Functional Study Of Populus Trichocarpa Aspartic Protease Genes AP17 And AP64 During Wood Formation

Wood is an important and green renewable resource with great social,economic and ecological value.Wood formation mainly goes through many complex biological processes such as secondary vascular cambium cell division,xylem cell expansion and elongation,secondary cell wall thickening and programmed cell death.At present,some plant hormones,kinases,transcription factors and wood component synthases have been identified to be involved in the formation of wood,but the complete molecular regulatory network has not been clarified,and whether there are new regulatory factors remains to be studied.Aspartic protease(AP)is an important proteolytic enzymes gene family,which is widely involved in plant growth and development,whether the involvement of APs in wood formation remains to be identified.In this study,we analyzed the genome-wide characteristics of APs gene family in the Populus trichocarpa,and several APs that related to wood formation were screened.By the genetics,wood anatomy,physiological and biochemical analyses,we elucidated the functions of high xylem abundance expression of typical AP17/45 and atypical AP64 during wood formation.The main results are as follows:(1)Identify APs associated with wood formation.67 APs were identified in the genome of Populus trichocarpa,based on gene structure and duplication,evolutionary relationship,protein subcellular localization and glycosylation site analyses,APs were divided into three types:typical,nucellin-like and atypical.12 APs with specific or high abundance expression in xylem of stem were screened by gene electronic expression data,RT-qPCR expression and analysis of cis-elements related to wood formation.In combination with the tissue expression characteristics of GUS reporter driven by the APs gene promoters,the typical AP 17/45 and atypical AP64 were suggested the key APs that regulating wood formation.AP17 and AP45 are localized in vacuole,while AP64 is localized in cytoplasm.(2)Typical AP17 and AP45 were found to be functional redundancy,AP17/45 are the positive regulators of secondary xylem fibres programmed cell death(PCD).Mutant plants of ap17,ap45 and ap17ap45 were created by the CRISPR/Cas9 gene editing technology.Knockouts of AP17 and AP45 did not affect plants growth,but delayed xylem fibres PCD.Further studies showed that AP17 and AP45 are functional redundancy and AP17 plays a major role.The delayed xylem PCD phenotype of ap17ap45 was supported by changes in transcription levels of genes related to xylem intracellular vesicular trafficking,plasma membrane oxidation and repair(APYRASE 2,Annexins and BAG).Overexpression of AP17 did not affects plants growth,while resulted in premature death of xylem fibres.(3)Genetic perturbation of AP17/45 mediated xylem fibres PCD that alter secondary cells wall synthesis and make wood cell walls more susceptible to glycosylation.Knockouts of AP17 and AP45 did not affect secondary walls structure,but increased cellulose content and decreased lignin content.Overexpression of AP17 results in thinning of secondary xylem fibre cells wall,and reduction of lignin content.Heterologous overexpression of AP17 and AP45 induced vascular PCD and reduced secondary cells wall synthesis in Arabidopsis thaliana,and the conserved aspartic acid sites(D106 and D293)are critical to the functions of AP17 and AP45.Genetic perturbation of AP17 and AP45 made the wood cells wall more susceptible to glycosylation,and the glycosylation efficiency of overexpression plants of AP17 was higher.(4)It was revealed that the functionally specialized atypical AP64 regulates secondary vascular cambium activity and wood tissue formation.Through the analysis of ap64 mutants and AP64 overexpression plants,we found that genetic perturbation of AP64 could inhibit plants growth,weaken the cambium division,reduce the expansion and elongation of xylem cells,and reduce the thickness of secondary xylem cells wall,but significantly increase the vessel density of secondary xylem.Heterologous transformation of A.thaliana revealed that the conserved aspartic acid sites(D167 and D368)are critical to the vivo activity of AP64.(5)5 interacting proteins of atypical AP64 were identified.Based on yeast two-hybrid and bimolecular fluorescence complementarity assay,screening five interaction proteins of AP64:carboxypeptidyase(CP),protein phosphatase(PP2A),pathogenesis-related protein like(PRPL),sucrose transporter(SWEET2a)and ATP-dependent chromosomal remodeling protein(SWIB).Further analysis of endogenous sugar content confirmed that genetic perturbation of AP64 affected sucrose transport and starch accumulation,the correlation between the action of AP64 and the function of its interacting protein(SWEET2a)was confirmedIn conclusion,we preliminarily analyzed the genome-wide characteristics of APs in P.trichocarpa,identified 12 APs with high xylem abundance expression,and speculated these genes might differentially regulate wood formation.Typical AP17 and AP45 are functional redundancy,and AP17/45 are the positive regulators of xylem fibres PCD;Overexpression of AP17 changes the wood cells wall composition,making it more susceptible to saccharification.While the atypical AP64 was functionally specially regulated secondary vascular cambium activity and wood tissue formation,genetic perturbation of AP64 alter wood structure.In this study,we identified the functions of key APs with high abundance expression in xylem,and preliminarily explained the differences of their biological functions and potential breeding value that involved in wood formation.This study not only enriched the molecular basis of wood formation,but also provided candidate genes for genetic improvement of forest trees.