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The gated induction system of a systemic oral inhibitor, antiorigen, determines obligate short-day owering in chrysanthemums Yohei Higuchi a , Takako Narumi b , Atsushi Oda a , Yoshihiro Nakano a , Katsuhiko Sumitomo a , Seiichi Fukai b , and Tamotsu Hisamatsu a,1 a NARO Institute of Floricultural Science, National Agriculture and Food Research Organization (NARO), Fujimoto, Tsukuba, Ibaraki 305-8519, Japan; and b Faculty of Agriculture, Kagawa University, Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795, Japan Edited by George Coupland, Max Planck Institute for Plant Breeding Research, Cologne, Germany, and approved September 6, 2013 (received for review April 23, 2013) Photoperiodic oral induction has had a signicant impact on the agricultural and horticultural industries. Changes in day length are perceived in leaves, which synthesize systemic owering inducers (origens) and inhibitors (antiorigens) that determine oral initiation at the shoot apex. Recently, FLOWERING LOCUS T (FT) was found to be a origen; however, the identity of the corresponding antiorigen remains to be elucidated. Here, we report the identi- cation of an antiorigen gene, Anti-origenic FT/TFL1 family pro- tein (AFT ), from a wild chrysanthemum (Chrysanthemum seticuspe) whose expression is mainly induced in leaves under noninductive conditions. Gain- and loss-of-function analyses demonstrated that CsAFT acts systemically to inhibit owering and plays a predomi- nant role in the obligate photoperiodic response. A transient gene expression assay indicated that CsAFT inhibits owering by di- rectly antagonizing the ower-inductive activity of CsFTL3, a C. seticuspe ortholog of FT, through interaction with CsFDL1, a basic leucine zipper (bZIP) transcription factor FD homolog of Arabi- dopsis. Induction of CsAFT was triggered by the coincidence of phytochrome signals with the photosensitive phase set by the dusk signal; owering occurred only when night length ex- ceeded the photosensitive phase for CsAFT induction. Thus, the gated antiorigen production system, a phytochrome-mediated response to light, determines obligate photoperiodic owering response in chrysanthemums, which enables their year-round commercial production by articial lighting. T he transition from the vegetative to the reproductive phase is one of the most important developmental stages in the plant life cycle. The timing of owering during the year, which is an important adaptive trait that strongly inuences reproductive tness, is affected by both endogenous and environmental fac- tors. Changes in day length (photoperiod) are among the most important and reliable seasonal signals to plants to reproduce at favorable times of the year. In 1920, Garner and Allard (1) demonstrated that several plant species ower in response to changes in day length and described this phenomenon as pho- toperiodism.Plants are classied according to their photoperi- odic responses as short-day plants (SDP), in which owering occurs when the night length is longer than a critical minimum, long-day plants (LDP), in which owering occurs when the day becomes longer than some crucial length, and day-neutral plants. Within the SDP and LDP, there are obligate (qualitative) and facultative (quantitative) types of photoperiodic responses. Ob- ligate-type plants are those in which a particular photoperiod is an absolute requirement for the occurrence of a response. Chrysanthemum has become one of the most important horti- cultural crops since the discovery of photoperiodism because the owering time of this obligate SDP can be strictly controlled by the use of blackouts or articial lighting, day-length extension, or illumination during the middle of the long night [night break (NB)] to meet the demand for marketable owers throughout the year. In 1936, Chailakhyan proposed the concept of the owering stimulus origenfrom an experiment using chrysanthemum (2). Recent studies have demonstrated that FLOWERING LOCUS T (FT) and its orthologs, which are synthesized in the leaves of several species, act as origens (36). In Arabidopsis, FT moves into the shoot apical meristem (SAM) via the phloem and forms a transcriptional complex with a basic leucine zipper (bZIP) tran- scription factor, FD, at the SAM; transcription of oral regulator genes such as FRUITFULL (FUL) and APETALA 1 (AP1) is then activated, which leads to owering (7, 8). FT encodes a small pro- tein, origen, with similarity to phosphatidylethanol-aminebinding protein (PEBP). The PEBP gene family has evolved both acti- vators and repressors of owering. The FT family in Arabidopsis contains ve other members: TWIN SISTER OF FT (TSF ), TERMINAL FLOWER 1 (TFL1), BROTHER OF FT AND TFL1 (BFT ), MOTHER OF FT AND TFL1 (MFT ), and Arabidopsis thaliana CENTRORADIALIS homolog (ATC). FT and TSF are oral activators (911) whereas TFL1, ATC, and BFT are oral repressors (1214), and MFT is involved in seed germination (15). Although TFL1 and ATC are known to act non-cell-autonomously, they are expressed in the shoot apex and in vasculature tissue, re- spectively (16, 17). The concept of a oral repressor, or antiorigen, was pro- posed almost as early as that of a oral stimulus (18). A classical physiological study of many plant species suggested the existence of an antiorigenic stimulus synthesized in leaves (19). A grafting experiment using tobacco cultivars with different photoperiodic responses clearly indicated that antiorigenic signals synthesized in leaves under non-oral-inductive day-length conditions sys- temically inhibited owering (20). The existence of a oral inhibitor Signicance Photoperiodic oral initiation is thought to be regulated by a systemic owering inducer (origen) and inhibitor (anti- origen) produced in the leaves. Here, we show the discovery of an antiorigen (CsAFT) from chrysanthemum, which is pro- duced in the leaves under a noninductive photoperiod to sys- temically inhibit owering. This antiorigen production system prevents precocious owering and enables the year-round supply of marketable owers by manipulation of day length. Author contributions: Y.H. and T.H. designed research; Y.H., T.N., A.O., and T.H. per- formed research; Y.N., K.S., and S.F. contributed new reagents/analytic tools. Y.H. and T.H. analyzed data; and Y.H. and T.H. wrote the paper. The authors declare no conict of interest. This article is a PNAS Direct Submission. Data deposition: Sequence data obtained in this study have been deposited in the DNA Data Bank of Japan database (DDBJ), www.ddbj.nig.ac.jp/index-e.html [accession nos. AB839766 (CsAFT ), AB839767 (CsTFL1), AB839768 (CsFDL1), AB839769 (CsFDL2), and AB839770 (CsAFL2). 1 To whom correspondence should be addressed. E-mail: [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1307617110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1307617110 PNAS | October 15, 2013 | vol. 110 | no. 42 | 1713717142 PLANT BIOLOGY

