Dynamic and specificity of CUC transcription factors during leaf development : Towards a high- resolution Gene Regulatory Network model – LEAFNET

A combination of genetic, molecular biology, biochemistry, imaging, biophysics and computational biology approaches has been designed to unravel the GRN controlling leaf morphology. The development of new technologies makes such multidisciplinary approaches possible. New sequencing technologies have shortened grandly mapping strategies; improvement of ChIP-Seq methods allow to locate precisely TF-DNA interactions; biophysical model of TF binding preferences allow to predict accurately binding sites at whole-genome level. We propose to take advantage of these technologies to generate a high-resolution GRN model of leaf development.
This project will use cutting edge genomics technologies to harness regulatory information in order to fill the gaps in our understanding of leaf shape development. We will focus on leaf morphology as a fundamental and experimentally tractable biological system. Using genomic and post-genomic tools integrated in a system biology approach, we aim (i) to reveal the dynamic of CUC homo/hetero dimers formation (ii) to understand the molecular contribution of CUCs transcription factors to leaf form, (iii) to set-up the binding specificities of CUC transcription factors and (iv) to identify new component downstream of CUC genes in this pathway.

- Task 1 : CUCs dimers
Plants expressing tagged version of CUC2 and CUC3 proteins were produced (35S::CUCs-Tag) to perform Co-Immunoprecipitation experiments.
We started interactions analysis studying homo-dimerization of CUC2 using Fluorescent Anisotropy techniques with pCUC2:CUC2-VENUS lines produced in the lab.
- Task 2 : CUCs Targets.
We produced plants with pCUC2:GR-CUC2 or pCUC3:GR-CUC3 in cuc2; cuc3 mutants background. DEX induction have to be carried out to test these inducible lines and crosses have to be done to obtain inducible CUCs constructs in simple mutants.
We want to use lines produced for Task 4 to perform ChIP experiments (35S::CUCs-Tag). We are currently testing whether these lines are suitable for ChIP experiments.
- Task 3 : CUCs binding preferences
This task will partially dependent on the success of the Task 2. Therefore, Task3 will be started when results will be obtained in Task2.. It is worth to notice that we planned alternative strategies to overcome a failure of Task 2.
- Task 4 : Genetic screen
To identify new components controlling leaf margins shape downstream of CUCs, we initiated a genetic screen in a pCUC2::CUC2gm4 transgenic background. These lines show highly serrated leaves and we are looking for suppressors exhibiting wild-type phenotype. To do so, Seeds of the pCUC2::CUC2gm4 line were mutagenized with EMS and a population of 6000 M1 was generated.
Up to now, we screened half of the population and identified approximately 108 putative mutants. We confirmed, in the progeny, the suppressor phenotype for 8 mutants.
As planned in the proposal, the mutations responsible for the phenotype will be identified using the SHOREmap strategy. Crosses necessary for these approaches were made and the F1 generations are currently growing.

The identification of new components in the leaf shape GRN and the generation of computational model of the regulatory interactions will strengthen current knowledge on leaf development and widen the perspective for crop improvement, having therefore a high impact in the area of growth and development.

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The main goal of this project is to understand the regulatory logic underlying the control of leaf shape in plant focussing on a set of key regulators, the CUCs transcription factors.
The diversity of shape seen in the plant kingdom is astonishing. How from small groups of cells encompassed in a primordium such variety of shape can be generated? The answer is maybe lying in the intricate regulatory network controlling cell fate and boundaries. It is believed indeed that changes in gene regulatory network (GRN) grandly contribute to morphological changes and therefore represent large evolutionary targets potentially producing important effects with limited pleiotropy (Doebley JF et al., 2006; Carroll SB, 2008; Arnaud et al., 2011). The proposed project is an opportunity to understand the regulatory logic of transcription factors, the combinatorial regulatory code governing a developmental process. To understand how genes control shape - a question relevant to all aspect of developmental biology and with important implications on crop improvement - we propose to focus on one of the simplest plant structure, the leaf. Grasping the mechanisms regulating leaf development will require the understanding of the regulatory logic of CUP-SHAPED COTYLEDON (CUCs), which are master regulators of leaf shape.
This fundamental research project will use a highly integrative approach combining molecular, genetic and computational approaches to gather information relative to GRN controlling leaf serration. Such a multi-disciplinary approach will allow us to define the GRN controlling leaf margins at the molecular level, and use these data to generate a high-resolution model of the GRN that control leaf morphology. The identification of new components in the leaf shape GRN and the generation of computational model of the regulatory interactions will strengthen current knowledge on leaf development and widen the perspective for crop improvement, having therefore a high impact in the area of growth and development.