Photobody Localization of Phytochrome B Is Tightly Correlated with Prolonged and Light-Dependent Inhibition of Hypocotyl Elongation in the Dark.

Photobody localization of Arabidopsis (Arabidopsis thaliana) phytochrome B (phyB) fused to green fluorescent protein (PBG) correlates closely with the photoinhibition of hypocotyl elongation. However, the amino-terminal half of phyB fused to green fluorescent protein (NGB) is hypersensitive to light despite its inability to localize to photobodies. Therefore, the significance of photobodies in regulating hypocotyl growth remains debatable. Accumulating evidence indicates that under diurnal conditions, photoactivated phyB persists into darkness to inhibit hypocotyl elongation. Here, we examine whether photobodies are involved in inhibiting hypocotyl growth in darkness by comparing the PBG and NGB lines after the red light-to-dark transition. Surprisingly, after the transition from 10 μmol m-2 s-1 red light to darkness, PBG inhibits hypocotyl elongation three times longer than NGB. The disassembly of photobodies in PBG hypocotyl nuclei correlates tightly with the accumulation of the growth-promoting transcription factor PHYTOCHROME-INTERACTING FACTOR3 (PIF3). Destabilizing photobodies by either decreasing the light intensity or adding monochromatic far-red light treatment before the light-to-dark transition leads to faster PIF3 accumulation and a dramatic reduction in the capacity for hypocotyl growth inhibition in PBG. In contrast, NGB is defective in PIF3 degradation, and its hypocotyl growth in the dark is nearly unresponsive to changes in light conditions. Together, our results support the model that photobodies are required for the prolonged, light-dependent inhibition of hypocotyl elongation in the dark by repressing PIF3 accumulation and by stabilizing the far-red light-absorbing form of phyB. Our study suggests that photobody localization patterns of phyB could serve as instructive cues that control light-dependent photomorphogenetic responses in the dark.


INTRODUCTION
Plant growth and development are extremely plastic in response to environmental light cues (Franklin and Quail, 2010;Kami et al., 2010). This light-dependent phenotypic plasticity is best exemplified by the photoinhibition of hypocotyl elongation during the seedling development of dicotyledonous plants, such as the reference plant species Arabidopsis thaliana. Plants perceive light through a number of photoreceptors, including the red (R) and far-red (FR) sensing phytochromes (phys). Phys are bilin-containing proteins that consist of two domains: an N-terminal photosensory/signaling domain and a C-terminal dimerization/localization domain (Rockwell et al., 2006;Nagatani, 2010). Because the phytochromobilin chromophore is buried inside the polypeptide moiety of the N-terminal domain, isomerization of the chromophore by R or FR light absorption triggers photo-conversion between two relatively stable phy conformers: a red (R) light-absorbing, inactive, Pr form, and a far-red (FR) light-absorbing, active, Pfr form (Rockwell et al., 2006;Nagatani, 2010;Ulijasz and Vierstra, 2011). In addition to photoconversion between the Pr and Pfr forms, the Pfr form is thermodynamically unstable and can spontaneously revert back to the Pr form in the dark in a process termed dark reversion (Furuya and Song, 1994;Nagy and Schafer, 2002). Therefore, photo-conversion and dark reversion together determine the equilibrium percentage of phy in the active Pfr form, which transmits signals to regulate downstream photomorphogenetic responses, such as the inhibition of hypocotyl growth. The Arabidopsis genome encodes five phytochrome genes, named phyA-E (Sharrock and Quail, 1989). Among the five phys, phyB is the main phy that mediates the perception of continuous R light and changes in the R-to-FR ratio (Chen et al., 2004).
First, most PIF proteins are stable only in the dark; in the light, photoactivated phys bind directly to PIFs and trigger their phosphorylation and subsequent degradation in the light (Al-Sady et al., 2006;Lorrain et al., 2008;Shen et al., 2008;Leivar and Quail, 2011;Ni et al., 2013 phys can inhibit the transcriptional activity of PIF1 and PIF3 by removing them from the promoters of their target genes (Park et al., 2012).
At the cellular level, one of the earliest responses to light is the translocation of photoactivated phys from the cytoplasm to subnuclear foci called phy speckles or photobodies (Yamaguchi et al., 1999;Kircher et al., 2002;Chen and Chory, 2011;Van Buskirk et al., 2012).
