Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS

Basic research on cell cycle genes

In Arabidopsis, the D-type cyclin CYCD3 is rate-limiting for transition of the G(1)/S boundary, and is transcriptionally unregulated at this point in cells re-entering the cell cycle in response to plant hormones and sucrose. However, little is known about the regulation of plant cell-cycle regulators at the protein level. We show here that CYCD3;1 is a phosphoprotein highly regulated at the level of protein abundance, whereas another D-type cyclin CYCD2;1 is not. The level of CYCD3;1 protein falls rapidly on sucrose depletion, correlated with the arrest of cells in G(1) phase, suggesting a rapid turnover of CYCD3;1. Treatment of exponentially growing cells with the protein synthesis inhibitor cycloheximide (CHX) confirms that CYCD3;1 is normally a highly unstable protein, with a half-life of approximately 7 min on CHX treatment. In both sucrose-starved and exponentially growing cells, CYCD3;1 protein abundance increases in response to treatment with MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal), a reversible proteasome inhibitor, but not in response to the cysteine protease inhibitor E-64 or the calpain inhibitor ALLN (N-acetyl-leucyl-leucyl-norleucinal).

The increase on MG132 treatment is because of de novo protein synthesis coupled with the blocking of CYCD3;1 degradation. Longer MG132 treatment leads to C-terminal cleavage of CYCD3;1, accumulation of a hyper-phosphorylated form and its subsequent disappearance. It can be concluded that CYCD3;1 is a highly unstable protein whose proteolysis is mediated by a proteasome-dependent pathway, and whose levels are highly dependent on the rate of CYCD3;1 protein synthesis. (Planchais et al., 2004; Differential stability of Arabidopsis D-type cyclins: CYCD3;1 is a highly unstable protein degraded by a proteasome-dependent mechanism (Planchais et al., Plant J. 2004 May; 38(4):616-25.)

In addition, tools have been developed for studying the role of eIF4E/ S6/ S6kinase signaling pathwayin cell cultures and intact plants. This was done in collaboration with CropDesign, Heberle-Bors's group and Murray's groups. CropDesign has found T-DNA insertion mutants in S6K, S6II, and CBP. Murray's group has over-expressed eIF4E and eIF4E2(iso) under control of the 35S promoter. T2 seeds of the 35S: eIF4E are available. Interestingly, leaves and siliques of these plants were purple. T0 seeds of 35S: eIF4E2(iso) did not germinate suggesting that over-expression of this gene perturbs embryogenesis and/or germination processes. Murray's group has made tobacco BY-2 cells, in which eIF4E1 and eIF4E2(iso) can be over-expressed in an inducible manner.

This construct induced aberrant phenotype in tobacco with abnormal cell morphology and more elongated cells. In Arabidopsis, plant lines have been produced with EiF4E1 and 2iso but not analysed yet. CropDesign's data on S6kinase indicate also negative phenotypes in arabidopsis.

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James MURRAY, (Head of Unit)
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