purification and characterization of an endopolygalacturonase produced by colletotrichum...

Physiological and Molecular Plant Pathology (1989) 35, 121-133

Purification and characterization of anendopolygalacturonase produced byColletotrichum gloeosporioides

D. PRUSKY* , S. GOLD and N . T. KEENtDepartment of Plant Pathology, University of California, Riverside, 92521 U .S.A .

(Accepted for publication January 1989)

An endopolygalacturonase from C. gloeosporioides was purified 130-fold to apparent homogeneityusing phenyl sepharose chromatography followed by isoelectric focusing . The purified preparationscontained two polygalacturonase species of 68 and 62 kD based on SDS gel electrophoresis andthin layer gel electrofocusing . These apeared to be glycosylation adducts of a single protein sincetheir amino acid composition and N-terminal amino acid sequences were identical and treatmentwith endoglycosidase F altered their migration to yield a single band on SDS gel electrophoresis .The purified C . gloeosporioides endopolygalacturonase efficiently macerated avocado and cucumbertissue . Epicatechin at 20 and 80 pg ml - ' inhibited enzyme activity by 6. 5 % and 43 %,respectively . The concentration of epicatechin in peel of unripe avocado fruits ranged between430-1970 µg g- ' Er wt (570-2600 µg ml- ') but decreased in ripe susceptible fruits to 6-25 pg g- 'fr wt (approx . 8-30 µg ml- ') . These observations raised the possibility that epicatechin mightregulate the activity of the fungal endopolygalacturonase in infected avocado fruits .

INTRODUCTION

Colletorichum gloeosporioides infects immature avocado fruits in the orchard but remainsquiescent until fruit ripening occurs [5] . Then the fungus renews development, causingbrown symptoms on the peel and soft-rotting of the flesh [21] . The factors responsiblefor such tissue maceration are generally cell-wall degrading enzymes [4] . Poly-galacturonases are considered important in the pathogenicity and virulence of severalfungi [10], but much of the evidence is contradictory and controversial [11, 13, 14] .Uncertainty persists because there is little genetic evidence for the role of the enzymes .Additionally, the mutants employed have been developed by chemical agents or UVlight with the attendant risks of multiple mutations .

Barash & Khazzan [2] reported the production of endopolygalacturonase andpectin lyase activities by C. gloeosporioides during fruit attack . As the first step towarddetermining the importance of these pectic enzymes in the pathogenicity of C .gloeosporioides, we have purified and characterized the major endopolygalacturonase

Abbreviations used in text : endo-PG, endopolygalacturonase ; PG, polygalacturonase ; SDS, sodiumdodecyl sulphate, IEF, isoelectric focusing .

*Permanent address : Department of Storage of Agricultural Products, Volcani Center, Bet Dagan, 50250Israel .

Author to whom correspondence should be addressed .

0885-5765/89/080121 + 13 $03 .00/0

© 1989 Academic Press Limited

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produced in culture . It is our intention to attempt to molecular clone the gene encodingthis enzyme in order to permit its mutagenesis by a gene replacement experiment . Thisand related molecular genetic experiments should provide a definitive assessment of therelative importance of pectic enzymes in fungal pathogenicity .

MATERIALS AND METHODS

Fungal isolates and growth conditionsA single spore isolate of C . gloeosporioides from avocado fruits was used for enzymepurification [21] . This isolate was maintained by inoculation of avocado fruits everymonth or on potato dextrose agar plates incubated at 25 °C. Water suspensionscontaining 2-4 x 10 6 spores per ml were used to initiate cultures . Fruit inoculationswere carried out as described previously [21] .

Other pectic enzymesA pectate lyase (PLe) was obtained from periplasmic fractions of Escherichia coli HB101cells carrying the cloned Erwinia chrysanthemi EC 16 pelE gene in plasmid pPEL748 [16] .Periplasmic fractions were dialysed against 10 mm Tris-HCl, pH 8 .0, before use. Apectate lyase (PL153) obtained from the periplasmic fraction of E. coli D1210.1 cellscontaining the cloned pell53 gene from Erwinia carotovora EC153 was also used [29] . Anadditional polygalacturonase was purified from a citrus isolate of Geotrichum candidum bymethods similar to [3], except that isoelectric focusing replaced affinity chromatography(Gold et al ., unpublished data) .