Since the initial observation of photobodies fifteen years ago (Yamaguchi et al., 1999), the necessity of photobodies in phy signaling has been debated. Accumulating evidence supports the notion that photobody localization of phyB is required for phy-mediated inhibition of hypocotyl growth (Van Buskirk et al., 2012). For example, in continuous R light, the steady-state photobody localization pattern (size and number) of phyB fused to GFP (PBG) is directly determined by the percentage of phyB in the Pfr form (Yamaguchi et al., 1999;Chen et al., 2003). Light conditions that shift the Pfr/Pr equilibrium in favor of the Pfr form promote the localization of phyB to large photobodies. Consistent with this notion, in high-intensity R light, PBG localizes exclusively to a few large photobodies with diameters between 1 μm and 2 μm, and seedlings are correspondingly short (Chen et al., 2003;Chen et al., 2010). By contrast, in dim R light or in light with a low R to FR ratio, PBG is localized to many small photobodies or is evenly dispersed in the nucleoplasm, and seedlings are taller (Chen et al., 2003). Together these results support the idea that the localization of phyB to photobodies correlates tightly with the degree of hypocotyl growth inhibition (Chen et al., 2003).
Genetic analyses of mutants with abnormal phyB photobody morphology also support the correlation between photobody localization of phyB and the inhibition of hypocotyl growth.
Most loss-of-function phyB alleles are defective in phyB localization to large photobodies and are taller than wild-type (Kircher et al., 2002;Chen et al., 2003;Matsushita et al., 2003), whereas in gain-of-function phyB mutants, phyB localizes to large photobodies under dim light and hypocotyl elongation is more restricted than in wild-type (Adam et al., 2011;Medzihradszky et al., 2013;Zhang et al., 2013). In the most extreme case, phyB Y276H (YHB), a constitutively active phyB mutant, localizes to large photobodies regardless of light conditions and can inhibit hypocotyl growth even in the dark (Su and Lagarias, 2007). In the extragenic hmr mutant, PBG fails to localize to large photobodies and localizes to small photobodies instead; hmr mutants have correspondingly longer hypocotyls than wild-type in R light (Chen et al., 2010 Although these results support the significance of photobodies in phy signaling, one line of evidence stands out against this model. Studies of photobody localization using Arabidopsis phyB have shown that the C-terminal domain of phyB is involved in dimerization and is sufficient for both nuclear and photobody localization (Matsushita et al., 2003;Chen et al., 2005). When the C-terminal domain of phyB is replaced with a dimerization domain, an SV40 nuclear localization signal, and GFP, the chimeric protein, NGB, does not localize to photobodies (Matsushita et al., 2003). However, NGB is hyperactive in inhibiting hypocotyl growth in the light, suggesting that photobodies are dispensable and might even play a negative role in the photoinhibition of hypocotyl elongation in the light (Matsushita et al., 2003;Palagyi et al., 2010).
To reconcile these contradictory conclusions about photobodies' roles in phy signaling and hypocotyl growth inhibition, a more detailed comparison between the PBG and NGB lines in hypocotyl growth regulation is warranted. In particular, recent studies show that although plants perceive light during the day, under short days, the maximum hypocotyl growth rate occurs at the end of the night (Nozue et al., 2007). Hypocotyl growth during the dark period is mediated by PIF3, as well as by PIF1, PIF4 and PIF5; the levels PIFs are coincidentally regulated by the circadian clock and light (Nozue et al., 2007;Leivar et al., 2012;Soy et al., 2012;Soy et al., 2014). The circadian clock mediates an increase in PIF4 and PIF5 gene expression at the end of the night (Nozue et al., 2007;Leivar et al., 2012;Soy et al., 2012). In parallel, because of the slow dark reversion rate of phyB (Hennig et al., 1999;Rausenberger et al., 2010), photoactivated phyB persists into the night to inhibit the accumulation of PIFs (Nozue et al., 2007;Soy et al., 2012;Soy et al., 2014). Based on these recent results, it would be interesting to investigate whether photobodies are required for phyB-mediated hypocotyl growth inhibition and PIF stability in the dark.
Here, by comparing the kinetics of hypocotyl growth, PIF3 accumulation, and the expression of PIF target genes between the PBG and NGB lines following the red-to-dark (R-to-D) transition, we show that PBG can repress hypocotyl growth for a prolonged period of time in the dark. In contrast, and contrary to its hyperactivity in hypocotyl growth inhibition in the light, NGB has substantially less capacity for repressing hypocotyl growth in the dark. We show a close correlation between the photobody localization of PBG, PIF3 degradation, and hypocotyl growth inhibition in the dark. Our results support the model that photobodies are required for the prolonged, light-dependent inhibition of hypocotyl elongation in the dark by stabilizing the Pfr form of phyB and by repressing PIF3 accumulation.

