The Ankyrin-Repeat Gene GmANK114 Confers Drought and Salt Tolerance in Arabidopsis and Soybean

Abstract

Ankyrin repeat (ANK) proteins are essential in cell growth, development, and response to hormones and environmental stresses. In the present study, 226 ANK genes were identified and classified into nine subfamilies according to conserved domains in the soybean genome (Glycine max L.). Among them, the GmANK114 was highly induced by drought, salt, and abscisic acid. The GmANK114 encodes a protein that belongs to the ANK-RF subfamily containing a RING finger (RF) domain in addition to the ankyrin repeats. Heterologous overexpression of GmANK114 in transgenic Arabidopsis improved the germination rate under drought and salt treatments compared to wild-type. Homologous overexpression of GmANK114 improved the survival rate under drought and salt stresses in transgenic soybean hairy roots. In response to drought or salt stress, GmANK114 overexpression in soybean hairy root showed higher proline and lower malondialdehyde contents, and lower H₂O₂ and O^2- contents compared control plants. Besides, GmANK114 activated transcription of several abiotic stress-related genes, including WRKY13, NAC11, DREB2, MYB84, and bZIP44 under drought and salt stresses in soybean. These results provide new insights for functional analysis of soybean ANK proteins and will be helpful for further understanding how ANK proteins in plants adapt to abiotic stress.

Keywords: ankyrin repeat protein; drought and salt tolerance; genome-wide analysis; responsive mechanism; soybean.

FIGURE 1

Distribution of 223 GmANK genes on the soybean chromosomes. (A) The physical location of each member in soybean. The scale on the left is in megabases (Mb). The chromosome number (Chr1–Chr20) is indicated at the top of each chromosome. The tandem duplicated gene clusters are marked by the red rectangle. (B) The numbers of ANK genes were distributed on 20 chromosomes.

FIGURE 2

Distribution of segmentally duplication ANK genes on soybean chromosomes. Gray lines indicate all synteny blocks in whole soybean genome, and red lines indicate duplicated ANK gene pairs.

FIGURE 3

Domain compositions of representative GmANK proteins from each subfamily. The subfamily name of each corresponding protein and gene ID of a representative protein of the family are given on the left. Different domains are indicated with different colors and abbreviations. The length, order and number of domains represent the actual situation in each protein. Domain abbreviations are: ANK, ankyrin repeat domain; TM, transmembrane; TPR, tetratricopeptide repeat domain; RING, ring finger domain; ZnF-C3H1, zinc finger; BTB, BTB/POZ domain; CG-1, calmodulin-binding transcription activator; IQ, calmodulin-binding domain; PH, pleckstrin homology domain; ArfGap, putative GTP-ase activating proteins for the small GTPase; AAA, ATPase family associated with various cellular activities; FYVE, FYVE zinc finger; cNMP, cyclic nucleotide-binding domain; CHROMO, chromatin organization modifier domain.

FIGURE 4

Phylogenetic analyses of ANK proteins in soybean. The complete amino acid sequences of the 226 GmANK proteins were aligned via Clustal X and were manually corrected. The phylogenetic tree was constructed with MEGA 7 in conjunction with the neighbor-joining method. This tree is drawn to scale with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Six discrete groups (Cluster I–VI) were highlighted in different colors.

FIGURE 5

Heat map of expression profiles (in log2-based FPKM) of soybean ANK-RF subgroup genes in different tissues (root, root hairs, stem, leaf, nodules, flower and seed). The gene names are on the left and the tissue names are on the top of the figure, and the expression abundance of each transcript is represented by the color bar: Red, higher expression; blue, lower expression.

FIGURE 6

Transcriptome analysis-based soybean RNA sequencing data under drought, NaCl, and ABA. The expression abundance of each transcript is represented by the color bar: Red, higher expression; blue, lower expression.

FIGURE 7

qRT-PCR analysis of 17 soybean ANK-RF genes under drought, NaCl and ABA treatments. The expression levels were normalized to that of CYP2. The mean and SD calculated from three biological replicates.

FIGURE 8

Overexpression of GmANK114 enhanced seed germination rates under PEG6000 treatment. (A) Seed germination analysis of different lines seeds under 6% and 9% PEG6000 to simulated drought treatment. (B–D) Seed germination rates of WT and GmANK114 transgenic Arabidopsis seeds at different time points. Date for each time point are means of three independent replicates.

FIGURE 9

Germination rates of seeds in the presence or absence of NaCl in transgenic Arabidopsis. Values are means from three experiments. (A) Seed germination analysis of different lines seeds under 75, 100 and 125 mM NaCl to simulated salt treatment. (B–E) Seed germination rates of WT and GmANK114 transgenic Arabidopsis seeds at different time points. Date for each time point are means of three independent replicates. The error bars indicate SD.

FIGURE 10

GmANK114 improves drought stress tolerance in transgenic soybean hairy roots. (A) Phenotypes of GmANK114-overexpression and 3301-control transgenic soybean plants under before and after drought treatment. (B) DAB (top) and NBT (bottom) staining of OE and 3301 plant leaves under drought treatments. (C) Relative GmANK114 expression in hairy roots of overexpressing GmANK114 and control as shown by qRT-PCR. (D) Survival rate of normal and drought-stressed plants. (E) MDA and (F) proline content were detected in OE and 3301 plants under drought or normal growth condition. (G)The content of H₂O₂ and (H) O^2– in the leaves of GmANK114-OE and 3301-control plants after drought or normal condition for one week. The data were means ± SDs of three experiments. ANOVA test demonstrated that OE were significant differences (*P < 0.05 and **P < 0.01) compared with the corresponding controls.

FIGURE 11

Phenotype and physiological indexes analysis of GmANK114 transgenic soybean hairy roots under salt stress. (A) Phenotypes was evaluated in transgenic soybean plants after NaCl treatment. (B) DAB (top) and NBT (bottom) staining of leaves of OE and 3301 plants under NaCl treatments. (C) Survival rate of soybean hairy roots grown with salt treatment. (D–G) MDA, proline, H₂O₂ and O^2– contents in leaves of plants with transgenic hairy roots were measured under salt treatment for 7 days. All values are represented means for three biological replicates (n = 18). The error bars indicate SD. Significant differences (*P < 0.05 and **P < 0.01) are indicated by asterisks above the columns.

FIGURE 12

The relative transcript levels of WRKY13, NAC11, DREB3, MYB84, bZIP1 and bZIP44 in transgenic GmANK114 and 3301 soybeans. qRT-PCR was used to detect expression levels under normal and stress treatment and GmCYP2 was used as an internal control. Values are means and SD obtained from three biological replicates. The asterisks indicate a statistical significance (*P < 0.05 and **P < 0.01) compared with the corresponding controls.