Photobody Localization of Phytochrome B Is TightlyCorrelated with Prolonged and Light-DependentInhibition of Hypocotyl Elongation in the Dark1[W][OPEN]

Elise K Van Buskirk Amit K Reddy Akira Nagatani and Meng Chen

Department of Biology Duke University Durham North Carolina 27708 (EKVB AKR MC) andDepartment of Botany Graduate School of Science Kyoto University Sakyo-ku Kyoto 606ndash8502 Japan (AN)

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 greenfluorescent protein (NGB) is hypersensitive to light despite its inability to localize to photobodies Therefore the significance ofphotobodies in regulating hypocotyl growth remains debatable Accumulating evidence indicates that under diurnal conditionsphotoactivated phyB persists into darkness to inhibit hypocotyl elongation Here we examine whether photobodies are involved ininhibiting hypocotyl growth in darkness by comparing the PBG and NGB lines after the red light-to-dark transition Surprisinglyafter the transition from 10 mmol m22 s21 red light to darkness PBG inhibits hypocotyl elongation three times longer than NGB Thedisassembly of photobodies in PBG hypocotyl nuclei correlates tightly with the accumulation of the growth-promoting transcriptionfactor PHYTOCHROME-INTERACTING FACTOR3 (PIF3) Destabilizing photobodies by either decreasing the light intensity oradding monochromatic far-red light treatment before the light-to-dark transition leads to faster PIF3 accumulation and a dramaticreduction in the capacity for hypocotyl growth inhibition in PBG In contrast NGB is defective in PIF3 degradation and itshypocotyl growth in the dark is nearly unresponsive to changes in light conditions Together our results support the model thatphotobodies are required for the prolonged light-dependent inhibition of hypocotyl elongation in the dark by repressing PIF3accumulation and by stabilizing the far-red light-absorbing form of phyB Our study suggests that photobody localization patternsof phyB could serve as instructive cues that control light-dependent photomorphogenetic responses in the dark

Plant growth and development are extremely plastic inresponse to environmental light cues (Franklin and Quail2010 Kami et al 2010) This light-dependent phenotypicplasticity is best exemplified by the photoinhibition ofhypocotyl elongation during the seedling development ofdicotyledonous plants such as the reference plant speciesArabidopsis (Arabidopsis thaliana) Plants perceive lightthrough a number of photoreceptors including the redlight (R)- and far-red light (FR)-sensing phytochromesPhytochromes are bilin-containing proteins that consist oftwo domains an N-terminal photosensorysignalingdomain and a C-terminal dimerizationlocalization do-main (Rockwell et al 2006 Nagatani 2010) Because thephytochromobilin chromophore is buried inside the poly-peptide moiety of the N-terminal domain isomerization of

the chromophore by R or FR absorption triggers pho-toconversion between two relatively stable phytochromeconformers the inactive Pr and the active Pfr (Rockwellet al 2006 Nagatani 2010 Ulijasz and Vierstra 2011) Inaddition to photoconversion between Pr and Pfr Pfr isthermodynamically unstable and can spontaneouslyrevert back to Pr in the dark in a process termed darkreversion (Furuya and Song 1994 Nagy and Schaumlfer2002) Therefore photoconversion and dark reversiontogether determine the equilibrium percentage of phyto-chrome in the active Pfr which transmits signals to reg-ulate downstream photomorphogenetic responses suchas the inhibition of hypocotyl growth The Arabidopsisgenome encodes five phytochrome genes named phyAto phyE (Sharrock and Quail 1989) Among the five phy-tochromes phyB is the main phytochrome that mediatesthe perception of continuous R and changes in the R-to-FRratio (Chen et al 2004)

Phytochromes inhibit hypocotyl elongation by an-tagonizing a group of basic helix-loop-helix transcriptionfactors the phytochrome-interacting factors (PIFs Leivarand Quail 2011) Most PIFs including PIF1 PIF3 PIF4PIF5 and PIF7 promote hypocotyl growth (Huq andQuail 2002 Fujimori et al 2004 Huq et al 2004Khanna et al 2004 Oh et al 2004 Al-Sady et al 2008Lorrain et al 2009 Li et al 2012) the quadruple pif1pif3-pif4pif5 (pifq) mutant exhibits reduced hypocotyl growth inthe dark (Leivar et al 2008 2009 Shin et al 2009) Thecurrent model suggests that phytochromes inhibit PIFs by

1 This work was supported by the National Institutes of Health(grant no R01GM087388 to MC) the National Science Foundation(grant no IOSndash1051602 to MC) and a Grant-in-Aid for Scientific Re-search on Innovative Areas from the Ministry of Education CultureSports Science and Technology Japan (grant no 22120002 to AN)

Address correspondence to mengchendukeeduThe author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (wwwplantphysiolorg) isMeng Chen (mengchendukeedu)

[W] The online version of this article contains Web-only data[OPEN] Articles can be viewed online without a subscriptionwwwplantphysiolorgcgidoi101104pp114236661

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at least two mechanisms First most PIF proteins arestable only in the dark in the light photoactivatedphytochromes bind directly to PIFs and trigger theirphosphorylation and subsequent degradation in thelight (Al-Sady et al 2006 Lorrain et al 2008 Shen et al2008 Leivar and Quail 2011 Ni et al 2013) Secondphytochromes can inhibit the transcriptional activity ofPIF1 and PIF3 by removing them from the promoters oftheir target genes (Park et al 2012)

At the cellular level one of the earliest responses tolight is the translocation of photoactivated phytochromesfrom the cytoplasm to subnuclear foci called phyto-chrome speckles or photobodies (Yamaguchi et al 1999Kircher et al 2002 Chen and Chory 2011 Van Buskirket al 2012) Since the initial observation of photobodies15 years ago (Yamaguchi et al 1999) the necessity ofphotobodies in phytochrome signaling has been de-bated Accumulating evidence supports the notion thatphotobody localization of phyB is required for thephytochrome-mediated inhibition of hypocotyl growth(Van Buskirk et al 2012) For example in continuous Rthe steady-state photobody localization pattern (sizeand number) of phyB fused to GFP (PBG) is determinedby the percentage of phyB in Pfr (Yamaguchi et al1999 Chen et al 2003) Light conditions that shift thePfrPr equilibrium in favor of Pfr promote the locali-zation of phyB to large photobodies Consistent withthis notion in high-intensity R PBG localizes exclu-sively to a few large photobodies with diameters be-tween 1 and 2 mm and seedlings are correspondinglyshort (Chen et al 2003 2010) By contrast in dim R orin light with a low R-to-FR ratio PBG is localized tomany small photobodies or is evenly dispersed in thenucleoplasm and seedlings are taller (Chen et al 2003)Together these results support the idea that the locali-zation of phyB to photobodies correlates tightly with thedegree of hypocotyl growth inhibition (Chen et al 2003)

Genetic analyses of mutants with abnormal phyBphotobody morphology also support the correlationbetween the photobody localization of phyB and theinhibition of hypocotyl growth Most loss-of-functionphyB alleles are defective in phyB localization to largephotobodies and are taller than the wild type (Kircheret al 2002 Chen et al 2003 Matsushita et al 2003Nito et al 2013) whereas in gain-of-function phyBmutants phyB localizes to large photobodies underdim light and hypocotyl elongation is more restrictedthan in the wild type (Aacutedaacutem et al 2011 Medzihradszkyet al 2013 Zhang et al 2013) In the most extreme casephyBY276H (YHB) a constitutively active phyB mutantlocalizes to large photobodies regardless of light con-ditions and can inhibit hypocotyl growth even in thedark (Su and Lagarias 2007) In the extragenic hemera(hmr) mutant PBG fails to localize to large photobodiesand localizes to small photobodies instead hmr mutantshave correspondingly longer hypocotyls than the wildtype in R (Chen et al 2010)

