Light serves as both the energy source for plant photosynthesis and critical environmental signal regulating plant growth and development (Jiao et al., 2007; Kami et al., 2010). Unlike animals, plants cannot perceive sunlight with their eyes, but they use a series of photoreceptors to perceive different qualitative and quantitative of ambient light. Among them, the far-red/red light photoreceptors phytochromes (PHYs, phyA to phyE), blue/UV-A light photoreceptors cryptochromes (CRYs, CRY1, and CRY2) and phototropins (PHOTs, PHOT1, and PHOT2), and UV-B light photoreceptor UV RESISTANCE LOCUS 8 (UVR8) are well studied in Arabidopsis (Cashmore et al., 1999; Briggs et al., 2001; Quail, 2002; Rizzini et al., 2011). These photoreceptors are involved in the light-dependent regulation of a variety of physiological responses, such as seedling photomorphogenesis, stomatal opening and development, photoperiodic flowering, and circadian rhythms (Ahmad and Cashmore, 1993; Guo et al., 1998; Deng and Quail, 1999; Yang et al., 2000; Kinoshita et al., 2001; Quail, 2002; Sullivan and Deng, 2003; Mao et al., 2005; Liu et al., 2008; Kang et al., 2009).

There are five phytochromes in Arabidopsis designated phyA to phyE. Among them, phyA mainly acts as the far-red light photoreceptor under far-red light to promote photomorphogenesis, while phyB is the primary red light photoreceptor that mediates red light-induced photomorphogenesis under red light (Quail, 2002). Phytochromes perceive light with the covalently attached linear tetrapyrrole chromophore and exist in two different conformations: the red light-absorbing Pr form and the far red light-absorbing Pfr form (Rockwell et al., 2006). Phytochromes are in the inactive Pr form in the cytoplasm when synthesized, and then translocate into the nucleus as the active Pfr form upon light irradiation (Kircher et al., 1999; Li et al., 2011). Once activated, they interact with a group of PHYTOCHROME-INTERACTING-FACTORS (PIFs), which are basic helix-loop-helix transcription factors and act as the key negative regulators in the phytochrome signaling cascade (Ni et al., 1998; Leivar and Quail, 2011). These interactions lead to the phosphorylation, ubiquitination and degradation of PIFs (Al-Sady et al., 2006; Shen et al., 2007, 2008), and eventually the activation or repression of their downstream target genes (Leivar and Quail, 2011). The RING-finger E3 ubiquitin ligase COP1 is the master negative regulator of light signaling, which interacts with and targets the degradation of many transcription factors in light signaling, such as HY5 and CONSTANS (CO), to control photomorphogenesis and flowering, respectively (Deng et al., 1992; Osterlund et al., 2000; Jang et al., 2008; Liu et al., 2008). Phytochromes interact with COP1, and its enhancer SPAs (SPA1–4), to repress the activity of COP1 and promote the photomorphogenesis (Jang et al., 2010; Lu et al., 2015; Sheerin et al., 2015). Cryptochromes and UVR8 regulate blue and UV-B light signaling by interacting with COP1/SPA1, respectively (Favory et al., 2009; Lian et al., 2011; Liu et al., 2011; Zuo et al., 2011). Moreover, cryptochromes mediate responses to low intensity blue light or high temperature by interacting with PIFs (Ma et al., 2016; Pedmale et al., 2016).

Brassinosteroids (BRs) are growth-promoting plant steroid hormones regulating many aspects of physiological processes of plants, including photomorphogenesis (Clouse, 2011; Lv and Li, 2020). BR is perceived by the receptor BRI1 (BRASSINOSTEROID INSENSITIVE1) and the co-receptor BAK1 (BRI1-ASSOCIATED RECEPTOR KINASE1), a membrane-localized receptor kinase complex, and then the activated BRI1 initiates the BR signal cascade through a series of phosphorylation and dephosphorylation events (Li and Chory, 1997; Li et al., 2002; Nam and Li, 2002; Kim et al., 2009; Zhu et al., 2013). BES1/BZR1 (BRI1-EMS SUPPRESSOR 1/BRASSINAZOLE-RESISTANT 1) are the key transcription factors of BR signaling, and the ratios of phosphorylated BES1/BZR1 (pBES1/pBZR1) to dephosphorylated BES1/BZR1 (dBES1/dBZR1) serve as the read-out of the BR signaling status (Wang et al., 2002; Yin et al., 2002). BIN2 (BRASSINOSTEROID INSENSITIVE2) is a GSK3-like protein kinase that acts as a negative factor in BR signaling to phosphorylate BES1/BZR1 and repress the expression of thousands of BR response genes (Li et al., 2001; Zhao et al., 2002), while protein phosphatase 2A acts as a positive regulator in BR signaling by dephosphorylating BES1/BZR1 (Tang et al., 2011).

