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.     

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.     

Immunocytochemical characterization of the cell walls of bean cell suspensions during habituation and dehabituation to dichlobenil

The effects of the cellulose inhibitor dichlobenil on the cell wall composition and structure during the habituation/dehabituation process of suspension‐cultured bean cells were assessed. A range of techniques were used including cell wall fractionation, sugar analysis, immunofluorescence and fluorochrome labelling of resin‐embedded sections, and immunodot assays (IDAs) of cell wall fractions. The cell walls from bean cell suspensions with initial levels of habituation to dichlobenil had decreased levels of cellulose, but this effect lessened with increasing numbers of subcultures. All cell walls analysed showed calcofluor‐stained appositions. However, in habituated and dehabituated cells, appositions were not recognized by an anticallose antibody. This finding suggested the accumulation of an extracellular polysaccharide different to callose, probably a 1,4‐β‐glucan in these cell lines. Appositions in habituated cells also contained homogalacturonan (HG) with a high degree of methyl esterification (DE), rhamnogalacturonan (RG) and xyloglucan. Habituated cell walls were also enriched in pectins, particularly HG, with a low DE, and RG. The levels of extensin epitope that colocalized with RG in habituated cells also diminished with the increasing number of subcultures. Habituated cells also liberated less extensin into the medium. In habituated cells, a decrease in the cell wall arabinogalactan protein (AGP) labelling was observed both in cell walls and in the culture medium. The increase in the number of subcultures in 0.3 µM dichlobenil was accompanied by an increment in some pectic epitopes (JIM5 and LM5) and a decrease in other pectic and in protein epitopes (JIM7, PAM1, LM6, LM2 and MAC207), indicating a re‐structuring of cell walls throughout the habituation procedure. Dehabituated cells showed an overall composition similar to that of non‐habituated cells, with exception of an increase in glucose in hemicellulosic fractions tightly bound to cellulose. However, these cells also showed reduced levels of extensin and AGP labelling. These differences could be related to the high tolerance to dichlobenil observed in dehabituated cells.     

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.Key words: Quinclorac, herbicide, Phaseolus vulgaris, cell culture habituation, primary cell wall, cellulose, FTIR spectroscopy     

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 ≥1 μM; I ₅₀ value for the effect on inhibited fresh weight gain was 1.5 μM. By increasing the DCB concentration in the culture medium, DCB-habituated cells became 13 times more tolerant of the inhibitor (I ₅₀: 20 μM). 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 β-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.     

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.     

Altered pectin composition in primary cell walls of korrigan, a dwarf mutant of Arabidopsis deficient in a membrane-bound endo-1,4-β-glucanase

Korrigan (kor) is a dwarf mutant of Arabidopsis thaliana (L.) Heynh. that is deficient in a membrane-bound endo-1,4-beta-glucanase. The effect of the mutation on the pectin network has been studied in kor by microscopical techniques associated with various probes specific for different classes of pectic polysaccharides. The localisation of native crystalline cellulose was also examined using the cellobiohydrolase I-gold probe. The investigations were focused on the external cell walls of the epidermis, a cell layer that, in a number of plant species, has been shown to be growth limiting. Anionic sites associated with pectic polymers were quantified using the cationic gold probe. Homogalacturonans were quantified using polyclonal anti-polygalacturonic acid/rhamnogalacturonan I antibodies recognising polygalacturonic acid, and monoclonal JIM7 and JIM5 antibodies recognising homogalacturonans with a high or low degree of methyl-esterification, respectively. Rhamnogalacturonans were quantified with two monoclonal antibodies, LM5, recognising beta-1,4 galactan side chains of rhamnogalacturonan I, and CCRCM2. Our results show a marked increase in homogalacturonan epitopes and a decrease in rhamnogalacturonan epitopes in kor compared to the wild type. A substantial decrease in cellobiohydrolase I-gold labelling was also observed in the mutant cell walls. These findings demonstrate that a deficiency in an endo-1,4-beta-glucanase, which is in principle not directly implicated in pectin metabolism, can induce important changes in pectin composition in the primary cell wall. The changes indicate the existence of feedback mechanisms controlling the synthesis and/or deposition of pectic polysaccharides in primary cell walls.     

