red light-mediated degradation of constans by the e3 ...red light-mediated degradation of constans...

Red Light-Mediated Degradation of CONSTANS by the E3Ubiquitin Ligase HOS1 Regulates PhotoperiodicFlowering in Arabidopsis

Ana Lazaro,1 Alfonso Mouriz, Manuel Piñeiro, and José A. Jarillo2

Centro de Biotecnología y Genómica de Plantas, INIA-UPM, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria,28223 Madrid, Spain

The regulation of CONSTANS (CO) gene expression is crucial to accurately measure changes in daylength, which influencesflowering time in Arabidopsis thaliana. CO expression is under both transcriptional and posttranslational controlmechanisms. We previously showed that the E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVEGENES1 (HOS1) physically interacts with CO in Arabidopsis. This interaction is required to precisely modulate the timing ofCO accumulation and, consequently, to maintain low levels of FLOWERING LOCUS T expression during the first part of theday. The data presented here demonstrate that HOS1 is involved in the red light-mediated degradation of CO that takes placein the early stages of the daylight period. Our results show that phytochrome B (phyB) is able to regulate flowering time,acting in the phloem companion cells, as previously described for CO and HOS1. Moreover, we reveal that phyB physicallyinteracts with HOS1 and CO, indicating that the three proteins may be present in a complex in planta that is required tocoordinate a correct photoperiodic response in Arabidopsis.

INTRODUCTION

The timing of flowering to favorable seasons of the year is crucialfor reproductive success of plants and is regulated by a number ofenvironmental factors, including daylength (photoperiod), lightquality, and temperature (Verhage et al., 2014). Genetic ap-proaches in the model species Arabidopsis thaliana have shownhow the underlying responses to changes in daylength are con-ferred and how they converge to create a robust seasonal re-sponse (Andrés and Coupland, 2012). Flowering time in thisspecies, which flowers earlier under long-day (LD) conditions thanunder short-day (SD) conditions, is regulated by several inter-connected pathways. Environmental cues such as daylength, lowwinter temperatures, and ambient temperature influence flower-ing through the photoperiod, vernalization, and temperaturepathway, respectively. In contrast, endogenous signals are in-tegrated in the control of flowering timebasedonplant age andviathe autonomous and gibberellin pathways (Fornara et al., 2010).Information from the vernalization and autonomous pathwaysmodulate the central floral repressor FLOWERING LOCUS C(FLC), aMADS box protein (Amasino, 2010). Eventually, the entirenetwork converges in the regulation of FLOWERING LOCUS T(FT), TWIN SISTER OF FT (TSF ), and SUPPRESSOR OF OVER-EXPRESSION OF CONSTANS1 genes, which act as a switch offlowering initiation (Fornara et al., 2010).

Different core components of the photoperiod pathway, such asCONSTANS (CO) and FT, are widely conserved among plant spe-cies (Kobayashi andWeigel, 2007; Turck et al., 2008; Serrano et al.,2009). In Arabidopsis, the expression of the floral integrator geneFT is activated in the phloem companion cells of the leaves by CO,a B-box-type transcription factor (An et al., 2004; Jackson, 2009;Tiwari et al., 2010). Under LDs, amobile signal translocates from theleaves to the shoot apex, promoting the floral transition. FT protein,and possibly TSF, are an essential part of this so called florigen(Corbesier et al., 2007; Jaeger and Wigge, 2007; Jang et al., 2009).The amount of FT strongly influences the timing of flowering inArabidopsis (Andrés and Coupland, 2012; Golembeski et al., 2014).Different layers of CO regulation at the transcriptional and

posttranslational level are crucial for the ability of Arabidopsis toperceive and respond to photoperiod. The coincidence of COexpression with the light in LD conditions is essential for thepromotion of flowering. In addition, different light qualities playantagonistic roles in the photoperiodic flowering response: whileblue and far-red light (F-RL) promote flowering, red light (RL)delays it (Valverde et al., 2004). Circadian clock-regulated com-ponents, such as GIGANTEA (GI), CYCLING DOF FACTORS(CDFs), and theF-boxprotein FLAVINBINDING,KELCHREPEAT,F-BOX1 (FKF1), regulate daily CO expression profiles (Imaizumiet al., 2005; Sawa et al., 2007; Fornara et al., 2009). Blue lightpromotes the binding of the E3 ubiquitin ligase FKF1 to GI; thisinteraction is necessary for the degradation of a family of COrepressors, the CDFs (Sawa et al., 2007). As a result, under in-ductive photoperiods, CO transcription peaks in the eveningfollowing thedegradation of theCDFsat the endof a LD.A secondpeak of CO expression can be observed during the night and theearly morning. In contrast, under SDs, the evening peak of COexpression disappears and only the night peak remains (Suárez-López et al., 2001; Song et al., 2013).

1 Current address: Botanical Institute, University of Cologne, ZülpicherStr. 47B, 50674 Cologne, Germany; Max Planck Institute for PlantBreeding Research, Carl-von-Linne Weg 10, 50829 Cologne, Germany.2 Address correspondence to [email protected] author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: José A. Jarillo ([email protected]).www.plantcell.org/cgi/doi/10.1105/tpc.15.00529

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In addition, CO protein stability is controlled by various lightsignals during the day (Valverde et al., 2004; Jang et al., 2008). Atnight, the RING finger protein CONSTITUTIVE PHOTOMOR-PHOGENIC1 (COP1), together with members of the SUPPRES-SOROF PHYA-105 family (SPA1, SPA3, and SPA4), promote COdegradation (Laubinger et al., 2006; Jang et al., 2008). In theevening, blue light prevents CO proteolysis by COP1 (Jang et al.,2008; Liu et al., 2008; Yu et al., 2008). The photoreceptorsCryptochrome 1 (Cry1) and Cry2 use different mechanisms torepress COP1-SPA function in a blue light-dependent manner(Lian et al., 2011; H. Liu et al., 2011; Zuo et al., 2011). Recently, anadditional role for the blue light photoreceptor FKF1 in CO reg-ulation has been proposed, as it stabilizes CO protein in theevening under LD (Song et al., 2012).

In order to precisely regulate the timing of CO accumulation, anadditional mechanism of CO degradation takes place in themorning (Jang et al., 2008). This mechanism depends on the RLphotoreceptor phytochrome B (phyB), but the E3 ubiquitin ligaseCOP1 is not involved in this process (Valverde et al., 2004; Janget al., 2008). Previous results fromour group revealed that anotherRING finger protein, HIGH EXPRESSION OF OSMOTICALLYRESPONSIVE GENES1 (HOS1), participates in the control ofphotoperiodic flowering in Arabidopsis by negatively regulatingCO abundance in the morning (Lazaro et al., 2012). The earlyflowering phenotype of hos1 mutants is due to the precociousupregulation of FT expression levels and is completely sup-pressed by mutations in the CO gene in the Landsberg erecta(Ler) background (Lazaro et al., 2012). Moreover, we showed thatHOS1 physically interacts with CO in Arabidopsis and that theCOprotein abnormally accumulates in the hos1 mutant during theearly hours of the daylight period (Jung et al., 2012; Lazaro et al.,2012). Altogether, these observations suggest that a complexregulatory mechanism ensures that CO accumulates only at theend of a LD, allowing a correct seasonal response. In addition, COactivity in the early morning is inhibited by the TARGET OF EAT1protein. This mechanism adds a new layer in the regulation of FTexpression that ensures that flowering only takes place after thedaylength reaches a threshold (Zhang et al., 2015).

