The Secondary Defense In Masson Pine Induced By Methyl Jasmonate

Pinus massoniana Lamb.is a major native afforestation tree with important economic and ecological value in southern China.However,pine wilt disease(PWD),pine caterpillars as well as other pests and diseases has seriously lead to the death of a large number of masson pine and result in great damage to society,the economy and the environment.Methyl jasmonate(Me JA)is considered to be a signaling substance that induces resistance in many plants.If the resistance of P.massoniana could be improved by methyl jasmonate,it might effectively prevent the invasion of pests and diseases.The resistance mechanism in P.massoniana induced by Me JA is not clear yet.In this study,two-year-old P.massoniana seedlings were treated with 10 m M Me JA solution,after that,the tramatic resin duct formation of P.massoniana was observed by paraffin section,the composition and content changes of induced terpenoids were detected by gas chromatography-Flame Ionization Detector/Mass Spectrometer(GC-FID/MS),the molecular mechanism of terpenoid production in P.massoniana was further studied by transcriptome sequencing.Furthermore,the effects of the secondary metabolites on insect-Monochamus alternatus was tested by comparing the differences of feeding area and enzyme activity between the beetles that fed with Me JA treated masson pine and the untreated ones.Methyl jasmonate treatment can induce secondaey defense that change the tissue anatomy of P.massoniana,including the expansion of polyphenolic parenchyma cell,the increasing size of cortical resin duct,and the formation of traumatic resin ducts.The development of the traumatic resin duct can be divided into the primitive cell stage,the interstitial formation stage,the lumen expansion stage and the resin duct maturation stage.The traumatic resin duct epithelial cells differentiated 6-9 days post methyl jasmonate treatment;the cell wall in the center of the original cell mass swelled,dissolved and formed a gap 9-21 days post treatment;the mature traumatic resin duct finally formed 24 days post treatment.The defense response of P.massoniana was influenced by the concentration of Me JA.In this study,the masson pine was treated with Me JA at a concentration of 0.1 m M,1 m M,and 10 m M respectively.The number of wound resin ducts gradually increased with the Me JA concentration increasing.Significantly more resin ducts(P <0.01)were formed post Me JA treatment compared to 0.1 m M and 1 m M Me JA treatment..1.10 monoterpenoids were detected by GC-MS: α-decene,terpene,(1S)-(-)-β-pinene,myrcene,hydrocelene,linalool,(-)-camphor,linalyl acetate,Terpinolen,L-acetate borneol;4sesquiterpenoid:(-)-α-copaene,longipiene,caryophyllene,α-caryophyllene;7 diterpenoids:1-Phenanthrenecarboxylic acid,L-Pimalamate,pimaric acid,methyl rosinate,methyl neodecanoate,methyl palustrate,neo-abietic acid.Within 30 days after treated with Me JA,the content of terpenoids in treatments were more than control,there were 2 evident rises during 0-3days and 15-21 days,the first one might due to the resin stored in the resin channel released after being stimulated by Me JA,the second rise probably result from the secretion of induced traumatic resin.3.This study used the Illumina HiSeq sequencing platform to sequence the P.massoniana after the spraying of methyl jasmonate in 9 days and 21 days,the sequence was assembled by Trinity under the default parameters.A total number of 592784492 raw reads were obtained.The transcripts were clustered and compared with Nr,Swiss-Prot,KEGG,COG and GO databases to obtain 374,905 unigenes,which the length of N50 is 950 bp,and length of E90N50 is 1991 bp.Among these unigenes,there are 35,407 sequences with a length greater than 1k,accounting for12.79%.Venn analysis showed that there were 107 differentially expressed genes related to secondary metabolism in the two groups of samples from 9 days and 21 days after treatment with methyl jasmonate,these 107 genes were subjected to GO and KEGG enrichment analysis,and the metabolic processes of the steroids,the isoprenoid biological processes,the isoprenoid metabolism process in the GO term were significantly enriched,and the skeletal skeleton synthesis,the flavonoid biosynthesis,phenylpropane biosynthesis and biguanide biosynthesis are also significantly enriched in KEGG.4.22 differentially expressed genes were identified by transcriptomic study in two pathways of terpenoid synthesis,including: enzymes that synthesis the co-precursor IPP and its isomer DMAP,such as hydroxymethylglutaryl coenzyme A reductase(HMGR),1-deoxy-D-xylulose、-5-phosphate synthase(DXS)and 1-deoxy-D-xylulose-5-phosphate reductoisomerase(DXR);enzymes that synthesis direct precursors GPP,FPP and GGPP,however,geranyl geranyl diphosphate synthase(GGPPS)was the only one that differently expressed;the enzymes directly synthesis,such as the monoterpenoid synthase,sesquiterpenoid synthase and diterpenoid synthase.Genes related to terpenoid synthesis treatment were up-regulated compared with the control after methyl jasmonate treatment.α-terpineol synthase,limonene synthase,(+)-α-pinene synthase,α-humulene synthase,levo-hippine diene synthase,(-)-β-pinene synthase and(-)-α/β-pinene synthase immediately up-regulated within 1-3 days post Me JA treatment,morever,the(-)-α-pinene synthase,(-)-α/β-pinene synthase,(-)-limonene synthase and 1(10),5-germacradien-4-ol synthase showed another up-regulation 9 days post treatment.5.M.alternatus fed on P.massoniana for 24 hours which were 1 day,3 days,6 days and 12 days later post 10 m M Me JA treatment.The feeding area of M.alternatus that exposed to P.massoniana which were 6 days post Me JA treatment were different from the beetles that fed on control masson pine.The activities of SOD,POD and Ach E of M.alternatus that fed on treated tree were increased compared to the beetles that fed on control masson pine while the activities of CAT and GSTs were decreased.No significant difference were detected between treatment and control.When M.alternatus continuously fed on masson pine which were 12 days post Me JA treatment for 12 h,24h,36 h and 48 h respectively,the activites of SOD,CAT and GSTs all declied with different levels while the activites of POD and Ach E increased.There was no significant difference between the treatment and the control group.