Low nitrogen conditions accelerate flowering by modulating the phosphorylation state of FLOWERING BHLH 4 in Arabidopsis

Significance

Nitrogen availability has a large impact on plant biomass and crop production. Both depletion and excessive application of nitrogen affect crop yields and quality and disturb important ecosystems. Thus, understanding the mechanisms by which nitrogen regulates developmental transitions affecting reproduction, such as flowering, is important to sustainably improve crop yields. Our study identified FBH4 as a key transcription factor mediating nitrogen-responsive flowering in Arabidopsis. Nitrogen levels control the FBH4 phosphorylation state that modulates transcriptional activity as a molecular switch to induce flowering. The cellular fuel sensor SnRK1 catalyzes FBH4 phosphorylation and negatively regulates florigen levels in high nitrogen conditions. These findings will enable new molecular breeding strategies to achieve better crop yields through optimized flowering time under different nitrogen conditions.

Abstract

Nitrogen (N) is an essential nutrient that affects multiple plant developmental processes, including flowering. As flowering requires resources to develop sink tissues for reproduction, nutrient availability is tightly linked to this process. Low N levels accelerate floral transition; however, the molecular mechanisms underlying this response are not well understood. Here, we identify the FLOWERING BHLH 4 (FBH4) transcription factor as a key regulator of N-responsive flowering in Arabidopsis. Low N-induced early flowering is compromised in fbh quadruple mutants. We found that FBH4 is a highly phosphorylated protein and that FBH4 phosphorylation levels decrease under low N conditions. In addition, decreased phosphorylation promotes FBH4 nuclear localization and transcriptional activation of the direct target CONSTANS (CO) and downstream florigen FLOWERING LOCUS T (FT) genes. Moreover, we demonstrate that the evolutionarily conserved cellular fuel sensor SNF1-RELATED KINASE 1 (SnRK1), whose kinase activity is down-regulated under low N conditions, directly phosphorylates FBH4. SnRK1 negatively regulates CO and FT transcript levels under high N conditions. Together, these results reveal a mechanism by which N levels may fine-tune FBH4 nuclear localization by adjusting the phosphorylation state to modulate flowering time. In addition to its role in flowering regulation, we also showed that FBH4 was involved in low N-induced up-regulation of nutrient recycling and remobilization-related gene expression. Thus, our findings provide insight into N-responsive growth phase transitions and optimization of plant fitness under nutrient-limited conditions.

Nitrogen (N) is an essential nutrient for plant growth and development (1–5). Limited N availability (low N) leads to changes in global gene expression and metabolic reprogramming to increase N uptake and N use efficiency (6–9). Remobilization of N between source and sink tissues is also required for sustainable homeostasis and better growth under low N conditions (8, 10, 11). In addition to metabolic adaptation, the timing of flowering is also modulated by N availability (5, 12–15). Applying nitrogen fertilizer delays flowering in crop species (5, 16, 17). Flowering is initiated by a developmental transition from the vegetative growth phase to the reproductive growth phase at the shoot apical meristem. This process is accompanied by drastic changes in metabolism as well as nutrient transport between source and sink tissues (8, 18). Therefore, the timing of flowering must be coordinated with cellular metabolic conditions to ensure reproductive success, especially under nutrient-limited conditions (8, 19, 20). However, the molecular link between flowering time regulation and metabolic state still remains largely unknown.

Flowering time is controlled by the coordinated effects of vernalization, photoperiod, gibberellins, autonomous, and endogenous/age pathways (21–24). In Arabidopsis, the signals from these pathways converge on the transcriptional regulation of FLOWERING LOCUS T (FT) gene in leaf phloem companion cells (25–28). Once FT is expressed, FT protein moves from leaves to the shoot apical meristem and activates gene expression to initiate the floral transition (29–34). The CONSTANS (CO)/FT module is the core component in the photoperiodic flowering under long-day conditions (23, 35–37). CO is a transcription factor that directly activates FT transcription. Although the light-dependent CO protein stability regulation is crucial, the photoperiod-dependent complex transcriptional regulation of CO also plays an important role in this module (35, 38–40). CYCLING DOF FACTOR (CDF) proteins are transcription factors that repress CO expression in the morning (41, 42). A photoreceptor E3 ubiquitin ligase, FLAVIN-BINDING KELCH REPEAT F-BOX 1 (FKF1), and its interactor GIGANTEA (GI) cooperatively regulate diurnal protein levels of CDFs (43). As for the transcriptional activators of the CO gene, the FLOWERING BHLH (FBH) and the class II TEOSINTE BRANCHED 1/ CYCLOIDEA/ PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR (TCP) transcription factors were identified (44, 45). The FBHs belong to the basic helix–loop–helix (bHLH) transcription factor family, and the Arabidopsis genome encodes at least four FBH genes, FBH1, FBH2, FBH3, and FBH4 (44). They redundantly regulate CO transcription by directly binding to the E-box elements in the CO promoter. When overexpressed at similar levels throughout the day, FBH proteins strongly increased the CO levels without affecting the circadian clock-regulated diurnal CO pattern (44), suggesting the presence of unknown posttranslational regulatory mechanisms to fine-tune FBH transcriptional activities throughout the day.

Several studies demonstrated that flowering was promoted under low N conditions in Arabidopsis (12, 13, 46, 47). Interestingly, the low N conditions increase the expression levels of CO (46, 47), indicating that CO transcriptional regulation may be one of the crosstalk points where the information of N conditions may be integrated into the flowering pathway. Cryptochrome 1 (CRY1) is involved in low N-induced flowering by modulating the amplitude of circadian clock gene expression, which affects the CO transcription (47). The gibberellin signaling pathway directly represses FT expression under high N conditions (48). However, the detailed molecular mechanisms mediating low N signal in the flowering regulation still remained elusive.

