The Sesquiterpene Metabolism And Response To Diverse Biotic Stresses In Tea Plant

Terpenoids,also known as isoprenes,is the largest class secondary metabolites,and more than 55,000 terpenoids have been found in various organisms.Many plant-derived terpenoids play important roles in plant-environment interactions.The lower molecular weights of monoterpene,sesquiterpene and a few diterpenes,triterpene have lower vapor pressure,so,they can be released in the form of gaseous from plants.These volatile terpenoids play key roles in plant population,plant-insects communications as essential chemical signals.The purpose of this study is to investigate the terpenoid volatiles in tea plant Camellia sinensis(L.)O.Kuntze,to explore the genes involved terpenoids synthesis,to indentify the function of terpene synthase,and to reveal the molecular mechanism of terpene metabolism response to various stress.In this study,the constitutive terpenoid volatiles were indentified from flowers of tea plants(C.sinensis var.assamica and C.sinensis var.sinensis);the induced terpenoid volatiles were analyzed under abiotic and biotic stresses;the transcriptomes of flowers form the two varieties of tea plants and herbivore damaged leaves were sequenced;the key genes involved in MVA and MEP metabolic pathway of terpene were isolated and their function was further investigated;ten terpene synthase were cloned from tea plant and their functions were anylzed by enzyme activity tests;the function of tea plant terpene synthase gene 1(CsTPS1)was verified using transgenic Arabidopsis thaliana;the responses to the chewing and sucking pests damage were compared,and JAs was proved to be the dominant signals of terpene metabolism induced by herbivores.The main results are as follows:1.Flower is the main organ that synthesizes and releases terpenoids in tea plant.The Pu-erh tea variety releases great amount of terpenoids including about 10 monoterpenes and 26 sesquiterpenes,and sesquiterpenes are the major terpene products.While tea variety germplasm releases much less terpene volatiles,only a few monoterpenes,especially linalool oxide,and basically no sesquiterpene products were detected.Whether the Pu-erh tea variety germplasm planted in Hangzhou or distributed in Yunnan,their flowers contain nearly the same terpene composition,which indicated that C.sinensis var.assamica and C.sinensis var.sinensis germplasms showed significant evolutionary difference at both phenotype and genetics level and it is not influenced by environmental factors.Biotic and abiotic stresses could both induce tea plant release terpene volatiles,but their inducing effects were significantly different.Herbivore damage could extremly induce two sesquiterpenes ?-farnesene and trans-nerolidol,and trans-nerolidol is its unique inducing terpene products.2.Transcriptomes of herbivore damaged leaves and tea plant flowers were sequenced,the genes involved terpenoid synthesis was explored via de novo splicing method.Ten terpene synthase were identified,and eight of them were sesquiterpene synthase genes,one had the characteristics of monoterpene synthase and one was diterpene synthase gene.Two genes CsHMGR1-2 encoding the rate-limiting step enzyme HMGR in MVA pathway and CsDXR gene encoding the rate-limiting step enzyme DXR in MEP pathway were isolated from tea plant.Other genes,including DXS in terpene MEP pathway,and COI1a-b,JAZ1-2 and CsMYC2a-b responsible for JAs signaling pathway were isolated.The acting elements involved JAs signal transduction might help us to further understand the regulation mechanism of induced terpene metabolism in tea plant.The qRT-PCR showed that CsHMGR1 can be significantly induced by geometrid(Ectropis obliqua)or green leafhopper(Empoasca onukii MATSUDA)feeding,which promotes the formation of terpenoids.CsDXR can dramatically increase carotenoids accumulation when co-expression with pAC-BETA plasmid in E.coli,which indicated CsDXR is a functional gene for synthesizing terpene precursor.All terpene synthase genes isolated from the transcriptomes of tea flowers and insect damaged leaves are homologous,some members expressed both in flowers and damaged leaves,and several genes can just be detected under insect induced conditions,while others expressed in flowers exclusively and they may be responsible for the monoterpenes and sesquiterpenes synthesis.3.Although all the terpene synthase genes obtained in this study possess typical plant terpene synthase characteristics,their prokaryotic expressed enzymes showed weak TPS activity.Based on the terpene volatiles from C.sinensis var.assamica and C.sinensis var.sinensis and TPS studies in other plants,terpene synthases have occurred functional divergence due to the mutations of active sites during the evolution of tea plant,which caused the degeneration of sesquiterpene synthesis of Cs TPSs in small-leaf varieties.The function of CsTPS1 was verified in transgenic Arabidopsis thaliana,gene over ecpressed lines release more longifolene up to 6-26 times compared with wild type plants under the treatment of Plutella xylostella,and the transgenic lines also exhibited stronger resistance to insect.A squalene synthase gene(CsSQS)was identified from tea plant and its function was verified by enzyme tests;the mutant1 sequence with truncated transmembrane domain can produce squalene by a condensation reaction with two FPP moleculars.The squalene volatile was screened in tea plant germplasm,and one special varity containing a large amount squalene was indentified.4.The inducing effect of chewing and sucking pests on volatile terpenes in tea plant was different,and chewing insect damage could induce more terpenes compared with sucking damage by leafhopper in tea plant.Tea geometrids could induce monoterpene 3-Carene and sesquiterpene nerolidol in tea plant,while leafhopper could not induce these two terpenes.Terpene inducing in tea plant by herbivores was also influenced by tea plant age,growth status,insect feeding ability,environmental factors and so on.Lovastatin,a HMGR inhibitor,can suppress two sesquiterpenes longicyclene and longifolene in tea plant seedlings when damged by geometrids.It indicated that inhibitor treatment can regulate tea plant terpene metabolism.The two terpenes were also significantly suppressed using JAs signal inhibitor ibuprofen in tea plant seedling under herbivore damage.It proved that JAs was the dominant signal regulating terpene metabolism.