Characterization of an extracellular endopolygalacturonase from the saprobe Mucor ramosissimus Samutsevitsch and its action as trigger of defensive response in tropical plants

In recent years, interest in the ability of non-pathogenic microorganisms to induce resistance in plants has grown, particularly with respect to their use as environmentally safe controllers of plant disease. In this study, we investigated the capacity of Mucor ramosissimus Samutsevitsch to release pectinases able to degrade cell walls of Palicourea marcgravii St. Hil., a tropical forest native Rubiaceae on which the spores of this saprobic fungus have been found. The fungus was grown in liquid culture medium containing pectin as the sole carbon source and filtrates were analyzed for pectinase activity. An endopolygalacturonase was partially purified by ion exchange chromatography, gel filtration, and preparative isoelectrofocusing, and characterized. This enzyme was more active upon pectic substrates with a low degree of methyl esterification. The products of hydrolysis of different pectic substrates (including pectin from P. marcgravii) by the action of this endopolygalacturonase elicited to different extents the phytoalexin production in soybean cotyledons. Also, the enzyme itself and the products of its action on the pectic fraction of P. marcgravii elicited the production of defensive compounds in the leaves of the plant. These results suggest that, besides the role in recycling organic matter, saprobes may also play an important role in the induction of defensive mechanisms in wild plants by enhancing their non-specific resistance against pathogens. Furthermore, they set the stage for future studies on the role of saprobic fungi in inducing resistance of host plants to pathogens.     

Pectic substances,pectic enzymes and haze formation in fruit juices

The pectic substances, located primarily in the middle lamella between cells in higher plant tissues, are complex polysaccharides. They include the negatively charged rhamnogalacturonans, and the neutral arabinogalactans I and II and l-arabinans. These polysaccharides add viscosity to juices but may also form hazes and precipitates and retard maximum recovery of juices from the fruit. The rhamnogalacturonans are degraded by the enzymes pectin methylesterase and polygalacturonase normally present in plant tissues and by these enzymes and pectate lyase in microbially derived commercial pectic enzymes added during processing. The presence of a?abinofuranosidase, which degrades l-arabinans, in commercial pectic enzyme preparations, can cause haze formation in juices such as apple and pear.     

Stimulation by Erwinia carotovora of the synthesis of ethylene in cauliflower tissue

The synthesis of ethylene by cauliflower floret tissue was increased when the tissue was inoculated with the soft-rot bacterium Erwinia carotovora. This effect was clearly associated with the production of pectic enzymes by the micro-organism. These enzymes, acting together with the plant enzymes, stimulated the production of ethylene from methionine. The increased synthetic activity was due to the release and increased activity of a glucose oxidase enzyme apparently attached to plant cell-wall material and liberated by the action of pectic enzymes of the bacterium.     

A simple procedure for the synthesis of alpha-32P-nucleoside triphosphates.

Chemical analysis of grapefruit (Citrus paradisi) pectic polysaccharides demonstrated that galacturonic acid constitutes 78% by weight of the total carbohydrates found. The remaining 22% was accounted for by a number of sugars which include galactose, glucose, arabinose, xylose, and mannose and, by weight, galactose accounted for almost 50% of the total neutral sugar components found in these pectic polysaccharides. Treatment of pectic polysaccharides with galactose oxidase followed by reduction of oxidized galactose residues with tritiated potassium borohydride resulted in the labeling of pectic polysaccharides. Analysis of the labeled polysaccharides demonstrated that of the total radioactivity incorporated more than 90% was recovered in the galactose residues. These results clearly demonstrate the successful utilization of the galactose oxidase/tritiated potassium borohydride method in labeling plant pectic polysaccharide.     

Purification and Properties of Endo-polygalacturonase from Aspergillus japonicus

An endo-polygalacturonase from culture extracts of Aspergillus japonicus was purified about 34-fold by ammonium sulfate fractionation, SE-Sephadex column chromatography and gel filtration. The purified enzyme was homogeneous on ultracentrifugation and disc electrophoresis. Using gel filtration a molecular weight of 35,500 was estimated for the enzyme. The enzyme rapidly reduced the viscosity of pectic acid and released reducing groups in a random manner, yielding a mixture of mono-, di- and trigalacturonic acids as end products. The pH optimum of the enzyme for viscosity-reducing activity was 4.5 with pectin and pectic acid as substrates, and that for releasing reducing groups was also 4.5 with various pectic substances. The purified enzyme was able to macerate various kinds of plant tissues by itself.     

