Polysaccharides from Hemileia vastatrix uredospores

EXPERIMENTAL

MYCOLOGY

7, 82-89 (1983)

Polysaccharides J. ANTONIO Znstituto

LEAL,

de Znmunologia

from Hemileia

vastatrix

BEGONA

GOMEZ-MIRANDA,

y Biologia

Microbiana,

CSZC

Accepted for publication

Uredospores

AND PILAR RUPEREZ Vel&zquez,

144, Madrid-6,

Spain

September 21, 1982

J. A., GOMEZ-MIRANDA, B., AND I&PEREZ, P. 1983. Polysaccharides from Hemileia uredospores. Experimentuf Mycology 7, 82-89. The polysaccharidic fractions isolated from vastatrix uredospores by alkali treatment, expressed as a percentage of the initial uredospore weight, gave the following yields: 1 M NaOH soluble at 22°C. (7.1); 1 M NaOH soluble at 60°C (5.0); and insoluble residue (7.6). Both alkali-soluble fractions contained mannose and glucose as the major constituents, with glycosidic linkages of the p-1 -+ 4 and p-1 + 3 types. The alkaliinsoluble residue contained predominantly glucosamine, and had infrared and X-ray spectra indistinguishable from those of crustacean chitin. Electron microscope observations revealed that the insoluble residue consisted of the cell wall spines connected by a thin layer of microfibtils. INDEX DESCRIPTORS: Hemileia vastatrix: rust fungi; uredospore spines; polysaccharides; glucomannans; chitin. LEAL,

vastatrix Hemileiu

The composition of the uredospore wall of the obligately parasitic rust fungi has been little studied. A glucomannan was extracted from wheat stem rust (Puccinia graminis tritici) uredospores with boiling 60% potassium hydroxide. This polysaccharide consists predominantly of pmannopyranosyl units joined by equal numbers of 1 + 4 and 1 -+ 3 bonds with D-glucose as a minor constituent sugar (Prentice et al., 1959). Prolonged extraction of P. graminis tritici uredospores with hot water affords polysaccharide material containing mannose, galactose, and glucose. A levorotatory mannan, separated from this material by copper complexing, possessed a highly ordered structure, in which isolated p- 1 + 4- and p- 1 + 3-linked units alternated along the linear chain. The insoluble spore residue contained pentoses and glucosamine (Kloker et al., 1965). Glucose, mannose, and glucosamine were the only carbohydrate components identified in Uromyces phaseoli var typica uredospore walls by Trocha et al. (1974). They found that glucosamine is associated with a red pigment in uredospore walls and with chitin-like polymers in uredospore germ tubes. Trocha and Daly (1974) found that a

linear (1 + 3)glucan, containing minor quantities of 1 + 6 linkages, may account for most of the glucose in the uredospore wall. They also isolated from the uredospore wall a glucomannan consisting of approximately equal numbers of 1 + 3 and 1 + 4 mannosidic linkages with glucose as a minor component. The structure and development of the uredospore walls and spines of various rust fungi have been studied by several workers (Thomas and Isaac, 1967; Ehrlich and Ehrlich, 1969; Littlefield and Bracker, 1971). We report here on the isolation and constitution of the carbohydrate polymers of Hemileia vastatrix uredospore and light and electron microscopy of the alkaliinsoluble residue. MATERIAL

Copyright @I 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

METHODS

Microorganism. Uredospores of mixed races of H. vastatrix Berk. & Br. were obtained from infected leaves of glasshousegrown Coffea arabica from the Centro de Investigacao das ferrugens do Cafeeiro. Estacao Agrondmica National, Oeiras, Portugal. Polysaccharide extraction from uredospores. Dry spores (2 g) were successively 82

0147-5975183/010082-08$03.00/O

AND

H.vastntrix

Chemical Composition

Fraction A B C

POLYSACCHARIOES

3

TABLE 1 of the Polysaccharide Fractions Obtained from Uredospores of N. vasta&ix

Treatment 1 M NaOH at 22°C 1 M NaOH at 60°C Insoluble residue

Percentage of fraction

Yield” (Fit%)

Carbohydrates

Glucosamine

Protein

Phosphate ion

7.1 5.0 7.6

91.3 89.8 6.4

0.0 0.0 83.5

6.8 3.6 0.3

o,o 0.0 0.0

a Values refer to the percentage of the fractions in uredospores. All the values are averages of tripkate determinations.

