Covering: Up to the end of 2019. Agarwood is a resinous portion of Aquilaria trees, which is formed in response to environmental stress factors such as physical injury or microbial attack. It is very sought-after among the natural incenses, as well as for its medicinal properties in traditional Chinese and Ayurvedic medicine. Interestingly, the chemical constituents of agarwood and healthy Aquilaria trees are quite different. Sesquiterpenes and 2-(2-phenethyl)chromones with diverse scaffolds commonly accumulate in agarwood. Similar structures have rarely been reported from the original trees that mainly contain flavonoids, benzophenones, xanthones, lignans, simple phenolic compounds, megastigmanes, diterpenoids, triterpenoids, steroids, alkaloids, etc. This review summarizes the chemical constituents and biological activities both in agarwood and Aquilaria trees, and their biosynthesis is discussed in order to give a comprehensive overview of the research progress on agarwood.

Aquilaria sinensis (Lour.) Spreng is one of the important plant resources involved in the production of agarwood in China. The agarwood resin collected from wounded Aquilaria trees has been used in Asia for aromatic or medicinal purposes from ancient times, although the mechanism underlying the formation of agarwood still remains poorly understood owing to a lack of accurate and high-quality genetic information. We report the genomic architecture of A. sinensis by using an integrated strategy combining Nanopore, Illumina, and Hi-C sequencing. The final genome was ∼726.5 Mb in size, which reached a high level of continuity and a contig N50 of 1.1 Mb. We combined Hi-C data with the genome assembly to generate chromosome-level scaffolds. Eight super-scaffolds corresponding to the 8 chromosomes were assembled to a final size of 716.6 Mb, with a scaffold N50 of 88.78 Mb using 1,862 contigs. BUSCO evaluation reveals that the genome completeness reached 95.27%. The repeat sequences accounted for 59.13%, and 29,203 protein-coding genes were annotated in the genome. According to phylogenetic analysis using single-copy orthologous genes, we found that A. sinensis is closely related to Gossypium hirsutum and Theobroma cacao from the Malvales order, and A. sinensis diverged from their common ancestor ∼53.18–84.37 million years ago. Here, we present the first chromosome-level genome assembly and gene annotation of A. sinensis. This study should contribute to valuable genetic resources for further research on the agarwood formation mechanism, genome-assisted improvement, and conservation biology of Aquilaria species.

Agarwood is the fragrant resinous heartwood obtained from certain trees in the genus Aquilaria belonging to the family Thymelaeaceae. 2-(2-Phenylethyl)chromones and characteristic sesquiterpenes are the main classes of aromatic compounds isolated from agarwood. Although there are many sesquiterpenes, relatively few 2-(2-phenylethyl)chromones have been determined in agarwood by GC-MS. After analysis of the MS spectra of eighteen 2-(2-phenylethyl)chromone derivatives isolated from agarwood and identified by NMR spectroscopy, together with the reported MS data and characteristic of structures of 2-(2-phenylethyl)chromones, the MS characterization, fragmentation patterns and characteristic fragment peaks for the compounds were deduced and a table summarizing MS characterization of 2-(2-phenylethyl)chromones in agarwood is presented. All the 2-(2-phenylethyl)chromones previously reported in agarwood are substituted by methoxy or/and hydroxy groups, except for one compound. Due to the fact they all possess the same basic skeleton (molecular weight: 250) and similar substituent groups (methoxy or hydroxy groups), a formula (30m + 16n = MW − 250) is provided to calculate the number of methoxy (m) or hydroxy (n) groups according to molecular ion peak or molecular weight (MW). We deduced that the characteristic fragmentation behaviors of the 2-(2-phenylethyl)chromones are the cleavages of the CH₂-CH₂ bond between chromone moiety and phenyl moiety. Thus, characteristic fragment ions, such as m/z 91 [C₇H₇], 107 [C₇H₆+OH], 121 [C₇H₆+OCH₃], 137 [C₇H₅+OH+OCH₃] are formed by different substituted benzyl moieties, while characteristic fragment ions such as m/z 160 [C₁₀H₈O₂], 176 [C₁₀H₇O₂+OH], 190 [C₁₀H₇O₂+OCH₃], 220 [C₁₀H₆O₂+OCH₃×2] are formed by different substituted chromone moieties. Furthermore, rules regarding to the relationship between the positions of hydroxy or methoxy groups and the relative abundances of benzyl and chromone fragment ions have been deduced. Elucidation of how the positions of hydroxy or methoxy groups affect the relative abundances of benzyl and chromone fragment peaks is also provided. Fifteen unidentified compounds of an artificial agarwood sample analyzed by GC-MS, were preliminary determined as 2-(2-phenylethyl)chromones by analysis of their MS characterization and by comparison of their MS spectra with those of 18 standard compounds or 2-(2-phenylethyl)chromones reported in literature according to the above-mentioned methods and rules. This report will be helpful for the analysis and structural elucidation of 2-(2-phenylethyl)chromones in agarwood by GC-MS, and provides fast and reliable characterization of the quality of agarwood.

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.