Cellulose and Callose Biosynthesis in Higher Plants (I. Solubilization and Separation of (1->3)- and (1->4)-[beta]-Glucan Synthase Activities from Mung Bean).

(1->3)- and (1->4)-[beta]-glucan synthase activities from higher plants have been physically separated by gel electrophoresis in nondenaturing conditions. The two glucan synthases show different mobilities in native polyacrylamide gels. Further separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a different polypeptide composition in these synthases. Three polypeptides (64, 54, and 32 kD) seem to be common to both synthase activities, whereas two polypeptides (78 and 38 kD) are associated only with callose synthase activity. Twelve polypeptides (170, 136, 108, 96, 83, 72, 66, 60, 52, 48, 42, and 34 kD) appear to be specifically associated with cellulose synthase activity. The successful separation of (1->3)- and (1->-4)-[beta]-glucan synthase activities was based on the manipulation of digitonin concentrations used in the solubilization of membrane proteins. At low dipitomin concentrations (0.05 and 0.1%), the ratio of the cellulose to callose synthase activity was higher. At higher digitonin (0.5-1%) concentrations, the ratio of the callose to cellulose synthase activity was higher. Rosette-like particles with attached product were observed in samples taken from the top of the stacking gel, where only cellulose was synthesized. Smaller (nonrosette) particles were found in the running gel, where only callose was synthesized. These findings suggest that a higher level of subunit organization is required for in vitro cellulose synthesis in comparison with callose assembly.

[beta]-Glucan Synthesis in the Cotton Fiber (IV. In Vitro Assembly of the Cellulose I Allomorph).

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.

Characterization of genes in the cellulose-synthesizing operon (acs operon) of Acetobacter xylinum: implications for cellulose crystallization.

The synthesis of an extracellular ribbon of cellulose in the bacterium Acetobacter xylinum takes place from linearly arranged, membrane-localized, cellulose-synthesizing and extrusion complexes that direct the coupled steps of polymerization and crystallization. To identify the different components involved in this process, we isolated an Acetobacter cellulose-synthesizing (acs) operon from this bacterium. Analysis of DNA sequence shows the presence of three genes in the acs operon, in which the first gene (acsAB) codes for a polypeptide with a molecular mass of 168 kDa, which was identified as the cellulose synthase. A single base change in the previously reported DNA sequence of this gene, resulting in a frameshift and synthesis of a larger protein, is described in the present paper, along with the sequences of the other two genes (acsC and acsD). The requirement of the acs operon genes for cellulose production was determined using site-determined TnphoA/Kanr GenBlock insertion mutants. Mutant analysis showed that while the acsAB and acsC genes were essential for cellulose production in vivo, the acsD mutant produced reduced amounts of two cellulose allomorphs (cellulose I and cellulose II), suggesting that the acsD gene is involved in cellulose crystallization. The role of the acs operon genes in determining the linear array of intramembranous particles, which are believed to be sites of cellulose synthesis, was investigated for the different mutants; however, this arrangement was observed only in cells that actively produced cellulose microfibrils, suggesting that it may be influenced by the crystallization of the nascent glucan chains.

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

In vitro [beta]-glucan products were synthesized by digitonin-solubilized enzyme preparations from plasma membrane-enriched fractions of cotton (Gossypium hirsutum) fiber cells. The reaction mixture favoring [beta]-1,4-glucan synthesis included the following effectors: Mg2+, Ca2+, cellobiose, cyclic-3[prime]:5[prime]-GMP, and digitonin. The ethanol insoluble fraction from this reaction contained [beta]-1,4-glucan and [beta]-1,3-glucan in an approximate ratio of 25:69. Approximately 16% of the [beta]-1,4-glucan was resistant to the acetic/nitric acid reagent. The x-ray diffraction pattern of the treated product favoring [beta]-1,4-glucan synthesis strongly resembled that of cellulose II. On the basis of methylation analysis, the acetic/nitric acid reagent-insoluble glucan product was found to be exclusively [beta]-1,4-linked. Enzymic hydrolysis confirmed that the product was hydrolyzed only by cellobiohydrolase I. Autoradiography proved that the product was synthesized in vitro. The 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 [beta]-1,3-glucanase-treated sample, and an average DP of 141 for the acetic/nitric acid reagent-insoluble product. On the basis of product analysis, the positive identification of in vitro-synthesized cellulose was established.

Gravity effects on cellulose assembly.

The effect of microgravity on cellulose synthesis using the model system of Acetobacter xylinum was the subject of recent investigations using The National Aeronautics and Space Administration's Reduced Gravity Laboratory, a modified KC-135 aircraft designed to produce 20 sec of microgravity during the top of a parabolic dive. Approximately 40 parabolas were executed per mission, and a period of 2 x g was integral to the pullout phase of each parabola. Cellulose biosynthesis was initiated on agar surfaces, liquid growth medium, and buffered glucose during parabolic flight and terminated with 2.0% sodium azide or 50.0% ethanol. While careful ground and in-flight controls indicated normal, compact ribbons of microbial cellulose, data from five different flights consistently showed that during progression into the parabola regime, the cellulose ribbons became splayed. This observation suggests that some element of the parabola (the 20 sec microgravity phase, the 20 sec 2 x g phase, or a combination of both) was responsible for this effect. Presumably the cellulose I alpha crystalline polymorph normally is produced under strain, and the microgravity/hypergravity combination may relieve this stress to produce splayed ribbons. An in-flight video microscopy analysis of bacterial motions during a parabolic series demonstrated that the bacteria continue to synthesize cellulose during all phases of the parabolic series. Thus, the splaying may be a reflection of a more subtle alteration such as reduction of intermicrofibrillar hydrogen bonding. Long-term microgravity exposures during spaceflight will be necessary to fully understand the cellulose alterations from the short-term microgravity experiments.