In this study, we aimed to reveal a molecular mechanism underlying N-responsive flowering. We identified FBH4 as a transcription factor of which phosphorylation states changed by different N conditions. Because FBH4 was originally characterized as the CO activator (44), we hypothesized that N conditions might affect CO transcription and flowering time partly by modulating the phosphorylation state of the FBH4 protein. Here, we showed that the phosphorylation state of FBH4 was altered by N availability, and this regulation plays a role in flowering under low N conditions. In addition, we found that FBH4 also controlled gene expression related to N recycling and remobilization, which may be a part of metabolic regulation that occurs during the floral transition under low N conditions.

Results

FBH4 Is Involved in the Low N-Induced Acceleration of Flowering through the CO/FT Photoperiodic Pathway.

Low N stress conditions accelerate flowering, but the underlying mechanisms remain largely unknown. As protein phosphorylation is an important mechanism to transmit information regarding N availability in plants (14, 49–52), we searched our carbon/nitrogen nutrients-responsive phosphoproteome dataset (53) for transcription factors that may affect flowering. We identified FBH4 as a top candidate, as several Ser residues in FBH4 are phosphorylated, and the level of Ser26 phosphorylation changes with carbon and nitrogen availability (53). FBH4 regulates flowering time by activating CONSTANS (CO) transcription (44). To examine the phosphorylation state of FBH4 protein in Arabidopsis, we performed a Phos-tag sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis using the FLAG-tagged FBH4 expressing plants (FLAG-FBH4) grown under the control N conditions (30 mM N: 10 mM KNO₃ and 10 mM NH₄NO₃). We used this N concentration for the control N in our agar-based assays, which is largely equivalent to the N concentration of the half strength Murashige and Skoog (1/2×MS) media (30 mM N: see detailed explanation in SI Appendix, Materials and Methods). This N concentration was indicated as the normal N agar media in the previous research (47). Immunoprecipitated FLAG-FBH4 proteins produced multiple slow migrating bands in the Phos-tag SDS-PAGE gel, suggesting that FBH4 exists in multiphosphorylated forms under the 30 mM N conditions (Fig. 1A). Lambda phosphatase (λPP) treatment resulted in loss of the highly shifted bands and increased the migration speed (Fig. 1A), confirming that FBH4 is phosphorylated in Arabidopsis grown under common growth conditions.

Fig. 1.

FBH4 regulates low N-induced acceleration of flowering through CO transcription. (A) Phos-tag SDS-PAGE analysis of FBH4 phosphorylation state. Total protein extracts from WT plants and FLAG-FBH4 overexpressors grown on the control 30 mM N plates were immunoprecipitated with anti-FLAG antibody beads and incubated in the absence (−) or presence (+) of lambda phosphatase (λPP). Purified FLAG-FBH4 proteins were separated by standard (Bottom) or Phos-tag SDS-PAGE (Top) followed by immunoblot analysis using anti-FLAG antibody. (B) Representative images of WT and fbh qd mutant plants grown in hydroponic solution containing 3 mM N (H) [1 mM KNO₃ + 1 mM NH₄NO₃, (H) indicates hydroponics] or 0.3 mM N (H) (0.1 mM KNO₃ + 0.1 mM NH₄NO₃). Pictures were taken when plants were 25 d old. (Scale bar, 1 cm.) (C and D) Plots of flowering rate (C) and number of days to bolting (D) of WT and fbh qd mutant plants. (D) Each box is located between the upper and the lower quartiles, and the whiskers indicate the 1.5 times interquartile ranges. The line in each box represents the median (n = 9). The different letters on top of each bar denote significant differences (Tukey’s honestly significant difference [HSD], P < 0.05). (E) Expression of CO and FT genes in WT and fbh qd mutant plants. Plants were grown on 30 mM N (10 mM KNO₃ + 10 mM NH₄NO₃) agar media for 8 d in long days and then transferred and grown on 30 mM or 0.3 mM N agar media for 3 d in long days and harvested at zeitgeber time 16. Expression levels were normalized to 18S ribosomal RNA and are shown relative to WT in 30 mM N. The results represent the means ± SD (n = 3) with different letters that denote significant differences (Tukey's HSD, P < 0.05). For all the bar graphs in this manuscript, individual values of biological replicates are superimposed onto each bar. (F and G) Plots of flowering rate (F) and number of days to bolting (G) of WT and the co-101 mutant hydroponically grown with different N conditions. Different letters in G denote significant differences (n = 8, Tukey's HSD, P < 0.05).

To assess whether FBH4 is involved in low N-induced acceleration of flowering, we first analyzed the mutant phenotype using a hydroponic culture system. The hydroponic culture was chosen for long-running flowering experiments because of prolonged stability of N concentrations in the media (i.e., lack of spatial N depletion around plant roots). Because the different FBH homologs (FBH1-4) redundantly control flowering time (44), we analyzed the phenotype of the fbh1/2/3/4 quadruple mutant (fbh qd) to test the possible involvement of FBHs on flowering under different N levels. Wild-type (WT) plants and the fbh qd mutants were hydroponically grown in 3 mM N (H) [control N in our hydroponic assay (H), note that the hydroponic assay requires less N to obtain the same effect observed in the agar media (13)] or 0.3 mM N (H) (hydroponic low N) conditions. Our hydroponic N conditions were chosen to study the effects of decreasing N availability [0.3 mM N (H)] without severely stressing plants by N starvation comparing with ample N conditions [3 mM N (H)] on flowering and also by being consistent with our previously published conditions (12, 13, 53–55). WT plants hydroponically grown in low N flowered earlier with a small stature than the control plants (Fig. 1 B–D and SI Appendix, Fig. S1 and Table S1). Flowering time of the fbh qd mutants was delayed in both conditions. Although the fbh qd mutants still responded to the low N conditions, the flowering time difference of fbh qd between two N conditions was smaller than that of WT (Fig. 1 B–D and SI Appendix, Fig. S1 and Table S1). This result suggests that four FBH proteins may be involved in the acceleration of flowering under the low N conditions. As FBH proteins activate transcription of CO (44), which regulates FT, we next measured the expression levels of CO and FT genes in these plants under the low N conditions. Both CO and FT levels were increased in the WT plants grown under the low N conditions (Fig. 1E and SI Appendix, Fig. S2), while the low N-induced increase in CO and FT levels was significantly attenuated in the fbh qd mutant (Fig. 1E). These results indicate that the FBH proteins are important for the low N-induced increase of CO and FT expression.