PBG represses hypocotyl growth for a substantially longer period than NGB in the dark
To examine whether photobodies are involved in phyB's function in inhibiting hypocotyl growth in the dark, we wanted to design a R-to-D transition assay to measure the capacity of a seedling to inhibit hypocotyl growth in the dark. Previous studies have shown that seedlings germinated under either light or dark conditions look similar during the first two days (Wei et al., 1994). Under both conditions, a burst of hypocotyl growth occurs mainly between the third and the fifth day post-germination, but this burst is much more pronounced in dark-grown seedlings (Wei et al., 1994;Gendreau et al., 1997). As a result, dark-grown seedlings exhibit elongated hypocotyls 4 d after seed germination, while hypocotyl growth in light-grown seedlings is comparatively limited (Wei et al., 1994); much of this growth inhibition in R light is due to the action of phyB (Reed et al., 1994). Given these reports, an assay was designed to determine whether photobodies are involved in regulating the magnitude of this growth burst in the dark. In this assay, seedlings were first exposed to 10 µmol m -2 s -1 of continuous R light for 48 h, which allows for seed germination and for phyB to localize to large photobodies (Chen et al., 2003).
Then, the seedlings were transferred to the dark just before the presumed growth burst for an additional 144 h (6 d), during which time the seedling growth kinetics were monitored by measuring hypocotyl length at various time points ( Figure 1A). We reasoned that if photobodies play a role in regulating hypocotyl growth in the dark, then the PBG and NGB lines should exhibit different hypocotyl growth kinetics in this assay. Consistent with previous reports (Wei et al., 1994;Gendreau et al., 1997), hypocotyl growth in both the PBG and NGB lines occurred mainly during the first 2 d after the R-to-D transition (equivalent to the third and fourth days after stratification) ( Figure 1B,C). Interestingly, PBG seedlings were able to repress hypocotyl growth more efficiently and thus were much shorter than NGB seedlings in this condition ( Figure   1B,C).
Because our initial experiments showed that hypocotyl growth occurs mainly during the first 2 d after the R-to-D transition, we refined the assay and performed more detailed hypocotyl growth kinetics analysis on PBG and NGB during the first 48 h after the R-to-D transition  Figure 1D,E). We defined the capacity to repress hypocotyl growth as the period of time that a seedling could maintain its hypocotyl length to within an arbitrary threshold of 1.3-fold that of time zero. These experiments showed that the PBG line was able to repress hypocotyl growth for 18 h in the dark. In contrast, the NGB line was only able to repress hypocotyl growth for 6 h ( Figure 1E). Therefore, under this experimental condition, the PBG line could repress hypocotyl growth three times longer than the NGB line. This result was surprising because in terms of hypocotyl growth inhibition, the NGB line is hypersensitive to light (Matsushita et al., 2003).
Because the protein levels of PBG and NGB remained relatively constant during the course of the assay (Supplemental Figure 1), and because a major difference between PBG and NGB is that PBG, but not NGB, can localize to photobodies, these results suggest that photobody localization of PBG might be the cause of the difference in the capacity for hypocotyl growth inhibition between the PBG and NGB lines.
Because both PBG and NGB are transgenic lines overexpressing either PBG or NGB, we wanted to confirm that hypocotyl growth in the R-to-D transition assay is also repressed by phyB and promoted by PIFs in the wild-type. We therefore examined the growth kinetics of the wildtype Col-0, phyB-9, and pifq in our assays ( Figure 1F). Col-0 seedlings were able to repress hypocotyl growth for 6 h, and this hypocotyl growth repression was almost completely lost in phyB-9 ( Figure 1F), suggesting that hypocotyl growth repression during the R-to-D transition is phyB-dependent. The fact that PBG can repress hypocotyl growth longer than the WT Col-0 ( Figure 1E,F) suggests that the amount of phyB is important in determining the capacity for hypocotyl growth inhibition in the dark. In contrast to Col-0 and phyB-9, pifq was impaired in growth after the R-to-D transition ( Figure 1F), indicating that the hypocotyl growth after the Rto-D transition is mediated by PIFs.

