Accumulation Characteristics And Formation Mechanisms Of Podophyllotoxins In Juniperus Sabina L.

Juniperus sabina L.is an evergreen shrub of Juniperus of Cupressaceae,which is the main Tibetan and Uygur medicine.J.sabina is rich in essential oils,podophyllotoxins and lipids,of which podophyllotoxins are one of the main secondary metabolites of J.sabina.The plant has identified 59 species of podophyllotoxin and their analogues,and as many as22 species of podophyllotoxins and their derivatives have been isolated and identified from the leaves alone,making the diversity of podophyllotoxins richer than that of plants in the Podohylloideae.In addition to the common podophyllotoxin and deoxypodophyllotoxin,J.sabina also contains epipodophyllotoxin,which can be synthesized from etoposide in just one step of structural modification.J.sabina is an important "native tree" for sand management in the arid and semi-arid sand areas of Northwest China.It is evergreen and resistant to pruning,and is often used as a landscape tree,but its pruning residues are often disposed of as waste,resulting in a waste of resources.Therefore,J.sabina can be used as a new plant resource as a source of podophyllotoxins.At present,the distribution patterns,accumulation dynamics and molecular formation mechanisms of the podophyllotoxins in J.sabina at different ages and in different parts of the tree are not clear,and most studies on their activity have focused on insecticidal aspects,and their medicinal value has not been efficiently utilized.In this study,we evaluated the accumulation and distribution characteristics of the podophyllotoxins of J.sabina and their biological activities in terms of age,part and growth period,to elucidate the accumulation and distribution characteristics of the podophyllotoxins of J.sabina,to analyze the dynamic changes of metabolites of J.sabina leaves during different growth periods,and to clarify the efficient medicinal uses of J.sabina.The results of this study will provide a basis for the efficient utilization of the resources of J.sabina,the targeted cultivation of J.sabina with a high content of podophyllotoxins and the creation of new germplasm.The main research results are as follows:(1)The accumulation and distribution of J.sabina podophyllotoxins varied significantly by age and part.The accumulation and distribution of podophyllotoxins in J.sabina showed a pattern of fifteen annual > seven annual > biennial(tree age),leaves >branches > stems > roots(part),with the highest amount of podophyllotoxin in leaves reaching 7.58 ± 0.21 mg/g DW,more than 33 times the amount in roots;the accumulation of podophyllotoxins in fifteen annual J.sabina was about 7.5 mg/g DW higher than that in biennial J.sabina.The results of the cluster analysis showed that fifteen annual J.sabina leaves were clustered into a single group as a dominant source of podophyllotoxins due to their richness in podophyllotoxins.(2)May to September is a critical period for the transformation and accumulation of podophyllotoxin in fifteen annual J.sabina current year’s leaves.The accumulation pattern of total lignin and podophyllotoxin in the current year leaves of J.sabina showed a "W"-shaped change with seasonal alternating up and down,with the rapid accumulation of podophyllotoxin in J.sabina current year’s leaves from May to September.(3)The differential distribution of the active compounds in different ages and parts of J.sabina is the key to the differences in their biological activities.The fifteen annual J.sabina current year’s leaves show a good advantage in terms of accumulation of podophyllotoxin and anticholinesterase activity,with activity comparable to galanthamine,with podophyllotoxin playing a key role in the anticholinesterase activity.In addition,the biennial J.sabina branches and fifteen annual J.sabina roots extracts have good potential for application due to their significant anti-α-glucosidase and anti-drug-resistant bacterial activities.This study emphasizes the importance of investigating the content of podophyllotoxins and defining their highly effective medicinal parts and medicinal period before J.sabina is used as medicine.(4)The composition and accumulation characteristics of the secondary metabolites in the J.sabina current year’s leaves during different growth periods were revealed.The accumulation of secondary metabolites in J.sabina current year’s leaves may have been interconverted during the different growth and development processes,with the accumulation of flavonoids decreasing and shifting to the accumulation of phenols and lignans,which belong to the same phenol-propane metabolic pathway,for synthesis.8podophyllotoxins and their precursor compounds were identified.The accumulation of all 5podophyllotoxins increased with the growth of J.sabina current year’s leaves,and the accumulation of the precursors and intermediate compounds involved in their synthesis,coniferol and matairesinol,all showed varying degrees of decline.The precursors and intermediate compounds involved in the synthesis of podophyllotoxins may be enzymatically involved in the synthesis of downstream podophyllotoxins and their derivatives.Studies targeting the genes encoding these key enzymes could further reveal the mechanism of accumulation and formation of podophyllotoxins.(5)The mechanism of accumulation and formation of podophyllotoxins in J.sabina current year’s leaves at different growth stages was elucidated.The genes encoding 8 key enzymes,PAL,HCT,CCR,C4 H,DIR,PLR,OMT and CYP82,which are positively associated with the biosynthesis of podophyllotoxins in J.sabina,were identified.The function of Js PLR,a key downstream structural gene that catalyzes a two-step reduction reaction,may play a positive regulatory role in the podophyllotoxins biosynthesis of J.sabina current year’s leaves.Transcription factor predictions suggest that AP2/ERF-ERF,b HLH,MYB and MYB-related transcription factors may be involved in regulating the accumulation and synthesis of podophyllotoxin in J.sabina at different growth stages.(6)The cloning and functional validation of the Js PLR gene for the biosynthesis of podophyllotoxin in J.sabina was completed.The sequence of Js PLR gene was cloned for the first time from J.sabina,and the overexpression vector of Js PLR gene was constructed.The expression of Js PLR gene was successfully induced in J.sabina current year’s leaves by transient transformation technology,which could significantly increase the expression level of the gene and the accumulation of key compounds of the podophyllotoxin synthesis pathway.The results of the q RT-PCR validation analysis indicated that the differential expression of Js PLR gene in J.sabina current year’s leaves at different ages and growth stages may be a key factor contributing to the differences in the accumulation characteristics of its podophyllotoxin.