A soybean sodium/hydrogen exchanger GmNHX6 confers plant alkaline salt tolerance by regulating Na+/K+ homeostasis

Abstract

Alkaline soil has a high pH due to carbonate salts and usually causes more detrimental effects on crop growth than saline soil. Sodium hydrogen exchangers (NHXs) are pivotal regulators of cellular Na⁺/K⁺ and pH homeostasis, which is essential for salt tolerance; however, their role in alkaline salt tolerance is largely unknown. Therefore, in this study, we investigated the function of a soybean NHX gene, GmNHX6, in plant response to alkaline salt stress. GmNHX6 encodes a Golgi-localized sodium/hydrogen exchanger, and its transcript abundance is more upregulated in alkaline salt tolerant soybean variety in response to NaHCO₃ stress. Ectopic expression of GmNHX6 in Arabidopsis enhanced alkaline salt tolerance by maintaining high K⁺ content and low Na⁺/K⁺ ratio. Overexpression of GmNHX6 also improved soybean tolerance to alkaline salt stress. A single nucleotide polymorphism in the promoter region of NHX6 is associated with the alkaline salt tolerance in soybean germplasm. A superior promoter of GmNHX6 was isolated from an alkaline salt tolerant soybean variety, which showed stronger activity than the promoter from an alkaline salt sensitive soybean variety in response to alkali stress, by luciferase transient expression assays. Our results suggested soybean NHX6 gene plays an important role in plant tolerance to alkaline salt stress.

Keywords: abiotic stress; alkaline salt tolerance; natural variation; promoter; sodium bicarbonate; sodium hydrogen exchanger; soybean.

Figure 1

Phylogenetic analysis and subcellular localization of GmNHX6. (A) Phylogenetic analysis of GmNHX6 (XP_028181010.1) from soybean Glycine max and NHX proteins from Arabidopsis thaliana including AtNHX1 (NP_198067.1), AtNHX2 (NP_187154.1), AtNHX3 (NP_200358.1), AtNHX4 (NP_187288.2), AtNHX5 (NP_175839.2), AtNHX6 (NP_178079.2), AtNHX7 (NP_178307.2), AtNHX8 (NP_172918.2). The sequences of NHX proteins were downloaded from the Phytozome database. Unrooted phylogenetic tree was constructed using MEGA6.0 based on the Maximum Likelihood algorithm with 1,000 bootstraps. (B) Subcellular localization of GmNHX6. GFP protein and GmNHX6-GFP fusion protein expressed under the control of CaMV 35S promoter in Arabidopsis protoplasts were observed under a confocal microscope. Bar, 10 μm. (C) Images of Arabidopsis mesophyll protoplasts co-expressing GmNHX6-GFP fusion protein and a mCherry Golgi apparatus marker (Man1-mCherry). Bars = 10 μm.

Figure 2

Phenotype and relative expression of GmNHX6 in two soybean varieties in response to alkali stress. (A) Phenotypes of two soybean varieties at 0, 24, 48, and 72 h after 90 mM NaHCO₃ treatment. The 12-day-old seedlings were subjected to treatment. Bar = 5 cm. (B,C) Relative expression of GmNHX6 in response to alkali stress in roots and leaves, respectively. Soybean seedlings were treated with 0 or 90 mM NaHCO₃ for 3, 6, 9, 12, 24, 48, and 72 h. Roots and leaves receiving 0 mM NaHCO₃ treatment at each time point were used as controls. Data represents the mean ± standard deviation of three biological replications with three repeats within each replication (n = 3 × 3 = 9). Differences between two soybean varieties were evaluated using the two-tailed Student’s t-tests (n.s., not significant; ^*p < 0.05; ^**p < 0.01).

Figure 3

Alkaline salt tolerance analyses of transgenic soybean composite plants. (A) Identification of positive transgenic soybean lines by green fluorescence using a stereoscopic fluorescence microscope. Bar = 1 cm. (B) Representative phenotype of transgenic soybean composite plants (25-day-old) under 0 mM or 90 mM NaHCO₃ for 7 days. Bar = 12 cm. (C) Relative expression of GmNHX6 gene in the roots of soybean composite plants at 24 h after 0 or 90 mM NaHCO₃ treatment by qRT-PCR. For relative expression calculation, the sample from soybean composite plants with empty vector (35S:GFP) under 0 mM NaHCO₃ was used as control and GmUKN1 was the reference gene. Data represents the mean ± standard deviation of three biological replications with three repeats within each replication (n = 3 × 3 = 9). Bars with the same letter in lowercase above bars indicate no significant difference according to Duncan’s multiple range test at 0.05 level. (D,E) The Soil and Plant Analysis Development (SPAD) value for chlorophyll content, and leaf relative water content (LRWC) of transgenic soybean composite plants under 0 mM or 90 mM NaHCO₃ for 7 days, respectively. Data represents the mean ± standard deviation of three biological replications and each repeat contained five independent transgenic plants for each genotype (n = 3 × 5 = 15). Soybean variety of “TianLong1” was used. Differences were evaluated using the two-tailed Student’s t-tests (n.s., not significant; ^**p < 0.01).

