A salt stress-activated GSO1-SOS2-SOS1 module protects the Arabidopsis root stem cell niche by enhancing sodium ion extrusion

Abstract

Soil salinity impairs plant growth reducing crop productivity. Toxic accumulation of sodium ions is counteracted by the Salt Overly Sensitive (SOS) pathway for Na⁺ extrusion, comprising the Na⁺ transporter SOS1, the kinase SOS2, and SOS3 as one of several Calcineurin-B-like (CBL) Ca^{2 +} sensors. Here, we report that the receptor-like kinase GSO1/SGN3 activates SOS2, independently of SOS3 binding, by physical interaction and phosphorylation at Thr16. Loss of GSO1 function renders plants salt sensitive and GSO1 is both sufficient and required for activating the SOS2-SOS1 module in yeast and in planta. Salt stress causes the accumulation of GSO1 in two specific and spatially defined areas of the root tip: in the endodermis section undergoing Casparian strip (CS) formation, where it reinforces the CIF-GSO1-SGN1 axis for CS barrier formation; and in the meristem, where it creates the GSO1-SOS2-SOS1 axis for Na⁺ detoxification. Thus, GSO1 simultaneously prevents Na⁺ both from diffusing into the vasculature, and from poisoning unprotected stem cells in the meristem. By protecting the meristem, receptor-like kinase-conferred activation of the SOS2-SOS1 module allows root growth to be maintained in adverse environments.

Keywords: Arabidopsis; SOS pathway; meristem; receptor-like kinase; salt stress.

Figure 1. Arabidopsis gso1 mutants are hypersensitive to salt stress

1. A

   Phenotypes of WT (Col‐0), two gso1 mutant alleles (gso1‐1, gso1‐3) and two independent complementation lines (com‐1, com‐2). Seedlings were grown on 1/2 MS for 6 days and then transferred to 1/2 MS medium supplemented with or without 100 mM NaCl for 10 days.

2. B, C

   Primary root length and seedling fresh weight of seedlings depicted in (A) were measured at day 10 after transfer (mean ± SEM, n = 10, P < 0.05, two‐way ANOVA).

Source data are available online for this figure.

Figure EV1. The SALK_103965 line harbors the T‐DNA insertion in GSO1 rendering these plants hypersensitive to salt stress

1. A–C

   Phenotypes of indicated plant lines. Seedlings were grown on 1/2 MS medium for 6 days and then transferred to 1/2 MS medium with 100 mM NaCl for 7 days. Primary root length (B) and seedling fresh weight (C) of seedlings depicted in (A) were measured at day 7 after transfer (mean ± SEM, n = 10, P < 0.05, two‐way ANOVA).

2. D

   Gene structure of At4g20140 (GSO1) (closed box: exon; line: intron; triangle, T‐DNA insertion site for indicated SALK lines).

3. E

   qRT‐PCR analyses of GSO1 expression in WT (Col‐0), two gso1 mutants and two independent complemented lines (com‐1, com‐2). UBQ10 was used as reference.

4. F–H

   Phenotypes of WT (Col‐0) and gso1 mutants. Seedlings were grown on 1/2 MS medium for 6 days and then transferred to 1/2 MS medium supplemented with 100 mM NaCl for 10 days. Primary root length (G) and seedling fresh weight (H) of seedlings depicted in (F) were measured at day 10 after transfer (mean ± SEM, n = 10, P < 0.05, one‐way ANOVA).

Figure 2. GSO1 interacts with SOS2

1. A

   Co‐IP of GSO1 and SOS2 using Arabidopsis protoplasts transiently expressing Myc‐GSO1KD (GSO1 kinase domain) and Flag‐SOS2, Flag‐HA‐SOS3 or Flag‐SCaBP8. Protein extracts were immunoprecipitated with anti‐Myc antibody and immunoblotted with anti‐Myc antibody or anti‐Flag antibody.

2. B

   BiFC analyses involving GSO1, SOS2, SOS3 in N. benthamiana. Scale bars: 50 μm. BiFC analyses of YNE‐SOS2 with SOS3‐YCE or GSO1‐YCE in N. benthamiana leaves. SOS3‐YNE co‐expressed with GSO1‐YCE as negative control. CBL1n‐OFP was co‐expressed as plasma membrane maker. Scale bars: 50 μm (BF: brightfield).

3. C

   High resolution localization analyses of SOS2 and GSO1 expressed under their respective native promoter at 1.5 mm from the quiescent center. Propidium iodide (PI) was used to stain the cell wall. Scale bars: 100 μm (BF: brightfield).

