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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)

   Summary 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. MPK12 in stomatal CO₂ signaling: function beyond its kinase activity

   https://doi.org/10.1111/nph.18913  

   Yeh, Chung‐Yueh; Wang, Yuh‐Shuh; Takahashi, Yohei; Kuusk, Katarina; Paul, Karnelia; Arjus, Triinu; Yadlos, Oleksii; Schroeder, Julian I.; Ilves, Ivar; Garcia‐Sosa, Alfonso T.; et al (April 2023, New Phytologist)

   Summary Protein phosphorylation is a major molecular switch involved in the regulation of stomatal opening and closure. Previous research defined interaction between MAP kinase 12 and Raf‐like kinase HT1 as a required step for stomatal movements caused by changes in CO₂concentration. However, whether MPK12 kinase activity is required for regulation of CO₂‐induced stomatal responses warrants in‐depth investigation.We apply genetic, biochemical, and structural modeling approaches to examining the noncatalytic role of MPK12 in guard cell CO₂signaling that relies on allosteric inhibition of HT1.We show that CO₂/HCO₃⁻‐enhanced MPK12 interaction with HT1 is independent of its kinase activity. By analyzing gas exchange of plant lines expressing various kinase‐dead and constitutively active versions of MPK12 in a plant line whereMPK12is deleted, we confirmed that CO₂‐dependent stomatal responses rely on MPK12's ability to bind to HT1, but not its kinase activity. We also demonstrate that purified MPK12 and HT1 proteins form a heterodimer in the presence of CO₂/HCO₃⁻and present structural modeling that explains the MPK12:HT1 interaction interface.These data add to the model that MPK12 kinase‐activity‐independent interaction with HT1 functions as a molecular switch by which guard cells sense changes in atmospheric CO₂concentration. 

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3. 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)

   Summary 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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4. Distinct guard cell–specific remodeling of chromatin accessibility during abscisic acid– and CO ₂ -dependent stomatal regulation

   https://doi.org/10.1073/pnas.2310670120  

   Seller, Charles A; Schroeder, Julian I (December 2023, Proceedings of the National Academy of Sciences)

   In plants, epidermal guard cells integrate and respond to numerous environmental signals to control stomatal pore apertures, thereby regulating gas exchange. Chromatin structure controls transcription factor (TF) access to the genome, but whether large-scale chromatin remodeling occurs in guard cells during stomatal movements, and in response to the hormone abscisic acid (ABA) in general, remains unknown. Here, we isolate guard cell nuclei fromArabidopsis thalianaplants to examine whether the physiological signals, ABA and CO₂(carbon dioxide), regulate guard cell chromatin during stomatal movements. Our cell type–specific analyses uncover patterns of chromatin accessibility specific to guard cells and define cis-regulatory sequences supporting guard cell–specific gene expression. We find that ABA triggers extensive and dynamic chromatin remodeling in guard cells, roots, and mesophyll cells with clear patterns of cell type specificity. DNA motif analyses uncover binding sites for distinct TFs enriched in ABA-induced and ABA-repressed chromatin. We identify the Abscisic Acid Response Element (ABRE) Binding Factor (ABF) bZIP-type TFs that are required for ABA-triggered chromatin opening in guard cells and roots and implicate the inhibition of a clade of bHLH-type TFs in controlling ABA-repressed chromatin. Moreover, we demonstrate that ABA and CO₂induce distinct programs of chromatin remodeling, whereby elevated atmospheric CO₂had only minimal impact on chromatin dynamics. We provide insight into the control of guard cell chromatin dynamics and propose that ABA-induced chromatin remodeling primes the genome for abiotic stress resistance. 

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5. Editorial: Stomatal Biology and Beyond

   https://doi.org/10.3389/fpls.2022.848811  

   Ye, Wenxiu; Dong, Juan; Kinoshita, Toshinori (February 2022, Frontiers in Plant Science)