Molecular Mechanism Of PtrOBP1 Regulating Secondary Cell Wall Formation In Populus Trichocarpa

The plant-specific Dof gene family plays a significant role as transcription factors in many biological processes of plants.OBP1 is a member of the D2 subtribe of the Dof family.Previous experiments showed that OBP1 had biological functions in the formation of secondary cell wall(SCW)in herbaceous plants.Ptr OBP1,which is homologous to At OBP1,was targeted as the object of this study based on the bioinformatics analysis and RNA-Seq data analysis.Its basic characteristics and functions in the metabolic mechanisms of SCW was analyzed and determined.The results are as follows:(1)By using bioinformatics tools,a total of 45 Dof gene family members in Populus trichocarpa were identified.They can be classified into eight subfamilies and unevenly located on 19 chromosomes.By using RNA-Seq and q RT-PCR,it showed differential expression levels of 18 Dof genes between different internodes in Populus trichocarpa.The results showed that Pt OBP1 had the highest expression level in xylem in Populus trichocarpa.(2)Ptr OBP1 protein was located in the nucleus and exhibited transcriptional activation activity.The transcriptional activation domain at the C-terminal of the protein specifically recognized three cis-acting elements,including AAAAGG-motif,the secondary NAC binding element(SNBE),and secondary MYB responding element(SMRE).(3)With the method of transgenic transformation in Populus trichocarpa,a total of 20 Ptr OBP1 ovexpressed lines(p ROKII-Ptr OBP1)and 21 Ptr OBP1 suppressed lines(p ROKIIPtr OBP1-RNAi)were gained.The phenotype and secondary growth indexes of transgenic plants showed that on the one hand,overexpression of Ptr OBP1 improved the content of lignin,reduced the content of hemicellulose,and thickened SCW,and on the other hand,overexpression of Ptr OBP1 caused dwarf phenotype by inhibiting the elongation of fiber cells.(4)The transcriptome analysis between WT and the overexpressed lines showed 3 592 differentially expressed genes,including 373 transcriptional factors in 39 transcriptional factor families,such as MYB,WRKY,and ERF.Further analysis showed that 14 downstream target genes were possibly related to the formation of SCW,including genes concerning to SCW biosynthesis(Ptr PAL4,Ptr CAld5H2,Ptr GT8 F,and Ptr GT47C),genes concerning to gibberellin biosynthesis(Ptr GA2 ox A,Ptr GA20ox2 A,and Ptr GA20ox2B),and transcription factors(Ptr ERF110,Ptr LBD30,Ptr MYB108,Ptr SND1-A1,Ptr SND1-B2,Ptr MYB152,and Ptr MYB156).(5)Yeast one-hybrid essay and chromosome immuncoprecipitation experiment proved that Ptr OBP1 protein regulated the expression of target genes by specifically recognizing the three cis-acting elements and binding to the 14 predicted promoter sequences.The results of effect and reporter vector co-transmission experiment and estradiol induction experiment showed that Ptr OBP1 up-regulated the expression level of Ptr PAL4,Ptr CAld5H2,Ptr GA2 ox A,Ptr ERF110,Ptr MYB108,Ptr SND1-B2,and Ptr MYB152,down-regulated the expression level of Ptr GT8 F,Ptr GT47 C,Ptr GA20ox2 A,Ptr GA20ox2 B,Ptr LBD30,Ptr SND1-A1,and Ptr MYB156.In conclusion,the Molecular regulatory mechanism model of Ptr OBP1 is proposed in this study.The functions of Ptr OBP1 on the formation of SCW can be summarized as follows:firstly,Ptr OBP1 directly regulates the expression levels of the genes concerning to SCW biosynthesis,and then causes SCW thickening,reduced cell swelling,and dwarven plants;secondly,Ptr OBP1 affects the formation of SCW by regulating the expression of related transcription factors;thirdly,Ptr OBP1 affects the formation of SCW and the elongation of fiber cells by regulating the expression level of the genes concerning to gibberellin biosynthesis.This study identified an important transcription factor,which is involved in the formation of SCW in Populus trichocarpa,and analyzed its molecular mechanism.All the results are hoped to provide a reference for improving wood quality via genetic engineering.