Genome-Wide Characterization of the ANN Gene Family in Corydalis saxicola Bunting and the Role of CsANN1 in Dehydrocavidine Biosynthesis

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. <i>Corydalis saxicola</i> 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 <i>C. saxicola</i> plants cope with abiotic properties, such as calcareous soils, are largely unknown. Here, nine <i>CsANN</i> genes were identified from <i>C. saxicola</i>, 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 <i>CsANN</i> genes were located on five chromosomes, and their expansions were mainly attributed to tandem and whole-genome duplications. The <i>CsANN</i> 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 <i>C. saxicola</i> 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 <i>CsANN</i> gene family and paves the way for unraveling the molecular mechanism of BIA accumulation, which is regulated by Ca²⁺ through CsANNs.