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Cellulose

, Volume 11, Issue 3–4, pp 313–327 | Cite as

In vitro synthesis of (1→3)-β-D-glucan (callose) and cellulose by detergent extracts of membranes from cell suspension cultures of hybrid aspen

  • Audrey Colombani
  • Soraya Djerbi
  • Laurence Bessueille
  • Kristina Blomqvist
  • Anna Ohlsson
  • Torkel Berglund
  • Tuula T. Teeri
  • Vincent BuloneEmail author
Article

Abstract

The aim of this work was to optimize the conditions for in vitro synthesis of (1→3)-β-D-glucan (callose) and cellulose, using detergent extracts of membranes from hybrid aspen (Populus tremula ×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 vitroβ-glucans were identified by hydrolysis of radioactive products, using specific enzymes. 13C-Nuclear magnetic resonance spectroscopy and transmission electron microscopy were also used for callose characterization. The (1→3)-β-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.

(1→3)-β-D-Glucan (callose) and cellulose synthases Hybrid aspen (Populus tremula × tremuloidesIn vitro synthesis of callose and cellulose Plant cell walls Suspension cultures 

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Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Audrey Colombani
    • 1
  • Soraya Djerbi
    • 2
  • Laurence Bessueille
    • 1
  • Kristina Blomqvist
    • 2
  • Anna Ohlsson
    • 2
  • Torkel Berglund
    • 2
  • Tuula T. Teeri
    • 2
  • Vincent Bulone
    • 1
    Email author
  1. 1.Equipe “Organisation et Dynamique des Membranes Biologiques”, UMR CNRS 5013, Bâtiment ChevreulUniversité Claude Bernard Lyon IVilleurbanne CedexFrance
  2. 2.Department of Biotechnology, Royal Institute of TechnologyAlbaNova University CenterStockholmSweden

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