Degree of pectin methyl esterification in endosperm cell walls is involved in embryo bending in Arabidopsis thaliana

The endosperm is a transitory structure involved in proper embryo elongation. The cell walls of mature seed endosperm are generally composed of a uniform distribution of cellulose, unesterified homogalacturonans, and arabinans. Recent studies suggest that changes in cell wall properties during endosperm development could be related to embryo growth. The degree of methyl esterification of homogalacturonans may be involved in this endosperm tissue remodelling. The relevance of the degree of homogalacturonan methyl esterification during seed development was determined by immunohistochemical analyses using a panel of probes with specificity for homogalaturonans with different degrees of methyl esterification. Low-esterified and un-esterified homogalacturonans were abundant in endosperm cells during embryo bending and were also detected in mature embryos. BIDXII (BDX) could be involved in seed development, because bdx-1 mutants had misshapen embryos. The methyl esterification pattern described for WT seeds was different during bdx-1 seed development; un-esterified homogalacturonans were scarcely present in the cell walls of endosperm in bending embryos and mature seeds. Our results suggested that the degree of methyl esterification of homogalacturonans in the endosperm cell wall may be involved in proper embryo development.     

Characterization of the cell-wall polysaccharides of Arabidopsis thaliana leaves.

The cell-wall polysaccharides of Arabidopsis thaliana leaves have been isolated, purified, and characterized. The primary cell walls of all higher plants that have been studied contain cellulose, the three pectic polysaccharides homogalacturonan, rhamnogalacturonan I and rhamnogalacturonan II, the two hemicelluloses xyloglucan and glucuronoarabinoxylan, and structural glycoproteins. The cell walls of Arabidopsis leaves contain each of these components and no others that we could detect, and these cell walls are remarkable in that they are particularly rich in phosphate buffer-soluble polysaccharides (34% of the wall). The pectic polysaccharides of the purified cell walls consist of rhamnogalacturonan I (11%), rhamnogalacturonon II (8%), and homogalacturonan (23%). Xyloglucan (XG) accounts for 20% of the wall, and the oligosaccharide fragments generated from XG by endoglucanase consist of the typical subunits of other higher plant XGs. Glucuronoarabinoxylan (4%), cellulose (14%) and protein (14%) account for the remainder of the wall. Except for the phosphate buffer-soluble pectic polysaccharides, the polysaccharides of Arabidopsis leaf cell walls occur in proportions similar to those of other plants. The structure of the Arabidopsis cell-wall polysaccharides are typical of those of many other plants.     

An extensin-rich matrix lines the carinal canals in Equisetum ramosissimum, which may function as water-conducting channels

Background and Aims

The anatomy of Equisetum stems is characterized by the occurrence of vallecular and carinal canals. Previous studies on the carinal canals in several Equisetum species suggest that they convey water from one node to another.

Methods

Cell wall composition and ultrastructure have been studied using immunocytochemistry and electron microscopy, respectively. Serial sectioning and X-ray computed tomography were employed to examine the internode–node–internode transition of Equisetum ramosissimum.

Key Results

The distribution of the LM1 and JIM20 extensin epitopes is restricted to the lining of carinal canals. The monoclonal antibodies JIM5 and LM19 directed against homogalacturonan with a low degree of methyl esterification and the CBM3a probe recognizing crystalline cellulose also bound to this lining. The xyloglucan epitopes recognized by LM15 and CCRC-M1 were only detected in this lining after pectate lyase treatment. The carinal canals, connecting consecutive rings of nodal xylem, are formed by the disruption and dissolution of protoxylem elements during elongation of the internodes. Their inner surface appears smooth compared with that of vallecular canals.