The gated induction system of a systemic florigen, … · The gated induction system of a systemic floral inhibitor, antiflorigen, determines obligate short-day flowering in chrysanthemums

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The gated induction system of a systemic floralinhibitor, antiflorigen, determines obligate short-dayflowering in chrysanthemumsYohei Higuchia, Takako Narumib, Atsushi Odaa, Yoshihiro Nakanoa, Katsuhiko Sumitomoa, Seiichi Fukaib,and Tamotsu Hisamatsua,1

aNARO Institute of Floricultural Science, National Agriculture and Food Research Organization (NARO), Fujimoto, Tsukuba, Ibaraki 305-8519, Japan;and bFaculty of Agriculture, Kagawa University, Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795, Japan

Edited by George Coupland, Max Planck Institute for Plant Breeding Research, Cologne, Germany, and approved September 6, 2013 (received for reviewApril 23, 2013)

Photoperiodic floral induction has had a significant impact on theagricultural and horticultural industries. Changes in day length areperceived in leaves, which synthesize systemic flowering inducers(florigens) and inhibitors (antiflorigens) that determine floralinitiation at the shoot apex. Recently, FLOWERING LOCUS T (FT) wasfound to be a florigen; however, the identity of the correspondingantiflorigen remains to be elucidated. Here, we report the identi-fication of an antiflorigen gene, Anti-florigenic FT/TFL1 family pro-tein (AFT), from a wild chrysanthemum (Chrysanthemum seticuspe)whose expression is mainly induced in leaves under noninductiveconditions. Gain- and loss-of-function analyses demonstrated thatCsAFT acts systemically to inhibit flowering and plays a predomi-nant role in the obligate photoperiodic response. A transient geneexpression assay indicated that CsAFT inhibits flowering by di-rectly antagonizing the flower-inductive activity of CsFTL3, a C.seticuspe ortholog of FT, through interaction with CsFDL1, a basicleucine zipper (bZIP) transcription factor FD homolog of Arabi-dopsis. Induction of CsAFT was triggered by the coincidenceof phytochrome signals with the photosensitive phase set bythe dusk signal; flowering occurred only when night length ex-ceeded the photosensitive phase for CsAFT induction. Thus, thegated antiflorigen production system, a phytochrome-mediatedresponse to light, determines obligate photoperiodic floweringresponse in chrysanthemums, which enables their year-roundcommercial production by artificial lighting.

The transition from the vegetative to the reproductive phase isone of the most important developmental stages in the plant

life cycle. The timing of flowering during the year, which is animportant adaptive trait that strongly influences reproductivefitness, is affected by both endogenous and environmental fac-tors. Changes in day length (photoperiod) are among the mostimportant and reliable seasonal signals to plants to reproduce atfavorable times of the year. In 1920, Garner and Allard (1)demonstrated that several plant species flower in response tochanges in day length and described this phenomenon as “pho-toperiodism.” Plants are classified according to their photoperi-odic responses as short-day plants (SDP), in which floweringoccurs when the night length is longer than a critical minimum,long-day plants (LDP), in which flowering occurs when the daybecomes longer than some crucial length, and day-neutral plants.Within the SDP and LDP, there are obligate (qualitative) andfacultative (quantitative) types of photoperiodic responses. Ob-ligate-type plants are those in which a particular photoperiodis an absolute requirement for the occurrence of a response.Chrysanthemum has become one of the most important horti-cultural crops since the discovery of photoperiodism because theflowering time of this obligate SDP can be strictly controlled bythe use of blackouts or artificial lighting, day-length extension, orillumination during the middle of the long night [night break (NB)]to meet the demand for marketable flowers throughout the year.

In 1936, Chailakhyan proposed the concept of the floweringstimulus “florigen” from an experiment using chrysanthemum (2).Recent studies have demonstrated that FLOWERING LOCUST (FT) and its orthologs, which are synthesized in the leaves ofseveral species, act as florigens (3–6). In Arabidopsis, FT movesinto the shoot apical meristem (SAM) via the phloem and forms atranscriptional complex with a basic leucine zipper (bZIP) tran-scription factor, FD, at the SAM; transcription of floral regulatorgenes such as FRUITFULL (FUL) and APETALA 1 (AP1) is thenactivated, which leads to flowering (7, 8). FT encodes a small pro-tein, florigen, with similarity to phosphatidylethanol-amine–bindingprotein (PEBP). The PEBP gene family has evolved both acti-vators and repressors of flowering. The FT family in Arabidopsiscontains five other members: TWIN SISTER OF FT (TSF),TERMINAL FLOWER 1 (TFL1), BROTHER OF FT AND TFL1(BFT), MOTHER OF FT AND TFL1 (MFT), and Arabidopsisthaliana CENTRORADIALIS homolog (ATC). FT and TSF arefloral activators (9–11) whereas TFL1, ATC, and BFT are floralrepressors (12–14), and MFT is involved in seed germination (15).Although TFL1 and ATC are known to act non-cell-autonomously,they are expressed in the shoot apex and in vasculature tissue, re-spectively (16, 17).The concept of a floral repressor, or antiflorigen, was pro-

posed almost as early as that of a floral stimulus (18). A classicalphysiological study of many plant species suggested the existenceof an antiflorigenic stimulus synthesized in leaves (19). A graftingexperiment using tobacco cultivars with different photoperiodicresponses clearly indicated that antiflorigenic signals synthesizedin leaves under non-floral-inductive day-length conditions sys-temically inhibited flowering (20). The existence of a floral inhibitor

Significance

Photoperiodic floral initiation is thought to be regulated bya systemic flowering inducer (florigen) and inhibitor (anti-florigen) produced in the leaves. Here, we show the discoveryof an antiflorigen (CsAFT) from chrysanthemum, which is pro-duced in the leaves under a noninductive photoperiod to sys-temically inhibit flowering. This antiflorigen production systemprevents precocious flowering and enables the year-roundsupply of marketable flowers by manipulation of day length.

