Cell wall development in maize coleoptiles

The physical bases for enhancement of growth rates induced by auxin involve changes in cell wall structure. Changes in the chemical composition of the primary walls during maize (Zea mays L. cv WF9 × Bear 38) coleoptile development were examined to provide a framework to study the nature of auxin action. This report documents that the primary walls of maize cells vary markedly depending on developmental state; polymers synthesized and deposited in the primary wall during cell division are substantially different from those formed during cell elongation.

The embryonal coleoptile wall is comprised of mostly glucuronoarabinoxylan (GAX), xyloglucan, and polymers enriched in 5-arabinosyl linkages. During development, both GAX and xyloglucan are synthesized, but the 5-arabinosyls are not. Rapid coleoptile elongation is accompanied by synthesis of a mixed-linked glucan that is nearly absent from the embryonal wall. A GAX highly substituted with mostly terminal arabinofuranosyl units is also synthesized during elongation and, based on pulse-chase studies, exhibits turnover possibly to xylans with less substitution via loss of the arabinosyl and glucuronosyl linkages.

     

Tracing cell wall biogenesis in intact cells and plants : selective turnover and alteration of soluble and cell wall polysaccharides in grasses

Cells of proso millet (Panicum miliaceum L. cv Abarr) in liquid culture and leaves of maize seedlings (Zea mays L. cv LH51 × LH1131) readily incorporated d-[U-¹⁴C]glucose and l-[U-¹⁴C]arabinose into soluble and cell wall polymers. Radioactivity from arabinose accumulated selectively in polymers containing arabinose or xylose because a salvage pathway and C-4 epimerase yield both nucleotide-pentoses. On the other hand, radioactivity from glucose was found in all sugars and polymers. Pulse-chase experiments with proso millet cells in liquid culture demonstrated turnover of buffer soluble polymers within minutes and accumulation of radioactive polymers in the cell wall. In leaves of maize seedlings, radioactive polymers accumulated quickly and peaked 30 hours after the pulse then decreased slowly for the remaining time course. During further growth of the seedlings, radioactive polymers became more tenaciously bound in the cell wall. Sugars were constantly recycled from turnover of polysaccharides of the cell wall. Arabinose, hydrolyzed from glucuronoarabinoxylans, and glucose, hydrolyzed from mixed-linkage (1→3, 1→4)β-d-glucans, constituted most of the sugar participating in turnover. Arabinogalactans were a large portion of the buffer soluble (cytoplasmic) polymers of both proso millet cells and maize seedlings, and these polymers also exhibited turnover. Our results indicate that the primary cell wall is not simply a sink for various polysaccharide components, but rather a dynamic compartment exhibiting long-term reorganization by turnover and alteration of specific polymers during development.     

Changing extracellular matrix of cells during development of suspension culture of Triticum timopheevii Zhuk

A study was made of the contents of the main polysaccharide fractions in the cell wall, and extracellular polysaccharides, and of the activity of cell wall enzymes during cultivation of suspension culture of cells of the winter wheat Triticum timopheevii Zhuk. It was shown that within 3 days of cultivation (a phase enriched in dividing cells), on the background of increased callose contents in plant cells, amounts of pectins and hemicelluloses extracted by 4N alkali decreased. The content of polysaccharides reached its initial level by the end of culturing. A parallel analysis of glycosidase activity in cell walls has shown their considerable activation at the stage enriched by dividing cells, which decreased at a transition of culture into the stationary level. The increased activity of hydrolyzing enzymes was combined with an increased efflux of extracellular polysaccharides into culture medium. The detected changes in polysaccharide composition of the cell wall at the first phase indicate its qualitative changes during cell wall reconstruction at the beginning of cytokines, whereas extensive expansion of cell wall was seen on the phase of elongation.     

