Metabolome And Transcriptome Analysis Of TIAs Biosynthesis In Catharanthus Roseus Induced By Exogenous Ethylene

In this paper, growth index, photosynthetic index and alkaloids content of Catharanthus roseus under ethephon treatment were measured. In addition, metabolome and transcriptome of control group and ethephon treatment group were also measured and analyzed, using HPLC-QTOF/MS and RNA-seq technique, to determine the effect of ethephon on growth and terpenoid indole alkaloids (TIAs) content and to investigate the regulating mechanism of TIAs biosynthesis in molecular level. The following progresses were made in this topic:Ethephon inhibited the plant height, root length and biomass of Catharanthus roseus, but enhanced stem diameter. These growing statuses were related to "triple responses" which were induced by exogenous ethylene. Ethephon suppressed photosynthesis of Catharanthus roseus, so we speculated that the reason of the growth inhibition was the suppression of efficiency of photosynthesis. Furthermore, the decrease of net photosynthetic rate was induced by non-stomatal factor. Moreover, ethephon regulated the accumulation of pigments content, including chlorophyll content and carotenoid content. The decrease of chlorophyll content and increase of carotenoid content which induced the decrease of net photosynthetic rate indirectly suggested that it was another reason for inhibition of plant growth. For four kinds of terpenoids indole alkaloids, ethephon promote the synthesis of strictosidine and vinblastine, but for the precursor of vinblastine including catharanthine and vindoline, their content change were different. Although the content of vindoline declined slightly, another precursor catharanthine and the end product vinblastine content were rising.Ethephone treatment suppressed primary metabolite UDP-L-rhamnose, meanwhile it promoted primary metabolites L-phenylalanine and 1-hydroxy-2-methy1-2-(E)-butenyl 4-diphosphate which closely related to secondary metabolic pathway. L-phenylalanine was the important product in phenylpropanoid metabolic pathway as well as the intermediate of shikimate pathway and 1-hydroxy-2-methy1-2-(E)-butenyl 4-diphosphate was involved in MEP pathway which also the upstream of shikimate pathway. The TIAs biosynthetic pathway was a downstream pathway, and the results revealed that many alkaloids in this pathway were up-regulated by ethephon treatment. Some metabolites contents that related to biosynthesis of TIAs were significantly up-regulated, such as strictosidine, serpentine, catharanthine and vinblastine. All results revealed that metabolic flux trended to secondary metabolism from primary metabolism.Differentially expressed genes mainly related to metabolic pathways and secondary metabolic pathways were up-regulated by ethephon treatment, so this is the reason that ethephon accelerated the synthesis of some intermediate and final products. The results of transcriptome information showed, in metabolic process, in total 30061 transcripts involved in 401 KEGG metabolic pathways and we screened 195 genes related to synthesis of secondary metabolism induced by ethephon. Though transcriptome splicing,195 specific sequences were divided into four major categories, including terpenoids indole alkaloids synthetic genes, transcription factors genes, ABC transporters and MATE transporters genes, which involved in the biosynthesis of secondary metabolism pathway, especially the genes related to monoterpene indole alkaloids synthetic pathway. The results of further analysis of these 195 genes showed that 32 genes were up-regulated and 35 were down-regulated. The functions of these 32 genes were metal ion binding and lyase activity and that of 35 genes was methyl esterase activity in molecule function. So we speculated that these results in molecule function were related to high expression of genes in the TIAs biosynthetic pathway, but it needed to be verified.By the results of principal component analysis of relationship between expression of catalytic enzyme genes in biosynthetic pathway of TIAs or genes of transcription factors and contents of TIAs, it can be seen that transcription level of CrWRKY1 gene was obviously related to that of ZCT1, ZCT2 ORCA3, therefore we deduced that WRKY genes feedback regulation was widely existed and its transcription activity was regulated by other signaling pathway factor. The expression of MAPK gene was significantly related with other transcription factors, and could regulate key enzyme genes related to upstream synthetic ways of terpenoids indole alkaloids with other regulatory factors such as promoters and inhibitors. Then these transcription factors regulated the synthesis of TIAs.Relationship analysis between expression of catalytic enzyme genes in biosynthetic pathway of TIAs or genes of transcription factors and contents of TIAs exhibited that expression of transcription factor genes ORCA3 and ZCT1, catalytic gene T16H and serpentine content had significant correlations. Expression of ORCA3 and ZCT1 had significant negative correlation, so it was deduced that competitive effect of transcript factors feedback regulated the expression of these genes. Contents of serpentine and vinblastine, expression of catalytic enzyme gene in downstream of TIAs synthetic pathway and transcript factor genes have significant correlation. It formed a positive feedback circle between transcript factors and biosynthetic pathway of TIAs. When exogenous ethylene activated biosynthetic pathway, alkaloids content were promoted.