The Soybean Gene J Contributes to Salt Stress Tolerance by Up-Regulating Salt-Responsive Genes

Abstract

Soybean [Glycine max (L.) Merr.] is an important crop for oil and protein resources worldwide, and its farming is impacted by increasing soil salinity levels. In Arabidopsis the gene EARLY FLOWERING 3 (ELF3), increased salt tolerance by suppressing salt stress response pathways. J is the ortholog of AtELF3 in soybean, and loss-of-function J-alleles greatly prolong soybean maturity and enhance grain yield. The exact role of J in abiotic stress response in soybean, however, remains unclear. In this study, we showed that J expression was induced by NaCl treatment and that the J protein was located in the nucleus. Compared to NIL-J, tolerance to NaCl was significantly lower in the NIL-j mutant. We also demonstrated that overexpression of J increased NaCl tolerance in transgenic soybean hairy roots. J positively regulated expression of downstream salt stress response genes, including GmWRKY12, GmWRKY27, GmWRKY54, GmNAC, and GmSIN1. Our study disclosed a mechanism in soybean for regulation of the salt stress response. Manipulation of these genes should facilitate improvements in salt tolerance in soybean.

Keywords: J; RNA-seq; Soybean; hairy roots; salt tolerance; transcription factor.

FIGURE 1

J gene expression and protein localization. (A) J expression levels in response to NaCl treatment in soybean seedlings as revealed by qRT-PCR analysis. Significant differences were analyzed based on the results of three biological replications (Student’s t test: **P < 0.01). Bars indicate standard error of the mean. The presence of the same lowercase letter above the histogram bars in a–e denotes non-significant differences across the two panels (P > 0.05). (B) J expression in various organs of soybean plants. (C) Subcellular localization of J protein in in tobacco leaf cells. DAPI, fluorescence of 4′,6-diamidino-2-phenylindole; Merge, merge of GFP and DAPI.

FIGURE 2

Phenotype identification of J under NaCl treatment in NIL-J and NIL-j soybean plants. (A) Phenotypes of 14 days NIL-J and NIL-j seedings after planting, treated with 200 mM NaCl for 3 days, Bottom photos: second leaves. N = 12. (B) Fresh weight of NIL-J and NIL-j soybean plants with 0 mM or 200 mM NaCl treatment. (C) Proline contents in NIL-J and NIL-j soybean seedlings under 0 mM or 200 mM NaCl treatment. (D) MDA contents in NIL-J and NIL-j soybean seedlings under 0 mM or 200 mM NaCl treatment. Error bars, s.e.m. Data were analyzed using Student’s t test. NS, not significant. **P < 0.01.

FIGURE 3

Phenotype identification of J under salt treatment in transgenic hairy roots. (A) Phenotypes of transgenic hairy roots expressing either GFP or J with or without NaCl treatment. Photos were taken 2 weeks after treatment. (B) Fresh weight of hairy roots with or without NaCl treatment. N = 12. Error bars, s.e.m. Data were analyzed using Student’s t test. NS, not significant. **P < 0.01.

FIGURE 4

Transcriptomic analysis of NIL-J and NIL-j soybean plant. (A) Numbers of genes showing differential expression between NIL-J and NIL-j soybean plant in non-NaCl-stressed seedings. (B) GO terms that were statistically enriched in differentially expressed genes in NIL-J and NIL-j RNA-seq assay. The numbers near the columns indicate the number of differentially expressed genes. (C,D) The heat map of differential expression of WRKY, bHLH, MYB, and NAC family genes in NIL-J and NIL-j. The numerical values for the blue-to-red gradient bar represent log2-fold change relative to the control sample.

FIGURE 5

J positively regulating the expression of salt stress–tolerant genes in soybean. (A) The transcription levels of GmWRKY12, GmWRKY27, GmWRKY54, GmNAC11, and GmSIN1 in 15-day-old seedlings of NIL-J and NIL-j soybean plant exposed to either 0 mM (mock) or 150 mM NaCl for 6 h; data obtained by qRT-PCR. (B) The transcription levels of GmWRKY12, GmWRKY27, GmWRKY54, GmNAC11, and GmSIN1 in J or GFP (Control) overexpressing soybean hairy root and exposed to either 0 mM (mock) or 150 mM NaCl; data obtained by qRT-PCR. Significant differences were analyzed based on the results of three biological replications (Student’s t-test: **P < 0.01). Bars indicate standard error of the mean.