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EPR Studies on Coconut Milk: Effect of Ethylendiamine Tetra-Acetic Acid and some Growth Regulators on Manganese Content
Biochem. Physiol. Pflanzen (BPP), Bd. 165, S. 536-540 (1974)
Botany Department, Gujarat University, Ahmedabad, India
EPR Studies on Coconut Milk: Effect of Ethylendiamine Tetra-Acetic Acid and some Growth Regulators on Manganese Content By J. J. CHINOY, Y. D. SINGH and M. M. LALORAYA With 2 figures (Received January 8, 1974)
Summary EPR spectra of coconut milk and developing kernel have shown that whilll the "milk" shows the characteristic six peaked, symmetrical signal of manganese (Mn++), the semisolid and the solid kernel (endosperm) exhibit a decrease in signal height and a change in the symmetry of the signal with development. High concentrations of EDTA (10~2 and 10~4M) when added to coconut milk inhibit the Mn++ signal while lower concentration, particularly 10~6M, stimulates signal height. Similar stimulation was observed at 10~2M ascorbic acid. The significance of these effects is discussed.
Introduction
Coconut milk has araised the interest of biologists because it exhibits some of the properties of the living cell and also because it is known to contain a number of growth regulating substances (SHANTZ and STEWARD 1964). The free floating nuclei have been used in experiments to demonstrate auxin induced nuclear RNA synthesis (ROYCHOUDHURY and SEN 1964). Considerable significance has been ascribed in recent years to the paramagnetic behaviour of actively growing plant and animal tissues as well as of other substances of biological interest (BLOIS et al. 1961; COMMONER et al. 1957). An examination of coconut milk in EPR spectrometer gave a dominant signal which resembled that of manganese (Mn++). This signal was equivalent to 10~5M concentration of Manganese chloride (fig. 1a and b). Since no organic free radical signals were detected an attempt was made to chelate the manganese to unmask any of the overlapping organic free radicals. High concentrations (10- 2 to 10-4 M) of ethylene diamine tetracetic acid (EDTA) chelated most of the manganese and reduced signal height but at 10-6 M concentration a stimulation in the height of the signal was observed. As EDTA at this concentration is known to promote growth (BENNET-CLARK 1956; HEATH and CLARK 1956), it was considered of interest to compare the effect of EDTA on manganese with other growth regulators viz. indole-3-acetic acid (IAA), gibberellic acid (GA) and ascorbic acid (AA).
537
J. J. CHINOY, Y. D. SINGH and M. M. LALORAYA
Material and Methods
/
/
Large bunches of green coconut having about 8-10 coconuts were selected to ensure homogeneity of the material and coconuts of about same size were picked up for experiments. "Milk" obtained from freshly cut coconuts was transferred to aqueous cell (E-248) accessory and the EPR spectrum was recorded by a Varian E-4 EPR-spectrometer. Requisite quantities of EDTA, IAAGA and AA were added to the coconut "milk" from their stock solutions, - 10-1M solution, to obtain concentrations ranging from 10-2M to 10-10 M. The EPR recordings were started 5 minutes after mixing the solutions in the aqueous cell accessory at room temperature (25° ± 2°C). Taking the signal height of the original coconut milk as 100 percent, reduction or stimulation in para-
b
Fig. 1. Pattern of EPR spectra in (a) 10-5M MnCl 2; (b) Coconut milk; (c) Coconut kernel - 5 mm thick; (d) Coconut kernel - 10 mm thick; recorded at 25°C ± 2°C; magnetic fields at 3400 Gauss and scanning field of 1000 Gauss.
538
EPR Studies on Coconut Milk
magnetism caused by the addition of various concentrations of EDTA or growth regulators was worked out. These experiments were repeated a number of times with practically the same results. Results of a typical experiment are presented in this paper.
