Intracellular consequences of SOS1 deficiency during salt stress

Abstract

A mutation of AtSOS1 (Salt Overly Sensitive 1), a plasma membrane Na(+)/H(+)-antiporter in Arabidopsis thaliana, leads to a salt-sensitive phenotype accompanied by the death of root cells under salt stress. Intracellular events and changes in gene expression were compared during a non-lethal salt stress between the wild type and a representative SOS1 mutant, atsos1-1, by confocal microscopy using ion-specific fluorophores and by quantitative RT-PCR. In addition to the higher accumulation of sodium ions, atsos1-1 showed inhibition of endocytosis, abnormalities in vacuolar shape and function, and changes in intracellular pH compared to the wild type in root tip cells under stress. Quantitative RT-PCR revealed a dramatically faster and higher induction of root-specific Ca(2+) transporters, including several CAXs and CNGCs, and the drastic down-regulation of genes involved in pH-homeostasis and membrane potential maintenance. Differential regulation of genes for functions in intracellular protein trafficking in atsos1-1 was also observed. The results suggested roles of the SOS1 protein, in addition to its function as a Na(+)/H(+) antiporter, whose disruption affected membrane traffic and vacuolar functions possibly by controlling pH homeostasis in root cells.

Fig. 1.

Visualization of sodium and endocytosis. (A, B) Confocal planes showing young cortex cells at the root tip region of Col3 (A) or sos1-1 (B) after a 8 h treatment with 100 mM NaCl. Seedlings were stained with CoroNa Green (green) and FM4-64 (red). (C–F) Cells at the root meristem of Col3 (C, D) or sos1-1 (E, F) under non-stress condition (C, E) or after 8 h treated with 100 mM NaCl (D, F). Shown were pictures taken at 30 min after application of FM4-64 at the same confocal setting.

Fig. 2.

Effects of long-term stress. CoroNa Green (green) was applied to visualize sodium and propidium iodide (red) to stain the cell wall and dead cells. (A–D) Root tip region after 14 h (A, B) and 20 h (C, D) treatment with 100 mM NaCl. Confocal planes showing epidermis and cortex cells of the Col3 (A, C) or sos1-1 (B, D). (E–F) Root hair zone of Col3 (E) and sos1-1 (F) after 14 h treatment with 100 mM NaCl. Confocal planes showing epidermis (Ep) and cortex (Co) cells. (G) Magnification of images in (E). (H) Magnification of images in (F). (I, J) Older parts of Col3 (G) and sos1-1(H) roots after 20 h treatment with 100 mM NaCl. Confocal planes showing epidermis (Ep), cortex (Co) layers, and stele (St).

Fig. 3.

Changes in intracellular pH under salt stress. (A–D) Carboxyl SNARF-AM, which shifts the emission wavelength to red at higher pH, was applied to visualize the intracellular pH. Confocal planes collected from GFP and RFP channel were superimposed. Epidermis and cortex cells at the root tip region of Col3 (A, C) or sos1-1 (B, D) under non-stress condition (A, B) or after 8 h treatment with 100 mM NaCl (C, D). (E) Analyses of pH at the cytosol (c) or vacuole (v) from cells of (C) and (D). The pH values were deduced from the ratio of RFP and GFP channel intensities as described in the Materials and methods section; also see Supplementary Fig. S4 at JXB online.

Fig. 4.

Visualization of calcium after salt stress. Fluo-4 was used for staining calcium in the root cells without stress (A, B) or after 2 h treatment of 100 mM NaCl (C, D). Shown are the cortex cells at the elongation zone of Col3 (A, C) and sos1-1 (B, D). (This figure is available in colour at JXB online.)

Fig. 5.

Expression pattern of genes encoding pH, membrane trafficking-related proteins, and aquaporins. Gene expression was compared by qPCR between roots of Col3 and sos1-1 after 0, 2, and 8 h treatment with 100 mM NaCl. Shown are fold changes compared to the Col3 under non-stress condition. Error bars indicate standard deviations from six repeats (two biological×three analytical). Selected genes encoding pH (A), membrane trafficking-related proteins (B) and aquaporins (C). For the complete list of analyses, see Supplementary Table S1 at JXB online. (This figure is available in colour at JXB online.)

Fig. 6.

Expression pattern of genes encoding ion transporters. Selected genes of NHX family (A), Ca²⁺ transporters (B) and K⁺ transporters. For the complete list of analyses, see Supplementary Table S1 at JXB online. (This figure is available in colour at JXB online.)

Fig. 7.

Consequences of SOS1 mutation in the root cell. A diagram summarizing cellular events of wild-type (A) and sos1-1 (B) root cells under salt stress. The coloured arrows indicated the transcriptional regulation of ion transporters, red; induction, black; no change, green; inhibition of gene expression. The size of the arrows depicts the magnitude of regulation comparing wild type and sos1-1.