The ESR2–HDA6 complex exerts negative feedback regulation of auxin biosynthesis to delay callus initiation from Arabidopsis leaf explants during tissue culture

Plants exhibit an astonishing ability to regulate organ regeneration upon wounding. Excision of leaf explants promotes biosynthesis of indole-3-acetic acid (IAA), which is polar-transported to excised regions, where cell fate transition leads to specification of root founder cells to induce de novo root regeneration. The regeneration capacity of plants has been utilized to develop in vitro tissue culture technology. Here, we report that IAA accumulation near wounded site of leaf explants is essential for induction of callus on 2,4-dichlorophenoxyacetic acid (2,4-D)-rich callus-inducing medium (CIM). Notably, a high concentration of a synthetic auxin, 2,4-D, does not compensate for IAA action because of its limited efflux; rather, it lowers IAA biosynthesis via a negative feedback mechanism at an early stage of in vitro tissue culture, delaying callus initiation. The auxin negative feedback loop in CIM-cultured leaf explants is mediated by an auxin-inducible AP2 transcription factor, ENHANCER OF SHOOT REGENERATION 2 (ESR2), and its interacting partner HISTONE DEACETYLASE 6 (HDA6). The ESR2–HDA6 complex binds directly to, and removes the H3ac mark from, the YUCCA1 (YUC1), YUC7, and YUC9 loci, consequently repressing auxin biosynthesis and inhibiting cell fate transition on 2,4-D-rich CIM. These findings indicate that negative feedback regulation of auxin biosynthesis by ESR2 and HDA6 interferes with proper cell fate transition and callus initiation.