Metabolomics of Red Light-Induced Stomatal Opening in Arabidopsis thaliana: Coupling with Abscisic Acid and Jasmonic Acid Metabolism.

Environmental stimuli-triggered stomatal movement is a key physiological process that regulates CO₂ uptake and water loss in plants. Stomata are defined by pairs of guard cells that perceive and transduce external signals, leading to cellular volume changes and consequent stomatal aperture change. Within the visible light spectrum, red light induces stomatal opening in intact leaves. However, there has been debate regarding the extent to which red light-induced stomatal opening arises from direct guard cell sensing of red light vs. indirect responses as a result of red light influences on mesophyll photosynthesis. Here we identify conditions that result in red light-stimulated stomatal opening in isolated epidermal peels and enlargement of protoplasts, firmly establishing a direct guard cell response to red light. We then employ metabolomics workflows utilizing gas chromatography mass spectrometry (GC-MS/MS) and liquid chromatography mass spectrometry (LC-MS/MS) for metabolite profiling and identification of Arabidopsis guard cell metabolic signatures in response to red light in the absence of the mesophyll. We quantified 223 metabolites in Arabidopsis guard cells, with 104 found to be red light responsive. These red light-modulated metabolites participate in the tricarboxylic acid (TCA) cycle, carbon balance, phytohormone biosynthesis, and redox homeostasis. We next analyzed selected Arabidopsis mutants and discovered that stomatal opening response to red light is correlated with a decrease in guard cell ABA content and an increase in JA content. The red light-modulated guard cell metabolome reported here provides fundamental information concerning autonomous red light signaling pathways in guard cells.