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Title: Calcium specificity signaling mechanisms in abscisic acid signal transduction in Arabidopsis guard cells

A central question is how specificity in cellular responses to the eukaryotic second messenger Ca2+ is achieved. Plant guard cells, that form stomatal pores for gas exchange, provide a powerful system for in depth investigation of Ca2+-signaling specificity in plants. In intact guard cells, abscisic acid (ABA) enhances (primes) the Ca2+-sensitivity of downstream signaling events that result in activation of S-type anion channels during stomatal closure, providing a specificity mechanism in Ca2+-signaling. However, the underlying genetic and biochemical mechanisms remain unknown. Here we show impairment of ABA signal transduction in stomata of calcium-dependent protein kinase quadruple mutant plants. Interestingly, protein phosphatase 2Cs prevent non-specific Ca2+-signaling. Moreover, we demonstrate an unexpected interdependence of the Ca2+-dependent and Ca2+-independent ABA-signaling branches and the in planta requirement of simultaneous phosphorylation at two key phosphorylation sites in SLAC1. We identify novel mechanisms ensuring specificity and robustness within stomatal Ca2+-signaling on a cellular, genetic, and biochemical level.

 
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NSF-PAR ID:
10000386
Author(s) / Creator(s):
 ;  ;  ;  ;  ;  ;  ;  ;  ;  ;  
Publisher / Repository:
eLife Sciences Publications, Ltd.
Date Published:
Journal Name:
eLife
Volume:
4
ISSN:
2050-084X
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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  2. 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/HCO3and 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. Summary

    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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  4. null (Ed.)
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  5. <bold>Summary</bold>

    Cytosolic calcium concentration ([Ca2+]cyt) and heterotrimeric G‐proteins are universal eukaryotic signaling elements. In plant guard cells, extracellular calcium (Cao) 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 Caoresponses: 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 Cao. By contrast, stomatal movements ofagb1mutants andagb1/gpa1double‐mutants, as well as those of theagg1agg2 Gγ double‐mutant, were insensitive to Cao. 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 Cao, 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 Caoinduced InsP3 production in Col but not inagb1. In sum, G‐protein signaling viaAGB1/AGG1/AGG2 is essential for Cao‐regulation of stomatal apertures, and stomatal movements in response to Caoapparently require Ca2+‐induced Ca2+release that is likely dependent on Gβγ interaction withPLCs leading to InsP3 production.

     
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