Genome-wide identification and co-expression network analysis provide insights into the roles of auxin response factor gene family in chickpea

Abstract

Auxin response factors (ARFs) are the transcription factors that regulate auxin responses in various aspects of plant growth and development. Although genome-wide analysis of ARF gene family has been done in some species, no information is available regarding ARF genes in chickpea. In this study, we identified 28 ARF genes (CaARF) in the chickpea genome. Phylogenetic analysis revealed that CaARFs can be divided into four different groups. Duplication analysis revealed that 50% of CaARF genes arose from duplication events. We analyzed expression pattern of CaARFs in various developmental stages. CaARF16.3, CaARF17.1 and CaARF17.2 showed highest expression at initial stages of flower bud development, while CaARF6.2 had higher expression at later stages of flower development. Further, CaARF4.2, CaARF9.2, CaARF16.2 and CaARF7.1 exhibited differential expression under different abiotic stress conditions, suggesting their role in abiotic stress responses. Co-expression network analysis among CaARF, CaIAA and CaGH3 genes enabled us to recognize components involved in the regulatory network associated with CaARFs. Further, we identified microRNAs that target CaARFs and TAS3 locus that trigger production of trans-acting siRNAs targeting CaARFs. The analyses presented here provide comprehensive information on ARF family members and will help in elucidating their exact function in chickpea.

Figure 1

Phylogenetic relationship among ARF proteins from chickpea and Arabidopsis. The deduced full-length amino acid sequences of chickpea (CaARF) and Arabidopsis (AtARF) genes were aligned by MUSCLE and the phylogenetic tree was constructed by MEGA (v7.0) using the neighbour joining (NJ) method. The numbers on the nodes represent bootstrap values from 100 replicates. Grouping of ARFs has been indicated in different colors.

Figure 2

Gene structure and motif organization of ARF genes in chickpea. Left panel illustrates the exon–intron organization of ARF genes. Exons and introns are represented by boxes (blue) and lines (black), respectively. Right panel shows motif organization in ARF proteins. Motifs 1, 2, 9 and 11 represent B3-domain; Motifs 4, 6, 7, 12, 13 and 14 represent ARF-domain; Motifs 8 and 10 represent PB1-domain; Motifs 3 and 5 are unknown novel motifs.

Figure 3

Mapping of duplicated CaARF genes on chickpea chromosomes. Grey ribbons indicate collinear relationship among the blocks in whole genome and blue ribbons show CaARF paralogs. The chickpea chromosomes are arranged in a colored arc and the size of each arc corresponds to the size of respective chromosome (Mb). Genes with black asterisk represent segmentally duplicated genes and red asterisks highlight the tandemly duplicated genes.

Figure 4

Expression profiles of CaARF genes in different tissue/organs. (a) Heatmap showing the normalized RNA-seq expression (FPKM) transformed into Z-score. Hierarchical clustering was conducted in R using the pheatmap package with dissimilarity metrics based on Euclidean distance with complete linkage rule. Color key at the bottom represents row wise Z-score. (b) RT–qPCR analysis of CaARF genes in various tissue/stages of development. Expression of germinating seedling (GS) was taken as a reference to determine relative mRNA level in all other tissues for each gene. Error bars indicate standard error (SE) of mean. GS, germinating seedling; ML, mature leaf; YL, young leaf; SAM, shoot apical meristem; FB1–4, stages of flower bud; FL1–5, stages of flower; YP, young pod.

Figure 5

Expression profiles of CaARF genes under abiotic stress conditions. (a) Heatmap showing differential expression of CaARFs based on RNA-seq data. Color key at the bottom represents log₂ fold change. (b) RT–qPCR analysis of CaARF genes under various stress treatments. Root and shoot control were taken as a reference to determine relative mRNA level under stress conditions. Error bars indicate standard error (SE) of mean. DS, desiccation; SS, salinity; CS, cold stress, CT, control.

Figure 6

Co-expression network analysis of CaARF, CaIAA and CaGH3 genes. (a) Cluster dendrogram showing correlation (0 to 1) between the modules. A high degree of correlation between modules is indicated by dark red color. (b) Module tissue correlation. Each row corresponds to a module. The number of genes in each module is indicated on the left. Each column corresponds to specific tissue labeled below. The color of each cell at row-column intersection indicates the correlation coefficient from −1 to 1. A high degree of correlation between a specific module and the tissue-type is indicated by dark red.

Figure 7

Co-expression modules and network of CaARF, CaIAA and CaGH3 genes. Left panel shows heatmap depicting the expression of all genes included in each module. Color range from blue (lowest expression) to yellow (highest expression) represent expression level. Right panel shows co-expression network of genes in each module. Network is visualized in ViSANT. Spheres (nodes) represent genes, and green and red color lines (edges) represent positive and negative correlation, respectively.

Figure 8

Target sites of tasiRNA in CaARF genes. Diagram showing TAS3 locus with miRNA390 target sites (blue and purple boxes) and tasiARF biogenesis site (red box). tasiARF targets on CaARFs are shown by red lines.