Introduction

Higher plants respond flexibly to their surroundings as sessile organisms. Among environmental cues, light is an essential element, not only for photosynthesis to produce the necessary energy as autotrophs, but also for plant growth and development by responding to fluctuating environments (Jing and Lin 2020). The monitoring of light in plants is mediated by various photoreceptors, including phytochromes (Legris et al. 2019). Phytochromes are red (R) and far-red (FR) photoreceptors that regulate various plant photoresponses, such as germination, de-etiolation, shade avoidance, leaf senescence, and flowering (Tripathi et al. 2019). Phytochromes are known to function as a molecular switch with physiologically active FR light-absorbing (Pfr) and inactive R light-absorbing (Pr) forms (Li et al. 2011). Upon absorbing light, the Pr-to-Pfr photoactivation induces a highly regulated signaling network for plant growth and development in response to light environments, which includes the translocation of phytochromes into the nucleus and interaction of phytochromes with a wide array of signaling partners (Hoang et al. 2019; Legris et al. 2019).

There are tens of known signaling partners that physically interact with phytochromes, suggesting that phytochromes regulate plant light signaling via protein–protein interactions (Bae and Choi 2008). Among them, phytochrome-interacting factors (PIFs) are suggested as central players in phytochrome-mediated light signaling networks (Leivar and Quail 2011). PIFs belong to bHLH (basic helix-loop-helix) transcription factors with conserved active phytochrome-binding motifs in the N-terminal domain, and eight PIFs have been identified in Arabidopsis thaliana (Pham et al. 2018a; Favero 2020; Oh et al. 2020). They usually promote seedling skotomorphogenesis, i.e., etiolated growth in the dark, such as long hypocotyls, closed cotyledons, and no chlorophyll biosynthesis. In contrast, PIFs repress seedling photomorphogenesis, including seed germination and de-etiolation, through the regulated expression of more than a thousand genes (Shin et al. 2009; Pham et al. 2018b). As another important signaling component for phytochrome-mediated light signaling, a member of bZIP (basic leucine zipper) transcription factor family, elongated hypocotyl 5 (HY5) is a master transcription factor that promotes photomorphogenesis downstream to photoreceptors (Lee et al. 2007; Gangappa and Botto 2016). Therefore, the principal regulatory mechanism of phytochromes for light signaling might be the transcriptional regulation of photoresponsive genes via the inactivation of negative transcriptional factors such as PIFs and via the accumulation of positive transcriptional factors such as HY5 (Tripathi et al. 2019; Jing and Lin 2020).

Among PIFs, phytochrome-interacting factor 3 (PIF3) is the founding member that negatively regulates phytochrome-mediated light signaling (Ni et al. 1998; Kim et al. 2003). The interaction of PIF3 with photoactivated phytochromes leads to its phosphorylation and subsequent degradation via the ubiquitin/26S proteasome pathway (Al-Sady et al. 2006). More recently, phytochromes are reported to function as protein kinases that can directly phosphorylate PIF3 (Shin et al. 2016). In addition, PIF3 phosphorylation can be occurred due to the influence of other kinases (Ni et al. 2017). Collectively, a regulatory model for phytochrome-mediated photomorphogenesis has been suggested. Upon light exposure, photoactivated phytochromes move to the nucleus, where they physically interact with PIFs and induce their phosphorylation and protein degradation, contributing to the initiation of the photomorphogenic development of plants (Hoang et al. 2019; Favero 2020). Therefore, the phytochrome-mediated removal of PIFs, the negative regulators of photomorphogenesis, might play a critical role in plant light signaling.

Although PIFs were originally discovered in the phytochrome-mediated light signaling pathways, they may also have functions in integrating multiple signaling pathways (Paik et al. 2017). In particular, PIF3 functions in chlorophyll biosynthesis, the regulation of diurnal hypocotyl elongation, and the modulation of cotyledon opening during de-etiolation (Shin et al. 2009; Soy et al. 2016; Dong et al. 2019). In addition, PIF3 affects plant hormone signaling, including ethylene, auxin, gibberellin, and abscisic acid, and also regulates cold tolerance by regulating the expression of C-repeat binding factors (Bours et al. 2015; Li et al. 2016; Yu and Huang 2017; Jiang et al. 2020; Liang et al. 2020). Furthermore, PIF3 is involved in regulating the circadian response and protecting seedlings from reactive oxygen species (Chen et al. 2013; Soy et al. 2016). Therefore, the important function of PIF3 is becoming increasingly apparent.

