Identification and Characterization of the AREB/ABF Gene Family in Three Orchid Species and Functional Analysis of DcaABI5 in Arabidopsis

Abstract

AREB/ABF (ABA response element binding) proteins in plants are essential for stress responses, while our understanding of AREB/ABFs from orchid species, important traditional medicinal and ornamental plants, is limited. Here, twelve AREB/ABF genes were identified within three orchids' complete genomes and classified into three groups through phylogenetic analysis, which was further supported with a combined analysis of their conserved motifs and gene structures. The cis-element analysis revealed that hormone response elements as well as light and stress response elements were widely rich in the AREB/ABFs. A prediction analysis of the orchid ABRE/ABF-mediated regulatory network was further constructed through cis-regulatory element (CRE) analysis of their promoter regions. And it revealed that several dominant transcriptional factor (TF) gene families were abundant as potential regulators of these orchid AREB/ABFs. Expression profile analysis using public transcriptomic data suggested that most AREB/ABF genes have distinct tissue-specific expression patterns in orchid plants. Additionally, DcaABI5 as a homolog of ABA INSENSITIVE 5 (ABI5) from Arabidopsis was selected for further analysis. The results showed that transgenic Arabidopsis overexpressing DcaABI5 could rescue the ABA-insensitive phenotype in the mutant abi5. Collectively, these findings will provide valuable information on AREB/ABF genes in orchids.

Keywords: ABA INSENSITIVE 5 (ABI5); AREB/ABF genes; abscisic acid (ABA); cis-regulatory element (CRE) analysis; gene expression; gene family; orchid.

Figure 1

Multiple-sequence alignment of AREB/ABF members from D. catenatum, A. shenzhenica, and P. equestris. The positions of C1 to C4 conserved domains and basic bZIP regions are represented with different colors. Potential phosphorylated residues (R-S-SX/T) of the characteristic phosphorylation sites are indicated with dash boxes and red stars.

Figure 2

Phylogenetic analysis of the AREB/ABF proteins. The diverse groups of the AREB/ABF proteins are indicated with different colored arcs. Proteins from D. catenatum, A. shenzhenica, P. equestris, rice (Oryza sativa), and Arabidopsis are indicated using black stars, blue triangle, yellow squares, purple circles, and red stars, respectively.

Figure 3

Architecture of conserved motifs and gene structures of AREB/ABFs. (A) The neighbor-joining phylogenetic tree was produced using MEGA using the neighbor-joining method with 1000 bootstrap replicates. Schematic represents the conserved motifs of the AREB/ABFs identified using MEME. Each motif is indicated by a colored box, number, and sequence. (B) Intron/exon structures of AREB/ABF genes. Exon(s), intron(s), and UTR(s) are represented with yellow boxes, black lines, and blue arrows, respectively.

Figure 4

Analysis of cis-elements in the promoter regions of AREB/ABF genes from orchids. (A) The distribution of cis-elements to each AREB/ABF gene. Different colored blocks represent the corresponding cis-elements. (B) Evaluation of cis-elements of each AREB/ABF gene. The number of individual elements is indicated with a colorful circle.

Figure 5

The putative TF regulatory network analysis of AREB/ABFs from orchid plants. (A) An overview of the cluster of enriched TF regulators for all AREB/ABF genes. Highly enriched TFs are indicated with dash boxes. (B) The distribution of TF regulators for individual AREB/ABF genes using pie charts and word clouds. The font size is positively correlated with the number of corresponding TF regulators. (C) Venn diagram showing the overlapping TF regulators among AREB/ABFs from three orchid genomes.

Figure 6

Expression pattern analysis for orchid AREB/ABF gene family. (A–C) Expression profiles of the AREB/ABF genes in different tissues/organs from indicated orchid species: D. catenatum (A), A. shenzhenica (B), and P. equestris (C). The heat map was constructed from the transcriptome data using TBtools-II with the log2-transformed RPKM values of each gene. The expression level was shown in color as the scale. (D) Expression patterns of DcaABI5 in indicated tissues (specifically expressed in orchid pollinium highlighted with yellow asterisk). DcaActin7 was used as the control. Three independent biological experiments, each with three technical replicates, were performed.

Figure 7

DcaABI5 is able to rescue ABI5 mutation in Arabidopsis. (A) Confocal microscopy images for the subcellular localization of DcaABI5 in tobacco leaf. (B) Representative images for 7-day-old seedlings of Col-0, abi5, DcaABI5 overexpression lines in WT (DcaABI5^OE) and abi5 (DcaABI5^OE/abi5) germinated on half MS medium supplemented without or with 0.5 μM ABA. (C) Statistical analysis of seed germination and greening cotyledon percentages of the various genotypes in response to ABA. Seed germination was recorded after 3 days of stratification, and cotyledon greening was recorded 7 days after stratification on half MS medium supplemented without or with 0.5 μM ABA. Data indicate mean ± SD (n = 3) with at least 100 seeds for each replicate of each genotype. (D,E) DcaABI5 regulated expression of stress-responsive genes. RT-qPCR analysis was performed to examine the relative transcript levels of EM1 and RAB18 in indicated plants treated without or with 10 μM ABA. Actin2 was used as the control. Three independent biological experiments were carried out, each with three technical replicates. Bars with different letters indicate significant differences from the control as determined using one-way ANOVA, p-value < 0.05.