Ncl Synchronously Regulates Na+, K+, and Cl- in Soybean and Greatly Increases the Grain Yield in Saline Field Conditions

Abstract

Salt stress inhibits soybean growth and reduces gain yield. Genetic improvement of salt tolerance is essential for sustainable soybean production in saline areas. In this study, we isolated a gene (Ncl) that could synchronously regulate the transport and accumulation of Na(+), K(+), and Cl(-) from a Brazilian soybean cultivar FT-Abyara using map-based cloning strategy. Higher expression of the salt tolerance gene Ncl in the root resulted in lower accumulations of Na(+), K(+), and Cl(-) in the shoot under salt stress. Transfer of Ncl with the Agrobacterium-mediated transformation method into a soybean cultivar Kariyutaka significantly enhanced its salt tolerance. Introgression of the tolerance allele into soybean cultivar Jackson, using DNA marker-assisted selection (MAS), produced an improved salt tolerance line. Ncl could increase soybean grain yield by 3.6-5.5 times in saline field conditions. Using Ncl in soybean breeding through gene transfer or MAS would contribute to sustainable soybean production in saline-prone areas.

Figure 1. Map based cloning and expression of the salt tolerance gene Ncl.

(a) Fine mapping delimits Ncl to a 16.6-kp region between SSR25.8 and CAPS42.4 on chromosome 3. ^amarker position (kb), ^bmarker position (bp). Red and blue bars represent homozygous chromosome segments for tolerance and sensitivity, respectively. Na⁺, K⁺, and Cl⁻ leaf contents (n = 3) and shoot dry weight (n = 5–8) for each recombinant line after a treatment with 100 mM NaCl for approximately three weeks in a hydroponic condition. Data are presented as means ± s.d. **significant difference at P < 0.01 level versus NILs18-28-14-S (Dunnett’s multiple comparison test). (b) Expression of Ncl analyzed by semi-quantitative RT-PCR in the roots at one and three days after treatment with 100 mM and 0 mM (Control) NaCl in a hydroponic condition. The actin gene was used as a control.

Figure 2. Overexpression of Ncl in transgenic lines enhanced salt tolerance.

(a) Real-time quantitative RT-PCR analysis of Ncl expression levels in the transgenic soybean lines and control materials after treatments with 100 mM NaCl for 24 hours in a hydroponic condition. 54-1-1, 34-2-7, 20-1-4, and 16-1-8 are T₂ 35S:Ncl transgenic lines. Kariyutaka: wild-type soybean cultivar. GFP is 35S:GFP transgenic line (T₆). NILs18-T and NILs18-S are tolerant and sensitive near isogenic lines. Values represent means from three biological replicates. (b,c) Leaf SPAD values and shoot dry weight for the transgenic lines after treatments with 100 mM NaCl for approximately three weeks in a hydroponic condition. (d) Na⁺, K⁺, and Cl⁻ leaf contents for the transgenic lines after treatments with 100 mM NaCl and 0 mM NaCl (Control) for approximately three weeks in a hydroponic condition. (e) Comparison of salt tolerance between 35S:Ncl plants and null plants of the T₂ transgenic line 20-1-4. Data are expressed as means ± s.d. **indicate significant difference at P < 0.01 levels versus from Kariyutaka (wild-type) (Dunnett’s multiple comparison test).

Figure 3

Effect of Ncl on salt tolerance in terms of leave SPAD value (a), shoot dry weight (b), and ion (Na⁺, K⁺, and Cl⁻ leaf contents (c) in BC₄F₃ lines developed by MAS by introducing the tolerance allele from a wild soybean accession JWS156-1 into Jackson. BC₄F₃-J1T and BC₄F₃-J1S was derived from progeny of self-pollination of a BC₄F₂ plant, which was heterozygous in the Ncl region. (d) Performance of BC₄F₃-J1T and BC₄F₃-J1S after treatment with 100 mM NaCl for approximately three weeks in a hydroponic condition. Data are shown as mean ± s.d. (n = 11). **indicate significant difference at P < 0.01 levels versus Jackson (Dunnett’s multiple comparison test).

Figure 4. Yield performances of lines carrying Ncl in a saline field condition in Miyagi Prefecture, Japan.

(a) Top view of NILs25-S and NILs25 T grown in a salt stress field in 2009. (b) Top view of N18-61 and N18-99 grown in a salt stress field in 2012. (c) Grain yield result of the three sets of NILs in 2011 field test. (d) Grain yield result of six recombinant lines derived from an F₈ residual heterozygous line (RHL18-28) in 2012 field test. N18-39, N18-99, and N18-122 are lines carrying tolerant allele of Ncl, whereas N18-9, N18-61, N18-180 are lines that had the sensitive allele. (e) Na⁺, K⁺, and Cl⁻ leaf contents for the three sets of NILs in 2011 field test. Data are shown as mean ± s.d. from three replicates. **Significant difference (P < 0.01) based on ANOVA (Tukey’s multiple comparison test).