Although there is such increasing data on the importance of PIF3 in plant growth and development being available, its study has been limited due to the lack of an authentic PIF3 antibody. In particular, PIF3 protein shows dynamic behaviors in plants, so temporal and spatial analyses of the PIF3 protein are required. Epitope tagging could be used to detect PIF3 in plants, but these methods require a considerable amount of time to generate transgenic plants and the behaviors of the tagged-PIF3 might not reflect the exact intrinsic function. Therefore, in this study, a polyclonal antibody with a high affinity to PIF3 was produced in rabbits through immunization to the electroeluted recombinant protein using inclusion bodies. The antibody specifically interacted with PIF3 but not with the seven other PIFs in Arabidopsis. With this antibody, light-dependent degradation and accumulation behaviors of PIF3 could be successfully analyzed in plants, and the elongated growth phenotype of phytochrome B (phyB)-deficient Arabidopsis could be explained by a higher PIF3 level at night than that in the control plant. Therefore, the PIF3 antibody obtained in this study could be helpful to determine the multiple functions of PIF3 in plant growth and development in the future.

Results and Discussion

Production of a Specific Polyclonal Antibody against PIF3

Previously, Arabidopsis GIGANTEA antibody (α-GI) was successfully produced with electroeluted proteins using inclusion bodies (Khaleda et al. 2017). Although the protein is denatured, this method provides large amounts of antigens with high purity, possibly inducing effective immune responses for antibody production. Thus, PIF3 was expressed as inclusion bodies (Supplementary Fig. S1), and used for polyclonal antibody production in rabbits after electroelution (hereafter, α-PIF3). Then, the specificity of the α-PIF3 antibody was investigated using different PIFs. For this, eight PIFs identified in Arabidopsis have been prepared using Escherichia coli protein expression systems (Supplementary Fig. S2 and Table S1). As PIFs are transcriptional factors, they are usually expressed as inclusion bodies, which makes it difficult to purify the recombinant proteins. To overcome this challenge, a two affinity-tag system has been developed and used for the purification of PIFs, in which GST- and strep-tags are fused to N- and C-termini, respectively (Jeong et al. 2016; Shin et al. 2016). With this system, recombinant proteins of six PIFs (PIF1, PIF3, PIF4, PIF6, PIF7, and PIF8) were expressed successfully and purified by streptavidin affinity chromatography (see the SDS-PAGE gel in Fig. 1). However, the other two PIFs (PIF2 and PIF5) were not expressed in this system, so a cold-inducible promoter system (pCold TF vector) was used for their expression, including PIF3. Finally, all eight PIF proteins were successfully purified and used to test the specificity of the produced antibody. The results showed that the antibody interacted specifically only with PIF3 but not with the other seven PIFs (Fig. 1).

Fig. 1
figure 1

Specificity test of the α-PIF3 antibody using recombinant proteins of eight PIFs in Arabidopsis thaliana. Six PIFs (PIF1, PIF3, PIF4, PIF6, PIF7, and PIF8) were expressed and purified as GST/strep-tagged proteins, and three PIFs (PIF2, PIF3, and PIF5) as TF/2B8/strep-tagged proteins (labeled as TF in parenthesis). It is notable that two purified PIF3 proteins with either GST/strep-tag or TF/2B8/strep-tag were used in this analysis. Approximately 1 μg of each purified recombinant protein was run on 10% SDS-PAGE and western blotting was performed with the purified α-PIF3 antibody generated in this study (1:3000 dilution). Asterisks (*) in red indicate protein bands of the corresponding PIFs