Although these results support the significance ofphotobodies in phytochrome signaling one line of evi-dence stands out against this model Studies of photobody

localization using Arabidopsis phyB have shown thatthe C-terminal domain of phyB is involved in dimeriza-tion and is sufficient for both nuclear and photobodylocalization (Matsushita et al 2003 Chen et al 2005)When the C-terminal domain of phyB is replaced with adimerization domain a Simian Vacuolating Virus40nuclear localization signal and GFP the chimeric proteinNGB (for the N-terminal half of phyB fused to GFP) doesnot localize to photobodies (Matsushita et al 2003)However NGB is hyperactive in inhibiting hypocotylgrowth in the light suggesting that photobodies aredispensable and might even play a negative role in thephotoinhibition of hypocotyl elongation in the light(Matsushita et al 2003 Palaacutegyi et al 2010)

To reconcile these contradictory conclusions aboutthe roles of photobodies in phytochrome signaling andhypocotyl growth inhibition a more detailed com-parison between the PBG and NGB lines in hypocotylgrowth regulation is warranted In particular recentstudies show that although plants perceive light duringthe day under short days the maximum hypocotylgrowth rate occurs at the end of the night (Nozue et al2007) Hypocotyl growth during the dark period ismediated by PIF3 as well as by PIF1 PIF4 and PIF5 thelevels of PIFs are coincidentally regulated by the cir-cadian clock and light (Nozue et al 2007 Leivar et al2012a Soy et al 2012 2014) The circadian clock medi-ates an increase in PIF4 and PIF5 gene expression at theend of the night (Nozue et al 2007 Leivar et al 2012aSoy et al 2012) In parallel because of the slow darkreversion rate of phyB (Hennig et al 1999 Rausenbergeret al 2010) photoactivated phyB persists into the nightto inhibit the accumulation of PIFs (Nozue et al 2007Soy et al 2012 2014) Based on these recent results itwould be interesting to investigate whether photobodiesare required for phyB-mediated hypocotyl growth inhi-bition and PIF stability in the dark

Here by comparing the kinetics of hypocotyl growthPIF3 accumulation and the expression of PIF targetgenes between the PBG andNGB lines following the redlight-to-dark (R-to-D) transition we show that PBG canrepress hypocotyl growth for a prolonged period oftime in the dark In contrast and contrary to its hy-peractivity in hypocotyl growth inhibition in the lightNGB has substantially less capacity for repressing hy-pocotyl growth in the dark We show a close correlationbetween the photobody localization of PBG PIF3 deg-radation and hypocotyl growth inhibition in the darkOur results support the model that photobodies arerequired for the prolonged light-dependent inhibitionof hypocotyl elongation in the dark by stabilizing thePfr of phyB and by repressing PIF3 accumulation

RESULTS

PBG Represses Hypocotyl Growth for a SubstantiallyLonger Period Than NGB in the Dark

To examine whether photobodies are involved inphyBrsquos function in inhibiting hypocotyl growth in the

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dark we wanted to design an R-to-D transition assay tomeasure the capacity of a seedling to inhibit hypocotylgrowth in the dark Previous studies have shown thatArabidopsis seedlings germinated under either light ordark conditions look similar during the first 2 d (Weiet al 1994) Under both conditions a burst of hypocotylgrowth occurs mainly between days 3 and 5 post-germination but this burst is much more pronounced indark-grown seedlings (Wei et al 1994 Gendreau et al1997) As a result dark-grown seedlings exhibit elon-gated hypocotyls 4 d after seed germination while hy-pocotyl growth in light-grown seedlings is comparativelylimited (Wei et al 1994) much of this growth inhibitionin R is due to the action of phyB (Reed et al 1994) Giventhese reports an assay was designed to determinewhether photobodies are involved in regulating themagnitude of this growth burst in the dark In this assayseedlings were first exposed to 10 mmol m22 s21 contin-uous R for 48 h which allows for seed germination andfor phyB to localize to large photobodies (Chen et al2003) Then the seedlings were transferred to the darkjust before the presumed growth burst for an additional144 h (6 d) during which time the seedling growth ki-netics were monitored by measuring hypocotyl length atvarious time points (Fig 1A) We reasoned that if pho-tobodies play a role in regulating hypocotyl growth inthe dark then the PBG and NGB lines should exhibitdifferent hypocotyl growth kinetics in this assay Con-sistent with previous reports (Wei et al 1994 Gendreauet al 1997) hypocotyl growth in both the PBG and NGBlines occurred mainly during the first 2 d after the R-to-Dtransition (equivalent to days 3 and 4 after stratificationFig 1 B and C) Interestingly PBG seedlings were ableto repress hypocotyl growth more efficiently and thuswere much shorter thanNGB seedlings in this condition(Fig 1 B and C)Because our initial experiments showed that hypo-

cotyl growth occurs mainly during the first 2 d after theR-to-D transition we refined the assay and performedmore detailed hypocotyl growth kinetics analysis onPBG and NGB during the first 48 h after the R-to-Dtransition (Fig 1 D and E) We defined the capacity torepress hypocotyl growth as the period of time that aseedling could maintain its hypocotyl length to withinan arbitrary threshold of 13-fold that of time zeroThese experiments showed that the PBG line was ableto repress hypocotyl growth for 18 h in the dark Incontrast the NGB line was only able to repress hypo-cotyl growth for 6 h (Fig 1E) Therefore under thisexperimental condition the PBG line could represshypocotyl growth three times longer than the NGBline This result was surprising because in terms ofhypocotyl growth inhibition the NGB line is hyper-sensitive to light (Matsushita et al 2003) Because theprotein levels of PBG and NGB remained relativelyconstant during the course of the assay (SupplementalFig S1) and because a major difference between PBGand NGB is that PBG but not NGB can localize tophotobodies these results suggest that photobody lo-calization of PBG might be the cause of the difference

in the capacity for hypocotyl growth inhibition betweenthe PBG and NGB lines

Because both PBG and NGB are transgenic linesoverexpressing either PBG or NGB we wanted toconfirm that hypocotyl growth in the R-to-D transitionassay is also repressed by phyB and promoted by PIFsin the wild type Therefore we examined the growthkinetics of the wild-type Columbia-0 (Col-0) phyB-9and pifq in our assays (Fig 1F) Col-0 seedlings wereable to repress hypocotyl growth for 6 h and this hy-pocotyl growth repression was almost completely lostin phyB-9 (Fig 1F) suggesting that hypocotyl growthrepression during the R-to-D transition is phyB depen-dent The fact that PBG can repress hypocotyl growthlonger than the wild-type Col-0 (Fig 1 E and F) sug-gests that the amount of phyB is important in deter-mining the capacity for hypocotyl growth inhibition inthe dark In contrast to Col-0 and phyB-9 pifq was im-paired in growth after the R-to-D transition (Fig 1F)indicating that the hypocotyl growth after the R-to-Dtransition is mediated by PIFs

PBG But Not NGB Can Repress PIF3 Accumulation inBoth the Light and the Dark

Among the PIFs PIF3 plays an import role in pro-moting hypocotyl growth in the dark under diurnalconditions in particular PIF3 is not regulated at thetranscriptional level by the circadian clock but mainlyat the step of protein degradation by photoactivatedphytochromes (Soy et al 2012) Therefore we decided touse PIF3 as a model to examine whether phytochrome-mediated PIF3 degradation is differentially regulated inthe PBG and NGB lines during the R-to-D transition Inthe PBG line PIF3 was undetectable in the light andremained that way until 18 h after the R-to-D transition(Fig 2 A and B) Consistent with the model that PIF3promotes hypocotyl growth the appearance of PIF3 wasperfectly correlated with the increase in hypocotylgrowth in both PBG and Col-0 (Fig 2B) In strikingcontrast PIF3 accumulated in NGB and phyB-9 in con-tinuous R and after the R-to-D transition (Fig 2B) ourobservation that the NGB line fails to degrade PIF3 in thelight is consistent with a recent report from Park et al(2012) Because the steady-state mRNA levels of PIF3were comparable between PBG and NGB seedlingsduring the R-to-D transition (Supplemental Fig S2) thedifference in PIF3 abundance between these two lines ismost likely due to differences in PIF3 degradation byPBG and NGB Together these results show that PBGbut not NGB can repress PIF3 accumulation both in Rand in darkness