PBG, but not NGB, can repress PIF3 accumulation in both the light and the dark
Among the PIFs, PIF3 plays an import role in promoting hypocotyl growth in the dark under diurnal conditions; in particular, PIF3 is not regulated at the transcriptional level by the circadian clock, but mainly at the step of protein degradation by photoactivated phys (Soy et al., 2012). Therefore, we decided to use PIF3 as a model to examine whether phy-mediated PIF3 degradation is differentially regulated in the PBG and NGB lines during the R-to-D transition. In the PBG line, PIF3 was undetectable in the light and remained that way until 18 h after the R-to- Van Buskirk et al., page 10 D transition (Figure 2A ,B). Consistent with the model that PIF3 promotes hypocotyl growth, the appearance of PIF3 was perfectly correlated with the increase in hypocotyl growth in both PBG and Col-0 ( Figure 2B). In striking contrast, PIF3 accumulated in NGB and phyB-9 in continuous R light and after the R-to-D transition ( Figure 2B); our observation that the NGB line fails to degrade PIF3 in the light is consistent with a recent report by Giltsou Choi's laboratory (Park et al., 2012). Because the steady-state mRNA levels of PIF3 were comparable between PBG and NGB seedlings during the R-to-D transition (Supplemental Figure 2), the difference in PIF3 abundance between these two lines is most likely due to differences in PIF3 degradation by PBG and NGB. Together, these results show that PBG, but not NGB, can repress PIF3 accumulation both in R light and in darkness.
Next, to assess the transcriptional activity of PIF3 in the PBG and NGB lines at various time points during the R-to-D transition, we determined the expression levels of four wellcharacterized PIF target genes: PIL1, IAA29, XTR7, and ATHB2 (Leivar et al., 2009;Hornitschek et al., 2012;Leivar et al., 2012). None of these genes were activated in pifq ( Figure 2C), confirming that, in this assay condition, their expression is dependent on PIFs. The expression of the PIF3 target genes was also correlated with the PIF3 levels in Col-0 and phyB-9, as all four genes were induced between 6 and 12 h after the R-to-D transition in Col-0, and they were induced in phyB-9 compared with Col-0 at time 0 ( Figure 2C). In PBG, the four PIF target genes were induced between 18 and 24 h after the R-to-D transition ( Figure 2B,D). Therefore, the timing of this induction corresponded faithfully with the increase in PIF3 levels and the initiation of hypocotyl growth ( Figure 2B,D and Figure 1E,F). In contrast, the induction of PIF targets did not coincide with PIF3 protein accumulation in the NGB line; although PIF3 was present at all time points, the PIF targets were only induced between 6 and 12 h after the R-to-D transition ( Figure 2B,D). Therefore, in NGB, it is not the PIF protein level, but rather the activity of PIF3, that correlates with the initiation of hypocotyl growth ( Figure 2B,D and Figure 1E). These results support the notion that there are at least two mechanisms by which phyB represses hypocotyl elongation in the dark: repression of PIF3 accumulation and inhibition of PIF3 transcriptional activity. Interestingly, PBG seems to inhibit hypocotyl growth mainly by repressing PIF3 accumulation or by regulating both PIF3 abundance and transcriptional activity simultaneously, whereas NGB inhibits hypocotyl growth primarily by inhibiting PIF transcriptional activity.

Photobody disassembly in PBG correlates with PIF3 accumulation and hypocotyl growth
The discrepancies between PBG and NGB in hypocotyl growth kinetics, PIF3 accumulation, and the expression of PIF target genes in the R-to-D transition assay provided an opportunity to precisely determine photobodies' roles in these processes in the dark. Previous studies have utilized two main parameters to describe the dynamics of photobodies: the percentage of nuclei with or without photobodies and the average size/number of photobodies per nucleus (Yamaguchi et al., 1999;Kircher et al., 2002;Chen et al., 2003;Chen et al., 2010).
In the past, the size and number of photobodies have been measured primarily by using 2D maximum projection images derived from stacks of images of optical sections. Although this approach is useful for the analysis of nuclei with only a few large photobodies, it does not work well for nuclei with many small photobodies; because small photobodies from different optical sections might overlap in the projected image, the information on the size and number of photobodies could be lost or mis-represented in the projection.
To circumvent this problem, we analyzed photobodies from 3D stacks of confocal images using the object analysis tool of Huygens Essential software (Scientific Volume Imaging). Using the software, we determined the number of large and small photobodies per nucleus and the size distribution of the small photobodies. All objects smaller than 0.5E-3 μm 3 in volume (0.1 μm in estimated diameter, assuming that photobodies are spherical) were excluded from our measurements because these objects were beyond our detection limit. We arbitrarily defined "large photobodies" as those with a volume equal to or greater than 0.2 μm 3 (0.72 μm in estimated diameter) and "small photobodies" as those with a volume between 0.5E-3 μm 3 and 0.2 μm 3 (0.1 μm to 0.72 μm in estimated diameter).
