Biological Activity of Reducing-End-Derivatized Oligogalacturonides in Tobacco Tissue Cultures

The biological activity of reducing-end-modified oligogalacturonides was quantified in four tobacco (Nicotiana tabacum) tissue culture bioassays. The derivatives used were oligogalacturonides with the C-1 of their reducing end (a) covalently linked to a biotin hydrazide, (b) covalently linked to tyramine, (c) chemically reduced to a primary alcohol, or (d) enzymatically oxidized to a carboxylic acid. These derivatives were tested for their ability to (a) alter morphogenesis of N. tabacum cv Samsun thin cell-layer explants, (b) elicit extracellular alkalinization by suspension-cultured cv Samsun cells, (c) elicit extracellular alkalinization by suspension-cultured N. tabacum cv Xanthi cells, and (d) elicit H₂O₂ accumulation in the cv Xanthi cells. In all four bioassays, each of the derivatives had reduced biological activity compared with the corresponding underivatized oligogalacturonides, demonstrating that the reducing end is a key element for the recognition of oligogalacturonides in these systems. However, the degree of reduction in biological activity depends on the tissue culture system used and on the nature of the specific reducing-end modification. These results suggest that oligogalacturonides are perceived differently in each tissue culture system.     

Galacturonic acid-induced changes in strawberry plant development <Emphasis Type="Italic">in vitro</Emphasis>

The oligogalacturonides derived from trifluoroacetic acid hydrolysis and their monomer galacturonic acid were tested for their effect on development of strawberry explants (Fragaria × ananassa) in vitro. The addition of oligogalacturonides or galacturonic acid in the plant tissue culture medium at concentrations of 0.1 or 1.0 mM increased shoot and leaf number in the presence of added benzyl adenine. The response to oligogalacturonides or galacturonic acid was maintained in the absence of benzyl adenine but was lessened. Conversely, root elongation was increased in the absence of benzyl adenine. A combined analysis of these experiments also indicated the increase in shoot or leaf number. High-performance anion exchange chromatography of the bioassayed oligogalacturonide samples using a pulsed amperometric detector revealed that the sample consisted of galactouronides with a degree of polymerization from 1 to 5 and the proportion of the monomer was very high (94%).     

Host-Pathogen Interactions : XXXIII. A Plant Protein Converts a Fungal Pathogenesis Factor into an Elicitor of Plant Defense Responses

This paper describes the effect of a plant-derived polygalacturonase-inhibiting protein (PGIP) on the activity of endopolygalacturonases isolated from fungi. PGIP's effect on endopolygalacturonases is to enhance the production of oligogalacturonides that are active as elicitors of phytoalexin (antibiotic) accumulation and other defense reactions in plants. Only oligogalacturonides with a degree of polymerization higher than nine are able to elicit phytoalexin synthesis in soybean cotyledons. In the absence of PGIP, a 1-minute exposure of polygalacturonic acid to endopolygalacturonase resulted in the production of elicitor-active oligogalacturonides. However, the enzyme depolymerized essentially all of the polygalacturonic acid substrate to elicitor-inactive oligogalacturonides within 15 minutes. When the digestion of polygalacturonic acid was carried out with the same amount of enzyme but in the presence of excess PGIP, the rate of production of elicitor-active oligogalacturonides was dramatically altered. The amount of elicitor-active oligogalacturonide steadily increased for 24 hours. It was only after about 48 hours that the enzyme converted the polygalacturonic acid into short, elicitor-inactive oligomers. PGIP is a specific, reversible, saturable, high-affinity receptor for endopolygalacturonase. Formation of the PGIP-endopolygalacturonase complex results in increased concentrations of oligogalacturonides that activate plant defense responses. The interaction of the plant-derived PGIP with fungal endopolygalacturonases may be a mechanism by which plants convert endopolygalacturonase, a factor important for the virulence of pathogens, into a factor that elicits plant defense mechanisms.     