Next to assess the transcriptional activity of PIF3 in thePBG andNGB lines at various time points during the R-to-Dtransition we determined the expression levels of four well-characterizedPIFtargetgenesPHYTOCHROME-INTERACTINGFACTOR3-Like1 (PIL1) INDOLE-3-ACETICACIDINDUCIBLE29(IAA29)XYLOGLUCANEDOTRANSGLYCOSYLASE7 (XTR7)and ARABIDOPSIS THALIANA HOMEOBOX PROTEIN2

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(ATHB2) (Leivar et al 2009 2012b Hornitschek et al2012) None of these genes were activated in pifq (Fig2C) confirming that in this assay condition their ex-pression is dependent on PIFs The expression of thePIF3 target genes was also correlated with the PIF3levels in Col-0 and phyB-9 as all four genes were in-duced between 6 and 12 h after the R-to-D transition inCol-0 and they were induced in phyB-9 compared withCol-0 at time zero (Fig 2C) In PBG the four PIF targetgeneswere inducedbetween18 and24hafter theR-to-Dtransition (Fig 2 B and D) Therefore the timing of thisinduction corresponded faithfully to the increase in PIF3levels and the initiation of hypocotyl growth (Figs 1 Eand F and 2 B and D) In contrast the induction of PIFtargets did not coincidewith PIF3 protein accumulation

in the NGB line although PIF3 was present at all timepoints the PIF targetswere only induced between 6 and12 h after the R-to-D transition (Fig 2 B and D)Therefore in NGB it is not the PIF protein level butrather the activity of PIF3 that correlates with the ini-tiation of hypocotyl growth (Figs 1E and 2 B and D)These results support the notion that there are at leasttwo mechanisms by which phyB represses hypocotylelongation in the dark repression of PIF3 accumulationand inhibition of PIF3 transcriptional activity Interest-ingly PBG seems to inhibit hypocotyl growthmainly byrepressing PIF3 accumulation or by regulating bothPIF3 abundance and transcriptional activity simulta-neously whereas NGB inhibits hypocotyl growth pri-marily by inhibiting PIF transcriptional activity

Figure 1 PBG represses hypocotyl growth substantially longer than NGB after the R-to-D transition A Schematic of the R-to-Dtransition experiment Seedlings were collected at the indicated time points after the R-to-D transition and hypocotyl lengthswere measured B Absolute hypocotyl lengths of PBG (black bars) andNGB (gray bars) at the time points shown in A Error barsindicate the SD of at least 30 seedlings C Hypocotyl lengths of PBG (black line) and NGB (gray line) relative to those at timezero D Schematic of the experimental conditions for assessing the fine-scale growth kinetics of PBG and NGB E Growthkinetics of PBG (black line) and NGB (gray line) seedlings grown in the conditions shown in D The horizontal dotted lineindicates a relative hypocotyl length of 13 the threshold for considering a seedling as having grown The black and gray arrowsindicate the time points at which PBG and NGB cross this threshold 18 and 6 h respectively after the R-to-D transition Errorbars represent the SE of three independent experiments F Growth kinetics of Col-0 phyB-9 and pifq seedlings grown in theconditions shown in D The horizontal dotted line indicates a relative hypocotyl length of 13 The arrow indicates the timepoint at which Col-0 crosses this threshold 6 h after the R-to-D transition Error bars represent the SD of at least 15 seedlings

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Photobody Disassembly in PBG Correlates with PIF3Accumulation and Hypocotyl Growth

The discrepancies between PBG and NGB in hypo-cotyl growth kinetics PIF3 accumulation and the ex-pression of PIF target genes in the R-to-D transitionassay provided an opportunity to precisely determinethe roles of photobodies in these processes in the darkPrevious studies have utilized two main parameters todescribe the dynamics of photobodies the percentageof nuclei with or without photobodies and the averagesizenumber of photobodies per nucleus (Yamaguchiet al 1999 Kircher et al 2002 Chen et al 2003 2010)In the past the size and number of photobodies havebeen measured primarily by using two-dimensionalmaximum projection images derived from stacks of im-ages of optical sections Although this approach is usefulfor the analysis of nuclei with only a few large photo-bodies it does not work well for nuclei with many smallphotobodies because small photobodies from differentoptical sections might overlap in the projected image theinformation on the size and number of photobodiescould be lost or misrepresented in the projectionTo circumvent this problem we analyzed photobodies

from three-dimensional stacks of confocal images usingthe object analysis tool of Huygens Essential software(Scientific Volume Imaging) Using the software we de-termined the number of large and small photobodies pernucleus and the size distribution of the small photo-bodies All objects smaller than 05E-3 mm3 in volume(01mm in estimated diameter assuming that photobodies

are spherical) were excluded from our measurementsbecause these objects were beyond our detection limitWe arbitrarily defined large photobodies as those witha volume equal to or greater than 02 mm3 (072 mm inestimated diameter) and small photobodies as those witha volume between 05E-3 and 02 mm3 (01ndash072 mm inestimated diameter)

Because the major differences between PBG andNGB in hypocotyl growth repression occur during thefirst 24 h after the R-to-D transition (Fig 1E) we fo-cused on the dynamics of PBG and NGB localizationduring this time period As expected PBG seedlingsgrown in continuous R for 2 d had photobodies in allhypocotyl nuclei on average there were between sixand eight large photobodies per nucleus (Fig 3) Somenuclei also had a few small photobodies but thesenuclei were rare (Fig 3) After the R-to-D transitionthe photobody morphology in PBG went through twomajor transitions The first transition took place overthe first 12 h in darkness during this period althoughmost of the hypocotyl nuclei had photobodies thelarge photobodies disassembled and began to disap-pear and the number of small photobodies increased(Fig 3) The second transition occurred between 12and 18 h after the R-to-D transition during this periodphotobodies were completely lost from about 97 ofhypocotyl nuclei (Fig 3) The disappearance of pho-tobodies at 18 h coincides with the accumulation ofPIF3 (Fig 2B) and the initiation of hypocotyl growth inPBG (Fig 1E) Therefore these data support the modelthat photobodies are required for inhibiting hypocotyl

Figure 2 PBG but not NGB can re-press PIF3 in the light and the dark ASchematic of the growth conditions andcollection time points for the assay BPIF3 abundance in PBG NGB Col-0and phyB-9 PIF3 abundance relative tothe mean overall PIF3 level within eachline is shown below the blots RPN6was used as a loading control Lane Dshows a dark-grown control C Ex-pression of four well-defined PIF targetgenes in Col-0 phyB-9 and pifq Datawere normalized to the expression ofPP2A D Expression of four well-definedPIF target genes in PBG and NGB Datawere normalized to the expression ofPP2A Error bars in C and D indicate theSD of three replicates

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growth by repressing PIF3 in the dark The analysis ofthe NGB line further supports this notion As shown inFigure 3 the majority of nuclei in NGB did not containany photobodies Inconsistent with the report byMatsushita et al (2003) a small fraction of nuclei didcontain small photobodies under these experimentalconditions Nonetheless because NGB failed to localizeto photobodies in more than 80 of nuclei (Fig 3) theseresults are consistent with the idea that photobodies arerequired for PIF degradation in the light and the pro-longed repression of PIF accumulation in the dark Ourdata also suggest that the repression of PIF3 activity byNGB can occur in the absence of photobodies

