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The original publication is available at www.springerlink.com.

Plant growth and development depends on controlled cell expansion. This, in itself, is determined by the plant cell wall, a structural matrix of polysaccharides encasing the plant cell. One line of investigation that has proven particularly successful in elucidating the components of the plant cell wall machinery has been the forward genetic screens of cell wall mutants. In this study, the molecular and cellular characterisation of sloughy, a cell separation mutant in Arabidopsis thaliana, was commenced. This mutant has a striking phenotype, with files of elongating epidermal cells snaking away from the adjacent epidermal cells and from the underlying cortex, loosing contact from the side walls while remaining attached at the cell ends, in a manner reminiscent of border-like cells in the root cap of arabidopsis. The sloughy mutation was fine mapped to a short region on chromosome I using high resolution melt point analysis. On sequencing all five genes in this region, a single nucleotide mutation, introducing a stop codon, was detected in exon 2 in the previously-described heat shock transcription factor SCHIZORIZA that results in a truncated protein missing several conserved domains essential for activity. SCHIZORIZA acts as a cell fate determinate in the root meristem to promote cortex fate, while suppressing epidermal and root cap fate in the mature ground tissue. Although the literature on schizoriza mutants has focused on the developing root meristem, with little documentation on the cell separation phenotype further up in the roots, the investigation of a collection of schizoriza TILLING mutants revealed that aberrant cell separation was ubiquitous to schizoriza mutants with a severely truncated protein. To investigate cell identity in the mature roots, sloughy was crossed to GAL4-GFP enhancer trap lines that act as cell-specific markers. Epidermal identity lines revealed that sloughy possessed a supernumerary ground tissue layer with epidermal identity. A cortex and endodermal line revealed that these two identities are restricted to the endodermal layer and the next ground tissue layer out. There was no indication of root cap identity in the mature root with any of the root cap lines used, although partial lateral root cap identity has been previously described in the epidermal and subepidermal cell layers in the meristem of schizoriza mutants expressing SOMBRERO-GFP, a lateral root cap-specific transcription factor. Immunolabelling of cell wall epitopes revealed that the JIM13 antibody, which specifically labels arabinogalactan-proteins in wild-type root caps, often labelled the epidermal cells and surrounding mucilage further up the in the roots of sloughy. The aberrant cell separation present in sloughy is thought to be a consequence of epidermal cells possessing partial lateral root cap identity. The data on sloughy/schizoriza is sufficient to generate a model on how a meristem developmental gene can generate a cell separation phenotype in the mature roots. Loss of SCHIZORIZA causes confused cell identity in the root meristem that results in an epidermal and subepidermal layer possessing mixed epidermal and lateral root cap identity. The distinctive properties of border-like cells in the root cap of arabidopsis have been linked to unique cell wall maturation and developmental processes, implicating the cellulases CEL3 and CEL5, the pectin glycosyltransferase QUA1, the pectin methyltransferase QUA2 and other pectolytic enzymes. The ectopic expression of these cell wall enzymes in the epidermal and subepidermal layers of sloughy roots result in reduced adhesion along the sides of the cell, while the ends remain attached, causing the observed cell separation phenotype.

Le comportement mécanique d’un sol constitue un des facteurs prépondérants affectant la croissance et le développement racinaire. L’augmentation de la résistance mécanique d’un sol affecte l’architecture du système racinaire, la croissance axiale et radiale des racines, et l’orientation de la croissance. Bien que ces réponses soient décrites sur diverses espèces végétales dans la littérature, leur mise en place et les mécanismes de signalisation sous-jacents restent à ce jour méconnus. L’objectif principal de cette thèse est d’étudier la mise en place des réponses des racines primaires d’Arabidopsis thaliana à des variations de contraintes mécaniques de son substrat. La littérature fait état jusqu’ici de deux voies de signalisation mécanique possibles, l’une impliquant une mécanoperception au niveau de la coiffe racinaire, et l’autre faisant intervenir entres autres l’activation de canaux mécano-sensibles au niveau de l’apex racinaire. Pour étudier leur implication respective, nous avons caractérisé les réponses à une augmentation des contraintes mécaniques de racines primaires issues de la lignée Arabidopsis thaliana sauvage et de lignées mutantes affectées au niveau de l’organisation de la coiffe (fez-2, smb-3, brn1-1 brn2-1) ou au niveau de canaux ioniques mécanosensibles (mca1-null, mslΔ5). Nous avons développé un système expérimental original basé sur le suivi de croissance et d’orientation des racines dans des milieux de culture à base de Phytagel. Ces milieux de culture permettent de mimer d’une part une augmentation à long terme et simultanée des contraintes mécaniques axiales, radiales et des frottements et d’autre part une augmentation à court terme des contraintes mécaniques axiales. Tout d’abord, nous avons observé que la mise en place des réponses de croissance des racines primaires d’Arabidopsis thaliana à ces augmentations de contraintes mécaniques reposait sur une interaction entre des processus mécaniques (flambement) et biologiques (régulations de la zone de croissance). Ensuite, nous avons mis en évidence que la coiffe racinaire participait à la mise en place des réponses des racines d’Arabidopsis thaliana face à une augmentation des contraintes mécaniques. Nos résultats suggèrent que la coiffe pourrait constituer un siège de la mécanoperception. Par ailleurs, nos résultats sur les lignées mca1-null et mslΔ5 suggèrent que les canaux ioniques MCA1 et MSLs participeraient également à la mise en place des réponses racinaires à l’augmentation des contraintes mécaniques. / Root growth and development are highly modulated by soil mechanical properties such as texture, structure and bulk density. Increases in soil penetration resistance affect root system architecture, root cell production and elongation, root diameter, and root tip orientation. Although root responses to changes in mechanical stresses are well described in several plant species, their establishment and the signaling pathways underlying these responses remains misunderstood. The main objective of this thesis is to study the establishment of Arabidopsis thaliana primary root responses to changes in mechanical stresses. So far, studies reported two distinct putative mechanical signaling pathways involving either (i) the root cap as a main mechanosensing site or (ii) the activity of stretch-activated ion channels localized on the cell plasma membranes along the root apex. According to previous studies, we studied the implication of the root cap and of the stretch-activated channels of the MCA and MSL families in the root responses to increases in mechanical stresses. We developed an original experimental set-up based on the monitoring of growth and orientation of Arabidopsis thaliana primary roots in one-layer and two-layer Phytagel based growth media. The one-layer growth media mimicked a long-term increase in frictions and in radial and axial mechanical stresses whereas the two-layer growth media mimicked a short-term increase in axial mechanical stresses. We characterized penetration abilities, growth rate and tip orientation of Arabidopsis thaliana wild type roots (Col-0) and mutant roots showing defects in root cap organization (fez-2, smb-3, brn1-1 brn2-1) or in stretch-activated ion channels activity (mca1-null, mslΔ5) in one-layer and two-layer media. Firstly, we observed that the establishment of root growth responses to increases in mechanical stresses involved both mechanical (buckling) and biological (growth zone modulations) mechanisms. Then, our experiments demonstrated that alterations in root cap organization led to altered root growth responses to increases in mechanical stresses. Our results suggest that the root cap acts as a mechanosensing site. Moreover, the results obtained with the mca1-null and mslΔ5 Arabidopsis lines suggest that the MCA1 and MSLs ion channels also participate in the establishment of root responses to increases in mechanical stresses.