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Characterization of two Sclerotinia sclerotiorum polygalacturonases with different abilities to elicit glyceollin in soybean
Plant Science, 83 (1992) 7-13 Elsevier Scientific Publishers Ireland Ltd.
7
Characterization of two Sclerotinia sclerotiorum polygalacturonases with different abilities to elicit glyceollin in soybean Francesco Favaron, Paolo Alghisi and Paola Marciano lstituto di Patologia vegetale, Universit?t di Padova (Italy)
(Received May 30th, 1991; revision received December 19th, 1991; accepted December 23rd, 1991)
Two endo-polygalacturonase isoenzymes (PG-II and PG-IV), with masses of 34 and 30 kDa respectively, were purified from soybean hypocotyls infected by Sclerotinia sclerotiorum. The pH optimum for both isoenzymes was about 4.6, but PG-IV exhibited a broader range of pH activity. PG-IV showed a much higher affinity for pectin than did PG-I1. PG-II hydrolyzed polygalacturonic acid in a more random fashion than PG-IV. Oligouronides produced by PG-II showed a higher phytoalexin elicitor activity. PG-IV produced a large degree of maceration of soybean hypocotyls releasing a significant amount of uronides. The properties of PG-II and PG-IV are discussed in relation to the different ability of the two isoenzymes to elicit glyceollin in soybean. Key words: polygalacturonase isoenzymes; glyceollin elicitation; uronide oligomers; Sclerotinia sclerotiorum
Introduction
Soybean tissues infected by Sclerotinia sclerotiorum (Lib.) de Bary may display a hypersensitive response to infection and accumulate the phytoalexin glyceollin [11. The involvement in glyceollin elicitation of the two major endopolygalacturonases (PG-II and PG-IV), produced by the B-24 isolate of S. sclerotiorum in soybean hypocotyls, was reported in a previous paper [2]. The release of heat stable elicitors of glyceollin from purified soybean cell walls by both PG-II and PG-IV, was consistent with the pectic nature of the elicitors as reported by Bruce and West [3] and Davis et al. [4]. When the elicitor activity of uronides released by both isoenzymes from purified cell walls of soybean hypocotyls was compared, the activity of PG-II cell wall-derived uronides was higher than those of PG-IV. In contrast to this, when etiolated soybean hypocotyls Correspondence to: F. Favaron, Istituto di Patologia vegetale, via Gradenigo 6, 35131 Padova, Italy. Abbreviations: PG, polygalacturonase; PGA, polygalacturonic acid.
were directly exposed to isoenzymes, PG-IV induced a higher glyceollin accumulation [2]. In the present work we show the possibility that the behaviour of the two isoenzymes might be related to the differences in their biochemical and physiological properties. Materials and Methods
Polygalacturonase (PG) production and isoenzyme purification Six-day-old etiolated soybean seedlings (Glycine max [L.] Merr., cv. Canton) were grown and inoculated with the B-24 isolate of S. sclerotiorum as previously described [2]. PGs were extracted from infected hypocotyls 24 h after inoculation and purified by isoelectrofocusing and FPLC ionexchange chromatography as reported [2]. Protein was determined by the method of Lowry et al. [5], with bovine serum albumin as the standard. Gel electrophoresis SDS-PAGE was performed as described by Laemmli [6]. Protein bands were stained with
0168-9452/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
Comassie blue R-250. Molecular weight standards were phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (42.7 kDa), carbonic anhydrase (31.0 kDa) and soybean trypsin inhibitor (21.5 kDa). '~
Enzyme assay PG activity was determined as an increase of reducing end-groups with respect to time. Reducing end-groups were measured by the method described by Avigad [7] using D-galacturonic acid (Fluka, Switzerland) as a standard. One unit of PG activity (RU) was defined as that producing at 30°C 1 #mol of reducing groups min -l on 0.5'7,, (w/v) polygalacturonic acid (PGA, Sigma, U.S.A.) in sodium acetate buffer (100 mM, pH 4.6). To ascertain the effect of pH on PG activity, 100 mM sodium acetate or 100 mM citrate-phosphate buffers were used. In this case reducing groups were determined by the Nelson-Somogyi method [8]. The activity was also assayed by measuring the decrease in relative viscosity at 30°C of a 75% methylated apple pectin (Pektin B, Roth, Germany), previously washed according to Kertesz [9], or PGA, in 100 mM sodium acetate buffer (pH 4.6), in a micro-Ostwald viscosimeter (i.d. 0.70 mm). Decrease in relative viscosity was monitored by an AVS 310 system (Schott Gerate, Germany). One relative viscosimetric unit (RVU) was defined as that causing in 1 h a 50% reduction in the relative viscosity of 2 ml of the reaction mixture.
Release and fractionation of oligogalacturonides from PGA P G A (0.5%) at pH 4.6 was incubated at 30°C with 0.015 R U ml -l of PG-II or PG-IV. At 0.5-, 1-, 2-, 4-, 8- and 24-h aliquots of the incubation mixture were removed, immediately boiled for 20 rain at 100°C and stored at -20°C until used. Aliquots of 0.5 ml were analyzed by FPLC using an anion-exchange Mono Q H R 5/5 column (Pharmacia, Sweden) equilibrated with 20 mM Tris-HC1 buffer at pH 7.9. After loading the sample, the column was eluted with 10 ml of the equilibrating buffer and then with a linear gradient of NaCI (0.0-0.5 M) in the Tris-HC1 buffer. Before the start of the gradient 0.5-ml fractions were collected, then the eluent was monitored at
214 nm and each peak was collected in a separate fraction. The fractions were assayed for the uronide concentration by the m-hydroxydiphenyl method [10] using D-galacturonic acid as a standard. The fraction corresponding to the second uronide peak eluted after the start of the NaCI gradient was desalted on a Sephadex G-10 colunm (1.6 x 40 cm) equilibrated and eluted with water. The oligomer was then passed through an aromatic sulfonic acid disposable column (Baker, Holland) in the H + form and then lyophilized. A sample of about 0.2 mg of the lyophilized oligomer was dissolved in thioglycerol and analyzed by fast atom bombardment mass spectrometry (FAB-MS) in the positive mode.
