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Influence of Phenoxy Acetic Acids on the Amino Acid Composition of Germinating Seeds of Cyamopsis Tetragonoloba
Biochem. Physiol. Pflanzen (BPP), Bd. 162, S. 466-469 (1971) Department of Botany, Jodhpur University, Jodhpur ,India
Short Communication
Influence of Phenoxy Acetic Acids on the Amino Acid Composition of Germinating Seeds of Cyamopsis Tetragonoloba By U. N.
CHATTERJI
(Received March 30, 1971)
Summary Effects of 2,4-D and 2,4,5-T on the free and protein-bound amino acid composition in the seedlings from treated seeds of Cyamopsis tetragonoloba have been studied semi-quantitatively by thin layer chromatography. Sixteen amino acids could be detected in the free state in the seedlings; the number of amino acids detected in acid hydrolysate of protein was seventeen. In general 2,4-D caused increased accumulation of amino acids in the free state, but it caused depletion of amino acids as found in protein hydrolysate. The content of amino acids both in the free state and in the protein-bound state declined as a result of treatment of seeds with 2,4,5-T.
Introduction
Since the discovery of 2,4-dichlorophenoxyacetic acid (2,4-D) by HAMNER and TUKEY (1944), a large number of papers have appeared dealing with the physiological and biochemical effects of this weedicide on a number of plants. Recenttly WORT (1964) has reviewed the physiological and biochemical effects of 2,4-D and certain other weedicides on plants. The present investigation was undertaken to study the effect of 2,4-D and 2,4,5-T on the composition of free and protein-bound amino acids in germinating seeds of Cyamopsis tetragonoloba. Materials and Methods The seeds of Cyamopsis tetragonoloba were soaked in aqueous solutions of 25 ppm each 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) for six hours. The control seeds were soaked in distilled water for six hours. After six hours of soaking the seeds were washed and kept for germination in petridishes lined with two layers of filter papers moistened with distilled water. The petridishes were kept in an incubator in dark at a temperature of 30 ± 2°C. The amino acids were analysed after 120 hours in control seedlings and those of treated seeds by the method described by KATHJU and TEWARI (1969). The amino acids were extracted in 80 per cent ethanol. The extract was centrifuged and the supertant was made fat-free with the chloroform and was directly used for the analysis of free amino acids by thin layer chromatography. The protein was washed with ethanol and hydrolysed
467
Influence of Phenoxy Acetic Acids etc.
with hydrochloric acid at 110°C under 15 pound pressure for six hours. The hydrolysate was filtered and the clear solution obtained was analysed for the protein-bound amino acids. Phenol: water (3: 1) and n-butanol: acetic acid: water (4: 1 :1) were used as the solvents in the first and the second directions respectively. The plates were sprayed with 0.3 per cent ninhydrin in butanol having 3.0 ml glacial acetic acid. The amino acids were identified with the help of standards prepared from known amino acids.
Results and Discussion
The chromatogram revealed that the seedlings of Cyamopsis tetragonoloba from untreated seeds contained 16 free amino acids and 17 protein-bound amino acids. As would be evident from the Table I, alanine, glycine, hydroxyproline and threonine were present in larger quantities followed by phenylalanine, glutamici acid, aspartic acid, cystine, histidine, arginine, proline, tryptophan, leucine/isoleucine. Valine and aspergine were present only in traces. All the sixteen amino acids mentioned above were also present in the seedlings developed from the seeds pretreated with Table 1
Effect of 2,4-D and 2,4,5-T on the free and protein bound amino acids composition in seedlings of Cyamopsis tetragonoloba.
Amino acid
Distribution pattern of free and protein-bound amino acids Free amino acids
cystine arginine histidine aspartic acid glutamic acid hydroxyproline alanine glycine threonine valine aspergine proline phenylalanine tryptophan leucine/isoleucine lysine serine tyrosine
Bound amino acids
Control
2,4-D
++ ++ ++ +++ +++ 1-+++ ++++ ++++ ++++
++ ++ ++ +++ +++ +++ ++++ ++++ +++++ +++ ++ +++ +++ +++ +++
_L
--t-
++ +++ ++ ++
2,4,5-T
++++ ++ ++ +++ +++ ++++ ++++ +++ +++ +++ ++ ++ ++ +
- absent; + present (traces); ++ present (poor); (intense); +++++ present (very intense).
