Protoplasma (1995) 187:117-126 PI 21OIKASMA 9 Springer-Verlag 1995 Printed in Austria Cell cycle dependent distribution of phosphorylated proteins in microspores and pollen of Brassica napus L., detected by the monoclonal antibody MPM-2 G. Hause 1, J. H. G. Cordewener 2, M. Ehrmanova 3, B. Hause 4, P. Binarova 5, M. M. Van Lookeren Campagne 2, and A. A. M. Van Lammeren 3'* 1Institute of Genetics, Martin-Luther-University, Halle, 2 Department of Developmental Biology, Centre for Plant Breeding and Reproduction Research (CPRO-DLO), Wageningen, 3 Department of Plant Cytology and Morphology, Wageningen Agricultural University, Wageningen, 4 Institute of Plant Biochemistry, Halle, and 5 Institute of Experimental Botany, Norman Borlaug Centre for Plant Science De Montfort Univer- sity, Olomouc Received October 2, 1994 Accepted February 16, 1995 Summary. The monoclonal antibody MPM-2, which interacts with a mitosis-specific phosphorylated epitope, has been used to study phosphorylation of proteins in microspores and pollen of Brassica napus. One- (l-D) and two-dimensional (2-D) immunoblots revealed that MPM-2 recognized a family of phosphorylated proteins in fresh- ly isolated microspores and pollen. The same set of phosphorylated proteins was found after 8 h of culture at embryogenic (32 ~ and non-embryogenic (18~ conditions. Two major spots were observed on 2-D immunoblots, one of which (Mrs75 kDa, pI~5.1) co-localized with the 70 kDa heat shock protein. Immunolabelling of sectioned microspores and pollen showed that MPM-2 reactive epi- topes were predominantly observed in the nucleoplasm from G1 until G2-phase, and in the cytoplasm during mitosis. This may be due to a cell cycle related translocation of phosphoproteins from the nucleus to the cytoplasm, or alternate phosphorylation and dephosphoryla- tion in nucleus and cytoplasm. Detectability of epitopes on sections depended on the embedding procedure. Cryo processing revealed epitope reactivity in all stages of the cell cycle whereas polyethylene glycol embedded material showed no labelling in the cytoplasm dur- ing mitosis. Processing might reduce the antigenicity of cytoplasmic MPM-2 detectable proteins, probably due to dephosphorylation. The MPM-2 detectable epitope was observed in all cells investigated, irrespective of culture conditions, and its intracellular distribution depended on the cell cycle stage and was not related to the develop- mental fate of the microspores and pollen. Keywords: Androgenesis; Brassica napus; Microspore culture; Pol- len embryogenesis; Protein phosphorylation. * Correspondence and reprints: Department of Plant Cytology and Morphology, Wageningen Agricultural University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands. Introduction When placed in culture, isolated late microspores and early bicellular pollen from Brassica napus cv. Topas can be induced to switch their developmental fate from gametophytic development to sporophytic development, resulting in the formation of androgen- ic embryos in up to 70% of the cells (Pechan and Kel- ler 1988, Telmer et al. 1992). The early stages of this sporophytic development are characterized by an altered pattern of cell division: a symmetric cell divi- sion instead of an asymmetric first pollen mitosis, when starting from late microspores, or the participa- tion of the vegetative cell instead of the generative cell in cell division, when starting from early bicellu- lar pollen. The redirection of gametophytic develop- ment to embryogenesis is irreversibly induced by ele- vating the culture temperature to 32 ~ (Hause et al. 1993, Clusters et al. 1994) for a period of at least 8 h. The 8 h inductive temperature treatment has been reported to coincide with (1) rearrangements of the microtubular cytoskeleton, such as changing orienta- tions of mitotic spindles in late microspores and redis- tribution of cytoplasmic microtubules in young bicel- lular pollen (Hause et al. 1992, 1993), (2) re-entry into the cell cycle of the vegetative cell from 4 h of culture onwards (Binarova et al. 1993 b), and (3) changes in the synthesis of 25 proteins detected on