Physiological properties Phytoalexin elicitor activity of the incubation mixtures (see above) at different times during PGA digestion, was assayed using the soybean cotyledon bioassay proposed by Hahn et al. [11]. 0.2 mg ml -l of streptomycin sulfate were added to each sample, Cotyledons were obtained from 7-day-old etiolated soybean plants (cv. Canton) and aliquots of sample of 50 /xl/ cotyledon were used. Three sets of 10 cotyledons were used for each treatment. Elicitor activity was measured as the A286of the wound-droplet solution. Tissue maceration was evaluated on 7-day-old etiolated soybean seedlings cut 5 cm below the cotyledons. The seedlings were washed, arranged in microvials (0.3 ml) and incubated as previously reported for the hypocotyl bioassay [2]. The microvials were filled with 0.2 ml of enzyme sample (0.4 R U m l -1) in 5 mM acetate buffer (pH 4.6) with 0.2 mg ml -l of streptomycin sulfate and 0.1 mg m1-1 of bovine serum albumin added. After 24 h of incubation the 1 cm terminal part of each hypocotyl was cut transversely into slices 2 mm thick. Tissue maceration was assessed as reported by Keon et al. [12] by measuring the weight required to cause the segments to collapse when placed between two glass microscope slides. Control consisted of hypocotyls treated with 5 mM buffer. The amount of uronides released from the hypocotyls was measured in the bathing solution by the m-hydroxydiphenyl method [10]. Five seedlings for each treatment were used. The experiment was repeated 3 times.
Results 100
PG-II and PG-IV were purified 48- and 95-fold, respectively, and their specific activity was 352 and 701 R U mg -] protein, respectively. On SDS-PAGE, PG-II and PG-IV showed only one band corresponding to a molecular mass of 34 kDa and 30 kDa, respectively (Fig. 1). These values are similar to those obtained by Superose-12 permeation chromatography (data not shown) where the PG-II and PG-IV molecular masses were found to be 35 kDa and 29 kDa, respectively. The activity of both isoenzymes was lower in citrate-phosphate buffer than in acetate buffer, with P G - I I showing a stronger reduction in activity (Fig. 2). Preliminary experiments had shown that the inhibitory effect was due to the citrate anion (data not reported). In acetate buffer both isoenzymes showed optimal activity at about pH 4.6. In both buffers PG-IV maintained its activity over a broader range o f p H values, and, at pH 6.0, PG-IV still showed an appreciable activity while PG-II was completely inactive. S0.5 values, determined from reducing-groups assays on different substrate concentrations, were 0.45 and 2.30 mg ml -] for PG-II on PGA and
1
2
3 k Da • 97.4
!
~
...... • 6 6 . 2
~,i~iii
4 42.7
< 31.0
•
21.5
Fig. 1. SDS-electrophoresis of PG-II (lane 1), PG-IV (lane 2) and protein standard (lane 3). Approx. 5 tzg of purified PG-II and 12 t~g of PG-IV were loaded.
80
._>
60 40
E
>,
20
N C
E
0 100
t~
E
80 60
"6 ae
40 20 0 3
3.5
4
4,5
5
5.5
6
6.5
7
pH Fig. 2. Effect of pH on PG activity. For both isoenzymes the maximum velocity corresponds to 0.015/~mol of galacturonic acid equivalents min -I ml -t of reaction mixture. Each data point is the average of 3 determinations. O, acetate: A citratephosphate.
pectin, respectively. Instead, the S0. 5 of PG-IV for PGA and pectin were 0.35 and 1.10 mg m1-1, respectively. Results of activity measured viscosimetrically (Fig. 3) showed that PG-II had a higher activity with PGA while PG-IV was always more active with pectin. Determinations of reducing end-groups at a time corresponding to a 50% loss in relative viscosity (T50) showed that PG-II cleaved 1.3% of the glycosidic bonds of PGA compared to 2.1% cleaved by PG-IV. When pectin was used as a substrate these values were 0.5% for PG-II and 0.9% for PG-IV. PG-IV, therefore, hydrolyzed both substrates less randomly than PG-II although both isoenzymes hydrolyzed the pectin in a more random manner than PGA. P G A was incubated for different times with the two isoenzymes. The oligomers released were analysed by a Mono Q column as described in Materials and Methods. At least 12 well resolved peaks were fractionated (figure not shown). The FAB-MS analysis of the second uronide peak
10 5~
200
PG-II
.
4
-
PGA
e
~
-
Pectin
(a)
150
~
pG-II
[~
PG-IV
3
100
rr
2 50
v
1 L
I
-
-
I
L
I
-
0 200
-
PG-IV
(b)
0
E
#
N4 U.I 3 ¢-
150
~=,, lOO 2
5O
1
0 0
--
i
1
--
~
L
I
i
2
3
4
5
[Substrate] (mg/ml) Fig. 3, Viscosimetricactivity of PG-II and PG-IV at different concentrations of PGA or pectin. Two milliliters of substrate containing 6 × 10-3 RU were used for each determination. Each data point is the average of 3 determinations. Zk, PGA; O, pectin.
