Purification and Partial Characterization of a 8-Glucan Fragment That ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists,

Vol. 259, No. 18, Issue of September 25, pp. 11312-11320,1984 Printed in U.S.A.

Inc.

Purification and Partial Characterization ofa 8-Glucan Fragment That Elicits PhytoalexinAccumulation in Soybean* (Received for publication, February 13,1984)

Janice K. Sharp$, Barbara Valent, and Peter Albersheimt From the Department of Chemistry, University of Colorado, Boulder, Colorado 80309

The synthesis of phytoalexins (antibiotics) in plant cells is induced by molecules called elicitors. Partial acid hydrolysis of mycelial walls of Phytophthora megasperma f. sp. glycinea solubilized a multicomponent mixture of elicitor-active and elicitor-inactive oligoglucosides. One elicitor-active and seven elicitor-inactive hexa@-D-glucopyranosyl)-D-glucitols were purified by gel-filtration, normal-phasepartition,and reversed-phase liquid chromatography after reduction with NaBH4. The elicitor-active and all but one of the elicitor-inactive hexa@-D-glucopyranosy1)-D-glucitols consisted of 3-, 6-, and 3,g-linked glucopyranosyl residues. Theeighth hexa@-D-glucopyranosyl)-D-glucitol consisted of 8-4-linked glucopyranosyl residues. The of of the similarity of the structural characteristics six elicitor-inactive hexa@-D-glucopyran0syl)-D-glucitols to the elicitor-active hexa@-D-glucopyranosy1)-D-glucitolsuggestedthatahighlyspecificcarbohydrate structure was required forelicitor activity.

The most studied defense mechanism of plants is the accumulation of phytoalexins at the site of infection (1).Phytoalexins, which are absent inhealthy plants, arehydrophobic compounds of low molecular weightthat are staticor toxic to a broad range of microorganisms (2). The biosynthesis of phytoalexins is induced by molecules called elicitors (3),which may be of abiotic or biotic origin. Elicitors of biotic origin, some or all of which probably play a role in host-pathogen interactions (4), include fungal mycelial wall carbohydrates (5-lo),enzymes that hydrolyze pectic polysaccharides of plant cell walls (11-13),fungal lipids (14),and plant cell-wall oligogalactosyluronides(11, 15, 16). The accumulation of phytoalexins in response to elicitors in true bean (17,18),parsley (19-21),pea (22), and soybean (23) is a result of de novo synthesis of the enzymes that catalyze the synthesis of the phytoalexins. For example, in soybean, elicitors trigger increases in the activities of those enzymes of phenylpropanoid metabolism that catalyze the biosynthesis of the pterocarpan phytoalexins of soybean. The enzymes are coordinately induced and synthesized de novo upon elicitation (23, 24).

* This work was supported by The Rockefeller Foundation Grant RF 81042-2 and Department of Energy Contract DE-AC02-76ERO1426. This paper is number XXVI in a series, Host-Pathogen Interactions. For the preceding paper, see Ref. 11. This paper was based in part on the 1983 Ph.D. thesis of J. K. Sharp, University of Colorado, Boulder, 80309. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Present address, Rijksuniversiteit Gent, Laboratorium voor Genetika, Ledeganckstraat 35, B-900 Gent, Belgium. § To whom correspondence should be addressed.

The mRNAs that encode the enzymes are also synthesized de novo (25, 26), establishing that elicitors are molecules capable of regulating specific metabolic pathways at thelevel of DNA transcription. The mechanism by which elicitors regulate gene expression is of great interest, andthese systems are among the very best available for the study of gene expression in plants. One of the most potent biotic elicitors-a 8-glucan fragment of the mycelial walls of the fungal pathogen Pmgl-was one of the first elicitors discovered (5-7).As little as 10 ng of the Pmg 8-glucan applied to 1 g of plant tissue elicited sufficient quantities of phytoalexins to inhibit microbial growth (27). Therefore, the elucidation of the structure of this physiologically important elicitor would bea steptoward understanding the mechanisms underlying elicitation of phytoalexins. The 8-glucan elicitor was first isolated from the culture filtrate of Pmg and was found to be a carbohydrate structurally similar to portions of the mycelial wall (5). Characterization of a partially purified elicitor-active fraction from Pmg mycelial walls has shown that the elicitor-active molecules were 8-glucans consisting of terminal, 3-, 6-, and 3,6-linked glucosyl residues (6, 7). The general nature of the @-glucan elicitor was determined, but its exact structure and, indeed, the exactness of its structure had not been elucidated. The purification and structural analysis of the smallest elicitoractive and some structurally related elicitor-inactive 8-glucan fragments are reported here and in the following paper. MATERIALS AND METHODS* RESULTS

