UC San Diego

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.