The phytochrome family of photoreceptors (in ArabidopsisphyA to phyE) perceives the red and far-redwavelengths (Franklinand Quail, 2010). Phytochromes can exist in two photoreversibleforms: a RL-absorbing Pr form (biologically inactive) and a F-RL-absorbing Pfr form (biologically active) (Rockwell et al., 2006).Photoactivation of the cytoplasmic-localized photoreceptorstriggers their translocation into the nucleus, where several sig-naling components regulate different light responses (Kircheret al., 2002; Nagy and Schäfer, 2002; Casal, 2013). In the ger-mination, hypocotyl elongation, and shade avoidance responses,nuclear Pfr phyB induces the rapid phosphorylation and deg-radation of negative regulators known as PHYTOCHROME-INTERACTING FACTORs (PIFs). Seven members of this subfamilyof basic helix-loop-helix transcription factors (PIF1/PIF3-LIKE5[PIL5], PIF3, PIF4, PIF5/PIL6, PIF6/PIL2, PIF7, and PIF8) havebeen shown to physically interact with phyB (Leivar and Quail,2011; Casal, 2013). Moreover, phyB active form can be revertedto the inactive Pr form by F-RL or in the dark (D). More recently, ithas been described that an additional receptor desensitizationmechanism mediated by COP1 exists in Arabidopsis. A fraction of

the Pfr form of phyB becomes degraded by COP1 in the nucleus,and the interaction between COP1 and phyB is enhanced bydifferent PIFs (Jang et al., 2010). In addition, PIF3 has also beenreported to recruit the LIGHT-RESPONSE BTB (LRB) proteins tothe phyB-PIF3 complex and trigger the degradation of phyB andPIF3 itself (Ni et al., 2014). The LRB1 and LRB2 proteins are targetrecognition adaptors of multimeric E3 ubiquitin ligases, and mu-tations in both loci delay flowering in Arabidopsis (Christians et al.,2012). LRBproteins negatively influencephyBaction by regulatingits stability, thus providing another attenuation mechanism of phyBsignaling (Christians et al., 2012; Ni et al., 2014).In contrast, only a few signaling components have been

identified in the regulation of the floral transition by phyB. In re-sponse to RL, phyB downregulates CO mRNA levels throughfactors such as PHYTOCHROME AND FLOWERING TIME1(PFT1). PFT1 is an activator of CO transcription that has beenproposed to act as a negative regulator of phytochrome signaling(Cerdán and Chory, 2003; Wollenberg et al., 2008; Iñigo et al., 2012).In addition, several factors have been shown to interact withphyBand regulatephotoperiodic flowering. ThePHYTOCHROME-DEPENDENT LATE FLOWERING (PHL) nuclear protein accele-rates flowering by suppressing the inhibitory effect of phyB onflowering time (Endo et al., 2013). PHL interacts physically withphyB in a RL-dependent manner and, in the presence of activephyB, PHL interacts with CO as well (Endo et al., 2013). TheVASCULAR PLANT ONE-ZINC FINGER1 (VOZ1) and VOZ2transcription factors are required for proper regulationof floweringtime and cold stress responses in Arabidopsis (Yasui et al., 2012;Celesnik et al., 2013; Nakai et al., 2013). The voz1 voz2 doublemutant plants display a late flowering phenotype under LD con-ditions, which is suppressed by mutations in PHYB (Yasui et al.,2012; Celesnik et al., 2013).Moreover, interaction of VOZproteinswith phyB was shown to occur in the cytoplasm under F-RL, andthe VOZ proteins are degraded in the nucleus under F-RL and Dconditions in a phytochrome-dependent manner (Yasui et al.,2012). Therefore, it has been proposed that the partial trans-location of VOZ proteins from the cytoplasm to the nucleus maymediate the initial step of the phyB signal transduction pathwaythat regulates flowering (Yasui et al., 2012).However, the molecular mechanismmediating the RL-induced

degradation of CO that regulates photoperiodic flowering inArabidopsis remains to be elucidated. A recent report has shownthat intermittent cold treatments trigger the degradation of COthrough a HOS1-mediated ubiquitination process (Jung et al.,2012). Interestingly, cold-induced regulation of CO abundanceoccurs independently of phyB because the effects of cold on COdegradation aremaintained in thephyB-9background (Junget al.,2012, 2014). Here, we present evidence indicating that the deg-radation ofCOunder RL is prevented in the hos1mutant at normalgrowth temperatures; therefore,wepropose thatHOS1 is involvedin the RL-mediated degradation of CO. Complementation studiesreveal that phyB plays a key role in the vascular tissue to regulateflowering time, similarly to CO and HOS1 (An et al., 2004; Lazaroet al., 2012). Moreover, we demonstrate that phyB physicallyinteracts in vivo with HOS1 and CO, suggesting that the mech-anismbywhichCO isdegradedunderRL involves the formationofa complex between these proteins that may be essential tomodulate a correct photoperiodic response in Arabidopsis.

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RESULTS

HOS1 and PHYB Regulate Flowering Time Additively

Previous results have shown that both the hos1-3 and phyB-9mutantsdisplayanearlyfloweringphenotypeunderLDconditionscompared with Col (Goto et al., 1991; Reed et al., 1993; Lazaroet al., 2012; Lee et al., 2012), and our results show that the hos1mutant flowers, on average, when it has two fewer leaves thanphyB (Figures 1A and 1B). Under SD photoperiods, the earlyflowering phenotype of hos1 mutant is accentuated comparedwith phyB (Figures 1A and 1C). Interestingly, the double mutanthos1 phyB flowers even earlier than any of the singlemutants bothin LD and SD, revealing an additive role ofHOS1 andPHYB genesin the regulation of flowering time (Figures 1A to 1C).

To assess if the early flowering phenotype of both hos1 andphyBmutants depends on the presence of a functional CO gene,we obtained double and triple mutants combining mutant allelesofHOS1, phyB, andCO. This analysis allowed us to confirm that thedouble mutant phyB-9 co-10 showed the same flowering time asco-10 in LDs, confirming an epistasis ofCO gene over phyB (Iñigo

et al., 2012). On the other hand, the double mutant hos1-3 co-10and the triple mutant hos1-3 co-10 phyB-9 showed that hos1mutation is still able to accelerate co late flowering phenotype in allcombinations tested (Figures 1D and 1E), indicating that HOS1has an additional role in flowering time control besides regulatingCO. This is consistent with previous data demonstrating thatHOS1 is required to maintain high expression levels of the floralrepressor FLC (Lee et al., 2001; Lazaro et al., 2012; Jung et al.,2013).WeanalyzedFLC transcript levels in the single, double, andtriple mutant plants and observed that the hos1 mutation con-sistently reduces FLC expression in every combination analyzedcompared with the wild type (Supplemental Figure 1). Hence, weconclude that the regulatory effect of HOS1 over FLC could ac-count for the additive flowering phenotype observed in the hos1phyB double mutant plants, as well as for the acceleration of thelate flowering phenotype of co in the hos1 co combinations.We also analyzed the genetic relationship between HOS1 and

the genes encoding the VOZ transcription factors. The voz1 voz2double mutant plants show a delay on flowering time under LDconditions that is suppressedbyphyBmutation (Yasui et al., 2012;Celesnik et al., 2013). Taking into account the involvement of

Figure 1. HOS1 and PHYB Regulate Flowering Time Additively.

(A) to (C)Flowering timephenotypeofCol,hos1-3 andphyB-9 singlemutants, and hos1-3phyB-9doublemutant plants. Plants in (A)were grown for 18d inLD conditions and for 60 d in SD. Data in (B) and (C) are shown asmean6 SE andwere derived from two experiments in which aminimum of 20 plants werescored for each genotype. Student’s t test *P < 0.01 when compared with hos1-3 phyB-9.(D) and (E) Flowering time phenotype of Col and hos1-3, phyB-9, and co-10 double and triple mutant combinations. Plants in (D)were grown for 42 d in LDconditions. Data in (E) are shown as mean6 SE and were derived from two experiments in which a minimum of 15 plants were scored for each genotype.Student’s t test *P < 0.01 when compared with co-10.

HOS1 Regulates CO Abundance in Red Light 2439

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http://www.plantcell.org/cgi/content/full/tpc.15.00529/DC1

HOS1 and VOZ proteins in common biological processes (Leeet al., 2001; Lazaro et al., 2012; Yasui et al., 2012; Celesnik et al.,2013; Nakai et al., 2013), we decided to generate the triple mutanthos1-3 voz1-1 voz2-1 and analyze its flowering time. The triplemutant showed an intermediate phenotype in LDs between theearlyflowering timeofhos1and the lateflowering timeof the voz1-1voz2-1 double mutant (Supplemental Figure 2). This result couldindicate that HOS1 and the VOZ proteins do not regulate the floraltransition through the same pathway, although we cannot rule outthe possibility that a genetic interaction might be masked by thephotoperiod-independent effect of HOS1 on flowering time(Supplemental Figure 1).