A sensitive and rapid bioassay of homogalacturonan synthase using 2-aminobenzamide-labeled oligogalacturonides

Polygalacturonate 4-alpha-galacturonosyltransferase (GalA T) activity was detected in the microsomal fraction isolated from pumpkin (Cucurbia moschata Duchesne, cv. Tokyou-Kabocha) seedlings using UDP-GalA and 2-aminobenzamide (2AB)-labeled oligogalacturonides. A 2AB-labeled undecagalacturonide was elongated by the attachment of galacturonic acid (GalA) residues to give 2AB-labeled oligogalacturonides with a degree of polymerization (DP) between 12 and 17. Exogenous 2AB-labeled oligogalacturonide acceptors with a DP >3 are effective acceptor molecules for pumpkin GalA T.     

Synthesis and characterization of tyramine-derivatized (1 å 4)-linked α-d-oligogalacturonides

The reducing end C-1 of (1 → 4)-linked α-d-oligogalacturonides (oligogalacturonides), with degrees of polymerization (dp) 3 and 13, was coupled to tyramine via reductive amination in the presence of sodium cyanoborohydride. These derivatives were purified in milligram quantities and structurally characterized. Tyramination of trigalacturonic acid proceeded to completion. The yield of apparently homogeneous tyraminated trigalacturonic acid after desalting was 35%. Derivatization of tridecagalacturonide with tyramine was incomplete. The tyraminated tridecagalacturonide was purified to apparent homogeneity using semipreparative high-performance anion-exchange chromatography (HPAEC) with a yield of 30%. The structures of the derivatized oligogalacturonides were established by ¹H NMR spectroscopy and electrospray mass spectrometry.     

Oligogalacturonides inhibit the formation of roots on tobacco explants

α-1,4-Oligogalacturonides with degrees of polymerization (DPs) ranging from 6 to 18 or 2 to 8 were added to tobacco leaf explants and root formation was evaluated after 15 days of incubation. Auxin-induced formation of roots was inhibited by oligogalacturonides with DPs 6–18 but not by the oligogalacturonides with DPs 2–8. The inhibition of root formation by the larger oligogalacturonides was prevented by increasing the amount of auxin present in the medium. Oligogalacturonides (DPs 6–18) also inhibited root formation when added to tobacco thin cell-layer (TCL) explants in a medium that is known to induce the formation of roots. The addition of size-homogeneous oligogalacturonides, to either tobacco leaf explants or TCLs, established that oligogalacturonides with DPs between 10 and 14 were most active in inhibiting the formation of roots. These data suggest that oligogalacturonides of the same size as those known to elicit plant defense responses, and to affect floral development and membrane functions, also inhibit the induction of root morphogenesis in tobacco.     

Microbial utilization and selectivity of pectin fractions with various structures

To evaluate the fermentation properties of oligosaccharides derived from pectins and their parent polysaccharides, a 5-ml-working-volume, pH- and temperature-controlled fermentor was tested. Six pectic oligosaccharides representing specific substructures found within pectins were prepared. These consisted of oligogalacturonides (average degrees of polymerization [DP] of 5 and 9), methylated oligogalacturonides (average DP of 5), oligorhamnogalacturonides (average DP of 10 as a disaccharide unit of galacturonic acid and rhamnose), oligogalactosides (average DP of 5), and oligoarabinosides (average DP of 6). The influence of these carbohydrates on the human fecal microbiota was evaluated. Use of neutral sugar fractions resulted in an increase in Bifidobacterium populations and gave higher organic acid yields. The Bacteroides-Prevotella group significantly increased on all oligosaccharides except oligogalacturonides with an average DP of 5. The most selective substrates for bifidobacteria were arabinan, galactan, oligoarabinosides, and oligogalactosides.     