Purification of Elicitor-active and -inactive Oligosaccharides Release of Elicitor-active Oligosaccharides from Pmg Mycelial Walk-Pmg mycelial walls accounted for 3% of the wet weight of the harvested mycelia. These walls contained 93% carbohydrate and 7% protein (27). Partial acid hydrolysis of the Pmg mycelial walls solubilized 50% of the carbohydrate present in the walls. Protein accounted for 2.6% of the solubilized material as determined by ‘The abbreviations used are: Pmg, Phytophthora megasperma f. sp. glycinea; FAB-MS, fast-atom bombardment mass spectrometry; ‘H NMR, proton nuclear magnetic resonance; HPLC, high-performance liquid chromatography; ODs, octadecylsilyl; PAC, polar aminocyano; RPLC, reversed-phase liquid chromatography; TMs, trimethylsilyl. * “Materials and Methods” are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 84M-0441, cite the authors, and include a check or money order for $3.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

11312

Purification of a Heptaglucoside Elicitor of Phytoalexins the ratio of the A m to theAzso(33). Carbohydrate accounted for 97%of the solubilized material of which 3.7%was mannose and 96.3% was glucose,as determined by GLC analysis of the alditol-acetate derivatives (34). The acid-solubilized material had a specific elicitor activity of 420 unitslpg of Glc (Table I). Purification of elicitor active and -inactive oligoglucosides from 10 g of Pmg mycelial walls was done three times. The data presented below, unless otherwise indicated, are from thethird purification andare representative of all three purifications. Low-resolution Gel-filtration Chromatography of the Acidsolubilized Material from Pmg Mycelial Walls-The acid-solubilized material was fractionated on the low-resolution BioGel P-2 column (Fig. 1). The mixtures of oligosaccharides composed of six sugars and larger had elicitor activity (Fig. 1). The pooled fractions (Fig. 1, shaded region) contained oligosaccharides of degrees of polymerization of 5 to 9 and had a specific elicitor activity of 83 units/pg of Glc (Table I). These elicitor-active oligosaccharides contained 3% of the elicitor activity and 12%of the carbohydrate of the totalacidsolubilized material. High-resolution Gel-filtration Chromatography of the Lawresolution P-2-purified Elicitor-active Oligosaccharides-The elicitor-active oligosaccharides were further purified on the high-resolution Bio-Gel P-2 column (Fig. 2). The per cent protein of the P-2 purified oligoglucoside fractions was determined by the ninhydrin assay using glycine as anamino acid standard (35). The void material was 50% protein and contained some elicitor-active oligosaccharides (data notshown). The material in the partially included delineated peaks contained less than 1% protein and the carbohydrate was 100% glucose as determined by GLC analysis of the alditol-acetate derivatives (34). However, verylow amounts of mannose (less than 0.5%) may not have been detected. The three peaks labeled 6,7, and 8 (Fig. 2) were shown by FAB-MS to contain predominantly hexa-, hepta-, andoctaglucosides,respectively. The hexaglucoside fraction and all of the larger oligoglucoside fractions exhibited elicitor activity. The octaglucoside fraction had a specific elicitor activity of 63 units/pg of Glc; the heptaglucoside fraction, 25 units/pg of Glc; and the hexaglucoside fraction, 2.5 units/pg of Glc. NaBH, Reduction of the Hem- and Heptaglucoside Fractions-The hexaglucoside fraction contained the smallest oligoglucosideswith elicitor activity. Considering the incomplete separation of hexaglucosides from heptaglucosides (Fig. 2), the elicitor activity of the hexaglucoside fraction was possibly

