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

气孔是通过植物叶片不透水的角质层提供气体交换的气孔。它们打开和关闭，以平衡光合作用对二氧化碳进入的要求，以及减少水蒸气的蒸腾和防止叶子干燥的需要。气孔运动是由气孔周围保卫细胞的细胞膜上的溶质运输--以及由此引起的水分吸收/流失--驱动的。气孔蒸腾是水资源供应和作物生产危机的核心，预计这场危机将在未来20-30年内展开：在过去100年里，全球农业用水量增加了6倍，是人类用水量的两倍，预计在2030年前将再翻一番。因此，气孔代表了对操纵作物表现感兴趣的育种者的一个重要目标。值得注意的是，面对环境波动，尤其是光的变化，气孔反应往往延迟。提高水分利用效率(=光合作用中固定的碳量/蒸散量)应该是可能的，而不需要花费光合作用中的碳同化，如果气孔对光的反应增强的话。然而，保卫细胞运输的复杂性及其与气体交换和蒸腾作用的耦合为操作提供了一个强大的障碍，因此遗传改良的努力通常被证明是有限的。合成方法提供了一种从生理上增强气孔功能的方法。此外，与定量系统分析相结合，它们提供了一个机会，以获得对植物细胞系统内稳态运输的协调的基本见解。我之前开发了OnGuard软件，用于对保卫细胞进行定量动态建模。OnGuard模型明确地建立在保卫细胞运输和新陈代谢的丰富的分子、生物物理和动力学知识的基础上；它们适应了不同植物物种的气孔，涵盖了迄今为止在实验室研究的所有条件；它们已被证明纳入了指导细胞和生理水平的实验所需的实际预测能力，这些实验始于硅胶中的分子操作。作为下一个重要步骤，我希望基于对气孔保卫细胞的深入了解，以及光驱动离子泵和通道工作的发展，建立用于设计气孔功能的定向综合策略。我现在建议使用OnGuard软件来建模和探索增强气孔动力学的最有效的方法。与此同时，我的实验室将开发一种合成方法，利用所谓的光遗传工具--光驱动的泵和通道--操纵保卫细胞的运输，使用这些工具来测试和验证模型预测。最重要的是，建模和实验相结合的方法将把分子成分与气孔的生理特性联系起来。它们将加强对保卫细胞运输的理解，并将使人们能够对水的利用及其与光合碳同化的平衡进行新的探索。这些研究将以基于OnGuard模型预测的选定光遗传工具的表达为目标。所获得的知识将有助于完善这些模型，并将允许探索质膜和液泡膜系列膜之间的动态和稳态协调。这种协调作用是控制气孔开度的基础，但其机制尚不清楚。最后，这些研究将有助于确定利用合成方法提高植物水分利用效率的潜力，以减少气孔和光合作用之间动态环境反应的不匹配。

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.