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Next, endogenous PIF3 in plants was detected using the produced antibody with different dilutions. For this, dark-grown seedlings were used for the extraction of total proteins, because PIF3 is degraded in light in a phytochrome-dependent manner (Al-Sady et al. 2006). In this experiment, Col-0 (wild-type Arabidopsis), pif3 (PIF3-deficient Col-0), and PIF3:eGFP (Col-0 expressing eGFP-fused PIF3) were included. As expected, an endogenous PIF3 band (524 aa; ~ 57.6 kDa) was detected in Col-0 but not in the pif3 plant (Fig. 2a). In the PIF3:eGFP plant, endogenous PIF3 and eGFP-fused PIF3 (763 aa; ~ 83.8 kDa) bands were both detected. Interestingly, this result suggests that the endogenous expression level of PIF3 is higher than that in the transgenic plant with cassava vein mosaic virus (CsVMV) promoter. Thus, the expression level of PIF3 in transgenic plants with constitutive promoters, such as 35S and CsVMV, will be lower than that in wild-type plants, which may be useful for interpreting the results of transgenic pif3 plants expressing PIF3. Moreover, these experiments were conducted with different dilutions of the purified α-PIF3 antibody, and results showed that the 1:10,000 dilution was sufficient to detect endogenous PIF3 in Col-0 (Fig. 2a). These results indicate that the antibody produced in this study had high sensitivity to detect the antigen. Considering background noise in western blots, a dilution of 1:3000 was used for further analyses.

Fig. 2
figure 2

Sensitivity and specificity tests of the α-PIF3 antibody in plants. a Western blots showing the sensitivity of α-PIF3 using 3.5-day-old dark-grown seedlings. Col-0, wild-type Arabidopsis; pif3, PIF3-deficient Arabidopsis; PIF3:eGFP, transgenic Col-0 expressing eGFP-fused PIF3. 80 μg of plant extract was run on 10% SDS-PAGE, and western blotting was performed using α-PIF3 with 1:1000, 1:3000, 1:5000, and 1:10,000 dilutions. A. thaliana translationally controlled tumor protein (AtTCTP; At3g16640) was used as the loading control. b Western blot showing the specificity of α-PIF3 using different pif mutants. 80 μg of plant extracts obtained from 3.5-day-old dark-grown seedlings of four pif mutants (pif1, pif3, pif4, and pif5) was used for western blotting with α-PIF3 (1:3000). AtTCTP was used as the loading control

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Among the eight PIFs, four (PIF1, PIF3, PIF4, and PIF5) play major roles in plant growth and development (Leivar et al. 2008; Shin et al. 2009; Pham et al. 2018a). Thus, immunoblotting was performed using the total extracts from pif1, pif3, pif4, and pif5 plants with the α-PIF3 antibody, and the result confirmed in vivo specificity toward PIF3 (Fig. 2b). Collectively, an authentic α-PIF3 antibody with high specificity and sensitivity was successfully produced in the present study.

Phytochrome-Induced Degradation of PIF3 in Plants

PIF3 is phosphorylated and degraded in a phytochrome-dependent manner (Al-Sady et al. 2006; Shin et al. 2016). There are five phytochromes (phyA to phyE) in Arabidopsis, and it is known that phyA regulates FR light signaling, while phyB to phyE regulate R light signaling (Mathews 2010). Accordingly, PIF3 is degraded under FR light conditions in a phyA-dependent manner, and degradation under R light conditions is mainly mediated by phyB. To test this, immunoblotting was undertaken with plant extracts from Ler (wild-type Arabidopsis), phyA-201 (phyA-deficient Ler), phyAB (i.e., phyA-201phyB-5; phyA- and phyB-deficient Ler), and phyABCDE (all five phytochrome-deficient Ler). In the Ler plant, PIF3 was detected in the dark, but degraded under R, FR, and WL conditions (Fig. 3). In contrast, degradation of PIF3 in the phyABCDE plant was not observed. In the phyA-201 plant, PIF3 was degraded under R and WL conditions but not under the FR condition, confirming that phyA is necessary for FR-mediated PIF3 degradation. On the other hand, PIF3 in the phyAB plant was degraded under the WL condition but not under the FR condition, suggesting the role of phyB in R-mediated PIF3 degradation. However, PIF3 was partially degraded in the phyAB plant under the R condition, which might be due to the presence of phyC–phyE that can also mediate R-light signaling (Franklin et al. 2003; Adam et al. 2013).

