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Scholarly Works (3 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2022)

As atmospheric carbon dioxide levels continue to rise, agricultural yields will be impacted worldwide. Our laboratory obtained evidence that specific MAPKs inhibit the activity of HT1, a negative regulator of CO2-induced stomatal closure. The findings showed that HT1 can phosphorylate CBC1/2 but not whether HT1 regulates CBC1/CBC2’s activity. This study analyzed stomatal conductance responses to changing CO2 levels with a cbc1/cbc2 double knockout line, a dominant ht1(A109V) line with a permanently active HT1 protein, and a cbc1/cbc2/ht1(A109V) triple mutant line with knocked out CBC1 and CBC2 genes and a constantly active HT1 protein. The cbc1/cbc2/ht1(A109V) mutant showed stomatal conductance values similar to cbc1/cbc2 samples, not ht1(A109V). The results suggest that the CBC1/CBC2 are epistatic to and function downstream of HT1 in the signaling pathway. Next, mutant lines of pGC1:MPK12(K70R)-GFP/mpk12, with a K70R mutation in MPK12, and pGC1:MPK12-GFP/mpk12, a rescue line, both showed stomatal conductance responses similar to Col-0. Meanwhile, mpk12 samples showed responses to changes in CO2 but higher stomatal conductance levels compared to Col-0. The data suggests that the kinase activity of MPK12 is not required to inhibit HT1. The final experiments used a line with two mutations in mapped HT1-mediated CBC1 phosphorylation sites, pGC1:CBC1(T256A/S280A)-GFP/cbc1cbc2, showed stomatal conductance similar to the cbc1/cbc2 samples while the rescue line pGC1:CBC1-GFP/cbc1cbc2 with a wildtype CBC1 gene, showed stomatal responses similar to Col-0. This suggests that the two mutated sites could be important for HT1 phosphorylation. The information gathered here can contribute to developing agricultural lines that can withstand rising levels of CO2.

    Cover page: Genetic modification and mutant construction for proteins involved in the CO2 signaling pathway of Arabidopsis thaliana
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2023)
    Little is known about long-distance mesophyll-driven signals that regulate stomatal conductance. Soluble and/or vapor-phase molecules have been proposed. In this study, the involvement of the gaseous signal ethylene in the modulation of stomatal conductance in Arabidopsis thaliana by CO2 /abscisic acid (ABA) was examined. We present a diffusion model which indicates that gaseous signaling molecule/s with a shorter/direct diffusion pathway to guard cells are more probable for rapid mesophyll-dependent stomatal conductance changes. We, therefore, analyzed different Arabidopsis ethylene-signaling and biosynthesis mutants for their ethylene production and kinetics of stomatal responses to ABA/[CO2 ]-shifts. According to our research, higher [CO2 ] causes Arabidopsis rosettes to produce more ethylene. An ACC-synthase octuple mutant with reduced ethylene biosynthesis exhibits dysfunctional CO2 -induced stomatal movements. Ethylene-insensitive receptor (gain-of-function), etr1-1 and etr2-1, and signaling, ein2-5 and ein2-1, mutants showed intact stomatal responses to [CO2 ]-shifts, whereas loss-of-function ethylene receptor mutants, including etr2-3;ein4-4;ers2-3, etr1-6;etr2-3 and etr1-6, showed markedly accelerated stomatal responses to [CO2 ]-shifts. Further investigation revealed a significantly impaired stomatal closure to ABA in the ACC-synthase octuple mutant and accelerated stomatal responses in the etr1-6;etr2-3, and etr1-6, but not in the etr2-3;ein4-4;ers2-3 mutants. These findings suggest essential functions of ethylene biosynthesis and signaling components in tuning/accelerating stomatal conductance responses to CO2 and ABA.
      Cover page: A role for ethylene signaling and biosynthesis in regulating and accelerating CO2 - and abscisic acid-mediated stomatal movements in Arabidopsis.
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2022)
      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.
        Cover page: Stomatal CO2/bicarbonate sensor consists of two interacting protein kinases, Raf-like HT1 and non-kinase-activity activity requiring MPK12/MPK4
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