Enzyme and maceration assaysPolygalacturonase was assayed by monitoring the release of reducing groups frompolygalacturonate as measured by the arsenomolybdate method of Nelson [2] .Reaction mixtures consisted of 750 pl of 0 . 5 °,ô sodium polypectate in 0 . 1 M sodiumacetate buffer, pH 5 .0, 700 gl of distilled water and 50 gl of enzyme and were incubatedat 31 °C. Samples (100 µl) were withdrawn at zero time and every 5 min thereafter andtransferred to the Nelson alkaline copper reagent . Nelson analyses were then completedwith galacturonic acid as a standard and rate curves for reducing sugar release weredrawn . One unit of enzyme activity was defined as that liberating 1 µmole of reducingequivalents min"' . Polygalacturonase activity was also assayed by monitoring changesin substrate viscosity . Reduction in viscosity was measured in a Cannon-Fenskeviscometer (size 100) at room temperature with 4 . 5 ml of 0 . 5 % sodium polypectate in25 mm sodium acetate, pH 5 .0 and 1 .5 ml of appropriately diluted enzyme . Pectatelyase activity was assayed according to Keen & Tamaki [16] and 1 unit was definedas that liberating 1 µmole of unsaturated galacturonide min - '. This assay, involvingthe measurement of absorbance change at 232 nm at pH 8 .5 was verified bycomparison with the reducing sugar assay . It was observed that the two methods forproduct measurement agreed within 10% . Viscosimetric assays were also performedwith PLe of E . chrysanthemi EC16 as described in [29] .

Maceration activity was determined by incubating five thin wedges approx . 0 . 2 by0 .4 cm and 0 . 5 to 0 . 6 cm thick, consisting of exo- and mesocarp of cucumber (marketbought) or avocado fruits (freshly harvested cv . Hass), with enzyme in 600 pi of

Purification and characterization of an endopolygalacturonase

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25 mm sodium acetate, pH 5 .0, for 5-6 h at 31 °C. Pectate lyases obtained fromEscherichia coli cells with the cloned Erwinia chrysanthemi EC 16 pelE and E. carotovoraEC153 pe1153 genes were assayed in the same way except in 10 mm Tris hydrochloride,pH 7 .5 [16] . Buffers alone were used as controls in all maceration assays . Maceratedcucumber wedges exhibited the loss of tissue turgidity and integrity . Maceration ofavocado wedges started from the outside of the flesh and proceeded into the centre ofthe wedge, finally releasing the peel from the macerated tissue . Data were expressed asrelative enzyme concentrations which completely macerated avocado wedges duringthe observation period .

Enzyme purificationC. gloeosporioides was grown in 2 1 Erlenmeyer flasks (containing I 1 of medium) at28 °C with shaking until the pH of the medium increased to 7 .5-7 . 7. The mediumconsisted of (g I - ') : polygalacturonic acid, sodium salt, 5 g ; citrus pectin, 5 g ; KNO3 ,5 g ; KH2PO4 , 4 g ; MgSO4 .7H2O, 2 g; CaCl2.7H2O, 0.3 g; asparagine, 2 . 5 g ; FeCl 3 ,0. 01 g and glucose, 0 . 01 g. Most of the hyphae were removed from approx . 5 1 ofculture by filtration through four layers of cheese cloth . The culture was thencentrifuged at 10000 g at 4 °C for 15 min and the supernatant fluids were concentratedto about 200 ml on a rotary evaporator at 40 °C and then centrifuged for 10 min at10000 g. The supernatant was dialysed overnight at 4 °C against two changes of25 mm sodium acetate, pH 6.0 (101 each) . The dialysed fluids were centrifuged at10000g for 15 min and the supernatant was adjusted to 30% (w/v) (NH 4 ) 2SO4 andsubjected to hydrophobic interaction chromatography using a phenyl-sepharose CL4Bcolumn (15 by 15 cm) previously equilibrated with 30" 0 (w/v) ammonium sulphatein 25 mm sodium acetate, pH 6 . 0. Bound proteins were then eluted with a lineargradient of 30 % (w/v) to 0 % ammonium sulphate (200 ml total volume) in 25 mmsodium acetate, pH 6 . 0, and fractions of 2-3 ml were collected .