Regulation of FT Expression by HOS1 and phyB Dependson CO

Previous results from our group showed that the early floweringphenotype of hos1mutant requires a functional FT gene and thatHOS1 is required to repress the expression of FT in the first part ofthe daylight period under LDs (Lazaro et al., 2012), when the in-volvement of phyB in the modulation of CO stability has beenproposed (Valverdeet al., 2004; Janget al., 2008). In a time-courseexpression analysis over a 24-h period, we observed that thehos1-2 mutant showed an unusual peak of FT expression in thesubjective morning, mainly at Zeitgeber time 4 (ZT4), but also atZT8,whenCO transcript levels are barely detectable in themutant(Lazaro et al., 2012). In order to investigate the behavior of the FTtranscript in plants lackingHOS1,phyB, and/orCO, weperformedexpression analyses in the hos1, phyB, and co double and triplemutant combinations. Seedlings were grown in LD for 11 d andharvested at ZT4 and ZT16. During the early stage of daylight(ZT4), mutations in HOS1, phyB, or both cause an increase in FTexpression compared with the wild type (Figure 2A). As wepreviously showed for the hos1-2 mutant (Ler background), thehos1-3mutant (Col) displays high FT levels at this time point (Lazaroet al., 2012). In addition, when these mutations were combined withamutation inCO,FTexpressionwasreducedto the levelsobservedin Col plants, or even lower in the case of phyB co double mutant(Figure 2A). In contrast, during the subjective evening (ZT16), onlythe phyB-9 single mutant and the hos1-3 phyB-9 double mutantshowed higher FT expression than Col, while all the mutantcombinations affected inCO showed a clear decrease in FT levelscompared with the wild type (Figure 2B).

The variation detected in FT expression in the different mutantcombinations analyzed correlates with the flowering time phe-notypes shown in Figure 1. In addition, these results corroboratethat HOS1 regulation of FT expression depends on CO and thatthe additive flowering phenotype observed in the hos1 phyB andhos1 co double mutants, as well as in the hos1 phyB co triplemutant, could be explained by the effect of HOS1 on additionalflowering time pathways mediated by FLC (SupplementalFigure 1).

Mutations in HOS1 Affect Several Developmental ProcessesRegulated by RL

Light quality regulates many developmental processes in plantssuch as flowering time, shade avoidance, hypocotyl elongation,

and seed germination (Kami et al., 2010; Casal, 2013). In order togain a deeper insight in the implication of HOS1 in the RL-dependent regulation of several of these developmental tran-sitions, we analyzed the phenotypic alterations present in thehos1 mutant and the hos1 phyB double mutant.As previously described, phyB mutant plants grown under

continuous RL displayed a conspicuous early flowering pheno-type (Reed et al., 1993; Endo et al., 2013), indicating that thiswavelength delays flowering time in Arabidopsis. We have alsogrown Col, hos1, and the double hos1 phyB mutant under con-tinuous RL, and we observed that hos1 flowering time is not asdelayed as in the wild type, but later than in the phyB mutant(Figure 3A). In addition, the hos1 phyB double mutant flowersearlier than any of the single parents. Both results indicate thatHOS1 is mediating RL signal transduction in the flowering re-sponse, although the early flowering phenotype of hos1mutantsunder RL is not totally dependent on PHYB.To check the effect of HOS1 in hypocotyl elongation, we grew

Col, hos1, phyB, and hos1 phyB mutant seedlings in continuousRL for 5 d. In accordance with the inhibitory effect of RL on thisprocess,wild-type seedlings displayed hypocotyls;60%shorterthan in D conditions, while phyB mutants were completely in-sensitive to RL, showing no inhibition of hypocotyl elongation

Figure2. RegulationofFTExpressionbyHOS1andphyBDependsonCO.

FT expression analysis in the hos1, co, and phyBmutant combinations.Eleven-day-old seedlings grown in LDs were harvested at ZT4 (A) andZT16 (B), and FT expression was detected by RT-qPCR. Relative ex-pression levels were normalized to UBC21 expression. Each data pointwasderived from threebiological replicates and is shownasmean6 SE ofthree technical replicates.

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under this wavelength compared with D. We found that RL inhib-itshypocotyl growth inhos1 to a smaller extent than inCol,;50%,which is consistent with previous observations (MacGregor et al.,2013) and that this phenotype totally depends on a functionalPHYB gene because the double mutant hos1 phyB is as in-sensitive to RL as phyB (Figures 3B and 3C). These findingssuggest thatHOS1 participates in the PHYB-dependent pathwaythat controls hypocotyl elongation under RL conditions.

In contrast to the repressive roleofRLon thefloral transition andhypocotyl elongation, this light wavelength promotes seed ger-mination, as shown by the failure to germinate of the phyBmutantunder short pulses of RL or D conditions (Shinomura et al., 1994;Hennig et al., 2002; Oh et al., 2004). Unexpectedly, when weperformed germination assays, we observed that hos1 mutantseeds showedahigh germination rate in theD (on average 50%ofthe seeds germinate), conditions in which Col seeds do notgerminate at all (Figures 3D to 3G). As expected, phyB seeds arecompletely incompetent to germinate in D conditions, while thedouble mutant hos1 phyB displayed a low germination ratecompared with the hos1mutant after 5 d in the D (Figures 3D and3E). Still, some of the double mutant seeds (an average of 9%)germinated in D conditions, indicating that the hos1 germinationphenotype depends to some extent on the PHYB gene.

To further investigate the role of HOS1 in the regulation of seedgermination, we obtained the double mutant hos1-3 pil5-2. PIL5,also known as PIF1, has been proposed to act as a molecularswitch that connects light perception to hormonal pathways thatregulate seed germination (Oh et al., 2004; Lau and Deng, 2010).Previous studies indicate that some phytochromes activate seedgermination by promoting the degradation of PIL5 protein (Oh et al.,2006); therefore, we investigated whether HOS1 could be involvedin the same repressingmechanismas PIL5. As shown in Figures 3Fand 3G,HOS1 andPIL5 act additively regarding seed germinationbecause almost every seed of the double mutant hos1-3 pil5-2germinatedafter 2d inD,while thegermination rateof thehos1andthe pil5 single mutants was around 56 and 33%, respectively.

We also examined the germination rate of hos1 and hos1 phyBunder continuous RL. In accordance with the role of RL in pro-moting seed germination, almost everyCol and hos1mutant seedgerminated after 2 d under this light condition (Figures 3H and 3I).As expected,phyBmutant seeds showed lower germination ratesthan Col and hos1, but still, three out of four seeds germinated,probably due to the effect of other RL-absorbing phytochromes.Interestingly, the loss of HOS1 overcomes the incompetence togerminate of one-quarter of the phyB mutant seeds, as almosteveryhos1phyBseedgerminatedunderRL (Figures3Hand3I). Torule out that these results could be due to low seed viability, weconfirmed that the germination rate in every seed batch used was100% after stratifying the seeds and exposing them to white light(WL) LDs for 5 d (Figure 3J).

Altogether, these observations led us to conclude thatHOS1 isinvolved in repressing seed germination in D and RL conditions.The hos1 mutant shows a constitutive activation of seed germi-nation even in the absence of light, and the effect ofPHYB in seedgermination under continuous RL is fully dependent on a func-tional HOS1 gene. Furthermore, our results demonstrate thatHOS1 is involved in the regulation of an array of developmentalresponses influenced by RL.

The Level and Subcellular Localization of HOS1 AreIndependent of Light Conditions

Since HOS1 is necessary for the regulation of several de-velopmental processes that are also controlled by RL, we decidedto test whether HOS1 protein stability or subcellular localization ismodulated by light. For that, we studied HOS1 protein regulation inWL, D, and RL conditions in transgenic lines harboring a 35S:HOS1-GFP construct in the hos1-3 mutant background. We pre-viously confirmed that these transgenic plants showed a delay inflowering time when compared with hos1-3, indicating that thefusion protein was functional (Lazaro et al., 2012). In order to studythe accumulation of HOS1 protein, we collected transgenic seed-lings grown for 10 d in LD for theWL samples and for 9 d in LD plus1 d in D conditions or continuous RL for the D and RL samples,respectively. All samples were harvested at the same time in theearly morning. Immunoblot analysis using an anti-GFP antibodyrevealed that HOS1 protein levels were higher in all the conditionstested when we added the proteasome inhibitor MG-132 duringthe extraction process (Figures 4A and 4B). This result indicatesthat HOS1 protein is regulated posttranslationally by degradationthrough the ubiquitin-proteasome proteolytic pathway. In addition,we observed in MG-132-treated samples that HOS1 levels werelower in RL than in WL (ZT4), probably due to the degradation of theprotein once it has accomplished its function (Figures 4Aand4B). Thisis a common feature in the regulation of E3 ubiquitin ligases activityto avoid its overfunctioning (Jurado et al., 2008; Yan et al., 2013).Previously,wehave shown that the fusion proteinHOS1-GFP is