Because the major differences between PBG and NGB in hypocotyl growth repression occur during the first 24 h after the R-to-D transition ( Figure 1E), we focused on the dynamics of PBG and NGB localization during this time period. As expected, PBG seedlings grown in continuous R light for 2 d had photobodies in all hypocotyl nuclei; on average there were between 6 and 8 large photobodies per nucleus (Figure 3). Some nuclei also had a few small photobodies, but these nuclei were rare (Figure 3). After the R-to-D transition, the photobody morphology in PBG went through two major transitions. The first transition took place over the first 12 h in darkness; during this period, although most of the hypocotyl nuclei had photobodies, the large photobodies disassembled and began to disappear, and the number of small photobodies  (Figure 3). The second transition occurred between 12 and 18 h after the R-to-D transition; during this period, photobodies were completely lost from about 97% of hypocotyl nuclei ( Figure 3). The disappearance of photobodies at 18 h coincides with the accumulation of PIF3 ( Figure 2B) and the initiation of hypocotyl growth in PBG ( Figure 1E). Therefore, these data support the model that photobodies are required for inhibiting hypocotyl growth by repressing PIF3 in the dark. The analysis of the NGB line further supports this notion. As shown in Figure 3, the majority of nuclei in NGB did not contain any photobodies. Inconsistent with the report by Matsushita et al. (Matsushita et al., 2003), a small fraction of nuclei did contain small photobodies under these experimental conditions. Nonetheless, because NGB failed to localize to photobodies in more than 80% of nuclei (Figure 3), these results are consistent with the idea that photobodies are required for PIF degradation in the light and the prolonged repression of PIF accumulation in the dark. Our data also suggest that the repression of PIF3 activity by NGB can occur in the absence of photobodies.

Decreased light intensity leads to the faster disassembly of photobodies and a reduced capacity for hypocotyl growth inhibition in PBG in the dark
To further test the model that photobody morphology determines the capacity for PIF3 repression and hypocotyl inhibition in the dark, we asked whether we could alter these two latter processes by manipulating photobody morphology. Because the steady-state pattern of photobodies is directly regulated by light intensity (Chen et al., 2003, Van Buskirk et al., 2012, we modified our assay condition by growing seedlings in a reduced R light intensity of 1 µmol m -2 s -1 for 2 d before the R-to-D transition. As reported previously (Chen et al., 2003), in the dimmer light condition, PBG was localized to both large and small photobodies (Figure 4).
Compared with the 10 µmol m -2 s -1 R light treatment, the dimmer light treatment led to the faster disassembly of PBG photobodies in the dark; this difference was most obvious between the 6 h and12 h time points (Figure 4). At the 6 h time point, the percentage of nuclei with photobodies had already dropped to approximately 69%; at the 12 h time point, photobodies were completely lost from more than 80% of all nuclei. Therefore, the photobody disassembly process in PBG was at least 6 h faster after the 1 µmol m -2 s -1 R light treatment compared with the 10 µmol m -2 s -1 R light treatment. In contrast, the localization pattern of NGB was quite similar after both the strong and dim R light treatments (Figure 3 and Figure 4). To test whether the change in photobody dynamics in PBG leads to changes in the kinetics of PIF3 accumulation, PIF transcriptional activity, and hypocotyl growth, we examined these responses in both PBG and NGB during the 1 µmol m -2 s -1 R-to-D transition ( Figure 5A).
As shown in Figure 5, consistent with the loss of photobodies in PBG after 12 h in darkness, the PBG seedlings grown in 1 µmol m -2 s -1 R light were only able to repress hypocotyl growth, PIF3 accumulation, and the expression of PIF targets for approximately 10-12 h ( Figure 5B-D). In contrast, just as NGB exhibited similar localization patterns between the 1 and 10 µmol m -2 s -1 treatments (Figure 3 and Figure 4), NGB seedlings showed similar hypocotyl growth kinetics, PIF3 accumulation, and PIF target gene induction between the two light conditions (Figures 1E,   2B, 2D and 5B-D). Together, these data suggest that the steady-state pattern of photobodies in PBG prior to the R-to-D transition correlates with the capacity to fine-tune hypocotyl growth inhibition and PIF3 repression in the dark. Consistent with this notion, NGB, which does not localize to photobodies in the majority of nuclei, does not respond to differences in light quantity before the dark period.  Figure 6B). The small fraction of cells with some small photobodies in in NGB also lost their photobodies within the first hour in the dark,

The prolonged hypocotyl growth inhibition in
indicating that the small photobodies in NGB are also dependent on its Pfr form.
