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Growth and Certain Metabolic Aspects in Relation to Sodium Diethyl Dithiocarbamate Effects in Phaseolus Seedlings
Biochem. Physiol. Pflanzen 171, S. 43-47 (1977)
Growth and Certain Metabolic Aspects in Relation to Sodium Diethyl Dithiocarbamate Effects in Phaseolus Seedlings D. BALASIMHA, M. N. TEWARI and G. RAM Plant Biochemistry Laboratory, Department of Botany. University of Jodhpur, Jodhpur, India Key Term Index: Sodium diethyl dithiocarbamate, herbicide, seedling growth, sulfhydryl, chlorophylls, peroxidase, aldolase; Phaseolus radiatus.
Summary The action of the herbicide, sodium diethyl dithiocarbamate on activities of peroxidase (EC 1.11.1.17) and aldolase (EC 4.1.2.7), chlorophyll and sulfhydryl content has been studied in the germinating seeds of Phaseolus radiatus L. The herbicide caused an inhibition of growth accompanied by an increase in peroxidase activity. The sulfhydryl content was decreased. An attempt have been made to correlate the growth and the redox-potential of the seedlings in relation to the herbicide. The chemical enhanced the chlorophyll content in the first formed leaves showing a lack of correlation between chlorophylls and growth parameters. The inhibition of aldolase activity by the chemical has been shown to be due to specific inhibition of protein synthesis.
Introduction
Among the various groups of substances reported which have herbicidal action, dithiocarbamates occupy an important place (MORELAND 1967; ASHTON and GRAFTS 1973). The common dithiocarbamates which are used as herbicides are sodium diethyl dithiocarbamate (SDEG), 2-chloroallyl diethyl dithiocarbamate (CDEG) and sodium methyl dithiocarbamate (Metham). CDEC is reported to chelate copper and possibly to inhibit tyrosinase and cytochrome oxidase (ANON 1970). Only a limited amount of work has been carried out on the metabolic pathways in dithiocarbamate treated plants. The present investigation was undertaken to study the effects of SDEG on growth and some aspects of metabolic changes during early growth of Phaseolus radiatus L. seedlings. Material and Methods Seeds of Phaseolus radiatus L. were imbibed in water (control) or SDEC (2000 mgjl) for a period of 12 h. After this period the seeds were washed in distilled water, transferred to sterilized petridishes lined with single layer of filter paper moistened with distilled water, and allowed to germinate in normal laboratory conditions at room temperature (25°-30°C). Growth measurements were performed at 1 day intervals. For the estimation of sulfhydryl group the tissues were extracted in 3 % metaphosphoric acid and estimated by nitroprusside reaction (GRUNERT and PHILLIPS 1951). Chlorophylls were extracted in 80% acetone and estimated according to the method of ARNON (1949). For peroxidase activity the tissues were extracted in 0.1 M sodium phosphate buffer, pH 6.0, and assayed with o-dianisidine (WORTHINGTON ENZYME MANUAL 1972). For aldolase tissues were extracted in 0.05 M tris-
44
D. BALASIMHA, M. N. TEWARI and C. RAM
HCl buffer and assayed according to SIBLEY and LEHNINGER (1949). Protein was determined by folin-phenol reagent (LOWRY et al. 1951). All the experiments were done in triplicate and repeated thrice. Results and Discussion
Sodium diethyl dithiocarbamate inhibited the growth of hypocotyl and radicle, but did not affect the epicotyl growth (Table 1): In contrast to growth the peroxidase activity increased significantly in both cotyledon and seedling axis due to SDEG treatment (Table 4). The dithiocarbamate herbicides have been reported to inhibit shoot growth of several weeds (DAWSON 1963). The studies on P. radiatus seedlings showed Table 1. Effect of sodium diethyl dithiocarbamate (SDEC) on hypocotyl (H), radicle (R) and epicotyl (E) length in mm Values are mean ± Standard deviation Treatment
2
3
4 Days
H
R
E
H
R
E
H
R
E
Control
11.8 ± 1.9
17.9 ± 5.1
1.5 ± 0.5
34.8 ± 5.2
39.1 ± 10.3
5.7 ±1.1
68.5 ± 11.1
53.9 ± 12.9
21.6 ± 2.5
SDEC
10.8 ± 1.4
12.3 ± 2.6
1.4 ± 0.6
17.5 ± 3.9
25.7 ± 5.3
20.3 ± 2.1
42.5 ± 12.1
48.0 ± 7.9
22.6 ± 4.8
