The Plant Cell, Vol. 30: 1277–1292, June 2018, www.plantcell.org © 2018 ASPB.

INTRODUCTION

Phytochromes are plant photoreceptors that regulate light responses ranging from seed germination, seedling photomor-phogenesis, and shade avoidance, to leaf senescence (Rockwell et al., 2006). Multiple phytochromes (i.e., phytochrome A [phyA] through phyE in Arabidopsis thaliana and phyA through phyC in rice [Oryza sativa]) mediate the responses of plants to light with very low to high fluence and light of different spectra (Takano et al., 2005; Franklin and Quail, 2010). For example, phyA me-diates both very low fluence response, which is the response to very low fluence light of any wavelength, and far-red high irradiance response, which is the response to prolonged far-red light exposure. In contrast, phyB mediates low fluence re-sponse, which is the response to low fluence red light that is reversible following far-red light exposure. When they absorb light of activating wavelengths, both phyA and phyB translocate into the nucleus either with or without the assistance of inter-acting proteins such as FAR-RED ELONGATED HYPOCOTYL1 (FHY1) and FHY1-LIKE (FHL) for phyA and phytochrome- interacting factors (PIFs) for phyB (Sakamoto and Nagatani, 1996; Kircher et al., 1999; Hiltbrunner et al., 2006; Pfeiffer et al., 2012). After entering the nucleus, phytochromes form nuclear foci called phy nuclear bodies—intense phy-GFP-positive puncta that increase in size with increasing light intensity (Chen et al., 2003). These phy nuclear bodies are thought to be sites of PIF

degradation and transcriptional regulation (Van Buskirk et al., 2012; Kaiserli et al., 2015) Phy promotes light responses via multiple pathways. In one pathway, phy interacts with and inhibits the function of the CON-STITUTIVELY PHOTOMORPHOGENIC1 (COP1)/SUPPRESSOR OF phyA-105 (SPA) ubiquitin E3 ligase complex by either ex-cluding COP1 from the nucleus, by disrupting the interaction of COP1 with SPAs, or by promoting the degradation of SPA1 and SPA2 (Osterlund and Deng, 1998; Pacín et al., 2014; Chen et al., 2015; Lu et al., 2015; Sheerin et al., 2015; Balcerowicz et al., 2017). In the light, the inhibition of the COP1/SPA complex by phy causes substrate transcription factors such as ELONGATED HYPOCOTYL5 (HY5), LONG HYPOCOTYL IN FAR-RED (HFR1), and the B-BOX ZINC FINGER PROTEINs accumulate (Osterlund et al., 2000; Jang et al., 2005; Yang et al., 2005; Datta et al., 2008; Chang et al., 2011; Fan et al., 2012; Xu et al., 2016). These, in turn, reprogram gene expression to promote light responses. In an-other pathway, phy interacts with a group of bHLH transcription factors, PIF1 to 8, which mainly repress light responses (Leivar and Quail, 2011; Lee and Choi, 2017). The interaction of phy with the PIFs, which occurs through active phyA binding or active phyB binding, leads to PIF degradation (Bauer et al., 2004; Khanna et al., 2004; Park et al., 2004; Al-Sady et al., 2006; Oh et al., 2006; Nozue et al., 2007; Shen et al., 2007, 2008,; Lorrain et al., 2008). Upon light exposure, the PIFs are rapidly phosphorylated either by phy or by other kinases, including PHOTOREGULATORY PROTEIN KINASEs (PPKs)/MUT9-like kinases (Al-Sady et al., 2006; Shin et al., 2016; Ni et al., 2017). Several phosphorylated PIFs are ubiquitinated by E3 ligases (i.e., PIF3 by the LIGHT- RESPONSE BTB PROTEINs [LRBs] and EIN3 BINDING F BOX PROTEINs, PIF4 by the BLADE-ON-PETIOLE PROTEINs, and PIF1 by the COP1/SPA complex) and subsequently degraded by the 26S proteasome (Ni et al., 2014; Zhu et al., 2015; Dong

Phytochrome B Requires PIF Degradation and Sequestration to Induce Light Responses across a Wide Range of Light Conditions

Eunae Park,1 Yeojae Kim,1 and Giltsu Choi2

Department of Biological Sciences, KAIST, Daejeon 34141, Korea

ORCID IDs: 0000-0002-1816-2732 (E.P.); 0000-0001-9962-8321 (G.C.)

Phytochrome B (phyB) inhibits the function of phytochrome-interacting factors (PIFs) by inducing their degradation and sequestration, but the relative physiological importance of these two phyB activities is unclear. In an analysis of published Arabidopsis thaliana phyB mutations, we identified a point mutation in the N-terminal half of phyB (phyBG111D) that abolishes its PIF sequestration activity without affecting its PIF degradation activity. We also identified a point mutation in the phyB C-terminal domain, which, when combined with a deletion of the C-terminal end (phyB990G767R), does the opposite; it blocks PIF degradation without affecting PIF sequestration. The resulting phyB proteins, phyB990G767R and phyBG111D, are equally capable of inducing light responses under continuous red light. However, phyBG111D, which exhibits only the PIF degradation activity, induces stronger light responses than phyB990G767R under white light with prolonged dark periods (i.e., diurnal cy-cles). In contrast, phyB990G767R, which exhibits only the PIF sequestration activity, induces stronger light responses in flick-ering light (a condition that mimics sunflecks). Together, our results indicate that both of these separable phyB activities are required for light responses in varying light conditions.

1 These authors contributed equally to this work.2 Address correspondence to gchoi@kaist.edu.The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Giltsu Choi (gchoi@kaist.edu).www.plantcell.org/cgi/doi/10.1105/tpc.17.00913