4. D

   Phenotypes of WT (Col‐0), gso1‐3, sos2‐2 and gso1‐3 sos2‐2. Seedlings were grown on 1/2 MS medium for 6 days and then transferred to 1/2 MS supplemented with or without extra NaCl for 10 days.

5. E, F

   Primary root length and fresh weight of seedlings depicted in (D) were measured at day 7 after transfer (mean ± SEM, n = 10, P < 0.05, two‐way ANOVA).

Source data are available online for this figure.

Figure EV2. GSO1 expresses in specific cell files of the root and contributes to salt stress tolerance

1. A

   qRT‐PCR analyses of GSO1 expression. Total RNA was extracted from roots, stems, leaves, and flowers. Actin2 was used as reference.

2. B–D

   GUS activity under the promoter of GSO1 indicated by blue color.

3. E–H

   pSOS2‐driven GUS activity.

4. I

   pSOS3‐driven GUS activity.

5. J

   Localization analyses of GSO1‐mVenus in the root. ClearSee treatment was used to improve the detection. Scale bars: 100 μm.

6. K–M

   Phenotypes of WT (Col‐0), gso1‐3 and sos2‐2. Seedlings were grown on 1/2 MS medium for 6 days and then transferred to 1/2 MS medium supplemented with or without the indicated concentrations of NaCl for 10 days. Primary root length (L) and seedling fresh weight (M) were measured at day 10 after transfer (mean ± SEM, n = 10, P < 0.05, two‐way ANOVA).

Figure 3. GSO1 kinase activity is essential for plant salt tolerance and activates SOS2 independent of SOS3

1. A

   Yeast strain JP837 cells transformed with empty vectors (control) or the indicated combinations of Arabidopsis genes were grown on AP medium supplemented with or without 100 mM NaCl for 3 days at 28°C (M: myristoylation motif, KD: kinase domain).

2. B

   Yeast strain JP837 cells transformed with the indicated combinations of genes were grown on AP medium supplemented with or without 75 mM NaCl for 4 days at 28°C (SOS2T/DΔ308: constitutively active form SOS2, M: myristoylation motif, KD: kinase domain).

3. C

   Phenotypes of WT (Col‐0), gso1‐3, sos3 and gso1‐3 sos3 grown on 1/2 MS medium for 6 days and then transferred to 1/2 MS medium supplemented with or without 75 mM NaCl for 10 days.

4. D, E

   Primary root length and fresh weight of seedlings depicted in (C) were measured at day 10 after transfer (mean ± SEM, n = 12, P < 0.05, two‐way ANOVA).

5. F

   Yeast strain JP837 cells expressing SOS2T/DΔ308 were transformed with M‐GSO1KD or M‐GSO1KD^K979N and grown on AP medium as in (B).

6. G–L

   Phenotypes, primary root length and seedling fresh weight of indicated plant lines grown on 1/2 MS medium for 6 days and then transferred to 1/2 MS medium supplemented with 100 mM NaCl for 7 days (mean ± SEM, n = 10, P < 0.05, one‐way ANOVA).

Source data are available online for this figure.

Figure 4. GSO1 phosphorylates SOS2 at Thr16

1. Coomassie Brilliant Blue stain (CBB) and autoradiograph of in vitro kinase assay combining the indicated combinations of SOS2/SOS2^K40N and GSO1KD/GSO1KD^K979N.

2. Immunoblot using anti‐Myc antibody and CBB and autoradiograph from kinase assays combining Myc‐SOS2 with SOS1C300 as substrate. Ten‐day‐old seedlings stably expressing Pro35S:6Myc‐SOS2 in Col‐0 or gso1‐3 were treated with or without 100 mM NaCl for 12 h, and Myc‐SOS2 protein immunoprecipitated with anti‐C‐Myc antibody‐conjugated agarose from roots was used in the assays. Signal strength of the phosphorylation bands has been calculated relative to the left lane.

3. CBB and autoradiograph of in vitro kinase assays combining GSO1KD (kinase domain) with SOS2^K40N or SOS2^T16AK40N.

4. CBB and autoradiograph of in vitro kinase assays using SOS2 p‐site variants addressing autophosphorylation activity.

5. Yeast strain JP837 cells expressing SOS1 plus the indicated combinations of genes were grown on AP medium supplemented with or without 70 mM NaCl for 4 days at 28°C (SOS2T/DΔ308: constitutively active form SOS2, M: myristoylation motif, KD: kinase domain).

Source data are available online for this figure.