Decreased Light Intensity Leads to the Faster Disassemblyof Photobodies and a Reduced Capacity for HypocotylGrowth Inhibition in PBG in the Dark

To further test the model that photobody morpho-logy determines the capacity for PIF3 repression andhypocotyl inhibition in the dark we asked whether wecould alter these two latter processes by manipulatingphotobody morphology Because the steady-state pattern

of photobodies is directly regulated by light intensity(Chen et al 2003 Van Buskirk et al 2012) we mod-ified our assay condition by growing seedlings in areduced R intensity of 1 mmol m22 s21 for 2 d beforethe R-to-D transition As reported previously (Chenet al 2003) in the dimmer light condition PBG waslocalized to both large and small photobodies (Fig 4)Compared with the 10 mmol m22 s21 R treatment thedimmer light treatment led to the faster disassembly ofPBG photobodies in the dark this difference was mostobvious between the 6- and 12-h time points (Fig 4)At the 6-h time point the percentage of nuclei withphotobodies had already dropped to approximately69 at the 12-h time point photobodies were com-pletely lost from more than 80 of all nuclei There-fore the photobody disassembly process in PBG wasat least 6 h faster after the 1 mmol m22 s21 R treatmentcompared with the 10 mmol m22 s21 R treatment Incontrast the localization pattern of NGB was quitesimilar after both the strong and dim R treatments(Figs 3 and 4)

To test whether the change in photobody dynamics inPBG leads to changes in the kinetics of PIF3 accumulationPIF transcriptional activity and hypocotyl growth we

Figure 3 Loss of photobodies correlates with the accumulation of PIF3 Top schematic of experimental conditions and samplingtime points Bottom representative confocal images of PBG (top row) and NGB (bottom row) localization along with thequantification of photobody number and size in conditions in which at least 50 of nuclei have photobodies After 12 h indarkness PBG begins to accumulate PIF3 and PIF target genes are induced (broken vertical red line Fig 2) In the confocal imagesthe percentage value indicates the mean percentage of all analyzed nuclei with the phenotype shown in the image (with or withoutphotobodies means 6 SE of at least three independent experiments) n indicates the total number of nuclei analyzed to generatethe percentage and bars = 5 mm In the graphs the error bars represent SD and n indicates the number of nuclei analyzed togenerate the distribution The blue bars indicate small photobodies (diameters between 01 and 072 mm volumes between 00005and 019 mm3) and the yellow bars plotted on the secondary axis indicate large photobodies (diameters greater than 072 mmvolumes of 02 mm3 or greater) Bins represent the following volume ranges with a to i representing small photobodies and Lrepresenting large photobodies a 00005 to 00019 b 0002 to 00049 c 0005 to 0099 d 001 to 0019 e 002 to 0039f 004 to 0079 g 008 to 0119 h 012 to 0159 i 016 to 0199 and L 020 mm3 or greater

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examined these responses in both PBG and NGB duringthe 1 mmol m22 s21 R-to-D transition (Fig 5A) As shownin Figure 5 consistent with the loss of photobodies inPBG after 12 h in darkness the PBG seedlings grown in1 mmol m22 s21 R were only able to repress hypocotylgrowth PIF3 accumulation and the expression of PIFtargets for approximately 10 to 12 h (Fig 5 BndashD) Incontrast just as NGB exhibited similar localization pat-terns between the 1 and 10 mmol m22 s21 treatments(Figs 3 and 4) NGB seedlings showed similar hypocotylgrowth kinetics PIF3 accumulation and PIF target geneinduction between the two light conditions (Figs 1E 2 Band D and 5 BndashD) Together these data suggest thatthe steady-state pattern of photobodies in PBG prior to theR-to-D transition correlates with the capacity to fine-tunehypocotyl growth inhibition and PIF3 repression in thedark Consistent with this notion NGB which does not lo-calize to photobodies in the majority of nuclei does not re-spond todifferences in lightquantitybefore thedarkperiod

The Prolonged Hypocotyl Growth Inhibition in PBG IsLikely Due to Enhanced Stabilization of the Pfr of phyB

Why compared with NGB can PBG repress PIF3accumulation and hypocotyl growth for a prolonged

period of time in the dark One possible explanationcould come from differences in the stability of the Pfr ofphyB Although the dark reversion rate of NGB is sim-ilar to that of full-length phyB in vitro (Oka et al 2004)the dark reversion rate of full-length phyB in vivo is muchslower it has been proposed that in vivo photobodiescan stabilize the Pfr form of phyB (Rausenberger et al2010) To test this hypothesis we treated PBG and NGBseedlings with a 15-min FR pulse to convert PBG andNGB to their respective Pr before transferring them todarkness Because photobody localization of phyB is Pfrdependent FR treatment should trigger the fast disas-sembly of photobodies in the dark (Rausenberger et al2010 Aacutedaacutem et al 2011) To monitor this rapid change inphotobody disassembly we examined photobody dy-namics at time points immediately after the FR treatment(Fig 6A) Consistent with previous reports (Rausenbergeret al 2010 Aacutedaacutem et al 2011) almost all photobodies inPBG disassembled within 1 h of the FR treatment (Fig6B) The small fraction of cells with some small photo-bodies in NGB also lost their photobodies within 1 hindicating that the small photobodies in NGB are alsodependent on its Pfr

Measuring hypocotyl growth kinetics after FR treatmentshowed that the FR pulse treatment caused virtually no

Figure 4 Seedlings grown in a lower fluence rate of light lose photobodies more quickly than in a higher fluence rate of light Topschematic of experimental conditions and sampling time points Bottom representative confocal images of PBG (top row) andNGB (bottom row) localization along with the quantification of photobody number and size in conditions in which at least 50 ofnuclei have photobodies After 6 h in darkness PBG loses photobodies from more than 50 of nuclei (broken vertical red line) Inthe confocal images the percentage value indicates the mean percentage of all analyzed nuclei with the phenotype shown in theimage (with or without photobodies means 6 SE of at least three independent experiments) n indicates the number of nucleianalyzed to generate the percentage and bars = 5 mm In the graphs the error bars represent SD and n indicates the number ofnuclei analyzed to generate the distribution The blue bars indicate small photobodies (diameters between 01 and 072 mmvolumes between 00005 and 019 mm3) and the yellow bars plotted on the secondary axis indicate large photobodies (diametersgreater than 072 mm volumes of 02 mm3 or greater) Bins represent the following volume ranges with a to i representing smallphotobodies and L representing large photobodies a 00005 to 00019 b 0002 to 00049 c 0005 to 0099 d 001 to 0019e 002 to 0039 f 004 to 0079 g 008 to 0119 h 012 to 0159 i 016 to 0199 and L 020 mm3 or greater

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change in the growth kinetics of NGB which still beganto grow 6 h after the red light-to-far-red light-to-dark(R-FR-D) transition (Fig 6B) suggesting that the mini-mum time required for a 30 increase in hypocotyl lengthmight be approximately 6 h In contrast PBG respondedstrongly to the FR treatment hypocotyl growth wasinhibited for only approximately 9 h which is half thetime of that without FR treatment (Figs 1E and 6B) Thisresult suggests that the prolonged hypocotyl growth re-pression in PBG (18 h compared with 6 h in NGB) ismainly due to Pfr stabilization in the dark However evenafter the FR treatment PBG was still able to repress hy-pocotyl growth 3 h longer than NGB (Fig 6B) suggestingthat there must be other mechanisms that account for thisdifference in hypocotyl growth repression

