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1. 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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2. Jasmonate‐mediated stomatal closure under elevated CO 2 revealed by time‐resolved metabolomics

   https://doi.org/10.1111/tpj.13296  

   Geng, Sisi ; Misra, Biswapriya B. ; de Armas, Evaldo ; Huhman, David V. ; Alborn, Hans T. ; Sumner, Lloyd W. ; Chen, Sixue ( October 2016 , The Plant Journal)

   Foliar stomatal movements are critical for regulating plant water loss and gas exchange. Elevated carbon dioxide (CO₂) levels are known to induce stomatal closure. However, the current knowledge onCO₂signal transduction in stomatal guard cells is limited. Here we report metabolomic responses ofBrassica napusguard cells to elevatedCO₂using three hyphenated metabolomics platforms: gas chromatography‐mass spectrometry (MS); liquid chromatography (LC)‐multiple reaction monitoring‐MS; and ultra‐high‐performanceLC‐quadrupole time‐of‐flight‐MS. A total of 358 metabolites from guard cells were quantified in a time‐course response to elevatedCO₂level. Most metabolites increased under elevatedCO₂, showing the most significant differences at 10 min. In addition, reactive oxygen species production increased and stomatal aperture decreased with time. Major alterations in flavonoid, organic acid, sugar, fatty acid, phenylpropanoid and amino acid metabolic pathways indicated changes in both primary and specialized metabolic pathways in guard cells. Most interestingly, the jasmonic acid (JA) biosynthesis pathway was significantly altered in the course of elevatedCO₂treatment. Together with results obtained fromJAbiosynthesis and signaling mutants as well asCO₂signaling mutants, we discovered thatCO₂‐induced stomatal closure is mediated byJAsignaling.

    

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3. Molecular and electrophysiological characterization of anion transport in Arabidopsis thaliana pollen reveals regulatory roles for pH , Ca 2+ and GABA

   https://doi.org/10.1111/nph.15863  

   Domingos, Patrícia ; Dias, Pedro N. ; Tavares, Bárbara ; Portes, Maria Teresa ; Wudick, Michael M. ; Konrad, Kai R. ; Gilliham, Matthew ; Bicho, Ana ; Feijó, José A. ( May 2019 , New Phytologist)

   We investigated the molecular basis and physiological implications of anion transport during pollen tube (PT) growth inArabidopsis thaliana(Col‐0).

   Patch‐clamp whole‐cell configuration analysis of pollen grain protoplasts revealed three subpopulations of anionic currents differentially regulated by cytoplasmic calcium ([Ca²⁺]_cyt). We investigated the pollen‐expressed proteinsAtSLAH3,AtALMT12,AtTMEM16 andAtCCCas the putative anion transporters responsible for these currents.

   AtCCC‐GFPwas observed at the shank andAtSLAH3‐GFPat the tip and shank of thePTplasma membrane. Both are likely to carry the majority of anion current at negative potentials, as extracellular anionic fluxes measured at the tip ofPTs with an anion vibrating probe were significantly lower inslah3^−/−andccc^−/−mutants, but unaffected inalmt12^−/−andtmem16^−/−. We further characterised the effect ofpHandGABAby patch clamp. Strong regulation by extracellularpHwas observed in the wild‐type, but not intmem16^−/−. Our results are compatible withAtTMEM16 functioning as an anion/H⁺cotransporter and therefore, as a putativepHsensor.GABApresence: (1) inhibited the overall currents, an effect that is abrogated in thealmt12^−/−and (2) reduced the current inAtALMT12 transfectedCOS‐7 cells, strongly suggesting the direct interaction ofGABAwithAtALMT12.

   Our data show thatAtSLAH3 andAtCCCactivity is sufficient to explain the major component of extracellular anion fluxes, and unveils a possible regulatory system linkingPTgrowth modulation bypH,GABA, and [Ca²⁺]_cytthrough anionic transporters.

    

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4. 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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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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