The PtrVCS2 Transcription Factor Forms Regulatory Nexus With A Histone Acetylation System For Vascular Cambium Development In Populus Trichocarpa

Wood is an important raw material for construction,pulp and paper making,furniture making,and its formation originates from the vascular cambium’s division and differentiation activities.The vascular cambium cells undergo asymmetric periclinal division to produce xylem cells in the centripetal direction and phloem cells in the centrifugal direction.The activity of the vascular cambium is finely orchestrated by phytohormones,peptides,transcription factors(TFs)and other regulatory factors.Although a few transcription factors have been shown to regulate cambium activity in trees,the knowledge of the underlying molecular mechanism is still in the early stage,and the exploration of potential transcription factors that play key regulatory roles in cambium development is far from enough.In previous studies,our team has systematically identified 95 transcription factors(VCS TFs)specifically expressed in the vascular cambium of Populus trichocarpa.The PtrWOX4 a transcription factor with the highest transcriptional abundance has a key function in regulating the cambium development in Populus trichocarpa.In this study,we identified the regulatory function of PtrVCS2 in vascular cambium development,which is second to PtrWOX4 a in transcriptional abundance in the cambium,and carried out detailed studies on its molecular mechanism.We overexpressed PtrVCS2 and its homolog PtrVCS2-h(Potri.017G082700)in Populus trichocarp respectively,and created double mutants ptrvcs2/ptrvcs2-h that knocked out PtrVCS2 and PtrVCS2-h in Populus trichocarpa through CRISPR/Cas9 technology.Phenotype analysis,histochemistry and histological analysis showed that overexpression of PtrVCS2 and overexpression of PtrVCS2-h in P.trichocarpa resulted in similar phenotypes,that is an abbreviated cell-proliferation zone in the cambium and a thinner tree stems.While the ptrvcs2/ptrvcs2-h mutants exhibit contrary phenotypes,that is an expanded cell-proliferation zone in the cambium and a thickened tree stem.The results indicate that PtrVCS2 and PtrVCS2-h negatively regulate the cambium proliferation and radial growth of trees.Analyzing the RNA-seq and the PtrVCS2 Ch IP-seq data from the PtrVCS2 overexpression transgenic plants,we identified 905 regulated target genes of PtrVCS2 in the vascular cambium,which involving PtrWOX4 a.RT-q PCR analysis and m RNA in situ hybridization assays confirmed the negative regulation of PtrVCS2 on PtrWOX4 a transcription.Genetic analysis shows that in mutants that knockout PtrWOX4 a and its homologous gene PtrWOX4b(ptrwox4a/ptrwox4b),the number of cell layers in cambium proliferation zone was significantly reduced,which was similar to the changes in the cambium of PtrVCS2 overexpression.The results suggest that the downstream target gene PtrWOX4 a of PtrVCS2 plays the opposite function with PtrVCS2 in regulating cambium proliferation of Populus trichocarpa.Analysis of the conserved domain of PtrVCS2 protein shows that the interaction with other transcription factors is needed for PtrVCS2 associating to target genes.We performed yeast two-hybrid(Y2H)assays,in vivo bimolecular fluorescence complementation(Bi FC)assays and Pull-down assays,and found that PtrVCS2 interacted with PtrWOX13 a.The electrophoretic mobility shift assays(EMSA)and Ch IP-q PCR assays further suggest that the PtrWOX13a–PtrVCS2 dimers associate to PtrWOX4 a promoter via the direct binding of PtrWOX13 a to CAATCAC motif on PtrWOX4 a promoter,thus forming a PtrVCS2–PtrWOX13a–PtrWOX4 a regulatory system.Glucocorticoid-receptor-based inducible gene expression assays confirmed the direct activation of the PtrWOX4 a transcription by PtrWOX13 a.Analysis of the histone acetylation modification levels at the PtrWOX4 a promoter revealed that overexpression of PtrVCS2 resulted in a downregulation of levels of acetylated lysine residues 9,14 and 27 of histone H3 at the PtrWOX4 a promoter,while knockout of PtrVCS2 and PtrVCS2-h resulted in an upregulation,indicating that PtrVCS2 negatively regulates histone acetylation modification of PtrWOX4 a.By Y2 H,Bi FC and Pull-down assays,we found that PtrVCS2 and PtrWOX13 a form a tetramer with the histone acetyltransferase complex PtrGCN5-1–PtrADA2b-3,PtrWOX13a–PtrVCS2–PtrGCN5-1–PtrADA2b-3.The level of PtrWOX4 a transcription and levels of acetylated lysine residues 9,14 and 27 of histone H3 at the PtrWOX4 a promoter were significantly downregulated in cambium from PtrGCN5-1-RNAi transgenics and ptrgcn5-1/ptrgcn5-2 mutants,and the number of cell layers in cambium proliferation zone was less than that in control wild-type,suggesting that PtrGCN5-1 positively regulates the histone acetylation modification levels and transcription level of PtrWOX4 a,thus promotes the cambium proliferation.Protein interaction analysis and HAT activity test demonstrated that PtrVCS2 attenuates the interactions within the members of the tetramer,thus affecting the histone acetyltransferase PtrGCN5-1’s HAT activity for the target sites.In summary,the tetrameric protein complex PtrWOX13 a PtrVCS2–PtrGCN5-1–PtrADA2b-3 binds to the PtrWOX4 a promoter via PtrWOX13 a,which subsequently recruits the HAT complex PtrGCN5-1–PtrADA2b-3.The abundance of PtrVCS2 controls the stability and HAT activity of the tetramer,thus regulating the dynamics of histone acetylation at PtrWOX4 a promoter and the dynamics of PtrWOX4 a transcription,for regulating the normal development of the vascular cambium for wood formation in Populus trichocarpa.This study revealed the molecular mechanism of a tetramer–PtrWOX4 a pathway coordinating genetic and epigenetic modification machinery to maintain the regular vascular cambium development for wood formation,and provides a new epigenetic perspective for tree molecular breeding to improve wood yield and wood quality.