We next determined the patterns of PIF3 accumula-tion and the expression of PIF targets in both PBG andNGB after the R-FR-D transition In PBG PIF3 began toaccumulate within 1 h after the FR treatment (Fig 6C)this result is consistent with a previous report on PIF3dynamics (Monte et al 2004) The accumulation of PIF3in the PBG line again correlated perfectly with photobodydisassembly (Fig 6 A and C) Because PIF3 degradationis triggered by the Pfr of phyB the dynamic changes inPIF3 levels could serve as a readout for the presence ofthe Pfr of phyB in PBG Based on this readout the Pfrof phyB-GFP in PBG lasts for approximately 18 h afterthe 10 mmol m22 s21 R-to-D transition (Fig 2B) and forabout 12 h after the 1 mmol m22 s21 R-to-D transition(Fig 5C) As predicted in NGB PIF3 was detectable incontinuous R and remained detectable for the durationof the experiment showing little change in abundanceafter FR treatment (Fig 6C)

Surprisingly although PIF3 began to accumulate inPBG within 1 h of the FR treatment the expression of

PIF targets remained repressed for 9 h (Fig 6D) Incontrast in NGB the expression of all four PIF targetswas induced immediately after FR treatment (Fig 6E)These data suggest that the repression of PIF activity inNGB in the dark is mainly dependent on the Pfr ofNGB however in PBG the repression of the expres-sion of PIF targets could be mediated by an unknownmechanism that is independent of photobodies and ofthe Pfr of phyB This offers an explanation for thedifference in hypocotyl growth inhibition between thePBG and NGB lines during the R-FR-D transition (Fig6B) Because the expression of PIF target genes is Pfrdependent in NGB the repression of PIF target genescan be used as a readout for the presence of the Pfr ofNGB based on this readout the Pfr of NGB can last forapproximately 6 h after both the 10 and 1 mmol m22 s21

R-to-D transitions (Figs 2D and 5D) Together theseresults suggest that the Pfr of PBG can last 12 h longerthan that of NGB

DISCUSSION AND CONCLUSION

Although a growing body of evidence supports thebiological importance of photobodies in the phytochrome-mediated photoinhibition of hypocotyl growth (VanBuskirk et al 2012) comparisons between the photobody-localized PBG and nucleoplasm-localized NGB showedthat in the light NGB is hyperactive in inhibitinghypocotyl growth suggesting that photobodies areunnecessary and might even play a negative role in thelight-dependent inhibition of hypocotyl elongation(Matsushita et al 2003 Palaacutegyi et al 2010) Here wedeveloped an R-to-D transition assay to examine therelationship between dynamic changes in photobody

Figure 5 PBG has a reduced capacity for hypocotyl growth inhibition and PIF3 repression after a dimmer 1 mmol m22 s21

R-to-D transition A Schematic of the growth conditions and sampling time points for the assay B Growth kinetics of PBG(black line) andNGB (gray line) The horizontal dotted line indicates the threshold value of 13 and black and gray arrows pointto where PBG and NGB cross that threshold (at 12 and 6 h respectively) Error bars represent the SE of three independentexperiments C Western blots showing PIF3 abundance in PBG (top) and NGB (bottom) PIF3 levels relative to the mean overallPIF3 level within each line are shown below the blots RPN6 was used as a loading control Lane D shows a dark-growncontrol D Transcript levels of four well-defined PIF target genes in PBG (black lines) and NGB (gray lines) Data were nor-malized to PP2A Error bars represent the SD of three replicates

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morphology the molecular events of PIF3 accumula-tion and the expression of PIF targets as well as therepression of hypocotyl growth between the PBG andNGB lines Our results demonstrate a tight correlationbetween the photobody localization of PBG and therepression of PIF3 accumulation in both light and darkconditions Our data support a model in which pho-tobodies mediate prolonged light-dependent hypo-cotyl growth inhibition in the dark by stabilizing thePfr of phyB and repressing PIF3 accumulation (Fig 7)

Photobody Localization of phyB Tightly Correlates withthe Repression of PIF3 Degradation

Accumulating evidence suggests that phytochromes in-hibit hypocotyl growth both by triggering the degradation

of multiple PIFs and by inhibiting the PIFsrsquo transcrip-tional activity Our data suggest that photobody locali-zation of phyB is specifically required for repressing PIF3accumulation in the light and after the light-to-darktransition This conclusion is supported by the followingtwo lines of evidence first by comparing PIF3 levels in2-d-old light-grown PBG and NGB seedlings we showthat PIF3 accumulation is repressed in PBG but not inNGB (Fig 2B) This result confirms the observations fromprevious reports by Choi and colleagues who showedusing transgenic lines overexpressing His- and Myc-tagged PIF3 that NGB fails to degrade PIF3 in contin-uous R (Park et al 2004 2012) Second in all threeconditions tested the 10 mmol m22 s21 R-to-D transition(Figs 2B and 3) the 1 mmol m22 s21 R-to-D transition(Figs 4 and 5C) and the R-FR-D transition (Fig 6

Figure 6 Photobody localization of PBG correlates with the repression of PIF3 accumulation but not the repression of PIF3activity after the R-FR-D transition A Top schematic of the experimental growth conditions and sampling time points Bottomrepresentative confocal images of PBG (top row) and NGB (bottom row) localization One hour after the FR treatment allphotobodies are gone from PBG (broken vertical red line) The percentage values indicate the percentage of all analyzed nucleiwith the phenotype shown in the image (with or without photobodies means 6 SD of at least two independent experiments)n indicates the number of nuclei analyzed to generate the percentage and bars = 5 mm B Growth kinetics of PBG (black line)and NGB (gray line) The horizontal dotted line is the threshold value of 13 and black and gray arrows point to where PBG andNGB cross this threshold after FR treatment 9 and 6 h respectively Error bars indicate the SE of three independent experimentsC Western blot showing PIF3 abundance in PBG (top) and NGB (bottom) after FR treatment PIF3 levels relative to the meanoverall PIF3 level within each line are given below the blots RPN6 was used as a loading control Lane D shows a dark-growncontrol D Transcript levels of four well-defined PIF target genes in PBG (black lines) and NGB (gray lines) after FR treatmentData were normalized to PP2A Error bars represent the SD of three replicates

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A and C) the disassembly of photobodies in hypocotylnuclei correlates tightly with the accumulation of PIF3The R-FR-D experiment is particularly informative asthis treatment demonstrates that the regulation of PIFdegradation and the repression of PIF activity can beseparated even in the PBG background

The tight correlation between the photobody locali-zation of PBG and PIF3 degradation provides strongevidence supporting the model that photobody locali-zation of phyB is required for PIF3 degradation (Fig 7)Although we still cannot completely exclude the possi-bility that photobody localization of phyB and PIF3degradation are two parallel consequences of phyB ac-tivation the conclusion that photobodies are required forPIF degradation is consistent with and supported bypreviously published results First the constitutively ac-tive phyB allele YHB localizes to photobodies and cantrigger PIF3 degradation in the dark (Su and Lagarias2007 Galvatildeo et al 2012) Second the photobody-deficient mutant hmr is also defective in the degrada-tion of PIF1 and PIF3 in the light (Chen et al 2010) Inaddition in the YHBhmr-1 double mutant YHB fails tolocalize to large photobodies in the dark and the re-pression of PIF3 accumulation by YHB is also reversed(Galvatildeo et al 2012) Third PIF3 localizes to photobodiesprior to its degradation (Bauer et al 2004 Al-Sady et al2006) and mutations in phyB that abrogate its interac-tion with PIFs do not affect phyBrsquos localization to pho-tobodies but are not functional in the photoinhibition ofhypocotyl elongation (Oka et al 2008 Kikis et al 2009)suggesting that recruiting PIFs to photobodies is re-quired for PIF degradation and phytochrome signalingTaken together the results from this and previousstudies support the most likely model that photobodiesare required for PIF degradation