Brassinosteroid is involved in inhibiting photomorphogenesis, as both BR biosynthesis and signaling deficient mutants display dwarf hypocotyl phenotype during photomorphogenesis (Chory et al., 1991; Li and Chory, 1997), and the gain-of-function mutant of BIN2, bin2-1, also displays a shortened hypocotyl phenotype in both darkness and light (Li et al., 2001). BIN2 integrates multiple signaling pathways by phosphorylating a range of proteins including the components in light signaling. For example, PIF3, PIF4, and PIF5 can be phosphorylated by BIN2 and then degraded via the 26S proteasome pathway to control transcription and hypocotyl growth (Bernardo-García et al., 2014; Ling et al., 2017). Meanwhile, light regulates the kinase activity of BIN2 through the COP1/SPA complex and HY5 (Ling et al., 2017; Li et al., 2020). Specifically, the COP1/SPA complex represses the activity of BIN2 and interferes with BIN2-PIF3 interaction in the dark to inhibit BIN2-mediated PIF3 destabilization (Ling et al., 2017), whereas HY5 enhances the activity of BIN2 via direct interaction in the light (Li et al., 2020). It is known that BR induced-degradation of BIN2 is dependent on the F-box protein KIB1 (Zhu et al., 2017), and that the repressor of BR signaling, the Nuclear Factor YCs (NF-YCs), interact with BIN2 to stabilize BIN2 (Zhang et al., 2021). However, whether light affects the stability of BIN2 to repress BR signaling is still unknown.

Previous studies have demonstrated that light represses BR signaling and promote photomorphogenesis through different mechanisms. For examples, both CRY1 and phyB interact with the dephosphorylated form of BES1 to inhibit its DNA-binding activity and repress BR signaling (Wang et al., 2018; Wu et al., 2019). In addition, CRY1 interacts with both BIN2 and BZR1 to bring them together and regulate hypocotyl elongation (He et al., 2019). Furthermore, phyB is shown to promote the amount of the phosphorylated BZR1 (Kim et al., 2014). In this study, we show that phyA and phyB repress BR signaling by inhibiting BR-induced degradation of BIN2 and promoting the phosphorylation of BES1. We demonstrate by protein-protein interaction studies that phyA and phyB interact directly with BIN2, which may promote the accumulation of BIN2 protein, as well as the interaction between BIN2 and BES1, leading to the phosphorylation of BES1. Our results provide a new insight into the molecular mechanism by which phyA and phyB regulate BR signaling and photomorphogenesis.

To explore whether BIN2 might regulate photomorphogenic development in far-red and red lights, we examined the hypocotyl phenotype of the gain-of-function mutant of BIN2, bin2-1 (Li et al., 2001), under red and far-red lights, respectively. The results showed that bin2-1 seedlings developed significantly shorter hypocotyls than WT in far-red and red lights (Figures 2A–D). We then analyzed anthocyanin contents in bin2-1 mutant grown in far-red and red lights, and found that bin2-1 seedlings accumulated significantly more anthocyanin than WT in far-red or red light (Figures 2E,F). These results suggest that BIN2 is involved in promoting photomorphogenesis in far-red and red lights, respectively.

