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Functional analysis of cell cycle genes

Inze's group was one of the most productive groups within the ECCO project. Together with CropDesign they have conducted an important gene discovery task. Instrumental for the genomic analysis of cell cycle control in Arabidopsis was the identification and classification of the core cell cycle constituents (Vandepoele et al., 2002). The highlight of the Gene Discovery research was however the genome-wide analysis of cell cycle phase specific transcripts. Using cDNA-AFLP technology, 1340 different transcripts have been identified that show differential expression during the plant cell cycle (Breyne et al., 2002). In parallel to this transcript-profiling project in tobacco BY-2 cell cultures, significant progress was made in the synchronization of Arabidopsis cell cultures. This has resulted in the successful deployment of Arabidopsis microarrays for the study of phase-specific transcripts during the cell cycle. As a spin-off from these projects, Inze's and Dudits's groups have jointly initiated a similar cDNA-AFLP approach for the study of cell cycle phase specific transcripts in rice.

Several proteins have been identified as key control points for cell cycle progression. Seven members of the KRP family of cell cycle inhibitor proteins have been characterized (CropDesigna anf Inze's group). KRPs bind and inactivate cyclin-dependent kinases (CDKs), which are considered to be the central regulators of cell cycle progression. Each of these KRP protein members is able to partially block cell division, leading to plants with much less but larger cells (De Veylder et al., 2001). Interestingly, knockout mutants in single KRP proteins did not show any apparent phenotype indicating functional redundancy within this protein family.

Activation of cyclin-dependent kinases by cyclins releases Rb and thus leads to activation of the E2F/DP complex. Over-expression studies have now revealed the dramatic effects of E2F/DP deregulation on cell division activity and overall plant development, thus confirming the key role of this complex in the cell cycle (De Veylder et al., 2002). To gain a better insight into the phenotypic behaviour of E2Fa-DPa transgenic plants and to identify E2Fa-DPa target genes, a transcriptomic microarray analysis was performed (Vlieghe et al., 2003). Out of 4,390 unique genes, a total of 188 had a twofold or more up- (84) or down-regulated (104) expression level in E2Fa-DPa transgenic plants compared to wild-type lines. Detailed promoter analysis allowed the identification of novel E2Fa-DPa target genes, mainly involved in DNA replication. Secondarily induced genes encoded proteins involved in cell wall biosynthesis, transcription and signal transduction or had an unknown function.

A large number of metabolic genes were modified as well, among which, surprisingly, many genes were involved in nitrate assimilation. Data suggest that the growth arrest observed upon E2Fa-DPa over-expression results at least partly from a nitrogen drain to the nucleotide synthesis pathway, causing decreased synthesis of other nitrogen compounds, such as amino acids and storage proteins. While over-expression of E2F or DP alone had only minor effects on plant development, increasing the expression levels of both proteins simultaneously caused a reiteration of G1/S transitions and the production of either very small or very large cells. This dichotomy in cell size was attributed to either the presence or absence of a mitosis-inducing factor. When present, G1/S reiteration would be ensued by mitosis, leading to enhanced rates of cell division and thus to more but smaller cells. In the absence of such mitosis-inducing factor, G1/S reiteration would cause consecutive rounds of DNA replication without cell division and thus result in large, endoreduplicating cells. The combination of both in a single plant clearly disrupted normal plant development, as illustrated by the fact that all plants over-expressing both E2F and DP exhibited an irreversible premature arrest in development. This mitosis¿inducing factor was subsequently identified as being a special type of CDK, named CDKB1;1, that is unique to plants (Boudolf et al., 2004).

With respect to the control of the G2/M transition, the picture is still more fragmentary. CDKs and cyclins clearly also control this phase transition, but the nature of the CDKs and cyclins is different for G1/S and G2/M. Degradation of mitotic cyclins, such as B2-type cyclins, will block G2/M transition and is essential for the onset of endoreduplication, a process of consecutive DNA replication rounds without intermittent mitosis. Interestingly, also KRP proteins appear to play a role in the control of endoreduplication and a simulation model has been proposed to explain how KRPs can arrest mitosis while G1/S transition is still maintained (Verkest et al., 2005).

Inze's group has published more that 13 papers in international peer reviewed journals.

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VIB, Department of Plant Systems Biology
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