ACTIVE CDC2-GENES AND CELL-CYCLE PHASE-SPECIFIC CDC2-RELATED KINASE COMPLEXES IN HORMONE-STIMULATED ALFALFA CELLS

Hormones as auxins and cytokinins trigger the division cycle in differ entiated plant cells and are required for the maintenance of prolifera tion in cultured cells in vitro. Northern analysis showed that the exp ression pattern of alfalfa cdc2 genes is significantly different in ce lls of primary explants exposed to hormone treatment and of rapidly cy cling suspension culture. Transcription of at least one of the cdc2 ge nes is activated by hormones in cultured leaf mesophyll protoplasts or in root tissues treated with auxins and cytokinins. In a suspension c ulture of alfalfa cells, cdc2 transcripts are at a constitutively high level, irrespective of actual cell cycle phase. In addition to the tr anscriptional control of cdc2 genes, in hormone-induced cells cdc2-rel ated kinase complexes were identified as potential phase-specific comp onents 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 cycl e regulatory protein kinases from alfalfa. Alfalfa suspension cells we re synchronized either for G1 phase by double-phosphate-starvation or for S phase by hydroxyurea treatment. The p13suc1-Sepharose affinity m atrix bound two cdc2 protein-related complexes, one with increased his tone H1 kinase activity in S, the other in G2/M phase. The complex fro m S phase cells showed higher kinase activity than the G2/M phase comp lex. 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. Im munoprecipitation 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 re sults suggest that a multi-component regulatory system is in control o f the cell cycle in plants, which includes a hormone-activated transcr iptional control of cdc2 genes and phase-specific cdc2-related kinase complexes.