Characterization of cell walls in bean (Phaseolus vulgaris L.) callus cultures tolerant to dichlobenil

doi:10.1016/S0168-9452(00)00397-6

Plant Science

Volume 160, Issue 2, 5 January 2001, Pages 331-339

https://doi.org/10.1016/S0168-9452(00)00397-6 Get rights and content

Abstract

The increase in dry weight during the culture of bean callus cultures was inhibited by the herbicide dichlobenil (2,6-dicholorobenzonitrile) with an I₅₀ of 0.5 μM. However bean calli became tolerant to a concentration of 12 μM by a stepwise increase in the concentration of the inhibitor in each subculture. Tolerant calli growing in 2,6-dicholorobenzonitrile developed with hollow protuberances. Groups of cells in these protuberances had irregular cell walls surrounded by a thicker cell wall with a lamellate structure and without a differentiated middle lamella. FTIR spectra of tolerant cell walls revealed an increase in both esterified and non-esterified pectins. Cell wall fractionation showed that in tolerant cell walls the xyloglucan–cellulose network of non-tolerant cell walls was partly replaced by a pectin-rich network mainly formed of cross-linked polyuronides with a large proportion of homogalacturonan. These modifications are comparable to those described for bean calli tolerant to isoxaben, pointing to a related mechanism of tolerance for both herbicides.

Introduction

The herbicide dichlobenil (2,6-dichlorobenzonitrile) is an effective and specific inhibitor of cellulose synthesis in higher plants ([1] and references therein) that inhibits the polymerisation of Glc into cellulose with little or no short-term effects on other physiological processes [2].

Cell suspensions of tomato [3], tobacco, and barley [4] have been adapted to grow on dichlobenil once they have acquired a non-target site mechanism of tolerance. The adapted cells are able to grow on dichlobenil because they develop the capacity to divide and expand under inhibitions of cellulose synthesis. Tomato and tobacco cells adapted to dichlobenil contain markedly reduced levels of cellulose and hemicellulose and are enriched in pectins extracted with calcium-chelating agents. Barley cells, whose walls have low pectin levels, show very different alterations in cell wall composition in response to adaptation to dichlobenil. In the absence of most of the load-bearing cellulose–xyloglucan network, Ca²⁺-bridged pectates would represent replacement. Fourier transform infrared microspectroscopy supports the biochemical results and confirms the existence of a large proportion of non-esterified pectins [5]. Strong changes in pectin levels and distribution, and an increase and colocalisation of extensin with pectin in tobacco cells tolerant to dichlobenil were revealed by immunodetection [6]. Tobacco cells tolerant to dichlobenil have more celA1 protein that the control cells, suggesting that celA1 protein is stabilised upon dichlobenil binding and the crystallisation of cellulose microfibrils is simultaneously inhibited [7].

Isoxaben, N-3[-(1-ethyl-1-methylpropyl)-5-isoxazolyl]-2,6-dimethoxy benzamide, is another pre-emergence herbicide that inhibits the incorporation of glucose into cellulose [8]. The mode of action of this herbicide is also largely unknown. In this case, both target [9], [10], [11] and non-target [12] tolerance mechanisms have been described. Cell suspensions of soybean selected for growth on lethal concentrations of isoxaben, do not show any quantitative or qualitative differences in the metabolism of isoxaben suggesting that the cause of resistance is at the level of the cellular target of the herbicide [9]. However, bean calli tolerant to isoxaben have cell walls modified in a similar way to that described in the above dichlobenil tolerant cultures [12].

The results obtained with cellulose biosynthesis inhibitors indicate that plant cells are able to develop different strategies to tolerate lethal concentrations of these compounds. In addition, recently mutants whose cellulose biosynthesis pathway is inhibited, such as PROCUSTE [13] KORRIGAN [14] of Arabidopsis thaliana, show similarities to dichlobenil-tolerant cells since they have a modified cell wall with a reduced amount of cellulose and are enriched in pectins. Likewise, the adaptation of cell suspensions to different types of environmental stress, such as saline or osmotic stress in tobacco [15], [16] induces different changes in the composition and structure of the cell wall.

The above studies reflect the flexibility of plant cells in tolerating changes induced in cell wall structure. Analysis of such modifications, some of which might be of commercial importance, should shed light on the relative contributions of the different polymers in the primary cell wall structure and their putative functions.