3¢, O t~
o
0
200
(c)
150!
1oo 5oi
eluting after the start o f the gradient gave a mass spectra revealing a m a j o r peak at 568 rn/: and two minor peaks at 590 and 612 re~z, respectively. These correspond to the mono-, di- and trisodium salts, respectively, of a trimer of galacturonic acid. Since, in a separate experiment, a standard o f galacturonic acid was eluted before the start o f the salt gradient, the first uronide peak eluted after the start of the gradient was considered to correspond to a dimer. Therefore, in accordance with other authors [13,14], the uronide peaks from 3 to 12 might correspond to oligomers with 4 - 1 3 galacturonosyl groups. The a m o u n t o f the oligomers with 1-13 galacturonosyl residues produced by P G - I I and P G - I V increased up to 4 h of digestion, P G - I I producing a greater a m o u n t o f oligomers with 3 to 13 residues than P G - I V (Fig. 4). With both isoenzymes, a prolonged digestion (8 and 24 h) produced mostly an increasing a m o u n t o f
0 20o
150
loo
50
0
Uronide size
Fig. 4. Size distribution of uronides released from PGA by PG-I! and PG-IV, after 0.5-, I-, 2- and 4-h incubation (a, b, c and d, respectively). II, PG-tl; Q, PG-IV. oligomers with 1 - 4 residues while oligomers with more than 7 - 9 residues declined at 8 h and were absent at 24 h (data not shown). In a first experiment the elicitor activity of the
11
digestion mixtures at 4, 8 and 24 h was compared. A decreasing elicitor activity was observed from 4 to 24 h of incubation without significant differences between the two isoenzymes (Table I). Therefore, in a second experiment the glyceollin elicitor activity of digestion mixtures up to 4 h of incubation was assessed. The elicitor activity increased from 30 min to 4 h of incubation for both isoenzymes (Table I). At 2 and 4 h of incubation the digestion products obtained by PG-II showed a significantly higher elicitor activity than those released by PG-IV. PG-IV produced a large degree of hypocotyl maceration and released a significant amount of uronides whilst the effect of PG-II on hypocotyls was not much different from the control (Table II).
Table II.
Table I. Elicitor activity of uronides obtained by digestion of PGA with PG-II or PG-IV.
Discussion
Treatment
Digestion time a
Elicitor activity b
(h)
(A286)
4 8 24 4 8 24
0.06 a c 0.19d 0.12 c 0.08 ab 0.12c 0.09 bc 0.07 ab
1st experiment Undigested PGA PG-II
PG-IV
2nd experiment Undigested P G A PG-II
PG-IV
0.5 1 2 4 0.5 1 2 4
0.05 0.07 0.10 0.16 0.21 0.06 0.06 0.07 0.11
a ab b c d ab ab ab b
a0.5% P G A at pH 4.6 was incubated at 30°C with 0.015 R U m1-1 of PG-II or PG-IV. At various times aliquots were taken from the incubation mixtures and heated at 100°C for 20 min. bA 50-/~1 aliquot of the incubation mixture was applied to the wounded surface of each cotyledon. Three sets of 10 cotyledons were used for each treatment. Elicitor activity was measured as the A286 of the wound-droplet solution. cWitbin each experiment the means followed by the same letter are not significantly different (P = 0.05) according to the Student-Newman-Keuls test.
Effect of PG-il and PG-IV on soybean hypocotyls.
Treatment
Macerating force (g)a
Uronides released (#g per hypocotyl)
Control b PG-ii PG-IV
350 a c 289 a 160 b
0.7 a 1.7 a 48.0 b
aThe macerating force is expressed as the weight required to collapse the hypocotyl slices. bControl consisted of hypocotyls treated with 5 m M sodium acetate buffer. cWithin columns, data followed by the same letter are not significantly different (P = 0.01) according to the Student Newman-Keuls test.
PG-II and PG-IV, the major endo-PG isoenzymes produced in soybean seedlings by the B-24 isolate of S. sclerotiorum, present differences in some biochemical and physiological properties. PG-IV shows a higher specific activity, a lower molecular mass, a more extended pH range of activity and a higher affinity for pectin compared to PG-II. PG-IV, therefore, possesses more advantageous properties than PG-II in the attack of the plant tissue: the enzyme might show a higher activity since the pH of the cell wall is neutral or subacid and the galacturonosyl carboxylate groups in the plant cell wall are partially methylated [15]. These properties could account for the large release of uronides and the tissue maceration. They might also be responsible for the higher glyceollin elicitation in soybean seedlings obtained with PG-IV when used at lower, nonmacerating doses [2]. The two isoenzymes exhibited different patterns of degradation of PGA. Uronides obtained by PGII showed higher elicitor activity. These findings are in agreement with the results obtained from soybean cell walls where the oligomers released by PG-II showed a higher elicitor activity than those released by PG-IV [2]. Nothnagel et al. [16] reported that, from the citrus pectin products obtained by partial acid hydrolysis, the greater elicitor activity in soybean is due to dodecagalac-
12
turonide. The higher elicitor activity observed using the digestion products obtained by PG-II may be partially explained by the greater presence of dodecagalacturonide in the PG-II incubation mixtures. However the significant presence of this oligomer also in the PG-IV uronide digestion pattern seems to suggest a more complex mechanism of action of galacturonides where the interference of the smaller size uronides may play an important role [17]. The different macerating abilities of the two isoenzymes and their different glyceollin elicitor activities observed on the soybean hypocotyls [2] could also be explained by a different level of inhibition exerted against the 2 isoenzymes by a cell wall factor such as a PG-inhibiting protein (PG1P). This is based on the findings of several authors [18-22] who purified and characterized PGIPs from different Leguminosae species. Although a Phaseolus vulgaris PGIP (gift of Prof. Cervone, University of Rome, Italy) inhibited both PG-II and PG-IV to a similar extent (unpublished results), the possibility of a differential inhibition cannot be excluded until a soybean PGIP be purified and assayed against the two S. sclerotiorum isoenzymes.