Methods for obtaining neutral and acid oligosaccharides from flax pectins

Esterified acid soluble pectins from flax (Linun usitatissimum L.) were degraded either with HCl or pectin lyase. Centrifugation and 2-propanol precipitation led to the isolation of two low molecular weight polygalacturonates after acid hydrolysis of pectins. However, after pectin lyase digestion and purification by size-exclusion HPLC, ¹H NMR analyses indicated that acetylated hairy regions, large methylated and acetylated oligogalacturonides together with small unsubstituted oligogalacturonides were produced. Thus, in a few steps, a panel of substituted neutral and acidic oligosaccharides was produced from a raw plant material. Such oligosaccharides could be useful for further fractionations such as chemical saponification and enzymatic removal of neutral sugar chains from the hairy regions. The procedures used for pectin extraction, for degradation, and for the purification of fragments seem appropriate for large-scale production of biologically active oligosaccharides from flax.Revisions requested 24 September 2004; Revisions received 4 November 2004     

Fluorescent labeling of pectic oligosaccharides with 2-aminobenzamide and enzyme assay for pectin

Oligogalacturonides [oligomers composed of (1-->4)-linked alpha-D-galactosyluronic acid residues] with degrees of polymerization (DP) from 1 to 10, and a tri-, penta-, and heptasaccharide generated from the backbone of rhamnogalacturonan I (RG-I) were labeled at their reducing ends using aqueous 2-aminobenzamide (2AB) in the presence of sodium cyanoborohydride in over 90% yield. These derivatives were analyzed by high-performance anion-exchange chromatography (HPAEC) and structurally characterized by electrospray-ionization mass spectrometry (ESIMS) and by 1H and 13C NMR spectroscopy. The 2AB-labeled oligogalacturonides and RG-I oligomers are fragmented by endo- and exo-polygalacturonase and by Driselase, respectively. 2AB-labeled oligogalacturonide is an exogenous acceptor for galacturonosyltransferase of transferring galacturonic acid from UDP-GalA. Thus, the 2AB-labeled oligogalacturonides and RG-I oligomers are useful for studying enzymes involved in pectin degradation and biosynthesis and may be of value in determining the biological functions of pectic fragments in plants.     

Effects of Methyl Jasmonate on Shikonin and Dihydroechinofuran Production in Lithospermum Cell Cultures

Methyl jasmonate, when administered to Lithospermum erythrorhizoncell suspension cultures, was found to induce the productionof shikonin derivatives (the red naph-thoquinone pigments ofthe root) and dihydroechinofuran (an abnormal metabolite ofgeranylhydroquinone). Culture experiments showed that methyljasmonate caused a rapid increase in the activities of enzymesinvolved in the biosynthesis of shikonin such as p-hydroxybenzoategeran-yltransferase, which was followed by the rapid accumulationof dihydroechinofuran and the delayed production of shikonin.The induction patterns observed were similar to those elicitedby oligogalacturonides in Lithospermum cells, suggesting thatjasmonic acid or its derivative may act as a signaling moleculein the elicitation of shikonin biosynthesis. Interestingly,however, the copper ion, which is essential for inducing shikoninbiosynthesis by oligogalacturonides, was not required for shikonininduction by methyl jasmonate ¹Present address: Laboratory of Molecular & Cellular Biology,Department of Agricultural Chemistry, Kyoto University, Kitashirakawa,Kyoto, 606-01 Japan     

Partial demethylation of oligogalacturonides by pectin methyl esterase 1 is required for eliciting defence responses in wild strawberry (Fragaria vesca)

In addition to the role of the cell wall as a physical barrier against pathogens, some of its constituents, such as pectin-derived oligogalacturonides (OGA), are essential components for elicitation of defence responses. To investigate how modifications of pectin alter defence responses, we expressed the fruit-specific Fragaria  ×  ananassa pectin methyl esterase FaPE1 in the wild strawberry Fragaria vesca . Pectin from transgenic ripe fruits differed from the wild-type with regard to the degree and pattern of methyl esterification, as well as the average size of pectin polymers. Purified oligogalacturonides from the transgenic fruits showed a reduced degree of esterification compared to oligogalacturonides from wild-type fruits. This reduced esterification is necessary to elicit defence responses in strawberry. The transgenic F. vesca lines had constitutively activated pathogen defence responses, resulting in higher resistance to the necrotropic fungus Botrytis cinerea . Further studies in F. vesca and Nicotiana benthamiana leaves showed that the elicitation capacity of the oligogalacturonides is more specific than previously envisaged.     

