Abstract
Culture temperature determines the developmental fate of isolated microspores from Brassica napus L. At 18°C, tricellular pollen develops, whereas culture at 32°C for 8 h leads to the quantitative and synchronous induction of embryogenesis, and ultimately to the formation of embryos. We investigated the changes in protein synthesis that are associated with this 8-h inductive period by using in-situ [35S]methionine labeling, followed by two-dimensional (2-D) gel electrophoretic analysis of the radiolabeled proteins. Qualitative and quantitative computer analyses of 2-D [35S]methionine protein patterns showed six polypeptides specifically labeled under embryogenic culture conditions. Eighteen polypeptides incorporated [35S]methionine at a statistically significant higher rate under embryogenic culture conditions (32°C) than in the controls (18°C), whereas one protein was preferentially labeled under non-embryogenic culture conditions (18°C). These results indicate that only a limited number of proteins detectable in the 2-D gels of microspore extracts are associated with the early induction of embryogenesis. The reproducible identification of the differentially radiolabeled proteins in the 2-D gels allow the sequencing of representative peptides and the isolation of the corresponding cDNAs. This may lead to the identification and characterization of proteins associated with the very first stages of plant embryogenesis.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Abbreviations
- 2-D:
-
two-dimensional
Reference
Binarova, P., Straatman, K., Hause, B., Hause, G., VanLammeren, A.A.M. (1993) Nuclear DNA synthesis during the induction of embryogenesis in cultured microspores and pollen of Brassica napus L. Theor. Appl. Genet. 87, 9–16
Blum, H., Beier, H., Gross, H.J. (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8, 93–99
Celis, J.E., Rasmussen, H.H., Madsen, P., Leffers, H., Honore, B., Dejgaard, K., Gesser, B., Olsen, E., Gromov, P., Hoffmann, H.J., Nielsen, M., Celis, A., Basse, B., Lauridsen, J.B., Ratz, G.P., Nilsen, H., Andersen, A.H., Walbum, E., Kjaergaard, I., Puype, M., VanDamme, J., Vandekerckhove, J. (1992) The human keratinocyte two-dimensional gel protein database (update 1992): towards an integrated approach to the study of cell proliferation, differentiation and skin diseases. Electrophoresis 13, 893–959
Chuong, P.V., Beversdorf, W.D. (1985) High frequency embryogenesis through isolated microspore culture in Brassica napus L. and B. carinata. Braun. Plant Sci. 39, 219–226
Custers, J.B.M., Cordewener, J.H.G., Nöllen, Y., Dons, J.J.M., VanLockeren Campagne, M.M. (1994) Temperature controls both gametophytic and sporophytic development in microspore cultures of Brassica napus. Plant Cell Rep. 13, 267–271
DeVries, S.C., Booij, H., Meyerink, P., Huisman, G., Wilde, D.H., Thomas, T.L., VanKammen, A. (1988) Acquisition of embryogenic potential in carrot cell-suspension cultures. Planta 176, 196–204
Dudits, D., Bögre, L., Györgyey, J. (1991) Molecular and cellular approaches to the analysis of plant embryo development from somatic cells in vitro. J. Cell Sci. 99, 475–484
Garrels, J.I. (1989) The QUEST system for quantitative analysis of two-dimensional gels. J. Biol. Chem. 264, 5269–5282
Garrels, J.I., Franza, B.R., Chang, C., Latter, G. (1990) Quantitative exploration of the REF52 protein database: cluster analysis reveals the major protein expression profiles in responses to growth regulation, serum stimulation, and viral transformation. Electrophoresis 11, 1114–1130
Garrido, D., Eller, N., Heberle-Bors, E., Vicente, O. (1993) De novo transcription of specific messenger RNAs during the induction of tobacco pollen embryogenesis. Sex. Plant Reprod. 6, 40–45
Giometti, C.S., Taylor, J., Tollaksen, S.L. (1992) Mouse liver protein database: a catalog of proteins detected by two-dimensional gel electrophoresis. Electrophoresis 13, 970–991
Görg, A., Postel, W., Gunther, S. (1988) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 9, 531–546
Hause, B., Hause, G., Pechan, P., VanLammeren, A.A.M. (1993) Cytoskeletal changes and induction of embryogenesis in microspore and pollen cultures of Brassica napus L. Cell Biol. Int. 17, 153–168
Hochstrasser, D.E., Frutiger, S., Paquet, N., Bairoch, A., Ravier, F., Pasquali, C., Sanchez, J.C., Tissot, J.D., Bjellqvist, B., Vargas, R., Appel, R.D., Hughes, G.J. (1992) Human liver protein map: a reference database established by microsequencing and gel comparison. Electrophoresis 13, 992–1001
Johnston, R.F., Pickett, S.C., Barker, D.L. (1990) Autoradiography using storage phosphor technology. Electrophoresis 11, 355–360
Kyo, M., Harada, H. (1986) Control of the developmental pathway of tobacco pollen in vitro. Planta 168, 427–432
Lichter, R. (1982) Induction of haploid plants from isolated pollen of Brassica napus. Z. Pflanzenphysiol. 105, 427–434
Nover, L., Neumann, D., Scharf, K.D. (1989) Heat shock and other stress response systems of plants. Springer, Berlin
Pechan, P.M., Bartels, D., Brown, D.C.W., Schell, J. (1991) Messenger-RNA and protein changes associated with induction of Brassica microspore embryogenesis. Planta 184, 161–165
Pechan, P.M., Keller, W.A. (1988) Identification of potentially embryogenic microspores in Brassica napus. Physiol. Plant. 74, 377–384
Rasmussen, H.H., VanDamme, J., Puype, M., Gesser, B., Celis, J.E., Vandekerckhove, J. (1992) Microsequences of 145 proteins recorded in the two-dimensional gel protein database of normal human epidermal keratinocytes. Electrophoresis 13, 960–969
VanBogelen, R.A., Sankar, P., Clark, R.L., Bogan, J.A., Neidhardt, F.C. (1992) The gene-protein database of Escherichia coli: edition 5. Electrophoresis 13, 1014–1054
Vicente, O., Garrido, D., Zarsky, V., Eller, N., Rihova, L., Berenyi, M., Tupy, J., Heberle-Bors, E. (1992) Induction of embryogenesis in isolated pollen cultures of tobacco. In: Angiosperm pollen and ovules, pp. 279–284, Ottaviano, E., Mulcahy, D.L., Sari Gorla, M., Bergamini Mulcahy, G., eds. Springer, New York
Yun, M., Wu, W., Hood, L., Harrington, M. (1992) Human cerebrospinal fluid protein database: edition 1992. Electrophoresis 13, 1002–1013
Author information
Authors and Affiliations
Corresponding author
Additional information
We would like to thank Dr. H. Van Steeg (Rijks Instituut voor Milieubeheer (RIVM), Bilthoven, The Netherlands) for use of the PhosphorImager apparatus. This research was carried out as part of the EC-Bridge project ‘Regulation of the inductive phase of microspore embryogenesis’ and EC-Science project ‘The role of mitotic and cytoskeletal genes in the induction of plant cell division’.
Rights and permissions
About this article
Cite this article
Cordewener, J.H.G., Busink, R., Traas, J.A. et al. Induction of microspore embryogenesis in Brassica napus L. is accompanied by specific changes in protein synthesis. Planta 195, 50–56 (1994). https://doi.org/10.1007/BF00206291
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00206291