Sub-cellular location of H2O2, peroxidases and pectin epitopes in control and hyperhydric shoots of carnation

In carnation shoots (Dianthus caryophyllus cv. Killer), hyperhydricity was induced in in vitro culture using a low agar concentration. Using transmission electron microscopy, cytochemical techniques and immunolocation of JIM5 and JIM7 pectin epitopes, we followed the sub-cellular modifications of cell walls in relation to peroxidase activity and hydrogen peroxide accumulation during hyperhydricity induction. Peroxidase activity revealed a significant induction of the stomatal and epidermal cells as well as of the intercellular spaces of hyperhydric leaves. Similarly, hydrogen peroxide accumulated in the epidermal cell walls and the intercellular spaces of hyperhydric leaves. Immunolocation of an epitope recognised by the JIM5 antibody revealed the main unesterified nature of the cell walls. Such an epitope was located in the epidermal cell walls as well as in the corners of cell junctions in control leaves. However, hyperhydric leaves showed a total reduction of JIM5 labelling in the corners of cell junctions and a significant reduction of the intercellular spaces and the middle lamella. Highly-methylsterified pectin, recognised by the JIM7 antibody, was present to a slight extent in cell walls in control and hyperhydric leaves. We propose that the altered anatomy observed in hyperhydric carnation leaves could be regulated by the concomitant actions of pectin methyl esterases and free radicals, modifying the structure of the pectin and polysaccharides of the cell walls.     

Overexpression of a pectin methylesterase inhibitor in Arabidopsis thaliana leads to altered growth morphology of the stem and defective organ separation

The methylesterification status of cell wall pectins, mediated through the interplay of pectin methylesterases (PMEs) and pectin methylesterase inhibitors (PMEIs), influences the biophysical properties of plant cell walls. We found that the overexpression of a PMEI gene in Arabidopsis thaliana plants caused the stems to develop twists and loops, most strongly around points on the stem where leaves or inflorescences failed to separate from the main stem. Altered elasticity of the stem, underdevelopment of the leaf cuticle, and changes in the sugar composition of the cell walls of stems were evident in the PMEI overexpression lines. We discuss the mechanisms that potentially underlie the aberrant growth phenotypes.     

Effects of snow-load and shading by vascular plants on the vertical growth of hummocks formed by Sphagnum papillosum in a mire of northern Japan

The growth of hummock Sphagnum species in bogs depends on hydrological and climatic conditions, with different hummock morphologies being found across geographical regions. We investigated how plant shade and winter snow-load regulate the growth pattern and height of Sphagnum papillosum hummocks in a cool-temperate bog in Japan. Hummocks were subjected to four treatments across 2?years (1 hummock per treatment per year): no treatment (control), snow-load-free (S), vascular plant trimming (T), and snow-load-free and vascular plant trimming in combination (S&T). Further, the effects of vascular plant cover and snow -load on the vertical growth and height of hummocks were examined. Annual growth rates of S. papillosum were higher for the control (34?C41?mm) than S&T treatment, and were intermediate for separate S and T treatments. In contrast to vascular plant-growing hummocks, vascular plant-trimmed hummocks showed a negative correlation between water-table depth (measured from the capitulum to the water-table) and Sphagnum growth. Hence, in summer, shading by vascular plants may prevent desiccation and facilitate the growth of Sphagnum. Snow-loaded hummocks were weighed down by 3?C11?cm. After snowmelt, the shoots continued to grow within the water-table depth range that allowed growth. Hence, heavy winter snow-loads may depress the surfaces of hummocks closer to the water-table, which stimulates Sphagnum growth, resulting in the recovery of hummock height. Thus, the water stress caused by summer desiccation is critical in regulating the upper limit of hummock height in bogs subject to dry summer conditions and heavy winter snowfall.     

