Functional analysis of cyclin-depdendent kinase inhibitors in Arabidopsis thaliana cell suspension cultures

This research project is situated in the field of plant cell cycle analysis. The study of the plant cell cycle is of interest to learn more about the general cell cycle machinery and to understand how cell division is integrated with environmental signals and with cellular processes such as growth and differentiation. Furthermore, the knowledge of the cell cycle and its regulation might result in applications of common interest like the increase in yield of agricultural crops and the production of alternative energy resources. At the end of the cell cycle, one cell divides in two, with each new cell containing the complete cell content including the genetic information or DNA. This cell cycle process consists of four successive phases: (1) the G1 phase, in which the cell prepares the new round of cell division, (2) the S phase, in which the DNA is duplicated, (3) the G2 phase, in which all other cell material is doubled and (4) the M phase, in which mitosis and cytokinesis or cell division occur. Because the correct succession of the different cell cycle phases is essential for proper growth and development, the cell cycle process is regulated by several mechanisms. Cell cycle is driven by cyclin-dependent kinases (CDKs) that require binding with cyclins to perform their functions. CDK/cyclin complexes are activated by phosphorylation through CDK-activating kinases (CAKs) and by dephosphorylation through the CDC25 phosphatase, whereas their inactivation is mediated by degradation of regulatory proteins such as cyclins, by phosphorylation through the WEE1 kinase and by binding with CDK inhibitors (CKIs) (Chapter 2). We focus on cell cycle control performed by CKIs and compare their functions in yeast, animals and plants (Chapter 2). This comparison shows similarities and differences in regulation and functions of CKIs and strengthens their importance in orchestrating cell cycle progression and in linking cell proliferation with differentiation. Furthermore, Chapter 2 reveals that CKIs in yeast, animals and plants participate in conserved eukaryotic mechanisms, but that their functions are adapted toward the specific needs of each type of organism. Chapter 3 describes the comparison of antibody technology and epitope tagging to study protein accumulation of KRPs. Recombinant monoclonal antibodies against KRP2, KRP4 and KRP6 are isolated from a naive phage display library and their use as detection agents is compared with polyclonal sera produced against KRP2, KRP4 and KRP6. Furthermore, transgenic cell suspension cultures with HA-tagged KRP2 and KRP4 are produced and evaluated to study protein accumulation of these KRPs. KRP2 and KRP4 protein levels are studied during growth of Arabidopsis cell suspension cultures, in response to sucrose and during cell cycle progression and we reveal that KRP2 and KRP4 are constitutively present in growing cell cultures and during cell cycle progression. In Chapter 4 we describe how cell suspension cultures are used to study the function of KRP6 independently from development. First, we compare the effect of overexpression of KRP2 and KRP6 on cell cycle progression and we show that they affect the cell cycle process differently. Because the effect of KRP2 overexpression correlates with its function as a cell cycle inhibitor, while the effect of KRP6 overexpression is unexpected for a putative inhibitor, our further research concentrates on the role of KRP6. A kinematic analysis of KRP6 overexpressing cell cultures is performed and the phenotype of KRP6 overexpressing cells is analyzed by confocal microscopy and flow cytometry. We reveal that overexpression of KRP6 inhibits cell divisions and results in cell cultures consisting of a reduced number of larger cells with increased dry weight. A detailed phenotypic analysis of KRP6 overexpressing cell cultures shows that a percentage of the cells is multinucleated or has misshaped nuclei and that overexpression of KRP6 causes genome alterations. In addition, KRP6 overexpressing plants were analyzed and we demonstrate that overexpression of KRP6 reduces plant growth and increases DNA ploidy levels in some KRP6 overexpressing plants. In Chapter 5, the role of KRP6 is further investigated by molecular analysis. The involvement of KRP6 in the DNA damage checkpoint is examined. Furthermore, the interactions of KRP6 with CDKs and cyclins are analyzed and the effect of recombinant KRP6 on CDKA;1 kinase activity is studied. We demonstrate that KRP6 does not interfere with the DNA damage checkpoint. Furthermore, we show that KRP6 binds with CDKA;1 and CYCD3;1 and not with CDKB1;1 or CYCB1;1 and that recombinant KRP6 inhibits CDKA;1 kinase activity. Finally, a summary is presented that covers the diverse chapters