- Email: [email protected]
Influence of gibberellic acid on the incorporation of 8-14C adenine into adenosine 3′,-5′-cyclic phosphate in barley aleurone layers
PRELIMINARY NOTES
511
BBA 21278
Influence of gibberellic acid on the incorporation of 8-14C adenine into adenosine 3',-5'-cyclic phosphate in barley aleurone layers Several communications have presented evidence that adenosine 3',5'-cyclic phosphate (cyclic AMP) is involved in the production of amylase b y barley half seeds treated with gibberellic acid1, ~. In an extension of earlier studies3, ~ on some of the other manifold responses of the phytohormone in these tissues it has also been observed in this laboratory that the nucleotide mimics gibberellic acid in effecting the secretion of several metabolites and enzymes. To m y knowledge, however, neither the occurrence of cyclic AMP nor its metabolism has been investigated in higher plants. I t seemed necessary therefore to investigate these in a plant tissue which responded to both the hormone and to cyclic AMP as well as to note the influence of the hormone on the level of the nucleotide in the tissues. Such information is requisite for assessing the roles and interrelationships of both substances in the cells. Hypochlorite sterilized layers prepared from dehusked seeds which had imbibed water for 24 h at room temperature followed by imbibition of water at 3 ° for an additional 24 h were used. They were incubated in 5-1o ml of labeled adenine solution containing 72 mM CaC12, 250/~g/ml streptomycin sulfate and where needed I #g/ml gibberellic acid. After incubation they were removed, homogenized with cold 5% trichloroacetic acid and centrifuged. After extraction with benzene and ether the supernatant solution was treated with charcoal. Labeled material was eluted from the charcoal with 5O~o ethanol containing 2% NH4OH, concentrated and cochromatographed with authentic cyclic AMP, eluted with 50% ethanol, dried and counted in a scintillation counter. Thin-layer chromatography was effected in one dimension on 5 cm × 20 cm plates of silica gel GF (Analtech, Inc., Wilmington, Del.) of 250 # thickness or in two dimensions on 2o cm x 20 cm plates of the same thickness or preparative plates of IOOO # thickness. The thin-layer solvents were: (A) n-butanol-methanol-ethyl acetate-NH4OH (7:3:4:4, by vol.); (B) isopropanol-methanol-NH~OH-water (io: 1:5:2, b y vol.) 5. The paper chromatographic solvents were: (C) 75% e t h a n o l - I o % satd. (NH~)2SO4 impregnated paper; (D) isobutyric acid-NH4OH-water (66:1:33, by vol.) ; (E) same as system A; (F) 95~o ethanol-i M ammonium acetate (75:30, by vol.), p H 7-5- The electrophoretic system (with a Durrum type unit) was o.oi M tetrasodium E D T A containing 0.34 ml pyridine per 1 and adjusted to pH 3.5 with acetic acid. Using FS-l~C]adenine as a precursor, the cyclic AMP isolated from gibberellic acid treated tissues contained as much as twice the amount of radioactivity as that from control (Table I). Critical evidence for the identity of the labeled cyclic AMP is as follows: after an initial chromatographic separation in System A more than 90% of the radioactivity migrates as a single area corresponding to the ultraviolet absorbing area of the unlabeled carrier in all of the chromatographic systems above, electrophoresis, as well as o t h e r s - - a total of io systems. Likewise, the material migrated with authentic cyclic AMP after successive chromatography in System E, electrophoresis and subsequent chromatography in System D followed by System F. Hydrolysis of the labeled material in the presence of unlabeled carrier with satd. Ba(OH)2 at ioo ° for I h resulted Biochim. Biophys. Acta, 2Ol (i97 o) 511-512
512
PRELIMINARY NOTES
TABLE I EFFECT OF GIBBERELLIC ACID ON THE RADIOACTIVITYIN CYCLIC AMP OF BARLEY ALEURONE LAYERS INCUBATED WITH [8-14C]ADENINE I n c u b a t i o n s of 2.5, 1.25 and 2. 5 #C [8-14C]adenine (Calbiochem; 12 mC/mmole) in E x p t s . i, 2 and 3 with 24, 20 and 15 layers per group, respectively. Whereas 2.0 /~C [8A4C]adenine (Schwarz BioResearch; 52.6 mC/mmole) and 22 layers per g r o u p employed in E x p t . 4-
Experiment. No.
