abi5-binding protein2 coordinates constans …abi5-binding protein2 coordinates constans to delay...

ABI5-BINDING PROTEIN2 Coordinates CONSTANS toDelay Flowering by Recruiting the TranscriptionalCorepressor TPR21

Guanxiao Chang,a Wenjuan Yang,b Qili Zhang,b Jinling Huang,c,d Yongping Yang,a,2 andXiangyang Hua,b,2,3

aCAS Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany,Chinese Academy of Sciences, Kunming 650201, ChinabShanghai Key Laboratory of Bio-Energy Crops, Research Center for Natural Products, Plant Science Center,School of Life Sciences, Shanghai University, Shanghai 200444, ChinacKey Laboratory of Plant Stress Biology, State Key Laboratory of Cotton Biology, School of Life Sciences,Henan University, Kaifeng 475001, ChinadDepartment of Biology, East Carolina University, Greenville, North Carolina 27858

ORCID ID: 0000-0002-8009-4419 (X.H.).

ABI5-BINDING PROTEIN2 (AFP2) negatively regulates the abscisic acid signal by accelerating ABI5 degradation during seedgermination in Arabidopsis (Arabidopsis thaliana). The abscisic acid signal is reported to delay flowering by up-regulatingFlowering Locus C expression, but the role of AFP2 in regulating flowering time is unknown. Here, we found that floweringtime was markedly delayed and CONSTANS (CO) expression was reduced in a transgenic Arabidopsis line overexpressingAFP2 under LD conditions. Conversely, the loss-of-function afp2 mutant showed slightly earlier flowering, with higher COexpression. These data suggest that AFP2 negatively regulates photoperiod-dependent flowering time by modulating the COsignal. We then found that AFP2 exhibited circadian expression rhythms that peaked during the night. Furthermore, theC-terminus of AFP2 interacted with CO, while its N-terminal ethylene response factor–associated amphiphilic repressionmotif interacted with the transcriptional corepressor TOPLESS-related protein2 (TPR2). Thus, AFP2 bridges CO and TPR2 toform the CO-AFP2-TPR2 complex. Biochemical and genetic analyses showed that AFP2 mediated CO degradation during thenight. AFP2 also recruited histone deacetylase activity at Flowering Locus T chromatin through its interaction with TPR2. Takentogether, our results reveal an elaborate mechanism by which AFP2 modulates flowering time through coordinating the activityand stability of CO.

Flowering is a critical phase in the life cycle of plantsand heralds the transition from vegetative to reproduc-tive growth. Plants have evolved complex mechanismsto ensure that flowering occurs at an appropriate time inresponse to environmental cues (such as photoperiod)and endogenous signals (Andrés and Coupland, 2012;

Johansson and Staiger, 2015; Song et al., 2015). InArabidopsis (Arabidopsis thaliana), environmental pho-toperiod information is processed by the core factorCONSTANS (CO), which is transcribed at the plant apexor in the leaf vasculature (Putterill et al., 1995; Suárez-López et al., 2001).In leaves, CO activates the expression of florigen

Flowering Locus T (FT). Loss-of-function mutants ofFT are late flowering, whereas overexpression ofFT promotes flowering (Corbesier et al., 2007; Jaegerand Wigge, 2007; Mathieu et al., 2007). The expressionlevel of CO is controlled by the circadian clock andlight signaling pathway (Suárez-López et al., 2001;Valverde et al., 2004; Turck et al., 2008). The CO tran-scriptional level oscillates rhythmically, peaking in theafternoon in long days but at dusk in short days(Hayama and Coupland, 2003). The Cycling DNAbinding with one finger (DOF) Factor (CDF) transcrip-tion factors, including CDF1, CDF3, CDF4, and CDF5,synergistically bind to the DOF binding sites in the COpromoter to suppress FT expression in the morning,while the transcription of CDF is regulated by the cir-cadian genes CIRCADIAN CLOCK ASSOCIATED1 and

1This work was supported by start-up funding from ShanghaiUniversity, the National Natural Science Foundation of China (grantnos. 31470348 and 31570279), the Applied Basic Research Project ofYunnan Province (grant no. 2016FA015), and the 13th Five-Year In-formatization Plan of the Chinese Academy of Sciences (grant no.XXH13506).

2 Senior authors.3Author for contact: [email protected] 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 (www.plantphysiol.org) is:Xiangyang Hu ([email protected]).

X.H. and Y.Y. conceived and supervised this study; X.H. and G.C.provided resources; X.H. and J.H. designed the experiments; G.C., W.Y.,and Q.Z. performed the research; X.H. and J.H. wrote the original draft;and all authors reviewed and edited the manuscript.

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LATEELONGATED HYPOCOTYL in the morning andsuppressed by PSEUDO RESPONSE REGULATOR5(PRR5), PRR7, and PRR9 in the afternoon (Nakamichiet al., 2007; Fornara et al., 2009). The blue light receptorFlavin Binding, Kelch Repeat, F-box acts together withGIGANTEA to degrade CDF under long-day (LD)conditions (Sawa et al., 2007; Song et al., 2014). OnceCDF has been removed from theCO promoter, the basichelix-loop-helix transcriptional activators FBH1, FBH2,FBH3, and FBH4 recognize the E-box cis-elements inthe CO promoter to activate its transcription (Ito et al.,2012; Nagel et al., 2014).

In addition to regulation at the transcriptional level,CO protein stability is tightly controlled by an elaboratemechanism. CO is degraded by the ring finger E3 ligaseCONSTITUTIVE PHOTOMORPHORGENIC1 and itspartner SUPPRESSOR OF PHYA1 in the night (Janget al., 2008; Liu et al., 2008; Zuo et al., 2011), or by an-other E3 ligase, HIGH EXPRESSION OF OSMOTI-CALLY RESPONSIVE GENES1, in the morning (Lazaroet al., 2012; Joon Seo et al., 2013). Recently, Goralogiaet al. reported that CDF recruits the transcriptional co-repressor TOPLESS (TPL) to reduce CO expression andthat two small B-boxmicroProteins namedmicroProtein1a (miP1a) and miP1b form a bridge between CO andTPL, thereby suppressing the FT signal (Goralogia et al.,2017). These findings suggest that TPL has an essentialfunction in photoperiod-mediated flowering time. Nor-mally, TPL and its homolog TOPLESS-RELATED PRO-TEINS are associated with the histone deacetylasecomplex (HDAC) during plant development and immu-nity (Long et al., 2006; Wang et al., 2013a; Oh et al., 2014;Ryu et al., 2014), and CO is required for periodic modi-fication of the histone acetylation level at FT chromatinthroughHDACactivity (Gu et al., 2013).Nevertheless, themechanism by which CO recruits TPL/TPRs to epige-netically modulate FT expression remains largelyunknown.

The phytohormone signal also affects flowering time(Davis, 2009; de Wit et al., 2016); for example, mutationof the jasmonic acid signal receptor CORONATINEINSENSITIVE1 promotes flowering, while over-expression of COI-targeted JASMONATE-ZIM-DO-MAIN PROTEIN1 delays flowering (Zhai et al., 2015).Further investigation has shown that JASMONATE-ZIM-DOMAIN PROTEIN1 orchestrates the activity ofthe APETALA2 family members Target of Eat1 (TOE1)and TOE2, which repress the transcription of FT. TOE1and TOE2 also form a complex with CO in the morning(Zhang et al., 2015) or with FKF1 in the afternoon (Songet al., 2012; Zhang et al., 2015) to suppress CO activity.Gibberellic acid-mediated activation of the FT signal,which results in early flowering, depends on CO, andthe GA signal repressor DELLA interacts with CO tosuppress its activity (Wang et al., 2016; Xu et al., 2016).The bZIP transcription factor ABSCISIC ACID-IN-SENSITIVE MUTANT5, which plays an essential rolein ABA-inhibited seed germination, also binds to theABRE/G-box promoter elements of the Flowering LocusC (FLC) promoter to up-regulate FLC expression and

delay flowering (Wang et al., 2013b). Arabidopsis ABI5-BINDING PROTEIN1 (AFP1) was first reported as anegative regulator of ABA signaling that promotesABI5 degradation during seed germination. Over-expression of AFP1 enhances tolerance to ABA andreduces ABI5 accumulation, whereas afp1 mutants aresensitive to ABA and have high levels of ABI5 (Lopez-Molina et al., 2003). There are three AFP1 homologs (i.e.AFP2, AFP3, and AFP4) in the Arabidopsis genome,and their expression is differentially induced by envi-ronmental stress (Garcia et al., 2008). AFP2, similar toAFP1, functions epistatically to ABI5, and mutation ofAFP2 leads to increased sensitivity to ABA stress(Lopez-Molina et al., 2003). AFP2/3 also interact withthe corepressor TPL/TPRs to impair ABA-regulatedgene expression (Pauwels et al., 2010; Lynch et al.,2017). We have recently reported that AFP2 interactswith ABI5 to enhance seed germination under hightemperature stress (Chang et al., 2018), but otherphysiological functions mediated by AFPs and theirTPL/TPR partner remain to be investigated.

Although it has been established that ABI5 delaysflowering by up-regulating FLC (Wang et al., 2013b), itis unknown whether and how AFPs affect floweringtime. In this study, we sought to determine the potentialrole of AFP2 in controlling flowering time. We showedthat overexpression of AFP2 (AFP2-ox) substantiallydelayed flowering under LD conditions by inactivatingthe CO signal; conversely, the afp2 mutant exhibitedearly flowering at a higher CO level. Additional bio-chemical analysis demonstrated that AFP2 bridged COand TPR2 to form a complex, and AFP2 not onlyinteractedwith TPR2 to dampen FT expression throughHDAC activity at the transcriptional level but alsointeracted with CO to promote ubiquitin-mediatedproteolysis of CO at the post-transcriptional level.Thus, our findings unravel an elaborate mechanism bywhich AFP2 recruits the corepressor TPR2 to coordi-nate the CO signal and thereby ensure that floweringoccurs at an appropriate time.