Response of Sphagnum papillosum and Drosera rotundifolia to Reduced and Oxidized Wet Nitrogen Deposition

We transplanted Sphagnum ??turfs?? containing abundant Drosera rotundifolia into an existing nitrogen deposition experiment at Whim Moss near Edinburgh. These mesocosms received simulated N deposition as either NH ₄ ⁺ or NO ₃ ^- , to give total N deposition rates of approximately 8, 16 or 32, or 64?kg?N?ha^-1?year^-1. Simulated N deposition was added in a realistic way (i.e., with rainfall throughout the year). The ??¹⁵N of this added N was elevated relative to background N. We measured the tissue chemistry and ??¹⁵N of Sphagnum papillosum and D. rotundifolia over two years after transplant. Our aim was to determine uptake of the deposited N and the impact on S. papillosum tissue chemistry and D. rotundifolia tissue chemistry and ecology. We found clear, significant impacts of N deposition on S. papillosum, with increased capitula N content and reduced C:N ratio. Increased ??¹⁵N indicated uptake of deposited N. The response of D. rotundifolia was less clear with impacts only at the highest rate of N deposition. There was no evidence of differential uptake of reduced or oxidized wet N deposition by either S. papillosum or D. rotundifolia. Using the natural abundance stable isotope method we estimated the minimum contribution of prey N to the total N in D. rotundifolia to be 35%. The results suggest that differences in the uptake of reduced or oxidized wet N deposition might not be ecologically significant when wet N deposition is added realistically. They also support the suggestion that a model of N dynamics in Sphagnum-dominated ecosystems that includes the role of Sphagnum as a small-scale ecosystem engineer, is required to predict vascular plant responses to N deposition accurately.     

The Role of beta-Galactosidases in the Modification of Cell Wall Components during Muskmelon Fruit Ripening

The changes in activities of soluble β-galactosidase and two forms of wall-bound β-galactosidases extracted with NaCl and EDTA were investigated throughout the development of muskmelon (Cucumis melo L. cv Prince) fruits. DEAE-cellulose ion-exchange chromatography of soluble β-galactosidase revealed the presence of two isoforms. Soluble isoform I was detected in all stages throughout the fruit development, whereas soluble isoform II appeared around 34 d after anthesis when fruit ripening initiated. Both NaCl- and EDTA-released β-galactosidase activities also increased as ripening proceeded. The soluble and wall-bound forms behaved differently upon ion-exchange chromatography. Enzymological properties such as optimum pH, optimum temperature, K_m values for p-nitrophenyl β-d-galactopyranoside, and inhibition by metal ions were nearly similar in all forms. Molecular sizes of pectic polymers and hemicelluloses extracted from fruit mesocarp cell walls were shifted from larger to smaller polymers during ripening, as determined by gel filtration profiles. NaCl-released β-galactosidase from cell walls of ripe fruits had the ability to degrade in vitro the pectin extracted from preripe fruit cell walls to smaller sizes of pectin similar to those that were observed in ripe cell walls in situ. Both soluble isoform I and II were able to degrade in vitro the 5% KOH-extractable hemicellulose from preripe fruit cell walls to sizes of molecules similar to those that were observed in ripe cell walls in situ. Soluble isoform I and the NaCl-released form from ripe fruits were able to modify in vitro 24% KOH-extractable hemicellulose from preripe cell walls to sizes of molecules similar to those that were observed in ripe fruits in situ.     

Distribution of cell wall components in<Emphasis Type="Italic"> Sphagnum</Emphasis> hyaline cells and in liverwort and hornwort elaters

Spiral secondary walls are found in hyaline cells of Sphagnum, in the elaters of most liverworts, and in elaters of the hornwort Megaceros. Recent studies on these cells suggest that cytoskeletal and ultrastructural processes involved in cell differentiation and secondary wall formation are similar in bryophytes and vascular plant tracheary elements. To examine differences in wall structure, primary and secondary wall constituents of the hyaline cells of Sphagnum novo-zelandicum and elaters of the liverwort Radula buccinifera and the hornwort Megaceros gracilis were analyzed by immunohistochemical and chemical methods. Anti-arabinogalactan–protein antibodies, JIM8 and JIM13, labeled the central fibrillar secondary wall layer of Megaceros elaters and the walls of Sphagnum leaf cells, but did not label the walls of Radula elaters. The CCRC-M7 antibody, which detects an arabinosylated (16)-linked -galactan epitope, exclusively labeled hyaline cells in Sphagnum leaves and the secondary walls of Radula elaters. Anti-pectin antibodies, LM5 and JIM5, labeled the primary wall in Megaceros elaters. LM5 also labeled the central layer of the secondary wall but only during formation. In Radula elaters, JIM5 and another anti-pectin antibody, JIM7, labeled the primary wall. The distribution of arabinogalactan–proteins and pectic polysaccharides restricted to specific wall types and stages of development provides evidence for the developmental and functional regulation of cell wall composition in bryophytes. Monosaccharide-linkage analysis of Sphagnum leaf cell walls suggests they contain polysaccharides similar to those of higher plants. The most abundant linkage was 4-Glc, typical of cellulose, but there was also evidence for xyloglucans, 4-linked mannans, 4-linked xylans and rhamnogalacturonan-type polysaccharides.Abbreviations AGP Arabinogalactan–protein - Araf Arabinofuranose - Fucp Fucopyranose - GalAp Galacturonopyranose - Galp Galactopyranose - GlcAp Glucuronopyranose - HGA Homogalacturonan - Manp Mannopyranose - RG Rhamnogalacturonan - Rhap Rhamnopyranose - XG Xyloglucan - Xylp Xylopyranose     

