Growth and Development of higher plants

The linear organization of a root tip, with cells organised in discrete files radiating from the quiescent centre and with successive regions of meristem, elongation zone and mature cells make it an ideal system to investigate the cellular and molecular basis of plant growth regulation. Moreover, the minimal number of cell layers in the roots of Arabidopsis limits its thickness to only 150 microns, which facilitates microscopical analysis by means of light (using DIC optics) and confocal microscopy (using fluorescent dyes or GFP tagged lines). To investigate the relationships between the cellular processes cell division and cell expansion and growth of the root as a whole, kinematic analyses have been developed based on fluid dynamics. P1C has successfully pioneered the practical use of these methods for the studies of cell division and cell expansion rates in Arabidopsis roots analyzing the effect of stress conditions (salt stress), natural (ecotypes) and engineered genetic variation (mutants and transgenic lines). This state-of-the-art kinematic platform has been extensively used during the previous phase of this network by various partners and will be again crucial for understanding root growth phenotypes obtained in this project.
These analyses unequivocally established the importance of cell division parameters (cell division rate and size of meristem) in determining root growth rates. Therefore P1C conducted a gene discovery experiment using genome-wide Affymetrics microarrays, analyzed gene expression in three zones of the root tip (meristem, elongation zone and mature tissue) and compared with corresponding stages of developing leaves. This way P1C was able to identify 430 genes that were specifically expressed in proliferating tissues. A large number of those genes are already known and almost without exception have a direct link to cell division. Therefore the unknown genes in this selection are strong candidates for new functions in cell cycle and plant growth regulation and they will be functionally analyzed in the framework of this project.
In this workpackage, P1C will generate overexpressing lines, knock-outs or knock-downs using publicly available T-DNA mutant collections of a selection of these putative new 430 cell division genes. Preferentially unknown genes will be selected based on criteria such as fold-change, number of homologs in Arabidopsis, presence of homologs in the human genome and type of the protein that is encoded (e.g. transcription factors, kinases and completely unknowns). These genes will be functionally analyzed. The phenotype of such lines will be studied using an array of tools available, among others including kinematic analysis, transcript profiling and flow-cytometry that will facilitate the identification of the function of the gene involved.
This analysis will no doubt increase further the already large number of genes involved in cell cycle regulation. Together these gene products form a regulatory network that is complex and often behaves in unexpected ways. To start addressing this complexity in the context of a growing multi-cellular organ, it is necessary to develop computer models that simulate experimentally determined and hypothetical interaction mechanisms (see WP 6, P1A, P1B, P4C, EU1).
Furthermore, P1C will continue performing kinematic analysis of genetic material and environmental or physiological treatments that are relevant to the project (P5, EU1).

Endoreduplication is a form of polyploidization that occurs in the somatic cells of many eukaryotes. It is a modified cell cycle in which part or all of the genome is replicated without the subsequent sister chromatid separation, nuclear division and cytokinesis. In animals, endoreduplication occurs in a variety of cells and tissues, where it is thought to be related to differentiation, cell size, and the level of gene expression. In plants, endoreduplication occurs in cells of vegetative and reproductive tissues. Despite its widespread occurrence in plants, the mechanisms which direct the switch from the mitotic cell cycle into an endocycle are still poorly understood. The developmental context and the regulation of endoreduplication in Arabidopsis roots have not been investigated in detail. In addition, the physiological relevance of endoreduplication in plants is still subject of debate.
P1B aims to identify regulatory pathways for the switch from the mitotic cycle to the endocycle in the root. For this purpose we will focus on the Arabidopsis root as an experimental model and we aim to benefit from the vast amount of genetic tools generated by the Arabidopsis community, in particular the opportunity of genome-wide expression profiling and the availability of tissue specific cell tagging. In addition, we aim to explore the developmental significance of the occurrence or absence of endoreduplication in the various root tissues.
First, P1B will generate a ploidy map of the Arabidopsis root. Using transgenic lines expressing green fluorescent proteins in different root tissues or cells within different stages of cellular differentiation will permit fluorescence activated cell sorting (FACS). Using this high resolution sampling technique in combination with DNA flow cytometry, we will create a detailed map of the nuclear ploidy within the different root tissues and examine the extent of endoreduplication in the different cell types with respect to their position along the root axis. These measurements can be backed up by confocal cytometry. Currently, different enhancer trap lines which direct GFP expression to specific root cells and tissues are publicly available (http://www.plantsci.cam.ac.uk/Haseloff/geneControl/GAL4Frame.html). In addition, UGent has a collection of cell cycle gene reporter lines which can be used to this end (in collaboration with P1A).
Secondly, the role of endoreduplication in the differentiation of different root tissues will be investigated by P1B. The transcriptional suppressor DEL1 has been identified as a factor that suppresses the endoreduplication cycle in Arabidopsis. The overproduction of DEL1 protein levels by targeted expression of a DEL1OE construct to different root cell types by means of enhancer trap driving the GAL4 activator, will provide us with a means to manipulate endoreduplication within the different root tissues. This enables us to investigate the role of endoreduplicaton in the differentiation and cellular growth of the various tissues, and to examine the role of endoreduplication in these tissues in relation to the overall growth of the root (in collaboration with P1C and P1D).

During the previous network P1A developed a research strategy to unravel the molecular mechanisms controlling the branching of roots. This resulted in a unique dataset reflecting the transcriptional changes associated with the initiation of lateral roots. An additional transcriptome analysis based on cell sorting refined the still extensive list of candidate “lateral root initiation” genes to a set of potential regulatory genes involved in asymmetric cell division, a hallmark of the onset of many vital developmental processes in both plants and animals.
In the present work package, P1A will continue on the same path and concentrate on the functional analysis of potential regulators of lateral root initiation and/or asymmetric cell division. Classically, functional analysis of regulatory genes relies almost entirely on the study of knock-out mutants. However, inherent to vital regulatory genes, such mutations will most likely be lethal. In the coming phase of the network we want to exploit the benefits of a chemical genetics approach to intercept key regulators of the process of lateral root initiation (see Integrated Advanced Techniques).
Chemical genomics makes use of small molecules to disturb signal transduction cascades allowing the identification of the involved proteins. The project aims at the identification of inhibitory or activating compounds of cell cycle activation in the root. A chemical library of 10 000 compounds will be tested on the potential effect of certain chemicals during the very early phases of lateral root formation using a well established lateral inducible system.
Once candidate compounds, negatively or positively influencing the process of asymmetric cell division, will be identified and validated, the bioactive aspects can be worked out further in collaboration with the other partners of the network.
In a first instance, the changes in the proteome upon application of the bioactive compound will be analysed. Such analysis will contribute seriously towards the elucidation of the target-pathway of the compound.
Within this project the isolated compounds can easily be tested for their role in or interference with other root developmental processes such as endoreduplication (P1B), hormonal signalling cascades (P1D), primary root growth rate (P1C), cell elongation (P5) and root/shoot crosstalk during floral induction (P2).
Furthermore, the experimental set-up and the library of the chemical genomics platform can made available for the partners of the network to perform their own screenings using the appropriate conditions and markers related to their projects.
Finally, in the recent post-genomic era the accumulation of an enormous amount of data necessitates the implementation of modelling. Therefore, we want to model the early cell division patterns of lateral root initiation in spatially-structured models combined with a modelling of the molecular networks involved (see WP6, in collaboration with P1C, P4C, EU1).