Host-Pathogen Interactions - NCBI

Plant Physiol. (1986) 80, 568-577 0032-0889/86/80/0568/10/$0 1.00/0

Host-Pathogen Interactions' XXIX. OLIGOGALACTURONIDES RELEASED FROM SODIUM POLYPECTATE BY ENDOPOLYGALACTURONIC ACID LYASE ARE ELICITORS OF PHYTOALEXINS IN SOYBEAN Received for publication June 10, 1985 and in revised form October 15, 1985

KEITH R. DAVIS2, ALAN G. DARVILL2, PETER ALBERSHEIM*2, AND ANNE DELL

Department of Molecular, Cellular and Developmental Biology and Department of Chemistry, University of Colorado, Campus Box 215, Boulder Colorado 80309 (K.R.D., A.G.D., P.A.); and Department of Biochemistry, Imperial College of Science and Technology, London SW7 2AZ, England (A.D.) toalexins (1, 1 1). Phytoalexins are a diverse group of compounds of low mol wt that have broad antibiotic activity against many prokaryotic and eukaryotic organisms (32, 36). In some hostpathogen combinations, phytoalexins accumulate at the site of attempted infection rapidly enough and in sufficient concentration to prevent further growth of the invading microorganism (19, 24). Phytoalexin accumulation is also induced by molecules called elicitors. Various compounds isolated from microorganisms have been found to have elicitor activity (reviewed in 1, 2, 6, 37). Molecules derived from plant tissues also induce phytoalexin

ABSTRACT Recent studies have demonstrated that an apparently homogeneous preparation of an a-1,4-D-endopolyplacturonic acid lyase (EC 4.2.2.2) isolated from the phytopathogenic bacterium Erwinia carotovora induced phytoalexin accumulation in cotyledons of soybean (Glycine max jL.1 Merr. cv Wayne) and that this pectin-degrading enzyme released heatstable elicitors of phytoalexins from soybean cell walls, citrus pectin, and sodium polypectate (KR Davis et al. 1984 Plant Physiol 74: 52-60). The present paper reports the purification, by anion-exchange chromatography on QAE-Sephadex columns followed by gel-permeation chromatography on a Bio-Gel P6 column, of the two fractions with highest specific elicitor activity present in a crude elicitor-preparation obtained by lyase treatment of sodium polypectate. Structural analysis of the fraction with highest specific elicitor activity indicated that the major, if not only, component was a decasaccharide of a-1,4-D-galactosyluronic acid that contained the expected product of lyase cleavage, 4-deoxy-,6-L-5-threohexopyranos-4-enyluronic acid (4,5-unsaturated galactosyluronic acid), at the nonreducing terminus. This modified decagalacturonide fraction exhibited half-maximum and maximum elicitor activity at 1 microgram/ cotyledon (6 micromolar) and 5 micrograms/cotyledon (32 micromolar) plactosyluronic acid equivalents, respectively. Reducing 90 to 95% of the carboxyl groups of the galactosyluronic acid residues abolished the elicitor activity of the decagalacturonide fraction. The second most elicitor-active fraction contained mostly undeca-a-1,4-D-galactosyluronic acid that contained 4,5-unsaturated gaactosyluronic acid at the nonreducing termini. This fraction exhibited half-maximum and maximum elicitor activity at approximately 3 micrograms/cotyledon (17 micromolar) and 6 micrograms/cotyledon (34 micromolar) galactosyluronic acid equivalents, respectively. These results confirm and extend previous observations that oligogalacturonides derived from the pectic polysaccharides of plant cell walls can serve as regulatory molecules that induce phytoalexin accumulation in soybean. These results are consistent with the hypothesis that oligogalacturonides play a role in disease resistance in plants.