Conclusions

The carinal canals in E. ramosissimum have a distinctive lining containing pectic homogalacturonan, cellulose, xyloglucan and extensin. These canals might function as water-conducting channels which would be especially important during the elongation of the internodes when protoxylem is disrupted and the metaxylem is not yet differentiated. How the molecularly distinct lining relates to the proposed water-conducting function of the carinal canals requires further study. Efforts to elucidate the spatial and temporal distribution of cell wall polymers in a taxonomically broad range of plants will probably provide more insight into the structural–functional relationships of individual cell wall components or of specific configurations of cell wall polymers.     

An Arabidopsis Cell Wall Proteoglycan Consists of Pectin and Arabinoxylan Covalently Linked to an Arabinogalactan Protein

Plant cell walls are comprised largely of the polysaccharides cellulose, hemicellulose, and pectin, along with ∼10% protein and up to 40% lignin. These wall polymers interact covalently and noncovalently to form the functional cell wall. Characterized cross-links in the wall include covalent linkages between wall glycoprotein extensins between rhamnogalacturonan II monomer domains and between polysaccharides and lignin phenolic residues. Here, we show that two isoforms of a purified Arabidopsis thaliana arabinogalactan protein (AGP) encoded by hydroxyproline-rich glycoprotein family protein gene At3g45230 are covalently attached to wall matrix hemicellulosic and pectic polysaccharides, with rhamnogalacturonan I (RG I)/homogalacturonan linked to the rhamnosyl residue in the arabinogalactan (AG) of the AGP and with arabinoxylan attached to either a rhamnosyl residue in the RG I domain or directly to an arabinosyl residue in the AG glycan domain. The existence of this wall structure, named ARABINOXYLAN PECTIN ARABINOGALACTAN PROTEIN1 (APAP1), is contrary to prevailing cell wall models that depict separate protein, pectin, and hemicellulose polysaccharide networks. The modified sugar composition and increased extractability of pectin and xylan immunoreactive epitopes in apap1 mutant aerial biomass support a role for the APAP1 proteoglycan in plant wall architecture and function.     

Disruption of cellulose synthesis by 2,6‐dichlorobenzonitrile affects the structure of the cytoskeleton and cell wall construction in Arabidopsis

Cellulose is the major component of plant cell walls and is an important source of industrial raw material. Although cellulose biosynthesis is one of the most important biochemical processes in plant biology, the regulatory mechanisms of cellulose synthesis are still unclear. Here, we report that 2,6‐dichlorobenzonitrile (DCB), an inhibitor of cellulose synthesis, inhibits Arabidopsis root development in a dose‐ and time‐dependent manner. When treated with DCB, the plant cell wall showed altered cellulose distribution and intensity, as shown by calcofluor white and S4B staining. Moreover, pectin deposition was reduced in the presence of DCB when immunostained with the monoclonal antibody JIM5, which was raised against pectin epitopes. This result was confirmed using Fourier transform infrared (FTIR) analysis. Confocal microscopy revealed that the organisation of the microtubule cytoskeleton was significantly disrupted in the presence of low concentrations of DCB, whereas the actin cytoskeleton only showed changes with the application of high DCB concentrations. In addition, the subcellular dynamics of Golgi bodies labelled with N‐ST‐YFP and TGN labelled with VHA‐a1‐GFP were both partially blocked by DCB. Transmission electron microscopy indicated that the cell wall structure was affected by DCB, as were the Golgi bodies. Scanning electron microscopy showed changes in the organisation of cellulose microfibrils. These results suggest that the inhibition of cellulose synthesis by DCB not only induced changes in the chemical composition of the root cell wall and cytoskeleton structure, but also changed the distribution of cellulose microfibrils, implying that cellulose plays an important role in root development in Arabidopsis.     