Fractions showing the highest polygalacturonase activity were pooled and dialysedovernight against 10 mm sodium acetate, pH 6 .0. The dialysed solution was subjectedto preparative IEF using an LKB 8100-1 column with 1 °p carrier ampholytes (pH 3 . 0to 10 or 4 to 6 . 5) . The sample was applied in a linear sucrose gradient 50-5 % (w/v)and electrophoresed at constant power of 5 W for 48 h and a final 1700-2000 V . ThepH, absorbance at 280 nm and enzyme activity were measured in 2 ml fractions .

In some cases the active fractions obtained from the IEF column were run on aSephacryl S-200 column (60 x 1 cm) at room temperature in 25 mm sodium acetatebuffer, pH 6. 0, and I ml fractions were collected .

Native and SDS-polyacrylamide gel electrophoresisThe cationic system of Reisfeld et al . [24] was used with 12 .5 % polyacrylamide fornative gels. Polygalacturonase activity in these gels was detected with sodiumpolypectate-agarose overlays, as described later . Proteins were also analysed ondenaturing sodium dodecyl sulphate-polyacrylamide gels (12 .0% acrylamide and1-2",, bis-acrylamide) according to the method of Laemmli [17], with carbonicanhydrase (31 kD), ovalbumin (45 kD), bovine serum albumin (66 kD) andphosphorylase B (97 kD) as molecular weight standards (Sigma) . Gels were stainedwith Coomassie brillant blue R250 . Analytical IEF was preformed on 5 % acrylamide

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D. Prusky et al.gels, 0.8 mm-thick, with a Bio-Rad Biophoresis unit according to the proceduredescribed by the manufacturer ; pH 3-10 or 4-6 ampholytes (Bio-Rad) were used .NaOH (IN) was used as the cathode buffer and the anode buffer was 1 N phosphoricacid. The following standards (Bio-Rad) were used : phycocyanin (pI 4.65), B-lactoglobulin B (pi 5 . 10), bovine carbonic anhydrase (pi 6 .0), human carbonicanhydrase (pI 6 .5), equine myoglobin (pI 7 .0), whale myoglobin (pI 8 .05) andcytochrome C (pI 9.6) . The standards were prefocused and 5-10 pl of enzyme samplewere then placed on a 1 x 1 cm square filter paper which was adpressed to the basic sideof the gel .

Thin (approx . lmm) pectate-agarose overlay gels for enzyme detection were caston Gel-fix (Ephortec) as described by Ried & Collmer [25] . The overlay solutioncontained 1 % agarose (Bethesda Research Laboratory), 0 . 1 % polygalacturonic acidand 25 mm sodium acetate buffer, pH 5 . 0. The overlays were incubated at 31 °C forapprox. 10 min and developed in 0. 1 % ruthenium red for 20 min [18] . The overlayswere dried by blotting on Whatman 3MM paper overnight .

Protein concentration in solution was determined by measuring the absorbance at280 nm or by the method of Bradford [6] with bovine serum albumin as the standard .

Amino acid sequencing and compositionThe purified polygalacturonase sample obtained from the isoelectric focusing columnwas run on a SDS-PAGE gel as described above in the presence of prestained markers .The proteins were then transferred to an Immobilon PVDF transfer membrane byelectro-blotting at a constant 20 volts for 30-45 min [28] . The membrane was thenstained with Coomassie Blue R250 and, after destaining, the desired protein bandswere cut from the membrane and used for amino acid sequencing by automated micro-Edman degradation using an Applied Biosystems Model 470-A vapour-phasesequencer .

