Sheremet I. Investigation of functional role of microtubules phosphorylation in plant cells.

The thesis is dedicated to the investigation of the effects of inhibitors of serine/threonine Са2+-dependent (Са2+-calmodulin dependent protein kinases, protein kinase C) and cyclin-dependent protein kinases and also tyrosine kinases as well as serine/threonine and tyrosine phosphatases on mitosis progression in cells of synchronized tobacco BY-2 suspension culture, Arabidopsis thaliana primary root growth, morphology and organization of different microtubular arrays in cells of synchronized suspension BY-2 culture and in cells of different growth zones of A. thaliana primary root. Stably transformed lines of A. thaliana (GFP-MAP4) and suspension BY-2 culture (GFP-MBD) for in vivo study of the microtubular organization in different types of plant cells were used in this research. It was found that alterations of microtubules organization in primary root cells as a result of inhibition of cyclin- and Са2+-calmodulin dependent protein kinases and also protein kinase C led to subsequent disturbances in primary root growth and differentiation. The mitosis transition in synchronized BY-2 cells was sensitive to the level of phosphorylation on serine/threonine residues. Inhibition of cyclin-dependent protein kinases and protein kinase C led to the retardation of the interphase/prophase transition. We showed an important role of tyrosine phosphorylation in the maintenance of the microtubules organization in A. thaliana primary root cells. It was found that phosphorylation of microtubular proteins on tyrosine residues participate in the regulation of A. thaliana primary root differentiation process. In synchronized BY-2 suspension cells inhibition of tyrosine kinases caused no visible disturbances of microtubular organization but have reduced the frequency of the mitotic figures occurrence. It is suggested that the decrease of tyrosine phosphorylation level could cause the slowing down of the interphase/prophase transition. Probably this is the result of alterations in dynamic parameters of plant microtubules in dividing cells via regulation of phosphorylation level of microtubular proteins during cell cycle specific stages transition. Immunofluorescence microscopy employing polyclonal anti-phosphotyrosine (P-Tyr), monoclonal anti-alpha-tubulin (TU-01) and anti-beta-tubulin (TUB 2.1) antibodies revealed tyrosine phosphorylation of cortical and mitotic microtubules in Arabidopsis cells. Anti-phosphotyrosine antibody staining was markedly enhanced if the cells were pretreated with sodium orthovanadate, a potent inhibitor of protein tyrosine phosphatases. As a result, we have established a certain functional interaction between the levels of phosphorylation on tyrosine residues and sensitivity of cortical microtubules to cold.