RESULTS

AFP2 and AFP3 Modulate Flowering Time

To investigate the functions of AFP genes in regu-lating flowering time, we obtained several T-DNA in-sertion mutants from the Arabidopsis BiologicalResource Center, including afp1-1 (Salk-020158), afp1-2(Salk-005054), afp2-1 (Salk-131676), afp2-2 (Salk-145086),afp3-1 (Salk-037555), afp3-2 (Salk-052114), afp4-1 (GABI-019E07), and afp4-2 (Salk-208284). For each mutant, theT-DNA transposon was inserted into the exons of thecorresponding gene (Supplemental Fig. S1, A and B),and the functional transcript was nearly undetectableby RT-qPCR analysis (Supplemental Fig. S1C). We thengenerated several individual transgenic lines in whichthe full-lengthAFPswere fused to a Flag tag and drivenby the cauliflower mosaic virus 35S promoter (named

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AFP1-ox, AFP2-ox, AFP3-ox, and AFP4-ox, respectively;Supplemental Fig. S2). Immunoblot analysis using anti-Flag antibody revealed strong immunoblot bands inthese transgenic lines, but not in the Col wild type,confirming their overexpression (Supplemental Fig. S2).We then compared the flowering times of the differ-

ent lines under LD conditions (16 h light/8 h darkness).As shown in Figure 1, A and B, and SupplementalFigure S3, the afp4-1 and afp4-2 mutants and AFP4-oxtransgenic lines had similar flowering times to the wild

type, suggesting thatAFP4 does not affect the floweringtime (Supplemental Fig. S3). The flowering time of theafp1-1 and afp1-2mutants was not different from that ofthe wild type, whereas the afp2-1 (hereafter namedafp2), afp2-2, afp3-1, and afp3-2 mutants showed earlierflowering times than the wild type, with the afp1-1/afp2-1/afp3-1 triple mutant flowering even earlier(Supplemental Fig. S3), suggesting a functional redun-dancy of AFP2 and AFP3 in flowering time regulation.Correspondingly, AFP2-ox or AFP3-ox markedlydelayed flowering time, whereas overexpression ofAFP1 was only slightly later than that of wild-typeCol (Supplemental Fig. S3), suggesting that AFP2and AFP3 are key regulators of flowering time un-der LD conditions.We also examined their flowering times under short-

day (SD) conditions (8 h light/16 h darkness), and bothafp2 and AFP2-ox showed a similar late flowering timeto the wild-type Col line (Supplemental Fig. S4, A andB). Similarly, the flowering time was not significantlydifferent among the afp3-1, afp1-1/afp2/afp3-1 triplemutant, AFP3-ox transgenic, and Col lines under SDconditions (Supplemental Fig. S4B), indicating thatAFP2 and AFP3 mainly delay flowering time throughmodulating the photoperiodic pathway. We also mea-sured the transcriptional level of AFP2 during the dayand night in 2-week-old wild-type seedlings under LDconditions (16L/8D) and found that the transcriptionallevel of AFP2 presented a circadian rhythm, with agradual increase during the day and peak during thenight at zeitgeber time 20 (ZT20), followed by a rapiddecline to a very low level at the next dawn (ZT0;Fig. 1C).

AFP2 Interacts with TPR2

To investigate the role ofAFP2 in regulating floweringtime, we used AFP2 as the bait to screen an Arabidopsis-normalized cDNA library by a yeast two-hybrid (Y2H)assay. After two rounds of screening, we obtained sev-eral positive clones that interacted with AFP2 in yeast.After sequencing these clones, we identified a gene en-coding TPR2, a member of the TPL/TPR transcriptionalcorepressor family. To confirm their interaction, wefused full-length TPR2 into the prey vector (AD-TPR2)andAFP2 into the Gal4 DNA-binding domain of the baitvector (BD-AFP2). As shown in Supplemental Figure S5,A and B,we observed a strong interaction betweenAFP2and TPR2 in yeast. As the control, AFP2 did not interactwith the empty AD vector in yeast cells.Bioinformatics analysis using SMART software

(http://smart.embl-heidelberg.de/) showed that AFP2contained three functional motifs: EAR, NINJA, and JAS(Supplemental Fig. S5A). We then expressed a truncatedversion of AFP2 that lacked the EAR motif (AFP2ΔE),NINJA motif (AFP2ΔN), or JAS motif (AFP2ΔJ), respec-tively, and tested the interaction with TPR2 in yeast. Wefound that TPR2 interactedwithAFP2ΔNorAFP2ΔJ, butnot with AFP2ΔE (Supplemental Fig. S5, A and B),

Figure 1. AFP2 regulates flowering time under LD conditions. A,Flowering phenotype of the wild-type (Col), afp2mutant, and AFP2-oxlines. The photograph was taken 25 d after germination under LDconditions. Bar = 2 cm. B, Flowering time, as indicated by the totalrosette leaf number, under LD conditions. Data are means6 SD of threebiological replicates; for each line, 20 plants were scored. Bars withdifferent letters are significantly different at P , 0.05 (Tukey’s test). C,RT-qPCR analysis ofAFP2 transcriptional levels in thewild-type Col linegrown under LD conditions for 14 d. IPP2 was used as an internalcontrol. Data are means 6 SD of three biological replicates. White anddark bars above the x axis indicate light and dark periods, respectively.

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suggesting that the EAR motif is required for the inter-action between AFP2 and TPR2.

Next, we determined the cellular localization of AFP2and TPR2 by fusing AFP2 with a cyan fluorescenceprotein (AFP2-CFP) and by fusing TPR2 to a yellowfluorescence protein (TPR2-YFP). The fusionswere thentransiently expressed in Nicotiana benthamiana leaves.The strong CFP or YFP fluorescence was observed inthe nucleus (Fig. 2A). We then examined the interac-tion between AFP2 and TPR2 in planta via bimolecu-lar fluorescence complementation (BiFC). Full-lengthAFP2 and AFP2 lacking the EAR, NINJA, or JAS mo-tif (AFP2ΔE, AFP2ΔN, AFP2ΔJ) were fused to theN-terminal half of YFP (named AFP2-nYFP, AFP2ΔE-nYFP, AFP2ΔN-nYFP, and AFP2ΔJ-nYFP), while full-length TPR2 was fused to the C-terminal half of YFP(cYFP-TPR2). Coexpression of AFP2-nYFP, AFP2ΔN-nYFP, or AFP2ΔJ-nYFP with cYFP-TPR2 causedstrong YFP fluorescence, whereas coexpression ofAFP2ΔE-nYFP with cYFP-TPR2 did not. As a control,we coexpressed empty nYFP with cYFP-TPR2. As ex-pected, this did not yield any YFP fluorescence (Fig. 2B;Supplemental Fig. S5C), suggesting that AFP2, specifi-cally the EAR motif, interacts with TPR2 in planta.

In agreement with this finding, the co-immunoprecipitation (Co-IP) results showed that full-length AFP2, or the truncated AFP2 without the NINJAor JAS motif (AFP2ΔN or AFP2ΔJ) fused to a Flag tag,could be coimmunoprecipitated by anti-GFP resinwhen coexpressed with TPR2 fused to GFP (TPR2-GFP), while no Co-IP signal could be detected whenAFP2ΔE fused to Flag tagwas coexpressedwithTPR2-GFP

(Fig. 2, C andD). These data indicate that the EARmotif ofAFP2 interacts with TPR2 in living cells.

AFP2 Depends on TPR2 To Delay Flowering Time

Given that AFP2 interacts with TPR2 in vitro andin vivo and thatAFP2-ox delays flowering time, we nextevaluated the role of TPR2 in flowering time regulation.We identified two T-DNA insertion mutants, tpr2-1(Salk_112730) and tpr2-2 (Salk_079848), in which theT-DNA insertions were located in the 13th and 20thexons, respectively (Supplemental Fig. S6A). The cor-responding upstream or downstream primers for the13th (F1/R1 primers pair) exon and the 20th (F2/R2primers pair) exon could successfully amplify the cor-rect bands using wild-type Col DNA as template,whereas they failed to amplify the bands with mutantDNA templates. In contrast, the primer pair of LB/R1and LB/R2 successfully amplified the mutant bandfrom the Salk_112730 and Salk_079848 lines, respec-tively. LBwas a primer located on T-DNA (SupplementalFig. S6B). RT-qPCR analysis also showed that the tran-script of TPR2 was significantly reduced in these twomutants (Supplemental Fig. S6C), confirming that tpr2-1(hereafter named tpr2) and tpr2-2 were loss-of-functionmutants. The flowering time of tpr2 or tpr2-2 was sub-stantially earlier than that of the wild-type Col line underLD conditions (Fig. 3, A and B; Supplemental Fig. S6, Dand E).

To investigate the function of TPR2, we expressedTPR2 in Escherichia coli and used the purified TPR2 as

Figure 2. AFP2 interacts with TPR2 in vitro and in vivo. A, Colocalization ofAFP2 and TPR2 in the nucleus.AFP2-CFP and TPR2-YFP were transiently cotransformed into N. benthamiana leaf epidermal cells, and the localization of AFP2 and TPR2 wereobserved based on CFP and YFP fluorescence, respectively. From top to bottom, CFP fluorescence, YFP fluorescence, mergedimage of CFPand YFP. Bar = 10 mm. B, BiFC analysis of the interaction of AFP2 and TPR2 in planta. Full-length or truncated AFP2was fused to nYFP (AFP2-nYFP or AFP2ΔE-nYFP) and full-length TPR2 was fused to cYFP (cYFP-TPR2), and both constructs werecotransformed intoN. benthamiana epidermal cells and YFP fluorescence was monitored. Bar = 10 mm. C and D, Co-IPanalysisof the AFP2 and TPR2 interaction. Flag-tagged full-length AFP2 or truncated AFP2 andGFP-tagged TPR2were cotransformed intoN. benthamiana epidermal leaves. TPR2-GFP was immunoprecipitated with GFP-TRAP beads and detected with anti-Flag an-tibody. TPR2-GFP in the immunoprecipitates was detected using an anti-GFP antibody.


