Increase in XET activity in bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) cells habituated to dichlobenil

Bean (Phaseolus vulgaris L.) cells have been habituated to grow in lethal concentrations of dichlobenil (DCB), a specific inhibitor of cellulose biosynthesis. Bean callus cells were successively cultured in increasing DCB concentrations up to 2 μM. The 2-μM DCB habituated cells were impoverished in cellulose and xyloglucan, had an increased xyloglucan endotransglucosylase (XET; EC 2.4.1.207) activity, together with an increased growth rate and a decreased molecular size of xyloglucan. However, the application of lethal concentrations of two different cellulose-biosynthesis inhibitors (DCB and isoxaben) for a short period of time produced little effect on XET activity and xyloglucan molecular size. We propose that the weakening of plant cell wall provoked by decrease in cellulose content might promote the xyloglucan tethers and increase the ability of xyloglucan to bind to cellulose in order to give rigidity to the wall.     

Early habituation of maize (Zea mays) suspension‐cultured cells to 2,6‐dichlorobenzonitrile is associated with the enhancement of antioxidant status

The cellulose biosynthesis inhibitor 2,6‐dichlorobenzonitrile (DCB) has been widely used to gain insights into cell wall composition and architecture. Studies of changes during early habituation to DCB can provide information on mechanisms that allow tolerance/habituation to DCB. In this context, maize‐cultured cells with a reduced amount of cellulose (～20%) were obtained by stepwise habituation to low DCB concentrations. The results reported here attempt to elucidate the putative role of an antioxidant strategy during incipient habituation. The short‐term exposure to DCB of non‐habituated maize‐cultured cells induced a substantial increase in oxidative damage. Concomitantly, short‐term treated cells presented an increase in class III peroxidase and glutathione S‐transferase activities and total glutathione content. Maize cells habituated to 0.3–1 µM DCB (incipient habituation) were characterized by a reduction in the relative cell growth rate, an enhancement of ascorbate peroxidase and class III peroxidase activities, and a net increment in total glutathione content. Moreover, these cell lines showed increased levels of glutathione S‐transferase activity. Changes in antioxidant/conjugation status enabled 0.3 and 0.5 µM DCB‐habituated cells to control lipid peroxidation levels, but this was not the case of maize cells habituated to 1 μM DCB, which despite showing an increased antioxidant capacity were not capable of reducing the oxidative damage to control levels. The results reported here confirm that exposure and incipient habituation of maize cells to DCB are associated with an enhancement in antioxidant/conjugation activities which could play a role in incipient DCB habituation of maize‐cultured cells.     

The phenolic profile of maize primary cell wall changes in cellulose-deficient cell cultures

Cultured maize cells habituated to grow in the presence of the cellulose synthesis inhibitor dichlobenil (DCB) have a modified cell wall in which the amounts of cellulose are reduced and the amounts of arabinoxylan increased. This paper examines the contribution of cell wall-esterified hydroxycinnamates to the mechanism of DCB habituation. For this purpose, differences in the phenolic composition of DCB-habituated and non-habituated cell walls, throughout the cell culture cycle and the habituation process were characterized by HPLC. DCB habituation was accompanied by a net enrichment in cell wall phenolics irrespective of the cell culture phase. The amount of monomeric phenolics was 2-fold higher in habituated cell walls. Moreover, habituated cell walls were notably enriched in p-coumaric acid. Dehydrodimers were 5–6-fold enhanced as a result of DCB habituation and the steep increase in 8,5′-diferulic acid in habituated cell walls would suggest that this dehydrodimer plays a role in DCB habituation. In summary, the results obtained indicate that cell wall phenolics increased as a consequence of DCB habituation, and suggest that they would play a role in maintaining the functionality of a cellulose impoverished cell wall.     

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 [³H]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 [³H]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 β-glucan synthesis in plants.     