Changes in cell wall polysaccharides during elongation in a 2, 4-D free medium in a carrot suspension culture

Cell elongation occurred when carrot (Daucus carota L. ev. Kurodagosun) cells subcultured through sieving (Y. Ozeki and A. Komamine, Physiol. Plant. 53: 570-577. 1981) were transferred to a medium lacking auxin, while the cells showed no elongation in a medium containing 2, 4-D. Changes in polysaccharides of the cell walls and in their sugar composition during elongation were investigated. All wall components, EDTA-soluble pectic substance, 5 and 24%, KOH-soluble hemicelluloses and cellulose increased markedly during elongation. The increase of hemicelluloses correlated especially with elongation. In the 5% KOH-soluble hemicellulose, galactose and arabinose contents in the walls increased significantly both in amounts (per fresh weight) and relative contents (% in total neutral sugars) during elongation, while the relative contents of glucose and xylose decreased rapidly in the 5 and 24% KOH-soluble hemicelluloses. The methylation analysis tentatively indicated that larger amounts of galactan and/or arabinogalactan and lower amount of xyloglucan were found as components of the two hemicelluloses of elongating cells than those of non-elongating cells. The amounts of total carbohydrate and of uronic acid of extracellular polysaccharides secreted into the medium increased to a larger extent in the elongation culture than in the non-elongation culture. The contents of galactose and arabinose in extracellular polysaccharides increased rapidly in the elongation culture. The biochemical aspects of cell elongation in the absence of auxin were discussed from the viewpoint of the results obtained here.     

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.     

Tensile strength of cell walls of living cells

A gas decompression technique was used to determine the breaking strength of cell walls of single cells. Breaking strengths of the bacterium Salmonella typhimurium and the unicellular green alga Chlamydomonas eugametos were 100 and 95 atmospheres, respectively, while those of sporophytes of the water mold Blastocladiella emersonii were 65 atmospheres, and those of suspension cultured cells of carrot were only 30 atmospheres. Estimation of wall tensile stress based on breaking pressures, cell radii, and estimation of wall thickness, indicates that microfibrillar walls are not necessarily stronger than walls of primitive organisms. Hence, alternative hypotheses for their evolution must be considered.     

Influence of growth conditions on the composition of cell wall polysaccharides from cultured tobacco cells

The sugar composition of cell wall polysaccharides of two tobacco varieties obtained from mesophyll, regenerating protoplasts and cells grown under various conditions were compared. Regenerating protoplasts developed an unusual cell wall with a low cellulose and a high non-cellulosic glucan content. In the presence of different phytohormones compact and friable calli were obtained with cell walls containing low and high arabinose/xylose ratios. The cell walls of compact calli were comparable to those of genuine mesophyll cells. The sugar constituents of cell walls obtained from cells grown in liquid media were different from those of solid calli. The cell wall composition of suspension cultured cells was hardly affected by various combinations of phytohormones, but was altered by high osmolarity of the medium.     

Cell wall polysaccharides from cell suspension cultures of the Atlantic Forest tree Rudgea jasminoides (Rubiaceae)

Rudgea jasminoides (Rubiaceae) is a tropical tree species native of the Atlantic Forest in the south of Brazil. Previous studies with leaf cell walls of R. jasminoides showed a different proportion of cross-linked glycans compared to what is usually reported for eudicots. However, due to the difficulties of working with whole plant organs, cell suspensions of R. jasminoides, consisting of predominantly undifferentiated cells with mainly primary cell walls, were used to examine cell walls and extracellular soluble polysaccharides (EP) released into the culture medium. Sugar composition and linkage analysis showed homogalacturonans, xylogalacturonans and arabinogalactans to be the predominant EP. In the cell wall, homogalacturonans and arabinogalactans are the major pectins, and xyloglucans and xylans are the major cross-linking glycans. The presence of xylogalacturonans in the R. jasminoides cell cultures seems to be related to the occurrence of a homogeneous cell suspension with loosely attached cells. Although all alkali extractions from the cell walls yielded amounts of xyloglucan that exceed those of the xylans, the latter was found in a proportion that is higher than what has been usually reported for primary cell walls of most eudicots. The xyloglucan from cell walls of cell suspension cultures of R. jasminoides has low fucosylation levels and high proportion of galactosyl residues, a branching pattern commonly found in storage cell-wall xyloglucans.     

