Pectic epitopes are differentially distributed in the cell walls of potato (Solanum tuberosum) tubers

Six monoclonal antibodies (mAbs) were used to map the distribution of pectic epitopes in the cell walls of potato ( Solanum tuberosum L. cvs Kardal and Karnico) tuber tissue in both light and electron microscopes. Unesterified (mAb JIM 5 epitope) and methyl-esterified (mAb JIM 7 epitope) pectins were abundant and equally distributed in all parenchymal and vascular cell walls. Homogalacturonans (HGAs) involved in Ca²⁺-cross-linking (mAb 2F4 epitope) were localised to the middle lamella and abundant at cell corners. The tuber cortex was densely labelled, but parenchymal cell walls in the perimedullary region contained few epitopes of calcium pectate except at corners and pit fields. In contrast, pectic side-chains were not detectable in the middle lamella of all parenchymal cell walls, except in the cortex where mAb LM6 (arabinan epitope) labelled the entire wall. The galactan epitope (mAb LM5) was localised to a zone very close to the plasmalemma in cortical cell walls and was also less abundant at pit fields and in vascular cell walls. MAb CCRC-M2 (rhamnogalacturonan I epitope) did not cross-react. Our results show that the cell walls of potato tubers are not homogeneous structures and that the pectic composition of the walls is spatially regulated.     

Disorganized distribution of homogalacturonan epitopes in cell walls as one possible mechanism for aluminium-induced root growth inhibition in maize

Background and Aims

Aluminium (Al) toxicity is one of the most severe limitations to crop production in acid soils. Inhibition of root elongation is the primary symptom of Al toxicity. However, the underlying basis of the process is unclear. Considering the multiple physiological and biochemical functions of pectin in plants, possible involvement of homogalacturonan (HG), one of the pectic polysaccharide domains, was examined in connection with root growth inhibition induced by Al.

Methods

An immunolabelling technique with antibodies specific to HG epitopes (JIM5, unesterified residues flanked by methylesterifed residues; JIM7, methyl-esterified residues flanked by unesterified residues) was used to visualize the distribution of different types of HG in cell walls of root apices of two maize cultivars differing in Al resistance.

Key Results

In the absence of Al, the JIM5 epitope was present around the cell wall with higher fluorescence intensity at cell corners lining the intercellular spaces, and the JIM7 epitope was present throughout the cell wall. However, treatment with 50 µm Al for 3 h produced 10 % root growth inhibition in both cultivars and caused the disappearance of fluorescence in the middle lamella of both epitopes. Prolonged Al treatment (24 h) with 50 % root growth inhibition in ‘B73’, an Al-sensitive cultivar, resulted in faint and irregular distribution of both epitopes. In ‘Nongda3138’, an Al-resistant cultivar, the distribution of HG epitopes was also restricted to the lining of intercellular spaces when a 50 % inhibition to root growth was induced by Al (100 µm Al, 9 h). Altered distribution of both epitopes was also observed when of roots were exposed to 50 µm LaCl₃ for 24 h, resulting in 40 % inhibition of root growth.

Conclusions

Changes in HG distribution and root growth inhibition were highly correlated, indicating that Al-induced perturbed distribution of HG epitopes is possibly involved in Al-induced inhibition of root growth in maize.Key words: Al toxicity, cell wall, homogalacturnonan, immunofluorescence, methylesterification, pectin     

Pectic polysaccharides of rice endosperm cell walls

Two pectic polysaccharide fractions were purified from rice endosperm cell walls. Methylation analysis including carboxyl-reduction and also selective     

Celery (Apium graveolens) parenchyma cell walls: cell walls with minimal xyloglucan

The primary walls of celery ( Apium graveolens L.) parenchyma cells were isolated and their polysaccharide components characterized by glycosyl linkage analysis, cross-polarization magic-angle spinning solid-state ¹³C nuclear magnetic resonance (CP/MAS ¹³C NMR) and X-ray diffraction. Glycosyl linkage analysis showed that the cell walls consisted of mainly cellulose (43 mol%) and pectic polysaccharides (51 mol%), comprising rhamnogalacturonan (28 mol%), arabinan (12 mol%) and galactan (11 mol%). The amounts of xyloglucan (2 mol%) and xylan (2 mol%) detected in the cell walls were strikingly low. The small amount of xyloglucan present means that it cannot coat the cellulose microfibrils. Solid-state ¹³C NMR signals were consistent with the constituents identified by glycosyl linkage analysis and allowed the walls to be divided into three domains, based on the rigidity of the polymers. Cellulose (rigid) and rhamnogalacturonan (semi-mobile) polymers responded to the CP/MAS ¹³C NMR pulse sequence and were distinguished by differences in proton spin relaxation time constants. The arabinans, the most mobile polymers, responded to single-pulse excitation (SPE), but not CP/MAS ¹³C NMR. From solid-state ¹³C NMR of the cell walls the diameter of the crystalline cellulose microfibrils was determined to be approximately 3 nm while X-ray diffraction of the cell walls gave a value for the diameter of approximately 2 nm.     