localized in the nuclear envelope of Arabidopsis root cells (Lazaroet al., 2012). This observation is in agreement with the finding thatHOS1 interacts with the RNA EXPORT FACTOR1 (RAE1) nucle-oporin (Tamura et al., 2010). To analyze if the subcellular locali-zation ofHOS1changedunderRL conditions,wegrew transgenicseedlings for 7 d in LD. We then exposed the plants to F-RL for30 min and kept them in the D overnight. Half of the plates werethen exposed to RL for 4 h, and the rest were kept in D. Confocalmicroscope images of Arabidopsis roots and leaf petiolesshowed that the HOS1-GFP fusion protein did not change itssubcellular localization under RL conditions and could be clearlyobserved in the nuclear envelope in the different light conditionsused (Figures 4C and 4D). In addition, we analyzed HOS1-GFPlocalization in etiolated seedlings and we concluded that HOS1also accumulates in the nuclear envelope in plants that have neverbeen exposed to the light (Supplemental Figure 3). These resultsled us to conclude that RL does not directly regulate either HOS1accumulation or nuclear localization.

phyB Acts in the Phloem to Regulate Flowering Time

Photoperiod is perceived in the leaves, which are composed ofdifferent tissues, such as the epidermis, the mesophyll, and thevascular bundles. It has been reported that the expression ofa PHYB-GFP fusion protein in Arabidopsis mesophyll cells ofphyB-9 mutants substantially delayed flowering (Endo et al.,2005).However, someessential componentsof thephotoperiodicflowering pathway are known to act in the vascular tissue, in-cluding CO andHOS1 (An et al., 2004; Lazaro et al., 2012). To testwhether the presence of phyB in other leaf tissues may also


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Figure 3. Mutations in HOS1 Affect Several Developmental Processes Regulated by RL.

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regulate floral transition, we analyzed flowering time in trans-genic phyB-9 mutant lines expressing a PHYB-YFP constructunder the control of the phloem companion cell SUCROSE H+

SYMPORTER2 (SUC2) promoter (Imlau et al., 1999), theb-CARBONIC ANHYDRASE1 (bCA1) promoter (Hu et al., 2010),which preferentially directs PHYB-YFP expression in mesophyllcells, and the constitutive 35S promoter (Casson and Hetherington,2014). In agreement with previous reports, the presence of phyB inthemesophyll delayedphyB-9 flowering time (Figures 5A, 5B, and5E). The constitutive expression of PHYB under the control of the35S promoter also delayed the early flowering time phenotype ofphyB-9 (Figures 5A, 5B, and 5D). Interestingly, our results revealthat the expression of PHYB in the phloem companion cells alsosubstantially delays flowering time and causes a partial com-plementation of the early-flowering phenotype of the phyB-9mutant, similar to that displayed by the bCA1:PHYB-YFP lines(Figures 5A to 5C), indicating that phyB acts in the phloem toregulate flowering time.

As previously reported, YFP expression levels are much higherin the 35S:PHYB-YFP lines than when PHYB-YFP is expressedonly in the phloem or the mesophyll (Figure 5F) (Casson andHetherington, 2014). Similar results were obtained when PHYBexpressionwasdeterminedbyRT-qPCR in these lines (Figure5G).Two independent transgenic lines that were analyzed per pro-moter construct behaved similarly (Casson and Hetherington,2014). Moreover, the level of FT expression in the SUC2:PHYB-YFP and the bCA1:PHYB-YFP transgenic lines was the same asthat observed in Col and correlates with the flowering time ob-served in these lines (Figure 5H). These results show that whenphyB is present in the phloem or the mesophyll, FT expression isreducedcomparedwith thephyB-9mutant. Theexpressionof thisfloral integrator is slightly lower in the 35S:PHYB-YFP plants thanin the other transgenic lines studied and Col, but considerablylower than in the phyB-9 mutant (Figure 5H).

HOS1, CO, and phyB Proteins Interact in Planta

Previous results showed that HOS1 and CO proteins physicallyinteract in planta (Jung et al., 2012; Lazaro et al., 2012). To explorethe possibility of a direct interaction between phyB and HOS1 orCO, we expressed the C-terminal part of HOS1 (cHOS1), theC-terminal part of phyB (cPHYB), and full-length CO and COP1 inyeast cells to perform two-hybrid experiments (Yu et al., 2008).Based on the evidence that the C-terminal domain of phyB is

directly involved in interactions with signaling partners (Quailet al., 1995; Ni et al., 1998) and that only cHOS1 interacts with COin yeast (Jung et al., 2012), we used these fragments instead ofthe full-length proteins in the yeast two-hybrid assays. As shownin Figure 6A, we observed that when we coexpressed cHOS1 to-gether with either COor cPHYB, yeast cells could grow in selectivemedium, revealing a protein interaction between them, while thecoexpressionofcHOS1andCOP1didnotallowyeastcells togrow,meaning that no direct interaction takes place between these twoE3 ubiquitin ligases. In addition, CO and cPHYB proteins also in-teracted in yeast cells that could grow in medium supplementedwith 10 mM 3-amino-1,2,4-triazole (3-AT) (Figure 6A).To further investigate interactions between these proteins and

to determine if they also occur in planta, we performed in vivocoimmunoprecipitation (co-IP) assays in wild tobacco (Nicotianabenthamiana) plants infiltrated with CO-TAP or HOS1-GFP andHA-PHYB fusion proteins. We detected a physical interactionbetween HOS1 and phyB, as well as between CO and phyB, inprotein extracts incubated under RL conditions (Figures 6B and6C). As expected, the immunoprecipitation assays showed nointeractions in plants infiltrated only with HOS1, CO, or phyB. Wealso performed the co-IP assays in protein extracts incubated inDand, although the interaction was slightly weaker, we could alsopull down the phyB protein with HOS1-GFP, indicating that thephysical interactions detected in wild tobacco are not dependenton the light treatment of the protein extracts (Supplemental Figure4). The fact thatphyBphysically interactswithCOandHOS1whenthey are coexpressed in wild tobacco leaves could indicate thatCO, HOS1, and phyB may be present in a complex in planta.It had been reported that a fraction of the light-activated form of

phyB is degraded by the E3 ubiquitin ligase COP1 in the nucleus(Jang et al., 2010; Ni et al., 2014). To check if HOS1 could also beinvolved in the regulation of phyB abundance, we analyzed phyBlevels in total protein extracts using an anti-phyB antibody(Somers et al., 1991). We could not detect any change in phyBlevels when we compared Col, the hos1 mutant, and the HOS1-overexpressing plants, suggesting that HOS1, in contrast toCOP1, is not involved in a phyB desensitization mechanism(Supplemental Figure 5).

HOS1 Regulates CO Stability under RL Conditions

CO protein stability has been shown to play a central role in thecontrol of photoperiodic flowering, and previous results showed

Figure 3. (continued).

(A) Flowering time phenotype of Col, hos1-3, phyB-9, and hos1-3 phyB-9 double mutant plants grown in continuous RL. Data are shown as mean6 SE. Aminimum of 15 plants were scored for each genotype. Student’s t test *P < 0.01 when compared with hos1-3 phyB-9.(B) Hypocotyl length in Col, hos1-3, phyB-9, and hos1-3 phyB-9 double mutant seedlings grown in continuous RL or D for 5 d.(C) Quantification of the percentage of inhibition of hypocotyl elongation in Col, hos1-3, phyB-9, and hos1-3 phyB-9 double mutant seedlings. Data areshown as mean 6 SE and were derived from three experiments in which a minimum of 25 seedlings were scored for each genotype.(D) and (E) Germination phenotype of Col, hos1-3, phyB-9, and hos1-3 phyB-9 double mutant seeds after 5 d in D.(F) and (G)Germination phenotype of Col, hos1-3, pil5-2, and hos1-3 pil5-2 double mutant seeds after 2 d in D. Arrowsmark emerging radicles from pil5-2mutant seeds.(H) and (I) Germination phenotype of Col, hos1-3, phyB-9, and hos1-3 phyB-9 double mutant seeds after 2 d in continuous RL.(J)GerminationphenotypeofCol,hos1-3,phyB-9,hos1-3phyB-9,pil5-2, andhos1-3pil5-2 stratifiedseedsafter 5d inWLLDs (viability). Data are shownasmean 6 SE and were derived from three experiments in which a minimum of 50 seeds were scored for each genotype.


