Inter- and Intraspecific Relationship between Two Native Greek Diploid Aegilops Species by Esterase Electrophoresis

Biochem. Physiol. Pflanzen (BPP), Bd. 167, S. 389-398 (1975)

Inter- and Intraspecific Relationship between Two Native Greek Diploid Aegilops Species by Esterase Electrophoresis S. S. KARATAGLIS Botanical Institute, University of Thessaloniki, Thessaloniki, Greece Key Term Index: enzymes, electrophoresis, esterase patterns, phylogenetic relationships, species specificity; Aegi!ops caudata, Ae. comosa.

Summary Esterase patterns of the species Aegi!ops caudata and Ae. comosa were studied by means of horizontal starch gel electrophoresis, in shoot and root of seedlings, in an attempt to detect the phylogenetic relationships within and between the two groups. A high degree of electrophoretic similarities was observed within the two varieties of Ae. caudata in the shoot as well as in the root. More electrophoretic differences were detected within the comosa group, so we studied them in more detail. Electropherograms were evaluated by means of spectrophotometry. Based on consideration of the present enzymograms and cytological and morphological information, it may be postulated that: 1. The similarity between subventricosa and biaristata was greater than those found between thessa!ica and the two varieties of the he!dreichii (subventricosa and biaristata). 2. Considering the varieties of Ae. he!dreichii and Ae. thessa!ica we found that a higher electrophoretic similarity existed between biaristata and thessalica than that of subventricosa and thessalica. 3. A low degree of electrophoretic similarity existed between the two species Ae. caudata and Ae. comosa as compared with interspecific differences.

Introduction The variability of zymograms within and between living organisms is of special importance from a genetical and evolutionary point of view (SCANDALIOS 1969, 1974; BOULTER et al. 1966). Genotypic differences have as a result variations in the chemical structure and the physicochemical behaviour of particular enzymes or proteins. The appropriate utilization of these physicochemical properties enables one to make inferences about the degree of rciationship of species at the molecular level. One of the most recent and useful methods for the study of enzyme properties is electrophoresis. By this method we can estimate with more accuracy the degree of the phylogenetic inter- and intraspecies relationship (SMITH et al. 1970; CONKLIN et al. 1971; CHERRY et al. 1972; BERRY et al. 1973). Similarities or dissimilarities in the electropherograms constitute a measure of the degree of phylogenetic relationship between species. In this way the classification of vDrious genera has been supplemented or verified. The respective studies have been carried out by SMITH et al. (1970), SHEEN (1970) for Nicotiana, by MACDANIEL (1970). 27*

390

S. S. KARATAGLIS

MITRA et al. (1970) for Hordeum, by JOHNSON et al. (1970) for Cossypi7tm, by MITRA et al. (1971) for Triticum, by CONKLIN et al. (1971) for Datura, and others. The aim of the present study is the examination of the native Greek diploid species of Aegilops in order to determine the degree of the phylogenetic I:elationship by establishing enzymic ratios. Two native species of the genus Aegilops, were used, namely Ae. caudata and Ae. comOSa comprising five varieties in all. The esterase pattern of two fundamental organs, i. e. of the root and shoot were examined. By the comparative study of their results we ascertained the degree of the intra- and interspecific relationship of the samples investigated.

Material and Methods Five native Greek varieties of the genus Aegilops (Ae. caudata var. typica, Ae. caudata var. polyathera, Ae. comosa ssp. eucomosa var. thessalica, Ae. comosa ssp. heldreichii var. biaristata and Ae. comosa ssp. heldreichii var. subventricosa), were studied at their initial ontogenetic stages. The bigges and heaviest seed of all these varieties (DATA et al. 1972) germinated in Petri-dishes at 24 + 1 °C in the dark. Seedlings were harvested after 4 days and were homogenized with distilled water in the radio of 2: 1 w/v. The extracts were subsequently electrophorized or were frozen at -15°C, for later use; they were plased on Whatman 3 MM filter paper strips (dimensions 7 X 6 mm) according to the technique used by BECKMAN and JOHNSON (1964). Horizontal starch gel electrophoresis was carried out as employed by SMITHIES (1955) and modified by KARATAGLIS (1973). During electrophoresis the potential difference was kept constant at 240 V, whereas the current intensity fluctuated between 60 and 90 rnA. Electrophoresis was interrupted when the front had migrated a distance of 8-8,5 cm from the origin. During the whole process of electrophoresis the temperature of the gel was kept constant at 7-8°C by means of circulating cold water. The gel bearing the segregated enzymes was stained at room temperature for about 1/2-1 hour in a solution having the following composition: a) 40 ml of distilled water. b) 50 ml 0,2 M NaH 2 P0 4 (PH = 4,6). c) 10 ml 0,2 M Na2 HP0 4 (PH = 8,8). d) 2 mil % a-naphthyl-acetate in an aqueous solution of 50 % acetone (v/v). e) 20 mg of dye Fast Red TR salt (BREWBAKER et al. 1968). Areas of gel where esterase activity was observed were stained brown red.

