Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
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.
KTH, School of Biotechnology (BIO), Glycoscience.ORCID iD: 0000-0002-5368-4296
KTH, School of Biotechnology (BIO), Glycoscience.
KTH, School of Biotechnology (BIO), Glycoscience.
Show others and affiliations
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.

Place, publisher, year, edition, pages
2006. Vol. 228, no 4, 221-9 p.
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:kth:diva-14261DOI: 10.1007/s00709-006-0156-4ISI: 000240615000007PubMedID: 16838081Scopus ID: 2-s2.0-33748917572OAI: oai:DiVA.org:kth-14261DiVA: diva2:331971
Note
QC20100729Available from: 2010-07-29 Created: 2010-07-29 Last updated: 2017-12-12Bibliographically 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
Opponent
Supervisors
Note
QC 20101005Available from: 2005-09-13 Created: 2005-09-13 Last updated: 2010-10-05Bibliographically approved
2. 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.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2010:1
Keyword
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
Identifiers
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)
Opponent
Supervisors
Note
QC20100730Available from: 2010-01-21 Created: 2010-01-21 Last updated: 2010-07-30Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textPubMedScopusProtoplasma

Search in DiVA

By author/editor
Ohlsson, Anna B.Djerbi, SorayaWinzell, AndersStåldal, VeronikaLi, XinguoBlomqvist, KristinaTeeri, Tuula T.Berglund, Torkel
By organisation
Glycoscience
In the same journal
Protoplasma
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 93 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf