Evolution-guided multiomics provide insights into the strengthening of bioactive flavone biosynthesis in medicinal pummelo.

Pummelo (Citrus maxima or Citrus grandis) is a basic species and an important type for breeding in Citrus. Pummelo is used not only for fresh consumption but also for medicinal purposes. However, the molecular basis of medicinal traits is unclear. Here, compared with wild citrus species/Citrus-related genera, the content of 43 bioactive metabolites and their derivatives increased in the pummelo. Furthermore, we assembled the genome sequence of a variety for medicinal purposes with a long history, Citrus maxima 'Huazhouyou-tomentosa' (HZY-T), at the chromosome level with a genome size of 349.07 Mb. Comparative genomics showed that the expanded gene family in the pummelo genome was enriched in flavonoids-, terpenoid-, and phenylpropanoid biosynthesis. Using the metabolome and transcriptome of six developmental stages of HZY-T and Citrus maxima 'Huazhouyou-smooth' (HZY-S) fruit peel, we generated the regulatory networks of bioactive metabolites and their derivatives. We identified a novel MYB transcription factor, CmtMYB108, as an important regulator of flavone pathways. Both mutations and expression of CmtMYB108, which targets the genes PAL (phenylalanine ammonia-lyase) and FNS (flavone synthase), displayed differential expression between Citrus-related genera, wild citrus species and pummelo species. This study provides insights into the evolution-associated changes in bioactive metabolism during the origin process of pummelo.