Author contributions: Y.H. and T.H. designed research; Y.H., T.N., A.O., and T.H. per-formed research; Y.N., K.S., and S.F. contributed new reagents/analytic tools. Y.H. andT.H. analyzed data; and Y.H. and T.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: Sequence data obtained in this study have been deposited in the DNAData Bank of Japan database (DDBJ), www.ddbj.nig.ac.jp/index-e.html [accession nos.AB839766 (CsAFT ), AB839767 (CsTFL1), AB839768 (CsFDL1), AB839769 (CsFDL2), andAB839770 (CsAFL2).1To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1307617110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1307617110 PNAS | October 15, 2013 | vol. 110 | no. 42 | 17137–17142

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in chrysanthemum leaves under non-floral-inductive day-lengthconditions was also suggested (21). These reports strongly sup-port the idea that an antiflorigenic signal produced in leaves maycontribute to photoperiodic responses in some plant species.Here, by using a reverse-genetic approach, we identified a

systemic floral inhibitor produced in the noninductive leaves ofchrysanthemum. Our results demonstrated that the phytochrome-mediated and gated production system of antiflorigen determinesobligate flowering response in chrysanthemum, which enablesyear-round commercial production of flowers by using artificiallighting.

ResultsReverseGenetic Screening of Systemic Floral Inhibitors in Chrysanthemum.To understand the molecular mechanisms of flowering in chry-santhemum, we applied a diploid wild chrysanthemum, Chrysan-themum seticuspe f. boreale (C. seticuspe), as a model system formolecular-genetic study. C. seticuspe shows an obligate photope-riodic flowering response in which flowering occurs under a >12-hdark period and is inhibited under a <10-h dark period (Fig. 1A).Recently, chrysanthemum orthologs of FT were identified, andit was confirmed that short day (SD)-induced CsFTL3 encodesa systemic floral inducer in C. seticuspe (22). To test whethernoninduced leaves produce a floral inhibitory signal, we conducteda localized photoperiodic treatment. Localized NB treatment com-pletely suppressed flowering of C. seticuspe, suggesting the existenceof an antiflorigenic stimulus produced in the leaves under non-floral-inductive conditions (Fig. 1B). Based on RNA-seq data, wedesigned a custom array to screen differentially expressed genesin leaves under SD and NB conditions (Datasets S1 and S2).Among genes with higher expression under NB, we identifiedone clone (CAC02.24543.C1) that showed high homology withTFL1C of Vitis vinifera, and named it as Anti-florigenic FT/TFL1family protein (AFT). Phylogenetic analysis revealed that CsAFTbelongs to the large TFL1/CEN/BFT clade (Figs. S1 and S2A). Wescreened >60,000 contig sequences derived from RNA-seq, andfound five FT/TFL1-like genes in C. seticuspe: three FT-like genes(CsFTL1, CsFTL2, CsFTL3), one TFL1/CEN-like gene (CsTFL1),

and CsAFT (Fig. S1). Among these, only CsFTL3, a florigen genein C. seticuspe (22), is highly expressed in the leaves under SDconditions (Fig. 1 C and D, Fig. S3A, and Dataset S1). The ab-solute expression level of CsFTL2 was very low under both SDand long-day (LD) conditions, and there were no remarkabledifferences in temporal expression of this gene between SD andNB conditions (Fig. 1C and Fig. S3A). CsTFL1, which is closelyrelated to Arabidopsis TFL1, was preferentially expressed inroot and shoot tips and was expressed at very low levels in leaves(Fig. S3B); there were no remarkable differences in CsTFL1expression under SD and LD conditions in leaves and shoot tips(Fig. 1C, Fig. S3B, and SI Text). CsFTL1 and CsAFT were highlyexpressed in the leaves under flower noninductive NB or LDconditions (Fig. 1 C and D and Fig. S3 A and B); however,constitutive expression of CsFTL1 in Chrysanthemum morifoliumrevealed that CsFTL1 has weak florigenic activity (Fig. S4). CsFTL1might function as an LD florigen similar to RICE FLOWERINGLOCUS T1 (RFT1) as suggested in rice, a facultative SDP (23).

CsAFT Acts Systemically to Inhibit Flowering. Sequence comparisonsbetween CsAFT and FT/TFL1 family proteins from other plantspecies showed that the CsAFT protein carries the functionallyimportant residues for TFL1-like activity, His88 (H) (24) andAsp144 (D) (25) (Fig. S2A). Constitutive expression of CsAFT(CsAFT-ox) in C. seticuspe and C. morifolium resulted in ex-tremely late flowering under SD conditions, indicating thatCsAFT has strong antiflorigenic activity (Fig. 2 A–C and Fig.S5A). Overexpression of CsAFT in Arabidopsis also resulted inlate flowering and morphological changes similar to TFL1-ox(14), suggesting that CsAFT acts as a floral inhibitor in Arabidopsis(Fig. S5G). To test the long-distance transmission of CsAFT geneproducts, we conducted a grafting experiment using CsAFT-oxplants. Wild-type plants grafted onto CsAFT-ox rootstock delayedflowering under SD conditions (Fig. 2E). Moreover, CsAFT pro-tein was detected in shoot tips of WT scions grafted onto trans-genic rootstock that overexpress HA-tagged CsAFT (CsAFT-HA),clearly demonstrating that CsAFT protein acts as a systemic floralinhibitor (Fig. 2F). Further, loss of function of CsAFT by RNAi

