Integrative Metabolomic and Transcriptomic Profiling Reveals the Chemical and Molecular Basis for Aroma Divergence in <i>Isatis</i> Fruits

ABSTRACT The fruits of Isatis species hold significant but underexplored potential in flavor and phytochemical applications. This study comprehensively investigated the volatile organic compounds (VOCs) and their transcriptional regulation underlying aroma divergence in the fruits of three Isatis species. Using headspace solid‐phase microextraction coupled with gas chromatography–mass spectrometry (HS‐SPME–GC–MS), a total of 1042 VOCs were identified, with terpenoids, esters, and ketones being the most abundant classes. Principal component and differential analyses revealed distinct, species‐specific VOC profiles. Quantitative analysis showed that Isatis oblongata had the highest total VOC content, characterized by esters and alcohols, whereas Isatis tinctoria and Isatis indigotica exhibited balanced aldehyde‐terpenoid and ketone‐enhanced profiles, respectively. The application of relative odor activity value (rOAV) modeling identified 44 key aroma‐active compounds, defining the sensory profiles: I. oblongata as mushroom dominant (driven by the high rOAV of 1‐octen‐3‐one), I. tinctoria as fatty‐green‐earthy (driven by C9 aldehydes), and I. indigotica as sweet‐green with a spicy undertone (associated with benzene, (isothiocyanatomethyl)‐). Integrated transcriptomic analysis and weighted gene co‐expression network analysis (WGCNA) uncovered the molecular basis for this divergence. The fatty‐green character of I. tinctoria was linked to a NAC transcription factor (Iin12486) that co‐expressed with specific lipoxygenase ( LOX ) genes (Iin24854, Iin09829). Conversely, the mushroom aroma of I. oblongata was associated with a different NAC TF (Iin05720) co‐expressed with a hydroperoxide lyase ( HPL ) gene (Iin21448). This multiomics study deciphers the coordinated transcriptional regulation underlying aroma divergence in Isatis , providing a scientific basis for chemotaxonomy, quality control, and the targeted utilization of aroma resources in these species.