Changes in cell wall polysaccharides associated with growth

Changes in the polysaccharide composition of Phaseolus vulgaris, P. aureus, and Zea mays cell walls were studied during the first 28 days of seedling development using a gas chromatographic method for the analysis of neutral sugars. Acid hydrolysis of cell wall material from young tissues liberates rhamnose, fucose, arabinose, xylose, mannose, galactose, and glucose which collectively can account for as much as 70% of the dry weight of the wall. Mature walls in fully expanded tissues of these same plants contain less of these constituents (10%-20% of dry wt). Gross differences are observed between developmental patterns of the cell wall in the various parts of a seedling, such as root, stem, and leaf. The general patterns of wall polysaccharide composition change, however, are similar for analogous organs among the varieties of a species. Small but significant differences in the rates of change in sugar composition were detected between varieties of the same species which exhibited different growth patterns. The cell walls of species which are further removed phylogenetically exhibit even more dissimilar developmental patterns. The results demonstrate the dynamic nature of the cell wall during growth as well as the quantitative and qualitative exactness with which the biosynthesis of plant cell walls is regulated.     

Comparison of cell wall regeneration on maize protoplasts isolated from leaf tissue and suspension cultured cells

Summary The cell wall regeneration on protoplasts derived from maize mesophyll cells was compared with wall regeneration on protoplasts derived from suspension cultured cells using light microscopy, transmission electron microscopy, and mass spectrometry. The time course of cell wall regeneration has shown that the mesophyll protoplasts regenerated walls much slower than the protoplasts derived from cultured cells. Moreover, cell wall materials on the mesophyll protoplasts were often unevenly distributed. Electron microscopy has further demonstrated that the mesophyll protoplasts have less organized and compact walls than the protoplasts from cultured cells. Chemical analysis revealed that the mesophyll protoplasts had a lower ratio ofβ-(1–3)-glucan toβ-(1–4)-glucan than protoplasts from cultured cells. The significance of these results for the viability and development of protoplasts in culture is discussed. National Research Council of Canada paper no. 32458.     

Aromatic aldehyde constituents of graminaceous cell walls

p-Hydroxybenzaldehyde, vanillin and syringaldehyde were released as their sodium salts from graminaceous cell walls by treatment with sodium hydroxide. Treatment of the walls with ‘cellulase’ having both cellulase and hemicellulase activity released the aldehydes in bound form apparently linked at their phenolic groups to the wall polysaccharides. These findings are discussed in relation to tests for lignin using phloroglucinol-HC1 and alkaline nitrobenzene reagents.     

Heterogeneity of silica and glycan-epitope distribution in epidermal idioblast cell walls in Adiantum raddianum laminae

Laminae of Adiantum raddianum Presl., a fern belonging to the family Pteridaceae, are characterised by the presence of epidermal fibre-like cells under the vascular bundles. These cells were thought to contain silica bodies, but their thickened walls leave no space for intracellular silica suggesting it may actually be deposited within their walls. Using advanced electron microscopy in conjunction with energy dispersive X-ray microanalysis we showed the presence of silica in the cell walls of the fibre-like idioblasts. However, it was specifically localised to the outer layers of the periclinal wall facing the leaf surface, with the thick secondary wall being devoid of silica. Immunocytochemical experiments were performed to ascertain the respective localisation of silica deposition and glycan polymers. Epitopes characteristic for pectic homogalacturonan and the hemicelluloses xyloglucan and mannan were detected in most epidermal walls, including the silica-rich cell wall layers. The monoclonal antibody, LM6, raised against pectic arabinan, labelled the silica-rich primary wall of the epidermal fibre-like cells and the guard cell walls, which were also shown to contain silica. We hypothesise that the silicified outer wall layers of the epidermal fibre-like cells support the lamina during cell expansion prior to secondary wall formation. This implies that silicification does not impede cell elongation. Although our results suggest that pectic arabinan may be implicated in silica deposition, further detailed analyses are needed to confirm this. The combinatorial approach presented here, which allows correlative screening and in situ localisation of silicon and cell wall polysaccharide distribution, shows great potential for future studies.     