These results also led us to investigate the role of CO in this regulation. We next examined the N-responsive flowering phenotype of the co mutant. The low N conditions did not cause acceleration of flowering time (as days of flowering) in the co mutant plants, although low N reduced leaf emergence rate (Fig. 1 F and G and SI Appendix, Fig. S3 and Table S2). Taken together, these results suggest that FBH proteins play an important role in N-responsive flowering through CO transcriptional regulation.

FBH4 Phosphorylation Modulates the Transcriptional Activation of CO.

Since FBH4 is phosphorylated in vivo (Fig. 1A), we speculated that the phosphorylation state of FBH4 may affect its activity on CO transcription in low N conditions. To test this possibility, we analyzed the effect of substituting the Ser residues in FBH4 identified by our phosphoproteome study with Ala (Fig. 2A and SI Appendix, Fig. S4) (53). We first generated the expression constructs that possess genes encoding mutated FBH4 proteins of which one Ser residue (FBH4 S26A) or three Ser residues (Ser26 and nearby Ser22 and Ser25, FBH4 3A) were substituted with Ala. We tested the functionality of these FBH4 variants using a transient expression assay in Arabidopsis leaf mesophyll protoplasts. We confirmed that ectopic expression of the FLAG-tagged WT FBH4 enables to induce CO expression in mesophyll protoplasts (SI Appendix, Fig. S5) and then analyzed transcriptional activity of the mutated FBH4 proteins in this system (SI Appendix, Fig. S5). The single S26A mutation did not change the activity of FBH4; however, the FBH4 with the triple mutations (FBH4 3A) induced CO levels slightly higher than the native FBH4 (SI Appendix, Fig. S5), indicating that the phosphorylation state of FBH4 may affect its function. Because multiple Ala substitutions are often required for changing the function of phosphorylated proteins, especially when the protein is multiphosphorylated (56, 57), we further substituted Ser/Thr residues conserved in the FBH family (Ser22, Ser25, Ser26, Ser46, Thr50, Ser51, Ser60, Ser135, Ser136, and Ser137; referred to as FLAG-FBH4 10A) with Ala (Fig. 2A and SI Appendix, Fig. S4) (44). Interestingly, transient expression of the FBH4 10A protein significantly increased CO expression levels, comparing with the effect of the WT FBH4 (Fig. 2B). In addition, as detected by Phos-tag SDS-PAGE analysis, the number of multiphosphorylated bands of the FBH4 10A protein was dramatically reduced to the two faster migrating forms (Fig. 2C). These results suggest that these 10 Ser/Thr residues contain phosphorylation sites and that the phosphorylation of these residues negatively regulates FBH4 transcriptional activity. Since we expressed all FBH4 Ala substitution variants in the WT background, the impact of those variants may also be influenced by some interactions with endogenous FBH4 and other FBHs.

Fig. 2.

FBH4 phosphorylation state affects its transcriptional activity. (A) Schematic structure of FBH4 10A protein. Black boxes represent the mutation sites of Ser or Thr in FBH4 10A. bHLH is a basic helix–loop–helix domain. (B) CO transcriptional activity of FBH4 10A in Arabidopsis protoplasts. Endogenous CO expression levels were analyzed in mesophyll protoplast cells transiently expressing FLAG-FBH4 or FLAG-FBH4 10A. Protoplasts were prepared from 5 wk old soil grown plants. CO expression levels were normalized to 18S ribosomal RNA (rRNA) and FBH4 and are shown relative to the FLAG-FBH4 sample. Control, protoplasts treated in the same way without plasmids. N.D., not detected. Results represent ± SD (n = 3, Student's t test, **P < 0.01). (C) Phosphorylation state of FBH4 10A in Arabidopsis protoplast cells. FLAG-FBH4 or FLAG-FBH4 10A proteins were immunoprecipitated with anti-FLAG antibody beads and subjected to Phos-tag SDS-PAGE followed by immunoblot analysis. An asterisk denotes nonspecific signal. Control, protoplasts treated in the same way without plasmids. (D) Expression levels of CO and FT in FLAG-FBH4 or FLAG-FBH4 10A (#3 and #5) plants grown on the control 30 mM N media. Expression levels were normalized to 18S rRNA and calculated relative to WT. Results represent the means ± SD (n = 3, Tukey's honestly significant difference, P < 0.05).

To corroborate and further examine the effect of the 10A mutations on FBH4 function, we generated Arabidopsis transgenic plants constitutively expressing FLAG-tagged FBH4 10A (FLAG-FBH4 10A). Even though the transcript levels of FLAG-FBH4 and FLAG-FBH4 10A in these transgenic plants resemble each other, the expression levels of CO were significantly higher in the FLAG-FBH4 10A plants under the control N (30 mM N) conditions (Fig. 2D), which is consistent with the result of our transient expression analysis. Accordingly, FT expression levels were also increased more in the FLAG-FBH4 10A plants than in the FLAG-FBH4 plants (Fig. 2D). These results suggest that the transcriptional activity of FBH4 on CO regulation is regulated by the phosphorylation state of multiple Ser/Thr residues of FBH4.

The Phosphorylation State of FBH4 Alters Its Subcellular Localization.