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Acknowledgements 13
The authors thank Dr. U. Vettori, CNR Padova, for FAB-MS analysis. Research work supported by C.N.R., Italy. Special grant RA1SA, Sub-project 2, Paper N. 285.
References 1
2
3
4
D.C. Sutton and B.J. Deverall, Phytoalexin accumulation during infection of bean and soybean by ascospores and mycelium of SclerothTia sclerotiorum, Plant Pathol., 33 (1984) 377-383. F. Favaron, P. Alghisi, P. Marciano and P. Magro, Polygalacturonase isoenzymes and oxalic acid produced by Sclerotinia sch, rotiorum in soybean hypocotyls as elicitors of glyceollin, Physiol. Mol. Plant Pathol., 33 (1988) 385-395. R.J. Bruce and C.A. West, Elicitation of casbene synthetase activity in castor bean. The role of pectic fragments of the plant cell wall in elicitation by a fungal endopolygalacturonase, Plant Physiol., 69 (1982) 1181-1188. K.R. Davis, G.D. Lyon, A.G. Darvill and P. Albersheim,
14
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18
Host-pathogen interactions. XXV. Endopolygalacturonic acid lyase from Erwinia carotovora elicits phytoalexin accumulation by releasing plant cell wall fragments, Plant Physiol., 74 (1984) 52-60. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. V.K. Laemmti, Cleavage of structural proteins during the assembly of the head ot" bacteriophage T4, Naturc, 227 (1970) 680-685. G. Avigad, Colorimetric assay for hexuronic acids and some keto sugars, in: W.A. Wood (Ed.), Methods in Enzymology, Vol. XLI, Academic Press, New York. 1975, pp. 29-3 I. N. Nelson, A photometric adaptation of the Somogyi method for determination of glucose, J. Biol. Chem., 153 (1944) 375-380. Z.I. Kertesz, Preparation and determination of pectic substances, in: S.P. Colowick and N.O. Kaplan (Eds,), Methods in Enzymology, Vol. Ill, Academic Press, New York, 1957, pp. 27-30. N. Blumenkrantz and G. Asboe-Hansen, New method for quantitative determination ot" uronic acids, Anal. Biochem., 54 (1973) 484-489. M.G. Hahn, A.G. Darvill and P. Albersheim, Hostpathogen interactions. XIX. The endogenous elicitor, a fragment of a plant cell wall polysaccharide that elicits phytoalexin accumulation in soybeans, Plant Physiol., 68 (1981) 1161-1169. J . P R . Keon, G. Waksman and J.A. Bailey, A comparison of the biochemical and physiological properties of a polygalacturonase from two races of Collemtrichum lindemuthianum, Physiol. Mol. Plant Pathol., 37 (1990) 193-206. D.F. Jin and C.A. West, Characteristics of galacturonic acid oligomers as elicitors of casbene synthetase activity in castor bean seed[ings, Plant Physiol., 74 (1984) 989-992. B. Robertsen, Elicitors of the production of lignin-Iike compounds in cucumber hypocotyls, Physiol. Mol. Plant Pathol., 28 (1986) 137-148. M. McNeil, A.G. Darvill, S.C. Fry and P. Albersheim, Structure and function of the primary cell walls of plants, Annu. Rev. Biochem., 53 (1984) 625-663. E.A. Nothnagel, M. McNeil, P. Albersheim and A. Dell, Host-pathogen interactions. XXII. A galacturonic acid oligosaccharide from plant cell walls elicits phytoalcxins, Plant Physiol., 71 (1983) 916-926. K.R. Davis, A.G. Darvill, P. Albersheim and A. Dell, Host-pathogen interactions. XXIX. Oligogalacturonides released from sodium polypectate by endopolygalacturonic acid lyase are elicitors of phytoalexins in soybean, Plant Physiol., 80(1986) 568-577. A.J. Anderson and P. Albersheim, Host-pathogen interactions. V. Comparison of abilities of proteins isolated from three varieties of Phaseolus vulgaris to inhibit the en-dopolygalacturonase secreted by three races of Colletotrichum lindemuthianum, Physiol. Plant Pathol., 2 ( 1972} 339-346.
13 19
20
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F. Cervone, G. De Lorenzo, L. Degra', G. Salvi and M. Bergami, Purification and characterization of a polygalacturonase-inhibiting protein from Phaseolus vulgaris L., Plant Physiol., 85 (1987) 631-637. L. Degra', G. Salvi, D. Mariotti, G. De Lorenzo and F. Cervone, A polygalacturonase-inhibiting protein in alfalfa callus cultures, J. Plant Physiol., 133 (1988) 364-366. R.M. Hofl'man and J.G. Turner, Partial purification of
22
proteins from pea leaflets that inhibit Aschochyta pisi endopolygalacturonase, Physiol. Plant Pathol., 20 (1982) 173-187. C. Lafitte, J.P. Barthe, J.k. Montillet and A. Touze', Clycoprotein inhibitors of Colletotrichum limtemuthianum endopolygalacturonase in near isogenic lines ot" Phaseolus vulgaris resistant and susceptible to anthracnose. Physiol. Plant Pathol., 25 (1984) 39-53.