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et al., 2017; Zhang et al., 2017). In addition, HEMERA (HMR), a phy-interacting RAD23-like protein that is localized in both the nucleus and in chloroplasts, promotes the formation of large phy nuclear bodies and the degradation of PIF proteins (Chen et al., 2010). Apparently, the transcriptional activation motif of HMR is necessary not only for the expression of PIF target genes but also for PIF1 and PIF3 protein degradation, suggesting that PIF degradation and PIF transcriptional activation activity are coupled (Qiu et al., 2015). Casein kinase II was also found to phosphor-ylate PIF1 in vitro and promote its degradation in vivo (Bu et al., 2011). Since PIFs are transcription factors that regulate gene expression in vivo, their degradation by phy alters gene expres-sion patterns, making them more suitable for growth in the light. It should be noted, however, that phy does not always in-hibit the function of its binding partners. Phy interacts with several other proteins, including CONSTANS (CO), EARLY FLOWERING3, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 (HOS1), PHOTOPERIODIC CONTROL OF HYPOCOTYL1 (PCH1), PHYTOCHROME-DEPENDENT LATE- FLOWERING (PHL), TANDEM ZINC-FINGER/PLUS3 (TZP), and VASCULAR PLANT ONE–ZINC FINGER, as well as a few circadian clock components, such as CIRCADIAN CLOCK ASSOCIATED, GIGANTEA, LATE ELONGATED HYPOCOTYL, LUX ARRHYTHMO, and TIMING OF CAB (TOC1) (Liu et al., 2001; Yasui et al., 2012; Endo et al., 2013; Yeom et al., 2014; Kaiserli et al., 2015; Lazaro et al., 2015; Huang et al., 2016). Of these, TZP possesses a tandem zinc-finger domain and a PLUS3 domain, both of which are thought to bind protein, RNA, and even DNA. TZP forms dynamic nuclear bodies in the presence of phyB that can be disrupted by inhibiting transcription with actinomycin D (Kaiserli et al., 2015). This suggests TZP nuclear bodies are associated with either tran-scription or RNA processing. Consistent with this hypothesis, TZP directly targets the FLOWERING LOCUS T (FT) promoter and increases FT mRNA expression to promote flowering in long-day conditions. Unlike its interaction with the PIFs, phyB

neither destabilizes nor inhibits the targeting of TZP. Instead, phyB facilitates the targeting of TZP to the FT promoter with-out directly binding the FT promoter itself. Similarly, phyB binds PCH1 to repress the expression of genes like HFR1, ARABIDOPSIS THALIANA HOMEOBOX PROTEIN2, and PIF4 (Huang et al., 2016). It binds PHL to activate the expression of FT by forming a phyB-PHL-CO tripartite complex, and it in-teracts with HOS1 to promote the degradation of CO protein (Endo et al., 2013; Lazaro et al., 2015). phyA and phyB can also target various gene promoters to directly regulate gene expression (Chen et al., 2014; Jung et al., 2016). Studies have shown that phyB inhibits PIFs not only by in-ducing their degradation, but also by sequestering them from their target promoters. The N-terminal half of phyB, consist-ing of the N-terminal extension as well as the PAS, GAF, and PHY domains fused to GUS, GFP, and NLS (NGB) interacts with PIF1 and PIF3 and inhibits their binding to their target promoters without promoting their degradation (Park et al., 2012). Because NGB cannot induce the degradation of PIF3, when the NGB line is transferred to the dark, it resumes PIF3 target gene expression and hypocotyl elongation faster the full-length phyB-GFP (PBG) control line (Van Buskirk et al., 2014). Here, we asked whether any previously reported phyB mutations selectively show only one of phyB’s two PIF-related inhibitory functions, PIF degradation or PIF sequestration. We found an N-terminal missense mutation that selectively dis-ables PIF sequestration and a C-terminal missense mutation combined with a deletion of the C-terminal end that selectively disables PIF degradation. By analyzing these two mutants, we demonstrate here that phyB’s PIF sequestration function al-lows it to activate light responses in a flickering light condition that mimics sunflecks. We also show that phyB’s PIF degrada-tion function allows it to prolong light responses in a dark pe-riod. In other words, phyB requires both its PIF sequestration and degradation functions to activate light responses across a wide range of light conditions.

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RESULTS

PIF Activity Is More Strongly Linked to PIF DNA Binding Than PIF Protein Levels in Red Light

We previously showed both NGB and full-length phyB inhibit the binding of PIF1 and PIF3 to target promoters (Park et al., 2012). This suggests PIF protein levels do not necessarily reflect PIF activity. To verify this hypothesis, we measured endogenous PIF protein levels and hypocotyl lengths in the wild type and phy mutants. Continuous red light (Rc) strongly reduces endog-enous PIF1 protein levels in the wild type, phyA mutants, and phyB mutants, but not in phyA phyB double mutants (Figure 1A). This reduction is not caused by reduced PIF1 mRNA levels (Figure 1B), suggesting both phyA and phyB promote the deg-radation of PIF1 proteins in Rc. Interestingly, wild-type and phyB mutant plants have similarly low levels of PIF1 proteins in Rc (Figure 1A). Furthermore, both wild-type and phy mutant plants show even higher levels of PIF4 protein level in Rc than in the dark (Figure 1C; Supplemental Figure 1), which is inconsistent with the shorter or similar hypocotyls in Rc than in the dark. The higher PIF4 protein levels in Rc are likely due to combined ef-fects of higher PIF4 mRNA levels and PIF4 protein degradation in Rc than in the dark (Figure 1B). Together, these results indi-cate that higher PIF proteins levels do not necessarily translate into longer hypocotyl lengths. Since high PIF protein levels do not necessarily indicate high PIF function, we hypothesized that hypocotyl length should still show a strong correlation with assessments of PIF activity. To gauge PIF activity, we measured the expression of PIF target genes in wild-type and phyB mutant plants. Wild-type plants express lower levels of PIF target gene mRNAs than phyB mu-tant plants in Rc (Figure 1D), suggesting the PIFs are less active in wild-type than in phyB mutant plants. These lower levels of PIF activity may be caused by the phyB-induced detachment of PIFs from their target promoters in Rc. Consistent with this hypothesis, we found in a chromatin immunoprecipitation (ChIP) analysis that endogenous PIF1 binds its target gene promoters more strongly in phyB mutant plants than in wild-type plants in Rc. In wild-type plants, although PIF4 protein levels are higher in Rc than in the dark (Figure 1C; Supplemental Figure 1), we observed similar or lower levels of PIF4 enrichment at its tar-get gene promoters in Rc than in the dark, but much stronger enrichment in phyB mutants (Figure 1E; Supplemental Figure 2). Together, these results indicate that PIF DNA binding rather than PIF protein level are a better indicator of PIF activity in vivo. These results also suggest PIF sequestration by phyB plays an important role inhibiting PIFs in vivo.