For amino acid composition analyses, 200-300 µl of active fraction from the IEFcolumn (approx . 90 µg of protein) were run on a Sephacryl S-200 column as describedbefore and pooled active fractions were dialysed against 4 mm ammonium acetatebuffer, pH 6 . 0. Alternatively, a portion of the proteins from the transfer blot above wasanalysed . All samples were hydrolysed in 6 N HC1 and I % phenol at 150 °C in the gasphase under argon for 90 min .

Determination of K m and inhibition constants .For determination of the apparent Kon polygalacturonic acid (av. mol . wt 5000 ;polygalacturonide content 85-90% ; Sigma), the substrate concentration was variedfrom 0.025 to 0 .5 mg ml-1 in the standard assay. Epicatechin (Aldrich) at finalconcentrations up to 100 p.g ml-1 was also added to reaction mixtures with 0 .25polygalacturonate and containing a final 1 % ethanol . Ethanol alone did not affect thevelocity of the enzyme reaction . In all cases, standard reciprocal plots were drawn todetermine the constants .

Deglycosylation of glycoproteinsThis was done essentially by the method of Tarentino et al. [27] . A stock solution of theendopolygalacturonase was boiled for 3 min in the presence of 1 % SDS and 0 .25 M


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sodium acetate, pH 5 .0. The reaction mixture (100 pl) contained boiled endo-polygalacturonase (30-40 µg), 20 mm EDTA and 10 units of endo-ß-N-acetyl-glucosaminidase F (Endoglycosidase F, Boehringer Mannheim Biochemica) . Mixtureswere incubated at 37 °C for at least 25 h and samples were then analysed by SDS-PAGE.

Extraction and quantitative analysis of epicatechin in avocado fruits .Epicatechin in avocado peel and flesh was extracted and quantitated according to themethod described by Prusky et al. [22] . Fruit firmness, a parameter inverse to fruitripeness, was also determined as previously described [21] .

RESULTS

Purification of endopolygalacturonasePolygalacturonase activity was detected in the culture medium of C. gloeosporioides after2-3 days . The pH of the culture filtrate increased from 4 . 3 initially to about 8-4 during8 days of growth . As the pH increased to 7 . 8, the enzyme activity of the culture filtrateincreased as well. Further increase in the pH, however, resulted in decreased enzymeactivity . Several methods were tested to concentrate the endopolygalacturonaseactivity of culture fluids harvested at approx . pH 7 . 8 : cold acetone precipitation, anAmicon hollow fibre cartridge and rotary evaporation at 40 °C. The first two methodsresulted in approx . 50% loss of activity, but rotary evaporation increased the specificactivity without significantly affecting enzyme yield . This method was thereforeroutinely used .

Ammonium sulphate was added to the concentrated culture filtrate which was thenpassed through a phenyl-sepharose column . The bulk of the protein did not adsorb andinstead passed through the column . The adsorbed endopolygalacturonase activity wasthen eluted in one main peak by decreasing the salt concentration to about 14ammonium sulphate (Fig . 1) . A minor peak of activity eluted at ca . 5 % ammoniumsulfate. A second purification method was also tested in preliminary experiments ;endo-PG precipitated with 66 % acetone was loaded onto a DEAE-Sephacel columnin 25 mm Tris-HCl buffer at pH 8 .0. The column was washed with 25 mm Tris-HCI,pH 5 .0, and PG activity was eluted with 25 mm sodium acetate buffer, pH 4 . 2. Asimilar pattern to that observed in Fig . 1 was obtained with a main active peakappearing first followed with a small peak of activity. However, losses of PG wereexcessive and the method was not routinely used .

Active fractions from the phenyl-sepharose column were pooled and applied to apreparative isoelectric focusing column . This yielded one major peak of poly-galacturonase activity with an isoelectric point of 4 . 95 (Fig. 2) . The yield, degree ofenrichment and specific activity of C. gloeosporioides endopolygalacturonase for a typicalpurification experiment are summarized in Table 1 . Specific activities of the finalpreparation in five different experiments were 1930±350 units mg protein' .

