Cellulose Synthesis Inhibitor Research Articles

Effects of isoxaben on sensitive and tolerant plant cell cultures: II. Cellular alterations and inhibition of the synthesis of acid-insoluble cell wall material

The herbicide isoxaben is selectively phytotoxic to dicotyledonous plants, whereas most monocots are tolerant. We previously selected a soybean cell culture tolerant to isoxaben. Some effects of the herbicide on wild-type soybean cells, tolerant soybean cells, and wheat cells were compared. Cytological observations showed that isoxaben induced some disorganization of sensitive soybean cells, especially at the plasma membrane-cell wall interface. Tolerant soybean cells appeared normal in the presence of isoxaben. The growth of wild-type soybean cells was roughly equally sensitive to isoxaben as to dichlobenil, a cellulose synthesis inhibitor. By comparison, the selected soybean line and a wheat cell culture were less sensitive to isoxaben than to dichlobenil. Glucose incorporation into acid-insoluble cell wall material was more inhibited by isoxaben than by dichlobenil in the wild-type soybean cell culture. In the tolerant soybean cell culture, the incorporation was slightly inhibited by isoxaben, but remained sensitive to dichlobenil. In the wheat cell culture, dichlobenil was also more inhibitory but only at high concentrations. Other compounds, inhibitors of cellulose biosynthesis, of glycosylation of lipids or protein, or of cell division, either had no effect on the synthesis of acid-insoluble cell wall material or exerted apparently unspecific inhibitions. The results are consistent with isoxaben inhibiting the synthesis of a cell wall polysaccharide, which could be cellulose.

Identification of a Receptor Protein in Cotton Fibers for the Herbicide 2,6-Dichlorobenzonitrile

The herbicide 2,6-dichlorobenzonitrile (DCB) is an effective and apparently specific inhibitor of cellulose synthesis in higher plants. We have synthesized a photoreactive analog of DCB (2,6-dichlorophenylazide [DCPA]) for use as an affinity-labeling probe to identify the DCB receptor in plants. This analog retains herbicide activity and inhibits cellulose synthesis in cotton fibers and tobacco cells in a manner similar to DCB. When cotton fiber extracts are incubated with [(3)H]DCPA and exposed to ultraviolet light, an 18 kilodalton polypeptide is specifically labeled. About 90% of this polypeptide is found in the 100,000g supernatant, the remainder being membrane-associated. Gel filtration and nondenaturing polyacrylamide gel electrophoresis of this polypeptide indicate that it is an acidic protein which has a similar size in its native or denatured state. The amount of 18 kilodalton polypeptide detectable by [(3)H]DCPA-labeling increases substantially at the onset of secondary wall cellulose synthesis in the fibers. A similar polypeptide, but of lower molecular weight (12,000), has been detected upon labeling of extracts from tomato or from the cellulosic alga Chara corallina. The specificity of labeling of the 18 kilodalton cotton fiber polypeptide, coupled with its pattern of developmental regulation, implicate a role for this protein in cellulose biosynthesis. Being, at most, only loosely associated with membranes, it is unlikely to be the catalytic polypeptide of the cellulose synthase, and we suggest instead that the DCB receptor may function as a regulatory protein for beta-glucan synthesis in plants.

THE EFFECTS OF SELECTED INHIBITORS ON CELLULOSE MICROFIBRIL ASSEMBLY IN BOERGESENIA FORBESII (CHLOROPHYTA) PROTOPLASTS

Protoplasts of Boergesenia forbesii (Harvey) were treated with inhibitors of protein synthesis in order to investigate their effects on cellulose synthesis. Cellulose synthesis was reversibly inhibited by 10 μM cycloheximide as assayed by fluorescence microscopy of Tinopal binding to cellulose. Freeze fracture and image analysis of cycloheximide- treated cells indicated a reduction in the number of intramembrane particles; however, the terminal synthesizing complexes remained at all times. Treatment with 10 μM actinomycin D, when applied during the first hour of protoplast formation, irreversibly inhibits cellulose synthesis and terminal complex formation. De novo protein synthesis is required for cell wall regeneration by protoplasts. The data suggest that the structural subunits visualized in the terminal complex do not undergo significant turnover, but that there may exist an essential proteinaceous component of cellulose synthesis which must be continually renewed.

