Linking Duplication of a Calcium Sensor to Salt Tolerance in Eutrema salsugineum

Abstract

The SALT-OVERLY-SENSITIVE (SOS) pathway in Arabidopsis (Arabidopsis thaliana) functions to prevent the toxic accumulation of sodium in the cytosol when plants are grown in salt-affected soils. In this pathway, the CALCINEURIN B-LIKE10 (AtCBL10) calcium sensor interacts with the AtSOS2 kinase to activate the AtSOS1 plasma membrane sodium/proton exchanger. CBL10 has been duplicated in Eutrema (Eutrema salsugineum), a salt-tolerant relative of Arabidopsis. Because Eutrema maintains growth in salt-affected soils that kill most crop plants, the duplication of CBL10 provides a unique opportunity to functionally test the outcome of gene duplication and its link to plant salt tolerance. In Eutrema, individual down-regulation of the duplicated CBL10 genes (EsCBL10a and EsCBL10b) decreased growth in the presence of salt and, in combination, led to an even greater decrease, suggesting that both genes function in response to salt and have distinct functions. Cross-species complementation assays demonstrated that EsCBL10b has an enhanced ability to activate the SOS pathway while EsCBL10a has a function not performed by AtCBL10 or EsCBL10b Chimeric EsCBL10a/EsCBL10b proteins revealed that the specific functions of the EsCBL10 proteins resulted from changes in the amino terminus. The duplication of CBL10 increased calcium-mediated signaling capacity in Eutrema and conferred increased salt tolerance to salt-sensitive Arabidopsis.

Figure 1.

Eutrema is salt tolerant during vegetative and reproductive development. A, Arabidopsis and Eutrema seeds were germinated on and grown in soil for 1 week and then seedlings were left untreated (Control) or treated with NaCl in 50 mm increments every 3 d until the indicated, final concentration was reached. Photographs were taken 3 weeks after the start of the treatment. Bar = 1 cm for all images. B, Arabidopsis and Eutrema were germinated on and grown in soil for 3 weeks and then, when inflorescence development began, were left untreated (Control) or treated with NaCl in 50 mm increments every 3 d until the indicated, final concentration was reached. Photographs were taken 4 weeks after the start of the treatment. Bar = 7 cm for all images.

Figure 2.

Expression of EsCBL10a and EsCBL10b differs. Transcript accumulation is shown for SOS pathway genes from Arabidopsis and Eutrema in roots (R), leaves (L), and stage 14 flowers (F). ELONGATION FACTOR 1α (EF1-α), Loading control. One representative image of three replicates is shown.

Figure 3.

Reduced expression of EsCBL10a and EsCBL10b results in seedling hypersensitivity to salt. AmiRNAs were designed to reduce expression of EsCBL10a (E10a) and EsCBL10b (E10b) individually (a1 and b1 and b2, respectively) and in combination (ab1 and ab2) and growth in the absence (Control) and presence of salt (200 mm NaCl) was monitored. WT, Wild type; -, no amiRNA construct. A, Photographs of wild type and amiRNA lines. Bar = 1 cm for all images. B, Fresh weight was measured to quantify growth. Data are means ± se of at least 30 seedlings per genotype grown in three independent experiments. C, Reverse transcription quantitative PCR. mRNA levels were normalized to EsACTIN2 (ΔC_T) and the fold change in expression relative to the wild type was calculated (2^−ΔΔCT). Data are means ± se of three biological replicates with two technical replicates each. For all graphs, different letters indicate significant differences between genotypes (two-way ANOVA, Tukey-Kramer honestly significant difference [HSD], P ≤ 0.05).

Figure 4.

EsCBL10a and EsCBL10b complement the Atcbl10 salt-sensitive phenotype. EsCBL10a and EsCBL10b were expressed in Atcbl10 and growth in the absence (Control) or presence of salt (125 mm NaCl) was monitored. A, Photographs of wild type (WT), Atcbl10, and Atcbl10 expressing AtCBL10 (A10), EsCBL10a (E10a), or EsCBL10b (E10b). Bar = 1 cm for all images. B, Fresh weight and the length of the primary root were measured to quantify growth. Data are means ± se of at least 24 seedlings per genotype grown in three independent experiments. For all graphs, different letters indicate significant differences between genotypes (two-way ANOVA, Tukey-Kramer HSD, P ≤ 0.05). C, Transcript accumulation. ACTIN, Loading control. One representative image of three replicates is shown.