The structure of plant cell walls: v. On the binding of xyloglucan to cellulose fibers

Cell wall strength is decreased by both auxin treatment and low pH. In a recently proposed model of the plant cell wall, xyloglucan polymers are hydrogen-bonded to cellulose fibrils, forming the only noncovalent link in the network of polymers which cross-link the cellulose fibers. The decreased strength of the cell wall seen upon lowering the pH might be due to an effect of hydrogen ions on the rate of xyloglucan creep along cellulose fibers. This paper investigates binding of xyloglucan fragments to cellulose. At equilibrium, the per cent of nine- and seven-sugar xyloglucan fragments which are bound to cellulose is sensitive to both temperature and the concentration of nonaqueous solvents. However, neither the per cent of xyloglucan fragments bound to cellulose at equilibrium, nor the rate at which the xyloglucan fragments bind to cellulose, is sensitive to changes in hydrogen ion concentration. These results support the hypothesis that, within the cell wall, xyloglucan chains are connected to cellulose fibers by hydrogen bonds, but these results suggest that this interconnection between xyloglucan and cellulose is unlikely to be the point within the wall which regulates the rate of cell elongation.     

Boron in plant cell walls

Boron is an essential element for higher plants, yet the primary functions remain unclear. In intact tissues of higher plants, this element occurs as both water soluble and water insoluble forms. In this review, the intracellular localisation of B and possible function of B in cell walls of higher plants are discussed. The majority of the water soluble B seems to be localised in the apoplastic region as boric acid. The water insoluble B is associated with rhamnogalacturonan II (RG-II) and the complex is ubiquitous in higher plants. In the Brassicaceae, Apiaceae, Chenopodiaceae, Asteraceae, Amaryllidaceae, and Liliaceae, nearly all the cell wall B is associated with RG-II, while in the Cucurbitaceae, only half of the cell wall B is in this complex. In duckweed, a different type of B-polysaccharide complex has been identified.Analysis of the structure of the B–RG-II complex reveals that the complex is composed of boric acid and two chains of monomeric RG-II. Boric acid does not merely bind to sugars but crosslinks two chains of pectic polysaccharide at the RG-II region through borate-diester bonding, thus forming a network of pectic polysaccharides in cell walls. The B–RG-II complex is reconstituted in vitro only by mixing monomeric RG-II and boric acid at pH 4.0. In the in vitro reconstitution, germanic acid can substitute for boric acid to some extent. The RG-II epitope, which cross reacts with the antibody toward the B-RG-II complex, is detected in walls of every cell in radish roots. The epitope is also detected in growing pollen tube cell walls, which are claimed to require B.Whilst it is now clear that boric acid links some cell wall components, it is not yet clear whether there is a structural requirement for B in cell wall function.     

Pectic polysaccharide breakdown of cell walls in cucumber roots grown with calcium starvation

Pectic polysaccharides from the roots of cucumber (Cucumis sativus L.) grown in liquid culture medium with or without calcium (1 mm CaCl₂) were studied after extraction successively by hot water and Na hexametaphosphate solution. The Ca²⁺ starvation-treatment caused a striking reduction in content of extracted pectic polysaccharide; from an equivalent weight of cell walls, only 33.1% of the control level was extracted from root cell walls of plants cultured under Ca²⁺ deficiency. The extracted pectic polysaccharides were fractionated into neutral and acidic polymers by a DEAE-Sephadex column. The acidic polymers, which represented more than 76% of the yield, appeared to be a major fraction of extracted pectic polysaccharides. The changes of molecular size and glycosyl residue composition of this fraction were compared for the control and Ca²⁺-deprived samples. The results indicate that Ca²⁺ deficiency caused structural changes which could involve both branching pattern and extent of contiguous galacturonosyl units in the water-solubilized pectic polysaccharides. Ca²⁺ starvation also led to a notable decrease in molecular size of the hexametaphosphate-solubilized polysaccharides and, to a lesser extent, of the water-solubilized fraction as well. In addition, polygalacturonase activity in tissue homogenates increased remarkably with the Ca²⁺ deficiency, whereas β-galactosidase activity did not undergo a change. Thus, it appears that one major effect of Ca²⁺ deprivation was to stimulate polygalacturonase activity, an effect which could be involved in the control of the breakdown of pectic polysaccharides in the cell walls.     

