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.

Fourier transform mid infrared spectroscopy applications for monitoring the structural plasticity of plant cell walls.

Fourier transform mid-infrared (FT-MIR) spectroscopy has been extensively used as a potent, fast and non-destructive procedure for analyzing cell wall architectures, with the capacity to provide abundant information about their polymers, functional groups, and in muro entanglement. In conjunction with multivariate analyses, this method has proved to be a valuable tool for tracking alterations in cell walls. The present review examines recent progress in the use of FT-MIR spectroscopy to monitor cell wall changes occurring in muro as a result of various factors, such as growth and development processes, genetic modifications, exposition or habituation to cellulose biosynthesis inhibitors and responses to other abiotic or biotic stresses, as well as its biotechnological applications.

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 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.

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-throughput microarray profiling of cell wall polymers during hydrothermal pre-treatment of wheat straw.

Lignocellulosic plant material is potentially a sustainable source of fermentable sugars for bioethanol production. However, a barrier to this is the high resistance or recalcitrance of plant cell walls to be hydrolyzed. Therefore, a detailed knowledge of the structural features of plant cell walls that contribute to recalcitrance is important for improving the efficiency of bioethanol production. In this work we have used a technique known as Comprehensive Microarray Polymer Profiling (CoMPP) to analyze wheat straw before and after being subjected to hydrothermal pre-treatments at four different temperatures. The CoMPP technique combines the specificity of monoclonal antibodies with the high-throughput capacity of microarrays. Changes in the relative abundance of cell wall polysaccharides could be tracked during processing, and a reduction in xylan, arabinoxylans, xyloglucan, and mixed-linked glucan epitopes was detected at the two highest temperatures of pre-treatment used. This work demonstrates the potential of CoMPP as a complementally technique to conventional methods for analyzing biomass composition.

Characterization of the primary cell walls of seedlings of Brachypodium distachyon--a potential model plant for temperate grasses.

The genome of Brachypodium distachyon, also known as purple false brome, was fully sequenced in 2008 largely in response to the demand for a model plant for temperate grasses. A comparative study of the primary cell walls of seedlings of B. distachyon, Hordeum vulgare and Triticum aestivum was carried out. The cell walls of the three species were characterized by similar relative levels of, and developmental changes in, hemicelluloses. The occurrence of (1,3;1,4)-beta-D-glucans was correlated with phases of growth involving cell elongation. Expression profiling of the genes involved in (1,3;1,4)-beta-D-glucan synthesis (cellulose synthase-like F family (CSLF), CSLH and a putative synthase gene CSLJ) did not show a transcriptional regulation that corresponded to the abundance of (1,3;1,4)-beta-D-glucans. CSLF6 transcripts were similarly highly expressed in all three grasses, and were much more abundant than any of the other transcripts. The CSLH transcript was relatively abundant in B. distachyon but almost undetectable in the other species. The deposition of arabinoxylans increased steadily during seedling growth in all three grasses, but they became less substituted and more cross-linked into the wall matrix during cell maturation. Moreover, arabinoxylans in B. distachyon differed from the two other grasses in having a lower degree of arabinose substitution, a higher percentage of ferulic acid in form of dimers and a larger proportion of ester-linked p-coumaric acid.

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.

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.

Novel type II cell wall architecture in dichlobenil-habituated maize calluses.

Growth of maize (Zea mays L.) callus-culture cells was inhibited using dichlobenil (2,6 dichlorobenzonitrile, DCB) concentrations > or =1 microM; I (50) value for the effect on inhibited fresh weight gain was 1.5 microM. By increasing the DCB concentration in the culture medium, DCB-habituated cells became 13 times more tolerant of the inhibitor (I (50): 20 microM). In comparison with non-habituated calluses, DCB-habituated calluses grew slower, were less friable and were formed by irregularly shaped cells surrounded by a thicker cell wall. By using an extensive array of techniques, changes in type II cell wall composition and structure associated with DCB habituation were studied. Walls from DCB-habituated cells showed a reduction of up to 75% in cellulose content, which was compensated for by a net increase in arabinoxylan content. Arabinoxylans also showed a reduction in their extractability and a marked increase in their relative molecular mass. DCB habituation also involved a shift from ferulate to coumarate-rich cells walls, and enrichment in cell wall esterified hydroxycinnamates and dehydroferulates. The content of polymers such as mixed-glucan, xyloglucan, mannans, pectins or proteins did not vary or was reduced. These results prove that the architecture of type II cell walls is able to compensate for deficiencies in cellulose content with a more extensive and phenolic cross-linked network of arabinoxylans, without necessitating beta-glucan or other polymer enhancement. As a consequence of this modified architecture, walls from DCB-habituated cells showed a reduction in their swelling capacity and an increase both in pore size and in resistance to polysaccharide hydrolytic enzymes.

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.