SOS1 tonoplast neo-localization and the RGG protein SALTY are important in the extreme salinity tolerance of Salicornia bigelovii

Abstract

The identification of genes involved in salinity tolerance has primarily focused on model plants and crops. However, plants naturally adapted to highly saline environments offer valuable insights into tolerance to extreme salinity. Salicornia plants grow in coastal salt marshes, stimulated by NaCl. To understand this tolerance, we generated genome sequences of two Salicornia species and analyzed the transcriptomic and proteomic responses of Salicornia bigelovii to NaCl. Subcellular membrane proteomes reveal that SbiSOS1, a homolog of the well-known SALT-OVERLY-SENSITIVE 1 (SOS1) protein, appears to localize to the tonoplast, consistent with subcellular localization assays in tobacco. This neo-localized protein can pump Na⁺ into the vacuole, preventing toxicity in the cytosol. We further identify 11 proteins of interest, of which SbiSALTY, substantially improves yeast growth on saline media. Structural characterization using NMR identified it as an intrinsically disordered protein, localizing to the endoplasmic reticulum in planta, where it can interact with ribosomes and RNA, stabilizing or protecting them during salt stress.

Fig. 1. S. bigelovii growth and ion accumulation under different salinities.

Plants were grown for 5 weeks prior to the addition of 0, 50, 200, or 600 mM NaCl. a Plants 1 week after treatment. b Length of individual plants 1 week after treatment. c, Plants 6 weeks after treatment. d Length of individual plants 6 weeks after treatment. e Shoot cross-sections. f Shoot cross-sectional area g, Na⁺ concentration in shoots and roots. h Cl^- concentration in shoots and roots. i K⁺ concentration in shoots and roots. j Water content in shoots and roots. n = 3 (f) and n = 9 (g–j) biologically independent samples per treatment. Mean differences were compared within each tissue through two-sided t-tests and the FDR was controlled with the Benjamini-Hochberg procedure at an α = 0.05, significant differences are indicated as different letters. Boxes: represent the interval between the 25th and 75th percentile with the median shown as a horizontal line. Whiskers: represent the maximum and minimum values or 1.5-fold the interquartile range when the data point is outside this range. Outliers are shown as black dots. Plant images (a) and (c) are representative images of three tray replicates.

Fig. 2. Conservation of orthologous proteins across families and species.

a Number of orthogroups shared across the families Brassicaceae (Arabidopsis thaliana, Brassica rapa), Amaranthaceae (Beta vulgaris, Chenopodium quinoa, S. bigelovii, S. europaea, Spinacia oleracea), Fabaceae (Glycine max, Phaseolus vulgaris), Solanaceae (Solanum lycopersicum, Solanum melongena), and Poaceae (Sorghum bicolor). b Number of orthogroups shared across the Amaranthaceae species: Beta vulgaris, Chenopodium quinoa, S. bigelovii, S. europaea, and Spinacia oleracea. c Phylogenetic reconstruction of NHX proteins. In bold, Salicornia proteins. The phylogenetic tree was generated with RAxML-NG and visualized with iTOL, values at branching nodes represent transfer bootstrap expectation (TBE) values based on 1,000 replicates.

Fig. 3. S. bigelovii transcriptional responses to NaCl.

a Hierarchical clustering of differentially expressed genes in shoots of S. bigelovii plants treated with 0, 50, 200, and 600 mM NaCl for 1 and 6 weeks. b–c Representative GO enriched terms relative to 0 mM NaCl treated plants. b upregulated upon the addition of NaCl, c, upregulated in 0 mM NaCl treated plants. d–f Gene expression of proteins commonly associated with Na⁺ transport. Yellow, plants treated for 1 week; Blue, plants treated for 6 weeks. d SOS1, e HKT1, f, NHX2. Gene expression differences were compared within DESeq2 at an α = 0.05, significant differences are indicated as different letters. n = 3 biologically independent samples per treatment. Boxes: represent the interval between the 25th and 75th percentile with the median shown as a horizontal line. Whiskers: represent the maximum and minimum values or 1.5-fold the interquartile range when the data point is outside this range. Outliers are shown as black dots.

Fig. 4. Spatial protein profiles and validation of marker abundance profiles generated with pRoloc from membrane preparations from shoots of S. bigelovii plants treated with 200 mM NaCl.

a Western blot analyses of organelle marker proteins for the plasma membrane (H⁺-ATPase), thylakoid (PsbA-D1), and tonoplast (V-ATPase) on 5 µg of proteins from each of the 12 density fractions. Representative image of 3 replicates. b Relative protein abundance profiles identified with pRoloc of organelle marker proteins on the 12 density fractions. c Principal component analysis of protein predicted subcellular localization by pRoloc. The size of the symbol represents confidence values of localization. Plasma membrane: red, markers as circles; endoplasmic reticulum: gray, markers as open triangles; chloroplast: green, markers as squares; mitochondrion: blue, markers as open circles; tonoplast: orange, markers as open squares; tonoplast marker proteins, encircled in black: Vacuolar ATPase subunits (a, b, c and d) Vacuolar pyrophosphatase, and Tonoplast intrinsic protein 1–3; and red star, SOS1. d–g Relative protein abundance profiles of SOS1 (red) and proteins allocated to the tonoplast (gray) at different treatments. d 0 mM NaCl. e 50 mM NaCl. f 200 mM NaCl. g 600 mM NaCl.

Fig. 5. Subcellular localization assays of S. bigelovii and Arabidopsis SOS1 in tobacco leaves.

a–f, SbiSOS1 subcellular localization assays. a SbiSOS1-eGFP. b mCherry-AtVAMP711 (vacuolar marker). c, Merged image. d SbiSOS1-eGFP. e mCherry-AtPIP1;4 (plasma membrane marker). f Merged image. g–l AtSOS1 subcellular localization assays. g AtSOS1-eGFP. h mCherry-AtVAMP711. i Merged image. j SbiSOS1-eGFP. k mCherry-AtPIP1;4. l Merged image. m–o eGFP control. m eGFP. n mCherry-AtVAMP711. o Merged image. Cells were plasmolyzed with 300 mM mannitol to aid in the visualization of the plasma membrane and tonoplast. T Tonoplast, V Vacuole, H Hechtian strands. All scale bars at 5 µm. Representative images of two independent plant inoculations per construct.

Fig. 6. Yeast spot assays in AXT3 strain at different NaCl treatments.

a genes increasing salt tolerance. b genes decreasing salt tolerance. c RGG proteins at high NaCl. Assays were done in SD medium pH 5.6 and supplemented with 2% galactose for gene induction. Representative images of three replicates.

Fig. 7. Structure prediction for SbiSALTY and its subcellular localization in rice protoplasts.

a Intrinsically disordered domain prediction. Dotted lines represent random expectation values for each model. b Coiled-coils prediction. c CD measurements of SbiSALTY at 30°C. Wavelength range from 190 nm to 260 nm showing secondary structure and wavelength range from 250 nm to 300 nm showing tryptophan and tertiary structure. d, Temperature melting curve analysis of 201 nm, 208 nm and 222 nm of SbiSALTY in CD ranging from 20 – 92 °C. e ¹H-¹⁵N HSQC spectrum of SALTY-WT in 800 MHz NMR analysis. f–l Protein expression in rice protoplasts. Representative images of three independent transfections per construct. f SbiSALTY-EGFP. g ER-marker ER-rk (AtWAK2 signal peptide-mCherry-ER retention signal). h Bright field. i Merged image. j empty vector expressing EGFP. k Bright field. l Merged image.