Pectinases from boll weevil larvae, Anthonomus grandis

J. Insect Physiol., 1972,Vol. 18, pp. 1295to 1301.Pergamon Press. Printed in Great Britain

PECTINASES FROM BOLL WEEVIL ANTHONOMIJS GRANDIS

LARVAE,

E. E. KING Plant Science Research Division, Agricultural Research Service, United States Department of Agriculture, State College, Mississippi 39762 (Received 11 January

1972)

Abstract-Assays were conducted throughout a wide pH range to measure the ability of extracts from third instar boll weevil larvae to degrade pectic substances. Pectin was deesterified and was depolymerized by both hydrolytic and trans-eliminative mechanisms. Sodium polypectate (deesterified pectin, or pectic acid) was depolymerized solely by trans-elimination. Addition of Ca2+ failed to stimulate the trans-eliminative cleavage of either pectin or polypectate. It is possible that one or more of the pectinases from boll weevil larvae contribute to the abscission of infested cotton flower buds.

INTRODUCTION OCCURRENCEof

pectin-degrading enzymes has been widely reported among microorganisms and plants where they have been implicated in plant disease and decay, but there are few references to their elaboration by insects or other invertebrates (BATEMAN and MILL-, 1966). The boll weevil has not been cited in this respect. This paper reports some of the characteristics of the enzymatic degradation of pectic substances by boll weevil larvae, Anthonomus grandis Boh. In addition to pectin methylesterase (pectin pectylhydrolase, E.C. 3.1.1.11) which removes the methoxyl groups of pectin or pectinic acid, there are two other enzymes or groups of enzymes which degrade pectic substances. They are distinguished on the basis of the mechanism by which the ol-1,4-glycosidic bonds of the polymers are split. Those acting in a hydrolytic manner are designated poly-cJ,4-galacturonide glycan hydrolase, E.C. 3.2.1.15, whereas those accomplishing a trans-eliminative cleavage are designated poly-cu-1,4-D-galacturonide lyase, E.C. 4.2.99.3. The two groups may be further subdivided (BATEMANand MILLAR, 1966) on the bases of substrate preference (pectin or pectic acid) and random vs. terminal degradation. Enzymes acting preferentially on pectin are termed polymethylgalacturonases and pectin methyl lyases; those acting preferentially on deesterified pectin (pectic acid) are termed polygalacturonases and polygalacturonate lyases. The prefixes endo- and exo- designate random and terminal attack respectively. 1295

1296

E. E. KING MATERIALS

AND

METHODS *

Boll weevil larvae

Third instar larvae of A. grandis were obtained from the mass rearing facility of the U.S. Department of Agriculture Boll Weevil Research Laboratory. Larvae were reared under antiseptic conditions on Gast’s larval medium (GAST and DAVICH, 1966). Preparation of extracts

Larvae were rinsed free of medium, homogenized for 1 min at 5°C in a Servall Omni-Mixer, and lyophilized. The lyophilized material was then rinsed exhaustively with cold ( -25°C) acetone on a Buchner funnel, air-dried, and stored in a vacuum desiccator at -25°C. Two g of the acetone powder were extracted for 1 hr in 55 ml water and centrifuged for 15 min at 27,OOOg, both at 5°C. The extract was then passed through a 5 x 50 cm column of Bio-Gel P-10 eluted with water at 5°C. Recovered protein was lyophilized and stored in a vacuum desiccator at -25°C until needed, when portions were dissolved in water for use in enzyme assays. Enzyme assays

Pectin methylesterase activity was assayed at 25°C by a modification of the continuous titration method of KERTESZ(1951). Reaction mixtures contained 5 ml 1% citrus pectin in O-1 M NaCl, 3 ml water, and 1 ml protein solution (1 mg/ml). Activity was determined at 1.0 pH intervals from pH 4-O to 9.0 ; titrations with 0.01 N NaOH were continued for 10 min following addition of enzyme. To compensate for spontaneous deesterification, controls including water instead of protein solution were titrated at each pH value, and blank values subtracted from experimental results. Data are expressed as m-moles of NaOH consumed per min per mg protein. Viscosity reduction was measured at 30°C in size 300 Canon-Fenske viscometers. Activity against pectin and sodium polypectate (NaPP) was measured with and without added Ca2+ throughout the pH range 3.5 to 9.0. Reaction mixtures contained 3 ml 2.4% substrate, 2.5 ml 0.01 M citrate, or Tris buffer, and 0.5 ml protein solution (2 mg/ml). When Ca2+ was added, it was included in the buffer as 6 mM CaCl,. Enzyme activities are expressed as the reciprocal of the time in minutes to achieve 50 per cent viscosity loss x 1000 (BATEMAN,1966). Hydrolysis of pectic substances was measured at 30°C using the same reaction mixtures, less Cat+, as employed in the measurement of viscosity reduction. Samples were taken at the start of the reaction and at 10 min, and mixed with dinitrosalicylic acid reagent for reducing sugar (MILLER, 1959). n-Galacturonic acid standards were used; data are expressed as pg galacturonic acid equivalents liberated per min per mg protein. * Mention of specific trade names is made for identification only, and does not imply endorsement by the United States Department of Agriculture.

