EPF2 and the Molecular Regulation of Stomatal Development

Stomata are pores on the surface of leaves which allow exchange of gas for photosynthesis and loss of water vapour via transpiration between the interior of the plant and the atmosphere. The evolution of stomata, and their ability to control water loss, is widely believed to have underpinned the colonisation of the land by plants and their subsequent spread throughout terrestrial environments. Today, stomatal behaviour underpins our crop productivity, and it has never been more important to understand the pathways that control stomatal development and function. Although plants are well known to adapt to stresses such as drought by closing their stomata, under longer term exposure to environmental change they adapt further by adjusting the number of stomata that develop on their new leaves. For leaves to function efficiently the frequency and spacing of their stomata must be optimal, and plant scientists have begun to identify some of the genetic factors that regulate this. We have recently characterised a factor which like the mammalian hormone insulin, is a secreted peptide. Plants lacking this peptide develop extra stomata, and many extra stomatal precursor cells are formed in the leaf surface. Plants manipulated to overproduce the peptide develop almost no stomata and grow slowly. Thus, the peptide we have studied is an inhibitor of stomatal development. In this project we will find out more about how this peptide signal works. We will find out whether the peptide works together with other factors identified as regulating stomatal development, and we will investigate whether it, like insulin, is activated by an enzyme that first breaks it into smaller fragments.

Stomata are microscopic pores on the surface of leaves. Each pore is formed by a pair of guard cells that expand and contract to control pore aperture, and regulate plant CO2 uptake and water loss. Work from a number of laboratories over the past decade indicates that the initiation of stomatal development is controlled by a signalling pathway involving a processing protease SDD1, ERECTA-family LRR receptor kinase(s), a receptor-like protein TMM, a MAP kinase cascade and downstram transcription factors. However, many stomatal development components remain to be identified. Until recently, one of the components that has been missing from this pathway was the secreted peptide ligand predicted to be processed by the SDD1 protease and to activate the TMM/ERECTA LRR receptor complex. Our recently published results identify a regulator of stomatal initiation that is a strong candidate for the secreted peptide ligand for the TMM and/or ERECTA-family receptors, but suprisingly this peptide is not processed by SDD1 (Hunt & Gray, Current Biology 2009). The EPF2 peptide regulates cell fate early in the developing epidermis and somehow controls whether cells enter the stomatal development pathway or whether they become epidermal pavement cells. The major aim of the work described in this proposal is to better understand how EPF2 controls epidermal cell fate decisions. The described experiments will identify the specific regions of EPF2 responsible for regulating stomatal development, identify which of the four putative receptor components interact with EPF2, and by carrying out a transcriptomic experiment identify other 'missing' components in the EPF2 signalling pathway. We have recent, unpublished results suggesting that two other members of the EPF peptide family can also restrict stomatal development, probably by inhibiting slightly different steps in the cell division programme. We therefore propose to include EPFL2 and EPFL7 in our studies too.

Our research will be of importance to academics with an interest in plant physiology and development. We will use the usual routes for disseminating scientific results through the publication of papers in international peer-reviewed journals, conference presentations and invited lectures at UK universities departments and government research institutes, and disseminate our work to a wider audience via public understanding of science events. Within six months of completion of the project, resources generated will be deposited in public databases or stock centres as appropriate. All completed datasets will be made publicly available as soon as feasible. We will play an active role in discussing our findings with the general public to ensure effective outreach. Prof Gray has been involved in scientific outreach activities for many years as a BBSRC Local Schools Liaison Officer and recently hosted the RCUK Darwin200 exhibition in Sheffield. Training impacts: Although the PDRA Dr Hunt is an experienced plant scientist this project presents a number of diverse training opportunities that will enhance his specific and generic skills. These include associated bioinformatic and genomic techniques, experimental design, data analysis and manuscript preparation. Dr Hunt will be encouraged to participate in Sheffield Research Leaders' Programme - a coherent framework of skills training provision for University of Sheffield research staff. The technician will be encouraged to be closely involved with the direction and planning of the research as well as its execution, and will be given training in areas as appropriate. As detailed in the proposal, this project will produce plants with a range of stomatal densities and clustering patterns and, for the first time, in the same genetic background. Analysis of these stomatal variant plants could enable us to identify optimal stomatal development characteristics for a particular environment (e.g. optimise plants for current or future atmospheric CO2 levels). Indeed, more complete understanding of stomatal development and function will be important for enhancing global food security in a changing climate. It is clear from the 'Living with Environmental Change' agenda and the recently published Royal Society 'Reaping the Benefits' policy report that the efficient use of water will become a key political and economical challenge on a global scale over the next decades. Even at a local scale, lack of water in the East of England is set to become the main limiting factor for UK grain production, and thus food security. Understanding and optimising water use by both crop plants and non-crop plants will be essential in efforts to meliorate global warming. Stomata are key in allowing plants to regulate water loss. Understanding their development and spacing may afford new perspectives on how to optimise water use so that a sustainable and equitable means of food production is achieved. At the molecular level, further understanding of stomatal patterning may lead to the identification of novel targets for both breeders and agrochemical producers to increase water efficiency.