The present work addresses the effect of dichlobenil on the growth of bean calli and the selection and characterisation of a bean cell line able to grow in the presence of high concentrations of the inhibitor. In order to shed light on the mode of action of cellulose biosynthesis inhibitors, dichlobenil tolerant cell walls were isolated and fractionated and their composition compared with that obtained for isoxaben-tolerant bean calli.

Section snippets

Chemicals

Plant tissue culture medium was purchased from Sigma Co. and agar was obtained from ROKO. Dichlobenil (>98%) was supplied by Fluka and was dissolved in ethanol.

Callus culture and accomodation to dichlobenil

The first pair of leaves from 10-day old bean (Phaseolus vulgaris) seedlings were aseptically cultured at 27°C for 30 days on Murashige and Skoog salt mixture [17], solidified with 8 g l⁻¹ agar, containing 30 g l⁻¹ sucrose and 10 μM 2,4-D. Calli were removed from the explants and routinely subcultured for 30 days on identical medium

Growth and accomodation of callus cultures

Fig. 1 shows the effect of increasing concentrations of dichlobenil on dry weight gain after 30 days of bean callus culture. The growth of NT bean calli was progressively diminished by dichlobenil concentrations equal or more than 0.1 μM and the I₅₀ was 0.5 μM. Growth was totally inhibited at a dichlobenil concentration of 0.7 μM or above, at which the calli turned brown and died.

Selection of dichlobenil T calli was accomplished by repeated transfer and culture of calli on solid media with

Discussion

The above results led us to assume that the accomodation to dichlobenil is accompanied by a modification of the cell wall. Differences were observed among callus cultures tolerant to various concentrations of dichlobenil, indicating that the changes in cell wall composition were reached gradually from T0.5 to T8. However, few differences between T8 and T12 callus cultures were seen.

Cell wall analysis revealed quantitative and qualitative changes in the pectin levels of T cells, pointing to

Acknowledgements

This work was supported by a Junta de Castilla y León grant (LE35/99).

