Immunohistochemical dynamics of cell wall matrix polymers during tomato autograft healing.

A large part of the production of tomato plants is grafted. Although it has recently been described that cell walls play an important role in tomato graft healing, the spatiotemporal dynamics of cell wall changes in this critical process remains largely unknown. The aim of this work was to immunolocalize changes in the major cell wall matrix components of autograft union tissues throughout the course of healing, from 1 to 20 days after grafting (DAG). Homogalacturonan was de novo synthetized and deposited in the cut edges, displaying the low methyl-esterified homogalacturonan a stronger labelling. Labelling of galactan side chains of rhamnogalacturonan increased until 8 DAG, although remarkably a set of cells at the graft union did not show labelling for this epitope. Changes in xylan immunolocalization were associated to the xylem vasculature development throughout, while those of xyloglucan revealed early synthesis at the cut edges. Arabinogalactan proteins increased up to 8 DAG and showed scion-rootstock asymmetry, with a higher extent in the scion. The combination of these changes appears to be related with the success of the autograft, specifically facilitating the adhesion phase between scion-rootstock tissues. This knowledge paves the way for improved grafting using methods that facilitate appropriate changes in the time and space dynamics of these cell wall compounds.

Production of Encecalin in Cell Cultures and Hairy Roots of Helianthella quinquenervis (Hook.) A. Gray.

Plant cell and organ cultures of Helianthella quinquenervis, a medicinal plant whose roots are used by the Tarahumara Indians of Chihuahua, Mexico, to relieve several ailments, were established to identify and quantify some chromenes with biological activity, such as encecalin, and to evaluate their potential for biotechnological production. Gas chromatography-mass spectrometry (GC-MS) analysis corroborated the presence of quantifiable amounts of encecalin in H. quinquenervis cell cultures (callus and cell suspensions). In addition, hairy roots were obtained through three transformation protocols (prick, 45-s sonication and co-culture), using wild type Agrobacterium rhizogenes A4. After three months, cocultivation achieved the highest percentage of transformation (66%), and a comparable production (FW) of encecalin (110 µg/g) than the sonication assay (120 µg/g), both giving far higher yields than the prick assay (19 µg/g). Stable integration of rolC and aux1 genes in the transformed roots was confirmed by polymerase chain reaction (PCR). Hairy roots from cocultivation (six months-old) accumulated as much as 1086 µg/g (FW) of encecalin, over three times higher than the cell suspension cultures. The production of encecalin varied with growth kinetics, being higher at the stationary phase. This is the first report of encecalin production in hairy roots of H. quinquenervis, demonstrating the potential for a future biotechnological production of chromenes.

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.

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.

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.

Elucidating compositional factors of maize cell walls contributing to stalk strength and lodging resistance.

Lodging is one of the causes of maize (Zea mays L.) production losses worldwide and, at least, the resistance to stalk lodging has been positively correlated with stalk strength. In order to elucidate the putative relationship between cell wall, stalk strength and lodging resistance, twelve maize inbreds varying in rind penetration strength and lodging resistance were characterized for cell wall composition and structure. Stepwise multiple regression indicates that H lignin subunits confer a greater rind penetration strength. Besides, the predictive model for lodging showed that a high ferulic acid content increases the resistance to lodging, whereas those of diferulates decrease it. These outcomes highlight that the strength and lodging susceptibility of maize stems may be conditioned by structural features of cell wall rather than by the net amount of cellulose, hemicelluloses and lignin. The results presented here provide biotechnological targets in breeding programs aimed at improving lodging in maize.

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.

Mineral stress affects the cell wall composition of grapevine (Vitis vinifera L.) callus.

Grapevine (Vitis vinifera L.) is one of the most economically important fruit crops in the world. Deficit in nitrogen, phosphorus and sulfur nutrition impairs essential metabolic pathways. The influence of mineral stress in the composition of the plant cell wall (CW) has received residual attention. Using grapevine callus as a model system, 6 weeks deficiency of those elements caused a significant decrease in growth. Callus CWs were analyzed by Fourier transform infrared spectroscopy (FT-IR), by quantification of CW components and by immunolocalization of CW epitopes with monoclonal antibodies. PCA analysis of FT-IR data suggested changes in the main components of the CW in response to individual mineral stress. Decreased cellulose, modifications in pectin methyl esterification and increase of structural proteins were among the events disclosed by FT-IR analysis. Chemical analyses supported some of the assumptions and further disclosed an increase in lignin content under nitrogen deficiency, suggesting a compensation of cellulose by lignin. Moreover, polysaccharides of callus under mineral deficiency showed to be more tightly bonded to the CW, probably due to a more extensive cross-linking of the cellulose-hemicellulose network. Our work showed that mineral stress impacts the CW at different extents according to the withdrawn mineral element, and that the modifications in a given CW component are compensated by the synthesis and/or alternative linking between polymers. The overall results here described for the first time pinpoint the CW of Vitis callus different strategies to overcome mineral stress, depending on how essential they are to cell growth and plant development.

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.

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.