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.

Annexins (ANNs), which are calcium (Ca2+)-dependent and phospholipid-binding protein families, are implicated in the regulation of plant growth and development as well as protection from various stresses. However, little is known about ANNs in Corydalis Saxicola, an endangered benzylisoquinoline alkaloid (BIA)-riched herbaceous plant widely used in traditional Chinese medicine and endemic to the calciphilic karst region in China. Here, nine CsANN genes were identified from C. saxicola, and they were divided into three subfamilies based on the phylogenetic analysis. 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 duplication and segmental duplication. The CsANN transcripts showed variable organ-specific and Ca2+-responsive expression patterns. Further transient overexpression assays showed that CsANN1 could positively regulate the accumulation of BIA compounds in C. saxicola leaves, probably through directly interacting with key BIA-biosynthesis-pathway enzymes or by interacting with BIA-biosynthesis-regulating factors, such as MYBs. This study sheds light on profiles and functions of the CsANN gene family and paves the way for unraveling the molecular mechanism of BIA accumulation regulated by Ca2+ through CsANNs.