Structural characterization of cell wall polysaccharides from two plant species endemic to central Africa,Fleurya aestuans and Phragmenthera capitata

Fleurya aestuans (Linnaeus) Miquel and Phragmenthera capitata (Spreng) are two plants endemic to central Africa that are used in traditional medicine. However, information on their molecular constituents is lacking. In the present study and as part of our research on the structure/bioactivity relationship of plant cell wall molecules, we investigated the structure of polysaccharides isolated from leaf cell walls of both plant species. To this end, we used sequential extraction of polysaccharides, gas chromatography, matrix assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) and immuno-dot assays. Our data indicate the presence of both pectin and hemicellulosic polysaccharides in the cell walls of both plants. In particular, cell wall of F. aestuans leaves appears to contain much more pectin than those of P. capitata. Structural analysis of hemicellulosic polysaccharides revealed differences in the structure of xyloglucan isolated from both species. While only the XXXG-type was found in P. capitata, both XXXG and XXGG types were detected in F. aestuans. No arabinosylated subunits were found in any of the xyloglucan isolated from both plant species. In addition, xylan structure with non methylated-α-d-glucuronic acid on side chains was only detected in F. aestuans leaf cell walls. Finally, structural analysis of rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) shows that unlike RG-II, RG-I is qualitatively different between F. aestuans and P. capitata leaves.     

Cell wall cementing materials of grass leaves

Treatment of grass leaves with either a purified pectin lyase of Aspergillus japonicus or a purified xylanase of Trichoderma viride could lead to the isolation of some single leaf cells. However, a mixture of pectin lyase and xylanase brought about more rapid isolation of single cells than did either of the two enzymes alone, indicating a synergistic effect. Analysis of the components released from oat cell walls by the enzymes indicated that both homogalacturonans with a high degree of esterification and a kind of glucuronoarabinoxylan with ferulic acid ester may play a role in cell wall cementing in grass leaves.     

Interacting effects of elevated atmospheric CO<Subscript>2</Subscript> and hydrology on the growth and carbon sequestration of <Emphasis Type="Italic">Sphagnum</Emphasis> moss

Peatlands are a critical carbon store comprising 30% of the Earth’s terrestrial soil carbon. Sphagnum mosses comprise up to 90% of peat in the northern hemisphere but impacts of climate change on Sphagnum mosses are poorly understood, limiting development of sustainable peatland management and restoration. This study investigates the effects of elevated atmospheric CO₂ (eCO₂) (800 ppm) and hydrology on the growth of Sphagnum fallax, Sphagnum capillifolium and Sphagnum papillosum and greenhouse gas fluxes from moss–peat mesocosms. Elevated CO₂ levels increased Sphagnum height and dry weight but the magnitude of the response differed among species. The most responsive species, S. fallax, yielded the most biomass compared to S. papillosum and S. capillifolium. Water levels and the CO₂ treatment were found to interact, with the highest water level (1 cm below the surface) seeing the largest increase in dry weight under eCO₂ compared to ambient (400 ppm) concentrations. Initially, CO₂ flux rates were similar between CO₂ treatments. After week 9 there was a consistent three-fold increase of the CO₂ sink strength under eCO₂. At the end of the experiment, S. papillosum and S. fallax were greater sinks of CO₂ than S. capillifolium and the ? 7 cm water level treatment showed the strongest CO₂ sink strength. The mesocosms were net sources of CH₄ but the source strength varied with species, specifically S. fallax produced more CH₄ than S. papillosum and S. capillifolium. Our findings demonstrate the importance of species selection on the outcomes of peatland restoration with regards to Sphagnum’s growth and GHG exchange.     