that growth inhibition was accompanied by rise in the peroxidase activity. Studies on the action of SDEG in Trigonella foenum grawum L. seedlings have shown that the activity of polyphenol oxidase was inhibited, while peroxidase and indoleacetic acid (IAA) oxidase activities were unaffected (BALASIMHA and TEWARI 1976). These authors have shown that inhibition of polyphenol oxidase activity might possibly be due to chelation of SDEC to copper molecule of the enzyme. Peroxidase has been shown to increase dramatically in plant tissues and organs under conditions which impose restriction on growth (LAVEE and GALSTON 1968; SHANNON et al. 1971; GALSTON et al. 1968). This might be explained on the IAA oxidase activity of peroxidase thereby decreasing the auxin level in the tissues (ENDO 1968). Further PILET and ZRYD (1965) have shown that SH concentrations were highest in young tissues of Lens roots and that the SH concentration was parallel to the IAA gradient. In P. radiatus the SH content declined Table 2. Effect of SDEC on SH content in cotyledon and seedling axis Numbers are Ilg/g fresh weight Treatment
Tissue
Days 1
2
3
4
Control
Cotyledon Seedling axis
1.18 2.99
1.08 1.05
1.76 0.42
2.39 0.41
SDEC
Cotyledon Seedling axis
0.70 2.87
1.10 0.60
1.70 0.29
1.99 0.24
y d
t e t d
l
n iA) e n se on is he wn H ed
Sodium Diethyl Dithiocarbamate Effects in Phaseolus
45
Table 3. Effect of SDEC on chlorophyll content in first formed leaves on 4th day of growth Numbers are mg/g fresh weight Treatment
Chlorophyll a
Chlorophyll b
Chlorophyll a
Control SDEC
0.225 0.370
0.112 0.160
0.337 0.530
+b
as a result of SDEC treatment (Table 2). In seedling axis SH rapidly declined with the progress of growth in contrast to increase in peroxidase activity. These results are in agreement with the earlier observations in carrot tissues (PILET and DUBOIS 1958a, b). Thus peroxidase might possibly oxidize SH compounds, apart from being acting as IAA-oxidases. SDEC might then, regulate the redox potential of the juvenile tissue. BETZ (1963) has discussed in detail the gradient of reducing power along the axis of higher plants which correlated inversely with IAA-oxidase activity. Table 4. Effect of SDEC on peroxidase activity in cotyledon and seedling axis. Enzyme activity expressed as LI A/min/mg protein Treatment
Tissue
Days 1
2
3
4
Control
Cotyledon Seedling axis
1.03 2.16
4.03 14.85
5.71 14.02
8.53 12.44
SDEC
Cotyledon Seedling axis
1.28 4.96
7.36 17.55
14.62 18.89
8.64 14.82
The levels of chlorophyll pigments was increased by SDEC (Table 3). Several growth retarding chemicals have been reported to delay loss of chlorophyll from detached leaves (MISHRA and :MISRA 1968). SEN and SHARMA (1972) found a lack of correlation between cblorophylls and the growth parameters of Merremia aegiptica cotyledons. In the present investigation also no direct correlation between growth and chlorophylls could be obtained. The chlorophyll content increased as a result of SDEC treatment, while seedling growth was inhibited. Table 5. Effect of SDEC on aldolase activity Enzyme activity expressed as LI A/15 min/mg protein Treatment
Tissue
Days 1
2
3
4
Control
Cotyledon Seedling axis
0.121 0.818
0.042 0.500
0.145 0.745
0.246 1.374
SDEC
Cotyledon Seedling axis
0.023 0.723
0.018 0.288
0.283 0.450
0.075 1.209
46
D. BALASIMHA, M. N. TEWARI and C. RAM
Table 6. Effect of SDEO on aldolase protein Numbers are mg proteinjg fresh weight Treatment
Tissue
Days 1
2
3
4
Control
Cotyledon Seedling axis
20.0 60.0
19.9 12.5
20.6 7.2
18.3 3.5
SDEC
Cotyledon Seedling axis
17.1 15.3
14.6 7.0
15.3 5.5
12.4 2.8
Very little work has been done on the effect of growth regulators on the activity aldolase in germinating seeds. The activity was significantly inhibited by SDEC (Table 5) accompanied by decrease in supernatant protein content (Table 6). Thus, it appeared that SDEC specifically inhibited certain functional proteins. The inhibition of aldolase might possibly be as a result of inhibition of protein synthesis as is shown by a decrease in protein content of supernatants of the extracts used for aldolase assays. Acknowledgement The authors are thankful to Prof. Dr. H. C. ARYA for encouragement and facilities.