Although plants are exposed to a multitude of potentially pathogenic microorganisms, successful infections are rare. Plants confronted with a potential pathogen use a variety of defense mechanisms (2). One well-characterized defense mechanism is the synthesis and accumulation of stress metabolites called phy-

accumulation (14-16, 23, 28). Recent work has demonstrated that a heat-labile elicitor of phytoalexins produced by the phytopathogenic bacterium Erwinia carotovora var carotovora is a pectin-degrading enzyme, PGA lyase3 (7). An apparently homogeneous preparation of the PGA lyase was shown to induce phytoalexin accumulation in soybean cotyledons and also to release uronic-acid rich, heatstable elicitors from soybean cell walls, citrus pectin, and sodium polypectate (7). Similar results have been obtained by West et al. (4, 18, 20, 21) with an endopolygalacturonase produced by the fungus Rhizopus stolonifer. The R. stolonifer endopolygalacturonase induced accumulation of casbene synthetase (an enzyme involved in the synthesis of the phytoalexin, casbene) in castor bean seedlings (20) and released heat-stable, GalUA-rich elicitors of casbene synthetase from particulate fractions of castor bean seedling homogenates, citrus pectin, and polygalacturonic acid (4, 18). The studies presented in this paper describe the purification and characterization of elicitor-active oligogalacturonides obtained by treating sodium polypectate with a purified PGA lyase from Erwinia carotovora. The results demonstrated that the oligogalacturonides with the highest specific elicitor activity were closely related, but not identical, to those obtained by partial acid hydrolysis of soybean cell walls and citrus pectin (28) and by treatment of polygalacturonic acid with endopolygalacturonin 1985 by K. R. D. The paper is number XXIX in a series, "HostPathogen Interactions." The preceding paper is "Comparison of the structures and elicitor activities of a synthetic and a mycelial-wall-derived hexa (#-D-glucopyranosyl)-D-glucitol", by J. K. Sharp, P. Albersheim, P. Ossowski, A. Pilotti, P. Garegg, and B. Lindberg 1984 Journal of Biological Chemistry 259: 11341-11345. 2 Present address: Complex Carbohydrate Research Center, University of Georgia, P. 0. Box 5677, Athens GA 30613. 'Abbreviations: PGA lyase, a-1,4-D-endopolygalacturonic acid lyase (EC 4.2.2.2); CMC, 1-cyclohexyl-342-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate; 6, chemical shift in ppm; FAB-MS, fast atom bombardment-mass spectrometry; m/z, mass-to-charge ratio.

'Supported by the United States Department of Energy (DE-AC0276ERO-1426) and The Rockefeller Foundation (RF79049). This paper is based on part of a Ph.D. thesis presented to the University of Colorado 568

OLIGOGALACTURONIDES ARE ELICITORS OF PHYTOALEXINS ase from

Rhizopus stolonifer (18). Preliminary reports of this

work have been published (8, 9).

