The Molecular Basis Of Terpene-mediated Cotton Induced Defense Against Apolygus Lucorum

The green plant bug(GPB;Apolygus lucorum)has become a severe pest of cotton in China.Cotton plants attacked by insect herbivory including A.lucorum emit large amounts of characteristic volatile terpenoids,which can directly repel and inhibit growth of attacking insects(i.e.direct defense)and are also implicated in plants ‘crying for help' to natural enemies of the attacking insects(i.e.indirect defense).However,little is known about volatile biosynthesis for defense response that GPB trigger in cotton plants and how defense mechanisms are orchestrated in the context of other biological processes.Here we monitored the volatile characteristics and transcriptome changes of cotton plants in response to GPB adults infestation,and characterize terpene synthase(TPS)genes involved in the induced defense of cotton against the GPB with integrated functional genomics analyses.The results are as follows:Using headspace collection coupled with GC-MS analysis,quality and relative quantity of cotton volatiles induced by the green plant bug(GPB;Apolygus lucorum)for the time series of 12,24 and 36 h were measured.The volatile profile of GPB-damaged rice plants was dramatically different from that of control plants.Control cotton plants emitted only trace amounts of volatiles,which include a-pinene,?-caryophyllene and a-humulene.These constitutive terpenoids stored in glands located in the cotton leaves elevated level of emission at the start of GPB feeding.Other volatiles were detected only from GPB-damaged cotton plants.And 15 volatile compounds were detected at 12 h after GPB infestation,18 at 24 h and 26 at 36 h.(E)-?-ocimene was the most abundant compound in GPB-induced volatiles.GPB-induced cotton can be categorized into three major groups: terpenes,green leaf volatiles and shikimic acid-derived metabolites(indole and MeSA).High levels of constitutive terpenoids and green leaf volatiles including(Z)-3-hexenyl acetate,(Z)-2-hexenyl butyrate,(Z)-3-hexenyl 2-methylbutanoate,(Z)-1-ethoxy-4-methyl-2-pentene and were emitted at 12 h after GPB infestation and then slowly declined thereafter.As damage proceeded,other acyclic terpenes including(E)-?-ocimene,linalool,DMNT and ?-farnesene,as well as indole,methyl benzoate,methyl anthranilate and MeSA,started to increase and peaked at 36 h,indicating that these volatiles are biosynthesized de novo following insect damage.Furthermore,we analyzed the difference between feeding by the phloem feeder GPB and Helicoverpa armigera with regard to the induction of volatile compounds from cotton plants.When challenged by the two insect herbivores simultaneously,relative quantity of some volatile compounds,including(E)-2-hexenol,hexyl acetate,(Z)-2-hexenyl acetate,1-decyne,(E)-2-hexenyl propionate,(Z)-3-hexenyl 2-methylbutanoate,(Z)-1-ethoxy-4-methyl-2-pentene,(E)-2-hexenyl tiglate and MeJA was significantly more than for plants infested with only GPB or only H.armigera.The three most abundant compounds were(Z)-3-hexenyl acetate,(E)-?-ocimene and indole in H.armigera-induced volatiles,while(E)-?-ocimene,DMNT and linalool were the three most abundant compounds in GPB-induced volatiles.Interestingly,MeJA was a specific GPBinduced volatile compound,which indicating SA pathway plays a pivotal role in GPB-induced defenses.To identify target genes and key pathways involved in volatile biosynthesis for herbivory-induced defense in cotton.we performed dynamic transcriptome analysis of cotton plants fed upon by A.lucorum adults using RNA-seq.Samples for the time series of 12 h,24 h and 36 h of GPB feeding were analyzed to explore the biosynthesis and regulatory mechanisms underlying such defense.Transcriptional levels of 57,345 genes in cotton young leaves were altered at 12 h after GPB feeding,57,015 at 24 h and 64,324 at 36 h.Genes associated with a cut-off of Probability >0.5 and the value of log2|Ratio| >2 considered to show significantly different expression.3,626(6%)transcripts were differentially expressed at 12 h,in which the numbers of up-regulated and down-regulated genes were 2,124 and 2,421 respectively.2,083(3%;1517 up-and 507 down-regulated)at 24 h,while 23,780(36%;23,616 up-and 164 downregulated)at 36 h after GPB feeding.Among these,121 gene were differentially expressed at three time points.Gene functions were classified using Gene Ontology(GO)analysis.In total,43 GO terms were attributed to one of the three GO ontologies.In the cellular components GO ontology,the top two terms were cell and cell part.Meanwhile,most of the genes were classified into the function ontology of binding and catalytic