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

气孔是一种可以打开和关闭的气孔，以防止叶片干燥，同时使二氧化碳进入叶片进行光合作用。当需求超过供水量时，它们可以限制50%或更多的光合作用，它们对世界的水和碳循环起着重要的控制作用。气孔是未来20-30年淡水供应和作物生产危机的中心。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；甚至在英国，灌溉面积在过去30年里也扩大了10倍。据估计，2010-12年和2018年的干旱仅给英国农民造成了12亿英镑的损失，而在过去五年里，全球每年的损失估计达到数千亿英镑。大多数植物的气孔都是根据叶片光合作用对二氧化碳的直接需求而形成的，在光照下打开，在黑暗中关闭。然而，与光合作用相比，气孔响应较慢。日光的自然波动，例如云层从头顶经过，降低了光合作用的碳同化和水利用效率，这主要是因为气孔反应通常落后于光的变化。我们知道，通过加速气孔运动可以大大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护细胞力学及其与叶肉来源的二氧化碳变化的整合，以便为工程气孔动力学提供信息。保卫细胞的运输是控制气孔开度的必要条件。气孔周围的保护细胞响应包括光和CO2在内的一系列细胞外信号来调节气孔孔径。保护细胞协调多种转运体活动的变化，特别是促进K+和Cl-离子通量的离子通道，并重塑细胞膜。气孔运动既需要离子通量的变化，也需要膜的重塑。尽管如此，了解这些变化是如何产生和协调的，尤其是二氧化碳的变化，仍然是一个挑战。我们已经发现，主要的Cl-通道SLAC1选择性地结合在一个多蛋白复合物内，该复合物包含了对细胞膜重塑至关重要的所谓SNARE蛋白SYP121，并与碳酸酐酶β - ca4结合。碳酸酐酶是已知的保护细胞中与二氧化碳结合的少数蛋白质之一，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构共同调节K+离子通量。我们现在发现SYP121与β - ca4和SLAC1的组装赋予了Cl-通道对近环境CO2变化的强烈依赖。这些正是长期寻求的二氧化碳介导的Cl-通量和气孔运动增强机制的特征。他们指出，多蛋白复合物在膜重构中协调Cl-和K+通量，并在这些事件中直接赋予CO2敏感性。我们在此提出解决β - ca4 - syp121 - slac1相互作用的机制，以了解二氧化碳如何调节气孔关闭的这些事件。因此，我们的主要目标是定量了解这种新型sla1超复合体的机制，以及它对保护细胞中二氧化碳反应的生理调节。除此之外，我们希望解决SYP121与β - ca4和SLAC1结合的关键蛋白结构域，它们对CA和SLAC1活性的影响，以及它们对SLAC1的二氧化碳依赖性的贡献。提出的研究是针对基础知识的。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.