The polysaccharide structure of potato cell walls: Chemical fractionation

Cell walls of potato tubers were fractionated by successive extraction with various reagents. A slightly degraded pectic fraction with 77% galacturonic acid was extracted in hot, oxalate-citrate buffer at pH 4. A further, major pectic fraction with 38% galacturonic acid was extracted in cold 0.1 M Na₂CO₃ with little apparent degradation. These two pectic fractions together made up 52% of the cell wall. Most of the oxalate-citrate fraction could alternatively be extracted with cold acetate-N,N,N-tetracetic acid (CDTA) buffer, a non-degradative extractant which nevertheless removed essentially all the calcium ions. This fraction was therefore probably held only by calcium binding, and the remainder of the pectins by covalent bonds. Electrophoresis showed that both pectic fractions contained a range of molecular types differing in composition, with a high arabinose: galactose ratio as well as much galacturonic acid in the most extractable fractions. From methylation data, the main side-chains were 1,4-linked galactans and 1,5-linked arabinans, with smaller quantities of covalently attached xyloglucan. Extraction with NaOH-borate removed a small hemicellulose fraction and some cellulose. The main hemicelluloses were apparently a galactoxyloglucan, a mannan or glucomannan and an arabinogalactan.Abbreviations GLC gas-liquid chromatography - MS mass spectrometry - V₀ void volume - MW weight-average molecular weight - DMSO dimethylsulphoxide - EDTA ethylenediamine tetraacetic acid - TFA trifluoroacetic acid - CDTA N,N,N-tetraacetic acid     

Intracellular feruloylation of pectic polysaccharides

The pectic polysaccharides of spinach cell walls carry feruloyl groups on arabinose and galactose residues. The following experiments were designed to discover whether the arabinose residues are feruloylated intra-or extracellularly. Cultured spinach cells started to incorporate exogenous [³H]arabinose into polymers at a linear rate after a lag period of approx. 3–4 min, although radioactive polysaccharides and extensin did not start to appear outside the plasmalemma until after an approx. 25-min lag. In the same cells, polysaccharide-bound feruloyl-[³H]arabinose units starded to accumulate radioactivity at a linear rate after a lag period of approx. 4–5 min. Therefore, arabinose residues of polysaccharides began to be feruloylated while still intracellular. The rate of formation of polysaccharide-bound feruloyl-[³H]arabinose units did not appreciably increase after 25 min, showing that any additional extracellular feruloylation of the polysaccharide was relatively slow. This conclusion was supported by two different types of pulse-chase experiments, one of which was designed to detect feruloylation of polysaccharides up to 6 d after synthesis.Abbreviations Ara₂ 3-O–-L-arabinopyranosyl-L-arabinose - BAW butan-1-ol/acetic acid/water (12:3:5, by vol.) - BEW butan-1-ol/ethanol/water (20:5:11, by vol.) - EPW ethyl acetate/pyridine/water (8:2:1, by vol.) - Fer-Ara₂ 3-O–(3-O–feruloyl--L-arabinopyranosyl)-L-arabinose - Fer-Gal₂ 4-O–(6-O–feruloyl--D-galactopyranosyl)-D-galactose     

Pectins as mediators of wall porosity in soybean cells

The non-invasive technique of fluorescence redistribution after photobleaching was employed on soybean (Glycine max (L.) Merr.) root cells grown in suspension culture to examine macromolecular transport across plant cell walls. Using both fluorescently derivatized dextrans and proteins of graded size, a functional range of diameters for putative trans-wall channels was determined to be 6.6–8.6 nm. A mild treatment with pectinase apparently enlarged the channels, without adversely affecting cell viability, enabling significantly larger molecules to pass through the wall. Treatment of the cells with cellulysin or protease did not have this enlargement effect. It appears that the organization of pectic substances is a major control element in defining the sieving properties of the wall.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Fl-dextran fluorescein-derivatized dextran - FRAP fluorescence redistribution after photobleaching - kDa kilodalton     

