Dissecting novel mechanically mediated growth co-ordination pathways in seeds – mécanograine

This multidisciplinary project uses a variety of approaches

1) Imaging. We will develop techniques for imaging developing seeds, and for extracting information from the images which we are able to obtain. Most of the imaging in this project will involve the use of classical confocal microscopes,

2) Transcriptome analysis. We will use whole transcriptome sequencing to undertsand the changes in gene expression caused by various genetic manipulations. The output from this analysis will inform genetic analysis, and permit the formulation of genetic models of the processes underlying the co-ordination of seed development

3) Molecular Genetics. We will combine classical mutant analysis with transgenic approaches, including the production of inducible genetic constructs, in order to understand the genetic networks underlying the co-ordination of seed development.

4) Gene Expression analysis. We will use imaging approaches (marker lines) and in situ hybridization to study the spatial and temporal regulation of gene expression.

5) Biomechanics techniques. We will test the utility of a range of methods for introducing mechanical perturbations into the seed system, including indentors, and drug treatments. This will permit the development of physical models of seed development.

So far we have been able to elucidate a molecular signalling pathway linking endosperm development (an in particular the function of the master endosperm regulator ZHOUPI) to the development of the embryonic surface. These results have been validated in two publications.

In addition we have identified a totally novel protein partner of ZHOUPI, which is, like ZHOUPI, required for endosperm degradation.

Our transcriptome analysis, in combination with imaging of seeds from various mutant backgrounds, has enabled us to elucidate the molecular mechanism underlying the biomechanical interplay between the developing embryo and endosperm.

Finally, we have advanced our analysis of a potential mechanosensor (DEK1) and in particular have identified some of its direct and indirect molecular targets.

Perspectives involve the generation of detailed images of developing seeds, in particular using novel lines expressing fluorescent proteins recently generated in the laboratory. This will allow the production of a physical framework on which to superimpose gene expression data which we have already generated

We will continue our biomechanical studies, and in particular measure the physical characteristics of seeds from a range of genetic backgrounds using a tissue indentor.

Finally we will continue our analysis of genetic networks identified in both the endosperm and testa which may be involved in responses to mechanical signals and which in turn, regulate the mechanical properties of developing seed tissues.

1. ZHOUPI controls embryonic cuticle formation via a signalling pathway involving the subtilisin protease ABNORMAL LEAF-SHAPE1 and the receptor kinases GASSHO1 and GASSHO2. Xing Q, Creff A, Waters A, Tanaka H, Goodrich J, Ingram GC. Development. 2013 Feb;140(4):770-9.
2. “What we’ve got here is failure to communicate” – zou mutants and endosperm cell death in seed development: Andrew Waters A, Creff A, Goodrich J and Ingram G. Accepted in Plant Signalling and Behaviour, 2013

The co-ordinated growth of different cell and tissue types is critical for the development of functional plant tissues and organs. Nowhere is this truer than in angiosperm seeds, where successful development necessitates exquisite co-ordination between three genetically and developmentally distinct tissues; the seed coat, the embryo and the endosperm. Despite its evolutionary and agronomic importance, the mechanistic basis for growth co-ordination in seeds, or indeed more globally during plant development, remains poorly understood. In particular the mechanisms underlying the perception of mechanical cues, which have long been proposed to play key roles in regulating plant growth, have proved elusive.
Disrupting pathways regulating plant growth co-ordination causes severe and pleiotropic defects throughout development. Such phenotypes are difficult to interpret possibly explaining why such important pathways remain so poorly elucidated. The Arabidopsis seed is a system in which some of these pathways can be dissected without affecting basic plant growth (for example the functioning of meristems). Particular attributes making the seed a good system for this type of study include:
1) The small size, relative accessibility, and determinate growth of the seed.
2) The genetic independence of the zygotic and maternal compartments.
3) The symplastic isolation of the embryo, endosperm and seed coat.
4) The existence of multiple seed-tissue specific promoters for use in tool development.
In this project we will make use of these attributes to develop the seed as a novel system in which to study the mechanics of seed tissue growth from both a physical and a genetic perspective. Firstly, we will generate detailed descriptions of early seed development which will provide a physical framework for the integration of mechanical and genetic information. We will investigate new techniques for assessing mechanical forces within the developing seed. In addition, we will develop novel tools for dissecting gene function at the level of individual seed tissues, which can be applied to the study of both seed specific and globally important pathways identified during previous research in the Ingram lab. Finally we will make use of approaches being developed in the host laboratory for modelling developmental processes in plant tissues, to generate “virtual seeds”; simple physical and genetic models of aspects of seed development. The emergent properties of these models will allow us to make non-intuitive predictions about the outcomes of physical and genetic manipulations, thus informing ongoing research in the laboratory.
In addition to their fundamental roles in plant development, mechanical cues are known to influence agronomically important traits. Our preliminary results show that mechanically regulated co-ordination of tissue growth in seeds could influence traits such as seed size and yield. More globally mechanical stress is known to influence traits such as cell wall thickness, which could be of key importance for increasing cellulosic biomass, and for modifying stem strength, for example in the generation of lodging-resistant cereals. For this reason the proposed research will be of long term interest in terms of providing potential targets for crop engineering. In this project we will, in direct collaboration with the host laboratory, investigate the possibility of transferring of our most important findings, into a cereal crop (maize).