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The characterisation of cell walls from DCB-habituated cells has demonstrated that plant cells develop the capacity to cope with the inhibitor through the acquisition of a modified cell wall. Changes in cell wall composition/structure greatly depended on the type of cell wall (Type I or II) and on the degree of cellulose lacking (Shedletzky et al., 1990; Encina et al., 2001, 2002; García-Angulo et al., 2006, 2009; Mélida et al., 2009, 2015). Maize cells habituated to high DCB concentrations (long-term habituation, 12 μM) showed a 75% reduction in cellulose.
The habituation of cultured cells to cellulose biosynthesis inhibitors such as dichlobenil (dichlorobenzonitrile, DCB) has proven a valuable tool to elucidate the mechanisms involved in plant cell wall structural plasticity. Our group has demonstrated that maize cells cope with DCB through a modified cell wall in which cellulose is replaced by a more extensive network of highly cross-linked feruloylated arabinoxylans. In order to gain further insight into the contribution of phenolics to the early remodelling of cellulose-deficient cell walls, a comparative HPLC-PAD analysis was carried out of hydroxycinnamates esterified into nascent and cell wall polysaccharides obtained from non-habituated (NH) and habituated to low DCB concentrations (1.5 μM; H) maize suspension-cultured cells.
Incipient DCB-habituated cell walls showed significantly higher levels of esterified ferulic acid and p-coumaric acid throughout the culture cycle. In terms of cell wall fortification, ferulic acid is associated to arabinoxylan crosslinking whereas the increase of p-coumaric suggests an early lignification response. As expected, the level of hydroxycinnamates esterified into nascent polysaccharides was also higher in DCB-habituated cells indicating an overexpression of phenylpropanoid pathway. Due to their key role in cell wall strengthening, special attention was paid into the dimerization pattern of ferulic acid. A quantitative comparison of diferulate dehydrodimers (DFAs) between cell lines and cell compartments revealed that an extra dimerization took place in H cells when both nascent and mature cell wall polysaccharides were analysed. In addition, qualitative differences in the ferulic acid coupling pattern were detected in H cells, allowing us to suggest that 8-O-4′-DFA and 8-5′-DFA featured the ferulic acid dimerization when it occurred in the protoplasmic and cell wall fractions respectively. Both qualitative and quantitative differences in the phenolic profile between NH and H cells point to a regioselectivity in the ferulate dehydrodimerization.
Quinclorac-habituation of bean (Phaseolus vulgaris) cultured cells is related to an increase in their antioxidant capacity
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We have previously observed that bean calluses can be habituated to herbicides following one or more mechanisms. The habituation of bean calluses to dichlobenil was associated with a high scavenging capacity of ROS, mainly by CIII-POX activity (García-Angulo et al., 2009) and also with the capacity of the cells to divide and expand with a modified cell wall in which the xyloglucan-cellulose network had been partially replaced by pectins (Encina et al., 2001, 2002). However, quinclorac-habituated bean cells have a non-modified cell wall (Alonso-Simón et al., 2008) and, as was observed in this study, this habituation seemed to be related to a high antioxidant capacity.
The habituation of bean cells to quinclorac did not rely on cell wall modifications, contrary to what it was previously observed for the well-known cellulose biosynthesis inhibitors dichlobenil or isoxaben. The aim of the present study was to investigate whether or not the bean cells habituation to quinclorac is related to an enhancement of antioxidant activities involved in the scavenging capacity of reactive oxygen species. Treating non-habituated bean calluses with 10 μM quinclorac reduced the relative growth rate and induced a two-fold increase in lipid peroxidation. However, the exposition of quinclorac-habituated cells to a concentration of quinclorac up to 30 μM neither affected their growth rate nor increased their lipid peroxidation levels. Quinclorac-habituated calluses had significantly higher constitutive levels of three antioxidant activities (class-III peroxidase, glutathione reductase, and superoxide dismutase) than those observed in non-habituated calluses, and the treatment of habituated calluses with 30 μM quinclorac significantly increased the level of class III-peroxidase and superoxide dismutase. The results reported here indicate that the process of habituation to quinclorac in bean callus-cultured cells is related, at least partially, to the development of a stable antioxidant capacity that enables them to cope with the oxidative stress caused by quinclorac. Class-III peroxidase and superoxide dismutase activities could play a major role in the quinclorac-habituation. Changes in the antioxidant status of bean cells were stable, since the increase in the antioxidant activities were maintained in quinclorac-dehabituated cells.
Early cell-wall modifications of maize cell cultures during habituation to dichlobenil
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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.
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.
Changes in cinnamic acid derivatives associated with the habituation of maize cells to dichlobenil
2011, Molecular Plant
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 [¹⁴C]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 [¹⁴C]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 [¹⁴C]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.
Plasticity of xyloglucan composition in bean (Phaseolus vulgaris)-cultured cells during habituation and dehabituation to lethal concentrations of dichlobenil
2010, Molecular Plant
Citation Excerpt :
In this sense, the loosening of xyloglucan in poplar trees has been proved to increase the ethanol production from its lignocellulosic material (Kaida et al., 2009), making further studies of this cell wall polysaccharide even more interesting. Some dicotyledonous cell cultures have been habituated to grow in lethal concentrations of herbicides that inhibit cellulose biosynthesis, such as dichlobenil (2,6-dichlorobenzonitrile, also called DCB) (Shedletzky et al., 1990, 1992; Encina et al., 2001, 2002; Alonso-Simón et al., 2004; García-Angulo et al., 2006) and isoxaben (N-3[-(1-ethyl-1-methylpropyl)-5-isoxazolyl]-2,6-dimethoxybenzamide) (Díaz-Cacho et al., 1999; Manfield et al., 2004). In all cases, habituated cells exhibited reductions in the amounts of cellulose and hemicelluloses, and compensated for these reductions with increased amounts of pectins.
Bean cells that have been habituated to grow in a lethal concentration (12 μM) of 2,6-dichlorobenzonitrile (dichlobenil or DCB, a cellulose biosynthesis inhibitor) are known to have decreased cellulose content in their cell walls. Xyloglucan, which is bound to cellulose and together with it forms the main loading network of plant cell walls, has also been described to decrease in habituated cells, but whether the change on cellulose affects the xyloglucan structure besides its abundance has not been analyzed. Fragmentation analysis with xyloglucan-specific endoglucanase (XEG) and endocellulase revealed that habituation to DCB caused a change in the fine structure of xyloglucan, namely a decrease in fucosyl residues attached to the galactosyl–xylosyl residues along the glucan backbone. After the removal of herbicide from the medium (dehabituated cells), xyloglucan recovered its fucosyl residues. In addition, some cello-oligosaccharides could be detected only in habituated cells' xyloglucan digested by XEG and endocellulase, corresponding to a glucan covalently bound or co-precipitated with the hemicelluloses. These results show that structural flexibility of cell walls relies in part on the plasticity of xyloglucan composition and opens up new perspectives to further research in this field.
High peroxidase activity and stable changes in the cell wall are related to dichlobenil tolerance
2009, Journal of Plant Physiology
Citation Excerpt :
Calluses were removed from the explants and routinely subcultured for 30 d in a Murashige and Skoog medium (Murashige and Skoog, 1962) supplemented with 10 μM 2,4-D. Cell suspensions were obtained from calluses cultured in liquid Murashige and Skoog medium containing 5 μM 2,4-D and rotary shaken. Cell suspensions were habituated to growth in dichlobenil by stepwise transfers into growth mediums containing increasing concentrations of the herbicide as previously described (Encina et al., 2001). Habituated cell suspensions growing on 12 μM dichlobenil (Sh12) were dehabituated by subculture in a medium lacking dichlobenil and are referred to as Sdn, where n indicates the number of subcultures in the absence of dichlobenil.
Suspension-cultured bean cells habituated to growth in a lethal concentration of dichlobenil were cultured for 3–5 years in a medium lacking the inhibitor in order to obtain long-term dehabituated cell lines. The growth parameters, cell morphology and ultrastructure of cells in the absence of dichlobenil reverted to that of non-habituated cells. The cellulose content and Fourier transform infrared (FTIR) spectra of crude cell walls from long-term dehabituated cells were also similar to those of non-habituated cells. However, long-term dehabituated cells showed three times more tolerance to dichlobenil than non-habituated cells. The incorporation of [¹⁴C]Glc into cellulose was reduced by 40% in dehabituated cells when compared with non-habituated cells. However, the addition of dichlobenil to dehabituated cells increased the incorporation of [¹⁴C]Glc into cellulose 3.3-fold with respect to that of non-habituated cells. Dehabituated cells showed a constitutively increased peroxidase activity when compared with non-habituated cells. Results reported here indicate that the habituation of bean cultured cells to dichlobenil relied partially on a stable change in the cellulose biosynthesis complex and is associated with high guaiacol peroxidase activity.

