Shade-induced nuclear localization of PIF7 is regulated by phosphorylation and 14-3-3 proteins in Arabidopsis

Shade avoidance syndrome enables shaded plants to grow and compete effectively against their neighbors. In Arabidopsis, the shade-induced de-phosphorylation of the transcription factor PIF7 (PHYTOCHROME-INTERACTING FACTOR 7) is the key event linking light perception to stem elongation. However, the mechanism through which phosphorylation regulates the activity of PIF7 is unclear. Here, we show that shade light induces the de-phosphorylation and nuclear accumulation of PIF7. Phosphorylation-resistant site mutations in PIF7 result in increased nuclear localization and shade-induced gene expression, and consequently augment hypocotyl elongation. PIF7 interacts with 14-3-3 proteins. Blocking the interaction between PIF7 and 14-3-3 proteins or reducing the expression of 14-3-3 proteins accelerates shade-induced nuclear localization and de-phosphorylation of PIF7, and enhances the shade phenotype. By contrast, the 14-3-3 overexpressing line displays an attenuated shade phenotype. These studies demonstrate a phosphorylation-dependent translocation of PIF7 when plants are in shade and a novel mechanism involving 14-3-3 proteins, mediated by the retention of PIF7 in the cytoplasm that suppresses the shade response.


Introduction
Because chlorophyll preferentially absorbs light in the red and blue ranges but not in the far-red range of the light spectrum, a perceived decrease in the ratio of red/far-red (R/FR) radiation, and thus in photosynthetically active radiation (PAR) of between 400 and 700 nm, provides a signal that shading by other plants is imminent. Shade-intolerant plants, such as Arabidopsis thaliana, sense this reduction and initiate the shade avoidance syndrome (SAS). In the SAS, energy resources are reallocated from storage organs to stem-like organs, including hypocotyls and petioles, thereby enabling plants to initiate a rapid growth response (Cole et al., 2011;Casal, 2012). Prolonged shade exposure leads to reduced branching, early flowering and seed set, and reduced yield (Ballaré, 1999;Franklin and Whitelam, 2005;Procko et al., 2014).
The R/FR-absorbing photoreceptor phytochrome B (phyB) plays the most dominant role during the SAS (Reed et al., 1993). In an open environment under sunlight (where the ratio of R/FR is about 1.2-1.5), most of the phyB is in the far-red-absorbing (Pfr) active form and moves to the nucleus, where it interacts with basic helix-loop-helix (bHLH) proteins known as PHYTOCHROME-INTERACT-ING FACTORS (PIFs) (Duek and Fankhauser, 2005;Leivar and Quail, 2011). The photoactivation of phyB induces the rapid phosphorylation of PIF1/3/4/5 prior to their degradation (Shen et al., 2005;Al-Sady et al., 2006;Shen et al., 2007), although the short half-lives of these PIFs impedes the tracing of the phosphorylated forms. When plants are in the shade, phyB is mostly in the inactive red-absorbing (Pr) cytosolic form, which facilitates the accumulation of PIFs and restricts the growth of the hypocotyl (Lorrain et al., 2008;Leivar and Quail, 2011).
PIF7 is a major regulator of the shade response, as shown by the severe shade-defective phenotype of pif7 mutants (Li et al., 2012;de Wit et al., 2015;Mizuno et al., 2015). PIF7 is less vulnerable than PIF1/3/4/5 to the rapid turnover of induced by the Pfr form of phyB (Leivar et al., 2008). Instead, the activity of PIF7 is controlled by rapid de-phosphorylation in response to shade, which leads to its binding to G-boxes in the promoters of auxin biosynthesis genes, causing an increase in auxin levels and a rapid growth response (Li et al., 2012).
The 14-3-3 proteins are highly conserved in all eukaryotes. Research in recent years has revealed several putative 14-3-3 targets in plants (Jaspert et al., 2011;Wang et al., 2011;Yoon and Kieber, 2013;Zhou et al., 2014). These studies have revealed that 14-3-3 proteins can interact with the phosphorylated forms of their client proteins in response to certain signals, and that this binding finalizes the signaling event by enabling a change in the subcellular localization, protein stability or intrinsic enzymatic activity of the client, or by promoting an association between the client and other proteins. The cellular 14-3-3 'pool' enables these proteins to react to altered signaling cues in an immediate and precise way through dynamic interactions with their clients.
Here, we demonstrate a shade induction of the nuclear localization of dephosphorylated PIF7 and a role for the 14-3-3 proteins in the cytoplasmic retention of PIF7 in Arabidopsis. Our work reveals a novel mechanism that rapidly switches PIF7 function in response to light conditions and the role of 14-3-3 proteins in SAS.

