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1. Identification of SLAC1 anion channel residues required for CO 2 /bicarbonate sensing and regulation of stomatal movements

   https://doi.org/10.1073/pnas.1807624115  

   Zhang, Jingbo ; Wang, Nuo ; Miao, Yinglong ; Hauser, Felix ; McCammon, J. Andrew ; Rappel, Wouter-Jan ; Schroeder, Julian I. ( October 2018 , Proceedings of the National Academy of Sciences)

   Increases in CO₂concentration in plant leaves due to respiration in the dark and the continuing atmospheric [CO₂] rise cause closing of stomatal pores, thus affecting plant–water relations globally. However, the underlying CO₂/bicarbonate (CO₂/HCO₃⁻) sensing mechanisms remain unknown. [CO₂] elevation in leaves triggers stomatal closure by anion efflux mediated via the SLAC1 anion channel localized in the plasma membrane of guard cells. Previous reconstitution analysis has suggested that intracellular bicarbonate ions might directly up-regulate SLAC1 channel activity. However, whether such a CO₂/HCO₃⁻regulation of SLAC1 is relevant for CO₂control of stomatal movements in planta remains unknown. Here, we computationally probe for candidate bicarbonate-interacting sites within the SLAC1 anion channel via long-timescale Gaussian accelerated molecular dynamics (GaMD) simulations. Mutations of two putative bicarbonate-interacting residues, R256 and R321, impaired the enhancement of the SLAC1 anion channel activity by CO₂/HCO₃⁻inXenopusoocytes. Mutations of the neighboring charged amino acid K255 and residue R432 and the predicted gate residue F450 did not affect HCO₃⁻regulation of SLAC1. Notably, gas-exchange experiments withslac1-transformed plants expressing mutated SLAC1 proteins revealed that the SLAC1 residue R256 is required for CO₂regulation of stomatal movements in planta, but not for abscisic acid (ABA)-induced stomatal closing. Patch clamp analyses of guard cells show that activation of S-type anion channels by CO₂/HCO₃⁻, but not by ABA, was impaired, indicating the relevance of R256 for CO₂signal transduction. Together, these analyses suggest that the SLAC1 anion channel is one of the physiologically relevant CO₂/HCO₃⁻sensors in guard cells.

    

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2. Abscisic acid-independent stomatal CO 2 signal transduction pathway and convergence of CO 2 and ABA signaling downstream of OST1 kinase

   https://doi.org/10.1073/pnas.1809204115  

   Hsu, Po-Kai ; Takahashi, Yohei ; Munemasa, Shintaro ; Merilo, Ebe ; Laanemets, Kristiina ; Waadt, Rainer ; Pater, Dianne ; Kollist, Hannes ; Schroeder, Julian I. ( October 2018 , Proceedings of the National Academy of Sciences)

   Stomatal pore apertures are narrowing globally due to the continuing rise in atmospheric [CO₂]. CO₂elevation and the plant hormone abscisic acid (ABA) both induce rapid stomatal closure. However, the underlying signal transduction mechanisms for CO₂/ABA interaction remain unclear. Two models have been considered: (i) CO₂elevation enhances ABA concentrations and/or early ABA signaling in guard cells to induce stomatal closure and (ii) CO₂signaling merges with ABA at OST1/SnRK2.6 protein kinase activation. Here we use genetics, ABA-reporter imaging, stomatal conductance, patch clamp, and biochemical analyses to investigate these models. The strong ABA biosynthesis mutantsnced3/nced5andaba2-1remain responsive to CO₂elevation. Rapid CO₂-triggered stomatal closure in PYR/RCAR ABA receptor quadruple and hextuple mutants is not disrupted but delayed. Time-resolved ABA concentration monitoring in guard cells using a FRET-based ABA-reporter, ABAleon2.15, and ABA reporter gene assays suggest that CO₂elevation does not trigger [ABA] increases in guard cells, in contrast to control ABA exposures. Moreover, CO₂activates guard cell S-type anion channels innced3/nced5and ABA receptor hextuple mutants. Unexpectedly, in-gel protein kinase assays show that unlike ABA, elevated CO₂does not activate OST1/SnRK2 kinases in guard cells. The present study points to a model in which rapid CO₂signal transduction leading to stomatal closure occurs via an ABA-independent pathway downstream of OST1/SnRK2.6. Basal ABA signaling and OST1/SnRK2 activity are required to facilitate the stomatal response to elevated CO₂. These findings provide insights into the interaction between CO₂/ABA signal transduction in light of the continuing rise in atmospheric [CO₂].

    

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3. The Arabidopsis heterotrimeric G‐protein β subunit, AGB 1, is required for guard cell calcium sensing and calcium‐induced calcium release

   https://doi.org/10.1111/tpj.14318  

   Jeon, Byeong Wook ; Acharya, Biswa R. ; Assmann, Sarah M. ( April 2019 , The Plant Journal)

