Abstract
The subcellular site of α-amylase (EC 1.6.2.1) synthesis and transport was studied in barley aleurone layers incubated in the presence or absence of gibberellic acid (GA3). Using [35S]methionine as a marker, the site of amino-acid incorporation into organelles isolated from aleurone layers incubated with and without GA3 was determined following purification by isopycnic sucrose-density-gradient centrifugation. Incorporation of radioactivity into trichloroacetic-acid-insoluble proteins was greatest in those fractions exhibiting activity of an endoplasmic reticulum (ER) marker enzyme. Further fractionation of densitygradient fractions by sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis showed that a major portion of the radioactivity in the ER fractions was present in a protein co-migrating with marker α-amylase. This protein was identified as authentic α-amylase by immunoadsorbent chromatography and affinity chromatography. The newly synthesized α-amylase associated with the ER was shown to be sequenstered within the lumen of the ER by experiments which showed that the enzyme was resistant to proteolytic degradation. The labelled α-amylase sequestered in the ER can be chased from this organelle when tissue is incubated in unlabelled methionine following a 1-h pulse of labelled methionine. The isoenzymic forms of α-amylase found in tissue homogenates and incubation media of aleurone layers incubated with and without GA3 were characterized after chromatography on diethylaminoethyl cellulose. In homogenates of GA3-treated aleurone layers, five peaks of α-amylase activity were detected, while in homogenates of aleurone layers incubated with-out GA3 only three peaks of activity were found. In incubation media, four isoenzymes were found after GA3 treatment and two were found after incubation without GA3. We conclude that at least five α-amylase isoenzymes are synthesized by the ER of barley aleurone layers and that this membrane system is involved in the sequestration and transport of four of these isoenzymes.
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Abbreviations
- CHA:
-
cyclohepataamylose
- DEAE-cellulose:
-
diethylaminoethyl-cellulose
- ER:
-
endoplasmic reticulum
- GA3 :
-
gibberellic acid
- SDS-PAGE:
-
sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis
References
Bennett, P.A., Chrispeels, M.J. (1972) De novo synthesis of ribonuclease and B-1,3-glucanase by aleurone cells of barley. Plant Physiol. 49, 445–447
Boston, R.S., Miller, T.J., Mertz, J.E., Burgess, R.R. (1982) In vitro synthesis and processing of wheat α-amylase. Plant Physiol. 69, 150–154
Buckhout, T.J., Gripshover, B.M., Morré, D.J. (1981) Endoplasmic reticulum formation during germination of wheat seeds. Plant Physiol. 68, 1319–1322
Chen, R.F., Jones, R.L. (1974a) Studies on the release of barley aleurone cell proteins: kinetics of labelling. Planta 119, 193–206
Chen, R.F., Jones, R.L. (1974b) Studies on the release of barley aleurone cell proteins: autoradiography. Planta 119, 207–220
Chrispeels, M.J., Varner, J.E. (1967) Gibberellic acid-enhanced synthesis and release of α-amylase and ribonuclease by isolated barley aleurone layers. Plant Physiol. 42, 398–406
Firn, R.D. (1975) On the secretion of α-amylase of barley aleurone layers after incubation in gibberellic acid. Planta 125, 227–233
Gibson, R.A., Paleg, L.G. (1972) Lysosomal nature of hormonally induced enzymes in wheat aleurone cells. Biochem. J. 128, 367–375
Gibson, R.A., Paleg, L.G. (1975) Further experiments on the α-amylase-containing lysosomes of wheat aleurone cells. Aust. J. Plant Physiol. 2, 41–49
Gibson, R.A., Paleg, L.G. (1976) Purification of GA3-induced lysosomes from wheat aleurone cells. J. Cell Sci. 22, 413–426
Haberlandt, G.F.J. (1884) Physiologische Pflanzenanatomie, 1st edn. Engelmann, Leipzig
Heslop-Harrison, Y., Heslop-Harrison, J. (1980) Chloride ion movement and enzyme secretion from the digestive glands of Pinguicula. Ann. Bot. 45, 729–731
Higgins, T.J.V., Jacobsen, J.V., Zwar, J.A. (in press) Gibberellic acid and abscisic acid modulate protein synthesis and mRNA levels in barley aleurone layers. Plant Mol. Biol.
