Phytochromes enhance SOS2-mediated PIF1 and PIF3 phosphorylation and degradation to promote Arabidopsis salt tolerance

Abstract

Soil salinity is one of the most detrimental abiotic stresses affecting plant survival, and light is a core environmental signal regulating plant growth and responses to abiotic stress. However, how light modulates the plant's response to salt stress remains largely obscure. Here, we show that Arabidopsis (Arabidopsis thaliana) seedlings are more tolerant to salt stress in the light than in the dark, and that the photoreceptors phytochrome A (phyA) and phyB are involved in this tolerance mechanism. We further show that phyA and phyB physically interact with the salt tolerance regulator SALT OVERLY SENSITIVE2 (SOS2) in the cytosol and nucleus, and enhance salt-activated SOS2 kinase activity in the light. Moreover, SOS2 directly interacts with and phosphorylates PHYTOCHROME-INTERACTING FACTORS PIF1 and PIF3 in the nucleus. Accordingly, PIFs act as negative regulators of plant salt tolerance, and SOS2 phosphorylation of PIF1 and PIF3 decreases their stability and relieves their repressive effect on plant salt tolerance in both light and dark conditions. Together, our study demonstrates that photoactivated phyA and phyB promote plant salt tolerance by increasing SOS2-mediated phosphorylation and degradation of PIF1 and PIF3, thus broadening our understanding of how plants adapt to salt stress according to their dynamic light environment.

Figure 1.

Phytochromes promote plant salt stress tolerance in the light. A) Germination rate measurement. Imbibed seeds of WT (Col) were sown on 1/2 MS medium containing 0 or 125 mM NaCl, grown in white light (W) or dark (D) conditions, and then seed germination rates were calculated at the indicated times. Germination was defined as the first sign of radicle tip emergence and scored daily until the 6th day of incubation. Data are the means ± SE (n = 4, each pool containing at least 45 seeds). **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student's t-test; Supplemental Data Set 2) for the comparations for Col treated with 125 mm NaCl between light and dark conditions. B) Germination rate measurement. Imbibed WT (Col) seeds were sown on 1/2 MS medium containing 0 or 125 mm NaCl, grown in light or dark conditions for 7 d, and then seed germination rates were calculated. Germination was defined as the first sign of radicle tip emergence (n = 4, each pool containing at least 45 seeds). Different letters represent significant differences determined by two-way ANOVA with Tukey's post hoc test (P < 0.05; Supplemental Data Set 2). C) Arabidopsis seedlings displayed enhanced salt tolerance in the light than in the dark. WT (Col) seedlings were grown vertically on 1/2 MS medium containing 0 or 125 mm NaCl in light or dark conditions for indicated times. Scale bar = 1 cm. D) Seedling establishment rate measurements. Col seedlings were grown on 1/2 MS medium containing 0 or 125 mm NaCl in light and dark conditions for 7 d. (n = 7, each pool containing 40 seedlings). Different letters represent significant differences determined by two-way ANOVA with Tukey's post hoc test (P < 0.05; Supplemental Data Set 2). The rates of seedling establishment were measured based on the criteria recently used by previous studies (Yadukrishnan et al. 2020; Peng et al. 2022) that hypocotyls and cotyledons should emerge completely in established seedlings. E) The shoot and root Na⁺ contents of plants grown under control and 100 mm NaCl in the light or in the dark. The seedlings were first grown vertically on 1/2 MS without NaCl in continuous white light for 7 d, then transferred to 1/2 MS medium containing 0 or 100 mm NaCl and grown in light or dark conditions for additional 6 d, and then the shoots and roots were harvested separately. A pool containing more than 60 individual plants represented 1 biological replicate (n = 6), P-value was determined by Student's t-test (Supplemental Data Set 2). F–H) Phenotypes (F), root lengths (G), and fresh weights (H) of Col, phyA-211, and ProPHYA:phyA-NLS-GFP phyA-211 seedlings grown on 0 or 100 mm NaCl in white light. The seedlings were first grown vertically on 1/2 MS medium without NaCl in continuous white light for 7 d, and then transferred to 1/2 MS medium containing 0 or 100 mm NaCl and grown in continuous white light for additional 6 d. In (F), scale bar = 1 cm. In (G), n = 15, and in (H), n = 5 (5 plates from 5 independent assays, with 3 seedlings on each plate weighed together). Different letters represent significant differences determined by two-way ANOVA with Tukey's post hoc test (P < 0.05; Supplemental Data Set 2).

Figure 2.

