Identification and characterization of the Quinoa AP2/ERF gene family and their expression patterns in response to salt stress

Abstract

The APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) transcription factors play crucial roles in plant growth, development, and responses to biotic and abiotic stresses. This study was performed to comprehensively identify and characterize the AP2/ERF gene family in quinoa (Chenopodium quinoa Willd.), a highly resilient pseudocereal crop known for its salinity tolerance. A total of 150 CqAP2/ERF genes were identified in the quinoa genome; these genes were unevenly distributed across the chromosomes. Phylogenetic analysis divided the CqAP2/ERFs into five subfamilies: 71 ERF, 49 DREB, 23 AP2, 3 RAV, and 4 Soloist. Additionally, the DREB and ERF subfamilies were subdivided into four and seven subgroups, respectively. The exon-intron structure of the putative CqAP2/ERF genes and the conserved motifs of their encoded proteins were also identified, showing general conservation within the phylogenetic subgroups. Promoter analysis revealed many cis-regulatory elements associated with light, hormones, and response mechanisms within the promoter regions of CqAP2/ERF genes. Synteny analysis revealed that segmental duplication under purifying selection pressure was the major evolutionary driver behind the expansion of the CqAP2/ERF gene family. The protein-protein interaction network predicted the pivotal CqAP2/ERF proteins and their interactions involved in the regulation of various biological processes including stress response mechanisms. The expression profiles obtained from RNA-seq and qRT-PCR data detected several salt-responsive CqAP2/ERF genes, particularly from the ERF, DREB, and RAV subfamilies, with varying up- and downregulation patterns, indicating their potential roles in salt stress responses in quinoa. Overall, this study provides insights into the structural and evolutionary features of the AP2/ERF gene family in quinoa, offering candidate genes for further analysis of their roles in salt tolerance and molecular breeding.

Keywords: Chenopodium quinoa; AP2/ERF transcription factors; DREB; Gene Duplication; Regulatory Network; Salinity.

Fig. 1

Phylogenetic tree of the AP2/ERF family members in Chenopodium quinoa (Cq), and A. thaliana (At). ERF, DREB, AP2, RAV, and Soloist subfamilies were presented in different colors.

Fig. 2

The phylogenetic relationships (A), compositions of conserved protein motifs (B), positions of protein domains (C), and the exon-intron structures (D) of quinoa AP2/ERFs.

Fig. 3

Chromosomal distributions of the quinoa AP2/ERF (CqAP2/ERF) genes. The ERF subfamily genes are represented in black; the DREB subfamily genes in blue; the AP2 subfamily genes in green, the RAV subfamily genes in red, and the Soloist subfamily genes in brown.

Fig. 4

Distribution of duplicated CqAP2/ERF genes on quinoa chromosomes. The ERF subfamily genes are represented in black; the DREB subfamily genes in blue; the AP2 subfamily genes in green, the RAV subfamily genes in red, and the Soloist subfamily genes in brown.

Fig. 5

Syntenic relationships between the quinoa CqAP2/ERF genes and their homologs from three other Amaranthaceae species (Beta vulgaris, Amaranthus hypochondriacus, and Spinacia oleracea) and Arabidopsis. thaliana.

Fig. 6

Analysis of cis-acting elements detected within the promoters of CqAP2/ERF. The numbers in the grids indicate the number of cis-regulatory elements related to the specified functions.

Fig. 7

(a) Protein-protein interaction network among CqAP2/ERF proteins predicted based on the Arabidopsis AP2/ERF orthologous proteins using the STRING software (b) The highly interactive CqAP2/ERF proteins, identified and visualized by Cytoscape software.

Fig. 8

Gene ontology (BP: Biological process; CC: Cellular component; MF: Molecular function) and KEGG pathways enriched for CqAP2/ERF genes.

Fig. 9

Heatmap representation of expression profiles of differentially expressed CqAP2/ERF genes in salt-stressed shoots and roots of quinoa.

Fig. 10

qRT-PCR expression profiles of six CqAP2/RRF genes in the leaves and roots of quinoa, collected at 0 (non-stress control), 6, and 24 h after applying salt treatment (300 mM NaCl). Error bars represent the standard deviations of the means for three biological replicates. Different letters over the bars indicate significant differences between mean values according to Tukey’s test (p ≤ 0.05).