References ANON, Herbicide handbook of the Weed Science Society of America, 368 pp., W. F. Humphrey Press Inc., Geneva, N. Y. 1970. ARNON, D. 1., Copper enzymes in isolated chloroplasts, polyphenol oxidase in Beta vulgaris. Plant PhysioI. 24, 1-15 (1949). ASHTON, F. M., and CRAFTS, A. S., Mode of action of herbicides, pp.504, John Wiley and Sons . Inc., 1973. BALASIMHA, D., and TEWARI, M. N., Growth and activities of certain oxidases of Trigonella foenum graecum L. seedlings in relation to sodium diethyldithiocarbamate. Geobios, In press (1976). BETZ, A., Ascorbinsaure, NADH, cystein und glutathion hemmen den durch Peroxydase katalysierten oxydativen Abbau von p-Indolylessigsaure, Z. Bot. 51, 424-433 (1963). * DAWSON, J. H., Weeds 11, 60 (1963). ENDO, T., Indole acetate oxidase activity of horse radish peroxidase and other plant peroxidase isozymes. Plant and Cell PhysioI. 9, 333-341 (1968). GALSTON, A. W., LAVEE, S., and SIEGEL, B. Z., In: Biochemistry and Physiology of plant growth substances. (Ed. WIGHTMAN, F., and SETTERFIELD, G.), p. 455, Runge Press, Ottawa 1968. GRUNERT, R. R., and PHILLIPS, P. H., A modification of the nitroprusside method of analysis for glutathione. Arch. Biochem. Biophys. 30, 217-255 (1951). LAVEE, S., and GALSTON, A. W., Hormonal control of peroxidase activity in cultured Pelargonium pith. Am. J. Botany 05, 890-893 (1968). LOWRY, O. H., ROSEBOROUGH, N. J., F ARR, A. L., and RANDALL, R. J., Protein measurement with Folin phenol reagent. J. BioI. Chem. 193, 265-275 (1951). MISHRA, D., and MISRA, B., Effect of growth regulating chemicals on degradation of chlorophyll and starch in detached leaves of crop plants. Z. PflanzenphysioI. 1i8, 207-211 (1968). MORELAND, D. E., Mechanism of action of herbicides. Annu. Rev. Plant PhysioI. 18, 365-386 (1967).
Sodium Diethyl Dithiocarbamate Effects in Phaseolus
ty C it on by ys.
rey
ant
ons
num 76). aly-
dase wth
s for
nium
with and
-386
47
PILET, P. E., and DUBOIS, J., Composes sulfhydriles, auxines et cutivite auxineoxydasique de tissus cultives in vitro. PhysioI. veg. 6, 269-278 (1968 a). - - Variations du taux en composes sulfhydriles acidosolubles de tissus cultives in vitro. PhysioI. Plant 21,445-454 (1968b). - and ZRYD, J. P., Distribution des composes sulfhydriles ands les mcines. Ann. PhysioI. Veg. 7, 243-250 (1965). SEN, D. N., and SHARMA, K. D., Role of growth retardins on excised cotyledons of Merremia aegiptica (LINN.) and their metabolism. Biochem. PhysioI. Pflanzen 163, 229-232 (1972). SHANNON, L. M., URITANI, I., and IMASEKI, I., De novo synthesis of peroxidase isoenzymes in sweet potato slices. Plant PhysioI. 47, 493-498 (1971). SIBLEY, J. A., and LEHNINGER, A. L., Determination of aldolase in animal tissues. J. BioI. Chem. 177, 859-872 (1949). WORTHINGTON ENZYME MANUAL: Enzymes,enzyme reagents, related biochemiCals. Worthington Biochemical Corporation, Freehold, New Jersey, U.S.A. 1972. * Original not seen. Recevied August 9, 1976. Authors' address: D. BALASIMHA, M. N. T.EWARI and C. RAM, Plant Biochemistry Laboratory, Department of Botany, University of JodhTmr, Jodhpur - 342001, India.