Overexpression of citrus polygalacturonase-inhibiting protein in citrus black rot pathogen Alternaria citri

The rough lemon (Citrus jambhiri) gene encoding polygalacturonase-inhibiting protein (RlemPGIPA) was overexpressed in the pathogenic fungus Alternaria citri. The overexpression mutant AcOPI6 retained the ability to utilize pectin as a sole carbon source, and the overexpression of polygalacturonase-inhibiting protein did not have any effect on the growth of AcOPI6 in potato dextrose and pectin medium. The pathogenicity of AcOPI6 to cause a black rot symptom in citrus fruits was also unchanged. Polygalacturonase-inhibiting protein was secreted together with endopolygalacturonase into culture filtrates of AcOPI6, and oligogalacturonides were digested from polygalacturonic acid by both proteins in the culture filtrates. The reaction mixture containing oligogalacturonides possessed activity for induction of defense-related gene, RlemLOX, in rough lemon leaves.     

Elicitation of H2O2 production in cucumber hypocotyl segments by oligo-1,4-α-D-galacturonides and an oligo-β-glucan preparation from cell walls of Phythophthora megasperma f. sp. glycinea

The production of H₂O₂ by cucumber hypocotyl segments ( Cucumis sativus L. cv. Wisconsin SMR 58) in response to α-1,4-linked oligomers of galacturonic acid and oligo-β-glucans from the cell walls of Phytophthora megasperma f. sp. glycinea was studied. Oligogalacturonides with degrees of polymerization of 9 to 13 elicited H₂O₂ production, the most effective being the deca-, undeca- and dodecamers. A similar relationship between size and effect was previously obtained when oligogalacturonides were tested for their ability to elicit lignification in cucumber hypocotyls. The oligogalacturonide-induced increase in H₂O₂ concentration was detected after 4 h, reaching a maximum after 10 h of incubation. The glucan elicitor induced lignification at a 100-fold lower concentration than the oligogalacturonides, but yielded only 10% of the maximum H₂O₂ accumulation seen with oligogalacturonides. The glucan elicitor-induced H₂O₂ production was detectable after 2 h, and reached a maximum after 4 to 6 h. Catalase abolished the elicitation of both phenol red oxidation and lignification in cucumber hypocotyls. At least part of the oligogalacturonide-induced H₂O₂ production appeared to be dependent upon de novo protein synthesis.     

Oligogalacturonides: novel signaling molecules in Rhizobium-legume communications

Oligogalacturonides are pectic fragments of the plant cell wall, whose signaling role has been described thus far during plant development and plant-pathogen interactions. In the present work, we evaluated the potential involvement of oligogalacturonides in the molecular communications between legumes and rhizobia during the establishment of nitrogen-fixing symbiosis. Oligogalacturonides with a degree of polymerization of 10 to 15 were found to trigger a rapid intracellular production of reactive oxygen species in Rhizobium leguminosarum bv. viciae 3841. Accumulation of H(2)O(2), detected by both 2',7'-dichlorodihydrofluorescein diacetate-based fluorescence and electron-dense deposits of cerium perhydroxides, was transient and did not affect bacterial cell viability, due to the prompt activation of the katG gene encoding a catalase. Calcium measurements carried out in R. leguminosarum transformed with the bioluminescent Ca(2+) reporter aequorin demonstrated the induction of a rapid and remarkable intracellular Ca(2+) increase in response to oligogalacturonides. When applied jointly with naringenin, oligogalacturonides effectively inhibited flavonoid-induced nod gene expression, indicating an antagonistic interplay between oligogalacturonides and inducing flavonoids in the early stages of plant root colonization. The above data suggest a novel role for oligogalacturonides as signaling molecules released in the rhizosphere in the initial rhizobium-legume interaction.     