Figure 4

Effect of GmNHX6 overexpression on Arabidopsis tolerance to NaHCO₃ treatment. (A) Seed germination assay of different Arabidopsis lines on 1/2 MS medium and 1/2 MS supplied with 8 mM NaHCO₃. Photographs were taken 7 days after NaHCO₃ treatment. WT: wild type; OE-1, OE-18, OE-9: Arabidopsis lines overexpressing GmNHX6. Bar = 1.5 cm. (B) Seed germination rates of different Arabidopsis lines as shown in A. Data represents the mean ± standard deviation of three biological replications and each repeat contained 36 seeds per line for each treatment (n = 36 × 3 = 108). (C) Phenotypes of Arabidopsis lines subjected to 0 and 8 mM NaHCO₃ treatment, respectively. Seeds were grown vertically on 1/2 MS agar plates supplemented with 0 or 8 mM NaHCO₃ for 10 days after seven days of normal growth. Bar = 1.5 cm. (D,E) Fresh weight and root length of Arabidopsis lines as shown in (C). Data represents the mean ± standard deviation of three biological replications and each repeat contained four plants per line for each treatment (n = 12). Bars with the same letter in lowercase above bars indicate no significant differences at the 0.05 level between lines under same treatment according to Duncan’s multiple range tests.

Figure 5

Na⁺ content (A), K⁺ content (B), and Na⁺/K ⁺ ratio (C) in the leaves and roots of Arabidopsis lines. Arabidopsis lines receiving 0 mM or 8 mM NaHCO₃ treatment for 10 days, respectively. Data represents the mean ± standard deviation of three biological replications and each repeat contained 10 plants per line for each treatment (n = 30). Bars with the same letter in lowercase above bars indicate no significant differences at the 0.05 level between lines under same treatment according to Duncan’s multiple range tests.

Figure 6

Natural variation and activity of soybean NHX6 promoter. (A) Boxplot of STR for two alleles of SNP₋₅₆₀ in GmNHX6 promoter among 60 soybean accessions. n denotes number of accessions. Statistical significance was detected by two-sided Wilcoxon test. The center bold line represents the median; box edges indicate the upper and lower quantiles; whiskers show the 1.5 × interquartile range. STR: sodic (alkaline salt) tolerance rating. (B,C) Promoter activities of two types of GmNHX6 promoters by luciferase (LUC) transient expression assays in tobacco leaves after alkaline salt (100 mM NaHCO₃) treatment for 16 h. Pro-M8206 contains SNP₋₅₆₀-C and Pro-ZY contains SNP₋₅₆₀-T. The LUC reporter gene was driven by each type of promoter. The photos were taken using an in vivo plant imaging system. Bar = 3 cm. Data represents the mean ± standard deviation of four biological replications with two repeats within each replication (n = 4 × 2 = 8). Differences were evaluated using the two-tailed Student’s t-tests (^**p < 0.01; n.s., not significant).

Figure 7

The effect of GmNHX6 overexpression on the salt tolerance of Arabidopsis. (A) Seed germination assay of different Arabidopsis lines on 1/2 MS medium supplied with 0 or 150 mM NaCl. Photographs were taken 7 days after treatment. WT: wild type; OE-1, OE-18, OE-9: Arabidopsis lines overexpressing GmNHX6. Bar = 1.5 cm. (B) Seed germination rates of different Arabidopsis lines as shown in A. Data represents the mean ± standard deviation of three biological replications and each repeat contained 36 seeds per line for each treatment (n = 36 × 3 = 108). (C) Arabidopsis seeds were grown vertically on 1/2 MS agar plates supplemented with 0 or 150 mM NaCl treatment for 10 days after 7 days of normal growth, respectively. Bar = 1.5 cm. (D,E) The fresh weight and root length of Arabidopsis lines shown in (C). Data represents the mean ± standard deviation of three biological replications and each repeat contained four plants per line for each treatment (n = 12). (F) Na⁺ content, (G) K ⁺ content, (H) Na⁺/K ⁺ ratio in the leaves and roots of Arabidopsis lines shown in (C). For (F–H), data represents the mean ± standard deviation of three biological replications and each replication has four repeats (n = 12), and 10 plants were pooled together for each sample. Bars with the same letter in lowercases above bars indicate no significant differences at the 0.05 level between lines under each condition according to Duncan’s multiple range test.