Characterization of the purified polygalacturonaseThe active fractions obtained from the electofocusing column showed two Coomassieblue-staining bands corresponding to apparent molecular masses of 62 and 68 kD

126

0.8

0 .6

aa0 . 2

1 .4

1 .2

00 1 .0NQ

0 8cOnô 0 .6DO

0.4Q

0 . 2

0_ )

I

I

I ii I

I

1

_ 00 10 20 30 60 70 80 90 100 110

Fraction numberFIG. 1 . Phenyl sepharose column chromatography of endopolygalacturonase from C .

gloeosporioides . The enzyme was loaded in 25 mm sodium acetate with 30 % ammonium sulphate andeluted with a gradient of 30-0 °ô ammonium sulphate ( ). Absorbance of fractions at 280 nmis denoted by (•--•) and endo-PG activity by (Q--Q) . Fractions 89-97 between the arrowswere pooled and saved . Note the occurrence of minor active peaks in fractions 87-89 and fractions102-108 .

00 4 8 12 16 20 24 28 32 36

Fraction number

D . Prusky et al.

TjtOo)NOO~- N

O GornTOovcW

FIG . 2. Column electrofocusing of C. gloeosporioides endopolygalacturonase from the phenylsepharose column . The enzyme preparation was run in 1 °,;, ampholytes, pH 3-10, for 36 h to afinal 2000 V . Fraction pH is indicated by (Q - [1), absorbance at 280 nm by (O O) and endo-PG activity by (0e) .

when subjected to SDS-polyacrylamide gel electrophoresis (Fig . 3) . When the purifiedsample was run on a native polyacrylamide gel, only one active band was detected withthe polygalacturonate overlay technique (data not shown) .Samples of the purified polygalacturanase decreased the viscosity of sodium

polypectate (data not shown) . In comparison with a pectate lyase from Erwiniachrysanthemi (PLe), equivalent activities of the C. gloeosporioides PG (as based on thereducing sugar assay) produced more rapid viscosity loss . Since the E. chrysanthemienzyme cleaves randomly [16, 29], the results suggest that the C. gloeosporioides enzyme

16

14T

12UO

10 10 N,O -.

8 8 7 Nûo

6

-

o.

6 irn

O4 - 4 ô

vc2 2 w

0 0


TABLE 1Purification of endopolygalacturonase from C . gloeosporioidess

"Five litres of culture fluids utilized .

97 kO

66 kD

45kD

31 kD

FIG. 3 . SDS-polyacrylamide gel electrophoresis of C . gleosporioides polygalacturonase recoveredfrom an electrofocusing column . Lanes A and B are from two different purified preparations . LaneC shows molecular weight standards : phosphorylase B, 97 kD ; bovine serum albumin, 66 kD ;ovalbumin, 45 kD and carbonic anhydrase, 31 kD . Arrows denote bands used for determinationof amino acid composition and N-terminal amino acid sequencing .

is an endo-PG. No pectate lyase activity was detected in samples of the purified C .gloeosporioides PG preparation and 1 mm EDTA did not affect the velocity of the endo-PG on polypectate .

The apparent pH optimum for the endopolygalacturonase was approximately 5 . 1 .The velocity of the reaction did not decrease as the temperature was increased up to49 °C. The enzyme was stable when incubated at 42 °C for 10 h in 25 mm sodiumacetate buffer, pH 6-0, without substrate. The isoelectric point of the enzyme asdetermined by column electrofocusing was 4-95 (Fig . 2) . This was confirmed by gelelectrofocusing, but substrate overlays disclosed two bands of polygalacturonaseactivity at pI 4-95 and 5-00 (Fig . 4) . Similar results were obtained when the gels wererun using pH 3-10 or 4-6 ampholytes, but the 4-6 ampholytes resulted in broaderactive bands. SDS-polyacrylamide gel electrophoresis of the purified enzyme treatedwith endoglycosidase F showed one single band at Mr of 62 kD (Fig . 6) .

