Mechanism of molecules crosstalk and the critical role of jasmonic acid on triterpenoid synthesis in Cyclocarya Paliurus cells under Aspergillus niger elicitor

Cyclocarya paliurus ( C. paliurus ) leaves contain multiple health beneficial metabolites, and therefore are often used in functional foods and Chinese herbal medicines. However, breeding difficulties confines its utilization. Consequently, cell suspension cultures were developed to produce the bioactive secondary metabolites of C. paliurus leaves, but the content was comparatively low. In the present paper, Aspergillus niger elicitor (ANE) was used to stimulate the synthesis of triterpenoids in the suspension cultured C. paliurus cells, and the signal molecules crosstalk involved in this elicitation was further studied to interpret the underlying mechanism. Total triterpenoids accumulation in the cultured C. paliurus cells elicited by 200 µg/mL ANE was 9.17 times higher than that of the untreated cells. Nitric oxide (NO), hydrogen peroxide (H 2 O 2 ) and jasmonic acid (JA) played important roles in the elicitation as signal molecules. Under the ANE stimulation, the concentrations of NO, H 2 O 2 and JA all increased significantly, but presented different profiles and peaked at different time points. Based on series experiments on NO quenching by C-PTIO, H 2 O 2 blocking by DMTU, and JA synthesis inhibition by IBU and NDGA, together with exogenous NO, H 2 O 2 and JA addition experiments, it was concluded that ANE stimulated triterpenoid biosynthesis in the suspension culture of C. paliurus via a complex signal transduction network, in which three deduced and three hypothetical signal transduction pathways might be involved. JA was the critical point in the entire signal network, which connected with NO and H 2 O 2 signal pathways. RNA-seq analysis showed that a total of three candidate JA synthesis pathway genes, including one LOX and two OPR genes, were significantly up-regulated under the ANE stimulation, along with five down-regulated JAZs and one up-regulated JAR1 regulating to JA signal transduction, which further validated the pivotal role of JA in ANE elicitation.