extracted with water and 80% ethanol at 22°C to get the soluble material. Afterward, proteins were extracted from the uredospore residue with 1 M NaOH for 16 h at 22°C; alkali was removed by washing with water until the pH of the wash was neutral. Free and bound lipids were extracted with ether-ethanol and 0.12 N HCI in ether, respectively, according to Bartnicki-Garcia and Nicker-son (1962). The residue (20% of the whole uredospore) was shaken with 1 M NaGH (100 ml) at 22°C for 30 min. The suspension was centrifugated and the supernate was treated with an equal volume of ethanol. Extraction was continued until no precipitate was formed. The precipitates were washed with 50% ethanol until the supernate was free from alkali, and then with acetone and desiccated at 80°C (fraction A). The residue from the previous treatment with 1 M NaOH at 22°C was suspended in 1 M NaOH (100 ml) at 60°C for 30 min and treated as described above. The material solubilized in this treatment constituted fraction B. The spore residue (insoluble in alkali) was washed with distilled water until the supernate was free from alkali, ethanol, and acetone and desiccated at 80°C (fraction C). Chemical and structural analysis. The different fractions were hydrolyzed with 0.5 M H,SO, at 105°C for different periods of time in sealed evacuated tubes, and neutralized with barium hydroxide. After centrifugation, reducing sugars were determined with a copper reagent (Somogyi, 1952) and the arsenomolybdate chromogen

of Nelson (1944). The polysaccharides were also hydrolyzed with HCI of different concentrations at 105°C and for different periods of time in sealed evacuated tubes. Amino sugars were determined i HCI hydrolysates by the method o and Morgan (1955). Phosphate was determined in the 2 M HCl hydrolysates according to Rand er al. (1975). Total hexosan was determined by the anthrone ~ro~e~~~~ (Dreywood, 1946) with glucose an nose as standards. The sugars we tified in the hydrolysates by d~s~e~d~~ paper chromatography on Whatman No. filter paper. The solvent system was nbutanol:pyridine:O.Ol M HCI (53: 3.5). The elution time was 40 h. The spots were veloped with silver nitrate (Hough Jones, 1962). The identity of the sugars, converted into the corresponding aidit acetates (Lame et al., 19723,was co~~r~~ and quantified by gas-liquid chromatography (GLC)l on 3% SP-2340 on 100-120 Supelcoport. A 2-m x 2-mm glass column was used at 200 to 230°C with a tern~erat~~~ rise of 10”C/min, a 3-min initial hold, and a final temperature time of 14 mm for ~~~t~a~ sugars and 44 min for amino sugars. The N, flow rate was 30 mUmin. A flame ionization detector, sensitivity 10-l*, sample size 3 ~1, was used in a Perkin-Elmer Sigma 10 and Sigma 3 gas chromatograph. Peaks were identified on the basis OS sample coincidence with the relative retention times of 1 Abbreviation raphy.

used: GLC, gas-liquid

chromatog-

84

LEAL,

GOMEZ-MIRANDA,

AND

RUPEREZ

l&L

L

2

3

2 3 L

L

2 3 L

4

3

1

,

I

I

I

I

I

/

5

I

I

I

/

/

IO

I

I

I

I

15 TIME

s

III.

20

I

I

30

I

40

(minutes)

1. Gas chromatograms of the acetyl derivatives of H. vastufrti polysaccharide fractions after acid hydrolysis. With 0.5 M H,SO, for 8 h: (A) material extracted with 1 A4 NaOH at 22°C; (B) material extracted with 1 M NaOH at 60°C; (C) insoluble residue. With 6 M HCI for 2 h: (C*) insoluble residue. 1, mannose; 2, glucose; 3, inositol (internal standard); 4, glucosamine. FIG.

TABLE 2 Molar Ratios of the Sugars Determined as Alditol Acetates by GLC in the Polysaccharide Fractions of H. vastatrir Uredospores Molar ratio bmol (k SD)] Fraction

Ribose

Xylose

Mannose

Galactose

Glucose

Glucosamine

A B C

0.40 (0.038) 0.54 (0.066) 0.00

0.39 (0.030) 0.47 (0.052) 0.00

86.39 (2.292) 78.21 (2.059) 4.02 (0.466)

Trace” 0.00 0.00

12.82 (1.193) 20.77 (1.61) 2.82 (0.307)

0.00 0.00 93.16 (2.377)

DRepresents less than 0.1 pmol.

