Spatial regulation of benzylisoquinoline alkaloid biosynthesis in lotus ( <i>Nelumbo nucifera</i> ) is controlled coordinately through the NnMYC2-NnMYB14-NnCYP80 modules

Plant benzylisoquinoline alkaloids (BIAs) are a group of plant-specialized metabolites with significant pharmacological properties. In lotus (<i>Nelumbo nucifera</i>), 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 <i>CYP80</i> genes discovered two tandemly arrayed yet tissue-specific <i>NnCYP80s</i> that are identical to the previously reported <i>NnCYP80G</i> and <i>NnCYP80A</i>, respectively. <i>NnCYP80G</i> is expressed primarily in the lotus laminae, while <i>NnCYP80A</i> 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 (<i>R</i>)-reticuline substrate into corytuberine. In addition, we characterized an R2R3 MYB transcription factor (TF) NnMYB14, which binds directly to the <i>NnCYP80G</i> and <i>NnCYP80A</i> 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 <i>NnMYB14</i> promoter and inducing its expression. Our results resolved that the organ-specific accumulation of BIAs in lotus is attributed to the tissue-specially expressed <i>NnCYP80G</i> and <i>NnCYP80A</i> genes, and the NnMYC2-NnMYB14 TF module could positively regulate the <i>NnCYP80G</i> and <i>NnCYP80A</i> expression and the lotus BIA biosynthesis.