Structural analysis of the cell walls regenerated by carrot protoplasts

A procedure was developed to isolate protoplasts rapidly from carrot (Daucus carota L. cv. Danvers) cells in liquid culture. High purity of cell-wall-degrading enzymes and ease of isolation each contributed to maintenance of viability and initiation of regeneration of the cell wall by a great majority of the protoplasts. We used this system to re-evaluate the chemical structure and physical properties of the incipient cell wall. Contrary to other reports, callose, a (1 3)-d-glucan whose synthesis is associated with wounding, was not a component of the incipient wall of carrot protoplasts. Intentional wounding by rapid shaking or treatment with dimethyl sulfoxide initiated synthesis of callose, detected both by Aniline blue and Cellufluor fluorescence of dying cells and by an increase in (1 3)-linked glucan quantified in methylation analyses. Linkage analyses by gas-liquid chromatography of partially methylated alditol-acetate derivatives of polysaccharides of the incipient wall of protoplasts and various fractions of the cell walls of parent cells showed that protoplasts quickly initiated synthesis of the same pectic and hemicellulosic polymers as normal cells, but acid-resistant cellulose was formed slowly. Complete formation of the wall required 3 d in culture, and at least 5 d were required before the wall could withstand turgor. Pectic substances synthesized by protoplasts were less anionic than those of parent cells, and became more highly charged during wall regeneration. We propose that de-esterification of the carboxyl groups of pectin uronic-acid units permits formation of a gel that envelops the protoplast, and the rigid cellulose-hemicellulose frame-work forms along with this gel matrix.Abbreviations DEAE Diethylaminoethyl - DMSO dimethyl sulfoxide - ECP extracellular polymers - EDTA ethylenediaminetetraacetic acid - HGA nomogalacturonan - RG rhamnogalacturonan - Tes N-tris(hydroxymethyl)methyl-2-amino-ethanesufonic acid - TFA trifluoroacetic acid Journal paper No. 11,776 of the Purdue University Agriculture Experiment Station     

Distribution of pectic polysaccharides throughout walls of suspension-cultured carrot cells

Summary A monoclonal antibody (2 F 4) recognizing a conformational epitope of polygalacturonic acid was used for immunogold localization of pectins in walls of suspension-cultured carrot (D. carota L.) cells at the electron microscopic level. In microcolonies of young or mature cells, polygalacturonic acid was essentially located on the middle lamella material expanded at three-way junctions between cells or lining intercellular spaces but was not found in primary walls. Middle lamellae far from junction zones and intercellular spaces were not recognized. Largely esterified pectic polymers, only detected by the 2 F 4 antibodies after on-grid de-esterification treatment by pectin methyl esterases, were present within all primary cell walls. Golgi bodies and associated vesicles were also labeled by the 2 F 4 antibodies only after de-esterification treatment, which indicates that pectic polymers are synthesized and secreted in a highly esterified form. A decrease of pectin esterification, which results probably from an in situ enzymatic de-esterification of the pectic polymers of the primary walls, was observed in senescent cells. These results are discussed in relation to biochemical analyses showing changes of the methyl ester content of pectins during the cell-wall growth.     

Effects of boron deficiency in cell suspension cultures of Populus alba L.

Cell suspension cultures of Populus alba L. (original cells) require at least 10 M boron for appropriate growth. Using original cells we established a cell line, T-5B, which can grow in a medium containing low levels of boron (5 M). The level of boron localized in the cell walls of T-5B cells was one-half that found in the cell walls of original cells maintained in medium containing 100 M boron, and the level of the rhamnogalacturonan II dimer, cross-linked by a borate ester, also decreased in the former. The sugar composition of whole cell walls of the T-5B cell line was similar that of the original cells, however pectic polysaccharides composed of arabinose or galacturonic acid were easily extracted from T-5B cell walls with 50 mM trans-1,2-cyclohexanediamine-N,N,N,N-tetraacetic acid. Our results suggest that boron deficiency causes a weakening of the interaction among pectic polysaccharides due to a decrease in boron-rhamnogalacturonanII cross-linkage.     