activity.The GO analysis also showed that both the cellular process and metabolic process GO categories were most abundant in the biological process ontology.Numerous studies have demonstrated that Phytohormones act as central players in plant defenses in the context of growth and development,which are well supported by the fact that lots of genes involved in phytohormone biosynthesis and signaling pathways such as jasmonic acid(JA),salicylic acid(SA),Ethylene(ET)were differentially expressed in response to GPB infestation.These findings indicated that JA,SA and ET are the main plant hormones playing pivotal roles in induced defense resistance against GPB.Moreover,224 genes annotated within ‘disease resistance family protein',‘protein involved in defense response' and ‘stress-related protein' were differentially expressed in response to GPB feeding,which are reported to be involved in plant defense in response to various pathogens and insects.We further analyzed the expression patterns of genes of secondary metabolite pathways that are known to be affected by herbivory,specifically phenylpropanoid,flavonoid and terpenoid metabolism.Many genes from these pathways were differentially expressed after GPB feeding.To elucidate the molecular basis for the biosynthesis of volatiles involved in induced defenses of cotton to GPB,we mainly addressed terpenoid metabolism.Nine genes encoding the crucial enzyme of the non-mevalonate pathway,1-deoxyD-xylulose 5-phosphate synthase were up-regulated by GPB feeding.Meanwhile,two hydroxymethylglutaryl-CoA reductases of the mevalonate pathway were up-regulated.In addition,31 terpene synthases were represented,in which five cadinene synthases,a sesquiterpene synthases and the catalyst for gossypol biosynthesis,were up-regulated.Overall,the present results indicated that the massive reprogramming of primary and secondary metabolic processes and rapid changes in signaling and other regulatory processes in cotton plants in response to GPB feeding.Volatile terpenes are the most abundant in GPB-induced volatiles in rice,which are synthesized by the action of TPS.The integrated information from volatile profiling,RNA-seq data and genome mining helped us identify 46(Cot1_TPS1-46)and 48(Cot2_TPS1-48)putative functional TPS genes in upland cotton Genome 1 and upland cotton Genome 2,respectively.In cotton,the terpene synthase genes were classed into class I,or III types based on grouping by physical similarities of gene architectures.Similar to those found in other plants such as Arabidopsis and Eucalyptus,the majority of cotton TPS genes are class-III type TPS genes,which consist of seven exons that are conserved in sequence and interrupted by six introns.Among class-III type TPS genes,32 out of 41 Cot1_TPS and 25 out of 41 Cot2_TPS consist of seven exons that are conserved in sequence and interrupted by six introns.The phase of introns(position in the coding frame)is also rigorously conserved.The order of intron phases(listed from the 5' end to the 3' end of the coding region)is: 0 for the first intron;1 for the second;2 for the third;2 for the fourth;0 for the fifth;and 0 for the sixth intron.In contrast,Genes from class I contain between 10 and 15 introns,which is highly variable.Phylogentic analysis showed that cotton TPS gene family was divided into two classes and six subfamilies: Class I consists of TPS c(copalyl diphospate),TPS e(ent-kaurene)and TPS f(other diterpenes)and class III of TPS a(sesquiterpenes),TPS b(cyclic monoterpenes)and TPS g(acyclic monoterpenes such as linalool).The finding is similar to those reported for other dicotyledonous and monocotyledonous plants.The most genes were classified into TPS a subfamily.It was difficult to attempt to clone all 33 TPS in cotton due to several reasons including high levels of plant secondary metabolites present in leaves.Only 17 TPS genes were successfully cloned,in which one was characterized previously,two were pseudogenes and two were not involved in volatile biosynthesis.Thus,the remaining 12 TPS were heterologously expressed in E.coli were assayed with the potential substrates NPP,GPP,FPP and GGPP to investigate the catalytic activity.Finally,8 out of 12 were able to produce significant amounts of terpenes,which were detected in GPB-induced volatiles,except in the case of GhTPS9,which converted FPP to germacreneD and germacrene D-4-ol.Transcription level of these 8 TPS gene increased after A.lucorum feeding,which consistent with cotton VOCs changes in GPB-infested plants,which indicating that the formation of herbivore-induced volatile terpenes in cotton is mainly regulated by transcript accumulation of multiple TPS genes.