Results
Fig. 1 gives a comparison between the EPR spectrum of coconut milk and that of manganese chloride. In all details viz. in the six characteristic peaks, in signal splitting and in the width of the signal the coconut milk gives a signal which is identical to that of Mn++. Thc results with EDTA (fig. 2a and b) show that while EDTA inhibits the signal height by 100 % and 80 %, respectively at concentrations of 10- 2M and 10- 4 M; at 10-6 M EDTA concentration 20 % stimulation of signal height is recorded. No broadening of the signals could be observed. Lower concentrations viz. lO- sM and 10- 1oM EDTA cause less marked effects; only 8 % stimulation is obtained at lO- sM EDTA, while the effect of 10-1oM was almost insignificant. EDTA added to manganese chloride solution inhibits the signal at all concentrations; the inhibition obtained at 10-6 to lO- loM EDTA was found to be between 10-13% while 10- 2 and 10- 4 EDTA inhibited it by 100% and 90 % respectively. Thus the effect of EDTA on Mn++ signals of coconut milk differs significantly from its effect upon that of MnCI2 • Fig. 2 c shows the effect of varying concentrations of ascorbic acid. It will be observed that at 10-2M AA a stimulation similar to that obtained at 10-6M EDTA is obtained. Lower concentrations viz. 10-4 M to 1O-10M have no significant effect on the signal height. Addition of other growth regulators like IAA and GA has no effect on Mn++ signal of the coconut milk. In several of the plant materials examined earlier the EPR spectrum of Mn++ showed differences in the symmetry of spectrum and it was thought to exist in complexed form. As coconut milk signal was found to be perfectly symmetrical, almost exactly comparable with that of manganese chloride, the changes in the EPR spectrum with kernel formation was studied. Result-s are shown in fig. 1 c and d. It will be seen that the symmetry of the signal changes with the stage of kernel development and the height of the signal progressively declines without any broadening of the signals. EFFECT OF COCONUT MILK
EDTA
EFFECT
OF AA
COCONUT MILK
130
-=
~ IOO~rl··"'· C>
W :I:
~I02 ..
D",4~
IffiIlo· ..
• ,oe .. ~IO'° ..
Fig. 2. Effect of EDTA and ascorbic acid (AA) on the manganese signal height of coconut "milk" compared with that of MnCl z.
EPR Studies on Coconut Milk
539
Discussion
Manganese (Mn++) has been recorded in the EPR studies on various types of biological materials and it has been suggested that it may be present in complexed form (CHINOY et al. 1971; NICHOLAS et al. 1962; RANDOLPH and HABER 1961; and SHIELDS et al. 1956). The manganese signal peaks overlap in most cases with some paramagnetic species present in these materials resulting in the changed symmetry of the signal. The observation that the coconut 'milk' signal is perfectly symmetrical while that of the jelly and kernel shows change in the symmetry, tends to suggest that the assymetry is brought about by some 'Structural' signal rather than due to any overlapping cytoplasmic signal. The enhancement of signal height of Mn++ with EDTA (10- 6 M) and ascorbic acid (10-2M) araise some interesting questions on the mechanism of action of two substances. EDTA (BENNET-CLARK 1956; HEATH and CLARK 1956) as well as ascorbic acid are known to cause growth effects similar to auxin (CHINOY et al. 1957; TON ZIG and MARRE 1961) and it has been suggested that the action of the former may be related to its chelating properties of essential metals notably iron, for which it has great affinity (BURSTROM 1963). However, it has been shown that at least in roots, the effect of auxin and EDTA are different (BURSTROM 1961, 1963) and at times quite opposite. EDTA is thought to act by chelating the Fe++ and thus inhibiting the meristematic activity. Chelates are known to possess differential binding affinities towards different metals and it has been suggested that the removal of an essential metal from the plant by an added chelate may be counterbalanced by the releasing action of the other metal (BROWN et al. 1960; BURSTROl\1 1963). The increased height of the manganese signal at 10-6M EDTA may thus be due to such differential actions of Fe++ and Mn++ in coconut milk system. Ascorbic acid on the other hand is a very powerful reducing agent and is able to reduce some of the existing manganese (Mn++++) present in the coconut milk to Mn++. The two substances thus may have different mode of action. The lad thaI widely different concentrations of the two substances bring about the same effect also suggests their different mode of action. Manganese occupies an unique position in growth reactions notably because of its requirement in IAA oxidase reaction (MAHADEVAN 1964) and its ability to substitute tor a wide variety of transition elements in biochemical reactions essential for growth. References BENNET-CLARK, T. A., Salt accumulation and mode of action of auxin. In: The chemistry and mode of action of plant growth substances. Eds. WAIN, R. L., and WIGHTMAN, F., pp. 284-291; Butterworth, London 1956. BLOIS, M. S., Jr., BROWN, H. W., LEMMON, R. M., LINDBLOM, R. 0., and WEISBLUTH, M. (Eds.): Free radicals in biological systems. Academic Press, New York 1961. BROWN, J. C., TIFFIN, L. 0., and HOLMES, R. S., Competition between chelating agents and roots as factors affecting absorption of iron and other ions by plant species. Plant Physiol. 31i, 878886 (1960).
