Stomatal-based systems analysis of water use efficiency

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 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. 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. Significantly, stomatal responses are slow compared to photosynthesis 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 speed of stomatal responses, especially to light, can be enhanced. However, the complexity of guard cell transport and its coupling to gas exchange and transpiration has presented a formidable barrier to systematic reverse-engineering aimed at enhancing stomatal responses through genetic manipulation and other means.

Quantitative systems analysis offers an effective approach in silico to exploring the link between microscopic gene function and the macroscopic characteristics of assimilation and transpiration. As a first step to bridging this gap in understanding, we 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 that drive stomatal movement; 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. The next major step towards establishing in silico strategies for crop design, based on our deep knowledge of stomatal guard cells, will be to establish and validate this computational link to incorporate carbon assimilation and water use efficiency at leaf and whole-plant levels.

We propose now to develop such a strategy in models of the leaf, and scaling to the crop in the field, that capture CO2 uptake and transpiration. We will build the next-generation OnGuard models that incorporate CO2 uptake and transpiration, and we will incorporate computational statistical methods to accelerate model construction. Most important, the models will provide the essential micro-macro link to connect molecular function with physiological traits of the whole plant in water use and photosynthetic carbon assimilation and will enable scaling to the crop in the field. We will test this second generation of OnGuard models and validate their outputs to examine the longstanding hypothesis that significant erosion in the efficiency of water use by plants arises because of the mismatch in dynamic environmental responses between stomata and photosynthesis. Additionally, we will explore the connection of these traits with oscillations known to occur in stomatal aperture and in the signalling events (e.g. cytosolic-free [Ca2+]) previously documented at the cellular level in single guard cells. All studies will focus on the crop plant Vicia for which there is much data at the single-cell and whole-leaf levels, and on Arabidopsis for which we have mutants with well-defined effects on stomatal kinetics.

We will incorporate transpiration and carbon fixation explicitly within the OnGuard software, and will test, by in silico modelling and experimental validation, the hypothesis that significant erosion in the efficiency of water use by plants arises because of the mismatch in environmental responses between stomata and photosynthesis. These studies will explore also the connection to oscillations known to occur in stomatal aperture and in the signalling events (e.g. cytosolic-free [Ca2+]) previously documented at the cellular level in single guard cells and reproduced in the first-generation OnGuard models. The modelling and experimental methods proposed are independent, but their combination gives added value to both. Model development will incorporate recent advances in Bayesian inference to enable fast, automated searching for computational solutions. These are proven methods and naturally compute the confidences in model predictions and their relation to observed data. Bayesian methods also address questions such as 'Which parameters are critical to defining a specific set of behaviours?', questions that are important in directing model development. The experimental work will build on methods at the research forefront in analysing water use efficiency in real time from single plants and in scaling for data capture in the field by eddy flux (covariance) and stable isotope discrimination analysis. Experiments will assess stomatal response to light, humidity and CO2 to extract stomatal kinetics and their association with oscillations in transpiration and carbon assimilation. We will use these data to parameterise models and to test the micro-macro link of the models in predicting transpiration and water use efficiency. Thus, we fully expect new and exciting insights into the behaviour of stomata in the leaf and crop canopy, much as our previous modelling efforts provided hitherto unexpected insights into the physiology of the isolated guard cell.

This proposal is for fundamental research developing new concepts at the core of ideas emerging within the international plant and systems biology communities. The research will stimulate thinking around strategies for systems modelling, especially in relation to membrane transport, plant growth, development and pathology, and it should facilitate in silico methodologies for the reverse-engineering of crops with improved traits. Thus, the research is expected to benefit fundamental researchers as well as agriculture and industry through conceptual developments as well as the introduction of new computational technologies for the analysis of 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 of this proposal. Finally the research will help guide future efforts in applications to agricultural/industrial systems. The applicants have 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.