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antigen to prepare an antibody against TPR2. As shownin Supplemental Figure S7A, our TPR2 antibody spe-cifically recognized endogenous TPR2 in wild-type Col,but not in the tpr2mutant, demonstrating the specificityof our TPR2 antibody. We then crossed the earlyflowering tpr2mutant with the late flowering AFP2-oxline to obtain AFP2-ox/tpr2 and confirmed the genotypeof AFP2-ox/tpr2 by immunoblot analysis (SupplementalFig. S7B). In contrast to the late flowering phenotype ofAFP2-ox, knockout of TPR2 in the AFP2-ox/tpr2 linemarkedly promoted flowering (Fig. 3, A and B), sug-gesting that TPR2 is required for the late floweringphenotype of the AFP2-ox line.

AFP2 Represses the CO-Mediated EarlyFlowering Phenotype

Because the flowering time ofAFP2-oxwas late underLD conditions but could not be distinguished fromwild-typeCol under SD conditions (Supplemental Fig. S4,A and B), we hypothesized that the AFP2-regulatedflowering time was dependent on the photoperiodicpathway. We thus compared the transcriptional level ofCO and its downstream targets FT and SOC1 in the Colwild-type, afp2, and AFP2-ox lines under LD conditions.We observed the circadian expression of CO in Col underLD conditions (Fig. 3C). CO also presented similar circa-dian expression in the afp2 and AFP2-ox lines, but the COcircadian altitude was slightly higher in afp2 and strik-ingly lower in AFP2-ox (Fig. 3C). Consistent with thesefindings, FT and SOC1 expressionwere clearly reduced in

the AFP2-ox line but higher in afp2 than in the wild type.These data suggest that AFP2 represses the transcriptionof CO and its downstream targets FT and SOC1 to delayflowering under LD conditions. Because both tpr2 andAFP2-ox/tpr2 presented early flowering but AFP2-oxshowed a late flowering time, we also compared the FTexpression patterns among AFP2-ox, tpr2, AFP2-ox/tpr2,and wild-type Col as the control. The expression levels ofFT at ZT16 in tpr2 or AFP2-ox/tpr2 were significantlyhigher than in AFP2-ox and Col (Fig. 3B, bottom panel),suggesting that the inhibitory effect of AFP2 on FT ex-pression required TPR2.TPL interacts with CO andmicroproteinmiP1a/1b as

a trimer complex to regulate flowering time (Graeffet al., 2016), and our above result suggested thatAFP2 interacts with TPR2. It is possible that AFP2 in-teracts with CO to delay flowering time. To test thispossibility, we examined whether there was an inter-action between CO protein lacking activation domains(deleted N-terminal amino acids 1–175, termed “CO-CT”) and AFP2 using Y2H analysis. As shown inFigure 4A, we found that CO-CT interacted with thefull-length AFP2, as well as with the truncated AFP2lacking the EAR or NINJA domain, but not with thetruncated AFP2 lacking the JAS domain, suggestingthat the JAS domain in AFP2 is required for the inter-action between AFP2 and CO. Consistent with thisfinding, a Co-IP assay using transiently transformedtobacco leaves revealed the strong interaction betweenCO and full-length AFP2, or truncated AFP2 lackingthe EAR or NINJA motif, but not between CO andtruncated AFP2 lacking the JAS motif (Fig. 4B),

Figure 3. The tpr2 mutation reduces floweringtime in the later-flowering AFP2-ox background.A, The flowering phenotype of wild type (Col),tpr2, AFP2-ox, and AFP2-ox/tpr2. Bar = 2.5 cm.The photos were taken at 18 d after seeds germi-nation. B, The different flowering times and FTtranscriptional levels in Col, tpr2, AFP2-ox, andAFP2-ox/tpr2. The flowering times of these linesare indicated by the total rosette leaf number un-der LD conditions. The FT transcriptional levelwas measured by RT-qPCR analysis in 10-d-oldseedlings under LD conditions. Data are themeans6 SD of three biological replicates. For eachline, 20 plants per line were observed. Bars withdifferent letters are significantly different at P ,0.05 (Tukey’s test). C, RT-qPCR analysis of CO, FT,and SOC1 expression in 10-d-old wild-type, afp2,and AFP2-ox lines under LD conditions, at a 4-hresolution. Actin was used as an internal control.Data are means6 SE of three biological replicates.Bars with different letters are significantly differentat P , 0.05 (two-way ANOVA with Tukey’s test).










demonstrating that the JAS motif is required for theinteraction between AFP2 and CO in vitro and in vivo.

ABI5 was previously reported as the target protein ofAFP2 during seed germination (Lopez-Molina et al.,2003), and overexpression of ABI5 activates the floweringnegative factor FLC at the transcriptional level to repressflowering (Wang et al., 2013b). Thus, we compared thetranscription of FLC in Col, afp2, and AFP2-ox under LDconditions. We did not observe any obvious differencesamong the FLC transcript levels in these lines(Supplemental Fig. S8); this suggests that AFP2-mediated late flowering occurs independently of FLC.

Furthermore, wemeasured the effects of AFP2 on COactivity and flowering time by genetic analysis. The

SUC2::CO-HA line (expressing CO fused to an HA tagand driven by the SUC2 promoter, hereafter termedCO-HA) showed earlier flowering than the wild-typeCol line. We crossed the CO-HA line with AFP2-ox togenerate the CO-HA/AFP2-ox line and confirmed theselines by immunoblot analysis using anti-HA and anti-GFP antibodies (Supplemental Fig. S9). In comparisonto the early floweringCO-HA line, the flowering time ofthe CO-HA/AFP2-ox lines was markedly later, similarto that of AFP2-ox (Fig. 4, C and D). In agreement withthis finding, the FT transcriptional level in CO-HA/AFP2-ox was also lower than that of CO-HA(Supplemental Fig. S10A). The co mutant showing alater flowering time was crossed with the earlier

Figure 4. AFP2 influences the ability of CO to activate FT expression. A, AFP2 interacts with CO-CT in yeast. Full-length ortruncated AFP2was fused toGAL4-AD, and CO-CT, in which the N-terminal 1-175 amino acid of CO was deleted, was fused toGAL4-BD, and both constructs were transformed into yeast cells. The interaction of AFP2 or truncated AFP2 with CO-CT wasassessed on selectivemedia lacking Leu, Trp, His, or Ade. Gal4-BDwas used as negative controls. Photographswere obtained 3 dafter incubation at 28°C. B, Co-IP analysis of the interaction between AFP2 and CO. Flag-fused AFP2 and GFP-fused CO weretransiently cotransformed intoN. benthamiana leaves, and CO-GFP was immunoprecipitated with GFP-TRAPand detected withanti-Flag antibody. The protein input was detected with anti-GFPand anti-Flag, respectively. C, The flowering phenotype of Col,AFP2-ox,CO-HA, andAFP2-ox/CO-HA. Bar = 3 cm. The photoswere taken at 18 d after seeds germination. D, Flowering times ofCol, AFP2-ox, CO-HA, and AFP2-ox/CO-HA based on the rosette leaf number under LD conditions. Data are the means6 SD ofthree biological replicates. For each line, 20 plants were observed. Bars with different letters are significantly different at P, 0.05(Tukey’s test). E and F, Circadian accumulation of CO-HA in the CO-HA and AFP2-ox/CO-HA lines. The CO-HA and AFP2-ox/CO-HA lines were grown under LD conditions for 2 weeks, and samples were collected every 4 h from ZT0. Total proteins wereextracted to detect CO-HA accumulation using anti-HA antibody. Antitubulin was used as the loading control. The intensity ratioof the CO-HA signal to that of tubulin is shown in (F). Data are themeans6 SD of three biological replicates. G, Flowering times ofCO-HA,CO-HA/AFP2-ox, cop1, andCO-HA/AFP2-ox/cop1 indicated by the total rosette leaf number under LD conditions. Dataare the means 6 SD of three biological replicates. For each line, 20 plants were observed. Bars with different letters are signifi-cantly different at P , 0.05 (Tukey’s test).


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flowering afp2mutant to obtain the afp2/co line. Similarto the later flowering co mutant, the afp2/co line pre-sented a late flowering phenotype (Supplemental Fig.S10B). These data suggest that CO is epistatic to AFP2,and AFP2 represses the early flowering phenotype ofCO-HA by negatively regulating the CO signal.AFP2 has been reported to accelerate ABI5 degra-

dation (Lopez-Molina et al., 2003), and AFP2-ox sup-pressed the activating activity of CO in CO-HA/AFP2-ox lines, suggesting that AFP2 not only sup-presses the transcription of CO (Fig. 3C), but also pos-sibly affects the stability of the CO protein itself. To testthis possibility, we compared CO protein accumulationdynamics between the CO-HA and CO-HA/AFP2-oxlines during the day and night under LD conditions.As shown in Figure 4, E and F, the protein accumulationlevel of CO in both CO-HA and CO-HA/AFP2-ox os-cillated with the same rhythm, peaking at ZT16, but theCO protein content during the evening and night, es-pecially at ZT16 and ZT20, was higher in the transgenicCO-HA line than in the crossed CO-HA/AFP2-ox line,supporting the notion that AFP2 promotes the degra-dation of CO during the night.We then found that AFP2-induced CO degradation

was partially inhibited by pretreatment with the pro-teasome inhibitor MG132 (Supplemental Fig. S11),suggesting that COdegradation potentially depends onthe ubiquitin proteasome system pathway. A previousstudy has shown that COP1 interacts with AFP2 in thenucleus (Lopez-Molina et al., 2003), and COP1 as the E3ligase mediates the degradation of CO during the night(Jang et al., 2008). It is possible that AFP2-promoteddegradation of CO requires COP1, and therefore wecrossed the cop1-4 (termed cop1 hereafter) mutant withCO-HA/AFP2-ox to generate the CO-HA/AFP2-ox/cop1 line. Similar to the earlier flowering CO-HA, theflowering time of the CO-HA/AFP2-ox/cop1 line wasearlier than that of CO-HA/AFP2-ox under LD condi-tions (Fig. 4E). Consistent with this finding, the COprotein content at ZT16 and ZT20 was higher inCO-HA/AFP2-ox/cop1 than in CO-HA/AFP2-ox(Supplemental Fig. S12). These findings indicate thatAFP2-dependent CO degradation and the late floweringphenotype require COP1.

