14-3-3 Proteins and Other Candidates form Protein-Protein Interactions with the Cytosolic C-terminal End of SOS1 Affecting Its Transport Activity

Abstract

The plasma membrane transporter SOS1 (SALT-OVERLY SENSITIVE1) is vital for plant survival under salt stress. SOS1 activity is tightly regulated, but little is known about the underlying mechanism. SOS1 contains a cytosolic, autoinhibitory C-terminal tail (abbreviated as SOS1 C-term), which is targeted by the protein kinase SOS2 to trigger its transport activity. Here, to identify additional binding proteins that regulate SOS1 activity, we synthesized the SOS1 C-term domain and used it as bait to probe Arabidopsis thaliana cell extracts. Several 14-3-3 proteins, which function in plant salt tolerance, specifically bound to and interacted with the SOS1 C-term. Compared to wild-type plants, when exposed to salt stress, Arabidopsis plants overexpressing SOS1 C-term showed improved salt tolerance, significantly reduced Na⁺ accumulation in leaves, reduced induction of the salt-responsive gene WRKY25, decreased soluble sugar, starch, and proline levels, less impaired inflorescence formation and increased biomass. It appears that overexpressing SOS1 C-term leads to the sequestration of inhibitory 14-3-3 proteins, allowing SOS1 to be more readily activated and leading to increased salt tolerance. We propose that the SOS1 C-term binds to previously unknown proteins such as 14-3-3 isoforms, thereby regulating salt tolerance. This finding uncovers another regulatory layer of the plant salt tolerance program.

Keywords: 14-3-3 proteins; Arabidopsis; membrane transporter; salt tolerance; salt-overly sensitive (SOS1).

Figure 1

Expression and purification of the recombinant AtSOS1 C-terminus (aa978–1146). (A) Model of the SALT-OVERLY-SENSITIVE1 (SOS1) antiporter; the target region used for the pull-down assay is marked in red. (B) SDS–PAGE and Western blot of purified AtSOS1 C-terminus (aa978–1146). Glutathione-sepharose chromatography was performed using the GST-tagged fusion protein. 1: total proteins from AtSOS1 C-terminus (aa978–1146) expressing E. coli cells; 2: supernatant (after centrifugation at 10,000× g); 3: cell pellet (after centrifugation at 10,000× g); 4: flowthrough; 5: washing step 1; 6: washing step 2; 7: elution 1 (apparent molecular mass was 44 kDa). The molecular mass (kDa) of the marker proteins is indicated.

Figure 2

Bimolecular fluorescence complementation analysis in N. benthamiana. Plasmids containing the generated yfp_NT and yfp^CT constructs were transiently co-transformed into N. benthamiana leaves using A. tumefaciens mediated transformation. Left image: YFP fluorescence, middle image: transmitted light and right image: merge of YFP fluorescence, chlorophyll fluorescence and transmitted light. The YFP fluorescence signal was detected using a Leica TCS SP5II confocal laser scanning microscope system. Bar = 100 µm. (A) 14-3-3 υ::yfp^CTT and sos1C-term::yfp^NT. (B) 14-3-3 ω::yfp^CT and sos1C-term::yfp^NT. (C) 14-3-3 κ::yfp^CT and sos1C-term::yfp^NT. (D) 14-3-3 λ::yfp^CT and sos1C-term::yfp^NT.

Figure 3

Peptide-spot binding assay with AtSOS1 C-terminus. Fifteen amino acid long peptides were spotted on a cellulose membrane and incubated with recombinant (10xHis)-labelled At14-3-3ω (A). The spots correspond to the respective region of AtSOS1 C-terminus as indicated. (B) Anti-poly-Histidine-antibody in combination with an HRP-coupled anti-mouse antibody, ECL™ and luminescence imaging were employed using an Odyssey^® Fc Imaging System (LI-COR Biosciences, Lincoln, NE, USA).

Figure 4

Relative overexpression levels of SOS1-C-terminus in Arabidopsis shoot and root tissue, revealed by quantitative RT-PCR. (A) Relative overexpression of SOS1-C-terminus in shoots, (B) relative overexpression of SOS1-C-terminus in roots. Arabidopsis plants were grown in hydroponic culture for five weeks, prior to watering for three days with 150 mM NaCl. Data represent means of six independent biological replicates. Data normalized to the housekeeping gene pp2a (At1g13320). Error bars are ±SE. Data with *** are significant from Wt at p < 0.001, data with ** are significant from Wt at p < 0.01 (two-way ANOVA).

Figure 5

Analysis of plant development on soil of Wt and p35s::SOS1^c-termStrepII mutants. Plants were grown on soil for three weeks prior to transfer to long-day conditions. For the following two weeks, plants were treated with the same volume of either tap water (A) or tap water containing 150 mM NaCl (B) every three days. Pictures were taken after seven weeks of growth. (C) Relative transcript levels of Flowering Locus T in Arabidopsis leaves under salt stress conditions. (D) Relative transcript levels of CONSTANS in Arabidopsis leaves under salt stress conditions. Data represent means of three independent biological replicates, +/- SE. Data with *** are significant from Wt at p < 0.001, data with ** are significant from Wt at p < 0.01 and data with * are significant from Wt at p < 0.05 (two-way ANOVA) Data are normalized to the housekeeping gene pp2a (At1g13320).

Figure 6

Sodium content in Wt and p35s::SOS1^c-termStrepII plants. Wt and p35s::SOS1^c-termStrepII plants were grown on soil for three weeks prior to transfer to long-day conditions. For the following two weeks, plants were treated with the same volume of either 150 mM NaCl solution or tap water every three days. Samples for ion quantification were taken after an additional two weeks. Na⁺ concentrations in leaves under control (A) and under salt stress conditions (B), quantified by Ion chromatography. Values represent the mean of 3 biological replicates, each with 2 technical replicates. Error bars are ±SE. Data with *, ** are significant from Wt at p < 0.05 or p < 0.01 (two-way ANOVA).

Figure 7

Sodium content and relative expression levels of the salt-stress indicator gene wrky25 in Wt and p35s::SOS1^c-termStrepII plants. Wt and p35s::SOS1^c-termStrepII plants were grown in hydroponic culture for five weeks and watered with ± 100 mM NaCl 72 h prior to measurement. Na₊ concentrations in leaves (A) and roots (B) under control and under salt stress conditions (leaves C, roots D), quantified by IC. Relative transcript levels of wrky25 in Arabidopsis shoot and root tissues under salt stress conditions (E). Data are normalized to the housekeeping gene pp2a (At1g13320). Values represent the mean of 3 biological replicates, each with 2 technical replicates. Error bars are ± SE. Data with *, **, *** are significant from Wt at p < 0.05, p < 0.01 or p < 0.001 (two-way ANOVA).

Figure 8

Metabolite accumulation in Arabidopsis leaves during 72 h of salt stress. (A) glucose, (B) fructose, (C) sucrose, (D) starch (in glucose equivalents), (E,F) proline. Wt plants and 35S::SOS1 C-terminus overexpression lines were grown in hydroponic culture for five weeks and watered ±100 mM NaCl 72 h prior to measurement. Sugar accumulation was determined in leaves under control and under salt stress conditions, quantified by IC. Proline accumulation was quantified by HPLC. Values represent the mean of 3 biological replicates, each with 2 technical replicates. Error bars are ±SE. Data with *, **, *** are significant from Wt at p < 0.05, p < 0.01 or p < 0.001 (two-way ANOVA).