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1. A role for calcium‐dependent protein kinases in differential CO 2 ‐ and ABA‐controlled stomatal closing and low CO 2 ‐induced stomatal opening in Arabidopsis

   https://doi.org/10.1111/nph.17079  

   Schulze, Sebastian ; Dubeaux, Guillaume ; Ceciliato, Paulo H. O. ; Munemasa, Shintaro ; Nuhkat, Maris ; Yarmolinsky, Dmitry ; Aguilar, Jaimee ; Diaz, Renee ; Azoulay‐Shemer, Tamar ; Steinhorst, Leonie ; et al ( December 2020 , New Phytologist)

   Low concentrations of CO₂cause stomatal opening, whereas [CO₂] elevation leads to stomatal closure. Classical studies have suggested a role for Ca²⁺and protein phosphorylation in CO₂‐induced stomatal closing. Calcium‐dependent protein kinases (CPKs) and calcineurin‐B‐like proteins (CBLs) can sense and translate cytosolic elevation of the second messenger Ca²⁺into specific phosphorylation events. However, Ca²⁺‐binding proteins that function in the stomatal CO₂response remain unknown.

   Time‐resolved stomatal conductance measurements using intact plants, and guard cell patch‐clamp experiments were performed.

   We isolatedcpkquintuple mutants and analyzed stomatal movements in response to CO₂, light and abscisic acid (ABA). Interestingly, we found thatcpk3/5/6/11/23quintuple mutant plants, but not other analyzedcpkquadruple/quintuple mutants, were defective in high CO₂‐induced stomatal closure and, unexpectedly, also in low CO₂‐induced stomatal opening. Furthermore, K⁺‐uptake‐channel activities were reduced incpk3/5/6/11/23quintuple mutants, in correlation with the stomatal opening phenotype. However, light‐mediated stomatal opening remained unaffected, and ABA responses showed slowing in some experiments. By contrast, CO₂‐regulated stomatal movement kinetics were not clearly affected in plasma membrane‐targetedcbl1/4/5/8/9quintuple mutant plants.

   Our findings describe combinatorialcpkmutants that function in CO₂control of stomatal movements and support the results of classical studies showing a role for Ca²⁺in this response.

    

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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. Stomatal behaviour moderates the water cost of CO 2 acquisition for 21 boreal and temperate species under experimental climate change

   https://doi.org/10.1111/pce.14559  

   Stefanski, Artur ; Butler, Ethan E. ; Bermudez, Raimundo ; Montgomery, Rebecca A. ; Reich, Peter B. ( February 2023 , Plant, Cell & Environment)
4. Stomatal CO 2 /bicarbonate sensor consists of two interacting protein kinases, Raf-like HT1 and non-kinase-activity requiring MPK12/MPK4

   https://doi.org/10.1126/sciadv.abq6161  

   Takahashi, Yohei ; Bosmans, Krystal C. ; Hsu, Po-Kai ; Paul, Karnelia ; Seitz, Christian ; Yeh, Chung-Yueh ; Wang, Yuh-Shuh ; Yarmolinsky, Dmitry ; Sierla, Maija ; Vahisalu, Triin ; et al ( December 2022 , Science Advances)

   Plant stomata sense CO2 via reversible interaction of the Raf-like HT1 protein kinase with non-activity requiring MAP kinase. 

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5. 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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