Genome-wide survey and comprehensive expression profiling of Aux/IAA gene family in chickpea and soybean

Abstract

Auxin plays a central role in many aspects of plant growth and development. Auxin/Indole-3-Acetic Acid (Aux/IAA) genes cooperate with several other components in the perception and signaling of plant hormone auxin. An investigation of chickpea and soybean genomes revealed 22 and 63 putative Aux/IAA genes, respectively. These genes were classified into six subfamilies on the basis of phylogenetic analysis. Among 63 soybean Aux/IAA genes, 57 (90.5%) were found to be duplicated via whole genome duplication (WGD)/segmental events. Transposed duplication played a significant role in tandem arrangements between the members of different subfamilies. Analysis of Ka/Ks ratio of duplicated Aux/IAA genes revealed purifying selection pressure with restricted functional divergence. Promoter sequence analysis revealed several cis-regulatory elements related to auxin, abscisic acid, desiccation, salt, seed, and endosperm, indicating their role in development and stress responses. Expression analysis of chickpea and soybean Aux/IAA genes in various tissues and stages of development demonstrated tissue/stage specific differential expression. In soybean, at least 16 paralog pairs, duplicated via WGD/segmental events, showed almost indistinguishable expression pattern, but eight pairs exhibited significantly diverse expression patterns. Under abiotic stress conditions, such as desiccation, salinity and/or cold, many Aux/IAA genes of chickpea and soybean revealed differential expression. qRT-PCR analysis confirmed the differential expression patterns of selected Aux/IAA genes in chickpea. The analyses presented here provide insights on putative roles of chickpea and soybean Aux/IAA genes and will facilitate elucidation of their precise functions during development and abiotic stress responses.

Keywords: Aux/IAA; abiotic stress; chickpea; gene duplication; gene expression; gene family; soybean; transposed duplication.

FIGURE 1

Phylogenetic relationship among Aux/IAA genes from chickpea, soybean, and Arabidopsis. The deduced full-length amino acid sequences of chickpea (CaIAA), soybean (GmIAA), and Arabidopsis (AtIAA) genes were aligned by MAFFT and the phylogenetic tree was constructed by CLC Genomics Workbench using the UPGMA method. The members of each Aux/IAA subfamily are shown in different colors. The numbers on the nodes represent bootstrap values from 1000 replicates.

FIGURE 2

Gene structure and motif organization of Aux/IAA family members in chickpea and soybean. Left panel illustrates the exon–intron organization of Aux/IAA genes in chickpea and soybean. The exons and introns are represented by boxes and lines, respectively. Right panel shows motif organization in chickpea and soybean Aux/IAA proteins. Motifs of Aux/IAA proteins were investigated by MEME web server. Six motifs representing four domains I, II, III, and IV of Aux/IAA proteins are displayed at the bottom.

FIGURE 3

Mapping of segmentally duplicated GmIAA genes on soybean chromosomes. Gray ribbons indicate collinear relationships among the blocks in whole genome and blue ribbons show GmIAA paralogs. The soybean chromosomes are arranged in a red circle and the size of each arc show the size of respective chromosome (Mb).

FIGURE 4

Expression profiles of chickpea Aux/IAA genes. (A) The normalized RNA-seq expression data was used to construct heatmap. Hierarchical clustering was conducted in R using the pheatmap package with a dissimilarity metrics based on Euclidean distance with complete linkage algorithm. Color key at the bottom represents row wise Z-score. (B) qRT–PCR analysis of CaIAA genes in various tissue/stages of development. Expression of germinating seedling (GS) was taken as a reference to determine relative mRNA level in other tissues for each gene. Error bars indicate SE of mean. R (root), S (shoot), ST (stem) ML (mature leaf), SAM (shoot apical meristem), FB1-4 (stages of flower bud), FL1-5 (stages of flower), YP (young pod). Data points marked with asterisk (^∗P ≤ 0.05, ^∗∗P ≤ 0.01, and ^∗∗∗P ≤ 0.001) indicates statistically significant difference between control (GS) and other tissues.

FIGURE 5

Expression profiles and the evolutionary patterns of soybean Aux/IAA genes. The normalized RNA-seq expression data of 19 tissues was used to construct heatmap. Three samples from soybean seed compartments: GloEP (globular stage embryo proper), EmSCP (early maturation seed coat parenchyma), and GloS (globular stage suspensor); 10 other tissues samples: Gs (globular stage seed), Hs (heart stage seed), Cs (cotyledon stage seed), Es (early maturation stage seed), Ds (dry Seed), R (root), ST (stem), L (trifoliate leave), FB (floral bud), and WS (whole seedling 6 days after imbibition); three cotyledon development samples: CoM (mid-maturation cotyledon), CoL (late maturation cotyledon), and CoS (seedling cotyledon); three early maturation seed parts: EcoEm (early maturation embryonic cotyledon), EmEA (early maturation embryonic axis), and EmSC (early maturation seed coat). The lines show the syntenic blocks containing the corresponding GmIAA genes, which experienced the WGD events. Gene names in red show dispersed duplicates. Color key at the bottom represents row wise Z-score.

FIGURE 6

Expression profiles of CaIAA and GmIAA genes under abiotic stress conditions. (A) Heatmap shows differential expression of CaIAA genes based on RNA-seq data. (B) qRT–PCR analysis of CaIAA genes under various stress treatments. Root and shoot control (CTR) was taken as a reference to determine relative mRNA level under stress conditions. Error bars indicate standard error of mean. Data points marked with asterisk (^∗P ≤ 0.05, ^∗∗P ≤ 0.01, and ^∗∗∗P ≤ 0.001) indicate statistically significant difference between control and stress treatments. (C,D) Differential expression of GmIAA genes in response to drought and salinity stress conditions. Color scale shows log₂ fold change relative to control sample. DS (desiccation), SS (salinity), CS (cold stress), V6 (vegetative stage leaves), R2 (reproductive stage leaves).