PhyA and phyB physically interact with SOS2 to promote SOS2 kinase activity in the light. A) Germination rate measurement. Imbibed seeds of the WT (Col) or sos2-T1 mutants were sown on 1/2 MS medium containing 0, 100, or 125 mm NaCl, grown in light or dark conditions, and then seed germination rates were calculated at the indicated times. Germination was defined as the first sign of radicle tip emergence and scored daily until the 6th day of incubation. Data are the means ± SD (n = 4). B) Growth of Col and sos2-T1 seedlings grown on different concentrations of NaCl in white light or dark conditions. Col and sos2-T1 seeds were sown on 1/2 MS medium containing 0, 100, or 125 mm NaCl and grown in continuous white light or dark conditions for 5 d. Scale bar = 1 cm. C) Seedling establishment rate measurements. Col and sos2-T1 seedlings were grown on 1/2 MS medium containing 0, 100, or 125 mm NaCl in light and dark conditions for 5 d; (n = 8), each pool containing 40 seedlings. Significant differences were determined by two-way ANOVA with Tukey's post hoc test (Supplemental Data Set 2). ns, not significant. D) Salt-induced SOS2 kinase activity more predominantly in the light than in the dark. Pro35S:Myc-SOS2 seedlings were grown in darkness or continuous FR, R, B, and W light conditions for 4 d, and then treated with mock or 100 mm NaCl for 12 h. The top panel shows the immunoprecipitated Myc-SOS2 proteins by immunoblotting, the middle panel shows Coomassie Brilliant Blue (CBB)-stained SDS-PAGE gel containing His-SCaBP8 proteins, and the bottom panel shows autoradiograph indicating SOS2 kinase activity. Numbers below the autoradiograph indicate the relative band intensities of SOS2 kinase activity normalized to those of the immunoprecipitated Myc-SOS2 proteins, respectively. The ratio of the first band was set to 100 for the gel. E) Statistical analysis of the relative kinase activity values shown in (D). Error bars indicated the means ± SD (n = 3). Different letters represent significant differences determined by two-way ANOVA with Tukey's post hoc test (P < 0.05; Supplemental Data Set 2). F) PhyA and phyB enhance salt-induced SOS2 kinase activity in the light. Pro35S:Myc-SOS2 and Pro35S:Myc-SOS2 phyA phyB seedlings were grown in continuous W light for 4 d, and then treated with mock or 100 mm NaCl for 12 h. The top panel shows the immunoprecipitated Myc-SOS2 proteins by immunoblotting, the middle panel shows CBB-stained SDS-PAGE gel containing His-SCaBP8 proteins, and the bottom panel shows autoradiograph indicating SOS2 kinase activity. Numbers below the autoradiograph indicate the relative band intensities of SOS2 kinase activity normalized to those of the immunoprecipitated Myc-SOS2 proteins, respectively. The ratio of the first band was set to 100 for the gel. G) Statistical analysis of the relative kinase activity values shown in (F). Error bars indicated the means ± SD (n = 7). Different letters represent significant differences determined by two-way ANOVA with Tukey's post hoc test (P < 0.05; Supplemental Data Set 2). H) Pull-down assays show that GST-tagged SOS2, but not GST alone, was able to pull down His-PHYA in vitro. Input, 5% of the purified His-tagged target proteins were loaded. I) LCI assays show the interactions between phyA and SOS2 in plant cells. The indicated combinations of constructs were transfected into N. benthamiana leaves, respectively, and the LUC activity was detected 3 d after infiltration. The infiltration areas were circled with red dotted lines. The color gradient scale represents the relative intensities of the luciferase signal provided by the cold CCD camera (Nikon-L936; Andor Tech). J) Statistical analysis of the relative fluorescence intensity values shown in (I). At least 3 independent assays were performed, with 8 N. benthamiana leaves for each assay. n = 20, different letters represent significant differences determined by one-way ANOVA with Tukey's post hoc test (P < 0.05; Supplemental Data Set 2). K) Co-IP assays show that SOS2 interacts with phyA in vivo. Myc-SOS2 and phyA-GFP fusion proteins were transiently expressed in Arabidopsis leaf protoplasts. The total proteins were extracted and incubated with anti-Myc Affinity Gel (Sigma-Aldrich). The total (1% input) and precipitated proteins were examined by immunoblotting using anti-Myc and anti-GFP antibodies, respectively. L) Co-IP assays show that SOS2 exhibits a higher affinity to active phyA in vivo. Four-day-old etiolated Col and Pro35S:Myc-SOS2 were extracted and treated with 5 min of R light (active phyA) or with 5 min of R light plus 5 min of FR light (inactive phyA) and then incubated with anti-Myc Affinity Gel (Sigma-Aldrich). The total (1% input) and precipitated proteins were examined by immunoblotting using anti-Myc and anti-phyA antibodies, respectively. M and O) BiFC assays show that SOS2 interacts with phyA in the nucleus of N. benthamiana leaf cells. H2A-mCherry, the nuclear localization marker. An unrelated protein (GUS) was used as the negative control. Scale bar = 20 μm. N and P) The fluorescence intensities (YFP and mCherry signals) over the white lines shown in (M) and (O) were scanned using the ImageJ plot profile tool. The y-axes indicate relative pixel intensity. Distance indicates the relative positions on the white lines.