The N-Terminal Half of phyB Inhibits PIFs without Triggering PIF Protein Degradation

We previously showed NGB does not promote the degradation of PIF1 and PIF3 proteins but still inhibits the function of two PIFs by detaching them from DNA (Park et al., 2012). To deter-mine whether a larger NGB fusion (GUS-GFP) can prevent PIF degradation in Rc, we generated transgenic lines expressing the N-terminal half of phyB (650 amino acids) fused to an NLS and a

short dimerization motif from GCN4 (35 amino acids) or HY5 (40 amino acids) in phyB or phyA phyB mutant background (Sup-plemental Figures 3 and 4). These will collectively be referred as PHYBN. As a control, we also expressed full-length PHYB in the phyA phyB mutant background. In Rc, the hypocotyls of both PHYB-OX and PHYBN-OX are shorter than those of phyA phyB mutants (Supplemental Figure 3). Although PHYBN-OX produces higher levels of phyBN protein than PHYB-OX produces of phyB protein, PHYBN-OX has longer hypocotyls than PHYB-OX. These results indicate phyBN is biologically active, but less so than full-length phyB. We next asked whether phyBN promotes the degradation of PIFs in red light. Since PIFs are also degraded by phyA in Rc, we introduced PHYBN into the phyA phyB double mutant background and measured PIF degradation. Using immuno-blotting, we found phyBN does not promote the degradation of PIF3-myc in Rc, but full-length phyB does (Supplemental Figure 5A). Similarly, phyBN does not reduce endogenous PIF1 protein levels in Rc, but full-length phyB does (Supplemen-tal Figure 5B). Since PIF1 mRNA levels are similar between PHYBN-OX and PHYB-OX (Supplemental Figure 5C), these results indicate phyBN does not promote the degradation of endogenous PIF1 in Rc. We then wondered whether phyBN induces light responses by sequestering PIFs. Using ChIP, we found that red light strongly reduces PIF3-myc targeting to two promoters in PHYBN-OX but not in phyA phyB mutant plants (Supplemental Figure 5D). Since red light does not reduce PIF3-myc protein levels in either PHYBN-OX or phyA phyB mutants, these results suggest phyBN inhibits PIF3 function by sequestering PIF3 from its target promoters. Consistent with this hypothesis, two PIF target genes are expressed at lower levels in PHYBN-OX than in phyA phyB mutant plants (Supplemen-tal Figure 5E). Together, these results indicate phyBN, like NGB, induces light responses by sequestering PIFs from their target promoters rather than by promoting PIF degradation.

The Two Different phyB Activities Are Separable by Mutations

Our analysis of PHYBN-OX suggests the N-terminal half of phyB is sufficient to sequester PIFs from their target promoters, while its C-terminal half is required for promoting PIF protein degra-dation. To investigate further whether these two different phyB activities are separable, we asked whether any of the previously reported phyB mutations can selectively disrupt either PIF seg-regation or PIF degradation. We chose a missense mutation in phyB’s N-terminal half (G111D), a missense mutation in its C-terminal half (G767R), and a deletion of its C-terminal 182 amino acids (990). According to previous reports, the G111D mutation abolishes the activity of NGB but not full-length phyB, the G767R mutation abolishes nuclear translocation of phyB, and the deletion of phyB’s C-terminal end reduces phyB’s ac-tivity (Krall and Reed, 2000; Matsushita et al., 2003; Oka et al., 2008). We introduced the G111D mutation into either PHYBN (PHYBNG111D) or full-length PHYB (PHYBG111D). PHYBNG111D-OX has long hypocotyls with closed cotyledons in Rc, like the phyA phyB mutant. PHYBG111D-OX, in contrast, has short hypocotyls

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with open cotyledons (Figures 2A and 2B). PHYBG111D-OX has longer hypocotyls than PHYB-OX, even though both lines pro-duce similar levels of phyB protein (Figure 2C). phyBG111D also represses PIF target gene expression in Rc, but phyBNG111D does not (Figure 2D). These results indicate that while the G111D mu-tation weakens but does not abolish full-length phyB activity, it abolishes phyBN activity.

Because the G111D mutation prevents phyBN’s induction of light responses and because phyBN functions by sequestering PIFs, we asked whether the G111D mutation selectively disables phyB’s ability to sequester PIFs without affecting its ability to induce PIF degradation. Using immunoblotting, we found that phyBG111D promotes the degradation of both PIF3-myc and PIF1 protein in Rc, whereas phyBNG111D does not (Figure 2E). This is

Figure 1. Hypocotyl Lengths under Red Light Increase with Increased PIF Binding to Target Promoters.

Four-day-old seedlings grown either in continuous dark (Dc) or red light (Rc) were used for this analysis. (A) PIF protein levels in the wild type and phy mutants. PIF1 and PIF4 protein levels were quantified with antibodies against PIF1 (α-PIF1), PIF4 (α-PIF4), and β-tubulin (α-TUB). Col-0, wild type; phyA, phyA-211 mutant; phyB, phyB-9 mutant; phyA phyB, phyA-211 phyB-9 double mutant. Arrow indicates PIF4. (B) PIF1 and PIF4 mRNA levels in the wild type and phy mutants. mRNA levels were measured by real-time PCR. Error bar indicates sd (n = 3 biological replicates, ANOVA, Tukey’s HSD, P < 0.05). (C) Hypocotyl lengths of the wild type and phy mutants in red light. (D) PIF target gene mRNA levels in the wild type and phy mutants. Error bar indicates sd (n = 3 biological replicates, ANOVA, Tukey’s HSD, P < 0.05). (E) PIF targeting to promoters, as determined by ChIP. For PIF targeting, dark or red light-grown wild-type and phyB mutant seedlings were used for ChIP analysis with an anti-PIF1 or anti-PIF4 antibody. Immunoprecipitated DNA fragments were quantified by real-time PCR, and the data were nor-malized using either the dark values for PIF1 targeting or PIF4 targeting to the corresponding promoters except for rDNA, which was normalized by the dark value for the PIL1 promoter. Error bar indicates sd; n = 3 technical replicates. A biological replicate is in Supplemental Figure 2.

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Figure 2. phyB with a Mutation in the N-Terminal Domain Does Not Inhibit PIF Binding to DNA but Still Promotes PIF Degradation.

Four-day-old seedlings grown either in continuous dark (Dc) or red light (Rc) were used for the analysis. Col-0, wild type; phyA phyB, phyA-211 phyB-9 double mutant; B #6, PHYB-OX phyA phyB #6; BN #2, PHYBN-GCN-OX phyA phyB #2, BG111D #21, #11, PHYBG111D-OX phyA phyB #21 #11; BNG111D #13, PHYBNG111D-HY5dd-OX phyA phyB #13; BNG111D #11, PHYBNG111D-GCN-OX phyA phyB #11. (A) The inhibition of hypocotyl elongation by phyBG111D but not by phyBNG111D in red light. (B) Quantification of hypocotyl lengths in red light. Error bar indicates sd (n > 20 seedlings, ANOVA, Tukey’s HSD, P < 0.05). (C) Similar phyB protein levels in transgenic plants. FLAG-tagged phyB or phyBN protein levels were determined in dark-grown seedlings with an antibody against FLAG (α-Flag) or an antibody against β-tubulin (α-TUB). (D) The repression of two PIF target gene mRNAs by phyBG111D but not by phyBNG111D in red light. Error bar indicates sd (n = 3 biological replicates, *P < 0.05 by Student’s t test). BG111D, PHYBG111D-OX phyA phyB #21; BNG111D, PHYBNG111D-HY5dd-OX phyA phyB #13.(E) The degradation of PIF3-myc and endogenous PIF1 proteins by phyBG111D but not by phyBNG111D in red light. PIF protein levels were determined with antibodies against myc (α-Myc) for PIF3-myc, PIF1 (α-PIF1), and β-tubulin (α-TUB). phyA phyB background (B, PHYB-OX phyA phyB #6; BN, PHYBN-GCN-OX phyA phyB #2; BG111D, PHYBG111D-OX phyA phyB #21; BNG111D, PHYBNG111D-HY5dd-OX #13), phyA phyB PIF3OX3 background (B, PHYB-OX #2; BN, PHYBN-HY5dd-OX #2; BG111D, PHYBG111D-OX #4; BNG111D, PHYBNG111D-HY5dd-OX #7).