The role of cell wall synthesis in sustained auxin‐induced growth

The dependence of auxin‐induced growth on continued cell wall synthesis was investigated in stem segments of etiolated pea (Pisum sativum L. cv. Alaska) seedlings using the cell wall synthesis inhibitors monensin and 2,6‐dichlorobenzonitrile (DCB). Monensin (5 μM) potently inhibited indole‐3‐acetic acid (IAA)‐induced growth, particularly during the second hour of treatment, whereas growth in fusicoccin (FC) was inhibited much less effectively. Incorporation of [14C]‐glucose into both matrix and cellulose fractions of the wall showed a sharp increase beginning after about 60 min, this rise being promoted by both IAA and FC. Monensin inhibited this rise in incorporation of label and completely removed the promotion of this by IAA, although some promotion by FC remained. Monensin inhibited incorporation into cellulose in a manner similar to that into matrix, but the use of the apparently specific cellulose synthesis inhibitor DCB showed that cellulose synthesis could be strongly inhibited without effect on growth, at least in the short term. The results support the view that sustained auxin‐induced growth depends upon the incorporation of new matrix cell wall components into the wall.

Influence of external factors on callose and cellulose synthesis during incubation in vitro of intact cotton fibres with [14C]sucrose

Seed clusters, with adhering fibres, from individual locules of 36-d-old fruit capsules of Gossypium arboreum L. were fed with [(14)C]sucrose in vitro. The fibres synthesised, under standard conditions, (1→3)-β-D-glucan (callose) and (1→4)-β-D-glucan (cellulose) in the ratio of approx. 2:1. Under a great variety of different conditions this product ratio remained more or less constant, even when total glucan synthesis was strongly inhibited with 2,4-dinitrophenol or phloretin, or when stimulated with abscisic acid. In attempts to favour cellulose synthesis, no conditions were found where the ratio was substantially reduced. On the other hand, the ratio could be appreciably increased by inhibiting cellulose synthesis, e.g. with 2,6-dichlorobenzonitrile or coumarin, by anionic detergents such as sodium dodecyl sulphate, by low temperatures, or by increasing the osmotic strength of the incubation medium up to conditions causing plasmolysis. Specific degradation of callose, during incubation of the seed clusters, by exogenous exo-(1→3)-β-D-glucanase significantly diminished incorporation of radioactivity into cellulose.

New approaches to the study of cellulose biosynthesis

Examples of a variety of approaches for studying the mechanism and regulation of cellulose biosynthesis are presented. Attempts to demonstrate conclusively a cellulose synthase activity using membrane preparations derived from higher plants have not been successful; the predominant UDP-glucose: beta-glucan-beta-glucosyltransferase detected in these preparations is a beta-(1----3)-glucan synthase that is dependent upon Ca2+ and a beta-glucobiose, such as laminaribiose or cellobiose, for activity. Xyloglucan glucosyltransferase activity is detected in all plant preparations examined and, in cotton fibres, the activity of this enzyme correlates well with the level of xyloglucan found in the fibre. Low activity for a Mg2+-dependent beta-(1----4)-glucan synthase is found in extracts from soybeans and mung beans, but not cotton fibres; this enzyme could represent either a dissociated form of xyloglucan glucosyltransferase or a partially latent form of cellulose synthase. In vitro translation of RNA from developing cotton fibres shows notable increases in the level of several relatively abundant translatable mRNAs associated with the time of onset of secondary-wall cellulose synthesis. In order to determine whether these apparent changes in gene expression represent enhanced expression of specific genes required for cellulose synthesis, several strategies are being developed for identification of specific polypeptides required for this process. One strategy involves the successful development of a technique for detection of glucan synthase activity in acrylamide gels, a technique that should prove useful for characterization of the polypeptide composition of such enzymes. We have also synthesized a photoaffinity analogue of 2,6-dichlorobenzonitrile (DCB), a potent and specific inhibitor of cellulose synthesis. The analogue is also an effective inhibitor in vivo, and upon ultraviolet irradiation of extracts in the presence of the radioactive analogue, we observe a relatively specific labelling of a polypeptide of 18 X 10(3)Mr. Finally, we have studied the spatial regulation and structural requirements for cellulose synthesis in internode cells of the alga Chara corallina. Cellulose deposition in vivo shows spatial localization, which correlates with acid and base bands along the cell. Using internodes perfused with solutions containing UDP-[14C]glucose and subsequently ligated, we were able to demonstrate synthesis of a highly insoluble cell-wall-localized glucan, thus offering hope that Chara can be developed as another useful system for studying the mechanism and regulation of cellulose synthesis.