Characterization of long-term extension of isolated cell walls from growing cucumber hypocotyls

Walls from frozen-thawed cucumber (Cucumis sativus L.) hypocotyls extend for many hours when placed in tension under acidic conditions. This study examined whether such creep is a purely physical process dependent on wall viscoelasticity alone or whether enzymatic activities are needed to maintain wall extension. Chemical denaturants inhibited wall creep, some acting reversibly and others irreversibly. Brief (15 s) boiling in water irreversibly inhibited creep, as did pre-incubation with proteases. Creep exhibited a high Q₁₀ (3.8) between 20° and 30°C, with slow inactivation at higher temperatures, whereas the viscous flow of pectin solutions exhibited a much lower Q₁₀ (1.35). On the basis of its temperature sensitivity, involvement of pectic gel-sol transitions was judged to be of little importance in creep. Pre-incubation of walls in neutral pH irreversibly inactivated their ability to creep, with a half-time of about 40 min. At 1 mM, Cu²⁺, Hg²⁺ and Al³⁺ were strongly inhibitory whereas most other cations, including Ca²⁺, had little effect. Sulfhydryl-reducing agents strongly stimulated creep, apparently by stabilizing wall enzyme(s). The physical effects of these treatments on polymer interactions were examined by Instron and stress-relaxation analyses. Some treatments, such as pH and Cu²⁺, had significant effects on wall viscoelasticity, but others had little or no apparent effect, thus implicating an enzymatic creep mechanism. The results indicate that creep depends on relatively rugged enzymes that are firmly attached to or entangled in the wall. The sensitivity of creep to SH-reducing agents indicates that thiol reduction of wall enzymes might provide a control mechanism for endogenous cell growth.Abbreviations DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetraacetic acid - Hepes N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid     

Cell walls as reservoirs of potassium ions for reversible volume changes of pulvinar motor cells during rhythmic leaf movements

The laminar pulvinus of primary leaves of Phaseolus coccineus L. was investigated with respect to the total K⁺ content, the apoplastic K⁺ content, and the water potential of extensor and flexor sections in relation to the leaf positions in a circadian leaf-movement cycle, as well as the cation-exchange properties of isolated extensor- and flexor-cell walls. Turgid tissue showed a high total but low apoplastic K⁺ content, shrunken tissue a low total but high apoplastic K⁺ content. Thus, part of the K⁺ transported into and out of the swelling or shrinking protoplasts is shuttled between the protoplasts and the surrounding walls, another part between different regions of the pulvinus. The K⁺ fraction shuttled between protoplasts and walls was found to be 30–40% of the total transported K⁺ fraction. Furthermore, 15–20% of the total K⁺ content of the tissue is located in the apoplast when the apoplastic reservoir is filled, 5–10% when the apoplastic reservoir is depleted. The ion-exchange properties of walls of extensor and flexor cells appear identical in situ and in isolated preparations. The walls behave as cation exchangers of hhe weak-acid type with a strong dependence of the activity of fixed negative charges as well as of the K⁺-storing capacity on pH and [K⁺] of the equilibration solution. The high apoplastic K⁺ contents of freshly cut tissues reflect the cation-storing capacity of the isolated walls. We suggest that K⁺ ions of the Donnan free space are used for the reversible volume changes (mediating the leaf movement) mainly by an electrogenic proton pump which changes the pH and-or the [K⁺] in the water free space of the apoplast.Abbreviations and symbols DFS Donnan free space - DW dry weight - pK negative logarithm of the equilibrium constant K of the acidic group - WFS water free space - water potential; Indices - cw cell wall - t tissue     

Effect of auxin and abscisic acid on cell wall extensibility in maize coleoptiles