Results

During electrophoresis of shoot extracts of the investigated materials, bands were observed, which can be classified in four main areas: a) Area of slow electrophoretic mobility b) Area of medium electrophoretic mobility c) Area of fast electrophoretic mobility d) Area of very fast electrophoretic mobility Among them area (b) indicated the greatest number of bands and variations of intensity. All the enzymes showing esterase activity were found to move towards the anode.

391

Esterase Relationships between Two ",iegilops Species

By comparing the zymograms of the meterials studied we ascertained a variation in the number of the bands ranging from ten to thirteen. Thus in the group caudata, the variety polyafhera displays eleven bands in all, while the variety typica shows two more bands. In the group comosa, twelve bands were observed in the variety thessalica, whereas the varieties of the subspecies heldreichii, subventricosa and biaristata showed ten and eleven bands respectively.

Group caudata

Ae. caudata var. typica Ae. caudata var. polyathera The zymograms of the shoot display great similartiy in the areas of high and low electrophoretic mobility. This similarity concerns the number and the mobility of the bands and their intensity, with the exception of two bands only, which display higher intensity in the variety typica than in polyathera (area of low electrophoretic mobility). In the area of medium electrophoretic mobility the main difference consists in the appearance of two extra bands in the variety typica (Fig. 1). Similar results were obtained, when we compared the zymograms of the root. Thus we found similarities in both materials of low and high electrophoretic mobility, whereas differences were observed in the area of medium mobility consisting of: a) the appearance of two extra bands in typica and in b) the presence of two bands each showing different intensities (Fig. 2).

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2 1 Fig. 1. Schematic representation of zymograms showing the electrophoretic patterns of shoot-esterase in Ae. caudata var. polyathera, Ae. caudata var. typica. Fig. 2. Schematic representation of zymograms showing the electrophoretic patterns of root-esterase in Ae. caudata var. polyathera, Ae. caudata var. typica.

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Fig. 3. Photograph of starch gel zymogram showing shoot-esterase patterns in Ae. comosa var. thessalica, Ae. comosa var. biaristata, Ae. comosa var. subventricosa.

Group comosa

heldreichii var. subventricosa heldreichii var. biaristata There is a relative similarity in the electropherograms regardind the number and mobility. The area of low electrophoretic mobility does not have a constant number of bands; differences also occur in the intensity. This variation in the intensity of the bands mar possibly be related to the various stages of activation (SUSSMAN 1964; BHATIA et al. 1969) of the genes. In the area of medium electrophoretic mobility one more band

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Ae. comosa var. subventricosa, Ae. comosa var. biaristata, Ae. comosa var. thessalica.

Esterase Relationships bet\\"een Two Llegilops Species

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Fig. 6. Photograph of starch gel zymogram sholcing root-esterase patterns in Ae. comosa var. subv~ntri­ cosa, Ae. comosa var. thessalica, Ae. comosamr. biaristata.

394

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(6a) appears in the variety biaristata and a second one (7 a) with a slight difference in the intensity when compared with (8a) of subventicosa. The remaining bands exhibit the same mobility but they have different intensities (Fig. 3, 4, 5). In the area of high electrophoretic mobility, there is the same number of bands with the same mobility and intersity. Biaristata, also, shows an area of very high mobility, with only one band of low activity and intersity, which, however, is absent in subventricosa. As regards the electropherograms of the root in the area of low electrophoretic mobility both materials display different mobilities. There is a greater similarity in the area of medium mobility owing to the fact, that we have two bands of the same mobility but of different intensity. The area of high mobility is absolutely similar (Fig. 6, 7, 8).

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Fig. 7. Schematic representation of zymograms showing the electrophoretic patterns of root-esterase in Ae. comosa var. biaristata, Ae. comosa var. thessalica, Ae. comosa var. subventricosa. Fig. 8. Curves of absorption and migration velocity for Ae. comosa var. subventricosa, Ae. comosa vaT. biaristata, Ae. comosa var. thessalica, obtained from electrophoretic gels illustrated in Fig. 6.