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Fig. 1. Photoperiod-dependent expression of FT/TFL1-like genes in chrysanthemum. (A) Flowering response of C. seticuspe grown for 7 wk under various daylengths. Data are means ± SEM (n = 10). (B) Schematic representation and flowering response under localized photoperiodic treatment. Black and white ovalsindicate leaves exposed to SD (8L/16D) and NB (SD plus 2 h NBwith red light), respectively. Thefigures indicate the number of SD or NB leaves. For NB0/SD10 andNB3/SD10, the lower part of each plant was covered with a polyvinyl chloride pipe and aluminum foil to prevent exposure to red light. Data aremeans± SEM (n=10). (C) Temporal expression patterns of FT/TFL1-like genes in leaves grown under SD and NB conditions for 7 d. Data are means ± SEM of three replicates. (D)Spatial expression of CsFTL3 and CsAFT genes under SD and long-day (LD) photoperiods. Plants were grown under SD or LD conditions for 12 d, and each tissuewas harvested at ZT4 (4 h after lights-on). Data are means ± SEM of 3 replicates.

17138 | www.pnas.org/cgi/doi/10.1073/pnas.1307617110 Higuchi et al.

resulted in reduced sensitivity to NB and promoted flowering(Fig. 2 G and H) but did not have any effect under strongly in-ductive SD conditions (Fig. S5B), indicating that CsAFT is es-sential for floral inhibition under noninductive NB conditions.Recently, antagonistic function for two FT paralogs has beenshown in sugar beet (Beta vulgaris) (26). BvFT1 prevents flow-ering during SDs and before vernalization by repressing the ex-pression of BvFT2, an activator of flowering. In CsAFT-ox plantsgrown under SD conditions, expression levels of CsFTL3 in leaveswere similar to those of the wild type (Fig. S5C) whereas expres-sion of CsAFL1, an AP1/FUL-like gene, and CsM111, the homologof AP1, was suppressed in shoot tips (Fig. 2D). This result suggeststhat late flowering of CsAFT-ox plants is not caused by suppressionof CsFTL3 induction in leaves and that the ratio of the floweringinducer (florigen) and inhibitor (antiflorigen) at the SAM maydetermine floral initiation.

CsAFT Antagonizes CsFTL3 Through Interaction with CsFDL1. The FT–FD protein complex triggers a cascade of positive transcriptionalsignals in floral induction (7, 8, 27) whereas the TFL1–FD pro-tein complex negatively regulates flowering (28). Two FD-likegenes (CsFDL1 and CsFDL2) (Fig. S2B and SI Text) were foundin C. seticuspe expressed sequence tags (ESTs). To examine theways in which interactions among CsAFT, CsFTL3, CsFDL1,and CsFDL2 affect several downstream genes, we developed

a transient gene expression system in protoplasts derived frommesophyll cells of C. seticuspe leaves (SI Text). We then exam-ined the subcellular localization and interaction of these proteinsin C. seticuspe protoplasts. Subcellular localization of CsFTL3and CsAFT was observed in both the cytoplasm and nucleuswhereas CsFDL1 and CsFDL2 clearly localized to the nucleus(Fig. S6A). Protein–protein interactions among CsFTL3, CsAFT,CsFDL1, and CsFDL2 revealed by bimolecular fluorescencecomplementation (BiFC) showed that CsFTL3 and CsAFT clearlyinteracted with both CsFDL1 and CsFDL2 in the nucleus (Fig.3A). This result indicates that CsAFT and CsFTL3 may functionin the same transcriptional complex.To evaluate the effect of transcriptional complexes composed

of CsFTL3, CsAFT, CsFDL1, and CsFDL2 on the regulation offloral-meristem identity genes, AP1/FUL-like genes (CsAFL1and CsAFL2) were selected for gene-expression analysis. Up-regulation of CsAFL1 and CsAFL2 in the SAM is one of theearliest events during the transition to the reproductive phase(22, 29) (Fig. S3C). Expression of CsAFL1 was up-regulatedseveralfold when CsFTL3 and CsFDL1 were coexpressed inprotoplasts, but none of the other combinations induced CsAFL1expression (Fig. 3B). Moreover, up-regulation of CsAFL1 byCsFTL3/CsFDL1 was partially suppressed by the addition ofCsAFT expression vectors (Fig. 3B). Expression of CsAFL2, an-other AP1/FUL-like gene, was strongly up-regulated by CsFTL3/CsFDL1 coexpression but was partially suppressed by the addi-tion of CsAFT expression vectors. Coexpression of CsFTL3/CsFDL2 also induced CsAFL2 expression, but the effect wasweaker than that observed in the CsFTL3/CsFDL1 combination.In transgenic plants overexpressing CsFDL1 fused to a tran-scriptional repressor domain (CsFDL1-SRDX), flowering wasseverely suppressed under SD conditions (Fig. S5E). Moreover,BiFC competition assay revealed that CsFTL3–CsFDL1 complexformation was repressed by CsAFT coexpression (Fig. S6B).These results suggest that the CsFTL3/CsFDL1 complex acti-vates expression of floral-meristem identity genes and that CsAFTantagonizes CsFTL3 function via interaction with the same inter-acting partner, CsFDL1, and thus inhibits subsequent flowerinitiation (SI Text).Interestingly, endogenous CsFTL3 expression was also in-

duced in our transient gene expression system when CsFTL3 andCsFDL1 were coexpressed (Fig. S7B). A recent study in potatosuggested an autoregulatory mechanism of StSP6A, an FT-like gene,in the photoperiodic regulation of tuberization (30). These findingssuggest that a positive feedback loop for CsFTL3 expression by

