Annexins (ANNs) are a family of calcium (Ca²⁺)-dependent and phospholipid-binding proteins, which are implicated in the regulation of plant growth and development as well as protection from biotic and abiotic stresses. Corydalis saxicola Bunting, an endangered benzylisoquinoline alkaloid (BIA)-rich herbaceous plant, widely used in traditional Chinese medicine, is endemic to the calciphilic karst region of China. However, whether and how ANNs are involved in the biosynthesis pathway of BIAs and/or help C. saxicola plants cope with abiotic properties, such as calcareous soils, are largely unknown. Here, nine CsANN genes were identified from C. saxicola, and they were divided into three subfamilies, namely subfamilies I, II, and IV, based on the phylogenetic tree. The CsANNs clustered into the same clade, sharing similar gene structures and conserved motifs. The nine CsANN genes were located on five chromosomes, and their expansions were mainly attributed to tandem and whole-genome duplications. The CsANN transcripts displayed organ-specific and Ca²⁺-responsive expression patterns across various tissues. In addition, transient overexpression assays showed that CsANN1 could positively regulate the accumulation of BIA compounds in C. saxicola leaves, probably by directly interacting with key BIA-biosynthetic-pathway enzymes or by interacting with BIA-biosynthetic regulatory factors, such as MYBs. This study sheds light on the profiles and functions of the CsANN gene family and paves the way for unraveling the molecular mechanism of BIA accumulation, which is regulated by Ca²⁺ through CsANNs.

Huperzine A (HupA) is a natural Lycopodium alkaloid known for its potent neuroprotective properties through the inhibition of acetylcholinesterase. Nevertheless, the limited understanding of its biosynthesis restricts its broader application. This study integrates full-length and second-generation transcriptomes with the quantification of HupA and its precursor, huperzine B, across various tissues of Huperzia serrata, the primary source plant. By employing phylogenetic clustering, expression profiling, and correlation analysis between gene expression and metabolite abundance, we identified 71 candidate genes from seven enzyme families potentially involved in the synthesis of the HupA backbone, including lysine/ornithine decarboxylases, copper amine oxidases (CAOs), chalcone synthases, and cytochrome P450 monooxygenases. Additionally, 28 genes from two families were identified for modification reactions, specifically 2-oxoglutarate/Fe(II)-dependent dioxygenases and caffeoyl shikimate esterases. Comparative analysis between young and mature leaves revealed 3801 genes with higher expression in young leaves, with 84 showing a high correlation with HupA content across seven families. Protein–protein interaction network analysis indicated possible interactions with transcription factors from the MYB, NF-YC, GRAS, ERF, BHLH, and SAP families. Functional validation of two candidate CAOs in planta confirmed their catalytic roles in amine/alkaloid metabolism. This study provides a theoretical foundation and a set of candidate genes for elucidating the biosynthetic pathway of HupA and related alkaloids in H. serrata.

Huperzine A (HupA) is a natural Lycopodium alkaloid known for its potent neuro-protective properties through the inhibition of acetylcholinesterase. Nevertheless, the limited understanding of its biosynthesis restricts its broader application. This study integrates full-length and second-generation transcriptomes with the quantification of HupA and its precursor, huperzine B, across various tissues of Huperzia serrata, the primary source plant. By employing phylogenetic clustering, expression profiling, and correlation analysis between gene expression and metabolite abundance, we identified 71 candidate genes from seven enzyme families potentially involved in the synthesis of the HupA backbone, including lysine/ornithine decarboxylases, copper amine oxidases (CAOs), chalcone synthases, and cytochrome P450 monooxygenases. Additionally, 28 genes from two families were identified for modification reactions, specifically 2-oxoglutarate/Fe(II)-dependent dioxygenases and caffeoyl shikimate esterases. Comparative analysis between young and mature leaves revealed 3,801 up-regulated genes in young leaves, with 84 showing a high correlation with HupA content across seven families. Protein–protein interaction network analysis indicated interactions with transcription factors from the MYB, NF-YC, GRAS, ERF, BHLH, and SAP families. Functional validation of two candidate CAOs in planta confirmed their catalytic roles in amine/alkaloid metabolism. This study provides a theoretical foundation and a set of candidate genes for elucidating the biosynthetic pathway of HupA and related alkaloids in H. serrata.