The transcription factor ENHANCER OF SHOOT REGENERATION 2 orchestrates cytokinin dynamics leading to developmental reprograming and green callus formation

Abstract Callus formation and shoot regeneration are naturally triggered by stress and damage to the plant. They are also key components of tissue culture, which is crucial for gene editing, transformation, propagation, and other technologies in many species. Thus, the study of callus formation and shoot regeneration provides valuable insights into plant development. The transcription factor ENHANCER OF SHOOT REGENERATION 2/DORNROSCHEN-LIKE/BOLITA/SUPPRESSOR OF PHYTOCHROME B-4 2 (ESR2/DRNL/BOL/SOB2) promotes green callus formation in roots as well as shoot regeneration when overactive, and the phythormone cytokinin plays a prominent role in both the processes. Yet, the role of ESR2 in the cytokinin pathway has not been previously described in Arabidopsis (Arabidopsis thaliana). We found that cytokinin content and the expression of the cytokinin biosynthesis gene ISOPENTENYLTRANSFERASE 5 (IPT5) are greater in plants with high ESR2 activity. ESR2 also regulates the cytokinin signaling repressor ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6), and surprisingly, ESR2-stimulated green callus formation requires IPT5 and AHP6. Therefore, ESR2 promotes both cytokinin biosynthesis and the inhibition of cytokinin signaling and, paradoxically, requires a combination of these 2 effects for green callus induction. These findings provide a foundation to better understand the processes involved in tissue reprograming toward callus formation and the role of ESR2 in callus formation and shoot regeneration.