Investigation into Sustainability of WEE1 Kinase in Plants

Cook, Gemma S. (2012) Investigation into Sustainability of WEE1 Kinase in Plants. PhD thesis, Cardiff University and the University of Worcester.

Abstract

Phosphoregulation is essential for the control of cell division. In yeasts and animals, premature entry into mitosis is prevented by the inhibitory phosphorylation of CDK by WEE1 kinase. WEE1 homologues have been identified in several species of higher plant, including Arabidopsis and tobacco. However, while WEE1 function has been confirmed in the DNA replication checkpoint in higher plants, a role for the protein in the G2/M transition during an unperturbed plant cell cycle is yet to be identified. To address this issue, the further characterisation of Arabidopsis WEE1 was completed, particularly focussing on the localisation and stability of the protein. A GFP-Arath;WEE1 construct under the 35S promoter was transformed into both Arabidopsis plants and the tobacco BY-2 cell line, and a nuclear localisation of the protein at interphase was confirmed. Additionally, the study of WEE1 subcellular localisation in different cell cycle phases revealed that the protein was absent during metaphase. Interestingly, levels of WEE1 degradation varied in different Arabidopsis root tissues, and the protein was absent in lateral root primordia. The proteasome inhibitor MG132 was used to demonstrate that Arath;WEE1 is degraded via the 26S proteasome pathway, as in yeasts and animals. Bimolecular fluorescence complementation confirmed an interaction between Arath;WEE1 and the F-box protein Arath;SKIP1 in vivo, which may target Arath;WEE1 for degradation. Tobacco BY-2 cells were stably co-transformed with BiFC constructs to facilitate the study of any changes in this interaction during the cell cycle. There was again no evidence of the interaction during metaphase, but a return of the signal during anaphase and telophase. The root phenotype of an Arath;SKIP1 knockdown line suggested that this F-box protein may target Arath;WEE1 for degradation early in development, but this requires confirmation. The work presented in this thesis describes, to my knowledge, the first investigation into the stability of Arath;WEE1 protein.

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