540
J. J. CHINOY, Y. D. SINGH and M. M. LALORAYA, EPR Studies on Coconut Milk
BUSTROM, H., Growth action of EDTA in light and darkness. Physiol. Plant. 14, 354-377 (1961). - Growth regulation by metals and chelates. In: Advances in Botanical Research, Ed. PRESTON, R. D., pp. 73-100. Academic Press, New York 1963. CHINOY, J. J., GROVER, R., and SIROHI, G. S., A study of the interaction of ascorbic acid and indole3-acetic acid in the growth of Avena coleoptile sections. Physiol. Plant. 10, 92 -99 (1957). - GURUMURTI, K., SINGH, Y. D., DAVE, I. C., and SAXENA, O. P., Paramagnetic behaviour of the differentiating reproductive apex of wheat. Biophysik 7, 157 -162 (1971). COMMONER, B., HEISE, J. J., LIPPINCOTT, B. B., NORBERG, R. E., PASSONEAU, J. V., and TOWNSEND, J., Biological activity of free radicals. Science 126, 57 -63 (1957). HEATH, O. V. S., and CLARK, J. E., Chelating agents as plant growth substances. Nature (Lond.) 177,1118-1121 (1956). MAHADEVAN, S., Enzymes involved in the synthesis and breakdown of indole acetic acid. In: Modern Methods of Plant Analysis, Vol. VII. Eds.: LINSKENS, M. F., SANWAL, B. D., TRACEY, M. V., pp. 238-258. Springer Verlag, Berlin 1964. NICHOLAS, D. J. D., WILSON, P. W., HEINEN, W., PALMER, G., and BEINERT, H., Use of electron paramagnetic resonance spectroscopy in investigation of functional metal components in microorganisms. Nature (Lond.) 196, 433-436 (1962). RANDOLPH, M. L., and HABER, A. H., Production and decay of free radicals induced by X-irradiation of dry lettuce seeds. In: Effects of ionizing radiations on seeds. IAEA, Vienna, 57 -65 (1961). ROYCHOUDHURY, R., and SEN, S. P., Studies on the mechanism of auxin action: Auxin regulation of nucleic acid metabolism in pea internodes and coconut milk nuclei. Physiol. Plant. 17, 352362 (1964). SHANTZ, E. M., and STEWARD, F. C., Growth promoting substances from the environment of the embryo. II. The growth-stimulating complexes of coconut milk, Corn, and Aesculus. In:Regulateurs Naturels de la Croissance Vegetable. Ed. NITSCH, J. P., Centre National de la Recherche Scientifique, 59 -75. Paris 1964. SHIELDS, H., ARD, W. B., and GODRY, W., Microwave detection of metabolic ions and organic radicals in plant materials. Nature (Lond.) 177, 984-985 (1956). TONZIG, S., and MARRE, E., Ascorbic acid as a growth hormone in plant growth regulation. Proc. IV. Internat. Conf. Plant Growth Regulation. The Iowa State University Press, pp. 725-734, Iowa 1961. Authors' address: Dr. J. J. CHINOY, Dr. Y. D. SINGH and Dr. M. M. LALORAYA, Botany Department, Gujarat University, Ahmedabad-9 (India).
Verantwortlich fiir die Redaktion: Dr. B. Parthier, 401 Halle (Saale). Verlag: VEB Gustav Fischer Verlag, 69 Jena, ViIIengang 2, Telefon 24141,24142. Alleinige Anzeigenannahme: DEWAG-Werbung Leipzig, 701 Leipzig, BriihI34-40, Telefon 29740. Fiir Auslandsanzeigen: Interwerbung GmbH, DDR - 104 Berlin, Tucholskystralle 40, Postfach 230. Satz und Druck: Druckerei "Magnus Poser", 69 Jena. - VerOffentlicht unter der Lizenznummer 1067 a des Presseamtes beim Vorsitzenden des Ministerrates der Deutschen Demokratischen Republik. Aile Rechte beim Verlag. Nachdruck (auch auszugsweise) nur mit Genehmigung des Verlages und des Verlassers sowie mit Angabe der Quelle gestattet. Printed in the German Democratic Republic.