Fig. 3
figure 3

Analysis of the light-induced degradation of PIF3 using α-PIF3 in plants. Ler, wild-type Arabidopsis; phyA-201, phyA-deficient Arabidopsis; phyAB, phyA- and phyB-deficient Arabidopsis (phyA-201phyB-5); phyABCDE, all five phytochrome (phyA to phyE)-deficient Arabidopsis. Total proteins were extracted from 3.5-day-old dark-grown seedlings (D), or after irradiation with red (R, 10 μmol m−2 s−1), far-red (FR, 5 μmol m−2 s−1) or white light (WL, 100 μmol m−2 s−1) for 30 min. 80 μg of extracted protein was used for western blotting with α-PIF3 (1:3000). AtTCTP was used as the loading control

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To further investigate phytochrome-mediated PIF3 degradation in plants, PIF3 degradation was analyzed in a time-dependent manner. In these experiments, A-OX (transgenic phyA-201 expressing wild-type phyA) and B-OX (transgenic phyB-5 expressing wild-type phyB) were included. FR-dependent PIF3 degradation was observed in Ler and A-OX, but not in phyA-201 and phyABCDE (Fig. 4a). When the degradation rates were estimated from immunoblots, PIF3 degradation was faster in A-OX than in Ler (see the graph in Fig. 4a). This result indicates that the expression level of phyA in transgenic plants is higher than the endogenous phyA level, which is consistent with the observed shorter hypocotyls of A-OX in previous studies (Jeong et al. 2016; Shin et al. 2016). Under R conditions, PIF3 degradation was observed in Ler and B-OX, but not in phyABCDE (Fig. 4b). In the case of phyB-5 plant, PIF3 degradation was observed with a much slower rate, which is also explained by the presence of phyC–phyE. In addition, shifted PIF3 bands were observed during the degradation, indicating the occurrence of the phosphorylated forms (marked as PIF3-P). Overall, the present study demonstrates that FR- and R-dependent PIF3 degradation is mediated by phyA and phyB–phyE, respectively.

Fig. 4
figure 4

Phytochrome-mediated PIF3 degradation and phosphorylation in plants. a FR-dependent degradation of PIF3. A-OX, transgenic phyA-201 expressing wild-type phyA. b R-dependent degradation of PIF3. phyB-5, phyB-deficient Arabidopsis; B-OX, transgenic phyB-5 expressing wild-type phyB. 3.5-day-old dark-grown seedlings (D) were irradiated with FR (5 μmol m−2 s−1) or R (10 μmol m−2 s−1) light for 5, 10, or 15 min before harvesting. 80 μg of extracted protein was used for western blotting with α-PIF3 (1:3000). PIF3-P indicates the phosphorylated PIF3 form. AtTCTP was used as the loading control. Graphs show relative PIF3 levels to AtTCTP in the western blots, assuming the PIF3/AtTCTP level in the dark is 1

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PIF3 Accumulation in the Dark for Elongated Growth in phyB-Deficient Arabidopsis

Among phytochrome-deficient Arabidopsis, the phyB plant shows the most dramatic differences in growing phenotype, i.e., constitutive shade avoidance responses (Casal 2013; Martinez-Garcia et al. 2014). Under a canopy habitat, plants rapidly elongate their stems in search of light at the expense of leaf growth and reproductive development (Franklin and Whitelam 2005). Among these shade avoidance responses, the stimulation of elongation is remarkably rapid, usually causing dramatically accelerated flowering. Thus, the phyB plants show elongated phenotypes with early flowering. When the photoresponses of the phyB-5 plant were compared with those of the control plant (Ler) by measuring hypocotyl lengths, phyB-5 seedlings showed elongated hypocotyls under both continuous R and WL conditions, being 2.1- and 1.9-fold longer than Ler seedlings, respectively (Fig. 5a). In addition, 4-week-grown phyB-5 plants flowered earlier than Ler (Fig. 5b). These phenotypes can be explained by the constitutive shade avoidance responses in the phyB-deficient Arabidopsis.

Fig. 5
figure 5

Analysis of PIF3 accumulation in the dark. a Hypocotyl de-etiolation of representative Ler and phyB-5 seedlings under different light conditions. Seedlings were grown for 4.5 days in the dark (D) or under continuous red (cR, 5 μmol m−2 s−1), or continuous white light (cWL, 100 μmol m−2 s−1). Scale bar = 5.0 mm. b Representative photographs of 4-week-grown plants. Plants were grown in a culture room (21 °C) under long day conditions (16 h light/8 h dark). The numbers of rosette leaves at bolting are shown at the bottom of the images. Scale bar = 3.0 cm. c Western blots showing PIF3 accumulation in the dark. 3.5-day-old seedlings grown under the long day conditions (0 h) were transferred into darkness and incubated further for the indicated times. d Relative PIF3 levels to AtTCTP in c. e–f Expression analysis of genes involved in elongated growth. RNA was extracted from 3.5-day-old seedlings grown under long day conditions (0 h) or transferred and incubated in the dark for 3 and 12 h. The transcript levels of YUC8 (e) and IAA29 (f) were analyzed by real-time PCR, using that of ACT2 for normalization. Relative expression levels were estimated with the transcript level in Ler at 0 h set to 1, and data are expressed as means ± s.d. (n = 3)

