14-3-3 Proteins Participate in Light Signaling through Association with PHYTOCHROME INTERACTING FACTORs

14-3-3 proteins are regulatory proteins found in all eukaryotes and are known to selectively interact with phosphorylated proteins to regulate physiological processes. Through an affinity purification screening, many light-related proteins were recovered as 14-3-3 candidate binding partners. Yeast two-hybrid analysis revealed that the 14-3-3 kappa isoform (14-3-3κ) could bind to PHYTOCHROME INTERACTING FACTOR3 (PIF3) and CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1). Further analysis by in vitro pull-down assay confirmed the interaction between 14-3-3κ and PIF3. Interruption of putative phosphorylation sites on the 14-3-3 binding motifs of PIF3 was not sufficient to inhibit 14-3-3κ from binding or to disturb nuclear localization of PIF3. It was also indicated that 14-3-3κ could bind to other members of the PIF family, such as PIF1 and PIF6, but not to LONG HYPOCOTYL IN FAR-RED1 (HFR1). 14-3-3 mutants, as well as the PIF3 overexpressor, displayed longer hypocotyls, and a pif3 mutant displayed shorter hypocotyls than the wild-type in red light, suggesting that 14-3-3 proteins are positive regulators of photomorphogenesis and function antagonistically with PIF3. Consequently, our results indicate that 14-3-3 proteins bind to PIFs and initiate photomorphogenesis in response to a light signal.

A basic helix-loop-helix (bHLH) transcription factor, PHYTOCHROME INTERACTING FACTOR3 (PIF3), is a key repressor of photomorphogenesis in the dark. PIF3 specifically binds to a cis-acting regulatory element, G-box, in the promoters of various light-regulated genes [16] to regulate their expression. Targets of PIF3 include LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1), the components required for a functional circadian clock [17]. PIF3 also negatively regulates light responses through interaction with HISTONE DEACETYLASE15 (HDA15) and synergistic transcriptional repression of genes related to chlorophyll biosynthesis and photosynthesis in the dark [18]. PIF3 is regulated at both the transcriptional and the post-translational levels. A noncoding RNA, HIDDEN TREASURE (HD), has recently been identified as repressing PIF3 transcription by associating with the chromatin of the first intron of PIF3 and promoting photomorphogenesis in continuous red (cR) light [19]. At the post-translational level, light rapidly induces the conversion of red/far-red (R/FR) light photoreceptors, phytochrome A (phyA) and phyB, to their active forms and consequent nuclear import and promotes direct interaction with DNA-bound PIF3 [20][21][22]. It has been reported that nuclear transport of phyB is mainly facilitated by PIF1, PIF3, PIF4 and PIF5, whereas that of phyA is facilitated by FAR-RED ELONGATED HYPOCOTYL1 (FHY1) and FHY1-LIKE (FHL) [23][24][25]. Upon PIF3-phy interaction in nuclear speckles, PIF3 is phosphorylated, and both PIF3 and phyB are ubiquitinated by Bric-a-Brack/Tramtrack/Broad (BTB)-Cullin3-type E3 ligases, LIGHT-RESPONSE BTBs (LRBs) and degraded by the 26S proteasome [26][27][28]. It has been shown recently that the N-terminal half of phyB is capable of inhibiting PIF3 from binding to its target DNA, but is not sufficient for speckle localization, PIF3 degradation nor inhibition of skotomorphogenesis [29,30]. In parallel with LRB-dependent degradation, PIF3 promotes interaction between phyB and another E3 ligase, CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), which is a negative regulator of photomorphogenesis, and COP1 ubiquitinates phyB (not PIF3), which is thereby degraded by the 26S proteasome [31]. By contrast, PIF3 degradation by R/FR light requires phyA, phyB and phyD, not COP1, but PIF3 accumulation in the dark requires COP1 [32]. Light-induced phosphorylation of PIF3 is crucial for its rapid degradation and consequent initiation of photomorphogenesis. A recent study has indicated that multiple Ser/Thr phosphorylation events in response to light collectively promote PIF3 degradation [33].
Our previous work has reported the involvement of 14-3-3κ and χ isoforms in plant nutrient metabolic pathways [7]. From the screening conducted [7], some light-related proteins, including PIF3, were also recovered. Although a proteomic analysis has revealed that PIF4 co-purifies with the 14-3-3ω isoform [6], direct interaction between 14-3-3 proteins and PIFs has not been confirmed. In this work, the role of 14-3-3 proteins in the light signaling pathway is discussed. Yeast two-hybrid and in vitro pull-down assays indicated the interaction between a 14-3-3 protein and light-related proteins, such as PIF3 and COP1. Mutants of 14-3-3s, as well as a PIF3 overexpressor showed longer hypocotyl lengths compared to the wild-type in cR light, suggesting that 14-3-3 proteins are important for the functional regulation of photomorphogenesis, likely the result of the interaction with phosphorylated PIF3 and the phyB-PIF3-COP1 signalosome.