Deficiency of adenosine kinase activity affects the degree of pectin methyl-esterification in cell walls of Arabidopsis thaliana

Pectin methyl-esterification is catalysed by S-adenosyl-l-methionine (SAM)-dependent methyltransferases. As deficiency in adenosine kinase (ADK; EC 2.7.1.20) activity impairs SAM recycling and utilization, we investigated the relationship between ADK-deficiency and the degree of pectin methyl-esterification in cell walls of Arabidopsis thaliana. The distribution patterns of epitopes associated with methyl-esterified homogalacturonan in leaves and hypocotyls of wild-type (WT) and ADK-deficient plants were examined using immunolocalization and biochemical techniques. JIM5 and LM7 epitopes, characteristic of low esterified pectins, were more irregularly distributed along the cell wall in ADK-deficient plants than in WT cell walls. In addition, epitopes recognized by JIM7, characteristic of pectins with a higher degree of methyl-esterification, were less abundant in ADK-deficient leaves and hypocotyls. Since de-esterified pectins have enhanced adhesion properties, we propose that the higher abundance and the altered distribution of low methyl-esterified pectin in ADK-deficient cell walls lead to the leaf shape abnormalities observed in these plants.     

Restoration of pool margin communities in cutover peatlands

Two experiments were conducted for developing restoration techniques for pool margin communities in cutover peatlands. We first aimed to measure the regeneration potential of a typical edge pool liverwort, Cladopodiella fluitans (Nees) H. Buch. We introduced C. fluitans in floating baskets in a restored peatland. We tested three fragment sizes (patches of 2 cm², stretched patches and shredded fine fragments of 0.1-1 mm), two introduction densities (ratio between surface of collected areas and surface of restored areas of 1:5 and 1:10) as well as the effect of a straw mulch. After two years, the percentage covers of C. fluitans were five times larger in experimental units protected with straw than in those without protection. Yet, the fragment sizes and the densities tested had no effect on the regeneration of the liverwort. The second experiment aimed to test a moss layer transfer approach to restore plant diversity around pool margins. We tested four communities, dominated by (1) Sphagnum cuspidatum Hoffman, (2) Sphagnum fallax (H. Klinggraff) H. Klinggraff, (3) Sphagnum papillosum Linberg as well as (4) a mixed community composed of equal quantities of C. fluitans, S. cuspidatum and S. papillosum. We introduced plant material in two density ratios (1:5 and 1:10). Sphagnum mosses did colonize pool margins, and showed even more than 60% cover for some treatments after three growing seasons, but the recovery of the introduced vascular plants remained below 5% for most species. The establishment of pool vascular species thus seems to be more intricate than for bryophytes and specific introduction techniques might be needed.     

Developmental changes in guard cell wall structure and pectin composition in the moss Funaria: implications for function and evolution of stomata

Background and Aims

In seed plants, the ability of guard cell walls to move is imparted by pectins. Arabinan rhamnogalacturonan I (RG1) pectins confer flexibility while unesterified homogalacturonan (HG) pectins impart rigidity. Recognized as the first extant plants with stomata, mosses are key to understanding guard cell function and evolution. Moss stomata open and close for only a short period during capsule expansion. This study examines the ultrastructure and pectin composition of guard cell walls during development in Funaria hygrometrica and relates these features to the limited movement of stomata.

Methods

Developing stomata were examined and immunogold-labelled in transmission electron microscopy using monoclonal antibodies to five pectin epitopes: LM19 (unesterified HG), LM20 (esterified HG), LM5 (galactan RG1), LM6 (arabinan RG1) and LM13 (linear arabinan RG1). Labels for pectin type were quantitated and compared across walls and stages on replicated, independent samples.

Key Results

Walls were four times thinner before pore formation than in mature stomata. When stomata opened and closed, guard cell walls were thin and pectinaceous before the striated internal and thickest layer was deposited. Unesterified HG localized strongly in early layers but weakly in the thick internal layer. Labelling was weak for esterified HG, absent for galactan RG1 and strong for arabinan RG1. Linear arabinan RG1 is the only pectin that exclusively labelled guard cell walls. Pectin content decreased but the proportion of HG to arabinans changed only slightly.

Conclusions

This is the first study to demonstrate changes in pectin composition during stomatal development in any plant. Movement of Funaria stomata coincides with capsule expansion before layering of guard cell walls is complete. Changes in wall architecture coupled with a decrease in total pectin may be responsible for the inability of mature stomata to move. Specialization of guard cells in mosses involves the addition of linear arabinans.     

