Agarwood is highly valued for its uses as incense, perfume, and medicine. However, systematic analyses of dynamic changes of secondary metabolites during the process of agarwood formation have not yet been reported. In this study, agarwood was produced by transfusing the agarwood inducer into the trunk of Aquilaria sinensis, and changing patterns of chemical constituents, especially 2-(2-phenylethyl)chromones (PECs), in wood samples collected from the 1st to 12th month, were analyzed by GC-EI-MS and UPLC-ESI-MS/MS methods. Aromatic compounds, steroids, fatty acids/esters, sesquiterpenoids, and PECs were detected by GC-MS, in which PECs were the major constituents. Following this, UPLC-MS was used for further comprehensive analysis of PECs, from which we found that 2-(2-phenylethyl)chromones of flindersia type (FTPECs) were the most abundant, while PECs with epoxidated chromone moiety were detected with limited numbers and relatively low content. Speculation on the formation of major FTPECs was fully elucidated in our context. The key step of FTPECs biosynthesis is possibly catalyzed by type III polyketide synthases (PKSs) which condensate dihydro-cinnamoyl-CoA analogues and malonyl-CoA with 2-hydroxy-benzoyl-CoA to produce 2-(2-phenyethyl)chromone scaffold, or with 2,5-dihydroxybenzoyl-CoA to form FTPECS with 6-hydroxy group, which may serve as precursors for further reactions catalyzed by hydroxylase or O-methyltransferase (OMT) to produce FTPECs with diverse substitution patterns. It is the first report that systematically analyzed dynamic changes of secondary metabolites during the process of agarwood formation and fully discussed the biosynthetic pathway of PECs.

A total of fifty-six chromones, including seven 5,6,7,8-tetrahydro-2-(2-phenylethyl)-chromones (THPECs), five 5,6-epoxy-2-(2-phenylethyl)chromones (EPECs), seven 5,6:7,8-diepoxy-2-(2-phenylethyl)chromones (DEPECs) and thirty-seven 2-(2-phenylethyl)chromones of the flidersia type (FTPECs), were characterized by HPLC/DAD/ESI/MS/MS in three agarwood samples (from Aquilaria crassna) induced by artificial holing with different holing times. The characteristic fragmentation behavior of DEPECs and EPECs, and the methods to distinguish these four types of chromones by MS analysis were described for the first time. In addition, it was found that the relative contents of DEPECs and EPECs were down-regulated, while the relative contents of THPECs and FTPECs were up-regulated for the samples from two, four and five years of the agarwood formation time. However, the relative contents of six most widespread and abundant FTPECs presented roughly upward based on the formation time. These results could be referenced to distinguish different agarwood samples collected from different formation time.

The Dalbergia genus, a morphologically diverse group within the Fabaceae family, encompasses species of significant value in furniture production and medicinal and aromatic applications. The taxonomy of Dalbergia has relied on morphological traits, chloroplast (cp) DNA fragments, and cp genomic data. However, genomic resources for tropical liana species within this genus remain scarce. In this study, we assembled and analyzed the cp genomes of 3 liana species—Dalbergia peishaensis, D. pinnata, and D. tsoi—and compared them with those of 26 other Dalbergia species to explore their cp genome characteristics and evolutionary patterns. We employed a combination of traditional cp genome analysis and methods adapted from plant whole-genome sequencing. Phylogenetic analysis revealed that D. peishaensis has a close relationship with D. cultrata, forming a recently diverged clade, whereas D. tsoi and D. pinnata are positioned within a basal clade of the Dalbergia genus, suggesting an earlier divergence. The Dalbergia cp genomes exhibit considerable variation in size, with evidence of pseudogenization, gene loss, and duplication observed in the three liana species. Notably, the infA gene, previously reported as absent in the chloroplast genomes of Dalbergia species, was identified in the cp genomes of these three liana Dalbergia species. A total of 4533 simple sequence repeats (SSRs) were identified, providing valuable insights into cp genome evolution and facilitating future population genetics studies, particularly when combined with the high structural variation observed in the genus through whole-genome analysis methods. Additionally, seven highly divergent regions were identified as potential DNA barcode hotspots. This study enhances the genomic characterization of liana Dalbergia species and offers a robust framework for future plant cp genome analyses by integrating methodologies originally developed for whole-genome studies.