Shade induces the rapid nuclear localization of PIF7
To monitor the cellular localization of PIF7, we generated 35S::GFP-PIF7 transgenic plants and analyzed the GFP signal in white-light-grown seedlings before and after shade treatment. Impressively, GFP-PIF7 rapidly accumulated in the nucleus when plants were placed in the shade, as observed in the cotyledon and the hypocotyl of transgenic lines. The extent of this shade response decreased gradually from the top to the bottom of the hypocotyls (Figure 1-figure supplement 1a). At the top of hypocotyls of two independent transgenic lines, the nuclear/cytoplasmic ratio of GFP-PIF7 increased within 5 min of moving the plants into shade and continued to increase for 45 min (Figure 1a,b). The localization of GFP, which was used as the control, was not affected by shade (Figure 1a . Shade treatment resulted in an increase of PIF7 in the nucleus and a decrease of PIF7 in the non-nuclear fraction, indicating that the increased nuclear fraction of PIF7 was probably translocated from the cytoplasmic compartment.

PIF7 interacts with 14-3-3 proteins
To identify potential PIF7-binding proteins, we conducted a yeast two-hybrid (Y2H) screen using PIF7 as bait. Interestingly, this study identified the phosphopeptide-binding protein 14-3-3 k as a binding partner of PIF7. As we have shown, 14-3-3 l and 14-3-3 k can interact with PIF7 when coexpressed in the yeast system ( Figure 2a). A bimolecular fluorescence complementation (BiFC) assay in Nicotiana benthamiana cells also supported the interaction between PIF7 and 14-3-3 l/k ( Figure 2b). In fact, there are at least six 14-3-3 proteins (14-3-3 l, k, c, g, m and e) that can interact with PIF7 in Y2H and BiFC assays (Figure 2a The 14-3-3 proteins are well known to bind phosphopeptides. When GST (glutathione S-transferase)À14-3-3 fusion proteins were used to pull down the protein lysate from the 35S::PIF7-Flash transgenic line grown under white-light and shade conditions, more PIF7 protein was enriched in the white-light-grown seedlings ( Figure 2c). Moreover, a co-immunoprecipitation experiment further confirmed that 14-3-3 proteins are precipitated with PIF7 from white-light-grown transgenic seedlings (Figure 2d), probably because more PIF7 is phosphorylated in white-light-grown seedlings.
Phosphorylation sites of PIF7 mediate its binding to 14-3-3 proteins There are two types of specific 14-3-3 binding motifs in mammalian and plant systems, mode I, R/ KXXpSX, and mode II, R/KXXXpSXP (where X = any amino acid, R = arginine, K = lysine, pS = phosphoserine and p=proline) (Muslin et al., 1996;Muslin and Xing, 2000;Schoonheim et al., 2007). Although an obvious interaction occurred between PIF7 and the 14-3-3 proteins, no typical mode I or mode II motifs can be identified in PIF7. We reasoned that non-canonical motifs, such as RXXS, might mediate the interaction between PIF7 and 14-3-3 proteins, as observed in PHOT1 (Figure 3-figure supplement 1a) (Kinoshita et al., 2003). Transgenic Arabidopsis expressing GFP-PIF7 or GFP was grown on 1/2 MS medium under white light for 5 days. Seedlings were treated with shade for 5, 15, or 25 min, and images of the GFP signal were obtained using confocal microscopy. White scale bar represents 25 mm. (b) Kinetics of the shade-induced nuclear accumulation of GFP-PIF7. GFP-PIF7 or GFP seedlings were treated as in (a). ImageJ was used to quantify the fluorescence intensities. Ratios of the nuclear and cytoplasmic signal intensities were calculated from 10 cells for each treatment. Error bars represent standard deviations. (c) Shade induces the nuclear localization of dephosphorylated PIF7. Immunoblot of the PIF7-Flash proteins using anti-Myc antibody in the total, nuclear and non-nuclear fractions from white-lightand shade-treated seedlings. Histone H3 is a nuclear marker, and the RuBisCO large subunit (RbcL), a chloroplast protein, is a non-nuclear fraction marker. DOI: https://doi.org/10.7554/eLife.31636.002 The following source data and figure supplement are available for figure 1: Source data 1. Source files for the ratios of the nuclear and cytoplasmic signal intensities in Figure 1b. In order to identify the phosphorylation sites of PIF7, we immunoprecipitated the PIF7 complex in 35S::PIF7-Flash transgenic plants and then performed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) experiment. The results showed that PIF7 was phosphorylated at S139 and S141 in seedlings grown under while light and in seedlings treated with 5 min of shade, but not in seedlings treated with 1 hr of shade ( Figure 3-figure supplement 1b). These phosphorylation sites constituted the putative 14-3-3 binding sequence (RSGSET). Because the LC-MS/MS experiment did not cover the entire protein, we also used the online software NetPhos 2.0 to predict the potential phosphorylation sites of PIF7, and the results showed the following high-score sites: S78, S80, S125, S139 and S141 (Figure 3-figure supplement 1c). On the basis of the 14-3-3-binding sequence in CDC25C (KTVSLC) (Chan et al., 2011), the sequence KDGSCS (75-80) of PIF7 may be another possible 14-3-3 binding motif ( Figure 3-figure supplement 1a).

