Genome-Wide Analysis of the GRAS Gene Family and Functional Identification of GmGRAS37 in Drought and Salt Tolerance

Abstract

GRAS genes, which form a plant-specific transcription factor family, play an important role in plant growth and development and stress responses. However, the functions of GRAS genes in soybean (Glycine max) remain largely unknown. Here, 117 GRAS genes distributed on 20 chromosomes were identified in the soybean genome and were classified into 11 subfamilies. Of the soybean GRAS genes, 80.34% did not have intron insertions, and 54 pairs of genes accounted for 88.52% of duplication events (61 pairs). RNA-seq analysis demonstrated that most GmGRASs were expressed in 14 different soybean tissues examined and responded to multiple abiotic stresses. Results from quantitative real-time PCR analysis of six selected GmGRASs suggested that GmGRAS37 was significantly upregulated under drought and salt stress conditions and abscisic acid and brassinosteroid treatment; therefore, this gene was selected for further study. Subcellular localization analysis revealed that the GmGRAS37 protein was located in the plasma membrane, nucleus, and cytosol. Soybean hairy roots overexpressing GmGRAS37 had improved resistance to drought and salt stresses. In addition, these roots showed increased transcript levels of several drought- and salt-related genes. The results of this study provide the basis for comprehensive analysis of GRAS genes and insight into the abiotic stress response mechanism in soybean.

Keywords: GRAS protein; abiotic stress; genome-wide analysis; hairy root assay; soybean.

Figure 1

Chromosomal distribution of 117 GRAS genes in soybean. The scale bar on the left indicates the size of the chromosomes.

Figure 2

Phylogenetic tree of GRAS proteins from soybean, Arabidopsis, maize, and rice. The complete amino acid sequences of GRAS proteins were aligned by ClustalW, and the phylogenetic tree was constructed using the maximum-likelihood method in MEGA7. The ten groups are represented by different colors.

Figure 3

Multiple sequence alignment of nine GRAS proteins from tomato, rice, Brassica rapa, and soybean. Amino acid sequences were aligned using DNAMAN. Black shading represents 100% amino acid similarity, blue shading represents >75% similarity, and pink shading represents >50% similarity. The red rectangles indicate residues R-E, W-G, and W-W.

Figure 4

Phylogenetic relationships and structures of the 117 GmGRAS proteins. The phylogenetic tree was constructed using MEGA7.0 software; the different classes of GRAS proteins make up separate clades. The schematic diagram indicates the gene structure. Introns and exons are indicated by black lines and yellow boxes, respectively. The lengths of introns and exons of each gene are displayed proportionally.

Figure 5

Putative motifs in each GmGRAS protein. Conserved motifs were identified using MEME and TBtools software. Ten putative motifs are indicated by colored boxes. The length of each protein can be estimated using the scale at the bottom.

Figure 6

Distribution of segmentally duplicated GmGRAS genes on soybean chromosomes. Green lines indicate duplicated GRAS gene pairs.

Figure 7

Histogram of distribution frequency of pairwise Ka/Ks ratios for pairs of homologous genes.

Figure 8

Heat map of the expression profiles of all GmGRAS genes in different soybean tissues. The expression abundance (in log10-based FPKM) of each transcript is represented by the color: red, higher expression; green, lower expression. Expression levels in 14 different tissues are shown: young leaves, flowers, one cm pods, pod shells at different days after flowering (DAF), roots, and nodules.

Figure 9

Heat map of expression profiles of all GmGRAS genes under different abiotic stresses. The expression abundance of each transcript (in log10-based FPKM) is represented by the color: red, higher expression; blue, lower expression; white, miss value.

Figure 10

Expression patterns of GmGRAS genes under drought, salt, abscisic acid (ABA), and brassinosteroid (BR) treatment. (A–D) Expression levels of six GmGRAS genes, as measured using quantitative real-time PCR (qRT-PCR), under drought (A), NaCl (B), ABA (C), and BR (D) treatment applied for 0, 0.5, 1, 2, 4, 8, 12, and 24 h. qRT-PCR data were normalized using the soybean Actin gene (U60506) and are displayed relative to 0 h. The x-axes show the duration of treatment and y-axes depict relative expression level (error bars indicate SD). The data are shown as means of three biology repeats ± SD.

Figure 11

Subcellular localization of GmGRAS37-16318hGFP fusion protein. 35S::GFP was used as a control. The scale bar indicates 10 μm.

Figure 12

Analysis of the function of soybean GmGRAS37 under normal conditions and drought and salt stresses. (A–C) Phenotypes of transgenic soybean hairy root composite GRAS37-RNAi, EV-Control (expressing the pCAMBIA3301 empty vector), and GmGRAS37-overexpression (35S::GRAS37) plants under normal conditions (A), drought stress (B), and salt stress (C). (D–F) The roots of transgenic soybean hairy root composite GRAS37-RNAi, EV-Control, and 35S: GRAS37 plants under normal conditions (A), drought stress (B), and salt stress (C). (G,I) Trypan blue staining of leaves of transgenic soybean hairy root composite GRAS37-RNAi, EV-Control, and 35S: GRAS37 plants under drought (G) and salt stress (I); the dead cells can be strained, but living cells cannot. (H,J) Nitroblue tetrazolium (NBT) staining of the leaves of transgenic soybean hairy root composite GRAS37-RNAi, EV-Control, and 35S: GRAS37 plants under drought (H) and salt stress (J). The intensity of color indicates the concentration of O₂⁻ in the leaves. (K–M) CAT (K), POD (L), and SOD (M) activities of transgenic soybean hairy root composite GRAS37-RNAi, EV-Control, and 35S: GRAS37 plants under drought conditions. (N,O) Chlorophyll content (N) and MDA content (O) of transgenic soybean hairy root composite GRAS37-RNAi, EV-Control, and 35S: GRAS37 plants under drought conditions. (P,Q) The fresh weights of the aerial parts (P) and roots (Q) of transgenic soybean hairy root composite GRAS37-RNAi, EV-Control, and 35S: GRAS37 plants under drought conditions. (R) Relative electrical conductivity of transgenic soybean hairy root composite GRAS37-RNAi, EV-Control, and 35S: GRAS37 plants under drought conditions. Vertical bars indicate ±SD of three replicates. ∗ (p < 0.05) and ∗∗ (p < 0.01) indicate significant differences determined by Student’s t-test.

Figure 13

Expression levels of six stress-responsive genes in transgenic GmGRAS37 soybean hairy root plants under normal conditions, drought stress, and 250 mM NaCl treatment determined by qRT-PCR. Vertical bars indicate ±SD of three replicates. ∗ (p < 0.05) and ∗∗ (p < 0.01) indicate significant differences determined by Student’s t-test.