Analysis of biosynthetic pathways to areca alkaloids in Areca catechu

Areca alkaloids, including arecoline and its metabolites, are abundant in areca nuts, the fruit produced by Areca catechu . Arecoline is considered the fourth most widely used psychoactive substance after nicotine, alcohol, and caffeine. However, the biosynthetic pathway and enzymes related to these alkaloids have remained largely unexplored until now. Using stable isotope tracer techniques, we identified trigonelline as a key precursor in arecoline biosynthesis and proposed a comprehensive biosynthetic pathway for this compound. We also integrated genomic and transcriptome data to identify candidate genes related to arecoline biosynthesis. Methylation events played a crucial role in the synthesis of arecoline and its derivatives, contributing to pyridine alkaloid diversification in A . catechu . Furthermore, whole genome duplication events facilitated the evolution of specific metabolites in A . catechu , while tandem duplication events contributed to the expansion of methyltransferase coding genes in its genome. We cloned and functionally characterized methyltransferase genes, including AcNMT8 , AcOMT11 , AcOMT16 , and AcOMT19 , and identified hub transcription factors, particularly the WRKY family, that may regulate pyridine alkaloid synthesis. Overall, our findings provide a foundational understanding for further elucidating the arecoline biosynthetic pathway and advancing molecular breeding efforts in A . catechu . • Trigonelline is a key biosynthetic precursor of arecoline. • The biosynthetic pathway of arecoline was proposed. • Several key genes related to arecoline biosynthesis were identified. • Methylation reaction plays a key role in the synthesis of arecoline. • The production of arecoline in areca was promoted by whole genome duplication and tandem duplication events.