11313

from carry-over of elicitor-active heptaglucosides. Therefore, both fractions were further analyzed. The hexaglucoside and heptaglucoside fractions were reduced with N&Hl to convert' all of the oligoglucosides to their alditol forms. This was done to reduce the number of molecular species in each fraction for the subsequent purification steps (see below). The specific elicitor activity of the hexaglucosyl-glucitols prepared from the heptaglucoaide fraction was 25 units/pg of Glc and was indistinguishable from that of the unreduced heptaglucoside fraction (Fig. 3). The specific elicitor activity of the pentaglucosyl-glucitols prepared from the hexaglucoside fraction was 1.25 units/pg of Glc,half that of the hexaglucoside fraction. The specific elicitor activity of the pentaglucosyl-glucitol fraction was too low to confidently follow in further purification steps of the elicitor-active hexaglucoside component. Therefore, the elicitor-active component of the hexaglucosyl-glucitol fraction was selected for further purification and characterization. Normal-phase Partition Chromatography of Heptaglucoside andHexaglucosyl-glucitolFractionsonthe Whatman PAC Column-Both heptaglucoside and hexaglucosyl-glucitol fractions were further purified on the PAC column to determine which mixture was better separated. The carbohydrate and elicitor-activity profiles of heptaglucoside and hexglucosylglucitol fractions were similar, although both recovery of carbohydrate and peak resolution were slightly better with the hexaglucosyl-glucitol fraction (data notshown). Five major peaks were obtained (Fig. 41, of which the most elicitoractive was pooled (Fig.4, shaded region).The pooled fractions had a specific elicitor activity of 71 units/pg of Glc-a %fold increase over the specific elicitor activity of the hexaglucosylglucitol mixture applied to thecolumn (Table I). The most concentrated elicitor activity was in the pooled fractions, although only 30% of the original elicitor activity was recovered (Fig.4, shaded region). Other, less concentrated elicitor activity in surrounding fractions was due primarily to dispersion of the major elicitor-active component and accounted for another 30% of the elicitor activity applied to the column (data not shown). Routine losses of up to 50% of applied carbohydrate were observed with the PAC column and accounted for the remainder of the elicitor activity lost in this purification step. Reversed-phase Liquid Chromatography of the Elicitor-active PAC-purified Hexaglucosyl-glucitok on Supelco Spherisorb-5 ODs Columns-The separation of oligosaccharides on reversed-phase columns in water (36) is complicated by the separation of the a and /3 anomers of each oligosaccharide.

TABLE I Purification of the elicitor-active hexaglucosyl-glucitol from the partidy hydrolyzedPmg cell walls Glucose equivalents (A), half-maximal elicitor activity, and specific elicitor activity ( B ) weredetermined aa described in the text. The per cent activity recovered was determined by the equation E , X A,,/E,, X An-,. The purification factor was calculated by dividing the specific elicitor activity of step n by step n + 1. The per cent activity recovered for each step could be determined only after the high-resolution P-2-purificationstep because of the presence of an undetermined amount of larger, elicitor-active oligoglucosides in previous purification steps. Sample

Cells walls oligosaccharides Acid-solubilized Low-resolution458 P-2 oligoglucosides High-resolutionP-2 hexaglucosyl-glu100 citols 2hexaglucosyl-glucitols PAC-purified Hexaglucosyl-glucitolS-IV -V Hexaglucosyl-glucitol

Glucose equivalents

Half-maximal elicitor activity

Specific elicitor activity

Elicitor activity recovered

mg

nglcotyledon

unhjpg Glc

%

F'urification factor

5200 25 15

68.0

0.280 11 0.100

12 40 1.1

83

71 910 1800

30

3

38 76

r+

11314

Purification of a Heptuglucoside Elicitor of Phytoalexins

Y

t

0 900

IO"

0.l300~

0.700

A620

A -

08"

Amax

06"

0.500.-

0.4"

0400-.

0.300" 1

0

0.200..

I

1

1

,

40

80

120

160

CARBOHYDRATE

I

200

(ng/cot)

FIG.3. Elicitor activity of a dilution series o f high-resolu200

500

300

400

ELUTION

VOLUME

600

(ml)

FIG.1. Low-resolution P-2 gel-filtration chromatography of 700 mg of acid-solubilized oligoglucosides from Pmg mycelial walls. The shaded region represents the elicitor-active oligoglucoside-containing fractions that were pooled for further purification. The amount of carbohydratein 5 p1 of each fraction was determined by the anthronemethod and is represented by the AS20(0) (see "Materials and Methods").

tion P-2 purified heptaglucosides (0)and hexaglucosyl-glucitols (0).The data is plotted as the A2&of the sample divided by the A, for the experiment (A/A,,,d. The amount of the hexaglucosyl-glucitols was determined by the Dische method as described in the text (see "Materials and Methods"). cot, cotyledon.

nlClTOR ACTIVITY 7

ELUTION

Glc

VOLUME

(ml)

FIG.4. Normal-phase partition-chromatography purification of 12 mg of high-resolution P-2 purified hexaglucosylglucitols on the Whatman Magnum-9 PAC column.The elicitor activity (0,Am) of one-100th of each fraction was determined as described in the text. The first two eluting peaks contained no measurable elicitor activity (data not shown). The fractions in the shaded region were pooled for further purification. The refractometer was set at anattenuation of 8X.

:I

r

5-

1

I

z

0

50

60

70

80

ELUTION

90

Kx)

110

120

130

VOLUME

(rnl) FIG.2. High-resolution P-2 gel filtration chromatography of 90 mg of low-resolution P-2 purified elicitor-active oligoglucosides. Each peak is labeled according to the degree of polymerization of the major components of that peak as determined by FAB-MS.The heptaglucoside fraction that was pooled for further purification is shown in the shaded region. The refractometer was set at an attenuationof 64X.