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consistent with the degradation of PIF3 protein by PBGG111D (Qiu et al., 2017). We next examined whether the G111D mutation affects PIF sequestration. Using ChIP, we found phyBNG111D does not inhibit the targeting of PIF1 or PIF3-myc to the PIL1 promoter in Rc (Figure 2F), but phyBN does (Supplemental Figure 4). PIF targeting is also reduced in PHYBG111D-OX in red light, probably due to a reduction in the level of PIF proteins. Consistent with this hypothesis, when we blocked PIF3 protein degradation with a 26S proteasome inhibitor, we observed increased PIF3 pro-tein levels and increased PIF3 targeting (Figure 2G). This sug-gests phyBG111D promotes PIF3 degradation but does not inhibit its binding to target promoters. Together, these results indicate that the G111D mutation selectively disables phyB’s ability to sequester PIFs but not its ability to degrade them. We next introduced two mutations (G767R and B990) as well as their combination (B990G767R) into the C-terminal half of full-length phyB. Since G767R affects the nuclear translocation of phyB, we included a NLS with the G767R mutation. Both PHYBG767R-OX and PHYB990-OX have short hypocotyls with open cotyledons in Rc (Figure 3A). PHYB990G767R-OX also has short hypocotyls with open cotyledons in Rc. We found PHYB990G767R-OX has longer hypocotyls than PHYBG767R-OX or PHYB990-OX, which both, in turn, have longer hypocotyls than PHYB-OX (Figure 3B). All lines express similar phyB protein levels (Figure 3C), suggesting the G767R and B990 mutations combine to reduce but not abolish phyB activity. Since the C-terminal half of phyB is necessary for PIF degra-dation, we asked whether the weaker activities of the C-terminal mutants are due to a reduction in their ability to induce PIF deg-radation. Using immunoblotting, we found both phyBG767R and phyB990 promote the degradation of PIF1 and PIF3-myc in Rc but to a lesser extent than wild-type phyB. The phyB990G767R, which combines the G767R and B990 mutations, shows a syner-gistic disruption of PIF1 and PIF3-myc degradation (Figure 3D). phyB990G767R still interacts with PIF3 in a Pfr-dependent manner (Figure 3E), suggesting phyB990G767R retains its ability to seques-ter PIFs. Indeed, using ChIP, we found phyB990G767R inhibits the binding of PIF1 and PIF3-myc to PIF target gene promoters in Rc (Figure 3F) and represses target gene expression (Figure 3G). Together, these results indicate that phyB990G767R selectively dis-rupts phyB’s ability to induce PIF degradation without affecting its ability to induce PIF sequestration. We next investigated the functional consequences of com-bining the G111D and G767R mutations. Since the G111D mu-tation disrupts phyB’s PIF sequestration activity and G767R partially disrupts its PIF degradation activity, the G111DG767R combination should combine to fully disrupt phyB function. As

expected, phyBG111DG767R does not repress hypocotyl elongation in Rc (Figures 4A and 4B). This near total loss of phyB activity, however, is not due to a reduction in phyB expression levels in PHYBG111DG767R-OX (Figure 4C). This suggests phyBG111DG767R is a very weak, if not dead, mutant. We further asked whether the G111DG767R mutation disrupts both PIF degradation and PIF sequestration. Using immunoblotting, we found phyBG111DG767R shows only partial degradation of PIF3-myc and endogenous PIF1 proteins in red light (Figure 4D). In addition, we found using ChIP that phyBG111DG767R does not inhibit the binding of PIF1 to its target promoters in Rc (Figure 4E). Consequently, phyBG111DG767R does not or only mildly represses target gene mRNA expression (Figure 4F). Together, these results indicate phyBG111DG767R is a virtually dead mutant with additive defects in both PIF seques-tration and PIF degradation.

The Two Different phyB Activities Induce Light Responses Rapidly and Persistently

Of the two different phyB activities, PIF sequestration seems to require fewer steps than PIF degradation. Thus, PIF sequestra-tion likely acts faster than PIF degradation in response to red light. On the other hand, the effect of PIF degradation likely lasts longer, even beyond the conversion of Pfr to Pr after the tran-sition to darkness. This is because PIF protein levels take time to recover in the dark. We found that phyBG111D represses PIF target gene mRNA expression longer than phyB990G767R after the transition to darkness (Figure 5A). However, phyB990G767R represses PIF target gene expression faster than phyBG111D after the transition to red light (Figure 5B). The longer repression of PIF target genes by phyBG111D is consistent with a previous re-port that showed a full-length phyB-GFP (PBG) represses PIF target genes longer than NGB after the transition to darkness (Van Buskirk et al., 2014). Together, these results indicate phyB regulates gene expression rapidly by PIF sequestration and per-sistently by PIF degradation.

The Two phyB Activities Regulate Light Responses under Varying Light Conditions

To better understand phyB’s induction of light responses under naturally occurring conditions, we looked for light conditions whose induction of light responses proceeds preferentially via either PIF degradation or PIF sequestration. Since PIF degradation maintains low levels of PIF for some time after the transition to darkness, we expected that PIF degradation is more important for conditions characterized by light and prolonged periods of

(F) The inhibition of PIF targeting by phyBG111D but not by phyBNG111D in red light. ChIP assays were performed with an antibody against myc (α-Myc) for PIF3-myc and against PIF1 (α-PIF1). Error bars indicate sd, n = 3 technical replicates. A biological replicate is in Supplemental Figure 2. phyA phyB PIF3OX3 background (BG111D, PHYBG111D-OX #4; BNG111D, PHYBNG111D-HY5dd-OX #7), phyA phyB background (BG111D, PHYBG111D-OX #21; BNG111D, PHYBNG111D-HY5dd-OX #13). (G) Increased PIF3-myc targeting by blocking PIF3-myc degradation in red light. PIF3-myc degradation was blocked by treatment with the 26S pro-teasome inhibitors MG132 and Epoxomicin. The right panel indicates an immunoblot analysis showing increased PIF3-myc protein levels induced by inhibitor treatment in red light. ChIP assays and immunoblot analyses were performed with an antibody against myc (α-Myc) for PIF3-myc. Error bars indicate sd; n = 3 technical replicates. A biological replicate is in Supplemental Figure 2. BG111D, PHYBG111D-OX phyA phyB PIF3OX3 #4.

