PtRR13 Regulates Poplar Meristem Regeneration By Mediating PtWUS Expression

Regeneration of shoot apical metistem(SAM)is the key step of plant regeneration,and provides a foundation for in vitro propagation and genetic engineering.Previous studies have shown that WHCSHEL(WUS)plays crucial roles in the regeneration and maintenance of SAM in Arabidopsis.Type-B ARABIDOPSIS RESPONSE REGULATORS(ARRs)are involved in shoot regeneration via direct regulation of WUS.However,the mechanisms underly in shoot regeneration in woody plants remain unclear.In this study,we studied the regulatory roles of type-B ARRs and WUS in shoot regeneration of poplar,the woody model species.Using TCS::GUS reporter lines,the distribution patterns of cytokinin response signals during shoot regeneration were analyzed.The results showed that cytokinin response signal were uniformly distributed at the edge of callus at the early stage of shoot induction,and gradually swiched to the central region of the regenerated shoot meristem.After the formation of SAM,GUS signals were distributed throughout the meristem except L1 cell layer.The results indicate that the distribution patterns of cytokinin response signals during shoot regeneration are similar to those in Arabidopsis.According to the sequence homology,PtRR12 and PtRR13,homologs of Arabidopsis ARR1 were cloned.In situ hybridization showed that the expression patterns of PtRR12 and PtRR13 during shoot regeneration were consistent with that of TCS::GUS.Overexpressing PtRR13 did not significantly change shoot regeneration.However,shoot regeneration frequencies were significantly increased in gPtRR13::GFP transgenic lines,wheares those of 35S::PtRR13-SRDX lines were repressed.The results suggest that PtRR13 positively regulated shoot regeneration via an expression-patterns dependent way.PtWUS1 and Pt WUS2,homologs of Arabidopsis WUS in poplar were cloned according to the sequence homology.The expression and function of these two genes were analyzed.The results showed that PtWUS1 was specifically expressed in the organizing center of the shoot meristem.Pt WUS2 was expressed at the tip of leaf primordium and in the central region of shoot meristem.The overexpressing PtWUS1 partially rescued the phenotypes of Arabidopsis wus mutants,suggesting that Pt WUS1 and Arabidopsis WUS have similar functions.Overexpression of PtWUS1 inhibits both cell division and differentiation.Enhanced expression of PtWUS1 through pPt WUS1::LHG4-pOp::PtWUS1 vector promoted shoot regeneration and branching.These results indicate that Pt WUS1 positively regulated shoot regeneration via an expression-patterns dependent manner.The expression levels of Pt WUS1 and Pt WUS2 were significantly improved in gPtRR13::GFP transgenic lines,and reduced in 35S::PtRR13-SRDX transgenic lines,indicating that PtRR13 positively regulated the transcription of PtWUS1 and Pt WUS2.ChIP analysis using gPtRR13::GFP regenerated shoots showed that PtRR13 bound different fragments of Pt WUS1 and PtWUS2 promoter regions,respectively.These results indicate that PtRR13 positively regulated expression through binding its promoter.In together,the results of this study indicate that PtRR13 mediated the expression of PtWUS1 and regulated the regeneration of shoot meristem in poplar.