Trichome and root hair patterning are governed by a conserved cassette of bHLH and MYB factors, the WD40 protein TTG1, and six single-repeat MYB R3 factors that are thought to counteract them. In this work we focus on the single-repeat R3 factor ETC3 and show that its major role is in the regulation of trichome density in a redundant manner. Diversification of the ETC3 gene has occurred at the promoter level, as etc3 mutants can be rescued by expressing ETC3 under the control of the TRY or CPC promoter. ETC3 movement was detected between epidermal cells as well as between the epidermis and underlying tissues. Finally, we found marked differences in the ability of the single-repeat R3 factors to interfere with the dimerisation of GL1 and GL3 in a yeast threehybrid system, with CPC being the most potent inhibitor followed by ETC1, TRY, ETC3 and ETC2. Mathematical analysis predicts that this behaviour has a major impact on protein mobility, suggesting a tight reverse correlation between inhibitory function and the diffusion/transport range of the inhibitors. This prediction is supported by a comparison of CPC and ETC3 mobility in egl3 gl3 double mutants and 35S:GL3 lines.

Trichome patterning in Arabidopsis serves as a model system for de novo pattern formation in plants.It is thought to typify the theoretical activator-inhibitor mechanism, although this hypothesis has never been challenged by a combined experimental and theoretical approach. By integrating the key genetic and molecular data of the trichome patterning system, we developed a new theoretical model that allows the direct testing of the effect of experimental interventions and in the prediction of patterning phenotypes. We show experimentally that the trichome inhibitor TRIPTYCHON is transcriptionally activated by the known positive regulators GLABRA1 and GLABRA3. Further, we demonstrate by particle bombardment of protein fusions with GFP that TRIPTYCHON and CAPRICE but not GLABRA1 and GLABRA3 can move between cells. Finally, theoretical considerations suggest promoter swapping and basal overexpression experiments by means of which we are able to discriminate three biologically meaningful variants of the trichome patterning model. Our study demonstrates that the mutual interplay between theory and experiment can reveal a new level of understanding of how biochemical mechanisms can drive biological patterning processes.

SUMMARYTrichome and root hair patterning is governed by a gene regulatory network involving TTG1 and several homologous MYB and bHLH proteins. The bHLH proteins GL3 and EGL3 are core components that serve as a regulatory platform for the activation of downstream genes. In this study we show that a homologue of GL3 and EGL3, AtMYC1, can regulate the intracellular localisation of GL1 and TRY. AtMYC1 protein is predominantly localised in the cytoplasm and can relocate GL1 from the nucleus into the cytoplasm. Conversely, AtMYC1 can be recruited into the nucleus by TRY and CPC, concomitant with a strong accumulation of TRY and CPC in the nucleus. When AtMYC1 is targeted to the nucleus or cytoplasm by nuclear localisation or export signals (NLS or NES), respectively, the intracellular localisation of GL1 and TRY also changes accordingly. The biological significance of this intracellular localisation is suggested by the finding that the efficiency of rescue of trichome number is significantly altered in NES and NLS fusions as compared with wild-type AtMYC1. Genetic analysis of mutants and overexpression lines supports the hypothesis that AtMYC1 represses the activity of TRY and CPC.

Individual plant cells are rather complex mechanical objects. Despite the fact that their wall mechanical strength may be weakened by comparison with their original tissue template, they nevertheless retain some generic properties of the mother tissue, namely the viscoelasticity and the shape of their walls, which are driven by their internal hydrostatic turgor pressure. This viscoelastic behavior, which affects the power-law response of these cells when indented by an atomic force cantilever with a pyramidal tip, is also very sensitive to the culture media. To our knowledge, we develop here an original analyzing method, based on a multiscale decomposition of force-indentation curves, that reveals and quantifies for the first time the nonlinearity of the mechanical response of living single plant cells upon mechanical deformation. Further comparing the nonlinear strain responses of these isolated cells in three different media, we reveal an alteration of their linear bending elastic regime in both hyper- and hypotonic conditions.