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Isoenzyme Pattern of Peroxidase, Polyphenol Oxidase and Catalase During Germination and Early Plant Development of Wheat (Triticum aestivum L.)
Biochem. Physiol. Pflanzen (BPP), Ed. 166, S. 233-237 (1974)
Chemistry and Biochemistry Department, Haryana Agricultural University, Hissar, India
Isoenzyme Pattern of Peroxidase, Polyphenol Oxidase and Catalase During Germination and Early Plant Development of Wheat (Triticum aestivum L.) By RANDHIR SINGH and DHARAM SINGH With 3 figures Key Term Index: isoenzymes, peroxidase, polyphenol oxidase, catalase, germination; Triticum aestivum.
Summary Isoenzymes of peroxidase, polyphenol oxidase and catalase at different intervals during germination and in different plant parts during early development of K-227 variety of wheat were investigated. The number of isoenzymic bands of the three enzymes and their intensities increased progressively with the concomitant increase in the stage of germination and early development. The isoenzymic patterns, in general, were more clear and better developed during the later stages of germination. Different tissues or organs of the wheat plant possessed specific isoenzmic patterns of these enzymes.
Introduction
The changes in enzyme levels have long been studied presumably as a means of rationalizing the nature of growth events. Most of the work of this nature was built on the implicit assumption that each enzyme was but a single molecular entity and maintenance of enzyme activity, would therefore, be considered as an accurate reflection of a stable enzyme level. Since enzyme multiplicity is now known to be a wide spread, perhaps general phenomenon (FuRNESS 1961), the above assumption could no longer be taken for granted. The isoenzymes of the same enzyme might, therefore, be changing and influencing metabolism even when gross assay shows little change. CouLSON and SIM (1965) were the first to report the changes in the electrophoretic patterns of soluble proteins of wheat at different stages of growth. MACKO et al. (1967) and NAINAWATEE et al. (1974) have also reported the changes in the protein spectrums of wheat seedlings during early growth periods. Although studies have been made of the multiple molecular forms and differential electrophoretic mobilities of enzyme components (HoNOLD et al. 1966; MACKO et al. 1967 and BHATIA and NELSON 1968) in the early growth stages of wheat, yet very little information is available on the systematic study of the isoenzymes of these oxidative enzymes during germination and early plant development of wheat. At the same time, since changes in the patterns of growth and differentiation at the cellular level must be preceded by quantitative or qualitative changes at the molecular level, it is pertinent to examine the kinds of the
234
R. SINGH and D. SINGH
various oxidative enzymes which plants may contain during the various stages of germination and early development. With these objectives in view, the present study was undertaken in which the isoenzymes of peroxidase, polyphenol oxidase and catalase have been reported during the major morphological changes in germination and early development of wheat.
Material and Methods Seeds of Triticum aestivum LINN. variety K-227 were treated with 0.1% mercuric chloride for minutes and then repeatedly washed with distilled water. The treated seeds were sown in petriplates; under sterilized conditions in a germinating chamber at 25 oc. During germination, the samples were drawn at 24, 48 and 72 hours, and for early developmental studies, 5 and 7 day old seedlings were taken, which were further separated into roots, shoots and whole plants. Extraction was done by grinding the samples in a mortar using 0.5 M phosphate buffer (pH 6.8). The slurry was centrifuged at 10,000 x g for 20 minutes. Extraction of samples, centrifugation and electrophoresis etc. were all conducted at 4 °C. Samples containing 250 p,g of protein were layered on 7.5% polyacrylamide gel and electrophoresis was done using anionic system (DAVIs 1964). Isoenzymes of peroxidase, polyphenol oxidase and catalase were detected by zymogram staining techniques of MAcKo et al. (1967), V ANLEAR and SMITH (1970) and ScANDALIOS (1969), respectively. Protein content of the extracts was determined using Folin phenol reagent (LoWRY et al.1951). fiv~
Results and Discussion
Results presented in figs. 1-3 show only one isoenzymic band with Rf 0. 76, 0. 79 and 0. 78, respectively for peroxidase, polyphenol oxidase and catalase in dormant seeds. Germination, however, brought about an increase in the number of isoenzymic bands and isoenzymes with Rf 0.03 and 0.76, 0.74 and 0.79 and 0.68 and 0.78 for each of the three enzymes, respectively, were observed at 24 hours interval during germination. At RF 0.()3
0.09 0.13
0.38
0.56 0.71 0.76
0.93
2
3
4
5
6
7
8
Fig. 1. Polyacrylamide gels showing peroxidase isoenzyme patterns during germination and early plant development.
