walls by large membrane-bound protein complexes proposed to contain several copies of the catalytic subunit of the cellulose synthase, CesA. Here we report identification of 10 distinct CesA genes within a database of 100,000 ESTs of the hybrid aspen, Populus tremula (L.) x P. tremuloides (Michx.). Expression analyses in normal wood undergoing xylogenesis and in tension wood indicate xylem specific expression of four putative CesA isoenzymes, PttCesA1, PttCesA3-1, PttCesA3-2 and PttCesA9. Both the protein sequences and the expression profiles of PttCesA3-1 and PttCesA3-2 are very similar, and they may thus represent redundant copies of an enzyme with essentially the same function. Further, one of the generally more constitutively expressed CesA genes, PttCesA2, seems to be activated on the opposite side of a tension wood induced stem, while PttCesA6 appears to be more specific for leaf tissues. The rest of the hybrid aspen CesA genes were found to be relatively evenly expressed over the poplar tissues hereby studied.

Keyword
cellulose synthesis, expression profiling, hybrid aspen, plant cell wall, tension wood
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6244 (URN)10.1023/B:CELL.0000046408.84510.06 (DOI)000225573000004 ()
Note
QC 20100916Available from: 2005-09-13 Created: 2005-09-13 Last updated: 2017-12-14Bibliographically approved
2. The genome sequence of black cottonwood (Populus trichocarpa) reveals 18 conserved cellulose synthase (CesA) genes
Open this publication in new window or tab >>The genome sequence of black cottonwood (Populus trichocarpa) reveals 18 conserved cellulose synthase (CesA) genes
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2005 (English)In: Planta, ISSN 0032-0935, E-ISSN 1432-2048, Vol. 221, no 5, 739-746 p.Article in journal (Refereed) Published
Abstract [en]

The genome sequence of Populus trichocarpa was screened for genes encoding cellulose synthases by using full-length cDNA sequences and ESTs previously identified in the tissue specific cDNA libraries of other poplars. The data obtained revealed 18 distinct CesA gene sequences in P. trichocarpa. The identified genes were grouped in seven gene pairs, one group of three sequences and one single gene. Evidence from gene expression studies of hybrid aspen suggests that both copies of at least one pair, CesA3-1 and CesA3-2, are actively transcribed. No sequences corresponding to the gene pair, CesA6-1 and CesA6-2, were found in Arabidopsis or hybrid aspen, while one homologous gene has been identified in the rice genome and an active transcript in Populus tremuloides. A phylogenetic analysis suggests that the CesA genes previously associated with secondary cell wall synthesis originate from a single ancestor gene and group in three distinct subgroups. The newly identified copies of CesA genes in P. trichocarpa give rise to a number of new questions concerning the mechanism of cellulose synthesis in trees.

Keyword
Populus, paleopolyploids, cellulose synthase, gene duplication, paralogues
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6245 (URN)10.1007/s00425-005-1498-4 (DOI)000230490200013 ()2-s2.0-22444446783 (Scopus ID)
Note
QC 20101001. Uppdaterad från In press till Published (20101001).Available from: 2005-09-13 Created: 2005-09-13 Last updated: 2017-12-14Bibliographically approved
3. Cell suspension cultures of Populus tremula x P. tremuloides exhibit a high level of cellulose synthase gene expression that coincides with increased in vitro cellulose synthase activity.
Open this publication in new window or tab >>Cell suspension cultures of Populus tremula x P. tremuloides exhibit a high level of cellulose synthase gene expression that coincides with increased in vitro cellulose synthase activity.
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2006 (English)In: Protoplasma, ISSN 0033-183X, E-ISSN 1615-6102, Vol. 228, no 4, 221-9 p.Article in journal (Refereed) Published
Abstract [en]

Compared to wood, cell suspension cultures provide convenient model systems to study many different cellular processes in plants. Here we have established cell suspension cultures of Populus tremula L. x P. tremuloides Michx. and characterized them by determining the enzymatic activities and/or mRNA expression levels of selected cell wall-specific proteins at the different stages of growth. While enzymes and proteins typically associated with primary cell wall synthesis and expansion were detected in the exponential growth phase of the cultures, the late stationary phase showed high expression of the secondary-cell-wall-associated cellulose synthase genes. Interestingly, detergent extracts of membranes from aging cell suspension cultures exhibited high levels of in vitro cellulose synthesis. The estimated ratio of cellulose to callose was as high as 50 : 50, as opposed to the ratio of 30 : 70 so far achieved with membrane preparations extracted from other systems. The increased cellulose synthase activity was also evidenced by higher levels of Calcofluor white binding in the cell material from the stationary-phase cultures. The ease of handling cell suspension cultures and the improved capacity for in vitro cellulose synthesis suggest that these cultures offer a new basis for studying the mechanism of cellulose biosynthesis.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-14261 (URN)10.1007/s00709-006-0156-4 (DOI)000240615000007 ()16838081 (PubMedID)2-s2.0-33748917572 (Scopus ID)
Note
QC20100729Available from: 2010-07-29 Created: 2010-07-29 Last updated: 2017-12-12Bibliographically approved
4. In vitro synthesis of (1→3)-β-D-glucan (callose) and cellulose by detergent extracts of membranes from cell suspension cultures of hybrid aspen
Open this publication in new window or tab >>In vitro synthesis of (1→3)-β-D-glucan (callose) and cellulose by detergent extracts of membranes from cell suspension cultures of hybrid aspen
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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.

Keyword
(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:nbn:se:kth:diva-6247 (URN)10.1023/B:CELL.0000046404.25406.19 (DOI)000225573000005 ()
Note
QC 20100917Available from: 2005-09-13 Created: 2005-09-13 Last updated: 2017-12-14Bibliographically approved

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