Measuring hypocotyl growth kinetics after FR light treatment showed that the FR pulse treatment caused virtually no change in the growth kinetics of NGB, which still began to grow 6 h after the R-FR-D transition darkness ( Figure 6B), suggesting that the minimum time required for a 30% increase in hypocotyl length might be approximately 6 h. In contrast, PBG responded strongly to the FR treatment; hypocotyl growth was inhibited for only approximately 9 h, which is half the time of that without FR treatment ( Figure 6B and Figure 1E). This result suggests that the prolonged hypocotyl growth repression in PBG -18 h, compared with 6 h in NGB -is mainly due to Pfr stabilization in the dark. However, even after the FR treatment, PBG was still able to repress hypocotyl growth 3 h longer than NGB ( Figure 6B), suggesting that there must be other mechanisms that account for this difference in hypocotyl growth repression.
We next determined the patterns of PIF3 accumulation and the expression of PIF targets in both PBG and NGB after the R-FR-D transition. In PBG, PIF3 began to accumulate within 1 h after the FR treatment ( Figure 6C); this result is consistent with a previous report on PIF3 dynamics (Monte et al., 2004). The accumulation of PIF3 in the PBG line again correlated again perfectly with photobody disassembly (Figure 6A,C). Because PIF3 degradation is triggered by the Pfr form of phyB, the dynamic changes in PIF3 levels could serve as a readout for the presence of the Pfr from of phyB in PBG. Based on this readout, the Pfr form of phyB-GFP in PBG lasts for approximately 18 h after the 10 µmol m -2 s -1 R-to-D transition ( Figure 2B) and for about 12 h after the 1 µmol m -2 s -1 R-to-D transition ( Figure 5C). As predicted, In NGB, PIF3 was detectable in both continuous R light and remained detectable for the duration of the experiment, showing little change in abundance after FR treatment ( Figure 6C). Surprisingly, although PIF3 began to accumulate in PBG within 1 h of the FR treatment, the expression of PIF targets remained repressed for 9 h ( Figure 6D). In contrast, in NGB, the expression of all four PIF targets was induced immediately after FR treatment ( Figure 6E). These data suggest the repression of PIF activity in NGB in the dark is mainly dependent on the Pfr form of NGB; however, in PBG, the repression of the expression of PIF targets could be mediated by an unknown mechanism that is independent of photobodies and of the Pfr from of phyB. This offers an explanation for the difference in hypocotyl growth inhibition between the PBG and NGB lines during the R-FR-D transition ( Figure 6B). Because the expression of PIF target genes is Pfr-dependent in NGB, the repression of PIF target genes can be used as a readout for the presence of the Pfr form of NGB; based on this readout, the Pfr form of NGB can last for approximately 6 h after both the 10 µmol m -2 s -1 and 1 µmol m -2 s -1 R-to-D transitions ( Figure   2D and Figure 5D). Together, these results suggest that the Pfr form of PBG can last 12 h longer than that of NGB.

DISCUSSION AND CONCLUSIONS
Although a growing body of evidence supports the biological importance of photobodies in the phy-mediated photoinhibition of hypocotyl growth (Van Buskirk et al., 2012), comparisons between the photobody-localized PBG and nucleoplasm-localized NGB showed that in the light, NGB is hyperactive in inhibiting hypocotyl growth, suggesting that photobodies are unnecessary and might even play a negative role in the light-dependent inhibition of hypocotyl elongation (Matsushita et al., 2003;Palagyi et al., 2010). Here, we developed a R-to-D transition assay to examine the relationship between dynamic changes in photobody morphology and the molecular events of PIF3 accumulation and the expression of PIF targets, as well as the repression of hypocotyl growth between the PBG and NGB lines. Our results demonstrate a tight correlation between the photobody localization of PBG and the repression of PIF3 accumulation in both light and dark conditions. Our data support a model in which photobodies mediate prolonged, light-dependent hypocotyl growth inhibition in the dark by stabilizing the Pfr from of phyB and repressing PIF3 accumulation (Figure 7).

Photobody localization of phyB tightly correlates with the repression of PIF3 degradation
Accumulating evidence suggests that phys inhibit hypocotyl growth both by triggering the degradation of multiple PIFs and by inhibiting PIFs' transcriptional activity. Our data suggest that photobody localization of phyB is specifically required for repressing PIF3 accumulation in the light and after the light-to-dark transition. This conclusion is supported by the following two lines of evidence: first, by comparing PIF3 levels in 2-d old light-grown PBG and NGB seedlings, we show that PIF3 accumulation is repressed in PBG but not in NGB ( Figure 2B). conditions tested: the 10 µmol m -2 s -1 R-to-D transition ( Figure 2B and Figure 3), the 1 µmol m -2 s -1 R light to D transition (Figure 4 and Figure 5C), and the R-FR-D transition ( Figure 6A,C), the disassembly of photobodies in hypocotyl nuclei correlates tightly with the accumulation PIF3.
