Servizio Comunitario di Informazione in materia di Ricerca e Sviluppo - CORDIS

Tools to study plant cell cycle

Many cell cycle genes are expressed at very low levels and it was unclear at the start of the project whether the sensitivity of microarrays would be sufficient for detecting modulations in expression of low-abundant cell cycle genes. The results that have been obtained here demonstrate the suitability of this method and data on cell cycle gene expression in different organs and in four cell cycle mutant lines have been generated. The current array is focused on cell cycle genes, which has the advantage that results can be analysed without the need for extensive bioinformatics support and at relative low cost. Complementary to this cell cycle array, a genome-wide array is accessible to the consortium (Affymetrix, Gruissem's group).

In the course of the project, Meyer's group has optimised individual steps of microarray preparation and sample labelling, which allows the collection of statistically sound data and has improved the sensitivity of the microarray assay using a linear amplification method. Inflorescence tissue and suspension culture cells were identified as suitable material for microarray analysis of cell cycle genes.

Meyer's group used inflorescence RNA to characterise the expression profile of cell cycle genes in 8 homozygous insertion lines, which were obtained from CropDesign. These 8 lines have T-DNA insertions in E2F5, KRP6, FKH2, KRP1, KRP3, S6b, DPa, S6kinase respectively. It is noteworthy that HAP genes are often upregulated in KRP altered TDNA lines; HAPs are transcription factors of the NF-Y family known to control several important processes such as cell cycle (animals), embryo development and flowering in plants. This finding corroborates results obtained in the animal field. It seems that for only 1 line (Dp1a) the decrease of target gene expression is visible on the macroarray. The other lines could be upregulation lines (as suggested by the increased expression of the target genes on the macro).

Bergounioux's group has submitted the CDC45 RNAi construct to Meyer's group to monitor the expression of selected cell cycle genes in its plants. Results indicate that 5 of the 12 clones that were down-regulated are cyclin-dependent kinase inhibitors (KRPs). Other clones that were down-regulated include CDC6 of the prereplication complex, DP1a and E2F-like factors of transcription involved in activating genes of the G1/S transition, and a wee1 homolog, a negative regulator of entry into mitosis. Genes that were upregulated include HAP transcription factors (homologs of yeast transcription factors that bind the CCAAT box in many eukaryotic promoters and that exists in multiple forms in Arabidopsis [Edwards et al., 1998]) and genes involved in the G1/S transition or S-phase, such as CDC2 or histone H4, respectively (Stevens et al., 2002).

In addition Meyer's group has performed some analysis of salt stressed arabidopsis cell cultures using the cell cycle macroarray. This study pointed out some new and interesting data.
S6ribosomal 2 and histone H4 are upregulated in two different situations of salt stress. This corroborates earlier data by Mizoguchi et al (1995) on the induction of S6 kinases after cold and salt stress treatment indicating that protein synthesis is a way to adapt plants to different stresses (need for new proteins as a stress response and an adaptation mechanism). By adapting the protein translation machinery the cell can modulate the transcripts that are preferentially expressed. Translational control would thus permit a rapid switch from cell division to stress defence, even before new transcripts are produced. Along the same line, it has also been shown that the over-expression of the sugar beet eIF1A specifically increased the sodium and lithium salt tolerance of yeast (Rausell et al., 2003). In addition, KRP1, Fbox, cyclin C2 and various CDKs were down regulated after salt treatment with 250mM salt whereas Y2H GT-1 transcription factor, cyclin D3, CDC20 are down-regulated after mild treatment. Several cyclins, CDKs are down regulated after both mild and strong stress treatment which is in line with a model predicting a switch in transcription from cell division genes to stress defence genes. The down-regulation of KRP1, a CDK inhibitor seems contradictory at first sight. Yet KRPs have a function during cell division as a modulator of CDK activity. When CDKs are not expressed, there is no role for KRPs either.

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Reported by

University of Leeds
University of leeds Institute for plant biotechnology and agriculture, Woodhouse Lane
LS29JT Leeds
United Kingdom