It is known that BES1 and BZR1 are the important transcription factors of BR signaling, and the ratios of phosphorylated BES1/BZR1 (pBES1/pBZR1) to dephosphorylated BES1/BZR1 (dBES1/dBZR1) is the read-out of the BR signaling status (Wang et al., 2002). BIN2 phosphorylates BES1 and BZR1 to inhibit their DNA-binding activities, thus blocking the transduction of the BR signaling (Vert and Chory, 2006). Based on our demonstration that phyA mediates far-red light inhibition of the degradation of BIN2, we asked whether phyA might mediate far-red light modulation of the phosphorylation of BES1 to regulate BR signaling. To test this possibility, we firstly used the etiolated transgenic seedlings over-expressing BES1 fused to Flag in the WT background (BES1-Flag-OX/WT) (Wang et al., 2018) exposed to different lengths of time of far-red light, and examined the phosphorylation status of BES1. The results showed that the ratio of pBES1/dBES1 was increased as the far-red light exposure time increased (Figure 3A and Supplementary Figures 3, 4). We also found that red light is able to promote the accumulation of the phosphorylated of BES1, and the ratio of pBES1/dBES1 was also increased as the red light exposure time increased (Figure 3B and Supplementary Figures 3, 4). We then generated the transgenic seedlings overexpressing BES1-Flag in the phyA mutant background (BES1-Flag-OX/phyA), and analyzed the phosphorylation status of BES1 protein in BES1-Flag-OX/WT and BES1-Flag-OX/phyA seedlings adapted in the dark and then exposed to far-red light for different lengths of time, respectively. The results showed that the ratio of pBES1/dBES1 was increased faster in the WT background than in the phyA mutant background as the far-red light exposure time increased (Figures 3C,D and Supplementary Figures 3, 4). These results indicate that phyA mediates far-red light-induced accumulation of the phosphorylated BES1.

It is shown that LiCl inhibits the GSK3-like kinase activity of BIN2 and its homologs, leading to the accumulation of dephosphorylated BZR1/BES1 in vivo (Li et al., 2017). To further determine whether phyA would affect BES1 phosphorylation in a BIN2-dependent manner, we first performed immunoblotting analyses using BES1-Flag-OX/WT seedlings grown in darkness for 5 days, and then transferred to the liquid medium containing 10 mM LiCl adapted in darkness or exposed to far-red or red light for different lengths of time. The results showed that, in the presence of LiCl, the phosphorylated BES1 failed to accumulate as the far-red or red light exposure time increased, and the ratio of pBES1/dBES1 remained at a similar low level (Figures 3E,F and Supplementary Figures 3, 4). Moreover, in the presence of both LiCl and BL, the phosphorylated BES1 also hardly accumulated upon far-red or red light irradiation, and the ratio of pBES1/dBES1 was lower than in the presence of BL only or in the absent of both LiCl and BL (Supplementary Figures 1, 5). We then perform the same assays to evaluate the phosphorylation status of BES1 in the WT and phyA mutant backgrounds, respectively. The results showed that the treatment of LiCl reduced the accumulation of phosphorylated BES1 in both WT and phyA mutant backgrounds within 3 h (Figures 3G,H and Supplementary Figures 3–5). After 6 h treatment, the effects of LiCl became weak, and the ratio of pBES1/dBES1 increased faster in the WT than in the phyA mutant (Figures 3G,H and Supplementary Figures 3–5). After 24 h treatment, LiCl barely had effects, probably because the treatment was too long, which may trigger other processes affecting the phosphorylation of BES1 (Figures 3G,H and Supplementary Figures 3–5). Taken together, these results indicate that phyA-mediated far-red light promotion of the accumulation of the phosphorylated BES1 is dependent on BIN2.

To explore the genetic relationships between BIN2 and PHYA, we introgressed bin2-1 into phyA mutant background by genetic crossing and generated the phyA bin2-1 double mutant. Photomorphogenic phenotype analysis showed that the phyA bin2-1 double mutant developed significantly shorter hypocotyls than phyA mutant in far-red light (Figures 4A,B), and accumulated more anthocyanin than phyA mutant under far-red light (Figure 4C). However, the phyA bin2-1 double mutant developed a little longer hypocotyls, but produced much lower levels of anthocyanins than the bin2-1 mutant. These results indicate that BIN2 may act as an important downstream component of phyA signaling to regulate hypocotyl elongation in far-red light, but play a minor role in mediating phyA signaling to regulate anthocyanin accumulation.

Given our previous demonstrations that CRY1 or phyB inhibits auxin and GA signaling by interacting with AUX/IAA and DELLA proteins to stabilize them, respectively (Xu et al., 2018, 2021), we explored whether phyA and phyB might affect the stability of BIN2 by interacting with BIN2. To verify this assumption, we performed pull-down experiments with the recombinant His-TF-phyA-N (phyA N-terminus), His-TF-phyA-C (phyA C-terminus), His-TF-phyB-N (phyB N-terminus) and His-TF-phyB-C (phyB C-terminus) fusion proteins as preys, and GST-BIN2 as a bait. As shown in Figures 5A,B and Supplementary Figure 5, His-TF-phyA-N, His-TF-phyA-C, His-TF-phyB-N and His-TF-phyB-C were all pulled down by GST-BIN2, but not by the His-TF control, indicating that both the N and C termini of phyA and phyB interact with BIN2 protein in vitro.

