Studies On The Molecular Mechanism Of PtrTCP3 In Regulating Secondary Cell Wall Formation In Populus Trichocarpa

The TCP gene family is a plant-specific transcription factors family,which play a critical role in plant growth and development.Little is known about the function of TCP genes in poplar growth and development,and their role in secondary cell wall formation remains to be investigated.In this study,the biological functions of PtrTCP3 and the molecular mechanisms regulating secondary cell wall formation were comprehensively analyzed using molecular genetics,anatomy,and physiological and biochemical analyses in poplar,with the following results:1.34 PtrTCPs were identified from the genome in Populus trichocarpa,which can be divided into two subfamilies,Class I and Class II;PtrTCP3 belongs to Class I subfamily and is highly expressed in secondary stems and xylem;PtrTCP3 is localized in the nucleus,and the transcriptional activation region is located between 226-274 aa;the PtrTCP3 promoter driven GUS genes were mainly expressed in the vascular of petioles,leaf veins and in the progressively lignified stem nodes.2.Creation of overexpressing PtrTCP3 transgenic poplar and Cas9/g RNA-based knockout ptrtcp3 mutants by Agrobacterium-mediated genetic transformation in P.trichocarpa.The length,width and cell wall thickness,cellulose content and hemicellulose content of fiber and vessel cells of overexpressed PtrTCP3 transgenic poplar were significantly increased compared with the wild-type control,while the length,width and cell wall thickness,cellulose content and hemicellulose content of fiber and vessel cells of ptrtcp3 mutant were significantly decreased compared with the wild-type control.3.617 differentially expressed genes were identified by RNA-seq from overexpressing PtrTCP3 transgenic poplars,which were GO functionally enriched to biological processes such as secondary cell wall biosynthesis,xylan biosynthesis and cellulose fraction synthesis.Four functional genes associated with secondary cell wall biosynthesis,including Ptr XTH23,Ptr IRX15,Ptr IRX9-1 and Ptr Ces A8 B,were initially identified as downstream regulatory target genes of PtrTCP3 and were positively regulated by PtrTCP3.Chromatin immunoprecipitation and yeast one-hybrid demonstrated that PtrTCP3 could regulate the expression of downstream target genes by binding to their promoters.4.Yeast two-hybrid,bimolecular fluorescence complementation and luciferase complementation experiments showed that PtrTCP3 can form homodimers by itself and interacts with 13 homologous PtrTCPs proteins.In summary,the expression patterns of PtrTCP gene family members are diverse,PtrTCP3 is specifically expressed during secondary cell wall formation,overexpression PtrTCP3 promotes secondary cell wall formation,and knockdown PtrTCP3 inhibits secondary cell wall formation.The analysis of the biological functions and regulatory mechanisms of PtrTCP3 in regulating secondary cell wall formation can lay the foundation for elucidating the role of TCP genes in tree growth and development,especially in secondary cell wall formation.