Figure 3.

PhyA regulates plant salt tolerance in the light by promoting SOS2 kinase activity. A–C) Phenotypes (A), root lengths (B), and fresh weights (C) of WT (Col), phyA-211, sos2-T1, and phyA sos2-T1 mutant seedlings grown on different concentrations of NaCl in white light. The seedlings were first grown vertically on 1/2 MS medium without NaCl in continuous white light for 7 d, and then transferred to 1/2 MS medium containing 0, 35, or 100 mM NaCl and grown in continuous white light for additional 5 d. In (A), scale bar = 1 cm. In (B), n = 12, and in (C), n = 4 (4 plates from 4 independent assays, with 3 seedlings on each plate weighed together). Different letters represent significant differences determined by two-way ANOVA with Tukey's post hoc test (P < 0.05; Supplemental Data Set 2). D) Nuclear localization of NLS-YFP-SOS2 and NLS-YFP-SOS2^T168D in the root, hypocotyl, and cotyledon cells of phyA-211 mutant seedlings. The seedlings were grown in continuous white light for 4 d and then examined using fluorescence microscopy. Scale bar = 10 μm. E–G) Phenotypes (E), root lengths (F), and fresh weights (G) of Col, phyA-211, and 2 homozygous ProUBQ10:NLS-YFP-SOS2^T168D phyA-211 lines grown on different concentrations of NaCl in white light. The seedlings were first grown vertically on 1/2 MS medium without NaCl in continuous white light for 7 d, and then transferred to 1/2 MS medium containing 0 or 100 mm NaCl and grown under continuous white light for additional 7 d. In (E), scale bar = 1 cm, in (F), n = 12, and in (G), n = 4 (4 plates from 4 independent assays, with 3 seedlings on each plate weighed together). Different letters represent significant differences determined by two-way ANOVA with Tukey's post hoc test (P < 0.05; Supplemental Data Set 2).

Figure 4.