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that CO degradation is impaired in the phyB mutant under RL(Valverde et al., 2004). Given that HOS1 interacts with phyB andCO, we used a 35S:CO-LUC construct to analyze the stability of

CO protein in RL conditions. One representative line expressingthe CO-LUC fusion protein was crossed into the hos1-3 mutantbackground. The plants overexpressing the CO-LUC fusion were

Figure 4. The Level and Subcellular Localization of HOS1 Are Independent of Light Conditions.

(A) Immunodetection of HOS1-GFP fusion protein in hos1-3 transgenic seedlings in different light conditions. TheWL samples were grown in LDs for 10 d.The RL and D samples were grown in LDs for 9 d and then transferred to RL or D for 24 h, respectively. Protein extraction and immunoprecipitation wereperformed in the presence or absence of the proteasome inhibitor MG-132.(B) Quantification of HOS1-GFP protein levels in the three different light conditions tested. Data are shown as mean 6 SE of two biological replicates.(C) and (D)Confocal images of 7-d-old transgenic plants containing aHOS1-GFPconstruct inhos1-3mutant background. All seedlingsweregrown in LDs,treated with F-RL for 30 min, and kept in D overnight. The RL image was taken from seedlings further exposed to RL for 4 h.(C) Arabidopsis roots were stained with propidium iodide.(D) Arabidopsis leaf petioles. The chloroplast autofluorescence and the bright field are merged with the GFP signal. Bar = 25 mm.

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early flowering, but the 35S:CO-LUC/hos1-3 plants floweredeven earlier than the 35S:CO-LUC/Col plants, and earlier than thehos1-3 mutant, indicating that the CO-LUC construct was func-tional (Supplemental Figure 6). To carry out the analysis, we grewtransgenic seedlings for 8 d in LD conditions and detected lu-minescence at ZT3 and ZT13 (WL ZT3 and WL ZT13). The D and RLimages in Figure 7 showplants grown for 7 d in LDand then kept inDconditionsorexposed toRL for24h.Wechecked thatCOmRNA

levelswere similar inCol andhos1-3 seedlingsoverexpressing theCO-LUC fusion, and this allowed us to presume that the changesthat might be observed in CO protein accumulation were not dueto the transcriptional regulation of CO (Supplemental Figure 7).As expected from our previous results, plants grown in LD

conditions showed higher levels of CO protein in hos1 mutantbackground than in the wild type at ZT3 (Lazaro et al., 2012)(Figures 7A and 7C). Moreover, consistent with the previously

Figure 5. phyB Acts in the Phloem to Regulate Flowering Time.

(A) and (B) Flowering time phenotype of Col, phyB-9mutant,SUC2:PHYB-YFP phyB-9, 35S:PHYB-YFP phyB-9, andbCA1:PHYB-YFP phyB-9 transgenicplants grown for 21 d in LD conditions. Data in (B) are shown as mean6 SE and were derived from two experiments in which a minimum of 25 plants werescored for each genotype. Student’s t test *P < 0.01 when compared with phyB-9.(C) to (E) Confocal images of cotyledons of SUC2:PHYB-YFP phyB-9, 35S:PHYB-YFP phyB-9, and bCA1:PHYB-YFP phyB-9 transgenic plants. Bar =100 mm.(F) to (H) Expression analysis in Col, the phyB-9 mutant, and the SUC2:PHYB-YFP phyB-9, 35S:PHYB-YFP phyB-9, and bCA1:PHYB-YFP phyB-9transgenicplants.Eleven-day-oldseedlingsgrown inLDswereharvestedatZT16,andYFP (F),PHYB (G), andFT (H)expressionwasdetectedbyRT-qPCR.Relative expression levelswere normalized toUBC21expression. Eachdata pointwasderived from threebiological replicates and is shownasmean6 SE ofthree technical replicates.


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described daily pattern of CO accumulation, at the end of a LD(ZT13),COprotein levels inColweremuchhigher than theywere inthe first part of the day, and only slight differences are observedbetween the wild type and hos1 (Figures 7B and 7C). In contrast,when the plants were kept in D for 24 h, CO expression was verylow in both genetic backgrounds (Figures 7Dand7F). This result isin accordance with the previously reported role of COP1 in COdegradation that takesplaceduring thenight (Janget al., 2008; Liuet al., 2008). Interestingly, when the plants were exposed to RL for24 h, we could clearly detect higher CO accumulation in the hos1mutant background than inCol (Figures 7Eand7F), indicating thatHOS1, like phyB, regulates CO stability under RL.

The hos1 Mutation Does Not Further Upregulate FTExpression in phyB-Deficient Plants under RL Conditions

To determine if HOS1 and phyB could be acting together in theregulation of flowering time in RL,we analyzed FT transcript levelsin Col and in mutant plants lacking HOS1, phyB, or both proteins.Seedlings were grown for 8 d in LD conditions and exposed tocontinuous RL for 24 h. In accordance with the role of RL in COdegradation, we did not detect significant FT expression in Col

plants, and as expected, the impaired degradation of CO in thehos1 and the phyBmutant under RL led to the upregulation of FTtranscript to detectable levels (Figure 8). Moreover, we found thatFT expression was not further upregulated in the hos1 phyBdouble mutant compared with hos1 and phyb single mutantsunder RL conditions (Figure 8), suggesting that HOS1 and phyBregulate FT expression through the same genetic mechanism.Altogether, our results indicate that HOS1 is involved in the

regulation of CO accumulation in the early stage of the daylightperiodand that it alsoparticipates in theRL-mediateddegradationof CO required to maintain low levels of FT expression during theearly part of the day in LDs. Given that phyB interacts with HOS1and CO, these three proteins may be part of functional complex(es)that could be indispensable to properly modulate the photope-riodic response in Arabidopsis.

DISCUSSION

HOS1 was initially described as a negative regulator of lowtemperature responses, but additional evidence suggested thatthe function of this E3 ubiquitin ligase was not restricted to coldacclimation (Ishitani et al., 1998;Leeetal., 2001;Dongetal., 2006).

Figure 6. HOS1, CO, and phyB Proteins Interact in Yeast Two-Hybrid Assays and in Planta.

(A) Yeast two-hybrid assay between CO and the C-terminal part of HOS1 (cHOS1), CO and cPHYB, cPHYB, and cHOS1, and cHOS1 and COP1. Physicalinteractions are detected for CO and cHOS1, as well as for CO and cPHYB, on minimal growth medium without Leu, Trp, His, and Ade (-LWHA), sup-plemented with 10 mM 3-AT. A physical interaction is also detected for cPHYB and cHOS1 on minimal growth medium -LWHA.(B) and (C) Co-IP experiments in agroinfiltrated N. benthamiana plants.(B)HOS1-GFPandHA-PHYB fusion proteinswere produced alone or in combination inwild tobaccoplants, andprotein extractswere incubatedwithGFP-Trap under RL. Immunodetection was performed with anti-HA and anti-GFP antibodies.(C) CO-TAP and HA-PHYB fusion proteins were produced alone or in combination in wild tobacco plants, and protein extracts were incubated with IgGSepharose under RL. Immunodetection was performed with anti-HA and anti-protein A antibodies.