Immunoprofiling of pectic polysaccharides

An assay is described for the rapid identification of unbranched homogalacturonan and branched components occurring in samples of pectic polysaccharides using anti-pectin monoclonal antibodies. The assay involves the immunodetection of pectic polysaccharides after separation into two components during the application in small volumes to nitrocellulose. In this system, homoglacturonan-rich components migrate further on the nitrocellulose in contrast to pectic components with abundant side chains, resulting in a clear separation and discrete rings of distinct polysaccharides that can be identified using specific antibodies. This procedure is also directly applicable to preparations of plant material without the need for isolation of pectic polysaccharides.     

Ultrastructure of the Interaction of Cells of Pseudomonas phaseolicola with Cell Walls of a Resistant and Susceptible Bean Cultivar

Cells of Pseudomonas phaseolicola were observed entrapped against plant cell walls in both susceptible (Red Kidney) and resistant (Red Mexican) cultivars of French bean (Phaseolus vulgaris). After staining of samples with ruthenium red for electron microscopy pectic polysaccharide within plant cell walls became particularly well contrasted as did fibrillar material connecting bacteria to the plant cell walls. In places this fibrillar material appeared to emanate from the pectic polysaccharide in the plant cell wall, and the plant cell wall surface was eroded at such points. Ruthenium red also stains acidic, bacterial extracellular polysaccharide (EPS) and some of the fibrillar material in intercellular spaces is probably from this source. It is possible that bacteria become attached through an interaction between EPS and Pectic polysaccharide in plant cell walls.     

Labeling and chemistry of grapefruit pectic substances

The labeling of a number of polysaccharides found in grapefruit (Citrus paradisi) was achieved by feeding labeled myo-inositol to ripening grapefruit through their cut fruit stem, and allowing 4 days for the metabolism of label. The pectic polysaccharides were isolated by successive extraction of the labeled grapefruit with 80% ethanol, chloroform-methanol (1:1) and finally with 0.2 M Na₂ EDTA to solubilize pectic polysaccharides. The incorporation of label from myo-inositol into galacturonosyl, arabinosyl, xylosyl and galactosyl residues of pectic polysaccharides via myo-inositol oxidation pathway was demonstrated. Ion exchange chromatography of these labeled pectic polysaccharides using DE-52 cellulose resulted in the elution of eight totally or partially resolved polysaccharides with increasing salt concentration. The results suggest that, like other plant tissues, the myo-inositol oxidation pathway is also operative in ripening grapefruit and this metabolic pathway could be successfully utilized to achieve labeling of a number of pectic polysaccharides.     

Pectin: cell biology and prospects for functional analysis

Pectin is a major component of primary cell walls of all land plants and encompasses a range of galacturonic acid-rich polysaccharides. Three major pectic polysaccharides (homogalacturonan, rhamnogalacturonan-I and rhamnogalacturonan-II) are thought to occur in all primary cell walls. This review surveys what is known about the structure and function of these pectin domains. The high degree of structural complexity and heterogeneity of the pectic matrix is produced both during biosynthesis in the endomembrane system and as a result of the action of an array of wall-based pectin-modifying enzymes. Recent developments in analytical techniques and in the generation of anti-pectin probes have begun to place the structural complexity of pectin in cell biological and developmental contexts. The in muro de-methyl-esterification of homogalacturonan by pectin methyl esterases is emerging as a key process for the local modulation of matrix properties. Rhamnogalacturonan-I comprises a highly diverse population of spatially and developmentally regulated polymers, whereas rhamnogalacturonan-II appears to be a highly conserved and stable pectic domain. Current knowledge of biosynthetic enzymes, plant and microbial pectinases and the interactions of pectin with other cell wall components and the impact of molecular genetic approaches are reviewed in terms of the functional analysis of pectic polysaccharides in plant growth and development.     

