Pectin-Methylesterases: Isozymes, Properties and Detection in Polyacrylamide1)

Biochem. Physiol. Pflanzen 168, S. 607 -615 (1975)

Pectin-Methylesterases: Isozymes, Properties and Detection in Polyacrylamide Gels!) L. ROEB and H. STEGEMANN2) Institut fur Biochemie, Biologische Bundesanstalt, Braunschweig Key Ter mIn d e x: electrophoresis

pectin-methylesterase, isoenzymes, enzyme purification, polyacrylamide

Summary A new, sensitive and specific method for the detection of pectin-methyl esterases (PME) in polyacrylamide gel has been described. The method is based on the observation that while the substrate pectin included in the polyacrylamide is barely stained by methylene blue, pectic acid which is enzymatically produced strongly adsorbs the dye. The advantage of the method is that PME activity can be detected in crude extracts, without prior purification. The isoenzyme patterns obtained from different fruits, vegetables, and parasitic fungi and also the influence of certain cations have been studied.

Introduction

Pectin methyl esterase (PME; EC 3.1.1.11) is widely distributed in plants and microorganisms. Though this enzyme has often been investigated in connection with its role in the fungal and bacterial pathogenesis of plants its precise role is not yet completely understood. According to SMITH (1958) PME activity substantially aggravates plant diseases, while BATEMAN und MILLAR (1966) are of the opinion that it prevents the tissue disintegration. The activity of the PME is determined by measuring the liberated methanol or more often by titration of the newly formed carboxyl groups in the pH-Stat. However, both methods are not very sensitive and often give no reproducible results, since ions influence the enzyme activity. The present work describes a highly sensitive method for the detection of PME in plants and microorganisms. The detection is based on the observati3n that while the pectin included in polyacrylamide (PAA) is barely stained by methylene blue, the pectic acid, produced by the PME, adsorbs the dye strongly resulting in a deep blue color where the enzyme is active (STEGEMANN 1967,1968). After the electrophoresis isozymes can be detected even in nanogramm quantities in crude extracts. This enabled us to study some properties of the enzyme from different fruits, vegetables and parasitic fungi which so far has not been reported. 1) This publication is part of the doctoral thesis. 2) Dedicated to Prof. Dr. K. MOTHES on his 75th birthday.

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Material and Methods Materials. - Pectin from citrus (NBCo now ICN, control-No. 2237, Cleveland, Ohio, (USA); Pectic acid (Fluka, control-No. 399.267, Buchs, Switzerland) were used in the experiments. Enzymes were purchased: PME from tomato (NBCo, control-No. 5025, activity 24.3 u/mg) was mostly used because of the good isozymespectrum. PME from tomato (Worthington, control-No. PE 33 M 920, activity 34.0 u/mg, Freehold, New Jersey, (USA). PME from potatoes. - Four different varieties of potatoes, viz. HANS A, MENSA, SIEGLINDE and VORAN, previously stored for 9 months at 10°C, were thoroughly washed, peeled and cut into pieces. The slices were then divided into two aliquots; one of them was homogenized in a blendor for one min at room temperature in distilled water (GROSSMANN 1968) and the other in 0.1 M NaC!. Browning was avoided by adding 0.02 ml of a sulfite solution with 3.75% Na 2 S2 0 5 and 5% Na 2 S0 3 per ml of the homogenate (STEGEMANN et a!. 1973). The homogenate was centrifuged for 15 minutes at 22,000 g at 4°C. The supernatants of all the samples were dialyzed against TRIS/glycine 0.005 M, pH 8.3 (MAURER 1971) and the dialysed solution was directly used for the investigations. PME from unripe and ripe fruits. - Plums, varieties: Hauszwetsche, Graf Althanns; Apples, varieties: Cox orange, Golden Delicious, Boskoop. Pears, variety: Williams christ. Cucumber, variety: Delikatess. The treatment was the same as described above substituting, however, the water by 0.5 M acetate buffer, pH 8.0. PME from Fusarium sulphureum. - (Schleckt, isolate-No. 704) from a liquid medium with 2 % citrus pectin and the following salts (p. a. Merck, Darmstadt): 0.2 % NaN 0 3 ; 0.05 % KCI; 0.05 % MgS0 4 • 7 H 2 0; 0.1 % KH 2 P04 ; 0.001 % FeS0 4 • 7 H 2 0. The medium was adjusted to pH 5.2 with Na'oH. Unless otherwise stated the fungus was grown in a shake culture at 40 rpm in the dark at 22°C for 6 weeks. After removing the mycelium the crude filtrate was used in all experiments and stored frozen. Multiple freezing and thawing did not affect the results. If the samples are stored at 4°C for more than 14 days, the electrophoretic zones tend to diffuse. PME and Polygalacturonase (PG) were taken from Rhizoclonia solani (Kuehn) (isolate-No. C 7); grown on agar plates according to ROEB (1974). Age-dependent enzyme synthesis. - For the above experiments Rhizoctonia solani grown at 28°C for four days was used. The electrophoretic separation was done as described earlier and stained for PG (STEGEMANN 1967, 1968) and PME. Electrophoresis and staining of the gels. - The electrophoretic separations were done in vertical slab electrophoresis according to STEGEMANN et a!. 1973). Unless otherwise stated 0.025 % citruspectin was included in the gel (5 % Cyanogum 41, Serva, Heidelberg), as done in substrate inclusion techniques (STEGEMANN 1967, 1968). The following buffers with and without 0.002 M EDTA were employed for the preparation of the gels, electrophoresis and incubation: a) TRIS/boric acid b) TRIS/glycine