To elucidate the mechanism by which FLAG-FBH4 10A became a stronger transcriptional activator of CO, we first analyzed whether the FLAG-FBH4 10A protein levels were increased under the control N conditions. Unexpectedly, the amount of FLAG-FBH4 10A protein was significantly lower than that of FLAG-FBH4 (SI Appendix, Fig. S6), indicating that this change could not explain the increased transcriptional activity of FLAG-FBH4 10A. We also saw that the FLAG-FBH4 10A band was shifted slightly lower than the FBH4 band even in a standard SDS-PAGE gel (SI Appendix, Fig. S6A).

As phosphorylation state sometimes affects nucleocytoplasmic distribution patterns of transcription factors (51, 58, 59), we next analyzed whether the phosphorylation state of FBH4 alters its nucleocytoplasmic distribution under different N conditions. Analysis of cytoplasmic and nuclear fractions of protein lysates from the FLAG-FBH4 plants shows that FBH4 was mainly localized in the cytoplasm in the control 30 mM N conditions (Fig. 3A and SI Appendix, Fig. S7), while the amount of FLAG-FBH4 in the nuclear fraction increased under the low N conditions (Fig. 3 A and B and SI Appendix, Fig. S7). We wondered whether the change in FBH4 intracellular distribution patterns correlates with its phosphorylation state. We therefore investigated the phosphorylation state of FLAG-FBH4 in the nuclear and cytoplasmic fractions using Phos-tag SDS-PAGE. Interestingly, FLAG-FBH4–specific band patterns differed distinctively between cytoplasmic and nuclear fractions under both control N and low N agar media and hydroponic growth conditions (Fig. 3C and SI Appendix, Fig. S8A), indicating that FLAG-FBH4 proteins exist in different phosphorylation forms in the different intracellular locations. The cytoplasmic fractions from both N conditions contain a number of FLAG-FBH4–specific bands that migrated more slowly (which means highly phosphorylated), and these bands were not detected in the nuclear fractions (Fig. 3C and SI Appendix, Fig. S8A). In addition, the signal intensities of these slow migrating FBH4 bands were weaker in the low N conditions than in the control N conditions (Fig. 3 C and D and SI Appendix, Fig. S8). Nuclear localized FLAG-FBH4 proteins were also detected as multiple phosphorylated forms, but these bands migrated faster than those of cytoplasmic FLAG-FBH4 bands, and the signals of faster migrating bands were higher in the low N conditions (Fig. 3 C and D and SI Appendix, Fig. S8). These results indicate that cytoplasmic FBH4 is more highly phosphorylated, while nuclear FBH4 is less phosphorylated. To verify whether lower phosphorylation levels triggered nuclear accumulation of FBH4, we examined FBH4 10A localization under the control 30 mM N conditions. Although the overall protein amount of FBH4 10A is lower than the wild version, our result clearly demonstrated that a higher proportion of FBH4 10 A indeed localized in the nucleus even under the control N conditions (Fig. 3 E and F and SI Appendix, Fig. S9). Taken together, these results indicate that lower N conditions reduce FBH4 phosphorylation, which may lead to nuclear accumulation. This mechanism may facilitate FBH4 to increase CO expression levels in response to the reduced N availability.

Fig. 3.

FBH4 phosphorylation state affects its nuclear localization. Plants were grown on 30 mM N agar media for 10 d and then transferred to 30 mM or 0.3 mM N agar media for 5 d. (A) Representative immunoblot results of cytoplasmic (Cyto) and nuclear (Nuc) fractions of FLAG-FBH4 protein. In A and E, signals derived from anti-PEPC (α-PEPC) and anti-histone H3 (α-H3) antibodies serve as controls for cytoplasmic and nuclear fractions, respectively. Asterisks denote nonspecific bands. (B) Quantification of nuclear/cytoplasmic ratio of FLAG-FBH4 proteins under different N conditions detected in A. Results represent the means ± SD (n = 3). (C) Phos-tag SDS-PAGE analysis of FLAG-FBH4 proteins in cytoplasmic and nuclear fractions. Open or closed arrowheads indicate bands specific to the FLAG-FBH4 extracts (cytoplasm or nucleus, respectively) compared with WT background bands. (D) Quantification of signal intensity and migration distance of FLAG-FBH4 proteins detected in C. To measure FLAG-FBH4 specific signals, the signals of WT background bands were subtracted from those in the FLAG-FBH4 sample bands for each condition. (E and F) Cytoplasmic and nuclear localization pattern of FLAG-FBH4 10A protein. Plants were grown on 30 mM N agar media for 15 d. (E) A representative immunoblot image of Cyto and Nuc fractions. (F) Quantification of nuclear/cytoplasmic ratio of FLAG-FBH4 and FLAG-FBH4 10A proteins detected in E. Results represent the means ± SD (n = 3, Student's t test, *P < 0.05).

The Low N Conditions Decrease SnRK1 Activity In Vivo.

To further investigate the regulatory mechanism of low N-induced acceleration of flowering, we aimed to identify a kinase that mediates FBH4 phosphorylation in response to the N level changes. Based on databases and previous studies for phosphorylation sites (60, 61), we found that FBH4 contains the putative target motifs of the SNF1-RELATED KINASE 1 (SnRK1) kinase (SI Appendix, Fig. S4). SnRK1 is the plant homolog of cellular fuel sensors, mammalian AMP-ACTIVATED KINASE (AMPK) and yeast SUCROSE NON-FERMENTING 1 (SNF1) (62–64). In addition, it was suggested that SnRK1 is involved in the N-responsive flowering regulation (47). We therefore inferred that SnRK1 may phosphorylate FBH4 under some N conditions. To test this possibility, we first examined whether SnRK1 activity is responsive to the N levels in Arabidopsis plants.