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Previous results obtained in our group demonstrated that HOS1is required for proper regulation of photoperiodic flowering inArabidopsis by modulating the stability of the CO protein in thesubjective morning under LDs (Lazaro et al., 2012). HOS1 alsomediates CO degradation at cold temperatures in the eveningunder LDs (Junget al., 2012). Thecold-induceddegradationofCOprotein was significantly reduced in hos1 mutants, which exhibitedcold-insensitive early flowering. Notably, the effects of cold stresson CO degradation were maintained in phyB mutants, showingthat the HOS1-mediated degradation of CO protein at low tem-peratures was independent of phyB (Jung et al., 2012). Here, weshow that HOS1 is involved in the RL-mediated degradation ofCO, which regulates photoperiodic flowering in Arabidopsis. Wedemonstrate that the degradation of CO under RL is substantiallyreduced in the hos1mutant at normal growth temperature (Figure7). Moreover, FT expression is not further upregulated in the hos1phyB double mutant compared with the single mutants underRL (Figure 8) or in the first part of the day (ZT3) under LD

photoperiods (Figure 2), suggesting that HOS1 and phyB regu-late FT expression through the same genetic mechanism.Recent studies indicate that HOS1 also plays nonproteolytic

roles in gene expression regulation (Jung et al., 2014; MacGregorand Penfield, 2015). We and other groups have shown that HOS1is required for full activation of the floral repressor gene FLC andthat the early flowering phenotype of hos1 mutants partially de-pends on FLC (Ishitani et al., 1998; Lee et al., 2001, 2012; Lazaroet al., 2012; Jung et al., 2013) (Supplemental Figure 1). FLC alsoplays a role in flowering time control under short-term cold stress,conditions in which flowering is delayed (Seo et al., 2009). Theeffects of intermittent cold treatments on FLC expression andflowering time are strongly diminished in hos1mutants (Seo et al.,2009; Jung et al., 2013), indicating that the activation of the FLCgene by cold stress is mediated by HOS1 (Jung et al., 2014).Recently, it has been shown that HOS1 binds to FLC chromatinand that this binding is significantly induced by cold stress (Junget al., 2013). Arabidopsis flowering under cold stress is also

Figure 7. HOS1 Regulates CO Stability under RL Conditions.

(A), (B), (D), and (E) Noninvasive in vivo luciferase imaging of 35S:CO-LUC/Col and 35S:CO-LUC/hos1-3 transgenic seedlings.(A) and (B) WL ZT3 and WL ZT13 pictures show 8-d-old seedlings grown in LDs, 3 and 13 h after the lights were turned on, respectively.(C) Quantification of the luciferase activity in LDs in both genetic backgrounds expressed as counts per second (cps) per plant.(D) and (E)D and RL pictures show 7-d-old seedlings grown in LDs and then transferred to D or RL for 24 h. The color scale in the right side of each picturerefers to the counts per second.(F)Quantification of the luciferase activity inDandRLconditions in both genetic backgrounds expressed as counts per secondper plant. All quantificationswere done at the same time in the early morning. Each data point was derived from three biological replicates and is shown asmean6 SE of three technicalreplicates.


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mediated by FVE (Kim et al., 2004). The fve mutant exhibits en-hanced freezing tolerance and late flowering due to FLC upre-gulation (Ausín et al., 2004; Kim et al., 2004). HOS1 interacts withFVE and HISTONE DEACETYLASE6 (HDA6) and inhibits thebinding of HDA6 to FLC chromatin, resulting in increased histoneacetylation levels in the FLC locus and subsequent activation ofFLC expression. Therefore, HOS1 activates FLC transcription throughchromatin remodeling under short-term cold stress conditions(Jung et al., 2013). HOS1 also binds themicroRNA geneMIR168band promotes its transcription (Wang et al., 2015). MicroRNA168a/b targetsARGONAUTE1 (AGO1) transcript to regulateAGO1protein level, which is important for the function of all microRNAsin Arabidopsis. Given that HOS1 can function through both pro-teolytic and nonproteolytic modes of action, and based on thewide range of genes that are influenced by mutations in the HOS1gene (MacGregor et al., 2013), it is evident that HOS1 playsversatile roles in gene expression regulation (Jung et al., 2014).

Since HOS1 mediates responses to both RL and cold, it hasbeen proposed that this protein could function as amolecular hubthat integrates light and temperature signals to modulate cellularprocesses that regulate plant development (Jung et al., 2014;MacGregor and Penfield, 2015). However, how these environ-mental cues influence HOS1 activity at the transcriptional orprotein level remains unknown. In our previous work, we studiedHOS1expressionpatternover a24-hperiod inLDsandconcludedthat HOS1 transcript did not show a diurnal oscillation in the Lerbackground (Lazaro et al., 2012). In this study, we also addressedthe effect of different light qualities in the regulation of HOS1protein levels or subcellular localization. Our results demonstratethat there is no difference in HOS1 accumulation between LD-grown or D-treated seedlings overexpressing HOS1 and that

plants exposed to RL for 24 h show a slight decrease in HOS1protein levels compared with seedlings grown in LDs (Figures 4Aand4B).Moreover,ourprevious results revealed thataHOS1-GFPfusion protein localizes in the nuclear envelope of Arabidopsis rootcells (Lazaro et al., 2012). This is consistent with previous ob-servations showing that HOS1 interacts with components of thenuclear pore complex, such as RAE1 and the nucleoporin Nup43(Tamura et al., 2010). The nuclear pore complex plays an essentialrole in gene expression regulation by mediating nucleocyto-plasmic mRNA export. Moreover, it has been reported thatpolyadenylatedmRNAs accumulate to a high level in the nucleusofHOS1-deficientmutants and thatHOS1 is required tomaintaincircadian periodicity over a wide range of light and temperatureenvironments through the promotion of mRNA export betweenthe nucleus and the cytoplasm (MacGregor et al., 2013). In thiswork, we demonstrate that HOS1 nuclear localization does notchange in response to RL or D treatment in different tissues(Figures 4C and 4D). However, it is still possible that HOS1activity might be differentially regulated by light; additional ex-periments will have to be performed to explore this point.RL regulates a number of developmental processes, including

seedgermination,hypocotylelongation,chloroplastdevelopment,shade avoidance, and flowering (Goto et al., 1991; Chory et al.,1996; Li et al., 2011). Distinct phyB signaling pathways regulatehypocotyl elongation and floral initiation (Botto and Smith, 2002;Cerdán and Chory, 2003), although there are some componentsinvolved in phyB signaling shared by several developmentalprocesses regulated by RL. Our results indicate that HOS1 pro-motes the destabilization of CO protein during the first part of thedaylight period and that HOS1 participates in the RL-dependentdegradation of CO, which precisely regulates photoperiodicflowering in Arabidopsis (Figures 7 and 8). Moreover, we havedetermined that HOS1 also plays a role in hypocotyl elongation inresponse to RL, corroborating previous observations (MacGregoret al., 2013), and that the effect ofHOS1on this response dependson PHYB (Figures 3B and 3C). Our data also revealed that this E3ubiquitin ligaseacts asa repressor of seedgermination inDandRLconditions (Figures 3D, 3E, 3H, and 3I). Particularly under RL, theeffect of PHYB is fully dependent on HOS1, suggesting that bothloci could act in the same genetic pathway to regulate seed ger-mination.PIL5/PIF1 is also involved in the light-mediatedcontrolofseed germination (Oh et al., 2004), but our results indicate that itworks independently of HOS1 in this developmental transition(Figures 3F and 3G). Interestingly, HOS1 acts antagonistically tophyB in seed germination, but HOS1 and phyB function as re-pressors of the floral transition and hypocotyl elongation. This isalso the case for PIF4, which has the opposite effect to phyB ongrowth responses, but acts in the same way as phyB in stomatalindex responses (Casson et al., 2009; Casal, 2013).Altogether, these results suggest that HOS1 and phyB proteins

participate in common molecular regulatory mechanisms. Incontrast to previous observations (Endo et al., 2005), we haveshown that phyB is able to regulate flowering time in the phloemcompanion cells (Figure 5). We observed a complementation ofthe early flowering phenotype of the phyB-9mutant in the SUC2:PHYB-YFP phyB-9 line, as well as the downregulation of FT ex-pression to Col levels (Figure 5). Moreover, we detected physicalinteractions between HOS1 and phyBwhenwe coexpressed both

Figure 8. The hos1Mutation Does Not Further Upregulate FT Expressionin phyB-Deficient Plants under RL Conditions.

FT expression analysis in Col, the hos1 and the phyB single mutants, andthehos1phyBdoublemutant. Eight-day-old seedlingsgrown in LDswheretransferred to continuous RL for 24 h and FT expression was detected byRT-qPCR. Relative expression levels were normalized to UBC21 ex-pression. Each data point was derived from three biological replicates andis shown as mean 6 SE of three technical replicates.