Growth and Certain Metabolic Aspects in Relation to Sodium Diethyl Dithiocarbamate Effects in Phaseolus Seedlings D. BALASIMHA, M. N. TEWARI and G. RAM Plant Biochemistry Laboratory, Department of Botany. University of Jodhpur, Jodhpur, India Key Term Index: Sodium diethyl dithiocarbamate, herbicide, seedling growth, sulfhydryl, chlorophylls, peroxidase, aldolase; Phaseolus radiatus.
Summary The action of the herbicide, sodium diethyl dithiocarbamate on activities of peroxidase (EC 1.11.1.17) and aldolase (EC 4.1.2.7), chlorophyll and sulfhydryl content has been studied in the germinating seeds of Phaseolus radiatus L. The herbicide caused an inhibition of growth accompanied by an increase in peroxidase activity. The sulfhydryl content was decreased. An attempt have been made to correlate the growth and the redox-potential of the seedlings in relation to the herbicide. The chemical enhanced the chlorophyll content in the first formed leaves showing a lack of correlation between chlorophylls and growth parameters. The inhibition of aldolase activity by the chemical has been shown to be due to specific inhibition of protein synthesis.
Introduction
Among the various groups of substances reported which have herbicidal action, dithiocarbamates occupy an important place (MORELAND 1967; ASHTON and GRAFTS 1973). The common dithiocarbamates which are used as herbicides are sodium diethyl dithiocarbamate (SDEG), 2-chloroallyl diethyl dithiocarbamate (CDEG) and sodium methyl dithiocarbamate (Metham). CDEC is reported to chelate copper and possibly to inhibit tyrosinase and cytochrome oxidase (ANON 1970). Only a limited amount of work has been carried out on the metabolic pathways in dithiocarbamate treated plants. The present investigation was undertaken to study the effects of SDEG on growth and some aspects of metabolic changes during early growth of Phaseolus radiatus L. seedlings. Material and Methods Seeds of Phaseolus radiatus L. were imbibed in water (control) or SDEC (2000 mgjl) for a period of 12 h. After this period the seeds were washed in distilled water, transferred to sterilized petridishes lined with single layer of filter paper moistened with distilled water, and allowed to germinate in normal laboratory conditions at room temperature (25°-30°C). Growth measurements were performed at 1 day intervals. For the estimation of sulfhydryl group the tissues were extracted in 3 % metaphosphoric acid and estimated by nitroprusside reaction (GRUNERT and PHILLIPS 1951). Chlorophylls were extracted in 80% acetone and estimated according to the method of ARNON (1949). For peroxidase activity the tissues were extracted in 0.1 M sodium phosphate buffer, pH 6.0, and assayed with o-dianisidine (WORTHINGTON ENZYME MANUAL 1972). For aldolase tissues were extracted in 0.05 M tris-
44
D. BALASIMHA, M. N. TEWARI and C. RAM
HCl buffer and assayed according to SIBLEY and LEHNINGER (1949). Protein was determined by folin-phenol reagent (LOWRY et al. 1951). All the experiments were done in triplicate and repeated thrice. Results and Discussion
Sodium diethyl dithiocarbamate inhibited the growth of hypocotyl and radicle, but did not affect the epicotyl growth (Table 1): In contrast to growth the peroxidase activity increased significantly in both cotyledon and seedling axis due to SDEG treatment (Table 4). The dithiocarbamate herbicides have been reported to inhibit shoot growth of several weeds (DAWSON 1963). The studies on P. radiatus seedlings showed Table 1. Effect of sodium diethyl dithiocarbamate (SDEC) on hypocotyl (H), radicle (R) and epicotyl (E) length in mm Values are mean ± Standard deviation Treatment
2
3
4 Days
H
R
E
H
R
E
H
R
E
Control
11.8 ± 1.9
17.9 ± 5.1
1.5 ± 0.5
34.8 ± 5.2
39.1 ± 10.3
5.7 ±1.1
68.5 ± 11.1
53.9 ± 12.9
21.6 ± 2.5
SDEC
10.8 ± 1.4
12.3 ± 2.6
1.4 ± 0.6
17.5 ± 3.9
25.7 ± 5.3
20.3 ± 2.1
42.5 ± 12.1
48.0 ± 7.9
22.6 ± 4.8
