Genome-wide analysis and expression profiling suggest diverse roles of GH3 genes during development and abiotic stress responses in legumes

Abstract

Growth hormone auxin regulates various cellular processes by altering the expression of diverse genes in plants. Among various auxin-responsive genes, GH3 genes maintain endogenous auxin homeostasis by conjugating excess of auxin with amino acids. GH3 genes have been characterized in many plant species, but not in legumes. In the present work, we identified members of GH3 gene family and analyzed their chromosomal distribution, gene structure, gene duplication and phylogenetic analysis in different legumes, including chickpea, soybean, Medicago, and Lotus. A comprehensive expression analysis in different vegetative and reproductive tissues/stages revealed that many of GH3 genes were expressed in a tissue-specific manner. Notably, chickpea CaGH3-3, soybean GmGH3-8 and -25, and Lotus LjGH3-4, -5, -9 and -18 genes were up-regulated in root, indicating their putative role in root development. In addition, chickpea CaGH3-1 and -7, and Medicago MtGH3-7, -8, and -9 were found to be highly induced under drought and/or salt stresses, suggesting their role in abiotic stress responses. We also observed the examples of differential expression pattern of duplicated GH3 genes in soybean, indicating their functional diversification. Furthermore, analyses of three-dimensional structures, active site residues and ligand preferences provided molecular insights into function of GH3 genes in legumes. The analysis presented here would help in investigation of precise function of GH3 genes in legumes during development and stress conditions.

Keywords: GH3 genes; RNA-seq; gene family; homology modeling; legumes; substrate specificities.

FIGURE 1

Exon-intron organization of chickpea and soybean GH3 genes. Boxes and lines represent exons and introns, respectively. The numbers 0, 1, and 2 represent phase 0, 1, and 2 introns, respectively.

FIGURE 2

Phylogenetic relationship among chickpea, soybean, Medicago, Lotus, and Arabidopsis GH3 proteins. Multiple sequence alignment of all GH3 proteins from chickpea (CaGH3), soybean (GmGH3), Medicago (MtGH3), Lotus (LjGH3) and Arabidopsis (AtGH3) was performed and tree was generated by UPGMA method. FigTree was used for visualization of the tree. The value at the nodes represents bootstrap values from 1000 replicates. Different groups of GH3 proteins are labeled.

FIGURE 3

Expression profiles of CaGH3 genes during development. (A) Heatmap showing expression profiles of CaGH3 genes based on RNA-seq data in various tissues/development stages. Heatmap was generated based on log₂ FPKM. (B) Real-time PCR analysis of CaGH3 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. YL, young leaf; ML, mature leaf; FB, flower bud; UOF, unopened flower; FBO, flower bud open; MF, mature flower; YP, young pod. Data points marked with asterisk (*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001) indicate statistically significant difference between control (GS) and other tissues.

FIGURE 4

Expression profiles and gene duplication of GmGH3 genes. (A) Heatmap showing expression profiles of soybean GH3 genes at various stages of development. Heatmap was generated based on log₂ RPKM. (B) Mapping of GmGH3 genes and duplication between them are shown on the soybean chromosomes. Duplication was determined using Plant Genome Duplication Database. Genes and their duplications were mapped on chromosomes using Circos tool. Soybean chromosomes have been arranged in circle and duplications are represented by lines.

FIGURE 5

Expression profiles of CaGH3 genes under abiotic stress conditions. (A) Heatmap showing expression of CaGH3 genes based on RNA-seq data. Heatmap was generated based on log₂ FPKM. (B) Real-time PCR analysis of CaGH3 genes under various stress treatments. Root control (CTR-R) and shoot control (CTR-S) was taken as a reference to determine relative mRNA level under stress conditions. Error bars indicate standard error of mean. DS-R: desiccation stressed root, SS-R, salt stressed root; CS-R, cold stressed root; DS-S, desiccation stressed shoot; SS-S, salt stressed shoot; CS-S, cold stressed shoot. Data points marked with asterisk (*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001) indicate statistically significant difference between control and stress treatments.

FIGURE 6

Predicted structures of GH3 proteins. Ribbon diagram showing the N- and C-terminal domains of chickpea (CaGH3-3 and CaGH3-8) and soybean (GmGH3-8 and GmGH3-25) GH3 protein with α-helices, β-strands and loops colored cyan, magenta, and gold, respectively. Ligands AIEP, JA-Ile, AMP are shown as space-filling model in blue, coral, and green colors, respectively.

FIGURE 7

Hormone and nucleotide binding residues in GH3 proteins. Ribbon diagram showing hormone binding residues in magenta, nucleotide (ATP/AMP) binding residues in yellow, and residues in pink determine amino-acid preferences.

FIGURE 8

Proposed substrates of GH3 proteins based on conserved amino acid residues. Protein sequences of all the identified GH3 genes were aligned using MAFFT. Green and blue boxes represent nucleotide (ATP/AMP) and hormone-binding motifs/residues, respectively. Magenta boxes represent residues determining amino-acid preferences. Only sequences with complete C- and N-terminal domains were included. Star across the top of the alignment indicates conserved residues in pocket forming active site. Numbering at the top corresponds to CaGH3-12.