The Research On Circadian Rhythmic And Light-induced Emission Of Methyl Jasmonate In Cymbidium Ensifolium

Cymbidium ensifolium is one kind of terrestrial orchid,cherished by people for its elegant flower shape and rich aroma.Previous studies have shown that sepals and lateral petals are the main parts of aroma emission in Cymbidium flowers.The main aroma components in C.ensifolium is methyl jasmonate(MeJA)that followed circadian rhythm under natural conditions by releasing high amount at day time and lowest at night.To understand the molecular mechanism of the circadian emission of MeJA in C.ensifolium,a transcriptome of flowers was constructed and a large amount of transcript information was obtained and established the basis for follow-up study.The genes involved in MeJA synthesis and circadian clock pathways were cloned.Bioinformatics,subcellular localization and characteristics of genes expression,were also studied.The interaction of CeCOP1,CeMYC2 and their relationship with the synthesis of jasmonic acid was also discussed.The main objectives of our study are enlisted below:1.To verify emission of floral substance from Cymbidium flowers followed a circadian rhythm or not under artificial photoperiod,the flowers were subjected towards different light treatment and quantification of fragrance was carried out for two days using GC-MS.Under natural photoperiod(12 h light/12 h dark),MeJA emission from Cymbidium flowers follows a circadian rhythm peaking at day time and lowest during night.However,the emission of MeJA was ceased when the plants were transferred towards continuous dark.Interestingly,the emission of MeJA was detected in all measuring times when the plants were transferred from normal photoperiod to continuous light and remain release in circadian rhythm but the amplitude and phase of rhythm was changed.The blue light was used to illuminate the Cymbidium flowering plants after treated by 48 h dark,and the results shows that the emission of MeJA of Cymbidium flowers was significantly induced by blue light.The releasing rate increased first and then decreased,and the maximum amount of MeJA was detected after 6 h treatment with blue light.2.The Cymbidium transcriptome data was analysed classified and summarized by RNA-Seq technology and bioinformatics methods.According to the characteristic of flower fragrance released from C.ensifolium,genes function in the biosynthetic pathway of MeJA and those involved in circadian clock were deeply analyzed.In total,there were 18 genes mining from MeJA synthesis pathway and 66 genes from plant circadian clock pathway.3.CeLOX,CeAOC and CeJMT are key genes in the biosynthetic pathway of MeJA.Using Hi-TAIL PCR and genome walking method,we isolated the promotor sequences of CeLOX,CeAOC and CeJMT.The results of promoter prediction showed that the three sequences had the characteristics of promoter,and possessed many cis elements include light response,hormone induction and stress induction.4.Eight key genes of circadian clock including CeCCA1,CeLHY,CePRRs(CePRR1/CeTOC1,CePRR73,CePRR59,CePRR95),CeGI and CeELF3 were cloned using RT-PCR method.The results from bioinformatics analysis showed that these genes had high homology with known circadian clock genes of other plants in the database,and were clustered together with the monocotyledon plants in the phylogenetic tree.5.Five representative circadian clock genes including CeLHY,CeTOC1,CePRR73,CeGI and CeELF3 were selected for further anlaysis.Subcellular localization results showed that the proteins encoded by all above mentioned genes were located in the nucleus.In terms of gene expression characteristics,5 genes were all showed circadian rhythm in fragrance released parts of Cymbidium flowers under normal light/dark cycles.Among them,the peak gene expression of CeLHY,CePRR73 and CeGI was in the light period of day,and such expression pattern was similar to the gene expression of floral function genes such as CeLOX,CeAOS,CeAOC and CeJMT,which is involved in the synthesis of methyl jasmonate.Whereas,the peak expression of CeTOC1 and CeELF3 was in the dark period,which was contrary to that of floral function genes.The expression of CeGI was induced by blue light,which was consistent with the expression patterns of floral function genes and they had the characteristics of collaborative expression.In addition to CeGI,the expression of the other 4 circadian clock genes was higher in the sepals and petals,and was low in non-flower organs.6.Transcription factors of CeCOP1 and CeMYC2 were cloned from Cymbidium flowers.Subcellular localization results showed that both CeMYC2 and CeCOP1 are localized in the nucleus.In vitro experiment results showed that they can interact with each other in yeast and onion epidermal cells.Furthermore,CeMYC2 can also be bound to the G-box region of CeAOC promotor in vitro.The expression of CeCOP1 and CeMYC2 was induced by blue light and the expression level increased at first and then decreased.The CeCOP1 expression was higher,whereas the expression of CeMYC2 was low as compared to the control group in all stages.Furthermore,CeCOP1 expression was highest in calyx and CeMYC2 expression in pseudobulb.In short,these results gave us the basis for the further study of the roles of TFs in the molecular mechanism fragrance emission in Cymbidium flowers.