Comprehensive Genome-Wide Identification and Expression Profiling of bHLH Transcription Factors in Areca catechu Under Abiotic Stress

Abstract

The basic helix-loop-helix (bHLH) transcription factor (TF) family, the second-largest among eukaryotes, is known for its evolutionary and functional diversity across plant species. However, bHLH genes have not yet been characterized in Areca catechu. In this study, we identified 76 AcbHLH genes, which exhibit a variety of physicochemical properties. Phylogenetic analysis revealed evolutionary relationships between Arabidopsis thaliana bHLH genes (AtbHLH) and their counterparts in A. catechu (AcbHLH). These analyses also highlighted conserved amino acid motifs (S, R, K, P, L, A, G, and D), conserved domains, and evolutionary changes, such as insertions, deletions, and exon gains or losses. Promoter analysis of AcbHLH genes revealed 76 cis-elements related to growth, phytohormones, light, and stress. Gene duplication analysis revealed four tandem duplications and twenty-three segmental duplications, while AcbHLH63 in the Areca genome exhibited significant synteny with bHLH genes from A. thaliana, Vitis vinifera, Solanum lycopersicum, Brachypodium distachyon, Oryza sativa, and Zea mays. Furthermore, relative expression analysis showed that under drought stress (DS), AcbHLH22, AcbHLH39, AcbHLH45, and AcbHLH58 showed distinct upregulation in leaves at specific time points, while all nine AcbHLH genes were upregulated in roots. Under salt stress (SS), AcbHLH22, AcbHLH39, AcbHLH45, and AcbHLH58 were upregulated in leaves, and AcbHLH22, AcbHLH34, and AcbHLH39 exhibited differential expression in roots at various time points. This study provides valuable insights into the bHLH superfamily in A. catechu, offering a solid foundation for further investigation into its role in responding to abiotic stresses.

Keywords: Areca catechu; abiotic stresses; bHLH gene family; expression pattern; genome-wide analysis.

Figure 1

A phylogenic tree illustrates the relationship among bHLH domains of A. catechu and A. thaliana. The black color presents (AtbHLH) A. thaliana, and red represents AcbHLH of A. catechu bHLH protein.

Figure 2

Multiple sequence alignments of A. catechu and 24 subgroups A. thaliana. A. catechu is devoid of subgroup 24. The location and boundaries of the bHLH domain are indicated at the top of each subgroup.

Figure 3

Comparative analysis of A. catechu AcbHLH gene phylogeny, structure, and motifs. (A) Phylogeny was inferred based on the NJ method with 1000 bootstrap replicates. (B) Introns and exons are visually represented as yellow and black lines. (C) Amino acid motifs (1–10) are indicated by colored, relative protein lengths represented with black lines.

Figure 4

(A) Cis-regulatory elements in the 2000 bp upstream region of AcbHLH gene promoters. (B) Distribution of cis—regulatory elements of AcbHLH gene members. Colored rectangles visually depict the various cis-acting elements.

Figure 5

(A) Location of 76 AcbHLH genes across 16 A. catechu chromosomes. The left-hand scale indicates chromosomal length. (B) The schematic diagram represents the distribution of A. catechu chromosomes and interchromosomal interaction. Distinct colored lines within the diagram represent gene pairs. Red lines indicate AcbHLH gene pairs. A. catechu are labeled outside the chromosome circles, while chromosome numbers are indicated within.

Figure 6

Gene AcbHLH duplication events. (A) Length distribution of AcbHLHs across five duplication events. (B–F) distribution of Ka, Ks, and Ka/Ks value in duplicated genes across five duplication events. (B–F) DSD, WGD, TRD, TD and PD event, respectively. Further details on the duplicated genes across the five duplication events are provided in Table S9.

Figure 7

(A–G) Comparative synteny analysis of AcbHLH genes between A. catechu and six representative plant species (A. thaliana, V. vinifera, S. lycopersicum, B. distachyon, O. sativa sub sp. indica, Z. mays and C. nucifera). Gray lines show conserved syntenic blocks between A. catechu and other plant genomes. Red lines indicate that AcbHLH pairs of genes are syntenic across species.

Figure 8

Heatmap of AcbHLH gene expression in A. catechu during salt stress (SS) (A) and drought stress (DS) (B). Red indicates larger log2FPKM values, whereas blue indicates lower values.

Figure 9

(A) The presentation of phenotypic changes observed on Day 1 and Day 28 of abiotic stress. (B) The physiological changes observed on Day 1 and Day 28 under abiotic stress (salt and drought): (a) plant height, (b) plant fresh weight, and (c) plant dry weight for CK, SS, and DS groups. Different lowercase letters represent the significant statistical level p < 0.05.

Figure 10

(A–D) The effects of abiotic stress (NaCl and PEG) detected by qRT-PCR on the expression of nine AcbHLH genes in roots and leaves of young A. catechu seedlings (0, 7, 14, 21, and 28 h) (p < 0.05). (E) Heatmap of nine AcbHLH genes. Different letters indicate statistically significant group differences (p < 0.05, LSD).