The Molecular Ecological Mechanism Of Pyrethrum-aphid-ladybird Mediated By (E)-beta-farnesene

Pyrethrum(Tanacetum cinerariifolium),a perennial ornamental plant belonging to the composiate family,is famous for the natural insecticide pyrethrins accumulated in the flower head.Previous research mostly focused on the extraction of natural pyrethrins and functional characterization of related enzyme involved in the pyrethrin biosynthesis.For earlier insect survey in the pyrethrum field,we found high density of ladybird beetles were attracted to the early flowering plants without its aphid preys.Subsequently we found a dominant(E)-beta-farnesene(EβF)emitted from the young flower bud.EβF is a sesquiterpene volatile of aphid alarm pheromone released from aphid cornicles upon attack,which is used to alarm other nearby conspecies to avoid the potential danger.EβF is also a common kairomone used by aphid enemy,such as ladybird beetles,to locate their potential preys.Here we suspect that EβF is the key factor responsible for beetle attraction.We performed the headspace volatile collection of intact pyrethrum plant and hexane extraction of different organs and do GC-MS analysis to identify the emission and storage pattern of EβF.Then we did cloning,expression analysis and functional characterization of Eb FS gene and its promoter.We also did beetles behavior assay,aphid probing hehavior(EPG)and aphid honeydew analysis to demonstrate the relationship between pyrethrum plants and insects.Here we try to reveal the molecular ecological mechanism of pyrethrum defense system based on the release of aphid alarm pheromone.Main results are as below:1.Cloning,functional characterization and expression analysis of the EbFS genes and its promoter in pyrethrumTwo allele genes of E-beta-farnesene synthase(Eb FS)were cloned from pyrethrum,which were named as Eb FS1 and Eb FS2 individually.These two genes encode 575 Aa and 577 Aa,respectively.For bacterial expression of Eb FS1 and Eb FS2,nearly 100 KD recombinant proteins were purified and done the catalysis analysis with the substrate FPP.Both Eb FS1 and Eb FS2 produce a major sesquiterpene EβF by catalyzing the FPP.Subcellular localization analysis of Eb FS1 and Eb FS2 showed that these two proteins have cytoplasm localization.Expression analysis of the Eb FS genes showed it has a peak expression in the S1 flower bud,moreover,the expression was specifically limited in the flower stem and receptacle of the S3 flower.Meanwhile,the 2.2 Kb promoter sequence of Eb FS gene was cloned in pyrethrum,and fused with the GUS reporter gene and did transformation in chrysanthemum.GUS staining analysis showed that the promoter was specifically expressed in cortex cells around the vascular bundles.These results suggested that EβF was biosynthesized by a sesquiterpene synthase and its production was limited in the cortex cells.2.EβF emission was regulated by the plant development and induced by mechanical damageHeadspace volatile analysis of pyrethrum plants at different developmental stages showed that EβF was the main volatile only at S0-2 stage,which was masked by another sesquiterpene germacrene D in the later flowering stages.Mechanical damage could dramatically increase the emission of EβF,from 130ng/h/plant to 5000 ng/h/plant.Moreover,EβF emission was only induced from the wounded site and it lasted for only a short period.Aphid infestation did not induce the emission of EβF.These results suggested that EβF emission was regulated by the plant development and wounding could induce its release.3.EβF was largely stored in the cortex cells around the vascular bundlesNADI staining of fresh sections of flower heads and stems showed that terpenes were abundant in the cortex tissues closing to the sieve elements of the phloem.Secondary metabolites analysis of different pyrethrum plant tissues at different developmental stages showed that EβF was the most abundant terpene stored in the flower stem and receptacle.4.Cortex-stored EβF was released from the honeydew of the aphids when they fed on the pyrethrumGC-MS analysis of the aphid honeydew showed that EβF was the only terpene when aphids fed on the pyrethrum,however,no EβF was detected in the honeydew when aphids fed on control benthamiana plant.These results revealed that aphid released the plant-derived EβF stored in the cortex cells during the intracellular penetration phase.In order to elucidate the effect of EβF-containing honeydew on aphid behavior,we performed the aphid alarm behavior experiment with artificial honeydew containing 10 ng/μl EβF.It showed that EβF-containing honeydew could repel aphids before kicking it away.5.Reveal the ecological relationship among pyrethrum,aphid and ladybird beetlesAphids could produce some habituated offsprings on different host plants.Aphid adapted/unadapted to pyrethrum flowers were inoculated with pyrethrum heads for behavior observation and recording.It showed that unadapted aphids were more active on the pyrethrum heads and showed obvious uneasiness compared with aphids pre-adapted to pyrethrum.Furthermore,the EPG electropuncture system was used to monitor the aphid feeding behavior on different organs of pyrethrum(flower stem,leaf and petiole).Complete feeding wave pattern was found when aphids fed on these plant organs,which indicated that aphids could survive on pyrethrum.To further reveal the effect of pyrethrum flower volatiles on aphid behavior,we performed an aphid alarm behavior experiment.Aphids adapted/unadapted to pyrethrum flowers and aphids adapted/unadapted to EβF were used.The results showed that aphids became adapted to pyrethrum were due to prior exposure to pyrethrum and habituated to EβF in the pyrethrum volatiles blend.Behavior choice assays of ladybird beetles showed that pyrethrum plants at early flowering stages(S0-2)significantly attractted beetles in lab controlled conditions and wounding could promote this attraction.In conclusion,we revealed that specific attraction of beetles for S2 flower was closely correlated with the dominant EβF emission from early stage flower head.EβF was the key volatile factor responsible for the ladybird beetle attraction and aphid repellence.EβF was specifically biosynthesized and accumulated in the cortex cells of the flower stem and receptacle,which are the most vulnerable tissues for aphid infestation.Cortex-stored EβF could be released from the aphid honeydew when aphids fed on the pyrethrum flower stem,which repeled aphids by mimicking aphids to release aphid alarm pheromone.It probably helps beetles locate the preys more effectively in the field.This research firstly reveal the double defense level of EβF emission occurring in pyrethrum by releasing EβF both from the flowering plant and aphid honeydew,which help us understand the evolution of plant,herbivores and carnivores and also guide us to apply this plant-derived pheromone in the aphid defense.