The purified enzyme preparation exhibited polygalacturonase activity with a K . onpolygalacturonate of 0-095 mg ml - ' and a Vmax of 0-75 units min' . The N-terminal

1 27

Specific SpecificTotal Total activity Recovery of activity

activity protein (units mg activity enrichment(units) (mg) protein') "o) (fold)

Culture filtrate 4500 303 . 7 14.8 100Phenyl sepharose 2720 25 .0 108 .0 60 75IEF column 425 0 . 22 1931 .0 9 . 5 130

1 28

D . Prusky et al.

.• 4 .6 5- 5.10

-• 6.00

-0- 6 .50

40 . 7 .00

E- 8 .05

FIG. 4 . Gel electrofocusing ofpurifed endo-PG detected by enzymatic activity . The electrofocusedgel was incubated for 10 min at 31 ° C with an overlay of 0 . 1 % polygalacturonate in 25 mm sodiumacetate, pH 5 . 0 . Two active bands are denoted by the left arrows and the position of standardsand their pl values are shown on the right .

amino acid sequence of the two protein bands blotted from the SDS gel in Fig . 3 wereidentical . Ala-Pro-Pro-Asp-Ile-Lys-Ala-Ala-Pro-Phe- . The amino acid compositions ofthe same bands were also indistinguishable (Table 2 .)

Maceration capability of the C . gloeosporioides endo-PG .

The C. gloeosporioides endopolygalacturonase was a better macerator than a pectatelyase from E. carotovora EC153 but was less efficient than the endopolygalacturonasefrom G. candidum (Table 3) . A pectate lyase (PLe) from E. chrysanthemi, however, wasthe most efficient macerating enzyme, based on equivalent units of PG or PL activityassayed under optimal conditions .

Inhibitors of the polygalacturonaseThe addition of 80, 60 and 20 .tg ml -1 of epicatechin to endo-PG reaction mixturesinhibited enzyme activity by 43, 23 and 6.5 %, respectively . Double reciprocal plotspermitted determination of the epicatechin K i as 0.29 mm. The addition of propylgallate or butylated hydroxy anisole to the reaction mixture at 5 x 10_ 4

M did notsignificantly affect enzyme activity .

Changes in epicatechin concentration in the peel and flesh of ripening avocado fruitsThe firmness of cv . Fuerte fruits decreased from 10 .7 to 0 . 2 Kg at 6 days after harvest(Table 4) . During the same period the concentration of epicatechin decreased in thepeel from 433 to 6 gg g - ' fresh weight and disease symptoms were recorded whenepicatechin reached the latter level . In cv . Hass, firmness was still 8 . 5 kg at 7 days afterharvest and decreased to 0 . 5 Kg only 6 days later . The epicatechin concentration inHass fruit was still 519 tg g - ' fresh weight at 10 days after harvest and decreased to15 jig after 13 days at which time disease symptoms appeared . The epicatechin


TABLE 2Amino acid composition and physical properties of C . gloeosporioides endopolygalacturonase

s Serine and threonine values were corrected for losses during hydrolysis .'To the nearest integer . Three different samples (enzyme from electrofocusing or bands 1

or 2 from SDS gel-electrophoresis, [Fig. 3]) did not show more than 5% differences in theresidue values .

For these estimations, one half of the aspartate and glutamate residues were assumed to beamidated ; data for tryptophan were not available .

TABLE 3Maceration of avocado fruit cv . Hass wedges at 37 °C by various pectic enzymes

'E. chrysanthemi EC16 [16] PLe and E. carotovora PL153 [29] were assayed in 10 mm Tris-HCIbuffer, pH 7.5 ; G . candidum and C. gloeosporioides endo-PGs were assayed in sodium acetate,25 mM, pH 6.0.

° A series of five two fold dilutions were prepared for each enzyme preparation of approx .concentrations that completely macerated the tissue during the observation period . Thedegree of maceration was determined once every 2 h and the relative activities of the variousenzymes required for complete maceration of the avocado wedges is reported . Thus, the EC 16enzyme was approximately 40 times more active than that from EC153 .

