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

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 Ca~(2+) and 8 mM Mg~(2+). 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 nmlmin. 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.