3 research outputs found
Stomatal CO2/bicarbonate sensor consists of two interacting protein kinases, Raf-like HT1 and nonkinase-activity activity requiring MPK12/MPK4
Publisher Copyright: © 2022 The Authors.The continuing rise in the atmospheric carbon dioxide (CO2) concentration causes stomatal closing, thus critically affecting transpirational water loss, photosynthesis, and plant growth. However, the primary CO2 sensor remains unknown. Here, we show that elevated CO2 triggers interaction of the MAP kinases MPK4/MPK12 with the HT1 protein kinase, thus inhibiting HT1 kinase activity. At low CO2, HT1 phosphorylates and activates the downstream negatively regulating CBC1 kinase. Physiologically relevant HT1-mediated phosphorylation sites in CBC1 are identified. In a genetic screen, we identify dominant active HT1 mutants that cause insensitivity to elevated CO2. Dominant HT1 mutants abrogate the CO2/bicarbonate-induced MPK4/12-HT1 interaction and HT1 inhibition, which may be explained by a structural AlphaFold2- and Gaussian-accelerated dynamics-generated model. Unexpectedly, MAP kinase activity is not required for CO2 sensor function and CO2-triggered HT1 inhibition and stomatal closing. The presented findings reveal that MPK4/12 and HT1 together constitute the long-sought primary stomatal CO2/bicarbonate sensor upstream of the CBC1 kinase in plants.Peer reviewe
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The utilization of genetic modification and biochemical analysis involved in stress response in the model organism Arabidopsis thaliana.
Abiotic stresses such as drought, salinity, and temperature trigger an increased production of the phytohormone abscisic acid (ABA), which plays several important roles in plant development, such as drought resistance, seed germination, stomatal response, and water use efficiency. A recent study showed that Raf-like MAPKK-kinases (M3Ks) are required to activate OST1/SnRK2 protein kinases after PP2Cs-dependent dephosphorylation in the presence of ABA. However, the function of M3Ks in stomata remains largely unknown. Previously no guard cell-targeted gene induction system has been reported. Here, to further examine the role of the M3Ks genes, a strategy for overexpression and induction lines was developed and transgenic lines were generated using a strong Guard Cell-specific Promoter (pGC1) and green fluorescent protein (GFP) tag. The overexpression constructs were created using the Multisite Gateway Cloning technology and inducible versions of M3Ks genes have been generated using USER Cloning. Both constructs were transformed into Arabidopsis thaliana using Agrobacterium transformation. Seeds were collected and transgenic plants went through Basta (overexpression) or Hygromycin (inducible) antibiotic selection, genotyping using DNA extraction, and confocal microscopic analyses to determine green fluorescent protein (GFP) activity. Two truncated constitutively active (CA) versions of Raf-Kẟ1 constructs were generated using Gateway (overexpression) and USER (ethanol inducible) cloning technology. An increase in ABA concentration has been shown to lead to stomatal closure, the loss of the ability to transpire water will result in the release of water vapor, which will show warmer leaf temperatures and can be measured by thermal imaging. Thermal imaging and stomatal conductance experiments suggest that both overexpression line and inducible line have a higher leaf temperature when compared with the wild type plants and similar temperature of the closed stomata mutant controls “High Leaf Temperature 1” (ht1-2). These preliminary data suggest that upon increasing the expression of the M3Ks gene, transgenic plants are able to close stomata more than the wild type, which potentially allows plants to display higher water use efficiency
Recommended from our members
The utilization of genetic modification and biochemical analysis involved in stress response in the model organism Arabidopsis thaliana.
Abiotic stresses such as drought, salinity, and temperature trigger an increased production of the phytohormone abscisic acid (ABA), which plays several important roles in plant development, such as drought resistance, seed germination, stomatal response, and water use efficiency. A recent study showed that Raf-like MAPKK-kinases (M3Ks) are required to activate OST1/SnRK2 protein kinases after PP2Cs-dependent dephosphorylation in the presence of ABA. However, the function of M3Ks in stomata remains largely unknown. Previously no guard cell-targeted gene induction system has been reported. Here, to further examine the role of the M3Ks genes, a strategy for overexpression and induction lines was developed and transgenic lines were generated using a strong Guard Cell-specific Promoter (pGC1) and green fluorescent protein (GFP) tag. The overexpression constructs were created using the Multisite Gateway Cloning technology and inducible versions of M3Ks genes have been generated using USER Cloning. Both constructs were transformed into Arabidopsis thaliana using Agrobacterium transformation. Seeds were collected and transgenic plants went through Basta (overexpression) or Hygromycin (inducible) antibiotic selection, genotyping using DNA extraction, and confocal microscopic analyses to determine green fluorescent protein (GFP) activity. Two truncated constitutively active (CA) versions of Raf-Kẟ1 constructs were generated using Gateway (overexpression) and USER (ethanol inducible) cloning technology. An increase in ABA concentration has been shown to lead to stomatal closure, the loss of the ability to transpire water will result in the release of water vapor, which will show warmer leaf temperatures and can be measured by thermal imaging. Thermal imaging and stomatal conductance experiments suggest that both overexpression line and inducible line have a higher leaf temperature when compared with the wild type plants and similar temperature of the closed stomata mutant controls “High Leaf Temperature 1” (ht1-2). These preliminary data suggest that upon increasing the expression of the M3Ks gene, transgenic plants are able to close stomata more than the wild type, which potentially allows plants to display higher water use efficiency