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

气孔是在植物叶片的不透水角质层上提供气体交换的气孔。它们的打开和关闭是为了平衡光合作用所需的二氧化碳进入与减少水蒸气蒸腾和防止叶子干燥的需要。气孔运动是由溶质运输驱动的——以及随之而来的水的吸收/损失——穿过气孔周围的保护细胞的细胞膜。气孔蒸腾是水资源供应和作物生产危机的核心，预计将在未来20-30年内展开：在全球范围内，农业用水量在过去100年中增加了6倍，是人口增长速度的两倍，预计到2030年将再翻一番。因此，对于有意操纵作物性能的育种者来说，气孔是一个重要的目标。值得注意的是，面对环境的波动，尤其是光的波动，气孔反应往往会延迟。提高水分利用效率（=光合作用中固定的碳量/蒸腾的水量）应该是可能的，而不以光合作用中吸收的碳为代价，如果气孔对光的响应增强。然而，保护细胞运输的复杂性及其与气体交换和蒸腾的耦合对操作提出了一个巨大的障碍，因此遗传改进的努力通常被证明是有限的。合成方法为生理上增强气孔功能提供了一种途径。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稳态中运输协调的基本见解。我以前开发了OnGuard软件，用于保护细胞的定量动态建模。OnGuard模型明确建立在丰富的分子，生物物理和动力学知识的保护细胞运输和代谢；迄今为止，在实验室研究的所有条件下，它们都能容纳不同植物物种的气孔；它们已经被证明包含了真正的预测能力，需要在细胞和生理水平上指导从硅分子操作开始的实验。下一步，我希望基于对气孔保护细胞的深入了解，以及光驱动离子泵和通道的研究进展，建立气孔功能设计的定向合成策略。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，利用所谓的光遗传学工具（光驱动泵和通道）来操纵保护细胞的运输，并使用这些工具来测试和验证模型预测。最重要的是，模型和实验相结合的方法将把气孔的分子成分与生理特性联系起来。它们将加强对保护细胞运输的理解，并将使水利用及其与光合作用碳同化平衡的新探索成为可能。这些研究将针对基于OnGuard模型预测的选定光遗传学工具的表达。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张力质体系列膜之间的动力学和稳态协调。这种协调是控制气孔孔径的基础，但其机制尚不清楚。最后，这些研究将有助于建立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响应的不匹配。

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.