The Mechanism Of Endophytic Fungus Gilmaniella Sp.AL12 Promotion Of Plant Growth And Sesquiterpenoid Accumulation In Atractylodes Lancea

Medicinal plants are rich in active compounds such as paclitaxel,artemisinin,ginseng saponin,which are important sources of modern drugs for human.Atractylodes lancea is an endangered traditional Chinese medicinal herb.Its bioactive component,the sesquiterpenoids,possesses various pharmacology properties such as antibacterial,immunomodulation,and antitumour abilities.In the past few years,natural sources of the herb have been in short supply because of the excessive exploitation and slow growth rate.The medicinal source of A.lancea mainly derives from artificial cultivation,but the yield and quality of the artificial cultivated sources are relatively low.At present,it is urgent to improve the quality and quantity of the herb as the market demand for A.lancea is increasing on a daily basis.The fungal endophyte Gilmaniella sp.AL12 was isolated from the stem of A.lancea,and could establish a symbiotic association with the host plant,also promoted host plant growth and three kinds of sesquiterpenoids accumulation.Consistent with this phenomenon,AL12 promoted plant growth and three kinds of sesquiterpenoids accumulation in A.lancea within two years in the field experiments.In this study,firstly,we analyzed the effect of AL12 on host plant biomass and total content of seven sesquiterpenoids(?-caryophyllene,zingiberene,?-sesquiphellandrene,caryophyllene oxide,hinesol,?-eudesmol,and atractylone),and confirmed the“dual promotion”effect of AL12 on A.lancea.Therefore,the beneficial symbiotic association of AL12 with A.lancea will provide a good example for promoting plant growth and secondary metabolite biosynthesis in other medicinal plants.Then,we elaborated the mechanisms underlying AL12 promoting plant growth and sesquiterpenoids accumulation in A.lancea from multiple aspects.Firstly,we analyzed some plant physiological parameters,such as root viability,element contents,Rubisco content,and some phytohormones contents to uncover the mechanism of AL12 promotion of plant growth.Results showed that AL12 improved root viability and some certain elements(P,K,Mg,and etc.)accumulation in shoots of A.lancea.Also,AL12 improved plant chlorophyll content and Rubisco content,and enhanced net photosynthetic rate.And,cytokinin content increased after AL12inoculation.Thus,the above results showed that the growth promotion effect induced by AL12 on A.lancea may depend on the increased nutrient content,the improved photosynthesis and the increased cytokinin content.Secondary metabolite biosynthesis consumes substance and energy during plant growth.As indicated by real-time quantitative PCR,AL12 promoted sesquiterpenoids biosynthesis by activating the mevalonic acid(MVA)pathway.Then,we analyzed plant physiological parameters,such as contents of sesquiterpenoids biosynthesis precursors and tricarboxylic acid(TCA)cycle related organic acids to identify the mechanism of AL12 promotion of plant sesquiterpenoids accumulation.AL12 promoted the content of total carbohydrate,pyruvate,and acetyl-Co A,thus accelerating glycolysis and TCA cycle.And,AL12 steadily increased the amounts of several organic acids in TCA cycle,accelerating TCA cycle.These results showed that AL12 improved plant photosynthesis,glycolysis,and TCA cycle,thus providing sufficient carbon and energy for sesquiterpenoids biosynthesis.In brief,this part uncovered the primary mechanism of AL12 promotion of plant growth and sesquiterpenoids accumulation in A.lancea.Based on this primary mechanism,we employed transcriptomics and proteomics on AL12-inoculated and AL12-free plants to better understand the impact of AL12 on plant metabolism and related regulatory processes in shoots of A.lancea at the transcriptional and translational levels.Results showed that plant genes involved in plant-pathogen interactions and signaling were suppressed,similar to the plant response caused by some other fungal endophyte and biotroph pathogen.The downregulated plant immunity may contribute to plant-endophyte symbiosis.Additionally,genes and proteins related to primary metabolism(carbon fixation,carbohydrate metabolism and energy metabolism)tended to be upregulated after AL12 inoculation.And,AL12 upregulated genes involved in terpene skeleton biosynthesis,and upregulated genes annotated as?-farnesene synthase and?