Figure 2. (continued).

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Figure 3. phyB with Mutations in the C-Terminal Domain Does Not Promote PIF Degradation but Inhibits PIF DNA Binding.

Four-day-old seedlings grown either in continuous dark (Dc) or red light (Rc) were used for the analysis. BG767R, PHYBG767R-OX phyA phyB; B990, PHYB990-OX phyA phyB; B990G767R, PHYB990G767R-OX phyA phyB. Other notations are as in Figure 2. (A) The inhibition of hypocotyl elongation by phyBG767R, phyB990, and phyB990G767R in red light. (B) Quantification of hypocotyl lengths in red light. Error bar indicates sd (n > 20 seedlings, ANOVA, Tukey’s HSD, P < 0.05). (C) Immunoblot analysis showing phyB protein levels in dark-grown transgenic plants. (D) Reduced degradation of PIF3-myc and endogenous PIF1 proteins by phyBG767R, phyB990, and phyB990G767R in red light. (E) In vitro binding assay showing a preferential binding between Pfr of phyB990G767R and GST-PIF3. “Input” compares recombinant phyB protein levels used for the binding assay. B and B990G767R indicate recombinant wild-type full-length phyB-myc and phyB990G767R-myc proteins, respectively. (F) The inhibition of PIF3-myc and endogenous PIF1 targeting to promoters by phyB990G767R. The degree of PIF targeting is expressed as values relative to Dc. Error bars indicate sd; n = 3 technical replicates. A biological replicate is in Supplemental Figure 2. PHYB990G767R-OX phyA phyB PIF3OX3 #16 and PHYB990G767R-OX phyA phyB #4. (G) The repression of PIF target gene mRNAs by phyBG767R, phyB990, and phyB990G767R in red light. Error bars indicate sd (n = 3 biological replicates, ANOVA, Tukey’s HSD, P < 0.05). PHYB990G767R-OX phyA phyB #4.

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darkness. To investigate this possibility, we measured PHYBG111D- OX and PHYB990G767R-OX hypocotyl lengths under conditions of differing daylength. In continuous dark or white light conditions, both PHYBG111D-OX and PHYB990G767R-OX produce hypocotyls of similar length. With shorter days, however, PHYB990G767R-OX produces longer hypocotyls than PHYBG111D-OX, reaching more than twice as long in the short-day condition (i.e., 8 h light/16 h dark) (Figure 6A, right panel). These results indicate hypocotyl elongation is preferentially inhibited by phyBG111D rather than by

phyB990G767R under light conditions characterized by prolonged periods of darkness. On the other hand, since PIF sequestration inhibits PIFs rap-idly in response to light, PIF sequestration is probably the more prominent mechanism at work in light conditions that generate short-lived Pfr. One such condition is sunflecks. Sunflecks are fluctuating light conditions found under a forest canopy. They are created by fluttering leaves or movement of the sun. To mea-sure the effect of sunfleck-like conditions, we measured the

Figure 4. phyBG111DG767R Is a Virtually Dead Mutant.

Four-day-old seedlings grown either in continuous dark (Dc) or red light (Rc) were used for the analysis. BGG and BG111DG767R, PHYBG111DG767R-OX phyA phyB. Other notations are as in Figure 2. (A) No inhibition of hypocotyl elongation by phyBG111DG767R in red light. (B) Quantification of hypocotyl lengths in red light. Error bar indicates sd (n > 20 seedlings, ANOVA, Tukey’s HSD, P < 0.05). (C) phyB protein levels in transgenic plants. (D) Reduced degradation of PIF3-myc and endogenous PIF1 proteins by phyBG111DG767R in red light. PHYBG111DG767R -OX phyA phyB #11 and PHYBG111DG767R-OX phyA phyB PIF3OX3 #3. (E) ChIP assay showing no inhibition of PIF1 targeting by phyBG111DG767R in red light. PHYBG111DG767R-OX phyA phyB #11. A biological replicate is in Supplemental Figure 2. (F) No repression of PIF target gene mRNAs by phyBG111DG767R in red light. Error bars indicate sd (n = 3 biological replicates, ANOVA, Tukey’s HSD, P < 0.05). PHYBG111DG767R-OX phyA phyB #11.

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hypocotyls of plants grown under conditions of rapidly alternat-ing 1-min pulses of red and far-red light of equal fluence. Due to the higher photoconversion rate coefficient of Pr by red light than that of Pfr by far-red light, some Pfr should remain after each far-red light pulse unless the fluence of the far-red light is significantly higher than that of the red light. Under this flickering light con-dition, wild-type plants produce short hypocotyls with open cotyledons, whereas phyA phyB double mutants produce long hypocotyls with closed cotyledons (Figures 6B and 6C). Consis-tent with the importance of the PIFs in phytochrome signaling, the pifQ mutant also produces short hypocotyls under flicker-ing light. Interestingly, unlike what we observed in the short-day condition, PHYB990G767R-OX produces shorter hypocotyls than PHYBG111D-OX under flickering light. Consistent with their hypo-cotyl lengths, we found stronger repression of PIF target genes in PHYB990G767R-OX than in PHYBG111D-OX (Figure 6D). These results indicate phyB990G767R is more active than phyBG111D under flickering light conditions. Finally, we asked whether other light responses are also preferentially regulated by one of these two phyB activities. We chose leaf senescence as a model response. Previous studies showed that PIF4 and PIF5 accelerate leaf senes-cence when seedlings are transferred to darkness by directly targeting and increasing the expression of ORE1, which en-codes a NAC-type transcription factor (Sakuraba et al., 2014). phyB delays leaf senescence by inhibiting PIF4 and PIF5. We measured senescence in two PHYB variants by measuring

chlorophyll levels and senescence marker gene expression under differing light conditions. When we transferred de-tached shoots to darkness for 5 d, PHYBG111D-OX remains greener and retains more chlorophyll than PHYB990G767R-OX (Figures 7A and 7B). When we transferred detached shoots to the flickering light condition, however, it is PHYB990G767R- OX that remains greener and retains more chlorophyll than PHYBG111D-OX (Figures 7C and 7D). PHYBG111D-OX seed-lings also express lower levels of senescence marker gene mRNAs than PHYB990G767R-OX in a prolonged dark condition, whereas PHYB990G767R-OX seedlings express lower levels of senescence marker gene mRNAs than PHYBG111D-OX in the flickering light condition (Figure 7E). This is consistent with phyBG111D showing higher activity in the prolonged dark con-dition than in the flickering light condition. Together, these results indicate PIF degradation (phyBG111D) is the more prom-inent mechanism directing light responses under conditions that include prolonged periods of darkness, while PIF seques-tration (phyB990G767R) is more prominent under conditions characterized by flickering light.