Although SnRK1 activity is frequently studied using target gene expression changes, this method provides a rather indirect readout of the SnRK1 activity. Mammalian AMPK kinase activity is typically assessed by the auto-phosphorylation of its catalytic domain T-loop (T172) and/or phosphorylation of its direct target enzyme acetyl-CoA carboxylase (ACC). However, while the AMPK T-loop phosphorylation correlates well with the AMPK activity, such correlation is much less obvious in plants, as SnRK1 is differently regulated (65, 66). Thus, we established a synthetic reporter system to test the SnRK1 activity more directly using the (GFP- and 2×HA-tagged) rat ACC1 peptides as a conserved target for AMPK/SNF1/SnRK1 phosphorylation. In this system, we can quantify the phosphorylation levels of the synthetic ACC reporter using the phospho-specific ACC antibody (α-ACC pS79) (Fig. 4A) (67). The specific rat ACC1 peptide sequence is not conserved in plants, avoiding interference with endogenous ACC signaling. A transient expression assay in Arabidopsis leaf mesophyll protoplasts confirmed that FLAG-tagged SnRK1α1, but not the kinase dead SnRK1α1 K48A mutant protein (which is defective in the kinase’s ATP-binding pocket), phosphorylated the ACC Ser79 (SI Appendix, Figs. S10A and S11A). This result was also consistent with the induction of SnRK1 target genes [DIN6 and SEN1 (62)] expression under the same conditions (SI Appendix, Fig. S10B). In addition, we compared the ACC Ser79 phosphorylation levels in leaf mesophyll protoplasts isolated from WT and the dexamethasone (DEX)-inducible snrk1α knockdown (snrk1α1i/α2) plants (68). Our immunoblot result showed that the ACC pSer79 signal was significantly decreased in the DEX-treated snrk1α1i/α2 cells compared with WT (SI Appendix, Figs. S10C and S11B), confirming the SnRK1 specificity of the reporter. Thus, we generated transgenic Arabidopsis plants constitutively expressing the ACC reporter construct (2×ACC-GFP-2×HA) for in planta SnRK1 activity assays. We grew the ACC reporter lines in the control N media and then transferred them to the low N media. The phosphorylation level of the reporter significantly decreased in the low N conditions, whereas the HA signals (that show the expression levels of the reporter) were similar in both conditions. These results indicate that SnRK1 activity is reduced under the low N conditions in Arabidopsis (Fig. 4 A and B and SI Appendix, Fig. S12).

Fig. 4.

SnRK1 activity is reduced in the low N conditions and affects the transcriptional activity of FBH4 in CO regulation. (A and B) In vivo SnRK1 activity under low N was assessed using the ACC reporter. (A) A schematic diagram of the synthetic ACC reporter and the immunoblot analysis showing SnRK1 activity in three biological replicates under different N conditions. Plants were grown on 30 mM N agar media for 10 d and then transferred to 30 mM or 0.3 mM N agar media for 3 d. The immunoblot with anti-HA antibody serves as the expression control of the ACC reporter. (B) Quantification of SnRK1 activity using the immunoblot shown in A. Band intensities detected with anti-ACC pS79 antibody were normalized to those with anti-HA antibody. Results represent the means ± SD (relative to the values in the 30 mM N condition, n = 3, Student's t test, *P < 0.05). (C) Expression level of CO and FT genes in the snrk1α1i/α2 (line #4) mutant grown on 30 mM N agar media in the absence (−) or presence (+) of DEX. Expression levels were normalized to 18S ribosomal RNA (rRNA) and are shown relative to WT. Results represent the means ± SD (n = 3) with different letters that denote significant differences (Tukey’s honestly significant difference [HSD], P < 0.05). Another line of the snrk1α1i/α2 (line #3) was analyzed, and the same results were obtained (SI Appendix, Fig. S13). (D) CO expression levels in Arabidopsis mesophyll protoplasts expressing FLAG-FBH4 or FLAG-FBH4 10A with SnRK1α1. Expression levels were normalized to 18S rRNA and FBH4 and are shown relative to the FLAG-FBH4 sample. Results represent the means ± SD (n = 3, Tukey's HSD, P < 0.05). (E and F) Protein amounts (E) and phosphorylation state (F) of FLAG-FBH4 proteins in the FLAG-FBH4 snrk1α1i/α2 (line #4). The indicated plants were grown on 30 mM N agar media in the absence (−) or presence (+) of DEX. The anti-AMPK pT172 antibody detects both SnRK1α1 phosphorylated at Thr175 and SnRK1α2 phosphorylated at Thr176, respectively. Signals from anti-tubulin antibody serve as a loading control. (F) Total protein extracts were immunoprecipitated with anti-FLAG antibody and subjected to Phos-tag SDS-PAGE.

SnRK1 Catalyzes FBH4 Phosphorylation and Affects the CO/FT Pathway.

We showed that the low N conditions increase CO and FT levels and accelerate flowering (Fig. 1) and that this is likely mediated partly by the nuclear localized FBH4 protein with reduced phosphorylation levels (Figs. 2 and 3). As FBH4 possesses putative phosphorylation sites for SnRK1, of which the activity is reduced under low N conditions (Fig. 4 A and B), we hypothesized that the low N conditions reduce the phosphorylation levels of FBH4 by suppressing the SnRK1 activity. This phosphorylation state change of FBH4 may facilitate elevation of CO levels and subsequent early flowering. To test this possibility, we analyzed the expression levels of CO and FT in the snrk1α1i/α2 mutant plants under the control N conditions. Consistently, knocking down SnRK1 expression in the snrk1α1i/α2 plants significantly increased the expression levels of CO and FT (Fig. 4C and SI Appendix, Fig. S13).

