Effect of ancymidol on cell wall metabolism in growing maize cells.

MAIN CONCLUSION: Ancymidol inhibits the incorporation of cellulose into cell walls of maize cell cultures in a gibberellin-independent manner, impairing cell growth; the reduction in the cellulose content is compensated with xylans. Ancymidol is a plant growth retardant which impairs gibberellin biosynthesis. It has been reported to inhibit cellulose synthesis by tobacco cells, based on its cell-malforming effects. To ascertain the putative role of ancymidol as a cellulose biosynthesis inhibitor, we conducted a biochemical study of its effect on cell growth and cell wall metabolism in maize cultured cells. Ancymidol concentrations ≤ 500 µM progressively reduced cell growth and induced globular cell shape without affecting cell viability. However, cell growth and viability were strongly reduced by ancymidol concentrations ≥ 1.5 mM. The I50 value for the effect of ancymidol on FW gain was 658 µM. A reversal of the inhibitory effects on cell growth was observed when 500 µM ancymidol-treated cultures were supplemented with 100 µM GA3. Ancymidol impaired the accumulation of cellulose in cell walls, as monitored by FTIR spectroscopy. Cells treated with 500 µM ancymidol showed a ~ 60% reduction in cellulose content, with no further change as the ancymidol concentration increased. Cellulose content was partially restored by 100 µM GA3. Radiolabeling experiments confirmed that ancymidol reduced the incorporation of [14C]glucose into α-cellulose and this reduction was not reverted by the simultaneous application of GA3. RT-PCR analysis indicated that the cellulose biosynthesis inhibition caused by ancymidol is not related to a downregulation of ZmCesA gene expression. Additionally, ancymidol treatment increased the incorporation of [3H]arabinose into a hemicellulose-enriched fraction, and up-regulated ZmIRX9 and ZmIRX10L gene expression, indicating an enhancement in the biosynthesis of arabinoxylans as a compensatory response to cellulose reduction.

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.

Purification and characterization of a soluble ß-1,4-glucan from bean (Phaseolus vulgaris L.)-cultured cells dehabituated to dichlobenil.

Bean cells habituated to grow in the presence of dichlobenil exhibited reduced cellulose and hemicellulose content and an increase in pectic polysaccharides. Furthermore, following the extraction of pectins and hemicelluloses, a large amount of neutral sugars was released. These sugars were found to be part of a soluble ß-1,4-glucan in a preliminary characterization, as reported by Encina et al. (Physiol Plant 114:182-191, 2002). When habituated cells were subcultured in the absence of the herbicide (dehabituated cells), the release of neutral sugars after the extraction of pectins and hemicelluloses was maintained. In this study, we have isolated a soluble ß-1,4-glucan from dehabituated cells by sonication of the wall residue (cellulose fraction) remaining after fractionation. Gel filtration chromatography revealed that its average molecular size was 14 kDa. Digestion of the sample with endocellulase revealed the presence of cellobiose, cellotriose, and cellotetraose. Methylation analysis showed that 4-linked glucose was the most abundant sugar residue, but 4,6-linked glucose, terminal arabinose and 4-linked galactose for xyloglucan, and arabinogalactan were also identified. NMR analysis showed that this 1,4-glucan may be composed of various kinds of substitutions along the glucan backbone together with acetyl groups linked to the OH group of sugar residues. Thus, despite its relatively high molecular mass, the ß-glucan remains soluble because of its unique configuration. This is the first time that a glucan with such characteristics has been isolated and described. The discovery of new molecules, as this ß-glucan with unique features, may help understand the composition and arrangement of the polymers within plant cell walls, contributing to a better understanding of this complex structure.

Cellulose biosynthesis inhibitors: comparative effect on bean cell cultures.

