Regulation Of Alkaloids Biosynthesis Through JA-Ile Synthase And JA-Ile Receptor CoI1 In Catharanthus Roseus

As a medicinal plant,Catharanthus roseus can produce more than 130 indole alkaloids,including the most widely used natural plant anti-tumor drugs,vinblastine and vincristine.However,these indole alkaloids are distributed in different tissues and organs in C.roseus,and chemical synthesis of these compounds is difficult due to their complex structures.Therefore,the production of such compounds is mainly based on plant extraction.As a plant hormone,jasmonicacid is a growth regulating substance widely present in plants.In plants,jasmonates can act as regulatory signals to regulate plant defense responses by accumulating secondary metabolites.Jasmonicacid and its derivatives can regulate the expression of multiple genes,thereby affecting the accumulation of secondary metabolites in plants.In plants,jasmonicacid is converted to jasmonicacid-isoleucine under the catalysis of jasmonicacid-isoleucine synthetase,JAR1,and then jasmonicacid-isoleucine binds to the receptor COI1 to activate the signaling pathway.Therefore,the receptor COI1 and JA-Ile synthase are key components in jasmonate signaling transduction pathway.Our previous works showed that overexpression of COI1 and JA-Ile synthase could activate JA signaling pathway and increase the accumulation of catharanthine and vindoline in C.roseus.In order to investigate the mechanism of CrCOI1 and CrJAR1 on regulating C.roseus alkaloid biosynthesis,virus induced gene silencing of CrCOI1 and CrJAR1 in leaves of C.roseus was performed for examining their effects on pathway gene expression and alkaloids accumulation.Bioinformatics analysis of CrC OI1 and CrJAR1 sequences predicted that proteins might be localized in the cytoplasm and phylogeny analysis showed that CrCOI1 is close to Ipomoea batatas and Capsicum annuum COI1,while CrJAR1 is close to Cucubita maxima and Carica papaya JAR1.Using virus induced gene silencing technology,CrCOI1 and CrJAR1 genes in the leaves of C.roseus were successfully silenced,and the contents of catharanthine and vindoline in the infected leaves of C.roseus were detected by high performance liquid chromatography.The results showed that compared with the control leaves,the accumulation of catharanthine was reduced by 3 times,and the accumulation of vindoline was reduced by 2 times.This indicates that silencing of CrCOI1 and CrJAR1 genes reduces the accumulation of catharanthine and vindoline in C.roseus.At the same time,real-time quantitative PCR was applied to analyze the expression of key pathway genes for catharanthine and vindoline biosynthesis.Results indicated that the expression levels of pathway genes were significantly lower than those in control leaves.When CrJAR1 and CrCOI1 genes were silenced,the expression of CrTDC was decreased about 10-fold and the rest of pathway genes were decarased about 20-fold,which indicated that silencing of CrJAR1 and CrCOI1 genes could down-regulate the expression of pathway genes for catharanthine and vindoline biosynthesis.Me JA could clearly induced the expression of pathway genes in 6 h in C.roseus leaves,but leaves silenced with CrJAR1 and CrCOI1 showed retarded response to Me JA treatment in comparison with control leaves.In summary,silencing of CrJAR1 and CrCOI1 could reduce the amounts of JA-Ile and CrCOI1,thereby weaken JA signaling pathway and lead to low expression of pathway genes for catharanthine and vindoline biosynthesis in C.roseus.