To further corroborate our hypothesis, we examined the effects of SnRK1 on FBH4-dependent transcriptional activation of CO using the mesophyll protoplast transient expression system. Intriguingly, coexpressing SnRK1 with FLAG-FBH4 remarkably repressed the CO activation induced by FLAG-FBH4 (Fig. 4D). SnRK1 also partly repressed the CO activation by FLAG-FBH4 10A; however, the negative effect of SnRK1 was weaker, and the CO expression level was still higher than that induced by FLAG-FBH4 coexpressed with SnRK1 (Fig. 4D). This result suggests that SnRK1 affects the transcriptional activity of FBH4 through phosphorylation of the sites mutated in FBH4 10A and potentially additional target sites. We then analyzed FBH4 phosphorylation levels in the conditional snrk1α knockdown line expressing the FLAG-FBH4 protein. We found a dramatic decrease in FLAG-FBH4 protein amounts in the DEX-induced snrk1αi1/α2 plants (Fig. 4E). Phos-tag SDS-PAGE analysis demonstrated that migration patterns of FLAG-FBH4 were shifted down in the DEX-induced snrk1αi1/α2 (Fig. 4F), indicating that FLAG-FBH4 is less phosphorylated. These results indicate that SnRK1 can change the phosphorylation state of FBH4 in planta.

To assess whether SnRK1 can directly phosphorylate FBH4, we examined the catalytic activity of SnRK1 on FBH4 in vitro. The SnRK1α1-FLAG protein was immunoprecipitated from the FLAG-tagged SnRK1α1-expressing plants (SnRK1α1-FLAG) and mixed with the recombinant GST-FBH4 protein or GST in vitro followed by analysis of GST-FBH4 phosphorylation. We observed a clear band shift of GST-FBH4 after 1 h incubation with SnRK1α1-FLAG (Fig. 5A and SI Appendix, Fig. S14A). In addition, in the Phos-tag SDS-PAGE analysis, a clear pattern of shifting GST-FBH4 bands was observed in the presence of SnRK1α1-FLAG, indicating that GST-FBH4 is phosphorylated by SnRK1 on multiple residues (Fig. 5B). Moreover, incubating phosphorylated GST-FBH4 with λPP eliminated the GST-FBH4 shift in the standard SDS-PAGE, which also confirmed that it is caused by phosphorylation (Fig. 5C and SI Appendix, Fig. S14B). These results indicate that SnRK1 can directly phosphorylate FBH4.

Fig. 5.

SnRK1 directly phosphorylates FBH4 in vitro. (A and B) In vitro phosphorylation assay. Recombinant GST or GST-FBH4 was incubated with immunoprecipitated proteins from WT or plants expressing SnRK1α1-FLAG. Samples were subjected to immunoblot analysis (A) and Phos-tag SDS-PAGE (B). GST-FBH4+P indicates GST-FBH4 with phosphorylation. (C) In vitro dephosphorylation assay. Recombinant GST-FBH4 was incubated with immunoprecipitated proteins from plants expressing SnRK1α1-FLAG in the presence or absence of lambda phosphatase (λPP).

Together with other results shown here, our results indicate that SnRK1 negatively regulates FBH4 transcriptional activity through direct phosphorylation.

FBH4 Is Involved in the Regulation of Gene Expression Related to Nutrient Recycling and Remobilization under Low N Conditions.

Nutrient recycling and remobilization are important metabolic events induced under low N conditions to repurpose limited N sources for vegetative growth (10, 11). During the floral transition, the nutrients are also transported from leaves (source tissues) to floral organs (sink tissues) (8, 18). A recent transcriptome study suggested that FBH4 may be involved in gene expression of multiple N-responsive genes (69). We therefore wondered whether FBHs, especially FBH4, also play a role in N metabolic change, which may occur simultaneously with floral transition. To test this possibility, we examined the gene expression related to nutrient recycling and remobilization in the fbh qd mutant. In addition, we included transgenic plants expressing a dominant repressor form of FBH4 (FBH4-SRDX) in which FBH4 coding sequence was translationally fused to the Superman repression domain modified ver. X (SRDX) sequences (70). Adding the SRDX domain made FBH4 a transcriptional repressor, as it was shown by the reduction of CO and FT expression levels under the low N conditions in the FBH4-SRDX plants (SI Appendix, Fig. S15). In the WT plants, the low N conditions induced the expression of the cytoplasmic glutamine synthetase isoenzyme genes, GLN1;1 and GLN1;4, involved in cellular ammonium recycling and glutamine synthesis (11, 71) (Fig. 6A). The low N-specific induction of these genes was attenuated in both fbh qd and FBH4-SRDX plants (Fig. 6A). A similar effect was observed for the induction of ATG8A and ATG8C genes required for autophagosome formation and important for cellular nutrient recycling (72, 73) (Fig. 6A). In addition, the induction of the high-affinity nitrate transporter NRT2.5 and low-affinity transporter NRT1.7 genes, which are expressed in the phloem of older leaves (source tissues) to supply nitrate to young leaves (sink tissues) (7, 74), was reduced in the fbh qd and the FBH4-SRDX plants (Fig. 6A). Moreover, we observed that the fbh qd mutants and the FBH4-SRDX plants showed the decrease in vegetative growth under both N conditions (SI Appendix, Fig. S16). These results indicate that FBH4 (and possibly other FBHs) is involved in the induction of the genes important for N recycling and remobilization, which may affect growth, under the low N conditions.

Fig. 6.

FBH4 is involved in the regulation of N recycling- and remobilization-related gene expressions. Expression levels of genes related to N recycling and remobilization in WT, fbh qd, and FBH4-SRDX (lines #5) (A) and WT and co-101 (B) plants. Plants were grown on 30 mM N agar media for 10 d and then transferred to 30 mM or 0.3 mM N agar media for 5 d. Expression levels were normalized to 18S ribosomal RNA and shown relative to WT grown in 30 mM N conditions. Results represent the means ± SD (n = 3) with different letters that denote significant differences (Tukey’s honestly significant difference, P < 0.05).

