- Email: [email protected]
Effect of β-Indoleacetic Acid on the Level of 2-Chloroethyl Trimetyl-Ammonium Chloride in Epicotyls of Pea Seedlings1)
Biochem. Physiol. Pflanzen, Bd. 161, S. 178--182 (1970)
Kurze Mitteilung Institute of Biology, eollege of Agrieulture, Brno, Jihlava Department (CSSR)
Effect of p-Indoleacetic Acid on the Level of 2-Chloroethyl Trimetyl-Ammonium Chloride in Epicotyls of Pea Seedlings 1) By
JAN HRADILIK
and
JIRi SEBANEK
With 4 figures (Received January 1,1970)
Summary A question of eee content was solved in pea epicotyls treated with IAA in comparison with the control without exogenous IAA. In the epicotyls of variants treated with IAA a provable eee decrease could be observed. For colorimetrical eee assay a method was described utilizing a colour complex produced by dipicrylamine with eee on paper chromatograms.
A number of papers have dealt with the relation between 2-chloroethyl trimethylammonium chloride (eee) and auxin (both endogenous and exogenous). eee is capable to increase the level of endogenous auxin as well as that of other indole compounds (NORRIS 1966). It may be there either a destruction of auxin (HALEVY 1963) or a fall of auxin biosynthesis (REED et al. 1965). SEBANEK (1967) gave a morphogenetic evidence for the decrease of endogenous auxin level due to eee. Low eee concentrations are able to decrease directly also the inhibiting influence of {J-indoleacetic acid (IAA) - (SEBANEK 1968), and the positive reaction of IAA in the pea test has been decreased or quite disturbed by the effect of eee applied simultaneously (SEBANEK and HRADILIK 1969); thus eee acts antagonistically evidently not only with respect to the endogenous auxin, but also with respect to the exogenous one. The present work has been focused on the opposite relation - the effect of exogenous IAA on the eee level. Materials, methods, and results Epicotyls of one-week-old pea plants (Pisum sativum), variety Raman, grown in the incubator in the dark at 25 °e were cut tightly above the cotyledons and divided into two variants. In the first variant, the epicotyls were immersed with their basal parts in the distilled water for Hi hr and thereafter in the water solution of eee at 5000 ppm concentration for 5 hr. The epicotyls of the second variant were similarly immersed in the water solution of IAA at 100 ppm 1) Dedicated to the Honour of 85 th Birthday of Professor Dr. R. DOSTAL, Brno.
Effect of P-Indoleacetic Acid on the Level etc.
179
for 15 hr and thereafter in the eee solution at 5000 ppm for 15 hr. In both the variants the epicotyls were transferred from the eee solution to the distilled water where they were immersed with their bases for 24 hr. Then the samples were taken for eee assay (Fig. 1). 4 g of epicotyls - _ _-,~
~
2g
2g
t
t IAA t eee t
water
t eee t
time
o t
15
t
water
water
20
homogenate +10ml of methyl alcohol
homogenate +10ml of methyl alcohol
t
t
extraction
t
0.25 ml for each spot
I
44
t
extraction
t
0.25 ml for each spot
"
56
Fig. 1. The scheme of the procedure at the application of regulants and at eee extraction from pea epicotyls.