DISCUSSION

In this study, we generated phyB mutants that exhibit only a sin-gle phyB activity—either PIF degradation or PIF sequestration—and we identified light conditions that induce light responses preferentially via only one of these two activities. We found

Figure 5. The Two Inhibitory Activities of phyB Induce Rapid and Enduring PIF Target Gene Expression during a Light-Dark Transition.

Four-day-old dark- or red light-grown seedlings were transferred to the indicated light conditions. BG111D and B990G767R indicate PHYBG111D-OX and PHYB990G767R-OX, respectively. (A) Stronger, more prolonged repression of PIF target gene mRNAs by phyBG111D than phyB990G767R during a light-to-dark transition. DxH indicates x hours after the transition. Error bars indicate sd (n = 3 biological replicates, *P < 0.05 by Student’s t test). B990G767R, PHYB990G767R-OX phyA phyB #4; BG111D, PHYBG111D-OX phyA phyB #21. (B) Faster repression of PIF target gene mRNAs by phyB990G767R than phyBG111D during a dark-to-red light transition. Rxm indicates x minutes after the transition. Error bars indicate sd (n = 3 biological replicates, *P < 0.05 by Student’s t test).

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phyBG111D disrupts PIF sequestration without affecting PIF degradation, whereas phyB990G767R disrupts PIF degradation without affecting PIF sequestration. When expressed in Arabi-dopsis, both of these alleles similarly inhibit hypocotyl elonga-tion, but they do so less effectively than wild-type phyB under continuous red light. This suggests wild-type phyB induces light responses via both PIF degradation and PIF sequestration un-der continuous red light. Different light conditions, however, can

induce light responses preferentially via one of these two phyB activities. We found phyBG111D is more active than phyB990G767R in light conditions characterized by prolonged periods of dark-ness (i.e., diurnal cycles). In contrast, phyB990G767R is more ac-tive than phyBG111D in conditions of alternating red and far-red light pulses, which induce short-lived Pfr. Our results thus imply phyB uses both PIF degradation and PIF sequestration to inhibit PIFs across a wide range of light conditions (Figure 8).

Figure 6. The Two phyB Activities Differentially Regulate Hypocotyl Elongation in Different Light Conditions.

Four-day-old seedlings grown in the indicated condition were used for the analysis. Notations are as in Figure 2. (A) A graph showing hypocotyl lengths of PHYBG111D-OX and PHYB990G767R-OX in conditions of differing daylengths. The x axis indicates hours of white light per 24 h of light-dark cycle. White light fluence: 100 μmol m−2 s−1. Error bar indicates sd; n > 20 seedlings. The right panel is a redrawing of hypo-cotyl lengths of PHYBG111D-OX and PHYB990G767R-OX in the short-day condition (8H). Error bar indicates sd (n > 20 seedlings, ANOVA, Tukey’s HSD, P < 0.05). B990G767R, PHYB990G767R-OX phyA phyB #4; BG111D, PHYBG111D-OX phyA phyB #21. (B) Stronger inhibition of hypocotyl elongation by phyB990G767R than phyBG111D in a flickering red and far-red light condition. (C) Quantification of hypocotyl lengths in the flickering light condition. Error bar indicates sd (n > 20 seedlings, ANOVA, Tukey’s HSD, P < 0.05). (E) Stronger repression of PIF target genes by phyB990G767R than phyBG111D in the flickering light condition. Error bar indicates sd (n = 3 biological replicates, ANOVA, Tukey’s HSD, P < 0.05).

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Figure 7. The Two phyB Activities Differentially Regulate Leaf Senescence in Different Light Conditions.

Notations are as in Figure 2. (A) Stronger inhibition of leaf senescence by phyBG111D than phyB990G767R in a prolonged dark condition. Senescence was induced by transferring shoots of 7-d-old light-grown seedlings to darkness for 5 d. B990G767R, PHYB990G767R-OX phyA phyB #4; BG111D, PHYBG111D-OX phyA phyB #21. (B) Quantification of chlorophyll levels of senescing leaves in (A). Error bar indicates sd (n > 30 cotyledons, *P < 0.05 by Student’s t test). (C) Stronger inhibition of leaf senescence by phyB990G767R than phyBG111D in the flickering light condition. Senescence was induced by transferring shoots of 7-d-old light-grown seedlings to the flickering light condition for 7 d. (D) Quantification of chlorophyll levels of senescing leaves in (C). Error bar indicates sd (n > 30 cotyledons, *P < 0.05 by Student’s t test). (E) mRNA expression analysis showing repression of senescence marker genes by phyBG111D or phyB990G767R in different light conditions. Error bars indicate sd (n ≥ 3 biological replicates, *P < 0.05 by Student’s t test).

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Our data show the PIF sequestration activity of phyB may explain some reported discrepancies between PIF protein lev-els and hypocotyl lengths. As reported, both phyA and phyB promote the degradation of endogenous PIF1 and 35S-driven PIF3-myc in response to red light, reducing their protein levels. In contrast, red light dramatically increases endogenous PIF4 pro-tein levels by inducing increased PIF4 mRNA expression. How-ever, these increased PIF4 protein levels do not lead to increased binding of PIF4 to target promoters. Instead, red light strongly inhibits the binding of PIF4 to its target gene promoters. In other words, the extent to which PIF binds DNA is better correlated with hypocotyl length than PIF protein levels, further supporting PIF sequestration as an important mechanism by which phyB regulates light responses. PIF sequestration may also explain a few reported discrepan-cies between PIF protein levels and hypocotyl length under red light. Previous studies showed that both phyB and PIF3 proteins are degraded upon phosphorylation by PPKs followed by ubiq-uitination by LRBs in red light (Ni et al., 2014, 2017). phyB and PIF3 proteins are stabilized in ppk multiple mutants and in lrb multiple mutants. Curiously, however, hypocotyl lengths in ppk multiple and lrb multiple mutants are shorter than those of wild-type seedlings in red light. In other words, there is a discrep-ancy between PIF3 protein levels and hypocotyl lengths. Since hypocotyl lengths are determined not only by PIF3 but also by other PIFs, the increased phyB may reduce overall levels of PIF proteins. Alternatively, the increased phyB may inhibit PIFs not just by PIF degradation but also by PIF sequestration. Similar discrepancies have been observed with other PIFs. When etio-lated seedlings are transferred to red light, 35S-driven PIF4 and PIF5 proteins are degraded, reaching minimum levels one hour after the transfer, but PIF protein levels quickly reaccumulate to the levels observed in the dark (Lorrain et al., 2008). Since hy-pocotyl lengths are short in red light, this accumulation of PIF4 and PIF5 proteins implies PIF4 and PIF5 are inhibited by means other than PIF degradation. Neither does increased PIF1 stability