Phosphorylation sites of PIF7 are important for its localization and function
To investigate the roles of the phosphorylation sites on the cellular localization of PIF7, we generated 35S::GFP-PIF7 derivatives in which the serine residues were mutated to alanine residues (PIF7 [2A] and PIF7[5A] or to aspartic acid residues (PIF7[2D] and PIF7[5D]). When over-expressed in tobacco leaf cells, the wildtype GFP-PIF7 localized in both the cytoplasm and the nucleus. The GFP-PIF7(2A), GFP-PIF7(5A) and GFP-PIF74 mutants exhibited strong nuclear signals, whereas the GFP-PIF7(2D) and GFP-PIF7(5D) mutants showed stronger cytoplasmic signals than did GFP-PIF7 (Figure 4a; Figure 4-figure supplement 1). To further examine the effect of the phosphorylation sites on PIF7 localization in Arabidopsis, we generated transgenic plants expressing GFP-PIF74 and GFP-PIF7(5A). Consistent with the findings in tobacco, GFP-PIF74 and GFP-PIF7(5A) displayed stronger nuclear signals than did wildtype GFP-PIF7 ( Figure 4b). The shade treatment caused substantial translocation of the wildtype GFP-PIF7, whereas its effects on GFP-PIF74 and GFP-PIF7(5A) were minimal (Figure 4b).
To  Figure 4g). Notably, the effects of PIF7(2A) overexpression were greater than those of PIF7 overexpression, indicating the nuclear localization is critical for the function of PIF7. It is also noteworthy that the shade-induced effects on gene expression and hypocotyl elongation were not totally abolished in the PIF7(2A) transgenic lines (Figure 4f,g), implying that other phosphorylation sites or other mechanisms that regulate PIF7 may exist. For example, shade may regulate additional essential factors that interact with PIF7.