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Fig. 2. CsAFT acts as a systemic floral inhibitor. (A) Expression of CsAFT inWT and CsAFT-ox plants under LD (16L/8D) conditions. Leaves were har-vested at ZT0. (B and C) Overexpression of CsAFT dramatically delayedflowering under SD conditions. (D) Expression of CsAFL1 and CsM111 inshoot tips of WT and CsAFT-ox plants grown under SD conditions for 2 wk.(E) Flowering response of WT scions grafted onto WT, CsAFT-ox #2, andCsAFT-ox #16. Plants were grown under SD (8L/16D) conditions for 6 wk.Data are means ± SEM (n = 9 or 10). (F) Detection of CsAFT-3×HA protein atthe shoot tips of WT scions grafted onto CsAFT-HA plants, revealed by im-munoblot analysis. M, molecular marker. (G) Expression of CsAFT in WT andCsAFT-RNAi lines. Plants were grown under NB conditions for 2 wk. Leaveswere harvested at ZT4. (H) Flowering response of WT and CsAFT-RNAi linesunder NB conditions. Plants were grown for 5 wk under NB (8L/16D plus15 min of red light at middle of dark period) conditions. Red circles indicatethe positions of flower buds. Data are means ± SEM (n = 6).

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Fig. 3. Antagonistic action of CsAFT on flower induction by CsFTL3-CsFDL1complex. (A) BiFC assays showing interactions of CsFTL3-CsFDL1, CsFTL3-CsFDL2, CsAFT-CsFDL1, and CsAFT-CsFDL2. (Scale bar: 10 μm.) (B) Transientexpression of CsFTL3 and CsFDL1 induced expression of CsAFL1 and CsAFL2but was partially antagonized by CsAFT. Data are means ± SD (n = 7).

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the CsFTL3/CsFDL1 complex, or by downstream target genes,may exist in chrysanthemum (Fig. S7C and SI Text).

Phytochrome B Mediates NB-Induced Inhibition of Flowering. Inchrysanthemum, NB with red light effectively inhibits flowering,which is repromoted by subsequent exposure to far-red light,suggesting the involvement of phytochromes in this response (31,

32). NB response in C. seticuspe to differing light quality was alsotested with four light-emitting diode (LED) panels. NB withpeak irradiance at 530 nm (green) or 660 nm (red) light effec-tively inhibited flowering and expression of CsFTL3, and inducedexpression of CsAFT (Fig. S8). Moreover, the effects of red lighton flowering and expression of CsFTL3 and CsAFT were partiallyreversed by subsequent exposure to far-red light (Fig. S8), sug-gesting that red/far-red reversible types of phytochromes, such asphyB, are involved in this reaction. As expected, knock-down ofchrysanthemum PHYB (CsPHYB) (33) resulted in reduced sen-sitivity to NB by red light and in extremely early flowering (Fig. 4A and B). CsFTL3 was up-regulated in CsPHYB-RNAi plantswhereas CsAFT was down-regulated under NB conditions (Fig. 4C).Therefore, expression of both CsFTL3 and CsAFT is regulated bylight-signaling mediated byCsPHYB, and up-regulation ofCsFTL3and down-regulation ofCsAFTmay have resulted in the synergisticeffect on dramatic flowering phenotype observed in CsPHYB-RNAi lines.

Gated Induction of CsAFT and Dark-Dominant Flowering ofChrysanthemum. Because expression of CsAFT was up-regulatedunder NB or LD conditions, we expected that CsAFT wouldrespond acutely to light exposure under noninductive conditions.In rice, phytochrome-mediated induction of the floral repressorGhd7 (Grain number, plant height, and heading date 7) is gated bycircadian clock action (34). Ghd7 is acutely induced when phy-tochrome signals coincide with a photosensitive phase set dif-ferently by distinct photoperiods. We examined the effect of ashort light pulse on the induction of CsAFT mRNA. Plants weregrown under SD or LD conditions and shifted to continuousdarkness (DD), and the effects of a short red-light pulse on acuteinduction of CsAFT were tested at different time points. UnderSD conditions, CsAFT expression showed clear gating responsesto red-light pulses, with peak expression between 8 and 10 h afterdusk (Fig. 5A and Fig. S9). Under LD conditions, although rel-atively high expression levels were observed even in the early

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Fig. 4. Phytochrome-dependent regulation of NB response and CsAFTexpression. (A and B) Flowering response of WT and CsPHYB-RNAi plantsgrown under SD or NB conditions. Plants were pinched when transferredinto the growth chamber and were grown under SD (8L/16D) or NB (SD plus10 min of red light at the middle of each dark period) for 54 d. Data aremeans ± SEM (n = 14 or 16). (C) Expression of CsPHYB, CsFTL3, and CsAFTin leaves of WT and CsPHYB-RNAi lines. Plants were grown under NB for 7 d.Average values and SD from three RT-PCR datasets are shown. Data arerepresentative of two independent experiments.

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Fig. 5. Gated induction of CsAFT and dark-dominant flowering of chrysanthemum. (A) Analysis of gated expression of CsAFT. Plants were entrained underSD or LD conditions for 7 d. Plants were then transferred to continuous dark (DD) at dusk and exposed once to 10 min of red light at times differing by 2 h.Induction of CsAFT expression in leaves was analyzed 6 h after each exposure. Black and gray bars represent subjective night and day. Average values and SDfrom three RT-PCR datasets are shown. Data are representative of two independent experiments. (B) Effect of daily 10-min NB given at different times of thenight. Numbers on the horizontal axis indicate the length of dark period before NB was given. Data were collected 35 d after treatments started. Data aremeans ± SD (n = 10–12). (C) Expression of CsAFT and CsFTL3 in leaves grown for 7 d under various photoperiods. Data are means ± SEM of three replicates.Black and white bars represent dark and light periods, respectively. (D) Flowering response under non-24-h photoperiod. Plants were grown under 10L/14D(24-h cycle), 16L/14D (30-h cycle), and 22L/14D (36-h cycle) for 30 d. Data are means ± SEM (n = 10). Data are representative of three independent experiments.(E) Expression of CsAFT and CsFTL3 in leaves grown under normal SD (10L/14D), 16L/14D (30-h cycle), and LD (14L/10D) conditions for 7 cycles. Data are means ±SEM of three replicates.