PECTINASES FROMBOLLWEEVIL LARVAE

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Changes in absorbance at 230 nm were monitored to measure release of unsaturated end products from pectin and NaPP (BATEMAN,1966). Reaction mixtures at 25°C contained 1 ml 0.3% substrate, 1.5 ml 0.01 M citrate, or Tris buffer, pH 3.5 to 9.0, with and without 6 mM CaCI,, and O-5 ml protein solution (2 mg/ml). Reactions were monitored for 10 min; data are expressed as AA,,, per min per mg protein. RESULTS Pectin methylesterase activity was present at a high level throughout the pH range 4 to 9, with a peak at pH 8 (Fig. 1). Although the spontaneous deesterification of pectin proceeds rapidly in alkaline solution (KERTESZ, 1951), the blank value correction employed in the assay procedure and the decrease in activity at pH 9 support the validity of the pH 8 peak.

FIG. 1. Effect of pH on the activity of pectin methylesterase. Conditions of assay as described in Materials and Methods.

Results of the viscosity reduction assays are presented in Fig. 2. Activity was generally greater against NaPP than against pectin except at pH 7.5, where there was a moderate peak of activity against pectin. Addition of CaCI, produced essentially no effect on the reduction of pectin viscosity. Addition of CaCI, to NaPP solutions was precluded because of increased viscosity, gel formation, and consequent plugging of viscometers. Analysis of reaction mixtures containing NaPP by the dinitrosalicylic acid technique indicated that the cleavage of this polymer was not hydrolytic, since no reaction product was formed at any pH. Conversely, measurable hydrolysis of pectin occurred at every pH tested with the exception of pH 7.5 (Fig. 3). Pectin methyl lyase activity, as measured by increase in absorption at 230 m-n, was present throughout the examined pH range. Definite peaks of activity occurred at pH 4.0 and 6.5, with a lesser peak at pH 7.5 (Fig. 4). Truns-eliminative cleavage of NaPP proceeded most rapidly in the pH range 4-O to 5.0, with a lesser peak of activity at pH 6.5 (Fig. 5).

E. E. KING

1298

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3

4

5

6

7

8

9

PH

FIG. 2. Effect of pH on viscosity loss of pectin and NaPP solutions. Conditions of assay and units of measure as described in Materials and Methods. A-A, NaPP; a-0, pectin; O------O, pectin + 2.5 mM CaCl,.

0 3

4

5

6

7

6

9

*H

FIG. 3. Effect of pH on the hydrolysis of pectin. Conditions of assay as described in Materials and Methods.

1299

PECTINASBS FROM BOLL WEEVIL LARVAE

040 -

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FIG. 4. Effect of pH on the @ans-eliminative cleavage of pectin. Conditions of assay as described in Materials and Methods. a-0, pectin; O--------O, pectin+ 3 mM CaCl,.

3

4

5

6 i)H

7

8

9

FIG. 5. Effect of pH on the truns-eliminative cleavage of NaPP. Conditions of assay as described in Materials and Methods. A- A, NaPP; A-------- A, NaPP + 3 mM CaCl,.