Botany Department, Gujarat University, Ahmedabad, India
EPR Studies on Coconut Milk: Effect of Ethylendiamine Tetra-Acetic Acid and some Growth Regulators on Manganese Content By J. J. CHINOY, Y. D. SINGH and M. M. LALORAYA With 2 figures (Received January 8, 1974)
Summary EPR spectra of coconut milk and developing kernel have shown that whilll the "milk" shows the characteristic six peaked, symmetrical signal of manganese (Mn++), the semisolid and the solid kernel (endosperm) exhibit a decrease in signal height and a change in the symmetry of the signal with development. High concentrations of EDTA (10~2 and 10~4M) when added to coconut milk inhibit the Mn++ signal while lower concentration, particularly 10~6M, stimulates signal height. Similar stimulation was observed at 10~2M ascorbic acid. The significance of these effects is discussed.
Introduction
Coconut milk has araised the interest of biologists because it exhibits some of the properties of the living cell and also because it is known to contain a number of growth regulating substances (SHANTZ and STEWARD 1964). The free floating nuclei have been used in experiments to demonstrate auxin induced nuclear RNA synthesis (ROYCHOUDHURY and SEN 1964). Considerable significance has been ascribed in recent years to the paramagnetic behaviour of actively growing plant and animal tissues as well as of other substances of biological interest (BLOIS et al. 1961; COMMONER et al. 1957). An examination of coconut milk in EPR spectrometer gave a dominant signal which resembled that of manganese (Mn++). This signal was equivalent to 10~5M concentration of Manganese chloride (fig. 1a and b). Since no organic free radical signals were detected an attempt was made to chelate the manganese to unmask any of the overlapping organic free radicals. High concentrations (10- 2 to 10-4 M) of ethylene diamine tetracetic acid (EDTA) chelated most of the manganese and reduced signal height but at 10-6 M concentration a stimulation in the height of the signal was observed. As EDTA at this concentration is known to promote growth (BENNET-CLARK 1956; HEATH and CLARK 1956), it was considered of interest to compare the effect of EDTA on manganese with other growth regulators viz. indole-3-acetic acid (IAA), gibberellic acid (GA) and ascorbic acid (AA).
537
J. J. CHINOY, Y. D. SINGH and M. M. LALORAYA
Material and Methods
/
/
Large bunches of green coconut having about 8-10 coconuts were selected to ensure homogeneity of the material and coconuts of about same size were picked up for experiments. "Milk" obtained from freshly cut coconuts was transferred to aqueous cell (E-248) accessory and the EPR spectrum was recorded by a Varian E-4 EPR-spectrometer. Requisite quantities of EDTA, IAAGA and AA were added to the coconut "milk" from their stock solutions, - 10-1M solution, to obtain concentrations ranging from 10-2M to 10-10 M. The EPR recordings were started 5 minutes after mixing the solutions in the aqueous cell accessory at room temperature (25° ± 2°C). Taking the signal height of the original coconut milk as 100 percent, reduction or stimulation in para-
b
Fig. 1. Pattern of EPR spectra in (a) 10-5M MnCl 2; (b) Coconut milk; (c) Coconut kernel - 5 mm thick; (d) Coconut kernel - 10 mm thick; recorded at 25°C ± 2°C; magnetic fields at 3400 Gauss and scanning field of 1000 Gauss.
538
EPR Studies on Coconut Milk
magnetism caused by the addition of various concentrations of EDTA or growth regulators was worked out. These experiments were repeated a number of times with practically the same results. Results of a typical experiment are presented in this paper.