Plastic and elastic in-vitro extensibilities (E _pland E _el ) of cell walls from growing maize (Zea mays L.) coleoptile segments were measured by stretching frozen-thawed tissue, pre-extended to its in-vivo length, at constant force (creep test) in a custom-buildt extensiometer, equipped with a linear-displacement transducer. The indole-3-acetic acid (IAA)-induced change of E _pl (E _pl ) is strictly correlated with the growth rate for a period of 3–4 h. Subsequently, E _plremains constant while the growth rate is slowing down. Since this discrepancy can be accounted for by a growth-dependent reduction of osmotic pressure, it is concluded that E _plrepresents quantitatively the relative increase of in-vivo extensibility (cell wall loosening) involved in IAA-mediated cell growth over a much longer time. On the other side it is argued that the growth rate may not be strictly correlated with wall extensibility during long-term growth. Abscisic acid (ABA) inhibits segment growth induced by auxin, fusicoccin, or exogenous acid, and this effect can be quantitatively attributed to an ABA-mediated reduction of cell wall extensibility as determined by the E _plmeasurement. Both, IAA and ABA have no effect on total protein synthesis, RNA synthesis, and amount of osmotic solutes. Fusicoccin-induced proton excretion is only slightly inhibited by ABA. In contrast to ABA, growth inhibition by cycloheximide (CHI) is always much larger than the concomitant reduction of E _pl , indicating that a further growth parameter is also involved in the inhibition of cell growth by CHI. E _el is not affected by either IAA, ABA, or CHI. It is concluded that E _pl as determined by the applied method, represents a relative measure of the actual in-vivo extensibility of the growing cell wall at the very moment when the tissue is killed, rather than an average extensibility accumulated over some immediate-past period of time as suggested by Cleland (1984, Planta 160, 514–520). Hence, we further draw the conclusion that IAA and ABA control of cell growth can entirely be attributed to a modulation of cell wall extensibility by these hormones in maize coleoptiles.Abbreviations ABA ±abscisic acid - CHI cycloheximide - E _el , E_pl elastic and plastic in vitro extensibilities, respectively (E _el+E_pl=E_tot>) - FC fusicoccin - IAA indole-3-acetic acid     

Cell-wall tension of the inner tissues of the maize coleoptile and its potential contribution to auxin-mediated organ growth

Plant organs such as maize (Zea mays L.) coleoptiles are characterized by longitudinal tissue tension, i.e. bulk turgor pressure produces unequal amounts of cell-wall tension in the epidermis (essentially the outer epidermal wall) and in the inner tissues. The fractional amount of turgor borne by the epidermal wall of turgid maize coleoptile segments was indirectly estimated by determining the water potential ^* of an external medium which is needed to replace quantitatively the compressive force of the epidermal wall on the inner tissues. The fractional amount of turgor borne by the walls of the inner tissues was estimated from the difference between -^* and the osmotic pressure of the cell sap (_i) which was assumed to represent the turgor of the fully turgid tissue. In segments incubated in water for 1 h, -^* was 6.1–6.5 bar at a _i of 6.7 bar. Both -^* and _i decreased during auxin-induced growth because of water uptake, but did not deviate significantly from each other. It is concluded that the turgor fraction utilized for the elastic extension of the inner tissue walls is less than 1 bar, i.e. less than 15% of bulk turgor, and that more than 85% of bulk turgor is utilized for counteracting the high compressive force of the outer epidermal wall which, in this way, is enabled to mechanically control elongation growth of the organ. This situation is maintained during auxin-induced growth.Abbreviations and Symbols _i osmotic pressure of the tissue - ₀ external water potential - ^* water potential at which segment length does not change - IAA indole-3-acetic acid - ITW longitudinal inner tissue walls - OEW outer epidermal wall - P turgor Supported by Deutsche Forschungsgemeinschaft (SFB 206).     