Early cell-wall modifications of maize cell cultures during habituation to dichlobenil

Studies involving the habituation of plant cell cultures to cellulose biosynthesis inhibitors have achieved significant progress as regards understanding the structural plasticity of cell walls. However, since habituation studies have typically used high concentrations of inhibitors and long-term habituation periods, information on initial changes associated with habituation has usually been lost. This study focuses on monitoring and characterizing the short-term habituation process of maize (Zea mays) cell suspensions to dichlobenil (DCB). Cellulose quantification and FTIR spectroscopy of cell walls from 20 cell lines obtained during an incipient DCB-habituation process showed a reduction in cellulose levels which tended to revert depending on the inhibitor concentration and the length of time that cells were in contact with it. Variations in the cellulose content were concomitant with changes in the expression of several ZmCesA genes, mainly involving overexpression of ZmCesA7 and ZmCesA8. In order to explore these changes in more depth, a cell line habituated to 1.5 μM DCB was identified as representative of incipient DCB habituation and selected for further analysis. The cells of this habituated cell line grew more slowly and formed larger clusters. Their cell walls were modified, showing a 33% reduction in cellulose content, that was mainly counteracted by an increase in arabinoxylans, which presented increased extractability. This result was confirmed by immunodot assays graphically plotted by heatmaps, since habituated cell walls had a more extensive presence of epitopes for arabinoxylans and xylans, but also for homogalacturonan with a low degree of esterification and for galactan side chains of rhamnogalacturonan I. Furthermore, a partial shift of xyloglucan epitopes toward more easily extractable fractions was found. However, other epitopes, such as these specific for arabinan side chains of rhamnogalacturonan I or homogalacturonan with a high degree of esterification, seemed to be not affected.     

Phenolic metabolism and molecular mass distribution of polysaccharides in cellulose‐deficient maize cells

As a consequence of the habituation to low levels of dichlobenil (DCB), cultured maize cells presented an altered hemicellulose cell fate with a lower proportion of strongly wall‐bound hemicelluloses and an increase in soluble extracellular polymers released into the culture medium. The aim of this study was to investigate the relative molecular mass distributions of polysaccharides as well as phenolic metabolism in cells habituated to low levels of DCB (1.5 μM). Generally, cell wall bound hemicelluloses and sloughed polymers from habituated cells were more homogeneously sized and had a lower weight‐average relative molecular mass. In addition, polysaccharides underwent massive cross‐linking after being secreted into the cell wall, but this cross‐linking was less pronounced in habituated cells than in non‐habituated ones. However, when relativized, ferulic acid and p‐coumaric acid contents were higher in this habituated cell line. Feasibly, cells habituated to low levels of DCB synthesized molecules with a lower weight‐average relative molecular mass, although cross‐linked, as a part of their strategy to compensate for the lack of cellulose.     

Localization of the Xyloglucan in Cell Walls in a Suspension Culture of Tobacco by Rapid-Freezing and Deep-Etching Techniques Coupled with Immunogold Labelling

Localization of xyloglucan in cell walls regenerated from tobaccoprotoplasts (Nicotiana tabacum L.; cv. BY-2) is visualized byrapid-freezing and deep-etching (RFDE) electron microscopy coupledwith immunogold electron microscopy. Xyloglucan was alreadydeposited in the cell wall 3 h after culture initiation. Xyloglucanwas mainly localized along microfibrils with a lesser amountin intersections between two crossed microfibrils in 120-hour-oldcells. These data support the previous hypothesis of Keegstraet al. (1973) that propose an interconnection between xyloglucanand cellulose. (Received May 22, 1998; Accepted July 13, 1998)     

The Mode of Expression and Promoter Analysis of the arcA Gene, an Auxin-Regulated Gene in Tobacco BY-2 Cells

