The phytochrome B signaling regulates salt-mediated seedling growth in the dark

Abstract

Light is an essential environmental factor that facilitates the robust upward growth of post-germinative seedlings emerging from buried seeds that is partly mediated by the photoreceptors. Salinity stress hampers plant growth and development and reduces yield. However, the involvement and regulatory role of photoreceptors and light signaling factors to salt stress are largely unknown. Here, we report that mutants of the phytochrome B (phyB) photoreceptor showed reduced sensitivity to salt-inhibited hypocotyl elongation in darkness, and that PHYTOCHROME-INTERACTING FACTOR 3 (PIF3) acts downstream of phyB in regulating this process in Arabidopsis thaliana. We also show that SALT OVERLY SENSITIVE 2 (SOS2) regulates phyB protein accumulation under salt stress in darkness. Surprisingly, salt treatment induces phyB nuclear body formation in darkness. Moreover, we found that the phosphorylation at residue Ser-86 of phyB is essential for its function, and the scaffold protein 14-3-3κ is involved in the regulation of phyB under salt stress in darkness. Taken together, our study reveals a regulatory role of the phyB–PIF3 module in mediating post-germination growth in darkness in response to salt stress.

Introduction

Successful emergence from the soil is critical for terrestrial seed plants lifecycle, particularly the initial growth stages. Rapid elongation of the hypocotyl helps to emergence from the soil, which is actively regulated by internal factors and passively influenced by external environmental conditions. Phytochrome B (phyB) photoreceptor switches between an active form (Pfr) and an inactive form (Pr) in response to changes in red/far-red light (Rockwell et al. 2006, Medzihradszky et al. 2013, Cheng et al. 2021). The active Pfr form initiates the phyB signaling pathway, interacting with transcription factors such as phytochrome-interacting factors (PIFs) in the nucleus to regulate plant responses to light (Park et al. 2018). This network influences various processes such as seed germination, hypocotyl elongation, seedling establishment, shade avoidance, and circadian clock (Chen and Chory 2011, Paik and Huq 2019, Yadav et al. 2020, Yang et al. 2020, Cheng et al. 2021). In addition, phyB acts as a temperature sensor (Casal and Balasubramanian 2019, Klose et al. 2020) and transfers from Pfr to Pr, leading to changes in gene expression related to thermomorphogenesis (Jung et al. 2016, Legris et al. 2016). It has been revealed that phosphorylation of Ser-86 is vital for the transition between Pfr and Pr (Medzihradszky et al. 2013).

Plants employ many adaptive strategies to survive a stress episode, the Salt Overly Sensitive (SOS) pathway as a primary defense mechanism against saline–alkali stress, rapidly responding to salt stress by facilitating the removal of excess sodium ions (Na⁺), maintaining intracellular K⁺/Na⁺ ion balance, and enhancing plant salt tolerance (Yang and Guo 2018, Gong et al. 2020). Essential components of the pathway include the calcium-binding proteins SOS3 and SOS3-like calcium-binding protein 8 (SCaBP8) (Liu and Zhu 1998, Quan et al. 2007), the plasma membrane and vacuoles-localized Na⁺/H⁺ antiporter SOS1 (Shi et al. 2000, Ramakrishna et al. 2025), and the serine/threonine protein kinase SOS2 (Liu et al. 2000). During exposure to salt stress, SOS3 and SCaBP8 sense calcium signals and interact with SOS2 to facilitate its recruitment to the plasma membrane (Quintero et al. 2011); SOS2 then activates SOS1 by phosphorylating its self-inhibitory domain and improves plant salt tolerance (Yang and Guo 2018). Besides the core SOS components, other key regulators, such as the GSK3-like kinase Brassinosteroid Insensitive 2 (BIN2), GIGANTEA (GI), 14-3-3 proteins, SOS2-like Protein Kinase 5 (PKS5), and ANNEXIN4 (ANN4) have been identified, exerting positive or negative influences on the SOS signaling cascade (Kim et al. 2013, Ma et al. 2019, Yang et al. 2019, Li et al. 2020). Recent studies suggest that light stimulation activates phyA/phyB, boosting SOS2 kinase activity in response to salt stress, which improves plant salt tolerance through phosphorylation and degradation of PIF1/PIF3 proteins (Ma et al. 2023). However, the function and mechanism of SOS2 and phyB in regulating post-germinative hypocotyl elongation under salt stress in darkness remain unclear.

Extensive research has been conducted on the 14-3-3 protein family owing to their functional diversity and distinctiveness, particularly in signaling pathways (Camoni et al. 2018, Huang et al. 2021). Recent studies have revealed their regulatory roles in light signaling. In skotomorphogenesis, 14-3-3ν displays a similar phenotype to phyB mutants (Mayfield et al. 2007). In photomorphogenesis, a 14-3-3 protein acts as a positive regulator by interacting with PIF3 and counteracting its effects on phototransduction (Adams et al. 2014). Further evidence indicates that the 14-3-3λ/κ protein can bind to phosphorylated PIF3 in darkness and also photoactivate phyB by red light, facilitating the formation of a phyB-PIF3 photoregulatory protein kinase complex to accelerate the degradation of PIF3 (Song et al. 2022). Moreover, 14-3-3 proteins are implicated in blue light-induced de-etiolation and phototropic growth (Hloušková et al. 2019, Reuter et al. 2021, Sullivan et al. 2021). Furthermore, 14-3-3 proteins play a crucial role in the salt stress response by regulating SOS2 activity in Arabidopsis (Zhou et al. 2014, Yang et al. 2019). However, the role of 14-3-3 proteins in regulating seedling etiolation under salt stress remains to be elucidated.

