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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

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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.

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  1. Equipe “Organisation et Dynamique des Membranes Biologiques”, UMR CNRS 5013, Bâtiment Chevreul, Université Claude Bernard Lyon I, 43 Boulevard du 11 Novembre 1918, F-69622, Villeurbanne Cedex, France

    Audrey Colombani, Laurence Bessueille & Vincent Bulone

  2. Department of Biotechnology, Royal Institute of Technology, AlbaNova University Center, SE-106 91, Stockholm, Sweden

    Soraya Djerbi, Kristina Blomqvist, Anna Ohlsson, Torkel Berglund & Tuula T. Teeri

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  1. Audrey Colombani
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  2. Soraya Djerbi
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  3. Laurence Bessueille
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  7. Tuula T. Teeri
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  8. Vincent Bulone
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Colombani, A., Djerbi, S., Bessueille, L. et al. In vitro synthesis of (1→3)-β-D-glucan (callose) and cellulose by detergent extracts of membranes from cell suspension cultures of hybrid aspen. Cellulose 11, 313–327 (2004). https://doi.org/10.1023/B:CELL.0000046404.25406.19

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