0.04 M/O.l M 0.009 M/0.04 M

pH 8.2 or pH 8.3

Unless otherwise stated TRIS/glycine in presence of EDTA was used and was circulated by a pump. After electrophoresis for 4.5 h at 500 volts and 80 rnA at 0 °C, the PME-activity was detected by incubating the gels in the electrophoretic buffer, containing 0.003 % methylene blue. Proteins were stained with Supranolcyanin (STEGEMANN et a!. 1973). When the substrate was not included in the gel, the gels were incubated for 12 hours at 22°C in a solution of 0.1 % citruspectin in the electrophoresis buffer adjusted to pH 8.3 with TRIS prior to methylene blue staining. The method is less' sensitive and the bands tend to blurr when the substrate is added to the incubation medium after the electrophoresis. The PME in the crude extracts was determined titrimetricly according to HILLS and MOTTERN (1947).

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Peetin-:\lethylesterases: Isozymes, Properties and Deteetion

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The preparation of the molecular sieve gradient. - 6-23% polyacrylamide was prepared in the same buffer as described in the foregoing section, generally following the procedure of ",fARGOLIS and KENRICK (1968) The concentration of citrnspectin was 0.0125 %. The electrophoretical run was 88.5 h.

Results

1. Proof of the enzymatic nature of the reaction In order to differentiate the enzymatic reaction from nonspecific staining with methylene blue the following experiments were done: (1) Electrophoresis of tomato and potato PME were carried out without the inclusion of the substrate in the gel. After electrophoretic separation and incubation no staining could be detected. Likewise no stained zones could be observed, if boiled enzyme solutions were separated in a gel containing the substrate. By incubation of gels loaded with the enzyme at 23 DC, 45 DC, 50 DC, 55 DC, 60 DC, 70 DC, 80 DC and 100 cC for 1 h, the inactivating temperature was found to be 60 DC which is in agreement with the irreversible denaturation of most of the proteins. Although all the isozymes from tomatoes (Fig. 1) were very active till 50 DC, they lost half of their activity at 55 cC. The results of the PME from Fusarium sulphureum are similar, but only till 50 DC all the 4 isoenzymes could be detected. (2) Synthesis of PME by Fusarium sulphureum was checked in a similar manner. An autoclaved (at 120 DO) medium, not inoculated by the fungus was electrophoretically separated as a blank. Due to boiling a part of the pectin was cleaved to pectic acid, and appears as homogeneous light blue background in the whole electropherogram. The addition of EDTA to the incubation medium minimized the background staining. Electrophoresis of culture filtrates of the fungus yielded sharp bands of enzymatic activity. 2. Sensitivity of the method 10- 7 g of a crude preparation of PME (NBCo No 5025) with an activity of 24.3 units!mg according to HILLS and MOTTERN (1947) could be detected. As the protein content of the sample was found to be 17 % according to Biuret (GORNALL et al. 1949) and Folin (STEGRMANN 1960) determination and as the enzyme protein is less than 10 % of the total protein as determined by Supranolcyanin staining in the PAA gel, the actual sensitivity of our method is 2 X 10- 9 g. After electrophoresis and incubation for 12 h in TRISjEDTA-glycine-buffer pH 8.3 at 45 DC the enzymatic activity was detectable even at lower concentrations. Similar results were obtained ming TRISjEDTAj boric acid pH 8.2 and even 0.2,ug of the crude preparation could be detected. TRISj citric acid (0.05 MjO.05 M; pH 5-10), glycine/boric acid (0.2 M/0.2 M; pH 5.3), TRISj boric acid (0.03 M/0.074 M; pH 7.9), TRISjboric acid (0.125 MjO.019 M; pH 8.9), TRIS HCI(0.3 MjO.15 M; pH 8.9), NaOHjboric acid (0.02 MjO.02 M;pH 11) buffers were found to be unsatisfactor~T. 39