1300

E. E. KING

In numerous instances, Ca2+ has been shown to be either essential or stimulatory to the action of pectin and polypectate lyases (BATEMANand MILLAR, 1966). The lyases studied here, however, respond quite differently to Ca2+. In addition to the insignificant effect of the ion on viscosity reduction, Ca2+ severely inhibited the production of unsaturated end products from both pectin and NaPP (Figs, 4, 5). DISCUSSION The fact that pectin methyl esterase is present in boll weevil larvae and is active throughout a wide pH range (Fig. 1) is of greater significance than the pH 8 optimum exhibited in assay. Such an optimum is not rational in the sense that it is advantageous to the insect. Boll weevil larvae feed exclusively on anthers while in cotton flower buds, and the pH of an homogenate of these floral organs is in the vicinity of 5.8 to 6.0. Indeed, a pH of 8 is not to be found in any part of the flower bud. Also, analyses of the gut in both starved and feeding larvae indicate a pH range of approximately 5.8 to 7.3 from crop through midgut (M. R. Bell, personal communication). The pH range throughout which viscosity reduction of pectin and NaPP solutions is catalysed (Fig. 2) is more compatible with the range of pH encountered in the flower bud and in the larval gut. Comparison of Figs. 2 to 5 permits some identification of the enzymes involved in the cleavage of the pectic substances. In the pH range 3.5 to 5.5, data from Figs. 2 to 5 indicate the viscosity loss of NaPP solutions to be the result of polygalacturonate lyases. There was, in fact, no measurable hydrolysis of NaPP throughout the pH range investigated. The high rate of viscosity reduction, together with the high rate of unsaturated reaction product liberation at acid pH, implicate both endo- and exo-polygalacturonate lyases. Had there been only random degradation of the substrate, the ratio of viscosity reduction to end product release would have been high as in the case of NaPP at pH 5.5 to 7.5 ; the reverse would be true if only terminal degradation had taken place. Below pH 6.5, pectin was evidently degraded by a combination of pectin methyl At pH 6.5, since the ratio of viscosity lyases and polymethylgalacturonases. reduction to end product release is low (Figs. 2, 4), an exo-pectin methyl lyase is implicated. Comparison of Figs. 2 to 4 suggests that at pH 7-O and above pectin was degraded primarily by hydrolysis. The apparent failure of Ca 2+ to stimulate the lyases is highly unusual, if not unique (BATEMANand MILLAR, 1966 ; MULLEN and BATEMAN,1971; WANG and PINCKARD,1971). A possibility, of course, is that the concentrations employed (2.5-3.0 mM) were supraoptimal and thus inhibitory. This phenomenon has been described (WANG and PINCKARD,1971), but in another instance (STARRand MORAN,1962) inhibition of lyase activity at Ca2+ concentrations greater than 1 mM Precipitation did not occur in reaction was ascribed to substrate precipitation. mixtures assayed in this study. The results of this study may have significance beyond the demonstration that as phytophagous insects, boll weevil larvae are well suited, at least with respect to

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pectic substances, to their dietary rCgime. Pectinases of various types have been shown in a number of cases to contribute to the tissue degeneration symptomatic of certain plant diseases caused by bacteria and fungi (BATEMANand MILLAR, 1966). Host plant injury caused by Lygus hespems Knight, including abscission of reproductive organs, is due principally to a polygalacturonase secreted from the insect during feeding (STRONG, 1970). Bud abscission is the ultimate consequence of cotton flower bud infestation by boll weevil larvae (COAKLEYet al., 1969); protein(s) extracted from larvae can cause this abscission (KING and LANE, 1969). It is reasonable then, to consider the possibility that one or more of the pectinases of boll weevil larvae may contribute to cotton flower bud abscission. Acknowledgements-1 am grateful to Miss PATRICIAMCCLENDONfor her skilful technical assistance, to Mr. NORMANMITLIN for valuable discussion and aid with manuscript preparation, and to Mr. OLIVERLINDIG for supplying boll weevil larvae.

REFERENCES BATEMAND. F. (1966) Hydrolytic and trans-eliminative degradation of pectic substances by extracellular enzymes of Fusarium solani f. phaseoli. Phytopathol. 56, 238-244. BATEMAND. F. and MILLAR R. L. (1966) Pectic enzymes in tissue degradation. A. Rev. Phytopathol. 4, 119-146. COAKLEYJ. M., MAXWELL F. G., and JENKINSJ. N. (1969) Influence of feeding, oviposition, and egg and larval development of the boll weevil on abscission of cotton squares. J. econ. Ent. 62, 244-245. GAST R. T. and DAVICHT. B. (1966) Boll weevils. In Insect Colonization and Mass Production (Ed. by SMITH C. N.), pp. 405-418. Academic Press, New York. KRRTRSZZ. I. (1951) The Pectic Substances, pp. 362-363. Interscience, New York. KING E. E. and LANE H. C. (1969) Abscission of cotton flower buds and petioles caused by protein from boll weevil larvae. PI. Physiol. 44, 903-906. MILLER G. L. (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analyt. Chem. 31, 426-428. MULLEN J. M. and BATEMAND. F. (1971) Production of an endopolygalacturonate transeliminase by a potato dry-rot pathogen, Fusarium roseum ‘Avenicum’, in culture and in diseased tissue. Physiol. Pl. Pathol. 1, 363-373. STARRM. P. and MORANF. (1962) Eliminative split of pectic substances by phytopathogenic soft-rot bacteria. Science, Wash. 135, 920-921. STRONGF. E. (1970) Physiology of injury caused by Lygus hesperus. J. econ. Ent. 63, 808814. WANG S. C. and PINCKARDJ. A. (1971) Pectic enzymes produced by Diplodia gossypina in vitro and in infected cotton bolls. Phytopathol. 61, 1118-1124.