Plant & CellPhysiol. 21 (8): 1657-1660 (1980)
Short communication
The effect of acetylcholine on hypocotyl elongation in soybean Ilabanta Mukherjee!
(Received January 11, 1980)
Acetylcholine (Ach) and gibberellic acid (GA 3) treatment of germinating seeds stimulated hypocotyl elongation in the soybean cultivar Otootan. Ethrel which evolves ethylene in plant tissues, markedly inhibited hypocotyl elongation. Ethrel also inhibited the Ach- and GA3-induced stimulation of hypocotyl elongation. The effects of Ach and GA3 were not additive. Key words: Acetylcholine - Ethrel- Gibberellic acid - Hypocotyl elongationSoybean.
The field emergence of soybeans is seriously affected by hypocotyl elongation (2, 7), and temperature in the germinating environment has been demonstrated to be an important factor in their hypocotyl elongation (2, 6-8). An anomalous temperature dependence of hypocotyl elongation in some cultivars of soybeans also has been demonstrated (6, 14). Recently, soil resistance also has been reported to affect hypocotyl elongation and the emergence of soybeans (12, 13). The role of plant growth regulators in hypocotyl elongation in soybeans has been studied, and ethylene has been reported to inhibit hypocotyl elongation and to mimic the temperature effect on hypocotyls. Auxins and kinetin affect the hypocotyls of soybeans in the same manner as ethylene (10,14).
Ach has been demonstrated to have regulatory properties in some phytochromemediated responses of plants (11). Ach has been shown to overcome the CCCinduced inhibition of growth in wheat seedlings (3). Thus the attempt has been made to observe the effects of Ach and other plant growth regulators on the germination and hypocotyl elongation of soybeans. Seeds of the soybean Glycine max (L.) Merr. cultivars Vicoya, Bossier, Disoy and Otootan were obtained from the USDA, Beltsville, Maryland. Seeds were surface-sterilized with 0.1 % HgCl 2 solution for 5 min then rinsed several times with sterile distilled water before subjecting them to different growth regulator treatments. Fifteen surface-sterilized seeds were placed on filter paper in petri-dishes, Abbreviations: Ach, acetylcholine; GA3, gibberellic acid; CCC, 2-chloroethyltrimethyl ammonium chloride; lAA, indole-3-acetic acid. 1 Present address: Department of Agronomy, College of Agriculture and Forestry, University of Liberia, Monrovia, Liberia. 1657
Downloaded from http://pcp.oxfordjournals.org/ at University of Michigan on September 5, 2015
Plant Physiology Section, Crops Research Institute, P.O. Box 3785, Kumasi, Ghana
1658
I. Mukherjee
Table 1 EffectofAch, GAs andEthrelongermination andhypocotyl elongation in Otootan soybeans a, b Experiment 1 Treatment
Water control Ethrel (120 mgfliter) GAs (10 mgjliter) Ach (10 mu) Ethrel + GAs Ethrel-l-Ach GAs+Ach
Average germination percentage
Average hypocotyl length (cm)
58.3 65.0 71. 7 80.0 80.0 73.3 68.3
3.95 1.98 8.30 6. 79 6.22 3.54 8. 10
LSD between growth regulator treatments
(0.01) 1.83
(0.05) 1.20
Experiment 2 Average Average germination hypocotyl percentage length (cm)
63.3 65.0 55.0 75.0 58.3 60.0 61.7
4.32 2.15 7.51 6.52 5.38 3.68 6. 72 (0.01) (0.05) 2. 19 1.56
a Surface-sterilized seeds were treated with distilled water, Ethrel (120 mgjliter), GAs (10 mgjliter) or Ach (10 mM) for 96 hr in petri-dishes. b During germination and seedling growth the temperature varied between 24 and 29°C.
