Presence and activity of Trypsin and chymotrypsin isoinhibitors in the annual species of Cicer

BiochemicalSystematicsandEcology, Vol.9, No. 1, pp. 23-25,1981. Printedin GreatBritain.

0305-1978/81/010023-03 $02.00/0 © 1981PerqamonPressLtd.

Presence and Activity of Trypsin and Chymotrypsin Isoinhibitors in the Annual Species of Cicer PATRICIA SMIRNOFF, MIRIAM ROTMAN and GIDEON LADIZINSKY Faculty of Agriculture, The Hebrew University, Rehovot, Israel

Key Word Index-Trypsin-chymotrypsin inhibitor; Cicer; chickpea; chemotaxonomy. Abstract-The trypsin and chymotrypsin isoinhibitors of eight annual species of Cicer, including the cultigen, C. arietinum, had the same migration rate in gel electrophoresis. According to the relative inhibitory activity found in 'the various species, they were grouped in three classes. These were identical to those based on the intercrossing potential of the annual chickpeas. The suggestions made in the literature regarding the value of protease inhibitors in seeds as defence mechanisms against insects were assessed and discussed according to the situation in the annual species of Cicer. m

Introduction Trypsin and chymotrypsin inhibitors are naturally occurring substances in seeds of various plants [1-3] and also occur in other plant organs [4]. The molecular weight, the number of amino acids and the composition of these proteins vary considerably from different sources [5]. Thus, the migration rate from these inhibitors on gel electrophoresis can be expected to be a useful diagnostic characteristic in plant systematics [6]. The genus Cicer L. contains 40 species, of which nine are annual. The genetic affinities among these species were examined recently [7] and the wild progenitor of the cultivated species was identified [8]. Lately trypsin and chymotrypsin isoinhibitors from the cultivated chickpea were studied and characterized [3]. The present paper reports the trypsin and chymotrypsin isoinhibitors in annual species of Cicer and discusses some of the ideas regarding the role of these inhibitors as a defence mechanism against insects.

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FIG. 1. TRYPSIN AND CHYMOTRYPSIN ISOINHIBITORS IN THE SEED PROTEIN PROFILESOF THE ANNUAL SPECIES OF ClCER. A, Pureisoinhibitorextractedfrom the cultivatedchickpeas;B, cultivated species C. ariednum; C, C reticulaturn; D, C. echinospermum; E, C. t#jugum; F, C. judaicum; G, C1 pinnatifidum; H. C. yamashirae; I. C. cuneatum.

indicates that the seed protein fraction with trypsin and chymotrypsin inhibitory activity is remarkably stable in related annual chickpeas. This is in contrast with the morphological and genetical diversity among these species. Thus, for example, by its leaf morphology, growth habit and seed dispersal mechanism, C. cuneatum Rich appears to be closer to the genus Vicia than to the annual chickpeas and is cross-incompatible with the latter [7]. Unfortunately, no information is available regarding trypsin and chymotrypsin inhibitors in Vicia or the tribe Viciae and no further conclusions can be drawn regarding the phylogeny of these protease inhibitors. In the genus Glycine, on the other hand, the situation is different and species belonging to different sections can be

Results and discussion The purified trypsin and chymotrypsin isoinhibitors of C. arietinum, the cultivated chickpea, form two bands on 7.5% acrylamide gel (pH 4.5) at 2.4 and 2.8 cm from the origin [3] (Fig. 1). No inhibitory activity was found in other sections of the gel. The two bands were detected in four lines of the cultigen originating from Ethiopia, India, Israel and Turkey. These two specific bands were also found in seven annual wild species of Cicer that were examined in the present study (Fig. 1). This (Received 26 February 1980) 23

24

PATRICIA SMIRNOFF, MIRIAM ROTMAN AND GIDEON LADIZlNSKY

identified and characterized by the migration rate of their trypsin inhibitor in the gel [6]. The presence of trypsin and chrymotrypsin isoinhibitors was determined not only by the existence of the two specific bands in the gel but also by their inhibitory activity. This activity was examined both in the crude meal obtained by grinding the seeds and in the inhibitor extracted from the gels. As the protein content varied considerably in the various species (Table 1), the TABLE 1. INHIBITORY ACTIVITY AGAINST TRYPSIN AND CHYMOTRYPSIN IN GROUND SEEDS OF ANNUAL SPECIES OF

