Integrated transcriptome and metabolome analysis reveals the podophyllotoxins accumulation and formation mechanisms in Juniperus sabina L.needles

Podophyllotoxin is an important secondary metabolite because of its remarkable anti-cancer and anti-Condyloma acuminatum activities.Juniperus sabina L.(J.sabina)has been proven as a new plant source of podophyllotoxins.However,the mechanism of podophyllotoxins accumulation and synthesis in J.sabina is unclear,which greatly limits their applications.Based on this,transcriptomic and metabolomic analyses were performed on different developmental stages of J.sabina needles to determine the molecular mechanism of the biosynthesis of podophyllotoxins in this study.Firstly,1022 compounds were identified from metabolomic analysis of J.sabina needles at different developmental stages,which contained 8 podophyllotoxins.The total lignans and podophyllotoxin contents were determined by HPLC,clarifying that May to September was the critical period for the accumulation of podophyllotoxins in J.sabina needles.Transcriptome and metabolic analysis showed that 47 DEGs encoded 16 key enzymes involved in podophyllotoxin biosynthesis,among which 8 key enzyme genes PAL,HCT,CCR,C4H,DIR,PLR,OMT and CYP82 were identified.Furthermore,JsPLR was proven as a key rate-limiting enzyme gene,with overexpression significantly increasing matairesinol and podophyllotoxin content.Subcellular localization results indicated that JsPLR is a cytoplasmic localization gene.The results of qRT-PCR study indicated that the differential expression of JsPLR gene might be a key factor leading to the differential accumulation of podophyllotoxin.Overall,the study laid a theoretical foundation for revealing the accumulation and formation mechanisms of podophyllotoxins and the comprehensive development and utilization of J.sabina resources.