translate into longer hypocotyl lengths in stable pif3 (spf3), spf4, and spf5 mutants (Zhu et al., 2016a). It should be noted that phyB is not the only protein that regu-lates the ability of PIFs to bind DNA. First, a group of HLH proteins (e.g., HECATE [HEC], HFR1, and PHYTOCHROME RAPIDLY REGULATED1 [PAR1]), which do not bind DNA themselves, heterodimerize with PIF1 (HEC and HFR1) or PIF4 (HFR1 and PAR1) to inhibit the binding of the PIFs to DNA (Hornitschek et al., 2009; Hao et al., 2012; Zhu et al., 2016b). Since PIFs ac-tivate the expression of these HLH genes by directly binding their promoters, these HLH proteins are components of nega-tive feedback loops that reduce the activity of the PIFs. Second, HY5, a bZIP transcription factor stabilized by light, competitively binds the same G-box elements as the PIFs, effectively reducing PIF DNA binding in the light (Toledo-Ortiz et al., 2014). Third, DELLA proteins, which inhibit GA signaling, also inhibit PIFs by sequestering PIF3 and PIF4 from their target promoters (de Lucas et al., 2008; Feng et al., 2008). Curiously, DELLA proteins also promote PIF degradation independent of light (Li et al., 2016). This suggests the DELLA proteins, similarly to phyB, in-hibit PIFs both by PIF sequestration and PIF degradation. Finally, group A bZIP transcription factors including ABA INSENSITIVE5, which promote abscisic acid signaling, interact with and assist PIF1 in binding specific promoters in vivo. This regulation of PIF DNA binding by the DELLAs and group A bZIPs provides a mechanism by which light and two hormone signaling path-ways merge to regulate common biological processes (Kim et al., 2016). In addition to PIF DNA binding, PIF transcriptional activation is also regulated by PIF-interacting proteins, such as ETHYLENE INSENSITIVE3, FHY3, REDUCED POTASSIUM DEPENDENCY3/HISTONE DEACETYLASE1-type histone deacetylase15, and TOC1 (Tang et al., 2012; Jeong et al., 2016; Soy et al., 2016; Gu et al., 2017). Thus, the reported discrepan-cies between PIF protein levels and light responses are likely due to the combined effects of PIF sequestration by phyB and the modulation of PIF activities by other proteins.

Figure 8. A Diagram Describing the Different Roles the Two phyB Activities Play in Different Light Conditions.

phyB’s PIF degradation activity is used preferentially to induce light responses in conditions characterized by prolonged periods of darkness (left panel), while its PIF sequestration activity is used preferentially to induce light responses in flickering light conditions (right panel). The PIF degradation activity of phyB maintains low PIF protein levels during extended periods of darkness, virtually lengthening the duration of the daytime, whereas the PIF sequestra-tion activity of phyB rapidly and reversibly inhibits PIFs from binding their target promoters, allowing faster responses to rapidly fluctuating light.

phyB Requires Both Inhibitory Activities 1289

Accumulating evidence suggests that, rather than acting as a mere appendage on the more functional parts of the protein, the C-terminal half of phy is actually an important signaling site. The C-terminal half of phyB is necessary for dimerization, nuclear localization, and photobody formation, but its roles in phyB function were thought to be either dispensable or replaceable by heterologous NLS and dimerization motifs (Matsushita et al., 2003; Oka et al., 2004). Our data, however, clearly indicate that the C-terminal half of phyB, although dispensable for PIF se-questration, is necessary for PIF degradation. Recently, Qiu et al. (2017) showed that the C-terminal half alone (i.e., without its N-terminal photosensory half) is sufficient to promote the deg-radation of PIF3 protein and to activate the expression of photo-synthetic genes including CAB2 even in the dark. These findings further support an essential role for the C-terminal half of phyB in phyB signaling. At this point, it is unknown whether the C-terminal half of phyA is necessary and sufficient for PIF degra-dation. Previous studies have shown that the N-terminal half of phyA (1–406, 1–651, or 1–686 amino acids) fused to an NLS and a dimerization motif is either constitutively photomorphogenic due to its interaction with and inhibition of COP1 (1–406 amino acids) or is very weakly active (1–651 and 1–686) (Mateos et al., 2006; Viczián et al., 2012). This suggests the N-terminal half of phyA is insufficient for full phyA activity. Although the role the C-terminal half of phyA plays in PIF degradation is unknown, one recent study showed that the C-terminal domain of phyA is required for the inhibition of AUX/IAA protein degradation by auxin (Yang et al., 2018). This suggests an important role for the C-terminal domain of phyA in phyA signaling. Finally, our data show that phyB preferentially induces light responses via PIF sequestration in flickering light; phyB990G767R induced stronger light responses than phyBG111D when grown un-der alternating pulses of red and far-red light. We designed our flickering light condition to mimic the sunfleck conditions found under forest canopies and characterized by repeated abrupt in-creases of unfiltered light caused by fluttering leaves. The dura-tions of sunflecks in an aspen forest span 0.2 to 50 s with 0.4 to 0.8 s as the most common (Roden and Pearcy, 1993). To assess the effects of sunfleck-like light conditions, we gave repeated 1-min pulses of red light (10 μmol m−2 s−1) and 1-min pulses of far-red light (10 μmol m−2 s−1). Under this condition, phyA phyB double mutants produce longer hypocotyls than the wild type, but pifQ mutants produce shorter hypocotyls than the wild type. This implies that, in sunfleck conditions, phytochromes induce light responses at least partly by inhibiting PIFs. When we compared our two phyB mutant alleles in our flickering light condition, phyB990G767R induces shorter hypocotyls and slower leaf senescence than phyBG111D. In conditions characterized by prolonged periods of darkness, phyBG111D induces stronger light responses than phyB990G767R. These results suggest that, in sunfleck conditions, phyB induces light responses primarily via PIF sequestration, while in conditions characterized by pro-longed periods of darkness, it acts primarily via PIF degrada-tion. phyA and phyB are also implicated in the light responses of Arabidopsis in another sunfleck-mimicking condition in which continuous shade under Viburnum is interrupted by 2 h of sun-light. HY5 and HYH inhibit hypocotyl elongation downstream of phyA and phyB in this sunfleck condition (Sellaro et al., 2011).