Ethylene-Induced Lateral Expansion in Etiolated Pea Stems

Treatment of etiolated pea (Pisum sativum L.) internode tissue with ethylene gas inhibits elongation and induces lateral expansion. Precise kinetics of the induction of this altered mode of growth of excised internode segments were recorded using a double laser optical monitoring device. Inhibition of elongation and promotion of lateral expansion began after about 1 hour of treatment and achieved a maximum by 3 hours. Similar induction kinetics were observed after treating internodes with colchicine and 2,6-dichlorobenzonitrile, an inhibitor of cellulose synthesis. In sealed flask experiments, ethylene had no detectable effect on incorporation of label from [(14)C]glucose into any of the classical pectin, hemicellulose, or cellulose wall fractions. Ethylene inhibited fresh weight increase (total cell expansion) of both excised internode segments (in sealed flasks) and intact seedlings. Ethylene treatment resulted in an increase in cell sap osmolality in those tissues (intact and excised) which are inhibited by the gas. A model for ethylene-induced inhibition of elongation and induction of lateral expansion is presented.

An Ultrastructural Study of the Relationship between Microtubules and Microfibrils in Cotton (Gossypium Hirsutum L.) Cell Wall Reversals

The arrangement of cellulosic fibrils in the cell walls of cotton fibers is very unusual; rather than exhibiting a continuous spiraling in one direction, they intermittantly reverse their gyre. Microtubules that line the periphery of the protoplasm, subjacent to the plasmalemma, tend to parallel the deployment of the cell wall microfibrils. It was not known whether this parallelism persisted through the reversal. By studying tangential sections of the cell wall/protoplasmic interfaces at the reversals, we show that congruity continues even through the reversals. Colchicine treatment did not appear to inhibit cellulose synthesis but it did abolish microtubules in the cotton fiber cells and deranged normal cell wall microfibrillar orientation. Previously, cotton fibers have been shown to possess most of the familiar organelles, but we found two new features not reported heretofore. They are microfilaments and peculiar “polygonal structures” that appear to be associated with the plasma membrane.

AN ULTRASTRUCTURAL STUDY OF THE RELATIONSHIP BETWEEN MICROTUBULES AND MICROFIBRILS IN COTTON (GOSSYPIUM HIRSUTUM L.) CELL WALL REVERSALS

The arrangement of cellulosic fibrils in the cell walls of cotton fibers is very unusual; rather than exhibiting a continuous spiraling in one direction, they intermittantly reverse their gyre. Microtubules that line the periphery of the protoplasm, subjacent to the plasmalemma, tend to parallel the deployment of the cell wall microfibrils. It was not known whether this parallelism persisted through the reversal. By studying tangential sections of the cell wall/protoplasmic interfaces at the reversals, we show that congruity continues even through the reversals.Colchicine treatment did not appear to inhibit cellulose synthesis but it did abolish microtubules in the cotton fiber cells and deranged normal cell wall microfibrillar orientation. Previously, cotton fibers have been shown to possess most of the familiar organelles, but we found two new features not reported heretofore. They are microfilaments and peculiar “polygonal structures” that appear to be associated with the plasma membrane.

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-(14)C]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.

On the inhibition of cellulose biosynthesis by coumarin

Previous work showed that coumarin is an effective inhibitor of cellulose biosynthesis. In the present study parallel determinations of cell production and cellulose formation by Acetobacter xylinum as a function of coumarin concentration in the medium suggested that the effect of coumarin is one of a general cell poison, rather than a specific inhibitor of cellulose synthesis. Because coumarin had no effect on cellulose biosynthesis in Glaser's cell-free particulate suspension from A. xylinum, the point(s) of general inhibition must be before the formation of uridine diphosphoglucose. Nonetheless, the pathway to cellulose in green plants must be more sensitive to coumarin than the pathways to hemicellulose or pectin.

Animal-specific membrane components visualised on the surface of animal/plant heterokaryons

Immunofluorescence was used to demonstrate the presence and distribution of animalspecific membrane components within the plasma membranes ofXenopus/carrot heterokaryons. The inhibitor of cellulose synthesis, 2,6-dichlorobenzonitrile, was used to impair cell wall regeneration so that the plasma membranes of cultured heterokaryons would remain accessible to antibodies.Xenopus-Specific surface components were observed in heterokaryons after 14 days of culture.