Figure EV3. Analysis of salt sensitivity of SOS2 T16 p‐site mutants in planta

1. A–F

   Phenotypes of Col‐0, sos2‐2, SOS2^T16A #3 and SOS2^T16A #4 (A), SOS2^T16D #1 and SOS2^T16D #2 (B). Seedlings were grown on 1/2 MS medium for 6 days and then transferred to 1/2 MS medium with or without 50 mM NaCl for 10 days. Primary root length (C and E) and seedling fresh weight (D and F) of seedlings at day 10 after transfer (mean ± SEM, n = 10, P < 0.05, two‐way ANOVA).

Figure 5. GSO1 dually functions in Casparian strip development and salt stress tolerance/SOS pathway activation

1. A

   Phenotypes of WT (Col‐0), gso1‐3, esb1, and casp1 casp3. Seedlings were grown on 1/2 MS for 6 days and then transferred to 1/2 MS medium with or without 100 mM NaCl for 10 days.

2. B, C

   Primary root length and seedling fresh weight of seedlings depicted in (A) were measured at day 10 after transfer (mean ± SEM, n = 12, two‐way ANOVA, P < 0.05).

3. D, E

   Na⁺ content in roots and shoots of the indicated genotypes. Seedlings were grown on 1/2 MS medium for 6 days and then transferred to 1/2 MS medium with or without 100 mM NaCl for 8 days (DW: dry weight, mean ± SEM, n = 3, P < 0.05, two‐way ANOVA).

4. F

   Na⁺ content of xylem sap of indicated genotypes. Seedlings were grown in soil for 2 weeks and then treated with 1/2 MS medium or 1/2 MS medium supplemented with NaCl for 1 day (mean ± SEM, n = 3, P < 0.05, two‐way ANOVA).

5. G

   Phenotypes of WT (Col‐0), cif1 cif2, and gso1‐3. Seedlings were grown on 1/2 MS medium for 6 days and then transferred to 1/2 MS medium supplemented with or without 100 mM NaCl for 10 days.

6. H, I

   Primary root length and seedling fresh weight of seedlings depicted in (A) were measured at day 10 after transfer (mean ± SEM, n = 10, P < 0.05, two‐way ANOVA).

7. J

   Quantification of Fluorol Yellow fluorescence intensity indicating amounts of suberin accumulation in maximum projections of z‐stacks in the first mm of the continuously suberized endodermis in the indicated genotypes. Seedlings were grown on 1/2 MS for 5 days and then treated with the indicated NaCl concentrations for 2 days (mean ± SEM, n = 3–5, P < 0.05, one‐way ANOVA and post hoc Tukey Kramer test, different letters indicate significant difference).

Source data are available online for this figure.

Figure EV4. GSO1 functions in endodermis development and salt tolerance

1. A–I

   Phenotypes of WT (Col‐0), gso1‐3, cif1 (A), cif2 (D), and sgn1 (G). Seedings were grown on 1/2 MS medium for 6 days and then transferred to 1/2 MS medium supplemented with or without 100 mM NaCl for 10 days. Primary root length (B, E and H) and seedling fresh weight (C, F, and I) of seedlings depicted in (A) at day 10 after transfer (mean ± SEM, n = 10, P < 0.05, two‐way ANOVA).

2. J

   Fluorol yellow staining of suberin in roots. WT (Col‐0), gso1‐3, sos2‐2 and casp1 casp3 were grown on 1/2 MS medium with or without NaCl for 5 days and the suberin amount in the DZ was analyzed. Scale bars: 50 μm.

Figure 6. Salt stress enhances the expression SOS2 and GSO1 in specific zones of the root

1. A–D

   Protein accumulation (A, C, D) or gene expression (B) of SOS2, CIF2, GSO1, and SGN1 displayed as false color fluorescence intensity. Seedlings from the indicated plant lines were grown on 1/2 MS for 4–5 days and then transferred to 1/2 MS with or without 100 mM NaCl for 1 day. Scale bars: 200 μm.

2. E–H

   Longitudinal quantification of protein accumulation or gene expression depicted in (A–D) represented as 8‐bit fluorescence intensity (Mean ± SEM, n = 4–5).

Source data are available online for this figure.

Figure EV5. SOS2 integrates salt stress signaling with development

Molecular model of SOS2 as a regulatory hub in salt stress signaling (FISL: Phe‐Ile‐Ser‐Leu motif, PPI: protein phosphatase interaction motif).

Figure 7. Model of salt tolerance signaling and adaptation

1. Organ‐scale model. Interrupted lines indicate CS formation, continuous lines indicate a closed functional CS. Protein accumulation of GSO1 and SOS3 is represented by color intensity (QC: quiescent center, MZ: meristematic zone, EZ: elongation zone, DZ: differentiation zone, SIF: Salt‐Induced Factor).

2. Molecular model.