14-3-3 proteins regulate the localization and dephosphorylation of PIF7
As PIF7 translocates to the nucleus under shade conditions and is able to bind 14-3-3 proteins, we hypothesized that 14-3-3 proteins sequester phosphorylated PIF7 in the cytoplasm.
Previous studies have shown that the interactions of 14-3-3 proteins with their client proteins can be disrupted by the R18 peptide (Wang et al., 1999). We therefore took advantage of this peptide to discover that the shade-induced nuclear localization of GFP-PIF7 (Figure 5a  14-3-3 proteins are negative regulators of the shade response Consistently, treatment with R18 significantly promoted shade-induced hypocotyl elongation ( Figure 6a) and shade-induced gene (IAA19 and YUCCA8) expression ( Figure 6b). However, this promotion is blunted in R18-treated pif7-1. We also determined whether a loss of 14-3-3 l or 14-3-3 k function would affect shade-induced hypocotyl elongation in Arabidopsis. Single mutants for each gene and the double mutant displayed enhanced shade responses, whereas the 14-3-3 l transgenic lines (14-3-3 l OE and 35S::FLAG-HA-14-3-3 l) showed a reduced shade response (Figure 6c (Figure 6d). Moreover, with R18-treated pif7-1, the hypocotyl length of the double mutant of pif7-1 and 14-3-3 lÀ2 was consistently more like that in pif7-1 (Figure 6c), suggesting that the function of 14-3-3 proteins is mediated by PIF7. Overall, the phenotype and gene expression analysis demonstrated that 14-3-3 l and 14-3-3 k negatively regulate the shade response through PIF7.