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In general, the elongated growth of plants is regulated with a diurnal cycle, i.e., elongated growth at night and repression in the daylight. Based on PIF3 function for the induction of the elongated growth (Soy et al. 2016), it can be hypothesized that the PIF3 level in the phyB-5 plant might be higher than that in the control plant (Ler), especially with the expectation of a greater accumulation of PIF3 at night. To test this hypothesis, seedlings grown in long day conditions were transferred to darkness, and PIF3 accumulation were investigated. The results showed that the protein levels of PIF3 in Ler increased with the length of incubated time in the dark, demonstrating PIF3 accumulation in the dark or at night (Fig. 5c). Up to 9 h after transfer into darkness, PIF3 accumulation was low. In contrast, PIF3 accumulation in the phyB-5 plant was much higher than that in the Ler plant, in which the PIF3 level at 3 h after the transfer into darkness was similar to that at 12–15 h after the transfer in Ler (Fig. 5d). Accordingly, YUCCA8 (YUC8) and INDOLE-3-ACETIC ACID INDUCIBLE 29 (IAA29), two genes involved in elongated growth (Kim et al. 2017), were expressed higher in the phyB-5 plant than in the Ler plant (Fig. 5e, f). These results were consistent with the observed phenotypes of the phyB-5 plant. Therefore, the present results help explain why the phyB-deficient plant exhibits elongated growth and suggest the importance of the α-PIF3 antibody in studying the phytochrome function in plants.

Applications for Immunoprecipitation Assays using α-PIF3

In general, antibodies can be used to analyze proteins’ behaviors, such as post-translational modifications (e.g., phosphorylation), protein–protein interactions (e.g., IP) and protein–DNA interaction (e.g., chromatin IP). Thus, the α-PIF3 antibody was applied for the protein–protein interaction analysis between phyB and PIF3. First, using phyAB and phyB:eGFP (transgenic phyAB expressing eGFP-fused wild-type phyB) seedlings, a Co-IP was conducted with α-PIF3. In this experiment, total extracts were obtained from seedlings in the dark (for the Pr form of phyB) or under R light (for the Pfr form). Results showed a Pfr-specific interaction of phyB with PIF3 in plants (Fig. 6a). In addition, the PIF3 bands after Co-IP were upper-shifted and smeared, which can be explained by the occurrence of PIF3 phosphorylated forms under R light. Moreover, protein–protein interaction analysis with recombinant proteins was also performed via IP. When the Pr and Pfr forms of recombinant phyB protein were mixed with recombinant PIF3 protein and immunoprecipitated using α-PIF3, the Pfr-specific interaction was obtained (Fig. 6b). Therefore, the α-PIF3 antibody produced in this study can be used as a valuable tool to study the multi-faceted functions of PIF3 in plants.

Fig. 6
figure 6

Interaction analysis between PIF3 and phyB using α-PIF3. a Co-immunoprecipitation (Co-IP) analysis. 3.5-day-old dark-grown seedlings of phyAB and phyB:eGFP (transgenic phyAB with eGFP-fused wild-type phyB) were pretreated with 50 μM MG132 for 4 h, then kept in the dark (D) or exposed to 5 min of red light (R, 10 μmol m−2 s−1) before harvesting. Extracted proteins were mixed with the PIF3 antibody-bound Dynabeads™ Protein G beads, and western blotting was then performed with α-GFP and α-PIF3 to detect phyB and PIF3, respectively. b In vitro interaction analysis by immunoprecipitation (IP). Pr, red light-absorbing form of phyB (i.e., inactive form); Pfr, far-red light-absorbing form (i.e., active form). 2.0 μg of PIF3 and full-length phyB (either Pr or Pfr) recombinant proteins were mixed with α-PIF3 bound Dynabeads™ Protein G beads, and western blotting was then performed with α-phyB and α-PIF3 to detect phyB and PIF3, respectively

