Atractylodes macrocephala Koidz. is a medicinal plant in high clinical demand due to its pharmacological efficacy. However, research on its quality dynamics across different growth years remains limited, primarily focusing on transcriptomics, microbiome analysis, and photosynthetic capacity, with studies only extending to three-year-old plants. This study examines A. macrocephala over a broader growth span (1, 2, 3, 5, and 10 years), integrating morphological, microstructural, metabolomic, spatial metabolite distribution, and transcriptomic analyses. Morphologically, rhizome weight and length increased with age, and the characteristic “Hejing” structure became more pronounced. Microstructural analysis revealed progressively developed xylem. Metabolomic profiling indicated a decline in sucrose content, alongside increased accumulation of bioactive sesquiterpenoids and phenolic acids over time. Transcriptomic analysis showed that genes involved in the biosynthesis of these active compounds—terpenoid backbone (HMGR, DXS, idi, GPS, and GGPS), phenylpropanoid (PAL, 4CL, and HCT), and sucrose metabolism (SPS and SPP)—were upregulated in older plants. Desorption electrospray ionisation mass spectrometry imaging (DESI-MSI) revealed an expanded distribution of key metabolites with increasing growth years. The findings suggest that A. macrocephala aged 5 and 10 years exhibit superior quality, with a more distinct “Hejing” morphology and higher levels of bioactive compounds. The quality stabilises after five years, indicating that older plants may possess enhanced medicinal value.

4-Coumarate:CoA ligases (4CLs) are a group of essential enzymes involved in the pathway of phenylpropanoid-derived compound metabolisms; however it is still difficult to identify orthologs and paralogs of these important enzymes just based on sequence similarity of the conserved domains. Using sequence data of 20 plant species from the public databases and sequences from Lonicera japonica, we define 1252 adenosine monophosphate (AMP)-dependent synthetase/ligase sequences and classify them into three phylogenetic clades. 4CLs are in one of the four subgroups, according to their partitioning, with known proteins characterized in A. thaliana and Oryza sativa. We also defined 184 non-redundant sequences that encode proteins containing the GEICIRG motif and the taxonomic distribution of these GEICIRG-containing proteins suggests unique catalytic activities in plants. We further analyzed their transcription levels in L. japonica and L. japonica. var. chinensis flowers and chose the highest expressed genes representing the subgroups for structure and binding site predictions. Coupled with liquid chromatography-mass spectrometry (LC-MS) analysis of the L. japonica flowers, the structural study on putative substrate binding amino acid residues, ferulate, and 4-coumaric acid of the conserved binding-site of LJ4CL1 leads to a conclusion that this highly expressed protein group in the flowers may process 4-coumarate that represents 90% of the known phenylpropanoid-derived compounds. The activity of purified crude LJ4CL1 protein was analyzed using 4-coumarate as template and high activity indicating that 4-coumarate is one of the substrates of LJ4CL1.