The molecular mechanism by which photobodiesare involved in PIF degradation is still unclear There isstill no direct evidence showing that PIF3 degradationoccurs on photobodies Therefore we cannot excludethe possibility that photobodies are only involved in aposttranslational modification of PIFs such as phos-phorylation (Al-Sady et al 2006 Lorrain et al 2008Shen et al 2008 Bu et al 2011 Ni et al 2013) andthat PIF degradation occurs elsewhere (Van Buskirket al 2012) It is also important to note that althoughour data show a clear separation between PIF3 deg-radation and the regulation of its transcriptional ac-tivity in the R-FR-D transition (Fig 6 C and D) thesetwo events occurred simultaneously in the other twoR-to-D conditions (Figs 2 B and D and 5 C and D)Therefore it is possible that PIF degradation and theregulation of PIF target genes are closely linked in thepresence of photobodies (Fig 7)

It is worth pointing out that the repression of PIF3activity after the R-FR-D transition in PBG appears to bemediated by a different mechanism from the NGB-dependent repression of PIF3 activity It has previouslybeen shown that NGB inhibits PIF3 activity by removingPIF3 from the promoters of its target genes and that thisactivity of NGB is Pfr dependent (Park et al 2012) Ourresults are consistent with this report in that an FR pulsecan release the repression of PIF targets by NGB (Fig6D) In contrast the repression of PIF targets in PBG afterthe R-FR-D transition was clearly not mediated by thePfr of PBG as the FR pulse quickly converted PBG to itsPr and promoted PIF accumulation but the repression ofPIF targets persisted (Fig 6D) Therefore the repressionof PIF activity in PBG in the assay is mediated by a yetunknown mechanism One possibility is that anothertranscriptional repressor protein for the PIF targets ispresent only in PBG and the delay in the induction of thePIF targets reflects the time required for the removal ofthis additional protein An alternative model is that thechromatin state of the PIF targets is different between thePBG and NGB lines in the light while the PIF targetscould be primed for induction in NGB in PBG theymight be in a silenced state that requires additional timefor activation Future experiments on the chromatinstatus of the PIF targets in these two lines might help toreveal this unknownmechanism for the repression of PIFtarget genes after the R-FR-D transition

Photobodies Mediate Prolonged Hypocotyl GrowthInhibition in the Dark Likely by Stabilizing the Pfrof phyB

It is well known that the Pfr of phyB has a relativelyslow dark reversion rate and that it can persist intodarkness these properties of phyB play a pivotal rolein hypocotyl growth inhibition under diurnal condi-tions (Hennig et al 1999 Rausenberger et al 2010)This is best demonstrated by end-of-day FR treatmentin which an FR light pulse is applied at dusk to inac-tivate phyB to its Pr End-of-day FR treatment leads to

Figure 7 Model for the function of photobodies in regulating seedlinggrowth in the dark The Pfr of both PBG and NGB can persist into thedarkness to repress hypocotyl growth There are two main differencesbetween the photobody-localized PBG and the nucleoplasmic NGB(1) Photobodies are required for PIF degradation photobody locali-zation of PBG inhibits both PIF accumulation and PIF transcriptionalactivity whereas NGB only represses PIF activity (2) Photobody lo-calization of PBG stabilizes its Pfr and extends its life in the darkconsequently PBG can inhibit hypocotyl elongation (cell growth) for aprolonged period of time This photobody-dependent mechanism ofPfr stabilization enables seedlings to convey perceived light cues intodarkness to fine-tune hypocotyl growth accordingly

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a dramatic decrease in hypocotyl growth inhibition un-der diurnal conditions (Elich and Chory 1997) Recentlyit has been shown that the Pfr of phyB is responsible forrepressing PIF1 and PIF3 accumulation and conse-quently hypocotyl growth in the dark (Soy et al 20122014) Interestingly the in vivo dark reversion rate ofphyB is much slower than the rate measured in vitroand photobodies have been proposed through mathe-matical modeling to be involved in stabilizing the Pfr ofphyB in the dark (Rausenberger et al 2010) Here weprovide experimental evidence supporting this hypoth-esis Our data show that the localization of PBG tophotobodies dramatically increases the stability of its Pfrcompared with that of the nucleoplasmic NGB Firstbecause the function of the Pfr of PBG is closely associ-ated with the repression of PIF3 accumulation we canestimate the presence of PBG Pfr based on PIF3 levelsSimilarly because the function of the Pfr of NGB isclosely linked to the repression of PIF target gene ex-pression we can estimate the presence of NGB Pfr basedon the induction of PIF targets Based on the comparisonbetween the 10 mmol m22 s21 R-to-D transition ex-periment (Fig 2 B and D) and the R-FR-D transitionexperiment (Fig 6 C and D) the Pfr of PBG lasts forabout 18 h compared with only 6 h for the Pfr of NGBBecause PBG and NGB have similar dark reversion ratesin vitro (Oka et al 2004) our data suggest that the lifeof the Pfr of PBG is extended three times longer than thatof NGB in vivo This dramatic decrease in the capacityfor hypocotyl growth inhibition in PBG between the10 mmol m22 s21 R-to-D transition (Fig 1E) and the R-FR-Dtransition (Fig 6B) demonstrates that the stabilization ofthe Pfr of PBG contributes substantially to the prolongedinhibition of hypocotyl growth in the dark The stabilityof PBG Pfr can also be fine-tuned by the light intensityand photobody localization pattern before the light-to-dark transition in the 1 mmol m22 s21 R-to-D transitionexperiment PBG localized to large and small photo-bodies and the life of the Pfr was reduced to 9 h (Figs 4and 5) In contrast the life of the Pfr of NGB seemsto remain the same in both the 10 and 1 mmol m22 s21

R-to-D transitions (Figs 2D and 5D) Together theseresults suggest that the photobody localization of phyBis required for the prolonged inhibition of hypocotylgrowth in the dark by stabilizing the Pfr of phyB andthat this photobody-dependent Pfr stabilization could bea mechanism to fine-tune hypocotyl elongation in thedark (Fig 7)Although our experiments were not performed under

diurnal conditions the mechanism involved in hypo-cotyl growth regulation appears to be similar betweenthe R-to-D transition and the day-to-night transitionunder both conditions hypocotyl growth is promotedby PIFs and repressed by active phyB (Figs 1F and 2 Band C Soy et al 2012 2014) Because photobodies alsoundergo similar disassembly dynamics during the day-to-night transition (Kircher et al 2002) in diurnalconditions the photobody localization of phyB may beinvolved in the PIF repression and hypocotyl growthinhibition seen in the early evening We propose that

this photobody-dependent hypocotyl growth repressionmechanism allows seedlings to carry light cues perceivedduring the day into the evening to fine-tune photo-morphogenetic responses accordingly (Fig 7)

MATERIALS AND METHODS

Plant Materials Growth Conditions andHypocotyl 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 of Arabidopsis (Arabidopsisthaliana) were as described previously Seeds were surface sterilized with arinse in 70 (vv) ethanol followed by 15 min in 50 (vv) bleach supple-mented with 002 (vv) Triton X-100 Seeds were rinsed five times withdistilled deionized water prior to plating on one-half-strength Murashige andSkoog medium supplemented with B vitamins (Caisson) and containing 06(wv) phyto agar (Caisson) Seeds were stratified for 5 d in darkness prior tobeing treated with the indicated light conditions The light intensity in the Rand FR light-emitting diode chambers (Percival Scientific) was measured us-ing a fiber-optic probe and SpectraWiz software (StellarNet) Seedling imageswere obtained by laying seedlings on a transparency and scanning using theEpson Perfection V700 photo scanner Hypocotyl lengths were measured us-ing ImageJ software (httprsbwebnihgovij)