Reversed-phase fractionation of oligosaccharide-glucitolsin water results in better peak shapes and resolution because converting the reducing-end aldose of each oligosaccharide to an alditol eliminates anomerization of the oligosaccharides. Therefore, PAC-purified heptaglucosides were converted to hexaglucosyl-glucitols before fractionation. The PAC-purified hexaglucosyl-glucitol fractions were fractionated on two Supelco Spherisorb-5 ODS analytical columns connected in series. Under the specified flow rate and back pressure (see "Materials and Methods"), the 2-fold increase in column length provided a 4-fold improvement in resolution compared to that obtained with a single column (data notshown) (37). When applied to theSupelco columns,

W

t U

A286

LL

0 20 0 10 0

W

a 0

30

66 RETENTION

133

100 TIME

166

lmln)

FIG.5. Reversed-phase liquid chromatography purification of 3 mg of PAC-purified hexaglucosyl-glucitols on theSupelco Spherisorb-5 ODS columns. Elicitor activity (0,A m ) of one2000th of each indicated fraction was determined as described in the text. All other fractions had no detectable elicitor activity (data not shown). The fractions in the shaded regions were pooled for further purification. The refractometer was set at an attenuationof 4X. the elicitor-active hexaglucosyl-glucitol fractions-whether reduced before or after PAC purification-gave similar elution profiles that contained the same major components (Fig. 5). The fractions designated S-I, S-11, S-111, S-IV,S-V, and SVI (Fig. 5, shaded regions) were pooled separately for further purification. Approximately 50% of the elicitor activity was recovered after each reversed-phase liquid chromatography step due to the routine loss of approximately 50% of the

Purification of a Heptaglucoside Elicitor

60

1.1

-

1.0

-

0

0

0

30

100

133

166

f

I /

1

0.5

1.0

CARBOHYDRATE

60

-m

0

0.9

0

S

a .

0

0.8 -

30

30

11315

of Phytoalexins

1.5

I

I

2.0

2.5

I

3.0

(ng/cot)

FIG. 7. Elicitor activity of hexaglucosyl-glucitol A-V. Three ) were assayed on separate days for dilution series ofA-V (0,0, . elicitor activity as described under “Materials and Methods.” Each point is the average of two separate plates of 10 cotyledons. The average specific elicitor activity was 0.55 ng/cotyledon (nglcot).

I, A-11,A-I11

and A-IV,A-V,A-VI, and A-VII, from the fractionation of each of the hexaglucosyl-glucitol fractions SFIG. 6. Reversed-phase liquid chromatography purification I, S-11, S-111, S-IV, S-V, and S-VI, were structurally characof Supelco-purified hexaglucosyl-glucitol fractions S-I terized. The retention times and yields of hexaglucosyl-gluthrough S-VI on the Altex columns. The refractometer was set citols A-I through A-VI1 are listed in Table 11. a t an attenuation of 4 x for S-I, S-11, and S-VI and at ZX for S-IIIOnly hexaglucosyl-glucitol A-V had significant elicitor acS-V. The units of elicitor activity (0)were determined as described in the text and were proportional to the amount of carbohydrate in tivity; it had a specific elicitor activity of 1800 units/pg of Glc each fraction. Shaded regions indicate the fractions pooled for struc- (Fig. 7 and Table I).Hexaglucosyl-glucitolA-I11 had a specific tural characterization. Retention times were calculated with respect elicitor activity of 13 units/pg of Glc, most likely accounted to internal standards tocorrect for pump and solvent variation. for by less than 1%contamination of A-I11 by A-V (see S-I11 and S-IV in Figs. 5 and 6). The specific elicitor activity of TABLEI1 hexaglucosyl-glucitol A-V was unchanged by the addition of Elution volumes and yields of the Altex-purified hexaglucosyl-glucitok as much as 20-fold greater amounts of each of hexaglucosylEach Supelco-purified fraction was obtained by purification of a glucitols A-I, A-11, A-111, A-IV, A-VI, or A-VII. total of 6 mgof PAC-purified hexaglucosyl-glucitols. The AltexThe presence of phytoalexins in the wound droplets of purified fractions were obtained from the specified Supelco-purified cotyledon assays of hexaglucosyl-glucitol A-V was confirmed fractions. The retention times of the Altex-purified fractions were calculated relative to theretention time of an oligoglucoside standard by thin-layer chromatography. An ethyl acetate extractof the because solvent strength and HPLC pump speed varied slightly for wound droplets from a cotyledon assay of hexaglucosyl-glucitol A-V waspartially purified by thin-layer chromatography, each purification. as described (38). The RF values of the UV-absorbing spots Origin of Yield Retention Altex Altex were identical to the RF values of authentic samples of glytime (Glcf fraction fractions ceollin and glycinol, the pterocarpan phytoalexinsof soybean min PL? (38, 39). To confirm the antimicrobial properties of the UV 373 A-I (S-I) 90 spots in the ethyl acetate extracts, potato-dextrose agarcon49 A-I1 (S-11) 130 taining the spores of the fungus Cladosporium cucumerinum 76 A-I11 (S-111) 137 was sprayed onto the thin-layer-chromatography plate, and 49 A-IV (S-111) 143 the plate incubated in high humidity in the dark for 4 days 100 A-V (S-IV) 137 (8).The fungus grew over the entireplate except over the UV 56 A-VI (S-V) 143 89 spots that migrated as glyceollin and glycinol. A-VI1 (S-VI) 150 RETENTION