Discussion
In the current study, we improved the model of shade signal transduction by demonstrating a shade-sensitive subcellular localization of PIF7, which is conferred by interactions with 14-3-3 proteins ( Figure 7). Under white-light conditions, phosphorylation of PIF7 results in the cytoplasmic location of this protein and enables its binding to 14-3-3 proteins. When plants sense shade conditions, unknown phosphatases remove the phosphorylation of PIF7. De-phosphorylated PIF7 does not interact with 14-3-3 proteins and translocates to the nucleus, where it promotes the expression of downstream genes, leading to shade-induced phenotypic changes. 14-3-3 proteins retain phosphorylated PIF7 in the cytoplasm to regulate shade-induced hypocotyl elongation negatively. Current and previous studies have provided several lines of evidence to strongly support the importance of the phosphorylation of PIFs for the transcriptional activity of these proteins. Several phosphorylation sites have been identified in PIF1/3/4 (Bu et al., 2011;Ni et al., 2013;Bernardo-García et al., 2014). These phosphorylation events lead to apparent ubiquitylation and degradation through the ubiquitin-proteasome system (Al-Sady et al., 2006). In sustained light, PIF1/3/4/5 are maintained at a relatively low steady-state level. After subsequent exposure to shade light, new protein synthesis is required for the accumulation of these PIF proteins (Lorrain et al., 2008). PIF7, however, is a light-stable bHLH factor (Leivar et al., 2008;Li et al., 2012).
In the current study, at least two phosphorylation sites (S139 and S141) in PIF7 are found to be critical for its activity and for hypocotyl elongation. These sites also mediate the binding of PIF7 with 14-3-3 l and k. Shade-induced nuclear accumulation of PIF7 and the constitutive nuclear localization of PIF7(2A) mutants in transgenic plants suggest that the interaction of 14-3-3 proteins and PIF7 is involved in determining the subcellular distribution of PIF7. Although there was a lack of interaction in the BiFC and pull-down assays, S-to-D substitution does enhance the interaction between 14-3-3 proteins and PIF7 in vivo. Moreover, PIF7(2D)-Flash was mainly localized in cytoplasm and was unable to complement the shade-defective gene expression and phenotype of pif7-1, which functionally reflects the phosphorylation defects in the in vivo system. The phosphorylation state of PIF7 determines its localization and function, and also affects its ability to bind to 14-3-3 proteins. 14-3-3 proteins have been reported to regulate transcription factors by sequestering them in the cytoplasm; for example, BZR1 and RSG are regulated by the binding of 14-3-3 l, w or m (Igarashi et al., 2001;Gampala et al., 2007). Although functional redundancy and different combinations of 14-3-3 isoforms bring difficulties in clarifying the specific roles of 14-3-3 proteins (Jaspert et al., 2011), the involvement of 14-3-3 proteins in light signaling has been illustrated by the elongated hypocotyls (relative to those of Col-0 seedlings) of 14-3-3 k, n and c mutants grown in red light (Mayfield et al., 2007;Adams et al., 2014). In our work, there was no significant effect of R18 treatment and 14-3-3 mutations on PIF7's localization and phosphorylation state under white light, which could be due to the strong light radiance, the inhibitory potency of R18 or the redundancy of the 13 14-3-3 proteins in Arabidopsis. By contrast, 14-3-3s significantly delay the shadeinduced translocation and de-phosphorylation of PIF7 ( Figure 5), and consequently enhance shadeinduced hypocotyl elongation which is dependent on PIF7 (Figure 6). The weak shade phenotype might be caused by functional redundancy of 14-3-3 proteins. It is also possible that a compensatory increase in other isoforms occurs in 14-3-3 lk. It is possible that 14-3-3 proteins sequester phosphorylated PIF7 in cytoplasm by protecting the PIF7 proteins from phosphatases during the transition from white light to shade.
PIF7 is a major controller for shade-induced hypocotyl elongation, as demonstrated by the severe shade-defective phenotype of pif7 mutants (Li et al., 2012). It is known that Arabidopsis grows rapidly in response to the shade stimulus, with an induction of PIL1 transcript levels detectable after only 8 min of low R/FR and growth measurable after just 30 or 45 min of exposure to shade (Salter et al., 2003;Cole et al., 2011). A quick shade regulatory mechanism is required to achieve this rapid response. Phosphorylation-dependent translocation of PIF7 is such a quick mechanism that can give rise to efficient photomorphogenesis. Moreover, several negative regulators of PIF7 have been shown to reduce the transcriptional activity of light-responsive genes and to prevent exaggerated shade responses (Hornitschek et al., 2009;Galstyan et al., 2011;Hao et al., 2012;Li et al., 2014). In our current study, the binding of 14-3-3 proteins delays the de-phosphorylation and nuclear import of PIF7 in response to shading, forming another layer of regulation to determine the appropriate SAS.
A conserved 14-3-3-binding motif has been identified in PIF3 (RNPSPP), and PIF3 has been found to be a 14-3-3 interaction partner in an affinity-purification assay using His-tagged 14-3-3-coated beads (Adams et al., 2014). However, the disruption of putative phosphorylation sites on the 14-3-3-binding motifs of PIF3 did not prevent 14-3-3 from binding to PIF3 or disturb the nuclear localization of PIF3 (Adams et al., 2014). Although the 14-3-3-binding sites of PIF7 are not typical of those of the other PIFs, the functional analysis of mutations of these sites (from Ser to Ala) illustrated their critical roles in PIF7 function.
The expression level and localization of 14-3-3 l protein remain stable after shading is introduced ( Figure 6-figure supplement 1), suggesting that 14-3-3 proteins probably exert their cargo function constantly. The activity of PIF7 is mostly determined by its phosphorylation status, which is controlled by a kinase and a phosphatase that are light-dependent. To date, CK2 (Bu et al., 2011), PPKs (Ni et al., 2017) and BIN2 (Bernardo-García et al., 2014) have been reported to be the kinases of PIF1, PIF3 and PIF4, and TOPP4 has been reported to be the phosphatase of PIF5 (Yue et al., 2016); however, no specific kinase or phosphatase of PIF7 has yet been identified. The shade-induced localization response of PIF7 varied in the different tissues (Figure 1-figure supplement 1), implying that the phosphorylation of PIF7 is probably regulated by upstream signals that have tissue and/or developmental specificity. One important goal for the future is to identify these upstream kinases, as well as the phosphatase(s) responsible for the de-phosphorylation of Ser 139 and 140, and hence for the disassociation of 14-3-3 proteins in response to shading. Col-0 seedlings grown in plates containing 200 mg/ml R18 or R18(Lys) peptide under white light or shade conditions. Seedlings were grown under white light for 4 days and maintained in white light or transferred to shade for next 5 days before the measurement of hypocotyl length. More than 20 seedlings were measured. Significant differences between two treatments are shown as asterisks. ***p<0.001 by Student's t-test. (b) IAA19 and YUCCA8 expression level in the Col-0 and pif7-1 seedlings treated with R18 and R18(Lys) under white light or shade. Seedlings were grown with 1/2 MS medium containing 200 mg/ml R18 or R18(Lys) under white light for 5 days. Then, the seedlings were kept in white light or transferred to shade for 1 hr. Mean ± SE from three independent biological replicates, after normalization to the internal control AT2G39960, are shown. Significant differences between two treatments are indicated by asterisks. ***p<0.001, by Student's t-test. (c) Quantification of the hypocotyl length of Col-0, pif7-1, 14-3-3 mutants and the overexpression line grown under white light or shade. More than 20 seedlings were measured. Bars marked with different letters denote significant differences (p<0.05) of the means of hypocotyl length. (d) Shade induction of IAA19 and YUCCA8 in Col-0, pif7-1, 14-3-3 mutants and 14-3-3 l OE. The seedlings were grown under white light for 5 days. Then, the seedlings were kept in white light or transferred to shade for 1 hr. The expression levels were normalized to a reference gene (AT2G39960) and then normalized to the expression under white light condition. The relative shade inductions were shown. Significant differences between mutants and Col-0 are indicated by asterisks. *p<0.05, **p<0.01,***p<0.001 by Student's t-test. DOI: https://doi.org/10.7554/eLife.31636.018 The following figure supplement is available for figure 6: Our findings offer novel insights into the mechanism through which a key transcription factor is activated by light. We already knew that a high R/FR light ration promotes the nuclear import of phyB, which positively regulates photomorphogenesis. Here, we propose that shade promotes the nuclear import of PIF7, which promotes SAS. Subcellular translocations contribute to the antagonistic action of phyB and PIF7. More detailed cooperation on light-mediated development will be necessary in the future.