7
Characterization of two Sclerotinia sclerotiorum polygalacturonases with different abilities to elicit glyceollin in soybean Francesco Favaron, Paolo Alghisi and Paola Marciano lstituto di Patologia vegetale, Universit?t di Padova (Italy)
(Received May 30th, 1991; revision received December 19th, 1991; accepted December 23rd, 1991)
Two endo-polygalacturonase isoenzymes (PG-II and PG-IV), with masses of 34 and 30 kDa respectively, were purified from soybean hypocotyls infected by Sclerotinia sclerotiorum. The pH optimum for both isoenzymes was about 4.6, but PG-IV exhibited a broader range of pH activity. PG-IV showed a much higher affinity for pectin than did PG-I1. PG-II hydrolyzed polygalacturonic acid in a more random fashion than PG-IV. Oligouronides produced by PG-II showed a higher phytoalexin elicitor activity. PG-IV produced a large degree of maceration of soybean hypocotyls releasing a significant amount of uronides. The properties of PG-II and PG-IV are discussed in relation to the different ability of the two isoenzymes to elicit glyceollin in soybean. Key words: polygalacturonase isoenzymes; glyceollin elicitation; uronide oligomers; Sclerotinia sclerotiorum
Introduction
Soybean tissues infected by Sclerotinia sclerotiorum (Lib.) de Bary may display a hypersensitive response to infection and accumulate the phytoalexin glyceollin [11. The involvement in glyceollin elicitation of the two major endopolygalacturonases (PG-II and PG-IV), produced by the B-24 isolate of S. sclerotiorum in soybean hypocotyls, was reported in a previous paper [2]. The release of heat stable elicitors of glyceollin from purified soybean cell walls by both PG-II and PG-IV, was consistent with the pectic nature of the elicitors as reported by Bruce and West [3] and Davis et al. [4]. When the elicitor activity of uronides released by both isoenzymes from purified cell walls of soybean hypocotyls was compared, the activity of PG-II cell wall-derived uronides was higher than those of PG-IV. In contrast to this, when etiolated soybean hypocotyls Correspondence to: F. Favaron, Istituto di Patologia vegetale, via Gradenigo 6, 35131 Padova, Italy. Abbreviations: PG, polygalacturonase; PGA, polygalacturonic acid.
were directly exposed to isoenzymes, PG-IV induced a higher glyceollin accumulation [2]. In the present work we show the possibility that the behaviour of the two isoenzymes might be related to the differences in their biochemical and physiological properties. Materials and Methods
Polygalacturonase (PG) production and isoenzyme purification Six-day-old etiolated soybean seedlings (Glycine max [L.] Merr., cv. Canton) were grown and inoculated with the B-24 isolate of S. sclerotiorum as previously described [2]. PGs were extracted from infected hypocotyls 24 h after inoculation and purified by isoelectrofocusing and FPLC ionexchange chromatography as reported [2]. Protein was determined by the method of Lowry et al. [5], with bovine serum albumin as the standard. Gel electrophoresis SDS-PAGE was performed as described by Laemmli [6]. Protein bands were stained with
0168-9452/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
Comassie blue R-250. Molecular weight standards were phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (42.7 kDa), carbonic anhydrase (31.0 kDa) and soybean trypsin inhibitor (21.5 kDa). '~
Enzyme assay PG activity was determined as an increase of reducing end-groups with respect to time. Reducing end-groups were measured by the method described by Avigad [7] using D-galacturonic acid (Fluka, Switzerland) as a standard. One unit of PG activity (RU) was defined as that producing at 30°C 1 #mol of reducing groups min -l on 0.5'7,, (w/v) polygalacturonic acid (PGA, Sigma, U.S.A.) in sodium acetate buffer (100 mM, pH 4.6). To ascertain the effect of pH on PG activity, 100 mM sodium acetate or 100 mM citrate-phosphate buffers were used. In this case reducing groups were determined by the Nelson-Somogyi method [8]. The activity was also assayed by measuring the decrease in relative viscosity at 30°C of a 75% methylated apple pectin (Pektin B, Roth, Germany), previously washed according to Kertesz [9], or PGA, in 100 mM sodium acetate buffer (pH 4.6), in a micro-Ostwald viscosimeter (i.d. 0.70 mm). Decrease in relative viscosity was monitored by an AVS 310 system (Schott Gerate, Germany). One relative viscosimetric unit (RVU) was defined as that causing in 1 h a 50% reduction in the relative viscosity of 2 ml of the reaction mixture.
Release and fractionation of oligogalacturonides from PGA P G A (0.5%) at pH 4.6 was incubated at 30°C with 0.015 R U ml -l of PG-II or PG-IV. At 0.5-, 1-, 2-, 4-, 8- and 24-h aliquots of the incubation mixture were removed, immediately boiled for 20 rain at 100°C and stored at -20°C until used. Aliquots of 0.5 ml were analyzed by FPLC using an anion-exchange Mono Q H R 5/5 column (Pharmacia, Sweden) equilibrated with 20 mM Tris-HC1 buffer at pH 7.9. After loading the sample, the column was eluted with 10 ml of the equilibrating buffer and then with a linear gradient of NaCI (0.0-0.5 M) in the Tris-HC1 buffer. Before the start of the gradient 0.5-ml fractions were collected, then the eluent was monitored at
214 nm and each peak was collected in a separate fraction. The fractions were assayed for the uronide concentration by the m-hydroxydiphenyl method [10] using D-galacturonic acid as a standard. The fraction corresponding to the second uronide peak eluted after the start of the NaCI gradient was desalted on a Sephadex G-10 colunm (1.6 x 40 cm) equilibrated and eluted with water. The oligomer was then passed through an aromatic sulfonic acid disposable column (Baker, Holland) in the H + form and then lyophilized. A sample of about 0.2 mg of the lyophilized oligomer was dissolved in thioglycerol and analyzed by fast atom bombardment mass spectrometry (FAB-MS) in the positive mode.