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part of the night (due to induced expression of this gene underLD), peak photo-inducibility of CsAFT occurred at 8 or 10 hafter dusk (Fig. 5A and Fig. S9). Thus, the gate for maximalinduction of CsAFT opened at constant time after dusk regard-less of the entrained photoperiod conditions. In the floweringresponse of C. seticuspe, the most sensitive phase to NB occurs8–11 h after dusk (Fig. 5B). Because at least 12 h of uninter-rupted darkness are needed for minimum flowering, and a darkperiod of <10 h was fully inhibitory (Fig. 1A), we further ex-amined the photoperiod-dependent induction of CsAFT. Ex-pression levels of CsAFT were highly correlated with the extentof flower inhibition; CsAFT was strongly up-regulated under LDconditions (16L/8D, 14L/10D) and moderately induced under12L/12D, but was suppressed under SD (10L/14D, 8L/16D)conditions (Fig. 5C). These results suggest that gated expression ofCsAFT is responsible for NB response under SD conditions and forcritical day-length response. We also found that the flowering re-sponse of C. seticuspe under non-24-h light/dark cycles consistingof constant dark periods (16L/14D or 22L/14D) was not dra-matically attenuated compared with that under a 24-h SD (10L/14D) photoperiod (Fig. 5D). Moreover, expression of CsAFTand CsFTL3 under the non-24-h light/dark cycles was similar tothat under the 24-h SD photoperiod (Fig. 5E), indicating thatperception of day length in chrysanthemum relies on the absoluteduration of darkness rather than on the appropriate timing ofa photoperiodic response rhythm set by the dawn signal (Fig. 6A).

DiscussionAntiflorigenic Signaling Determines Obligate Short-Day Flowering inChrysanthemums. The physiological concept that inductive pho-toperiods cause leaves to synthesize a floral stimulus (“florigen”)is widely accepted. However, it has also been proposed that anantiflorigenic signal produced in leaves may regulate photope-riodic floral induction; the appropriate day-length would thenlead to removal of an antiflorigen (18, 19). Here, we reveal theexistence of an antiflorigenic FT/TFL1 family protein, AFT, inC. seticuspe, and clearly demonstrate that CsAFT protein acts asa systemic floral inhibitor—an antiflorigenic signal produced inleaves under noninductive conditions (Figs. 1B and 2). Thesefindings provide insight into the importance of systemic in-hibition of photoperiodic flowering by antiflorigens in manyplant species. Chrysanthemum is an obligate SDP that maintainsa vegetative state under noninductive LD photoperiod con-ditions (Fig. 1A). Unlike chrysanthemum, rice (Oryza sativa),a facultative SDP, can ultimately flower even under noninductiveLD conditions. Rice uses two florigen genes (Hd3a and RFT1)depending on day length, and RFT1 is suggested as an LD flo-rigen (23). In chrysanthemum, CsFTL1, which has weak flori-genic activity (Fig. S4), could function as an LD florigen similarto RFT1 in rice. It can be assumed that residual CsFTL3 andincreased CsFTL1 activity under noninductive photoperiodconditions (Fig. 1C and Fig. S3A) eventually make the plantflower, but the transition from vegetative to reproductive de-velopment is strictly suppressed under those conditions. There-fore, the photoperiodic transcriptional regulation of florigensalone is not sufficient to explain the obligate flowering responseof chrysanthemum; it is reasonable to assume that a systemicantiflorigen, which maintains the vegetative state at the shootapex during noninductive photoperiods, may be needed. Thenecessity of the antiflorigenic signal (CsAFT) to maintaining thevegetative state is supported by analysis of photoperiodic reac-tions of CsAFT-RNAi plants (Fig. 2 G and H). Thus, althoughphotoperiodic regulation of florigenic stimuli must be importantto achieve flowering (SI Text), the phyB-mediated antiflorigenproduction system (Fig. 6A) plays a predominant role in theobligate photoperiodic flowering response in chrysanthemum,allowing strict maintenance of the vegetative state under non-inductive photoperiod conditions (Fig. 6 B and C).

Possible Model of Day-Length Measurement in Photoperiodic Floweringof Chrysanthemum. Day length in many plant species is measuredthrough a circadian clock, which is an endogenous time-keepingcomponent that is reset by dawn and dusk signals. In Arabidopsis,expression of CONSTANS (CO), a critical activator of FT, isregulated by the circadian rhythm set by the dawn signal, andincreases toward evening (35, 36). Under LD conditions, whenhigh CO expression coincides with external light signals in theevening, the CO protein is stabilized and activates the tran-scription of FT (36, 37). In rice, flowering of wild-type plantswas extremely delayed under non-24-h light/dark cycles (24L/12Dor 36L/12D) compared with that under SD and LD photoperiods(38). Moreover, this complete inhibition of flowering in wild-typeplants under atypical entrainment conditions was lost in the se1(hd1) mutant, a rice counterpart of Arabidopsis co. This resultindicates that SE1 (Hd1) function is evening phase-specific andthat a circadian rhythm set by the dawn signal is critical for day-length recognition. These observations suggest that the dawnrather than the dusk signal plays a critical role for phase settingof the photoperiodic response rhythm in these two species. Wedemonstrated that, in C. seticuspe, if long night conditions (14 h)were given, flowering was successfully induced regardless of theday length (10, 16, or 22 h) (Fig. 5D). Moreover, expression ofCsAFT and CsFTL3 under atypical entrainment conditions (16L/14D) was similar to that under a 24-h SD (10L/14D) photoperiod(Fig. 5E), indicating that perception of day length in chrysan-themum relies on the absolute duration of darkness rather thanon the appropriate timing of a photoperiodic response rhythm setby the dawn signal. Our finding that the gate for maximal