AFP2 Recruits TPR2 To Suppress CO Expression andDelay Flowering

Microproteins miP1a/1b bridge TPL and CO to formthe complex (Graeff et al., 2016). Because AFP2 interactswith CO or TPR2, it is possible that AFP2 bridges TPR2and CO to form the CO-AFP2-TPR2 complex. To testthis possibility, we conducted a yeast three-hybrid(Y3H) analysis to examine the interactions among CO,AFP2, and TPR2. Cotransformation of empty pDRplasmid with AD-CO and BD-TPL did not enablegrowth on selection medium, but in the presence of theAFP2 plasmid, the cotransformed yeast grew well onselective medium (Fig. 5A). Yeast cotransformed with

truncated AFP2 lacking either EAR or JAS did not growwell, supporting the idea that full-length AFP2 is re-quired to reinforce the interaction between CO andTPR2 (Fig. 5A).The presence of the CO-AFP2-TPR2 complex was

further supported by an in vitro pull-down assay. Asshown in Figure 5B, MBP-CO and GST-TPR2 did notcoprecipitate when incubated in the absence of HIS-AFP2, but they did coprecipitate in the presence of HIS-AFP2, suggesting formation of the CO-AFP2-TPR2complex. We also tested whether CO and TPR2 couldinteract with each other in the presence of truncatedAFP2 and found that deletion of the EAR or JAS do-main blocked the interaction between CO and TPR2(Fig. 5B). These findings suggest that these two do-mains are required for the formation of the CO-AFP2-TPR2 complex in vitro.To test whether the CO-AFP2-TPR2 complex ex-

ists in vivo, we first generated the AFP2-ox/afp2(overexpressing full-length AFP2-ox in the afp2 mu-tant background), AFP2ΔE-ox/afp2 (overexpressingAFP2ΔEN-ox in the afp2 mutant background), andAFP2ΔJ-ox/afp2 (overexpressing AFP2ΔJ-ox in theafp2 mutant background) lines, and then crossedthem to obtain AFP2-ox/CO-HA/afp2, AFP2ΔE-ox/CO-HA/afp2, AFP2ΔN-ox/CO-HA/afp2, and AFP2ΔJ-ox/CO-HA/afp2. Using the TPR2 antibody, we foundthat CO-HA coprecipitated with TPR2 in the crossedAFP2-ox/CO-HA/afp2 line, but not in the crossedAFP2ΔE-ox/CO-HA/afp2 or AFP2ΔE-ox/CO-HA/afp2line (Fig. 5C). These data suggest that full-length AFP2reinforces the interaction between CO and TPR2 in vivothrough the EAR and JAS domains.We further evaluated the function of the CO-AFP2-

TPR2 complex in modulating flowering time. Both afp2and CO-HA/afp2 flowered early, while AFP2-ox/afp2 flowered late. The AFP2-ox/afp2 and CO-HA/AFP2-ox/afp2 lines showed later flowering than the afp2mutant (Fig. 5, D and E), suggesting that AFP2 over-expression suppresses the early flowering of CO-HA.However,CO-HA/AFP2ΔE-ox/afp2 orCO-HA/AFP2ΔJ-ox/afp2 flowered early, similar to CO-HA/afp2, butCO-HA/AFP2ΔN-ox/afp2 showed a similar floweringtime to CO-HA/AFP2-ox/afp2 (Fig. 5, D and E), sug-gesting that the EAR and JAS domains are required forAFP2-mediated inhibition of CO activity. In agreementwith this notion, FT transcript levels were higher inCO-HA/afp2, CO-HA/AFP2ΔE/afp2, and CO-HA/AFP2ΔJ/afp2 and lower in CO-HA/AFP2-ox/afp2 andCO-HA/AFP2ΔN/afp2 (Fig. 5F).

AFP2 and TPR2 Suppress CO Transcriptional Activity ByReducing the Chromatin Acetylation Level at the FT Locus

Having shown that AFP2 reinforces the interactionbetween CO and TPR2, we next investigated the effectsof AFP2 and TPR2 on the transcriptional activity of COusing an Arabidopsis mesophyll protoplast analysis.Transient transformation of CO-HA/afp2 protoplasts









with the FT::LUC reporter construct resulted in highbioluminescence emission with a high Luc/Ren ratio,because CO-HA activates FT transcription (Fig. 6,A and B). The CO-HA-induced increase in the Luc/Renratio could be limited by cotransformation with full-length AFP2-ox or AFP2ΔN-ox, but not by AFP2ΔE-oxor AFP2ΔJ-ox, suggesting that AFP2, mainly the EARand JAS domains, suppresses the transcriptional acti-vation effect of CO on FT expression. Cotransformationof AFP2-ox or AFP2ΔN-ox with TPR2-ox further sup-pressed the bioluminescence and Luc/Ren ratio, butcotransformation of AFP2ΔE-ox or AFP2ΔJ-ox with

TPR2-ox did not suppress the CO-induced increase ofbioluminescence, suggesting that functional AFP2 isrequired for the corepressive activity of TPR2 on thetranscriptional activation activity of CO on FT.

Members of the TPL/TPR family recruit HDAC tosuppress target gene transcription through deacetyla-tion (Long et al., 2006; Zhu et al., 2010; Oh et al., 2014;Ryu et al., 2014). Our above results showed that TPR2suppressed CO-dependent transcription, possibly byrecruiting HDAC to FT chromatin and reducing histoneH3 acetylation at this region. Thus, we examined thehistone acetylation level at the promoter region of FT

Figure 5. AFP2 reinforces CO and TPR2 to coordinate flowering time. A, Y3H analysis to detect the formation of the CO-AFP2-TPR2 complex. Yeast cotransformed with these three constructs could grow well on the nonselective medium lacking Leu, Trp,and uracil (2L/2W/2U), but only yeast harboring constructs that had positive interactionswere able to growon restrictive growthmedium supplementedwith 10mM 3-aminotriazole plus 2% (w/v) Gal and lackingHis/Leu/Trp/Ura (2H/2L/2W/2U). B, In vitropull-down analysis of the interactions among CO, AFP2, and TPR2. Recombinant GST-TPR2 and MBP-CO proteins were pro-duced in E. coli. After cell lysis, cell extracts of GST-TPR2 and MBP-COwere mixed with HIS-AFP2, HIS-AFPΔE, or HIS-AFP2ΔJ,respectively, and then incubated with magnetic anti-His-coupled magnetic beads. His-tagged full-length or truncated AFP2 wasprecipitated andwashed using a magnetic stand, eluted by boiling in SDS loading buffer, and separated by SDS-PAGE. GST-TPR2and MBP-CO were detected by immunoblotting. C, Co-IP analysis of the CO-AFP2-TPR2 complex in vivo. The CO-HA/afp2transgenic line was crossed with AFP-ox/afp2 to obtain CO-HA/AFP2-ox/afp2, which was subjected to Co-IP analysis. Totalproteins were extracted from CO-HA/AFP2-ox/afp2 and immunoprecipitated with anti-Flag agarose beads, and the immuno-precipitated proteins were detected with anti-TPR2 antibody. D, Flowering phenotype of the afp2 mutant and the indicatedtransgenic lines in the afp2mutant background. The photos were taken at 18 d after seeds germination. Bar = 3 cm. E, Floweringtimes based on the total rosette leaf number under LD conditions. Data are means 6 SD of three biological replicates. For eachline, 20 plants were observed. Bars with different letters are significantly different at P, 0.05 (Tukey’s test). F, RT-qPCR analysis ofCO transcript levels in the afp2mutant and different transgenic lines in the afp2 background. IPP2was used as an internal control.Data are means6 SD of three biological replicates. Bars with different letters are significantly different at P, 0.05 (Tukey’s test).3AT, 3-aminotriazole.


Chang et al.



chromatin. The histone H3 acetylation level in theproximal region of the FT promoter in the CO-HA/AFP2-ox and CO-HA/AFP2ΔN-ox lines was lower thanthat in the control wild-type Col line, as well as inthe CO-HA/AFP2ΔE-ox and CO-HA/AFP2ΔJ-ox lines,in agreement with the lower FT transcriptional leveland late flowering time of the CO-HA/AFP2-ox andCO-HA/AFP2ΔN-ox lines (Fig. 6C). The histone H3acetylation level in CO-HA/AFP2-ox/tpr2 was higherthan that in CO-HA/AFP2-ox, suggesting that theHDAC activity-mediated low H3 acetylation level inCO-HA/AFP2-ox requires TPR2. Thus, these data in-dicate that the AFP2-CO-TPR2 complex suppressesflowering time through HDAC-mediated reduction ofthe H3 acetylation level at the FT promoter.

DISCUSSION

AFP2 Delays Flowering Time via CO

In this study, we report that AFP2 is a regulator offlowering time. By comparing the flowering timesamong various afp mutants and AFP overexpressionlines, we determined that AFP2 and AFP3mainly affectflowering time, as the flowering time of afp1 was onlyslightly earlier, but knocking out or overexpressing

AFP4 did not markedly affect flowering time (Fig. 1;Supplemental Figs. S3 and S4). These phenotypes sug-gest that the different AFP isoforms have evolved di-verse functions to differentially regulate Arabidopsisgrowth and development, or the response to environ-mental stress. For example, only AFP1 and AFP2 weresubstantially induced by salt and ABA stress (Garciaet al., 2008). A previous study has shown that AFP1promotes the degradation of ABI5 during seed germi-nation and that ABI5 binds to the FLC promoterto up-regulate FLC expression and thereby delayflowering (Wang et al., 2013a, 2013b). However, herewe found that FLC transcript levels in AFP2-ox weresimilar to those in the wild type (Supplemental Fig. S8),which excludes the possibility thatAFP2 delaysfloweringthrough an ABI5-dependent FLC signal.Furthermore, the delaying effect on flowering time in

the AFP2-ox line was obvious under LD but not SDconditions, suggesting that the later flowering pheno-type of the AFP2-ox lines was dependent on the pho-toperiod time. In support of this proposal, our Y2H,in vitro pull-down, and in vivo Co-IP experimentsconfirmed the interaction between CO and AFP2(Supplemental Fig. S5); AFP2-ox also reduced the ex-pression level of CO and of its downstream targets FTand SOC1, while the afp2 mutant showed slightlyhigher transcription of CO, FT, and SOC1 (Fig. 3C).

