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

Sucrose-non-fermenting-1-related protein kinase-2s (SnRK2s) are critical for plant abiotic stress responses, including abscisic acid (ABA) signaling. Here, we develop a genetically encoded reporter for SnRK2 kinase activity. This sensor, named SNACS, shows an increase in the ratio of yellow to cyan fluorescence emission by OST1/SnRK2.6-mediated phosphorylation of a defined serine residue in SNACS. ABA rapidly increases FRET efficiency in N. benthamiana leaf cells and Arabidopsis guard cells. Interestingly, protein kinase inhibition decreases FRET efficiency in guard cells, providing direct experimental evidence that basal SnRK2 activity prevails in guard cells. Moreover, in contrast to ABA, the stomatal closing stimuli, elevated CO2 and MeJA, did not increase SNACS FRET ratios. These findings and gas exchange analyses of quintuple/sextuple ABA receptor mutants show that stomatal CO2 signaling requires basal ABA and SnRK2 signaling, but not SnRK2 activation. A recent model that CO2 signaling is mediated by PYL4/PYL5 ABA-receptors could not be supported here in two independent labs. We report a potent approach for real-time live-cell investigations of stress signaling. 

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.

 

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].

 

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.

 

Jasmonic acid (JA) and salicylic acid (SA) regulate stomatal closure, preventing pathogen invasion into plants. However, to what extent abscisic acid (ABA), SA and JA interact, and what the roles of SA and JA are in stomatal responses to environmental cues, remains unclear. Here, by using intact plant gas‐exchange measurements in JA and SA single and double mutants, we show that stomatal responsiveness to CO₂, light intensity, ABA, high vapor pressure deficit and ozone either did not or, for some stimuli only, very slightly depended upon JA and SA biosynthesis and signaling mutants, includingdde2, sid2, coi1,jai1,myc2andnpr1alleles. Although the stomata in the mutants studied clearly responded to ABA, CO₂, light and ozone, ABA‐triggered stomatal closure innpr1‐1was slightly accelerated compared with the wild type. Stomatal reopening after ozone pulses was quicker in thecoi1‐16mutant than in the wild type. In intact Arabidopsis plants, spraying with methyl‐JA led to only a modest reduction in stomatal conductance 80 min after treatment, whereas ABA and CO₂induced pronounced stomatal closure within minutes. We could not document a reduction of stomatal conductance after spraying with SA. Coronatine‐induced stomatal opening was initiated slowly after 1.5–2.0 h, and reached a maximum by 3 h after spraying intact plants. Our results suggest that ABA, CO₂and light are major regulators of rapid guard cell signaling, whereas JA and SA could play only minor roles in the whole‐plant stomatal response to environmental cues in Arabidopsis andSolanum lycopersicum(tomato).

 

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.

 

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.