Structure of Plant Cell Walls : XXVI. The Walls of Suspension-Cultured Sycamore Cells Contain a Family of Rhamnogalacturonan-I-Like Pectic Polysaccharides

Considerable information has been obtained about the primary structures of suspension-cultured sycamore (Acer pseudoplatanus) cell-wall pectic polysaccharides, i.e. rhamnogalacturonan I, rhamnogalacturonan II, and homogalacturonan. However, these polysaccharides, which are solubilized from the walls by endo-α-1,4-polygalacturonase, account for only about half of the pectic polysaccharides known to be present in sycamore cell walls. We now report that, after exhaustive treatment with endo-α-1,4-polygalacturonase, additional pectic polysaccharides were extracted from sycamore cell walls by treatment with Na₂CO₃ at 1 and 22°C. These previously uncharacterized polysaccharides accounted for ~4% of the cell wall. Based on the glycosyl and glycosyl-linkage compositions and the nature of the products obtained by treating the quantitatively predominant NaCO₃-extracted polysaccharides with lithium metal dissolved in ethylenediamine, the polysaccharides were found to strongly resemble rhamnogalacturonan I. However, unlike rhamnogalacturonan I that characteristically had equal amounts of 2- and 2,4-linked rhamnosyl residues in its backbone, the polysaccharides extracted in Na₂CO₃ at 1°C had markedly disparate ratios of 2- to 2,4-linked rhamnosyl residues. We concluded that polysaccharides similar to rhamnogalacturonan I but with different degrees of branching are present in the walls of suspension-cultured sycamore cells.     

A cytochemical and immunocytochemical analysis of the wall labyrinth apparatus in leaf transfer cells in Elodea canadensis

Background and Aims

Transfer cells are plant cells specialized in apoplast/symplast transport and characterized by a distinctive wall labyrinth apparatus. The molecular architecture and biochemistry of the labyrinth apparatus are poorly known. The leaf lamina in the aquatic angiosperm Elodea canadensis consists of only two cell layers, with the abaxial cells developing as transfer cells. The present study investigated biochemical properties of wall ingrowths and associated plasmalemma in these cells.

Methods

Leaves of Elodea were examined by light and electron microscopy and ATPase activity was localized cytochemically. Immunogold electron microscopy was employed to localize carbohydrate epitopes associated with major cell wall polysaccharides and glycoproteins.

Key Results

The plasmalemma associated with the wall labyrinth is strongly enriched in light-dependent ATPase activity. The wall ingrowths and an underlying wall layer share an LM11 epitope probably associated with glucuronoarabinoxylan and a CCRC-M7 epitope typically associated with rhamnogalacturonan I. No labelling was observed with LM10, an antibody that recognizes low-substituted and unsubstituted xylan, a polysaccharide consistently associated with secondary cell walls. The JIM5 and JIM7 epitopes, associated with homogalacturonan with different degrees of methylation, appear to be absent in the wall labyrinth but present in the rest of cell walls.

Conclusions

The wall labyrinth apparatus of leaf transfer cells in Elodea is a specialized structure with distinctive biochemical properties. The high level of light-dependent ATPase activity in the plasmalemma lining the wall labyrinth is consistent with a formerly suggested role of leaf transfer cells in enhancing inorganic carbon inflow. The wall labyrinth is a part of the primary cell wall. The discovery that the wall ingrowths in Elodea have an antibody-binding pattern divergent, in part, from that of the rest of cell wall suggests that their carbohydrate composition is modulated in relation to transfer cell functioning.     

Developmental localization and methylesterification of pectin epitopes during somatic embryogenesis of banana (Musa spp. AAA)

Background

The plant cell walls play an important role in somatic embryogenesis and plant development. Pectins are major chemical components of primary cell walls while homogalacturonan (HG) is the most abundant pectin polysaccharide. Developmental regulation of HG methyl-esterification degree is important for cell adhesion, division and expansion, and in general for proper organ and plant development.