Results
Fig. 1 gives a comparison between the EPR spectrum of coconut milk and that of manganese chloride. In all details viz. in the six characteristic peaks, in signal splitting and in the width of the signal the coconut milk gives a signal which is identical to that of Mn++. Thc results with EDTA (fig. 2a and b) show that while EDTA inhibits the signal height by 100 % and 80 %, respectively at concentrations of 10- 2M and 10- 4 M; at 10-6 M EDTA concentration 20 % stimulation of signal height is recorded. No broadening of the signals could be observed. Lower concentrations viz. lO- sM and 10- 1oM EDTA cause less marked effects; only 8 % stimulation is obtained at lO- sM EDTA, while the effect of 10-1oM was almost insignificant. EDTA added to manganese chloride solution inhibits the signal at all concentrations; the inhibition obtained at 10-6 to lO- loM EDTA was found to be between 10-13% while 10- 2 and 10- 4 EDTA inhibited it by 100% and 90 % respectively. Thus the effect of EDTA on Mn++ signals of coconut milk differs significantly from its effect upon that of MnCI2 • Fig. 2 c shows the effect of varying concentrations of ascorbic acid. It will be observed that at 10-2M AA a stimulation similar to that obtained at 10-6M EDTA is obtained. Lower concentrations viz. 10-4 M to 1O-10M have no significant effect on the signal height. Addition of other growth regulators like IAA and GA has no effect on Mn++ signal of the coconut milk. In several of the plant materials examined earlier the EPR spectrum of Mn++ showed differences in the symmetry of spectrum and it was thought to exist in complexed form. As coconut milk signal was found to be perfectly symmetrical, almost exactly comparable with that of manganese chloride, the changes in the EPR spectrum with kernel formation was studied. Result-s are shown in fig. 1 c and d. It will be seen that the symmetry of the signal changes with the stage of kernel development and the height of the signal progressively declines without any broadening of the signals. EFFECT OF COCONUT MILK
EDTA
EFFECT
OF AA
COCONUT MILK
130
-=
~ IOO~rl··"'· C>
W :I:
~I02 ..
D",4~
IffiIlo· ..
• ,oe .. ~IO'° ..
Fig. 2. Effect of EDTA and ascorbic acid (AA) on the manganese signal height of coconut "milk" compared with that of MnCl z.
EPR Studies on Coconut Milk
539
Discussion
Manganese (Mn++) has been recorded in the EPR studies on various types of biological materials and it has been suggested that it may be present in complexed form (CHINOY et al. 1971; NICHOLAS et al. 1962; RANDOLPH and HABER 1961; and SHIELDS et al. 1956). The manganese signal peaks overlap in most cases with some paramagnetic species present in these materials resulting in the changed symmetry of the signal. The observation that the coconut 'milk' signal is perfectly symmetrical while that of the jelly and kernel shows change in the symmetry, tends to suggest that the assymetry is brought about by some 'Structural' signal rather than due to any overlapping cytoplasmic signal. The enhancement of signal height of Mn++ with EDTA (10- 6 M) and ascorbic acid (10-2M) araise some interesting questions on the mechanism of action of two substances. EDTA (BENNET-CLARK 1956; HEATH and CLARK 1956) as well as ascorbic acid are known to cause growth effects similar to auxin (CHINOY et al. 1957; TON ZIG and MARRE 1961) and it has been suggested that the action of the former may be related to its chelating properties of essential metals notably iron, for which it has great affinity (BURSTROM 1963). However, it has been shown that at least in roots, the effect of auxin and EDTA are different (BURSTROM 1961, 1963) and at times quite opposite. EDTA is thought to act by chelating the Fe++ and thus inhibiting the meristematic activity. Chelates are known to possess differential binding affinities towards different metals and it has been suggested that the removal of an essential metal from the plant by an added chelate may be counterbalanced by the releasing action of the other metal (BROWN et al. 1960; BURSTROl\1 1963). The increased height of the manganese signal at 10-6M EDTA may thus be due to such differential actions of Fe++ and Mn++ in coconut milk system. Ascorbic acid on the other hand is a very powerful reducing agent and is able to reduce some of the existing manganese (Mn++++) present in the coconut milk to Mn++. The two substances thus may have different mode of action. The lad thaI widely different concentrations of the two substances bring about the same effect also suggests their different mode of action. Manganese occupies an unique position in growth reactions notably because of its requirement in IAA oxidase reaction (MAHADEVAN 1964) and its ability to substitute tor a wide variety of transition elements in biochemical reactions essential for growth. References BENNET-CLARK, T. A., Salt accumulation and mode of action of auxin. In: The chemistry and mode of action of plant growth substances. Eds. WAIN, R. L., and WIGHTMAN, F., pp. 284-291; Butterworth, London 1956. BLOIS, M. S., Jr., BROWN, H. W., LEMMON, R. M., LINDBLOM, R. 0., and WEISBLUTH, M. (Eds.): Free radicals in biological systems. Academic Press, New York 1961. BROWN, J. C., TIFFIN, L. 0., and HOLMES, R. S., Competition between chelating agents and roots as factors affecting absorption of iron and other ions by plant species. Plant Physiol. 31i, 878886 (1960).
