Evolutionary tuning of molecular charge state of UBP24 shapes eukaryotic responses to high temperature

Protein evolution is shaped by sequence variation that modulates protein properties, e.g., through gain or loss of post-translational modifications. Among these, reversible phosphorylation alters a protein’s overall electrical charge and enables organisms to dynamically respond to environmental fluctuations. In plants, the hydro-active opening of stomata, microscopic pores that regulate gas exchange and leaf temperature, is governed by phosphorylation-dependent signalling. Here, we identify a mechanism involving the deubiquitylase UBIQUITIN-SPECIFIC PROTEASE 24 (UBP24) that promotes stomatal opening in Arabidopsis thaliana under heat. UBP24 is phosphorylated at serine 360 by the kinase OPEN STOMATA 1 (OST1), which is activated by B4 RAF kinases in response to heat stress. This phosphorylation stabilizes UBP24, enabling deubiquitylation of a plasma membrane H⁺-ATPase to promote stomatal opening. This reveals a novel heat-responsive signalling pathway that evolved in vascular plants to regulate stomatal function. Strikingly, a similar evolutionary feature exists in Saccharomyces cerevisiae, where the UBP24 homolog Ubp3 requires a constitutively negatively charged residue at the homologous site to support growth after heat shock. Our findings uncover a conserved molecular mechanism in which negative charge, via phosphorylation or acidic residues, modulates deubiquitylase function, supporting adaptive thermal responses across eukaryotes and highlighting how charge-based regulation promotes cellular resilience under stress.