The arcA, a member of the G protein rß-subunit family,was isolated from tobacco BY-2 cells as an auxin-responsivegene. Characterization of arcA, which should help to elucidatethe function of the gene product in the plant cells, was performedwith emphasis on the mode of expression and the analysis ofits promoter. Accumulation of the arcA message was detectedonly after treatments with auxins and not after treatments withother phytohormones or CdCl₂, implying that responsiveness ofarcA was exclusive to auxin. The putative arcA promoter regionwas fused to a reporter gene for rß-glucuronidase(GUS), and transient expression was analyzed in tobacco BY-2cells. Two series of arcA promoter/GUS chimeric genes were constructed.One consisted of a set of 5' nested deletions of the arcA promoterconnected to the gene for GUS and the other consisted of a varietyof the arcA promoter fragments fused to a minimal promoter-GUSconstruct. The results indicated that the promoter sequencecovering four sets of direct repeats (– 562 to –167)was necessary for the sufficient response of arcA promoter toauxin in BY-2 cells. Moreover, irrespective of auxin treatment,elevated activity of GUS driven by this promoter fragment wasdetected, a result that implies that this region behaves anenhancer in BY-2 cells. (Received September 30, 1995; Accepted March 1, 1996)     

Disruption of cellulose synthesis by 2,6‐dichlorobenzonitrile affects the structure of the cytoskeleton and cell wall construction in Arabidopsis

Cellulose is the major component of plant cell walls and is an important source of industrial raw material. Although cellulose biosynthesis is one of the most important biochemical processes in plant biology, the regulatory mechanisms of cellulose synthesis are still unclear. Here, we report that 2,6‐dichlorobenzonitrile (DCB), an inhibitor of cellulose synthesis, inhibits Arabidopsis root development in a dose‐ and time‐dependent manner. When treated with DCB, the plant cell wall showed altered cellulose distribution and intensity, as shown by calcofluor white and S4B staining. Moreover, pectin deposition was reduced in the presence of DCB when immunostained with the monoclonal antibody JIM5, which was raised against pectin epitopes. This result was confirmed using Fourier transform infrared (FTIR) analysis. Confocal microscopy revealed that the organisation of the microtubule cytoskeleton was significantly disrupted in the presence of low concentrations of DCB, whereas the actin cytoskeleton only showed changes with the application of high DCB concentrations. In addition, the subcellular dynamics of Golgi bodies labelled with N‐ST‐YFP and TGN labelled with VHA‐a1‐GFP were both partially blocked by DCB. Transmission electron microscopy indicated that the cell wall structure was affected by DCB, as were the Golgi bodies. Scanning electron microscopy showed changes in the organisation of cellulose microfibrils. These results suggest that the inhibition of cellulose synthesis by DCB not only induced changes in the chemical composition of the root cell wall and cytoskeleton structure, but also changed the distribution of cellulose microfibrils, implying that cellulose plays an important role in root development in Arabidopsis.     

Inhibition and Recovery of Cell Wall Formation in Root Hairs of Ceratopteris thalictroides

The processes occurring in root hairs of Ceratopteris thalictroidesduring recovery after 10 min incubation in cellulose crystallizationinhibitors (Congo Red and Calcofluor White) and in cellulosesynthesis inhibitors (Coumarin and Dichlorobenzonitrile) werestudied using light and electron microscopy. All these drugscause reversible cessation of growth of the root hairs. AfterCR and CW treatment the nuclei proceed in their normal directionof movement, whereas after Cm and DCB treatment the directionof movement is reversed immediately. After CR and CW treatment the organization of the cytoplasmin the root hair tip is temporarily altered. Cell wall synthesiscontinues, although the resulting wall has a different appearance.After Cm and DCB treatment, the cytoplasm in the root hair tiphas disintegrated and cell wall synthesis is stopped. Recoveryof cell wall synthesis results in a new cell wall between thedisintegrated cytoplasm and the remaining cytoplasm. The effects of the drugs on cytoplasmic organization are discussedin relation to their specificity in inhibiting cell wall synthesis. Key words: Ceratopteris thalictroides, root hairs, cellulose synthesis, inhibition of cell wall formation     