9 ~n
10
wheat at different intervals
(1) 0 hour; (2) 24 hours; (3) 48 hours; (4) 72 hours; (5) 5 day roots; (6) 5 day shoots; (7) 5 day whole plants; (8) 7 day roots; (9) 7 day shoots; (10) 7 day whole plants.
23!)
Isoenzyme Pattern of Peroxidase etc.
48 hours of germination, two new bands with Rf 0.09 and 0.71 appeared for peroxidase. The intensity of these bands was, however, less in comparison to that of the previously existing isoenzymes. Similarly, for polyphenol oxidase and catalase, a new band with Rf 0.06 and 0.22, respectively appeared at this interval. However, the bands were relatively less intense in comparison to the earlier ones. At 72 hours interval during germination, another less intense isoperoxidase with Rf 0.93 became apparent on the gels. Three additional bands for polyphenol oxidase with Rf 0.15, 0.65 and 0. 70 and one band for catalase with Rf 0.35 also became visible at this interval. These bands were again less intense in comparison to the previously existing bands. The catalase band with Rf 0.22 became more intense at this interval. Seven isoperoxidases with Rf 0.03, 0.09, 0.38, 0.56, 0. 71, 0. 76 and 0.93 were observed in the extracts of the roots of 5 day old seedlings. However, the bands with Rf 0.38, 0.56 and 0.93 were relatively poor in intensity when compared to the other ones. Another RF
0.06
0.1S
0.65 0.70
0.74 o.79
2
3
5
6
7
8
9
10
Fig.c2. Polyacrylamide gels showing polyphenoloxidase isoenzyme patterns in wheat at different intervals duri"'ng germination and early plant development. (1) 0 hour; (2) 24 hours; (3) 48 hours; (4) 72 hours; (5) 5 day roots; (6) 5 day shoots; (7) 5 day whole plants; (8) 7 day roots; (9) 7 day shoots; (10) 7 day whole plants.
0.78
o.ao
2
3
5
6
7
8
9
10
Fig. 3. Polyacrylamide gels showing catalase isoenzyme patterns in wheat at different intervals during germination and early plant development. (1) 0 hours; (2) 24 hours; (3) 48 hours; (4) 72 hours; (5) 5 day roots; (6) 5 day shoots; (7) 5 day whole plants; (8) 7 day roots; (9) 7 day shoots; (10) 7 day whole plants.
236
R.
SINGH
and D.
SINGH
additional isoperoxidase with Rf 0.13 appeared in roots of 7 days old seedlings, thus raising the total strength of isoperoxidases to 8. In shoots of 5 and 7 day old seedlings, the isoperoxidases with Rf 0.03, 0.09, 0.38, 0.71 and 0.76 were present, whereas the other three isoperoxidases with Rf 0.13, 0.56 and 0.93 observed in roots were absent in the extracts of shoots. The patterns of isoperoxidases in 5 and 7 day whole plants were almost similar to that of their respective roots patterns except the minor differences in the intensity of the bands. The bands were generally less intense in extracts of 5 and 7 day whole plants. The isoenzymic pattern of polyphenol oxidase in roots of 5 day old seedlings was identical to that obtained at 72 hours germination. In roots of 7 day old seedlings, the bands with Rf 0.15 and 0.70 were absent when compared to the pattern obtained at 5 days. Four polyphenol oxidase bands with Rf 0.06, 0.65, 0.74 and 0.79 were observed in 5 day old shoots. On 7th day, the band with Rf 0.65 disappeared with simultaneous decrease in the intensity of previously existing bands. Although, the isoenzymic pattern for polyphenol oxidase was similar in 5 and 7 day whole plants, the band with Rf 0. 70 was notably absent in 7 day whole plants. The isoenzymic pattern of catalase in roots of 5 and 7 day old seedlings was almost similar except that an additional band with Rf 0.80 appeared in roots of 7 day old plants which was earlier absent at 5 days. Similarly, the patterns in shoots of 5 and 7 day seedlings was almost identical except that the isoenzyme with Rf 0.80 appeared in shoots at 7 days. In whole plants of 5 and 7 day old seedlings, the similar situation existed. The intensity of the band with Rf 0. 