Protein Extraction and Western Blot

Total protein was extracted from seedlings using a mortar and pestle and 3volumes of extraction buffer containing Bromphenol Blue as described pre-viously (Galvatildeo et al 2012) Protein samples were run on 8 (wv) Bis-Tris-SDS-acrylamide gels and transferred to nitrocellulose membranes (Bio-Rad)Polyclonal antibodies against PIF3 (Chen et al 2010) were used at a 1500dilution and polyclonal antibodies against REGULATORY PARTICLE NON-ATPASE6 (Enzo Life Sciences catalog no BML-PW8370-0025) were used at a11000 dilution Secondary antibodies were horseradish peroxidase-conjugatedgoat anti-rabbit IgG (Bio-Rad) and were used at a 15000 dilution Blots werevisualized on x-ray film using SuperSignal West chemiluminescent substrate(Thermo Fisher Scientific) PIF3 and GFP levels were quantified using QuantityOnesoftware (Bio-Rad) followed by a multistep normalization after backgroundsubtraction the intensity of the PIF3 or GFP band was divided by the intensity ofthe corresponding REGULATORY PARTICLE NON-ATPASE6 band Then themean PIF3 or GFP intensity was calculated and the normalized PIF3 or GFPintensity for each individual time point within each line was divided by thismean This is the value that is reported in the figures

Quantitative Reverse Transcription-PCR

Seedlings were flash frozen in liquid nitrogen and ground to a powder usinga plastic mortar and wooden pestle Total RNA was then extracted using theSpectrum Plant Total RNA kit (Sigma-Aldrich catalog no STRN-250) with on-column DNase treatment (Sigma-Aldrich catalog no DNASE10-1SET) andyield was quantified using a NanoDrop ND-1000 spectrophotometer (ThermoFisher Scientific) Five micrograms of total RNA was then used for comple-mentary DNA synthesis using SuperScript II Reverse Transcriptase (LifeTechnologies catalog no 18064-014) using oligo(dT)12-18 primers (Life Tech-nologies catalog no 18418-012) according to the manufacturerrsquos instructionsQuantitative reverse transcription-PCR was performed using FastStart Uni-versal SYBR Green (Roche Applied Science catalog no 04913914001) and aMastercycler ep realplex qPCR machine with realplex software (Eppendorf)Primers are listed in Supplemental Table S1

Confocal Live-Cell Imaging and Quantification ofPhotobody Morphology

Seedlings were mounted on Superfrost slides (VWR catalog no 48311-600)using distilled deionized water and 22- 3 40-mm coverslips (no 15 VWRcatalog no 48393-172) Nuclei from hypocotyl epidermal cells were imagedusing a Zeiss LSM 510 inverted confocal microscope (Carl Zeiss) GFP wasdetected using a 1003 Plan-Apochromat oil-immersion objective 488-nm

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excitation from an argon laser and the manufacturerrsquos default Green onlydetection setting (505- to 550-nm bandpass detector) Images were collectedusing LSM 510 software version 42 Images were processed using AdobePhotoshop CS5 software (Adobe Systems)

To calculate the overall proportion of nuclei possessing photobodies maximumprojections of optical sections of the hypocotyl cells were generated using LSMImage Browser software version 420121 (Carl Zeiss) Then the number of nucleiwith photobodies was manually scored To determine the size and number ofphotobodies stacks of optical sectionswere loaded intoHuygens Essential software(Scientific Volume Imaging) The object analyzer tool was used to threshold theimage and to calculate the number and the volume of photobodies in the image Foreach nucleus the photobodies were sorted by size and thenmanually binned usingMicrosoft Excel

Supplemental Data

The following materials are available in the online version of this article

Supplemental Figure S1 The protein levels of PBG and NGB remain rel-atively constant during the course of the R-to-D transition assay

Supplemental Figure S2 The mRNA levels of PIF3 in PBG NGB Col-0and phyB-9 lines during the R-to-D transition

Supplemental Table S1 List of quantitative reverse transcription-PCRprimers used in this study

ACKNOWLEDGMENTS

We thank Dr Joanne Chory for the anti-PIF3 antibody Dr Peter Quail forthe pifq seeds and Drs Sam Johnson and Yasheng Gao for technical assistancewith the live-cell imaging

Received February 4 2014 accepted April 25 2014 published April 25 2014

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Al-Sady B Kikis EA Monte E Quail PH (2008) Mechanistic duality oftranscription factor function in phytochrome signaling Proc Natl AcadSci USA 105 2232ndash2237

Al-Sady B Ni W Kircher S Schaumlfer E Quail PH (2006) Photoactivatedphytochrome induces rapid PIF3 phosphorylation prior to proteasome-mediated degradation Mol Cell 23 439ndash446

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Bu Q Zhu L Dennis MD Yu L Lu SX Person MD Tobin EM BrowningKS Huq E (2011) Phosphorylation by CK2 enhances the rapid light-induceddegradation of phytochrome interacting factor 1 in Arabidopsis J Biol Chem286 12066ndash12074

Chen M Chory J (2011) Phytochrome signaling mechanisms and the con-trol of plant development Trends Cell Biol 21 664ndash671

Chen M Chory J Fankhauser C (2004) Light signal transduction in higherplants Annu Rev Genet 38 87ndash117

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Chen M Schwab R Chory J (2003) Characterization of the requirementsfor localization of phytochrome B to nuclear bodies Proc Natl Acad SciUSA 100 14493ndash14498

Chen M Tao Y Lim J Shaw A Chory J (2005) Regulation of phytochromeB nuclear localization through light-dependent unmasking of nuclear-localization signals Curr Biol 15 637ndash642

Elich TD Chory J (1997) Biochemical characterization of Arabidopsis wild-type and mutant phytochrome B holoproteins Plant Cell 9 2271ndash2280

Franklin KA Quail PH (2010) Phytochrome functions in Arabidopsis de-velopment J Exp Bot 61 11ndash24

Fujimori T Yamashino T Kato T Mizuno T (2004) Circadian-controlledbasichelix-loop-helix factor PIL6 implicated in light-signal transduc-tion in Arabidopsis thaliana Plant Cell Physiol 45 1078ndash1086

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Galvatildeo RM Li M Kothadia SM Haskel JD Decker PV Van Buskirk EKChen M (2012) Photoactivated phytochromes interact with HEMERAand promote its accumulation to establish photomorphogenesis inArabidopsis Genes Dev 26 1851ndash1863

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Hennig L Poppe C Unger S Schaumlfer E (1999) Control of hypocotyl elongationin Arabidopsis thaliana by photoreceptor interaction Planta 208 257ndash263

Hornitschek P Kohnen MV Lorrain S Rougemont J Ljung K Loacutepez-Vidriero I Franco-Zorrilla JM Solano R Trevisan M Pradervand Set al (2012) Phytochrome interacting factors 4 and 5 control seedlinggrowth in changing light conditions by directly controlling auxin sig-naling Plant J 71 699ndash711

Huq E Al-Sady B Hudson M Kim C Apel K Quail PH (2004)Phytochrome-interacting factor 1 is a critical bHLH regulator of chlo-rophyll biosynthesis Science 305 1937ndash1941

Huq E Quail PH (2002) PIF4 a phytochrome-interacting bHLH factorfunctions as a negative regulator of phytochrome B signaling in Arabi-dopsis EMBO J 21 2441ndash2450

Kami C Lorrain S Hornitschek P Fankhauser C (2010) Light-regulatedplant growth and development Curr Top Dev Biol 91 29ndash66

Khanna R Huq E Kikis EA Al-Sady B Lanzatella C Quail PH (2004) Anovel molecular recognition motif necessary for targeting photo-activated phytochrome signaling to specific basic helix-loop-helix tran-scription factors Plant Cell 16 3033ndash3044