Yeast Two-Hybrid Analysis of 14-3-3 with Light-Related Proteins
In our previous report, we identified 14-3-3 interacting partners through an affinity purification method using His-tagged 14-3-3-coated beads and elution by adding 14-3-3 binding phosphopeptides followed by sequencing through trypsin digestion and mass spectrometry. 14-3-3 proteins have been shown to bind to nitrogen-and sulfur-related proteins and play roles in nutrient metabolic pathways in Arabidopsis [7]. Proteins involved in potassium and phosphate transport have also been suggested as the binding partners of 14-3-3 proteins. From the same affinity purification screening, we found a series of light-related proteins that might interact with 14-3-3 proteins (Table 1). PHOT2 and phyB are B and R/FR light photoreceptors, respectively [34,35], and COP1 and PIF3 are known to interact with phyB [20,31]. PHOT2 is also reported to bind with COP1 [36]. RGA1 and RGL2 are members of DELLA proteins and reported to bind to PIF3 [37]. Table 1. List of light-related 14-3-3 candidate partners recovered from the affinity purification strategy [7]. The Arabidopsis Genome Identification (AGI) and presence of the 14-3-3 binding motif in each protein are also indicated.

In Vitro Pull-Down of 14-3-3 with PIFs
To confirm the interaction between 14-3-3κ and PIF3, in vitro pull-down was performed. His-tagged 14-3-3κ was successfully pulled down with maltose-binding protein (MBP)-tagged PIF3 and MBP-PIF3ala1+2, but not with the MBP negative control ( Figure 1B), suggesting that 14-3-3κ binds to PIF3 and that the disturbance of these phosphorylation sites does not interrupt this interaction, being consistent with the yeast two-hybrid results. Other members of the PIF family were also investigated for their ability to bind with 14-3-3 proteins. PIF1 and PIF6 were shown to interact with 14-3-3κ, but not LONG HYPOCOTYL IN FAR-RED1 (HFR1), according to the in vitro pull-down assay ( Figure 1C,D).

Subcellular Localization of GFP-PIF3ala1+2
The light-related transcription factor PIF3 is known to localize in the nucleus, where it binds to phyB in response to light [32]. In order to investigate the integrity of PIF3 with deactivated putative phosphorylation sites, GFP-fused Arabidopsis PIF3 constructs were created, and subcellular localization of each protein was analyzed in onion cells through transient expression. In contrast to the GFP control, whose signal was distributed throughout the cytoplasm, GFP-PIF3 showed clear localization in the nuclei ( Figure 2). This nuclear localization was maintained for GFP-PIF3ala1 and GFP-PIF3ala1+2.