Degradation of different pectins by fungi: correlations and contrasts between the pectinolytic enzyme sets identified in genomes and the growth on pectins of different origin

ABSTRACT: BACKGROUND: Pectins are diverse and very complexe biomolecules and their structure depends on the plant species and tissue. It was previously shown that derivatives of pectic polymers and oligosaccharides from pectins have positive effects on human health. To obtain specific pectic oligosaccharides, highly defined enzymatic mixes are required. Filamentous fungi are specialized in plant cell wall degradation and some produce a broad range of pectinases. They may therefore shed light on the enzyme mixes needed for partial hydrolysis. RESULTS: The growth profiles of 12 fungi on four pectins and four structural elements of pectins show that the presence/absence of pectinolytic genes in the fungal genome clearly correlates with their ability to degrade pectins. However, this correlation is less clear when we zoom in to the pectic structural elements. CONCLUSIONS: This study highlights the complexity of the mechanisms involved in fungal degradation of complex carbon sources such as pectins. Mining genomes and comparative genomics are promising first steps towards the production of specific pectinolytic fractions.     

Cell Surface Interactions between Bean Leaf Cells and Colletotrichum lindemuthianum: Cytochemical Aspects of Pectin Breakdown and Fungal Endopolygalacturonase Accumulation

After a brief period of biotrophic growth, the anthracnose fungus Colletotrichum lindemuthianum (Sacc. et Mgn.) Bri et Cav. develops extensively in bean leaf cells, causing severe wall alterations and death of the host protoplast. Aplysia gonad lectin, a polygalacturonic acid-binding agglutinin, was complexed to gold and used to study the extent of pectin breakdown during the necrotrophic phase of the infection process. In view of its specific binding properties for the endopolygalacturonase produced by C. lindemuthianum, a polygalacturonase-inhibiting protein isolated from bean cell walls was successfully tagged with gold particles and used for localizing the sites of enzyme accumulation in infected host tissues. The basal level of endopolygalacturonase produced by C. lindemuthianum grown in culture was found to increase severalfold when the fungus developed in host plant tissues. The enzyme was able to diffuse freely in the host cell wall, causing drastic degradation of the pectic material of primary walls and middle lamella matrices. The enzymatic alteration of plant cell walls was accompanied by the release of pectic fragments and by the accumulation of pectic molecules at specific sites, such as intercellular spaces and aggregated cytoplasm of infected host cells. The occurrence of pectic molecules at those sites where fungal growth is likely to be restricted is discussed in relation to their origin and their implication in the plant's defense system.     

Effect of nutritional stress on plant composition

Summary Pectic substances were determined and characterized in marc prepared from leaf-petiole, stem, and root fractions of alfalfa grown in nutrient cultures containing adequate and inadequate sulfur. The percent pectin (as AUA) was somewhat greater in the sulfur-deficient tissues, the stem fractions showing the widest difference. The degree of methyl esterification of the pectic substances was not too dissimilar, however, the sulfur-deficient tissues appeared to be slightly more esterified, especially the leaf-petiole fraction. Acetyl values were not affected by the sulfur nutrition but considerable variance in percent acetyl was found in the different plant parts. The data is interpreted with respect to nutritional effects on the metabolism of pectic substances in plant tissue, and on the degree of methyl esterification of pectin in relation to methionine and sulphur nutrition.     

The immobility of pectic substances in injured tomato leaves and its bearing on the identity of the wound hormone

It has been suggested that pectic polysaccharides (or oligosaccharides cleaved from them) are liberated from the cell wall upon wounding of leaf tissue, and that they act as long-distance hormones evoking a defence response in neighbouring uninjured leaves (P.D. bishop et al. 1981, Proc. Natl. Acad. Sci. USA 78, 3536–3540, and cited literature). We have tested this hypothesis by infiltration of radioactive pectic fragments (rhamnogalacturonans and homogalacturonans of degrec of polymerisation down to 6) into wounds on tomato leaves. No radioactivity was exported from the treated leaf. [¹⁴C]Sucrose, applied in the same way, was effectively translocated, probably via the phloem. We suggest that pectic substances are not themselves long-distance wound hormones. The possibility remains that pectic substances, solubilised on wounding, act in the immediate vicinity of the wound to stimulate the dispatch of a second messenger, which would be the long-distance wound hormone.Abbreviations DP degree of polymerisation - PI proteinase inhibitor protein - PIIF PI inducing factor - QAE quaternary aminoethyl - TLC thin-layer chromatography     

picA, a novel plant-inducible locus on the Agrobacterium tumefaciens chromosome.

We used the transposon Mu dI1681 to identify genes on the Agrobacterium tumefaciens chromosome that are inducible by extracts from carrot roots. One such locus (picA, for plant inducible chromosomal), harbored by A. tumefaciens At156, was inducible 10- to 50-fold by these extracts. Mutation of picA had no detectable effect upon bacterial growth or virulence under laboratory assay conditions. However, A. tumefaciens cells harboring a mutated picA locus aggregated into long "ropes" when incubated with pea root tip cells. Such aggregation was not displayed by the parental strain A. tumefaciens A136. A preliminary characterization of the inducing compound in the carrot root extract suggests that the active substance is an acidic polysaccharide that is most likely derived from the pectic portion of the plant cell wall.     

