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Drought is a major environmental stress that threatens crop production. Therefore, identification of genes involved in drought stress response is of vital importance to decipher the molecular mechanism of stress signal transduction and breed drought tolerance crops, especially for maize. Clade A PP2C phosphatases are core abscisic acid (ABA) signaling components, regulating ABA signal transduction and drought response. However, the roles of other clade PP2Cs in drought resistance remain largely unknown. Here, we discovered a clade F PP2C, ZmPP84, that negatively regulates drought tolerance by screening a transgenic overexpression maize library. Quantitative RT-PCR indicates that the transcription of ZmPP84 is suppressed by drought stress. We identified that ZmMEK1, a member of the MAPKK family, interacts with ZmPP84 by immunoprecipitation and mass spectrometry analysis. Additionally, we found that ZmPP84 can dephosphorylate ZmMEK1 and repress its kinase activity on the downstream substrate kinase ZmSIMK1, while ZmSIMK1 is able to phosphorylate S-type anion channel ZmSLAC1 at S146 and T520 in vitro. Mutations of S146 and T520 to phosphomimetic aspartate could activate ZmSLAC1 currents in Xenopus oocytes. Taken together, our study suggests that ZmPP84 is a negative regulator of drought stress response that inhibits stomatal closure through dephosphorylating ZmMEK1, thereby repressing ZmMEK1-ZmSIMK1 signaling pathway., (© 2022 The Authors New Phytologist © 2022 New Phytologist Foundation.)

Abiotic stresses, including drought and salinity, trigger a complex osmotic-stress and abscisic acid (ABA) signal transduction network. The core ABA signalling components are snf1-related protein kinase2s (SnRK2s), which are activated by ABA-triggered inhibition of type-2C protein-phosphatases (PP2Cs). SnRK2 kinases are also activated by a rapid, largely unknown, ABA-independent osmotic-stress signalling pathway. Here, through a combination of a redundancy-circumventing genetic screen and biochemical analyses, we have identified functionally-redundant MAPKK-kinases (M3Ks) that are necessary for activation of SnRK2 kinases. These M3Ks phosphorylate a specific SnRK2/OST1 site, which is indispensable for ABA-induced reactivation of PP2C-dephosphorylated SnRK2 kinases. ABA-triggered SnRK2 activation, transcription factor phosphorylation and SLAC1 activation require these M3Ks in vitro and in plants. M3K triple knock-out plants show reduced ABA sensitivity and strongly impaired rapid osmotic-stress-induced SnRK2 activation. These findings demonstrate that this M3K clade is required for ABA- and osmotic-stress-activation of SnRK2 kinases, enabling robust ABA and osmotic stress signal transduction.

Stomatal pores are formed between a pair of guard cells and allow plant uptake of CO 2 and water evaporation. Their aperture depends on changes in osmolyte concentration of guard cell vacuoles, specifically of K + and Mal 2- Efflux of Mal 2- from the vacuole is required for stomatal closure; however, it is not clear how the anion is released. Here, we report the identification of ALMT4 (ALUMINUM ACTIVATED MALATE TRANSPORTER4) as an Arabidopsis thaliana ion channel that can mediate Mal 2- release from the vacuole and is required for stomatal closure in response to abscisic acid (ABA). Knockout mutants showed impaired stomatal closure in response to the drought stress hormone ABA and increased whole-plant wilting in response to drought and ABA. Electrophysiological data show that ALMT4 can mediate Mal 2- efflux and that the channel activity is dependent on a phosphorylatable C-terminal serine. Dephosphomimetic mutants of ALMT4 S382 showed increased channel activity and Mal 2- efflux. Reconstituting the active channel in almt4 mutants impaired growth and stomatal opening. Phosphomimetic mutants were electrically inactive and phenocopied the almt4 mutants. Surprisingly, S382 can be phosphorylated by mitogen-activated protein kinases in vitro. In brief, ALMT4 likely mediates Mal 2- efflux during ABA-induced stomatal closure and its activity depends on phosphorylation., (© 2017 American Society of Plant Biologists. All rights reserved.)

Increases in CO2 concentration 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/HCO3- regulation of SLAC1 is relevant for CO2 control 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/HCO3- in Xenopus oocytes. Mutations of the neighboring charged amino acid K255 and residue R432 and the predicted gate residue F450 did not affect HCO3- regulation of SLAC1. Notably, gas-exchange experiments with slac1-transformed plants expressing mutated SLAC1 proteins revealed that the SLAC1 residue R256 is required for CO2 regulation of stomatal movements in planta, but not for abscisic acid (ABA)-induced stomatal closing. Patch clamp analyses of guard cells show that activation of S-type anion channels by CO2/HCO3-, but not by ABA, was impaired, indicating the relevance of R256 for CO2 signal transduction. Together, these analyses suggest that the SLAC1 anion channel is one of the physiologically relevant CO2/HCO3- sensors in guard cells.

Global climate change‐caused drought stress, high temperatures and other extreme weather profoundly impact plant growth and development, restricting sustainable crop production. To cope with various environmental stimuli, plants can optimize the opening and closing of stomata to balance CO2 uptake for photosynthesis and water loss from leaves. Guard cells perceive and integrate various signals to adjust stomatal pores through turgor pressure regulation. Molecular mechanisms and signaling networks underlying the stomatal movements in response to environmental stresses have been extensively studied and elucidated. This review focuses on the molecular mechanisms of stomatal movements mediated by abscisic acid, light, CO2, reactive oxygen species, pathogens, temperature, and other phytohormones. We discussed the significance of elucidating the integrative mechanisms that regulate stomatal movements in helping design smart crops with enhanced water use efficiency and resilience in a climate‐changing world. [ABSTRACT FROM AUTHOR]