Incubation time (miD)
Control
Plus gibberellic acid
i
24o
2
3oo
3 4
16o 300
36oo * 4635 ** 45oo * 35oo *** I92O§ 80o§§
796o* 75oo** 8o8o* 8600*** 2750§ 168o§§
* Counted ** Counted *** Counted § Counted §§ Counted
after after after after after
separation in one dimension in System A. separation in two dimensions; S y s t e m s A and 13. separation in one dimension in S y s t e m t3. separation in S y s t e m A followed b y C. Separation in S y s t e m E followed b y electrophoresis.
in the production of 3'-AMP and 5'-AMP. The labeled material was deaminated to cyclic IMP with acetic acid and NAN02 (ref. 6). The purified labeled compound and carrier authentic cyclic AMP were also converted to adenosine by a preparation from carrot leaves (the 40-80% (NH4)~SO, saturation fraction of ref. 7) which contained phosphodiesterase and nucleotidase activities. These results merely demonstrate that labeled adenine may be converted to cyclic AMP in barley layers and the amount of radioactivity found in the nucleotide of gibberellic acid treated tissue m a y be twice that of controls. More extensive studies on this novel approach to the mode of action of gibberellic acid, now in progress, were justifiable only after these facts were established. This work was done while the author was on sabbatical leave at the National Institutes of Health.
Department of Botany and Plant Pathology, Michigan State University, East Lansing, Mich. 48823 and Laboratory of Chemistm, N.I.A.M.D., National Institutes of Health, Bethesda, Md. 2OOl4 (U.S.A.)
CLIFFORD J . POLLARD
C. M. DUFFUS AND J. H. DUFFUS, Experentia, 25 (1969) 581. A. G. GALSKY AND J. A. LIPPINCOTT, Plant Cell Physiol., io (1969) 607. C. J. POLLARD AND B. iNT. SINGH, Biochem. Biophys. Res. Commun., 33 (1968) 321C. J. POLLARD, Plant Physiol., 44 (1969) 1227. H. SHIMIZU, J. W. DALY AND C. R. CREVELING, J. Neuroehem., 16 (1969} 16o9. N. O. KAPLAN, in S. P. COLOWlCK AND lX]-. O, KAPLAN, Methods in Enzymology, Vol. 3, Academic Press, New York, 1957, P. 873. 7 V. E. BECKER AND C. J. POLLARD, Plant Physiol., 44 (1969) 978.
I 2 3 4 5 6
Received January 5th, 197o Biochim. Biophys. Aeta, 2Ol (197 o) 5 I I - 5 1 2
511
BBA 21278
Influence of gibberellic acid on the incorporation of 8-14C adenine into adenosine 3',-5'-cyclic phosphate in barley aleurone layers Several communications have presented evidence that adenosine 3',5'-cyclic phosphate (cyclic AMP) is involved in the production of amylase b y barley half seeds treated with gibberellic acid1, ~. In an extension of earlier studies3, ~ on some of the other manifold responses of the phytohormone in these tissues it has also been observed in this laboratory that the nucleotide mimics gibberellic acid in effecting the secretion of several metabolites and enzymes. To m y knowledge, however, neither the occurrence of cyclic AMP nor its metabolism has been investigated in higher plants. I t seemed necessary therefore to investigate these in a plant tissue which responded to both the hormone and to cyclic AMP as well as to note the influence of the hormone on the level of the nucleotide in the tissues. Such information is requisite for assessing the roles and interrelationships of both substances in the cells. Hypochlorite sterilized layers prepared from dehusked seeds which had imbibed water for 24 h at room temperature followed by imbibition of water at 3 ° for an additional 24 h were used. They were incubated in 5-1o ml of labeled adenine solution containing 72 mM CaC12, 250/~g/ml streptomycin sulfate and where needed I #g/ml gibberellic acid. After incubation they were removed, homogenized with cold 5% trichloroacetic acid and centrifuged. After extraction with benzene and ether the supernatant solution was treated with charcoal. Labeled material was eluted from the charcoal with 5O~o ethanol containing 2% NH4OH, concentrated and cochromatographed with authentic cyclic AMP, eluted with 50% ethanol, dried and counted in a scintillation counter. Thin-layer chromatography was effected in one dimension on 5 cm × 20 cm plates of silica gel GF (Analtech, Inc., Wilmington, Del.) of 250 # thickness or in two dimensions on 2o cm