Plant benzylisoquinoline alkaloids (BIAs) are a group of plant-specialized metabolites with significant pharmacological properties. In lotus (Nelumbo nucifera), BIAs accumulate primarily in the leaf blade and plumule organs. The two organs, however, accumulate quite different types of BIAs, within the former primarily aporphine-type BIAs, while the latter predominantly bis-BIAs. Herein, we demonstrate that the spatial regulation of BIA biosynthesis in lotus is coordinately controlled through the NnMYC2-NnMYB14-NnCYP80 modules. Genome-wide screening of lotus CYP80 genes discovered two tandemly arrayed yet tissue-specific NnCYP80s that are identical to the previously reported NnCYP80G and NnCYP80A, respectively. NnCYP80G is expressed primarily in the lotus laminae, while NnCYP80A is expressed particularly in the plumules. Our enzyme assays confirmed the proaporphine synthase activity of NnCYP80G and the bis-BIA synthase activity of NnCYP80A, and revealed the aporphine synthase activity of NnCYP80G by efficiently converting the (R)-reticuline substrate into corytuberine. In addition, we characterized an R2R3 MYB transcription factor (TF) NnMYB14, which binds directly to the NnCYP80G and NnCYP80A promoters and positively regulates their expression. NnMYC2, the core regulator in the JA signaling pathway, acts very upstream of NnMYB14, by binding directly to the NnMYB14 promoter and inducing its expression. Our results resolved that the organ-specific accumulation of BIAs in lotus is attributed to the tissue-specially expressed NnCYP80G and NnCYP80A genes, and the NnMYC2-NnMYB14 TF module could positively regulate the NnCYP80G and NnCYP80A expression and the lotus BIA biosynthesis.

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