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Title: Identification of SLAC1 anion channel residues required for CO 2 /bicarbonate sensing and regulation of stomatal movements

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/HCO3regulation 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/HCO3inXenopusoocytes. Mutations of the neighboring charged amino acid K255 and residue R432 and the predicted gate residue F450 did not affect HCO3regulation 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 more » 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/HCO3sensors in guard cells.

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Publication Date:
Journal Name:
Proceedings of the National Academy of Sciences
Page Range or eLocation-ID:
p. 11129-11137
Proceedings of the National Academy of Sciences
Sponsoring Org:
National Science Foundation
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  1. 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 havemore »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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  5. <bold>Summary</bold>

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