78 was relatively less at 7 days than at 5 days. The results presented here indicate that germination brings about an increase in the number of isoenzymic components of the three enzymes and in general, the isoenzymic fractions were more clear and better developed at 72 hours of germination. Different tissues and organs of the same plant possess specific isoenzymic pattern of these oxidative enzymes at a particular stage of growth. The increase in multimolecular forms of these oxidative enzymes during germination may be because of de novo protein synthesis as a result of enhanced metabolism. The appearance or disappearance of a particular isoenzymic component at a particular stage of growth and development of a plant may be explained on the basis of a switching on - off mechanism of the protein synthesizing machinery of a cell depending upon the metabolic requirements of the plant at that particular stage of growth. At the same time, since it is well known that the different isoenzymic fractions of these enzymes have specific substrate requirements and catalyse specific oxidation reactions (MARCU et al. 1969), this general phenomenon can also go a long way in explaining the organ specificity of these enzymes. These results show a general resemblance to those of earlier workers (MACKO et al. 1967; UPADHYA and YEE 1968; YAASKA and YAASKA 1968; MARCU et al. 1969 and HAGIMA and LoANA 1970) who reported that the activity of oxidative enzymes is generally low in dormant seeds and the process of germination brings about both qualitative and quantitative increase in these oxidative enzymes.
Isoenzyme Pattern of Peroxidase etc.
237
Acknowledgement The authors are thankful to Dr. B. M. LAL, Professor of Biochemistry, for providing the facilities and to SH. D. R. VASUDEVA, Economic Botanist (Wheat}, for providing the materials.
References BHATIA, C. R., and NELSON, J.P., Isoenzyme changes accompanying germination of wheat seeds. Biochem. Genet. 3, 207-214 (1968). CouLsoN, C. B., and SIM, A. K., Wheat proteins. II. Changes in the protein composition of Triticum vulgare during the life cycle of plant. J. Sci. Fd. Agri. 16, 499-508 (1965). DAVIS, B. J., Disc electrophoresis. II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121, 404-427 (1964). FuRNESS, F. N., Multiple molecular forms of enzymes. Ann. N. Y. Acad. Sci. 94, 655-666 (1961). HAGIMA, C., and LoAN A, P., Peroxidase in maize during the early developmental stages. Stud. Cereet. Biochem. 13, 47-53 (1970). HoNOLD, G. R., FARKAS, G. L., and STAHMANN, A., The oxidation reduction enzymes of wheat. 1. Qualitative investigation of the dehydrogenases. Cereal chem. 43, 517-529 (1966). LOWRY, 0. H., RosEBROUGH, N.J., FARR, A. L., and RANDALL, R. J., Protein measurement with the Folin-phenol reagent. J. Bioi. Chern. 193, 265-275 (1951). MACKO, 0. H., HoNOLD, G. R., and STAHMANN, M. A., Soluble proteins and multiple enzyme forms in early growth of wheat. Phytochem. 6, 465-471 (1967). MARCU, Z., BRAD, 1., and NwuLEscu, S.M. E., Differences in peroxidase isoenzymes pattern as a function of the structural peculiarities of the hydrogen donor. Stud. Cereet. Biochem. 12, 265-272 (1969). NAINAWATEE, H. S., MEHTA, S. L., and DAs, N. B., Electrophoretic changes in wheat proteins during germination. Biochem. Physiol. Pflanzen (BPP) 165, 93-99 (1974). ScANDALIOs, J. G., Genetic control of multiple molecular forms of enzymes in plants. A. Rev. Biochem. Genet. 3, 37 -79 (1969). UPADHYA, M.D., and YEE, L., Isoenzyme polymorphism in flowering plants. VII. Isoenzyme variations in tissues of barley seedlings. Phytochem. 7, 937-943 (1968). VANLEAR, D. H., and SMITH, W. H., Soluble proteins and enzyme pattern in shoots of slashpine under different nutritional regimes. Phytochem. 9, 1929-1932 (1970). YAASKA, V., and YAASKA, N., Phosphorylase and peroxidase isoenzymes in wheat seedlings. Izv. Akad. Nauk. Est. USSR. Ser. Bioi. 17, 164-169 (1968). Received: March 4, 1974. Authors' address: Dr. RANDHIR SINGH and DHARAM SINGH, Department of Chemistry and Biochemistry, Haryana Agricultural University, Hissar, Haryana (India).