Hydrogen and Oxygen Isotopic Fractionation During Heterotrophic Cellulose Synthesis

Hydrogen and oxygen isotopic fractionation relative to mediumwater for two different carbohydrate metabolic pathways leadingto cellulose synthesis were measured. This was accomplishedby analysing stable hydrogen and oxygen isotope ratios of waterand cellulose for seedlings. The seedlings had been germinatedand heterotrophically grown in closed vessels from species havingstarch (Triticum aestivum L. and Hordeum vulgare L.) and lipids(Ricinus communisL. and Arachis hypogaea L.) as the primarysubstrate. Isotopic fractionation factors occurring during enzyme-mediatedexchange of carbon-bound hydrogen with water or the additionof carbon-bound hydrogens from water during the synthesis ofcellulose from either starch or lipids were similar (rangingfrom +144 to +166%). About 34% and 67% of carbon-bound hydrogenswere derived from water during the synthesis of cellulose fromstarch and lipid, respectively. Thus, the greater deuteriumenrichment in cellulose from oil seed species associated withgluconeogenesis was caused by a greater proportion of water-derivedcarbon-bound hydrogens and not because of differences in fractionationfactors. The proportion of carbon-bound hydrogens derived fromwater during these metabolic pathways was similar to that ofoxygen derived from water. These results may explain the variabilityin D/H ratios of cellulose nitrate from terrestrial and aquaticplants. Key words:     

Carbon partitioning to cellulose synthesis

This article discusses the importance and implications of regulating carbon partitioning to cellulose synthesis, the characteristics of cells that serve as major sinks for cellulose deposition, and enzymes that participate in the conversion of supplied carbon to cellulose. Cotton fibers, which deposit almost pure cellulose into their secondary cell walls, are referred to as a primary model system. For sucrose synthase, we discuss its proposed role in channeling UDP-Glc to cellulose synthase during secondary wall deposition, its gene family, its manipulation in transgenic plants, and mechanisms that may regulate its association with sites of polysaccharide synthesis. For cellulose synthase, we discuss the organization of the gene family and how protein diversity could relate to control of carbon partitioning to cellulose synthesis. Other enzymes emphasized include UDP-Glc pyrophosphorylase and sucrose phosphate synthase. New data are included on phosphorylation of cotton fiber sucrose synthase, possible regulation by Ca²⁺ of sucrose synthase localization, electron microscopic immunolocalization of sucrose synthase in cotton fibers, and phylogenetic relationships between cellulose synthase proteins, including three new ones identified in differentiating tracheary elements of Zinnia elegans. We develop a model for metabolism related to cellulose synthesis that implicates the changing intracellular localization of sucrose synthase as a molecular switch between survival metabolism and growth and/or differentiation processes involving cellulose synthesis. Abbreviations: CesA, cellulose synthase; Csl, cellulose-like synthase (genes); DCB, dichlobenil; DPA, days after anthesis; SPS, sucrose phosphate synthase; SuSy, sucrose synthase; P-SuSy, particulate SuSy; S-SuSy, soluble SuSy     

Inhibition of the synthesis of microbial cellulose by coumarin

Coumarin, a specific inhibitor of the biosynthesis of celluloseof higher plant cell walls, inhibited the cellulose formationof Acetobacter xylinum. The degree of inhibition reached 55%in the presence of 1 mM coumarin, which causes 70% inhibitionin the case of plant cellulose. (Received April 12, 1976; )     

Habituation of bean (Phaseolus vulgaris) cell cultures to Quinclorac and analysis of the subsequent cell wall modifications

Background and Aims

The herbicide quinclorac has been reported to inhibit incorporation of glucose both into cellulose and other cell wall polysaccharides. However, further work has failed to detect any apparent effect of this herbicide on the synthesis of the wall. In order to elucidate whether quinclorac elicits the inhibition of cellulose biosynthesis directly, in this study bean cell calli were habituated to grow on lethal concentrations of the herbicide and the modifications in cell wall composition due to the habituation process were analysed.