Biochem. Physiol. Pflanzen, Bd. 168

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STEGE~[AN)!

Fig. 1. Thermostability of 0.5 fl9 crude extract from tomato tL'itk pecti n-metkyl-esterase activity (N BOo control-No. 5025). Separated by polyacrylamide electrophoresis for 4.5 hat 500 volts and 70 mA.; buffer: 0.009 M TRISj glycine with 0.002 l\I EDT A pH 8.3. Citruspectin (0.025 %) was included in the gel as substrate. Cathode on top. - From left to right: The samples were incubated at 23 DC, 45 DC, 50 DC, 55 DC, 60 DC and 70 DC in 0.005 M TRISjglycine pH 8.3 for 1 h, prior to electrophoresis. Incubation after separation for 12 h in 200 ml electrophoretic buffer with 0.003 % methylene blue at room temperature.

3. Factors influencing the enzymatic activity Incubation of tomato PME in media containing 10~4 M NaCI, KCI, CaCI 2 or MgCI2 and without EDTA activates PME. Higher salt concentrations inhibit the enzyme. The inhibition caused by [) X 10~2 M NaCI is so strong, t)1at the gel-electrophoretic detection is impossible even at a concentration as high as 100 ftg. Similar results were obtained with 1O~2 M CaCI 2 and MgCI 2. With KCI a concentration of 1O~1 M Was required to cause a total inhibition of enzymatic activity. Studies with the Fusarium PME showed that the enzyme is remarkably more sensitive to MgCI 2. A solution of 1O~3 M MgCI2 inhibits the enzyme completely. 0.002 M EDT A has no inhibitory effect on PME, but on the contrary it sharpens the bands. Since EDTA at the same concentration inhibits the PG from Fusarium sulphureum only the PME enzymes can be detected. If the EDTA concentration is increased to 0.008 M both the fungal enzymes are inhibited. 4. Enzyme-su bstra te interaction

PME from tomato interacts with citrus pectin. This interaction becomes more pronounced at higher substrate concentrations. If the substrate concentration is raised to

Pectin-:\Iethylesterases: Isozymes, Properties amI Deteetion

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3 Fig. 2. Pectin-methyl-esterase from fruas. Separation in a gradient gel (6- 23 % PAA) for 88.5 h at 500 volts and 65 rnA; buffer: 0.04 M TRISI boric-acid with 0.002 ~i EDTA pH 8.2. The concentration of the included substrate was 0.0125 % citruspectin. Cathode on top. Incubation as described in Fig. 1 using, however, 0.04 M TRIS/boricacid pH 8.2 with 0.002 :\i EDTA. - From left to right: Unripe and ripe Hauszwetsche, unripe Golden Delicious, unripe Cox orange. Fig. 3. Exudation of polygalacturonase and pectin methyl-esterase tcith respect to the age of the mycelium from Rhizoctonia solani according to ROEB (1974). a) PG: Synthesis in accordance with our demands only from the young mycelium; only cathodic migration. Electrophoresis, incubation and staining according to STEGEMANX (1967). b) P)IE: On the contrary to PG the main components of PME are synthesized by the old mycelium, where we can detect 3 isoenzymes; only anodic migration. Electrophoresis and incubation as described in Fig. 1.