In this study, we show that PIF3 regulates hypocotyl elongation in darkness under salt stress in a phyB-dependent manner. Our genetic analysis reveals that the regulatory effect of phyB on hypocotyl elongation under salt stress in darkness is contingent upon SOS2. Collectively, our findings uncover the role of the phyB-PIF3 signaling pathway in controlling post-germination growth under dark conditions in response to salt stress.

Results

Hypocotyl elongation of the phyB mutant shows reduced sensitivity to salt in darkness

We obtained a T-DNA insertion mutant of phyB (SALK_069700) from the Arabidopsis Biological Resource Center (Supplementary Fig. 1a and b). The phyB transcripts were greatly reduced and its protein was undetectable in this mutant and the mutant showed very long hypocotyl under continuous red light (Supplementary Fig. 1c–f), confirming that it was a loss-of-function allele of phyB. We therefore designated this mutant as phyB-69. We investigated the effect of salt (NaCl treatment) on seedling growth under dark conditions for 4 days. The hypocotyl elongation of Columbia (Col-0) wild-type (wild-type) seedlings was increasingly inhibited by the increased concentrations of NaCl. Notably, the phyB-69 mutant seedlings displayed slightly but significantly long hypocotyls compared to Col-0 (Fig. 1a and b). The phyB-9 (Col-0 ecotype) mutant exhibited a similar reduced salt sensitivity in darkness to phyB-69 (Supplementary Fig. 1e and f). Furthermore, NaCl treatment stimulated the opening of apical hook of the Col-0 seedlings in darkness, and this effect was enhanced in phyB-69 (Fig. 1c and d). Interestingly, we found that the phyB mutants also exhibited reduced salt sensitivity under weak red light (10 μmol m^–2 s^–1, Supplementary Fig. 2a and b). Although salt inhibited root elongation in the dark, the phyB-69 mutant showed similar root length to Col-0 (Supplementary Fig. 2c and d). Additionally, we also investigated the phenotype of a phyB transgenic line in the phyB-9 background (35S:phyB-GFP/phyB-9). This line exhibited a distinct constitutive morphotype under red light and had higher sensitivity to NaCl treatment than Col-0 (Supplementary Fig. 2e and f).

Figure 1.

Salt-mediated inhibition of hypocotyl elongation is less pronounced in the phyB mutant. (a) Seedling phenotypes of the Col-0 WT and the phyB-69 mutant grown in 1/2 MS medium with varying concentrations of salt in the dark for 4 days. Bars, 2 mm. (b) Hypocotyl length of the seedlings shown in (a). (c) Representative apical hook phenotypes of seedlings as shown in (a). Bars, 500 µm. (d) Apical hook curvature of the seedlings as shown in (c). (e) Seedling phenotypes of the Col-0 WT and different photoreceptor mutants grown in 1/2 MS medium with 150 mM NaCl in the dark for 4 days. (f) Hypocotyl length of the seedlings as shown in (e). Data are shown as means ± SD of more than 20 seedlings. Asterisks indicate significant differences between genotypes by a two-sided Student’s t-test (*P < 0.05, **P < 0.01, ns: no significance,) in (b and d). Letters indicate significant differences as determined by a two-sided Fisher’s LSD test (P < 0.05) in (f).

Next, we examined whether the other photoreceptors, including phyA, CRY1, CRY2, and PHOT1, are involved in this process. The hypocotyl length of the phyA-211, cry1 cry2, or phot1 mutants did not show significant difference under salt stress compared to the wild type (WT) in the dark conditions as tested (Fig. 1e and f). Collectively, these findings suggest that phyB may be specifically involved in regulating salt-inhibition of hypocotyl elongation in darkness.

PIF3 reduces salt-mediated inhibition of hypocotyl elongation in darkness

Previous studies have established that PIF proteins act downstream of phyB in regulating hypocotyl growth during morphogenesis (Al-Sady et al. 2006, Leivar and Quail 2011). We investigated the role of PIFs and found that seedlings overexpressing PIF3 (35S:PIF3-GFP) exhibited moderately longer hypocotyls than the WT seedlings under salt treatment in darkness (Fig. 2a and b). In contrast, transgenic seedlings overexpressing several other PIF genes, including PIF1, PIF4, or PIF5, did not display significant difference in hypocotyl growth under the conditions tested (Fig. 2a and b). In addition, the 35S:PIF3-GFP seedlings showed smaller apical hook curvature than the WT seedlings under 150 mM NaCl treatment in the dark (Fig. 2c and d). These results suggest that PIF3 reduces the sensitivity of hypocotyl growth to salt stress in darkness.

Figure 2.

Overexpressing PIF3 reduces salt-mediated inhibition of hypocotyl elongation. (a) Seedling phenotypes of the Col-0 WT and the 35S:PIF1-GFP, 35S:PIF3-GFP, 35S:PIF4-GFP, and 35S:PIF5-GFP transgenic lines grown in 1/2 MS medium with 150 mM NaCl in the dark for 4 days. Bars, 2 mm. (b) Hypocotyl length of the seedlings as shown in (a). (c) Representative apical hook phenotypes of Col-0 and 35S:PIF3-GFP under 150 mM NaCl treatment in the dark for 4 days. Bars, 200 µm. (d) Apical hook curvature of the seedlings shown in (c). Data are shown as means ± SD of more than 20 seedlings. Letters indicate significant differences as determined by a two-sided Fisher’s LSD test (P < 0.05) in (b and d).