Control
2,4-D
2,4,5-T
+ ++ +++ ++ +++
+ + ++ +++ +++
+ +++ ++ +++
++ +++++ + +++ + ++ +
-L
++ ++ + + ++ +
+ +++ + + + ++ +
+++ ++ +++ ++
++ ++ ++ ++
++ ++ ++ ++
I
+++ present (moderate); ++++ present
468
U. N.
CHATTERJI
2,4-D. In the seedlings of the seeds preteated with 2,4,5-T aspergine could not be detected in the free state. The treatment of the seeds with 2,4-D caused an increase in the threonine, valine, aspergine, proline, tryptophan, leucine/isoleucine contents in the seedlings, while the contents of hydroxyproline decreased as a result of the treatment with 2,4-D. Treatment of seeds with 2,4,5-T caused increased accumulation of cystine and valine in the free state in the seedlings from treated seeds, while the concentrations of amino acids like glycine, threonine, phenylalanine and leucine/ isoleucine declined. Of the seventeen amino acids detected in proteins of the seedlings of Cyamopsis tetragonoloba from untreated seeds, glycine was present in the largest quantity. Histidine, glutamic acid, alanine, valine, leucine/isoleucine, serine, arginine, aspartic acid, proline, lysine and tyrosine were also present in quite appreciable quantities, while cystine, threonine, aspergine and phenylalanine could be detected in acid hydrolysate of proteins of Cyamopsis tetragonoloba seedlings from both treated and untreated seeds. Lysine, serine and threonine, which did not exist in the free state in the seedlings from untreated seeds were present in quite appreciable quantities in proteins in the bound form. The contents of arginine, histidine, alanine, glycine, valine, leucine/isoleucine and serine declined in the seedlings obtained from 2,4-D treated seeds, while amino acids like aspartic acid and threonine indicated a quantitative increase. The treatment of seeds with 2,4,5-T caused a complete disappearance of cystine from the protein hydrolysate of control seedlings and also in the seedlings of the 2,4-D treated seeds. The contents of some amino acids like arginine, alanine, glycine, valine, leucine/isoleucine and serine in acid hydrolysate of protein of seedlings obtained from 2,4,5-T treated seeds decreased. Threonine was present in a greater amount in the proteins of seedlings of 2,4,5-T treated seeds than that in those of the control seedlings. SELL et al. (1949) have observed an increased content of protein and also amino acids in beans as a result of 2,4-D treatment. In the present case also 2,4-D caused an increase in free amino acid content, while the contents of protein-bound amino acids declined. PAYNE et al. (1952) working on potato have observed a decrease in contents of eleven amino acids in the tubers, but there was an increase in the content of glutamic acid in the free state. But in the present case 2,4-D caused depletion in the amount of hydroxyproline only in the free state. It would seen that 2,4-D and 2,4,5-T did not influence the amount of glutamic acid in the free or protein-bound state. WELLER et. al. (1950) noted that treatment of bean plants with 2,4-D resulted in a considerable reduction of amino acids and proteins in the roots and leaves of this plant. It was suggested that the increase in stem proteins was the result of translocation of nitrogenous material to the stems from these two organs. In the present case 2,4-D caused increased accumulation of free amino acids. This discrepancy might possibly be traced to the difference in the stage of development of the plant material
Influence of Phenoxy Acetic Acids etc.
469
analysed; in the case of Cyamopsis tetragonolo ba seedlings were involved, while the values reported by WELLER et al. (1950) relate to those obtaining different plant organs. Acknowledgement The author is thankful to Dr. M. N. TEWARI and Dr. S. KATHJU for help and cooperation.
Literature 1. HAMNER, C. L., and TUKEY, H. B., Science 100, 164-155 (1944). 2. KATHJli, S., and TEWARI, M. N., In "Maximization of Agricultural Production" (P. K. Sen, ed.), pp. 57-61, Calcutta 1969. 3. P.\YNE, M. G., FULTS, J. L., and HAY, R. L., American Potato J. 32, 144-149 (1952). 4. SELL, H. M., LUECKE, R. W., TAYLOR, B. M., and HAMNER, C. L., Plant Physiol. 24, 295299 (1949). 5. WELLER, L. E., LUCKE, R. W., HAMNER, C. L., and SELL, H. M., Plant Physiol. 20, 289293 (1950). 6. WORT, D. J., In "Physiology and biochemistry of herbicides" (L. J. Audus, ed.), pp. 291-334, Academic Press, I,ondon 1964.