Acid and alkaline invertases in suspension cultures of sugar beet cells

Alkaline invertase was induced during the initiation of suspension cultures of single cells from leaf explants of sugar beets in Murashige-Skoog liquid medium which contained benzyladenine. This activity was barely detectable in the leaves themselves. In suspension cultures, the presence of both acid and alkaline invertases was detected; alkaline invertase was only present in the cytoplasm of the cultured cells, whereas acid invertase was present in the cytoplasm and cell walls, and was also detected in the culture medium. The cell wall contained at least three types of acid invertase; two of these activities were solubilized by saline (saline-released) and EDTA (EDTA-released), respectively, and the third remained tightly associated with the cell wall. Saline-released and EDTA-released invertases from the cell wall showed the significant differences in their properties: the saline-released enzyme had the highest affinity for sucrose among the invertases tested, and was easily bound to cell walls, to DNA, and to a cation exchanger, unlike the EDTA-released enzyme. Sucrose is the source of carbon for plant cells in suspension culture and is probably degraded in the cell wall by the saline-released invertase, which had the highest activity and the highest affinity for sucrose. Hexose products of this degradation would be transported to cytoplasm. Soluble invertase, EDTA-released invertase from the cell wall, and one of two extracellular invertases behaved similarly upon chromatography on DEAE-cellulose. They had similar activity profiles with changing pH, and similar K_m values for sucrose. Thus it appears that they are identical. Two extracellular invertases found in the growth medium of the suspension cultures were probably identical with those in the soluble fraction of callus and seedlings of sugar beets, because they showed similar behaviors during chromatography on DEAE-cellulose, and had similar activity profiles with changing pH and K_m values for sucrose.     

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.     

Structure of Plant Cell Walls : XIX. Isolation and Characterization of Wall Polysaccharides from Suspension-Cultured Douglas Fir Cells

The partial purification and characterization of cell wall polysaccharides isolated from suspension-cultured Douglas fir (Pseudotsuga menziesii) cells are described. Extraction of isolated cell walls with 1.0 m LiCl solubilized pectic polysaccharides with glycosyl-linkage compositions similar to those of rhamnogalacturonans I and II, pectic polysaccharides isolated from walls of suspension-cultured sycamore cells. Treatment of LiCl-extracted Douglas fir walls with an endo-α-1,4-polygalacturonase released only small, additional amounts of pectic polysaccharide, which had a glycosyl-linkage composition similar to that of rhamnogalacturonan I. Xyloglucan oligosaccharides were released from the endo-α-1,4-polygalacturonase-treated walls by treatment with an endo-β-1,4-glucanase. These oligosaccharides included hepta- and nonasaccharides similar or identical to those released from sycamore cell walls by the same enzyme, and structurally related octa- and decasaccharides similar to those isolated from various angiosperms. Finally, additional xyloglucan and small amounts of xylan were extracted from the endo-β-1,4-glucanase-treated walls by 0.5 n NaOH. The xylan resembled that extracted by NaOH from dicot cell walls in that it contained 2,4- but not 3,4-linked xylosyl residues. In this study, a total of 15% of the cell wall was isolated as pectic material, 10% as xyloglucan, and less than 1% as xylan. The noncellulosic polysaccharides accounted for 26% of the cell walls, cellulose for 23%, protein for 34%, and ash for 5%, for a total of 88% of the cell wall. The cell walls of Douglas fir were more similar to dicot (sycamore) cell walls than to those of graminaceous monocots, because they had a predominance of xyloglucan over xylan as the principle hemicellulose and because they possessed relatively large amounts of rhamnogalacturonan-like pectic polysaccharides.     

Kinetin induced changes in cell wall composition of tobacco callus

Culture of tobacco callus on high or low kinetin in light or darkness leads to changed tissue texture and associated changes in cell wall composition. In particular, friable callus (low kinetin, darkness) cell walls have a greater extensin content and an altered composition of arabinose and xylose containing hemicelluloses compared with cell walls of compact callus (high kinetin, darkness). The possible importance of these differences in determining callus friability is discussed.     