Cell wall components affect mechanical properties: studies with thistle flowers

Pollen presentation of Cirsium horridulum depends partially on the thigmonastic contraction of staminal filaments. Although the elastic cuticle is a major component in filament elasticity, it is not clear how the cell wall copes with the shape change. Based on mechanical studies, FT-IR spectroscopy and biochemical analyses we investigated the relationship between cell wall composition and elastic properties using thistle floral tissues as a model. EDTA-extractable pectin correlated with the increased elasticity of the filament and the basal style, suggesting that pectin plays a major role in the elastic behavior of soft tissues. In contrast, covalently linked pectin contributes to the stiffness of the upper style and corolla. Mechanical tests contrasting the soft basal and rigid apical parts of the style after incubation in solutions designed to alter the pectin network confirmed these results. The rigid corolla contained more cellulose than the softer style and filaments. The cellulose-associated xyloglucan of the style and filament cell walls increase the flexibility of cell walls.     

Variation in the Degree of Pectin Methylesterification during the Development of Baccharis dracunculifolia Kidney-Shaped Gall

Insect galls may be study models to test the distribution of pectins and arabinogalactan-proteins (AGPs) and their related functions during plant cell cycles. These molecules are herein histochemically and immunocitochemically investigated in the kidney-shaped gall induced by Baccharopelma dracunculifoliae (Psyllidae) on leaves of Baccharis dracunculifolia DC. (Asteraceae) on developmental basis. The homogalacturonans (HGAs) (labeled by JIM5) and the arabinans (labeled by LM6) were detected either in non-galled leaves or in young galls, and indicated stiffening of epidermal cell walls, which is an important step for cell redifferentiation. The labeling of HGAs by JIM7 changed from young to senescent stage, with an increase in the rigidity of cell walls, which is important for the acquaintance of the final gall shape and for the mechanical opening of the gall. The variation on the degree of HGAs during gall development indicated differential PMEs activity during gall development. The epitopes recognized by LM2 (AGP glycan) and LM5 (1–4-β-D-galactans) had poor alterations from non-galled leaves towards gall maturation and senescence. Moreover, the dynamics of pectin and AGPs on two comparable mature kidney-shaped galls on B. dracunculifolia and on B. reticularia revealed specific peculiarities. Our results indicate that similar gall morphotypes in cogeneric host species may present distinct cell responses in the subcelular level, and also corroborate the functions proposed in literature for HGAs.     

Pectin Biosynthesis Is Critical for Cell Wall Integrity and Immunity in Arabidopsis thaliana

Plant cell walls are important barriers against microbial pathogens. Cell walls of Arabidopsis thaliana leaves contain three major types of polysaccharides: cellulose, various hemicelluloses, and pectins. UDP-d-galacturonic acid, the key building block of pectins, is produced from the precursor UDP-d-glucuronic acid by the action of glucuronate 4-epimerases (GAEs). Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) repressed expression of GAE1 and GAE6 in Arabidopsis, and immunity to Pma ES4326 was compromised in gae6 and gae1 gae6 mutant plants. These plants had brittle leaves and cell walls of leaves had less galacturonic acid. Resistance to specific Botrytis cinerea isolates was also compromised in gae1 gae6 double mutant plants. Although oligogalacturonide (OG)-induced immune signaling was unaltered in gae1 gae6 mutant plants, immune signaling induced by a commercial pectinase, macerozyme, was reduced. Macerozyme treatment or infection with B. cinerea released less soluble uronic acid, likely reflecting fewer OGs, from gae1 gae6 cell walls than from wild-type Col-0. Although both OGs and macerozyme-induced immunity to B. cinerea in Col-0, only OGs also induced immunity in gae1 gae6. Pectin is thus an important contributor to plant immunity, and this is due at least in part to the induction of immune responses by soluble pectin, likely OGs, that are released during plant-pathogen interactions.     