The variety of bioassays developed to evaluate different inhibition responses for cellulose biosynthesis inhibitors makes it difficult to compare the results obtained. This work aims (i) to test a single inhibitory assay for comparing active concentrations of a set of putative cellulose biosynthesis inhibitors and (ii) to characterize their effect on cell wall polysaccharides biosynthesis following a short-term exposure. For the first aim, dose-response curves for inhibition of dry-weight increase following a 30 days exposure of bean callus-cultured cells to these inhibitors were obtained. The compound concentration capable of inhibiting dry weight increase by 50% compared to control (I(50)) ranged from subnanomolar (CGA 325'615) to nanomolar (AE F150944, flupoxam, triazofenamide and oxaziclomefone) and micromolar (dichlobenil, quinclorac and compound 1) concentrations. In order to gain a better understanding of the effect of the putative inhibitors on cell wall polysaccharides biosynthesis, the [(14)C]glucose incorporation into cell wall fractions was determined after a 20 h exposure of cell suspensions to each inhibitor at their I(50) value. All the inhibitors tested decreased glucose incorporation into cellulose with the exception of quinclorac, which increased it. In some herbicide treatments, reduction in the incorporation into cellulose was accompanied by an increase in the incorporation into other fractions. In order to appreciate the effect of the inhibitors on cell wall partitioning, a cluster and Principal Component Analysis (PCA) based on the relative contribution of [(14)C]glucose incorporation into the different cell wall fractions were performed, and three groups of compounds were identified. The first group included quinclorac, which increased glucose incorporation into cellulose; the second group consisted of compound 1, CGA 325'615, oxaziclomefone and AE F150944, which decreased the relative glucose incorporation into cellulose but increased it into tightly-bound cellulose fractions; and the third group, comprising flupoxam, triazofenamide and dichlobenil, decreased the relative glucose incorporation into cellulose and increased it into a pectin rich fraction.

The role of cell wall phenolics during the early remodelling of cellulose-deficient maize cells.

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.

A novel surgical technique focused on the study of the ileum: The preduodenal ileal transposition.

AIMS: Our main goal is to study the effects on the carbohydrate metabolism. Thus, we designed various experimental surgical models on healthy non-obese Wistar rats to reproduce several conditions. In this sense, we report a new experimental model. It is well known that bariatric surgery has important effects on the control of Type 2 Diabetes Mellitus. The underlying reasons are yet unknown, although the different theories focused in the release of different hormones after the pass of the nutrients through the tract. These released hormones have opposite effects that come together in a balanced glycemic metabolism. MATERIALS AND METHODS: After bariatric surgical techniques, the modified anatomy resulted in an imbalance of the secreted hormones. Wistar rats were randomized in two groups Sham and surgical group. Our model consisted on the transposition of the terminal ileum right after the pylorus. Weight gain, food intake, and basal glycemia were measured weekly. RESULTS: We did not obtain significant differences between both groups for these functional variables. CONCLUSIONS: This technique involved an early pass of the bolus through the ileum. The change on the luminal pH, along with the lack of enzymes to absorb the content, or the changes in the release of several hormones must be variables to the study. The mortality rate was assumable considering it was an experimental model on animals.

OBJETIVO: Crear un nuevo modelo quirúrgico experimental en ratas Wistar sanas no obesas para estudiar los efectos del metabolismo glucídico. Es bien sabido que las técnicas de cirugía bariátrica tienen un efecto importante sobre la resolución de la diabetes mellitus tipo 2. Se han invocado diferentes hipótesis, algunas centradas en el papel que tienen distintas hormonas secretadas por el propio tubo digestivo tras el paso de los nutrientes a su través, pero las razones últimas subyacentes permanecen desconocidas. El efecto contrapuesto de dichas hormonas consigue un efecto de control glucémico. El desequilibrio hormonal tras las alteraciones anatómicas de las cirugías bariátricas podría estar en la base de dicha mejora del metabolismo glucídico final. MATERIAL Y MÉTODOS: Las ratas fueron operadas en dos grupos (control quirúrgico y experimental) y se procedió a disponerles el íleon anastomosado al antro pilórico, previo al esfínter pilórico. Medimos distintos parámetros funcionales (ganancia de peso, ingesta y glucemias semanales). RESULTADOS: No obtuvimos diferencias significativas en la evolución de estos parámetros. CONCLUSIONES: Este modelo será útil para nuestro propósito de estudiar el íleon, en su componente secretor de enterohormonas, cuando el paso de los nutrientes se produzca tempranamente. La mortalidad fue asumible, dada la innovación técnica realizada.

Characterization of structural cell wall polysaccharides in cattail (Typha latifolia): Evaluation as potential biofuel feedstock.

