J Integr Plant Biol. ›› 2010, Vol. 52 ›› Issue (2): 221-233.DOI: 10.1111/j.1744-7909.2010.00919.x

• Research Articles • Previous Articles     Next Articles

Biosynthesis of Callose and Cellulose by Detergent Extracts of Tobacco Cell Membranes and Quantification of the Polymers Synthesized in vitro

Carolina Cifuentes1, Vincent Bulone2 and Anne Mie C. Emons1,3*   

  1. 1Laboratory of Plant Cell Biology, Wageningen University, Droevendaalsesteeg, 1,6708 PB Wageningen, The Netherlands
    2Division of Glycoscience, School of Biotechnology, Royal Institute of Technology, AlbaNova University Center, SE-106 91 Stockholm, Sweden
    3Department of Biomolecular Systems, FOM Institute for Atomic and Molecular Physics, Science Park 104, 1098 SG Amsterdam, The Netherlands
  • Received:2009-10-30 Accepted:2009-12-13 Published:2010-02-01
  • About author:*Author for correspondence Tel: + 31-317-484329; Fax: + 31-317-418094; E-mail: annemie.emons@wur.nl

Abstract:

The conditions that favor the in vitro synthesis of cellulose from tobacco BY-2 cell extracts were determined. The procedure leading to the highest yield of cellulose consisted of incubating digitonin extracts of membranes from 11-day-old tobacco BY-2 cells in the presence of 1 mM UDP-glucose, 8 mM Ca2+ and 8 mM Mg2+. Under these conditions, up to nearly 40% of the polysaccharides synthesized in vitro corresponded to cellulose, the other polymer synthesized being callose. Transmission electron microscopy analysis revealed the occurrence of two types of structures in the synthetic reactions. The first type consisted of small aggregates with a diameter between 3 and 5 nm that associated to form fibrillar strings of a maximum length of 400 nm. These structures were sensitive to the acetic/nitric acid treatment of Updegraff and corresponded to callose. The second type of structures was resistant to the Updegraff reagent and corresponded to straight cellulose microfibrils of 2–3 nm in diameter and 200 nm to up to 5 μm in length. In vitro reactions performed on electron microscopy grids indicated that the minimal rate of microfibril elongation in vitro is 120 nm/min. Measurements of retardance by liquid crystal polarization microscopy as a function of time showed that small groups of microfibrils increased in retardance by up to 0.047 nm/min per pixel, confirming the formation of organized structures.

Cifuentes C, Bulone V, Emons AMC (2010) Biosynthesis of callose and cellulose by detergent extracts of tobacco cell membranes and quantification of the polymers synthesized in vitro. J. Integr. Plant Biol. 52(2), 221–233.

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