Author's address. Prof. Dr. U. N. CHATTERJI, Botany Department University of Jodhpur New Campus, Bhagat-Ki-Kothi, Jodhpur (India).
Short Communication
Influence of Phenoxy Acetic Acids on the Amino Acid Composition of Germinating Seeds of Cyamopsis Tetragonoloba By U. N.
CHATTERJI
(Received March 30, 1971)
Summary Effects of 2,4-D and 2,4,5-T on the free and protein-bound amino acid composition in the seedlings from treated seeds of Cyamopsis tetragonoloba have been studied semi-quantitatively by thin layer chromatography. Sixteen amino acids could be detected in the free state in the seedlings; the number of amino acids detected in acid hydrolysate of protein was seventeen. In general 2,4-D caused increased accumulation of amino acids in the free state, but it caused depletion of amino acids as found in protein hydrolysate. The content of amino acids both in the free state and in the protein-bound state declined as a result of treatment of seeds with 2,4,5-T.
Introduction
Since the discovery of 2,4-dichlorophenoxyacetic acid (2,4-D) by HAMNER and TUKEY (1944), a large number of papers have appeared dealing with the physiological and biochemical effects of this weedicide on a number of plants. Recenttly WORT (1964) has reviewed the physiological and biochemical effects of 2,4-D and certain other weedicides on plants. The present investigation was undertaken to study the effect of 2,4-D and 2,4,5-T on the composition of free and protein-bound amino acids in germinating seeds of Cyamopsis tetragonoloba. Materials and Methods The seeds of Cyamopsis tetragonoloba were soaked in aqueous solutions of 25 ppm each 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) for six hours. The control seeds were soaked in distilled water for six hours. After six hours of soaking the seeds were washed and kept for germination in petridishes lined with two layers of filter papers moistened with distilled water. The petridishes were kept in an incubator in dark at a temperature of 30 ± 2°C. The amino acids were analysed after 120 hours in control seedlings and those of treated seeds by the method described by KATHJU and TEWARI (1969). The amino acids were extracted in 80 per cent ethanol. The extract was centrifuged and the supertant was made fat-free with the chloroform and was directly used for the analysis of free amino acids by thin layer chromatography. The protein was washed with ethanol and hydrolysed
467
Influence of Phenoxy Acetic Acids etc.
with hydrochloric acid at 110°C under 15 pound pressure for six hours. The hydrolysate was filtered and the clear solution obtained was analysed for the protein-bound amino acids. Phenol: water (3: 1) and n-butanol: acetic acid: water (4: 1 :1) were used as the solvents in the first and the second directions respectively. The plates were sprayed with 0.3 per cent ninhydrin in butanol having 3.0 ml glacial acetic acid. The amino acids were identified with the help of standards prepared from known amino acids.
Results and Discussion
The chromatogram revealed that the seedlings of Cyamopsis tetragonoloba from untreated seeds contained 16 free amino acids and 17 protein-bound amino acids. As would be evident from the Table I, alanine, glycine, hydroxyproline and threonine were present in larger quantities followed by phenylalanine, glutamici acid, aspartic acid, cystine, histidine, arginine, proline, tryptophan, leucine/isoleucine. Valine and aspergine were present only in traces. All the sixteen amino acids mentioned above were also present in the seedlings developed from the seeds pretreated with Table 1
Effect of 2,4-D and 2,4,5-T on the free and protein bound amino acids composition in seedlings of Cyamopsis tetragonoloba.
Amino acid
Distribution pattern of free and protein-bound amino acids Free amino acids
cystine arginine histidine aspartic acid glutamic acid hydroxyproline alanine glycine threonine valine aspergine proline phenylalanine tryptophan leucine/isoleucine lysine serine tyrosine
Bound amino acids
Control
2,4-D
++ ++ ++ +++ +++ 1-+++ ++++ ++++ ++++
++ ++ ++ +++ +++ +++ ++++ ++++ +++++ +++ ++ +++ +++ +++ +++
_L
--t-
++ +++ ++ ++
2,4,5-T
++++ ++ ++ +++ +++ ++++ ++++ +++ +++ +++ ++ ++ ++ +
- absent; + present (traces); ++ present (poor); (intense); +++++ present (very intense).
