## 3 Discussion  
The present study employed GC-MS-based metabolomics to detect and analyze the variations in volatile compound content among different plant parts (root, stem, leaf, seed, husk, and callus) of _A. sinensis_. A total of 367 metabolites were detected, with 84 terpenoids identified as the predominant compounds. In addition, a comprehensive analysis yielded a total of 331 differentially accumulated metabolites (DAMs) from 15 control groups. The primary volatile metabolites in stem, seed, husk, and leaf callus were terpenoids and ketones. In leaves, ketones and esters dominate as the main volatile metabolites. Meanwhile, roots exhibited terpenoids and heterocycles as their principal volatile metabolites. In this study, the majority of the differential metabolites, particularly terpenoids, exhibited higher expression levels in the callus, whereas chromosomes displayed lower expression levels in all non-resin parts compared to other sections. The application of salicylic acid (SA) and methyl jasmonate (MJ) has been employed to stimulate the production of aromatic compounds in callus and suspension cells of agarwood species. The results demonstrated that both SA and MJ significantly stimulated the biosynthesis of sesquiterpenoids, while they did not directly induce the production of chromosomes. Further investigation unveiled that sesquiterpenoids are synthesized in living cells, whereas chromosomes are likely derived from dying cellular debris [58]. Sesquiterpenes, which belong to the class of terpenoids, are considered crucial defensive secondary metabolites synthesized by agarwood in response to both biotic and abiotic stresses. Under mechanical stress, plants synthesize key molecules such as hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)), jasmonic acid (JA), and salicylic acid (SA) [59], which trigger the biosynthesis of sesquiterpenes in agarwood, subsequently regulating defense responses against various abiotic and biotic stimuli [60; 61; 62; 63]. A total of 43 sesquiterpenoids were isolated from six tissue sites of _A. sinensis_, which were categorized into 11 distinct types based on their backbone structures, predominantly comprising bisabolanes (8) and cadinanes (9) types. The accumulation of germaranes was found to be significant in leaves, stems, and seeds. On the other hand, agaroseiprane showed significant accumulation, specifically in the husk and leaf callus. Additionally, RNA-seq was employed to conduct transcriptome sequencing across various tissues for further analysis of the metabolic regulatory network associated with variations in volatile component content among different tissues, as well as for identification  
Figure 9: Nine quadrant diagram showing the correlation of differentially expressed genes and differentially accumulated metabolites between husk and leaf callus libraries. of potential key genes involved in this process. The differential expression analysis showed that the number of differentially expressed genes identified in each control group surpassed 808. The annotation identified a total of 58 unigenes involved in the biosynthesis of sesquiterpenenes. The main biosynthetic pathway of sesquiterpenenes is the MVA pathway. ACAT is predominantly expressed in leaf callus, seed, and root tissues, while HMGS shows predominant expression in stem and leaf tissues. The gene HDR, involved in the MEP pathway for monoterpene, diterpene, and triterpene biosynthesis, exhibits high expression levels in the husk tissue. The observed outcome demonstrated a positive correlation with the accumulation of sesquiterpenes in the metabolome. The efficacy of sesquiterpenes in conferring resistance against biotic and abiotic stresses has been extensively investigated, leading to numerous studies aimed at enhancing sesquiterpene production. In addition to inducing physical or chemical trauma, studies have also been conducted to enhance sesquiterpene formation by modulating the activity of rate-limiting enzymes in the biosynthetic pathway.