| Wood is one of the most abundant renewable resources in the world,which is a natural environmental protection and low energy consumption material.It plays an important role during the history of human civilization,and has long been used as raw material for construction,pulp,paper production and as a feed-stock for biofuels.Wood formation is a complex process which is the result of continuous deposition and accumulation of secondary xylem generated by the division and differentiation of cambium cells.Wood formation is controlled by transcriptional regulatory networks(TRNs)involving regulatory homeostasis determined by combinations of transcription factor(TF)–DNA and TF–TF interactions.Functions of TF–TF interactions in wood formation are still in the early stages of identification.PtrMYB074 is a woody dicot-specific TF in a TRN for wood formation in Populus trichocarpa.Therefore,PtrMYB074 was selected to study the regulation mechanism of TF-TF interaction in wood formation.Through analysis of transcriptome data from different tissues(root,stem,leaf and shoot)of poplar,996 transcription factors with high abundance expression in xylem were identified.We successfully cloned 221 TF to obtain a xylem abundance library of transcription factors.Here,the interaction between 221 TF and PtrMYB074,a key factor in the TRN of wood formation,was analyzed by yeast two-hybrid(Y2H).We identified 54 TFs that interact with PtrMYB074.In the xylem protoplast of poplar,bimolecular fluorescence complementation(Bi FC)assay was used to further prove the interactions in poplar.On this basis,the molecular mechanism of how PtrWRKY19,the most highly expressed as well as xylem-specific transcription factor,interacts with PtrMYB074 to synergistically regulate wood formation,was studied.Firstly,overexpressing PtrMYB074 and PtrWRKY19 transgenics were generated by Agrobacterium Tumefaciens-mediated genetic transformation.Phenotypic analysis showed that overexpression of PtrMYB074 and PtrWRKY19 genes resulted in retarded plant growth,increased lignification,increased total lignin and guaiacyllignin,and a higher proportion of smaller stem vessels.Then,by chromatin immunoprecipitation combined with high-throughput sequencing(Ch IP-seq)analysis,we identified the common target genes of PtrMYB074 and PtrWRKY19.Among the target genes related to wood formation,PtrbHLH186 was the xylem-specific one.Secondly,using chromatin immunoprecipitation and electrophoretic mobility shift assays,we demonstrated that PtrWRKY19 alone could bind directly to the PtrbHLH186 promoter,while PtrMYB074 alone cannot bind to PtrbHLH186 promoter.PtrMYB074 interacts with PtrWRKY19 to form a heterodimer,and PtrMYB074 is recruited by PtrWRKY19 to the PtrbHLH186 promoter Wbox to enhance the expression level of PtrbHLH186.In addition,using transient and CRISPRmediated transgenesis in P.trichocarpa coupled,we demonstrated that co-expression of PtrMYB074 and PtrWRKY19 is required for the transcriptional activation of PtrbHLH186.The gene function of PtrbHLH186 transcription factor was further analyzed,and overexpressing of PtrbHLH186 transgenics in P.trichocarpa were generated.The transgenic phenotypes were further analyzed.Overexpressing PtrbHLH186 in P.trichocarpa resulted in retarded plant growth,increased total lignin and guaiacyl-lignin,reduced polysaccharide,a higher proportion of smaller stem vessels,and strong drought-tolerance phenotypes.In conclusion,we found that the molecular module “PtrMYB074-PtrWRKY19-PtrbHLH186” regulation influences secondary xylem development through TF-TF interaction and TF-DNA interaction,changing the structure and number of vessel cells and wood composition to adapt poplar secondary growth to drought conditions.The discovery provided a new clue for the genetic improvement of forest trees by molecular breeding. |