Composition of the walls of pollen grains of the seagrass Amphibolis antarctica

The composition of walls isolated from pollen grains of the seagrass Amphibolis antarctica was determined. Glucose, galactose, and rhamnose were the major neutral monosaccharides in the wall polysaccharides, and fucose, arabinose, xylose, and mannose were present in minor proportions. No apiose, a monosaccharide present in the wall polysaccharides of the vegetative parts of the seagrass Heterozostera tasmanica, was found. Large amounts of uronic acid (mainly as galacturonic acid) were found in the walls. The monosaccharides were probably present in cellulose and pectic polysaccharides, the latter comprising neutral pectic galactans, and rhamnogalacturonans containing high proportions of rhamnose. The walls contained a small amount of protein; glycine and lysine were the amino acids present in the highest proportions. Histochemical examination of isolated walls confirmed the presence of polyanionic components (pectic polysaccharides), -glucans (cellulose), and protein. The composition of the walls is discussed in relation to analyses of the walls of pollen grains and vegetative organs of other plants.     

A monoclonal antibody to feruloylated-(1→4)-β-<Emphasis Type="SmallCaps">d</Emphasis>-galactan

We report the isolation and characterization of a monoclonal antibody, designated LM9, against feruloylated-(14)--d-galactan. This epitope is a structural feature of cell wall pectic polysaccharides of plants belonging to the family Amaranthaceae (including the Chenopodiaceae). Immuno-assays and immunofluorescence microscopy indicated that LM9 binding is specific to samples and cell walls obtained from species belonging to this family. In a series of competitive-inhibition enzyme-linked immunosorbent assays with potential oligosaccharide haptens, the most effective inhibitor was O-[6-O-(trans-feruloyl)--d-galactopyranosyl]-(14)-d-galactopyranose (Gal₂F). LM9 is therefore a useful antibody probe for the analysis of phenolic substitution of cell wall pectic polymers and of cell wall structure in the Amaranthaceae including sugar beet (Beta vulgaris L.) and spinach (Spinacia oleracea L.).Abbreviations DA Degree of acetylation - DM Degree of methyl esterification - ELISA Enzyme-linked immunosorbent assay - IDA Immunodot assay     

Distribution of pectins in cell walls of maize coleoptiles and evidence against their involvement in auxin-induced extension growth

Summary Aiming to elucidate the possible involvement of pectins in auxin-mediated elongation growth the distribution of pectins in cell walls of maize coleoptiles was investigated. Antibodies against defined epitopes of pectin were used: JIM 5 recognizing pectin with a low degree of esterification, JIM 7 recognizing highly esterified pectin and 2F4 recognizing a pectin epitope induced by Ca²⁺. JIM 5 weakly labeled the outer third of the outer epidermal wall and the center of filled cell corners in the parenchyma. A similar labeling pattern was obtained with 2F4. In contrast, JIM 7 densely labeled the whole outer epidermal wall except the innermost layer, the middle lamellae, and the inner edges of open cell corners in the parenchyma. Enzymatic de-esterification with pectin methylesterase increased the labeling by JIM 5 and 2F4 substantially. A further increase of the labeling density by JIM 5 and 2F4 and an extension of the labeling over the whole outer epidermal wall could be observed after chemical de-esterification with alkali. This indicates that both methyl- and other esters exist in maize outer epidermal walls. Thus, in the growth-controlling outer epidermal wall a clear zonation of pectin fractions was observed: the outermost layer (about one third to one half of wall thickness) contains unesterified pectin epitopes, presumably cross-linked by Ca²⁺ extract. Tracer experiments with³H-myo-inositol showed rapid accumulation of tracer in all extractable pectin fractions and in a fraction tightly bound to the cell wall. A stimulatory effect of IAA on tracer incorporation could not be detected in any fraction. Summarizing the data a model of the pectin distribution in the cell walls of maize coleoptiles was developed and its implications for the mechanism of auxin-induced wall loosening are discussed.Abbreviations CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid - CWP cell-wall pellet - IAA indole-3-acetic acid - LSE low-salt extract - TCA trichloroacetic acid; Tris tris-(hydroxy-methyl)aminoethane     

An oat coleoptile wall protein that induces wall extension in vitro and that is antigenically related to a similar protein from cucumber hypocotyls

