Dichlobenil Research Articles

Simultaneous determination of bifenox, dichlobenil and diclofop methyl by hollow carbon nanospheres enhanced magnetic carboxylic multi-walled carbon nanotubes

A novel analytical method based on magnetic solid-phase extraction (MSPE) coupled with high performance liquid chromatography-diode array detection (HPLC-DAD) was established for three herbicides simultaneous determination. The key of the method was the explored extractant, which was a composite of hollow carbon nanospheres (HCSs) enhanced magnetic carboxylic MWCNTs (HCSs@Fe3O4-MWCNTs-COOH). MWCNTs-COOH was inserted in the pores of HCSs by oscillation and sonication with the capillary forces, making the carboxylic groups and structure intervals of the composite increase as compared to MWCNTs-COOH, therefore the extractant exhibited enhanced hydrophilicity, dispersibility, adsorptivity and selectivity. The extractant was sensitive to analytes with the structure of multi-aromatic ring, more hydrogen bond acceptor and large molecular polar surface area, which can be attributed to hydrogen bonding and π-π electron-donor-acceptor (EDA) interactions. The morphology, structure and magnetic property of the extractant were characterized. Then the explored extractant based MSPE-HPLC-DAD method was applied for bifenox (BFO), dichlobenil (DCB) and diclofop methyl (DCM) determination from wheat flour samples. Prior to real sample analysis, critical extraction parameters such as solution pH, extraction time, salt addition and temperature were investigated and optimized, and the analytical method was evaluated. It was indicated that the method had satisfactory linearities with the linear coefficients above 0.99, good precision with the RSD less than 3.5%, desirable recoveries ranged from 88.8% to 96.6%, and low limits of detection (LOD) that were 0.39, 0.24, and 0.68 ng/g for DCB, BFO and DCM, respectively. The established MSPE-HPLC-DAD method has great potentials for trace polar herbicides selective determination from complex matrix samples.

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.

Ectopic lignification in primary cellulose-deficient cell walls of maize cell suspension cultures.

Maize (Zea mays L.) suspension-cultured cells with up to 70% less cellulose were obtained by stepwise habituation to dichlobenil (DCB), a cellulose biosynthesis inhibitor. Cellulose deficiency was accompanied by marked changes in cell wall matrix polysaccharides and phenolics as revealed by Fourier transform infrared (FTIR) spectroscopy. Cell wall compositional analysis indicated that the cellulose-deficient cell walls showed an enhancement of highly branched and cross-linked arabinoxylans, as well as an increased content in ferulic acid, diferulates and p-coumaric acid, and the presence of a polymer that stained positive for phloroglucinol. In accordance with this, cellulose-deficient cell walls showed a fivefold increase in Klason-type lignin. Thioacidolysis/GC-MS analysis of cellulose-deficient cell walls indicated the presence of a lignin-like polymer with a Syringyl/Guaiacyl ratio of 1.45, which differed from the sensu stricto stress-related lignin that arose in response to short-term DCB-treatments. Gene expression analysis of these cells indicated an overexpression of genes specific for the biosynthesis of monolignol units of lignin. A study of stress signaling pathways revealed an overexpression of some of the jasmonate signaling pathway genes, which might trigger ectopic lignification in response to cell wall integrity disruptions. In summary, the structural plasticity of primary cell walls is proven, since a lignification process is possible in response to cellulose impoverishment.

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 1.5μM 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.In conclusion, the early modifications occurring in maize cell walls as a consequence of DCB-habituation involved quantitative and qualitative changes of arabinoxylans, but also other polysaccharides. Thereby some of the changes that took place in the cell walls in order to compensate for the lack of cellulose differed according to the DCB-habituation level, and illustrate the ability of plant cells to adopt appropriate coping strategies depending on the herbicide concentration and length of exposure time.

Enhanced resistance to the cellulose biosynthetic inhibitors, thaxtomin A and isoxaben in Arabidopsis thaliana mutants, also provides specific co-resistance to the auxin transport inhibitor, 1-NPA.

BackgroundThaxtomin A (TA) is a phytotoxin produced by plant pathogenic Streptomyces spp. responsible for potato common scab. TA inhibits cellulose biosynthesis in expanding plant tissues and is essential for disease induction. Auxin treatment of various plant tissues has been repeatedly demonstrated to inhibit TA toxicity and to reduce common scab. This work utilises Arabidopsis thaliana mutants with resistance to cellulose biosynthesis inhibitors (CBIs) to investigate the interaction between TA, other CBIs and auxins.ResultsThree CBI resistant A. thaliana mutants; txr1-1 (tolerance to TA), ixr1-1 (tolerance to isoxaben - IXB) and KOR1 (cellulose deficiency), showed no altered root growth response to treatment with natural or synthetic auxins, nor with the auxin efflux transport inhibitor 2,3,5-Triiodobenzoic acid (TIBA). However, all mutants had significantly enhanced tolerance to 1-napthylphthalamic acid (NPA), another auxin efflux transport inhibitor, which blocks polar auxin transport at a site distinct from TIBA. NPA tolerance of txr1-1 and ixr1-1 was further supported by electrophysiological analysis of net H+ fluxes in the mature, but not elongation zone of roots. All three mutants showed increased tolerance to IXB, but only txr1-1 showed tolerance to TA. No mutant showed enhanced tolerance to a third CBI, dichlobenil (DCB).ConclusionsWe have demonstrated that plant tolerance to TA and IXB, as well as cell wall synthesis modifications in roots, have resulted in specific co-resistance to NPA but not TIBA. This suggests that CBI resistance has an impact on polar auxin efflux transport processes associated with the NPA binding protein. We also show that NPA inhibitory response in roots occurs in the mature root zone but not the elongation zone. Responses of mutants to CBIs indicate a similar, but not identical mode of action of TA and IXB, in contrast to DCB.