Discussion
PIF3 is a bHLH transcription factor, which serves as a negative regulator of photomorphogenesis. Since perception of and response to light signals are of the utmost importance in plants, the mechanisms by which PIF3 is regulated have been intensively studied. Recent findings have filled some gaps in our knowledge, and what appears to be the whole picture of this mechanism has been revealed. That is, PIF3 binds to the promoter of the light-related genes and regulates their transcription in the dark [16]. Upon light signaling, phytochromes are activated and translocated to the nuclear speckle, where they bind to PIF3 [20][21][22]. PIF3 is phosphorylated, ubiquitinated by E3 ligases, called LRBs, and degraded by the 26S proteasome, so that the light-related processes can be induced [26][27][28]. Here, we propose 14-3-3 proteins serving as yet another layer of the regulatory components of PIF-dependent light signaling in plants.
According to our yeast two-hybrid and in vitro pull-down results, it is suggested that 14-3-3κ directly binds to PIF3, as well as to PIF1 and PIF6. Due to their extremely short half-life in light [38], phosphorylated forms of PIFs were not detected in vivo. Thus, further confirmation of an interaction between 14-3-3κ and PIFs by an in vivo co-immunoprecipitation assay has not been successful so far. Although PIF3 is considered to be the predominant repressor of photomorphogenesis, other PIFs, including PIF1 and PIF6, are known to bind with phytochromes in response to light and participate in the repression of photomorphogenesis in the dark [38][39][40][41]. It is therefore assumed that 14-3-3κ is a general regulatory component of light signaling through the interaction with PIFs. By contrast, our data suggest that 14-3-3κ does not bind with HFR1, an atypical bHLH protein. Although HFR1 shares sequence similarity with PIF3, it has an atypical basic region and cannot bind to phytochromes [42]. Recent findings have indicated that HFR1, together with PIF3-LIKE1 (PIL1), function antagonistically to PIFs and promote photomorphogenesis [43]. These results, together with our results, suggest that 14-3-3 proteins specifically associate with PIFs to regulate a photomorphogenic response. Since 14-3-3 proteins selectively bind to phosphorylated proteins, putative phosphorylation sites on the 14-3-3 binding motifs of PIF3 were disrupted by exchanging Ser with Ala (PIF3ala1+2). However, PIF3ala1+2 protein was capable of proper nuclear localization and 14-3-3 binding. PIF3ala1 (Ser-23 exchanged with Ala) and PIF3ala2 (each of Ser-115 and Ser-116 exchanged with Ala) regions are the only sites on PIF3 that harbor the complete 14-3-3 binding motif, RXXSXP. According to a previous report, the phosphopeptide signals of S23 and S116 are approximately 20% in both dark and R light [33]. These findings suggest that incomplete motifs, such as RXXS/T, may be the actual 14-3-3 binding sites of PIF3 ( Figure S1), as is the case for PHOT1 [11]. It has recently been reported that multiple light-induced phosphorylation sites in PIF3 are collectively required for rapid degradation of PIF3 and phyB [33]. It is speculated that ala1+2 was not sufficient to cancel PIF3's function or that other phosphorylation sites are important for its localization and 14-3-3 binding.
Our phenotypical data show that 14-3-3 mutants have longer hypocotyls in cR light, suggesting their reduced sensitivity to R light. This phenotype is similar to that of the PIF3 overexpressor and opposite to that of the pif3 mutant, consistent with the previously reported data [44]. From these results, the involvement of 14-3-3κ in light signaling and the antagonistic role to that of PIF3 can be inferred. The reduced sensitivity of 14-3-3 mutants is not as much as that of phyB, since the phyB mutant shows much longer hypocotyls in cR light. It is possible that members of the 14-3-3 family function redundantly or that the participation of other components is significant in light signaling.
The summary of our findings, together with the previous findings, is depicted in Figure 4. In response to light signaling, 14-3-3κ is predicted to bind to phosphorylated-PIFs or possibly bridge PIFs-COP1, according to our yeast two-hybrid analysis. It can be speculated that 14-3-3κ either escorts LRBs to the Phy-PIF complex or stabilizes the LRB-Phy-PIF complex, so that PIFs are degraded by the 26S proteasome and photomorphogenesis initiates, serving as positive regulators of light signaling. The role of 14-3-3 proteins in protein degradation through ubiquitin E3 protein ligases has been reported in Arabidopsis. A stress-responsive F-box protein, FBS1, has been shown to interact with 14-3-3 proteins, and this interaction is important for FBS1 degradation by the 26S proteasome [45]. 14-3-3 proteins also interact with components of an E3 ligase, ETO1/ELOs, and promote their degradation in ethylene biosynthesis [46], suggesting that regulation of protein degradation by 14-3-3 proteins may be a general mechanism in plants. The involvement of 14-3-3 proteins in light signaling can be elaborated further by investigation of the other light-related proteins identified in our analysis detailed above. The list of candidate proteins recovered from affinity purification analysis contains photoreceptors phyB and PHOT2 and DELLA proteins, such as RGA1 apart from PIF3 and COP1. PHOT2 has been reported to interact with the 14-3-3λ isoform [12], which has strong sequence similarity to 14-3-3κ. The interaction of 14-3-3 proteins with phyB could be indirect through the PIF-COP1 complex. RGA1 is involved in GA signaling and known to interact with PIF3 to inhibit its transcriptional activity [37]. Hence, 14-3-3 proteins might regulate GA light signaling through direct or indirect binding to RGA1 or the PIF3-RGA1 complex, respectively. Taken together, the data presented here indicate that 14-3-3 proteins bind to PIFs and initiate photomorphogenesis in response to light signaling, possibly through PIF degradation. I. Upon light illumination, phytochromes (Phy) rapidly convert to an active form and translocate into a nuclear speckle where they bind to negative regulators of light signalling, PHYTOCHROME INTERACTING FACTORs (PIFs). An E3 ligase, CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) polyubiquitinates Phy, but not PIFs, to be degraded by the 26S proteasome, serving as a negative feedback mechanism in light signaling; II. Phy-bound PIFs are phosphorylated, E3 ligases, LIGHT-RESPONSE BTBs (LRBs), polyubiquitinate both Phy and PIFs to be degraded by the 26S proteasome, and consequently photomorphogenesis is initiated. 14-3-3κ is predicted to bind with phosphorylated-PIFs and either escort LRBs to the Phy-PIF complex or stabilize the LRB-Phy-PIF complex to promote PIFs degradation as positive regulators of light signalling.