that growth inhibition was accompanied by rise in the peroxidase activity. Studies on the action of SDEG in Trigonella foenum grawum L. seedlings have shown that the activity of polyphenol oxidase was inhibited, while peroxidase and indoleacetic acid (IAA) oxidase activities were unaffected (BALASIMHA and TEWARI 1976). These authors have shown that inhibition of polyphenol oxidase activity might possibly be due to chelation of SDEC to copper molecule of the enzyme. Peroxidase has been shown to increase dramatically in plant tissues and organs under conditions which impose restriction on growth (LAVEE and GALSTON 1968; SHANNON et al. 1971; GALSTON et al. 1968). This might be explained on the IAA oxidase activity of peroxidase thereby decreasing the auxin level in the tissues (ENDO 1968). Further PILET and ZRYD (1965) have shown that SH concentrations were highest in young tissues of Lens roots and that the SH concentration was parallel to the IAA gradient. In P. radiatus the SH content declined Table 2. Effect of SDEC on SH content in cotyledon and seedling axis Numbers are Ilg/g fresh weight Treatment
Tissue
Days 1
2
3
4
Control
Cotyledon Seedling axis
1.18 2.99
1.08 1.05
1.76 0.42
2.39 0.41
SDEC
Cotyledon Seedling axis
0.70 2.87
1.10 0.60
1.70 0.29
1.99 0.24
y d
t e t d
l
n iA) e n se on is he wn H ed
Sodium Diethyl Dithiocarbamate Effects in Phaseolus
45
Table 3. Effect of SDEC on chlorophyll content in first formed leaves on 4th day of growth Numbers are mg/g fresh weight Treatment
Chlorophyll a
Chlorophyll b
Chlorophyll a
Control SDEC
0.225 0.370
0.112 0.160
0.337 0.530
+b
as a result of SDEC treatment (Table 2). In seedling axis SH rapidly declined with the progress of growth in contrast to increase in peroxidase activity. These results are in agreement with the earlier observations in carrot tissues (PILET and DUBOIS 1958a, b). Thus peroxidase might possibly oxidize SH compounds, apart from being acting as IAA-oxidases. SDEC might then, regulate the redox potential of the juvenile tissue. BETZ (1963) has discussed in detail the gradient of reducing power along the axis of higher plants which correlated inversely with IAA-oxidase activity. Table 4. Effect of SDEC on peroxidase activity in cotyledon and seedling axis. Enzyme activity expressed as LI A/min/mg protein Treatment
Tissue
Days 1
2
3
4
Control
Cotyledon Seedling axis
1.03 2.16
4.03 14.85
5.71 14.02
8.53 12.44
SDEC
Cotyledon Seedling axis
1.28 4.96
7.36 17.55
14.62 18.89
8.64 14.82
The levels of chlorophyll pigments was increased by SDEC (Table 3). Several growth retarding chemicals have been reported to delay loss of chlorophyll from detached leaves (MISHRA and :MISRA 1968). SEN and SHARMA (1972) found a lack of correlation between cblorophylls and the growth parameters of Merremia aegiptica cotyledons. In the present investigation also no direct correlation between growth and chlorophylls could be obtained. The chlorophyll content increased as a result of SDEC treatment, while seedling growth was inhibited. Table 5. Effect of SDEC on aldolase activity Enzyme activity expressed as LI A/15 min/mg protein Treatment
Tissue
Days 1
2
3
4
Control
Cotyledon Seedling axis
0.121 0.818
0.042 0.500
0.145 0.745
0.246 1.374
SDEC
Cotyledon Seedling axis
0.023 0.723
0.018 0.288
0.283 0.450
0.075 1.209
46
D. BALASIMHA, M. N. TEWARI and C. RAM
Table 6. Effect of SDEO on aldolase protein Numbers are mg proteinjg fresh weight Treatment
Tissue
Days 1
2
3
4
Control
Cotyledon Seedling axis
20.0 60.0
19.9 12.5
20.6 7.2
18.3 3.5
SDEC
Cotyledon Seedling axis
17.1 15.3
14.6 7.0
15.3 5.5
12.4 2.8
Very little work has been done on the effect of growth regulators on the activity aldolase in germinating seeds. The activity was significantly inhibited by SDEC (Table 5) accompanied by decrease in supernatant protein content (Table 6). Thus, it appeared that SDEC specifically inhibited certain functional proteins. The inhibition of aldolase might possibly be as a result of inhibition of protein synthesis as is shown by a decrease in protein content of supernatants of the extracts used for aldolase assays. Acknowledgement The authors are thankful to Prof. Dr. H. C. ARYA for encouragement and facilities.