Plant materials and growth conditions
All of the Arabidopsis thaliana plants used in this study were of the Columbia-0 ecotype. The mutants of the 14-3-3 l T-DNA lines (Salk_075219C and CS482153) and the 14-3-3 k T-DNA lines (Salk_148929C and Salk_071097) were obtained from ABRC. The 14-3-3 l and 14-3-3 k double mutant (Salk_071097X Salk_075219C), the 35S::FLAG-HA-14-3-3 l transgenic plants (14-3-3 l OE) (Zhou et al., 2014) and the 35S::PIF7-Flash plants (Li et al., 2012) have been described previously. For the phenotypic analysis, seeds were germinated on 1/2 Murashige and Skoog (MS) medium (Duchefa Biochemie, Haarlem, The Netherlands) plates with 1% agar (Sangon, Shanghai, China) and without sucrose. After stratification, the plates were incubated in growth chambers under continuous white light for 4 days at 22˚C, and then the plates were either left in white light or transferred to canopy shade for 5 days before hypocotyl measurements were performed. HR-350 (HiPoint, Taiwan) was used to measure the light conditions. The shade conditions were as described for previous studies (Tao et al., 2008;Li et al., 2012). Nicotiana benthamiana plants were grown at 26˚C under 16-hr-light long-day conditions.

Identification of PIF7-interacting proteins by LC-MS/MS
The different light-treated 35S::PIF7-Flash seedlings were ground to a fine powder in liquid nitrogen and solubilized with lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 2 mM DTT, 1% NP40, 10% glycerol and protease inhibitor cocktail) (Roche, USA). The extracts were incubated at 4˚C for 1 hr on a rotating wheel, and the insoluble material was removed by centrifugation at 20,000 x g at 4 C for 15 min three times until the supernatant was clear. The supernatant was incubated with a prewashed anti-FLAG M2 agarose gel (Cat# A2220 RRID: AB_10063035, Sigma-Aldrich, USA) at 4 C for 3 hr in the rotating wheel. The beads were recovered by centrifugation at 800 rpm at 4˚C for 2-5 min. After six washes with lysis buffer, SDS-loading buffer was added to the pellet fraction. The samples were boiled for 5 min, centrifuged at maximum speed for 15 min, and then loaded onto an SDS-PAGE gel. After running the gel halfway, the gel was cut into 1 mm 3 cubes and sent for MS analysis. The trypsin-digested peptides were concentrated and analyzed using a Finniqan LTQ mass spectrometer (Thermoquest, San Jose, USA) coupled with a surveyor HPLC system.