Elongation Growth of Setae of Pellia (Bryophyta): Effects of Auxin and Inhibitors

Summary Growth of setae of the liverwort, Pellia epiphylla, was monitored with intact plants and with excised stalks in aqueous solutions. Growth of sporangiophores of intact plants was linear for nearly 48 hours at an average rate of about 0.6 mm/h. Excised setae in the presence of a near optimal concentration of 0.1 mM indole-3-acetic acid (auxin) grew at a steady-state rate of 0.7-1.2 mm/h, greater than that of the sporangiophores of the intact plant. Elongation was not a simple passive phenomenon since growth of excised setae was inhibited by cycloheximide, puromycin, fluorophenylalanine, anti auxins, coumarin, and heavy water as well as ionophores. Colchicine inhibited auxin-induced but not basal elongation as did the cellulose synthesis inhibitor dichlobenil. Both basal and auxin-induced elongation were sensitive to cytochalasin B. The inhibitory effects of cytochalasin B are obvious only in short pieces of setae. As shown by the application of radioactive cytochalasin B, this is due to its poor penetration. The results are significant in that they demonstrate an example of auxin-promoted elongation where the rate of growth of excised setae equals or exceeds that of the intact plant and is clearly not an example of a passive thinning of auxin-loosened walls. Rather growth is dependent upon maintenance of organized structure and macromolecule synthesis.

THE RELATIONSHIPS AMONG UMBELLIFERONE, GROWTH, AND PEROXIDASE LEVELS IN CUCUMBER ROOTS

Umbelliferone decreases the elongation rate and initiates a swelling response in cucumber roots. Associated with these growth effects are increases in peroxidase activity in water soluble, salt soluble, and wall bound fractions. The effect on elongation is reversible and is separated in time from the swelling response. Increased peroxidase levels appear after the elongation rate has been reduced but coincident with the swelling response. It is suggested that umbelliferone effects axial elongation and radial expansion through different mechanisms. As IS TRUE of a number of coumarin compounds (Goodwin and Taves, 1950), umbelliferone (7hydroxycoumarin) retards root growth. Coumarin typically upsets growth polarity (Avers and Goodwin, 1956; Svensson, 1971 and 1972), and results in increased peroxidase activity (Goren and Tomer, 1971). Many chemically diverse compounds alter the transverse microfibrillar pattern and evoke a swelling response (Green, 1969). In addition, many such compounds have been shown to elicit increased peroxidase levels. Hypotheses involving an alteration of the microfibrillar pattern (Apelbaum and Burg, 1971), weakening of the cell wall by an inhibition of cellulose synthesis (Hogetsu, Shibaoka, and Shimokoriyama, 1974), and an involvement of increased peroxidase levels (Ridge and Osborne, 1970), have been offered to explain the mode of action of these compounds. The present paper describes the effect of umbelliferone on cucumber primary roots. Growth data and enzyme assays suggest that neither a possible altered microfibrillar pattern nor increased peroxidase levels are involved in the umbelliferone-imposed inhibition of axial growth. However, the results are consistent with any or a combination of these mechanisms being involved in radial expansion.

Involvement of cellulose synthesis in actions of gibberellin and kinetin on cell expansion. 2,6-Dicholorobenzonitrile as a new cellulose-synthesis inhibitor

Involvement of cellulose synthesis in actions of gibberellin and kinetin on cell expansion. 2,6-Dicholorobenzonitrile as a new cellulose-synthesis inhibitor

THE RELATION BETWEEN CELLULOSE SYNTHESIS AND AN UNIDENTIFIED GLUCOSE-COMPLEX IN PEA STEM SEGMENTS

SUMMARY An as yet unidentified glucose-complex is formed in the cells of 5 mm stem segments cut from the third internode at 1 mm from the tip of etiolated pea seedlings. In segments growing in solutions containing 10 μg/ml IAA and various concentrations of glucose the quantities of the unknown glucose-complex and cellulose are proportional. Increasing the IAA concentration at a constant glucose concentration of 0.1% causes an increase in the glucose-complex and in the synthesis of cellulose up to a concentration of 10 μg/ml. At IAA concentrations higher than 50 μg/ml cellulose synthesis decreases to the value obtained when no IAA is present, whereas the amount of the glucose-complex is increased. Galactose inhibits cellulose synthesis by way of interfering with the glucose absorption. In low concentrations of glucosecobalt chloride promotes sugar absorption, the formation of the glucose-complex, and cellulose synthesis. In high sugar concentrations it does not affect these processes. The results obtained indicate a close relationship between cellulose synthesis and the formation of the glucose-complex. It is suggested that the latter is an intermediate in the synthesis of cellulose.