PIF3 acts downstream of phyB to regulate hypocotyl growth under salt stress in darkness

We next examined the genetic relationship between phyB and PIF3 by crossing the phyB-9 mutant with the pif3 mutant to generate the pif3 phyB-9 double mutant. Phenotypic analysis revealed that under dark conditions, the pif3 single mutant displayed pronounced salt sensitivity, characterized by significantly shortened hypocotyls compared to the Col-0 WT in the dark. Interestingly, the hypocotyl length of the pif3 phyB-9 double mutant was indistinguishable from that of the pif3 single mutant but was shorter than phyB-9 after NaCl treatment (Fig. 3a and b). Similarly, under red light conditions, the salt sensitivity of the pif3 phyB-9 double mutant resembled that of pif3 (Fig. 3c and d). Furthermore, we crossed the 35S:PIF3-GFP overexpression line with phyB-9 and found that the 35S:PIF3-GFPphyB-9 line exhibited similar salt sensitivity as the parent lines in darkness but had longer hypocotyl length than the parent lines upon NaCl treatment under weak red light (Supplementary Fig. 3a–d). The PIF3-GFP protein levels were increased in the phyB-9 mutant (Supplementary Fig. 3e). These findings collectively suggest a genetic relationship where PIF3 functions downstream of phyB in the context of salt stress response in darkness.

Figure 3.

PIF3 acts downstream of phyB. (a) Seedling phenotypes of Col-0, pif3, phyB-9, and pif3 phyB-9 under 150 mM NaCl treatment in the dark for 4 days. Bars, 2 mm. (b) Hypocotyl length of the seedlings shown in (a). (c) Seedling phenotypes with 150 mM NaCl treatment under low-intensity red light (10 μmol m^–2 s^–1) for 4 days. Bars, 2 mm. (d) Hypocotyl length of the seedlings as shown in (c). Letters indicate significant differences by a two-sided Fisher’s LSD test (P < 0.05). (e and f) Relative transcript levels of four genes associated with cell elongation (e) and three genes related to salt stress (f). Letters indicate significant differences by Tukey’s HSD test (P < 0.05). Seedlings were grown in the dark for 4 days and then treated with or without 150 mM NaCl for 6 h. Data are shown as means ± SD, n = 3.

To further characterize the response at the molecular level, we assessed the gene expression of canonical downstream targets of PIFs (Zhang et al. 2014, Hayes et al. 2019, Li et al. 2022). Following 6 h of NaCl treatment, the expression levels of several cell elongation-related genes, including INDOLE-3-ACETIC ACID INDUCIBLE 19 (IAA19), IAA29, YUCCA 8 (YUC8), and IAA6, were significantly reduced in Col-0 compared to mock control in darkness. We observed that the transcript levels of these genes in 35S:PIF3-GFP, phyB-9, and 35S:PIF3-GFP phyB-9 lines were much higher than those in Col-0 after NaCl treatment (Fig. 3e). Furthermore, three marker genes associated with salt stress, such as DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2 (DREB2A), RESPONSIVE TO DESICCATION 29A (RD29A) and COLD REGULATED 15A (COR15A) were notably induced in Col-0 after NaCl treatment, whereas this salt-induced transcript levels were markedly reduced in 35S:PIF3-GFP, phyB-9, and 35S:PIF3-GFPphyB-9 lines compared to Col-0 (Fig. 3f). These findings suggest that the phyB-PIF3 module might respond to salt stress by modulating the expression of downstream genes associated with cell elongation.

SOS 2 regulates phyB protein accumulation under salt stress

As phyB physically interacts with SOS2 (Ma et al. 2023) and the SOS pathway plays vital roles in regulating plant growth under salt stress (Ali et al. 2023, Yang and Guo 2018), we explored the potential genetic relationship between phyB and SOS2 by crossing phyB-69 with sos2-2, a loss-of-function mutant of SOS2 (Liu et al. 2000). The hypocotyl phenotypes of the sos2-2 phyB-69 double mutant seedlings resembled those of the sos2-2 mutant when grown in the presence of 150 mM NaCl in darkness or under low red light (Fig. 4a and b and Supplementary Fig. 4a and b), implying that the phenotype of the phyB mutant under salt stress depends on SOS2.

Figure 4.

NaCl regulates phyB accumulation in a SOS2-dependent manner. (a) Seedling phenotypes of Col-0, sos2-2, phyB-69, and sos2-2 phyB-69 with 150 mM NaCl treatment in the dark for 4 days. Bars, 2 mm. (b) Relative hypocotyl length of the seedlings as shown in (A). Letters indicate significant differences by a two-sided Fisher’s LSD test (P < 0.05). (c and d) phyB protein levels in Col-0 (c) and in the 35S:phyB-GFP/phyB-9 line (d) with 150 mM NaCl treatment in the dark. (e) Relative transcript levels of PHYA, PHYB, SOS2, and SOS3. Data are shown as means ± SD, n = 3. Asterisks indicate significant differences by a two-sided Student’s t-test (**P < 0.01, *P < 0.05, ns: no significance). (f) phyB protein levels in Col-0 and sos2-2 with 150 mM NaCl treatment in the dark. A star indicates nonspecific bands. Immunoblotting was performed using anti-phyB antibody, anti-Actin served as a loading control. The values indicate the ratio of gray values of the protein bands corresponding to phyB and Actin (c, d, and f). Seedlings were grown in the dark for 4 days and then treated with or without 150 mM NaCl for the indicated time (c–f).