In Vitro Pull-Down
MBP-tagged PIFs were created using the pMAL system (New England BioLabs, Ipswich, MA, USA). Primers used for cloning are PIF1f: CGGAATTCATGCATCATTTTGTCCCTGCA, PIF1r: GCCTGCAGTTAACCTGTTGTGTGGTTTCC, PIF3f: CACCGAATTCATGCCTCTGTTTGAGC TTTT, PIF3r: GTCGACTCACGACGATCCACAAAACTG, PIF6f: CGGAATTCATGATGTTC TTACCAACCGAT, PIF6r: GCCTGCAGTCATCTGTTAGTTTTCCTTGA, HFR1f: CGGAATT CATGTCGAATAATCAAGCTTTC and HFR1r: GCCTGCAGTCATAGTCTTCTCATCGCATG. The PIF3 coding sequence with the enzyme sites was amplified by Phusion High-Fidelity DNA Polymerase (New England BioLabs), cloned into pENTR (Invitrogen), and the sequence was confirmed. The PIF3 fragment and the pMAL vector were cut by the restriction enzymes, EcoRI and SalI (Takara), and recombined by T4 DNA ligase (Takara). For PIF1, PIF6 and HFR1, the coding sequences with the enzyme sites were amplified by Ex Taq DNA polymerase (Takara), ligated into pGem-T Easy vector (Promega, Fitchburg, WI, USA), and the sequence was confirmed. The genes in the pGEM-T Easy vector and the pMAL vector were cut by the restriction enzymes, EcoRI and PstI (Takara), and recombined by T4 DNA ligase (Takara). pMAL-PIFs were transformed into Escherichia coli BL21 (DE3), induced by 0.3-0.5 mM isoprophyl β-D-1-thiogalactopyranoside (IPTG) at 25-28 °C for 5-6 h and purified using amylose resin according to the manufacturer's instructions (New England BioLabs). The details of the His-tagged 14-3-3 proteins were described previously [9]. For the pull-down assay, 0.3-20 µg of 14-3-3κ protein were incubated with equivalent or half the amount of MBP or 5-10-times more MBP-PIFs in the MBP column buffer at 4 °C in dark on a rotator for 5-6 h, followed by incubation with amylose resin (New England BioLabs) overnight. The sample mixture was washed and eluted with elution buffer containing 100 µM MG132 and 10 mM maltose.
Retained proteins were resolved by SDS-PAGE and detected using anti-MBP antibody (New England BioLabs) and anti-His antibody (Covance, Princeton, NJ, USA).

Subcellular Localization of PIF3 in Onion
GFP-PIF3 was created using pGW6 [36], as mentioned in Section 4.1. Purified gold particles were coated with GFP-PIF3 DNA and bombarded into a freshly prepared onion (Allium cepa) epidermis layer using Biolistic Particle Delivery System PDS-1000/He (Bio-Rad, Hercules, CA, USA). Onion cells were incubated at 22 °C in the dark overnight and observed under a fluorescence microscope, BX51 (Olympus, Tokyo, Japan).