Effect of osmotic stress on the growth of epicotyls of Cicer arietinum in relation to changes in cell wall composition

The inhibition of growth by polyethlene glycol (PEG)-induced osmotic stress led to modifications in the changes taking place in cell wall composition during normal growth of epicotyls of Cicer arietinum L. cv. Castellana. Epicotyls growing under normal conditions showed a decrease in the amount of pectic fractions and an increase in the hemicellulosic fractions and α-cellulose that led to an increase in the rigidity and a loss in growth capacity. Among the hemicellulosic fractions, the KI-2 fraction (insoluble fraction of 10% KOH-extracted hemicelluloses) seemed to be the only one related to the elongation process and subsequent rigidity. During normal growth a decrease was observed in the total amount of galactose, mainly from the pectic fractions. The inhibition of elongation led to an increase in the amount of the cell walls, due to inhibition of cellular elongation. PEG prevented the increase in the hemicelluloses and the α-cellulose, indicating that these changes were related to elongation. Thus, during the inhibition of elongation there is probably an inhibition of new synthesis that prevents cell wall rigidity and maintains cell wall growth capacity. Changes in the pectic fractions during growth were not affected by the inhibition of elongation, showing that these fractions are related to cell wall loosening rather than to elongation. Study of the cell wall composition confirms the idea that the autolytic process is regulated by changes in the cell wall structure during epicotyl growth     

Breaking strengths of pollen grain walls

50% breaking point pressures of pollen grain walls of eleven species were determined. The breaking point pressures of most pollen grain walls are equivalent to those reported in the literature for other types of living cell walls such as bacterial spore coats, algal cell walls, mold sporophyte cells, and dicot suspension culture cells. The strongest pollen grain walls are two or three orders of magnitude stronger, however. Pollen grain walls are proportionately very thick in comparison to other types of cell walls. It is this thickness, not the construction or physical properties of the pollen grain wall, that most probably accounts for their strength.     

Illuminating the wall: Using click chemistry to image pectins in Arabidopsis cell walls

Plant cell walls are the most abundant biomaterials on Earth and serve a multitude of purposes in human society. These complex extracellular matrices are mainly composed of polysaccharides, including cellulose, hemicelluloses, and pectins, which cannot be cytologically examined using conventional techniques. Click chemistry, which exploits a bio-orthogonal cycloaddition reaction between alkynyl and azido groups, has proven to be useful for the metabolic incorporation and detection of modified sugars in polysaccharides in animals, fungi, and bacteria, but its use to interrogate the biosynthesis or dynamics of plant cell walls has not been previously reported. Recently, we found that an alkynylated analog of fucose can be metabolically incorporated into Arabidopsis thaliana cell walls and click labeled with fluorescent probes, facilitating imaging of cell wall carbohydrates. Despite the presence of fucose in several classes of wall polysaccharides, fucose-alkyne was primarily incorporated into rhamnogalacturonan-I, a type of pectin. Using timecourse and pulse-labeling experiments, we observed the dynamics of pectin delivery and reorganization in expanding cell walls. The use of click chemistry to investigate plant cell wall architecture should help bridge the gap between biochemical characterization of isolated cell wall components and an understanding of how those components interact in intact cell walls.     

A survey of the pectic content of nonlignified monocot cell walls

The primary cell walls of graminaceous monocots were known to have a low content of pectin compared to those of dicots, but it was uncertain how widespread this feature was within the monocots as a whole. Nonlignified cell walls were therefore prepared from 33 monocot species for determination of their pectin content. It was not possible to solubilize intact pectins quantitatively from the cell walls, and the pectin content was assessed from three criteria: the total uronic acid content; the content of α-(1,4′)-D-galacturonan isolated by partial hydrolysis and characterized by electrophoresis and degradation by purified polygalacturonase; and the proportion of neutral residues in a representative pectic fraction solubilized by sequential β-elimination and N,N,N′N′-cyclohexanediaminetetraacetic acid extraction. Low galacturonan contents were restricted to species from the Gramineae, Cyperaceae, Juncaceae, and Restionaceae. Other species related to these had intermediate galacturonan contents, and the remainder of the monocots examined had high galacturonan contents comparable with those of dicots. The other criteria of pectin content showed the same pattern.