Since we showed that FBH4 induces CO under the low N conditions, we tested whether FBH4 regulates the expression of these N metabolic genes through the change in CO levels. We therefore analyzed the expression of these genes in the co mutants under the same N conditions. The expression levels of the GLN1;1/1;4, ATG8A/8C, NRT2.5, and NRT1.7 genes in the co mutants were similar to those in WT under different N conditions (Fig. 6B), indicating that FBH4 is independently involved in the induction of N recycling and remobilization genes in addition to the CO induction under the low N conditions.

Discussion

Reduced Phosphorylation of FBH4 Mediates Low N-Induced Acceleration of Flowering.

Plants have evolved complicated mechanisms to modulate the timing of the growth phase transition in response to various environmental stimuli for successful reproduction. N is one of the most abundant nutrients required for plant growth and affects plant developmental regulation including flowering. The effects of N on the flowering regulation differ depending on the concentrations, as both a depletion and an excess of N cause a delay of flowering (13). However, the mechanisms that cause flowering delay may be different, as these two N conditions induce contrasting growth patterns. In addition, the chemical property of N sources also plays a role in the N-dependent flowering regulation. Nitrate affects flowering time as a signaling molecule at the shoot apical meristem in addition to acting as a major N source for plants (15). These findings indicate the presence of complex N signaling/metabolic networks that affect flowering time. In this study, we identified one of these network modules that exist in the leaf phloem. FBH4 is the transcriptional activator of the CO gene in the leaf phloem (44). Our genetic analyses demonstrate that N nutrient signaling interacts with the CO/FT photoperiodic pathway through adjusting the phosphorylation state of FBH4 to modulate flowering time (Fig. 1 B–E and SI Appendix, Figs. S1 and S2). A previous study showed that N levels alter the expression levels of circadian clock genes as well as the CO gene, resulting in early flowering in low N conditions in Arabidopsis, although the detailed underlying mechanism remained unclear (47). The delayed flowering phenotype of the co mutants under the low N conditions confirms the importance of the CO regulation to control low N-induced flowering promotion (Fig. 1 F and G and SI Appendix, Fig. S3). In addition, we revealed that FBH4 phosphorylation is a critical regulatory mechanism to modulate CO transcription levels. A total of 10 Ser/Thr residues (Ser22, Ser25, Ser26, Ser46, Thr50, Ser51, Ser60, Ser135, Ser136, and Ser137) were identified as putative phosphorylation sites involved in the modulation of FBH4 activity on the CO transcription (Fig. 2). Decreased levels of FBH4 phosphorylation promote its nuclear localization and activation of CO transcription (Figs. 2 and 3). Furthermore, we identified SnRK1 as a kinase directly catalyzing FBH4 phosphorylation. The SnRK1 protein kinase is the ortholog of mammalian AMPK and yeast SNF1 and functions as a conserved cellular fuel sensor in plants required for maintaining metabolic homeostasis (62–64). As such, SnRK1 regulates many aspects of plant growth and development, including flowering regulation (75, 76). However, the exact function of SnRK1 in flowering remained unclear. Our biochemical and genetic analyses showed that SnRK1 directly catalyzes the phosphorylation of FBH4 and negatively regulates CO and FT expression levels (Figs. 4 and 5). Our findings suggest that the SnRK1-FBH4 module integrates the information of nitrogen availability into flowering time regulation. Under the low N conditions where SnRK1 activity is down-regulated, the phosphorylation levels of FBH4 are decreased. The reduction of the FBH4 phosphorylation likely promotes FBH4 nuclear localization and transcriptional activation of the CO/FT pathway, thereby accelerating flowering in Arabidopsis (SI Appendix, Fig. S17). Considering the redundant functions of the FBH family proteins in CO transcription (44) and the high conservation of the 10 Ser/Thr residues mutated in FBH4 10A among the FBH proteins, it is possible that the other FBH proteins also participate in the low N-induced acceleration of flowering through the same phosphorylation-dependent mechanisms.

Phos-tag western blot analysis showed that FBH4 existed in highly phosphorylated forms with different degrees of phosphorylation, especially under higher N conditions (Figs. 1A and 3C). The phosphatase treatment could not completely remove all phosphate moieties from FBH4 (Fig. 1A), suggesting that some phosphorylation sites might be structurally protected from the phosphatase. Although mutating 10 Ser/Thr residues (FBH4 10A) decreased FBH4 phosphorylation levels, some FBH4 10A proteins still existed as certain phosphorylated forms (Fig. 2C). A previous study showed that ABA-induced phosphorylation of Ser/Thr residues located near the C-terminal bHLH domain of FBH3 (also designated as ABA-RESPONSIVE KINASE SUBSTRATES 1) inhibits dimerization of FBH3 in stomatal guard cells (77, 78). FBH3 homodimerizes and also heterodimerizes with FBH4 and other related bHLH proteins (78). The Ser/Thr residues important for controlling the FBH3 dimerization are conserved in FBH4 (78), suggesting that this mechanism may also participate in the phosphorylation-induced reduction of the FBH4 transcriptional activity. Detailed analyses of phosphorylation dynamics and identification of unidentified kinases and phosphatases of the FBH4 protein will further facilitate our understanding of how the N conditions regulate FBH4 activity through phosphorylation state change in the low N-induced acceleration of flowering. In addition, multiple mitogen-activated protein kinases could phosphorylate FBH1 and FBH2 proteins in vitro (79). Together with our work, these findings indicate that FBH group proteins may work as molecular switches for flowering time regulation, which are fine-tuned by multiple phosphorylation states catalyzed by various kinases under different stress conditions.

SnRK1 Activity Is Affected by N Availability.