Figure 5.

EsCBL10b strongly activates the Arabidopsis and Eutrema SOS pathways. A and B, Yeast (AXT3K) was transformed with SOS1 and SOS2 from Arabidopsis (A) or Eutrema (B) in combination with AtCBL10 (A10), EsCBL10a (E10a), or EsCBL10b (E10b). Serial decimal dilutions of yeast cells were spotted onto control media or media containing 125 mm NaCl (A) or 250 mm NaCl (B). Two independently transformed colonies were assayed in three biological replicates; one representative image is shown. C, Transcript accumulation. 18S, Loading control. One representative image of three replicates is shown.

Figure 6.

EsCBL10b and AtCBL10 interact more strongly with AtSOS2 than EsCBL10a. AtCBL10 (A10), EsCBL10a (E10a), EsCBL10b (E10b), and AtSOS3 (A3) were fused to the GAL4 DNA-activation domain (AD) and interaction with AtSOS2 fused to the GAL4 DNA-binding domain (BD) was assessed using yeast two-hybrid assays. Serial decimal dilutions of diploid yeast harboring both constructs were spotted onto synthetic defined media (SD) minus Leu (L) and Trp (W), minus LW and His (H), minus LWH and adenine (A), or with the addition of 0.5 mm 3-amino-1,2,4-triazole (3AT). Two independently mated colonies were assayed in two biological replicates; one representative image is shown.

Figure 7.

EsCBL10a but not EsCBL10b complements the Atsos3 salt-sensitive phenotype. EsCBL10a, EsCBL10b, and EsSOS3 were expressed in Atsos3 and growth in the absence (Control) and presence of salt (75 mm NaCl) was monitored. A, Photographs of the wild type (WT), Atsos3, and Atsos3 expressing AtCBL10 (A10), EsCBL10a (E10a), EsCBL10b (E10b), AtSOS3 (A3), or EsSOS3 (E3). Bar = 1 cm for all images. B, Fresh weight and the length of the primary root were measured to quantify growth. Data are means ± se of at least 24 seedlings per genotype grown in three independent experiments. For all graphs, different letters indicate significant differences between genotypes (two-way ANOVA, Tukey-Kramer HSD, P ≤ 0.05). C, Transcript accumulation. ACTIN, Loading control. One representative image of three replicates is shown.

Figure 8.

Four Arabidopsis CIPKs interact with EsCBL10a. AtCBL10 (A10), EsCBL10a (E10a), EsCBL10b (E10b), and AtSOS3 (A3) were fused to the GAL4 DNA-activation domain (AD) and interaction with the Arabidopsis CIPK proteins fused to the GAL4 DNA-binding domain (BD) was assessed using yeast two-hybrid assays. Serial decimal dilutions of diploid yeast harboring both constructs were spotted onto synthetic defined media (SD) minus Leu (L) and Trp (W) or minus LW and His (H). Two independently mated colonies were assayed in two biological replicates; one representative image is shown.

Figure 9.

The first eight amino acids of EsCBL10b are important for activation of the SOS pathway. A, Yeast (AXT3K) was transformed with Arabidopsis SOS1 and SOS2 in combination with AtCBL10 (A10), EsCBL10a (E10a), EsCBL10b (E10b), or the chimeric genes. Red, E10a protein; blue, E10b protein; I, insertion of seven amino acids in E10a; HD, hydrophobic domain; V, variable sequence of amino acids; Ca, calcium-binding domains; S, Ser phosphorylation site. Serial decimal dilutions of yeast cells were spotted onto control media or media containing 125 mm NaCl. Two independently transformed colonies were assayed in three biological replicates; one representative image is shown. B, Transcript accumulation. 18S, Loading control. One representative image of three replicates is shown. C, Amino acids in the N3 fragment were aligned and color coded based on side chain properties: blue, nonpolar; magenta, polar; green, negatively charged; orange, positively charged.