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Kircher S Gil P Kozma-Bognaacuter L Fejes E Speth V Husselstein-MullerT Bauer D Adaacutem E Schaumlfer E Nagy F (2002) Nucleocytoplasmicpartitioning of the plant photoreceptors phytochrome A B C D and Eis regulated differentially by light and exhibits a diurnal rhythm PlantCell 14 1541ndash1555

Leivar P Monte E Cohn MM Quail PH (2012a) Phytochrome signaling ingreen Arabidopsis seedlings impact assessment of a mutually negativephyB-PIF feedback loop Mol Plant 5 734ndash749

Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH(2008) Multiple phytochrome-interacting bHLH transcription factorsrepress premature seedling photomorphogenesis in darkness Curr Biol18 1815ndash1823

Leivar P Quail PH (2011) PIFs pivotal components in a cellular signalinghub Trends Plant Sci 16 19ndash28

Leivar P Tepperman JM Cohn MM Monte E Al-Sady B Erickson EQuail PH (2012b) Dynamic antagonism between phytochromes and PIFfamily basic helix-loop-helix factors induces selective reciprocal re-sponses to light and shade in a rapidly responsive transcriptional net-work in Arabidopsis Plant Cell 24 1398ndash1419

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Lorrain S Trevisan M Pradervand S Fankhauser C (2009) Phytochromeinteracting factors 4 and 5 redundantly limit seedling de-etiolation incontinuous far-red light Plant J 60 449ndash461

Matsushita T Mochizuki N Nagatani A (2003) Dimers of the N-terminal do-main of phytochrome B are functional in the nucleus Nature 424 571ndash574

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Phosphorylation of phytochrome B inhibits light-induced signaling viaaccelerated dark reversion in Arabidopsis Plant Cell 25 535ndash544

Monte E Tepperman JM Al-Sady B Kaczorowski KA Alonso JM Ecker JRLi X Zhang Y Quail PH (2004) The phytochrome-interacting transcriptionfactor PIF3 acts early selectively and positively in light-induced chloroplastdevelopment Proc Natl Acad Sci USA 101 16091ndash16098

Nagatani A (2010) Phytochrome structural basis for its functions CurrOpin Plant Biol 13 565ndash570

Nagy F Schaumlfer E (2002) Phytochromes control photomorphogenesis bydifferentially regulated interacting signaling pathways in higher plantsAnnu Rev Plant Biol 53 329ndash355

Ni W Xu SL Chalkley RJ Pham TN Guan S Maltby DA BurlingameAL Wang ZY Quail PH (2013) Multisite light-induced phosphorylationof the transcription factor PIF3 is necessary for both its rapid degrada-tion and concomitant negative feedback modulation of photoreceptorphyB levels in Arabidopsis Plant Cell 25 2679ndash2698

Nito K Wong CC Yates JR 3rd Chory J (2013) Tyrosine phosphorylationregulates the activity of phytochrome photoreceptors Cell Rep 3 1970ndash1979

Nozue K Covington MF Duek PD Lorrain S Fankhauser C Harmer SLMaloof JN (2007) Rhythmic growth explained by coincidence betweeninternal and external cues Nature 448 358ndash361

Oh E Kim J Park E Kim JI Kang C Choi G (2004) PIL5 a phytochrome-interacting basic helix-loop-helix protein is a key negative regulator ofseed germination in Arabidopsis thaliana Plant Cell 16 3045ndash3058

Oka Y Matsushita T Mochizuki N Quail PH Nagatani A (2008) Mutantscreen distinguishes between residues necessary for light-signal per-ception and signal transfer by phytochrome B PLoS Genet 4 e1000158

Oka Y Matsushita T Mochizuki N Suzuki T Tokutomi S Nagatani A(2004) Functional analysis of a 450-amino acid N-terminal fragment ofphytochrome B in Arabidopsis Plant Cell 16 2104ndash2116

Palaacutegyi A Terecskei K Adaacutem E Kevei E Kircher S Meacuterai Z Schaumlfer E NagyF Kozma-Bognaacuter L (2010) Functional analysis of amino-terminal domains ofthe photoreceptor phytochrome B Plant Physiol 153 1834ndash1845

Park E Kim J Lee Y Shin J Oh E Chung WI Liu JR Choi G (2004) Degra-dation of phytochrome interacting factor 3 in phytochrome-mediated lightsignaling Plant Cell Physiol 45 968ndash975

Park E Park J Kim J Nagatani A Lagarias JC Choi G (2012) Phyto-chrome B inhibits binding of phytochrome-interacting factors to theirtarget promoters Plant J 72 537ndash546

Rausenberger J Hussong A Kircher S Kirchenbauer D Timmer J Nagy FSchaumlfer E Fleck C (2010) An integrative model for phytochrome B mediatedphotomorphogenesis from protein dynamics to physiology PLoSONE 5 e10721

Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome Aand phytochrome B have overlapping but distinct functions in Arabi-dopsis development Plant Physiol 104 1139ndash1149

Reed JW Nagpal P Poole DS Furuya M Chory J (1993) Mutations in thegene for the redfar-red light receptor phytochrome B alter cell elon-gation and physiological responses throughout Arabidopsis develop-ment Plant Cell 5 147ndash157

Rockwell NC Su YS Lagarias JC (2006) Phytochrome structure and sig-naling mechanisms Annu Rev Plant Biol 57 837ndash858

Sharrock RA Quail PH (1989) Novel phytochrome sequences in Arabi-dopsis thaliana structure evolution and differential expression of a plantregulatory photoreceptor family Genes Dev 3 1745ndash1757

Shen H Zhu L Castillon A Majee M Downie B Huq E (2008) Light-induced phosphorylation and degradation of the negative regulatorPHYTOCHROME-INTERACTING FACTOR1 from Arabidopsis dependupon its direct physical interactions with photoactivated phytochromesPlant Cell 20 1586ndash1602

Shin J Kim K Kang H Zulfugarov IS Bae G Lee CH Lee D Choi G(2009) Phytochromes promote seedling light responses by inhibitingfour negatively-acting phytochrome-interacting factors Proc Natl AcadSci USA 106 7660ndash7665

Soy J Leivar P Gonzaacutelez-Schain N Sentandreu M Prat S Quail PHMonte E (2012) Phytochrome-imposed oscillations in PIF3 protein a-bundance regulate hypocotyl growth under diurnal lightdark condi-tions in Arabidopsis Plant J 71 390ndash401

Soy J Leivar P Monte E (January 13 2014) PIF1 promotes phytochrome-regulated growth under photoperiodic conditions in Arabidopsis to-gether with PIF3 PIF4 and PIF5 J Exp Bot http dxdoiorg101093jxbert465

Su YS Lagarias JC (2007) Light-independent phytochrome signaling me-diated by dominant GAF domain tyrosine mutants of Arabidopsis phy-tochromes in transgenic plants Plant Cell 19 2124ndash2139

Ulijasz AT Vierstra RD (2011) Phytochrome structure and photochemis-try recent advances toward a complete molecular picture Curr OpinPlant Biol 14 498ndash506

Van Buskirk EK Decker PV Chen M (2012) Photobodies in light signal-ing Plant Physiol 158 52ndash60

Wei N Kwok SF von Arnim AG Lee A McNellis TW Piekos B DengXW (1994) Arabidopsis COP8 COP10 and COP11 genes are involved inrepression of photomorphogenic development in darkness Plant Cell 6629ndash643

Yamaguchi R Nakamura M Mochizuki N Kay SA Nagatani A(1999) Light-dependent translocation of a phytochrome B-GFP fusionprotein to the nucleus in transgenic Arabidopsis J Cell Biol 145 437ndash445

Zhang J Stankey RJ Vierstra RD (2013) Structure-guided engineering ofplant phytochrome B with altered photochemistry and light signalingPlant Physiol 161 1445ndash1457

Plant Physiol Vol 165 2014 607

The Function of Photobodies in the Dark

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