Induction of defense responses in cultured parsley cells by plant cell wall fragments

Cell suspension cultures of parsley (Petroselinum crispum) accumulated coumarin phytoalexins and exhibited increased β-1,3-glucanase activity when treated with either a purified α-1,4-d-endopolygalacturonic acid lyase from Erwinia carotovora or oligogalacturonides solubilized from parsley cell walls by endopolygalacturonic acid lyase. Coumarin accumulation induced by the plant cell wall elicitor was preceded by increases in the activities of phenylalanine ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL) and S-adenosyl-l-methionine:xanthotoxol O-methyltransferase (XMT). The time courses for the changes in these three enzyme activities were similar to those observed in cell cultures treated with a fungal glucan elicitor. The plant cell wall elicitor was found to act synergistically with the fungal glucan elicitor in the induction of coumarin phytoalexins. As much as a 10-fold stimulation in coumarin accumulation above the calculated additive response was observed in cell cultures treated with combinations of plant and fungal elicitors. The synergistic effect was also observed for the induction of PAL, 4CL, and XMT activities. These results demonstrate that plant cell wall elicitors induce at least two distinct biochemical responses in parsley cells and further support the role of oligogalacturonides as important regulators of plant defense.     

Analysis of pectic epitopes recognised by hybridoma and phage display monoclonal antibodies using defined oligosaccharides, polysaccharides, and enzymatic degradation

The structure of epitopes recognised by anti-pectin monoclonal antibodies (mAbs) has been investigated using a series of model lime-pectin samples with defined degrees and patterns of methyl esterification, a range of defined oligogalacturonides and enzymatic degradation of pectic polysaccharides. In immuno-dot-assays, the anti-homogalacturonan (HG) mAbs JIM5 and JIM7 both bound to samples with a wide range of degrees of methyl esterification in preference to fully de-esterified samples. In contrast, the anti-HG phage display mAb PAM1 bound most effectively to fully de-esterified pectin. In competitive inhibition ELISAs using fully methyl-esterified or fully de-esterified oligogalacturonides with 3-9 galacturonic acid residues, JIM5 bound weakly to a fully de-esterified nonagalacturonide but JIM7 did not bind to any of the oligogalacturonides tested. Therefore, optimal JIM5 and JIM7 binding occurs where specific but undefined methyl-esterification patterns are present on HG domains, although fully de-esterified HG samples contain sub-optimal JIM5 epitopes. The persistence of mAb binding to epitopes in pectic antigens, with 41% blockwise esterification (P41) and 43% random esterification (F43) subject to fragmentation by endo-polygalacturonase II (PG II) and endo-pectin lyase (PL), was also studied. Time course analysis of PG II digestion of P41 revealed that JIM5 epitopes were rapidly degraded, but a low level of PAM1 and JIM7 epitopes existed even after extensive digestion, indicating that some HG domains were more resistant to cleavage by PG II. The chromatographic separation of fragments produced by the complete digestion of P41 by pectin lyase indicated that a very restricted population of fragments contained the PAM1 epitope while a (1-->4)-beta-D-galactan epitope occurring on the side chains of pectic polysaccharides was recovered in a broad range of fractions.     

Enzymatic properties and deduced amino acid sequence of a high-alkaline pectate lyase from an alkaliphilic Bacillus isolate

