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 regener-ation competence of the callus in Arabidopsis thaliana.We showed that hypoxia-repressed plant regener-ation is mediated by the RELATED TO APETALA2.12(RAP2.12)protein,a memberof the Ethylene Response Factor VIl(ERF-Vll)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 find-ings uncover that the cell proliferation-dependent hypoxic microenvironment reduces cellular pluripotency and plant regeneration through the RAP2.12-SID2 module.

ESR2–HDA6 complex negatively regulates auxin biosynthesis to delay callus initiation in Arabidopsis leaf explants during tissue culture

Plants exhibit an astonishing ability to regulate organ regeneration upon wounding.Excision of leaf explants promotes the biosynthesis of indole-3-acetic acid(IAA),which is polar-transported to excised regions,where cell fate transition leads to root founder cell specification to induce de novo root regeneration.The regeneration capacity of plants has been utilized to develop in vitro tissue culture technologies.Here,we report that IAA accumulation near the wounded site of leaf explants is essential for callus formation on 2,4-dichlorophenoxyacetic acid(2,4-D)-rich callus-inducing medium(CIM).Notably,a high concentration of 2,4-D does not compensate for the action of IAA 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 APETALA2 transcription factor,ENHANCER OF SHOOT REGENERATION 2(ESR2),along with 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.

Arabidopsis TOR signaling is essential forsugar-regulated callus formation

Dedifferentiation is a remarkable process that produces pluripotent stem cells from differentiated somatic cells to ensure developmental plasticity.Plants have evolved the ability of cellular dedifferentiation,and signaling cascades related to auxin and cytokinin-dependent callus formation have been extensively investigated.However,the molecular mechanism underlying sugar-dependent callus format:ion remains unknown.Here,we show that sugar-dependent callus formation is mainly regulated by the TOR-E;Fa module in Arabidopsis.Sugar-activated TOR kinase phosphorylates and stabilizes E;Fa proteins to transcriptionally activate S-phase genes during callus formation.In parallel,E;Fa is transcriptionally regulated by the ARF-LBD transcription cascade.Mult:i-layered regulation of E;Fa by sugar and auxin is likely to shape balanced cellular dedifferentiation capability in Arabidopsis.