Control
2,4-D
2,4,5-T
+ ++ +++ ++ +++
+ + ++ +++ +++
+ +++ ++ +++
++ +++++ + +++ + ++ +
-L
++ ++ + + ++ +
+ +++ + + + ++ +
+++ ++ +++ ++
++ ++ ++ ++
++ ++ ++ ++
I
+++ present (moderate); ++++ present
468
U. N.
CHATTERJI
2,4-D. In the seedlings of the seeds preteated with 2,4,5-T aspergine could not be detected in the free state. The treatment of the seeds with 2,4-D caused an increase in the threonine, valine, aspergine, proline, tryptophan, leucine/isoleucine contents in the seedlings, while the contents of hydroxyproline decreased as a result of the treatment with 2,4-D. Treatment of seeds with 2,4,5-T caused increased accumulation of cystine and valine in the free state in the seedlings from treated seeds, while the concentrations of amino acids like glycine, threonine, phenylalanine and leucine/ isoleucine declined. Of the seventeen amino acids detected in proteins of the seedlings of Cyamopsis tetragonoloba from untreated seeds, glycine was present in the largest quantity. Histidine, glutamic acid, alanine, valine, leucine/isoleucine, serine, arginine, aspartic acid, proline, lysine and tyrosine were also present in quite appreciable quantities, while cystine, threonine, aspergine and phenylalanine could be detected in acid hydrolysate of proteins of Cyamopsis tetragonoloba seedlings from both treated and untreated seeds. Lysine, serine and threonine, which did not exist in the free state in the seedlings from untreated seeds were present in quite appreciable quantities in proteins in the bound form. The contents of arginine, histidine, alanine, glycine, valine, leucine/isoleucine and serine declined in the seedlings obtained from 2,4-D treated seeds, while amino acids like aspartic acid and threonine indicated a quantitative increase. The treatment of seeds with 2,4,5-T caused a complete disappearance of cystine from the protein hydrolysate of control seedlings and also in the seedlings of the 2,4-D treated seeds. The contents of some amino acids like arginine, alanine, glycine, valine, leucine/isoleucine and serine in acid hydrolysate of protein of seedlings obtained from 2,4,5-T treated seeds decreased. Threonine was present in a greater amount in the proteins of seedlings of 2,4,5-T treated seeds than that in those of the control seedlings. SELL et al. (1949) have observed an increased content of protein and also amino acids in beans as a result of 2,4-D treatment. In the present case also 2,4-D caused an increase in free amino acid content, while the contents of protein-bound amino acids declined. PAYNE et al. (1952) working on potato have observed a decrease in contents of eleven amino acids in the tubers, but there was an increase in the content of glutamic acid in the free state. But in the present case 2,4-D caused depletion in the amount of hydroxyproline only in the free state. It would seen that 2,4-D and 2,4,5-T did not influence the amount of glutamic acid in the free or protein-bound state. WELLER et. al. (1950) noted that treatment of bean plants with 2,4-D resulted in a considerable reduction of amino acids and proteins in the roots and leaves of this plant. It was suggested that the increase in stem proteins was the result of translocation of nitrogenous material to the stems from these two organs. In the present case 2,4-D caused increased accumulation of free amino acids. This discrepancy might possibly be traced to the difference in the stage of development of the plant material
Influence of Phenoxy Acetic Acids etc.
469
analysed; in the case of Cyamopsis tetragonolo ba seedlings were involved, while the values reported by WELLER et al. (1950) relate to those obtaining different plant organs. Acknowledgement The author is thankful to Dr. M. N. TEWARI and Dr. S. KATHJU for help and cooperation.
Literature 1. HAMNER, C. L., and TUKEY, H. B., Science 100, 164-155 (1944). 2. KATHJli, S., and TEWARI, M. N., In "Maximization of Agricultural Production" (P. K. Sen, ed.), pp. 57-61, Calcutta 1969. 3. P.\YNE, M. G., FULTS, J. L., and HAY, R. L., American Potato J. 32, 144-149 (1952). 4. SELL, H. M., LUECKE, R. W., TAYLOR, B. M., and HAMNER, C. L., Plant Physiol. 24, 295299 (1949). 5. WELLER, L. E., LUCKE, R. W., HAMNER, C. L., and SELL, H. M., Plant Physiol. 20, 289293 (1950). 6. WORT, D. J., In "Physiology and biochemistry of herbicides" (L. J. Audus, ed.), pp. 291-334, Academic Press, I,ondon 1964.
Author's address. Prof. Dr. U. N. CHATTERJI, Botany Department University of Jodhpur New Campus, Bhagat-Ki-Kothi, Jodhpur (India).