Bisbenzylisoquinoline and aporphine alkaloids are the two main pharmacological compounds in the ancient sacred lotus (Nelumbo nucifera). The biosynthesis of bisbenzylisoquinoline and aporphine alkaloids has attracted extensive attention because bisbenzylisoquinoline alkaloids have been reported as potential therapeutic agents for COVID-19. Our study showed that NnCYP80A can catalyze C-O coupling in both (R)-N-methylcoclaurine and (S)-N-methylcoclaurine to produce bisbenzylisoquinoline alkaloids with three different linkages. In addition, NnCYP80G catalyzed C-C coupling in aporphine alkaloids with extensive substrate selectivity, specifically using (R)-N-methylcoclaurine, (S)-N-methylcoclaurine, coclaurine and reticuline as substrates, but the synthesis of C-ring alkaloids without hydroxyl groups in the lotus remains to be elucidated. The key residues of NnCYP80G were also studied using the 3D structure of the protein predicted using Alphafold 2, and six key amino acids (G39, G69, A211, P288, R425 and C427) were identified. The R425A mutation significantly decreased the catalysis of (R)-N-methylcoclaurine and coclaurine inactivation, which might play important role in the biosynthesis of alkaloids with new configurations.

Benzylisoquinoline alkaloids in lotus (Nelumbo nucifera) seed plumules and leaves exhibit significant tissue specificity for their pharmacological effects and potential nutritional properties. Herein, 46 benzylisoquinoline alkaloids were identified via UPLC-QTOF-HRMS, of which 9 were annotated as glycosylated monobenzylisoquinoline alkaloids concentrated in the seed plumules. The spatial distribution of targeted benzylisoquinoline alkaloids in leaves, seed plumules, and milky sap was determined via MALDI–MSI. Furthermore, 37 Nelumbo cultivars were investigated using targeted metabolomics to provide insights into functional tea development. While aporphine alkaloids comprised the main compounds present in lotus leaves, bisbenzylisoquinoline alkaloids were the main compounds in lotus plumules, where glycosylation primarily occurs. These findings can help understand the distribution of benzylisoquinoline alkaloids in lotus tissue and the directional breeding of varieties enriched with specific chemical functional groups for nutritional and pharmacological applications.

The edible rhizome variant of Nelumbo nucifera with various cultivars has a long history of use as a food in East Asia. In this study, 48 target metabolites were untargeted and identified in 212 rhizome cultivars (tropical and temperate types) using ultraperformance liquid chromatography-electrospray ionization quadrupole time-of-flight high-resolution mass spectrometry; among these, 32 compounds were newly reported in the rhizome. Combined with the browning phenotype of 212 lotus rhizomes, (epi) catechin, norarmepavine, and N-feruloyl-3-methoxytyramine were used as predominant chemical markers to separate different degrees of lotus rhizome browning. p-Coumaroyltyramine and N-trans-feruloyltyramine were selected as predominant chemical markers to investigate the differential expression between tropical and temperate lotus using principal component analysis and orthogonal partial least squares discriminant analysis. Shared and unique structure plots were used to compare the outcomes of the ecotype and browning OPLS model, showing that variation in tropical lotus rhizome browning is not obvious; this will be of great importance for genetic improvement by providing a hereditary basis.