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Conclusions

Among the eight PIF members identified in A. thaliana, PIF3 is the first to be discovered to function as a negative regulator in plant photomorphogenesis. After this discovery, the roles of PIF3 in plant growth and development were extensively studied, and expanding roles of PIF3 in multiple processes, such as hormone signaling and cold tolerance, have recently been reported (Jiang et al. 2020; Leivar et al. 2020; Xu and Deng 2020). Thus, the antibody against PIF3 with high sensitivity and specificity is desired to study the multiple functions of PIF3 in plants. In this study, a specific and sensitive polyclonal antibody against PIF3 was successfully produced using recombinant proteins expressed as inclusion bodies. The purified antibody detected only PIF3 but no other PIFs, and exhibited a high sensitivity to detect the endogenous PIF3 level in wild-type Arabidopsis, such as Col-0 and Ler. Using this antibody, phytochrome-mediated PIF3 phosphorylation and degradation was confirmed, in which PIF3 degradation under FR light was mediated by phyA and that under R light was mediated by other phytochromes, including phyB. Moreover, it was demonstrated that the elongated growth phenotype of phyB-deficient Arabidopsis is positively co-related with the elevated PIF3 level in the plant, especially at night. The antibody was also successfully used to determine the light-dependent interaction of phyB with PIF3 both in vivo and in vitro. Therefore, the PIF3 antibody produced in this study can be used as a powerful tool for elucidating the molecular mechanisms of PIF3 in multiple processes in plants, including photomorphogenesis, hormone signaling, and abiotic/biotic stress responses, such as cold tolerance.

Materials and Methods

Expression of PIF3 as Inclusion Bodies for Antibody Production

Previously, the Arabidopsis α-GI antibody was successfully produced using inclusion bodies (Khaleda et al. 2017). Thus, Arabidopsis PIF3 (At1g09530) was also expressed as inclusion bodies. For this, the PIF3 gene was subcloned into pET28a (Invitrogen) to express the his-tagged recombinant PIF3 protein. The primers and restriction enzymes used for the cloning are listed in Supplementary Table S2. The constructs were transformed into E. coli strain BL21(DE3)-CodonPlus and expressed as previously described (Shin et al. 2016). After sonicating the cells, the pellet (ppt) was harvested and washed with TE buffer (100 mM Tris, pH 7.8, 1 mM EDTA). Then, the suspended ppt was run on 10% SDS-PAGE and the concentration of the PIF3 inclusion bodies was estimated using BSA as a standard. Overall, 10 mg ppt was used for the elution of PIF3 proteins to produce PIF3 polyclonal antibody (i.e., α-PIF3).

Generation of Polyclonal Antibodies in Rabbits

The PIF3 antibody was produced according to the methods of a previous study (Khaleda et al. 2017). Briefly, PIF3 protein bands were excised from 10% SDS-PAGE gels and eluted using an Electro-Eluter (Bio-Rad). Then, the eluted protein (0.5 mg) was mixed with complete Freund’s adjuvant at a 1:1 (v/v) ratio and the antigen was injected into rabbits with triple immunization. Rabbits were housed in accordance with the ethical principles and experimental procedures to minimize suffering, following a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at Gyeongsang National University (GNU-151006-B0058). After obtaining anti-serum from the blood samples of the immunized rabbits, the polyclonal antibody was further purified using antigen-specific affinity purification with the recombinant PIF3 protein. For this, the purified PIF3 proteins were separated on 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride (PVDF) membranes. After staining with Ponseaus S solution for 1 min, the PIF3 protein bands were excised and blocked with 1% (w/v) BSA in 1 × TBS (50 mM Tris, 150 mM NaCl, pH 7.6). Subsequently, the excised blot strips were mixed with the antiserum (diluted 1 mL antiserum in 9 mL 1% BSA) and incubated overnight at 4 °C. After washing with 1 × TBS, PIF3-bound polyclonal antibodies were eluted with 900 μL of 0.1 M glycine (pH 2.5) and immediately neutralized with 100 μL of 2 M Tris–HCl (pH 8.0). The concentration of the purified PIF3 antibody was approximately 10 μg/μL, and α-PIF3 was stored at 4 °C until further use.

