Krystyna Kudlicka

β-Glucan synthesis in the cotton fiber: I. Identification of β-1,4- and β-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.

Β-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.

Further evidence from sectioned material in support of the existence of a linear terminal complex in cellulose synthesis

SummaryTransmembrane linear terminal complexes considered to be involved in the synthesis of cellulose microfibrils have been described in the plasma membrane ofBoergesenia forbesii. Evidence for the existence of these structures has been obtained almost exlusively using the freeze etching technique. In the present study an attempt has been made to complete these studies using conventional fixation, staining, and sectioning procedures. In developing cells ofBoergesenia forbesii, strongly stained structures traversing the plasma membrane and averaging 598.9 nm ± 171.3 nm in length, 28.7 nm ± 4.2 nm in width, and 35.2 nm ± 6.6 nm in depth have been demonstrated. These structures are considered to be linear terminal complexes. At their distal (cell wall) surface, they appear to be closely associated with cellulose microfibrils. At the proximal (cytoplasmic) surface, they are associated with microtubules and polysomes. A model of the possible interrelation of the terminal complexes and microtubules leading to the generation of cell wall microfibrils is proposed.

Organelle Aggregations During Microsporogenesis in Stangeria, Nymphaea, and Malva

Microsporocytes of Stangeria and Nymphaea acquire a polarizedappearance for a short part of prophase I, when the areas on the opposite sides of the nucleus differ in their organelle contents. By the end of prophase I in microsporocytes of Nymphaea plastids and mitochondria form a group which after telophase I stretches into the equatorial plane, and after telophase II separates a tetrad into four regions. During microsporogenesis in Malva organelles form a dense coat around the late prophase I nucleus and later around each nucleus in dyads and tetrads.