Fluorescence Properties of Guard Cell Chloroplasts: EVIDENCE FOR LINEAR ELECTRON TRANSPORT AND LIGHT-HARVESTING PIGMENTS OF PHOTOSYSTEMS I AND II

The presence of chloroplasts in guard cells from leaf epidermis, coleoptile, flowers, and albino portions of variegated leaves was established by incident fluorescence microscopy, thus confirming the notion that guard cell chloroplasts are remarkably conserved. Room temperature emission spectra from a few chloroplasts in a single guard cell of Vicia faba showed one major peak at around 683 nanometers. Low-temperature (77 K) emission spectra from peels of albino portions of Chlorophytum comosum leaves and from mesophyll chloroplasts of green parts of the same leaves showed major peaks at around 687 and 733 nanometers, peaks usually attributed to photosystem II and photosystem I pigment systems, respectively. Spectra of peels of V. faba leaves showed similar peaks. However, fluorescence microscopy revealed that the Vicia peels, as well as those from Allium cepa and Tulipa sp., were contaminated with non-guard cell chloroplasts which were practically undetectable under bright field illumination. These observations pose restrictions on the use of epidermal peels as a source of isolated guard cell chloroplasts. Studies on the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive variable fluorescence kinetics of uncontaminated epidermal peels of C. comosum indicated that guard cell chloroplasts operate a normal, photosystem II-dependent, linear electron transport. The above properties in combination with their reported inability to fix CO₂ photosynthetically may render the guard cell chloroplasts optimally suited to supply the reducing and high-energy phosphate equivalents needed to sustain active ion transport during stomatal opening in daylight.     

Identification of enzymes that are effective for isolating protoplasts from grass leaves

Cellulase C₁, cellulase Cx, and xylanase were isolated and purified from a cellulase preparation of Trichoderma viride as enzymes effective in the isolation of protoplasts from oat leaves. Pectin lyase which is specific for methyl-galacturonide linkages was also found to be a useful enzyme for the isolation of protoplasts from the tissues. This suggested that pectic polysaccharides with a high degree of esterification may play an important role in cell walls of Gramineae. It was necessary to use the mixture of cellulase C₁, cellulase Cx, xylanase, and pectin lyase for the rapid isolation of protoplasts, while a small amount of protoplasts could be isolated from oat leaves by cellulase C₁ plus xylanase or cellulase C₁ plus pectin lyase. The mixture of four enzymes also was effective in the isolation of protoplasts from the leaves of wheat, barley, and corn.     

Arabinoxylan, β‐glucan and pectin in barley and malt endosperm cell walls: a microstructure study using CLSM and cryo‐SEM

The architecture of endosperm cell walls in Hordeum vulgare (barley) differs remarkably from that of other grass species and is affected by germination or malting. Here, the cell wall microstructure is investigated using (bio)chemical analyses, cryogenic scanning electron microscopy (cryo‐SEM) and confocal laser scanning microscopy (CLSM) as the main techniques. The relative proportions of β‐glucan, arabinoxylan and pectin in cell walls were 61, 34 and 5%, respectively. The average thickness of a single endosperm cell wall was 0.30 µm, as estimated by the cryo‐SEM analysis of barley seeds, which was reduced to 0.16 µm after malting. After fluorescent staining, 3D confocal multiphoton microscopy (multiphoton CLSM) imaging revealed the complex cell wall architecture. The endosperm cell wall is composed of a structure in which arabinoxylan and pectin are colocalized on the outside, with β‐glucan depositions on the inside. During germination, arabinoxylan and β‐glucan are hydrolysed, but unlike β‐glucan, arabinoxylan remains present in defined cell walls in malt. Integrating the results, an enhanced model for the endosperm cell walls in barley is proposed.     

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.     

Visualization of halos in the epidermal cell wall of Allium cepa caused by Colletotrichum dematium f. circinans and Botrytis allii using fluorochromes

Halos were detected with epifluorescence microscopy around penetration sites of Colletotrichum dematium f. circinans and Botrytis allii in onion epidermal cell walls as areas of less intense fluorescence or negatively stained areas in fluorescing cell walls following treatments with berberin sulphate and acridine orange but not with brilliant sulphaflavine (which stained the cell wall), ninhydrin, dansylchloride, or analine blue. Since pectin, pectic acid, avacil (microcrystaline cellulose super fine), filter paper, and Sephadex G-100–120 fluoresced with acridine orange and berberin sulphate, it was inferred that the halos were negatively stained or appeared as areas with less intense fluorescence because enzymes from these pathogens degraded cell wall pectin and cellulose at the point of penetration. Spores of both pathogens fluoresced when stained with brilliant sulphaflavine, acridine orange, ninhydrin, and dansylchloride. These stains and berberin sulphate caused germ tubes, appressoria, and primary infection mycelia to fluoresce. Nuclei in these fungal structures fluoresced when stained with acridine orange and brilliant sulphaflavine.