Figure 6. AFP2 and TPR2 repress the transcriptional activation activity of CO on FT. A, Schematic of the reporter and effectorsused in the transient protoplast transformation assay. B, Transient dual-luciferase reporter analysis of the inhibitory effect of AFP2and TPR2 on CO-induced FT expression. Error bars indicate SD from three biological replicates. C, ChIP-qPCR analysis of thehistone H3 acetylation levels in the indicated region (denoted as FT-P, FT-E, and FT-L locus) of FT chromatin in Col, CO-HA,AFP2-ox, and the various crossed lines. An antiacetyl-histone H3 antibody was used for immunoprecipitation. TUB2was used asan internal control, and the relative histoneH3 acetylation levels of FTwere normalized to those of TUB2. The detailed position ofFT-P, FT-E, and FT-L are indicated by bars below the FT gene. Exons are shown as solid boxes and introns as solid lines. Data aremeans6 SD of triplicate experiments. For (B) and (C), bars with different letters are significantly different at P, 0.05 (Tukey’s test).








These results support the notion that AFP2 interactswith CO to repress CO activity and the expression of FTand SOC1, ultimately delaying flowering time.

Arabidopsis AFP1 has been reported to colocalizewith the E3 ligase COP1 in the nucleus (Lopez-Molinaet al., 2003), and COP1 induces proteasome-mediatedCO degradation in the dark (Jang et al., 2008).OsbZIP46is the rice (Oryza sativa) homolog of Arabidopsis ABI5,while OsMODD is the rice homolog of ArabidopsisAFP2. The transcriptional activity of OsbZIP46 is sup-pressed byOsMODD by recruiting HADC toOsbZIP46chromatin to reduce its acetylation level. The proteinstability of OsbZIP46 is also regulated by OsMODDthrough recruitment of the U-box-type ubiquitin E3 li-gase OsPUB7 (Tang et al., 2016). Consistent with thisfinding, we detected a lower COprotein level in theCO-HA/AFP2-ox line than in the CO-HA line during thenight (Fig. 4E), and the flowering time of the CO-HA/AFP2-ox line was later than that of the CO-HA line(Fig. 4, C and D). It is possible that AFP2 recruits the E3ligase COP1 and thereby destabilizes CO to delayflowering. The progeny of CO-HA/AFP2-ox/cop1-4flowered earlier than CO-HA/AFP2-ox (Fig. 4E),and the CO protein content at ZT20 was higher inCO-HA/AFP2-ox/cop1-4 than in CO-HA/AFP2-ox(Supplemental Fig. S12), supporting the notion thatCOP1 is required for AFP2-mediated CO proteinstability during the night. Thus, the detailed mech-anism by which AFP2 combines with COP1 to trig-ger CO degradation or CO stabilization meritsfurther investigation.

AFP2 Recruits TPR2 To Suppress CO Activity, Resulting inDelayed Flowering

The EAR motif characterized by the consensus se-quence of LxLxL or DLNxxP is a principal transcrip-tional repression motif in plants (Causier et al., 2012a,2012b). Several studies have demonstrated that EARmotif-containing proteins interact with the corepressorTPL/TPR, which in turn recruits histone deacetylases(HDAC), including HDA6 or HDA19, to promotechromatin compaction and transcriptional inactivation(Long et al., 2006; Zhu et al., 2010; Tao et al., 2013; Ohet al., 2014; Ryu et al., 2014). The interactions of AFP2and AFP3 with TPL was reported previously. Y2H

analysis showed that the EAR motif of AFP2 and AFP3is required for the observed interaction between AFPsand TPL (Pauwels et al., 2010; Causier et al., 2012a).AFP2 also interacts with all four TPR proteins, butAFP3 interacts with all except TPR4. However, unlikeAFP2 and AFP3, AFP1 only shows weak interactionwith TPL or TPR1/2/3 (Lynch et al., 2017). In agree-ment with these earlier findings, we found that AFP2interacts with TPR2 in yeast and in planta, mainlythrough the EAR motif (Supplemental Fig. S5, A and B;Fig. 2, C and D).

Furthermore, we found that the late flowering phe-notype of AFP2-ox could be reversed by theTPR2 mutation (Fig. 3, A and B). Transient protoplasttransformation experiments showed that AFP2-oxinhibited CO-mediated FT transcription, while over-expression of TPR2 enhanced the inhibitory effect ofAFP2 on FT transcription (Fig. 6, A and B). The acet-ylation level at the FT locus of CO-HA/AFP2-ox waslower than in CO-HA (Fig. 6C), in agreement with thelower FT transcript levels in CO-HA/AFP2-ox(Fig. 6B). Thus, AFP2 may recruit the TPR2-HADCcomplex to increase deacetylation levels at the FT lo-cus and thereby delay flowering. This result supportsthe critical role of TPR2 in controlling histone acety-lation levels at FT chromatin through the AFP2-COcomplex. Gu et al. reported that SAP30 FUNCTION-RELATED1 (AFR1) and AFR2 are the components ofHDAC complexes that modulate the periodic histoneacetylation level of FT chromatin (Gu et al., 2013). Itremains to be determined whether TPR2 recruitsAFR1/2 to delay flowering in the AFP2-ox line.

AFP2 Reinforces CO and TPR2 To Form a Complex thatRepresses the FT Signal

Although TPL suppresses CO activity, TPL does notinteract directly with CO. Previous studies have shownthat a microprotein called miP1a/b can bridge CO andTPL and recruit TPL, which suppresses CO activity(Graeff et al., 2016). In this study, AFP2 was found tointeract with CO through the C terminus JAS motif(Fig. 4, A and B), as well as with TPR2 through the EARmotifs (Fig. 2; Supplemental Fig. S5); however, we didnot detect a direct interaction between CO and TPR2(Fig. 5A). Similar to miP1a/b, Y3H analysis showed

Figure 7. Proposed model to illustrate the role ofAFP2 in regulating the circadian transcription ofFT. In this model, AFP2 restrains CO-induced FTtranscription by degrading CO and recruits TPR2to remove histone acetylation at the FT chromatin,leading to lower FT transcript levels during theevening hours.


Chang et al.






that AFP2 mediated the interaction between CO andTPR2. Furthermore, pull-down and Co-IP experimentsshowed that both the EAR and JAS motifs were re-quired for the formation of the CO-AFP2-TPR2 com-plex (Fig. 5, B and C). Consistently, the flowering timeof the CO-HA/AFP2-ox/afp2 or CO-HA/AFP2ΔN-ox/afp2 line was later than that of the CO-HA/afp2, CO-HA/AFP2ΔE-ox/afp2, or CO-HA/AFP2ΔJ-ox/afp2 line(Fig. 5, D and E), but the flowering time of theAFP2-ox/tpr2 line was earlier than that of the AFP2-ox line(Fig. 3A).CO activity has been reported to be required for

AFR2 recruitment to FT chromatin, thereby dampeningFT expression by reducing histone acetylation levels atthe FT locus (Gu et al., 2013). Here, we found that theCO-AFP2-TPR2 complex recruited HDAC activity toreduce the histone acetylation level at the FT promoter,which led to lower FT expression and a delayedflowering time in the CO-HA/AFP2-ox line (Fig. 5F).Conversely, histone acetylation was restored to nor-mal levels in the CO-HA/AFP2-ox/tpr2 line (Fig. 6C),confirming that AFP2 delays flowering through TPR2-recruited HDAC activity and that AFP2 mediates theindirect interaction between CO and TPR2 and sub-sequently suppresses CO-dependent activation of FTexpression.Consistent with our findings, a previous study has

shown that HDAC activity participates in the AFP2-mediated inhibition of ABA-responsive gene expres-sion and ABA-induced seed dormancy (Lopez-Molinaet al., 2003; Wang et al., 2013a; Tang et al., 2016; Lynchet al., 2017). AFP2 may not be the sole componentlinking CO and TPL/TPR. For example, TPL interactswith TOE1/TOE2, overexpression of TOE1/TOE2 re-duces FT expression and delays flowering, and TPL/TPR is required for TOE1/2-mediated repression of FTexpression (Causier et al., 2012a). TOE1/TOE2 also in-teract with CO to induce its degradation at the post-translational level (Zhang et al., 2015); thus, similar toAFP2, it is possible that TOE1/2 bridges the interactionbetween CO and TPL to repress FT expression. Furtherstudies should investigate how AFP2 and TOE1/2 co-ordinate to recruit TPL/TPR and tightly regulate the FTsignal and flowering time. Similarly, although CO ac-tivity is required for AGL18- or AFR1/2-mediated pe-riodic histone deacetylation at FT chromatin (Gu et al.,2013), CO did not interact directly with AGL18 andAFR1/2. Thus, whether or howAFP2 integrates CO andAFR1/2 for FT expression requires further investigation.In conclusion, our findings demonstrate that AFP2

controls flowering time by recruiting the corepressorTPR2 to silence CO activity and the downstream FTsignal. Based on our data, we propose a workingmodelto illustrate the regulatory mechanism by whichAFP2 fine-tunes CO activity and flowering time (Fig. 7).In the early morning, there is insufficient CO to effi-ciently activate FT transcription. CO protein levelsgradually accumulate and peak in the afternoon, atwhich point CO efficiently activates FT transcription.Some other protein, such as FKF, also stabilizes CO

during this period.However, upon arrival of the eveningor night, more AFP2 accumulates and interacts with CO.On the one hand, AFP2 induces protein degradation ofCO, possibly coordinating with COP1; on the other,AFP2 recruits TPR2 andHDAC to reduce the acetylationlevel at the promoter of the FT locus, subsequentlysuppressing FT transcription during the evening ornight. Therefore, the CO-AFP2-TPR module is a majorregulator that fine-tunes photoperiodic flowering timeby temporally modulating histone deacetylation at aregion of chromatin underlying FT expression.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

All the Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutants, in-cluding afp1-1 (Salk-020158), afp1-2 (Salk-005054), afp2-1 (Salk-131676), afp2-2(Salk-145086), afp3-1 (Salk-037555), afp3-2 (Salk-052114), afp4-1(GK-019E07),afp4-2 (Salk-208284), TPR2 (Salk_112730), and TPR2-2 (Salk_079848), wereobtained from the Arabidopsis Biological Resource Center. The SUC2::CO-HAseeds were kindly provided by Dr. Brigitte Koop and Prof. George Coupland(Max Planck Institute for Plant Breeding Research) and by Prof. Takato Imaizumi(University of WA, Seattle). The plants were grown under a white lightphotoperiod at 22°C under LD (16-h light [100 mmol22 s21]/8-h darkness) orSD (8-h light [100 mmol22 s21]/16-h darkness) conditions. Flowering timewas determined by counting the number of rosette leaves at the time whenthe inflorescence had grown to a height of 3–5 cm.