Self-assembly of plant cell walls

The object of this paper is to define criteria for distinguishing between self-assembly and template-based assembly in plant cell walls. The example of cellulose shows that cell wall polymers biosynthesized at a membrane may retain parallel chain packing arrangements that are thermodynamically unstable and cannot be reproduced in vitro, making the experimental testing of the self-assembly hypothesis difficult. Also, natural cellulose is ordered on a number of scales of pattern, each of which may be constructed by either self- or template-based assembly independently of the rest. These conceptual problems apply equally to the self-assembly of complete cell walls and other cell wall polymers. It is suggested that the self-assembly concept should be applied only to one stage or level in the synthesis of a cell wall, and that an additional concept of parallel assembly may be useful for understanding the synthesis of some polysaccharides.     

Microfibril orientation in plant cell walls

Summary The distribution of particles on the surface of the plasmalemma in the collenchyma of Apium graveolens was studied by the freeze-etching technique. The aim was to determine whether the distribution of particles was related to the known longitudinal or transverse orientation of cellulose microfibrils in different layers of the walls of these cells. Preliminary statistical studies have shown no obvious correlation between particle distribution and microfibril orientation although the distribution appeared uniform rather than random. Qualitatively, the particle distribution on the plasmalemma of differentiating xylem fibres of Eucalyptus maculata and of the cortical parenchyma of Avena sativa coleoptiles appeared to be similar to that observed on the plasmalemma of Apium. No correlation between the particle distribution and the microfibril orientation known to exist in the walls of these cells could be discerned.The orientation of microtubules in the cytoplasm of collenchyma cells of Apium graveolens was parallel to the microfibril orientation in many instances, but exceptions were noted. A possible interpretation for this variation is discussed. It is concluded that the microtubules are the structures which are most likely to be involved in determining microfibril orientation in the cell wall.     

Glycoprotein conformation in plant cell walls

The major structural glycoprotein of the cell wall of Chlamydomonas reinhardii has a protein core, at least 50% of which is in the unusual polyproline II conformation. This has been demonstrated by examining the circular dichroism of the cell wall, its constituent glycoproteins, and thermolysin released wall glycopeptides. One of these glycopeptides, T2, has a high hydroxyproline and sugar content, and possesses upward of 85% polyproline II structure. The main extracellular matrix glycoprotein therefore has a rigid, rod-like structure and the significance of this and its relation to higher plant cell wall glycoproteins is discussed. The unusual conformation appears to confer great stability on the glycoprotein as it is unchanged either by certain denaturing agents or during the transition from protomer to assembled cell wall.Abbreviations CD circular dichroism - HP 4-hydroxy-L-proline - PP poly-L-proline - SDS sodium dodecylsulphate This is the eight paper in a series entitled Structure, Composition and Morphogenesis of the Cell Wall of Chlamydomonas reinhardii. The last paper in this series was Catt et al. (1978)     

Separation of vascular cell walls from cortical cell walls of plant roots

Summary The cortex was physically separated from the stele of corn roots. The isolated walls from cortical cells were less dense than the walls isolated from stelar cells. The cell walls from each tissue were centrifuged on a step gradient composed of 50 and 60% sucrose for 5 min at 900 g. After the short centrifugation time, the cortical cell walls banded at the 50/60% interface while the vascular tissue walls pelleted through 60% sucrose. An aliquot of vascular cell walls was then marked with cytochromec. The marked cell walls were mixed with cortical cell walls and centrifuged on a 50/60% sucrose gradient and after 5 min, the vascular tissue walls were cleanly separated from the cortical cell walls. The experiment was repeated without prior separation of tissue types with another corn variety, carrot roots grown in culture, and pea roots. A clean separation of cell wall types was achieved after homogenization of intact roots in liquid nitrogen, extrusion from a nitrogen bomb, and centrifugation in sucrose gradients.     