SOS2 relieves the repressive effect of PIF1 and PIF3 on plant salt tolerance by mediating their degradation in response to salt stress. A) Germination rate measurement. Imbibed seeds of WT (Col) and pifq mutants were sown on 1/2 MS medium containing 0 or 125 mm NaCl in darkness, and then seed germination rates were calculated at the indicated times. Germination was defined as the first sign of radicle tip emergence and scored daily until the 6th day of incubation. Data are the means ± SE (n = 4, each pool containing at least 45 seeds). ****P < 0.0001 (Student's t-test; Supplemental Data Set 2) for the indicated pairs of seeds. B) Growth of pifq seedlings is less sensitive to NaCl. WT (Col) and pifq seedlings were grown vertically on 1/2 MS medium containing 0 and 125 mm NaCl in the dark for 6 d. C) Seedling establishment rate measurements. Col and pifq seedlings were grown on 1/2 MS medium containing 0 or 125 mm NaCl in the dark conditions for 6 d. (n = 6), each pool containing 40 seedlings. Different letters represent significant differences determined by two-way ANOVA with Tukey's post hoc test (P < 0.05; Supplemental Data Set 2). D) Germination rate measurement. Imbibed seeds of Col, sos2-T1, pifq, and sos2-T1 pifq seeds were sown on 1/2 MS medium containing 0 or 125 mM NaCl in darkness, and then seed germination rates were calculated at the indicated times. Germination was defined as the first sign of radicle tip emergence and scored daily until the 6th day of incubation. Error bars represent SD (n = 4, each pool containing at least 45 seeds). **P < 0.01 (Student's t-test; Supplemental Data Set 2) for the comparisons between sos2-T1 and sos2-T1 pifq seeds. E and F) Phenotypes (E) and root lengths (F) of Col, sos2-T1, pifq, and sos2-T1 pifq seedlings grown on control (1/2 MS) or NaCl (35 mM) media in white light. The seedlings were first grown vertically on 1/2 MS medium without NaCl in continuous white light for 7 d, and then transferred to 1/2 MS medium containing 0 or 35 mm NaCl and grown under continuous white light for additional 5 d. In (E), scale bar = 1 cm, in (F), n = 12. Different letters represent significant differences determined by two-way ANOVA with Tukey's post hoc test (P < 0.05; Supplemental Data Set 2). G and H) Immunoblots show that SOS2 mediates the degradation of the translated PIF3-Myc proteins in response to NaCl treatments. Super:PIF3-Myc and Super:PIF3-Myc sos2-T1 seedlings grown in darkness for 4 d were treated with CHX, together with or without 100 mm NaCl, for 6 or 12 h. Representative pictures are shown in (G) and the relative levels of PIF3-Myc proteins are shown in (H). I and J) Immunoblots showing that MG132 could efficiently inhibit the degradation of endogenous PIF3 proteins in salt-treated Col seedlings. Col and sos2-T1 mutants grown in darkness for 4 d were treated with MG132, together with or without 100 mm NaCl, for 12 h. Representative pictures are shown in (I) and the relative levels of PIF3 proteins are shown in (J). K and L) Immunoblots showing that the PIF3-Myc proteins were degraded faster upon light exposure in the seedlings pre-treated with salt stress in the dark. Super:PIF3-Myc seedlings grown in darkness for 4 d were treated with CHX, together with or without 100 mm NaCl, and incubated in darkness for additional 6 h, and then transferred to white light for the indicated times. Representative pictures are shown in (K) and the relative levels of PIF3 proteins are shown in (L). M and N) Immunoblots show that the degradation of PIF3-Myc was much slower upon light exposure in the absence of SOS2. Super:PIF3-Myc pif3-3 and Super:PIF3-Myc sos2-T1 grown in darkness for 4 d were treated with CHX and 100 mm NaCl, and incubated in darkness for additional 10 h, and then transferred to white light for the indicated times. Representative pictures are shown in (M) and the relative levels of PIF3 proteins are shown in (N). In (G), (I), (K), and (M), anti-Actin was used as a sample loading control. Relative band intensities of endogenous PIF3 or PIF3-Myc proteins normalized to those of the loading controls, respectively. The error bars in (H), (J), (L), and (N) represent SE from 3 or 4 independent assays, with each assay using a different pool of seedlings. Different letters represent significant differences by one-way ANOVA with Tukey's post hoc test (P < 0.05; Supplemental Data Set 2).

Figure 5.

SOS2 physically interacts with PIF1 and PIF3. A and B) Pull-down assays showing that GST-tagged SOS2 and SOS2^KD, but not GST-tagged SOS2^RD or GST alone, could pull down His-tagged PIF1 (A) and PIF3 (B) in vitro. C) Yeast two-hybrid assays show that both PIF1 and PIF3 interacted with SOS2 in yeast cells. D) Semi-in vivo pull-down assays showing that MBP-PIF1 and MBP-PIF3 proteins were coprecipitated with Myc-SOS2. Total proteins were extracted from 4-d-old Pro35S:Myc-SOS2 seedlings grown in white light, and then equivalent amounts of protein extract (500 µg each sample) were incubated with 2 µg of MBP-PIF1, MBP-PIF3, or MBP for 2 h. Then, the mixtures were incubated with anti-Myc Affinity Gel (Sigma-Aldrich), and the total and precipitated proteins were subjected to immunoblot analyses with antibodies against Myc and MBP, respectively. The asterisks denote the nonspecific bands; the arrow denotes the corresponding protein bands of PIF proteins. E) Co-IP assays showing that SOS2 interacted with PIF1 and PIF3 in vivo. Flag-SOS2 and PIF1-Myc or PIF3-Myc fusion proteins were transiently expressed in Arabidopsis leaf protoplasts. The total proteins were extracted and incubated with anti-Myc Affinity Gel (Sigma-Aldrich). The total (1% input) and precipitated proteins were examined by immunoblotting using anti-Myc and anti-Flag antibodies, respectively. F) BiFC assays showing the interactions between SOS2 and PIF1/PIF3 in N. benthamiana leaf cells. H2A-mCherry, the nuclear localization marker. The regulatory domain of SOS2 (SOS2^RD) was used as the negative control. Scale bar = 20 μm. G) The fluorescence intensities (YFP and mCherry signals) over the white lines shown in (F) were scanned using the ImageJ plot profile tool. The y-axes indicate relative pixel intensity. Distance indicates the relative positions on the white lines. H) BiFC assays show that SOS2 co-localizes with both phyB and PIFs in the nucleus of N. benthamiana leaf cells. The regulatory domain of SOS2 (SOS2^RD) was used as the negative control. Scale bar = 20 μm. I) The fluorescence intensities (YFP and mCherry signals) over the white lines shown in (H) were scanned using the ImageJ plot profile tool. The y-axes indicate relative pixel intensity. Distance indicates the relative positions on the white lines.

