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

Abstract

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.

Keywords: Corydalis saxicola Bunting; annexin; benzylisoquinoline alkaloid; calcium.

Figure 1

The heatmap of the subcellular localizations of CsANNs ((navy blue signifies absence, blue suggests minimal distribution presence, dark pink suggests distribution presence, and pink indicates significantly greater distribution in the specific region) (A)) and multiple sequence alignments of deduced amino acid sequences of ANN proteins from Corydalis saxicola Bunting and Arabidopsis thaliana (B).

Figure 2

The phylogenetic tree of the ANN proteins. The phylogenetic tree was constructed using the neighbor-joining method tree with 1000 bootstrap replicates. The numbers at the nodes indicate the bootstrap values from 1000 replicates. The red font is used to denote CsANNs. At: A. thaliana; Bo: Brassica oleracea; Br: B. rapa; Ct: C. tomentella; Cy: C. yanhusuo; Hv: Hordeum vulgare; Os: Oryza sativa; Pt: Populus tremula × Populus.

Figure 3

The phylogenetic relationship (A), conserved motifs (B), ANN domains of CsANN proteins (C), and the intron and exon compositions (D) of CsANN genes. The phylogenetic tree was constructed using the neighbor-joining method tree with 1000 bootstrap replicates. The conserved motifs of CsANNs were identified using the MEME Suite 5.5.7, with the maximum number of motifs being set to 10. The intron and exon regions of CsANNs were analyzed using the visualize gene structure program in TBtools v2.096. In the gene structures, the dark blue boxes represent the untranslated regions (UTRs), with the orange boxes representing exons and the black lines representing introns. I, II and IV represent the subfamilies of ANNs displayed in Figure 2.

Figure 4

The localized distribution of CsANN genes on the C. saxicola chromosomes (A) and synteny analysis of interchromosomal relationships between CsANN genes (B). Nine CsANN genes were distributed on five of eight chromosomes. For synteny analysis of CsANN genes, red lines and gray ones represented the collinear gene pair and the syntenic blocks, respectively.

Figure 5

The extra-genomic collinearity related to CsANN genes in A. thaliana, C. saxicola, C. tomentella, H. vulgare, O. sativa, and P. tremula. Red lines and gray ones represented the collinear gene pairs and the syntenic blocks, respectively.

Figure 6

The distribution of cis-acting elements in the promoters of CsANNs. The promoter region was defined as a 2.0 kb sequence upstream of the translation initiation codon of the CsANN gene. Different types of cis-acting elements in the promoters are represented by ellipses of different colors.

Figure 7

The relative expression levels of CsANNs in different tissues of C. saxicola. R: roots; S: stems; LS: lateral stems; ML: mature leaves; YL: young leaves; FP: fruit pods; F: flowers. The a, b, c and d represent statistically significant differences.

Figure 8

The effects of exogenous CaCl₂ treatment on C. saxicola seedlings. (A–C): The concentrations of calcium, proline, and soluble sugars in mature leaves of C. saxicola, which were exposed to different CaCl₂ concentrations. (D–F): The DHCA levels in the stems, mature leaves, and roots of C. saxicola, which were treated with different CaCl₂ concentrations. (G–O): The relative expression levels of CsANNs treated with different concentrations of CaCl₂. The lowercase letters represent statistically significant differences, and ns represents no statistically significant difference.

Figure 9

The transient overexpression and functional analysis of CsANN1. (A) A schematic diagram of the infiltration of C. saxicola leaves with either CsANN-pCAMBIA1301 or EV; EV: the empty pCAMBIA1301 plasmid vector, serving as a negative control. (B) Expression profiles of CsANN1 and other genes in C. saxicola leaves infiltrated with either CsANN1-pCAMBIA1301 or EV. (C) Changes in BIA concentrations in C. saxicola leaves infiltrated with CsANN1-pCAMBIA1301 or EV. (D) Statistical analysis of BIA levels in C. saxicola leaves infiltrated with either CsANN1-pCAMBIA1301 or EV. Note: *: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: p ≥ 0.05.

Figure 10

Yeast two hybrid assays and protein interaction networks of the CsANNs. (A): Transactivation assay. SD/-Trp: SD medium without Trp; SD/-Trp/+X/+A: SD medium without Trp supplemented with X-α-gal at a concentration of 40 ng/mL; AbA at a concentration of 100 ng/mL. (B): Yeast two hybrid assay. DDO: SD medium without Trp and Leu; TDO: SD medium without Trp, Leu, and His; QDO: SD medium without Trp, Leu, His and Ade. (C): The protein interaction networks of the CsANNs.