MATERIALS AND METHODS Chemicals. Imidazole (Grade I), QAE Sephadex (A-25-120), Sephadex G-10, Dowex 50W-X8, CMC, streptomycin sulfate, and Tris were from Sigma Chemical Co. Bio-Gel P-6 gel (200400 mesh) was from Bio-Rad Laboratories. High purity (+)-2butanol and (-)-2-butanol were from Aldrich Chemical Co. TriSil silanizing reagent and 0.3 ml Reacti-Vials were from Pierce Chemical Co. Deuterium oxide (99.7-99.8 atom % 2H) was from Aldrich Chemical Co. or Stohler Isotope Chemicals (Waltham, MA). NaB(QH)4 was from Alfa Products, m-hydroxydiphenol was from Eastman Kodak, and Sep-Pak C18 cartridges were from Waters Associates. Solvents used for carbohydrate structural analyses were of Spectroanalyzed or HPLC grade and were purchased from Fisher Scientific Co. All other chemicals and solvents were of reagent grade or better. Carbohydrates. Sodium polypectate (sodium salt of polygalacturonic acid; Grade II) and D-galactose were from Sigma Chemical Co. D-GalUA was from Aldrich Chemical Co. and D-glucose was from Fisher Scientific Co. a- 1 ,4-Trigalactosyluronic acid was prepared as described (12). Galactaric acid and a-D-GalUA-( 1 -* 4)-a-D-GalUA41 -- 4)-a-galactaric acid were prepared from D-GalUA and a- 1 ,4-trigalactosyluronic acid, respectively, by bromine oxidation as described (33). A high mol wt mixture of phytoalexin elicitor-active fl-D-glucans, partially purified from a partial acid hydrolysate of cell walls of the phytopathogenic fungus, Phytophthora megasperma f. sp. glycinea (the void fraction from the low resolution P-2 column described in [30]), was provided by J. K. Sharp. Enzymes. Homogeneous a-1,4-D-endopolygalacturonic acid lyase (EC 4.2.2.2) was prepared from cultures of Erwinia carotovora var carotovora (ATCC No. 495) as described (7). The enzyme activity of the PGA lyase was measured spectrophotometrically (7). One unit of PGA lyase activity was the amount required to release 1 umol unsaturated products/min at 30°C. Colorimetric Assays. The m-hydroxydiphenyl method (3), with GalUA as the standard, was used to determine uronic acid concentrations. The anthrone method (10), with glucose or galactose as a standard, was used to measure hexose concentrations. Assay for Phytoalexin Elicitor Activity. The modification of the soybean-cotyledon assay previously described was used to determine elicitor activities (7). All samples assayed for elicitor activity were desalted by gel permeation chromatography on Sephadex G- 10 columns (1.0 x 18 cm) equilibrated with deionized H20. Samples were desalted twice, unless the original salt concentration was less than 50 mM; in that case, they were desalted once. The data are expressed as the A286 of the test sample relative to the A286 of a high mol wt glucan elicitor assayed at 0.18 gg/cotyledon, a concentration of glucan that induces maximum phytoalexin accumulation (A286/A286max). The A286 is directly proportional to the amount of pterocarpan phytoalexins present in the cotyledon wound-droplet solutions (14). The specific phytoalexin elicitor activity of different fractions was computed by converting the A286/A286max values to decagalacturonide eq per amount of GalUA applied to each cotyledon with a standard curve prepared from the data shown in Figure 4 (plotted as log concentration GalUA eq [x] versus A286max [y]). This conversion corrects for the nonlinear response of the cotyledons to different concentrations of elicitor (14). Analysis of Glycosyl-Residue and Glycosyl-Linkage Compositions. Glycosyl-residue compositions were determined by preparing the trimethylsilyl methyl glycoside, methyl ester derivatives (5), which were analyzed by flame-ionization GLC and by GLC-MS. GLC analyses of these derivatives were performed on

569

a 0.25 mm x 15 m fused-silica capillary column (DB-1; J and W Scientific Co.) fitted in a Hewlett-Packard 5880A gas chromatograph with the following temperature program: 2 min at

the injection temperature of 120°C, 15°C/min to 150°C, 2°C/ min to 190°C, 30°C/min to 240°C, and then 10 min at 240°C. Peak areas were quantitated and adjusted for differences in yield and detection with correction factors obtained by analyzing known amounts of the appropriate sugar standards. The relative amount of each glycosyl residue, expressed as weight percent (wt %), was calculated by dividing the peak areas of the derivatives of a particular glycosyl residue by the total area of all the derivatives detected and multiplying by 100. GLC-MS analyses were performed with a Hewlett-Packard model 5985 GLC-MS system fitted with a 0.33 mm x 15 m fused-silica capillary column (DB-1; J and W Scientific Co.). Samples were injected in the on-column mode and separations were achieved with the following temperature program: 2 min at the injection temperature of 50C, 30°C/min to 140C, and then 4°C/min (or, in some cases, 2°C/min) to 200°C. The eluted compounds were detected by either electron impact-mass spectrometry (EI-MS) or chemical ionization-mass spectrometry (CI-MS) with isobutane as the ionization gas. Glycosyl-linkage compositions were determined by the methylation analysis method as described by Waeghe et al. (35). Prior to being per-O-methylated, the underivatized, desalted oligogalacturonides were subjected to carboxyl reduction by a modification of published procedures (34). Oligogalacturonides (500 ,ug GalUA eq) were dissolved in 1.0 ml of 2H20. A 10-fold molar excess of CMC was added and an automatic titrator (Radiometer Copenhagen) was used to maintain the pH at 4.75 with 0.1 M HCI in 2H20. After the CMC reaction was complete, 2.0 ml of 7 M NaB2H)4 in 2H20 was added dropwise. The pH of the reaction mixture was automatically maintained at 7.0 with 4 M HCI in 2H20. The reaction vessel was kept in a cool water bath (- I 5C) to dissipate heat generated during the reduction. The reduction was judged complete when hydrogen gas evolution ceased. The pH was adjusted to 5.0 and the reaction mixture (8-10 ml total) was desalted by gel permeation chromatography on a Sephadex G-10 column (2.0 x 18.5 cm) equilibrated with deionized H20. The desalted material was concentrated to approximately 2 ml by rotoevaporation under reduced pressure and transferred to a test tube, where the remaining H20 was removed by evaporation under a stream of filtered air. Borate-esters were hydrolyzed by dissolving the carboxyl-reduced oligosaccharides in 0.2 ml H20, combining the dissolved oligosaccharides with 0.5 ml of 10% acetic acid/methanol (v/v), and evaporating to dryness with filtered air at room temperature. This was repeated three times, except that the volume was reduced to half that of the first evaporation. Two evaporations with 0.5 ml 10% acetic acid/ methanol (v/v) and three evaporations with 0.5 ml of methanol removed the remaining borate as trimethyl borate. Previous studies indicated that including several evaporations in which reduced oligosaccharides are dissolved in H20 before acidic methanol is added hydrolyzes borate-esters more effectively than evaporating directly from acidic methanol (26). Salt was removed by passing the borate-free, reduced oligosaccharides through Dowex 50 (H+ form) equilibrated with deionized H20; the desalted material was lyophilized. GLC analysis of the trimethylsilyl methyl glycoside derivatives of the carboxyl-reduced oligosaccharides demonstrated that 90 to 95% of the GalUA residues were reduced to the corresponding 6,6-dideuterio-labeled galactosyl residues by this procedure. The reduced oligosaccharides were subjected to per-O-methylation, a second carboxylreduction with NaB(2H)4 in 95% ethanol/oxalane (v/v), a second per-O-methylation, and hydrolysis as described (35). The resulting, partially O-methylated aldoses were then reduced to the corresponding, partially O-methylated alditols and O-acetylated

