Analysis Of PtrbHLH186 Transcription Factor Regulating PtrLBD12 To Affect Wood Formation In Populus Trichocarpa

Wood is one of the most important raw materials for pulp,paper,timber and energy.Wood formation is a continuous and complex process which is resulted from the development of xylem in woody plants.Transcription factors play important regulatory roles in this process.b HLH186（basic helix-loop-helix 186）is an important transcription factor involved in wood formation.In this study,we used the woody model plant Populus trichocarpa to identify the target genes of PtrbHLH186 transcription factor and analyze the regulation mechanism of PtrbHLH186 on wood formation.In this study,RNA-seq was carried out using xylem tissue of PtrbHLH186 overexpression transgenic plants.2272 downstream genes were identified,including 179 transcription factors.Based on analysis of wood cell-based chromatin-binding assays,we obtained 615 target genes,including 38 transcription factors.Integrative analysis of Ch IP-seq and RNA-seq identified 5downstream target transcription factors.Among these transcription factors,PtrLBD12 which belongs to LBD（Lateral Organ Boundaries Domain）family,has a higher transcriptional level in the secondary xylem.Ch IP-q PCR and EMSA assays demonstrated that PtrbHLH186 could specifically recognize and bind to E-box（5’-CANNTG-3’）sequences of the PtrLBD12 promoter.Moreover,as a downstream target gene of PtrbHLH186,PtrLBD12 may also play roles in wood formation.The analysis of PtrLBD12 expression in different tissues of P.trichocarpa showed that,it had a higher expression level in the secondary xylem.Subcellular localization indicated that PtrLBD12 was expressed only in nucleus,which is the characteristic of transcription factors.CRISPR/Cas9 gene editing technology was used to create the PtrLBD12loss-of-function mutants.PtrLBD12 overexpression transgenic plants were also generated.Phenotypic analysis showed that the transgenic plants had stunted growth with a reduced height,stem basal diameter,length of 8^th internode,and a number of internodes.Further analysis showed that transgenic plants promote lignin deposition and secondary growth.They had more fibers and more smaller vessels cells.RT-q PCR results showed that transgenic plants increased the expression of lignin monomer synthase genes.In conclusion,PtrbHLH186 specifically recognizes and binds to E-box（5’-CANNTG-3’）sequences of the PtrLBD12 promoter to activate its transcription.PtrLBD12 regulates the expression of monolignol genes to modify wood formation,including lignin deposition and cellular organization.We discovered that PtrbHLH186 regulates PtrLBD12 to affect wood formation,which provides new clues for tree species breeding with better traits.