Resolving CO2 regulation of the SLAC1 Cl- channel in guard cell ion transport and photosynthetic carbon assimilation

气孔是打开和关闭的气孔，以防止叶片干燥，同时使二氧化碳进入叶片进行光合作用。当供不应求时，它们可以将光合作用限制50%或更多，它们对世界上的水和碳循环起着重要控制作用。气孔是淡水供应和作物生产危机的中心，预计未来20-30年内将出现这场危机。全球农业用水量在过去100年里增长了6倍，是人口增长速度的两倍；即使在英国，灌溉在过去30年里也增长了10倍。仅2010-12年和2018年的干旱就给英国农民造成了约1.2B英镑的损失，在过去五年中，全球每年的成本估计高达数千亿英镑。大多数植物的气孔通过叶片的光合作用来跟踪对二氧化碳的即时需求，在光下打开，在黑暗中关闭。然而，与光合作用相比，气孔反应较慢。日光的自然波动，例如当云从头顶掠过时，会降低光合作用的碳同化和水分利用效率，主要是因为气孔的反应通常滞后于光的变化。我们知道，通过加速气孔运动，可以大幅提高碳同化和水分利用效率，但我们需要了解二氧化碳如何影响保卫细胞机制及其与叶肉衍生的二氧化碳变化的整合，以便为气孔动力学工程提供信息。保卫细胞运输是控制气孔开度所不可或缺的。保卫细胞包围气孔孔，对包括光和二氧化碳在内的一系列细胞外信号做出反应，以调节气孔开度。保卫细胞协调许多转运蛋白活性的变化，特别是促进K+和Cl-离子流动的离子通道的活性变化，并重塑细胞膜。离子通量的变化和膜的重塑都是气孔运动所必需的。然而，挑战仍然是理解这些变化是如何发生和协调的，特别是在二氧化碳的作用下。我们已经发现，占主导地位的氯通道SLAC1选择性地结合在一个多蛋白复合体中，该复合体结合了一个所谓的SNARE蛋白SYP121，它对细胞膜的重塑至关重要，并与碳酸酐酶β-CA4结合。碳酸酐酶是保卫细胞中已知的少数蛋白质之一，它与二氧化碳结合，因此能够直接对二氧化碳做出反应。SYP121还结合K+通道的一个子集，在气孔运动过程中通过膜重塑来共同调节K+离子通量。我们现在发现，SYP121与β-CA4和SLAC1的组装使Cl-通道对CO2附近环境的变化有很强的依赖性，这正是人们期待已久的CO2介导的氯离子通量和气孔运动增强机制的特征。他们指出，多蛋白质复合体通过膜重塑来协调氯离子和钾离子的流动，并直接赋予这些事件二氧化碳敏感性。我们在这里建议解决β-CA4-SYP121-SLAC1相互作用的机制，以了解二氧化碳如何调节这些气孔关闭事件。因此，我们的主要目标是对这种新的SLAC1超复合体的机制以及它对保卫细胞中二氧化碳反应的生理协调调节有一个定量的了解。其中，我们想要解决SYP121与β-CA4和SLAC1结合的关键蛋白质结构域，它们对CA和SLAC1活性的影响，以及它们对SLAC1的二氧化碳依赖的贡献。所提出的研究是为了基础知识。然而，它对作物改良具有更长期的相关性，对生产者、消费者和环境都有好处。

Stomata are pores that open and close to protect against leaf drying while enabling CO2 entry into the leaf for photosynthesis. They can limit photosynthesis by 50% or more when demand exceeds water supply and they exert a major control on water and carbon cycles of the world. Stomata are at the centre of a crisis in fresh water availability and crop production that is expected over the next 20-30 years. Global agricultural water usage has increased 6-fold in the past 100 years, twice as fast as the human population; even in the UK irrigation has expanded 10-fold in the past 30 years. The droughts of 2010-12 and 2018 cost UK farmers alone an estimated £1.2B and worldwide costs year-by-year are estimated in the hundreds of billions of pounds over the past five years.Stomata in most plants track the immediate demand for CO2 by photosynthesis in the leaf, opening in the light and closing in the dark. However, stomatal responses are slow by comparison with that of photosynthesis. Natural fluctuations in daylight, for example as clouds pass overhead, degrade photosynthetic carbon assimilation and water use efficiencies, principally because stomatal responses generally lag behind changes in light. We know that substantial gains in carbon assimilation and water use efficiencies are possible by accelerating stomatal movements, but we need to understand how CO2 affects guard cell mechanics and its integration with mesophyll-derived changes in CO2 in order to inform efforts in engineering stomatal kinetics.Guard cell transport is integral to controlling stomatal aperture. Guard cells surround the stomatal pore and respond to an array of extracellular signals, including light and CO2, to regulate stomatal aperture. Guard cells coordinate changes in the activities of a number of transporters, notably of ion channels that facilitate K+ and Cl- ion fluxes, and they remodel the cell membrane. Both the changes ion flux and membrane remodelling are needed for stomatal movements. Nonetheless, the challenge remains to understand how these changes arise and are coordinated, especially by CO2.We have discovered that the dominant Cl- channel, SLAC1, binds selectively within a multi-protein complex that incorporates a so-called SNARE protein, SYP121, that is vital for remodelling of the cell membrane, and with the carbonic anhydrase beta-CA4. The carbonic anhydrase is one of a small number of proteins known in the guard cells that bind with, and hence are capable of responding to CO2 directly. SYP121 also binds a subset of K+ channels to co-regulate K+ ion flux with membrane remodelling during stomatal movements. We find now that the assembly of SYP121 with beta-CA4 and SLAC1 confers a strong dependence of the Cl- channel on near-ambient changes in CO2.These are precisely the characteristics expected for the long-sought mechanism of CO2-mediated enhancement in Cl- flux and stomatal movements. They point to the multi-protein complex in coordinating Cl- as well as K+ flux with membrane remodelling and in conferring a CO2 sensitivity directly on these events. We propose here to resolve the mechanics of beta-CA4-SYP121-SLAC1 interactions in order to understand how CO2 regulates these events for stomatal closure. Thus, our primary goal is to develop a quantitative understanding of the mechanics of this novel SLAC1 supercomplex and the coordinate regulation it confers on the physiology of CO2 responses in guard cells. Among others, we want to resolve the key protein domains for binding of SYP121 with beta-CA4 and SLAC1, their impact on CA and SLAC1 activities, and their contributions to the CO2-dependence of SLAC1. The research proposed is for fundamental knowledge. It nonetheless holds longer-term relevance for crop improvement with benefits for producers, consumers, and the environment.