Towards sustainable agarwood production: integrating microbial interactions, anatomical changes, and metabolite biosynthesis.

Agarwood is a highly valuable non-timber forest product mainly derived from the Aquilaria genus, widely traded in the perfumery, religious items, and traditional medicine industries. Naturally, agarwood forms within the xylem as part of the tree's defense mechanism against environmental stressors and microbial infection. The escalating demand for agarwood has led to the overexploitation of Aquilaria species, with some now classified as critically endangered. Despite advancements in artificial induction methods for sustainable agarwood supply, the intricate links between physiological and molecular mechanisms governing its formation remain poorly understood. This review addresses these knowledge gaps by examining the interplay between morphological changes in xylem structure during tylose formation and molecular alterations, particularly the biosynthesis of 2-(2-phenylethyl)chromones (PECs), key compounds in agarwood. Additionally, it integrates findings from multi-omics approaches including genomics, transcriptomics, proteomics, and metagenomics to reveal how secondary metabolite biosynthesis, including PECs and terpenes, is regulated across various Aquilaria species, regions, and induction techniques. The role of microbial communities, particularly endophytes such as Fusarium, in regulating agarwood formation is also discussed, emphasizing their involvement in both natural and artificial induction strategies. Furthermore, this review explores the role of reactive oxygen species in mediating morphological and biochemical defense responses, alongside the functions of transcription factors (TFs), protein kinases, and signaling molecules in balancing defense and growth. However, the crosstalk between key genes such as chalcone synthases, MAPK, cytochromes, NADPH oxidases, TFs, and miRNAs require further study to fully understand the complex defense mechanisms in Aquilaria trees. Overall, this review aims to bridge the current knowledge gaps by linking morphological and biochemical changes in agarwood formation, particularly PEC biosynthesis, while proposing metabolite engineering using microbial hosts as a promising tool for sustainable and technology-driven agarwood production. One-Sentence Summary: This review explores the physiological and molecular processes behind agarwood formation in Aquilaria malaccensis, highlighting the roles of tyloses, microbial interactions, secondary metabolite biosynthesis particularly 2-(2-phenylethyl)chromones and the integration of biotechnology for sustainable production and metabolic engineering.