TIME

(mln)

carbohydrate applied to the columns. Hexaglucosyl-glucitol Preliminary Characterizationof the Hexaglucosyl-glucitols fraction S-IV was the only elicitor-active fraction observed and hada specific elicitor activity of 910 units/pg of Glc, 13The primary structures of hexaglucosyl-glucitols A-I fold greater than that of the PAC-purified hexaglucosyl-glu- through A-VI1 can be defined by elucidation of a number of their characteristics (40, 41). These include their glycosylcitol fraction (Table I). Reversed-phase Liquid Chromatography of the Supelco-pu- residue composition; the absolute configuration, D or L, of rifiedHexaglucosyl-glucitolFractions on Altex Ultrasphere each of their glycosyl residues; the ring form, furanosyl or O D s Columns-The Supelco-purified fractions S-I through pyranosyl, of each of their glycosyl residues; their glycosylS-V were purified separately in 2.00% acetonitrile in water linkage composition; the anomeric configuration, CY or @,of (v/v) on two Altex Ultrasphere ODS analytical columns con- each of their glycosyl residues; the identity and points of nected in series (Fig. 6). Supelco-purified fraction S-VI was attachment of any nonglycosyl substituents; and thesequence purified in 2.10% acetonitrilk in water (v/v). Components A- of their variously-linked glycosyl residues. With the exception

Purification of a Heptaglucoside Elicitor of Phytoalexins

11316

of glycosyl-sequence analysis, the results of analyses to determine all these characteristics for hexaglucosyl-glucitols A-I through A-VI1 follows. FAB-MS Analysis of Hexaglucosyl-glucitokr A-I through AVII-FAB mass-spectral analysis of the underivatized hexaglucosyl-glucitol fractions A-I through A-VI1 showed molecular ions (M H) of m/z 1155. This was consistent with molecules having 1 hexitol and 6 hexosylresidues. These spectra established that no noncarbohydrate moieties were covalently attached to any of the hexaglucosyl-glucitols. FABMS of the per-0-methylated derivatives of hexaglucosyl-glucitols A-I through A-VI1 showeda molecular ion (M + NHI) of m/z 1508, which was consistent with a per-0-methylated molecule consisting of 1 hexitol and 6 hexosyl residues. Glycosyl-residue Composition and Absolute Configuration of the Glycosyl Residues of Hexagtwosyl-glucitols A-I through AVII-The identity and absolute configuration of the glycosyl residues of hexaglucosyl-glucitols A-I through A-VI1were determined. GLC analysis of the TMSderivatives of the (+)2-butylglycosides of each hexaglucosyl-glucitol yielded only the derivatives corresponding to D-glucose and glucitol. No (+)-2-butylglycoside TMS derivatives corresponding to Lglucose, D-mannOSe, or L-mannose were observed. Therefore, all 6 glucosyl residues of the hexaglucosyl-glucitols analyzed were D-glucose, and all contained a single residue of glucitol. This agreed with alditol-acetate analyses of less pure, unreduced fractions that showed that the only sugar in the fragments was glucose. Glycosyl-linkage Composition andRing Form of the Glycosyl Residues of Hexa(D-glucosyl)-D-glucitokr A - I through A- VIIGlycosyl-linkage analysis of hexa(D-glucosy1)-D-ghcitolsA-I through A-VI1 determined the ring form of the glucosyl residues, the glucosyl-linkage composition, andthe relative amounts of the differently linked glucosyl residues.All of the glucosyl residues of hexa(D-ghcosyl)-D-glucitolsA-11 through A-VI1were shown to be in the pyranoid ring-form by the presence of acetate groups on C-1 andC-5 of all the partially methylated alditol-acetate derivatives except those derived from the prereduced glucitol (Table 111). Hexa(D-glucosyl)-D-glucitolA-I consisted only of 4-linked glucopyranosyl or 5-linked glucofuranosylresidues (Table 111) (42, 43). These were later shown to be all 4-linked glucopyranosyl residues by 'H NMR analysis and by further analysis of the products of glycosyl-residue sequence analysis (44,45).

