Studies On The Molecular Regulation Mechanism Of NF-YA6 In SCW Formation Of Populus Trichocarpa

The plant NF-Y gene family is involved in a variety of biological processes including hormone response,stress response and growth and development.The PtrNF-YA6 gene of Populus trichocarpa belongs to the NF-YA subfamily,which was found to be involved in the secondary cell wall formation process in a previous study of Arabidopsis thaliana.In this study,we analyzed the role of PtrNF-YA6 gene in the formation of secondary cell wall(SCW)in Populus trichocarpa by bioinformatics and molecular biology techniques and analyzed the molecular mechanism of PtrNFYA6 regulating SCW formation in Populus trichocarpa.The main findings are as follows:(1)A total of 52 NF-Y family members,containing 13 NF-YA,21 NF-YB,and 18 NF-YC,were identified from Populus trichocarpa,and these family members were unevenly distributed on 17 chromosomes.RNA-seq analysis revealed that the expression of PtrNF-YA6 in Populus trichocarpa was tissue-specific,with higher expression in roots and secondary xylem.(2)PtrNF-YA6 protein is localized in the nucleus,has no transcriptional self-activating activity,and specifically recognizes and binds SNBE and SMRE elements.(3)12 PtrNF-YA6 overexpression transgenic lines and 16 suppression expression transgenic lines were obtained by the agrobacterium-mediated stem segment transformation method.Phenotypic observations and secondary trait analysis of the transgenic lines revealed that overexpression PtrNF-YA6 had an inhibitory effect on the growth and development of the transgenic plants and could significantly inhibit the hemicellulose synthesis and SCW thickening process of the transgenic plants.(4)By RNA-seq,405 differentially expressed genes(DEGs)were identified from overexpressing PtrNF-YA6 transgenic lines,and their expression trends were consistent with the results of RT-q PCR validation.Yeast single hybridization and Ch IP-PCR experiments demonstrated that PtrNF-YA6 was able to inhibit plant hemicellulose synthesis and SCW thickening process by regulating downstream genes Ptr4CL3,Ptr GT47A-1,Ptr GT8 C,Ptr GT8 F,Ptr GT43 B,Ptr GT47 C,Ptr GT8 A,Ptr GT8 B,AT-EXP1 expression.(5)All mi R169 family members were found to potentially affect PtrNF-YA6 gene expression by upstream mi RNA prediction of PtrNF-YA6.The expression of PtrNF-YA6 in the secondary xylem was demonstrated to be regulated by mi R169 o and mi R169 y by RNA-seq,degradome sequencing,and effector vector and reporter vector cotransformation experiments.In summary,PtrNF-YA6 can inhibit plant hemicellulose synthesis and SCW thickening by regulating the expression of downstream SCW formation-related target genes,while its expression level during wood formation is regulated by mi R169 o and mi R169 y.This study provides new evidence for the pivotal role of transcription factors in the regulatory network of SCW formation in woody plants and the mechanisms of transcriptional regulation.