The bZIP Transcription Factor GmbZIP15 Negatively Regulates Salt- and Drought-Stress Responses in Soybean

Abstract

Soybean (Glycine max), as an important oilseed crop, is constantly threatened by abiotic stress, including that caused by salinity and drought. bZIP transcription factors (TFs) are one of the largest TF families and have been shown to be associated with various environmental-stress tolerances among species; however, their function in abiotic-stress response in soybean remains poorly understood. Here, we characterized the roles of soybean transcription factor GmbZIP15 in response to abiotic stresses. The transcript level of GmbZIP15 was suppressed under salt- and drought-stress conditions. Overexpression of GmbZIP15 in soybean resulted in hypersensitivity to abiotic stress compared with wild-type (WT) plants, which was associated with lower transcript levels of stress-responsive genes involved in both abscisic acid (ABA)-dependent and ABA-independent pathways, defective stomatal aperture regulation, and reduced antioxidant enzyme activities. Furthermore, plants expressing a functional repressor form of GmbZIP15 exhibited drought-stress resistance similar to WT. RNA-seq and qRT-PCR analyses revealed that GmbZIP15 positively regulates GmSAHH1 expression and negatively regulates GmWRKY12 and GmABF1 expression in response to abiotic stress. Overall, these data indicate that GmbZIP15 functions as a negative regulator in response to salt and drought stresses.

Keywords: GmbZIP15; RNA-seq; drought stress; salt stress; soybean; transcription factor.

Figure 1

GmbZIP15 negatively regulates salt-stress resistance in soybean. (A) Phenotype observation of transgenic soybean seedlings in response to salt stress. The pictures were obtained before or after 200 mM NaCl treatment for 2 weeks. Numbers in the panels denote the frequencies of the phenotypes shown. (B) Diaminobenzidine (DAB) staining of the soybean leaves. All the plants were treated with 200 mM NaCl for 4 days and then the leaves were harvested. The depth of color shows the H₂O₂ content in leaves. Bar = 1 cm.

Figure 2

GmbZIP15 negatively regulates drought-stress resistance in soybean. (A) Phenotype observation of transgenic soybean seedlings in response to drought stress. The pictures were obtained under normal conditions; thereafter, the plants were not watered for 2 weeks, then rewatered for 3 days. Numbers in the panels denote the frequencies of the phenotypes shown. (B) DAB staining of the soybean leaves. All the plants were not watered for 4 days and then the leaves were harvested. The depth of color shows the H₂O₂ content in the leaves. Bar = 1 cm.

Figure 3

Changes in the stomatal aperture in GmbZIP15 transgenic soybean plants under salt- and drought-stress conditions. (A,B) Comparison of stomatal aperture with width over length before or after 200 mM NaCl treatment for 1 h (A) or before or after 300 mM mannitol treatment for 1 h (B). Data were calculated from 100 stomata of the leaves of three different soybean plants. The experiments were performed three times with similar results. Bar = 10 μm. Errors bars indicate ± SD of three biological replicates. Significant differences between samples labeled a, b, and c were determined by one-way ANOVA, p < 0.05.

Figure 4

GmbZIP15-overexpressed Arabidopsis is hypersensitive to salt and drought stresses. (A) Phenotype observation of wild-type (WT) and GmbZIP15 transgenic Arabidopsis plants under normal and stress conditions. All the seeds were germinated on the 1/2 Murashige and Skoog Medium (MS) medium under normal conditions or supplemented with 150 mM NaCl or 300 mM mannitol for 1 week. (B) Transcript level detection of GmbZIP15 in transgenic Arabidopsis plants. (C) Quantification of the cotyledon green rate. (D,E) GmbZIP15 regulates stress-responsive gene expression in WT and GmbZIP15 transgenic Arabidopsis plants. Gene expression levels of AtCOR6-6, AtDREB2A, and AtRD29A were quantified by qRT-PCR assays after 150 mM NaCl treatment for 0.12, and 24 h (D). Gene expression levels of AtWRKY33, AtDREB2A, and AtRD29A were quantified by qRT-PCR assays after 300 mM mannitol treatment for 0.12, and 24 h (E). Errors bars indicate ± SD of three biological replicates. Significant differences between samples labeled a, b, and c were determined by one-way ANOVA, p < 0.05.

Figure 5

Transcriptomic analysis of OX-GmbZIP15 transgenic soybean plants. (A) Number of specific and common salt- and drought-responsive differentially expressed genes (DEGs) in the WT and OX-GmbZIP15-16 soybean plants. (B) Number of specific and common DEGs in the OX-GmbZIP15-16 soybean plants after salt and drought-stress treatment. (C,D) gene annotation (GO) analysis of the DEGs downregulated in OX-GmbZIP15-16 soybean plants after salt stress: (C) biological process; (D) molecular function. (E,F) GO analysis of the DEGs downregulated in OX-GmbZIP15-16 soybean plants after drought stress; (E) biological process; (F) molecular function. The numbers next to the columns indicate the number of DEGs with corresponding annotation and the p-value, respectively (C–F).

Figure 6

Phenotypic analysis of GmSAHH1-, GmWRKY12-, and GmABF1-overexpressed Arabidopsis plants in response to salt and drought stresses. (A) Transcript level detection of GmSAHH1, GmWRKY12, and GmABF1 in transgenic Arabidopsis plants. Errors bars indicate ± SD of three biological replicates. Significant differences between samples labeled asterisks were determined by one-way ANOVA, p < 0.05. (B) Growth observation of WT and overexpression of GmSAHH1, GmWRKY12, and GmABF1 Arabidopsis seedlings under either normal conditions or 150-mM-NaCl- and 300-mM-mannitol-supplemented 1/2 MS medium.

Figure 7

A schematic model of GmbZIP15 mediated abiotic-stress tolerance in soybean. GmbZIP15 negatively modulates the abiotic-stress tolerance: GmbZIP15 positively regulates the expression of GmSAHH1 and negatively regulates the expression of GmWRKY12 and GmABF1 in response to abiotic stresses. The arrows indicate induction or positive modulation; the blunt-end arrows represent block or suppression.