Abstract
We have purified an abundant, 33000-dalton polypeptide (P33) from cultured pith parenchyma tissue of Nicotiana tabacum L. cv. Havana 425. The accumulation of P33 in culture is inhibited by the cytokinin kinetin (N6-furfuryl-amino purine). When tissues are subcultured on auxin-containing medium, the P33 content measured by rocket immunoelectrophoresis increases by 10-fold from 9 to 90 μg·mg-1 soluble protein over a 7-d period. This increase is blocked when kinetin is added to the culture medium. There is strong evidence that P33 is a β-1,3-glucanase (EC 3.2.1.39): i) Purified P33 specificially promotes the endo-type hydrolysis of β-1,3-glucans and has essentially the same moleculear weight, pH optimum, and sensitivitiy to heavy metals as the β-1,3-glucanase isolated by others from tobacco. ii) Glucanase activity is inhibited by specific antibodies against P33. iii) P33 and glucanase activity co-purify and cannot be separated by affinity chromatography using the β-1,3-glucanase substrate pachyman. iv) P33 content and glucanase activity are strongly correlated in tissues grown under inductive and non-inductive conditions. The pattern of glucanase synthesis estimated by [35S]methionine incorporation parallels changes in the amount of glucanase. This indicates that cytokinin acts, at least in part, by blocking synthesis of the enzyme.
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Abbreviations
- IgG:
-
immunoglobulin G
- P33:
-
33000-dalton polypeptide
- SDS-PAGE:
-
sodium-dodecyl-sulfate polyacryl-amide-gel electrophoresis
References
Abeles, F.B., Forrence, L.E. (1970) Temporal and hormonal control of β-1,3-glucanase in Phaseolus vulgaris L. Plant Physiol. 45, 395–400
Axelos, M., Péaud-Lenoël, C. (1980) The apoprotein of the light-harvesting chlorophyll a/b complex of tobacco cells as a molecular marker of cytokinin activity. Plant Sci. Lett. 19, 33–41
Avrameas, S., Guilbert, B. (1971) A method for quantitative determination of cellular immunoglobulins by enzyme-labelled antibodies. Eur. J. Immunol. 1, 394–396
Bevan, M., Northcote, D.H. (1981) Subculture-induced protein synthesis in tissue cultures of Glycine max and Phaseolus vulgaris. Planta 152, 24–31
Boller, T. (in press) Induction of hydrolases as a defense reaction against pathogens. UCLA Symp. Molec. and Cell. Biol., ed(s). Liss, New York
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254
Darvill, A.G., Albersheim, P. (1984) Phytoalexins and their elicitors — A defense against microbial infection in plants. Annu. Rev. Plant Physiol. 35, 243–275
Denault, L.J., Allen, W.G., Boyer, E.W., Collins, D., Kramme, D., Spradlin, J.E. (1978) A simple reducing sugar assay for measuring β-glucanase activity in malt and various microbial enzyme preparations. Am. Soc. Brew. Chem. J. 36, 18–23
Eichholz, R., Harper, J., Felix, G., Meins, F., Jr. (1983) Evidence for an abundant 33000-dalton polypeptide regulated by cytokinins in cultured tobacco tissues. Planta 158, 410–415
Fosket, D.E., Volk, M.J., Goldsmith, M.R. (1977) Polyribosome formation in relation to cytokinin-induced cell division in suspension cultures of Glycine max (L.) Merr. Plant Physiol. 60, 554–562
Goding, J.W. (1976) Conjugation of antibodies with fluorochromes: modifications to the standard methods. J. Immunol. Methods. 13, 215–226
Gomori, G. (1952) Microscopic histochemistry University of Chicago, Chicago
Jouanneau, J.-P. (1970) Renouvellement des proteines et effet specifique de la kinetine sur des cultures de cellules de tabac. Physiol. Plant. 23, 232–244
Katô, K., Yamada, A., Noguchi, M (1973) Purification and some properties of β-1,3-glucanase of suspension-cultured tobacco cells. Agric. Biol. Chem. 37, 1269–1275
Laurell, C.B. (1965) Antigen-antibody crossed electrophoresis. Anal. Biochem. 10, 359–361
Laurell, C.B., McKay, E.J. (1981) Electroimmunoassay. Methods Enzymol. 73, 339–369
Linsmaier, E., Skoog, F. (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant. 18, 100–127
Meins, F., Jr., Lutz, J. (1980) The induction of cytokinin habituation in primary pith explants of tobacco. Planta 149, 402–407
Meyer, Y., Chartier, Y. (1981) Hormonal control of mitotic development in tobacco. Two-dimensional distribution of newly synthesized proteins. Plant Physiol. 68, 1273–1278
Molano, J., Duran, A., Cabib, E. (1977) A rapid and sensitive assay for chitinase using tritiated chitin. Anal. Biochem. 83, 648–656
Moore, A.E., Stone, B.A. (1972) Effect of senescence and hormone treatment on the activity of a β-1,3-glucan hydrolase in Nicotiana glutinosa leaves. Planta 104, 93–109
Nelson, N. (1944) A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153, 275–280
Philpot, W.A., Chapman, J.M. (1977) A new improved method for the assay of endo-β-1,3-glucanase. Anal. Biochem. 79, 257–262
Shinshi, H., Kato, K. (1983) Physical and chemical properties of β-1,3-glucanse from cultured tobacco cells. Agric. Biol. Chem. 47, 1455–1460
Somogyi, M. (1952) Notes on sugar determination. J. Biol. Chem. 195, 19–23
Towbin, H., Staehelin, T., Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedures and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354
Voller, A., Bidwell, D.E., Barlett, A. (1976) Enzyme immunoassay in diagnostic medicine. Bull. World Health Organ. 53, 55–65
Wang, T.L., Everett, N.P., Gould, A.R., Street, H.E. (1981) Studies on the control of the cell cycle in cultured plant cells. III. The effects of cytokinin. Protoplasma 106, 23–35
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Felix, G., Meins, F. Purification, immunoassay and characterization of an abundant, cytokinin-regulated polypeptide in cultured tobacco tissues. Planta 164, 423–428 (1985). https://doi.org/10.1007/BF00402956
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DOI: https://doi.org/10.1007/BF00402956