The R-FR-D experiment is particularly informative, as this treatment demonstrates that the regulation of PIF degradation and the repression of PIF activity can be separated even in the PBG background.
The tight correlation between the photobody localization of PBG and PIF3 degradation provides strong evidence supporting the model that photobody localization of phyB is required for PIF3 degradation (Figure 7). Although we still cannot completely exclude the possibility that photobody localization of phyB and PIF3 degradation are two parallel consequences of phyB activation, the conclusion that photobodies are required for PIF degradation is consistent with and supported by previously published results. First, the constitutively active phyB allele, YHB, localizes to photobodies and can trigger PIF3 degradation in the dark (Su and Lagarias, 2007;Galvão et al., 2012). Second, the photobody-deficient mutant, hmr, is also defective in the degradation of PIF1 and PIF3 in the light (Chen et al., 2010). In addition, in the YHB/hmr-1 double mutant, YHB fails to localize to large photobodies in the dark and the repression of PIF3 accumulation by YHB is also reversed (Galvão et al., 2012). Third, PIF3 localizes to photobodies prior to its degradation (Bauer et al., 2004;Al-Sady et al., 2006), and mutations in phyB that abrogate its interaction with PIFs do not affect phyB's localization to photobodies but are not functional in the photoinhibition of hypocotyl elongation (Oka et al., 2008;Kikis et al., 2009), suggesting that recruiting PIFs to photobodies is required for PIF degradation and phy signaling.
Taken together, the results from this and previous studies support the most likely model that photobodies are required for PIF degradation.
The molecular mechanism by which photobodies are involved in PIF degradation is still unclear. There is still no direct evidence showing that PIF3 degradation occurs on photobodies.
Therefore, we cannot exclude the possibility that photobodies are only involved in a posttranslational modification of PIFs, such as phosphorylation (Al-Sady et al., 2006;Lorrain et al., 2008;Shen et al., 2008;Bu et al., 2011;Ni et al., 2013), and that PIF degradation occurs elsewhere (Van Buskirk et al., 2012). It is also important to note that although our data show a clear separation between PIF3 degradation and the regulation of its transcriptional activity in the R-FR-D transition ( Figure 6C It is worth pointing out that the repression of PIF3 activity after the R-FR-D transition in PBG appears to be mediated by a different mechanism from the NGB-dependent repression of PIF3 activity. It has previously been shown that NGB inhibits PIF3 activity by removing PIF3 from the promoters of its target genes and that this activity of NGB is Pfr-dependent (Park et al., 2012). Our results are consistent with this report in that a FR pulse can release the repression of PIF targets by NGB ( Figure 6D). In contrast, the repression of PIF targets in PBG after the R-FR-D transition was clearly not mediated by the Pfr form of PBG, as the FR pulse quickly converted PBG to its Pr form and promoted PIF accumulation, but the repression of PIF targets persisted ( Figure 6D). Therefore, the repression of PIF activity in PBG in the assay is mediated by a yet unknown mechanism. One possibility is that another transcriptional repressor protein for the PIF targets is present only in PBG and that the delay in the induction of the PIF targets reflects the time required for the removal of this additional protein. An alternative model is that the chromatin state of the PIF targets is different between the PBG and NGB lines in the light; while the PIF targets could be primed for induction in NGB, in PBG, they might be in a silenced state that requires additional time for activation. Future experiments on the chromatin status of the PIF targets in these two lines might help to reveal this unknown mechanism for the repression of PIF target genes after the R-FR-D transition.

Photobodies mediate prolonged hypocotyl growth inhibition in the dark likely by stabilizing the Pfr form of phyB
It is well known that the Pfr form of phyB has a relatively slow dark reversion rate and that it can persist into darkness; these properties of phyB play a pivotal role in hypocotyl growth inhibition under diurnal conditions (Hennig et al., 1999;Rausenberger et al., 2010). This is best demonstrated by end-of-day FR treatment, in which a FR light pulse is applied at dusk to inactivate phyB to its Pr form. End-of-day FR treatment leads to a dramatic decrease in the in vivo dark reversion rate of phyB is much slower than the rate measured in vitro, and photobodies have been proposed through mathematical modeling to be involved in stabilizing the Pfr form of phyB in the dark (Rausenberger et al., 2010). Here, we provide experimental evidence supporting this hypothesis. Our data show that the localization of PBG to photobodies dramatically increases the stability of its Pfr form compared with that of the nucleoplasmic NGB. PBG is extended three times longer than that of NGB in vivo. This dramatic decrease in the capacity for hypocotyl growth inhibition in PBG between the 10 µmol m -2 s -1 R-to-D ( Figure 1E) and the R-FR-D transitions ( Figure 6B) demonstrates that the stabilization of the Pfr form of PBG contributes substantially to the prolonged inhibition of hypocotyl growth in the dark. The stability of PBG Pfr can also be fine-tuned by the light intensity and photobody localization pattern before the light-to-dark transition; in the 1 µmol m -2 s -1 R-to-D transition experiment, PBG localized to large and small photobodies and the life of the Pfr form was reduced to 9 h ( Figure 4 and Figure 5). In contrast, the life of the Pfr form of NGB seems to remain the same in both the 10 µmol m -2 s -1 and 1 µmol m -2 s -1 R-to-D transitions ( Figure 2D and Figure 5D).
