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New techniques to study cell cycle

Traas' group has focused his work on the understanding of the role of cell cycle genes on meristem organisation and plant growth. A first requirement for studying the effects of cell cycle genes on meristem function is the development of a reproducible method for studying cell division dynamics in the shoot apical meristem. A method was developed that allows following individual cells in the living shoot apex. Plants are germinated on NPA, an inhibitor of auxin transport. These plants form naked inflorescence stems, without any primordia. When transferred to a medium without inhibitor, these naked meristems will subsequently form primordia.

These regenerating meristems can be directly stained with vital dyes and viewed in the confocal microscope. Using this technique, Traas's group was able to follow cell divisions throughout the shoot apex during several days (Grandjean et al., 2004). This method, combined with green fluorescent protein marker lines and vital stains, allows us to follow the dynamics of cell proliferation, cell expansion, and cell differentiation at the shoot apex. Using primordium promoters (ANT and LFY) driving GFP expression, the recruitment of meristematic cells in the incipient flower primordia was followed. This suggested that cells preferentially activated the reporter genes just after cell division, i.e. while they are in early G1. Using this approach, the effects of several mitotic drugs on meristem development were studied. Oryzalin (depolymerising microtubules and blocking the cells in G2/M) very rapidly caused cell division arrest. Nevertheless, both cell expansion and cell differentiation proceeded in the treated meristems.

Interestingly, DNA synthesis was not blocked, and the meristematic cells went through several rounds of endoreduplication in the presence of the drug. We next treated the meristems with two inhibitors of DNA synthesis, aphidicolin and hydroxyurea. In this case, cell growth and, later, cell differentiation was inhibited, suggesting an important role for DNA synthesis in growth and patterning. In addition, cells at the periphery of the meristem and in the young primordia expanded much faster than those at the meristem centre. This showed that differential cell expansion rates and cell differentiation do not necessarily depend on the cell cycle. Traas's group has been also working on a model in which auxin gradient drives the expression of certain genes such as Aintegumenta, and PIN1 as well as certain cell division events (use of cell cycle blockers).

Traas's group has also concentrated on the analysis of the expression of 7 CDK inhibitors, called CKI 1-7 (or KRP1-7), using in situ hybridisation in order to determine the localisation of CKI 1 to 7mRNAs in the Shoot Apical Meristem. From the 7 genes studied, P8 showed that CKI 2 and CKI 3mRNAs accumulate in the SAM at different developmental stages. CKI 2mRNA accumulates in young primordia at the vegetative stage and in the SAM at the inflorescence stage. CKI 3mRNA accumulates in the central cylinder of mature embryos and under the rib zone in vegetative and inflorescence SAMs. The other CKIs were below the detection level. At this stage, we do not intend to continue the in situ hybridisation studies.

A total of 25 T-DNA insertion mutants have been identified in cell cycle genes in collaboration with CropDesign. Subtile phenotype has been observed on petal cell size in KRP mutants. In addition, another cell cycle mutant in E2F5 gene was shown to have a leaf phenotype. The analysis of these mutants by Meyer's group has already identified target genes that could be related with the observed phenotypic defects.

The struwwelpeter (swp) mutant in Arabidopsis shows reduced cell numbers in all aerial organs (Autran et al., 2002). In certain cases, this defect is partially compensated by an increase in final cell size. Although the mutation does not affect cell cycle duration in the young primordia, it does influence the window of cell proliferation, as cell number is reduced during the very early stages of primordium initiation and a precocious arrest of cell proliferation occurs. In addition, the mutation also perturbs the shoot apical meristem (SAM), which becomes gradually disorganized. SWP encodes a protein with similarities to subunits of the Mediator complex, required for RNA polymerase II recruitment at target promoters in response to specific activators. To gain further insight into its function, we over-expressed the gene under the control of a constitutive promoter. This interfered again with the moment of cell cycle arrest in the young leaf. Our results suggest that the levels of SWP, besides their role in pattern formation at the meristem, play an important role in defining the duration of cell proliferation.

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Jan TRAAS, (Head of Unit)
Tel.: +33-4-72728614
Fax: +33-4-72728600
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