5

MPP 35

129

Amino acids Mol . wt. % (mm 100 g - ')Integer'

(residues mole - t)

ASX 92 . 35 60GLX 139 . 75 91SER 241 . 53 157GLY 222 . 38 145HIS 20 . 70 13ARG 13 . 88 9THR 51 . 75 34ALA 93 .03 60PRO 32 . 80 21TYR 13 . 88 9VAL 35 . 54 23MET 2 . 42 2CYS 4. 44 3ILE 23 . 40 15LEU 30 . 14 20PHE 8 . 75 6LYS 28 . 60 19Total 687Molecular weight From amino acid composition` PC 65000Molecular weight Sephacryl S-200 66000

SDS-Gel electrophoresis 68000-62000Isoelectric point Electofocusing 4 . 95

From amino acid composition 5 . 13

Sources PIMr(kD)

Relativemacerationcapability'

E . chrysanthemi EC16 (PLe) [16] 9. 8 38 40G . candidum [3] 7 . 8 38 13C. gloeosporioides 4. 95 68-62 4E. carotovora EC153 (PL153) [29] 8. 7 61 1

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D. Prusky et al.

TABLE 4Effect offruit ripening on epicatechin concentration, fruit firmness and the appearance of symptoms induced

by Colletotrichum gleosporioides infection in two avocado cultivars

cv . Fuerte

cv. Hass

'Fruits were incubated at 20 'C .° Epicatechin determinations represent the means of three extractions . The standard

deviations of mean vaues presented was not larger than 10% .

concentration in the flesh of both cultivars was significantly lower than in the peel andaveraged 21 Itg g- ' fresh weight in cv. Hass fruits and 7 .8 µg g -t fresh weight in cv .Fuerte fruits .

DISCUSSION

The major polygalacturonase of C . gloeosporioides was purified and appears to be anendoenzyme because it exhibited marked activity in both the viscosimetric andreducing sugar assays. Specific activities of the purified preparations were similar tothose reported for other fungal polygalacturonases [10] . Since the purified endo-PG ranas a single peak on a gel filtration column at approx . 66 kD (Fig. 5), it appears thata single polypeptide chain is sufficient for activity . This value is similar to thatcalculated from amino acid composition data (Table 2) . Two molecular species wereconsistently observed in the purified endo-PG preparations by SDS gel electrophoresis(Fig. 3) and IEF gels (Fig . 4) . However, these are believed to represent different degreesof glycosylation of a single protein species because : (i) the amino acid composition andN-terminal amino acid sequences of the two bands were the same ; and (ii) endo-Ftreatment caused both species to run as a single band of 62 kD on SDS-Polyacrylamidegels . (Fig . 6) .The C. gloeosporioides endo-PG is larger than most reported polygalacturonases, but

it should be noted that C . lindemuthianum has previously been reported to produce anendo-PG with a molecular weight of 70 kD [10] and Botrytis cinerea an endo-PG of70 kD [30] . We also detected one or more minor polygalacturonases in the culturefluids of C. gloeosporioides (see Fig . 1). A protein of approximately 40 kD, from culturefluids, was recognized by antibodies prepared to an endo-PG from Geotrichum candidum,

Days afterharvest'

Epicatechin°(µg g-, fr . wt.) First

Firmness

symptom(kg)

appearance

Epicatechin °(µg g -, fr . wt.) First

Firmness

symptom(kg)

appearancePeel Flesh Peel Flesh

1 433 4 10. 72 456 22 12 . 33 344 9 5 . 3 1970 30 12 . 56 12 6 0 . 2

+7 591 28 8 . 59 517 15 8 . 1

10 519 28 1 .013 15 25 0 . 5

+18 I 9 0 . 5


60

50

40

E 30

20

10

4.2 4 .4 4 .6 4 .8 5 .0 5.2log Mr

FIG . 5 . Molcular weight determination of purified endopolygalacturonase from C. gloeosporioideson a Sephacryl S-200 column . The molecular weight was estimated from a standard plot of elutionvolume vs . log Mr . Standards were : 1, alcohol dehydrogenase ; 2, ovalbumin ; 3, pepsin ; 4, ß-lactoglobulin and x, purified endopolygalacturonase .

