Unraveling the Spatiotemporal Dynamics of Alkaloid Biosynthesis During Red Chicory Heading Through Integrated Multi-omics

Red chicory (Cichorium intybus L.) is a nutritionally and medicinally important leafy vegetable, yet the metabolic pathways of alkaloids and their regulatory mechanisms during head formation remain largely uncharacterized. This study aimed to systematically elucidate the dynamic changes in alkaloid profiles and to identify key transcriptional regulators controlling alkaloid biosynthesis during red chicory heading. An integrated multiomics strategy combining metabolomic and transcriptomic analyses was applied across three developmental stages: seedling (SS), rosette (RS), and heading (HS). Gene–metabolite correlation networks were constructed to uncover regulatory relationships, and candidate genes were validated by qRT-PCR. A total of 43 alkaloids, distributed across four structural subclasses, namely, phenolamines, pyridine and piperidine alkaloids (PPAs), indole alkaloids (IAs), and other alkaloids, were identified, showing distinct stage-specific accumulation patterns. Transcriptomic analysis revealed 46 differentially expressed genes (DEGs) associated with alkaloid biosynthesis. Integration of transcriptomic and metabolomic data showed that 40 DEGs were positively correlated with differentially accumulated metabolites (DAMs), whereas 39 DEGs displayed negative correlations. A single transcription factor (Cint.ptg000005l.g315, a C2H2 family member) displayed both positive and negative correlations with multiple DAMs. These results indicate that red chicory heading is governed by a complex transcriptional network that coordinately modulates alkaloid biosynthesis through both activation and repression of metabolic pathways. This work provides a comprehensive characterization of alkaloid metabolism during red chicory head development and offers valuable genetic resources for future functional genomics and quality improvement efforts in leafy vegetables.