A B

C

Fig. 6. Anti-florigenic regulation of obligate photoperiodic flowering re-sponse in chrysanthemum. (A) Model for induction of CsAFT in response tonatural day length extension and artificial lighting. The gate for maximalinduction of CsAFT mRNA opens at a constant time after dusk regardless ofthe entrained photoperiod. As the night becomes shorter, red-light signalsin the morning coincide with the photo-inducible phase of CsAFT and theninhibit flowering. Under NB conditions, midnight illumination coincides withthe photo-inducible phase of CsAFT. (B) Transcriptional regulation of CsAFTand CsFTL3 by photoperiod. High accumulation of CsAFT under noninductive(LD/NB) photoperiod overcomes residual floral-inductive activity of CsFTL3,which enables maintenance of the vegetative state. Upon a shift from LD toSD photoperiod, the CsAFT level rapidly decreases, whereas CsFTL3 is grad-ually induced by repeating SD cycles. (C) Systemic regulation of flowering byanti-florigen (AFT) and florigen (FTL3). AFT is synthesized in leaves undernoninductive photoperiods (LD/NB) and is translocated to the shoot apexwhere it then inhibits flowering. FTL3 is produced in leaves under inductive(SD) photoperiod independently of the production of AFT and then movesto the shoot apex where it induces flowering.

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induction of CsAFT opens at a constant time after dusk, regard-less of the entrained photoperiod (Fig. 5A and Fig. S9), alsosupports this notion. The mechanism for gated induction ofCsAFT mRNA appears similar to, yet differs from, that of Ghd7in rice. The gate for Ghd7 under SD photoperiods opens maxi-mally at 4 h after dusk whereas it opens around dawn (10 h afterdusk) under LD photoperiods (34). Therefore, timing for Ghd7gate opening is not determined solely by time elapsed from thebeginning of the dark period but is also affected by day length.In Pharbitis, flowering of which is induced by a single exposure todarkness, induction of PnFT occurs at a constant time after thelight-to-dark transition, regardless of the day length preceding theinductive dark period (39). Therefore, as in the case of Pharbitis,some time-keeping component set by the dusk signal may be in-volved in dark-time measurement in chrysanthemum. The gatefor CsAFT induction, which is initiated by light-to-dark transitionat dusk, is crucial for determining critical day length in chrysan-themum because CsAFT can be triggered by the coincidence ofmorning light with the photosensitive phase only when the nightlength becomes shorter than a critical minimum (Fig. 6A).The work reported here provides the molecular basis for a

long-standing physiological observation: that flowering is inhibi-ted by an antiflorigenic stimulus produced in noninductive leaves(Fig. 6 B andC). The gated induction of a floral repressor,CsAFT,a phyB-mediated response to light, determines the obligate pho-toperiodic flowering response in chrysanthemums. The mechanismfor gated induction of CsAFT has a strong link between flowering

and critical day length in chrysanthemums; flowering occurs onlywhen night length exceeds the duration of the photosensitive phasefor CsAFT induction. Elucidation of the mechanisms underlyingphotoperiodic flowering in chrysanthemums makes an importantcontribution to understanding of this plant’s reproductive successin its native environments. In addition, these findings will aid inachieving economic benefits from a stable year-round supply ofmarketable flowers that can be produced by manipulation ofphotoperiod using artificial lighting or blackouts. Further, iden-tification of the AFT gene will lead to improved understanding ofthe exquisite coordination that exists within the photoperiodic-flowering gene network for various plant species.

Materials and MethodsDetails are described in SI Materials and Methods. These details include in-formation on plant materials and growth conditions, localized photoperi-odic treatments, grafting experiments, RNA-seq and custom array analyses,the gene-expression study, plasmid construction, protoplast preparation,subcellular localization and BiFC assays, and immunoblot analyses. Primersused for expression analyses are provided in Dataset S3.

ACKNOWLEDGMENTS. We thank T. Nakagawa for the binary vectors andBiFC vectors and N. Mitsuda for the transient expression vector. We alsothank S. Kamei and T. Hashimoto for technical assistance. This work wassupported by the grant “Elucidation of biological mechanisms of photores-ponse and development of advanced technologies utilizing light” from theMinistry of Agriculture, Forestry and Fisheries of Japan.

1. Garner WW, Allard HA (1920) Effect of the relative length of day and night and otherfactors of the environment on growth and reproduction in plants. J Agric Res18:553–606.

2. Chailakhyan MK (1936) New facts in support of the hormonal theory of plant de-velopment. Dokl Akad Nauk SSSR 13:79–83.

3. Kobayashi Y, Weigel D (2007) Move on up, it’s time for change—mobile signalscontrolling photoperiod-dependent flowering. Genes Dev 21(19):2371–2384.

4. Zeevaart JA (2008) Leaf-produced floral signals. Curr Opin Plant Biol 11(5):541–547.5. Turnbull C (2011) Long-distance regulation of flowering time. J Exp Bot 62(13):

4399–4413.6. McGarry RC, Ayre BG (2012) Manipulating plant architecture with members of the

CETS gene family. Plant Sci 188-189:71–81.7. Abe M, et al. (2005) FD, a bZIP protein mediating signals from the floral pathway

integrator FT at the shoot apex. Science 309(5737):1052–1056.8. Wigge PA, et al. (2005) Integration of spatial and temporal information during floral

induction in Arabidopsis. Science 309(5737):1056–1059.9. Kobayashi Y, Kaya H, Goto K, Iwabuchi M, Araki T (1999) A pair of related genes with

antagonistic roles in mediating flowering signals. Science 286(5446):1960–1962.10. Kardailsky I, et al. (1999) Activation tagging of the floral inducer FT. Science 286(5446):

1962–1965.11. Yamaguchi A, Kobayashi Y, Goto K, Abe M, Araki T (2005) TWIN SISTER OF FT (TSF )

acts as a floral pathway integrator redundantly with FT. Plant Cell Physiol 46(8):1175–1189.

