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

Reference	BB/W001217/1
Principal Investigator / Supervisor	Professor Michael Blatt
Co-Investigators / Co-Supervisors	Dr Rucha Karnik
Institution	University of Glasgow
Department	College of Medical, Veterinary, Life Sci
Funding type	Research
Value (£)	629,255
Status	Current
Type	Research Grant
Start date	21/02/2022
End date	20/02/2025
Duration	36 months

Abstract

We propose a strategy of parallel and synergistic studies to analyse the mechanism and physiological consequences of SLAC1 Cl- channel assembly in complex with the beta-CA4 carbonic anhydrase and the vesicle-trafficking protein SYP121. Our aims are (1) to establish the binding relations and higher-order dynamics between SYP121, the ion channels, and carbonic anhydrase, (2) to resolve the functional consequences for transport and secretory traffic regulation by CO2, and (3) to place the mechanics of the assembly in context of stomatal coordination with the demand of photosynthesis for CO2 within the leaf. Our overarching goal is to inform on approaches to enhance stomatal movements with photosynthetic demand. As part of this work will develop and validate new sets of genetic tools based around the mutant proteins with modified interaction characteristics. Experiments will employ methodologies we have used successfully in the past to resolve SYP121 interactions with the K+ channels KAT1 and KC1, and they will build on evidence gathered to date that shows the beta-CA4-SYP121-SLAC1 assembly confers a strong dependence of the SLAC1 current on near-ambient changes in CO2. We will combine molecular biology, protein-interaction and electrophysiological analyses, and trafficking-marker studies, and we will follow this work with in vivo gas exchange and biomass analysis to assess the consequences of manipulating beta-CA4-SYP121-SLAC1 interactions in vivo. Among others, we will assess the impacts of these interactions on the dynamics of stomatal responses, carbon assimilation and whole-plant water use efficiency under fluctuating light conditions that are commonly found in nature. These studies will expand our fundamental understanding of stomatal mechanics and their coupling to the demand for CO2 by photosynthesis, and they will help identify bona fide targets for future efforts towards crop improvement.

Summary

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.

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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