A B

FIG . 6 . SDS-polyacrylamide gel electrophoresis of the purified endopolygalacturonase from C .gloeosporioides (A) and the same preparation following treatment with 20 units of endoglycosidaseF iB) . Arrows denote molecular weights of 68 (upper) and 62 kD (lower) .

but it is not known whether this is in fact a polygalacturonase (data not shown) . TheG. candidum antibody showed no reactivity to the major 62 kD C. gloeosporioides endo-PG .

Polygalacturonases produced by fungal plant pathogens vary in their isoelectricpoints [3, 10, 26, 30, 32], but the C . gloeosporioides PG is somewhat unusual in having apI of about 5 . 0 . Despite the low pI value, the enzyme is a relatively efficient maceratorof plant tissue (Table 2) . Although less active than PLe of Erwinia chrysanthemi and theendo-PG of Geotrichum candidum, which have much higher pI values, it was more activethan a PL from Erwinia carotovora which also has a basic pI (Table 2) .

With the exception of certain plant protein inhibitors, specific and effective naturalinhibitors of fungal polygalacturonases are not known . It was therefore somewhatsurprising that epicatechin inhibited the C. gloeosporioides endo-PG . While the inhibitionconstant was relatively high (Kt = 0.29 mm), epicatechin nevertheless is a relativelygood inhibitor of endo-PG . Inhibition of endo-PG activity may be of importance in thedisease caused by C. gloeosporioides on avocado fruits because the peel of unripe fruitscontains an epicatechin concentration ranging between 450-2000 p.g g - ' fresh weight

131

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D. Prusky et al.

which decreases to 12-15 gg g- ' fresh weight in ripe, symptom expressing fruits(Table 4) . The onset of decay occurs in ripe fruits only when epicatechin decreases toapprox 25 .tg g- ' fresh weight . Since epicatechin inhibited endo-PG at concentrationshigher than 30 gg ml - ', it may be one factor accounting for fungal quiescence in unripefruits . Epicatechin changes during fruit ripening were determined in several avocadocultivars and it was concluded that cultivar susceptibility was always correlated withdecreases in epicatechin concentration [23] . Epicatechin also appears to be involved inthe regulation of lipoxygenase-mediated oxidation of an antifungal diene [22] .However, the present results suggest a second possible function for epicatechin-inhibition of the major endo-PG produced by the fungus .

Verhoeff [31] proposed that the negation of enzymatic function in pathogens mightbe a possible cause for quiescent infections of unripe fruits . Brown & Adikaram [1, 8]and Labavitch and co-workers [1] have suggested such a role for proteinaceous enzymeinhibitors of fungal decay in tomato fruits and pears, respectively . On the other hand,the significance of phenolic inhibitors has been generally neglected because of theirrelatively low activity. However, it was previously noted that certain phenolics mayinhibit fungal growth [19] and catechin and oxidized phenols were also shown toinhibit pectic enzymes [9, 15] . The inhibition of fungal pectolytic enzymes by phenoliccompounds was also suggested in grapes [12] . In this case, decreased concentrations ofphenolic compounds present in the fruit were correlated with the onset of diseasedevelopment. Since unripe avocado fruit peel contains significant amounts ofepicatechin [22, 23], this phenolic may also function physiologically to inhibit the endo-PGs produced by pathogens .

This work was supported by a BARD grant and USDA grant 86-CRCR-1-2233 as wellas a State of California Biotechnology Training Grant . We acknowledge the help of DrD . Kobiler for his suggestions during the initial stages of enzyme purification . Dr GaryHathaway of the UCR Biotechnology Instrumentation Facility kindly performed theamino acid analyses and N-terminal sequencing .

REFERENCES1 . ABU-GOUKH, A . A ., GREVE, L. C . & LABAVITCH, J. M . (1981) . Purification and partial characterization

of" Bartlett" pear fruit polygalacturonase inhibitors . Physiological Plant Pathology 23, 111-122 .2 . BARASH, I. & KHAZZAM, S. (1970) . The relationship and properties of pectic glycosidases produced by

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purification and characterization of an endopolygalacturonase produced by colletotrichum...

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