   Cytosolic calcium concentration ([Ca²⁺]_cyt) and heterotrimeric G‐proteins are universal eukaryotic signaling elements. In plant guard cells, extracellular calcium (Ca_o) is as strong a stimulus for stomatal closure as the phytohormone abscisic acid (ABA), but underlying mechanisms remain elusive. Here, we report that the sole Arabidopsis heterotrimeric Gβ subunit,AGB1, is required for four guard cell Ca_oresponses: induction of stomatal closure; inhibition of stomatal opening; [Ca²⁺]_cytoscillation; and inositol 1,4,5‐trisphosphate (InsP3) production. Stomata in wild‐type Arabidopsis (Col) and in mutants of the canonical Gα subunit,GPA1, showed inhibition of stomatal opening and promotion of stomatal closure by Ca_o. By contrast, stomatal movements ofagb1mutants andagb1/gpa1double‐mutants, as well as those of theagg1agg2 Gγ double‐mutant, were insensitive to Ca_o. These behaviors contrast withABA‐regulated stomatal movements, which involveGPA1 andAGB1/AGG3 dimers, illustrating differential partitioning of G‐protein subunits among stimuli with similar ultimate impacts, which may facilitate stimulus‐specific encoding.AGB1knockouts retained reactive oxygen species andNOproduction, but lostYC3.6‐detected [Ca²⁺]_cytoscillations in response to Ca_o, initiating only a single [Ca²⁺]_cytspike. Experimentally imposed [Ca²⁺]_cytoscillations restored stomatal closure inagb1. Yeast two‐hybrid and bimolecular complementation fluorescence experiments revealed thatAGB1 interacts with phospholipase Cs (PLCs), and Ca_oinduced InsP3 production in Col but not inagb1. In sum, G‐protein signaling viaAGB1/AGG1/AGG2 is essential for Ca_o‐regulation of stomatal apertures, and stomatal movements in response to Ca_oapparently require Ca²⁺‐induced Ca²⁺release that is likely dependent on Gβγ interaction withPLCs leading to InsP3 production.

    

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4. Mitogen‐activated protein kinases MPK 4 and MPK 12 are key components mediating CO 2 ‐induced stomatal movements

   https://doi.org/10.1111/tpj.14087  

   Tõldsepp, Kadri ; Zhang, Jingbo ; Takahashi, Yohei ; Sindarovska, Yana ; Hõrak, Hanna ; Ceciliato, Paulo H. O. ; Koolmeister, Kaspar ; Wang, Yuh‐Shuh ; Vaahtera, Lauri ; Jakobson, Liina ; et al ( October 2018 , The Plant Journal)

   Respiration in leaves and the continued elevation in the atmosphericCO₂concentration causeCO₂‐mediated reduction in stomatal pore apertures. Several mutants have been isolated for which stomatal responses to both abscisic acid (ABA) andCO₂are simultaneously defective. However, there are only few mutations that impair the stomatal response to elevatedCO₂, but not toABA. Such mutants are invaluable in unraveling the molecular mechanisms of earlyCO₂signal transduction in guard cells. Recently, mutations in the mitogen‐activated protein (MAP) kinase,MPK12, have been shown to partially impairCO₂‐induced stomatal closure. Here, we show thatmpk12plants, in whichMPK4is stably silenced specifically in guard cells (mpk12 mpk4GChomozygous double‐mutants), completely lackCO₂‐induced stomatal responses and have impaired activation of guard cell S‐type anion channels in response to elevatedCO₂/bicarbonate. However,ABA‐induced stomatal closure, S‐type anion channel activation andABA‐induced marker gene expression remain intact in thempk12 mpk4GCdouble‐mutants. These findings suggest thatMPK12 andMPK4 act very early inCO₂signaling, upstream of, or parallel to the convergence ofCO₂andABAsignal transduction. The activities ofMPK4 andMPK12 protein kinases were not directly modulated byCO₂/bicarbonatein vitro, suggesting that they are not directCO₂/bicarbonate sensors. Further data indicate thatMPK4 andMPK12 have distinguishable roles in Arabidopsis and that the previously suggested role ofRHC1 in stomatalCO₂signaling is minor, whereasMPK4 andMPK12 act as key components of early stomatalCO₂signal transduction.

    

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5. A role for ethylene signaling and biosynthesis in regulating and accelerating CO2 ‐ and abscisic acid‐mediated stomatal movements in Arabidopsis

   https://doi.org/10.1111/nph.18918  

   Azoulay‐Shemer, Tamar ; Schulze, Sebastian ; Nissan‐Roda, Dikla ; Bosmans, Krystal ; Shapira, Or ; Weckwerth, Philipp ; Zamora, Olena ; Yarmolinsky, Dmitry ; Trainin, Taly ; Kollist, Hannes ; et al ( April 2023 , New Phytologist)

   Little is known about long‐distance mesophyll‐driven signals that regulate stomatal conductance. Soluble and/or vapor‐phase molecules have been proposed. In this study, the involvement of the gaseous signal ethylene in the modulation of stomatal conductance inArabidopsis thalianaby CO₂/abscisic acid (ABA) was examined.

   We present a diffusion model which indicates that gaseous signaling molecule/s with a shorter/direct diffusion pathway to guard cells are more probable for rapid mesophyll‐dependent stomatal conductance changes. We, therefore, analyzed different Arabidopsis ethylene‐signaling and biosynthesis mutants for their ethylene production and kinetics of stomatal responses to ABA/[CO₂]‐shifts.

   According to our research, higher [CO₂] causes Arabidopsis rosettes to produce more ethylene. An ACC‐synthase octuple mutant with reduced ethylene biosynthesis exhibits dysfunctional CO₂‐induced stomatal movements. Ethylene‐insensitive receptor (gain‐of‐function),etr1‐1andetr2‐1, and signaling,ein2‐5andein2‐1, mutants showed intact stomatal responses to [CO₂]‐shifts, whereas loss‐of‐function ethylene receptor mutants, includingetr2‐3;ein4‐4;ers2‐3,etr1‐6;etr2‐3andetr1‐6, showed markedly accelerated stomatal responses to [CO₂]‐shifts. Further investigation revealed a significantly impaired stomatal closure to ABA in the ACC‐synthase octuple mutant and accelerated stomatal responses in theetr1‐6;etr2‐3, andetr1‐6, but not in theetr2‐3;ein4‐4;ers2‐3mutants.

   These findings suggest essential functions of ethylene biosynthesis and signaling components in tuning/accelerating stomatal conductance responses to CO₂and ABA.

    

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