Figure 10.

The hydrophobic domain of EsCBL10a is important for complementation of Atsos3. Chimeric EsCBL10a (E10a)/ EsCBL10b (E10b) proteins were expressed in Atsos3 and growth in the absence (Control) and presence of salt (75 mm NaCl) was monitored. A, Schematic representation of the chimeric proteins. Red, E10a protein; blue, E10b protein; I, insertion of seven amino acids in E10a; HD, hydrophobic domain; V, variable sequence of amino acids; Ca, calcium-binding domains; S, Ser phosphorylation site. B, Photographs of wild type (WT), Atsos3, and Atsos3 expressing E10a, E10b, or the chimeric proteins. Bar = 1 cm for all images. C, Fresh weight and length of the primary root were measured to quantify growth. Data are means ± se of at least 24 seedlings per genotype grown in three independent experiments. For all graphs, different letters indicate significant differences between genotypes (two-way ANOVA, Tukey-Kramer HSD, P ≤ 0.05). D, Amino acids in the amino terminus of E10a, E10b, AtCBL10 (A10), and AtSOS3 (A3) proteins were aligned and color coded based on side chain properties: blue, nonpolar; magenta, polar; green, negatively charged; orange, positively charged. Major element, Portion of the N2 fragment conferring full ability to complement Atsos3; Minor element, portion of the N1 fragment conferring partial ability to complement Atsos3; black boxes, amino acid differences that might underlie complementation.

Figure 11.

EsCBL10a and EsCBL10b in combination can confer salt tolerance. Atcbl10 plants strongly expressing EsCBL10a and EsCBL10b individually were crossed to generate heterozygous plants expressing both genes and growth in the absence (Control) or presence of salt (125 mm NaCl) was monitored. A, Photographs of wild type (WT), Atcbl10, and Atcbl10 expressing EsCBL10a (E10a), EsCBL10b (E10b), or both genes (abD). Bar = 1 cm for all images. B, Fresh weight and the length of the primary root were measured to quantify growth. Data are means ± se of at least 24 seedlings per genotype grown in three independent experiments. For all graphs, different letters indicate significant differences between genotypes (two-way ANOVA, Tukey-Kramer HSD, P ≤ 0.05). C, Transcript accumulation. ACTIN, Loading control. One representative image of three replicates is shown.

Figure 12.

EsCBL10a and EsSOS3 in combination confer salt tolerance. Atsos3 plants expressing EsCBL10a and EsSOS3 individually were crossed to generate heterozygous plants expressing both genes and growth in the absence (Control) or presence of salt (75 mm NaCl) was monitored. A, Photographs of wild type (WT), Atsos3, and Atsos3 expressing EsCBL10a (E10a), EsSOS3 (E3), or both genes (a3D). Bar = 1 cm for all images. B, Fresh weight and the length of the primary root were measured to quantify growth. Data are means ± se of at least 24 seedlings per genotype grown in three independent experiments. For all graphs, different letters indicate significant differences between genotypes (two-way ANOVA, Tukey-Kramer HSD, P ≤ 0.05). C, Transcript accumulation. ACTIN, Loading control. One representative image of three replicates is shown.

Figure 13.

The duplication of EsCBL10 increased the complexity of signaling in response to salt. In Arabidopsis, AtSOS3 and AtCBL10 interact with and activate the protein kinase AtSOS2, which, in turn, activates the sodium-proton exchanger AtSOS1 (AtSOS pathway) in the plasma membrane (PM). AtSOS1 then transports sodium out of the cell, preventing its toxic accumulation. AtSOS3 and AtCBL10 have additional functions outside of activation of the AtSOS pathway. Homologs were identified in Eutrema and one gene, EsCBL10, has been duplicated. EsCBL10b has an enhanced ability to activate the SOS pathway and complements the Atcbl10 salt-sensitive phenotype. EsCBL10a complements both the Atcbl10 and Atsos3 salt-sensitive phenotypes but only weakly activates the SOS pathway. EsSOS3 activates the SOS pathway and complements the Atsos3 salt-sensitive phenotype.