Comparison of proteinase inhibitor-inducing activities and phytoalexin elicitor activities of a pure fungal endopolygalacturonase, pectic fragments, and chitosans

Rhizopus stolonifer endopolygalacturonase, an elicitor of casbene synthetase activity in castor bean seedlings, was found to be a potent elicitor of the phytoalexin pisatin in pea pods and of proteinase Inhibitor I in tomato leaves. The enzyme was an active elicitor or inducer only in its active native state; heat-denatured enzyme was inactive in all three systems. The activities of (a) the tomato pectic polysaccharide proteinase inhibitor-inducing factor, (b) a partially acid hydrolyzed proteinase inhibitor-inducing factor, (c) citrus pectic fragments, and (d) chitosan, were also compared in the three bioassay systems. The four oligosaccharide preparations were active in all three systems, but with different degrees of potency. In tomato leaves and pea pods, chitosans were most active, whereas in castor beans, the citrus pectic fragments were the best elicitors. The data presented support the hypothesis that plant and fungal cell wall fragments are important signals in mobilizing a wide variety of biochemically different types of plant defense responses, and that endopolygalacturonases play a key role in releasing the plant cell wall fragments during pest attacks.     

Physico-chemical characteristics of cell walls from Arabidopsis thaliana microcalli showing different adhesion strengths

Changes in the composition and structure of cell walls and extracellular polysaccharides (ECP) were studied during the growth of suspension-cultured Arabidopsis thaliana microcalli. Three growth phases, namely the cell division phase, the cell expansion phase, and the stationary phase, were distinguished and associated with a decreasing cell cluster adhesion strength. Degradation of the homogalacturonan pectic backbone and of linear pectic side chains (1,4)-beta-D-galactan were observed concomitantly with the cell expansion and stationary phases and the decrease in cell adhesion. Also, in the stationary phase, branched (1,5)-alpha-L-arabinans were linearized. The AGP content of the culture medium increased while it decreased in the cell wall during cell growth and as cell adhesion decreased. These data suggest that, in addition to homogalacturonan, pectic side chains and AGP are involved in plant cell development and particularly in cell-cell attachment.     

Studies on Intercellular Pectic Strands of Leaf Palisade Parenchyma

Regular rows (‘scala’) of pectic strands of uniformthickness interconnect palisade cells of leaves of a wide rangeof plant species, the leaf lamina of which possesses a regularpalisade layer. As the strands can be viewed by scanning electronmicroscopy of fractured, uncoated, fresh frozen leaves, theyare not artefacts. The techniques of electron probe analysis have been used toexamine fresh frozen leaves of holly in which there are regularpectic scala. Evidence is presented to support the view alreadyput forward, to explain the origin of the strands, that theyare deficient in calcium. The strands are shown to be rich inpotassium which, like the potassium in leaf cells, can be readilyleached with water. The advantages of fresh frozen plant material for elementalelectron probe analyses as well as problems arising from surfaceirregularities and surface charging, are discussed. pectic strands, leaf palisade parenchyma, electron-probe micro analysis, calcium, potassium     

Pectins: structure, biosynthesis, and oligogalacturonide-related signaling.

Pectin is a family of complex polysaccharides present in all plant primary cell walls. The complicated structure of the pectic polysaccharides, and the retention by plants of the large number of genes required to synthesize pectin, suggests that pectins have multiple functions in plant growth and development. In this review we summarize the current level of understanding of pectin primary and tertiary structure, and describe new methods that may be useful to study localized pectin structure in the plant cell wall. We also discuss progress in our understanding of how pectin is biosynthesized and review the biological activities and possible modes of action of pectic oligosaccharides referred to as oligogalacturonides. We present our view of critical questions regarding pectin structure, biosynthesis, and function that need to be addressed in the coming decade. As the plant community works towards understanding the functions of the tens of thousands of genes expressed by plants, a large number of those genes are likely to be involved in the synthesis, turnover, biological activity, and restructuring of pectin. A combination of genetic, molecular, biochemical and chemical approaches will be necessary to fully understand the function and biosynthesis of pectin.