Moleular Mechanism Of JA Signaling Pathway Involved In The Regulation Of Agarwood Sesquiterpene Biosynthesis

Jasmonic acid (JA) and its derivates, collectively known as jasmoantes (JAs), are essential signaling molecules that coordinate the plant response to biotic and abiotic challenges, and they are involved in several growth and developmental processes. The research on the function and mechanism of JA signaling has become one of the host spots in the life science for a long time. Aquilaria sinensis (Lour.) Gilg is the main species of Aquilaria that is one of the main and also as the only certified plant source for agarwood production in China. Agarwood is widely used as a Traditional Chinese Medicine and has many therapeutic properties, such as anti-emetic, carminative, sedative and so on.Agarwood only formed when the A. sinensis tree is wounded, and the production of agarwood is also identified as a result of A. sinensis defensive response. Sesquiterpenes and phenylethyl chromone derivatives are the main fragrant compounds of agarwood. The biosynthetic pathway of sesquiterpenes is comparetively clear, but its regulatory pathway is largely unclear. In our previous study, MeJA treatment could significantly induce the biosynthesis and accumulation of sesquiterpenes in A. sinensis tree and calli, suggesting that JA plays some role in agarwood sesquiterpenes biosynthesis. While whether there is a direct relationship between JA signaling pathway and agarwood sesquiterpenes biosynthesis, and the exact regulatory mechanism by JA signaling pathway is unknown. The main conclusions obtained from this study are as follows:1. JA sifinal molecule is involved in the regulation of agarwood sesquiterpenes biosynthesis. Combining transcriptome gene profiles analysis and qRT-PCR detection, we found that the key genes in JA signaling pathway significantly responded to stresses, and all of the detective genes are early-wounding-responsive genes. Heat treatment could induce the rapid and high-yield biosynthesis of plant endogenous hormone JA and agarwood sesquiterpenes (a-Guaiene, a-Humulene and d’-Guaiene); JA biosynthesis inhibitor could repress the biosynthesis of JA and sesquiterpenes; and there was no sesquiterpene detected within 12 h after heat treatment, and only a small quantity of sesquiterpene was detected afer 24 h.2. Based on the gene fragments from transcriptome data, the full-length cDNA sequence of five key genes (LOX、COI1、NINJA、JAZ and MYC2) in JA signaling was firstly cloned and named AsLOX1、AsCOI1、AsNINJA1、AsJAZ1 and AsMYC2, respectively. The gene sequence was analyzed by informatics tools, which lay the foundation for the study on JA signaling pathway in the process of sesquiterpene synthesis.3. Basically identifying the function of AsJAZ1 gene, and AsJAZ1 was very likely one of the negative regulatory factors of agarwood sesquiterpene synthase gene. Subcellular localization analysis showed that A. sinensis AsJAZ1 was located in nucleus. The transcripts of AsJAZl gene was highest in leaves, followed by brantches, and lowest in roots and stems. Over-expression AsJAZl gene in wild type Arabidopsis plants could repress the expression of Arabidopsis sesquiterpene synthase genes TPS21 and TPS11.4. Basically identifying the function of AsMYC2, and it was very likely a positive regulatory transcription factor of agarwood sesquiterpene synthase gene. Subcellular localization analysis showed that A. sinensis AsMYC2 was located in nucleus. The transcripts of AsMYC2 gene was highest in stems and roots, followed by branches, and lowest in leaves. Over-expression AsMYC2 gene in A. thaliana myc2-2 mutant and wild type plants could up-regulate the expression of A. thaliana sesquiterpene synthase genes TPS21 and TPS11.5. Defining that AsJAZ1 interacted with AsMYC2 protein in vitro. We successfully constructed the prokaryotic expression vector of AsJAZ1 and AsMYC2 gene separately, expressed them in prokaryotic expression system, and purified the His-AsJAZ1 and GST-AsMYC2 fusion proteins; High quality protein will provide materials for polyclonal antibody preparation in our next work. The interaction between these two proteins identified by pull-down assay will help us to reveal the molecular mechanism of JA signaling pathway involved in the regulation of agarwood sesquiterpene biosynthesis.In this study, we preliminarily confirmed that JA signaling pathway is involved in the regulation of agarwood sesquiterpenes biosynthesis. In this paper, we carried on a systematic investigation about the regulatory mechanism of JA signaling pathway in Aquilaria plants for the first time. This study offered a good reference for researching other signal molecular pathway, and provided some molecule proofs to reveal the mechanism of wound-induced agarwood formation in A. sinensis defense reponse.