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Characterization of cell walls in bean (Phaseolus vulgaris L.) callus cultures tolerant to dichlobenil

Abstract

Introduction

Section snippets

Chemicals

Callus culture and accomodation to dichlobenil

Growth and accomodation of callus cultures

Discussion

Acknowledgements

Pestic. Biochem. Physiol.

Pestic. Biochem. Physiol.

Anal. Biochem.

Anal. Biochem.

Carbohydr. Res.

Cellulose biosynthesis

Annu. Rev. Plant Physiol.

Cellulose biosynthesis: exciting times for a difficult field of study

Annu. Rev. Plant Physiol. Plant Mol. Biol.

Adaptation and growth of tomato cells on the herbicide 2,6-dichlorobenzonitrile leads to production of unique cell walls virtually lacking a cellulose–xyloglucan network

Plant Physiol.

Cell wall structure in cells adapted to growth on the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile

Plant Physiol.

Structural features of cell walls from tomato cells adapted to growth on the herbicide 2,6-dichlorobenzonitrile

J. Microsc.

Structural and immunocytochemical characterization of the walls of dichlobenil-habituated BY-2 tobacco cells

Int. J. Plant Sci.

Increase in the amount of celA-1 protein in tobacco BY-2 cells by a cellulose biosynthesis inhibitor, 2,6-dichlorobenzonitrile

Plant Cell Physiol.

Isoxaben, a new selective herbicide for use in cereals

Weeds

Mechanism of isoxaben tolerance in Agrostis palustris var. Penncross

J. Exp. Bot.

Cell wall modifications in bean (Phaseolus vulgaris) callus cultures tolerant to isoxaben

Plant Physiol.