Presentation Information
[1ASPR-20]Identification of Downstream Factors of MAP4K1/2 in ABA Signaling and Physiological Approach to Functional Analysis in the Guard Cell Extracellular Environment
○Taiki Yanagisawa1 (1. Tokyo University of Agriculture and Technology (Japan))
Keywords:
MAP4K1/2,Stomatal closure,Abscisic acid (ABA),Phosphoproteomics,Gas exchange,Vapor Pressure Deficit (VPD)
[Purpose] Stomatal opening and closing in plants is precisely regulated by abscisic acid (ABA). Recent studies from our laboratory have revealed that Arabidopsis MAP4K1 and MAP4K2 are key regulators in ABA-induced stomatal closure. However, the direct downstream substrates of these MAP4K1/2 enzymes remain undiscovered, and the full scope of their signaling pathways is not yet understood. Furthermore, although these factors are expressed throughout the plant body, their functions outside guard cells remain largely unknown. Therefore, this study aims to establish a physiological phenotyping analysis workflow through the ASPIRE program to evaluate the functions of the identified MAP4K1/2 multifacetedly, thereby gaining insights that bridge the molecular level with the tissue and plant body levels.
[Methods] To master cutting-edge physiological phenotyping techniques, we spent approximately one month at Professor Tracy Lawson′s laboratory at the University of Illinois. We focused primarily on establishing two techniques. First, for dynamic gas exchange measurements, we used the portable photosynthesis measurement system LI-6800 to learn real-time measurement techniques for stomatal conductance and CO2 uptake rate. Additionally, a rapid multi-plant screening method using the LI-600 was implemented. Next, for stomatal morphology analysis, a pipeline was constructed to automatically calculate stomatal density (SD) from leaf surface impressions using the AI image analysis tool ″Biodock.″ This enables objective evaluation of whether MAP4K1/2 mutations affect stomatal morphology or influence dynamic physiological responses.
[Results and Discussion] Given the short duration of this one-month assignment, the primary achievement was establishing measurement protocols optimized for mutant analysis in Arabidopsis thaliana. Preliminary measurements using LI-6800 confirmed sufficient sensitivity to capture subtle dynamic changes in stomatal closure. Furthermore, integrating AI analysis via Biodock established a method to clearly separate morphological and physiological factors. These technical foundations are essential for linking our laboratory's phosphoproteome technology with physiological phenomena—the actual behavior of plants.
[Conclusion] Through training at the University of Illinois, we acquired a pipeline to objectively evaluate whether MAP4K1/2 mutations affect either the morphology or the dynamics (physiological response) of stomata. Moving forward, we plan to measure dynamic responses to changes in water potential deficit (VPD) in our mutant lines and elucidate the role of MAP4K1/2 in the apical parenchyma. By integrating physiology and molecular biology, we aim to identify unknown downstream substrates and fully elucidate stress adaptation strategies centered on MAP4K1/2.
[Methods] To master cutting-edge physiological phenotyping techniques, we spent approximately one month at Professor Tracy Lawson′s laboratory at the University of Illinois. We focused primarily on establishing two techniques. First, for dynamic gas exchange measurements, we used the portable photosynthesis measurement system LI-6800 to learn real-time measurement techniques for stomatal conductance and CO2 uptake rate. Additionally, a rapid multi-plant screening method using the LI-600 was implemented. Next, for stomatal morphology analysis, a pipeline was constructed to automatically calculate stomatal density (SD) from leaf surface impressions using the AI image analysis tool ″Biodock.″ This enables objective evaluation of whether MAP4K1/2 mutations affect stomatal morphology or influence dynamic physiological responses.
[Results and Discussion] Given the short duration of this one-month assignment, the primary achievement was establishing measurement protocols optimized for mutant analysis in Arabidopsis thaliana. Preliminary measurements using LI-6800 confirmed sufficient sensitivity to capture subtle dynamic changes in stomatal closure. Furthermore, integrating AI analysis via Biodock established a method to clearly separate morphological and physiological factors. These technical foundations are essential for linking our laboratory's phosphoproteome technology with physiological phenomena—the actual behavior of plants.
[Conclusion] Through training at the University of Illinois, we acquired a pipeline to objectively evaluate whether MAP4K1/2 mutations affect either the morphology or the dynamics (physiological response) of stomata. Moving forward, we plan to measure dynamic responses to changes in water potential deficit (VPD) in our mutant lines and elucidate the role of MAP4K1/2 in the apical parenchyma. By integrating physiology and molecular biology, we aim to identify unknown downstream substrates and fully elucidate stress adaptation strategies centered on MAP4K1/2.
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