To further prove whether phyA and phyB interact with BIN2 in vivo, we performed split luciferase complementation (split-LUC) assays in Nicotiana benthamiana leaves. phyA or phyB and BIN2 were fused to the N- and C-terminal halves of firefly luciferase (cLUC-phyA or cLUC-phyB and BIN2-nLUC), respectively. We found that the luciferase activity was reconstituted when cLUC-phyA or cLUC-phyB was co-expressed with BIN2-nLUC, but not when cLUC-phyA or cLUC-phyB was co-expressed with nLUC or when BIN2-nLUC was co-expressed with cLUC (Figures 5C,D). These results indicated that both phyA and phyB interact with BIN2 in vivo.

To investigate whether light quality may affect the interactions of phyA and phyB with BIN2, we performed semi-in vivo pull-down experiments. The recombinant GST-BIN2 protein was incubated with the protein extracts prepared from transgenic Arabidopsis seedlings overexpressing phyA-YFP-OX or Myc-phyB-OX that were dark-adapted before exposure to far-red or red light. As shown in Figures 5E,F and Supplementary Figure 5, BIN2 pulled down phyA and phyB from the extracts prepared from seedlings exposed to far-red and red lights, respectively, but not from those prepared from dark-adapted seedlings. These results suggest that phyA and phyB may interact with BIN2 in a far-red and red light-dependent manner, respectively.

Based on our demonstrations that phyA and phyB interact with BIN2 (Figure 5) and that phyA and phyB promote BIN2 accumulation and BES1 phosphorylation in far-red and red lights (Figures 1, 3), we explored whether phyA and phyB might promote the interaction of BIN2 with BES1. To do this, we first performed split-LUC assay in N. benthamiana leaves and confirmed the interaction between BIN2 with BES1 (Supplementary Figure 2A). We then detected the interaction strength between BIN2 and BES1 in the presence of Myc-phyA, Myc-phyB, and the control protein Myc-GUS, respectively. The results showed that the interaction of BIN2 with BES1 was enhanced significantly by phyA and phyB, but not by GUS (Figures 6A,B,D,E). Western blot assays showed that Myc-GUS was expressed at higher or similar levels compared with Myc-phyA and Myc-phyB (Figures 6C,F and Supplementary Figure 5). BIN2-nLUC and cLUC-BES1 were expressed at only slightly lower levels when co-expressed with Myc-GUS than when co-expressed with Myc-phyA (Supplementary Figures 2B, 5) or Myc-phyB (Supplementary Figures 2C, 5). These results indicate that phyA and phyB may promote the interaction of BIN2 with BES1.

We then performed cell-free pull-down assays with recombinant GST-BIN2, His-BES1 proteins and the protein extracts prepared from phyA, phyB, and phyA-YFP-OX or Myc-phyB-OX seedlings adapted in darkness and exposed to red and far-red light, respectively. We first tested this system by confirming the interaction between BIN2 and BES1, and found that the strength of the interaction between BIN2 and BES1 was enhanced as the amounts of His-BES1 increased (Supplementary Figures 2D, 5), which is consistent with the previous study (Zhao et al., 2002). We then added the protein extracts prepared from phyA, phyB, and phyA-YFP-OX or Myc-phyB-OX seedlings, and found that much more His-BES1 was pulled down by GST-BIN2 in the presence than in the absence of the activated phyA or phyB (Figures 6G,H and Supplementary Figure 5). These results indicate that phyA and phyB may promote the interaction of BIN2 with BES1 in a far-red and red light-dependent manner, respectively.

Given that the BR-deficient mutant det2 (de-etiolated 2) was initially identified in the genetic screening for mutants showing constitutive photomorphogenic phenotype in darkness (Chory et al., 1991), it is likely that light and BR signaling are connected in some way. Through 30 years’ study, some impressive progresses have been made in dissecting the molecular mechanism of the crosstalk between light and BR signaling. It is known that BES1/BZR1 and BIN2 function at the heart of BR signaling networks, as BES1/BZR1 can directly regulate the expression of thousands of the downstream BR-responsive genes, and BIN2 can phosphorylate and destabilize BES1/BZR1 to repress BR signaling (Li et al., 2001; Wang et al., 2002; Yin et al., 2002; Vert and Chory, 2006). The multiple photoreceptors and the negative regulators of light signaling can mediate light repression of BR signaling by regulating the protein stability and the DNA-binding ability of BES1 and BZR1. Specifically, UVR8, phyB and CRY1 interact with dephosphorylated BES1 and BZR1 specifically to inhibit the DNA-binding ability of BES1 and repress the expression of their target genes (Liang et al., 2018; Wang et al., 2018; Wu et al., 2019). COP1 degrades the phosphorylated BZR1 in darkness, while SINAT E3 ligases are involved in degrading dephosphorylated BES1 protein in the light (Kim et al., 2014; Yang et al., 2017). It is shown that light regulates the kinase activity of BIN2 through the COP1/SPA1 complex and HY5 (Ling et al., 2017; Li et al., 2020). However, whether light regulates BR signaling through modulating the BIN2 protein stability is not known.