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(35). The resulting, partially O-methylated alditol acetates were analyzed by GLC-MS as described (35). Determination of Absolute Configurations. The absolute configurations of glycosyl residues were determined by preparing the trimethylsilyl butyl glycoside derivatives and analyzing the derivatives by GLC (13) as previously described (28). 'H-NMR Spectrometry. 'H-NMR spectra were recorded on a Bruker WM-250 Fourier transform spectrometer operating at 250 MHz. Prior to analysis, samples (250 or 500 ,g GalUA eq) were lyophilized in the presence of imidazole-HCl buffer-three times from 99.7 to 99.8 atom 2H20 and once from 99.96 atom % 2H20. The samples were then dissolved in 0.5 ml of 99.96 atom % 2H20 and transferred to an NMR tube for analysis. The final concentration of imidazole-HCl was approximately 0.2 M (pH 7.0). A standard containing 0.2 M imidazole-HCl (pH 7.0) and sodium 2,2,3,3-tetradeuterio-4,4-dimethyl-4-silapentanoate was used to calibrate the spectrometer so that the chemical shift of sodium 2,2,3,3-tetradeuterio-4,4-dimethyl-4-silapentanoate was equal to 0.00. The temperature of the sample was maintained at 75C while data was collected. In some cases, solvent suppression was used to minimize the intensity of signals resulting from 2HHO and imidazole. FAB-MS. The mol wt of the underivatized oligogalacturonides were determined by FAB-MS by a modification of a described procedure (28). Prior to analysis, the samples were dissolved in 5 to 10 gl of 5% (v/v) aqueous acetic acid; 1 ,ul of the dissolved sample was added to a drop of a thioglycerol-glycerol mixture on the target. After partial evaporation of the solvent in the vacuum of the mass spectrometer source, a 1 ,ul aliquot of 0.1 M HCI was added to the target. All analyses were performed in the negative-ion mode. Adding 0.1 M HCI to the sample greatly inhibited salts from forming and thereby promoted the detection of molecular ions ([M-H]-). UV-Visible Spectrophotometry. UV-visible spectra were obtained with a scanning UV-visible spectrophotometer (Perkin Elmer Model 330, kindly provided by Dr. A. Staehelin). Spectra of desalted samples diluted with distilled H20 to a final concentration of 1.8 x l0-' M were obtained at 20°C. Preparation of Oligogalacturonide Elicitors. Oligogalacturonide elicitors were prepared by adding 1.6 units of PGA lyase to each of five plastic bottles containing 150 ml of 1 mg/ml sodium polypectate in buffer (5 mm Tris-HCI, 1 mm CaCl2 [pH 8.5]). The reaction mixtures were incubated, with agitation, at 30C in a water bath. The reaction in each bottle was monitored by removing 0.1 ml aliquots, diluting each aliquot to a total volume of 1.0 ml with distilled H20, and measuring the amount of 4,5unsaturated GalUA by determining the A at 235 nm. Previous experiments indicated that maximum production of elicitor activity correlated with a net increase in the A235 of 1.0 (corrected for the 1:10 dilution) (7; KR Davis, AG Darvill, P Albersheim,