To further elucidate the response of phyB to salt, we assessed changes of phyB protein accumulation. The application of NaCl led to an increase in phyB protein levels in both the WT and the 35S:phyB-GFP/phyB-9 line in darkness (Fig. 4c and d). In contrast, the phyB protein appears to be unaffected by NaCl under conditions of red, white, and far-red light (Supplementary Fig. 4c). However, the transcript level of PHYB was not drastically regulated by NaCl in darkness (Fig. 4e). Notably, we observed a significant high accumulation of phyB protein in the sos2-2 mutant in darkness compared to that in the WT. Furthermore, the accumulation of phyB induced by NaCl was compromised in the sos2-2 mutant (Fig. 4f). Similarly, the sos2-2 mutant seedlings accumulated a high amount of phyB protein when the plants were grown under red and white light conditions (Supplementary Fig. 4d). These results suggest that salt promotes phyB protein accumulation and this process is likely dependent on SOS2.

NaCl promotes phyB nuclear body formation in the dark

Previous studies reveal that phyB can form nuclear bodies (NBs) upon light activation via liquid–liquid phase separation within the nucleus (Chen et al. 2022, Kim et al. 2023, Du et al. 2024, Kwon et al. 2024). We then observed the GFP fluorescence of the 35S:phyB-GFP/phyB-9 transgenic line using a confocal microscope. In the dark, phyB NB was barely detected in the nucleus, exhibiting a uniform distribution in the nucleoplasm (Fig. 5a). As expected, red light exposure induced phyB NB formation (Fig. 5a). Surprisingly, phyB NBs were also formed upon 3 h of 150 mM NaCl treatment in darkness (Fig. 5a). The number of phyB NBs formed after salt stress treatment either in darkness or under red light was significantly more than those under red light without NaCl treatment (Fig. 5b). However, the NB size under salt stress was smaller than that under red light (Fig. 5c). These results suggest that salt stress promotes the formation of phyB NB in darkness.

Figure 5.

NaCl promotes phyB NB formation in the dark. (a) GFP fluorescence of phyB-GFP. The 35S:phyB-GFP/phyB-9 seedlings were grown in darkness for 4 days and subsequently treated with 150 mM NaCl in darkness or/and exposed to red light (30 μmol m^–2 s^–1) for 3 h. Representative pictures are shown. Bars, 10 µm. (b) Number of phyB-GFP NBs per nucleus. Approximately 15 nuclei were counted. (c) Diameter of phyB-GFP NBs. Approximately 25 nuclei were counted. Letters indicate significant differences as determined by a two-sided Fisher’s LSD test (P < 0.05).

PhyB phosphorylation is involved in its response to salt stress

Previous studies demonstrated that the phyB NB formation partly depends on the level of Pfr of phyB (Van Buskirk et al. 2012, 2014, Liu et al. 2023) and phosphorylation of the serine 86 (S86) residue regulates the transition between Pfr and Pr (Medzihradszky et al. 2013, Viczián et al. 2020). We then investigated the response of the classic S86 phosphorylation of phyB to salt stress. Under red light, the non-phosphorylation-mimicking mutant S86A of phyB (35S:phyB^S86A-YFP/phyB-9) exhibited significant constitutive photomorphogenesis with shortened hypocotyls, akin to the phyB WT transgenic line (35S:phyB-GFP/phyB-9), whereas the phosphorylation-mimicking mutant S86D (35S:phyB^S86D-YFP/phyB-9) showed a slight weak photomorphogenic phenotype as previously reported (Fig. 6a and b; Viczián et al. 2020). Our result showed that transgenic seedlings of S86A were less sensitive to salt-inhibition of hypocotyl elongation than WT and Col-0 in darkness, whereas S86D displayed increased salt sensitivity similar to 35S:phyB-GFP compared with Col-0 (Fig. 6a and b). These data suggest that phosphorylation at S86 is required for the function of phyB in response to salt stress in darkness, whose action mode is opposite to its function in photomorphogenesis under red light.

Figure 6.

Phosphorylation modification of phyB in response to salt stress. (a) Seedling phenotypes of Col-0, WT (35S:phyB-GFP/phyB-9), S86A (35S:phyB^S86A-YFP/phyB-9), and S86D (35S:phyB^S86D-YFP/phyB-9) with 150 mM NaCl treatment in the dark or under low-intensity red light (10 μmol m^–2 s^–1) for 4 days. Bars, 2 mm. (b) Hypocotyl length of the seedlings shown in (a). Letters indicate significant differences by a two-sided Fisher’s LSD test (P < 0.05). (c) GFP/YFP fluorescence was detected after salt treatment. Seedlings were grown in darkness for 4 days and subsequently treated with 150 mM NaCl for 3 h. Representative pictures are shown. Bars, 20 µm. (d) Number of phyB NBs per nucleus. Approximately 15 nuclei were counted. (e) Analysis of phyB phosphorylation. Col-0 or transgenic plants expressing the phyB-GFP fusion protein were grown in darkness for 4 days and then treated with H₂O (Mock) or NaCl (150 mM) for 3 h. Total proteins were extracted and separated by 8% SDS-PAGE. Phosphorylation of phyB was examined using anti-phosphoserine antibody. The lanes contain equal amounts of total protein as shown by the comparable levels of Actin.