CCC assay 2 g of pea epicotyls (12 pieces) were homogenized in the piston homogenizer, the homogenate was quantitatively washed with methyl alcohol into a 10-ml vessel. After 12-hr extraction at o°e and after controlling the temperature methyl alcohol was added to complete the volume. 0.25 ml of the solution gained in this way wasfjlNltfitatively applied on the origin of Whatman No.1 chromatography paper. The analysis W"aif}*frlormed by descending chromatography with the mixture n-butyl alcohol: glacial acetida-d4: water (40: 12: 28). The chromatographic separation was replicated three times. AfttI!=fJikYperfect drying the chromatograms were detected with the following solution:
D.200
aooo~36~O~---4~O~O------~~5TO--------5~O~O WAVELENGTH, nm
Fig. 2. Absorption curve of dipicrylamine-eee complex. 12*
J. HRADILIK and J. SEBANEK
180
0.2 g of dipicrylamine (hexanitrodiphenylamine Mg [NCaH2) (N0 2)a)2)]2 is dissolved in 50 ml of acetone and redistilled water is added to the volume of 100 m!. The chromatogram was sprayed with this solution thoroughly on both sides. Immediately after the spraying, CCC gives with this reagent clearly redcoloured spots on a yellow background. For quantitative determination of CCC the yelloow background of the spots was gradually washed out with 1 % NH 4 0H solutin until it was nearly white. The red spots of CCC remained preserved. The chromatogram was dried in this form on a glass plate in a stream of air. On the dried chromatograms the individual CCC spots were outlined into the rectangles so that their area should be uniform for the whole series. After cutting out of the paper these rectangles with the spots were cut into small pieces and extracted with 10ml of methyl alcohol on a shaking apparatus for about 30 minutes. The solutions gained were measured on the spectrocolorimeter at 415 nm in 1-cm cuvettes (Fig. 3), or with a small amount of CCC in the sample in a 5-cm cuvette (Fig. 4). The calibration curve was constructed identically from the CCC standards which were chromatographically separated for each series of samples anew. It was demonstrated that IAA decreased the CCC content, as can be seen from Table 1. Table 1
The results from the determination of CCC content replicated three times in epicotyls of pea plants treated first with IAA and thereafter with CCC, and in those treated first with water and thereafter with CCC
>
Epicotyls incubated in solutions of
{tg of CCC in 0.05 g of fresh material
water CCC
115 114 122
•
IAA CCC
90 67
94
i
1.000
>, {COO
<:0800
...S2 ~a600 i::
~CMOO 0200
0200
aoOO~~----~------------~~ fa 30 50 70 90 HO f30 13 CCC
OOOOL...-,.--~-~-----~~
o
f 2
a
-I 5 6 ., 8 9 fO,n CCC
Fig. 3. Calibration curve for CCC (415 nm; 1-cm cuvette). Fig. 4. Calibration curve for CCC (415 nm; 5-cm cuvette).
Effect of fJ- Indoleacetic Acid on the Leval etc.
181
Discussion
Regarding the necessity to determine quantitatively the eee content in the present study, we were concerned with the question of assaying this regulant and none of the methods described by SKopiK and CERVINKA (1967), BAYZER (1967), JIRACEK (1968), HRADILiK (1969) was quite convenient for this purpose. Therefore we tried a new methodical process originating from the knowledge by AUGUSTINSSON and GRAHM (1953) that dipicrylamine gives a red spot with quarternal bases on the chromatogram. We found that the colour complex arose also from the eee reaction with dipicrylamine, that it was extractable with methyl alcohol, and that a yellow background of the mentioned red spots could be washed out with 1 % solution of NH 4 0H, the sensitibility of the method becoming thus increased. Absorption maximum of colour intensity due to the reaction of dipicrylamine with eee was at 415 nm (Fig. 2) and the values of extinction in dependence on the concentration are in accordance with Lambert-Beer's law. With the use of other cuvettes (5-cm cuvette) it is possible to modify the method for eee assay even with the amounts of 2-20 ftg of eee. Employing 1-cm cuvette it is possible to determine the eee content in this manner in samples with 10-150 ftg of eee (Fig. 3). The results achieved at determining the eee content in pea epicotyls give evidence for some antagonism between IAA and eee. It may be there either the decrease of eee content due to IAA directly in the plant tissue, or an inhibition of eee uptake due to IAA.