Methodology/Principal Findings

Developmental localization of pectic homogalacturonan (HG) epitopes and the (1→4)-β-D-galactan epitope of rhamnogalacturonan I (RG-I) and degree of pectin methyl-esterification (DM) were studied during somatic embryogenesis of banana (Musa spp. AAA). Histological analysis documented all major developmental stages including embryogenic cells (ECs), pre-globular, globular, pear-shaped and cotyledonary somatic embryos. Histochemical staining of extracellularly secreted pectins with ruthenium red showed the most intense staining at the surface of pre-globular, globular and pear-shaped somatic embryos. Biochemical analysis revealed developmental regulation of galacturonic acid content and DM in diverse embryogenic stages. Immunodots and immunolabeling on tissue sections revealed developmental regulation of highly methyl-esterified HG epitopes recognized by JIM7 and LM20 antibodies during somatic embryogenesis. Cell walls of pre-globular/globular and late-stage embryos contained both low methyl-esterified HG epitopes as well as partially and highly methyl-esterified ones. Extracellular matrix which covered surface of early developing embryos contained pectin epitopes recognized by 2F4, LM18, JIM5, JIM7 and LM5 antibodies. De-esterification of cell wall pectins by NaOH caused a decrease or an elimination of immunolabeling in the case of highly methyl-esterified HG epitopes. However, immunolabeling of some low methyl-esterified epitopes appeared stronger after this base treatment.

Conclusions/Significance

These data suggest that both low- and highly-methyl-esterified HG epitopes are developmentally regulated in diverse embryogenic stages during somatic embryogenesis. This study provides new information about pectin composition, HG methyl-esterification and developmental localization of pectin epitopes during somatic embryogenesis of banana.     

