Integrated proteomic and metabolomic analyses reveal the salt-induced agarwood formation mechanism of Aquilaria sinensis

Aquilaria sinensis (Agarwood) is a precious Chinese medicine commonly used in clinical practice. Previous researches about agarwood mainly focused on chemical composition, pharmacological activities and artificial induction. The molecular processes responsible for agarwood formation remain largely unknown. This study performs an integrated proteomic and metabolomic analysis of agarwood after treatment with fire (F) or fire plus different concentrations of salt (FSH, FSM, FSL) for 4 and 12 months. A total of 1611 differential metabolites and 9148 differentially expressed proteins were detected in 30 samples of agarwood. The fire plus 0.4 mol/L NaCl (FSM) treatment induced more differential metabolites compared to fire plus 0.04 mol/L NaCl (FSL) and fire plus 4 mol/L NaCl (FSH) at both 4 and 12 month. The primary differential categories included organic acids, sesquiterpenoids, phenolic acids, flavonoids, and chromones. KEGG analysis indicated significant enrichment of secondary metabolite biosynthesis, ABC transporters, and flavonoid biosynthesis pathways across all comparison groups. Additionally, all six comparison groups revealed different levels of 2-(2-phenylethyl) chromones (PECs), the distinctive constituents of agarwood. Nevertheless, the fire plus 0.04 mol/L NaCl (FSL) treatment produced the most differentially expressed proteins compared to fire plus 0.4 mol/L NaCl (FSM) and fire plus 4 mol/L NaCl (FSH). Sesquiterpene synthases were significantly upregulated in the F1(fire drill in the 4th month)_vs_FSH1(fire drill + 4 mol/L NaCl in the 4th month) and F1_vs_FSL1(fire drill + 0.04 mol/L NaCl in the 4th month) group, but downregulated in the F3(fire drill in the 12th month)_vs_FSH3(fire drill + 4 mol/L NaCl in the 12th month) group. Besides, the varying expression levels of these candidate proteins (HMGS, PMK and TPS) involved in the sesquiterpene biosynthesis pathway across the different stress treatments provide valuable insights into their roles in biological mechanism of agarwood formation. Proteomics and metabolomics analyses of agarwood provide essential insights into its formation mechanisms and support quality enhancement initiatives.