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proteins in yeast or in plant cells (Figure 6). Also, we demonstratedthat phyB binds to CO in yeast two-hybrid assays and in co-IPexperiments in N. benthamiana (Figure 6). In addition, previousresults obtained in our group revealed that HOS1 and CO proteinsinteract physically in Arabidopsis (Lazaro et al., 2012). The fact thatCO,HOS1,andphyBregulatefloweringtime inthesameleaf tissue,together with the physical interactions detected in co-IP experi-ments, supports that the three proteins may be present in a com-plex inplanta (Anetal., 2004;Lazaroetal., 2012).Howthiscomplexis shaped in response to environmental cues remains to be elu-cidated. It is well established that phyB moves to the nucleus inresponse to light (Kircheretal., 2002),whereasHOS1andCOseemto be constitutively nuclear localized (Robson et al., 2001; Lazaroet al., 2012). It is tempting to speculate that HOS1 could assist inthe translocation of phyB to the nucleus. Alternatively, it is alsoconceivable that light-activated phyB might mediate CO recogni-tion by HOS1 in the nucleus. Thus, under RL, the three proteins arenuclear localized, enabling the formation of a complex that couldmodulate the photoperiodic flowering response in Arabidopsis.

As described above, the light-activated form of phyB is de-graded in the nucleus after ubiquitination by the E3 ubiquitin ligaseCOP1 (Jang et al., 2010; Ni et al., 2014). In vitro pull-down assaysshowed that the N-terminal region of phyB strongly interacts withCOP1, whereas weak binding was detected with the C-terminalregion (Jang et al., 2010). In this work, we have shown that theC-terminal fragment of HOS1 interacts with the C-terminal domainof phyB in yeast cells (Figure 6) and that HOS1 is not able to bindCOP1 (Figure 6). In addition, our results indicate that HOS1 is notinvolved in phyB degradation (Supplemental Figure 5), and wepreviously demonstrated thatHOS1 andCOP1 regulate floweringtime additively (Lazaro et al., 2012). Altogether, these data sug-gest that HOS1 participates in phyB signaling, while COP1 me-diates a receptor desensitization mechanism.

Light signaling pathways control CO protein activity to induceFT under favorable conditions for flowering (Song et al., 2013).Wedemonstrated thatHOS1 regulation of FT expression depends onCO (Figure 2) and that the additive flowering phenotype observedin the hos1 phyB and hos1 co double mutants, as well as in thehos1 phyB co triple mutant, could be due to the effect of HOS1on an additional flowering time pathway mediated by FLC(Supplemental Figure 1). The regulation of FT by HOS1 seems tobe crucial in the first part of the day, when an unusual peak of FTexpression can be observed in the hos1mutant at ZT3 (Figure 2)(Lazaro et al., 2012). In contrast, in the late-flowering phl-1mutant,FT expression is substantially reduced compared with Col at ZT15(Endo et al., 2013). Endo and coworkers performed a co-IP assayto test if PHL could interactwith both phyBandCO. Theco-IPwasperformed with tagged versions of PHL and phyB proteins ex-tracted from Arabidopsis transgenic lines combined with extractscontaining CO-tdTomato prepared fromwild tobacco leaves. Theresults obtained indicate that PHL can bridge phyB and CO ina RL-dependent manner (Endo et al., 2013), indicating that phyBand CO should be present in the same plant tissue, although nodirect interaction was demonstrated for phyB and CO. In-terestingly, we present sound evidence supporting the notion thatphyB and CO, and also phyB and HOS1, physically interact inplanta (Figure 6), andwe reveal that phyB regulates flowering timein the phloem (Figure 5). Moreover, we observed that the hos1

mutation does not further upregulate FT expression in PHYB-deficient plants under RL conditions (Figure 8). Therefore, HOS1and phyB might prevent precocious flowering by mediating thedegradation of CO protein in the morning (Valverde et al., 2004;Lazaroetal., 2012),whereasPHLwouldstabilize it in theafternoon(Endo et al., 2013), shaping the accumulation of COprotein duringthe daylight period in LDsandcontributing to the fine-tuning of theseasonal flowering response in Arabidopsis.According to the current photoperiodic flowering model, red

and blue light have antagonistic functions in the regulation of theflowering activator CO. Direct regulation of CO stability by Cry1,Cry2, andFKF1has recently been reported (Lianet al., 2011;B. Liuet al., 2011; Zuo et al., 2011; Song et al., 2012). However, how RLregulates CO degradation remains unclear. The results presentedhere, together with previous observations (Lazaro et al., 2012),allow us to propose that CO, HOS1 and phyB could be at leasttransiently bound in a complex in planta and that HOS1mediatesthe RL-mediated degradation of CO in Arabidopsis. RL regulatesflowering time in a variety of LD and SD species. Indeed, pea(Pisum sativum) phyB mutants flower earlier in SDs, rice (Oryzasativa) phyB mutants flower early under both photoperiodicconditions, and sorghum (Sorghum bicolor) genotypes lackingPHYB mainly flower earlier in LD (Weller and Reid, 1993; Childset al., 1997; Takano et al., 2005). Because HOS1, CO, and phyBhomologs are found in all plant species, the interaction revealedheremaybegenerallypresent inallfloweringplants. Theexistenceof this protein complex provides a framework that will help elu-cidate how flowering time is regulated by environmental signals,particularly by RL.

METHODS

Plant Material and Growing Conditions

The Arabidopsis thalianamutant seed stocks used were in the Col geneticbackground and were obtained from the ABRC of Ohio State University(Columbus, OH) and personal donations. The monogenic mutants used inthis work were described previously: hos1-3 (Lazaro et al., 2012), co-10(Laubinger et al., 2006), phyB-9 (Reed et al., 1998), pil5-2 (Penfield et al.,2005), and voz1-1 and voz2-1 (Yasui et al., 2012).

Plants were grown in plastic pots containing a mixture of substrate andvermiculite (3:1). For in vitroculture, seedlingsweregrown inMS (MurashigeandSkoog)mediumsupplementedwith0.8%(w/v)agar,withorwithout1%(w/v) sucrose. Controlled environmental conditions were provided ingrowth chambers at 22°C and 70% relative humidity. Plants were illumi-natedwithcool-whitefluorescent lights (;120mmolm–2s–1). LDconditionsconsisted of 16 h of light followed by 8 h of D; SD conditions consisted of 8 hof light followedby 16hof D. ContinuousRL treatments consisted of a flowrate of 32 mmol m–2 s–1 and F-RL consisted of a flow rate of 5mmol m–2 s–1

(E-30LEDL3 monochromatic growing chamber; Percival Scientific).

Phenotypic Analysis

Flowering Time Measurement

Plants were grown on soil under LD or SD photoperiods or continuous RL.Total leaf number was scored as the number of leaves in the rosette (ex-cluding cotyledons) plus the number of cauline leaves in the inflorescence atthe time of opening of the first flower (Koornneef, 1991). A minimum of 15plants were scored for each genotype. Data are shown as mean 6 SE.


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Hypocotyl Measurement

Seeds were plated in MS medium supplemented with 0.8% (w/v) agar,stratified, and exposed toWL for two and a half hours before keeping themunder continuous RL for 5 d. The inhibition of hypocotyl elongation wascalculated as the ratio between the hypocotyl length in RL and the hy-pocotyl length in D. The mean and SE were calculated from three in-dependent replicates.

Germination Rate Experiments

Seeds were sterilized with ethanol, plated on MS medium supplementedwith 0.8% (w/v) agar, and kept in D or continuous RL from imbibition untilgerminationwasquantified. Thepercentageof germinationwas calculatedas the ratio between germinated and nongerminated seeds normalizedto the viability. In all experiments, the viability (germination in WL afterstratification) of each genotype was 100%. The mean and SE were cal-culated from three independent replicates.

RNA Extraction and RT-qPCR

Plants were grown in MS medium supplemented with 1% (w/v) sucroseand 0.8% (w/v) agar and harvested at ZT4 and ZT16 after 11 d in LDs. TotalRNA was isolated using the E.Z.N.A. Plant RNA Kit (OMEGA Bio-Tek) andtreated with RQ1 DNase (Promega). Retrotranscription to cDNA wasperformed using M-MLV RT (Invitrogen). RT-qPCR analyses were per-formed using FastStart Universal SYBR Green Master (Roche) in a Light-Cycler 480 (Roche). Primers already described for analyzing the expressionof FT, FLC, PHYB, YFP, and the UBIQUITIN-CONJUGATING ENZYME21(UBC21) genes were used (Czechowski et al., 2005; Chiang et al., 2009;Morris et al., 2010; Strange et al., 2011; Crevillén et al., 2013; Casson andHetherington, 2014). Each data point was derived from three biologicalreplicates and is shown as mean 6 SE.

