Bilateral NSF/BIO-BBSRC: Unravelling the Grass Leaf

Flowering plants exhibit two major growth strategies. The dicot strategy is for the growing tip of the plant to climb upward by producing an elongating stem below it. Leaf buds are also generated at the growing tip and eventually these grow out from the stem to form fully grown leaves. The monocot strategy is for the growing tip to stay protected at the base of the plant and produce a series of concentric leaves that rise above it. Leaf blades emerge and bend outwards at the top of the concentric cylinder or leaf bases. Only at later stages does the growing tip itself rise upwards through elongation of the stem to produce the flowering structures. This monocot strategy has the advantage of protecting the growing tip at the base of the plant for much of its life history. It enables grasses to survive extensive grazing and is the growth strategy that underlies cereals like wheat, maize and rice.

Despite its ecological and agronomic importance, the monocot strategy is much less well understood than the dicot strategy. In particular, it is unclear how monocot leaf buds grow to form concentric cylinders topped by outwardly bending blades. By using computational modelling we have developed some preliminary hypotheses for how this might work. A key idea is that growth is oriented by a polarity field; analogous to the way a magnetic field can be used to orient directions of navigation. The observed growth and shape changes of the monocot leaf can then be explained by simple changes in the polarity field and pattern of growth rates it orients. The main aim of this proposal is to test and further build upon this model to determine whether the fields and rates of growth it predicts are correct or not. This will be achieved using the maize monocot system which has the advantage of having well developed genetics and associated technologies. By looking at markers that highlight the presumed polarity fields and determining the growth rates in different regions of the leaf we hope to test predictions of the model. Models will also be tested by analysing the mutants in which key transitions of development are disrupted. These studies will be made quantitative by writing computer programs that extract the relevant measures automatically. New computational methods will also be developed and applied to this system so that the processes can be understood at different levels, from cellular to tissue scale.

This type of study, which integrates computational and experimental approaches, should provide a rigorous and quantitative understanding of the mysteries behind the monocot growth strategy. The understanding it generates may also allow us to further modulate the shape and disposition of leaves in crops. The angle at which the leaf blade bends out, for example, depends on growth at the blade junction, and has an important effect on yield because it influences the amount of light that can be harvested for photosynthesis. Knowing how this process works and is controlled by genes may therefore help breeders improve crop performance.

Through a combination of experimental and modelling approaches we aim to produce a mechanistic model for how the key developmental transitions underlying the monocot strategy are generated and genetically controlled. Hypotheses for key developmental and shape transitions will be tested by visualising maize PIN auxin transporter proteins, which act both as markers for tissue cell polarity and as readouts for auxin-based polarity coordination mechanisms. Further tests will be carried out by following the expression of genes known to affect PIN1 polarity and key developmental switches in maize. We will also introduce Cre-Lox reporters to enable induction and visualisation of clonal markers at various stages, allowing hypotheses about growth patterns to be tested. Both Confocal and Optical Projection Tomography imaging will be used to obtain information from the cellular to whole organ scales. We will apply the above methods to wild type and mutants which affect key transitions and determine whether the results confirm or refute particular hypotheses. We will also use the Cre-Lox system to generate timed clonal sectors for genes such as KN1 and analyse their consequences on polarity and growth. In parallel, computational methods will be developed to extract salient features and parameters from images generated by immunolocalisation, GFP markers, clonal analysis and live tissue tracking. The results will provide quantitative measures for hypothesis testing. In conjunction with the experiments described above, hypotheses for key developmental transitions will be further formulated and modified using the GPT computational framework, which allows tissues to be modelled as 2D sheets that deform in 3D. This framework will also be elaborated to allow cellular level models for polarity coordination to be explored, and models for the formation of volumetric 3D outgrowths, such as ligules, to be incorporated.

This project will benefit non-academic beneficiaries, in the following ways:

1. Breeders will benefit from knowledge that will facilitate the selection of candidate genes for improving crop growth and yield by conventional or transgenic approaches. The expected time frame for this beneficial impact will be 10 years after the start of the project.

2. Biotech industries will benefit from our work in the long term, through the greater fundamental understanding of processes that underlie tissue properties in plants and animals. This may open up new avenues to exploit and manipulate growing tissues. They will also benefit from the tools developed in the project such as integrating computational methods with bioimaging approaches that may become applicable to their research and development programmes. The time frame for this type of impact is expected to be 10-20 years.

3. The general public and school children will benefit directly from this project through the proposed hands-on training events and through dissemination of latest research findings in an accessible way via media routes like youtube videos and press articles. They will also benefit in the longer term because of the contribution that this project will make to maintaining and developing forward-looking scientific research that provides the foundations of a modern healthy and growing economy.

4. BBSRC will benefit because the project is directly relevant to the research priority Systems Biology and the general area of food security. It will also benefit from the integration of expertise in developmental genetics of monocots from a leading lab in the USA, with computational modelling and image analysis in the UK, enhanced by leverage of funds from NSF.