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 longterm (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.Key words: cell-wall, cellulose, dichlobenil, habituation-dehabituation, Fourier transform infrared spectroscopy, principal component analysisThe habituation of cell cultures to the presence of lethal concentrations of cellulose biosynthesis inhibitors illustrates the ability of cells to survive with a modified cell wall and is therefore a valuable experimental technique for gaining an insight into the plasticity of plant cell wall composition and structure. Dichlobenil-habituated cultures usually display some common features: slower growth rates, irregularly shaped cells, a trend to grow in clumps when cultured in suspension and compensation of reduced cell wall cellulose content with other cell wall components.^1–3Most of the cell wall changes induced during the habituation to dichlobenil reverted when cells were dehabituated by culturing them in an inhibitor-free medium.^4–7 However, even in long term (3–5 years) dehabituated cell cultures, some habituation-induced cell wall modifications remain, such as altered extractability of pectins, a decrease in arabinogalactan proteins and hydroxyproline-rich glycoproteins epitopes, and the presence of a soluble β-(1,4)-glucan, although cellulose levels were restored.^5–7 Most remarkably, in addition to these stable changes in cell wall architecture, bean dehabituated cells retained a high capacity to cope with lethal concentrations of dichlobenil, as dehabituated cells were forty times more tolerant to dichlobenil than non-habituated cells.⁵ In an attempt to explain the dichlobenil resistance of dehabituated cells it was found that they had a constitutively increased peroxidase activity, indicating a positive relationship between habituation to dichlobenil and antioxidant capacity.⁷If most of the cell wall modifications induced during the habituation to dichlobenil eventually revert to those of non-habituated cells during the dehabituation process, a question arises: is dehabituation simply the inverse of habituation, in the same way that the cloth woven by Penelope during the day was unwoven during the night, as narrated in Homer''s The Odyssey?Principal Component Analysis applied to Fourier Transformed InfraRed spectra of cell walls has been demonstrated to be a powerful technique for conducting comparative analysis of a wide range of cell wall samples.^3,8 Therefore, a suitable approach to answering this question consists in comparison of cell walls from dichlobenil-habituated and dehabituated bean cell lines using this technique.Clearly, FTIR spectra of cell walls from dehabituated cells with few subcultures in the absence of the herbicide resemble those from cultures habituated to high dichlobenil concentrations.⁵ However, the spectra from cells habituated to low inhibitor concentrations and from cells dehabituated for long periods of time⁷ were more similar to those from non-habituated ones. In fact, when Principal Component Analysis is applied to the entire range, Principal Component 2 (PC2) discriminates between Sh₁₂ (corresponding to cells habituated to high dichlobenil concentration) and the rest of the spectra, which is indicative of the above-mentioned similarity (Fig. 1). Nevertheless, PC1 clearly discriminates between spectra from long-term dehabituated cell walls (located at the positive side) and those from cells habituated to low dichlobenil concentrations (at the negative side). This indicates that progression towards dehabituation follows a different path to that of habituation.Open in a separate windowFigure 1Principal Component Analysis of spectra of cell walls from different calluses. A plot of the first two Principal Components scores is represented based on the FTIR spectra of cell walls from non-habituated cells (Snh, ○), cells habituated to different dichlobenil concentrations (Sh, ▲), and cells previously habituated to 12 µm dichlobenil, with a different number of subcultures in the absence of the herbicide (Sd, ◆). Subindexes indicate dichlobenil concentrations in the growth media of habituated cells (0.3, 0.4 or 12 µm); superindexes indicate number of subcultures in the same media. Arrows indicate the different pathways followed by dichlobenil habituation and dehabituation: black arrows, from non-habituated to habituated cells (habituation), and white arrows, from habituated to non-habituated cells (dehabituation).With the aim of identifying those factors which determine this different pathway, PC1 and PC2 loading factors were analyzed (Fig. 2). This analysis indicated that PC2 (explaining 26.4% of total variance) has a positive correlation with wavenumbers attributed to uronic acids (1,420 and 1,600 cm⁻¹) and galactose (950 cm⁻¹), and a negative correlation with wavenumbers associated with cellulose (1,040, 1,060, 1,175, 1,320 and 1,370 cm⁻¹) and xyloglucan (1,125 cm⁻¹). Thus, Sh₁₂ cell walls (clearly located at the positive side of PC2) are pectin enriched and cellulose/xyloglucan impoverished. As explained above, PC1 discriminates between cell walls from dehabituated cell lines and those from cells habituated to low concentrations of dichlobenil. PC1 (accounting for 42.55% of total variance) has a negative correlation with wavenumbers associated with methylester groups (negative peaks at 1,250 and 1,720 cm⁻¹), and a positive correlation with the so called “fingerprint” region (980–1,200 cm⁻¹). Therefore, cell walls from dehabituated cells (those located at the positive side of PC1) would have lower methyl-esterified pectins when compared with cells habituated to low concentrations of dichlobenil.Open in a separate windowFigure 2Loadings for PC1 and PC2 corresponding to Figure 1. White arrowheads point wavenumbers associated with methyl-esterification; black arrowheads, those associated with cellulose and hemicelluloses, and grey arrowheads indicate wavenumbers associated with uronic acids and galactose.Previous results had revealed that dichlobenil habituated cells experienced a progressive reversion in their cell wall composition when they were subcultured in an inhibitor-free medium, gradually increasing their xyloglucan and cellulose content,^5,6 and that both dichlobenil habituated and dehabituated cells showed changes in the distribution of pectin among cell wall fractions: cell suspensions with a low habituation level had cell walls with a higher amount of pectins, and these were more methyl-esterified.⁶Now, FTIR spectroscopy in association to Principal Component Analysis has shown that, although some of the changes observed in the first steps of habituation and in the last steps of dehabituation are common (i.e., reversion of cellulose content), some other changes affect habituated and dehabituated cells differently, and that these changes involve mainly pectin composition and organization. A more detailed analysis of cell walls focusing on the first steps of habituation will serve to identify which specific modifications are responsible for the differences observed in the pectic component and, consequently, responsible for the fine modulation of cell wall architecture.     

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 de ficiency 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 cellulosede ficient 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-de ficient cell walls showed a fivefold increase in Klason-type lignin.Thioacidolysis/GC-MS analysis of cellulose-de ficient 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 shortterm 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.     

The distribution of cell wall polymers during antheridium development and spermatogenesis in the Charophycean green alga, Chara corallina

Background and Aims

The production of multicellular gametangia in green plants represents an early evolutionary development that is found today in all land plants and advanced clades of the Charophycean green algae. The processing of cell walls is an integral part of this morphogenesis yet very little is known about cell wall dynamics in early-divergent green plants such as the Charophycean green algae. This study represents a comprehensive analysis of antheridium development and spermatogenesis in the green alga, Chara corallina.

