The main objective is to identify the key elements involved in grain development. These key elements may correspond to critical phases or stages that are sensitive to climate change, to crucial cell compartments (endosperm or peripheral layers), and also to genes and metabolic pathways of importance to determining grain size under optimum conditions and under abiotic stress. Of the different grain compartments, we are focusing firstly on the endosperm, its size and its content are important components in yield, and secondly on the peripheral layers (cross-cells, tube cells) which are hypothesized to exert mechanical constraints that restrict grain growth. To summarise, we wish to combine studies at the cellular and tissue levels (endosperm, peripheral layers) and at the scale of the organ (final grain size). These studies will be performed on the Récital wheat line, cultivated under optimum conditions or under high temperature/water stress conditions, by varying the durations, intensities and stages of application of these constraints
We propose to pursue a study initiated during the previous four-year period on the effect of climate constraints on potential and final grain size and on different grain components, by focusing on the controlled application of high temperatures (under a variety of modes) and post-flowering water stress (with or without interactions). By multiplying the methods of application of environmental constraints, in this way, we hope to: (1) establish laws on how grain size responds to environmental factors, (2) develop, in collaboration with the BIG team, a simulation model based on SiriusQuality1, that can simulate the effect of the environmental factors associated with climate change, and (3) propose early-stage and pertinent cytomorphological variables correlated with final grain size. This approach, and the variables thus identified, will be used to characterise transgenic plants produced by the Unit's transgenesis platform that over-express the genes implicated in grain growth processes (cell division/cell elongation) and identified using the molecular approach , or isogenic lines that differ in terms of a grain size QTL. This phenotyping will be facilitated by the acquisition at the end of 2010 of an X-ray micro-CTscanner that enables the non-destructive 3-D visualisation of grain compartments, with an excellent spatial resolution (to a few µm) and rapid acquisition times.
In addition, these initial studies, combined with data from the literature, suggest that the peripheral layers are involved in determining potential grain size by limiting the internal volume available for storage accumulation. Furthermore, studies in sunflower have shown that high temperatures during grain development cause major modifications to the peripheral layers, leading to a marked reduction in grain mass. Thus the hypothesis of a role for the peripheral layers in controlling potential and final grain size will be studied by monitoring the kinetics of the sclerenchymatisation of certain components in the peripheral layers (cross cells, tube cells of the internal pericarp) using photon microscopy or UV spectrophotometry (in collaboration with the BIA in Nantes). These kinetics will be supplemented by in situ hybridisations of the genes implicated in the biosynthesis of lignins under normal versus high temperatures, in order to determine the importance (timetable and localisation) of the lignification of the peripheral layers of a grain to the establishment of its internal volume.
The development of an organ such as a wheat grain is a complex process that requires the sequential or combined intervention of a very large number of genes and metabolic pathways. Identifying all these pathways, and modelling their roles, is an important research objective. However, this requires the accumulation of a very large quantity of data and experimental results. In addition, it will not be possible to achieve this completely until the wheat genome has been fully sequenced. Of these metabolic pathways, that of carbon metabolism (and particularly of sucrose degradation and glycolysis ), phytohormone signaling pathways, the Ubiquitin/Proteasome 26S pathway (UPS) and the cell cycle appear to play an important role in the grain weight and yield of cereals. In the context of our project, we will focus on the phytohormone, UPS pathways and on the cell cycle. The approach chosen can be summarised in the following stages:
1- Transcriptomic analysis of early developmental stages, identified using the cytomorphological approach under both optimum conditions and high temperature stresses.
2- The differentially expressed genes identified will be confirmed and then localised using a high-throughput in situ hybridisation facility (in collaboration with the GRED Research Unit in Clermont-Ferrand). Only genes expressed in the endosperm and peripheral layers will be retained.
3- A biochemical analysis will be made on proteins coded by these genes, and notably the regulatory enzymes E3 ligases (UPS pathway). This analysis will include either measuring enzyme activity or searching for interactions using protein-protein interaction techniques (Y2H, BiFC, Immunoprecipitations, Pull-Down, etc.).
4- These genes will undergo functional validation, firstly by association genetics using the populations available within the team and the Unit. Genes that display a co-localisation with QTLs for grain weight will then be validated as a priority using transgenesis (RNAi and over-expression) by the Unit's "Functional Validation" platform. Collections of mutants (EMS and irradiation) may also be screened. The phenotyping of these lines will be performed on the basis of pertinent variables resulting from the cytomorphological approach.
This work will enable: (1) the identification of genes regulating endosperm and peripheral layer development, and (2) the establishment of relationships between these different signaling pathways and the proposal of functional and predictive models for wheat grain size.
The success of this project is conditional upon the availability of several tools that will be developed by the team: (1) the cultivation of wheat cells, (2) exhaustive information on the full sequences of genes (5' extremities) that can be obtained either by constructing "cDNA full-length" libraries or by massive sequencing of the RNASeq type in collaboration with the Genome team (C. Feuillet), and (3) the development of high-throughput tools for the analysis of protein-protein interactions, including the construction of several Y2H libraries and the initiation of Y2H or BiFC high-throughput screening.