[beta]-Glucan Synthesis in the Cotton Fiber (I. Identification of [beta]-1,4- and [beta]-1,3-Glucans Synthesized in Vitro)

Abstract

In vitro B-glucan products were synthesized by digitonin-solubilized enzyme preparations from plasma membrane-enriched fractions of cotton (Cossypium hirsutum) fiber cells. The reaction mixture favoring &1,4-glucan synthesis included the following effedors: MgZ+, Caz+, cellobiose, cyclic-3':5'-CMP, and digitonin. The ethanol insoluble fraction from this reaction contained &1,4-glucan and 8-1,3-glucan in an approximate ratio of 25:69. Approximately 16% of the &1,4-glucan was resistant to the acetic/nitric acid reagent. The x-ray diffraction pattern of the treated produd favoring &l,4-glucan synthesis strongly resembled that of cellulose II. O n the basis of methylation analysis, the acetic/nitric acid reagent-insoluble glucan produd was found to be exclusively 8-1,4-linked. Enzymic hydrolysis confirmed that the produd was hydrolyzed only by cellobiohydrolase 1. Autoradiography proved that the produd was synthesized in vitro. l h e degree of polymerization (DP) of the in vitro product was estimated by nitration and size exclusion chromatography; there were two average DPs of 59 (70%) and 396 (30%) for the @-1,3-glucanase-treated sample, and an average DP of 141 for the acetic/nitric acid reagent-insoluble produd. O n the basis of product analysis, the positive identification of in vitro-synthesized cellulose was established.Cellulose is the most abundant macromolecule on earth (Brown, 1985). It serves a major structural role in the cell wall of plants, some algae, and certain fungi and is the primary component of economical products such as wood, cotton, and paper. Because of its tremendous abundance and its physiological and economical importance, many attempts at in vitro cellulose synthesis have been made with cell-free systems from various sources during the past three decades Brown, 1989b;Read and Delmer, 1991). The greatest progress has been made using Acetobacter xylinum as an experimental model (Ross et al., 1987;Brown, 1989a;Lin and Brown, 1989;Lin et al., 1990;Saxena et al., 1990;Wong et al., 1990;Mayer et al., 1991;Saxena et al., 1991). In higher plants, however, numerous failures to detect activity for in vitro cellulose synthase have continued because it has remained impossible to make preparations from higher plant cells capable of synthesizing true microfibrillar cellulose or even appreciable quantities of (3-1,4-glucan (Read and Delmer, 1991).Severa1 methods for the characterization of products synthesized in vitro, such as solubility properties, acid hydrolysis, acetolysis, specific enzymic digestion, and linkage analysis after periodate oxidation or methylation, have been used either alone or in various combinations (Fry, 1988). Insolubility in strong alkali (eg. 24% KOH) has been used for the detection of p-1,4-glucan, but (3-1,3-glucan synthesized in vitro may also be present in the alkali-insoluble fraction (Hayashi et al., 1987). The AN reagent (Updegraff, 1969) has been used for the selection of cellulose because only crystalline cellulose can remain after treatment with this reagent. The mo...

Cellulose in Cyanobacteria. Origin of Vascular Plant Cellulose Synthase?

“…These microfibrils were strongly labeled with CBHIgold, indicating that they are composed of ␤-1,4-glucans (Okuda et al, 1993;Tomme et al, 1995;Fig. 1).…”

“…A, Oriented bundles of microfibrils from Gloeocapsa sp. L795, many of which are stained with the CBHI-gold complex, which specifically binds to the surface of crystalline cellulose (Okuda et al, 1993;Tomme et al, 1995). B, Cellulose microfibrils from N. muscorum UTEX 2209.…”

“…Slime tube material collected from motile filaments on Formvar (polyvinyl formyl; Monsanto, St. Louis)-coated grids was labeled without further treatment. CBHI-gold labeling was performed es-sentially as described previously (Okuda et al, 1993) with the following exceptions: (1) 10 nm gold was used for the CBHI-gold complex, (2) rather than floating grids, 8-L drops of enzyme complex were added to Formvar grids with the EPS isolates, and (3) enzyme complex and product were incubated for 3 min at room temperature (RT). Grids were negatively stained with 2% (w/v) uranyl acetate.…”

Tools for Cellulose Analysis in Plant Cell Walls

“…In addition, further complication is due to the unavoidable concomitant extraction of callose synthase from the PM. This enzyme uses the same substrate as cellulose synthase (UDP-Glc) and exhibits high activity in vitro (Okuda et al, 1993;Lai Kee Him et al, 2001Colombani et al, 2004), even though callose is normally a minor cell wall component that forms transiently at the cell plate prior to cell division or in specialized cells (e.g. pollen tubes) or cell structures (e.g.…”

Immunogold Labeling of Rosette Terminal Cellulose-Synthesizing Complexes in the Vascular Plant Vigna angularis

“…The plasma membrane proteins from 14 and 24 DPA cotton fibers as well as Arabidopsis and V. radiata were prepared according to Okuda et al (1993).…”