New insights into the control of stomatal aperture and development by CO2

Stomata are pores found on the surfaces of leaves that open and close and thereby control the uptake of carbon dioxide (essential for photosynthesis and dry matter accumulation) and the loss of water vapour from the plant (minimising water loss is important in drought tolerance). The aperture of the stomatal pore and the number of stomata that develop on the surface of the leaf are controlled by environmental factors, such as light intensity and the concentration of carbon dioxide in the environment. The acquisition of stomata is considered to be a key element in the evolution of land plants as it allowed them to inhabit a range of different, often fluctuating environments, and still control water content. Stomata exert major controls on the water and carbon cycles of the world. This can be readily appreciated at the local level where a one hectare crop of wheat in the UK will lose 60 tonnes of water a day through its stomata during the summer months. Accordingly, understanding how stomata work is important both for agriculture, especially in the context of soil water conservation/crop water use efficiency, and for predicting the impacts of global environment change (impact on water/C cycles). The objective of this application is to understand how carbon dioxide controls stomatal aperture and the number of stomata that form on the leaf surface. In the absence of internationally binding legislation global atmospheric concentrations of carbon dioxide are set to increase. We know that increased concentrations of carbon dioxide cause a) less stomata to form on leaves and b) stomatal pores to decrease in aperture. We are specifically interested in finding out the molecular details of how these two processes happen. Our application is based on preliminary work in which we have identified plants unable to respond appropriately to increased concentrations of carbon dioxide (they either fail to close their stomata in response to carbon dioxide or are 'super-sensitive', while some fail to adjust the number of stomata that develop on the surface of the leaf). We will use genetic approaches to find out which genes are disrupted in these individuals and use physiological experiments to understand what cellular processes are damaged and thereby cause the failure to respond. The results of our work will provide us with new insights into how carbon dioxide controls the number of stomata that form on the leaf surface and the way that they open and close in response to this important greenhouse gas. Understanding how these processes occur is likely to be of benefit to plant breeders interested in producing new varieties of crop better able to cope with growing in a climate characterised by increased concentrations of carbon dioxide. Our work also fits well with UK Government Research Programmes 'Living With Environment Change' and 'Global Food Security'.

We aim to understand how CO2 controls stomatal pore aperture and density. We will use three new HIC loci to address these objectives. Objective 1. Characterise new loci involved in the control of stomatal development and function by CO2 and place them in the signalling networks controlling stomatal function and development. 1.1. To identify the causal mutations in hic3 and hic4 genetic mapping coupled to whole genome sequencing will be used. 1.2. To characterize the role of the HIC3 and HIC4 loci we shall use stomatal function and development bioassays. 1.3. To characterise why hic2 is compromised in CO2-induced but not ABA-induced closure we shall investigate membrane trafficking. 1.4. We will place the new HIC loci relative to the CO2-induced increase in [Ca2+]cyt using cameleon technology. 1.5. We shall use epistatic analysis to place the HIC genes relative to one another and known components in the stomatal CO2 signalling pathway. Objective 2. Test the hypothesis that ROS are involved in the control of stomatal development and function by CO2. 2.1. We shall quantify H2DCTDA fluorescence in CO2 - treated stomata and investigate whether CO2 -induced closure is compromised in Atrboh mutants. 2.2. We shall investigate stomatal density in Atrboh mutants grown under ambient and elevated CO2 . Objective 3. Test the hypothesis that stomatal density in developing leaves is controlled by transpiration rates in mature leaves and that ABA is a signal involved in this process. 3.1. We will investigate whether ABA is a long distance negative regulator of stomatal development. 3.2. We will place the HIC loci relative to ABA biosynthesis in the stomatal development pathway. On completion we will have doubled the number of characterised loci known to be involved in stomatal CO2 -specific signalling and as such made a major contribution to this important area in plant biology.

Who will benefit from this research? Beyond academic audiences where the impact is obvious in terms of increased understanding of how a model plant single cell signalling system works, the three beneficiaries of the work described in this proposal are the commercial private sector, the public sector and the wider public. There are main mechanisms to engage these groups. To engage the commercial and public sectors the work will be presented either as poster or oral presentation format during a Research Day of the University of Bristol, Lady Emily Smyth Agricultural Research Station. In addition to the external Advisory Board we shall specifically invite representatives of the agrichemical and plant breeding industries together with representatives from DEFRA. We have existing contacts with these groups through previous (Syngenta) or ongoing (DEFRA) collaborations. Engaging with the wider public will be undertaken through the University of Bristol Botanic Garden. This is a popular visitor attraction in Bristol. Here we will produce a series of display boards introducing the general public to the use of water in plants (including crops), the requirement to produce more water use efficient crops (food security/climate change) and how modifying stomatal behaviour is likely to impact on this process. At Sheffield Prof Gray already does extensive outreach work with local schools and as described in the impact plan the work in this proposal sits within the Sheffield Project Sunshine and will benefit from existing infrastucture. The commercial arms of both Universities will be engaged if patent worthy property results from our work with Bristol taking the lead. How will they benefit? In the case of industry the likely benefit is increased understanding of the parameters affecting water use efficiency. Increased atmospheric carbon dioxide is likely to influence crop WUE and the work in this application has the potential to be exploited by plant breeders aiming to produce more WUE crops tailored to an increased carbon dioxide world. Water and crops is a key strategic target for Syngenta. If our work results in patentable intellectual property our Research and Enterprise Division and its counterpart at Sheffield will discuss protection with industrial partners such as Syngenta. Similarly, with a Government body such as DEFRA who are contributors to the LWEC and Food Security programmes the data we produce will contribute towards the process of gathering 'evidence' that forms the basis of advice to Ministers. The general public will benefit by a greater knowledge of how plants work and an understanding of how tax payers money is being used tohelp contribute towards global food security.