Plant cell walls expand considerably during cell enlargement, but the biochemical reactions leading to wall expansion are unknown. McQueen-Mason et al. (1992, Plant Cell 4, 1425) recently identified two proteins from cucumber (Cucumis sativus L.) that induced extension in walls isolated from dicotyledons, but were relatively ineffective on grass coleoptile walls. Here we report the identification and partial characterization of an oat (Avena sativa L.) coleoptile wall protein with similar properties. The oat protein has an apparent molecular mass of 29 kDa as revealed by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis. Activity was optimal between pH 4.5 and 5.0, which makes it a suitable candidate for acid growth responses of plant cell walls. The oat protein induced extension in walls from oat coleoptiles, cucumber hypocotyls and pea (Pisum sativum L.) epicotyls and was specifically recognized by an antibody raised against the 29-kDa wall-extension-inducing protein from cucumber hypocotyls. Contrary to the situation in cucumber walls, the acid-extension response in heat-inactivated oat walls was only partially restored by oat or cucumber wall-extension proteins. Our results show that an antigenically conserved protein in the walls of cucumber and oat seedlings is able to mediate a form of acid-induced wall extension. This implies that dicotyledons and grasses share a common biochemical mechanism for at least part of acid-induced wall extensions, despite the significant differences in wall composition between these two classes of plants.Abbreviations ConA concanavalin A - CM carboxymethyl - DEAE diethylaminoethyl - DTT dithiothreitol - Ex29 29-kDa expansin     

Molecular weight distribution of cellulose in primary cell walls

The distribution pattern of the degree of polymerization (DP) of cellulose present in the cell walls of mesophyll- and suspension-cultured cells of tobacco was compared to that of newly synthesized ¹⁴C-labeled cellulose from regenerating tobacco protoplasts and suspension-cultured cells. The cellulose was nitrated, and, after fractionation according to differences in solubility in acetone/water, the DP pattern of labeled or unlabeled cellulose nitrate was determined by viscosity measurements. A low (DP<500) and high DP-fraction (DP>2500) of cellulose were predominant in the cell walls of protoplasts, suspension — cultured cells, and mesophyll cells. The average DP of the high molecular weight fraction of cellulose in the cell walls of mesophyll was higher (DP4,000) than in protoplasts or suspension — cultured cells (DP 2,500-3,000). In all cell walls tested, minor amounts of cellulose molecules with a broad spectrum of a medium DP were present. Pulse — chase experiments with either protoplasts or suspension —cultured cells showed that a large proportion of the low and medium DP-cellulose are a separate class of structural components of the cellulose network. The results are discussed in relation to the organization of cellulose in the primary cell wall.Abbreviations DP degree of polymerisation - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid     

Cell-wall-bound lytic activity in Chlorella fusca: function and characterization of an endo-mannanase

A cell-wall-degrading activity was solubilized from young cells and from mother cell walls of Chlorella fusca by treatment with LiCl. The cytoplasmic enzyme hexokinase was not detectable in these extracts. The LiCl-solubilized activity increased in the cell cycle parallel to the release of autospores. The enzyme was purified on a chromatofocusing column followed by gel filtration. Sodium dodecyl sulfate/polyacryl amide gel electrophoresis of the purified enzyme revealed a molecular weight of 44 kDa, whereas gel filtration indicated a molecular weight of 25 kDa. Cell-wall-lytic activity and -1,4-mannanase activity coeluted in gel filtration and were separated from -d-fucosidase activity. The enzyme degraded isolated cell walls and ivory nut mannan primarily to oligosaccharides with an estimated degree of polymerization 6. The soluble degradation products of the cell wall consisted of 92–96% mannose and 4–8% glucose. It is concluded that the cell-wall-lytic activity is caused by an endo-mannanase. In vivo, this enzyme probably degrades the mother cell wall and, after autospore release, remains bound to it as well as to the surface of the daughter cells by ionic forces. The identity of this bound enzyme with a soluble wall-degrading enzyme previously obtained from mother cells is discussed.     