Second generation bioethanol produced from lignocellulosic biomass is attracting attention as an alternative energy source. In this study, a detailed knowledge of the composition and structure of common cattail (Typha latifolia L.) cell wall polysaccharides, obtained from stem or leaves, has been conducted using a wide set of techniques to evaluate this species as a potential bioethanol feedstock. Our results showed that common cattail cellulose content was high for plants in the order Poales and was accompanied by a small amount of cross-linked polysaccharides. A high degree of arabinose-substitution in xylans, a high syringyl/guaiacyl ratio in lignin and a low level of cell wall crystallinity could yield a good performance for lignocellulose saccharification. These results identify common cattail as a promising plant for use as potential bioethanol feedstock. To the best of our knowledge, this is the first in-depth analysis to be conducted of lignocellulosic material from common cattail.

Quinclorac-habituation of bean (Phaseolus vulgaris) cultured cells is related to an increase in their 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.

The use of FTIR spectroscopy to monitor modifications in plant cell wall architecture caused by cellulose biosynthesis inhibitors.

Fourier Transform InfraRed (FTIR) spectroscopy is a powerful and rapid technique for analysing cell wall components and putative cross-links, which is able to non-destructively recognize polymers and functional groups and provide abundant information about their in muro organization. FTIR spectroscopy has been reported to be a useful tool for monitoring cell wall changes occurring in muro as a result of various factors, such as growth and development processes, mutations or biotic and abiotic stresses. This mini-review examines the use of FTIR spectroscopy in conjunction with multivariate analyses to monitor cell wall changes related to (1) the exposure of diverse plant materials to cellulose biosynthesis inhibitors (CBIs), and (2) the habituation/dehabituation of plant cell cultures to this kind of herbicides. The spectra analyses show differences not only regarding the inhibitor, but also regarding how long cells have been growing in its presence.

Biological responses of a simulated marine food chain to lead addition.

This investigation sought to assess the biological responses to Pb along a simplified four-level food chain, from the primary producer, the microalgae Tetraselmis suecica, grown in a control medium with < 1 µg/L of Pb and exposed to a sublethal dose (20 µg/L of Pb) and used as the base of a simulated food chain, through the primary-, secondary-, and tertiary-level consumers, namely, the brine shrimp, Artemia franciscana; the white-leg shrimp, Litopenaeus vannamei; and the grunt fish, Haemulon scudderi, respectively. Growth of Pb-exposed T. suecica was 40% lower than that of the control cultures, and survival of A. franciscana fed this diet was 25 to 30% lower than the control. No differences in the growth rates of Pb-exposed and control shrimp and fish and no gross morphological changes were evident in the exposed specimens. However, the exposed shrimp and fish had 20 and 15% higher mortalities than their controls, respectively. In addition, behavioral alterations were observed in exposed shrimp and fish, including reduction in food consumption or cessation of feeding, breathing air out of the water, reduction of motility, and erratic swimming. The negative correlation between Pb concentration in whole body of shrimp and fish and Fulton's condition factor suggested also that the exposed organisms were stressed because of Pb accumulation.

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.

High peroxidase activity and stable changes in the cell wall are related to dichlobenil tolerance.

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 [(14)C]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 [(14)C]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.

Habituation and dehabituation to dichlobenil: simply the equivalent of Penélope's weaving and unweaving process?

The habituation of cell cultures to cellulose biosynthesis inhibitors constitutes a valuable method for learning more about the plasticity of plant cell wall composition and structure. The subculture of habituated cells in the absence of an inhibitor (dehabituation) offers complementary information: some habituation-associated modifications revert, whereas others remain, even after long-term (3-5 years) dehabituation processes. However, is dehabituation simply the opposite to the process of habituation, in the same way that the cloth woven by Penélope during the day was unwoven during the night? Principal Component Analysis applied to Fourier Transformed Infrared (FTIR) spectra of cell walls from dichlobenil-habituated and dehabituated bean cell lines has shown that dehabituation follows a different pathway to that of habituation. Principal component loadings show that dehabituated cells have more pectins, but that these display a lower degree of methyl-esterification, than those of habituated ones. Further analysis of cell walls focusing on the first steps of habituation would serve to identify which specific modifications in pectins are responsible to the fine modulation of cell wall architecture observed during the habituation/dehabituation process.