Esterase Relationships between Two A.egilops Species

395

heldreichii - thessalica The area of low electrophoretic mobility of the zymograms of the shoot shows very few differences, owing to the fact that the number of the bands in this area varies between two and three. In the area of medium electrophoretic mobility differences are observed regarding the number of the bands and their intensity (in thessalica, bands 7a, 8a, 9a are absent, whereas new additional bands, 5a, lla and 14a make their appearance). The only band of very high mobility in thessalica appears with a slightly lower mobility than the respective band of biaristata (Fig. 1, 3, 5). In the area of low electrophoretic mobility of the zymograms in the root of sub-· ventricosa, biaristata and thessalica, differences are observed regarding the mobility of the bands as well as their intensity. In the area of medium electrophoretic mobility differences are also observed concernig the mobility and intensity of the bands. Among the above three materials, in the same area the bands of thessalica and subventricosa showed greater similarities between each other as regards their mobility and intensity than in the case of biaristata and thessalica. In the area of high electrophoretic mobility differences were revealed in the intensity and the activity of the frontal bands of thessalica as compared with those of biaristata and subventricosa, whereas the other band of thessalica migrates more slowly and shows lower intensity.

Discussion

A. Aegilops caudata There is great similarity in the electropherograms of the shoot of the two varieties of Ae. caudata. Very few differences are located in the area of medium electrophoretic mobility, where typica shows two more bands than polyathera. The same differences were also observed in the zymograms of the root, namely in the root and shoot of typica there were two more bands (Fig. 1, 2). The presence of two more bands in typica in comparison to polyathera, at the same stage of ontogenetic evolution (4-day-old plants) presupposes the existence of one or perhaps several responsible sites (loci) regulating the synthesis of these enzymic forms. The absence of these enzymic forms in polyathera at the respective stage is perhaps due to the lack of a responsible genetic site (or sites) or to the repression of its activity (or their activities) (silent genes). As regards the appearance of bands of the same mobility but of different intensity this may be attributed to the fact that the responsible gene or genes are at different activation stages (SUSSMAN 1964; BHATIA et al. 1969). The differences observed in the two fundamental organs (shoot and root) as regards intensity, are due to the fact that the responsible genes of these organs display a different activation time. From the above observations it follows that the electrophoretic similarities are criteria of the degree of relationship of the plants. In fact, as table 1 shows, the two varieties of Ae. caudata display a close phylogenetic relationship, which can also be verified by morphological and cytological data (KARATAGLIS, in press).

396

S. S.

KAUATAGLIS

B. Aegilops como sa Comparative studies of the zymograms of the shoot between biaristata and subventricosa have demonstrated that these zymograms display some enzymic differences. Thus the area of low electrophoretic mobility in subventricosa shows one more band and there are also differences as regards the intensity of the other bands. In the· area of medium electrophoretic mobility, biaristata display one more band, while another one shows a slight delay in its mobility as compared with subventricosa. In the area of high electrophoretic mobility the similarity is absolute. Finally, the area of very high electrophoretic mobility, which is found only in biaristata, shows only one band of very low intensity (Fig. 3, 4, 5). In the zymograms of the root obtained from the same materials a marked difference was located in the area of low electrophoretic mobility, as the respective bands exhibited variations regarding their mobility. In the area of medium electrophorE'tic mobility, the difference was smaller, whereas absolute similarity was observed in the area of high electrophoretic mobility (Fig. 6, 7, 8). Comparing the zymograms of the shoot of Ae. comosa var. thessalica with those of Ae. comosa ssp. heldreichii, we notice slight deviatiom in the area of low electrophoretic mobility. In the area of medium electrophoretic mobility, thessalica displays two more additional bands, whereas one of the bands of heldreichii is absent from it. As regards the zymograms of the root, the existing differences in the area of medium electrophoretic mobility are more significant. In fact, we have in thessalica three additional bands as compared with subventricosa and two more than biaristata.

C. Inter- and intraspecific comparisons The electrophoretic behaviour constitutes a measure for the estimation of the degree of phylogenetic relationship within and between the species. This relationship can be calculated by means of a method employed by SMITHet al. (1970), CONKLIN et al. (1971) and KARATAGLIS et al. (1975) in the case of Nicotiana and Datura, taking into consideration phenotypic ratios of the zymograms examined. ' According to this method we use the following equation:

where P(i) is the probality under randon matching of obtaining i common bands, Kiis the number of bands in one species, K2 is the number of bands in the other species, n' is the total number of bands possible. By applyHig the above equation we can find out the probability under random matching of obtaining the observed number of matching bands (the electropherograms being compared two at a time). Thus a low probability means a close phylogenetic

397

Esterase Relationships between Two Aegilops Speeies n~latioIlShip,

i. a. the matching of many bands of the same mobility constitutes a Cl·iterium of closer genetic relationship and cannot be considered as a result of random matching. According to the above statements and on the basis of the formula applied we can estimate the exiflting phylogenetic relationship between the species, as shown in the Table 1. Table 1. Probability of random band matching intra- and interspecific of Ae. caudata and Ae. comosa varieties

Ty

P

S

B

Ph

typica (Ty) polyathera (P) subventricosa (S) biaristata (B) thessalica (Th)