Chemistry and Biochemistry Department, Haryana Agricultural University, Hissar, India
Isoenzyme Pattern of Peroxidase, Polyphenol Oxidase and Catalase During Germination and Early Plant Development of Wheat (Triticum aestivum L.) By RANDHIR SINGH and DHARAM SINGH With 3 figures Key Term Index: isoenzymes, peroxidase, polyphenol oxidase, catalase, germination; Triticum aestivum.
Summary Isoenzymes of peroxidase, polyphenol oxidase and catalase at different intervals during germination and in different plant parts during early development of K-227 variety of wheat were investigated. The number of isoenzymic bands of the three enzymes and their intensities increased progressively with the concomitant increase in the stage of germination and early development. The isoenzymic patterns, in general, were more clear and better developed during the later stages of germination. Different tissues or organs of the wheat plant possessed specific isoenzmic patterns of these enzymes.
Introduction
The changes in enzyme levels have long been studied presumably as a means of rationalizing the nature of growth events. Most of the work of this nature was built on the implicit assumption that each enzyme was but a single molecular entity and maintenance of enzyme activity, would therefore, be considered as an accurate reflection of a stable enzyme level. Since enzyme multiplicity is now known to be a wide spread, perhaps general phenomenon (FuRNESS 1961), the above assumption could no longer be taken for granted. The isoenzymes of the same enzyme might, therefore, be changing and influencing metabolism even when gross assay shows little change. CouLSON and SIM (1965) were the first to report the changes in the electrophoretic patterns of soluble proteins of wheat at different stages of growth. MACKO et al. (1967) and NAINAWATEE et al. (1974) have also reported the changes in the protein spectrums of wheat seedlings during early growth periods. Although studies have been made of the multiple molecular forms and differential electrophoretic mobilities of enzyme components (HoNOLD et al. 1966; MACKO et al. 1967 and BHATIA and NELSON 1968) in the early growth stages of wheat, yet very little information is available on the systematic study of the isoenzymes of these oxidative enzymes during germination and early plant development of wheat. At the same time, since changes in the patterns of growth and differentiation at the cellular level must be preceded by quantitative or qualitative changes at the molecular level, it is pertinent to examine the kinds of the
234
R. SINGH and D. SINGH
various oxidative enzymes which plants may contain during the various stages of germination and early development. With these objectives in view, the present study was undertaken in which the isoenzymes of peroxidase, polyphenol oxidase and catalase have been reported during the major morphological changes in germination and early development of wheat.
Material and Methods Seeds of Triticum aestivum LINN. variety K-227 were treated with 0.1% mercuric chloride for minutes and then repeatedly washed with distilled water. The treated seeds were sown in petriplates; under sterilized conditions in a germinating chamber at 25 oc. During germination, the samples were drawn at 24, 48 and 72 hours, and for early developmental studies, 5 and 7 day old seedlings were taken, which were further separated into roots, shoots and whole plants. Extraction was done by grinding the samples in a mortar using 0.5 M phosphate buffer (pH 6.8). The slurry was centrifuged at 10,000 x g for 20 minutes. Extraction of samples, centrifugation and electrophoresis etc. were all conducted at 4 °C. Samples containing 250 p,g of protein were layered on 7.5% polyacrylamide gel and electrophoresis was done using anionic system (DAVIs 1964). Isoenzymes of peroxidase, polyphenol oxidase and catalase were detected by zymogram staining techniques of MAcKo et al. (1967), V ANLEAR and SMITH (1970) and ScANDALIOS (1969), respectively. Protein content of the extracts was determined using Folin phenol reagent (LoWRY et al.1951). fiv~
Results and Discussion
Results presented in figs. 1-3 show only one isoenzymic band with Rf 0. 76, 0. 79 and 0. 78, respectively for peroxidase, polyphenol oxidase and catalase in dormant seeds. Germination, however, brought about an increase in the number of isoenzymic bands and isoenzymes with Rf 0.03 and 0.76, 0.74 and 0.79 and 0.68 and 0.78 for each of the three enzymes, respectively, were observed at 24 hours interval during germination. At RF 0.()3
0.09 0.13
0.38
0.56 0.71 0.76
0.93
2
3
4
5
6
7
8
Fig. 1. Polyacrylamide gels showing peroxidase isoenzyme patterns during germination and early plant development.