ClCER

Species

C. arietinum C. ~ticulatum C..echinospermum C bijugum C. judaicum C. pinnatifidum C. yamashitae C. cuneatum

Protein content (%)

Inhibition of trypsin (%)

Inhibition of chymotrypsin (%}

26 26 28

53.7 60.0 50.5

48. I 47.5 37.1

30

22.5

30.0

20 20 20

13.7 16.0 trace

25.2 6.2 13.0

30

11.0

20.0

samples taken for determining the inhibitory activity were calculated to give similar amounts of protein for the various species. The greatest inhibitory activity was found in the cultigen C. reticulatum Ladiz. and C. echinospermum Davis. This activity was much lower in the rest of the species and only traces of activity against trypsin were detected in C. yamashitae Kitam. Except in C. arietinum, no consistency was found between the inhibitory activity against trypsin and chymotrypsin. The activity against trypsin was higher than against chymotrypsin in C. reticu/atum, C. echinospermum and C. pinnatifidum Jaub. et Spach.; the opposite was the case in the rest of the species (Table 1). When the trypsin and chymotrypsin isoinhibitors were extracted from the gels, the inhibitory activity was similar to that observed in the meal of C. aEetinum, C. reticulatum, C. echinospermum and, to a lesser degree, in C. bijugum Rech. In other species a remarkable increase in the inhibitorv activity was noticed (Table 2). The different inhibitory activity found in the meal of the various species can be attributed to several factors such as activity per se, concentration, presence of promotors or reducers in the meal, etc. The inhibitory activity of the trypsin and chymotrypsin extracted from the gels of the various species indicates that the differences observed are due, primarily, to a different inhibitory potential. On the other hand, the role of other

TABLE 2. INHIBITORY ACTIVITY OF CHICKPEA TRYPSIN AND CHYMOTRYPSIN ISOINHIBITOR EXTRACTED FROM GELS

Species

C. arietinum C. reticulatum C. echinospermum C. bilugum C judaicum C. pinnatifidum C. yamashitae C. cuneatum

Inhibition of trypsin (%)

Inhibition of chymotrypsin (%)

58.7 62.5 57.5 28.7 35.0

47.5 50.0 . 48.7 28.7 31.2 27.5 27.5 15.0

36.0 28.7 12.5

factors should not be overlooked. The difference between the activity in the meal and of the pure inhibitor, at least in C. yamashitae, could be interpreted as a consequence of concentration or presence of some factors in the meal limiting the inhibitory activity. The relative activity of the trypsin and chymotrypsin isoinhibitors agrees well with the genetic affinities among the annual species of Cicer as indicated by their intercrossing potential. The cultivated species C. reticulatum and C. echinospermum form one cross-ability group [7], and each of them manifests more or less the same inhibitor activity. Similarly, C. bijugum, C.judaicum Boiss and C. pinnatifidum are members of the same group and recently it was found that C. yamashitae belongs to that group as well (G. Ladizinsky, unpublished results). While the level of the inhibitory activity among these species is quite similar, it differs conspicuously from the level of the cultigen and its related species and from that of C. cuneatum Hochst. Birk [5] had advanced the hypothesis that protease inhibitors in the seed had evolved as a defence mechanism against insects. On the other hand, soybean trypsin inhibitor at the level found in soybean meal added to the diet of Tribolium confusum had not Significantly affected the growth of the larvae [9]. If the presence of proteinase inhibitors in the seeds is indeed a defence mechanism and manifests a better selective value, one might expect higher activity of these proteinase inhibitors in the seeds of wild species which can be reached easily by various insects in nature. The marked differences in the inhibitory activity found in the annual species of Cicer is inconsistent with such an assumption. That activity was low in the wild species C. judaicum, C. pinnatofidum and hardly exists in the meal of C. yamashitae. Furthermore, it is unlikely that these differences are the result of geographic variation or have been devised because of differential vulnerability. The distribution of C.