Acetyl- and methyl-esterification of pectins of friable and compact sugar-beet calli: consequences for intercellular adhesion

Monoclonal antibodies (2F4), specific for a conformational epitope of homopolygalacturonic acid induced by calcium ions, were used to compare the nature and the distribution of the pectic polysaccharides in cell walls of compact and friable sugar-beet (Beta vulgaris L. var. altissima) calli, at the electron-microscope level. Labelings performed before or after de-esterification pretreatments of callus sections enabled three major types of pectic polysaccharides to be distinguished within compact calli: (i) acidic pectins, probably with few acetyl ester groups, detected without any de-esterification treatment in expanded areas of cell separation but never on middle lamellae between tightly associated cells; (ii) highly methyl-esterified pectins with an expected low acetyl ester content, recognized by the 2F4 antibodies after pectin methylesterase de-esterification, and mostly located on intercellular junctions and on middle lamellae in the central zones of the calli; (iii) highly methyl-esterified and largely acetylated pectins, only localized after alkaline de-esterification, in all primary walls of the compact calli. By contrast, all pectins of friable calli were highly methyland acetyl-esterified. This was consistent with an average degree of methyl-esterification of about 60% measured in both calli, and a higher average degree of acetylation for the friable callus line (85%) compared to the compact one (60%). Accordingly, the pectic fraction (acid-soluble) predominant in both calli was acetyl-esterified to 85% in friable callus and to 22% in compact callus cell walls. Friability of sugar-beet callus is thus correlated with an increase in acetylation of its pectin. Labelings of the Golgi apparatus indicate that the pectic polymers of both callus types are synthesized in dictyosomes in a highly methyl-esterified form and are probably subsequently acetyl-esterified.Abbreviations AIR alcohol-insoluble residue - DA degree of acetylation - DM degree of methyl-esterification - MAbs monoclonal antibodies - PME pectin methylesterase Many thanks are due to Mrs. Ch. Devignon (Unité interfacultaire de microscopie électronique, FUNDP, Namur, Belgium) for her technical assistance. F.L. gratefully acknowledges Dr. J.-F. Thibault (Laboratoire de Biochimie et Technologie des glucides, INRA, Nantes, France) for allowing her to stay in his laboratory and Dr. C. Renard for her help with biochemical analyses and her comments on the results. Appreciation is also expressed to P. Vandersmissen (Unité Cell, I.C.P., Bruxelles, Belgium) and to P. Cambier and C. Vinals (FUNDP) for their contribution. The work reported here was supported in part by grants from IRSIA and CGRI, Belgium, to F.L.     

Tissue-related changes in methyl-esterification of pectic polysaccharides in cauliflower (Brassica oleracea L. var. botrytis) stems

Pectic substances are a major component of cell walls in vegetable plants and have an important influence on plant food texture. Cauliflower (Brassica oleracea L. var. botrytis) stem sections at different regions of the mature plant stem have been monitored for tissue-related changes in the native pectic polysaccharides. Chemical analysis detected appreciable differences in the degree of methyl-esterification (ME) of pectic polysaccharides. About 65% of galacturonic acid (GalpA) residues were methyl-esterified in floret tissues. Relative ME showed a basipetal decrease, from 94% in the upper stem to 51% in the lower-stem vascular tissues. The decrease was not related to a basipetal increase in glucuronic acid (GlcpA) residues. The monoclonal antibodies, JIM 5 and JIM 7, produced distinct labelling patterns for the relatively low-methyl-esterified and high-methyl-esterified pectin epitopes, respectively. Labelling was related to cell type and tissue location in the stem. Floret cell walls contained epitopes for both JIM 5 and JIM 7 throughout the wall. Stem vascular tissues labelled more strongly with JIM 5. Whereas pith parenchyma in the upper stem labelled more strongly with JIM 7, in the lower-stem pith parenchyma, JIM 5 labelling predominated. Localization of pectic polysaccharide epitopes in cell walls provides an insight into how structural modifications might relate to the textural and nutritional properties of cell walls. Received: 16 August 1997 / Accepted: 20 December 1997     

Expression and localization of polygalacturonase during the outgrowth of lateral roots in Allium porrum L.