0.05 % the isozymes are retarded, and at a substrate concentration of 0.2 % they cannot be differentiated any more. An even stronger interaction takes place in case of PME from Fusarium sulphureum. The pronounced retardation is obvious even at a pectin concentration as low as 0.025 %. The interaction could not be prevented even if the enzyme was electrodialyzed or purified by ammoniumsulfate fractionation prior to electrophoresis.

5. PME from fruits Electrophoresis of fruit PME was done in the gradient gel (6 -23 % P AA) by including 0.0125 % pectin. Differences in the isozyme spectrum could only be detected be-

612

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ROEB

and H.

STEGEMANN

tween the several fruits but not between the single varieties, if they had the same state of ripening. However, if we compare unripe and ripe fruits of one variety, we could observe one more band in the ripe fruits (Fig. 2). 6. PME from potatoes PME from four potato varieties namely HANS A, MENSA SIEGLINDE and VORAN were extracted in presence of 0.1 M NaCI. The isozyme spectra were the same in all the varieties and in all five isozymes could be detected. There were, however, activity differences, especially in the two faster moving isozymes. 7. Age dependent enzyme synthesis of the mycelium With a two dimensional method (ROEB 1974) it was detected that a number of phytopathogenic fungi synthesize PG only in the young mycelium (Fig. 3a). This is in accordance with our hypothesis that the PG is an important enzyme in the initial steps in pathogenesis to destroy pectic components of the cell wall. The question was, at what state PME was synthesized by the fungus. Fig. 3b shows, that three isozymes of PME are produced by the old mycelium. Discussion

As the role of PME is not yet fully understood contradictory informations are cited in the literature. According to SMITH (1958) and SCHULZ (1972) PME is a prerequisite for the PG action and is therefore an important enzyme in pathogenesis. Though the importance of PME has been attributed to its role in the degradation of the plant tissues, it ha~ also been suggested (BATEMAN and MILLAR 1966) that the enzymatic reaction counteracts the disintegration of the plant tissues. The newly formed carboxyl groups of the polygalacturonic acid chains may interact with Ca++ and other multivalent cations leading to a stronger binding between the chains. An important aspect of this work is the development of a more sensitive method for the detection and studying the properties of PME isozymes. The former methods ( MEYER et al. 1964, GARBER and BEHARA 1966, KNOSEL an~ GARBER 1968) employed indicators for the pH-shift, which were not very sensitive and influenced by the ionic environments. Using starch gel-electrophoresis (MARCOVIC 1974) could detect five enzymes after a tedious operation prior to electrophoresis. However, the isoenzyme pattern of tomato PME is the same. If the enzyme was obtained from a fungus with low PME producing activity detection was not at all possible. Therefore it is not surprising that no PME activity could be found, where it was expected (SCHULZ 1972, KNOSEL and GARBER 1968). The new procedure is based upon the substrate technique (STEGEMAN 1967, 1968) as the citruspectin included in the gel serves as substrate for the PME. In contrast to all the techniques described till now (proteinases, nucleases etc.) the backbone molecule is not split, but only an adjacent group is removed resulting in a strong and a relatively specific adsorption of the dye. This procedure of staining pectic acid within the gel is about thousandfold more sensitive than the conventional determination.