Methods

Fourier transform infrared spectroscopy associated with multivariate analysis, cell wall fractionation techniques, biochemical analyses and the immunolocation of different cell wall components with specific monoclonal antibodies were used to characterize the cell walls of quinclorac-habituated cells.

Key Results

Quinclorac-habituated cells were more irregularly shaped than non-habituated cells and they accumulated an extracellular material, which was more abundant as the level of habituation rose. Habituated cells did not show any decrease in cellulose content, but cell wall fractionation revealed that changes occurred in the distribution and post-depositional modifications of homogalacturonan and rhamnogalacturonan I during the habituation process. Therefore, since the action of quinclorac on the cell wall does not seem to be due to a direct inhibition of any cell wall component, it is suggested that the effect of quinclorac on the cell wall could be due to a side-effect of the herbicide.

Conclusions

Long-term modifications of the cell wall caused by the habituation of bean cell cultures to quinclorac did not resemble those of bean cells habituated to the well-known cellulose biosynthesis inhibitors dichlobenil or isoxaben. Quinclorac does not seem to act primarily as an inhibitor of cellulose biosynthesis.Key words: Quinclorac, herbicide, Phaseolus vulgaris, cell culture habituation, primary cell wall, cellulose, FTIR spectroscopy     

Cellulose biosynthesis pathway is a potential target in the improved treatment of <Emphasis Type="Italic">Acanthamoeba</Emphasis> keratitis

Acanthamoeba is an opportunistic protozoan pathogen that can cause blinding keratitis as well as fatal granulomatous encephalitis. One of the distressing aspects in combating Acanthamoeba infections is the prolonged and problematic treatment. For example, current treatment against Acanthamoeba keratitis requires early diagnosis followed by hourly topical application of a mixture of drugs that can last up to a year. The aggressive and prolonged management is due to the ability of Acanthamoeba to rapidly adapt to harsh conditions and switch phenotypes into a resistant cyst form. One possibility of improving the treatment of Acanthamoeba infections is to inhibit the ability of these parasites to switch into the cyst form. The cyst wall is partially made of cellulose. Here, we tested whether a cellulose synthesis inhibitor, 2,6-dichlorobenzonitrile (DCB), can enhance the effects of the antiamoebic drug pentamidine isethionate (PMD). Our findings revealed that DCB can block Acanthamoeba encystment and may improve the antiamoebic effects of PMD. Using in vitro assays, the findings revealed that DCB enhanced the inhibitory effects of PMD on Acanthamoeba binding to and cytotoxicity of the host cells, suggesting the cellulose biosynthesis pathway as a novel target for the improved treatment of Acanthamoeba infections.     

The biosynthesis and wall-binding of hemicelluloses in cellulose-deficient maize cells:An example of metabolic plasticity

Cell-suspension cultures(Zea mays L.,Black Mexican sweet corn) habituated to 2,6-dichlorobenzonitrile(DCB) survive with reduced cellulose owing to hemicellulose network modification. We aimed to de fine the hemicellulose metabolism modifications in DCB-habituated maize cells showing a mild reduction in cellulose at different stages in the culture cycle. Using pulse-chase radiolabeling, we fed habituated and non-habituated cultures with [3H]arabinose,and traced the distribution of 3H-pentose residues between xylans, xyloglucans and other polymers in several cellular compartments for 5 h. Habituated cells were slower taking up exogenous [3H]arabinose. Tritium was incorporated into polysaccharide-bound arabinose and xylose residues, but habituated cells diverted a higher proportion of their new [3H]xylose residues into(hetero) xylans at the expense of xyloglucan synthesis. During logarithmic growth, habituated cells showed slower vesicular traf ficking of polymers,especially xylans. Moreover, habituated cells showed a decrease in the strong wall-binding of all pentose-containing polysaccharides studied; correspondingly, especially in log phase cultures, habituation increased the proportion of 3H-hemicelluloses([3H]xylans and [3H]xyloglucan) sloughed into the medium. These findings could be related to the cel walls' cellulose-deficiency, and consequent reduction in binding sites for hemicelluloses; the data could also re fl ect the habituated cells' reduced capacity to integrate arabinox ylans by extra-protoplasmic phenolic cross-linking, as well as xyloglucans, during wall assembly.     