Downloaded from http://pcp.oxfordjournals.org/ at University of Michigan on September 5, 2015
then 15 ml of distilled water or hormone solution was pipetted into the petri-dish. To minimise infection, sterile distilled water and sterile glassware were used in all the experiments, and all the operations were performed in a transfer chamber. During and after treatment, the experimental materials were maintained on laboratory benches under natural light conditions. The laboratory temperature varied between 24 and 29°C. After 4 days the germination count and the length of the hypocotyl were recorded. The experiment involving continuous treatment with different growth regulators, was designed statistically with three replicates per treatment and was repeated once within a month. Preliminary experiments involving germination and hypocotyl elongation revealed that as far as viability and hypocotyl growth were concerned, the cultivar Otootan was the best followed by Disoy > Vicoya > Bossier. Hence, subsequent experiments were performed with the cultivar Otootan. The effects of Ach, Ethrel, GAs and their combinations were examined in two trials each. The results of the two experiments are presented in Table 1. Similar effects were observed in the two experiments. Although the results show that there were differences in the percentage of germination due to the different hormone treatments, these differences were not statistically significant in any of the two experiments. In contrast, the differences in hypocotyl length due to different hormone treatments were highly significant in each experiments. The experimental results reported here clearly demonstrate that hypocotyl elongation in the Otootan soybean was inhibited markedly by Ethrel which evolves ethylene in plant tissues. These results agree with earlier findings that ethylene inhibits hypocotyl elongation in soybean (10, 14). The results also demonstrate that GAs alone promoted hypocotyl elongation significantly, and that the presence of Ethrel reduced the GAs-induced stimulation of hypocotyl growth. Conversely, GA3 not only overcame the Ethrel-induced inhibition of hypocotyl growth, but it enhanced it to a level significantly higher than that of the water control. Holm
Acetylcholine and hypocotyl elongation
1659
I wish to thank Mr.]. E. Blankson for his technical assistance and Dr. G. A. Paku for the statistical analysis. References ( 1) Burg, S. P. and E. A. Burg: The interaction between auxin and ethylene and its role in plant growth. Proc. Natl. Acad.Sci. 55: 262-269 (1966). ( 2) Burris,]. S. and K. H. Knittle: Partial reversal of temperature-dependent inhibition of soybean hypocotyl elongation by cotyledon excision. Crop Sci. 15: 461-462 (1975). ( 3) Dekhuihzen, H. M.: The effect of acetylcholine on growth and on growth inhibition by eee in wheat seedlings. Planta 111: 149-156 (1973). ( 4) Evans, M. L.: Promotion of cell elongation in Avena coleoptiles by acetylcholine. Plant Physiol. 50: 414-416 (1972). ( 5) Fuchs, Y. and M. Lieberman: Effects of kinetin, lAA and gibberellin on ethylene production and their interaction on growth of seedlings. ibid. 43: 2029-2036 (1968). ( 6) Gilman, D. F., W. R. Fehr and]. S. Burris: Temperature effects on hypocotyl elongation of soybeans. Crop Sci. 13: 246-249 (1973). ( 7) Grabe, D. F. and R. B. Metzer: Temperature-induced inhibition of soybean hypocotyl elongation and seedling emergence. ibid. 9: 331-333 (1969). ( 8) Hatfield,]. L. and D. E. Egli: Effect of temperature on the rate of soybean hypocotyl elongation and field emergence. ibid. 14: 423-426 (1974). ( .9) Holm, R. E. and F. B. Abeles: The role of ethylene in 2,4-D-induced growth inhibition. Planta 78: 293-304 (1968). (10) Holm, R. E. and]. L. Key: Hormonal regulation of cell elongation in hypocotyl of rootless soybean: an evaluation of the role of DNA synthesis. Plant Physiol. 44: 1295-1302 (1969).