540
J. J. CHINOY, Y. D. SINGH and M. M. LALORAYA, EPR Studies on Coconut Milk
BUSTROM, H., Growth action of EDTA in light and darkness. Physiol. Plant. 14, 354-377 (1961). - Growth regulation by metals and chelates. In: Advances in Botanical Research, Ed. PRESTON, R. D., pp. 73-100. Academic Press, New York 1963. CHINOY, J. J., GROVER, R., and SIROHI, G. S., A study of the interaction of ascorbic acid and indole3-acetic acid in the growth of Avena coleoptile sections. Physiol. Plant. 10, 92 -99 (1957). - GURUMURTI, K., SINGH, Y. D., DAVE, I. C., and SAXENA, O. P., Paramagnetic behaviour of the differentiating reproductive apex of wheat. Biophysik 7, 157 -162 (1971). COMMONER, B., HEISE, J. J., LIPPINCOTT, B. B., NORBERG, R. E., PASSONEAU, J. V., and TOWNSEND, J., Biological activity of free radicals. Science 126, 57 -63 (1957). HEATH, O. V. S., and CLARK, J. E., Chelating agents as plant growth substances. Nature (Lond.) 177,1118-1121 (1956). MAHADEVAN, S., Enzymes involved in the synthesis and breakdown of indole acetic acid. In: Modern Methods of Plant Analysis, Vol. VII. Eds.: LINSKENS, M. F., SANWAL, B. D., TRACEY, M. V., pp. 238-258. Springer Verlag, Berlin 1964. NICHOLAS, D. J. D., WILSON, P. W., HEINEN, W., PALMER, G., and BEINERT, H., Use of electron paramagnetic resonance spectroscopy in investigation of functional metal components in microorganisms. Nature (Lond.) 196, 433-436 (1962). RANDOLPH, M. L., and HABER, A. H., Production and decay of free radicals induced by X-irradiation of dry lettuce seeds. In: Effects of ionizing radiations on seeds. IAEA, Vienna, 57 -65 (1961). ROYCHOUDHURY, R., and SEN, S. P., Studies on the mechanism of auxin action: Auxin regulation of nucleic acid metabolism in pea internodes and coconut milk nuclei. Physiol. Plant. 17, 352362 (1964). SHANTZ, E. M., and STEWARD, F. C., Growth promoting substances from the environment of the embryo. II. The growth-stimulating complexes of coconut milk, Corn, and Aesculus. In:Regulateurs Naturels de la Croissance Vegetable. Ed. NITSCH, J. P., Centre National de la Recherche Scientifique, 59 -75. Paris 1964. SHIELDS, H., ARD, W. B., and GODRY, W., Microwave detection of metabolic ions and organic radicals in plant materials. Nature (Lond.) 177, 984-985 (1956). TONZIG, S., and MARRE, E., Ascorbic acid as a growth hormone in plant growth regulation. Proc. IV. Internat. Conf. Plant Growth Regulation. The Iowa State University Press, pp. 725-734, Iowa 1961. Authors' address: Dr. J. J. CHINOY, Dr. Y. D. SINGH and Dr. M. M. LALORAYA, Botany Department, Gujarat University, Ahmedabad-9 (India).
Verantwortlich fiir die Redaktion: Dr. B. Parthier, 401 Halle (Saale). Verlag: VEB Gustav Fischer Verlag, 69 Jena, ViIIengang 2, Telefon 24141,24142. Alleinige Anzeigenannahme: DEWAG-Werbung Leipzig, 701 Leipzig, BriihI34-40, Telefon 29740. Fiir Auslandsanzeigen: Interwerbung GmbH, DDR - 104 Berlin, Tucholskystralle 40, Postfach 230. Satz und Druck: Druckerei "Magnus Poser", 69 Jena. - VerOffentlicht unter der Lizenznummer 1067 a des Presseamtes beim Vorsitzenden des Ministerrates der Deutschen Demokratischen Republik. Aile Rechte beim Verlag. Nachdruck (auch auszugsweise) nur mit Genehmigung des Verlages und des Verlassers sowie mit Angabe der Quelle gestattet. Printed in the German Democratic Republic.