PLANT PHYSIOLOGY , Vol 107, Issue 1 111-123, Copyright © 1995 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
[beta]-Glucan Synthesis in the Cotton Fiber (IV. In Vitro Assembly of the Cellulose I Allomorph)
K. Kudlicka, R. M. Brown Jr, L. Li, J. H. Lee, H. Shin and S. Kuga
Department of Botany, The University of Texas at Austin, Austin, Texas 78713-7640
In vitro assembly of cellulose from plasma membrane extracts of the cotton
(Gossypium hirsutum) fiber was enriched by a combination of
3-(N-morpholino)propanesulfonic acid extraction buffer and two independent
digitonin solubilization steps consisting of 0.05% digitonin (SE1) followed
by 1% digitonin (SE2). Glucan synthase activity assays revealed that,
although the SE2 fraction possessed higher activity, only 8.6% of the in
vitro product survived acetic/nitric acid treatment. On the other hand, the
SE1 fraction was less active, but 32.1% of the total glucan in vitro
product was resistant to acetic/nitric acid. In vitro products synthesized
from the SE1 fraction contained [beta]-1,3-glucan and fibrillar cellulose
I, whereas the SE2 fraction produced [beta]-1,3-glucan and cellulose II.
Both celluloses assembled in vitro were labeled with cellobiohydrolase
I-gold complex, and the electron diffraction patterns of both products from
SE1 and SE2 revealed cellulose I and cellulose II, respectively.
Contamination of native cellulose was ruled out by extensive evidence from
autoradiography of the ethanol-insoluble and acetic/nitric acid-insoluble
materials, including three different controls.