Confocal Microscope Images

Previously described mutant hos1-3 plants containing a 35S:HOS1-GFPconstruct were grown in MS medium supplemented with 1% (w/v) sucroseand 1% (w/v) plant agar in Petri dishes placed vertically (Lazaro et al., 2012).Seven-day-old transgenic plants grown in LDs were treated with F-RL for30minandkept inDovernight.Half of theplateswere further exposed toRLfor 4 h, and theother halfwere kept inD.Rootswere stainedwithpropidiumiodide before the confocal microscopy images were taken (Leica SP8).

Protein Extraction and Immunodetection

Protein extraction of Arabidopsis seedlings and wild tobacco (Nicotianabenthamiana) leaves was performed using co-IP buffer (50 mM Na-phosphate, pH 7.4, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 1 mM DTT,0.1%Triton X-100, 2mMNa3VO4, and 2mMNaF) plus Complete proteaseinhibitor cocktail tablets (Roche) and 50 mMproteasome inhibitor MG-132(Peptide Institute) (Song et al., 2012). Plant tissue was ground with liquidnitrogen, resuspended in co-IP buffer, and centrifuged twice to obtain thetotal soluble protein extract.

Immunodetection of HOS1-GFP Fusion Protein in hos1-3 TransgenicSeedlings

We grew plants in MS medium supplemented with 1% (w/v) sucrose and0.8% (w/v) agar for 10d in LDphotoperiod for theWLsample and9d in LDsplus 24 h of D or continuous RL for the D and RL samples, respectively.

Proteins were extracted with and without the proteasome inhibitorMG-132. Extractswere incubatedwithGFP-Trap_A (ChromoTek) for 2 h at4°C, agarose beads were washed three times with co-IP buffer, andproteins bound to the anti-GFP antibody were separated by SDS-PAGE.Immunodetection was performed with a monoclonal anti-GFP antibodydiluted 1/1000 (Roche catalog number 11814460001). Coomassie BrilliantBlue staining was used to quantify the amount of proteins in each sample.The mean and SE were calculated from two biological replicates.

Co-IP Experiments in N. benthamiana

We used a pCTAPi-CO construct (Song et al., 2012) and the 35S:HOS1-GFP construct for the co-IP experiments in wild tobacco. The full-lengthcDNA of PHYB was obtained by PCR and cloned using Gateway re-combinant technologies (Life Technologies) in the pEarlyGate201 vector.We used 3-week-old N. benthamiana plants to produce CO-TAP, HOS1-GFP, and HA-PHYB fusion proteins. Infiltration was performed in 10 mMMES, pH 5.6, 10 mM MgCl, and 150 mM acetosyringone, at an opticaldensity of 0.3 for each of the Agrobacterium tumefaciens cultures trans-formed with the single constructs. Wild tobacco leaves were collected 3 dafter agroinfiltration. Protein extracts were incubated with either GFP-Trap_A (ChromoTek) or IgG Sepharose 6 Fast Flow (GE Healthcare) for 1 hinRLorDconditions at room temperature. Beadswerewashed three timeswith co-IP buffer and resuspended in Laemmli buffer. Proteins were re-solved in 8% SDS-PAGE gels. As an input, 0.2 to 2.5% of the total extractwas loaded. Immunodetections were performed with monoclonal anti-bodies anti-GFPdiluted1/1000 (Rochecatalognumber11814460001) andanti-HA clone 3F10 diluted 1/3000 (Roche catalog number 11867423001),and anti-protein A diluted 1/5000 (Sigma-Aldrich catalog number P3775),using a SuperSignal West Femto Trial Kit (Thermo Scientific).

Immunodetection of phyB in Total Protein Extracts

Immunodetection was performed with a 1:1000 dilution of themonoclonalanti-phyB B1 antibody (Somers et al., 1991).

Yeast Two-Hybrid Assays

Yeast two-hybrid interaction analyses were conducted in the pJ69-4aSaccharomyces cerevisiae strain. The C-terminal part of the protein en-coded by the HOS1 cDNA (amino acids 457 to 927) was obtained by PCRand cloned using Gateway recombinant technologies (Life Technologies)to generate the GBD or GAD fusion constructs (Jung et al., 2012). TheC-terminal part of phyB contains amino acids 645 to 1210 (Ni et al., 1998).cHOS1was cloned both in the pGAD and the pGBT8 vectors, cPHYBwascloned in the pGADT7 and pGBKT7 vectors, and CO and COP1 werecloned in the pGAD and the pGBKT7 vectors, respectively. Selection wasperformed on synthetic complete minimal mediumwithout Leu and Trp, orwithout Leu, Trp, His, andAde, supplementedwith 10mM3-AT if required.

Luciferase Activity Assays

The 35S:CO-LUC construct was transformed in Col plants and homozy-gous lines were established. Several independent transgenic plants ex-hibiting early flowering phenotype were selected and one representativeline was crossed with hos1-3 plants. For noninvasive in vivo luciferase(LUC) imaging, seedlings were grown in MS plates for 8 d in LD conditionsandsprayedwith200mMluciferin (Biosynth) 3and13hafterdawn.Both theDandRL imageswere takenafter growing seedlings for 7d in LDs and thenkeeping them in D or treating themwith RL for 24 h. Image acquisition andquantification of luciferase activity were performed with NightOWL II

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(Berthold Technologies) and IndiGO software provided by the cameramanufacturer. Luciferase activity is expressed as counts per second perplant. The mean and SE are calculated from three biological replicates.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis GenomeInitiative or GenBank/EMBL databases under the following accessionnumbers: HOS1 (At2g39810), PHYB (At2g18790), CO (At5g15840), VOZ1(At1g28520), VOZ2 (At2g42400), FT (At1g65480), FLC (At5g10140),UBC21 (At5g25760), PIL5 (At2g20180), and COP1 (At2g32950).

Supplemental Data

Supplemental Figure 1. FLC expression in the hos1, co, and phyBmutant combinations.

Supplemental Figure 2. Flowering time of hos1 voz1 voz2 triplemutant plants.

Supplemental Figure 3. Confocal images of etiolated transgenicplants expressing the 35S:HOS1-GFP construct in the hos1-3 mutantbackground.

Supplemental Figure 4. Co-IP assays in protein extracts incubatedin D from N. benthamiana plants infiltrated with HOS1-GFP andHA-PHYB constructs.

Supplemental Figure 5. Immunodetection of phyB in protein extractsfrom Col, the phyB-9 and the hos1-3 mutant, and HOS1-GFP trans-genic seedlings grown in LDs for 10 days.

Supplemental Figure 6. Flowering time of transgenic Col and hos1-3plants expressing the 35S:CO-LUC construct.

Supplemental Figure 7. CO expression in Col and hos1-3 plantsexpressing the 35S:CO-LUC construct.

ACKNOWLEDGMENTS

voz1 and voz2 mutants were kindly provided by T. Kohchi (Kyoto University,Kyoto, Japan), the transgenic plants expressing PHYB-YFP under thecontrol of the SUC2, bCA1, and 35S promoters were kindly donated byCasson (University of Sheffield, UK), the C-terminal clone of phyB waskindly provided by S. Prat (CNB, CSIC, Madrid, Spain), the full-lengthCOP1 cDNAwas kindly donated byV. Rubio (CNB,CSIC,Madrid, Spain),the pCTAPi-CO construct was kindly donated by T. Imaizumi (Universityof Washington, Seattle, WA), the 35S:CO-LUC construct was kindly pro-vided by H.-Q. Yang (Shanghai Institute for Biological Sciences, ChineseAcademy of Science, Shanghai, China), and the anti-phyB antibody waskindly provided by Peter Quail (University of California, Berkeley, CA). Wethank L. Narro (CBGP, UPM-INIA, Madrid, Spain) for establishing homo-zygous pil5-2 mutant lines from the SALK collection (SALK_131872) andJ.C. del Pozo (CBGP, UPM-INIA, Madrid, Spain) and S. Prat (CNB, CSIC,Madrid, Spain) for helpful discussions.We also thankM. Albani (UniversityofCologne,Germany) for her kindhelp. ThisworkwassupportedbyGrantsBIO2010-15589 andBIO2013-43098R to J.A.J. andM.P. from theSpanishMinisterio de Ciencia e Innovación and Ministerio de Economia y Com-petitividad, respectively.

AUTHOR CONTRIBUTIONS

A.L. performed all the experiments, except for the expression analysis inFigure 5, which was done by A.M. A.L., M.P., and J.A.J. designed theresearch, analyzed data, and wrote the article.

Received June 15, 2015; revised August 12, 2015; accepted August 22,2015; published September 15, 2015.

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