H. vastatrix

POLYSACCHARIDES

TABLE 4 Estimated Types of Linkages Calculated from the Results of Periodate Oxidation of the Polysacc~ar~de Fractions from Uredospores of H. vastatrix

TABLE 3 Periodate Consumption and Production of Formic Acid and Ammonia (moUmo1 Hexose Residue) of Polysaccharide Fractions from Uredospores of H. vastratrix Fraction

Periodate

Formic acid

Ammonia

A B C

0.62 0.65 0.41

0.10 0.03 0.00

0.10

Note. Similar results were obtained with polysaccharide fractions from different extractions.

standards. Inositol was used as internal standard. Nitrogen was determined by the Kjeldahl method using a 1007 digestor and 1001 distilling unit from Tecator Instruments. Total protein was measured by the method of Lowry et al. (1951), using bovine serum albumin as the standard.

5

Linkage (%) Fraction

1+6

A B c

10 3 10

l-+4

l-+3

42 59 21

48 38 69

Periodate oxidation was performed according to Aspinall and Ferrier (2957). The formic acid formed was determined by the method of Kabat and Mayer (1961). Ammonia was determined in the oxidation mixture by distillation, after addition of 40% NaQH, in the 1001 distilling unit. The distillate was collected in a flask

r

‘ 4ow

3500

I

I

3ooo

2500

2ow

1800

1600

WAVENUMBER

1400

I200

1800

a00

608

(cm41

FIG. 2. Infrared spectra of H. vnsf&-ix. (U) uredospores: (W) uredospore walls; (A) material e%tracted with 1 M NaOH at 22°C: (B) material extracted with I M NadH at 60°C; (C) insoluble residue.

86

LEAL,

GOMEZ-MIRANDA,

AND RUPEREZ

NaOH treatment and their chemical compositions are presented in Table 1. The alkali-soluble fractions precipitated with Fehling solution (Kocourek and Ballou, 1969). The polysaccharide recovered from the copper precipitates had an ir spectrum and chemical composition similar to those of the crude polysaccharides. The gas chromatograms of the acetyl derivatives of H. vastatrix polysaccharide fractions after acid hydrolysis are shown in Fig. 1. Molar ratios of the sugars, determined by GLC in the polysaccharide fractions of H. vastatrix uredospores, are presented in Table 2.

.ch A

FIG. 3. X-ray diffraction patterns of crustacean chitin (Ch) and insoluble residue (C).

a solution of 4% boric acid and 1.7 ml of Tashiro’s indicator and was titrated with 0.01 A4 HCI. Infrared (ir) spectra were obtained by the KBr technique on a Perkin-Elmer 457 ir spectrophotometer. X-Ray diffraction. X-Ray diagrams of the alkali-insoluble fraction and commercial chitin were obtained with a PW 1065 Philips recording diffractometer. Nickel-filtered CuKa X radiation was employed. The X-ray tube was operated at 36 kV and 20 mA. Electron microscopy. A portion of the water-washed insoluble residue (alkali free), which was not washed with alcohol and acetone, was suspended in distilled water and drops of a diluted suspension were transferred to specimen grids bearing carbon films. The grids were then dried in a desiccator over anhydrous CaSO,, prior to shadowing with gold-palladium (60:40). Shadowed specimens were examined in a JEOL JEM-1OOB electron microscope. RESULTS

Extraction and Constitution of Polysaccharide Fractions The yield of the polysaccharide fractions obtained from H. vastatrix uredospores by

Periodate

Oxidation

The periodate consumed by the polysaccharide fractions and the formic acid released (mole/mole of anhydrohexose residue) are presented in Table 3. Ammonia was also determined in fraction C since it is produced by oxidation of glucosamine. The linkage types calculated from these results according to Rankin and Jeanes (1954) are presented in Table 4. Spectral Analysis The ir spectrum of the uredospore (Fig. 2U) has an absorption band at 1750 cm-l due to the carbonyl -CO- group of lipids. This band is absent in the uredospore wall (Fig. 2W). The uredospore and its wall have bands at 1650 and 1550 cm-l due to the NH-CO group present in N-acetylglucosamine . The alkali-soluble polysaccharides gave ir spectra (Figs. 2A, B) with absorption bands in the region 812-805 cm-’ characteristic of mannose-containing polysaccharides (Marchessault, 1962). The band at 890 cm-l and the lack of a band at 850 cm-l are characteristic of the P-linked polysaccharides (Barker et al., 1956). The ir spectrum of the insoluble fraction (Fig. 2C) is characteristic of /3chitin (Michell and Scurfield, 1970). The insoluble fraction has an X-ray dif-