Together, these results suggest that photobody localization of phyB is required for the prolonged inhibition of hypocotyl growth in the dark by stabilizing the Pfr form of phyB, and that this photobody-dependent Pfr stabilization could be a mechanism to fine-tune hypocotyl elongation in the dark (Figure 7).
Although our experiments were not performed under diurnal conditions, the mechanism involved in hypocotyl growth regulation appears to be similar between the R-to-D transition and the day-to-night transition; under both conditions, hypocotyl growth is promoted by PIFs and repressed by active phyB ( Figure 1F and Figure  transition (Kircher et al., 2002), in diurnal conditions, photobody localization of phyB may be involved in the PIF repression and hypocotyl growth inhibition seen in the early evening. We propose that this photobody-dependent hypocotyl growth repression mechanism allows seedlings to carry light cues perceived during the day into the evening to fine-tune photomorphogenetic responses accordingly (Figure 7).

Plant materials, growth conditions, and hypocotyl measurement
The PBG (Yamaguchi et al., 1999), NGB (Matsushita et al. 2003), phyB-9 (Reed et al., 1993), and pifq (Leivar et al., 2008 lines were as previously described. Seeds were surface sterilized with a rinse in 70% EtOH, followed by 15 min in 50% bleach supplemented with 0.02% Triton-X-100. Seeds were rinsed 5 times with ddH 2 O prior to plating on half-strength Murashige and Skoog medium supplemented with B vitamins (Caisson) and containing 0.6% phyto agar (Caisson). Seeds were stratified for 5 d in darkness prior to being treated with the indicated light conditions. The light intensity in the R and FR light LED chambers (Percival Scientific, Perry, IA) was measured using a fiber optic probe and SpectraWiz software (StellarNet, Inc., Tampa, FL). Seedling images were obtained by laying seedlings on a transparency and scanning using an Epson Perfection V700 photo scanner. Hypocotyl lengths were measured using ImageJ software (http://rsbweb.nih.gov/ij/).

Protein extraction and western blot
Total protein was extracted from seedlings using a mortar and pestle and 3 volumes of extraction buffer containing bromophenol blue as described previously (Galvão et al., 2012).
Protein samples were run on 8% Bis-tris-SDS-acrylamide gels and transferred to nitrocellulose membranes (Bio-Rad). Polyclonal antibodies against PIF3 (Chen et al. 2010) were used at a 1:500 dilution, and polyclonal antibodies against RPN6 (Enzo Life Sciences, Farmingdale, NY, Cat. #BML-PW8370-0025) were used at a 1:1000 dilution. Secondary antibodies were horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad) and were used at a 1:5000 dilution. Blots were visualized on X-ray film using SuperSignal West chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA). PIF3 and GFP levels were quantified using QuantityOne software (Bio-Rad), followed by a multi-step normalization: after background subtraction, the intensity of the PIF3 or GFP band was divided by the intensity of the corresponding RPN6 band. Then, the mean PIF3 or GFP intensity was calculated, and the normalized PIF3 or GFP intensity for each individual time point within each line was divided by this mean. This is the value that is reported in the figures.

qRT-PCR
Seedlings were flash-frozen in liquid nitrogen and ground to a powder using a plastic  Supplemental Table S1.     the Pfr forms of PBG and NGB can persist into the darkness to repress hypocotyl growth. There are two main differences between the photobody-localized PBG and the nucleoplasmic NGB: (1) photobodies are required for PIF degradation; photobody localization of PBG inhibits both PIF accumulation and PIF transcriptional activity, whereas NGB only represses PIF activity. (2) Photobody localization of PBG stabilizes its Pfr form and extend its life in the dark; consequently, PBG can inhibit hypocotyl elongation (cell growth) for a prolonged period of time.
This photobody-dependent mechanism of Pfr stabilization enables seedlings to convey perceived light cues into darkness to fine-tune hypocotyl growth accordingly.