Downloaded from http://pcp.oxfordjournals.org/ at University of Michigan on September 5, 2015
and Key (10) showed that GAs enhanced hypocotyl elongation in auxin-treated Hawkeye soybean by inhibiting endogenous as well as auxin-induced ethylene evolution. The results reported further demonstrate that Ach alone significantly stimulated hypocotyl elongation in the Otootan soybean, but Ethrel in combination with Ach also depressed the stimulatory effect of Ach. The degree of inhibition of Ach-induced stimulation by Ethrel is about the same as that for Ethrel treatment alone. Conversely, Ach treatment together with Ethrel caused almost the same degree of stimulation on Ethrel-inhibited growth as that of Ach treatment alone. This may mean that Ethrel and Ach affect elongation of the hypocotyl independent of each other. Although growth promoting effects of Ach on wheat seedlings and Avena coleoptiles (3, 4) have been reported, no report on stimulation of hypocotyl elongation in soybeans due to Ach treatment has yet appeared in the literature. Inhibitory effects of ethylene on different types of auxin and gibberellin-induced plant responses have been reported (1, 15). The present findings clearly show that ethylene inhibits both gibberellin- and Ach-induced stimulation of hypocotyl elongation in soybean. Stimulation of hypocotyl elongation and partial reversion of the 25°C symptoms in the Clark soybean due to GAs treatment have been reported by Samimy and Lamotte (14). However, the GAs stimulation of pea shoot elongation inhibited by IAA or ethylene was not found to be due to the effects of GAs on ethylene generation. In view of the present findings of the stimulation of hypocotyl elongation by Ach, it would be interesting to investigate whether this Ach action is due to its effect on ethylene generation in soybean seedlings.
1660
I. Mukherjee
(11) ] affe, M.].: Evidence for the regulation of phytochrome mediated processes in bean roots by the neurohumor, acetylcholine. ibid. 46: 768-777 (1970). (12) Knittle, K. H. and]. S. Burris: Soybean hypocotyl growth under field conditions. Crop Sci. 19: 37-41 (1979). (13) Knittle, K. H. and]. S. Burris: Effect of downward force on soybean hypocotyl growth. ibid. 19: 47-51 (1979). (14) Samimy, C. and C. E. La Motte: Anomalous temperature dependence of seedling development in some soybeans (Glycine max (L.) Merr.). Plant Phvsiol. 58: 786-789 (1976). (15) Scott, P. C. and A. C. Leopold: Opposing effects of gibberellin and ethylene. ibid. 42: 1021-1022 (1967). Downloaded from http://pcp.oxfordjournals.org/ at University of Michigan on September 5, 2015
Short communication
The effect of acetylcholine on hypocotyl elongation in soybean Ilabanta Mukherjee!
(Received January 11, 1980)
Acetylcholine (Ach) and gibberellic acid (GA 3) treatment of germinating seeds stimulated hypocotyl elongation in the soybean cultivar Otootan. Ethrel which evolves ethylene in plant tissues, markedly inhibited hypocotyl elongation. Ethrel also inhibited the Ach- and GA3-induced stimulation of hypocotyl elongation. The effects of Ach and GA3 were not additive. Key words: Acetylcholine - Ethrel- Gibberellic acid - Hypocotyl elongationSoybean.
The field emergence of soybeans is seriously affected by hypocotyl elongation (2, 7), and temperature in the germinating environment has been demonstrated to be an important factor in their hypocotyl elongation (2, 6-8). An anomalous temperature dependence of hypocotyl elongation in some cultivars of soybeans also has been demonstrated (6, 14). Recently, soil resistance also has been reported to affect hypocotyl elongation and the emergence of soybeans (12, 13). The role of plant growth regulators in hypocotyl elongation in soybeans has been studied, and ethylene has been reported to inhibit hypocotyl elongation and to mimic the temperature effect on hypocotyls. Auxins and kinetin affect the hypocotyls of soybeans in the same manner as ethylene (10,14).