0

0,002 0

0.996 0.974 0

0.926 0.860 0.010 0

0.934 0.970 0.407 0.263 0

It follows from table I that there is a greater degree of relationship between subventricasa and biaristata than between subventricosa and thessalica or biaristata and thessalica. Furthermorp, is shows that there exist a closer relationship between biaristata and thessalica than between subventricosa and thessalica. This fact, in connection with the limited distribution of biaristata leads us to the assumption, that the variety biaristata constitutes perhaps the connecting link between subventricosa and thessalica (Fig. 9). In conclusion, the comparison of the electropherograms of Ae. caudata and Ae. comosa that there is a lower degree of phylogenetic relationship between these two species, whereas there exists a high degree of phylogenetic relationship among the varieties of the same species (see also Table 1).

Fig. 9. Schematic representation of relationships within the varieties of Ae. comosa.

References L., and JOHNSON, F. ~I., Esterase variation in Drosophila melanogaster Hereditas ill, 212-220 (1964). BERRY, J. A., and FRANKE, R. G., Taxonomic significance of intraspecific. isozyme patterns of the slime mold Fuligo septica produced by disc electrophoresis. Amer. J. Bot. 60, 976-986 (1973).

BECK)UN,

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S. S. KARATAGLIS, Esterase Relationships between Two A.egilops Species

BHATIA, C. R., and ::\fILSON, J. P., Isoenzyme changes accompanying germination of wheat seeds. Biochem. Genetics 3, 207-214 (1969). BOULTER, D., THURMAN, D. A., and TURNER, B. L., The use of disc electrophoresis of plant proteins in systematics, Taxon 15, 135-143 (1966). BREWBAKER, J. L., UPADHYA, M. D., MAKINEN, Y., and MACDoNALD, T., Isoenzyme polymorphism in flowering Plants. III. Gel electrophoretic methods and applications. Physio!. Plant 21, 930-940 (1968). CHERRY, J. P., KATTERMAN, F. R. H., and ENDRIZZI, J. E., Seed esterases, leucine aminopaptidases and catalases of species of the genus. Gossypium Theoret. App!. Genetics 42, 218-226 (1972). CONKLIN, M., and SMITH, H., Peroxidase isozymes: A measure of molecular variation in ten herbaceous species of Datura. Amer. J. Bot. 58, 688-696 (1971). DATTA, S. C., GUTTERMAN, Y., and EVENARI, M., The influence of the origin of the mother plant on yield and germination of their carypopses in A.egilops ovata. Planta 105, 155-164 (1972). JOHNSON, B. L., and THEIN, M. M., Assessment of evolutionary affinities in Gossypium by protein electrophoresis. Amer. J. Bot. 57, 1081-1092 (1970). KARATAGLIS, S. S., Comparative research upon the caryotype and the electrophoretic enzymes of esterases from some Greek native species of the genus A.egilops. Ph. D. Thesis, University of Thessaloniki (1973). - Karyotype analysis on some diploid native Greek. A.egilops species. Caryologia 1975 (in press). - and TSEKOS, I., Electrophoretic variation in esterases of native Greek A.egilops species ( A.e. ovata, A.e. biuncialis and A.e. triuncialis). Protoplasma 83, 311-325 (1975). MACDANIEL, R., Electrophoretic characterization of proteins in Hordeum. J. Heredity 61,243-247. (1970). MITRA, R., JAGANNATH, D. R., and BHATIA, C. R., Disc electrophoresis of analogous enzymes in Hordeum. Phytochem. 9, 1843-1950 (1970). MITRA, R., and BHATIA, C. B., Isoenzymes and polyploidy. I. Qualitative and quantitative isoenzyme studies in the Triticinae. Gent. Res., Comb. 18, 57-69 (1971). SCANDALIOS, J. G., Genetic control of multiple molecular forms of enzymes in plants: A review, Biochem. Genet. 3, 37-79 (1969). - Isoenzymes in development and differentiation. Ann. Rev. Plant Physiol. 21), 225-258 (1974). SHEEN, S. J., Peroxidases in the genus Nicotiana. Theoret. App!. Gent. 40, 18-25 (1970). SMITH, H. H., HAMILL, D. E., WEAVER, E. A., and THOMPSON, K. S., Multiple molecular forms of peroxidases and esterases among Nicotiana species and amphiploids. J. Heredity 61, 203-212 (1970). SMITHIES, 0., Zone electrophoresis in starch gels: Group variations in the serum proteins of normal human adults. Biochem. J. 61, 629-641 (1955). SUSSMAN, M., Growth and Development. 2nd edition Englewood cliffs, N. J. Pretire-Hall ING pp. 105 (1964). Received February 6, 1975. Author's address: Dr. S. S. KARATAGLIS, Botanical Institute, University of Thessaloniki, Thessaloniki (Greece).