+

Glycosyl-linkage analysis of hexa(D-glucopyranosyl)-D-glucitols A-I through A-VI1 (Table 111) showed that the prereduced glucitol of A-I was 4-linked, whereas the prereduced glucitols of A-I1 through A-VI1 were 6-linked. Although the predominant glucitol in A-I11 was 6-linked, a small amount of the derivative corresponding to a 3,6-linked prereduced glucitol was also observed and probably arose from the presence of a small amount of a second hexa(D-glucopyranosy1)D-glucitol. The low yields of the penta-0-methylglucitol acetate derivatives (Table 111) derived fromthe prereduced glucitol of each of the hexa(D-ghcopyranosyl)-D-glucitols were a result of their relatively high volatility (32) and of incomplete methylation of the prereduced glucitol. Undermethylation occurred predominantly at 0-3 and 0-4 of the 6-linked prereduced glucitol and at 0-1and 0-3 of the 4-linked prereduced glucitol. Attempts to eliminate undermethylation of the glucitols without significant loss or degradation of the hexa(D-glucopyranosy1)-D-glucitols were unsuccessful. The undermethylation was shown by FAB-MS analysis not to result from covalent substitution of these positions. Therefore, the mole per cents of the tri-0-acetyl and tri-0-methyl-glucitol derivatives of the undermethylated prereduced glucitols of the hexa(D-glucopyranosy1)-D-glucitols wereadded to theexpected mole per cents of the penta-0-methyl glucitol acetate derivatives (Table 111). The glycosyl-linkage compositions of hexa(D-glucopyranosyl)-D-glucitols A-I1 through A-VI1showed that all of the linkages were at the 0-3 and 0-6 positions of the glucosyl residues. Hexa(D-glucopyranosyl)-D-glucitolsA-11,A-111 and A-IV had indistinguishable glycosyl-linkage compositions (Table 111). Hexa(D-glucopyranosy1)-D-glucitolsA-V,A-VI, and A-VI1 also had indistinguishable glycosyl-linkage compositions (Table 111). Anomeric Configuration of the Glycosyl Residues of thePer0-methylated Hexa(D-glucopyranosyI)-o-glucitols A-I through A-VII-The 'HNMR spectra of the per-0-methylated derivatives of hexa(D-glucopyranosyl)-D-glucitolsA-I through AVI1 contained anomeric proton signals at chemical shifts and coupling constants that were consistent with p-anomeric protons of glucopyranosyl residues (Table IV) (46). The 'H NMR spectrum of hexa(P-D-g1ucopyranosyl)-Dglucitol A-I11 confirmed the presence of another hexa(p-Dglucopyranosy1)-D-glucitolbecause less-intense anomeric proton signals, in addition to the sixmajor anomeric proton

TABLE 111 Glycosyl-linkage analysisof hezaglucosyl-glucitols A-I through A- VII The mole per cent of each per-0-methylated alditol-acetate derivative was calculated as described (47). The numbers in parentheses are estimates of the number of each differently linked glucosyl residue in the intact hexaglucosyl-glucitol and are based,in part, on the molecular weights ofthe hexaglucosyl-glucitols.The mole per cents of the prereduced alditols are the sum of the fully methylated and undermethylated derivatives(see text). The data for A-111, A-IV, A-V, and A-VI are averages of two glycosyl linkageanalyses of two separate purifications of these hexaglucosyl-glucitols. Positions of 0-methyl groups

1.4.5 2.3.

n-.I.."d -"I.-

-l..",.":A:g,ycvr.rruI.

linkage

Prereduced 6-linked Prereduced 4-linked

(2,5,6) 1,2,4, 5 Terminal 2,3,4, 6 2,4,6 2,3,6 2,394 2,4

A-I

A-I1 A-VI

A-VI1

0.5 (1)

0.5 (1)

0.6 (1)

0.5 (1)

0.4 (1)

0.8 (1)

2.0 (2) 1.1 (1)

0.2 2.4 (2) 1.1 (1)

2.3 (2) 1.2 (1)

2.8 (3)

2.8 (3)

3.5 (3)

1.5 (2) 0.8 (1)

1.9 (2) 0.9 (1)

1.8 (2) 1.0 (1)

1.0 (1) 1.9 (2)

1.0 (1) 1.8 (2)

1.0 (1) 1.8 (2)

0.6 (1)

Prereduced 3,6-linked 1.0 (1) 3-linked 4-linked 6-linked 3,g-linked

Hexaglucosyl-glucitols A-111 A-V A-IV

5.1 (5)

Purification of a Heptaglucoside Elicitor of Phytoalexins

11317

TABLE IV Chemical shifts and coupling constantsof the anomeric protonsignals of the per-0-methylatedhemglucosyl-glucitl A-I throughA-VI1 Number of Hexaglucosylglucitol

