Deborah P. Delmer

Determination of the Pore Size of Cell Walls of Living Plant Cells

The limiting diameter of pores in the walls of living plant cells through which molecules can freely pass has been determined by a solute exclusion technique to be 35 to 38 angstroms for hair cells of Raphanus sativus roots and fibers of Gossypium hirsutum, 38 to 40 angstroms for cultured cells of Acer pseudoplatanus, and 45 to 52 angstroms for isolated palisade parenchyma cells of the leaves of Xanthium strumarium and Commelina communis. These results indicate that molecules with diameters larger than these pores would be restricted in their ability to penetrate such a cell wall, and that such a wall may represent a more significant barrier to cellular communication than has been previously assumed.

Characterization of inhibitors of cellulose synthesis in cotton fibers.

Several compounds were tested for their ability to inhibit the in-vivo synthesis of cellulose and other cell-wall polysaccharides in fibers of cotton (Gossypium hirsutum L.) developing on in-vitro cultured ovules. Inhibitory effects were measured by the ability of the compounds to inhibit the incorporation of radioactivity from [U-14C]glucose into these cell-wall polymers. Of the compounds surveyed, 2,6-dichlorobenzonitrile (DCB) was the most effective and specific one for its effects on cellulose synthesis when compared to its effect on the synthesis of other cell-wall components. At 10 μM DCB caused 80% inhibition of cellulose synthesis, and the effect was reversed upon removal of the DCB, with recovery to 90% of the control rate. Two analogs of DCB, 2-chloro-6-fluorobenzonitrile and 2,6-dichlorobenzene carbothiamide, were as specific and nearly as effective as DCB with respect to their effects on cellulose synthesis. Coumarin, generally regarded as an inhibitor of cellulose synthesis in other plant systems, was effective in cotton fibers in millimolar concentrations and, like DCB, was relatively specific with regard to its effect on cellulose synthesis. DCB and coumarin inhibited the synthesis of both primary and secondary wall cellulose. Bacitracin, an inhibitor of the cycling of phosphorylated polyprenols involved in cell-wall synthesis in bacteria, and ethylenediaminetetracetic acid (EDTA) and ethyleneglycol-bis-(β-amino-ethylether)-N,N′-tetracetic acid (EGTA), chelators of civalent cations, were also effective, although only at relatively high concentrations, in inhibiting incorporation of radioactivity into cellulose.

Biosynthesis of cellulose

Publisher Summary This chapter discusses the biosynthesis of cellulose. It analyzes the older, fragmented literature and assesses its major contributions; concentrates extensively on new findings and coordinates their interpretations; and examines the existence of gaps in the knowledge of this complex process. The polymerization of D -glucan chains occurs by way of a multi-subunit, enzyme complex embedded in the plasma membrane. An almost simultaneous association, by means of hydrogen bonds, of the newly formed chains results in the formation of partially crystalline microfibrils. This mechanism of polymerization and crystallization results in the creation of microfibrils whose chains are oriented parallel (cellulose I). In A. xylinum, the complex is apparently immobile, but, in cells in which cellulose is deposited as a cell-wall constituent, it seems probable that the force generated by the polymerization of the relatively rigid microfibrils propels the complex through the fluid-mosaic membrane. The direction of motion may be guided through the influence of microtubules. The chapter discusses the cytological investigations of cellulose biosynthesis and elaborates the mechanism of polymerization.

The cotton kinesin-like calmodulin-binding protein associates with cortical microtubules in cotton fibers

Microtubules in interphase plant cells form a cortical array, which is critical for plant cell morphogenesis. Genetic studies imply that the minus end-directed microtubule motor kinesin-like calmodulin-binding protein (KCBP) plays a role in trichome morphogenesis in Arabidopsis. However, it was not clear whether this motor interacted with interphase microtubules. In cotton (Gossypium hirsutum) fibers, cortical microtubules undergo dramatic reorganization during fiber development. In this study, cDNA clones of the cotton KCBP homolog GhKCBP were isolated from a cotton fiber-specific cDNA library. During cotton fiber development from 10 to 21 DPA, the GhKCBP protein level gradually decreases. By immunofluorescence, GhKCBP was detected as puncta along cortical microtubules in fiber cells of different developmental stages. Thus our results provide evidence that GhKCBP plays a role in interphase cell growth likely by interacting with cortical microtubules. In contrast to fibers, in dividing cells of cotton, GhKCBP localized to the nucleus, the microtubule preprophase band, mitotic spindle, and the phragmoplast. Therefore KCBP likely exerts multiple roles in cell division and cell growth in flowering plants.

Cellulose and 1,3-glucan synthesis during the early stages of wall regeneration in soybean protoplasts.

