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CO2进入叶片进行光合作用。当需求超过供水时，它们可以将光合作用限制50%或更多，并且它们对世界的水和碳循环施加主要控制。气孔是未来20-30年淡水供应和作物生产危机的核心。在过去的100年里，全球农业用水量增加了6倍，是人口增长速度的两倍;即使在英国，灌溉也在过去30年里扩大了10倍。2010-12年和2018年的干旱仅英国农民就损失了约12亿英镑，在过去的五年里，全球每年的损失估计为数千亿英镑。大多数植物的气孔通过叶片中的光合作用来跟踪对二氧化碳的直接需求，在光照下打开，在黑暗中关闭。然而，气孔的反应与光合作用相比是缓慢的。日光的自然波动，例如云从头顶经过，降低了光合作用的碳同化和水的利用效率，主要是因为气孔的反应通常滞后于光的变化。我们知道，碳同化和水利用效率的实质性收益是可能的，通过加速气孔运动，但我们需要了解CO2如何影响保卫细胞力学及其整合与叶肉衍生的变化CO2，以告知工程气孔动力学的努力。保卫细胞运输是不可或缺的控制气孔孔径。保卫细胞围绕气孔周围，并响应一系列细胞外信号，包括光和CO2，以调节气孔开度。保卫细胞协调许多转运蛋白（特别是促进K+和Cl-离子通量的离子通道）活性的变化，并重塑细胞膜。气孔运动需要离子流的变化和膜的重塑。尽管如此，挑战仍然是要了解这些变化是如何产生和协调的，特别是通过CO2。我们已经发现，占主导地位的Cl-通道，SLAC 1，选择性地结合在一个多蛋白质复合物内，该复合物包含一个所谓的SNARE蛋白，SYP 121，这对细胞膜的重塑至关重要，并与碳酸酐酶β-CA 4结合。碳酸酐酶是保卫细胞中已知的少数蛋白质之一，其与CO2结合，因此能够直接响应CO2。SYP 121还结合K+通道的子集，以在气孔运动期间与膜重塑共调节K+离子通量。我们发现SYP 121与beta-CA 4和SLAC 1的组装赋予了Cl-通道对近环境CO2变化的强烈依赖性。这些特征正是人们长期以来对CO2介导的Cl-通量和气孔运动增强机制的预期。他们指出，多蛋白质复合物在协调Cl-以及K+通量与膜重塑，并直接赋予这些事件的CO2敏感性。我们建议在这里解决β-CA 4-SYP 121-SLAC 1相互作用的机制，以了解CO2如何调节气孔关闭这些事件。因此，我们的主要目标是发展一个定量的了解这种新的SLAC 1超复合物的力学和协调调节它赋予的生理CO2反应在保卫细胞。其中，我们希望解决SYP 121与β-CA 4和SLAC 1结合的关键蛋白结构域，它们对CA和SLAC 1活性的影响，以及它们对SLAC 1的CO2依赖性的贡献。所提出的研究是为了基础知识。尽管如此，它对作物改良具有长期意义，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.