Ach has been demonstrated to have regulatory properties in some phytochromemediated responses of plants (11). Ach has been shown to overcome the CCCinduced inhibition of growth in wheat seedlings (3). Thus the attempt has been made to observe the effects of Ach and other plant growth regulators on the germination and hypocotyl elongation of soybeans. Seeds of the soybean Glycine max (L.) Merr. cultivars Vicoya, Bossier, Disoy and Otootan were obtained from the USDA, Beltsville, Maryland. Seeds were surface-sterilized with 0.1 % HgCl 2 solution for 5 min then rinsed several times with sterile distilled water before subjecting them to different growth regulator treatments. Fifteen surface-sterilized seeds were placed on filter paper in petri-dishes, Abbreviations: Ach, acetylcholine; GA3, gibberellic acid; CCC, 2-chloroethyltrimethyl ammonium chloride; lAA, indole-3-acetic acid. 1 Present address: Department of Agronomy, College of Agriculture and Forestry, University of Liberia, Monrovia, Liberia. 1657
Downloaded from http://pcp.oxfordjournals.org/ at University of Michigan on September 5, 2015
Plant Physiology Section, Crops Research Institute, P.O. Box 3785, Kumasi, Ghana
1658
I. Mukherjee
Table 1 EffectofAch, GAs andEthrelongermination andhypocotyl elongation in Otootan soybeans a, b Experiment 1 Treatment
Water control Ethrel (120 mgfliter) GAs (10 mgjliter) Ach (10 mu) Ethrel + GAs Ethrel-l-Ach GAs+Ach
Average germination percentage
Average hypocotyl length (cm)
58.3 65.0 71. 7 80.0 80.0 73.3 68.3
3.95 1.98 8.30 6. 79 6.22 3.54 8. 10
LSD between growth regulator treatments
(0.01) 1.83
(0.05) 1.20
Experiment 2 Average Average germination hypocotyl percentage length (cm)
63.3 65.0 55.0 75.0 58.3 60.0 61.7
4.32 2.15 7.51 6.52 5.38 3.68 6. 72 (0.01) (0.05) 2. 19 1.56
a Surface-sterilized seeds were treated with distilled water, Ethrel (120 mgjliter), GAs (10 mgjliter) or Ach (10 mM) for 96 hr in petri-dishes. b During germination and seedling growth the temperature varied between 24 and 29°C.
Downloaded from http://pcp.oxfordjournals.org/ at University of Michigan on September 5, 2015
then 15 ml of distilled water or hormone solution was pipetted into the petri-dish. To minimise infection, sterile distilled water and sterile glassware were used in all the experiments, and all the operations were performed in a transfer chamber. During and after treatment, the experimental materials were maintained on laboratory benches under natural light conditions. The laboratory temperature varied between 24 and 29°C. After 4 days the germination count and the length of the hypocotyl were recorded. The experiment involving continuous treatment with different growth regulators, was designed statistically with three replicates per treatment and was repeated once within a month. Preliminary experiments involving germination and hypocotyl elongation revealed that as far as viability and hypocotyl growth were concerned, the cultivar Otootan was the best followed by Disoy > Vicoya > Bossier. Hence, subsequent experiments were performed with the cultivar Otootan. The effects of Ach, Ethrel, GAs and their combinations were examined in two trials each. The results of the two experiments are presented in Table 1. Similar effects were observed in the two experiments. Although the results show that there were differences in the percentage of germination due to the different hormone treatments, these differences were not statistically significant in any of the two experiments. In contrast, the differences in hypocotyl length due to different hormone treatments were highly significant in each experiments. The experimental results reported here clearly demonstrate that hypocotyl elongation in the Otootan soybean was inhibited markedly by Ethrel which evolves ethylene in plant tissues. These results agree with earlier findings that ethylene inhibits hypocotyl elongation in soybean (10, 14). The results also demonstrate that GAs alone promoted hypocotyl elongation significantly, and that the presence of Ethrel reduced the GAs-induced stimulation of hypocotyl growth. Conversely, GA3 not only overcame the Ethrel-induced inhibition of hypocotyl growth, but it enhanced it to a level significantly higher than that of the water control. Holm