x 20 cm plates of the same thickness or preparative plates of IOOO # thickness. The thin-layer solvents were: (A) n-butanol-methanol-ethyl acetate-NH4OH (7:3:4:4, by vol.); (B) isopropanol-methanol-NH~OH-water (io: 1:5:2, b y vol.) 5. The paper chromatographic solvents were: (C) 75% e t h a n o l - I o % satd. (NH~)2SO4 impregnated paper; (D) isobutyric acid-NH4OH-water (66:1:33, by vol.) ; (E) same as system A; (F) 95~o ethanol-i M ammonium acetate (75:30, by vol.), p H 7-5- The electrophoretic system (with a Durrum type unit) was o.oi M tetrasodium E D T A containing 0.34 ml pyridine per 1 and adjusted to pH 3.5 with acetic acid. Using FS-l~C]adenine as a precursor, the cyclic AMP isolated from gibberellic acid treated tissues contained as much as twice the amount of radioactivity as that from control (Table I). Critical evidence for the identity of the labeled cyclic AMP is as follows: after an initial chromatographic separation in System A more than 90% of the radioactivity migrates as a single area corresponding to the ultraviolet absorbing area of the unlabeled carrier in all of the chromatographic systems above, electrophoresis, as well as o t h e r s - - a total of io systems. Likewise, the material migrated with authentic cyclic AMP after successive chromatography in System E, electrophoresis and subsequent chromatography in System D followed by System F. Hydrolysis of the labeled material in the presence of unlabeled carrier with satd. Ba(OH)2 at ioo ° for I h resulted Biochim. Biophys. Acta, 2Ol (i97 o) 511-512
512
PRELIMINARY NOTES
TABLE I EFFECT OF GIBBERELLIC ACID ON THE RADIOACTIVITYIN CYCLIC AMP OF BARLEY ALEURONE LAYERS INCUBATED WITH [8-14C]ADENINE I n c u b a t i o n s of 2.5, 1.25 and 2. 5 #C [8-14C]adenine (Calbiochem; 12 mC/mmole) in E x p t s . i, 2 and 3 with 24, 20 and 15 layers per group, respectively. Whereas 2.0 /~C [8A4C]adenine (Schwarz BioResearch; 52.6 mC/mmole) and 22 layers per g r o u p employed in E x p t . 4-
Experiment. No.
Incubation time (miD)
Control
Plus gibberellic acid
i
24o
2
3oo
3 4
16o 300
36oo * 4635 ** 45oo * 35oo *** I92O§ 80o§§
796o* 75oo** 8o8o* 8600*** 2750§ 168o§§
* Counted ** Counted *** Counted § Counted §§ Counted
after after after after after
separation in one dimension in System A. separation in two dimensions; S y s t e m s A and 13. separation in one dimension in S y s t e m t3. separation in S y s t e m A followed b y C. Separation in S y s t e m E followed b y electrophoresis.
in the production of 3'-AMP and 5'-AMP. The labeled material was deaminated to cyclic IMP with acetic acid and NAN02 (ref. 6). The purified labeled compound and carrier authentic cyclic AMP were also converted to adenosine by a preparation from carrot leaves (the 40-80% (NH4)~SO, saturation fraction of ref. 7) which contained phosphodiesterase and nucleotidase activities. These results merely demonstrate that labeled adenine may be converted to cyclic AMP in barley layers and the amount of radioactivity found in the nucleotide of gibberellic acid treated tissue m a y be twice that of controls. More extensive studies on this novel approach to the mode of action of gibberellic acid, now in progress, were justifiable only after these facts were established. This work was done while the author was on sabbatical leave at the National Institutes of Health.
Department of Botany and Plant Pathology, Michigan State University, East Lansing, Mich. 48823 and Laboratory of Chemistm, N.I.A.M.D., National Institutes of Health, Bethesda, Md. 2OOl4 (U.S.A.)
CLIFFORD J . POLLARD
C. M. DUFFUS AND J. H. DUFFUS, Experentia, 25 (1969) 581. A. G. GALSKY AND J. A. LIPPINCOTT, Plant Cell Physiol., io (1969) 607. C. J. POLLARD AND B. iNT. SINGH, Biochem. Biophys. Res. Commun., 33 (1968) 321C. J. POLLARD, Plant Physiol., 44 (1969) 1227. H. SHIMIZU, J. W. DALY AND C. R. CREVELING, J. Neuroehem., 16 (1969} 16o9. N. O. KAPLAN, in S. P. COLOWlCK AND lX]-. O, KAPLAN, Methods in Enzymology, Vol. 3, Academic Press, New York, 1957, P. 873. 7 V. E. BECKER AND C. J. POLLARD, Plant Physiol., 44 (1969) 978.
I 2 3 4 5 6
Received January 5th, 197o Biochim. Biophys. Aeta, 2Ol (197 o) 5 I I - 5 1 2