Mechanism of Isoxaben Tolerance in Agrostis palustris var. Penncross

Previous work has demonstrated that isoxaben tolerant mutantsof Arabidopsis thaliana var. Columbia are most likely alteredat the site of isoxaben binding. The salient question becomeswhether or not species selectivity to this herbicide might alsobe a result of differential target site binding. Grasses aregenerally more tolerant to isoxaben than dicots. In this communicationwe show that Agrostis palustris var. Penncross, a grass, is83-fold more tolerant in a soil incorporation test and 170-foldmore tolerant to inhibition of glucose incorporation into cellulosethan is Arabidopsis, a dicot. Cell wall fractionation of Agrostisshows a specific effect on cellulose biosynthesis. At most,5-fold of the 170-fold tolerance exhibited by Agrostis in termsof cellulose biosynthesis can be attributed to decreased isoxabenuptake under the test conditions. Furthermore, Agrostis is unableto metabolize isoxaben to any significant degree. Therefore,we suggest that the major portion of the tolerance in Agrostismight be due to differences in isoxaben binding. Key words: Isoxaben, cellulose, Arabidopsis, Agrostis, herbicide tolerance     

Recent progress in cellulose biosynthesis

Cellulose comprises the major polymer of the plant cell wall. It consists of a set of parallel chains composed of glucans and these chains are highly oriented to form a structure known as a microfibril. The orientation of the microfibrils controls the extension of the direction of the plant cell. Extensive studies on the cellulose biosynthesis have been carried out for over three decades, and recently (1996) genes for cellulose biosynthesis in plants (CesA) were isolated. In the year 2002, a specific primer for cellulose biosynthesis reaction has been discovered and cellulose synthetic activity has been also confirmed by recombinant protein derived from the plant CesA gene. Furthermore, other proteins involved in cellulose biosynthesis besides CesA proteins were also proposed at the same time. One of these proteins, Korrigan cellulase, was suggested to act by removing sitosterol from the primer for biosynthesis reaction of cellulose. A membrane-bound sucrose synthase was also suggested to provide UDP-glucose as a substrate for cellulose biosynthesis. On the basis of these results, a new pathway for cellulose biosynthesis was proposed. Now, the research field of cellulose biosynthesis is facing a major turning point. Electronic Publication     

2, 6-dichlorobenzonitrile Causes Multiple Effects on Pollen Tube Growth beyond Altering Cellulose Synthesis in Pinus bungeana Zucc

Cellulose is an important component of cell wall, yet its location and function in pollen tubes remain speculative. In this paper, we studied the role of cellulose synthesis in pollen tube elongation in Pinus bungeana Zucc. by using the specific inhibitor, 2, 6-dichlorobenzonitrile (DCB). In the presence of DCB, the growth rate and morphology of pollen tubes were distinctly changed. The organization of cytoskeleton and vesicle trafficking were also disturbed. Ultrastructure of pollen tubes treated with DCB was characterized by the loose tube wall and damaged organelles. DCB treatment induced distinct changes in tube wall components. Fluorescence labeling results showed that callose, and acidic pectin accumulated in the tip regions, whereas there was less cellulose when treated with DCB. These results were confirmed by FTIR microspectroscopic analysis. In summary, our findings showed that inhibition of cellulose synthesis by DCB affected the organization of cytoskeleton and vesicle trafficking in pollen tubes, and induced changes in the tube wall chemical composition in a dose-dependent manner. These results confirm that cellulose is involved in the establishment of growth direction of pollen tubes, and plays important role in the cell wall construction during pollen tube development despite its lower quantity.