Methods

Microarrays of cell wall components and immunocytochemical methods were employed in order to analyse cell wall macromolecules during antheridium development.

Key Results

Cellulose and pectic homogalacturonan epitopes were detected throughout all cell types of the developing antheridium including the unique cell wall protuberances of the shield cells and the cell walls of sperm cell initials. Arabinogalactan protein epitopes were distributed only in the epidermal shield cell layers and anti-xyloglucan antibody binding was only observed in the capitulum region that initially yields the sperm filaments. During the terminal stage of sperm development, no cell wall polymers recognized by the probes employed were found on the scale-covered sperm cells.

Conclusions

Antheridium development in C. corallina is a rapid event that includes the production of cell walls that contain polymers similar to those found in land plants. While pectic and cellulosic epitopes are ubiquitous in the antheridium, the distribution of arabinogalactan protein and xyloglucan epitopes is restricted to specific zones. Spermatogenesis also includes a major switch in the production of extracellular matrix macromolecules from cell walls to scales, the latter being a primitive extracellular matrix characteristic of green plants.     

Immunolocalization of beta-D-glucans,pectins, and arabinogalactan-proteins during intrusive growth and elongation of nonarticulated laticifers in Asclepias speciosa Torr

Nonarticulated laticifers are latex-containing cells that elongate indefinitely and grow intrusively between the walls of meristematic cells. To identify biochemical mechanisms involved in the growth of nonarticulated laticifers, we have analyzed the distribution of various polysaccharides and proteoglycans in walls of meristematic cells in contact with laticifers, nonadjacent to laticifers, and in laticifer walls. In the shoot apex of Asclepias speciosa, the levels of callose and a (1-->4)-beta-galactan epitope are lower in meristematic walls in contact with laticifers than in nonadjacent walls. In contrast, we did not detect a decline in xyloglucan, homogalacturonan, and arabinogalactan-protein epitopes upon contact of meristematic cells with laticifers. Laticifer elongation is also associated with the development of a homogalacturonan-rich middle lamella between laticifers and their neighboring cells. Furthermore, laticifers lay down walls that differ from those of their surrounding cells. This is particularly evident for epitopes in rhamnogalacturonan I. A (1-->5)-alpha-arabinan epitope in this pectin is more abundant in laticifers than meristematic cells, while the opposite is observed for a (1-->4)-beta-galactan epitope. Also, different cell wall components exhibit distinct distribution patterns within laticifer walls. The (1-->5)-alpha-arabinan epitope is distributed throughout the laticifer walls while certain homogalacturonan and arabinogalactan-protein epitopes are preferentially located in particular regions of laticifer walls. Taken together, our results indicate that laticifer penetration causes changes in the walls of meristematic cells and that there are differences in wall composition within laticifer walls and between laticifers and their surrounding cells.     

Temporal and spatial regulation of pectic (1-->4)-beta-D-galactan in cell walls of developing pea cotyledons: implications for mechanical properties

Modifications in cell wall pectic polysaccharides are thought to influence cell-cell adhesion and the mechanical properties of plant tissues. Monoclonal antibodies to epitopes occurring in homo- galacturonan and side chains of rhamnogalacturonan I have been used in an immunolocalization study of cell wall architecture of developing pea cotyledons. Pectic (1-->4)-beta-D-galactan appears in cotyledon cell walls at a defined stage late in development (approximately 26-30 days after anthesis), whereas homogalacturonan and pectic (1-->5)-alpha-L-arabinan are present in cotyledon cell walls throughout development. (1-->4)-beta-galactan was restricted to a distinct thin layer at the plasma membrane face of the cell wall. Anion exchange and immunoaffinity chromatography indicated that the (1-->4)-beta-galactan was associated with acidic pectic components. Mechanical compressive testing of pea cotyledons, before and after (1-->4)-beta-galactan appearance, indicated that the cotyledons with the galactan-rich cell wall layer were twice as firm as those with no detectable (1-->4)-beta-galactan.