Protein evolution is shaped by sequence variation that modulates protein properties—for example, through the 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, which is activated by B4 RAF kinases in response to heat stress. This phosphorylation stabilizes UBP24, enabling the deubiquitylation of a plasma membrane H+-ATPase to promote stomatal opening. This reveals a 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 homologue 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 in plants and yeast and highlighting how charge-based regulation promotes cellular resilience under stress.