Literature AUGUSTINSSON, K. B., GRAHN, M., 1953. The separation of choline esters by paper chromatography. Acta Chern. Scand. 7, 906-912. BAYZER, H., 1967. Nachweis und quantitative Bestimmung von Chlorcholinchlorid inbiologischem Material. Monatsh. Chemie 98, 1826-1831. HALEVY, A. H., 1963. Interaction of growth retarding compounds and gibberellin on IAA oxidase and peroxidase of cucumber seedlings. Plant Physiol. 38, 731-737. HRADILiK, J., 1969. The CCC morphoregulant, its extraction, proof and determination. Acta Univ. Agric. Brno, D, 1, 85-91 (in czech.) .. JIRACEK, V., 1968. Determination of chlorocholinechloride (CCC) in plant material by means of paper chromatography. Rostlinna vyroba, 6, 597-602. (in czech.). NORRIS, R. F., 1966. Effect of(2-chloroethyl)trimethylammonium chloride on the level of endogenous indole compounds in wheat seedlings. Canad. J. Bot. 44, 675-684. REED, D. J., MOORE, T. C., ANDERSON, J. D., 1965. Plant growth retardant B-995: A possible mode of action. Science 148, 1469-1471. SEBANEK, J., 1967. Die Verliingerung des erst en Internodiums und die Aszidienbildung bei Leinkeimlingen als ein morphogenetischer Beweis des Antagonismus zwischen CCC und endogenem Auxin. Flora A, 108, 271-276.
182
J. HRADILIK and J. SEBANEK, Effect of p-Indoleacetic Acid on the Leval etc.
SEB1I.NEK, J., 1968. The antagonism of (2-chloroethyl) trimethylammoniumchloride and indole 3-acetic acid in epicotyl and axillary buds growth in the pea seedlings. BioI. Plant. 10, 449-454. HRADILlK, J., 1969. On the interaction of growth retardants with IAA and kinetin. BioI. Plant. 11 (3), 356-365. SKoPIK, P., CERVINKA, M., 1967. Study of the effect of eee (2-chloroethyl-trimethyl-ammonium chloride) by cereals. 1. Movement and transformation of eee after the application on leaves by wheat and barley. Rostlinna vyroba, 40, 547 -558 (in czech.). Author's address: Dr. JAN HRADILlK and Prof. Dr. In''d SEBANEK, Agricultural eollege, Jihlava, Tolsteho CSSR).
Kurze Mitteilung Institute of Biology, eollege of Agrieulture, Brno, Jihlava Department (CSSR)
Effect of p-Indoleacetic Acid on the Level of 2-Chloroethyl Trimetyl-Ammonium Chloride in Epicotyls of Pea Seedlings 1) By
JAN HRADILIK
and
JIRi SEBANEK
With 4 figures (Received January 1,1970)
Summary A question of eee content was solved in pea epicotyls treated with IAA in comparison with the control without exogenous IAA. In the epicotyls of variants treated with IAA a provable eee decrease could be observed. For colorimetrical eee assay a method was described utilizing a colour complex produced by dipicrylamine with eee on paper chromatograms.
A number of papers have dealt with the relation between 2-chloroethyl trimethylammonium chloride (eee) and auxin (both endogenous and exogenous). eee is capable to increase the level of endogenous auxin as well as that of other indole compounds (NORRIS 1966). It may be there either a destruction of auxin (HALEVY 1963) or a fall of auxin biosynthesis (REED et al. 1965). SEBANEK (1967) gave a morphogenetic evidence for the decrease of endogenous auxin level due to eee. Low eee concentrations are able to decrease directly also the inhibiting influence of {J-indoleacetic acid (IAA) - (SEBANEK 1968), and the positive reaction of IAA in the pea test has been decreased or quite disturbed by the effect of eee applied simultaneously (SEBANEK and HRADILIK 1969); thus eee acts antagonistically evidently not only with respect to the endogenous auxin, but also with respect to the exogenous one. The present work has been focused on the opposite relation - the effect of exogenous IAA on the eee level. Materials, methods, and results Epicotyls of one-week-old pea plants (Pisum sativum), variety Raman, grown in the incubator in the dark at 25 °e were cut tightly above the cotyledons and divided into two variants. In the first variant, the epicotyls were immersed with their basal parts in the distilled water for Hi hr and thereafter in the water solution of eee at 5000 ppm concentration for 5 hr. The epicotyls of the second variant were similarly immersed in the water solution of IAA at 100 ppm 1) Dedicated to the Honour of 85 th Birthday of Professor Dr. R. DOSTAL, Brno.
