Magyar Z, Bakó L, Bogre L, Dedeoglu D, Kapros T, Dudits D
Active Cdc2-Genes and Cell-Cycle Phase-Specific Cdc2-Related Kinase Complexes in Hormone-Stimulated Alfalfa Cells
Plant Journal: 1993 4:151-161
Hormones as auxins and cytokinins trigger the division cycle in differentiated plant cells and are required for the maintenance of proliferation in cultured cells in vitro. Northern analysis showed that the expression pattern of alfalfa cdc2 genes is significantly different in cells of primary explants exposed to hormone treatment and of rapidly cycling suspension culture. Transcription of at least one of the cdc2 genes is activated by hormones in cultured leaf mesophyll protoplasts or in root tissues treated with auxins and cytokinins. In a suspension culture of alfalfa cells, cdc2 transcripts are at a constitutively high level, irrespective of actual cell cycle phase. In addition to the transcriptional control of cdc2 genes, in hormone-induced cells cdc2-related kinase complexes were identified as potential phase-specific components of post-transcriptional regulation of the cell cycle. The known specific interaction between eukaryotic p34cdc2 protein complexes and the yeast p13suc1 protein was exploited for purification of cell cycle regulatory protein kinases from alfalfa. Alfalfa suspension cells were synchronized either for G1 phase by double-phosphate-starvation or for S phase by hydroxyurea treatment. The p13suc1-Sepharose affinity matrix bound two cdc2 protein-related complexes, one with increased histone H1 kinase activity in S, the other in G2/M phase. The complex from S phase cells showed higher kinase activity than the G2/M phase complex. Immunoblotting of p13suc1-Sepharose-bound protein complex showed the presence of a 33-34 kDa doublet that is recognized by anti-PSTAIR antibodies and the co-purification of two proteins (apparent molecular mass 65 and 42 kDa) cross-reacting with human cyclin A antibodies. Immunoprecipitation with these cyclin A antibodies allowed the detection of a third cdc2-related kinase complex that appeared during G1/S and early S phases of the cell cycle and phosphorylated histone H1. The results suggest that a multi-component regulatory system is in control of the cell cycle in plants, which includes a hormone-activated transcriptional control of cdc2 genes and phase-specific cdc2-related kinase complexes.
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