to 0.1

A Dell, unpublished results). When a net increase in the A235 of

Table I. Elicitor Activity and GalUA Content of Fractions Obtained by QAE-Sephadex Anion-Exchange Chromatography of Sodium Polypectate Treated with PGA Lyase Total GalUA eq Fraction' Elicitor Activityb Recovered mg III 0.37 9.2 IV 19.9 0.04 V 0.42 176.0 VI 0.23 75.0 VII 0.08 104.5 VIII 0.07 16.1 a Fractions are as described in Figure 1. All samples were assayed at b The data shown for each sample is the 10 ,g/cotyledon GalUA eq. average A286/A286max obtained from 40 cotyledons, except fractions VII and VIII, which are the averages of 20 cotyledons.

%

between 0.99 to 1.14 was observed (approximately 2 h incubation), the enzyme reactions were stopped by adjusting the pH of the reaction mixtures to between 4.8 and 5.2 with 1 M HCI. The reaction mixtures in the five bottles were combined and heated at 65 to 80°C for 45 min to inactivate the PGA lyase. This heattreated material constituted the PGA-lyase digest of sodium polypectate used for the studies described below.

RESULTS Purification of the most elicitor-active components in the PGA-lyase digest of sodium polypectate was accomplished by modification of methods used to purify oligogalacturonides released from soybean cell walls and from citrus pectin by partial acid hydrolysis (28). Anion-Exchange Chromatography of the Sodium-Polypectate Digest. The PGA-lyase digest of sodium polypectate was brought

M imidazole-HCl and applied to a QAE-Sephadex anionexchange column. The bound compounds were eluted stepwise with increasing concentrations of imidazole-HCI buffer, and the hexose and uronic acid contents ofthe fractions were determined (Fig. 1). Aliquots of the fractions were desalted and assayed for elicitor activity (Table I). The elicitor activities of the void material and fractions I, II, and IX could not be determined because insufficient material was recovered. Fraction V possessed the highest specific elicitor activity, while fractions III and VI exhibited 67 and 25%, respectively, ofthe specific elicitor activity of fraction V. Fractions IV, VII, and VIII had little elicitor activity. Approximately 83% of the original GalUA eq applied to the

2.0

0.125 M

LOADING

1.6

0.95M

0.75 M

0.55 M

2.OM

URONIC ACID HEXOSE

E z

i.2

0

1

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1.8

ELUTION VOLUME (liters) FIG. 1. Anion-exchange chromatography of the PGA-lyase digest of sodium polypectate. The PGA-lyase-treated sodium polypectate (480 mg GalUA eq in a total volume of 820 ml), adjusted to 0.1 M imidazole-HCl by the addition of 100 ml of 0.95 M imidazole-HCl (pH 7.0), was applied to a QAE-Sephadex column (3.0 x 13.5 cm) equilibrated with 0.125 M imidazole-HCl (pH 7.0). The column was washed with 200 ml of 0.125 M imidazole-HCl (pH 7.0) and then washed stepwise, with 200 ml each of 0.55, 0.75, 0.95, and 2.0 M imidazole-HCl (pH 7.0). Fractions (100150 ml) were collected as shown and assayed colorimetrically for uronic acid and hexose content, with GalUA and glucose, respectively, as standards. The concentrations of hexose shown have not been corrected for the response of uronic acid in the hexose assay (GalUA gives -5% of the response of glucose). The elicitor activities of the fractions are presented in Table I.