H. vastatrix POLYSACCHARIDES

FIG. 4. Phase-contrast micrographs of the insoluble residue ofM. vastatrix uredospores showing the spines (sp) (x928). FIGS. 5 AND 6. Electron micrographs of the insoluble residue of H. vasratrix uredospores shadowed with gold-palladium. Spines (sp); microfibrilar layer (m). Fig. 5, Spines showing microfibrils (~28,160). Fig. 6. Thin layer joining the spines (X 16,425).

fraction pattern (Fig. 3) indistinguishable from that of ordinary crustacean chitin. Electron Microscopy of the Insoluble Residue

A water suspension of the insoluble residue observed by phase-contrast micros-

copy revealed that the uredospores wail had lost its normal appearance, but spines remained arranged in a somehow regular pattern that maintained the size and shape of the uredospores (Fig. 4). Electron microscopy of a shadowed preparation of this material revealed the microfibrilar ar-

88

LEAL,

GOMEZ-MIRANDA,

chitecture of the spines (Fig. 5). A thin layer with spines is shown in Fig. 6. DISCUSSION

The polysaccharides isolated from H. uredospores amounted to a 20% of the uredospore wet weight. The alkalisoluble fractions were identified as p-glucomannans with the following glucose-mannose molar proportions: fraction A (13:86) and fraction B (21:78). In both polysaccharides, small proportions of ribose and xylose were detected. Differences were also found in the glycosidic linkage types of these polysaccharides. Fraction A contained about equal numbers of 1 -+ 4 and 1 + 3 linkages (42:48) while in fraction B the ratio 1 + 4 to 1 -+ 3 linkages was 59:38. The composition and structure of these two fractions were fairly similar to glucomannans extracted from Puccinia graminis f. sp tritici (Prentice et al., 1959), and Uromyces phaseoli (Trocha and Daly, 1974). The alkali-soluble polysaccharides precipitated with copper. The polysaccharide recovered from the copper complex ha@ composition identical to that of the crude polysaccharide, indicating that glucose and mannose were in the same molecule. Klbker et al. (1965) isolated, by copper complexing, a mannan from a polysaccharide containing mannose, galactose, and glucose in P. graminis tritici uredospores. The insoluble residue of H. vastatrix uredospores amounted to 7.6% and contained predominantly glucosamine (83%). The infrared spectrum (Fig. 2C) and X-ray diffraction pattern (Fig. 3C) of this material corresponded to those of chitin. Kliiker et al. (1965) found glucosamine in the insoluble spore residue of P. graminis tritici. The liberation of ammonia during the periodate oxidation of the insoluble residue, indicated that it has free amino groups. The main type of linkages must be 1 + 4, since N-acetylamino groups do not consume vastatrix

AND

RUPEREZ

NaIO,, even when they are 1 -+ 4 linked (Bardalaye and Nordin, 1976; Rup&ez and Leal, 1981). Microscopic observations of the insoluble residue showed the spines arranged in a regular pattern (Figs. 4-6). The spines were continuous with and had the same microfibrilar architecture of a wall layer (Fig. 6). This structural arrangement is in accordance with the findings of Ehrlich and Ehrlich (1969) in P. graminis uredospores. ACKNOWLEDGMENTS

Thanks are given to Dr. L. Rijo of the Centro de InvestigaFao das ferrugens do Cafeeiro, Oeiras, Portugal for collecting the uredospores for us, to Dr. I. Fonseca of the Institute de Quimica Fisica “Rocasolano” for the X-ray spectra, and to Mr. E. Blanc0 for the technical assistance in the electron microscope work. This investigation was supported by a grant from the CAICYT. P. Rup&ez and B. GbmezMiranda thank the Consejo Superior de Investigaciones Cientificas for their fellowships. REFERENCES FERRIER, R. J. 1957. A spectrophotometric method for the determination of periodate consumed during the oxidation of carbohydrates. Chem. Ind. (N. Y.) 1957: 1216. BARDALAYE, P. C., AND NORDIN, J. H. 1976. Galactosaminogalactan from cell walls of Aspergillus Niger. J. Bacterial. 125: 6.55-669. BARKER, S. A., BOURNE, E. J., AND WHIFFEN, D. H. 1956. Use of infrared analysis in the determination of carbohydrate structure. In Mefhods of Biochemical Analysis (D. Glick, Ed.), Vol. 3, pp. 213-245. Interscience, New York. BARTNICKI-GARCIA, S., AND NICKERSON, W. J. 1962. Isolation, composition and structure of cell walls of filamentous and yeast-like forms of Mucor rouxii. Biochim. Biophys. Acta 58: 102-119. DREYWOOD, R. 1946. Qualitative test for carbohydrate material. Ind. Eng., Chem. Anal. Ed. 18: 499. EHRLICH, M. A., AND EHRLICH, H. G. 1969. Uredospore development in Puccinia graminis. Canad. J. Bot. 47: 2061-2064. ASPINALL,

