Apoplastic metabolomics reveals sugars as mesophyll messengers regulating guard cell ion transport under red light

Abstract

Guard cell pairs in the leaf epidermis enclose stomata, microscopic pores mediating CO2 uptake and water loss. Historical data suggest that signals from interior mesophyll tissue may modulate guard-cell regulation of stomatal apertures, but the molecular identity of any metabolite-based signals has remained elusive. We discovered that extracellular (apoplastic) fluid from Arabidopsis thaliana and Vicia faba enhances red-light-induced stomatal opening. Our extensive metabolomics analyses identified 448 apoplastic metabolites; among these, both sugars (photosynthetic products) and maleic acid increased under red light and caused enhanced stomatal opening. Immunohistochemical assays demonstrated sucrose upregulation of H+-ATPase phosphorylation, indicating increased ATPase activity. Patch clamp assays revealed that sucrose inhibits slow anion currents, thus opposing anion efflux. These impacts occurred at sucrose concentrations matching those present endogenously under red light. These regulatory influences promote guard-cell solute import and retention, which drive stomatal opening. Our research thus addresses the decades-long question concerning the existence, identity and mechanistic impact of mesophyll messengers that coordinate photosynthesis with stomatal response. Guard cells define microscopic stomatal pores for CO2 uptake and water loss. Characterization of the extracellular metabolome revealed sugars as ‘mesophyll messengers’ from the leaf interior that enhance stomatal opening via regulation of the guard-cell H+-ATPase and anion channels.