Generation of Transgenic Plants

To generate the transgenic lines overexpressing full-length or truncatedAFP2, the full-length CDS sequence or truncated versions of AFP2 lacking theEAR, NINJA, or JAS domains were amplified using the primers listed inSupplemental Table S1. The amplified fragments were cloned into the SalI/BamHI (New England Biolabs) sites of the pRI101-6Flag vector [6xFlag taginserted in the EcoRI/SacI site of the pRI101-AN vector (3262; Takara)] using theIn-fusion HDCloning Kit (638911, Clontech), upstream of the 6xFlag and drivenby the 35S constitutive promoter. The constructs were named pRI101-AFP2-6Flag, pRI101-AFP2ΔE-6Flag, pRI101ΔN-AFP2-6Flag, and pRI101-AFP2ΔJ-6Flag,respectively. Similarly, the full-length sequences ofAFP1,AFP3, andAFP4wereamplified and cloned into pRI101-6Flag to obtain pRI101-AFP1-6Flag, pRI101-AFP3-6Flag, and pRI101-AFP4-6Flag, and full-length TPR2 was cloned into theSalI/BamHI sites of the pRI101-GFP vector (GFP tag inserted in the NdeI/SalIsite of the pRI101-AN vector [3262; Takara]) to obtain the pRI101-GFP-TPR2constructs. These constructs were transformed into Arabidopsis plants usingthe Agrobacterium tumefaciens-mediated floral dip method (Clough and Bent,1998).

Y2H and Y3H Assays

To generate full-length AFP2 and TPR2, or truncated TPR2, the primerslisted in Supplemental Table S1 were used to amplify the coding sequencesfrom Arabidopsis cDNA template. These PCR products were then cloned intothe prey vector pGADT7 (630442, Clontech) and bait vector pGBKT7 (630443,Clontech) using In-fusion Cloning Technology (Clontech). Yeast strains Y187andAH109were transformedwith the prey and bait vectors, respectively, usingpolyethylene glycol-mediated yeast transformation as described in Hu et al.(2014). After screening on 2Trp medium (630413, Clontech) or 2Leu me-dium (630414, Clontech), three independent clones were mated and grown on2Trp/2Leumedium (630417, Clontech) for 3 d to confirmmating success, andthe corresponding clones were transferred to 2TPR/2Leu/2His/2Ade me-dium (630428, Clontech) to measure their growth status.

For the Y3H assay, the TPR2 coding sequence was recombined into apGBKT7 vector (pGBKT7-TPR2) and CO into the pGADT7 vector (pGADT7-CO).The full-length or truncatedAFP2were cloned into the pYES2 vector (Invitrogen).Yeast strains Y187 and AH109 were cotransformed with pGBKT7-TPR2 and theempty pYES2 or pYES2 with full-length (or truncated) AFP2 as described above,







and positive clones were screened on 2Trp/2Ura medium (630427, Clontech).pGADT7-COwas transformed into the yeast strain AH109 and selected on2Leumedium. The selected yeast strains Y187 and AH109 were then mated for 2 d at28°C and then selected on 2Trp/2Leu/2Ura medium (630426, Clontech). Theinteraction of the TPR2-AFP2-CO complex was determined by screening themated yeast cell on2Trp/2Leu/2Ura/2His selectivemedium (630425, Clontech)supplemented with 2% (w/v) Gal and 10 mM 3-aminotriazole.

BiFC Analysis

BiFC experiments were performed using 3-week-old Nicotiana benthamianaplants according to a method reported in both Lee et al. (2008) and Hu et al.(2014). The full-lengthAFP2 and TPR2 sequences and truncated AFP2 sequencewere recombined into the binary BIFC vector pSPYNE or pSPYCE, respectively(Walter et al., 2004), so that AFP2was fused to the N-terminal vector and TPR2was fused to the C-terminal vector. The constructs were transformed into A.tumefaciens–competent LBA4404 (9115, Clontech), and the cultures were incu-bated in a rotator at 300 rpm for 6 h at 28°C. After culture, Agrobacteria har-boring nYFP or cYFPwere mixed together and centrifuged at 300 rpm for 5 minat 4°C, and the pellets were dissolved in injection solution (10 mM Tri-HClbuffer, 25 mM MgCl2, pH 5.6) to an OD600 of 0.1. Then, the Agrobacteria solu-tion was injected into N. benthamiana leaves, and YFP fluorescence was ob-served 3 d after injection using a confocal microscope (LSM710; Zeiss).

In Vitro Pull-Down Analysis

In vitro pull-down analysis was carried out as described in Hu et al. (2014).In brief, full-length AFP2 or the truncated AFP2 coding sequence was clonedinto the EcoRI/XhoI sites of pET28a to generate theHIS-AFP2,HIS-AFPΔE,HIS-AFP2ΔN, and HIS-AFP2ΔJ constructs. The full-length coding region of CO wascloned into the EcoR1/XhoI site of pMAL-C2 to generateMBP-CO, and the full-length coding region of TPR2 was cloned into pGEX-4T-1 to generateGST-TPR2. All the constructs were manipulated using the In-Fusion Cloningtechnique (Clontech), and primer information is provided in SupplementalTable S1. The Escherichia coli BL21 (DE3) strain harboring an expression con-struct was incubated at 37°C for 2 h, shifted to 22°C, and then incubated for anadditional 12 h after induction with 1 mM isopropyl b-D-1-thio-galactopyr-anoside (V900917; Sigma-Aldrich). HIS-, GST-, or MBP-tagged recombinantproteins were purified using glutathione- or MBP-Sepharose according to themanufacturers’ protocols (GE Healthcare for GST-tagged protein, New Eng-land Biolabs for MBP-tagged protein).

For the in vitro pull-down assays, 100–300 mg of MBP-CO and GST-TPR2proteins were incubated with 100 mg of HIS-AFP2 (or its truncated protein) and2 mg of glutathione-Sepharose 4B beads (50 mL) in PBS buffer (13 PBS, 13protease inhibitor cocktail; Roche) with 0.1% (v/v) NP-40 for 2 h at 4°C. Thepulled-down proteins were extensively washed with buffer (50 mM Tris-HCl,pH 7.4, 100 mM NaCl, and 0.6% [v/v] Triton X-100) before the samples wereresolved on 8% (w/v) SDS-PAGE gels and analyzed by protein gel blot analysisusing anti-MBP (1:5000, New England Biolabs), anti-HIS (1:300, Qiagen), andanti-GST (1:3000, Invitrogen), followed by amouse secondary antibody (1:5000,Promega) and the ECL system (Invitrogen).

Antibody Production and Immunoblot Analysis

The TPR2 antibody was produced commercially (YouKe Biotech). A regionencoding a polypeptide from amino acids 152–353 of TPR2 was subcloned intothe prokaryotic expression vector pET28a. The polypeptide was expressed,purified, and used as antigen to raise polyclonal antibodies in rabbits. To obtaina purified antibody against TPR2, the immunity serum was further applied toHis-TPR2-Sepharose, and the bound antibodies were eluted with 0.2 M Gly-HClbuffer, pH 2.2. Thewashed antibodies were then preserved in 10mMPBS buffer,pH 7.4 with 0.01% (w/v) NaN3 and 20% (v/v) glycerol for further immunoblotanalysis.

For immunoblot analysis, 2-week-old seedlingswere rapidly frozen in liquidN and ground in extraction buffer (50 mM sodium P pH 7.0, 100 mMNaCl, 5 mM

EDTA, 0.1% [v/v] Triton X-100, 0.1% [w/v]sodium deoxycholate, and proteaseinhibitor tablet; Roche). The supernatant was collected after centrifugation at21,000g for 5min at 4°C. Approximately 10mg of total protein was separated ona 12% (w/v) SDS-polyacrylamide electrophoresis gel and transferred to a ni-trocellulose membrane, which was then probed with the appropriate primaryantibody (anti-GFP, 1:3000, Clontech; anti-Flag, 1:3000, Sigma-Aldrich; anti-HA,

1:3000, Roche) and horseradish-conjugated goat anti-mouse secondary antibody(1:3000, Promega). Signals were detected using an ECL Kit (Invitrogen).

Co-IP Analysis

Co-IP was performed as described in Hu et al. (2014). The leaves of three-week-old N. benthamiana seedlings were injected with Agrobacterium LBA4404harboring 35S:AFP2-6Flag and 35S:TPR2-GFP, or the truncated 35S:AFP2ΔE-6xFlag or 35S:AFP2ΔJ-6xFlag with 35S:TPR2-GFP, respectively, for 3 d. Totalproteinswere extracted using extraction buffer (50mM sodiumPpH 7.0, 100mM

NaCl, 5 mM EDTA, 0.1% [v/v] Triton X-100, 0.1% [w/v] sodium deoxycholate,20 mg mL21 MG132, and a protease inhibitor tablet) and incubated with GFP-Trap-A (Chromotek) for 4 h at 4°C. The beads were then washed three timesusingwashing buffer (50 mM sodium P pH 7.0, 100 mMNaCl, 5 mM EDTA, 0.1%[v/v] Triton X-100, 20 mg mL21 MG132 and a protease inhibitor tablet). Theimmunoprecipitates were separated on a 12% (w/v) SDS-polyacrylamide geland detected by immunoblot analysis with anti-GFP (Clontech) or anti-Flag(Sigma-Aldrich) antibodies, and the immunoblot signals were detected usingan ECL Kit (Invitrogen).