The presence of polygalacturonase and its correlation with the formation of lateral roots in leek (Allium porrum L.) seedlings have been investigated. During root growth, a steady increase in polygalacturonase activity was associated with that of the lateral root primordia. Fractionation of root extract by fast protein liquid chromatography resolved at least two polygalacturonase isoforms. One of the isoforms, a 75-kdalton protein, strongly reacted on Western blots probed with a polyclonal antibody raised against tomato polygalacturonase. It also reacted with both polyclonal and monoclonal antisera raised against Fusarium moniliforme polygalacturonase. In situ localization with these three antibodies showed that polygalacturonase was present over the meristems of lateral root primordia. Antibodies against pectins (Knox et al. 1990, Planta 181, 512–521) detected large amounts of pectic material filling the area between the apex of the primordium and the mother root tissues. We suggest that a polygalacturonase plays an important role in leek root morphogenesis, particularly during lateral root outgrowth.Abbreviations FPLC fast protein liquid chromatography - RGU one unit of polygalacturonase activity - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis The Authors are grateful to Dr. Dean Della Penna (Department of Vegetable Crops, University of California, Davis, Calif., USA) for generously providing the polyclonal antibody raised against the tomato polygalacturonase. This research was supported by National Research of Italy, Special project RAISA, Subproject N2, N360.     

Ion exchange properties of plant root cell walls

Acid-base properties and the swelling capacity of wheat, lupin and pea root cell walls were investigated. Roots of seedlings and green plants of different age were analysed by the potentiometric method. The ion exchange capacity (S _i) and the swelling coefficient (K ^cw) of root cell walls were estimated at various pH values (from 2 to 12) and at different ionic strength (between 0.3 and 1000 mM). To analyse the polysigmoid titration curves pH_i = f (S _i), the Gregor's equation was employed. It was shown that the Gregor's model fits well the experimental data. The total number of the cation exchange (S _t ^cat) and the anion exchange (S _t ^an) groups were determined in the root cell walls. The number of the functional group of each type (S ^j) was estimated, and the corresponding values of pK _a ^j were calculated. It was shown that for all types of cation exchangeable groups arranged in the cell wall structure the acid properties are enhanced by the increasing concentration of electrolyte. For each ionogenic group the coefficients of Helfferich's equation [pK _a ^j = f (C ^K+)] were determined. It was found that the swelling of root cell walls changes with pH, C ^K+ and strongly depends on plant species. Within the experimental pH and C ^K+ range the swelling coefficient changes as follows: lupin > pea > wheat. The obtained results show that for the plant species under investigation the differences in the swelling coefficients originate from (a) the differences in the cross-linking degrees of polymeric chains arranged in the cell wall structure, (b) the differences in the number of carboxyl groups and (c) the differences in the total number of functional groups. Based on the estimated swelling coefficients in water it could be inferred that for wheat the cross-linking degree of the polymeric chains in the root cell walls is higher than those for lupin or pea. It has been emphasized that the calculated parameters (S ^j, pK _a ^j, K ^cw), the equation {pK _a ^j = f (C^K+)} and the dependencies {K ^cw = f (C^K+, pH)} allow to estimate quantitatively the changes in the ion exchange capacity of the root cell walls in response to the changes in an ionic composition of an outer solution. The results of these estimations allow to suggest that (a) the root apoplast is a compartment where the accumulation of cations takes place during the first stage of cation uptake from an outer medium, and (b) the accumulation degree is defined by pH and ionic composition of an outer solution. On the basis of the literature review and the results of the present experimental study it was proposed that the changes in the cell wall swelling in response to variances of environmental or experimental conditions could lead to a change of the water flow through a root apoplast. It has been supported that there is direct relationship between the swelling of root cell walls and the water flow within the plant root apoplast.