9 ~n
10
wheat at different intervals
(1) 0 hour; (2) 24 hours; (3) 48 hours; (4) 72 hours; (5) 5 day roots; (6) 5 day shoots; (7) 5 day whole plants; (8) 7 day roots; (9) 7 day shoots; (10) 7 day whole plants.
23!)
Isoenzyme Pattern of Peroxidase etc.
48 hours of germination, two new bands with Rf 0.09 and 0.71 appeared for peroxidase. The intensity of these bands was, however, less in comparison to that of the previously existing isoenzymes. Similarly, for polyphenol oxidase and catalase, a new band with Rf 0.06 and 0.22, respectively appeared at this interval. However, the bands were relatively less intense in comparison to the earlier ones. At 72 hours interval during germination, another less intense isoperoxidase with Rf 0.93 became apparent on the gels. Three additional bands for polyphenol oxidase with Rf 0.15, 0.65 and 0. 70 and one band for catalase with Rf 0.35 also became visible at this interval. These bands were again less intense in comparison to the previously existing bands. The catalase band with Rf 0.22 became more intense at this interval. Seven isoperoxidases with Rf 0.03, 0.09, 0.38, 0.56, 0. 71, 0. 76 and 0.93 were observed in the extracts of the roots of 5 day old seedlings. However, the bands with Rf 0.38, 0.56 and 0.93 were relatively poor in intensity when compared to the other ones. Another RF
0.06
0.1S
0.65 0.70
0.74 o.79
2
3
5
6
7
8
9
10
Fig.c2. Polyacrylamide gels showing polyphenoloxidase isoenzyme patterns in wheat at different intervals duri"'ng germination and early plant development. (1) 0 hour; (2) 24 hours; (3) 48 hours; (4) 72 hours; (5) 5 day roots; (6) 5 day shoots; (7) 5 day whole plants; (8) 7 day roots; (9) 7 day shoots; (10) 7 day whole plants.
0.78
o.ao
2
3
5
6
7
8
9
10
Fig. 3. Polyacrylamide gels showing catalase isoenzyme patterns in wheat at different intervals during germination and early plant development. (1) 0 hours; (2) 24 hours; (3) 48 hours; (4) 72 hours; (5) 5 day roots; (6) 5 day shoots; (7) 5 day whole plants; (8) 7 day roots; (9) 7 day shoots; (10) 7 day whole plants.
236
R.
SINGH
and D.
SINGH
additional isoperoxidase with Rf 0.13 appeared in roots of 7 days old seedlings, thus raising the total strength of isoperoxidases to 8. In shoots of 5 and 7 day old seedlings, the isoperoxidases with Rf 0.03, 0.09, 0.38, 0.71 and 0.76 were present, whereas the other three isoperoxidases with Rf 0.13, 0.56 and 0.93 observed in roots were absent in the extracts of shoots. The patterns of isoperoxidases in 5 and 7 day whole plants were almost similar to that of their respective roots patterns except the minor differences in the intensity of the bands. The bands were generally less intense in extracts of 5 and 7 day whole plants. The isoenzymic pattern of polyphenol oxidase in roots of 5 day old seedlings was identical to that obtained at 72 hours germination. In roots of 7 day old seedlings, the bands with Rf 0.15 and 0.70 were absent when compared to the pattern obtained at 5 days. Four polyphenol oxidase bands with Rf 0.06, 0.65, 0.74 and 0.79 were observed in 5 day old shoots. On 7th day, the band with Rf 0.65 disappeared with simultaneous decrease in the intensity of previously existing bands. Although, the isoenzymic pattern for polyphenol oxidase was similar in 5 and 7 day whole plants, the band with Rf 0. 70 was notably absent in 7 day whole plants. The isoenzymic pattern of catalase in roots of 5 and 7 day old seedlings was almost similar except that an additional band with Rf 0.80 appeared in roots of 7 day old plants which was earlier absent at 5 days. Similarly, the patterns in shoots of 5 and 7 day seedlings was almost identical except that the isoenzyme with Rf 0.80 appeared in shoots at 7 days. In whole plants of 5 and 7 day old seedlings, the similar situation existed. The intensity of the band with Rf 0. 78 was relatively less at 7 days than at 5 days. The results presented here indicate that germination brings about an increase in the number of isoenzymic components of the three enzymes and in general, the isoenzymic fractions were more clear and better developed at 72 hours of germination. Different tissues and organs of the same plant possess specific isoenzymic pattern of these oxidative enzymes at a particular stage of growth. The increase in multimolecular forms of these oxidative enzymes during germination may be because of de novo protein synthesis as a result of enhanced metabolism. The appearance or disappearance of a particular isoenzymic component at a particular stage of growth and development of a plant may be explained on the basis of a switching on - off mechanism of the protein synthesizing machinery of a cell depending upon the metabolic requirements of the plant at that particular stage of growth. At the same time, since it is well known that the different isoenzymic fractions of these enzymes have specific substrate requirements and catalyse specific oxidation reactions (MARCU et al. 1969), this general phenomenon can also go a long way in explaining the organ specificity of these enzymes. These results show a general resemblance to those of earlier workers (MACKO et al. 1967; UPADHYA and YEE 1968; YAASKA and YAASKA 1968; MARCU et al. 1969 and HAGIMA and LoANA 1970) who reported that the activity of oxidative enzymes is generally low in dormant seeds and the process of germination brings about both qualitative and quantitative increase in these oxidative enzymes.