Next, we investigated the impact of S86 point mutations on phyB NB formation by salt treatment. Under dark conditions, nuclear GFP/YFP fluorescence levels were comparable among WT, S86A, and S86D. Upon NaCl treatment, S86A displayed many small NBs, resembling the pattern observed in WT. In contrast, S86D exhibited a uniform distribution in the nucleoplasm (Fig. 6c and d), which is consistent with previous findings implicating phyB phosphorylation in photobody disassembly under red light (Medzihradszky et al. 2013). Additionally, an immunoblotting assay with anti-Phosphoserine antibody showed that the overall phyB phosphorylation levels were decreased by NaCl treatment (Fig. 6e). These results suggest that NaCl reduces phyB phosphorylation, which in turn leads to increased stability of the phyB protein.

Phytochrome B and 14-3-3κ protein may regulate salt-inhibited hypocotyl elongation in the same pathway

Previous studies have shown that 14-3-3 proteins primarily interact with the phosphorylated forms of their client proteins, assisting in functions such as conformational changes and trans-localization (Liu et al. 2017, Zhao et al. 2021, Huang et al. 2022). Additionally, previous studies have highlighted the involvement of 14-3-3κ and 14-3-3λ proteins in interaction with SOS2, phyB, and PIF3 in salt and light signaling pathways (Song et al. 2022). We hypothesized that 14-3-3 might regulate the growth of dark-grown seedling under salt stress by interacting with phyB. We therefore investigated the role of 14-3-3κ and 14-3-3λ and found that, following NaCl treatment, the hypocotyl length of the 14-3-3κ single mutant and the 14-3-3κ 14-3-3λ double mutant, but not the 14-3-3λ single mutant, was longer than that of Col-0 (Fig. 7a and b). However, overexpression of 14-3-3κ (35S:14-3-3κ-GFP) did not show significant difference in hypocotyl elongation compared to the WT (Fig. 7a and b). These results indicate that 14-3-3κ is involved in regulating salt-mediated hypocotyl growth in darkness.

Figure 7.

phyB and 14-3-3 regulate hypocotyl elongation under salt stress likely in the same pathway. (a) Seedling phenotypes of Col-0, 14-3-3 mutants (κ-2, λ-2, and κ-2 λ-2), and a line overexpressing 14-3-3 (14-3-3κ-GFP) with 150 mM NaCl treatment in the dark for 4 days. Bars, 2 mm. (b) Hypocotyl length of the seedlings shown in (a). (c) Seedling phenotypes of Col-0, 14-3-3 double (κ-2 λ-2), phyB-9, and 14-3-3 phyB double (κ-2 phyB-9) mutants with 150 mM NaCl treatment in the dark for 4 days. Bars, 2 mm. (d) Hypocotyl length of the seedlings shown in (c). Letters indicate significant differences by a two-sided Fisher’s LSD test (P < 0.05) in (b and d). (e and f) Relative transcript levels of three genes associated with cell elongation (e) and three genes related to salt stress (f). Letters indicate significant differences by Tukey’s HSD test (P < 0.05). Seedlings were grown in the dark for 4 days and then treated with or without 150 mM NaCl for 6 h. Data are shown as means ± SD, n = 3.

We then generated the 14-3-3κ phyB double mutant and found that its hypocotyl length was similar to the parent phyB-9 mutant upon NaCl application in darkness or under weak red-light conditions (Fig. 7c and d; Supplementary Fig. 5). Consistent with the observed phenotype, the expression levels of IAA19 and YUC8 were higher in the 14-3-3κ-2 and 14-3-3κ phyB mutant lines than those in the WT after NaCl treatment in darkness (Fig. 7e). Under NaCl treatment, the expression levels of DREB2A, RD29A, and COR15A were lower in 14-3-3κ-2, and 14-3-3κ phyB mutants than those in Col-0 (Fig. 7f). These findings suggest that 14-3-3κ and phyB may have a synergistic regulatory role on seedling growth under salt stress environments.

Discussion

Previous studies have explored the role of phytochromes in regulating plant growth under salt stress during photoperiod conditions (Kwon et al. 2018, Wang et al. 2021, Liu et al. 2023), demonstrating that low red/far-red light (R:FR) ratios enhance growth in tomato (Solanum lycopersicum) plants under NaCl treatment (Wang et al. 2021). Mutations in phyB have been linked to improved salt tolerance in Arabidopsis and rice (Kwon et al. 2018, Liu et al. 2023). Hypocotyl elongation is essential for the vigorous emergence of post-germinative terrestrial flowering plant seedlings from buried seeds, enabling them to grow upward in darkness toward the soil surface. Previous studies have revealed the role of phyB and PIFs in regulating photomorphogenesis under salt stress (Han et al. 2023, Ma et al. 2023). These findings suggest that phyB and PIFs play specific roles in germination and hypocotyl elongation. However, the role of the phytochrome signaling under dark conditions remains largely unknown. Therefore, our study aims to investigate the function of the phyB-PIF module in response to salt stress in darkness.