HOUGH,

L.,

G. O.,

AND

AND

JONES,

J. K.

W.

1962.

Chromatogra-

phy on paper. In Methods in Carbohydrate Chernistry (R. L. Whistler and M. L. Wolfrom, Eds.), Vol. 1, pp. 21-31. Academic Press, New York/London. KABAT, E. A., AND MAYER, M. M. 1961. Periodate oxidations. In Kabat and Mayer’s Experimental

H. vast&-ix

POLYSACCHARIDES

Immunochemistry, pp. 542-550. C. C. Thomas, Springfield, 111. KOCOUREK, J., ANDBALLOU, C.E.1969.Methodfor fingerprinting yeast cell wall mannans. J. Bacterial. 100: 1175-1181. KLOKER, W.,LEWINGHAM,G.A.,ANWPERLIN, A.S. 1965. Polysaccharides, nucleic acids, and protein of wheat stem rust (Puccinia graminis tritici) uredospores. Canad. J. Biochem. 43: 1387- 1395. LAINE, R. A., ESSELMAN, W. J., AND SWEELEY, C. C. 1972. Gas-liquid chromatography of carbohydrates. In Methods in Enzymology (S. P. Colowick and N. 0. Kaplan, Ed.), Vol. 28, pp. 159- 167. Academic Press, New York/London. LITTLEFIELD, L. J., AND BRACKER, C. E. 1971. Ultrastructure and development of urediospore ornamentation in Melampsora lini. Canad. J. Bot. 49: 2067-2073. LOWRY, 0. II., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265- 275. MARCHESSAULT, R. H. 1962. Application of infrared spectroscopy to cellulose and wood polysaccharides. Pure Appl. Chem. 5: 107-129. MICHELL, A. J., AND SCURFIELD, G. 1970. An assessment of Infrared spectra as indicators of Fungal Cell wall composition. Aust. J. Biol. Sci. 23: 345-360. NELSON, N. 1944. A photometric adaptation of the Somogyi method to the determination of glucose. J. Biol. Chem. 153: 375-380.

9

PRENTICE, N., CUENWET, L. S., GE~DES, W. F., AND SMITH, F. 1959. The constitution of a glucomannan from wheat stem rust (Puccinia graminis tritici) urediospores. J. Amer. Chem. Sot. 31: 684-688. RAND, M. C., G~REENBERG,P. E., AND TARAS, M. J. 1975. Phosphate ascorbic acid method. In Standard Methods for the Examination of Water and Wastewater, pp. 481-483. APHAIAWWAIWPCF. RANKIN, J. C., AND ~EANES, A. 1954. ~va~~at~o~ of the periodate oxidation method for structurzl analysis of dextrans. /. Amer. Chem. Sot. ‘76~ 4435-4441. RONDLE, C. J. M., AND MORGAN, W. T. J. 1955. The hexosamine ratios in blood group substances. Biochem. J. 59: xiii-xiv. RUPEREZ, P., AND LEAL, J. A. 1981. Extracellular galactosaminogalactan fromAspergil1u.r parasiticus. Trans. Brit. Mycol. Sot. 77: 621-625. SOMOGYI, M. 1952. Notes on sugar determination. J. Biol. Chem. 195: 19-23. THOMAS, P. L., ANW ISAAC, P. K. 1967. The development of echinulation in uredospores of wheat stem rust. Canad. J. Bot. 45: 287-289. TROCHA, P., AND DALY, J. M. 1974. Cell walls of germinating uredospores. II. Carbohydrates polymers. Plant Physiol. 53: 527-532. TROCHA, P., DALY,J.M., AND LANGENBACH, K.7. 1974. Cell walls of germinating uredospores. I. Amino acid and carbohydrate constituents. P/unt Physiol. 53: 519-526.