Pectin-J\{ethylesterases: Isozymes, Properties and Detection

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During electrophoresis the activity of the enzyme is low because of the low temperature. At higher temperatures PME transforms pectin into pectic acid (and methanol), which in contrast to pectin is intensively stained with methylene blue, and the enzyme can be located as a dark blue zone against a light blue background. Although the detection is only qualitative it can be made quantitative by comparing known concentrations of the enzyme. The advantage of the method is that qualitative assesments can be made about the isozyme patterns, since the initial separation is an integral part. The results are reproducible and can be controlled. In earlier methods the strict control was difficult due to the presence of ions. Ions, however, have inhibitory and/or activating effect on PME activity depending upon the concentrations. Furthermore, the titration of weakly acidic groups is erratic, especially in a buffered medium. The extraction of PME in saline solution on one hand, the analytical determination in the P AA gel on the other hand, are two separate steps and allow the determination and evaluation of the properties of individual isozymes without any environmental influence. Earlier findings about contradictory influence of ions can be explained if one considers the mixed influence of a better extraction with lower activity in the range of 0.1-0.01 M NaCI solutions. A minor disadvantage of the new method in crude extracts is the possibility that P AA included pectin is first transformed by PME to pectic acid and this product could be further degraded to smaller units by PG, in case these two enzymes are present in the same location after electrophoresis. But (1) this superimposition of PME and PG or transeliminase (PTE) can be avoided by varying the P AA concentration and/or the pH of the electrophoretic buffer. The presence of PG and PTE are indicated by blank spots in the light blue gel. A homogeneously stained gel indicates the absence of all the three enzymes. - (2) the PG activity can be inhibited by 0.002 M EDTA. That the staining with methylene blue is really a specific one for PME and not for PTE we know from the fact, that methylene blue stains only long chain molecules. PTE, however, split the chain into small pieces and the reaction products therefore cannot be stained with this reagent. Frequently reported observations (HANCOCK and MILLAR 1965, WANG et al. 1971, HANCOCK 1968, BATEMAN 1963) that alkaline earth and alkaline ions, especially Na+ in high concentrations activate PME are contradictory to our observations. We have observed highest activity in presence of 10- 4 M of the above mentioned ions. PME from tomatoes is ten times less sensitive to MgCl 2 than the PME from Fusarium sulphureum. This criterium can be employed for the differential diagnosis of plant and fungal enzymes. If the incubation buffer is made 10- 3 M with respect to MgCI 2 , then PME from Fusarium sulphureum will be inhibited while the PME from tomatoes will not be influenced. EDTA in low concentrations has no inhibitory effect on PME. Besides, it has the advantage that it sharpens the bands, probably by preventing the aggregation of the pectin molecules. The observation of KERTESZ (1951) that there is no optimum pH for the PME was confirmed. We found that the activity strongly depends on the salts and buffer components. In suitable incubation buffers, such as glycine/boric acid, TRIS/glycine or TRIS/

614

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STEGE~UXX

boric acid, PME is active in the pH range 5.5 to 10. In unsuitable incubation buffers such as TRIS/citric acid, where citric acid is the unfavourable component the enzyme was not active in the pH range 5.5-7. At higher pH values where TRIS was the main component, activity could be observed. The enzyme substrate interaction of the fungal PME is stronger than that of the tomato PME complex. This points to the fact, that the PME of the fungus acts also upon the pectin of the host. When we compared the fruit PME isozymes migration rate from different varieties, we could not detect any difference neither in the conventional slab-electrophoresis nor in the porosity gradient. Therefore, there does not exist neither charge differences nor differences in molecular weights. PME is not suitable for variety diagnosis. But the fact, that there is one more band in ripe fruits may be an indication to the role of the enzyme during ripening. The age dependent PlVIE synthesis of plant pathogenic fungi has been shown to be especially active with old mycelium possibly indicating that the PG plays the leading role (ALBERSHEIM and ANDERS OX 1973), because this enz~Tme is exudated by the young mycelium. Acknowledgements We thank ;\irs. R. KX.~CKSTEDT for photographs, Dr. E. L.~XGERFELD and Dr. J. M.~RTIX, Biologische Bnndesanstalt, Braunschweig, for supplying fungal cultures. We are also grateful to Dr. R. KOEXIG and Dr. V. SHANKAR for their help in translating the manuscript.