RNA Isolation and RT-qPCR

Total RNA was extracted from 2-week-old seedlings using TRIzol reagent(Invitrogen) according to the manufacturer’s instructions. First-strand cDNAwassynthesized from 1.5 mg DNase-treated RNA in a 20-mL reaction volume usingM-MuLV reverse transcriptase (Takara) with oligo(dT)18 primers. The relativetranscript level of each gene was quantified by qPCR using SYBR Green I MasterMix (04 707 516 001, Roche) and a Light Cycler 480 Real-Time PCR machine(Roche) according to a method described in Hu et al. (2014). At least three bio-logical replicates for each sample were performed to confirm the gene expressionpattern. IPP2 was used as an internal gene expression control. The gene-specificprimers used to detect the transcripts are listed in Supplemental Table S1.

Transient Transactivation Assay

The fragment containing the 2,675-bp region upstreamof the start codon of FTwas amplified and cloned into the pGreenII 0800-LUC vector to generate the re-porter construct. The full-lengthAFP2 orTPR2, or the truncatedAFP2 region, wasamplified and cloned into pGreenII-SK to generate different effector constructs. Allprimers used to generate these constructs are listed in Supplemental Table S1.Preparation of Arabidopsis mesophyll protoplasts from CO-HA/afp2 leaves andsubsequent transfections were performed as described in Yoo et al. (2007) withminormodifications.A dual-luciferase reporter assay system (Promega)was usedtomeasurefireflyLuc andRenilla luciferase (REN) activities. The RENgeneunderthe control of the cauliflower mosaic virus 35S promoter and the LUC gene were inthe pGreenII 0800-LUC vector. Relative REN activity was used as an internalcontrol, and LUC/REN ratios were calculated.

Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) experiments were carried out asdescribed in Gu et al. (2013) withminormodifications. Briefly, 5 g of 2-week-oldseedlings grown in LD conditions was fixed in fixation buffer (10 mM Tris-HCl,pH 8.0, 0.4 M Suc, 1 mM EDTA, 1 mM PMSF, 0.25% [v/v] Triton X-100, and 1%[v/v] formaldehyde) for 10–15 min under a vacuum. The fixation procedurewas then stopped by the addition of 0.125 M Gly for 10 min under a vacuum.After washing three times with 50 mL cold water, the fixed seedlings wereground to a fine powder in liquidN and suspended in extraction buffer I (10mM

Tris-HCl, pH 8.0, 0.4 M Suc, 5 mM EDTA, 5 mM b-mercaptoethanol, 1 mM PMSF,5mg/mL leupeptin, 1mg/mL aprotinin, 5mg/mL antipain, 1mg/mLpepstatin,and 50 mM MG-132). After filtration through Miracloth (EMD Millipore), thecells were centrifuged at 1000g for 20 min and washed with extraction buffer II(10 mM Tris-HCl, pH 8.0, 0.25 M Suc, 10 mM MgCl2, 1% [v/v] Triton X-100, 5 mM

EDTA, 5 mM b-mercaptoethanol, 1 mM PMSF, 5 mg/mL leupeptin, 1 mg/mLaprotinin, 5 mg/mL antipain, 1 mg/mL pepstatin, and 50 mM MG-132) to purifythe nuclei. The nuclei were then resuspended in 1 mL of nuclei lysis buffer(10 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% [w/v] SDS, 5 mM b-mercaptoeth-anol, 1 mM PMSF, 5 mg/mL leupeptin, 1 mg/mL aprotinin, 5 mg/mL antipain,1 mg/mL pepstatin, and 50 mM MG-132) and sonicated for 5 min (30 s on, 30 soff, low intensity) with a sonicator (Bioruptor; Diagenode). Approximately20 mg of chromatin was then diluted in 1 mL of ChIP dilution buffer (15 mM


Chang et al.







Tris-HCl, pH 8.0, 167 mM NaCl, 1 mM EDTA, 1% [v/v] Triton X-100, 1 mM

PMSF, 5 mg/mL leupeptin, 1 mg/mL aprotinin, 5 mg/mL antipain, 1 mg/mLpepstatin, and 50 mM MG-132) and incubated with rabbit polyclonal anti-acetylated histone H3 (with acetyl K9+K14+K18+K23+K27, catalog no.ab47915; Abcam) overnight at 4°C. The protein-DNA complexes were col-lected by incubation with 50 mL of equilibrated Protein G beads (Dynabeads;Invitrogen) for 2–3 h. The beads were sequentially washed with low-saltbuffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% [v/v] Triton X-100, 1 mM

PMSF, 5 mg/mL leupeptin, 1 mg/mL aprotinin, 5 mg/mL antipain, 1 mg/mLpepstatin, and 50 mM MG-132), high-salt buffer (low-salt buffer replacing150 mM NaCl with 500 mM NaCl), LiCl buffer (low-salt buffer replacing150 mM NaCl with 250 mM LiCl), and TE buffer (10 mM Tris-HCl, pH 8.0, and1 mM EDTA). Protein-DNA complexes were released by incubation with300 mL of ChIP elution buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 50 mM

NaCl, 1% [w/v] SDS, and 50 mg/mL proteinase K) for 1 h at 65°C. Immu-noprecipitated DNAwas purified using a PCR Purification Kit (New EnglandBiolabs), and qPCRwas conducted tomeasure the amounts of FT fragment ona Light Cycler 480 Real-Time PCR machine (Roche) using SYBR Green PCRMaster Mix. TUB2 was used to normalize the qPCR results in each ChIPsample. The primers used are specified in Supplemental Table S1.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis TAIR da-tabase under the following accession numbers: AFP1, At1g69260; AFP2,At1G13740; AFP3, At3g29575; AFP4, At3g02140; TPL, At1g15750; TPR2,At1g04130; CONSTANS, At5g15840; FT, At1g65480.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Identification of T-DNA insertion mutants ofAFP1, AFP2, AFP3, and AFP4.

Supplemental Figure S2. Immunoblot analysis of transgenic lines over-expressing AFP1, AFP2, AFP3, and APF4.

Supplemental Figure S3. Flowering times of mutants lacking various AFPgenes and of transgenic lines overexpressing these genes under LDconditions.

Supplemental Figure S4. Flowering times of mutants lacking various AFPgenes and of transgenic lines overexpressing these genes under SDconditions.

Supplemental Figure S5. The interaction of AFP2 and TPR2 in yeast andplants.

Supplemental Figure S6. Identification of T-DNA insertion mutantsof TPR2.

Supplemental Figure S7. Confirmation of the specificity of the anti-TPR2antibody and measurement of the protein accumulation of TPR2 andAFP2 in tpr2, AFP2-ox, and its crossed line.

Supplemental Figure S8. Comparison of the transcriptional level of FLC inCol, afp2, and AFP2-ox plants.

Supplemental Figure S9. Verification of the transgenic line harboring CO-HA/AFP2-ox.

Supplemental Figure S10. FT transcription levels and flowering time dif-ference among Col, afp2, AFP2-ox, CO-HA, and their crossed lines.

Supplemental Figure S11. MG132 treatment suppressed the degradationof CO-HA in transgenic CO-HA seedlings.

Supplemental Figure S12. CO-HA protein accumulation in CO-HA, AFP2-ox, cop1, and their crossed line.

Supplemental Table S1. Primers used in this study.

ACKNOWLEDGMENTS

We thank Professors George Coupland (Max Planck Institute for PlantBreeding Research, Cologne, Germany), Takato Imaizumi (University of

Washington, Seattle), and Ruth Finkelstein (University of California at SantaBarbara) for contributing related seeds. We also thank all the former colleaguesand students for their work at the beginning of this project.

Received July 13, 2018; accepted November 27, 2018; published December 4,2018.

LITERATURE CITED

Andrés F, Coupland G (2012) The genetic basis of flowering responses toseasonal cues. Nat Rev Genet 13: 627–639

Causier B, Ashworth M, Guo W, Davies B (2012a) The TOPLESS inter-actome: A framework for gene repression in Arabidopsis. Plant Physiol158: 423–438

Causier B, Lloyd J, Stevens L, Davies B (2012b) TOPLESS co-repressorinteractions and their evolutionary conservation in plants. Plant SignalBehav 7: 325–328

Chang G, Wang C, Kong X, Chen Q, Yang Y, Hu X (2018) AFP2 as thenovel regulator breaks high-temperature-induced seeds secondarydormancy through ABI5 and SOM in Arabidopsis thaliana. Biochem Bi-ophys Res Commun 501: 232–238

Clough SJ, Bent AF (1998) Floral dip: A simplified method forAgrobacterium-mediated transformation of Arabidopsis thaliana. Plant J16: 735–743

Corbesier L, Vincent C, Jang S, Fornara F, Fan Q, Searle I, Giakountis A,Farrona S, Gissot L, Turnbull C, et al (2007) FT protein movementcontributes to long-distance signaling in floral induction of Arabidopsis.Science 316: 1030–1033

Davis SJ (2009) Integrating hormones into the floral-transition pathway ofArabidopsis thaliana. Plant Cell Environ 32: 1201–1210

de Wit M, Galvão VC, Fankhauser C (2016) Light-mediated hormonalregulation of plant growth and development. Annu Rev Plant Biol 67:513–537

Fornara F, Panigrahi KC, Gissot L, Sauerbrunn N, Rühl M, Jarillo JA,Coupland G (2009) Arabidopsis DOF transcription factors act redun-dantly to reduce CONSTANS expression and are essential for a photo-periodic flowering response. Dev Cell 17: 75–86