Immunogold localization of xyloglucan and rhamnogalacturonan I in the cell walls of suspension-cultured sycamore cells

Plant cell walls serve several functions: they impart rigidity to the plant, provide a physical and chemical barrier between the cell and its environment, and regulate the size and shape of each cell. Chemical studies have provided information on the biochemical composition of the plant cell walls as well as detailed knowledge of individual cell wall molecules. In contrast, very little is known about the distribution of specific cell wall components around individual cells and throughout tissues. To address this problem, we have produced polyclonal antibodies against two cell wall matrix components; rhamnogalacturonan I (RG-I), a pectic polysaccharide, and xyloglucan (XG), a hemicellulose. By using the antibiodies as specific markers we have been able to localize these polymers on thin sections of suspension-cultured sycamore cells (Acer pseudoplatanus). Our results reveal that each molecule has a unique distribution. XG is localized throughout the entire wall and middle lamella. RG-I is restricted to the middle lamella and is especially evident in the junctions between cells. These observations indicate that plant cell walls may have more distinct chemical (and functional?) domains than previously envisaged.     

Molecular domains of the cellulose/xyloglucan network in the cell walls of higher plants

Cellulose and xyloglucan (XG) assemble to form the cellulose/XG network, which is considered to be the dominant load-bearing structure in the growing cell walls of non-graminaceous land plants. We have extended the most commonly accepted model for the macromolecular organization of XG in this network, based on the structural and quantitative analysis of three distinct XG fractions that can be differentially extracted from the cell walls isolated from etiolated pea stems. Approximately 8% of the dry weight of these cell walls consists of XG that can be solubilized by treatment of the walls with a XG-specific endoglucanase (XEG). This material corresponds to an enzyme-susceptible XG domain, proposed to form the cross-links between cellulose microfibrils. Another 10% of the cell wall consists of XG that can be solubilized by concentrated KOH after XEG treatment. This material constitutes another XG domain, proposed to be closely associated with the surface of the cellulose microfibrils. An additional 3% of the cell wall consists of XG that can be solubilized only when the XEG- and KOH-treated cell walls are treated with cellulase. This material constitutes a third XG domain, proposed to be entrapped within or between cellulose microfibrils. Analysis of the three fractions indicates that metabolism is essentially limited to the enzyme-susceptible domain. These results support the hypothesis that enzyme-catalyzed modification of XG cross-links in the cellulose/XG network is required for the growth and development of the primary plant cell wall, and demonstrate that the structural consequences of these metabolic events can be analyzed in detail.     

Polygalacturonase inhibiting protein in plant cell walls

Polygalacturonase inhibiting protein (PGIP) is localized in plant cell walls and plays an important role both in pectic substance metabolism and in prevention of the penetration of phytopathogenic microorganisms. Apparently, PGIP is responsible for the specificity of cell--cell interactions during pollination or inoculation by fungi nonpathogenic for the particular plant. PGIPs from different plants share a basic common structure. They are rather thermostable glycoproteins enriched with leucine and contain about 20% carbohydrates; the molecular weight varies between 37-54 kD. The synthesis of PGIP is encoded by one gene, and its expression is stimulated by injury and fungal infection. The resistance of plant tissues to infection frequently correlates with PGIP expression and with inhibiting action on fungal PG. Thus, PGIP is believed to be useful for gene engineering to obtain transgenic plants resistant to fungal infection or retaining commercial value during storage.     

Pollen tube cell walls of wild and domesticated tomatoes contain arabinosylated and fucosylated xyloglucan