TRYPSIN AND CHYMOTRYPSIN ISOINHIBITORS OF CICER

,bijugum and C. echinospermum overlaps considerably. In fact, the seed of the two species used in the present study were collected in sites 5 km apart in more or less the same habitat formed by deep basaltic soil. No insect barriers could be distinguished there and it is reasonable to assume that the selection pressure by seed-fed insects there is similar on both species. Yet, the activity of the trypsin and chymotrypsin isoinhibitors in C. echinospermum seeds is twice that in C. bijugum. The Tribolium protease inhibitor of wheat is only partially effective as a defence mechanism, but the relatively high level of protease inhibitor in modern wheat cultivars, in comparison with the wild progenitor of wheat, was suggested to be a consequence of selection under domestication [10]. The results obtained in the present study indicate that a different situation exists in chickpea. The inhibitory activity found in C. reticulatum, the wild progenitor of the cultigen [7] was higher than in four cultivars originating from different geographical areas. While the biological significance of the trypsin and chymotrypsin isoinhibitors in the seed is not yet clear, it will be very interesting to find out if this proteinase inhibitor is useful for characterizing taxonomic units above the genus level, at least in the tribe Viciae. Experimental Four accessions of the cultigen C. arietinum, three of C. judaicum and C. pinnatifidum, two of C. bijugum and C. echinospermum and a single accession of C. cuneatum, C. reticulatum and C. yamashitae were employed. Protein content of the extracts of ground seeds was determined according to the Lowry method [11]. Disc electrophoresis was performed in 7.5% acrylamide gel at pH 4.5 with a current of 7.5 mA per tube per 2 h at room temp.

25 Inhibition of the esterolytic activity of trypsin and chymotrypsin was determined titrimetrically in a Radiometer pH-Stat Model TTTI in which 0.1 N NaOH was used as titrant. The reactions were carried out at 30 ° in buffer (pH 8.0) made of 0.03 M Tris, 0.045 M KCI and 0.015 M CaCI2 • 2 H20.0.015 M p-tosyl-L-arginine methyl ester (TAM E) was used as substrate for trypsin and 0.019 M N-acetyl-L-tyrosine ethyl ester (ATEE) for chymotrypsin. Inhibition of esterolysis was expressed in inhibition units defined as pmol of substrate hydrolysed by 1 mg of enzyme for 1 min of reaction. Electrophoretic elution of the inhibition from gel slices was performed by the modified Braatz and Mclntire technique [12]. Acrylamide gels were run in 0.035 M 6-alanine pH 4.5 and the gel slices containing the protein to be eluted were put on the top of the gel. The glass tubes were inserted into the electrophoresis chamber and the electrodes were reversed so that the protein migrated toward the buffer chamber. Reversed electrophoresis was carried out at 4 ° for 3 h at 7.5 mA per tube. The upper buffer was collected, lyophilized and dialysed several times against H20.

References 1. Kunitz, M. (1946) J. Gen. Physiol. 29, 149. 2. FrankeI-Conrat, M., Bean, R. C., Ducay, E. D. and Olcott, H, S. (1952)Arch. Biochem. Biophys. 37,393. 3. Smirnoff, P., Khalef, S., Birk, Y. and Applebaum, S. W. (1976) Biochem. J. 157, 745. 4. Ryan, C. A. and Balls, A. K. (1962) Proc. Natl. Acad. Sci. U.S.A. 48, 1839. 5. Birk, Y. (1968)Ann. N.Y. Acad. Sci. 146,388. 6. Mies, D. W. and Hymowitz, T. (1973) Bot. Gaz. 134, 121. 7. Ladizinsky, G. and Adler, A. (1976) Theor. AppL Genet. 48, 197. 8. Ladizinsky, G. and Adler, A. (1975) Euphytica 25, 211. 9. Lipke, H., Frankel, G. S. and Liener, I. E. (1954) Agric. Food Chem. 2, 410. 10. Applebaum, S. W. and Konijn, A. M. (1966) J. Insect PhysioL 12, 665. 11. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193,265. 12. Braatz, J. A. and Mclntire, K. R. (1977) Prep. Biochem. 7, 495.