571

OLIGOGALACTURONIDES ARE ELICITORS OF PHYTOALEXINS column were recovered in fractions III through VIII, inclusive (401 mg). Approximately 43% of the original elicitor activity applied to the column was recovered in these fractions. The loss of 57% of the original elicitor activity probably resulted from a loss of activity of the larger oligogalacturonides that became insoluble after QAE-Sephadex purification and desalting. Test samples containing purified larger oligogalacturonides (QAE fractions V-IX) were turbid, which suggested that the oligogalacturonides were forming insoluble aggregates. Test samples prepared from the crude elicitor were less turbid than those of the more purified large oligogalacturonides. Since these larger oligogalacturonides accounted for the majority of GalUA eq recovered from the sodium-polypectate digest, a loss of elicitor activity due to gelation of these molecules would account for the apparent loss of total elicitor activity. Similar observations were made during the purification of oligogalacturonide elicitors obtained by partial acid hydrolysis of soybean cell walls and citrus pectin (28). To investigate the possibility that removal of smaller oligogalacturonides during QAE-Sephadex chromatography decreased the elicitor activity of large oligogalacturonides, small-scale purifications of the crude elicitor preparations on QAE-Sephadex columns were conducted. Approximately 1 mg GalUA eq of the PGA-lyase digest of sodium polypectate was applied to a QAESephadex column (0.7 x 5.0 cm) equilibrated with 0.125 M imidazole-HCl (pH 7.0). The column was washed with 5 ml of 0.125 M imidazole-HCl (pH 7.0) and the bound material was eluted from the column stepwise with 4 ml of 1 M imidazoleHCI (pH 7.0). The fractions obtained were concentrated, desalted, and assayed for uronic acid content and elicitor activity. In each of three separate experiments, approximately 80% of the GalUA eq applied to the column was recovered in the 1 M imidazole-HCl wash. The specific elicitor activity of the 1 M imidazole-HCl wash was equivalent to that of the sodium-polypectate digest applied to the column. These results support the hypothesis that the larger oligogalacturonides remain soluble and elicitor-active in the presence of smaller oligogalacturonides. These results also rule out the possibility that unidentified components with elicitor activity or factors that promote the elicitor activity of oligogalacturonides were removed during QAE-Sephadex chromatography. High-Resolution Anion-Exchange Chromatography. Further purification was conducted to identify the elicitor-active components in the QAE fractions described above. Fractions III, IV, V, and VI from the first anion-exchange column (Fig. 1) were pooled, diluted, and applied to a high-resolution QAE-Sephadex anion-exchange column. The compounds that bound to the column were eluted with a linear gradient of increasing concentrations of imidazole-HCl buffer (Fig. 2). Aliquots from selected fractions were assayed for hexose and uronic acid content. Approximately 13 different uronic acid-rich fractions were separated (peaks 1-13). FAB-MS analyses of high-resolution QAE-Sephadex column fractions demonstrated that each successive, major peak contained oligogalacturonides that had one more GalUA residue than the oligogalacturonides in the preceding peak (28; KR Davis, AG Darvill, P Albersheim, A Dell, unpublished results). In the elution profile in Figure 2, peak 1 contains trigalactosyluronic acid; peak 3, tetragalactosyluronic acid; and so on. Minor peaks 2 and 7 do not follow this pattern; they contain oligosaccharides, of undetermined sizes, that contain significant quantities of glycosyl residues other than GalUA. Aliquots of selected fractions from each of the major peaks were desalted and assayed for elicitor activity (Table II). Peak 10 (fractions 211-216) possessed the highest specific elicitor activity. Peak 1I1 (fractions 219-222), the fractions with the second highest specific elicitor activity, possessed approximately 50% of the

specific elicitor activity of peak

10.

Lower specific elicitor activity

I.

0.6

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cr~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I 2tA

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90

170

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250

FRACTION NUMBER FIG. 2.

High-resolution

anion-exchange

chromatography of QAE

fractions III, IV, V, and VI (Fig. 1). The pooled fractions diluted with distilled H20 to a conductivity equivalent to that of 0.125 imidazole-HCI (-5 mhmos) and applied to high-resolution QAEwere

m

a

Sephadex (A-25-120) column (1.7

x

45.0 cm)

imidazole-HCI (pH 7.0). Approximately 274 onto the column in a total volume

with 200 ml of 0. 125 were m

m

eluted with 2.4 L of

m

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loaded

was

washed

of 1.8 L and the column

imidazole-HCI (pH 7.0).