We found that the phyB mutants displayed a longer hypocotyl length than Col-0 under salt stress in darkness (Fig. 1 and Supplementary Fig. 1 and 2), which indicates that phyB negatively regulates hypocotyl elongation in response to salt stress. PIF proteins have been extensively studied and are known to be involved in phyB regulation and responses to various stresses (Castillon et al. 2007, Sharma et al. 2023, Sun et al. 2024). Transgenic rice and pepper (Capsicum annuum) plants overexpressing specific PIF genes exhibit enhanced salt tolerance (Gao et al. 2015, Mo et al. 2020, Yang et al. 2021). In our study, transgenic lines overexpressing PIF3, but not other PIFs, displayed salt-insensitive phenotypes similar to those of the phyB mutants under NaCl treatment in darkness (Fig. 2; Ma et al. 2023), suggesting that PIF3 functions predominantly in salt-mediated hypocotyl elongation in the dark. This is consistent with a previous work showing that the key function of PIF3 in regulating hypocotyl elongation under a low illumination condition (Wang et al. 2020). Moreover, both pif3 phyB-9 and 35S:PIF3-GFP phyB-9 showed a similiar phenotype compared to pif3 and 35S:PIF3-GFP, respectively (Fig. 3a–d and Supplementary Fig. 3), indicating that PIF3 regulates the growth response to salt stress downstream of phyB. Collectively, our data not only identifies the specific function of phyB and PIF3 in regulating etiolated seedling growth in response to salt but also highlights the dependency of PIF3’s function on phyB in skotomorphogenesis under salt stress.

The SOS pathway is critical for plant growth against saline–alkali stress both in the light and dark conditions (Zhou et al. 2014). A previous study demonstrates that phyB interacts with SOS2 (Han et al. 2023, Ma et al. 2023). We showed that SOS2 plays a role in negatively modulating the protein stability of phyB and salt treatment induces phyB protein accumulation in a SOS2-dependent manner. Furthermore, genetic evidence showed that the SOS2 mutation fully restores the phyB mutant phenotype, implying a genetic linkage between SOS2 and phyB (Fig. 4 and Supplementary Fig. 4). Intriguingly, we found that salt promotes the formation of small NBs of phyB in darkness, although the NBs were not as typical as those induced by red light. Additionally, the application of NaCl under red light results in the formation of many small NBs (Fig. 5), indicating that red light is the predominant factor in the assembly of large NBs by phyB, with NaCl exerting a minor effect. phyB protein abundance in the nucleus was increased under salt stress, especially upon a transition from light to dark (Liu et al. 2023). Our data and previous work together suggest that the accumulation and NB formation of phyB regulate hypocotyl elongation in response to salt stress in the dark conditions.

In darkness, phyB is in the inactive Pr form, which involves self-inhibition by the interaction between its N- and C-terminals (Chen et al. 2022, Du et al. 2024). The ratio of Pfr/Pr may regulate the size and numbers of phyB NBs (Medzihradszky et al. 2013, Kim et al. 2023). We hypothesize that salt-induced NB formation of phyB in darkness could be due to its conformational changes. The S86 residue can regulate the transition between the Pfr and Pr isoforms, and phosphorylation of S86 accelerates the transition of Pfr into Pr (Medzihradszky et al. 2013). We showed that dark-grown seedlings overexpressing 35S:phyB^S86A-YFP/phyB-9 displayed longer hypocotyl than 35S:phyB^S86D-YFP/phyB-9 and Col-0 under salt stress (Fig. 6), which is opposite to their responses under red light, suggesting that phosphorylation at S86 affects salt-mediated hypocotyl elongation of dark-grown seedlings rather than the Pfr and Pr isoform transition.

Previous studies have shown that the phosphorylation sites of phyB within the N-terminal extension (NTE) have been identified, especially at S80, S86, Y104, and S106 (Medzihradszky et al. 2013, Nito et al. 2013, Viczián et al. 2020, Liu et al. 2023, Zhao et al. 2023). Notably, phosphorylation at S86 accelerates the dark reversion of phyB’s Pfr form, thereby attenuating phyB function (Medzihradszky et al. 2013, Viczián et al. 2020). Seedlings with a mutation at S86A showed hypersensitivity, while those with a phospho-mimic mutation S86D were less sensitive to phytochrome signaling (Medzihradszky et al. 2013). Y104 phosphorylation interferes with phyB-PIF3 binding and destabilizes the nuclear photobody (Nito et al. 2013). In line with a recent study, the phosphorylation at S106 and S227 of phyB by FER has been found to promote the dissociation of phyB photobody and reduce the accumulation of phyB protein in the nucleus after light-to-dark transition (Liu et al. 2023). Two calcium-dependent protein kinases, CPK6 and CPK12, phosphorylate phyB at S80 and S106, which are required for phyB nuclear import in etiolated seedlings (Zhao et al. 2023). The existing evidence suggests that different kinases selectively phosphorylate specific sites on phyB and the phosphorylation sites of phyB are diverse in different biological processes (Viczián and Nagy 2023). Understanding phosphorylation of phyB is crucial for unraveling the intricate regulatory mechanisms of plant signaling pathways.

Based on the above discussion, we hypothesize that the phosphorylation at S86 leads to distinct phenotypic responses to red light and NaCl stress, potentially due to distinct kinases-mediated phosphorylation of phyB under these two conditions. In our experiment, we observed the formation of many small NBs in darkness under NaCl treatment, a pattern reminiscent of what occurs under low light conditions (Chen 2008). This suggests that phyB^S86A leads to more NBs under salt stress, which may increase phyB accumulation. A study has shown that phyB^S86A binds less effectively to PIF3 (Medzihradszky et al. 2013), indicating that phyB^S86A might affect phyB stability rather than its activity or role in PIF3 degradation. PIF3 is known to promote hypocotyl elongation (Kim et al. 2003, Dong et al. 2017). The effect of phyB phosphorylation on phyB turnover within NBs is also important. The phyB^S86D variant speeds up phyB turnover, leading to faster degradation of phyB and associated proteins like PIFs, which explains why phyB^S86D NBs were not observed in our study. On the other hand, the phyB^S86A variant slows down the turnover, delaying the degradation of PIFs, resulting in higher PIF levels and longer hypocotyls under salt stress.

