The Roles Of Strigolactone In Resistance Of Chrysanthemum To Aphid

Chrysanthemum(Chrysanthemum morifolium),one of ten traditional Chinese famous flowers and one of four cut flowers over the world,of high ornamental and economic values.Aphids are the main pests of chrysanthemum,causes serious damage though the whole growth season is very serious.As a kind of sucking insects,aphids mainly feed on phloem sap,consume plant nutrients,transmit virus disease and induce pathogen disease,which seriously affects the yield and ornamental quality of chrysanthemum.Chemical application fails to control the aphid efficiently,and aphid infestation has become a bottleneck of chrysanthemum industry.Strigolactones(SLs)are a new class of carotenoid-derived phytohormones,which has established an essential role in developmental processes including branching,root architecture and root hair development,adventitious rooting,vascular secondary growth and mesocotyl elongation.Recently,a role of SL,crosstalk with other resistant related hormones in abiotic stress response like drought and salinity,and defense of pathogen have been reported.However,how SL regulates the resistance of aphid,remains unknown.Thus,we set up to illucidate if SL plays any roles in resistance of chrysanthemum to aphid?And how it regulates the aphid resistance?The main contents and conclusions on this research are as follows:1.CmCCD8 was isolated from chrysanthemum ’Jinba’ using degenerate primers and RACE-PCR.Phylogenetic analysis showed that CmCCD8 was most closely related to PhCCD8/DAD1 from Petunia.CmCCD8 transcripts were detectable in all the tissues.The expression level in the root is highest,followed by stem and leaves,and its expression was strongly induced by GR24 and aphid treatment.Chrysanthemum genetic transformation expression vector of overexpression pBIG-CmCCD8 were constructed,and were introduced into chrysanthemum cultivar ’Jinba’ via an Agrobacterium tumefaciens EHA105-mediated leaf disc transformation method.PCR analysis showed that the target fragment in the positive plant was successfully inserted into the genome.The content of endogenous SL analog in wild type and CmCCD8 overexpression lines showed that SL analogue 361 in transgenic chrysanthemum CmCCD8 overexpressing lines was significantly higher than that in wild type plants,while contents of analogue 363 and 371 are lower that wild type.Further indicating that CmCCD8 encoding enzymes catalyzing the biosynthesis of SL.2.Aphid inoculation assay showed that GR24 treatment and CmCCD8 overexpression enhanced the aphid density compared with control plants.To correlate the colonization and reproduction of the aphids with their feeding behavior,aphid feeding activity was detected by EPG The feeding activities of the tested aphids on the CmCCD8-OX plants differed from those on the WT plants.The time to the first cell puncturing,the total duration of the non-puncturing phase and the time to the first pathway phase in both CmCCD8-OX plants were shorter than those in the WT plants,and the total duration of the pathway phase in CmCCD8-OX plants was longer than in WT plants.The microstructure and anatomical structure of the leaf epidermis showed that the decrease in aphid resistance in the transgenic lines was related to the lower epidermal trichome density and thinner leaf thickness,palisade tissue thickness and lower epidermis thickness.The content of lignin,cellulose and hemicellulose in leaves of transgenic lines were decreased,which weakened the mechanical defense of aphids.Transcriptome analysis revealed that the expression of lignin biosynthesis genes Cm4CL2,CmCCR2,CmCAD6,cellulose biosynthesis genes CmCesA3,CmCesA8 and the transcription factors CmMYB19,CmMYB46 and CmNAC2 genes in lignin biosynthesis of transgenic lines were significantly lower than those of wild-type plants.Metabolites from the leaves of wild-type and overexpression lines were analyzed using metabonomics method.The results showed that the content of the primary metabolites such as acetol,palmitoleic acid,3,7,12-Trihydroxycoprostane,N(alpha),N(alpha)-dimethyl-L-histidine and 2,4-diaminobutyric acid and kyotorphin,in the leaves of two transgenic lines was significantly lower than that of the wild type.In summary,strigolactone regulated the resistance of chrysanthemum to aphid by weakening mechanical defense and reducing the toxic components in the primary metabolites.3.To determine if SL affect the aphid feeding choice,the olfactometer bioassay was performed.The results showed that aphids tended to choose 10 μM GR24 treated plants and transgenic lines rather than acetone-treated plants(control)and wild-type plants,suggesting that SL plays negative roles in the response of chrysanthemum to aphids,at least partially by impairing aphid repellence.Gas chromatography-mass spectrometry(GC-MS)analysis showed there were 14 kinds of volatile components was changed from the leaves of CmCCD8 overexpression transgenic lines compared with the wild type,and the content of(E)-β-Farnesene or β-caryophyllene as the repellence for aphid was significantly reduced in CmCCD8 overexpression transgenic plants.Exogenous application of(E)-β-Farnesene orβ-Caryophyllene to WT,OX-1 and OX-2 repelled the aphids,but a solvent control(acetone or ethyl acetate treatment)did not.Transcriptome analysis revealed that CmTPSl and CmTPS2 genes were significantly lower in OX-1 and OX-2 plants compared with wild-type plants.The suppression of the ABA biosynthesis gene CmNCED3a by GR24 and CmCCD8 overexpression was observed.The ABA contents in the CmCCD8-OX plants were much lower than in the WT plants.Application of ABA to WT,OX-1 and OX-2 increased repellence to aphids,in agreement with an induction of CmTPSl and CmTPS2 by ABA.The expression of SA biosynthesis gene CmICS increased upon GR24 treatment and CmCCD8 overexpression.Quantification of these two defense phytohormones showed that the content of SA was elevated in OX-1 and OX-2 plants.However,the application of SA increased the repellence to aphids.Additionally,SA suppressed the expression of CmTPSl and CmTPS2.The CmAOS and CmLOX2 genes,which are involved in JA biosynthesis,remained stable upon GR24 treatment and in CmCCD8-OX plants compared with WT.Quantification of this defense phytohormone showed that the content of JA in CmCCD8-OX plants remained at a level comparable to that of WT plants.Exogenous application of JA increased the repellence to aphids and increased the expression of CmTPS1 and CmTPS2.Silencing plants,TRV2e-CnCCD8-1,2,3,4 groups all showed increased repellence to aphids.The expression of CnFAS and CnCAS increased in CnCCD8-silenced plants,consistently,the content of(E)-β-Famesene or β-Caryophyllene increased in CnCCD8-silenced plants.These data again support the notion that SL negatively regulates the repellence to aphids,likely via regulating the biosynthesis of(E)-β-Farnesene or β-Caryophyllene.Taken together,decreased repellence in transgenic plants might be a consequence of higher content of SL,or crosstalk between SL,ABA and SA.