m

were

The bound materials

gradient of imidazole-HCI (0. 125-0.75

m imidazole-HCI (pH 7.0), and finally imidazole-HCI (pH 7.0). Fractions of 9.8 ml were collected

[pH 7.0]) then 150

150 ml 0.95

equilibrated with 0. 125

mg GalUA eq

ml of 0.75

and aliquots assayed colorimetrically for uronic acid and hexose content, with GalUA and glucose, respectively, as standards. The hexose concentrations shown have not been corrected for the response of uronic acids in the hexose assay (GalUA gives -5% of the response.of glucose). Elicitor activities of these fractions are presented in Table II. Table II. Elicitor Activity and GalUA Content of Fractions Obtained by High-Resolution QAE-Sephadex Anion-Exchange Chromatography of Pooled QAE Fractions III to VI Elicitor Total GalUA eq Activityb Recovered

mg 1 103-105 0.03 5.2 133-135 3 0.00 4.7 4 154-155 0.07 8.6 169-170 5 0.10 12.0 184-185 6 0.19 6.9 7 190-191 0.26 2.3 195-196 8 0.14 4.7 204-206 9 0.22 4.8 211-216 10 0.52 7.9 219-222 11 0.44 6.2 225-227 12 0.38 5.4 231-232 13 0.18 3.7 a b All samples were assayed at 10 gg/cotyledon GalUA eq. The data shown for each sample is the average A286/A286, obtained from 20

cotyledons. was associated with other fractions that eluted before and after peaks 10 and 11. Components that eluted from the high-resolution QAE-Sephadex column in 0.75 and 0.95 M buffer washes had less than 10% of the specific elicitor activity of peak 10. The GalUA eq applied to the high-resolution QAE column were recovered quantitatively. The fractions comprising peaks 1

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DAVIS ET AL.

to 13 contained approximately 25% of the GalUA eq applied to the column and approximately 13% of the total elicitor activity applied to the column. Fractions 237 to 277 contained approximately 75% of the GalUA eq applied to the column and approximately 13% of the total elicitor activity applied to the column. As discussed above, the apparent loss of elicitor activity was probably due to the insolubility of the later eluting fractions (> fraction 220) under the bioassay conditions (that is, after the removal of smaller oligogalacturonides and imidazole buffer). The glycosyl composition ofaliquots ofdesalted fractions from QAE peaks 5 to 13 was determined. These results are shown in Table III and are compared to the composition of sodium polypectate. All the peaks were rich in GalUA, which was expected because GalUA residues accounted for 87% (w/w) of the carbohydrate in the starting material. In comparison to sodium polypectate, peaks 7 to 12 were enriched for xylose and galactaric acid (the oxidized C-1-carboxyl derivative of GalUA). All the peaks examined contained small amounts of rhamnosyl, galactosyl, and glucosyl residues. The trimethylsilyl methyl glycoside derivatives of the 4,5-unsaturated GalUA residues were not identifiable; most likely the 4,5-unsaturated GalUA residues were degraded during methanolysis. However, the oligosaccharides absorbed strongly at 235 nm, which established that the lyasegenerated, 4,5-unsaturated GalUA residue was certainly present before methanolysis (17). The compounds present in peaks 10 and 11, the fractions with the highest specific elicitor activity, contained significant amounts of rhamnosyl, xylosyl, and galactaric acid residues. This raised the possibility that one of these residues might be present in the elicitor-active molecules and could be required for elicitor activity. To investigate this possibility, QAE peaks 10 and 11 were further fractionated by gel permeation chromatography. Bio-Gel P-6 Gel Permeation Chromatography of QAE Peaks 10 and 11. The remaining portions of fractions 211 to 216 from peak 10 and fractions 219 to 222 from peak 11 were separately pooled, concentrated, and desalted. The desalted samples were applied individually to a Bio-Gel P-6 gel permeation column. The elution profiles obtained for peaks 10 and 11 are shown in Figure 3, A and D, respectively. Peak 10 was fractionated into three major components: a void fraction and two partially included fractions, peaks A and B. The fractions were pooled as indicated by the bars (Fig. 3A). Analysis of the uronic acid content of the pooled fractions demonstrated that the void fraction, peak A, and peak B contained approximately 27, 53, and 20% (w/w), respectively, of the original GalUA eq applied to the column. The total amount of elicitor activity applied to the P-6 column was recovered quantitatively in the three fractions (void, peak A, and peak B); greater than 95% of the elicitor activity was recovered in peak A. The pooled fractions for peaks A and B were concentrated and reapplied separately to the P-6 column. The elution profiles obtained for peaks A and B are shown in Figure 3, B and C, respectively. The