Background and Aims In flowering plants, fertilization relies on the delivery of the sperm cells carried by the pollen tube to the ovule. During the tip growth of the pollen tube, proper assembly of the cell wall polymers is required to maintain the mechanical properties of the cell wall. Xyloglucan (XyG) is a cell wall polymer known for maintaining the wall integrity and thus allowing cell expansion. In most angiosperms, the XyG of somatic cells is fucosylated, except in the Asterid clade (including the Solanaceae), where the fucosyl residues are replaced by arabinose, presumably due to an adaptive and/or selective diversification. However, it has been shown recently that XyG of Nicotiana alata pollen tubes is mostly fucosylated. The objective of the present work was to determine whether such structural differences between somatic and gametophytic cells are a common feature of Nicotiana and Solanum (more precisely tomato) genera.Methods XyGs of pollen tubes of domesticated (Solanum lycopersicum var. cerasiforme and var. Saint-Pierre) and wild (S. pimpinellifolium and S. peruvianum) tomatoes and tobacco (Nicotiana tabacum) were analysed by immunolabelling, oligosaccharide mass profiling and GC-MS analyses.Key Results Pollen tubes from all the species were labelled with the mAb CCRC-M1, a monoclonal antibody that recognizes epitopes associated with fucosylated XyG motifs. Analyses of the cell wall did not highlight major structural differences between previously studied N. alata and N. tabacum XyG. In contrast, XyG of tomato pollen tubes contained fucosylated and arabinosylated motifs. The highest levels of fucosylated XyG were found in pollen tubes from the wild species.Conclusions The results clearly indicate that the male gametophyte (pollen tube) and the sporophyte have structurally different XyG. This suggests that fucosylated XyG may have an important role in the tip growth of pollen tubes, and that they must have a specific set of functional XyG fucosyltransferases, which are yet to be characterized.     

Measurement of pectin methylation in plant cell walls

A procedure was developed to measure the degree of pectin methylation in small samples of isolated cell walls from nonlignified plant tissues or pectin solutions. Galacturonic acid was determined colorimetrically with the 3,5-dimethylphenol reagent. Methylation was measured by base hydrolysis of galacturonic acid methyl esters, followed by gas chromatographic determination of released methanol. Estimates of the precision of analysis of pectin and cell wall samples were made. The coefficient of variation for estimates of the pectin esterification in cell walls isolated from 10-g samples of cucumber tissue ranged from 7.7 to 13.2%.     

Modulation of the degree and pattern of methyl-esterification of pectic homogalacturonan in plant cell walls. Implications for pectin methyl esterase action, matrix properties, and cell adhesion

Homogalacturonan (HG) is a multifunctional pectic polysaccharide of the primary cell wall matrix of all land plants. HG is thought to be deposited in cell walls in a highly methyl-esterified form but can be subsequently de-esterified by wall-based pectin methyl esterases (PMEs) that have the capacity to remove methyl ester groups from HG. Plant PMEs typically occur in multigene families/isoforms, but the precise details of the functions of PMEs are far from clear. Most are thought to act in a processive or blockwise fashion resulting in domains of contiguous de-esterified galacturonic acid residues. Such de-esterified blocks of HG can be cross-linked by calcium resulting in gel formation and can contribute to intercellular adhesion. We demonstrate that, in addition to blockwise de-esterification, HG with a non-blockwise distribution of methyl esters is also an abundant feature of HG in primary plant cell walls. A partially methyl-esterified epitope of HG that is generated in greatest abundance by non-blockwise de-esterification is spatially regulated within the cell wall matrix and occurs at points of cell separation at intercellular spaces in parenchymatous tissues of pea and other angiosperms. Analysis of the properties of calcium-mediated gels formed from pectins containing HG domains with differing degrees and patterns of methyl-esterification indicated that HG with a non-blockwise pattern of methyl ester group distribution is likely to contribute distinct mechanical and porosity properties to the cell wall matrix. These findings have important implications for our understanding of both the action of pectin methyl esterases on matrix properties and mechanisms of intercellular adhesion and its loss in plants.     

Pectic polysaccharides of growing plant tissues

1. The polysaccharide compositions of the cell walls of sycamore cambium and sycamore callus tissue have been analysed and found to be directly comparable. 2. Electrophoretic analyses of the whole pectins prepared from actively growing callus and cambial tissue have shown that these preparations contain, in addition to the neutral and weakly acidic components present in apple fruit, a strongly acidic polygalacturonic acid component. 3. The weakly acidic component of all the pectins was directly comparable with that of the pectinic acid of apple fruit. 4. The components of the whole pectin of sycamore callus tissue have been partially purified and analysed. The neutral and weakly acidic components also found in apple fruit were isolated. 5. The pattern of the composition of the neutral sugars present in the pectins of actively growing tissues of cambium and callus has been compared with those present in apple-fruit pectinic acid. 6. The presence of rhamnose linked as galacturonosyl-(1-->2)-rhamnose has been found in sycamore whole pectin. 7. The difference in the pectins of callus, cambium and fruit appears not to be that of species difference but is more characteristic of the nature of the growth and growth conditions of the cells. This is discussed in relation to the problems of the control and mechanism of plant-cell growth and differentiation.