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Identification and functional characterization of the chloride channel gene, GsCLC-c2 from wild soybean.
Wei P
,
Che B
,
Shen L
,
Cui Y
,
Wu S
,
Cheng C
,
Liu F
,
Li MW
,
Yu B
,
Lam HM
.
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BACKGROUND: The anionic toxicity of plants under salt stress is mainly caused by chloride (Cl-). Thus Cl- influx, transport and their regulatory mechanisms should be one of the most important aspects of plant salt tolerance studies, but are often sidelined by the focus on sodium (Na+) toxicity and its associated adaptations. Plant chloride channels (CLCs) are transport proteins for anions including Cl- and nitrate (NO3-), and are critical for nutrition uptake and transport, adjustment of cellular turgor, stomatal movement, signal transduction, and Cl- and NO3- homeostasis under salt stress.
RESULTS: Among the eight soybean CLC genes, the tonoplast-localized c2 has uniquely different transcriptional patterns between cultivated soybean N23674 and wild soybean BB52. Using soybean hairy root transformation, we found that GsCLC-c2 over-expression contributed to Cl- and NO3- homeostasis, and therefore conferred salt tolerance, through increasing the accumulation of Cl- in the roots, thereby reducing their transportation to the shoots where most of the cellular damages occur. Also, by keeping relatively high levels of NO3- in the aerial part of the plant, GsCLC-c2 could reduce the Cl-/NO3- ratio. Wild type GsCLC-c2, but not its mutants (S184P, E227V and E294G) with mutations in the conserved domains, is able to complement Saccharomyces cerevisiae △gef1 Cl- sensitive phenotype. Using two-electrode voltage clamp on Xenopus laevis oocytes injected with GsCLC-c2 cRNA, we found that GsCLC-c2 transports both Cl- and NO3- with slightly different affinity, and the affinity toward Cl- was pH-independent.
CONCLUSION: This study revealed that the expression of GsCLC-c2 is induced by NaCl-stress in the root of wild soybean. The tonoplast localized GsCLC-c2 transports Cl- with a higher affinity than NO3- in a pH-independent fashion. GsCLC-c2 probably alleviates salt stress in planta through the sequestration of excess Cl- into the vacuoles of root cells and thus preventing Cl- from entering the shoots where it could result in cellular damages.
31671604 National Natural Science Foundation of China, U1603111 National Natural Science Foundation of China, AoE/M-403/16 Research Grants Council, University Grants Committee, VCF2014004 CUHK VC Discretionary Fund
Fig. 1. Genome-wide chromosomal locations of soybean CLC-homologous genes. Number on the right of the chromosome indicate the position of the gene in megabases (Mb). Max: total length of the chromosome
Fig. 2. Relative expressions of CLC-homologous genes in G. max N23674 and G. soja BB52 15-day-old seedlings. a Under normal conditions. All expressions were normalized to the expression of GmCLC1 in root of N23674. Expression of GmCLC1 in root in N23674 was set as 1 for comparison. b-i Expression of different CLC-homologs under 150 mM NaCl stress for 0, 2, 4, 8, 12, and 24 h. g, b1, b2, c1, c2, d1 and d2 are CLC homologs. The transcript levels have been normalized against those of GmEF1α2a. Expression of the gene in root of N23674 was set as 1 for comparison. The results were presented as mean ± SD (3 ≤ n ≤ 5). Letters indicate groups with statistically significant differences (P ≤ 0.05) using Duncan’s test after one-way ANOVA
Fig. 3. Effects of expressing wild type or mutant GsCLC-c2 on salt-sensitive yeast mutant Δgef1 under salt treatments. a Survival in YPD and YPG media, and YPG media supplemented with 1 M KCl or 1 M NaCl. b Cl− contents in yeast cells under 1 M NaCl treatment. S184P, E227V, E294G, C638F, A746T and I71V/C165Y are mutants with specific amino acid substituted. YPD:1% yeast extract/2% peptone/2% dextrose; YPG:1% yeast extract/2% peptone/2% galactose. Each bar represents mean ± SD of ion contents of three independent cultures of each strain. Letters indicate groups with statistically significant differences (P ≤ 0.05) using Duncan’s test after one-way ANOVA
Fig. 4. Subcellular co-localization of GsCLC-c2-GFP and δ-TIP-RFP recombinant proteins in leaf protoplasts of Arabidopsis seedlings. GsCLC-c2-GFP and δ-TIP-RFP were expressed in Arabidopsis leaf protoplasts under the control of CaMV-35S promoter. δ-TIP-RFP was used as the tonoplast marker
Fig. 5. Comparisons of salt stress-related physiological parameters in GsCLC-c2- and GmCLC1-transgenic soybean hairy root-composite seedlings. a Photo showing the hairy root-composite seedlings with or without NaCl treatment. b Fresh weight, (c) root vigor, (d) leaf area, (e) relative water content (RWC) of the 1st trifoliate, (f) root relative electrolyte leakage (REL), and (g) leaf REL were compared between vector-transformed-, GsCLC-c2- and GmCLC1-transgenic soybean hairy root-composite seedling with or without NaCl treatment. Results are presented as mean ± SD (n = 3). Data of different lines under control and NaCl treatment were analyzed together by one-way ANOVA followed by Duncan’s test. Letters indicate groups with statistically significant differences P ≤ 0.05
Fig. 6. Ion content in different tissues of transgenic hairy root-composite plant. a Cl− content, (b) NO3− content, (c) Cl−/NO3− ratio, (d) Na+ content, (e) K+ content, and (f) Na+/K+ ratio of transgenic hairy root-composite plants. Composite plants with hairy root transformed with vector, GmCLC1, and GsCLC-c2 were employed in this study. Results are presented as mean ± SD (n = 3). Data of different lines within the same tissue under control and NaCl treatment were analyzed together by one-way ANOVA followed by Duncan’s test. Letters indicate groups with statistically significant differences P ≤ 0.05
Fig. 7. Voltage clamp analyses of GsCLC-c2 expressions in Xenopus laevis oocytes. a Effects of NPPB. Water- or GsCLC2-c2- cRNA injected oocytes were incubated in standard bath solution (96 mM NaCl) with or without the anion channel blocker NPPB. b Effects of Cl− concentrations. GsCLC2-c2 cRNA injected oocytes were incubated in bath solution containing 0, 3, 30 or 96 mM NaCl. c pH effect. GsCLC2-c2 cRNA injected oocytes were incubated in standard bath solution containing 96 mM NaCl at pH 5.5, 7.5, or 8.5. d Halide ion selectivity. Cells were kept in a bath solution containing 96 mM NaCl, NaBr or NaI. Results are presented as mean ± SD (n ≥ 4)
Fig. 8. Electrophysiological comparisons of Xenopus laevis oocytes expressing GsCLC-c2 and GmCLC1. a Ion transport activity in a bath solution containing 96 mM NaCl. b Cl− and NO3− selectivity in a bath solution containing 96 mM NaCl or and 96 mM NaNO3, respectively. Results are presented as mean ± SD (n ≥ 5). Upper panel: oocytes were injected with water; middle panel: oocytes were injected with GmCLC1 cRNA; lower panel: oocytes were injected with GsCLC-c2 cRNA
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