Isoenzyme Pattern of Peroxidase etc.
237
Acknowledgement The authors are thankful to Dr. B. M. LAL, Professor of Biochemistry, for providing the facilities and to SH. D. R. VASUDEVA, Economic Botanist (Wheat}, for providing the materials.
References BHATIA, C. R., and NELSON, J.P., Isoenzyme changes accompanying germination of wheat seeds. Biochem. Genet. 3, 207-214 (1968). CouLsoN, C. B., and SIM, A. K., Wheat proteins. II. Changes in the protein composition of Triticum vulgare during the life cycle of plant. J. Sci. Fd. Agri. 16, 499-508 (1965). DAVIS, B. J., Disc electrophoresis. II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121, 404-427 (1964). FuRNESS, F. N., Multiple molecular forms of enzymes. Ann. N. Y. Acad. Sci. 94, 655-666 (1961). HAGIMA, C., and LoAN A, P., Peroxidase in maize during the early developmental stages. Stud. Cereet. Biochem. 13, 47-53 (1970). HoNOLD, G. R., FARKAS, G. L., and STAHMANN, A., The oxidation reduction enzymes of wheat. 1. Qualitative investigation of the dehydrogenases. Cereal chem. 43, 517-529 (1966). LOWRY, 0. H., RosEBROUGH, N.J., FARR, A. L., and RANDALL, R. J., Protein measurement with the Folin-phenol reagent. J. Bioi. Chern. 193, 265-275 (1951). MACKO, 0. H., HoNOLD, G. R., and STAHMANN, M. A., Soluble proteins and multiple enzyme forms in early growth of wheat. Phytochem. 6, 465-471 (1967). MARCU, Z., BRAD, 1., and NwuLEscu, S.M. E., Differences in peroxidase isoenzymes pattern as a function of the structural peculiarities of the hydrogen donor. Stud. Cereet. Biochem. 12, 265-272 (1969). NAINAWATEE, H. S., MEHTA, S. L., and DAs, N. B., Electrophoretic changes in wheat proteins during germination. Biochem. Physiol. Pflanzen (BPP) 165, 93-99 (1974). ScANDALIOs, J. G., Genetic control of multiple molecular forms of enzymes in plants. A. Rev. Biochem. Genet. 3, 37 -79 (1969). UPADHYA, M.D., and YEE, L., Isoenzyme polymorphism in flowering plants. VII. Isoenzyme variations in tissues of barley seedlings. Phytochem. 7, 937-943 (1968). VANLEAR, D. H., and SMITH, W. H., Soluble proteins and enzyme pattern in shoots of slashpine under different nutritional regimes. Phytochem. 9, 1929-1932 (1970). YAASKA, V., and YAASKA, N., Phosphorylase and peroxidase isoenzymes in wheat seedlings. Izv. Akad. Nauk. Est. USSR. Ser. Bioi. 17, 164-169 (1968). Received: March 4, 1974. Authors' address: Dr. RANDHIR SINGH and DHARAM SINGH, Department of Chemistry and Biochemistry, Haryana Agricultural University, Hissar, Haryana (India).