The 14-3-3κ single mutants and 14-3-3κ phyB double mutants exhibited elongated hypocotyls under salt stress, suggesting a potential shared pathway between phyB and 14-3-3 (Fig. 7 and Supplementary Fig. 5). phyB may interact with PIF3 within the nucleus in darkness under salt stress via 14-3-3κ protein. These findings indicate that 14-3-3 is involved in regulating the growth of dark-grown seedlings under salt stress. This involvement affects the function of phyB, thereby facilitating the degradation of PIF3 (Mo et al. 2020, Ma et al. 2023). Overall, salt stress induces the formation of phyB NBs in the nucleus, which is influenced by its phosphorylation state. Nuclear-localized and phosphorylated phyB may undergo co-degradation with PIF3 mediated by the 14-3-3 protein.

In summary, our study reveals an interaction between the light signal transduction pathway and the SOS pathway in fine-tuning the plant’s post-germinative growth in darkness in response to salt stress. Our findings uncover the mechanism underlying the specific function of the phyB-PIF3 module in regulating hypocotyl elongation under salt stress in darkness, which advances our understanding of how phyB-PIF3 coordinate specific growth stages and stress responses under adverse environmental conditions (Supplementary Fig. 6). Additionally, we provide an example of how a protein translocation occurs in response to external cues through phosphorylation and interaction with scaffold proteins.

Materials and Methods

Plant materials and growth conditions

WT Arabidopsis thaliana plants used in this study were of the Columbia (Col-0) accession. The phyB-69 (SALK_069700) (Mayfield et al. 2007), pif3 (SALK_030753), 14-3-3λ-2 (SALK_075219C), 14-3-3κ-2 (SALK_071097) (Song et al. 2022), phyB-9 (Reed et al. 1993), phyA-211 (Reed et al. 1994), cry1 cry2 (Mockler et al. 1999), photo1 (Alonso et al. 2003), sos2-2 (in gl1 background, Liu et al. 2000), 35S:PIF1-GFP, 35S:PIF3-GFP (Guo et al. 2023), 35S:PIF4-GFP, 35S:PIF5-GFP (Huai et al. 2018), 35S:phyB-GFP/phyB-9 (Chen et al. 2022), and 35S:phyB^S86A-YFP/phyB-9, 35S:phyB^S86D-YFP/phyB-9 (Medzihradszky et al. 2013) lines were reported previously. All mutants were validated using PCR genotyping and sequencing. The double mutants pif3 phyB-9, 35S:PIF3-GFPphyB-9, 14-3-3κ-2 14-3-3 λ-2, 14-3-3κ phyB-9, and sos2-2 phyB-69 were generated by genetic crosses. The accuracy of crosses was confirmed through phenotyping, antibiotic selection, PCR genotyping, and/or sequencing.

Seeds were surface sterilized before being planted on 1/2 Murashige and Skoog (MS) medium supplemented with 1% (w/v) sucrose and 0.8% (w/v) agar. Following a 3-day period of stratification in darkness at 4°C, seeds were exposed to either darkness or red light. Red light, emitted at an intensity of 30/10 μmol m^–2 s^–1, was generated by light-emitting diodes. Adult plants were cultivated in close proximity in soil within a growth chamber with an extended photoperiods consisting of 16 h of light and 8 h of darkness, while maintaining humidity levels at 60%–70% and a temperature of 22 ± 2°C.

Plasmid construction and generation of transgenic plants

To generate a transgenic vector overexpressing 14-3-3κ, the coding sequence of 14-3-3κ was amplified from Col-0 cDNA using KOD DNA Polymerase (TOYOBO). The resulting DNA was inserted into the pEASY-Blunt Simple vector (TransGen) and verified by sequencing. Subsequently, the full coding sequence of 14-3-3κ was amplified from the pEASY-plasmid and integrated into the EcoRI and SalI sites of PRI-GFP through recombination using a Seamless Assembly Cloning kit (CloneSmarter) to create PRI-GFP-14-3-3κ construct. The binary construct was introduced into Agrobacterium tumefaciens strain GV3101 via electroporation and then transferred into WT Arabidopsis plants using the floral dip method. Transgenic plants were screened on MS plates supplemented with kanamycin. Homozygous lines were utilized in all experiments. Primers employed for cloning, along with the corresponding restriction sites, are provided in Supplementary Table S1.

Salt sensitivity assays

For phenotypic observations and gene expression analysis, seeds of WT, mutant, and transgenic plants underwent a 3-day stratification process at 4°C in darkness. Subsequently, they were exposed to 8 h of white light (80 μmol m^–2 s^–1) before being transferred to darkness at 22°C for 3 h. To better distinguish the germination and growth responses to salt stress in darkness, we conducted a “true dark” pre-treatment before NaCl treatment to minimize the impact of light during the experiment following the methodology described previously (Leivar et al. 2008). Briefly, the plates were exposed to far-red light (1 μmol m^–2 s^–1) for 5 min, maintained in darkness at 22°C for 24 h, and then subjected to far-red light for another 5 min. One-day-old seedlings were then transferred to 1/2 MS medium with specified NaCl concentrations for salt sensitivity analysis under darkness or low-intensity red light (10 μmol m^–2 s^–1). Protein detection was conducted on 4-day-old seedlings in the dark, with or without 150 mM NaCl treatment for the indicated time.