A high-alkaline pectate lyase (pectate trans-eliminase, EC 4.2.2.2.) from alkaliphilic Bacillus sp. strain KSM-P7, designated Pel-7, was purified to homogeneity. The purified Pel-7 had a molecular mass of approximately 33 kDa as determined by SDS-polyacrylamide gel electrophoresis. The isoelectric point was close to or higher than pH 10.5. In the presence of Ca2+ ions, Pel-7 trans-eliminated polygalacturonate in random manner to generate oligogalacturonides; it exhibited optimal activity at pH 10.5 and around at 60 to 65 degrees C in glycine-NaOH buffer. Mn2+ and Sr2+ ions can serve as cofactors at almost the same level of Ca2+ ions. It also exhibited a protopectinase-like activity, liberating soluble pectin and/or oligogalacturonides from cotton fibers. The pel gene was cloned and sequenced, and the deduced amino acid sequence of mature Pel-7 (302 amino acids, 33, 355 Da) showed some conserved regions in Pel superfamily, although homology to amino acid sequences of known Pels with 27 to 32% identity. Furthermore, Pel-7 appears to have similar core structure of parallel beta-helix and active site topology with other Pels as revealed by secondary structure prediction in the Pel proteins. These results suggest that Pel-7 is basically grouped into Pel superfamily although the enzymatic and molecular properties are different.     

Separation and quantitation of galacturonic acid oligomers from 3 to over 25 residues in length by anion-exchange high-performance liquid chromatography

A method for separation and quantitation of galacturonic acid oligomers from 3 to over 25 residues in length is described. Oligomers were labeled at the reducing end with 2-aminopyridine and then analyzed by anion-exchange high-performance liquid chromatography using a sodium acetate gradient. The amount of each oligogalacturonide present was determined by comparison to the response of an internal reference oligogalacturonide over a range from 0.5 to 20 nmol per oligomer. At least 5 h of incubation in the 2-aminopyridine reagent was required to obtain maximum and oligomer length-independent derivatization. To be analyzed using this technique, oligogalacturonides must possess a reducing terminus, they should be deesterified prior to derivatization if identification of the actual galacturonide chain length is desired, and they should fall within the range of 3 to over 25 galacturonide residues per oligomer. The wide range of oligogalacturonides separable, sensitivity of detection, ease of quantitation of chromatographic data, and ability to hydrolyze the 2-aminopyridinyl group from sugars makes this technique of potential use for numerous applications ranging from simple characterization of oligogalacturonide mixtures to purification of oligomers for use in bioassays.     

Several biotic and abiotic elicitors act synergistically in the induction of phytoalexin accumulation in soybean

Summary Plants often respond to microbial infection by producing antimicrobial compounds called phytoalexins. Plants also produce phytoalexins in response to in vitro treatment with molecules called elicitors. Specific elicitors, including a hexa--glucosyl glucitol derived from fungal cell walls, the pectin-degrading enzyme endopolygalacturonic acid lyase, and oligogalacturonides obtained by either partial acid hydrolysis or enzymatic degradation of plant cell walls or citrus polygalacturonic acid, induce soybean (Glycine max. L.) cytoledons to accumulate phytoalexins. The experiments reported here demonstrate that the elicitor-active hexa--glucosyl glucitol acts synergistically with several biotic and abiotic elicitors in the induction of phytoalexins in soybean cotyledons. At concentrations below 50 ng/ml, the hexa--glucosyl glucitol does not induce significant phytoalexin accumulation. When assayed in combination with either endopolygalacturonic acid lyase or with a decagalacturonide released from citrus polygalacturonic acid by this lyase, however, the observed elicitor activity of the hexa--glucosyl glucitol is as much as 35-fold higher than the sum of the responses of these elicitors assayed separately. A similar synergism was also demonstrated for the combination of the hexa--glucosyl glucitol with dilute solutions of sodium acetate, sodium formate, or sodium propionate buffers. These buffers are thought to damage or kill plant cells, which may cause the release of oligogalacturonides from the plant cell wall. The results suggest that oligogalacturonides act as signals of tissue damage and, as such, can enhance the response of plant tissues to other elicitor-active molecules during the initiation of phytoalexin accumulation.Supported by the United States Department of Energy DE-ACO2-84ER13161. This paper is number XXXI in a series, Host-Pathogen Interactions. The preceding paper, Host-Pathogen Interactions XXX is Characterization of elicitors of phytoalexin accumulation in soybean released from soybean cells by endopolygalacturonic acid lyase, by K. R. Davis, A. G. Darvill, P. Albersheim, and A. Dell. Zeitschrift für Naturforsschung, in press.