5Plant Physiol. Vol. 80, 1986

fractions from the second gel permeation chromatography of peaks A and B were pooled as indicated by the bars (Fig. 3, B and C) and designated P-6-purified fractions Alo and Blo. QAE peak 11 was fractionated into two major components: a void fraction and partially included peak A (Fig. 3D). The void fraction and peak A contained approximately 19 and 81%, respectively, of the original GalUA eq applied to the column. The total amount of elicitor activity applied to the column was recovered quantitatively in these two fractions, with greater than 85% of the elicitor activity recovered in peak A. The fractions were pooled as indicated by the bars (Fig. 3D). The peak A fractions were concentrated and reapplied to the P-6 column. The elution profile obtained is shown in Figure 3E. The fractions were pooled as indicated by the bars (Fig. 3E) and designated P6-purified fraction A,. Aliquots from the concentrated P-6-purified fractions from QAE peaks 10 and 1 1 were desalted and the elicitor activities were determined (Table IV). Fraction A1o was the most elicitoractive fraction obtained from QAE peak 10. Similarly, fraction A,, was the most elicitor-active fraction obtained from QAE peak 11. The response of soybean cotyledons to different amounts of fractions A,o and Al, is shown in Figure 4. Reduction of 90 to 95% of the GalUA carboxyl groups of fraction Alo abolished its elicitor activity (Fig. 4). The glycosyl compositions of the P-6 purified fractions from QAE peaks 10 and 11 are presented in Table V. The elicitoractive fractions A.0 and A,1 were very rich in GalUA and contained only small amounts of xylosyl, rhamnosyl, and glucosyl residues-the sum of which was not nearly enough to constitute a single residue per oligosaccharide. The elicitor-inactive void fractions contained 60 to 70% GalUA and significant amounts of xylosyl, galactosyl, and glucosyl residues. Fraction B1o contained 89% GalUA and 8% (w/w) galactaric acid, about 8-fold more galactaric acid, on a per weight basis, than that present in sodium polypectate. These results strongly suggest that the elicitor-active molecules were pure oligogalacturonides. Further structural studies were done on the elicitor-inactive fraction B1o and the elicitor-active fractions A0o and Al, to define more precisely the structural features necessary for elicitor activity. Glycosyl-Linkage-Composition Analysis of Fractions Alo, Blo, and Al1. The glycosyl-linkage composition of the GalUA residues present in fractions A1o, B1o, and A,, was examined by methylation analysis (35). The results obtained indicated the presence of only 4-linked and terminal GalUA residues in all three samples. Significant undermethylation of C-6 (10-20%) was noted in all analyses. The reason for the relatively high level of undermethylation of C-6 in these compounds is not known; the undermethylated derivatives clearly originated from GalUA residues, however, since they contained the two deuterium atoms expected in carboxyl-reduced residues. No derivatives of the unsaturated GalUA residues were detected. The presence of the unsaturated residue within the oligogalacturonides in fraction Alo was con-

Table III. Glycosyl Composition of Sodium Polypectate and Fractions Obtained by High-Resolution QAESephadex Anion-Exchange Chromatography of Pooled QAE Fractions III to VI Sodium

Polypectate

5

6

7

QAE Peak Numbera 8 9 10

11

12

13

wt % 97.9 95.7 85.0 91.6 82.6 90.7 92.8 93.9 94.9 87.3 Galactosyluronic acid 2.4 3.2 1.6 1.2 2.9 1.5 1.2 1.2 1.3 1.4 Rhamnosyl 2.3 5.2 9.2 5.3 4.3 0.4 2.0 7.3 3.0 3.1 Xylosyl 1.0 0.6 0.6 0.7 0.6 0.7 0.5 4.1 1.8 0.8 Galactosyl 0.4 0.2 0.2 0.4 0.8 T 0.4 0.5 1.9 T" Glucosyl 2.9 1.1 0.8 5.6 0.8 0.4 3.4 1.0 NDC ND Galactaric acid b Trace amounts (