Plants Adaptive Evolution And Utilization Of Essential Oil Resources On The Qinghai

The Qinghai-Tibet Plateau(QTP),known as the roof of the world,is one of the biodiversity hotspots in the world.It is very important to study the adaptive evolution of plants and the sustainable utilization of the biological resources in this region.Our recent work has showed that an Arabidopsis thaliana(Tibet-0)collected on the QTP is more ancient than other Arabidopsis ecotypes,and it is the highest-altitude Arabidopsis ecotype ever found from Tibet.As the most mature model of plant biology research,the discovery of the new ecotype of A.thaliana will provide a new model system and reference for the study of plant's phenotypic adaptability and molecular evolution mechanism on the QTP.In additon,as we know that essential oils as secondary metabolites of plants in response to biological and abiotic interactions,which have important ecological functions and broad-spectrum antibacterial properties.The medicinal excavation of plant essential oil resources that grown in the extreme habitats of the QTP has also been another research hotspot.Therefore,in order to improve our understanding of plant adaptive evolution and sustainable utilization of plant essential oil resources on the QTP,we performed the common garden experiment and the positive selection analysis of genomic functionally encoded genes using A.thaliana(Tibet-0).Furthermore,we also studied the molecular identification of Artemisia dracunculus var.qinghaiensis using it's ITS sequence,and the chemical composition and antimicrobial activity of it's essential oil.The specific contents and main results are as following:1.We selected a total of eight ecotypes of A.thaliana to conduct common gardenexperiments,which are from China's Tibet(Lhasa/Tibet-0),Xinjiang(Xqh),Shaanxi(Scg),Chongqing(Ctl),Jiangxi(Jnf),Nanjing(Jnj),United States(Col)and Cape Verde(Cvi).In the Lhasa common garden experiment site,thephenotypic traits of Tibet-0 had a significant differentiation with other sevenecotypes(P < 0.05).Tibet-0 exhibited the smallest rosette diameter(7.86 cm),the lowest plant height(16.42 cm),the highest number of basal rosettebranches(3.31),the lowest number of rosette leaves(9.40),the earliest bolting(45.40 days),the earliest flowering(57.00 days),the heaviest 1000-seedsweight(0.02407g),the highest seed yield per plant(0.0455g),and the lowestseed germination rate(5.00%).Based on these phenotypic differences,it isspeculated that Tibet-0 had a higher relative fitness than other seven ecotypesin its original habitat.And that early flowering and deep seed dormancymediated life cycle regulation strategies possibly promoted its adaptability tothe shorter growing season of the QTP.Principal component analysis andfactor analysis of phenotypic characteristics of A.thaliana also found thatrosette leaf number,bolting date,flowering date and the number of basalrosette branches had significant impact on the adaptability of A.thaliana onthe QTP(the cumulative interpretation rate was 73.05%).Meanwhile,we alsoconducted parallel common garden experiments in the laboratory.Two-factorvariance analysis showed that genetic factors significantly determined thenumber of basal rosette branches(F =49.909,P < 0.001),indicating that Tibet-0 has adaptively evolved a stable basal rosette branch genetic controlmechanism.In conclusion,Tibet-0 had evolved a series of the phenotypiccharacteristics of dwarfism,more basal rosette branches,early flowering anddeep seed dormancy,which promoted its adaptability on the QTP.2.We used the branch-site model of PAML to perform the positive selectionanalysis.We analyzed 5611 single-copy orthologous genes shared by Tibet-0and other 19 A.thaliana ecotypes and A.lyrata.A total of 65 genes werepossibly subject to positive selection in the Tibet-0(? > 1,P < 0.05).FurtherGO analysis in TAIR showed that these genes were mainly enriched in thestress response,developmental processes,metabolic processes and regulatingDNA or RNA metabolic and expression.These genes included some stressresponse genes,such as temperature-induced posttranscriptional genesilencing gene SGS3,the calcium signaling genes BAG6 and IQD20,and theimmune genes LOX5 and LYK5;some developmental genes,such as suppressshoot branching gene MAX4,seed dormancy gene DOG1,the root patterndevelopment related genes CASPL1A1,XEG113 and SIEL;and themitochondrial RNA editing genes MEF3 and PPR19,replication factor csubunit 3(RFC3),histone acetyltransferase complex gene HDP2 and tRNAsposttranscriptional thiolation gene CTU2 which mediate the regulation ofDNA or RNA metabolic and expression.The metabolic related genes includedglycoproteins processing genes HEXO2 and FUC1,and antioxidant damagegene GSTU8.Furthermore,the molecular mechanism analysis of 27 of all thepositive selection genes showed that they mainly improve the stress resistanceof A.thaliana.In summary,these positively selected mutations occurred instress response,developmental processes,metabolic processes and DNA orRNA expression regulation related genes which possibly improved theadaptability of Tibet-0 to the extreme high-altitude environments on the QTP.3.The phylogenetic tree(NJ)constructed using ITS sequences indicated A.dracunculus var.qinghaiensis was sister to other species of the A.dracunculuscomplex,which could provide some basis for the molecular identification ofA.dracunculus var.qinghaiensis.The essential oil extracted byhydrodistillation from the fresh aerial parts of A.dracunculus var.qinghaiensiswas 0.67%(v/w).We identified a total of 37 compounds from this essentialoil using Gas Chromatography/Mass Spectrometry(GC/MS),accounting for60.88% of the whole essential oil.This essential oil was mainly consisted ofmonoterpene hydrocarbons(42.12%).The most abundant components weresabinene(19.19%),?-terpinene(8.94%),terpinen-4-ol(3.83%).This essentialoil also contained various low concentrations of antimicrobial ingredients.Wealso used the agar diffusion method to evaluate the antimicrobial activity ofthe essential oil against 13 common pathogens.The essential oil exhibitedinhibitory activity against Staphylococcus aureus,Micrococcus luteus,Bacillus subtilis,Klebsiella pneumoniae,Pseudomonas aeruginosa,Salmonella paratyphi and Saccharomyces cerevisiae with minimum inhibitoryconcentration(MIC)values ranged from 1.25 to 5.00 ?l/ml.Meanwhile,theessential oil showed moderate inhibitory effects against these susceptiblemicrobial strains with an inhibition zone diameter in the range of 10.04-13.59mm produced by the concentration of 10.00 ?l/ml.In addition,no significantdifferences were observed in the antimicrobial activity between the uses of theessential oil in the 1.25–5.00 ?l/ml concentrations and the respectiveantibiotics(ampicillin,gentamicin,or amphotericin B for different microbialstrains)in the 15.00 ?g/ml concentration.Therefore,these studies couldprovide a preliminary basis for the antimicrobials production and resourcesutilization of A.dracunculus var.qinghaiensis.In summary,this study takes the model plants A.thaliana(Tibet-0)and A.dracunculus var.qinghaiensis endemic to the QTP as the research material.The results preliminarily revealed the phenotypic adaptation of Tibet-0 with dwarfism,more branches and short growth cycle and its anti-adversity adaptive evolution at molecular level.In the meantime,assisted molecular identification of A.dracunculus var.qinghaiensis with ITS molecular sequence was carried out.The studies of chemical composition and antimicrobial activity found that its essential oil had a high proportion of sabinene and moderate antimicrobial activity against seven common pathogens,and could be used as a potential antimicrobial medicinal resource.These results provide new insights and patterns for studying plant adaptive evolution and utilization of plant essential oil resources on the QTP.