Callus proliferation-induced hypoxic microenvironment decreases shoot regeneration competence in Arabidopsis

Plants are aerobic organisms that rely on molecular oxygen for respiratory energy production. Hypoxic conditions, with oxygen levels ranging between 1% and 5%, usually limit aerobic respiration and affect plant growth and development. Here, we demonstrate that the hypoxic microenvironment induced by active cell proliferation during the two-step plant regeneration process intrinsically represses the regeneration competence of the callus in Arabidopsis thaliana. We showed that hypoxia-repressed plant regeneration is mediated by the RELATED TO APETALA 2.12 (RAP2.12) protein, a member of the Ethylene Response Factor VII (ERF-VII) family. We found that the hypoxia-activated RAP2.12 protein promotes salicylic acid (SA) biosynthesis and defense responses, thereby inhibiting pluripotency acquisition and de novo shoot regeneration in calli. Molecular and genetic analyses revealed that RAP2.12 could bind directly to the SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2) gene promoter and activate SA biosynthesis, repressing plant regeneration possibly via a PLETHORA (PLT)-dependent pathway. Consistently, the rap2.12 mutant calli exhibits enhanced shoot regeneration, which is impaired by SA treatment. Taken together, these findings uncover that the cell proliferation-dependent hypoxic microenvironment reduces cellular pluripotency and plant regeneration through the RAP2.12-SID2 module.