Preparation of A. thaliana PIFs

Eight PIFs in A. thaliana were expressed and purified to investigate the specificity of the PIF3 antibody produced in this study. For the cloning of PIF1, PIF3, PIF4, PIF6, PIF7, and PIF8, pGEX 4T-1 (GE Healthcare) with the streptavidin affinity-tag (SAWRHPQFGG; strep-tag) at the 3′ end was used (hereafter, named pStrep). The primers and restriction enzymes used for the cloning are listed in Supplementary Table S2. The E. coli strain BL21(DE3)-CodonPlus cells with these constructs were used for the expression of recombinant proteins which were purified by streptavidin affinity chromatography, as previously described (Shin et al. 2016). In this system, the recombinant proteins were expressed with fusions of glutathione S-transferase (GST) and strep affinity-tags to their N- and C-termini, respectively (Supplementary Fig. S2). For the cloning of PIF2, PIF3, and PIF5, pCold TF (TaKaRa) with 2B8 (RDPLPFFPP; BioJane, Korea) and strep affinity-tags at the 3′ end was used. In this system, the recombinant proteins were expressed with fusions of his-affinity (6 × His) and TF (trigger factor) tags to N-terminus and 2B8- and strep-affinity tags to C-terminus (Supplementary Fig. S2). For the expression of recombinant proteins, E. coli strain BL21(DE3)-CodonPlus cells were incubated at 37 °C until OD600 reached 0.4–0.6, and then transferred to 15 °C. After incubation for 1 h, IPTG was added to a final concentration of 1 mM and the culture was further incubated overnight at 15 °C. After the cells were harvested and resuspended in ice-cold TE buffer, protein extracts were obtained by repeated sonication of the cells, followed by centrifugation. Then, the supernatant was filtrated with a 0.45 μm microfilter to remove insoluble particles, and the recombinant proteins were purified by streptavidin affinity chromatography. The concentrations of the recombinant proteins were determined using a Quant-iT Protein Assay Kit (Invitrogen).

Western Blot Analysis Using Purified PIF3 Antibody

For western blots with recombinant proteins, 1 μg of each purified protein was run on 10% SDS-PAGE and electroblotted on to a PVDF membrane. Immunoblot analysis was performed with the rabbit α-PIF3 antibody with 1:3000 dilution and peroxidase-conjugated donkey anti-rabbit IgG (Na934v, GE healthcare). The PIF3 protein was detected by chemiluminescence using a Clarity Max Western ECL Substrate (Bio-Rad). For western blots with plant extracts, 10 μL of seeds were germinated and grown for 3.5 days in the dark before collecting the seedlings. The seedlings were then frozen in liquid nitrogen and ground using TissueRuptor (Qiagen) with 250 μL of an extraction buffer (100 mM Tris–HCl, pH 8.0, 100 mM NaH2PO4, 8 M Urea, 10 μM MG132, 1 mM PMSF, 1 × protease inhibitor cocktail). The supernatants were used to determine protein concentrations of total plant extracts using a BCA Protein Assay Kit (TaKaRa). Then, 80 μg of the extracts was used for western blot analysis with α-PIF3 (1:3000) to detect PIF3. For loading controls, A. thaliana translationally controlled tumor protein (AtTCTP; At3g16640) was immunodetected with the rabbit α-AtTCTP antibody (1:10,000), as previously described (Kim et al. 2012).

Analysis of PIF3 Degradation and Accumulation in Plants

To detect PIF3 in plants, seeds were surface-sterilized and stratified at 4 °C for 3 d in the dark, and were sown on 0.6% phytoagar plates containing half-strength MS salts and vitamins. The seeds were then exposed to white light (WL; 100 μmol m−2 s−1) for 4 h to promote germination, returned to darkness (21 °C) for 24 h, and grown for 3.5 days in the dark. The seedlings were then kept in the dark or exposed to R (10 μmol m−2 s−1), FR (5 μmol m−2 s−1) or WL (100 μmol m−2 s−1) for the indicated time. After harvesting seedlings, western blotting was performed with α-PIF3 (1:3000) and α-AtTCTP (1:10,000), as described above. For PIF3 accumulation analysis in plants, after growth under long day conditions (16 h light/8 h dark photoperiod) for 3.5 days, the seedlings under fluorescent light conditions were transferred to dark conditions (set to 0 h) and incubated for sampling. Seedlings were collected at the indicated times and immediately frozen in liquid nitrogen. Then, the plant extracts were used for western blotting to detect PIF3.

