GmANKTM21 Positively Regulates Drought Tolerance and Enhanced Stomatal Response through the MAPK Signaling Pathway in Soybean

Abstract

Drought stress is one of the significant abiotic stresses that limit soybean (Glycine max [L.] Merr.) growth and production. Ankyrin repeat (ANK) proteins, being highly conserved, occupy a pivotal role in diverse biological processes. ANK genes were classified into nine subfamilies according to conserved domains in the soybean genome. However, the function of ANK-TM subfamily proteins (Ankyrin repeat proteins with a transmembrane domain) in the abiotic-stress response to soybean remains poorly understood. In this study, we first demonstrated the subcellular localization of GmANKTM21 in the cell membrane and nucleus. Drought stress-induced mRNA levels of GmANKTM21, which encodes proteins belonging to the ANK-TM subfamily, Transgenic 35S:GmANKTM21 soybean improved drought tolerance at the germination and seedling stages, with higher stomatal closure in soybean, lower water loss, lower malondialdehyde (MDA) content, and less reactive oxygen species (ROS) production compared with the wild-type soybean (Dongnong50). RNA-sequencing (RNA-seq) and RT-qPCR analysis of differentially expressed transcripts in overexpression of GmANKTM21 further identified potential downstream genes, including GmSPK2, GmSPK4, and GmCYP707A1, which showed higher expression in transgenic soybean, than those in wild-type soybean and KEGG enrichment analysis showed that MAPK signaling pathways were mostly enriched in GmANKTM21 overexpressing soybean plants under drought stress conditions. Therefore, we demonstrate that GmANKTM21 plays an important role in tolerance to drought stress in soybeans.

Keywords: Glycine max; GmANKTM21; ankyrin repeat protein; drought stress.

Figure 1

Subcellular localization of GmANKTM21 in tobacco leaf lower epidermal cells. Green fluorescent protein (GFP) fluorescence, fluorescence, bright field images, and merged images are displayed from left to right. Fluorescence was observed by confocal microscopy. Scale bar is 50 μm.

Figure 2

Germination of GmANKTM21-OE (Line2 and Line13) and wild-type (DN50) seeds under different concentrations of PEG6000. (A) T-DNA region map of recombinant vector; (B) RT-qPCR detection of GmANKTM21 gene expression level in soybean overexpression plants. Values indicate the means ± SD (n = 3); (C) Germination phenotype of soybean seeds under drought; (D) Hypocotyl length; (E) Plant fresh weight, values indicate the means ± SD (n = 6); (F) Germination rate; (G) Germination potential. Values indicate the means ± SD (n = 6); the differences were statistically assessed using the Student’s t test; asterisks indicate significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001) compared to those of the control samples (DN50).

Figure 3

Seedling phenotype of soybeans with overexpression of the GmANKTM21 gene under different drought simulation conditions. (A) PEG600 treatment; (C) Water cut-off treatment and restoration; (B,D) Leaf wilting rate. Values indicate the means ± SD (n = 6); asterisks indicate significant differences (*** p < 0.001) compared to that of the control samples (DN50).

Figure 4

Water retention ability of plants overexpressing the GmANKTM21 gene. (A) Leaf wilting phenotype; (B) Water loss rate; values indicate the means ± SD (n = 6); (C) Stomatal phenotype before and after drought treatment; (D) Stomatal opening. In total, 100 stomata were evaluated in each replicate; (E) Stomatal density; (F) Wax observation; (G) Toluidine blue staining. Values indicate the means ± SD (n = 6). The differences were statistically assessed using the Student’s t test; asterisks indicate significant differences (* p < 0.05, ** p < 0.01) compared to those of the control samples (DN50).

Figure 5

Analysis of physiological parameters of different soybean plants under drought treatment. (A) CAT activity; (B) POD activity; (C) SOD activity; (D) MDA content; (E) NBT staining; (F) DAB dyeing. Values indicate the means ± SD (n = 6). The differences were statistically assessed using the Student’s t test; asterisks indicate significant differences (** p < 0.01, *** p < 0.001) compared to those of the control samples (DN50).

Figure 6

Analysis of the KEGG pathway and verification of drought tolerance genes. (A) KEGG pathway analysis of DEGs of GmANKTM21-OE and DN50 under the drought treatment; (B–G) Analysis of expression patterns of selected genes under the drought treatment by RT-qPCR. Values indicate the means ± SD (n = 3). The differences were statistically assessed using the Student’s t test; asterisks indicate significant differences (** p < 0.01, *** p < 0.001) compared to those of the control samples (DN50).