Garcia ME, Lynch T, Peeters J, Snowden C, Finkelstein R (2008) A smallplant-specific protein family of ABI five binding proteins (AFPs) regu-lates stress response in germinating Arabidopsis seeds and seedlings.Plant Mol Biol 67: 643–658

Goralogia GS, Liu TK, Zhao L, Panipinto PM, Groover ED, Bains YS,Imaizumi T (2017) CYCLING DOF FACTOR1 represses transcriptionthrough the TOPLESS co-repressor to control photoperiodic flowering inArabidopsis. Plant J 92: 244–262

Graeff M, Straub D, Eguen T, Dolde U, Rodrigues V, Brandt R, Wenkel S(2016) MicroProtein-mediated recruitment of CONSTANS into a TOP-LESS trimeric complex represses flowering in Arabidopsis. PLoS Genet12: e1005959

Gu X, Wang Y, He Y (2013) Photoperiodic regulation of flowering timethrough periodic histone deacetylation of the florigen gene FT. PLoS Biol11: e1001649

Hayama R, Coupland G (2003) Shedding light on the circadian clock andthe photoperiodic control of flowering. Curr Opin Plant Biol 6: 13–19

Hu X, Kong X, Wang C, Ma L, Zhao J, Wei J, Zhang X, Loake GJ, Zhang T,Huang J, et al (2014) Proteasome-mediated degradation of FRIGIDAmodulates flowering time in Arabidopsis during vernalization. PlantCell 26: 4763–4781

Ito S, Song YH, Josephson-Day AR, Miller RJ, Breton G, Olmstead RG,Imaizumi T (2012) FLOWERING BHLH transcriptional activators con-trol expression of the photoperiodic flowering regulator CONSTANS inArabidopsis. Proc Natl Acad Sci USA 109: 3582–3587

Jaeger KE, Wigge PA (2007) FT protein acts as a long-range signal inArabidopsis. Curr Biol 17: 1050–1054

Jang S, Marchal V, Panigrahi KC, Wenkel S, Soppe W, Deng XW,Valverde F, Coupland G (2008) Arabidopsis COP1 shapes the temporalpattern of CO accumulation conferring a photoperiodic flowering re-sponse. EMBO J 27: 1277–1288

Johansson M, Staiger D (2015) Time to flower: Interplay between photo-period and the circadian clock. J Exp Bot 66: 719–730



















Joon Seo P, Jung JH, Park MJ, Lee K, Park CM (2013) Controlled turnoverof CONSTANS protein by the HOS1 E3 ligase regulates floral transitionat low temperatures. Plant Signal Behav 8: e23780

Lazaro A, Valverde F, Piñeiro M, Jarillo JA (2012) The Arabidopsis E3ubiquitin ligase HOS1 negatively regulates CONSTANS abundance inthe photoperiodic control of flowering. Plant Cell 24: 982–999

Lee LY, Fang MJ, Kuang LY, Gelvin SB (2008) Vectors for multi-colorbimolecular fluorescence complementation to investigate protein-protein interactions in living plant cells. Plant Methods 4: 24

Liu LJ, Zhang YC, Li QH, Sang Y, Mao J, Lian HL, Wang L, Yang HQ(2008) COP1-mediated ubiquitination of CONSTANS is implicated incryptochrome regulation of flowering in Arabidopsis. Plant Cell 20:292–306

Long JA, Ohno C, Smith ZR, Meyerowitz EM (2006) TOPLESS regulatesapical embryonic fate in Arabidopsis. Science 312: 1520–1523

Lopez-Molina L, Mongrand S, Kinoshita N, Chua NH (2003) AFP is anovel negative regulator of ABA signaling that promotes ABI5 proteindegradation. Genes Dev 17: 410–418

Lynch TJ, Erickson BJ, Miller DR, Finkelstein RR (2017) ABI5-bindingproteins (AFPs) alter transcription of ABA-induced genes via a varietyof interactions with chromatin modifiers. Plant Mol Biol 93: 403–418

Mathieu J, Warthmann N, Küttner F, Schmid M (2007) Export of FTprotein from phloem companion cells is sufficient for floral induction inArabidopsis. Curr Biol 17: 1055–1060

Nagel DH, Pruneda-Paz JL, Kay SA (2014) FBH1 affects warm temperatureresponses in the Arabidopsis circadian clock. Proc Natl Acad Sci USA111: 14595–14600

Nakamichi N, Kita M, Niinuma K, Ito S, Yamashino T, Mizoguchi T,Mizuno T (2007) Arabidopsis clock-associated pseudo-response regu-lators PRR9, PRR7 and PRR5 coordinately and positively regulateflowering time through the canonical CONSTANS-dependent photo-periodic pathway. Plant Cell Physiol 48: 822–832

Oh E, Zhu JY, Ryu H, Hwang I, Wang ZY (2014) TOPLESS mediatesbrassinosteroid-induced transcriptional repression through interactionwith BZR1. Nat Commun 5: 4140

Pauwels L, Barbero GF, Geerinck J, Tilleman S, Grunewald W, Pérez AC,Chico JM, Bossche RV, Sewell J, Gil E, et al (2010) NINJA connects theco-repressor TOPLESS to jasmonate signalling. Nature 464: 788–791

Putterill J, Robson F, Lee K, Simon R, Coupland G (1995) The CONSTANSgene of Arabidopsis promotes flowering and encodes a protein showingsimilarities to zinc finger transcription factors. Cell 80: 847–857

Ryu H, Cho H, Bae W, Hwang I (2014) Control of early seedling devel-opment by BES1/TPL/HDA19-mediated epigenetic regulation of ABI3.Nat Commun 5: 4138

Sawa M, Nusinow DA, Kay SA, Imaizumi T (2007) FKF1 and GIGANTEAcomplex formation is required for day-length measurement in Arabi-dopsis. Science 318: 261–265

Song YH, Smith RW, To BJ, Millar AJ, Imaizumi T (2012) FKF1 conveystiming information for CONSTANS stabilization in photoperiodicflowering. Science 336: 1045–1049

Song YH, Estrada DA, Johnson RS, Kim SK, Lee SY, MacCoss MJ,Imaizumi T (2014) Distinct roles of FKF1, GIGANTEA, and ZEITLUPEproteins in the regulation of CONSTANS stability in Arabidopsis pho-toperiodic flowering. Proc Natl Acad Sci USA 111: 17672–17677

Song YH, Shim JS, Kinmonth-Schultz HA, Imaizumi T (2015) Photope-riodic flowering: Time measurement mechanisms in leaves. Annu RevPlant Biol 66: 441–464

Suárez-López P, Wheatley K, Robson F, Onouchi H, Valverde F,Coupland G (2001) CONSTANS mediates between the circadian clockand the control of flowering in Arabidopsis. Nature 410: 1116–1120

Tang N, Ma S, Zong W, Yang N, Lv Y, Yan C, Guo Z, Li J, Li X, Xiang Y,et al (2016) MODD mediates deactivation and degradation of OsbZIP46to negatively regulate ABA signaling and drought resistance in rice.Plant Cell 28: 2161–2177

Tao Q, Guo D, Wei B, Zhang F, Pang C, Jiang H, Zhang J, Wei T, Gu H,Qu LJ, et al (2013) The TIE1 transcriptional repressor links TCP tran-scription factors with TOPLESS/TOPLESS-RELATED corepressors andmodulates leaf development in Arabidopsis. Plant Cell 25: 421–437

Turck F, Fornara F, Coupland G (2008) Regulation and identity of florigen:FLOWERING LOCUS T moves center stage. Annu Rev Plant Biol 59:573–594

Valverde F, Mouradov A, Soppe W, Ravenscroft D, Samach A, CouplandG (2004) Photoreceptor regulation of CONSTANS protein in photope-riodic flowering. Science 303: 1003–1006

Walter M, Chaban C, Schütze K, Batistic O, Weckermann K, Näke C,Blazevic D, Grefen C, Schumacher K, Oecking C, et al (2004) Visual-ization of protein interactions in living plant cells using bimolecularfluorescence complementation. Plant J 40: 428–438

Wang H, Pan J, Li Y, Lou D, Hu Y, Yu D (2016) The DELLA-CONSTANStranscription factor cascade integrates gibberellic acid and photoperiodsignaling to regulate flowering. Plant Physiol 172: 479–488

Wang L, Kim J, Somers DE (2013a) Transcriptional corepressor TOPLESScomplexes with pseudoresponse regulator proteins and histone deace-tylases to regulate circadian transcription. Proc Natl Acad Sci USA 110:761–766

Wang Y, Li L, Ye T, Lu Y, Chen X, Wu Y (2013b) The inhibitory effect ofABA on floral transition is mediated by ABI5 in Arabidopsis. J Exp Bot64: 675–684

Xu F, Li T, Xu PB, Li L, Du SS, Lian HL, Yang HQ (2016) DELLA proteinsphysically interact with CONSTANS to regulate flowering under longdays in Arabidopsis. FEBS Lett 590: 541–549

Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: Aversatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572

Zhai Q, Zhang X, Wu F, Feng H, Deng L, Xu L, Zhang M, Wang Q, Li C(2015) Transcriptional mechanism of jasmonate receptor COI1-mediateddelay of flowering time in Arabidopsis. Plant Cell 27: 2814–2828

Zhang B, Wang L, Zeng L, Zhang C, Ma H (2015) Arabidopsis TOE pro-teins convey a photoperiodic signal to antagonize CONSTANS andregulate flowering time. Genes Dev 29: 975–987

Zhu Z, Xu F, Zhang Y, Cheng YT, Wiermer M, Li X, Zhang Y (2010)Arabidopsis resistance protein SNC1 activates immune responsesthrough association with a transcriptional corepressor. Proc Natl AcadSci USA 107: 13960–13965

Zuo Z, Liu H, Liu B, Liu X, Lin C (2011) Blue light-dependent interaction ofCRY2 with SPA1 regulates COP1 activity and floral initiation in Ara-bidopsis. Curr Biol 21: 841–847


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