Plant Materials

In this study, pif3 (Col-0 background) and PIF3:eGFP (transgenic Col-0 expressing eGFP-fused PIF3) plants as well as Col-0 were used to analyze the sensitivity of α-PIF3. Among the eight PIFs, the pif1, pif3, pif4, and pif5 plants were used to analyze the specificity of α-PIF3. Phytochrome-deficient or overexpressing plants, phyA-201, phyB-5, phyAB (phyA-201phyB-5), phyABCDE (Strasser et al. 2010), A-OX (transgenic phyA-201 expressing Avena sativa phyA; Jeong et al. 2016), and B-OX (transgenic phyB-5 expressing A. thaliana phyB; Jeong et al. 2016), were used to analyze phytochrome-mediated PIF3 degradation. For the comparative analysis of photoresponses between Ler and phyB-5, seedlings were grown at 21 °C for 4.5 days in the dark (D) or under continuous R (cR, 5 μmol m−2 s−1) light or continuous WL (cWL, 100 μmol m−2 s−1), and images of the seedlings were obtained. To compare growth phenotype, Arabidopsis plants were grown on soil in a culture room (21 °C with a 16-h photoperiod) and flowering time was estimated by counting the leaf numbers at bolting. Images of 4-week-grown plants were then obtained.

Gene Expression Analysis

After growth under long day conditions for 3.5 days, the seedlings under fluorescent light conditions were transferred to darkness and incubated further for 3 and 24 h. Then, RNA was extracted from the seedlings using a FavorPrep™ Plant Total RNA Mini Kit (Favorgen) and cDNA was synthesized from 1 μg of RNA using RNA to cDNA EcoDry™ Premix (TaKaRa). Quantitative real-time RT-PCR analysis was performed using Stratagene MX3005p with Brilliant III Ultra-Fast SYBR Green Q-PCR Master Mix (Agilent Technologies). The transcript levels of YUC8 and IAA29 were normalized to that of ACT2. The primers used for this analysis are listed in Supplementary Table S2.

Co-Immunoprecipitation (Co-IP)

For Co-IP analysis, 3.5-day-old dark-grown seedlings of phyAB and phyB:eGFP (transgenic phyAB with eGFP-fused wild-type phyB) plants were pretreated with 50 μM of MG132 for 4 h and then kept in the dark or exposed to R light (10 μmol m−2 s−1) for 5 min. Seedlings were immediately frozen in liquid nitrogen and total proteins were extracted using TissueRuptor (Qiagen) in a Co-IP buffer (50 mM Tris–HCl, pH 7.5, 200 mM NaCl, 1 mM EDTA, 10% glycerol, 0.1% Twin 20, 1 mM DTT, 10 μM MG132, 1 mM PMSF, 1 × protease inhibitor cocktail, 10 mM NaF, and 2 mM Na3VO4). After centrifugation, supernatants were used for Co-IP. For this, 1 μg of α-PIF3 was incubated with Dynabeads™ Protein G (Invitrogen) for 1 h at 4 °C, and α-PIF3-bound beads were washed with the Co-IP buffer (without 10 μM MG132, 1 mM PMSF, 1 × protease inhibitor cocktail, 10 mM NaF, and 2 mM Na3VO4) three times. Subsequently, the supernatants were mixed with the washed α-PIF3-bound beads for 30 min at 4 °C. After washing with a buffer (1 × PBS, 1% Triton X-100, 3% BSA) three times, SDS loading buffer was added and western blot analysis was performed with α-PIF3 (1:3,000) and α-GFP (1:5,000; sc-9996, Santa Cruz Biotechnology).

In vitro Protein–Protein Interaction Analysis

For the interaction analysis between phyB and PIF3 in vitro, recombinant protein of full-length strep-tagged phyB was expressed in Pichia pastoris cells and purified by streptavidin affinity chromatography, as previously described (Shin et al. 2014, 2016). Then, Pr and Pfr were prepared by irradiating FR (50 μmol m−2 s−1 for 15 min) or R light (20 μmol m−2 s−1 for 30 min). For in vitro interaction analysis by IP, phyB (2.0 μg) and PIF3 (2.0 μg) were mixed and incubated for 30 min at 4 °C in 500 μL of 1 × TBS, and α-PIF3-bound Dynabeads™ Protein G beads were added and incubated for an additional 30 min. PIF3 and phyB in the input and immunoprecipitated fractions were detected using Clarity Max Western ECL Substrate (Bio-Rad) with α-PIF3 (1:3000) and α-phyB (1:2000; aN-20, Santa Cruz Biotechnology).