References ALBERSHEDI, P., ANDERSOX, A., Susceptibility of plant cell walls to enzymatie degradation by fungal pathogens. 2nd Internat. Congress of Plant Pathology. Abstr. of Papers. No. 1078, :\Iinneapolis, :\Iinnesota, Sept. 5 -12 (1973). B.~TEMAX, D. F., Pectolytic activities of culture filtrates of Rhizoctonia solam' and extracts of RhizoctOllia-infected tissues on bean. Phytopathology 03,197-204 (1963). - :\hLL.~R, R. L., Pectic enzymes in tissue degradation. Ann. Rev. Phytopath. 4,119-146 (196G). GARBER, E. D., BERAHA, L., Genetics of phytopathogenic fungi. Pectolytie enzymes of virulent and avirulent strains of three phytopathogenic penicillia. Cari. J. Bot. 44, 1645 -1649 (1966). GOR:'HLL, A. G., BARDAWILL, C. S., DAVID, M. M., Determination of serum proteins by means of the biuret reaction. J. Bio!. Chern. 177, 751-766 (1949). GROSS)UXX, F., Zur Bildung pektischer und zellulolytischer Enzyme durch Phytophthorainfestal1s de By. Phytopath. Z. 63, 15-22 (1968). H.\xcoc](, J. G., Degradation of pectic substances during pathogenesis by Fusarium solam' f. sp. cucurbitae. Phytopathology 08, G2 - 69 (1968). :\hLL\R, R. L., Relative importance of polygalacturonate trans-eliminase and other pectolytic enzymes in southern anthracnose, spring black stem, and Stemphylium leaf spot of alfalfa. Phytopathology 00, 346-355 (1965). HILLS, C. H., MOTTERN, H. H., Properties of tomato pectase. J. Bio!. Chern. 168, 651-659 (1947). KERTESZ, Z. I., The Pectic Substances, pp. 628. Interscience Pub!., :'lew York-London (1951). KXiisEL, D., G.\RBER, E. D., Separation of pectolytic and cellulolytic enzymes in culture filtrates of phytopathogenic bacterial species by starch gel zone electrophoresis. Phytopath. Z. 61, 292-298 (1968).

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L,INGE, E., KNOSEL, D., Zur Bedeutung pektolytiseher, cellulolytiseher und proteolytiseher Enzyme fiir die Virulenz phytopathogener Bakterien. Phytopath. Z. 69, 315 -329 (1970). ~L\RGOLIS, J., KENRICK, K. G., Polyacrylamide gel electrophoresis in a eontinuous moleeular sieve gradient. Anal. Biochem. 25, 347 -3G2 (19G8). ~IARKOVIC, 0., Tomato pectin esterase. Characterisation of one of its multiple forms. Collect. Czech. Chern. Commun. 39, 908-919 (1974). ~LWRER, R. R., Disc Electrophoresis, 2nd edn. pp. 222. de Gruyter, Berlin 1971. ~[EYER, J. A., GARBER, E. D., SHAEFFER, S. G., Genetics of phytopathogenic fungi. XII. Detections of esterases and phosphatases in culture filtrates of Fusarium oxysporum and Fusarium xylarioides by starch-gel zone electrophoresis. Botan. Gaz. 125 (4),298-300 (19G4). ROEB, L., Eine neue Technik zur in-situ-Bestimmung von Enzymen in Pilzkulturen, dargestellt an Polygalakturonasen. Phytopath. Z. 79, 359-363 (1974). SCHL:LZ, F. A., Untersuchungen iiber die Bildung pektinolytischer und zellulolytischer Enzyme durch Gloeosporium perennans. Phytopath. Z. 74, 97 -108 (1972). S)[JTH, W. K., A survey of the production of pectic enzymes by plant pathogenic and other bacteria. J. gen. Minobiol. 18, 33-41 (1958). STEGEM,INN, R., Formamidolyse von Proteinen. Die Einwirkung von Formamid auf Gelatine. Z. Physiol. Chemie 319, G4-8G (19GO). Enzym-Elektrophorese in EinschluB-Polymerisaten des Acrylamids. Z. Physiol. Chemie 348, 951-952 (19G7). Die PrimereinschluB-Technik zum Enzymnachweis bis 10-12 g nach Polyacrylamid-Elektrophorese, dargestellt an Phosphorylasen. Z. anal. Chemie 243, 573-578 (1968). Apparatur zur thermokonstanten Eleketrophorese oder Fokussierung und ihre Zusatzteile. Z. anal. Chemie 261, 388-391 (1972). FRA:"!CKSEN, R., MACKO, V., Potato proteins: Genetic and physiological changes evaluated by one- and two-dimensional PAA-Gel-techniques. Z. Naturforsch. 28c, 722-732 (1973). W,I:"!G, SY-YING, C., PrxcKARD, J. A., Pectic enzymes produced by Diplodia gossypina in vitro and in infected cotton bolls. Phytopathology 61, 1118 -1124 (1971). Received }Iarch 24, Un5. Authors' address: Dr. L. ROEB and Prof. Dr. R. Bundesanstalt, 33 Braunschweig.

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Institut fiir Biochemie, Biologische

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