MPK3- and MPK6-mediated phosphorylation of STOP1 triggers its nuclear stabilization to modulate hypoxia responses in Arabidopsis.

In plants, responses to hypoxia include activation of fermentation pathways, cytosolic acidification, and other metabolic shifts. In Arabidopsis (Arabidopsis thaliana), the transcription factor SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) contributes to regulating cellular responses to low-oxygen stress; however, the underlying mechanism remains largely unknown. Here, we showed that transgenic lines overexpressing STOP1 exhibited improved tolerance of hypoxia and submergence, whereas knockout of STOP1 reduced tolerance. STOP1 accumulated during hypoxia and was degraded during post-hypoxia reoxygenation via ubiquitination by PLANT U-BOX-TYPE UBIQUITIN LIGASE 24 (PUB24). Under hypoxia, MITOGEN-ACTIVATED PROTEIN KINASE 3 (MPK3) and MPK6 interacted with and phosphorylated STOP1 to compete with its PUB24-mediated ubiquitination, thus stabilizing STOP1 in the nucleus, where it activated the transcription of GLUTAMATE DEHYDROGENASE 1 (GDH1) and GDH2 for cellular homeostasis of acidic metabolism during hypoxia. Mutating three phosphorylated residues in STOP1 to alanine attenuated its nuclear accumulation and diminished STOP1-mediated hypoxia tolerance. Moreover, we identified the lipid phosphatidic acid as a critical modulator of the MPK3/6-STOP1 association. Overall, these findings uncover an antagonistic biochemical mechanism in which MPK3/6-dependent phosphorylation and PUB24-dependent ubiquitination of STOP1 modulate its nuclear accumulation to control hypoxia responses in Arabidopsis.