Confocal microscopy and quantitation of the fluorescent protein signal
The fluorescence images of GFP and YFP expression were obtained with a Leica confocal microscope (Leica SP8) at 488 and 514 nm. GFP-PIF7 transgenic lines were grown in white light and then transferred to the shade for 0, 5 15, 25, and 45 min. The fluorescence at each time point was recorded using a 40 Â 1.3 objective lens. ImageJ (http://rsb.info.nih.gov/ij/) was used to quantify the fluorescence intensities. The images were converted to an 8-bit format, and the fluorescence intensity was integrated from all pixels in the selected area. To measure the ratio between the nuclear and cytoplasmic signals for each cell, the entire cellular and nuclear area was selected for quantification of fluorescence intensity. The cytoplasmic intensity was calculated by subtracting the value for the nuclear area from that for the whole cell. The ratio between the nuclear and cytoplasmic signals was calculated for 10 cells, and three repeated measurements were performed in each condition.

Yeast two-hybrid screens and assay
A Matchmaker Gold Yeast Two-Hybrid system was used. The CDS of PIF7 was cloned into a pGBKT7 vector and used as bait to identify interacting proteins from a cDNA library, which was prepared by Oebiotech (China) with RNA from Col-0 seedlings grown under white-light conditions for 4 days. The cDNA synthesized from this material was cloned into the bait vector pGADT7. The interactions were tested on SD medium without Leu, Trp, and His but with 5 mM 3-amino-1,2,4-triazole (3AT, Sigma), using the yeast strain AH109 according to the manufacturer's manual (Clontech).
To confirm the interaction between PIF7 and 14-3-3 proteins, the CDS of PIF7, PIF7(2A) and PIF7 (2D) was cloned into pGADT7 and 14-3-3s were cloned into pGBKT7. Interactions were tested on SD medium without Leu, Trp and His but with 5 mM 3AT.

Semi-in vivo pull-down assay
Plant materials were ground with liquid nitrogen and re-suspended in extraction buffer (100 mM Tris-HCl [pH 7.5], 300 mM NaCl, 2 mM EDTA, 1% Trion X-100, 10% glycerol, and protease inhibitor cocktail). Protein extracts were centrifuged at 20,000 x g for 10 min, and the resulting supernatant was incubated with pretreated GST-14-3-3 beads for 2 hr. GST was used as a negative control. Beads were re-suspended with SDS-PAGE loading buffer and analyzed by SDS-PAGE and immunoblotting.

Generation of transgenic plants
For the overexpression of PIF7 fused with GFP, the full-length CDS of PIF7 and mutated PIF7 (4, 5A) were cloned into pMDC43 and transformed into a Col-0 background. To generate transgenic plants that overexpressed mutated PIF7 in the pif7-1 background, the mutated PIF7(2A) and PIF7 (2D) were created in a plasmid of 35S::PIF7-Flash using site-directed mutagenesis. All the constructs were transformed into Agrobacterium GV3101. The primers are listed in Supplementary file 1.

Hypocotyl measurements
Quantitative measurements of hypocotyls were performed on scanned images of seedlings using ImageJ software. For measurements of mutants and stable transgenic lines, at least 20 seedlings were used per treatment or genotype. For hypocotyl analysis of T1 transgenic lines under shade, the numbers of seedlings with elongated hypocotyls were counted.

Gene expression analysis by quantitative real-time RT-PCR
Total RNA was extracted using an RNApre Plant Kit (TIANGEN, China), and the first-strand cDNA was synthesized using a FastQuant RT kit (with gDNase) (TIANGEN, China). Real-time PCR was performed with a Biorad CFX Connect system. All of the oligonucleotide primers were listed in Supplementary file 1.