Functional Analysis Of PtrDLT And PtrWOX4 Genes In The Development Of Vascular Cambium In Populus Trichocarpa

Wood remains the world's most abundant and renewable resource,and an alternative to fossil fuels.Wood production is dependent on the activity of the vascular cambium.Wood formation is a complex developmental process involving differentiation of secondary xylem cells from the vascular cambium,followed by thickening of the cell wall.The differentiation of the vascular cambium,particularly in woody species,is regulated at many levels by transacting molecules following well-structured regulatory hierarchies.Transcription factors play a pivotal role in this process.In this study,we focused on DWARF AND LOW-TILLERING(DLT)and WUSCHEL-RELATED HOMEOBOX4(WOX4)genes,which are specifically expressed in the vascular cambium of Populus trichocarpa.The discovery of this study may offer new clues to revealing the functional regulation of the vascular cambium in woody plants.Firstly,the coding regions of DLT and WOX4 were amplified from P.trichocarpa plants.DLT has two P.trichocarpa orthologs with high protein sequence identity,PtrDLT-A and PtrDLT-B(94.1%).WOX4 also has two P.trichocarpa orthologs with high protein sequence identity,PtrWOX4-A and PtrWOX4-B(97.2%).These results suggest that the two pairs of genes may have functional redundancy in P.trichocarpa,respectively.Knockout mutants of the genes were generated using the CRISPR-Cas9 system.The guide RNA sequence targeting these genes was designed and inserted into the pEgP237 vector.Genetic transformation of P.trichocarpa was carried out by using a rapid genetic transformation method infected by Agrobacterium.The mutants of both PtrDLT-A and PtrDLT-B and the mutants of both PtrWOX4-A and PtrWOX4-B were obtained.The results show that the mutants of PtrDLT genes are thinner,shorter,and have fewer stem nodes.The fiber cells are closely packed in the xylem.The ray cell has a wide cross section.The mutants of PtrWOX4 genes is extremely short,thin,and the leaves are very small.The top buds and roots were underdeveloped.A large number of branches grew out of the bottom.The vascular cambium is destroyed,the arrangement of the xylem is disordered,and the cell wall becomes thin.Then RNA-seq analysis was performed using the mutants and wildtype P.trichocarpa plants.Mutation of PtrDLT genes affect only a few genes related to vascular cambium function,but almost all cell wall synthetase-related genes are down-regulated in xylem,indicating that the deletion of PtrDLT gene has an effect on xylem development.The mutants of PtrWOX4 genes lead to changes in the transcription of a large number of specific genes in vascular cambium,and also affect a large number of vascular cambium functional genes.Therefore,PtrWOX4 genes are key genes that regulate the development of vascular cambium in P.trichocarpa.At the same time,it is found that the expression of some stem cell-related genes in the apical meristem are also changed.Therefore,PtrWOX4 genes also participate in the development of apical meristem stem cells.In conclusion,we have demonstrated that the PtrDLT genes in P.trichocarpa are involved in regulation of xylem differentiation but have a small effect on the development of vascular cambium;While PtrWOX4 genes are the key genes that regulate the development of vascular cambium,and are likely to be involved in apical meristem stem cell-related functions.These data can be useful for studying the mechanism of their functions in the development of the vascular systems.