Physiological properties Phytoalexin elicitor activity of the incubation mixtures (see above) at different times during PGA digestion, was assayed using the soybean cotyledon bioassay proposed by Hahn et al. [11]. 0.2 mg ml -l of streptomycin sulfate were added to each sample, Cotyledons were obtained from 7-day-old etiolated soybean plants (cv. Canton) and aliquots of sample of 50 /xl/ cotyledon were used. Three sets of 10 cotyledons were used for each treatment. Elicitor activity was measured as the A286of the wound-droplet solution. Tissue maceration was evaluated on 7-day-old etiolated soybean seedlings cut 5 cm below the cotyledons. The seedlings were washed, arranged in microvials (0.3 ml) and incubated as previously reported for the hypocotyl bioassay [2]. The microvials were filled with 0.2 ml of enzyme sample (0.4 R U m l -1) in 5 mM acetate buffer (pH 4.6) with 0.2 mg ml -l of streptomycin sulfate and 0.1 mg m1-1 of bovine serum albumin added. After 24 h of incubation the 1 cm terminal part of each hypocotyl was cut transversely into slices 2 mm thick. Tissue maceration was assessed as reported by Keon et al. [12] by measuring the weight required to cause the segments to collapse when placed between two glass microscope slides. Control consisted of hypocotyls treated with 5 mM buffer. The amount of uronides released from the hypocotyls was measured in the bathing solution by the m-hydroxydiphenyl method [10]. Five seedlings for each treatment were used. The experiment was repeated 3 times.
Results 100
PG-II and PG-IV were purified 48- and 95-fold, respectively, and their specific activity was 352 and 701 R U mg -] protein, respectively. On SDS-PAGE, PG-II and PG-IV showed only one band corresponding to a molecular mass of 34 kDa and 30 kDa, respectively (Fig. 1). These values are similar to those obtained by Superose-12 permeation chromatography (data not shown) where the PG-II and PG-IV molecular masses were found to be 35 kDa and 29 kDa, respectively. The activity of both isoenzymes was lower in citrate-phosphate buffer than in acetate buffer, with P G - I I showing a stronger reduction in activity (Fig. 2). Preliminary experiments had shown that the inhibitory effect was due to the citrate anion (data not reported). In acetate buffer both isoenzymes showed optimal activity at about pH 4.6. In both buffers PG-IV maintained its activity over a broader range o f p H values, and, at pH 6.0, PG-IV still showed an appreciable activity while PG-II was completely inactive. S0.5 values, determined from reducing-groups assays on different substrate concentrations, were 0.45 and 2.30 mg ml -] for PG-II on PGA and
1
2
3 k Da • 97.4
!
~
...... • 6 6 . 2
~,i~iii
4 42.7
< 31.0
•
21.5
Fig. 1. SDS-electrophoresis of PG-II (lane 1), PG-IV (lane 2) and protein standard (lane 3). Approx. 5 tzg of purified PG-II and 12 t~g of PG-IV were loaded.
80
._>
60 40
E
>,
20
N C
E
0 100
t~
E
80 60
"6 ae
40 20 0 3
3.5
4
4,5
5
5.5
6
6.5
7
pH Fig. 2. Effect of pH on PG activity. For both isoenzymes the maximum velocity corresponds to 0.015/~mol of galacturonic acid equivalents min -I ml -t of reaction mixture. Each data point is the average of 3 determinations. O, acetate: A citratephosphate.
pectin, respectively. Instead, the S0. 5 of PG-IV for PGA and pectin were 0.35 and 1.10 mg m1-1, respectively. Results of activity measured viscosimetrically (Fig. 3) showed that PG-II had a higher activity with PGA while PG-IV was always more active with pectin. Determinations of reducing end-groups at a time corresponding to a 50% loss in relative viscosity (T50) showed that PG-II cleaved 1.3% of the glycosidic bonds of PGA compared to 2.1% cleaved by PG-IV. When pectin was used as a substrate these values were 0.5% for PG-II and 0.9% for PG-IV. PG-IV, therefore, hydrolyzed both substrates less randomly than PG-II although both isoenzymes hydrolyzed the pectin in a more random manner than PGA. P G A was incubated for different times with the two isoenzymes. The oligomers released were analysed by a Mono Q column as described in Materials and Methods. At least 12 well resolved peaks were fractionated (figure not shown). The FAB-MS analysis of the second uronide peak
10 5~
200
PG-II
.
4
-
PGA
e
~
-
Pectin
(a)
150
~
pG-II
[~
PG-IV
3
100
rr
2 50
v
1 L
I
-
-
I
L
I
-
0 200
-
PG-IV
(b)
0
E
#
N4 U.I 3 ¢-
150
~=,, lOO 2
5O
1
0 0
--
i
1
--
~
L
I
i
2
3
4
5
[Substrate] (mg/ml) Fig. 3, Viscosimetricactivity of PG-II and PG-IV at different concentrations of PGA or pectin. Two milliliters of substrate containing 6 × 10-3 RU were used for each determination. Each data point is the average of 3 determinations. Zk, PGA; O, pectin.
3¢, O t~
o
0
200
(c)
150!