Acetylcholine and hypocotyl elongation
1659
I wish to thank Mr.]. E. Blankson for his technical assistance and Dr. G. A. Paku for the statistical analysis. References ( 1) Burg, S. P. and E. A. Burg: The interaction between auxin and ethylene and its role in plant growth. Proc. Natl. Acad.Sci. 55: 262-269 (1966). ( 2) Burris,]. S. and K. H. Knittle: Partial reversal of temperature-dependent inhibition of soybean hypocotyl elongation by cotyledon excision. Crop Sci. 15: 461-462 (1975). ( 3) Dekhuihzen, H. M.: The effect of acetylcholine on growth and on growth inhibition by eee in wheat seedlings. Planta 111: 149-156 (1973). ( 4) Evans, M. L.: Promotion of cell elongation in Avena coleoptiles by acetylcholine. Plant Physiol. 50: 414-416 (1972). ( 5) Fuchs, Y. and M. Lieberman: Effects of kinetin, lAA and gibberellin on ethylene production and their interaction on growth of seedlings. ibid. 43: 2029-2036 (1968). ( 6) Gilman, D. F., W. R. Fehr and]. S. Burris: Temperature effects on hypocotyl elongation of soybeans. Crop Sci. 13: 246-249 (1973). ( 7) Grabe, D. F. and R. B. Metzer: Temperature-induced inhibition of soybean hypocotyl elongation and seedling emergence. ibid. 9: 331-333 (1969). ( 8) Hatfield,]. L. and D. E. Egli: Effect of temperature on the rate of soybean hypocotyl elongation and field emergence. ibid. 14: 423-426 (1974). ( .9) Holm, R. E. and F. B. Abeles: The role of ethylene in 2,4-D-induced growth inhibition. Planta 78: 293-304 (1968). (10) Holm, R. E. and]. L. Key: Hormonal regulation of cell elongation in hypocotyl of rootless soybean: an evaluation of the role of DNA synthesis. Plant Physiol. 44: 1295-1302 (1969).
Downloaded from http://pcp.oxfordjournals.org/ at University of Michigan on September 5, 2015
and Key (10) showed that GAs enhanced hypocotyl elongation in auxin-treated Hawkeye soybean by inhibiting endogenous as well as auxin-induced ethylene evolution. The results reported further demonstrate that Ach alone significantly stimulated hypocotyl elongation in the Otootan soybean, but Ethrel in combination with Ach also depressed the stimulatory effect of Ach. The degree of inhibition of Ach-induced stimulation by Ethrel is about the same as that for Ethrel treatment alone. Conversely, Ach treatment together with Ethrel caused almost the same degree of stimulation on Ethrel-inhibited growth as that of Ach treatment alone. This may mean that Ethrel and Ach affect elongation of the hypocotyl independent of each other. Although growth promoting effects of Ach on wheat seedlings and Avena coleoptiles (3, 4) have been reported, no report on stimulation of hypocotyl elongation in soybeans due to Ach treatment has yet appeared in the literature. Inhibitory effects of ethylene on different types of auxin and gibberellin-induced plant responses have been reported (1, 15). The present findings clearly show that ethylene inhibits both gibberellin- and Ach-induced stimulation of hypocotyl elongation in soybean. Stimulation of hypocotyl elongation and partial reversion of the 25°C symptoms in the Clark soybean due to GAs treatment have been reported by Samimy and Lamotte (14). However, the GAs stimulation of pea shoot elongation inhibited by IAA or ethylene was not found to be due to the effects of GAs on ethylene generation. In view of the present findings of the stimulation of hypocotyl elongation by Ach, it would be interesting to investigate whether this Ach action is due to its effect on ethylene generation in soybean seedlings.
1660
I. Mukherjee
(11) ] affe, M.].: Evidence for the regulation of phytochrome mediated processes in bean roots by the neurohumor, acetylcholine. ibid. 46: 768-777 (1970). (12) Knittle, K. H. and]. S. Burris: Soybean hypocotyl growth under field conditions. Crop Sci. 19: 37-41 (1979). (13) Knittle, K. H. and]. S. Burris: Effect of downward force on soybean hypocotyl growth. ibid. 19: 47-51 (1979). (14) Samimy, C. and C. E. La Motte: Anomalous temperature dependence of seedling development in some soybeans (Glycine max (L.) Merr.). Plant Phvsiol. 58: 786-789 (1976). (15) Scott, P. C. and A. C. Leopold: Opposing effects of gibberellin and ethylene. ibid. 42: 1021-1022 (1967). Downloaded from http://pcp.oxfordjournals.org/ at University of Michigan on September 5, 2015