Effect of P-Indoleacetic Acid on the Level etc.
179
for 15 hr and thereafter in the eee solution at 5000 ppm for 15 hr. In both the variants the epicotyls were transferred from the eee solution to the distilled water where they were immersed with their bases for 24 hr. Then the samples were taken for eee assay (Fig. 1). 4 g of epicotyls - _ _-,~
~
2g
2g
t
t IAA t eee t
water
t eee t
time
o t
15
t
water
water
20
homogenate +10ml of methyl alcohol
homogenate +10ml of methyl alcohol
t
t
extraction
t
0.25 ml for each spot
I
44
t
extraction
t
0.25 ml for each spot
"
56
Fig. 1. The scheme of the procedure at the application of regulants and at eee extraction from pea epicotyls.
CCC assay 2 g of pea epicotyls (12 pieces) were homogenized in the piston homogenizer, the homogenate was quantitatively washed with methyl alcohol into a 10-ml vessel. After 12-hr extraction at o°e and after controlling the temperature methyl alcohol was added to complete the volume. 0.25 ml of the solution gained in this way wasfjlNltfitatively applied on the origin of Whatman No.1 chromatography paper. The analysis W"aif}*frlormed by descending chromatography with the mixture n-butyl alcohol: glacial acetida-d4: water (40: 12: 28). The chromatographic separation was replicated three times. AfttI!=fJikYperfect drying the chromatograms were detected with the following solution:
D.200
aooo~36~O~---4~O~O------~~5TO--------5~O~O WAVELENGTH, nm
Fig. 2. Absorption curve of dipicrylamine-eee complex. 12*
J. HRADILIK and J. SEBANEK
180
0.2 g of dipicrylamine (hexanitrodiphenylamine Mg [NCaH2) (N0 2)a)2)]2 is dissolved in 50 ml of acetone and redistilled water is added to the volume of 100 m!. The chromatogram was sprayed with this solution thoroughly on both sides. Immediately after the spraying, CCC gives with this reagent clearly redcoloured spots on a yellow background. For quantitative determination of CCC the yelloow background of the spots was gradually washed out with 1 % NH 4 0H solutin until it was nearly white. The red spots of CCC remained preserved. The chromatogram was dried in this form on a glass plate in a stream of air. On the dried chromatograms the individual CCC spots were outlined into the rectangles so that their area should be uniform for the whole series. After cutting out of the paper these rectangles with the spots were cut into small pieces and extracted with 10ml of methyl alcohol on a shaking apparatus for about 30 minutes. The solutions gained were measured on the spectrocolorimeter at 415 nm in 1-cm cuvettes (Fig. 3), or with a small amount of CCC in the sample in a 5-cm cuvette (Fig. 4). The calibration curve was constructed identically from the CCC standards which were chromatographically separated for each series of samples anew. It was demonstrated that IAA decreased the CCC content, as can be seen from Table 1. Table 1
The results from the determination of CCC content replicated three times in epicotyls of pea plants treated first with IAA and thereafter with CCC, and in those treated first with water and thereafter with CCC
>
Epicotyls incubated in solutions of
{tg of CCC in 0.05 g of fresh material
water CCC
115 114 122
•
IAA CCC
90 67
94
i
1.000
>, {COO
<:0800
...S2 ~a600 i::
~CMOO 0200
0200
aoOO~~----~------------~~ fa 30 50 70 90 HO f30 13 CCC
OOOOL...-,.--~-~-----~~
o
f 2
a
-I 5 6 ., 8 9 fO,n CCC
Fig. 3. Calibration curve for CCC (415 nm; 1-cm cuvette). Fig. 4. Calibration curve for CCC (415 nm; 5-cm cuvette).