-caryophyllene synthase.Based on the above results,we proposed that endophyte-plant associations may improve production(biomass and sesquiterpenoids accumulation)by increasing the source(photosynthesis),expanding the sink(glycolysis and TCA cycle),and enhancing the metabolic flux(sesquiterpenoids biosynthesis pathway)in A.lancea.Thus,this part uncovered the“dual promotion”effect of AL12 on A.lancea at the transcriptional and translational levels.The accumulation of secondary metabolites is a common plant response for adapting biotic/abiotic environmental stresses.Second messenger is involved in regulating plant secondary metabolite accumulation.The above results showed that AL12 induced ethylene(ET)accumulation in A.lancea.As an important signal in plant,ET was involved in inducing plant secondary metabolites accumulation.In this part,the effect of ET on the promotion of sesquiterpene accumulation by AL12 was studied.Blocking ET biosynthesis suppressed AL12 induced sesquiterpenoids accumulation,indicating that ET was involved in AL12 induced sesquiterpenoids accumulation in A.lancea.Additionally,the upstream and downstream relationship between ET with jasmonic acid(JA),salicylic acid(SA),hydrogen peroxide(H₂O₂)and nitric oxide(NO)was described in the signaling cascade mediated AL12-induced sesquiterpenoids accumulation of A.lancea.Results showed that ET acted as an upstream signal of JA and SA in AL12-induced sesquiterpenoids accumulation of A.lancea.And,ET also acted as a downstream signal in NO or H₂O₂-mediated sesquiterpenoids accumulation induced by AL12 in A.lancea.In summary,ET acted as an important signal molecular mediating sesquiterpenoids accumulation of A.lancea induced by AL12.Plant signaling cascades were triggered after recognizing the fungal endophyte,thus inducing the expression of genes involved in secondary metabolites biosynthesis.We investigated the regulation of AL12 on the terpenoid skeleton synthesis pathway and sesquiterpene synthase pathway of A.lancea.Firstly,we described the functions of Al CPS1 and Al CPS2 induced by AL12 and identified them as?-caryophyllene synthase coding genes.AL12 induced Al CPS1 and Al CPS2 expression and?-caryophyllene accumulation.And,AL12 induced?-caryophyllene biosynthesis by activating the 2-C-methyl-D-erythritol 4-phosphate(MEP)pathway.Thus,we proposed that AL12 induced?-caryophyllene biosynthesis by activating the MEP pathway,and?-caryophyllene synthase pathway.On this basis,we explored whether ET,JA and SA were involved in AL12 induced?-caryophyllene biosynthesis.The results indicated that JA and SA mediated AL12 induced?-caryophyllene accumulation mainly by activating the MEP pathway and?-caryophyllene synthase pathway.Additionally,JA and SA activated many transcription factors coding genes,indicating the transcriptional regulatory role of JA and SA.In summary,this part describes the transcriptional regulatory role of JA and SA mediated AL12 induced?-caryophyllene biosynthesis in A.lancea.The expression of genes involved in secondary metabolite biosynthesis were regulated at transcriptional or post-transcriptional levels.Micro RNAs is an important signaling molecule involved in mediating biotic/abiotic stresses induced terpenoids biosynthesis at post-transcriptional levels.In this part,we identified AL12-responsive mi RNAs in A.lancea and analyzed the function of their targets.A total of 73upregulated and 95 downregulated mi RNAs were identified in A.lancea after AL12inoculation.Subsequently,we predicted 2 280 and 3 165 targets for the upregulated and downregulated mi RNAs,respectively.Gene ontology(GO)enrichment analysis was performed for these targets.For biological process,targets of downregulated mi RNAs enriched in intracellular signal transduction.By inspecting the genes involved in hormone biosynthesis,we found that genes involved in the biosynthesis of zeatin,brassinosteroids and JA were primarily targeted by the downregulated mi RNAs.Based on our previous studies,we proposed that mi RNAs may affect terpenoids biosynthesis by regulating signaling transduction.Further,by inspecting the genes involved in sesquiterpenoids biosynthesis,we found that novel?mir?45 and novel?mir?144 targeted genes coding for 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGR)and 1-deoxy-D-xylulose 5-phosphate synthase(DXS),respectively.Moreover,novel?mir?144 could target genes encoding RNA polymerase,replication factor,DNA repair protein,and many transcription factors,indicating that novel?mir?144 might well play important regulatory role at transcriptional level.Based on the above results,we proposed that AL12 might well upregulate genes related to signaling,transcription factors,and terpene skeleton biosynthesis by downregulating multiple mi RNAs,thus inducing sesquiterpenoids biosynthesis in A.lancea.In brief,this part speculated the first elaborate glimpse of AL12 promotion of plant sesquiterpenoids biosynthesis at micro RNAs expression level in A.lancea.In conclusion,this study used fungal endophyte AL12-A.lancea association as the research object to study the promotion mechanism of AL12 inoculation on plant growth and sesquiterpenoids accumulation from multiple aspects.This study provides a theoretical reference for using of the fungal endophyte to promote plant growth and secondary metabolites biosynthesis in other medicinal plants,and also helps to understand plant-fungal endophyte interactions.