Figure 6.

SOS2 directly phosphorylates PIF1 and PIF3 and decreases the stability of PIF3 in response to light. A) In vitro kinase assays show that SOS2 directly phosphorylates PIF1 and PIF3 proteins. The top panel shows CBB-stained SDS-PAGE gel containing His-SOS2, MBP-PIF1/PIF3, and MBP proteins, and the bottom panel shows autoradiograph indicating SOS2 autophosphorylation (bottom bands) and MBP-PIF1/PIF3 phosphorylation (top bands). B and C) In vitro kinase assays show that SOS2 predominantly phosphorylates the N-terminal domains of PIF3 (B) and PIF1 (C). The top panel shows CBB-stained SDS-PAGE gel containing His-SOS2, MBP, full-length and truncated PIF1/PIF3 proteins, and the bottom panel shows autoradiograph indicating SOS2 autophosphorylation (bottom bands) and MBP-PIF1/PIF3 phosphorylation (top bands). D) In vitro kinase assays show that SOS2 phosphorylates 4 sites (Ser-151, Ser-152, Ser-153, and Ser-307) of PIF3. The top panel shows CBB-stained SDS-PAGE gel containing His-SOS2, MBP, WT, and mutated PIF3 proteins (PIF^4A: non-phosphorylatable variant; PIF^4D: phosphomimic variant), and the bottom panel shows autoradiograph indicating SOS2 autophosphorylation (bottom bands) and MBP-PIF1/PIF3 phosphorylation (top bands). E) Growth of Col, pif3-3, Super:PIF3-Myc, Super:PIF3^4A-Myc pif3-3, and Super:PIF3^4D-Myc pif3-3 seedlings under the treatment of NaCl in the light. Seeds of indicated genotypes were sown on ½ MS medium containing 0, 100, or 125 mm NaCl, and then grown in the light for 7 d. Scale bar = 1 cm. F and G) Immunoblots and quantification showing that the degradation of PIF3^4A-Myc was much slower than that of PIF3-Myc and PIF3^4D-Myc proteins upon light exposure. Super:PIF3-Myc, Super:PIF3^4A-Myc pif3-3, and Super:PIF3^4D-Myc pif3-3 seedlings grown in darkness for 4 d were treated with CHX and 100 mm NaCl, and incubated in darkness for additional 6 h, and then transferred to white light for the indicated times. Anti-Actin was used as a sample loading control. Numbers below the immunoblot indicate the relative band intensities of PIF3-Myc proteins normalized to those of the loading controls, respectively. The ratio of the first band was set to 100 for the blot. Representative pictures are shown in (F), and the relative levels of Myc-tagged proteins are shown in (G). Error bars in (G) represent SE from 3 independent assays. Different letters represent significant differences by one-way ANOVA with Tukey's post hoc test (P < 0.05; Supplemental Data Set 2). H and I) qPCR analyses showing the expression levels of RD29A(H) and SAUR-AC1(I) in Col, sos2-T1, and pifq seedlings grown in white light or dark conditions for 5 d and then treated with mock or 100 mm NaCl for 6 h. Error bars represent the SD of 3 technical replicates. Data are normalized to tubulin3. Different letters represent significant differences by two-way ANOVA with Tukey's post hoc test (P < 0.05; Supplemental Data Set 2).

Figure 7.

A working model showing that phytochromes promote plant salt tolerance by enhancing SOS2-mediated phosphorylation and degradation of PIF1 and PIF3 in the light. In the dark, SOS2 kinase activity is mildly induced by salt stress, and salt-activated SOS2 interacts with and phosphorylates PIF1 and PIF3, thus relieving their repressive effect on plant salt tolerance. In the light, photoactivated phytochromes (Pfr; mainly phyA and phyB) translocate into the nucleus, interact with SOS2, and enhance SOS2 kinase activity in response to salt stress. Highly activated SOS2 phosphorylates PIF1 and PIF3 and facilitates the more rapid turnover of PIF1 and PIF3 under salt stress in the light. On the other hand, photoactivated phytochromes may also promote SOS2 activity in the cytosol, thereby leading to enhanced SOS1 activity at the plasma membrane (PM).