We identified SnRK1 as one of the kinases that phosphorylate FBH4 in response to high N conditions. SnRK1/AMPK/SNF1 kinases are evolutionarily conserved in eukaryotes, all of which form heterotrimeric complexes with a catalytic α-subunit and regulatory β- and γ-subunits (65, 80, 81). However, the regulation of the plant SnRK1 kinase likely differs from that of the animal and yeast counterparts (66, 82, 83). Activations of AMPK and SNF1 need phosphorylation of specific Thr residues in the T-loop of α-subunit by upstream kinases and also require the complex formation with regulatory β- and γ-subunits (80, 81). On the other hand, SnRK1 possesses significant auto-phosphorylation of the T-loop and a background activity of the α-subunit (66, 82). Thus, the kinase activity of SnRK1 could not be determined by the detection of the T-loop phosphorylation levels. Although it is useful and widely used, measuring the expression levels of the genes regulated by SnRK1-dependent phosphorylation is a much more indirect way to assess the SnRK1 activity. To evaluate the SnRK1 activity in vivo, we generated the ACC peptide-based reporter system (Fig. 4A and SI Appendix, Fig. S10) and found that SnRK1 activity was down-regulated under the low N conditions (Fig. 4 A and B). While SnRK1/AMPK/SNF1 kinases are well characterized as key regulators of sugar and energy signaling, their roles in N signaling were unclear. Several recent studies suggest that AMPK plays an important role under N starvation stress conditions in yeast and mammals. The decreased availability of glutamate, the N source for fission yeast, activates the AMPK (84). The T-loop phosphorylation of the AMPK is up-regulated by upstream kinases under glutamate depletion, which promotes cell division at a reduced cell size in fission yeast (84). Glutamine depletion also activates the AMPK in mammalian cells, resulting in reduced proliferation (85). Plants uptake inorganic nitrogen such as nitrate and ammonium from the soil and also possess complex N metabolism networks, which is distinct from yeast and mammal ones (1, 2, 6, 11). This also suggests that plants develop unique mechanisms to sense and mediate N starvation signals in plant cells. Our development of the ACC reporter system helped us to monitor the SnRK1 activity more directly in planta, which contributes to understanding detailed SnRK1 functions in the N signaling. In addition, according to the recent report, trehalose-6-phosphate (T6P), which functions as a sugar signaling molecule in plants, binds to SnRK1α to inhibit the physical interaction of SnRK1α with upstream kinases (86). Besides, the SnRK1 activity is modulated by the redox state (87). The redox state plays a role in regulating N starvation responses in plants (88). In addition, nitric oxide, a readily diffusible free radical, affects flowering time (89). These findings suggest the possibility that N levels may be directly sensed by SnRK1 by binding to some N signaling–related metabolites and/or proteins that are involved in the regulation of intercellular N availability.

FBH4 Is Involved in Nutrient Recycling and Remobilization-Related Gene Expressions in Low N.

The floral developmental transition requires optimal nutrient allocation from source tissues (leaves) to newly developing sink tissues (floral organs). N recycling and remobilization are crucial for plant adaptation to low N conditions. Low N promotes changes in global gene expression to enhance uptake of N sources from the soil and activate metabolism for N recycling and remobilization, which increases N use efficiency (7, 10). Thus, transcriptional control of these genes is important for better reproductive growth and crop yields, especially under low N conditions. Recent research progress uncovered the detailed molecular mechanisms of primary nitrate signaling and identified several gene families of transcription factors important for the regulation (51, 52, 90–93). However, the signaling mechanisms mediating cellular N starvation are not well understood. Although negative regulators, NITRATE-INDUCIBLE, GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1/HYPERSENSITIVE TO LOW Pi-ELICITED PRIMARY ROOT SHORTENING 1 (NIGT1/HRS1), for low N-induced gene expression were identified recently (94–96), transcriptional activators of low N-induced gene expression were not known. We found that FBH4 (and possibly other FBHs) control low N-induced gene expression related to N recycling and remobilization. The recent advanced transcriptome coupled with genome-wide promoter binding analyses also suggested the potential involvement of FBH4 in the transcriptional regulation of multiple N-responsive genes (69). Therefore, FBH4 likely functions as the transcriptional activator of nutrient recycling- and remobilization-related gene expression under low N conditions.

The fbh qd mutant rosettes look larger compared with WT under the low N conditions in some figures (Fig. 1B and SI Appendix, Fig. S1). However, as the fbh qd mutants showed decreased biomass during the vegetative growth phase (SI Appendix, Fig. S16), the larger rosettes of fbh qd at later developmental stages are likely due to that the fbh qd mutants continued to grow for a longer period than WT because of the delayed flowering time. The floral transition by itself also may affect N-related metabolisms and rosette growth (6, 18). An alternative explanation is that these differences were caused by different growth conditions (hydroponic versus agar media). These explanations are not mutually exclusive. Currently, we are studying the more precise effects of FBH4 on N-associated metabolism and growth.

In summary, we revealed the crucial roles of FBH transcription factors, especially FBH4, as signaling components for low N-induced acceleration of flowering through CO and FT expression. Additionally, we found that FBH4 may regulate nutrient recycling- and remobilization-related gene expression. In response to the N deficiency, Arabidopsis plants seem to precisely coordinate both developmental and metabolic reprogramming that occurs during floral transition in part through multiple phosphorylation events of the FBH4 protein. The FBH family is conserved in various plant species including major crops (44). Because both flowering time and N use efficiency strongly impact crop yields, controlling FBH activities might be an effective way to sustainably increase agricultural production.

Materials and Methods

Detailed materials and methods for plant materials and growth conditions, flowering time, diurnal gene expression, phosphorylation analyses in hydroponics, transient expression in leaf mesophyll protoplasts, cytoplasmic and nuclear fractionation, immunoprecipitation of the FBH4 protein, in vivo SnRK1 activity assay, recombinant protein expression and purification, in vitro phosphorylation assay, immunoblot analysis, Phos-tag SDS-PAGE, and transcript analysis are provided in SI Appendix, Materials and Methods. All primer sequences used in this study are listed in SI Appendix, Table S3.

Data Availability

All study data are included in the article and/or SI Appendix.