BcBNl

BCcumulated

Amount of carbohydratea (Glc)

Coupling Chemical shift (6)

4.49

A-I1

12,703 3,621

82 20 7.6

A-I11 Major component Minor A-IV A-V

A-VI A-VI1

14,230

19

Integration factor

J1S

Asaid anomeric configuration

Hz

rB

A-I

constant

4.41 4.37 4.74 4.72 4.40 4.39 4.37 4.73 4.71 4.37 4.84 4.49

7.9 7.9 7.9 7.6 7-6 7.9 7.6 7.9 7.6 7.6 7.9 7.9 7.6 7.6 7.6 7.6 7.6 7.9 7.6 7.3 7.6 7.9 7.9 7.6 7.9

1.0 4.0 1.0 1.0 1.0 1.0 1.0 2.2

1.0 1.0 4.0 0.2 0.2 4.73 50,827 3.4 1.0 4.72 1.0 b 4.2 14,238 22 4.74 2.1 4.39 1.0 4.38 2.1 4.33 1.0 2,000 5.5 4.74 2.0 4.39 2.0 4.33 2.0 964 31 4.74 2.0 4.38 2.0 4.33 2.0 Amount of carbohydratewas determined fromthe glycosyl-linkage compositionanalysis (32,47). Multiple signals centered at 4.37.

B B B B B B B B B B B B B B B

B

B B B B B B B B B B

ysis established that no noncarbohydrate groups were covalently attached to any of the hexa(j3-D-glucopyranosyl)-Dglucitols. Determination of the absolute configuration of the glycosyl residues of each hexa(@-D-ghcopyranosyl)-D-glucitol established that all were composed of only D-glucose. Glycosyl-linkage analysis established that A-I1 through A-VI1 were composed of 3-, 6-, and 3,6-linked glucopyranosyl residues. Hexa(~-D-glucopyranosyl)-D-glucitol A-I was composed only of 4-linked glucopyranosyl residues and, therefore, was probably derived fromcellulose, a known component of Pmg mycelial walls (42,43). The glycosyl-linkage compositions of hexa(j3-D-glucopyranosy1)-D-glucitolsA-I1 through A-VI1 suggested that two sets of glycosyl-linkage isomers, that is structural isomers with the same glycosyl-linkage composition, had been isolated. The first set of glycosyl-linkage isomers, hexa(j3-D-glucopyranosyl)-D-glucitols A-11, A-111, and A-IV, contained one 3-linked, one 3,6-linked, two 6-linked, and two terminal glucosyl residues. Noneof these hexa(j3-D-ghcopyranosyl)-D-glucitols had detectable elicitor activity. The glycosyl-linkage composition of the minor component of A-I11 was not obvious, except that a 3,6-linked prereduced glucitol (Table IV) was probably the glucitol end of the minor component. The second set of DISCUSSION glycosyl-linkage isomers, hexa(@-D-glucopyranosy1)-D-gluciComposition of Elicitor-active and Elicitor-inactive Oligoglu- tols A-V, A-VI, and A-VII, had no 3-linked glucosyl residues, cosyl-glucitols-Seven highly purified hexa(j3-D-glucopyrano- but, instead, contained two 3,6-linked,one 6-linked, and three syl)-D-ghcitol structural isomers were isolated from the oli- terminal glucosyl residues. Only hexa(j3-D-glucopyranosy1)-Dgoglucosides released from Pmg mycelial walls bypartial acid glucitol A-V had elicitor activity. hydrolysis. Analysis by 'H NMR established that hexa(j3-DHexa(/3-D-glucopyranosyl)-D-glucitol A-V was a potent elglucopyranosy1)-D-glucitolsA-I through A-VI1 were ,%linked icitor. Its specific elicitor activity of 1800units/wg of Glc and further indicated that all the hexa(8-D-glucopyranosy1)- means that only 1pmol in a tissue volume of about 1ml ( D-ghcitols except A-I11 were relatively pure. FAB-MS anal- M)is sufficient to induce phytoalexin accumulation in soybean

signals, were seen at chemical shifts of4.84 and 4.49 ppm (Table IV). The 'H NMR analysis of A-IV was done with a small amount of per-0-methylated A-IV due to loss during the Sep-Pak C1, cleanup after methylation. Thus, the 'H NMR spectrum of A-IV was of less resolution then theother spectra, making the coupling constants in the 4.37-ppm region difficult to measure. The chemical shifts and coupling constants of the proton signals at 4.74 ppm in the spectra of A-I1 through A-VI1 were consistent with those expected for the B-anomeric protons of glucopyranosyl residues linked to 0-3 of other glucosyl residues (46). The chemical shifts and coupling constants of the proton signals centered at 4.38 ppm were consistent with those of j3-anomeric protons of glucopyranosyl residues linked to 0-6 of other glucopyranosyl residues (46). The chemical shifts and coupling constants of the proton signals center around 4.41 ppm in the spectrum of A-I were consistent with j3-anomeric protons of glucopyranosyl residues linked to 0-4 of another glucopyranosyl residue. The integration of the signals of these protons were consistent with the expected number of residues linked to 0-3 and 0-6 in each of the hexa(8-D-glucopyranosyl)-D-glucitols(Tables 111 and IV).