References ANON, Herbicide handbook of the Weed Science Society of America, 368 pp., W. F. Humphrey Press Inc., Geneva, N. Y. 1970. ARNON, D. 1., Copper enzymes in isolated chloroplasts, polyphenol oxidase in Beta vulgaris. Plant PhysioI. 24, 1-15 (1949). ASHTON, F. M., and CRAFTS, A. S., Mode of action of herbicides, pp.504, John Wiley and Sons . Inc., 1973. BALASIMHA, D., and TEWARI, M. N., Growth and activities of certain oxidases of Trigonella foenum graecum L. seedlings in relation to sodium diethyldithiocarbamate. Geobios, In press (1976). BETZ, A., Ascorbinsaure, NADH, cystein und glutathion hemmen den durch Peroxydase katalysierten oxydativen Abbau von p-Indolylessigsaure, Z. Bot. 51, 424-433 (1963). * DAWSON, J. H., Weeds 11, 60 (1963). ENDO, T., Indole acetate oxidase activity of horse radish peroxidase and other plant peroxidase isozymes. Plant and Cell PhysioI. 9, 333-341 (1968). GALSTON, A. W., LAVEE, S., and SIEGEL, B. Z., In: Biochemistry and Physiology of plant growth substances. (Ed. WIGHTMAN, F., and SETTERFIELD, G.), p. 455, Runge Press, Ottawa 1968. GRUNERT, R. R., and PHILLIPS, P. H., A modification of the nitroprusside method of analysis for glutathione. Arch. Biochem. Biophys. 30, 217-255 (1951). LAVEE, S., and GALSTON, A. W., Hormonal control of peroxidase activity in cultured Pelargonium pith. Am. J. Botany 05, 890-893 (1968). LOWRY, O. H., ROSEBOROUGH, N. J., F ARR, A. L., and RANDALL, R. J., Protein measurement with Folin phenol reagent. J. BioI. Chem. 193, 265-275 (1951). MISHRA, D., and MISRA, B., Effect of growth regulating chemicals on degradation of chlorophyll and starch in detached leaves of crop plants. Z. PflanzenphysioI. 1i8, 207-211 (1968). MORELAND, D. E., Mechanism of action of herbicides. Annu. Rev. Plant PhysioI. 18, 365-386 (1967).
Sodium Diethyl Dithiocarbamate Effects in Phaseolus
ty C it on by ys.
rey
ant
ons
num 76). aly-
dase wth
s for
nium
with and
-386
47
PILET, P. E., and DUBOIS, J., Composes sulfhydriles, auxines et cutivite auxineoxydasique de tissus cultives in vitro. PhysioI. veg. 6, 269-278 (1968 a). - - Variations du taux en composes sulfhydriles acidosolubles de tissus cultives in vitro. PhysioI. Plant 21,445-454 (1968b). - and ZRYD, J. P., Distribution des composes sulfhydriles ands les mcines. Ann. PhysioI. Veg. 7, 243-250 (1965). SEN, D. N., and SHARMA, K. D., Role of growth retardins on excised cotyledons of Merremia aegiptica (LINN.) and their metabolism. Biochem. PhysioI. Pflanzen 163, 229-232 (1972). SHANNON, L. M., URITANI, I., and IMASEKI, I., De novo synthesis of peroxidase isoenzymes in sweet potato slices. Plant PhysioI. 47, 493-498 (1971). SIBLEY, J. A., and LEHNINGER, A. L., Determination of aldolase in animal tissues. J. BioI. Chem. 177, 859-872 (1949). WORTHINGTON ENZYME MANUAL: Enzymes,enzyme reagents, related biochemiCals. Worthington Biochemical Corporation, Freehold, New Jersey, U.S.A. 1972. * Original not seen. Recevied August 9, 1976. Authors' address: D. BALASIMHA, M. N. T.EWARI and C. RAM, Plant Biochemistry Laboratory, Department of Botany, University of JodhTmr, Jodhpur - 342001, India.