12. Bradley D, Ratcliffe O, Vincent C, Carpenter R, Coen E (1997) Inflorescence commit-ment and architecture in Arabidopsis. Science 275(5296):80–83.

13. Mimida N, et al. (2001) Functional divergence of the TFL1-like gene family in Arabi-dopsis revealed by characterization of a novel homologue. Genes Cells 6(4):327–336.

14. Yoo SJ, et al. (2010) BROTHER OF FT AND TFL1 (BFT ) has TFL1-like activity andfunctions redundantly with TFL1 in inflorescence meristem development in Arabi-dopsis. Plant J 63(2):241–253.

15. Xi W, Liu C, Hou X, Yu H (2010) MOTHER OF FT AND TFL1 regulates seed germinationthrough a negative feedback loop modulating ABA signaling in Arabidopsis. PlantCell 22(6):1733–1748.

16. Conti L, Bradley D (2007) TERMINAL FLOWER1 is a mobile signal controlling Arabi-dopsis architecture. Plant Cell 19(3):767–778.

17. Huang NC, Jane WN, Chen J, Yu TS (2012) Arabidopsis thaliana CENTRORADIALIShomologue (ATC) acts systemically to inhibit floral initiation in Arabidopsis. Plant J72(2):175–184.

18. Lang A, Melchers G (1943) Die photoperiodische reaktion von Hyoscyamus niger.Planta 33:653–702.

19. Thomas B, Vince-Prue D (1997) Photoperiodism in Plants (Academic, London).20. Lang A, Chailakhyan MK, Frolova IA (1977) Promotion and inhibition of flower for-

mation in a dayneutral plant in grafts with a short-day plant and a long-day plant.Proc Natl Acad Sci USA 74(6):2412–2416.

21. Tanaka T (1967) Studies on the regulation of Chrysanthemum flowering with specialreference to plant regulators. I. The inhibiting action of non-induced leaves on floralstimulus. J Jpn Soc Hortic Sci 36:77–85.

22. Oda A, et al. (2012) CsFTL3, a chrysanthemum FLOWERING LOCUS T-like gene, is a keyregulator of photoperiodic flowering in chrysanthemums. J Exp Bot 63(3):1461–1477.

23. Komiya R, Yokoi S, Shimamoto K (2009) A gene network for long-day flowering acti-vates RFT1 encoding a mobile flowering signal in rice. Development 136(20):3443–3450.

24. Hanzawa Y, Money T, Bradley D (2005) A single amino acid converts a repressor to anactivator of flowering. Proc Natl Acad Sci USA 102(21):7748–7753.

25. Ahn JH, et al. (2006) A divergent external loop confers antagonistic activity on floralregulators FT and TFL1. EMBO J 25(3):605–614.

26. Pin PA, et al. (2010) An antagonistic pair of FT homologs mediates the control offlowering time in sugar beet. Science 330(6009):1397–1400.

27. Taoka K, et al. (2011) 14-3-3 proteins act as intracellular receptors for rice Hd3a flo-rigen. Nature 476(7360):332–335.

28. Hanano S, Goto K (2011) Arabidopsis TERMINAL FLOWER1 is involved in the regu-lation of flowering time and inflorescence development through transcriptional re-pression. Plant Cell 23(9):3172–3184.

29. Nakano Y, Higuchi Y, Sumitomo K, Hisamatsu T (2013) Flowering retardation by hightemperature in chrysanthemums: Involvement of FLOWERING LOCUS T-like 3 generepression. J Exp Bot 64(4):909–920.

30. Navarro C, et al. (2011) Control of flowering and storage organ formation in potatoby FLOWERING LOCUS T. Nature 478(7367):119–122.

31. Cathey HM, Borthwick HA (1957) Photoreversibility of floral initiation in Chrysan-themum. Bot Gaz 119:71–76.

32. Sumitomo K, et al. (2012) Spectral sensitivity of flowering and FT-like gene expressionin response to a night break treatment in the chrysanthemum cultivar ‘Reagan’J Hortic Sci Biotechnol 87:461–469.

33. Higuchi Y, Sumitomo K, Oda A, Shimizu H, Hisamatsu T (2012) Day light quality affectsthe night-break response in the short-day plant chrysanthemum, suggesting differentialphytochrome-mediated regulation of flowering. J Plant Physiol 169(18):1789–1796.

34. Itoh H, Nonoue Y, Yano M, Izawa T (2010) A pair of floral regulators sets critical daylength for Hd3a florigen expression in rice. Nat Genet 42(7):635–638.

35. Suárez-López P, et al. (2001) CONSTANS mediates between the circadian clock andthe control of flowering in Arabidopsis. Nature 410(6832):1116–1120.

36. Yanovsky MJ, Kay SA (2002) Molecular basis of seasonal time measurement in Ara-bidopsis. Nature 419(6904):308–312.

37. Valverde F, et al. (2004) Photoreceptor regulation of CONSTANS protein in photo-periodic flowering. Science 303(5660):1003–1006.

38. Izawa T, et al. (2002) Phytochrome mediates the external light signal to repress FTorthologs in photoperiodic flowering of rice. Genes Dev 16(15):2006–2020.

39. Hayama R, Agashe B, Luley E, King R, Coupland G (2007) A circadian rhythm set bydusk determines the expression of FT homologs and the short-day photoperiodicflowering response in Pharbitis. Plant Cell 19(10):2988–3000.

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