Changes in Cinnamic Acid Derivatives Associated with the Habituation of Maize Cells to Dichlobenil

The habituation of cell cultures to cellulose biosynthesis inhibitors such as dichlobenil (DCB) represents a valuable tool to improve our knowledge of the mechanisms involved in plant cell wall structural plasticity. Maize cell lines habituated to lethal concentrations of DCB were able to grow through the acquisition of a modified cell wall in which cellulose was partially replaced by a more extensive network of arabinoxylans. The aim of this work was to investigate the phenolic metabolism of non-habituated and DCB-habituated maize cell cultures. Maize cell cultures were fed [(14)C]cinnamate and the fate of the radioactivity in different intra-protoplasmic and wall-localized fractions throughout the culture cycle was analyzed by autoradiography and scintillation counting. Non-habituated and habituated cultures did not markedly differ in their ability to uptake exogenous [(14)C]cinnamic acid. However, interesting differences were found in the radiolabeling of low- and high-M(r) metabolites. Habituated cultures displayed a higher number and amount of radiolabeled low-M(r) compounds, which could act as reserves later used for polysaccharide feruloylation. DCB-habituated cultures were highly enriched in esterified [(14)C]dehydrodiferulates and larger coupling products. In conclusion, an extensive and early cross-linking of hydroxycinnamates was observed in DCB-habituated cultures, probably strengthening their cellulose-deficient walls.

Deepening into the proteome of maize cells habituated to the cellulose biosynthesis inhibitor dichlobenil

Cellulose biosynthesis inhibitors, such as dichlobenil (DCB), have become a valuable tool for the analysis of structural and compositional plasticity of plant cell walls. By stepwise increasing the concentration of DCB in the culture medium, we obtained maize cells able to cope with DCB through the acquisition of a modified cell wall in which cellulose was partially replaced by a more extensive network of feruloylated arabinoxylans. Recently we demonstrated that the expression of several Cellulose Synthase and phenylpropanoid-related genes is altered in DCB-habituated cells. In addition, by using a proteomic approach we identified several proteins induced or repressed in DCB-habituated cells. After a more in-depth analysis, some new proteins induced (two inhibitors TAXI-IV, an α-1,4-glucan-protein synthase, and a pectinesterase inhibitor) or repressed (a chaperonin 60, a fructokinase-1 and a spermidine synthase 1) were identified, and their possible role in the context of DCB-habituation is discussed.

Unraveling the Biochemical and Molecular Networks Involved in Maize Cell Habituation to the Cellulose Biosynthesis Inhibitor Dichlobenil

The biochemical and molecular processes involved in the habituation of maize cells to growth in the presence of the cellulose biosynthesis inhibitor dichlobenil (DCB) were investigated. DCB affects the synthesis of cellulose both in active and stationary growth phases and alters the expression of several CesA genes. Of these, ZmCesA5 and ZmCesA7 seem to play a major role in habituating cells to growth in the presence of DCB. As a consequence of the reduction in cellulose, the expression of several genes involved in the synthesis of hydroxycinnamates is increased, resulting in cell walls with higher levels of ferulic and p-coumaric acids. A proteomic analysis revealed that habituation to DCB is linked to modifications in several metabolic pathways. Finally, habituated cells present a reduction in glutathione S-transferase detoxifying activity and antioxidant activities. Plant cell adaptation to the disturbance of such a crucial process as cellulose biosynthesis requires changes in several metabolic networks, in order to modify cell wall architecture and metabolism, and survive in the presence of the inhibitor. Some of these modifications are described in this paper.

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.

Increase in XET activity in bean (Phaseolus vulgaris 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 microM. The 2-microM 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.

Structural and Immunocytochemical Characterization of the Walls of Dichlobenil‐Habituated BY‐2 Tobacco Cells

BY‐2 tobacco cells, grown in the presence of the cellulose biosynthesis inhibitor herbicide dichlobenil (DCB), change in morphology from finely dispersed clumps and filaments to large aggregates after 6 mo. In this article, we utilize structural, cytochemical, and immunocytochemical techniques to characterize the cell wall changes that accompany the habituation of the cells to DCB. Wall structure is altered radically in the habituated cells, revealing a lamellate structure with no distinct middle lamella, even at cell junctions. Plasmodesmata traverse the cell wall by more circuitous routes than in the control cells. Habituated cells also produce unusual appositions of the wall that extend into the cytoplasm. Immunogold localization of various pectin epitopes reveals a 30‐fold‐greater density of gold particles detected in the walls of habituated cells compared to the controls and less density when the labeling of the middle lamellae is included. Antiextensin labeling was also found along the strands of pectic‐reactive material, and the density of immunogold labeling exhibits a similar increase to that for pectin. Cellulose, detected by cellulase‐gold, revealed only 2% of the reactivity of the controls, while callose detection was slightly greater compared to controls. Despite the loss of cellulose, xyloglucan is still detected, loosely associated with the strands of pectin or trapped between the pectin lamellae. Cortical microtubule arrays in habituated cells were apparently normal, despite a virtual absence of cellulose, although callose fibrils did parallel this array. These data indicate that long‐term habituation to DCB results in the production of cell walls in which a pectin‐extensin network has replaced cellulose as the major component of the cell wall. These changes have profound effects on wall ultrastructure and the gross morphology of the cultures.

Effect of Water on the Persistence of Dichlobenil*

Effect of Water on the Persistence of Dichlobenil*