11318

Purification of a Heptaglucoside Elicitor

cotyledons. This is the highest specific elicitx activity yet reported for any elicitor of phytoalexins. None of the structurally similar inactive hexa(B-D-ghcopyranosyl)-D-glucitols inhibited or enhanced the elicitor activity of A-V, suggesting that theelicitor-active structure is highly specific. The smallestelicitor-active oligoglucoside released from Pmg mycelial walls was probably a hexaglucoside. The structure of an elicitor-active hexaglucoside in the P-2 purified hexaglucosides was sufficiently altered by reduction of its reducing-end glucose residue to at least diminish its specific elicitor activity by half. The remaining elicitor activity of the pentaglucosyl-glucitol fraction may have been due to either contaminatingelicitor-active hexaglucosyl-glucitols inthe mixture or residual activity of the alteredelicitor-active pentaglucosyl-glucitol. In contrast, the elicitor activity of the P2 purified heptaglucosides was unchanged by the reduction of its reducing-end glucose. It is probable that thehexaglucoside that lost activity upon reduction had the same structure as the 6 glucosyl residues of hexaglucosyl-glucitol A-V. Hexa(p-D-glucopyranosyl)-D-glucitol A-V was the only elicitor-active heptaglucoside present in the mixture of heptaglucosides released from Pmg mycelial walls. Approximately 30 heptaglucosides could be distinguished in the reversedphase elution profile of one-half of one of five peaks purified on the PAC column. If one assumes, conservatively, that each of the five PAC peaks was composed of 30 distinct heptaglucosides, then the elicitor-active heptaglucoside was one of among at least 150 structurally distinct elicitor-inactive heptaglucosides; it could be one of 300. This observation,in conjunction with the observation that two elicitor-inactive glycosyl-linkage isomers were also isolated, established that the structural requirements of elicitor-active hexa(0-D-glucopyranosyl)-D-glucitol A-V were very precise. Determination of the complete primary structures of hexa(@-D-ghcopyranoSyl)-D-glUCitOlSA-I through A-VI1 is presented in the following two papers. Comments on Methods-The high-resolution reversedphase liquid chromatographyseparationmethodreported here is a valuable technique for separating structurally similar oligosaccharides. It is worth noting a few chromatographic parameters and how they caneffect such separations. HexaW D-glucopyranosy1)-D-gjucitols that coeluted on one manufacturer's column were 'separated on another manufacturer's column. Increasesin the acetonitrileconcentration of the chromatography solvent by as little as 0.2% caused decreases in the elution volume of the hexa(0-D-glucopyranosy1)-Dglucitols by as much as 1 k' unit (data not shown). On the other hand, structurally diverse oligosaccharide-glucitols required very different solvent conditions for separation. For instance, 4-linked oligo-a-glucosides, which separate well in the water on ODS columns (36), were notretainedunder solvent conditions used to separate these hexa(P-D-glucopyranosy1)-D-glucitols (data not shown). Thus, high-resolution separations of oligosaccharides by reversed-phase liquid chromatography in water and in low-percentage organic solvents in water is a very selective method for purifying structurally similar neutral oligosaccharides. Acknowledgments-We would like to thank Connie Winans for technical assistance, Tom Waeghe, Martin Ashley, Mike McNeil, and Keith Davis for helpful discussions, and Barbara Sloane and Leigh Kirkland for editing and preparing the manuscripts. REFERENCES 1. Mansfield, J. W. (1982) in Phytoalexins (Bailey, J. A., and Mansfield, J. W., eds) pp. 253-288, John Wiley and Sons, New York

of Phytoalexins

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Purification of a Heptaglucoside Elicitorof Phytoalexins 45. Darvill, A. G., McNeil, M., and Albersheim, P. (1980) Carbohydr. Res. 86,309-315 46. Haverkamp, J., de Bie, M. J. A., and Vliegenthart, J. F.G . (1974)

11319

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11320

Purification of a Heptaglucoside Elicitor of Phytoulexins

Note Added in Proof-Before the last sentence of the section entitled "Preparation of Pmg Mycelial Walls," the following sentence should be inserted This procedure was repeated six times against80 volumes of distilled water.