Nitraria sibirica is a shrub that can survive in extreme drought environments. The auxin-response factors (ARFs) are a class of transcription factors that are widely involved in plant growth and development, as well as in the regulation of stress resistance. However, the genome-wide identification of the ARF gene family and its responses to environmental stresses, especially drought stress, in N. sibirica has not yet been reported. Here, we identified a total of 12 ARF genes in the genome of N. sibirica, which were distributed over 10 chromosomes and divided into three clades. Intragenome synteny analysis revealed one collinear gene pair in the ARF gene family, i.e., NsARF9a and NsARF9b. Cis-acting element analysis showed that multiple hormones and stress-responsive cis-acting elements were found in the promoters of NsARFs, suggesting that NsARFs may be involved in multiple biological processes. Quantitative real-time PCR (qRT-PCR) showed that many NsARFs had tissue-specific expression patterns, with the highest expression of NsARF16 in the seedlings of N. sibirica. In addition, most of the NsARFs that were upregulated under drought were independent of endogenous ABA biosynthesis, whereas the response of NsARF5 and NsARF7a to drought was disrupted by the ABA-biosynthesis inhibitor fluridone. These studies provide a basis for further research into how NsARFs in N. sibirica respond to hormonal signaling and environmental stresses. [ABSTRACT FROM AUTHOR]

Summary: Respiration in leaves and the continued elevation in the atmospheric CO2 concentration cause CO2‐mediated reduction in stomatal pore apertures. Several mutants have been isolated for which stomatal responses to both abscisic acid (ABA) and CO2 are simultaneously defective. However, there are only few mutations that impair the stomatal response to elevated CO2, but not to ABA. Such mutants are invaluable in unraveling the molecular mechanisms of early CO2 signal transduction in guard cells. Recently, mutations in the mitogen‐activated protein (MAP) kinase, MPK12, have been shown to partially impair CO2‐induced stomatal closure. Here, we show that mpk12 plants, in which MPK4 is stably silenced specifically in guard cells (mpk12 mpk4GC homozygous double‐mutants), completely lack CO2‐induced stomatal responses and have impaired activation of guard cell S‐type anion channels in response to elevated CO2/bicarbonate. However, ABA‐induced stomatal closure, S‐type anion channel activation and ABA‐induced marker gene expression remain intact in the mpk12 mpk4GC double‐mutants. These findings suggest that MPK12 and MPK4 act very early in CO2 signaling, upstream of, or parallel to the convergence of CO2 and ABA signal transduction. The activities of MPK4 and MPK12 protein kinases were not directly modulated by CO2/bicarbonate in vitro, suggesting that they are not direct CO2/bicarbonate sensors. Further data indicate that MPK4 and MPK12 have distinguishable roles in Arabidopsis and that the previously suggested role of RHC1 in stomatal CO2 signaling is minor, whereas MPK4 and MPK12 act as key components of early stomatal CO2 signal transduction. Significance statement: We show that MPK4 and MPK12 are required for CO2‐triggered stomatal responses but not for ABA‐induced stomatal closure. These results suggest that these two MAP kinases function upstream of the convergence of CO2 and ABA signaling. However, activities of MPK4 and MPK12 were not modulated by CO2/bicarbonate and, thus, direct HCO3−/CO2 sensing mechanisms remain to be identified. [ABSTRACT FROM AUTHOR]

Summary: We conducted an infrared thermal imaging‐based genetic screen to identify Arabidopsis mutants displaying aberrant stomatal behavior in response to elevated concentrations of CO2. This approach resulted in the isolation of a novel allele of the Arabidopsis BIG locus (At3g02260) that we have called CO2insensitive 1 (cis1). BIG mutants are compromised in elevated CO2‐induced stomatal closure and bicarbonate activation of S‐type anion channel currents. In contrast with the wild‐type, they fail to exhibit reductions in stomatal density and index when grown in elevated CO2. However, like the wild‐type, BIG mutants display inhibition of stomatal opening when exposed to elevated CO2. BIG mutants also display wild‐type stomatal aperture responses to the closure‐inducing stimulus abscisic acid (ABA). Our results indicate that BIG is a signaling component involved in the elevated CO2‐mediated control of stomatal development. In the control of stomatal aperture by CO2, BIG is only required in elevated CO2‐induced closure and not in the inhibition of stomatal opening by this environmental signal. These data show that, at the molecular level, the CO2‐mediated inhibition of opening and promotion of stomatal closure signaling pathways are separable and BIG represents a distinguishing element in these two CO2‐mediated responses. [ABSTRACT FROM AUTHOR]

Summary: We conducted an infrared thermal imaging‐based genetic screen to identify Arabidopsis mutants displaying aberrant stomatal behavior in response to elevated concentrations of CO2. This approach resulted in the isolation of a novel allele of the Arabidopsis BIG locus (At3g02260) that we have called CO2insensitive 1 (cis1). BIG mutants are compromised in elevated CO2‐induced stomatal closure and bicarbonate activation of S‐type anion channel currents. In contrast with the wild‐type, they fail to exhibit reductions in stomatal density and index when grown in elevated CO2. However, like the wild‐type, BIG mutants display inhibition of stomatal opening when exposed to elevated CO2. BIG mutants also display wild‐type stomatal aperture responses to the closure‐inducing stimulus abscisic acid (ABA). Our results indicate that BIG is a signaling component involved in the elevated CO2‐mediated control of stomatal development. In the control of stomatal aperture by CO2, BIG is only required in elevated CO2‐induced closure and not in the inhibition of stomatal opening by this environmental signal. These data show that, at the molecular level, the CO2‐mediated inhibition of opening and promotion of stomatal closure signaling pathways are separable and BIG represents a distinguishing element in these two CO2‐mediated responses. [ABSTRACT FROM AUTHOR]