Function Analysis Of PtrVCS2 Gene In Wood Formation In Populus Trichocarpa

Wood derives from the vascular cambium,which differentiates into xylem cells with the biosynthesis and deposition of three major cell-wall components,cellulose,hemicelluloses,and lignin.Transcription factors(TFs)play key regulatory roles in the process of wood formation.In previous research work,we dentified 87 TF genes,which are specifically expressed in the vascular cambium of Populus trichocarpa.These TFs may be involved in regulating wood formation.In this study,we focused on the VCS2(Vascular Cambium Specific 2)TF gene and analyzed its regulatory functions in wood formation.In this study,coding sequences of PtrVCS2 were fused with GFP protein to investigate the subcellular localization in the stem differentiating xylem(SDX)protoplasts of P.trichocarpa.The results showed that PtrVCS2 is located in the nucleus,cytoplasm,and cell membrane,indicating that it may have unique transcriptional activity.At the same time,the expression pattern of Ptr VCS2 was detected by in situ hybridization.The results showed that the PtrVCS2 gene mainly expresses in the cambium and was also partially expressed in the xylem and phloem.In this study,we then investigated the in vivo functions of PtrVCS2 in P.trichocarpa.We generated the overexpression vectors and CRISPR vectors of PtrVCS2.Rapid transformation was applied to the transgenics production,and the transgenic plants were tested at different levels and were confirmed by sequencing.We finally obtained over-expression plants and mutant plants of PtrVCS2 gene.Phenotypic analysis of the transgenic plants revealed that PtrVCS2 overexpression plants had shorter plant height and the mutants had higher plant heigh,comparing wild-type plants.Moreover,tissue sections were applied to the transgenic plants.The results showed that the cell walls of PtrVCS2 overexpression plants were thicker than wild-type plants and the xylem cells of the transgenic plants were lignified in advance,which indicates that overexpression of PtrVCS2 accelerates xylem differentiation.We examined lignin biosynthesis-related genes in the xylem of the transgenic plants,and found that Ptr VCS2 repressed their expression.Furthermore,yeast two-hybrid assay was carried out by using the TF genes that are specifically expressed in the xylem tissue as prey and using PtrVCS2 as bait.The results showed that PtrMYB221 and PtrWOX13 interacted with PtrVCS2.Bimolecular fluorescence complementation assays confirmed the presence of the interactions in vivo using P.trichocarpa SDX protoplasts.These results suggest that PtrVCS2 may function in transcriptional regulation as heterodimers.In summary,we have demonstrated that the PtrVCS2 gene is involved in regulation of wood formation in P.trichocarpa.These data can be useful for studying the specific mechanism of its functions in wood formation.