Developing a synthetic approach to manipulating guard cell membrane transport and stomatal control

Stomata are pores that provide for gaseous exchange across the impermeable cuticle of plant leaves. They open and close to balance the requirement for CO2 entry for photosynthesis against the need to reduce the transpiration of water vapour and prevent leaf drying. Stomatal movements are driven by solute transport - and consequent uptake/loss of water - across the cell membrane of the guard cells which surround the stomatal pore. Stomatal transpiration is at the centre of a crisis in water availability and crop production that is expected to unfold over the next 20-30 years: globally, agricultural water usage has increased 6-fold in the past 100 years, twice as fast as the human population, and is projected to double again before 2030. Thus stomata represent an important target for breeders interested in manipulating crop performance. Significantly, stomatal responses are often delayed in the face of environmental fluctuations, especially of light. Improving water use efficiency (=amount of carbon fixed in photosynthesis/amount of water transpired) should be possible, without a cost to carbon assimilated in photosynthesis, if the stomatal response to light are enhanced. However, the complexity of guard cell transport and its coupling to gas exchange and transpiration has presented a formidable barrier to manipulations so that efforts at genetic improvements have generally proven constrained.

Synthetic methods offer one approach to physiologically enhancing stomatal function. Furthermore, in combination with quantitative systems analysis they present an opportunity to gain fundamental insights into the coordination of transport in the homeostasis of a plant cell system. I developed previously the OnGuard software for quantitative dynamic modelling of the guard cell. OnGuard models build explicitly on the wealth of molecular, biophysical and kinetic knowledge for guard cell transport and metabolism; they accommodate stomata of different plant species, over the full range of conditions studied in the laboratory to date; and they have been shown to incorporate the real predictive power needed to guide experiments at the cellular and physiological levels that start with molecular manipulations in silico. As the next major step, I wish to establish directed synthetic strategies for design of stomatal function, based on this deep knowledge of stomatal guard cells, and on developments in work with light-driven ion pumps and channels.

I propose now to use the OnGuard software to model and explore the most effective approaches to enhancing stomatal kinetics. In parallel, my laboratory will develop a synthetic approach to manipulate guard cell transport with so-called optogenetic tools - light-driven pumps and channels - using these to test and validate the model predictions. Most important, the combined modelling and experimental approaches will connect the molecular components with physiological properties of stomata. They will reinforce an understanding of guard cell transport and will enable novel explorations of water use and its balance with photosynthetic carbon assimilation. These studies will target expression of selected optogenetic tools based on OnGuard model predictions. The knowledge gained will aid in refining these models and will allow exploration of the kinetic and homeostatic coordination between the serial membranes of the plasma membrane and tonoplast. This coordination is known to be fundamental to the control of stomatal aperture, but its mechanism remains unknown. Finally, the studies will serve to establish the potential for improving the efficiency of water use by plants drawing on synthetic methods to reduce the mismatch in dynamic environmental responses between stomata and photosynthesis.

These studies will utilise targeted expression of light-driven ion pumps and channels - including bacteriorhodopsins, halorhodopsins and channelrhodopsins - to probe the coordination of transport between the plasma membrane and tonoplast of stomatal guard cells. They will build on the OnGuard software, previously developed in this laboratory, that has proven effective in uncovering a hitherto unrecognised homeostatic connection in K+ and Cl- channel regulation. We will test, by in silico modelling and experimental validation, the hypothesis that transport coordination between these serial membranes is emergent from the interactions implicit in the sharing of transported substrates/products. Additionally, we will explore the potential for synthetic manipulation to accelerate stomatal kinetics for improved water use efficiency. The experimental work will focus on Arabidopsis for which we have a substantial molecular toolchest. The modelling and experimental methods proposed are independent, but their combination gives added value to both. We will build on methods at the research forefront in so-called optogenetics that have enabled substantial advances in understanding of neural networks. Electrophysiological and related methods will be used quantify transport at the cellular and subcellular levels, and will assess stomatal responses, especially to light, to relate the consequences of targeted pump and channel expression on stomatal kinetics and their association with transpiration and carbon assimilation. We will use these data to parameterise models and to test the micro-macro link of the models in predicting enhanced stomatal behaviour and its consequences for water use efficiency. Thus, I fully expect new and exciting insights into guard cell transport and its coordination through synthetic manipulations, much as our previous modelling efforts provided hitherto unexpected insights through the mutatiional analysis.

This proposal is for fundamental research developing new concepts at the core of ideas emerging within the international plant, systems and synthetic biology communities. The research will stimulate thinking around strategies for systems modelling and synthetic applications, especially in relation to membrane transport, plant growth, development and pathology, and it should strengthen in silico methodologies for approaching crop engineering. Thus, the research is expected to benefit fundamental researchers and, in the longer-term agriculture and industry, through conceptual developments as well as synthetic approaches to improving plant water use efficiency and productivity. The research will feed into higher education training programmes through capacity building at the postgraduate and postdoctoral levels. Additional impact is proposed through public displays and the development of teaching resources building on the background work for this proposal. Finally the research will help guide future efforts in applications to agricultural/industrial systems. The applicant has established links with industrial/technology transfer partners and research institutes to take advantage of these developments. Further details of these, and additional impacts will be found in Part 1 of the Case for Support and in the attached Impact Pathways.