Phenotypic analysis

To measure hypocotyl length and hook angle, 25–30 seedlings were arranged horizontally on a plate and photographed using a digital camera (Olympus). Lengths and angles were then assessed using the freely available software ImageJ (http://rsbweb.nih.gov/ij/). The hook angle was measured as previously described (Zhang et al. 2018).

Fluorescence observation

35S:phyB-GFP/phyB-9, 35S:phyB^S86A-YFP/phyB-9, and 35S:phyB^S86D-YFP/phyB-9 seedlings were cultivated in darkness for 4 days and were then either kept in darkness or transferred to red light (30 μmol m^–2 s^–1) for 3 h with or without 150 mM NaCl treatment. GFP/YFP fluorescence was examined using a Zeiss LSM 980 with Elyra7 confocal microscope (settings: GFP, excitation 489 nm, emission 500–550 nm). An observation site near the hook of the hypocotyl was chosen. The number and diameter of photobodies were quantified and depicted spatially using ZEN3.0 software.

RNA isolation and reverse transcription–quantitative PCR

RNA isolation and reverse transcription–quantitative PCR (RT-qPCR) were performed as previously described (Li et al. 2022). Four-day-old seedlings were promptly harvested and frozen in liquid nitrogen. Total RNA extraction was performed using an RNAprep Pure Plant Kit (Tiangen), followed by treatment with RNase-free DNase I (Tiangen). First-strand cDNA synthesis was accomplished using 1 μg of total RNA, oligo(dT) primers, and reverse transcriptase (Invitrogen). RT-qPCR was conducted using the cDNA samples with SYBR Green I Master (Takara) on a Light-Cycler 480 (Roche) following the manufacturer’s instructions. Data analysis and calculations were carried out using the 2^–ΔΔCT method on three independent samples, with each sample normalized to the UBQ1 internal control. Primer sequences for RT-qPCR are provided in Supplementary Table S1.

Protein immunoblotting

For immunoblots targeting anti-GFP and anti-phyB, total protein extraction was performed as previously described (Dong et al. 2020). Four-day-old seedlings were homogenized in extraction buffer comprising 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 150 mM NaCl, 1 mM EDTA, 10% (w/v) glycerol, 0.1% (w/v) Tween 20, 20 mM dithiothreitol (DTT), 1 mM phenylmethanesulfonyl fluoride (PMSF), 1× EDTA-free protease inhibitor cocktail (Roche), and 50 μM MG132. Protein concentration was determined using the Bradford assay with Bradford assay reagent (Bio-Rad). Subsequently, equal amounts of total proteins from each sample were boiled with 10× SDS loading buffer for 10 min.

Proteins were analyzed by immunoblotting using primary antibodies against phyB (1:1000 dilution), GFP (TransGen Biotech, 1:1000 dilution), phosphoserine (Millipore 1:1000 dilution), or Actin (Cwbio, CW0264M, 1:10 000 dilution), followed by detection with a secondary antibody conjugated with horseradish peroxidase (Cwbio, CW0102S, 1:10 000 dilution). Immunoblot bands were detected using a chemiluminescence imaging system (Biostep).

Statistical analysis

The values are presented as means ± standard error. Asterisks denote significance levels: *P < 0.05, **P < 0.01. Statistical significance was determined using Origin 2019, employing various tests including Student’s t-test, one-way ANOVA with Fisher’s LSD multiple comparisons test, and two-way ANOVA with Tukey’s HSD multiple comparisons test.

Accession numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: phyB (AT2G18790), phyA (AT1G09570), SOS2 (AT5G35410), SOS3 (AT5G24270), 14-3-3 κ (AT5G65430), 14-3-3λ (AT5G10450), PIF1 (AT2G20180), PIF3 (AT1G09530), PIF4 (AT2G43010), PIF5 (AT3G59060), IAA19 (At3G15540), IAA29 (AT4G32280), IAA6 (AT1G52830), YUC8 (AT4G28720), DREB2A (AT5G05410), RD29A (AT5G52310), COR15A (AT2G42540), and UBQ1 (AT3G52590).

Acknowledgments

We thank Dr Shangwei Zhong (Peking University) for providing 35S:phyB-GFP/phyB-9 seeds, Dr Andras Viczian (Institute of Plant Biology, Biological Research Center, Hungary) for providing 35S:phyB^S86A-YFP/phyB-9 and 35S:phyB^S86D-YFP/phyB-9 seeds, Dr Yan Guo (China Agricultural University) for providing sos2-2 mutants, and Dr Jigang Li (China Agricultural University) for providing phyB antibody and 14-3-3 T-DNA insertion lines.

Author Contributions

R.L. conceived the study. P.Q. and W.M. conducted the experiments and analyses and contributed equally to this work. W.M. and P.Q. wrote the manuscript and R.L. revised the manuscript.

Supplementary Data

Supplementary Data is available at PCP online.

Conflict of Interest

The authors have no conflicts of interest to declare.

Funding

This work was supported by a grant from the National Natural Science Foundation of China (32030009) and the Key R&D Program of Zhejiang (2024SSYS0100) to R.L.

Data Availability

Accession information of sequence data and all other data are available in the article and online suppementry data.

Author notes

‡

contribute equally to this work.

4

Current address: Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100093, China.