At this point, it is unclear whether these two sunfleck-mimicking conditions induce light responses via the same phyB activities. Further comparative analyses will be necessary to delineate the various phytochrome signaling pathways induced by differing sunfleck conditions.

METHODS

Plant Materials and Growth Conditions

Arabidopsis thaliana plants were grown in a growth room with a 16-h- light/8-h-dark cycle at 22 to 24°C for general growth and seed harvesting.

To generate mutated PHYB, site-directed mutations were introduced by the inverse PCR method. For PHYBN, amino acids 1 to 650 from PHYB were ligated to an adaptor encoding a GCN4 dimerization mo-tif and cloned into the phCF vector. An NLS (PKKKRKV) was fused to PHYBN, PHYBG767R, PHYBG111DG767R, and PHYB990G767R. The mutated PHYBs were cloned into phCF vectors (Nguyen et al., 2015) and intro-duced into either phyA-211/phyB-9 or PIF3-OX/phyA-211/phyB-9 (Park et al., 2012). Homozygous lines were selected and used for phenotypic analysis. The oligomers used for construction are listed in Supplemental Table 1.

Hypocotyl Length Measurement

To measure hypocotyl lengths in red light, surface-sterilized seeds were plated on half-strength Murashige and Skoog (MS) media (2.2 g MS, 0.5 g MES, 8 g phytoagar, 1 liter, pH 5.7), imbibed for 3 d at 4°C in the dark, irradiated with white light (100 μmol m−2 s−1) for 6 h, and grown 4 d either in continuous red light (9 μmol m−2 s−1) or in darkness. To measure hypo-cotyl lengths in a flickering light condition, plated seeds were irradiated with white light for 16 h and grown 4 d in a light condition consisting of repeated 1-min pulses of red light (10 μmol m−2 s−1) and 1-min pulses of far-red light (10 μmol m−2 s−1). Hypocotyl lengths of at least 20 seedlings were measured using ImageJ.

Leaf Senescence Assay

Seedlings were grown for 7 d in white light (100 μmol m−2 s−1) and their shoots were cut at the upper hypocotyls with a scissor. The shoots were then transferred to sterilized distilled water in 6-well cell culture plates and incubated for 5 d in darkness or 7 d in the flickering light condition. Chlorophylls were extracted with 80% ice-cold acetone and quantified by measuring absorbance at 663.2 and 646.8 nm.

In Vitro Binding Assay

Recombinant myc-tagged phytochrome B proteins were purified using an Escherichia coli system harboring both phyB and phycocyanobilin biosynthetic cassette vectors as previously described (Gambetta and Lagarias, 2001; Park et al., 2012). GST-tagged PIF3 proteins were purified using GST resins. An in vitro binding assay between phyB and PIFs was performed as previously described (Oh et al., 2004). Briefly, 1 μg phyto-chrome B proteins was exposed to red (9 μmol m−2 s−1) or far-red light (3 μmol m−2 s−1) for 10 min before mixing with 2 μg of GST-PIF3 proteins. GST-PIF3 proteins were pulled down and the bound phyB proteins were detected using a myc antibody (Santa Cruz Biotechnology).

ChIP Assay

ChIP assays were performed as previously described (Park et al., 2012). Briefly, seedlings were grown in darkness or red light (9 µmol m–2 s–1) for

1290 The Plant Cell

4 d. Fixed and sonicated extracts were immunoprecipitated with either a PIF1 antibody (Lee et al., 2014), a PIF4 antibody (Agrisera; AS12 1860 and AS16 3157), or a myc antibody (Cell Signaling; 2276). The immuno-precipitated promoter fragments were quantified by real-time PCR using specific primer sets (Supplemental Table 1). For the 26S proteasome in-hibitor treatments, PHYBG111D PIF3OX3 phyA phyB seedlings were grown for 4 d in red light and treated with 50 μM MG132 and 0.5 μM Epoxomicin for 16 h in red light before sampling for the ChIP assays. ChIP assays were performed using at least two biological replicates. For biological replicates, we prepared seedling samples independently and analyzed them independently. For technical replicates, we repeated the same anal-ysis using the same sample.

mRNA Expression Analysis

Seedlings were grown on half-strength MS media plus 1% sucrose for 4 d in red light. Total mRNAs were extracted using the Spectrum plant total RNA kit (Sigma-Aldrich) and converted to cDNAs using MMLV-RTase (Promega) according to the manufacturer’s protocols. The relative transcript level for each gene was determined by quantitative PCR and normalized to that of PP2A. Quantitative PCR was performed using CFX Connect (Bio-Rad). Each gene expression pattern was confirmed using at least three biological replicates. For biological replicates, we prepared seedling samples independently and analyzed them independently.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: PHYB (AT2G18790), PHYA (AT1G09570), PIF1 (AT2G20180), PIF3 (AT1G09530), PIF4 (AT2G43010), PIL1 (AT2G46970), PIL2 (AT3G62090), FHL (AT5G02200), IAA29 (AT4G32280), ORE1 (At5G39610), NYE1 (At4G22920), PPH (At5G13800), and PP2A (AT1G13320).

Supplemental Data

Supplemental Figure 1. PIF4 protein levels are higher in red light than in the dark.

Supplemental Figure 2. Biological replicates of ChIP assay data.

Supplemental Figure 3. The N-terminal half of phyB (phyBN) inhibits hypocotyl elongation in red light.

Supplemental Figure 4. phyBN forms a dimer.

Supplemental Figure 5. phyBN induces light responses not by PIF protein degradation but by PIF sequestration.

Supplemental Table 1. List of primers.

ACKNOWLEDGMENTS

We thank TAIR and NASC for providing information and mutant seeds. This work was supported in part by grants from the National Research Foundation of Korea (2015R1A2A1A05001091 and 2011-0031955) to G.C.

AUTHOR CONTRIBUTIONS

E.P., Y.K., and G.C. designed experiments. E.P. and Y.K. performed ex-periments. E.P. and G.C. wrote the article.

Received November 27, 2017; revised March 23, 2018; accepted May 10, 2018; published May 15, 2018.

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DOI 10.1105/tpc.17.00913; originally published online May 15, 2018; 2018;30;1277-1292Plant Cell

Eunae Park, Yeojae Kim and Giltsu ChoiWide Range of Light Conditions

Phytochrome B Requires PIF Degradation and Sequestration to Induce Light Responses across a

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References /content/30/6/1277.full.html#ref-list-1

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