Effect of fJ- Indoleacetic Acid on the Leval etc.
181
Discussion
Regarding the necessity to determine quantitatively the eee content in the present study, we were concerned with the question of assaying this regulant and none of the methods described by SKopiK and CERVINKA (1967), BAYZER (1967), JIRACEK (1968), HRADILiK (1969) was quite convenient for this purpose. Therefore we tried a new methodical process originating from the knowledge by AUGUSTINSSON and GRAHM (1953) that dipicrylamine gives a red spot with quarternal bases on the chromatogram. We found that the colour complex arose also from the eee reaction with dipicrylamine, that it was extractable with methyl alcohol, and that a yellow background of the mentioned red spots could be washed out with 1 % solution of NH 4 0H, the sensitibility of the method becoming thus increased. Absorption maximum of colour intensity due to the reaction of dipicrylamine with eee was at 415 nm (Fig. 2) and the values of extinction in dependence on the concentration are in accordance with Lambert-Beer's law. With the use of other cuvettes (5-cm cuvette) it is possible to modify the method for eee assay even with the amounts of 2-20 ftg of eee. Employing 1-cm cuvette it is possible to determine the eee content in this manner in samples with 10-150 ftg of eee (Fig. 3). The results achieved at determining the eee content in pea epicotyls give evidence for some antagonism between IAA and eee. It may be there either the decrease of eee content due to IAA directly in the plant tissue, or an inhibition of eee uptake due to IAA.
Literature AUGUSTINSSON, K. B., GRAHN, M., 1953. The separation of choline esters by paper chromatography. Acta Chern. Scand. 7, 906-912. BAYZER, H., 1967. Nachweis und quantitative Bestimmung von Chlorcholinchlorid inbiologischem Material. Monatsh. Chemie 98, 1826-1831. HALEVY, A. H., 1963. Interaction of growth retarding compounds and gibberellin on IAA oxidase and peroxidase of cucumber seedlings. Plant Physiol. 38, 731-737. HRADILiK, J., 1969. The CCC morphoregulant, its extraction, proof and determination. Acta Univ. Agric. Brno, D, 1, 85-91 (in czech.) .. JIRACEK, V., 1968. Determination of chlorocholinechloride (CCC) in plant material by means of paper chromatography. Rostlinna vyroba, 6, 597-602. (in czech.). NORRIS, R. F., 1966. Effect of(2-chloroethyl)trimethylammonium chloride on the level of endogenous indole compounds in wheat seedlings. Canad. J. Bot. 44, 675-684. REED, D. J., MOORE, T. C., ANDERSON, J. D., 1965. Plant growth retardant B-995: A possible mode of action. Science 148, 1469-1471. SEBANEK, J., 1967. Die Verliingerung des erst en Internodiums und die Aszidienbildung bei Leinkeimlingen als ein morphogenetischer Beweis des Antagonismus zwischen CCC und endogenem Auxin. Flora A, 108, 271-276.
182
J. HRADILIK and J. SEBANEK, Effect of p-Indoleacetic Acid on the Leval etc.
SEB1I.NEK, J., 1968. The antagonism of (2-chloroethyl) trimethylammoniumchloride and indole 3-acetic acid in epicotyl and axillary buds growth in the pea seedlings. BioI. Plant. 10, 449-454. HRADILlK, J., 1969. On the interaction of growth retardants with IAA and kinetin. BioI. Plant. 11 (3), 356-365. SKoPIK, P., CERVINKA, M., 1967. Study of the effect of eee (2-chloroethyl-trimethyl-ammonium chloride) by cereals. 1. Movement and transformation of eee after the application on leaves by wheat and barley. Rostlinna vyroba, 40, 547 -558 (in czech.). Author's address: Dr. JAN HRADILlK and Prof. Dr. In''d SEBANEK, Agricultural eollege, Jihlava, Tolsteho CSSR).