The 2-(2-phenethyl)chromones (PECs) are the signature constituents responsible for the fragrance and pharmacological properties of agarwood. O-Methyltransferases (OMTs) are necessary for the biosynthesis of methylated PECs, but there is little known about OMTs in Aquilaria sinensis. In this study, we identified 29 OMT genes from the A. sinensis genome. Expression analysis showed they were differentially expressed in different tissues and responded to drill wounding. Comprehensive analysis of the gene expression and methylated PEC content revealed that AsOMT2, AsOMT8, AsOMT11, AsOMT16, and AsOMT28 could potentially be involved in methylated PECs biosynthesis. In vitro enzyme assays and functional analysis in Nicotiana benthamiana demonstrated that AsOMT11 and AsOMT16 could methylate 6-hydroxy-2-(2-phenylethyl)chromone to form 6-methoxy-2-(2-phenylethyl)chromone. A transient overexpression experiment in the variety ‘Qi-Nan’ revealed that AsOMT11 and AsOMT16 could significantly promote the accumulation of three major methylated PECs. Our results provide candidate genes for the mass production of methylated PECs using synthetic biology.

The 2-(2-phenylethyl) chromones are the main characteristic constituents for the fragrance and pharmacological activities of agarwood. Iron and methyl jasmonate (MeJA) are two important elicitors that affect the production of secondary metabolites in plants. With the aim to evaluate the response of eight 2-(2-phenylethyl)chromones constituents of Aquilaria sinensis to ferrous sulfate (FeSO4) and MeJA treatment, the content of eight 2-(2-phenylethyl)chromones in A. sinensis callus tissues treated with 0

N6-methyladenosine (m6A) RNA modification is a conserved mechanism to regulate gene expression that plays vital roles in the development of plants. However, the m6A RNA modification in forest trees remains limited. Here, we performed a complete analysis of m6A writers, erasers and readers in Poplar 84K, including gene location, gene structures, conserved motifs, phylogenetic relationships, promoter analysis, expression profiles and the homology modeling. We have identified 61 m6A pathway genes in Poplar 84K (Populus alba × Populus glandulosa), including 14 m6A writers, 14 m6A erasers and 33 m6A readers. Phylogenetic analysis indicated that the m6A writers and erasers were clustered into four groups and m6A readers were clustered into two groups. Promoter analysis showed that m6A pathway genes were mainly responsive to low oxygen followed by ABA and ethylene. The expression of the identified m6A pathway genes showed tissue-specific expression patterns in leaves, xylem, phloem and roots. Moreover, 17 genes were significantly up-regulated and 13 genes were significantly down-regulated in poplar overexpressing the transcription factor LBD15. Homology modeling and molecular docking results suggested that PagFIP37b was most likely to be regulated by LBD15, and the qPCRshowed that PagFIP37b were up-regulated in the LBD15-oe plants. The results provide insights that aid in the future elucidation of the functions of these m6A pathway genes and the epigenetic regulation mechanism of these genes in Poplar 84K.

N6-methyladenosine (m6A) plays an important role in the gene expression regulation. Previously, we found an ortholog of Arabidopsis LBD15 that showed xylem preferential expression and involved in leaf development in Poplar 84 K. In order to investigate whether m6A modification affects the function of LBD15, the m6A-immunoprecipitation sequence and the matched input RNA sequence for non-transgenic plants (CK) and the LBD15 overexpression (LBD15-oe) plants were compared and analyzed. As a result, 7,156 differential m6A peaks were identified, with 2,896 upregulated m6A peaks and 4,260 downregulated m6A peaks. Correlation analysis of differential expression genes and differential m6A peaks indicated that a total of 119 differently methylated genes showed a negative correlation with the differentially expressed genes. Among them, Nudix hydrolase, LRR receptor-like serine/threonine-protein kinase, tubulin, vacuole membrane protein KMS1, and MYB family transcription factor PHL11 may be involved in the posttranscriptional gene regulation in LBD15 overexpression plants. The expression of ten m6A-modified genes was validated by qRT-PCR. Our results will provide a basis for the further elucidation of the regulatory mechanism of m6A modification and the epigenetic regulation of LBD15.