1oo 5oi
eluting after the start o f the gradient gave a mass spectra revealing a m a j o r peak at 568 rn/: and two minor peaks at 590 and 612 re~z, respectively. These correspond to the mono-, di- and trisodium salts, respectively, of a trimer of galacturonic acid. Since, in a separate experiment, a standard o f galacturonic acid was eluted before the start o f the salt gradient, the first uronide peak eluted after the start of the gradient was considered to correspond to a dimer. Therefore, in accordance with other authors [13,14], the uronide peaks from 3 to 12 might correspond to oligomers with 4 - 1 3 galacturonosyl groups. The a m o u n t o f the oligomers with 1-13 galacturonosyl residues produced by P G - I I and P G - I V increased up to 4 h of digestion, P G - I I producing a greater a m o u n t o f oligomers with 3 to 13 residues than P G - I V (Fig. 4). With both isoenzymes, a prolonged digestion (8 and 24 h) produced mostly an increasing a m o u n t o f
0 20o
150
loo
50
0
Uronide size
Fig. 4. Size distribution of uronides released from PGA by PG-I! and PG-IV, after 0.5-, I-, 2- and 4-h incubation (a, b, c and d, respectively). II, PG-tl; Q, PG-IV. oligomers with 1 - 4 residues while oligomers with more than 7 - 9 residues declined at 8 h and were absent at 24 h (data not shown). In a first experiment the elicitor activity of the
11
digestion mixtures at 4, 8 and 24 h was compared. A decreasing elicitor activity was observed from 4 to 24 h of incubation without significant differences between the two isoenzymes (Table I). Therefore, in a second experiment the glyceollin elicitor activity of digestion mixtures up to 4 h of incubation was assessed. The elicitor activity increased from 30 min to 4 h of incubation for both isoenzymes (Table I). At 2 and 4 h of incubation the digestion products obtained by PG-II showed a significantly higher elicitor activity than those released by PG-IV. PG-IV produced a large degree of hypocotyl maceration and released a significant amount of uronides whilst the effect of PG-II on hypocotyls was not much different from the control (Table II).
Table II.
Table I. Elicitor activity of uronides obtained by digestion of PGA with PG-II or PG-IV.
Discussion
Treatment
Digestion time a
Elicitor activity b
(h)
(A286)
4 8 24 4 8 24
0.06 a c 0.19d 0.12 c 0.08 ab 0.12c 0.09 bc 0.07 ab
1st experiment Undigested PGA PG-II
PG-IV
2nd experiment Undigested P G A PG-II
PG-IV
0.5 1 2 4 0.5 1 2 4
0.05 0.07 0.10 0.16 0.21 0.06 0.06 0.07 0.11
a ab b c d ab ab ab b
a0.5% P G A at pH 4.6 was incubated at 30°C with 0.015 R U m1-1 of PG-II or PG-IV. At various times aliquots were taken from the incubation mixtures and heated at 100°C for 20 min. bA 50-/~1 aliquot of the incubation mixture was applied to the wounded surface of each cotyledon. Three sets of 10 cotyledons were used for each treatment. Elicitor activity was measured as the A286 of the wound-droplet solution. cWitbin each experiment the means followed by the same letter are not significantly different (P = 0.05) according to the Student-Newman-Keuls test.
Effect of PG-il and PG-IV on soybean hypocotyls.
Treatment
Macerating force (g)a
Uronides released (#g per hypocotyl)
Control b PG-ii PG-IV
350 a c 289 a 160 b
0.7 a 1.7 a 48.0 b
aThe macerating force is expressed as the weight required to collapse the hypocotyl slices. bControl consisted of hypocotyls treated with 5 m M sodium acetate buffer. cWithin columns, data followed by the same letter are not significantly different (P = 0.01) according to the Student Newman-Keuls test.
PG-II and PG-IV, the major endo-PG isoenzymes produced in soybean seedlings by the B-24 isolate of S. sclerotiorum, present differences in some biochemical and physiological properties. PG-IV shows a higher specific activity, a lower molecular mass, a more extended pH range of activity and a higher affinity for pectin compared to PG-II. PG-IV, therefore, possesses more advantageous properties than PG-II in the attack of the plant tissue: the enzyme might show a higher activity since the pH of the cell wall is neutral or subacid and the galacturonosyl carboxylate groups in the plant cell wall are partially methylated [15]. These properties could account for the large release of uronides and the tissue maceration. They might also be responsible for the higher glyceollin elicitation in soybean seedlings obtained with PG-IV when used at lower, nonmacerating doses [2]. The two isoenzymes exhibited different patterns of degradation of PGA. Uronides obtained by PGII showed higher elicitor activity. These findings are in agreement with the results obtained from soybean cell walls where the oligomers released by PG-II showed a higher elicitor activity than those released by PG-IV [2]. Nothnagel et al. [16] reported that, from the citrus pectin products obtained by partial acid hydrolysis, the greater elicitor activity in soybean is due to dodecagalac-
12
turonide. The higher elicitor activity observed using the digestion products obtained by PG-II may be partially explained by the greater presence of dodecagalacturonide in the PG-II incubation mixtures. However the significant presence of this oligomer also in the PG-IV uronide digestion pattern seems to suggest a more complex mechanism of action of galacturonides where the interference of the smaller size uronides may play an important role [17]. The different macerating abilities of the two isoenzymes and their different glyceollin elicitor activities observed on the soybean hypocotyls [2] could also be explained by a different level of inhibition exerted against the 2 isoenzymes by a cell wall factor such as a PG-inhibiting protein (PG1P). This is based on the findings of several authors [18-22] who purified and characterized PGIPs from different Leguminosae species. Although a Phaseolus vulgaris PGIP (gift of Prof. Cervone, University of Rome, Italy) inhibited both PG-II and PG-IV to a similar extent (unpublished results), the possibility of a differential inhibition cannot be excluded until a soybean PGIP be purified and assayed against the two S. sclerotiorum isoenzymes.