In this study, we report that phyA and phyB mediate light regulation of BR signaling and photomorphogenesis by modulating the stability of BIN2 protein. we show by protein expression assays that phyA- and phyB-mediated far-red and red lights signaling inhibits BL-induced degradation of BIN2 protein (Figure 1), and that the accumulated BIN2 promotes the phosphorylation of BES1 (Figure 3). Analysis of the phenotype of bin2-1 mutant in monochromatic light conditions demonstrates that BIN2 acts to promote photomorphogenesis in far-red and red lights, respectively (Figure 2). Genetic interaction analysis indicates that BIN2 acts partially downstream of phyA to regulate photomorphogenesis under far-red light (Figure 4). We show by protein-protein interaction studies that phyA and phyB interact directly with BIN2 (Figure 5), and that phyA and phyB may promote the interaction between BIN2 and BES1 (Figure 6), which may in turn enhance the phosphorylation of BES1 by BIN2. These results, combined with the previous studies, suggest that phytochromes may mediate light inhibition of BR signaling through modulation of the activity of both BES1 and BIN2. On the one hand, they interact with the dephosphorylated BES1 to inhibit its DNA-binding ability (Wu et al., 2019); On the other hand, they stabilize BIN2 to promote the accumulation of the phosphorylated BES1, thus inhibiting its DNA-binding ability to promote photomorphogenesis (this report).

How do phyA and phyB regulate BIN2 protein stability? Previous studies have shown that phyB mediates red light induction of the degradation of EIN3 by enhancing the binding of the EBF1/EBF2 E3 ligase to EIN3 and regulate ethylene response (Shi et al., 2016), and that blue light-triggered interactions of CRY1 with AUX/IAA proteins or DELLA proteins inhibit the associations of SCF^TIR1 with AUX/IAA proteins or the interactions of the GA receptor GID1 and SCF^SLY1 with DELLA proteins to repress auxin or GA signaling (Xu et al., 2018, 2021). Given these demonstrations and our results showing that phyA and phyB interact with BIN2 directly (Figure 5), we speculate that the interactions of phyA and phyB with BIN2 may promote the dissociation of BIN2 from its E3 ligase, such as KIB1 (Zhu et al., 2017), to inhibit the ubiquitination and degradation of BIN2. Since it is known that NF-YCs promotes the autophosphorylation of BIN2 to protect BIN2 degradation, while the phosphorylated BSU1 dephosphorylates BIN2 to trigger the degradation of BIN2 via the 26S proteasome (Kim et al., 2009; Ryu et al., 2010; Zhang et al., 2021), it is also possible that the interactions of phyA and phyB with BIN2 may result in the inhibition of the dephosphorylation and degradation of BIN2. These possibilities will be worth exploring in future studies. In sum, we propose that, in darkness and in the presence of BR, as phyA and phyB are at their inactive Pr form, and localized in the cytoplasm, they cannot interact with BIN2 and BES1. BIN2 is ubiquitinated by KIB1 and degraded through the 26S proteasome, thus BES1 is dephosphorylated, and able to bind to its target genes to promote their expression and skotomorphogenesis (Figure 7A); Upon light irradiation, phyA and phyB are activated and translocate from the cytoplasm to the nucleus, where they may inhibit the association of BIN2 with KIB1 to stabilize BIN2 and enhance the interaction of BIN2 with BES1. As a result, BIN2 accumulates and the phosphorylation of BES1 is enhanced, and BES1 binding to its target genes is inhibited, leading to the inhibition of BR signaling and the eventual promotion of photomorphogenesis (Figure 7B).

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

WW and H-QY conceived the project and wrote the manuscript. JZ, GY, and WW designed the research plan. JZ and GY carried out the most of the experiments. All authors contributed the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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