Study On Transcriptional Regulation Of Wood Formation In Populus Trichocarpa

Wood is one of the world’s most important raw materials for timber,pulp,and energy.Wood formation is a complex process that involves cell differentiation,cell expansion,secondary wall deposition,and programmed cell death.Vascular tissue-specific transcription factors(TFs)play an important role in the process of wood formation.Identifying these transcription factors and analyzing their functions in regulating wood formation will provide important clues for revealing the molecular mechanism of wood formation and creating new wood species with excellent material properties.Wood formation is regulated by a molecular network mediated by transcription factors.Research on the rapid and efficient construction of a transcriptional regulatory network for wood formation will help systematically reveal the molecular mechanism of wood formation.In this study,the woody model plant Populus trichocarpa was used as the research object to identify TFs specifically expressed in the vascular tissue of P.trichocarpa,analyze the gene functions of some TFs,and create a transcription regulation.The technical system of network construction has laid the experimental foundation for the establishment of the subsequent wood formation network.The main research results obtained in this study are as follows:(1)Optimized laser capture microdissection(LCM)technology suitable for the collection of forest vascular tissue,using this technology to accurately collect the vascular cambium,xylem and phloem of P.trichocarpa,and identify the vascular tissue-specific TFs.A total of 95 xylem-specific TFs,95 cambium-specific TFs and 139 phloem-specific TFs were identified.(2)The gene functions of some vascular cambium and xylem specific expression TFs were analyzed.Cambium-specific expression TFs of PtrB3 and PtrOBP1,and xylem-specific TF of PtrMYB161 were overexpressed in transgenic P.trichocarpa,the transgenic plants phenotypes were further analyzed.Studies have found that overexpression of PtrB3 and PtrOBP1 genes did not cause the division and differentiation of vascular cambium cells and the development process from cambium to xylem.Overexpression of the PtrMYB161 gene resulted in a decrease in the number of xylem fiber cells and a thinning of fiber secondary call wall,but an increase in the number of vessel cells and no significant change in the thickness of vessel secondary cell wall.At the same time,overexpression of the PtrMYB161 seriously affects the wood composition of the transgenic plants,making the content of glucose and xylose which were respectively the main component of cellulose and hemicellulose were both reduced by-50%.Compared with wild type,S(Syringyl)lignin was reduced by-57%and G(Guaiacyl)lignin was increased by～115%in OE-PtrMYB161 transgenic plants.(3)Transcriptomics analysis of xylem of PtrMYB161 overexpression transgenic plants showed that overexpression of PtrMYB161 gene significantly reduced the expression of secondary cell wall synthase genes in transgenic plants.Integrative analysis of wood cell-based chromatin-binding assays with OE-PtrMYB161 transcriptomics revealed a feedback regulation system in the PtrSND1-TRN,where PtrMYB161 represses all four top-layer regulators and one second-layer regulator,PtrMYB021,possibly affecting many downstream TFs in,and likely beyond,the TRN,to generate the observed phenotypic changes.(4)The expression of members of the silencing cofactor histone deacetylase PtrHDT3s subfamily was significantly up-regulated in the PtrMYB161 overexpression transgenic P.trichocarpa.The PtrMYB161 protein does not contain any known inhiBitory domains,and its inhibitory function may work by silencing cofactors.Using CRISPR/Cas9 technology to create a PtrMYB161 loss-of-function mutant,the mutant showed a phenotype similar to the wild type,indicating that the regulatory function of PtrMYB161 is redundant with other TFs in the wood formation network.(5)Established a meiosis-mediated Yeast-one hybrid(Y1H)system to construct TF-mediated wood formation TRN.The commercial Y1HGold yeast strain was modified by introducing two magic markers for mating type screening and mating type modification to produce Y1 HGold-MM yeast strain.Hybridize the YIHGold and Y1 HGold-MM yeast strains containing DNA and TF respectively to produce diploid yeast cells,and then screen the haploid cells containing TF-DNA by the method of meiosis for subsequent yeast one-hybrid screening.(6)Cloning the promoter of secondary cell wall synthetase gene in P.trichocarpa,and adopting meiosis-mediated Y1H and traditional haploid transformation,diploid hybrid Y1Hmethods to identify the interaction relationship Between the secondary cell wall synthetase gene of the TFs related to wood formation.The screening efficiency and working time of the three Y1H methods are compared,and the results show that the meiosis-mediated Y1H system has the highest TF-DNA screening efficiency and shorter working time.(7)The positive combination of TF-DNA identi fied by the three yeast one-hybrid methods was verified by using wood cell-based chromatin-binding assays of P.trichocarpa,the results indicate that the Y1H system mediated by meiosis has the highest incidence in vivo.In summary,this study optimized LCM system suitable for the collection of forest vascular tissues,and used this system combined with transcriptomics analysis to isolate and identify vascular cambium-specific,xylem-specific and phloem-specific TFs of P.trichocarpa.The specifically expressed TFs provide candidate genes for the subsequent in-depth study of specific wood formation regulatory mechanisms.Our research revealed the feedback regulation mechanism of wood formation mediated by PtrMYB161,and discovered the molecular network of TFs that finely regulate wood formation,which provides new clues for accurately creating new forest tree species with excellent traits through molecular breeding.At the same time,a new meiosis-mediated Y1H technology system has been established,which has broken through the long-standing problems of Y1H system that cannot balance the screening efficiency and working time,and laid an important experimental foundation for the construction of a wood formation regulatory network at the whole genome level.