5
6
7
8
9
10
11
12
Acknowledgements 13
The authors thank Dr. U. Vettori, CNR Padova, for FAB-MS analysis. Research work supported by C.N.R., Italy. Special grant RA1SA, Sub-project 2, Paper N. 285.
References 1
2
3
4
D.C. Sutton and B.J. Deverall, Phytoalexin accumulation during infection of bean and soybean by ascospores and mycelium of SclerothTia sclerotiorum, Plant Pathol., 33 (1984) 377-383. F. Favaron, P. Alghisi, P. Marciano and P. Magro, Polygalacturonase isoenzymes and oxalic acid produced by Sclerotinia sch, rotiorum in soybean hypocotyls as elicitors of glyceollin, Physiol. Mol. Plant Pathol., 33 (1988) 385-395. R.J. Bruce and C.A. West, Elicitation of casbene synthetase activity in castor bean. The role of pectic fragments of the plant cell wall in elicitation by a fungal endopolygalacturonase, Plant Physiol., 69 (1982) 1181-1188. K.R. Davis, G.D. Lyon, A.G. Darvill and P. Albersheim,
14
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18
Host-pathogen interactions. XXV. Endopolygalacturonic acid lyase from Erwinia carotovora elicits phytoalexin accumulation by releasing plant cell wall fragments, Plant Physiol., 74 (1984) 52-60. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. V.K. Laemmti, Cleavage of structural proteins during the assembly of the head ot" bacteriophage T4, Naturc, 227 (1970) 680-685. G. Avigad, Colorimetric assay for hexuronic acids and some keto sugars, in: W.A. Wood (Ed.), Methods in Enzymology, Vol. XLI, Academic Press, New York. 1975, pp. 29-3 I. N. Nelson, A photometric adaptation of the Somogyi method for determination of glucose, J. Biol. Chem., 153 (1944) 375-380. Z.I. Kertesz, Preparation and determination of pectic substances, in: S.P. Colowick and N.O. Kaplan (Eds,), Methods in Enzymology, Vol. Ill, Academic Press, New York, 1957, pp. 27-30. N. Blumenkrantz and G. Asboe-Hansen, New method for quantitative determination ot" uronic acids, Anal. Biochem., 54 (1973) 484-489. M.G. Hahn, A.G. Darvill and P. Albersheim, Hostpathogen interactions. XIX. The endogenous elicitor, a fragment of a plant cell wall polysaccharide that elicits phytoalexin accumulation in soybeans, Plant Physiol., 68 (1981) 1161-1169. J . P R . Keon, G. Waksman and J.A. Bailey, A comparison of the biochemical and physiological properties of a polygalacturonase from two races of Collemtrichum lindemuthianum, Physiol. Mol. Plant Pathol., 37 (1990) 193-206. D.F. Jin and C.A. West, Characteristics of galacturonic acid oligomers as elicitors of casbene synthetase activity in castor bean seed[ings, Plant Physiol., 74 (1984) 989-992. B. Robertsen, Elicitors of the production of lignin-Iike compounds in cucumber hypocotyls, Physiol. Mol. Plant Pathol., 28 (1986) 137-148. M. McNeil, A.G. Darvill, S.C. Fry and P. Albersheim, Structure and function of the primary cell walls of plants, Annu. Rev. Biochem., 53 (1984) 625-663. E.A. Nothnagel, M. McNeil, P. Albersheim and A. Dell, Host-pathogen interactions. XXII. A galacturonic acid oligosaccharide from plant cell walls elicits phytoalcxins, Plant Physiol., 71 (1983) 916-926. K.R. Davis, A.G. Darvill, P. Albersheim and A. Dell, Host-pathogen interactions. XXIX. Oligogalacturonides released from sodium polypectate by endopolygalacturonic acid lyase are elicitors of phytoalexins in soybean, Plant Physiol., 80(1986) 568-577. A.J. Anderson and P. Albersheim, Host-pathogen interactions. V. Comparison of abilities of proteins isolated from three varieties of Phaseolus vulgaris to inhibit the en-dopolygalacturonase secreted by three races of Colletotrichum lindemuthianum, Physiol. Plant Pathol., 2 ( 1972} 339-346.
13 19
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F. Cervone, G. De Lorenzo, L. Degra', G. Salvi and M. Bergami, Purification and characterization of a polygalacturonase-inhibiting protein from Phaseolus vulgaris L., Plant Physiol., 85 (1987) 631-637. L. Degra', G. Salvi, D. Mariotti, G. De Lorenzo and F. Cervone, A polygalacturonase-inhibiting protein in alfalfa callus cultures, J. Plant Physiol., 133 (1988) 364-366. R.M. Hofl'man and J.G. Turner, Partial purification of
22
proteins from pea leaflets that inhibit Aschochyta pisi endopolygalacturonase, Physiol. Plant Pathol., 20 (1982) 173-187. C. Lafitte, J.P. Barthe, J.k. Montillet and A. Touze', Clycoprotein inhibitors of Colletotrichum limtemuthianum endopolygalacturonase in near isogenic lines ot" Phaseolus vulgaris resistant and susceptible to anthracnose. Physiol. Plant Pathol., 25 (1984) 39-53.