Protoplasts isolated from cultured soybean cells (Glycine max (L.) Merr., cv. Mandarin) were used to study polysaccharide biosynthesis during the initial stages of cell wall-regeneration. Within minutes after the protoplasts were transferred to a wall-regeneration medium containing [14C]glucose, radioactivity was detected in a product which was chemically characterized as cellulose. The onset and accumulation of radioactivity into cellulose coincided with the appearance fibrils on the surface of protoplasts, as seen under the electron microscope. At these early stages, a variety of polysaccharide-containing polymers other than cellulose were also synthesized. Under conditions where the protoplasts were competent to synthesize cellulose from glucose, uridine diphosphate-[14C]glucose and guanosine diphosphate-[14C]glucose did not serve as effective substrates for cellulose synthesis. However, substantial amounts of label from uridine diphosphate glucose were incorporated into 1,3-glucan.

The Biosynthesis of Cellulose and Other Plant Cell Wall Polysaccharides

In recent years we have observed very rapid progress toward achieving an understanding of the structure of plant cell walls. In large part, this seems to be due to wise application by researchers of many newly-developed techniques for analysis of complex carbohydrates. Unfortunately, the area of cell wall biosynthesis cannot yet claim comparable progress. It is difficult to study the biosynthesis of polymers whose structures and interconnections have been poorly understood until quite recently. However, new knowledge concerning cell wall structure, availability of many new radioactive precursors, and recent advances in the isolation of plant organelles, all indicate that the subject of plant cell wall biosynthesis will be an exciting field of research in the years ahead.

Stimulation of membrane-associated polysaccharide synthetases by a membrane potential in developing cotton fibers

Conditions which induce a transmembrane electrical potential, positive with respect to the inside of membrane vesicles, result in a substantial (4–12-fold) stimulation of the activity of membrane-associated β-glucan synthetases in a membrane preparation derived from the developing cotton (Gossypium hirsutum L.) fiber. Induction of electrical potentials which are negative with respect to the inside of the membrane vesicle results in little or no stimulation of β-glucan synthesis. Those products whose synthesis is stimulated are mainly β-1,3-glucan, but there is also a considerable increase in β-1,4-glucan. No α-1,4-glucan (starch) was detected in the reaction products. A transmembrane pH gradient was found to have no effect on β-glucan synthesis. The results indicate that a transmembrane electrical potential can influence, either directly or indirectly, the activity of membrane-associated polysaccharide synthetases.

Seed reserve-protein glycosylation in an in vitro preparation from developing cotyledons of Phaseolus vulgaris

A particulate preparation from developing cotyledons of Phaseolus vulgaris L. was incubated with uridine-5′-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc; [6-3H]glucosamine), and by polyacrylamide gel electrophoretic analysis it was shown that the labeled (N-acetyl)glucosamine (GlcNAc) was incorporated into the principal reserve protein of the cotyledons, vicilin, and also into phytohemagglutinin. Some of the labeled product also reacted with antiserum to vicilin from mature seeds. In contrast it was not possible to detect the incorporation of labeled mannose from guanosine-5′-diphospho-D-mannose (GDP-mannose; [U-14C]mannose) into either of these proteins by gel-electrophoretic analysis of the mannose-labeled products, but we did observe a low incorporation of mannose into material which reacted with antiserum to vicillin. The predominant glycosylation reaction in vitro was therefore probably a transfer of GlcNAc alone, rather than in combination with mannose as preformed oligosaccharide.

Protein Glycosylation in Higher Plants: Recent Developments

Within the last few years, very rapid progress has been made in the investigation of the biosynthesis of the most common type of glycoproteins, i.e., those having an N-glycosidic link between the oligosaccharide(s) and the amide nitrogen of asparagine residues on the protein. Pathways that involve the use of glycolipid intermediates have been delineated, and new, unexpected, phenomena such as oligosaccharide processing have been discovered. In this chapter, we shall review developments in this area with respect to higher plants, concentrating on the transfer of sugars to protein rather than on details of the synthesis of glycolipid precursors. An excellent review on the formation of lipid-linked sugars in plants and their functions in glycoprotein synthesis was recently published by Elbein (1980). The reader interested in the synthesis of O-glycosidically linked oligosaccharides of plant glycoproteins is referred to papers by Karr (1972) on the glycosylation of extensin and Soliday and Kolattukudy (1979) on the glycosylation of fungal cutinase as examples. We shall also exclude from this chapter the protein-linked carbohydrates proposed as primers or intermediates in polysaccharide synthesis. The existence, structure, and functioning of these are still controversial issues, and evidence is limited [for example, their consideration with respect to cellulose synthesis will be found in the paper by Hopp et al. (1978) and for starch synthesis in the paper by Lavintman et al., (1974)].