Higgins, T.J.V., Spencer, D. (1977) Cell-free synthesis of pea seed proteins. Plant Physiol. 60, 655–661
Jacobsen, J.V., Higgins, T.J.V. (in press) Characterization of the α-amylases synthesized by aleurone layers of Himilaya barley in response to GA3. Plant Physiol.
Jacobsen, J.V., Knox, R.B. (1973) Cytochemical localization and antigenicity of α-amylase in barley aleurone tissue. Planta 112, 213–224
Jacobsen, J.V., Scandalios, J.G., Varner, J.E. (1970) Multiple forms of amylase induced by gibberellic acid in isolated barley aleurone layers. Plant Physiol. 45, 367–371
Jones, R.L. (1969a) Gibberellic acid and the fine structure of barley aleurone layers. I. Changes during the lag phase of α-amylase synthesis. Planta 87, 119–133
Jones, R.L. (1969b) Gibberellic acid and the fine structure of barley aleurone layers. II. Changes during the syntehsis and secretion of α-amylase. Planta 88, 73–86
Jones, R.L. (1972) Fractionation of the enzymes of the barley aleurone layer: evidence for a soluble mode of enzyme release. Planta 103, 95–109
Jones, R.L. (1980a) The isolation of endoplasmic reticulum from barley aleurone layers. Planta 150, 58–69
Jones, R.L. (1980b) Quantitative and qualitative changes in the endoplasmic reticulum of barley aleurone layers. Planta 150, 70–81
Jones, R.L., Price, J.M. (1970) Gibberellic acid and the fine structure of barley aleurone layers. III. Vacuolization of the aleurone cell during the phase of ribonuclease release. Planta 94, 191–202
Locy, R., Kende, H. (1978) The mode of secretion of α-amylase in barley aleurone layers. Planta 143, 89–99
Mans, R.J., Novelli, G.D. (1961) Measurement of the incorporation of radioactive amino acids into protein by a filterpaper disc method. Arch. Biochem. Biophys. 94, 48–53
Obata, T., Suzuki, H. (1976) Gibberellic acid-induced secretion of hydrolases in barley aleurone layers. Plant Cell Physiol. 17, 63–71
Ouchterlony, O. (1967) Immunodiffusion and immunoelectrophoresis. In: Handbook of experimental immunology, pp. 655–706, Weir, D.M., ed. Blackwell Scientific Publications, F.A. Davis, Co., Philadelphia
Paleg, L.G., Hyde, B. (1964) Physiological effects of gibberellic acid. VII. Electron microscopy of barley aleurone cells. Plant Physiol. 39, 673–680
Robyt, J., French, D. (1963) Action pattern and specificity of an amylase from Bacillus subtillis. Arch. Biochem. Biophys. 100, 451–467
Silvanovich, M.P., Hill, R.D. (1976) Affinity chromatography of cereal α-amylase. Anal. Biochem. 73, 430–433
Spencer, D., Higgins, T.J.V., Button, S.C., Davey, R.A. (1980) Pulse labelling studies on protein synthesis in developing pea seeds and evidence of a precursor form of legumin small subunit. Plant Physiol. 66, 510–515
van der Eb, A.A., Nieuwdorp, P.J. (1967) Electron microscopic structure of the aleurone cells of barley during germination. Acta Bot. Neerl. 15, 690–699
Varner, J.E., Ho, D.T. (1976) The role of hormones in the integration of seedling growth. In: The molecular biology of hormone action, pp. 173–194, Papaconstantinou, J., ed. Academic Press, New York
Vigil, E.L., Ruddat, M. (1973) Effect of gibberellic acid and actinomycin D on the formation and distribution of rough endoplasmic reticulum in barley aleurone cells. Plant Physiol. 51, 549–558
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Jones, R.L., Jacobsen, J.V. The role of the endoplasmic reticulum in the synthesis and transport of α-amylase in barley aleurone layers. Planta 156, 421–432 (1982). https://doi.org/10.1007/BF00393313
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DOI: https://doi.org/10.1007/BF00393313